hematology education

the education program for the annual congress of the European Hematology Association

ISSN 1872-5503

Education program for the

14th congress of the
European Hematology
Association

Berlin, Germany,
June 4-7, 2009
hematology education
the education program for the annual congress of the European Hematology Association

Education program for the

14th congress of the
European Hematology
Association

Berlin, Germany,
June 4-7, 2009
Copyright Information

©2009 by European Hematology Association. All rights reserved.

ISSN 1872-5503

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hematology education
the education program for the annual congress of the European Hematology Association

EHA Executive Board B Huntly, United Kingdom

W Fibbe, President, The Netherlands J Ingerslev, Denmark
R Foà, President Elect, Italy S Izraeli, Israel
E Hellström-Lindberg, Past President, Sweden B Lämmle, Switzerland
H Döhner, Treasurer, Germany L Malcovati, Italy
I Roberts, Secretary, United Kingdom M Manz, Switzerland
R Pieters, The Netherlands
EHA Councilors M Piris, Spain
E Berntorp, Sweden J San Miguel, Spain
C Camaschella, Italy B Schlegelberger, Germany
C Chomienne, France R Schlenk, Germany
U Jäger, Austria M Trneny, Czech Republic
C Lacombe, France A Urbano Ispizua, Spain
J Sierra, Spain A Waage, Norway
R Skoda, Switzerland
I Touw, The Netherlands EHA Education Committee
A Green, Chair, United Kingdom
EHA Local Organizing Committee 14th Congress M Arat, Turkey
R Hehlmann, Congress President, Germany E Berntorp, Sweden
B Dörken, Germany C Chomienne, France
D Niederwieser, Germany C Craddock, United Kingdom
E Thiel, Germany W Fibbe, The Netherlands
E Hellström-Lindberg, Sweden
EHA Scientific Program Committee 14th Congress D Jasmin, France
R Skoda, Chair, Switzerland F Lo Coco, Italy
H Büller, The Netherlands H Servé, Germany
C Chomienne, France
J Cools, Belgium EHA Press Committee 2009
F Lo Coco, Italy A Hagenbeek, Chair, The Netherlands
C Niemeyer, Germany W Fibbe, The Netherlands
I Peake, United Kingdom R Hehlmann, Germany
G Salles, France I Roberts, United Kingdom
M Theobald, The Netherlands R Skoda, Switzerland
S Thein, United Kingdom
EHA Publication Committee
EHA Scientific Program Committee Advisory W Fibbe, Chair, The Netherlands
Board 14th Congress M Cazzola, Italy
N Avent, United Kingdom R Foà, Italy
M Baccarani, Italy C Lacombe, France
C Beaumont, France S McCann, Ireland
E Berntorp, Sweden
J Cornelissen, The Netherlands European Hematology Association
C Craddock, United Kingdom E-mail: info@ehaweb.org,
N Cross, United Kingdom Website: www.ehaweb.org
T Enver, United Kingdom
C Gachet, France
G Gaidano, Italy
P Ghia, Italy
W Hofmann, Germany
Education Book Reviewers

We would like to thank the reviewers of the manuscripts for the Education Book.

M André, Belgium E Hellström-Lindberg, Sweden

N Avent, United Kingdom W Hofmann, Germany
M Baccarani, Italy S Izraeli, Israel
E Berntorp, Sweden M Laffan, United Kingdom
A Biondi, Italy T Lamy, France
M Björkholm, Sweden B Lämmle, Switzerland
F Caligaris-Cappio, Italy F Lo Coco, Italy
A Cohen, United Kingdom H Pahl, Germany
J Cools, Belgium R Pieters, The Netherlands
J Cornelissen, The Netherlands M Piris, Spain
C Craddock, United Kingdom P Rousselot, France
H Einsele, Germany G Salles, France
T Enver, United Kingdom J San Miguel, Spain
W Fibbe, The Netherlands R Schlenk, Germany
G Gaidano, Italy I Roberts, United Kingdom
A Gratwohl, Switzerland S Thein, United Kingdom
A Green, United Kingdom M Theobald, The Netherlands
Word of welcome

On behalf of the EHA Board and the Scientific Program Committee we would like to welcome you to
the beautiful city of Berlin. The EHA Congress is the largest and most comprehensive hematology meet-
ing in Europe with a world-class program of invited speakers and selected abstracts. The Education
Program covers a large spectrum of basic, translational and clinical research in hematology that will be pre-
sented by distinguished internationally recognized speakers. In addition to enjoying the presentations, we
hope you will find the peer-reviewed manuscripts in this Education Book a useful source of information
and references.

Radek Skoda Rüdiger Hehlmann

Chair Scientific Program Committee Congress President

© www.visitberlin.de
 hematology education: the education program for the annual congress
of the European Hematology Association - 2009; volume 3, issue 1

Table of Contents

Acute lymphoblastic leukemia Clinical trial design

1-7 Genomic profiling of acute lymphoblastic leukemia 89-95 Designing and interpreting quality-of-life and other
C.G. Mullighan patient-reported outcomes in clinical trials
F. Efficace, M.A. Sprangers
8-12 Genetic and epigenetic programs in MLL-rearranged acute
lymphoblastic leukemia 96-100 The use of genomics in clinical trial design
S.A. Armstrong R. Simon
13-16 The acute lymphoblastic leukemias of Down syndrome 101-105 Do commonly-used clinical trial designs reflect
S. Izraeli clinical reality?
E. Estey

Acute myeloid leukemia

Diffuse large B-cell lymphoma
17-23 Acute myeloid leukemia biology and leukemia stem cells
E. Gudgin, B. Huntly 106-111 Molecular basis of aggressive B-cell non-Hodgkin
lymphomas
24-29 Minimal residual disease detection in acute myeloid E. Hartmann, A. Rosenwald
leukemias
G. Saglio, E. Messa, D. Cilloni 112-117 Using molecular signatures to identify rational therapeutic
targets in diffuse-large B-cell lymphomas
30-37 Risk-adapted treatment for adult acute myeloid leukemia B. Chapuy, M. Shipp
J. Sierra, S. Brunet
118-122 Treatment options for relapsing patients with diffuse large
B cell lymphoma
C. Gisselbrecht
Bleeding disorders
38-43 von Willebrand factor: its role in hemostasis
and thrombosis Hematopoiesis
S.F. De Meyer, K. Vanhoorelbeke
123-127 Anatomy of the hematopoietic stem cell niche during
44-49 Phenotype, genotype and classification development
in von Willebrand disease E. Dzierzak
A. Goodeve
128-132 Population dynamics of hematopoietic stem cells
50-54 Treatment options in inherited von Willebrand disease H. Takizawa, M.G. Manz
A.B. Federici
133-139 Dynamic interactions between the nervous and immune
systems with the microenvironment regulate
hematopoietic stem cell migration and development.
Chronic lymphocytic leukemia The essential roles of SDF-1 and CXCR4
K. Lapid, T. Lapidot
55-60 Cellular origin of chronic lymphocytic leukemia
U. Klein

61-67 Clinical implications of novel biological markers in Hodgkin’s disease

chronic lymphocytic leukemia
R. Rosenquist 140-143 Nodular lymphocyte-predominant Hodgkin’s lymphoma
in children and adults
68-71 Risk-adapted chronic lymphocytic leukemia management J. Landman-Parker
P. Hillmen
144-150 The prognostic role of positron emission tomography
scan in Hodgkin’s lymphoma
A. Gallamini
Chronic myeloid leukemia
151-154 Recent advances in the treatment of early-stage
72-77 Stem cell persistence in chronic myeloid leukemia Hodgkin’s lymphoma
M.W. Deininger D.A. Eichenauer, A. Engert

78-82 Dynamics of mutated clones in chronic myeloid

leukemia patients
A. Hochhaus
Multiple myeloma

83-88 Second generation tyrosine kinase inhibitors: 155-160 Pathobiology, genetics and clinical outcome
which and when? of myeloma
G. Morgan, K. Boyd
H. Kantarjian, J. Cortes

Education program for the 14th congress of the European Hematology Association, Berlin, Germany, June 4-7, 2009
 hematology education: the education program for the annual congress
of the European Hematology Association - 2009; volume 3, issue 1

Table of Contents

161-165 Experimental agents knocking at the door of myeloma

treatment Stem cell transplantation
N.C. Munshi
242-249 Immunobiology of reduced intensity conditioning
166-171 Treatment options for myeloma in 2009 in hematopoietic stem cell transplantation
J. Bladé, L. Rosiñol B. Sandmaier, F. Baron

250-255 Reduced intensity conditioning as a platform for

immunotherapy
Myelodysplastic syndromes J. Barrett

172-176 Pathogenesis and basic aspects of myelodysplastic

syndromes 256-260 The role of reduced intensity conditioning in
N. Galili, A. Raza allogeneic hematopoietic stem cell transplantation
A. Gratwohl
177-180 Molecular pathogenesis of myelodysplastic syndromes:
identifying new therapeutic targets based on biology
S.D. Nimer
Thrombosis
181-187 Risk-adapted treatment of myelodysplastic syndromes
M. Cazzola, L. Malcovati 261-265 Genetics of thrombosis
P.H. Reitsma

266-270 Venous thrombosis: from bench to bedside and back

Myeloproliferative disorders T. Baglin

188-191 The role of somatic and hereditary genetic factors 271-275 New anticoagulants
in the pathogenesis of myeloproliferative neoplasms A. Kleinjan, H.R. Büller
R. Kralovics

192-199 Risk adapted approach to the treatment of primary

myelofibrosis Transfusion
J. Mascarenhas, R. Hoffman
276-281 Transfusion policies and practices in alloimmunized sickle
200-207 Interferon in the treatment of myeloproliferative disorders cell patients
J.-J. Kiladjian J.M. Moulds

282-287 Transfusion problems in stem cell transplant patients

D.H. Pamphilon
Pediatric hematological malignancies
288-293 Transfusion issues in neonatal and pediatric hematology
208-213 Myelodysplastic syndromes in children and adolescents
N.L.C. Luban
C.M. Niemeyer

214-220 Pediatric acute myeloid leukemia

G.J.L. Kaspers Uncommon lymphoproliferative disorders
221-226 Pediatric myeloproliferative neoplasms 294-301 Natural killer cell disorders
M.L. Randi, M.C. Putti
G. Semenzato, R. Zambello

302-307 Immunological and clinical features of

T cell large granular lymphocyte leukemia
Red cell disease
E. Matutes
227-232 Red cell membrane disorders
308-313 The treatment of hairy cell leukemia
N. Mohandas
D. Catovsky, M. Else
233-237 Predicting clinical severity in sickle cell anemia
M.H. Steinberg 314 Index of authors
238-241 Diagnosis and management of erythrocytosis
M. F. McMullin

Education program for the 14th congress of the European Hematology Association, Berlin, Germany, June 4-7, 2009
 Acute lymphoblastic leukemia

Genomic profiling of acute lymphoblastic leukemia

C.G. Mullighan A B S T R A C T

Department of Pathology, St Jude
Children’s Research Hospital,
Memphis, Tennessee, USA
Hematology Education:
the education program
for the annual congress of the
European Hematology Association
2009;3:1-7
Until recently, our understanding of the genetic basis of acute lymphoblastic leukemia (ALL) has
relied primarily on the identification of gross chromosomal changes. However, the full repertoire of
genetic changes involved in leukemogenesis has been incompletely understood. Microarray profiling
of genetic alterations across the genome, coupled with gene expression profiling and cancer gene
resequencing has transformed our understanding of ALL, and has identified new prognostic markers
and potential pathways for therapeutic intervention. Genetic alterations targeting key cellular path-
ways are common in ALL, and are significantly associated with disease subtype. Notably, genes regu-
lating lymphoid development are mutated in over two-thirds of B-progenitor ALL cases, and con-
tribute to leukemogenesis in experimental models. Specific genetic alterations are associated with
prognosis, notably deletion or sequence mutation of the early lymphoid transcription factor IKAROS
(IKZF1). Alteration of IKZF1 is common in BCR-ABL1 lymphoid leukemia, predicts very poor outcome
in BCR-ABL1 negative leukemia, with recurring new abnormalities at the time of relapse. Future stud-
ies will utilize next-generation sequencing approaches to perform genome-wide analysis of epigentic
alterations, sequence variations, and small RNA expression. Together, this will provide a comprehen-
sive understanding of the genetic alterations in ALL, and enable the development of rationally target-
ed therapies to improve outcome in this disease.

cute lymphoblastic leukemia (ALL) is have identified additional lesions (e.g., dele-

A the commonest childhood cancer, and

although associated with a generally
favorable prognosis,1,2 a substantial minority
of children fail therapy and relapse, with a
very poor outcome.3,4 ALL is associated with
a much worse prognosis in adults, in part
due to the high frequency of unfavorable
alterations, such as the Philadelphia chromo-
some.5-7 Moreover, with the exception of
imatinib, existing therapies are associated
with substantial short and long-term toxici-
ties. To improve outcome in both childhood
and adult ALL, novel targeted therapies
against rational therapeutic targets are
required.
Identification of suitable pathways for
therapeutic intervention has been limited by
our incomplete understanding of the genetic
alterations contributing to leukemogenesis.
ALL is characterized by multiple recurring
chromosomal abnormalities detectable on
cytogenetic analysis, including karyotypic
analysis and FISH, including aneuploidy
(e.g., high hyperdiploidy and hypodiploidy)
and translocations (e.g., t(12;21) ETV6-
RUNX1, t(1;19) TCF3-PBX1, t(9;22) BCR-
ABL1 and MLL rearrangement in B-progeni-
tor ALL), and rearrangement of the TLX1,
TLX3, LYL1, TAL1 and MLL genes in
T-ALL.8-12 However, several of these alter-
ations may be detected for years prior to the
onset of leukemia, and commonly do not
alone induce leukemia in experimental mod-
els, 13,14 suggesting that additional genetic or
epigenetic alterations must contribute to
leukemogenesis. Candidate gene studies
tion of ETV6 in B-ALL and CDKN2A/B and
NOTCH1 in T-ALL).15-18 The completion of
the human genome project and development
of microarray-based approaches has now
enabled identification of structural genetic
abnormalities in ALL on a genome-wide
scale at high resolution, and this has been
exceptionally informative in ALL.
Microarray analysis of genetic alterations in
acute lymphoblastic leukemia
Microarray platforms for analysis of
genetic alterations include bacterial artificial
chromosome (BAC) and oligonucleotide
arrays,19 in which these probes are either
spotted or synthesized directly onto the
array. BAC arrays reliably detect large alter-
ations and have been extensively used in
studies of cancer genomics,20-34 however the
probes used are large (often over 100 kb),
and consequently BAC arrays have a limited
ability to detect focal DNA copy number
alterations common in ALL.35-37 Oligonu-
cleotide arrays use short (20 to 50+)
nucleotide probes and can interrogate copy
number alterations at extremely high resolu-
tion. Two main designs are most widely
used: two color oligonucleotide array-CGH
platforms (e.g., Agilent, Roche Nimblegen),
and single channel arrays that genotype sin-
gle nucleotide polymorphisms (SNPs; e.g.,
Affymetrix, Illumina).38,39 Both provide high
quality, high-resolution data, but SNP array
platforms combine measurement of DNA
copy number state in addition to SNP geno-
typing. This allows the detection of both

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 1 |
14th Congress of the European Hematology Association
copy number alterations and copy-neutral loss of het-
erozygosity (copy-neutral LOH, also referred to as
acquired uniparental disomy) that may indicate redupli-
cation of a mutated or aberrantly methylated cancer
gene in the region of copy-neutral LOH. The majority of
published analyses in ALL have reported acquired
genetic changes; however, oligonucleotide arrays are
also being used to investigate the role of inherited
genetic variants, including copy number polymor-
phisms and SNP genotypes, in tumor susceptibility,
treatment responsiveness, and outcome.
The first studies profiling genome-wide copy number
alterations in ALL utilized BAC arrays, and demonstrat-
ed the ability of this approach to identify sub-micro-
scopic lesions,21,22,24-34,40 including deletions of ETV6, dele-
tion of CDKN2A/B and 9q34 duplication in T-ALL.
They also mapped complex structural changes, such as
translocation breakpoints24,29 and large amplifications
(e.g., intrachromosomal amplification of chromosome
21).24,25,27,29 The first report of SNP array analysis of ALL
examined ten cases, and identified multiple regions of
LOH, most commonly CDKN2A/B.41
Although these studies provided valuable proof of the
utility of genome-wide profiling of ALL, a limitation
was the low resolution of both array platforms used,
which prevented comprehensive identification of copy
number alterations. In the last four years, there has been
a dramatic increase in the feature density, and conse-
quently in the resolution, of oligonucleotide arrays.
Most reported SNP array studies in ALL have used
arrays incorporating 100,000 to 500,000 probes, with an
average intermarker resolution of 5 to 25 kb. Current
platforms employ millions of probes: the SNP 6.0 plat-
form (Affymetrix) incorporates approximately 900,000
SNP probes, and 900,000 non-SNP copy number probes.
Copy number data can be extracted from both probe
types and combined to identify copy number alterations
at sub kilobase resolution. Current oligonucleotide
array-CGH platforms employ over two million probes
(e.g., Roche NimbleGen). It is important to note that the
SNP array platform was initially designed as a genotyp-
ing tool to facilitate genetic association studies, and the
distribution of markers is not even across the genome,
resulting in suboptimal coverage of many genes, partic-
ularly with lower resolution arrays.42 Assessment of
tumor DNA copy number status requires careful bioin-
formatic analysis, in order to: (i) account for gross genet-
ic abnormalities, such as aneuploidy, during the normal-
ization process; (ii) accurately and robustly identify all
lesions, many of which are very focal and may be
missed by visual inspection of large genomic regions;
(iii) accurately distinguish inherited from somatic copy
number variants. We and others have developed and
adapted tools to perform these analyses.37,43,44
Consequently, the choice of analysis approach should
be carefully considered when performing and interpret-
ing SNP array studies in cancer.
Genetic alterations at diagnosis in acute lymphoblastic
leukemia
Several groups have now performed genome-wide
profiling of copy number alterations and LOH in
leukemic samples obtained at diagnosis in B-progenitor
and T-lineage ALL, and have identified multiple novel
| 2 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
regions of recurring genetic alterations targeting critical
cellular pathways, including lymphoid differentiation,
cell cycle, apoptosis, tumor suppression, oncogenes,
lymphoid signaling, and drug responsiveness.37,45-49 In a
study of 192 B-progenitor and 50 T-lineage ALL sam-
ples, we used three Affymetrix SNP arrays examining
over 350,000 markers at a resolution of approximately 5
kb.37 This identified over 50 recurring regions of copy
number alterations. An important finding was that,
unlike many solid tumors, large genomic abnormalities
and high level amplification were uncommon, with the
exception of the large whole and sub chromosomal
gains characteristic of hyperdiploid ALL and unbalanced
translocations (e.g., 1q+ in TCF3-PBX1 ALL). The
majority of recurring copy number alterations were
focal (less than 1 Mb), commonly with a single or few
genes in the minimal region of deletion. Few focal gains
were seen, notable exceptions including amplification
of MYB/AHI1, and gains flanking the NUP214-ABL1
translocation at 9q in T-lineage ALL. The recurring dele-
tions identified targeted lymphoid development (dis-
cussed below), tumor suppressors (NF1, PTEN, RB1,
ATM), apoptosis regulators (BTG1), putative lymphoid
signaling molecules (BTLA/CD200, TOX), micro-RNAs
(mir-15/16), steroid receptors (NR3C1, NR3C2), and a
number of genes with unknown function in oncogene-
sis and leukemia (C20orf94/MKKS, ADD3, DMD).
This study examined the principle recurring cytoge-
netic subtypes of ALL (high hyperdiploid, hypodiploid,
ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, MLL-
rearranged, and normal/miscellaneous karyotype) and
observed significant differences in the frequency and
nature of copy number alterations across ALL subtypes.
Overall, approximately six lesions per case were seen,
suggesting that gross genomic instability is not present
in the majority of cases. Less than one deletion per case
was observed in MLL-rearranged ALL, suggesting that
few additional genetic alterations are required to induce
this disease. In contrast, both ETV6-RUNX1 and BCR-
ABL1 ALL harbored over six lesions per case, many of
which were recurring and distinct between these sub-
types, suggesting an absolute requirement for these
lesions in the pathogenesis of each leukemia. Deletions
involving ADD3, C20orf94, ERG, ETV6, the fragile histi-
dine triad gene FHIT, TBL1XR1, a histone cluster at
6p22.22 were common in B-ALL but rare in T-ALL,
whereas CDKN2A/B deletion was more frequent in T-
ALL (72%) than B-ALL (34%).37
A key finding was alteration of genes regulating lym-
phoid development in over 40% of B-progenitor ALL.
Genes involved included the transcription factors PAX5
(one third of cases), the IKAROS family (IKZF1, IKZF2,
IKZF3), early B cell factor (EBF1), TCF3, LEF1, the
recombinase activating genes RAG1/2, B-cell linker
(BLNK) and the pre-B cell receptor gene VPREB1.
Several observations suggest that these alterations are
important in leukemogenesis. B-progenitor ALL is char-
acterized by a block in differentiation at the pro- to pre-
B cell stage of development, and the genes targeted by
copy number alterations are most commonly transcrip-
tion factors required for B-cell commitment and differ-
entiation. Targeted deletion of Pax5 and Ebf1 in mice
results in arrested lymphoid development at this pro- to
pre-B stage of development. Furthermore, PAX5, which
 Berlin, Germany, June 4-7, 2009

is required for normal B-lymphoid commitment and dif-

ferentiation50-53 is targeted by deletions, focal internal
amplification, sequence mutations, and novel transloca-
tions,48,54-59 each of which impair the normal transactivat-
ing function of PAX5 in vitro.37 Furthermore, the frequen-
cy and pattern of lesions in this pathway is again asso-
ciated with ALL subtype. All hypodiploid ALL cases
have PAX5 deletions, and commonly concomitant PAX5
point mutations, as well as other lesions in the B cell
development pathway. Notably, hypodiploidy is associ-
ated with poor outcome, and studies in other cohorts
have observed that an increasing number of lesions tar-
geting B lymphoid development are associated with
poor outcome.60 Furthermore, deletion of IKZF1
(IKAROS) is a near obligate lesion in BCR-ABL1 lym-
phoid leukemia,46 and is associated with poor prognosis
in BCR-ABL1 negative ALL, suggesting a central role of
IKAROS in leukemogenesis and responsiveness to ther-
apy.60 Finally, experimental modeling of individual genes
in the B-cell development pathway has established a
role of these lesions in leukemogenesis. In a retroviral
bone marrow transplant model of BCR-ABL1 ALL, Pax5
haploinsufficiency increases the penetrance and reduces
the latency of ALL.61 Similarly, following chemical or
retroviral mutagenesis, Pax5 haploinsufficiency dramat-
ically increases the penetrance of B-ALL.62 In both mod-
els, the development of leukemia is accompanied by the
acquisition of additional genetic changes also observed
in the analogous human leukemias, such as deletion of
Cdkn2a/b, and deletion or sequence mutation of the sec-
ond Pax5 allele, suggesting that these lesions are
required for acquisition of the full leukemic phenotype.

Genome wide profiling of T-lineage acute lymphoblastic

leukemia Figure 1. DNA copy number alterations in ALL. (A) Log2-
ratio copy number heatmap of Affymetrix SNP array wide
Genome wide profiling has also been revealing in T- copy number data in 126 representative pediatric B-pro-
lineage ALL. BAC and oligonucleotide arrays have been genitor and T-lineage ALL cases. Blue is deletion, red is
gain. (B) Copy number heatmap of 61 pediatric ALL cases
used to identify copy number alterations accompanying with copy number alterations involving PAX5 at 9p13.2.
novel structural abnormalities, such as the NUP214- Data is taken from ALL patients reported in (37), and the
ABL1 and SET-NUP214 fusions,63,64 and focal copy num- figure is reproduced with permission from Seminars in
ber alterations involving and dysregulating TAL1, Hematology 2009;46:3-15, Copyright Elsevier.
LMO2, RB1, PTEN, FBXW7 and MYB.37,65-70 Recently, a
novel subtype was of T-ALL was identified with aber-
rant immunophenotype reminiscent of early thymic
progenitors and very poor outcome.71 These atypical T-
ALL cases have an unusually high burden of copy num-
ber alterations on SNP array analysis suggestive of
genomic instability, although the underlying genetic
defect remains to be identified.

Genomic analysis of BCR-ABL1 leukemia

An important area of research is the use of genomic
approaches to identify novel genetic alterations con-
tributing to poor outcome in ALL. One example is
leukemia associated with the constitutively active
kinase, BCR-ABL1, which is a hallmark of chronic
myeloid leukemia (CML) and which typically responds
favorably to tyrosine kinase inhibitors,72 and BCR-ABL1
de novo ALL, which has a poor prognosis.73 The mecha-
nisms responsible for these distinct biologic and clinical
phenotypes are incompletely understood, and previous-
ly have been attributed, at least in part, to the BCR-
ABL1 isoform (p185/p190 v. p210)74 and the lineage and
maturational stage of the leukemia initiating cell.75,76
Figure 2. Mutations in genes regulating B-lymphoid devel-
opment in B-progenitor ALL. The differentiation of
hematopoietic stem cells to mature B cells is regulated by
a controlled hierarchy of transcription factors that enforce
B-lineage commitment and differentiation, and suppress
differentiation to alternate (T-lymphoid, myeloid) lineages.
These transcription factors are targeted by genetic alter-
ations in over 60% of B-progenitor ALL cases.37,46,60 Notably,
the genetic alterations affect transcription factors control-
ling differentiation up to the pre-B cell stage of develop-
ment, at which differentiation is arrested in many B-ALL
cases. Additional known or putative B-lymphoid regulatory
genes targeted less frequently in ALL include BLNK, IKZF2,
IKZF3, MEF2C, RAG1/2 and SOX4.

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 3 |
14th Congress of the European Hematology Association
SNP array studies of ALL and CML incorporating 43
BCR-ABL1 de novo ALL cases, and 128 samples obtained
from 90 CML patients at key stages of disease, includ-
ing chronic phase (N=64), accelerated phase (N=15) and
blast crisis (22 myeloid and 9 lymphoid), have identified
lesions significantly associated with lineage and disease
progression in BCR-ABL1 leukemia.77,78 Notably, few
copy number alterations are present in chronic phase
CML, and the only recurring lesions are deletions or
gains involving the breakpoints of BCR and ABL1. In
contrast, deletion of IKZF1 (IKAROS) is very common
in de novo BCR-ABL1 ALL, and at the progression of
CML to lymphoid blast crisis, but not chronic phase
CML.78 Moreover, serial analysis of CML samples
demonstrated that progression to lymphoid blast crisis
is accompanied by the acquisition of deletions also seen
at similar frequency in de novo BCR-ABL1 ALL, including
deletions of CDKN2A/B, PAX5, C20orf94, RB1, MEF2C
and EBF1.78-79
The high frequency of IKZF1 deletion in BCR-ABL1
ALL (36 of 43 cases) is notable, as prior studies had
observed a high frequency of expression of aberrant
IKZF1 transcripts in ALL.80-86 IKZF1 has two zinc finger
domains: N-terminal zinc fingers that mediate DNA-
binding, and C-terminal zinc fingers that mediate
dimerization.87 One third of ALL cases with IKZF1 dele-
tion have a focal lesion involving the N-terminal zinc
fingers. This results in expression of an internally trun-
cated isoform, Ik6, that cannot bind DNA yet retains
the C-terminal zinc fingers and may still dimerize, and
act as a dominant negative isoform. The high frequency
of expression of this isoform in BCR-ABL1 ALL and
lymphoid blast crisis CML suggests a central role in the
pathogenesis and poor outcome of these diseases.
Genomic profiling of high-risk leukemia
Relapse is most frequent in specific ALL subtypes,
including BCR-ABL1 positive, MLL-rearranged and
hypodiploid ALL, but is seen across the spectrum of
ALL. Two approaches have been used to identify the
genetic basis of treatment failure: genomic analysis of
samples obtained at diagnosis from children with high-
risk ALL, and comparative profiling of matched diagno-
sis and relapsed ALL samples.
In a study that incorporated SNP array profiling, gene
expression analysis, and candidate gene resequencing in
a cohort of 221 children with high-risk ALL (the
Children’s Oncology Group P9906 cohort, that exclud-
ed known high-risk subtypes, such as BCR-ABL1 and
hypodiploid ALL), we identified a high significant asso-
ciation between deletion or sequence mutation of
IKZF1 and poor outcome.60 Children with IKZF1 alter-
ation had a five-year cumulative incidence of relapse of
73.4% compared to 25% without IKZF1 alteration.
This was confirmed in a validation cohort of St Jude
childhood ALL cases. Furthermore, IKZF1 alteration
was significantly associated with high levels of minimal
residual disease (MRD) early in therapy in both P9906
and St Jude cohorts. Moreover, IKZF1 alteration
remained significantly associated with poor outcome in
multivariable analyses incorporating age, leukocyte
count, subtype and levels of MRD, suggesting that
detection of IKZF1 alteration will be useful in the initial
management of newly diagnosed ALL cases. An addi-
| 4 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
tional finding was that the poor outcome, IKZF1 mutat-
ed BCR-ABL1 negative cases in this study shared a sim-
ilar gene expression profile to BCR-ABL1 positive ALL.
A similar subtype of “BCR-ABL1-like” childhood ALL
cases with poor outcome has also been recently report-
ed by den Boer et al.88 This suggests that IKZF1 is an
important determinant of the biology of both BCR-
ABL1 positive and negative disease, and suggests that
BCR-ABL1 negative cases have hitherto unidentified
kinase mutations. Ongoing efforts profiling genomic
alterations in adult ALL, which has a substantially
worse outcome than pediatric ALL, will be of great
interest.
The studies described above demonstrate that genet-
ic alterations present in diagnosis ALL samples are
important in leukemogenesis, and predict risk of subse-
quent relapse. A logical next step was to compare the
genetic alterations present at relapse with those present
at the time of diagnosis. We analyzed 61 matched diag-
nosis-relapse B-progenitor and T-lineage ALL cases, and
demonstrated that over 92% of patients exhibited dif-
ferences in the patterns of copy number alterations from
diagnosis to relapse. Importantly, profiling of the pat-
tern of deletions at antigen receptor loci demonstrated a
common clonal origin of the diagnosis and relapse sam-
ples.89 Of these cases, only one third showed simple
clonal evolution, with retention of all copy number
alterations detected at diagnosis, and the acquisition of
new lesions. Over 50% of cases acquired new lesions
and lost copy number alterations present at diagnosis,
but had a common clonal origin. Moreover, backtrack-
ing of relapse-acquired lesions using genomic PCR
showed that in most cases, the relapse clone was pres-
ent at diagnosis, but at low levels not detectable using
the SNP arrays. This has provided important insights
into the mechanism of relapse. Specifically, many
patients harbor multiple clones at the time of diagnosis,
and the lesions present in each clone confer resistance to
therapy and promote subsequent relapse. Also, among
the commonest deletions acquired at the time of relapse
were those targeting CDKN2A/B and IKZF1 (and relat-
ed IKAROS family members), again suggesting that
these lesions have important roles in resistance to ther-
apy.
Future directions
High resolution, genome wide profiling of genetic
alterations using microarray methodologies has provid-
ed important insights into the mechanisms of leukemo-
genesis and therapeutic resistance in ALL that had pre-
viously been unsuspected from low-resolution genetic
approaches. It is likely that ongoing genomic profiling
studies will continue to provide valuable insights. The
ability to identify copy number alterations using
microarray approaches is critically dependent on array
design and resolution. Notably, several key lesions in
ALL, such as deletion of IKZF1, are poorly detected
using arrays that incorporate as many as 250,000
probes, and are only robustly detected using newer,
multimillion feature arrays.90 Consequently, the full
repertoire of copy number alterations in ALL remains to
be defined and tiling analysis of leukemia genomes is
required. Furthermore, these data have examined only
type of genetic alteration in a genome wide fashion.

Given the tremendous insights gained from analysis of
copy number alterations, it is now logical to perform
genome-wide analysis of sequence variation, epigenetic
changes, and gene expression, and this is a major focus
of many cancer genomics projects. Acquisition, analy-
sis, and integration of these massive datasets will be
challenging, but is becoming attainable with the rapid
advances in next generation sequencing technologies
that may eventually supplant microarray analysis of
genetic alterations.91-93 Together, these studies offer the
promise of new targets, new therapies, and improved
outcome in ALL.
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92. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B.
Mapping and quantifying mammalian transcriptomes by
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93. Shendure J. The beginning of the end for microarrays? Nat
Methods 2008;5:585-7.
 Acute lymphoblastic leukemia

Genetic and epigenetic programs in MLL-rearranged

acute lymphoblastic leukemia

S.A. Armstrong A B S T R A C T

Division of Hematology/Oncology,
Children’s Hospital, Department of
Pediatric Oncology, Boston, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
Mixed lineage leukemia (MLL)-rearranged leukemias represent a subset of human leukemias that
often have a poor prognosis. Recent studies have focused on characterization of the aberrant gene
expression and epigenetic programs have begun to provide insight into the mechanisms by which
MLL-translocations induce both acute lymphoblastic leukemia and acute myelogenous leukemia.
Wildtype MLL is a histone methyltransferase that modifies histone H3 on lysine 4. However, MLL-
fusion proteins generated by the MLL-translocations interact with other histone modifying enzymes
to direct inappropriate histone modifications as part of the mechanism by which they induce aberrant
gene expression. Future studies promise to guide us to therapeutics that target the epigenetic pro-
grams in these difficult to treat leukemias.

2009;3:8-12

eukemias bearing translocations involv- Subsequently, over 30 different transloca-

L ing chromosome 11q23 are of particular

interest due to unique clinical and bio-
logical characteristics. 11q23 rearrangements
are found in over 70% of leukemias in
infants of less than 1 year of age whether the
immunophenotype is more consistent with
acute lymphoblastic leukemia (ALL) or acute
myelogenous leukemia (AML).1 11q23
rearrangements are also found in a subset of
ALL cases diagnosed in adults. Importantly,
infants diagnosed with ALL harboring an
11q23 translocation have a particularly poor
prognosis with an overall survival of less
than 50%, whereas children with ALL har-
boring other translocations have an overall
survival of over 80%. Leukemias that devel-
op as a result of treatment with topoiso-
merase II inhibitors frequently harbor 11q23
rearrangements and have a poor prognosis.2
Some infant leukemias express cell surface
antigens characteristic of both lymphoblasts
and monoblasts, and are often designated
Acute Biphenotyic Leukemias (ABL). The
association of 11q23 rearrangements with
either ALL, AML, or ABL is unique in that
most other chromosomal rearrangements
tend to be associated with leukemias of a
particular hematopoietic lineage. These
observations led to the name mixed lineage
leukemia (MLL) for the gene that resides on
11q23.
The most common MLL translocation
found in lymphoblastic leukemias is the
t(4;11)(q21;q23). Multiple groups cloned the
MLL-AF4 gene formed as a result of this
translocation in the early 1990s.3-6 MLL-AF4
encodes a protein of 2304 amino acids, with
the NH2-terminal with approximately 1400
amino acids derived from MLL on chromo-
some 11, and COOH-terminal amino acids
from the AF4 gene on chromosome 4.
tions have been identified, all of which
appear to produce a fusion protein possess-
ing the NH2-terminus of MLL fused in-frame
to the COOH-terminus of the fusion part-
ner.7 Of interest, a subset of t(4;11) ALLs also
express the reciprocal product (AF4-AML),
suggesting this fusion protein might also
have pathogenic importance.8 While MLL
translocations can be found in either ALL or
AML, particular translocations show lineage
specificity with the t(4;11) found most often
in ALL and the t(9;11)(p21;q23) found most
often in AML. This points to the transloca-
tion partner as having a role in the disease
phenotype, and to functional heterogeneity
of MLL-fusions, but the molecular details of
these associations are unclear.
Mixed lineage leukemia function in
hematopoiesis
The MLL gene encodes a 3969 amino acid
DNA-binding protein that possesses multi-
ple recognizable protein motifs, including an
NH2-terminal DNA binding domain, tran-
scriptional activation and repression
domains, and a COOH-terminal SET
domain that contains histone H3 lysine 4
(H3K4) methyltransferase activity.9,10 Recent
biochemical studies have identified MLL as a
member of a large multi-protein complex
that contains members involved in chro-
matin modification/remodeling. Notably,
the complex includes histone deacetylases
(HDACs) and members of the Swi/Snf chro-
matin remodeling complex.10 Also, MLL is
recruited to the promoters of select cell cycle
regulatory genes by the protein product of
the MEN1 tumor suppressor gene, suggest-
ing a role for MLL in tumor suppression and
cell cycle control.11,12 These data support the
hypothesis that the MLL protein regulates

|8| Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

gene expression via chromatin modification.
Analysis of MLL knockout mice suggests that MLL
plays an important role in development and
hematopoiesis through maintenance of appropriate
homeotic (Hox) gene expression.13,14 Detailed studies
assessing the specific role of MLL in hematopoietic
development have shown that it is necessary for defini-
tive hematopoiesis and expansion of hematopoietic
progenitors and stem cells found in the aorta-gonad-
mesonephros (AGM) region of the developing
embryo.15 Also, MLL plays a critical role in adult HSC.16
The defect in hematopoietic progenitor expansion can
be rescued by re-expression of Hox genes confirming
the importance of MLL-mediated Hox gene expression
during hematopoiesis.
Multiple studies have demonstrated the ability of Hox
genes to induce leukemia in mice, and the t(7;11)
(p15;p15) translocation found in some human AML
results in a fusion of the HOXA9 gene to the nucleo-
porin NUP98.17,18 Given the apparent importance of Hox
genes in leukemogenesis, it seems likely that transloca-
tions involving MLL, a known regulator of HOX genes,
alters expression of HOX genes that are important for
leukemogenesis. Further support for HOX genes as cen-
tral regulators of MLL-induced leukemogenesis comes
from gene expression studies that have found multiple
HOXA cluster genes more highly expressed in MLL-
rearranged myelogenous and lymphoblastic leukemias
as compared to MLL-germline leukemias,19-22 and exper-
iments using RNAi, which demonstrate a continued
requirement for HOXA cluster genes and MEIS1.23,24
Gene expression programs in mixed lineage leukemia-
rearranged acute lymphoblastic leukemia
Gene expression studies of human MLL-rearranged B-
precursor ALL demonstrated that hundreds of genes are
differentially expressed when compared to other B-pre-
cursor ALLs.19,20,22,25 Based on the magnitude of the differ-
ences in gene expression, we proposed that MLL-
translocations specify a unique lymphoblastic leukemia.
Other large gene expression studies have also shown
that ALLs with distinct chromosomal rearrangements
have unique gene expression profiles, thus providing
support for this hypothesis.20 Genes relatively highly
expressed in MLL-rearranged B-precursor ALL are those
associated with hematopoietic progenitors and devel-
oping myeloid cells, whereas the genes expressed at
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 9 |
Berlin, Germany, June 4-7, 2009
lower levels are associated with lymphoid identity.
Recent studies have also defined specific gene expres-
sion signatures associated with MLL-translocations in
primary human AML blasts.21 Of interest, even though
there are clear differences in expression of lineage asso-
ciated genes between MLL-rearranged ALL and MLL-
rearranged AML, there appears to be a core gene expres-
sion profile found in all MLL-rearranged human
leukemias independent of the lineage markers.21
Presumably, MLL-fusion proteins directly regulate a
subset of these genes. This is further supported by the
fact that this MLL-associated signature consists of mul-
tiple highly expressed HOX genes.
Histone methylation and mixed lineage leukemia-fusions
Recent data suggests that many MLL fusion partners
belong to a network involved in transcriptional regula-
tion through histone modification.26,27 An example of
this is the MLL-EEN fusion protein that requires an argi-
nine methyltransferase for its transforming activity.28
Also, the MLL fusion partner AF10 associates with the
DOT1L histone methyltransferase that methylates
lysine 79 residues in histone H3 (H3K79).29 Expression
of an MLL-DOT1L fusion immortalized hematopoietic
progenitors, an activity not observed with either MLL
or DOT1L alone.29 Further evidence that MLL-fusions
may be involved in regulation of gene expression via
histone modification came from studies assessing the
MLL-ENL fusion. Upon expression of MLL-ENL, H3K79
levels associated with the HoxA9 and Meis1 promoters
increase.30 More recent studies have also demonstrated
physical association between MLL-ENL and DOT1L.31
Thus, at least for these fusions, association with a
unique histone methyltransferase appears to be impor-
tant for leukemogenic transformation. As studies have
linked methylation of H3K79 to positive transcriptional
regulation,32,33 the loss of H3K4 methyltransferase activ-
ity in an MLL-fusion might be compensated by acquisi-
tion of H3K79 methyltransferase activity or arginine
methyltransferase activity. Furthermore, as different
methylation marks (H3K4, H3K36, H3K79, etc.) may
positively regulate transcription in unique ways,32,34 the
replacement of H3K4 activity in wild-type MLL with a
different histone methyltransferase activity in the MLL-
fusion complex could further perturb transcriptional
control (Figure 1).
Figure 1. Histone methylation and
MLL-fusions. H3K4 methylation is
associated with transcriptional initia-
tion and other marks such as H3K79
and H3K36 are associated with tran-
scriptional elongation. Biochemical
studies have demonstrated binding of
MLL-fusion partners to the H3K79
methyltransferase DOT1L. This leads
to the hypothesis that abnormal
recruitment of DOT1L to MLL target
genes is an important part of MLL-
fusion mediated oncogenesis.
Reprinted with permission from refer-
ence 27.
14th Congress of the European Hematology Association
H3K79 methylation in mixed lineage leukemia-AF4 ALL
Since AF4 also associates with the DOT1L methyl-
transferase,35 we assessed whether there might be dif-
ferences in H3K79 methylation between MLL-AF4 ALLs
and normal B-cell progenitors.36 Using a newly devel-
oped mouse model of MLL-AF4 ALL, we performed
ChIP-chip analysis on mouse MLL-AF4 pre-B cell ALLs
and three normal pre-B cell samples. These studies
demonstrated an increase in H3K79 dimethylation asso-
ciated with the HoxA cluster in leukemia cells, and in
approximately 1100 other promoter regions, whereas
only 285 promoter regions were associated with
enhanced H3K79 dimethylation in normal pre-B cells.
Comparison of H3K79 methylation and gene expres-
sion demonstrated that ectopic H3K79me2 in MLL-AF4
leukemias is associated with enhanced gene expression.
Next, we assessed whether similar differences in
H3K79me2 exist in human MLL-rearranged ALLs and
found elevated H3K79me2 associated with 1378 pro-
moter regions in the leukemias and elevated H3K79me2
associated with 562 promoter regions in normal sam-
ples, thus demonstrating more genes associated with
increased H3K79me2 in the MLL-rearranged leukemias
as compared to normal cells. Similar to the mouse
model, we found enhanced H3K79 methylation associ-
ated with the HOXA cluster in human leukemia cells
(Figure 2). Furthermore, there is a strong correlation
between elevated H3K79 methylation and abnormal
gene expression in human MLL-rearranged ALL. A
recent independent study similarly demonstrated wide-
spread abnormal H3K79 methylation in MLL-
rearranged ALL cell lines.37 These data demonstrate that
ectopic H3K79me2 is associated with gene expression
in human leukemias as is the case in mouse leukemias.
Identification of histone methyltransferases as impor-
tant for MLL-fusion mediated leukemias provides hope
that therapeutics targeting these enzymes may be par-
ticularly useful in these leukemias.
Multi-step pathogenesis of mixed lineage leukemia-
rearranged leukemias?
Mouse models of leukemia predict multiple genetic
events are necessary for the development of acute
leukemias. “Knock-in” models of MLL-rearranged AML
have been developed where the fusion genes generated
by translocations found in human leukemias are under
Figure 2. H3K79 Methylation in Human MLL-rearranged ALL. Identically scaled average tracks from ChIP-chip analysis
of H3K79me2 modifications associated with HOXA cluster loci in human CD34/19+ cells (n=5) or MLL-rearranged ALL
(MLL-r ALL ) (n=5). Reprinted with permission from reference 36.
| 10 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
the control of the endogenous promoter. Mice contain-
ing an MLL-AF9 fusion gene under control of the MLL
promoter spontaneously develop AML with a latency of
4 months to over 1 year.38 This is widely interpreted as
a requirement for a second genetic event during leuke-
mogenesis. Other models, such as an MLL-Cbp knock-
in or an AML1-Eto knock-in model do not spontaneous-
ly develop leukemia.39,40 In these models, either irradia-
tion or chemical mutagenesis is necessary in order to
induce leukemias, thus clearly requiring multiple events
for the development of leukemia.
Multiple lines of evidence now points to a multi-step
pathogenesis of human acute leukemia. Elegant epi-
demiologic studies suggest that childhood leukemias
require at least two and probably more genetic events to
occur for the development of leukemia. In particular,
lymphoblastic leukemias with TEL-AML1 rearrange-
ments appear to develop after a multi-step process. TEL-
AML1 rearrangements are often detected in blood taken
at birth from a child who will develop ALL 3 to 5 years
later. This suggests that TEL-AML1 rearrangements are
the first genetic event, but also that other mutations are
required for the development of ALL.41 Similar studies
have been performed on blood spots from children that
would develop MLL-rearranged ALL and the MLL-
translocations clearly develop in utero while the
leukemias become apparent sometime during the first
year of life.42
Receptor tyrosine kinases are attractive candidates as
signaling molecules that may cooperate with transloca-
tion-associated fusion proteins during leukemogenesis.
Ever increasing evidence suggests that activated kinases
play a central role in the pathogenesis of leukemias and
myeloproliferative syndrome.43 The most dramatic evi-
dence for such a role is activation of the ABL tyrosine
kinase by the BCR-ABL fusion produced by the t(9;22),
and its inhibition by imantinib (Gleevec).44. Other
mutant kinases frequently identified in AML are the
receptor tyrosine kinases FLT3 and c-KIT.43 Recent
mouse experiments support the hypothesis that DNA-
binding fusion proteins generated by leukemia associat-
ed translocations perform different functions than acti-
vated tyrosine kinases.45 DNA-binding fusions either
induce a block in differentiation or activate self-renewal
in developing hematopoietic progenitors, while activat-
ed kinases may provide a survival or proliferation signal.

This has prompted the hypothesis that at least two dif-
ferent classes of mutations are necessary for leukemoge-
nesis.46
Identification of FLT3 mutations in MLL-rearranged
ALLs suggest that a multi-step process may also be nec-
essary for MLL-rearranged leukemias ALL.47 However,
given the epigenetic dysregulation described above in
MLL-rearranged ALLs, the question arises as to whether
these leukemias might not require further genetic
changes and that the majority of oncogenic “progres-
sion” in MLL-rearranged ALL is a result of epigenetic
changes that are driven by the MLL-fusion. This is sup-
ported by recent genome wide studies that have
demonstrated very few gains or losses of chromosomal
regions in MLL-rearranged ALLs.48 If MLL-rearranged
ALLs are largely driven by epigenetic changes, then
therapeutics targeting chromatin structure might be par-
ticularly useful in this dreadful disease. Further genetic,
genomic and biochemical studies should help us deter-
mine how best to exploit this knowledge for therapeu-
tic benefit.
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 Acute lymphoblastic leukemia

The acute lymphoblastic leukemias of Down syndrome

S. Izraeli A B S T R A C T

Sheba Medical Center and Tel Aviv
University Medical School, Israel
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:13-16
Children with Down syndrome have a markedly increased risk for acute lymphoblastic leukemia (DS-
ALL). These leukemias are exclusively of the B cell precursor phenotype and occur in a similar age to
“common” sporadic ALLs. Recent studies reveal that DS-ALLs are heterogeneous and differ from spo-
radic ALLs with acquired trisomy or tetrasomy 21. Acquired mutations in Arginine 683 of JAK2, result-
ing in a constitutive activation of JAK/STAT signaling, characterize about 20% of DS-ALLs and suggest
the potential therapeutic utility of JAK2 inhibitors. In general, the prognosis of DS-ALL is inferior to
sporadic ALL, mainly because of increased treatment toxicity and possibly because of the lack of prog-
nostically favorable genetic abnormalities. Recent data of improved prognosis on more intensified reg-
imens implies that excessive chemotherapy dose reductions may not be appropriate for these patients.
Current international surveys aimed at detailed characterization of patients’ clinical outcome and on-
going cooperative biological studies could lead to improved therapy for this high-risk disease.

hildren with Down syndrome (DS) with DS do not develop leukemia, such

C have a markedly enhanced incidence

of myeloid (ML-DS) and lymphoid
(DS-ALL) leukemias. The higher risk of
leukemias in DS is striking in light of the
reduced risk of most solid tumors.1,2 This sug-
gests a leukemogenic role of constitutional
trisomy 21. The ML-DS is a defined entity,
unique to DS, with a clear clinical presenta-
tion and course, excellent response to
chemotherapy and a relatively well deci-
phered molecular pathogenesis.3,4 Much less
is known about DS-ALL, and here the cur-
rent understanding of its pathogenesis and
clinical management is reviewed.
The risk of DS-ALL has been estimated to
be 10-20 times higher than sporadic ALL
and it is the most common leukemia in chil-
dren with DS.1,5 In most published multi-
institutional ALL protocols, DS-ALL com-
prises about 1 to 3% of total patients.6-8 The
peak age is about 5 years, slightly older than
the peak age of sporadic ALLs, possibly
related to absence of DS-ALL below the age
of 1 year. The immunophenotype is typical
for B cell precursor ALL5,9,10 with the notable
absence of T and pro-B ALLs.
Pathogenesis
The excess of ALLs in DS raises several
general questions:
a) Are these leukemias unique to DS (like
the ML-DS) or do children with DS have
a general increased risk for childhood
“common” B cell precursor ALL?
b) What is the role of trisomy 21? Which
are the genes on trisomy 21 that confers
increased risk for ALL?
c) What is the nature of the acquired
somatic genetic events that cooperate
with constitutional trisomy 21 in the
evolution to ALL? Since most children
progression events are necessary for
leukemogenesis. Almost all the ML-DS
have an acquired mutation in the
megakaryocytic transcription factor
GATA1.11,12 Does a similar cooperative
genetic event, unique to DS, exist in DS-
ALL?
There is high interest in the pathogenesis
of DS-ALL also because trisomy 21 (or
sometimes tetrasomy 21) is the most com-
mon acquired somatic chromosomal abnor-
malities in sporadic ALL.13 It is mostly found
in high hyperdiploid ALL (HHD-ALL), a sub-
type of ALL characterized by more than 50
chromosomes, always involving chromo-
some 21. Hence, it is tempting to speculate
that constitutional and somatic trisomy 21
may facilitate leukemogenesis in a similar
fashion and, therefore, the study of DS-ALL
may have direct implications for sporadic
childhood ALL.
However, there are fundamental differ-
ences between constitutional and somatic tri-
somies that could explain the uniqueness of
DS leukemias. The former exists in all body
cells, whereas the latter is acquired and only
exists in transformed cells. Thus, constitu-
tional trisomy can predispose to cancer in a
variety of ways. It may exert a direct onco-
genic activity in a cell autonomous manner,
similar to the somatic trisomy. Alternatively,
the trisomy could promote leukemia
because of aberrant effects on immediate
micro-environment; for example, on the bone
marrow’s or fetal liver’s stroma cells that reg-
ulate proliferation and differentiation of
hematopoietic stem cells. More complex
may be the influence of the trisomy on the
macro-environment. For example, viral
infections and the immunological response
have been suggested to have a role in the

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 13 |
14th Congress of the European Hematology Association
pathogenesis of childhood common ALL.14,15 The
markedly increased risk of ALL in DS could also be
caused by the immunodeficiency, altered immunologi-
cal environment and the increased infection rate that
characterize DS.
Another fundamental difference between constitu-
tional and somatic trisomies is the timing of their occur-
rence during ontogeny. Constitutional trisomy 21 exists
since conception. Thus, it is possible that constitutional
trisomy 21 could lead to an expansion of “primitive”
fetal hematopoietic cells that are later transformed by
acquired somatic oncogenic events. Thus, the fetal ori-
gin of leukemia associated with constitutional trisomy
21 may differ from the sporadic leukemias. This is prob-
ably the case with ML-DS where expansion of primitive
megakaryoblastic precursors have been shown in-vivo
in human livers from DS fetuses.4,16,17 These abnormal
precursors may be especially sensitive to the mutation
in GATA1, universally observed in ML-DS.18 Similarly,
DS-ALL may be caused by an expansion of a “special”
fetal lymphoid precursor by constitutional trisomy 21.
Recent studies demonstrate that unlike ML-DS, DS-
ALL is a heterogeneous disease suggesting complex
pathogenesis (Figure 1). Overall, there is a significantly
lower prevalence of the common genetic subtypes of B
cell precursor ALL (BCR/ABL, TEL/AML1 and HHD) in
DS.10,19-24 This was confirmed in the recent largest cyto-
genetic study of 215 DS children with ALL.25 The preva-
lence of TEL/AML1 and hyperdiploid ALLs was lower
in DS patients compared to sporadic ALLs. However, if
one takes into account the 20-fold increased risk of ALL
in DS,1 then there may be an absolute increase in the
incidence of these common subtypes of childhood
leukemias in DS. Similarly, genomic studies by CGH or
SNP arrays have revealed similar acquired copy number
variations in DS and TEL/AML1 ALL.26,27
However, it emerges that the majority of the DS-ALLs
differ from the sporadic ALLs containing excess chro-
mosome 21. The most common cytogenetic abnormal-
ity in DS-ALL is an extra chromosome X, observed in
close to half the patients.25 An additional copy of chro-
mosome X is usually present in HHD sporadic ALL, dis-
playing additional copies of multiple chromosomes
including chromosomes 21, X, 6 and occasionally chro-
mosomes 10, 14, 17 and 18.13 However, the combina-
tion of trisomy 21 and extra chromosome X as a single
cytogenetic abnormality seems unique to DS-ALL, and sug-
gests a yet unknown collaborating event between gene
(s) on chromosome 21 and X.
Another recurrent cytogenetic abnormality t(8;14)
(q11;q32) is associated with DS-ALL.25,28 This rare
translocation displaces the C/EBPδ gene into the IGH
locus. The C/EBP transcription factors induce myeloid
differentiation. Presumably their expression in B cell
precursor ALL blocks lymphoid differentiation. Of 44
patients reported with this translocation, 27% had DS-
ALL.28
The JAK2 kinase participates in signal transduction of
growth promoting signals induced by several cytokines.
Activating mutations in JAK2 are commonly found in
myeloproliferative neoplasms.29,30 All are missense
mutations in the JAK2 pseudokinase domain, causing its
constitutive activation leading to cytokine independent
growth. Following one case report,31 several large stud-
| 14 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Figure 1. Cooperating genetic events in DS leukemias. The
myeloid leukemias of DS (ML-DS) are universally character-
ized by an acquired mutation in GATA1 (Additional somatic
genetic aberrations are also required). The lymphoid
leukemias (DS-ALL) are more heterogeneous. About 15% of
DS-ALLs carry similar aberrations of sporadic ALLs, namely
TEL/AML1 translocation or hyperdiploidy (HHD). Most of DS-
ALLs have cooperating events that are relatively unique to
DS. About 20% have an acquired JAK2 mutation, another
40% and additional X chromosome. Translocations into the
IgH locus at 14q32 are also more common in DS-ALL.
ies identified that acquired mutations in the JAK2 kinase
characterize about a fifth of all DS-ALLs.26,32,33 The
largest study from the iBFM group, involving 88 chil-
dren with DS-ALL, revealed that children with JAK2
mutations were significantly younger (mean 4.5 vs. 8.6
yrs, p<0.001) at diagnosis. As this was not confirmed in
a smaller study from the Children’s Oncology Group,33
it is presently unclear if the patients with JAK2 mutation
constitute a defined clinical subgroup. All mutant alleles
are centered around a highly conserved arginine residue
(R683) within the JAK2 pseudokinase domain. The
mutations immortalize primary mouse hematopoietic
progenitors, and cause constitutive JAK/STAT activation
and cytokine independent growth hematopoietic cells
that are sensitive to pharmacological inhibition of
JAK/STAT signaling. Similar to the GATA1s mutations
in DS myeloid malignancies, the novel mutations in R683
of JAK2 are unique to DS-ALL and are not detected nei-
ther in myeloproliferative neoplasms (MPNs) nor in
sporadic childhood ALL. Modeling of JAK2 pseudoki-
nase domain revealed that R683 is situated in an
exposed conserved region separated from the one
involved in MPNs.
The association between a specific mutation in JAK2
and DS-ALL is fascinating from several aspects, not only
relevant for DS but for the general understanding of
leukemogenesis and the biology of JAK signaling.
Acquired somatic point mutations in two different sites
(V617 and R683) in the same domain of JAK2 leading to
apparently the same biochemical outcome (constitutive
activation of JAK2) are strictly associated with hemato-
logical malignancies arising in different lineages
(myeloid vs. B lymphoid, respectively). Importantly, the
V617F mutation occurs in an early hematopoietic pro-
genitor and is also detected in lymphocytes in patients
with MPNs,34-37 yet it causes an expansion only of the
myeloid compartment. One potential mechanism could

be the presence of lineage specific regulators of JAK2,
whose activity is disrupted by the specific point muta-
tions. The second fascinating observation is the tight
association between constitutional trisomy 21 and the
R683 mutated JAK2. This association does not simply
depend on the presence of extra chromosome 21 since
it is not observed in HHD sporadic ALL. Rather it has to
do with (most likely) some lymphoid developmental
effects of constitutional trisomy 21 or with, as yet
unknown, a specific type of genomic instability con-
ferred by constitutional trisomy 21. Finally, these results
not only provide the first molecular specific lesion of
DS-ALL but also suggest that these leukemias are candi-
dates for therapy with the novel JAK2 inhibitors that are
being explored in early clinical trials for myeloprolifera-
tive neoplasms.
Clinical course and therapy
Unlike ML-DS that is uniquely sensitive to
chemotherapy, in particular to ARA-C, the prognosis of
DS-ALL is less favorable in most of the clinical tri-
als.9,10,21,22,24,38-41 Marked toxicity manifested by increased
mucositis, infections and death during intensive periods
of chemotherapy is observed. DS patients may be espe-
cially sensitive to the toxic effects of Methotrexate, a
drug that is not used in AML, due to the excess activity
of the folate transporter coded by a gene on chromo-
some 21.42 However, severe toxicity is also observed to
anthracyclines and to the marked immunosuppressive
effect of ALL therapy.
Importantly, marked reduction of chemotherapy may
be a mistake in DS-ALL. Unlike “common” ALL (HHD
and TEL/AML1 ALLs), many of the DS-ALLs fall by
NCI clinical criteria into a higher risk group.24 A recent
Children’s Cancer Group study demonstrated a surpris-
ingly good survival in children with DS-ALL treated by
risk adjusted intensive chemotherapy protocols. Event-
free (56% vs. 74%; p<0.001) and disease-free (55% vs.
73%; p<0.001) survival at 10 years was significantly
lower in the standard-risk DS-ALL population com-
pared with ALL in non DS, but not in high-risk DS-ALL
population (event-free survival, 62% vs. 59%; p=0.9;
disease-free survival, 64% vs. 59%; p=0.09). In addition,
these differences persisted, regardless of treatment era
(early era [1983-1989] vs. recent era [1989-1995]).
Multivariate analysis revealed that presence of DS
demonstrated an independent significant adverse prog-
nostic effect for the standard-risk population, but not
for the high-risk patients.9 These results suggest that
intensification of therapy for patients with DS-ALL is
needed to maintain outcome comparable with those of
ALL in non DS patients. Similarly, a recent survey of
eight children with DS-ALL who underwent bone mar-
row transplantation reported that relapse and not treat-
ment related toxicity were the major causes for treat-
ment failure. Indeed, the only surviving patients were
those that were treated by myeloablative chemothera-
py.39
To understand better the response to therapy, toxicity
and treatment outcome, an international retrospective
survey of the fate of children with DS-ALL treated on
major cooperative ALL protocols has been initiated.
Meanwhile, clinicians should be careful in dose reduc-
tion of DS-ALL patients. Prospective clinical trials and
incorporation of potentially less toxic drugs such as
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 15 |
Berlin, Germany, June 4-7, 2009
JAK2 inhibitors are required for this specific group of
leukemia prone children.
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 Acute myeloid leukemia

Acute myeloid leukemia biology

and leukemia stem cells

E. Gudgin
B. Huntly
Department of Haematology,
Cambridge Institute for Medical
Research, UK
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:17-23
A B S T R A C T
Acute myeloid leukemia (AML) is an extremely heterogeneous malignancy with many distinct sub-
groups defined by specific morphological, cytogenetic, molecular and gene expression characteristics.
This heterogeneity is also mirrored in the variability of the prognosis of individual patients with AML;
however, the majority of patients still die from the disease. AML was the first malignancy in which a
cancer stem or initiating cell was demonstrated, and it is likely that this cellular compartment is
responsible for disease relapse and secondary resistance. The AML leukemia stem cell (LSC) is, there-
fore, a critical target for disease eradication. Evidence also exists for biological and phenotypic het-
erogeneity within this AML LSC compartment, and this in part, may relate to the molecular complex-
ity of the disease. It may also reflect the contribution of the target cell transformed to initiate the
leukemia. This review will address the cellular pathogenesis of AML and the current controversies sur-
rounding the LSC in AML, such as its identity and the initial target cell of transformation. We will then
discuss the molecular pathogenesis of AML, particularly focusing on recurrent aberrations, which alter
self-renewal and differentiation, two defining characteristics of normal stem cells. A greater under-
standing of LSC biology may allow molecular targeting of these LSC specific pathways, or of the LSC
itself, and should facilitate improved outcomes in AML.

ancer is caused by the acquisition of questions and discuss the identity and ori-

C multiple mutations within a cell,

with their interaction and cumulative
effects resulting in the full malignant pheno-
type. Self-renewal, either inherent within
the cell initially transformed, or acquired
early through somatic mutation, is an
absolute requirement in this process. The
evolution of the phenotype requires long-
lived cells, which can accumulate the requi-
site number of mutations. Other require-
ments, such as increased proliferative
potential, evasion of apoptosis and differen-
tiation arrest occur over time with the
acquisition of further mutations.1 Acute
myeloid leukemia (AML) is an excellent
example of this process, with numerous
cytogenetic and molecular abnormalities
described which alter these critical cellular
functions. In addition, proof-of-principle for
the cancer stem cell hypothesis was first
demonstrated in AML.2-4 This hypothesis
proposed that the malignant clone is
arranged as a hierarchy, with a small popu-
lation of cancer stem or initiating cells con-
tinuously replenishing the bulk of the
tumour, which itself lacks self-renewal abil-
ity. Subsequently, cancer stem cells (CSCs)
have been observed in other leukemias and
solid-organ tumours.5 However, recent stud-
ies are questioning some assumptions about
CSCs.6-8 Furthermore, the origins of self-
renewal, where this self-renewal resides
within the cellular hierarchy of AML, and
how these properties either relate to the
combination of transforming mutations or
to the cell initially transformed are also
unclear. In this review, we address these
gins of the leukemia stem cell (LSC) in AML.
We will then discuss critical molecular path-
ways that alter self-renewal and differentia-
tion and likely control LSC biology and the
AML hierarchy. We will also discuss the
potential for therapeutically modulating
these pathways.
The cellular biology of acute myeloid leukemia
Stem cells were first demonstrated in the
hematopoietic system and are, by defini-
tion, able to self-renew and differentiate
into multiple lineages. They sit at the apex
of a hierarchy of cells and their progeny pass
through lineage restricted progenitor popu-
lations before differentiating into fully
mature, functional effector cells. This
process of differentiation is accompanied by
a rapidly decreasing self-renewal potential,
with no self-renewal potential occurring
outside of the stem cell compartment
(Figure 1A). Stem cells themselves are
organised in a hierarchy, with long-term
self-renewing hematopoietic stem cells
(HSC) giving rise to short-term self-renew-
ing HSC, and then multipotent progenitors.
By analogy, cancers have long been pro-
posed to also organise themselves in disor-
dered hierarchies, and this cancer stem cell
hypothesis was first demonstrated in AML.
The identity of the leukemia stem cell in acute
myeloid leukemia
In seminal experiments over a decade ago,
Dick and colleagues from Toronto identified
cells which could transfer disease in xeno-
transplant experiments into immunocom-

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 17 |
14th Congress of the European Hematology Association
promised (NOD/SCID) murine recipients.2,3 These cells
only comprised a fraction of the total tumour bulk and
demonstrated a surface phenotype, lineage negative
(Lin–), CD34+/CD38– similar to that of normal HSC,
which reconstitutes non-malignant hematopoiesis
within the same recipients.2 Moreover,
Lin–/CD34+/CD38+ cells did not transfer the disease.
However, refinement of the phenotype of LSC in AML
has also suggested some important differences
between LSC and normal HSCs. Antigens present on
normal HSC but not on LSC include Thy1 (CD90) and
c-KIT (CD117),9 while conversely, antigens present on
LSC, but not or only at low level on HSCs, include the
IL3 receptor chain alpha (CD123),10 the C-type lectin-
like molecule 1(CLL-1),11 the cell adhesion molecule
CD44,12 and the immunoglobulin superfamily member,
CD96.13 Importantly, extending the proven clinical util-
ity of antibody therapy in hematological malignan-
cies,14 these antigens, and others that are present or
upregulated in AML LSC by comparison with normal
HSC, could serve as a specific means to therapeutically
target LSC.15
However, recent work has introduced a note of cau-
tion in the interpretation of many of these experi-
ments.6 It had been previously accepted that the
immunocompromised mice used as recipients had little
or no residual immune function and that the antibodies
used to define surface phenotypes were neutral. Bonnet
and colleagues have demonstrated that in AML,
NOD/SCID mice, the strain of mouse used in many of
the above functional experiments to initially define and
further refine the AML LSC surface phenotype, may
still be able to immunologically clear cells bound by
commercially available antibodies, specifically CD38.
In contrast, this did not occur in the more immunocom-
promised NOD/SCID/IL2rγnull strain.6 Furthermore, out-
side of the hematopoietic system, and again using
NOD-SCID-IL2rγnull recipient mice, up to 27% of unse-
lected human melanoma cells formed tumours, com-
pared to less than 0.001% in NOD-SCID mice.8
Therefore, the choice of immunocompromised recipi-
ent mouse strain, the differing choice of markers used
to define LSC populations, and perhaps even the com-
mercial antibodies to these markers have influenced
both the perceived identity of the LSC in AML and the
likely size of this population. However, although there
is likely to be variability between different malignan-
cies in the proportion of cancer-initiating cells, in AML,
the current evidence supports LSCs as being only a
small proportion of the malignant population.
Transplantation experiments have shown that engraft-
ment is increased in NOD-SCID-IL2rγnull mice, but,
where quantitated, still only represents only around
1% of the total cells.16
The origins of the leukemia stem cell in acute myeloid
leukemia
There are theoretical arguments for both primitive
and more differentiated cells as being the origin of
LSCs in AML.4,15 Early studies suggested that the LSC in
AML has a surface phenotype, Lin–/CD34+/CD38–.
However, in recent experiments, when the inhibitory
effects of the CD38+ antibody on engraftment were
corrected for by use of the NOD/SCID/IL2rγnull strain,
| 18 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Figure 1. The balance of self-renewal and differentiation in
normal hematopoiesis and transformation. (A) This
demonstrates that during progression through the stem
cell compartment and into the progenitor compartment,
self-renewal is lost while differentiation program s are
being initiated. (B) This process is finely balanced, with
self-renewal and differentiation inversely correlated and
possibly antagonistic to each other. This is demonstrated
in CEBPA deficient mice, which not only demonstrate a
defect in myeloid differentiation but also show increased
self-renewal in reconstitution assays.
the CD34+/CD38+ compartment was shown to contain
the majority of the LSC activity in many AML
patients.6 In normal human hematopoietic ontogeny,
cells that are CD34+/CD38+ correspond to a more
mature committed progenitor cell population.
Furthermore, studies prospectively conducted in
murine leukemia models also suggest that usually LSCs
can develop from progenitor cells. In these experi-
ments, populations of myeloid committed cells that
lacked self-renewal properties but expressed a variety
of leukemia-associated fusion genes or mutations
(MOZ-TIF2,17 MLL-ENL,18 MLL-AF919 and CEBPA20)
were purified by flow cytometry, and subsequently
able to generate AML when transplanted into mice.
Evidence for progenitor transformation also exists in
other forms of leukemia, such as acute lymphoblastic
leukemia (ALL), where the LSC may be positive for the
progenitor maker CD1921,22 and in myeloid blast trans-
formation of chronic myeloid leukemia (CML), where
the LSC appears to have the phenotype of a granulo-
cyte-monocyte precursor (GMP).23,24 Taken all together,
these data suggest that there is a greater variability in
the immune-phenotype of LSCs than previously
thought, and that the LSC may arise from progenitor as
well as stem cells (Figure 2).
The molecular biology of acute myeloid leukemia
The heterogeneity of AML is reflected in the spec-
trum of mutations with which it is recurrently associat-
ed.25 These include numerical chromosome abnormali-
ties, such as trisomies and monosomies (e.g. trisomy 8
and monosomy 7), chromosomal rearrangements gen-
erating translocations [e.g. t(8;21) and t(15;17)], intersti-
tial deletions (del 5q and 7q) and amplifications (MLL-
PTD, FLT3-ITD), and point mutations of specific critical
genes (NPMc, CEBPA, FLT3-TKD).25,26 In addition to
these genomic aberrations, it is becoming increasingly
clear that epigenetic abnormalities are also important in
the transcriptional dysregulation characteristic of AML.
 Berlin, Germany, June 4-7, 2009

Alteration of self-renewal pathways

Important pathways in normal HSC self-renewal
include the clustered HOX genes, the WNT/β-
CATENIN pathway, the PTEN/AKT/FOXO axis, BMI1
and polycomb group proteins and the NOTCH and
HEDGEHOG pathways.4,33 These same pathways are
also important for LSC self-renewal, but recent reports
have described differences in some critical aspects that
may potentially be exploited therapeutically in the

Figure 2. A proposed roadmap for LSC induction. The right

side of the figure demonstrates current interpretations of
the roadmap of normal myeloid ontogeny, with critical tran-
scription factors whose transcriptional program s control
the process shown. The left side of the panel speculates on
where transforming events occur within the stem and pro-
genitor compartments to generate LSC in AML evolution.

Figure 3. Reductionist model of AML generation. This is a

simplified model of the induction of AML, where the collab-
These lesions alter critical cellular processes and collab- oration of type 1 mutations, which confer a proliferative
orate to generate the full malignant phenotype. A sim- and survival advantage on cells, and type 2 mutations,
which impair differentiation and/or increase self-renewal,
plified system has been proposed, where the mutations lead to the development of AML. Selected representative
fall into complementation groups, predominantly examples of type 1 and type 2 mutations are shown.
affecting growth and survival (type 1 mutations) and
differentiation and self-renewal (type 2 mutations).27
Examples of these mutations are given in Figure 3.
While this model has proven helpful in our understand-
ing of such a complicated disease and has provided us
with critical processes to focus on therapeutically, it is
widely accepted to be an oversimplification. Many
mutations may have both type 1 and type 2 functions
directly, such as FLT3 mutations, which provide growth
and survival signals,28 yet also impair differentiation via
phosphorylation of CEBPA.29 In addition, DNA damage
caused by oncogenic tyrosine kinases30 and some chro-
mosomal fusion products, such as AML-ETO and PML-
RARA,31,32 may indirectly generate random mutations
which fall into a different complementation group. We
have chosen to focus on lesions, which alter the two
defining properties of stem cells: unlimited self-renewal
and multi lineage differentiation.
In order to maintain hematopoiesis throughout a life-
time, self-renewal must maintain the pool of HSCs,
whereby one or both daughter cells remain undifferen-
tiated and able to continue self-renewing. All stem cells
must regulate the relative balance between stem cell
maintenance and differentiation to replenish the
mature cells of an organ. If a stem cell is triggered to
begin differentiating, genes that maintain self-renewal
are switched off, and genes that enforce differentiation
are switched on (Figure 1 B, 2). A large network of stim-
ulatory and inhibitory genes, which are mostly tran-
scription factors, then directs these cells to their ulti- Figure 4. Alterations of critical self-renewal and differenti-
ation pathways in AML. Alterations of self-renewal and dif-
mate fate. Disruption of this balance leads to the unreg- ferentiation cooperate to generate AML. (A) An illustration
ulated self-renewal and block in differentiation seen in of how some critical self-renewal pathways are dysregulat-
AML. It is likely that these two processes are inversely ed in AML, and their relationship to recurrent genetic
lesions. Potential therapeutics, which might abrogate
regulated (Figure 1 A, B), as loss of self-renewal occurs these pathways, are shown in red. (B) An illustration of how
around the same time that differentiation begins, and it recurrent genetic lesions in AML target the expression and
is possible that the transcriptional program s which function of the master-regulators of myeloid differentiation
PU.1 and CEBPA. Potential therapeutics are shown in red.
drive these processes may be antagonistic.

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 19 |
14th Congress of the European Hematology Association
future (Figure 4 A).
HOX genes. The HOX family of homeobox genes in
which mammals are divided into four genomic clusters
(A-D), encode DNA-binding transcription factors. In
normal bone marrow, HOX genes are predominantly
expressed in HSCs, and downregulated during terminal
differentiation.34 They play a central role in develop-
mental hematopoiesis and HSC self-renewal.35
However, HOX genes are also globally dysregulated in
the majority of patients with AML,36-38 and substantial
evidence implicates them in the pathogenesis of AML.39
Dysregulation of HOX genes by AML-associated muta-
tions may be a central mechanism underlying the
abnormal self-renewal of LSCs. Alterations of the
mixed lineage leukemia gene MLL, either transloca-
tions or partial tandem duplications, collectively occur
in 5-10% of cases of AML, and are all associated with
HOX gene dysregulation.36,40 In addition, it has also
been shown that other rarer leukemia-associated fusion
proteins, such as CALM-AF10 and MOZ-CBP (MYST3-
CREBBP) also upregulate HOX genes, and that individ-
ual HOX genes, such as HOXA9 and A10 are them-
selves occasionally rearranged with the NUP98 gene in
AML patients.39 Gene expression profiling of AML
patients with mutated nucleo-phosmin (NPMc), the
commonest acquired molecular abnormality in AML,
are highly associated with a stem cell molecular signa-
ture that includes activation of HOX genes and the
HOX-cofactor MEIS1.41 Furthermore, members of the
Drosophila Caudal-like CDX family, CDX2 and CDX4,
which regulate HOX genes in murine development,42
have recently been shown to be significantly upregulat-
ed in the majority of human AML samples and may
represent a master regulator of HOX gene
induction.43-45 However, despite their apparent impor-
tance, little is known about the downstream targets of
HOX dysregulation, and this remains an important area
for future study. Interestingly, a recent report showed
that MLL rearranged leukemic cells are susceptible to
inhibition by GSK3-B inhibitors through a mechanism
which destabilises p27Kip1.46 Whether this treatment is
MLL specific or may be applicable to all AML cases
with HOX dysregulation remains to be seen.
PI3-K pathway. In normal HSC, quiescence and cell
cycle entry are very tightly controlled. Central to this
appears to be the PI3K-AKT-FoxO pathway, mediated
through effectors, such as p21.47-49 Constitutive activa-
tion of the PI3K pathway through targeted deletion of
PTEN, the major lipid phosphatase attenuating PI3K
signaling, results in both HSC depletion and in the
development of acute leukemia in mice.47 The mice
demonstrated an initial myeloproliferative phase that
was quickly followed by an acute leukemia that was
transplantable to secondary recipients.47 Inhibition of
this pathway with the mTOR inhibitor rapamycin
restored normal function to PTEN-/- HSCs and prevent-
ed the development of leukemia. In addition, in trans-
plant experiments, rapamycin also depleted the num-
ber of leukemia-initiating cells and, importantly, pro-
longed survival, even when administered to mice with
established leukemia. This shows that mechanistic dif-
ferences between LSCs and HSCs may provide a ther-
apeutic target, without destruction of the HSC com-
partment. Similarly, constitutive activation of the PI3K
| 20 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
pathway is common in AML patients and is down-
stream of AML-associated mutations, such as those
affecting RAS and the FLT-3 and C-KIT oncogenic tyro-
sine kinases.50 Inhibition of this pathway causes in vitro
apoptosis but also reduced clonagenic potential in AML
blasts51 and reduced NOD-SCID engraftment in vivo,
suggesting a LSC specific effect.51,52 Furthermore, as a
single agent, rapamycin induced clinically significant
responses in 4 of 9 patients with poor risk AML in a
phase I trial.52 Taken together, these data have prompt-
ed the inclusion of mTOR/PI3K inhibitors in ongoing
combination chemotherapy studies.
WNT/β-catenin pathway. The WNT/β-catenin
(CTNNB1) pathway has been demonstrated to be con-
stitutively activated in the majority of cases of human
AML.53 This activation has been experimentally
demonstrated to occur downstream of AML-associated
mutations, such as FLT3-ITD,56 AML1-ETO, PML-
RARA and PML-PLZF.54,55 In addition, α, β and γ catenin
have all been implicated in the malignant self-renewal
of AML and myeloid blast crisis CML.23,55,57 Moreover,
retroviral transduction of the LEF1 transcription factor,
an effector of the WNT/β-catenin pathway, generates
AML in a mouse model.58 This suggests potential ther-
apeutic options and small molecule inhibitors exist,
which target the interaction of β-catenin and LEF/TCF
transcription factors necessary for induction of the
WNT/β-catenin pathway.59 Initial concerns that these
agents would have unacceptable side-effects on normal
stem cell function may prove unfounded as mouse
models have recently questioned the requirement for
WNT/β-catenin signaling in homeostatic
hematopoiesis.60,61 Therefore, similar agents may prove
therapeutically useful in some forms of AML.
Alteration of normal differentiation
Differentiation block of varying degrees is a cardinal
feature of AML,62 with the degree of block the major
determinant of the AML phenotype. The process of lin-
eage commitment and differentiation is tightly coordi-
nated by distinct transcriptional program s, which are
in turn controlled by master-regulators. In the process
of myeloid determination and differentiation, the
major master-regulators are the CEBPA and PU.1 tran-
scription factors.63
CEBPA. The CCAAT/enhancer-binding-protein-α
(CEBPA) is a basic region leucine zipper (bZIP) tran-
scription factor critical for myelopoiesis and growth
control.64 It regulates a program of genes critical to
establishing myeloid identity and differentiation, such
as G-CSF and secondary granule protein genes,65 and
also limits proliferation via E2F mediated inhibition of
c-MYC.64 The protein is expressed in two isoforms,
with differing transcriptional start sites, a longer p42
protein and a shorter p30 protein. Both contain the pro-
tein-protein interaction and DNA binding domains, but
they differ in that only p42 contains all of the transac-
tivation domains.64 CEBPA expression is detectable first
in committed myeloid progenitors, and its expression is
upregulated as cells commit to mature granulocytes65,66
(Figure 2). Mice deficient in CEBPA die perinatally due
to a defect in glucose homeostasis, but they also com-
pletely lack mature neutrophilic and eosinophilic gran-
ulocytes.67 The inverse correlation of self-renewal and

differentiation previously alluded to is demonstrated
by the fact that mice deficient in CEBPA have an
increased self-renewal and repopulating capacity,
although they do not develop leukemia.68
CEBPA may be dysregulated by a number of mecha-
nisms in AML (Figure 4B). Mutations of the CEBPA
gene, both acquired and inherited, occur. They are
described sporadically in around 10% of AML cases,69,70
and are associated with a relatively good prognosis.71
There are multiple mutations described which mainly
lead to the generation of only the p30 isoform or alter
the DNA binding of the p42 protein. Their unifying
feature appears to be a down-modulation of, or domi-
nant negative effect on CEBPA function.64 Mouse mod-
els engineered to expressed only p30 CEBPA or mutant
CEBPA developed AML with complete penetrance.72,73
Expression of CEBPA is also downregulated through
hypermethylation of CpG islands at the genes promot-
er in a significant proportion of patients with AML.74-76
In addition, leukemia associated mutations can also tar-
get the translation of CEBPA, as occurs in myeloid
transformation of CML, where high levels of BCR-ABL
induce the RNA binding protein hnRNP E2, which
facilitates degradation of the CEBPA transcript.77
Recurrent mutations associated with AML also disrupt
the function of CEBPA. These include the PML-RARA,
AML1-ETO (RUNX1-RUNX1T1) and AML1/EVI1 gene
rearrangements associated with the t(15;17), t(8;21) and
t(3;21) translocations. They physically interact with
CEBPA and either recruit transcriptional repressors,
prevent its binding with co-factors or impair its DNA
binding, thereby blocking its transcriptional activity at
target loci.78,79 Finally, CEBPA function can also be
blocked by post-translational modification, such as
occurs in FLT3 mutant AML cells.29 This block appears
to be dependent upon ERK1/2 phosphorylation of
residue serine 21 and as can be reversed by FLT3 or
MEK1 inhibitors,29 which may be a useful therapeutic
strategy.
PU.1. The PU.1 transcription factor is a hematopoiet-
ic specific ETS family member, involved in the develop-
ment of all hematopoietic lineages.63 PU.1 is highly
expressed in myelomonocytic cells and GMPs, with
more primitive cells such as common myeloid progen-
itors(CMP), and HSC expressing PU.1 at lower levels66
(Figure 2). Many genes critical for myeloid differentia-
tion are described as targets for PU.1 and include G-
CSF, M-CSF and GM-CSF.62 Complete loss of PU.1 is
embryonically lethal, and evidence also suggests that
PU.1 is required for HSC function.83,84 However,
although conditional loss of PU.1 does not lead to
leukemia, a mouse engineered to express a graded
reduction of PU.1 to around 20% does develop AML,85
suggesting that at least some PU.1 function is required
to mandate myeloid fate, malignant self-renewal or
both.
Many different processes in AML (Figure 4 B) target
PU.1. These include direct mutation, which occurs in
around 7% of patients. In general, these mutations alter
the interaction of PU.1 with its protein partners AML1
and JUN, leading to decreased transcriptional activity.86
Although no germ line mutations have been described
for PU.1, a polymorphism in its upstream regulatory
element (URE) has been demonstrated at a higher fre-
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 21 |
Berlin, Germany, June 4-7, 2009
quency in AML patients with a complex karyotype. In
the homozygous state, this polymorphism abolishes
binding of the chromatin remodelling agent SATB1,
reduces the enhancer activity of the URE and decreases
the expression of PU.1.87 PU.1 is also targeted by
leukemia-associated mutations, such as FLT3-ITD,
AML1-ETO and PML-RARA. AML1-ETO physically
binds to PU.1 and displaces its co-activator JUN, there-
by downregulating its transcriptional activity,88 where-
as PU.1 expression is downregulated by FLT3-ITD,89
and PML-RARA.90
The paradigm of differentiation therapy already
exists for AML, where ATRA has revolutionized the
treatment of the PML-RARA-associated promyelocytic
variant. As an extension to this paradigm, therapeutic
upregulation of CEBPA and PU.1 should alter the AML
phenotype and Figure 4B shows the potential mecha-
nisms to achieve this. Furthermore, in the future, it may
be possible to design peptodomimetics of CEBPA and
PU.1, or to block the interference by leukemia-associat-
ed fusion genes by redirection therapy91 or by systemic
delivery of shRNA.63
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dependent transcription and blocks granulocytic differentia-
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Scott EW, Klug CA et al. The ETS family transcription factor
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differentiation. Blood 2005;106:1590-600.
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Okuno Y et al. Acute myeloid leukemia induced by graded
reduction of a lineage-specific transcription factor, PU.1. Nat
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Minden MD et al. Heterozygous PU.1 mutations are associat-
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 Acute myeloid leukemia

Minimal residual disease detection

in acute myeloid leukemias

G. Saglio
E. Messa
D. Cilloni
Department of Clinical and
Biological Sciences, University of
Turin, Italy
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:24-29
A B S T R A C T
Monitoring of residual disease (MRD) in patients with acute myeloid leukemia (AML) is now recog-
nized as an important diagnostic tool for assessing the response to treatment and the individual risk
of relapse. The study of minimal residual disease has been greatly promoted by the technologic advent
of multiparameter flow cytometry (MFC), polymerase chain reaction (PCR), and by the identification
of the genetic lesions involved in AML pathogenesis. Several studies have demonstrated that quanti-
tative assessment of MRD by MFC is feasible in most AML cases, and may be of prognostic signifi-
cance. In parallel, using available molecular markers, including the recently recognized NPM1 muta-
tions in addition to fusion genes, MRD assessment by real-time quantitative PCR is now possible in
more than half the AML patients with a sensitivity of at least 10–4. Finally, over-expression of the
Wilms’ tumor gene (WT1) occurs in most AML patients, and represents a reliable marker for MRD
assessment and quantification in 80-85% of AML patients. Recently, a large European Leukaemia
Netstudy has shown incorporating early assessment of MRD through a standardized WT1 assay may
help to enhance the risk stratification of AML patients.

urrent treatment protocols for acute titative real-time quantitative PCR (RQ-

C myeloid leukemia (AML) are partly

based on prognostic factors, including
age, white blood cell (WBC) count,
immunophenotypic profiles, specific chro-
mosomal aberrations and other molecularly
detectable abnormalities, such as FLT3 and
NMP1 gene defects.1-8 Individual patient out-
come, however, cannot always be reliably
predicted just on the basis of these classical
parameters, and it is well known that
response to initial therapy may provide a
further prognostic marker. Monitoring of
minimal residual disease (MRD) in AML
patients is now recognised as an important
diagnostic tool that can be used to assess the
response to treatment and to establish the
risk of relapse in the individual patient.9
During the last 20 years, different tech-
niques have enabled the detection of
leukemic cells beyond the threshold of mor-
phology or conventional cytogenetics,
which has generally had a detection of
around 5%. The newer techniques, which
are principally based on immunophenotypic
or polymerase chain reaction (PCR) analy-
sis,10,11 have, in most instances, the sensitivi-
ty to detect at least one leukemic cell out of
103 background cells. The advent of multipa-
rameter flow cytometry (MFC) greatly
improved immunophenotypic analysis.
MFC allows the identification of leukemia-
associated aberrant immunophenotypes
(LAIPs) at diagnosis.12-16 Similarly, the study
of MRD was originally fuelled by the pro-
gressive identification of the recurrent
molecular lesions that are associated with
AML and by the advent of PCR technology
that includes either qualitative reverse-tran-
scriptase-PCR (RT-PCR) or, especially, quan-
PCR).17-18
In most AMLs, very sensitive, end-point
qualitative RT-PCR techniques are used to
reveal the presence and persistence of the
residual disease shortly after the end of
induction and consolidation therapy. A
notable exception includes acute promyelo-
cytic leukemia (APL), for which RT-PCR still
represents the gold standard for MRD deter-
mination by exploiting the presence of the
PML-RARa fusion transcripts associated
with the presence of the t(15;17) transloca-
tion. However, positive results from these
assays do not necessarily imply an increased
risk of relapse.19-22 RQ-PCR, which intro-
duced an easy and rapid way to rank the
PCR-positive patients into different sub-cat-
egories according to the amount of their
MRD, was able to fill this gap. Standardised
RQ-PCR procedures for the most common
types of fusion transcripts present in AML
have also been developed, allowing the
application of this innovative technology to
large-scale MRD studies within multi-centre
therapeutic trials.18
The most common fusion genes (FG) tran-
scripts are present in only about 30% of all
AML cases, and are generally associated
with corresponding well-known cytogenetic
abnormalities. However, approximately
50% of AMLs display a normal karyotype
(NK).23 This problem has been partly over-
come by the discovery of new frequent
molecular defects preferentially found in
normal karyotype AML, such as FLT3 inter-
nal tandem duplication (ITD) and NPM1
mutations.7,24 Apart from serving as impor-
tant prognostic markers, mutations in the
NMP1 gene have been demonstrated to rep-

| 24 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

resent reliable and robust markers for MRD testing and
quantification, and efforts to standardize these proce-
dures are currently ongoing.25
Given that approximately 30-40% of all AML cases
may still lack a specific and stable genetic target, such as
those described previously, genes that are highly and
preferentially over-expressed in AML have been investi-
gated as suitable markers for MRD analysis. In particu-
lar, in approximately 85% of AML cases,26 the amount
of Wilms’ tumor gene (WT1) gene transcript in AML cells
is at least three logs higher than in normal bone marrow
(BM). Since RQ-PCR may clearly distinguish between
normal and abnormal levels of WT1 expression, WT1
has been proposed as a type of universal marker for
MRD assessment in AML, particularly in those cases
that lack another suitable genetic marker.27 Recent data
from our laboratory, as well as data obtained from a
large European standardization study, recently promot-
ed by the European Leukaemia Net, support the notion
that assessment of WT1 transcript levels in both periph-
eral blood (PB) and BM specimens collected post-consol-
idation may bear important and independent prognostic
significance in terms of the risk of relapse.28
Multiparameter flow cytometry
Flow cytometry may be the most accessible method
for MRD detection, because of its wide availability in
most hematological centres. Using an accurate combi-
nation of monoclonal antibodies (MoAbs) that are con-
jugated to different fluorochromes, immunophenotypic
detection of MRD in AML can be performed by identi-
fying abnormal combinations or expressions of markers
on malignant cells at diagnosis.12,14 These markers would
either be not present or very infrequently present on
normal blood or BM cells.13-15 These are referred to as
leukemia-associated aberrant immunophenotypes
(LAIPs), and can be subsequently used during follow-up
to track the amount and behavior of the residual
leukemic clone(s).29,30 LAIPS mainly derive from asyn-
chronous antigen expression, antigen over-expression,
lineage infidelity or the absence of lineage-specific anti-
gens. With respect to acute lymphoblastic leukemia
(ALL) cells, which show more homogeneous pheno-
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 25 |
Berlin, Germany, June 4-7, 2009
types, AMLs may simultaneously show more than a
single LAIP. The choice of LAIPs for MRD detection,
therefore, depends on several factors, including i) speci-
ficity, such that the percentage of LAIP expression on
normal BM cells should ideally be less than 0.1%; ii)
sensitivity, such that the percentage of LAIP expression
on the leukemic blast population at diagnosis should be
at least 10%; iii) stability, such that LAIPs should not
undergo phenotypic shifts during the time course of the
disease.12-16 Although infrequent, in some cases, the last
is known to occur between diagnosis and relapse.31-34
This aspect, along with the fact that it is not as specific
as PCR, represents one of the major limitations of this
method. Conversely, this method has several advan-
tages, such as being relatively easy, cheap, rapid and
applicable for most cases (>80%).29,30 Although studies
of MRD detection by MFC in AML are limited and may
show some discrepancies when compared with ALL,
they indicate that MFC may represent a useful method
for predicting relapse. In particular, most studies per-
formed on AML samples from specific clinical trials
show that the amount of MRD present after consolida-
tion and, in some studies, after induction, strongly cor-
relates with relapse-free survival (RFS).35-46 At the
moment, the lack of a sufficient degree of standardisa-
tion among different laboratories hampers a precise
identification of the best and most common LAIPs to
use. In addition, another major limitation of this tech-
nology, if it is to be widely used in common clinical
practice outside specialised centres and controlled trials,
is the lack of precise thresholds at specific time-points
during follow-up, which define the significant MRD
amounts for the risk of relapse.
Reverse-transcriptase polymerase chain reaction
Several clinical studies have shown that chromosomal
aberrations and the corresponding fusion genes that are
present in AML can be used for risk group classification
(Figure 1).2-47 In addition to their potential use for the
cytogenetic/molecular classification of acute leukemia
at diagnosis, chromosomal aberrations with leukemia-
specific fusion-gene regions can also be used as PCR tar-
gets for the detection of MRD during follow-up, with
Figure 1. Comparison of the sen-
sitivity of methods currently used
to detect and to quantify mini-
mal residual disease in AML.
14th Congress of the European Hematology Association
sensitivities that generally reach 10–4 to 10–5.17 PCR
analysis of fusion genes is based upon the design of
oligonucleotide primers located on opposite sides of the
breakpoint fusion regions, so that the PCR product con-
tains the tumour-specific fusion sequences. In most
types of chromosomal aberrations with fusion genes,
the breakpoints in different patients are spread over 10
kb or more, which represent distances that are difficult
to span on a routine basis by PCR on DNA. However,
in many acute leukemias, the fusion gene is transcribed
into a fusion mRNA, which can serve as the PCR target
after reverse transcription into cDNA.48-49
The persistence of leukemia-specific hybrid tran-
scripts in the bone marrow and peripheral blood of
patients with a t(8;21) or inv(16) AML at the end of the
conventional therapy is normal, and apparently compat-
ible with a durable hematological remission.50-52 This has
favoured the shift towards RQ-PCR for monitoring
most cases of AML.53,54 Despite this observation, RT-
PCR still remains the method of choice, not only for
confirmation of APL at diagnosis, but also to monitor
MRD in this type of leukemia.19,55 Standardised condi-
tions for RT-PCR analysis of PML-RARa FG transcripts
have been developed by the BIOMED-1 Concerted
Action, and different primer sets designed that allow
the detection of the various PML-RARa FG transcripts.17
In the last decade, the availability of differentiation
therapy with all-trans retinoic acid (ATRA) has pro-
duced a remarkable improvement in the outcome of
patients with APL. The challenge is how to identify the
relatively small subgroup of patients, who have a partic-
ular risk of relapse, and cannot be reliably distinguished
based on pre-treatment characteristics, and could poten-
tially benefit from more intensive or alternative treat-
ments during the first remission. During the last few
years, there has been general agreement from a number
of studies that a positive PML-RARa test after consolida-
tion is a strong predictor of subsequent hematological
relapse, whereas repeatedly negative results are associ-
ated with long-term survival in the majority of these
patients.55-59 The Italian GIMEMA group reported that
recurrence of positive PCR results, which were detected
by three-month BM surveillance performed after the
completion of therapy, was highly predictive of relapse.
The use of this strategy successfully predicted approxi-
mately 70% of relapses.60 Although the benefit of early
treatment at the time of molecular relapse has still to be
definitely proven, preliminary evidence supports such a
strategy. In addition, considering that there have been
relatively few studies that report the use of RQ-PCR in
APL patients,61 the molecular monitoring of APL by tra-
ditional RT-PCR must be regarded as one of the most
relevant examples of the impact of molecular genetics in
clinical hematology.
Real-time quantitative polymerase chain reaction
RQ-PCR is now the method of choice in PCR-based
quantification in AML patients. Although RQ-PCR has
been reported to be less sensitive than nested RT-PCR,
it allows for the exact determination of the amount of
residual disease and provides the sensitivity to detect
the residual disease within a given sample. This can be
established by measuring the amount of a so-called
“control gene,” which is a gene that has a stable and
| 26 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
constant expression level in all cell types at any stage of
maturation in BM and peripheral blood. A large
European cooperative study demonstrated that ABL,
GUS and β2 microglobulin might represent optimal con-
trols.18 This method avoids a number of false negative
results that can occur due to RNA degradation, one of
the major pitfalls of RT-PCR. RQ-PCR targets may
include fusion-gene transcripts, specific gene defects,
specific mutations and aberrantly expressed genes.
Real-time quantitative-PCR for quantitative assessment of
fusion genes
AML1-ETO and CBFB-MYH11 fusion transcripts,
which are associated respectively with the t(8;21)
translocation and the inv(16), are the most common
fusion genes present in AML. These two fusion tran-
scripts are also generally associated with favourable
outcomes. Up to 40% of these patients, however, may
experience relapse, and so early identification of
patients at high risk of relapse is extremely important to
determine clinical and therapeutic decisions. Several
RQ-PCR studies have clearly shown that a log reduction
decrease in the fusion transcripts, present at diagnosis,
at distinct time points of the treatment are prognostical-
ly important. In some studies,62-64 the prognostic value of
post-induction AML1-ETO levels has been shown in
both childhood and adult AML, but other studies sug-
gest that the post-consolidation level may be more rele-
vant than the one achieved post-induction.65,66 Specific
thresholds of FG amounts after post-induction and post-
consolidation therapy have also been suggested to cor-
relate with risk relapse. In contrast to AML1-ETO posi-
tive AML, positive PCR results that occur, even after
several years of continuous complete remission (CCR),
have been described in CBFB-MYH11-positive AMLs,67
with only those patients who finally achieve a negative
PCR result being able to persist with CCR. Long-term
monitoring studies have suggested that a consistent
increase in the FG amounts at any time during follow-
up is indicative of an impending relapse.68-70
Real-time quantitative-PCR for quantification of aberrant
FLIT3 and NPM1 transcripts
FLT3-ITD is the most frequent genetic lesion in AML,
accounting for 25-30% and reaching a 40% frequency
in AMLs that have a normal karyotype, and is associat-
ed with a bad prognosis.71 Investigational protocols with
FLT3 tyrosine-kinase inhibitors are currently ongoing. A
large study demonstrated that FLT3-ITD transcripts
could be used as markers for monitoring treatment
response by PCR.72 However, the necessity to use
patient specific primers in order to achieve a sufficient
sensitivity, as well as the instability of this marker at
relapse observed in some cases, are both pitfalls pre-
venting widespread use of this analysis.
NPM1 mutations are the most frequent genetic
changes in AMLs that have a normal karyotype.6 Many
different NPM1 mutation variants have been described,
but the most common variant (MutA) represents 75%
of all cases, with the inclusion of two other mutations
accounting for more than 90% of the total. Therefore,
the use of three different primer-specific assays provides
a reliable and highly sensitive RQ-PCR test for MRD
monitoring, which can be performed in most patients

with NPM1 mutations.26 Recently, some studies have
speculated on the prognostic significance of this analy-
sis, so we are still waiting confirmation from large
prospective clinical studies that use a standardized
approach, such as the one currently planned within the
context of the European Leukaemia Net.
Real-time quantitative-PCR for Wilms’ tumor gene overex-
pression
The presence of AML patients who present with
detectable genetic aberrations prompted several investi-
gators to validate the use of alternative markers for
MRD detection that are suitable for use in the vast
majority of AML patients. The Wilms’s tumor gene codes
for a transcription factor has been shown to be highly
expressed in several hematopoietic tumours, including
AMLs.27-28,73-76 Given that WT1 is expressed in normal BM
and peripheral blood, studies that use qualitative RT-
PCR have provided conflicting results on the clinical
value of this marker. However, the most recent investi-
gations by RQ-PCR have been able to clearly distin-
guish WT1 transcript levels found in AML cells, normal
hematopoietic and post-chemotherapeutic cells, and
regenerating normal BM cells.28 Therefore, longitudinal
RQ-PCR analysis of WT1 transcript levels may prove to
be clinically relevant for AML monitoring. Since WT1
expression in normal PB is about one log lower than in
normal BM, with the majority of normal PB samples
scoring negative, a sequential RQ-PCR study of WT1
expression in PB may further improve the sensitivity of
MRD evaluation in AML, and also favour compliance
and sample availability. A study, which analyzed WT1
expression levels at diagnosis and during follow-up in
82 AML patients who were treated with standard
chemotherapy protocols, provided important prognos-
tic information in AML by earlier identification of
patients who were at a higher risk of relapse early after
induction chemotherapy.77 A recent, large European
cooperative study clearly demonstrated that WT1 is
overexpressed when compared to controls in more than
85% of AML cases. In cases with high-level expression,
MRD assessment following induction showed that a
greater response in terms of WT1 transcript reduction
was associated with a reduced risk of relapse, and that
this correlation remained significant after adjusting for
age, WBC and cytogenetics. In addition, the failure to
normalise WT1 transcript levels by the post-consolida-
tion time-point predicted a significantly increased risk
of relapse with a highly significant prognostic value.
Conclusions
Quantification of MRD provides the opportunity to
improve prognostication significantly in AML and plays
a major role as a stratification parameter in guiding risk-
adapted therapy. MFC may already be applied to most
patients with AML with a clinically relevant sensitivity,
but RQ-PCR is more sensitive, and the identification of
new suitable molecular markers is leading to increase
further its future applicability. Prospective studies, how-
ever, are needed to better define which checkpoints in
specific therapeutic protocols are the most appropriate
for the risk stratification of AML patients in response to
treatment.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 27 |
Berlin, Germany, June 4-7, 2009
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14th Congress of the European Hematology Association
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38. Kern W, Haferlach T, Schoch C, Loffler H, Gassmann W,
Heinecke A, et al. Early blast clearance by remission induction
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39. Coustan-Smith E, Ribeiro RC, Rubnitz JE, Razzouk BI, Pui
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40. Sievers EL, Lange BJ, Alonzo TA, Gerbing RB, Bernstein ID,
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after induction therapy predicts relapse: results from a
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41. Kern W, Voskova D, Schoch C, Hiddemann W, Schnittger S,
Haferlach T. Determination of relapse risk based on assess-
ment of minimal residual disease during complete remission
by multiparameter flow cytometry in unselected patients with
acute myeloid leukemia. Blood 2004;104:3078-85.
42. Kern W, Voskova D, Schoch C, Schnittger S, Hiddemann W,
Haferlach T. Prognostic impact of early response to induction
therapy as assessed by multiparameter flow cytometry in
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43. Venditti A, Maurillo L, Buccisano F, Del Poeta G, Mazzone C,
Tamburini A, et al. Pretransplant minimal residual disease
level predicts clinical outcome in patients with acute myeloid
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44. Gianfaldoni G, Mannelli F, Baccini M, Antonioli E, Leoni F,
Bosi A. Clearance of leukaemic blasts from peripheral blood
during standard induction treatment predicts the bone mar-
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Haematol 2006;134:54-7.
45. Buccisano F, Maurillo L, Gattei V, Del Poeta G, Del Principe
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patients with acute myeloid leukemia. Leukemia
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46. Laane E, Derolf AR, Bjorklund E, Mazur J, Everaus H,
Söderhäll S, et al. The effect of allogeneic stem cell transplan-
tation on outcome in younger acute myeloid leukemia
patients with minimal residual disease detected by flow
cytometry at the end of post-remission chemotherapy.
Haematologica 2006;91:833-6.
47. Szczepanski T, Orfao A, van der Velden V, San Miguel JF, van
Dongen JJ. Minimal residual disease in leukaemia patients.
Lancet Oncol 2001;2:409-17.
48. Claxton DF, Liu P, Hsu HB, Marlton P, Hester J, Collins F, et al.
Detection of fusion transcripts generated by the inversion 16
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83:1750-6.
49. Perego RA, Marenco P, Bianchi C, Cairoli R, Urbano M, Nosari
AM, et al. PML/RAR alpha transcripts monitored by poly-
merase chain reaction in acute promyelocytic leukemia during
complete remission, relapse and after bone marrow transplan-
tation. Leukemia 1996;10:207-12.
50. Nucifora G, Larson RA, Rowley JD. Persistence of the 8;21
translocation in patients with acute myeloid leukemia type
M2 in long-term remission. Blood 1993;82:712-5.
51. Tobal K, Johnson PR, Saunders MJ, Harrison CJ, Liu Yin JA.
Detection of CBFB/MYH11 transcripts in patients with inver-
sion and other abnormalities of chromosome 16 at presenta-
tion and remission. Br J Haematol 1995;91:104.
52. Costello R, Sainty D, Blaise D, Gastaut JA, Gabert J, Poirel H,
et al. Prognosis value of residual disease monitoring by poly-
merase chain reaction in patients with CBF/MYH11-positive
acute myeloblastic leukemia. Blood 1997;89:2222.
53. Schnittger S, Weisser M, Schoch C, Hiddemann W, Haferlach
T, Kern W. New score predicting for prognosis in PML-
RARA+, AML1-ETO+, or CBFBMYH11+ acute myeloid
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2003;102:2746-55.
54. Tobal K, Liu Yin JA. Monitoring of minimal residual disease by
quantitative reverse transcriptase-polymerase chain reaction
for AML1-MTG8 transcripts in AML-M2 with t(8;21). Blood
1996;88:3704-9.
55. Perego RA, Marenco P, Bianchi C, Cairoli R, Urbano M, Nosari
AM, et al. PML/RAR alpha transcripts monitored by poly-
merase chain reaction in acute promyelocytic leukemia during
complete remission, relapse and after bone marrow transplan-
tation. Leukemia 1996;10:207-12.
56. Miller Jr WH, Levine K, DeBlasio A, Frankel SR, Dmitrovsky
E, Warrell Jr RP. Detection of minimal residual disease in acute
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Blood 1993;82:1689-94.
57. Koller E, Karlic H, Krieger O, Mistrik M, Michlmayr G, Gadner
H, et al. Early detection of minimal residual disease by reverse
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58. Korninger L, Knobl P, Laczika K, Mustafa S, Quehenberger P,
Schwarzinger I,et al. PML-RAR alpha PCR positivity in the
bone marrow of patients with APL precedes haematological
relapse by 2-3 months. Br J Haematol 994;88:427-31.
59. Diverio D, Pandolfi PP, Biondi A, Avvisati G, Petti MC,
Mandelli F, et al. Absence of reverse transcription-polymerase
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199;82:3556-9.
60. Diverio D, Rossi V, Avvisati G, De Santis S, Pistilli A, Pane F,
et al. Early detection of relapse by prospective reverse tran-
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PML/RARalpha fusion gene in patients with acute promyelo-
cytic leukemia enrolled in the GIMEMA-AIEOP multicenter
‘‘AIDA’’ trial. GIMEMA-AIEOP Multicenter ‘‘AIDA’’ Trial
Blood 1998;92:784-9.
61. Gallagher RE, Yeap BY, Bi W, Livak KJ, Beaubier N, Rao S, et
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62. Sugimoto T, Hiranmoy D, Shion I, et al. Quantitation of min-
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63. Fujimaki S, Funato T, Harigae H, et al. A quantitative reverse
transcriptase polymerase chain reaction method for the detec-
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64. Viehmann S, Teigler-Schlegel A, Bruch J, et al. Monitoring of
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65. Perea G, Lasa A, Aventin A, et al. Prognostic value of minimal
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66. Schnittger S, Schoch C. Quantitative PCR based minimal
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67. Marcucci G, Caligiuri MA, Bloomfield CD. Defining the
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 Acute myeloid leukemia

Risk-adapted treatment for adult acute myeloid

leukemia

J. Sierra
S. Brunet
Hematology Department, Hospital de
la Santa Creu i Sant Pau,
Autonomous University of Barcelona,
Spain
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:30-37
A B S T R A C T
Advances in the understanding of the pathophysiology of acute myeloid leukemia (AML) and the
recognition of new prognostic categories underline the need for risk-adapted treatment. Cumulative
experience with cytotoxic agents has been helpful in defining who may benefit from intensive
chemotherapy. Elderly AML patients and those with severe comorbidities are normally candidates for
conservative approaches or investigational agents with low extra-hematological toxicity. Intensior
chemotherapy is indicated in the remainder. In the absence of favourable genetic markers, most stud-
ies support performing HLA-identical sibling transplantation in the first remission of AML. If an HLA-
compatible relative is not available, unrelated donors or cryopreserved umbilical cord blood are alter-
natives when the risk of AML recurrence is high. Of note, reduced intensity conditioning extends
hematopoietic transplantation to elderly and debilitated patients. Efforts are being made for further
characterizing AML cases by novel molecular techniques. These techniques could also be applied to
predict sensitivity to treatment and to anticipate individual toxicities. Therapy against antigen, genet-
ic, epigenetic and vascular targets is also under investigation. Given the complexity and interactions
of molecular pathways involved in AML, progress with this approach could be slower than other hema-
tologic disorders. However, this is an exciting challenge for the next years.

cute myeloid leukemia (AML) is now novel agents is actively being investigated

A recognised as a disease with a wide

clinical and biological diversity.1
Discovery of this heterogeneity is the con-
sequence of substantial progress in the
understanding of its pathophysiology. In
the recently published World Health
Organization classification of AML, the
variety of genetic findings in AML has been
taken into account.2 Many of the included
entities constitute new prognostic cate-
gories. With the introduction of antigen
and/or molecular targeted therapies, the
relevance of the precise characterization of
the disease is further emphasised.3 After
initial treatment or during follow-up, it is
now time to adjust AML therapy, not only
to classical factors, such as age and preced-
ing hematologic disorders, but to individual
co-morbidities, genetics of leukemic cells
and the presence of minimal residual dis-
ease (MRD). After many years of homoge-
neous treatment with a modest improve-
ment in outcome, it is now becoming
apparent that an individualised therapeutic
approach for AML could be the most
appropriate strategy.
This review summarizes current knowl-
edge useful for tailoring treatment in adult
AML. Pediatric AML and acute promyelo-
cytic leukemia (APL) are excluded, even
though the latter is the paradigm for how
targeted treatment may overcome the
pathogenetic events leading to leukemia.
Based on the success in APL, the strategy of
combining classic cytotoxic drugs with
in other AML categories. The goal is to cure
most cases of AML or transform the disease
into a chronic disorder.
Initial risk-adapted decision
It is generally accepted that reaching
complete remission (CR) is the requisite to
prolong survival in AML patients.4 Conse-
quently, intensive chemotherapy is usually
recommended, except in patients of very
advanced age and/or those with severe
comorbidities. In practice, patients over 80
years old usually only receive palliative
measures and their life expectancy is not
longer than a few weeks. Decisions con-
cerning the intensity of treatment for AML
patients between 65 and 80 years of age
depend on the anticipated probability of
death due to therapy complications. The
results expected from combination
chemotherapy in this age interval have not
been clearly defined, as published trials fre-
quently have the selection bias of including
the healthiest patients. In such trials, CR
rates range from 40-60%.5,6 The poor toler-
ance to intensive treatment and the com-
mon development of chemoresistance in
this age group justify the investigation of
innovative approaches with low extra-
hematological toxicity, particularly in
patients with low leukemic burden. Within
clinical trials, demethylating drugs such as
5-azacytidine or decitabine with/without
histone deacetylase inhibitors (valproic
acid, vorinostat), and antiangiogenic agents,

| 30 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

such as lenalidomide are being investigated.5,7,8,9
Hematologic improvement is observed in approxi-
mately 30-40% of AML patients treated with these
agents. Preliminary experiences suggest that the
response to demethylating drugs may not be affected
by the cytogenetics of leukemic cells. These agents
may prolong survival, even in the absence of CR crite-
ria, and pose fewer life-threatening complications than
combinations of cytotoxic drugs; furthermore, they
often allow outpatient management. Clofarabine, a
new purine analog, has remarkably low extra-hemato-
logical toxicity. It is presently being tested as a single
drug, and in combination with classic and novel agents
in adult patients with adverse prognosis AML.10
Gemtuzumab ozogamycin (GO) is another option to
consider in patients ineligible for intensive combina-
tion chemotherapy, although prolonged myelosup-
pression is constant, and liver toxicity is to be expect-
ed in a number of cases.11,12 In Europe, access to this
agent is restricted to a compassionate basis. A more
classical alternative without significant extra-hemato-
logical toxicity is to administer subcutaneous low dose
cytarabine. In patients unfit for intensive chemothera-
py, this drug prolongs survival when compared to
hydroxyurea.13
AML patients up to the age of 60-65 years are candi-
dates for intensive induction treatment, preferably
within cooperative clinical trials. The proportion of CR
in these younger adults ranges between 65% and 85%,
with regimens including an anthracycline or anthra-
cenodione for 3 days, and conventional-dose cytara-
bine for 7-10 days.3,4 Twenty to thirty-five percent of
patients need two courses of induction treatment to
achieve CR, and this group has an increased risk of
leukemia recurrence. In most cases, a higher dose of
cytarabine or the addition of a third drug has no
impact on the CR rate. In the HOVON experience, G-
CSF priming has been shown to be of benefit in the
intermediate-risk cytogenetic group by improving the
quality of remission, thereby, leading to a decrease in
relapse incidence.14 This favourable effect has not been
observed in other studies.15 By adding GO to induction
chemotherapy, the Medical Research Council (MRC)
of the United Kingdom observed a positive impact, not
on CR rates, but on leukemia-free and overall survival
of intermediate-risk AML patients.16 Within clinical tri-
als, intensified CT, in case of slow blast clearance is
another option that may improve the CR propor-
tion.17,18 With the same objective, the addition of mol-
ecularly-targeted therapy to induction CT is being
investigated in several trials.19
The causes for not achieving a CR (induction failure)
are toxic death and refractoriness of leukemic cells. As
mentioned, advanced age has an adverse impact on
induction mortality and overall survival when consid-
ered as a continuous variable and at different age cut-
offs (Figure 1). During induction, patients older than 70
years have a probability of dying in the order of 30-
50%.5,6,20 Recently published scoring systems that
include preceding diseases and concurrent morbidities
have been developed to predict mortality after
hematopoietic transplantation.21 These and other pre-
dictive scores are starting to be applied to AML
patients receiving induction chemotherapy, and may
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 31 |
Berlin, Germany, June 4-7, 2009
Figure 1. Survival of patients with primary acute myeloid
leukemia treated with intensive chemotherapy according
to the Spanish CETLAM protocols: effect of age at diagno-
sis.
be of particular interest in determining treatment of
elder patients.22,23 Specific scores predicting death due
to tumour lysis syndrome have also been reported.24
Patients at high-risk for this complication should be
carefully monitored during CT, and supportive meas-
ures are of special relevance.
Chemoresistance is more frequent in elderly AML
patients, mainly because they have a higher propor-
tion of adverse cytogenetics.4,5,6 Currently, the cytoge-
netics of AML cells are the most determinant factor on
short- and long-term sensitivity to chemotherapy in
any age group.25,26,27 Several classifications of cytogenet-
ic findings based on prognostic categories in AML
have been published by cooperative groups in Europe
and the United States. These include the MRC,26,27
EORTC-GIMEMA,28 HOVON-SAKK,29 SWOG-
ECOG,30 and CALGB.31,32 Patients with adverse cytoge-
netics have a probability of refractoriness to
chemotherapy as high as 40-50%. The poor outcome
of a complex karyotype harbouring three or more
abnormalities could be further related to the presence
of monosomies (two monosomies or one monosomy
together with a structural abnormality) than to the
number of chromosomal alterations.33 Other factors
predicting refractoriness have been identified in some
studies. Single nucleotide polymorphisms (SNPs)
affecting DNA-repair genes, such as xeroderma pig-
mentosa A are associated with decreased sensitivity to
induction chemotherapy.34 Overexpression or
rearrangements involving the EV1 gene also favour
refractoriness.35 In contrast, recently communicated
data indicate that this group of AML could be sensitive
to demetilating agents.36 Slow clearance of leukemic
cells from peripheral blood and/or marrow by mor-
phologic, or flow cytometry examination is also pre-
dictive of decreased sensitivity to CT.17,18,37,38,39,40 In con-
trast, few patients with favourable cytogenetic and
molecular features experience resistance to induction
treatment. Core binding factors (CBF) AML in non-eld-
erly adults are associated with more than 85% proba-
bility of CR achievement, with less than 5% of cases
14th Congress of the European Hematology Association
being refractory (Figure 2).3,4,5 The results in this
favourable category may improve even further if clas-
sic chemotherapy regimens are intensified with novel
agents. The results of a recent report from the MRC
combining chemotherapy with GO are encouraging,
although confirmation is needed.16 Other AML with
high sensitivity to initial chemotherapy are the recent-
ly identified good molecular abnormalitites, that is,
NPM1 and CEBPA mutations without associated
adverse genetic features (Figure 2).40
Since so many factors must be considered, the ques-
tion is whether it is necessary to wait for the results of
a full biologic evaluation of the disease before starting
CT. The available data indicate that delaying treat-
ment adversely affects patients up to 60 years old but
not above that age.41 Therefore, young adults with
AML should be treated shortly after diagnosis. Since
some trials combining chemotherapy and targeted
treatment require molecular typing before inclusion,
substantial efforts must be made here to obtain short-
term results of laboratory studies. In patients between
60 and 80 years of age, it seems reasonable to delay the
decision until the cytogenetics of AML is known. The
therapeutic approach in this age group should also take
into consideration whether the patient is a potential
candidate for allogeneic hematopoietic transplantation
after reduced intensity conditioning. Therefore,
despite the presence of AML with poor-risk features, it
makes sense to administer intensive CT if the patient
may benefit from a subsequent transplantation while
in complete or partial remission. It is important to note
that if a significant reduction in blasts is not achieved
after a first course of CT, recent experiences support
proceeding directly to sequential regimens, combining
intensive rescue CT with reduced intensity condition-
ing transplants.42,43 With such an approach, 40% of
refractory or relapsed patients may become long-term
survivors in remission.
| 32 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Figure 2. Results of induc-
tion chemotherapy in the
different genetic categories
of acute myeloid leukemia.
Experience of the Spanish
CETLAM group.
adv: adverse; mk: monoso-
mal karyoptype; mut: muta-
tion; pos: positive; wt: wild
type.
Postremission chemotherapy
After achieving CR, the standard approach is to
administer consolidation CT. In practice, the excep-
tions are patients who required three or more courses
of induction CT to achieve CR with an HLA-compati-
ble donor. In this circumstance, consolidation treat-
ment is frequently omitted in order to proceed to
hematopoietic transplantation as soon as possible. The
aim of this strategy is to avoid the early recurrence of
leukemia, which is particularly frequent in AML
patients with adverse cytogenetics, and to take advan-
tage of the conditioning regimen and graft versus
leukemia effects. All other patients are candidates for
consolidation CT, which, in patients of up to 60 years,
usually includes high-dose cytarabine. In contrast, in
patients above this age, it is safer to administer con-
ventional or intermediate-doses of this drug to avoid
an excess of neurologic and gastrointestinal toxicities.44
The number of consolidation courses to be adminis-
tered has not been clearly defined. In the case of non-
CBF leukemia, most patients receive one to three
courses. Patients with CBF leukemia have a decreased
relapse rate if they receive three or four courses of
high-dose cytarabine, rather than one or two.45,46 These
repeated consolidation courses lead to 70-80% of CBF
leukemia patients becoming long-term survivors in
remission. There are, however, certain patients with
CBF leukemia who relapse, despite high-dose cytara-
bine; those with high WBC counts at diagnosis and/or
c-KIT mutations.47,48 Tyrosine kinase inhibitors, such as
dasatinib in c-KIT mutated cases and hematopoietic
transplantation are being investigated to improve the
results in these cases. As in CBF leukemias, it has been
suggested that AML from the intermediate cytogenet-
ics group with a favourable molecular profile may also
benefit from repeated high-dose cytarabine consolida-
tion cycles.40 This group includes AML patients with
mutated nucleophosmin and without the internal tan-
dem duplication of the FLT3 gene (NPM1+/FLT3–), and
those with CEBPA mutations.49-52 In these cases, the
 Berlin, Germany, June 4-7, 2009

possible favourable impact from high-dose cytarabine

should be further investigated. Also in NPM1 positive
AML, recent studies indicate that the addition of
ATRA to CT should be explored, since this agent sig-
nificantly decreased the relapse risk in one study.53
In contrast to these favourable molecular markers,
others confer an adverse outcome to intermediate-risk
cytogenetics AML. These include the internal tandem
duplication of the FLT3 gene (FLT3/ITD),54-58 rearrange-
ments and the partial tandem duplication of the MLL
gene,59-62 mutations of the Wilms’ tumor 1 (WT1)63 and
over-expression of the BAALC64-66 and/or ERG
genes.67,68
Different combinations of postremission CT have
been administered in these unfavourable cases belong-
ing to the intermediate-risk cytogenetics category, and
in AML with adverse karyotype. So far, none of them
has shown that high-doses of cytarabine improve the
results compared to intermediate or conventional
doses. Figure 3. Relapse-free survival in the donor (black line)
To further exploit the antileukemic effect of CT, and no donor (red line) groups in AML patients with normal
some groups administered maintenance treatment. cytogenetics and mutations of NPM1 without FLT3-ITD.
This approach was effective in occasional studies;69 Adapted from Schlenk R et al. N Engl J Med 2008;358:
1909-1918; with permission.
therefore, novel maintenance strategies deserve inves-
tigation. In this sense, the CALGB recently reported
the use low-dose subcutaneous interleukin-2.70
Unfortunately, a protective effect on the development
of leukemia relapse was not observed. Other alterna-
tives, such as the administration of maintenance,
including GO or demethylating agents, are currently
being explored.71

Autologous transplantation
The role of autologous hematopoietic cell transplan-
tation (auto-HCT) in the first CR of AML remains con-
troversial.72 In many centers in the United States, and
in some European countries, this treatment has been
abandoned for this disease, whereas for other institu-
tions and cooperative groups, it is the treatment of
choice for patients without an HLA compatible donor.
Large meta-analyses are ongoing to define whether
autologous transplantation offers an advantage com-
pared with further consolidation CT or abstention. Figure 4. Leukemia-free survival in the donor (green line)
Reported series comparing this treatment versus and no donor (blue line) groups in AML patients from the
chemotherapy have the caveat of the relatively high intermediate and poor risk cytogenetics categories and
transplant-related mortality reported (10%) that does without favorable molecular markers. Experience of the
Spanish CETLAM group.
not reflect the improvement observed nowadays.73
The relapse rate after autografting for adverse cytoge-
netics AML is very high, and for this reason, this treat-
ment is being abandoned in this group of patients. In
contrast, the role of autologous transplantation, if any, availability or not of an HLA-identical sibling are need-
in favourable categories, such as CBF leukemia, ed to draw conclusions. Some studies have recently
NPM1+/FLT3– and CEBPA+ AML, particularly if high been published and others are currently ongoing.29,75,76
WBC counts or other adverse indicators are present, Knowing the strong impact of age and cytogenetics on
deserves investigation within clinical trials. the prognosis of AML, comparisons between treat-
ments have to take these factors into account.74,77
Allogeneic transplantation from HLA-identical siblings Ideally, molecular information with impact on progno-
It is clear that allogeneic hematopoietic cell trans- sis should also be available, but this is the case in only
plantation (allo-HCT) decreases the relapse rate com- a few reports.78,79 In summary, information from
pared to CT or auto-HCT.74 In many studies, this does prospective trials and published meta-analyses indi-
not translate into significantly improved leukemia-free cate that patients with CBF leukemia and
survival and/or overall survival as there is a 15-30% NPM1+/FLT3– AML have equal survival in donor and
transplant related mortality. As in the case of auto- no donor groups (Figure 3).29,75-77,79 In contrast, in most
HCT versus CT, large meta-analyses based on the studies, patients in the intermediate cytogenetics

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 33 |
14th Congress of the European Hematology Association
group without a favourable molecular profile, and
those in the adverse karyotype category benefit from
allogeneic transplantation (Figure 4). Some experiences
state that the advantage is limited to young patients,
up to the age of 35 years, since in older patients, trans-
plant related mortality counterbalances the decrease in
relapse rate observed after allograft.29 In patients aged
above this cut-off, an individualized decision concern-
ing the administration of reduced intensity condition-
ing may be an option when comorbidity scores are
high. Reduced intensity conditioning has allowed
extending the benefit of the graft versus leukemia
effect to patients up to the age of 75.80-82 To define the
impact of this treatment on survival of elderly adults
with AML in first CR, a prospective randomized com-
parison versus no transplantation is ongoing in Europe.
Alternative donor and sources for hematopoietic trans-
plantation
Since the majority of AML patients who are poten-
tial candidates for allo-HCT lack an HLA-identical sib-
ling, the use of alternative donors and stem cell sources
has increased greatly over the last two decades.83,84
Regarding unrelated transplants from adult donors
(URD), results have improved due to the better selec-
tion of donor-recipient pairs based on high-resolution
molecular HLA-typing.84 URD transplants were initial-
ly restricted to patients with an extremely high-risk of
relapse due to increased transplant related mortality
(TRM), as compared to those from HLA-identical sib-
lings. Currently, the indications of the two procedures
are similar since the risk of death due to complications
is the same in experienced centres. Transplantation of
hematopoietic progenitors from unrelated cord blood
is also an option for adult patients with AML, although
cell dose remains a limitation for this procedure.85,86
Due to the risk of graft failure or delayed engraftment,
this type of transplant is usually restricted to patients
with a high risk of leukemia relapse and who need an
allogeneic procedure on a short-term basis. In this cir-
cumstance and within clinical trials, haploidentical
family transplantation is another possibility.87
Different approaches to improve immune recovery
after the procedure, based on ex vivo or in vivo depletion
of alloreactive cells, are being explored.
New approaches for tailoring treatment
Gene expression profiling (GEP) is a recently intro-
duced method to identify molecular pathways
involved in AML and to select new therapeutic tar-
gets.88-91 GEP studies have shown to be useful by recog-
nizing known AML subtypes, detecting new varieties,
and separating otherwise not identifiable prognostic
groups. This method may be useful in the near future
to adjust treatment based on patterns of expression
and genes involved. Methylation status of a large num-
ber of genes may also be studied using this technique.92
This may identify those patients who are more suscep-
tible to responding to demethylating agents. The
recent discovery of the impact of certain micro RNAs
on the development and prognosis of AML justifies
further investigation on this subject.93 For a better dis-
crimination of susceptibility to treatment, additional
studies on individual genetic polymorphisms are nec-
| 34 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
essary. Improved methods for quantitative evaluation
of MRD after treatment in AML, and their role in spe-
cific molecular subgroups are being explored.94,95 Based
on the results, additional targeted treatment or intensi-
fication with hematopoietic transplantation may be
decided. Finally, a large variety of new agents with
potential activity in AML is under investigation in clin-
ical trials.96,97 Many drugs to treat AML are in at differ-
ent stages of development in pharmaceutical compa-
nies. All these mentioned advances indicate that
research in biology, prognosis and risk-adapted treat-
ment in AML will continue to be a challenging and
evolving area in the coming years.
This work was supported in part by grants from the
Instituto de Salud Carlos III, Spain (07/EC90065 and
06/0020/0101RD).
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 Bleeding disorders

von Willebrand factor: its role in hemostasis and

thrombosis

S.F. De Meyer
K. Vanhoorelbeke
Laboratory for Thrombosis Research,
IRC, KULeuven Campus Kortrijk,
Belgium
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
A B S T R A C T
A key player in thrombosis and hemostasis is von Willebrand factor (VWF), a multimeric glycopro-
tein that is essential for the recruitment of circulating platelets to damaged vessels and for protect-
ing the anti-hemophilic coagulation factor VIII from rapid degradation. As a result, quantitative or
qualitative defects in VWF lead to the most common inherited bleeding disorder – von Willebrand dis-
ease. On the other hand, the presence of over-reactive VWF multimers may result in severe thrombot-
ic complications. While our knowledge on this intriguingly large hemostatic factor is increasing, its
pivotal role in maintaining the fine balance between bleeding and thrombosis is being revealed. In this
article, we will discuss the role of VWF in hemostasis and give a concise overview of how our grow-
ing understanding of VWF biology can be used in the design of treatment modalities for both bleed-
ing and thrombotic complications.

2009;3:38-43

Structure of von Willebrand factor
von Willebrand factor (VWF) is a large
adhesive, multimeric glycoprotein that ful-
fils two crucial roles in primary hemostasis.
First, VWF functions as a bridge between
subendothelial structures in the injured ves-
sel wall and circulating blood platelets,
thereby supporting the formation of a
platelet-rich plug that prevents excessive
bleeding and promotes wound healing.
Second, by serving as a carrier protein for
factor VIII (FVIII), it protects rapid degrada-
tion of this clotting factor. VWF is synthe-
sized exclusively in endothelial cells and
megakaryocytes as pre-proVWF subunits.1
Each subunit consists of four types of repeat-
ed domains that are arranged in the
sequence D1-D2-D’-D3-A1-A2-A3-D4-B1-
B2-B3-C1-C2-CK2,3 (Figure 1). After removal
of the signal peptide, pro-VWF becomes gly-
cosylated and dimerizes through the forma-
tion of disulfide bonds between C-terminal
CK domains in the rough endoplasmic retic-
ulum.4 Upon transportation through the
Golgi complex and the post-Golgi compart-
ments, pro-VWF is sulphatated; carbohy-
drates are further processed and multimers
are formed through head-to-head disulfide
bonds between D3 domains.5,6 Concomitant
with the multimerization, the propeptide
(D1D2 domains, Figure 1) is proteolytically
removed from mature VWF. The modular
structure of VWF forms the basis of its mul-
tifunctional properties (Figure 1). The N-ter-
minal D’ and D3 domains contain binding
sites for FVIII7 and heparin.8 The A1 domain
is the essential binding site for the platelet
glycoprotein (GP) Ib/IX/V-complex9 and has
multiple binding sites for a variety of other
molecules while the A3 domain comprises
the major binding sites for fibrillar colla-
gen.10,11 Finally, VWF interacts with the
platelet receptor αIIbβ3 via its C1 domain.12
von Willebrand factor size regulation
Endothelial VWF is either constitutively
secreted into the blood or is stored in
Weibel-Palade bodies,13 from which it is
released upon stimulation or, as recent evi-
dence suggests, via an unstimulated basal
pathway.14 VWF present in platelets is stored
in α-granules and is only released after
platelet activation.15 Whereas VWF storage
granules contain ultra-large (UL) VWF multi-
mers (>10.000 kDa) for acute and local
release where needed, circulating VWF is
composed of smaller multimers ranging
from 2 (500 kDa) to up to 40 monomers (up
to 10.000 kDa).16 The size of the VWF multi-
mers is an important regulator of their reac-
tivity since the largest multimers are the
most active ones. Hence, since the circula-
tion of UL (and thus hyper-reactive) VWF
multimers can cause spontaneous intravas-
cular thrombosis, as occurs in the devastat-
ing disease thrombotic thrombocytopenic
purpura, their size must be tightly regulated
upon release from endothelial cells and
platelets. Multimer size is controlled by the
metalloprotease ADAMTS13 (a disintegrin
and metalloprotease with thrombospondin
type 1 repeats, 13),17,18 which digests the
released UL-VWF into the smaller multimers
found in plasma. Interestingly, the proteolyt-
ic site of VWF (Y1605-M160619) is buried in
the A2 domain, and becomes exposed after
conformational changes induced by, for
example, shear or denaturating agents.20-22
Once in the bloodstream, VWF multimers
circulate with a half-life of 8 to 12 hours.
The exact clearance mechanism of VWF is
not yet fully understood, but recent evidence

| 38 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

Figure 1. Structure of VWF. VWF is synthesized as a pre-pro-VWF that comprises a 22-amino acid (aa) residue signal pep-
tide (SP), a 741 aa-residue pro-peptide and the 2050 aa-residue mature subunit. The pro-peptide and the mature sub-
unit compose pro-VWF (2791 residues) consisting of four types of repeated domains which are indicated. After removal
of the signal peptide from pre-pro-VWF, pro-VWF translocates into the endoplasmic reticulum, where pro-VWF subunits
associate to ‘tail-to-tail’-dimers by the formation of disulfide-bonds between the cysteine-rich carboxyl-terminal CK
domains, which in turn, are transported to the Golgi. There, the ‘tail-to-tail’-dimers multimerize by forming ‘head-to-head’
disulfide bonds between the aminoterminal cysteine-rich D3 domains. The main binding sites that are important for the
hemostatic function of VWF are indicated together with the ADAMTS13 cleavage site. Major regions in which mutations
have been found that are associated with VWD type 1 and 2 are also shown.

suggests a role for macrophages in the removal of the

VWF/FVIII complex in both liver and spleen.23

von Willebrand factor and its role in platelet adhesion and

aggregation
Under (venous) low shear conditions (<1000 s–1), the
first steps of platelet adhesion at sites of vascular injury
are established through direct interactions of the
platelet collagen receptors α2β1 and GPVI with the
exposed collagen. However, under higher shear condi-
tions (>1000 s–1) initial platelet adhesion becomes more
dependent on VWF to capture fast-flowing platelets.
Upon exposure of subendothelial structures at vascu-
lar lesions, VWF binds to fibrillar collagens through its
A3 domain (Figure 2).10,11 Elegant studies revealed that a
single high affinity binding site in collagen type III24
interacts with the front face of the VWF A3 domain.25,26
Fluid dynamic forces on collagen-bound VWF induce a
conformational alteration, stretching the globular form
of VWF to a more elongated structure.27,28 This results in
the abolition of the shielding of the A1 domain by the
A2 and/or D’D3 domains;29-31 thereby exposing its
GPIbα binding sites, which are situated in two separate
areas in the A1 domain.32,33 The interaction between the
A1 domain and the platelet receptor GPIbα is character-
ized by fast on- and off-rates, allowing transient tether-
ing and translocation of platelets on the damaged vessel
wall.34-37 This deceleration mechanism permits platelets
to establish more firm interactions between their colla-
gen receptors and the exposed subendothelium, eventu-
ally leading to final platelet arrest at the site of injury
(Figure 2). Platelet adhesion is followed by platelet acti-
vation and aggregation involving interaction of activat-
ed αIIbβ3 with RGD sequences in fibrinogen and VWF.
Recently, it was suggested that platelet aggregation can
Figure 2. Platelet adhesion. Under high shear conditions,
platelet adhesion is initiated by the transient tethering of
the platelets to VWF, which is anchored to the damaged
vessel wall. This reversible interaction between the
platelets and collagen-bound VWF slows down the
platelets and allows the formation of firm interactions
between the collagen receptors α2β1 and GPVI and the
exposed collagen. The concomitant platelet activation
results in granular release and activation of integrin
αIIbβ3.
occur in the absence of platelet activation at pathologi-
cal shear rates that exceed 10,000 s–1 found, for example,
in stenotic coronary arteries. Above this threshold,
active A1 domains become exposed in soluble VWF
multimers, promoting additional platelet recruitment
via GPIbα binding.38 Apparently, plasma VWF does not
only bind to exposed collagen, but also to the first layer
of collagen-bound VWF multimers.39,40 This process of
reversible self-association between immobilized VWF
and plasma VWF modulates the adhesive properties of
immobilized VWF.39

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 39 |
14th Congress of the European Hematology Association
Physiological importance of von Willebrand factor
von Willebrand disease
VWD is a bleeding disorder caused by inherited VWF
abnormalities. The prevalence of VWD patients that
need treatment is estimated to be 0.002-0.01%,
although low VWF-levels may be found in up to 1% of
the population.41-43 VWD is commonly characterized by
mucosal hemorrhages, such as epistaxis, menorrhagia,
gastro-intestinal and gingival bleeding, and often pro-
longed bleeding after trauma to skin or mucous mem-
branes, especially during or after surgery. The most
severely affected patients, who also have low FVIII lev-
els, additionally suffer from spontaneous soft-tissue
bleeding. Although the more than 250 mutations found
in VWD (database: www.sheffield.ac.uk/vwf), provide
useful information on the cause of the bleeding pheno-
type, current VWD classification44,45 is based on the
underlying pathophysiological defects, indicative for
optimal clinical treatment. Partial or complete quantita-
tive deficiency of VWF results in type 1 and 3 VWD
respectively, and qualitative defects in type 2 VWD.
Type 1 von Willebrand disease
This is found in 60-80% of all VWD cases, and is due
to mild/severe quantitative defects of VWF and FVIII.
Although low levels can be due to various causes, such
as ABO blood group, recent large studies indicated that
most are associated with mutations within VWF.46-48
Highly penetrant dominant mutations, mostly single
amino acid mutations in the D3-domain, either affect
production following disturbed intracellular transport of
dimeric proVWF or provoke rapid clearance from the
circulation.49
Type 2 von Willebrand disease
Subtype 2A VWD is the most common qualitative vari-
ant with generally an autosomal dominant inheritance.
The large and intermediate multimers are lost due to
mutations in either the propeptide or the C-terminal cys-
tein-knot-domain, resulting in defective assembly and/or
in increased susceptibility to ADAMTS13.50 As a conse-
quence, both platelet- and collagen-binding are dis-
turbed. Isolated defects within the A1-domain, causing
an impaired binding to platelet GPIbα51 give rise to sub-
type 2M VWD, an autosomal dominant variant with
(near) normal multimeric distribution. One family has
been identified with a mutation in the A3-domain,
resulting in defective binding to collagen,52 also possibly
causing type 2M VWD. In contrast, in subtype 2B VWD,
usually inherited as an autosomal dominant trait, various
mutations within the A1-domain cause an increased and
frequent spontaneous binding of VWF to GPIbα.51 The
larger multimers are lost from plasma as binding to
platelets facilitates proteolysis by ADAMTS13 and/or
results in faster clearance of platelet-VWF complexes,
which often gives rise to thrombocytopenia.53 Together,
they explain the apparently paradoxical bleeding diathe-
sis in these “gain-of-function” phenotypes. Subtype 2N
VWD, an autosomal recessive disorder, due to a
decreased affinity of VWF for FVIII, is caused by some
20 homozygous or compound heterozygous missense
mutations in the D’ or D3 FVIII binding-domain. In case
of severe FVIII reduction, type 2N VWD resembles mild
or moderate hemophilia A.
| 40 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Type 3 Von Willebrand disease
Type 3 VWD is the most severe VWD form, charac-
terized by recessive inheritance with a prevalence rang-
ing from 0.5 to 5 per million, with virtually completely
absent VWF and in addition low levels of FVIII. Some 90
associated mutations51 are scattered over the entire gene
comprising partial/total deletions, small insertions,
exon/intron boundary splice site mutations which result
in exon skipping or aberrant splicing, missense and
frameshift mutations.
Treatment of von Willebrand disease
Current treatments
The main goal in treatment of VWD is the correction
of hemostatic deficits caused by VWF and FVIII defi-
ciency. At present, two different treatment modalities
make up the mainstay of VWD management: adminis-
tration of desmopressin, which induces the secretion of
endogenous VWF from endothelial cells, and direct
administration of exogenous VWF contained in plasma
derived concentrates.49,54-56
Future treatments
Recently, new strategies to treat VWD are being
explored, including the development of a recombinant
VWF preparation, the use of interleukin (IL)-11 and
attempts to provide a long-term cure for VWD by VWF
gene transfer. Use of a standardized preparation of
recombinant VWF (rVWF) produced in CHO cells under
plasma-free conditions57 would eradicate the risk of
blood-borne pathogen transmission in patients and
would bypass the high degree of variability present
among the FVIII/VWF concentrates. Recent observa-
tions that IL-11 increases VWF and FVIII levels in mice,58
dogs59 and humans60 (by a not yet identified mechanism)
stimulate further research on the use of recombinant IL-
11 to treat VWD patients.58,60,61 Since VWD is a mono-
genic disease, it is an appealing candidate for gene ther-
apy.62-66 Indeed, instead of repetitive on-demand replace-
ment of the defective protein, a permanent correction of
the underlying genetic defect would provide a life-long
cure that would dramatically improve the quality of life
of severe bleeders. Original concerns on the incorpora-
tion of the large VWF cDNA (8,4kb) into integrating
viral vectors have been eliminated by the recent con-
struction of a lentiviral vector accommodating full
length VWF.65 Furthermore, using this vector, the pheno-
type of blood outgrowth endothelial cells isolated from
dogs suffering from VWD type 3 could be completely
corrected. Since lentiviral vectors would predominantly
target the liver upon systemic administration, transgene
encoded VWF would be synthesized in liver cells; how-
ever, this is an ectopic site for VWF expression.
Interestingly, using the technique of hydrodynamic
gene transfer in a murine model of severe VWD, we and
others demonstrated that the liver is capable of produc-
ing VWF containing the full range of multimers.63,66,67
Transgene encoded, liver expressed VWF restored FVIII
levels, and most importantly, reversed the prolongation
of the tail bleeding time,63,66,67 and in a FeCl3-induced
thrombosis model, restored the platelet plug forming
capacity, still one of the main functions of VWF in
hemostasis.66,68 It is evident that these first steps towards
gene therapy for VWD are only the beginning. More

studies in the future will undoubtedly reveal the possi-
bilities and limitations of VWF gene therapy to cure
severe VWD.
von Willebrand factor as antithrombotic target
Because of the pivotal role of VWF in hemostasis and
thrombosis, there is an increasing interest in targeting
VWF as antithrombotic strategy. Indeed, recently, many
compounds have been developed that inhibit the bio-
logical activity of VWF. As outlined above, one of the
major roles of VWF is mediating platelet adhesion at
sites of vascular injury. Recruitment of platelets is
accomplished by the binding of VWF to both platelets
(GPIbα) and exposed subendothelial collagen.
Consequently, interfering with the binding of VWF to
collagen, as well as inhibiting the binding of VWF to
GPIbα are appealing strategies for antithrombotic ther-
apy. Since these interactions are first steps in platelet
adhesion without any known redundancy, inhibition is
expected to have a good antithrombotic potency.
Moreover, it is anticipated that inhibition of platelet
adhesion might induce less clinical bleeding problems
compared to the antiplatelet drugs currently used in the
clinic. Indeed, since VWF has a dominant role in platelet
adhesion, essentially at high shear stress, interfering
with VWF-mediated platelet adhesion would be more
specific for high-shear pathological (stenosed) arterial
systems, leaving the hemostatic situation in venous sys-
tems rather intact.69-72
During the late nineties, two antithrombotic anti-
human VWF antibodies were developed in two differ-
ent groups; one targeting the VWF A1, thus inhibiting
its binding to GPIb and the other one targeting the VWF
A3 domain, thereby inhibiting VWF binding to collagen.
The first, monoclonal antibody AJvW2, was developed
by Yoshimoto and colleagues and was found to inhibit
the VWF-GPIbα interaction in various in vitro assays.73
When tested in experimental thrombosis models in ani-
mals, AJvW2 demonstrated a potent antithrombotic
effect with no indication for severe bleeding complica-
tions.73-75 The second antibody, murine monoclonal anti-
body 82D6A3, was developed in our group and has an
epitope that overlaps with the collagen binding region
in the VWF A3-domain;76,77 thereby, inhibiting the bind-
ing of VWF to fibrillar collagen type I and III under stat-
ic and flow conditions.76,78 The in vivo antithrombotic
efficacy of 82D6A3 has been demonstrated in baboons
using a modified Folts model,79 where a strong reduction
of thrombus formation was observed after administra-
tion of 300 µg/kg 82D6A3, without significantly pro-
longing the bleeding time. In the mean time, both
AJvW2 and 82D6A3 have been humanized80,81 for test-
ing in humans. Humanized AJvW2 (named AJW200)
was well tolerated when administered to 24 male
healthy humans and ristocetin cofactor activity was sig-
nificantly reduced until 12 hours after infusion of 0.01-
0.05 mg/kg AJW200, without changing template bleed-
ing times.82 In pursuit of these first studies, several other
components have been developed, all targeting the
VWF A1 domain. GPG-290 is a recombinant fragment
of human GPIbα with two gain-of-function mutations
(G233V/ M239V) grafted on the Fc part of a human
IgG1, and expressed as a homodimer linked by the two-
disulfide bonds in the Fc hinge region. Its antithrombot-
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 41 |
Berlin, Germany, June 4-7, 2009
ic efficacy has been shown in two experimental throm-
bosis models in dogs.83,84 ALX-0081 is a cameloid anti-
VWF A1-domain bivalent nanobody that reduces
thrombus formation in a modified Folts model in the
baboon femoral artery and ex vivo ristocetin induced
platelet aggregation.85 ARC1779 is a synthetically man-
ufactured, modified DNA/RNA aptamer, which by
binding to the VWF A1 domain, inhibits its interaction
with GPIbα. This compound prevents the formation of
occlusive thrombi in cynomolgus macaques during con-
tinuous electrical injury for six hours with only modest
prolongation of the bleeding time at the optimal
antithrombotic dose.86 In a placebo-controlled study in
47 healthy volunteers, ARC1779 was well tolerated,
and maintained its ex vivo activity, without bleeding
complications.87
It is clear that a potent antithrombotic effect is
obtained at inhibitor doses that do not markedly affect
bleeding times when the collagen-VWF-GPIbα axis is
blocked. However, it is not yet fully clear whether
reduced bleeding times will also translate into a lower
bleeding risk in patients. Nevertheless, all data clearly
indicate that the therapeutic window with these kind of
compounds is significantly larger than with the current-
ly available antiplatelet agents. In analogy with this, we
and others recently demonstrated that, in a murine
ischemic stroke model, deficiency of VWF or blocking
GPIbα protected the mice from brain ischemia without
inducing excessive bleeding.88,89 If this can be confirmed
in man, this class of compounds might even become the
treatment of choice for ischemic stroke.
S.F.D.M. and K.V. are postdoctoral fellows of the FWO
(Fonds voor Wetenschappelijk Onderzoek, Belgium).
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 Bleeding disorders

Phenotype, genotype and classification

in von Willebrand disease

A. Goodeve A B S T R A C T

The common autosomally-inherited, mucocutaneous bleeding disorder von Willebrand disease

Academic Unit of Haematology,
University of Sheffield School of
Medicine and Biomedical Science
and Sheffield Diagnostic Genetics
Service, Sheffield Children’s NHS
Foundation Trust, Sheffield, UK
(VWD) displays a range of different phenotypes and patterns of inheritance, which can be categorised
into three different types. Types 1 and 3 describe partial and virtually complete quantitative deficien-
cies of plasma von Willebrand factor (VWF), whereas type 2 comprises four subtypes differentiated by
the specific VWF functions that they perturb. Mutation type and location responsible for each VWD
type are explored, along with a look at the mechanisms by which mutations result in disease.

Hematology Education:
the education program for the
annual congress of the European
Hematology Association

2009;3:44-49

on Willebrand factor (VWF) is a large the normal complement of high molecular

v multimeric plasma glycoprotein essen-

tial for primary hemostasis. When dam-
age to blood vessels occurs, it binds to colla-
gen in exposed sub-endothelium, and fol-
lowing a conformational change, to the
platelet Glycoprotein 1b (Gp1b). This initi-
ates platelet thrombus formation at the site
of vascular damage. Additionally, VWF
binds to the coagulation factor VIII (FVIII),
protecting it from premature proteolytic
degradation in the circulation and delivering
it to injury sites. von Willebrand disease
(VWD), a common autosomally-inherited
bleeding disorder results from perturbation
of these functions through quantitative
(types 1 and 3) or qualitative (type 2) defects.
The disorder clinically affects approximately
1 in 10,000 individuals,1 with symptoms
ranging from nose bleeds and easy bruising
through to bleeding into joints and gastroin-
testinal bleeding, with menorrhagia and
bleeding associated with childbirth being
common symptoms in affected females.
The VWF protein is composed of repeated
domains in the order D1-D2-D’-D3-A1-A2-
A3-D4-B1-B2-B3-C1-C2-CK. This large pro-
tein, comprising 2,813 amino acids,2 under-
goes carboxyl terminal dimerisation through
disulphide bonds joining CK domains, fol-
lowed by multimerisation involving inter-
molecular disulphide bonding of adjacent
D3 domains, prior to cleavage of the propep-
tide (D1-D2 domains).3 Multimers, compris-
ing up to 40 dimeric subunits4 are present in
the circulation and are required for full
hemostatic function. SDS-agarose gels can
be used to assess the extent of multimerisa-
tion of VWF present in plasma, and whether
weight (HMW) forms are present.5 Upon
secretion from endothelial cells or platelets,
VWF is cleaved by its protease, a disintegrin
and metalloproteinase with throm-
bospondin motifs (ADAMTS13) between
residues p.Tyr1605_Met1606 to reduce its
size and thrombogenic potential.
von Willebrand disease phenotype
Bleeding assessment
Assessment of whether an individual
bleeds more than is normal can be difficult
to assess and a number of clinical tools for
taking a detailed bleeding history have been
developed. Tosetto and colleagues devised
an extensive bleeding score, for which utili-
ty was demonstrated in two European stud-
ies on type 1 VWD.6,7 Scores are given to
each of several different bleeding symptoms,
including epistaxis, bruising and bleeding
after tooth extraction and surgery. Each
symptom is given increasingly positive
scores where intervention is required for
cessation of bleeding. In the Molecular and
Clinical Markers for the Diagnosis and
Management of Type 1 VWD (MCMDM-
1VWD) study, scores ranged from -1 to +4
for each symptom. Possible scores ranged
from -3 (no bleeding following tooth extrac-
tion and two surgeries) to +45. Median
scores were -1 in healthy controls and 9 in
type 1 VWD index cases.7 The majority of
index cases had significantly higher scores
than healthy controls did. The recently pub-
lished modified MCMDM-1VWD bleeding
score8 uses symptoms shown to best dis-
criminate bleeders from non-bleeders and
can be completed in 5 to 10 minutes. Such a

| 44 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

score may become part of an initial standardised assess-
ment of possible VWD patients in the future.
Laboratory assessment
Determination of VWF quantity and function in indi-
viduals investigated for possible VWD requires a num-
ber of tests: (i) amount of protein can be quantified
using immunological analysis (VWF:Ag); (ii) ability to
aggregate platelets assessed following conformational
change mimicking binding to subendothelium stimulat-
ed by the antibiotic ristocetin (VWF:RCo); (iii) quantity
of FVIII through its activity in the coagulation cascade
(FVIII:C). The normal range for each of these parame-
ters is approximately 50 to 200 IU/dL. Specialist tests to
differentiate specific subtypes are described in a subse-
quent section.
Genotype
The VWF gene (VWF) is located near the tip of chro-
mosome 12 at 12p13.3. The gene covers 178 kb of
genomic DNA organised into 52 exons. A partial
pseudogene VWFP on chromosome 22 (22q11.22-
11.23), which is 97% similar in sequence to VWF,9 com-
plicates genetic analysis and promotes gene conversion
mutations.10 Due to the very polymorphic nature of
VWF,11 mutation scanning techniques, including dena-
turing high performance liquid chromatography
(dHPLC), conformation sensitive gel electrophoresis
(CSGE) and single strand conformation polymorphism
(SSCP) analysis do not work well in mutation detection.
This is because they frequently detect heterozygous sin-
gle nucleotide polymorphisms (SNPs), as well as the
mutation sought, but do not indicate the nature of the
variant identified.12 Therefore, DNA sequencing is the
preferred genetic analysis method. Determination of
whether a sequence variant is pathogenic can be chal-
lenging; in vitro expression has a useful role in demon-
strating the likely pathogenicity of sequence variants.
Types 2 and 3 VWD have been examined for muta-
tions for nearly 20 years. As mutations in type 2 VWD
affect specific functional domains, and an isolated sec-
tion of VWF can be targeted for analysis, they have been
more accessible to examination, and many mutations
have been reported, helping to define the extent of func-
tional domains. A web-based database, compiled on
behalf of the International Society Thrombosis and
Haemostasis Scientific and Standardisation Committee
(ISTH-SSC) on VWF (VWFdb) is a repository for infor-
mation on VWF sequence variation.11
von Willebrand disease
Type 1 von Willebrand disease
This most common form of VWD (up to 70% of
cases) comprises patients with a partial quantitative
VWF deficiency, where the proportion of HMW multi-
mers is not significantly decreased. Plasma levels of
VWF range from approximately 5 to 50 IU/dL, with
VWF:Ag and VWF:RCo being similarly reduced, and
FVIII:C levels typically 1.5 times higher than those of
VWF (Table 1). Bleeding symptoms are often moderate

Table 1. Phenotype, genotype and mechanisms involved in von Willebrand disease.

VWD Type Description Phenotype* Genotypes Inheritance† & Mechanism

responsible penetrance

1 Partial quantitative VWF VWF:RCo & VWF:Ag Missense Generally AD Under investigation, but include
deficiency, HMW multimers reduced in parallel Other in-frame Reduced penetrance increased clearance
not significantly decreased VWF:Ag >5 IU/dL alterations common Enhanced ADAMTS13
Null alleles Small proportion AR susceptibility
Interaction with ABO
blood group O
Contribution of other
inherited/environmental factors
2A Reduced VWF-dependent VWF:RCo disproportionately Missense 2A (IIA & IIE) AD Enhanced ADAMTS13
platelet adhesion plus selective reduced to VWF:Ag Other in-frame Fully penetrant susceptibility
HMW multimer deficiency alterations 2A (IID) mostly AD Disrupted dimerisation/
Null alleles 2A (IIC) AR multimerisation
2B Increased affinity for platelet GpIb Enhanced RIPA sensitivity Missense AD Spontaneous GP1b binding
Variable VWF levels Other in-frame Fully penetrant Enhanced ADAMTS13 susceptibility
Platelet count often reduced alterations
2M Decreased VWF-dependent platelet VWF:RCo or VWF: Missense AD Reduced GP1b or collagen binding
adhesion without CB very disproportionately Other in-frame Fully penetrant
selective HMW multimer deficiency reduced to VWF:Ag alterations
2N Markedly decreased VWF:RCo & VWF: Missense AR Reduced FVIII binding
FVIII binding affinity Ag normal/reduced Null alleles
FVIII:C <30IU/dL
3 Virtually complete VWF VWF:RCo & VWF: Null alleles AR Lack of secretion
deficiency Ag <5IU/dL Missense
FVIII:C <10IU/dL
* levels;† AD: Autosomal dominant; AR: autosomal recessive.

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 45 |
14th Congress of the European Hematology Association
to mild, but their severity is not closely linked to VWF
level. Three recent systematic studies of type 1 VWD,
which together have analysed about 300 patients, pro-
vided insight into this VWD type.13-15 Sixty to seventy
percent of cases have a mutation identified. Candidate
mutations have been reported throughout VWF, from
the 5’ untranslated region (UTR), (c.-2731) through to
exon 52 (p.C2804). Figure 1 shows mutation location.
The majority are missense changes (70%),16 with 5’
UTR, splice (leading to either exon skipping or to a non-
expressed (null) allele), small deletion, nonsense and
small insertion/duplication mutations, respectively
comprise the remainder, in order of frequency of occur-
rence. Pathogenicity of 5’ UTR variants has yet to be
demonstrated. Large deletions have recently been
recognised to contribute to the spectrum of mutations.17
Two of thirty-four British patients were heterozygous
for an exon 4-5 deletion, first identified in British type 3
VWD patients. Mutations vary from fully penetrant,
dominantly inherited variations, where all individuals
who inherit a particular sequence variant are affected by
disease, for example the “Vicenza” p.R1205H mutation,
to incompletely penetrant alterations, such as
p.Y1584C, where an interaction with blood group O is
required for disease.16
After the coding region of VWF plus 3kb of 5’ UTR
had been analysed, a significant proportion (30-40%) of
type 1 VWD patients had no candidate mutations iden-
tified.13-15 The majority of these cases had VWF levels
greater than 20 IU/dL. Bleeding in these patients was
not significantly different from that in patients with a
candidate VWF mutation, who generally had lower
VWF levels.14 It is, therefore, likely that other hemostat-
ic defects also contribute to bleeding in patients diag-
nosed with “type 1 VWD.”
Type 1 is not subdivided into different categories.
However, different mutation mechanisms resulting in
the disease are beginning to be identified. The most
recognisable is enhanced clearance rate of VWF from
the plasma. Mutations in the D3 domain, notably those
affecting p.C1130, p.C1149 and p.R1205,18,19 plus
residues elsewhere in VWF (e.g., p.S2179F),20 have a
plasma residence time that is up to ten times shorter
| 46 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
than normal. Determination of the steady-state ratio of
VWF propeptide (VWFpp) to VWF:Ag or of dynamics of
VWF response following desmopressin infusion21 pro-
vide information on VWF clearance. Ultra-large VWF
multimers can sometimes highlight individuals with
clearance mutations, as ADAMTS13-VWF cleavage is
reduced by rapid VWF clearance from the plasma.22
p.Y1584C, present in approximately 1% of normal
Caucasian populations,23 but very enriched in type 1
VWD (8-25% of IC are heterozygous for the variant,
homozygotes are rare) has been demonstrated to have
slightly enhanced ADAMTS13 cleavage,24 not leading to
multimer abnormality. The p.C1584 variant is cleared
more rapidly than p.Y1584, but the increase in clearance
rate is much less overt than for the above mutations.
ABO blood group O also results in slightly enhanced
clearance of VWF from plasma, and co-inheritance of
both blood group O and p.C1584 leads to more rapid
clearance of VWF from plasma (up to 1.8 times higher
than for group A p.Y1584 homozygotes).25 This results
in a greater likelihood of low VWF levels and diagnosis
of type 1 VWD. In the MCMDM-1VWD study, 85% of
family members heterozygous for p.Y1584C were
classed as affected, but index cases had been selected
due to their bleeding symptoms. This variant, therefore,
shows incomplete penetrance, a long-recognised feature
of type 1 VWD for which explanations are beginning to
be made.
Type 2 von Willebrand disease
Twenty to thirty percent of all VWD patients can be
classified as having type 2 disease, where VWF function
is affected largely as a result of missense alterations.
Type 2A von Willebrand disease
Decreased VWF-dependent platelet adhesion and
selective deficiency of HMW VWF multimers charac-
terise this VWD type.4 Levels of VWF:Ag and VWF:RCo
are reduced, whereas FVIII may be normal (Table 1).
The deficiency of large multimers can result from
defects in multimer assembly4 or from enhanced sensi-
tivity to ADAMTS13 cleavage.26 An earlier classification
based on multimer abnormalities can be useful to subdi-
Figure 1. von
Willebrand factor pro-
tein showing func-
tional domains and
exons encoding
them, binding sites
and location of muta-
tions in each VWD
type.

vide type 2A,4 and correlates to an extent with function-
al impairment. Previous IIA comprises two groups of
mutations, located in and close to the A2 domain,
where they cluster around the p.Tyr1605_Met1606
ADAMTS13 cleavage site. Group I mutations impair
multimer assembly and enhance proteolysis, whereas
group II mutations do not affect large multimer secre-
tion but enhance proteolysis. However, mutation mech-
anism cannot be predicted by location. A small number
of common missense mutations make up about 50% of
all VWFdb reports,11 with mutations affecting p.S1506
and p.R1597 being the most common. These mutations
are generally dominantly inherited and fully penetrant.
Figure 1 shows mutation location.
VWF must dimerise and then multimerise prior to
secretion for full hemostatic activity. Other type 2A
mutations specifically interfere with this process. The
CK domain at the carboxyl terminal of VWF is involved
in the formation of “tail-to-tail” dimers through disul-
phide bond formation. Mutations at, or affecting these
cysteines, result in reduced or failed dimerisation (IID).
Multimer profiles show a characteristic pattern with
intermediate sized bands truncated by a monomer.27
Mutations are mostly dominantly inherited, but some
homozygous recessive cases have been reported.11,28
Mutations in the D3 domain result in a characteristic
multimer pattern with a smeary appearance, lacking
obvious multimer satellite “triplet” bands. This IIE phe-
notype29 often results from mutations affecting cysteine
residues, which may be involved in inter-molecular
disulphide bonds between adjacent D3 domains.
Mutations in the D1 and D2 domains may also inter-
fere with disulphide bonding between D3 domains,
catalysed by two CGLC disulphide isomerase sites
(p.159_162 and 521_524), thus affecting multimer for-
mation. Recessively inherited homozygous/compound
heterozygous missense mutations in exons 12-16 result
in a characteristic 2A (IIC) multimer pattern, devoid of
satellite “triplet” bands.29
Type 2B von Willebrand disease
Qualitative variants with increased affinity for
platelet GPIb are classified as type 2B. Increased platelet
agglutination at low ristocetin concentrations (deter-
mined in the ristocetin induced platelet agglutination
(RIPA) assay) identifies such patients. Most patients
have a reduction in HMW multimers, and the multimer
profile can demonstrate proteolysis of the VWF sub-
units, resulting from increased ADAMTS13 susceptibil-
ity.26 Following secretion, VWF may spontaneously bind
to platelets facilitating ADAMTS13 proteolysis. The
characteristic platelet binding, seen in many 2B patients,
can result in thrombocytopenia, which may be exacer-
bated by pregnancy, stress and use of desmopressin.
Dominantly inherited missense mutations lie within or
close to the A1 domain, with mutations affecting only
15 different amino acid residues having been reported
on the VWFdb (p.1266_1461).11 Mutations generally lie
within the amino terminal of the A1 domain at the
“base” of the globular structure, in contrast to 2M muta-
tions, which cluster at the “top” of the domain.30
Patients with p.P1266L mutations (previously referred
to as type IB New York/Malmo31), which result from
gene conversion where a section of the VWF pseudo-
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 47 |
Berlin, Germany, June 4-7, 2009
gene, VWFP on chromosome 22 invades and replaces a
short section of the gene sequence10,32 may show a dif-
ferent phenotype. Thrombocytopenia and loss of HMW
multimers may not feature, and enhanced RIPA at low
concentration may be the only clue as to VWF involve-
ment in their symptoms.33,34
A phenocopy of 2B VWD, platelet type VWD,35
results from a reciprocal defect in platelet Glycoprotein
1b. Mutations in exon 1 of the GP1BA gene encode mis-
sense or in-frame alterations leading to enhanced affini-
ty for VWF.36,37 The two disorders, which are both dom-
inantly inherited and fully penetrant, can be most read-
ily discriminated by genetic analysis, or alternatively by
plasma-platelet mixing studies.
Type 2M von Willebrand disease
This subtype is comprised of qualitative variants with
decreased VWF-dependent platelet adhesion but with-
out a selective deficiency of HMW multimers.
Mutations disrupt VWF binding to either platelets or
subendothelium.4 This VWD type can be difficult to dis-
criminate from both type 1 and 2A VWD. Good multi-
mer analysis and the VWF-collagen binding assay
(VWF:CB) can be helpful. Very reduced VWF:RCo com-
pared to VWF:Ag indicates defective binding to platelet
Gp1b, whereas a low VWF:CB to VWF:Ag ratio indi-
cates that binding to collagen is disrupted. Due to diffi-
culties in identifying this subtype, relatively few muta-
tions are reported on the VWFdb, only p.V1279I and
p.I1425F having been reported more than once. The
majority of mutations reduce Gp1b binding and are
located in exon 28 (p.1266_1467). A single mutation
resulting in 2M VWD due to reduced collagen binding
has been reported.38 Mutations are dominantly inherited
and fully penetrant.
Type 2N von Willebrand disease
FVIII level is decreased disproportionately to VWF:Ag
in this VWD type and multimers are normal, with
minor exceptions (outlined below). It is often challeng-
ing to discriminate the disorder from mild hemophilia A
in males, or from carriership for hemophilia A in
females. Unless the pedigree is sufficiently large, deter-
mining clear X-linked or autosomal recessive inheri-
tance may not be possible. Diagnosis is ideally achieved
through measuring the ability of patient plasma VWF to
bind to FVIII using an immunoassay (VWF:FVIIIB).
However, this test is not widely available and genetic
analysis of the VWF:FVIIIB region of VWF or of the F8
gene may be required to discriminate the disorders and
facilitate relevant genetic counselling. Two mutations
are necessary for 2N VWD, patients may be homozy-
gous for a missense change, compound heterozygous
for two different VWF:FVIIIB missense mutations or
more commonly compound heterozygous for one
VWF:FVIIIB mutation, with a mutation resulting in a
lack of VWF expression on the second allele. Relative
levels of VWF:Ag and FVIII can give clues as to which
situation is more likely; VWF levels are lower where a
null mutation is present. More than 80% of the muta-
tions reported are located in exons 18-20 (D’) domain,
with p.R854Q, p.R816W and p.T791M being particular-
ly common. p.R854Q is present at polymorphic fre-
quencies in the Caucasian population.39 A much smaller
14th Congress of the European Hematology Association
proportion of mutations are found in exons 17 plus 24-
27 (p.1053_1225).11 They may indirectly affect
VWF:FVIIIB through altering the confirmation of the
FVIII binding site. A small proportion of mutations,
including those affecting cysteine residues also have an
effect on VWF multimer profile. Examples include
p.C788R and p.C1225G.40,41 A patient’s symptoms most-
ly result from their reduced FVIII level (typically 10-30
IU/dL), thus resembling mild hemophilia A. Individuals
heterozygous for a single VWF:FVIIIB missense muta-
tion are rarely symptomatic.
Type 3 von Willebrand disease
This virtually complete deficiency of VWF, where
VWF:RCo and VWF:Ag are less than 5 IU/dL and
FVIII:C is less than 10 IU/dL, is recessively inherited.
When the FVIII:C level is particularly low, patients can
experience joint bleeding, in addition to a range of
mucocutaneous symptoms. Most heterozygous rela-
tives are unaffected by bleeding. Disease prevalence is
greatly increased in the offspring of consanguineous
partnerships, rising from about 0.5/million to 6/million
in these circumstances.42 Mutations result in lack of
VWF expression, either through affecting its production
(nonsense, small deletion, splice, large deletion and
small insertion mutations are responsible, in decreasing
order of frequency) or by missense mutations affecting
VWF secretion. Most of the latter affect dimerisation
(CK domain) or the propeptide and VWF multimerisa-
tion (D1-D3 domains). Other mutations are located
throughout VWF, from p.47_2804. There are no partic-
ularly common mutations, but p.P812fs, p.R1659X and
p.R2535X have each been reported in several patients.
Founder effect, where the same mutant allele causes
disease in a population, is evident for a small number of
type 3 VWD mutations, and is likely to be under-recog-
nized as haplotype of mutated alleles is not commonly
reported. A recently described large deletion of exons 1-
3 is responsible for about half of type 3 VWD cases in
Hungary,43 and a complete 253kb VWF deletion has
been identified in both German and Italian type 3
patients.44 Deletion of exons 4-5 contributes to both
type 3 and type 1 VWD in England,17 and the p.P812fs
mutation is found in type 3 VWD in Scandinavia and
northern Europe.45-47
Summary
The above disease types allow the majority of
patients to be categorised by their VWD phenotype and
this informs management and genetic counselling of
families. However, co-inheritance of different types of
mutations may lead to complex phenotypes that are
challenging to classify, and novel mutations that do not
appear to readily fit into the classification continue to be
identified.48 Investigation of such instances enhances
understanding of the complex biology of VWF.
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41. Jorieux S, Gaucher C, Goudemand J, Mazurier C. A novel
mutation in the D3 domain of von Willebrand factor marked-
ly decreases its ability to bind factor VIII and affects its multi-
merization. Blood 1998;92:4663-70.
42. Rodeghiero F. von Willebrand disease: still an intriguing disor-
der in the era of molecular medicine. Haemophilia 2002;8:292-
300.
43. Mohl A, Marschalek R, Masszi T, Nagy E, Obser T, Oyen F et
al. An Alu-mediated novel large deletion is the most frequent
cause of type 3 von Willebrand disease in Hungary. J Thromb
Haemost 2008;6:1729-35.
44. Schneppenheim R, Castaman G, Federici AB, Kreuz W,
Marschalek R, Oldenburg J et al. A common 253-kb deletion
involving VWF and TMEM16B in German and Italian patients
with severe von Willebrand disease type 3. J Thromb Haemost
2007;5:722-8.
45. Zhang ZP, Falk G, Blomback M, Egberg N, Anvret M. A single
cytosine deletion in exon 18 of the von Willebrand factor gene
is the most common mutation in Swedish vWD type III
patients. Hum Mol Genet 1992;1:767-8.
46. Zhang ZP, Blomback M, Nyman D, Anvret M. Mutations of
von Willebrand factor gene in families with von Willebrand
disease in the Aland Islands. Proc Natl Acad Sci USA 1993;90:
7937-40.
47. Schneppenheim R, Krey S, Bergmann F, Bock D, Budde U,
Lange M et al. Genetic heterogeneity of severe von Willebrand
disease type III in the German population. Hum Genet
1994;94:640-52.
48. Lanke E, Kristoffersson AC, Philips M, Holmberg L, Lethagen
S. Characterization of a novel mutation in the von Willebrand
factor propeptide in a distinct subtype of recessive von
Willebrand disease. Thromb Haemost 2008;100:211-6.
 Bleeding disorders

Treatment options in inherited von Willebrand disease

A.B. Federici A B S T R A C T

Angelo Bianchi Bonomi Haemophilia
and Thrombosis Centre, Department
of Medicine and Medical
Specialities, University of Milan,
Milan, Italy
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:50-54
von Willebrand disease (VWD) is the most common inherited bleeding disorder and is due to quan-
titative or qualitative defects of the von Willebrand factor (VWF). VWD is inherited by autosomal dom-
inant or recessive patterns, but women with mild forms are symptomatic. There are six types of VWD
with peculiar phenotype and genotype: VWD1, VWD2A, VWD2B, VWD2M, VWD2N and VWD3. The ris-
tocetin cofactor activity (VWF:RCo) is the most useful test for diagnosis because it can mimic the
interactions between the VWF and platelets. Two therapeutic approaches are available to manage
patients with VWD: the release of endogenous VWF from endothelial compartments induced by
desmopressin (DDAVP); and the transfusion of exogenous VWF contained in VWF/FVIII plasma-derived
concentrates. DDAVP is the treatment of choice for VWD1 because it can induce the release of nor-
mal VWF from endothelial cells: it can also be useful in mild-moderate VWD2A, VWD2M and VWD2N.
In VWD3, and in severe forms of VWD1 and VWD2, DDAVP is not effective, and plasma-derived
VWF/FVIII concentrates should be used in bleedings, surgery and secondary-long term prophylaxis.
Only the VWF/FVIII concentrates that have been evaluated in pharmacokinetic (PK) trials, as well as in
large retrospective or prospective efficacy studies, must be recommended to treat VWD patients.

Definition, epidemiology and classification Treatment and prevention of bleeding

von Willebrand disease (VWD) is the
most common inherited bleeding disorder
and is due to quantitative or qualitative
defects of the von Willebrand factor (VWF).
VWD is inherited by autosomal dominant
or recessive patterns, but women with mild
forms are more symptomatic.1 In popula-
tion-based studies, the prevalence of VWD
is very high (0.81%),2 but the clinical rele-
vance of many of these cases is uncertain. If
we consider only patients referred for clini-
cal manifestations of bleeding, the actual
prevalence is 66-100 cases per million of the
general population.
The most updated classification of VWD
has proposed six different types: VWD1,
VWD3, VWD2A, VWD2B, VWD2M,
VWD2N.3 In the past, VWD1 was reported
to be the most frequent form, accounting
for approximately 70% of cases. In a recent
study based on the reappraisal of VWD
diagnoses after 10 years (1998-2008),
VWD1 cases were only 671/1234 (55%).4
This is because many cases previously diag-
nosed as VWD1 were re-diagnosed as
VWD2 due to discrepant VWF measure-
ments [ratio of ristocetin cofactor activity
(VWF:RCo) to VWF:Ag <0.6)], as previous-
ly recommended.5 Age and VWD type dis-
tribution of the 1,234 patients enrolled in
the Italian registry on VWD are shown in
Figure 1 A-B. The presence of qualitative
defects of VWF in previously diagnosed
VWD1 has also been reported in 154 fami-
lies evaluated prospectively by a European
Study entitled Molecular and Clinical
Markers for Diagnosis and Management of
VWD1.6
The goal of treatment is to correct the dual
defects of hemostasis, that is, abnormal
platelet adhesion due to low or defective
VWF, and abnormal intrinsic coagulation
due to low FVIII.7 Two main therapeutic
approaches are available: (i) desmopressin
(DDAVP) that releases endogenous VWF
from endothelial cells; (ii) exogenous VWF
contained in VWF/FVIII concentrates. Table 1
summarizes the choice of these two
approaches according to VWD types.
Desmopressin: definitions of biological
response versus clinical efficacy
Desmopressin
Desmopressin (1-deamino-8-D-arginine
vasopressin, DDAVP) is a synthetic analogue
of vasopressin that it is relatively inexpensive
and carries no risk of transmitting blood-
borne infectious agents. DDAVP, infused
intravenously at a dose of 0.3 µg/kg diluted
in 50 mL saline over 30 minutes, usually
increases plasma VWF and FVIII three to five
times above baseline levels within 30 to 60
minutes and, in general, high VWF and FVIII
levels last for 6-8 hours.8 The drug is also
available in concentrated forms for subcuta-
neous and intranasal administration, which
can be convenient for home treatment.9 At
the time of diagnosis, a test dose of DDAVP
is recommended in VWD patients to estab-
lish the individual patterns of biological
response, and to predict clinical efficacy dur-
ing bleeding, since the responses in a given
patient are consistent on different occasions.5
Ideally, this infusion trial should be always
organized in non bleeding patients to avoid
any interference of VWF consumption due to

| 50 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

bleeding or surgery. For this infusion trial, DDAVP must
be administered at 0.3 µg/kg body weight, either by sub-
cutaneous or intravenous route (in this case the com-
pound is diluted in 50-100 mL of isotonic saline and
infused over 30 minutes). The time of sampling is sched-
uled by taking the time of subcutaneous injection, or the
beginning of intravenous infusion, as time 0. Plasma
samples should be obtained prior to infusion and at 60,
120 and 240 minutes after administration of DDAVP
from at least one affected member of each family, prefer-
ably the index case. In pediatric patients, excessive with-
drawal of blood should be avoided and the evaluation
should be limited at baseline and 240 minutes. The pro-
tocol of the DDAVP infusion test with laboratory param-
eters to assess the biological response has been described
previously10,11 and is summarized in Table 2.
In clinical practice, DDAVP infusions can be repeated
every 12-24 hours, depending on the type and severity
of the bleeding episode. The efficacy of DDAVP in
achieving hemostasis should be assessed using the same
approach as for VWF/FVIII concentrates, and according
to the following criteria: hemostasis clinically not differ-
ent from normal (excellent); mildly abnormal hemostasis,
partial or delayed control of spontaneous bleeding or
slight transient oozing from surgical wounds (good);
moderately abnormal hemostasis, that is, bleeding not
fully controlled but no need of additional therapy (mod-
erate); and no improvement at all with continuation of
bleeding and need of additional or alternative therapies
(poor).12 The list and description of the published
prospective or retrospective studies on biological
response and clinical efficacy of DDAVP in inherited
VWD have been recently reported.13 Despite the wide-
spread use of DDAVP in VWD, there are only a few
prospective clinical trials in large number of cases on
DDAVP efficacy and safety aimed at determining bene-
fits and limits of this therapeutic approach. For these
reasons, an investigator driven observational prospec-
tive study on clinical efficacy of DDAVP in 200 patients
with VWD1 and VWD2 has been organized recently:

Table 1. Treatment of different types of von Willebrand dis-

ease.

Treatment of choice Alternative Adjunctive therapy

VWD1 Desmopressin Antifibrinolytics, estrogens
VWD2A VWF/FVIII concentrates Desmopressin for mild bleeding
VWD2B VWF/FVIII concentrates
VWD2M Desmopressin VWF/FVIII concentrates
VWD2N* Desmopressin VWF/FVIII concentrates
VWD3 VWF/FVIII concentrates Desmopressin, platelet concentrates
VWD3 with Recombinant factor VIII Recombinant activated factor VII
alloantibodies
* Not all VWD2N patients can respond to Desmopressin

Table 2. Recommendations for the infusion test with DDAVP.

Infusion test with desmopressin

Infusion protocol Administer in 30 minutes 0.3 µg/kg of

Desmopressin in 50 mL of saline.
The same dosage can also be
administered subcutaneously
Clinical and laboratory parameters VWF and FVIII activities must be measured
before and 0.5, 1, 2 and 4 hours after
Figure 1. Age distribution of the 1,234 patients enrolled in the starting Desmopressin infusion;
Italian Registry on VWD. (A) Each decade reports the frequen- check platelet count before and at least 2
cy of VWD1, VWD2 and VWD3. (B) VWD type distribution of
the same population, as classified in 2008 according to the hours after infusion.
recommendations reported in references (2). Note that the Patients with VWD2B should be excluded
percentage of VWD1 is lower than that found ten years before
by the Italian Association of Hemophilia Centers. The reason Definition of responsiveness VWD patients should be considered
of this lower percentage is related to the demonstration of responsive to Desmopressin if after 2
abnormal ratio (<0.6) between VWF:RCo and VWF:Ag. All the hours they show increases of baseline
patients were characterized by decreased RIPA (risto- levels of VWF:RCo and FVIII by at least
cetin>1.6 mg/ml) and by only partial loss of high molecular
weight multimers in plasma (VWD2M); however, mutations three times, with levels of at least 30 U/dL 10
could be identified only in 60% of cases. For this reason we
cannot exclude in this group of patients some rare cases of An updated definition of biological response for VWD1 has been proposed:
VWD2A (subgroup IIC, IIE, IID, IIF) and we have identified these Complete responders: Both VWF:RCo and FVIII:C ≥50 IU/dL after DDAVP;
patients as VWD2M/2A. Partial responders: VWF:RCo or FVIII:C <50 IU/dL but increased at least 3-fold;
Non responders: None of the above.11

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 51 |
14th Congress of the European Hematology Association

the effectiveness and safety of DDAVP will be evaluat-
ed prospectively for 24 months during bleeding
episodes and minor or major surgeries in VWD patients
who were exposed to an infusion trial at enrollment.
Side effects of DDAVP are usually mild tachycardia,
headache and flushing. These are attributed to the vaso-
motor effects of the drug and can often be attenuated by
slowing the rate of infusion. Hyponatremia and volume
overload due to the anti-diuretic effects of DDAVP are
relatively rare and usually occur in young children.14
Though no thrombotic episodes have been reported in
VWD patients treated with DDAVP, this drug should be
used with caution in elderly patients with atherosclerot-
ic disease and hypertension, as reported in patients with
mild hemophilia A.15 In the past, DDAVP was used to
manage bleeding in VWD2B patients. Even though
there are no reported cases of thrombosis, DDAVP
should be avoided in VWD2B because of the transient
thrombocytopenia due to the enhanced interaction of
increased levels of VWF2B with platelet glycoprotein Ib
(GpIb). More importantly, we cannot exclude in princi-
ple that such enhanced VWF2B-GpIb interactions caus-
ing circulating platelet aggregates might predispose rel-
atively old patients with VWD2B to thrombosis.13
Adjuvant treatments
Antifibrinolytic amino acids (AAA) are synthetic drugs
that interfere with the lysis of newly formed clots by
saturating the binding sites on plasminogen, thereby
preventing its attachment to fibrin and making plas-
minogen unavailable within the forming clot. Epsilon
aminocaproic acid (50 mg/kg, four times a day) and
tranexamic acid (15-25 mg/kg, three times a day) are the
most frequently used antifibrinolytic amino acids. Both
can be administered orally, intravenously or topically,
and are useful alone or as adjuncts in the management
of oral cavity bleeding, epistaxis, gastrointestinal bleed-
ing and menorrhagia. They should be avoided in the
management of urinary tract bleeding. AAA are given as
adjuvants, because they help to reduce the total amount
of factors by stabilizing the already formed fibrin clot. A
typical example is dental surgey, in which these drugs
can also be used locally as mouthwashes. We recom-
mend the use of AAA together with DDAVP for the pre-
viously mentioned reasons. Even though DDAVP
induces a short-term increase of t-PA, there is no evi-
dence that this effect affects hemostasis in treated
patients.7
von Willebrand factor/FVIII concentrates
VWF/FVIII concentrates are indicated in VWD3 and in
VWD2B because DDAVP can induce transient thrombo-
cytopenia, and in all VWD1 or VWD2 patients who are
not responsive to DDAVP or who may have contraindi-
cations to its use (Table 1). Minimal requirements for
plasma-derived VWF/FVIII concentrates in VWD manage-
ment are: (i) they must contain biologically active VWF
that could correct the primary hemostasis defect and
stabilize the endogenous FVIII molecule (the latter
objective can be achieved independently of the content
in exogenous FVIII); (ii) they should be treated by viru-
cidal methods; (iii) before clinical use, they should be
tested for pharmacokinetics (PK) and efficacy in retro-
spective or prospective clinical trials in relatively large
numbers of VWD patients.7 Among the many
VWF/FVIII concentrates available in the market, only a
few meet these requirements (Table 3). VWF/FVIII con-
centrates can be given to stop bleeding episodes when
they occur (treatment on demand), to prevent bleeding
during surgery (prophylaxis for surgery), or to prevent
recurrent bleeding at specific sites (secondary long-term
prophylaxis). The PK and clinical efficacy results of the
first prospective study in VWD were published in
2002.16 This study included 53 patients receiving treat-
ment with a doubly virus-inactivated VWF/FVIII concen-
trate (Alphanate®) for 87 bleeding episodes and in 39
patients receiving treatment for 71 surgical or invasive
diagnostic procedures. A good clinical response with
this VWF/FVIII concentrate was observed in 86% of the
spontaneous bleeding episodes, and in 71% of surgical

Table 3. Plasma-derived concentrates containing von Willebrand factor.

A. Concentrates with published activity in von Willebrand disease subjects.

Concentrate Purification procedures Virucidal Rx VWF:RCo/Ag VWFRCo/FVIII Available in Manufacturer

Alphanate Heparin ligand CT SD; Dry heat 0.6 1.2 GE, IT, UK, US Grifols (US)
Biostate Precipitation/Heparin ligand CT SD:Dry heat 0.8 2.0 AU, ASIA CslBehring
Fanhdi Precipitation, heparin ligand CT SD; Dry heat 0.6 1.6 SP-IT Grifols (Sp)
Haemate-P† Polyelectrolyte precipitations Pasteurization 0.8 2.5 ASIA, EUR, US CslBehring
Wilate Affinity CT, size exclusion SD; Dry heat 0.7 0.8 GE Octapharma
Wilfactin Ion-exchange, affinity CTs SD; NF; Dry heat 0.7 60 FR LFB (Lille)
B. Concentrates with limited activity or no published studies in von Willebrand disease subjects.

Concentrate Purification procedures Virucidal Rx VWF:RCoAg* VWF:RCo/FVIII* Available in Manufacturer

Emoclot Ion-exchange CT SD; Dry heat 0.5 1.2 BR, IT Kedrion

Immunate Ion-exchange CT SD; Vapor heat 0.2 0.2 EUR Baxter
Koate DVI Precipitations, size exclusion SD; Dry heat 0.5 1.2 US Talecris
8Y Heparin/Glycine precipitations Dry heat 0.3 0.8 UK BioProducts
CT: chromatography; SD: solvent-detergent (t-N-butyl-PO4 with polysorbate, Tween, or otoxynol, Triton); nf: nanofiltration; na: not applicable (multiple countries).
*Ratio of ristocetin cofactor activity (VWF:RCo) to VWF:Ag or FVIII activity expressed as IU/mL (or % of a normal pool). †Humate P in US.

| 52 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

or invasive procedures.16 Two retrospective and one
prospective study have also been performed using
Fandhi®, a concentrate manufactured using a process
very similar to that of Alphanate®17,18 (A. Federici et al.,
unpublished data, 2009).
Haemate-P®/Humate-P®, an intermediate-purity
VWF/FVIII concentrate, has been widely used in VWD.
This product was introduced into clinical practice in
Europe (Haemate-P®) in 1984 and in the United States
(Humate-P®) in 1999. The first PK study of Haemate-P®,
published in 1998, was a single-centre evaluation
involving six VWD3 patients.20 Results of a large retro-
spective study organized by the Canadian Hemophilia
Centers were published in 2002.21 Other published stud-
ies include two retrospective analyses of Haemate
P®/Humate-P® efficacy and safety in Italian VWD
patients,12,22 as well as two prospective, multicenter,
open-label, non-randomised studies conducted in the
USA on Haemate-P®/Humate-P® used in urgent bleeding
and urgent surgical events.23,24 The results of another
prospective study in elective surgery with
HaemateP®/Humate-P® with dosing based on PK have
been recently published.25
The use of Wilate® in VWD management has been
reported in Germany since 200526 and the results of effi-
cacy and safety in acute bleeding episodes, in surgical
interventions and in secondary long-term prophylaxis
will be published within a few months. Data have been
also reported on the PK and clinical efficacy of Biostate®,
a VWF/FVIII concentrate available in Australia and
Asia.27,28 A peculiar plasma-derived VWF concentrate
with low FVIII levels was introduced in France in 1992
and the first PK study in VWD3 was published in 1996.26
An improved version of this concentrate (Wilfactin®),
which is almost devoid of FVIII, was evaluated in two
large French and European studies and data on PK have
been already published.30 Results in VWD3 show no
major differences in VWF:RCo and VWF:Ag for the con-
centrates that did or did not contain FVIII; the only dif-
ference was an approximate 6-hour delay in FVIII
increase with the concentrate devoid of FVIII.
Therefore, administration of exogenous FVIII is recom-
mended in VWD3 cases with acute life-threatening
bleeding episodes or emergency surgeries.30 Clinical effi-
cacy results of the French and European studies have
been recently reported.31
On the whole, there is no evidence from retrospective
or prospective clinical studies that the six VWF/FVIII
concentrates (Alphanate®, Biostate®, Fandhi®, Haemate-
P®/Humate-P®, Wilate®, Wilfactin®) reported in Table 3
differ with regards to efficacy, because no head-to-head
clinical study has been carried out. Therefore, all these
VWF/FVIII concentrates can be effective to manage or
prevent bleeding in patients with VWD.
Treatment of patients with alloantibodies to von Willebrand
factor
For the rare patients with VWD3 who develop anti-
VWF allo-antibodies after multiple transfusions, the use
of VWF/FVIII concentrates is ineffective, but may even
cause post-infusion anaph.32 These reactions may be life
threatening. To overcome this drawback, a patient
undergoing emergency abdominal surgery was treated
with recombinant FVIII, because this product contain-
ing no VWF, could not cause anaphylactic reactions. In
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 53 |
Berlin, Germany, June 4-7, 2009
view of the very short half-life of FVIII without its VWF
carrier, recombinant FVIII had to be administered by
continuous intravenous infusion at very large doses, to
keep FVIII levels above 50 U/dL for 10 days after sur-
gery.32 Another possible therapeutic approach is the
recombinant activated factor VII (rFVIIa) that can be
used in VWD with allo-antibodies according to the
same dosage and regimens as for hemophilia A patients
with inhibitors.33,34
Secondary long-term prophylaxis
Patients with severe forms of VWD may have fre-
quent hemarthroses, especially when FVIII levels are
below 10 U/dL, so that some of them develop target
joints similar to patients with moderate hemophilia A.
Some patients have recurrent gastrointestinal (GI)
bleeding, often without lesions in the GI tract and need
treatment every day or every other day. Finally, there
are children who frequently have epistaxis and are
severe enough to cause anemia. In these bleeders, the
optimal therapy may be regular prophylaxis with VWF
concentrates rather than on demand treatment during
bleeding episodes. The largest experience on secondary
prophylaxis in VWD has been collected in Sweden in 35
patients with severe forms of VWD.35 Secondary pro-
phylaxis was also implemented in a cohort of Italian
patients with VWD.36 These two retrospective studies
suggest that cost-effectiveness of these prophylaxis reg-
imens versus on demand therapy should be further eval-
uated in larger prospective studies.
Future perspectives
Overall, the treatments currently available for VWD
are quite satisfactory. For patients unresponsive to
DDAVP, VWF/FVIII concentrates are the only form of
available treatment. The fact that they are fractionated
from plasma is of concern for some, even if more than
one viral inactivation method is used for most concen-
trates in the manufacturing process. Haemate P/Humate
P is the only concentrate that uses only one viral inacti-
vation method (pasteurization), but the safety record of
this product is impeccable. This favorable situation
notwithstanding, there are advanced plans to develop a
therapeutic preparation of recombinant VWF. This
product containing only VWF will require the concomi-
tant administration of FVIII for the control of acute
bleeding episodes and for the prevention of excessive
bleeding at the time of major surgery. Attempts to cor-
rect VWD partially through gene replacement therapy
are in progress.37
The author would like to thank the work of Dr. Luigi
Flaminio Ghilardini, who prepared the figures reported in this
manuscript.
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concentrate (Humate-P): use of the ristocetin cofactor assay
(VWF:RCo) to measure potency and to guide therapy. Haemo-
philia 2003;9:688-95.
24. Thompson AR, Gill JC, Ewenstein BM, Mueller-Velten G,
Schwartz BA. Successful treatment for patients with von
Willebrand Disease undergoing urgent surgery using factor
VIII/von Willebrand Factor concentrate (Humate-P). Haemo-
philia 2004;10:42-51.
25. Lethagen S, Kyrle PA, Castaman G, Haertel S, Mannucci PM et
al. von Willebrand factor/factor VIII concentrate (Haemate P)
dosing based on pharmacokinetics: a prospective multicenter
trial in elective surgery. J Thromb Haemost 2007;5:1420-30.
26. Stadler M, Gruber G, Kannicht C, Biesert L, Radomski KU,
Suhartono H et al. Characterisation of a novel high-purity,
double virus inactivated von Willebrand factor and factor VIII
concentrate (Wilate®). Biologicals 2006;34:281-8.
27. Favaloro EJ, Llyod J, Rowell J, Baker R, Rickard K, Kershaw G
et al. Comparison of the pharmacokinetics of two von
Willebrand factor concentrates [Biostate and AHF (High
Purity)] in people with vpn Willebrand disorder. Thromb
Haemost 2007;97:922-30.
28. Shortt J, Dunkley S, Rickard K, Baker R, Street A et al. Efficacy
and safety of a high purity, double virus inactivated factor
VIII/von Willebrand factor concentrate (Biostate) in patients
with von Willebrand disorder requiring invasive or surgical
procedures. Haemophilia 2007;13:144-8.
29. Menache D, Aronson DL, Darr F, Montgomery RR, Gill JC,
Kessler CM et al. Pharmacokinetics of von Willebrand factor
and factor VIIIC in patients with severe von Willebrand dis-
ease (type 3 VWD): estimation of the rate of factor VIIIC syn-
thesis. Br J Haematol 1996;94:740-5.
30. Goudemand J, Scharrer I, Berntorp E, Lee CA, Borel-Derlon A,
Stieltjes N et al. Pharmacokinetic studies on Wilfactin, a von
Willebrand factor concentrate with a low factor VIII content
treated with three virus-inactivation/removal methods. J
Thromb Haemost 2005;3:2219-27.
31. Borel-Derlon A, Federici AB, Rouseel-Robert V, et al
Treatment of severe von Willebrand disease with a high-puri-
ty von Willebrand factor concentrate (Wilfactin): a prospective
study of 50 patients. J Thromb Haemost 2007;5:1115-24.
32. Bergamaschini L, Mannucci PM., Federici AB, Coppola R,
Guzzoni S, Agostoni A. Posttransfusion anaphylactic reaction
in a patient with severe von Willebrand disease: role of com-
plement and alloantibodies to von Willebrand factor. J Lab
Clin Med 1995;125:348-55.
33. Ciavarella N, Schiavoni M, Valenzano E, Mangini F, Inchingolo
F. Use of recombinant factor VIIa (Novoseven) in the treat-
ment of two patients with type III von Willebrand disease and
an inhibitor against von Willebrand factor. Haemostasis 1996;
26:150-4.
34. Boyer-Neumann C, Dreyfus M, Wolf M, Veyradier A, Meyer
D. Multi-therapeutic approach to manage delivery in an
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Thromb Haemost 2003;1:190-2.
35. Berntorp E, Petrini P. Long-term prophylaxis in von Willebrand
disease. Blood Coagul Fibrinolysis 2005;16:S23-S6.
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37. De Meyer SF, Vanhoorelbeke K, Chuah MK, Pareyn I, Gillijns
V, Hebbel RP et al. Phenotypic correction of von Willebrand
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4728-36.
 Chronic lymphocytic leukemia

Cellular origin of chronic lymphocytic leukemia

U. Klein A B S T R A C T

Institute for Cancer Genetics and
Herbert Irving Comprehensive
Cancer Center, Columbia University,
New York, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:55-60
Chronic lymphocytic leukemia (CLL) has long been recognized as a morphologically homogeneous
disease of mature B-lymphocytes. However, the unique phenotypic and genotypic features of this
tumor precluded a conclusive assignment of the CLL tumor precursor cell to a specific normal B-cell.
First, the immunophenotype of CLL cells characterized by the expression of the cell surface antigens
CD5, CD23, CD27 and low level of surface immunoglobulin (Ig) are different from that of any normal
B-cell.1,2 Second, CLL cases are heterogeneous at the genetic level, since the rearranged Ig variable
region (IgV) genes of CLL cases can be either somatically hypermutated or unmutated,3-6 suggesting
that the corresponding tumor precursor cells may originate from distinct B-cell developmental stages.
Over the past several years, results obtained from global gene expression profiling (GEP) analyses, as
well as insights into the expressed antibody repertoire of CLL from large scale IgV gene sequencing
studies, have led to the accepted view that all CLL cases, independent of their IgV gene hypermuta-
tion status, originate from the oncogenic transformation of a common cellular precursor, which
appears to be an antigen-experienced B-lymphocyte.

B-cell development and cellular origin of
B-cell malignancies
A major interest in the study of B-cell
malignancies is the identification of the nor-
mal cellular counterpart from which a partic-
ular tumor type originates. Knowing the
developmental stage a B-cell malignancy
corresponds to has obvious implications for
understanding its pathogenesis. It may
become possible to dissociate the compo-
nents of the malignancy that reflect the nor-
mal differentiation stage from which the
tumor originated from truly disease-associ-
ated genes, thus potentially providing
insights into the mechanism of transforma-
tion. Equally important, identified tumor-
specific gene products may be developed
into valuable new diagnostic markers or
therapeutic targets.
The investigation of the peripheral B-cell
differentiation at the phenotypic and molec-
ular levels led to the establishment of a B-cell
developmental “scheme” that was useful in
assigning the various lymphoma subtypes to
specific differentiation stages.7,8 The hall-
mark of antibody-mediated immunity is the
generation of memory and plasma cells – the
cell types that form the basis of the immuno-
logical memory against pathogens – in the
germinal center (GC) microenvironment of
the peripheral lymph nodes.9,10 The precur-
sors of the GC response are bone marrow-
derived, antigen-inexperienced (naïve) B-
cells, which are activated by foreign antigens
with the help of T-cells and other immune
cells. Those cells move to the GC, where
they begin to divide and proliferate. In the
course of the GC-reaction, the antibody
genes of the B-cells are modified by the
process of immunoglobulin variable region
(IgV) somatic hypermutation, which intro-
duces mainly point mutations into the
rearranged antibody gene with the aim of
generating high affinity antibodies. GC B-
cells with improved antigen binding are pos-
itively selected and differentiate into memo-
ry B-cells or plasma cells. GC B-cells also
undergo Ig class switching from the expres-
sion of IgM to IgG, A, or E by a somatic
DNA recombination mechanism. In the
human, memory B-cells can be distinguished
from naïve B-cells by the expression of the
cell surface antigen CD27. While in the
mouse, CD5+ B-cells comprise a functionally
well-defined B-cell subset (B1 cells) that pro-
duces natural antibodies, CD5+ B-cells in
human adults do not seem to exert the same
role.
The descendents of the GC response –
memory and plasma cells – have somatically
mutated IgV genes and frequently express
class switched isotypes. When the various
subtypes of mature B-cell lymphomas were
analyzed for their level of IgV gene somatic
hypermutation during the 1990s, it was
intriguing to find that almost all lymphoma
subtypes expressed hypermutated antibody
genes (Figure 1).8,11 Most notably, this catego-
ry included follicular lymphoma, Burkitt
lymphoma, and diffuse large B-cell lym-
phoma (DLBCL), and virtually every case
displayed IgV hypermutations. These find-
ings suggested that the precursor cells of
most lymphoma subtypes represent either a
GC B-cell that actively undergoes somatic
hypermutation, or a cell that has passed
through the GC-microenvironment. From
this finding, the concept developed that the
GC-reaction plays a critical role in the gene-
sis of B-cell lymphomas,8,11 and that an acci-

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 55 |
14th Congress of the European Hematology Association

dental “misfiring” of the somatic hypermutation and
class switch processes may cause potentially transform-
ing genomic mutations.12,13 Only few B-cell tumor sub-
types showed both somatically mutated, as well unmu-
tated IgV genes, namely mantle cell lymphoma, with
approximately 80% of the cases expressing unmutated
IgV genes,14,15 and chronic lymphocytic leukemia (CLL),
which expresses both somatically mutated and unmu-
tated IgV genes at about equal percentages.3
Evidently, in the light of peripheral B-cell develop-
ment (Figure 1), the fact that CLL can express IgV
mutated or unmutated IgV genes suggests that this
tumor subtype may have precursors of different devel-
opmental stages, namely the antigen-inexperienced
(naïve) and antigen-experienced (memory/marginal
zone) stages. Further, pointing towards a biological dif-
ference between the two genetically defined subtypes
was the observation that IgV mutated CLL cases gener-
ally show a more benign clinical course than unmutated
CLL.16,17 However, as will be discussed in the following
paragraphs, the evidence accumulating over the last sev-
eral years was inconsistent with the notion that the
major CLL subtypes originate from different cellular
precursors.
Chronic lymphocytic leukemia shows a homogeneous gene
expression profile, suggesting a common cellular precursor
GEP analyses revealed that all CLL displayed a com-
mon gene expression profile that is independent of the
level of IgV somatic hypermutation18-20 or of the expres-
sion of CD38,21 a cell surface antigen that is differential-
ly expressed on CLL cases. With more sensitive analysis
methods, one could extract subtle differences in the
genotypically defined subsets (as described in the fol-
lowing paragraph). These observations strongly suggest
that CLL represents a phenotypically homogeneous dis-
ease. From this, it follows that CLL is markedly differ-
ent from a disease entity, such as DLBCL,22 which is
characterized by an extensive phenotypic heterogeneity
and whose individual subtypes originate from develop-
mentally distinct B-cell subpopulations (e.g., GC-type
from GC B-cells and activated B-cell (ABC)-type DLBCL
from plasmablasts). Instead, the available evidence sug-
gest that all CLL originate from a common cellular pre-
cursor as a result of a common pathogenetic mecha-
nis.18,19
By applying more stringent criteria in the biostatisti-
cal (supervised) analysis of the GEP data, it is possible to
identify a small number of transcripts differentially
expressed between somatically mutated versus unmu-
tated CLLs.18,19 Recently, a group of differentially
expressed microRNAs was added to this list.23 This spe-
cific set of genes (molecular signature) allowed the clas-
sification of an independent panel of CLL cases into the
two subgroups, demonstrating the existence of a subtle
but consistent phenotypic difference. One of those
analyses revealed that the IgV unmutated CLL subgroup
seems to express high mRNA levels of genes that are

Figure 1. Germinal center reaction and lymphomagenesis. (A) B-cell development and Ig remodeling processes. Antigen-
inexperienced naïve B-cells are generated throughout life in the bone marrow where they rearrange their antigen recep-
tor genes by somatic DNA recombination of the variable (V), diversity (D), and joining (J) gene segments. Upon antigen
activation, and with the help of T-cells and other immune cells, naïve B-cells are driven into the germinal center (GC)
response, where they modify their rearranged IgV genes at the centroblast stage by somatic hypermutation, and at the
centrocyte stage by Ig class switch recombination. Apotosis eliminates GC B-cells that have acquired unfavorable somat-
ic hypermutations; antibody mutants with a higher affinity against the antigen are selected into the post-GC repertoire
and differentiate into memory B-cells or, via a plasmablast stage, into plasma cells. In an antigen recall response, mem-
ory B-cells quickly differentiate into plasma cells. (B) B-cell malignancies and the level of somatic hyper-mutation in their
rearranged IgV genes. Virtually all cases of diffuse large B-cell, Burkitt, follicular, Hodgkin and AIDS-associated lym-
phomas and multiple myeloma carry somatically mutated IgV genes, indicating that the corresponding tumor precursor
cells represent GC B-cells or have passed through the GC. However, mantle cell lymphoma is mainly characterized by the
expression of unmutated IgV genes, and CLL express mutated and unmutated IgV genes at almost equal percentages.
Acute lymphoblastic leukemia is a tumor of developing B-cells and expresses unmutated IgV genes.

| 56 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

known to be activated as a result of in vitro B-cell recep-
tor (BCR)-mediated stimulation;19 therefore, suggesting
that CLL belonging to this subgroup are subjected to
signaling through the BCR in vivo. However, the situa-
tion is more complex. While the GEP pattern that sepa-
rates somatically mutated and unmutated CLL allows
the classification of CLL cases into either subgroup at a
high confidence level, it is not absolute. In agreement
with this notion, CLL cases with a VH3-21 gene
rearrangement show a low overall poor survival irre-
spective of the level of IgV hypermutation,24 and also
display a GEP, which is distinct from that of VH3-21-
negative cases.25 These observations suggest a crucial
role for a common antigen in the pathogenesis of VH3-
21-expressing CLL cases. In conclusion, the small but
measurable expression differences between IgV mutat-
ed and unmutated, or VH3-21-expressing and non-
expressing CLL cases, seem to reflect the physiological
consequences of external stimuli, such as the interaction
of BCR with specific antigen rather than a particular
developmental stage of the tumor precursor cell.
Somatically mutated and unmutated chronic lymphocytic
leukemia express stereotype antigen-receptors with
evidence of antigen-experience
The realization that a subgroup of CLL carry somati-
cally mutated antibody genes was a first indication that
a sizable fraction of CLL may have undergone the GC-
reaction and express selected BCRs;4 thus, hinting at the
possibility that antigenic selection may play a role in the
development of CLL. Analyses of larger panels of cases
then found that in both somatically mutated and unmu-
tated cases, certain IgV gene family members are
expressed much more frequently in CLL than would be
expected from their expression in the antibody reper-
toire of normal B-cells6 [for review, see (3)]. This obser-
vation suggested that CLL express restricted sets of anti-
gen receptors irrespective of the level of IgV gene hyper-
mutation, and led to the conclusion that many CLL
seem to be derived from previously stimulated B-cells.6
Further studies that compared the IgV gene repertoires
expressed by CLL with those of elderly normal adults
provided conclusive evidence that the CLL-characteris-
tic repertoire indeed seems to be the result of antigen-
selection and does not reflect the aging process.26,27
Intriguingly, the general picture emerged that CLL cases
from unrelated patients can have almost identical
BCRs.24,28-32 This BCR ‘stereotypy’ occurs at the levels of
IgV gene usage, V-D-J junctional regions (heavy chain
CDR3s), and combination of particular heavy chain
CDR3s with light chain CDR3s [summarized in (33)].
Thus, CLL represents a tumor characterized by the
expression of stereotyped BCRs. This skewed BCR
repertoire seems to be a result of an antigen-mediated
selection. In support of this notion, it recently became
possible to identify specific antigens that are recognized
by BCRs expressed by CLL; these antigens represent
autoantigens derived from “body” cells undergoing
apoptosis, and some of the recognized epitopes show
similarities to microbial antigens.34,35
As eluded in the previous section, results from GEP
analysis suggest the existence of active BCR-stimulation
in the IgV gene unmutated CLL subset. Thus, the phys-
iological consequences of the binding of a particular
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 57 |
Berlin, Germany, June 4-7, 2009
BCR with its specific antigen may have an impact on
the clinical prognosis of CLL. A recently proposed
model attempts to integrate the findings that all CLL
seem to be subjected to BCR stimulation on the one
hand, and that CLL subtypes have different clinical
prognoses on the other. It suggests that some CLL may
be continuously stimulated by antigen which renders
them “activated” (likely representing bad-prognosis
CLLs), whereas others may have acquired an anergic,
“quite” state.36 Taken together, the results of the large-
scale IgV gene repertoire analyses provide strong evi-
dence that antigen plays a critical role in the develop-
ment of CLL.
The gene expression profiling of chronic lymphocytic
leukemia is related to that of antigen-experienced B-cells
The immunophenotype of the CLL tumor cells, CD5+
CD23+ CD27+ and sIglow, does not resemble that of any
normal B-cell.1,2 In addition, CLL tumor cells have a
characteristic cell surface phenotype resembling that of
(antigen)-activated B-cells.37 Thus, although it was rec-
ognized early on that CLL is a tumor of mature B-cells,
the particular immunophenotype of the tumor cells pro-
vided no direct clues about the normal cellular counter-
part of this disease. Global GEP, which simultaneously
measures the expression of thousands of marker genes
of a cell population, turned out to be extremely useful in
dissecting the cellular origin of B-cell malignancies, and
indeed provided new information on the cellular origin
of CLL. As discussed in a previous section, the results of
the GEP analyses suggest that all CLL originate from a
common cellular precursor. A more specific GEP-based
approach using supervised analysis methods for a com-
parison of GEP data from CLL with those of the various
normal B-cell subsets provides further insights into the
identity of the putative normal counterpart of CLL. The
results suggest that all CLL are mostly related in their
GEP to that derived from a specific B-cell subset, the
CD27+ B-cells,18 which comprises a heterogeneous sub-
set of antigen-experienced B-cells. This includes classi-
cal memory B-cells, as well as marginal zone B-cells (see
below). Noticeably, the CLL-profile did not show any
similarity to those derived from CD5+ cord blood,
CD27– (naïve), or GC B-cells.18,19
CD27+ B-cells comprise up to 40% of B-cells in the
peripheral blood (PB) of adults, and are found in the lym-
phoid tissues, predominantly in the marginal zone and
the tonsillar subepithelium, which both represent major
sites of antigen-entry. The majority of CD27+ B-cells (80-
90%) expresses somatically mutated IgV genes, while
CD27– B-cells are almost exclusively unmutated.38,39 In
vitro, CD27+ cells, in contrast to CD27– cells, and irrespec-
tive of their Ig isotype, respond quickly by differentiat-
ing into antibody-secreting cells.40-42 CD27+ B-cells can be
class switched, IgM-only, or IgM+IgD+.38,43 IgM-express-
ing CD27+ cells respond to T-independent antigens,42,44
which is consistent with the notion that the marginal
zone comprises B-cells that are rapidly activated to
secret Ig against invading microorganisms.45 Thus, a
common feature of CD27+ memory/marginal zone B-
cells is that they have been specifically generated to react
against exogenous antigens. Together with the observa-
tion that the BCRs of CLL show evidence of antigenic
selection, the unique functional characteristics of CD27+
14th Congress of the European Hematology Association
B-cells provide additional evidence for a phenotypic rela-
tion of the CLL precursor cell to this cell type.
Relation of chronic lymphocytic leukemia to other B-cell
lymphomas
The notion that CLL is a tumor of antigen-experi-
enced B-cells related to memory/marginal zone B-cells
suggests that the multistep process of leukemogenesis
begins in these cells.18 Additional evidence for this
hypothesis stems from the particular pattern of cytoge-
netic abnormalities in CLL, which differs markedly
from that found in most types of mature B-cell malig-
nancies. The major subtypes of B-cell lymphoma, that
is, Burkitt lymphoma, follicular lymphoma, and DLBCL
characteristically show recurrent balanced chromoso-
mal translocations, typically involving the translocation
of a proto-oncogene into the Ig loci (e.g., BCL2, c-MYC,
BCL6).12 These translocations are thought to be a direct
result of errors during the processes of VDJ recombina-
tion, class switch recombination, or somatic hypermu-
tation.11,12 However, CLL does not typically show recur-
rent chromosomal translocations, but is instead associ-
ated with chromosomal deletions and amplifications,
namely trisomy of chromosome 12 (16%), and dele-
tions of chromosomal regions 17p (p53; 7%), 11q
(ATM; 18%), and 13q14 (55%).46 This is similar to the
pattern of genomic aberrations found in solid tumors
(although the occurrence of non-recurrent, imbalanced
chromosomal translocations in a subgroup of CLL has
been reported.47 The general lack of reciprocal balanced
translocations involving the Ig loci in CLL indicates that
the causative Ig loci-remodeling mechanisms are not
active in the tumor precursor cell. Instead, genomic
aberrations eventually leading to clonal expansion of a
CLL precursor cell may occur in the developmental
stage following the completion of these processes, pre-
sumably during the antigen-driven proliferation of the
corresponding B-cell. Hierarchical cluster analyses of
GEP data representing the various types of B-cell lym-
phomas revealed that CLL clusters separately from the
lymphomas of putative GC origin.18,19,22 These biostatis-
tical analyses somewhat unexpectedly revealed that
CLL cluster along with hairy cell leukemia (HCL).48
| 58 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
While the morphological and phenotypic characteristics
of HCL are clearly distinct from that of any other B-cell
malignancy, including CLL,49 HCL resembles CLL in that
its GEP is most closely related to that of CD27+ B-cells.48
Moreover, both tumor entities characteristically lack
chromosomal translocations.50,51 These findings suggest
that the tumor precursor cells of both CLL and HCL
may be related to CD27+ B-cells. Thus, CLL and HCL
may belong to a distinct group of B-cell malignancies
originating from the oncogenic transformation of anti-
gen-experienced B-cells.
A model for the cellular derivation of chronic lymphocytic
leukemia
This review discusses the evidence suggesting that
CLL is a tumor of a functionally characterized subgroup
of (CD5–) B-lymphocytes, namely the antigen-experi-
enced B-cells, which in the human comprise a sizable
fraction in the lymphoid organs and the PB (~40% in
the PB). As suggested by the restricted BCR repertoire
expressed by CLL cases, among this huge reservoir of
antibody specificities only few cells would represent
actual targets for oncogenic transformation. Since virtu-
ally all CD27+ cells are CD5–, in this scenario the CLL-
associated expression of CD5 would be a consequence
of the activation-phenotype of the tumor cell, as has
previously been suggested.52 Alternative scenarios are
based on the assumption that the putative CLL precur-
sor cell corresponds to a minor, yet uncharacterized, B-
cell subset. Thus, a unique CD5+ B-cell population
occurring at low frequency in the lymphoid organs has
been identified that shows some features of the CLL
tumor cell and has hence been invoked as a putative
normal counterpart of CLL.53 Also, the causal relation-
ship between CLL and monoclonal B-cell lymphocyto-
sis, which represents a benign clonal proliferation of
CD5+ B-cells in the PB of adults with phenotypic resem-
blance to the CLL tumor cells, as well as a CLL-associ-
ated pattern of genetic aberrations, remains to be deter-
mined.54-56 Figure 2 depicts a model for CLL pathogene-
sis that reflects the possible cellular origin of the tumor
cell precursor based on its phenotypic relationship to
CD27+ B-cells. A naïve B-cell is driven by antigen stim-
Figure 2. A model for the cellular der-
ivation of CLL. Naïve B-cells (antigen-
inexperienced) may be driven either
into a T cell-dependent or T-inde-
pendent immune response (top two
branches). A separate pathway (bot-
tom branch) has been suggested in
which somatically mutated IgM-
expressing B-cells are generated in
an antigen-independent fashion.
Upon completion of either the GC-
response or the T-independent
response (a low level of hypermuta-
tion in T-independent responses has
been observed in an animal model)61
the cells now represent antigen-
experienced B-cells. These B-cells
may be continuously activated by
chronic antigen stimulation and
acquire genetic alterations that may
eventually cause oncogenic transfor-
mation and development of CLL.

ulation either into a GC-response where it undergoes
IgV hypermutation, or into a T-independent response
without undergoing hypermutation. Both developmen-
tal pathways are generally thought to give rise to mem-
ory/marginal zone B-cells.9,57 While IgM-expressing,
somatically mutated B-cells are considered to represent
memory/marginal zone B-cells that have undergone the
GC without isotype switching,38,39 a recently proposed
alternative scenario invokes generation of these cells in
an antigen-independent fashion by an extrafollicular
developmental pathway58,59 (bottom part of Figure 2).
Regardless of the generation of those IgM+ cells, it is
thought that chronic antigen stimulation may keep the
CLL precursor cell in a constant state of activation, and
over time, mutations may accumulate in the genome of
those cells that eventually lead to oncogenic transforma-
tion. Taken together, it emerges that memory/marginal
zone B-cells display features consistent with a putative
CLL precursor cell. All CLL express CD27 on the cell
surface,60 and the pattern of Ig isotype distribution
among CLL cases (class switched, IgM-only, IgM+IgD+)
resembles that of CD27+ cells. Moreover, the IgV genes
of both mutated and unmutated CLL display evidence
of antigen-experience. Thus, the available evidence is
compatible with the notion that CLL develops through
the oncogenic transformation of antigen-experienced B-
cells.
The author would like thank Riccardo Dalla-Favera,
Andrea Califano, Gustavo Stolovitzky, Yuhai Tu, and Katia
Basso for their essential involvement in some of the GEP exper-
iments described here.
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21. Dürig J, Nückel H, Hüttmann A, Kruse E, Hölter T, Halfmeyer
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22. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosen-
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23. Fulci V, Chiaretti S, Goldoni M, Azzalin G, Carucci N,
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24. Tobin G, Thunberg U, Johnson A, Eriksson I, Soderberg O,
Karlsson K, et al. Chronic lymphocytic leukemias utilizing the
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and homologous CDR3s: implicating recognition of a com-
mon antigen epitope. Blood 2003;101:4952-7.
25. Falt S, Merup M, Tobin G, Thunberg U, Gahrton G, Rosen-
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26. Potter KN, Orchard J, Critchley E, Mockridge CI, Jose A,
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27. Widhopf GF, 2nd, Kipps TJ. Normal B cells express 51p1-
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28. Widhopf GF, 2nd, Rassenti LZ, Toy TL, Gribben JG, Wierda
WG, Kipps TJ. Chronic lymphocytic leukemia B cells of more
than 1% of patients express virtually identical immunoglobu-
lins. Blood 2004;104:2499-504.
29. Messmer BT, Albesiano E, Efremov DG, Ghiotto F, Allen SL,
Kolitz J, et al. Multiple distinct sets of stereotyped antigen
receptors indicate a role for antigen in promoting chronic lym-
phocytic leukemia. J Exp Med 2004;200:519-25.
30. Tobin G, Thunberg U, Karlsson K, Murray F, Laurell A,
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31. Stamatopoulos K, Belessi C, Moreno C, Boudjograh M, Guida
G, Smilevska T, et al. Over 20% of patients with chronic lym-
phocytic leukemia carry stereotyped receptors: Pathogenetic
implications and clinical correlations. Blood 2007;109:259-70.
32. Murray F, Darzentas N, Hadzidimitriou A, Tobin G, Boudjogra
M, Scielzo C, et al. Stereotyped patterns of somatic hypermu-
tation in subsets of patients with chronic lymphocytic
leukemia: implications for the role of antigen selection in
leukemogenesis. Blood 2008;111:1524-33.
33. Ghia P, Chiorazzi N, Stamatopoulos K. Microenvironmental
influences in chronic lymphocytic leukaemia: the role of anti-
gen stimulation. J Intern Med 2008;264:549-62.
34. Chu CC, Catera R, Hatzi K, Yan XJ, Zhang L, Wang XB, et al.
14th Congress of the European Hematology Association
Chronic lymphocytic leukemia antibodies with a common
stereotypic rearrangement recognize nonmuscle myosin
heavy chain IIA. Blood 2008;112:5122-9.
35. Catera R, Silverman GJ, Hatzi K, Seiler T, Didier S, Zhang L, et
al. Chronic lymphocytic leukemia cells recognize conserved
epitopes associated with apoptosis and oxidation. Mol Med
2008;14:665-74.
36. Muzio M, Apollonio B, Scielzo C, Frenquelli M, Vandoni I,
Boussiotis V, et al. Constitutive activation of distinct BCR-sig-
naling pathways in a subset of CLL patients: a molecular sig-
nature of anergy. Blood 2008;112:188-95.
37. Damle RN, Ghiotto F, Valetto A, Albesiano E, Fais F, Yan XJ, et
al. B-cell chronic lymphocytic leukemia cells express a surface
membrane phenotype of activated, antigen-experienced B
lymphocytes. Blood 2002;99:4087-93.
38. Klein U, Rajewsky K, Küppers R. Human immunoglobulin
(Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell
surface antigen carry somatically mutated variable region
genes: CD27 as a general marker for somatically mutated
(memory) B cells. J Exp Med 1998;188:1679-89.
39. Tangye SG, Liu YJ, Aversa G, Phillips JH, de Vries JE.
Identification of functional human splenic memory B cells by
expression of CD148 and CD27. J Exp Med 1998;188:1691-
703.
40. Agematsu K, Nagumo H, Yang FC, Nakazawa T, Fukushima
K, Ito S, et al. B cell subpopulations separated by CD27 and
crucial collaboration of CD27+ B cells and helper T cells in
immunoglobulin production. Eur J Immunol 1997;27:2073-9.
41. Kindler V, Zubler RH. Memory, but not naive, peripheral
blood B lymphocytes differentiate into Ig-secreting cells after
CD40 ligation and costimulation with IL-4 and the differenti-
ation factors IL-2, IL-10, and IL-3. J Immunol 1997;159:2085-
90.
42. Werner-Favre C, Bovia F, Schneider P, Holler N, Barnet M,
Kindler V, et al. IgG subclass switch capacity is low in
switched and in IgM-only, but high in IgD+IgM+, post-germi-
nal center (CD27+) human B cells. Eur J Immunol 2001;31:
243-9.
43. Agematsu K, Hokibara S, Nagumo H, Komiyama A. CD27: a
memory B-cell marker. Immunol Today 2000;21:204-6.
44. Dono M, Zupo S, Massara R, Ferrini S, Melagrana A,
Chiorazzi N, et al. In vitro stimulation of human tonsillar
subepithelial B cells: requirement for interaction with activat-
ed T cells. Eur J Immunol 2001;31:752-6.
45. Martin F, Kearney JF. Marginal-zone B cells. Nat Rev Immunol
2002;2:323-35.
46. Döhner H, Stilgenbauer S, Döhner K, Bentz M, Lichter P.
Chromosome aberrations in B-cell chronic lymphocytic
leukemia: reassessment based on molecular cytogenetic analy-
sis. J Mol Med 1999;77:266-81.
47. Mayr C, Speicher MR, Kofler DM, Buhmann R, Strehl J, Busch
R, et al. Chromosomal translocations are associated with poor
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48. Basso K, Liso A, Tiacci E, Benedetti R, Pulsoni A, Foa R, et al.
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notype related to memory B cells with altered expression of
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49. Harris NL, Jaffe ES, Stein H, Banks PM, Chan JK, Cleary ML,
et al. A revised European-American classification of lymphoid
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50. Haglund U, Juliusson G, Stellan B, Gahrton G. Hairy cell
leukemia is characterized by clonal chromosome abnormali-
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E, Makropoulos V, Pantelias GE, et al. Clonal chromosome
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and review of literature. Cancer Genet Cytogenet 2001;
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activation by crosslinking of surface IgM or ligation of CD40
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lial B cells found in human tonsils. Eur J Immunol 2007;37:
2138-47.
54. Rawstron AC, Bennett FL, O'Connor SJ, Kwok M, Fenton JA,
Plummer M, et al. Monoclonal B-cell lymphocytosis and
chronic lymphocytic leukemia. N Engl J Med 2008;359:575-83.
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R, et al. The immunoglobulin gene repertoire of low-count
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immune responses generate memory B cells. J Exp Med 2006;
203:305-10.
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ME, et al. Human blood IgM "memory" B cells are circulating
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O, Peter HH, et al. Human immunoglobulin M memory B cells
controlling Streptococcus pneumoniae infections are generat-
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 Chronic lymphocytic leukemia

Clinical implications of novel biological markers

in chronic lymphocytic leukemia

R. Rosenquist A B S T R A C T

Department of Genetics and
Pathology, Uppsala University,
Uppsala, Sweden
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:61-67
Considering that chronic lymphocytic leukemia (CLL) is an inherently heterogeneous disease with
varying clinical outcome, there has been great interest throughout the last 10 years in establishing
reliable prognostic markers to predict its outcome. The immunoglobulin heavy-chain variable (IGHV)
gene mutation status and FISH analysis of recurrent genomic aberrations represents strong and robust
genetic markers that currently can be applied in CLL prognostication. A plethora of other biological
markers has been proposed at the protein (CD38, ZAP-70), DNA (TP53 mutations, telomere length) and
RNA (CLLU1, LPL) level, where many of these are promising for outcome prediction. To be applied in
the clinical setting, however, it is very important to not only select biomarkers that have been repeat-
edly confirmed in independent cohorts, preferably in prospective trials, but also ones that can be ana-
lyzed by standardized assays. In this overview, the pros and cons of a number of biological, prognos-
tic markers will be discussed. Many of these markers will require further evaluation, particularly in
relation to new treatment protocols. Ultimately, we may be able to create a ‘molecular prognostic
index’ in CLL comprised of the most informative and robust markers. By studying the mechanisms
behind these various markers, further insights into CLL pathogenesis will also be unraveled.

major breakthrough in chronic lym- unmutated B-cells, yet it was not until the

A phocytic leukemia (CLL) prognostics

was the observation that the somatic
hypermutation status of the immunoglobu-
lin heavy-chain variable (IGHV) genes could
group CLL into two main categories;1,2 IGHV
mutated CLL with an expected long overall
survival and IGHV unmutated CLL, having
an inferior outcome. Moreover, the presence
of certain genomic aberrations was also
demonstrated to have great impact on CLL
prognosis,3,4 where poor-risk markers such
as deletion of 11q and 17p are strongly asso-
ciated with short survival. Following these
discoveries, there has been a tremendous
interest in prognostic indicators in CLL,
which is reflected by the numerous biologi-
cal markers suggested throughout the last
decade. These different markers could be
proteins, both surface-bound and intracellu-
lar (CD38, ZAP-70, CD49d), RNA-based
(LPL, CLLU1, micro-RNA) or DNA-based
[TP53 mutations, single nucleotide polymor-
phisms (SNPs), telomere length].1,5-10 It is
clear that we need prognostic markers in
CLL to subdivide this heterogeneous dis-
ease; however, the essential question is
which markers/combination of markers
should be applied in clinical trials and in clin-
ical routine. In this overview, the clinical and
biological role of many of these new mark-
ers will be discussed, with a focus on biolog-
ical markers that have repeatedly been
shown to be good predictors of prognosis in
CLL.
Immunoglobulin gene analysis
For many years, it was assumed that CLL
cells would originate from CD5+, naïve,
mid-nineties that Schroeder and Dighiero
reported that CLL cells may display a more
diverse somatic hypermutation status than
anticipated.11 In 1998, Fais et al. demonstrat-
ed that roughly 50% of their CLL patients
had mutated IGHV genes,12 and this was-
closely followed by two simultaneous publi-
cations verifying the existence of two sub-
sets of CLL, i.e., IGHV mutated and unmu-
tated,1,2 both similar in size. Importantly,
these latter reports illustrated that CLL cells
carrying unmutated IGHV genes (>98%
identity to the corresponding germline
IGHV gene) had a significantly shorter over-
all survival compared to those cases show-
ing somatically hypermutated IGHV genes
(<98% identity). Furthermore, in line with
the poor survival, IGHV unmutated CLL dis-
played a more progressive disease, showed
poor-risk genomic aberrations more fre-
quently and required more chemotherapy
than mutated CLL.1,2 Many groups have sub-
sequently verified the strong predictive
capacity of the IGHV mutational status in
multivariate analysis, both in more “aggres-
sive” and in more “benign” cohorts (Figure
1).13-15
The cutoff for dividing mutated and
unmutated CLL cases was initially set at
98% identity in order to avoid counting
polymorphisms as true mutations.
However, since most polymorphisms are
currently known, this issue is not as relevant
today. Although the 98% cutoff has repeat-
edly been shown to be the best statistical
level for clinical discrimination of unmutat-
ed and mutated CLL and is widely accepted,
this is probably not as straightforward at a

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 61 |
14th Congress of the European Hematology Association

biological level. Firstly, the IGHV mutational status is

not absolute at the individual level. For instance, CLL
patients with borderline identity (97-98%) may have a
heterogeneous disease course, where some patients will
experience a more aggressive clinical course despite
being classified as “mutated”.15,16 Furthermore, excep-
tions to the mutated/unmutated classification have
been shown, for instance IGHV3-21 gene usage (see
below).17
The IG gene analysis is very stable and this test can be
performed at any time point during the course of the
disease since the IGHV mutational status remains con-
stant. That notwithstanding, there are many technical
aspects to consider when performing IG gene analysis
in CLL. In order to standardize this procedure, eight
European laboratories recently published recommenda-
tions for successful analysis18 and several educational
workshops focused on this topic. Despite the time-con-
suming nature of IGHV mutation analysis, it has now
become standard in many laboratories worldwide and
currently represents one of the most reliable prognostic
markers in CLL.

Immunoglobulin heavy-chain variable gene usage and

B-cell receptor “stereotypy”
In 2002, our group reported an exception to the
mutated and unmutated subcategorization. Examina-
tion of our cohort revealed a novel subset of CLL cases
utilizing the IGHV3-21 gene at a frequency of approxi-
mately 10%.17 The majority of these IGHV3-21 cases
carried mutated IGHV genes but still followed a disease
course similar to unmutated CLL cases. We verified this
finding in a follow-up study, where two-thirds of cases
had mutated IGHV genes and showed poor outcome.19
Interestingly, this subset had peculiar characteristics,
such as predominant λ expression and restricted IG
gene features; an extremely short and almost identical
complementarity determining region 3 (CDR3) together
with usage of one lambda light-chain gene, IGLV3-21, in
greater than 50% of patients.19 The finding of a “stereo-
typed” B-cell receptor in non-related IGHV3-21 CLL
patients has been confirmed worldwide,20-22 and indi-
cates that antigens may be involved in CLL develop-
ment.
Thereafter, we and others have identified several
additional subsets with stereotyped heavy-chain
CDR3s in combination with restricted light-chain gene
usage, such as the IGHV1-69/IGKV3-20 and the IGHV4-
39/IGKV2-30 subset,23-26 further supporting the concept
of antigen-driven leukemogenesis A major advance-
ment in this area was made in 2007, when Stama-
topoulos and colleagues investigated a large Mediter-
ranean CLL cohort and demonstrated that at least 48
different subsets existed, with more than 20% of their
CLL cases belonging to one of these ‘stereotyped’ sub-
sets.27 Interestingly, they showed that stereotypy, inde-
pendent of mutation status, may influence clinical out-
come in CLL, a finding which has lately been verified by
others.28 For instance, stereotyped IGHV3-21 cases (des-
ignated subset 2) had an inferior prognosis than non-
stereotyped IGHV3-21 cases, with shorter time to pro-
gression, as well as the presence of poor-risk mark-
ers.21,27-28 Conversely, stereotyped IGHV4-34 cases (sub-
set 4) had a superior prognosis when compared to non-
Figure 1. (A) Time to treatment and (B) overall survival
data in a subset of cases (n=202) from a population-based
Scandinavian CLL cohort. The high proportion of IGHV
mutated (63%) versus unmutated (37%) cases reflects its
unselected nature.
stereotyped cases.27 Stereotyped IGHV4-34 cases also
displayed unique characteristics, such as a low median
age at diagnosis and IgG switched receptors.27
Moreover, a subgroup expressing various IGHV1/5/7
genes but with similar CDR3 sequences (subset 1, the
“mixed” subset) had a very poor survival compared to
non-stereotyped cases.27 This apparent relationship
between stereotypy and clinical outcome may therefore
be useful and applicable in the foreseeable future in clin-
ical reporting, in conjunction with the IGHV mutation-
al status. We now have to continue performing large
multi-center studies to fully elucidate the clinical rele-
vance of various stereotyped subsets, which today com-
prises of more than 100 subsets.29
The search for surrogate markers
CD38
Since the discovery of the clinical impact of the IGHV
mutational status in CLL, many attempts have been
made to find surrogate markers to substitute the IG
gene analysis, preferably markers suitable for flow cyto-
metric analysis. In 1999, Damle and colleagues showed

| 62 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

that expression of CD38 correlated well with the IGHV
mutation status and could be suitable as a surrogate
marker.1 Using a 30% cutoff, they demonstrated that all
CD38+ cases had unmutated IGHV genes, whereas most
of the CD38– cases carried mutated IGHV genes. Several
reports have reproduced this initial finding;30,31 however,
we and others have shown a less stringent correlation
between the markers in up to one-third of patients.13,32,33
Furthermore, despite being easily measured by flow
cytometry, a consensus cutoff level has never been
reached, and various cutoffs have been described (rang-
ing from any positivity to 30%)1,13,31,34 In addition, CD38
levels have been reported to vary over time in up to a
quarter of CLL cases,35 although this has not been
demonstrated by others.34 In a recent combined analysis
of CD38, ZAP-70 and IGHV mutation status, CD38 did
not improve the ability to predict time to treatment
compared to the other two markers,36 which is in line
with our own observation.15
Lipoprotein lipase
Gene expression profiling also revealed that several
other genes were differently expressed in mutated and
unmutated CLL.9 One such gene was lipoprotein lipase
(LPL) which was highly expressed in unmutated CLL.
Using real-time PCR, several independent groups have
confirmed this finding and shown a strong correlation
with poor outcome in CLL.8,43,44 We recently investigat-
ed the LPL levels in 140 patients, both at the RNA and
protein level, and confirmed high levels in unmutated
CLL (Figure 2),45 where multivariate analysis revealed
LPL as a strong independent marker for survival predic-
tion.

ZAP-70
In 2001, Rosenwald and colleagues performed large-
scale gene expression profiling of unmutated and mutat-
ed CLL and surprisingly found rather few differently
expressed genes between the groups.9 One very inter-
esting finding was the upregulation of Zeta-Associated
Protein 70 (ZAP-70) in unmutated CLL. ZAP-70 was,
until then, well-studied in T-cells but had not been ana-
lyzed in B-cells. Several follow-up papers verified the
upregulation of ZAP-70 in unmutated CLL, and ZAP-70
analysis using flow-cytometry was proposed as a surro-
gate assay for IG gene analysis.37-39 For instance, Crespo
et al. showed that all cases with unmutated IGHV genes
were ZAP-70+, whereas most IGHV mutated cases were
ZAP-70–.37 In contrast, Rassenti et al. reported that up to Figure 2. Prognostic impact of low versus high LPL expres-
a quarter of cases had discordant mutation status and sion in 136 Swedish CLL patients (based on the threshold
value 0.0001 as defined by ROC curve analysis).45
ZAP-70 expression.40 These inconsistencies could in part
be attributed to the observation that patients with 11q
or 17p deletion could be ZAP-70– despite being IGHV
unmutated, whereas mutated IGHV3-21 CLL patients
could show high ZAP-70 expression.41
Although ZAP-70 may not be an ideal surrogate mark- Table 1. Established and potential biological, prognostic
er for the IGHV mutation status at the cohort or individ- markers in chronic lymphocytic leukemia.
ual level, it has repeatedly been shown to be an inde- Immunoglobulin gene analysis IGHV gene mutation status
pendent prognostic marker for time to progression and IGHV gene usage
overall survival in CLL.37,38,40 Some studies have also B-cell receptor ‘stereotypy’
shown that ZAP-70 may even be the superior marker for
estimating prognosis in CLL.36,40 However, since its dis- Flow cytometry CD38
covery there has been an intense debate how to perform ZAP-70
ZAP-70 analysis in a standardized way. This relates to CD49d
the fact that ZAP-70 (i) is an intracellular protein; (ii) is FCRL2
expressed not only by T-cells but also by normal B- Realtime-PCR LPL
cells:42 (iii) that different protocols have been employed CLLU1
using various antibodies and cutoffs. A multi-center Protein- or RNA-based MCL1
effort initiated by the European Research Initiative on TCL1
CLL (ERIC) has aimed at finding a standardized proto- HS1
col, which probably will be reported during 2009.
Genetic aberrations CLL-FISH
Newly proposed markers (various techniques) TP53 mutations
In addition to CD38 and ZAP-70, several other inter- ATM mutations
esting markers have recently been proposed to predict Clonal evolution
Genomic complexity
outcome in CLL. Brief presentations of four selected
Telomere length
markers follow, chosen from the rather extensive litera-
micro-RNAs 13-miR signature, miR-29c, miR-223
ture that has emerged lasting recent years (Table 1).

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 63 |
14th Congress of the European Hematology Association
CLLU1
Using differential display technique, Buhl and col-
leagues identified a new gene expressed in unmutated
CLL.5 This gene was cloned and found not to be
expressed in any other tissue type or in other hemato-
logical malignancies, and could thus be described as a
CLL-specific gene, CLL up-regulated gene 1 (CLLU1).
Furthermore, high CLLU1 expression correlated with
short time to treatment and overall survival, and com-
plemented several other prognostic markers, such as
stage, mutation status and ZAP-70 expression.5,46 To
date, no “CLLU1” protein has been identified; however,
such a protein could make an interesting new therapeu-
tic target.
TCL-1
TCL-1 is an anti-apoptotic protein which was first
described in T-cell leukemia47 and more recently high
levels of TCL-1 have been demonstrated in unmutated
CLL and in patients with 11q deletion.48 By applying
realtime-PCR we confirmed high levels of TCL-1 in
unmutated CLL, however TCL-1 did not remain inde-
pendent following multivariate analysis,49 possibly due
to its close association to IGHV mutation status and 11q
deletion. Interestingly, we observed that the poor-prog-
nostic IGHV3-21 group displayed high levels similar to
the high ZAP-70 levels also observed it this group.41
Micro-RNA
Micro-RNAs (miR) are short non-coding RNA mole-
cules where each miR is believed to silence the expres-
sion of other target genes. Recently, genome-wide miR
analysis revealed a CLL-specific profile of 13 different
miRs. Interestingly, a combination of these miRs corre-
lated with other prognostic markers and clinical out-
come.50 Down-regulation of two individual miRs,
miR29c and miR223, was also recently associated with
inferior outcome in CLL.51 In the coming years, we will
hopefully learn much about the role of specific micro-
RNAs, which will help us to unravel important aspects
of CLL pathogenesis.
Genetics in chronic lymphocytic leukemia
As with many other B-cell lymphoma entities, there is
no genetic aberration that is common to all CLL
patients. Several recurrent aberrations have been
described, the most frequent being deletion of 13q14,
followed by deletion of 11q22-23, trisomy 12 and dele-
tion of 17p13.3,4 The 13q deletion has been intensively
analyzed during the last 10 to 15 years without the suc-
cessful identification of any candidate genes. However,
it has lately been shown that miR15 and miR16, which
are encoded in the deleted region, are involved in the
pathogenesis of CLL.50 In addition, the 11q22-23 dele-
tion spans the ATM gene, a very important regulator of
cell division and DNA repair, whereas the 17p13 dele-
tion covers the TP53 gene, a tumor suppressor gene cru-
cial in cell cycle regulation.
CLL-FISH
In 2000, Döhner et al. proposed the hierarchical model
for classification of CLL, where patients with 13q dele-
tion, as a single aberration, were shown to have superior
outcome to patients with 11q and in particular 17p dele-
| 64 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
tions, whilst trisomy 12 patients had a more intermedi-
ate risk-profile.3 Since conventional cytogenetics tradi-
tionally had a low success rate, fluorescence in situ
hybridization (FISH) analysis rapidly became the gold
standard in CLL using a commercial panel of FISH probes
to screen for 11q-, 13q-, +12 and 17p-. Patients with 11q
or 17p deletion usually carry unmutated IGHV genes and
they generally respond poorly to current treatment pro-
tocols, in particular the 17p- group.13 For the latter group,
treatment regimes using alemtuzumab and prednisolone
appear especially promising.52 FISH analysis is now con-
sidered mandatory as a predictive marker, not only in
clinical trials but also in routine practice,53 particularly
when treatment is planned with the aim of long-term
remission. Since clonal evolution is a known phenome-
non in CLL,54 where poor-risk aberrations can emerge
over time, it is also important to perform FISH analysis
when there is a change in the patient’s clinical course or
if the patient responds poorly to therapy.
TP53 mutations
Recently, the significant role of TP53 mutations was
reinforced in CLL. By screening the TP53 gene, Zentz et
al. demonstrated that most cases with 17p deletion also
displayed an accompanying TP53 mutation on the other
allele.10 Importantly, they also reported that a proportion
of cases (4-5%) displayed a TP53 mutation in the
absence of a 17p deletion, and such cases had a poor
prognosis similar to cases with 17p deletion. This find-
ing has now been confirmed by others.55 Different tech-
niques are available for detection of TP53 mutations,
such as direct sequencing and denaturing high-pressure
liquid chromatography (dHPLC). dHPLC gives a higher
sensitivity, although direct sequencing is more readily
available in most laboratories. In addition, it has been
shown that assessment of p53 dysfunction, using for
example, flow cytometry, predicts poor prognosis in
CLL.56 Considering the very poor prognosis in the TP53
mutated group, it is reasonable to assume that mutation
screening will soon be added to the FISH panel,
although the most appropriate technique to apply has
yet to be agreed upon.
Genomic complexity
Difficulties exist culturing CLL cells whilst attempting
to obtain proper metaphases for karyotyping. In recent
years, however, new culturing protocols have been
applied that have improved the success rate significant-
ly.57,58 By adding IL2 and CpG oligonucleotides, Haferlach
and colleagues achieved successful cultures in most of
their 500 cases and detected clonal aberrations in more
than 80%.59 Interestingly, several reports have now
shown that an increasing number of aberrations and the
presence of translocations, especially unbalanced, will
predict poor outcome in CLL.57-59 Hence, we may consid-
er applying these new culturing protocols when analyz-
ing for genomic aberrations in CLL.
Recent studies using high-resolution array-based tech-
nology have detected few novel recurrent aberrations in
CLL.60,61 However, considering the recent advances with
oligonucleotide arrays and SNP-arrays, it is still possible
that further important aberrations will be detected. In
line with recent cytogenetic studies, the finding of higher
genomic complexity (≥3 aberrations) using genomic
 Berlin, Germany, June 4-7, 2009

arrays was also predictive of poor outcome in CLL.62 We

recently investigated part of a population-based
Scandinavian cohort (203 of ~600 cases) using Affymetrix
250K SNP-arrays, where we confirmed the prognostic
impact of the recurrent aberrations and increasing com-
plexity (Figure 3).63 That notwithstanding, one has to
keep in mind that most of the cases with higher complex-
ity are cases with 11q/17p deletion.59 Although of great
interest, the application of whole-genome arrays is
expensive and the procedure remains to be standardized
before it can be used in the clinical setting.

SNP analysis
During the last seven years, numerous new correla-
tions between single SNPs and clinical outcome have
been proffered. Notably, genetic variants within the
P2X7, BCL-2, BAX, MCL1, and GNAS1 genes have been
reported,64-67 although none of these SNPs have subse-
quently been confirmed in independent materials.68-71 For
instance, the MDM2 SNP309 was shown to correlate
with poor prognosis in CLL,72 whereas a separate study
and our own unpublished data did not reveal any corre-
lation.73 The lesson learnt from all these studies is that a
SNP, which appears to give prognostic information in
CLL, requires re-confirmation in a second, independent
cohort before proposing it as a clinically applicable
prognostic marker.

Telomeres
Telomeres, the ends of the chromosomes, are com-
posed of repetitive DNA sequences (TTAGGG). During
each cell division, the telomere shortens, and when the
telomeres eventually become too short, the cell under-
goes apoptosis. However, in certain cells types, such as
stem cells and germ cells, and in cancer cells, the
enzyme telomerase restores telomere length, thereby
allowing these cells an extended replicative capacity. In
CLL, we and others have shown that short telomeres
correlate strongly to unmutated IGHV genes and poor
outcome, whereas longer telomeres are associated with
good prognosis and mutated IGHV genes.7,74-77 In unmu-
tated CLL, the short telomeres may reflect a higher pro-
liferative activity of the progenitor cells, although the
telomeres will remain intact due to high telomerase
activity.77 Recently, we demonstrated that CLL patients
with complex karyotype and/or presence of 11q-/17p-
also demonstrated significantly shorter telomeres.76
Initially, the assay used to determine the length of
telomeres was the time-consuming and cumbersome
Southern blot technique.74 However, a simple PCR-
based assay is now available, although standardization
between laboratories has to be performed if this test is
to be routinely applied.7
Conclusions
During the last decade, several prognostic markers
have been demonstrated to be solid and reproducible
markers to predict outcome in CLL, such as the IGHV
mutation status and FISH analysis. In addition to this, a
very extensive list of markers has been proposed to be
of prognostic significance in CLL, some of which have
been detailed in this paper and which appear promising.
The next step is to determine which markers hold most
prognostic value, but are reasonably easy to perform in
Figure 3. Genomic data achieved by applying high-resolu-
tion SNP-arrays (Affymetrix, 250K) in 203 CLL cases from
a Scandinavian population-based cohort. (A) Survival
analysis according to the hierarchical classification (13q-,
no aberration, +12, 11q- and 17p-). (B) Survival analysis in
relation to increasing genomic complexity, when including
copy-number aberrations larger than 5 Mb. CNA, copy-num-
ber aberration.
a clinical setting. Firstly, for all markers, it is important
to perform extensive analysis, not only retrospectively
but also prospectively, in independent, large cohorts and
to evaluate them against already established markers.
Hopefully, this will render a “molecular prognostic
index” in CLL, where the most suitable and strongest
markers are combined. Furthermore, it is very essential
to define markers that can predict therapy response in
CLL, especially considering ongoing and new upcoming
strategies for therapy. To date, a few studies have exten-
sively compared predictive capacity of prognostic mark-
ers in large materials. In one of them, as mentioned,
ZAP-70 was shown to be the strongest predictive mark-
er of time to treatment, followed by the IGHV mutation
status, whereas CD38 did not add to the other two
markers.36 Similar prospective studies are now necessary
to guide us through this “jungle” of prognostic markers.
As a final note, one may say that we currently have
too many proposed markers in CLL. Conversely, while
many of these will never be applied in the clinics, hope-
fully we will learn more about CLL biology by trying to
understand the reasoning behind their deregulation.

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 65 |
14th Congress of the European Hematology Association
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 Chronic lymphocytic leukemia

Risk adapted chronic lymphocytic

leukemia management

P. Hillmen A B S T R A C T

Leeds General Infirmary, Leeds,
United Kingdom
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:68-71
The understanding of chronic lymphocytic leukemia (CLL) and the treatment of patients with this
disease has changed dramatically over the last 10 years. There are now more effective therapies result-
ing in higher remission rates with prolonged progression free survival. In addition, the use of reduced
intensity conditioning allogeneic stem cell transplantation as a potentially curative procedure is being
more widely applied for selected patients. This has resulted in the development of risk-adapted ther-
apy in CLL. We are beginning to predict which patients will respond to which therapies (for example,
patients with p53 deletion will not respond well to purine analogues or conventional chemotherapy
but appear to respond well to alemtuzumab), and to tailor therapies to an individual patient’s disease
characteristics, as well as according to their other comorbidities and their response to therapy.
Intensive potentially curative allogeneic stem cell transplantation is being used in patients with poor
prognostic CLL at an earlier time-point in their disease. In this article, the current approaches to
patient directed therapies will be reviewed.

ver the last decade, our understand- Epidemiology and End Results) Registry is

O ing of chronic lymphocytic

leukemia (CLL) has changed dra-
matically. We now have a clearer under-
standing of the pathophysiology of the dis-
ease including the dynamics of the CLL
clone,1 its molecular biology,2 the impor-
tance of the interaction between the CLL
cell, and its micro-environment and critical
role of antigen stimulation in the develop-
ment of CLL in some patients.3 In addition,
the therapeutic options in CLL have devel-
oped considerably over the last decade
with the advent of combination immuno-
chemotherapy.4-6 Previously, when single
agent alkylating agents or purine analogues
were used but this is now being superseded
by the use of monoclonal antibodies alone
(alemtuzumab7) or in combination with
chemotherapy (rituximab8) and with an
increasing and selective use of reduced
intensity conditioning allogeneic stem cell
transplant.9,10 We are now witnessing the
introduction of novel agents, such as
lenalidomide.11,12 Largely due to the avail-
ability of more effective therapies, assays
that can detect extremely low levels of CLL
have been developed in order to assess the
presence of detectable minimal residual dis-
ease (MRD) to as low as a single CLL cell in
10,000 leucocytes.13,14 This rapid increase in
our understanding of the disease and in the
therapeutic agents and end-points alters our
approach to the treatment of patients with
CLL and the era of risk-adapted therapy is
now with us.
Factors influencing risk-adapted therapy
Patient comorbidity
The median age at diagnosis of CLL in
North America on the SEER (Surveillance
72 years (http://seer.cancer.gov/statfacts/
html/clyl.html). At this age, many individu-
als have comorbidities (mean of 3.6 comor-
bid conditions between 65 and 74 years
old), and these will impact the patient’s
ability to tolerate more intensive
chemotherapeutic regimes. In addition, cer-
tain drugs, such as fludarabine, are cleared
by the kidneys, and therefore, specific
comorbidities, such as renal dysfunction
will directly contraindicate the use of flu-
darabine. Therefore, therapy in CLL needs
to be adapted to the individual patient’s fit-
ness. Thus, there is no single therapy that is
currently the “gold standard” standard in
CLL, as it varies depending on the patient’s
fitness.
Pathophysiology of chronic lymphocytic
leukemia
It appears that there are at least two bio-
logical sub-types of CLL, which can be dif-
ferentiated by the presence or absence of
somatic mutations in the immunoglobulin
gene of the CLL clone.15,16 A proportion of
patients with mutated CLL presenting with
Binet Stage A disease are destined to
remain stable and never to require therapy.
In contrast, almost all patients with unmu-
tated CLL will need treatment at presenta-
tion or a short time after presentation. It
could be considered that patients with “VH
mutated” CLL have genetically “stable” dis-
ease, whereas those with “VH unmutated”
CLL have a genetically “unstable” disease,
as patients with a genetic evolution, such as
deletion of the p53 oncogene, usually have
unmutated CLL (Figure 1). However,
although the outcomes for patients with
unmutated CLL after their initial therapy

| 68 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

are inferior to those with mutated CLL, there is no evi-
dence that stratifying patients to different therapies
according to their mutational status is beneficial. A key
unanswered question is whether patients with poor
risk CLL should receive treatment when they present
with Stage A disease before their CLL progresses. The
data that supports a “watch and wait” strategy in CLL
is based on large randomised trials in unselected early
stage patients,17-20 some of whom would never have
progressed, and using relatively ineffective therapies,
such as monotherapy with alkylating agents. At pres-
ent, the German and French CLL Study Groups are
participating in a large randomised trial comparing
early treatment with FCR against “watch and wait”
(the CLL7 trial) in patients with biologically poor-risk
Stage A CLL who would not conventionally receive
therapy. This trial, which is recruiting well, will prob-
ably provide an answer to this important question in
the near future. An alternative approach maybe to use
a therapy that does not have the potential to damage
DNA, and thus, result in genetic evolution of a “genet-
ically unstable” CLL clone. Such therapies may include
monoclonal antibodies as single agents or agents, such
as lenalidomide. Trials with these agents in early stage
poor risk CLL are being considered.
VH mutational status is an integral part of the CLL
clone that is a marker of the transformed original cell
and will not alter throughout the duration of a
patient’s disease. In contrast, patients can acquire
genetic abnormalities as part of the evolution of their
disease. The best-characterized abnormality that can
evolve is the loss or mutation of the p53 oncogene.
This is usually identified by the loss of the p53 locus or
deletion of the short arm of chromosome 17 (17p dele-
tion).21 In most patients with 17p deletion, the p53
gene on the other chromosome 17 has a mutation, and
therefore, there is no active normal p53 in the CLL cell.
The chemotherapeutic agents conventionally used in
CLL, either alkylating agents or purine analogues,
damage DNA (or prevent its repair), leading to apopto-
sis or cell cycle arrest via the p53 pathway. Thus, CLL
cells with a dysfunctional p53 pathway cannot
respond in a “normal” way to chemotherapy in that
they are unable to undergo apoptosis or cell cycle
arrest. This means that the CLL cell suffers DNA dam-
age but is unable to die – clearly an inherently danger-
ous situation. It is entirely plausible that patients with
p53 dysfunction are not only resistant to conventional
chemotherapy, but that when they are exposed to
DNA damaging chemotherapy, their CLL cells develop
mutations in critical oncogenes and tumour suppressor
genes. If true, this would result in the chemotherapeu-
tic insult, leading to the development of more prolifer-
ative or resistant sub-clones, and thus, a poorer prog-
nosis. Therefore, it is sensible that conventional
chemotherapy should be avoided in patients with CLL
with a deletion p53 and agents that do not depend on
the p53 pathway for their function should be
employed. These agents include monoclonal antibod-
ies, particularly alemtuzumab, and steroids. Trials are
ongoing to study the combination of monoclonal anti-
bodies and steroids in p53 deleted CLL. Now, the stan-
dard therapy for these patients who cannot be recruit-
ed into clinical trials should be alemtuzumab.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 69 |
Berlin, Germany, June 4-7, 2009
Figure 1. The prognostic markers in CLL indicate that there
are at least two sub-types of the disease. These are simi-
lar at an immunophenotypic level, which is used to estab-
lish the diagnosis. A minority of patients requiring therapy
have what might be considered “genetically stable” CLL,
whereas the majority have “genetically unstable” CLL. This
difference may be defined by the mutational status of the
immunoglobulin genes and the presence or absence of
other poor prognostic features. Both groups have high
response rates to initial combination chemotherapy, but
patients with “genetically stable” CLL rarely develop p53
pathway abnormalities, or resistant disease at relapse in
contrast to “genetically unstable” CLL, in which evolution to
resistant disease is common at relapse. A minority of
patients present with p53 pathway abnormalities and are
inherently resistant to conventional chemotherapy.
However, in patients with very large lymph nodes
(individual nodes greater than 5 cm in diameter), the
response rates to alemtuzumab are low,7 with very few
patients achieving a complete remission. In these
patients, it is worth considering the use of therapies to
reduce the size of the lymph nodes, such as high dose
methyl prednisolone,22 followed by alemtuzumab.
The deletion of 17p is the worst biological prognos-
tic marker but is relatively uncommon in patients
when they initially require therapy. In contrast, the
deletion of the long arm of chromosome 11 (11q del)
occurs in as many as 20% of patients prior to their ini-
tial therapy and is also a poor prognostic marker.4,23 It
is thought that the loss of the ataxia telangiectasia
mutated (ATM) gene from chromosome 11 results in
the poor prognosis for these patients, who frequently
present with bulky lymphadenopathy. The initial
reports of the use of FCR from the front-line German
CLL8 Trial suggest that the beneficial effects of the
addition of rituximab to FC are particularly marked for
patients with 11q del and that these patients may even
show a survival advantage with FCR.24,25
Response to adapt therapy
It is becoming increasingly evident that remission
duration, treatment-free survival and overall survival is
dependent on the depth of remission. Patients who
achieve a complete remission by IWCLL Criteria26
have a better survival than those who do not. Even
more evident is that patients who have no detectable
14th Congress of the European Hematology Association
CLL at the end of therapy, those who are minimal
residual disease (MRD) negative, have a far superior
survival independent of the therapy used to achieve
MRD negativity.23 This means that the response to
therapy is an important factor affecting further treat-
ment. For example, there are now many ongoing stud-
ies testing the use of alemtuzumab and other therapies
as consolidation following conventional immuno-
chemotherapy in order to eradicate detectable MRD.
Also, many allogeneic stem cell regimes include the
use of donor lymphocyte infusions in patients who
have detectable MRD post-transplant. Therefore, we
are now risk-adapting therapy according to MRD.
Optimal therapy for chronic lymphocytic leukemia
Immunochemotherapy
Three large randomised Phase III trials have previ-
ously demonstrated that the combination of fludara-
bine plus cyclophosphamide (FC) is superior to single
agent therapy with fludarabine or chlorambucil.4,5,6
However, the patients in these trials were either young
and/or were considered fit enough to tolerate fludara-
bine-based combination therapy. Thus, the logical
backbone chemotherapy with which to combine other
therapies, such as monoclonal antibodies, is FC.
Rituximab was added to FC (FCR) by the group at the
MD Anderson Cancer Center.8,27 When compared to
historical controls from the same institution, it
appeared that the addition of rituximab almost dou-
bled the complete remission rate and improved overall
survival. Recently, the results of two randomised Phase
III trials comparing FC to FCR have been presented.
One of these trials, the German CLL Study Group
CLL8 Trial,26 involved over 800 patients with CLL who
required therapy by conventional criteria but were
previously untreated, and the trial randomly assigned
them to receive either FC or FCR. In the GMCLLSG
CLL8 trial, the addition of rituximab to FC doubled the
complete response rate (27% for FC compared to 52%
for FCR) and improved the median progression-free
survival by approximately 10 months, thus meeting
the trial’s primary end-point. In addition, the REACH
Study28 also compared FCR with FC but in patients
who had previously received one line of therapy.
Again, the REACH study showed a significant advan-
tage in terms of complete response rate and progres-
sion free survival for the addition of rituximab.
Therefore, we can now consider FCR as the standard
therapy for younger fitter patients. However, there are
situations where FCR is not appropriate:
Elderly patient or those with significant comorbidity
In many patients, fludarabine and FC are contraindi-
cated due to the intensity of the treatment, renal dys-
function and/or previous fludarabine-induced autoim-
mune hemolysis. In these patients, there is no “gold
standard” therapy but chlorambucil as a single agent
remains the most frequently used treatment.
Theoretically, the addition of rituximab is likely to be
synergistic with chlorambucil, as appears to be the case
with most chemotherapies in other B-cell malignancies,
such as follicular lymphoma or diffuse large B-cell lym-
phoma. However, there are no studies to date that
report this combination and no randomised trials sup-
| 70 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
porting the use of rituximab in addition to any other
chemotherapeutic combination except for FC.
Patients with deletion of chromosome 17
It is clear that fludarabine-based combination chemo-
therapy is ineffective, and potentially deleterious, for
patients with p53 dysfunction. In these patients, alem-
tuzumab, either alone29,30 or in combination.31 In addi-
tion, patients with 17p deletion should be considered
for an allogeneic stem cell transplant; preferably, when
they attain a remission.9 This is because even if patients
reach a good remission, these tend to be short-lived
and then the re-induction of a further remission may
not be possible. In addition, there is good evidence that
the graft-versus-leukemia is as effective against 17p-
deleted CLL compared to other cases, and the outcome
from allogeneic stem cell transplantation appears to be
equally good.
MRD status
Patients who are MRD negative at the end of thera-
py have a better survival than those who have
detectable disease.7,13 This raises the question whether
consolidation therapy with the aim of eradicating
MRD post-chemotherapy is a reasonable approach. At
present, there are a number of trials looking at the use
of a variety of agents, such as alemtuzumab or lenalido-
mide as consolidation, but to date an effective and safe
schedule has not been established.
Conclusion
Over the last decade, there have been huge strides in
our understanding and treatment of CLL. We are now
just entering the era where biological features of a
patient’s CLL are being used to define the most appro-
priate therapy. In addition, we are now exploring the
tailoring of therapy to individual patients based on
their comorbidity and/or response to previous therapy.
It is likely that over the next 5-10 years, the risk adap-
tion of therapy in CLL will become the norm.
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Warzocha K, Loscertales J, et al. Rituximab, Fludarabine, and
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29. Stilgenbauer S, Zenz T, Winkler D, Bühler A, Busch R, Fin-
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30. Lozanski G, Heerema NA, Flinn IW, Smith L, Harbison J,
Webb J, et al. Alemtuzumab is an effective therapy for chron-
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31. Pettitt AR, Matutes E, Oscier D. Alemtuzumab in combina-
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M.W. Deininger
Oregon Health & Science University,
Portland, OR, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:72-77
| 72 |
Chronic myeloid leukemia
Stem cell persistence in chronic myeloid leukemia
Clinical and molecular features of chronic
myeloid leukemia
Chronic myeloid leukemia (CML) is char-
acterized by a 2-phase clinical course. In the
developed world, most patients are diag-
nosed in the chronic phase, which is charac-
terized by grossly increased output of
myeloid cells, whose differentiation and
function is largely maintained.1 Historical
data suggest that the median duration of the
chronic phase, in the absence of any effec-
tive therapy, is 2-3 years, after which there is
a progressive loss of cellular differentiation
that culminates in the blastic phase, an acute
leukemia of myeloid or lymphoid pheno-
type. While complications in the chronic
phase are rare, blast crisis is a rapidly fatal
disease. Thus, the foremost aim of CML
therapy is to prevent the progression from
the chronic to the blastic phase.
The cytogenetic hallmark of CML is the
Philadelphia chromosome (Ph), the conse-
quence of the t(9;22)(q34;q11). At the molec-
ular level, the translocation leads to the
fusion of BCR genetic sequences upstream
of the ABL gene on chromosome 9. The
resulting BCR-ABL fusion protein is that
activates multiple signaling pathways,
including mitogen activated protein kinases
(MAPK), phosphotidyl inositol 3’ kinase
(PI3K) and Janus kinase/signal transduction
and activation of transcription (JAK/STAT)
signaling, amongst many others.
Collectively, these pathways increase prolif-
eration, inhibit apoptosis, induce genetic
instability and perturb the interaction
between the CML cells and their microenvi-
ronment. The identification of non-redun-
dant components in this complex signaling
network has proved difficult, consistent
with a high degree of redundancy. However,
it is clear that the tyrosine kinase activity of
BCR-ABL is conditio sine qua non of leuke-
mogenesis.2
It has been a matter of debate whether
BCR-ABL is sufficient to induce the chronic
phase of CML. Early studies based on glu-
cose 6 phosphate dehydrogenase (G6PD)
isoenzyme expression in EBV-transformed
Ph-negative B cell lines from CML patients
had suggested that a clonal state predates
the acquisition of Ph.3-4 Over the years, sub-
stantial evidence has accumulated in support
of the notion that chronic phase CML does
not require mutations in addition to Ph.
Thus, chromosomal abnormalities in Ph-
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
positive cells, referred to as clonal evolution,
are rare in newly diagnosed chronic phase
patients.5 Moreover, polyclonal hemato-
poiesis is restored in most patients with a
complete cytogenetic response (CCyR) to
imatinib.6 Lastly, bone marrow infected with
BCR-ABL retrovirus causes a polyclonal
CML-like myeloproliferative disease upon
transplantation into lethally irradiated syn-
geneic recipients, consistent with a single hit
pathogenesis.7 What these data cannot
exclude is that in human CML, a mutation
may be acquired before BCR-ABL, but with-
out causing clonal expansion. This notion
has attracted renewed interest from observa-
tions in Ph-negative JAK2V617F-positive MPD,
where acute leukemia may arise from
JAK2WT cells, consistent with a JAK2V617F-neg-
ative abnormal cell at increased risk of trans-
formation to acute leukemia.8 Moreover, the
clinical course of newly diagnosed patients
with seemingly exchangeable disease pre-
sentations can be very different, indicating a
level of disease heterogeneity that is not
appreciated by the resolution of morpholo-
gy and cytogenetics.9
Cell of origin
Studies using infection of immunopheno-
typically and functionally defined murine
hematopoietic progenitor and stem cells have
revealed that BCR-ABL does not confer self
renewal capacity to progenitor cells, unlike
AML-related fusion genes, such as MOZ-
TIF2.10 This implies that the BCR-ABL translo-
cation must be acquired by a hematopoietic
stem cell that is able to self-renew. In support
of this, BCR-ABL has been demonstrated in all
hematopoietic lineages and in lineage-nega-
tive CD34+/CD38– cells.11-12 For mechanisms
that are not well understood, cellular expan-
sion is, however, limited to the myeloid
compartment, particularly granulocytes and
their precursors. Interestingly, the degree to
which the “stem cell” compartment is pene-
trated by the BCR-ABL-positive cell clone is
extremely variable.12 Although precise data
are unavailable, it is believed that a more
complete replacement of the stem cell com-
partment with leukemic cells is a feature of
long-standing disease. From a clinical view,
the paucity of residual normal cells present
in the marrow of patients with CML of
many years’ duration may be responsible for
the frequent cytopenias of such patients on
imatinib therapy. From the fact that CML

originates in a hematopoietic stem cell, we may deduce
that the organization of the leukemia follows the same
hierarchical structure as normal hematopoiesis. This
would imply that acquisition of Ph by a cell further
down in the hierarchy, and without self-renewal capac-
ity, will not produce disease. This would explain, why
BCR-ABL transcripts are detectable at very low level in
many healthy individuals, most of whom will never
develop CML.13 In contrast to the chronic phase, there is
evidence that myeloid blast crisis may originate in a
granulocyte macrophage progenitor (GMP) that
acquires self-renewal capacity through activation of β-
catenin.14-15 This implies that there may be many more
leukemia stem cells at the time of blastic transformation
compared to chronic phase, which may explain the
treatment-refractory nature of blastic phase.
The difficulty of eradicating the Ph+ cell clone with
chemotherapy
The notion that BCR-ABL is acquired by a hemato-
poietic stem cell fits very well to the old clinical obser-
vation that it is virtually impossible to eradicate CML
with chemotherapy. Thus, even with intensive multia-
gent regimens, Ph-negativity (as assessed by cytogenet-
ics) is only transient; suggesting that killing the Ph-pos-
itive cell clone would require killing the entire
hematopoietic system. The only way to accomplish this
is myeloablative regimens used in allogeneic stem cell
transplantation. While there is no doubt that a T cell
dependent graft versus leukemia effect mediates the
potent graft versus leukemia effect, the contribution of
the conditioning regimen should not be underestimat-
ed.16 Otherwise, it would be difficult to explain the sus-
tained responses in some recipients of identical twin
allografts.17 On the other hand, a “PCR undetectable”
state is not equivalent to the absence of residual
leukemia and even years post allograft, there continues
to be a small but not negligible risk of relapse.18 These
data are in contrast to patients with acute leukemia,
where a substantial subset of patients is curable with
non-myeloablative chemotherapy regimens based on
cytarabine and anthracyclins. This suggests that AML in
long-term responders may have originated at the level
of a hematopoietic progenitor rather than a stem cell.
CML cells, on the other hand, are likely to command
the vast armentarium of defenses that allow normal
stem cells to weather the toxic stress of a lifetime.
Chronic myeloid leukemia stem cells
The major obstacle to a better understanding of CML
stem cells is the lack of distinct phenotypic and precise
functional markers. Studies by the Vancouver group
have identified CML stem cells in the lineage-
/CD34+/CD38– cell population of CML cells, based on
their ability to engraft immunodeficient mice.
Importantly, engraftment and serial transplantation
capacity includes the quiescent (non-cycling) cells with-
in this population.19 Despite these stem cell features, it
should be noticed that enraftment is low, and the mice
do not develop leukemia. Yet, no surface marker has
been identified that would distinguish CML stem cell
from their normal counterparts, preventing the isolation
of such cells for prospective studies. The other widely
used system to study stem and progenitor cells are long
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 73 |
Berlin, Germany, June 4-7, 2009
term culture initiating cell (LTC-IC) assays on irradiated
human or murine stromal cells. While these cultures are
relatively easy to standardize, they favor the outgrowth
of normal over CML progenitor and stem cells; a fact
that formed the basis for in vitro purging strategies.20-21
This is in obvious contrast to the human disease, and
clearly indicates that the assay system impacts on the
parameter it intends to measure, much in the sense of
Heisenberg’s paradigm in physics. Thus, for the time
being, the study of CML stem cells is limited by the
availability of accurate assays that do not affect func-
tional properties, and all available data need to be inter-
preted with caution.
Residual disease in patients treated with BCR-ABL
inhibitors
Between 80-90% of patients with newly diagnosed
chronic phase CML will achieve CCyR on therapy with
imatinib, with considerably lower rates in patients with
advanced CML.22-23 However, residual disease remains
detectable in the majority of patients with a CCyR.24
Since recurrence is almost inevitable in these individu-
als, the current recommendation is to continue therapy
indefinitely. This clinical observation corroborates labo-
ratory data showing persistence of BCR-ABL expressing
clonogenic cells.25-26 Only a minority of patients become
“PCR undetectable” on imatinib treatment, a state
referred to as a complete molecular response (CMR). In
anecdotal reports, a subset of these patients has main-
tained responses even after discontinuation of ima-
tinib,27 and several clinical trials are currently underway
to investigate this question in a systematic fashion.
Interestingly, most recurrences occurred rather early
after discontinuation of imatinib, while the recurrence
free survival curves seem to stabilize over time. While
very preliminary, these data are intriguing and suggest
that a subset of patients may be ‘curable’ with imatinib.
What we currently do not know is whether the disease
burden in these patients is zero, or whether they contin-
ue to harbor residual leukemia that is below the detec-
tion limit of the clinical BCR-ABL assays. Reassuringly,
complete molecular responses were readily achieved
upon re-challenge with imatinib.
Mathematical modeling has been applied to explain
the dynamics of BCR-ABL mRNA in patients on ima-
tinib, and to predict the drug’s ability or inability to
eliminate the disease. One study found a biphasic
course, where a rapid reduction of leukemia burden is
followed by a slow decline.28 The first phase is thought
to reflect the killing of differentiating cells, while the
second is thought to mirror the reduction of leukemic
progenitor cells. This model has concluded that eradica-
tion of the CML clone is impossible during the lifetime
of the host. In contrast, another mathematical model
came to a different conclusion, suggesting that imatinib
has the general potential to eradicate the Ph-positive cell
clone and that agents stimulating cell cycle entry may
accelerate this process.29
Mechanisms of chronic myeloid leukemia stem cell
resistance to tyrosine kinase inhibitors
The fact that almost all patients achieve a complete
hematologic response and most a complete cytogenetic
response indicates that differentiating CML cells are
14th Congress of the European Hematology Association
dependent on BCR-ABL kinase activity, an observation
that is well supported by in vitro data. Conceptually,
another possible explanation is that the kinase inactive
BCR-ABL generated by imatinib is toxic to these cells.
In contrast, CML stem cells are apparently much more
resistant to imatinib. A number of mechanisms have
been proposed the ability of these cells to withstand
imatinib (Figure 1). In vitro studies by the Glasgow group
have suggested that quiescent CML cells are resistant to
imatinib and also the more potent BCR-ABL inhibitors
nilotinib and dasatinib, suggesting that neither of these
agents has activity against the most critical cell popula-
tion.30-32 One has to concede, however, that quiescence
is a functional state rather than a mechanistic explana-
tion for resistance. Furthermore, we cannot be certain
that the quiescent lineage-/CD34+/CD38– cells isolated
from newly diagnosed patients are identical to the cells
that survive in vivo in patients treated with imatinib,
and proof is lacking that they exhibit the functional
characteristics of stem cells; that is, the ability to engraft
immunodeficient mice and transplantability in serial
transplantation assays. Other explanations for the TKI
resistance of CML stem cells are their much higher lev-
els of BCR-ABL in comparison to more differentiated
progenitor cells.33 This would be similar to overt resist-
ance caused by BCR-ABL gene amplification or
increased mRNA expression. Furthermore, one study
found a high frequency of kinase domain mutations in
patients with CCyR, a finding that was, however, not
confirmed in subsequent studies and may be due to a
particularly poor risk population with a high risk of
relapse.34-35 Lastly, drug transport proteins may decrease
intracellular imatinib levels below a critical threshold.
This could involve drug resistance proteins, such as Pgp,
but also hOCT-1, a cation pump that transports ima-
tinib into the cells. Several studies have shown correla-
tions between high levels of hOCT-1 expression or
function and cytogenetic response.36-38 However, infor-
mation on hOCT-1 expression in the most primitive lin-
eage-/CD34+/CD38– cells is currently lacking.
Despite all these data, it seems that the critical ques-
tion has not yet been resolved, namely whether or not
| 74 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
BCR-ABL is active in CML stem cells treated with tyro-
sine kinase inhibitors. Studies from different laborato-
ries have come to discordant conclusions, probably
reflecting the difficulty of acutely measuring BCR-ABL
activity in the relevant cells.31-33 In our own experience,
FACS using antibodies directed against phosphorylated
CrkL (pCrkL) or pan-phosphotyrosine requires
extremely stringent controls to rule out non-specific
binding, and immunoblot analysis is difficult due to the
usually small cell numbers. However, from a therapeu-
tic perspective, there is no more important question to
answer. If BCR-ABL is not inhibited, then efforts need
to be directed at improving BCR-ABL inhibition, by
developing yet more potent inhibitors or by overcoming
drug transport mechanisms that prevent achieving a
critical threshold of BCR-ABL inhibition in the relevant
cells. Conversely, if imatinib (and the more potent sec-
ond-line inhibitors) suppress BCR-ABL in the most
primitive CML cells, and these cells still survive in cul-
ture despite BCR-ABL inhibition, this would indicate
that CML stem cells are not addicted to BCR-ABL and
that disease persistence is not a BCR-ABL dependent
process. If so, then biochemically targeting BCR-ABL
will not suffice to annihilate the Ph+ cell clone, despite
the profound suppression of CML progenitor cells, and
disease eradication will require a fundamentally differ-
ent approach. At this point, it seems that more evidence
is accumulating to support the second possibility.39
Perhaps this should not come as a surprise. If we consid-
er how close chronic phase CML cells are to their nor-
mal counterparts in terms of function – there are just too
many of them. Shutting off BCR-ABL may be expected
to reverse them to normality rather than kill them, and
one may speculate that this may be particularly true for
the most primitive cells. Thus, CML stem cells, when
confronted with BCR-ABL inhibition, may be able to
utilize the same physiological growth and survival path-
ways as normal cells. On the other hand, we could hope
that BCR-ABL-inhibited CML stem cells will never be
quite like normal cells, since they harbor a large, kinase-
dead protein that is lacking from their normal equiva-
lents and may interfere with physiological signaling.
Figure 1. Mechanisms
potentially involved in
the resistance of CML
stem cell to BCR-ABL
kinase inhibitors. FN -
fibronectin.

Some evidence for this comes from studies in cell lines
transformed to cytokine-independence because of BCR-
ABL expression. While many of these lines can be res-
cued from imatinib-induced apoptosis by exogenous
growth factors, the rescue is incomplete in others, such
as BCR-ABL expressing Mo7e cells.40 This raises two
questions. First, can we intercept such exogenous sur-
vival factors to target CML stem cells over normal stem
cells? Secondly, are there other, cell-intrinsic pathways
that are uniquely relevant to the survival or self-renew-
al of CML stem cells, but not to normal cells?
Pathways relevant to chronic myeloid leukemia stem cell
survival
Studies into the pathways critical to CML stem cell
have mainly relied on murine models, either transgenic
models or the standard transduction/transplantation
model. The implicated pathways or molecules include
Wnt/β-catenin, Hedgehog and PML.
β-catenin is a multifunctional protein with distinct
roles in cell adhesion and as a transcriptional co-activa-
tor. β-catenin transcriptional activity is negatively regu-
lated through proteasomal degradation that requires
phosphorylation by GSK3β. Binding of Wnt proteins to
their cognate surface receptors inhibits GSK3β and
induces β-catenin stabilization, nuclear translocation
and activation of target genes. Many solid tumors
exhibit constitutive β-catenin activity because of muta-
tions in negative regulators, such as APC and axin, or in
β-catenin itself. In hematopoietic stem cells, β-catenin is
required for self-renewal.41 Thus, there is more rapid
exhaustion of hematopoietic stem cells in serial trans-
plantation assays of mice lacking β-catenin. As noted
above, active (nuclear) β-catenin was shown in granulo-
cyte-macrophage progenitor cells from CML patients in
myeloid blastic phase, but not chronic phase, suggesting
that this may underlie the acquisition of self-renewal
capacity by this cell population.15 Conditional knockout
of β-catenin in transgenic mice attenuated CML-like
MPD.41 The mechanism of β-catenin activation in
human CML has not been defined conclusively, but
may involve direct phosphorylation by BCR-ABL that
prevents binding to axin, thereby stabilizing the pro-
tein.42 Altogether, this data suggests that targeting β-
catenin may be useful for the treatment of CML.
Unfortunately, despite efforts by many groups and com-
panies, until now no clinical β-catenin inhibitors have
been identified.
Hedgehog signaling has also been implicated in the
self-renewal of CML stem cells.43-44 The Hedgehog fam-
ily of proteins has three members, Sonic, Desert and
Indian Hedgehog. Binding to their receptor, patched
activates a signaling cascade the leads to stabilization of
the transcription factor Smoothen (SMO). Mice trans-
planted with BCR-ABL transduced cells lacking SMO
had significantly prolonged survival compared with
SMO+/+ cells. Furthermore, expression of an activated
form of SMO accelerated the disease in the same model.
Conversely, cyclopamine, which inhibits Hedghog sig-
naling by stabilizing an inactive form of SMO, pro-
longed survival of leukemic mice and reduced colony
formation by human CML cells, suggesting that target-
ing the Hedgehog pathway may be therapeutically
effective. SMO inhibitors are in development by sever-
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 75 |
Berlin, Germany, June 4-7, 2009
al pharmaceutical companies.
Another molecule implicated in CML stem cell sur-
vival is PML, the fusion partner of the retinoic acid
receptor α (RARA) in patients with acute promyelocyt-
ic leukemia and the t(15;17).45 Interestingly, targeting
PML may be possible using Arsenic trioxide, an agent
with a long history in the treatment of CML and an
approved drug. As a note of caution though, trials com-
bining imatinib with As2O3 in patients who had failed
imatinib showed rather limited success, and this
approach is not being pursued further at the current
time. Whether targeting any of these pathways will
eradicate the Ph+ stem cells remains to be seen in clini-
cal studies, but they clearly provide a therapeutic ration-
ale. What frequently remains unaddressed in the murine
studies is the question to which extent the activation of
these pathways in CML cells is dependent on BCR-ABL
or not. This will obviously decide whether their inhibi-
tion will add to the effects of BCR-ABL inhibitors, and
whether they should be used simultaneously or sequen-
tially.
Empirical approaches to target chronic myeloid leukemia
stem cells
The Glasgow group has tested a number of drugs and
compounds for their ability to reduce the quiescent lin-
eage-/CD34+/CD38– fraction of cells that survive the in
vitro exposure to imatinib.46 The only agent with this
ability was farnesyl transferase inhibitors (FTI), which
on its own, and in combination with imatinib, had
modest clinical efficacy in patients with active CML.
Building on these data, they then tested BMS214662, an
FTI with previously described activity against quiescent
cells, for its ability to inhibit primitive quiescent CML
cells alone or in combination with imatinib. These stud-
ies showed a significant reduction of BCR-ABL-positive
LTC-IC compared to imatinib alone in short-term liquid
culture assays.47 Interestingly, the effects of BMS214662
do not seem to depend on its FTI activity, but involve
another yet unknown pathway. Unfortunately, for rea-
sons that are unclear, the manufacturer has decided not
to develop this compound any further.
A number of other drugs have been tested in the lab-
oratory, including proteasome inhibitors, histone
deacetylase (HDAC) inhibitors and DNA methytrans-
ferase inhibitors, for their ability to target CML stem
cells alone or in conjunction with imatinib. A clinical
trial is planning to test LBH589, a potent HDAC
inhibitor in combination with imatinib, in patients with
molecular disease persistence.
Targeting stem cells rather than their pathways
The unprecedented ability of imatinib to reduce dis-
ease burden in CML to very low levels has greatly stim-
ulated interest in vaccination strategies. Non-specific
approaches are mainly based on Interferon-α, the drug
therapy standard of care before the arrival of imatinib.
Patients with CCyR to IFN-α, but to imatinib were
shown to harbor cytotoxic T-cell directed against PR-3,
a peptide derived from myeloblastin, a protein that is
overexpressed on CML cells.48 This suggests that
responses to IFN-α are immunologically based. The
combination of imatinib and interferon-α was tested in
several phase 1/2 studies, without clear benefit but sig-
14th Congress of the European Hematology Association
nificant toxicity. In contrast, a recent interim analysis
from the French multicenter trial showed a significantly
higher rate of major molecular responses for the inter-
feron/imatinib combination compared to imatinib and
imatinib/cytarabine.49 A second approach is vaccines
based on peptides derived from the BCR-ABL junction.
Various types of vaccines have been tested in several
clinical trials, with a suggestion of activity in patients
with low residual disease burden.50-51 Unfortunately, as
all trials were single-armed, these data await confirma-
tion in a prospective controlled study. Yet another
approach is vaccines based on a leukemia cell line
(K562) expressing GM-CSF, with promising preliminary
but as yet unconfirmed results.52
Conclusion
Imatinib has changed the face of CML from a deadly
disease to a chronic ailment, manageable with drug
therapy, and in many patients compatible with good or
at least acceptable quality of life. As a result, CML
prevalence has increased considerably and is set to
increase even more over the next several decades.
Estimates are that in 2040 there will be 250,000 CML
patients in the US alone. One might argue that we do
not need to worry, given the low risk of relapse in
patients with a CCyR and the apparent lack of late tox-
icities. However, despite generally good tolerability,
many patients do experience side effects that impair
their quality of life. For women of childbearing age, the
inability to have children is a significant burden. Lastly,
the health-economic burden of lifelong therapy is con-
siderable. Resistance to imatinib, while still a problem,
affects only a minority of patients, so the focus has
rightly shifted to the question of disease eradication and
the ability to stop medication. At this point, the most
promising finding is that some patients in CMR have
stopped the drug and maintained PCR negativity, sug-
gesting that the drug may cure a few patients, although
more follow-up is still warranted. How this is accom-
plished is unknown, and what sets the disease in these
patients apart from others remains to be determined.
Does one simply have to treat long enough to deplete
the Ph+ cell clone? This would be supported by the fact
that the median levels of BCR-ABL transcripts in the
IRIS study continue to decline.53 On the other hand,
there are many patients in whom the levels of residual
disease stabilize at a low level, without achieving PCR
negativity, and who would have a rapid disease recur-
rence after stopping imatinib. Thus, much work
remains to be done to understand better the hetero-
geneity of the disease and its response to BCR-ABL-tar-
geted therapy. At this point, it seems that imatinib may
be curative for a minority of patients, but that a funda-
mentally different approach will be required to eradi-
cate the disease in the remaining patients.
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of cancer stem cells in myeloid leukaemia. Nature 2009
45. Ito K, Bernardi R, Morotti A, Matsuoka S, Saglio G, Ikeda Y et
al. PML targeting eradicates quiescent leukaemia-initiating
cells. Nature 2008;453:1072-8.
46. Jorgensen HG, Allan EK, Graham SM, Godden JL, Richmond
L, Elliott MA et al. Lonafarnib reduces the resistance of primi-
tive quiescent CML cells to imatinib mesylate in vitro.
Leukemia 2005;19:1184-91.
47. Copland M, Pellicano F, Richmond L, Allan EK, Hamilton A,
Lee FY et al. BMS-214662 potently induces apoptosis of chron-
ic myeloid leukemia stem and progenitor cells and synergises
with tyrosine kinase inhibitors. Blood 2007;
48. Burchert A, Wolfl S, Schmidt M, Brendel C, Denecke B, Cai D
et al. Interferon-α, but not the ABL-kinase inhibitor imatinib
(STI571), induces expression of myeloblastin and a specific T-
cell response in chronic myeloid leukemia. Blood 2003;101:
259-4.
49. Guilhot F, Mahon FX, Guilhot J, et al. Randomized compari-
son of Imatinib versus Imatinib combination therapies in
newly diagnosed chronic myeloid leukaemia (CML) patients
in chronic phase (CP): First Results of the Phase III (SPIRIT)
Trial from the French CML Group (FI LMC). Blood 2008;112:
183.
50. Bocchia M, Gentili S, Abruzzese E, et al. Effect of a p210 mul-
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657-62.
51. Cathcart K, Pinilla-Ibarz J, Korontsvit T, Schwartz J, Zakhaleva
V, Papadopoulos EB, Scheinberg DA. A multivalent bcr-abl
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52. Smith BD, Kasamon YL, Miller CB, et al. K562/GM-CSF
Vaccination Reduces Tumor Burden, Including Achieving
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ABL Transcript Levels at 6, 12, and 18 Months (mo) Correlates
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112:334.
 Chronic myeloid leukemia

Dynamics of mutated clones in chronic myeloid

leukemia patients

A. Hochhaus A B S T R A C T

III Medizinische Klinik,
Universitätsmedizin Mannheim,
Universität Heidelberg, Mannheim,
Germany
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:78-82
Point mutations of the BCR-ABL tyrosine kinase domain are considered the predominant cause of
imatinib resistance in chronic myeloid leukemia patients. The expansion of mutant BCR-ABL positive
clones during treatment with tyrosine kinase inhibitors (TKI) is based on a clonal selection of less sen-
sitive cells. Eventually, clonal selection leads to molecular, cytogenetic and hematologic resistance and
to advanced stages of the disease. Time to relapse after first appearance of the mutation on low level
is variable and depends on the degree of resistance and the enzymatic activity of the mutated BCR-
ABL protein. Data have been reported on the reversibility of clonal selection after cessation of thera-
py with TKI. Response to second generation TKI is closely related to the specific sensitivity of the indi-
vidual mutation. Knowledge of the sensitivities of mutations to different inhibitors may allow selec-
tion of the second line therapy based on molecular data. The availability of highly sensitive methods
to detect mutation allows for modeling the dynamics of mutated clones. However, there is no prospec-
tive data suggestion that all low level mutation will eventually cause relapse. In the case of newly
detected, previously unpublished BCR-ABL sequence abnormalities, polymorphisms of ABL should be
excluded for the individual patient.

esistance to imatinib and second ge- tage of the mutated clone over clones con-

R neration tyrosine kinase inhibitors

(TKI) during the treatment of chronic
myeloid leukemia (CML) is frequently asso-
ciated with point mutations in the BCR-ABL
gene encoding the ATP binding region likely
to cause disease relapse.1 Early diagnosis and
monitoring of these mutations may be
important in order to prevent the expansion
of resistant clones. Mutations may be cate-
gorized into four groups, based upon the
crystallographic structure of cABL: (i) those
which directly impair imatinib, binding to
the catalytic domain of the oncogenic pro-
tein; (ii) those within the phosphate binding
site (“P-loop”); (iii) those within the activa-
tion loop, preventing the kinase from
achieving the inactive conformation
required for imatinib binding; (iv) those
within the catalytic domain.2
Mutation detection may aid in risk stratifi-
cation and molecular-based treatment deci-
sions. Hence, in addition to conventional
sequencing, various high sensitive methods
have been developed to screen for mutations
in case of lack of optimal response or se-
condary relapse. Examples are Denaturating
High Performance Liquid Chromatography
(D-HPLC),3-4 Ligation-PCR,5-6 sequencing of
clones,7-8 ARMS (amplification refractory
mutation system) assays9 and quantitative
mutation specific PCR assays.10 Sensitive
methods were used to determine BCR-ABL
mutations in subpopulations of cells harbo-
ring intrinsic resistance, for example, per-
sistent leukemic stem cells.11
Dynamics of clones harboring mutated
BCR-ABL depend on the selective pressure
by the TKI used and the proliferative advan-
taining unmutated BCR-ABL or normal
hematopoiesis with wild-type ABL; that is,
at least three different cell types should be
considered in any model of TKI resistance.
Dynamics of resistant clones during imatinib
therapy
In 2001, Gorre et al. described 11 patients
treated with imatinib for CML blast crisis or
Ph+ acute lymphoblastic leukemia (ALL)
who relapsed on treatment. BCR-ABL gene
amplification was detected in three patients.
Sequencing of the ATP-binding pocket and
the activation loop of the kinase domain
showed an identical cytosine to thymidine
mutation at ABL nucleotide 944 in six of
nine assessable patients. This mutation
resulted in a single amino-acid change at
position 315, designated T315I. Threonine
315 forms a crucial hydrogen bond with
imatinib and the absence of an oxygen atom
in the substituted isoleucine prevented bond
formation.7,12
However, T315I was not the only muta-
tion seen in imatinib resistant CML
patients.12-14 Up to now, more than 80
mutants15 have been described, some at a
higher frequency than others: 15 amino-acid
substitutions account for more than 85% of
the mutations, and the mutations responsi-
ble for 66% of reported cases occur at seven
sites only (G250, Y253, E255, T315, M351,
F359, H396). Furthermore, different amino-
acid substitutions can occur at the same
residue – for example, F317C or – L or – V –
and can confer different imatinib sensitivi-
ties. Certain mutations seem to occur more
often in different disease phases. For exam-

| 78 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

ple, substitutions at M244, L248, F317, H396, and S417
are more likely to occur in patients with chronic phase
(CP) disease, whereas those at Q252, Y253, E255, T315,
E459, and F486 are often associated with advanced-
phase disease.12
The systematic and regular analysis of mutations by
conventional sequencing in a cohort of 319 patients
with CML-CP (171 in early and 148 in late CP) who
were treated with imatinib revealed a 5-year cumulative
incidence of mutations of 6.6% and 17% for early and
late CP, respectively. Of the 319 patients, 214 (67%)
achieved complete cytogenetic responses (CCyR). The
identification of a mutation without other evidence of
imatinib resistance was highly predictive for loss of
CCyR and for progression to advanced phase, though
the intervals from first identification to loss of CCyR
and disease progression were relatively long (median,
21 and 16 months, respectively). Mutations in the P-
loop were associated with a higher risk of progression
than mutations elsewhere.16
To test whether kinase domain mutations are a com-
mon mechanism of disease persistence, patients in sta-
ble CCyR were studied. Mutations were demonstrated
in 8 of 42 (19%) cases. Four patients with mutations had
a concomitant rise of BCR-ABL transcript levels, two of
whom subsequently relapsed; the remaining four did
not have an increase in transcript levels and follow-up
samples, when amplifiable, showed native BCR-ABL.
Thus, BCR-ABL-kinase domain mutations in patients
with a stable CCyR are infrequent, and their detection
does not consistently predict relapse.17
Prior to hematologic or cytogenetic relapse, dynamics
of mutated clones in CML patients were investigated in
95 patients by D-HPLC and direct sequencing. In
patients harboring mutations, hematologic relapse
occurred after a median of 12.9 months (range, 0.9-
44.2), and BCR-ABL mutations first became detectable
at a median of 5.8 months (range, 0-30.5) after starting
imatinib therapy. The median interval of the first
detectability of mutations and hematologic relapse cor-
related with the degree of resistance of the mutated
clone to imatinib, expressed as the increase of the half
maximal inhibitory concentration (IC50) of imatinib to
cell lines. Nine patients showed evidence of BCR-ABL
mutations prior to imatinib therapy (T315I, n=4;
M351T, n=3; M244V and Y253H, n=1 each). The sensi-
tive detection of small numbers of mutated clones could
provide clinical benefit by triggering early therapeutic
interventions.4
Dynamics of mutations after imatinib cessation
Prior to the advent of second-generation TKI patients
with resistance to imatinib were treated with non-spe-
cific drugs, like hydroxyurea. After stopping imatinib
due to lack of efficacy in case of a BCR-ABL mutation,
absolute and relative reduction of the mutated clone has
been observed in the majority of patients.18-19 This is
explained by the proliferative advantage of clones har-
boring unmutated BCR-ABL in the absence of the selec-
tive pressure of TKI. Deselection of mutant BCR-ABL
positive clones after cessation of tyrosine kinase
inhibitor therapy is a common and reproducible phe-
nomenon. However, BCR-ABLT315I clones tend to
account for a persisting proportion of BCR-ABL expres-
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 79 |
Berlin, Germany, June 4-7, 2009
sion after cessation although decreasing in size due to a
gradually declining BCR-ABL ratio. The reduction of the
proportion of BCR-ABLT315I expression under the thre-
shold of detection might offer the therapeutic option to
resume at least temporarily a TKI treatment.19
Quantification of the mutated clone to evaluate treat-
ment efficacy should consider the total BCR-ABL load,
that is, the proportion of mutated BCR-ABL in total
BCR-ABL should be multiplied by the BCR-ABL tran-
script level.
Omacetaxine (Homoharringtonine, HHT), a cephalo-
taxine ester that has demonstrated clinical activity in
CML, inhibits protein synthesis and induces cell diffe-
rentiation and apoptosis. The absence of cross-resist-
ance between HHT and imatinib in resistant leukemic
cell lines has been demonstrated, and a possible syner-
gy between the two compounds has been observed in
vitro. HHT decreases the production of the oncogenic
kinase BCR-ABL, by inhibition of transcription and
translation in the same way in BCR-ABLT315I mutant and
in cell lines harboring native BCR-ABL. BCR-ABLT315I
may completely disappear during treatment with HHT,
even after persistence of the clone during hydroxyurea
therapy.20
In vitro models on the dynamics of mutations on second
generation inhibitors and combinations of inhibitors
Dasatinib, nilotinib and bosutinib are potent alternate
ABL inhibitors with activity against many imatinib-
resistant BCR-ABL kinase domain mutants, except
T315I. Ethyl-N-nitrosourea (ENU)-exposed Ba/F3-
p210BCR-ABL cells were used to compare incidence and
types of KD mutants emerging in the presence of ima-
tinib, dasatinib, and nilotinib, alone and in dual combi-
nations. Resistant clones were almost exclusively BCR-
ABL KD mutant at relevant concentrations of nilotinib
and dasatinib, consistent with a central role of KD
mutations for resistance to these drugs. Twenty diffe-
rent mutations were identified with imatinib, ten with
nilotinib and nine with dasatinib. At intermediate drug
levels, the spectrum narrowed to F317V and T315I for
dasatinib and Y253H, E255V, and T315I for nilotinib.
Thus, cross resistance in vitro is limited to T315I, which
is also the only mutant isolated at drug concentrations
equivalent to maximal achievable plasma trough levels.
With drug combinations, maximal suppression of resist-
ant clone outgrowth was achieved at lower concentra-
tions compared with single agents, suggesting that such
combinations may be equipotent to higher dose single
agents.21 Through saturation mutagenesis, ten BCR-ABL
mutations resistant to dasatinib were identified, eight of
which occurred at drug contact residues. Some mutants
were unique to dasatinib, whereas others also conferred
imatinib resistance. The combination of imatinib plus
dasatinib greatly reduced the recovery of drug-resistant
clones.22
To identify mutations in BCR-ABL that could result in
resistance to nilotinib, a cDNA library of BCR-ABL
mutants was introduced into Ba/F3 cells followed by
selection in nilotinib. Eighty-six individual, drug-re-
sistant colonies were recovered, and the SH3, SH2, and
kinase domains of BCR-ABL were sequenced. Forty-six
colonies had single point mutations in BCR-ABL, with
seventeen different mutations, all within the kinase
14th Congress of the European Hematology Association
domain. Each of the 17 single point mutants were
reconstructed by site directed mutagenesis of native
BCR-ABL and found to be approximately 2.5- to 800-
fold more resistant to nilotinib than native BCR-ABL.
The mutations included six known imatinib–resistant
mutations, including T315I, which showed complete
resistance to nilotinib. Most nilotinib-resistant mutants
were also resistant to imatinib.23
Indeed, results from in vitro models predicted most of
the resistance mutations that were later detected in clini-
cal trials with different kinase inhibitors. A practical
measure of the sensitivity of a given mutation to a TKI
is its IC50 for that specific inhibitor, determined either on
cellular or biochemical assays. There are two relevant
consequences of IC50 determination of ABL mutations.
First, it is possible to modify the clinical strategy in
treatment resistant patients with a kinase domain muta-
tion based on the IC50 of drugs for the mutation; that is,
choosing whether to increase the dose of imatinib or
replace it with a second generation tyrosine kinase
inhibitor. In the case of the pan-resistant T315I muta-
tion, allogeneic stem cell transplantation could be con-
sidered. Second, when finding a mutation in a resistant
patient, it is important to search for its IC50 in a muta-
tion database to link it to the clinical resistance, since
some mutations may be completely resistant to an
inhibitor whilst others are “innocent bystanders” that
accompany other mechanisms of resistance. A compre-
hensive list of the in vitro IC50 to imatinib, dasatinib, nilo-
tinib, and bosutinib was published recently.24 Such
tables, however, do not mirror the clinical activity com-
pletely, since data on the pharmacokinetics of the va-
rious inhibitors and the cellular availability are not con-
sidered.
The identification of a specific type of mutation in
relapsed patients seems to be of prognostic relevance
since the different types of mutation correlate with the
risk of evolution of relapsed patients.25 The presence
and the type of a mutation, should, therefore, help to re-
evaluate the treatment strategy, particularly now that
second generation TKI are available. Mutations with
limited sensitivities have been described in vitro and in
vivo for both dasatinib and nilotinib. Screening for muta-
tions should always be carried out in patients who fail
to respond or who have a suboptimal response to ima-
tinib, as defined by the recommendations of the
European LeukemiaNet.26
Dynamics of mutations during treatment with second
generation inhibitors
Among patients with CML-CP who developed ima-
tinib resistance after prior treatment with interferon α,
a BCR-ABL mutation was reported in 31-42% of
patients. In patients with imatinib-resistant advanced-
phase CML or Ph+ ALL, a BCR-ABL mutation was
detected in 30% to 83% of patients. Although more
than 90 different BCR-ABL mutations have been repor-
ted affecting more than 55 amino acids, 15 amino-acid
substitutions account for more than 85% of reported
mutations, and 7 amino acids (G250, Y253, E255, T315,
M351, F359, H396) are responsible for two-thirds.
Mutation types vary by disease phase; in patients with
CML-CP, the most commonly mutated amino acids
(M244, M351, G250) are different to those found in
| 80 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
advanced-phase CML (T315, E255, Y253).12 In the nilo-
tinib phase II study after imatinib resistance, among
imatinib-resistant patients, the frequency of mutations
at baseline was 55%. In patients without baseline muta-
tions, MCyR was achieved in 60%, CCyR in 40%, and
major molecular response (MMR) in 29% of patients
after 12 months of therapy versus 49%, 32%, and 22%
of patients with mutations. Responses in patients har-
boring mutations with high in vitro sensitivity to nilo-
tinib (IC50<150 nM), or mutations with unknown nilo-
tinib sensitivity, were equivalent to those for patients
without mutations. Patients with mutations that were
less sensitive to nilotinib in vitro (IC50>150 nM; Y253H,
E255V/K, F359V/C) had less favorable responses with
13%, 43%, and 18%, respectively, achieving MCyR;
none achieved CCyR.27 Similar to nilotinib, a correlation
of the in vitro IC50 (<vs>3 nM) and response to dasatinib
was observed in CP CML patients after failure to ima-
tinib. Except resistance to T315I, response was poor in
patients with F317L mutations, as predicted in vitro.28
Resistance to second generation inhibitors
Analysis of BCR-ABL genotypes in CML patients
who relapsed after sequential treatment with the ABL
inhibitors imatinib and dasatinib revealed evolving
resistant BCR-ABL kinase domain mutations in all
cases. Twelve patients relapsed with the pan-resistant
T315I mutation, whereas six patients developed novel
BCR-ABL mutations predicted to retain sensitivity to
imatinib based on in vitro studies. Three of these patients
were retreated with imatinib or nilotinib and respon-
ded; however, selection for compound mutants (2 or 3
BCR-ABL mutations in the same molecule) can substan-
tially limit the potential effectiveness of retreating
patients with inhibitors that have previously failed.
Furthermore, drug-resistant mutations, when com-
pounded, can increase oncogenic potency relative to the
component mutants in transformation assays. These
findings demonstrate the potential hazards of sequen-
tial kinase inhibitor therapy and suggest a role for a
combination of ABL kinase inhibitors to prevent the
outgrowth of cells harboring drug-resistant BCR-ABL
mutations.29 In vivo, however, the level of most muta-
tions is fluctuating individually suggesting two different
clones instead of one compound clone containing two
mutations.
In CP CML patients after dasatinib therapy, among
1,043 patients analyzed, 174 had a mutational assess-
ment at the time of dasatinib discontinuation. In these
174 patients, 54 new mutations occurred in 47 patients
(7 patients developed more than one new mutation and
42 patients had new mutations with an IC50 to dasatinib
>3 nM). Mutations at the time of dasatinib resistance
were more common in patients with versus without
mutations at the time of resistance to imatinib.28 The
expansion of a mutant Ph-positive clone that responds
initially to a second generation TKI may be due either to
late acquisition of a second mutation in the originally
mutated clone, such as the T315I, or to acquisition of a
completely new mutant clone, such as F317L.30
Gatekeeper mutation BCR-ABLT315I
The BCR-ABLT315I mutation represents a major me-
chanism of resistance to TKI. CML patients with a BCR-

ABLT315I mutation have been reported to have poor pro-
gnosis. Out of 27 patients with T315I, 20 developed
T315I after imatinib failure (representing 11% of 186
patients with imatinib failure), and 7 of 23 who devel-
oped new mutations after second TKI with a median
follow-up from mutation detection of 18 months. At
the time of T315I detection, 10 were CP, and 17 in
advanced disease. Except for the lack of response to sec-
ond TKIs, there was no difference in patient characte-
ristics and outcome between patients with T315I and
those with other or no mutations. Patients in CP had a
2-year survival rate of 87%. Although the T315I muta-
tion is resistant to currently available TKIs, survival of
patients with T315I remains mostly dependent on the
stage of the disease, with many CP patients having an
indolent course.31
The objectives of an observational study were to esti-
mate outcome for Ph+ ALL and CML patients with
T315I mutation. Median time between TKI treatment
start and T315I mutation detection was 29 months for
CP, 15 for AP, 6 for BP, and 9 for Ph+ ALL. Median OS
from T315I mutation detection was 22.4, 28.4, 4.0, and
4.9 months, and median PFS was 11.5, 22.2, 1.8, and 2.5
months, respectively, for CP, AP, BP, and Ph+ ALL
patients. These results confirm that survival of patients
harboring a T315I mutation is dependent on the disease
phase at the time of T315I mutation detection.32
ABL polymorphisms
The BCR-ABLK247R change is based on a rare single
nucleotide polymorphism (SNP) of ABL occurring like-
wise in healthy controls and non-hematologic cell
types. Despite its juxtaposition to the P-loop, functio-
nal analysis showed no alteration compared to non-
mutated BCR-ABL. In order to investigate if other
changes in the BCR-ABL kinase domain should be con-
sidered as SNP rather than acquired mutations, 911
CML patients after failure or suboptimal response to
imatinib were screened for BCR-ABL kinase domain
mutations. SNP analysis was based on the search for
nucleotide changes in corresponding normal, non-
translocated ABL alleles by ABL allele-specific PCR fol-
lowing mutation analysis. In addition to the K247R
polymorphism, five new SNPs within the BCR-ABL
kinase domain were uncovered; two of them led to
amino acid changes. SNPs could theoretically contribute
to primary but not to secondary resistance to TKI and
must, therefore, be distinguished from acquired muta-
tions. Novel point mutations should be confirmed by
analyzing the normal ABL alleles to exclude polymor-
phisms.15
Sensitive methods for mutation detection
Several methods have been used to detect the pre-
sence of kinase domain mutations in CML patients.
Some of these give a quantitative or semi-quantitative
estimation of the relative proportion of the mutated
clone, and the sensitivity in some cases is very high
(1/10,000). Direct sequencing, which is now often per-
formed using automated high-throughput instruments,
is still the most widespread method applied in the rou-
tine monitoring of patients. The main disadvantage of
this technique is its low sensitivity (around 20%).
Techniques based upon the use of allele-specific PCR
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 81 |
Berlin, Germany, June 4-7, 2009
have a higher sensitivity, which can reach 0.1%. They
are, however, directed to the search for specific muta-
tions and do not screen the entire kinase domain region
of the BCR-ABL gene. Various groups use D-HPLC as a
routine approach to screen for the presence of kinase
domain mutations, which will then be confirmed and
semi-quantified by sequence analysis. This method has
been shown to be applicable to the routine monitoring
of CML patients and to have a sensitivity of 0.5-5% in
mutation detection. Nevertheless, from a clinical stand-
point, the significance of very small subclones carrying
kinase domain mutations has yet to be demonstrated.
In contrast to CP CML, BCR-ABL mutations are com-
mon in Ph+ acute lymphoblastic leukemia (ALL) patients
at diagnosis and prior to imatinib therapy. Using D-
HPLC and allele specific PCR, Pfeifer et al. detected
BCR-ABL mutations in a minor subpopulation of
leukemic cells in 40% of newly diagnosed and imatinib-
naïve Ph+ ALL patients. At relapse, the dominant cell
clone harbored an identical mutation in 90% of cases;
the overall prevalence of mutations at relapse was 80%.
P-loop mutations predominated and were not associa-
ted with an inferior hematologic or molecular remission
rate or shorter remission duration compared with
unmutated BCR-ABL. BCR-ABL mutations conferring
high-level imatinib resistance are present in a substan-
tial proportion of patients with de novo Ph+ ALL and
eventually give rise to relapse.33
A recent study on consecutive unselected CML
patients found correlations between the pre-treatment
presence of low-level mutations and both stage of di-
sease and clonal cytogenetic evolution, but not with the
probability of response to imatinib. Even the complete-
ly resistant T315I mutant, when detected at a low level
prior to treatment, did not prove to be selected during
treatment.34 These findings are confirmed by observa-
tions on the kinetics of the mutated clones in a small
group of early and late chronic phase CML patients
showing a lack of correlation between the residual di-
sease and the presence of a predominant kinase domain
mutation.35 As a practical consequence, screening with
high sensitivity methods for low-titer kinase domain
mutations in newly diagnosed patients should be
reserved for clinical investigations, since the long term
significance of the pre-treatment low titer mutations
that may constitute a biological marker of the clone’s
instability still needs to be assessed in prospective clini-
cal trials. On the other hand, the issue of evaluating
kinase domain mutations during follow-up deserves
more attention.2
The author was supported by the German José-Carreras-
Foundation (DJCLS H 03/01).
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U, et al. Roots of clinical resistance to STI-571 cancer therapy.
Science 2001;293:2163.
15. Ernst T, Hoffmann J, Erben P, Hanfstein B, Leitner A,
Hehlmann R, et al. ABL single nucleotide polymorphisms may
masquerade as BCR-ABL mutations associated with resistance
to tyrosine kinase inhibitors in patients with chronic myeloid
leukemia. Haematologica 2008;93:1389-93.
16. Khorashad JS, de Lavallade H, Apperley JF, Milojkovic D, Reid
AG, Bua M, et al. Finding of kinase domain mutations in
patients with chronic phase chronic myeloid leukemia
responding to imatinib may identify those at high risk of di-
sease progression. J Clin Oncol 2008;26:4806-13.
17. Sherbenou DW, Wong MJ, Humayun A, McGreevey LS,
Harrell P, Yang R, et al. Mutations of the BCR-ABL-kinase
domain occur in a minority of patients with stable complete
cytogenetic response to imatinib. Leukemia 2007;21:489-93.
18. Müller MC, Lahaye T, Hochhaus A. [Resistance to tumor spe-
cific therapy with imatinib by clonal selection of mutated
cells]. Dtsch Med Wochenschr 2002;127:2205-7.
19. Hanfstein B, Mueller MC, Kreil S, Schenk T, Lorentz C,
Schwindel U, et al. Dynamics of mutant BCR-ABL positive
clones after cessation of imatinib treatment. Haematologica
2008;93:43.
20. Legros L, Hayette S, Nicolini FE, Raynaud S, Chabane K,
Magaud JP, et al. BCR-ABL(T315I) transcript disappearance in
an imatinib-resistant CML patient treated with homoharring-
tonine: a new therapeutic challenge? Leukemia 2007;21:2204-
6.
21. Bradeen HA, Eide CA, O'Hare T, Johnson KJ, Willis SG, Lee
FY, et al. Comparison of imatinib mesylate, dasatinib (BMS-
354825), and nilotinib (AMN107) in an N-ethyl-N-nitrosourea
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(ENU)-based mutagenesis screen: high efficacy of drug combi-
nations. Blood 2006;108:2332-8.
22. Burgess MR, Skaggs BJ, Shah NP, Lee FY, Sawyers CL.
Comparative analysis of two clinically active BCR-ABL kinase
inhibitors reveals the role of conformation-specific binding in
resistance. Proc Natl Acad Sci USA 2005;102:3395-400.
23. Ray A, Cowan-Jacob SW, Manley PW, Mestan J, Griffin JD.
Identification of BCR-ABL point mutations conferring resist-
ance to the Abl kinase inhibitor AMN107 (nilotinib) by a ran-
dom mutagenesis study. Blood 2007;109:5011-5.
24. Redaelli S, Piazza R, Rostagno R, Magistroni V, Perini P, Marega
M, et al. Activity of bosutinib, dasatinib, and nilotinib against
18 imatinib-resistant BCR/ABL mutants. J Clin Oncol 2009;
27:469-71.
25. Soverini S, Martinelli G, Rosti G, Bassi S, Amabile M, Poerio
A, et al. ABL mutations in late chronic phase chronic myeloid
leukemia patients with up-front cytogenetic resistance to ima-
tinib are associated with a greater likelihood of progression to
blast crisis and shorter survival: a study by the GIMEMA
Working Party on Chronic Myeloid Leukemia. J Clin Oncol
2005;23:4100-9.
26. Baccarani M, Saglio G, Goldman J, Hochhaus A, Simonsson B,
Appelbaum F, et al. Evolving concepts in the management of
chronic myeloid leukemia: recommendations from an expert
panel on behalf of the European LeukemiaNet. Blood 2006;
108:1809-20.
27. Hochhaus A, Kim DW, Martinelli G, Hughes TP, Soverini S,
Branford S, et al. Nilotinib efficacy according to baseline BCR-
ABl mutations in patients with imatinib-resistant chronic
myeloid leukemia in chronic phase (CML-CP). Blood 2008;
112:1103-4.
28. Müller MC, Cortes J, Kim DW, Druker BJ, Erben P, Pasquini R,
et al. Dasatinib efficacy in patients with chronic myeloid
leukemia in chronic phase (CML-CP) and pre-existing BCR-
ABL mutations. Blood 112:171-2.
29. Shah NP, Skaggs BJ, Branford S, Hughes TP, Nicoll JM, Pa-
quette RL, et al. Sequential ABL kinase inhibitor therapy
selects for compound drug-resistant BCR-ABL mutations with
altered oncogenic potency. J Clin Invest 2007;117:2562-9.
30. Khorashad JS, Milojkovic D, Mehta P, Anand M, Ghorashian
S, Reid AG, et al. In vivo kinetics of kinase domain mutations
in CML patients treated with dasatinib after failing imatinib.
Blood 2008;111:2378-81.
31. Jabbour E, Kantarjian H, Jones D, Breeden M, Garcia-Manero
G, O'Brien S, et al. Characteristics and outcomes of patients
with chronic myeloid leukemia and T315I mutation following
failure of imatinib mesylate therapy. Blood 2008;112:53-5.
32. Nicolini F, Martinelli G, Kim DW, Hochhaus A, Mauro MJ,
Cortes J, et al. Epidemiological study on survival of chronic
myeloid leukemia (CML) and Ph+ acute lymphoblastic
leukemia (ALL) patients with T315I mutation. Final analysis.
Blood 2008;112:76-7.
33. Pfeifer H, Wassmann B, Pavlova A, Wunderle L, Oldenburg J,
Binckebanck, A et al. Kinase domain mutations of BCR-ABL
frequently precede imatinib-based therapy and give rise to
relapse in patients with de novo Philadelphia-positive acute
lymphoblastic leukemia (Ph+ALL). Blood 2007;110:727-34.
34. Willis SG, Lange T, Demehri S, Otto S, Crossman L,
Niederwieser D, et al. High-sensitivity detection of BCR-ABL
kinase domain mutations in imatinib-naive patients: correla-
tion with clonal cytogenetic evolution but not response to
therapy. Blood 2005;106:2128-37.
35. Khorashad JS, Anand M, Marin D, Saunders S, Al Jabary T,
Iqbal A, et al. The presence of a BCR-ABL mutant allele in
CML does not always explain clinical resistance to imatinib.
Leukemia 2006;20:658-63.
 Chronic myeloid leukemia

Second generation tyrosine kinase inhibitors:

which and when?

H. Kantarjian
J. Cortes
Department of Leukemia, The
University of Texas, MD Anderson
Cancer Center, Houston, TX, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:83-88
A B S T R A C T
Patients with newly diagnosed chronic myeloid leukemia (CML) treated with imatinib have an esti-
mated 7-year survival of 86% and event-free survival of 81%. The annual failure rate on imatinib is
2-4%. New treatment options are available for these patients. Second generation tyrosine kinase
inhibitors (dasatinib, nilotinib, bosutinib) have shown significant efficacy in CML after imatinib fail-
ure, and responses appear to be durable. Although all three agents are tyrosine kinase inhibitors, they
have different biochemical structures, and spectra and mechanisms of action. The clinical significance
of these differences is not clear. As patients receive sequential therapies, some have developed failure
to two or more tyrosine kinase inhibitors or have the multi-resistant T315I mutation. Therapy in these
instances is mostly ineffective. New agents are being developed for these patients with promising
results.

matinib has revolutionized the manage- as poor as that with imatinib failure.5 The

I ment of chronic myeloid leukemia

(CML). In the IRIS trial, 82% of patients
achieved a complete cytogenetic remission
(CCyR). The estimated 7-year survival is
86% and event-free survival (EFS) 81%.1
This outcome compares favorably to the his-
torical median survival of 5 years. A subset
of patients do not have an optimal response
with imatinib. The minimum acceptable
response on imatinib therapy should be a
CCyR. In the IRIS trial, 18% of patients did
not achieve a CCyR, 11% later lost CCyR,
and 8% discontinued therapy for safety rea-
sons. Thus, approximately 30% of patients
need alternative therapies. The criteria for
diagnosing failure on imatinib therapy must
be clear and precise. The European
LeukemiaNet have provided definitions of
what constitutes failure to imatinib therapy
(Table 1).2 These definitions identify patients
with a significantly inferior outcome (medi-
an survival of 5 years).3 In this setting, a
change of therapy is clearly indicated. Early
intervention may improve outcome:
patients treated after loss of a major cytoge-
netic response (MCyR) have a significantly
better probability of achieving a CCyR
(72%) compared to those in whom alterna-
tive treatment is not initiated until loss of a
complete hematologic response (CHR)
(CCyR 42%). This translates into a signifi-
cantly better 24-month EFS with earlier
treatment (EFS 89% vs. 29%).4 The
European LeukemiaNet definitions of sub-
optimal response (less than CHR at 3
months; less than partial cytogenetic
response at 6 months; less than CCyR at 12
months; less than major molecular response
[MMR] at ≥18 months) also identify inferior
outcomes compared to those with optimal
response, although the outcome may not be
“suboptimal response” population is more
heterogenous and the management recom-
mendations are less well defined. There
have been no prospective trials comparing
increasing doses of imatinib versus changing
therapy. Here, we focus on patients with
imatinib failure.
Treatment approaches post imatinib failure
Imatinib dose escalation
Before the availability of second genera-
tion tyrosine kinase inhibitors (TKI), ima-
tinib dose escalation was a preferred option
for patients with imatinib failure. Imatinib
dose escalation to 800 mg in patients treated
with 400 mg resulted in a CCyR rate of 50%
in patients with cytogenetic failure to ima-
tinib; patients with hematologic failure have
only a 5% probability of obtaining such a
response.6 Responses are durable and the 2-
year EFS rate is 65%. Dose escalation is par-
ticularly beneficial for patients who had a
prior cytogenetic response to standard dose
imatinib. In these instances, the CCyR prob-
ability was 73% and 2-year EFS rate 74%.
The introduction of second generation TKIs
has changed the role of imatinib dose escala-
tion, which is now most frequently consid-
ered for patients with suboptimal response
to standard dose imatinib.
Second generation tyrosine kinase inhibitors
Two of these TKIs, dasatinib and nilotinib
have already been approved by regulatory
agencies around the world for this indica-
tion, and others are under development.
Dasatinib
Dasatinib is a potent Bcr-Abl tyrosine
kinase inhibitor with an IC50 of less than 1
nM. Dasatinib also inhibits other kinases,

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14th Congress of the European Hematology Association

notably the Src family of kinases (SFKs, IC50 0.2-1.1 nM),
as well as PDGFRβ (IC50 28 nM) and c-KIT (IC50 13 nM).7
Dasatinib inhibits most BCR-ABL kinase domain
mutants with the exception of T315I. Different mutants
have different levels of sensitivity to the inhibitor.8-9 A
phase I study selected a dasatinib dose of 70 mg twice
daily (BID) for phase II studies based on the short half-
life of dasatinib, and recovery of kinase inhibition 8 to
12 hours after administration. Phase II studies demon-
strated the efficacy of dasatinib in CML in all phases
post imatinib resistance or intolerance (Table 2). In a
study of 387 patients with CML in chronic phase who
were intolerant (n=99) or resistant (n=288) to imatinib,
a MCyR was achieved in 52% of patients resistant to
imatinib; it was complete in 40%. Corresponding rates
for patients with imatinib intolerance were 80% and
75%, respectively.10 The probability of MCyR was high-
er with a prior cytogenetic response to imatinib com-
pared with no prior cytogenetic response (72% vs.
42%). The progression-free survival rate was 90% after
a median follow-up of 15.2 months. A study compared
dasatinib to imatinib dose escalation among patients
who had failed prior therapy with imatinib. Patients
were randomized to dasatinib 70 gm BID (N=101) or
imatinib 400 mg BID (N=49). With a median duration of
treatment of 14 months for the dasatinib arm and 3
months for the imatinib arm (crossover allowed for lack
of MCyR at 12 weeks, intolerance, or progression), the
rates of CCyR were 30% with dasatinib and 7% with
imatinib.11 Treatment with dasatinib at this dose was
well tolerated. Common adverse events were grade 3 to
4 neutropenia (49%) and thrombocytopenia (48%).
Pleural effusions were noted in 27% but were grade 3 to
4 in only 6%.10 Increased risk of pleural effusions is
noted with hypertension, prior cardiac conditions, and a
twice daily dose schedule.12
The phase I study suggested that single daily dose
schedules and doses of 100 mg daily might be as effec-
tive at BID schedules and higher doses. A randomized
trial of four dose schedules of dasatinib was conducted:
100 mg QD, 50 mg BID, 140 mg QD, and 70 mg BID.13
This study showed that dasatinib 100 mg single daily
dose resulted in equivalent response rates and showed a
Table 1. Failure on imatinib therapy according to the
European Leukemia Net.
Months of treatment Failure response
3 No hematologic response
6 Ph-positive 100%
12 Ph-positive>35%
18 Any Ph-positivity
Any time post ≥18 months Loss of hematologic reponse or complete
cytogenetic response
decreased probability of adverse events. Grade 3 to 4
thrombocytopenia occurred in 22% of patients treated
with 100 mg QD compared to 32% with 50 mg BID and
37 to 40% with 140 mg either in a single or divided dose
schedule. The incidence of pleural effusions was 7%
with the QD schedule. This resulted in a change in the
standard dose of dasatinib in CML chronic phase to 100
mg QD. Dasatinib was effective in the advanced CML
phases, with high rates of hematologic and cytogenetic
responses.14-15 However, responses were less durable. In
advanced phase disease, a study testing 70 mg BID ver-
sus 140 mg QD showed an improved toxicity profile
with equivalent response rates, but the long-term effica-
cy has not been established.
Nilotinib
Nilotinib is a phenylamino-pyrimidine derivative
designed based on the crystal structure of imatinib in
complex with the ABL kinase.16 Nilotinib has a 20 to 30-
fold increase selectivity and improved affinity against
unmutated BCR-ABL and activity against most clinical-
ly relevant BCR-ABL mutants, with the exception of
T315I.16 Based on its relatively long half life, nilotinib
was first used with a single daily dose schedule. Dose
proportionality was observed at doses up to 400 mg
QD, but reached a plateau beyond that dose. With BID
administration, the area under the curve increased pro-
portional to doses up to 600 mg BID.17 The maximum
tolerated dose was 600 mg BID; 400 mg BID was select-
ed for phase II studies.

Table 2. Response to dasatinib, nilotinib and bosutinib after imatinib resistance.

Percent response
Response Dasatinib Nilotinib Bosutinib

Chronic Accelerated Blastic Chronic Accelerated Blastic ChronicAccelerated Blastic

N=387 n=174 n=157 n=321 N=137 N=136 N=146 N=51 N=38

Median follow-up (mo) 15 14 12+ 19 9 3 7 6 3

% Resistant 74 93 88-91 70 80 82 69 NR NR
Hematologic 79 40-50 94 56 19-22 85 54 36
CHR 91 45 27-29 76 31 11-13 81 54 36
NEL - 19 7 - 12 1 - 0 0
Cytogenetic NR 44 36-52 NR NR NR NR NR
Complete 49 32 26-46 44 20 29-32 34 27 35
Partial 11 7 7 15 12 10-16 13 20 18
% Survival (at x months) 96 (15) 82 (12) 50 (12) 88 (24) 67 (24) 42 (12) 98 (12) 60 (12) 50 (10)
NR: not reported; +Minimum follow-up. Data extracted from references 10, 14, 15, 18, 20, 22, 23, 38. Response ranges are for myeloid and lymphoid blastic phase subsets.

| 84 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

Table 3. Characteristics of second generation tyrosine kinase inhibitors.

Parameter Dasatinib Nilotinib Bosutinib

Potency (vs IM) 325 30 20-50

Target Dual Src & Abl Abl Dual Src & ABL
BCR-ABL binding Active + Inactive Inactive Intermediate
Resistant mutations T315I, T315I, E255V T315I
Mutations with intermediate sensitivity E255K/V, V299L, F317L E255K/V, Y253F/H, Q252H, F359V F317L, E255V/K
Standard dose (CP) 100 mg QD 400 mg BID 500 mg QD
Grade 3-4 neutropenia & thrombocytopenia 33%/22% 31%/33% 12%/21%
Other notable toxicities Pleural effusion, bleeding Bilirubin, lipase elevation Diarrhea, rash
C-kit inhibition (vs imatinib) Increased Similar None
PDGFR inhibition (vs imatinib) Increased Similar None
Clinical activity Highly active Highly active Highly active

In a phase II trial of nilotinib, 321 patients with CP

Table 4. In vitro sensitivity of different BCR-ABL mutants to
CML resistant or intolerant to imatinib were treated different tyrosine kinase inhibitors.
with 400 mg BID (Table 2). A CCyR and partial cytoge-
netic response (PCyR) occurred in 44% of patients and IC50-fold increase (WT=1)
15%, respectively.18 MCyR was sustained in 84% of
Imatinib Bosutinib Dasatinib Nilotinib
patients after 12 months. The 24-month survival rate
was 88%. Nilotinib was well tolerated. Common WT 1 1 1 1
adverse events were neutropenia and thrombocytope- L248V 3.54 2.97 5.11 2.80
nia, which were grade 3 or 4 in 31% and 33%, respec- G250E 6.86 4.31 4.45 4.56
tively. Other adverse events were mostly transient bio- Q252H 1.39 0.31 3.05 2.64
chemical abnormalities including hypophosphatemia, Y253F 3.58 0.96 1.58 3.23
hyperglycemia, indirect hyperbilirubinemia, and elevat- E255K 6.02 9.47 5.61 6.69
ed lipase. Only three patients developed pancreatitis. E255V 16.99 5.53 3.44 10.31
Hyperbilirubinemia occurred mostly in patients with D276G 2.18 0.60 1.44 2.00
the genotype associated with Gilbert’s syndrome.17 E279K 3.55 0.95 1.64 2.05
Nilotinib has the potential to prolong QTc, but signifi- V299L 1.54 26.10 8.65 1.34
T315I 17.50 45.42 75.03 39.41
cant prolongations (>500 msec) only occurred in three
F317L 2.60 2.42 4.46 2.22
patients.19
M351T 1.76 0.70 0.88 0.44
Nilotinib has also demonstrated activity in patients F359V 2.86 0.93 1.49 5.16
with accelerated phase20 and blastic phase (Table 2). L384M 1.28 0.47 2.21 2.33
Nilotinib is approved in CML for chronic and accelerat- H396P 2.43 0.43 1.07 2.41
ed phases after imatinib failure. H396R 3.91 0.81 1.63 3.10
G398R 0.35 1.16 0.69 0.49
Bosutinib F486S 8.10 2.31 3.04 1.85
Bosutinib (SKI-6060) is an orally bioavailable inhibitor
of Bcr-Abl and SFK. In contrast to imatinib, nilotinib and Mutations can be classified as sensitive (IC50 fold increase ≤2), resistant (between
2.01 and 10 or highly resistant (>10); T315I mutation). Data from reference 39.
dasatinib, it has little if any inhibitory activity against c-
KIT and PDGFR.21 A phase II study of bosutinib in CML
CP post imatinib resistance or intolerance is ongoing.
Among 146 patients with resistance (N=101) or intoler- differences is uncertain, (e.g., ABL configuration to
ance (N=45) to imatinib treated with bosutinib for a which the agent binds, or the inhibition of Src family of
median of only 7 months, the CCyR was 32% and kinases). Although these inhibitors are active in vitro
40%, respectively, with similar response rates for against most ABL mutants, the relative potency against
patients with and without mutations.22 Over 80% of particular mutants varies (Table 4). These differences
patients are projected to maintain their response after may be useful in selecting therapy.
24 months. Diarrhea occurred frequently but was grade Changing therapy should be considered for patients
3 to 4 in only 8%. Other toxicities included nausea, who have CML post imatinib failure as defined above.
abdominal discomfort, rash and vomiting; the only sig- An important recent question arising from the success
nificant grade 3 to 4 toxicity was a rash in 7%. of new generation TKIs is whether allogeneic SCT
Neutropenia and thrombocytopenia grade 3 to 4 should be always considered as the optimal second-line
occurred in 12% and 21% of patients, respectively. therapy post imatinib failure or whether one can rely on
Bosutinib has shown activity in advanced CML phase new generation TKI as a definitive second-line therapy,
(Table 2).23 before considering allogeneic SCT. The decision should
rely on several factors: (ii) the CML phase at the time of
Selecting second generation tyrosine kinase inhibitors imatinib failure; (ii) the patient age and source of SCT
Table 3 shows some of the differences between the (matched related, unrelated, mismatched); (iii) the pres-
different TKIs. The clinical significance of some of the ence of particular mutations; (iv) the initial response to

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14th Congress of the European Hematology Association
the new generation TKIs. In general, patients who
progress on imatinib with accelerated or blastic phase
should consider allogeneic SCT immediately, regardless
of the risk, and use new-generation TKIs as a temporary
approach to debulk CML disease, hoping to improve
outcome post allogeneic SCT; these patients should be
considered for TKI maintenance post allogeneic SCT.
Patients with imatinib failure who remain in chronic
phase may consider allogeneic SCT as second line ther-
apy if its risks are acceptable (younger age, fully
matched sibling donor). Otherwise, they may consider
new generation TKIs as a more definitive therapy and
evaluate this choice based on the particular identified
mutations, early response to the new TKIs, and the gen-
eral condition and age of the patient. Older patients (age
≥65-70 years) and those with significant commodities,
may elect to continue on the new generation TKIs and
forgo the potential of a cure with allogeneic SCT in
favor of a durable control of the disease (functional cure)
and a better quality of life. Failure to achieve a cytoge-
netic response and particular mutations with high IC50s
are associated with shorter durations of disease control.
Patients with T315I mutations (10-20% of patients post
imatinib failure; 1-3% of all patients) should consider
immediate allogeneic SCT.
Within the choice of the second-generation TKIs, the
decision is based first on the mutations detected, and
second on the patient’s history (Table 5). Patients with a
mutation with a high IC50 to one TKIs should be treated
with other alternatives (Table 5). In patients with F317L,
dasatinib and probably bosutinib are less likely to be
effective than nilotinib. In patients with Y253F muta-
tions, dasatinib and bosutinib may be better choices.
Patients with a history of pleural effusions or chronic
lung disease may not be good candidates for dasatinib.
Patients with a history of pancreatits, severe diabetes,
and QTc prolongation may not be good candidates for
nilotinib. Still, for the majority of patients there are no
clear features that can help with the decision. Evidence
of good clinical activity is available for all agents, mak-
ing them reasonable choices for most patients.
Third line therapy
With second generation TKI, nearly half of the
patients do not achieve a CCyR; others eventually lose
their response. Patients who do not achieve a MCyR by
12 months from the start of therapy have a probability
of progression of 17% over the next 12 months com-
pared to only 3% for those with MCyR by 12 months.24
Prognosis can be established even at earlier timepoints.
Among patients with no cytogenetic response at 3
months, only 7% achieved a MCyR by 12 months,
compared to 67% for those with at least a minor cyto-
genetic response. These landmarks can be used to deter-
mine when a patient can be considered to have primary
resistance to second generation TKI therapy. Treatment
choices for patients who fail two TKI are limited. Some
reports have suggested that using one more second gen-
eration TKI can be effective in some patients after fail-
ure to imatinib and one other TKI. Dasatinib used after
imatinib and nilotinib failure induced a response in 57%
among patients in all phases, although MCyR was
achieved in only 26%.25 Nilotinib after imatinib and
dasatinib failure resulted in responses.26 Unfortunately,
| 86 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Table 5. Choice of a second generation tyrosine kinase
inhibitors.
Disease characteristics
- AP/BP: favor dasatinib (?) and combinations
- chronic: see below
Mutations
- T315I → none
- nilotinib IC50 > 150 nM → avoid
- dasatinib IC > 3 nM → avoid
Patient Hx
- Hypertension, CHF, lung problems, COPD → avoid dasatinib
- Severe diabetes, pancreatitis Hx → avoid nilotinib
- QTc problems → be cautious with all (?)
responses may not be very durable, with the median
time to failure of 2-4 months for patients in advanced
stages and 20 months for patients treated in chronic
phase.27 New treatment options are needed for these
patients. Obviously patients who have failed two TKIs
should be considered for allogeneic SCT if they have a
suitable donor
XL-228
XL228 is a potent, multi-kinase inhibitor with activi-
ty against BCR-ABL, SFK FGFR1-3, the aurora kinases
(A, B and C), and insulin-like growth factor receptor 1
(IGF-1R). In vitro XL-228 inhibits T315I-mutated BCR-
ABL at nanomolar concentrations and inhibits prolifera-
tion of Ph+ CML and ALL cell lines with IC50s of less
than 100 nM. A phase I study is in progress. Preliminary
results indicate that XL228 is well tolerated with no
MTD reported to date at doses up to 10.8 mg/kg.
Importantly, hematologic and cytogenetic responses
have been reported in 7 of 35 (20%) patients who have
failed multiple prior therapies, (2 CCyR, 2 PCyR and 2
minor cytogenetic responses, and 1 return to chronic
phase). Three of the nine patients with T315I respond-
ed (1 CCyR, 1 MCyR, and 1 minor cytogenetic
response).29
PHA-739538
PHA-739538 is an aurora kinase inhibitor with signif-
icant in vitro activity against BCR-ABL, even in the pres-
ence of mutations, including T315I.30 There is synergy
between PHA-739358 and imatinib in cells that have
some remaining sensitivity to imatinib. Preliminary
reports of a phase II study in 7 patients treated (6 with
T315I), showed 2 cytogenetic response (1 CCyR, 1
minor).31
AP24534
AP24534 is a potent multikinase inhibitor with in vitro
activity against unmutated BCR-ABL (IC50 0.5 nM) as
well as several mutated isoforms (IC50 0.5-35.7 nM),
including T315I (IC50 11.4 nM). It also has significant
activity against Flt3 and c-kit among others. AP24534
has shown significant activity in animal models of CML
and AML. In a mutagenesis assay, single-agent AP24534
at concentration of 40 nM was able to suppress the
growth of colonies with mutant BCR-ABL.32 This is in
contrast with similar experiments with dasatinib or

nilotinib, where colony growth with T315I occurred
even at high concentrations. AP24534 may thus prevent
the development of resistance. A phase I study of
AP24534 in patients with CML who have failed at least
2 TKI or with T315I mutations, as well as other hema-
tologic malignancies, is ongoing.
DCC-2036
DCC-2036 is a multikinase inhibitor that binds to dis-
tinct structural pockets that the ABL kinase uses to
switch between the inactive and active confirmations
(i.e., “switch pockets”). DCC-2036 is selective for ABL,
Flt3, TIE2 and SFK. It inhibits phosphorylation of ABL
via a non-ATP competitive mechanisms, and impairs
proliferation and induces apoptosis of cells transfected
with unmutated BCR-ABL, or with a wide variety of
mutants, including T315I.33 In mice models, DCC-2036
prolonged survival of animals with BCR-ABL-induced
disease, both myeloid and lymphoid. First-in-man, dose
finding, phase I studies with DCC-2036 are starting.
Omacetaxine (Homoharringtonine)
Omacetaxine is a cephalotaxine ester with activity in
CML.34 The mechanism of action is the inhibition of
protein synthesis. In BCR-ABL cell lines, HHT reduced
the Bcr-Abl protein level and the sequential administra-
tion of omacetaxine and imatinib was synergistic in this
model.35 The effect was seen in cells with imatinib-
resistant BCR-ABL mutants, including T315I.35
Omacetaxine has clinical activity in CML post imatinib
failure.36 Omacetaxine has been used to treat patients
who have failed therapy with at least two TKIs or who
have developed T315I mutations. In an ongoing study
for patients with T315I mutations, 7 of 25 (28%)
patients with CML in chronic phase had a cytogenetic
response to omacetaxine. The mutated clone became
undetectable in 64% of patients.37
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Bruzzi J, Munden R, Cortes J et al. Pleural effusion in patients
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13. Shah NP, Kantarjian HM, Kim DW, Réa D, Dorlhiac-Llacer PE,
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ty in imatinib-resistant and -intolerant chronic-phase chronic
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14. Cortes J, Kim DW, Raffoux E, Martinelli G, Ritchie E, Roy L et
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intolerant patients with chronic myeloid leukemia in blast
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16. Weisberg E, Manley PW, Breitenstein W, Brüggen J, Cowan-
Jacob SW, Ray A et al. Characterization of AMN107, a selec-
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17. Kantarjian H, Giles F, Wunderle L, Bhalla K, O'Brien S,
Wassmann B et al. Nilotinib in imatinib-resistant CML and
Philadelphia chromosome-positive ALL. N Engl J Med 2006;
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18. Kantarjian H, Giles F, Bhalla K, et al. Nilotinib in Chronic
Myeloid Leukemia Patients in Chronic Phase (CML-CP) with
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of a Phase 2 Study. Blood 2008;112:3238.
19. Kantarjian HM, Giles F, Gattermann N, Bhalla K, Alimena G,
Palandri F et al. Nilotinib (formerly AMN107), a highly selec-
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with Philadelphia chromosome-positive chronic myelogenous
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20. Le Coutre P, Giles F, Hochhaus A, et al. Nilotinib in Chronic
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with Imatinib Resistance or Intolerance: 2-Year Follow-up
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21. Puttini M, Coluccia AM, Boschelli F, Cleris L, Marchesi E,
Donella-Deana A et al. In vitro and in vivo activity of SKI-606,
a novel Src-Abl inhibitor, against imatinib-resistant Bcr-Abl+
neoplastic cells. Cancer Res 2006;66:11314-22.
22. Cortes J, Kantarjian H, Kim D-W, et al. Efficacy and Safety of
Bosutinib (SKI-606) in Patients with Chronic Phase (CP) Ph+
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Intolerance to Imatinib Blood 2008;112:1098.
23. Gambacorti-Passerini C, Pogliani E, Baccarani M, et al. Bosu-
tinib (SKI-606) Demonstrates Clinical Activity and Is Well
Tolerated in Patients with AP and BP CML and Ph+ ALL. Blood
2008;112:1101.
24. Tam CS, Kantarjian H, Garcia-Manero G, Borthakur G,
O'Brien S, Ravandi F et al. Failure to achieve a major cytoge-
netic response by 12 months defines inadequate response in
patients receiving nilotinib or dasatinib as second or subse-
quent line therapy for chronic myeloid leukemia. Blood 2008;
112:516-8.
25. Quintas-Cardama A, Kantarjian H, Jones D, Nicaise C,
O'Brien S, Giles F et al. Dasatinib (BMS-354825) is active in
Philadelphia chromosome-positive chronic myelogenous
leukemia after imatinib and nilotinib (AMN107) therapy fail-
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ure. Blood 2007;109:497-9.
26. Giles F, Le Coutre P, Bhalla K, et al. Efficacy and Tolerability of
Nilotinib in Chronic Myeloid Leukemia Patients in Chronic
Phase (CML-CP) Who Failed Prior Imatinib and Dasatinib
Therapy: Updated Results of a Phase 2 Study. Blood 2008;
112:3234.
27. Garg RJ, Kantarjian H, O'Brien SM, et al. The Use of 2nd gen-
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TKI: Long-Term Follow-up. Blood 2008;112:2119.
28. Velev N, Cortes J, Champlin R, et al. Stem Cell Transplant
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29. Cortes J, Paquette R, Talpaz M, et al. Preliminary Clinical
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A, Pesenti E et al. Simultaneous targeting of Aurora kinases
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 Clinical trial design

Designing and interpreting quality-of-life and other

patient-reported outcomes in clinical trials

F. Efficace1
M.A. Sprangers2
1 ingly reported as important outcome measures in cancer clinical trials. While these types of outcomes
Health Outcomes Research Unit,
Italian Group for Adult Hematologic
Disease, GIMEMA Data Center,
Rome, Italy
2
Department of Medical Psychology,
Academic Medical Center, University
of Amsterdam, The Netherlands
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
A B S T R A C T
Health-related quality of life and other types of patient-reported outcomes (PRO) are being increas-
have been mainly used in studies involving patients with solid tumours, there is now increased empha-
sis in also using PRO in trials of patients with hematologic diseases. A number of potentially less toxic
drugs are now available and newer treatments can potentially offer patients with hematologic dis-
eases the possibility to be treated less aggressively, making PRO more critical in evaluating treatment
effectiveness. In addition to traditional clinical endpoints, PRO can provide valuable information to
make clinical decision-making better. However, assessing PRO in clinical trials requires careful consid-
eration of a number of methodological issues already at the protocol design stage. Robust methodol-
ogy and accurate reporting of results are crucial when evaluating PRO. This is in order to provide the
scientific community and health care providers with a clear and transparent message about the
impact of a given drug on a patient’s health status. This paper provides basic guidance to all those
involved in hematologic clinical trials, and gives real-world examples on the added value of including
PRO for a better evaluation of overall treatment effectiveness.

2009;3:89-95

n 1996, the American Society of Clinical PRO as it applies to a wider set of outcomes

I Oncology (ASCO) pointed to the key

role health-related quality-of-life
(HRQOL) assessment plays in cancer
research.1 Indeed, major research organiza-
tions routinely consider inclusion of such
outcomes in randomized controlled trials
(RCTs).2 One of the National Cancer
Institute Strategic Objectives is to ensure
the best outcome for all, including improv-
ing the “quality of life for cancer patients,
survivors and their families”.3 The World
Health Organization (WHO) also points out
that the main goal of cancer control is “a
reduction in the incidence of the disease
and of the associated morbidity and mortal-
ity, as well as improved quality of life for
cancer patients and their families”.4
Health-related quality of life and patient
reported outcomes: what to measure?
It is important to clarify what is meant by
HRQOL and Patient-Reported Outcomes
(PRO), both of which are now often report-
ed in the medical literature. Defining
HRQOL is challenging but there is now
general agreement that it refers to key
areas, including minimally physical, psy-
chological and social functioning, as well as
symptoms induced by the disease and its
treatment.5-6
More recently, the term “patient-reported
outcome” (PRO) has been introduced in the
literature to describe a broader set of
parameters self-reported by the patient.7
Here, we use the broader definition of
(including HRQOL). The US Food and Drug
Administration (FDA) define a PRO as a
“measurement of any aspect of a patient’s
health status that comes directly from the
patient (i.e., without the interpretation of
the patient’s responses by a physician or
anyone else)”.8 PRO include a wide spec-
trum of measures, ranging from single item
instruments assessing a specific health
domain (e.g., pain or fatigue) to broader
multidimensional constructs, such as
HRQOL. It is important to make such a dis-
tinction, as readers will find both terms in
the literature.
PRO are now highly valued by stakehold-
ers. The FDA recently published a docu-
ment with the main goal of providing the
medical community its view on PRO
instruments as effective endpoints in clini-
cal trials, and providing medical product
developer guidance on using PRO data used
to support claims in product labeling.8
Using PRO as an outcome measure in a
clinical trial is, in essence, the only way of
obtaining evidence-based data from the
patient’s view on the effect of a treatment.
As stated by the FDA, some “treatment
effects are known only to the patient,” and
such information can be lost when the
patient’s perspective “is filtered through a
clinician’s evaluation of the patient’s
response to clinical interview questions”.8
There is now robust evidence indicating
that PRO, for example, are independent
prognostic factors of survival beyond and

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 89 |
14th Congress of the European Hematology Association

above clinical and laboratory data in patients with
advanced diseases including those with hematological
malignancies.9-11
What is the added-value of measuring PRO in hematologi-
cal clinical trials?
Several RCTs in patients with solid tumors have suc-
cessfully implemented PRO and have provided addi-
tional robust data to understand better the overall
treatment effectiveness from the patients’ perspective.
Some of these data have also served as a basis for drug
approval (or in support of) by the FDA.12-14
The number of trials conducted in patients with
hematological diseases is substantially lower. There
are several reasons to explain this gap.15-17 Recently,
however, much emphasis is being placed on PRO
issues in hematological research. For example, recent
international recommendations for various hematolog-
ic diseases (including acute leukemia, chronic lympho-
cytic leukemia, immune thrombocytopenic purpura
and myelodysplastic syndromes) are paying greater
attention to HRQOL issues and are advocating more
research into this area.18-22
Although little research has been conducted in
hematology, and the number of studies has only start-
ed to increase over the last few years, PRO assessment
has been shown to be feasible and of value in provid-
ing unique information. Table 1 provides a brief sum-
mary of the key findings related to the added-value of
measuring PRO in selected RCTs of three major hema-
tological disease sites.
Chronic lymphocytic leukemia
Eichhorst and colleagues23 recently conducted a large
RCT comparing Fludarabine (F) versus F plus
cyclophosphamide (FC) as first line therapy in younger

Table 1. Selected prospective RCTs, including PRO in patients with leukemia and myelodysplastic syndromes. (Adapted
from Efficace et al.17 and Caocci et al.53)

Authors Year of Overall no. of Disease Treatment PRO PRO measures Summary of traditional Summary of PROs
publication patients outline endpoint used clinical outcomes

Eichhorst et al.23,24 2007 375 CLL Fludarabine (F) Secondary EORTC QLQ-C30 F plus C is more effective Overall, HRQOL
with or without than F alone as first was comparable
Cyclophosphamide (C) line therapy for CLL patients between treatment arms
in terms of: complete remission;
overall response; PFS;
treatment-free survival time
Catovsky et al. 25 2007 777 CLL Fludarabine versus Secondary EORTC QLQ-C30 Better complete and Overall, HRQOL was
Fludarabine plus overall response rates, comparable amongst
Cyclophosphamide versus as well as progression-free treatment arms
Chlorambucil survival at 5 years for patients
with Fludarabine plus
Cyclophosphamide than Fludarabine
and better than with Chlorambucil
Hahn et al.27 2003 1,106 CML Imatinib (IM) versus Secondary FACT-BRM; IM significantly superior The Trial Outcome
and O’Brien26 interferon (IFN) -alfa plus Euro QoL-5D to IFN-alfa plus Ara-C Index of the FACT-BRM
low dose cytarabine in terms of CHR, MCR, was much higher in the IM
CCR, FFP, toxicity Group throughout
all study period
Kornblith et al. 29 2002 191 MDS Azacitidine (AZA) Secondary EORTC QLQ- C30; AZA yielded a higher Patients on the AZA
and Silverman et al.28 versus supportive care Mental health inventory; response rate, arm experienced
patient’s perception of reduced risk of leukemic significantly greater
improvement transformation and improvement in fatigue,
improved survival physical functioning,
positive affect, psychological
distress.Also patients
who crossed over from the
supportive care arm to the
azacitidine arm had
significantly improved QoL
Kantarijan et al.30 2006 170 MDS Decitabine plus Secondary EORTC QLQ-C30 Higher overall response Decitabine yielded
supportive care versus rate and longer median statistically better
supportive care alone time trend to AML in patients results for global health
treated with decitabine compared status, fatigue, and
to those on supportive care dyspnea in comparison
with supportive care
FACT-BRM: functional assessment of cancer therapy-biologic response modifiers; EORTC QLQ-C30: european organization for research and treatment of cancer quality
of life questionnaire; CML: chronic myeloid leukemia; CLL: chronic lymphocytic leukemia; MDS: myelodysplastic syndromes.

| 90 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

patients with CLL showing better response and pro-
gression-free survival for the FC arm. Nevertheless,
toxicity did not favor the FC arm, showing worse data,
mainly in terms of leukocytopenia and myelotoxicty.
In this trial, HRQOL was used as a secondary end-
point, and the results obtained were of particular value
in understanding that worse toxicity did not translate
into worse HRQOL of patients treated in the FC arm.
The full analysis of HRQOL over time between the
two treatment arms showed no significant differences,
allowing the conclusion that the beneficial clinical
effects of FC was not obtained at the expense of detri-
mental effects on patient’s HRQOL.24 Such informa-
tion could have been lost if this outcome had not been
included in the original trial. Catovsky and colleagues25
compared F versus FC versus Chlorambucil and includ-
ed HRQOL as a secondary endpoint in the trial. While
FC arm patients reported better clinical outcomes, a
preliminary analysis showed that HRQOL was gener-
ally comparable among treatment arms. The authors
explicitly mentioned that a full HRQOL report would
follow, thus providing additional details on this analy-
sis. However, preliminary HRQOL data of this trial
lent additional support that FC treatment in CLL
patients can be obtained with no major impact on
patient’s HRQOL when compared to other standard
treatments.
Chronic myeloid leukemia
O’Brien and colleagues26 included HRQOL as a sec-
ondary endpoint in a trial comparing standard inter-
feron treatment with Imatinib (IRIS study), with the
main goal of providing evidence-based data of the
possible benefits in terms of HRQOL for the newer
treatment. When the full HRQOL analysis was pub-
lished in a separate report by Hahn and colleagues,27 it
was clear that the use of Imatinib not only provided
superior traditional outcomes over the interferon-
based treatment, but also had greater beneficial effects
in a number of HRQOL areas. The effect size found
for the difference in HRQOL outcomes between treat-
ment arms was one of the largest ever shown in any
previous HRQOL trial-based report.27 Interestingly,
PRO data of this study were also used to support FDA
approval of Imatinib for use in newly diagnosed,
chronic phase CML.12
Myelodysplastic syndromes
Silverman and colleagues28 included HRQOL as a
secondary endpoint in a large trial comparing azaciti-
dine (AZA) versus supportive care, demonstrating that
treatment with AZA provided a delayed time to dis-
ease progression and death. A separate HRQOL paper
was also published by Kornblith and colleagues,29 indi-
cating that treatment with AZA also provided benefi-
cial effects in terms of reduced fatigue, physical func-
tioning, positive effect and psychological distress.
Another RCT compared decitabine plus supportive
care versus supportive care alone,30 led to the conclu-
sions that various HRQOL aspects can be improved by
using decitabine in addition to supportive care.
However, this trial reported few details on the design
of HRQOL assessment and related outcomes, thus it is
currently challenging to draw definitive conclusions
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 91 |
Berlin, Germany, June 4-7, 2009
about the benefit of decitabine on patient’s HRQOL.
Overall, these studies provide good examples of
how PRO implementation in a RCT setting is feasible
and can augment investigators’ knowledge of treat-
ment effectiveness.
Practical considerations in designing and reporting PRO
in clinical trials
Information about side effects, symptoms and treat-
ment options are important to cancer patients as they
enable them to make informed treatment decisions.
Cancer patients require information related to survival
estimates, as well as HRQOL issues.31 Providing
patients with such evidence-based information is,
therefore, of paramount importance. Thus, including
PRO as an effectiveness endpoint in a clinical trial set-
ting could potentially provide invaluable information
related to treatment side effects from the patients’ per-
spectives.
Measuring PRO should always follow the same rigor
as applied to measuring any other traditional clinical
endpoint in clinical research. In phase I trials, PRO is
not a relevant outcome measure but, in some phase II
studies, measuring the patient’s perspective can be of
value (e.g., in hypothesis generating for future phase III
studies). In phase III studies, however, inclusion of
PRO can always be of great value and should be con-
sidered as a possible study outcome. There is also gen-
eral agreement that PRO inclusion is relevant in virtu-
ally all symptom management trials.32 The following
paragraphs mainly address issues relevant in phase III
studies. If PRO are to fulfill their potential of allowing
health-care providers to make informed decisions
about the overall value and impact of a given treat-
ment, investigators should pay careful attention to a
number of methodological issues. Actually, evaluating
PRO in clinical trials requires making a number of chal-
lenging decisions. It is fair to mention that previous
work investigating the quality of PRO assessment in
oncology over the last 20 years have found a number
of methodological drawbacks that, in many cases,
have hampered a critical appraisal of results.33-36 An edi-
torial which appeared in May 2002 in the official jour-
nal of the ASCO37 stated that it is “disappointing” that,
despite the fact that thousands of patients enrolled in
cancer clinical trials with a PRO component, “there are
relatively few examples of formal quality of life meas-
urement that have influenced individual patient deci-
sion-making or treatment policies.” However, a signif-
icant learning curve, in terms of accuracy of PRO
reporting since 1990, has been recently demonstrated
in cancer clinical trials conducted in major solid
tumors,38 thus supporting the hypothesis that results
from recent studies are more likely to support clinical
decision-making than earlier published studies.
Some administrative and methodological decisions
should be taken already at the time of protocol writ-
ing, while others are mainly relevant when reporting
and disseminating PRO results.
At the stage of protocol writing
PRO can either be used as a secondary or primary
endpoint in a given trial but, in both instances, they
should be an integral part of the research protocol,
14th Congress of the European Hematology Association
with a number of issues well addressed and planned at
this early stage. The first step is formulating a ration-
ale for PRO assessment in the particular trial and spec-
ifying what this type of outcome would add to the pri-
mary endpoint of the study (in case PRO is planned as
a secondary endpoint). Selecting the most appropriate
instrument (e.g., questionnaire) for the particular trial
deserves attention and must be justified in the proto-
col. Selecting the “right” questionnaire is a fundamen-
tal step in designing and conducting a PRO assessment
and needs careful evaluation of various aspects.
Questions need to be asked, such as: is the content of
the questionnaire appropriate to the research ques-
tion? Does the questionnaire yield robust psychomet-
ric properties, in terms of validity, reliability and
responsiveness? How interpretable are the scores? Is
the questionnaire easy to administer? Besides these,
the cultural validity of the questionnaire has also to be
verified, in case it has not been validated for the same
population under study. The choice of instruments is
analogous to choosing laboratory tests, that is, it is
important to choose the most appropriate and sensi-
tive (laboratory) test for the purpose at hand.7 Detailed
guidelines on how to make this choice have been pub-
lished and will assist investigators when designing
future studies.39
Nevertheless, the choice of instrument is only one
aspect of the multi-faceted process of obtaining valid
and reliable data in a clinical trial. Another relevant
issue is the problem of missing data. Difficulties with
data collection and compliance have historically been
considered the major barriers to the successful imple-
mentation of PRO in clinical trials.40 PRO data are col-
lected at different time points during the course of the
study and missing data (i.e., questionnaires not com-
pleted and/or returned) from different scheduled
assessments are unavoidable, mainly, due to patients’
health conditions and/or administrative failures.
However, as lost data might not be missing at random,
they cannot be ignored without introducing bias in
outcome interpretation. If the pattern of missing data
is systematically different between treatment arms,
such information should alert investigators in further
exploring this issue and check, for example, if this
could be associated with the specific treatment (e.g.,
more toxic) of that trial arm. Given the importance of
the representativeness of the data of the trial popula-
tion, investigators are recommended to take actions to
minimize the number of missing data during the
study.40 A number of steps can be taken, including:40-42
- the collection of PRO data should not be presented
as an “optional” part of the study rather as an inte-
gral and mandatory part of the trial;
- avoid a PRO assessment including an unnecessary
large number of items. Clearly, the number of items
needs to be evaluated in light of the target popula-
tion (e.g., patient’s age and disease), frequency of
assessments and resources available. For example,
older and more ill patients, a study design with
many assessments, and a study with few resources,
would require a brief questionnaire;
- provide brief guidelines to all investigators stressing
the importance of PRO data collection and giving
practical and brief indication for PRO administration
| 92 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
to patients (e.g., when and how);
- identify one responsible person for PRO data collec-
tion in each participating institution to facilitate liai-
son with the coordinating center;
- provide regular feedback about study progress on
compliance to study co-investigators.
It is worthy of note, that, on the basis of these rec-
ommendations, recently conducted trials obtained a
good level of compliance, also in patients with
advanced hematological malignancies.10
Another issue to be addressed during protocol devel-
opment is the a priori definition of what constitutes a
“minimally important difference” (MID) in the PRO
measure of interest, and thus, to address clinical signif-
icance of outcomes. Using PRO poses unique problems
inherent to their subjective nature. For example, what
is the meaning of a given statistically significant differ-
ence in terms of HRQOL from a patient’s perspective?
Does a statistically significant difference in a HRQOL
domain between treatment arms necessarily reflect a
subjectively meaningful difference perceived by
patients? The challenging issue is how to evaluate “a
tangible benefit (improvement in health) with an
intangible construct (HRQOL)”.43 Since statistical sig-
nificance is dependent on sample size, it is possible
that a 2 to 3 unit change on a 0 to 100 PRO scale,
results in a significant p-value if the results were based
on a large sample. Most likely, this finding would not
be clinically meaningful. As a comprehensive discus-
sion on assessing clinical significance is beyond the
scope of this chapter, we would like to highlight that a
number of methods are available, which can be divid-
ed into anchor-based and distribution-based approach-
es.44-45 To illustrate, previous work has shown that the
MID for one of the most widely used cancer-specific
questionnaires, the EORTC QLQ-C30, equals a 10-
point shift on its 0-100 response scale.46
For further details on the main steps to be taken into
consideration at an early stage of protocol writing to
ensure a successful PRO implementation, we refer the
reader to other relevant documents in this area.42,47-48
At the stage of reporting and disseminating PRO findings
Some issues need to be addressed, particularly at the
time of disclosing and disseminating PRO findings to
ensure consistency of reporting. First, when collecting
PRO data in a trial, the amount of information would
be better published also in a separate report from the
main clinical findings. In this scenario of disclosing
PRO (when this has been used as a secondary endpoint
of a trial), the recommendation would be to include
the major PRO results in the main original paper, and
additionally publish a separate paper providing full
details of PRO analysis; thus, allowing the medical
community a critical appraisal of the results.
In Table 2, we report a number of practical basic
issues that investigators should consider when report-
ing PRO assessment in clinical trials. We do not intend
to provide a comprehensive list of all detailed issues
that need to be ideally reported, rather we provide a
brief pragmatic guide on the main topics deserving
attention when reporting PRO results. These issues are
taken from the Minimum standard checklist for evaluating
HRQOL outcomes in cancer clinical trials that has been
 Berlin, Germany, June 4-7, 2009

Table 2. Minimum standard criteria for PRO reporting in clinical trials. (Adapted from Efficace et al.49)

Items related to the conceptual design

A priori hypothesis stated

(Do the authors have a pre-defined PRO endpoint and/or stated expected changes due to the specific treatment?) Yes r No r N/A r†
Rationale for instrument reported
(Do the authors report a rationale for selecting a specific PRO measure?) Yes r No r

Items related to the Measurement

Psychometric properties reported*

(Assessed if a previously validated measure was used or psychometric properties were reported or referenced in the paper) Yes r No r
Cultural validity verified
(Assessed if the measure was validated for the specific study population) Yes r No r N/A r‡
Adequacy of domains covered
(Assessed if the measure covered, at least, the main PRO dimensions relevant for a generic cancer population and/or
according to the specific research question) Yes r No r

Items related to the methodology

Instrument administration reported

(Do the authors report who and/or in which clinical setting the PRO instrument was administered?) Yes r No r
Baseline compliance reported*
(Do the authors report the number of patients by treatment arms providing the initial PRO assessment?) Yes r No r
Timing of assessments documented
(Do the authors report the PRO timing of assessment during the trial?) Yes r No r
Missing data documented*
(Do the authors report the extent of PRO missing data during the trial between treatment arms?) Yes r No r

Items related to the Interpretation of outcomes

Clinical significance addressed

(Do the authors discuss PRO data being clinically significant from a patient’s perspective and not simply statistically significant?) Yes r No r
Presentation of results in general
(Do the authors discuss PRO findings giving any comment regardless of the results) Yes r No r
Each item can be scored as ‘yes’ (giving a score of 1) or ‘no’ (giving a score of 0), with higher sum scores indicating more robustness of the outcomes. * Although some
items might have different importance according to the specific study questions, these basic items should always be documented in the report; †if a study explicitly states an
exploratory PRO evaluation; ‡ if the PRO measure is validated in the same population as the one of the trial.

previously developed based on good practice in report-
ing HRQOL studies and was published in the Journal of
Clinical Oncology in 2003.49 It was purposely devised to
help both investigators and readers in general. This
checklist is primarily intended for reviewing and facil-
itating a critical appraisal and interpretation of PRO
reports and for guiding investigators when designing,
and most importantly, writing PRO reports from a clin-
ical trial. The items are devised to have a dichotomous
answer (yes or no), and each can be scored as “yes”
(giving a score of 1) or “no” (giving a score of 0), with
higher sum scores indicating more robustness of the
outcomes. This tool is of pragmatic use, and has been
adopted in several studies to evaluate consistency and
level of reporting. It has been shown to be sensitive in
picking up differences in quality reporting over
time.38,50-52 It is important to note that good reports may
have different emphases and some issues might have
different importance according to the specific study
questions. For example, reporting “timing of assess-
ment” and possibly time windows for PRO assessment
is very important in studies adopting a “time-based
design,” where the exact timing of assessments is
defined since baseline, for example, every second
week since baseline. Conversely, timing of assess-
ments may be less critical in an “event-based design,”
where the assessments are defined in relationship to
events, for example, at the end of each chemotherapy
cycle, or following relapse and/or recurrence. Finally, it
is worth noting that the investigator should use this
checklist as a flexible and basic tool, putting more
emphasis on various items on a case-by-case basis.
Conclusions
In conclusion, robust methodology and accurate
reporting of data is crucial when evaluating PRO in
clinical trials in order to provide the scientific commu-
nity and health care providers with a clear and trans-
parent message about the impact of a given drug on
the patient’s health status. Caution is required when
interpreting PRO data. Publications in this area stem-
ming from a poor study design or simply reporting
inadequate information can potentially mislead read-
ers when interpreting study outcomes; educational
efforts in this area are very important. By providing
basic guidance on the main issues deserving attention

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 93 |
14th Congress of the European Hematology Association
when interpreting the robustness of studies in this
area, clinicians (and readers in general) will be able to
evaluate PRO results critically. It is our hope that such
practical guidelines will stimulate stringent and robust
PRO-based RCTs in hematologic cancer.
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National Cancer Institute
Bethesda, Maryland 20892-7434,
USA
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the education program for the
annual congress of the European
Hematology Association
2009;3:96-100
| 96 |
Clinical trial design
The use of genomics in clinical trial design
Importance of prognostic and predictive
classifiers
Genomics offers great opportunities to
improve cancer therapeutics by identifying
key molecular targets and by selecting the
right treatment for the right patient. In this
paper, we shall focus on the latter objective.
Current practice in oncology involves treat-
ing many patients who do not benefit for
each one who does. For example, patients
with an early stage solid tumor may have an
80% or better long-term disease free survival
with local therapy. There would often be
substantial interest in identifying a
chemotherapy regimen that increased the
chance from 80-85%. This would represent
a 25% relative reduction in the hazard of
recurrence. However, 80% of the patients
do not require systemic therapy, and the
absolute improvement of 5 percentage
points results in treating 20 patients for each
one who benefits.
Because cancer is a life threatening dis-
ease, treating 20 patients to benefit 1 is
acceptable, if the benefit is sufficiently great.
However, it would be better for the patients
if we could prospectively identify those
patients whose prognosis with local therapy
alone is sufficiently good that they do not
require systemic therapy. The OncotypeDx
and Mammaprint tests are examples of com-
mercially available prognostic classifiers for
patients with early stage breast cancer.1-2
It would also be useful to be able to classi-
fy patients accurately whose prognosis with
local therapy is not as good into those who
are and those who are not likely to benefit
from a specific therapeutic regimen. Such a
treatment specific test is generally called a
predictive classifier. This is not an accurate use
of language, as prognostic classifiers are used
for predicting outcome, but it is well estab-
lished to reserve the phrase predictive classi-
fier or predictive marker for those tests asso-
ciated with predicting benefit for a specific
therapy.
Useful prognostic classifiers are them-
selves treatment specific, but not in quite the
same way as predictive classifiers. The
OncotypeDx prognostic classifier was devel-
oped for patients with axillary node nega-
tive, estrogen receptor positive breast cancer
who received local therapy plus tamoxifen.
It does not identify the patients whose
tumors respond to tamoxifen. It identifies
the node negative ER positive patients who
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
have good outcome whether or not they
needed or benefited from tamoxifen.
Developing a prognostic classifier using a
group of patients that are homogeneous
with regard to both treatment and the prog-
nostic factors that are incorporated into cur-
rent practice standards is very important for
ensuring that the classifier has potential
medical utility to aid in treatment selection.
We use the term classifier to indicate any
categorization of tumors based one or more
biological measurements. If there are two
categories, such as good prognosis or poor
prognosis, then the classifier is called a bina-
ry classifier. A binary predictive classifier
would categorize patients into those who
are likely benefit from the specified thera-
peutic versus those less likely to benefit. A
prognostic or predictive index provides a grad-
ed or continuous value based on one or more
biological measurements. For example, both
OncotypeDx and Mammaprint tests are
prognostic indices. For purposes of clinical
trial design or patient management, cut-
points are often introduced to convert a
prognostic index to a prognostic classifier.
Prognostic or predictive classifiers or
indices may be based on single genes, single
proteins, or on expression levels of many
genes. For example, OncotypeDx is based
on expression of 21 genes. In order to have a
classifier or index, however, it is essential
that the multiple components be combined
in a completely defined manner. Many gene
expression indices are weighted averages of
expression levels of multiple genes with the
weights explicitly defined. In order to use a
classifier for new patients, it is not sufficient
just to have a set of genes. The patient can-
not be classified unless the test defines how
the expression levels are to be combined and
what cut-points to use for grouping the
index values into prognostic categories.
Many publications reporting the develop-
ment of prognostic gene expression signa-
tures provide only a set of genes, not a well-
defined classifier that can be tested on inde-
pendent data.
Although gene expression profiling is
actively used to develop prognostic classi-
fiers, much new drug development is based
on single gene predictive biomarkers.
Estrogen receptor expression is a classic pre-
dictive biomarker for effectiveness of
tamoxifen in breast cancer. Her2 amplifica-
tion is an important predictive biomarker for

Table 1. Definitions.
Term
Biomarker
Predictive biomarker
Prognostic biomarker
Prognostic or predictive classifier
Prognostic or predictive index
Gold standard
Analytical validation
Clinical validation/correlation
Medical/Clinical utility
Enrichment design
effectiveness of trastuzumab. In fact, over-expression of
the Her2 protein was critical to the development of
trastuzumab in patients with metastatic breast cancer. If
the phase III clinical trial used for establishing the effec-
tiveness of trastuzumab in metastatic breast cancer had
not used Her2 testing to focus the analysis on patients
whose tumors over-expressed Her2, the effectiveness of
the antibody would almost surely not have been estab-
lished.3 This is because only about 25% of patients have
tumors that over-express Her2 and the benefit of
trastuzumab in those patients would have been diluted
and obscured by the lack of effectiveness of the anti-
body in the remaining 75% of patients in an overall
analysis.
Trastuzumab had a very low response rate in
metastatic breast cancer. Had the phase II studies not
separately evaluated patients whose tumors over-
expressed Her2, the phase III trial would probably never
have been initiated. But comparing response rates to
trastuzumab for patients whose tumors are Her2 posi-
tive versus those whose tumors are Her2 negative does
not establish the medical utility of trastuzumab. Since
partial tumor response is not an indication of patient
benefit or drug approval in metastatic breast cancer,
comparing response rates between Her2 negative and
Her2 positive patients cannot establish the medical util-
ity of the therapeutic. Comparing survivals between
Her2 positive patients who receive trastuzumab and
Her2 negative patients who receive trastuzumab would
also not establish medical utility of trastuzumab. It
would only establish that Her2 testing is prognostic for
survival in patients treated with trastuzumab. That in
itself would be of no real medical value.
To establish the medical utility of trastuzumab
required a randomized clinical trial with a control group
of patients not receiving trastuzumab. The randomized
trial that led to the FDA approval of trastuzumab in
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 97 |
Berlin, Germany, June 4-7, 2009
Definition
Biological measurement
Biomarker measured before treatment to identify patients
likely to benefit from a specific treatment
Biomarker measured before treatment to provide information about
long-term outcome; often incorporates tumor factors and treatment sensitivity
Algorithm that provides a category (e.g., high vs low)
prediction based on one or more biological measurements
Algorithm that provides a score based on several biological measurements
Biological measurement of perfect accuracy
Establishing reproducibility and robustness of test.
If gold standard exists, establishing accuracy of test for measuring biological target
Establishing accuracy of test for predicting clinical outcome
Establishing that use of the test enables better treatment decisions to be made
that result in improved patient outcome
RCT comparing new treatment to control restricting entry to patients most likely (or
least unlikely) to benefit from the new treatment compared to the control
metastatic breast cancer restricted entry to patients
whose tumors over-expressed the Her2 protein.3
Restricting entry in that way was important for several
reasons. It meant that the primary analysis was prospec-
tively defined as evaluation of trastuzumab plus
chemotherapy versus chemotherapy alone in patients
whose tumors over-expressed Her2. Restricting entry
based on Her2 made it clear that the analysis was not
the result of post-hoc data dredging. It also meant that
the trial was sized to have adequate statistical power for
the evaluation of trastuzumab versus control in Her2
positive patients.
Today, there appears to be greater resistance by regu-
lators to clinical trials that restrict patient entry based on
a putative predictive biomarker. This is based on con-
cern that the therapeutic might also benefit test negative
patients. Sometimes the credentials of the predictive
biomarker are based on the understanding of the biolo-
gy of the therapeutic target and data from pre-clinical
models. Understanding mechanisms is often imperfect
or seriously flawed, and regulators would often prefer
to see human data that establish the credentials of the
predictive classifier. If the pre-clinical evidence is not
compelling, it is often possible to include test negative
patients at least in phase II studies of the therapeutic. It
may be important to include such patients in phase II
studies to establish a cut-point for the test. Showing
that there is a gradation in response rate with test result
in phase II studies may support exclusion of test nega-
tive patients from the phase III trial. However, there
may not be sufficient phase II data to justify excluding
test negative patients from the phase III trial. In some
cases, however, the pre-clinical data will be compelling
and it will not be satisfactory to include test negative
patients in phase III trials in order to demonstrate that
the drug is ineffective for them.
14th Congress of the European Hematology Association
Validation of prognostic and predictive tests
The term validation has several meanings for prognos-
tic and predictive classifiers.
Analytical validation
First is the analytical validation of an assay for meas-
uring the classifier. Analytical validation has traditional-
ly meant that the assay accurately measures what it
claims to measure. This presumes, however, the exis-
tence of a gold standard way of measuring the true
value. For gene expression based predictive classifiers,
there is usually no gold standard. Whereas an RT-PCR
assay might be accepted as a gold standard for measuring
gene expression of an RNA sample, this ignores ques-
tions of the representativeness of the RNA sample for
the target tissue prior to biopsy. For assays in which
there is no gold standard, analytical validation generally
means reproducibility of the assay and robustness to tis-
sue handling and laboratory variations. Careful develop-
ment of an analytically validated assay is important for
all later steps of validation.
Clinical validation/correlation
Most reports describing the development of a predic-
tive classifier based on gene expression address clinical
correlation, which is sometimes called clinical validation.
Clinical validation involves evaluating the degree of
accuracy of the classifier for predicting some clinical
outcome. For classifiers developed from microarray
gene expression data, it is essential to separate the cases
used for developing the classifier from the cases used for
evaluating the classifier. With traditional regression
modeling, the number of candidate variables is much
less than the number of cases and separation of training
and validation cases is often not practiced. Failure to
observe this key separation principle with microarray
based classifiers, however, results in enormous bias in
the resulting estimate of accuracy.4
The most straightforward way of preserving the key
separation principle is the split-sample method of parti-
tioning the set of samples into a training set and a test
set. Cross-validation is an alternative to the split sample
method of estimating prediction accuracy5 while pre-
serving the cardinal separation principle. Molinaro et al.6
describe and evaluate many variants of cross-validation
and bootstrap re-sampling for classification problems
where the number of candidate predictors vastly
exceeds the number of cases. The cross-validated pre-
diction error is an estimate of the prediction error asso-
ciated with application of the algorithm for model
building to the entire dataset. The model building
process must be repeated from scratch for each loop of
the cross-validation and so the process must be com-
pletely algorithmic. Simon et al.4 showed that cross-val-
idating the fitting of the prediction model after selection
of differentially expressed genes from the full data set
results in a highly biased estimate of prediction accura-
cy. This is one of the most common and most serious
errors made in using cross-validation.7
Medical utility
The third level of validation of a predictive classifier is
that of determining whether the classifier has medical
utility. A classifier generally has medical utility only if it
| 98 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
enables physicians to make better treatment decisions.
Many classifiers are developed using convenience sam-
ples of available specimens not selected for purposes of
addressing a question of medical decision-making. For
such classifiers there may be an insurmountable gap
between clinical correlation and medical utility.
Clinical trials for establishing the medical utility of prog-
nostic classifiers
A prognostic classifier can have medical utility if it
enables physicians to withhold a debilitating or expen-
sive treatment from patients whose prognosis is suffi-
ciently good with local therapy alone or a less intensive
standard approach. To evaluate utility of such a classifi-
er one would screen eligible patients using the prognos-
tic classifier and register patients whose prognostic cat-
egory was sufficiently favorable, using a prospectively
defined cutoff. The therapy being addressed would be
withheld from the registered patients. If the long-term
outcome of the registered patients is sufficiently good,
the prognostic classifier is considered to have medical
utility. This is the design being used in the TAILORx
clinical trial for prospective validation of OncotypeDx.8
The MINDACT clinical trial randomizes patients pre-
dicted to be good risk by Mammaprint but for whom
the standard of care involves chemotherapy between
withholding or not-withholding chemotherapy.
However, the primary analysis and sample size for
MINDACT is not based on the randomized compari-
son. As in TAILORx, it is based on evaluating the long-
term disease-free survival of good prognosis category
patients for whom chemotherapy has been withheld.9
The effect of chemotherapy is expected to be very small
and the primary analysis is one of evaluating the test as
a prognostic classifier in the absence of chemotherapy.
Some people consider the gold standard evaluation of
medical utility of a new test to involve randomizing
patients to treatment determination based on practice
standards or based on the test. The test is considered to
have medical utility if treatment outcome is improved
overall for the group randomized to test based treat-
ment assignment. The test would also be considered to
have medical utility if outcome is the same for the two
randomized groups but the patients randomized to test
determined treatment have reduced adverse events,
inconvenience or expense. This kind of prospective clin-
ical trial design is very inefficient and rarely practical. It
generally requires an enormous sample size because
many or most patients in both randomization groups
receive the same treatment.10-11 It is generally better to
screen all relevant patients using the test and to evaluate
the implications of acting or not acting on the test in
subsets of patients defined by the test and current prac-
tice standards.
Clinical trials for establishing the medical utility of predic-
tive classifiers
Simon et al.12-14 discussed prospective clinical trial
designs for the co-development of new drugs and com-
panion diagnostics. The objective of a phase III pivotal
clinical trial is to evaluate whether a new drug, given in
a defined manner, has medical utility for a defined set of
patients. Pivotal trials test pre-specified hypotheses
about treatment effectiveness in specified patient popu-

lation groups. The role of a predictive biomarker classi-
fier is to specify the population of patients. The process
of classifier development may be exploratory and sub-
jective, but the use of the classifier in the pivotal trial
must not be.
With an enrichment design, a diagnostic test is used
to restrict eligibility for a randomized clinical trial of a
regimen containing a new drug to a control regimen.15-17
This approach was used for the development of
trastuzumab in which patients with metastatic breast
cancer whose tumors expressed HER2 in an immuno-
histochemistry test were eligible for randomization.3
Simon and Maitournam13,18-19 studied the efficiency of
this approach relative to the standard approach of ran-
domizing all patients without measuring the diagnostic.
They found that the efficiency of the enrichment design
depended on the prevalence of test positive patients and
on the effectiveness of the new treatment in test nega-
tive patients. When fewer than half of the patients are
test positive and the new treatment is relatively ineffec-
tive in test negative patients, the number of randomized
patients required for an enrichment design is often dra-
matically smaller than the number of randomized
patients required for a standard design. Zhao and Simon
have made the methods of sample size planning for the
design of enrichment trials available on line at
http://linus.nci.nih.gov/brb/.
The enrichment design does not provide data on the
effectiveness of the new treatment compared to control
for test negative patients. Consequently, unless there is
preliminary data or compelling biological evidence that
the new drug is not effective in test negative patients,
the enrichment design may not be adequate to support
approval of the test as a medical device, and designs that
include both test positive and test negative patients
should be considered.
When both test positive and test negative patients are
included in the clinical trial, it is essential that an analy-
sis plan be pre-defined in the protocol for how the pre-
dictive classifier will be used in the analysis. It is not suf-
ficient just to stratify the randomization with regard to
the classifier without specifying a complete analysis
plan. The purpose of the pivotal trial is to evaluate the
new treatment in the subsets determined by the pre-
specified classifier. The purpose is not to modify or opti-
mize the classifier. If the classifier is a composite gene
expression based classifier, the purpose of the design is
not to re-examine the contributions of each component.
If one does any of this, then an additional phase III trial
may be needed to evaluate treatment benefit in subsets
determined by the new classifier. In moving from post-
hoc correlative science to reliable predictive medicine, it
is important to strictly separate the data used for devel-
oping classifiers from the data used for testing treatment
effects in subsets determined by those classifiers.
Reliable conclusions can only be achieved by strictly
honoring this principle. The process of classifier devel-
opment can be exploratory, but the process of evaluat-
ing treatments should not be; it should be based on test-
ing pre-specified hypotheses in pre-specified patient
groups.11 Simon14 describes a variety of analysis strate-
gies and Zhao and Simon have made the methods of
sample size planning for designs involving treatment of
both test positive and test negative patients available on
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 99 |
Berlin, Germany, June 4-7, 2009
line at http://linus.nci.nih.gov/brb/.
Jiang et al.20 reported on a “Biomarker Adaptive
Threshold Design” for situations where a predictive
index is available at the start of the trial, but a cut-point
for converting the index to a binary classifier is not
established. Freidlin and Simon21 proposed an adaptive
design for a phase III trial that can be used when no clas-
sifier is available at the start of the trial. The design pro-
vides for development of the classifier and evaluation of
treatment effects in subsets determined by the classifier
in a single trial. The analysis plan of the adaptive signa-
ture design is structured to preserve the principal of sep-
arating the data used for developing a classifier from the
data used for evaluating treatment in subsets deter-
mined by the classifier, although both processes are part
of the same clinical trial.
Establishing the clinical utility of a classifier for use of
an established treatment can be more difficult. Clinical
utility depends on a variety of factors including other
treatments available, availability of more easily meas-
ured predictive factors and practice standards. It may be
more difficult to conduct prospective trials that involve
withholding widely used treatments.
Using archived tumor specimens
Archived specimens are routinely used to establish
analytical validity and clinical validity of tests. The
more challenging issue is, however, under what condi-
tions are they useful for establishing medical utility of
prognostic and predictive classifiers. In some cases, it
may be possible to utilize archived tumor samples from
patients treated in a randomized clinical trial to simulate
reasonably the analysis that would have been per-
formed in a prospective trial. This is only a viable strat-
egy when archived specimens are available for a large
proportion of the patients in an appropriately designed
previously conducted randomized trial. The retrospec-
tive strategy is not credible unless the plan for the retro-
spective analysis is completely specified in writing prior
to performing assays on the archived specimens. The
classifier must be completely determined by data exter-
nal to the clinical trial used for retrospective analysis.
Because retrospective classification of archived speci-
mens does not accurately reflect the challenges of tissue
handling and assay performance encountered prospec-
tively in a time frame that enables real-world treatment
selection, it is important to separately establish the ana-
lytical validity of the assay for use with archived tissue.
References
1. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, et al. A
multigene assay to predict recurrence of tamoxifen-treated,
node-negative breast cancer. N Engl J Med 2004;351:2817-26.
2. van-de-Vijver MJ, He YD, Veer LJvt, Dai H, Hart AAM,
Voskuil DW, et al. A gene expression signature as a predictor
of survival in breast cancer. N Engl J Med 2002;347:1999-2009.
3. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Baja-
monde A, et al. Use of chemotherapy plus a monoclonal anti-
body against HER2 for metastatic breast cancer that overex-
presses HER2. N Engl J Med 2001;344:783-92.
4. Simon R, Radmacher MD, Dobbin K, McShane LM. Pitfalls in
the use of DNA microarray data for diagnostic and prognostic
classification. J Nat Cancer Inst 2003;95:14-8.
5. Radmacher MD, McShane LM, Simon R. A paradigm for class
prediction using gene expression profiles. J Comp Biol 2002;9:
505-12.
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6. Molinaro AM, Simon R, Pfeiffer RM. Prediction error estima-
tion: A comparison of resampling methods. Bioinformatics
2005;21:3301-7.
7. Dupuy A, Simon R. Critical review of published microarray
studies for cancer outcome and guidelines on statistical analy-
sis and reporting. J Nat Cancer Inst 2007;99:147-57.
8. Sparano JA, Paik S. Development of the 21-gene assay and its
application in clinical practice and clinical trials. J Clin Oncol
2008;26:721-8.
9. Bogaerts J, Cardoso F, Buyse M, Braga S, Loi S, Harrison JA, et
al. Gene signature evaluation as a prognostic tool: Challenges
in the design of the MINDACT trial. Nat Clin Prac Oncol
2006;3:540-51.
10. Sargent DJ, Conley BA, Allegra C, Collette L. Clinical trial
designs for predictive marker validation in cancer treatment
trials. J Clin Oncol 2005;23:2020-7.
11. Pusztai L, Hess KR. Clinical trial design for microarray predic-
tive marker discovery and assessment. Ann Oncol 2004;15:
1731-7.
12. Simon R. A roadmap for developing and validating therapeu-
tically relevant genomic classifiers. J ClinOncol 2005;23:7332-
41.
13. Simon R, Maitournam A. Evaluating the efficiency of targeted
designs for randomized clinical trials. Clin Cancer Res 2005;
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10:6759-63.
14. Simon R. Using genomics in clinical trial design. Clin Cancer
Res 2008;14:5984-93.
15. Temple RJ. Special study designs: Early escape, enrichment,
studies in non-responders. Communications in Statistical
Theory and Methods 1994;23:499-531.
16. Temple RJ. Enrichment designs: Efficiency in development of
cancer treatments. J ClinOncol. 2005;23:4838-9.
17. Schilsky RL. Target practice: oncology drug development in
the era of genomic medicine. Clin Trials 2007;4:163-6.
18. Simon R, Maitournam A. Evaluating the efficiency of targeted
designs for randomized clinical trials: Supplement and
Correction. Clin Cancer Res 2006;12:3229.
19. Maitournam A, Simon R. On the efficiency of targeted clinical
trials. Stat Med 2005;24:329-39.
20. Jiang W, Freidlin B, Simon R. Biomarker adaptive threshold
design: A procedure for evaluating treatment with possible
biomarker-defined subset effect. J Nat Cancer Inst 2007;99:
1036-43.
21. Freidlin B, Simon R. Adaptive signature design: An adaptive
clinical trial design for generating and prospectively testing a
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Res 2005;11:7872-8.
 Clinical trial design

Do commonly-used clinical trial designs

reflect clinical reality?

E. Estey A B S T R A C T

Fred Hutchinson Cancer Research
Center and University of Washington
School of Medicine, Seattle, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:101-105
This paper contends that commonly used clinical trial designs do not reflect clinical reality as viewed
by patients or physicians. Specifically, randomized phase 3 designs focus on improvements that are
more significant statistically than medically and put an emphasis on avoiding a false positive result
that is more appropriate for diseases that are curable, in contrast to acute leukemias. The resultant
large sample sizes needed for each treatment restrict the trial to one or two new treatments, although
historical reality suggests the difficulty in knowing, without clinical data, whether these are the best
of several new treatments. The p-value-based statistics discourage use of data from previous patients
in the trial to inform treatment of subsequent patients, contravening patients’ assumptions. Standard
phase 2 trials focus on a single outcome, ignoring the complexity of medical practice, and ignore prog-
nostic heterogeneity. Finally, although patients are more interested in whether a new treatment is bet-
ter than another is, rather than whether it is active, randomization between different treatments does
not begin until phase 2 trials have been completed. This paper proposes alternatives based on the
Bayesian statistical approach.
The thesis that I will develop here is that commonly used clinical trial designs are unrealistic in the
sense that they do not correspond well to patients’ views of medical practice and greatly over-simplify
such practice. By emphasizing Bayesian rather than p-value-based statistics and focusing on AML, I hope
to familiarize physicians with some of the many new published designs that address these problems.

The standard phase 3 trial no satisfactory treatment for most patients

These trials typically randomize approxi-
mately 400 patients between two therapies.1-3
This relatively large number is required to
detect relatively small improvements with a
false positive rate less than 5% (p<0.05) and
a false negative rate less than 20% (80%
power). For example, the trials in references
1-3 targeted increases in median EFS or sur-
vival of 6 to 12 months, in 2 year EFS or sur-
vival of from 10-20% and in CR rate from 50
-65%. Consider the relevance of a 6-month
improvement in survival to an otherwise
healthy 65-year-old man with untreated
AML. Such a patient might expect to live
another 15 years if he did not have AML but
only another one half-year if he is random-
ized to a standard treatment arm. In such a
case, he only retains 0.5/15 (3%) of his nor-
mally remaining life expectancy. If he is ran-
domized to the investigational arm and it is
“successful,” he gains another half-year and
now retains 1/15 (7%) of his life expectancy.
While statistically significant, I doubt many
patients would consider this result medically
significant. Hence, the targeted improve-
ment does not reflect clinical reality.
The choice of a false positive rate of 0.05
but a false negative rate of 0.20 implies a
preference for more protection against a
false positive than a false negative result.
This is quite sensible when satisfactory
treatment exists for the disease in question,
and hence, replacement of this standard
with a falsely positive new therapy is partic-
ularly undesirable. However because there is
with AML, the medical risk of a false posi-
tive is much less. Indeed, the near universal
choice of p=0.05 and power=80%, regard-
less of the disease in question, ignores the
reality that diseases vary considerably in cur-
ability.
Consequently, phase 3 AML trials should
perhaps seek more clinically meaningful
improvements and permit higher p-values.
Although this formulation would result in
loss of power to detect relatively small
advances, I question whether leukemia ther-
apeutics advances in such small increments.
In particular, it would appear that quantum
therapeutic advances are not infrequent, as
with all-trans retinoic acid (ATRA) and
arsenic trioxide (ATO) for APL, 2 chloro-
deoxyadenosine for hairy cell leukemia,
high-dose ara-C and likely gemtuzumab
ozogamycin for CBF AML, and imatinib for
CML. Even if there were value in retaining
sufficient power to detect small advances,
the added value may not justify the neces-
sary sample sizes, which prevent expedi-
tious completion of trials and simultaneous
investigation of a large number of new ther-
apies.
P-value-based versus bayesian approaches
(Table 1)
Patients naturally prefer “adaptive”
designs, those that permit treatment deci-
sions for subsequent patients in a trial to be
based on results in previous patients.
However, p-value-based designs tend to dis-

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 101 |
14th Congress of the European Hematology Association
Table 1. Definitions.
p value: the probability of observing the data or more extreme data under the null
hypothesis; the latter states that there is truly no difference between two treat-
ments. For example, assume the null hypothesis is that, among 10 people, 5 would
prefer red and 5 blue. In fact, 8 preferred red and 2 blue. The p-value is calculat-
ed as the probability that, under the null hypothesis, 8/10 would prefer red + the
probability that 9/10 would prefer red + the probability that all 10 would prefer
red.
Bayes theorem states:
P(B|A) = [P (A|B)] [P(B)]
[P (A|B)] [P(B)] + [P (A|not B)] [P (not B)]
where P is probability, B is a hypothesis, and A are observed data. P(B) is known as
the prior probability of the hypothesis, while P(B|A) is the posterior probability of the
hypothesis. Thus, the p-value is based on the probability of data given a hypothesis
while Bayesians compute the posterior probability of a hypothesis given data.
Physicians often mistakenly believe that a p-value is a Bayesian posterior probability
courage frequent examination of incoming data. This
reflects the inextricable link between p-value and trial
design, such that the same data can produce different p-
values depending on the particular design used (Table
1).4-7 For example, it is well-known that the probability
of finding an association at p<0.05 increases purely by
chance as the number of tests of significance that are
performed increases.8-9
Accordingly, interim analyses of clinical trials are gen-
erally performed at p-values much less than 0.05 in
order to preserve an approximately 0.05 level of signifi-
cance at the final analysis. For example, the design pro-
posed by Fleming et al. stops a trial, declaring one arm
superior, only with p-values of 0.005, 0.006, 0.007, and
0.009 at the 1st, 2nd, 3rd, and 4th of 4 interim analyses,
respectively. This of course makes it difficult to stop 1:1
randomization to an arm, even when the probability
that that arm is inferior is greater than 90%, leading
most patients to prefer randomization to the better arm.
The dependence of p-value on trial design is such
that, in a case when the final planned analysis yields a
p-value of 0.051, but in which subsequently obtained
data strengthen the evidence in favor of a difference,
these data cannot be used, since they were not obtained
as part of the planned experiment.
The Bayesian approach provides flexibility, and in
particular, encourages interim analyses. The approach
begins with parameters, such as the probability of CR
or, when comparing two treatments, the probability
that the relative risk of survival is greater than 1.0.
These parameters (denoted here by θ) are random quan-
tities, with probability distributions describing one’s
uncertainty about them. One begins with a prior distri-
bution, p(θ), that characterizes the uncertainty about θ
before observing any data. The second Bayesian quanti-
ty is the likelihood, L(data | θ), which describes the
probability of observing any specified data given any
value of θ; examples of likelihoods are the binomial dis-
tribution for binary events and the normal(bell-shaped)
distribution for continuous variables. Bayes’ theorem
multiplies the prior by the likelihood of observing the
data given the parameter to arrive at a “posterior” distri-
| 102 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Figure 1. Bayesian probability distributions using a trial of
a new therapy in relapsed AML as an example. The values
on the horizontal axis are different probabilities of CR. The
values on the vertical axis represent the weight assigned
to each CR probability. Prior to treatment, although the
average CR rate is thought to be 20%, some credence is
assigned to each probability of CR (prior probability distri-
bution, dotted line). After observing 5/10 CRS (first poste-
rior probability distribution, dashed line), the average CR
rate is close to 50% and no credence is given CR rates less
than 10% or greater than 90%, reflecting the impact of the
observed data on the prior. Thus, the posteriors become
successively more informative as the data accumulate,
and shift to reflect the overall average behavior of the
data. After observing 7 CRs in the next 30 patients (total
12 CRs in 40 patients), the average CR rate is approxi-
mately 30% and no credence is given a CR rate greater
than 60% (2nd posterior probability distribution solid line).
Computing the proportion of the area under the curve that
is to the right of a CR rate of 0.4 gives the current proba-
bility that the CR rate is greater than 0.4. This probability
can be used to make treatment decisions.
bution of θ, which describes uncertainty about θ after
observing the data (Figure 1). In contrast to p-value-
based methods, Bayesian inference is not affected by
the experimental design since data only enter inferences
through the likelihood function. Consequently, when
making decisions or inferences based on accruing data,
Bayes’s theorem may be repeatedly applied, with the
posterior at each stage becoming the prior for the next
stage. The probability distributions in this sequence
become increasingly informative about θ as the data
accumulate. This process, known as “Bayesian learn-
ing,” (Figure 1) is especially useful in sequential data
monitoring during a clinical trial. The current posterior
probability distribution may be used to modify doses,
unbalance a randomization in favor of a treatment with
relatively superior performance, or terminate a trial
early due to either superiority of a treatment or futility.
The Bayesian approaches’ flexibility can be appreciated
by contrasting its ability to incorporate data obtained
subsequent to trial completion with the p-value
approach’s inability to do this, as noted above.
A significant issue with the Bayesian approach is set-
ting prior probabilities. In Figure 1, we made the prior
non-informative reflecting a lack of any information
about response to the new drug. However, it might be
contended that the prior should be more informative,
incorporating knowledge of previous trials with other
drugs in relapsed AML. The choice of prior obviously
influences computation of the posterior – the more

informative the prior, the more data needed to influence
the posterior. The designs described below generally
use non-informative priors. A more detailed presenta-
tion would describe how selection of different priors
influences the posterior.
Adaptive randomization
Bayesian designs for adaptive randomization repeat-
edly use interim data to compute the probability that
one arm of a randomized trial is better than the other(s),
unbalancing the randomization to favor the likely better
treatment.10-11 If this probability crosses a pre-specified
boundary, the inferior arm is shut down before the
maximal sample size is reached. However, it may re-
open if further analyses indicate that results with the
open arm(s) are deteriorating such that the probability
that this arm(s) is superior has decreased.
A trial adaptively randomizing patients over age 50
with untreated AML among idarubicin + ara-C (IA, the
standard), troxacitabine + ara-C (TA), and troxacitabine
+ idarubcin (TI) illustrates the process.12 The first 15
patients were randomized fairly among the three arms.
As each patient after the 15th entered the trial, we com-
puted the posterior probability that the CR rate with IA
was greater than or equal to 10% better than that with
TA or TI. If this probability was less than 0.15 accrual to
IA was suspended. If in contrast the posterior probabil-
ity was greater than 0.85 that the CR rate with TA or TI
was greater than or equal to 10% worse with IA accru-
al to either TA or TI was suspended. Depending on
results in arms that remained open, a closed arm could
re-open. A maximum of 75 patients were to be random-
ized. The TI arm closed and remained closed after the
first five patients failed to respond, while the TA arm
closed and remained closed after the CR rate was 3/11,
at which time the CR rate in the IA arm was 10/18. If
the 34 patients who had been entered on the trial when
both TA and TI arms were closed had been randomized
fairly, 11 patients would have received each of TA, TI,
and IA. With adaptive randomization, only 16, rather
than 22 patients, received the inferior TA or TI arms,
probably corresponding with how patients visualize
clinical practice,
The possibility certainly exists that stopping arms so
early might lead to a false negative conclusion.
Beginning adaptive randomization only once 15 to 20
patients have been randomized equally among the var-
ious treatment arms reduces the problem. At any rate, it
is critical to examine how the design performs under
various clinical scenarios, that is, what are its operating
characteristics (OC). OC include the probabilities that
the design will correctly select a truly superior treatment
or incorrectly select a truly inferior treatment, as well as
the median number of patients treated on each arm. If
clinicians feel the OC are unsatisfactory, the parameters
above, such as the criterion probabilities of 0.15 or 0.85,
or the number of patients to be fairly randomized are
changed until desirable OC are obtained. Table 2 illus-
trates 1000 computer simulations for two scenarios in
the IA versus TA versus TI trial. In the first, the true CR
rates with TA, IA, and TI are 50%, 40%, and 30%,
respectively; hence, the correct conclusion is that TA is
superior. As parameterized above, the probability was
80% that the design would reach the correct conclusion,
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 103 |
Berlin, Germany, June 4-7, 2009
corresponding to a power of 80%. In contrast, if the true
CR rates were 30%, 40%, and 30% with TA, IA, and TI,
respectively, the probability that the design would cor-
rectly select IA as superior was only 10%. Hence, in this
case, the design provided much more protection against
a false negative than a false positive. The false positive
rate could have been decreased by eliminating the
requirement that, with high probability, TA or TI be at
least 10% worse than IA before either of these arms
would close. However, this would have also increased
the false negative rate contrary to the desire of the clin-
ical investigators to maintain a low false negative rate.
As outlined above, adaptive randomization fails to
account for the possible imbalance in prognostic covari-
ates between patients randomized on each arm. This
issue has recently been addressed, together with how
adaptive randomization may be used with censored
data as might arise when survival is the endpoint.13 In
any event, implementation of adaptive randomization
requires that patients only infrequently present for ran-
domization before there has been sufficient opportuni-
ty to observe the outcome in previous patients.
Accounting for prognostic heterogeneity in single arm trials
New drugs are typically tested in single-arm phase 2
trials before investigation in phase 3. The most com-
monly used design for single arm phase 2 trials is the
Simon 2-stage (S2S) design.14-15 Rates of no interest
(known as p0), typically corresponding to the historical
rate, and of interest (p1) typically 0.15 to 0.20 higher
than p0 are specified, together with maximum false pos-
itive and false negative rates (typically 0.10). These
parameters determine the number of patients treated in
the first stage and the minimum number of responses
needed to proceed to a second stage of specified num-
ber. After the latter is completed, a drug is accepted if
the number of responses is greater than a specified min-
imum.
The S2S unrealistically assumes that treated patients
have homogeneous prognoses. Certainly, in AML this is
unlikely to be the case.16 Hence reliance on the S2S risks
declaring drugs inactive when they might have been
found active had a better prognostic group been treated.
Conducting separate phase 2 trials in distinct prognostic
groups is time consuming and does not allow informa-
tion gained in one prognostic group to affect the trial in
a second prognostic group.
A method that accounts for treatment-prognostic sub-
group interactions has been proposed, specifically using
data from the trial to estimate the degree to which the
results in the different subgroups can be combined.17
There are two levels of prior probability distributions
(“hierarchical Bayes”). The first is the usual probability
of response to a drug in each of, for example, two prog-
nostic groups. The second quantifies prior belief that
the response in one prognostic group can inform the
probability of response in the other. As usual, these pri-
ors are updated as the trial proceeds.
Consider a hypothetical trial of a new drug in
relapsed AML. Actual historical data indicated a
response rate of 21% in 169 patients. This rate was 11%
(118 patients) if initial CR duration was less than 1 year
but 43% (51 patients) if initial CR duration was greater
than 1 year. The goal was to increase response rate to
14th Congress of the European Hematology Association

31% (absolute increase of 0.2) in the worse prognostic Table 2. Operating characteristics for IA vs. TA vs. TI trial.
group and to 58% (absolute increase of 15%) in the bet-
ter prognostic group. Since the historical data suggest True CR Rates Correct Probability
that 69% of patients will be in the worse group, the conclusion correct conclu-
overall targeted improvement is [0.20×0.69}+[0.15× sion
0.31]=0.18. Thus, an S2S design would set 21% as p0
and 0.21+0.18=0.39 as p1. Setting the nominal false pos- TA IA TI
itive and false negative rates at 0.10, the S2S would treat 50% 40% 30% TA superior 80%
22 patients in a first stage, and the trial would stop if less
than five responses occurred. If greater than four
responses occurred, an additional 21 patients would be
enrolled and the drug declared a success if responses Tables 3A and B Comparative operating characteristics of
were seen in more than 12/43 patients. Thus, to make STI and Simon 2 stage (S2S) designs.
the proposed design (hereafter, STI because it examines
Probability Mean
subgroup-treatment interactions) comparable to the True (Reject) # Pts
STS, we specify that STI will also take its first look after
22 patients have been evaluated and will also set its Subgroup CR Rate S-TI S2S S-TI S2S
false negative rate at 0.10. Better 0.58 0.10 0.75 21 10
Table 2 compares the operating characteristics of the Worse 0.11 0.90 0.75 19 25
STI and S2S designs. In Table 3A, the new drug achieves
its goal in the better but not the worse group. Because Probability Mean
the S2S does not consider interactions between prog- True (Reject) # Pts
nostic subgroups and treatment, it has the same proba-
bility (0.75) of rejecting the drug in both groups. In con- Subgroup CR Rate S-TI S2S S-TI S2S
trast, the STI is less likely to reject the drug in the better Better 0.43 0.50 0.26 13 11
group and more likely to reject it in the worse group. Worse 0.31 0.10 0.26 27 30
Furthermore, 52% of the patients treated with STI will
be in the better group versus only 29% with S2S. Table
3B illustrates that, in the case where the desired
improvement occurs in the worse but not the better
group, STI is more likely to accept and reject the drug in to stop if it appeared likely that they were only relevant
the appropriate subgroups. Although conducting sepa- for a small subset of the eligible population. Designs
rate Simon 2-stage designed trials in better and worse that monitor multiple outcomes are readily available.22-23
subgroups corrects this problem, S2S’s inability to allow
results in one subgroup to affect the conduct of the trial
in the other subgroup continues to result in a smaller Testing more new therapies and allowing earlier compari-
proportion of patients belonging to the group where son of these
treatment seems more effective relative to historical Patients are more interested in whether one therapy is
better than another than whether either therapy is
Monitoring multiple outcomes “active.” Because comparison is best done through ran-
The great majority of clinical trials specify one “pri- domization, it has been proposed that randomization
mary” outcome, such as toxicity, response rate, or sur- begin earlier than is now the case. In particular, selection
vival. Stopping rules are based only on the primary out- designs have been proposed in which a relatively small
come. This formulation appears unrealistic, ignoring the number of patients are randomized among several new
complexities of medical practice and clinical research. therapies.22,24 The rationale is that, although many new
For example, because phase 1 trials are often quite small therapies are available that may be tested in different
and, unrealistically, fail to account for covariates other schedules and combinations, pre-clinical rationale is an
than dose associated with toxicity, knowledge of toxic- imperfect guide to selecting which new drug to com-
ity is often incomplete after phase 1.18-20 It follows that it pare with a standard. Thus, a compelling pre-clinical
is desirable in phase 2 to formally measure both rationale did not exist for arsenic trioxide in APL, flu-
response and toxicity and allow stopping based on darabine in CLL, and cladribine in hairy cell leukemia,
either outcome. Consider also a trial of a new therapy, while many drugs that failed clinically were accompa-
postulated to be less toxic than standard 3+7, in older nied by seemingly unassailable rationales. A Bayesian
patients with untreated AML. While the reduced toxic- selection design randomizes 45 to 80 patients among
ity might improve survival relative to 3+7, it might also three to four therapies. Each therapy begins with the
reduce CR rate, with long-term survival most likely in same prior probability distribution. As patients are
patients achieving CR.21 However, some decrease in CR treated, the priors are updated with these posteriors
rate would be accepted provide survival increased. used to shut down accrual to an arm if, for example, the
Thus, the trial would formally monitor both survival probability that its true response rate is greater than
and CR, stopping if the decrement in CR rate appeared 20% worse than a competing arm is high. At the end of
too great or the increase in survival insufficient. The the trial, the arm with the highest response rate among
proportion of eligible patients who actually enrol on a those not shut down is selected for further study, per-
trial is often relatively low due to selection bias. The haps in comparison to standard therapy.
consequences of such bias might be reduced were trials Such selection designs are often criticized as “under-

| 104 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

powered phase 3 trials.” Examination of selection
designs’ operating characteristics indicate that, in a sce-
nario where three drugs have the same true response
rate and the fourth provides an absolute 20% improve-
ment, the probability of correctly selecting the fourth
drug (that is the probability that it will not stop early
plus the probability that it will have the highest
response rate at the end of the trial) is only about 60%.
This of course contrasts with the aforementioned 80%
power typical of randomized trials, involving, for exam-
ple, a new drug versus a standard. However, the 80%
figure is purely nominal, ignoring the process used to
select the new drug. Assume that four new therapies
were available for comparison with a standard, and that
because pre-clinical rationale cannot substitute for clini-
cal data in the selection process, each was equally like-
ly to be useful clinically. It follows that the probability
of correctly selecting the best drug was 25%. This 25%
is ignored in the computation of 80% power; if it were
not, the power of the trial would be 25%×80%=20%.
Thus, the selection design’s 60% probability of correct
selection should be viewed, not in relation to 80%
power, but in relation to the 25% probability of correct
selection that would obtain in the absence of the selec-
tion design. Recognizing these issues, the Medical
Research Council-sponsored trials in AML in the United
Kingdom are employing selection designs rather than
more conventional phase 3 designs.
References
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Caligiuri MA et al. Phase 3 study of the multidrug resistance
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Leukemia Group B Study 9720. Blood 2002;100:1224-32 .
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 Diffuse large B cell lymphoma

Molecular basis of aggressive B-cell

non-Hodgkin lymphomas

E. Hartmann
A. Rosenwald
Institute of Pathology, University of
Würzburg, Germany
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:106-111
A B S T R A C T
Diffuse large B-cell lymphomas (DLBCL) and Burkitt lymphomas (BL) represent major entities in the
World Health Organization (WHO) classification. Genetic alterations in DLBCL include translocations of
BCL6 (30-40%), BCL2 (20 to 30%) and MYC (10%), and inactivation of the tumor suppressor TP53
(20%); none of which are specific for this malignancy. Aberrant somatic hypermutation may target var-
ious proto-oncogenes, including PIM1, MYC and PAX5. On the gene expression level, the germinal cen-
ter B-cell type (GCB DLBCL) and the activated B-cell type (ABC DLBCL) can be discerned and show dif-
ferences in underlying genetic alterations, activation of oncogenic pathways and clinical behavior. The
more favorable clinical course for patients with GCB DLBCL treated with CHOP appears to remain sig-
nificant in the R-CHOP era. Microarray-based studies have suggested new potential therapeutic targets
in DLBCL, including PKC-β and SYK inhibitors. CARD11 shows oncogenic mutations in a subset of
DLBCL, and could be targeted to block aberrant NF-κB activation, a feature of ABC DLBCL. BL is char-
acterized by the translocation of the MYC oncogene. Gene expression and molecular studies suggest a
gray zone between DLBCL and BL that led to the new provisional category of “B-cell lymphoma, unclas-
sifiable, with features intermediate between DLBCL and BL” in the new WHO classification.

mong aggressive B-cell non-Hodgkin ly expressed in GC B-cells,5 but is down-reg-

A lymphomas (B-NHL), diffuse large B-

cell lymphomas (DLBCL) and Burkitt
lymphomas (BL) represent major entities in
the World Health Organization (WHO) clas-
sification scheme,1 and this review will focus
on the molecular basis of these clinically
important malignancies. In DLBCL, the
well-recognized widely varying molecular
features do not come as a surprise, given the
large number of its clinical variants, morpho-
logical and immunohistochemical subgroups
and differences in overall survival. In BL, the
molecular basis appears less heterogeneous
compared to DLBCL, but recent investiga-
tions have highlighted the problematic his-
tomorphological, genetic and molecular gray
zone between BL and DLBCL that may bear
important clinical implications.
Diffuse large B-cell lymphoma
The diverse clinical course of DLBCL is
also reflected on the molecular level. In con-
trast to other B-cell lymphomas, no unifying
primary genetic alteration has been identi-
fied so far in DLBCL. Instead, a large spec-
trum of genetic alterations may contribute to
the development and progression of this
lymphoma subtype (Figure 1).
The most frequently observed genetic
alterations in DLBCL lead to constitutive
expression of the BCL6 oncogene, and
include translocations of BCL6 in the chro-
mosomal band 3q27 (30-40% of DLBCL) as
well as mutations in the 5’ non-coding regu-
latory sequences of the BCL6 locus.1-3 BCL6
expression is necessary for the formation of
germinal centers (GC)4 and is physiological-
ulated upon terminal B-cell differentiation
into plasma cells. It acts as a transcriptional
repressor and targets genes involved in the
DNA damage response and cell cycle control
(including TP53, CDKN1B/p27 and
CCND2/Cyclin D2), thereby allowing DNA
remodeling events (e.g., somatic hypermuta-
tion, SHM) to occur in the GC environ-
ment.6-7 BCL6 expression prevents terminal
B-cell differentiation by suppressing
PRDM1/BLIMP1, a “master regulator” of
plasma cell differentiation.6,8 Taken together,
deregulated and sustained expression of
BCL6 may promote lymphomagenesis
through aberrant proliferation and survival
signals and a B-cell differentiation blockade.
Other frequently detected genetic alter-
ations in DLBCL include the translocation
t(14;18)(q32;q21), the hallmark translocation
of follicular lymphoma, that also occurs in
20-30% of DLBCL,1,9-10 and rearrangements
of the MYC gene locus in 8q24 detectable in
approximately 10% of cases.1,3,11-12 Moreover,
the BCL2/18q21 and MYC/8q24 gene loci,
as well as the REL gene located in 2p13, are
amplified in around 20% of cases.3,13
Inactivation of the tumor suppressor TP53
located in 17p13.1 has been reported in
approximately 20% of DLBCL and is associ-
ated with a poor prognosis.3,14-15 A misdirec-
tion of the somatic hypermutation machin-
ery might be another important mechanism
involved in the molecular pathogenesis of
DLBCL. While physiologically the somatic
hypermutation process affects predominant-
ly immunoglobulin genes, aberrant somatic
hypermutation has been observed in up to

| 106 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

50% of DLBCL targeting various proto-oncogenes
(including PIM1, MYC, RHOH/TTF and PAX5) leading
to potentially oncogenic mutations.16
Microarray-based gene expression profiling studies
also contributed to the elucidation of the molecular het-
erogeneity in DLBCL (Figure 2). Importantly, two bio-
logically and clinically distinct subgroups of DLBCL, the
germinal center B-cell like (GCB) DLBCL and the acti-
vated B-cell like (ABC) DLBCL, have been identified
based on differences in their gene expression pattern.17
In the treatment era of CHOP/CHOP-like chemothera-
py (Cyclophosphamide, Vincristine, Doxorubicin and
Prednisone), the clinical course of patients with GCB
and ABC DLBCL differed markedly, with a more favor-
able prognosis for patients with the GCB subtype.10
Additional gene expression signatures associated with
overall survival in the CHOP era include the “prolifera-
tion signature,” representing tumor cell proliferation,
the “lymph node signature,” capturing molecular
aspects of the microenvironment and the “MHC class II
signature.” High expression of the proliferation signa-
ture was associated with an unfavorable prognosis,
whereas increased expression of the lymph node and
MHC class II signatures correlated with increased sur-
vival times.10 Loss of the MHC class II protein expres-
sion on the tumor cells measured immunohistochemi-
cally predicts for an inferior clinical course, suggesting
that a diminished tumor immunosurveillance may neg-
atively impact on clinical outcome.18
The question whether the prognostic impact of the
molecular distinction of DLBCL into the GCB and ABC
DLBCL subgroups is still relevant in the R-CHOP treat-
ment era (CHOP combined with Rituximab) was
addressed in a recent study by Lenz and co-workers.19
This study demonstrated the predictive value of the
gene expression-based distinction of the GCB/ABC
DLBCL subgroups in a large series of DLBCL patients
treated with R-CHOP.19 Moreover, biological features of
the microenvironment associated with the clinical
course of the disease could be more precisely dissected.
Specifically, a “stromal-1” signature (including genes
that reflect extracellular matrix components and genes
expressed by cells derived from the monocytic lineage),
and a “stromal-2” signature (including genes related to
angiogenesis and tumor blood vessel density), were
identified that appear to function synergistically and to
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 107 |
Berlin, Germany, June 4-7, 2009
Figure 1. Molecular features of diffuse large B cell lym-
phomas (DLBCL), Burkitt lymphomas (BL) and the gray
zone between the two entities. SHM, somatic hypermuta-
tion; Ig, immunoglobulin genes.
predict the clinical course with high accuracy.19
The notion that the tumor microenvironment may
contribute importantly to the biological variability of
DLBCL was also substantiated by another gene expres-
sion profiling study.20 In this study, Monti and col-
leagues defined three prominent gene expression clus-
ters in DLBCL. One cluster reflected the host response
as a defining feature of a subset of DLBCLs (“host
response cluster, HR”), and two additional gene clusters
were found to be differentially expressed within their
cohort of DLBCL tumors, termed “oxidative phospho-
rylation (OxPhos)” and “B-cell receptor/proliferation
(BCR)”, the latter showing an increased expression of
genes involved in the BCR signaling cascade. This group
also proposed a 13 gene-based survival predictor in
DLBCL, amongst which the genes PDE4B and PKC-β
were found to be overexpressed in lymphoma speci-
mens from patients that followed a fatal clinical
course.21 The subdivision of DLBCL into the ABC and
GCB subtypes based on gene expression profiling stud-
ies is further corroborated by additional molecular fea-
tures that are distinctive between the two groups. The
analysis of the immunoglobulin gene mutational status
revealed an intraclonal variation in GCB DLBCL indicat-
ing ongoing somatic hypermutation as a marker of ger-
minal center (GC) derivation, whereas the ABC DLBCL
Figure 2. Gene expression features
of diffuse large B cell lymphoma
(DLBCL). Neoplastic B-cells vary in
the expression of the proliferation
signature, the major histocompatibil-
ity complex (MHC) II signature and
the germinal center B-cell (GCB) sig-
nature.10 Two additional signatures
were described by Monti at al.,20
namely the ‘oxidative phosphoryla-
tion, OxPhos’ and the ‘B cell recep-
tor/proliferation, BCR’ signatures.
Molecular features of the microenvi-
ronment are captured by the stromal
score, consisting of the synergistic
stromal-1 and stromal-2 signatures,19
as well as by the host response sig-
nature.20
14th Congress of the European Hematology Association
subtype usually displayed mutated immunoglobulin
genes without evidence for ongoing somatic hypermu-
tation suggesting a post germinal center phenotype.22
On the chromosomal level, alterations that preferential-
ly occur in GCB DLBCL include the translocation
t(14;18)(q32;q21), gains/amplifications in 2p including
the REL gene locus, as well as gains/amplifications of
12q.10,23 In contrast, ABC DLBCL show more frequently
losses of 6q, trisomy 3 or gains/amplifications of 3q, and
gains/amplifications of 18q.23 In addition, a recent study
using a high resolution array comparative genomic
hybridization (CGH) platform identified deletions in
9p21.3, the genomic locus of the INK4a/ARF tumor sup-
pressor, and a small gain in 19q, including the gene locus
of the transcription factor SPIB, to occur more frequent-
ly in ABC DLBCL.24 The combined analysis of data from
a genome-wide copy number and gene expression
analysis revealed the transcription factor SPIB as a high-
ly up-regulated gene in this small amplicon in 19q, and
knockdown of SPIB by RNA interference was found to
be toxic to ABC but not to GCB DLBCL cell lines.24
ABC DLBCL are further characterized by constitutive
activation of the antiapoptotic NF-κB signaling path-
way,25-27 and a loss-of-function RNA interference screen
in ABC and GCB DLBCL cell lines revealed CARD11 as
a key molecule contributing to the enhanced NF-κB-sig-
naling in ABC DLBCL.27 A subsequent study uncovered
missense mutations in the coiled-coil domain of
CARD11 as a mechanism of its oncogenic activation,
and reported that these mutations occur more frequent-
ly in ABC DLBCL.28 Therefore, therapeutic targeting of
molecules involved in NF-κB signaling and, in particular,
inhibition of CARD11 might be promising approaches
to improve treatment options for the prognostically
unfavorable ABC DLBCL subgroup.
Following the publications from gene expression pro-
filing studies in DLBCL, various attempts have been
made to translate the prognostic information into sim-
plified tests that may be more applicable for routine
clinical practice. Hans and colleagues studied 152
DLBCL tumors from patients treated with anthracyclin-
based chemotherapeutic regimens with a panel of
immunohistochemical markers, and reported that stain-
ings for CD10, BCL6 and IRF4/MUM1 may be suffi-
cient to subdivide DLBCL into GCB and non-GCB cases
retaining the prognostic information.29 However, vari-
ous subsequent studies using the same or slightly mod-
ified immunohistochemical marker panels in other
DLBCL cohorts yielded highly inconsistent results
demonstrating that it may be difficult to capture the
wealth of information provided by complex gene
expression signatures in just a few immunohistochemi-
cal stains. On the other hand, different selection criteria
and treatment protocols in the studied DLBCL cohorts,
as well as variations in the staining and scoring tech-
niques of different laboratories and observers may have
significantly contributed to the varying results.30-31 The
incorporation of new, promising markers, such as
GCET2/HGAL32 and LMO233 into existing immunohis-
tochemical algorithms may, however, improve our abil-
ity to discern the more favorable GCB DLBCL cases
from the remainder.
An interesting alternative to translate complex gene
expression signatures into routinely applicable tests
| 108 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
may be the use of quantitative RT-PCR-based tech-
niques. For example, a six-gene model including LMO2,
BCL6, FN1, CCND2, SCYA3 and BCL2 has been sug-
gested for routine application in DLBCL,34 and this test
also predicts survival if applied to formalin fixed and
paraffin embedded specimens from patients treated
with R-CHOP.35
Results from gene expression profiling and other
molecular studies also suggest a number of new poten-
tial therapeutic targets in DLBCL. One pathway may be
the NF-κB signaling cascade that is constitutively acti-
vated in the prognostically unfavorable ABC DLBCL
subgroup, and molecules, such as CARD11,28 or the very
recently described casein kinase 1α36 may represent
attractive new targets in this pathway. The recent find-
ing of an increased tumor blood vessel density in
DLBCL with an unfavorable clinical course suggests a
potential benefit of the addition of antiangiogenic
agents to current therapeutic regimens in these
patients,19 and this approach is currently being tested in
a large international trial. The finding by Margaret
Shipp’s group that a subset of DLBCL is characterized
by enhanced BCR signaling20 has led to the evaluation of
a SYK-inhibitor (R406) in DLBCL cell lines, with recent-
ly reported promising results.37 This group also evaluat-
ed the PKC-β inhibitor enzastaurin in a phase II study
for patients with relapsed and refractory DLBCL,38
demonstrating the immediate generation of hypotheses
for new treatments based on information that original-
ly came from gene expression profiling studies.
Burkitt lymphoma
The diagnosis of BL relies on the combination of dis-
tinct morphological, immunophenotypic and molecular
features. In Europe, the sporadic variant of BL prevails,
accounting for 30-50% of childhood lymphomas. EBV
infection has been recognized as an important co-factor
in the pathogenesis of BL, and is detectable in nearly
100% of patients with the endemic variant and in 20-
30% and 30-40% of the sporadic and immunodeficien-
cy-associated cases, respectively.1
On the molecular level, BL is characterized by chro-
mosomal rearrangements involving the MYC gene in
8q24. In around 80% of the cases, the t(8;14)(q24;q32)
can be detected, whereas the remaining 20% show vari-
ant translocations involving the light chain loci
(t(2;8)/t(8;22)), all resulting in transcriptional induction
of MYC by juxtaposition to immunoglobulin gene reg-
ulatory elements.39
The t(8;14) translocation breakpoints can be classified
according to their location with respect to both the
8q24/MYC and the 14q32/immunoglobulin heavy
chain gene (IgVH) loci. Three different breakpoint local-
izations within the MYC gene locus have been recog-
nized: the class I breakpoints within the first exon or
intron of MYC, class II breakpoints that occur immedi-
ately 5’ to MYC and the more distant class III break-
points. In the IgVH locus, breakpoints located in the
VDJ-region, as well as in the class switch region have
been detected. Interestingly, the locations of the break-
points differ between the epidemiologic variants of BL,
pointing to the formation of the translocation at differ-
ent stages of B-cell differentiation. In endemic BL, the
breakpoints in the MYC gene most frequently fall into

the class III, and the breakpoints in the immunoglobulin
locus usually occur in the VDJ region, indicating an ori-
gin of the translocation during erroneous VDJ-recombi-
nation in early B-cell stages in the bone marrow. In com-
parison, the sporadic and immunodeficiency-associated
forms show more frequently class I MYC breakpoints
and IgVH breakpoints located in the class switch region,
pointing to a rearrangement during immunoglobulin
class switching that occurs after activation of the B cell.39
Apart from MYC gene translocations, only few sec-
ondary chromosomal abnormalities have been detected
in BL using conventional CGH. Among the most com-
monly reported alterations are gains in chromosomes 1
(1q), 7 (7q) and 12, as well as losses in chromosome 6
and 17p.40-41
MYC is a key transcription factor that controls impor-
tant cellular processes, such as growth control, differen-
tiation and apoptosis. In the absence of additional sur-
vival signals, over-expression of MYC triggers apopto-
sis, either in a p53 dependent manner via the p14
ARF/MDM2/TP53 pathway or independently of p53,
for example, mediated through the pro-apoptotic BH3-
only protein BIM.42-43 In BL, several genetic and epigenet-
ic alterations that may counteract MYC-induced apop-
totic signals have been reported, such as inactivating
mutations of TP53 in around 30% of the cases, but also
inactivation of the CDKN2a locus, overexpression of
MDM2 or hypermethylation of p73.44 Furthermore, in a
substantial proportion of cases missense mutations in
the translocated MYC allele have been detected, that are
particularly located in the conserved MYC box1 ele-
ment,45 and, more recently, specific mutations in this
area of the MYC gene locus have been shown to
increase tumorigenicity by failure to induce BIM-medi-
ated apoptosis.46
By analyzing morphologic, immunophenotypic and
cytogenetic features, the clinically important distinction
between DLBCL and classical BL that fulfill all diagnos-
tic criteria of the WHO classification is usually unprob-
lematic. However, a subset of aggressive B-NHL can
show morphologic and immunophenotypic features
intermediate between BL and DLBCL, thereby prevent-
ing a straightforward classification into one diagnostic
category. Importantly, the presence of the
t(8;14)(q24;q32) involving MYC cannot be viewed as a
robust cytogenetic marker for this discrimination, since
MYC translocations also occur in a subset of bona fide
DLBCL.
In 2006, two microarray-based studies47-48 analyzed
the gene expression profiles of large cohorts of patients
with aggressive B-NHL in order to better characterize
BL on the molecular level and to identify transcriptional
characteristics of BL that might help to distinguish BL
from DLBCL. Dave and coworkers47 studied the gene
expression profiles of 303 patients diagnosed with
aggressive B-NHL, including BL and DLBCL, and devel-
oped a two-step gene expression-based molecular clas-
sifier that allowed assignment of all but one case to the
diagnostic categories of BL, ABC DLBCL, GCB DLBCL
and primary mediastinal B-cell lymphoma (PMBL).
Cluster analysis of the genes included in the BL classifi-
er revealed several prominent gene expression signa-
tures providing further insights into molecular features
of BL. One gene cluster that was more highly expressed
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 109 |
Berlin, Germany, June 4-7, 2009
in BL compared to DLBCL included MYC and its target
genes, which was not unexpected since a deregulation
of MYC expression is a key oncogenic event in BL. Two
signatures were expressed at lower levels in BL com-
pared to DLBCL, one including MHC class I genes and
another including NF-κB target genes.47 In another
study, Hummel and colleagues performed48 gene expres-
sion and genetic profiling in 220 tumor specimens from
patients diagnosed with BL, DLBCL and unclassifiable
mature B-NHL by using Affymetrix U133 A gene
expression arrays, as well as comparative genomic
hybridization. A statistical approach called “core group
extension” defined an expression signature of 58 genes
that allowed the subdivision of the lymphomas into
molecular BL (mBL; 22%), non-molecular BL (non-mBL;
58%) and a relatively large group of intermediate cases
(20%). In accordance with the results from Dave et al.,47
several genes of the NF-κB signaling pathway were
found to be expressed at lower levels in the mBL group.
On the genetic level, the mBL cases were characterized
by relatively few chromosomal alterations apart from
the MYC translocation (low chromosomal complexity).
Conversely, the non-mBL (DLBCL) and intermediate
cases harbored significantly more chromosomal imbal-
ances (high chromosomal complexity), and the correla-
tion with clinical parameters revealed that the presence
of a MYC translocation in these groups was associated
with poor survival.48 Taken together, both studies iden-
tified a characteristic transcriptional profile of BL that
differed from that of DLBCL. In addition, however, they
described a subset of lymphomas that – according to
current WHO criteria – were classified as DLBCL, but
showed a gene expression profile characteristic of BL
“discrepant BL”.40,47-48 A subsequent study that combined
gene expression profiling and conventional CGH detect-
ed a high number of chromosomal alterations among
“discrepant BL”, which is in contrast to the low chromo-
somal complexity observed in typical BL. Since the
chromosomal alterations in the “discrepant BL’ sub-
group also differed somewhat from the alterations
observed in typical DLBCL, these cases might constitute
a separate molecular subgroup of aggressive B-NHL.
The group of “discrepant BL” appears to include so-
called dual translocation cases, that is, aggressive B-NHL
carrying both the MYC and a BCL2 (and/or BCL6)
translocation.40,47-48 Uniformly, these lymphomas were
reported to have a devastating clinical course.49-51
In summary, recent studies have provided new
insights into the molecular basis of BL that sharpened
the molecular delineation between BL and DLBCL.
However, there remains a subset of cases that cannot be
clearly assigned to the Burkitt or DLBCL category with
currently available morphological, immunophenotypi-
cal and molecular techniques. To reflect this “molecular
gray zone,” the latest WHO classification also suggests
a “diagnostic gray zone” category termed “B-cell lym-
phoma, unclassifiable, with features intermediate
between DLBCL and BL.” Importantly, this category
should not be viewed as a new and separate diagnostic
lymphoma entity; instead, it is realized that this group
of aggressive lymphomas not meeting the criteria for
classical BL or DLBCL, is molecularly, genetically and
clinically heterogeneous and needs further molecular
and clinical evaluation.
14th Congress of the European Hematology Association
EH and AR are supported by the Interdisciplinary Center for
Clinical Research (IZKF), University of Würzburg, Germany.
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 Diffuse large B cell lymphoma

Using molecular signatures to identify rational

therapeutic targets in diffuse large B-cell lymphomas

B. Chapuy
M. Shipp
Dana Farber Cancer Institute,
Harvard Medical School,
Boston, MA, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
A B S T R A C T
Diffuse large B-cell lymphomas (DLBCL) are heterogeneous disorders with significant differences in
clinical outcome, multiple genetic abnormalities and recognized pathological subtypes. Although clin-
ical prognostic classifications can be used to identify patients who are less likely to be cured with
standard therapy, these algorithms do not identify alternative treatment strategies. Transcriptional
profiling and associated genetic and functional analyses have increased our understanding of recog-
nized pathological subtypes of DLBCL and additional groups of tumors with shared features and
reliance upon targetable survival pathways. This review will focus on rational therapeutic targets that
have been identified by comprehensive molecular analyses of DLBCLs and translated into clinical ini-
tiatives.

2009;3:112-117

iffuse large B-cell lymphoma dard chemotherapy were previously evalu-

D (DLBCL) is the most common lym-

phoid malignancy in adults, account-
ing for nearly 30-40% of all non-Hodgkin’s
lymphomas (NHLs) in the Western world.1-3
Although a substantial fraction of patients
can be cured with the combination of anti-
CD20 and empiric antracycline-based
chemotherapy, up to 40% of the patients
relapse and ultimately succumb to their dis-
ease.4-7 In addition to being clinically hetero-
geneous disorders, DLBCLs exhibit multiple
genetic lesions and include recognized
pathologic subtypes.1,3
Earlier analyses of biologic heterogeneity
in DLBCL focused on individual genes asso-
ciated with treatment outcome, known
function in other malignancies, and normal
lymphocyte development.1,8 With more
comprehensive whole-genome techniques,
coordinate transcriptional signatures of spe-
cific groups of large B-cell lymphomas have
been defined and used to characterize rela-
tionships between certain tumors and nor-
mal cells of origin, define clinically recog-
nized and previously unidentified subtypes,
identify subtype-associated survival path-
ways and delineate signatures of outcome.9-19
Signatures of outcome - PKCβ in poor progno-
sis diffuse large B-cell lymphoma
Clinical risk stratification models, such as
the international prognostic index (IPI) can
be used to identify DLBCL patients who are
less likely to be cured with current
therapy.20-21 However, these clinical models
do not provide insights into molecular het-
erogeneity, pathogenetic mechanisms or
associated rational therapeutic targets. For
this reason, the transcriptional profiles of
DLBCLs with different responses to stan-
ated.9 Two of the genes and pathways asso-
ciated with poor response to standard regi-
mens in this initial study9 – the cyclic adeno-
sine monophosphate (c-AMP)—specific
phosphodiesterase, PDE4B and protein
kinase Cβ – prompted further analysis and
clinical evaluation.22,23 The association of
PKCβ expression with poor outcome and
shortened survival was confirmed in an
independent series of primary DLBCLs eval-
uated by immunohistochemistry.24
PKCβ is a serine/threonine kinase that
phosphorylates the scaffolding protein
CARD11 (CARMA1), which brings the
kinases transforming growth factor-activat-
ed kinase 1 (TAK1) and Iκ-inhibitor kinase
(IκK), into close proximity. This allows
TAK1 to phosphorylate IκK, the initial step
in activating the pro-survival NFκB path-
way.25-29 PKCβ is also a critical component of
the VEGF signaling pathway,30-32 which is of
note because VEGF-mediated tumor angio-
genesis has been linked with poor prognosis
in DLBCL.33,34 The expression of PKCβ in
high-risk DLBCLs and the role of the kinase
in critical signaling pathways including
NFκB and VEGF-mediated tumor angiogen-
esis suggested that PKCβ might be a rational
therapeutic target in DLBCL and prompted
analysis of candidate target inhibitors.
Enzastaurin HCI is an adenosine triphos-
phate-competitive, selective inhibitor of
PKCβ that induced apoptosis and inhibited
the proliferation of DLBCL, glioblastoma,
and colon carcinoma cell lines and
xenografts at low micromolar doses.35-36
After the doses and safety profile were
determined in a phase I study,37 we conduct-
ed a phase II multicenter trial of oral
Enzastaurin in patients with relapsed/refrac-

| 112 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

tory DLBCL.23 Twelve of fifty-five patients with
relapsed DLBCL (22%; 95% CI, 13-46%) experienced
freedom from progression (FFP) for greater than or equal
to two cycles, and eight patients remained free from
progression for greater than or equal to four cycles
(15%; 95% CI, 6-27%).23 Of particular interest, four
patients (7%; 95% CI, 2-18%) continue to experience
FFP 20 plus to 50 plus months after study entry.23 These
pilot data prompted the development of additional mul-
ticenter phase III trials of standard induction therapies
(CHOP/rituxan) with or without Enzastaurin as initial
therapy in patients with high intermediate/high risk
DLBCL.
Signatures of large B-cell lymphoma subtypes – NFkB in
MLBCL
In additional studies, investigators used transcrip-
tional profiling to delineate the signature of primary
MLBCL, distinguish this clinically distinct entity from
DLBCL and identify shared features of MLBCL and
classical Hodgkin lymphoma (cHL).11,15 Like cHL, pri-
mary MLBCL exhibited near-uniform nuclear localiza-
tion of cREL-containing NFκB heterodimers (Figure 1),
increased expression of NFκB target genes and
increased NFκB activity in functional assays.11,38,39
Although many patients with primary MLBCL
respond well to current combined modality therapy,
CHOP-Rituxan and mediastinal radiation, the long-
term sequelae of radiation remain a concern.40 As spe-
cific NFκB inhibitors became available for clinical tri-
als, it will be of interest to determine whether such tar-
geted agents could replace radiation in MLBCL.
Developmental signatures – NFkB in ABC-type diffuse
large B-cell lymphomas
A transcriptional profile-based model has been
described that relates subsets of DLBCLs to certain
stages of normal B-cell development.12-14 “Germinal cen-
ter (GC)-type” tumors share certain features with nor-
mal GC B cells and “activated B-cell (ABC)-type”
DLBCLs have additional traits in common with in vitro-
activated post-germinal B cells.12-14 A third poorly
defined group of tumors, “others,” includes 17-40% of
DLBCLs in recent series.10,14 Of clinical interest, the
GCB-type DLBCLs have a more favorable outcome in
comparison to the ABC-type tumors.13,14,16 The outcome
differences in DLBCLs defined by cell of origin (COO)
have not been consistently captured with more limited
3 or 4-parameter immunohistochemical signatures,
underscoring the importance of the more comprehen-
sive transcriptional profiles.41,42 In previous studies,
developmentally defined “ABC-type” DLBCLs were
found to be particularly dependent upon the NFκB sur-
vival pathway and NFκB activation signals mediated by
the upstream pathway components, CARD11, BCL10
and MALT1.43-45 These observations raise the interesting
possibility that ABC-type DLBCLs may be more sensi-
tive to targeted inhibitors of the NFkB pathway.
Although the NFκB pathway (Figure 1) is a promising
rational treatment target, available candidate inhibitors

Figure 1. NFkB Pathway in DLBCL and MLBCL. (A) Canonical and alternative NFkB pathways. Multiple stimuli activate I-
kappa-B kinase 1 (IkK), a member of the serine/threonine protein kinase family. Activated IkK itself phosphorylates IkB
proteins in the canonical NFkB pathway. The phosphorylation of IkB-proteins results in their dissociation from the NFkB
heterodimers, c-REL/p50 or RELA/p50, within the cytosol and the subsequently polyubiquitination and proteasomal
degradation of IkkB proteins. The liberated NFkB heterodimers translocate to the nucleus and induce target gene expres-
sion. In the alternative NFkB pathway, activated IKK phosphorylates and targets NFkB2 for proteasomal degradation, lib-
erating RELB/p52 for translocation to the nucleus and target gene induction. (B) Nuclear cREL in primary MLBCL.
Primary mediastinal B-cell lymphomas exhibit constitutive activation of the canonical NFkB pathway and localization of
nuclear c-REL by immunohistochemistry.11,38-39

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 113 |
14th Congress of the European Hematology Association
have either not been potent enough for clinical develop-
ment or have multiple other likely mechanisms of
action. For example, the proteasomal inhibitor, borte-
zomib, blocks the proteasomal degradation of the
cytosolic inhibitor of kappaB, IκKB, but also affects
many other cellular proteins and pathways.46 It is also
important to note the complexity of NFκB activation.47
Whereas MLBCLs and Hodgkin lymphomas rely, at
least in part, on c-REL containing heterodimers and the
canonical NFκB activation pathway, ABC-type DLBCLs
do not exhibit a cREL heterodimer-dependent immuno-
histochemical pattern or activation signature.38,44 As tar-
geted inhibitors of the NFκB pathway are developed
and evaluated in specific lymphoid malignancies, the
composition of NFκB heterodimers and the relative con-
tributions of the canonical and alternative NFκB path-
ways (Figure 1) will need to be considered.
Signatures of comprehensive clusters
Given the striking genetic heterogeneity and recog-
nized histologic variants of DLBCL, our group rea-
soned that clinically relevant disease subtypes
remained to be defined. Transcriptional profiling, an
analytical approach that selects the most stable num-
ber of discrete tumor clusters (consensus clustering),48
and additional functional studies were used to identify
three groups of DLBCLs: “Host Response (HR),”
“Oxidative Phosphorylation (OxPhos)” and “B-cell
Receptor (BCR) tumors”.10,38,49-51 OxPhos tumors have
increased expression of genes regulating oxidative
phosphorylation, mitochondrial function, electron
transport, and proteasomal degradation.10 BCR tumors
have increased expression of multiple components of
the B-cell receptor (BCR) signaling cascade and addi-
tional B-cell specific/B-cell essential transcription fac-
tors, such as BCL6.10 In contrast to the other two
groups, Host Response tumors were largely character-
ized by their prominent immune/inflammatory
response, including a prominent T-cell/dendritic cell
infiltrate.10,38,49
Consistent with their morphological features, pro-
file-defined HR tumors included a subset previously
identified as the DLBCL subtype, T-cell/histiocyte-rich
BCL (T/HRBCL).3,10 HR tumors shared clinical features
with morphologically defined T/HRBCLs, including
increased involvement of liver, spleen and bone mar-
row and younger age at presentation.3,10,52 Like
T/HRBCLs, HR tumors also had fewer known genetic
abnormalities. Of interest, primary HR tumors also
had a robust NFκB target gene signature, as did
microdissected primary tumor cells from
T/HRBCLs.17,38
BCL6 in BCR-type diffuse large B-cell lymphomas
BCR DLBCLs have increased expression of BCL6, a
transcription repressor required for normal germinal
center B cell development;53 these tumors also have
more frequent translocations of the BCL6 locus.10 Of
interest, these tumors also exhibit selective coordinate
regulation of BCL6 target genes.51 Consistent with
these observations, BCR tumors were significantly
more sensitive to a highly selective BCL6 peptide
inhibitor, BPI (Figure 2).51,54 These data suggest that
patients with BCR DLBCLs may be good candidates
| 114 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Figure 2. DLBCL subtypes have different sensitivities to
BCL6 inhibitors. BCR and OxPhos DLBCL cell lines exhibit
differential sensitivity to the BCL6 peptide inhibitor BPI.51
for therapeutic trials of BCL6 inhibitors when such
agents are available for clinical trials.
Tonic B-cell signaling in BCR-type diffuse large B-cell
lymphomas
As noted, BCR DLBCLs have a transcriptional profile
notable for increased expression of multiple compo-
nents of the BCR signaling cascade including the SYK
tyrosine kinase.10,50 Recent studies highlight the role of
B-cell receptor (BCR)-mediated survival signals in nor-
mal B cells and B-cell lymphomas.50,55-58 Engagement of
the BCR induces phosphorylation of Igα and β
immunoreceptor tyrosine-based activation motif
(ITAMS) and recruitment and activation of SYK and
downstream pathways (Figure 3). Although BCR signal-
ing is triggered by antigen binding, emerging data high-
light the role of “tonic” BCR survival signals in the
absence of receptor engagement. In murine models,
which illustrated the role and consequences of tonic
BCR signaling, loss of the BCR or the excision of the Igα
ITAM triggered apoptosis of peripheral B cells.59-60
The protein tyrosine kinase SYK plays a critical role in
tonic BCR signaling, transmitting downstream events
and amplifying the original signal (Figure 3). The activi-
ty of SYK is tightly regulated by BCR-associated phos-
phorylation and protein tyrosine phosphatase-mediated
inhibition.56,57,61,62 In recent studies, we found that over-
expression of the SYK phosphatase, PTPROt, decreased
cellular proliferation and induced apoptosis of BCR-
dependent lymphomas in the absence of BCR crosslink-
ing.50,58 These studies indicate that these tumors depend
upon SYK-mediated tonic BCR survival signals.
For these reasons, we recently assessed the role of
SYK-dependent tonic BCR survival signals in primary
DLBCLs and DLBCL cell lines and evaluated the in vitro
efficacy of a competitive SYK inhibitor, R406 (Figure
4).50 R406-mediated SYK inhibition has already been
evaluated in murine models of allergen-induced airways
hyper-responsiveness63 and rheumatoid arthritis.64 R406
 Berlin, Germany, June 4-7, 2009

induced apoptosis in the majority of DLBCL cell lines

and primary tumors and specifically inhibited tonic
BCR signaling (Figure 4). The BCR-dependent, R406-
sensitive DLBCL cell lines were independently found to
have the “BCR-type” transcriptional profiles, suggesting
that BCR-dependent DLBCLs might be identified by
their coordinate molecular signature.50
The in vitro studies indicated that SYK-dependent
tonic BCR signaling was a targetable survival pathway
in certain B-cell lymphomas, prompting further clinical
investigation. A phase I/II trial of an oral version of the
SYK inhibitor, R788/fostamatimib disodium (FOS D),
was recently completed.65 After confirming an appropri-
ate dose for phase II testing, the oral SYK inhibitor was
evaluated in relapsed/refractory DLBCL, follicular lym-
phoma and additional B-cell malignancies. In this clini-
cal trial, there was clear evidence of activity in DLBCL.65
Additional clinical trials are planned.

Figure 3. B-cell-receptor signaling. B-cells and a subset of
DLBCLs rely on B-cell-receptor (BCR) mediated survival
signals. BCR signaling induces phosphorylation of Igα and
β immunoreceptor tyrosine activation motifs (ITAMs) by
SRC family kinases, such as LYN. ITAM phosphorylation
results in recruitment of the spleen tyrosine kinase SYK,
which initiates downstream events and amplifies the orig-
inal signal.
Future directions
Emerging data from the analyses of coordinate molec-
ular signatures of subsets of large B-cell lymphomas
suggest that groups of tumors rely on specific survival
pathways that may represent rational treatment targets.
As clinical trials of targeted agents are implemented, it
will be important to have practical methods of identify-
ing tumors that rely upon specific functional pathways.
To date, immunohistochemical signatures based on
components of coordinate transcriptional profiles have
been a major focus.39,42

Figure 4. Inhibition of
SYK-mediated BCR
signaling in DLBCL cell
lines and primary
DLBCLs. SYK inhibi-
tion selectively
induces apoptosis in
BCR-type DLBCL cell
lines.50 (A) Single cell
phosphoflow cytome-
try can be used to
assess SYK-dependent
phosphorylation of the
target protein BLNK in
the absence (green) or
presence (red) of BCR
crosslinking in R406-
sensitive and resistant
DLBCL cell lines.50 (B)
Primary DLBCLs.50 (C)
Only DLBCLs that
exhibit SYK-dependent
BCR-signaling are sen-
sitive to R406 mediat-
ed SYK inhibition.50

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 115 |
14th Congress of the European Hematology Association
Recent studies suggest that it may be possible to
obtain transcriptional profiles from fixed paraffin-
embedded tumor specimens.66 Another alternative is to
develop functionally relevant target signatures suggest-
ed by candidate survival pathways.50 Future directions
will likely include the analysis of rational targeted
agents in combination with standard empiric
chemotherapy, antibody therapy, and in association
with complementary specific target inhibitors.
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 Diffuse large B cell lymphoma

Treatment options for relapsing patients with diffuse

large B cell lymphoma

C. Gisselbrecht A B S T R A C T

Institut d’Hématologie,
Hopital Saint Louis, Paris, France
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:118-122
Salvage chemotherapy followed by high dose therapy and autologous stem cell transplantation
(ASCT) is the standard of treatment for chemosensitive relapses in diffuse large B cell lymphoma.
Selection of salvage regimen was never done by a randomized study. To answer this question, the
intergroup CORAL trial included lymphoma patients with DLBCL CD 20+ in first relapse or patients
refractory after first line therapy, who were randomized between R-DHAP and R-ICE. Responding
patients received BEAM and ASCT. Intent to treat analysis was made on 396 randomized patients (R
ICE:202; R DHAP:194). The overall response rate was 63%, with 38% complete remission. There was
no difference in response rate between R-ICE 63.5% and R-DHAP 62.8%. ASCT was performed for 206
patients. There was no significant difference between R-ICE and R-DHAP for 3 year EFS 26% vs. 35%
(p=0.6) and OS 47% versus 51% (p=0.5). EFS was affected by: prior treatment with rituximab; early
relapse less than 12 m; secondary IPI 2-3. On going studies are evaluating new conditioning regimens
with radioimmunotherapy, other combinations of chemotherapy and immunotherapy post transplant
as in CORAL study. Early relapses/ refractory patients to upfront rituximab-based chemotherapy have
a poor response rate and prognosis. New approaches are warranted. Chemotherapy has a poor
response rate and prognosis. A better biological understanding of these patients and new approaches
are required.

ggressive lymphoma patients who (p=0.076). In the overall cohort, patients

A relapse or fail to achieve a CR have a

poor outcome with a life expectancy
of 6 months. Since less than 10% of these
patients obtain long-term disease-free sur-
vival1 with a salvage regimen alone, it has
long been established that salvage chemo-
therapy should, whenever possible, be fol-
lowed by consolidation with HDT and then
ASCT in a chemosensitive patient.
All patients are now treated with front line
rituximab (R) and chemotherapy. The analy-
sis of registry data from patients treated
with R CHOP confirmed that a major
improvement in the treatment of diffuse
large B cell lymphoma is observed in the
general population.2
Fewer relapses are seen among patients
with 0-2 IPI factors (10-20%); however, 50%
of relapses are still seen for patients with
more than 2 IPI factors.
In the absence of transplantation, the out-
come of relapsing patients is still poor. In the
long-term analysis of data from the LNH 98-
5 trial, comparing CHOP and R CHOP3
among patients who received first-line treat-
ment with CHOP, those who received ritux-
imab-containing salvage therapy at relapse
achieved significantly longer survival than
those who underwent salvage therapy with
chemotherapy alone (p=0.00043). In con-
trast, among those patients who received R-
CHOP as first-line therapy, the survival of
patients who underwent salvage chemother-
apy plus rituximab at relapse did not differ
significantly from the survival of those who
received salvage chemotherapy alone
given salvage chemotherapy plus rituximab
achieved significantly longer survival at 2
years than those who received salvage
chemotherapy without rituximab (2-year
OS: 58% vs. 24%; p=0.00067).3
Selecting a salvage regimen
Various old and new drugs are treatment
options for DLBCL in the salvage setting.
The effectiveness of these agents has been
evaluated mainly in non-randomized stud-
ies, and the difficulty of obtaining a cure or
a prolonged disease-free period with con-
ventional salvage chemotherapy may
explain the large number of phase II studies
that have been conducted in this setting.
Consequently, salvage regimens outcomes
are generally expressed as response rates and
the possibility of collecting stem cells for
ASCT. Survival data very often represent a
mixture of transplanted patients and those
not eligible for transplantation. No clear
superiority of one regimen over the other
has been demonstrated in the absence of
randomized study.
Advances in salvage therapy are needed
for two reasons: first, to overcome resistance
to chemotherapy, enabling more patients to
achieve a CR, thus allowing suitable candi-
dates to proceed to transplantation and, sec-
ond, to optimize transplantation procedures.
The addition of rituximab to CHOP
chemotherapy has significantly improved
the CR rate, event-free and overall survival
rates compared to CHOP alone, as first-line
treatment of aggressive NHL, without

| 118 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

increasing toxicity.4 Thus, combining rituximab with
ICE (R-ICE), one of the most effective salvage regimens,
has been given to patients with relapsed/refractory dis-
ease. Results from the first 36 assessable patients who
had relapsed (n=23) or refractory (n=13) disease follow-
ing a single standard anthracycline-based treatment for
diffuse large B-cell lymphoma have been reported.5-6
The overall response rate was 78% and the CR rate
53%. The CR rate was significantly higher for patients
receiving R-ICE than historical controls, with a similar
second-line IPI given ICE (p=0.006). Patients with
relapsed disease had a significantly higher overall
response rate than those who had primary refractory
disease (96% vs. 46%; p=0.01). Several phase II studies
with various regimens have been reported going in the
same direction (Table 1).
The clear demonstration is provided by a prospective
randomized trial exploring the potential benefits associ-
ated with the addition of rituximab to platinum-based
salvage regimens. In the study conducted by the
HOVON group, 239 patients with relapsed or refracto-
ry DLBCL received a salvage regimen consisting of
DHAP-VIM-DHAP, with or without rituximab, fol-
lowed by ASCT. Analysis of the 225 patients evaluable
showed that after two courses of chemotherapy, PR/CR
was obtained in 54% of the patients in the DHAP arm
and 75% in the R-DHAP arm (p≤01; intention-to-treat
analysis). Post-transplantation PR/CR was obtained in
50% and 73% of the patients, respectively (p=0.003). A
marked difference in favour of the R-DHAP arm was
observed at 24 months for failure-free survival, 50%
versus 24% (p<0.001) but for OS 52% vs. 59%
(p=0.15).7 Cox regression analysis demonstrated a sig-
nificant effect of rituximab treatment on FFS and overall
survival when adjusted for time, since upfront treat-
ment, age, performance status and secondary age
adjusted or secondary IPI. However, less than 5% of the
patients had been previously exposed to rituximab.7
What is the optimal chemotherapy regimen to com-
bine with rituximab as salvage therapy for DLBCL? The
CORAL intergroup trial compared the association of rit-
uximab, Ifosfamide, etoposide, carboplatinum, R-ICE
and rituximab dexamethasone aracytine and cisplat-
inum R-DHAP. DLBCL CD 20+ in first relapse or
patients’ refractory after first line therapy were random-
ized between R-DHAP and R-ICE. Responding patients
received BEAM and ASCT and were randomized
between observation and maintenance with rituximab
for 1 year. Intent to treat analysis was made on the first
396 patients randomized in 11 countries (R ICE:202; R
DHAP:194).8 The median age was 55 years. In 225
patients, a relapse greater than 12 months was observed
after initial complete remission. In 166 cases, patients
did not achieve initial complete remission (refractory) or
had an early relapse in less than 12 months. Two hun-
dred and forty-four patients were treated with combi-
nation chemotherapy with prior exposure to rituximab.
At the time of inclusion in the study, there were 240
patients with Stage III to IV, 198 patients with elevated
LDH. At relapse, 226 patients had a secondary IPI 0 to 1
and 149 patients, sIPI 2 to 3. Patients with prior expo-
sure to rituximab had more refractory disease and
adverse prognostic factors. The overall response rate
was 63%, with 38% complete remission. There was no
difference in response rate between R-ICE 63.5% (CI:
56-70%) and R-DHAP 62.8% (CI: 55-69%), and in

Table 1. Rituximab in combination with platinum-containing regimens.

Regimen Disease status n ORR/CR ASCT performed Survival in ASCT patients

R-ICE5 DLBCL 36 78%/53% 70% 67% OS at 2 years

R-ICE26 Aggressive B NHL 8 75%/50% 100% Not given
R-various (mostly ICE)27 DLBCL 59 Not given 100% 81% OS at 2 years
R-ICE28 Aggressive B NHL 11 89%/67% 27% Not given
R-DHAP29 Aggressive B NHL 53 62%/32% 38% Median 20.4 months OS
DHAP/VIM/DHAP ± R7 Aggressive B NHL 225 75%/46% vs. 63% vs. At 2 years FFS 50% vs. 24% in
54%/35% 46% ± R, favour of R (p<0.001)
± R, respectively respectively
R-ESHAP30 DLBCL/MCL/HD 24 (15 = DLBCL) Not given 79% DFS=53%
R-ESHAP31 DLBCL/MCL 6 100%/67% Not done Not applicable
R-ESHAP32 Aggressive B NHL 26 92 %/46% 88% median OS and PFS not yet reached
R-ESHAP33 Aggressive B NHL 18 56%/28% 38% Not stated
R-ICE or R-DHAP34 DLBCL 10 60%/10% Not done Not applicable
R-ASHAP35 Aggressive B NHL 20 75%/45% 25% Not stated
R-DHAOX36 NHL 33 (10 were HIV+) 70%/27% Not stated Not stated
R-ICE or R-DHAP followed by Relapsed DLBCL 396 63%/38% 52% (n= 204) 49% OS at 3 years
HDT (BEAM) and ASCT ± R
maintenance8
*Planned interim analysis for the first 400 patients. ASCT: autologous stem cell transplant; ASHAP: doxorubicin, methylprednisolone, cytarabine, cisplatin; CR; com-
plete response, DFS: disease-free survival; FFS: failure-free survival; DHAOX: dexamethasone, oxaliplatin; DHAP: dexamethasone, cytarabine, cisplatin; DLBCL, diffuse
large B-cell lymphoma; ESHAP: etoposide, methylprednisolone, cytarabine, cisplatin; FU: follow-up; HD, Hodgkin’s disease; ICE: ifosfamide, carboplatin, etoposide;
HDT: high dose therapy; MCL, mantle-cell lymphoma; NHL, non-Hodgkin’s lymphoma; ORR: overall response rate, OS: overall survival; PFS: progression-free survival;
R: rituximab; VIM: etoposide, ifosfamide, methotrexate.

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 119 |
14th Congress of the European Hematology Association
mobilization adjusted response rate 52% vs. 54%.
Factors significantly affecting response (p<0.0001) were
refractory/relapse less than 12 months with a response
rate of 46% vs. 88%, secondary IPI greater than 1: 52%
vs. 71% and prior exposure to rituximab: 51% vs. 83%.
There were fewer serious adverse events in the RICE
regimen when compared to R DHAP.
From this first randomized study on relapses, there
was obviously no difference in response rate and the
ability to mobilize stem cell between the two major reg-
imens used around the world in DLBCL.
Prognostic factors at relapse
Only 206 ASCT were performed in the CORAL
study, with an unexpected drop out of 50%, mainly due
to tumor progression. The same drop out was observed
in the PARMA study. There was not significant differ-
ence between R-ICE and R-DHAP for 3-year event free
survival (EFS 26% vs. 35% p=0.6) and overall survival
OS (47% vs. 51%, p=0.5), respectively. Three years EFS
was affected by prior treatment with rituximab, 21% vs.
none 47% (p<0.0001); early relapse less than 12 months
20% versus greater than 12 months 45% (p<0.0001);
secondary IPI 2 to 3: 18% vs. 0 to 1: 40% (p=0.0001). In
the Cox model, all these parameters were significant
(p<0.0001) for EFS, PFS and OS but not the treatment
arm. As early relapse less than 12 months was the main
prognostic factor. We looked in the population with
relapse greater than 12 months if prior exposure to rit-
uximab will or will not affect the outcome. There was
no difference in EFS, OS between these two subgroups
with or without rituximab exposure. Obviously, the
response rate is also affected by other parameters such
as age, time to progression, less than 1 year or not and
secondary international prognostic index IPI9,10 but now
prior exposure to rituximab should be add.
Due to the introduction in the armamentarium of
very efficient monoclonal antibody, we are now experi-
encing patients with early relapse more refractory to
any available treatment.
PET scan prior to transplant
There is a need to improve salvage regimen with new
drugs or experimental strategy. PET scans have been
incorporated in the definition of response. The quality
of response before transplant is highly predictive of the
outcome. FDG-PET can also be of prognostic value
when performed prior to autologous stem cell therapy
(ASCT) in patients with NHL who had previously
achieved remission and were receiving consolidation
ASCT. Results indicated that a PET-positive result prior
to ASCT was associated with a poor duration of
response and a cumulatively higher risk of relapse, par-
ticularly if the post-ASCT was still PET positive.11
Conversely, a PET-negative result prior to ASCT would
predict a better duration of response and suggested that
further PET-based investigations post-ASCT would not
be mandatory.11 This method is more sensitive to pre-
dict a low rate of relapse in PET-negative patients.
Standardization of the procedure is in progress to avoid
false positive results. Nevertheless, it still provides a bet-
ter evaluation of response and who could be a candidate
for a more experimental approach.
| 120 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Improving conditioning regimen
Monoclonal antibodies, owing to their targeted effica-
cy, have now become established components of thera-
peutic regimens to combat hematological malignancies.
The development of radiolabelled immunotherapies,
such as 90Y-ibritumomab tiuxetan has capitalized on the
targeting ability of antibodies to deliver therapeutic
doses of radiation to disseminated tumour sites. In a
number of independent studies in the transplant setting,
90
Y-ibritumomab tiuxetan has been shown to have a
promising role as a component of conditioning regi-
mens, not only to augment standard chemotherapy reg-
imens (immunotherapy-enhanced conditioning) but
also as the sole conditioning agent, extending the option
of transplantation to a wider range of patients.12-19 In
ASCT, the use of 90Y-ibritumomab tiuxetan in combina-
tion with HDT has no effect on the speed of engraft-
ment and has a toxicity profile similar to conventional
conditioning regimens.13,17 Further phase II, and subse-
quent phase III, studies are now needed to evaluate the
role of 90Y-ibritumomab tiuxetan (Zevalin) in combina-
tion with HDT (e.g., Z-BEAM vs BEAM) or alone prior
to ASCT. These studies should investigate different
patient subgroups and analyse outcomes according to
histology.18-19
Rituximab as post-transplantation maintenance/
consolidation
In a study in which rituximab was used during stem
cell mobilization (for in vivo purging prior to stem cell
harvest) and for consolidation of response at days 1 and
8 after transplantation in patients with relapsed aggres-
sive NHL, the 2-year OS rate was 80%, compared with
53% in historical controls who underwent ASCT with-
out rituximab (p=0.002). Similarly, the DFS rate after a
median follow-up of 20 months was 67%, compared
with 43% in a historical control group (p=0.004).20
Rituximab can also consolidate the response to trans-
plantation when given once weekly at weeks 4 to 8
after transplantation. Of 26 patients with relapsed NHL
who received post-transplantation rituximab on this
schedule, 7 showed measurable responses in sites of
known disease involvement.21 Similarly, in a separate
study, rituximab was given once weekly at weeks 4 to 8
after salvage ASCT and repeated if needed over 4 weeks
at month 622 to 21 patients with relapsed or refractory
large-cell lymphoma. After a median follow-up of 30
months, the EFS rate was 81% and the OS rate was
85%.22 As most patients with DLBCL receive rituximab
as a component of their first-line treatment, the role of
adjuvant immunotherapy after salvage therapy with or
without ASCT should be defined more precisely
through randomized studies. In the CORAL trial,
patients with relapsed DLBCL were randomised after
ASCT to either rituximab maintenance for 1 year or
observation. The study is now completed and 240
patients have been randomized in the second part. A
longer follow up is necessary before providing results.8
Rituximab and other chemotherapy regimens without
transplantation
However, not all patients are candidates for transplan-
tation because of age, previous transplantation and/or
comorbidity. Therefore, effective and well-tolerated sal-

vage therapies with minimal toxicities are still needed.
More recently, oxaliplatin and gemcitabine have also
been shown to be active separately against relapsed or
refractory NHL. A study on 24 patients assessed the
efficacy and safety of oxaliplatin replacement of cis-
platin in the DHAP regimen for patients with relapsed
or refractory NHL.23 Because the objective response rate
was 77% for patients treated at first relapse with no
major renal and neurotoxicity, this regimen is particular-
ly suitable in elderly patients.23 In hematologic malig-
nancies, gemcitabine alone had some degree of efficacy
against aggressive NHL. The combination of rituximab-
platinum and gemcitabine, GEMOX, seems promising
and is currently being evaluated. In a single-arm study
involving 46 patients with aggressive B-NHL who
received up to eight cycles of rituximab, gemcitabine
and oxaliplatin in combination (R-GEMOX), 38 patients
(83%) responded after four cycles and 50% were in CR
or CRu by the end of treatment.24 With a median follow
up of 27 months, the 2-year and overall survival rates
were 53% and 66%, respectively. The estimated medi-
an time to progression (TTP) was 20 months (range 1.6
to >33.5 months) and median duration of response (DR)
was 18 months (range 0.5 to >31.4 months). In addition,
this platform was chosen to investigate the combination
of chemotherapy and enzastaurin. Enzastaurin is a
potent, selective inhibitor of PKC and (PI3K)/protein
kinase 3 (AKT) pathways known to promote tumor-
induced angiogenesis, as well as tumor-cell survival and
proliferation.25 A phase II trial with oral enzastaurin plus
GEMOX is ongoing. Oral enzastaurin is also tested as
maintenance after the end of chemotherapy. The devel-
opment of new monoclonal antibodies, new generation
of anti CD 20, anti CD 40, anti CD 22, or more sophis-
ticated immunoconjugate anti CD22 with calichea-
mycin (CMC 544) are now evaluated in the clinic, as
well as lenalidomide which has show promising activi-
ties in lymphoma. At present, no real “gold standard”
treatment exists for relapsed aggressive NHL. Because
the goal of salvage therapy for aggressive NHL is CR,
and because the major breakthrough in the treatment of
B-cell malignancies was the addition of rituximab to
chemotherapy for front-line treatment, most physicians
are in favor of including rituximab in salvage
chemotherapy regimens.
Conclusion
Most regimens are giving the same response rate and
there is no difference between the two most widely
used salvage treatments, R ICE and R DHAP. According
to the main prognostic factors, response rates range
from 80-50% and 3 years event free survival from 60-
30%. If the introduction of rituximab was the main
progress in the treatment of B cell lymphoma, patients
failing to respond to rituximab combination chemother-
apy have a very poor prognosis and can be candidates
for more experimental innovative treatments. Any
progress can be easily detected in a poor prognosis
patient. Enrolment in large randomized study will be
more problematic in the absence of significant progress
in response rate. From the CORAL study, we started,
with the help of pathobiologists a Bio-CORAL program,
as we need to better understand poor prognosis patient
and to design new approaches.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 121 |
Berlin, Germany, June 4-7, 2009
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 Hematopoiesis

Anatomy of the hematopoietic stem cell niche

during development

E. Dzierzak A B S T R A C T

Erasmus Medical Center, Erasmus
Stem Cell Institute, Dept of Cell
Biology
Rotterdam, The Netherlands
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:123-127
Stem cell microenvironments and their component elements, particularly the growth factors and
signalling pathways that play a role in the survival, expansion and/or physiologic function of stem cells
are of fundamental interest in the field of stem cell biology. Within the adult blood system,
hematopoietic stem cells (HSCs) are supported in the osteoblastic and vascular niches of the bone
marrow. Genetic manipulations within the mouse bone marrow microenvironment have led to the
identification of some of the molecules involved in HSC homing, maintenance and differentiation.
During ontogeny, in early stages of HSC development, several anatomically distinct tissues serve as the
hematopoietic-supportive microenvironment and include the yolk sac, aorta-gonad-mesonephros
(AGM) region, placenta and fetal liver. While the first HSCs are known to emerge autonomously in the
mouse AGM region at midgestation, little is known about the potent microenvironment that promotes
the generation of these stem cells. The current knowledge on the AGM microenvironment, its cellular
complexity and its relationship to the generation and maintenance of the first HSCs is presented here.

ematopoietic stem cells (HSC) are the niches.11,12 Some of the key molecular regula-

H foundation of the adult hematopoiet-

ic system and sustain the life long
production of all blood lineages. HSCs are
maintained in specialized microenviron-
ments throughout development. In the
adult, the bone marrow harbors the majori-
ty of HSCs.1 Genetic studies first demon-
strated the importance of the HSC – niche
interaction [reviewed in (2)]: mouse strains
deficient in the c-kit receptor tyrosine kinase
(W mice) and kit-ligand (KL; Steel mice)
showed that while bone marrow from W
mutant mice could not repopulate the
hematopoietic system of wild type irradiat-
ed recipient mice, bone marrow from Steel
mutant mice could.3 Yet, the Steel bone mar-
row was defective for the support of wild
type hematopoietic cells. Thus, within the
bone marrow microenvironment, the c-kit-
KL signalling axis is important for hemato-
poietic progenitor/stem cell maintenance
and function. Together with the develop-
ment of methods to culture this complex
microenvironment ex vivo,4-6 further studies
dissecting the cellular and molecular aspects
of the bone marrow microenvironment
were stimulated.
Adult hematopoietic supportive
microenvironment
The bone marrow microenvironment is
very complex, containing osteoblastic niches
and vascular niches within the trabecular
regions of the long bones.7-9 HSCs are sup-
ported and maintained in close association
to the so-called “stromal cells” of the niches10
and live-animal tracking of hematopoietic
progenitor/stem cells has shown the homing
ability of these cells to the bone marrow
tors within the bone marrow niches include
N-cadherin, CD150, BMPR1A, and the
SDF1/CXCR4, Notch, Wnt, Hedgehog,
Tie2/Angiopoietin, TGF and FGF signaling
pathways.13,14 Stromal cell lines derived from
the bone marrow and fetal liver tissues have
been instrumental in further characterization
of these hematopoietic supportive niches.
Many stromal cell lines have been generated
and are characteristically heterogeneous. A
few stromal cell lines maintain HSCs ex vivo
in co-cultures for long periods.6,15,16
Transcriptional profiling of HSC support-
ive/non-supportive stromal cell lines has
revealed a complex genetic program involv-
ing a wide variety of known molecules and
molecules whose function in hematopoiesis
is unknown,17-20 suggesting the concerted and
perhaps overlapping actions of stromal mol-
ecules/cells in supporting HSCs. These
results provide a basis for the study of the
microenvironment throughout develop-
ment.
Microenvironments important for hematopoi-
etic development in the conceptus
Prior to the necessity for a supportive
microenvironment, developmental microen-
vironments promote the generation of
hematopoietic progenitor and stem cells.
Several tissues early within the development
of the conceptus are able to promote the
generation of these cells from precursors
called hemangioblasts (the common meso-
dermal precursor to both endothelial and
hematopoietic lineages) or hemogenic
endothelium (endothelial cells that retain
potency for the hematopoietic lineage)
[reviewed in (21)]. Recent cell tracing exper-

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 123 |
14th Congress of the European Hematology Association

iments in the mouse have demonstrated that the

founder hematopoietic cells for the adult hematopoietic
system are generated during a specific window of devel-
opmental time – in the mouse, this is approximately
between embryonic day (E) 10 and E12.22 Both extraem-
bryonic tissues (yolk sac and placenta) and the intraem-
bryonic AGM are sites of hematopoietic cell genera-
tion,23-26 but it is as yet uncertain whether one or all of
these sites contribute cells to the adult. Following their
generation, the hematopoietic progenitor and stem cells
are thought to migrate to the fetal liver,27 where they are
maintained/expanded until the bone marrow microen-
vironment is established. As shown in Figure 1, HSCs
are found in all the hematopoietic sites of the concep-
tus.21 While the AGM has been shown to autonomous-
ly generate HSCs,25 it is as yet unclear whether the
extraembryonic sites can also generate HSCs.
Nonetheless, the fact that HSCs are found at in all the
Figure 1. Generation and migration of hematopoietic stem
hematopoietic sites of the conceptus, suggests migra- cells throughout development. Hematopoietic stem cells
tion between anatomically distinct tissues that provide (HSC) begin to be generated in the AGM region at embry-
HSC supportive microenvironments. As the site where onic day (E)10.5 in mouse gestation and between weeks 4
to 6 of gestation in the human embryo. HSCs generation
the first HSCs are generated, investigations have continues only during a short window of time – most like-
focused on the AGM midgestation microenvironment ly until E12/13 in the mouse conceptus. It is yet unknown
to provide molecular insights into the how HSCs are whether the extraembryonic tissues, yolk sac and placenta,
established. can generate HSCs. HSCs migrate and colonize the fetal
liver, and subsequently, the bone marrow at the indicated
times. Red dots indicate HSCs.
The aorta-gonad-mesonephros microenvironment
AGM HSCs are thought to be derived from aortic
endothelial cells, progressing through an endothelial cell
stage that expresses VE-cadherin.22 Cells expressing
hematopoietic markers28-30 are closely adherent to the
vascular endothelium of the aorta at midgestation and
are thought to bud from these endothelial cells into the
lumen as they take on HSC identity (Figure 2). Such
hematopoietic clusters appear at E10 in the mouse aorta
and the first adult-repopulating HSCs are autonomous-
ly generated in the aorta at E10.5 (>34 somite pairs).25,31,32
Although clusters are found both dorsally and ventrally,
HSC activity is localized exclusively to the ventral
aspect of the mouse midgestation aorta, suggesting a
strong positive ventral positional influence in the devel-
opment of AGM hematopoiesis.33 Indeed, hematopoiet-
ic transcription factors, such as Gata2 and Runx1
(required for HSC generation) are expressed in cells of Figure 2. Model for hematopoietic stem cell generation
from hemogenic endothelium. The first generation of
the ventral clusters and endothelium.29,33-35 Several devel- hematopoietic stem cells (HSC) has been localized to the
opmental factors and signaling molecules involved in ventral wall of the dorsal aorta. Based on endothelial and
these pathways are also expressed in the ventral aortic hematopoietic marker expression, HSC generation occurs
during the transition from an endothelial phenotype
region at midgestion, including BMP4.37,38 Within the (hemogenic endothelium) to a hematopoietic phenotype. It
normal physiology of the embryo, the AGM lies is thought that HSC emerge from the endothelial wall (blue
between the ventral tissue that includes the endoderm- cells) and bud into the lumen of the aorta, resulting in
derived gut and the dorsal tissue including the ecto- closely adherent clusters of hematopoietic cells (orange
cells). The inductive factors (blue and green arrows) driving
derm-derived notochord and neural tube. Using AGM the generation of HSCs are thought to come from the mes-
explant cultures, we have preliminary data suggesting enchymal cell layer (beige cells) underlying the aorta and
that dorsal tissues/signals repress AGM HSC activity from ventral endodermal tissues (not shown). These fac-
tors act in concert to drive the expression of transcription
and ventral tissues/signals enhance HSC emergence. factors, such as Gata2 and Runx1 to turn on an HSC genet-
Thus, morphogens and local signals emanating from the ic program. h:hematopoietic cell; e:endothelial cell;
ventral endodermal tissues may be responsible for m:mesenchymal cell.
induction of HSCs.

Mesenchymal stromal cells

What are the cells that make up the hematopoietic are the stromal cells of this microenvironment. It is
generative and/or supportive microenvironment of the known that the AGM region contains different types of
conceptus? In the adult bone marrow, mesenchymal mesenchymal stem/progenitor cells. As reported by
stem/progenitor cells and their differentiating progeny Minasi et al.39 in the quail embryo, a population of aorta-

| 124 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

associated stem cells contributes to cartilage, bone and
muscle tissues and also to blood,when transplanted into
chick recipients. Based on its differentiation potential,
this cell was called a “meso-angioblast.” Cells with
more restricted lineage differentiation potential have
been localized to the mouse AGM region and to the
other major hematopoietic tissues during mouse
ontogeny. These are characteristically mesenchymal
stem/progenitor cells. Interestingly, mapping and fre-
quency analysis of mesenchymal progenitors in the
mouse conceptus show that mesenchymal progenitors,
with the potential to differentiate into cells of the
osteogenic, adipogenic and/or chondrogenic lineages,
reside in most of the sites harboring hematopoietic cells.
Mesenchymal progenitors appear in the AGM region at
the time of HSC emergence,40 suggesting a functional
coordination during development of the mesenchymal
and hematopoietic lineages. Many stromal cell lines
have been established from the AGM region, placenta
and fetal liver, and the yolk sac has yielded a few stro-
mal cell lines.16,41-47 We previously isolated and character-
ized stromal cell lines from the AGM subregions (aorta
and urogenital ridges) and also from the embryonic liver
and ventrally located gut.44,45 Most of these clones were
derived from transgenic mice expressing the thermola-
bile form of the SV40 Tag gene under the control of the
β-actin or PGK (phosphoglycerate kinase) promoters.
Phenotypic characterization places the AGM stromal
lines in the vascular smooth muscle cell hierarchy
(VSMC) in between a mesenchymal stem cell and a
VSMC.48 In vivo and in vitro assays show that some of the
AGM stromal clones are potent supporters of
hematopoietic progenitors and HSCs as compared to
adult bone marrow and fetal liver cell lines.44 Hence, the
AGM cell lines can provide important signals for the
maintenance of the first HSCs. Indeed, some of these
lines can support the hematopoietic differentiation of
embryonic stem cells.49 Similar to the mesenchymal dif-
ferentiation potential of bone marrow and fetal liver
stromal cell lines, AGM cell lines were able to give rise
to osteoblasts, adipocytes and endothelial cells.50 Even
after differentiation, osteoblastic stromal cells continue
to support the growth of hematopoietic cells.37,50 These
results suggest that the AGM hematopoietic microenvi-
ronment within the AGM region is, in some ways, very
similar to that in the bone marrow. Yet its ability in vivo
to provide HSC inductive signals suggests that it is more
complex. While one AGM cell line, AGM-S3, was
reported previously to promote the generation of HSCs
from early mesodermal precursors,51 this result has not
been repeated and no other AGM cell lines have been
shown to yield HSC inductive effects. Insights into the
molecular identity of the AGM microenvironment have
been provided by transcriptional profiling of closely-
related AGM stromal clones that differentially support
HSCs.20,37 Expression analyses identified putative HSC
regulatory factors - two novel regulators, β-NGF (a neu-
rotrophic factor) and MIP-1γ (a C-C chemokine family
member) and one known regulator, BMP4 (a TGF-β
family member). BMP4 was previously shown to act at
the mesodermal and primitive erythropoietic stages.52,53
When added to AGM explant cultures, these three fac-
tors enhance the in vivo repopulating ability of AGM
HSCs.37 E11 AGM HSCs express BMP receptors and can
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 125 |
Berlin, Germany, June 4-7, 2009
be inhibited in their activity by gremlin, a BMP antago-
nist. Interestingly, a ventral localization of BMP4 expres-
sion in the mesenchyme underlying HSC-containing
aortic clusters was found at E11 .37 This finding confirms
the ventral expression previously reported in the human
AGM region,38 and proves that BMP4 plays a relatively
late role in the regulation of HSCs as they emerge in the
midgestation AGM. BMP4 does not act as a survival fac-
tor but may promote the differentiation and/or expan-
sion of AGM HSCs. Earlier acting endodermal factors
are suggested to effect the transition of mesoderm to
hematopoietic differentiation. The formation of primi-
tive erythroblasts in the chick yolk sac requires the
inductive influence of endodermal cells,54 and avian
somatopleural mesoderm is respecified to exhibit
hematopoietic and vascular potential when briefly
exposed to endoderm.55 Remarkably in mouse embryo
explant grafts, visceral endoderm can induce erythroid
development in prospective neuroectoderm56 and
hedgehog proteins were shown to mimic these effects.57
Studies in Zebrafish embryos show that hedgehog is
involved at three distinct stages in dorsal aorta and
hematopoietic development.58 Taken together, these
studies suggest that ventral positional information in
the endoderm promotes hematopoietic induction. We
have tested the hypothesis that tissues positioned dor-
sal or ventral to the AGM affect HSC induction and test-
ed whether Hedgehog proteins may play a role in HSC
induction. Our preliminary data show that ventral tis-
sues have a positive influence on AGM HSC induction
and implicate Hedgehog protein as a potential positive
effector in the induction of AGM HSCs.
Summary
The hematopoietic system requires a supportive
microenvironment throughout development. In parallel
to the earliest stages of the hematopoietic cell generation
in the conceptus, the related microenvironment consist-
ing of mesenchymal stem/progenitor cells is undergoing
coordinate growth. Such cells are found in all the major
sites of hematpoietic cell generation – the yolk sac, AGM
and placenta. Stromal cell lines isolated from these
embryonic tissues have provided insights into the sig-
nalling molecules and pathways involved in the many
aspects of hematopoietic cell growth and maintenance.
Since the AGM region generates the first HSCs, study of
this tissue has revealed the importance and positive
influence of BMP4 and potentially other ventral and/or
endodermal factors on AGM HSC growth. Further pro-
filing of stromal cell lines, as well as novel complex ex
vivo cultures and mouse models in which such factors are
dysregulated, will provide information on additional
developmental factors, their downstream effectors and
the interactions/convergence of multiple signalling net-
works to direct the generation, expansion and mainte-
nance of the founder HSCs for the adult hematopoietic
system. These data should influence strategies for the
generation and/or expansion of HSCs ex vivo for use in
clinical cell replacement therapies.
This work is supported by the Landsteiner Society for Blood
Research (0614), NIH R37 (DK51077), Dutch BSIK Stem
Cells in Development and Disease (03038), Dutch BSIK
Tissue Engineering.
14th Congress of the European Hematology Association
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 Hematopoiesis

Population dynamics of hematopoietic stem cells

H. Takizawa1
M.G. Manz1,2
1 lifetime of an individual. Moreover, HSCs exist in “excess” as one individual can share its HSCs with
Institute for Research in
Biomedicine (IRB), Bellinzona,
Switzerland;
2
Department of Hematology, IOSI,
Bellinzona, Switzerland
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
A B S T R A C T
Hematopoietic stem cells (HSCs) continuously provide all hemato-lymphoid cells throughout the
another, in which they homeostatically expand and equally form blood for the respective lifetime. This
is demonstrated over several decades by clinical hematopoietic cell, or bone marrow transplantation,
the most advanced and broadly applied somatic stem cell therapy to date. However, as successful as
hematopoietic transplantation works, it is difficult to reveal HSC correlates and to study their biolo-
gy, especially in humans. Here, we here reflect on recent and mostly, in mice generated data on HSC
phenotype, numbers, homeostatic turn-over, and contribution to hematopoiesis, and try to translate
how this knowledge might impact on our concepts of human HSCs and hematopoiesis in steady-state,
hematopoietic challenges as severe bleeding, infections, or HSC transplantation.

2009;3:128-132

ematopoietic stem cells (HSCs) are (FACS) to isolate minute populations or sin-

H defined by their capacity to self-

renew and to give rise to all mature
cell types of the hemato-lymphoid system
for the lifetime of an individual. HSC-con-
taining bone marrow or peripheral blood cell
transplantation from one individual to
another, that is, allogeneic transplantation,
represents the prime example of successful
somatic stem cell therapy with about 20,000
transplants worldwide, and about 10,000
transplants in Europe per year.1,2 Although
tremendous headway has been made in
basic understanding of HSC biology, several
long-standing questions still await definitive
answers: (i) what identifies HSCs and allows
prospective isolation to purity; (ii) how
many HSCs are contained in a body, in
which niches do they reside, what deter-
mines expansion in growing organisms and
homeostatic maintenance at fairly constant
levels in adult organisms; (iii) how often and
how synchronous do HSCs divide in steady-
state; (iv) do all or only some HSCs con-
tribute to hematopoiesis at any given time,
or do HSCs engage sequentially and repeti-
tively to blood formation. Touching on these
questions, we discuss recent data generated
mostly in mouse models and try to extrapo-
late these findings to human HSCs and
hematopoiesis, an exercise that is intended
to stimulate fruitful basic-science to medical-
practice discussions.
Phenotypic identification, numbers, and loca-
tion of HSCs in mice
With the development of monoclonal
antibody technology in the seventies and
advances in fluorescent-activated cell sorting
gle live cells from bulk cellular populations,
purification of candidate HSCs became tech-
nically possible.3 Although various kinds of
culture assays have been established to
probe HSC activity in vitro, the gold-standard
assay to functionally identify HSCs contin-
ues to be in vivo transplantation followed by
long-term follow-up. That is, lifelong recon-
stitution of hematopoiesis in conditioned
(mostly irradiated) mice or in some cases,
serial transplantation several times into
another recipient; a task that requires self-
renewal and most likely homeostatic expan-
sion of HSCs. This assay, thus, asks for max-
imal engagement of HSCs, that is appropri-
ate homing to a most supportive environ-
ment and massive contribution to blood for-
mation, and represents a stress test similar to
human hematopoietic cell transplantation,
an engagement not likely to occur often in
normal adult HSC physiology. Using this
approach, HSCs have been found to be
included in adult bone marrow mature
hematopoietic lineage marker negative or
low (Lin-/lo) cell fractions, express c-Kit (the
receptor for stem cell factor, SCF) and stem
cell antigen 1 (Sca-1).4,5 This “LSK” popula-
tion accounts for about 0.08% of total bone
marrow nucleated cells and contains all
HSCs; however, the HSC frequency within
the LSK population was determined to be
only about 3.3%.6 Further refinement of
marker combinations determined HSC fre-
quency to be about 20% in CD34– LSK,7 and
about 50% in CD150+CD244–CD48– LSK
that account for 0.006% of total bone mar-
row nucleated cells.8 Beyond surface marker
expression, HSCs can also be enriched by

| 128 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

their biologic property to eliminate drugs efficiently, as
for example, DNA binding Hoechest33342, a property
that defines them as a “side population” by FACS.9
According to this and previous work, it is estimated that
the true HSC frequency in an adult mouse accounts for
0.003% of 5×108 whole body total bone marrow nucle-
ated cells, which translates into a total HSC number of
approximately 15000 per adult, 20-25 g body weight
mouse.10-12 HSCs are newly generated during intra-uter-
ine development until about mid-gestation, are expand-
ed with growth of the organism until young adulthood,
and are subsequently homeostatically maintained.13 Of
interest, one mouse contains about 500 HSCs in fetal
liver at day 14.5, about 1000 HSCs at birth, and as dis-
cussed above, about 15000 HSCs in adulthood. As mice
have a birth weight of about 1 g and gain weight to
adulthood to about 20-25 g, the expansion factor of
HSC numbers correlates reasonably with the factor of
body weight gain.14,15
Bone marrow (BM) has been known to be the primary
organ of adult hematopoiesis, providing a place to main-
tain HSCs called the HSC “niche.” Recent studies using
microscopic analysis revealed that osteoblastic cells lin-
ing along the bone, form the so-called osteoblastic HSC
niche.16-19 Also, it was suggested that vascular cells sur-
rounding sinusoids within the BM and spleen might
form a HSC niche, the so-called vascular niche, especial-
ly after gamma-irradiation or HSC mobilization.8,18
However, because of the close proximity of osteoblasts
to the perivascular region, a clear separation between
distinct osteoblastic and vascular niches seems to be
anatomically difficult.17 Of note, upon single cell i.v.
transplantation HSCs (Lin-/lo c-Kit+ Sca-1+CD34– within
the side population) can home to their niche with
almost 100% efficiency.20 This demonstrates that HSCs
are equipped with a very robust machinery that guides
homing to the BM niche upon intravenous injection via
circulation, transmigration through BM endothelium
barrier, and lodgment in proper HSC niche.21 Given this
efficacy, it seems very likely that blood to bone marrow
homing is a physiological process occurring on a regular
basis. Indeed, some HSCs (10-100) are found to leave
bone marrow and circulate in blood at any given time
and are rapidly cleared again, with some of them locat-
ing to the estimated about 1% free HSC niches in bone
marrow.22,23 This has been most conclusively demon-
strated by parabiosis experiments, where mice were sur-
gically joined to temporarily share their blood circula-
tion, which led to continuous both-sided engraftment of
hematopoiesis post separation of animals.23
What could be the physiological relevance of blood-
born HSCs? It has not been demonstrated definitively
yet, but as bone marrow is an organ with multiple loca-
tions, HSC circulation may be important to keep HSC
populations constant in different active marrow spaces
throughout life. In addition, circulating HSCs might be
able to feed in the thymus to provide T cell progenitors
and play a role in innate immunesurveillance by traf-
ficking through peripheral organs and giving rise to
myeloid lineage cells upon demand.24,25 Finally, circulat-
ing HSC allow for re-activating extramedullary
hematopoiesis, for example, in spleen and liver after
massive blood loss or in the case of critical bone mar-
row space reduction.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 129 |
Berlin, Germany, June 4-7, 2009
Hematopoietic stem cells turnover in mice
Evaluation of HSC turnover has been addressed by in
vivo labeling with 5-bromo-2-deoxyuridine (BrdU) that
is incorporated in DNA replicating cells.11,26 Animals
were administered with BrdU for 10-13 days and sub-
jected to BrdU-free chase. Results demonstrated that
about 6-8% of HSCs per day enter cell cycle in a ran-
dom fashion and almost all HSCs divide every 8 weeks.
Mathematical simulation provides a powerful tool to
estimate HSC behavior based on available data and dif-
ferent assumptions more precisely (i.e., replication,
apoptosis and differentiation). Based on the experimen-
tal data with BrdU retention, it was estimated that the
steady-state differentiation rate of HSCs to progenitors
is 0.01-0.02/day, their transition rate from a quiescent
status to a proliferative phase is 0.02-0.05/day and the
rate of apoptosis from proliferative phase is 0.07-
0.23/day.27 Assuming that all events related to HSCs
occur independently and randomly (i.e., a stochastic
model), the outcome compared closely to previous
studies using limiting-dilution, competitive repopula-
tion assays.28 However, direct functional evidence for
HSC turnover was lacking in these models, as the detec-
tion of BrdU requires membrane permeabilization, lead-
ing to cell death. Furthermore, it has been demonstrated
that BrdU retention is not HSC specific and that BrdU
has mitogenic effects, that is, it induces HSC prolifera-
tion.11,29 Importantly, BrdU labeling experiments are
based on the premise that HSCs take up label equally,
and estimates would go wrong if there are different
HSC populations that divide and contribute to
hematopoiesis at different frequencies. Thus, the divi-
sional dynamics of HSC populations have to be evaluat-
ed by assays possibly covering all HSCs and allowing,
based on sensitive divisional history, functional analysis
as a read out.
Recently, HSC turnover has been studied with more
sophisticated divisional tracking systems. One group
developed a technique to label cell surface proteins by in
vivo biotin injection, and showed that in the hematopoi-
etic compartment, slow dividing cells contain candidate
HSCs.30 In disagreement with previous studies, howev-
er, the observation was that no HSC activity was pres-
ent within a dormant cell population retaining high
biotin label at 3 weeks of chase. Other groups used
genetically engineered mouse strains expressing histon
H2B-green fluorescent protein (H2B-GFP) controlled by
a tetracycline-responsive regulatory element.29,31,32 These
transgenic mice were crossed with other strains
expressing the tetracycline transactivator protein under
a CMV promoter, an endogenous scl promoter, or
ROSA26 promoter. Subsequently, doxycycline treat-
ment was used to turn on and shut off H2B-GFP expres-
sion followed by chase. About 20% of cells with HSCs
phenotype retained H2B-GFP expression after 24-28
weeks and approximately 5% of HSCs were labeled for
more than 44 weeks. Mathematical simulation assum-
ing a uniform turnover rate of HSCs did not fit the
experimental data, while a two-population model
showed better fit. This indicated the presence of hetero-
geneous HSC pools, a dormant pool composed of 15-
20% of the entire HSC population that divides every 55
to 145 days, which is equivalent to one fifth of mouse
lifetime, and an actively cycling pool composed of 80-
14th Congress of the European Hematology Association
85% of all HSCs that divides every 9-36 days.
Transplantation experiments with these populations
showed that the majority of long-term HSC activity is
contained in the dormant HSC pool. Importantly, it was
shown that the dormant HSC pool can be activated to
cycle in response to myelosuppression by 5-FU or
gamma-irradiation, and subsequently can reach a dor-
mant state again after homeostasis is reestablished, con-
firming a reversible cycling phenotype of HSCs depend-
ing on the environmental context. From these studies,
the dormant HSC pool would play a role as reservoir for
catastrophic situations of hematopoiesis, such as exces-
sive bleeding or acute infections. Keeping some HSCs in
an inactive state by reducing the metabolic machinery
could be an evolved strategy to protect HSCs from
cycling and aging related changes33 and replication asso-
ciated DNA mutations, potentially leading to cell death
or cancer.
Some limitations to the above described new divi-
sional tracking systems remain. Although calculations
on HSC turnover rate were performed under the
assumption that more than five divisions are needed
until H2B-GFP expression becomes undetectable, it is
not feasible to evaluate the exact number of divisions.
This is because uniform fluorescent intensity cannot be
achieved at beginning of chase, and thus, numbers of
divisions are difficult to assess and might lead to over-
estimation of the dormant pool size, given dormancy
defines zero division. To address HSC cycling kinetics
on a one-by-one divisional basis, we employ CFSE (car-
boxyfluorescein diacetate succinimicyl ester) labeling of
HSCs and subsequent transfer into non-conditioned
recipients, thus mimicking steady-state HSC traveling
and turn-over as described above.
Questions that have not been answered yet in the
series of these studies are: (i) whether some actively
cycling HSCs might re-enter quiescence even in steady-
state; (ii) whether all HSCs at any given time contribute
to blood formation and (iii) to what extend distinct HSC
pools are regulated by intrinsic and extrinsic, environ-
mental programs in steady-state and upon challenge.
Implications for hematopoietic stem cells biology in
humans
Comparatively little is known about pool size, surface
phenotype, cycling, and localization of human HSCs
due to practical and ethical constrains, as well as due to
lack of representative and predictive animal models.
Both in vitro and in vivo experiments have been utilized
to identify human HSCs. Using the long-term culture-
initiating cell (LTC-IC) assay, where cells are cultured
for several weeks on bone marrow feeder cells and the
immunodeficient mouse repopulating assay, human
HSCs were found in the Lin–CD34+CD38-/lo frac-
tion.3,34,35 Recently, it was shown that successful long-
term, that is, at least 3 months engraftment was
observed in NOD/SCID/IL2Rγ-/- mice transplanted with
as few as 10 human HSCs enriched cells defined by Lin-
CD34+CD38–CD90+CD45RA– from umbilical cord
blood or bone marrow.36 However, it should be noted
that the NOD/SCID immunodeficient mouse repopu-
lating assay and its variants, which are currently used as
best human HSC surrogate assay, do not allow homeo-
static expansion of HSCs (as observed in mouse to
| 130 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
mouse or human to human transplants). In contrast,
although serial transplantation is possible, the number
of repopulating cells and chimerism declines over time
indicating that human HSCs are not maintained.34
Given the observation that HSC numbers increase
proportional to body weight in growing mice from
birth to adulthood, it seems reasonable to make the
assumption that HSC numbers correlate with body
weight, at least within the range of a normal body mass
index. An about 70 kg human adult contains about
1.5×1012 nucleated bone marrow cells as determined by
59
Fe distribution.37 Indeed human total bone marrow
cells to mouse total bone marrow cells reflect the same
ratio as human body weight to mouse body weight (70
kg-25 g). Given the assumption that human bone mar-
row contains the same HSC frequency as observed in
mice (0.003%, discussed above), an adult 70 kg human
being would contain about 45×106, and a 3.5 kg new-
born about 2.25×106 HSCs.
Similarly, calculations for HSC turn-over can be made.
If mouse HSC cycle once every 21-145 days, they divide
5 to 35 times within a 2-year lifetime of a mouse, num-
bers that seem reasonable given the assumption of the
Hayflick limit. If human HSCs would divide with the
same frequency in a 75-year lifetime, they would divide
188-1300 times. Alternatively, cycling frequency could
be kept constant and adapted to life expectancy. If this
holds true, human HSCs would divide 5-35 times in 75
years, accounting for a HSC division every 2-15 years.
Interestingly, measurement of telomere length in
humans from the age of 0-90 years are compatible with
15-30 HSC divisions in early life, followed by less than
one division per year in the following years, accounting
for about 70-100 divisions during a lifetime.38 Thus, the
second assumption seems to get relatively close to real-
life human HSC cycling frequency.
Nevertheless, it would be reasonable and valuable to
investigate larger animals to estimate human HSC biol-
ogy. Surprisingly, evaluation of HSCs in cats revealed
that the total number (11.400±5.400) is very close to
that of mice, which was taken as evidence that the
number of HSCs is conserved in mammals.39,40 However,
since the demand for blood production per day increas-
es with body size, HSCs in larger mammals need to pro-
duce more blood per cell. Further studies on baboons
and macaques (weigh; 11-26 kg and 5-8 kg, life span; 30-
45 years and 25 years, respectively) by retrovius-medi-
ated HSC gene marking experiments and telomere
length analysis revealed that the turnover rate of
baboon HSCs was once per 23-36 weeks. Extrapolation
of human HSC replication rate from this data resulted in
192 times turn-over during a life-time, which is similar
to the frequency observed by telomere-shortening in
human blood, and would be consistent with a con-
served HSC cycling number among species.38,41 While an
adult mouse contains about 2 mL, an adult human con-
tains about 5000 mL of blood, accounting for a factor of
2500 difference. If, however, the HSC and the HSC
cycling numbers are conserved in mammals, there must
be other mechanisms ensuring sufficient blood produc-
tion. This could be an increased active HSC pool size
contributing to hematopoiesis correlating with animal
size, a scaled number of committed progenitors produc-
ing differentiated cells, a longer time and burst size from

HSC to mature cells, and a reduced frequency of blood
cell death. Concerning the first possibility, the dilemma
of different blood cell demand with the same numbers
of HSCs was mathematically approached using “allo-
metric scaling” of the active hematopoietic stem cell
pool. In this model, the active HSC pool increases across
mammalian species (40, 385, 4690, and 9640 active
HSCs for cats, humans, pilot whales, and Asian ele-
phants, respectively).42 Of note, the size of the active
HSC pool was consecutively used to explain the higher
frequency of acquired HSC disorders in humans versus
cats or mice.43 However, given the recent data on mouse
HSC turn-over and active HSC pool size, other possible
explanations as replication time and size of each
hematopoietic compartment need to be addressed
experimentally and by computational analysis.44
Overall, we believe that, similar as scaling up gut crypt
stem cells in longer intestines in large versus small ani-
mals, scaling up HSC number with size and weight of
species would be the evolutionary most simple solution
to the problem.
Hematopoietic stem cell transplantation
In syngeneic mice, 2×105 nucleated bone marrow cells
are sufficient to rescue an animal from irradiation that
otherwise would lead to death from bleeding and ane-
mia within about 3 weeks45 and establish a lifelong
100% chimerism. In fully MHC mismatched mice,
about 10-fold more bone marrow cells are needed. This
correlates with 0.05% and 0.5% of total bone marrow
cells of a mouse, or 7 and 70 HSCs, respectively. These
HSCs re-establish by homeostatic expansion the HSC
pool within about 4 months,46 that is, the HSC pool
needs to expand 200 to 2000 fold.
In HLA matched human hematopoietic cell transplan-
tation, a marrow nucleated cell dose of 3.65×108/kg
body weight was reported to be associated with a
favorable outcome,47 and a median number of 2×108
bone marrow nucleated cells/kg body weight was
reported to be transplanted in the 2008 EBMT
Handbook of Hematopoietic Stem Cell Transplantation.
In a 70 kg individual, this correlates with about 2% and
1% of total nucleated marrow cells, respectively. If
humans, similar as mice have a HSC frequency of
0.003%, transplants contain 0.9×106 and 0.45×106 true
HSCs. If, however, the HSC number is conserved in
mammals, transplants would contain about 300 and 150
HSCs. In any case, the transplanted HSC pool needs to
expand 50-100 fold to re-establish normal homeostatic
HSC levels.
Of note, while humans receive 2-40 fold more bone
marrow cells compared to mice relative to their respec-
tive total marrow cell numbers, mice need to receive no
further treatment, while humans frequently need to be
substituted with red blood cells and thrombocytes. Thus,
there seems to be substantial differences in the dynamics
of blood formation between species, an issue that needs
to be accounted for in future comparative analysis.
Conclusions
HSCs exist in “excess” as one individual can share its
HSCs with another in which these homeostatically
expand and form blood for the respective lifetime.
While HSC transplantation has been proven successful
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 131 |
Berlin, Germany, June 4-7, 2009
in the clinic, understanding HSC population dynamics
in steady-state and upon transplantation has been
proven difficult and only made substantial progress
recently in small animal models. The challenge for the
future remains to translate this knowledge to clinical
settings in humans in order to bridge the gap between
basic research and clinical medicine.
This work was in part supported by a Postdoctoral
Fellowship of the Japanese Society for the Promotion of Science
for Research Abroad to H.T., the Swiss National Science
Foundation (310000-116637), the Oncosuisse (OCS-02019-
02-2007), and the Bill and Melinda Gates Foundation to
M.G.M.
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 Hematopoiesis

Dynamic interactions between the nervous and

immune systems with the microenvironment regulate
hematopoietic stem cell migration and development.
The essential roles of SDF-1 and CXCR4

K. Lapid
T. Lapidot
Department of Immunology,
Weizmann Institute of Science,
Rehovot, Israel
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:133-139
A B S T R A C T
Hematopoietic stem cell (HSC) migration and development are tightly regulated processes in order
to produce and replenish the circulation with immature and maturing blood cells on demand.
Physiological interactions between the nervous and immune systems, with local microenvironments in
various organs throughout the body are needed to maintain homeostasis. These interactions include
dynamic regulation of bone remodeling, orchestrating the evolving stem cell niches, which impact
indirectly on rare, HSC populations and their progeny as part of host defense and repair mechanism.
Signals by the nervous system also directly regulate the motility and proliferation of hematopoietic
stem and progenitor cells (HSPC) that hierarchically express functional neurotransmitter receptors,
which are also regulated by myeloid cytokines. Reciprocal effects of activated leukocytes, neuronal
and stromal cells via secretion of cytokines, chemokines, neurotransmitters and proteolytic enzymes
are part of the dynamic crosstalk between these overlapping systems, providing a stem cell regulato-
ry brain bone blood triad. Homing, retention, egress, recruitment and clinical mobilization of HSPCs
will be discussed in the context of this crosstalk, focusing on the essential roles of the chemokine
Stromal Derived Factor-1 (SDF-1) and its major receptor CXCR4 in the functional preclinical NOD/SCID
mouse model.

ost blood forming stem and progen- one amino acid and are cross reactive.13

M itor cells are located in the bone

marrow (BM), anchored to special-
ized niches; however, there are very low lev-
els of motile progenitors in the circulation as
part of homeostasis.1 These low levels are
dramatically amplified during alarm situa-
tions in response to stress signals due to
injury, bleeding, infections and inflamma-
tion, and as part of host defense and repair
mechanism.2,3 Stress induced recruitment of
progenitor cells to the circulation is mim-
icked in clinical mobilization protocols by
chemotherapy and/or repeated granulocyte
colony stimulating factor (G-CSF) stimula-
tions, in which immature cells with repopu-
lation potential are harvested from the blood
for autologous and allogeneic transplanta-
tions of myeloablated patients.2,4,5 HSPCs are
identified and characterized in functional
transplantation assays based on their ability
to migrate in the circulation, home to the
host BM and durably repopulate it with
immature and maturing leukocytes, which
in turn are released back to the circulation on
demand. The chemokine SDF-1 (also termed
CXCL12), and its major receptor CXCR4,
regulate many types of stem cells and play
pivotal roles during development. SDF-1 is
highly expressed in the BM by stromal cells,
including bone-lining osteoblasts, endothe-
lial, reticular and perivascular cells.6-8 Murine
embryos that lack SDF-1 or CXCR4 have
multiple lethal defects, including lack of fetal
BM seeding by hematopoietic progenitor
cells.9-11 In addition, cleavage of SDF-1 by
CD26 reduces homing of murine HSPC to
the BM.12 Human and murine SDF-1 differ in
Currently, this ligand is the only known
powerful chemotactic factor for human and
murine HSPCs14,15 In vivo migration of both
normal and leukemic human stem cells, and
their engraftment of the BM of transplanted
immune deficient mice are dependent on
SDF-1/CXCR4 interactions.8,14,16,17 Apart from
mediating hematopoietic stem cell motility,
SDF-1 plays roles in survival and quiescence,
and murine HSCs from inducible KO of
CXCR4 demonstrate uncontrolled cycling
and impaired repopulation ability.7,18 This
review will elaborate on the SDF-1/CXCR4
dependent regulation of normal and
leukemic human CD34+ progenitors, dis-
cussing both engraftment and induced mobi-
lization processes in chimeric NOD/SCID
mice and also wild type murine HSPC.
Furthermore, the emerging concept of the
microenvironmetal regulation of HSCs
mobilization and recruitment will be dis-
cussed, involving bone remodeling and sig-
nals from the nervous system.
Homing and engraftment of immature human
CD34+ cells in immune-deficient mice
Functional preclinical animal models for
engraftment of human HSPCs (derived
from human BM, cord blood or G-CSF
mobilized peripheral blood) were devel-
oped in pre-immune sheep and immune
deficient mice.19,20 Several immune deficient
strains have been developed, of which
Non-Obese Diabetic Immune Deficient
(NOD/SCID) is the most common model,
lacking mature T and B cells and having
weak innate immunity.21 Other strains that

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 133 |
14th Congress of the European Hematology Association
lack also NK cell activity, such as NOD/SCID/β2m22
and NOD/SCID/IL-2Rγ-/- (NOG)23 mice are routinely
used today. These models have identified primitive
human CD34+/CD38–/low cells with long term repopu-
lation in transplanted animal recipients.24,25 These pre-
clinical immune deficient mouse models can predict
clinical outcome in leukemia patients and repopulation
of normal cells in these mice reflects on their clinical
potential.26,27 Homing, engraftment and mobilization of
human CD34+ enriched progenitors in chimeric
NOD/SCID mice are dependent on CXCR4 signal-
ing,14,28,29 demonstrated by direct blockage of CXCR4
by neutralizing antibodies. Transplantation of
enriched human CD34+ cells overexpressing CXCR430
or in vivo pre-treatment with intravenously injected
SDF-1,31 result in enhanced homing and repopulation
capacity, further supporting the essential roles of SDF-
1/CXCR4 in the engraftment process. This significance
is manifested in stress situations as well; for instance,
when chemotherapy or irradiation are applied, it
results in increased SDF-1 expression in the BM of
recipient mice, thereby mediating increased repopula-
tion by transplanted cells.6 Of note, pre-conditioning
by irradiation or chemotherapy is essential for durable
high level reconstitution by transplanted cells. In vitro
SDF-1 induced migration capacity of mobilized CD34+
enriched human cells correlates with their in vivo
repopulation potential in autologous transplanted
patients32 and immune deficient mice.14 The lipid medi-
ator Sphingosine-1 Phosphate (S1P) also serves as a
modest chemoattractant for progenitor cells. Using an
agonist for its receptor (S1P1) augments SDF-1 induced
migration of human CD34+ cells, resulting in increased
homing,33 while overexpression of S1P1 leads to reduc-
tion in human CD34+ cell migration and homing by
downregulating CXCR4 expression.34 Various
cytokines, such as Hepatocyte Growth Factor (HGF),
interleukin-6, Stem Cell Factor (SCF) and FLT-3 lig-
and,29,35,36 as well as other factors, such as
prostaglandins,37 can upregulate CXCR4 expression or
enhance CXCR4 signaling in immature human CD34+
cells, and thus, improve their motility and repopula-
tion potential. Taken together, these findings imply
that not only SDF-1 expression is dynamically
expressed in the BM, but also CXCR4. Interestingly,
CXCR4 can be functionally transferred from platelet
microparticles to immature human CD34+ cells and
augment their SDF-1 mediated homing and engraft-
ment potential.38 Understanding the mechanisms gov-
erning SDF-1 and CXCR4 activity may assist in
improving current transplantation protocols. SDF-1
activates the major adhesion molecules LFA-1, VLA-4,
VLA-5 and CD44, which participate in human CD34+
cell homing and repopulation in transplanted
NOD/SCID mice.39-41 These adhesion molecules inter-
act with Extra-Cellular Matrix (ECM) components,
such as fibronectin and hyaluronan, which are also
expressed in the BM niches, as well as with adhesion
molecules, such as VCAM-1 and ICAM-1, expressed
by BM endothelial cells, reticular cells and osteoblasts.
All of which are important for active homing to the
BM and retention of HSPCs, which is pre-requisite for
subsequent successful repopulation.19 Of interest, neu-
tralization of CD44 prevents SDF-1 mediated activa-
| 134 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
tion of human progenitor cells, revealing a two way
cross talk in which inhibition of adhesion molecules
over-rules SDF-1 induced activation and migration,
which could lead to their retention in the BM.40
Altogether, it is concluded that by triggering chemo-
taxis and adhesion machineries, SDF-1/CXCR4 guide
human HSPCs into the BM of recipient chimeric mice,
enabling efficient homing and engraftment.
Nevertheless, the SDF-1/CXCR4 story does not end in
cell-autonomous mechanisms, and in fact, harbors a
broader concept. We have found that BM endothelial
cells, which also express CXCR4 on their surface, can
translocate functional SDF-1 from the circulation in a
CXCR4 dependent manner, controlling SDF-1 levels in
the BM, and thus actively guide transplanted HSPCs in
their course of homing to the BM.42 These findings
open a door to the involvement of other cellular fac-
tors in the environment, regulating availability of SDF-
1 and affecting indirectly stem cell function (will be
discussed later on).
The leukemic point of view
Utilization of the immune deficient mice models
enabled identification of human acute myeloid
leukemia (AML) and Pre-B acute lymphoblastic
leukemia (ALL) initiating cells, and more recently, other
types of human cancer stem cells.43,44 Human Pre-B ALL
cells home to the same SDF-1-rich vascular micro-
domains as murine HSPCs in the BM, and antagonizing
CXCR4 abolishes their homing.45 Moreover, leukemic
stem cells were recently found to utilize the same spe-
cialized stem cell niches rich area in the BM, the endos-
teum region, which provides chemoprotective signals to
the AML stem cells46 (also see Figure 1). Since leukemic
cells are derived from hematopoietic progenitors and
share overlapping features, it is plausible to assume that
SDF-1/CXCR4 contribute to their malignant pheno-
type. Indeed, blockage of CXCR4 reduces homing by
human Pre-B ALL and AML cells to the BM of recipient
NOD/SCID mice.16,27 Likewise, CXCR4 is important in
chronic myeloid leukemia pathogenesis.47 AML cells
were found to be more CXCR4 dependent than normal
human repopulating progenitor cells, and exhibited
poor engraftment upon anti-CXCR4 treatment in pre-
established chimeras.16 A similar observation has been
shown for human AML with unique anti-CD44 Ab
treatment, which eradicated AML in chimeric mice,
while only minimally affecting normal human
hematopoietic cells.48 It should be noted that CXCR4+
plasma microparticles contribute to the homing process
of human AML cells, in a similar fashion to normal
human HSPCs, by transferring membranal CXCR4, and
are, as a result, involved in the progression of the dis-
ease.38,49 Furthermore, high expression of CXCR4 corre-
lates with poor prognosis in AML patients.50 These find-
ings suggest that mechanisms governing homing and
engraftment of normal human HSPCs are also crucial
for understanding human leukemias.
Stem cell recruitment and mobilization
Recruitment of HSPCs to the peripheral blood (PB)
following treatment with chemotherapy, or cytokines,
is a clinical process termed mobilization.2 This process
mimics enhancement of the physiological release of

HSPCs from the BM reservoir in response to stress sig-
nals during injury and inflammation as part of host
defense and repair. Mobilization requires active naviga-
tion of stem cells across physical endothelial and ECM
barriers to the circulation, which requires proteolytic
enzymes activity and breakdown of adhesion interac-
tions. The SDF-1/CXCR4 axis plays a pivotal role in this
active navigation, since concomitant blockage of
CXCR4 or SDF-1 by neutralizing antibodies, during
repetitive G-CSF stimulations, prevents both human
and murine progenitor cell mobilization.28 Lack of mobi-
lization upon G-CSF treatment in murine CXCR4-/- BM
chimeras supports this observation.51 Interference with
CXCR4 signaling needed for HSPC retention in the BM,
also results in progenitor cell mobilization, as demon-
strated by inhibition of PKCζ and RAC1 activities.37,52,53
CXCR4 levels are increased on BM HSPCs following G-
CSF treatment, enhancing their migration potential
towards SDF-1.28 On the other hand, SDF-1 levels are
reduced in the BM and are transiently increased in the
PB following G-CSF treatment, implying that by con-
trolling SDF-1 levels, HSCs could be recruited to site of
demand.28 For example, upon liver injury or limb
ischemia, SDF-1 expression is increased, mediating spe-
cific recruitment of progenitor cells to these organs as
part of host defense and repair mechnaisms.54,55 In a sim-
ilar fashion, high levels of S1P in the blood and lymph
attract circulating murine HSPCs, which navigate their
ways to peripheral organ, where they can undergo
induced activation and differentiation.56 Repetitive daily
administrations of SDF-1 or a single dose administration
of the Met-SDF-1 analog, which has a longer half life in
vivo, trigger preferential mobilization of murine progen-
itor cells.57-59 Notably, during G-CSF induced mobiliza-
tion, SDF-1 levels in the BM are transiently increased,
followed by their eventual reduction at mRNA and pro-
tein levels.28,60 SDF-1 stimulations activate proteolytic
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 135 |
Berlin, Germany, June 4-7, 2009
(A) An illustration of a femur with
its regions. (B) DiR labeled human
pre-B ALL G2 cells were injected
into a NOD/SCID mouse. Homing
was examined 16 hours post trans-
plantation. The obtained femur was
quickly frozen and cut smoothly in
a cryostat device. The image was
taken using a CCD controlled fluo-
rescent microscope. Leukemic
cells localize closely to the endos-
teum along the diaphysis (yellow
arrow) and in trabeculae-rich
regions in the metaphysis and epi-
physis (green arrow).
enzymes, such as matrix metalloproteinases (MMPs),
which in turn deactivate SDF-1 by its cleavage. In fact,
proteolytic activity enables breakdown of adhesion
interactions in the BM and loss of retention in the
course of recruitment and mobilization processes,4 in
which SDF-1 induced MMP-9 plays a major role by
cleaving membrane bound SCF.61 The released soluble
SCF augments cell motility. G-CSF induced mobiliza-
tion is associated with neutrophilia,62 and activated neu-
trophils secrete serine proteases, such as neutrophil elas-
tase and cathespsin G, which have been shown to
cleave SDF-1,28 CXCR4,63 c-Kit64 and VCAM-1.65
Therefore, inhibition of elastase results in reduced G-
CSF induced mobilization.28 In spite of these observa-
tions, mice lacking these proteases still react normally to
G-CSF treatment and are capable of cleaving SDF-1,66
indicating redundancy among proteases activity.
Indeed, in addition to secreted proteases, membranal
proteases were found to participate in the SDF-1 cleav-
age activity, including CD26 and MT1-MMP, which are
also expressed by the progenitor cells themselves.67,68
The latter has also been shown to mediate G-CSF
induced mobilization of immature human CD34+ cells
by CD44 cleavage. Our data reveal that RECK, which is
the endogenous inhibitor of MT1-MMP and MMP2/9,
is inversely regulated on human CD34+ HSPCs and
mouse BM cells.67 Upon G-CSF treatment, MT1-MMP
expression is increased, while RECK expression is
decreased, emphasizing the importance of balanced
proteolytic activity in the BM. Since SDF-1 and CXCR4
interactions are essential for HSC retention, as observed
by increased circulating HSCs in inducible CXCR4 KO
mice,18 it is logical to assume that disruption of this
interaction would result in loss of retention. The bicy-
clam AMD3100 was found to cause rapid mobilization
of mouse, human and non-human primate HSPCs.69-71
AMD3100 treatment also synergistically augments G-
14th Congress of the European Hematology Association
CSF induced mobilization. Moreover, AMD3100 mobi-
lized progenitors demonstrate increased SDF-1 induced
migration in vitro and in vivo repopulation potential,69
synergized upon combination of AMD3100 and G-CSF
treatments. Interestingly, preliminary results reveal that
AMD3100 rapidly induces increased SDF-1 secretion
from the BM stromal cells to the circulation.72 Blocking
the SDF-1/CXCR4 axis, using neutralizing antibodies to
either SDF-1 or CXCR4, in steady state and in
AMD3100 treated mice, reduced mobilization of
HSPCs, but not mature WBCs,72 strengthening the
hypothesis that this pathway is relatively more selective
for immature cells. These results complicate the theory
of passive disruption of SDF-1/CXCR4 interactions by
AMD3100, suggesting that active SDF-1/CXCR4 is also
necessary for steady state egress and AMD3100 induced
progenitor cell mobilization. Our preliminary experi-
ments reveal that AMD3100 does not affect only
hematopoietic cells, but also BM stromal cells, which
also express CXCR4.42,72 In response to AMD3100, SDF-
1 secretion from BM endothelial cells and osteoblasts in
vitro and in vivo to the circulation is increased, further
contributing to transient, local SDF-1 gradients towards
the blood, after which, motile HSPCs follow. In conclu-
sion, the involvement of other cellular players, such as
neutrophils and BM stromal cells, in mobilization
processes, strongly imply that regulation of cell-
autonomous motility properties are only part of the pic-
ture that includes a complex regulation by the BM
microenvironment.
A cross-talk between bone remodeling and stem cell
egress, recruitment and mobilization
Bones are dynamic and continuously undergo forma-
tion by bone lining osteoblasts and resorption by the
stem cell derived fused-monocytes, termed osteoclasts,
throughout life. Osteoblasts are involved in maintaining
quiescent HSCs at the endosteal niches and play a role
in supporting BM hematopoiesis.73 Genetic manipula-
tions that cause an increase or a decrease in osteoblasts
numbers have led to an increase or a decrease in HSCs
numbers respectively.74-76 Following G-CSF administra-
tion, BM SDF-1 mRNA levels are reduced, and it was
discovered that reduction in numbers and activity of
osteoblasts, which produce SDF-1, is one of the major
reasons.60 Decrease in BM SDF-1 levels is correlated
with mobilization.28 The reduction in osteoblasts results
from stimulation of the sympathetic system during G-
CSF treatment77 (further discussed in next section).
Furthermore, osteoblasts isolated from cyclophos-
phamide + G-CSF treated mice could expand HSPCs in
vitro at higher rates than osteoblasts from non-treated
mice.78 One of the factors promoting HSC self-renewal
is interleukin-10, whose secretion by endosteal
osteoblasts is upregulated following irradiation induced
stress.79 Thus, it is suggested that BM niches are under-
going dynamic changes, during stress induced mobiliza-
tion, to allow proliferation and egress of HSPCs.
Following G-CSF administration, osteoclasts activity is
upregulated, explaining why G-CSF mobilized PB
donors show reduced bone mass.3,80,81 Osteoclasts
express both SDF-1 and CXCR4, and SDF-1 is involved
in recruitment of osteoclast precursors to the bone sur-
face and promotes immature osteoclast development
| 136 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
and survival.82,83 In addition, G-CSF, SDF-1 or HGF
administration induces both an increase in osteoclast
numbers and preferential mobilization of progenitors.57
Stress-induced situations, such as mild bleeding and LPS
treatment, also trigger HSPCs mobilization concomi-
tantly with osteoclast activity. Indeed, we have demon-
strated that osteoclasts are directly involved in inducing
progenitor cell egress by cathepsin K mediated degrada-
tion of major endosteal niche components, such as SDF-
1, osteopontin and SCF,57 linking bone remodeling with
mobilization of HSPCs. Osteoclast activation by
RANKL preferentially increases mobilization of imma-
ture cells, whereas osteoclasts inhibition by the hor-
mone clacitonin decreases G-CSF and LPS induced
mobilization, as well as homeostatic release of progeni-
tors. Of note, mobilization induced by repeated stimu-
lations with RANKL, HGF or SDF-1 was selective in
terms of mobilizing HSPCs to the PB, but not mature
leukocytes.57 Utilization of PTPε deficient young female
mice, which have transient and mild impairment of
osteoclast adhesion and resorption,84 also demonstrated
reduced numbers of circulating progenitors and capaci-
ty to mobilize, even in response to repeated RANKL
stimulations, despite normal levels of immature cells in
the BM.57 Another example for the involvement of
osteoclasts in the mobilization process is presented by
CD45 KO mice. CD45 is a pan leukocyte phosphatase
that is dynamically expressed only on leukocytes. CD45
KO mice demonstrate defective osteoclast activity,
altered metaphysial trabecular bone structure, resem-
bling mild osteopetrosis, and reduced BM pool of prim-
itive HSPCs.85 RANKL and suboptimal G-CSF induced
mobilization are consequently impaired in these mice.
These results reveal that HSPCs are involved in regulat-
ing their own levels and the dynamic BM microenviron-
ment via their osteoclast progeny. Parathyroid hormone
(PTH) maintains calcium homeostasis by accelerating
bone remodeling, demonstrated by proliferation of
osteoblasts that express its receptor, which is followed
by osteoclast activation. The effects of PTH on
osteoblasts has been shown to increase the HSC pool
size via the Notch pathway;74 however, PTH adminis-
tration also results in HSC mobilization.86,87 Moreover,
pre-treatment of mice with PTH five weeks prior to
administration of G-CSF, augments mobilization. It is
yet to be determined whether osteoclasts are involved
or not in PTH induced mobilization. Altogether, bone
remodeling has a strong impact on stem cell function
and the degree of both steady-state progenitor cell
egress and stress-induced mobilization by shaping the
dynamic BM microenvironment and the evolving stem
cell niches.
Direct and indirect regulation of stem cell egress, recruit-
ment and mobilization by the nervous system
Early in the 20th century, researchers found a marked
increase in the number of circulating leukocytes follow-
ing injection of adrenaline (epinephrine), a neurotrans-
mitter produced by the sympathetic system, into
healthy human volunteers.88 In rats, epinephrine admin-
istration stimulated the release of leukocytes from the
spleen, BM and lymphatic organs, contributing signifi-
cantly to leukocytosis.89 Recently, sympathetic system
induced mobilization of more primitive cells was docu-

mented (reviewed in 90). The BM is highly innervated
and sympathetic nerve endings localize, especially in
proximity to endothelial cells and the endosteum in the
epiphysis and metaphysis, regions known to be
enriched with stem cell niches, implying indirect control
over HSC function.91 Indeed, immature and primitive
progenitor cells, among other leukocytes, express β2
adrenergic receptors, as well as dopamine receptors.92
These receptors are upregulated in G-CSF mobilized
immature human CD34+ cells or upon in vitro treatment
with myeloid cytokines, also suggesting a direct role for
sympathetic system stimulation in inducing mobiliza-
tion of HSPCs. β2 adrenergic stimulation by norepi-
nephrine administration in mice resulted in increased
numbers of circulating HSPCs, while administration of
a β2 adrenergic antagonist, resulted in their reduced PB
numbers.92 The importance of the nervous system for
the mobilization process was determined by establish-
ing mice that display aberrant nerve conduction or that
underwent catecholaminergic ablation without affect-
ing the BM resident HSCs pool during steady state
homeostasis. Strikingly, G-CSF administration in these
mice had no effect on HSPCs mobilization.77 Our pre-
liminary data demonstrate that norepinephrine admin-
istration stimulates release of SDF-1 from BM stromal
cells to the PB, after which progenitors follow, conse-
quently increasing their numbers in the circulation.72
Interestingly, this norepinephrine induced mobilization
was selective, since no increase in circulating mature
leukocytes was observed. Recently, it was revealed that
neuronal stimulation plays a role in steady-state egress
of HSPCs and not only during stress situations. Both
human and murine circulating HSPCs exhibit robust
daily oscillations.93,94 In fact, β3 adrenergic stimulations
were affected by circadian rhythmic control over norep-
inephrine release, leading to downregulation of SDF-1
transcription in BM stromal cells. As a result, SDF-1 syn-
thesis fluctuates, triggering circadian rhythmic oscilla-
tions of PB circulating progenitor cells.93,94 Furthermore,
it seems that G-CSF-induced leukocytosis is also
dependent on the timing of G-CSF injection in both
mice95 and human patients.96 Unexpectedly, adrenergic
stimulation suppressed bone-lining osteoblasts, causing
a significant reduction in SDF-1 production,60,77 thereby
enabling cell egress. It was found that G-CSF induced
mobilization requires adrenergic signals, since G-CSF
administration in mice lacking catecholaminergic activi-
ty did not result in osteoblast suppression, SDF-1 down-
regulation in the BM or subsequent progenitor cell
mobilization.77 In addition to suppression of osteoblasts,
adrenergic stimulation positively affects osteoclasts,
leading to loss of bone mass.97 It is yet to be determined
whether adrenergic stimulation also exerts its effect on
HSCs mobilization via osteoclasts. Apart from showing
a direct induction of mobilization by the sympathetic
system, evidence for indirect effects are accumulating,
including circadian control over SDF-1 synthesis and
regulation of osteoblasts and osteoclast activity. These
new findings broaden the concept of complex microen-
vironmental regulation of progenitor cell egress, orches-
trating bone remodeling processes, dynamic nervous
system and immune system interactions with stromal
cells, regulating stem cell function.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 137 |
Berlin, Germany, June 4-7, 2009
Concluding remarks
The major aim of pre-clinical hematopoietic stem cell
research is to develop improved procedures to harvest
and transplant HSPCs, together with improved reconsti-
tution capacity for clinical protocols. Today, G-CSF
administration is the most common clinical procedure
to mobilize HSPCs intended for transplantation; how-
ever, the emergence of rapid mobilizing agents, such as
AMD3100, promises a development of improved clini-
cal procedures. Furthermore, they exhibit synergistic
mobilizing effects in combination with G-CSF treat-
ment.69,98 Apart from developing improved mobilization
protocols, improving the capacity of transplanted
HSPCs to engraft can be achieved by increasing cell
homing, for example, ex vivo treatments that enhance
CXCR4 signaling.19 Importantly, the recent discoveries
of dynamic interactions between the microenvironment
and the resident BM stem cells, which include the bone,
immune and nervous systems, provide a broader per-
spective over stem cell regulation and function. Stromal
dependent control over SDF-1 availability in the BM,
osteoblasts and osteoclasts activity and both direct and
indirect adrenergic stimuli dynamically regulate steady
state egress and recruitment of HSPCs to the circulation,
as well as maintenance in the BM. Thus, manipulation
of the brain bone blood triad may assist in developing
improved clinical protocols in the future.
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 Hodgkin disease

Nodular lymphocyte-predominant Hodgkin’s lymphoma

in children and adults

J. Landman-Parker A B S T R A C T

Nodular lymphocyte-predominant Hodgkin’s lymphoma is a rare subtype of CD20+ Hodgkin’s lym-

Université Pierre et Marie Curie-
Paris 6/Service d’hématologie et
d’oncologie pédiatrique Hôpital
Armand Trousseau APHP, Paris,
France
phoma characterized by indolent course, young male predominance early stage and overall good prog-
nostic. New understandings in the biology of the lymphocyte predominant cell biology are described.
The major clinical concerns are related to late effects of treatment described in patients and the
occurence of high grade lymphoma. We report current strategies in this context and emphasize the
need for clinical studies.

Hematology Education:
the education program for the
annual congress of the European
Hematology Association

2009;3:140-143

odular lymphocyte-predominant ly all cases, and epithelial membrane antigen

N Hodgkin’s lymphoma (NLPHL) is a

rare sub-type of CD20+ Hodgkin’s
lymphoma (HL), which represents 5-10% of
all HL cases. NLPHD is characterized by
indolent course, young male predominance,
early stage and overall good prognostic,
although late occurrence of high-grade lym-
phoma and late deaths related to treatment
are described in patients. Past two decades
have brought biological and clinical new
insights in NLPHL that lead to a new
approach of this rare entity.
Histopathology
In 1994, the REAL classification separated
NLPHD as a distinct clinicopathologic entity
from other subtypes of HD, which were
subsumed under the term “classical HD”
(cHD). This new classification was followed
by the World Health Classification (WHO)
classification in 2001.1 Subsequently, an
additional type of cHD with an abundance
of lymphocytes was recognized, named
“lymphocyte-rich form of classical HD”
(LRCHL). In the 2008 WHO classification of
tumors of hematopoietic and lymphoid tis-
sues, NLPHL is defined as a monoclonal B
cell neoplasm, characterized by a nodular, or
a nodular and diffuse proliferation of lym-
phocytic and histiocytic (L&H) cells (or pop-
corn cells), now named lymphocyte pre-
dominant (LP) cells.2,3 From a morphologic
point of view, the lymph node architecture
is totally or partially replaced by a nodular
infiltrate predominantly consisting of small
lymphocytes, histiocytes and LP cells, which
are few (less than 5% of the cells).
Immunophenotyping of LP cells are positive
for CD 20, CD 79a, CD45 and BCL6 in near-
(EMA) in more than 50%. The major differ-
ential diagnosis of NLPHL is LRCHD.3,4 In
contrast to Hodgkin Reed Sternberg cells
(HRS) of LRCHD, LP cells lack CD30, CD15
and EBV genome expression. Surrounding
cells are predominantly small B cells and
numerous CD4+/CD57+ T cells. The study
performed by the European Task Force on
lymphocyte predominant Hodgkin lym-
phoma demonstrated that 20% of the past
cases diagnosed as NLPHL were LRCHD in
a review of 521 cases.3 In this study, differen-
tiating between T-cell rich B cell lymphoma
and diffuse form of LPHD was uncommon
and only occurred in 2% of the cases.
Biology of lymphocyte predominant cells
LP cells are clonally related B-cell–derived
malignant cells.5-7 Analysis of isolated L&H
cells for the variable region of the heavy
chain of immunoglobulin (IGVH) genes
rearrangements revealed that these cells
present clonal rearrangements and carry a
high load of somatic mutations – a feature
they share with normal germinal center B
cells. The Ig VH rearrangements are usually
functional with mRNA detectable in the LP
cells.7 L&H cells express CD-20 and multiple
other B-lymphocyte markers, including ger-
minal centre B-cell differentiation antigens,
such as the transcription factors Bcl-6,8 acti-
vation-induced cytidine deaminase,9 and
centerine.10,11 Cytogenetic studies and molec-
ular analysis of microdissected HRS and LP
cells revealed frequent Bcl-6 gene rearrange-
ments.12 Aberrant somatic hypermutation,
identified as a mechanism for genomewide
instability in diffuse large B-cell lymphoma,
is demonstrated in LP cells.13 Aberrant

| 140 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

somatic hypermutation targets various protooncogenes
in LP cells, such as PAX5 or Myc, and more recently
SOCS1, which may alter their function, thereby con-
tributing to NLPHL pathogenesis.14 A recent gene
expression study of isolated LP cells indicated that these
cells resemble an intermediate developmental stage
between germinal centre and memory B cells, and
defines NLPHL as a distinct entity closely related to T
cell – rich B cell lymphoma and cHL. This study reports
a selection of 49 genes related to the phenotype of LP
cells, among them notably ubiquitin D, which may also
contribute to genome instability in NLPHL.15
Clinical presentation
Patients typically present with early stage disease,
mainly IA, with peripheral lymph nodes involvement,
that is, cervical, axillary, and inguinal, rather than
mediastinal involvement, which is rarely seen. There
is a striking male predominance. This particular clini-
cal presentation is described in the report of the
European Task Force on lymphocyte predominant
Hodgkin lymphoma and confirmed by others.3,16,17 In
their data set on adults patients with NLPHL, median
age distribution is 35 years and 70% of NLPHL
patients are male, which is three times more than in
cHD. In children, NLPHL is very rare. In a collaborative
European study, 85% of their 58 patients were male,
which is higher than in pediatric cHD, and median age
was 11 years.18,19 In the study of the European Project
on 219 patients with confirmed NLPHL, early stage I
was common with 53%, 28% stage II, 13% bulky dis-
ease, 10% B symptoms, 7% mediastinal mass, and 8%
had splenic involvement. Of 394 NLPHL patients
report by the German Hodgkin study Group, 63%
were in early the favorable stage, 16% were in early
unfavorable stage, and 21% were in advanced stage of
disease. LPHL patients often presented with normal
ESR (96%) and normal LDH (84%). A mediastinal
bulky tumor or extranodal involvement (6%) were
rarely seen.16 Staging procedures in cHD commonly
used FDG-PET scanning. NLPHL, despite indolent evo-
lution, has an avidity for FDG and, therefore, a signif-
icant place for staging in NLPHL.20,21 Few cases of
NLPHL are reported associated with neurological dis-
eases, such as cerebral angeitis, polyradiculonevritis or
cerebellar ataxia (personal observation).22,23 These obser-
vations emphasis the role of B lymphocytes in the
pathogeny of the disease.
Prognosis and treatment
Prognosis is favorable with an indolent course of dis-
ease and patients deaths are mostly related to secondary
(treatment related) malignancies or transformation to
aggressive B cell lymphoma. NLPHL patients have an
overall survival range from 83-96%, and a relapse free
survival dependent on treatment strategy, stage and fol-
low up, from 70-88%. Late and multiple relapses are
more frequent than in cHD. These previous results have
already been reviewed in 2006 and 2008.24,25
As clinical and pathological studies have suggested
that LPHL and cHL differ in pathology and clinical
behavior, treatment strategies for LPHL patients are
needed. Clinical questions on the best treatment for
these patients are still open. In the past, LPHL patients
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 141 |
Berlin, Germany, June 4-7, 2009
have often been treated with chemotherapy and radio-
therapy according to standard classical HL
protocols.16,26-28 Early stage patients treated with radio-
therapy alone usually received extended field radiation,
involving field radiation therapy with 30-36 Gray,29
chemotherapy,30 or more recently, monoclonal antibody
therapy using Rituximab or an anti-CD-20 radiolabeled
antibody the 90Y-ibritumomab tiuxetan for advanced
stages of LPHL.31
Adenectomy, followed by watch-and-wait strategy
Occasionally, patients with early stage LPHL have
been treated with surgery alone. In 1983, Miettinen et al.
reported an overall survival of 93% at five years in 31
adult LPHL cases of retrospectively diagnosed LPHL,
who remained untreated except for lymph-node exci-
sion because the original histology was reported as
benign.32 In 1984, Hansmann et al. reported on 24 cases
of LPHL that, for various reasons, had surgery only. Nine
of them achieved long-term remission.33 These reports
suggested that surgery alone might be an option in early
stage LPHL. This strategy was applied in children in two
studies with limited number of cases.34,35 An retrospec-
tive update of 58 cases has been recently published by
the European Network Euronet Pediatric Hodgkin lym-
phoma.19 The decision to use surgery alone in these
early stage patients was made on an individual basis by
the treating physicians in consultation with the parents
in an effort to limit treatment toxicity whilst maintain-
ing a successful outcome. There were 50 boys out of 58
patients, with a median age of 11 years (age range was
4-17); stage IA n=54, IIA n= 2, IIIA n=2. With a median
follow up of 43 months, overall survival is 100% and
PFS 57%. Fifty-one out of fifty-eight patients achieved
complete remission (CR) after surgery, although precise
CR definition was not controlled in this retrospective
study. In the CR group, overall PFS is 67% (95% CI
51%; 82%). All cases with residual disease after initial
surgery relapsed (p=0.003) (7/7). From 18 stage IA
patients with relapse, 11 had a local relapse and 7
relapsed at stage IIA. One patient (IIIA) presented with
high-grade B cell lymphoma (NHL) at 10 years of fol-
low-up. These results suggest that a substantial propor-
tion of patients stage IA with one or two involved nodes
might be cured after complete surgery. This approach is
currently explored by the Pediatric Euronet on Hodgkin
Lymphoma and the Children Oncology Group in US in
prospective ongoing studies. In adults, the French GELA
group opened a registry on NLPHL in 1993, and a sus-
tained CR was observed in 50% of the patients given a
watch-and-wait strategy after surgical excision of the
lymph node at NLPHL diagnosis (personal communication).
Consider-ing the limited data published, long-term
observat ion that is needed and indolent course of the
disease, it is still difficult to recommend a strategy. At
least two questions remains unsolved: is no further
treatment a safe option in patient with stage IA who
have obtained CR after initial adenectomy, and what is
the risk of a significant upstaging at relapse and subse-
quent transformation into a more aggressive B-cell lym-
phoma. Nevertheless, considering resection alone in a
very limited disease without associated risk factors and
complete excision controlled by modern imaging evalu-
ation is a realistic option.
14th Congress of the European Hematology Association
Low dose chemotherapy in NLPHL
In order to limit morbidity related to treatment, alter-
native chemotherapy to standard cHL treatment has
been proposed in children.36,37 With limited follow up,
French and UK pediatricians have reported efficacy of
low dose chemotherapy alone with vinblastine, low
dose cyclofosphamide and prednisone.38
Rituximab in NLPHL
As almost 100% of LP cells express CD-20, Rituximab
could be an effective approach, and treatment based on
Rituximab alone on a classical schedule has been report-
ed in a phase II and retrospective study, but very limit-
ed data are available. The German Hodgkin Study
group reported long-term results in relapse NLPHL in a
phase II study in 2008.39 With 63 months follow-up on
15 confirmed NLPHL patients, PFS is 50% and median
time to relapse 33 months. Deaths are related to high-
grade B cell lymphoma, stage IV and second malignan-
cies. Seven other patients are reported from in a single
institution, with 100% of CR and three relapses at a
median time of 27 months.40 Single cases have been
reported with successful results in literature but current-
ly no available studies on rituximab alone as first line
therapy in LP have been published, although efficacy is
probably high but seems limited in the time duration.
Rituximab combined with chemotherapy is reported in
relapsed or newly diagnosed cHL but available data are
very limited.41,42 Combined treatment with ABVD,
DHAP or gemcitabine could, therefore, be an interesting
option for NLPHL, particularly in rare advanced stage or
presence of adverse prognostic factors or relapse after
radiation therapy in early stage, but controlled studies
are recommended.
Multiples relapses and high grade B cell lymphoma
transformation
The rate of secondary diffuse large B cell lymphoma
and specifically T-cell rich B cell lymphoma is reported
although, if considering the new lymphoma classifica-
tions and the long-term outcome, more informative
data are necessary.43-45 It varies from 3-10% at 10 years,
but may be higher if prolonged observation is docu-
mented. The pattern of relapses is rather local but stages
or resistance may increase with time, leading to aggres-
sive strategies, including high doses chemotherapy with
stem cells support or radio labeled CD-20 monoclonal
treatment. This pattern of relapses emphasizes the need
of repeated biopsy to reconsider histology, as mixed or
T-cell rich B cell lymphoma may occur, and be misdiag-
nosed.
Conclusion
NLPHL is a rare entity related to germinal centers
transformation and classical Hodgkin disease. A con-
firmed diagnosis by hematopathologist in the case of
CD-20 HL is strongly recommended considering the
risk of misdiagnosis with LRCHD. Treatment strategy
adapted to early stage of the disease, as well as indolent
course and risk of subsequent malignancies is the major
challenge. Treatment according to early stage cHL is
associated with good results but the role of Rituximab
associated with chemotherapy with low rate of adverse
effects has to be investigated considering the high effi-
| 142 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
cacy of rituximab alone in relapse. The need of treat-
ment for patients in confirmed complete remission after
initial surgery for stage I disease is an option, particular-
ly in children, in order to limit treatment related mor-
bidity.
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 Hodgkin disease

The prognostic role of positron emission tomography

scan in Hodgkin’s lymphoma

A. Gallamini A B S T R A C T

Hematology Department and BMT
Unit, Cuneo, Italy
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:144-150
Several prognostic factors have been proposed for Hodgkin lymphoma (HL); however, their predic-
tive value in defining treatment outcome has been questioned. Positron emission tomography using
[18F]-fluoro-2-deoxy-D-glucose (FDG-PET) is an important tool for staging, treatment response assess-
ment, post-treatment prognosis definition of residual mass, and prediction of autologous stem cell
transplantation outcomes in HL. Moreover, FDG-PET has shown high accuracy and overall predictive
value in therapy response forecasts when performed very early during standard ABVD chemotherapy.
The treatment of HL has shown an improving efficacy during the last 30 years, and durable remissions
are now achieved in more than 85% of the patients; however, some concerns still exist about the tox-
icity and risk of over treatment. Therefore, new prognostic factors, such as FDG-PET, seem to play a
role in HL in defining a risk-adapted therapy for the single patient. Many trials are currently under-
way, both in limited and advanced HL, and are aimed at exploring the role of a PET-driven, risk-adapt-
ed therapy. However, many questions remain unanswered, such as the standardization criteria for early
PET scanning and interpretation, the role of interim-PET during the course of therapies with different
intensity, and during follow-up.

The clinical usefulness of prognostic factors in
Hodgkin lymphoma
The ABVD polychemotherapy regimen
has long been considered the golden stan-
dard for Hodgkin lymphoma (HL) therapy.
The recommended schedule is three or four
courses, plus Involved-Field (IF) radiothera-
py in limited-stage, and six courses plus
“consolidation” radiotherapy for bulky or
residual disease in advanced stage patients.1
However, 20-30% of the advanced-stage
patients fail to achieve durable remissions,
and ultimately die of recurrent/resistant
lymphoma.2
In the nineties, more aggressive regimens
were proposed, such as escalated BEACOPP.
Their efficacy has been shown superior to
ABVD in at least three randomized studies,
both in terms of progression-free and overall
survival.3-5 Diehl and colleagues for the
German Hodgkin Lymphoma Study Group
demonstrated a superiority of escalated
BEACOPP versus the COPP/ABVD regimen,
with an 8-year progression-free survival of
85% for the former, and 69% for the latter.
At the beginning of the millennium, the
same authors claimed that there was no
longer any need for prognostic factors, since
they had lost their predictive power, as treat-
ment was successfully adapted to disease
burden.6 Consequently, some clinicians con-
sider this regimen to be the standard therapy
for advanced-stage HL.
These brilliant results, however, have
been partially hampered by an increased
risk of secondary malignancies, with a
higher risk benefit ratio for a non-trivial
subset of the patients.7 The most common-
ly reported side effects were short-term
hematological toxicities and amenorrhoea,8
but the cumulative 5-year risk of secondary
acute leukemia or myelodysplastic syn-
drome was 2.2% vs. 0.4% for the standard
treatment. Moreover, nearly two-thirds of
the patients are exposed to the risks of over
treatment.
In limited-stage HL, the long-term seque-
lae of radiotherapy, such as cardiovascular
events or secondary neoplasm arising inside
the fields of radiation, are responsible for
most deaths in long-term survivors for HL.9
For these reasons, the search for new
parameters able to predict treatment out-
come has been spurred on in the last few
years, and prognostic markers regained
great popularity. They play now a central
role in planning a new therapeutic strategy
aimed to single out patients with a very
poor prognosis, candidates for an aggres-
sive front-line therapy, and to spare toxici-
ty to the majority of the patients.10 [18F]-flu-
oro-2-deoxy-D-glucose positron emission
tomography (FDG-PET) seems to play an
ideal role in this perspective. The FDG
uptake by the tumor is a consequence of an
intact metabolic activity and reflects the
viability of the neoplastic cells. Therefore,
in principle, PET scanning could give a visu-
al update of the state of the lymphoma at
every time point during treatment, and pos-
sibly identify patients with different
chemosensitivity and treatment response.
In this perspective, two major points are
currently being investigated in several cur-
rent clinical trials:
1. In the early-stages, could involved-field
radiotherapy be delivered only to
patients with a poor prognosis, as defined
by a positive interim-PET after two
ABVD courses?

| 144 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

2. In advanced-stages, could an aggressive poly-
chemotherapy be reserved for patients with a very
poor prognosis, as defined by a positive-interim-PET
after two ABVD courses?
A possible answer to question 1 could arise from the
H10 study of GELA-EORTC-IIL and from MRC
RAPID study; for question 2, the RATHL study, the
GHSH HD18 and the GITIL HD0607 will try to give an
answer (see the paragraph on open issues).
The role of FDG-PET in post-treatment evaluation
FDG-PET was first introduced in the management of
lymphomas in the early 1990s. It is now recognised as
an important tool for staging and treatment response
assessment in Hodgkin and non-Hodgkin lym-
phomas.14,15 At the end of therapy, the persistent FDG
uptake is considered a proof of survival of viable neo-
plastic cells.16,17
Moving from these concepts, several reports in the
literature have demonstrated the high sensitivity and
specificity of FDG-PET in tumor response assessment.
In a recent accurate meta-analysis from 13 studies on
408 HL patients, upon exclusion of other studies not
fulfilling the minimal requirements for review (full ring
of CT-PET, adequate follow-up, definition of the refer-
ence test), Zijlstra and colleagues were able to demon-
strate a pooled sensitivity and specificity of PET in
defining treatment outcomes of 84% and 90%, respec-
tively18 (Figure 2).
Thanks to these results, in 2007 FDG-PET was pro-
posed as an essential tool in defining treatment
response and it has been integrated in the previous
Figure 1. Sensitivity and specificity of FDG-PET for treatments response (adapted from Zijlstra, by permission)
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 145 |
Berlin, Germany, June 4-7, 2009
definition on CR, PR, stable or progressive disease by
the International Workshop Criteria on Treatment
Response in Lymphomas.15 So far, two experiences
have been published on the clinical consequences of
the application of these new criteria in assessing tumor
response to treatment. The concept of CRU has been
abolished, and patients defined in CR or CRU at the
end of treatment had an identical outcome; patients in
PR had a progression free survival similar to the ones
in stable or progressive disease.25,26
Despite the good response to therapy, treatment of
HL results in residual mass in up to 64-80% of the
patients, as shown by conventional restaging modali-
ties.22,27,28 After Jerusalem’s preliminary study,29 many
reports focused on the role of FDG-PET for post-treat-
ment evaluation of a residual mass in lymphoma.
Quite recently, Terasawa systematically reviewed all
the studies published so far on this issue, and reported
a sensitivity for HL patients ranging between 43% and
100%, and a specificity ranging between 67% and
100%30 (Figure 3).
With these premises, FDG-PET has been proposed
to drive the decision of delivering consolidation radio-
therapy in cases of single residual mass persistence at
the end of lymphoma treatment, and its role has been
proven essential.31,32 In the German Cooperative
Hodgkin Lymphoma Study Group’s experience, PET-
driven decisions of delivering consolidation radiother-
apy at the end of treatment for Hodgkin’s lymphoma
has allowed a significant reduction of its use from 70%
of the patients in HD nine trials to a 12% in the HD in
fifteen trials.32
14th Congress of the European Hematology Association
Figure 2. Sensitivity and specificity for treatment response in case of residual mass (adapted from Terasawa, by permission).
Early interim positron emission tomography during conven-
tional Hodgkin lymphoma treatment
More recently, PET has emerged as a useful prognos-
tic tool, which may predict treatment outcome when
performed very early during therapy (early PET). A
number of studies have focused on its prognostic role
after two33-35 or three36 courses of standard ABVD
chemotherapy, and some studies have also suggested
that PET can predict treatment response as early as
after one course of chemotherapy.37-40 Overall, in HL
the positive and negative predictive values have
ranged between 60-80% and 80-90%, respectively,
while in non-Hodgkin lymphoma (NHL) the positive
predictive value was lower (40-60%). In all studies, the
authors confirmed the prognostic role of early PET in
predicting treatment outcome and concluded that it is
useful and reliable for assessment of the treatment
response.
Nearly 80% of the HL patients show a complete nor-
malization of the PET scan after two courses of
ABVD.34,35 This phenomenon, that has been called
“metabolic CR”,41,42 can be explained by the peculiar
architecture and organization of the neoplastic tissue,
where only a few, scattered neoplastic cells (accounting
for less than 1% of the total cellular population) are sur-
rounded by a population of non-neoplastic mononu-
clear bystander cells. The latter are probably responsible
for the immortalisation of Hodgkin and Reed-Sternberg
(HRS) cells by stimulating cytokine production by CD
4+ lymphoid cells recruited by TARC (thymus and acti-
vation-related chemokines: paracrine loop), or by trig-
gering cytokine release by the HRS cells able to induce
survival of the same cells (autocrine loop).43 In cases pre-
senting with bulky lesions at diagnosis, a negative early
PET is often associated with a persisting bulky lesion of
more or less unchanged size. The explanation might be
that the chemotherapy switches off the production of
chemokines by the activated lymphoid cells, as
described for TARC. The latter can be measured in the
serum of HL patients and its level correlated to the qual-
ity of treatment response; for patients in CR, the levels
are much lower than in patients with stable or progress-
ing disease.44 The situation is different in NHL. In a
French study, a substantial fraction of diffuse, large B-
cell lymphoma (DLCL) patients (40%) showed a posi-
| 146 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Proposed Criteria for PET-2 interpretation after ABVD chemotherapy.
Negative
0 no uptake
1 uptake ≤ mediastinum
2 uptake ≥ mediastinum but ≤ liver
Positive
3 uptake > liver in some sites even if uptake ≤ liver or mediastinum
at other sites
4 uptake > liver in over 90% of sites or development of new uptake
consistent with progressive disease
Figure 3. Proposed semi-quantitative criteria for interim-
PET interpretation in HL (M Juweid and J Radford, person-
al communication, 2007).
tive early PET, but only 60% of those patients eventual-
ly experienced treatment failure.45 The same group ret-
rospectively re-evaluated the same cohort of patients
using SUV analysis and reported that when using an
SUV maximum reduction of 66% as the cut-off value,
the positive predictive value shifted from 43-83%.46
In HL, after two courses of ABVD, early PET is posi-
tive and minimally positive in 20% and 9-10% of the
patients, respectively.34-36,47 The patients with minimal
residual FDG uptake (MRU) on early PET are character-
ized by a very good prognosis36,47 and it has been pro-
posed that a PET scan with a faint FDG uptake consis-
tent with MRU be considered as negative.36,48 Since the
majority of patients displaying MRU on early PET still
have an excellent prognosis, this phenomenon could be
related to non-specific activity in macrophages and
other inflammatory cells infiltrating the tumor as a
response to the chemotherapy rather than to a chemo-
resistance.17 Hutchings and Mikhael first introduced the
term of MRU as a low-grade uptake of FDG (just above
background uptake) in a focus within an area of previ-
ous documented disease, reported by the nuclear medi-
cine physicians as unlikely to represent active malignan-
cy.36,49 Later, in a joint Italian and Danish study,48 MRU
was defined as a faint, non-focal FDG uptake in a site of
involvement at diagnosis, with an SUV lower or equal
to the one recorded in the mediastinal blood pool struc-

Table 1. Prognostic meaning of FDG-PET prior to ASCT.
Author N. Histology Indication
(ref) patients
Jabbour64 68 HL Rel/Pro
Schot 65 117 NHL/HL Rel/Pro
Spaepen66 60 NHL/HL Rel/Pro
Filmont67 20 NHL/HL Rel/Pro
Svoboda68 50 NHL/HL Rel/Pro; 3 line
Crocchiolo69 53 NHL/HL Rel/Pro
tures (MBPS). A similar semi-quantitative approach has
recently been proposed for interim PET interpretation in
the context of an International Protocol for advanced-
stage HL (Table 1).
The prognostic role of early chemotherapy sensitive-
ness assessment seems independent and more predic-
tive of treatment outcome than IPS. In the afore men-
tioned Italian-Danish study early PET proved to be the
only statistically significant predictor of treatment out-
come, whatever the IPS score in the single patient. From
this point of view, interim FDG-PET seems to represent
a step forward in the direction of a single-patient, risk-
adapted therapeutic strategy.48 It is noteworthy that, in
the same study patients with a negative early PET
showed a 2-year progression-free survival (PFS) equal or
even better than the one for the entire cohort of patients
treated with more aggressive (and more toxic) regimens
such as BEACOPPesc.
The ideal time for interim positron emission tomography
during therapy
Two questions concerning the ideal time for interim-
PET scanning are still unsettled:
1. What is the ideal time for interim-PET scan after
chemotherapy administration?
2. What is the ideal number of chemotherapy cycles
before interim-PET scan?
As far the point (1) is concerned, Spaepen has shown
in mice that FDG uptake by neoplastic cells and reactive
inflammatory macrophages was minimal 14 days after
chemotherapy administration.17 In a review of the pub-
lished experience of interim PET early during treatment,
Kasamon concluded that the optimal time for perform-
ing interim PET during chemotherapy ranged between
7 and 14 days after chemotherapy.50
As far as point (2) the answer could depend on the
aggressiveness of the tumor and the efficacy of the
chemotherapy. In a preliminary experience Iagaru, in a
small cohort of 20 HL and NHL patients found, by ∆
SUV variation analysis, found that both 18F-FDG
PET/CT scans obtained at two and four cycles correlat-
ed well (correlation coefficient 0.98 and 0.80, respective-
ly) with end-treatment response. Furthermore, scans
performed at two cycles did not differ significantly from
scans performed at four cycles in terms of ∆ SUV from
baseline to post-treatment.51 In HL, most experiences
have been performed after two courses of chemothera-
py, although promising preliminary reports have stress-
es the high predictive value after one cycle.38-40)
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 147 |
Berlin, Germany, June 4-7, 2009
Timing PPV PNV 2-y PFS for PET resp 2-y PFS for PET non-resp
PET
Pre ASCT 72% 76% 76% 27%
Pre ASCT 73% 67% 73% 25%
PreASCT 87% 90% 100% 24%
PreASCT 91% 87% 87% 7%
PreASCT 94% 46% 50% 12%
PreASCT 43% 72% 90% 55%
Early interim-positron emission tomography during BEA-
COPP or equivalent therapies
Very recently, during the Seventh International
Symposium on Hodgkin Lymphoma and the Fiftieth
Annual ASH meeting, preliminary results were present-
ed on the prognostic role of early PET after two courses
of BEACOPPesc in advanced HL.55-57 All these studies are
characterized by a relatively good outcome of patients
with a positive early interim scan. In particular, in both
studies, the number of false positive studies and the low
positive predictive value proved to be a problem; 10%,
20% and 83%, and 60%, 46% and 17%, respectively.
Despite the relatively good outcome of the interim-PET
positive patients, possibly due to a late “salvage” effect
of escalated BEACOPP; the higher proportion of posi-
tive scans (30%), as compared to the one observed after
ABVD (20%) points to the low specificity of PET during
BEACOPP. The mean time between the last administra-
tion of BEACOPPesc and early PET ranged between 11
and 17 days.55-56 Currently, we do not know the optimal
timing for early PET after BEACOPPesc, but one could
speculate that the risk of false-positive studies due to a
non-specific inflammatory effect is greater early after
BEACOPPesc than after ABVD, due to the more effi-
cient tumor kill. Other possible explanations include the
frequent use of growth factors (G-CSF) and the
increased rate of infections and use of antibiotics after
BEACOPPesc compared with after ABVD.
We do not know yet the prognostic role of early PET
after BEACOPPesc, since until now it has not been eval-
uated in prospective studies where no therapeutic
changes were based on early PET results. The ongoing
HD-18 study of the German Hodgkin Study Group will
specifically address this issue. Now, I do not recom-
mend early PET in routine clinical practice as a basis for
early therapeutic adaptation in patients treated with
BEACOPPesc outside a clinical trial.
Prognostic value of FDG-PET before high-dose chemother-
apy/autologous stem-cell transplantation
High-dose chemotherapy (HDT) followed by autolo-
gous stem-cell transplantation (ASCT) has been success-
fully used for relapsing or primary resistant HL.58-61 The
only significant prognostic factors were the duration of
response to first-line chemotherapy and the status of
the disease at transplant or, in other words, the
chemosensitivity of the tumor. In a review of the pub-
lished literature, evidence points to the high overall pre-
dictive value of pre-transplant FDG-PET. Some reports
compose a mixture of NHL and HL, while others deal
14th Congress of the European Hematology Association
exclusively with HL. In general, the predictive value is
higher in HL in respect to NHL and the positive predic-
tive value (PPV) is higher than the negative predictive
value (NPV) (see Table 3). In particular the PPV ranges
between 91% and 43%, while the PNV between 90%
and 46%. These wide-range fluctuations are mainly due
to the presence of a wide array of NHL subtypes that, as
already known, display different FDG avidity.62,63
The role of FDG-PET in follow-up
FDG-PET has been proposed in the follow-up of HL
patients. In a preliminary experience of follow-up per-
formed with serial FDG-PET scans every 6 months in 36
HL patients in CR after ABVD therapy, Jerusalem found
six false-positive studies, and no false-negative studies
out of 119 performed scans. In five other positive stud-
ies, FDG-PET preceded the relapse after a median of 3.5
(1-9) months.70 In a series of 57 HL patients who under-
went a systematic follow-up with FDG PET every 6
months for the first two years, and then once in a year
for the following three years, Zinzani et al found a rela-
tively high percentage of patients in which the relapse
suspicion was based on PET results: (21/57: 37%). In all
cases, the positive scans were recorded at mediastinal
site. All underwent histological confirmation by surgical
biopsy. HL relapse was found in only 10/21 (48%)
patients. More than three quarters of the positive scans
were found 6 and 12 months after treatment end.71 In
another experience where PET was performed every 6
months during follow-up in a cohort of 45 HL patients
in CR after six ABVD courses, Zuckerman found the
rate of false positive was 9/45 (20%).72 Based on these
results the use of FDG-PET in the follow-up of HL
patients cannot be routinely recommended and further
studies are warranted prior to any definite conclusion.73
Controversial problems
Many issues remain controversial, and important
questions regarding PET scanning standardization and
interpretation are still unanswered: (i) it is still unclear
whether a qualitative approach using visual assessment
only is preferable or whether a semi-quantitative evalu-
ation by SUV analysis should be applied; (ii) the differ-
ent FDG avidity and uptake patterns before and during
chemotherapy between Hodgkin and non-Hodgkin
lymphomas (NHL) prevents the use of common criteria
for PET interpretation for all lymphomas; (iii) it is still
uncertain if the prognostic value of early PET varies
between different therapeutic regimens; (iv) it is still
unknown whether a strategy based on early chemother-
apy intensification only in interim-PET positive patients
could turn out in improving the overall outcome of the
entire cohort of HL patients.
The role of early PET in HL has been extensively eval-
uated and a number of answers to the above-mentioned
issues have been proposed. Recently a number of “posi-
tion papers” have been published on the issue of stan-
dardization criteria for interim-PET interpretation,
focusing on the dilemma of qualitative or quantitative
assessment. The authors concluded that: (i) there is no
need for quantitative assessment in HL, and visual
assessment gives the best results, provided that some
“liberal” criteria for a negative scan interpretation are
accepted, including the cases of minimal residual
| 148 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
uptake; (ii) a semi-quantitative score with reference
organs, such as mediastinum and liver could be used to
better distinguish between unspecific residual uptake
and persisting positive lesions; (iii) a baseline study is
strongly recommended, since early PET interpretation is
based on a site-to-site comparison of FDG uptake both
before and after chemotherapy; (iv) in the case of visual
assessment, the inter-observer concordance was no
higher than 50% in absence of defined rules for PET
scan interpretation; (v) the quantitative approach seems
superior in terms of predictive power on treatment out-
come in NHL; (vi) the quantitative approach needs a
more stringent standardization since the SUV value
could be influenced by different variables; (vii) the rela-
tive SUVMAX reduction of an interim PET with respect to
the baseline scan was equivalent to the relative SUVMEAN
reduction or to the absolute SUVMAX calculation, but less
operator-dependent and less influenced by the different
PET scanners.52-54
Open Issues: ongoing trials
Limited-stage Hodgkin lymphoma
In the EORTC/GELA/IIL H10 trial, patients with lim-
ited stage IA-IIB are stratified in two risk classes and
evaluated for treatment response after two ABVD
courses; thereafter, they are randomised to receive a
PET-independent treatment, a PET-driven treatment
with chemotherapy intensification, or de-escalation in
PET positive and PET-negative patients, respectively.11 A
similar project is ongoing in the United Kingdom; a ran-
domised trial (RAPID) comparing no further treatment
with involved field RT following three cycles ABVD
and a “negative” (-ve) PET scan in limited-stage HL
patients.12
Advanced-stage Hodgkin lymphoma
Several trials are running worldwide aimed at assess-
ing the overall clinical impact of a strategy of early ther-
apy intensification (with BEACOPP or IGEV followed
by ASCT) in patients with an interim PET positive after
two courses of ABVD (RATHL from BMC Nordic
Group and GISL), trial HD 18 from GHSG, Trial HD
0607 from GITIL (Gruppo Italiano Terapie Innovative
nei Linfomi). Preliminary results have been presented
from the Italian GITIL group; interim-PET scans per-
formed after two ABVD courses in a cohort of 136
patients with stage IIB-IVB HL – 19/136 (14%) were
found to be positive with a semi-quantitative score sys-
tem. They were treated with BEACOPP (a escalated + 4
baseline courses) and after a mean follow-up of 14
months, 15/19 were in continuous CR.13
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ring lymphoma. Blood 2007;109:486-91.
66. Spaepen K, Stroobants S, Dupont P, Vandenberghe P, Maertens
J, Bormans G et al. Prognostic value of pretransplantation
positron emission tomography using fluorine 18-fluoro-
deoxyglucose in patients with aggressive lymphoma treated
with high-dose chemotherapy and stem cell transplantation.
Blood 2003;102:53-9.
67. Filmont JE, Czernin J, Yap C, Silverman DH, Quon A, Phelps
ME, Emmanouilides C. Value of F-18 Fluorodeoxyglucose
Positron Emission Tomography for Predicting the Clinical
Outcome of Patients With Aggressive Lymphoma Prior to and
After Autologous Stem-Cell Transplantation. Chest 2003;124;
608-13.
68. Svoboda J, Andreadis C, Elstrom R, Chong EA, Downs LH,
Berkowitz A et al. Prognostic value of FDG-PET scan imaging
in lymphoma patients undergoing autologous stem cell trans-
plantation. Bone Marrow Transplant 2006;38:211-6.
69. Crocchiolo R, Canevari C, Assanelli A, Lunghi F, Tassara M,
Stanghellini ML et al. Pre-transplant 18FDG-PET predicts out-
come in lymphoma patients treated with high-dose sequential
chemotherapy followed by autologous stem cell transplanta-
tion. Leuk Lymphoma 2008;49:727-33.
70. Jerusalem G, Beguin Y, Fassotte MF, Belhocine T, Hustinx R,
Rigo P, Fillet G et al. Early detection of relapse by whole-body
positron emission tomography in the follow-up of patients
with Hodgkin’s disease. Ann Oncol 2003;14:123-30.
71. Zinzani PL, Tani M, Trisolini R, Fanti S, Stefoni V, Alifano M
et al. Histological verification of positive positron emission
tomography findings in the follow-up of patients with medi-
astinal lymphoma. Haematologica 2007;92:771-7.
72. Zuckerman D, Lacasce A, Jacobsen E, et al. High False Positive
Rate with the Use of CT and FDG-PET in Post-Remission
Surveillance for Hodgkin Lymphoma. Blood 2007;110:2327.
73. Seam P, Juweid ME, Cheson BD. The role of FDG-PET scans
in patients with lymphoma. Blood 2007;110:3507-16.
 Hodgkin disease

Recent advances in the treatment

of early-stage Hodgkin’s lymphoma

D.A. Eichenauer
A. Engert
First Department of Internal
Medicine, University Hospital
Cologne, Cologne, Germany, and
German Hodgkin Study Group
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
A B S T R A C T
The standard of care for patients with early-stage Hodgkin lymphoma (HL) has changed from wide-
field radiotherapy alone to combined modality strategies consisting of a brief chemotherapy followed
by involved-field radiotherapy (IF-RT). Since the vast majority of patients treated with this approach
achieve permanent remissions and long-term survival rates beyond 90%, cure rates can hardly be fur-
ther improved. Therefore, the major goal of current clinical research is to minimize therapy-related
acute and late toxicities, such as secondary malignancies, infertility, heart failure and pulmonary dys-
function, without compromising treatment efficacy. Different strategies are being pursued. A possible
reduction of chemotherapy, as well as the minimization of radiation fields from IF to involved-node
(IN) and the value of risk-adapted strategies based on PET imaging is currently being investigated in
clinical trials worldwide.

2009;3:151-154

odgkin lymphoma (HL) is a malig- bined modality strategies were introduced

H nant disease of the lymphatic system

with an incidence of 2-3/100.000/
year in developed countries. Generally, HL
occurs in all age groups but young adults are
most often affected.1 Two major subtypes
can be distinguished, classical Hodgkin lym-
phoma (cHL), which accounts for about
95% of all cases, and nodular lymphocyte
predominant Hodgkin lymphoma (NLPHL),
representing about 5% of cases.2
Due to substantial treatment improve-
ments over the past decades, HL has
changed from an incurable disease to one of
the adult malignancies with the best cure
rates; a fact resulting in a steadily growing
number of long-term survivors. Since these
survivors often suffer from treatment-associ-
ated late toxicities, such as secondary malig-
nancies, infertility, heart failure and pul-
monary dysfunction, reducing the frequency
of long-term sequelae without compromis-
ing treatment efficacy has become the major
challenge of today’s clinical research in HL.3
This text aims at giving an overview on the
changes in the treatment of early-stage HL in
recent years and of current developments to
improve therapy further.
Radiotherapy alone
Early favorable and early unfavorable-
stage HL are distinguished by the presence
or absence of certain risk factors (Table 1).
For many years, wide-field radiotherapy
alone was considered the treatment of
choice for early favorable-stage HL. By this
approach, many patients achieved a com-
plete remission (CR) but the relapse rate was
high and overall survival (OS) was not satis-
fying.4 To improve treatment results, com-
and randomized trials demonstrated them to
be more effective than radiotherapy alone.5,6
Thus, there is currently no indication for the
use of radiotherapy alone in HL, except for
the treatment of patients with stage IA
NLPHL, without risk factors. This small sub-
set of patients, analyzed by the German
Hodgkin Study Group (GHSG) and the
European Organisation for Research and
Treatment of Cancer (EORTC), revealed no
significant outcome differences between
patients treated with 30 Gy involved-field
radiotherapy (IF-RT) and patients treated
with extended-field RT (EF-RT) or combined
modality strategies.7-8 Consequentially, the
least toxic approach, 30 Gy IF-RT, was
adopted as the standard of care.
Combined modality approaches for early favor-
able-stage Hodgkin lymphoma
Various randomized trials from different
study groups showed the superiority of
combined modality treatment over radio-
therapy alone in patients with early favor-
able HL.
The HD7 trial, conducted by the GHSG,
compared 30 Gy EF-RT plus 10 Gy to the IF
alone with two cycles of ABVD chemother-
apy followed by the same radiotherapy.
Tumor control was superior in patients treat-
ed with the combined modality approach,
resulting in a significantly better 7-year free-
dom from treatment failure (FFTF) (88% vs.
67%, p<0.001).6 The ability to reduce radia-
tion fields to IF without compromising treat-
ment efficacy has been shown in a number
of trials, so that combined modality treat-
ment, including a brief chemotherapy, such
as ABVD followed by IF-RT has become the

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 151 |
14th Congress of the European Hematology Association
accepted standard therapy in early favorable HL.9,10
In trials already closed but not fully analyzed, the pos-
sible reduction of either chemotherapy or radiotherapy
was evaluated. The purpose of the GHSG HD10 trial
was to investigate whether it is possible to reduce the
dose of IF-RT from 30 Gy to 20 Gy after chemotherapy
consisting of two or four cycles of ABVD. The interim
analyses performed so far revealed no significant differ-
ences between 20 Gy or 30 Gy IF-RT, and between two
or four cycles of ABVD chemotherapy.11 The final analy-
sis of the trial is expected later in 2009.
The GHSG follow-up trial HD13 aimed at decreasing
toxicity from the ABVD backbone by reducing the
number of drugs given. Patients were randomized
between two cycles of ABVD, ABV, AVD or AV
chemotherapy followed by 30 Gy IF-RT. A safety analy-
sis performed in June 2006 detected a fourfold increase
of events in the ABV and the AV arm, respectively, com-
pared to the ABVD standard arm. This increase could
not be explained by chance variation. Hence, the
ABV/AV arms were closed. The question whether AVD
is equivalent to ABVD will be answered in future analy-
ses of the trial.
Combined modality approaches for early unfavorable-stage
Hodgkin lymphoma
Patients with early unfavorable HL are commonly
treated with combined modality approaches. However,
the optimal chemotherapy regimen and the number of
chemotherapy cycles needed was a matter of debate.
In the EORTC/Groupe d’etude des Lymphomes de
l’adulte (GELA) H8U study, 996 patients were treated
with either six cycles of MOPP-ABV plus IF-RT, four
cycles of MOPP-ABV plus IF-RT or four cycles of
MOPP-ABV plus subtotal nodal irradiation (STNI). All
groups had similar 5-year event-free survival (EFS) (84%
vs. 88% vs. 87%) and 10-year OS estimates (88% vs.
85% vs. 84%). Thus, four cycles of chemotherapy fol-
lowed by IF-RT was proposed as the standard treatment
for patients with early unfavorable HL.12
In the EORTC/GELA follow-up H9U study, patients
were randomized into three treatment arms consisting
of four cycles of ABVD, six cycles of ABVD or four
cycles of BEACOPPbaseline, each followed by 30 Gy IF-
RT. An interim analysis at a median follow-up of four
years showed no significant differences regarding EFS
and OS between the treatment arms while increased
toxicity was observed with BEACOPPbaseline.13
In the GHSG HD14 trial, patients were randomly
assigned to either two cycles of BEACOPPescalated fol-
lowed by two cycles of ABVD chemotherapy or to four
cycles of ABVD chemotherapy. All patients received 30
Gy IF-RT. In 2008, a planned interim analysis of the
trial, including data from 1010 patients at a median
observation of three years was performed. This analysis
revealed a significant superiority of the treatment with
two cycles of BEACOPPescalated, followed by two
cycles of ABVD plus 30 Gy IF-RT compared to four
cycles of ABVD plus 30 Gy IF-RT in terms of FFTF (96%
vs. 90%). Therefore, the study arm consisting of four
cycles of ABVD followed by 30 Gy IF-RT was closed
and the regimen consisting of two cycles of
BEACOPPescalated plus two cycles of ABVD followed
by 30 Gy IF-RT was adopted as new standard for
| 152 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
patients with early unfavorable HL within the GHSG.14
Another new approach in the treatment of early unfa-
vorable HL is the PVAG14 regimen. The protocol con-
sisting of prednisone, vinblastine, doxorubicin and gem-
citabine administered in a 14-day cycle is currently
under investigation in a GHSG phase II trial.
Chemotherapy alone
Since it is well known that a number of severe long-
term sequelae, such as secondary malignancies, pul-
monary fibrosis or hypothyroidism can potentially be
caused by radiotherapy, several groups initiated trials
evaluating the impact of radiotherapy after adequate
chemotherapy.
In a trial conducted by the National Cancer Institute
(NCI) of Canada and the Eastern Cooperative Oncology
Group (ECOG), 399 patients with limited-stage HL
(nonbulky clinical stages IA and IIA) were enrolled.
They were randomly assigned to either receive four to
six cycles of ABVD chemotherapy alone or ABVD com-
bined with STNI. With a median follow-up of 4.2 years,
freedom from disease progression was superior in
patients receiving combined modality treatment (93%
in the combined modality vs. 87% in the chemotherapy
alone arm), indicating a better tumor control by addi-
tional radiation after chemotherapy. There were no sig-
nificant differences in terms of EFS and OS (88% vs.
86% and 94% vs. 96%, respectively).15 However, longer
observation and documentation of patients included in
this trial is required to draw final conclusions because
follow-up is still too short to assess, particularly the
radiotherapy-related long-term side effects.
Another study conducted at the Memorial Sloan-
Kettering Cancer Center (MSKCC) led to similar results.
A total of 152 patients with nonbulky clinical stages IA,
IB, IIA, IIB and IIIA were prospectively randomized to
either six cycles of ABVD chemotherapy alone or six
cycles of ABVD followed by radiotherapy. Although no
significant differences in terms of CR duration, freedom
from progression (FFP) and OS could be detected, a ten-
dency towards a superior outcome in patients receiving
combined modality treatment was observed. At 60
months, 91% of patients receiving combined modality
treatment and 87% of patients receiving chemotherapy
alone were still in CR (after 94% of patients in both
Table 1. EORTC and GHSG risk factors defining treatment
groups clinical stage I/II Hodgkin lymphoma.
Trial group Risk factors Treatment groups
EORTC/GELA 1) Large MM Early favourable:
2) Age ≥50 CS I or II without any RF
3) B symptoms and ESR ≥30 mm/h Early unfavourable:
or no B symptoms and ESR ≥50 mm/h CS I or II without any RF
4) ≥4 involved sites
GHSG 1) Large MM Early favourable:
2) Extranodal disease CS I or II without any RF
3) B symptoms and ESR ≥30 mm/h Early unfavourable:
or no B symptoms and ESR ≥50 mm/h CS I and IIA with any RF;
4) ≥3 involved sites CS IIB with RF 3 and/or 4
MM: mediastinal mass; ESR: erythrocyte sedimentation rate; CS: clinical stage; RF: risk
factor.

arms had initially achieved CR). FFP and OS rates were
86% and 97%, respectively, for patients treated with
chemotherapy plus radiotherapy compared to 81% and
90%, respectively, for patients who received chemo-
therapy alone.16
In the EORTC/GELA H9F study for patients with
early favorable HL, all patients received six cycles of
EBVP chemotherapy. Patients with CR/CRu were then
randomized between 36 Gy IF-RT, 20 Gy IF-RT and no
RT. The trial could not be completed according to plan
since the no RT arm had to be closed prematurely due
to an excessive rate of events.13
The results of these trials indicate that omission of
radiotherapy after an appropriate chemotherapy might
only be possible in carefully selected patients. Currently,
one of the most relevant questions is to identify and
possibly further reduce toxicity in these patients. Since
response assessment based on CT scans alone does not
seem adequate to distinguish between those patients
who may be sufficiently treated with chemotherapy
alone and those who require additional radiotherapy or
even more intensive treatment, ongoing trials focus on
the predictive value and possible stratification based on
the results of interim fluoro-deoxy-glucose positron
emission tomography (FDG-PET).
Response adapted therapy based on FDG-PET
As indicated by Danish and Italian groups, early inter-
im FDG-PET might be a good predictor for treatment
failure in HL patients.17-18 Furthermore, the results of the
GHSG HD15 trial for patients with advanced HL indi-
cate that FDG-PET is a valuable tool for the decision
whether patients with residual lymphoma after
chemotherapy should be irradiated or not. Patients with
residual lymphoma larger than 2.5 cm had PET scans.
Patients with negative PET were not irradiated; patients
with positive PET were irradiated. The negative prog-
nostic value (NPV), defined as the portion of patients
without progression, relapse or radiotherapy within 12
months, was 94%.19 Based on these findings, various
ongoing trials evaluate whether treatment of patients
with early-stage HL might be stratified based on the
results of an interim PET scan. In the EORTC/GELA
H10 trial, the treatment in the standard arm consists of
three cycles of ABVD chemotherapy for patients with
favorable risk profile and four cycles of ABVD
chemotherapy for patients with unfavorable risk pro-
file. Chemotherapy is followed by involved-node RT
(IN-RT), which means a reduction of the radiation fields
compared to the standard IF-RT technique.20 In the
experimental study arms, treatment is stratified based
on the results of a PET scan performed after the second
chemotherapy cycle. Patients with negative PET receive
two or three additional cycles of ABVD depending on
their risk profile. Patients with positive PET receive an
intensified treatment consisting of two cycles of
BEACOPPescalated followed by IN-RT. In the GHSG
HD16 trial for patients with early favorable HL, patients
receive two cycles of ABVD chemotherapy. Then, PET
scan is performed in all patients. In the experimental
arm, those patients with positive PET additionally
receive 30 Gy IF-RT, those with negative PET do not. In
the standard arm, all patients receive 30 Gy IF-RT sub-
sequent to chemotherapy irrespective of the PET result
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 153 |
Berlin, Germany, June 4-7, 2009
Figure 1. HD16 trial for early favorable-stage Hodgkin lym-
phoma.
(Figure 1). In a large British trial, which started recruit-
ment in 2003, patients with a negative PET after three
cycles of ABVD chemotherapy are randomized
between either no further treatment or IF-RT. Patients
with positive PET receive a fourth cycle of ABVD
chemotherapy followed by IF-RT.21
Conclusions
The introduction of combined modality approaches
has led to cure rates beyond 90% in patients with early-
stage HL. Since these cure rates can hardly be further
improved, current trials aim at evaluating whether a
response-adapted stratification of treatment based on
the risk profile of the individual patient is possible and
practicable. At the moment, FDG-PET is considered the
most promising tool to distinguish between those
patients who might receive a reduced treatment with-
out worsening the prognosis and those patients who
might benefit from an intensified treatment. However,
results from large randomized trials addressing this
issue are pending. Therefore, a brief chemotherapy fol-
lowed by IF-RT remains the standard treatment for
patients with early-stage HL.
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G. Morgan
K. Boyd
Brookes Lawley Building, Institute of
Cancer Research, Brookes Lawley
Building, 15 Cotswold Road, Sutton,
Surrey SM2 5NG, UK
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:155-160
Multiple myeloma
Pathobiology, genetics and clinical outcome
of myeloma
ultiple myeloma (MM) is presently
M an incurable malignancy that affects
over 3,000 new individuals each
year in the United Kingdom (UK). The inci-
dence increases with age, although approxi-
mately 40% of patients present under the
age of 60 years. The clinical picture involves
a combination of bone destruction, immune
deficiency, bone marrow, and renal failure
due to the accumulation of malignant plas-
ma cells within the bone marrow. The out-
look for patients is poor, with a median sur-
vival of approximately 5 years. Traditional
staging systems used in MM separate
patients into prognostic groups using simple
laboratory parameters. The first reliable sys-
tem to be devised, the Durie-Salmon staging
system, is a surrogate measurement of
tumor bulk and combines a number of fac-
tors including the extent of bone lesions, the
presence of hypercalcaemia, and the level of
paraprotein and hemoglobin.1 These basic
measurements enable clinicians to make a
rough estimate of prognosis, as patients with
stage IA disease have a median survival of 5
years compared to patients with stage IIIB
disease, who have a median survival of 14.7
months. In the intervening years, a number
of other prognostic factors have been identi-
fied: beta 2 microglobulin (β2m), a polypep-
tide that forms part of the extra cellular por-
tion of major histocompatibility complex
(MHC) class I molecules; the plasma cell
labelling index (PCLI), a measure of tumour
cell proliferation; plasmablastic morphology;
and cytogenetics. Currently, the most easily
reproducible and most widely used staging
system is the International Staging System
(ISS) which categorises patients into three
groups based on their presenting β2m and
albumin, with stage I patients having a
median survival of 62 months, compared to
29 months in stage III patients.2 These stag-
ing systems do not reliably predict outcome
in individual patients and have not been val-
idated in the era of novel agents.
Importantly, they are not based upon our
enhanced understanding of the biological
and molecular characterization of the MM
cell and the role of the bone marrow
microenvironment in disease pathogenesis.
Normal plasma cell differentiation
In order for a B cell to exit the germinal
centre and differentiate into a plasma cell, it
must downregulate the transcriptional
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 155 |
repressor BCL6, which results in the upregu-
lation of BLIMP1. BLIMP1 expression results
in repression of Pax5 and consequently
upregulation of XBP1. An important control
point of these events relies on the presence
of nascent immunoglobulin within the
endoplasmic reticulum, which requires cor-
rect folding mediated via the unfolded pro-
tein response (UPR). The UPR ensures cor-
rect folding, processing, export or degrada-
tion of proteins emerging from the endoplas-
mic reticulum (ER). Activation of the UPR
results in a bias of translation towards the
synthesis of chaperone proteins involved in
protein folding, and an increase in disposal
of misfolded proteins. At the same time,
there is activation of the NF-κB pathway,
which acts as a survival signal. A key regula-
tor of the UPR is the chaperone protein BiP,
which is associated with the luminal
domains of the serine/threonine kinases
PERK, IRE1 and the transcription factor
ATF6. In the presence of misfolded proteins,
this interaction is destabilised, resulting in
the release of PERK and IRE1, which simul-
taneously results in their autocatalytic acti-
vation. The most important molecule for
normal plasma cell development is IRE1.
IRE1 recruits TRAF2, which activates sig-
nalling via JNK and p38 with associated acti-
vation of NF-κB. For plasma cell develop-
ment, the most important function resides in
the kinase and endoribonuclease domains.
The endoribonuclease activity regulates the
activation of the transcription factor XBP1.
Activation of XBP1 by the cleavage of XBP1
mRNA to form XBP1s results in terminal dif-
ferentiation to a functional plasma cell. The
activation of XBP1 also provides a survival
signal for cells passing this point, which is
mediated by IL6, as well as a positive feed-
back signal to the ER allowing it to handle
more unfolded protein. PERK phosphoryla-
tion inhibits the general translation initiation
factor eIF2a, resulting in a general shutdown
of non-chaperone protein synthesis. In the
presence of unfolded proteins, the precursor
form of ATF6 is also released from BiP and
translocates to the nucleus, where it medi-
ates the expression of key genes involved in
the UPR response, including BiP and XBP1.
Following the recognition of misfolded pro-
teins, disposal is via the ubiquitin-protea-
some pathway. If there is failure to adapt to
the accumulation of unfolded protein in the
ER, the cell is deleted via apoptosis mediat-
14th Congress of the European Hematology Association
ed by a number of pathways. CHOP/GADD153 is acti-
vated by activation of PERK and ATF4 with consequent
suppression of bcl-2 transcription and the induction of
other pro-apoptotic genes. Induction of CHOP is itself
repressed by NFkB, demonstrating the tight control
within the UPR of survival and apoptotic mechanisms.
The environment and signalling pathways of plasma cells
Within the bone marrow, plasma cells exist in spe-
cialised niches in close proximity to accessory cells,
including stromal cells, osteoclasts, osteoblasts, and
microvessels. This proximity triggers “adhesion mole-
cule” mediated interactions, which generate important
autocrine and paracrine loops mediating long-term sur-
vival and proliferation of the myeloma clone. Key
cytokines mediating these processes, secreted by stro-
mal cells, include IL6, IGF1, VEGF, BAFF, FGF, SDF1 and
TNFα. Important molecules secreted by the plasma cell
include TNFα, VEGF and TGFβ, which further upregu-
late proliferative cytokines, such as IL6 secreted by the
stromal elements. The CD40/CD40L interaction upreg-
ulates adhesion molecules, and consequently, IL6 and
VEGF secretion further. Other key interactions are with
osteoclasts (HGF, IL1β and MIP1α), osteoblasts (DKK,
RANKL, OPG, sFRP2) and vascular elements (VEGF).
Either directly through cell adhesion or via growth fac-
tor engagement, there is activation of a network of
downstream signalling molecules. Amongst the best
understood of these pathways are the canonical and
non canonical NF-κB pathways, the JAK/STAT,
RAS/RAF/MAPK and PI3k/AKT pathways. Genetic
alterations in the myeloma clone can clearly impact
these plasma cell interactions, leading to significant
changes in cellular behaviour and, as such, characteriz-
ing these lesions can define the abnormal biology of the
myeloma clone, giving insights into clinical behavior.
They can also define therapeutic targets.
Genetic mechanisms and molecular modelling of myeloma
Myeloma is thought to result from the transformation
of a proliferative “plasmablastic” cell located in the ger-
minal centre. The progeny of this cell migrate to spe-
cialised niches in the bone marrow, where they mature
towards terminally differentiated antibody secreting
plasma cells. During this process of terminal differentia-
tion, it is essential that the plasma cell should exit from
cell cycle, and close linkage of terminal differentiation
with cell cycle exit is crucial to prevent the continued
expansion of an immortalized pre-myelomatous clone.
Molecular lesions leading to either abnormal differenti-
ation or abnormal cell cycle progression are, therefore,
two potentially relevant pathways to myeloma. In addi-
tion to the requirement for generating antibody produc-
ing plasma cells, ensuring long term immunological
memory is another important part of normal plasma cell
biology, which if deregulated, could lead to clonal
immortalization. The pathological activation of survival
pathways and a retained ability to proliferate in
response to antigen are two further potentially impor-
tant pathways to myeloma.
Monoclonal gammopathy of undetermined signifi-
cance (MGUS) is a clonal plasma cell dyscrasia charac-
terized by a monoclonal paraprotein but no evidence of
end organ damage. One percent of patients with MGUS
| 156 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
progress per year to myeloma, with the risk of progres-
sion remaining constant for a patient over a 25 year
period.3 The majority of patients, therefore, do not
progress to malignant disease, and it is useful to consid-
er the genetic mechanisms leading to the development
of myeloma within this framework. Some genetic
lesions are likely to be initiating events, leading to
immortalization of the clone and producing the MGUS
phenotype, whilst secondary lesions are required to
produce the proliferation and growth advantage
required to result in a malignant phenotype.
Two common genetic lesions in myeloma are IgH
translocations and hyperdiploidy. Normal plasma cells
undergo class switch recombination events centered on
the immunoglobulin heavy chain locus at 14q32 in
order to determine the class of immunoglobulin secret-
ed by the mature cell. Illegitimate class switch events
result in translocations occurring between 14q, with the
breakpoint at the IgH locus and a number of partner
chromosomes. These molecular events juxtapose the
strong IgH enhancer next to an oncogene, resulting in
up-regulated expression of the oncogene. Hyper-
diploidy is gain of the odd numbered chromosomes,
resulting in a complement of 48 to 74 chromosomes,
and the mechanisms underlying this are not well under-
stood. Both these lesions have been found with similar
frequency in MGUS and myeloma, so probably consti-
tute initiating events producing clonal immortalization
but are not enough in themselves to result in myeloma.4
Loss of heterozygosity (LOH) of an allele is a common
mechanism for disease progression, conceptually lead-
ing to under-expression of a tumor suppressor gene.
LOH of 1p, for example, is found in 4.5% of MGUS
cases but 15% of myeloma cases, suggesting an associ-
ation with disease progression.5 Homozygous deletions,
resulting in biallelic silencing, is a progressive form of
this method of gene inactivation and has been found to
be prevalent in myeloma. Another potential molecular
mechanism of disease progression is mutation, which
results in the silencing of one allele, where perhaps the
other allele is already down-regulated by LOH or anoth-
er mechanism. Mutations of TP53 on 17p, for example,
have never been described in MGUS, and are seen at
rate of less than 1% in myeloma cases as a whole.
However, in cases, which have LOH of 17p the muta-
tion rate is nearer 25%, which again demonstrates that
co-operating events are necessary to produce the malig-
nant phenotype. Another potential mechanism, where a
deleted region can lead to gene upregulation, is mediat-
ed via loss of microRNAs (miRNA), which are small (17-
25 bases) oligonucleotide sequences that regulate the
translation of other genes by complementary base pair-
ing to their mRNA. By this method, it has been estimat-
ed that they regulate the expression of 30% of the
genome. Methylation of tumor suppressor genes is
another epigenetic mechanism of gene silencing and has
been identified in myeloma where it has been shown to
be of prognostic importance.6
Gain of genetic material manifest as gain of whole or
interstitial chromosomal regions represents an alternate
means of tumor progression, which conceptually leads
to overexpression of oncogenes. Gain of 1q has been
identified as being a region that is commonly gained in
myeloma, and has prognostic significance.7

Classification of myeloma
Progress has been made in the classification of myelo-
ma based on its genetic make-up. Two main subgroups
of myeloma have been defined: hyperdiploid and non-
hyperdiploid. Hyperdiploid cases are genetically and
clinically heterogeneous but overall, the group appears
to have a slightly favorable prognosis. The non-hyper-
diploid group is characterized by a high frequency of
illegitimate class switch recombination events centred
on the IgH locus (14q32). Five main translocation part-
ners have been identified:
1)t(4;14)(p16;q32). This translocation is found in 10% of
myeloma patients and results in the dysregulation of
two genes.8 Firstly, on the derivative 14 (der(14))
fibroblast growth factor receptor 3 (FGFR3), a recep-
tor tyrosine kinase, is over-expressed and occasional-
ly mutated. In addition on the der(4) MMSET is over-
expressed. MMSET has been shown to regulate cell
cycle progression and cell adhesion.9-11 t(4;14) is posi-
tively associated with IgA myeloma and is an inde-
pendent predictor of poor outcome. The majority of
t(4;14) cases also have del(13).
2)t(6;14)(p21;q32). This rare translocation (4% of pri-
mary myeloma samples) results in CCND3 overex-
pression.
3)t(11;14)(q13;32). This occurs in 15% of myeloma
patients and leads to overexpression of cyclin D1
(CCND1), a cell cycle regulator. Outcome is neutral in
this group.8,12-14
4)t(14;16)(q32;q23). This results in overexpression of
the transcription factor c-MAF and is associated with
poor prognosis.15-17 Characterization of this group has
identified upregulation of Cyclin D2 (CCND2), a pro-
moter of cell cycle progression.18
5)The t(14;20)(q32;q21) results in the overexpression of
MAFB, a transcription factor that has similar down-
stream targets to c-maf including upregulation of
CCND2, and is also associated with poor progno-
sis.19,20
A global molecular classification strategy building on
knowledge of the IgH translocation partners and based
on the expression of D group cyclins has been pro-
posed. The rationale for the use of the D group cyclins
in this way is based on observations that the levels of
cyclin D1, D2 and D3 are high, combined with the prior
observations that there are switch translocations, which
dysregulate cyclin D1 or D3. It is possible to recognise
eight distinct groups using these two basic features.
Genome mapping
The completion of the Human Genome Project has
led to the generation of a range of new technologies that
have transformed the way genomic abnormalities in
myeloma can be interrogated. Single nucleotide poly-
morphism (SNP) micro-array chips focus on polymor-
phic nucleotides which are scattered throughout the
human genome, accounting for genetic variation
between individuals in a population. The arrays allow
detection of allelic loss or gain, both of which can be
important in tumor pathogenesis. Uniquely, they are
also able to identify uniparental disomy where both loci
are inherited from the same parent, the result of incom-
plete chromosomal segregation during mitosis or
recombination between chromatids. SNP array analysis
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 157 |
Berlin, Germany, June 4-7, 2009
Table 1. Relationship of cyclin D expression and transloca-
tion group.
Group Cyclin D Ig translocation (gene) %
1 3 6p21 (D3) 3.7
2 1 11q13 (D1) 17.3
3 2 16q23 (MAF) 8.6
4 2 4p16 (FGFR3/MMSET) 11.1
5 2 - 21.0
6 2 +1 (low) - 6.2
7 1 (low) - 32.1
8 none - 1.2
of newly diagnosed myeloma patients emphasizes the
complexity and heterogeneity of the myeloma genome.
In our data set, each patient had a median of 14 regions
of loss of heterozygosity of which three per patient
were due to UPD, the remainder being due to deletion
events.21 Most new information has arisen from this
technology platform, or from comparative genomic
hybridization arrays (CGH), which provide similar copy
number information.
Despite this apparent complexity, there are recurrent
non-random structural chromosomal abnormalities and
it is possible to define some key regions and genes that
are important in myeloma pathogenesis and have prog-
nostic impact.
Deletion of 1p is a frequent event in myeloma, occur-
ring in 30% of cases, and with four potential minimally
deleted regions with deletion of some regions confer-
ring an adverse prognosis.22,23 We have previously iden-
tified a region at 1p32.3, which had LOH in 15% of
cases of newly diagnosed myeloma, and using a FISH
probe for this region, hemizygous deletions were found
in 4.5% of MGUS cases and 10.3% of SMM cases, sug-
gesting that deletion of this region is associated with
disease progression.5 This region contains two genes,
whose expression is abrogated by deletion: CDKN2C
and FAF1. CDKN2C, also known as p18, is a negative
regulator of the G1/S cell cycle transition checkpoint,
and its deletion has been shown to induce cellular pro-
liferation.24 FAF1 is involved in the ubiquitin pathway
and inhibits the chaperone activity of the heat shock
proteins, Hsc70 and Hsp70,25,26 as well as being a nega-
tive regulator of the NF-κB pathway.27 Cases with hem-
izygous or homozygous deletion of CDKN2C/FAF1 had
a lower overall survival compared with cases with an
intact 1p32 region (20 months vs. 46 months).5
Gain of 1q is common in primary myeloma samples,
being found in approximately 35% of our cases.
Increased expression of genes mapping to chromosome
1q has been linked to shorter progression-free survival
and overall survival post autologous stem cell trans-
plant.28 1q21 has previously been identified as a mini-
mally amplified region, which contains the potential
candidate oncogenes CKS1B, PDZK1 and BCL9.7,29 We
find a minimally deleted region between 1q21.1-q23.2,
a region containing 679 genes. Of these, only ANP32E
has significantly increased expression levels and is
linked to adverse overall survival.
A third of our cases had deletion of 6q, the majority
being interstitial deletions with the most commonly
14th Congress of the European Hematology Association
deleted region centred on 6q25.3. Deletion of this region
was not associated with adverse prognosis in our
dataset, but there is the potential for genes located here
to have prognostic importance. Twenty-nine percent of
cases have deletion of 8p, and most of these are large
deletions encompassing more than 75% of the short
arm with small interstitial events being rare. Deletion of
this region was not linked to adverse outcome in this
data set. We have identified a high rate of homozygous
deletion of a small region of 11q22.2, which contains
BIRC1 and BIRC2, both of which play a role in NF-?B
regulation and are, therefore, very likely to be relevant
to myeloma pathogenesis, but which do not carry prog-
nostic significance when deleted.
Monoallelic loss of 13 is the commonest genetic
abnormality of myeloma cells, found in 44% of cases
and is usually caused by monosomy of the whole chro-
mosome as opposed to interstitial deletion of 13q.
When detected by conventional cytogenetics it has been
associated with poor prognosis.30 However, when
detected by FISH the prognostic significance lessens. In
our dataset, if cases which are strongly and independ-
ently associated with poor outcome are removed, that
is, 17p- and the poor risk IgH translocation [t(4;14),
t(14;16), t(14;20)], the effect of 13q deletion becomes
neutral. It is difficult to define a minimally deleted
region, but 13q14.11-q14.3 contains all but three of our
cases, and contains 16 significantly under-expressed
genes, of which RB1 shows the largest fold change. This
region also contains the microRNA genes mir-16-1 and
mir-15a which have also been implicated in myeloma
pathogenesis.31
NF-κB pathway activation has been linked to muta-
tions and deletions of several regulators of the canonical
and non-canonical pathway.32,33 The net result of these
abnormalities is inhibition of the central negative regu-
lator of the pathway, IKK, resulting in a lifting of its reg-
ulatory effect and, therefore, constitutive NF-κB activa-
tion. The single most common abnormality is inactiva-
tion of TRAF3, which maps to 14q32. Biallelic loss of
this locus has been demonstrated in 4% of myeloma
patients, whilst 12% were shown to have loss of het-
erozygosity. Moreover, decreased expression of TRAF3
was linked to clinical outcome based on retrospective
analysis of a trial of relapsed myeloma patients treated
with either dexamethasone or bortezomib. Patients
with low TRAF3 expression had high response rates to
bortezomib (89% vs. 40%) and low response rates to
steroids (10% vs. 30%).32
Deletion of 16q23 has been found in 20% of primary
myeloma samples. Overall survival at 3 years in cases
with del(16q) was 42% vs. 72% in those without dele-
tion, although longer follow-up data suggests a neutral
prognostic effect. Using SNP-based gene mapping com-
bined with gene expression data it is possible to identi-
fy two key genes which are dysregulated in these cases:
WWOX and CYLD.34 WWOX is a known tumor sup-
pressor gene deleted in other malignancies, such as
prostate and breast cancer.35,36 CYLD is a tumor suppres-
sor gene first identified in cylindroma tumors, a heredi-
tary skin cancer37 and is known to function as a negative
regulator of the NF-κB pathway.38,39 It is possible to iden-
tify mutations in CYLD in the cases with LOH, con-
firming its role as an important tumor suppressor gene
| 158 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
in myeloma and the central role of the NF-κB pathway
in myeloma pathogenesis.
Hemizygous deletion of 17p is detected in approxi-
mately 10% of primary myeloma cases. 17p deletion is
an independent adverse prognostic factor predicting sig-
nificantly shorter overall survival in patients treated
with an autologous transplant regimen (15 vs. 48
months),40 and we confirm this poor prognosis in our
dataset. Although the FISH probe targets the TP53 locus
at 17p13, mapping of samples with LOH of 17p demon-
strates that the majority of samples have hemizygous
deletion of a large part of 17p, and it is difficult to define
a minimally deleted region. Other groups have shown
that hemizygous deletion of 17p correlates with
decreased expression of TP53, and low expression of
TP53 to be associated with an inferior clinical out-
come.41 We do not find low TP53 expression in samples
with LOH in our dataset, but we have found a high
mutation rate in exons 5-9 of the remaining TP53 allele.
The mutation rate for cases with LOH of 17p is 25%,
compared to a background rate of less than 1% in cases
without 17p-, so it is likely that TP53 is the gene associ-
ated with the poor outcome in this cytogenetic group.
Relevance of deletional mapping
Biology of myeloma
This type of data has allowed us to understand some
of the important biological pathways, which include
abnormalities of cell cycle progression, aberrant survival
gene expression, abnormalities of terminal differentia-
tion and downstream activation of cell signalling path-
ways. Overexpression of a cyclin D family member is
recognized as being a recurrent abnormality and loss of
negative regulators of cell cycle progression have also
been identified as being important. Later phases of dis-
ease often have increased proliferation and MYC abnor-
malities. Survival abnormalities are encapsulated in
what we understand about the frequent events such as
17p- (TP53 mutation) and abnormalities of WWOX.
RAS mutations are frequent in late phase disease and
lead to IL6 independent survival. In an analogous fash-
ion, we also understand now that deregulation of the
NF-κB pathway by both deletional events and mutation
is important in disease progression. Examples of genes
affected in the NF-κB pathway are the CYLD, TRAF and
BIRC genes, which will become increasingly important
in predicting response to specific therapeutic modalities.
The overexpression of XBP1 and other abnormalities of
this key regulator gene and pathway in terminal plasma
cell differentiation, points the way to the relevance of
abnormalities of plasma cell differentiation to myeloma
cell survival and proliferation.
Clinical outcome of myeloma
Taken together this high-resolution gene mapping has
identified regions and genes with relevance to myeloma
pathogenesis, and some with prognostic significance.
However, few of these abnormalities are associated
with a sufficiently poor prognosis to consider changing
a patient from conventional treatment pathways. This
may change as the accrual of more mapping cases
changes levels of statistical significance, but we present-
ly have much larger and more robust data related to
genetic lesions detected by FISH. 17p- and the poor risk

translocations (t(4;14), t(14;16), t(14;20)) have a much
worse prognosis than cases without these lesions, and
would fit the criteria of high risk disease.
Gene expression signatures
An alternative approach to looking at individual
lesions is to use patterns of gene expression, termed
gene expression signatures. The first prognostic gene
expression signature to be defined in this way was
based on a patient set who received a tandem autotrans-
plantation-based regimen.28 A list of 70 genes was pro-
duced, the differential expression of which was able to
identify 13% of presenting patients at risk of early dis-
ease-related death, making it a more discriminating
prognostic indicator than the ISS. More recently, a 15
gene survival signature has been produced by a similar
method based on an IFM series of patients treated with
chemotherapy followed by high dose melphalan with
stem cell rescue.42 High-risk patients had overexpression
of genes involved in cell cycle progression and a 3 year
OS of 47%. Low risk patients displayed hyperdiploid
signatures and had a 3 year OS of 90%. Again, this sig-
nature discriminates patients with the best and worst
prognoses more effectively than the ISS. We have pro-
duced a prognostic gene expression signature by a dif-
ferent method. We mapped regions with homozygous
deletions and combined this with expression array data
to identify genes whose expression is abrogated by
deletion. We have used this approach to define a prog-
nostic signature based on the expression of six genes.
Summary of current status
Understanding the genetic abnormalities that are
associated with malignant behaviour in plasma cells is
important as they ultimately define the way that
myeloma behaves in an individual, and are, therefore,
likely to be the best way of predicting prognosis in the
individual. Our current data suggests that few single
genetic lesions have sufficient prognostic power to jus-
tify a change of treatment. The exceptions are 17p-,
where the median survival is dismal at approximately
22 months, and the unfavourable IgH translocations,
where the median survival is 26 months, compared to
50 months for those without the lesions. Several gene
expression signatures have recently been produced
which may have sufficient power to identify the high-
est risk patients. These may ultimately prove to be the
most powerful way of identifying high-risk patients.
However, they are relatively new in myeloma and are
derived assessments of the tumor as opposed to 17p-
and IgH translocations, which are anchored in the
tumor genome. They also rely on technology, which is
not widely available in the clinical setting so remain an
experimental tool. FISH abnormalities, possibly in com-
bination with independent risk factors such as β2m, are
currently the most accurate prognostic tool we have for
the newly presenting patient with myeloma.
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33. Annunziata CM, Davis RE, Demchenko Y, Bellamy W, Gabrea
A, Zhan F et al. Frequent engagement of the classical and alter-
native NF-kappaB pathways by diverse genetic abnormalities
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34. Jenner MW, Leone PE, Walker BA, Ross FM, Johnson DC,
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36. Iliopoulos D, Fabbri M, Druck T, Qin HR, Han SY, Huebner K.
Inhibition of breast cancer cell growth in vitro and in vivo:
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4798-805.

N.C. Munshi
Boston VA Healthcare System,
Jerome Lipper Multiple Myeloma
Center, Dana-Farber Cancer
Institute, Harvard Medical School,
Boston, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:161-165
Multiple myeloma
Experimental agents knocking at the door of myeloma
treatment
espite the advances in conventional
D and high dose chemotherapy, multi-
ple myeloma (MM) remains incur-
able. In order to overcome resistance to cur-
rent therapies and improve patient outcome,
novel biologically-based treatment
approaches are being developed based upon
targeting the MM cell, as well as its BM
microenvironment.1 Towards validating
novel targets and developing newer agents,
we and others have developed in vitro sys-
tems and in vivo animal models to study
growth, and survival mechanisms intrinsic
to MM cells and extrinsic factors (MM cell-
bone marrow stromal cell interactions,
cytokines, angiogenesis) promoting MM cell
growth, survival, drug resistance, and migra-
tion in the BM microenvironment.2-6 These
model systems have allowed for the devel-
opment of several promising biologically-
based therapies, which can target the MM
cells in their BM microenvironment, and
thereby, overcome classical drug resistance
in vitro, including thalidomide (Thal) and its
more potent immunomodulatory analogs,
lenalidomide, as well as proteasome
inhibitor bortezomib.7
Once in vitro promise of these novel agents
is demonstrated, they are rapidly tested in
murine models of human myeloma.8 Thal
and lenalidomide9,10 and bortezomib11 all
inhibit human MM cell growth, decrease
associated angiogenesis, and prolong host
survival in animal models. These agents
have already demonstrated marked clinical
anti-MM activity and have now been incor-
porated in the routine management of both
newly diagnosed, as well as relapsed multi-
ple myeloma. However, despite a high-level
of success of these new agents, patients
relapse, and there is a need for novel target-
ed therapies in myeloma.
Oncogenomics to identify novel targets
Understanding the molecular pathogene-
sis of MM by gene expression profiling has
demonstrated sequential genetic changes
from normal to malignant plasma cells and
highlighted specific pathways important in
the multistep transformation process of nor-
mal plasma cells to monoclonal gammopa-
thy of undetermined significance to MM.12
Moreover, a high-resolution array based
comparative genomic hybridization (aCGH)
analyzing recurrent copy number alterations
with integrated expression profiling data
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 161 |
from outcome-annotated clinical specimens
has allowed for further identification of
DNA based prognostic classification system.
It has defined discrete minimal common
regions and generated a refined list of MM
gene candidates residing within these MCRs
as potential therapeutic targets.13,14
Techniques, such as systematic RNA inter-
ference (RNAi) lethality screening of the
“druggable genome” in human myeloma cell
lines (HMCL), is now providing a compre-
hensive map of molecular vulnerabilities in
human MM. This technique identifies addi-
tional targets with functional significance in
MM cell survival.
Genomic and proteomic studies can also
provide the preclinical basis for clinical appli-
cation of novel targeted therapies. For exam-
ple, our in vitro studies demonstrated that
exposure of MM cells to bortezomib down
regulates DNA repair-related genes and
induces cleavage of DNA repair kinases,
such as DNA PKcs in a dose and time
dependent manner.15 For the first time, this
observation suggests that bortezomib
inhibits DNA repair. Subsequent in vitro stud-
ies demonstrated that coupling bortezomib
with DNA damaging agents (alkylating
agents and anthracyclines) can enhance sen-
sitivity or even restore sensitivity to these
agents in resistant MM cells.16 Already clini-
cal protocols coupling Velcade with Doxil
and with melphalan17,18 are demonstrating
very promising clinical results.
Understanding of the molecular signalling
pathways to identify novel targets
Understanding how the interaction and
adhesion of MM cells to bone-marrow stro-
mal cells (BMSCs) leads to adhesion- and
cytokine-induced signaling, and delineating
those pathways mediating tumor cell
growth, survival, and drug resistance in the
BM milieu has improved our understanding
of myeloma cell pathobioogy and provided
series of potential therapeutic targets. (Figure
1).1 MM cell binding to BMSCs induces
nuclear factor B (NF-κB) in BMSCs, which
upregulates adhesion molecules and cyto-
kines secretion, including interleukin-6 (IL-
6), insulin-like growth factor-1 (IGF1),
tumor-necrosis factor-α (TNF-α), and vascu-
lar endothelial growth factor (VEGF). Adhe-
sion and cytokines activate p42/44 mitogen-
activated protein kinase (MAPK), Janus
kinase (JAK)/signal transducer and activator
14th Congress of the European Hematology Association

Figure 1. Potential molecular targets in multiple myeloma cells and their interactions with the BM microenvironment.

of transcription 3 (STAT3), and/or phosphatidylinositol

Table 1. Cell surface targeting agents in clinical studies.
3-kinase (PI3K)/Akt, and their downstream pathways
that mediate MM cell growth, survival, and migration. Target Agent Phase
Specifically, the Ras/Raf/MAPK pathway mediates pro-
liferation of MM. JAK/STAT3, along with upregulation IL6 CNTO328 (mAb) I/II
of Bcl-XL and Mcl-1, mediates survival. PI3K/Akt, IL6R Altizumab (mAb) III
through downstream activation of BAD and NF-κB CD40 SGN-40 (mAb) I
and/or inactivation of caspase-9, mediates anti-apopto-
HCD122) (mAb) I
sis. NF-κB and forkhead in rhabdomyosarcoma (FKHR)
CD56 huN901-DM1 (mAb) I
modulate cyclin D and KIP1, thereby regulating cell-
cycle progression. Signalling through PI3K induces CD74 anti-CD74 mAb I/II
downstream protein kinase C (PKC) activity and MM CD138 BT-162 (mAb) I
cell migration (Hideshima et al. Nat Can Review 2007). CS1 HuLuc63 (mAb) I
RANKL AMG162 (mAb) I/II
Identification and validation of novel targeted multiple DKK-1 Anti-DKK-1 mAb I
myeloma therapies: cell surface targets MUC1 AR20.5 (BrevaRex)(mAb) I/II
Proteamic and genomic studies have identified a BAFFR (TACI) TACI-IG (mAb) I/II
number of cell surface molecules and have formed the CD52 Alemtuzumab (mAb) II
basis for therapeutic tareting. These targets are either TRAIL Ppo2L/TRAIL (Apo2 ligand) I
adhesion molecules or growth factor receptors on the Mapatumumab (HGS-ETR1) I/II
MM cells, or are cytokines present in the BM milieu IGF1, IGF1R CP-571 (mAb) I
with significant effect on myeloma cell behavior. Table EM164 (mAb) I
1 lists the molecular targets and agents directed at these VEGF, VEGFR Bevacizumab (mAb) II
cell surface targets19-21 in clinical studies. SU5416 (SMI) II
Majority of these molecules are targeted using mono- Vatalanib (PTK-787) (SMI) II
clonal antibodies (mAb), which are now established as Zactima (ZD6474) (SMI) II
an important therapeutic modality in various cancers, FGF, PDGF TKI258 (mAb) I
especially after the success of Rituximab targeting FGFR3 Dasatinib (SMI) II
CD20 in the treatment of B-cell non-Hodgkin’s lym-
phoma. Although there is still no mAb-based therapy Adapted from Hideshima et al. 2007 Nature Reviews Cancer .

| 162 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

approved for the treatment of MM, a number of agents Table 2. Intracellular molecule targeting agents in clinical
are at various stages of development. Many of these studies.
mAbs are now being tested as single agents but more
importantly, a number of them are being evaluated as a Target Agent Single agent/ Phase
conjugate molecule (CD56,22 CD38, CD13823) with the combination
ability to deliver potent chemotherapeutic agents
specifically to myeloma cells. Earlier studies have Proteasome NPI-0052 Single/Lenalidomide I/II
shown that Rituximab had minimal clinical activity due PR-171 Single II
Mitochondria GCS-100 Single I/II
to only few MM cells expressing CD20. The significant
CCI-779 Bortezomib II
increase in the number of antibodies in clinical trials are RAD001 Bortezomib II
now mainly attributed to better understanding of MM Akt Perifosine Bortezomib II/III
biology and identification of antigen expression pat- Histone deacetylase LBH589 Bortezomib II
terns by gene expression profiling and oncogenomics, as Vorinostat BortezomibI III
well as the advancement in mAbs engineering. Romidepsin Single I/II
It is also possible to combine monoclonal antibodies Belinostat (PDX101) Single II
with novel drugs to potentiate their activity. Lena- HSP90 KOS953 Bortezomib I/II
lidomide, for example, can markedly augment anti- IPI504 Single I/II
body-dependent cellular cytotoxicity (ADCC) induced SRC, RAC1, JNK Plitidepsin (Aplidin) Single/Bortezomib I/II
by anti-CD40 in MM.19,24 PKC Enzastaurin Bortezomib I/II
Heparanase PI-88 II
Targets within multiple myeloma cell BCL2 G3139 (Genasense) Dexamethasone III
We and others have utilized high resolution genomic Telomerase GRN163L Single I
profiles to both identify additional potential novel ther- Farnesyltransferase Tipifarnib (R115777) Bortezomib II
apeutic targets in tumor cells, as well as to define dis- MEK AZD6244 Single I/II
tinct clinical pathogenetic subgroups of MM. The sig- DNA Bendamustine Single II
naling cascades and molecular mechanisms whereby Temozolomide Single II
NF-κB,25 p38MAPK,26 telomerase,27 Ku86/70,28 and cave- O-6-benzylguanine Single II
olae29 mediate homing and adhesion to BM are now CDK Flavopiridol Single I
Multi-kinases Atiprimod Single II/II
delineated, providing the framework for therapeutic
VQD-001 Single I/II
strategies targeting these cascades. The understanding ZIO-101 Single I/II
that the MM cell growth is mediated via ERK/MAPK,26 Arsenic trioxide Single II
survival via JAK/STAT,30 drug resistance via PI3-K/Akt,31
and migration via PKC dependent signaling cascades,32 Adapted from Hideshima et al. 2007, Nature Reviews Cancer
respectively, has provided novel agents (Table 2). These
include the new proteasome inhibitors: VEGF receptor
tyrosine kinase inhibitor PTK78757, pan VEGF receptor
inhibitors GW786034B; multi-targeted kinase inhibitor
PKC-412; 2-methoxyestradiol (2ME); p38MAPK Profiling of gene and protein expression has provided
inhibitor; histone deacetylase (HDAC) inhibitors the preclinical rationale for clinical protocols combining
SAHA, romidopsine and LBH589; heat shock protein 90 novel targeted therapies. For example, our studies
(hsp90) inhibitor geldanamycin; telomerase inhibitors demonstrate that bortezomib treatment of MM cells in
GRN163; inosine monophosphate dehydrogenase vitro induces death signaling, down regulates survival
inhibitor VX944117; small molecule Akt inhibitor peri- signaling, and up regulates both ubiquitin/proteasome
fosine, PKCβ inhibitor enzastaurin; and MEK inhibitor and stress response gene transcripts. Specifically, borte-
AZD6244. The new proteasome inhibitors have irre- zomib induces Hsp90, which is not only a stress
versible proteasome inhibitor activity (PR171) or affect response protein but also plays a major role in the pro-
all three active sites in proteasome (NPI-0052), and both tein unfolding required before proteins can be degraded
have demonstrated activity against bortezomib resist- by the proteasome.33 In vitro studies show that Hsp90
ant MM cells. inhibitor 17AAG can block the Hsp90 stress response
induced by bortezomib, and thereby, increase MM cell
Development of combinations apoptosis.34 These gene microarray studies, therefore,
It is our hypothesis that drugs from multiple of these provided the framework for a clinical trial coupling
classes will need to be combined to achieve complete these agents in MM, which shows that Hsp90 inhibitor
eradication of MM cells. Our ongoing studies of their KOS953 can sensitize to, and even overcome, resistance
molecular mechanisms of action aim at providing a to bortezomib.35
framework for clinical trials of rationally designed com- In order to provide the framework for coupling these
binations to overcome drug resistance and improve novel agents in rational clinical trials, the apoptotic sig-
patient outcome. The overall goal is to develop: (i) com- naling cascades triggered in MM cells by both conven-
bination therapies optimized to enhance cytotoxicity, tional and novel agents have been characterized.7,36 For
overcome drug resistance, and allow for use of lower example, use of IMiDs with TRAIL provides dual trig-
doses with less toxicity; (ii) and provide a framework gering of caspase 8 death signaling, whereas treatment
for the development of the next generation of potent with IMiDs (lenalidomide) and bortezomib triggers
targeted therapies with higher selectivity and less toxic- both caspase 8 and caspase 9-mediated MM cell death,
ities. providing potential for synergistic activity. An ongoing

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 163 |
14th Congress of the European Hematology Association
clinical trial has demonstrated remarkable activity in
patients treated with lenalidomide/bortezomib, even in
patients resistant to either agent alone.37
mTOR inhibitor rapamycin sensitizes MM cells to
both conventional (dexamethasone) and novel thera-
pies;38 CCI-779 has anti-MM activity in xenograft mod-
els,39 which has led to combination studies. Bortezomib
inhibits growth (MEK/ERK) and survival (Jak/STAT) sig-
naling, but activates Akt, providing the preclinical
rationale for combining bortezomib with the Akt
inhibitor perifosine.40
Recent signaling studies have defined the role of the
aggresome in degrading ubiquitinated protein in MM,
and specifically used the HDAC6 inhibitor to inhibit its
transport to the aggresome for degradation. Blocking
the aggresome induces a compensatory upregulation of
the proteasome; conversely, blocking the proteasome
with bortezomib triggers a compensatory upregulation
of the aggresome. Importantly, blocking both the pro-
teasome and aggresome with bortezomib and HDAC6
inhibitor, respectively, induces synergistic toxicity.41
This strategy is now under intense investigation in both
phase II and III studies.
Finally, gene expression profiling in correlative science
studies of patients on clinical protocols will both identi-
fy targets of drug sensitivity versus resistance, and allow
for predicting those patients most likely to respond to
conventional and novel targeted therapies. For example,
gene expression profiling of patient tumor samples
showed genes upregulated in patients responding to
bortezomib versus those patients who did not respond.
Hsp27 upregulation correlated with intrinsic or acquired
bortezomib resistance. Preclinical studies showed that
p38MAPK inhibition downregulated Hsp27 expression
and restored bortezomib sensitivity in resistant MM cell
lines and patient samples,26,42,43 providing the basis for a
trial combining these two agents.
Ultimately, it may be possible to carry out gene and
protein expression profiling on individual patient sam-
ples to allow selection of those agents most likely to be
effective. For example, we recently compared the gene
profile of patient MM cells to normal twin plasma cells
and showed surprisingly few significant differences.44
This data may allow selection of those combinations of
agents targeting these gene products to optimize clinical
response.
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 Multiple myeloma

Treatment options for myeloma in 2009

J. Bladé
L. Rosiñol
Hematology and Oncology Institute,
Hematology Department. IDIBAPS,
Hospital Clínic, University of
Barcelona. Barcelona, Spain
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:166-171
A B S T R A C T
The incorporation of novel drugs with new mechanisms of action is resulting in an improved out-
come in patients with multiple myeloma. In the context of ASCT, the new induction regimens result
in a higher pre-transplant cytoreduction with a higher proportion of patients achieving CR after trans-
plant, this leading to a longer progression-free survival when compared with previous studies. The
Allo-RIC is a promising approach that deserves further investigation in prospective trials. Major
improvements are being accomplished in the treatment of elderly patients, particularly when combin-
ing melphalan/prednisone with thalidomide (MPT), bortezomib (MPV) or lenalidomide (MPR), or with
lenalidomide/dexamethasone. In the relapse setting, the availability of thalidomide, bortezomib and
lenalidomide has resulted in more effective salvage regimens. A number of phase III trials by different
national cooperative groups, both in eligible and non-eligible patients for ASCT, are ongoing.
Hopefully, the results of these trials will allow the selection of a more individualized anti-myeloma
therapy, likely based on the molecular genetic status.

ntil recently, the median survival of benefit of ASCT in terms of EFS and OS.

U patients with multiple myeloma

(MM) has been about 3 years and the
number of long-term survivors disappoint-
ingly low. The introduction of high-dose
therapy followed by stem cell support
(HDT/SCT), and the incorporation of novel
agents (i.e., thalidomide, lenalidomide and
bortezomib) are resulting in a higher anti-
myeloma effect and prolonged survival. This
improvement is due to a better initial thera-
py for eligible and non-eligible patients for
HDT/SCT and/or more effective rescue regi-
mens for patients with relapsed/refractory
disease plus better supportive measures. This
review focuses on: (i) treatment of patients
eligible for HDT/SCT; (ii) treatment of non-
eligible patients for HDT/SCT; (iii) treatment
of relapsed and/or refractory disease.
Treatment of patients eligible for high-dose ther-
apy/hematopoietic stem cell transplantation
Autologous stem cell transplantation
Autologous stem cell transplantation
(ASCT) is considered the gold standard as
part of the front-line therapy in patients with
MM younger than 65 years. The results of
ASCT with induction with “conventional
chemotherapy” and with induction incorpo-
rating new agents are summarized in the fol-
lowing sections.
Results with conventional chemotherapy induc-
tion regimens
Five trials comparing ASCT versus con-
ventional chemotherapy have been fully
reported.1 Two of them showed that ASCT
was superior in complete remission (CR),
event-free survival (EFS) and overall survival
(OS), while the remaining three showed no
The question of whether ASCT is beneficial
for the majority of patients or whether the
overall benefit results from certain sub-
groups of patients remains to be deter-
mined. It seems that the achievement of CR
is the most important step for a long-lasting
response and prolonged survival in patients
with MM.2,3 On the other hand, the sensitiv-
ity to the initial chemotherapy measured by
the M-protein type at the time of transplant
is the most reliable predictor of CR after
ASCT.2,3 Supporting this concept, in a recent
meta-analysis by Van de Velde et al.,4 a
strong association between maximal
response to the induction therapy and long-
term outcome was found. The pre-trans-
plant induction therapy with different con-
ventional chemotherapy regimens has
resulted in a pre- and post-transplant
immunofixation negative CR of 5-10% and
about 35%, respectively, as well as in a
median survival of approximately 6 years in
the best circumstances.
Results with novel pre-transplant induction regi-
mens
The ideal induction chemotherapy prior to
ASCT should consist of a regimen with high
anti-myeloma effect, no interference with
stem cell collection and acceptable toxicity.
The association of thalidomide/dexam-
ethasone (TD) replaced VAD and was
approved by the US Food and Drug
Administration for its use as pre-transplant
induction regimen. In addition, two large tri-
als showed that TD is superior to dexam-
ethasone alone.5,6 The overall response rate
(ORR) to TD ranges from 58-76%. However,
the CR rate is only about 5-7%. Furthermore,

| 166 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

Table 1. Post-induction and postransplant response in
patients with multiple myeloma eligible for ASCT.
Regimen CR before
ASCT (%)
Old regimens
Dexa/VAD 5 35
Cyclophosphamide/Dexa 7 32
VBMCP/VBAD 10
New regimens
Thalidomide/Dexa5,6 5-10
Bortezomib/Dexa8,9 12
Bortezomib/Adria/Dexa (PAD-1)11 24
Bortezomib/Adria/Dexa (PAD-2)12 11
Bortezomib/Thal/Dexa (VTD)13,14 21-30
thalidomide and dexamethasone would not be an opti-
mal regimen for patients with extramedullary disease,
due to the lack of effect of thalidomide on soft-tissue
plasmacytomas.7 The results of two phase II trials com-
bining bortezomib and dexamethasone showed a post-
induction response rate of about 65%, with 12%
immunofixation negative CR.8,9 The ORR post-trans-
plant was about 90% with 33% CR. In a large phase III
trial by the Intergroup Francophone du Myelome (IFM)
comparing bortezomib/dexamethasone versus VAD, the
immunofixation negative CR rate post-transplant was
significantly higher with bortezomib/dexamethasone
than with VAD (10% vs. 19%, p<0.01).10 Thalidomide
and bortezomib are increasingly used in combination
with dexamethasone or antracyclines, resulting in the
so-called triple regimens. The PAD-1 regimen (borte-
zomib –PS 341-, adriamycin, dexamethasone) resulted in
a pre-transplant ORR of 95% with 24% and in a post-
transplant CR rate of 43%.11 However, about 50% of the
patients developed peripheral neuropathy, leading to a
reduction in the dose of bortezomib to 1 mg/m2 (PAD-2)
in a subsequent study from the same group.12 With PAD-
2, the incidence of neuropathy significantly decreased,
while the ORR was still 89%. However, the CR rate
after induction and transplant were lower (11% and
37%, respectively) than with PAD-1.12 Cavo et al recent-
ly reported that bortezomib-Velcade-/thalidomide/dex-
amethasone (VTD) was superior to TD in terms of CR
rate both before (21% vs. 6%) and after (43% vs. 23%)
transplant.13 The Spanish PETHEMA group is comparing
TD versus VTD versus combination chemotherapy with
vincristine, BCNU, melphalan, cyclophosphamide, pred-
nisone (VBMCP)/vincristine, BCNU, adriamycin and
dexamethasone (VBAD) (four cycles) plus two cycles of
bortezomib as pretransplant induction therapy.14 The
preliminary results show that the best regimen is VTD
with a pre-transplant and post-transplant CR rate of
30% and 49%, respectively.14 Table 1 shows a summary
of the results before and after transplant achieved with
the so-called “old” and “new” induction regimens.
Results of single vs. tandem double autologous stem cell
transplant
Five prospective randomized studies comparing single
versus double ASCT have been conducted.15-19 Two tri-
als showed a significant increase in CR or near CR
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 167 |
Berlin, Germany, June 4-7, 2009
rate,16,18 three showed a prolonged EFS15,16,18 and only
one, a significantly longer OS in favour of tandem trans-
plant.15 Surprisingly, a recently published trial19 compar-
CR after ing single transplant followed by 6 months thalidomide
ASCT (%) maintenance versus double ASCT showed that the sin-
gle transplant arm was superior to tandem transplant in
both RR and survival.19 From two of these studies, it
seems that patients who benefit from tandem trans-
plant are only those failing to achieve at least very good
35
partial response (VGPR) with the first transplant.15,16 The
44* most consistent finding of these studies is the prolonga-
33 tion of EFS or PFS with double transplant.15,16,18,20 The role
43 of maintenance,21 as well as the concept of early intensi-
37 fication22 after ASCT with novel drugs are interesting
43-49 and deserve prospective further investigation.
Allogeneic stem cell transplantation
The best curative approach in multiple myeloma is
allogeneic transplantation. However, allogeneic trans-
plantation with myeloablative conditioning (MAC)
results in a transplant-related mortality (TRM) up to
50%, and the proportion of patients long-term disease-
free and possibly cured does not exceed 10-15%.23 For
this reasons, allogeneic transplantation with MAC has
almost universally been replaced by the so-called dose-
reduced intensity conditioning allogeneic transplanta-
tion (Allo-RIC).23-26 The current conditioning consists of
fludarabine/MEL-140 or fludarabine/low-dose total
body irradiation (TBI). The TRM has decreased by 10 to
20%, whereas the CR rate is about 40-50%. The inci-
dence of acute and chronic graft-versus-host disease
(cGVHD) is 30-50%, respectively. The most important
predictors of outcome are the development of cGVHD
and chemosensitive disease with a low tumor burden at
the time of transplant.23,24 Considering that an important
requisite for the success of Allo-RIC is to have achieved
a low tumor mass at the time of transplantation, the use
of ASCT to reduce tumor burden followed by Allo-RIC
in order to benefit from graft-versus myeloma (GVM)
effect has been recently investigated with promising
results.24,26 In this sense, the efficacy of a tandem ASCT
versus single autograft, followed by Allo-RIC in patients
with newly diagnosed multiple myeloma with an avail-
able donor have been compared in three studies.27-29 The
IFM group reported no difference between the two
approaches.27 In contrast, the Italian group found an
increased CR rate and a significant survival advantage in
favour of Allo-RIC with a survival plateau beyond 4
years of allografting, while the tandem ASCT group
observed no plateau.28 In a Spanish PETHEMA study,
there were no significant differences in EFS and OS
between the two groups. However, the curve of
patients who received an Allo-RIC showed an encour-
aging survival plateau beyond 3 years of follow-up.29
With the current data, it is evident that there is a need
for continuing the investigation of conditioning intensi-
ty and pos-transplant strategies aimed at enhancing the
GVM effect while minimizing the GVHD.30
Treatment of patients not eligible for high-dose
therapy/hematopoietic stem cell transplantation
In patients not eligible for HDT/ASCT (older than 65
years or younger with comorbidities), the conventional
therapy has consisted of alkylating-based regimens,
14th Congress of the European Hematology Association

Table 2. Front-line therapy studies for patients with myeloma not eligible for HDT/SCT.

Author, yr Regimen ORR (%) CR (%) EFS OS

Rajkumar et al.6 THAL/DEX vs. DEX 63 vs. 46 7.7 vs. 2.6 22.6 vs. 6.5 29 mos vs. not reached
Facon et al.32 MPT vs. MP 76 vs. 35 13 vs. 2 HR 0.56 (in favour of MPT) 51.6 vs. 33.2 mos.
Hulin et al.33 MPT vs. MP 62 vs. 31 7 vs. 1 24.1 vs 19 mos. 45.3 vs. 27.7 mos.
Palumbo et al.34 MPT vs MP 76 vs. 47 15.5 vs. 2.4 54 vs. 27% at 2 yrs 80 vs. 64% at 3 yrs
San Miguel et al.38 MPV vs. MP 71 vs. 36 30 vs. 4 24 vs. 16.6 mos. 82.6 vs 69.5% at 2 yrs
Palumbo et al.39 MPR 81 24 92% at 1 yr 100% at 1 yr
Ludwig et al.40 THAL/DEX vs. MP 68 vs. 51 14 vs. 7 43 vs. 25 mos. 58 vs. 45 mos.
Rajkumar et al.41 Len/Dex vs Len/dex 82 vs. 70- - 87 vs. 75% at 2 yrs
MP: melphalan/prednisone; MPT: melphalan/prednisone/thalidomide; MPV: melphalan/prednisone/bortezomib; THAL/DEX: thalidomide/dexamethasone;
LEN/DEX: lenalidomide/dexamethasone; LEN/dex: lenalidomide/low dose dexamethasone; MPR: melphalan/prednisone/lenalidomide; HR: hazard ratio;
Main toxicities: MP: myelosuppresion; MPT: myelosupresion, peripheral neuropathy, DVT; MPV: myelosupresion, thrombocytopenia, peripheral neuropathy;
THAL/DEX: DVT; LEN/DEX or LEN/dex: myelosupresion, DVT; MPR: myelosupresion, DVT.

In a phase II trial, the association of MP with borte-

Table 3. General considerations in the treatment of
relapsed myeloma.
Components of initial therapy
Alkylating-based
Dexamethasone-based
Hdt/scs
Efficacy of initial therapy
Degree of response
Timing of relapse
Patient status and circumstances of relapse
Age, ps
Aggressive vs “non-aggressive” relapse
mainly melphalan and prednisone (MP), dexametha-
sone-based therapies – VAD or VAD-like regimens – or
dexamethasone alone. With these regimens, the ORR
was about 50% with less than 5% CR with median sur-
vivals of about 3 years. With the incorporation of novel
agents, with different mechanisms of action than con-
ventional therapy, an improvement in response rate and
generally in survival are consistently reported.31
The French IFM group compared MP versus two
cycles of VAD, followed by two courses of intermediate
high-dose therapy with MEL-100 versus MP-thalido-
mide (MPT) in patients aged 65-75 years.32 The MPT
regimen resulted in a significantly higher ORR and CR
rate, as well as in significantly longer EFS and OS. The
same group compared MP versus MPT (with a dose of
thalidomide of 100 mg/day) in patients older than 75
years.33 The RR was significantly higher with MPT
(62% vs. 31%). Importantly, the PFS (median 24.1 vs. 19
months) and the OS (median 45.3 vs. 27.7 months) were
significantly longer with MPT.33 The Italian group also
reported a significantly higher ORR and CR rate, as well
as a longer PFS in favour of MPT versus MP.34 However,
the OS was not significantly different. The Nordic and
the HOVON groups found a significantly longer PFS in
favour of MPT versus MP, with no differences in OS.35,36
Peripheral neuropathy is the main concern in thalido-
mide-containing regimens and the risk of deep vein
thrombosis (DVT)/pulmonary embolism makes the use
of prophylactic anticoagulation mandatory when
thalidomide is combined with cytotoxic agents or with
high-dose glucocorticoids.
zomib (MPV) showed encouraging results.37 In a subse-
quent large phase 3 trial, MPV was compared with MP
– VISTA trial. 38 The ORR (71 vs. 36%), the CR rate (30
vs. 4%), the PFS (median 24 vs. 16.6 months) and the OS
(82.6% and 69.5% at 2 years follow-up) were all in
favour of MPV.38 MPV was superior to MP in all prog-
nostic subgroups, including patients with poor cytoge-
netics.
In a phase II study, the association of MP with
lenalidomide – Revlimid – (MPR) was very promising,
with 81% PR rate, including 24% immnofixation nega-
tive CR.39 The EFS and OS at 1 year was 92%.39 The EFS
and OS at 1 year were 92% and 100%, respectively. A
large phase III trial comparing MPR versus MP has been
recently completed.
Ludwig et al.40 compared TD versus MP and found a
significantly higher CR rate (68% vs. 51%). However,
the number of deaths during the first year was signifi-
cantly higher with TD, and there was a trend towards a
superior EFS and a significantly longer OS in favour of
the MP arm.40 A large ECOG trial compared lenalido-
mide/dexamethasone at full doses of dexamethasone
versus lenalidomide/dexamethasone with a weekly
dose of 40 mg of dexamethasone.41 Although the RR
was lower with low-dose dexamethasone,41 the OS at 1
and 2 years was significantly longer with lenalido-
mide/low-dose dexamethasone because a significantly
lower toxicity and mortality.
From the above, it seems that the association of an
old regimen, such as MP or dexamethasone with a new
agent (i.e., thalidomide, bortezomib or lenalidomide) is
becoming the treatment of choice in the elderly. It also
appears that MP combinations are resulting in higher
RR than dexamethasone-based combinations. Concern-
ing MPT versus MPV, the results in RR and survival are
very similar. Once they are approved for their use as up-
front therapy, the first choice will depend on age,
aggressiveness of the disease, cytogenetics and patient
preference. For patients with very advanced age or less
aggressive disease, MPT would be preferable. In con-
trast, for patients aged 65-75 years with clinically
aggressive disease (extensive symptomatic bone
involvement, extramedullary spread) or with poor cyto-
genetics, a bortezomib containing regimen could be
more appropriate. For patients with peripheral neuropa-

| 168 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

Table 4. Randomized trials on treatment of relapsed/refractory myeloma.
Author, yr Regimen
Richardson et al., 2005 APEX trial48 Bort. vs. Dex
Orlowski et al., 2007 49
Bort.+Doxil vs. Bort.
Weber et al., 2007 50
Len/Dex vs. Dex
Dimopoulos et al., 200751 Len/Dex vs. Dex
ORR: overall response rate; CR: complete response; TTP: time to progression; OS: overall survival; Bort: bortezomib; Dex: dexamethasone; Len: lenalidomide
thy, a lenalidomide-based regimen (lenalidomide/low
dose dexamethasone or MPR) would be preferable. For
patients with renal failure, bortezomib/dexamethasone
would be the initial treatment of choice.42-44 Table 2
shows a summary of the recent trials in the treatment of
MM in patients not eligible for HDT/SCT.
Treatment of relapsed/refractory myeloma
In patients with relapsed or refractory myeloma, the
choice of the salvage therapy must depend on: (i) the
components of the initial therapy; (ii) the degree and
duration of response to primary therapy (i.e., deep
responses with duration longer than 2 years, relapsing
off therapy might benefit from pre-treatment with the
initial option); (iii) performance status and age at the
time of relapse (older patients with poor performance
status should be treated with more gentle approaches);
(iv) type of relapse (aggressive relapses should be treat-
ed with bortezomib-containing regimens, while more
“indolent” relapses might be treated with thalidomide
or lenalidomide-based regimens, saving bortezomib for
subsequent relapses); (v) previous toxicity (i.e., patients
with significant peripheral neuropathy avoid thalido-
mide and bortezomib) (Table 3).
A recent review on thalidomide therapy in MM
emphasizes that in patients with refractory disease, the
RR to single agent thalidomide is between 30 and 40%,
that thalidomide/dexamethasone combinations induce
RR ranging from 40-65% and that soft-tissue plasmacy-
tomas rarely respond to thalidomide.45
In the SUMMIT study, bortezomib produced a
response rate of 35% in patients with advanced refrac-
tory myeloma.46 The APEX trial showed that borte-
zomib was superior to dexamethasone in RR, PFS and
OS.47 An extended follow-up of this study showed that
the OR and CR rate of bortezomib was 43 and 9%,
respectively and that higher response quality was asso-
ciated with longer response duration.48 A large phase 3
trial comparing bortezomib associated with pegylated
liposomal doxorubicin versus bortezomib alone
showed that the combination was superior in CR and
VGPR, PFS and OS.49
The combination of lenalidomide and dexamethasne
results in an ORR of about 60%, including 15% CR ver-
sus 20-24%, ORR with 1-4% CR with dexamethasone
alone.50,51 Compared with dexamethasone, the combina-
tion of lenalidomide and dexamethasone was superior
not only in RR but also in TTP and OS50,51 (Table 4).
An increasing number of three and four drug combi-
nations, including “old” and novel agents, showing a
high RR in patients with relapsed/refractory disease are
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 169 |
Berlin, Germany, June 4-7, 2009
ORR (%) CR (%) EFS OS
38 vs. 18 6 vs. 1 6.2 vs. 3.5 80 vs. 66% at 1 yr
44 vs. 41 4 vs. 2 9.3 vs. 6.5 76 vs. 65% at 15 months
61 vs. 19.9 14.1 vs. 0.6 11.1 vs. 4.7 29.6 vs. 20.2 months
60.2 vs. 24 15.9 vs. 3.4 11.3 vs. 4.7 Not reached vs. 20.6 months
being reported. However, these studies have a number
of shortcomings, such as low number of patients, possi-
ble favorable patient selection, short follow-up, consid-
erable toxicity, cost and limited rescue options at
relapse. The authors of this review are inclined to use a
sequential therapy approach in subsequent relapses.
The selection of therapy will depend on the previous
drug exposure, previous response, toxicity and type of
relapse (aggressive vs. “indolent”). In patients with
chemosensitive relapse, ASCT as rescue therapy should
be considered. Also, in patients relapsing after ASCT, a
second HDT/SCT could be of benefit, provided that the
duration of response with the first ASCT was longer
than 2 years. If there is an available HLA-matched
donor and the patient has chemosensitive relapse and is
younger than 65 years, an Alli-RC should be consid-
ered.23 There is an extensive review by the International
Myeloma Working Group on the prophylaxis of throm-
botic events in thalidomide and lenalidomide contain-
ing regimens.52
Two recent studies53,54 have shown a significant
improvement in the survival of patients with multiple
myeloma since the introduction of novel agents.
Although there is still a long way to go, the current data
already represent a significant advance and a real hope
for patients with multiple myeloma.
This work has been supported in part by grants
06/0020/0005 and 08/0147 from Instituto Carlos III
(Spanish Institute of Health).
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26. Crawley C, Iacobelli S, Björkstrand B, Apperley JF, Nieder-
wieser D, Gahrton G et al. Reduced-intensity conditioning for
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compared with myeloablative conditioning. Blood 2007;109:
3588-94.
27. Garban F, Attal M, Michallet M, Hulin C, Bourhis JH, Yakoub-
Agha I et al. Prospective comparison of autologous stem cell
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28. Bruno B, Rotta M, Patriarca F, Mordini N, Allione B, Carne-
vale-Schianca F et al. A comparison of allografting with auto-
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356:1110-20.
29. Rosiñol L, Pérez-Simón JA, Sureda A, de la Rubia J, de Arriba
F, Lahuerta JJ et al. A prospective PETHEMA study of tandem
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30. Cavo M. Nonmyeloablative allotransplantation for myeloma:
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31. San Miguel JF, Harousseau JL, Joshua D, Anderson K. Individ-
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et al. Melphalan and prednisone plus thalidomide versus mel-
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Palomera L, Fuertes M et al. Bortezomib plus melphalan and
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ma: up-dated time-to-events results and prognostic factors for
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myeloma. Blood 2007;110:31a.
39. Palumbo A, Falco P, Corradini P, Falcone A, Di Raimondo F,
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al. Thalidomide-dexamethasone compared to melphalan-
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Blood 2008; Oct 27 [Epub ahead of print]
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of lenalidomide plus high-dose dexamethasone versus
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nosed multiple myeloma (E4A03): a trial coordinated by the
Eastern Cooperative Oncology Group. J Clin Oncol 2008;26:
42. Chanan-Khan AA, Kaufmann JL, Metha J, Richardson PG,
Miller KC, Lonial S et al. Activity and safety of bortezomib in
multiple myeloma patients with advanced renal failure: a mul-
ticenter retrospective study. Blood 2007;109;2604-6.
43. Kastritis E, Anagnostopoulos A, Roussou M, Gika D,
Matsouka C, Barmparousi D et al. Reversibility of renal failure
in newly diagnosed multiple myeloma patients treated with
high dose dexamethasone and the impact of novel agents.
Haematologica 2007;92:546-9.
44. Ludwig H, Drach J, Graf H, Land A, Meran JG. Reversal of
acute renal failure by bortezomib-based chemotherapy in

patients with multiple myeloma. Haematologica 2007;92:
1411-4.
45. Palumbo A, Facon T, Sonneveld, Bladè J, Offidani M, Gay F et
al. Thalidomide for treatment of multiple myeloma: 10 years
later. Blood 2008;111:3968-77.
46. Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S,
Irwin D et al. A phase 2 study of bortezomib in relapsed,
refractory myeloma. N Engl J Med 2003;348:2609-17.
47. Richardson P, Sonneveld P, Schuster MW, et al. Bortezomib or
high-dose dexamethasone for relapsed multiple myeloma. N
Engl J Med 2005;352:2487-98.
48. Richardson PG, Sonneveld P, Schuster M, Irwin D, Stadtmauer
E, Facon T et al. Extended follow-up of a phase 3 trial in
relapsed multiple myeloma: final time-to-event results of the
APEX trial. Blood 2007;110:3557-60.
49. Orlowski RZ, Nagler A, Sonneveld P, Bladé J, Hajek R, Spencer
A et al. Randomized phase III study of pegylated liposomal
doxorubicin plus bortezomib compared with bortezomib
alone in relapsed or refractory multiple myeloma: combina-
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Berlin, Germany, June 4-7, 2009
tion therapy improves time to progression. J Clin Oncol 2007;
25:3892-901.
50. Weber D, Chen C, Niesvizky R, Wang M, Belch A, Stadt-
mauer EA et al. Lenalidomide plus dexamethasone for
relapsed multiple myeloma in North America. N Engl J Med
2007;357:2133-42.
51. Dimopoulos M, Spencer A, Attal M, et al. Lenalidomide plus
dexamethasone for relapsed or refractory multiple myeloma.
N Engl J Med 2007;357:2123-32.
52. Palumbo A, Rajkumar SV, Dimopoulos MA, Richardson PG,
San Miguel J, Barlogie B et al. Prevention of thalidomide- and
lenalidomide-associated thrombosis in myeloma. Leukemia
2008;22:414-23.
53. Brenner H, Gondos A, Pulte D. Recent major improvement in
long-term survival of younger patients with multiple myelo-
ma. Blood. 2008;111:2521-6.
54. Kumar SK, Rajkumar SV, Dispenzieri A, Lacy MQ, Hayman
SR, Buadi FK et al. Improved survival in multiple myeloma and
the impact of novel therapies. Blood 2008;111:2516-20.
 Myelodysplastic syndromes

Pathogenesis and basic aspects of myelodysplastic

syndromes

N. Galili
A. Raza
St. Vincent’s Comprehensive
Cancer Center, New York, USA
Hematology Education:
the education program
for the annual congress of the
European Hematology Association
2009;3:172-176
A B S T R A C T
The inherent heterogeneity of myelodysplastic syndromes (MDS) makes it difficult to obtain a sim-
plified description of the pathobiology leading to disease initiation and evolution of this universally
fatal disorder. However, it has become evident, that MDS is a result of an abnormal hematopoietic cell
interacting with an increasingly abnormal microenvironment, thus disrupting the delicate balance
between proliferative and apoptotic signals that control normal stem cell function. The hallmark of
the MDS marrow, high proliferation in the presence of increased apoptosis, is reflective of the devel-
opment of monoclonality with the appearance of dysplasia and cytopenia(s). Excessive inflammatory
cytokines from the MDS microenvironment stimulate angiogenesis, which helps to disseminate the
dysplastic cells. Epigenetic mechanisms coupled with genomic instability, lead to aberrant immune
response and high risk of transformation. It is the complex interplay of these and other, as of yet,
unknown factors that underlie the fatal course of MDS as described here.

yelodysplastic syndromes, a hetero- more lineages. A breakthrough in the under-

M geneous group of clonal stem cell

diseases resulting in peripheral
cytopenias with dysplastic bone marrows,
which primarily affects the elderly, has
become one of the most diagnosed hemato-
logical malignancies due to the increasingly
aging population. Initially, this group of dis-
eases was thought to be a form of pre-
leukemia, as many patients went on to devel-
op acute leukemia.1 It has become clear,
MDS is not solely a disease of the hemato-
poietic stem cell, but is a result of complex
interplay between factors derived from the
stromal cells of the bone marrow microenvi-
ronment and abnormal stem cells. Thus,
MDS has become a model for the seed and
soil hypothesis of tumor development.
Therefore, it is important to look at each
component of this intricate network in order
to understand why, despite many common-
alities found in the various subtypes, the
molecular basis of MDS pathology and ther-
apy is still evolving.
Clonality studies
Clonality studies using chromosomal
abnormalities together with X-chromosome
inactivation demonstrated that MDS is a
clonal disease which most typically origi-
nates in a pluripotent stem cell committed to
the myeloid lineage.2 Studies have shown,
however, that in some patients, the lym-
phoid lineage is also part of the diseased
clone, suggesting that in these patients the
originating MDS stem cell has not yet under-
gone lineage differentiation.2
Proliferation, apoptosis and cytokines
The MDS marrow is filled with rapidly
proliferating cells, yet paradoxically, the
patients present with cytopenia in one or
standing of the biology of the MDS marrow
came with the demonstration that, while the
marrow cells were rapidly proliferating,
there was simultaneous excessive intra-
medullary cytokine mediated apoptosis.3-7
Inflammatory cytokines, notably TNF-α and
TGF-β, secreted by the stromal cells of the
marrow microenvironment, as well as by
other immune reactive cells, have been espe-
cially implicated in a paracrine process.7
Additionally, levels of TNF-α ligand, as well
as apoptosis signaling receptors FAS and
TRAIL are also increased on the MDS
hematopoietic cells,8,9 inducing an autocrine
component to apoptosis. Activation of the
mitochondrial caspase cascade is triggered
by these signaling pathways, resulting in
apoptosis. The marrows of patients that
present or evolve into higher risk MDS tend
to have lower levels of apoptosis than those
with lower risk MDS, while retaining similar
rates of proliferation,10 and thus, are more
likely to transform. Decreased apoptosis is
attributed to accumulating mutations in this
molecular pathway. Additional genetic
mutations with continuing proliferation may
also explain the gradual increase in blast cells
and the resulting resistance to chemotherapy
that characterizes AML derived from MDS.
Immune response
The presence of an abnormal immune
response in patients with MDS was first sug-
gested by the observation that patients with
aplastic anemia (AA) may evolve to MDS
that has a trisomy 8 or monsomy 7 karyo-
type.11 Patients with AA respond to treat-
ment with immunosuppressive therapy,
including anti-thymocyte globulin (ATG)
and cyclosporine. MDS patients, specifically
those with trisomy 8, showed hematologic

| 172 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

improvement when treated with these drugs, suggest-
ing that a T-cell mediated response was active in these
patients. Studies later showed that MDS patients with
trisomy 8 have expanded CD8+ T-cells of one or more
T-cell receptor subfamilies, and that these cells actually
inhibit trisomy 8 cells in culture.12 Surprisingly, patients
with this subtype of MDS rarely transform to AML
with ATG therapy and their cytopenias improve. This
suggests that the trisomy 8 MDS cells retain their abili-
ty to mature and when cytotoxic T-cells are reduced or
eliminated by ATG, cytopenia can recover. Eventually,
however, continued genetic instability results in disease
progression. Deregulated cytokine balance (especially
TNFα), abnormal immune response in MDS and
increase in angiogenesis (see below) led to the use of
thalidomide in a clinical trial.13 Thalidomide is a strong
inhibitor of TNFα and, has numerous immumodulatory
effects and decreases angiogenesis. Hematologic
response was seen in 19% of all patients and 31% of
evaluable patients. Subsequently, Lenalidomide, a deriv-
ative of thalidomide, was shown to improve erythro-
cytopenia in transfusion dependant low-risk MDS
patients,14-16 and was especially effective in patients hav-
ing a 5q- karyotype. Lenalidomide has been shown to
modulate both NK cell and T-cell activity.17
Angiogenesis
Angiogenesis is mediated by a number of growth fac-
tors, including vascular endothelial growth factor
(VEGF), TNF-α and TGF-β, and a notable increase in
vascularity is found in the marrow of patients with
MDS.18 The cytokines that control normal marrow
angiogenesis are the very cytokines that balance prolif-
erative and apoptotic signaling in normal hemato-
poiesis; the pathways deregulated in MDS. It is possi-
ble, therefore, that in MDS, these cytokines act to stim-
ulate proliferation with excess apoptosis directly in the
MDS hematopoietic cell, while simultaneously stimu-
lating neo-angiogenesis to support the aberrant
microenvironment and to disseminate the dysplastic
cells. In addition, it is important to note that these
cytokines are also part of the inflammatory response
pathway, and thus, the pathological effects in MDS may
be associated with an initiating inflammatory stimula-
tion that cascades into eventual monoclonality and dis-
ease progression.
Specific gene mutations in myelodysplastic syndromes
TP53 mutations have been detected in 5-10% of MDS
patients.19-21 Wild type TP53 plays a pivotal role in the
induction of apoptotic pathway, and mutations have
been found predominantly in high-risk MDS patients;
an observation consistent with the decreased apoptosis
and high risk of transformation. In addition, TP53 muta-
tions are often associated with complex karyotypes in
MDS as part of chromosome 17 deletions or transloca-
tions. Since mutations of TP53 have been shown to
cause genetic instability, it is possible that the complex
karyotype is a secondary event in the evolution of
advanced disease. NRAS has been reported to be abnor-
mally activated in patients with MDS (5-20%).22 Point
mutations result in constitutive activation of the gene.
The family of RAS genes control signaling pathways for
proliferation, differentiation and apoptosis, and is thus
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 173 |
Berlin, Germany, June 4-7, 2009
likely to be mutated during disease progression. The
BCL2 family of proteins tightly controls the apoptotic
pathway as it includes both pro and anti-apoptosis pro-
teins. Levels of the pro-apoptotic proteins are increased
in low risk patients when compared to high risk, while
levels of the anti-apoptotic proteins are increased in the
high-risk patients.23 JAK2 mutations have been detected
in a high percentage of patients with myeloproliferative
disease, but rarely in most patients with MDS.24 This
gene, a tyrosine kinase, activates the JAK-STAT signal
transduction pathway controlling proteins essential for
multiple biologic functions. Many cytokines and
growth factors are regulated by this pathway. It has
recently been shown that JAK2 mutation is characteris-
tic of a subset of MDS patients with refractory anemia
with ringed sideroblasts and thrombocytosis.25,26 In con-
trast, FLT3, a stimulatory protein for hematopoietic cell
proliferation and differentiation often found (20-25%)
as tandem repeats in AML, is mutated in only 1-3% of
MDS patients.27,28 However, when present, the MDS
patient is likely to transform to AML. Thus, this muta-
tion may be an early indicator of poor prognosis and
transformation. Mutations of RAS and AML1 genes
have been associated with -7 MDS, which has a partic-
ularly poor prognosis. AML1 is a transcription factor
associated with normal hematopoiesis that has been
found to be mutated several types of leukemias, includ-
ing AML.29 These patients also show constitutive activa-
tion of the STAT/STAT5 signal transduction pathway
following treatment with granulocyte colony-stimulat-
ing factor, although the connection to chromosome 7
remains unknown. A high incidence of AML1 muta-
tions has been found in high-risk MDS patients and is
thought to be implicated in transformation to AML in
these patients.30,31 A number of other gene mutations,
including EVI1, PTPN11 and PDGFR-A are often mutat-
ed in AML and rarely in MDS.32-34 The commonality is
that they all are essential parts of proliferative, apoptot-
ic or differentiation pathways.
Epigenetics
While genomic mutations have been recognized as a
cause of malignant transformations since the discovery
of the Philadelphia chromosome, other biochemical
events can lead to changes in the DNA without disrupt-
ing the primary nuclei acid sequence. This modification,
referred to as epigenetics, has a profound role in genet-
ic regulation and control of gene expression. Abnormal
methylation of a gene has the same functional effect as
a genetic mutation, and thus provides an additional
mechanism for malignant transformation. Two
hypomethylating agents, 5-azacitidine and decitabine,
have been approved by the FDA for treatment of
MDS.35-37 The mechanism of action is not clear; it has
been assumed that silenced tumor suppressor genes are
reactivated upon treatment. While a number of genes
have been shown to be hypermethylated in patients
with MDS, including the tumor suppressor gene, p15,
response to therapy could not be correlated with
increase in p15 gene expression.37 Using methylation-
specific polymerase chain reaction to look at promoter
methylation of regulatory genes essential to the evolu-
tion of MDS, it was possible to identify gene-silencing,
specific for both cell lineage and differentiation states.38
14th Congress of the European Hematology Association

Other studies using high-resolution single nucleotide Table 1.

polymorphism (SNP) arrays have shown that genomic
methylation profiles may be useful for identifying direct Reference Patient samples Cell Type Results
or indirect targets of these drugs.39,40 Additionally, bio-
chemical modifications of the histone proteins compos-
ing the chromatin structure have been found to greatly Miyazato et al.45 RAEB AC133 MDS
alter the ability of transcriptional initiating complexes to and AML separated BM samples heterogeneous
assemble on gene promoters, and thus, are another hindering analysis;
form of epigentic control of gene expression. The access Dlk gene identified
to promoter DNA is influenced by the activities of his- as MDS specific
tone acetylating (HAC) enzymes, which relax the chro-
matin structure and histone deacetylating (HDAC) Ueda et al.46 all stages AC133 Identified poor vs.
MDS separated BM good prognosis genes
enzymes, which condense the chromatin. In theory, the
use of HDAC inhibitors should restore gene expression Hofmann et al.47 low risk CD34 Large number of
of normal genes (including tumor suppressor genes) that versus separated BM deregulate genes in MDS;
have been silenced during disease evolution. Clinical tri- high risk identified 11 genes
als with HDAC inhibitors are currently underway in as class predictors
patients with MDS.41
The development of high-resolution SNP arrays has Pellagatti et al.48 RA(5q-, Neutrophils High variability between
also enabled investigators to look at the DNA structure normal), MDS samples; few genes
of the largest sub-group of MDS patients, the 50% that AML commonly deregulated
have a normal karyotype.42-44 Surprisingly, a number of in MDS; 71 genes could
patients (10-15%) with no observable clonal abnormal- discriminate 5q-; only 1
ity by karyotype had DNA that contained regions of of 71 genes maps to
uniparental disomy (UPD; regions inherited from only CDR of 5q-
one parent resulting in a neutral loss of heterozygosity),
as well as small regions of amplification and/or dele- Chen et al.49 monsomy 7, CD34 separated BM; Small number
tions. While the UPDs were found to be constitutional, trisomy 8 pooled/individual commonly de-regulated
the amplifications and deletions were acquired and samples from genes; different pathways
associated with poorer disease outcome.43 These prelim- different patients implicated for each
inary studies indicate that perhaps, the patients with karyotype; very little
UPD have inherently increased genetic instability, and overlap with results
of other studies
thus, have an increased risk for developing MDS.
Further studies are needed to confirm these findings,
but if confirmed, they point to a method to screen for
individuals at high risk of developing MDS.

Gene expression studies mapped to the commonly deleted region or CDR of

DNA microarray technology offers an unprecedented
opportunity to define signature profiles and several
early studies have attempted to do this (Table 1). The
first study45 compared MDS with excess blasts (RAEB)
to cells of patients with de novo AML. Dlk gene encod-
ing the protein δ-like gene was identified as MDS spe-
cific. The individual variability of MDS samples, how-
ever, made further analysis very difficult. In a second
study,46 this same group used a transcriptosome approach
to examine differences between normal BM and those
of MDS patients at various stages of the disease, identi-
fying poor versus good prognosis genes, which were
thought to contribute to the progression of MDS.
Hoffman et al.47 compared CD34+ isolated BM cells of
low risk versus high risk MDS to normal control samples.
While 11 genes were identified as class (risk) predictors,
a large number of genes were found to be deregulated in
the MDS patients. Pellagatti et al.48 looked at the expres-
sion profile of neutrophils isolated from the peripheral
blood of 21 MDS patients [7 with del(5q) karyotype], 2
AMLs and normal controls. Similar to the other studies,
expression was highly variable but several genes were
commonly deregulated. Del(5q) patients could be dis-
criminated from refractory anemia (RA) patients with
normal karyotypes on the basis of 71 genes. Interesting-
ly, only 5 of the 71 genes mapped to 5q and only one
5q–. Chen et al.49 found a small number of deregulated
genes common to monosomy 7 and trisomy 8, but dif-
ferent cellular pathways were implicated in the profiles
of each karyotype.
The 5q deletion is the most frequent cytogenetic
abnormality in MDS and is found in 10-15% of patients.
The haplo-deletion is highly variable encompassing
5q31, but two smaller regions have been defined by
molecular studies to be associated with clinical fea-
tures.50 In a more recent expression study,51 it was noted
that two ribosomal genes, RBM22 and RPS14, mapping
to the CDR, were significantly down-regulated. Since
another ribosomal gene, RPS19, had already been impli-
cated in the congenital anemia of Blackfan-Diamond
syndrome,52 this group suggested that deregulation of
these genes, and the pathway that they were part of,
could be the causative abnormality in 5q- syndrome
patients. Shortly thereafter, using RNA inhibition,
RPS14 was indeed shown to be a causal gene for the 5q-
syndrome.53 To support their hypothesis that deregulat-
ed ribosomal and protein synthesis pathways are a fea-
ture of 5q- syndrome, Pellagatti et al.54 looked at the
expression of 579 ribosomal and translation related
genes in the CD34+ cells of 5q- syndrome patients com-
pared to RA patients with normal karyotype and
healthy controls. Fifty-five genes associated with ribo-

| 174 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

some and protein synthesis were differentially
expressed with the lowest expression levels found in
the 5q- syndrome patients. The rapid proliferation of
red blood cells requiring continuous protein synthesis
would make erythropoiesis highly sensitive to disrup-
tions in ribosome biosynthesis, and explains the severe
anemia seen in these patients. RPS14 haploinsufficiency
explains the paradox of anemia and thrombocytosis but
not the initial clonal expansion. Recent meta-analysis
comparing CD34+ expression profiles of 60 cases of
non-del(5q) MDS patients with 52 normal controls
using datasets from seven independent studies in the
NCBI GEO database showed that the most significant-
ly deregulated class of genes in the MDS group were the
ribosomal protein genes.55 Thus, deregulated expression
of genes in a specific functional pathway provides a uni-
fied mechanism for the pathology of a seemingly
diverse disease.
Our group, together with the Broad Institute took a
different bedside to bench approach to examine gene
expression in patients with MDS.56 Studies showed that
lenalidomide treatment elicited transfusion independ-
ence in 67% of patients that had a del(5q) karyotype15
and 26% of patients without del(5q).16 Using pre-thera-
py mononuclear bone marrow cells, a signature of 32
genes, which are normally expressed during later stages
of erythroid differentiation, was found to be down reg-
ulated in responders.56
MicroRNA studies
The expression of microRNAs has been shown to dif-
ferentiate between MDS subgroups.57 In patients with
5q- syndrome, two microRNAs (miR-145 and miR-146a
that target TIRAP and TRAF6) mapping to the CDR
appear to have biologic relevance to the disease pathol-
ogy, specifically affecting the 5q- syndrome pheno-
type.58 Although preliminary, these studies are paving
the way for an entirely novel area of investigation.
Conclusion
Multiple biologic pathways have been found to be
deregulated in MDS. The heterogeneity of the disease
precludes development of a single agent targeting a sin-
gle pathway as therapy for all patients and accounts for
the variation of response to a given therapy. New tech-
nologies that predict response or individualize therapeu-
tic strategies are promising weapons in the battle with
MDS. Continued basic studies into the biology of dis-
ease evolution may elucidate additional unifying char-
acteristics that will eventually lead to new therapies for
this devastating disease.
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Thalidomide produces transfusion independence in long-
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syndromes. Blood 2001;98:958-65.
14. List A, Kurtin S, Roe DJ, Buresh A, Mahadevan D, Fuchs D, et
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16. Raza A, Reeves JA, Feldman EJ, Dewald GW, Bennett JM,
Deeg HJ, et al. Phase 2 study of lenalidomide in transfusion-
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syndromes with karyotypes other than deletion 5q. Blood
2008;111:86-93.
17. Chang DH, Liu N, Klimek V, Hassoun H, Mazumder A, Nimer
SD, et al. Enhancement of ligand-dependent activation of
human natural killer T cells by lenalidomide: therapeutic
implications. Blood 2006;108:618-21.
18. Zorat F, Shetty V, Dutt D, Lisak L, Nascimben F, Allampallam
K, et al. The clinical and biological effects of thalidomide in
patients with myelodysplastic syndromes. Br J Haematol
2001;115:881-94
19. Padua RA, Guinn BA, Al-Sabah AI, Smith M, Taylor C,
Pettersson T, et al. RAS, FMS and p53 mutations and poor clin-
ical outcome in myelodysplasias: a 10-year follow-up. Leu-
kemia 1998;12:887-92.
20. Kita-Sasai Y, Horiike S, Misawa S, Kaneko H, Kobayashi M,
Nakao M, et al. International prognostic scoring system and
TP53 mutations are independent prognostic indicators for
patients with myelodysplastic syndrome. Br J Haematol 2001;
115:309-12.
21. Wattel E, Preudhomme C, Hecquet B, Vanrumbeke M, Que-
snel B, Dervite I, et al. p53 mutations are associated with
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22. Lu D, Nounou R, Beran M, Estey E, Manshouri T, Kantarjian
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23. Parker JE, Mufti GJ, Rasool F, Mijovic A, Devereux S, Pagliuca
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24. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg
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25. Steensma DP, Dewald GW, Lasho TL, Powell HL, McClure RF,
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14th Congress of the European Hematology Association
26. Wang SA, Hasserjian RP, Loew JM, Sechman EV, Jones D, Hao
S, et al. Refractory anemia with ringed sideroblasts associated
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27. Horiike S, Yokota S, Nakao M, Iwai T, Sasai Y, Kaneko H, et
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28. Shih LY, Huang CF, Wang PN, Wu JH, Lin TL, Dunn P, et al.
Acquisition of FLT3 or N-ras mutations is frequently associat-
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29. Niimi H, Harada H, Harada Y, Ding Y, Imagawa J, Inaba T, et
al. Hyperactivation of the RAS signaling pathway in
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30. Harada H, Harada Y, Niimi H, Kyo T, Kimura A, Inaba T. High
incidence of somatic mutations in the AML1/RUNX1 gene in
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31. Nakao M, Horiike S, Fukushima-Nakase Y, Nishimura M,
Fujita Y, Taniwaki M, et al. Novel loss-of-function mutations
of the haematopoiesis-related transcription factor, acute
myeloid leukaemia 1/runt-related transcription factor 1,
detected in acute myeloblastic leukaemia and myelodysplastic
syndrome Br J Haematol 2004;125:709-19.
32. Raza A, Buonamici S, Lisak L, Tahir S, Li D, Imran M, et al.
Arsenic trioxide and thalidomide combination produces multi-
lineage hematological responses in myelodysplastic syn-
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EVI1 expression. Leuk Res 2004;28:791-803.
33. Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J,
Jung A, et al. Somatic mutations in PTPN11 in juvenile myelo-
monocytic leukemia, myelodysplastic syndromes and acute
myeloid leukemia. Nat Genet 2003;34:148-50.
34. Christiansen DH, Desta F, Andersen MK, Pedersen-Bjergaard J.
Mutations of the PTPN11 gene in therapy-related MDS and
AML with rare balanced chromosome translocations. Genes
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35. Silverman LR, Demakos EP, Peterson BL, Kornblith AB,
Holland JC, Odchimar-Reissig R, et al. Randomized controlled
trial of azacitidine in patients with the myelodysplastic syn-
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Oncol 2002;20:2429-40.
36. Kantarjian H, Oki Y, Garcia-Manero G, Huang X, O'Brien
Kantarjian H, Oki Y, et al. Results of a randomized study of 3
schedules of low-dose decitabine in higher-risk myelodysplas-
tic syndrome and chronic myelomonocytic leukemia. Blood
2007;109:52-7.
37. Kantarjian H, Oki Y, Garcia-Manero G, Huang X, O'Brien
SO’Brien S, Cortes J, et al. Results of a randomized study of 3
schedules of low-dose decitabine in higher-risk myelodysplas-
tic syndrome and chronic myelomonocytic leukemia. Blood
2007;109:52-7.
38. Hopfer O, Komor M, Koehler IS, Schulze M, Hoelzer D, Thiel
E, et al. DNA methylation profiling of myelodysplastic syn-
drome hematopoietic progenitor cells during in vitro lineage-
specific differentiation. Exp Hematol 2007;35:712-23.
39. Figueroa ME, Reimers M, Thompson RF, Ye K, Li Y, Selzer RR,
et al. An integrative genomic and epigenomic approach for the
study of transcriptional regulation. PLOS 2008;3:e1882
40. Kerkel K, Spadola A, Yuan E, Kosek J, Jiang L, Hod E, et al.
Genomic surveys by methylation-sensitive SNP analysis iden-
tify sequence-dependent allele-specific DNA methylation. Nat
Genet 2008;40:904-8.
41. Griffiths EA, Gore SD. DNA methyltransferase and histone
deacetylase inhibitors in the treatment of myelodysplastic
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43. Gondek LP, Tiu R, O'Keefe O’Keefe CL, Sekeres MA, Theil
KS, et al. Chromosomal lesions and uniparental disomy
detected by SNP arrays in MDS, MDS/MPD, and MDS-
derived AML Blood 2008;111:1534-42.
44. Nowak D, Nolte F, Mossner M, Nowak V, Baldus CD, Hopfer
O, et al. Genome-wide DNA-mapping of CD34+ cells from
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disomy. Exp Hematol 2009;37:215-24.
45. Miyazato A, Ueno S, Ohmine K, Ueda M, Yoshida K, Yama-
shita Y, et al. Identification of myelodysplastic syndrome-spe-
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DNA microarray analysis of stage progression mechanism in
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47. Hofmann WK, de Vos S, Komor M, Hoelzer D, Wachsman W,
Koeffler HP. Characterization of gene expression of CD34+
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2002;100:3553-60.
48. Pellagatti A, Esoof N, Watkins F, Langford CF, Vetrie D,
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49. Chen G, Zeng W, Miyazato A, Billings E, Maciejewski JP,
Kajigaya S, et al. Distinctive gene expression profiles of CD34
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A, Malcovati L, et al. Gene expression profiling of CD34+ cells
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55. Sohal D, Yeatts A, Ye K, Pellagatti A, Zhou L, Pahanish P, et al.
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853].
 Myelodysplastic syndromes

Molecular pathogenesis of myelodysplastic

syndromes: identifying new therapeutic targets
based on biology

S.D. Nimer A B S T R A C T

Division of Hematologic Oncology
and Sloan-Kettering Institute,
Memorial Sloan-Kettering Cancer
Center, New York, NY, USA
Hematology Education:
the education program
for the annual congress of the
European Hematology Association
We are making slow but steady progress in advancing our knowledge of the molecular defects that
underlie acute leukemia and the myelodysplastic syndromes (MDS). MDS has been more difficult to
understand because of its heterogeneity. A common feature is morphologic evidence of dysplasia
within the blood and marrow compartments. The basis for this and other features of the disease is
unclear. Furthermore, the significant immunologic abnormalities identified in this disease, coupled
with the mixture of MDS stem cells within the dysplastic progenitor cells and normal stem and pro-
genitor cells in the marrow, has made it difficult to molecularly characterize this disease. Additionally,
the lack of “MDS” cell lines and the difficulty growing MDS stem or progenitor cells in vitro or in
mouse xenotransplantation studies have hampered our understanding of this disease.

2009;3:177-180

lthough the cause of MDS is usually t(3;21)(q26;q22) (also seen in patients with

A unknown, it can occur following

exposure to environmental toxins, to
alkylating agent or topoisomerase II
inhibitor therapy that is used to treat a pri-
mary malignancy. Indeed, further investiga-
tion of MDS arising in these settings may be
more fruitful in the years to come.
Secondary MDS caused by these agents has
two distinct time courses and often-distinct
cytogenetic abnormalities. These include the
loss of all or part of chromosomes 5 and 7
seen following alkylating agent therapy, and
abnormalities (generally translocations)
involving the chromosome 11q23 region
seen in patients exposed to topoisomerase
inhibitors leading to fusion of the MLL gene
with one of its more than 70 partner genes.1
The genes affected by chromosomal inter-
stitial deletions or numerical changes in
MDS have escaped characterization, even
though the recurring cytogenetic abnormali-
ties have been known for decades (e.g., 5q-,
7q-, 20q-, trisomy 8). Thus, for most MDS
patients, the molecular basis of their disease
is not known. However, in addition to obvi-
ous cytogenetic abnormalities in MDS, vari-
eties of modern molecular studies of genom-
ic integrity (e.g., SNP arrays) have shown
other recurrent genomic abnormalities (e.g.,
small regions of chromosome gain or loss
and uniparental disomy). Recently, we have
gleaned much information about certain
genes that may be critically involved in
MDS, and hopefully, we can use this knowl-
edge to rapidly develop better therapeutic
approaches for patients with this disease.
Cytogenetic abnormalities
One of the first chromosomal transloca-
tions described in MDS patients is the
blast crisis CML), which rearranges the
AML1 and EVI-1 genes, fusing the N termi-
nus of AML1 with nearly all of the
MDS1/EVI-1 protein. Both components of
the AML1-MDS1/EVI-1 fusion protein
appear to play critical roles in normal
hematopoiesis, and both are required for the
unique effects of the fusion protein. The
AML1 gene (now known officially as
RUNX1) is involved in a variety of chromo-
somal translocations, mutations and dele-
tions. Translocations involving RUNX1 (and
chromosome 21q22) are found in specific
subtypes of acute leukemias, while point
mutations or deletions are found in the M0
subtype of AML and in MDS, in particular in
therapy-related MDS.2,3 These mutations
generally serve as loss of function mutants,
but some have the potential to serve as dom-
inant negatives, affecting the function of the
remaining wild type AML1 protein. RUNX1
expression is required for the development
of definitive hematopoiesis in the mouse,
and RUNX1 regulates lymphocyte develop-
ment, progenitor cell numbers, and
megakaryocytic differentiation in the adult
mouse.4
While the role of AML1/RUNX1 in AML
and MDS is widely appreciated, the biology
of the EVI-1 gene, located on 3q26, is not.
The EVI-1 gene encodes both the MDS1-
EVI-1 and EVI-1 proteins via differential pro-
moter usage. EVI-1, but not MDS1-EVI-1, is
transforming and can block hematopoietic
differentiation, especially of the erythroid
lineage.5 EVI-1 contains zinc finger domains
that interact with DNA and others involved
in protein-protein interactions. MDS1-EVI-1
but not EVI-1 contains a PR domain, a region
shown to contain methyltransferase activity

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 177 |
14th Congress of the European Hematology Association
in other proteins. EVI-1 has been shown to impair
GATA-1 function via protein-protein interaction and
this has been postulated to play a critical role in the inhi-
bition of erythroid differentiation. However, EVI-1 has
also been reported to interact with RUNX1, blocking its
binding to DNA.6 Furthermore, EVI-1 overexpression
can cooperate with certain mutant forms of RUNX1 to
generate AML from MDS.7 MDS1-EVI-1 has been
shown to function as a transcriptional repressor and to
interact with several methyltransferases, such as G9a,8
Suv39H1,9 CtBP,10 and MBD3,11 a member of the Mi2-
NuRD repressor complex.
PRDM16 (aka MEL1) is an EVI-1 like gene12 and it is
also involved in a translocation with RUNX1.13
PRDM16 also interacts with the CtBP repressor com-
plex, which itself interacts with the LSD1 histone
demethylase (LSD1= lysine specific demethylase, a H3-
K4 demethylase).14 Thus, both the RUNX1-MDS1-EVI1
and the RUNX1-MEL1 (PRDM16) fusion proteins may
function, at least in part, by changing the normal H3-K4
methyl activation mark to a demethylated H3-K4 mark.
This would promote the H3-K9 methylation mark and
contribute to gene repression. If so, restoration of H3-
K4 trimethylation, and inhibition of LSD1 and H3-K9
methylation, could be a therapeutic approach for EVI-1
related, and AML1-MDS-EVI-1 related MDS or AML.
Epigenetic deregulation in myelodysplastic syndromes
In general, alterations in histone marks and DNA
methylation may also be the hallmarks of MDS and cer-
tain other types of malignances. Given the success of
DNA hypomethylating agents in treating MDS patients,
a clearer understanding of how gene expression is dys-
regulated in this disease is essential. Therefore, under-
standing how transcription factors (such as RUNX1,
EVI-1, PU.1, HoxA9) regulate normal (and malignant)
hematopoiesis will be a focus of this report. Potential
candidate MDS genes affecting chromatin structure are
being actively studied, as loss of these genes could have
pleiotropic effects and result in the epigenetic propaga-
tion of gene expression abnormalities from the MDS
initiating cell to its progeny. Yet, in addition to under-
standing gene transcriptional regulation, a strong work-
ing knowledge of transcriptional elongation, post-tran-
scriptional modification of protein function and riboso-
mal biogenesis are necessary to understand fully how
candidate genes contribute to MDS biology.
Silencing of gene expression has been largely ascribed
to the processes of histone deacetylation, DNA promot-
er hypermethylation and histone methylation. Thus,
the enzymes that catalyze these processes represent
potential therapeutic targets. They include: (i) the his-
tone deacetylases (HDACs), which remove acetyl
groups from lysine residues in histones; (ii) the DNA
methyltransferases, which physically interact with
HDAC complexes and catalyze methylation at CpG
islands within the promoters of critical growth regulato-
ry genes; and (iii) the histone methyltransferases
(HKMTs and PRMTs) that methylate arginine or lysine
residues within the tails or globular domains of the his-
tones and promote a closed chromatin conformation
and heterochromatin formation. Aberrant methylation
of cytosines within CpG promoter regions by DNA
methyltransferases (primarily DNMT1 and DNMT3b)
| 178 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
may silence key components of signal transduction,
apoptosis, cell cycle progression, or DNA repair gene
pathways. Hypomethylating agents like decitabine (5-
aza-2’-deoxycytidine) and 5-Azacytidine covalently
bind to the DNA methyltransferase enzymes, irre-
versibly inhibiting their function. This results in the pro-
gressive loss of methylation, which can trigger changes
in gene expression and induce cellular differentiation.
The ability of these agents to induce the DNA damage
response has also been shown. Thus, the basis for the
effectiveness of action of 5-azacitidine and decitabine in
MDS is not really known, nor is the reason why some
patients respond and others do not respond.
Hypomethylating agents are being combined with
histone deacetylase inhibitors in an attempt to affect
gene expression synergistically. For combination thera-
pies to synergize optimally, the dynamics of the
processes being inhibited needs to be considered.
Histone acetylation is a rapidly reversible process,
whereas DNA methylation is felt to be irreversible.
Thus far, histone methyltransferase inhibitors have not
been clinically tested. Histone methylation was thought
to be irreversible until multiple site-specific demethylases
were reported (e.g. JMJD6 and LSD1, which act on
methyl arginine and methyl lysine, respectively). Thus,
both the methyltransferases and the demethylases rep-
resent potential targets.
MLL, NSD1, and NSD3 all have methyltransferase
activity and all are involved in translocations in myeloid
disorders. These proteins contain a SET domain [named
because it is found in the Su(var)3-9; E(z); and Trithorax
proteins]. SET domains are found in several other can-
cer-related proteins, including the EZH2 gene which is
overexpressed in prostate cancer,15 the MMSET gene
which is overexpressed in multiple myeloma,16 and the
MLL gene. Generally, the SET domains contain the his-
tone methyltransferase (HMT) activity within a protein.
A second class of HMTs has also been identified; they
contain a PR domain rather than a SET domain.
MLL methylates lysine 4 in histone H3, a mark gener-
ally associated with gene activation. The region of MLL
protein lost in the 11q23 translocation includes the SET
domain, which contains its methyltransferase enzymat-
ic activity. Yet, in most cases, other activating histone
marks are still found on MLL regulated genes (e.g.,
H3K79me on the HoxA9 gene promoter) suggesting that
the MLL-fusion proteins still activate gene expression,
although they may have lost their ability to fine tune
gene expression.
Other processes implicated in myelodysplastic syndromes
The molecular basis of 5q-MDS is being explored,
especially because two-thirds of transfusion dependent
patients with 5q- MDS respond to lenalidomide, losing
their transfusion dependency and achieving major or
complete cytogenetic responses.17 Many genes have
been suggested to play a role in 5q-MDS via haploinsuf-
ficiency, a mechanism that has been invoked because
deletion of candidate genes has not been accompanied
by mutations in the remaining allele. RPS14 is a candi-
date 5q- gene,18 as is SPARC, a protein secreted by cells
into the extracellular matrix.19 So are α-catenin, an intra-
cellular protein that may regulate cell polarity and adhe-
sion,20 and the cell cycle regulator cdc25c, which con-

trols the movement of cells from G2 to M. Furthermore,
the EGR1 transcriptional regulator, which can function
as a tumor suppressor gene, and the NPM gene, which
encodes a nuclear-cytoplasmic shutting protein that reg-
ulates the p53-p14ARF pathway, also represent intrigu-
ing candidates.21,22 However, not all of these genes are
located within the commonly deleted region (the CDR)
on chromosome 5q. If multiple genes must be lost to
generate the 5q-phenotype, then the current approach
of studying genes one at a time will need adaptation in
order to describe the myelodysplastic syndromes fully.
Point mutations in activating transcription factors
(like AML1) have been found in MDS patients, but acti-
vated kinases, such as JAK2 or RAS, have also been
found. In fact activating RAS mutations may be more
frequent in patients with AML1 point mutations. The
RAS point mutations found in MDS patients generally
involve codon12 of H-RAS or N-RAS. In addition to
RAS mutations, loss of NF1 or PTPN11, proteins,
downregulate RAS signaling are found in CMML (or
juvenile CMML). Translocations that activate tyrosine
kinases have also been reported in MDS, but these tend
to be present in patients with more proliferative fea-
tures to their disease. Thus, the t(5;12) that generates a
TEL-PDGFRβ fusion protein with constitutive kinase
activity, frequently accompanies CMML with eosino-
philia. Like the TEL-PDGFRβ fusion, the JAK2V617F
mutation, when found in MDS, is associated with spe-
cific clinical syndromes, such as RARS with thrombocy-
tosis23 or in some patients with 5q- MDS.24 More than
50% of patients with the RARS-T syndrome may have
an activating JAK2 mutation; the frequency, in part,
depends on the threshold platelet count used to define
the syndrome.
Mouse models of myelodysplastic syndromes
Several mouse models of MDS now exist. One such
model is the heterozygous NPM +/- mouse, which
develops a myelodysplastic syndrome similar to refrac-
tory anemia (RA), with prominent dyserythropoiesis
and erythroid hyperplasia in the bone marrow. These
mice survive to 1 year of age, comparable to their wild
type littermates; transformation to acute leukemia has
now been reported. While NPM is mutated and aber-
rantly located the cytoplasm in 40% of AML cases with
normal cytogenetics,25,26 NPM mutations are generally
not found in patients with MDS.25
Another MDS model is based on transplanting
murine bone marrow cells that have been transduced
with an EVI-1 expressing retrovirus. After a significant
period of latency, the recipient mice develop a syn-
drome that mimics MDS,27 with significant erythroid
defects, including greatly impaired maturation. These
mice do not have increased blasts, nor do they develop
acute leukemia.
The NUP98-HOXD13 transgenic mouse model may
be particularly useful for studying the development and
progression of MDS. These mice develop refractory
anemia with excess blasts (RAEB) that can be fatal. Mice
that do not die of cytopenias go on to develop AML.28
Thus, this model seems to recapitulate the human dis-
ease best.
Another MDS model that demonstrates both an MDS
phase and progression to AML is one based on the
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 179 |
Berlin, Germany, June 4-7, 2009
expression of RUNX1 (AML1) point mutant proteins in
retrovirally transduced bone marrow cells.7 This model
has shown the potential synergism between EVI-1 over-
expression and loss of RUNX1 function.
Conclusions
Much remains to be defined in the molecular patho-
genesis of MDS. The syndromes are characterized by
defects in differentiation and defects in proliferation and
apoptosis, yet the path to their more successful treat-
ment is not clear. Further study of the MDS stem or ini-
tiating cell is essential, as is a better understanding of the
role of inflammatory mediators, and immune effector
cells within the bone marrow microenvironment in
inhibiting hematopoiesis. Only then will the defects
that lead to the cytopenias and the predisposition to
develop AML be understood.
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6. Senyuk V, Sinha KK, Li D, Rinaldi CR, Yanamandra S,
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7. Watanabe-Okochi N, Kitaura J, Ono R, Harada H, Harada Y,
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8. Spensberger D, Delwel R. A novel interaction between the
proto-oncogene Evi1 and histone methyltransferases,
SUV39H1 and G9a. FEBS Lett 2008;582:2761-7.
9. Cattaneo F, Nucifora G. EVI1 recruits the histone methyltrans-
ferase SUV39H1 for transcription repression. J Cell Biochem.
2008;105:344-52.
10. Palmer S, Brouillet JP, Kilbey A, Fulton R, Walker M, Crossley
M, et al. Evi-1 transforming and repressor activities are medi-
ated by CtBP co-repressor proteins. J Biol Chem 2001;276:
25834-40.
11. Spensberger D, Vermeulen M, Le Guezennec X, Beekman R,
van Hoven A, Bindels E, et al. Myeloid transforming protein
Evi1 interacts with methyl-CpG binding domain protein 3 and
inhibits in vitro histone deacetylation by Mbd3/Mi-2/NuRD.
Biochemistry 2008;47:6418-26.
12. Mochizuki N, Shimizu S, Nagasawa T, Tanaka H, Taniwaki
M, Yokota J, et al. A novel gene, MEL1, mapped to 1p36.3 is
highly homologous to the MDS1/EVI1 gene and is transcrip-
tionally activated in t(1;3)(p36;q21)-positive leukemia cells.
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13. Sakai I, Tamura T, Narumi H, Uchida N, Yakushijin Y, Hato T,
et al. Novel RUNX1-PRDM16 fusion transcripts in a patient
with acute myeloid leukemia showing t(1;21)(p36;q22). Genes
Chromosomes Cancer 2005;44:265-70.
14. Wang J, Scully K, Zhu X, Cai L, Zhang J, Prefontaine GG, et al.
Opposing LSD1 complexes function in developmental gene
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15. Varambally S, Dhanasekaran SM, Zhou M, Barrette TR,
Kumar-Sinha C, Sanda MG, et al. The polycomb group protein
EZH2 is involved in progression of prostate cancer. Nature
2002;419:624-9.
14th Congress of the European Hematology Association
16. Chesi M, Nardini E, Lim RS, Smith KD, Kuehl WM, Bergsagel
PL. The t(4;14) translocation in myeloma dysregulates both
FGFR3 and a novel gene, MMSET, resulting in IgH/MMSET
hybrid transcripts. Blood 1998;92:3025-34.
17. List A, Dewald G, Bennett J, Giagounidis A, Raza A, Feldman
E, et al. Lenalidomide in the myelodysplastic syndrome with
chromosome 5q deletion. Myelodysplastic Syndrome-003
Study Investigators. N Engl J Med 2006;355:1456-65.
18. Ebert BL, Pretz J, Bosco J, Chang CY, Tamayo P, Galili N, et al.
Identification of RPS14 as a 5q- syndrome gene by RNA inter-
ference screen. Nature 2008;451:335-9.
19. Pellagatti A, Jädersten M, Forsblom AM, Cattan H, Christens-
son B, Emanuelsson EK, et al. Lenalidomide inhibits the malig-
nant clone and up-regulates the SPARC gene mapping to the
commonly deleted region in 5q- syndrome patients. Proc Natl
Acad Sci USA 2007;104:11406-11.
20 Liu TX, Becker MW, Jelinek J, Wu WS, Deng M, Mikhalkevich
N, et al. Chromosome 5q deletion and epigenetic suppression
of the gene encoding alpha-catenin (CTNNA1) in myeloid cell
transformation. Nat Med 2007;13:78-83.
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Anastasi J, et al. Haploinsufficiency of EGR1, a candidate gene
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25. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci
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287-95.
 Myelodysplastic syndromes

Risk-adapted treatment of myelodysplastic syndromes

M. Cazzola
L. Malcovati
Department of Hematology
Oncology, University of Pavia
Medical School, Pavia, Italy
Hematology Education:
the education program
for the annual congress of the
European Hematology Association
2009;3:181-187
A B S T R A C T
Myelodysplastic syndromes (MDS) are usually characterized by clonal proliferation of hematopoiet-
ic cells, which partly retain their capacity to differentiate and maturate, but do so in an inefficient
manner. Their clinical heterogeneity is best illustrated by the observation that these disorders range
from indolent conditions with a near-normal life expectancy to forms approaching acute myeloid
leukemia (AML). A risk-adapted treatment strategy is mandatory for conditions showing a so highly
variable clinical course, and definition of the individual risk has been based so far on the use of prog-
nostic scoring systems. We have developed a prognostic model that accounts for the WHO categories,
cytogenetics and transfusion dependency. This WHO classification-based prognostic scoring system
(WPSS) is able to classify patients into five risk groups showing different survivals and probabilities of
leukemic evolution. WPSS predicts survival and leukemia progression at any time during follow-up,
and may therefore be used for implementing risk-adapted treatment strategies. The approach to a
patient with myelodysplastic syndrome should always begin with a period of observation, with
sequential peripheral blood counts – and sometimes bone marrow examinations – to assess the rate
of progression, if any. Several therapeutic tools have been proposed in the last decades but only few
survived the evidence-based criteria of efficacy. Patients with very low WPSS risk do not need any
treatment and can be just followed regularly. Transfusion dependent patients with low WPSS risk can
be treated with recombinant human erythropoietin. Responsive patients are mainly those with early
disease, inadequate endogenous erythropoietin production and low need for blood transfusion.
Transfusion-dependent patients with myelodysplastic syndrome associated with del(5q) may respond
to lenalidomide with cytogenetic remission and abolishment of transfusion requirement. However, in
2008 the EMEA Committee for Medicinal Products for Human Use was of the opinion that the bene-
fits of lenalidomide in the treatment of anemia of MDS with del(5q) did not outweigh its potential
risks, and therefore the drug has not been approved for this use in Europe. A recent survival study com-
paring azacitidine versus conventional care showed that treatment with azacitidine increases overall
survival in patients with higher-risk MDS. Thus, EMEA has recently approved azacitidine for the treat-
ment of adult patients with high-risk myelodysplastic syndrome who are not eligible for allogeneic
hematopoietic stem cell transplantation. The only treatment that can cure a patient with myelodys-
plastic syndrome is still allogeneic stem cell transplantation. It can be estimated that approximately
one third of patients receiving an allogeneic transplantation are cured with this treatment, but only a
minority of all MDS patients are eligible and have a donor.

yelodysplastic syndromes (MDSs) may be difficult to assess in a clinical set-

M are usually characterized by clonal

proliferation of hematopoietic
cells, which partly retain their capacity to
differentiate and maturate, but do so in an
inefficient manner.1 The bone marrow is
generally hypercellular while peripheral
blood cells are variably reduced (cytope-
nia), and several morphological abnormali-
ties are observed in these two compart-
ments.2 In addition, clones of hematopoiet-
ic cells carrying non-random cytogenetic
abnormalities are typically found in these
disorders.3 With time, there is a progressive
impairment in the capacity of hematopoiet-
ic cells to differentiate and maturate; blast
cells may appear in the bone marrow and
peripheral blood, and there is an increasing-
ly high risk of evolution to overt acute
myeloid leukemia (AML). The basic com-
ponents of the definition of MDS are clon-
ality, ineffective hematopoiesis and pre-
leukemic nature. These features, however,
ting, and – as stated in the World Health
Organization (WHO) classification4 –
MDSs remain among the most challenging
of the myeloid neoplasms for proper diag-
nosis and classification.
Diagnosis and classification
MDSs were defined and classified in 1982
by the French American British (FAB)
group.5 In 2001, the WHO classification of
myeloid neoplasms was developed.6 This
classification has been updated recently,7
and the 2008 WHO classification of myelo-
dysplastic syndromes/neoplasms is report-
ed in Table 1.
The current diagnostic approach includes
peripheral blood and bone marrow mor-
phology (to evaluate abnormalities of peri-
pheral blood cells and hematopoietic pre-
cursors), bone marrow biopsy (to assess
marrow cellularity, fibrosis and topogra-
phy), and cytogenetics (to identify non-ran-

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14th Congress of the European Hematology Association

Table 1. WHO classification of myelodysplastic syndromes Table 2. WHO classification-based Prognostic Scoring
and peculiar entities. System (WPSS) and bone marrow fibrosis as criteria for
predicting likelihood of survival and risk of leukemic evo-
Disease lution in the individual patient with myelodysplastic syn-
drome.
2008 WHO classification
Calculation of the WPSS score and evaluation of bone marrow fibrosis
Refractory anemia (RA), refractory neutropenia (RN), refractory thrombocytopenia
Variable Variable scores
(RT), refractory cytopenia with unilineage dysplasia (RCUD)

Refractory anemia with ringed sideroblasts (RARS) 0 1 2 3

Refractory cytopenia with multilineage dysplasia (RCMD) WHO category RA, RARS, MDS RCMD RAEB-1 RAEB-2
with isolated
Refractory anemiawith excess blasts-1 (RAEB-1) deletion (5q)

Refractory anemia with excess blasts-2 (RAEB-2) Karyotype* Good Intermediate Poor −

Myelodysplastic syndrome, unclassified (MDS-U) Transfusion Absent Present − −

requirement**
Myelodysplastic syndrome associated with isolated del(5q)
Bone marrow The presence of grade 2 to 3 bone marrow fibrosis involves
fibrosis*** a shift to a one-step more advanced risk group after accounting
Peculiar entities for WHO category, karyotype, and transfusion requirement

Hypocellular myelodysplastic syndrome Definition of the individual patient’s risk group

Myelodysplastic syndrome with marrow fibrosis Risk group Score Median Risk of leukemic
survival evolution

dom chromosomal abnormalities). The combination

Very low 0 >10 years <10% at 15 years
of overt marrow dysplasia and clonal cytogenetic
abnormality allows a conclusive diagnosis of myelo- Low 1 >5 years 10–20% at 5 years
dysplastic syndrome, but this is found in only a por-
tion of patients.1 Although bone marrow biopsy may Intermediate 2 (or score ~4 years 30-40% at 5 years
be considered too invasive for elderly patients, it pro- 1 plus
vides extremely useful diagnostic and prognostic infor- marrow fibrosis)
mation regarding cellularity,8 fibrosis,9 and CD34-posi-
tive cell topography.9 High 3-4 (or score ~2 years 30% at 3 years
2 plus
Risk assessment marrow fibrosis)
The clinical heterogeneity of myelodysplastic syn-
dromes is best illustrated by the observation that these Very high 5-6 (or score ~1 year >50% at 1 year
disorders range from indolent conditions with a near- 3 to 4 plus
normal life expectancy to forms approaching AML.1 A marrow fibrosis)
risk-adapted treatment strategy is mandatory for condi-
tions showing a highly variable clinical course, and def- *Good: normal, -Y, del(5q), del(20q); Poor: complex, chromosome 7 anomalies;
Intermediate: other chromosomal abnormalities. ** Transfusion dependency: at
inition of the individual risk has been based so far on least one transfusion every 8 weeks over a period of 4 months. ***Bone marrow
the use of prognostic scoring systems. In 1997, fibrosis should be evaluated according to the European consensus criteria.
Greenberg and co-workers10 developed the Intern-
ational Prognostic Scoring System (IPSS), based on mar-
row blasts, cytogenetic pattern, and number of cytope-
nias. The IPSS was derived from a multivariable analy- fusion dependency could be considered as an inde-
sis of hematological characteristics of 816 patients at pendent indicator of the severity of the disease, and
clinical onset, and included subjects with 20-30% mar- consequently an independent prognostic factor. These
row blasts, now considered as having AML. data are in agreement with the results of a recent study
In a study on the prognostic significance of the by Greenberg and coworkers,13 which showed that the
WHO classification,11 we found that the IPSS retained severity of anemia at diagnosis is of additive prognos-
significance within the WHO subgroups. However, tic value to IPSS in terms of survival.
when accounting for blast percentage using WHO cat- In collaboration with colleagues in Düsseldorf, we
egories, the only other IPSS variable adding prognostic developed a prognostic model that accounted for the
information was found to be cytogenetics. We also WHO categories, cytogenetics and transfusion
found that transfusion dependency had a significant dependency.14 This WHO classification-based prog-
effect on survival in multivariable analysis.11 Based on nostic scoring system (WPSS) was able to classify
this and other observations1,12 we concluded that trans- patients into five risk groups showing different sur-

| 182 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

Table 3. Comparison of two different risk assessment systems (IPSS and WPSS) in 258 patients with myelodysplastic
syndrome. Within each IPSS risk group, patients have been stratified according to WPSS criteria, taking into account mul-
tilineage dysplasia, transfusion dependency and bone marrow fibrosis as indicated in Table 2.

IPSS risk group No. of patients Median overall WPSS risk group No. of patients (%) Median overall
survival* survival**

Low 71 68 Very low 26 (37) 141

Low 23 (32) 66
Intermediate 16 (23) 48
High 6 (8) 26

Intermediate 1 122 42 Very Low 6 (5) 141

Low 23 (19) 66
Intermediate 42 (34) 48
High 33 (27) 26
Very high 18 (15) 9

Intermediate 2 47 14 Intermediate 1 (2) 48

High 25 (53) 26
Very high 21 (45) 9

High 18 5 High 1 (6) 26

Very high 17 (94) 9
*Data are from Greenberg et al.10 **Data are from Malcovati et al.14

useful in clinical decision making.9 Interestingly, with-

in patients stratified according to IPSS and WPSS cate-
gories, bone marrow fibrosis involved a shift to a one-
step more advanced risk group.9 As indicated in Table
2, grade 2 to 3 marrow fibrosis can be used to better
define the individual patient’s risk group within the
WPSS. Accounting for multilineage dysplasia, transfu-
sion dependency and bone marrow fibrosis within the
WPSS categorization allows us to define the prognosis
of the individual patient with MDS more accurately as
compared with the IPSS categorization. This is mainly
noticeable for patients belonging to the IPSS low or
intermediate-1 risk groups (Table 3), that is, those
patients who pose the most difficulties in therapeutic
decision making.

Therapeutic tools currently available for myelodysplastic

Figure 1. Cumulative risk of progressing from a WPSS risk
group to the next one (from very low to low, from low to
intermediate, from intermediate to high, and from high to
very high), or from the very high risk group to acute
myeloid leukemia. Data are from the patient population
that was analyzed for defining the WPSS.14
vivals and probabilities of leukemic evolution. WPSS
was shown to predict survival and leukemia progres-
sion at any time during follow-up so, therefore, may
be used for implementing risk-adapted treatment
strategies. Figure 1 illustrates the risk of progressing
from a WPSS risk group to the next one or to acute
myeloid leukemia over time. More recently we found
that bone marrow fibrosis identifies a distinct sub-
group of MDS with multilineage dysplasia, high trans-
fusion requirement, and poor prognosis, and repre-
sents an independent prognostic factor that may be
syndromes
Several therapeutic tools have been proposed in the
last decades but only a few survived the evidence-
based criteria of efficacy.1 In the past years, Italian15
and British16 guidelines for the therapy of myelodys-
plastic syndromes have been published. The therapeu-
tic tools currently employed in the treatment of these
disorders are reported in Table 4.
WPSS risk-adapted strategy
Our WPSS risk-adapted strategy is summarized in
Table 5.
A) Very low WPSS risk
The approach to a patient with myelodysplastic syn-
drome should always begin with a period of observa-
tion, with sequential peripheral blood counts – and
sometimes bone marrow examinations – to assess the
rate of progression, if any. Patients with very low

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14th Congress of the European Hematology Association

Table 4. Drugs and therapeutic tools currently employed in the treatment of myelodysplastic syndromes.

Therapeutic tool Current clinical use Approval by EMEA and FDA (or off-label use)

Allogeneic stem Curative treatment of relatively young Not required

cell transplantation patients with MDS who have a donor

Red cell transfusion Most widely employed treatment of anemia Not required

Iron chelation Treatment of transfusion iron overload Deferoxamine is approved for treatment of transfusion iron overload.
(deferoxamine, Deferasirox has been approved by FDA for first-line treatment
deferasirox) of transfusion iron overload and by EMEA for the treatment of this condition
when deferoxamine therapy is contraindicated or inadequate

Recombinant human Employed for treatment of anemia Despite the fact that epoetins are widely employed for treatment of
erythropoietin anemia in MDS patients, this represents an off-label use of these drugs
(different epoetins)

Granulocyte-colony Employed in combination with recombinant The use of G-CSF in the treatment of anemia in MDS patients
stimulating factor human erythropoietin for treatment of anemia. represents an off-label use of this drug
(G-CSF) Occasionally employed for treatment of granulocytopenia

Anti-thymocyte Immunosuppressive therapy in patients This represents an off-label use of both ATG and cyclosporine A
globulin (ATG) with hypocellular myelodysplastic syndrome
and cyclosporine

Lenalidomide Treatment of transfusion dependent patients
with myelodysplastic syndrome associated with deletion (5q)
In 2005, the FDA approved lenalidomide for the treatment of patients with
transfusion-dependent anemia due to low- or intermediate-1-risk MDS
associated with a deletion 5q cytogenetic abnormality with or without
additional cytogenetic abnormalities. In 2008, the EMEA Committee for
Medicinal Products for Human Use (CHMP) adopted a negative opinion,
recommending the refusal of marketing authorization for lenalidomide,
intended for treatment of transfusion-dependent patients with MDS
associated with del(5q).The EMEA CHMP concluded that the safety of
lenalidomide was difficult to assess, and that, in particular, it was difficult
to determine whether treatment with this drug increased the risk of
progression to acute myeloid leukemia

Azacitidine Employed mainly for treatment of patients with high-risk MDS FDA has approved azacitidine for the treatment of patients with the following
FAB MDS subtypes: refractory anemia (RA) or refractory anemia with ringed
sideroblasts (RARS) (if accompanied by neutropenia or thrombocytopenia or
requiring transfusions), refractory anemia with excess blasts (RAEB),
refractory anemia with excess blasts in transformation (RAEB-T), and
chronic myelomonocytic leukemia (CMMoL).
EMEA has approved azacitidine for the treatment of adult patients who are
not eligible for hematopoietic stem cell transplantation with:
- intermediate 2 and high risk MDS according to the IPSS;
- chronic myelomonocytic leukemia with 10-29% marrow blasts without
myeloproliferative disorder;
- acute myeloid leukemia with 20-30% blasts and multilineage dysplasia,
according to the WHO classification

Decitabine Employed mainly for treatment of patients with high-risk MDS FDA has approved decitabine for the treatment of patients with MDS
including previously treated and untreated, de novo and secondary MDS of
all FAB subtypes (RA, RARS, RAEB, RAEB-t, and CMML) and IPSS
intermediate-1, intermediate-2, and high-risk

| 184 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

Table 5. WPSS risk-adapted treatment strategy for
myelodysplastic syndromes.
WPSS risk-group defined Therapeutic options
as indicated in Table 2
Very low risk Watchful-waiting strategy
Low risk Patients with multilineage dysplasia or intermediate
risk cytogenetics without symptomatic cytopenia may
be just followed without any specific treatment
(watchful-waiting strategy)
Transfusion-dependent patients can be treated with
recombinant human erythropoietin: responsive
subjects are those with inadequate endogenous
erythropoietin productions (serum erythropoietin levels
lower than 100 to 200 mU/mL) and low need for
blood transfusion (less than 2 units per month).
Patients who fail to respond to recombinant human
erythropoietin may be considered for more aggressive
treatments (allogeneic stem cell transplantation and
azacitidine). Those who are given regular blood
transfusion should also receive iron chelation therapy
Transfusion-dependent patients with myelodysplastic
syndrome associated with deletion (5q) may respond
to lenalidomide treatment with abolishment of trans-
fusion requirement. In Europe, however, this not only
represents an off-label use of this drug but more
importantly a use that contrasts with the negative
opinion of EMEA (related to the currently uncertain
safety profile of lenalidomide in this condition)
Patients with hypoplastic myelodysplastic syndrome
under the age of 60 years may be treated with
anti-thymocyte globulin of horse origin (h-ATG) and
cyclosporine A
Intermediate, high Fit patients aged up to 65 years with intermediate to
and very high risk very high WPSS risk are candidates for allogeneic stem
cell transplantation
Patients who are not eligible for allogeneic stem cell
transplantation may be treated with azacitidine
Patients who are not eligible for allogeneic stem cell
transplantation and/or do not respond to azacitidine
may be considered for experimental treatments
(clinical trials)
Patients who do not fit into previous groups are given
supportive therapy (regular red cell transfusion)
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 185 |
Berlin, Germany, June 4-7, 2009
WPSS risk include those with refractory anemia, or
refractory anemia with ringed sideroblasts, or
myelodysplastic syndrome with isolated del(5q) and
no transfusion requirement. Their median life
expectancy is greater than 10 years, and as long as the
disease remains stable, is not significantly different
than that of the general population;14 these patients do
not need any treatment and can be followed regularly.
B) Low WPSS risk, including 5q- syndrome and hypoplas-
tic myelodysplastic syndrome
These patients may belong to this risk group (score
1) because of one of the following features: multilin-
eage dysplasia, intermediate risk cytogenetics, or
transfusion dependency.
Patients with multilineage dysplasia or intermediate
risk cytogenetics without symptomatic cytopenia may
be just followed without any specific treatment.
However, frequent controls (every 2 to 3 months) are
needed in these patients, as the 1-year risk of disease
progression is about 20-25%.
Transfusion dependent patients with low WPSS risk
can be treated with recombinant human erythropoi-
etin. Responsive patients are mainly those with early
disease, inadequate endogenous erythropoietin pro-
duction (serum erythropoietin levels lower than 100 to
200 mU/mL) and a low need for blood transfusion (less
than 2 units per month).
The issue of transfusion-dependent patients with
myelodysplastic syndrome associated with deletion
(5q) has been examined in detail elsewhere.17 In
December 2005, the US Food and Drug Administration
(FDA) approved lenalidomide for the treatment of patients
with transfusion-dependent anemia due to low- or intermedi-
ate-1-risk myelodysplastic syndromes associated with a dele-
tion 5q cytogenetic abnormality with or without additional
cytogenetic abnormalities. In a substantial portion of
patients with myelodysplastic syndrome and del(5q),
studies by List et al.18,19 indeed showed that lenalido-
mide is able to induce a cytogenetic remission and to
abolish transfusion requirement. On January 24, 2008,
the EMEA Committee for Medicinal Products for
Human Use (CHMP) adopted a negative opinion, rec-
ommending the refusal of the marketing authorization
for lenalidomide intended for the treatment of anemia
due to myelodysplastic syndromes, more specifically
for the treatment of transfusion-dependent patients
with MDS associated with del(5q), and with a low to
intermediate risk of progressing to leukemia or death.20
Following the applicant’s request for a re-examination
of the opinion, the CHMP confirmed the refusal of the
marketing authorization on May 30, 2008. The CHMP
concluded that the safety of lenalidomide was difficult
to assess; in particular because the study did not com-
pare the medicine with any other treatment, it was dif-
ficult to determine if treatment with this drug
increased the risk of progression to acute myeloid
leukemia. In conclusion, the CHMP was of the opinion
that the benefits of lenalidomide in the treatment of
anemia of MDS with del(5q) did not outweigh its
potential risks. Thus, in Europe, the administration of
lenalidomide to patients with myelodysplastic syn-
drome associated with del(5q) not only represents an
off-label use of this drug, but also clashes with the
14th Congress of the European Hematology Association
European authority’s opinion that the benefits of this
treatment do not outweigh its potential risks. In our
opinion, this places an enormous responsibility on the
prescribing physician’s shoulders and prevents any rec-
ommendations being made.
In patients with hypoplastic myelodysplastic syn-
drome, combined treatment with anti-thymocyte
globulin of horse origin (h-ATG) and cyclosporine A
may be considered, especially in those under the age of
60 years. About one third of these patients may obtain
a durable response.21
C) Intermediate, high or very high WPSS risk
At present, the only treatment that can cure a patient
with myelodysplastic syndrome is allogeneic stem cell
transplantation.22,23 It is estimated that approximately
one third of patients receiving an allogeneic transplan-
tation are cured with this treatment, but only about 8
to 10% of all patients are eligible and have a donor.1
The decision to perform allogeneic stem cell transplan-
tation can be based on WPSS risk, comorbidities, age,
and should be shared as much as possible with the
patient, whose risk aversion should be taken into
account. Fit patients aged up to 65 years with interme-
diate to very high WPSS risk are candidates for allo-
geneic stem cell transplantation. In a recent report,23 5-
year overall survival after transplantation was 63%,
40%, and 16% for the intermediate, high and very
high-risk groups, respectively. This may suggest that
patients who are eligible for allogeneic stem cell trans-
plantation should be transplanted before they progress
to the very high-risk group. In the estimation of the
individual risk/benefit balance for allogeneic stem cell
transplantation, besides defining disease-related and
demographic variables, a critical task is identifying and
assessing comorbidities that have a substantial impact
on non-relapse mortality. A Hematopoietic Cell Trans-
plantation-specific Comorbidity Index (HCT-CI) for
predicting risk of non-relapse mortality has been
developed and validated.24
DNA hypermethylation may play a role in the pa-
thogenesis of myeloid neoplasms.25 The DNA hypo-
methylating pyrimidine analogues 5-azacytidine might
reduce hypermethylation and induce re-expression of
key tumor suppressor genes. However, the action
mechanism of azacitidine in myelodysplastic syn-
dromes remains uncertain, and might be in part cyto-
toxic.
A prospective randomized trial has shown that
treatment of patients with myelodysplastic syndrome
with 5-azacytidine resulted in significantly higher
response rates, improved quality of life, reduced risk
of leukemic transformation, and improved survival
compared with supportive care.26,27 On May 19, 2004
the U.S. Food and Drug Administration approved
azacitidine as injectable suspension for the treatment
of patients with myelodysplastic syndrome (Table 4).
A survival study in patients with high-risk myelodys-
plastic syndrome comparing azacitidine versus con-
ventional care has been recently completed.28 This
study showed that treatment with azacitidine increas-
es overall survival in patients with higher-risk
myelodysplastic syndromes relative to conventional
care. On December 17, 2008, EMEA approved azaciti-
| 186 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
dine for the treatment of adult patients with high-risk
myelodysplastic syndrome who are not eligible for
allogeneic hematopoietic stem cell transplantation
(Table 4).
Patients who are not eligible for allogeneic stem cell
transplantation and those who do not respond to azac-
itidine should be considered for participation in a clin-
ical trial or given supportive therapy.
For most patients, red cell transfusions eventually
remain the mainstay of therapy. As regards this sup-
portive care, pre-transfusion hemoglobin levels may
range between 7 and 9 g/dL according to the patient’s
performance status and co-morbidities.
Conclusions
In the last few years, our ability to define the prog-
nosis of the individual patient with myelodysplastic
syndrome has improved, and a few drugs have been
approved by FDA and/or EMEA for treatment of MDS.
However, allogeneic stem cell transplantation remains
the only treatment capable of modifying the natural
history of these disorders.
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 Myeloproliferative disorders

The role of somatic and hereditary genetic factors in

the pathogenesis of myeloproliferative neoplasms

R. Kralovics A B S T R A C T

Center for Molecular Medicine
(CeMM), Austrian Academy of
Sciences, Vienna, Austria, and
Department of Internal Medicine I,
Division of Hematology and Blood
Coagulation, Medical University of
Vienna, Vienna, Austria
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
The variable presence of the JAK2 and MPL oncogenic mutations and diverse cytogenetic lesions in
myeloproliferative disorders (MPN) accounts for the genetic heterogeneity among patients and gen-
erates progenitors with different genotypes within each progenitor pool. Mutation acquisition in MPN
appears to be stochastic and mutations seem to be randomly combined during the genetic evolution
of the MPN stem cell clone. Patients with multiple acquisitions of JAK2 or MPL mutations are com-
mon. The currently available data suggests that MPN displays an increased mutational frequency con-
sistent with a mild form of a mutator phenotype. The genetic factors underlying the MPN mutator
phenotype are discussed.

2009;3:188-191

ncreased activity of the myeloid lineages occur in patients with PMF and ET with a

I is the hallmark of myeloproliferative dis-

orders (MPN) phenotypes. Three major
phenotypic subtypes of MPN are distin-
guished according to the predominant pres-
entation of the phenotype. In polycythemia
vera (PV), increased red cell mass, whereas in
essential thrombocythemia (ET), increased
platelet count dominate the phenotype. In
primary myelofibrosis (PMF), fibrosis of the
bone marrow is the phenotype defining dis-
ease feature. Since the clinical presentations
of all three MPN entities often overlap and a
number of reactive condition masquerade as
MPN, precise diagnosis of the individual
MPN entities is often challenging and
requires molecular and histopathological
evaluation of each case. The complications
associated with MPN include thrombosis
and bleeding,1,2 secondary bone marrow
fibrosis, and leukemic transformation.3-6
frequency between 1-9% depending on the
cohort examined.13-17 Up to now, five MPL
variants have been described (MPL-W515L,
MPL-W515K, MPL-S505N, MPL-A506T, and
MPL-A519T), all confined to exon 10 of the
MPL gene. Studies in animal models (bone
marrow transplant or transgenic) have clear-
ly shown that JAK2-V617F, JAK2-ex12, and
MPL-W515L induce a myelopriliferative
phenotype in vivo.7,11,13,18-23 In addition, animal
studies revealed several interesting features
of these mutations. Tiedt et al. observed that
high expression level of the JAK2-V617F
transgene induces a PV like phenotype in
mice, whereas low expression induces an ET
like phenotype.21 In a bone marrow trans-
plant model, Wernig et al. showed that the
myeloproliferative phenotype of mice is
modified depending on the genetic back-
ground of mice.18

Somatic mutations in myeloproliferative Cytogenetic aberrations

disorders
The discovery of a gain-of-function
somatic mutation in the JAK2 gene greatly
enhanced the understanding of the disease
pathogenesis and facilitated MPN diagno-
sis.7-11 The most frequent mutation of JAK2
(JAK-V617F) occurs in exon 14 in over 50%
of MPN cases but it is not restricted to a sin-
gle MPN entity. The less frequent exon 12
mutations (JAK2-ex12) were observed in PV
patients only with a frequency of up to
5%.11,12 Further examination of the
JAK/STAT signaling pathway and cytokine
receptors led to the discovery of various
gain-of-function mutations in the throm-
bopoietin receptor (MPL). MPL mutations
Monoclonal origin of the myeloid lineages
in MPN has been a fundamental part of the
disease pathogenesis and found to be shared
among all three types of MPN.24-28 Only a
proportion of ET patients are exceptions
from this rule.29,30 Some polyclonal ET
patients display positivity for the JAK2-
V617F mutation,31-33 ruling out the possibility
that this patient population represents a phe-
nocopy or an unrecognized reactive condi-
tion.
To explain clonal hematopoiesis in MPN,
extensive cytogenetic studies were under-
taken but no invariant aberration or MPN-
specific defect was found. Instead, cytoge-
netic lesions commonly seen in other

| 188 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

myeloid malignancies were detected, especially dele-
tions of chromosomes 5q, 13q and 20q, numerical aber-
rations of chromosomes 1, 8, 9, and frequent gains of
9p.34-43 Acquired uniparental disomy (or isodisomy) of
the short arm of chromosome (9pUPD) was found to be
a frequent lesion in about one third of PV patients.44
Detailed mapping studies of 9pUPD represented one of
the approaches that led to identification of the JAK2-
V617F mutation in MPN.10
Clonal diversity of the progenitor pool
Variability of oncogenic point mutations and the spo-
radic presence of cytogenetic lesions in MPN patients
create a number of genotypic classes of progenitors.
When in vitro cultured progenitor colonies are geno-
typed for the presence of JAK2-V617F or JAK2-ex12,
three genotypic classes (wild type, heterozygous and
homozygous for the mutation) are often observed in
variable ratios.45-47 The only exception are progenitor
clones from ET patients, in which homozygosity for
JAK2-V617F is rarely observed.45 Similar complexity is
observed in patients carrying cytogenetic aberrations,
such as deletions on chromosome 20q (del20q) and
JAK2 or MPL mutations.47 Schaub et al. observed a vari-
able acquisition order of del20q and JAK2-V617F in
patients carrying del20q and JAK2-V617F simultaneous-
ly.47 An even higher progenitor pool complexity consist-
ing of six different genotypic classes was reported
recently in a patient with del20q, del13q, 9pUPD, and
JAK2-V617F.48 These studies demonstrated that the
acquisition of mutations in MPN does not follow a pre-
determined order but appears to be stochastic.49
Variability of mutational burden
As quantitative assays became available for analysis
of JAK2-V617F mutational burden (mutant allele bur-
den), large differences were observed among patients.
Mutant allele burdens for JAK2-V617F range from 0 to
100% (homozygosity) in PV and PMF whereas patients
with ET carry burdens between 0 and 50% and only
rarely exceeds 50% (heterozygosity). Low JAK2-V617F
mutational burden in ET is largely due to the absence of
9pUPD in these patients.10,45 Another source of muta-
tional burden variability is the differences in population
size of mutant cells and variable ratio of heterozygous
and homozygous cells for JAK2-V617F. Allelelic burden
was shown to influence MPN-specific gene expression
along with a number of clinical variables, such as sec-
ondary fibrosis, thrombosis, hematocrit, leukocyte and
platelet counts.50-54 Presence of 9pUPD resulting in gain
of homozygosity along the short arm of chromosome 9
and gain of homozygosity for JAK2-V617F is the main
cause of high mutational burden in MPN. As a result,
secondary genetic effects might contribute to the phe-
notype of MPN patients with high mutational burden,
including dysregulation of imprinted genes and expres-
sion of rare recessive alleles of genes.49
Hereditary predispositions to myeloproliferative
disorders
Patients with a familial history of MPN are pheno-
typically and molecularly indistinguishable from spo-
radic MPN patients.55-58 All familial MPN cases tested
to date were carrying a somatic JAK2-V617F or JAK2-
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 189 |
Berlin, Germany, June 4-7, 2009
ex12.57,58 Thus, familial MPN exhibits a relatively
strong hereditary predisposition to acquire a disease-
causing somatic mutation, such as JAK2-V617F.
Although the JAK2-V617F mutation was never
observed in the germ line, the MPL-S505N mutation
was found to be both germ line inherited in a Japanese
family with autosomal dominant thrombocytosis59
and acquired somatically in patients with PMF or ET.15
Absence of germ line JAK2-V617F mutations might be
due to reduced embryonic viability observed in JAK2-
V617F transgenic mice.21
Recently, a population based study showed that first
degree relatives of patients with MPN have an
increased risk to develop MPN.60 The same study pro-
posed a recessive mode of inheritance in familial
MPN. The incomplete penetrance observed in MPN
families often makes it difficult to conclude the mode
of inheritance. The majority of familial MPN cases
represent either parent/child pairs or pedigrees with
affected members in each generation.55-58 Although an
autosomal dominant inheritance mode seems to be
more likely than a recessive one, this issue will be
resolved only after identification of the genetic factors
involved.
An interesting feature of MPN pathogenesis
emerged from screening patients for JAK2 and MPL
mutations. In certain cases, patients positive for two
different mutations were reported, for example, JAK2-
V617F and MPL-W515L, JAK2-ex12 and JAK2-V617F,
or two independently occurring JAK2-V617F muta-
tions.14,46,48 Considering the rarity of JAK2 and MPL
mutations in the population, the mathematical likeli-
hood of two such events in the same individual are
extremely low (one to three events in the entire world
population) if the mutagenesis of these genes is ran-
dom. Thus, we cannot rule out the possibility that
MPN exhibits a certain form of “mutator” phenotype
resulting in increased mutation frequency, which
would provide an explanation for the acquisition of
multiple mutations. In an attempt to determine the
frequency of multiple acquisition of JAK2-V617F
among MPN patients, an unequal distribution of
JAK2-V617F mutations was observed between the
two most common JAK2 gene haplotypes.48 One of
the haplotypes harbored 80% of all JAK2-V617F muta-
tions and was found to be a susceptibility locus for the
development of JAK2-V617F positive MPN. 48,61
However, the JAK2 locus was not shown to exhibit
linkage with MPN phenotype in families.55 It, there-
fore, appears unlikely to be responsible for the famil-
ial MPN predisposition, which seems to be stronger
than the effect exerted by the 46/1 or the GGCC MPN
susceptibility haplotype of JAK2.48,61
In conclusion, two types of hereditary predisposi-
tions for MPN were identified so far: one weaker pre-
disposition exerted by a specific JAK2 haplotype and a
stronger predisposition exerted by a yet unknown
mutation responsible for the familial MPN clustering.
Both of these changes represent examples of a germ
line “mutator” effect since they increase the likelihood
of JAK2 mutation acquisition. It remains to be seen
whether a somatic mutation can render a clone more
susceptible to acquire a JAK2 mutation and result in
the development of MPN.
14th Congress of the European Hematology Association
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34. Rege-Cambrin G, Mecucci C, Tricot G, Michaux JL, Louwagie
A, Van Hove W, et al. A chromosomal profile of polycythemia
vera. Cancer Genet Cytogenet 1987;25:233-45.
35. Diez-Martin JL, Graham DL, Petitt RM, Dewald GW.
Chromosome studies in 104 patients with polycythemia vera.
Mayo Clinic Proc 1991;66:287-99.
36. Mertens F, Johansson B, Heim S, Kristoffersson U, Mitelman F.
Karyotypic patterns in chronic myeloproliferative disorders:
report on 74 cases and review of the literature. Leukemia
1991;5:214-20.
37. Reilly JT, Snowden JA, Spearing RL, Fitzgerald PM, Jones N,
Watmore A, et al. Cytogenetic abnormalities and their prog-
nostic significance in idiopathic myelofibrosis: a study of 106
cases. Br J Haematol 1997;98:96-102.
38. Kanfer E, Price CM, Colman SM, Barrett AJ. Erythropoietin-
independent colony growth in polycythaemia vera is not
restricted to progenitor cells with trisomy of chromosome 8.
Br J Haematol 1992;82:773-4.
39. Westwood NB, Gruszka-Westwood AM, Pearson CE, Delord
CF, Green AR, Huntly BJ, et al. The incidences of trisomy 8,
trisomy 9 and D20S108 deletion in polycythaemia vera: an
analysis of blood granulocytes using interphase fluorescence
in situ hybridization. Br J Haematol 2000;110:839-46.
40. Najfeld V, Montella L, Scalise A, Fruchtman S. Exploring poly-
cythaemia vera with fluorescence in situ hybridization: addi-
tional cryptic 9p is the most frequent abnormality detected. Br
J Haematol 2002;119:558-66.
41. Sinclair EJ, Forrest EC, Reilly JT, Watmore AE, Potter AM.
Fluorescence in situ hybridization analysis of 25 cases of idio-
pathic myelofibrosis and two cases of secondary myelofibro-
sis: monoallelic loss of RB1, D13S319 and D13S25 loci associ-
ated with cytogenetic deletion and translocation involving
13q14. Br J Haematol 2001;113:365-8.
42. Bench AJ, Nacheva EP, Champion KM, Green AR. Molecular
genetics and cytogenetics of myeloproliferative disorders.
Baillieres Clin Haematol 1998;11:819-48.
43. Gangat N, Strand J, Lasho TL, Finke CM, Knudson RA, Parda-

nani A, et al. Cytogenetic studies at diagnosis in polycythemia
vera: clinical and JAK2V617F allele burden correlates. Eur J
Haematol 2008;80:197-200.
44. Kralovics R, Guan Y, Prchal JT. Acquired uniparental disomy
of chromosome 9p is a frequent stem cell defect in poly-
cythemia vera. Exp Hematol 2002;30:229-36.
45. Scott LM, Scott MA, Campbell PJ, Green AR. Progenitors
homozygous for the V617F mutation occur in most patients
with polycythemia vera, but not essential thrombocythemia.
Blood 2006;108:2435-7.
46. Li S, Kralovics R, De Libero G, Theocharides A, Gisslinger H,
Skoda RC. Clonal heterogeneity in polycythemia vera patients
with JAK2 exon12 and JAK2-V617F mutations. Blood 2008;
111:3863-6.
47. Schaub FX, Jager R, Looser R, Hao-Shen H, Hermouet S,
Girodon F, et al. Clonal analysis of deletions on chromosome
20q and JAK2-V617F in MPD suggests that del20q acts inde-
pendently and is not one of the pre-disposing mutations for
JAK2-V617F. Blood 2008;113:2022-7.
48. Olcaydu D, Harutyunyan A, Jäger R, Berg T, Gisslinger B,
Pabinger I, et al. A common JAK2 haplotype confers suscepti-
bility to myeloproliferative neoplasms. Nat Gen 2009;AOP.
49. Kralovics R. Genetic complexity of myeloproliferative neo-
plasms. Leukemia 2008;22:1841-8.
50. Kralovics R, Teo SS, Buser AS, Brutsche M, Tiedt R, Tichelli A,
et al. Altered gene expression in myeloproliferative disorders
correlates with activation of signaling by the V617F mutation
of Jak2. Blood 2005;106:3374-6.
51. Antonioli E, Guglielmelli P, Poli G, Bogani C, Pancrazzi A,
Longo G, et al. Influence of JAK2V617F allele burden on phe-
notype in essential thrombocythemia. Haematologica 2008;
93:41-8.
52. Vannucchi AM, Antonioli E, Guglielmelli P, Pardanani A,
Tefferi A. Clinical correlates of JAK2V617F presence or allele
burden in myeloproliferative neoplasms: a critical reappraisal.
Leukemia 2008;22:1299-307.
53. Vannucchi AM, Antonioli E, Guglielmelli P, Longo G,
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Berlin, Germany, June 4-7, 2009
Pancrazzi A, Ponziani V, et al. Prospective identification of
high-risk polycythemia vera patients based on JAK2(V617F)
allele burden. Leukemia 2007;21:1952-9.
54. Vannucchi AM, Antonioli E, Guglielmelli P, Rambaldi A,
Barosi G, Marchioli R, et al. Clinical profile of homozygous
JAK2 617V>F mutation in patients with polycythemia vera or
essential thrombocythemia. Blood 2007;110:840-6.
55. Kralovics R, Stockton DW, Prchal JT. Clonal hematopoiesis in
familial polycythemia vera suggests the involvement of multi-
ple mutational events in the early pathogenesis of the disease.
Blood 2003;102:3793-7.
56. Kralovics R, Skoda RC. Molecular pathogenesis of Phila-
delphia chromosome negative myeloproliferative disorders.
Blood Rev 2005;19:1-13.
57. Bellanne-Chantelot C, Chaumarel I, Labopin M, Bellanger F,
Barbu V, De Toma C, et al. Genetic and clinical implications of
the Val617Phe JAK2 mutation in 72 families with myeloprolif-
erative disorders. Blood 2006;108:346-52.
58. Rumi E, Passamonti F, Pietra D, Della Porta MG, Arcaini L,
Boggi S, et al. JAK2 (V617F) as an acquired somatic mutation
and a secondary genetic event associated with disease pro-
gression in familial myeloproliferative disorders. Cancer 2006;
107:2206-11.
59. Ding J, Komatsu H, Wakita A, Kato-Uranishi M, Ito M, Satoh
A, et al. Familial essential thrombocythemia associated with a
dominant-positive activating mutation of the c-MPL gene,
which encodes for the receptor for thrombopoietin. Blood
2004;103:4198-200.
60. Landgren O, Goldin LR, Kristinsson SY, Helgadottir EA,
Samuelsson J, Bjorkholm M. Increased risks of polycythemia
vera, essential thrombocythemia, and myelofibrosis among
24577 first-degree relatives of 11039 patients with myelopro-
liferative neoplasms in Sweden. Blood 2008;112:2199-204.
61. Jones AV, Chase A, Silver RT, Oscier D, Zoi K, Wang YL, et al.
JAK2 haplotype is a major risk factor for the development of
myeloproliferative neoplasms. Nat Genet 2009;41:446-9.
 Myeloproliferative disorders

Risk adapted approach to the treatment of primary

myelofibrosis

J. Mascarenhas
R. Hoffman
Tisch Cancer Institute, Mount Sinai
School of Medicine, New York, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:192-199
A B S T R A C T
Primary myelofibrosis is a clonal hematopoietic stem cell disorder associated with characteristic
clinical histopathologic and hematologic features. However, there is heterogeneity with regard to clin-
ical manifestations, clinical course and overall survival. It is paramount that patients be correctly
stratified according to their risk of developing life threatening outcomes based on accepted prognos-
tic features in order that treatment plans be developed to favor the benefit versus risk of a selected
therapy for an individual patient. With advances in the understanding of the molecular underpinnings
of the myeloproliferative neoplasms, the therapeutic options for such patients have greatly increased.
Treatment approaches range from watchful waiting, palliative/supportive care, implementation of rec-
ognized therapeutic treatments, testing experimental small molecule therapies and pursuing poten-
tially curative allogeneic stem cell transplantation. These alternative approaches available to the cli-
nician treating PMF require decision-making based on an understanding of the biology of the disease,
risk stratification, and the potential benefits as well as risks of each of these choices.

rimary myelofibrosis, PMF, is a ence of either a WBC greater than 30×106/µL

P Philadelphia chromosome negative

myeloproliferative neoplasm (MPN). In
2008, the World Health Organization
(WHO) revised the system for classifying
myeloid malignancies, changing the term
myeloproliferative disease or disorder to neo-
plasm. Primary myelofibrosis is one of the
nine MPN. The diagnosis of PMF requires
meeting all three major and at least two of
the minor diagnostic criteria (Table 1).1
Within the umbrella of MPN, the clinical fea-
tures of PMF are virtually identical to those
of patients with post polycythemia vera
myelofibrosis (post-PV MF) and post essen-
tial thrombocythemia myelofibrosis (post-
ET MF). Diagnostic criteria for these entities
have also been created by an International
Working Group on Myelofibrosis Research
and Treatment (IWG-MRT).2 It has been the
practice of most investigators to apply simi-
lar therapeutic strategies to patients suffer-
ing from each of these entities.
Risk stratification
A number of reports have identified risk
factors predictive of survival in PMF
patient.3-10 Progressive marrow failure, trans-
formation to acute myeloid leukemia and
the consequences of portal hypertension are
the major causes of death in PMF.3 After test-
ing the predictive value of a number of these
risk factors, prognostic scoring systems have
been created with which to predict survival.
Such systems are extremely valuable in
determining appropriate therapeutic strate-
gies to be used for the treatment of individ-
ual patients. The most widely used system is
the Lille scoring system, which classifies
patients into three groups based on the pres-
or less than 4 ×106/µL, and a hemoglobin less
than 10 g/dL.3 A point is given for each of
these risk factors to create a Lille score of
either 0, 1 or 2. Patients with Lille class 0, 1
or 2 disease have median survivals of 93, 26
and 13 months, respectively. Recently, the
IWG-MRT has reported a study assessing
prognostic variables in over 1000 PMF
patients from seven institutions and found
an overall median survival of 69 months.4 An
age greater than 65, presence of constitution-
al symptoms, hemoglobin less than 10 g/dL,
leukocytosis more than 25×106/µL and cir-
culating peripheral blood blast count greater
than or equal to 1% were all identified as
negative risk factors by multivariate analy-
sis. Four risk groups with distinct patterns of
survival were identified. Patients were classi-
fied as low risk, intermediate-1, intermedi-
ate-2, and high risk with median survivals of
135, 95, 48 and 27 months, respectively.
Importantly, JAK2V617F mutational status
was not associated with a particular prog-
nostic score or survival time. It is important
to emphasize that both the Lille and the
IWG-MRT prognostic scoring systems are
predictive of survival based on clinical fea-
tures at the time of diagnosis. Whether these
criteria are of use as a patient’s clinical course
changes over time and places the patient in a
different prognostic group remains
unknown. Although this is common prac-
tice, this approach has never been validated.
Whether these scoring systems are equally
effective in predicting the outcome of
patients with post-PV/ET MF has also not
been well documented. The median overall
survival of patients with post-PV/ET MF has
been reported to be 90 months.11 Older age,

| 192 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

anemia and prior history of PV have been reported to be

Table 1. Proposed criteria for primary myelofibrosis.
adverse prognostic characteristics. The presence of
unfavorable cytogenetic abnormalities (clones other
than 13q- or 20q-) at time of transformation from PV/ET
to MF has been identified as the most significant
adverse prognostic factor for survival of this patient
population.11
The relative utility of the Lille and IWG-MRT scoring
systems in clinical practice has yet to be determined.
During the remainder of this text, we will rely on the
Lille system for prognostication and risk stratification
for defining appropriate treatment plans.

Treatment
We hypothesize that the treatment of PMF patients
should be tailored to the individual patient based on the
risk stratification tools described. For the purposes of
this discussion and acknowledging that the various
prognostic scoring systems have never been compared
in a prospective fashion, we have chosen to use to the
Lille classification system to outline a risk based treat-
ment approach in PMF. There are no randomized trials
available at present to provide data, which could be
used to construct an evidence based algorithm for the
treatment of PMF. The recommendations made in this
manuscript represent the opinions of the authors and
the cumulative experience reported in the medical liter-
ature.

Standard of care
The present strategies employed as the standard of
care for patients with PMF are directed toward treating
the clinical symptoms and laboratory abnormalities as
they affect the quality of life of an individual patient.
None of these interventions is known to alter the natu-
ral history of this disease.
Anemia
Symptomatic patients with MF frequently present
with anemia and require intervention. It is important
that B12, folate, and iron deficiency as possible contrib-
utory factors be identified and corrected. Beyond trans-
fusional support for the acutely symptomatic patient,
various erythropoiesis stimulating agents (ESA) have
been used to minimize or eliminate the need for trans-
fusion therapy. The role of recombinant erythropoietin
(rEPO) in the amelioration of anemia and the associated
symptoms is somewhat controversial. Studies favoring
the use of rEPO suggest that MF patients with low
serum erythropoietin levels, limited transfusional
requirements, female sex and older age are most likely
to respond to therapy.12 In a study of 61 patients with
MF, 87% had serum erythropoietin levels appropriate to
the degree of anemia, supporting the concept that rEPO
would unlikely be beneficial to the majority of anemic
MF patients.13 However, a 40 to 55% overall response
rate using darbepoetin or rEPO in MF has been reported
in the literature. ESA should remain the first line thera-
py for anemic patients with serum erythropoietin levels
less than 125 U/L at a rEPO dose of at least 350
U/kg/week.13-15 Concomitant interferon alpha therapy
with rEPO has been associated with unexpected favor-
able response rate which deserves further prospective
evaluation.16
Androgen therapy has been shown to be an effective
class of agents in the treatment of anemia in MF.
Danazol, a synthetic attenuated androgen, given at an
initial dose of 600 mg/day and tapered to a minimum
effective dose at 6 months was associated with a
response rate of 37 to 55% with a median time to
response between 3 to 6 months.17,18 The absence of a
transfusion requirement and higher hemoglobin prior to
initiation of danazol were associated with a favorable
response to therapy. The most frequent toxicity of
transaminitis improved with dose reduction.17
An unexpected finding in a retrospective review of
311 PMF patients treated at the Mayo Clinic found that
prior therapy with ESA or danazol was associated with
a higher risk for leukemic transformation (LT).19 The
contention that the use of an ESA may, in fact, act as a
surrogate marker for a more aggressive form of PMF
may not be valid considering that other agents
employed to treat anemia were not found to be associ-
ated with LT. It remains uncertain whether ESA therapy
has an effect of the evolution of such patients to acute
leukemia.
Occasionally chemotherapeutic agents can alleviate
the degree of anemia in PMF patients. Oral melphalan,
for instance, has been reported to result in 60% of
severely anemic patients without transfusion require-
ments having a significant elevation of hemoglobin
while nearly 38% of transfusion dependent patients
becoming transfusion independent with such therapy.20
Based on the anti-angiogenic and immunomodulato-
ry activity of thalidomide, this agent has been used
alone and in combination with prednisone to treat PMF
patients with anemia. The use of this agent is tempered

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 193 |
14th Congress of the European Hematology Association
by a toxicity profile of sedation, constipation, neuropa-
thy, myelosuppression and increased thrombotic poten-
tial. A pooled-analysis of 5 phase II studies in MF using
standard dose thalidomide showed a reduction in 6
point severity score in 45% of patients, and worsening of
this score in 20%, with 66% discontinuing treatment
before 6 months due to intolerance of therapy.21 In a ran-
domized multicenter phase II B setting in MF, thalido-
mide at 400 mg daily failed to reach endpoints of
improving anemia and reducing transfusion require-
ments.22 Low dose thalidomide at 50 mg/day, combined
with a prednisone taper, demonstrated an objective clin-
ical response in 62% of patients with anemia with 95%
of patients able to complete 3 months of therapy.23,24
More recently, lenalidomide, a more potent thalidomide
analogue with greater myelosuppressive effects and less
sedative and neurotoxic effects has been explored in
clinical trials for MF. Phase II studies with lenalidomide
as a single agent in MF were initially seen to produce
overall response rates of 22%, 33%, and 50% for ane-
mia, splenomegaly and thrombocytopenia, respective-
ly.24,25 As expected, grade 3/4 neutropenia and thrombo-
cytopenia were observed, but increased thromboembol-
ic events were not seen. In a cooperative group study,
the combination of prednisone and lenalidomide did
not improve the response rate for anemia as compared
to lenalidomide alone. Lenalidomide therapy was com-
plicated by grade 3/4 myelosuppression in 45% of
cases.25 Given the degree of myelosuppression observed
in this study and the comparable response rates seen
with thalidomide therapy, these investigators conclud-
ed that lenalidomide should be reserved as second line
therapy for anemic patients that fail thalidomide thera-
py or posses the 5q- abnormality. Pomalidomide, CC-
4047, a newer IMiD, which has far less neurotoxicity is
being investigated in early clinical trials in patients with
MF. Pomalidomide alone or in combination with short
course prednisone showed a 25% response rate for ane-
mia and increased the platelet count by more than 50%
in 43% of patients with platelet counts less than
100×109/L.26 WBC less than 4×109/L were the most pow-
erful predictor of response but no patients experienced
a significant reduction in splenomegaly. It is sobering
that in this trial, a cohort of patients received pred-
nisone therapy alone, which was associated with a 19%
response rate in anemic patients. We conclude that
pomalidomide requires further careful evaluation.
There are no randomized trials specifically investigat-
ing the benefit of iron chelation therapy in PMF. Case
reports indicate that effective reduction in ferritin levels
can be achieved and additionally, this may enhance ery-
thropoiesis.27-29 A very recent analysis of transfusion
dependent PMF patients receiving iron chelation thera-
py, demonstrated a survival benefit favoring chelation
therapy over those not receiving chelation therapy.30
Based on the available data in PMF, iron chelation ther-
apy should be considered in transfusion dependent
patients with a ferritin more than 1000 and an extended
expected survival.
Organomegaly
Splenomegaly is a frequent complication of PMF,
which is associated with a hypercatabolic state and con-
stitutional symptoms that compromise the patient’s
| 194 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
quality of life (QOL). Hydroxyurea is a ribonucleotide
reductase inhibitor with cytoreductive potential with
the advantage of relieving constitutional symptoms and
reversing advancing splenomegaly in MF. Hydroxyurea
has not been shown to have any advantage in selective-
ly decreasing the malignant clone as assessed by
JAK2V617F allele burden31 and, therefore, does not hold
potential for modifying the natural history of this dis-
ease. The success of hydroxyurea in effectively control-
ling splenomegaly has not been well documented in the
literature. Its use is largely empiric and frequently com-
plicated by excessive hematopoietic toxicity, often
requiring the use of alternative strategies, including
splenectomy.
Splenectomy is associated with long-term improve-
ment in symptomatic splenomegaly, anemia, portal
hypertension, and severe thrombocytopenia in approx-
imately 80%, 50%, 40% and 30% of cases, respective-
ly.32 Splenectomy is not always a viable option in
patients that have a poor performance status and carries
a risk of postoperative bleeding of 14%, infection of
10%, and thrombosis of 10% that contribute to an over-
all fatal complication rate of approximately 7%.32-34 An
alternative approach is low dose radiation to the spleen,
which can potentially result in a transient reduction in
spleen size and improvement in symptoms in patients
that are considered poor candidates for splenectomy.
Rebound thrombocytosis and compensatory hepato-
megaly also complicate splenectomy, and postoperative
thrombocytosis can increase the risk of perioperative
thrombosis and compromise survival.34 A post splenec-
tomy rate of LT has been reported of 16.3% and the risk
of LT appears to be highest in MF patients with marked-
ly increased spleen size and thrombocytopenia prior to
surgery.34 Most patients require hydroxyurea therapy to
prevent postoperative compensatory hepatomegaly. In
patients resistant to hydroxyurea, 2-chlorodeoxyadeno-
sine (2-CdA), a purine nucleoside analogue has been
shown to decrease hepatomegaly and thrombocytosis
in post-splenectomy MF patients in a pilot study of nine
patients.35
Experimental therapies
Most clinical trials for patients with advanced forms
of MF do not discriminate between patients with PMF
or post-PV/ET MF. These clinical trials are designed for
the enrollment of intermediate/high risk or sympto-
matic patients. Response criteria created by the IWG-
MRT and the European Myelofibrosis Network (EUM-
NET) have enabled standardized evaluation of these
agents by objective endpoints.36,37
JAK2 inhibitors
JAK2 inhibitors are a class of small molecules that
block the JAK-STAT pathway resulting from the
JAK2V617F gene mutation, and to some degree, wild
type JAK2. Clinical trials with several candidate mole-
cules, including Cep-701, XL019, INCB018424,
TG101348 have been pursued. Each trial has reported
some success in reaching clinical endpoints, including
reduction in spleen size and improvement in constitu-
tional symptoms.38-40 The excitement about JAK2V617F
inhibition has been tempered by the realization that a
significant diminution in mutant allele burden or

improvement in the degree of anemia has been rarely
observed.38 The Myelofibrosis Symptom Assessment
Form (MFSAF) is a proposed new instrument designed
to better capture the disease related symptoms and
assess the QOL and can be utilized as a metric in future
therapeutic trials.41 These JAK2 inhibitors may also play
an important role in reversing anorexia and cancer
cachexia by suppressing the pro-inflammatory cytokine
state, reducing splenomegaly and reversing the hypo-
cholesterolemia characteristic of PMF.42
MF is a chronic inflammatory state with marked ele-
vation in pro-inflammatory cytokines irrespective of the
JAK2 status or degree of splenomegaly. INCB18424 has
been shown to dramatically and quickly reduce these
levels, which has been shown to correlate with
improvement in constitutional symptoms and cachex-
ia.43 Myelosuppression, gastrointestinal toxicity and
neurotoxicity have been reported with the use of specif-
ic JAK2 inhibitors. Although the primary effect of these
agents appears to be on hematopoiesis, JAK-STAT sig-
naling plays a role in the biological regulatory mecha-
nisms involving a variety of other organ systems, which
could account for the toxicities observed. Since the
presently evaluated inhibitors are not specific to the
mutated from of JAK2, these toxicities are not surpris-
ing. At this time, the relative clinical efficacy of each of
the JAK2 inhibitors has not been compared, thereby
making it impossible to identify the most effective
member of this class of drugs.
Chromatin modifying agents
The epigenetic silencing of genes through DNA
hypermethylation and histone deacetylation likely
plays a role in the pathogenesis of PMF and provides a
different set of therapeutic targets.44 The nucleoside ana-
logue 5-azacitidine (5-AZA), a DNA methyltransferase
inhibitor with proven clinical efficacy in MDS, had a
response rate of 24% in MF patients.45 5-AZA given
with an alternative dosing schedule for 5 days was also
attempted and failed to achieve even a single clinical
response.46 Decitabine in case reports47 and in our hands
(unpublished data) has demonstrated clinical activity
resulting in significant reduction in splenomegaly,
improvement in the degree of anemia and constitution-
al symptoms, and even a durable CR in a single patient
in LT. Myelosuppression is the major adverse event that
has been encountered. The sequential exposure of PMF
CD34+ cells in vitro to Decitabine and trichostatin A has
been reported to result in a reduction in total number of
assayable cells, CD34+ cells, hematopoietic progenitor
cells (HPC), and selective reduction in JAK2V617F+
HPCs and HPCs containing cytogenetic abnormalities.48
The sequential exposure of these agents also upregulat-
ed CD34+ CXCR4 expression, restoring the ability of
CD34+ PMF cells to migrate in response to the SDF-1.
These data provide a very compelling rationale to eval-
uate sequential chromatin modifying agents in future
clinical trials of PMF patients.
ITF2357, a class I/II HDAC inhibitor, was investigat-
ed in PV/ET and MF patients in a phase IIa study, result-
ing in a median reduction of JAK2V617F mutant allele
burden of 8% in 10 of 17 patients. Two major and two
moderate clinical responses were achieved by EUMNET
criteria in the 13 MF patients treated.49 The pan HDAC
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 195 |
Berlin, Germany, June 4-7, 2009
inhibitor, LBH589 is also being evaluated in phase I/II
studies in MF based on encouraging data using this oral
drug in a trial of patients with advanced hematological
malignancies. Two PMF patients in these trials experi-
enced a reduction in the degree of splenomegaly, while
one achieved transfusion independence.50
Anti-angiogenic agents
The tyrosine kinase (TK) associated with the VEGFR
is another potential PMF therapeutic target, since this
entity is associated with increased marrow micro-vessel
density and increased serum VEGF levels. SU5416 is an
intravenous small molecule TKI that can inhibit VEGFR,
among other TKs, and has been tested in refractory
MPNs, including a phase II study, which included three
PMF patients with one patient obtaining a partial
response.51 PTK787/ZK 222584 is a novel oral small mol-
ecule inhibitor of the VEGFR tyrosine kinase that was
shown in a pilot study of 29 MF patients to result in a
CR in a single patient and clinical improvement (CI) in
five patients.52 Bevacizumab, a recombinant humanized
monoclonal antibody against VEGF, is also currently
being investigated in a phase II trial. These agents will
likely have more clinical relevance in combination ther-
apy with other agents.
Aplidin, a marine-derived depsipeptide with both
anti-apoptotic and anti-angiogenic properties, has been
shown to reduce BM MVD in a murine MF model
through downregulation of VEGF and TGFβ and may
even impact megakaryocyte proliferation and the for-
mation of reticulin fibrosis.53 This drug will soon enter
clinical trials in MF.
Interferon
Recombinant interferon (IFN) has both anti-tumor
and immunomodulatory activity and has been investi-
gated extensively in various formulations in the treat-
ment of MPNs.54 Pegylated interferon alpha is produced
by covalent addition of ethylene glycol polymers to
rIFNα, resulting in a decreased immunogenicity and an
increased half-life due to decreased clearance. Both
therapy with rIFNα and PEG-IFN have not enjoyed the
same favorable clinical response rates in PMF as has
been achieved in PV and ET patients.54 Some investiga-
tors have suggested that treatment with IFN during
early phases of the disease, so called pre-fibrotic form,
might delay disease progression.55 Such an approach
will require testing in a randomized trial before it can be
implemented.
Combination therapy
Combination therapy in cancer treatment is a stan-
dard approach based upon the use of drugs that affect
different targets and at the same time have non-overlap-
ping organ toxicities. Such an approach has not been
previously explored in PMF. Since none of the standard
therapeutic options or experimental therapies outlined
above has similar therapeutic effectiveness as displayed
by imatinib, the paradigm of CML might not be appli-
cable to PMF. These obstacles have lead several investi-
gators to explore the use of combination therapy for
PMF. For instance, a combination of the pan HDAC
inhibitor LBH589 or heat shock protein (HSP90)
inhibitor AU922 with the JAK2 inhibitor TG101209 in
14th Congress of the European Hematology Association
primary MPD cells or JAK2V617F+ HEL cell line showed
enhanced and even synergistic levels of apoptosis and
attenuation of JAK2V617F, p-STAT-3, p-STAT-5, p-AKT,
Bcl-xl levels.56 In addition, upregulation of anti-apoptot-
ic proteins in erythroblasts and megakaryocytes have
been observed in MPN, suggesting to some that selec-
tive inhibitors of these pathways might be effective
components of a combination drug therapy for PMF.
These encouraging laboratory findings will surely be
followed by in vivo pilot studies attempting to improve
upon the current success seen with JAK2 inhibitors
alone in MF.
Stem cell transplantation
The only known curative approach for PMF is allo-
geneic transplantation (SCT). The first step is determin-
ing who is an appropriate candidate for SCT and the
second is determining the optimal time to proceed. The
effective completion of these decision-making process-
es requires an appreciation of the risks involved in the
procedure and the risk of disease evolution to a terminal
event. For the purposes of this discussion, we will use
the Lille prognostic score system to risk stratify in order
to optimize the application of SCT in MF patients. It
must be emphasized that, although very encouraging,
this modality of treatment remains experimental at this
time and that patients who are interested in being eval-
uated for SCT would likely be best served by participat-
ing in a clinical trial. Due to the high transplant related
mortality (TRM) (27-48%) and poor overall survival
(OS) (41-64%) associated with myeloablative SCT
(MA-SCT) in a disease that preferentially affects those
older than 45 years of age, reduced intensity condition-
ing SCT (RIC-SCT) offers a reduced treatment related
mortality (TRM) (10%). It has a favorable complete
remission rate (CR) and overall survival (OS) of 76%
and 86%, respectively.57-60 Initial reports of lower TRM
and successful engraftment with RIC-SCT61,62 prompted
the first prospective pilot study of 21 MF patients with
a median age of 53, and some as old as 70 years of age.63
Conditioning with fludarabine, busulphan and ATG and
utilizing related donors, if available, resulted in a medi-
an time to leukocyte engraftment of 16 days with 95%
complete donor chimerism at day 100. Treatment relat-
ed mortality was 16% at 1 year and 19% of patients
experienced grade III/IV acute GVHD while maintain-
ing a 3 year estimated OS of 84%. A busulfan/fludara-
bine based RIC regimen was investigated in a prospec-
tive study by the European Group for Blood and
Marrow Transplantation (EBMT) in 104 patients with a
median age of 55 years of age demonstrating a 3 year
OS and EFS of 70% and 55%, respectively.64
Additionally, a graft versus myelofibrosis effect has
been reported to occur following donor lymphocyte
infusions in patients with graft rejection.65,66
Most investigators have concluded that the improved
results with allogeneic stem cell transplantation for PMF
are a consequence of RIC and or the use of PBSC grafts.
However, a retrospective review of 100 MF patients
treated with SCT over 20 years in 26 Italian centers did
not observe an advantage in TRM or OS favoring RIC
over MA regimens. Interestingly, it is not clear that a
reduction in the TRM seen after 1996 was a result of the
introduction of RIC regimens or improved supportive
| 196 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
measures, antimicrobials and immunosuppressive ther-
apies. However, in a smaller review of SCT in 27 MF
patients in Sweden over 22 years, a TRM of 10% and
30% was seen in RIC and fully MA regimens, respec-
tively.67 One can conclude from these studies that both
MA and RIC allogeneic SCT can be curative for patients
with PMF. Future prospective trials are, therefore, clear-
ly required to define the choice of conditioning regimen,
the source of donor cells, and the selection of appropri-
ate PMF candidates for allogeneic SCT.
Currently, it is our practice to follow younger patients
with low risk disease for signs and symptoms of disease
progression and only resort to transplantation with dis-
ease progression as reflected with advancing Lille score.
The appropriateness of this strategy is uncertain. A long
interval between diagnosis and transplant has been
found to be a negative predictor of TRM in multivariate
analysis in a retrospective review and may actually sup-
port the use of SCT earlier in low risk patients before
progression of disease occurs.68 An argument could be
made that some young patients with low risk disease
may benefit from SCT upfront. At this time, we would
advocate watchful waiting (WW) for low risk disease in
any age group outside of a clinical trial. It remains
uncertain whether younger PMF patients with interme-
diate/high risk disease by Lille classification should pur-
sue a MA-SCT or RIC SCT; this question warrants a
prospective study.
Although the presence of the JAK2V617F mutation
has not been shown to influence either the relapse rate
or survival after SCT, JAK2V617F has been used to
assess minimal residual disease after SCT and the allele
burden is inversely correlated with donor-cell
chimerism.66,69 Kroger et al. demonstrated that in 22 MF
patients receiving SCT, 78% achieved real time PCR
negativity, while four of the five residual JAK2V17F pos-
itive patients still achieved a CR by IWG criteria.66 One
JAK2V617F positive patient post transplant was able to
eliminate the evidence of the mutation after receiving a
donor lymphocyte infusion. This data underscores the
potential role of monitoring the JAK2V617F gene muta-
tion as a clonal marker of the depth of response post-
transplant, and it may even be used to guide additional
immunotherapy.
Concern regarding donor cell sequestration and
increased transfusional requirements in patients with
MF and splenomegaly has prompted investigation into
the role of splenectomy prior to SCT. This has been
evaluated in a limited fashion and prior splenectomy
has not been shown to be associated with an obvious
survival advantage.70 Moreover, both a progressive
reduction in splenomegaly and the degree of marrow
fibrosis can be observed over a 12 month period follow-
ing RIC-SCT.71 A statistically significant prolongation in
ANC recovery was seen in patients with massive
splenomegaly (>30 cm) of 19 days compared to 13 days
in patients with smaller spleens. Full donor chimerism
was achieved in all the patients indicating successful
engraftment is possible, even in the face of extensive
splenomegaly. These results suggest that the risk associ-
ated with splenectomy may not be warranted despite a
longer period of neutropenia after RIC-SCT and we
would not recommend splenectomy prior to SCT unless
done to specifically address splenomegaly associated

Table 2. Proposed approach to treatment of primary
myelofibrosis in 2009.
Age Standard of Care Alternative options
Lille Class O <45 WW SCT on protocol
45-65 WW SCT on protocol
>65 WW SMT on protocol/rIFN
Lille Class I/II < 45 SCT on protocol SMT on protocol
45-65 SCT on protocol SMT on protocol
>65 Conventional therapy SMT on protocol/ SCT
on protocol up to age 70
WW: watchful waiting; SCT: stem cell transplant; SMT: small molecule therapy;
rIFN: recombinant interferon α.
signs and symptoms. The actuarial probability of devel-
oping LT in PMF over 100 months from diagnosis has
been estimated at approximately 20%, which is much
higher than that observed in PPV-MF or PET-MF at 9%
and 4%, respectively.72 The leukemic phenotype is usu-
ally myeloid and is uniformly resistant to standard
induction therapies with a median survival of 2.6
months.73 The role of SCT in transformed disease has
not been clarified and emphasis should be made on
timely institution of SCT prior to transformation.
In the era of rationally designed targeted molecular
therapeutics, one could envision the use of JAK2
inhibitors and/or chromatin modifying agents either as
a means to reduce splenomegaly and transfusion
requirements prior to SCT or as consolidative therapy
post transplant to suppress the re-emergence of minimal
residual disease. More than ever, it is imperative to refer
MF patients to centers offering early phase clinical trials
and transplant protocols. A collective attempt to share
experience and data in order to provide the best thera-
peutic option to each individual patient in a disease
characterized by biological and clinical heterogeneity
should be the goal of the international hematology com-
munity. Outlined in Table 2 is the approach of the
authors in providing care to patients with PMF. It is our
hope that new data generated in well-designed clinical
trials will allow us to provide evidence-based recom-
mendations in the future.
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 Myeloproliferative disorders

Interferon in the treatment of myeloproliferative

disorders

J.-J. Kiladjian
Assistance Publique, Hôpitaux de
Paris, Hôpital Avicenne, Service
d’Hématologie Clinique, Bobigny,
France; Assistance Publique,
Hôpitaux de Paris, Hôpital
Saint-Louis, Centre d’Investigation
Clinique, Paris, France; Université
Paris 7, Denis Diderot, Paris, France
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:200-207
A B S T R A C T
Interferon (IFN), the first cytokine discovered, has a wide range of biological properties, including
immunomodulatory, pro-apoptotic and anti-angiogenic activities that rapidly raised interest in its
therapeutic use in malignancies. In addition, IFN-receptor characterization was pivotal in the discov-
ery of the JAK/STAT signaling pathway. Among the large IFN family, IFNα2 is mainly used in therapy.
Many clinical trials have shown remarkable efficacy of IFNα in bcr-abl-negative myeloproliferative
disorders (MPD), especially polycythemia vera (PV), and essential thrombocythemia (ET). In those dis-
eases, IFNα induces hematological responses in about 80% of patients, and is able to reduce
splenomegaly, relieve pruritus and other constitutional symptoms. Yet, its use is limited by toxicity,
leading to early treatment discontinuation in about 20% of the patients. However, its lack of leuke-
mogenic potential, and its possible use during pregnancy have already made IFNα the drug of choice
for younger MPD patients. In addition, several studies have shown a probable selective effect of IFNα
on the malignant PV and ET clones. In an increasing number of patients, cytogenetic remissions, rever-
sions to polyclonal hematopoiesis, and more recently, induction of JAK2V617F complete molecular
remissions have been reported. These observations may widen the indications of IFNα in myeloprolif-
erative disorders.

nterferon (IFN) was the first cytokine dis- Janus kinase (JAK)/signal transducers and

I covered, identified 50 years ago by Isaacs

and Lindenmann.1 They found a secreted
factor produced by influenza-virus infected
activators of transcription (STAT) signaling
pathway.

chick cells able to transfer a virus-resistant Biology of interferons

state to previously uninfected cells. This fac-
tor was named IFN because of its ability to
interfere with viral growth. Soon, IFNs were
found to be produced in many animals, tis-
sues, and cells. About ten mammalian inter-
feron species have been discovered, includ-
ing seven in humans (Table 1).2 IFNs belong
to the class 2 α-helical cytokines that have
existed in early chordates for about 500 mil-
lion years.3 They play important roles in
innate and adaptative immunity, which
raised interest in their therapeutic use.
Production and purification of human leuko-
cyte IFNs obtained after infection of human
white blood cells (WBC) with viruses
already allowed their use in clinical trials in
the late seventies.4 Such preparation howev-
er, did not allow complete purity, and
although leukocyte interferon consisted pre-
dominantly of IFN-α, small amounts of
other IFN species were present. In 1980,
cloning of recombinant human IFN-α and
IFN-β allowed production of large amounts
of IFN for characterization, biological activi-
ty studies, and clinical trials.5-7 It is only 20
years after their discovery that 2 IFN species,
IFN-α and -β, could be satisfactorily purified
using high performance liquid chromatogra-
phy allowing their analysis and characteriza-
tion.8 In addition, early availability of recom-
binant IFNs offered an opportunity to inves-
tigate how cytokines induce gene expres-
sion, culminating in the discovery of the
The human family of IFNs includes seven
species.8 Interferon proteins are historically
characterized as type I (viral) IFNs, or type II
(immune) IFNs. The latter type consists of a
unique member, IFN-γ. Both types I and II
IFNs exert their actions through cognate
receptor complexes, IFNAR and IFNGR
respectively, present on cell surface mem-
branes.9,10 The most important and best stud-
ied among type I IFNs is IFN-α.
The human IFN-α family comprises 14
genes, including 2 pseudo-genes or allelic
forms (IFNAP22 and IFNA10). Thus in all,
there are 13 proteins expressed from these
genes, but only 12 distinct IFN-α and allelic
forms (the protein produced from IFNA13
gene being identical to that produced from
IFNA1). Of these, IFN-α2 has been very pre-
dominantly used as a therapeutic agent to
date.
IFNs elicit many biological effects that can
even be opposite in different cell types. For
example, type I IFN inhibits proliferation
and is proapoptotic for many cell types,11 yet
it prolongs the survival of memory T cells.12
Such a functional diversity can, at least in
part, be explained by diversity of the IFN
receptor itself, and that of the signaling path-
ways it may use.
The type I IFN receptor (IFNAR) complex
is unique among cytokine receptors in the
number of cognate ligands, including the 13
IFN-α subtypes, as well as β, w, ε, and κ

| 200 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

Table 1. Published trials of interferon in polycythemia Vera.

Author, year Number Reduction Freedom Discontinuations Discontinuations Type of IFN

of patients of PHL (%) from PHL (%) 1st year (%) total

Cacciola, 199169 11 9 (82) 5 (45) 0 NA NA

Cimino, 199370 13 10 (77) 4 (31) 0 NA α2b
Finelli, 199371 13 11 (85) 11 (85) 3 (23%) NA NA
Turri, 199172 11 7 (64) 4 (36) 0 0 α2a
Papineschi, 199473 11 9 (82) 8 (73) NA NA α2a
Sacchi, 199474 22 21 (95) 21 (95) 0 0 hl-IFN
Muller, 199575 15 7 (47%) NA 4 (27%) 6 (40%) α2b
Taylor, 199676 17 14 (82) 9 (53) 2 (12%) 6 (35%) NA
Foa, 199877 38 19 (50) 11 (29) 11 (29%) 16 (42%) α2a
Gilbert, 199878 31 NA NA NA 7 (23%) α2b
Stasi, 199879 18 17 (94) 11 (61) 0 NA hl-IFN
Heis, 199980 32 28 (87) 2 (6%) 4 (12%) 10 (31%) NA
Radin, 200381 12 5 (42) 1 (8) NA NA NA
Silver, 200634 55 55 (100) 53 (96) NA 8 (14%) α2a, α2b
Samuelsson, 200682 21 7/9 (78) 4/9 (44) NA 7/23 (30%) peg-α2b
Kiladjian, 200823 37 37 (100) 36 (97) 3 (8%) 13 (35%) peg-α2a
Total 349 260/318 (82%) 182/303 (60%) 27/227 (12%) 82/281 (29%)
NA: data not available, PHL: phlebotomy, hl-IFN: human leukocyte interferon.

species. Conventional type I IFN receptor is comprised
of two chains, IFNAR1 and IFNAR2c. IFN binding to
this complex induces activation by phosphorylation of
pre-associated JAKs in their cytoplasmic portions: Tyk2,
associated to IFNAR1, and Jak1, associated to IFNAR2c.
This provides docking sites on the receptor complex for
STAT-1 and -2 proteins that are in turn phosphorylated.
Activated STAT-1/STAT-2 heterodimers translocate to
the nucleus, associate with IRF-9 (IFN regulatory factor-
9) to form the trimeric ISGF-3 (IFN-stimulated gene fac-
tor-3) complex that binds to the ISRE (IFN-stimulated
response element) enhancer family element within the
promoters of interferon-regulated genes, leading to their
transcription. However, diversity of IFNAR signaling is
probably in part achieved by the activation of other
pathways, including other STATs (in particular STAT-3)
and non-STAT proteins.11 These alternative signaling
pathways include CrkL, Rap1, MAP-kinases, Vav,
RAC1, PI 3-kinase, IRS1 and -2, PMRT1, and Sin1.
Some mechanisms also exist to downregulate IFNs
signaling. Indeed, if the diversity of signals generated by
IFNs aim at protecting the host against infection and
malignant transformation, disproportionate signaling
may be harmful by inducing leukopenia, and autoim-
mune manifestations. Several negative regulatory mole-
cules have been identified, including SOCS-1
(Suppressor Of Cytokine Signaling-1), UBP43 (a type I
IFN-inducible cysteine protease), and SHP-2. SOCS-1
inhibits IFN signaling by binding the c-terminal region
of IFNAR1 and blocking the interaction with Tyk2,
UBP43 blocks the JAK1/IFNAR2 interaction, whereas
SHP-2 is implicated in nuclear STAT dephosphorylation,
which appears to be critical for STAT nuclear export,
another mechanism of regulation of STAT activity.
SOCS proteins are also thought to regulate JAKs activi-
ty by binding to their JH1 catalytic loop and targeting
those kinases for degradation.
Activities and therapeutic use of interferons
IFNs have a wide range of biological activities. They:
(i) stimulate cytotoxic activity of several immune cells
(T-cells, natural killer cells, monocytes, macrophages,
dendritic cells);13 (ii) increase the expression of tumor-
associated surface antigens and other surface molecules,
such as major histocompatibility complex (MHC) class
I antigens, amplifying the recognition of infected or
transformed cells by immune-effectors;14 (iii) induce
and/or activate proapoptotic genes and proteins includ-
ing TRAIL, caspases, Bak, and Bax; (iv) repress antiapop-
totic genes like Bcl-2, IAP (inhibitor of apoptosis pro-
tein); (v) modulate cell differentiation; (vi) display
antiangiogenic activity.2 Accordingly, the evaluation of
IFNs as therapeutic agents focused mainly on cancers
and viral diseases.
The U.S. Food and Drug Administration approved
human IFN-α2a and IFN-α2b (allelic versions of IFN-α2)
for the treatment of hairy cell leukemia in 1986. About
10 years later, IFN-β1a and IFN-β1b were approved for
the treatment of multiple sclerosis. Clinical trials for
IFN-α, before and after approval, focused on cancers
and viral diseases. To date, IFN-α is approved for the
treatment of hairy cell leukemia, malignant melanoma,
follicular lymphoma, condylomata acuminata (genital
warts), AIDS-related Kaposi sarcoma, and chronic hep-
atitis B and C. In addition, use of IFN-α is frequent in
many cancers, especially in bladder and renal cancers.
Rationale for IFN-α in the treatment of myeloproliferative
disorders
Many activities of IFN-α provide a rationale for its
therapeutic use in MPDs. First, IFN-α inhibits in vitro
proliferation of hematopoietic progenitors. It has been
shown that IFN-α can markedly reduce the colony-
forming ability of erythroid, granulocytic, and mega-
karyocytic progenitors in polycythemia vera (PV) and
primary myelofibrosis (PMF).15-17 Some of these in vitro

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 201 |
14th Congress of the European Hematology Association

studies even suggested that clonal MPD progenitors Table 2. Published trials of interferon in essential thrombo-
might be more sensitive to IFN-α than their normal cythemia.
counterparts might. This possible selective effect on the
MPD clone is further supported by several case reports Author, Year Number Response Discontinuation Type
showing reversion from monoclonal to polyclonal pat- of patients rate (%) n (%) of IFN
terns of hematopoiesis (based on X-chromosome inacti-
vation pattern studies) or disappearance of a chromoso-
Bellucci, 198827 12 NA 4 (33) α2a
mal abnormality present before treatment in IFN-α-
Giles, 198828 18 100 0 α2a &α2b
treated patients.18-21 Using quantification of JAK2V617F Gugliotta, 198983 10 100 NA α2a
mutated alleles in circulating granulocytes as a marker Lazzarino, 198984 26 86 9 (35) α2b
of minimal residual disease, our group has also demon- Giralt, 199185 13 69 NA α2b
strated that IFN-a markedly decreased the proportion of Gisslinger, 199186 20 85 10 (50) α2c
circulating mutated cells in a large cohort of patients.22,23 Sacchi, 199187 35 85 4 (11) α2b
The megakaryocytic lineage seems particularly sensitive Turri, 199172 10 70 1 (10) α2a
to IFN-α, with clear morphological and biochemical Seewann, 199188 19 80 6 (30) α2b
changes of megakaryocytes (MK) in IFN-treated Kasparu, 199289 14 86 0 α2b
patients.24 Furthermore, IFN-α is able to directly repress Rametta, 199490 25 92 NA α2b
megakaryopoiesis by inhibiting thrombopoietin- Berte, 199691 12 83 NA α2a & α2b
induced Mpl receptor signaling.25 Finally, IFN-α antago- Sacchi, 199892 11 100 1 (9) α
nizes platelet-derived growth factor (PDGF) and inhibits Radin, 200381 17 88% NA α2
the growth of marrow-derived fibroblasts, two activi- Alvarado, 200393 11 100 2 (18) peg-α2b
ties that may be useful in treating myelofibrosis. Saba, 200594 20 75 3 (15) α2a
Although the exact mechanisms of action of this Langer, 200595 36 75 13 (36) peg-α2b
cytokine on MPD clones are unknown, theoretically Samuelsson, 200682 21 70 11 (55) peg-α2b
IFN-a could, by acting at the level of the hematopoietic Jabbour, 200796 13 70 NA peg-α2b
stem cell and by enhancing the autologous immune Total 343 84 23
response against transformed cells, eradicate the MPD NA: data not available.
clone in selected cases.

Results of clinical trials using IFN-α in myeloproliferative
disorders
Clinical trials have shown clear efficacy of IFN-α in
the treatment of all types of MPDs. The pioneer study
of Silver in 1988 was followed by many studies demon-
strating the clinical efficacy of IFN-α in controlling
myeloid proliferation and relieving pruritus and other
constitutional symptoms in PV.26 Similar efficacy was at
the same time shown in ET.27,28 This review will focus
on the three main Ph-negative MPD: PV, Essential
Thrombocythemia (ET), and PMF, and will not analyze
IFN-α effects in other MPDs, including chronic myelo-
genous leukemia (CML), hypereosinophilic syndromes,
and systemic mastocytosis.
Many clinical studies have been performed since
those first works using various commercial forms of
IFN-α. Of note, to date none of these forms is registered
by FDA, EMEA or other countries’ health agencies for
Ph-negative MPD. Most of the published studies used
standard IFN-α2a or 2b, which were the first available
for therapy. There was no biological evidence to antici-
pate better efficacy of one of those isoforms over the
other in MPD, and, indeed, efficacy and toxicity profiles
of these two drugs when used in Ph-negative MPD
were similar (Tables 1 to 3). More recently, pegylated
forms of IFN have been developed. Addition of a poly-
ethyleneglycol (peg) tail produces several benefits,
including enhanced plasma half-life, lower toxicity, and
increased drug stability and solubility, without affecting
therapeutic activity.29 Use of pegylated IFN translated to
improvement for patients, mainly due to longer inter-
vals between administrations (weekly instead of every
24 to 48 hours). Surprisingly, although pegylation of
drugs usually results in a certain decrease in side effects
due to reduced relative peak concentration after each
injection, such benefit was not found for IFN as studies
comparing standard versus pegylated IFN in hepatitis,
and CML patients showed similar rates of toxicity and
treatment withdrawal using one or the other form.30,31
More than 700 PV, ET or PMF, treated with IFN have

Table 3. Published trials of interferon in primary myelofibrosis.

Author, Year Number of patients Response rate (%) Spleen size reduction Discontinuation (%) Type of IFN
(% of patients)

Gilbert, 199878 22 NA 58 46 α2b

Tefferi, 2001 (35) 11 0 18 64 α2
Heis-Vahidi-Fard, 2001 (97) 9 0 20 67 γ
Radin, 2003 (81) 31 3 33 NA α2
Jabbour, 2007 (96) 11 9 NA 26 peg-α2b
Total 84 3 32 51
NA: data not available.

| 202 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

been published, as shown in Tables 1, 2, and 3, which
summarize IFN studies that included more than 10
patients. Meta-analysis of these studies is difficult. First,
various forms of IFN-α were used, and one cannot
exclude (even though it is unlikely) a differential effect
of those forms on response rate and/or disease evolu-
tion. Furthermore, heterogeneous response criteria were
used. Harmonization of response criteria to therapy in
PV and ET, as published for PMF,32,33 would be very use-
ful, and the MPD working group of the European
LeukemiaNet has developed consensus response criteria
that may be used in future studies.
Results of published studies show that IFN-α is effec-
tive at controlling myeloproliferation in PV and ET. In
almost all studies, IFN-α was able to normalize platelet
counts and leukocytosis rapidly, and to reduce erythro-
cytosis, allowing reduction in the need for phle-
botomies within a few months. In both diseases, an
objective response was observed in about 80% of
patients, including complete freedom from phle-
botomies in PV in 60% of patients (Tables 1 and 2). In
addition, IFN-α was also able to reduce PV-associated
pruritus in a significant number of cases, and appears to
be the most efficient drug for this symptom. However,
toxicity of IFN was relatively significant, leading to
treatment discontinuation in almost one quarter of
patients (see below).
Although in vitro data suggested that IFN-α was a
good candidate drug for treating bone marrow fibrosis,
clinical trials in PMF have been disappointing (Table 3).
Almost no objective response was obtained in the 84
reported PMF patients. In 30% spleen size decreased on
treatment, while in almost the same proportion of
patients, it increased. Importantly, IFN toxicity in these
studies was much higher than in PV or ET studies, lead-
ing to rapid treatment discontinuation (usually after 3 to
6 months only) in more than 50% of the patients. One
of the main limiting toxicities of IFN in PMF, contrary to
PV or ET studies, appeared to be worsening of cytope-
nias.
Side effects of IFN-α
One of the main concerns regarding wide use of IFN-
α is toxicity. As shown in Tables 1 and 2, the average
rate of discontinuation because of toxicity in PV and ET
trials with IFN-α was around 25%, almost half of those
withdrawals occurring during the first year of therapy.
A wide range of side effects has been reported with
standard IFN-α.
Flu like symptoms
Common flu-like symptoms are experienced by most
patients, including headache, malaise, fever, chills,
fatigue, myalgia, back and joint pain. These symptoms
usually appear 1 to 3 hours after administration, disap-
pear within hours, and are generally manageable by pre-
ventive use of paracetamol. In addition, they gradually
decrease in intensity within a few weeks of treatment
initiation. These symptoms are clearly dose-dependent,
and can be prevented or at least limited by starting IFN
therapy at low doses.34 The frequency and intensity of
those flu-like symptoms are usually less marked using
pegylated forms of IFN.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 203 |
Berlin, Germany, June 4-7, 2009
Hematological toxicity
Hematological toxicity on erythroid and megakary-
ocytic lineages is commonly observed during IFN treat-
ment of hepatitis patients (even requiring sometimes
treatment with recombinant erythropoietin), but is very
rare in PV and ET. In contrast, PMF patients often expe-
rience worsening of anemia or thrombocytopenia dur-
ing IFN therapy, requiring treatment discontinuation in
a substantial number of cases. Neutropenia is rarely
reported as a limiting toxicity in MPD treatment, again
with the exception of PMF. The most common hemato-
logical side effect of IFN is lymphopenia, which is
reversed after IFN treatment discontinuation.8 However,
clinical complications potentially related to lymphope-
nia (like viral infections) were not reported in MPD
studies.
Other toxicities
Some chronic IFN toxicities are difficult to prevent or
control. They include fatigue and musculoskeletal pain,
two of the most frequent reasons for treatment with-
drawal. Depression is seen less often, but a history of
depression or psychiatric disorder constitutes a con-
traindication to IFN therapy. Less frequently reported,
but often noticed by patients’ community, is subtler
mood changes, like anxiety, irritability, and sullenness,
which may impact social life. Other rare side effects
include skin toxicity (mild injection site reactions, or
more generalized exanthema or urticaria), hair loss, gas-
trointestinal toxicity, including nausea, diarrhea, and
weight loss, liver, cardiac and neurologic toxicities.
An intriguing set of complications of chronic IFN ther-
apy is the development of autoimmune abnormalities,
ranging from single asymptomatic autoantibodies to
overt autoimmune disease. Hypothyroidism, for exam-
ple, is a rare but well-known complication of IFN treat-
ment. In addition, other types of autoimmune abnormal-
ities have been described in IFN-treated CML patients,
including antinuclear autoantibodies, immune-mediated
hemolysis or thrombocytopenia, polyarthritis, glomeru-
lonephritis, and connective tissue diseases.36-39 Some
authors have raised the hypothesis that development of
autoimmune phenomena in IFN-treated CML patients
could parallel and reflect the anti-leukemic efficacy of
the drug, as patients presenting with autoimmune
abnormalities had earlier and more frequent cytogenet-
ic responses.36,37 Outcome of those immunological
abnormalities is usually favorable, autoantibodies disap-
pearing after discontinuation of IFN-α, but they some-
times require specific treatment, such as hormone
replacement in patients developing thyroiditis.
IFN-α and leukemic evolution
One major advantage of IFN-α is that it clearly
appears to be non-leukemogenic, especially compared
to most available cytoreductive agents. Indeed, concern
has appeared in recent years in MPD regarding the long-
term risk of hematological evolution to acute leukemia
and myelodysplastic syndromes (AL/MDS). Increased
risk of AL/MDS has been found to be associated with
the use of alkylating agents and radioactive phosphorus
in randomized clinical trials.40-43 By contrast, a leuke-
mogenic potential of HU has not been demonstrated in
clinical trials, although there is evidence showing that
14th Congress of the European Hematology Association
sequential use of HU and other cytoreductive drugs
(alkylating agents, radioactive phosphorus) increases
the risk of AL/MDS.43-46 In addition, several retrospec-
tive studies did not find an increased risk of AL in PV
and ET treated with HU alone,47-49 and similar conclu-
sions were drawn from a recent meta-analysis of HU
therapy in sickle cell anemia.50 Still, very long term
results of prospective studies in HU treated MPD
patients with more than 10 years of follow-up showed
a cumulative incidence of AL/MDS of more than 10%
beyond 12 years of follow-up, suggesting that the risk
of leukemic evolution could be higher than previously
reported in studies with shorter follow-up.51 The rela-
tive contribution of HU and of the natural very long-
term evolution of the disease in the pathogenesis of
those late AL/MDS remains unclear. These findings
have, however, lead published guidelines in ET and PV
to recommend the use of clearly non-leukemogenic
drugs, like IFNα in younger patients requiring cytore-
ductive therapy.52,53
Has IFN-α a selective effect on myeloproliferative clones?
As for all hematological malignancies, a future pri-
mary objective of Ph-negative MPD treatment should
be cure; a goal that cannot be achieved with currently
available drugs, which have never been shown to influ-
ence the natural history of these diseases positively. The
situation may change with the development of JAK2
inhibitors54 but, to date, only IFNα has been able to
induce cytogenetic remissions or reversion from mono-
clonal to polyclonal hematopoiesis in some patients,18-21
suggesting that it could eradicate the malignant clone
and possibly cure the disease in selected cases. By mon-
itoring JAK2V617F mutation in 37 PV patients treated
with peg-IFNα-2a, we showed that V617F mutation
could be reduced to undetectable levels (using a PCR
assay with 1% sensitivity) in circulating granulocytes in
24% of patients (molecular remission).23 Such reduction of
the JAK2 mutated clone with IFNα may explain report-
ed cases of hematological remissions in ET and PV
patients lasting for months after treatment discontinua-
tion.55,56 Molecular complete remission with peg-IFNα-
2a has also been described in a case report,57 showing
that JAK2V617F was no longer detectable after only 2
months of treatment, (which was continued for an addi-
tional month) but rapidly re-appeared after peg-IFNα-2a
discontinuation. Comparison with our results suggests
that peg-IFNα-2a should not be discontinued as soon as
molecular CR is reached if one aims at eliminating the
mutated clone.
Reduction of the JAK mutated clone, however, does
not imply disease cure. First, the sensitivity limit of the
technique we used (1%) remained low. Furthermore,
studies comparing clonality (based on X-chromosome
inactivation patterns or coexisting cytogenetic abnor-
malities) to JAK2 mutant allele frequency suggest that
JAK2V617F could be a secondary genetic event in some
PV patients.58-61 If so, elimination of JAK2V617F would
only reflect the evolution of subclones. On the other
hand, a theoretical advantage of IFNa compared to more
specific inhibitors is that being non-specific of any genet-
ic alteration, it may have some activity against MPD
clones regardless of their underlying molecular defects.
Finally, experience with IFN or tyrosine-kinase
| 204 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
inhibitors in CML taught us that molecular complete
remission does not necessarily equate to cure.62
Reduction of the JAK2 mutated clone may, however,
impact disease evolution. For example, the percentage
of circulating mutated JAK2 allele was shown to be sig-
nificantly higher in PV and ET patients who developed
myelofibrosis, and may be significantly higher in
patients who develop vascular complications.63,64 Some
authors suggested that IFNα could delay myelofibrosis
development in PV patients34 based on in vitro data, a
benefit that could be mediated by reduction of the pro-
portion of JAK2V617F mutated cells. In addition, the
selective effect of IFNa on mutated granulocytes may
impact the incidence of thrombosis, as it has been
shown that leukocyte activation and hemostatic
changes were correlated with the presence of the JAK2
mutation.63,65,66 Accordingly, the incidence of thrombosis
in IFNα-treated patients was consistently found to be
lower than expected during follow-up.34,55,56 However,
such possible reduction in thrombotic risk with IFN has
yet to be demonstrated in a properly designed con-
trolled trial.
Conclusion and perspectives
Many studies over the last 20 years have shown
remarkable efficacy of IFNa in PV and ET, but its use
remained limited in clinical practice, due to side effects,
and maybe in some cases to the lack of experience of
physicians with management of IFNα treatment.
Indeed, in expert hands, low toxicity with long-term use
of this drug has been reported.34
Although many biological properties of IFNα may
account for its efficacy in MPD, the exact mechanisms
of action remain unknown.
To date, in published guidelines indications of IFNα
for the treatment of PV and ET have generally remained
limited to high risk patients younger than 40 years, and
to pregnancy.52,53 Indeed, when cytoreduction is neces-
sary during pregnancy, many publications have docu-
mented the safety of IFNα in MPD mothers, as well as
fetuses.67,68 However, there are very few data available
on the long-term safety of IFNα in children exposed to
this drug during pregnancy. Recent results suggest,
however, that indications of treatment with IFNα
should be broader. First, the possibility of a higher inci-
dence of progression to MDS and acute leukemia in the
very long-term follow-up of PV and ET51 suggests that
non-leukemogenic drugs like IFNα should be more
widely used in those disorders. An additional advantage
of IFNα may be its ability to reduce specifically the
MPD clone in a large proportion of PV patients, and
even to induce complete molecular remissions.23
Confirmation of those findings may widen the indica-
tions of IFNα in JAK2-mutated PV and ET, without
restricting treatment to patients at high risk of vascular
complications. At least, the large experience with IFN
accumulated for 20 years in MPD treatment we have
described in this review should prompt investigators to
initiate a direct comparison of this drug versus hydrox-
yurea in a randomized trial. Such a trial could answer
the question of the best current cytoreductive treatment
in MPDs, in terms of hematological and molecular
response, as well as reduction of short-term vascular
risk, and of long-term risk of evolution to AL/MDS.

The current development of JAK2 inhibitors raises the
hope that molecularly targeted therapy may benefit
MPD patients by reducing the kinase hyper-activity that
may lead to disease that is more aggressive with higher
incidence of vascular events and of hematological evo-
lution. Early results of phase 1 to 2 trials with these new
agents already seem to indicate that they will play a role
in the therapy of symptomatic PMF patients. Their role
in the treatment of PV and ET will certainly be studied,
but one should keep in mind that the vast majority of
ET and PV have excellent response rates and significant
reduction of the vascular risk using conventional pallia-
tive therapies, such as HU. The use of much more
expensive drugs with unknown long-term risk will be
justified only if they can cure or significantly alter natu-
ral disease history, and in particular, the very long-term
risk of evolution to AL/MDS.
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 Pediatric hematological malignancies

Myelodysplastic syndromes in children

and adolescents

C. M. Niemeyer A B S T R A C T

Accounting for 4% of hematopoietic neoplasia in childhood, myelodysplastic syndromes (MDS) in

Division of Paediatric Haematology
and Oncology, Department of
Paediatrics and Adolescent
Medicine, Albert-Ludwigs-University,
Freiburg, Germany
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:208-213
children are far less common than in adults. There is a great heterogeneity in presentation and clini-
cal course. Apart from inherited bone marrow failure disorders, the genetic changes predisposing chil-
dren to MDS are largely obscure. Monosomy 7 is the most common chromosomal aberration, often
occurring as sole abnormality. Refractory cytopenia is the most common subtype accounting for about
50% of the cases. The recent edition of the World Health Organization (WHO) classification of
hematopoietic neoplasia has incorporated refractory cytopenia (RC) of childhood as provisional enti-
ty. While children with RC and a normal karoytype or trisomy 8 can experience a long stable course
and may be candidates for a watch-and-wait strategy or immunosuppressive therapy, patients with
monosomy 7, 7q- or complex karoytypes generally have rapidly progressive disease and are candidates
for hematopoietic stem cell transplantation (HSCT) early in the course of their disease. Similarly, the
therapeutic approach in children for MDS with elevated blast count is primarily curative based on
HSCT. Research on genetic conditions predisposing to MDS in young age, such as inherited syndromes
with bone marrow failure may give important insights into MDS pathogenesis.

yelodysplastic syndromes (MDS) the subdivision in RAEB I (blasts 5-9%) and

M are a heterogeneous group of clonal

hematopoietic stem cells disorders
characterized by ineffective hematopoiesis,
morphological dysplasia and a variable risk
of transformation to MDS-related leukemia.
MDS belong to the most frequent bone mar-
row (BM) disorders in adults, with a crude
incidence of about 4/100.000/year.1 In chil-
dren and adolescents, MDS is rare, account-
ing 4% of hematopoietic malignancies
(Table 1) corresponding to an annual inci-
dence estimated at 1.8 children per million
children aged 0 to 14 years.2,3 The dypslastic
prodrome of acute myeloid leukemia (AML)
in infants with Down syndrome is excluded
from these population-based studies of MDS
and are classified with myeloid leukemia of
Down syndrome.4,5
As expected, there are a number of differ-
ences between adult and pediatric MDS.
Refractory anemia (RA) with ring siderob-
lasts and MDS associated with del(5q) chro-
mosome are exceedingly rare in children.
While in adults with RA, anemia is frequent-
ly the main presenting symptom, in child-
hood cases of MDS without increased blast
count, neutropenia and thrombocytopenia
are prominent.6 Therefore, refractory cytopenia
(RC) is felt more suitable for childhood MDS
without excess blast count. RC is the most
common subtype of childhood MDS,
accounting for more than half of the cases.7
The importance of multilineage dysplasia in
RC is unknown.
Refractory anemia with excess blasts
(RAEB) in children has similar morphological
and immunophenotypical features than that
observed in adults. It is unknown whether
RAEB II (blasts 10-19%), as suggested for
MDS in adults is useful in children. There is
consensus that the relationship between
advanced MDS and de novo AML is better
defined by biological and clinical behaviour
than by blast count.8 Consequently, myeloid
disease with low blast count and cytogenet-
ic abnormalities typically associated with de
novo AML is classified as AML. In addition,
disease with rapid increase in marrow blasts
or organ infiltration should be considered de
novo AML, which is by far the more com-
mon disorder. Children with RAEB generally
have relatively stable counts for weeks or
months. A similar biological behaviour is
noted in some children with myelodyspla-
sia, and blasts 20-29% are considered refrac-
tory anemia with excess blasts in transfor-
mation (RAEB-T) in the French-American-
British Cooperative Group classification.9
Because these children may not benefit from
intensive chemotherapy, upfront HCST has
been suggested by some investigators.8
MDS after prior chemo- or radiation ther-
apy, prior acquired aplastic anemia, in inher-
ited BM failure disorders and in familial dis-
ease is generally classified as secondary
MDS. All other cases are referred to as pri-
mary MDS, although it is reasonable to
assume that most of these disorders are sec-
ondary to some yet unknown genetic predis-
position. These presumed underlying genet-
ic changes may also give rise to subtle phe-
notypic abnormalities observed in many
children with primary MDS.
Genetics
Monosomy 7 is the most common cyto-

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 Berlin, Germany, June 4-7, 2009

genetic abnormality in childhood MDS, seen in Table 1. Incidence of hematological malignancies in chil-
approximately 30% of the cases.7,10-12 While mono- dren 0 to 14 years. Combined data from Denmark 1980-
somy 7 is associated with a poor prognosis in child- 1991 and British Columbia 1982–1996.2,3
hood AML13 and MDS in adults,14 it is not an unfavor-
able feature in childhood MDS.15 After monosomy 7, Annual incidence
trisomy 8 and trisomy 21 are the most common N % per million
numerical abnormalities. Constitutional trisomy 21 is
usually clinically obvious when present, whereas con-
ALL 815 79 38.5
stitutional trisomy 8 mosaicism may remain unrecog- AML1 115 11 5.4
nized16 and should be tested when trisomy 8 is found MDS1 38 4 1.8
in the bone marrow (BM). Chromosomal abnormali- Myeloid leukemia of DS 19 2 0.9
ties detected in children enrolled in Study 98 of the JMML 25 2 1.2
European Working Group of MDS in Childhood CML 13 1 0.6
(EWOG-MDS) are depicted in Table 3. PV/ET2 3 0 0.1
RAS mutations are detected in childhood MDS at Unclassified 3 0 0.1
low frequency.17 Mutations of the transcription factor, Total 1031 100 48.7
RUNX1 (AML1), with a consequential decrease in its
1
trans-activation potential have been described in AML Excluding Down syndrome; (DS) 2PV: polycythemia vera; ET: essential
thrombocythemia.
M0, therapy related AML, RAEB, RAEB-T, and AML
following MDS and suggests a loss of function as a
mechanism for transformation.18,19 MDS patients with
RUNX1 mutations frequently harbor somatic muta-
tions in genes of the RAS signaling pathway. topenia, and pancytopenia with radioulnar synostosis,
Mutations of TP53 are present in a significant propor- cannot be distinguished from RC by morphology or
tion of cases of MDS and are especially associated histology alone; they have to be excluded by careful
with therapy related MDS.20 physical examination for skeletal and other organ
abnormalities, past and family history and appropriate
Primary myelodysplastic symdrome without increase in laboratory tests. The distinction between RC and SAA
blast count (refractory cytopenia) may be challenging. In contrast to RC, SAA presents
Diagnosis with adipocytosis of the bone marrow spaces with
MDS with less than 5% blasts in the BM is particu- few scattered myeloid cells, absence of erythroid
larly difficult to diagnose, because dysplasia of islands with increased numbers of immature erythrob-
hematopoietic cells is frequently observed in associa- lasts and without any micromegakaryocytes.21
tion with infections, metabolic disorders, nutritional Following immunosuppressive therapy (IST), the his-
deficiencies, and a variety of other diseases. In the tological pattern of SAA can no longer be separated
absence of a cytogenetic marker, the clinical course has from that observed in RC.
to be carefully evaluated before a diagnosis of RC can
be established. Most children present with symptoms Therapy
related to pancytopenia, but in up to 20% of patients, The median time to progression for children with
no clinical signs or symptoms are reported.6 The mean RC and monosomy 7 is less than 2 years.6 Sponta-
corpuscular volume (MCV) of red cells and hemoglo- neous disappearance of monosomy 7 and cytopenia
bin F are usually elevated, but they are also high in the has been noted in some infants,22,23 but remains a rare
majority of patients with inherited BM failure disor- event. In contrast to monosomy 7, patients with tri-
ders, in some patients with acquired severe aplastic somy 8 and other karyotypes may experience a long
anemia (SAA) at diagnosis, and in most patients with stable course of their disease.6
SAA during their clinical course. On PB smears, red
cells usually are macrocytic with anisopoikilocytosis. Watch-and-wait strategy
There may be giant platelets. Neutrophils with pseudo For children with a normal karyotype or chromoso-
Pelger Huet nuclei and/or hypogranulation can often mal abnormalities other than monosomy 7, 7q- or a
be noted.21 complex karyotype, and absence of transfusion
Like in adults, MDS without increase in blast count dependency or neutropenia, a watch-and-wait strategy
in children can present with cytopenia and a hyper- can be appropriate.
plastic BM; however, three quarters of cases in chil-
dren are hypoplastic (Figure 1). The morphological pic- Immunosuppressive therapy
ture of RC with hypocellular BM is similar to that If in these above patients, cytopenia necessitates
observed in cases with normo- or hypercellualr BM. treatment, current therapy options include HSCT or
There is a patchy pattern of erythropoiesis with imma- IST. It has been suggested that autoimmunity con-
ture precursors accompanies by a sparse granulo- tributes to cytopenia of MDS,24 and approximately 35
poiesis and significantly decreased or absent mega- to 50% of adults with MDS have clinically relevant
karyocytes (Table 3). At least two trephine biopsies are responses to IST.25 In children with hypoplastic RC
recommended to detect the characteristic histological with normal karyotype treated with IST, overall and
pattern. Inherited BM failure disorders, such as failure-free survival at 3 years was 89% and 55%,
Fanconi anemia, dyskeratosis congenita, Shwachman respectively.26 However, the long-term outcome
Diamond syndrome, amegakaryocytic thrombocy- remains unknown.

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14th Congress of the European Hematology Association

Table 2. Results of cytogenetics in study EWOG-MDS 98 for

patients with primary myelodysplastic syndrome (MDS)
and secondary MDS following chemo- or radiation therapy,
inherited BM failure disorder, acquired aplastic anemia or
in familial disease (update of 55).

Karyotype Primary MDS (%) Secondary MDS (%)

RC RAEB/RAEB-T RC RAEB /RAEB-T

(N= 126) (N=86) (N=23) (N=44)

Normal 77 45 39 27
Monosomy 7 12 29 22 32
(+/- 1 additional)
Other aberrations (<3) 10 17 22 11
≥3 aberrations 1 8 17 30
Figure 1. Bone marrow cellularity and karyotype in chil- RC: refractory cytopenia; RAEB: refractory anemia with excess blasts;
dren with primary refractory cytopenia (unpublished data RAEB-T: RAEB in transformation; EWOG-MDS: European Working Group
from EWOG-MDS. of Myelodysplastic Syndrome in Childhood.

Hematopoietic stem cell transplantation New agents

For patients with RC and monosomy 7, 7q- or com-
plex karyotypes allogeneic HSCT from an HLA-identi-
cal sibling donor (MSD) or HLA-compatible unrelated
donor (UD) is recommended early in the course of the
disease. Following different high intensity regimens the
3 to 5-year Kaplan-Meier estimate of event-free survival
is 75 to 80%.27,28 Recurrence of the original disease is
rarely observed, while transplant-related mortality is
the major cause of failure.
HSCT with reduced intensity conditioning may be
able to reduce the risk of early and late toxicity. However,
this may be counterbalanced by an increased incidence of
delayed engraftment, graft failure, mixed chimerism and
relapse. Strahm et al.29 report on a pilot study on 19 chil-
dren with RC transplanted following a fludarabine-based
reduced intensity regimen. Overall and event-free sur-
vival at 3 years was 0.84 and 0.74, respectively. Infections
were the most frequently observed complication. These
results are comparable to those of patients transplanted
following high intensity preparative regimens.
Treatment of adults with MDS has changed dra-
matically after new agents, such as azacitidine,
decitabine and lenalidomie became available. Lenali-
domide, an immunomodulatory drug derived from
the parent compound thalidomide, has a dramatic
effect demonstrated in adult patients with del(5q), a
subtype unknown in pediatric MDS. In addition, it is
becoming increasingly clear that aberrant DNA
methylation contributes to the malignant phenotype
in MDS. Few investigators have started to tackle the
epigenetic profile of MDS in childhood.30,31 Because of
small cohorts, and the fact that, in contrast to adults,
most children with MDS will receive a hematopoiet-
ic transplant, results obtained in adult MDS will be
difficult to verify in the pediatric age group.
Nevertheless, it is plausible to believe that exciting
findings with new agents will be in store for child-
hood MDS as well.

Table 3. Minimal diagnostic criteria for refractory cytopenia of childhood.

Erythropoiesis Granulopoiesis Megakaryopoiesis

Bone marrow aspirate Dysplastic changes* Dysplastic changes# in at Unequivocal micromegakaryocytes,

and/or megaloblastoid least 10% of granulocytic other dysplastic changes§
changes in at least 10% precursors and neutrophils in variable numbers
of erythroid precursors

Bone marrow biopsy A few clusters of at least 20
erythroid precursors. Stop in
maturation with increased numbers of
proerythroblasts. Increasednumbers
of mitoses.
No minimal diagnostic criteria Unequivocal micromegakaryocytes;
immunohistochemistry is
obligatory (CD61, CD41),
other dysplastic changes§ in variable numbers

Peripheral blood Dysplastic changes# in at least

10% of neutrophils
*Abnormal nuclear lobulation, multinuclear cells, nuclear bridges; #Pseudo-Pelger-Huet cells, hypo-or agranularity, giant bands (in cases of severe neutropenia this cri-
teria may not be fulfilled). §Megakaryocytes of variable size with separated nuclei or round nuclei. The absence of megakaryocytes will not exclude refractory cytopenia
of childhood.

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Primary myelodysplastic syndrome with increased blast
count
Allogeneic HSCT is the treatment of choice in chil-
dren with MDS and increased blast count. Since most
patients receive HSCT, the International Prognostic
Scoring System (IPSS) for MDS in adults based upon
weighted data on BM blasts percentage, cytopenia and
cytogenetics is of limited value in children.32 Whether
intensive chemotherapy prior to stem cell transplanta-
tion should routinely be employed is highly controver-
sial.28,33-35 In the United States and United Kingdom, chil-
dren with RAEB and RAEB-T are generally included in
pediatric AML trials.33,34 For MDS patients enrolled,
most AML studies found significant morbidity and mor-
tality associated with induction chemotherapy, a com-
plete remission rate of less than 60%, many relapses,
and an overall survival of less than 30%.34,36,37 Data from
the European Working Group of MDS in Childhood
(EWOG-MDS) on 101 children with primary RAEB,
RAEB-T or MDR-AML given allogeneic HSCT after a
preparative regimen consisting of BU 16 mg/kg, CY 120
mg/kg and L-PAM 140 mg/m2 indicate that AML-type
therapy prior to the grafting procedure does not prolong
survival.35 Allogeneic HSCT can cure about half the chil-
dren with advanced primary MDS.27,38 Toxicity of the
procedure and relapse rate contribute equally to the
number of events. Children receiving a graft from an
unrelated donor have previously suffered a higher TRM
and a lower EFS rate, but more recent studies have
shown survival following UD-HSCT comparable to
MSD-HSCT.39
Secondary myelodysplastic syndrome
MDS in inherited bone marrow failure disorders
The risk of myeloid neoplasias in individuals with
inherited BM failure disorders depends on the specific
disorder and the underlying genetic lesion. Among the
inherited BM failure disorders Fanconi anemia (FA) is
most frequent, followed by hematopoietic neoplasia.
MDS or AML develop in as many as 50% of the patients
with FA before the age of 40 years.40 For patients with
severe congenital neutropenia, an incidence of
MDS/AML development of about 13% at 8 years of G-
CSF treatment has been reported.41 In most SCN patients
with MDS/AML, acquired mutations in the G-CSF
receptor are noted, and partial or complete loss of chro-
mosome 7 is found in more than half.42,43
MDS may occur in as many as one third of patients
with Shwachman-Diamond syndrome,44 but less fre-
quently in patients with Diamond-Blackfan anemia.45,46 It
is noteworthy that not all BM failure syndromes predis-
pose to the development of MDS; for example, patients
with dyskeratosis congenita develop BM failure in 95%
of the cases, but MDS is rare.47 Heterozygous mutations
of the transcription factor RUNX1, which is a common
target for chromosomal translocation in actue leukemia,
cause familial thrombocytopenia, with a propensity to
develop acute myelogenous leukemia (FTP/AML syn-
drome).48 There are no solid data indicating that avanced
MDS occurring in patients with inherited BM failure dis-
order other than Fanconi anemia differ from MDS in
other children. The patients should be included in series
of MDS and the type and the frequency of constitution-
al abnormalities should be reported.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 211 |
Berlin, Germany, June 4-7, 2009
Myelodysplastic syndrome after acquired aplastic anemia
MDS develops in 10 to 15% of those children with
aplastic anemia not treated with HSCT.49,50 The initial
diagnosis of SAA has to be questioned when secondary
MDS/AML is diagnosed within a few months after
presentation. Risk factors for clonal disease in SAA are
not well clarified, but some reports showed that older
age, splenectomy, multiple courses of IST, duration of
G-CSF therapy, and unresponsiveness to IST are related
with high risk of clonal evolution.49,50
Therapy-related myelodysplastic syndrome
Hematological, therapy-related secondary MDS
(tMDS) after chemo- or radiotherapy can manifest as
RC, high grade MDS, or CMML.15 Between 7% to 18%
of MDS cases in children are tMDS.7,37 The pathophysi-
ology of the development of tMDS is supposed to be
similar in children, adolescents and adults. However, in
children and adolescents, the classical distinction
between alkylator-type and topoisomerase II inhibitor-
type tMDS does neither exists clinically51 nor cytogenet-
ically.52 HSCT is the therapy of choice, but outcome
remains poor.51,53
Familial myelodysplastic syndrome
Familial occurrence of MDS, especially with -7/7q-,
has been reported in a number of cases. Some families
show discordance for -7;15 therefore, it is uncertain
whether -7 per se increase the risk for familiar cases.
The inherited predisposing locus in familial MDS or
AML with -7/7q- may not be located on chromosome
7.54 Familial MDS does also occur without -7/7q-.7
Outlook
Clinical progress in rare hematological disorder like
childhood MDS will only be obtained by enrolling
patients in clinical studies and preserving sufficient
material for bio banks. MDS in inherited BM failure dis-
orders and familial disease may serve as a model system
for studying the succeeding genetic changes in leukemic
stem cells.
The author wishes to acknowledge the relevant discussions
and contributions in literature of many colleagues who are
active in several committees and task forces, including that of
the Dutch Childhood Oncology Group, the International
Pediatric AML Group, the International BFM Study Group,
and the International Consortium on Childhood APL.
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54. Gao Q, Horwitz M, Roulston D, Hagos F, Zhao N, Freireich EJ,
et al. Susceptibility gene for familial acute myeloid leukemia
associated with loss of 5q and/or 7q is not localized on the
commonly deleted portion of 5q. Genes Chromosomes
Cancer 2000;28:164-72.
55. Pui CH, Schrappe M, Ribeiro RC, Niemeyer CM. Childhood
and adolescent lymphoid and myeloid leukemia. Hematology
Am Soc Hematol Educ Program 2004:118-45.
 Pediatric hematological malignancies

Pediatric acute myeloid leukemia

G.J.L. Kaspers
Pediatric Oncology/Hematology,
VU University Medical Center,
Amsterdam, the Netherlands
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:214-220
A B S T R A C T
Pediatric acute myeloid leukemia (AML) has become a curable disease, with the possibility of cur-
ing about two thirds of the patients. However, many events still occur; side-effects are very signifi-
cant, and late-effects can be severe and even life-threatening. Thus, treatment of pediatric AML still
needs to improve further. While most study groups agree on several principles of treatment, many
unanswered questions and even controversies remain. This also justifies further clinical studies on the
optimal treatment of pediatric AML. Fortunately, biotechnical developments provide novel treatment
targets and targeted drugs, and intensified international collaboration enables such clinical research.
This makes it realistic to expect major improvements in the treatment of pediatric AML in the next
10-20 years.

Pediatric acute myeloid leukemia (AML)
concerns up to 20% of all childhood
leukemias and has become a curable disease
over the past three to four decades. While
most children with AML still died in the
early 1970s, recent reports indicate probabil-
ities of long-term survival of more than
60%.1 This improvement, which unfortu-
nately is limited to the more privileged coun-
tries, and has certainly not been achieved
worldwide, cannot be attributed to the
introduction of many new antileukemic
drugs or to innovative treatment modalities.
In fact, the drugs to treat AML being used
nowadays, by most if not all groups have
been available for decades. Thus, other fac-
tors should explain the much higher cure
rates. These include the identification of typ-
ical AML-drugs, such as cytarabine and
anthracyclines, the treatment of patients
according to detailed protocols, improved
dosing and scheduling of the drugs, better
risk-group stratification and risk-adapted
therapy, and major improvements in sup-
portive care. The latter includes blood prod-
uct support, prevention and treatment of
infections, and temporary admissions to the
pediatric intensive care unit to support vital
body functions. All of this means that
improvements in the treatment of AML can
theoretically be achieved in less-privileged
countries as well, if the drugs and supportive
care are available for the majority of
patients. An international effort by different
study groups to report on long-term clinical
outcome in pediatric AML resulted in a large
number of excellent manuscripts.1 These
papers clearly illustrate the main events that
occur in pediatric AML. First, early death in
2-10% of patients is mainly caused by infec-
tious and bleeding complications. Second,
refractory or resistant disease in 3-19% (usu-
ally 5-10%) of patients is caused by
increased drug resistance by the bulk of
AML cells. Third, late mortality in 5-10% of
patients (in complete remission) is mainly
caused by treatment-related complications,
such as infections, bleeding, graft-versus-
host-disease, second malignancies and car-
diac failure. Fourth, relapsed disease is the
most frequent event, occurring in 30-40% of
patients. These four main types of events
also indicate how we should improve out-
come in pediatric AML.
Meanwhile, several main issues remain in
pediatric AML, which will be discussed here
to some extent. First, there is no consensus
on several elements in the treatment of pedi-
atric AML. These include the need for high-
dose cytarabine at induction, the need for a
third or even fourth drug in addition to
cytarabine and an anthracycline at induc-
tion, the optimal number of treatment cours-
es, the maximum cumulative dose of anthra-
cyclines to be given in relation to the risk of
cardiotoxicity (and for that matter, the safest
anthracycline), prophylaxis for central nerv-
ous system (CNS) relapse, the best risk-
group stratification, and the need for allo-
geneic hematopietic stem cell transplanta-
tion (allo-SCT), and if needed, in which
patients. Second, now that it seems that cure
rates are reaching a plateau, we need the
introduction of novel agents and innovative
treatment modalities. This is especially true
in view of the significant late-effects often
found in survivors of pediatric AML. Finally,
with improved outcome and the identifica-
tion of more and more subgroups, the need
for international collaboration increases,
which is also a challenge.
Since acute promyelocytic leukemia (APL)
and myeloid leukemia of Down syndrome
have become well-recognised separate enti-
ties, these will not be extensively discussed
in this contribution.

| 214 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

Current controversies in the treatment of newly diagnosed
pediatric acute myeloid leukemia
Do we need an additional drug at induction? Many groups
use etoposide, or etoposide plus thioguanine, in addi-
tion to the classical combination of cytarabine and
daunorubicin (or another anthracycline/anthracene-
dione). However, it has not convincingly been shown
that such additional drugs are required at induction.
Indeed, several pediatric groups still apply a two drug
induction. A direct comparison of etoposide and
thioguanine, added to cytarabine and daunorubicin, did
not reveal statistically significant differences in remis-
sion rates, event-free or overall survival.2 The issue of
additional drugs has been addressed in the past by sev-
eral groups but large, more recent randomized studies in
children are lacking. Study MRC AML15 recently ran-
domized conventional induction with cytarabine,
daunorubicin and etoposide against FLAG plus idaru-
bicin, enrolling both children and adults. In addition, the
value of adding gemtuzumab ozogamicin to conven-
tional induction chemotherapy was studied. Mature
results are eagerly awaited. Today, complete remissions
rates nor event-free or overall survival rates differ to
such an extent between study groups that the best strat-
egy becomes apparent. However, a comparison of dif-
ferent schedules in relation to the rate of resistant dis-
ease and prognosis suggested that a cumulative anthra-
cycline dose of less than 100 mg/m2 after one induction
course is associated with a worse outcome.3
Theoretically, we should aim for high quality complete
remissions with as low as possible amounts of minimal
residual disease. Then, it is reasonable to combine drugs
with different mechanisms of action, as long as toxicity
remains manageable.
What is the best moment for high-dose cytarabine? Most
groups now assume that high-dose cytarabine should be
incorporated in the treatment, although there are no
recent randomized studies in pediatric AML reported to
support that policy. An older study by the POG with
overall inferior outcome compared to currently achiev-
able survival rates did not reveal an improvement with
higher doses of cytarabine.4 However, in adult AML, it
has been shown that high-dose cytarabine at induction
results in better postremission outcome.5,6 High-dose
cytarabine as post-remission strategy benefited adults
especially, with core binding factor or normal karyotype
AML.7 Knowing that pediatric AML on average concerns
better risk, and thus, more chemosensitive patients than
adults with AML, it seems reasonable to extrapolate
these data and to indeed incorporate higher-dose of
cytarabine in our protocols. Moreover, preclinical studies
have shown that AML cells from some patients are more
resistant to cytarabine, and that in some patients the
active transport of cytarabine into the AML cells is lim-
ited; a mechanism which is necessary in case of lower
concentrations of cytarabine.8,9 Both mechanisms of
resistance theoretically can be overcome by higher dose
cytarabine. However, these studies do not identify the
optimal moment to use high-dose cytarabine. Again, the
recently closed study MRC AML15 may provide infor-
mation on this issue, since it does include a comparison
of standard low dose and high-dose cytarabine at induc-
tion, although unfortunately other differences in that
induction therapy may confound the results.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 215 |
Berlin, Germany, June 4-7, 2009
Table 1. Current controversies in the treatment of
pediatric acute myeloid leukemia.
Need for a third or even fourth drug at induction
Dose of cytarabine at induction
Total number of courses of combination chemotherapy
Optimal cumulative dose of anthracyclines
Best anthracycline (including anthracenediones) in terms of cardiotoxicity
Need for and type of prophylaxis for central nervous system relapses
Indications for allogeneic stem cell transplantation
How many courses of chemotherapy are required? Most
groups now use multiple courses of intensive
chemotherapy, first as induction, followed by consoli-
dation therapy. The minimum total number is four, as
applied by the MRC group, and results of that group are
among the best in the world. Study MRC AML12 did
not show a statistically significant benefit for five cours-
es as compared to four, but that question was continued
to be studied in AML15, and pediatric results have not
yet been reported. Higher-risk patients theoretically
may benefit from an additional (fifth) course of
chemotherapy, but such a risk-group adapted therapy
has not been proven better in pediatric AML. The
NOPHO is studying the role of two doses of gem-
tuzumab ozogamicine as post-consolidation therapy,
which one might consider an extra course of
chemotherapy. The Japanese group reported that they
could safely reduce the number of consolidation cours-
es from eight to six to five for standard-risk patients and
to six for higher-risk patients.10,11 One may conclude that
between four and six courses of chemotherapy in total
are usually needed in pediatric AML. Depending on the
content of each course, the number of courses will also
influence the cumulative dose of anthracyclines or relat-
ed drugs. It seems fair to state that the optimal cumula-
tive dose is unknown. On the other hand, it is quite
clear that cumulative doses of anthracyclines above 300
mg/m2 are associated with increasing risk of cardiotoxi-
city.12
Do we need maintenance chemotherapy? Two ran-
domised studies have shown that maintenance
chemotherapy did not improve overall survival.13,14
In fact, both studies even suggested that maintenance
might be worse because of a lower salvage rate of
relapsed AML cases. Therefore, maintenance therapy
should not be given, although one cannot exclude the
possibility that some patients might actually benefit
from it.
Do we need prophylaxis of CNS relapse? The incidence
of AML cells in the cerebrospinal fluid at initial diagno-
sis is different among different studies, but in the range
of 5-30%. Moreover, CNS involvement at relapse is
observed in up to 10% of patients, while it was up to
20% in studies in which CNS prophylaxis had not been
used.15 Therefore, there are good arguments to apply
prophylaxis of CNS relapse. Most groups successfully
use intrathecal chemotherapy, in that their CNS relapse
rates or cumulative incidence of any relapse do not
seem to be higher than that reported by the BFM-AML
group, which still uses cranial irradiation. The latter pol-
icy is based on a single randomized study, and cranial
14th Congress of the European Hematology Association

Table 2. Incidence and prognostic significance of FLT3-internal tandem duplication in pediatric acute myeloid leukemia.

Authors/ Patient FLT3-ITD FLT-ITD p value Remarks

Year number positivity negativity
and outcome and outcome

Iwai 94 5 (5%) => 89 (95%) => 0.004

1999 5-yr EFS 20% 5-yr EFS 60%

Kondo 64 7 (11%) => 57 (89%) => 0.003 FLT3-ITD positive patients especially more induction failures
1999 5-yr EFS 14% 5-yr EFS 69%

Xu 87 12 (14%) => 75 (86%) => ? FLT3-ITD positive patients: more than 50% induction failure rate
1999 4-yr OS 0% ?

Meshinchi 91 15 (16%) => 76 (84%) => FLT3-ITD positive subgroup more induction failures and more relapses
2001 8-yr EFS 7% 8-yr EFS 44% 0.002
8-yr OS 13% 8-yr OS 50% 0.002

Liang 80 9 (11%) 71 (89%) n.s. Only 6 non-APL patients; all 3 cases with FLT3-ITD and AR>2.0 died
2002

Arrigoni 45 10 (22%) => 35 (78%) => 0.033

2003 5-yr EFS 20% 5-yr EFS 49%

Zwaan 234 27 (12%) => 207 (88%) => FLT3-ITD positive patients with AR>median had a significantly worse outcome
2003 CR rate 70% CR rate 88% 0.01 than FLT3 wild type patients, which was not found for FLT3-ITD positive
5-yr EFS 29% 5-yr EFS 46% 0.005 patients with AR<median (2-yr EFS 44 vs 61%, p=0.26)
5-yr OS 32% 5-yr OS 58% 0.037

Kang 61 4 (7%) => DFS 57 (93%) => ?

2005 0% DFS 52%

Meshinchi 630 77 (12%) => 553 (88%) => <0.001 FLT3-ITD positive patients with AR >0.4 had PFS of 16% vs.72% in patients
2006 4-yr PFS 31% 4-yr PFS 55% with AR<0.4 (p=0.001)

Meshinchi 630 77 (12%) 553 (88%) FLT3-ITD positive patients with a longer ITD length had a worse RFS (19 vs 51%,
2008 p=0.035), OS from CR (21% vs. 67% (p=0.006), and OS at 4 yr (17% vs 51%,
p=0.006) than those with a shorter ITD length

Shimada 158 17 (13%) => 141 (87%) =>

2008 3-yr RR 52% 3-yr RR 30% <0.005
3-yr DFS 40% 3-yr DFS 67% <0.003
3-yr OS 35% 3-yr OS 84% <0.001

Hollink 276 53 (19%) => 223 (81%) => 0.05 Within patients with NPM1 mutation, FLT3-ITD presence or absence: no
2009 5-yr EFS 25% 5-yr EFS 44% 0.04 prognostic impact; similar outcome for ITD positive patients with AR >0.4 and
<0.4
5-yr OS 42% 5-yr OS 63%
APL: acute promyelocytic leukemia; AR: allelic ratio (amount of FLT3 with ITD divided by wild type FLT3); CR: complete remission; DFS: disease-free survival;
EFS: event-free survival; n.s.: not significant; OS: overall survival; PFS: progression-free survival; RFS: relapse-free survival; RR: relapse rate.

irradiation provided a statistically significant benefit
only when randomized and non-randomized patients
were pooled, and mainly concerned less bone marrow
relapses.16 Indeed, a St. Jude study showed that even
CNS leukemia at initial diagnosis can be successfully
treated with chemotherapy only.17 In conclusion, pro-
phylaxis of CNS relapse is indicated using intrathecal
chemotherapy, either single-agent cytarabine or cytara-
bine in combination with prednisolone and methotrex-
ate, while there seems no place for prophylactic cranial
irradiation in the setting of current intensive chemother-
apy.
Do we need allo-SCT in CR1? There have been no ran-
domized studies on this important question. There
seems to be an increasing difference in the actual use of
allo-SCT in CR1 between the USA and European
groups. While COG still advocates its use in newly
diagnosed AML according to its most recent protocol,
except for core-binding factor leukemia, European
groups tend to reserve allo-SCT for patients who
relapse. Several study groups have done intention-to-
treat analyses, comparing the outcome between
patients with and those without a suitable donor. Most
studies seem to demonstrate a decreased incidence of

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 Berlin, Germany, June 4-7, 2009

Table 3. Incidence and type of recurrent cytogenetic and molecular abnormalities in pediatric acute myeloid leukemia.
Actual percentages differ slightly between groups and original studies, except for the t(15;17) translocation reflecting
acute promyelocytic leukemia, the incidence of which is highly dependent on the ethnicity of the population being stud-
ied.

Type of abnormality Percentage of cases Clinical relevance

Cytogenetic subtype
Normal karyotype 20 prognosis average
t(8;21) 12 good prognosis, especially in terms of overall survival
t(9;11) 7 prognosis differs per study group and additional risk factors; MRD marker
trisomy 8 8 prognosis average
trisomy 21 6 prognosis excellent in case of Myeloid Leukemia of Down syndrome
other 11q23 abnormalities 6 heterogeneous group; MRD marker
Inv(16) 6 prognosis good; MRD marker
t(15;17) 5 (may be much higher) prognosis excellent; enables tailored therapy; MRD marker
monosomy 7 3 prognosis poor
7q deletion 2 prognosis average
t(6;9) 2 prognosis supposed to be poor
monosomy 5/5q- 1-2 prognosis supposed to be poor
t(9;22) 0-1 prognosis supposed to be poor
other 21 heterogeneous

Molecular subtype
RAS mutation 17 prognosis average; marker for MRD?
FLT3-ITD 12 prognosis poor; treatment target; MRD monitoring, but be aware of instability
WT1 mutation 12 prognosis poor; suitable for MRD monitoring
KIT mutation 11 prognosis poor in some studies; treatment target; marker for MRD?
NPM1 mutation 8 prognosis good, might depend on FLT3 status; marker for MRD?
CEBPα mutation 6 prognosis good in some recent (unpublished) studies; marker for MRD?
PTPN11 mutation 5 potential treatment target; marker for MRD?
FLT3-TKD 5 treatment target; marker for MRD monitoring?
MRD; minimal residual disease. For all potential treatment targets and MRD markers, the possibility of (in-) stability of the target/marker during the course of the
disease must be taken into account.

relapses in the donor group, but this favourable effect is
diminished by increased treatment-related mortality
and perhaps decreased salvageability of patients that
relapse after allo-SCT in CR1. Thus, overall survival
rates are similar (at least, at a statistical level) in “donor”
versus “no-donor” groups. This is especially true in
studies in which chemotherapy alone results in event-
free survival rates above 50% and in overall survival
rates above 60%. In other words, if chemotherapy
results are suboptimal, the chance that allo-SCT shows
a benefit increases and vice versa. Together with the
costs associated with allo-SCT and its more severe side-
effects and late effects than associated with chemother-
apy only,18,19 it seems preferable to apply allo-SCT in
CR2 only. This is further supported by recent analyses
reported by the BFM-AML Group, MRC and even
COG, that poor risk AML patients do not benefit from
allo-SCT.20-22 The COG group recently reported that
intermediate risk AML patients benefit from allo-SCT,
while good and poor risk patients did not.21 However,
the overall survival with allo-SCT in that intermediate
risk group of 62% seems to be in the same range that is
being achieved by other groups with chemotherapy
only.20,23,24 Obviously, innovative therapies should be
tested in patients identified to have poor risk AML at
initial diagnosis. This is very briefly discussed below.
Autologous “SCT” (reinfusion would be more appropri-
ate) is not discussed here. Randomized studies showed
it is not superior to chemotherapy only.25,26
What is a good risk and what a poor risk AML patient?
This discussion becomes especially important if risk-
group directed therapy is being used. Most groups only
use prognostic risk factors for the decision whether a
patient is eligible for an allo-SCT or not. Probably the
only exceptions are the clinical studies done by the St.
Jude’s consortium. In view of the potential use of risk
factors for risk-group directed therapy, it is remarkable
that various groups interpret the prognostic significance
of several such clinical and cell-biological factors differ-
ently. For instance, t(9;22) is considered to be a poor risk
factor by the BFM-AML Group, NOPHO and Japanese
JPSLG, but as average by the MRC. Also, the t(9;11) is
considered to be a poor risk factor by the BFM-AML
group, as good by NOPHO and as average by MRC and
JPLSG. As final example, NOPHO does not consider
FLT3-internal tandem duplication (ITD) to be a poor risk
factor, while the BFM-AML Group and JPLSG do. Table
2 summarizes the currently available studies on the
prognostic significance of FLT3-ITD in pediatric AML,
as an example of an intensively studied novel prognos-
tic factor.27-38 The studies clearly indicate a worse out-
come associated with FLT3-ITD. However, length of
ITD, allelic ratio and the concurrent NPM1 status
should probably be taken into account for risk-group
directed therapy based on FLT3 status.
Partly the differences between groups in how they

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 217 |
14th Congress of the European Hematology Association
interpret the prognostic significance of a biological fea-
ture reflect differences in implementation of novel data
in ongoing and new protocols. It is also likely that dif-
ferent conclusions are drawn based on own treatment
protocols, which could be explained by chance or by an
actual influence of the treatment itself on the prognostic
significance of a given factor. However, it also reflects
the lack of solid data on the prognostic significance of
many of these factors in pediatric AML, such as seems
true; for example, t(9;22), t(6;9) and multilineage dyspla-
sia. Therefore, international retrospective and prospec-
tive studies are necessary to clarify these issues. In fact,
such collaborative studies have been done and are ongo-
ing. A good example is the effort coordinated by Hasle
et al., on the prognostic significance of monosomy and
7q-.39 Ongoing analyses focus on MLL gene rearranged
AML and on the reasons for the clinical heterogeneity
among AML patients with t(8;21). In general, important
prognostic factors are initial treatment response and
cytogenetic and molecular abnormalities. The latter bio-
logical factors are summarized in Table 3.
Molecular abnormalities may also provide leukemia-
specific treatment targets, and have been studied more
recently.28,40-43 The definition of a good and a poor risk
patient will become more important if we can actually
design more tailored therapies, as briefly discussed
below in the section “Innovative treatment of pediatric
AML.” However, instability of treatment targets should
be taken into account, as has been described for FLT3-
ITD.44
Are there subgroups of AML that should be treated differ-
ently? There is compelling evidence that APL should be
treated with drugs, including ATRA, at induction, con-
solidation and maintenance therapy. Traditionally, APL
has been treated with high doses of anthracyclines.
However, cumulative doses of more than 600 mg/m2 of
anthracyclines do not seem justified in such a curable
disease, in view of the severe cardiac late effects that
may be the consequence. Therefore, the international
consortium on childhood (ICC) APL recently developed
ICC APL Study 01, in which ATRA is used intensively,
together with intermediate-dose cyarabine and anthra-
cyclines at maximum cumulative doses of 355 mg/m2
for standard risk patients and of 405 mg/m2 for high-risk
patients. In addition, minimal residual disease monitor-
ing will be used to enable treatment of disease persist-
ing or relapsing at the molecular level. The protocol
includes guidelines for such situations, as well as for
frank relapse and CNS relapses. The study will open in
2009. Another group now treated separately by most
groups according to an international protocol concerns
Myeloid Leukemia of Down syndrome. This entity typ-
ically occurs in children below the age of 5 years, who
have the GATA1 mutation in their AML cells. With that
approach, probability of survival for this special sub-
group is above 85%. In the near future, it is likely that
AML patients with activating type I mutations, such as
FLT3-ITD and KIT mutations, will be treated with
chemotherapy plus tyrosine kinase inhibitors. This will
be discussed below in the section “Innovative treatment
of pediatric AML”.
| 218 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Relapsed acute myeloid leukemia
The most frequent event for newly diagnosed
patients with AML is a relapse, occurring in about one
in three children. Bone marrow usually is involved, and
the CNS in up to 10% of cases (including combined
relapses). Arbitrarily defined as within or after 1 year
from initial diagnosis, about 50% of patients have an
early or late relapse. Outcome from relapse is signifi-
cantly better in case of a longer duration of CR1.45,46
However, the international study Relapsed AML
2001/01 showed early treatment response to be an even
more important prognostic factor.47 In that study, early
response is determined by morphological examination
of the bone marrow obtained at day 28 from start of
reinduction, and defined as good in case of less than
20% AML blasts, and poor in case of 20% or more of
such blasts. Day 28 in practice is between days 28 and
42 from the start of reinduction therapy. In addition to
treatment response, favorable cytogenetics t(8;21) and
inv(16), as initially identified in studies with newly diag-
nosed children, also seems to be a favorable prognostic
factor at relapsed AML (48 and unpublished data).
Study Relapsed AML 2001/01 and several other recent-
ly published studies report a probability of long-term
survival from relapsed AML of more than 30%, as sum-
marized by Goemans et al.18 Therefore, reinduction
chemotherapy should be offered to all children and ado-
lescents with relapsed AML who can tolerate intensive
treatment. However, patients that respond poorly to the
first course of reinduction chemotherapy, patients that
do not achieve a second complete remission, and
patients that relapse again should be offered more
experimental therapy in that setting of a very dismal
prognosis. Fortunately, innovative treatment seems
achievable within 10-20 years from now.
Innovative treatment of pediatric acute myeloid leukemia
Significant improvements have already been achieved
with conventional drugs that have been available for the
treatment of AML for decades. Therefore, it seems real-
istic to expect major advances with the development of
new drugs and especially novel treatment modalities.
Discussion of all of these challenges is beyond the scope
of this manuscript, but has been reviewed elsewhere.49
In brief, innovative treatment will consist of at least four
different modalities. First, the development of novel but
conventional drugs. A good example might be clofara-
bine, a relatively novel nucleoside analogue that is being
studied in both children and adults with AML. Second,
the design of drugs with novel mechanisms of action,
often targeted at leukemia-specific abnormalities.
Typical examples are monoclonal-antibody mediated
treatment, such as with gemtuzumab ozogamicin (tar-
geting CD33-positive cells) and with tyrosine kinase
inhibitors (targeting e.g., Flt3- or kit-mutated AML
cells). Third, improving the graft-versus-leukemia effect
of donor cells without an increase in graft-versus-host-
disease. Finally, the development of vaccination and
other immunotherapy approaches. Innovative treat-
ment will become more and more tailored and person-
alized, which necessitates the need for large-scale, and
thus, international collaboration. Such collaborative
efforts will be the only way to enrol a sufficient number
of patients with a specific subtype of AML in a given

study. Despite such an international setting, random-
ized studies in large cohorts of patients will become
more often impossible because of limited patient num-
bers eligible for subgrou p-directed therapy. Thus, we
also need innovative trial designs. Fortunately, interna-
tional large-scale collaboration has been realised in the
past 10-20 years, especially in the setting of the
International BFM Study Group, the so-called
International Pediatric AML Group, and the
International Consortium for Childhood APL. These
collaborations, together with the efforts of many pro-
fessionals in pediatric AML will undoubtedly further
improve the outcome of this disease in terms of cure,
side-effects and late effects.
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10. Tomizawa D, Tabuchi K, Kinoshita A, Hanada R, Kigasawa H,
Tsukimoto I, , et al. Repetitive cycles of high-dose cytarabine
are effective for childhood acute myeloid leukemia: long-term
outcome of the children with AML treated on two consecutive
trials of Tokyo Children’s Cancer Study Group. Tokyo
Children's Cancer Study Group. Pediatr Blood Cancer 2007;
49:127-32.
11. Tsukimoto I, Tawa A, Horibe K, et al. Intensive regimens with
an increasing dose density of cytarabine improves the out-
come in childhood acute myeloid leukemia. J Clin Oncol 2009
[Epub ahead of print]
12. Kremer LC, van Dalen EC, Offringa M, Ottenkamp J, Voûte
PA. Anthracycline-induced clinical heart failure in a cohort of
607 children: long-term follow-up study. J Clin Oncol 2001;
19:191-6.
13. Perel Y, Auvrignon A, Leblanc T, Michel G, Reguerre Y,
Vannier JP, et al. Treatment of childhood acute myeloblastic
leukemia: dose intensification improves outcome and mainte-
nance is of no benefit: multicenter studies of the French LAME
(Leucémie Aiguë Myéloblastic Enfant) Cooperative Group.
Leukemia 2005;19:2082-9.
14. Wells RJ, Woods WG, Buckley JD, et al. Treatment of newly
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15. Pui CH, Schrappe M, Ribeiro RC, Niemeyer CM. Childhood
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16. Creutzig U, Ritter J, Zimmermann M, Schellong G. Does cra-
nial irradiation reduce the risk for bone marrow relapse in
acute myelogenous leukemia? Unexpected results of the
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Clin Oncol 1993;11:279-86.
17. Abbott BL, Rubnitz JE, Tong X, Srivastava DK, Pui CH,
Ribeiro RC, et al. Clinical significance of central nervous sys-
tem involvement at diagnosis of pediatric acute myeloid
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17:2090-6.
18. Goemans BF, Tamminga RY, Corbij CM, Hählen K, Kaspers
GJ. Outcome for children with relapsed acute myeloid
leukemia in the Netherlands following initial treatment
between 1980 and 1998: survival after chemotherapy only?
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19. Leung W, Hudson MM, Strickland DK, Phipps S, Srivastava
DK, Ribeiro RC, et al. Late effects of treatment in survivors of
childhood acute myeloid leukemia. J Clin Oncol 2000;18:
3273-9.
20. Gibson BE, Wheatley K, Hann IN, Stevens RF, Webb D, Hills
RK, et al. Treatment strategy and long-term results in pediatric
patients treated in consecutive UK AML trials. Leukemia 2005;
19:2130-8.
21. Horan JT, Alonzo TA, Lyman GH, Gerbing RB, Lange BJ,
Ravindranath Y, et al. Impact of disease risk on efficacy of
matched related bone marrow transplantation for pediatric
acute myeloid leukemia: the Children’s Oncology Group. J
Clin Oncol 2008;26:5797-801.
22. Reinhardt D, Kremens B, Zimmermann M. No improvement
of overall-survival in children with high-risk acute myeloid
leukemia by stem cell transplantation in 1st complete remis-
sion. Blood 2006;108:99a[Abstract].
23. Creutzig U, Zimmermann M, Lehrnbecher T, et al. Less toxic-
ity by optimizing chemotherapy, but not by addition of gran-
ulocyte colony-stimulating factor in children and adolescents
with acute myeloid leukemia: results of AML-BFM 98. J Clin
Oncol 2006;24:4499-506.
24. Lie SO, Abrahamsson J, Clausen N, Graf N, Hermann J,
Niemeyer CM, et al. Long-term results in children with AML:
NOPHO-AML Study Group--report of three consecutive tri-
als. Leukemia 2005;19:2090-100.
25. Ravindranath Y, Yeager AM, Chang MN, Steuber CP, Krischer
J, Graham-Pole J, et al. Autologous bone marrow transplanta-
tion versus intensive consolidation chemotherapy for acute
myeloid leukemia in childhood, Pediatric Oncology Group. N
Engl J Med 1996;334:1428-34.
26. Woods WG, Neudorf S, Sanders J, Sanders J, Buckley JD,
Barnard DR, et al. A comparison of allogeneic bon marrow
transplantation, autologous bone marrow transplantation, and
aggressive chemotherapy in children with acute myeloid
leukemia in remission: a report from the Children’s Cancer
Group. Children's Cancer Group. Blood 2001;97:56-62.
27. Arrigoni P, Beretta C, Silvestri D, Rossi V, Rizzari C, Valsecchi
MG, et al. FLT3 internal tandem duplication in childhood
acute myeloid leukaemia: association with hyperleucocytosis
in acute promyelocytic leukaemia. Br J Haematol 2003;120:89-
92.
28. Hollink IH, Zwaan CM, Zimmermann M, Arentsen-Peters TC,
Pieters R, Cloos J, et al. Favorable prognostic impact of NPM1
gene mutations in childhood acute myeloid leukemia, with
emphasis on cytogenetically normal AML. Leukemia 2009;23:
262-70.
29. Iwai T, Yokota S, Nakao M, Okamoto T, Taniwaki M,
Onodera N, et al. Internal tandem duplication of the FLT3 gene
and clinical evaluation in childhood acute myeloid leukemia.
The Children’s Cancer and Leukemia Study Group, Japan.
Leukemia 1999;13:38-43.
30. Kang HJ, Hong SH, Kim IH, Park BK, Han KS, Cho HI, et al.
Prognostic significance of FLT3 mutations in pediatric non-
promyelocytic acute myeloid leukemia. Leuk Res 2005; 29:
617-23.
31. Kondo M, Horibe K, Takahashi Y, Matsumoto K, Fukuda M,
Inaba J, et al. Prognostic value of internal tandem duplication
of the FLT3 gene in childhood acute myelogenous leukemia.
Med Pediatr Oncol 1999; 33:525-9.
32. Liang DC, Shih LY, Hung IJ, Yang CP, Chen SH, Jaing TH, et al.
Clinical relevance of internal tandem duplication of the FLT3
14th Congress of the European Hematology Association
gene in childhood acute myeloid leukemia. Cancer 2002; 94:
3292-8.
33. Meshinchi S, Woods WG, Stirewalt DL, Sweetser DA,
Buckley JD, Tjoa TK, et al. Prevalence and prognostic signifi-
cance of Flt3 internal tandem duplication in pediatric acute
myeloid leukemia. Blood 2001;97:89-94.
34. Meshinchi S, Alonzo T, Stirewalt DL, Zwaan M, Zimmerman
M, Reinhardt D, et al. Clinical implications of FLT3 mutations
in pediatric AML. Blood 2006;108:3654-61.
35. Meshinchi S, Stirewalt DL, Alonzo TA, Boggon TJ, Gerbing
RB, Rocnik JL, et al. Structural and numerical variation of
FLT3/ITD in pediatric AML. Blood 2008;111:4930-3.
36. Shimada A, Taki T, Tabuchi K, Taketani T, Hanada R, Tawa
A, et al. Tandem duplications of MLL and FLT3 are correlated
with poor prognoses in pediatric acute myeloid leukemia: a
study of the Japanese childhood AML Cooperative Study
Group. Pediatr Blood Cancer 2008;50:264-9.
37. Xu F, Taki T, Yang HW, Hanada R, Hongo T, Ohnishi H, et al.
Tandem duplication of the FLT3 gene is found in acute lym-
phoblastic leukaemia as well as acute myeloid leukaemia but
not in myelodysplastic syndrome or juvenile chronic myeloge-
nous leukaemia in children. Br J Haematol 1999;105:155-62.
38. Zwaan CM, Meshinchi S, Radich JP, Veerman AJ, Huismans
DR, Munske L, et al. FLT3 internal tandem duplication in 234
children with acute myeloid leukemia (AML): prognostic sig-
nificance and relation to cellular drug resistance. Blood
2003;102:2387-94.
39. Hasle H, Alonzo TA, Auvrignon A, Behar C, Chang M,
Creutzig U, et al. Monosomy 7 and deletion 7q in children and
adolescents with acute myeloid leukemia: an international ret-
rospective study. Blood 2007;109:4641-7.
40. Goemans BF, Zwaan CM, Martinelli S, Harrell P, de Lange D,
Carta C, et al. Differences in the prevalence of PTPN11 muta-
tions in FAB M5 paediatric acute myeloid leukaemia. Br J
Haematol 2005;130:801-3.
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41. Goemans BF, Zwaan CM, Miller M, Zimmermann M, Harlow
A, Meshinchi S, et al. Mutations in KIT and RAS are frequent
events in pediatric core-binding factor acute myeloid
leukemia. Leukemia 2005;19:1536-42.
42. Hollink IH, van den Heuvel-Eibrink MM, Zimmermann M,
Balgobind BV, Arentsen-Peters ST, Alders M, et al. Clinical rel-
evance of Wilms’ tumor 1 gene mutations in childhood acute
myeloid leukemia. Blood 2009;[Epub ahead of print]
43. Liang DC, Shih LY, Huang CF, Hung IJ, Yang CP, Liu HC, et al.
CEBPα mutations in childhood acute myeloid leukemia.
Leukemia 2005;19:410-4.
44. Cloos J, Goemans BF, Hess CJ, van Oostveen JW, Waisfisz Q,
Corthals S, et al. Stability and prognostic influence of FLT3
mutations in paired initial and relapsed AML samples.
Leukemia 2006;20:1217-20.
45. Stahnke K, Boos J, Bender-Götze C, Ritter J, Zimmermann M,
Creutzig U. Duration of first remission predicts remission
rates and long-term survival in children with relapsed acute
myelogenous leukemia. Leukemia 1998;12:1534-8.
46. Webb DKH. Management of relapsed acute myeloid
leukaemia. Br J Haematol 1999;106:851-9.
47. Kaspers GJL, Zimmermann M, Reinhardt D. Prognostic signif-
icance of time to relapse in pediatric AML: results from the
International randomised Phase III Study Relapsed AML
2001/01. Blood 2008;112:976.
48. Webb D, Wheatley K, Harrison G, Stevens RF, Hann IM.
Outcome for children with relapsed acute myeloid leukemia
following initial therapy in the Medical Research Council
(MRC) AML 10 trial. MRC Childhood Leukaemia Working
Party. Leukemia 1999;13:25-31.
49. Kaspers GJL, Zwaan CM. Pediatric acute myeloid leukemia:
towards high-quality cure of all patients. Haematologica 2007;
92:1519-32.
 Pediatric hematological malignancies

Pediatric myeloproliferative neoplasms

M.L. Randi
M.C. Putti1
Dept of Medical and Surgical
Sciences, Internal Medicine;
1
Dept of Pediatrics, Clinic
Hemato-Oncology, University of
Padua Medical School, Italy
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:221-226
A B S T R A C T
The increasing knowledge accumulated over recent years in adults with Philadelphia-negative
myeloproliferative disorders (Ph-MPD) has prompted better evaluation of the rare pediatric essential
thrombocythemia (ET), polycythemia vera (PV) and primary idiopathic myelofibrosis (PMF). A few cases
of PV, ET and PMF in children were published in the English literature and an up-date of these works
is here given. The experience recently collected by the Italian Pediatric Hemato-Oncology Association
is also reported. Overall, the findings suggest that pediatric Ph-negative MPD are heterogeneous dis-
eases and before the diagnosis is made, hereditary disorders have to be excluded. Because JAK2 and
other genes mutations are only detectable in a minority of children with sporadic forms, complemen-
tary markers, such as clonality of hematopoieis, spontaneous erythroid colony growth, and so on
should be performed for the diagnosis and new tests have to be identified. Diagnostic criteria that fit
pediatric ET, PV and PMF have to be considered. Prognosis in children is still unknown and treatment
guidelines need to be established; therefore, prospective observations and clinical trials are needed.

hronic Philadelphia-negative myelo- detailed that these diseases are classified as

C proliferative diseases (Ph-MPD), now

better defined as myeloproliferative
neoplasms, are clonal1,2 pathologies charac-
terized by an increased proliferation of mul-
tipotent hematopoietic progenitors and a
prolonged clinical course. They are frequent-
ly complicated by thrombotic and hemor-
rhagic events and a possible transformation
to myelofibrosis and/or acute leukemia.3 In
the past, they were considered as orphan
diseases, but the recent identification of typ-
ical mutations of Ph-MPD in adults gave
new interest to these diseases. In the spring
of 2005, five different research groups4-8
identified the presence of the V617F muta-
tion in the gene of Janus Kinase 2 (JAK2) in
most Ph-MPD patients. This is a protein
involved in the signaling transduction. This
mutation determines a constitutive activa-
tion of JAK2, which continuously stimulates
STAT54-7 leading to proliferation of normally
maturing cells. Later, other less common
biological markers in Ph-MPD were found:
exon 12 JAK29 and thrombopoietin receptor
(MPL)10 mutations.
From a clinical point of view, the discov-
ery of the biological markers greatly
improved the specificity of the diagnostic
criteria for Ph-MPD. For a long time, the
available criteria were those proposed by the
Polycythemia Vera Study Group (PVSG).11,12
In 2001, the first World Health Organization
(WHO) criteria were published13 and they
have been improved recently.3 The relevance
given to JAK2V617F is justified by the
demonstration that this mutation is now
known to occur in virtually all patients
affected by Polycythemia Vera (PV) and in
half or little more of those affected by essen-
tial thrombocythemia (ET) and primary
myelofibrosis (PMF).14 The WHO13 also
chronic myeloproliferative neoplasm, as
well as chronic myelogenous leukemia and
less common forms (chronic neutrophilic
leukemia, chronic eosinophilic leukemia/
hypereosinophilic syndrome and unclassifi-
able chronic myeloproliferative diseases).
Regarding pediatric Ph-MPD, a greater
interest has emerged only recently, mainly
on ET. This is not surprising if we consider
that this is the Ph-MPD, which manifests
not only in median-advanced age but also in
young people.5,16,17 In about 20% of cases, it
is diagnosed in people below 40 years of
age.18
Essential thrombocythemia
ET is an acquired Ph-MPD characterized
by a sustained increase of platelet count over
the normal level. It is more common (dou-
ble) in females than in males; its incidence is
1.5/100,000 persons/year and the median
diagnostic age is 65-70 years.19 After the dis-
covery of JAK2V617F mutation in about
50% of ET14 and of MPLW515K mutation in
about 1%,10 the WHO3 developed new diag-
nostic criteria, which in the presence of a
sustained platelet count over 450x109/L,
large and mature megakaryocytes prolifera-
tion in bone marrow, without PV, PMF,
chronic myelogenous leukemia, myelodys-
plastic syndromes and causes of re-
active/secondary thrombocytosis, gave a
first line importance to the presence of a
clonal marker, such as JAK2V617F. An
increased platelet number over 500x109/L is
extremely common in children to such an
extent that a secondary/reactive thrombocy-
tosis is found in 5-6% of hospitalized chil-
dren.20,21 This is mainly due to asplenic con-
ditions (splenectomy) infections, Kawasaki
syndrome, autoimmunity, tumors, drugs,

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 221 |
14th Congress of the European Hematology Association
surgical procedures and iron deficiency. Some other
pediatric thrombocytosis are found in children from
kindreds with familial thrombocytosis.22 Finally, a small
number of children are affected by primary thrombocy-
tosis; it has been evaluated that 0.09/1,000,000 chil-
dren/year23 are affected by ET. This means that ET in
childhood is 60 times lower than in adults.18 The distri-
bution of ET diagnosis in different ages in our experi-
ence is shown in Figure 1.
The first report of hemorrhagic thrombocythemia in a
child was published in 1960.24 The patient was treated
with radiophosphorus (32P) and died of acute leukemia. A
total of 94 cases, 23 case reports and 10 small series with
less than 15 patients each25-34 and 2 reviews21,35 have been
published on the topic. The children are characterized by
young age, high platelets counts frequent positive familial
history and a quite severe clinical picture, with about
20% of hemorrhagic or thrombotic complications. Rare
cases of transformation are also reported, possibly related
to drugs use.24,36-38 Evolution to myelofibrosis was found in
three patients (one treated with hydroxyurea and two
untreated) and to acute leukemia in two (one treated with
busulphan and one with phosphorus).32
The mean patients’ age at diagnosis was 7.44 years
(range 2 months to 16 years) with only four infants. There
were some more females (1.5:1), the follow-up ranged
between 10 months and 16 years, the platelet was 600-
4000x109/L, with half cases with a platelet count over
1500x109/L. In 35 patients, splenomegaly was described.
It is important to note that some reported cases belong to
families with thrombocythemia and these children seem
to have less thrombotic events then those with sporadic
forms. The risk of hemorrhagic and thrombotic events
seem to be higher in children with a platelet count over
1.500-2.000x109/L. Symptoms were described in 40% of
children, the most common of which was headache. One
fifth of patients developed major vascular complications
(five arterial and nine venous, mainly in splancnic or cere-
| 222 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Figure 1. Age distribution at diag-
nosis of ET in 350 patients diag-
nosed in University of Padua over
the last 10 years. Pediatric
patients represent 3% of total;
while diagnoses in females are
distributed all over pediatric age,
all males were diagnosed in schol-
ar age. It has to be underlined that
our departments are third level
centres for myeloproliferative neo-
plasms
bral district) and two of them died (one myocardial infarc-
tion and one multiple vein thrombosis).39 Platelet func-
tional studies27 did not give evidence of significant alter-
ations as in adults,40 serum EPO was found to be within
normal limits,27,41 and no patients with spontaneous ery-
throid colony (EEC) growth in vitro were found.27
Moreover, in 2004 our group (42) evaluated seven spo-
radic cases of children with ET and a platelet count over
900x109/L with normal plasma thrombopoietin (TPO)
level, without finding any mutation of TPO or of MPL
genes.
The use of drugs was extremely variable: within the
patients in whom the therapy adopted was reported, 12
did not receive any drug, 1 received warfarin, 5 aspirin, 2
alpha-interferon (α-IFN) (in 1 case with aspirin), 5 hydrox-
yurea, 3 busulphan, 1 radiophosphorus, 2 anagrelide (in 1
case after α-IFN) and 1 polychemotherapy.
The Italian Pediatric Hemato-Oncology Association
(AIEOP) recently43 collected a retrospective cohort of 90
children (under 16 years of age), all diagnosed to be affect-
ed by ET in agreement with the criteria in use at the time
of the first diagnosis, followed in 14 different pediatric
centers, plus 16 cases pertaining to 6 different families.22
This represents the largest series ever published on this
topic. The main clinical features of Italian pediatric ET, as
well as the treatment adopted are summarized in Table 1.
Not all the diagnostic procedures were used in this
cohort, in particular, the bone marrow biopsy, which is
not so easy in children as in adults, because it needs a pro-
found sedation. The biological studies were all performed
in three centers (Padua, Rome and Turin) utilizing the cen-
tralization system in use for leukemia studies in Italy.
The work concluded that ET might be heterogeneous,
with different features from adult ET. The children seem
to have a mild clinical pictures (even with high platelets
counts and long follow-up) and headache was the com-
monest symptom. Vascular complications are infrequent
and do not correlate with a high leukocyte count at diag-
 Berlin, Germany, June 4-7, 2009

nosis in agreement with Passamonti44 but not with Table 1. Main clinical and laboratory data of 90 Italian
Carobbio45 in adult ET. The myeloproliferative origin of patients with ET.
ET in pediatric age is confirmed only in a minority of chil-
dren46,47 and its correlation with thrombosis, mainly in N° of patients 90
unusual veins, is suggestive as in adults ET.48 While about
one quarter of children with ET carry the JAK2V617F
mutation, no mutation of MPL515 was found, while in Males/Females 28/62
adults with sporadic ET, it is demonstrated in about 1 to Age at diagnosis (range) 6.75 years (1 month-17 years)
Follow-up (years)(range) 5 (2-11)
3% of cases.10 It is interesting to note that the presence of
Plts×109/L (range) 1260 (611-4020)
MPL mutations in ET seems to represent a significant risk WBC109/L 11.75
factor for microvessels disturbances.49 JAK2V617F 16/65 (24%)
Regarding treatment (Table 2), through interview to the EEC positive 17/28
different pediatricians in the different centers, we con- Monoclonality 15/32
cluded that, in absence of any guidelines for ET in chil- MPL/TPO mutation none
dren, the physician’s aims were: (i) to resolve symptoms; No signs, symtoms or complications 52 (55%)
(ii) to reduce platelets at least to 500 to 600 x109/L. Budd Chiari syndrome 2
Low dose aspirin (50-100 mg/daily) have been used Deep Venous Thrombosis 2
alone or associated with cytoreductive agents, mainly Headache 20
hydroxyurea; in one patient aspirin was suspended due to Paresthesia 3
epistaxis. Two patients refused the use of a-IFN and 1 Nosebleed 2
anagrelide for headache. Over a median follow up of 5 Abdominal pain 4
years, no case of transformation under hydroxyurea or Splenomegaly 19
other treatments was observed. Peripheral neuropathy 1
Not surprisingly, in this retrospective study, treatment
has been heterogeneous, not following any established
criteria50-52 and each pediatrician chose based on their own
experience. Aspirin was used mainly in patients with
headache, between cytoreductive drugs hydroxyurea38,53 Table 2. Treatment adopted in 90 Italian children with ET.
and anagrelide were preferred54,55 to α-IFN.56 It is remark- The extreme variability of the options of different physi-
able that a recent paper57 demonstrates a significant cians is evident.
reduction of JAK2V617F allele burden in ET patients after Treatment adopted N° of patients
first year of hydroxyurea therapy. Therefore, considering
that the leukemogenic potential of hydroxyurea in ET is None never 28
still now debated,58 we think that it may be useful to re- Low dose aspirin (ASA) only 16
consider the use of this drug in young patients with ET Warfarin only 1
and a previous major thrombotic event. α-IFN only 3
The observation of the results of these treatments dial Anagrelide only 2
in some relevant questions: Is anti-aggregation necessary? Hydroxyurea only 9
Is it dangerous with the common high level of platelets α-IFN + aspirin 2
counts of children? Is cytoreduction necessary? Which Anagrelide +ASA 3
long-term drug toxicity is predictable in ET children?59 Hydroxyurea + ASA 3
Hydroxyurea >anagrelide 5
Polycythemia vera Hydroxyurea > α-IFN > anagrelide 1
Polycthemias or erythrocytosis are characterized by the Hydroxyurea > anagrelide > α-IFN 2
expansion of erythrocyte compartment in the peripheral α-IFN > anagrelide 3
blood reflected by an increase of erythrocyte count, α-IFN > anagrelide >α-IFN 1
hemoglobin content and hematocrit. Absolute erythrocy- α-IFN > hydroxyurea 4
α-IFN > anagrelide >hydroxyurea 1
tosis,11,60 as defined by the increase of total red cell mass,
Hydroxyurea >α-IFN 3
must be distinguished from relative erythrocytosis caused Pipobroman > hydroxyurea > anagrelide 2
by a severe reduction of the plasma volume. Plateltpheresis > hydroxyurea > anagrelide >α-IFN 1
Absolute secondary erythrocytosis is driven by increase
of erythropoietin (EPO),61 which may be due to physio- α-IFN: α-interferon.
logic response to tissue hypoxia or abnormal EPO produc-
tion or a deregulation of the oxygen-dependent EPO syn-
thesis. The more common causes of secondary erythro- thropoietic compartment is expanded independently of
cytsis in children are renal diseases (cancers, transplanta- EPO level, is extremely rare in pediatric patients. In this
tions, and malformations), EPO-producing tumors group of diseases, primary familial and congenital poly-
(hemangioblastoma, hepatic adenoma) and Down syn- cythemia (PFCP) caused by a number of mutations in
drome.62 The congenital forms of secondary erythrocyto- cytoplasmic domain of the erythropoietin receptor (EPO-
sis may be due to abnormal hemoglobin with high oxy- R) gene70 and polycythemia vera (PV), the only clonal
gen affinity,63 to 2 to 3 bisphosphoglycerate deficiency,64 acquired primary erythrocytosis, are comprehended.62 PV
to altered oxygen-sensing pathway genes (HIF-1-α, is one of the Ph-MPD, its incidence is of 2/100,000 per-
PHD2, VHL).65-69 sons/year19 and almost all the patients carry the acquired
Primary erythrocytosis, a condition in which the ery- JAK2V617F point mutation;14 a somatic gain-of function

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14th Congress of the European Hematology Association
mutations affecting JAK2 exon 12 is identified in 25% of
PV patients.9,71 The 2008 WHO3 criteria established that
patients with increased hemoglobin or hematocrit, or ele-
vated RBC mass, in the presence of JAK2V617F or similar
mutation are affected by PV. In the few cases without any
mutation, a minor criterion is needed (1-bone marrow tri-
lineage myeloproliferation or 2-subnormal serum EPO
level, or 3-spontaneous EEC growth).
Few cases of PV in childhood have been published in the
English literature. Before 2005, about 30 cases in children
and adolescents were described.62 In recent years, we found
only 18 children with PV;72-75 all presented with plethora
and hematocrit levels over 65%. The research of JAK2
mutations were performed in 17 of these children: 10 had
classic V617F mutation, 2 had exon 12 mutations (in one
case PV developed after treatment for large cell anaplastic
lymphoma)75 and the remaining 5 were JAK2 wild type.
A new case of PV in a 13-year-old girl was found in
Padua University. The patient suffers for headache and
undergoes periodical phlebotomies. She has EEC sponta-
neous formation and carries JAK2V617F mutation with
an allele burden of 35%. Similarly, three other cases in
Rome have an allele burden lower than 50% significantly
lower than 13 ET patients.76
No indications are available regarding the best manage-
ment of PV in children. Most authors used phlebotomies
to maintain the Ht below 50%,60 but, for example, the use
of low-dose aspirin77 is scarce.
Myelofibrosis
PMF is a rare Ph-MPD (estimated incidence 0.4-
0.7/100,000 persons/year)79 usually affecting elderly peo-
ple reducing the life expectancy.79 PMF manifests clinical-
ly as anemia, splenomegaly due to extramedullary
hematopoiesis, leukoerythroblastosis and constitutional
symptoms.80,81 It implies an increase in the bone marrow
fiber with reticulin and/or collagen deposition,82 which
occurs in response to a clonal proliferation of hematopoi-
etic stem cells. It also leads to a profound hyperplasia of
morphologically abnormal megakaryocytes (MK) and
populations of monocytes that release fibrogenic growth
factors. PMF is characterized by the constitutive mobi-
lization of CD34+ cells, as well as endothelial progenitor
cells into the peripheral blood.83 Approximately 50% of
patients with PMF are JAK2V617F positive4 and 5% har-
bour MPLW515L mutations.84,85 As well as for other Ph-
MPD, and also for PMF, the WHO3 has recently devel-
oped diagnostic criteria. All major criteria are requested:
(i) proliferation of atypical megakaryocytes with reticulin
and/or collagen fibrosis or increased granulocytic prolifer-
ation with decreased erytrhopoiesis; (ii) not meeting
WHO criteria for chronic myelogenous leukemia, PV,
myelodysplasia or other myeloid neoplasms; (iii) demon-
stration of clonal marker or no evidence of reactive mar-
row fibrosis, together with at least 2 minor criteria ((i) leu-
coerythroblastosis; (ii) increased serum LDH; (iii) anemia;
(iv) palpable spleen). PMF and secondary myelofibrosis is
extremely rare in childhood.86 The last was described as
evolution of neoplastic diseases (myeloproliferative neo-
plasms, myelodysplastic syndromes, reticulum cell sarco-
ma, neuroblastoma, Fanconi anemia, acute leukemia,
mainly megakaryocytic leukemia carrying t(1;22) translo-
cation, Hodgkin disease, rabdomyosarcoma, systemic
| 224 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
mastocytosis, histiocytosis), rheumatic diseases (juvenile
rheumatoid arthritis, systemic lupus erythematosus),
sickle cell anemia, infectious diseases, vitamin D deficien-
cy rickets and severe combined immunodeficiency.
At present, PMF has been described, as our best knowl-
edge, in 11 children (7 males and 4 females),32,87-89 mainly
in children of Indian or neighboring countries origins.72,90,91
The age at diagnosis ranged between 3 months and 13
years, and almost all were in pre-school age. The numer-
ous neonatal cases suggested a congenital pathogenesis
beginning in utero.87 In all the published data, the diagno-
sis was performed with a dry tap and the observation a
hypercellular bone marrow with excess of reticulin and
abnormal megakaryocytes. Almost all children had ane-
mia (Hb 50-90 gr/L) with teardrops poikilocytosis. They
had normal to mildly elevated leucocyte counts (5 to
35x109/L), variable platelet counts (10 to 1970x109/L) and
extramedullary hematopoiesis with or without
splenomegaly and/or hepatomegaly, but no karyotype
alteration. In two patients, spontaneous EEC were found.
From a clinical point of view, two patients had spleno-
portal thrombosis and one girl died soon after diagnosis
for septicemia while other patients had a long (3 to 21
years) and relatively benign course. Therefore, the clinical
and laboratory findings suggested that PMF presenting in
infancy may represent a distinct entity compared with
the disease in adults and a conservative approach to clin-
ical management has been recommended.87
Currently, the AIEOP group has found only one 13-
year-old girl affected by PMF in cellular phase (WBC
19.42x109/L, platelets 2444x109/L, Hb 103 g/L). The diag-
nosis was performed in agreement with WHO criteria; in
particular, the patient had splenomegaly (16 cm longitudi-
nal diameter), teardrops poikilocytosis and increased cir-
culating CD34+ cells (73x106/L); her bone marrow was
hypercellular with a great increase of reticulin and the
presence of anomalous megakaryocytes in clusters. Both
JAK2 and MPL genes were wild type, spontaneous EEC
were positive while clonality evaluation resulted skew-
ing. At present, the girl is treated with anagrelide. A pre-
vious therapy with low dose aspirin was discontinued
because of gastric ulcer.92
The treatment of patients with PMF is largely palliative
and therapeutic interventions are mostly used in the
patients with symptoms. A number of drugs have been
used in PMF, but, at present, they have not been shown to
alter the natural history of PMF;93 however, targeted ther-
apies for PMF, that is, small molecule inhibitors of JAK2,
are now under evaluation to assess their efficacy and their
potential associated toxicities.94,95
Overall, the findings collected show that polycythemia,
thrombocythemia and myelofibrosis in childhood are
heterogeneous diseases. In the diagnostic screening, the
first step should be to investigate the possible presence of
a familial occurrence.96 Because genes mutations are
detectable in a minority of children, other tests have to be
performed (e.g., clonality, EEC) for the diagnosis and new
markers have to be searched; diagnostic criteria that fit for
pediatric ET, PV and PMF have to be considered.
Prognosis in children is still unknown and treatment
guidelines need to be established: therefore, prospective
observations and clinical trials are needed.

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 Red cell disease

Red cell membrane disorders

N. Mohandas A B S T R A C T

New York Blood Center,
New York, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:227-232
During the last three decades, molecular, biochemical and biophysical studies have enabled the
development of detailed insights into altered membrane function in inherited red cell membrane dis-
orders. This paper highlights our current understanding of disorders involving either altered membrane
assembly (hereditary spherocytosis, hereditary elliptocytosis and hereditary ovalocytosis) or altered
membrane transport function (hereditary stomatocytosis). Mutations in multiple genes account for
hereditary spherocytosis and elliptocytosis, while a single genetic defect accounts for all cases of
hereditary ovalocytosis. The structural basis for altered membrane function of spherocytosis and ellip-
tocytosis but not of ovalocytosis has been delineated. Loss of vertical linkage between lipid bilayer and
membrane skeleton leads to membrane loss in spherocytosis, while weakening of lateral linkages
between skeletal proteins leads to membrane fragmentation and surface area loss in elliptocytosis. The
severity of anemia in both these disorders is related to extent of membrane loss and splenectomy ame-
liorates the anemia. Ovalocytosis is associated with mild anemia. In terms of membrane transport
defects, two distinct phenotypes have been identified: dehydrated stomatocytosis with mild to mod-
erate anemia and overhydrated stomatocytosis with moderate to severe anemia. Molecular basis for
stomatocytoses has not been fully defined. Splenectomy is contraindicated in both forms of stomato-
cytosis.

n order to perform its function of oxygen

I
delivery, the discoid human red cell
undergoes extensive passive deformation
during repeated passage through the narrow
capillaries of the microvasculature during its
120-day life span in the circulation.
Decreased cellular deformability, not only
compromises the ability of the red cell to
perform its function of oxygen delivery opti-
mally, but also leads to its premature
removal from circulation by the spleen. The
importance of red cell deformability was
first recognized by Anton van Leeuwenhoek
in 1675 when he stated that when he was
greatly disordered, the globules of his blood
appeared hard and rigid, but grew softer and
more pliable as his health returned: whence he
infers that in a healthy body they should be soft
and flexible (Van Leeuwenhoek, 1675). This
truly prescient observation made more than
three centuries ago has since been extensive-
ly validated.
During the last four decades, comprehen-
sive biochemical, biophysical and molecular
biological studies on red cells from normal
individuals and individuals with various red
membrane disorders have enabled the devel-
opment of detailed molecular insights into
structural basis for membrane function in
health and disease.1-7 This presentation high-
lights our current understanding of the
molecular and structural basis for various red
cell membrane disorders, and discusses how
these new insights have contributed to the
understanding of their pathophysiology and
clinical manifestations.
Structural organization of normal red cell membrane
The structural organization of the mem-
brane is responsible for endowing the cell
with its ability to undergo repeated exten-
sive reversible deformations while main-
taining its structural integrity during its life
span in circulation. The red cell membrane
is highly elastic, responds rapidly to applied
stresses and is capable of undergoing large
membrane extensions without fragmenta-
tion. These unusual membrane material
properties are the result of a composite
structure, in which a plasma membrane
envelope composed of amphiphilic lipid
molecules is anchored to a two dimensional
elastic network of skeletal proteins through
tethering sites (transmembrane proteins)
embedded in the lipid bilayer (Figure 1).3,6,7
There is also evidence suggesting a direct
interaction of skeletal proteins with the
anionic phospholipids.8,9
The lipid bilayer is composed of equiva-
lent amounts of cholesterol and phospho-
lipids. A significant feature of bilayer lipid
organization is that various phospholipids
are asymmetrically distributed with phos-
phatidylcholine and sphingomyelin local-
ized predominantly in the outer monolayer,
while most of phosphatidylethanolamine
and all of phosphatidylserine (PS) and phos-
phoinositides are localized in the inner
monolayer.10 This asymmetric distribution
of phospholipids is functionally relevant
since both PS and phosphoinositides inter-
act with spectrin and protein 4.1R, thereby
anchoring the skeletal network to the bilay-

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14th Congress of the European Hematology Association
er. Recent studies documented that spectrin binding to
PS enhances membrane mechanical stability11 while
phosphoinositide binding to 4.1R regulates its interac-
tion with transmembrane proteins band 3 and gly-
cophorin C, and thus, linkage of the lipid bilayer to the
skeletal network.12
More than 50 transmembrane proteins of varying
abundance, ranging from a few hundred to approxi-
mately million copies per red cell, have been identi-
fied.13 Of direct relevance to structural integrity of the
membrane are membrane proteins, band 3, glycophrin
C and RhAG that link the bilayer to the spectrin based
membrane skeleton. Band 3 and RhAG link the bilayer
to the membrane skeleton through the interaction of
their cytoplasmic domains with ankyrin,14,15 while gly-
cophorin C links through its interaction with protein
4.1R.16-18 The linkages play a key role in regulating cohe-
sion between the bilayer and the membrane skeleton.19
Loss of linkages results in lipid loss and decreased
membrane surface area leading to decreased cellular
deformability, a key contributor to decreased cell sur-
vival.2,7 In contrast, an increased number of linkages
leads to increased membrane cohesion and increased
membrane rigidity.19-21
The major protein constituents of the membrane
skeletal network are α- and β-spectrin, actin, protein
4.1R, adducin, dematin, tropomyosin and tropomod-
ulin.7,22-23 α- and β-spectrin form an anti-parallel het-
erodimer through strong lateral interaction of the C-ter-
minus of α-spectrin with the N-terminus of β-spec-
trin.24 Spectrin tetramer, the major structural compo-
nent of the two-dimension skeletal network is formed
by the lateral interaction of the single helical repeat at
N-terminus of α-spectrin of one spectrin dimer with a
two helical repeat at C-terminus of β-spectrin of the
second dimer.25,26 The spectrin dimer-dimer interaction
is dynamically regulated in intact red cell membranes
and the loss of avidity of the interaction leads to
decreased membrane mechanical stability.27,28 The other
end of the 100nm long spectrin dimer forms a junction-
al complex with actin and protein 4.1R.7,29-31
Based on detailed analysis of normal and variously
modified red cells, the following concepts regarding the
structural basis for membrane material properties have
evolved. The unfolding and refolding of distinct spec-
trin repeats, as well as transient dissociation and re-
association of spectrin dimers accounts for remarkable
elasticity of the normal red cell membrane. The vertical
linkages between bilayer and membrane skeleton play
a critical role in maintaining membrane cohesion, while
the lateral linkages between spectrin dimers and
between spectrin-actin-protein 4.1R are the dominant
regulators of membrane mechanical stability.
Maintenance of both membrane cohesion and mem-
brane mechanical stability is critical for the red cell to
maintain its redundant surface area that is critical for it
to undergo extensive deformations. Mutations in vari-
ous membrane and skeletal proteins that result in either
decreased membrane cohesion or membrane mechani-
cal stability lead to membrane surface area loss,
decreased red cell life span and resultant anemia in a
variety of inherited red cell membrane disorders.
| 228 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Hereditary spherocytosis
Hereditary spherocytosis (HS) is a common inherited
hemolytic anemia that occurs in all racial ethnic groups
and is particularly common in individuals of northern
European ancestry, affecting approximately 1 person in
3000.32,33 HS is commonly associated with dominant
inheritance (75%), although nondominant and recessive
inheritance (25%) has been reported. According to com-
mon hematological parameters, including hemoglobin
and reticulocyte counts, disease severity is classified as
mild, moderate, moderately severe and severe. About
20% of HS patients have mild HS with compensated
hemolysis with minimal spherocytosis, near normal
hemoglobin levels, slight reticulocytosis (<6%), and
mild splemenomegaly. Many of these individuals
escape detection until adulthood when complications
related to chronic hemolysis, such as gallstones occur.
Moderate HS is the largest group of HS patients, com-
prising about 60% of cases. In this group, the hemoglo-
bin level is between 8-11 g/dL and reticulocytes in most
cases are greater than 8%. The incidence of palpable
splenomegaly varies from about 50% in young children
to 75 to 95% in older children and adults. A small group
of patients (approximately 10%) have moderately
severe HS with low hemoglobin values (6 to 8 g/dL),
reticulocytosis (>15%) and intermittent need for trans-
fusions. Approximately 3 to 5% have severe HS with
life-threatening anemia requiring regular transfusions.
They usually have recessive HS.
A common feature of all forms of HS is loss of mem-
brane surface area and resultant change in cell shape
from discocytes to stomatocytes to spherocytes. As red
cells with decreased membrane surface area and
reduced cellular deformability are unable to traverse
effectively through the splenic cords into the sinuses,
they are sequestered in the red pulp of the spleen and
removed from circulation. Importantly, the severity of
the disease is related to extent of decreased membrane
surface area. Splenectomy significantly reduces the
severity of anemia by increasing the circulatory life span
of spherocytes.
The mechanistic basis for membrane loss in HS is the
result of the defective anchoring of the skeletal network
to the membrane because of defects in several proteins
(Table 1). Reduced anchoring because of deficiencies of
transmembrane proteins that link the bilayer to mem-
brane skeleton (band 3 or RhAG), of anchoring proteins
(ankyrin or protein 4.2), or of spectrin leads to decreased
membrane cohesion with resultant loss of membrane
surface area. Thus, HS is the result of defects in any of
the protein components involved in vertical linkages
between skeletal network and the membrane (Figure 1).
Ankyrin deficiency is the most common cause of HS
in Northern European populations, accounting for
approximately 50-60% of cases but it is found in only 5-
10% of HS cases in Japan.32-34 Ankyrin mutations cause
both dominant and recessive HS, and patients with
ankyrin defects have prominent spherocytosis with
clinical severity ranging from mild to severe depending
on the extent of membrane loss. Since ankyrin links β-
spectrin to band 3, it is not surprising that ankyrin defi-
ciency leads to a proportional and secondary decrease in
membrane associated spectrin. Isolated spectrin defi-
ciency due to mutations in either α- or β-spectrin
 Berlin, Germany, June 4-7, 2009

Table 1. Gene mutation in inherited human red cell mem-
brane disorders.
Spherocytosis Elliptocytsis Ovalocytosis
Ankyrin 50/60% α- spectrin 65% Band 3 100%
α- and β- spectrin 20% β- spectrin 30%
Band 3 15-20% Protein 4.1 5%
Protein 4.2 <5%
Rh complex <1%
No defect identified 10%
account for 20% of HS cases.32,33 Patients with β-spectrin
defects typically have mild to moderately severe HS,
while patients with α-spectrin suffer from severe HS.
Band 3 deficiency is found in approximately 15-20% of
HS patients, presenting with a phenotype of a mild to
moderate anemia, although a few cases of severe HS
have also been reported.33,35-38 Band 3 mutations are
dominantly inherited. Mushroom-shaped or “pincered”
red cells, in addition to spherocytes may be seen on
peripheral blood smear. Recessive HS due to homozy-
gous mutations in protein 4.2 gene is common in Japan
but is rare in other populations, accounting for less than
5% of HS cases.33,39-40 Rh deficiency associated with
absent or markedly reduced RhAG expression is associ-
ated with mild to moderate hemolytic anemia associat-
ed with the presence of stomatocytes and spherocytes
on peripheral blood smears.41,42 Rh deficiency accounts
for less than 1% of HS cases. The molecular basis for
approximately 10% of HS cases has yet to be estab-
lished.
Hereditary elliptocytosis
Hereditary elliptocytosis (HE) is a relatively common,
clinically heterogeneous disorder characterized by pres-
ence of elliptically-shaped red cells on peripheral blood
smears,43 with prevalence approaching 2% in West
Africa, a malaria endemic region. Inheritance of HE is
autosomal dominant. The clinical presentation of HE
ranges from asymptomatic carrier to severe, life-threat-
ening anemia, with a few reported case of hydrops
fetalis. The overwhelming majority of HE is asympto-
matic but approximately 10% of patients have moder-
ate to severe anemia. Typically, individuals heterozy-
gous for an elliptocytic variant have asymptomatic ellip-
tocytosis while individuals with homozygous or com-
pound heterozygous for HE variants experience mild to
severe anemia. Poikylocytes and fragmented red cells, in
addition to elliptocytes is a feature of red cell morphol-
ogy in HE cases with severe anemia.
A distinguishing feature of all forms of HE is a
mechanically unstable membrane, which results in pro-
gressive transformation of cell shape from discocytes to
elliptocytes during circulation, and in severe cases to
membrane fragmentation and generation of cells with
reduced membrane surface area and abnormal morphol-
ogy. Red cells with decreased membrane surface area, as

Figure 1. A schematic representation of red cell membrane. The membrane is a composite structure in which a plasma
membrane envelope composed of amphiphilic lipid molecules is anchored to a two dimensional elastic network of skele-
tal proteins through tethering sites (transmembrane proteins) embedded in the lipid bilayer. Of particular relevance to
the present review is the vertical interaction involving cytoplasmic domains of band 3 and RhAG, ankyrin, protein 4.2 and
β-spectrin and the lateral linkages in the membrane skeletal network involving spectrin self-association and spectrin-
actin junctional complex.

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 229 |
14th Congress of the European Hematology Association
result of membrane fragmentation are sequestered and
removed from circulation by the spleen. As with HS,
the severity of the disease is related to the extent of the
decrease in membrane surface area. Since splenic
sequestration is the dominant mechanism responsible
for reduced life span of fragmented red cells with
reduced membrane surface area, splenectomy reduces
the severity of anemia by increasing the circulatory life
span of fragmented red cells.
The mechanistic basis for decreased membrane
mechanical stability in HE is weakened lateral linkages
in membrane skeleton due to either defective spectrin
dimer-dimer interaction or a defective spectrin-actin-
protein 4.1R junctional complex (Figure 1). Reduced
avidity of lateral interactions due to defects in α-spec-
trin, β-spectrin or protein 4.1R lead to decreased mem-
brane mechanical stability. Thus, HE is the result of
defects in any of the protein components involved in
lateral linkages in the skeletal network (Table 1).
Mutations in α-spectrin are the most common cause of
HE, accounting for approximately 65% of the cases.
Missense mutations in the amino-terminal region of α-
spectrin that is involved in spectrin dimer-dimer interac-
tion are the most frequent, while mutations in other
parts of α-spectrin, that are not directly involved in
spectrin self association have also been identified.43,44
Mutations is β-spectrin account for 30% of HE cases.43
Point mutations, as well as truncations in the carboxyl-
terminus of β-spectrin that impair spectrin self-associa-
tion have been identified. Heterozygous mutations in
this region of β-spectrin are associated with variable
clinical severity, while in the homozygous state they
are fatal or near-fatal. Importantly, in almost all cases of
spectrin mutations associated with HE, the degree of
spectrin self-association disruption correlates with clin-
ical severity – the larger the degree of disruption of
spectrin self-association, more severe the clinical phe-
notype. Quantitative deficiency of protein 4.1R, as well
as qualitative defects in protein 4.1R account for 5% of
HE cases43,45,46 In all these instances, the defects in pro-
tein 4.1R lead to a weakened spectrin-actin junctional
complex, leading to decreased membrane mechanical
stability.
When originally described, hereditary pyropoikilo-
cytes (HPP) was thought to be a distinct entity due to
increased thermal sensitivity of the red cells and the
unusual morphological features that were similar to
those seen in blood smears in severe thermal burns.47
However, subsequent molecular studies have estab-
lished that HPP is a subset of HE due to either homozy-
gous or compound heterozygous mutations in spectrin
leading to severe disruption of spectrin self-associa-
tion.43
Hereditary ovalocytosis
Hereditary ovalocytosis is very common in malaria
endemic areas in Melanesia, Malaysia, Philippines,
Indonesia and Southern Thailand. In endemic areas, its
prevalence ranges from 5-25%.48 Ovalocytosis charac-
terized by the presence of oval-shaped red cells on
blood smears is dominantly inherited. To date, only het-
erozygotes have been identified in high prevalent
regions implying that homozygosity leads to embryon-
ic or fetal lethality. Hereditary ovalocytosis provides
| 230 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
some protection against malaria.
Marked increase in membrane rigidity is a distin-
guishing feature of ovalocytic red cell membranes.20 In
terms of clinical manifestations, it is important to note
that, in spite of a marked increase in rigidity, most
affected individuals experience minimal hemolysis. In
all cases of ovalocytosis studied to date, only one muta-
tion has been identified – a genomic deletion of 27bp
encoding amino acids 400 to 408 located at the bound-
ary of the cytoplasmic and first transmembrane domain
of band 3.20,49,50 Thus, hereditary ovalocytosis is unique
among red cell membrane disorders in that the identical
mutation in a single gene is responsible for the pheno-
type (Table 1). Although a number of hypotheses have
been proposed regarding how mutation in band 3 could
lead to increased membrane rigidity, the precise mecha-
nistic basis for increased membrane rigidity of ovalo-
cytes has yet to be established.
Red cell membrane transport defects:
hereditary stomatocytoses
In addition to playing a critical role in regulating the
key membrane material properties of deformability and
membrane integrity, membrane proteins play a critical
role in regulating cell volume homeostasis. As with the
requirement for maintenance of normal membrane sur-
face area for optimal cell function, there is a similar
requirement for maintenance of normal cell volume.
Loss of the ability of the red cell to regulate its volume
compromises its ability to perform its function optimal-
ly. An increase in net cation content, with an accompa-
nying increase in cell water content leads to increased
cell volume, thereby reducing redundant surface area
and generation of undeformable stomatocytic and sphe-
rocytic red cells that are sequestered by the spleen. A
decrease in net cation content, with an accompanying
loss of cell water leads to decreased cell volume, and
hence, increased cytoplasmic viscosity that compromis-
es the ability of the cell to undergo rapid deformation
needed for optimal oxygen delivery. Dehydrated red
cells with increased cytoplasmic viscosity appear to tra-
verse the spleen albeit less effectively.
Two distinct phenotypes of inherited red cell mem-
brane disorders with membrane transport defects have
been identified: dehydrated hereditary stomatocytosis
(xerocytosis) (DHS) and overhydrated hereditary stom-
atocytosis (OHS).51 DHS may appear alone or in con-
junction with other clinical manifestations, including
pseudohyperkalemia and/or perinatal fluid effusions.
The inheritance pattern of DHS is autosomal dominant.
DHS alone is associated with well-compensated anemia
with borderline macrocytosis and a mild to moderately
enlarged spleen. Blood smears show stomatocytosis but
usually less than 10%. The distinctive feature of DHS is
cell dehydration with a resultant increase in MCHC and
decreased osmotic resistance. OHS is associated with
uncompensated hemolytic anemia with frank macrocy-
tosis and reticulocytosis. In contrast to DHS, stomato-
cytes are a major feature of red cell morphology on
blood smears. The distinctive feature of OHS is
increased cell hydration with resultant increase in MCV,
decreased MCHC and increased osmotic fragility. In
contrast to HS, the increased osmotic fragility is not the
result of cell surface area but increased cell volume with

normal surface area. The inheritance pattern of OHS is
autosomal dominant. In contrast to our detailed under-
standing of the molecular basis for HS, HE and heredi-
tary ovalocytosis, the molecular basis for HHS and OHS
has yet to be fully defined.52-54
While splenectomy is highly beneficial in the manage-
ment of HS and HE patients with moderately severe to
severe anemia, it is contraindicated in both forms of
hereditary stomatocytosis since venous thromboembol-
ic and other complications occur following splenecto-
my.55,56 The mechanistic basis for this complication has
yet to be defined.
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 Red cell disease

Predicting clinical severity in sickle cell anemia

M.H. Steinberg A B S T R A C T

Department of Medicine, Boston
University School of Medicine and
the Center of Excellence in Sickle
Cell Disease, Boston Medical Center,
Boston, MA., USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
Sickle cell anemia is noted for its clinical heterogeneity. Some patients almost continually seem to
suffer from disease-related complications. Others have far fewer complications. Some individuals die
young; others survive until the 8th or 9th decade. The ability to predict the phenotype of an individ-
ual with sickle cell anemia would allow a reliable prognosis and could guide therapeutic decision-
making. Some risk factors for individual disease complications are known but are insufficiently pre-
cise to use for individual prognostic purposes. Progress has been made toward estimating the overall
disease severity, at least in adults. Genetic association studies, which attempt to link gene polymor-
phisms with selected disease subphenotypes, may eventually provide useful means of predicting the
likelihood of certain complications and allow more individualized treatment.

2009;3:233-237

ickle cell anemia, a monogenic disorder interest was HbF, SNPs in this gene were

S caused by homozygosity for a single β-

globin gene (HBB) mutation (glu6val), is
phenotypically heterogeneous. Persuasive
evidence suggests that to a first approxima-
tion, the pathophysiology of disease has
components related to intravascular hemoly-
sis and proliferative vasculopathy, and to
sickle cell-related vasoocclusion. This pro-
vides many loci where the disease pheno-
type can be influenced by modifying genes
(Figure 1). Accurately forecasting the severi-
ty of sickle cell disease might require know-
ing which genes are associated with its
hemolytic and vascular complications, and
how variants of these genes interact among
themselves and with the environment to
determine the course of disease.
Fetal hemoglobin (HbF)
HbF (α2γ2) is the most thoroughly studied
and powerful genetic modulator of sickle
cell anemia. Its exclusion from the HbS poly-
mer inhibits HbS polymerization, the initia-
tor of most pathophysiology. Among
patients with sickle cell anemia, HbF con-
centrations vary from 0.1-30%. At least two
well-characterized quantitative trait loci
(QTL) loci on chromosomes 6q and 2p are
associated with HbF concentration, and it is
likely that others exist.1-3 In K562 cells, over-
expression of MYB inhibites γ-globin gene
expression. In northern European families,
polymorphisms within and 5’ to HBS1L are
strongly associated with F cell levels,
accounting for 17.6% of the F cell variance.4
BCL11A (2p16.1) codes for a zinc finger tran-
scription factor. In genome-wide association
studies (GWAS), where the phenotype of
associated with HbF concentration in nor-
mal individuals, patients with β thalassemia
trait, β thalassemia intermedia, and sickle
cell anemia.1-2,5 Patients with sickle cell ane-
mia, who were homozygous for the C allele
of SNP rs 766432 had an average of 7% HbF
compared with 3% HbF in patients
homozygous for the A allele.1 The associa-
tion of BCL11A with HbF in at least four dif-
ferent populations, are consistent with an
ancient variant that is highly conserved
among ancestral populations. SNPs at the
BCL11A and HBS1L-MYB loci, along with a
SNP 5’ to HBG2 creating an Xmn I restric-
tion site (rs7482144), accounted for more
than 20% of the variation in HbF and were
associated with painful events in two sickle
cell anemia populations.3
Hydroxyurea is the sole agent available for
specifically treating the complications of
sickle cell anemia. Predicting an individual’s
HbF response to hydroxyurea treatment
would aid in the selection of patients for
treatment and reduce toxicity from unfruit-
ful dose escalation.
Twenty-nine candidate genes were stud-
ied in sickle cell anemia patients treated with
hydroxyurea. SNPs in genes within the
6q22.3-23.2 and 8q11-q12 linkage peaks,
and the ARG2, FLT1, HAO2 and NOS1
genes were associated with the HbF
response to this agent.6
In GWAS of hydroxyurea-treated patients,
chromosome 20 had more significant SNPs
than expected at random, especially in
CST9, one of a family of protease
inhibitors.7 CST9 is tagged by three SNPs
(rs2983639, rs2983640, rs10485646); two of

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 233 |
14th Congress of the European Hematology Association
which were associated with significant positive changes
in HbF after treatment and one with significant negative
changes of HbF. Although individually these SNPs did
not reach genome-wide significance, cumulatively they
provide strong evidence of association, as the probabil-
ity that they are all simultaneously associated by chance
was 10-4. Furthermore, significant variants in other
genes that belong to the same family of type 2 cysteine
protease inhibitors, CTS3 and CTS5, also had SNPs
associated with HbF. Small sample size and the large
number of SNPs tested in GWAS suggests caution until
these results are replicated.
A small guanosine triphosphate (GTP)-binding pro-
tein, secretion-associated and RAS-related (SAR1A) pro-
tein is inducible by hydroxyurea, and might play a piv-
otal role in induction of γ-globin gene expression via its
role in erythroid maturation. Polymorphisms in the
SAR1A promoter were associated with differences in
HbF levels or the HbF response to hydroxyurea in
patients with sickle cell anemia.8
α thalassemia
In patients with sickle cell anemia and α thalassemia,
the concentration of HbS is reduced, lessening the poly-
merization potential of HbS and decreasing hemolysis.
Vasoocclusive events that are highly dependent on the
intensity of hemolysis, such as stroke, leg ulcer and pri-
apism benefit from the coexistence of α thalassemia.
Painful episodes, acute chest syndrome and osteonecro-
sis are either minimally affected, or their prevalence is
increased by coincident α thalassemia.9
Predictors of disease severity
The diversity of sickle cell anemia cannot be
explained by HbF and α-globin gene-linked modulation
alone. Other modifying genes must affect the pathobi-
ology of sickle cell anemia and while candidate genes
association studies have been informative, GWAS stud-
ies, with their promise of capturing more genetic vari-
ability, are starting to be reported. Based on candidate
| 234 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Figure 1. Pathophysiology of sickle
cell disease. The mutation, HBB
glu6val causes the production of HbS
that polymerizes when deoxygenated.
HbS polymer damages the membrane
cytoskeleton, causing reduced cation
and water content, and altered distri-
bution of membrane lipids. Sickle cells
interact with endothelium and other
blood cells, causing vasoocclusion and
a variable number lyse intravascularly
releasing hemoglobin that scavenges
plasma nitric oxide (NO). NO, by bind-
ing soluble guanylate cyclase, converts
cGTP to GMP, relaxes vascular smooth
muscle and causes vasodilatation.
Reduced endothelial NO bioavailabilty
also impairs inhibition of platelet acti-
vation and aggregation and transcrip-
tional repression of cell adhesion mol-
ecule genes. Oxygen radicals and pro-
tein nitration potentially further limit
NO bioavailability and activates
endothelium. Lysed red cells also liber-
ate arginase that destroys L-arginine,
the substrate for NO production.
gene studies, polymorphisms in genes of the TGF-
β/BMP pathway appear involved in several subpheno-
types of disease, and could reflect the hitherto unappre-
ciated role of this very large pathway in the pathobiol-
ogy of disease.10
Stroke
About 10-15% of patients with sickle cell anemia
have overt strokes and larger numbers have silent cere-
brovascular disease. A family predisposition to stroke in
sickle cell disease suggested that inherited modulation
of this phenotype was possible.11 Among the genes
associated with stroke in sickle cell anemia are vascular
adhesion molecule-1 (VCAM1),12-14 interleukin 4 recep-
tor gene (IL4R), tumor necrosis factor α gene (TNFA) and
α adrenergic receptor 2 (ADRB2), and low density
lipoprotein receptor (LDLR).13,15-16
To examine the interactions among genes and their
SNPs, and to develop a prognostic model for stroke in
sickle cell anemia, a Bayesian network was developed
to analyze SNPs in candidate genes in 1398 unrelated
subjects with sickle cell anemia.17 SNPs in 11 genes and
four clinical variables, including α thalassemia and HbF,
interacted in a complex network of dependency to
modulate the risk of a stroke. This network of interac-
tions included three genes, BMP6, TGFBR2, TGFBR3,
with a functional role in the TGF-β/BMP pathway and
P-selectin (SELP). The model predicted the occurrence
of a stroke in unrelated individuals with 98.2% accura-
cy. The predictive accuracy of this stroke model is a step
toward the development of prognostic tests better able
to identify patients at risk for stroke.
Gene expression studies have also contributed to
understanding the predisposition to stroke.18 When sub-
jects at risk for stroke, estimated by the presence of
Circle of Willis disease or having had a stroke, were
compared with controls, transcripts in genes of inflam-
mation-related pathways expressed in blood-outgrowth
endothelial cells were most strongly associated with
stroke or predisposition to stroke.

Priapism
Priapism occurs in about 40% of males with sickle cell
anemia. A strong association was found between the
prevalence of priapism, the severity of hemolysis and
the presence of α thalassemia.19 Polymorphisms in
Klotho (KL) showed an association with priapism.20 KL
has a role in NO biochemistry and directly or indirectly
promotes endothelial NO production. In another study,
priapism was associated with SNPs in TGFBR3, AQP1
and the adhesion molecule, ITGAV..21
Osteonecrosis
Osteonecrosis is found in nearly half of all adults with
sickle cell anemia. Its prevalence might be increased by
concurrent α thalassemia. When subjects with
osteonecrosis were compared with controls, significant
associations were observed with SNPS in BMP6,
ANXA2, TGFBR2, TGFBR3, EDN1, ERG, KL and ECE1.22
Some of these genes might play a role in bone metabo-
lism. KL is also a glycosyl hydrolase in a negative regu-
latory network of the vitamin D endocrine system.
Bone morphogenetic proteins, including BMP6, are
pleiotropic secreted proteins structurally related to TGF-
β and activins and are participate in bone formation and
development. ANXA2, a member of the calcium-
dependent phospholipid-binding protein family, is
involved in osteoblast mineralization.
Leg ulcers
Sickle cell leg ulcers develop in about 10% of patients
and are related to hemolysis. In candidate gene associ-
ation studies, associations were found with SNPs in KL,
TEK and several genes in the TGF-β/BMP signaling
pathway.23
Bacteremia
Infection, bacteremia and sepsis are common events
in sickle cell anemia. SNPs in candidate genes have been
associated with an increased risk of sepsis in other dis-
eases. In a case control study, bacteremia in sickle cell
anemia was associated with SNPs and haplotypes of
genes of the TGF-β/BMP pathway, such as BMP6,
TGFBR3, BMPR1A, SMAD6 and SMAD3.24
Renal disease
With aging, sickle nephropathy becomes common.
Patients with sickle cell anemia associated with a Bantu
haplotype were more likely to develop renal failure,
perhaps because they had the lowest levels of HbF.25,26
SNPs in selected candidate genes were also associated
with glomerular filtration rate (GFR). Tagging SNPs in
about 70 genes of the TGF-β/BMP pathway were stud-
ied and four SNPs in BMPR1B, a BMP receptor gene,
showed significant associations with GFR. The TGF-
β/BMP pathway has been associated with the develop-
ment of diabetic nephropathy, which has some features
in common with sickle cell nephropathy.27
Pulmonary disease
Pulmonary hypertension, estimated by tricuspid
regurgitant jet velocity (TRJV) is a major risk factor for
premature death in sickle cell anemia.28 Mutations in
bone morphogenetic protein receptor 2 (BMPR2) and a
few other genes were associated with both familial and
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 235 |
Berlin, Germany, June 4-7, 2009
sporadic pulmonary hypertension. In 111 sickle cell dis-
ease patients screened for pulmonary hypertension,
genes in the TGF-β/BMP superfamily, including
ACVRL1, BMPR2, and BMP6 were associated with
TRJV.29 One of these SNP in BMP6, (rs 449853), was also
associated with stroke and bacteremia in other stud-
ies.17,24
Acute chest syndrome is the second most frequent
disease complication, and occurs in about 60% of
cases.30 Both an increased and decrease risk of having
acute chest syndrome was associated with a T786C
SNP in the endothelial NO synthase gene (NOS3); how-
ever, this observation has not been replicated.31-33
Exhaled NO levels were reduced in patients with acute
chest syndrome compared with controls, and this was
associated with the number of AAT repeats in intron 20
of NOS1.34 In follow-up, the ATT repeat polymorphism
in NOS1 was associated with acute chest syndrome
only in patients without asthma (itself, a risk factor for
acute chest syndrome).33 A T8002C SNP in the endothe-
lin-1 gene (EDN1) was associated with an increased risk
of acute chest syndrome in 173 children with sickle cell
anemia detected at birth and followed longitudinally.32
These studies examined small numbers of cases and
controls, were limited to children, and few SNPs were
examined.
In 1422 subjects with sickle cell anemia, the popula-
tion was dichotomized into children aged less than or
equal to 5 years and older children and adults.35 There
were 170 acute chest syndrome cases and 884 controls
in patients aged less than 5 years and 388 cases and 819
controls in older individuals. Using time-to-first event in
an age, gender, leukocyte, reticulocyte and platelet
count-adjusted analysis, and controlling for the false dis-
covery rate, one SNP in TGFBR3 and one in LD with
SMAD7 were significantly associated with acute chest
syndrome in both patient groups.
Hyperbilirubinemia and gallstones
Promoter polymorphisms in the uridine diphosphate-
glucuronosyltransferase 1A (UGT1A) gene were associ-
ated with unconjugated hyperbilirubinemia and Gilbert
syndrome. Children with sickle cell disease had a signif-
icantly higher mean bilirubin level if they carried the 7/7
UGT1A genotype compared with the wild type 6/6 or
the 6/7 genotypes; patients with the 7/7 genotype were
more likely to have had a cholecystectomy.36-37
Erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD)
deficiency
G-6-PD deficiency was not associated with differen-
tial survival, increased hemolysis, or a higher incidence
of acute anemic episodes.38 Using DNA-based methods
to detect the GdA- allele of G6PD unequivocally, blood
counts were similar in patients with and without G-6-
PD deficiency, although the hemoglobin concentration
was lower in sickle cell anemia with the GdA- gene.39
While little, if any, modulation of the phenotype of
sickle cell anemia by coincident G-6-PD deficiency is
apparent, perhaps the right phenotype has not been
studied. Patients with sickle cell anemia have impaired
flow-dependant and independent vasodilation.40 This
might be a consequence of intravascular hemolysis and
oxidant stress. Adequate availability of G-6-PD is need-
14th Congress of the European Hematology Association
ed to maintain both NO levels and preserve the proper
redox milieu. A G-6-PD deficient phenotype could be
present in critical vascular tissues in G-6-PD deficient
individuals and perhaps even in sickle cell disease
patients with a normal G-6-PD genotype.41 Perhaps, the
hyperaldosteronism of sickle cell anemia might impair
vascular reactivity by decreasing endothelial G-6-PD
activity.42
Painful episodes
Besides HbF concentration and a possible role of α
thalassemia, a genetic basis for the heterogeneous distri-
bution of painful episodes among patients has not yet
been described. Patient response to opioid analgesics
varies considerably, and the efficacy of these drugs is
known to depend on genetic variability of their catabol-
ic enzymes and receptors.43
Compound phenotypes and integrated measure of disease
severity
Few genetic association studies have used compound
phenotypes. To produce an integrated disease pheno-
type, a Bayesian network model was developed that
describe the complex associations of 25 clinical and lab-
oratory variables, deriving a score to define disease
severity as the risk of death within five years.44 The
genetic basis of severity was examined by using this
score as a phenotype and studying the association of
795 SNPs in 320 candidate genes in mild and severe dis-
ease.45 Positive associations were validated in a smaller
independent patient sample. Among the SNPs in genes
associated with disease severity, some associations, like
with TGFBR3, confirmed previous findings and sup-
ported the conjecture that dysregulation of the TGF-
β/BMP signaling pathway might modulate disease
severity. Some associated genes had a less obvious role
in the pathobiology of disease, but were strongly asso-
ciated with the phenotype of severity. SNPs in these
genes were also associated with normal aging. Perhaps
increased oxidative stress and sickle vasculopathy cause
accelerated tissue damage that is modulated by genes
similar to those involved in the aging process.
In GWAS of patients aged over 18 years, 49 SNPs were
strongly associated with the severity score and 15 asso-
ciations were reproduced in an independent sample. To
boost the power of the sample size, a complementary
discovery approach was used to leverage the pleiotropic
effects of genes. As some complications of sickle cell
anemia resemble aging diseases, it was hypothesized
that protective gene variants associated with mild dis-
ease might also be associated with delayed aging in the
general population, and that deleterious gene variants
associated with severe disease might be associated with
early mortality. Significant associations identified in a
GWAS of exceptional longevity of more than 700 cente-
narians and 1800 referent subjects were compared with
the results in sickle cell anemia. Several SNPs that were
more frequent in long-lived individuals were also more
frequent in patients with mild disease. Among these
associations were SNPs in the gene KCNQ1 that is asso-
ciated with long and short QT syndrome.46 Prolonged
QT interval is more common in sickle cell anemia than
in controls and could be a risk factor for the unexpected
sudden death that is common in adults. Interestingly,
| 236 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
some SNPs discovered with this strategy tag genes asso-
ciated with susceptibility to diabetes (CDKN2A), and
regulation of insulin-like growth factor (IGF2BP1).
Conclusions
Personalized medicine is a goal of the future. By cap-
turing the genetic heterogeneity associated with the
complications of sickle cell anemia, it might be possible
very early in life to build predictive models that esti-
mate the likelihood of the clinical events most responsi-
ble for disease morbidity and mortality. These models
could be revised for different populations and environ-
ments, and take into account clinical events and labora-
tory measures. GWAS and deep sequencing of candi-
date genes to capture uncommon genetic variants
require large populations and careful validation studies,
not an easy task in a rare disease like sickle cell anemia.
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ty and future perspectives. Clin Pharmacokinet 2004;43:983-
1013.
44. Sebastiani P, Nolan VG, Baldwin CT, Abad-Grau MM, Wang
L, Adewoye AH, et al. A netwok model to predict the risk of
death in sickle cell disease. Blood 2007;110:2727-35.
45. Sebastiani P, Wang L, Perls T, et al. A repertoire of genes mod-
ifying the risk of death in sickle cell anemia. Blood 2007; 110:
52a[Abstract].
46. Sebastiani P, Timofeev N, Hartley SH, et al. Genome-wide
association studies suggest shared polymorphisms are associ-
ated with severity of sickle cell anemia and exceptional
longevity. Blood 2008;1446a[Abstract].
 Red cell disease

Diagnosis and management of erythrocytosis

M. F. McMullin A B S T R A C T

Department of Haematology,
Queen’s University Belfast, Belfast,
N. Ireland
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:238-241
An erythrocytosis is present when there is an increase in the number of circulating red cells.
Erythrocytoses are classified as primary where there is an intrinsic problem in the erythroid compart-
ment, and secondary where an external factor is driving the red cell production. They are also classi-
fied as congenital or acquired. Investigation starts with a clear history, examination and establishment
of a true erythrocytosis. The diagnosis of polycythemia vera should be made if current criteria are ful-
filled. Measurement of the erythropoietin level can then direct further investigation. A low erythro-
poietin level should lead to investigation of the erythropoietin-signalling pathway, whereas an elevat-
ed erythropoietin level would lead to investigations for all secondary causes. Management considera-
tions include administration of aspirin, venesection to reduce the hematocrit and a specific target
hematocrit for venesection but there is little objective evidence to support decisions.

he term erythrocytosis refers to an

T
increase in the number of circulating
differentiated red cells. This means
that the total amount of red cells (red cell
mass) is increased. As red cells make up the
vast majority of the circulating cells, the
hematocrit (Hct) is a reflection of the red cell
mass, and, as red cells contain hemoglobin
(Hb), an elevated Hct or Hb raises the possi-
bility of an erythrocytosis. A Hb above 18.5
dl in a male or 16.5 g/dL in a female, or a Hct
above 0.52 in a male or 0.48 in a female is
judged to be elevated, thus, raising the pos-
sibility of an erythrocytosis. However, to
establish the unequivocal presence of an
absolute erythrocytosis, it is necessary to
show that the measured red cell mass is
greater that 125% of predicted for an indi-
vidual’s height and weight.1 Those who
have a raised Hct but do not demonstrate an
increase in red cell mass have an apparent
erythrocytosis. It is clear than a Hct of 0.60
or more in a male or 0.56 or more in a female
is always associated with an increased red
cell mass,2 and, with these results, a red cell
mass investigation is not required.
Causes of an absolute erythrocytosis
An erythrocytosis can be classified as pri-
mary or secondary. In a primary erythrocy-
tosis, an intrinsic defect in the erythroid pro-
genitor cells is associated with an enhanced
response to cytokines. Whereas in a second-
ary erythrocytosis, the increased red cell
mass results from factors external to the ery-
throid progenitor cells, such as increased
erythropoietin (EPO) production from any
cause. Both primary and secondary causes
can be congenital or acquired (for a full list
see Table 1).
Primary erythrocytosis
A primary congenital erythrocytosis
results from mutations of the EPO receptor.
An abnormal receptor in the erythroid cell
behaves abnormally in response to EPO
(after it attaches to the receptor) and ulti-
mately results in increased red cells. A num-
ber of mutations, all of which lead to a trun-
cation of the receptor and failure to abort
EPO signalling, have been described in a
number of families.3
Polycythemia vera is an acquired primary
erythrocytosis, as there is an intrinsic defect
in the erythroid progenitor cell. In the vast
majority of cases, this has been shown to be
due to a mutation in the JAK2 gene (either
the V671F mutation or rarely an exon 12
mutation)4-8 leading to constitutively active
intracellular signalling and cell proliferation.
Secondary erythrocytosis
The cell has a mechanism for oxygen sens-
ing. In normoxia, oxygen activates the prolyl
hydroxlases (PHDs), which then hydroxlate
the a subunit of the transcription factor,
Hypoxia Inducible Factor (HIF). This then
leads to binding of the Von Hippel Lindau
(VHL) ubiquitination of HIF and destruction
of the proteins. However, in the presence of
hypoxia, instead of going down this path-
way, HIF-α associates with its b subunit and
the complex then binds to the hypoxia
response elements within the genome, acti-
vating downstream genes, including EPO.
Increased EPO leads to increased red cell
production, an appropriate response to the
hypoxia. Alteration in any of the genes in
the oxygen-sensing pathway may lead to
the protein behaving abnormally, and even
in normoxia, driving the HIF down the

| 238 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

Table 1. Causes of an absolute erythrocytosis.

Primary erythrocytosis
Congenital
Erythropoietin receptor mutation
Acquired
Polycythemia vera

Secondary erythrocytosis
Congenital
VHL gene mutation (Chuvash erythrocytosis)
PHD2 mutations
HIF-2α mutations
High oxygen-affinity hemoglobin
Bisphosphoglycerate mutase deficiency
Acquired
EPO mediated
Hypoxia driven Figure 1. An initial approach to the investigation of erythro-
Central hypoxic process cytosis.
Chronic lung disease
Right-to-left cardiopulmonary vascular shunts
Carbon monoxide poisoning
Smoker’s erythrocytosis erythrocytosis.14,15
Hypoventilation syndromes including sleep apnea Anything, which would shift the oxygen dissociation
(High-altitude habitat) curve to the left, resulting in hemoglobin with high oxy-
Local renal hypoxia gen affinity leads to tissue hypoxia and secondary ery-
Renal artery stenosis throcytosis. High oxygen affinity hemoglobins, of which
End-stage renal disease over 90 variants have been described, can have this effect.
Hydronephrosis There are also rare mutations of biphosphoglycerate
Renal cysts (polycystic kidney disease) mutase, leading to deficiency of 2, 3 biphosphoglycerate
Post-renal transplant erythrocytosis and shifting the oxygen dissociation curve to the left.16
Pathologic EPO production An acquired secondary erythrocytosis can arise from
Tumors any process, which results in hypoxia, thus increasing
Cerebellar hemangioblastoma EPO production. This can be a central hypoxia, for exam-
Meningioma ple, altitude, lung disease, right to left cardiopulmonary
Parathyroid carcinoma/adenomas shunts or smoking. The hypoxia can also arise at a local
Hepatocellular carcinoma
renal level from a variety of renal disorders, such as renal
Renal cell cancer
artery stenosis. Pathological production of EPO has been
Pheochromocytoma
Uterine leiomyomas described for a variety of different tumors, for example, in
Exogenous EPO cerebellar hemangioblastoma. Erythrocytosis will also
Drug associated result from exogenous EPO or androgen administration.17
Erythropoietin administration There then remains a group of individuals with an
Androgen administration absolute erythrocytosis in whom no cause can be identi-
Idiopathic erythrocytosis fied and this is termed idiopathic erythrocytosis.

EPO: erythropoietin. Diagnostic pathway

hypoxic pathway, ultimately leading to increased EPO
production. A number of mutations in the genes for the
proteins in the oxygen-sensing pathway have been
described in families with erythrocytosis. Thus, these
congenital abnormalities are the cause of the increased
red cell production. A large group of individuals
described in the Chuvashia region of Russia have a sin-
gle mutation in the VHL gene, C598T, which alters
amino acid 200 from arginine to tryptophan.9 A further
cluster with the same mutation was discovered in the
island of Ischia, Italy,10 and a disparate group, all of
Pakistani or Bangladeshi origin, have been identified.11
Several kindreds with a mutation in the PHD2 gene,
producing functionally abnormal protein, leading to
erythrocytosis have been described.12-13 Similarly a num-
ber of families and individuals with erythrocytosis have
been shown to a have mutated HIF-2a gene leading to
The first step in any diagnostic pathway is to undertake
a complete history and examination. During this process,
the causes of erythrocytosis should be considered and
likely factors explored. For example, the patients with
severe respiratory disease should be identified, and this
can dictate further investigation (Figure 1).
Secondly, the presence of an erythrocytosis must also
be clearly established. A consistently raised Hb and Hct
should be observed. In those with an isolated erythrocy-
tosis without an immediately obvious cause, further
investigation, including red cell mass is required to be sure
that an absolute erythrocytosis is present.2
The main primary cause is of course polycythemia vera
and the criteria for this diagnosis have been recently sim-
plified. The United Kingdom (UK) group examined the
evidence, and agreed that the presence of an elevated Hct
or red cell mass and a JAK2 mutation was sufficient to
make this diagnosis.18 The World Health Organization
(WHO) group also redefined their criteria, and require a

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 239 |
14th Congress of the European Hematology Association
raised Hb or red cell mass, a JAK2 mutation and one
minor criteria from bone marrow biopsy changes, serum
EPO below the normal range, or in vitro endogenous ery-
throid colony formation.19 In those without a JAK2 muta-
tion, more extensive criteria are required, including
changes in white cell and platelet counts.18,19
Therefore, screening for the JAK2 mutations should be
an initial investigation in those who do not have an obvi-
ous secondary cause and in all those in whom poly-
cythemia vera is a possibility. Bone marrow biopsy and
cytogenetics may not be required in those who have an
obvious polycythemia vera but should be considered if
there is any doubt about the diagnosis, and when further
investigating an erythrocytosis.
Having established the presence of an erythrocytosis
the next procedure, the EPO level should be considered.
A subnormal EPO level leads to the suspicion of an abnor-
mality in the EPO signalling pathway or a primary cause,
whereas an elevated EPO level or one, which is inappro-
priately normal for a raised Hb suggests an abnormality in
the oxygen sensing pathway or a secondary cause (Figure
1).
Further investigation in those with a subnormal EPO
level should include an EPO receptor mutation screen and
testing for JAK2 exon 12 mutations.
In those with a raised or inappropriately normal EPO
level, an extensive list of investigations can be considered
(Table 2). The order and extent to which these investiga-
tions should be carried out is driven by the history and
examination. In those with an obvious secondary cause,
further investigation may not be necessary, whereas in
the young patient with a marked erythrocytosis, exten-
sive investigation may be required in order to attempt to
identify the cause.
Management
Elevated red cell masses cause an increase in blood vis-
cosity, which may cause thromboembolic events.
Therefore, management must consider reduction of the
red cell mass, and therefore, reduction of the viscosity.
The Hct reflects the whole blood viscosity most acurate-
ly;20 therefore, the Hct level should be considered and the
target for reduction.
In polycythemia vera, a retrospective study found a
relationship between the Hct and thromboembolic events
and showed that there was an increased event rate unless
the Hct was reduced to less that 0.45.21 This study led to
the recommendation that, in polycythemia vera, the Hct
should be maintained below 0.45. Venesection can be
used to achieve this target. However, in the large
European Collaboration on Low-dose Aspirin in
Polycythemia Vera (ECLAP) prospective study, the Hct
did not correlate with the rate of occurrence of thrombot-
ic events.22 In this study, the incidence of thrombotic
events, mainly myocardial infarction, was related to
white cell counts greater that 15×109/L.23
Therapy in polycythemia vera is directed to reducing all
the blood counts with the aim of reducing the likelihood
of thromboembolic events, and transformation to
myelofibrosis or acute leukemia. Guidelines have been
developed providing advice on management, taking into
account the patient’s age and risk factors.24 However, the
JAK2 mutation provides a specific and measurable target
for therapy. Pegylated interferon has been shown to
| 240 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Table 2. Investigations of an erythrocytosis with a normal
or raised EPO level.
Oxygen sensing pathway gene mutations
VHL
PHD2
HIF-2α
Hemoglobin electrophoresis/globin gene sequencing
P50
Biphosphoglycerate mutase level
Pulse oximetry
Carboxyhemoglobin levels
CXR
Radiology of head, chest and abdomen
Sleep studies
reduce the JAK2 mutant level with induction of molecu-
lar remission.25 No vascular events occurred. A number of
JAK2 inhibitors are in development and are likely to be
available in the future for therapy of polycythemia vera.
TG101348 is a small, orally administered molecule,
which inhibits JAK2. In a phase myelofibrosis I study, it
showed reduction in splenomegaly.26 XL019 in another
oral highly selective JAK2 inhibitor, which in phase I stud-
ies in myelofibrosis, showed reduction in splenomegaly.27
In similar phase I/II studies, INCB018424, a potent selec-
tive JAK2 inhibitor, showed reduction in spenomegaly
and marked improvement in constitutional symptoms.28
CEP-701 has JAK2 inhibitory activity and is showing ini-
tially encouraging responses in a study of patient with
polycythemia vera and essential thrombocythemia.29A
histone deacetylase inhibitor ITF2357 has JAK2 inhibito-
ry activity has also shown encouraging responses in
myelofibrosis or polycythemia vera and essential throm-
bocythemia refractory to other therapy.30 More trials and
compounds are in development. In those with a pure ery-
throcytosis from other causes, there is a paucity of evi-
dence to guide management. However, some factors can
be considered. A study with controls of those homozy-
gous for the Chuvash VHL mutation found that those
with the mutation had an increased morbidity and mor-
tality from thromboembolic events. History of venesec-
tion did not significantly influence the event rate but the
numbers who were not venesected were very small.31
If venesection is to be undertaken, there is no guidance
about the target Hct to aim for and, therefore, the original
evidence to reduce to 0.45 is the only information avail-
able to guide management. This may be very difficult to
achieve and maintain in those starting with very elevated
Hct. Aspirin at low dose is also often considered in those
with a presumed thromboembolic risk. In the Chuvash
group, aspirin use was associated with an increased but
non-significant risk of thrombosis,31 whereas in the ran-
domised study ECLAP, it was shown to be beneficial.32
Low dose aspirin is relatively safe and it is likely that most
patients will be offered this therapy in the absence of a
specific contraindication. In many of the specific second-
ary erythrocytoses, there is limited evidence suggesting
some control of the Hct with venesection, taking into
account other events in the patient’s history. In the case of
familial defects, performance of other affected family
members may be beneficial. Judicious limits for the
reduction of Hct to much higher levels than 0.45 have

been suggested.24
In the congenital defects of the oxygen-sensing path-
way studies, including the largest cohort from Chuvashia,
no tumors were reported.31 However, a new mutation in
PHD2, H374R, was recently described in association with
a paraganglioma. There was loss of heterozygosity of
PHD2 in the tumor, suggesting the PHD2 could be a
tumor-suppressor gene.33 This patient was first seen with
erythrocytosis at the age of 30 years. The tumor was first
seen 13 years later. Many of the other recently described
patients with oxygen sensing pathway mutations are
much younger so observation and screening for tumors
may become an issue in management.
Conclusion
In polycythemia vera management, venesection and
low dose aspirin have benefits. Cytoreduction depend-
ing on the risk, group and age should be considered.
JAK2 inhibitors are likely to be used in the future. In
other erythrocytosis, venesection can be used to reduce
the Hct. There is conflicting evidence as to the benefits
of this strategy and how much the level of Hct should
be reduced. In the absence of other guidance, a thera-
peutic target of 0.45 should be considered. Low dose
aspirin may also be considered.
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 Stem cell transplantation

Immunobiology of reduced intensity conditioning

in hematopoietic stem cell transplantation

F. Baron1
B. Sandmaier2,3
1
Giga-research, division of
Hematology, University of Liège,
Belgium; 2Fred Hutchinson Cancer
Research Center; 3University of
Washington, Seattle, WA, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:242-249
A B S T R A C T
Reduced-intensity and truly nonmyeloablative conditioning regimens followed by allogeneic
hematopoietic stem cell transplantation (SCT) have been evaluated in patients with hematological
malignancies who were not considered candidates for conventional SCT because of age or medical
comorbidities. Reduced intensity conditioning regimens have been aimed at both eliminating host-
versus-graft reactions (graft rejection) and producing major anti-tumor effects. Conversely, nonmye-
loablative conditioning regimens have relied on optimization of pre- and post-transplant immunosup-
pression to overcome host-versus-graft reactions, thereby allowing engraftment and eradication of
tumors nearly exclusively via immune-mediated graft-versus-tumor effects. Remarkably, a minimally
toxic regimen of 2 Gy total body irradiation with or without fludarabine followed by postgrafting
immunosuppression with cyclosporine and mycophenolate mofetil has assured engraftment rates
almost similar to those after myeloablative conditioning. While nonmyeloablative and reduced-inten-
sity SCT have been associated with reduced regimen-related toxicities and have been curative for
many patients with otherwise fatal hematological malignancies, challenges have remained in regard
to acute graft-versus-host disease, infections, and disease progression.

Allogeneic hematopoietic stem cell of graft-versus-tumor effects came from the

transplantation and graft-versus-tumor effects
Most hematological malignancies display
a dose-response susceptibility to radiation
therapy and alkylating agents. Because mye-
loablation is the dose-limiting toxicity of
many of these agents, allogeneic hematopoi-
etic stem cell transplantation (SCT) was first
developed as a way to rescue patients from
severe myelosuppression occurring after
administration of high-dose chemoradio-
therapy.1 Despite major improvements in
supportive care in the last 40 years, the non-
hematopoietic toxicities of high-dose condi-
tioning has restricted its use to patients
younger than 55-60 years. Given that medi-
an ages at diagnosis for patients with most
hematologic malignancies range from 65-70
years,1 the majority of such patients could
not benefit from potentially curative allo-
geneic SCT. Further, some patients have
medical cormorbidities, which exclude them
from high-dose conditioning.
Since the late 1970s, it has become appar-
ent that an important part of the efficacy of
allogeneic SCT was mediated by immune
reactions of the graft itself against recipient
tumor cells, termed graft-versus-tumor
effects.2,3 Indeed, patients who developed
acute and/or chronic graft-versus-host dis-
ease (GVHD) had lower risks of relapse than
those who did not.2-4 In addition, patients
given syngeneic SCT and those given T-cell
depleted grafts had substantially higher risk
of relapse than those receiving unmanipulat-
ed grafts from allogeneic donors.4
Furthermore, a direct evidence of the power
observations by Kolb et al. and Slavin et al.
that donor lymphocyte infusions (DLI) were
able to eradicate the malignancy in a number
of patients who relapsed with chronic
myeloid leukemia5 or acute lymphoblastic
leukemia (ALL)6 after allogeneic SCT. Since
then, DLI has also proven to be effective in a
number of patients with acute myeloid
leukemia (AML),7 multiple myeloma (MM),8
chronic lymphocytic leukemia (CLL),9 non-
hodgkin lymphoma (NHL),10 and Hodgkin
lymphoma (HL).11
Nonmyeloablative and reduced-intensity
conditioning
Two key observations in 1997 led to the
emergence of nonmyeloablative and
reduced-intensity conditioning, respective-
ly. First, Storb et al. demonstrated that opti-
mizing post-transplant immunosuppression
could not only control GVHD but also graft
rejection.12 Secondly, Giralt et al. and Slavin
et al. showed that administration of purine
analogs, such as fludarabine provided suffi-
cient immunosuppression to allow sus-
tained engraftment of HLA-matched allo-
geneic stem cells.13,14 Based on these obser-
vations, several groups of investigators
developed a number of reduced intensity13-16
or truly nonmyeloablative17-20 conditioning
regimens for allogeneic SCT. Reduced inten-
sity conditioning regimens have been aimed
at eliminating host-versus-graft reactions
(graft rejections) and producing major anti-
tumor effects. Most reduced-intensity con-
ditioning regimens have combined fludara-

| 242 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

bine with relatively high doses of busulfan (8 mg/kg),21
melphalan (140 mg/m2),22 or thiotepa.16 Conversely,
nonmyeloablative conditioning regimens have relied
on optimization of pre- and post-transplant immuno-
suppression to overcome host-versus-graft reactions,
thereby allowing engraftment,12,23 while eradication of
tumors has depended nearly exclusively on the graft-
versus-tumor effects.24 Examples of nonmyeloablative
conditioning regimens include low-dose (2 Gy) TBI
with or without added fludarabine,18 8 Gy total lym-
phoid irradiation with anti-thymocyte globulin
(ATG),25 or fludarabine with cyclophosphamide.17
Figure 1 shows commonly used conditioning regimens
in relation to their immunosuppressive and myelosup-
pressive properties.
Typical complications of high-dose therapy, such as
nausea, vomiting, pancytopenias, mucositis, and new-
onset alopecia have been observed with most of
reduced-intensity conditioning regimens, and sinu-
soidal obstructive syndrome has also been seen,
although less frequently than after myeloablative con-
ditioning.21,22,26-29 Conversely, nonmyeloablative regi-
mens produced only mild myelosuppression and few
regimen-related toxicities,30 and have been associated
with a low 100-day incidence of nonrelapse mortality,
even in elderly patients and those with comorbid con-
ditions.18 Typically, many transplants following non-
myeloablative conditioning could be carried out entire-
ly in the outpatient setting.31
No randomized study thus far has compared non-
myeloablative versus reduced-intensity conditioning
regimens. A recent analysis by the Société Française de
Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC)
retrospectively compared outcomes in patients given
grafts after 2 Gy TBI with or without added fludarabine
(n=255; nonmyeloablative conditioning) or after flu-
darabine, busulfan plus ATG (n=465; reduced-intensity
conditioning) as treatment for various hematological
malignancies.32 Three-year overall and disease-free sur-
vivals were both similar in the two groups.
Engraftment kinetics
By definition, nonmyeloablative and reduced-intensi-
ty conditioning regimens usually lead to an initial state
of mixed chimerism defined as co-existence of
hematopoiesis of host and donor origin.33 Several factors
have been associated with faster donor T-cell engraft-
ment, including higher intensity of the conditioning reg-
imen,33,34 having received prior myelosuppressive
chemotherapy,35,36 the use of peripheral blood stem cells
(PBSC) instead of marrow as a stem cell source,20,37,38 a
high number of CD34+ and T-cells in the graft,35,37,38 and
intense post-grafting immunosuppression.39
Further, several authors have demonstrated correla-
tions between engraftment kinetics and clinical events.
Specifically, high levels (>50%) of donor T- and NK-cell
chimerism 1 month after SCT have each been associat-
ed with lower risk of graft rejection.18,36 While high day
14 to 42 donor T-cell chimerism levels were associated
with high incidence of acute GVHD,36,40 high donor NK
cell levels had no such association.41 Conversely, high
donor NK cell41 and T cell40,41 chimerism levels early after
SCT have each been associated with low relapse risk
and good progression-free survival.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 243 |
Berlin, Germany, June 4-7, 2009
Figure 1. Commonly used conditioning regimens in rela-
tion to their immunosuppressive and myelosuppressive
properties.83 Please note that this classification is not
based on direct experimentation, and is thus hypothetical.
Ale, alemtuzumab; ATG, antithymocyte globulin; Bu8,
busulfan 8 mg/kg; Bu16, busulfan 16 mg/kg; Cy,
cyclophosphamide; Cy120, cyclophosphamide 120
mg/kg; Cy200, cyclophosphamide 200 mg/kg; F, fludara-
bine; Flag-Ida, fludarabine/cytosine arabinoside/idaru-
bicin; M, melphalan; M 140, melphalan 140 mg/m2; M
180, melphalan 180 mg/m2; TBI, total body irradiation;
TLI, total lymphoid irradiation; TT, thiotepa. (From
Sandmaier BM, Storb R. "Reduced-intensity conditioning
followed by hematopoietic cell transplantation for hemato-
logic malignancies." In: Appelbaum FR, Forman SJ, Negrin
RS, Blume KG (eds): Thomas' Hematopoietic Cell
Transplantation. Oxford, UK: Wiley-Blackwell, p. 1043-
1058, 2009. Used with permission from the authors.
Non-myeloablative versus myeloablative conditioning
In order to determine the relative contributions of
conditioning intensity and graft-versus-host reactions to
transplant related complications, several retrospective
studies compared transplant-related toxicities after non-
myeloablative versus myeloablative SCT. Not surpris-
ingly, the hematological changes after nonmyeloabla-
tive conditioning were milder than those seen after
myeloablative conditioning.42 Specifically, neutrophil
counts remained above 500 throughout the transplant
period in a high proportion of patients given nonmye-
loablative conditioning, while temporary grade IV neu-
tropenia was the rule in patients given reduced-intensi-
ty or myeloablative conditioning. Further, patients given
nonmyeloablative or reduced-intensity conditioning
required less platelet and red blood cell transfusions
than those given myeloablative conditioning.43
Similarly, liver, kidney, gastro-intestinal, and lung toxic-
ities were significantly reduced with nonmyeloablative
conditioning.44-47
Three large retrospective studies from the European
Group for Blood and Marrow Transplantation (EBMT)
compared SCT outcomes of patients given various mye-
loablative versus various reduced-intensity/nonmye-
loablative conditioning regimens as treatment for
AML,48 myelodysplastic syndrome (MDS),49 or CLL50
(Figure 2). Obviously, these studies were limited by the
fact that fitter patients were probably more often pro-
posed myeloablative regimens, while older and sicker
patients were probably more often given nonmyeloab-
lative or reduced-intensity conditioning. Nevertheless,
14th Congress of the European Hematology Association
Figure 2. Retrospective studies comparing SCT outcomes
after reduced-intensity (RIC) or myeloablative conditioning
as treatment for myelodysplastic syndrome (MDS),49 acute
myeloid leukemia (AML),48 or chronic lymphocytic
leukemia (CLL).50 TRM, transplant related mortality. The
black bars show the hazard ratio and the grey bars the
95% confidence intervals.
these studies found similar disease-free and overall sur-
vival in the two groups of patients, since nonrelapse
mortality was lower in nonmyeloablative patients, but
relapse rate was lower in myeloablative recipients.48-50
Further, Scott et al. compared efficacy of SCT after
nonmyeloablative (with 2 Gy TBI with or without
added fludarabine; n=38) or myeloablative conditioning
(with busulfan [16 mg/kg, targeted to 800 to 900 ng/mL]
and cyclophosphamide [120 mg/kg; n=112]) in patients
with MDS over 40 years of age.51 In multivariate analy-
ses adjusted for IPSS group and comorbidity at SCT, 3-
year overall survival [(HR=1.2, p=0.56), progression-free
survival (HR=1.1, p=0.60), progression rate (HR=1.3,
p=0.43), and nonrelapse mortality (HR=1.0, p=0.94)] did
not differ significantly between the two groups of
patients.
More recently, Sorror et al. compared outcomes
among patients with lymphoma or CLL given either
nonmyeloablative (n = 152, consisting of 2 Gy TBI with
or without added fludarabine) or myeloablative (n = 68)
conditioning.52 Outcomes were stratified by the
hematopoietic cell transplantation (HCT)-specific co-
morbidity index (HCT-CI).53,54 Among patients without
comorbidity at SCT (HCT-CI = 0), survival and nonre-
lapse mortality were comparable for patients given non-
myeloablative or myeloablative conditioning (p=0.7 and
| 244 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Figure 3. (A) Impact of acute and chronic GVHD and of
achievement of full donor T-cell chimerism (Ach. FDC) on
relapse risk in 322 patients reported in ref. 24 given grafts
after 2 Gy TBI with or without fludarabine. (B) Impact of
chronic GVHD on relapse risk according to disease group
in the same study. The black bars show the hazard ratio
and the grey bars the 95% confidence intervals.
p=0.7, respectively). In contrast, among patients with
comorbidities (HCT-CI score ≥1) at SCT, the use of non-
myeloablative conditioning was associated with lower
nonrelapse mortality (HR: 0.19; p<0.001) and better
overall survival (HR: 0.33; p=0.007) in multivariate
analyses. These observations suggest that the HCT-CI
score might help in determining which patients might
benefit from nonmyeloablative or reduced-intensity
conditioning, and which others could safely receive
myeloablative regimens.
Graft-versus-host disease and graft-versus-tumor effects
after nonmyeloablative conditioning
Graft-versus-host disease
Several studies have shown slightly lower day-100
incidence of acute GVHD after nonmyeloablative than
after myeloablative conditioning.48,51,55,56 In contrast, the
incidence of chronic GVHD has been comparable in
patients given nonmyeloablative or myeloablative con-
ditioning.48,51,55,56
Some reduced-intensity conditioning regimens have
used in vivo T-cell depletion of the grafts (with either
anti-thymocyte globulin (ATG) or alemtuzumab) in
order to decrease the incidence of acute and chronic
GVHD. While these strategies achieved their goal,15,57
increased incidences of both infections (due to slower
 Berlin, Germany, June 4-7, 2009

immune recovery)58 and disease relapse59 were

observed, especially when high doses of these agents
were used.32 Based on murine experiments, the Stanford
University group investigated an innovative nonmye-
loablative regimen that favored the presence of a high
proportion of regulatory NK-T cells.60 This regimen
combined total lymphoid irradiation (TLI, 8 Gy) with
ATG (Thymoglobulin, 7.5 mg/kg total dose), and post-
grafting immunosuppression with mycophenolate
mofetil and cyclosporine. Initial results in 37 patients
with various hematological malignancies indicated that
this regimen was indeed associated with a low inci-
dence of acute and chronic GVHD, while graft-versus-
tumor effects were apparently preserved,25 some late
relapses did occur.61

Association between graft-versus-host disease and

graft-versus-tumor effects
There has been a close relationship between GVHD
and graft-versus-tumor responses after myeloablative
conditioning.2,4,62,63 We investigated whether such a rela-
tionship existed for patients given nonmyeloablative
conditioning in 322 patients given grafts from HLA-
matched related (n=192) or unrelated (n=130) donors
after 2 Gy TBI with or without added fludarabine.24
Grade II, III and IV acute GVHD occurred in 44%, 11%
and 3% of the patients, respectively, while extensive
chronic was seen in 56% of the patients. While grades
II and III-IV acute GVHD had no significant impact on
relapse/progression, they were associated with
increased non-relapse mortality and decreased progres-
sion-free survival. Conversely, extensive chronic GVHD
was associated with decreased relapse/progression
(p=0.006; Figure 3), and better progression-free survival
(p=0.003). The greatest reduction of relapse risk in
patients with chronic GVHD was observed in the sub-
group of patients with AML or MDS (p<0.001; Figure 3),
while a similar trend was observed in patients with
lymphoma or CLL (p=0.12). In contrast, the study failed
to show a significant association between GVHD and
relapse risk in patients with MM (p=0.41).
Confirming these observations, several groups of
investigators observed a significant association between
occurrence of chronic GVHD and low relapse risk and
high progression-free survival in patients with AML or
MDS. Specifically, Blaise et al. analyzed outcomes of 33
patients with AML in first complete remission (CR)
receiving allogeneic SCT from HLA-identical siblings
following reduced-intensity conditioning with fludara-
bine, busulfan and ATG.64 In a landmark analysis start-
ing on day 100, occurrence of chronic GVHD was asso-
ciated with a lower risk of relapse (0% versus 44%,
p=0.007) and better disease-free survival (95% versus
53%, p=0.007). More recently, Valcarcel et al. studied
data from 93 patients with AML (n=59) or MDS (n=34)
given allogeneic SCT after a reduced intensity condi-
tioning consisting of fludarabine and busulfan, and post-
grafting immunosuppression with cyclosporine plus
methotrexate or mycophenolate mofetil.65 The 4-year
overall and disease-free survival rates were 45% and
43%, respectively. The 4-year cumulative incidence of
chronic GVHD was 53% (45% extensive), and its devel-
opment was the major factor associated with lower
relapse incidence and improved overall and disease-free
Figure 4. Susceptibility of disease groups to graft-versus-
tumor effects. Relapse rates per patient year during the
first 2 years after SCT corrected for follow-up and compet-
ing nonrelapse mortality in a cohort of 834 consecutive
patients reported in ref 69 given grafts after 2 Gy TBI with
or without added fludarabine: (A) in patients in complete
remission (CR) at SCT. (B) In patients not in CR at SCT. CLL,
chronic lymphocytic leukemia; NHL-LG, low-grade non-
Hodgkin lymphoma; MM, multiple myeloma; MCL, mantle
cell lymphoma; NHL-HG, high-grade NHL; ALL, acute lym-
phoblastic leukemia; AML, acute myeloid leukemia; HL,
Hodgkin lymphoma; MDS-RA, myelodysplastic syndrome
with refractory anemia; MPD, myeloproliferative disease;
CMML, chronic myelomonocytic leukemia.
survival. In contrast, acute GVHD was not associated
with a lower risk of relapse.
The impact of chronic GVHD on relapse risk in
patients with MM has been more controversial. Indeed,
two studies found lower risk of relapse and better pro-
gression-free survival in MM patients who developed
chronic GVHD in time-dependent Cox analyses,59,66 while
two others failed to find such an association.67,68 Finally, a
recent analysis from the EBMT demonstrated in a land-
mark analysis that Hodgkin lymphoma patients who had
chronic GVHD before 9 months after SCT had lower risk
of relapse than patients who did not (p=0.008).11
Disease susceptibility to graft-versus-tumor effects
In order to assess the susceptibility of the different
hematological malignancies to graft-versus-tumor
effects, Kahl et al. analyzed the relapse risk according to

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 245 |
14th Congress of the European Hematology Association
disease characteristics in a cohort of 834 consecutive
patients (median age, 55 years; range, 5 to 74 years).
They were given related (n=498) or unrelated (n=336)
grafts after 2 Gy TBI alone (n=171) or combined with
fludarabine (90 mg/m2; n=663).69 Besides the variation in
tumor burden, which had a major impact on the relapse
risk, graft-versus-tumor effects were most impressive in
patients with indolent NHL, CLL and mantle cell lym-
phoma. Specifically, relapse rates per patient year (PY)
during the first 2 years after SCT (corrected for follow-
up and competing nonrelapse mortality) were between
0.0 and 0.24 in patients with CLL and MM in CR (most
of whom received tandem autologous/allogeneic SCT),
low-grade or mantle cell NHL in CR or in partial remis-
sion (PR), and high-grade NHL in CR (Figure 4). In con-
trast, patients with advanced myeloid and advanced
aggressive lymphoid malignancies had rates of more
than 0.50. Patients with lymphoproliferative diseases
not in CR (except Hodgkin lymphoma and high-grade
NHL) and myeloid malignancies in CR had rates of 0.26
to 0.37. The exact reasons for the variable graft-versus-
tumor effects are unclear, but might include variability
in kinetics of tumor cell growth, presentation and den-
sity of minor-histocompatibility (and perhaps tumor-
specific) target antigens on tumor cells, susceptibility of
tumor cells to cytotoxic cell kill, or access of tumor to
donor cytotoxic cells.
Nonmyeloablative or reduced-intensity conditioning for
cord blood or HLA-haploidentical stem cell transplantation
Given that HLA-matched donors can only be found
for 50-80% of patients, depending on their ethnic
group, there has been a considerable interest to extend
the use of nonmyeloablative or reduced-intensity condi-
tioning to cord blood or HLA-haploidentical SCT. Due
to greater degrees of histoincompatibility, the use of
such alternative donors has been associated with
increased risks of both graft rejection and GVHD.
Brunstein et al. investigated the feasibility of unrelated
cord blood transplantation after nonmyeloablative con-
ditioning consisting of fludarabine (200 mg/m2),
cyclophosphamide (50 mg/kg), and 2 Gy TBI.70 Data
from 110 patients (median age 51 [range, 17-69] years)
with hematologic malignancies (n=106) or aplastic ane-
mia (n=4) given one (n=17) or two (n=93) unrelated cord
blood units have been recently analyzed.70 Thirty-nine
patients not given myelosuppressive chemotherapy in
the 6 months preceding SCT were also given ATG.
Postgrafting immunosuppression consisted of MMF and
CSP. Cord blood units were predominantly 1- or 2-
HLA-antigen mismatched with the recipient. Primary
neutrophil recovery occurred in 92% of patients at a
median of 12 days (range, 0-32) after SCT, while the
cumulative incidence of sustained donor engraftment
was 85%. The cumulative incidence of grade II-IV and
grade III-IV acute GVHD were 59% and 22%, respec-
tively, while chronic GVHD was seen in 23% of
patients. Three-year overall and progression-free sur-
vivals were 45% and 38%, respectively. The most fre-
quent causes of death were disease relapse/progression
(n=27), infection (n=12) and GVHD (n=6).
O’Donnell et al. investigated feasibility of unmanipu-
lated HLA-haploidentical marrow transplantation after
nonmyeloablative conditioning combining fludarabine
| 246 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
(150 mg/m2), cyclophosphamide (29 mg/kg), and 2 Gy
TBI in 68 patients with advanced hematologic malig-
nancies (n=67), or paroxysmal nocturnal hemoglobin-
uria (n=1).71 Postgrafting immunosuppression consisted
of MMF, tacrolimus, and cyclophosphamide, the latter
given at a dose of 50 mg/kg on day 3 or on days 3 and
4 after SCT. Nine patients (13%) had graft rejection,
while 59 achieved sustained donor engraftment. The
200-day cumulative incidences of grades II-IV and III-IV
acute GVHD were 34% and 6%, respectively, while
chronic GVHD occurred in less than 30% of the
patients. With a median follow-up of 2 years, 2-year
overall and disease-free survivals were 36% and 26%,
respectively.
Conclusions and perspectives
Nonmyeloablative and reduced-intensity condition-
ing regimens have allowed older patients, those who
had failed a prior myeloablative SCT,72 and those with
comorbidity to benefit from the potentially curative
graft-versus-tumor effects. While graft-versus-tumor
effects were most impressive in patients with CLL,
indolent NHL or mantle cell lymphoma, patients with
chemosensitive aggressive lymphoma or Hodgkin lym-
phoma, and those with MDS or myeloprolifarative dis-
orders with less than 5% marrow blasts, or acute
leukemias in complete remission also had encouraging
results following nonmyeloablative or reduced-intensi-
ty conditioning.25 Anti-tumor responses in some disease
types required extended periods, with some patients
achieving a CR more than 1 year after SCT.18,24
Occurrence of chronic GVHD has been associated with
lower risk of relapse and better progression-free survival
in several studies, and particularly so in patients with
AML or MDS.
For patients with non-malignant diseases, ongoing
efforts are directed at further decreasing the intensity of
the conditioning regimen needed to achieve sustained
donor engraftment. Promising strategies aimed at reduc-
ing the intensity of host immune responsiveness include
pre-transplant administration of donor cells together
with T-cell costimulation blocking agents,73,74 or pre-
transplant administration of radiolabeled monoclonal
antibodies directed against T-cell receptors.75
For patients with hematological malignancies, ongo-
ing efforts are directed at better preventing acute
GVHD, better treating extensive chronic GVHD, and
preventing relapse in patients with aggressive malignan-
cies by reducing tumor burden at the time of SCT,
through preceding cytoreductive autologous SCT in the
cases of multiple myeloma,76 or the addition of radiola-
beled monoclonal antibodies against CD20 or CD45 in
case of advanced non-Hodgkin lymphoma,77 or myeloid
malignancies.78 The addition of disease-targeted thera-
py, such as imatinib, thalidomide, bortezomib, lenalido-
mide or monoclonal antibodies after transplant seems
also promising.9,78-81 Finally, recent progress in large scale
identification of minor-histocompatibility antigens82
might allow the adoptive transfer of T-cell populations
specifically directed against recipient minor-histocom-
patibility antigens expressed only by hematopoietic
(including tumor) recipient cells, thus increasing graft-
versus-tumor effects without inducing GVHD.

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58. Chakrabarti S, Mackinnon S, Chopra R, Kottaridis PD, Peggs
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59. Crawley C, Lalancette M, Szydlo R, Gilleece M, Peggs K,
Mackinnon S, et al. Outcomes for reduced-intensity allogene-
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60. Lan F, Zeng D, Higuchi M, Huie P, Higgins JP, Strober S.
Predominance of NK1.1+TCR αβ+ or DX5+TCR αβ+ T cells
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61. Lowsky R, Stockerl-Goldstein K, Laport G. Clinical outcomes
following allogeneic hematopoietic cell transplantation (HCT)
using nonmyeloablative host conditioning with total lym-
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62. Collins RH, Shpilberg O, Drobyski WR, Porter DL, Giralt S,
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63. Kolb HJ, Schmidt C, Barrett AJ, Schendel DJ. Graft-versus-
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64. Blaise DP, Boiron JM, Faucher C, Mohty M, Bay JO, Bardoux
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65. Valcarcel D, Martino R, Caballero D, Martin J, Ferra C, Nieto
JB, et al. Sustained remissions of high-risk acute myeloid
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66. Kennedy GA, Durrant S, Butler J, Morton J, Western R, Bartlett
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67. Bruno B, Rotta M, Patriarca F, Mattei D, Allione B, Carnevale-
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 Stem cell transplantation

Reduced intensity conditioning as a platform

for immunotherapy

J. Barrett A B S T R A C T

Allogeneic Stem Cell Transplantation
Section, Hematology Branch, NHLBI,
NIH, Bethesda, MD, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:250-255
Reduced intensity conditioning (RIC) regimens offer the patient a stem cell transplant (SCT) with
low regimen-related toxicity but preserved graft-versus-leukemia (GVL) effects. In the last decade, a
wide variety of regimens has been developed and it is now possible to refine protocol design to achieve
desired properties of suppression and intensity. RIC regimens have some unique immunological fea-
tures, which can favor GVL while controlling graft-versus-host disease (GVHD). Current regimens and
strategies provide GVL and graft-versus-tumor (GVT) effects whose efficacy varies according to dis-
ease, with chronic leukemia and lymphomas being the most susceptible to immune control. While
much has been learned through RIC SCT about ways to manipulate immune recovery and GVL/GVT
effects, both acute and chronic GVHD remain an issue requiring better ways of separating GVHD from
antimalignant effects. Here, concepts of RIC transplantation in relation to their immunotherapeutic
potential are discussed, and disease-specific RIC transplant approaches to maximize malignant dis-
ease control and cure are described.

Introduction malignant disease states and strategies to

Over 10 years ago, encouraged by grow-
ing evidence that the graft-versus-leukemia
(GVL) effect from donor lymphocyte infu-
sions (DLI) could be harnessed as a potent
immunological weapon, investigators began
to develop conditioning regimens for
leukemia stem cell transplantation (SCT),
which achieved lymphocyte engraftment
and the GVL effect without using myeloab-
lation to minimize residual malignancy.1
This conceptual change in perception of
how SCT cures malignancy is illustrated in
Figure 1. Reduced intensity conditioning
(RIC) regimens (whether or not myeloabla-
tion is achieved) have extended the use of
SCT to older and debilitated patients with
hematological malignancies, who cannot
withstand classical myeloablative regimens,
and to patients with solid tumors, where
intensive myeloablative treatment is ineffec-
tive and only adds unwanted toxicity.2
There are now over 40 published RIC reg-
imens varying in the relative degree of
immunosuppression or myelosuppression
they achieve.1 They fall into two broad cate-
gories: non-myeloablative (e.g., fludarabine
and cyclophosphamide) and partially or
completely myeloablative (e.g., fludarabine
or other purine analogs with melphalan,
busulfan or total body irradiation and non-
purine-analog regimens). In each of these
categories, additional treatments are some-
times included, such as antilymphocyte anti-
bodies. This diversity reflects the ability to
select separately the degree of immunosup-
pression and myelosuppression desired for
different transplant situations. Here, we dis-
cuss the nature of the immune recovery that
occurs after RIC regimens, the approaches
available to optimize GVL effects in specific
further refine and augment the GVL effect of
RIC regimens.
Immune reconstitution after reduced intensity
conditioning regimens
Immediately after allogeneic SCT, host
antigen presenting cells (APC) induce allore-
sponses in the incoming donor lymphocytes,
initiating in graft-versus-host disease
(GVHD) and GVL reactions. The cytokine
milieu (the so-called cytokine storm induced
by the conditioning regimen and a surge of
lymphocyte growth factors, notably IL-15
induced by lymphopenia)3 tends to boost the
alloresponse. Subsequently, alloresponding
and engrafting donor lymphocytes displace
any residual host immune cells and marrow
cells.4 Lineage-specific chimerism studies can
be used to track separate recovery of donor
lymphopoiesis and myelopoiesis and loss of
host cells.5 In children who have a function-
ing thymus before SCT, recovery of the thy-
mus after transplant speeds the acquisiton of
full T cell competence and favors donor-host
tolerance. Acute and chronic GVHD and the
immunosuppressive agents used to prevent
or treat the disease are the major limits to
complete and rapid immune reconstitution.4
RIC transplants have particular impact on
immune recovery, which can be manipulat-
ed to optimize GVL effects and transplant
outcome. It was thought that the lower
intensity conditioning might protect patients
from GVHD because it was argued that the
cytokine storm (from regimen-induced tis-
sue damage) would be less. In fact, acute
GVHD is not mitigated by the regimen, and
risk factors for acute GVHD appear to be
comparable after standard and RIC trans-
plants.6 One factor, which may increase the

| 250 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

risk and severity of acute and chronic GVHD after RIC Myelofibrosis
transplantation, is the less complete eradication of host This myeloproliferative disorder, common in older
APC by non-myeloablative regimens.7 While RIC regi- individuals, appears to be especially susceptible to erad-
mens may favor thymic recovery, T cell repertoire nor- ication by RIC regimens. Not only is the recipient mar-
malization and tolerance,7,8 it could be argued that the row rapidly replaced by engrafting donor cells but the
establishment of tolerance may, in fact, be unfavorable fibrosis resolves, making it unnecessary to perform
for GVL effects, which require intolerant T cells to attack splenectomy before SCT.21,22
leukemia cells.4 Chimerism has been extensively stud-
ied in RIC transplants. The patterns of T cell and
myeloid cell recovery are distinct and regimen-specific.
Pure immunoablative regimens result in an early switch
to 100% donor T cells, while hematopoietic recovery is
100% host, switching within a few months to 100%
donor myelopoiesis as the incoming donor immune
function eliminates host marrow through a graft-vs-
marrow mechanism.2,5 The switch to donor marrow
often heralds GVL and graft-versus-tumor (GVT)
effects.2 RIC regimens with more myelosuppressive
intensity achieve earlier and more complete donor
myeloid chimerism. Also, fully myeloablative RIC regi-
mens show 100% donor myeloid reconstitution imme-
diately on engraftment.9 Similarly, by varying the
immunosuppressive intensity of the conditioning the
degree of donor-host T cell, chimerism can be modulat-
ed. Figure 2 segregates conditioning regimens with dif-
ferent immunosuppressive and myeloablative capacity.

Reduced intensity conditioning regimens in specific malig-
nancies
Chronic myelocytic leukemia
The exquisite sensitivity of chronic myelocytic
leukemia (CML) relapsing after SCT to achieving a sec-
ond cure following DLI has led several investigators to
use RIC SCT for this leukemia.10-13 Disappointingly,
truly myeloablative regimens have not been found to
reliably eradicate chronic phase CML, although regi-
mens, including reduced doses of busulfan confer GVL
effects without increased toxicity.10 The arrival of tyro-
sine kinase inhibitors to treat CML has restricted SCT to
advanced CML where again RIC regimens appear
unsuitable.14 The attractive idea of combining targeted
treatment with imatinib with RIC SCT to eradicate
residual disease has not been formally explored.
However, the observation that combined treatment
with DLI and imatinib to treat relapsed CML is better
than either agent used alone would support the idea of
a useful synergy using both modalities.14,15
Figure 1. Classical and “reduced intensity” concepts of the
relationship between conditioning regimens, engraftment
and GVL effects. (A) Classical conditioning (e.g.,
Cyclophosphamide and TBI 12Gy) results in rapid and
complete engraftment of marrow and T cells (slower if the
graft is T cell depleted). Conditioning eliminates most of
the disease burden while the GVL effect is perceived only
to eliminate minimal residual disease. (B) In the reduced
intensity conditioning, a larger burden of leukemia
remains and is only eradicated after DLI which converts
mixed T cell and myeloid chimerism to full donor engraft-
ment. The neutrophil and platelet nadir after conditioning
may not be profound (depending on myeloablative intensi-
ty of the regimen) and the GVL effect is perceived to be a
significant component of disease eradication.

Acute myeloblastic leukemia and myelodysplastic syndrome

While there is universal agreement that RIC regimens
have made it possible to safely transplant Acute
myeloblastic leukemia (AML) and myelodysplastic syn-
drome (MDS) patients in their 60s, it is now clear that
lower transplant-related mortality (TRM) from reduced
intensity is offset by increased relapse,16 rendering RIC
approaches unsuccessful in advanced myeloid malig-
nancies.17 Strategies to reduce relapse in high risk AML
and MDS patients include pretreatment with chemo- Figure 2. Relationship between regimen intensity,
therapy (e.g., the fludarabine/amsacrine based FLAMSA immunosuppression and myelosuppression. Single agents
and low dose TBI are inadequate to ensure engraftment,
regimen) to reduce tumor burden,18 targeted therapy while immunoablative regimens ensure engraftment with-
with the anti CD33 immunotoxin gemtuzemab out myeloablation. Increasing intensity achieves increas-
ozogamicin prior to SCT,19 or radiolabled anti CD45 ing myelosuppression, such that some RIC regimens are
fully myeloablative without the extra toxicity of classical
given during conditioning.20 “full dose” regimens.

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 251 |
14th Congress of the European Hematology Association
Chronic lymphocytic leukemia
This chronic leukemia is very susceptible to the GVL
effect. Even advanced and high-risk forms of CLL with
bulky disease at transplant can show durable responses
to SCT. RIC SCT has been widely used to treat this
older population of patients.23,24 It is concluded that out-
come is favored because of a lower treatment-related
mortality (TRM) while the GVL effect can control dis-
ease. However, mixed chimerism delays disease
response and may predispose to relapse, which reached
31% in a large European series.25
Non-Hodgkin’s lymphoma
RIC SCT has been extensively explored in both low
grade (follicular) and high grade NHL.26,27 Low grade
NHL appears to be uniquely susceptible to GVL effects
after RIC transplants. Even patients with high-grade
NHL who have relapsed after autologous SCT can
achieve prolonged remissions and possible cure.28,29
Hodgkin’s disease
RIC regimens are often the only practical approach to
SCT in patients with HD where allogeneic SCT is used
in high risk patients with multiply relapsed and multi-
ply treated disease. There is clear evidence for a GVL
effect in HD but lack of prospective comparative stud-
ies makes it difficult to determine the regimen intensity
that confers the optimum balance between disease con-
trol and toxicity.30
Multiple myeloma
There is a modest GV-myeloma effect in MM.
Although progression free survivals are often compara-
ble, relapse and progression is greater after RIC regi-
mens than after full intensity regimens.31,32 The most
promising strategy to improve outcome has been to
uncouple the tumor reduction from the GV-myeloma
effect by prior intensive therapy and autologous SCT
followed by a RIC SCT.33
Solid tumors
Complete remissions in metastatic renal cell cancer
following fludarabine/cyclophosphamide conditioning
and allo SCT34,35 paved the way to numerous studies of
RIC SCT in breast,36 colon,37 ovarian,38 pancreatic,39
hepatic40 cancers and sarcomas.41 It is generally agreed
that there is a GVT effect in some of these advanced
malignancies. Notably relationships between GVHD
and GVT effects and regression of metastases after stop-
ping immunosuppression in the context of conditioning
regimens that are unlikely to cause tumor shrinkage is
strong evidence for GVT effects in many cancers.2
Unfortunately, only a minority of patients are respon-
ders and durable complete remissions appear to be con-
fined to patients with renal cancer.
What we have learned about immune function in reduced
intensity conditioning stem cell transplantation?
These results from a wide diversity of studies over 12
years (unfortunately mostly non-randomized and some
conflicting)1 allow us to draw some general conclusions
about the factors regulating immune function, GVHD
and GVL after RIC SCT, and point the way towards
optimizing disease-specific RIC transplant regimens.
| 252 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Not all reduced intensity conditioning regimens are safer
There is a limit to the amount of dose reduction in
RIC regimens. For example, early regimens using only
200 cGy total body irradiation were associated with
graft rejection. When fludarabine was combined with
low dose TBI, engraftment was improved.42 The safety
of RIC regimens hinges as much on the prophylactic
regimen for GVHD as the regimen-related toxicity, and
some RIC protocols have a higher risk of severe acute,
as well as chronic GVHD. In addressing safety of RIC
SCT, attention must be focused on the entire procedure
to include adequate GVHD prevention. In this regard,
correctly dosed alemtuzemab appears to offer a better
alternative to standard methotrexate/cyclosporine
GVHD prophylaxis.43
Myeloablation does not correlate with increased toxicity
Although non-myeloablative SCT may be sufficient
to achieve cures in some chronic leukemias and low
grade lymphomas, abundant data shows that in AML
and MDS, the RIC regimens reduce TRM only at the
price of increased relapse. While DFS may still be favor-
able because of the low TRM compared with higher
intensity regimens, it has to be concluded that regimen
intensity does contribute to leukemia control. The com-
bination of fludarabine and busulfan is being increasing-
ly used in RIC regimens. This regimen is myeloablative
but well tolerated at busulfan doses about 50% of stan-
dard regimens.44,45 Studies where the dose of busulfan is
cautiously advanced further are in order in high risk
patients receiving RIC SCT to find the optimum balance
between GVL effect and regimen related toxicity.
GVHD remains a major problem
Despite earlier assumptions to the contrary, it is now
agreed that RIC SCT carry at least as high as risk of
acute and chronic GVHD as standard SCT.6 Therefore,
the GVL effect is compromised by the need to give
GVHD prophylaxis. The negative impact of GVHD pro-
phylaxis on relapse is clear from two trials in myeloab-
lative SCT, which demonstrated a significant reduction
in relapse in patients receiving low dose CSA compared
with those who received standard dose CSA.46,47 The
humanized monoclonal antibody anti CD52 (alem-
tuzemab) has been used to deplete T cells in the donor
and the recipient. Studies have shown that alemtuzum-
ab decreases acute and chronic GVHD, while conserv-
ing GVL/GVT. However, the beneficial effect is sched-
ule and dose sensitive with best results seen in adminis-
tering the antibody early in conditioning at lower
doses.48 Other promising GVHD prophylactic regimens
are the combination of tacrolimus, sirolimus and low
dose methotrexate,49 and cyclosporine combined with
mycophenolate mofetil.50
Mixed chimerism has mixed benefits
Some investigators have used regimens, which initial-
ly caused a mixed chimeric T cell state (carrying a low
risk of GVHD development), followed by DLI to con-
vert to full donor T cell chimerism a few months later.51
While the DLI can deliver powerful antitumor effects,
the major problem with the DLI given in the first few
months after SCT is the rapid development of acute and
chronic GVHD. Thus, at worst both GVHD and GVL

are simply delayed. The persistence of mixed
chimerism carries a worse prognosis in some studies.52
Individual donor-host variations make it impossible to
design a regimen that reliably achieves mixed
chimerism and DLI does not always result in full donor
T cell reconstitution because a full lymphocyte compart-
ment (with reflexly low levels of lymphocyte growth
factors) is not favorable to further engraftment and
expansion of infused T cells.53 Reversal of mixed
chimerism can be achieved more reliably by treating
with fludarabine to create lymphopenia prior to DLI.54
However, mixed chimerism is inherently unstable, car-
rying the risk of graft rejection when donor T cells are
in the minority and acute GVHD when full chimerism
is achieved.
Preemptive DLI is more effective at controlling disease post-
SCT
Although DLI is highly effective at treating relapse in
chronic leukemias the control of relapsed acute
leukemias and other diseases disappointing. Giving DLI
preemptively is a logical means of improving the man-
agement of persisting disease that has not yet pro-
gressed to hematological relapse55-57 but randomized
studies to validate this approach are lacking.
Diseases vary in their susceptibility to GVT effects
As described above GVT/GVL effects vary widely in
different disease states. Thus, RIC treatment strategies
have to be tailored to specific disease states.
Control of disease benefits from pretransplant tumor control
Carella was the first to propose separating a prelimi-
nary intensive therapy with autologous SCT to reduce
bulky disease followed after a recovery period by a RIC
transplant to confer GVL.58 This approach appears
promising for treatment of multiple myeloma, a disease,
which historically had a high TRM after full intensity
SCT. But it is not yet clear whether this approach is
superior to the tandem autologous SCT.59 A similar
approach in older MDS patients appears promising.
Kolb et al. have used a fludarabine/amsacrine regimen
(Flamsa) followed after a 5 day rest period by a condi-
tioning regimen of fludarabine and melphalan.18
Although, this study was not randomized, the
leukemia-free survival of 73% for this high risk patient
group was extremely encouraging.
Targeted treatments are promising partners for RIC
regimens: in the last few years, non-chemotherapeutic
agents with low toxicity, which primarily target the
malignancy, have begun to be incorporated into RIC
SCT. In addition to incorporating them into the condi-
tioning regimen agents, such as imatinib for CML60 and
sirolumus which, as well as being a prophylactic agent
for GVHD, has a suppressive effect on B cell lym-
phomas61 and bortezomib for MM,62 can be given post
SCT to augment the effect of DLI.
Future developments
RIC regimens have made SCT available to a potential-
ly large cohort of older patients with hematological
malignancies and patients with solid tumors who do
not respond to high dose myeloablative treatments. In
susceptible diseases, such as selected low grade lym-
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 253 |
Berlin, Germany, June 4-7, 2009
phomas, they may also replace full intensity SCT in
younger patients. They are being further explored to
reduce toxicity in haploidentical SCT.63 Their persisting
limitation is one common to all allogeneic SCT
approaches, namely the separation of GVHD and GVL
effects.4 New approaches, such as selective T cell deple-
tion have already been explored in the context of RIC
SCT in older patients to reduce the risk from GVHD in
this high-risk group.64 Further boosting of GVL/GVT
effects could be achieved using vaccines or adoptive
transfer of NK cells or tumor specific T cells.4
Conclusions
Since their introduction more than a decade ago, RIC
regimens have come to be applied to a wide group of
malignant and non-malignant disorders. In the process,
we have developed a greater understanding of the rela-
tive roles of conditioning agents in achieving immuno-
ablation and myelosuppression and it is now possible to
refine protocol design to achieve desired properties of
suppression and intensity. Studies of immune reconsti-
tution after RIC SCT has taught us the impact of donor
chimerism on outcome and improved the way to con-
trol immune recovery and deliver DLI effectively. Future
regimens incorporating targeted agents and immuno-
therapy strategies, such as vaccines, adoptive cell trans-
fer, selective T cell depletion and administration of
cytokines should further improve outcome of this type
of transplant.
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 Stem cell transplantation

The role of reduced intensity conditioning in

allogeneic hematopoietic stem cell transplantation

A. Gratwohl A B S T R A C T

Hematology, University Hospital,
University of Basel, Basel,
Switzer-land
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:256-260
Reduced intensity conditioning (RIC) has obtained wide acceptance over the last decade in allo-
geneic hematopoietic stem cell transplantation (HSCT). It has extended this therapy to older patients
and opened new possibilities for patients with comorbidities. Nearly a quarter of all allogeneic HSCT
in Europe are currently prepared with RIC. RIC HSCT has given proof of principle for the power of
immunotherapy: on its own, a healthy immune system can control a malignant disease. RIC reduces
mortality due to the conditioning; it remains a full allogeneic transplant. It does not reduce risk of
death from GvHD or infection and is associated with an increased risk of rejection and relapse. Overall
survival is not necessarily improved compared to standard conditioning. RIC HSCT has its primary place
in situations where this benefit of reduced TRM is not offset by increased rejection or relapse, for
example, primarily in older patients with disease in remission. Its value is currently being tested in a
prospective randomised EBMT study for elderly patients with AML in their first complete remission.

Introduction occlusive syndrome, diffuse alveolar hemor-

Hematopoietic stem cell transplantation
(HSCT) has become an established therapy
for many congenital or acquired severe dis-
orders of the hematopoietic system, as well
as for chemo- radio-, and immuno-sensitive
malignancies.1,2 It is estimated that more
than 50,000 such procedures are performed
annually all over the world. The risk factors
have been defined and results have signifi-
cantly improved over the last decades.
Allogeneic and autologous stem cells from
bone marrow, peripheral blood or cord
blood are used depending on the disease,
clinical condition and availability of a donor.
Still, HSCT remains associated with signifi-
cant early and late morbidity and mortality,
and this is illustrated in Figure 1. Early mor-
tality increases with increasing degree of
mismatching, and many years might be
required to compensate for the early years of
life lost due to the immunological complica-
tions of the allogeneic stem stell product. In
contrast, relapse remains the key problemat-
ic of autologous HSCT. The important role
of the immunological complications is fur-
ther illustrated by the best results of syn-
geneic HSCT and the difference between
matched and mismatched allogeneic donors.
We still lack the tools to separate the detri-
mental consequences of GvHD from the
beneficial effects of graft-versus-disease
effects on a routine basis.1
Causes of death after an HSCT include
disease related (relapse) or transplant related
complications.1,5 The latter can be subdivid-
ed in immunological complications (rejec-
tion or graft-versus-host disease), death from
infection or death due to the conditioning
itself. High dose chemotherapy or total body
irradiation (TBI) can directly cause endothe-
lial damage syndromes, for example, veno
rhage. It can also induce severe mucositis or
failure of near every organ (Figure 2). There
has been substantial improvement in sur-
vival over the last decades.6,7 These
improved outcome results were primarily
due to a significant reduction of infectious
death. Disappointingly, there has been little
improvement in reduction of relapse and in
reduction of death from GvHD since the
early introduction of calcineurine inhibitors
in the early 1980s. It is understandable that
there is a substantial interest on reducing the
detrimental effects from conditioning.
History of reduced intensity conditioning
hematopoietic stem cell transplantation
The basic concepts of allogeneic HSCT
were outlined very early in the history and
they still hold true.8 The purpose of the con-
ditioning is threefold: (i) to immunosuppress
the recipient in order to accept the trans-
plant; (ii) to eradicate the disease; (iii) to cre-
ate space for the incoming hematopoietic
stem cells. Engraftment itself is a complex
process and influenced by the degree of
immunosuppression before and after the
transplant, and by the stem cell source, the
cell dose and the composition of the stem
cell product. Control of the disease is influ-
enced by the degree of conditioning, the
graft product, the degree of engraftment,
presence or absence of graft-versus-host
reactions and by the post-transplant treat-
ment.1,8 The importance of the role of both
conditioning and graft-versus-leukemia
effects to control the disease was shown
very early in history. It is nicely illustrated by
the first clinical description of the disappear-
ance of a refractory acute leukemia follow-
ing the transfusion of granulocytes for
patients with a severe infection in neutrope-

| 256 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

Figure 1. Outcome of HSCT by donor type. Results are
based on near 200,000 autologous and allogeneic HSCT
reported to thee EBMT.
nia, which was accompanied by a skin rash, liver abnor-
malities and diarrhea:9 graft-versus-host disease by its
own can induce graft-versus-leukemia and eliminate the
disease.10-13 Still, early results in the 1970s were disap-
pointing. Transplants for leukemia were primarily based
on single dose total body irradiation alone. The majori-
ty of the patients who did engraft and who did survive
the early complications eventually relapsed. It was the
concept of combining total body irradiation with
chemotherapy (cyclophosphamide and TBI) or giving
intensive radiomimetic chemotherapy (CyBu, BACT),
which led to the success.14 Subsequent concepts in the
1980s concentrated on increasing conditioning and find-
ing the maximum tolerated conditioning regimen. All of
the many attempts failed. They all successfully reduced
relapse. This occurred at the expense of increased trans-
plant related mortality and the net survival was never
improved.
The reobservation of the powerful effects of donor
lymphocyte infusion in the 1990s brought a complete
shift.15-19 Focus was on smarter rather than “stronger”
transplants.20 Carefully conducted animal studies
defined a minimal dose of conditioning required for sta-
ble engraftment in dogs. A dose as low as 200 cGy TBI
was found sufficient when accompanied by combined
immunosuppression either with cyclosporine and
methotrexate or mycophenolate mofetil.21 These exper-
iments formed the basis for the now traditional Seattle-
Leipzig mini-transplant conditioning regimen.22-25 The con-
cept was rapidly adopted when initial studies showed
surprisingly good early results, such as those published
very early on patients with chronic myeloid leukemia.26
It was followed by an over-abundance of re-duced
intensity conditioning regimens, to such an extent that
the diversity of RIC probably exceeds the number of
transplant teams. Initially, there was also a wealth of
names for this new form of conditioning, from mini-
transplants, micro-transplants, non-myelo-ablative
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 257 |
Berlin, Germany, June 4-7, 2009
Figure 2. Principle causes of death after allogeneic HSCT.
Curves represent survival and causes of death of patients
transplanted for early disease.
transplants up to metacrine transplants. There was also
an intensive debate between myeloablative versus
immunoablative conditioning strategies.27-32
Current status of reduced intensity conditioning
hematopoietic stem cell transplantation
Today, RIC HSCT is an established form of treatment
and nearly a quarter of all allogeneic HSCT performed
last year in Europe were RIC HSCT (Figure 3).33 The
concept is used all over Europe, even though some
countries apply it more and others to a lesser extent
(Figure 4). The general term RIC has become established
and the distinction is no longer between im-muno-sup-
pression or myelosuppression. All drugs have both
immuno- and myelosuppressive properties in common.
Focus is primarily on the total intensity. This explains
why the separation between standard and reduced
intensity conditioning is not a clear cutoff. It has also
become clear that, for different diseases, different arbi-
trary distinc-tions might be made.
Within its definition’s committee, the EBMT has
defined which regimens with doses equal or be-low
these limits should be classified as RIC HSCT
(www.ebmt.org). In brief, conditioning regimens, which
include total body irradiation below the dose of 600
cGy (fractionated) +/- a purine analog and +/- ATG or
with a dose of less than 60 mg/kg of cyclophosphamide
or alternatively, less than 8 mg/kg busulphan should be
considered as RIC for leukemias. The same applies for
patients with lymphoma, where in addition condition-
ing regimens, using melphalan of a dose of less than 140
mg/m2 +/- purine analogue +/- campath or ATG should
be considered as RIC HSCT. High dose fludarabine (90
mg/m2 i.v. with TBI 2 Gy) should also be considered as
RIC. For myelomas, melphalan less than 100 mg/m2, +/-
purine analogue +/- ATG is considered RIC. Any dose of
cyclophosphamide less than 1,200 mg/m2 +/- ATG is
con-sidered RIC for aplastic anemia and nonconstitu-
14th Congress of the European Hematology Association
Figure 3. Number of allogeneic HSCT reported to the EBMT
activity survey office from 1990 to 2007 and numbers of
RIC allogeneic HSCT.
tional bone marrow failures syndromes. For solid
tumors busulphan, less than 8 mg/kg +/- TBI up to 600
cGy +/- purine analogue and +/- ATG or cyclophos-
phamide less than 60 mg/kg should be considered as
RIC HSCT. There are no general recommendations for
other conditioning approaches or other disease cate-
gories. There are no prospective comparative studies.
Feasibility of RIC HSCT has been documented for
patients with acute leukemias, chronic leukemias,
myelodysplastic and myeloproliferative syndromes,
lymphomas and multiple myelomas.27-32,34 RIC can main-
tain complete remission for many years and induce
% RIC transplants 2007
no allogeneic hematopoietic stem cell
transplantation/no report
no RIC
1-25
25.1-40
>40
Figure 4. Proportion of RIC HSCT amongst allogeneic HSCT reported to the EBMT activity survey office in 2007.
| 258 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
complete remission in patients with active disease prior
to the transplant. RIC HSCT does reduce early toxicity
and has expanded the age limit. Still, there are no com-
parative studies yet between RIC and standard condi-
tioning regimens for similar patient categories. There
are indications that incidence of acute and chronic
GvHD is probably similar if patients without engraft-
ment are excluded.33 The use of RIC HSCT is associated
with a higher incidence of graft rejection, specifically if
bone marrow is used as stem cell source.22 Peripheral
blood is, therefore, the recommended stem cell source
for RIC HSCT in the lower intensity spectrum. There
are also indications that RIC HSCT has made little
impact on the rate of infectious complications. There is
one exception: the risk of lethal septicemias within the
first 30 days is lower in patients with RIC HSCT. There
is clearly a reduced rate of days with neutropenia in
patients with RIC compared to standard HSCT. There is
one early complication, potentially lethal, which is
more frequent in RIC HSCT. The risk of early acute
hemolysis should not be neglected in situations of a
minor blood group barrier. Specifically after peripheral
blood transplants, rapidly expanding donor lympho-
cytes can induce hyperacute red blood cell lysis.38 With
a follow-up of more than 5 years in many disease cate-
gories, RIC HSCT appears to be associated with a high-
er incidence of relapse. Few longterm remissions are
observed for patients transplanted with RIC in very
advanced disease.
Algeria
Iran
Israel
Lebanon
Saudi Arabia
South Africa
Tunisia

RIC HSCT can be applied to patients with high risks
as measured by EBMT risk score or to patients with
multiple comorbidities or a high Sorror score. However,
RIC HSCT does not abrogate the risk score or the
comorbidity or Karnofsky score. Patients with a high
risk score or a high comorbidity score have a worse out-
come than patients with a low risk score or with no
comorbidities, whether they receive a transplant with
standard or RIC approach.38
RIC HSCT has also been discussed as a new tool to
avoid late complications of an allogeneic HSCT in chil-
dren.39 Specifically, some late effects, such as cataract
formation, growth retardation or fertility are directly
affected by conditioning intensity. Feasibility of RIC
HSCT in children with malignant disorders has been
documented. RIC HSCT appears less promising in con-
genital disorders such as hemoglobinopathies, where
the risk of rejection remains high. It remains open
whether the improvement in late effects remains in bal-
ance with the risk of late relapse.
Outlook
The place of RIC appears most successful in patients
with advanced age where early transplant related mor-
tality caused by the conditioning reduces the probabili-
ty of a better survival compared to a conventional non-
transplant strategy the most. This applies primarily to
patients with acute leukemia above the age of 60 years.
Survival prospects without a transplant are limited but
not nil. If RIC can maintain complete remissions at the
expense of a limited amount of early toxicity and mor-
tality it might become standard of care. Preliminary
pilot data from non-controlled studies indicate that the
traditional 20% survival rate at 5 years without a trans-
plant could be increased to 40 to 50% with RIC HSCT.
No comparative data are available yet.34,40 A prospective
controlled European wide study has been organised by
the EBMT. Patients with AML in first complete remis-
sion after induction chemotherapy and with an HLA-
identical sibling or unrelated donor will be randomized
to early RIC HSCT in first CR or to conventional con-
solidation chemotherapy. If the study can successfully
be conducted, we might know in 5 years from now
whether RIC HSCT will hold the promises it gives
today.
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13. Barnes DW, Corp MJ, Loutit JF, NeaL FE. Treatment of murine
leukaemia with X rays and homologous bone marrow; prelim-
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14. Thomas ED, Buckner CD, Clift RA, Fefer A, Johnson FL,
Neiman PE, et al. Marrow transplantation for acute nonlym-
phoblastic leukemia in first remission. N Engl J Med 1979;
301:597-9.
15. Kolb HJ, Schattenberg A, Goldman JM, Hertenstein B,
Jacobsen N, Arcese W, et al. Graft-versus-leukemia effect of
donor lymphocyte transfusions in marrow grafted patients.
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Working Party Chronic Leukemia. Blood 1995;86:2041-50.
16. Kolb HJ. Graft-versus-leukemia effects of transplantation and
donor lymphocytes. Blood. 2008;112:371-83.
17. Giralt SA, Kolb HJ. Donor lymphocyte infusions. Curr Opin
Oncol 1996;8:96-102.
18. Slavin S, Naparstek E, Nagler A, Ackerstein A, Kapelushnik J,
Or R. Allogeneic cell therapy for relapsed leukemia after bone
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cytes. Exp Hematol 1995;23:1553-62.
19. Slavin S, Naparstek E, Nagler A, Ackerstein A, Samuel S,
Kapelushnik J, et al. Allogeneic cell therapy with donor periph-
eral blood cells and recombinant human interleukin-2 to treat
leukemia relapse after allogeneic bone marrow transplan-
tation Blood 1996;87:2195-204.
20. Slavin S. Immunotherapy of cancer with alloreactive lympho-
cytes. N Engl J Med 2000;14;802-3.
21. Storb R, Yu C, Wagner JL, Deeg HJ, Nash RA, Kiem HP, et al.
Stable mixed hematopoietic chimerism in DLA-identical litter-
mate dogs given sublethal total body irradiation before and
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tation. Blood 1997;89:3048-54.
22. Niederwieser D, Maris M, Shizuru JA, Petersdorf E, Hegenbart
U, Sandmaier BM, et al. Low-dose total body irra-diation (TBI)
and fludarabine followed by hematopoietic cell transplanta-
tion (HCT) from HLA-matched or mismatched unrelated
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24. Baron F, Storb R, Storer BE, Maris MB, Niederwieser D,
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25. Hegenbart U, Niederwieser D, Sandmaier BM, Maris MB,
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Shizuru JA, Greinix H, et al. Treatment for acute myelogenous
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tioning and hematopoietic cell transplantation from related
and unrelated donors. J Clin Oncol 2006;24:444-53.
26. Or R, Shapira MY, Resnick I, Amar A, Ackerstein A, Samuel S,
et al. Nonmyeloablative allogeneic stem cell transplantation
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phase. Blood 2003;101:441-5.
27. Rotta M, Storer BE, Sahebi F, Shizuru JA, Bruno B, Lange T, et
al. Long-term outcome of patients with multiple myeloma
after autologous hematopoietic cell transplantation and non-
myeloablative allografting. Blood 2009;113:3383-91.
28. Sobecks RM, Dean R, Rybicki LA, Chan J, Theil KS, Macklis
R, et al. 400 cGy TBI with fludarabine for reduced-intensity
conditioning allogeneic hematopoietic stem cell transplanta-
tion. Bone Marrow Transplant 2008;42:715-22.
29. Maris MB, Niederwieser D, Sandmaier BM, Storer B, Stuart
M, Maloney D, et al. HLA-matched unrelated donor hemato-
poietic cell transplantation after nonmyeloablative condition-
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102:2021-30.
30. Giralt S, Estey E, Albitar M, van Besien K, Rondón G, Ander-
lini P, et al. Engraftment of allogeneic hematopoietic progeni-
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31. Crawley C, Szydlo R, Lalancette M, Bacigalupo A, Lange A,
Brune M, et al. Chronic Leukemia Working Party of the EBMT.
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Chronic Leukemia Working Party of the EBMT. Blood 2005;
106:2969-76.
32. Crawley C, Lalancette M, Szydlo R, Gilleece M, Peggs K,
Mackinnon S, et al. Chromic Leukaemia Working Party of the
EBMT. Outcomes for re-duced-intensity allogeneic transplan-
tation for multiple myeloma: an analysis of prognostic factors
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Blood 2005;105:4532-9.
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European Group for Blood and Marrow Transplantation
(EBMT). Accreditation Committee Increasing use of reduced
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activity survey. Bone Marrow Transplant 2002;30:813-31.
34. Appelbaum FR. Allogeneic hematopoietic cell transplantation
for acute myeloid leuke-mia when a matched related donor is
not available. Hematology Am Soc Hematol Educ Program
2008;2008:412-7.
35. Mielcarek M, Martin PJ, Leisenring W, Flowers ME, Maloney
DG, Sandmaier BM, et al. Graft-versus-host disease after non-
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transplantation. Blood 2003;102:756-62.
36. Junghanss C, Marr KA, Carter RA, Sandmaier BM, Maris MB,
Maloney DG, et al. Incidence and outcome of bacterial and
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tion: a matched control study. Biol Blood Marrow Transplant
2002;8:512-20.
37. Sorror ML, Giralt S, Sandmaier BM, De Lima M, Shahjahan M,
Maloney DG, et al. Hematopoietic cell transplantation specif-
ic comor-bidity index as an outcome predictor for patients
with acute myeloid leukemia in first remission: combined
FHCRC and MDACC experiences. Blood 2007;110:4606-13.
38. Kimura F, Sato K, Kobayashi S, Ikeda T, Sao H, Okamoto S, et
al. Japan Marrow Donor Program. Impact of ABO-blood
group incompatibility on the outcome of recipients of bone
marrow transplants from unrelated donors in the Japan
Marrow Donor Program. Haematologica 2008;93:1686-93.
39. Yaniv I, Stein J. EBMT Paediatric Working Party. Reduced-
intensity conditioning in children: a reappraisal in 2008. Bone
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40. Niederwieser D, Lange T, Cross M, Basara N, Al-Ali H.
Reduced intensity conditioning (RIC) haematopoietic cell
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Haematol 2006;19:825-38.
 Thrombosis

Genetics of thrombosis

P.H. Reitsma A B S T R A C T

Einthoven Laboratory for
Experimental Vascular Medicine,
Departments of Thrombosis and
Hemostasis and Nephrology, Leiden
University Medical Center, Leiden,
The Netherlands
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:261-265
Venous thrombosis is a common episodic disease with a complex etiology. The mean annual inci-
dence of venous thrombosis is about 1:1000, but there is a steep age gradient. In the etiology, both
environmental and genetic risks are important. The environmental risk factors include medical condi-
tions, such as surgery, prolonged bed rest and cancer, but also life style conditions, varying from obe-
sity and oral contraceptive use to long-haul air travel. A variety of genetic risk factors is known for
venous thrombosis. Arbitrarily these are often divided between classical genetic risk factors and ‘other’
genetic risk factors. There are six classical risk factors and these include deficiencies of the natural
anticoagulants protein C, protein S and antithrombin, factor V Leiden, prothrombin 20210, and blood
group non-O. With the exception of blood group non-O, these risk factors are quite rare in the gener-
al population, but their associated relative risks are high, varying from 2 for blood group non-O to per-
haps 20 antithrombin deficiency. The remaining risk factors consist predominantly of single for
nucleotide polymorphisms (SNPs) in a variety of genes encoding coagulation proteins. The risk alleles
of these SNPs are often quite common, but the associated relative risks are low.

Introduction also in veins. Deep venous thrombosis

The blood coagulation system is responsi-
ble for stemming the loss of blood in the
case of vascular damage. In almost all indi-
viduals, the system is quite effective and
excessive blood loss in therefore uncom-
mon. On the other hand, the capacity to
form blood clots brings the risk that blood
clotting inadvertently leads to blood clots in
the circulation per se: thrombosis. Such
blood clots often occur in the arterial circu-
lation of individuals affected by atheroscle-
rosis.
Atherosclerosis is an inflammatory degen-
erative disease of the vessel wall. The degen-
eration of the vessel wall leads to so-called
atherosclerotic plaques with procoagulant
properties. The procoagulant potential of
the plaque becomes operational when the
endothelium on the plaque surface is dam-
aged, for example by plaque rupture, leading
to the formation of a blood clot. This clot
may occlude the artery leading to myocar-
dial infarction or ischemic stroke. In essence,
the blood clotting reactions on the damaged
plaque surface are not different from the
reactions that occur when a blood vessel is
damaged by a trauma, in which case the pri-
mary driving force is also the exposure of
strongly procoagulant proteins and surfaces
to blood. The reaction that follows is only to
a limited extend sensitive to subtle varia-
tions in the concentrations of the coagula-
tion proteins, which explains why levels of
coagulations proteins, whether determined
genetically or by environmental factors, only
play a minor role in determining risk for
arterial thrombosis.
Blood clots not only occur in arteries but
(DVT) and pulmonary embolism (PE), com-
monly referred to as venous thromboem-
bolism (VTE), are common complications
during and after hospitalization for acute
medical illness or surgery, but also often
occur spontaneously. The overall incidence
of VTE, which increases with age, is approx-
imately 1:1000 in Western Europe and the
United States (USA).1,2 The results of the
VITAE study reported that the annual bur-
den in the European Union (EU) of fatal and
non-fatal symptomatic VTE comprises
435,000 cases of PE and 684,000 cases of
documented symptomatic DVT.3 The yearly
number of VTE-related deaths was estimat-
ed at 543,500.
Pulmonary embolism accounts for 5 to 10%
of deaths in hospitalized patients, making
VTE the most common preventable cause of
in-hospital death.
In the United Kingdom, an independent
Government Health Select Committee
enquiry in 2007 estimated that VTE in hos-
pitalized patients causes 25,000 deaths each
year(http://www.dh.gov.uk/en/Publicationsand
statistics/Lettersandcirculars/Dearcolleagueletter
s/DH_073957). The Committee noted that
deaths from VTE in hospital exceed the
combined total of deaths from breast cancer,
AIDS and traffic accidents. This mortality is
also over 25 times greater than the annual
deaths from methicillin resistant
Staphylococcus aureus (MRSA) and more than
five times the total of all hospital-acquired
infections.
These data give a strong indication of the
burden of VTE in the EU, and underline that
actions to improve the prevention of VTE in

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 261 |
14th Congress of the European Hematology Association
primary, secondary and tertiary care are badly needed.
The need for action is also supported by the U.S. Surgeon
General’s Call to Action released 15 September 2008
(http://www.surgeongeneral.gov/topics/deepvein/calltoaction/cal
l-to-action-on-dvt-2008.pdf).
The risk of venous thrombosis is determined partly
by the delicate balance between procoagulant and anti-
coagulant pathways, but it also involves a poorly
defined anatomic substrate in the proximal leg veins.
The age-gradient of thrombosis incidence is steep,
increasing from less than 1:10,000 in the young to 1:100
per year in the elderly. Precise mechanisms for this age
gradient remain largely unknown.4 Prominent among
hypothesized factors are age related metabolic changes,
steroid hormone use, obesity and cancer.
The basal risk for thrombosis is strongly influenced
by inherited risk factors. The notion that genetics are
important for venous thrombosis dates back to observa-
tions on familial occurrence in 1956.5 In the ensuing 50
years, stepwise progress was made in the identification
of inherited gene variations that, at least partly,
explained the familial occurrence of thrombosis.
Moreover, we have learned now that in individuals
with an isolated thrombosis, genetic risk factors may
also play a role in determining the risk profile.
Natural anticoagulants
The procoagulant forces of the clotting system are
kept in balance by several anticoagulant mechanisms.
The first operates at the level of the initiation of blood
coagulation and involves the soluble plasma protein tis-
sue factor pathway inhibitor (TFPI).6 TFPI can bind to
the factor Xa when it is still in complex with tissue fac-
tor-factor VIIa on the cell membrane.
Second, there is a set of serine protease inhibitors (ser-
pins) that are capable of irreversible inhibition of acti-
vated coagulation enzymes. Best known among these is
antithrombin, that inhibits the activity of, not only the
procoagulant thrombin, but also of factors IXa and Xa.7
The inhibitory capacity of antithrombin is greatly
enhanced by heparin. Heparin cofactor II inhibits
thrombin rapidly in the presence of dermatan sulfate,
heparan sulfate, or heparin.8 Protein Z-dependent pro-
tease inhibitor inhibits factors Xa and XIa of the coagu-
lation cascade.9 Its name implies that it requires protein
Z, another circulating protein, to function properly, but
this only applies to its inhibition of factor Xa. Finally,
CI-inhibitor functions as a potent inhibitor for the coag-
ulation factors XIa and XIIa.10
Next, there is the so-called protein C anticoagulant
system.11 When the blood coagulation system is silent,
this anticoagulant system does not operate. It becomes
active when thrombin, the key enzymatic end-product
of the clotting system, binds to the endothelial cell
membrane protein thrombomodulin (TM). Once bound
to TM, thrombin loses its capacity to cleave fibrinogen.
Instead, thrombin will cleave protein C to form activat-
ed protein C. This reaction is enhanced by the endothe-
lial protein C receptor (EPCR). APC is able to proteolyt-
ically inactivate the procoagulant co-factors factor Va
and factor VIIIa in the presence of its co-factor protein
S.
Taken together, these anticoagulant mechanisms
cover each serine protease and activated co-factor of the
| 262 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
coagulation cascade except for factor VIIa, for which
there is no inhibitor when it is not in complex with tis-
sue factor and factor Xa.
Deficiencies of natural anticoagulants
The coagulation balance will shift towards increased
clot formation when coagulation inhibitors are present
in low amounts. Based on this, one would predict that
inhibitor deficiencies would increase the risk of throm-
botic disease. Indeed, this appears to be the case. In
1965, it was already established by Egeberg et al. that a
partial deficiency of antithrombin leads to an increased
risk of thrombosis.12 A complete deficiency of
antithrombin has not been observed in humans and is
generally considered to be incompatible with life.
A similar pattern is observed for deficiencies of pro-
tein C and protein S. Shortly after the discovery of these
anticoagulant proteins, it was established that complete
deficiency of either protein C or protein S leads to lethal
purpura fulminans immediately after birth, whereas a
partial deficiency increases the risk for venous thrombo-
sis.13-15
The magnitude of the increased risk of protein C, S,
and antithrombin deficiency is not known with certain-
ty but has been estimated to be 10 to 20-fold. This esti-
mate is mostly based on observations in anecdotal fam-
ilies rather than on large case-control studies. The rea-
son why these latter studies have failed to yield reliable
risk estimates is that the deficiency states are quite rare
in the normal population (1 in 500 to 1 in 1000 at most),
indicating that only very large studies will suffice for a
reliable estimate.16,17
After cloning of the genes encoding protein C, S and
antithrombin it became possible to search for the genet-
ic alterations that lead to a deficiency state. As expect-
ed, hundreds of different mutations have been docu-
mented that all lead to loss of function of the gene in
question.18-20 It almost appears as if every family has its
own private mutation, and in that respect, the genetic
architecture of deficiencies of natural anticoagulants is
very much like the genetic architecture of for example
hemophilia B. The spectrum of mutations is kept in
databases like the Human Gene Mutation Database
(http://www.hgmd.cf.ac.uk/ac/). The large variety of muta-
tions makes it difficult to do routine genetic testing for
protein C, S or antithrombin deficiency. Therefore, the
diagnosis of these deficiency states in a thrombophilia
screening wholly relies on plasma assays. This is quite
straightforward for antithrombin and protein C, but can
be unreliable for protein S. These complications are
beyond the scope of this review.
The other components of the natural anticoagulant
system are not clearly related to an increased risk of
venous thrombosis but they may be related to other dis-
eases. Levels of heparin cofactor II are probably associ-
ated with atherosclerosis.21 In some strains of mice,
heparin cofactor II deficiency is lethal, and neointima
formation is increased in heterozygotes.22 One early
report described deleterious nonsense mutations in ZPI,
which were associated with venous thrombosis.23 This
result could not be confirmed in a recent meta-analy-
sis.24 Complete deficiency of C1 inhibitor leads to
angioedema, probably because of effects on the comple-
ment system.25 Heterozygosity for C1 inhibitor defects

seems to be associated with age-related macular degen-
eration.26
Several studies have tried to relate levels of TFPI to
venous thrombosis. Low levels of TFPI seem to be relat-
ed to venous thrombosis, but homozygosity or het-
erozygosity for true deficiency states have not been
described.27
The other components of the protein C anticoagulant
system, thrombomodulin and EPCR have also been
studied, and again, no true deficiency states have been
documented in humans. Both are membrane receptors
of the endothelium. Shedding of these receptors into
the blood does occur and several assays are available to
estimate levels of soluble TM and EPCR. The relation-
ship between these levels and venous thrombosis is not
very strong. Several missense mutations and polymor-
phisms in the TM gene have been found, but the rela-
tionship with venous thrombosis has not been proven
unequivocally.28 There is one frequent polymorphism in
the EPCR gene that is quite strongly related to soluble
levels of EPCR that may be a weak risk factor for
venous thrombosis.29
Gain of function mutations in procoagulant proteins
Not only a (partial) deficiency of coagulation
inhibitors tips the hemostatic balance in the direction of
venous thrombosis. An excess of pro-coagulant factors
may do the same. It was not until the discovery of
Factor V Leiden that this notion became generally
accepted.
The discovery of this mutation was based on seminal
findings of Dahlbäck et al. in a family with what is now
called APC resistance.30 The unusual plasma phenotype
in this family became evident in assays where the acti-
vated partial thromboplastin time (APTT) was meas-
ured in both the absence and presence of activated pro-
tein C (APC). In most individuals, the addition of APC
markedly prolonged the APTT, but in members of the
family that Dahlbäck et al had identified the prolonga-
tion was much less. Later studies showed that APC
resistance is due to a mutation in coagulation factor V
within one of the cleavage sites for APC, and this muta-
tion was named factor V Leiden.31 Although activated
factor V Leiden can still be degraded by APC through
alternative cleavage sites, inactivation is less efficient.
From the perspective of protein function, this mutation
is a gain-of-function mutation because the pro-coagu-
lant potential of factor V Leiden is stronger than that for
normal factor V. Since there are fewer possibilities to
improve protein function, there is little or no hetero-
geneity in the genetic basis of APC resistance; in other
words, the plasma phenotype almost always goes with
the same mutation.
The relative risk for venous thrombosis associated
with factor V Leiden was first estimated in the Leiden
Thrombophilia Study (LETS) and found to be around
seven.31 Although the estimates vary somewhat in sub-
sequent studies, these original results were essentially
confirmed. Homozygotes for factor V Leiden have a rel-
atively mild phenotype, at least compared to the severe
phenotype of homozygotes for deficiencies of natural
anticoagulants.32 Compared to heterozygous deficiency
of natural anticoagulants, the risk of homozygous factor
V Leiden is quite potent.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 263 |
Berlin, Germany, June 4-7, 2009
The prevalence of factor V Leiden is high in the
Caucasian population. In LETS, the prevalence in the
Dutch population was estimated at 3%. In other ethnic
groups, like the Chinese and Japanese, factor V Leiden
is extremely rare.33
Not long after the discovery of APC resistance and
factor V Leiden, a second unique mutation in a gene
encoding a pro-coagulant protein, prothrombin, was
found.34 This mutation, often called FII G20210A, was
later shown to increase the level of prothrombin mRNA
by improving 3’ end formation of mature mRNA.35 This
effect on mRNA metabolism leads to elevated levels of
plasma prothrombin. The increase is about 20% in het-
erozygotes, and in such individuals, the relative risk for
venous thrombosis is increased by about a factor of
three.34 By analogy with factor V Leiden, the thrombot-
ic tendency in homozygotes is relatively modest, the
prevalence in the general population is quite high
(~1%), and the mutation is present primarily in Cau-
casians and not in other races.36
As noted above, the notion that procoagulant factors
could carry a risk for venous thrombosis was only gen-
erally accepted after the discovery of APC resistance
and factor V Leiden. This is surprising in light of the fact
that already in 1969, Jick et al. noted that ABO blood
group played an important role in venous thrombotic
risk, in particular within the context of oral contracep-
tive use.37 It appeared that non-O individuals had a high-
er risk than individuals with blood group O. This was
later confirmed in more formal studies and the risk asso-
ciated with non-O is estimated to be around two.38
A probable explanation for this increased risk can be
based on seminal observations that date back to 1964.
Preston and Barr reported in that year that coagulation
factor VIII levels were higher in individuals with non-O
blood group than in those with blood group O.39 Later
research showed that this was due to an effect of blood
group on levels of Von Willebrand Factor, the carrier
protein of factor VIII.
To summarize, genetically determined increases in
procoagulant function increase the risk for venous
thrombosis. Factor V Leiden, PT 20210A and non-O
blood group are the major risk factors with relative risks
varying between seven and two. The risk factors are
much more prevalent in the population than those
involved in anticoagulant function.
Single nucleotide polymorphisms
The genetic findings in procoagulant and anticoagu-
lant proteins described above were obtained in relative-
ly straightforward family and case-control studies using
simple genetic analysis tools. In the meantime, The
Human Genome Project has taught us that the human
genome contains millions of variations that set one indi-
vidual apart from another. Most of these differences
take the form of common single nucleotide polymor-
phisms (SNPs). In theory, some of the common SNPs
may alter the risk for venous thrombosis, and the chal-
lenge that is before us is to sift out these relatively few
functional SNPs from the millions of SNPs that are irrel-
evant. During the past years, SNPs that influence throm-
botic risk have indeed been found. The discovery plat-
forms used a combination of family and case-control
studies with either candidate gene or whole genome
14th Congress of the European Hematology Association
screening.
The results include SNPs in procoagulant genes, such
as fibrinogen (alpha, beta, and gamma), factor VIII,
Tissue Factor, factor IX, factor X, factor XI, factor XIII
(both the A and B subunit), glycoprotein 6, and
Thrombin Activated Fibrinolysis Inhibitor (TAFI). Also
common SNPs in the natural anticoagulants were found
for TFPI, TM, protein C, and antithrombin.40,41 Al-
together, there is now reasonably convincing data for
about 20 SNPs – mostly, if not exclusively, located in
coagulation genes – that influence thrombotic risk. As
the large genome wide association studies that are still
under way become finished this number is likely to
increase considerably and perhaps also SNPs in genes
not commonly associated with hemostasis and throm-
bosis will be found.
The risk SNPs that were discovered share two com-
mon features. Almost by definition, the risk alleles have
a relatively high prevalence in the population, with
extremes up to 80%. Secondly, the risk for venous
thrombosis that goes with these risk factors is relative-
ly low, that is, in the order of 1.5-fold.
Conclusions
This review discussed how research into the genetic
background of venous thrombosis slowly shifted from
discovering proteins and genes based on plasma assays
to whole-genome studies aimed at determining the risk
associated with common genetic variation in the form
of SNPs. At the same time, we have seen attention shift
from genetic abnormalities that go with high risk, for
example, 20-fold for antithrombin deficiency, to those
with a much smaller risk. The overall result is that we
now know about 20 genetic risk factors for venous
thrombosis. This is providing us with unprecedented
insight into the genetic architecture of this common but
complex disease. It is tempting to assume that the pres-
ent knowledge of so many genetic risk factors easily
leads to robust predictive models for venous thrombo-
sis. However, work in progress indicates that this is
almost certainly not the case. The challenge which
remains before us is to translate these insights into
approaches that can be used in the doctor’s office; for
example, for better tailoring of dose or duration of anti-
coagulant treatment.
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 Thrombosis

Venous thrombosis: from bench to bedside and back

T. Baglin A B S T R A C T

Department of Haematology,
Addenbrooke’s NHS Trust,
Cambridge, UK
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:266-270
The laboratory has a critical role in the diagnosis and management of venous thromboembolism.
Increasingly, diagnosis is dependent on diagnostic algorithms that incorporate D-dimer measurement.
Monitoring and adjusting the intensity of anticoagulation determines efficacy and safety, and recent
studies indicate that the quality of control of oral anticoagulant therapy determines the long-term risk
of post thrombotic syndrome. The identification of heritable and acquired thrombophilic defects has
helped us to understand the mechanism of venous thromboembolism and appreciate the importance
of gene-environment interaction, although testing for a limited number of heritable thrombophilic
defects has not been shown to have clinical utility so far. Recent studies indicate that measurement
of the global activity of the coagulation system, either using biomarkers or measuring the thrombin
generating potential, may have useful clinical predictive value for recurrent thrombosis.
Pharmacogenetic studies incorporating rapid genotyping may improve anticoagulant therapy with
vitamin K antagonists. Finally, whilst a simple dichotomous testing strategy for heritable throm-
bophilic defects has not been shown to have useful clinical predictive value, future assessment of the
intermediate phenotype by global coagulation tests combined with genome wide mutation and SNP
detection may provide a complimentary approach to the quantification of both thrombotic and bleed-
ing risks.

Introduction tive approach to reliance on imaging tech-

Venous thrombosis (VT), or venous
thromboembolism (VTE), comprises deep
vein thrombosis (DVT) with or without
symptomatic pulmonary embolus (PE). The
incidence of a first episode of VT is 1.5 per
1000 person-years,1 with a per-person life-
time incidence of 5%.2 The clinical utility of
the atology laboratory has advanced signifi-
cantly with laboratory tests having an
increasing clinical utility in diagnosis, moni-
toring and prognosis (clinical utility is
defined as the likelihood that a test will lead
to an improved health outcome). Venous
thrombosis is a multicausal disease.
Understanding the genetic risk factors and
gene-environment interactions is key to
understanding why an individual develops
thrombosis at a specific point in time.3 The
risk factors for venous thrombosis and arte-
rial atherothrombosis overlap but the mate-
rial contribution of each differs between the
two diseases. Hypercoagulability resulting
from variation in the genetic control of
blood coagulation produces a greater materi-
al contribution to venous thrombosis.
However, it is becoming increasingly recog-
nised that both genetic and acquired risk fac-
tors are common denominators for venous
and arterial thrombosis.4
Diagnosis
As VTE is common, diagnostic clinical sus-
picion is high and consequently the preva-
lence of the disease in suspected cases is low
at about 10% and 20%. Because of cost and
a high number of negative tests, an alterna-
niques alone for diagnosis and decision mak-
ing in suspected cases of VTE has been
adopted. This relies on integrated strategies
that incorporate clinical assessment and
measurement of D-dimer.5 The emphasis of
methods is on high sensitivity and the safe
exclusion of VTE in a significant number of
patients, thus reducing the need for imaging.
For measurement of D-dimer, fast single
samples assays are now available, producing
results in less than 30 minutes with sensitiv-
ity in excess of 95%. For high sensitivity
assays, the negative predictive value (NPV) is
close to 100% and for intermediate sensitiv-
ity assays, the NPV is close to 100% when
the assay is combined with a pre-test proba-
bility score.6
The cut-off level used for exclusion of VTE
is not the upper limit of normal in a healthy
population. Rather, each assay has a desig-
nated cut-off for VTE exclusion. For some
assays, the cut-off has been validated in
management studies. The specificity of
D-dimer is inevitably low because D-dimer
levels are elevated by infection, inflamma-
tion, cancer, surgery, trauma, atherosclerosis,
bleeding and pregnancy. Consequently, the
specificity is lower in certain populations,
including the elderly and hospitalised
patients. The Vidas® D-dimer is one of the
most extensively evaluated assays and con-
sistently produces a 100% NPV for either
DVT or PE regardless of the pre-test proba-
bility score.6 An extensive meta-analysis
completed in 2007 calculated assay perform-
ance from 184 articles, including 328

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D-dimer test evaluations, and showed that automated
quantitative ELISAs and turbidometric assays generally
have a higher sensitivity than whole blood agglutina-
tion assays.7
In patients with previous DVT or suspected PE and a
suspected recurrence a negative high sensitivity
D-dimer assay result safely excludes recurrence.8,9
Monitoring
Heparin
The usual treatment of VTE requires therapeutic anti-
coagulation, with additional thrombolysis in up to 5%
of patients presenting with PE. Immediate heparin ther-
apy results in a low risk of fatal PE and progression of
DVT.10 A therapeutic APTT can be achieved quicker by
commencing UFH according to the patient’s weight.11
However, with doses of UFH exceeding 30,000 units
per 24 hours, it is now uncertain if continued adjust-
ment of heparin dose in response to the APPT improves
safety and efficacy.12 Nevertheless, current guidelines
recommend monitoring and dose adjustment of thera-
py with UFH.13,14
In many countries, UFH has been superseded by low
molecular weight heparin (LMWH). Treatment with
LMWH is at least as effective as UFH, and low risk
patients can be treated as outpatients.14 The APTT is rel-
atively insensitive to LMWH. Anti-Xa activity can be
used to monitor LMWH but there are significant limita-
tions.13 It is a major advantage of LMWH that therapeu-
tic anticoagulation does not require routine monitoring
or dose adjustment. However, in patients with renal fail-
ure, pregnancy and young children monitoring with an
anti-Xa assay is advised. A target peak anti-Xa level of
1.0-2.0 IU/mL is suggested for once daily subcutaneous
administration and 0.6-1.0 IU/mL for twice daily.14
Oral vitamin K antagonists
Continued treatment with an oral vitamin K antago-
nist (VKA), such as warfarin, will prevent more than
95% of recurrent episodes of VTE.15,16 VKA therapy is
monitored by the International Normalised Ratio (INR),
with a target INR of 2.5 for the majority of patients. The
risk of VTE recurrence during treatment is higher when
the INR is subtherapeutic.16 It has recently been demon-
strated that achievement of a sustained intensity of
early anticoagulation is required to reduce the risk of
post thrombotic syndrome (PTS),17 and possibly also
prevent recurrent VTE after treatment is stopped.18 Point
of care testing and patient self-management offer
opportunities for improved anticoagulant care in some
locations.
Van Dongen et al. reported that patients who have an
INR less than 2.0 for more than 50% of the time are
almost three-times as likely to develop PTS after DVT.17
After adjustment, the single most important risk factor
for PTS was recurrent ipsilateral DVT but there was
almost a 3-fold increased risk when more than 50% of
the time was spent with an INR less than 2.0. Whilst
ipsilateral recurrence was the stronger risk, only 7% of
patients suffered ipsilateral recurrence whilst 30% of
the time was spent below an INR of 2.0. A relationship
between subtherapeutic anticoagulation and the devel-
opment of PTS was reported in a retrospective analysis
but VKA therapy was not standardised by the INR.19
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 267 |
Berlin, Germany, June 4-7, 2009
Pharmacogenetics
The aim of pharmacogenetic-based dosing of oral
vitamin K antagonists (VKA) is to improve safety and
effectiveness. Warfarin induction, rather than mainte-
nance therapy, is the time when the INR is most likely
to be out of range and when the risk of thrombosis and
bleeding is greatest.20,21 Previously discussed, early sub-
therapeutic anticoagulation also increases the risk of
recurrent VTE and the development of PTS.
Common single nucleotide polymorphisms (SNPs)
in CYP2C9 genes are associated with altered kinetic
activity of CYP2C9 with lower warfarin maintenance
dose requirements. Two common SNPs in the vitamin
K epoxide reductase gene (VKORC1 6853 and -1639)
correlate with the dose of warfarin required to achieve
therapeutic anticoagulation, as measured by the INR.22
In patients with mutant alleles, there is an increased
risk of overanticoagulation and bleeding during induc-
tion of therapy.23-26 The influence of mutant alleles on
overanticoagulation and bleeding during long term
therapy is less.27,28 Studies of the combined effect of
CYP2C9 and VKORC1 polymophisms indicate that
approximately 50-70% of the warfarin dose require-
ment is determined by these genetic variants but a
prospective randomized trial of genotype-guided ver-
sus standard warfarin dosing in patients initiating war-
farin did not demonstrate a difference in percentage of
INRs out of range.29 In a recent study comparing clini-
cal and pharmacogenetic induction algorithms in
patients undergoing orthopedic surgery the time in
therapeutic range, measured by linear interpolation,
was higher in the pharmacogenetic cohort than the
clinical cohort and this was associated with fewer
adverse clinical events.30 Commercial platforms for
rapid genotyping are now available.31 Further studies
will be needed to clarify the clinical utility of pharma-
cogenetic testing for induction and maintenance thera-
py with oral VKA.32
Prognosis
After a first episode of VTE, patients are 40 times
more likely to suffer a further event compared with pre-
viously unaffected individuals.33 Recurrent DVT or PE is
associated with a high case-fatality rate and an
increased likelihood of PTS and pulmonary hyperten-
sion, respectively. Therefore, secondary prevention of
VTE significantly reduces the burden of disease.
However, recurrent VTE is only prevented for as long as
anticoagulation is continued and so anticoagulation
must be continued indefinitely in patients at high risk.
In view of the risk of anticoagulant-related bleeding,
there is a need to improve risk stratification for recur-
rent VTE, and hence target long-term therapy at
patients considered to be at greatest risk. Similarly,
accurate individualised estimates of anticoagulant-relat-
ed bleeding are required in order to estimate the benefit
and risk trade-off with continued anticoagulation. The
risk of recurrent VTE in an individual can be determined
from a variety of interacting clinical, laboratory and
imaging results. It is becoming apparent that measure-
ment of the global activity of the coagulation system,
using either biomarkers34 or measuring the thrombin
generating potential,35-38 may have useful clinical predic-
tive value for VTE recurrence.
14th Congress of the European Hematology Association
Hypercoagulability (D-dimer and thrombin generation)
One month after completion of treatment of a first
episode of VTE, 40% of patients have increased levels
of D-dimer and these patients are at significantly greater
risk of recurrence.34,39 Measurement of D-dimer levels
following cessation of anticoagulant therapy has a high
negative predictive value (NPV) for recurrent venous
thrombosis with 96% of patients with a low D-dimer
being recurrence free at 2 years.34 The positive predic-
tive value (PPV) has less clinical utility. In patients with
a first episode of unprovoked venous thrombosis and an
elevated D-dimer after stopping anticoagulant therapy,
there is still a likelihood of being free from recurrence of
approximately 90% at 2 years.34 Therefore, it is debat-
able whether a high D-dimer result alone is an indica-
tion for prolonged anticoagulation.40,41 Patients at high
risk of recurrence might be better identified by a combi-
nation of clinical and laboratory factors.42,43 Furthermore,
the predictive value of D-dimer may be improved by
using different cut-offs according to patient characteris-
tics, including age.44 Additional cohort and management
studies are still required to determine the clinical utility
of D-dimer testing, including the value of measurement
during anticoagulant treatment.
D-dimer measurement reflects in vivo thrombin gener-
ation that has occurred. Potentially complimentary to
this measurement is analysis of the tissue factor-throm-
bin response coupling mechanism by measurement of
the in vitro thrombin generating capacity.45-47 This indi-
cates how much relative thrombin an individual may
generate in response to a pre-determined stimulus.
Several recent studies have demonstrated a higher rate
of recurrent VTE in patients with increased thrombin
generation after unprovoked VTE.35-38 Interestingly, D-
dimer and the thrombin generating potential are inde-
pendent predictors of risk of recurrence35,36 suggesting
they may be measuring different aspects of hypercoag-
ulability. Future studies will evaluate the clinical predic-
tive value of these measurements in conjunction with
clinical risk factors.
A strong association between a short APTT and the
risk of VTE is well recognised.48 The Activated throm-
boplastin Time (APTT) is a simple global assay of
thrombin generation, albeit of limited physiological rel-
evance. Nevertheless two studies have demonstrated an
association between short APTT values and recurrent
VTE.49,50 However, after correction for levels of factors
VIII, IX and XI there was no independent predictive
value. High factor VIII and IX levels have previously
been reported as independent predictors of VTE recur-
rence.51,52
Genetic testing
Heritable thrombophilia describes an inherited ten-
dency for venous thrombosis, with or without associat-
ed pulmonary embolus. In addition to case-control stud-
ies, family pedigree studies have reported a risk of
venous thrombosis in association with heritable throm-
bophilia in thrombosis-prone families. However, the
reported rates of thrombosis in these studies are consid-
ered to be overestimates of the true risk due to selection
bias. Nevertheless, a causal link between heritable
thrombophilia and venous thrombosis is unequivocal.3,53
In the 1980s and 1990s, thrombophilia testing became
| 268 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
common in unselected patients and their relatives,
despite the fact that clinical utility was unproven.54 It is
now apparent that:
(i) recurrence on treatment is not more likely in unse-
lected patients with heritable thrombophilia;55
(ii) testing for heritable thrombophilia does not use-
fully predict likelihood of recurrence after stopping anti-
coagulant treatment in unselected patients;56,57
(iii) testing for inherited thrombophilia has not been
shown to prevent VTE recurrence;58
(iv) systematic review of the risk of recurrent venous
thromboembolism in patients heterozygous for the fac-
tor V Leiden has shown a modest risk not justifying life
long anticoagulation;59
(v) in patients with deficiency of a natural anticoagu-
lant (antithrombin, protein C, protein S deficiency), the
risk of recurrence is uncertain but relative risks of recur-
rence in relation to deficiency of a natural anticoagulant
appear to be less than 2.0 in unselected patients;56,57,60
(vi) in young selected patients, the presence of defi-
ciency of antithrombin, protein C or protein S predicts
a high rate of recurrence61 but selection criteria need to
be defined;
(vii) cohort studies have demonstrated a low risk of
primary events in prospectively followed affected
asymptomatic relatives of patients with the factor V
Leiden or prothrombin mutations.62
Consequently indiscriminate testing for heritable
thrombophilia is not mandatory and decisions regarding
duration of anticoagulation (lifelong or not) should be
made with reference to whether or not an episode of
VTE was provoked and the presence of other risk fac-
tors, regardless of whether a heritable thrombophilic
defect is present.
Laboratory testing for acquired thrombophilia
Antiphospholipid syndrome (APS) is diagnosed when
thrombosis or pregnancy loss occurs in association with
positive laboratory tests for antiphospholipid antibody.
In contrast to heritable thrombophilia, thrombosis in
patients with APS commonly occurs in the arterial, as
well as venous circulation. Whilst there is a higher rate
of recurrent VTE in patients with APS, the risk-benefit
of long-term anticoagulation has not yet been clarified.
Therefore, it is recommended that the intensity and
duration of treatment should be determined on an indi-
vidual basis, taking into account the presence of addi-
tional risk is factors, the severity of the presenting event
and the risk of bleeding on warfarin, rather than just the
diagnosis of APS.63
Paroxysmal nocturnal oglobinuria (PNH) is associated
with venous and arterial thrombosis with the greatest
risk is in patients with large PNH clones (PNH granulo-
cytes >50%, 44% versus 5%).64 Oral anticoagulation
effectively prevents thrombosis and long-term anticoag-
ulation with a target INR of 2.5 is recommended.65
An acquired point mutation (1849G>T, JAK2V617F) is
present in the majority of Ph-negative myeloprolifera-
tive disorders (MPD).66 Thrombosis is the main cause of
mortality and morbidity in MPD and age over 60 years
is an additional risk factor. The contribution of co-exis-
tent heritable thrombophilia to thrombosis risk has not
yet been determined. In patients with MPD, the degree
of thrombocytosis does not predict thrombosis risk.67

This is also the case, even in essential thrombocytia.68
This does not exclude an effect of platelets, independent
of the actual count, on thrombosis risk. An elevated
white count is associated with thrombosis risk69 which
may relate to the observed reduction in thrombosis risk
with cytoreductive therapy. There is conflicting evi-
dence regarding the association between JAK2V617F
and thrombois risk in MPD patients and the association
requires clarification.
Conclusions
The association between the bench and bedside is a
two-way association. High quality clinical care is
dependent on a high standard of laboratory practice and
clinical practice informs research and development in
the laboratory. Given the importance of laboratory
results for diagnosis and management, continued satis-
factory performance in internal quality control and
external quality assurance is necessary to prevent
adverse clinical outcomes.
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 Thrombosis

New anticoagulants

A. Kleinjan
H.R. Büller
Department of Vascular Medicine,
Academic Medical Center,
Amsterdam, The Netherlands
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:271-275
A B S T R A C T
Although the efficacy of low molecular weight heparin and vitamin K antagonists is undisputed,
shortcomings of these anticoagulants prompted the search for new ones. New anticoagulants for the
prevention of venous thrombosis act upon different stages of the coagulation cascade. Targeting the
initiation phase is promising, but potential applications are limited. Many factor Xa inhibitors are
under development. Fondaparinux is one of the pentasaccharides, indirect factor Xa inhibitors, which
have been approved for several indications. Rivaroxaban is the direct factor Xa inhibitor in the most
advanced stage of development. Parenteral direct thrombin inhibitors have been approved for limited
indications. Dabigatran etexilate is the oral direct thrombin inhibitor in phase III stage of development.
Inhibitors of factors in the amplification phase of the coagulation could potentially be interesting
because they do not completely block thrombin generation. Drugs specifically targeted at factor XI are
also being developed. Recombinant activated protein C and thrombomodulin act upon natural antico-
agulant pathways. Because of the arrival of these new anticoagulants, the field of anticoagulant treat-
ment may change drastically within a few years.

Introduction inhibits factor Xa and thrombin. LMWH

Arterial and venous thromboembolic
events are frequently encountered and cause
significant morbidity and mortality. The
introduction of anticoagulants more than 50
years ago meant a significant step forward in
the therapeutic management of these dis-
eases. Anticoagulants currently used for the
prevention of venous thrombosis are low
molecular weight heparins (LMWH) and
fondaparinux. Vitamin K antagonists,
LMWH and fondaparinux are the main
drugs for the treatment of symptomatic
venous thrombosis. For the prevention and
treatment of arterial thrombosis anti-platelet
drugs and again vitamin K antagonists,
LMWH and fondaparinux are the current
drugs of choice.
The FDA approved Vitamin K antagonists
in 1954. These are taken orally and inhibit
the synthesis of the vitamin K dependent
clotting factors (factors II, VII, IX and X).
Because of the unpredictable anticoagulant
effect, the intensity has to be monitored,
with dose adjustments when necessary.
Vitamin K antagonists need four to five days
until a steady effect has been reached. In
case immediate protection is needed, bridg-
ing therapy with a parenteral anticoagulant
should be employed. The effect of vitamin K
antagonists can be reversed by oral vitamin
K and factor concentrates.
Discovered in 1916, unfractionated
heparin was the most commonly used par-
enteral anticoagulant until recently. Low
molecular weight heparin (LMWH) is a
derivative of unfractioned heparin, which is
given subcutaneously once or twice daily.
Both unfractioned heparin and LMWH
increase the activity of antithrombin, which
inhibits factor Xa more than thrombin. In
case of bleeding, protamine sulphate is an
antidote for unfractioned heparin and to a
lesser extent for LMWH.
Both compounds are highly active and
their efficacy in the prevention and treat-
ment of venous and arterial thrombosis is
undisputed. Limitations of existing parenter-
al anticoagulants, such as users’ conven-
ience, risk for bleeding and need for dose
adjustments, prompted the search for new
parenteral anticoagulants. Shortcomings of
existing oral anticoagulants, such as the need
for monitoring and bridging therapy and the
bleeding risk stimulated the search for new
oral antithrombotic agents. Because of the
better knowledge of how the coagulation
cascade functions, specifically targeted drugs
were developed.1
Here we will review the new anticoagu-
lants for the prevention and treatment of
venous thrombosis, with the coagulation
cascade as a guide (Figure 1).
The coagulation system
The process of clot formation is divided in
three parts, which do not occur consecutive-
ly but rather simultaneously: primary hemo-
stasis involving platelet activation and aggre-
gation, coagulation cascade activation result-
ing in fibrin formation and fibrinolysis.
Venous thrombi are formed under low shear
conditions and are mainly composed of fib-
rin and red blood cells. Therefore, to prevent
or treat venous thrombosis, targeting the
coagulation cascade is the most appropriate
approach.
The coagulation cascade consists of a few
overlapping stages, in which inactive

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 271 |
14th Congress of the European Hematology Association
enzymes become activated and subsequently activate
the next factor in the cascade (Figure 1). The initiation
of coagulation is triggered by tissue factor (TF), which
binds factor VIIa and as a complex, activates factor X.
Activated factor X then converts small amounts of pro-
thrombin to thrombin. These small amounts of throm-
bin are sufficient to activate the amplification loops via
factors V, VIII, IX and XI. Finally, thrombin converses
fibrinogen to fibrin. To prevent a hypercoagulable state,
procoagulant activity is regulated by anticoagulant
pathways, such as activated protein C, thrombomod-
ulin, antithrombin and tissue factor pathway inhibitor.2
Theoretically, new anticoagulants can be targeted at
each of these steps; however, not all steps are equally
attractive.
Classical evaluation of new anticoagulants
The clinical evaluation of new anticoagulant drugs
follows a typical pattern. First, the new drug is tested in
vitro, in animal models and in healthy volunteers.
Thereafter, clinical dose finding trials are set up in
orthopedic patients to evaluate the efficacy of the novel
compound for the prevention of thrombosis after knee
or hip replacement. In this group of patients, asympto-
matic thrombosis occurs frequently and can be easily
detected by venography 7-10 days postoperatively. This
allows the establishment, relative to control, of the
most effective dose. Furthermore, a non-safe dose is
also quickly identified. The second group of patients in
which drugs are subsequently evaluated, comprises of
patients with symptomatic venous thrombosis. Again,
usually initially with dose finding studies, followed by
large phase III studies. Finally, when the optimum dose
is known, the drug is tested in patients with or with a
risk for arterial thrombosis, such as atrial fibrillation and
the acute coronary syndrome.
Initiation
Conceptually, targeting TF, factor VIIa or the complex
is promising; however, most studies were disappointing
and potential applications are limited. A recombinant
TF-pathway inhibitor named tifacogin was evaluated in
sepsis patients, after animal studies showed an effect on
mortality. A large phase III trial in patients with severe
sepsis comparing tifacogin to placebo could not demon-
strate an effect of tifacogin on 28-day mortality, while
there was a significant higher risk of bleeding.3
NAPc2, a protein originally isolated from a hook-
worm, binds to factor X or Xa, and as a complex,
inhibits TF bound factor VIIa. Promising results were
found with recombinant NAPc2 in a phase II dose-
response trial in patients undergoing elective knee
replacement surgery and in a phase II study in arterial
thrombosis. No phase III studies have been set up in
these patient groups so far.4,5
The development of active site-blocked factor VIIa
has been halted after disappointing results in patients
undergoing elective percutaneous coronary intervention
(PCI).
Factor Xa
Many factor Xa inhibitors are under development.
Factor Xa inhibitors are divided into agents, which
directly target factor Xa and indirect drugs, which
| 272 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
increase the affinity for antithrombin. Direct factor Xa
inhibitors also target factor Xa within the prothrombi-
nase complex, while indirect inhibitors only act against
free factor Xa.
Indirect factor Xa inhibitors
All indirect factor Xa inhibitors are administered sub-
cutaneously; however, because of the long half-life of
some of the compounds, they may also be suitable for
long-term indications.
Fondaparinux is the protype of the pentasaccharides,
which are synthetic analogs of a part of the heparin
molecule, namely the five sugars that bind to
antithrombin. Fondaparinux was first tested in patients
after hip and knee operations in phase II and III trials. A
meta-analysis of four large phase III trials, including
7344 patients undergoing hip or knee replacement, con-
cluded that fondaparinux showed a benefit over enoxa-
parin without increasing the incidence of clinically rele-
vant bleeding. This beneficial effect of fondaparinux
was on total venous thromboembolism (VTE), includ-
ing asymptomatic VTE, while rates of symptomatic
VTE were comparable in most studies.6
The Matisse studies compared fondaparinux to
enoxaparin for the initial treatment of deep venous
thrombosis or pulmonary embolism. Both studies
found that fondaparinux was equally safe and effective
as enoxaparin or unfractioned heparin for this indica-
tion.7,8 Also, in post-operative prevention of venous
thrombo-embolism after general surgery, fondaparinux
was shown to be effective. Fondaparinux has been
approved for these indications. Fondaparinux has also
been approved in arterial thrombosis, most notably
acute coronary syndromes.9-11 A phase III trial in patients
undergoing bypass surgery is planned.
Idraparinux is a similar pentasaccharide with a long
half-life, which makes once weekly administration pos-
sible and, therefore, was evaluated for the treatment of
VTE. The Van Gogh DVT and PE trials were phase III
trials comparing idraparinux with LMWH or vitamin K
antagonists for 3 to 6 months in patients with sympto-
matic deep venous thrombosis (DVT) or pulmonary
embolism (PE). In the DVT patients, the rates of recur-
rent VTE were similar in both groups, while clinically
relevant bleeds were initially less frequent with idra-
parinux. In the PE patients, however, idraparinux was
less effective than LMWH and vitamin K antagonists.12
The Van Gogh extension study aimed to evaluate the
efficacy of idraparinux for an additional 6 months of
treatment after patients completed 6 months of treat-
ment with idraparinux or a vitamin K antagonist. In this
study, idraparinux produced a 73% relative reduction in
the frequency of recurrent VTE, but at the cost of a rate
of 3.7 % major bleeding. After this study, safety con-
cerns halted further development of idraparinux.13
Biotynalated idraparinux (SSR-126517-E) is derived
from idraparinux and, therefore, exhibits the same bio-
logical charactistics as idraparinux. The added biotin
molecule to idraparinux facilitates direct neutralisation
by the infusion of avidin, an egg protein, as was demon-
strated in the EQUINOX trial.14 SSR12517E is now
undergoing phase III evaluation in patients with symp-
tomatic PE, after bridging therapy with heparin or
LMWH. In addition, this drug is currently being com-

Figure 1. The new anticoagulants and the various levels in the coagulation cascade they act upon.
pared to warfarin for the prevention of stroke in a phase
III trial (Borealis study).
SR 123781A comprises of a pentasaccharide plus a
thrombin binding tetrasaccharide and, therefore, has
both thrombin and Xa inhibiting activity. This drug is in
phase II evaluation for patients after knee arthroplasty.15
Direct factor Xa inhibitors
A lot of attention is presently going to direct oral Xa
inhibitors. Rivaroxaban is the oral Xa inhibitor in the
most advanced stage of development. It has a rapid
onset of action and a half-life of 5 to 9 hours, which
does not necessitate the use of bridging with parenteral
anticoagulants. Four phase II trials evaluated rivaroxa-
ban in patients undergoing knee or hip surgery and
showed that rivaroxaban was at least as effective as
enoxaparin, while higher doses of rivaroxaban caused
more major bleedings.16
The RECORD phase III trials compared rivaroxaban
with enoxaparin in the same patient population. All
three studies demonstrated that rivaroxaban was signif-
icantly more effective than enoxaparin in the prophy-
laxis of VTE after knee or hip replacement surgery, with
comparable bleeding rates.17-19 Interestingly, rivaroxaban
in a dose of 10 mg once daily was started post-opera-
tively.
The evaluation of rivaroxaban for the treatment of
DVT is in an advanced stage. Phase II studies showed at
least non-inferiority of rivaroxaban compared to
LMWH plus vitamin K antagonists, with low bleeding
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 273 |
Berlin, Germany, June 4-7, 2009
rates. Phase III studies for the treatment of VTE and for
stroke prevention in patients with atrial fibrillation are
ongoing.
Apixaban is another direct oral Xa inhibitor, currently
in phase III development: one phase III trial in orthope-
dic surgery has just been completed and showed prom-
ising results.20 Other phase III studies for the prevention
of VTE in hospitalized patients, in stroke prevention
and treatment of VTE are currently recruiting patients.
Results will probably become available within two to
three years. Many other oral direct Xa inhibitors are in
phase II development.21
Thrombin
The new anticoagulants in this class are direct throm-
bin inhibitors. Indirect thrombin inhibitors, such as
LMWH and heparin who act by stimulating antithrom-
bin, will not be described here.
Parenteral thrombin inhibitors have been approved
for limited indications. Hirudin and argatroban are reg-
istered for the treatment of patients with heparin-
induced thrombocytopenia, and bivalirudin is licensed
as an alternative to heparin in percutaneous cutaneous
interventions. Two new parenteral direct thrombin
inhibitors are in phase II development. The oral direct
thrombin inhibitor, which provided proof of concept,
was ximelagatran. However, because of liver toxicity,
the drug was removed from the market, after having
been licensed briefly in Europe. Currently, dabigatran
etexilate is the most advanced in clinical development in
14th Congress of the European Hematology Association
this class of anticoagulants. Phase II studies in patients
undergoing hip or knee replacement surgery provided
evidence for the efficacy of dabigatran in this patient
group. After that, three phase III trials were undertaken.
The RE-MODEL trial included 2076 patients undergo-
ing knee replacement, which were randomized to dabi-
gatran or enoxaparin.22 The primary outcome, a combi-
nation of symptomatic and asymptomatic VTE and
total mortality, was not significantly different between
both groups. Also, bleeding rates were comparable. The
RE-NOVATE trial in 3494 patients undergoing hip
replacement reported efficacy and bleeding rates of
dabigatran similar to enoxaparin.23 The RE-MOBILIZE
in patients undergoing knee replacement surgery con-
cluded that dabigatran was inferior to enoxaparin, but
in this trial a higher (North America) dose of enoxaparin
was used compared to the other phase III trials.24 In
patients with atrial fibrillation, dabigatran was found to
be promising in phase II trials; therefore, the RE-LY trial
in patients with atrial fibrillation was designed. The
results of this study will become available soon. The
RE-COVER and RE-MEDY trials will compare dabiga-
tran with warfarin for the treatment and secondary pre-
vention of patients with acute symptomatic VTE.
Amplification phase
Inhibitors of actors in the amplification phase of the
coagulation cascade, mainly mediated through throm-
bin, could potentially be interesting, because they do
not completely block thrombin generation. Therefore,
they theoretically might have fewer bleeding complica-
tions.25-28
TB-402 is a partial inhibitor of factor VIII. Phase I
studies have been completed and a phase II study in
prophylaxis after total knee replacement is currently
underway. The results of this study may provide ‘proof
of principle’ for the efficacy of factor VIII inhibition as a
viable anticoagulant.
Two different factor IXa inhibitors have been evaluat-
ed. TTP 889 is an oral active direct factor IXa inhibitor.
The FIXIT study, a phase II randomized placebo con-
trolled study in patients after hip fracture surgery
demonstrated that TTP 889 was not effective in reduc-
ing the incidence of venous thromboembolism.
Strikingly, little bleeding complications were reported.
The development was subsequently stopped.29 RB006 is
another parenteral factor IXa inhibitor, with a specifical-
ly designed antidote called RB007. In phase I studies,
RB006 showed rapid anticoagulant activity which could
be reversed by RB007.30 Because of the potential for
rapid reversal, this drug is being developed, for example,
for use in cardiac surgery, and a phase II study is ongo-
ing in patients undergoing PCI.
Lately, factor XI is being investigated as a target for
anticoagulant therapy. In mice models, factor XI knock-
out resulted in significantly reduced thrombus forma-
tion, which was more profound than the effect of a high
dose of heparin, without an increase in bleeding risk.
Studies in humans show a correlation with high levels
of factor XI with arterial and venous thrombosis, while
this factor is thought to have a relatively small role in
normal hemostasis. Presently, different research groups
are developing drugs specifically targeted at factor XI.31
| 274 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Natural anticoagulant mechanisms
Activated protein C inactivates the cofactors Va and
VIIIa and therefore, is an other interesting target for
new antithrombotics. Activated protein C also has anti-
inflammatory actions, which makes it suitable for use in
sepsis patients, who suffer from both thrombotic and
inflammatory complications. Recombinant activated
protein C, called drotrecogin α, is already licensed for
use in severe sepsis. This approval was based on a trial
in 1690 patients with severe sepsis, which compared
drotrecogin alfa to placebo and found a 19% reduction
in mortality at 28 days. However, the rate of major
bleeding was higher in the treated group compared to
the placebo group (3.5% vs. 2%, p=0.06). After
approval, two additional phase III trials in adults with
sepsis and a low risk of death and in children with sep-
sis were prematurely halted because of lack of effect
with an increased risk of bleeding.32,33
Recombinant thrombomodulin (ART-123) binds to
thrombin and, thereby converts thrombin from a proco-
agulant enzyme into a potent activator of protein C. A
phase II dose-finding trial has been reported in patients
undergoing elective hip surgery. In this study, ART-123
seemed to have antithrombotic potential; however, the
real value of ART-123 needs to be determined in a ran-
domized controlled trial.34
The future of anticoagulant therapy
Although heparin and VKAs have been used for a
long time and, especially heparin, has been improved in
the last ten years, these drugs certainly have drawbacks.
At these points, anticoagulant therapy can be improved
by the new anticoagulants. Dabigatran, rivaroxaban and
apixaban are the drugs closest to implementation into
clinical practise. The first two have recently been
licensed for thromboprophylaxis in orthopedic surgery
in Europe. However, because of the limited patient
numbers in which these drugs have been tested, unex-
pected adverse effects may still arise. The most recent
example is ximelagatran, which first seemed to be very
promising, but was withdrawn from the market
because of unexpected liver toxicity. Another potential
drawback for most newly developed anticoagulants is
the lack of a specific antidote and the lack of a way to
monitor compliance. Also, costs for the health system
will likely rise with these new drugs.
It is difficult to predict whether LMWH and vitamin
K antagonists will keep playing an important role in the
future. In specific subgroups such as cancer patients,
LMWH will probably stay the preferred choice for long-
term treatment and secondary prevention, because of its
possible advantageous effects on cancer progression.
These additional advantageous effects remain to be
evaluated for the new anticoagulants.
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Lassen MR, et al. Prevention of venous thromboembolism:
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2. Furie B, Furie BC. Mechanisms of thrombus formation. N Engl
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3. Abraham E, Reinhart K, Opal S, Demeyer I, Doig C,
Rodriguez AL, et al. Efficacy and safety of tifacogin (recombi-

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5. Lee AY, Vlasuk GP. Recombinant nematode anticoagulant pro-
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6. Turpie AG, Bauer KA, Eriksson BI, Lassen MR. Fondaparinux
vs enoxaparin for the prevention of venous thromboembolism
in major orthopedic surgery: a meta-analysis of 4 randomized
double-blind studies. Arch Intern Med 2002;162:1833-40.
7. Buller HR, Davidson BL, Decousus H, Gallus A, Gent M,
Piovella F, et al. Subcutaneous fondaparinux versus intra-
venous unfractionated heparin in the initial treatment of pul-
monary embolism. N Engl J Med 2003;349:1695-702.
8. Buller HR, Davidson BL, Decousus H, Gallus A, Gent M,
Piovella F, et al. Fondaparinux or enoxaparin for the initial
treatment of symptomatic deep venous thrombosis: a ran-
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9. Oldgren J, Wallentin L, Afzal R, Bassand JP, Budaj A,
Chrolavicius S, et al. Effects of fondaparinux in patients with
ST-segment elevation acute myocardial infarction not receiv-
ing reperfusion treatment. Eur Heart J 2008;29:315-23.
10. Yusuf S, Mehta SR, Chrolavicius S, Afzal R, Pogue J, Granger
CB, et al. Effects of fondaparinux on mortality and reinfarction
in patients with acute ST-segment elevation myocardial
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11. Yusuf S, Mehta SR, Chrolavicius S, Afzal R, Pogue J, Granger
CB, et al. Comparison of fondaparinux and enoxaparin in
acute coronary syndromes. N Engl J Med 2006;354:1464-76.
12. Buller HR, Cohen AT, Davidson B, Decousus H, Gallus AS,
Gent M, et al. Idraparinux versus standard therapy for venous
thromboembolic disease. N Engl J Med 2007;357:1094-104.
13. Buller HR, Cohen AT, Davidson B, Decousus H, Gallus AS,
Gent M, et al. Extended prophylaxis of venous thromboem-
bolism with idraparinux. N Engl J Med 2007;357:1105-12.
14. Buller HR, Destors JM, Gallus AS, Prins MH, Raskob G.
Idrabiotaparinux, a Biotinylated Long-Acting Anticoagulant,
in the Treatment of Deep Venous Thrombosis (EQUINOX
Study): Safety, Efficacy, and Reversibility by Avidin. Blood
2008.
15. Savi P, Herault JP, Duchaussoy P, Millet L, Schaeffer P, Petitou
M, et al. Reversible biotinylated oligosaccharides: a new
approach for a better management of anticoagulant therapy. J
Thromb Haemost 2008;6:1697-706.
16. Eriksson BI, Borris LC, Dahl OE, Haas S, Huisman MV, Kakkar
AK, et al. A once-daily, oral, direct Factor Xa inhibitor, rivarox-
aban (BAY 59-7939), for thromboprophylaxis after total hip
replacement. Circulation 2006;114:2374-81.
17. Eriksson BI, Borris LC, Friedman RJ, Haas S, Huisman MV,
Kakkar AK, et al. Rivaroxaban versus enoxaparin for thrombo-
prophylaxis after hip arthroplasty. N Engl J Med 2008;
358:2765-75.
18. Kakkar AK, Brenner B, Dahl OE, Eriksson BI, Mouret P, Muntz
J, et al. Extended duration rivaroxaban versus short-term
enoxaparin for the prevention of venous thromboembolism
after total hip arthroplasty: a double-blind, randomised con-
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19. Lassen MR, Ageno W, Borris LC, Lieberman JR, Rosencher N,
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prophylaxis after total knee arthroplasty. N Engl J Med 2008;
358:2776-86.
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20. Buller H, Deitchman D, Prins M, Segers A. Efficacy and safety
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deep vein thrombosis. The Botticelli DVT dose-ranging study.
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21. Lassen MR, Davidson BL, Gallus A, Pineo G, Ansell J,
Deitchman D. The efficacy and safety of apixaban, an oral,
direct factor Xa inhibitor, as thromboprophylaxis in patients
following total knee replacement. J Thromb Haemost 2007;
5:2368-75.
22. Eriksson BI, Dahl OE, Rosencher N, Kurth AA, van Dijk CN,
Frostick SP, et al. Oral dabigatran etexilate vs. subcutaneous
enoxaparin for the prevention of venous thromboembolism
after total knee replacement: the RE-MODEL randomized
trial. J Thromb Haemost 2007;5:2178-85.
23. Eriksson BI, Dahl OE, Rosencher N, Kurth AA, van Dijk CN,
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late vs North American enoxaparin regimen for prevention of
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25. Gailani D, Renne T. The intrinsic pathway of coagulation: a
target for treating thromboembolic disease? J Thromb
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26. Doggen CJ, Rosendaal FR, Meijers JC. Levels of intrinsic coag-
ulation factors and the risk of myocardial infarction among
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27. Wang X, Smith PL, Hsu MY, Gailani D, Schumacher WA,
Ogletree ML, et al. Effects of factor XI deficiency on ferric
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28. Wang X, Cheng Q, Xu L, Feuerstein GZ, Hsu MY, Smith PL, et
al. Effects of factor IX or factor XI deficiency on ferric chloride-
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29. Eriksson BI, Dahl OE, Lassen MR, Ward DP, Rothlein R, Davis
G, et al. Partial factor IXa inhibition with TTP889 for preven-
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30. Chan MY, Rusconi CP, Alexander JH, Tonkens RM,
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BL, et al. Drotrecogin alfa (activated) for adults with severe
sepsis and a low risk of death. N Engl J Med 2005;353:1332-41.
33. Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF,
Lopez-Rodriguez A, et al. Efficacy and safety of recombinant
human activated protein C for severe sepsis. N Engl J Med
2001;344:699-709.
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962-8.
 Transfusion

Transfusion policies and practices in alloimmunized

sickle cell patients

J.M. Moulds A B S T R A C T

LifeShare Blood Centers,
Shreveport, LA, USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:276-281
The alloimmunization rate in patients with sickle cell disease is higher than in most transfused
patients and it appears this is the result of many factors. Clearly increasing patient age and number
of transfusions, as well as being female affect this rate. Limited blood group phenotype matching has
been controversial in the past but is becoming the standard of care in large centers, resulting in
decreased rates of alloimmunization once implemented. More recently, extended genotype matching
has become available due to advances in molecular technology. High throughput microarrays allow
for more complete patient blood group typing, as well as finding matched donors. Genotyping has the
added benefit of locating compatible donors for patients with complex antibodies. Since delayed
transfusion reactions and hyperhemolysis pose a severe risk for SCD patients, genotype matched
donors may provide a safer blood product. However, in some instances of hyperhemolysis, withhold-
ing transfusion or short-term corticosteroid treatment may be best for patient management. The
introduction of molecular genetics into the medical field has not only changed our approach to the
treatment of sickle cell disease (SCD) but promises to significantly improve the transfusion needs of
SCD patients and their quality of life in the future. This manuscript will cover three relevant areas,
including the causes of alloimmunization and its prevention, unusual alloantibody specificities that
may be unrecognized in the SCD patients and delayed transfusion reactions resulting in hyperhemol-
ysis – an often unrecognized syndrome in transfused SCD patients.

Alloimmunization in sickle cell disease Alloantibody frequency and specificity

In the United States, an estimated 80,000
individuals have been diagnosed with sick-
le cell disease (SCD), yet there remains a
significant lack of funding both for research
and clinical care.1 This has adversely affect-
ed the adoption of policies regarding stan-
dard of care for these patients. Accordingly,
specialized centers for Sickle Cell research
and treatment have been established and
are addressing some of the major health
issues of SCD; for example, strokes.
It is well established that strokes during
early childhood result in significant morbid-
ity and mortality among SCD patients.
Based on an abnormal transcranial doppler,
Adams et al.2 showed that blood transfusion
was very useful in preventing the first
stroke among children who were at great-
est risk. In fact, the results from the Stroke
Prevention Trial in Sickle Cell Anemia
(STOP) were so convincing regarding the
benefit of red cell transfusion that the study
was actually halted before its scheduled
completion date.
A follow-up study, in which transfusions
were discontinued, showed a high rate of
reversion to abnormal blood-flow velocities
and stroke.3-4
Thus, the continued use of red cell trans-
fusion has become generally accepted for
the treatment of high-risk SCD patients.
Chronic transfusion, however, carries risk
including transmission of infectious disease,
iron overload and alloimmunization to red
cell, platelet and HLA antigens. Published
figures regarding the incidence of red cell
alloimmunization in sickle cell patients vary
greatly ranging from 4-40%.5-9 It is clear that
many factors can influence the observed
alloimmunization rate, including increasing
age, number of transfusions, and racial dis-
parity, as well as the techniques used for
antibody detection. For example, Rh blood
group antibodies are enhanced using
enzyme treated reagent cells but this is not a
routine technique in all blood banks. Studies
that do not use this technique could under-
estimate the percent of immunized patients.
One constant among all of the transfusion
studies is the antibody specificities that
develop in these transfused patients. In
almost all studies, the most frequent anti-
bodies are anti-E, anti-Kell and anti-C.5,10-12
This is in part due to the high frequency (35-
40%) of the Ro (cDe) blood type in Blacks
making them at risk to develop anti-C and -
E. It has been a common assumption that
the high rate of allloimmunization in SCD
patients is due to racial differences between
patients and donors.10 In fact, some investi-
gators have suggested using intra-racial
transfusions for SCD patients to reduce the
exposure rate to foreign blood group anti-

| 276 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

gens.13,14 A study by Noumsi and Moulds,15 comparing
SCD patients in West Africa to the Southern United
States (US), found that anti-E and anti-C occurred with
a similar incidence in Mali where all the donors were
Black (Figure 1). This suggests that donors, Black or
White, must be matched for the Rh antigens in order to
avoid an antibody response. Another study from Sao
Paulo, Brazil reported that their mixed race donor pop-
ulation had a high incidence of the C antigen, and that
the most frequent antibodies in their SCD patients were
antibodies in the Rh and Kell systems.5
Phenotype matching
The argument for and against phenotype matching of
blood donors and patients has been ongoing for the last
20 years but with no consensus policy. Arguments
against matching cite cost as the major drawback.16-17
Reports from small or single institution studies suggest-
ed that matching of blood donors to recipient pheno-
type reduced the incidence of alloimmunization and
that cost was justified by improved clinical response.14,18
However, it was the publication from a large multi-cen-
ter trial that provided the most convincing data for phe-
notype matching.19 In the US, the National Institutes of
Health now recommend phenotype matching for multi-
ply transfused SCD patients (www.nhlbi.nih.gov/health/
prof/blood/sickle/sc_mngt.pdf) and almost all of the
Comprehensive Sickle Cell Centers (CSCC) routinely
perform patient phenotyping to include ABO, Rh, Kell,
Duffy, Kidd, Lewis, Lutheran, P1 and the MNS blood
group systems.20 Although providing at least C, E, and
Kell compatible red cells has become the standard of
care at these institutions, it is not widely accepted. A
survey of over 1,000 hospital laboratories in North
America found that only 1 in 4 performed phenotype
matching for SCD patients.21 Indeed, in our survey of
the 128 hospitals served by LifeShare Blood Centers
(which covers three states in the Southern US) only
26% provide phenotyped matched blood.22 Many labo-
ratories are beginning to adopt a two-tiered approach to
phenotype matching. All chronically transfused SCD
patients are matched for C, E and Kell (limited pheno-
type), which is not difficult to achieve. However, some
(including the CSCCs) provide more fully (extended)
matched units, that is, Duffy, Kidd, MNS, once the
patient has made an alloantibody.
Extended genotype matching
The common arguments against provision of pheno-
type matched units is that: (i) there is no way currently
to predict who will be a responder; (ii) it is not cost-
effective; (iii) the needed blood types are not readily
available for most blood centers.16,23-24 In pediatric SCD
patients, minimal matching can reduce alloimmuniza-
tion by 65%.17 Castro et al.16 demonstrated that limited
phenotype matched blood could have prevented alloim-
munization in 53.3% of SCD patients they studied,
while extended matching would have reduced this in
70.8%. However, these authors conclude that matching
was impractical for long term-use because it relied heav-
ily on phenotypes not found in the majority of
(Caucasian) blood donors. The rising cost of reagents
made these very real concerns in the US. However, the
advent of molecular techniques, especially microarrays
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 277 |
Berlin, Germany, June 4-7, 2009
Mali
USA
Figure 1. Comparison of the alloimmunization rate for spe-
cific antibody specificities in SCD patients from Mali and
the USA.
for rapid, high-throughput genotyping of patients and
donors has caused us to re-evaluate cost-related argu-
ments against phenotype matching. The BloodGen
project in Europe developed microarrays, not only for
ABO and Rh (including weak D and D variants), but
also for the other important minor blood groups, includ-
ing Kell, Duffy, Kidd, MNSs, Diego, Dombrock and
Colton. In the US and South America, the bead-chip
microarray is used for red cell, platelet and low-resolu-
tion HLA genotyping. The red cell chip does not have
ABO or D, but does include Cc, Ee, Kell, Duffy, Kidd,
MNSs, Diego, Dombrock, LW, Scianna, Colton and
HgbS. Our 2008 cost analysis study of the bead-chip
assay found that genotyping was actually less expensive
than serological typing, and more genotypes could be
determined in a shorter period of time. Ninety-five
donors could be typed in 6 hours, as opposed to 18
hours for serology; thus, allowing technologists to per-
form other revenue generating tasks.
Thus, the goal of extended genotyping matched
donors for multi-transfused SCD patients is now with-
in reach. Molecular techniques for genotyping have the
added advantage in that they are not influenced by
recent transfusions as are serological methods.25 Yet
there remains opposition to genotype matching because
of the assumption that donors will not be available.
Zhang et al.26 described a fulfillment model, which was
tested in two Southern US populations. Using actual
patient and donor data, they showed that the fulfill-
ment of blood orders using donors matched for 14 anti-
gens could be achieved in 48-53% for site A and 59 to
65% in site B. A feasibility study performed in Hong
Kong27 stated that, by providing genotyped matched
units, the antibody screen could be eliminated resulting
in a savings of approximately $14 million US dollars.
Other factors influencing alloimunization
Even though the STOP trials used prospective red cell
phenotype matching, alloimmunization was greatly
reduced but not entirely eliminated.19 This suggests that
other factors may be involved in the immune response.
From the investigation of over 3,000 patients enrolled in
14th Congress of the European Hematology Association
the Comprehensive Study for Sickle Cell Disease, Rosse
et al.28 reported that the immunization rate was affected
by increased age, female sex and the presence of other
abnormal hemoglobins. This was confirmed in a study
by Murao and Viana29 in Brazil, but not in the
Netherlands.30 Female sex is a also a risk factor for
alloimmunization in non-SCD patients,31 occurring in a
2:1 ratio in West Africa (G. Noumsi, unpublished data).
This most likely can be explained by the added anti-
genic exposure from pregnancies. In support of this idea
is the fact that there is no sex bias for alloimmunization
rates in pediatric patients.32
Investigations of HLA and immunization have been
contradictory with some investigators reporting a role
for HLA B-35.33,34 The presence of white blood cells,
which carry HLA antigens, appears detrimental by
affecting the type 2 immune response. Blumberg et al.35
postulated that by reducing the leukocytes and platelets
found in whole blood there would be a decreased fre-
quency of alloimmunization. These authors were
proved correct and leukoreduced blood products, that
are also hemoglobin S negative, have become the stan-
dard of care in the US for transfusion of SCD patients.
Finally, recent work by Hendrickson and colleagues
has demonstrated that inflammation may contribute to
the heightened immune response.18,36 Using a murine
model of transfusion, they showed that induction of
inflammation with poly(I:C) induced significant con-
sumption of transfused cells by splenic dendritic cells.
Inflammation has been well documented in SCD and its
role in alloimmunization, as well as hyperhemolysis
deserves further study.
Unique antibody specificities and blood group
phenotypes in Blacks
Autoantibodies
It is not unusual for SCD patients to have, at some
point in time, a demonstrable warm autoantibody,
sometimes having apparent Rh blood group specificity.37
This makes it difficult to identify underlying alloanti-
bodies and makes transfusion even riskier. Once again,
extended genotyped units can provide a safer alterna-
tive to random unit transfusions. Perhaps a more com-
pelling argument for genotype matching for SCD comes
from recent data showing that alloimmunization is a
contributing risk factor for autoimmune hemolytic ane-
mia.38,39 Furthermore, correctly identifying antibody
specificities in these complex cases may often be diffi-
cult due to unique blood group types found in Blacks,
some of which are described in the following sections.
RH blood group
This is not intended to be a review of blood groups
but rather a brief description of some of the unusual
genotypes found in SCD patients. These unusual geno-
types can result in the production of complex antibody
specificities in transfused SCD patients and can ulti-
mately delay obtaining compatible blood.
Partial D phenotypes are characterized by lost epi-
topes, and arise from the replacement of RHD exons by
their counterparts in the RHCE gene. Several of these
are found more frequently in Black SCD patients,
including DIIIa and DAR.40 The DAR gene has mutations
in exon 4, 5 and 7, resulting in the weak expression of
| 278 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
the D antigen; however, due to the lost epitopes they
can form allo-anti-D. In addition, the DAR gene appears
to be linked to a RHCE variant known as ceAR.
Individuals who are homozygous for these alleles have
the unique phenotype known as hrS–. The hrS– pheno-
type can also be the result of other variant alleles, for
example, ceMO.41,42 These individuals may produce an
anti e-like antibody when transfused and if so, geno-
typed-matched blood may only be available through
rare donor programs.43
Another variant RHD gene, known as r’s, is actually a
hybrid gene with exons 4-8 and part of exon 3 being
contributed by RHCE.44-46 If the r’s gene is paired with an
Ro or r gene, the patient will be genetically C negative
and may make allo anti-C, even though the RBCs may
type as C+.47,48 We have observed this situation in at
least a half dozen patients, so we now routinely geno-
type for r’s, and when this scenario occurs, we transfuse
C negative units. When a patient is homozygous for r,s
their red cells will lack the hrB antigen and they are also
at risk to make anti-hrB or other complex Rh antibodies.
R2R2 units will be compatible since they lack hrB but one
runs the risk of stimulating an anti-E. When this occurs,
rare E-hrB negative blood may only be obtainable
through rare donor programs.
Dombrock blood group
The major antigens in the Dombrock system include:
Doa, Dob, Gya, Hy and Joa, all of which have been iden-
tified at the molecular level.49 The Hy– and Jo(a–) phe-
notypes are rare but have been found more frequently
in Blacks descended from Africa.50,51 The antibodies are
often weak and difficult to identify but they can cause
hemolytic transfusion reactions.52 Compatible donors
are best identified by molecular methods. Using
microarray, we have found the frequency of Hy- and
Jo(a–) to be 1/1,000 and 1/100, respectively, in Southern
US Black blood donors (JMM, unpublished data). Of
note, we identified 3 Jo(a–) SCD patient in the first 75
that were genotyped.
MNSs blood group
The MNSs and U blood group antigens are carried on
glycophorin A and B. Deletion of the genes encoding
GYPB results in the S-s-U- phenotype. Some red cells
that type as S-s- may be weakly U+ and are known as
U+var. Both types are virtually exclusive to Blacks and,
thus, can pose a problem if found in a SCD patient
needing transfusions. Storry and Reid reported that anti-
U were heterogeneous depending if the individual had
no GYPB or a hybrid GYPB-GYPA protein.53 Allo anti-U
has caused both hemolytic and delayed hemolytic
transfusion reactions (DHTR). These are sometimes dif-
ficult to recognize in the SCD patient, often being con-
fused with acute chest syndrome or infection. DHTR
will be dealt with in more detail in the following sec-
tion.
Hyperhemolysis in transfused sickle cell patients
Incidence
There are many reports in the literature of SCD
patients having delayed transfusion reactions and hyper-
hemolysis, that is, the post-transfusion hemoglobin drops
lower than pre-transfusion.54-59 Hyperhemolysis can

Table 1. Sickle cell hemolytic transfusion syndrome.61
• Acute or delayed hemolytic transfusion reaction
• Marked reticulocytopenia
• Development of more severe anemia post-transfusion in the absence of
bleeding
• Immune hemolysis of autologous red cells
• Subsequent transfusions may exacerbate the anemia and be fatal
• Patients may have multiple alloantibodies or autoantibodies
• Serological studies may not provide an explanation for hemolysis
• Recovery, as manifested by reticulocytosis and increasing hematocrit occurs
following the withholding of additional transfusions
• Similar symptoms may occur with future transfusion
occur both in pediatric and adult patients and is often,
but not always the result of a delayed transfusion reac-
tion. Alloantibodies will appear in the serum 1 to 2
weeks post transfusion but the direct antiglobulin test
(DAT) may or may not be positive. Antibody specifici-
ties associated with DHTR include C, E, Fya, Jka, Jkb and
S, but in some cases there are no detectable antibod-
ies.17,59 It is important that the manifestation of this syn-
drome be recognized because failure to do so may result
in inappropriate patient management.
Clinical findings
Multiple factors contribute to the clinical picture
found in hyperhemolysis, including recent transfusion,
presence of allo or autoantibodies, persistent reticulocy-
topenia and splenic sequestration.17 Laboratory results
may include increased total bilirubin, LDH, RDW and
nucleated red cells in the peripheral circulation.60 The
patient often presents with pain and/or fever. Table 1
shows the criteria for hyperhemolysis as defined by
Petz et al.61 for the syndrome they call sickle cell hemolyt-
ic transfusion reaction. Often SCD patients having a
hemolytic transfusion reaction are misdiagnosed as hav-
ing typical vaso-occlusive or aplastic sickle cell crises.
However, a DHTR should be suspected when the
patient has recurrent or severe sickle cell crises follow-
ing transfusion. Treatment for patients having hyperhe-
molysis are; (i) withholding of further transfusions; (ii) a
short course of corticosteroids, for example, 1 to 2
mg/kg of prednisone daily.61,62 Although there is prom-
ising research and a few case reports in the literature
regarding the use of intravenous immune globulin for
the treatment of hyperhemolysis, a recent guideline
from Canada did not recommend its use.63
Theories regarding hyperhemolysis
The syndrome of hyperhemolysis may now be better
defined but its exact cause is unknown. Several theories
have been put forth including: (i) sequestration followed
by intravascular hemolysis secondary to adhesion to
endothelial cells via inflammatory mediators; (ii) reor-
ganization of the phospholipid bilayer of sickled cells
and possible exposure of crypt antigens; (iii) innocent
bystander hemolysis due to reactive lysis.62,64 The true
explanation may be a combination of these. For exam-
ple, it has been reported that phosphatidylserine is
exposed on the outer membrane surface of sickled red
cells leading to complement activation.58,65 This, of
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 279 |
Berlin, Germany, June 4-7, 2009
course, would add to the proinflammatory mileau.
Furthermore, Test and Woolworth66 found that sickled
cells had a defect in the regulation of the complement
membrane attack complex particularly in the most
dense or older cells. Thus, membrane perturbations aris-
ing from the release of oxygen radicals, paired with
defective complement regulatory control may con-
tribute to increased RBC destruction in the sickle cell
patient.
The hyperhemolysis syndrome bears striking similar-
ity to the severe anemia found in malaria where the fall
in hematocrit is more than can be explained by the rup-
ture of parasitized cells alone. In both situations, it
appears that there is reactive lysis or innocent bystander
hemolysis, that is, immune destruction of RBCs that are
not sensitized with immunoglobulin. Thompson and
Lachmann64 found that acute phase sera often exhibited
this phenomenon and showed that these sera could
activate C5-C6-C7 causing lysis without the early com-
plement components being bound.
Conclusions
The application of molecular genotyping to provide
better-matched red cell units to multiply transfused
sickle cell patients, holds promise to help reduce all-
loimmunization. However, this will require increased
public awareness of SCD, a better knowledge of moti-
vating factors for Black blood donors and targeted
recruitment programs.67 Furthermore, a consensus will
need to be achieved and standards developed regard-
ing the matching of red cell antigens.21,67 Delayed
transfusion reactions and hyperhemolysis, however,
remain a risk of multiple transfusions. Education of
physicians and medical staff regarding transfusion
medicine practices is paramount to providing better
care for the SCD patient, and the hematologist should
be an integral part of this effort.
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RM. The sickle cell hemolytic transfusion reaction syn-
drome. Transfusion 1997;37:382-92.
62. Wayne AS, Kevy SV, Nathan DG. Transfusion manage-
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ment of sickle cell disease. Blood 1993;81:1109-23.
63. Anderson D, Ali K, Blanchette V, Brouwers M, Couban S,
Radmoor P, et al. Guidelines on the use of intravenous
immune globulin for hematologic conditions. Transfus
Med Rev 2007;21 Suppl 1:S9-S56.
64. Thompson RA, Lachmann PJ. Reactive lysis: the comple-
ment-mediated lysis of unsensitized cells. I. The character-
ization of the indicator factor and its identification as C7.
J Exp Med 1970;131:629-41.
65. Mold C, Tamerius JD, Phillips G. Complement activation
during painful crisis in sickle cell anemia. Clin Immunol
Immunopathol 1995;76:314-20.
66. Test ST, Woolworth VS. Defective regulation of comple-
ment by the sickle erythrocyte: evidence for a defect in
control of membrane attack complex formation. Blood
1994;83:842-52.
67. Hassell K, Pace B, Wang W, Kulkarni R, Luban N, Johnson
CS, et al. Sickle cell disease summit: from clinical and
research disparity to action. Am J Hematol 2008:84:39-45.
 Transfusion

Transfusion problems in stem cell transplant patients

D.H. Pamphilon A B S T R A C T

NHS Blood and Transplant, Bristol,
UK
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:282-287
Stem cell transplant (SCT) patients may require intensive blood component support. Transfusions
can be complicated by transmission of viral and bacterial infections, transfusion-associated (TA)-
GVHD, febrile non-hemolytic transfusion reactions (FNHTR) and transfusion-related acute lung injury
(TRALI). Alloimmunisation (AI) to red cell antigens may cause difficulties in selecting compatible blood,
whilst AI to the human leukocyte antigens (HLA) present on platelets may cause refractoriness to sub-
sequent platelet transfusions. It is essential to define robust transfusion policies and procedures, and
these should be regularly audited. This article briefly reviews the blood components available for
transfusion and discusses the use of granulocyte transfusions in the setting of SCT, the impact of
reduced-intensity conditioning (RIC) transplantation on transfusion requirements, the prevention of
cytomegalovirus (CMV) transmission and the management of ABO-mismatched transplants.

This paper is modified from the

chapter on transfusion support first
published in The EBMT Handbook
2008 with permission of the EBMT
(ref 28).

Regulatory requirements for blood and blood
components in Europe
The European Union (EU) Directive
2002/98/EC sets standards for the collection,
testing, processing, storage and distribution
of human blood and blood components.1 It
requires that Blood Establishments should
be licensed, and this is of importance for
both Blood Centers in EU countries that
undertake these activities, as well as hospi-
tals that collect and issue, for example, gran-
ulocytes for transfusion. The most impor-
tant aspects of the Directive are: (i) the fate
of each unit of all blood components should
be recorded and this record kept for 30
years. i.e. vein-to-vein traceability; (ii) robust
quality systems should be in place; the pro-
cessing of blood and blood components
should be undertaken by licensed blood
establishments (see above); (iii) training
should be provided for hospital transfusion
laboratory staff, and (iv) hemovigilance sys-
tems should be established to include the
reporting of adverse events
Establishments are licensed by the
Competent Authorities in EU Member
States following inspection by a regulatory
body – in the United Kingdom (UK) this is
the Medicines and Healthcare Products
Regulatory Authority (MHRA). Reports of
compliance must be submitted.
Testing of donated blood for infectious
disease markers
A number of microbial agents may be
transmitted by blood transfusion. These
include hepatitis B and C, HIV-1 and 2,
HTLV-1 and 2, CMV and syphilis. Blood
services routinely test blood for:
• Hepatitis B: hepatitis B surface antigen
(HbsAg)*
• Hepatitis C: hepatitis C antibodies (anti-
HCV)*
• Human Immunodeficiency Virus 1 and 2:
HIV 1 + 2 antibodies (anti-HIV 1+2)*
• Human T- Lymphotropic Virus 1 and 2:
HTLV 1 + 2 antibodies (anti-HTLV-1+2)
• Syphilis
*These tests are mandated by the EU Blood
Directive (2002/98/EC).1
Blood donors may also be tested for anti-
HBc, that is, anti-hepatitis B core antigen,
alanine aminotransferase (ALT): a surrogate
marker of hepatitis C, HCV-RNA by PCR for
hepatitis C and p24 antigen for HIV-1.
Testing for anti-CMV antibody to identify
CMV seronegative donors is done on a pro-
portion of blood donations, and must be suf-
ficient to identify enough CMV seronegative
components for transfusion to those patients
for whom it is appropriate.
Bacterial contamination is a relatively
common occurrence, with an incidence esti-
mated at 0.05 to 0.5% of components.2 The
sources of bacteria are donor bacteremia and
contamination with bacteria present on the
skin at the time of donation or present in
blood packs. Screening tests for bacteria in
platelet concentrates (PC) using automated
blood culture systems, for example,
BacT/ALERT have been evaluated,2 and are
now used routinely by some transfusion
services. PC are not issued until at least 48
hours after collection but the storage period

| 282 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

may be extended to 7 days once sterility has been eval-
uated. The risk of bacterial transmission is also mini-
mized by careful donor selection, meticulous attention
to sterility during venepuncture, diversion of the first 30
mL of blood collected – which contains most of the bac-
teria – away from the primary collection pack, and
sterility during preparation of blood components.
Bacterial contamination should be suspected in any
patient who develops a febrile reaction characterized by
fever, chills and/or hypotension.
Microbiological testing does not completely remove
the risk of infection, although the chance of infection in
the UK after transfusion of screened blood components
from known/previously tested donors is estimated to be
less than 1 in 2×106 for HIV-1, HBV and HCV. This risk
will vary somewhat according to the donor selection
and testing policies that are operative within a Blood
Service.
Prevention of cytomegalovirus transmission
A proportion of SCT patients are CMV seropositive
pre-transplant or have seropositive donors. They
require regular screening by PCR and antigenemia test-
ing, together with ganciclovir therapy where appropri-
ate to minimize the impact of virus reactivation and
prevent clinical infection post-transplant. All CMV
seronegative SCT patients, with CMV seronegative
donors (neg/neg), and patients with hematological and
other disorders who are likely to proceed to a trans-
plant, should receive blood components that have a
minimal risk of causing CMV acquisition.3 Studies show
that the use of CMV seronegative components is asso-
ciated with an incidence of CMV infection in CMV
neg/neg SCT of between 1 to 4%.4-7 CMV is transmitted
via leucocytes and leucodepletion also minimizes the
risk of CMV transmission.4-6,8 CMV seronegative and
leucodepleted blood components are probably of equiv-
alent efficacy but this view is not generally accepted.6,9
Further evidence from prospective randomized con-
trolled studies (PRCT) using pre-storage leucodepleted
blood components is required. Centers must establish
their own policies.
Leucodepleted blood components
Transfused leucocytes cause alloimmunisation (AI) to
HLA class 1 antigens in a proportion of patients. This
may be manifested clinically as FNHTRs, although
these may also be caused by antibodies to neutrophils,
platelets, plasma proteins, and by cytokines such as
interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor
(TNF)-alpha, which accumulate in stored blood compo-
nents, especially PC. HLA AI may cause accelerated
destruction of HLA incompatible transfused platelets.
This is clinically manifest as a failure to achieve a satis-
factory increment after platelet transfusion (refractori-
ness). A summary of the adverse effects of transfused
leucocytes is shown in Table 1.10,11
Donor dendritic cells (DC), which are present in red
cell and platelet transfusions, appear to be responsible
for sensitisation to HLA. Studies show that removal of
leucocytes to less than 5×106 per blood component pre-
vents primary HLA AI in more than 97% of patients
with hematological malignancies. The use of leucode-
pleted components also reduces secondary AI and
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 283 |
Berlin, Germany, June 4-7, 2009
Table 1. Adverse effects of transfused leukocytes.
HLA alloimmunisation causing
FNHTR
Refractoriness to random donor platelets
Graft rejection
Shortened/red cell survival
Transmission of microorganisms
CMV
HTLV – 1 / 11
Toxoplasma gondii
Yersinia enterocolitica
Immunomodulation
GVHD
Activation of viruses in host cells e.g., HIV – 1
Immune suppression of T- and NK-cell functions
Affecting the quality of stored blood
Microaggregate formation
Metabolic deterioration during storage
refractoriness. Refractoriness is not always prevented,
since in more than 50% of cases, it results from
increased platelet destruction due to non-immune caus-
es, which include fever, splenomegaly, DIC and ampho-
tericin therapy.12
AI is also associated with a higher incidence of graft
failure in patients with severe aplastic anemia.
Filtration of blood or its components is best performed
in blood centers and hospital blood banks. Data from
studies where leucocytes were filtered from blood com-
ponents at the bedside show that this might be ineffec-
tive in preventing or reducing FNHTR, AI and refractori-
ness.13
Indications for leucodepleted blood components14
include: (i) pre-transplant in patients with SAA to
reduce the likelihood of graft failure; (ii) pre- and post-
transplant to prevent recurrent FNHTR; (iii) pre-and
post-SCT to minimise HLA AI and platelet refractori-
ness. This is optional since there is no evidence of a sig-
nificant impact on important clinical outcome meas-
ures, such as survival post-SCT, except in patients with
SAA. Nonetheless, many blood services have imple-
mented leucodepletion of a large proportion or, in some
cases, all of their blood components. In the UK, univer-
sal leucodepletion was implemented in 1999, with the
aim of minimizing the risk of transfusion-associated
transmission of the causative agent of variant
Creutzfeld-Jakob disease (vCJD) (iv) as an alternative to
CMV seronegative components.
Gamma-irradiation of blood components and TA-GVHD
HLA incompatible third party leucocytes contained
in donated blood components can engraft and initiate
an alloreactive response after transfusion. This can
cause TA-GVHD, manifest clinically by fever, rash,
diarrhea, jaundice and pancytopenia, and this is fatal in
more than 90% of cases, so prevention is essential.
Donor leucocytes are inactivated by gamma-irradiation
of 2500 cGy, and all components for SCT recipients
14th Congress of the European Hematology Association
should be irradiated from the time that conditioning
therapy is started. In addition, HLA matched PC should
be irradiated, as should those from family members,
since HLA haplotype sharing may result in TA-GVHD,
even in immunocompetent patients. A summary of the
indications for blood component irradiation is shown
in Table 2.
Platelets show normal functional characteristics
through 5 days storage after irradiation with doses up to
5000 cGy. Red cells leak potassium during storage and
this is made worse by irradiation. Therefore, storage is
limited to 14 days after 2500 cGy. TA-GVHD has been
shown to occur after 1500-2000 cGy, and this dose
range is not recommended.15
Red cell transfusion policies
Red cells should be matched for ABO and Rhesus D
type.1 Extended phenotyping may be necessary in
patients, e.g., those with sickle cell disease, who have
formed red cell alloantibodies after previous transfu-
sions. Red cells should be cross-matched against the
patient’s serum by standard techniques prior to transfu-
sion. Thresholds should be defined for hemoglobin and
PCV below which red cell transfusions are always
given. Suggested arbitrary cut-off points are Hb less
than 8.0 g/dL and PCV less than 25%.
In adults 1 unit of red cells raises the Hb by 1.0 g/dL,
whereas in children the volume of blood to be trans-
fused is derived from the formula: volume to transfuse
= increase in Hb (g/dL) required x 4 x weight (kg)
These policies apply to both SCT patients, as well as
being accepted recommendations for patients with
bone marrow failure who require supportive transfu-
sions.
Platelet transfusion policies
Current practice, based on the results of randomized
studies is to transfuse platelets concentrates (PC) pro-
phylactically when the platelet count is less than
10x109/L. A recent Cochrane Systematic Review con-
cluded that, whilst there is no reason to change current
practice, blood products may become scarce, and fur-
ther trials should be undertaken to compare prophylac-
tic versus therapeutic platelet transfusion; that is, PC
should only be given when there is clinical bleeding.16 In
autologous HPCT, this has been found to be safe.17
Fewer PC transfusions are required in RIC allografted
patients compared to those who receive full myeloabla-
tive conditioning.18 Best current practice is: PC should be
ABO and Rh compatible wherever possible since ABO
incompatibility may reduce the expected count incre-
ment (CI) by 10-30%.
Group O PC should be tested for high titre anti-A, B,
and if positive, should only be transfused to group O
recipients to avoid hemolysis caused by passive admin-
istration of the antibody.
If Rh D positive platelets are given to an Rh D nega-
tive patient, then give 250 iu polyclonal anti-Rh (D)
immunoglobulin. Since the chance of Rh immunization
is probably less than 5%, this may be omitted and the
patients serum screened for immune red cell antibodies,
or prior to a red cell transfusion. Studies show that a
threshold of 10×109/L in stable thrombocytopenic
patients is optimal for prophylactic platelet transfusion.
| 284 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Table 2. Indications for irradiated blood components.
Allogeneic HSC recipients from time of conditioning therapy for 6 months or
until the lymphocyte count is 1×109/L in the absence of chronic GVHD
Allogeneic HSC donors
Autologous HSC recipients (from 7 days before harvest until 3 months
post transplant)
All donations from HLA-matched donors or 1st or 2nd degree relatives
All patients with Hodgkins disease at any stage of therapy
All patients treated with purine analogues, e.g., fludarabine
All patients with congenital immunodeficiency states
A higher threshold of 20×109/L should be used in
patients with fever, sepsis, splenomegaly and other
well-established causes of increased platelet consump-
tion.
If an invasive procedure is planned, e.g. central line
insertion, the platelet count should be more than
50×109/L. PC should be transfused when there is signif-
icant clinical bleeding, irrespective of the platelet count.
PC is contraindicated in patients with TTP. In adults
the usual dose of platelets is 3 x 1011 (an adult therapeu-
tic dose – ATD) in a volume of 200 to 300 mL.
Children more than 30 kg receive one ATD. Children
less than 30 kg are given 10 mL/kg.
These policies apply to both SCT patients, as well as
being accepted recommendations for patients with
bone marrow failure who require supportive transfu-
sions.16-18
Platelet transfusions can be monitored by looking for
cessation of bleeding, measuring the platelet count the
following day (values <20×109/L suggest refractoriness)
or by measuring the platelet count at between 10 to 60
minutes post-transfusion where the corrected count
increment (CCI) should be more than 7.5 (absolute
value).
If the patient is refractory to transfusion of PC, sam-
ples should be taken to test for HLA antibodies. Testing
is not required pre-transplant in all patients, although
some centers recommend it as a screening test. If HLA
antibodies are detected, HLA-matched platelets collect-
ed by apheresis of HLA-typed donors should be used in
these patients. If the CCI is less than 7.5 following
transfusion of HLA matched PC and the patient is not
bleeding, then withhold all platelet transfusions. If the
CCI using well-HLA-matched PC is less than 7.5 and/or
bleeding persists, then possible causes include non-
immune causes of refractoriness or, less commonly,
platelet-specific antibodies. Platelet transfusions will be
required in bleeding patients – sometimes 2 to 3 times
daily to prevent life-threatening hemorrhage. There is
no evidence that therapies such as IVIG improve out-
come in this situation
Granulocyte transfusion
Granulocyte transfusions (GT) are prepared by pool-
ing buffy coats from whole blood donations or they
may be collected by the apheresis of steady state
healthy donors who may be family members or unre-
lated volunteers. Granulocyte apheresis donors must

have a full medical assessment and testing to select
those that are ABO compatible and CMV appropriate.
The granulocyte content is in the range 5-10×109 unit
for both these preparations. Mobilized granulocytes
can be collected from donors who receive G-CSF (5-10
µg/kg) and/or dexamethasone (8 mg) – both given 12 to
24 hours before – to increase the number that can be
collected during a standard apheresis procedure.
Current practice is to give only G-CSF, since the use of
steroids has been associated with the development of
posterior subcapsular cataracts,19 and the two methods
give similar yields.20 This strategy gives a granulocyte
yield of 10-100×109 per unit, and data available so far
indicates that significant granulocyte increments, for
example, 1-2×109/L can be obtained.21 By contrast, it is
unusual to observe such increments with buffy coat or
unmobilised granulocytes. All granulocyte products
must be irradiated prior to transfusion to prevent TA-
GVHD. Cross-matching is also required. A recent
Cochrane Systemic Review indicated that there is cur-
rently inconclusive evidence from PRCTs to support or
refute the use of GT in neutropenic patients. Further
PRCT are required before definitive recommendations
can be made.22 There is recent anecdotal evidence that
prophylactic administration of granulocytes may
reduce the incidence of severe fungal infections after
BMT, but currently few centers use such transfusions
and further studies are needed.21 Furthermore, granulo-
cyte transfusions increase the likelihood of HLA immu-
nization and platelet refractoriness. Granulocyte trans-
fusions are probably best reserved for patients with
granulocyte counts less than 0.2×109/L and document-
ed bacterial or fungal infections not responding to at
least 3 days of appropriate antimicrobial therapy, in sit-
uations where the granulocyte count is not expected to
recover within 7 days.
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 285 |
Berlin, Germany, June 4-7, 2009
Figure 1. Diagram showing
the blood groups of red cells
platelets and FFP used for
transfusion support in ABO
missmatched SCT.
Donor/recipient ABO incompatibility and transfusion
support
Approximately 15 to 25% of HLA identical sibling
donor/recipient pairs differ for ABO blood groups and
the figure is higher in alternative donor transplants. In
myeloablative transplants, ABO incompatibility is asso-
ciated with an increased risk of delayed red cell engraft-
ment, pure red cell aplasia (PRCA), hemolysis and
increased transfusion requirements.18 There are also
reports of increased platelet transfusion requirements.
ABO mismatch does not affect neutrophil engraftment,
the incidence of graft rejection, GVHD, disease progres-
sion or overall survival.23
RIC transplants are associated with decreased red cell
and PC, usage18 and it has been demonstrated with
chimerism studies that early erythroid progenitors
engrafted as promptly as myeloid progenitors.24
However, as with myeloablative SCT, engraftment of
mature red cells is delayed, and cases of PRCA have
been reported. ABO mismatch is associated with
increased red cell transfusion requirements.25 Recipient
plasma cells produce anti-donor ABO alloagglutinins
and after RIC SCT, the rate of decline of anti-donor
hemagglutinins takes twice as long, leading to more
hemolysis.26 In one report of 40 patients who had RIC
SCT, ABO mismatch was associated with one death
due to hemolysis, three cases of PRCA, six cases of
thrombotic microangiopathy (three fatal), an increase in
rehospitalisation days, relapse or disease progression
and higher TRM.27 By contrast, other reports do not
show an inferior outcome.24-26
Definitions
Major ABO incompatibility is defined as the presence
in the recipients plasma of anti- A, -B or -A,B alloagglu-
tinins reactive with the donor’s red cells, e.g., donor
14th Congress of the European Hematology Association
group A and recipient group O.
Minor ABO incompatibility is defined as the presence
of anti-A,-B or -A,B alloagglutinins in the donors plasma
reactive with the recipient’s red cells, e.g., donor group
O and recipient group A.
Major plus minor, also referred to as bidirectional,
ABO compatibility is defined as the presence in both
the donor and recipients plasma of anti-A, -B or -A,B
alloagglutinins reactive with recipient and donor cells
respectively, e.g., donor group A and recipient group B.
Incompatible stem cell graft infusion
If the titre of anti-A and/or anti-B, is less than 1:64,
then bone marrow or PBPC grafts may be infused with-
out any modification. At higher titres, red cells should
be removed from the graft. Marrow processing or HPC
collection on certain apheresis machines, such as the
GAMBRO Spectra, usually results in red cell contamina-
tion of less than 5 mL and ABO incompatibility may be
ignored. Likewise, stem cells that are separated on den-
sity gradients and washed can also be infused without
regard to ABO incompatibility.
Plasma may be removed from the transplant in cases
of minor ABO mismatch where the alloagglutinin titre
is high to avoid acute hemolysis in the recipient.
Delayed hemolytic transfusion reactions may follow
the infusion of donor HSC where there is a minor ABO
mismatch. This is because of a secondary (anamnestic)
immune response mediated via memory B cells in the
graft against recipient ABO antigens. A rise in anti-A, -B
or -A,B titre is seen together with anemia and jaundice.
This phenomenon is rarely, if ever, seen when bone
marrows are depleted of alloreactive T-lymphocytes
using strategies, such as alemtuzumab (CAMPATH-1)
antibody or CD34 positive cell selection since B-cells are
also depleted.
Blood groups used for transfusion support
Before transplant, recipient-type red cells and platelets
should be given but after the transplant, the situation is
more complicated (Figure 1).
For major ABO mismatch, use group O red cell prod-
ucts, irrespective of ABO group until recipient ABO
antibodies are undetectable, and the antiglobulin test is
negative, and platelets and plasma from donors of the
recipients ABO type until recipient red cells are no
longer detected.
For minor ABO mismatch, use red cells of the donor
type, i.e., group O throughout. Give platelets and plas-
ma of recipient type until recipient-type red cells are no
longer detected.
For major and minor ABO mismatch, use group O red
cells until recipient ABO antibodies are undetectable
and the antiglobulin test is negative, then switch to
donor type. For platelets and plasma use group AB until
recipient red cells are undetectable.
Following graft rejection, revert to recipient-type red
cells and platelets.
Conclusions
Transfusion support in BMT patients requires special
consideration and carefully defined policies. The use of
high quality blood components, which have a high
degree of microbiological safety, are also gamma-irradi-
| 286 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
ated and, in addition, may be CMV seronegative and
leucodepleted, provide optimum transfusion support
and minimize the chance of adverse effects.
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23. Helbig G, Stella-Holowiecka B, Wojnar J, Krawczyk M,
Krzemien S, Wojciechowska-Sadus M, et al. Pure red-cell apla-
sia following major and bi-directional ABO-incompatible allo- 27.
geneic stem cell transplantation: recovery of donor-derived
erythropoiesis after long-term treatment using different thera-
peutic strategies. Ann Hematol 2007;86:677-83.
24. Maciej Zaucha J, Mielcarek M, Takatu A, Little MT, Gooley T, 28.
Baker J, et al. Engraftment of early erythroid progenitors is not
delayed after non-myeloablative major ABO-incompatible
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 287 |
Berlin, Germany, June 4-7, 2009
haematopoietic stem cell transplantation. Br J Haematol 2002;
119:740-50.
Canals C, Muñiz-Díaz E, Martínez C, Martino R, Moreno I,
Ramos A, et al. Impact of ABO incompatibility on allogeneic
peripheral blood progenitor cell transplantation after reduced
intensity conditioning. Transfusion 2004;44:1603-11.
Griffith LM, McCoy JP Jr, Bolan CD, Stroncek DF, Pickett AC,
Linton GF, et al. Persistence of recipient plasma cells and anti-
donor isohaemagglutinins in patients with delayed donor ery-
thropoiesis after major ABO incompatible non-myeloablative
haematopoietic cell transplantation. Br J Haematol 2005; 128:
668-75.
Worel N, Kalhs P, Keil F, Prinz E, Moser K, Schulenburg A, et
al. ABO mismatch increases transplant-related morbidity and
mortality in patients given nonmyeloablative allogeneic HPC
transplantation. Transfusion 2003;43:1153-61
Pamphilon DH. Transfusion Policy in the EBMT Handbook.
5th Edition. Haemopoietic Stem Cell Transplantation 2008. p.
146-62.

N.L.C. Luban
George Washington University
School of Medicine and Health
Sciences Professor, Pediatrics
& Pathology, Children's National
Medical Center, Washington, DC,
USA
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:288-293
| 288 |
Transfusion
Transfusion issues in neonatal and pediatric
hematology
Introduction
Blood component transfusion is integral to
the treatment of infants and children cared
for by a wide range of generalists, pediatri-
cians, neonatologists, surgeons, intensivists,
and hematologists/oncologists. Technolo-
gical advances in blood collection, separa-
tion, preservation and storage have resulted
in red blood cell, platelet, white blood cell,
and plasma products, which are superior to
whole blood used in the past. Advances in
donor selection, infectious disease testing,
use of leukoreduction (LR) filters and gamma
irradiation ensure that products are less like-
ly to cause adverse reactions, but issues
remain. Areas of particular concern include
poorly defined indications, unintentional
adverse and long-term consequences, and
the need to alter products to meet the
unique needs of children with specific diag-
noses. Transfusion practices in infants less
than 4 months of age differ markedly from
transfusion in older children, which more
closely resemble adult practices.
Neonates have a small total blood volume
when compared to older children and adults
with high blood volume per kilogram of
body weight. Their immature organs and
relative immunologic immaturity predispos-
es them to specific hazards of transfusion
including metabolic complications, infec-
tions and immuno modulation.1 Their rapid
growth is coupled with a limited capacity to
expand their blood volume. The infant’s
response to stresses, such as hypothermia,
hypovolemia, hypoxia, acidosis, and comor-
bidities, warrant special considerations
before and during the transfusion process.
This monograph will concentrate on issues
relevant to newborns.
Transfusion of RBCs
RBC transfusions in newborns are admin-
istered to replace acute or chronic blood loss
or treat anemia of prematurity. Blood loss
can result from hemorrhage or phlebotomy.
While iatrogenic losses from phlebotomy
can be considerable, they can be minimized
by combining judicious testing strategies,
sampling from indwelling catheters, micro-
tizing laboratory assays and implementing
point-of-care testing.
Indications
In the first few days of life, full term
infants have an elevated hemoglobin (hgb)
concentration (14-20 gm/dL); however, due
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
to physiologic decreases in erythropoietin
(EPO) levels, the hgb concentrations decreas-
es to a nadir of 10-12 gm/dL at aproximately
2 to 3 months before a secondary rise. This
is referred to as the physiologic anemia of
infancy. In preterm infants, this anemia is
more significant with mean nadirs of 8
gm/dL in very low birth weight (VLBW)
infants (birthweight: 1.0-1.5 kg), and 7
gm/dL in extremely low birth weight
(ELBW) infants (birthweight: <1 kg). This is
the result of lower hgb concentrations at
birth, frequent blood sampling, low total
blood volume to blood sampling ratio,
increased risk for other comorbidities, and
diminished capacity of the premature infant
to increase EPO.2 Tissue hypoxia ensues
when peripheral vascular resistance increas-
es. This is, at least, partly due to the infant’s
inability to improve left ventricular stroke
volume without an increase in heart rate
when greater than 10% of blood volume has
been lost. Infants with significant cardiac or
respiratory disease generally receive more
aggressive RBC transfusion therapy.3,4
Normal ranges of hgb and hematocrit have
been established for full-term and premature
infants; however, guidelines for red cell
transfusion therapy remain controversial
because of a paucity of randomized con-
trolled studies addressing appropriate
neonatal transfusion triggers. Guidelines for
replacement transfusion therapy in neonates
are given in Table 1.
Two recent studies attempted to address
high versus low threshold transfusion guide-
lines based on level of respiratory support in
ELBW and VLBW infants. Although differ-
ent in design and outcome, neither study
clearly established an appropriate hemoglo-
bin target. While the multi-institutional
Canadian Premature Infants in Need of
Transfusion (PINT) study5 demonstrated no
advantage for liberal transfusion practices,
the American study6 suggested that restric-
tive transfusion was associated with, apneic
more episodes, intraparenchymal brain
hemmorhage, and periventricular leukoma-
lacia. Unfortunately, the question of long-
term neurodevelopmental outcome has not
yet been addressed by either study. The use
of recombinant human EPO (rhEPO) has
been proposed to decrease neonatal transfu-
sion burden, donor exposure, anemia of pre-
maturity, as well as other comorbidities.
Meta-analysis of late (>8 days of life) rhEPO
use involving 1302 preterm infants showed

Table 1. Transfusion guidelines for RBCs in infants less
than 4 months of age.1,24
Hematocrit <20% with low reticulocyte count and symptoms of anemia
(tachycardia, tachypnea, poor feeding)
Hematocrit <30% and any of the following:
• On <35% oxygen hood
• On oxygen by nasal cannula
• On continuous positive airway pressure and/or intermittent mandatory
ventilation on mechanical ventilation with mean airway pressure
<6 cm of water
• With significant tachycardia or tachypnea (heart rate>80 beats/minute for 24
hours, respiratory rate>80 beats/minute for 24 hours)
• With significant apnea or bradycardia (>6 episodes in 12 hours or 2 episodes
in 24 hours requiring bag and mask ventilation while receiving therapeutic
doses of methylxanthines)
• With low weight gain (<10 g/day observed over 4 days while
receiving >100 kcal/kg/day)
Hematocrit <35% and either of the following:
• On >35% oxygen hood
• On continuous positive airway pressure/intermittent mandatory ventilation
with mean airway pressure ≥6-8 cm of water
Hematocrit <45% and either of the following:
• On extracorporeal membrane oxygenation
• With congenital cyanotic heart disease
no clear benefit in terms of significantly decreasing
donor exposures, nor was there any effect on preva-
lence of cormorbidities. Although late administration of
rhEPO reduced the number of RBC transfusions and the
total transfused volume of RBCs per infant, the clinical
impact of these results is trivial (<1 transfusion per
infant and 7 mL/kg of RBCs). Avoiding the use of RBC
transfusions was not demonstrated because many
infants had received one or more transfusion prior to
receiving rhEPO at study entry.7 A subsequent meta-
analysis designed to assess the effectiveness and safety
of early initiation of rhEPO (<8 days of life) in 1825 pre-
mature infants also showed no evidence of any substan-
tial benefits with regard to donor blood exposure.
However, a statistically significant increased risk of
retinopathy of prematurity (>grade 3) was noted in
neonates who received early rhEPO therapy. Again,
RBC transfusion was not avoided when RBCs trans-
fused prior to study entry was considered.8,9 Therefore,
no conclusive evidence currently exists that either early
or late rhEPO use offers any clear benefit in regard to
donor exposure or improvement of morbidity and/or
mortality in preterm infants, and that early rhEPO use
may increase the risk of ROP in the VLBW neonatal
population. The use of rhEPO as a neuroprotectant is
under investigation.
Another area of interest is long-term outcomes follow-
ing transfusion. Patient outcomes, especially mortality,
have been studied in adults, leading to recommendations
for lowering transfusion triggers. Only a couple of stud-
ies have addressed pediatric patients10,11 but neither were
designed to specifically address neonates nor the long-
term adverse effects. This is an area of increasing interest,
which warrants large scale multi-institutional studies.
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Berlin, Germany, June 4-7, 2009
Table 2. Platelet transfusion guidelines in neonates and
older children.1,24
With Thrombocytopenia
• Platelet count 5,000 to 10,000/µL with failue of platelet production
• Platelet count <30,000/µL in neonate with failure of platelet production
• Platelet count <50,000/µL in stable premature infant with:
a. Active bleeding, or
b. Before an invasive procedure with failure of platelet production
• Platelet count <100,000/µL in sick premature infant with:
a. Active bleeding, or
b. Before an invasive procedure in patient with DIC
Without Thrombocytopenia
• Active bleeding in association with qualitative platelet defect
• Unexplained excessive bleeding in a patient undergoing cardiopulmonary
bypass
• Patient undergoing ECMO with:
a. A platelet count of <100,000/µL
b. Higher platelet counts and bleeding
DIC : disseminated intravascular coagulation; ECMO: extracorporeal membrane
oxygenation
Pretransfusion testing and product selection
Prior to RBC transfusion, a blood sample is obtained
for ABO and Rh determination (blood group and type)
and to screen for antibodies against blood group anti-
gens.12 Because antibodies identified in the neonate’s
blood are most often of maternal origin, maternal blood
can often serve as the source of serum/plasma for the
antibody screen. A newborn’s ABO group is assigned
solely on the testing of the patient’s RBCs for the A and
B antigens (forward typing), because the isohemagglu-
tinins anti-A and anti-B are not present in the serum at
birth. Cord blood specimens for infant blood type deter-
mination should not be used because contamination
with Wharton’s jelly often hinders correct grouping, and
because proper identification of the specimen in the
delivery room may be problematic.
If the newborn’s antibody screen is negative, ABO-
and Rh-specific RBCs may be transfused. Neither the
antibody screen nor crossmatching need to be per-
formed during the infant’s hospitalization during the
first 4 months of life. If the screening identifies passive-
ly acquired maternal blood group antibodies, as in Rh
hemolytic disease of the newborn, then O-negative red
cells should be transfused until repeat testing is negative
for antibodies reacting against the ABO- or Rh-specific
units. When antibodies to other RBC antigens other
than D are detected, blood should be selected that is
ABO and Rh specific but antigen negative for the iden-
tified antibody. In cases when reconstituted WB is need-
ed for large volume transfusion procedures (i.e.,
exchange transfusions, cardiopulmonary bypass,
ECMO), the neonate may be given plasma that is ABO
compatible with their RBCs, but receive RBCs that are
compatible with maternal serum. This may mean that
the ABO group of the RBC and FFP units are different.
In circumstances of multiple transfusions access ABO
group, we advise crossmatching of red blood cell units.
An alternative used by some transfusion centers entails
the use of low isohemagglutinin titer, group-O WB, if
available.2
14th Congress of the European Hematology Association
In infants older than 4 months of age, repeat testing
for blood group, Rh-type and antibody screening is per-
formed within 72 hours of each red blood cell transfu-
sion if the patient has received a transfusion during the
last 3 months, or if the history is uncertain or unavail-
able.2 Reverse ABO blood group testing, which detects
anti-A and anti-B antibodies, is frequently deferred for
the first 6 months of life because this testing is often
weak or nonreactive in babies less than 1 year of age.
RBC viability and functional activity require that
RBCs be preserved in additive solutions that support
their metabolic demands. All anticoagulant/preservative
(AP) solutions contain citrate, phosphate, and dextrose
(CPD), which function as an anticoagulant, a buffer and
a source of RBC metabolic energy, respectively; the
addition of mannitol and adenine as preservatives to
anticoagulant solutions increases the shelf life of RBCs
from 21 days (CPD) to 35 days (CPDA-1) and to 42 days
for newer AP solutions (AS1, AS3, AS5) by stabilizing
the RBC membrane and maintaining 2.3-diphospho-
glycerate (DPG) and adenosine triphosphate (ATP)
within the stored RBCs. Studies have shown that blood
collected in AP solutions is safe and as efficacious as
CPDA-1 RBCs in increasing the Hct for neonates receiv-
ing small volume (10-15 mL/kg) RBC transfusions.13,14
These products can be dispensed as small aliquots from
one RBC unit (300-350 mL) to one or more neonates
who require multiple transfusions in order to decrease
donor exposure and to conserve RBC inventory. Sterile
connecting devices ensure that the original RBC unit
remains a closed system and transfer packs or syringe
sets permit multiple aliquots to be removed. Studies
have shown that CPDA-1 and AS-3-preserved split RBC
packs effectively limit donor exposures, and are safe for
use in neonatal small volume transfusions until 35 days
of storage.15 However, clinical studies have neither con-
firmed or refuted the effect of an AP on metabolic
abnormalities in massive transfusion for the neonate.
Therefore, many experts recommend avoiding RBCs
stored in AP solutions for large volume transfusions
until such data have been published.16 Because hypo-
thermia can trigger hypoglycemia, metabolic acidosis
and apnea in line blood warmers should be used when
infants receive large volume transfusions.
Platelet transfusion
Indications
Platelet transfusions are administered to neonates
therapeutically or prophylactically to prevent the hem-
orrhagic complications of thrombocytopenia. Neonates
have different risks of bleeding given the same degree of
thrombocytopenia. For example, thrombocytopenic
neonates with neonatal alloimmune thrombocytopenia
(NAIT) have a high risk of major bleeding, whereas
those with sepsis or necrotizing enterocolitis, and those
with intra-uterine growth retardation have an interme-
diate and low risk of major hemorrhage, respectively.
Platelet functions either intrinsic or secondary to drugs
or comorbidities like coagulopathy are likely causes for
these discrepancies. The only randomized controlled
trial addressing whether platelet transfusions reduce
major bleeding in neonate found no benefit of maintain-
ing a normal platelet count (>150,000/uL) in preterm
neonates compared to those maintained at greater than
| 290 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Table 3. Tranfsuion guidelines for plasma products in
neonates and older children.1,24
FFP
• Support during treatment of DIC
• Replacement therapy:
a. When specific factor concentrates are not available, including but not limited
to, antithrombin, protein C or S deficiency, and Factor II, Factor V, Factor X, and
Factor XI deficiencies
b. During therapeutic plasma exchange when FFP is indicated (cryopoor asthma,
plasma from which the cryoprecipitate has been removed)
• Reversal of warfarin in an emergency situation, such as before an invasive
procedure with active bleeding
Note: FFP is not indicated for volume expansion or enhancement of wound
healing.
Cryoprecipitate
• Hypofibrinogenemia or dysfibrinogenemia with active bleeding
• Hypofibrinogenemia or dysfibrinogenemia, undergoing an invasive procedure
• Factor XIII deficiency with active bleeding or undergoing an invasive procedure
in the absence of Factor XIII concentrate
• Limited directed-donor cryoprecipitate for bleeding episodes in small children
with hemophilia A (when recombinant and plasma-derived Factor VIII products
are not available
• In the preparation of fibrin sealant
• von Willebrand’s disease with active bleeding but only when both of the
following are true:
a. Deamino-D arginine vasopressin (DDAVP) is contraindicated, not available, or
does not elicit response.
b. Virus-inactivated plasma-derived Factor VIII concentrate (which contains von
Willebrand factor) is not available
50,000/uL. However, this study did not address bleed-
ing risk or transfusion benefit for neonates with platelet
counts below 50,000/mL.17
A number of platelet transfusion guidelines for the
newborn have been proposed. Given the lack of clinical
trials addressing absolute bleeding risk in thrombocy-
topenia caused by different etiologies, these recommen-
dations are based on expert panel recommendations
garnered from clinical experience. Becasue of concerns
for the outcomes secondary to intraventricular hemor-
rhage (IVH) in the sick neonate, neonatologists have tra-
ditionally adopted an aggressive platelet threshold for
transfusion. Murray et al. retrospectively studied 53
neonates (44 preterm) with severe thrombocytopenia,
and concluded that a threshold of 30,000/mL, without
other risk factors or previous IVH, is safe for the major-
ity of neonates.18 Thus, a generally accepted transfusion
trigger for platelet count less than 30,000/mL has been
endorsed for neonates without other risk factors.
However, some propose a higher trigger (<50,000/mL)
for ELBW neonates within the first week of life, clinical-
ly unstable neonates, and neonates with NAIT (using
HPA-compatible platelet products).3,19-20 Platelet transfu-
sions are also indicated to treat hemorrhage associated
with acquired (i.e., ECMO, cardiopulmonary bypass,
uremia) or congenital qualitative platelet abnormalities
(i.e., Glanzman thrombasthenia, Bernard-Soulier syn-
drome), even when the platelet count is within the nor-
mal range.

Pretransfusion testing and product selection
Platelets express ABO but not Rh antigens. They
should be ABO-group specific whenever possible, due
to reports of intravascular hemolysis following transfu-
sion of ABO-incompatible platelets in infants and chil-
dren.21 Platelets do not routinely require crossmatching.
If ABO-incompatible platelets must be used, plasma
removal via volume reduction or washing, or selecting
low isohemagglutinin (anti-A, anti-B) titer units are
options. Routine volume reduction methods for all
neonates should be avoided because approximately
20% of platelets are lost in the final product, which is
resuspended in either saline or compatible plasma.22
Although Rh matching does not affect post-transfusion
platelet survival, RBCs are present in small amounts of
whole blood derived platelet concentrates, which can
cause Rh sensitization in an Rh-negative recipient.
Administration of Rh immune globulin (RhIG) should
be strongly considered for any Rh-negative neonate,
especially females, within 72 hours of exposure to Rh-
positive RBCs through a platelet transfusion. The rec-
ommended dose is 120 IU RhIG per mL of RBCs trans-
fused, administered intramuscularly (90 IU RhIG per mL
of RBCs intravenously).23 When aliquots of apheresis
platelets, which are virtually RBC free are used, there
should be limited to no concern for Rh sensitization.
Component definitions
A whole blood derived platelet unit contains at least
5.5×1010 platelets in 50- 70 mL of plasma. Apheresis
platelets , also called single-donor platelets (SDP), con-
tain a minimum of 3×1011 platelets in approximately 250
mL (range: 200-400 mL) of plasma; the equivalent of an
estimated six units of WB-derived platelets. SDPs are
split for neonatal use and offer the advantage of avoid-
ing multiple donor exposures. Platelets are optimally
stored at 20-24ºC under constant agitation, and have a
shelf life of 5 days after collection. Transfusion of 10-15
mL/kg of platelets for neonates, or 0.1-0.2 Unit/kg for
children over 10 kg, should yield a platelet increment of
50,000/mL to 100,000/mL if no predisposing risk factors
for refractoriness exist.24 It is important to account for
device related dead space (10-30 mL) when issuing the
product, as this can be considerable in relation to the
overall platelet dose. The expected rise in the platelet
count after transfusion may not be met because of
destructive thrombocytopenia, which occur with
splenomegaly, fever, sepsis, disseminated intravascular
coagulation (DIC), bleeding, or antibiotic therapy.
Immune-mediated causes of platelet refractoriness, such
as alloantibodies to platelet specific antigens (i.e. HPA-
1a, 5b) in NAIT, and autoantibodies to common platelet
antigens in ITP, require consideration in this population,
so that appropriate management can be initiated.2
Granulocyte transfusion
Indications
Indication VLBW neonates are susceptible to over-
whelming bacterial infection. Many factors contribute
to this risk, including disruption of mucosal barriers,
hypogammaglobulinemia, and qualitative neutrophil
defects. Furthermore, preterm neonates may become
neutropenic during sepsis due to a marginal myeloid
storage pool and poor myeloid progenitor cell prolifera-
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Berlin, Germany, June 4-7, 2009
tion and release. Transfusion of granulocytes has been
employed in the treatment of neonatal sepsis; however,
their efficacy is controversial. Meta-analysis of the safe-
ty and efficacy of granulocyte infusion adjunctive to
antimicrobial therapy in the treatment of septic neu-
tropenic neonates failed to show a reduction in morbid-
ity or mortality.25
Granulocyte preparations
Granulocyte concentrates for neonatal transfusion are
prepared by automated leukopheresis of healthy stimu-
lated (Dexamethasone +/-GCSF) donors, and contain 1-
2×109 neutrophils per kilogram in a volume of 10 to 15
mL/kg. Treatment should be continued daily until clini-
cal improvement or neutrophil count recovery (ANC
>3000/mL in first week of life; >1500/mL thereafter). All
granulocytes should be gamma irradiated and CMV
negative, since leukodepletion is contraindicated.
Because granulocyte concentrates have a significant
amount of RBCs (Hct: 15-20%), the component must be
ABO, Rh, and crossmatch compatible with the intend-
ed neonatal recipient. Because granulocytes must be
transfused within 24 hours of collection, FDA-mandat-
ed testing for blood products will not be completed
before the product is released for administration; there-
fore the risks and benefits of transfusing an untested
blood product must be weighed by the medical team
and the parents of the infant.2,24
Unique risks to granulocyte transfusion include pul-
monary reactions from mild transient respiratory dis-
tress, severe pulmonary edema, hypoxia, ARDS and a
high incidence of febrile transfusion reactions.
Pulmonary complications have been reported in 4% of
transfused infants, and severe pulmonary reactions
resembling TRALI have been reported.25,26 Mild to mod-
erate reactions occur in 25-50% and severe reactions in
about 1% of all granuloctye transfusions.2
Although granulocyte colony-stimulating factor (G-
CSF) and granulocyte-macrophage colony-stimulating
factor (GM-CSF) have been used successfully to stimu-
late neutrophil numbers, and IVIG has been attempted
to augment traditional antimicrobial therapy to support
septic neonates, data on efficacy are inconclusive.27,28
Due to the lack of clear consensus on their impact on
improving host defense mechanisms and improving
outcomes, these adjuncts to standard antimicrobial and
antifungal therapy need further investigation.
Transfusion of plasma
Indications
FFP is used primarily to treat acquired coagulation fac-
tor deficiencies because of DIC, liver failure, vitamin K
deficiency from malabsorbtion, biliary disease or war-
farin therapy, or dilutional coagulopathy from massive
transfusion. It can also be used for specific factor
replacement in congenital factor deficiencies (i.e., factor
II, V, X, XI, protein C, protein S, antithrombin, etc.)
when specific factor concentrates or recombinant prod-
ucts are either not manufactured or unavailable.2,3,12,24 FFP
is not indicated for volume expansion, enhancement of
wound healing, or as first-line treatment for congenital
factor deficiencies when either a virally-inactivated
plasma derived factor concentrate or recombinant fac-
tor is available.
14th Congress of the European Hematology Association
Plasma preparations
Plasma can be prepared by either WB separation or by
apheresis. When the plasma product is frozen to -18ºC
or colder within 8 hours of collection it is labeled as
fresh frozen plasma (FFP), (approximately 250-300 mL),
and can be stored at this temperature for up to 1 year.2
In order to conserve plasma inventory and limit donor
exposures for infants who receive only fractions of an
FFP unit, plasma can be separated into a system of mul-
tiple satellite bags, and frozen as aliquots. Once thawed,
FFP can be further subdivided into aliquots for multiple
neonates via sterile connecting devices, stored at 1-6ºC
for up to 5 days and transfused as thawed plasma (TP).21
Although TP has approximately 40% Factor V and VIII
(heat labile factors) activity, effective hemostasis is
maintained at this level, making TP clinically similar to
FFP. Plasma transfusions should be ABO-compatible
with the neonate’s RBCs to avoid passive transfer of iso-
hemagglutinins from ABO-incompatible plasma which
can result in hemolysis.21,24 Because the freezing process
renders the frozen-thawed plasma component free of
viable leukocytes, FFP is not screened for CMV anti-
body, nor are leukoreduction and irradiation necessary
for the prevention of CMV reactivation and TA-GVHD,
respectively. However, passive administration of anti-
body to CMV in FFP may cause CMV IgG testing to
become positive in the transfused neonate. This does
not represent CMV infection, and the antibody disap-
pears in a time course consistent with the 21-day half-
life of γ-globulin.29
FFP is typically dosed at 10-15 mL/kg and will replace
approximately 10-30% of most factors immediately fol-
lowing transfusion. However, the half lives of different
factor(s) vary which impacts dosing intervals.24
Furthermore, vitamin K dependent factors are lower in
neonates prolonging both the PT and aPTT. Correlation
of laboratory values to clinical status is important when
devising an FFP dosing schedule for a coagulopathic
infant.
Specialized products
Leukoreduction of blood components
Current third-generation LR filters consistently pro-
vide a 3-4 log or 99.9% reduction of white blood cell
content to less than 5×106 white blood cells and, with
some filters, less than 1×106 per product. This LR step is
best performed prestorage with good quality control
techniques. Febrile nonhemolytic transfusion reactions
are typically caused by reactions to donor white blood
cells or to cytokines present in the product, and are
reduced when in pre-storage LR products are used.30,31-32
Alloimmunization to foreign HLA class I antigens is also
reduced. LR has also been used to reduce transmission
of cytomegalovirus (CMV) in high-risk patient popula-
tions. Recipient groups at increased risk for post-trans-
fusion CMV related morbidity and mortality include:
• Premature, seronegative, neonates less than 1250 g,
who require blood component support
• Recipients of hematopoietic stem cell and solid-
organ transplants
• Fetuses who receive intrauterine transfusions
• Other severely immunocompromised individuals.1,24
Historically, the use of CMV-seronegative blood was
considered as the gold standard; such products are often
| 292 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
difficult to obtain depending on donor demographics.
Because CMV is harbored within WBC s, decreasing
leukocyte number and viability would theoretically
reduce transmission of CMV; LR (<5×106 WBCs per
unit) has been shown to be effective in preventing CMV
infection in adults with hematopoietic malignancies,
neonates and patients post stem cell transplant. It has
been widely debated whether LR is as efficacious as
CMV-seronegative blood. In a landmark study, Bowden
et al.33 found equivalent rates of post transfusion CMV
infection in an allogeneic hematopoietic stem cell popu-
lation (1.4% for seronegative versus 2.4% for LR).
Although this study’s conclusions have been debated
widely, no formal consensus on the debate of equiva-
lency has been formulated. A subsequent study by
Nichols et al.35 demonstrated that, although LR platelet
products were deemed similar to CMV-seronegative
products in regard to transfusion transmission of CMV,
LR depleted RBCs were not. The authors warned
against the practice of abandoning dual inventory blood
products for CMV-seronegative and seropositive units.
Nonetheless, variable practices currently exist.35 Many
institutions use algorithms based on pre-transplant
serostatus of recipient, serostatus of the hematopoietic
stem cell donor, and the donor demographics within the
area. It is unlikely that further randomized controlled
trials will be performed to assess comparability of CMV
seronegative versus leukoreduced products.
Transfusion-associated graft-versus-host-disease
Transfusion-associated graft versus host disease (TA-
GVHD) occurs when an immunosuppressed or immun-
odeficient patient receives cellular blood products,
which possess immunologically competent lympho-
cytes. The transfused donor lymphocytes are able to
proliferate and engraft in the immunologic incompetent
recipient because they are unable to detect and reject
foreign cells. The degree of similarity between HLA
antigens also increases the ability of donor lymphocytes
to engraft with the recipient. This fact explains why TA-
GVHD can occur in situations of directed donation
among family members. In the event where the donor
is homozygous and the recipient is heterozygous for an
HLA antigen, donor lymphocytes may escape immune
surveillance and engraft in the immunocompetent host,
resulting in TA-GVHD. This situation may also occur in
populations with limited HLA variability, thus necessi-
tating universal gamma-irradiation of all cellular compo-
nents in specific situations.
Clinical symptoms of TA-GVHD include fever and an
erythematous rash, which may progress to bullae and
desquamation and diarrhea developing within 3 to 30
days of receiving cellular blood components. Because
the hematopoietic progenitor cells are particularly
affected, severe cytopenia usually is present. Mild hep-
atitis to fulminant liver failure may occur. Mortality for
TA-GVHD is 90% in the pediatric population. Patients
at high risk of TA-GVHD include:
• Patients with congenital immunodeficiencies, par-
ticularly of cellular immunity
• Those receiving intrauterine transfusion followed
by neonatal exchange transfusion
• Bone marrow transplant recipients
• Recipients of HLA-matched cellular components, or

blood components from blood-related donors
• Patients with hematologic malignancies and cancer
patients undergoing intense chemotherapy or immuno-
modulatory therapy (i.e. Fludarabine, and other purine
analogs).35
Neonates, especially those who are extremely prema-
ture, are considered by many to be at high risk for TA-
GVHD. Whereas some neonatal centers irradiate all cel-
lular blood products for infants less than 4 months of
age, others only irradiate blood products given to
preterm infants born at less than 1.2 kg.36 However,
given the lack of clinical studies on the incidence of TA-
GVHD in the neonatal population, and concern for fail-
ing to recognize an infant with an undiagnosed congen-
ital immunodeficiency, there exists no standard of care
regarding irradiation of blood products for otherwise
non-high risk infants born greater than 1.2 kg.
TA-GVHD can be prevented by γ-irradiation of cellu-
lar blood components at a minimum of 2,500 cGy using
cesium, cobalt or linear accelerators as radiation
sources.2 Because in vivo recovery of irradiated RBCs is
decreased compared to non-irradiated RBCs at 42 days
of storage, the FDA recommends a 28-day expiration
for irradiated RBCs.12 Potassium and free hemoglobin
are also increased after irradiation and storage of RBCs.
Therefore, it is preferable to irradiate in a time frame
close to administration, especially for neonates who are
sensitive to large potassium loads. Irradiation of
platelets and granulocytes does not affect the function
of component products.
References
1. Luban NL, Wong EC. Hazards of transfusion. In: Arceci RJ,
Hann IM, Smith OP, editors. Pediatric Hematology. 3rd ed.
2006. p. 724-44.
2. Roseff SD, editor. Pediatric Transfusion: A physician’s hand-
book. 2nd edition. Bethesda (MD): American Association of
Blood Banks; 2006. p. 1-52.
3. Roseff SD, Luban NL, Manno CS. Guidelines for assessing
appropriateness of pediatric transfusion. Transfusion 2002; 42:
1398-413.
4. Widness JA. Treatment and prevention of neonatal anemia.
NeoRev 2008;9:e526-33.
5. Kirpalani H, Whyte RK, Andersen C, Asztalos EV, Heddle N,
Blajchman MA, et al. The Premature Infants in Need of
Transfusion (PINT) study: a randomized, controlled trial of a
restrictive (low) versus liberal (high) transfusion threshold for
extremely low birth weight infants. J Pediatr 2006;149:301-7.
6. Bell EF, Strauss RG, Widness JA, Mahoney LT, Mock DM,
Seward VJ, et al. Randomized trial of liberal versus restrictive
guidelines for red blood cell transfusion in preterm infants. J
Pediatr 2005; 115:1685-91.
7. Aher S, Ohlsson A. Late erythropoietin for preventing red
blood cell transfusion in preterm and/or low birth weight
infants. Cochrane Database Syst Rev 2006;3:CD004868.
8. Mainie P. Is there a role for erythropoietin in neonatal medi-
cine? Early Hum Dev 2008;84:525-32.
9. Ohlsson A, Aher SM. Early erythropoietin for preventing red
blood cell transfusion in preterm and/or low birth weight
infants. Cochrane Database Syst Rev 2006;3:CD004863.
10. Slonim AD, Joseph JG, Turenne WM, Sharangpani A, Luban
NL. Blood transfusion in children: a multi-institutional analy-
sis of practices and complications. Transfusion 2008;48:73-80.
11. Lacroix J, Hébert PC, Hutchison JS, Hume HA, Tucci M,
Ducruet T, et al. Transfusion strategies for patients in pediatric
intensive care units. TRIPICU Investigators; Canadian Critical
Care Trials Group; Pediatric Acute Lung Injury and Sepsis
Investigators Network. N Engl J Med 2007;356:1609-19.
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12. Price TH, editor. Standards for Blood Banks and Transfusion
services. 25th edition. Bethesda (MD, USA): American
Association of Blood Banks; 2008. p. 41-7.
13. Strauss RG, Burmeister LF, Johnson K, Cress G, Cordle D.
Feasibility and safety of AS-3 red blood cells for neonatal
transfusions. J Pediatr 2000;136:215-9.
14. Jain R, Jarosz C. Safety and efficacy of AS-1 red blood cell use
in neonates. Transfus Apher Sci 2001;24:111-5.
15. Mangel J, Goldman M, Garcia C, Spurll G. Reduction of donor
exposures in premature infants by the use of designated ade-
nine-saline preserved split red blood cell packs. J Perinatol
2001;6:363-7.
16. Luban NL, Strauss RG, Hume HA. Commentary on the safety
of red cells preserved in extended storage media for neonatal
transfusions. Transfusion 1991;31:229-35.
17. Andrew M, Vegh P, Caco C. A randomized, controlled trial of
platelet transfusions in thrombocytopenic premature infants, J
Pediatr 1993;123:285-91.
18. Murray NA, Howarth LJ, McCloy MP, Letsky EA, Roberts IA.
Platelet transfusion in the management of severe thrombocy-
topenia in neonatal intensive care unit patients. Transfus Med
2002;12:35-41.
19. Roberts I, Murray NA. Neonatal thrombocytopenia. Semin
Fetal Neonatal Med 2008;13:256-64.
20. Blanchette VS, Kühne T, Hume H, Hellmann J. Platelet trans-
fusion therapy in newborn infants. Transfus Med Rev 1995;
9:215-30.
21. Angiolillo A, Luban NL. Hemolysis following an out-of-group
platelet transfusion in an 8-month-old wih Langerhans cell his-
tiocytosis. J Pediatr Hematol Oncol 2004;26:267-9.
22. Moroff G, Friedman A, Robkin-Kline L, Gautier G, Luban NL.
Reduction of the volume of stored platelet concentrates for
use in neonatal patients. Transfusion 1984;24:144-6.
23, Kennedy MS. Perinatal issues in transfusion practice. In
Roback JD, editor. Technical Manual of the American
Association of Blood Banks. 16th ed. Bethesda (MD):
American Association of Blood Banks; 2008. p. 625-37.
24. Josephson CD. Neonatal and pediatric transfusion practice. In
Roback JD, editor. Technical Manual of the American
Association of Blood Banks. 16th ed. Bethesda (MD):
American Association of Blood Banks; 2008. p. 639-63.
25. O'Connor JC, Strauss RG, Goeken NE, Knox LB. A near-fatal
reaction during granulocyte transfusion of a neonate.
Transfusion 1988;28:173-6.
26. Sanchez R, Toy P. Transfusion related acute lung injury: A
pediatric perspective. Pediatr Blood Cancer 2005;45:248-55.
27. Sandberg K, Fasth A, Berger A, Eibl M, Isacson K, Lischka A,
et al. Preterm infants with low immunoglobin G have
increased risk of neonatal sepsis but do not benefit from pro-
phylactic immuoglobin G. J Pediatr 2000;623-8.
28. Carr R, Modi N, Doré C. G-CSF and GM-CSF for treating or
preventing neonatal infections. Cochrane Database Syst Rev
2003;3:CD003066.
29. Robitaille N, Hume HA, Blood components and fractionated
plasma products: preparations, indications, and administra-
tion. In: Arceci RJ, Hann IM, Smith OP editors. Pediatric
Hematology. 3rd edition. 2006. p. 693-706.
30. Yazer MH, Podlosky L, Clarke G, Nahirniak SM. The effect of
prestorage LR on the rates of febrile nonhemolytic transfusion
reactions to PC and RBC. Transfusion 2004;44:10-5.
31. Paglino JC, Pomper GJ, Fisch GS, Champion MH, Snyder EL.
Reduction of febrile but not allergic reactions to red cells and
platelets following conversion to universal prestorage LR.
Transfusion 2004;44:16-24.
32. King TE, Tanz W, Shirey S. Universal LR decreases the inci-
dence of febrile nonhemolytic transfusion reactions to red
cells. Transfusion 2004;44:25-9.
33. Bowden RA. A comparison of filtered leukocyte-reduced and
cytomegalovirus (CMV) seronegative blood products for the
prevention of transfusion-associated CMV infection after mar-
row transplant. Blood 2003;86:3598-603.
34. Nichols WG, Price TH, Gooley T, Corey L, Boeckh M.
Transfusion-transmitted cytomegalovirus infection after
receipt of leukoreduced blood products. Blood 2003;101:4195-
220.
35. Pisciotto PT, Luban NL. Complications of neonatal transfu-
sion. In: Popovsky MA, editor. Transfusion Reactions. 3rd edi-
tion. 2007;459-99.
36. Strauss RG. Data-driven blood banking practices for neonatal
RBC transfusions. Transfusion 2000;40:1528-40.
 Uncommon lymphoproliferative disorders

Natural killer cell disorders

G. Semenzato
R. Zambello
Padua University School of
Medicine, Department of Clinical
and Experimental Medicine,
Hematology and Clinical
Immunology Branch, Padova, Italy
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:294-301
A B S T R A C T
Natural killer (NK) cell neoplasms account for less than 5% of all groups of lymphoid malignancies.
The recently updated World Health Organization classification in 2008 has made a great effort in
revisiting and ordering a series of disorders previously included in the pool of NK cell neoplasms.
Accordingly, blastic NK-cell lymphoma, which in the previous classification likely included blastic plas-
macytoid dentritic cell neoplasms, has been provisionally changed into NK cell lymphoblastic
leukemia/lymphoma, since rare cases of bona fide immature NK lymphoid tumours have been report-
ed in the literature. In addition, the new provisional entity defined Chronic Lymphoproliferative
Disorders (CLPD) of NK cells has been added to the list of mature T-cell and NK-cell neoplasms that
already included Aggressive NK cell leukemia and Extranodal NK/T lymphoma, nasal type. These last
two NK cell tumours are more frequent in Asia and Central and South America, are often associated
with Epstein-Barr virus (EBV) infection and show a very aggressive clinical course. By contrast, CLPD
of NK cells are prevalent in Europe and USA, and show an indolent clinical course over a long period
of time, with some cases displaying a spontaneous remission. This paper focuses on recent concepts
and progress in clinicopathologic features, pathogenesis, treatment approaches and outcomes of NK
cell disorders.

Introduction Classification of natural killer disorders

Natural killer (NK) cells represent the
major component of innate immune system
and mediate a MHC non-restricted cytotox-
icity against tumor cells and bacterial or viral
infected cells.1-3 NK cells account for 10% of
peripheral blood lymphocytes, showing typ-
ically large granular lymphocyte (LGL) mor-
phology. NK cells are derived in bone mar-
row from hematopoietic stem cells through
the intermediate developmental stages of
lymphoid stem cells, bipotential T/NK pro-
genitor cells, and committed NK progenitor
cells.4-6 For this reason, NK cells express vari-
ably T-lineage-associated antigens (CD2
and/or CD7). By definition, NK cells are sur-
face CD3-negative and myeloperoxidase
(MPO)-negative, and have germline configu-
ration of T-cell receptor (TCR) and
immunoglobulin (Ig) genes.1-3 CD16, CD56,
and CD57 are NK-associated antigens.
Among these three markers, CD56 (a neural
cell adhesion molecule) is most consistently
expressed.3 Major advances in understanding
the actual role of NK cells in immune
defence have been related to the discovery
and molecular characterization of numerous
surface receptors that play a crucial role in
cell function.7 A key example is represented
by the HLA-class I specific Ig killer-like
receptors (KIR) that play a crucial role in reg-
ulating NK cell function and are also useful
in recognition of NK cell subsets. In this way,
the molecular mechanisms that allow NK
cell to spare normal cells and kill tumor- or
virus-infected cells have now been clarified.
Table 1 summarizes the nomenclature of
most relevant NK receptors.
NK cell neoplasms are a rare and hetero-
geneous group of disorders with a broad
spectrum of morphologic, immunopheno-
typic, and clinical features. The World
Health Organization (WHO) classification
encompasses four distinct entities, two of
them are still provisional: (i) NK cell lym-
phoblastic leukemia/lymphoma (provision-
al);8 (ii) chronic lymphoproliferative disor-
der of NK cells (provisional);9 (iii) aggressive
NK cell leukemia;10 (iv) extranodal NK/T-
cell lymphoma, nasal type.11 In addition,
based on morphology, immunophenotype,
functional NK cell activity, and expression
of cytotoxic molecules, NK cell neoplasms
can be divided into immature and mature
categories.12-16
In recent years, rare cases of lymphoblas-
tic lymphomas/leukemia (LBL) arising from
immature NK cells have been reported,13,15
although the lack of suitable markers for
immature NK cells mentioned above makes
it difficult to distinguish NK-cell lympho-
blastic lymphoma (LBL) from precursor T-
cell LBL. It is also worth mentioning that
the plasticity of hematopoietic-cell lineage
seems greater than previously thought, and
relations between phenotypically dissimilar
neoplastic disorders are being reassessed. In
contrast, it is believed that chronic lympho-
proliferative disorder of NK cells, aggressive
NK cell leukemia and extranodal NK cell
lymphoma and nasal type all originate from
mature NK cells.12,17,18

| 294 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
 Berlin, Germany, June 4-7, 2009

Precursor NK cell neoplasms Table 1. Nomenclature of natural killer receptor antigens.

NK cell lymphoblastic leukemia/lymphoma, provisional
A considerable confusion in the literature concerns NK Receptor specificity
this type of disorders, mostly due to the definition of CD designation* Gene Molecule mAb
NK cell leukemia according to the expression of CD56
antigen. Many cases reported as NK cell leukemia are Killer Immunoglobulin-like Receptors (KIR)
indeed cases of plasmacytoid dendritic cell leukemia19,20 CD158a KIR2DL1 p58.1 EB6
or acute myeloid leukemias.19,21 As a matter of fact, there CD158h KIR2DS1 P50.1 EB6
are rare reports of CD56+ immature lymphoid tumours CD158b1 KIR2DL2 p58.2 GL183
that are characterized by a blastic appearance of neo- CD158b2 KIR2DL3 p58.3 GL183
plastic cells with a CD4–/CD56+/CD13–/CD33– pheno- CD158j KIR2DS2 p50.2 GL183
type, lacking expression of CD3, CD19, CD20, and CD158i KIR2DS4 p50.3 FES172
MPO.22-25 These patients frequently presented with CD158d KIR2DL4 p49 ?
CD158e1 KIR3DL1 p70 Z27
leukemia and lymphadenopathy without skin involve-
CD158e2 KIR3DS1 p70 Z27
ment and were negative for EBV. TCR and/or Ig genes
CD158k KIR3DL2 p140 Q66
were in a germline configuration in all cases in which
the tests were performed. Outcomes of these patients Leukocyte Ig-like receptors/immunoglobulin like Ttranscripts (LIR/ILT)
were unfavorable. The immature morphology with NK CD85j LIR1/ILT2 LIR1/ILT2 F278
cell-associated phenotype and genotype suggests that CD85d LIR2/ILT4 LIR2/ILT4 42D1
these tumors represent true precursor NK cell neo-
plasms. Killer Lectin-like receptors (KLR)
Some well-characterized cases of NK precursor CD94 KP43 CD94 XA185
tumours with lymphomatous presentation that CD159a NKG2A/B NKG2A/B Z199
expressed CD94 1A transcripts have been reported.16 CD159c NKG2C NKG2C P25
Recent advances in the developmental biology of T cells CD314 NKG2D NKG2D BAT221
and NK cells indicate that both cell types are derived
from a common T/NK cell thymic precursor. Interleukin Natural cytotoxicity receptors (NCR)
15 (IL-15) and transcription factor ID2 are essential for CD335 NKp46 NKp46 BAB281
the NK cell lineage to diverge from the T cell lineage.15 CD336 NKp44 NKp44 Z231
CD94 1A, a distal promoter of the CD94 molecule (an CD337 NKp30 NKp30 AZZ20
NK cell receptor), is activated only by IL-15.26,27 Lin et al.16 NC NKp80 NKp80 MA152
recently reported that CD94 1A is the predominant
form found in immature NK cells and it is expressed in Co-receptors
CD244 2B4 2B4 C1.7.1
TCR-ve LBL (NK lineage LBL) but not in TCR+ve LBL (T
CD328 AIRM1 p75 Z176
lineage LBL). By studying 21 patients with LBL, and
based on the expression of CD94 1A transcripts and the NC: Not clustered. Regular characters indicate inhibiting molecules while italic
lack of TCR, these investigators identified seven denotes activating receptors. *CD are indicated according to 8th Human
Leukocyte Differentiation Workshop (HLDA8).91
patients with LBL of immature NK cell origin (CD94
1A+, TCR–). It is noteworthy that those NK-LBLs
occurred in young patients and had better outcomes as
compared with patients who had T-LBL (CD94 1A-, They are the most common type among primary
31,32

TCR+); none of the tumors were positive for CD56.
Thus, the use of CD94 1A associated with TCR appears
to be more precise than CD56 for identifying an imma-
ture NK cell neoplasm. It is hoped that a wider availabil-
ity of more specific NK markers, including panels of
KIR, KLR, and LIR1 will help to clarify this issue8 that
still remains provisional.
Mature natural killer cell neoplasms
Extranodal NK/T cell lymphoma, nasal type
Since they share the same histology, the WHO classi-
fication groups both nasal NK cell lymphoma and
extranasal NK cell lymphoma in the same category,11
even if these lymphomas might have different clinical
manifestations, treatment approaches, and prog-
noses.15,29 Although most cases are genuine NK-cell neo-
plasms, the term NK/T rather than NK is used because
this entity also includes cytotoxic T-cell neoplasms.30
Nasal and extranasal NK cell lymphoma are more preva-
lent in Asia, Mexico, and Central and South America11,29
and are rare in Western countries.
Nasal NK/T cell lymphomas refer to tumours that
occur in the nose and the upper aerodigestive tract.17,18,
lymphomas of the nasal cavity.33 The site of disease is
primarily in the midline and includes the nasal cavity in
more than 80% of cases. The tumour is locally invasive
and might infiltrate surrounding tissues and organs,
such as oropharynx, palate, orbits, untill the appearance
of the characteristic midfacial destructive lesions, the so
called lethal midline granuloma.31-33 Common symptoms
include nasal discharge, nasal obstruction, purulent rhi-
norrhea, epistaxis and local swelling of the nasal bridge.
The tumours may be destructive, leading to the highly
characteristic midline perforation.
Extranasal NK cell lymphomas represent the counter-
part of nasal NK cell lymphomas, and involve any other
part of the body. Men are predominantly affected, and
the median age of presentation is the fifth decade.
Primary sites of involvement include the skin, gastroin-
testinal tract, salivary glands, spleen, and testis.34
Patients with extranasal NK cell lymphoma are more
likely to exhibit an advanced stage of disease with sig-
nificantly higher general involvement, high levels of lac-
tate dehydrogenase and a significantly decrease in
hemoglobin and platelet count as compared with
patients who have nasal NK cell lymphoma.34 The his-

Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 295 |
14th Congress of the European Hematology Association
tologic features are similar, regardless of the involved
sites. Mucosal sites often show ulceration. A diffuse
infiltrate of lymphoid cells is found in association with
tissue necrosis and coagulation. An angiocentric and
angiodestructive growth pattern with associated fibri-
noid changes in the blood vessels is frequently
observed. In most patients, the neoplastic cells are char-
acterized by medium-sized cells or a mixture of small
and large lymphoid cells, with a moderate amount of
cytoplasm, irregular or elongated nuclei, granular or
vescicular chromatin, and inconspicous, small nucleoli.
These cells express the CD45+ sCD3–cCD3+CD56+, lack-
ing myeloid and B lymphoid markers. Association with
EBV can be demonstrated in nearly all patients.35 Using
in situ hybridization technique, EBV-encoded RNA can
be found in neoplastic cells and Southern Blot analysis
can detect monoclonal proliferation of EBV. Analysis of
the terminal repeat region of the EBV genome indicates
that the virus is in a clonal episomal form. Other than
providing an indirect proof of the clonal nature of the
lymphoid proliferation, this finding suggests that the
EBV might play an etiologic role in mature NK cell neo-
plasms rather than simply being a bystander.33,36,37
Aggressive natural killer cell leukemia
First characterized by Fernandez et al.38 aggressive NK
cell leukemia is a systemic disease, again more common
in Asians than in Caucasians.10 It is characterized by the
presence of neoplastic NK cells in the peripheral blood,
bone marrow, liver and spleen and by a rapidly progres-
sive clinical course with poor prognosis. There is an
equal sex incidence in men and women. The disease
typically affects young to middle-aged adults with a
median age in the third decade. At presentation,
patients usually are very compromised with systemic
symptoms, liver dysfunction, and hepatosplenomegaly
sometimes accompanied by systemic lymphadenopa-
thy. In contrast to extranodal NK cell lymphoma, skin
lesions are uncommon. Disseminated intravascular
coagulation and hemophagocytic syndrome are often
seen during the course of disease.17,18 The clinical pro-
gression is devastating despite treatment, and most
patients survive for only days to weeks. Morphological-
ly, in this disorder the leukemic cells are slightly larger
than normal LGLs.17 An ample amount of pale or slight-
| 296 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
Figure 1. Hypothesis on the
pathogenetic events leading to
CLPD of NK cell.
ly basophilic cytoplasm contains fine or coarse
azurophilic granules. Nuclei show slightly immature
chromatin pattern and inconspicuous or distinct nucle-
oli. These cells are sCD3–, cCD3+CD56+CD16+, CD57–,
CD94+ with a germline configuration of β and γ genes
of TCR. In tissue sections, the neoplastic infiltrate is dif-
fuse and destructive, with lymphoid cell population
usually appearing monomorphous.33 Necrosis, apopto-
sis, angioinvasion, and angiodestruction are common
findings.10,34,39,40 Although clonal EBV is found in tumour
cells in most patients, and EBV is considered to be the
etiological agent,17 little is known about the mechanisms
though which EBV infection triggers clonal proliferation
of NK cells. Several chromosomal abnormalities have
been reported and the finding of the same chromosomal
abnormality involving del(6q) in aggressive NK-cell
leukemia and in extranodal NK/T cell lymphoma pro-
vides a biological link between these two diseases.41,42
However, array-based comparative genomic hybridiza-
tion analysis demonstrated clear genetic differences
between aggressive NK-cell leukemia and extranodal
NK/T cell lymphoma, suggesting that they are two sep-
arate entities.43
Chronic lymphoproliferative disorder of natural killer cells
(provisional)
The chronic lymphoproliferative disorders of NK cells
(CLPD-NK) are included among the novelties of revised
WHO classification published in September 2008.9
These rare and heterogeneous disorders are character-
ized by a chronic expansion of mature looking NK-cells
(usually more than 2,000 µL) in peripheral blood for
more than 6 months,44-48 without a clearly identified
cause. Patients are usually adults with a mean age of 60
years without sexual and racial predisposition.49,50
Recently, several studies have been published focusing
on the pathogenetic mechanisms of this disease and the
current concepts on this topic are reported in Figure 1.
NK cell activation in response to an unknown stimulus,
likely of viral origin, is postulated to play a role in the
initial steps of CLPD-NK by selecting NK clones.51-54 In
particular, although no prototypical HTLV infection was
demonstrated in these patients, the evidence that sera
from a series of patients from Europe and USA reacted
with the recombinant HTLV env protein p21E suggests

Table 2. Clinical and biological features of natural killer cell neoplasms.
NK cell Extranodal NK/T-cell
lymphoblastic lymphoma, nasal type
leukemia/lymphoma
Age Pediatric patients
Geographic distribution Worldwide
Cell origin Immature NK cell
Relevant phenotype sCD3-CD4-CD13-
CD33- CD16-CD7+ cCD3ε+CD56+CD16-
CD2+CD56+CD3ε+
EBV association No
Clinical course Aggressive
Prognosis Poor
that exposure to a protein containing homology to
BA21 may be important in the pathogenesis of this lym-
phoproliferative disorder.55 In contrast with other
mature NK cell neoplasms, EBV is not usually detected
within pathological GL.50,52,54 It is believed that bone
marrow, which is frequently involved in CLPD-NK
patients, represents the setting where the putative incit-
ing antigen could reside, and dendritic cells (DC) have
been suggested to represent the target of infection in
these patients.56 In fact, analysis of bone marrow biop-
sies of patients demonstrated a topographic distribution
of DCs and NK cells that indicates contact between the
two cell types.56 DC are also likely to represent the
source of IL-15, which is crucial in the mechanisms sus-
taining the maintenance of NK proliferation.57 IL-15 has
been demonstrated to mediate its activity through tar-
geted proteasome degradation of Bid expression, thus
preventing apoptosis of NK cells. A genetic susceptibili-
ty for this disease has been suggested and related to the
detection in these patients of type B KIR gene reper-
toire, which is characterized by a high number of acti-
vating genes.58-60 In fact, the expression of NK receptors,
mostly represented by KIR, is altered in patients with
CLPD-NK. A restricted pattern of KIR expression has
usually been reported in these patients, which is charac-
terized either by a dominant expression of a relevant
KIR, or by a lack of KIR expression.58,60-65 A typical fea-
ture of these patients is the preferential expression of
the KIR activating receptor isoforms,58,60 and this pattern
correlates with a reduced expression of other activating
receptors,60 such as natural cytotoxicity receptors
(NCR). Together with a bias towards activating KIR
expression, a deep silencing of inhibitory KIR through
increased gene metylation has been reported.
Biochemical studies on the mechanisms sustaining the
growth of NK cells in these patients have demonstrated
a role of RAS farnesil transferase,65 with clinical implica-
tions.65 Pathological NK cells express CD16 and usually
low levels of CD56 and CD57. As expected, cells
espress TIA1, granzyme and perforins, which correlate
with the cytotoxic potential of these cells displayed in in
vitro cytotoxic assays. CD94 antigen is expressed at high
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 297 |
Berlin, Germany, June 4-7, 2009
Aggressive NK cell Chronic
leukemia lymphoproliferative
disorder of NK cells
Middle age adult Young to Middle aged adult Adults (6th decade)
Asia Asia Worldwide
Mature NK cell Mature NK cell Mature NK cell
CD2+sCD3- sCD3- CD3-
cCD3ε±CD56+CD16± CD8±CD16+CD56+CD57+
CD57- CD57-, CD94+ KIRs±CD94/NKG2A±
Yes Yes No
Aggressive Very aggressive Indolent
Poor Poor Good
density on patients’ NK cells; this antigen is usually
associated with the inhibitory subunit NKG2A,
although in some cases the association CD94/NKG2C
has been reported.66 Patients’ NK cells characteristically
express functional β and γ chains of IL-2/IL-15 receptor,
which are strictly related to the role of these cytokines
in the pathogenesis of disease.57
Most patients are asymptomatic, and the disease has
a chronic indolent clinical course, similar to that report-
ed for patients with T LGL leukemia.44,47,48 In some cases,
this disorder is associated with other conditions, includ-
ing pure red cell aplasia, vasculitic syndromes, solid and
hematologic tumors, splenectomy, neuropathy and
autoimmune disorders.47-51,53,67 Recently, in patients with
chronic myeloid leukemia, the association has been
reported between treatment with dasatinib and the
development of CLPD-NK; the hypothesis having been
suggested that the development of CLPD-NK might
have a therapeutical effect on Ph+ leukemic cells.68
Systemic symptoms, such as cytopenia (mostly neu-
tropenia and anemia), are rare. Lymphoadenopathy,
hepatomegaly, splenomegaly and cutaneous lesions are
uncommon.67 Occasionally, patients present with a
slowly progressive increase of peripheral blood NK cells
and with organ involvement. In rare cases, the disease
transforms to aggressive NK cell leukemia69 and cases
with EBV positive NK cells tend to evolve.17 These EBV
positive patients usually suffer from chronic active EBV
infection and should be carefully monitored for the
emergence of clonal cells.69,70 Several cases with a spon-
taneous complete remission have been reported.51,53,69,72
Cytologically, the circulating cells show typical GL mor-
phology, with moderate amount of pale cytoplasm that
contains more than or equal to three azurophilic gran-
ules.48,49 Bone marrow biopsy is characterized by inter-
stitial infiltration of cells with small nuclei and pale
cytoplasm, which are difficult to recognize without the
help of immunohistochemical techniques. Cytogenetic
is normal in most cases,53,67,69 and the germ line configu-
ration of TCR is demonstrated, as expected for normal
NK cells. Clonality of proliferating cells is difficult to
detect in these patients. As an indirect marker of clonal-
14th Congress of the European Hematology Association
ity, the analysis of restriction fragment length polymor-
phism (RLFP) has been used to demonstrate the clonali-
ty in some but not all patients.51,73-74 The evidence of a
restricted pattern of clonally distributed KIR genes
expression by proliferating NK cells might also provide
indirect demonstration of clonality.60,61 In rare cases, in
which EBV can be demonstrated in plasmid form with-
in NK cells, the clonality of cells might be easily exam-
ined by Southern Blot analysis using probes recognizing
the EBV terminal repeats.70,75
Within post-transplant lymphoproliferative disorders
(PTLD), WHO classification also identifies as provision-
al a group of conditions, which are indistiguishable
from that of the immunocompetent host apart from the
feature that they occur as a consequence of immuno-
suppression.76 Patients are usually recipients of a solid
organ, bone marrow, or stem cell allograft.76 Among
these, the group of monomorphic PTLD provisionally
includes B- and T/NK-cell disorders. Clinical features
and prognosis, morphology, immunophenotype and
genetics are consistent with those reported for the same
disorder in immunocompetent host.
Treatment and prognosis
NK cell lymphoblastic leukemia/lymphoma, provisional
A standard treatment protocol for immature NK cell
neoplasms has not been established because of the
paucity of patients. Current chemotherapy strategies for
non-Hodgkin’s lymphoma or acute lymphoblastic
leukemia (ALL) were the most commonly used.
However, the overall outcomes were dismal. Two pedi-
atric patients who received non-Hodgkin lymphoma
therapy and ALL therapy, respectively, followed by allo-
geneic bone marrow transplantation achieved complete
remission for 3 years.16 Patients who did not undergo
bone marrow transplantation died of disease between 6
months and 35 months. Further studies are necessary to
determine whether increased survival can be obtained
with aggressive chemotherapy followed by HSCT. It is
possible that allogeneic HSCT could provide additional
graft versus leukemia/lymphoma benefit.
Mature natural killer cell neoplasms
The clinical outcome of patients with nasal NK cell
lymphoma is variable. Most observational studies have
consistently demonstrated that radiotherapy is superior
to chemotherapy alone in patients with stage I/II dis-
ease.77,78 Some patients with early-stage disease are
cured by radiation therapy. It has been demonstrated
that radiotherapy, either as initial treatment or as part of
the chemotherapy regimen, is the single most important
key to a successful outcome.79-81 However, some
patients with early-stage disease have early local or sys-
temic recurrences and die of the disease. For patients
with stage III/IV disease, chemotherapy is the treatment
of choice.29 In several published series, the median sur-
vival of patients with advanced-stage disease was
approximately 12 months.82 Extranasal NK cell lym-
phomas are clinically aggressive. Because the disease
may be disseminated, chemotherapy usually is the ini-
tial choice of treatment. The response is poor, and most
patients die within 6 months after diagnosis. The long-
term remission rate with allogeneic bone marrow trans-
plantation is less than 10%.31 Other non-anthracycline
| 298 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)
drugs (in particular L-asparaginase) have been success-
fully used in relapsed or refractory patients83,84 and oth-
ers are currently under investigation,85-87 warranting
promising upfront use in extranasal and advanced nasal
disease.
Aggressive NK cell leukemia is a catastrophic disease
with an almost uniform mortality. A few patients have
a clinical response with conventional chemotherapy,29
although the response is typically transient. Survival is
measured in days to weeks. Allogeneic bone marrow
transplantation reportedly results in short-term remis-
sion in a few patients.80-82
Patients with CLPD-NK usually have an indolent clin-
ical course and respond to immunosuppressive therapy,
with cyclosporin (3-5 mg/kg/day), low doses
methotrexate (usually 15 mg/week) or cyclophos-
phamide (50 mg/day), associated or not to low doses of
steroid.44-49 Because of the potential long-term side
effects of immunosuppressive therapy, limiting specific
therapy only to patients with symptomatic disease is
recommended.
New therapeutic strategies
Considering the poor efficacy of current therapies for
NK cell neoplasms, novel approaches must be consid-
ered to improve survival. Chemotherapeutic agents cur-
rently being tested in cutaneous T-cell lymphoma
(CTCL) and peripheral T-cell lymphoma (PTCL), such
as gemcitabine, liposomal doxorubicin, as well as the
purine analogs, such as fludarabine and cladribine and
nelarabine,88 represent possible new agents to be consid-
ered for NK cell treatment protocols. Furthermore, the
use of histone deacetylase inhibitors (depsipeptide and
vorinostat) is being tested in CTCL.89 Monoclonal anti-
bodies like alemtuzumab have some activity in PTCL
and are being investigated in combination with other
therapies.88 In addition, a better understanding of the
signaling pathways activated in NK cell neoplasms
could identify other biologically targeted agents as
potential candidates for inclusion in NK cell treatment
protocols. Good responses have been obtained in
patients with symptomatic CLPD-NK with the use of
RAS farnesyltransferase inhibitor (FTI), tipifarnib
(Zarnestra®, Johnson & Johnson), in accordance to the
findings of constitutively active signaling of the
Ras/MAPK/ERK pathway.65 In addition, the proteasome
inhibitor bortezomib has been reported to display anti-
cancer activity against aggressive NK leukemia and
extranodal NK/T cell lymphoma in vitro and in vivo,85,86,90
opening new therapeutical perspectives for these
patients.
Conclusions and future perspectives
In order to get insight into the characteristics of NK-
lineage disorders, the understanding of development
pathways of normal NK cell is mandatory. Impressive
progress made in understanding the expression and
functional role of NKR has contributed to our knowl-
edge of the characteristics of proliferating cells in
patients with CLPD. The distinctive features between
cytotoxic T and NK cells are not fully understood and
DNA profiling using purified normal and pathological T
and NK cells will help to address this issue and would
likely help in understanding the differences of clinical

behavior between cytotoxic T and NK derived tumors.
Investigation of disease pathogenesis will lead to identi-
fy molecular targets and discovery of more efficacious
and less toxic treatments. The elucidation of mecha-
nisms by which EBV transforms NK cells might help to
identify new molecular targets. Furthermore, the mech-
anisms by which NK cell tumours are resistant to ther-
apy should be explored, and molecules and pathways
responsible for apoptosis of cells should be character-
ized. Finally, to extend the understanding of NK neo-
plasm pathophysiology and development of novel ther-
apeutics, physicians should be encouraged to assess
National Registries and stimulate patients to participate.
Funding
This work was supported by A.I.R.C. (Milan), by
Fondazione CARIPARO e CARIVERONA, and by a grant
from Fondazione Berlucchi per la Ricerca sul Cancro and by
Regione Veneto.
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and extranasal NK/T-cell lymphoma: a study of 136 cases
from the International Peripheral T-cell Lymphoma Project.
Blood 2008; [Epub ahead of print].
88. Greer JP. Therapy of peripheral T/NK neoplasms.
Hematology Am Soc Hematol Educ Program 2006;331-7.
89. Duvic M, Talpur R, Ni X, Zhang C, Hazarika P, Kelly C, et al.
Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic
acid, SAHA) for refractory cutaneous T-cell lymphoma
(CTCL). Blood 2007;109:31-9.
90. Shen L, Au WY, Guo T, Wong KY, Wong ML, Tsuchiyama J,
et al. Proteasome inhibitor bortezomib-induced apoptosis in
natural killer(NK)-cell leukemia and lymphoma: an in vitro
and in vivo preclinical evaluation. Blood 2007;110:469-70.
91. Zola H, Swart B, Nicholson I, Aasted B, Bensussan A,
Boumsell L, et al. CD molecules 2005: human cell differenti-
ation molecules. J Immunol 2005;106:3123-6.
 Uncommon lymphoproliferative disorders

Immunological and clinical features of T cell large

granular lymphocyte leukemia

E. Matutes A B S T R A C T

Haemato-Oncology Unit, Royal
Marsden Hospital and Institute of
Cancer Research, London, UK
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:302-307
T-cell large granular lymphocytes (LGL) disorders comprise a spectrum of conditions, ranging from
polyclonal to clonal asymptomatic and/or frank leukemic LGL proliferations. We will focus on the clon-
al LGL proliferations designated T-cell LGL leukemia. Its main features are cytopenias, splenomegaly
and autoimmune phenomena. Morphology, immunophenotype and molecular analysis are key diag-
nostic investigations. Most cases represent clonal expansions of TCRα/β+ LGL, displaying a CD8+ CD4-
or rarely a CD4+ CD8-/+dim phenotype with expression of cytotoxic T-cell antigens (CD57, CD16, TIA-1,
perforin and granzyme B); a minority are clonal expansions of TCRγ/δ+ LGL. The etiology is unknown
but over the last years, there have been advances in understanding its pathogenesis. There is evidence
that T-cell LGL leukemia results from an expansion of a terminal memory activated cytotoxic lympho-
cyte. This is due to a persistent stimulation by an antigen with failure to undergo activated induced
cell death because of an impaired apoptotic pathway. Gene profiling has shown deregulation of genes
involved in the apoptosis. In the rare form with a CD4+ CD8-/+dim phenotype, cytomegalovirus is the
agent involved in triggering this response on individuals with a genetic predisposition. The course is
usually chronic and transformation is rare. Treatment strategies comprise immunomodulatory agents,
purine analogs and antibody therapy.

Introduction and NK-cell derived diseases but the World

Proliferations of large granular lympho-
cytes (LGL) comprise a spectrum of condi-
tions, ranging from polyclonal and self-lim-
ited LGL expansions to asymptomatic
clonal LGL and frank leukemic sympto-
matic disease. They are defined by the
polyclonal, oligoclonal and/or clonal
expansion in the peripheral blood and bone
marrow, or rarely in other lymphoid and
non-lymphoid tissues of a lymphocyte
with a distinct morphology and for this
designated LGL. Polyclonal expansions of
LGL are usually transient and due to a viral
infection, such as Epstein Barr virus (EBV),
solid tumors, connective tissue diseases,
and so on,1 or develop post-splenectomy.
This is in contrast to clonal LGL prolifera-
tions that persist along time whether the
patients are or not symptomatic. Oligo-
clonal and clonal expansions of T-cell LGL
have been documented in different settings
such as in patients with B-cell neoplasms,2
following allogeneic bone marrow trans-
plant3 or Rituximab treatment for a B-cell
neoplasm possibly as a result from a pro-
found B-cell depletion.4 It has been suggest-
ed that a disturbance of the immunity with
mechanisms similar to those in T-cell LGL
leukemia occurs in these patients.
In normal individuals, a minority of cir-
culating blood lymphocytes display the
morphological characteristics of LGL. The
majority of these lymphocytes have a cyto-
toxic T-cell phenotype (CD3+ CD8+ CD4–),
while only a minority corresponds to natu-
ral killer (NK) cells (CD3– CD56+). Clonal
expansions of LGL may comprise T-cell
Health Organization (WHO) considers the
latter as a separate group designated chron-
ic lymphoid disorders of NK cells.5 The
majority of cases with a T-cell LGL
leukemia represent the clonal expansion of
a memory activated cytotoxic T-cell bear-
ing the T-cell receptor (TCR) α/β and are
CD8+ CD4–, rarely CD4+ CD8-/+dim; only a
minority are expansions of TCR γ/δ+ cells.
We will focus on the clinical features,
diagnosis, biology and molecular character-
istics of the clonal T-cell LGL proliferations
as expansions of NK cells are the topic of
another presentation.
Etiology
The etiology of T-cell LGL leukemia is
largely unknown. However, over the last
years, there have been major advances in
the understanding of the pathogenesis of
this disease. A number of reports strongly
support the role of a chronic/persistent anti-
genic stimulation by an auto- or foreign
infective antigen as an initial event. This
would lead to the expansion of a memory
effector cytotoxic LGL, which is not elimi-
nated from the system due to an impair-
ment on the apoptotic pathway.6,7 No single
infective agent has been identified in the
most common form of T-cell LGL leukemia
(CD8+ CD4–). Early data suggesting the
pathogenic role of EBV or human T-cell
leukemia viruses has not been confirmed. T-
cell LGL leukemia is more common in
patients with autoimmune disorders, such
as rheumatoid arthritis, Sjogrens syndrome,
Hashimoto’s thyroiditis and lupus erythe-

| 302 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

matosus, as well as in patients with bone marrow fail-
ure due to myelodysplasia; thus, reinforcing that the
immune system plays a role in its pathogenesis.8-10 A
history of rheumatoid arthritis is documented in about
25% of patients, and it has been shown that LGL
leukemia patients have the HLADR4 haplotype, as it
occurs in patients with rheumatoid arthritis, with a
higher frequency than in the normal population. This
suggests a similar immunogenetic basis for these two
conditions.11 Unlike in CD8+ CD4- T-cell LGL leukemia,
in the rare form with a CD4+, CD8–/CD8+dim phenotype
a persistent infection by cytomegalovirus (CMV),
appears to play a key role as an initiating event in
patients with a genetic predisposition.12
Clinical features
The most common form of T-cell LGL leukemia
preferentially affects adult patients and is rare in child-
hood. It is more common in Eastern than in Western
countries. Approximately one-third of patients are
asymptomatic at diagnosis, and the disease is discov-
ered on a blood count that shows lymphocytosis, per-
sistent mild neutropenia or both.1,13 These asympto-
matic patients may have associated other conditions,
such as myelofibrosis, eosinophilia, monoclonal gam-
mopathy, and so on. The term of T-cell clonopathy of
unknown significance (TLUS) has been suggested to
be more appropriate to designate these asymptomatic
patients that represent the benign end of the clonal T-
cell LGL proliferations.14 Symptomatic patients fre-
quently manifest with fever due to infections or
mouth ulcers, often related to neutropenia, arthralgias
and tiredness; the latter not attributable to the ane-
mia. A few patients may manifest with immune or no
immune thrombocytopenia and pulmonary artery
hypertension.8,15 Up to a half of the patients have
splenomegaly, around 20% skin lesions and, a minor-
ity have hepatomegaly; lymphadenopathy, is rare. In
some cases, computed tomography shows that the
spleen is enlarged. Patients with the CD4+ CD8-/+dim
phenotype rarely present with cytopenias and
autoimmune phenomena whilst its association with
neoplasms is frequent.16
The leukocyte count may be normal or slightly
raised but in most patients, there is an increase in cir-
culating LGL, even without having an absolute lym-
phocytosis; a few patients are lymphopenic but many
develop lymphocytosis after splenectomy. Neutro-
penia is the most frequent cytopenia and it is unrelat-
ed to the degree of marrow infiltration or hyper-
splenism. Anemia and thrombocytopenia are less fre-
quent and are present in around a third of patients.
Liver function tests may be impaired and the autoim-
mune screen reveals abnormalities, such as the pres-
ence of rheumatoid factor, antinuclear antibodies, cir-
culating immunocomplexes, and polyclonal hyper-
gammaglobulinemia; a few patients may have
hypogammaglobulinemia that relates to the suppres-
sor function of the LGL upon the B lymphocytes.
Diagnosis
Although the diagnosis of T-cell LGL can be suspect-
ed in patients with a persistent increase in LGL (greater
than 6 months and with >2000/µL circulating LGL), it
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Berlin, Germany, June 4-7, 2009
usually requires the integration of morphology,
immunophenotyping, and TCR rearrangement studies
by Southern-blot or the polymerase chain reaction
(PCR). Suggested criteria for diagnosis comprise: (i) per-
sistent and sustained expansions of LGL; (ii) demon-
stration of a characteristic immunophenotype; (iii) con-
firmation of clonality by molecular studies; (iv) integra-
tion of all these data with clinical features. Updated cri-
teria by Semenzato et al. stresses the fact that not all
these criteria may be present in some patients with low
LGL counts in whom an expansion of an LGL clone is
detected by molecular analysis.13
On blood films, LGLs have a nucleus with con-
densed chromatin, no visible nucleoli, and abundant
cytoplasm that contains coarse granules (Figure 1).
The bone marrow cellularity is variable and involve-
ment by LGL may be inconspicuous; there may be a
good hemopoietic reserve even with increase in the
myeloid precursors with left shift despite of the neu-
tropenia. The trephine biopsy shows interstitial or
patchy lymphoid infiltrates and/or intrasinusoidal
infiltration; there may be lymphoid nodules but these
are shown to be reactive by immunohistochemistry.17
Immunohistochemistry allows us to better estimate
the degree of involvement by highlighting the infil-
trates. In a few patients, there is a picture of pure red
cell aplasia or reticulin fibrosis. Immunohisto-
chemistry shows the cells to be CD3+CD8+ CD4- TIA-
1+. The reactive nodules are composed by B-cells
(CD20+) and non clonal CD4+ T-cells.17 Immuno-
histochemistry in the marrow trephine parallel find-
ings of flow cytometry in the blood and marrow aspi-
rates. Spleen histology shows red pulp involvement
with preservation of the white pulp or a reactive ger-
minal centre hyperplasia with mantle zone expansion;
unlike in T-cell prolymphocytic leukemia there is no
invasion of the white pulp. The neoplastic LGL
express TIA-1 and perforin but show a CD5–,
CD45RO– phenotype unlike the normal splenic red
pulp cells which are CD5+, CD45RO+ .18
In most cases, immunophenotyping by flow cytom-
etry shows a CD2, CD3, CD8, CD16, CD57 positive
phenotype (Figure 2A). CD5 and CD7 are weakly
expressed or negative; CD56 is rarely positive and it
has been suggested to be associated with a more
aggressive course.19 A small proportion of cases have a
CD4+ CD8-/+dim (Figure 2B) or have a double
CD4/CD8 negative phenotype. It has been suggested
that cases with a CD4+ CD8–/+dim phenotype are a dis-
tinct subgroup with a preferential Vb13.1 usage (44%
of cases) and associated with neoplasias.16 These cells
have a cytotoxic (CD56+, CD57+, granzyme B+) pheno-
type corresponding to that of a memory/activated
cell. Most cases represent expansions of T cells bear-
ing the TCR αβ and a very minority is derived from
TCRγδ+ cells, which have a Vγ9/δ2 or a Vnonγ9/δ1
genophenotypic profile equally restricted compared to
normal TCRγδ+ T-cells. It is uncertain as to whether
cases with TCRγδ+ T-cell LGL leukemia have different
clinical manifestations compared to the most common
form of TCRαβ+ T-cell LGL leukemia; it seems that
there is a higher proportion of cases in this cohort that
have a double CD4/CD8 negative phenotype.20 The
availability of a repertoire of Monoclonal antibodies
14th Congress of the European Hematology Association

Figure 1. Circulating lymphocyte dysplaying an eccentric

nucleus and abundant cytoplasm with coarse granules,
feaures typical of LGL.

(McAb) against NK cell antigens has proved to be

valuable for the diagnosis of LGL leukemia. These
McAb recognize receptors that belong to the
immunoglobulin (Ig) superfamily and comprise killer-
cell Ig-like receptors (KIRs) clustered under CD158a,
CD158b, CD158e and CD158i and C-type lectin
receptors (CD94, CD161). In healthy individuals, T-
cells do not express KIRs and a small proportion is
CD94+ and/or CD161+. By contrast, CD94, like CD57,
is expressed in clonal LGL, and some cases, may
express one or more KIR.21,22 Over-expression of KIRs
in LGL is aberrant and potentially diagnostic. CD52
has been documented to be expressed in cells from all
T-cell LGL leukemias investigated and there is a
potential for the use of Campath-1H in this disease.23
Flow cytometry analysis with McAb against the
various Vβ regions of the TCR allows establishing
clonality of the LGLs by showing the preferential use
of one or two TCR-Vβ segments. There is a correla-
tion between the PCR and flow cytometry findings,
with flow cytometry able to predict clonality with a
sensitivity of 93% and a specificity of 80%.24 Unlike
in CD4+ CD8–/+dim T-cell LGL leukemias, there is no
preferential use of a Vβ segment as the pattern of dis-
tribution is similar to that found in normal blood.
The diagnosis of T-cell LGL leukemia should be
confirmed by documenting clonal TCR rearrangement
of the β or γ chain genes by Southern blot or PCR
(Figure 3). These techniques should be applied to all
suspected cases and are useful in those patients in
which the absolute LGL count is not significantly
increased.13
The differential diagnosis arises with self-limited
polyclonal expansions of LGL and other post-thymic
T-cell disorders, namely prolymphocytic leukemia
and γ/δ T-cell lymphoma, myelofibrosis and
myelodysplasia.
Pathogenesis and molecular features
Cytogenetic studies in T-cell LGL leukemia are
scant; clonal abnormalities are infrequently detected,
without a recurrent genetic marker identified.1,5 Clonal
Figure 2. (A) Flow cytometry dot plots from a case with a
CD8+ T-cell leukemia showing expression on the leukemic
cells of CD2, CD5, CD45RA, CD8, CD11c, CD16, CD57,
CD94, granzyme B, perforin and HLA-Dr whilst are negative
for CD7, CD45RO and CD27. A residual normal T-cell pop-
ulation with a different phenotype can be also identified.
(B) Flow cytometry dot plots from a case with a CD4+ T-cell
leukemia showing expression of CD2, CD4, CD5, CD45RA,
CD45RO, CD56, granzyme B, perforin and partial expres-
sion of CD57 whilst cells are negative with CD16, CD161,
CD158a and CD27.
abnormalities include those involving rearrangements
of the TCR loci genes at 7p14-p15 and 14q11, inver-
sion of 12p, 5q deletion and trisomy 3, 8 and 14.5,25
It is now becoming evident that T-cell LGL
leukemia with a CD8+ CD4– phenotype results from
an expansion and accumulation of a CD8+ terminal
effector memory cytotoxic cell. This is due to a per-
sistent stimulation by an auto or foreign antigen with
a failure to undergo activate induced cell death
(AICD) consequent to a profound impaired apoptotic
pathway. Elimination of potentially harmful antigen-
stimulated effector memory T-cells after a successful
immune response physiologically occurs by triggering
Fas-mediated apoptosis through induction of a death-
inducing signaling complex (DISC). This allows main-

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Figure 3. PCR for the TCRβ and TCRγ chain genes showing
multiple bands in a control (polyclonal pattern) and a sin-
gle TCRβ (monoallelic) and two TCRγ bands (bi-allelic) on a
patient with T-cell LGL leukemia.
taining the T-cell homeostasis. Globally, leukemic
LGL are characterized by a profound deregulation of
the apoptosis and do not undergo to AICD. Although
clonal LGL constitutively express high levels of both
Fas (CD95) and Fas ligand (Fas-L), and do not harbor
mutations of the Fas receptor, they are resistant to
Fas-induced apoptosis due to a defective Fas apoptot-
ic pathway.26 Fas resistance can occur in cells with
over-expression of the DISC inhibitory protein c-FLIP.
c-FLIP is constituvely expressed in leukemic LGL pre-
venting DISC formation and Fas mediated apoptosis.27
Besides this, it is likely that several genes are involved
in such apoptotic failure as revealed by gene microar-
ray expression as discussed in the following para-
graph. Gene expression profiling has revealed that
leukemic LGLs have a different signature from that of
normal naïve and activated memory T-cells.28 Major
differences are found on the expression of genes
belonging to the categories of apoptosis, regulation of
TCR signaling and immune response. This supports
the notion that an uncoupling of activation and apop-
totic pathways is responsible for a failure of AICD.
One of the main findings is the deregulation in the
expression of genes involved in the apoptotic path-
way. Genes which are up-regulated in leukemic LGL
have anti-apoptotic functions whilst those which are
down-regulated are pro-apoptotic. Among the apop-
totic related genes, the gene that shows the highest
up-regulation compared to normal activated CD8+
lymphocytes is the tumour necrosis factor α induced
protein 3 (TNFAIP3).28 This is a NF-Kb inducible gene
that protects T-lymphocytes from undergoing TNF
induced apoptosis and, in physiological conditions is,
unlike in leukemic LGL, down-regulated in T-cells fol-
lowing activation. Genes belonging to the BCL-2 fam-
ily, such as the BCL-2 related X gene (BAX) are down-
regulated, whilst the myeloid cell factor (MCL-1) is
up-regulated. Recent data indicates that the sphin-
golipid-mediated signaling pathway plays a role on
the apoptotic deregulation in leukemic LGL. Key
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 305 |
Berlin, Germany, June 4-7, 2009
genes in the sphingolipid metabolism and signaling
which are up-regulated in LGL, include acid
ceraminidase and S1P5 receptor. Up-regulation of
ceramidase is found in many neoplasms and it has
been postulated to also be involved in the apoptosis
failure in LGL.
TCRα/β+ CD4+ CD8-/+dim LGL leukemias is an infre-
quent form. There is compelling evidence that chron-
ic stimulation of T-cells by CMV leads to a persistent
clonal expansion of CD4+/CD8-/+dim LGL, with a pre-
dominance of TCR VB13.1 usage in individuals with a
HLADRB1*0701 haplotype. Recent reports have
shown that the expanded T-cell clones have a similar
TCR with a high degree of homology in the comple-
mentary determinant region (CDR3) and similar com-
binatorial gene rearrangements.29 In vitro studies have
demonstrated that the expanded CD4+ LGL clones
have a characteristic and unique response to a CMV
peptide.12 The gene expression signature of these cells
is different from that of normal memory CD4+ LGL
with outstanding abnormalities in CD4+ leukemic
LGL encompassing a variety of genes that function in
cell cycle, resistance to apoptosis and genetic instabil-
ity. All these data strongly supports that a persistent
CMV stimulation is the responsible for triggering and
maintaining the activated/memory LGL clone in
patients with a genetic predisposition. Beside the
pathogenesis of the expansion of LGL, the underlying
mechanisms of the cytopenias may be multiple, such
as lack of erythroid precursors (PRCA), autoimmune
mediated or inhibition of the burst-forming and ery-
throid colony-forming units by the release of
cytokines by the LGL.30
Prognosis and survival
T-cell LGL leukemia usually runs a chronic course in
most patients with mortalities of 10 to 28% at 4 years
and a median survival greater than 10 years.1,31 Most
deaths are due to sepsis and rarely occur due to dis-
ease progression; transformation to large-cell lym-
phoma is exceedingly rare.32
Therapy
Up to a third of patients may will never require
treatment as they are asymptomatic and cytopenias
are mild; a watch and wait strategy is appropriate.1
Indications for treatment include severe neutropenia,
symptomatic anemia or thrombocytopenia and pro-
gressive disease, (i.e., organomegaly and/or rapidly
raising counts). The aim of treatment is to correct the
cytopenias, and this can be achieved without signifi-
cant reduction of the clone with immunomodulatory
drugs, such as methotrexate (10 mg/m2/weekly),
cyclosporine A (5-10 mg/kg/day) or low dose cyclo-
phosphamide (50 to 100 mg/day).31,33 Corticosteroids
may be useful as part of the initial treatment to accel-
erate response; growth factors are of value as they
may act synergistically with the immunosuppressive
therapy. There have not been randomized trials to
compare the efficacy of these agents. While all are
effective in improving cytopenias in up to two thirds
of patients, the malignant clone persists. Adverse
events are not severe and are more common with
cyclosporine A, particularly in elderly patients.
14th Congress of the European Hematology Association
Second malignancies are rare but close vigilance is rec-
ommended, particularly in cases with prolonged
immunosuppression. Splenectomy may be considered
as an adjuvant in patients with gross splenomegaly
and refractory cytopenias.34
Purine analogs have been only used in few patients
with response rates of 40 to 67% for pentostatin,35 flu-
darabine alone36 or in combination.37 The use of these
agents should be contemplated in young patients as
they allow achieving good remissions, including
reduction of bone marrow infiltration.
Newer agents such as the McAb anti-CD52
(Campath-1H), anti-CD122 and anti-CD2 are being
incorporated in the therapeutic scenario.38 There have
been anecdotal reports of successful treatment with
anti-CD52 antibody in refractory disease. In light of
the deregulation of the MAPK/Ras pathway in the
leukemic NK and T-cell LGL, the use of the farnesyl-
transferase inhibitor tipifarnib may offer therapeutic
utility.39 There are few ongoing clinical trials for T-cell
LGL leukemia, such as a phase II ECOG study of
methotrexate 10 mg/m2 weekly, with or without
prednisolone 30 mg/day with tapering doses, crossing
over to low-dose cyclophosphamide 100 mg daily for
non-responders, a phase II study of tipifarnib39 and a
phase I study of the anti-CD2 antibody siplizumab.
In current practice, the approach to treatment in T-
cell LGL leukemia is to start with an immunosuppres-
sive agent, such as methotrexate or cyclosporine A,
with or without prednisolone or growth factors.
Purine analogs, McAb and/or experimental drugs may
be considered in selected patients with the aim of
achieving a complete and durable remission.
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25. Wong KF, Chan JC, Liu HS, Man C, Kwong YL.
Chromosomal abnormalities in T-cell large granular lym-
phocyte leukemia: report of two cases and review of the lit-
erature. Br J Haematol 2002;116:598-600.
26. Lamy T, Liu JH, Landowski TH, Dalton WS, Loughran TP.
Dysregulation of CD95/CD95 ligand-apoptotic pathway in
CD3(+) large granular lymphocyte leukemia. Blood 1998;
92: 4771-7.
27. Yang J, Epling-Burnette PK, Painter JS, Zou J, Bai F, Wei S,
Loughran TP. Antigen activation and impaired Fas-induced
death-inducing signaling complex formation in T-large gran-
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28. Shah MV, Zhang R, Irby R, Kothapalli R, Liu X, Arrington T,
et al. Molecular profiling of LGL leukemia reveals role of
sphingolipid signaling in survival of cytotoxic lymphocytes.
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29. Garrido P, Ruiz-Cabello F, Barcena P, Sandberg Y, Canton J,
Lima M, et al. Monoclonal TCR-Vβ 13.1+/CD4+/NKa+/
CD8-/+dim T-LGL lymphocytosis: evidence for an antigen-
driven chronic T-cell stimulation origin. Blood 2007;109:
4890-8.
30. Handgretinger R, Geiselhart A, Moris A, Grau R, Teuffel O,
Bethge W, et al. Pure red-cell aplasia associated with clonal
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31. Osuji N, Matutes E, Tjonnfjord G, Grech H, Del Giudice I,

Wotherspoon A, et al. T-cell large granular lymphocyte
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32. Matutes E, Wotherspoon AC, Parker NE, Osuji N, Isaacson
PG, Catovsky D. Transformation of T-cell large granular
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33. Loughran TP, Kidd PG, Starkebaum G. Treatment of large
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trexate. Blood 1994;84:2164-70.
34. Subbiah V, Viny AD, Rosenblatt S, Pohlman B, Lichtin A,
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Blood 2008;112:4694-8.
 Uncommon lymphoproliferative disorders

The treatment of hairy cell leukemia

D. Catovsky
M. Else
Section of Haemato-Oncology
The Institute of Cancer Research,
Sutton, UK
Hematology Education:
the education program for the
annual congress of the European
Hematology Association
2009;3:308-313
A B S T R A C T
There has been significant progress in our understanding of the nature of hairy cell leukemia (HCL)
since the first description of the disease fifty years ago. In contrast to other B-cell lymphoprolifera-
tive disorders, progress in HCL treatment has been remarkable, with several agents, mainly the purine
analogs pentostatin and cladribine, able to achieve a high proportion of complete responses, which
are of long duration. We will illustrate this from our experience with these two agents from a series
of 233 patients treated over the last 22 years, with a median follow-up of 16 years. For the propor-
tion of relapsing patients (42% at ten years and 47% at fifteen years), as well as those initial non-
responders, there are now new treatment modalities in the form of monoclonal antibodies which,
either alone or in combination with either purine analog, could improve outcome. We will show that
rituximab (anti-CD20), combined with either pentostatin or cladribine, could achieve better respons-
es in relapsing patients than when used alone. This is not surprising, as the density of expression of
the CD20 antigen is the highest of all the B-cell malignancies. Anti-CD22, an antibody linked to an
immunotoxin, also offers new hope for the minority of relapsing/refractory patients.

Introduction
Exactly fifty years ago, a seminal paper by
Bouroncle et al.1 put hairy cell leukemia
(HCL) on the clinico-pathological map. The
report described clearly all the features we
now recognize in this distinct disease entity.
The term leukemic reticuloendotheliosis was
coined and the benefits of splenctomy, with
long-term remissions in some patients, were
noted. It is of historical interest that
Rosenthal described a similar entity a few
years earlier (1952) under the term reticular
cell leukemia but, because of the vagaries of
the peer review system, the paper was
rejected, and only published in 1969. Schrek
and Donnelly2 introduced the term hairy cell
later, when they compared hairy cells to
some flagellated cells seen in lymph nodes,
using phase contrast microscopy. Other dis-
tinct features of HCL were noted: the bone
marrow fibrosis in the presence of lympho-
cytes, the rather unique tartrate resistant
acid phosphatase (TRAP),3 the ribosome
lamella complex seen first by electron
microscopy and later by light microscopy4
and, finally, the distinct pattern of the bone
marrow histology.5
In the 1970s, hairy cells were shown to be
of B-cell origin; a fact confirmed in the 1980s
by showing clonal rearrangement of the
immunoglobulin (Igl) genes.
Diagnosis
HCL is now a well-recognized entity in
the World Health Organization (WHO) clas-
sification.6 Although the diagnosis is relative-
ly straightforward in the presence of cytope-
nias in middle-aged male patients with a pal-
pable spleen, it is important to be aware that
other splenic B-cell lymphomas with circu-
lating villous (or hairy-looking) lymphocytes
can present a diagnostic problem. There are
three conditions: (i) splenic marginal zone
lymphoma (formerly described as splenic
lymphoma with circulating villous lympho-
cytes); (ii) HCL-variant, which is a rare con-
dition and now included in the WHO classi-
fication as a provisional entity. It bears no
biological connection with typical HCL; (iii)
a recently described condition with some
similarities to splenic marginal zone lym-
phoma and HCL-variant, described as
splenic diffuse red-pulp B-cell lymphoma.
This is also a provisional entity in the new
WHO classification.7 The latter two condi-
tions have the involvement of the splenic red
pulp and the presence of circulating lympho-
cytes with villous projections in common
with HCL.
The key to HCL diagnosis is the bone mar-
row histology. Monoclonal antibodies
(MoAb) can facilitate the diagnosis. This can
be flow cytometry and/or immunohisto-
chemistry in bone marrow sections.
Characteristic MoAb used by flow cytome-
try are CD25, CD103, CD123, in addition to
markers seen in other B-cells, CD11c, CD19,
CD20, FMC7, and monotypic membrane
Igl.6,8 By immunohistochemistry, specific
markers are Annexin A1, TRAP (demonstrat-
ed by means of a MoAb), DBA44, CD20 and
CD11c (a formalin resistant epitope).9 The
importance of immunohistochemistry is not
just for diagnosis but also for its power to
identify minimal residual disease after thera-
py. Without this methodology, it is very dif-
ficult to identify small clusters of residual
hairy cells in the bone marrow by conven-
tional hemotoxylin and eosin staining. The

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antibody to Annexin A1, a very specific marker of HCL
(as distinct from other B-cell disorders), is less useful for
detecting residual disease because it also reacts with
myeloid cells.6
Clinical features and indications for treatment
In contrast to chronic lymphocytic leukemia (CLL),
most patients with HCL are diagnosed because of clini-
cal problems at presentation: cytopenias, infections
caused by neutropenia and/or monocytopenia,
splenomegaly. A chance diagnosis is rare. Lack of clini-
cal progression (in the absence of treatment) is also very
rare. In our experience, around 3% of patients may
remain stable without the need for treatment for long
periods of time.
Although treatment is seldom an emergency, infec-
tion is one of the main clinical indications, as well as
progressive splenomegaly, anemia or thrombocytope-
nia. A period of watch and wait, as in CLL, is possible
but very often the manifestations of the disease show
progressive features requiring an urgent initiation of
therapy.
A few studies are looking at the prognostic features at
presentation in HCL. A large French series of 238
patients10 identified a hemoglobin level less than 10 g/dL
and a white blood count less than 2.0×109/L as factors
influencing the achievement of a complete response
(CR). Both factors were also associated with reduced
survival.10 In our study of 233 patients, we also docu-
mented that hemoglobin less than 10 g/dL and platelets
less than 100×109/L at the start of treatment were, when
combined, associated with a shorter relapse-free sur-
vival (9 years) versus patients without these features
(20+ years).52 Figure 1 illustrates this. Therefore, as in
CLL, a poor bone marrow function, reflected in low
hemoglobin and platelet levels, seems to indicate a
worse prognosis. Such features may need to be re-
examined prospectively, since patients with poor bone
marrow function may require special attention for their
treatment plan.
In the era before effective therapy was available, it
was common to see patients with mycobacterial infec-
tions, both classic tuberculosis and atypical mycobacte-
ria, such as mycobacteria Kansasai. In patients present-
ing with sweating and/or unexplained fever, it is always
important to investigate such infections, sometimes
requiring a liver biopsy to establish the cause.
The issue of lymphadenopathy in HCL is of particu-
lar interest. Peripheral enlarged lymph nodes are not a
feature of the disease, as they are in CLL. In an earlier
study, we noticed a group of patients with advanced
HCL who had large abdominal masses of lymph
nodes.11 A study by Hakimian et al.12 reported a similar
finding in 25% of previously treated HCL cases, but
none in 19 newly-diagnosed patients screened by CT
scan. Maloisel et al.10 identified 8% of patients with sig-
nificant lymphadenopathy at diagnosis, and 7% previ-
ously treated. Large abdominal masses seem, in our
experience and that of others10,12 to be associated with
bulky disease, also manifested by a large spleen (which
has often been removed). In our series13 and that of the
French group,10 patients with abdominal nodes have a
worse outcome. Although it is not common practice to
perform abdominal CT scanning in newly diagnosed
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 309 |
Berlin, Germany, June 4-7, 2009
Figure 1. Relapse-free survival by hemoglobin and platelet
counts. Patients with hemoglobin <10 g/dL and/or
platelets <100×109/L had shorter relapse-free survival
than patients with higher counts, p<0.0001. (Royal
Marsden Hospital/Institute of Cancer Research series of
233 patients with hairy cell leukemia, updated 2009).52
patients, it should probably be a rule in relapsing cases,
and particularly in those who presented with large
spleens. Treating such patients without checking for
evidence of abdominal nodes may result in inadequate
clinical responses because of giving insufficient treat-
ment. It was of interest that out of our four patients
with abdominal lymphadenopathy who were deemed
to have become refractory to pentostatin, we docu-
mented a CR in two and a partial response (PR) in
another, using cladribine.13 This provided some evi-
dence for a lack of cross-resistance between these two
commonly used purine analogs.
Treatments for hairy cell leukemia
There are several treatment modalities, which can be
used in HCL although, for the last 20 years, the main
agents have been the nucleoside analogs pentostatin (2’-
deoxycoformycin) and cladribine (2-chlorodeoxyadeno-
sine) because of their ability to induce high response
rates and long remissions. A summary of some of the
large published series with both reagents is shown in
Table 1.
Splenectomy, first mentioned by Bouroncle et al.,1 can
still achieve spectacular results, with rapid improve-
ment of the cytopenias in close to 90% of cases. A
minority of such patients may not require any further
therapy for very long periods (e.g., 15-20 years) and
enjoy excellent quality of life. We have treated one such
patient whose clinical remission lasted 28 years before
he relapsed and achieved a complete response with two
courses of cladribine. Splenectomy is not a treatment of
choice in HCL, but it may benefit patients presenting
with massive splenomegaly (e.g., >10 cm below the
costal margin) and little or moderate bone marrow
involvement. Rarely, a splenectomy may be useful to
establish a diagnosis and to distinguish HCL from some
of the other forms of splenic B-cell lymphoma men-
tioned above. The first major advance in the treatment
of HCL was interferon-α (IFN-α), first published by
14th Congress of the European Hematology Association

Table 1. Responses to pentostatin and cladribine in HCL.

Ref. No. pts. % ORR % CR

Pentostatin
Cassileth et al. 1991 (14) 50 84 64
Golomb et al. 1994 (15) 85 84 42
Ribeiro et al. 1999 (16) 50 96 44
Rafel et al. 2000 (17) 78 88 72
Grever et al. 1995 (18) 154 79 76
Johnston et al. 2000 (19) 28 100 89
Maloisel et al. 2003 (10) 238 96 79
Else et al. 2005 (20) 185 96 81

Cladribine
Estey et al. 1992 (21) 46 89 78
Tallman et al. 1996 (22) 50 100 86
Hoffman et al. 1997 (23) 49 100 76
Cheson et al. 1998 (24) 861 87 50 Figure 2. Relapse-free survival by response to treatment.
Patients who achieved a complete response to treatment
Goodman et al. 2003 (25) 207 100 95 had longer relapse-free survival than patients who
Jehn et al. 2004 (26) 44 100 98 attained a partial response, p<0.0001. (Royal Marsden
Zinzani et al. 2004 (27) 37 100 81 Hospital/Institute of Cancer Research series of 233
Chadha et al. 2005 (28) 86 99 79 patients with hairy cell leukemia, updated 2009).52
Else et al. 2005 (20) 34 100 82
Robak et al. 2007 (29) 132 93 74
CR: complete response; ORR: overall response rate (CR + partial response).
tions. The number of injections was variable (range 2-
27; median 10) as it was guided by the target of achiev-
ing CR. CR is the aim of treatment in HCL, as it is
Quesada in 1984, with 3 out of 7 patients achieving associated with long disease-free survival, regardless
CR.30 In fact, IFN-α does not induce a high CR rate, but of the therapy used (as shown in Figure 2, which com-
the overall response rate in some series is 91%.31 The bines our data with both pentostatin and cladribine).
responses to IFN-α are rapid and are not associated The third major landmark in the treatment of HCL
with the myeloid and lymphoid toxicities seen with was cladribine, introduced by Piro et al.36 in 1990.
the purine analogs. There has been only one random- Cladribine has become very well established in the
ized trial comparing IFN-α with pentostatin18 and that USA, where it is the treatment of choice.
showed a very superior disease-free interval after ces- One important aspect of treatment with cladribine is
sation of therapy with pentostatin. Clearly, IFN-α that many physicians thought that one single course of
needs continuous treatment in order to maintain nor- treatment was sufficient and failed to look at the bone
mal counts, as HCL is still present in the bone marrow. marrow to assess response. We repeated a bone mar-
Some authors have tested the possibility of long-term row examination after 4 to 6 months and, if there was
maintenance, which prolongs progression-free sur- only a PR, a further course was given. This was neces-
vival.31 In fact, two such studies have shown that it is sary in 18% of patients and the majority of them
possible to apply such a program in 50-60% of switched from PR to CR after the second course.
patients.31,32 Because the CR rate with IFN-α is low and Cladribine is an easier agent to produce than pento-
there is discomfort associated with the side effects of statin and can be given in different modalities or routes
continuous maintenance, this agent is no longer used of administration. We have used the original dose of
as first line in HCL. One possible additional advantage 0.1 mg/kg/day by continuous infusion over 7 days.36
of using IFN-α for a short term (circa 3 months), prior Others have used short two-hour infusions at 0.12-
to giving a nucleoside analog, is that it may result in 0.15 mg/kg for 5 or 7 days, with apparently similar
fewer early infections;33 an observation that coincides response rates.37 More recently, Robak et al.29 compared
with our early experience.34 Whether there is still a role the daily short infusions for five days with weekly
for IFN-α in the treatment of HCL, for example, in infusions over six weeks, with no differences in
patients non-responsive to cladribine or pentostatin, is response rate or toxicities. A preparation for bolus sub-
an open question. The advent of MoAb with therapeu- cutaneous injections is now available and this has also
tic activity in this disease (see below) suggests that this been used with equal success with respect to the
is unlikely to be the case. response rate. What is missing in many of these stud-
The first nucleoside analog with activity in HCL was ies, which compare the various modalities for admin-
pentostatin. Spiers et al.35 reported a CR in 16 out of 27 istering cladribine, is data on whether the quality of
patients. This high CR rate was confirmed in several the response translates into a long disease-free interval.
other studies (Table 1), including our own, which now To this end, it is critical always to examine the bone
has a median follow-up of 16 years.52 One feature of marrow after the treatment is completed.
the way we have administered pentostatin is to use 4 We compared pentostatin and cladribine in our
mg/m2 as an intravenous push every two weeks until series of 233 patients and found no differences in
maximum response, followed by two additional injec- response rates, disease-free survival and overall sur-

| 310 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1)

Table 2. Responses to rituximab in HCL, as a single agent
and combined with pentostatin or cladribine.
Ref. No. pts. % ORR
Single agent
Zinzani et al. 2000 (39) 24 25
Lauria et al. 2001 (40) 10 50
Hagberg & Lundholm 2001 (41) 11 64
Nieva et al. 2003 (42) 24 25
Thomas et al. 2003* (43) 15 80
Zenhausern et al. 2008 (44) 26 80
Combination treatment
Ravandi et al. 2006* (45) 13 100
Else et al. 2007 (38) 8 100
Cervetti et al. 2008 (46) 27 100
*8 doses rituximab (previous standard: 4)
vival.20 Although this was not a randomized trial; nev-
ertheless, the characteristics of the patients were not
very different between the treatment groups and our
conclusion is that there is no major difference
between these two purine analogs. The choice will
depend on cost, ease of administration and, ultimate-
ly, quality of life and duration of remission. Survival at
ten years was 96% and 100% with pentostatin and
cladribine, respectively. For patients achieving CR and
still in CR at five years, the risk of relapse by 15 years
was only 25%.
From our data, at 16 years’ median follow-up from
diagnosis, only one fifth of patients have died, and this
is equivalent to the age/sex-matched death rate for the
general population.52 The incidence of HCL-related
deaths in our series was less than 4%. Damasio et al.31
reached a similar conclusion with the maintenance
schedule of IFN-α. Thus, in HCL the long-term sur-
vival is not a major issue. The main measures to assess
the benefits of any therapy are the duration and quali-
ty of remissions, as assessed by disease-free survival.
Treatment of relapsed/refractory disease
We have also investigated the effect of therapy with
either nucleoside analog after first and subsequent
relapses and in non-responding patients.52 The CR rate
after first line was 80%, after second line was 69%,
and after third line was 50%, clearly suggesting that
achieving a new response in relapsing patients
becomes more difficult. Nevertheless, when we
looked at disease-free survival in those achieving CR,
the duration of these remissions was equally long,
whether after first, second or third-line therapy. This
introduces the concept that new modalities, perhaps
adding MoAb to chemotherapy, may be used to
improve the CR rate in such relapsing/refractory
patients. Indeed, in a preliminary analysis, now updat-
ed to 12 patients relapsing after 2, 3, 4 or 6 lines of
therapy, we recorded a CR rate of 92% when ritux-
imab (Mabthera) was used in combination with either
cladribine or pentostatin.38
Table 2 shows the results of various small series using
this anti-CD20 MoAb. There is evidence that the den-
sity of CD20 in hairy cells is the highest of all the B-cell
Hematology Education: the education program for the annual congress of the European Hematology Association | 2009; 3(1) | 311 |
Berlin, Germany, June 4-7, 2009
malignancies.47 Also, adding this antibody to either
nucleoside analog does not increase toxicity and allows
the achievement of better responses in the
% CR relapse/refractory setting than with either cladribine or
pentostatin alone. Although it is not clear whether it is
better to use rituximab concurrently or sequentially
13
(after the initial response to the nucleoside analog), 9
10
54 out of our 12 patients were given the MoAb concur-
13 rently. By analogy with data in CLL,48 concurrently may
53 achieve a better synergistic effect. The issue also arises
32 as to the number of rituximab infusions and whether
this antibody should move to being used as part of the
first line therapy, as used by Ravandi et al.45 Because
100 best results may be achieved with six to eight ritux-
87 imab infusions, plus the lack of added toxicity, it is like-
89 ly that this number, rather than the conventional four
infusions, should be used for optimal effect. Now, we
would consider this combined therapy – nucleoside
analog plus rituximab – for the 42-47% who experience
a relapse within the first 10 -15 years after first line
therapy. The choice of nucleoside analog will depend
on which of the two was previously used. The exis-
tence of possible lack of cross-resistance mentioned
above suggests that the alternative agent, if available,
should be used with rituximab in such indications. The
issue of cost may determine whether such modalities
become the treatment of choice for all patients.
Immunotoxins in the treatment of hairy cell leukemia
Kreitman et al.49 reported promising results with a
recombinant immunotoxin containing anti-CD22 vari-
able domain fused to truncated Pseudomonas exotox-
in (BL22) in a series of patients resistant to cladribine.
A recent update of this series50 reported a CR rate of
61% and an overall response rate of 81%, with a medi-
an duration of remission of 36 months. Although
refractory HCL is not common, this modality could
also be considered in relapsing patients, not necessari-
ly refractory, and a clinical trial could perhaps be envis-
aged to compare BL22 with the combination of a
purine analog plus rituximab. Problems with BL22 are
hemolytic uremic syndrome in some patients, liver
dysfunction and hypoalbuminemia. Again, issues of
safety and cost will be paramount. Patients in whom
these new modalities may be used include: (i) non-
responders to first line pentostatin or cladribine (<5%);
(ii) partial responders to either drug (15-20%); (iii)
complete responders but who tend to relapse in the
first 5-10 years (circa 40%).
Second malignancies in hairy cell leukemia
There are uneven data in the literature as to whether
the rate of second malignancies in HCL may be
increased, either by the disease itself or by the use of
purine analogs and/or IFN-α. In our series, 31 patients
developed second malignancies (excluding non-
melanoma skin cancers).52 This was equivalent to the
incidence of cancer in the general population matched
by age and sex. Similar conclusions were reached by
two other large series of 238 patients10 and 241
patients;51 therefore, it seems unlikely that either the
disease or its treatment increases the likelihood of
other malignancies.
14th Congress of the European Hematology Association
Conclusions
The treatment of HCL has been remarkably success-
ful and new possibilities have appeared on the horizon,
in the form of MoAb for therapeutic use, which will
improve treatment further. For a relatively rare disease,
it is a major success story, achieved without random-
ized clinical trials. The outlook for patients with respect
to disease-free interval and overall survival has
improved dramatically since the disease was first
described fifty years ago.1
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 Berlin, Germany, June 4-7, 2009

Index of authors

Armstrong S.A. 8 Gratwohl A. 256 Mullighan C.G. 1

Gudgin E. 17 Munshi N.C. 161
Baglin T. 266
Baron F. 242 Hartmann E. 106 Niemeyer C.M. 208
Barrett J. 250 Hillmen P. 68 Nimer S.D. 177
Bladé J. 166 Hochhaus A. 78
Boyd K. 155 Hoffman R. 192 Pamphilon D.H. 282
Brunet S. 30 Huntly B. 17,83 Putti M.C. 221
Büller H.R. 271
Izraeli S. 13 Randi M.L. 221
Catovsky D. 308 Raza A. 172
Cazzola M. 181 Kantarjian H. 83 Reitsma P.H. 261
Chapuy B. 112 Kaspers G.J.L. 214 Rosenquist R. 61
Cilloni D. 24 Kiladjian J.-J. 200 Rosenwald A. 106
Cortes J. 83 Klein U. 55 Rosiñol L. 166
Kleinjan A. 271
De Meyer S.F. 38 Kralovics R. 188 Saglio G. 24
Deininger M.W. 72 Sandmaier B. 242
Dzierzak E. 123 Landman-Parker J. 140 Semenzato G. 294
Lapid K. 133 Shipp M. 112
Efficace F. 89 Lapidot T. 133 Sierra J. 30
Eichenauer D.A. 151 Luban N.L.C. 288 Simon R. 96
Else M. 308 Sprangers M.A. 89
Engert A. 151 Malcovati L. 181 Steinberg M.H. 233
Estey E. 101 Manz M.G. 128
Mascarenhas J. 192 Takizawa H. 128
Federici A.B. 50,89 Matutes E. 302
McMullin M.F. 238 Vanhoorelbeke K. 38
Galili N. 172 Messa E. 24
Gallamini A. 144 Mohandas N. 227 Zambello R. 294
Gisselbrecht C. 118 Morgan G. 155
Goodeve A. 44,78 Moulds J.M. 276

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