Cancer Genetics 206 (2013) 49e62

REVIEW ARTICLE
Chronic lymphocytic leukemia: a clinical
and molecular heterogenous disease

lez Dı́az a,b,
Ana E. Rodrı́guez-Vicente a, Marcos Gonza

s M. Herna
Jesu
ndez-Rivas a,b,*
a
IBSAL, IBMCC, Centro de Investigacio
n del Ca
ncer, Universidad de Salamanca-CSIC, Salamanca, Spain; b Servicio de
Hematologı́a, Hospital Clı́nico Universitario de Salamanca, Salamanca, Spain

The clinical heterogeneity that characterizes chronic lymphocytic leukemia (CLL), with survival
times ranging from months to decades, reflects its biological diversity. Our understanding of
the biology of CLL has helped us identify several markers of prognostic significance, by which
CLL can be differentiated into several distinct diseases. The presence of specific chromosomal
abnormalities is a prognostic indicator of disease progression and survival. Conventional cytoge-
netic analyses have revealed chromosomal aberrations in 40e50% of patients, but the detection
of abnormalities is limited by the low mitotic activity of CLL cells. Metaphase analysis has recently
undergone a “revival” because the metaphase yield has been improved by stimulation of CLL
cells with alternative methods. Fluorescence in situ hybridization identifies chromosomal
changes in approximately 80% of patients with CLL, and comparative genomic hybridization
using high-density arrays (i.e., array comparative genomic hybridization [aCGH]) enables high-
resolution genome-wide scanning for detecting copy number alterations in a single hybridization.
The mutational status of the immunoglobulin heavy chain variable (IGHV) genes identifies two
subsets of CLL with different outcomes. Unfortunately, the determination of IGHV mutation status
may not be practical in all laboratories, and for this reason characteristics that are correlated with
IGHV mutation status are neededdzeta-chain associated (TCR) protein kinase 70 kDa (ZAP-70)
being that most commonly used currently in routine clinical practice. Whole genome sequencing
has offered new insights into the mutational status of the disease, highlighting the role of several
genes previously unrelated to CLL. Of these, NOTCH1 and SF3B1 are the most frequently
mutated genes that predict poor prognosis. MicroRNA alterations are also involved in the initia-
tion and progression of CLL, and the expression levels of some microRNAs correlate with previ-
ously established prognostic markers such as IGHV mutation status or ZAP-70. In addition, both
global and gene-specific aberrant DNA methylation have been observed in CLL. Aberrant meth-
ylation has been described for genes that are specifically deregulated in CLL, such as BCL2,
TCL1, and ZAP-70. Expanding knowledge of aberrant methylation profiles in CLL has a potential
future impact on diagnosis, prognosis, and prediction of treatment response in CLL patients.
Keywords Chronic lymphocytic leukemia, FISH, cytogenetic aberrations, next-generation
sequencing
ª 2013 Elsevier Inc. All rights reserved.

Chronic lymphocytic leukemia (CLL) is a hematological
malignancy with marked clinical heterogeneity, due in part to
the genetic alterations of the leukemic cells (1). CLL is the
Received October 8, 2012; received in revised form January 21,
2013; accepted January 24, 2013.
* Corresponding author.
E-mail address: jmhr@usal.es
most common adult leukemia in Western countries and
mainly affects individuals >50 years of age (2,3). Its main
morphological feature is the accumulation of small B lym-
phocytes with a mature appearance in blood, bone mar-
row, lymph nodes, or other lymphoid tissues (4). CLL has
a distinctive repertoire of immunological markers that allow it
to be differentiated from other B cell lymphomas (5e7).
Our understanding of CLL has changed over the last
decade. It was once thought to be a homogeneous disease,

2210-7762/$ - see front matter ª 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.cancergen.2013.01.003
50 A.E. Rodrı́guez-Vicente et al.

in which mature B cells accumulated largely due to a lack Table 1 Most common cytogenetic aberrations in CLL
of normal cell death; however, several reports have shown detected by conventional cytogenetics, FISH, and aCGH
that the high lymphocyte count in CLL patients is caused not CC-TPA CC-DSP30/IL-2 FISH aCGH
only by the prolonged survival, but also by proliferating cells Aberration (%)a or CD40L (%)b (%)c (%)d
(8e10). Moreover, CLL has recently been established as
13q total 5e25 14.3e57 30e62 40e59
a disease of remarkable diversity and differences in cell
13q biallelic <1 7 14e24 e
morphology, immunophenotype, cytogenetics, and molec-
ular characteristics have been identified. This heterogeneity Trisomy 12 20e25 12.8e14 12e30 7e25
translates into the clinical course and the response to treat- del(11q) 12e18 9.7e15.9 7e19 9e13
ment (11): Approximately one-third of patients survive for 20 del(17p) 7e12 3.7e7.5 4e9 4e11
years or longer and never require treatment (12) and, alter- Translocations 5e20 4e34 1e13e e
natively, some patients may progress rapidly from the time of del(6q) 5e8 4e6.8 3.4e6 5.5
diagnosis and experience complications such as autoim- Clonality 20e40 21.5e83 77e82 65.6e90
mune hemolytic anemia, thrombocytopenia, and infection. a
Cumulative data from 1,109 cases (17,21).
Moreover, 3e10% of cases can develop an aggressive ly- b
Cumulative data from 1,322 cases (19,22,94,98,194,195).
mphoma during the course of the disease, which was origi- c
Cumulative data from 1,485 cases (18,20,119,196,197).
nally described as “Richte
rs syndrome” (13,14). CLL may d
Cumulative data from 2,141 cases (31,33,34,42,43,45e47,49,
also undergo transformation to a prolymphocytic leukemia, 50,96,97,198e200).
e
with a worsening clinical outcome in most cases, which is Only IGH translocations.
frequently associated with abnormalities of p53 (15) and
sometimes of c-MYC (16). most common being trisomy 12 and abnormalities of chro-
A number of clinical and biological markers of prognostic mosome band 13q14 (17,21).
relevance have been identified. These include clinical char- Recently, improved culture methods have been introduced
acteristics (e.g., age, stage, sex, and performance status) as and CLL cells can be stimulated by alternative means, such as
well as laboratory parameters: lymphocyte count, bone marrow CD40 ligand-expressing cells and IL-4 or the addition of CpG-
infiltration pattern, and serum parameters such as lactate oligodeoxynucleotides and IL-2 to cultures (19). This approach
dehydrogenase (LDH), b2-microglobulin, and thymidine kinase revealed that translocations occurred in one-third of patients with
(TK). Recently, progress has been made in identifying markers CLL (19,22). After stimulation with CpG-oligodeoxynucleotides
related to the biology of the disease, such as chromosomal and IL-2, the observed aberration rate was comparable to the
abnormalities, the proportion of zeta-chain associated (TCR) rate detected by parallel interphase fluorescence in situ hybrid-
protein kinase 70 kDa (ZAP-70)-positive cells, somatic hyper- ization (FISH) (Table 1). These studies demonstrated that
mutation of the variable region of the immunoglobulin heavy additional cases of complex aberrations can also be detected. In
chain (IGHV), and IGHV-3-21 usage. A major scientific goal is a larger series of >500 patients, aberrations were detected in
to find a biomolecular explanation for the heterogeneity of CLL 83% of cases by chromosome banding after stimulation with
prognosis that can provide clues to understanding the disease CpG-oligonucleotide DSP30 and IL-2. These new cytogenetic
etiology and its pathogenic mechanisms. techniques could provide a deeper understanding of the disease
and the prognostic subgroups (23).
Chromosomal abnormalities in CLL
FISH
The presence of cytogenetic abnormalities is a hallmark of
CLL. Indeed, several studies have shown that the type and FISH has a higher resolution than standard cytogenetics and
number of chromosomal aberrations are an independent allows the detection of chromosomal aberrations, irre-
predictor of prognosis in CLL (17e20), and cytogenetic spective of the cell’s ability to divide (24); however, the
analysis is now routinely performed in this disease. Recently, information gained by FISH analysis is limited to the targets
chromosomal microarrays (CMAs) have been validated as of the selected probes. For this reason, the technique
reliable tools for investigating global genetic abnormalities in underestimates genomic complexity.
CLL at a higher resolution. FISH panels contain probes for the most commonly
altered regions, which can be identified in approximately 80%
Cytogenetic techniques of CLL cases (Table 1). The most common recurrent chro-
mosomal abnormalities include trisomy 12 as well as dele-
tions of 13q 11q, 17p, and 6q (18).
As early as 1990, genomic aberrations were associated with
FISH has also revealed that clonal evolution is common
prognosis in CLL using G-banding techniques (17), which
in CLL. Thus, long-term studies in several patient cohorts
examine the patient’s chromosomes in a sample of cells,
demonstrated that clonal evolution occurred in 20e40% of the
counting the number and evaluating their structural aberra-
patients and corresponded to disease progression (25e30).
tions. Conventional G-banding provides a whole-genome
perspective, but the diagnostic yield may be low in tumors,
such as CLL, with low mitotic activity. Longer culture dura- Microarray-based molecular genetic methods
tions were introduced and several stimulating agents, such
as TPA, PHA, LPS, and PWM, were added to the culture The recent developments of comparative genome hybrid-
medium; however, clonal chromosomal aberrations were ization (CGH) and high-resolution single nucleotide poly-
detected in approximately 40% of cases (Table 1), with the morphism (SNP) arrays have led to a better understanding of
Molecular heterogeneity in CLL 51

the genetic profile of CLL. Considering the great heteroge- Table 2 Recurrent genomic aberrations in >2.5% of CLL
neity of CLL from genetic and prognostic points of view, patients by aCGH
array-based karyotyping is a powerful tool for the analysis of Chromosome Cytoband %
genetic alterations in CLL, as it yields dramatically higher
resolution than conventional cytogenetics (Table 1), does not Losses
require cell culture, and avoids subjective interpretation of 2 q37.1 3.4
fluorescent signals. Limitations of the technology include the 3 p21 8.3
inability to detect balanced rearrangements and decreased 6 2.7
performance at low levels of tumor. Several studies that have 6 q21 7.1
compared microarray results to FISH results for CLL have 8p 1.5e7.3
shown a high concordance rate (31e35). 8 p23.1ep21.2 4.7
Array-based karyotyping can be done with several 8 p21.2ep12 3.4
platforms. The arrays can be genome-wide (probes 8 p21.2 4.7
distributed over the entire genome) or targeted (probes for 8 p11 6.25
specific genomic regions). Array-CGH (aCGH) using 9 p21.3 3.4
bacterial artificial chromosome (BAC) clones as probes on 10q 3.6e3.9
the array has been a widely applied method for analyzing 10 q24 6.25
hematological malignancies (36e39) because it allows for 11 q22 16.7
high-resolution genome-wide scanning to detect copy Atypical loss 11q 15.2
number alterations (CNAs) in a single hybridization. The 14q 3.5
resolution is basically limited only by the number and 14 q24 8.3
genomic distribution of the arrayed elements of the 14 q32 25.5
particular platform. 17 p12ep11.2 14.9
Microarray technology has already been used to detect 17 p13.3ep11.2 10.1
novel CLL-associated genomic aberrations in >2,000 pa- 18q 3.1e7.3
tients (Table 2) (32,40e43). These studies have not only
18 q23 3.4
allowed for the detection of CNAs, but have also yielded
22 q11 15.0
additional information about genome-wide alterations in this
disease (31e33,41e44). Several studies have demonstrated Gains
2p 2.9
the presence of new, cytogenetically cryptic, and recurrent
2 p25.3ep22.3 3.4
chromosomal changes, such as the gain of 2p and deletions
2 p22.3 3.4
of 8p, 18q, and 22q. Our group recently identified a novel
recurrent region of gain in CLL, which was located on 20q13 2 p16.2ep14 3.4
in 19% of CLL patients (45). Gains in 20q13 in CLL did not 2 p16 5.7
occur as a single aberration. In fact, gains on 20q were 8 q23.3eq24.3 1.7e2.5
associated with genomic complexity. Notably, genomic 10q 3.6
complexity has a significant impact on cancer prognosis, and Cumulative data from 2,141 patients.
studies have described the presence of several genomic
changes as predictors of disease progression and chemo-
sensitivity in CLL (46,47). In addition, great genomic com- but better survival for patients with trisomy 12, normal
plexity has been associated with worse survival and is also karyotype, and deletion 13q as the sole abnormality (114,
closely related to markers of poor prognosis (42,46e49). 111, and 133 months, respectively) (18). The current Inter-
SNP array analysis has also enabled further character- national Workshop on Chronic Lymphocytic Leukemia
ization of previously identified abnormalities, providing infor- guidelines consider assessment of genomic aberrations by
mation about the exact location of breakpoints and insight FISH mandatory in clinical trials and desirable in general
into recombination mechanisms (33,50). SNP arrays have practice as a pre-treatment evaluation (51).
the additional advantage of detecting copy number-neutral
loss of heterozygosity (cnLOH) (33) or acquired uniparental 13qL
disomy (aUPD).
Loss of 13q14.3 is the most common chromosome aberra-
Cytogenetic abnormalities in CLL tion in CLL, with a prevalence of 40e60% (18,20,52). In
contrast to other recurrent aberrations, 13q14 deletions may
No single genetic abnormality responsible for CLL develop- be heterozygous (monoallelic in 76% of cases) or homozy-
ment has been identified. Instead, the disease is character- gous (biallelic in 24% of cases). Studies of serial samples
ized by a variety of chromosomal abnormalities. The most suggest that heterozygous deletion is an early event,
common recurrent chromosomal abnormalities include whereas deletion of the second copy of this region occurs at
deletion 13q, trisomy 12, and deletions 11q, 17p, and 6q a later stage (28,53).
(18). A subdivision based on these aberrations is important, Patients with losses on 13q14 as the only aberration,
as they are predictors of disease outcome. Five prognostic based on FISH data (13qe only), show a favorable prog-
categories have been identified in a hierarchical model, nosis; however, recent studies indicate that the situation may
showing poor survival in patients with 17p deletion and 11q be more complex, suggesting that the percentage of inter-
deletion (median survival 32 and 79 months, respectively) phase nuclei with deletions, as well as the size of the
52
deletion, could influence the outcome. Our group demon-
strated that the number of malignant cells carrying the 13q
deletion is strongly correlated with disease outcome (20) and
molecular characteristics (54). Thus, two prognostic groups
can be established on the basis of the percentage of cells
with 13qe. The patients with a high proportion (>80%) of
13qe cells had a shorter overall survival (OS) than patients
with <80% 13qe cells, as well as a shorter time to first
therapy. These results have been corroborated by two other
studies (55,56). In addition, the extensive characterization of
the 13q14 deletions has demonstrated that the breakpoints
are heterogeneous and the deleted region can vary sub-
stantially in size, ranging from only w300 kbp up to >70 mbp
(49,50,57e59). These studies led to the identification of
a minimal deleted region (MDR) (59,60), distal to RB1, that
comprises the deleted in leukemia 2 (DLEU2) gene and the
microRNA (miR) 15a/16-1 cluster (61). Moreover, recent
analysis of the 13q14 deletion architecture by SNP arrays
suggests that the extent of the deletion contributes to
disease characteristics (50,57,62,63). Thus, two types of
13q14 deletions are proposed: del(13q) type I (short), which
breaks close to the miR16/15a locus and does not involve
RB1; and del(13q) type II (larger), which includes the RB1
locus and has been proposed to be associated with greater
genomic complexity and a more aggressive course. The
large 13q deletions are most often monoallelic, whereas
a minor proportion carries biallelic deletions. It remains to be
established whether the presence of a homozygous versus
a heterozygous 13q deletion plays an additional role.
Patients with a monoallelic del(13q) show lower lymphocyte
growth kinetics than patients with biallelic deletions (33);
however, recent reports suggest that heterozygosity does
not affect time to first treatment or OS (55,64).
Various groups have attempted to identify a tumor
suppressor gene in 13q. No inactivation of candidate genes
by mutation has been demonstrated, but a complex epige-
netic regulatory tumor suppressor mechanism that controls
the expression of the whole region has been described (65).
Functional evidence has long suggested an involvement of
the miR-15a/16-1 deletion in CLL development (66e68). The
miR-15a/16-1 cluster was deleted or down-regulated in the
majority of CLL cases (68%) and seems to negatively regu-
late the expression of BCL2 and several other genes
involved in proliferation and apoptosis (66,69,70). Subse-
quent murine studies in which the MDR or the specific
miR15a/16-1 cluster was deleted have led to the develop-
ment of monoclonal B cell lymphocytosis and CLL with low
penetrance (69).
Trisomy 12
Trisomy 12 is among the most frequent aberrations in CLL,
occurring in 10e20% of cases. It has long been associated
with early progression (18). Initial FISH studies have sug-
gested that the outcome was intermediate for this group.
Recent analysis of prospective trials suggests that although
progression-free survival (PFS) may be shorter, OS is
favorable (71). Cases with trisomy 12 rarely show TP53
mutations and rarely acquire these over timeda finding that
may partly explain the benign course after treatment (72).
Trisomy 12 has been associated with an atypical
morphology or immunophenotype (73). A minimal common
A.E. Rodrı́guez-Vicente et al.
gained region has been confined to 12q13, small duplications
of 12q have been reported, and the murine double-minute 2
gene (MDM2), which is located at 12q15, is amplified in CLL
(74). Moreover, the CLL up-regulated gene 1 (CLLU1), which
is located at 12q22, has been proposed as a prognostic
marker in patients <70 years of age, since its higher level of
expression has been associated with shorter OS (75),
although over-expression of CLLU1 occurs irrespective of
trisomy 12 (76). The critical genes involved in this aberration,
therefore, remain unknown.
11qe
Approximately one-fifth of patients with treatment indications
exhibit 11q deletions, and ATM mutations have been re-
ported in 30% of patients with del(11q). Patients with an 11q
deletion are generally younger, have more B-symptoms and
a more rapid progression of the disease, and shorter OS
(77,78). Furthermore, the aberration is typically associated
with extensive lymphadenopathy (51); however, it has been
recently reported that de novo 11q deleted CLLs can exhibit
variable clinical outcomes (79).
The minimal consensus region in bands 11q22.3eq23.1
harbors the ATM gene in almost all cases. The ATM protein
kinase is a central component of the DNA damage pathway
and mediates cellular responses to DNA double-strand
breaks (DSBs). ATM activates cell cycle checkpoints, can
induce apoptosis in response to DNA breaks, and functions
directly in the repair of DNA DSBs (80). The role of other
genes in 11q22eq23.1 remains unresolved, but there is
evidence of a gene dosage effect as well as other genes in
the region being involved.
17pe
Del(17p) has been described in 3e8% of patients who are
na€ıve to treatment, although higher occurrences of up to 30%
have been reported in patients with advanced, relapsed
disease (18,77,81). Most cases with del(17p) show loss of
one copy and mutation of the remaining copy (82e84).
This aberration is usually associated with a very aggres-
sive clinical course and is predictive of lower PFS, lack of
response to therapy, short response duration, and short OS
(18,85,86). The clinical relevance of the number of cells
displaying del(17p) has been demonstrated in CLL patients.
Thus, the presence of >20% of cells with loss of TP53 has
been associated with an adverse prognosis, whereas
patients with <20% of cells with loss of TP53 had a prog-
nosis similar to that of the global series (78,87).
The deletion always contains the TP53 locus but usually
covers most of the short arm of chromosome 17, which has
prompted a consideration of other genes that might be tar-
geted by the deletion. The tumor suppressor p53 plays an
essential role in inducing apoptosis or cell cycle arrest after
DNA damage. Therapy with fludarabine and alkylating
agents is based on a p53-dependent mechanism, which
could explain why CLL patients with del(17p) or inactivating
mutations of TP53 are refractory to such chemotherapy (52);
however, fludarabine refractoriness is caused by TP53
disruption in approximately 40% of CLL patients who did not
respond to treatment, but, in a sizeable fraction, the
Molecular heterogeneity in CLL
molecular basis of this aggressive clinical phenotype still
remains unclear (88e90).
6qe
Deletion of the long arm of chromosome 6 is a rare cytoge-
netic abnormality seen in approximately 6% of CLL patients
(18,91,92). It is generally considered an intermediate-risk
feature, since it is associated with more prominent lympho-
cytosis with atypical morphology, splenomegaly, higher rates
of CD38 positivity, and no association with IGHV mutation
status (91).
Other abnormalities
Several other recurrent genomic aberrations have been
described in CLL, such as total or partial trisomy 3, trisomy 8,
trisomy 18, and trisomy 19dchanges that lead to gains of
2p24e25, 3q26e27, and 8q24. Two cases of patients with
a deletion of the long arm of chromosome 5 (5qe) and no
other cytogenetic abnormalities have been reported; these
patients were Binet stage A at 18 and 28 months after
diagnosis, which suggests that this aberration may be related
to an indolent course (93). These aberrations are rare in CLL
and their prognostic significance is unknown.
Translocations
Recurrent balanced translocations are rare in CLL, in
contrast to other types of leukemia or B cell lymphoma in
which specific and recurrent translocations deregulate known
oncogenes (18,19,94). Translocations involving immuno-
globulin genes are the most common recurrent trans-
locations in CLL, although they have only been observed in
<5% of cases. Recurrent partners include BCL2, BCL3,
BCL11A, and c-MYC, although unknown partner genes are
also frequent (95). Chromosomal translocations generally
have a negative effect on response to therapy and survival,
especially when unbalanced. Unbalanced non-reciprocal
aberrations are frequent and often seen in complex
karyotypes.
Complex karyotype
Although CLL is characterized by a relatively stable genome
and most CLL cases appear to carry few genomic aberra-
tions, a high number of CNAs (
3 per patient), termed
genomic complexity, is detected in a proportion of CLL
patients (w20%). Genomic complexity demarcates a CLL
subset with progressive and aggressive disease, short
survival, and decreased therapeutic efficacy (46,96,97). Very
recent data have shown that genomic complexity evaluated
by conventional karyotyping with DSP30/IL-2 stimulation (98)
can be helpful in the evaluation of the outcome of patients
with a normal FISH panel.
New chromosomal abnormalities can be acquired by the
CLL clone during the course of disease, leading to the
concept of “clonal evolution” (26,53,99,100). Thus, as
subsets of leukemic cells acquire additional chromosomal
abnormalities, subclones with different cytogenetic profiles
and gain or loss of function of molecular machinery may
53
develop. These subclones may have a survival advantage
that results in a more aggressive disease. CLL patients
should have FISH and/or a stimulated karyotype evaluation
by a reputable laboratory at diagnosis as well as before the
initiation of therapy to evaluate for del(17p), del(11q), or
a complex karyotype that may alter treatment decisions.
Cytogenetics and treatment
The presence or absence of cytogenetic defects can also
be used to guide treatment strategy and to predict response
to treatment (101,102). Patients with 17p deletions or TP53
mutations are known to be refractory to purine analogs (85)
and not benefit substantially from front-line standard-of-care
treatment with fludarabine, cyclophosphamide and rituximab
(71). Treatment with alemtuzumab specially combined with
methylprednisolone is highly effective in these patients, alth-
ough they still have shorter survival than patients with lower-
risk cytogenetics (81,103,104). Therefore, allogeneic stem
cell transplantation should be offered to patients with del(17p)
who are physically fit, if they achieve a remission. Moreover,
new agents that act independently of p53 as several kinase
inhibitors to target the proximal B cell receptor signaling
pathway (e.g., spleen tyrosine kinase inhibitor [fostamatinib]
and Bruton’s tyrosine kinase inhibitors [ibrutinib and AVL-263];
cyclin-dependent kinase inhibitor [flavopiridol] and BCL2
inhibitors [ABT-263 and oblimersen]) are being tested in clin-
ical trials with very promising results (105,106).
Regarding other cytogenetic defects, the treatment
with fludarabine, cyclophosphamide and rituximab improves
significantly the previous results in all molecular genetic
alterations, mainly in patients with trisomy 12, possibly
because of their higher levels of expression of surface CD20
(107), and in patients with del(11q), an aberration previously
associated with a poor prognosis (71).
Cytogenetics and differential diagnosis
In the context of a well-established diagnosis of CLL, FISH
analysis is essentially performed for prognostic purposes;
however, it can also be useful in cases in which the diagnosis
is uncertain. FISH studies that investigate the presence of
the translocation t(11;14)(q13;q32) that leads to BCL1 rear-
rangement, or the t(14;18) with BCL2 rearrangement, are
also useful to distinguish CLL from mantle cell lymphoma and
follicular lymphoma, respectively (108). Although the t(11;14)
has been described in B-cell prolymphocytic leukemia, most
of these cases were probably mantle-cell lymphomas in the
leukemic phase (109,110).
Somatic mutations in CLL
IGHV mutations
Over a decade ago, the simultaneous publication of two
papers seemed to explain the heterogeneity that clinicians
had observed in the natural history of CLL (111,112). Both
studies mutually corroborated the finding that IGHV muta-
tional status identifies two subsets of CLL: Patients with
a >2% difference in nucleotide sequences from germline
54
cells had a mutated clone, and those with a difference of
2% had an unmutated clone (113). When patients with
Binet stage A disease were stratified according to their IGHV
gene mutational status, significant survival differences were
observed. The median survival was 8e9 years for patients
with non-mutated IGHV genes (germline cells) but >24 years
for patients with somatically mutated IGHV genes. These
reports also noted a relationship between non-mutated IGHV
genes and the need for chemotherapy (111), response to
chemotherapy (110), and unfavorable cytogenetic abnor-
malities detected by FISH (112).
Somatic mutations of the variable gene region of the
heavy chain of immunoglobulins are present in approxi-
mately one-half of all CLL cases. Unmutated cases originate
from cells that probably underwent their final development
before entering the germinal center, whereas mutated CLL
cells probably transited the germinal center first and then
underwent final transformation. In any case, the cellular
origin of CLL remains unknown.
Although the biological significance of IGHV gene muta-
tion status is unclear (114), the prognostic significance of
IGHV mutation status has been confirmed (115e119) and
clinical trial data confirm the impact of IGHV mutation on
outcome (78,120). The value of IGHV status to predict
response to therapy has not been fully investigated. CLL
patients with non-mutated IGHV genes have a higher risk of
relapse after stem cell transplantation (121), and it has been
shown that Richter’s transformation occurs almost exclu-
sively in this group of patients (28,122). Additionally, unmu-
tated IGHV patients are also more likely to undergo clonal
evolution, with a greater tendency to acquire poor prognostic
cytogenetic abnormalities. IGHV mutational status and
cytogenetic abnormalities identified by FISH have a major
impact on the survival of patients with CLL, but although
cytogenetic changes during the course of the disease are
relatively common, IGHV mutational status remains constant
over time. Unfavorable aberrations (11qe, 17pe) occur more
frequently in IGHV-unmutated people, whereas favorable
aberrations (13q single) are more frequent in the IGHV-
mutated subgroup (116,119,123). This unbalanced distribu-
tion of genomic aberrations emphasizes the different
biological background of the CLL subgroups with mutated
or unmutated IGHV, but only partly explains their different
clinical course. Approximately two-thirds of the IGHV-
unmutated CLL cases have no unfavorable genomic aber-
rations, indicating a differential influence of these factors.
Work involving sequence comparison and assignment
to specific IGHV family genes has revealed additional
complexity. Irrespective of mutational status, some heavy
chain variable regions are associated with specific clinical
features and varying occurrences from country to country
(124). For example, IGHV3-21 usage is associated with
a worse prognosis and is less prevalent in southern European
countries. By contrast, patients expressing the IGHV3-72
gene have a favorable immunophenotypic profile (CD38e
and ZAP-70-negative) and a stable disease course (125,126),
whereas use of IGHV3-72 and IGHV3-30 indicate good clin-
ical outcomes, including spontaneous regression in anecdotal
cases (127). Moreover, the involvement of the IGHV1-69
family and the use of IGHV4-39 occur mainly in unmutated
cases, whereas IGHV4-34 and most cases of IGHV3 contain
mutated cases. Despite the advantages of this robust
A.E. Rodrı́guez-Vicente et al.
prognostic marker, determining the mutational status of the
IGHV gene can be laborious and requires certain laboratory
equipment. Therefore, several alternative biomarkers have
been explored as potential surrogate markers for patient
prognosis. CD38 expression on leukemic lymphocytes was
the first marker that was found to correlate with IGHV muta-
tions (111). Eventually, however, it was found that the rela-
tionship is not absolute and that, according to some studies,
CD38 expression may vary over time (112,128). CD38
expression and IGHV mutations are independent prognostic
factors.
Gene expression profiling demonstrated a distinct pattern
for CLL mutated and unmutated subtypes, with ZAP-70
standing out as the gene that most stringently separated the
subsets (129). Thus, the measurement of ZAP-70 expres-
sion levels was suggested as a surrogate marker for the
IGHV mutation status and this was further verified in
subsequent studies (130e132). The majority of mutated
cases are ZAP-70-negative, whereas unmutated forms are
ZAP-70-positive. Higher ZAP-70 expression predicts cases
with unfavorable clinical courses in terms of disease
progression and OS (130e132). In fact, ZAP-70 protein ex-
pression in CLL patients appears to have more predictive
value than IGHV mutations, and its expression level appears
to be constant during the course of disease (133). Never-
theless, ZAP-70 determination remains to be appropriately
standardized by flow cytometry.
Mutations of key tumor suppressor genes in CLL
TP53 mutations have been described in 4e12% of patients
with untreated CLL. Approximately 80e90% of cases with
a deletion of one copy of the TP53 locus will have a TP53
mutation on the remaining copy (82e84,134,135). Thus, very
few cases with a 17p deletion will have a functional p53
pathway. TP53 mutations are more prevalent in progressive
and refractory CLL (135). A TP53 mutation is an independent
predictor of poor prognosis and confers even shorter OS
than del(17p) in the absence of a TP53 mutation (83).
Mutations of the ATM gene may also have prognostic
implications independent of those associated with the dele-
tion of chromosome 11q. ATM gene mutations have been
described in approximately 12% of patients with CLL and
30% of patients with 11q deletion (136,137), and compared
with the ATM/TP53 wild-type, have a more aggressive
course and are more resistant to traditional chemothera-
peutic agents (136). Germline mutations in the ATM gene
have also been described in CLL patients, and these are
thought to be a predisposing factor for developing CLL (138).
Next-generation sequencing
Next-generation sequencing (NGS) techniques have pro-
vided a better knowledge of the genetic complexity and
heterogeneity of CLL. Whole genome sequencing has
provided new insights into the mutational status of the
disease, involving several genes previously unrelated to CLL
(Table 3) (139e141). Two unexpected pathways have been
found to be recurrently mutated in CLL, and indicate that
activated NOTCH1 signaling (139,142) and defects in the
Molecular heterogeneity in CLL
Table 3 Comparative analysis of the genes recurrently mutated in CLL by NGS
Gene Mutation
NOTCH1 (9q34) P2515Rfs*4
Q2503*
F2482Ffs*2
SF3B1 (2q33) Exon 14
Exon 15 (p.Lys700Glu)
Exon 16
POT1 (7q31-33) Exons 4e7
CHD2 (15q26) Exons 16, 17, 27, 30, 35, 36
LRP1B (2q21.2) Exons 41, 46, 59, 62, 86
MYD88 (3p22) L265P
XPO1 (2p15) E571K
E571G
TP53 (17p13) Exons 4e9
ATM (11q22) Scattered along the gene
splicing machinery (140,141) play a prominent role in the
development of specific subsets of CLL.
Activation of NOTCH1 in leukemia was first discovered
through the analysis of the chromosomal translocation
t(7;9)(q34;q34.3) in patients with T cell acute lymphoblastic
leukemia (T-ALL). Later, activating mutations in NOTCH1
were discovered in >50% of T-ALL patients. In CLL, Notch
signaling plays a critical role in CLL cell survival and apoptosis
resistance, due to its constitutive activation (143). To our
knowledge, NOTCH1 mutations occur in approximately 10%
of CLLs at diagnosis, with a higher frequency in advanced
disease such as in Richter’s syndrome (139,142). The muta-
tions mostly affect the functional PEST domain of NOTCH1,
leading to enhanced stability of the protein and to differential
gene expression patterns of target genes in CLL patients
(139). Most NOTCH1-mutated cases seem to carry unmu-
tated IGHV genes and are associated with poor prognoses
(139,141,144). Subsequent studies have demonstrated that
NOTCH1 mutations are an independent predictor of shorter
OS in CLL (145). NOTCH1 mutations have been significantly
associated with trisomy 12 (145,146) and they were particu-
larly common in cases harboring trisomy 12 as the sole genetic
abnormality (147). A recent study has reported that the
distribution of NOTCH1 mutations in CLL with trisomy 12 is
heterogeneous and that the presence of additional chromo-
somal abnormalities, such as trisomy 18, could modify the
prognoses of these patients (148). Interestingly, in addition to
NOTCH1 mutations, an exome sequencing study of 91 CLL
cases also identified mutations in FBXW7, a negative regu-
lator of NOTCH1 (141). These mutations were also associated
with trisomy 12, although the NOTCH1 and FBXW7 mutations
were present in independent samples, suggesting that they
may lead to aberrant Notch signaling in patients with trisomy
12 and unmutated IGHV. The possibility of targeting NOTCH1
with drugs currently under development in other clinical
contexts reinforces the relevance of these mutations (149).
Mutations in the splice factor SF3B1, a gene also
frequently mutated in myelodysplastic syndromes (MDS)
(150), were found in 9.8% of CLLs at diagnosis (140) and
15% of untreated and treated cases (Table 3) (141). SF3B1
somatic mutations are missense mutations clustered in hot-
spot codons 662, 666, and 700. These mutations are more
55
No. of mutations (%)
Puente et al., 2011 Quesada et al., 2011 Wang et al., 2011
31/255 (12.2) 4/91 (4)
27/279 (9.7) 14/91 (15)
5/105 (4.8) 0
5/105 (4.8) 0
5/105 (4.8) 0
9/310 (2.9) 9/91 (10)
4/165 (2.4) 0
0 15/91 (15)
0 9/91 (9)
frequent in later stages of CLL, are associated with markers
of poor clinical outcome (e.g., ZAP-70 expression), and
predict poor prognosis. Intriguingly, SF3B1 mutations seem
to confer a more favorable prognosis in MDS and are
associated with refractory anemia with ring sideroblasts. In
CLL, SF3B1 mutations have been associated with del(11q)
and unmutated IGHV status, but they were also detected in
samples with mutated IGHV, which suggests that it is an
independent risk factor for CLL (141). It is worth noting
that the investigation of the coding genome of fludarabine-
refractory CLL to identify genetic lesions associated with
chemorefractoriness has revealed recurrent mutations of
SF3B1 in 17% of CLL patients (151).
Recently, inactivating mutations in BIRC3 have also been
reported in fludarabine-refractory CLLs (152). These muta-
tions, together with BIRC3 deletions, are recurrently and
selectively associated with TP53 wild-type, although they
identified patients with survival similar to that of patients with
TP53 abnormalities.
The identification of SF3B1 and BIRC3 mutations in this
subset of patients helps elucidate the molecular basis of
a fraction of high-risk CLL patients, which remains unclear
given the absence of TP53 alterations. These genes may be
molecular markers for the early identification of chemo-
refractoriness among CLL patients.
By using a sequence capture method, recent reports have
confirmed the involvement of the B cell receptor signal in CLL
and defined the presence of mutations in the KRAS,
SMARCA2, NFKBIE, and PRKD3 genes. These findings
support the idea that B cell receptor signaling and related
pathways in CLL cells may depend on somatic mutations, at
least in part (153). Moreover, whole genome sequencing has
been applied to study the heterogeneity of clonal evolution in
CLL (154), where it has demonstrated the presence of
molecular changes (mutations and cytogenetics) in pre-
treatment, post-treatment, and relapse samples from three
CLL patients. The analysis enabled the identification of
a founder subclone in each patient who had a specific muta-
tional profile and defined the genetic composition of subclones
that later became dominant, even before initiation or relapse
treatment. This study also identified heterogeneous patterns of
clonal evolution in CLL based on the somatic mutation profiles.
56
NGS therefore appears to be a highly effective technique
for identifying new genetic lesions. It seems likely that future
studies will improve our understanding of disease onset and
evolution.
Epigenetic modifications
microRNAs
MicroRNAs (miRNAs or miRs) are non-coding RNA mole-
cules (w22 nucleotides) that have the capacity for simulta-
neous regulation of tens to hundreds of genes through direct
targeting of the untranslated regions (UTR) (155e157). The
first description of an miRNA associated with cancer was
reported in CLL. Calin et al. showed that the miR-15a/16-1
cluster was located in 13q14.3 and was either deleted or
down-regulated in 68% of patients with CLL. Further studies
found that these miRNAs target the mRNA encoding the anti-
apoptotic protein B-CLL/lymphoma 2 (BCL2), the up-
regulation of which is critical for CLL cell survival (70).
Importantly, the direct interaction of miR-15/miR-16 with
BCL2 transcripts delayed protein translation, induced
apoptosis, and reinforced the role of miRNAs as part of
a new class of tumor suppressor genes.
CLL has also been classified by miRNA expression
profiling (61,158e162). Interestingly, none of these miRNA
expression profiles for CLL are identical. These miRNA
signatures could act as surrogate prognostic biomarkers in
CLL, as the expression levels of these miRNAs correlate with
previously established prognostic markers such as IGHV
mutation status or ZAP-70. A recent study also demonstrated
the decrease of miR-29c and miR-223 levels in cells during
the progression of the disease (163). Over-expression of
miR-21 and low miR-181b expression have been reported as
unfavorable prognostic factors independent of other clinico-
pathologic factors (164). Interestingly, miR-181a expression
correlated with a shortened time to treatment as well as
distinguished aggressive from indolent disease in 17p dele-
tion cases (165).
The miR-34 family has been also implicated in CLL. MiR-
34a expression partially correlated with TP53 status, and
patients with TP53 mutations or 17pe in general showed
lower miR-34a expression; however, the link between TP53
mutation/deletion status and miR-34a has proven to be much
more complex, and further studies are required. Interestingly,
it was recently reported that the recurring deletion hotspots at
13q, 11q, and 17p actually represent nodes of a complex
regulatory network in CLL that integrates the miR-15a/miR-
16-1 and miR-34b/miR-34c clusters with the tumor sup-
pressor TP53 (166).
Given the relationship between cytogenetic abnormalities
in CLL and miRNA deregulation, miRNAs have become
attractive candidates as both biomarkers and therapeutic
targets for CLL. The interaction among miRNAs, target
genes, and pathways in CLL is clearly complex, as are the
links between genotype and phenotype. The rapid advan-
cement of miRNA research in just the past few years
suggests that the roles of many other miRNAs in CLL have
yet to be discovered. Thus, miRNAs remain one of the most
exciting new avenues for CLL research.
A.E. Rodrı́guez-Vicente et al.
Methylation
DNA from CLL patients is globally hypomethylated when
compared with DNA from peripheral blood mononuclear cells
from healthy individuals (167e169); however, in CLL the
expression of tumor suppressor genes is commonly silenced
by regional hypermethylation of gene promoters (170e172).
Gene-specific hypomethylation events have also been
described in CLL (173e175).
Aberrant methylation has been described for genes
that are specifically deregulated in CLL. Moreover, studies
involving epigenetic aberrations have accelerated the search
for affected genes in CLL, which was initially restricted to the
commonly deleted chromosomal regions. Many novel genes
that are epigenetically silenced in CLL have been identified,
such as TWIST-2, a transcription factor and well-known
silencer of p53 function in other malignancies, which was
shown to be methylated predominantly in IGHV-mutated
patients (176). In this group of CLLs, DNA methylation of
CD38 and BTG4 has been correlated with a favorable
outcome, whereas DNA methylation of HOXA4 has been
associated with poor outcome (171). Interestingly, the
expression levels of ZAP-70 correlate closely with the me-
thylation of specific CpG islands in the gene (177). Recently,
a comprehensive quantitative DNA methylation analysis of
the ZAP-70 gene allowed for the identification of important
regions responsible for its transcriptional regulation (178),
which may make this prognostic marker more clinically
relevant in the future. The evidence of down-regulation of the
death-associated protein kinase 1 (DAPK1, which is involved
in apoptotic cell death regulation) gene through promoter
CpG methylation in CLL indicate that both genetic and
epigenetic factors may define both the sporadic and inherited
forms of this disease (179). Of note, methylation of the
p16INK4A (180) and p15INK4B (181) genes has also been
described in a small proportion of CLL patients; however, the
clinical significance from these small studies is yet undefined.
Although the roles of aberrant DNA methylation and
individual DNA promoter methylation have been investigated
in CLL, studies on genome-wide epigenetic modifications are
limited. With the advent of higher-resolution microarray and
sequencing technologies, comprehensive studies dedicated
to uncover the methylation status on a global level will
provide new data on this field (182e184). Overall, DNA
methylation scores have been indicated as strong indepen-
dent predictors of CLL progression (182), and differences in
global methylation profiles between prognostic subsets of
CLL have been reported (184). Of note, a very recent study
integrates whole epigenome and genome sequencing of CLL
patients (183).
Interestingly, epigenetic regulation is likely to have a role
in altered miRNA expression in CLL (185,186). For instance,
it has been reported that down-regulation of DLEU2 and miR-
15a/16-1 expression in CLL cases without a 13q14 deletion
(61) could be explained by suppressive epigenetic mecha-
nisms (187). PLAG1, a putative oncogene in CLL, is over-
expressed due to miRNA deregulation and an inactivation
of miR-124-1 (188e190). Moreover, some members of the
miR-29 family also target the de novo DNA methyltran-
sferases (DNMTs) and can reactivate tumor suppressor
genes (191). Thus, loss of miR-29 family members could
Molecular heterogeneity in CLL
cause epigenetic changes associated with CLL and other
cancer types.
Epigenetic modifications are an attractive therapeutic
target because they are reversible. Several compounds,
including histone deacetylase inhibitors (HDACi), have been
tested on CLL cells in vitro and in clinical studies (192). To
date, however, targeting acetylation with HDACi as a mono-
therapy has shown modest activity (193).
Conclusions
CLL is a heterogeneous disease with a marked variable
clinical course, with survival ranging from months to de-
cades. The disease is also characterized by the presence of
a genetic heterogeneity that is becoming apparent through
studies of chromosomal alterations, the immunoglobulin
heavy chain gene, miRNA deregulation, and genetic lesions
identified by whole genome sequencing. Furthermore,
a strong relationship exists between specific genetic aber-
rations and the clinical course of CLL. Specifically, the
expression of unmutated immunoglobulin genes, some
cytogenetic abnormalities such as 17p and 11q deletions,
and the expression of ZAP-70 are associated with poor
prognoses. Mutations of different tumor suppressor genes,
such as TP53 and ATM, are associated with a more ag-
gressive course and resistance to traditional chemothera-
peutic agents. Therefore, genetic studies are mandatory in
the initial evaluation of CLL patients. More recently, emerging
evidence implies microRNAs and methylation alterations in
the pathogenesis of CLL. Thus, future research in these
fields should bring further insights into mechanisms of the
origin and development of the disease.
Acknowledgment
This work was partially supported by grants from the Spanish
Fondo de Investigaciones Sanitarias 02/1041, FIS 09/01543,
and PI12/00281; Caja de Burgos-Banca Cı́vica, Proyectos
de Investigacio
n del SACYL 106/A/06; and by the Accio
n
Transversal del Ca
ncer project, through an agreement
among the Instituto de Salud Carlos III (ISCIII), the Spanish
Ministry of Science and Innovation, the Cancer Research
Foundation of Salamanca University, and the Redes de
Investigacio
n RTIIC (FIS). Ana E. Rodrı́guez-Vicente was
fully supported by an Ayuda Predoctoral FIS de Formacio
n
en Investigacio
n by the Spanish Fondo de Investigaciones
Sanitarias.
References
1. Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic
leukemia. N Engl J Med 2005;352:804e815.
2. Rozman C, Montserrat E. Chronic lymphocytic leukemia.
N Engl J Med 1995;333:1052e1057.
3. Zwiebel JA, Cheson BD. Chronic lymphocytic leukemia:
staging and prognostic factors. Semin Oncol 1998;25:42e59.
4. Watson L, Wyld P, Catovsky D. Disease burden of chronic
lymphocytic leukaemia within the European Union. Eur J
Haematol 2008;81:253e258.
5. Dighiero G, Hamblin TJ. Chronic lymphocytic leukaemia.
Lancet 2008;371:1017e1029.
57
6. Matutes E, Owusu-Ankomah K, Morilla R, et al. The immu-
nological profile of B-cell disorders and proposal of a scoring
system for the diagnosis of CLL. Leukemia 1994;8:
1640e1645.
7. Matutes E, Morilla R, Owusu-Ankomah K, et al. The immuno-
phenotype of splenic lymphoma with villous lymphocytes and
its relevance to the differential diagnosis with other B-cell
disorders. Blood 1994;83:1558e1562.
8. Chiorazzi N. Cell proliferation and death: forgotten features of
chronic lymphocytic leukemia B cells. Best Pract Res Clin
Haematol 2007;20:399e413.
9. Messmer BT, Messmer D, Allen SL, et al. In vivo measure-
ments document the dynamic cellular kinetics of chronic
lymphocytic leukemia B cells. J Clin Invest 2005;115:755e764.
10. Sieklucka M, Pozarowski P, Bojarska-Junak A, et al. Apoptosis
in B-CLL: the relationship between higher ex vivo spontaneous
apoptosis before treatment in III-IV Rai stage patients and poor
outcome. Oncol Rep 2008;19:1611e1620.
11. Wierda WG, O’Brien S, Wang X, et al. Prognostic nomogram
and index for overall survival in previously untreated pa-
tients with chronic lymphocytic leukemia. Blood 2007;109:
4679e4685.
12. Dighiero G, Binet JL. When and how to treat chronic lympho-
cytic leukemia. N Engl J Med 2000;343:1799e1801.
13. Giles FJ, O’Brien SM, Keating MJ. Chronic lymphocytic
leukemia in (Richter’s) transformation. Semin Oncol 1998;25:
117e125.
14. Cuneo A, De AC, Roberti MG, et al. Richter’s syndrome
in a case of atypical chronic lymphocytic leukaemia with the
t(11;14)(q13;q32): role for a p53 exon 7 gene mutation. Br J
Haematol 1996;92:375e381.
15. Hercher C, Robain M, Davi F, et al. A multicentric study of 41
cases of B-prolymphocytic leukemia: two evolutive forms. Leuk
Lymphoma 2001;42:981e987.
16. Lens D, Coignet LJ, Brito-Babapulle V, et al. B cell prolym-
phocytic leukaemia (B-PLL) with complex karyotype and
concurrent abnormalities of the p53 and c-MYC gene.
Leukemia 1999;13:873e876.
17. Juliusson G, Oscier DG, Fitchett M, et al. Prognostic subgroups
in B-cell chronic lymphocytic leukemia defined by specific
chromosomal abnormalities. N Engl J Med 1990;323:720e724.
18. Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberra-
tions and survival in chronic lymphocytic leukemia. N Engl J
Med 2000;343:1910e1916.
19. Mayr C, Speicher MR, Kofler DM, et al. Chromosomal trans-
locations are associated with poor prognosis in chronic
lymphocytic leukemia. Blood 2006;107:742e751.
20. Hernandez JA, Rodriguez AE, Gonzalez M, et al. A high
number of losses in 13q14 chromosome band is associated
with a worse outcome and biological differences in patients with
B-cell chronic lymphoid leukemia. Haematologica 2009;94:
364e371.
21. Hernandez JM, Mecucci C, Criel A, et al. Cytogenetic analysis
of B cell chronic lymphoid leukemias classified according to
morphologic and immunophenotypic (FAB) criteria. Leukemia
1995;9:2140e2146.
22. Dicker F, Schnittger S, Haferlach T, et al. Immunostimulatory
oligonucleotide-induced metaphase cytogenetics detect chro-
mosomal aberrations in 80% of CLL patients: a study of 132
CLL cases with correlation to FISH, IgVH status, and CD38
expression. Blood 2006;108:3152e3160.
23. Zenz T, Mertens D, Kuppers R, et al. From pathogenesis to
treatment of chronic lymphocytic leukaemia. Nat Rev Cancer
2010;10:37e50.
24. Lichter P, Cremer T, Borden J, et al. Delineation of individual
human chromosomes in metaphase and interphase cells by in
situ suppression hybridization using recombinant DNA libraries.
Hum Genet 1988;80:224e234.
58
25. Finn WG, Kay NE, Kroft SH, et al. Secondary abnormalities of
chromosome 6q in B-cell chronic lymphocytic leukemia:
a sequential study of karyotypic instability in 51 patients. Am J
Hematol 1998;59:223e229.
26. Fegan C, Robinson H, Thompson P, et al. Karyotypic evolution
in CLL: identification of a new sub-group of patients with
deletions of 11q and advanced or progressive disease.
Leukemia 1995;9:2003e2008.
27. Cuneo A, Bigoni R, Rigolin GM, et al. Late appearance of the
11q22.3-23.1 deletion involving the ATM locus in B-cell chronic
lymphocytic leukemia and related disorders. Clinico-biological
significance. Haematologica 2002;87:44e51.
28. Stilgenbauer S, Sander S, Bullinger L, et al. Clonal evolution
in chronic lymphocytic leukemia: acquisition of high-risk genomic
aberrations associated with unmutated VH, resistance to therapy,
and short survival. Haematologica 2007;92:1242e1245.
29. Stilgenbauer S, Bullinger L, Lichter P, et al. Genetics of chronic
lymphocytic leukemia: genomic aberrations and V(H) gene
mutation status in pathogenesis and clinical course. Leukemia
2002;16:993e1007.
30. Berkova A, Zemanova Z, Trneny M, et al. Clonal evolution in
chronic lymphocytic leukemia studied by interphase fluores-
cence in-situ hybridization. Neoplasma 2009;56:455e458.
31. Lehmann S, Ogawa S, Raynaud SD, et al. Molecular allelo-
karyotyping of early-stage, untreated chronic lymphocytic
leukemia. Cancer 2008;112:1296e1305.
32. Schwaenen C, Nessling M, Wessendorf S, et al. Automated
array-based genomic profiling in chronic lymphocytic leukemia:
development of a clinical tool and discovery of recurrent
genomic alterations. Proc Natl Acad Sci U S A 2004;101:
1039e1044.
33. Pfeifer D, Pantic M, Skatulla I, et al. Genome-wide analysis of
DNA copy number changes and LOH in CLL using high-density
SNP arrays. Blood 2007;109:1202e1210.
34. Gunn SR, Mohammed MS, Gorre ME, et al. Whole-genome
scanning by array comparative genomic hybridization as
a clinical tool for risk assessment in chroniclymphocytic
leukemia. J Mol Diagn 2008;10:442e451.
35. Sargent R, Jones D, Abruzzo LV, et al. Customized oligonu-
cleotide array-based comparative genomic hybridization as
a clinical assay for genomic profiling of chronic lymphocytic
leukemia. J Mol Diagn 2009;11:25e34.
36. de Leeuw RJ, Davies JJ, Rosenwald A, et al. Comprehensive
whole genome array CGH profiling of mantle cell lymphoma
model genomes. Hum Mol Genet 2004;13:1827e1837.
37. Kohlhammer H, Schwaenen C, Wessendorf S, et al. Genomic
DNA-chip hybridization in t(11;14)-positive mantle cell
lymphomas shows a high frequency of aberrations and allows
a refined characterization of consensus regions. Blood 2004;
104:795e801.
38. Rubio-Moscardo F, Climent J, Siebert R, et al. Mantle-cell
lymphoma genotypes identified with CGH to BAC microarrays
define a leukemic subgroup of disease and predict patient
outcome. Blood 2005;105:4445e4454.
39. Tyybakinoja A, Saarinen-Pihkala U, Elonen E, et al. Amplified,
lost, and fused genes in 11q23-25 amplicon in acute myeloid
leukemia, an array-CGH study. Genes Chromosomes Cancer
2006;45:257e264.
40. Tyybakinoja A, Vilpo J, Knuutila S. High-resolution oligonucle-
otide array-CGH pinpoints genes involved in cryptic losses in
chronic lymphocytic leukemia. Cytogenet Genome Res 2007;
118:8e12.
41. Patel A, Kang SH, Lennon PA, et al. Validation of a targeted
DNA microarray for the clinical evaluation of recurrent abnor-
malities in chronic lymphocytic leukemia. Am J Hematol 2008;
83:540e546.
42. Grubor V, Krasnitz A, Troge JE, et al. Novel genomic alter-
ations and clonal evolution in chronic lymphocytic leukemia
A.E. Rodrı́guez-Vicente et al.
revealed by representational oligonucleotide microarray anal-
ysis (ROMA). Blood 2009;113:1294e1303.
43. Gunn SR, Bolla AR, Barron LL, et al. Array CGH analysis of
chronic lymphocytic leukemia reveals frequent cryptic mono-
allelic and biallelic deletions of chromosome 22q11 that include
the PRAME gene. Leuk Res 2009;33:1276e1281.
44. Forconi F, Rinaldi A, Kwee I, et al. Genome-wide DNA analysis
identifies recurrent imbalances predicting outcome in chronic
lymphocytic leukaemia with 17p deletion. Br J Haematol 2008;
143:532e536.
45. Rodriguez AE, Robledo C, Garcia JL, et al. Identification of
a novel recurrent gain on 20q13 in chronic lymphocytic
leukemia by array CGH and gene expression profiling. Ann
Oncol 2012.
46. Kujawski L, Ouillette P, Erba H, et al. Genomic complexity
identifies patients with aggressive chronic lymphocytic
leukemia. Blood 2008;112:1993e2003.
47. Kay NE, Eckel-Passow JE, Braggio E, et al. Progressive but
previously untreated CLL patients with greater array CGH
complexity exhibit a less durable response to chemo-
immunotherapy. Cancer Genet Cytogenet 2010;203:161e168.
48. Kipps TJ. Genomic complexity in chronic lymphocytic leukemia.
Blood 2008;112:1550.
49. Gunnarsson R, Mansouri L, Isaksson A, et al. Array-based
genomic screening at diagnosis and during follow-up in chronic
lymphocytic leukemia. Haematologica 2011;96:1161e1169.
50. Ouillette P, Erba H, Kujawski L, et al. Integrated genomic
profiling of chronic lymphocytic leukemia identifies subtypes of
deletion 13q14. Cancer Res 2008;68:1012e1021.
51. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the
diagnosis and treatment of chronic lymphocytic leukemia:
a report from the International Workshop on Chronic Lympho-
cytic Leukemia updating the National Cancer Institute-Working
Group 1996 guidelines. Blood 2008;111:5446e5456.
52. Van BF, Verhasselt B, Philippe J. Prognostic markers in chronic
lymphocytic leukemia: a comprehensive review. Blood Rev
2009;23:25e47.
53. Shanafelt TD, Witzig TE, Fink SR, et al. Prospective evaluation
of clonal evolution during long-term follow-up of patients with
untreated early-stage chronic lymphocytic leukemia. J Clin
Oncol 2006;24:4634e4641.
54. Rodriguez AE, Hernandez JA, Benito R, et al. Molecular
characterization of chronic lymphocytic leukemia patients with
a high number of losses in 13q14. PLoS One 2012;7:e48485.
55. Van Dyke DL, Shanafelt TD, Call TG, et al. A comprehensive
evaluation of the prognostic significance of 13q deletions in
patients with B-chronic lymphocytic leukaemia. Br J Haematol
2010;148:544e550.
56. Dal BM, Rossi FM, Rossi D, et al. 13q14 deletion size and
number of deleted cells both influence prognosis in chronic
lymphocytic leukemia. Genes Chromosomes Cancer 2011;50:
633e643.
57. Mosca L, Fabris S, Lionetti M, et al. Integrative genomics
analyses reveal molecularly distinct subgroups of B-cell chronic
lymphocytic leukemia patients with 13q14 deletion. Clin Cancer
Res 2010;16:5641e5653.
58. Bullrich F, Fujii H, Calin G, et al. Characterization of the 13q14
tumor suppressor locus in CLL: identification of ALT1, an
alternative splice variant of the LEU2 gene. Cancer Res 2001;
61:6640e6648.
59. Liu Y, Corcoran M, Rasool O, et al. Cloning of two candidate
tumor suppressor genes within a 10 kb region on chromosome
13q14, frequently deleted in chronic lymphocytic leukemia.
Oncogene 1997;15:2463e2473.
60. Migliazza A, Bosch F, Komatsu H, et al. Nucleotide sequence,
transcription map, and mutation analysis of the 13q14 chro-
mosomal region deleted in B-cell chronic lymphocytic leukemia.
Blood 2001;97:2098e2104.
Molecular heterogeneity in CLL
61. Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions
and down-regulation of micro-RNA genes miR15 and miR16 at
13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S
A 2002;99:15524e15529.
62. Parker H, Rose-Zerilli MJ, Parker A, et al. 13q deletion anatomy
and disease progression in patients with chronic lymphocytic
leukemia. Leukemia 2011;25:489e497.
63. Hanlon K, Ellard S, Rudin CE, et al. Evaluation of 13q14 status in
patients with chronic lymphocytic leukemia using single nucleotide
polymorphism-based techniques. J Mol Diagn 2009;11:298e305.
64. Garg R, Wierda W, Ferrajoli A, et al. The prognostic difference
of monoallelic versus biallelic deletion of 13q in chronic
lymphocytic leukemia. Cancer 2012;118:3531e3537.
65. Mertens D, Wolf S, Tschuch C, et al. Allelic silencing at the
tumor-suppressor locus 13q14.3 suggests an epigenetic tumor-
suppressor mechanism. Proc Natl Acad Sci U S A 2006;103:
7741e7746.
66. Calin GA, Cimmino A, Fabbri M, et al. MiR-15a and miR-16-1
cluster functions in human leukemia. Proc Natl Acad Sci U S
A 2008;105:5166e5171.
67. Garzon R, Calin GA, Croce CM. MicroRNAs in Cancer. Annu
Rev Med 2009;60:167e179.
68. Raveche ES, Salerno E, Scaglione BJ, et al. Abnormal
microRNA-16 locus with synteny to human 13q14 linked to CLL
in NZB mice. Blood 2007;109:5079e5086.
69. Klein U, Lia M, Crespo M, et al. The DLEU2/miR-15a/16-1
cluster controls B cell proliferation and its deletion leads
to chronic lymphocytic leukemia. Cancer Cell 2010;17:
28e40.
70. Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16
induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S
A 2005;102:13944e13949.
71. Hallek M, Fischer K, Fingerle-Rowson G, et al. Addition of rit-
uximab to fludarabine and cyclophosphamide in patients with
chronic lymphocytic leukaemia: a randomised, open-label,
phase 3 trial. Lancet 2010;376:1164e1174.
72. Zenz T, Vollmer D, Trbusek M, et al. TP53 mutation profile in
chronic lymphocytic leukemia: evidence for a disease specific
profile from a comprehensive analysis of 268 mutations.
Leukemia 2010;24:2072e2079.
73. Matutes E, Oscier D, Garcia-Marco J, et al. Trisomy 12 defines
a group of CLL with atypical morphology: correlation between
cytogenetic, clinical and laboratory features in 544 patients. Br
J Haematol 1996;92:382e388.
74. Merup M, Juliusson G, Wu X, et al. Amplification of multiple
regions of chromosome 12, including 12q13-15, in chronic
lymphocytic leukaemia. Eur J Haematol 1997;58:174e180.
75. Josefsson P, Geisler CH, Leffers H, et al. CLLU1 expression
analysis adds prognostic information to risk prediction in
chronic lymphocytic leukemia. Blood 2007;109:4973e4979.
76. Buhl AM, Jurlander J, Jorgensen FS, et al. Identification of
a gene on chromosome 12q22 uniquely overexpressed in
chronic lymphocytic leukemia. Blood 2006;107:2904e2911.
77. Dohner H, Stilgenbauer S, James MR, et al. 11q deletions
identify a new subset of B-cell chronic lymphocytic leukemia
characterized by extensive nodal involvement and inferior
prognosis. Blood 1997;89:2516e2522.
78. Catovsky D, Richards S, Matutes E, et al. Assessment of flu-
darabine plus cyclophosphamide for patients with chronic
lymphocytic leukaemia (the LRF CLL4 Trial): a randomised
controlled trial. Lancet 2007;370:230e239.
79. Marasca R, Maffei R, Martinelli S, et al. Clinical heterogeneity
of de novo 11q deletion chronic lymphocytic leukaemia: prog-
nostic relevance of extent of 11q deleted nuclei inside leukemic
clone. Hematol Oncol 2012 [Epub ahead of print].
80. Lavin MF. Ataxia-telangiectasia: from a rare disorder to
a paradigm for cell signalling and cancer. Nat Rev Mol Cell Biol
2008;9:759e769.
59
81. Fiegl M, Erdel M, Tinhofer I, et al. Clinical outcome of pre-
treated B-cell chronic lymphocytic leukemia following alemtu-
zumab therapy: a retrospective study on various cytogenetic
risk categories. Ann Oncol 2010;21:2410e2419.
82. Dicker F, Herholz H, Schnittger S, et al. The detection of TP53
mutations in chronic lymphocytic leukemia independently
predicts rapid disease progression and is highly correlated with
a complex aberrant karyotype. Leukemia 2009;23:117e124.
83. Rossi D, Cerri M, Deambrogi C, et al. The prognostic value of
TP53 mutations in chronic lymphocytic leukemia is independent
of Del17p13: implications for overall survival and chemo-
refractoriness. Clin Cancer Res 2009;15:995e1004.
84. Zenz T, Krober A, Scherer K, et al. Monoallelic TP53 inacti-
vation is associated with poor prognosis in chronic lymphocytic
leukemia: results from a detailed genetic characterization with
long-term follow-up. Blood 2008;112:3322e3329.
85. Dohner H, Fischer K, Bentz M, et al. p53 gene deletion predicts
for poor survival and non-response to therapy with purine
analogs in chronic B-cell leukemias. Blood 1995;85:
1580e1589.
86. Oscier D, Wade R, Davis Z, et al. Prognostic factors identified
three risk groups in the LRF CLL4 trial, independent of treat-
ment allocation. Haematologica 2010;95:1705e1712.
87. Tam CS, Shanafelt TD, Wierda WG, et al. De novo deletion
17p13.1 chronic lymphocytic leukemia shows significant clinical
heterogeneity: the M. D. Anderson and Mayo Clinic experience.
Blood 2009;114:957e964.
88. Zenz T, Eichhorst B, Busch R, et al. TP53 mutation and survival
in chronic lymphocytic leukemia. J Clin Oncol 2010;28:
4473e4479.
89. Gonzalez D, Martinez P, Wade R, et al. Mutational status of the
TP53 gene as a predictor of response and survival in patients
with chronic lymphocytic leukemia: results from the LRF CLL4
trial. J Clin Oncol 2011;29:2223e2229.
90. Zenz T, Habe S, Denzel T, et al. Detailed analysis of p53
pathway defects in fludarabine-refractory chronic lymphocytic
leukemia (CLL): dissecting the contribution of 17p deletion,
TP53 mutation, p53-p21 dysfunction, and miR34a in
a prospective clinical trial. Blood 2009;114:2589e2597.
91. Cuneo A, Rigolin GM, Bigoni R, et al. Chronic lymphocytic
leukemia with 6q- shows distinct hematological features and
intermediate prognosis. Leukemia 2004;18:476e483.
92. Stilgenbauer S, Bullinger L, Benner A, et al. Incidence and
clinical significance of 6q deletions in B cell chronic lymphocytic
leukemia. Leukemia 1999;13:1331e1334.
93. Karakosta M, Tsakiridou A, Korantzis I, et al. Deletion of 5q as
a rare abnormality in chronic lymphocytic leukemia. Cancer
Genet Cytogenet 2010;200:175e179.
94. Haferlach C, Dicker F, Schnittger S, et al. Comprehensive
genetic characterization of CLL: a study on 506 cases analysed
with chromosome banding analysis, interphase FISH,
IgV(H) status and immunophenotyping. Leukemia 2007;21:
2442e2451.
95. Cavazzini F, Hernandez JA, Gozzetti A, et al. Chromosome
14q32 translocations involving the immunoglobulin heavy chain
locus in chronic lymphocytic leukaemia identify a disease
subset with poor prognosis. Br J Haematol 2008;142:529e537.
96. Ouillette P, Collins R, Shakhan S, et al. Acquired genomic copy
number aberrations and survival in chronic lymphocytic
leukemia. Blood 2011;118:3051e3061.
97. Gunnarsson R, Isaksson A, Mansouri M, et al. Large but not
small copy-number alterations correlate to high-risk genomic
aberrations and survival in chronic lymphocytic leukemia:
a high-resolution genomic screening of newly diagnosed
patients. Leukemia 2010;24:211e215.
98. Rigolin GM, Cibien F, Martinelli S, et al. Chromosome aberra-
tions detected by conventional karyotyping using novel mito-
gens in chronic lymphocytic leukemia with “normal” FISH:
60
correlations with clinicobiologic parameters. Blood 2012;119:
2310e2313.
99. Dewald GW, Brockman SR, Paternoster SF, et al. Chromo-
some anomalies detected by interphase fluorescence in situ
hybridization: correlation with significant biological features of
B-cell chronic lymphocytic leukaemia. Br J Haematol 2003;121:
287e295.
100. Chevallier P, Penther D, vet-Loiseau H, et al. CD38 expression
and secondary 17p deletion are important prognostic factors in
chronic lymphocytic leukaemia. Br J Haematol 2002;116:
142e150.
101. Gribben JG. How I treat CLL up front. Blood 2010;115:
187e197.
102. Mougalian SS, O’Brien S. Adverse prognostic features in
chronic lymphocytic leukemia. Oncology (Williston Park) 2011;
25:692e696, 699.
103. Hillmen P, Skotnicki AB, Robak T, et al. Alemtuzumab
compared with chlorambucil as first-line therapy for chronic
lymphocytic leukemia. J Clin Oncol 2007;25:5616e5623.
104. Pettitt AR, Jackson R, Carruthers S, et al. Alemtuzumab in
combination with methylprednisolone is a highly effective
induction regimen for patients with chronic lymphocytic
leukemia and deletion of TP53: final results of the National
Cancer Research Institute CLL206 Trial. J Clin Oncol 2012;30:
1647e1655.
105. Reeder CB, Ansell SM. Novel therapeutic agents for B-cell
lymphoma: developing rational combinations. Blood 2011;117:
1453e1462.
106. Wiestner A. Emerging role of kinase-targeted strategies in
chronic lymphocytic leukemia. Blood 2012;120:4684e4691.
107. Tam CS, Otero-Palacios J, Abruzzo LV, et al. Chronic
lymphocytic leukaemia CD20 expression is dependent on the
genetic subtype: a study of quantitative flow cytometry and
fluorescent in-situ hybridization in 510 patients. Br J Haematol
2008;141:36e40.
108. Matutes E, Carrara P, Coignet L, et al. FISH analysis for BCL-1
rearrangements and trisomy 12 helps the diagnosis of atypical
B cell leukaemias. Leukemia 1999;13:1721e1726.
109. Brito-Babapulle V, Ellis J, Matutes E, et al. Translocation t(11;
14)(q13;q32) in chronic lymphoid disorders. Genes Chromo-
somes Cancer 1992;5:158e165.
110. Cuneo A, Bigoni R, Negrini M, et al. Cytogenetic and inter-
phase cytogenetic characterization of atypical chronic lymp-
hocytic leukemia carrying BCL1 translocation. Cancer Res
1997;57:1144e1150.
111. Damle RN, Wasil T, Fais F, et al. Ig V gene mutation status and
CD38 expression as novel prognostic indicators in chronic
lymphocytic leukemia. Blood 1999;94:1840e1847.
112. Hamblin TJ, Davis Z, Gardiner A, et al. Unmutated Ig V(H)
genes are associated with a more aggressive form of chronic
lymphocytic leukemia. Blood 1999;94:1848e1854.
113. Schroeder HW Jr, Dighiero G. The pathogenesis of chronic
lymphocytic leukemia: analysis of the antibody repertoire.
Immunol Today 1994;15:288e294.
114. Lanham S, Hamblin T, Oscier D, et al. Differential signaling via
surface IgM is associated with VH gene mutational status and
CD38 expression in chronic lymphocytic leukemia. Blood 2003;
101:1087e1093.
115. Magnac C, Porcher R, Davi F, et al. Predictive value of serum
thymidine kinase level for Ig-V mutational status in B-CLL.
Leukemia 2003;17:133e137.
116. Krober A, Seiler T, Benner A, et al. V(H) mutation status, CD38
expression level, genomic aberrations, and survival in chronic
lymphocytic leukemia. Blood 2002;100:1410e1416.
117. Jelinek DF, Tschumper RC, Geyer SM, et al. Analysis of clonal
B-cell CD38 and immunoglobulin variable region sequence
status in relation to clinical outcome for B-chronic lymphocytic
leukaemia. Br J Haematol 2001;115:854e861.
A.E. Rodrı́guez-Vicente et al.
118. Hamblin TJ, Orchard JA, Ibbotson RE, et al. CD38 expression
and immunoglobulin variable region mutations are independent
prognostic variables in chronic lymphocytic leukemia, but CD38
expression may vary during the course of the disease. Blood
2002;99:1023e1029.
119. Oscier DG, Gardiner AC, Mould SJ, et al. Multivariate analysis
of prognostic factors in CLL: clinical stage, IGVH gene muta-
tional status, and loss or mutation of the p53 gene are inde-
pendent prognostic factors. Blood 2002;100:1177e1184.
120. Grever MR, Lucas DM, Johnson AJ, et al. Novel agents and
strategies for treatment of p53-defective chronic lymphocytic
leukemia. Best Pract Res Clin Haematol 2007;20:545e556.
121. Ritgen M, Lange A, Stilgenbauer S, et al. Unmutated immuno-
globulin variable heavy-chain gene status remains an adverse
prognostic factor after autologous stem cell transplantation for
chronic lymphocytic leukemia. Blood 2003;101:2049e2053.
122. Smit LA, van MF, Langerak AW, et al. Antigen receptors and
somatic hypermutation in B-cell chronic lymphocytic leukemia
with Richter’s transformation. Haematologica 2006;91:
903e911.
123. Lin K, Sherrington PD, Dennis M, et al. Relationship between
p53 dysfunction, CD38 expression, and IgV(H) mutation in
chronic lymphocytic leukemia. Blood 2002;100:1404e1409.
124. Ghia P, Stamatopoulos K, Belessi C, et al. Geographic patterns
and pathogenetic implications of IGHV gene usage in chronic
lymphocytic leukemia: the lesson of the IGHV3-21 gene. Blood
2005;105:1678e1685.
125. Capello D, Guarini A, Berra E, et al. Evidence of biased
immunoglobulin variable gene usage in highly stable B-cell
chronic lymphocytic leukemia. Leukemia 2004;18:1941e1947.
126. Capello D, Zucchetto A, Degan M, et al. Immunophenotypic
characterization of IgVH3-72 B-cell chronic lymphocytic
leukaemia (B-CLL). Leuk Res 2006;30:1197e1199.
127. Dal-Bo M, Del GI, Bomben R, et al. B-cell receptor, clinical
course and prognosis in chronic lymphocytic leukaemia: the
growing saga of the IGHV3 subgroup gene usage. Br J Hae-
matol 2011;153:3e14.
128. Montillo M, Hamblin T, Hallek M, et al. Chronic lymphocytic
leukemia: novel prognostic factors and their relevance for risk-
adapted therapeutic strategies. Haematologica 2005;90:
391e399.
129. Rosenwald A, Alizadeh AA, Widhopf G, et al. Relation of gene
expression phenotype to immunoglobulin mutation genotype in
B cell chronic lymphocytic leukemia. J Exp Med 2001;194:
1639e1647.
130. Crespo M, Bosch F, Villamor N, et al. ZAP-70 expression as
a surrogate for immunoglobulin-variable-region mutations in
chronic lymphocytic leukemia. N Engl J Med 2003;348:
1764e1775.
131. Wiestner A, Rosenwald A, Barry TS, et al. ZAP-70 expression
identifies a chronic lymphocytic leukemia subtype with unmu-
tated immunoglobulin genes, inferior clinical outcome, and
distinct gene expression profile. Blood 2003;101:4944e4951.
132. Orchard JA, Ibbotson RE, Davis Z, et al. ZAP-70 expression
and prognosis in chronic lymphocytic leukaemia. Lancet 2004;
363:105e111.
133. Rassenti LZ, Huynh L, Toy TL, et al. ZAP-70 compared with
immunoglobulin heavy-chain gene mutation status as
a predictor of disease progression in chronic lymphocytic
leukemia. N Engl J Med 2004;351:893e901.
134. Wattel E, Preudhomme C, Hecquet B, et al. p53 mutations are
associated with resistance to chemotherapy and short survival
in hematologic malignancies. Blood 1994;84:3148e3157.
135. Cordone I, Masi S, Mauro FR, et al. p53 expression in B-cell
chronic lymphocytic leukemia: a marker of disease progression
and poor prognosis. Blood 1998;91:4342e4349.
136. Austen B, Skowronska A, Baker C, et al. Mutation status of the
residual ATM allele is an important determinant of the cellular
Molecular heterogeneity in CLL
response to chemotherapy and survival in patients with chronic
lymphocytic leukemia containing an 11q deletion. J Clin Oncol
2007;25:5448e5457.
137. Austen B, Powell JE, Alvi A, et al. Mutations in the ATM gene
lead to impaired overall and treatment-free survival that is
independent of IGVH mutation status in patients with B-CLL.
Blood 2005;106:3175e3182.
138. Bullrich F, Rasio D, Kitada S, et al. ATM mutations in B-cell
chronic lymphocytic leukemia. Cancer Res 1999;59:24e27.
139. Puente XS, Pinyol M, Quesada V, et al. Whole-genome
sequencing identifies recurrent mutations in chronic lympho-
cytic leukaemia. Nature 2011;475:101e105.
140. Quesada V, Conde L, Villamor N, et al. Exome sequencing
identifies recurrent mutations of the splicing factor SF3B1 gene
in chronic lymphocytic leukemia. Nat Genet 2011;44:47e52.
141. Wang L, Lawrence MS, Wan Y, et al. SF3B1 and other novel
cancer genes in chronic lymphocytic leukemia. N Engl J Med
2011;365:2497e2506.
142. Fabbri G, Rasi S, Rossi D, et al. Analysis of the chronic
lymphocytic leukemia coding genome: role of NOTCH1 muta-
tional activation. J Exp Med 2011;208:1389e1401.
143. Rosati E, Sabatini R, Rampino G, et al. Constitutively activated
Notch signaling is involved in survival and apoptosis resistance
of B-CLL cells. Blood 2009;113:856e865.
144. Villamor N, Conde L, Martinez-Trillos A, et al. NOTCH1
mutations identify a genetic subgroup of chronic lymphocytic
leukemia patients with high risk of transformation and poor
outcome. Leukemia 2012 [Epub ahead of print].
145. Rossi D, Rasi S, Fabbri G, et al. Mutations of NOTCH1 are an
independent predictor of survival in chronic lymphocytic
leukemia. Blood 2012;119:521e529.
146. Balatti V, Bottoni A, Palamarchuk A, et al. NOTCH1 mutations
in CLL associated with trisomy 12. Blood 2012;119:329e331.
147. Del Giudice I, Rossi D, Chiaretti S, et al. NOTCH1 mutations in
þ12 chronic lymphocytic leukemia (CLL) confer an unfavorable
prognosis, induce a distinctive transcriptional profiling and
refine the intermediate prognosis of þ12 CLL. Haematologica
2012;97:437e441.
148. Lopez C, Delgado J, Costa D, et al. Different distribution of
NOTCH1 mutations in chronic lymphocytic leukemia with iso-
lated trisomy 12 or associated with other chromosomal alter-
ations. Genes Chromosomes Cancer 2012;51:881e889.
149. Paganin M, Ferrando A. Molecular pathogenesis and targeted
therapies for NOTCH1-induced T-cell acute lymphoblastic
leukemia. Blood Rev 2011;25:83e90.
150. Papaemmanuil E, Cazzola M, Boultwood J, et al. Somatic
SF3B1 mutation in myelodysplasia with ring sideroblasts.
N Engl J Med 2011;365:1384e1395.
151. Rossi D, Bruscaggin A, Spina V, et al. Mutations of the SF3B1
splicing factor in chronic lymphocytic leukemia: association
with progression and fludarabine-refractoriness. Blood 2011;
118:6904e6908.
152. Rossi D, Fangazio M, Rasi S, et al. Disruption of BIRC3
associates with fludarabine chemorefractoriness in TP53
wild-type chronic lymphocytic leukemia. Blood 2012;119:
2854e2862.
153. Compagno M, Lim WK, Grunn A, et al. Mutations of multiple
genes cause deregulation of NF-kappaB in diffuse large B-cell
lymphoma. Nature 2009;459:717e721.
154. Schuh A, Becq J, Humphray S, et al. Monitoring chronic
lymphocytic leukemia progression by whole genome
sequencing reveals heterogeneous clonal evolution patterns.
Blood 2012.
155. Croce CM. Causes and consequences of microRNA dysregu-
lation in cancer. Nat Rev Genet 2009;10:704e714.
156. Grimson A, Farh KK, Johnston WK, et al. MicroRNA targeting
specificity in mammals: determinants beyond seed pairing. Mol
Cell 2007;27:91e105.
61
157. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and
function. Cell 2004;116:281e297.
158. Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling
reveals distinct signatures in B cell chronic lymphocytic leuke-
mias. Proc Natl Acad Sci U S A 2004;101:11755e11760.
159. Calin GA, Ferracin M, Cimmino A, et al. A MicroRNA signature
associated with prognosis and progression in chronic lympho-
cytic leukemia. N Engl J Med 2005;353:1793e1801.
160. Fulci V, Chiaretti S, Goldoni M, et al. Quantitative technologies
establish a novel microRNA profile of chronic lymphocytic
leukemia. Blood 2007;109:4944e4951.
161. Zanette DL, Rivadavia F, Molfetta GA, et al. miRNA expression
profiles in chronic lymphocytic and acute lymphocytic leukemia.
Braz J Med Biol Res 2007;40:1435e1440.
162. Zhang J, Jima DD, Jacobs C, et al. Patterns of microRNA
expression characterize stages of human B-cell differentiation.
Blood 2009;113:4586e4594.
163. Stamatopoulos B, Meuleman N, Haibe-Kains B, et al. micro-
RNA-29c and microRNA-223 down-regulation has in vivo
significance in chronic lymphocytic leukemia and improves
disease risk stratification. Blood 2009;113:5237e5245.
164. Rossi S, Shimizu M, Barbarotto E, et al. microRNA finger-
printing of CLL patients with chromosome 17p deletion identify
a miR-21 score that stratifies early survival. Blood 2010;116:
945e952.
165. Visone R, Rassenti LZ, Veronese A, et al. Karyotype-specific
microRNA signature in chronic lymphocytic leukemia. Blood
2009;114:3872e3879.
166. Fabbri M, Bottoni A, Shimizu M, et al. Association of a micro-
RNA/TP53 feedback circuitry with pathogenesis and outcome
of B-cell chronic lymphocytic leukemia. JAMA 2011;305:
59e67.
167. Stach D, Schmitz OJ, Stilgenbauer S, et al. Capillary electro-
phoretic analysis of genomic DNA methylation levels. Nucleic
Acids Res 2003;31:E2.
168. Lyko F, Stach D, Brenner A, et al. Quantitative analysis of DNA
methylation in chronic lymphocytic leukemia patients. Electro-
phoresis 2004;25:1530e1535.
169. Wahlfors J, Hiltunen H, Heinonen K, et al. Genomic hypo-
methylation in human chronic lymphocytic leukemia. Blood
1992;80:2074e2080.
170. Robertson KD, Jones PA. DNA methylation: past, present and
future directions. Carcinogenesis 2000;21:461e467.
171. Rahmatpanah FB, Carstens S, Hooshmand SI, et al. Large-
scale analysis of DNA methylation in chronic lymphocytic
leukemia. Epigenomics 2009;1:39e61.
172. Rush LJ, Raval A, Funchain P, et al. Epigenetic profiling in
chronic lymphocytic leukemia reveals novel methylation
targets. Cancer Res 2004;64:2424e2433.
173. Hanada M, Delia D, Aiello A, et al. bcl-2 gene hypomethylation
and high-level expression in B-cell chronic lymphocytic
leukemia. Blood 1993;82:1820e1828.
174. Yuille MR, Condie A, Stone EM, et al. TCL1 is activated by
chromosomal rearrangement or by hypomethylation. Genes
Chromosomes Cancer 2001;30:336e341.
175. Lipsanen V, Leinonen P, Alhonen L, et al. Hypomethylation of
ornithine decarboxylase gene and erb-A1 oncogene in human
chronic lymphatic leukemia. Blood 1988;72:2042e2044.
176. Raval A, Lucas DM, Matkovic JJ, et al. TWIST2 demonstrates
differential methylation in immunoglobulin variable heavy chain
mutated and unmutated chronic lymphocytic leukemia. J Clin
Oncol 2005;23:3877e3885.
177. Corcoran M, Parker A, Orchard J, et al. ZAP-70 methylation
status is associated with ZAP-70 expression status in chronic
lymphocytic leukemia. Haematologica 2005;90:1078e1088.
178. Claus R, Lucas DM, Stilgenbauer S, et al. Quantitative DNA
methylation analysis identifies a single CpG dinucleotide
important for ZAP-70 expression and predictive of prognosis in
62
chronic lymphocytic leukemia. J Clin Oncol 2012;30:
2483e2491.
179. Raval A, Tanner SM, Byrd JC, et al. Downregulation of death-
associated protein kinase 1 (DAPK1) in chronic lymphocytic
leukemia. Cell 2007;129:879e890.
180. Tsirigotis P, Pappa V, Labropoulos S, et al. Mutational and
methylation analysis of the cyclin-dependent kinase 4 inhibitor
(p16INK4A) gene in chronic lymphocytic leukemia. Eur J
Haematol 2006;76:230e236.
181. Papageorgiou SG, Lambropoulos S, Pappa V, et al. Hyper-
methylation of the p15INK4B gene promoter in B-chronic
lymphocytic leukemia. Am J Hematol 2007;82:824e825.
182. Yu MK, Bergonia H, Szabo A, et al. Progressive disease in
chronic lymphocytic leukemia is correlated with the DNA
methylation index. Leuk Res 2007;31:773e777.
183. Kulis M, Heath S, Bibikova M, et al. Epigenomic analysis
detects widespread gene-body DNA hypomethylation in
chronic lymphocytic leukemia. Nat Genet 2012;44:1236e1242.
184. Kanduri M, Cahill N, Goransson H, et al. Differential genome-
wide array-based methylation profiles in prognostic subsets of
chronic lymphocytic leukemia. Blood 2010;115:296e305.
185. Nana-Sinkam SP, Croce CM. MicroRNA in chronic lymphocytic
leukemia: transitioning from laboratory-based investigation to
clinical application. Cancer Genet Cytogenet 2010;203:127e133.
186. Nicoloso MS, Kipps TJ, Croce CM, et al. MicroRNAs in the
pathogeny of chronic lymphocytic leukaemia. Br J Haematol
2007;139:709e716.
187. Mertens D, Wolf S, Schroeter P, et al. Down-regulation of
candidate tumor suppressor genes within chromosome band
13q14.3 is independent of the DNA methylation pattern in B-
cell chronic lymphocytic leukemia. Blood 2002;99:4116e4121.
188. Pallasch CP, Patz M, Park YJ, et al. miRNA deregulation by
epigenetic silencing disrupts suppression of the oncogene
PLAG1 in chronic lymphocytic leukemia. Blood 2009;114:
3255e3264.
189. Patz M, Pallasch CP, Wendtner CM. Critical role of microRNAs
in chronic lymphocytic leukemia: overexpression of the onco-
gene PLAG1 by deregulated miRNAs. Leuk Lymphoma 2010;
51:1379e1381.
190. Wong KY, So CC, Loong F, et al. Epigenetic inactivation of the
miR-124-1 in haematological malignancies. PLoS One 2011;6:
e19027.
A.E. Rodrı́guez-Vicente et al.
191. Fabbri M, Garzon R, Cimmino A, et al. MicroRNA-29 family
reverts aberrant methylation in lung cancer by targeting DNA
methyltransferases 3A and 3B. Proc Natl Acad Sci U S A 2007;
104:15805e15810.
192. Mercurio C, Minucci S, Pelicci PG. Histone deacetylases and
epigenetic therapies of hematological malignancies. Pharmacol
Res 2010;62:18e34.
193. Blum KA, Advani A, Fernandez L, et al. Phase II study of the
histone deacetylase inhibitor MGCD0103 in patients with
previously treated chronic lymphocytic leukaemia. Br J Hae-
matol 2009;147:507e514.
194. Heerema NA, Byrd JC, Dal Cin PS, et al. Stimulation of chronic
lymphocytic leukemia cells with CpG oligodeoxynucleotide
gives consistent karyotypic results among laboratories: a CLL
Research Consortium (CRC) Study. Cancer Genet Cytogenet
2010;203:134e140.
195. Put N, Konings P, Rack K, et al. Improved detection of chro-
mosomal abnormalities in chronic lymphocytic leukemia by
conventional cytogenetics using CpG oligonucleotide and
interleukin-2 stimulation: a Belgian multicentric study. Genes
Chromosomes Cancer 2009;48:843e853.
196. Grever MR, Lucas DM, Dewald GW, et al. Comprehensive
assessment of genetic and molecular features predicting
outcome in patients with chronic lymphocytic leukemia: results
from the US Intergroup Phase III Trial E2997. J Clin Oncol
2007;25:799e804.
197. Quijano S, Lopez A, Rasillo A, et al. Association between the
proliferative rate of neoplastic B cells, their maturation stage,
and underlying cytogenetic abnormalities in B-cell chronic
lymphoproliferative disorders: analysis of a series of 432
patients. Blood 2008;111:5130e5141.
198. Hagenkord JM, Monzon FA, Kash SF, et al. Array-based kar-
yotyping for prognostic assessment in chronic lymphocytic
leukemia: performance comparison of Affymetrix 10K2.0, 250K
Nsp, and SNP6.0 arrays. J Mol Diagn 2010;12:184e196.
199. O’Malley DP, Giudice C, Chang AS, et al. Comparison of array
comparative genomic hybridization (aCGH) to FISH and cyto-
genetics in prognostic evaluation of chronic lymphocytic
leukemia. Int J Lab Hematol 2011;33:238e244.
200. Rinaldi A, Mian M, Kwee I, et al. Genome-wide DNA profiling
better defines the prognosis of chronic lymphocytic leukaemia.
Br J Haematol 2011;154:590e599.