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Chronic Lymphoproliferative Disorders: An

Integrated Point of View for the Differential
Diagnosis
a b a
Giovanni D'arena , Michael J. Keating & Mario Carotenuto
a
Division of Hematology, IRCCS Casa Sollievo della Sofferenza Hospital, San
Giovanni Rotondo, Italy
b
The MD Anderson Cancer Center, The University of Texas, Houston, TX, USA
Published online: 01 Jun 2015.

To cite this article: Giovanni D'arena, Michael J. Keating & Mario Carotenuto (2000) Chronic
Lymphoproliferative Disorders: An Integrated Point of View for the Differential Diagnosis, Leukemia &
Lymphoma, 36:3-4, 225-237

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 020OO OPA IOveraeu Puhliaherr Assin-Cation)N.V
Puhlirhed by licenw under
the Hanvood Academic Publishen imprinl.
part ufthe Gordon and Breach Puhlkhing Group.
Printed in Malayw

Chronic Lymphoproliferative Disorders: An Integrated

Point of View for the Differential Diagnosis
GIOVANNI D’ARENAa*, MICHAEL J. KEATINGb and MARIO CAROTENUTOa

“Division of Hemutology, IRCCS “Cascr Sollievo della Sofferenza ” Hospital, Sun Giovanni Rotondo, Italy and bThe M D Anderson Cancer
CenteK The Universio of Texas, Houston, TX,USA

(In final form June 30, 1999)

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Morphology is regarded as the principle basis for the identification of lymphoid neoplasms.
Sometimes, however, it fails to discriminate among several chronic lymphoproliferative dis-
orders (CLDs). Improved immunophenotyping has resulted in a better characterization of a
number of variants of these diseases, some of which may benefit from different therapeutic
approaches.
In particular, the proposal of scoring systems using a panel of monoclonal antibodies
(MoAbs) has represented a critical step in this field. In fact, to date, some MoAbs (CD5,
CD23, FMC7, CD22, CD79b, and surface immunoglobulin density) are able to distinguish
among several entities, thus allowing for a correct diagnosis in the majority of cases. How-
ever, there is still a small percentage of patients where the combined diagnostic approach
(morphology and immunophenotyping) should be further refined by other techniques, such as
cytogenetic and molecular Characterization. Here numerous questions are raised indicating
the need to more accurately differentiate the disease entities under discussion and better
understand some of their clinical manifestations.

Keywordr: morphology, immunology, chronic lymphoproliferative disorders, flow cytometry, diagnosis

INTRODUCTION chain ratio, while a clonal T-cell population is more

difficult to evaluate in absence of a distinctive immu-
Chronic lymphoproliferative disorders (CLDs) repre- nophenotype(’).
sent a heterogeneous group of diseases characterized Chronic lymphocytic leukemias (CLL), prolym-
by the expansion of mature B (rarely T) lymphocytes phocytic leukemias (PLL), hairy cell leukemias
in peripheral blood, bone marrow and other lymphoid (HCL), and the leukemic phase of low grade
tissues. Tumor cells are expression of a clonal prolif- non-Hodgkin lymphomas (NHL) are distinct CLD
eration. A clonal B-cell population is arbitrarely entities now recognized as peripheral B-cell neo-
defined as having greater than 3: 1 or less than 1:2 K/h plasms by the Revised European-American Lym-
surface membrane immunoglobulin (SmIg) light phoid neoplasms (REAL) classification (2).
* Corresponding Author: Giovanni D’Arena, MD Division of Hematology IRCCS “Casa Sollievo della Sofferenza” Hospital 7 1013 San
Giovanni Rotondo, Italy Tel. Int. +39.882.410539 FAX Int +39.882.410258 E-mail: ematologia@operapadrepio.it

225
 226 GIOVANNI D’ARENA er al.

TABLE I Relevant molecules in the immunological classification of chronic lymphoproliferative disorders

Designation Cell Type Antigen

CD5 Thymocytes, mature T cells, subpopulations of B cells Linked to T cell proliferation

CDlO c-ALL, lymphatic precursor cells, neutrophils, subset of Common acute leukemia antigen (CALLA),
mature B cells neutral endopeptidase (enkephalinase)
CDllc Monocytes, neutrophils, NK cells, subpopulation of B cells Adhesion molecule, gp 150/95
CD19 Precursor B cells, B cells Bridge for surface immunoglobulin signal
CD20 Subpopulation of precursor B cells, B cells Ion channel, protein kinase C substrate
CD22 Surface expression of B cells, cytoplasmic expression Related to neural cell adhesion molecule, bridge
in precursor B cells for surface immunoglobulin signal
CD23 Subpopulation of B cells Low affinity Fc-receptor for IgE
CD24 B lymphocytes, activated T lymphocytes, neutrophils 35-45 kDa glycosylphosphatidylinositol-linked
glycoprotein
CD25 Activated T and B lymphocytes Interleukin-2 receptor a-chain
CD79b Precursor B cells. B cells P-chain of B cell receptor complex (IgP)
CD103 Intraepithelial lymphocytes HML- 1
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FMC7 Differentiated B-lymphocytes 105 kDa glycoprotein

HLA-DR B lymphocytes, activated T lymphocytes, monocytes, Part of the MHC I1 complex
precursor cells
Kappa light chain Surface expression on B lymphocytes Immunoglobulin-light chain type kappa
Lambda light chain Surface expression on B lymphocytes Immunoglobulin-light chain type lambda

Cell morphology, as proposed by the French-Amer-
ican-British (FAB) study group, and immunological
markers are able to distinguish various diseases (374).
However, there may be some overlaps in morphology,
in particular between some entities which may mim-
ick other conditions, such as HCL, its variant form
(HCL-v), and splenic lymphoma with villous lympho-
cytes (SLVL), all characterized by the presence of cir-
culating lymphocytes with villous projections.
Therefore it is of crucial importance to make a correct
diagnosis because different treatments are required
for these diseases. Again, it is now clear that both
MCL and “atypical” CLL display a clinical behaviour
more aggressive than other low-grade NHL in leuke-
mic phase and “typical” CLL, respectively. In the past
years flow cytometry has gained a pivotal role in the
diagnosis of CLDs, making it possible to detect the
antigenic profile of neoplastic cells (5,6). AS also
shown in this report, a small panel of monoclonal
antibodies (MoAbs) may be used to perform accurate
immunophenotyping, which combined with morphol-
ogy, may permit a better definition of different CLDs
as distinct entities (798). A comprehensive list of the
most useful molecules in the immunological classifi-
cation of these diseases is given in Table I. Finally,
advances recently made in the genomic analysis pro-
vide additional details for its diagnostic application in
hematopathology (9).
CLL
A sustained marrow and blood accumulation (prob-
ably mediated by apoptosis inhibition) of well-differ-
entiated and long-lived lymphocytes with light chain
restriction characterizes B-CLL, a relatively common
disorder in Western C o ~ m t r i e s ( ’ ~ ’Small
~ ) . lympho-
cytes characterize “typical” CLL (Fig. 1). However, a
varying number of prolymphocytes and/or centro-
cytes (cleaved-cells) and/or larger lymphocytes may
be detected in circulating blood. As suggested by the
FAB cooperative group, when a mixture of these cells
and small lymphocytes is found the morphological
 DIFFERENTIAL DIAGNOSIS OF CHRONIC LYMPHOPROLIFERATIVE DISORDERS
FIGURE I B-CLL peripheral blood. Small lymphocytes (slightly
larger than a red cell) with clumped nuclear chromatin without visi-
ble nucleoli, and a high nuclear: cytoplasmic ratio (See Color
Plate I at the back of this issue)
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diagnosis of “atypical” CLL should be taken into
account (4). Two forms of “atypical” CLL have been
described: a) CLLPLL, which includes cases with
more than 10% and less than 55% prolymphocytes,
and b) mixed cell type CLL characterized by a spec-
trum of small to large lymphocytes with less than
10% prolymphocytes.
Several observations lead to the suggestion that
typical and atypical CLL are two separate entities
both in biological and in clinical features. In fact,
atypical morphology seem to be closely related to tri-
somy 12 and a poor prognosis Furthermore,
Criel et a1 considered atylical even those patients
with immunophenotypic deviations with respect to a
standard profile of reactivity (I4). However, Oscier et
a1 considered the atypical morphology as the only
important feature to define the treatment strategy (20).
Lymphocytes usually express pan-B antigens CD 19,
CD20, CD24, and CD23 as well as CD5, a T-cell
marker antigen that is also present in a subpopulation
of normal B lymphocytes (21,22). A weak expression
of SmIg and CD22, and the absence of the antigen
recognized by FMC7 and CD 103 are also typical fea-
tures of classical B-CLL(23-29). In addition, the mye-
loid marker CDllc is frequently present on B-CLL
cells and about one half of cases express CD25 (30).
The classical immunological profile in a B-CLL case
is depicted in Fig. 2.
227
TABLE II Matutes’s Scoring System for the differential diagnosis
of CLDs ( 3 ’ )
Points
Membrane marker
I 0
SmIg weak moderatelstrong
CD5 + -
CD23 + -
FMC7 - 4-
CD22 weakhegative moderate/strong
Scores range from 5 to 0.
Matutes, Catovsky and co-workers have recently
proposed a scoring system based on 5 markers (Smig
density, CD5, CD22, CD23 and FMC7) to enhance
the identification of CLL from other CLDs (31-33)
(Table 11). They found that the score for “typical”
CLL ranged from 5 to 4 in 85% of cases, whereas in
the other diseases the score ranged from 0 to 2 in 88%
of cases. Furthermore, some other MoAbs are helpful
to better classify the B cell chronic disorders. In fact,
additional information can be obtained from the
expression of CD79b (B29 protein), which along with
the mb- 1 protein (CD79a) constitute a heterodimeric
molecule non-covalently bound to membrane immu-
noglobulin to form the B-cell antigen receptor com-
plex (34). Indeed, the same British group recently
showed that in a series of 499 patients with a B-cell
lymphoproliferative disorder, only 5% of B-CLL was
found to be CD79b positive, unlike the 100% of
B-PLL, 92% of MCL, 83% of FL, and 74% of SLVL.
HCL and HCL-V,however, displayed CD79b positiv-
ity in 25% of cases (35). This has lead to the proposal
of revising the scoring system sobstituting CD79b for
CD22 In fact, such a change made it possible to
increase diagnostic accuracy from 91.6% to 96% (36).
In our series, including only CD5+CD23+ CLL cases,
we found 16% of positivity for CD79b, using CB3-1
clone (37). Moreover, other authors (Molica S, per-
sonal communication) found 15% of positivity using
the same MoAb (SN8) used by British Group, thus
suggesting that an higher proportion of CD79b CLL
cases may be seen with respect to previosuly reported
data, as also shown in a recent paper (38).
 228 GIOVANNI D'ARENA et al.

e
a

W23 PE

8-
-4J I v-.
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CD79b CD22
0 '
Q- s;
I Ml 0
I MI
0 '
a":
e
m
e "l c -
c . a .
0 -

s: 8s;

0
01

cD22 FlTc
0 7 % PE

FMC7

I M1

$L
0 loo lo1 lo2
FMc7 FlTc
lo3 1
4

FIGURE 2 Immunological profile of a typical B-CLL expression of CD5 and CD23, weak expression of CD22 and absence of FMC7 and
CD79b
 DIFFERENTIAL DIAGNOSIS OF CHRONIC LYMPHOPROLIFERATIVE DISORDERS 229

PLL

PLL was first described in 1974 by Galton et a1 (39) as

a separate clinico-pathological entity usually charac-
terized by a prominent splenomegaly, minimal or
absent lymphadenopathy and marked by lymphocyto-
sis with predominance of prolymphocytes (large cells
with a slight to moderate basophilic cytoplasm, a usu-

s
ally round or oval nucleus with coarse chromatin and
a typically prominent nucleolus) (Fig. 3). Subse-
quently (19891, the FAB cooperative group proposed
a definition of PLL as a disease with 55% or more 0 loo lo1 lo2 lo3 lo4
prolymphocytes (4).
KAPPA FlTC
Prolymphocytes express the pan-B cell markers
CD 19, CD20 and CD24. Usually negative for CD 1 I c
and CD23, they frequently do not display CD5 posi-
tivity (31-33,40). Abnormal B-cells in PLL typically
Downloaded by [] at 15:06 15 July 2015

express high density SmIg. Fig. 4 shows the different

pattern of SmIg light chain ( d h )expression in vari-
ous CLDs. Note the weak expression of smIg in
B-CLL as compared to the other cases. Finally, it is to
be considered that, in addition to the development of
aggressive non-Hodgkin lymphoma (Richter's syn-
drome) (41), a prolymphocytoid transformation may
occur during the course of CLL. In the latter case,
however, neoplastic cells may express CD5 and low
density SmIg as in CLL.

a-.
c
c .
C
.o'
0 '

KAPPA FlTC
FIGURE 3 B-PLL peripheral blood. Large cells with a basophilic
cytoplasm, a round nucleus with coarse chromatin and a typically FIGURE 4 Pattern of SmIg light chains ( d h )expression in B-CLL
prominent nucleuolus (See Color Plate 11 at the back of this issue) (A), HCL (B) and B-PLL (C)
 230 GIOVANNI D’ARENA et al.

HCL

HCL is a rare and well-recognized chronic lymphop-

roliferative disorder in which insidious onset, pancy-
topenia, splenomegaly, absence of
lymphadenomegaly, bone marrow involvement and
circulating malignant cells with irregular cytoplasmic
“hairy” projections on smear preparations are the
hallmarks of the disease (42-44) (Fig. 5). In addition,
HCL cells are usually stained for the tartrate-resistant
intracytoplasmic isoenzyme 5 acid phosphatase
(TRAP) (Fig. 6) (45*46). However, a variant form
(HCL-V) was described in which the cells exhibit a
FIGURE 5 HCL peripheral blood. Larger than normal lympho-
prominent single nucleolus similar to prolympho- cytes, HCL cells display an oval nucleus, reticular chromatin, and a
cytes, but with a villous cytoplasm(4749). pale cytoplasm with numerous fine projections (See Color Plate 111
So far, the most useful marker for distinguishing at the back of this issue)
HCL from other chronic lymphoproliferative disor-
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ders is CD103 @O). A strong expression of CD22,

CDllc, CD25 and FMC7 is also displayed. In addi-
tion, CD5 and CD23 negativity are common features
of both HCL and HCL-v (51-53).This latter form is
consistently CD25 negative.

SLVL

Splenomegaly, lymphocytosis (infrequently over 30 x

109/1) and absence of lymphadenopathy are usually
the main characteristics of SLVL, a rare but distinct
clinico-pathological entity (54-59). Even though an
accurate morphological smear examination may be
useful in performing a differential diagnosis (SLVL
cells, slightly larger than CLL cells, have a round
nucleus with a variable degree of basophilic cyto-
plasm and, characteristically, long cytoplasmic pro-
jections and numerous thin and short villi with a polar
distribution, different from HCL cells), abnormal vil-
lous lymphocytes are often mistaken for hairy cells
(60*61).A useful tool to distinguish between these two
chronic diseases is the TRAP staining (usually nega-
tive in SLVL). Membrane marker studies display pos-
itivity for B-cell markers such as CD19, CD20, and
CD79b, a strong expression of CD22, FMC7 and
SmIg, and lack of CD5 and CD23. Finally, CD25 is
expressed in at least 25% of SLVL cases while
CD103 is almost always absent (62*63).
FIGURE 6 TRAP-stained peripheral blood smear. Note the
TRAP-positive hairy cell with abundant cytoplasmic granules (See
Color Plate IV at the back of this issue)
MANTLE CELL LYMPHOMA (MCL)
IN LEUKEMIC PHASE
MCL is a relatively uncommon type of NHL account-
ing for approximately 5% of lymphomas in Western
Countries. It is clinically characterized by advanced
stage, frequent extranodal involvement and bone mar-
row infiltration in the majority of cases. So far, most
investigators look at MCL as a malignancy of inter-
mediate grade, with a median overall survival of 2 to
5 years, thus needing more aggressive therapeutical
 DIFFERENTIAL DIAGNOSIS OF CHRONIC LY MPHOPROLIFERATIVE DISORDERS 23 I

approaches (e.g., high-dose therapy followed by

peripheral blood progenitor cells rescue) (64).
Neoplastic cells are small to medium compared to
lymphoid cells, with a more dispersed chromatin,
scant pale cytoplasm, inconspicuous nucleoli, and
irregular or “cleaved” nucleus. In some cases, cells
resemble lymphoblastic lymphoma, justifying the
terms of “lymphoblastoid’ or “blastoid”, as proposed
by the REAL Working Group(2)(Fig. 7). MCL resem-
bles CLL, especially in the mixed cell type (4). Since
MCL is a more aggressive disease than CLL, the
immunological evaluation of the surface membrane FIGURE 7 “Blastoid” variant of MCL. A cluster of neoplastic cells
profile in these malignancies may offer a useful diag- displaying blastic features (large cells with high nuc1ear:cytoplas-
mic ratio, and disperded chromatin with nucleoli) in a bone marrow
nostic tool. Though MCL cells express B-cell anti- film (See Color Plate V at the back of this issue)
gens such as CD19, CD20 and FMC7 as well as the
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T-antigen CD5, they also strongly display restricted

SmIg light chain (but brighter than in CLL) and lack
CD23. This latter molecule, to date, seem to be the
most relevant marker in distinguish CLL and MCL
(65*66).Conflicting data are reported on CDlO and
CD 1 Ic expression which, however, frequently
appears to be absent.
Finally, the t( 11;14)(qI3;q32) cytogenetic aberra-
tion and its molecular counterpart Bcl- 1 rearrange-
ment are closely associated with MCL (about 70% of
cases) (67). This translocation, detectable by conven-
tional cytogenetics or by FISH, results in
PRADAIkyclin Dl gene overexpression (detectable
in over 95% of cases by Southern Blot or PCR)which
appears to be another highly specific and sensitive
molecular marker for MCL (68369).
FOLLICULAR LYMPHOMA (FL) IN
LEUKEMIC PHASE
markers positivity. CDlO is frequently, but not
always, present whereas CD5 and CDllc are usually
absent. Furthermore, neoplastic cells may react with
FMC7 while CD23 is frequently not expressed (70).
MARGINAL ZONE LYMPHOMA (MZL) IN
LEUKEMIC PHASE
Several entities, such as extranodal mucosa-associ-
ated lymphoid tissue (MALT) type lymphoma, nodal
monocytoid B-cell lymphoma, with or without villous
lymphocytes, are categorized as MZLs (2). These dis-
orders display a growth pattern and phenotypic fea-
tures reminiscent of the B follicle marginal zone (71).
B-cell associated markers (CD19, CD20, CD22) are
always expressed while CD5, CDIO, and CD23 are
absent. SmIgs are expressed at higher density.

FL neoplastic cells arise from germinal center cells T-CELL CHRONIC

with similar morphological features. Thus, a variable
mixture of centrocytes (small cleaved) and centro-
blasts (large noncleaved) is usually present. Abnor-
mal lymphocytes show intermediate to high density
SmIg along with CD19, CD20, and CD24 pan B-cell
LYMPHOPROLIFERATIVE DISORDERS
T-cell chronic leukemias are rare disorders whith no
morphological or clinical features allowing their rec-
ognition as different entities from B-cell chronic
 232 GIOVA"1 D'ARENA et al.

malignancies. The existence of a true T-CLL is dis-

cussed. Some authors assert that it does not exist and
suggest that B-CLL and CLL are synonymous and the
diseases incorrectly diagnosed as T-CLL belong to
what are now recognized as distinct clinico-patholog-
ical entities, such as large granular lymphocytic
(LGL) leukemia or adult T-cell leukemia (ATLL) and
T-PLL (72-75). The putative T-CLL has been described
to consistently show a mature T-cell immunopheno-
type (CD2+,CD3+,CD5+,CD7+); it lacks CD 1,
CD16, CD56, CD57 and expresses CD4 more often
than CD8 ('@. In T-PLL, prolymphocytes display a
TYPICAL CLL post-thymic phenotype: TdT negativity, CD2, CD3,
CD5 and CD7 positivity, and are CD4+ (70% of
cases) or CD8+ (10%) or both (20%) (77978). Further-
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more, ATLL, a HTLV-1-associated disease, as a rule,

expresses CD3, CD4, CD5, HLA-DR and CD25, but
not CD7 and CD8. LGL leukemias are rare and indo-
lent disorders in which an abnormal proliferation of
large and granulated lymphocytes occurs. Neoplastic
cells usually show CD2, CD3, CD8 positivity with
CD16 and CD57 natural killer antigen expression.
Less frequently they are CD56 positive (79*80).
Finally, Sezary's syndrome is characterized by
abnormal cells with typical cerebriform, large, and
ATYPICAL CLL clefted nuclei with fine chromatin and scanty cyto-
plasm. The membrane phenotype is typically CD2+,
CD3+,CD4+,CD5+,CD7-,CD8-
(33)

CONCLUSIONS

In Fig. 9 we have outlined a simple diagnostic deci-

MCL
FIGURE 8 Detection of the number of CD20 molecules/per cell
(ABC) in a case of classical CLL, atypical CLL and MCL. In atyp-
ical CLL is most often detected a greater CD20 ABC number than
in typical CLL
sional tree usually used in our practise to easily recog-
nize the most important subtypes of B-CLDs.
However, the complexity of clinico-biological mani-
festations of some of them clearly shows it is not
exhaustive. In fact, although morphology and immu-
nophenotyping may allow for a correct diagnosis in
the majority of cases, there is still a small percentage
of patients where this combined approach should be
further study in depth. In this setting, our personal
 DIFFERENTIAL DIAGNOSIS OF CHRONIC LYMPHOPROLIFERATIVE DISORDERS 233

I Clonal SmIg expression I

CD5+ on B-cells
Yes . No
hairy cells
CD5+CLDs CD5-CLDs splenomegaly
no node
m
TRAP+ TRAP-
CD23+ CD23- prolymphocytes>55% CD103+ CD103-

1 1
splenomegaly
V V no node
marked lymphocytosis
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CLL MCL
V
HCL SLVL
HCL-v

/\
morphology Cleaved cells
immunology t (14;18)
BCL2 overexpression
typical atypical
I

FIGURE 9 Decisional tree for the diagnosis of the B-CLDs

feeling is that two main issues could now be raised.
The first one: unfortunately, no marker has absolute
diagnostic value, nevertheless a combination of sev-
eral molecules is helpful even though, as evidenced
by Table 111, too +/- associated signs are still present.
The development of new more disease-specific
MoAbs will certainly provide to simplify this scheme
in the future. In addition, we firmly believe that a piv-
otal role will be played by the quantification of B-cell
antigens, such as CD20 (8 I,84). In our hands the anti-
body binding capacity (ABC) of B-cells is more rep-
resented in CDS-negative B-CLDs as well as in
B-CLL with deviation from the typical morphology
and immunophenotype with respect to the typical
forms of the disease (Fig. 8) (37). Finally, it is clear
that the questions raised indicate the need of an inte-
grated diagnostic approach to these disease entities
under discussion in order to differentiate more accu-
rately the several subtypes and better understand
some of their clinical manifestations in order to treat
them correctly.
 234 GIOVANNI D'ARENA et al.

TABLE 111 Immunophenotypes of B-cell CLDs

Condition Smlg CD5 CD22 CD23 FMC7 CD79b CD103 CD25 CDllc CDlO
CLL -I+ + -I+ + -I+ -I+
PLL ++ -I+ + -I+ + -

HCL ++ - + - + +

HCL-v ++ - + - + +

SLVL ++ -I+ + -I+ + +I-

FL ++ -I+ +I- - + -
MCL ++ + +I- +I- + -

MZL ++ - +I- - + +I-

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