Leukemia & Lymphoma, November 2013; 54(11): 23772384

2013 Informa UK, Ltd.

ISSN: 1042-8194 print / 1029-2403 online
DOI: 10.3109/10428194.2013.780653

REVIEW

The role of bone marrow biopsy examination at diagnosis of chronic

lymphocytic leukemia: a reappraisal
Panagiotis Baliakas1, George Kanellis2, Niki Stavroyianni1, Maria Fameli2, Achilles Anagnostopoulos1,
Kostas Stamatopoulos1,3 & Theodora Papadaki2
1Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece, 2Hematopathology Department,

Evangelismos Hospital, Athens, Greece and 3Institute of Applied Biosciences, Center for Research and Technology Hellas,
Thessaloniki, Greece

including the Toll-like receptors, CD40 and chemokine

receptors, as well as the antigen-specific B cell receptor (BcR),
also known as clonotypic immunoglobulin (BcR IG) [3]. The
importance of BcR-mediated signals in CLL ontogeny is
underscored by: (i) remarkable biases in the immunoglobulin (IG) gene repertoire [5,6]; (ii) the subdivision of CLL into
two major categories with different biological and clinical
behavior depending on the extent of somatic hypermutation
in the clonotypic rearranged IG heavy variable (IGHV) genes
[7,8]; and (iii) the existence of distinct BcR IGs repeated with
little or even no variation in different subsets of patients with
CLL (stereotyped BcRs) [6,911].
CLL is very heterogeneous in terms of both the clinical course and the response to treatment, likely reflecting
its underlying biological and genetic heterogeneity [12].
In recent years, the progressive increase in understanding
CLL has translated into a biologically oriented assessment
of clinical prognosis [13,14]. In particular, there has been
major progress in the identification of immunophenotypic
and molecular genetic markers that: (i) predict the tendency
for disease progression in CLL; and (ii) assist in the rational
design of risk-adapted therapies [1315].
Numerous biological markers, including genomic abnormalities identified by classic cytogenetic analysis, fluorescence in situ hybridization (FISH) or high-resolution single
nucleotide polymorphism (SNP) arrays, intracellular or
surface proteins, recurrent gene mutations (TP53, ATM,
NOTCH1, SF3B1 and BIRC3), many unveiled very recently
through the application of high-thoughput sequencing
technology [1624], and IGHV gene mutational status, have
been shown to predict outcome in early stage CLL. Of these,
mainly genomic aberrations and IGHV gene mutational status provide the most consistent information while, besides
their clinical utility, they also offer important insight into the
pathogenesis of the disease [25].
For instance, it is now established that patients with deletion of chromosome 17p [26] and/or mutations of the TP53

Abstract
According to the International Workshop on Chronic Lymphocytic
Leukemia/National Institutes of Health (iwCLL/NIH) guidelines
for the diagnosis and treatment of chronic lymphocytic
leukemia (CLL), bone marrow biopsy (BMB) is not required at
diagnosis, however recommended before initiating treatment.
That notwithstanding, histopathological examination of the
BMB has the potential to provide important information of
both clinical and biological significance. Here we attempt a
reappraisal of the role of BMB examination in the modern
diagnostic work-up of patients with CLL, based on both the
literature and our accumulated experience from the systematic
and multiparametric evaluation of a large series of BMB samples
taken at diagnosis of CLL. Overall, we argue that the study of BMB
offers important information not only for diagnostic purposes
but also for elucidating CLL pathobiology.
Keywords: CLL, bone marrow biopsy, immunohistochemistry,
autoimmunity, chronic lymphocytic leukemia

Introduction
Chronic lymphocytic leukemia (CLL) is a disease of aged
populations and the most common adult leukemia in the
Western world [1]. CLL is characterized by the in vivo accumulation of CD5  monoclonal B cells in peripheral lymphoid organs, bone marrow (BM) and peripheral blood (PB).
In recent years, the concept that CLL represents primarily an
accumulative disease has been fully revisited on the basis of
seminal findings showing that a sizeable proportion of the
entire malignant clone can be recycled daily [2]. Nowadays,
CLL is considered a dynamic disease driven by both intrinsic
(genetic) and extrinsic (microenvironmental) factors [3,4].
CLL development and evolution is promoted by interactions of the clonogenic progenitors or even the malignant
cells themselves with other cells and soluble factors within
their microenvironment through various receptor systems,

Correspondence: Kostas Stamatopoulos, MD, PhD, Hematology Department and HCT Unit, G. Papanicolaou Hospital, 57010 Exokhi, Thessaloniki, Greece.
Tel:  30-2313-307992. Fax:  30-2313-307579. E-mail: kostas.stamatopoulos@gmail.com
Received 18 August 2012; revised 21 February 2013; accepted 24 February 2013

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2378 P. Baliakas et al.

gene [27], disrupting the expression and function of the p53
protein, experience aggressive disease with a distinctive
resistance to standard chemotherapy regimens using alkylating drugs and/or purine analogs. Furthermore, genetic
dissection of other loci frequently implicated in genomic
aberrations in CLL has identified genes with important roles
in CLL biology; for example the mir-15a, mir-16-1, DLEU-2
and DLEU-7 genes, which are removed from chromosome 13
in cases carrying deletion of chromosome 13q, are now known
to be part of a relevant pathway in CLL development [28,29].
All that notwithstanding, as of current writing, the decision about which laboratory examinations and tests should
be considered essential for patients with CLL remains undefined, although relevant guidelines were published by the
International Workshop on Chronic Lymphocytic Leukemia/
National Insititutes of Health (iwCLL/NIH) in 2008 [30]. The
issue is confounded by the lack of large prospective studies
with long follow-up that include a range of both clinical and
biological markers. This is critical for eventually realizing the
objective of personalized treatment, thus maximizing clinical
benefit while minimizing unnecessary cost and unwanted
toxicities.

status quo at diagnosis. Third, it precludes a complete appreciation of the clinical significance of CLL-like monoclonal B
lymphocytosis (MBL), at least in cases with a large clonal size
(clinical MBL) [32,33].
Here we reappraise the role of BMB examination in the
diagnostic work-up of patients with CLL based on both the
literature and our extensive experience from the systematic
examination of diagnostic samples from a large and unselected CLL cohort (Supplemental material: Patient cohort
and Supplemental Tables I and II to be found online at http://
informahealthcare.com/lal/doi/10.3109/10428194.2013.
780653), well characterized from both a clinical and a biological perspective. The large size of the cohort and the multidisciplinarity of our approach provided a unique opportunity
to obtain some answers to the outstanding questions about
the significance and utility of BMB examination in CLL. This
opportunity is unprecedented, since published histopathological studies of BMB in CLL antedate the era of biologically
oriented patient stratification, and are also quite difficult
to recapitulate, at least in the foreseeable future, due to the
paucity of diagnostic specimens as a result of adoption of the
iwCLL/NCI guidelines [30].

Do we need examination of the bone marrow biopsy at

diagnosis of CLL? Past and present concepts

Value of bone marrow histopathology at diagnosis of CLL:

what can we learn?

Histopathological and immunohistochemical examination of the bone marrow biopsy (BMB) is of great value in
the diagnostic work-up and overall evaluation of B-cell
malignancies, including CLL [31]. Until the late 1990s,
BMB examination was considered essential in the diagnostic work-up of CLL. However, more recently, this practice
has gradually faded and been partially if not altogether
abandoned. In fact, the 2008 iwCLL/NCI guidelines [30]
proclaim that a marrow aspirate and biopsy generally
are not required for the diagnosis of CLL and should be
reserved for evaluating factors that might contribute to
cytopenias (anemia, thrombocytopenia) that may or may
not be directly related to leukemia-cell infiltration of the
marrow. According to the guidelines, further indications
for performing a BMB are: (i) clinical progression prior to
therapy initiation; and (ii) assessment of treatment, at least
within the context of clinical trials. With hindsight, a possible reason for this paradigm shift was the feeling that
the prognostic value of BM biopsy may now be superseded
by new prognostic markers (verbatim from the iwCLL/NCI
guidelines) [30]. However, it is relevant to mention that at
the time of publication of the guidelines, only limited published information was available regarding the precise role
of BMB examination in the frame of modern diagnostics
and prognostication of CLL.
As will hopefully be evident in the sections that follow,
although practical, the approach recommended by the
iwCLL/NCI leaves many open issues. First, it hinders the
in situ assessment of CLL within the compartment where the
clonal B cell population may arise, which is relevant for better appreciating the crosstalk of the neoplastic B cells with
other immune and non-immune cells. Second, it might give
a misleading view of tumor dynamics, as analyzing the BM at
disease progression is not necessarily representative of the

Although not required for the diagnosis of CLL (2008

iwCLL/NCI guidelines) [30], assessment of good-quality
BMB sections, both morphologically (architecture/cytology)
and immunohistochemically, is of help in the diagnostic
work-up of patients with CLL by identifying parameters with
complementary diagnostic and perhaps also prognostic
significance that cannot be reliably appreciated by other
methods. A thorough examination entails: (i) detailed characterization of the neoplastic lymphocytic infiltration; and
(ii) careful assessment of residual hematopoietic reserves.

Neoplastic lymphocytic inltration

Extent
The extent of the neoplastic infiltrate of the BM in CLL is
much more reliably assessed in core biopsy specimens
compared to aspirate smears due to the multifocal nature of
the lymphocytic infiltration with expected variability in the
lymphocytic content of the smears, especially if the CLL cells
constitute less than 30% of total cells.
In our series of 159 diagnostic BMB specimens from
unselected patients with CLL, all cases showed neoplastic
lymphocytic infiltration ranging from only 5% (single case)
to greater than 90% of BM cellularity (Supplemental Table II
to be found online at http://informahealthcare.com/lal/doi/
10.3109/10428194.2013.780653). Importantly, overall, 33/156
cases (20%) with an unequivocal diagnosis of CLL according to the 2008 iwCLL/NCI criteria [30], including clonal B
lymphocytosis of greater than 5.0  109/L, showed a neoplastic lymphocytic infiltration of less than 30% (Figure
1; Supplemental Table III to be found online at http://
informahealthcare.com/lal/doi/10.3109/10428194.2013.
780653). This finding alludes to distinct compartmentalization profiles of the clonal population and questions the value

Bone marrow biopsy in CLL

2379

Figure 1. Minimal neoplastic lymphocytic infiltration of the bone marrow at diagnosis of CLL. Two cases of CLL (A and B) with minimal BM
involvement and interstitial pattern of infiltration (CD23).

of mathematical cut-offs in describing a biological phenomenon. Further support for the latter notion is provided by the
fact that two-thirds of clinical MBL cases from our series
(clonal B cell count: 2.8 and 4.1  109/L, respectively) that
eventually progressed to CLL had pronounced neoplastic
lymphocytic infiltration at diagnosis of MBL.

Pattern of bone marrow infiltration

A great advantage of the BMB over the aspirate smear is
that only the former allows identification of the pattern of
neoplastic lymphocytic infiltration of the BM (diffuse versus
non-diffuse), which, when combined with immunophenotypic information, can be useful in both distinguishing CLL
from other neoplastic small cell lymphocytic infiltrations of
the BM and providing prognostic information.
In CLL, the BMB may exhibit different patterns of neoplastic infiltration, including: (i) focal, non-paratrabecular
nodules (nodular pattern) in which normal hematopoietic
tissue is replaced but the architecture is preserved, while
no interstitial pattern is observed; (ii) an interstitial infiltrate, in which CLL cells are admixed with hematopoietic
elements (interstitial pattern); and (iii) diffuse solid lesions
with complete replacement of both hematopoietic and fat
cells (diffuse pattern). It is common to see multiple patterns
co-exist in a single BMB specimen (mixed pattern) [3438].
Likewise, in our study of 159 cases, the patterns of infiltration
designated in BMBs were as follows (Figure 2, Supplemental
Table II to be found online at http://informahealthcare.com/
lal/doi/10.3109/10428194.2013.780653): (i) nodular: 21
cases; (ii) interstitial: 56 cases; (iii) diffuse: 40 cases, presenting with advanced disease (Binet stage B or C at diagnosis)
significantly more frequently (p  0.001) than cases assigned
to the other groups; and (iv) mixed: 42 cases. Cases with a
diffuse pattern of infiltration were characterized by extensive
neoplastic lymphocytic infiltration (median: 80%, range,
5098%); those with a nodular pattern had less involvement
(median: 50%, range, 1580%), while the mixed pattern
showed variable involvement (median: 50%, range, 595%).
It is relevant to mention that the interstitial pattern predominated among cases with less than 30% neoplastic lymphocytic infiltration (22/33 cases; Supplemental Table III to be
found online at http://informahealthcare.com/lal/doi/10.
3109/10428194.2013.780653).
Studies from the 1980s have reported that the diffuse pattern of infiltration is associated with a worse prognosis and

the non-diffuse (especially the nodular pattern) with a better

prognosis; however, thus far, limited biological sub-context
has been available for this observation [34,38,39]. We sought
to obtain more insight into this issue by searching for potential associations with biological features, available in all cases
of our series. From this analysis, it clearly emerged that cases
with nodular infiltrates had a distinctive biological profile
characterized by significantly more frequent expression of
mutated IGHV genes and, in contrast, significantly lower
prevalence of prognostically adverse biological prognostic
markers compared to cases of the other groups, especially
those with a diffuse pattern of infiltration (Supplemental
Table IV to be found online at http://informahealthcare.com/
lal/doi/10.3109/10428194.2013.780653). It should be emphasized, however, that the pattern of neoplastic lymphocytic
infiltration did not retain statistical significance as a prognostic parameter in a multiparametric analysis (multivariate
Cox regression analysis) including all factors with significant
associations, being superseded by IGHV gene mutational status and clinical stage. There are several possibilities for this,
including: (i) the relatively small (for statistical purposes)
number of cases in each subgroup; (ii) the fact that, despite
the observed associations, a sizeable minority of cases with
nodular pattern of infiltration carry unmutated IGHV genes,
while on the other hand, a proportion of cases with diffuse
pattern of infiltration are at early clinical stages at diagnosis.
Nevertheless, these results offer for the first time a biological explanation for the long appreciated favorable prognostic
implications of the nodular architectural pattern. Furthermore, they have an important practical implication in showing that an old-fashioned approach with relatively low cost
can yield prognostically useful information, to a large extent
equally valid to that obtained by more sophisticated techniques. Therefore, when determination of modern biological prognostic parameters is not possible (which is relevant
for, yet not necessarily limited to, the developing world), it
is reassuring to know that BMB examination may still offer
reliable hints as to the eventual clinical outcome.

Cytology/immunohistology
As defined in the World Health Organization (WHO) classification [31], CLL/small lymphocytic lymphoma (SLL) is a neoplasm of small lymphocytes, slightly larger than the normal
lymphocyte, with clumped chromatin, usually around the
nucleus, scant cytoplasm with low mitotic activity, admixed

2380

P. Baliakas et al.

Figure 2. Patterns of bone marrow infiltration in CLL. (A) Diffuse (CD79), (B) interstitial (CD20), (C) pure nodular (CD20) and (D) mixed nodular
and interstitial (arrow) (CD20).

with a very low number ( 2%) of prolymphocytoid cells

and scarce blast cells (paraimmunoblasts) forming proliferation centers in tissue infiltrates. Our findings are in keeping with these observations in that the vast majority of 159
evaluated cases exhibited neoplastic infiltration consisting
almost exclusively of small, well-differentiated lymphocytes
admixed with scattered ( 2%) medium and/or large-sized
lymphocytes [Figure 3(A)].
According to the WHO classification [31], CLL cells are
identified immunophenotypically as positive for: (i) monotypic surface immunoglobulin: weak expression; (ii) CD5,
CD23, CD19, CD79: strong expression; (iii) CD20, CD22:
weak expression. In contrast, they are generally negative for
cytoplasmic immunoglobulin (CIg) and CD11c, CD79, CD25
and FMC7. With very few exceptions, mainly atypical CLL
(see section below), our experience confirms these observations. Indeed, in almost all cases, the neoplastic lymphocytes
expressed CD20, CD79a, CD5 and CD23. Only two and three
cases were negative for CD5 and CD23, respectively; however,
none was double-negative and, collectively, all five such cases
tested positive for both markers in peripheral blood immunophenotyping by flow cytometry. This apparent discrepancy may be due to technical issues or, alternatively, linked
to modulation of cell surface marker expression in different
compartments and/or microenvironments. Interestingly,
great variation was observed regarding CD20 versus CD79a
positivity, both quantitatively (proportion of positive cells for
either marker) and qualitatively (intensity of staining); in particular, discordant results were obtained in 43 cases, which
were characterized by increased proportions and/or intensity
of staining of CD79 vs. CD20 cells (Figure 4).

The aforementioned morphologic and immunophenotypical findings characterize the so-called classical or prototypic
CLL. Recently, however, morphologic and immunophenotypic variants of CLL were recognized that deviate from the
morphology and immunophenotype of classical CLL [40]. The
identification of these variants is best assessed in good-quality
BMB specimens and is not only of pathobiological value
but also important for: (i) establishing the diagnosis of CLL
and its differential diagnosis from other diseases mimicking
normal CLL; and (ii) deciding the optimal therapeutic
approach. The most common morphological variants of CLL
include: (i) atypical CLL; and (ii) CLL with plasmacytoid
differentiation. In addition, very rarely, patients with CLL at
diagnosis may exhibit ReedSternberg cells (see below) [40].

Atypical CLL
The term atypical CLL refers to cases that morphologically
resemble CLL yet are also characterized by the presence of
subpopulations (1015%) of neoplastic cells that are larger,
exhibit greater nuclear irregularity with occasional monocytoid features [6/159 (3.7%) cases in our series; Figure 3(B)],
display a prominent nucleolus typical of prolymphocytes or
have plasmacytoid features [4143].
Cases with atypical morphology often exhibit an immunophenotypic profile that deviates from the prototypic CLL and
raise problems in differential diagnosis from other small cell
B-cell lymphomas, especially mantle cell lymphoma (MCL),
lymphoplasmacytic lymphoma or marginal zone lymphoma
[44]. Besides identification of the pattern of neoplastic infiltration, which is indicative (although not pathognomonic)
for the abovementioned entities, BMB immunohistology

Bone marrow biopsy in CLL

2381

Figure 3. Cytology of bone marrow infiltrate in CLL. (A) Predominantly small neoplastic lymphocytes with scant cytoplasm and a single
paraimmunoblast with dispersed chromatin and central nucleolus (arrow) (hematoxylin and eosin stain). (B) Monocytoid-like cells with clear
cytoplasm (hematoxylin and eosin stain). (C) Plasmacytoid differentiation (arrow) (IgM immunostain). (D) Plasmacytic differentiation with lambda
light chain restriction (plasma cell expressing kappa light chain shown in inset).

with a broad panel of monoclonal antibodies is of great help

in the differential diagnosis of atypical CLL from these entities, especially thanks to its unique advantage of confirming
monoclonal surface and/or cytoplasmic immunoglobulin.
One particularly challenging issue concerns the distinction
between atypical CLL and MCL, because some atypical CLL
cases may indeed have an immunophenotype that mimics
MCL. Assessment of cyclin D1 expression by immunohistology in paraffin-embedded BMB sections is required for this
distinction. Given that CLL/atypical CLL is almost always
cyclin D1-negative, the detection of cyclin D1 positivity in the
neoplastic lymphocytes in association with the other immunophenotypic markers helps in the confirmation of MCL,
with its well-known prognostic and therapeutic implications.

CLL with plasmacytoid differentiation

This rare CLL variant of the WHO classification is synonymous with lymphoplasmacytoid immunocytoma of the Kiel
classification [45]. It has been reported to account for a small
subset, rougly 3% of patients with CLL, usually positive for
serum IgM paraprotein at a low concentration ( 3 g/dL)
[46]. In our series, 8/159 (5%) cases exhibited plasmacytoid
differentiation; interestingly, 5/8 such cases carried unmutated IGHV genes and experienced rapid clinical progression with a median time to first treatment of 16 months.
Cases of CLL with plasmacytoid differentiation consist
predominantly of small lymphocytes, with up to 25% or more
exhibiting plasmacytoid differentiation [Figure 3(C)] [46,47].
The plasmacytoid cells are only marginally larger than normal

lymphocytes and, in contrast to normal lymphocytes, are

characterized by moderately-to-strongly basophilic cytoplasm. Such cells are difficult to recognize and, for this
reason, often overlooked in Giemsa-stained smears. The
histology and immunophenotype of this variant are identical
to those of classical CLL, with their main distinction being
the strong expression of CIg in the plasmacytoid lymphocytes [48]. Recently, there have been case reports of CLL with
plasmacytoid differentiation carrying the cytogenetic abnormality del(7)(q32) [49], but other abnormalities have been
reported as well, including trisomy 12 [26]. Although earlier
studies reported that patients with this variant of CLL have
an inferior survival compared with typical cases, other studies have not confirmed this observation [15,42,5054].
Discriminating CLL with plasmacytoid differentiation from lymphoplasmacytic lymphoma is mandatory
because of their distinct clinical courses and very different management. Again, morphology and immunohistology of BMB tissue sections offers important supportive
evidence to establish a correct diagnosis. In CLL with
plasmacytoid differentiation, BM immunohistology will
identify monoclonal plasmacytoid lymphocytes; however,
no plasma cells of the same clonality will be present. In
contrast, in lymphoplasmacytic lymphoma the neoplastic
lymphocytes and lymphoplasmacytoid lymphocytes are
always CD5  and rarely CD23, and by definition associated with clonal plasma cells of the same clonality as the
lymphocytes and plasmacytoid lymphocytes of the neoplastic infiltration.

2382

P. Baliakas et al.

Figure 4. Discordant expression of CD20 and CD79 in bone marrow CLL infiltrate. (A) CD20 expression: a number of neoplastic lymphocytes do
not express CD20 (membranous staining); and (B) increased CD79 expression (cytoplasmic staining).

Finally, there are cases of CLL/SLL with plasmacytic differentiation [Figure 3(D)], some of which are associated with
aberrations of chromosome 1p [55]. Their frequency is indeed
very low (only 3/159, 1.8%, cases of our series); however, they
also pose a problem of differential diagnosis from lymphoplasmacytic lymphoma.

Transformation at diagnosis of CLL

Transformation of CLL into a biologically more aggressive
neoplasm is a rare event [56,57]. However, its true incidence
may be higher than anticipated, as post-mortem examination is not performed in most patients today, thus underestimating occult disease. Although transformation in CLL
predominates in extramedullary sites and usually occurs
in patients with long-standing disease [56,57], in very rare
cases large-cell transformation can be detected in BMB
specimens taken at diagnosis of CLL, even in patients with
early stage before any treatment. In these cases, it can take
the form of discrete foci of large cell lymphoma infiltration
alongside typical CLL infiltrates, without peripheral blood
involvement. In a proportion of such cases, immunohistology can strongly indicate the clonal association of both entities by identifying the same monoclonal surface/cytoplasmic
immunoglobulin.
Examination of the BMB is the only diagnostic approach
that can reveal and confirm the very rare cases of CLL coexistence with Hodgkin lymphoma (HL), even in early stage
CLL. In such cases, great caution should be given to differentiating ReedSternberg (RS)-like cells sparsely admixed with
otherwise typical CLL cells, from HL transformation [5861].
In the former situation, RS-like cells are not associated with
a typical HL milieu. In sharp contrast, in HL transformation
at diagnosis of CLL, examination of the BMB confirms the
close association of CLL with foci of effacement of normal
BM architecture by a typical HL milieu, which is essential for
establishing the diagnosis and documentation of HL, especially in extranodal sites. In both situations, however, RS-like
cells can be positive for EpsteinBarr virus (EBV) RNA, indicating an association with EBV infection [56,59].

Immunohistological evaluation of residual

hematopoietic reserves
At diagnosis, most patients with CLL are asymptomatic and
have preserved hematopoietic reserves. Any case presenting

with anemia and/or thrombocytopenia is stratified to Binet

stage C/Ray stage IIIIV. In such cases, BMB immunohistology
is of great value in discriminating the implicated pathophysiologic mechanisms by offering a unique opportunity for reliable in situ evaluation of the hematopoietic reserves.
The underlying cause(s) of cytopenias in CLL may differ
from case to case, extending from pronounced reduction of
the hematopoietic marrow due to massive BM infiltration by
the neoplastic lymphocytic population to, less frequently,
immune-mediated destruction of one or more hematopoietic series. Among the wide spectrum of autoimmune phenomena reported in CLL, hematopoietic autoimmunity is by
far the most common, manifesting as autoimmune hemolytic anemia (AIHA), immune thrombocytopenic purpura
(ITP), AIHA  ITP (Evans syndrome) or pure red cell aplasia (PRCA) [62]. In CLL associated with either AIHA or ITP,
BMB immunohistology usually reveals hyperplasia of the
erythroid and megakaryocytic series. These morphological
findings at diagnosis of CLL, before any treatment, could be
interpreted as autoimmunity-related; however, in our experience, similar findings can often be identified in the diagnostic BMBs of CLL cases with no anemia or thrombocytopenia.
Overall, these observations in CLL recall what has previously
been reported for other low-grade B-cell malignancies, e.g.
hairy cell leukemia (HCL), lymphoplasmacytic lymphoma
and Hodgkin lymphoma [6365]. The underlying cause and
mechanism(s) are currently unknown and require further
systematic investigation, ideally in prospective cohorts.

Numerical cut-offs: do they mean something?

The fact that a sizeable proportion of cases in our series showed
a degree of infiltration of less than 30% is notable, given that all
but one fulfilled the iwCLL/NIH criteria [30] for typical CLL,
whereas the remaining case was classified as clinical MBL
with a clonal B cell count at diagnosis of 3.8  109/L. Although
this finding could potentially be related to sampling or processing issues, this possibility is rather remote for the following reasons: (i) all BMB samples were processed in a uniform fashion
throughout the duration of the study; and (ii) the BMB trephines
of all cases with limited neoplastic lymphocytic infiltration were
of good quality and deemed satisfactory for histopathological
evaluation. Hence, it would not be unreasonable to propose
that this finding might have a biological explanation, perhaps
reflecting a focal pattern of BM infiltration or, alternatively, a
distinctive compartmentalization of the neoplastic clone.

Bone marrow biopsy in CLL

The jury is still out as to which of these (or other, yet to
be defined) options could represent a plausible explanation.
That notwithstanding, these observations confirm the general rule that mathematical cut-offs should be viewed with
caution when dealing with biological phenomena. In addition, they suggest that a reappraisal of the 2008 WHO criteria
[31] for the histopathological diagnosis of CLL might be warranted. This is especially timely now that clinical MBL is separated from CLL solely on the basis of a mathematical cut-off
value [30,32,66], although, as evidenced by this and previous
studies, it may exhibit pronounced lymphocytic infiltration
of the BMB that would be considered as unequivocally
neoplastic on histopathological examination [67].

Conclusions
Detailed BMB examination at diagnosis of CLL contributes
to the accurate determination of the extent, pattern cytology
and immunohistology of neoplastic lymphocytic infiltration
of the BM, and the differential diagnosis of CLL from malignant and benign mimickers such as leukemic manifestations
of B-cell lymphomas with concordant or discordant BM
morphology and persistent polyclonal B-cell lymphocytosis
(PPBL), respectively. Furthermore, detailed immunohistology of the BM is of great importance in unraveling foci of
large cell lymphoma transformation even at diagnosis of
CLL before the administration of treatment, as well as the
co-existence of CLL with either other B- or T-lymphoproliferative disorders, or other malignant diseases of hematopoietic
or non-hematopoietic origin [3537,39].
Equally important, the reliable estimation of residual
hematopoietic reserves, realized only through histopathological examination of the BMB, has important practical
implications for the design of treatment in cases with cytopenias [30]. By logical extension, it is expected to contribute to
improved understanding of the mode of action of novel therapies targeting the interaction of CLL cells and their (micro)
environment [68,69].

Acknowledgements
The authors wish to thank Drs. Anastasia Athanasiadou,
Chrysanthi Vadikolia, Chrysavgi Lalayanni and Riad Saloum
for stimulating discussions and their long-standing collaboration, and Evangelia Stalika for expert technical support.
Potential conflict of interest: Disclosure forms provided
by the authors are available with the full text of this article at
www.informahealthcare.com/lal.
This work was supported in part by Cariplo Foundation
(Milan, Italy), and the ENosAI project (code 09SYN-13-880),
co-funded by the EU and the Hellenic General Secretariat for
Research and Technology.

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Details of patient cohort and tables showing results

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