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Leukemia & Lymphoma, November 2013; 54(11): 2377–2384 © 2013 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2013.780653


/ 1029-2403 online DOI: 10.3109/10428194.2013.780653 REVIEW The role of bone marrow biopsy examination at diagnosis of

The role of bone marrow biopsy examination at diagnosis of chronic lymphocytic leukemia: a reappraisal

Panagiotis Baliakas 1 , George Kanellis 2 , Niki Stavroyianni 1 , Maria Fameli 2 , Achilles Anagnostopoulos 1 , Kostas Stamatopoulos 1,3 & Theodora Papadaki 2

Kostas Stamatopoulos 1 , 3 & Theodora Papadaki 2 1 Hematology Department and HCT Unit, G.

1 Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece, 2 Hematopathology Department, Evangelismos Hospital, Athens, Greece and 3 Institute of Applied Biosciences, Center for Research and Technology Hellas, Thessaloniki, Greece

Abstract According to the InternationalWorkshop 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.

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 immunoglobu- lin (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) [6,9 – 11]. CLL is very heterogeneous in terms of both the clini- cal 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 [13–15]. Numerous biological markers, including genomic abnor- malities identified by classic cytogenetic analysis, fluores- cence 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 [16–24], and IGHV gene mutational status, have been shown to predict outcome in early stage CLL. Of these, mainly genomic aberrations and IGHV gene mutational sta- tus 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 dele- tion of chromosome 17p [26] and/or mutations of the TP53

Keywords: CLL , bone marrow biopsy , immunohistochemistry , autoimmunity , chronic lymphocytic leukemia


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 accu- mulation of CD5 monoclonal B cells in peripheral lym- phoid 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 interac- tions of the clonogenic progenitors or even the malignant cells themselves with other cells and soluble factors within their microenvironment through various receptor systems,

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 alky- lating 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 deci- sion about which laboratory examinations and tests should be considered essential for patients with CLL remains unde- fined, 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.

Do we need examination of the bone marrow biopsy at diagnosis of CLL? Past and present concepts

Histopathological and immunohistochemical examina- tion 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 diagnos- tic 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 pos- sible 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 pub- lished 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 bet- ter 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

status quo at diagnosis. Third, it precludes a complete appre- ciation 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 unse- lected CLL cohort (Supplemental material: Patient cohort and Supplemental Tables I and II to be found online at http://

780653), well characterized from both a clinical and a biolog- ical perspective. The large size of the cohort and the multidis- ciplinarity 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 histopatho- logical 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].

Value of bone marrow histopathology at diagnosis of CLL:

what can we learn?

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 char- acterization of the neoplastic lymphocytic infiltration; and (ii) careful assessment of residual hematopoietic reserves.

Neoplastic lymphocytic infiltration


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 10.3109/10428194.2013.780653). Importantly, overall, 33/156 cases (20%) with an unequivocal diagnosis of CLL accord- ing to the 2008 iwCLL/NCI criteria [30], including clonal B lymphocytosis of greater than 5.0 10 9 /L, showed a neo- plastic lymphocytic infiltration of less than 30% (Figure 1; Supplemental Table III to be found online at http://

780653). This finding alludes to distinct compartmentaliza- tion profiles of the clonal population and questions the value

Bone marrow biopsy in CLL


Bone marrow biopsy in CLL 2379 Figure 1. Minimal neoplastic lymphocytic infi ltration of the bone

Figure 1. Minimal neoplastic lymphocytic infi ltration 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 phenom-

enon. 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 10 9 /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 immunopheno- typic 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 neo- plastic 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 infil- trate, 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) [34–38].

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 lal/doi/10.3109/10428194.2013.780653): (i) nodular: 21 cases; (ii) interstitial: 56 cases; (iii) diffuse: 40 cases, present- ing 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, 50–98%); those with a nodular pattern had less involvement (median: 50%, range, 15–80%), while the mixed pattern showed variable involvement (median: 50%, range, 5–95%). It is relevant to mention that the interstitial pattern predomi- nated among cases with less than 30% neoplastic lympho- cytic infiltration (22/33 cases; Supplemental Table III to be found online at


Studies from the 1980s have reported that the diffuse pat- tern 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 poten- tial 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 lal/doi/10.3109/10428194.2013.780653). It should be empha- sized, however, that the pattern of neoplastic lymphocytic infiltration did not retain statistical significance as a prog- nostic parameter in a multiparametric analysis (multivariate Cox regression analysis) including all factors with significant associations, being superseded by IGHV gene mutational sta- tus 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 infi ltration 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 biologi- cal explanation for the long appreciated favorable prognostic implications of the nodular architectural pattern. Further- more, they have an important practical implication in show- ing 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 tech- niques. Therefore, when determination of modern biologi- cal 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.


As defined in the World Health Organization (WHO) classifi- cation [31], CLL/small lymphocytic lymphoma (SLL) is a neo- plasm 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.

2380 P. Baliakas et al. Figure 2. Patterns of bone marrow infi ltration in CLL. (A)

Figure 2. Patterns of bone marrow infi ltration in CLL. (A) Diff use (CD79 α ), (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 prolif- eration centers in tissue infiltrates. Our findings are in keep- ing 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) mono- typic surface immunoglobulin: weak expression; (ii) CD5, CD23, CD19, CD79α: strong expression; (iii) CD20, CD22:

weak expression. In contrast, they are generally negative for cytoplasmic immunoglobulin (CIg) and CD11c, CD79β, CD25 and FMC7. With very few exceptions, mainly “atypical” CLL (see section below), our experience confirms these observa- tions. 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 immu- nophenotyping by flow cytometry. This apparent discrep- ancy 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 par- ticular, discordant results were obtained in 43 cases, which were characterized by increased proportions and/or intensity of staining of CD79α vs. CD20 cells (Figure 4).

The aforementioned morphologic and immunophenotypi- cal findings characterize the so-called classical or prototypic CLL. Recently, however, morphologic and immunopheno- typic 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 Reed–Sternberg 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 (10–15%) of neoplastic cells that are larger, exhibit greater nuclear irregularity with occasional monocy- toid features [6/159 (3.7%) cases in our series; Figure 3(B)], display a prominent nucleolus typical of prolymphocytes or have plasmacytoid features [41–43]. Cases with atypical morphology often exhibit an immuno- phenotypic 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 infil- tration, which is indicative (although not pathognomonic) for the abovementioned entities, BMB immunohistology

Bone marrow biopsy in CLL


Bone marrow biopsy in CLL 2381 Figure 3. Cytology of bone marrow infi ltrate in CLL.

Figure 3. Cytology of bone marrow infi ltrate 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 enti- ties, 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 immunohistol- ogy 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 immu- nophenotypic 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 synony- mous 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 unmu- tated IGHV genes and experienced rapid clinical progres- sion 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 cyto- plasm. 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 lympho- cytes [48]. Recently, there have been case reports of CLL with plasmacytoid differentiation carrying the cytogenetic abnor- mality 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 stud- ies have not confirmed this observation [15,42,50–54]. Discriminating CLL with plasmacytoid differentia- tion from lymphoplasmacytic lymphoma is mandatory because of their distinct clinical courses and very diffe- rent management. Again, morphology and immunohisto- logy 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 associ- ated with clonal plasma cells of the same clonality as the lymphocytes and plasmacytoid lymphocytes of the neo- plastic infiltration.

2382 P. Baliakas et al.

2382 P. Baliakas et al. Figure 4. Discordant expression of CD20 and CD79 α in bone

Figure 4. Discordant expression of CD20 and CD79α in bone marrow CLL infiltrate. (A) CD20 expression: a number of neoplastic lymphocytes do not express CD20 (membranous staining); and (B) increased CD79α expression (cytoplasmic staining).

Finally, there are cases of CLL/SLL with plasmacytic dif- ferentiation [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 lymphop- lasmacytic 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 examina- tion is not performed in most patients today, thus under- estimating 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, immunohistol- ogy can strongly indicate the clonal association of both enti- ties 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 co- existence with Hodgkin lymphoma (HL), even in early stage CLL. In such cases, great caution should be given to differen- tiating Reed–Sternberg (RS)-like cells sparsely admixed with otherwise typical CLL cells, from HL transformation [58–61]. 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, espe- cially in extranodal sites. In both situations, however, RS-like cells can be positive for Epstein–Barr virus (EBV) RNA, indi- cating 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 III–IV. In such cases, BMB immunohistology

is of great value in discriminating the implicated pathophysi-

ologic mechanisms by offering a unique opportunity for reli- able 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 hematopoi- etic series. Among the wide spectrum of autoimmune phe- nomena reported in CLL, hematopoietic autoimmunity is by far the most common, manifesting as autoimmune hemo- lytic anemia (AIHA), immune thrombocytopenic purpura (ITP), AIHA ITP (Evans’ syndrome) or pure red cell apla- sia (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 experi- ence, similar findings can often be identified in the diagnos- tic 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 [63–65]. 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 classifi ed as “ clinical ” MBL with a clonal B cell count at diagnosis of 3.8 10 9 /L. Although this finding could potentially be related to sampling or process- ing issues, this possibility is rather remote for the following rea- sons: (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.

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 gen- eral rule that mathematical cut-offs should be viewed with caution when dealing with biological phenomena. In addi- tion, they suggest that a reappraisal of the 2008 WHO criteria [31] for the histopathological diagnosis of CLL might be war- ranted. This is especially timely now that clinical MBL is sep- arated 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].


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 malig- nant 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 immunohisto- logy 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-lymphoprolifer- ative disorders, or other malignant diseases of hematopoietic or non-hematopoietic origin [35–37,39]. Equally important, the reliable estimation of residual hematopoietic reserves, realized only through histopatho- logical examination of the BMB, has important practical implications for the design of treatment in cases with cytope- nias [30]. By logical extension, it is expected to contribute to improved understanding of the mode of action of novel ther- apies targeting the interaction of CLL cells and their (micro) environment [68,69].


The authors wish to thank Drs. Anastasia Athanasiadou, Chrysanthi Vadikolia, Chrysavgi Lalayanni and Riad Saloum for stimulating discussions and their long-standing collabo- ration, and Evangelia Stalika for expert technical support.

Potential confl ict of interest: Disclosure forms provided by the authors are available with the full text of this article at 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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