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small lymphocytic lymphoma
Human Pathology (2006) 37, 152 – 159
www.elsevier.com/locate/humpath
The proliferation center microenvironment and prognostic markers in chronic lymphocytic leukemia/small lymphocytic lymphoma Lorinda A. Soma, Fiona E. Craig, Steven H. Swerdlow MD* Division of Hematopathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213-2582, USA Received 19 July 2005; revised 27 September 2005; accepted 30 September 2005
Keywords: B cells; CD38; Chronic lymphocytic leukemia/small lymphocytic lymphoma; Lymphoma; MUM1/IRF4; Proliferation centers; ZAP-70
Summary Prognostication in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) based, in part, on ZAP-70 and CD38 expression, and to a lesser extent, on MUM1/IRF4 expression, is currently of great interest. The more aggressive type of CLL/SLL is reportedly characterized by neoplastic cells that are more responsive to B-cell signaling with proliferation centers (PCs), a potentially important site of neoplastic cell stimulation. To study the relationship of these markers to each other and to the pattern of PCs, immunohistochemical stains for ZAP-70 and MUM1/IRF4 were performed and the PC patterns assessed (where possible) in 29 tissue biopsies with CLL/SLL. CD38 expression was assessed in 18 cases using flow cytometry. Ten evaluable cases had a typical PC pattern and 16 an atypical pattern with larger or more confluent PCs and/or more numerous paraimmunoblasts/transformed cells. ZAP-70 was positive in 14 of 28 cases, including 3 with atypical PCs and enhanced PC staining. All 29 cases showed MUM1/IRF4 expression in PCs. Seven cases, none with atypical PC, also showed uniform positivity throughout, 14 showed weaker staining of surrounding lymphocytes, and 8 had PC staining only. CD38 was positive in 14 of 18 cases. The only significant association identified was between uniform MUM1/IRF4 positivity and typical PCs ( P = .004). These findings highlight the complex interrelationship of prognostic markers in CLL/SLL and demonstrate potentially important microenvironmental variations in their expression. They support the hypothesis that PCs are a site for B-cell receptor signaling, which helps explain reported site-dependent antigenic variation in CLL/SLL, and suggest that PC morphology may correlate with other biological features. D 2006 Elsevier Inc. All rights reserved.
1. Introduction Until recently, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) was considered a homoge-
* Corresponding author. E-mail address: [email protected] (S.H. Swerdlow). 0046-8177/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2005.09.029
neous baccumulativeQ rather than proliferative indolent B-cell lymphoid neoplasm characterized by defective apoptosis [1]. Although still considered a single disease entity, it is now known that there are at least 2 major types of CLL/SLL, one with mutated immunoglobulin heavy-chain variable region (IGHV) genes that is more indolent and one with unmutated IGHV genes that is more aggressive [1-7]. A more aggressive type of CLL/SLL is also recognized by its expression of the
Chronic lymphocytic leukemia/small lymphocytic lymphoma
153 Table 1 Comparison of the PC pattern with ZAP-70, MUM1/ IRF4, and CD38 expression Immunophenotypic marker ZAP-70 Positive Negative MUM-1 Positive PC positive with weaker staining of surrounding small lymphocytes PC only CD38 Positive Negative
Typical PC
Atypical PC
Total
3 7
10 6
13 13
5 3
0 11
5 14
2
5
7
5 2
9 1
14 3
Cases where PCs could not be evaluated are not included in this table.
activation marker CD38 and the T- and natural killer (NK) cell–associated ZAP-70 tyrosine kinase [3-5,7-13]. Cases that are positive for CD38 and especially those that are positive for ZAP-70 are most likely to be of the unmutated type, although the former is no longer considered a good surrogate marker for mutational status and the latter is still an imperfect one. Furthermore, although reports are conflicting, expression of the proto-oncogene multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) has also been suggested to be of prognostic importance in CLL/SLL [14,15]. With the recognition of these 2 subtypes of CLL/SLL and the prognostic significance of at least CD38 and ZAP-70 expression, there is now great interest in trying to understand the biological basis for these clinically important findings. It has been proposed that B-cell signaling pathways are more important in the proliferation of the neoplastic cells in the more aggressive CD38-positive, ZAP-70–positive, unmutated type of CLL/SLL than in the mutated cases [1,16]. Furthermore, it has been suggested that this stimulation may occur in the bpseudofollicularQ proliferation centers (PCs), the major site of neoplastic cell proliferation in tissues involved by CLL/SLL [17]. This would imply that PCs might be more important in the unmutated or at least the ZAP-70–positive cases. PCs are known to sometimes show differential antigenic expression compared with the surrounding CLL/SLL cells [11,17-19]. In addition, it is also known that CLL/SLL in lymph nodes and other tissues is heterogeneous in terms of
Fig. 1 PCs in CLL/SLL. A, Note the btypicalQ pattern with smallto medium-sized well-separated paler PC. B, In contrast, in this case, the batypicalQ PCs are much larger and focally confluent. C, In this atypical PC, there are numerous paraimmunoblasts and other transformed cells (hematoxylin-eosin, original magnification 4 [A, B], 100 [C]).
154 the prominence of the PCs and the abundance of paraimmunoblasts/large cells; however, these findings have not been considered to be of prognostic importance [17,18, 20,21]. It is also recognized that in a given patient, lymph nodes, possibly as well as the marrow, on average, have a greater proportion of CD38-positive cells than the peripheral blood [12,13]. There are no studies, to the best of our knowledge, that have attempted to look at a combination of the prognostic markers described above in CLL/SLL together with a morphological and immunoarchitectural evaluation of the PC. Therefore, the presence and morphological features of the PCs were assessed in 29 tissue biopsies with CLL/SLL, and the findings correlated with the immunoarchitectural distribution of ZAP-70 and MUM1/IRF4 expression and also with the flow cytometric evaluation of CD38.
2. Materials and methods Twenty-nine cases of CLL/SLL as defined in the World Health Organization classification of hematopoietic and lymphoid tumors [22] (22 lymph nodes, 2 tonsils, 2 spleens, 1 orbital soft tissue, 1 pleural biopsy, and 1 bone biopsy) from 2000 to 2004 with available tissue blocks were identified from the University of Pittsburgh Medical Center Presbyterian Hospital, 18 of which had previously performed flow cytometric immunophenotypic studies that included CD38. The patients included 21 men and 8 women aged from 38 to 85 years (median age, 68 years). The routine histological sections and available immunophenotypic studies were reviewed. All 28 cases tested were CD5-positive, and all 22 cases tested were CD23-positive. One additional patient had flow cytometric studies only performed on a subsequent pleural effusion which demonstrated a CD5-positive, CD23negative, FMC7-negative k monoclonal B-cell population with weak CD20 and surface immunoglobulin expression. After confirming the diagnosis of CLL/SLL, cases (except for the spleens and pleural biopsy) were morphologically divided into 1 of 2 categories independently by each of the authors with any discrepancies resolved by consensus. Those with predominantly uniform small- to intermediate-sized lymphocytes and relatively small, well-separated, or nondistinct PCs were considered of btypicalQ CLL/SLL type. This corresponds with the pseudofollicular and probably diffuse subtypes of CLL/SLL as defined by Lennert [23]. Cases were categorized as batypicalQ if the PCs were large and irregular, confluent, or if they had more paraimmunoblasts
L.A. Soma et al. and other large transformed cells than are usually present. This corresponds largely to the tumor-forming subtype as defined by Lennert [23]. Cases demonstrating at least 20% CD38 neoplastic cells as assessed from a retrospective review of flow cytometry reports were considered CD38-positive. The flow cytometric immunophenotypic studies were performed using previously described methods [24]. CD38 assessment was based on a 3-color combination that included CD38-phycoerythrin, CD19-PC5, and CD5-fluorescein isothiocyanate. Immunohistochemical stains were performed on deparaffinized, formalin-fixed tissue sections using antibodies for the following antigens: ZAP-70 (2F3.2, Upstate Biotechnology, Lake Placid, NY; 1:100 dilution after an initial subset of cases were stained at 1:500 dilution), MUM1/IRF4 (monoclonal antibody provided by B. Falini; 1:10 dilution after an initial subset of cases were stained at 1:100 dilution), CD3 (polyclonal, 1:250 dilution; Dako, Carpenteria, Calif), and CD20 (L26, 1:1000 dilution; Dako). CD3 and CD20 immunostains that had been performed using a streptavidinbiotin peroxidase detection system with the Ventana Automated Immunostainer (Ventana Medical Systems, Tucson, Ariz) were available in some cases and were not repeated. The remainder of the immunostains were performed manually using a streptavidin-biotin complex method (Vector Laboratories, Burlingame, Calif) as described previously [24]. The following antigen retrieval techniques were used: microwave antigen retrieval with citrate buffer for the ZAP70 and MUM1/IRF4 and 0.2% pepsin digestion for the CD3. After comparison with CD3 and CD20 stains to assess T/NKand B-cell distribution, and after determining that the T/NK cells were ZAP-70–positive as expected, cases were evaluated for nuclear ZAP-70 staining of the CLL/SLL as follows: positive (staining of majority of cells throughout), weak PC staining only, and negative. The last-named category included both the clearly negative cases plus cases that were indeterminate usually because of weak cytoplasmic staining without definite nuclear positivity. One case did not demonstrate T-cell staining and was not included in the ZAP-70 analyses. Cases were evaluated for nuclear MUM1/ IRF4 staining as follows: positive (majority of small lymphocytes and PCs with moderate to strong positivity), PC positive with additional weaker staining of the surrounding small lymphocytes, and weak/moderate to strong PC staining only. The staining category definitions were determined after an initial review of the cases. Statistical analyses were performed using the Fisher exact test and Prism software (GraphPad Software, San Diego, Calif). The
Fig. 2 ZAP-70 immunohistochemical staining with comparison to CD3 T/NK-cell stain. A, Note the distribution of the CD3-positive cells. B, In comparison with the CD3 stain in A, numerous CLL/SLL cells show nuclear ZAP-70 positivity. C, At higher magnification, note the numerous positive lymphoid cells but negative endothelial cells. D, In this case, only the PCs appear to be weakly ZAP-70–positive in this more dilute stain. E, See the distribution of the CD3-positive T/NK cells mostly around the central reactive germinal center, which can be compared with (F) the nuclear ZAP-70 staining in both the T/NK cells and CLL/SLL cells (CD3 and ZAP-70 immunoperoxidase stains with hematoxylin counterstain, original magnification 10 [A, B], 100 [C], 10 [D], 40 [E, F]).
Chronic lymphocytic leukemia/small lymphocytic lymphoma
155
156
L.A. Soma et al. Table 2 Comparison of MUM1/IRF4 expression to the PC pattern and ZAP-70 and CD38 expression MUM-1– positive
MUM-1 PC–predominanta
Total
Typical PC Atypical PC
5 0
5 16
10 16
ZAP-70–positive ZAP-70–negativeb
2 5
12 9
14 14
CD38-positive CD38-negative
3 1
11 3
14 4
a
The MUM1 PC–predominant category includes cases with PC staining and either absent or weak staining of the remaining lymphocytes. b The ZAP-70–negative category includes the totally negative cases and the indeterminate cases.
MUM1/IRF4 groups with weak or negative inter-PC staining were combined for comparison purposes.
3. Results All evaluable cases had PCs with 10 having a typical pattern (Fig. 1A) and 16 an atypical pattern (Fig. 1B,C). These patterns were correlated with the immunohistological staining patterns (Table 1). ZAP-70 was positive in 14 of 28 evaluable cases, including 3 that, with the ZAP-70 antibody used at a 1:500 dilution, showed apparent weak PC staining only (Fig. 2). No statistical correlations, however, could be documented between staining pattern and morphologic pattern or with any of the other markers tested. MUM1/IRF4 was uniformly expressed throughout in 7 cases, none with atypical PCs (where evaluable). An additional 14 cases had positive PCs plus weaker staining of surrounding lymphocytes and 8 had positive PCs only (Fig. 3). There was no significant correlation between MUM1/IRF4 and ZAP-70 or CD38; however, the difference in PC morphology between those with overall MUM1/IRF4 positivity versus all others was statistically significant ( P = .004) (Table 2). CD38 was positive in 14 of 18 cases with no statistically significant associations with ZAP-70, MUM1/IRF4, or PC pattern.
4. Discussion Although PCs are a unique and an almost invariable microenvironmental feature of CLL/SLL in tissue biopsies, Fig. 3 MUM1/IRF4 immunohistochemical staining. A, Numerous CLL/SLL cells have at least nuclear staining in this case. B, At higher magnification, note the numerous positive cells. C, In contrast, in this case, essentially only the PCs show positive staining (MUM1/IRF4 immunoperoxidase stain with hematoxylin counterstain, original magnification 10 [A, C], 100 [B]).
Chronic lymphocytic leukemia/small lymphocytic lymphoma their relationship to the new paradigm of CLL/SLL being divided into 2 major clinical categories based on a variety of immunophenotypic markers and immunoglobulin heavy chain mutational status has received little attention. For that reason, the immunoarchitectural features of tissue biopsies with CLL/SLL were studied using the ZAP-70 and MUM-1/ IRF4 prognostic markers, and the results correlated with the morphologic features of the PCs as well as with flow cytometrically determined CD38 expression. The observation that PCs may demonstrate antigenic differences compared with the surrounding neoplastic small lymphocytes in CLL/SLL is not a new one. In addition to frequently showing increased expression of proliferationassociated markers such as CD71 (OKT9, transferrin receptor) and Ki-67, PCs in a subset of CLL/SLL cases also show increased expression of HLA-DC (33.1), HLA-DR, CD20 (B1), and CD23 [17-19]. CD23, a low-affinity receptor for immunoglobulin E, is considered a B-cell bactivation markerQ and can lead to entry of resting CLL/ SLL cells into the cell cycle [25]. Higher serum levels of CD23 have also been reported to be an adverse prognostic indicator in CLL/SLL [26]. More recently, it has been reported that expression of survivin, an antiapoptotic protein, in lymph nodes with CLL/SLL is also only detected in PCs [19]. In contrast, other markers may be either downregulated in the PCs or up-regulated in non-PC cells, such as IgD [27], CD9 (BA-2, p24) [17], which modulates cell adhesion and migration [28], and the B-lineage–associated antigen identified by the PI-153 antibody [17]. Decreased CD9 expression in CLL has been associated with more pronounced bone marrow infiltration [28]. Although imperfect, there is a correlation between some of these in vivo changes and the changes induced in CLL/SLL cells exposed in vitro to the mitogen phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) [17]. TPA exposure also leads to inconsistent increased expression of CD38 (OKT10) [17] and to increased cytoplasmic immunoglobulin expression [29]. Two decades ago, these data were used to suggest that CLL/SLL bis representative of a precursor of secretory B cells (plasma cells), which may arise from differentiation pathways distinct from the T cell dependent development pathway related to germinal follicle formationQ [17]. These observations are consistent with the gene profiling studies, suggesting that CLL/SLL of all types is a neoplasm of memory-type B cells [30,31], consistent with the bpreferredQ model of Ferrarini and Chiorazzi [1] that even the bmutatedQ cases may arise from a T cell–independent pathway and consistent with the hypothesis that selective, continued microenvironmental stimulation of the CLL/SLL cells takes place in the PCs. Variations in PC extent and morphology are also well recognized. Although a direct association with prognosis has not been documented [17,21], more extensive PCs in follow-up compared with diagnostic biopsies have been reported [17] as well as a correlation between large numbers of paraimmunoblasts and prolymphocytes, large PC, and/or
157 nuclear irregularities in the neoplastic cells and a diagnosis of batypicalQ CLL/SLL in the blood or bone marrow [20]. Recently, there has been great interest in the assessment of ZAP-70, CD38, and, to a much lesser extent, MUM1/IRF4, both from a clinical and biological perspective in CLL/SLL [3-5,7-15]. ZAP-70, a protein tyrosine kinase [32-35], is currently considered the best phenotypic surrogate of mutational status in CLL/SLL, and positivity is associated with an adverse prognosis [8-11]. We demonstrated that, at least in a minority of cases, there was greater PC ZAP-70 staining than in the surrounding CLL/SLL cells. Carreras et al [11] reported stronger ZAP-70 staining, in general, in the larger cells of the PCs. We did not document an association with PC pattern. MUM1/IRF4 is a transcriptional regulator reported to play a role in gene expression in response to B-cell signaling [36]. Among normal B cells, its expression is associated with a late germinal center/postgerminal center phenotype and mutated IGHV [36]. MUM1/IRF4 expression in peripheral blood lymphocytes is up-regulated by mitogenic stimuli [37]. The role of MUM1/IRF4 expression in CLL/SLL prognostication is not well defined [14,15]. Ito et al [15], who reported MUM1/IRF4 to be an adverse prognostic indicator and whose study included lymph node and bone marrow biopsies, did not show a correlation with mutational status, or like in our series, with CD38 expression. Chang et al [14], who reported that MUM1/IRF4 expression was associated with a better prognosis and whose study included bone marrow biopsies only, did report that all but 1 of 8 nonmutated cases were MUM1/IRF4-negative compared with 7 of 14 mutated cases. Our study demonstrates that PCs uniformly express MUM1/IRF4, whereas the remaining small lymphocytes show more variable staining. Others have also reported preferential MUM1/IRF4 expression in PCs in at least some cases, with some bnegativeQ cases reported as well [15,36]. The constant presence of MUM1/IRF4 PC staining seen in our study supports the important role that this transcriptional regulator must play in PC cells, independent of whether the majority of the CLL/SLL cells are positive or not. It is also consistent with the concepts that B-cell signaling may have a role to play even in the ZAP-70– negative and mutated cases, that PCs largely mimic the phenotypic changes seen in CLL/SLL cells stimulated with TPA, and that PCs represent antigen-stimulated CLL/SLL cells. The significance of the fact that the most extensive MUM1/IRF4 staining was only seen in cases with typicalappearing PCs remains to be determined. Expression of the CD38 activation marker is another well-established adverse prognostic indicator in CLL/SLL [3-5]. Using flow cytometric analysis, neither we nor others have shown a statistically significant correlation between CD38 expression and PC morphology [21]. Given the reported increase in CD38 expression with in vitro activation of CLL/SLL cells [17] and consistent with the finding of greater CD38 expression in lymph nodes than in
158 peripheral blood samples [12,13], CD38 expression might be a feature especially of CLL/SLL cells exposed to the PC microenvironment. Although other explanations are also possible, the observation that the majority of the cases in this study (77%) showed a higher percentage of flow cytometrically evaluated CD38-positive cells in the larger compared with the smaller cell region would be consistent with this possibility (data not shown). This study, therefore, highlights the differential expression of some prognosis-associated antigens in the PCs of CLL/SLL, provides additional evidence that B-cell stimulation with activation and potentially B-cell signaling preferentially take place, at least in part, in the PC, and supports previous observations that phenotypic changes in PCs often mimic findings of mitogenic/immunologic stimulation of CLL/SLL cells. The constant MUM1/IRF4 expression in PCs is also consistent with the prevailing concept that even unmutated CLL/SLL is a neoplasm of memory-type B cells. Finally, the heterogeneity in the staining of CLL/SLL cells based, in part, on microenvironmental factors, is important not only in understanding the biology of this neoplasm but also in evaluating studies looking at the prognostic significance of these markers.
Acknowledgments The authors would like to thank Dr Brunangelo Falini for the gift of the MUM1/IRF4 antibody used in this study and Ms Mary Jo Buffo, Ms Lisa Fitchwell, and Ms Kimberly Fuhrer for their help with the organizational and technical aspects of this study.
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The proliferation center microenvironment and prognostic markers in chronic lymphocytic leukemia/small lymphocytic lymphoma Lorinda A. Soma, Fiona E. Craig, Steven H. Swerdlow MD* Division of Hematopathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213-2582, USA Received 19 July 2005; revised 27 September 2005; accepted 30 September 2005
Keywords: B cells; CD38; Chronic lymphocytic leukemia/small lymphocytic lymphoma; Lymphoma; MUM1/IRF4; Proliferation centers; ZAP-70
Summary Prognostication in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) based, in part, on ZAP-70 and CD38 expression, and to a lesser extent, on MUM1/IRF4 expression, is currently of great interest. The more aggressive type of CLL/SLL is reportedly characterized by neoplastic cells that are more responsive to B-cell signaling with proliferation centers (PCs), a potentially important site of neoplastic cell stimulation. To study the relationship of these markers to each other and to the pattern of PCs, immunohistochemical stains for ZAP-70 and MUM1/IRF4 were performed and the PC patterns assessed (where possible) in 29 tissue biopsies with CLL/SLL. CD38 expression was assessed in 18 cases using flow cytometry. Ten evaluable cases had a typical PC pattern and 16 an atypical pattern with larger or more confluent PCs and/or more numerous paraimmunoblasts/transformed cells. ZAP-70 was positive in 14 of 28 cases, including 3 with atypical PCs and enhanced PC staining. All 29 cases showed MUM1/IRF4 expression in PCs. Seven cases, none with atypical PC, also showed uniform positivity throughout, 14 showed weaker staining of surrounding lymphocytes, and 8 had PC staining only. CD38 was positive in 14 of 18 cases. The only significant association identified was between uniform MUM1/IRF4 positivity and typical PCs ( P = .004). These findings highlight the complex interrelationship of prognostic markers in CLL/SLL and demonstrate potentially important microenvironmental variations in their expression. They support the hypothesis that PCs are a site for B-cell receptor signaling, which helps explain reported site-dependent antigenic variation in CLL/SLL, and suggest that PC morphology may correlate with other biological features. D 2006 Elsevier Inc. All rights reserved.
1. Introduction Until recently, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) was considered a homoge-
* Corresponding author. E-mail address: [email protected] (S.H. Swerdlow). 0046-8177/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2005.09.029
neous baccumulativeQ rather than proliferative indolent B-cell lymphoid neoplasm characterized by defective apoptosis [1]. Although still considered a single disease entity, it is now known that there are at least 2 major types of CLL/SLL, one with mutated immunoglobulin heavy-chain variable region (IGHV) genes that is more indolent and one with unmutated IGHV genes that is more aggressive [1-7]. A more aggressive type of CLL/SLL is also recognized by its expression of the
Chronic lymphocytic leukemia/small lymphocytic lymphoma
153 Table 1 Comparison of the PC pattern with ZAP-70, MUM1/ IRF4, and CD38 expression Immunophenotypic marker ZAP-70 Positive Negative MUM-1 Positive PC positive with weaker staining of surrounding small lymphocytes PC only CD38 Positive Negative
Typical PC
Atypical PC
Total
3 7
10 6
13 13
5 3
0 11
5 14
2
5
7
5 2
9 1
14 3
Cases where PCs could not be evaluated are not included in this table.
activation marker CD38 and the T- and natural killer (NK) cell–associated ZAP-70 tyrosine kinase [3-5,7-13]. Cases that are positive for CD38 and especially those that are positive for ZAP-70 are most likely to be of the unmutated type, although the former is no longer considered a good surrogate marker for mutational status and the latter is still an imperfect one. Furthermore, although reports are conflicting, expression of the proto-oncogene multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) has also been suggested to be of prognostic importance in CLL/SLL [14,15]. With the recognition of these 2 subtypes of CLL/SLL and the prognostic significance of at least CD38 and ZAP-70 expression, there is now great interest in trying to understand the biological basis for these clinically important findings. It has been proposed that B-cell signaling pathways are more important in the proliferation of the neoplastic cells in the more aggressive CD38-positive, ZAP-70–positive, unmutated type of CLL/SLL than in the mutated cases [1,16]. Furthermore, it has been suggested that this stimulation may occur in the bpseudofollicularQ proliferation centers (PCs), the major site of neoplastic cell proliferation in tissues involved by CLL/SLL [17]. This would imply that PCs might be more important in the unmutated or at least the ZAP-70–positive cases. PCs are known to sometimes show differential antigenic expression compared with the surrounding CLL/SLL cells [11,17-19]. In addition, it is also known that CLL/SLL in lymph nodes and other tissues is heterogeneous in terms of
Fig. 1 PCs in CLL/SLL. A, Note the btypicalQ pattern with smallto medium-sized well-separated paler PC. B, In contrast, in this case, the batypicalQ PCs are much larger and focally confluent. C, In this atypical PC, there are numerous paraimmunoblasts and other transformed cells (hematoxylin-eosin, original magnification 4 [A, B], 100 [C]).
154 the prominence of the PCs and the abundance of paraimmunoblasts/large cells; however, these findings have not been considered to be of prognostic importance [17,18, 20,21]. It is also recognized that in a given patient, lymph nodes, possibly as well as the marrow, on average, have a greater proportion of CD38-positive cells than the peripheral blood [12,13]. There are no studies, to the best of our knowledge, that have attempted to look at a combination of the prognostic markers described above in CLL/SLL together with a morphological and immunoarchitectural evaluation of the PC. Therefore, the presence and morphological features of the PCs were assessed in 29 tissue biopsies with CLL/SLL, and the findings correlated with the immunoarchitectural distribution of ZAP-70 and MUM1/IRF4 expression and also with the flow cytometric evaluation of CD38.
2. Materials and methods Twenty-nine cases of CLL/SLL as defined in the World Health Organization classification of hematopoietic and lymphoid tumors [22] (22 lymph nodes, 2 tonsils, 2 spleens, 1 orbital soft tissue, 1 pleural biopsy, and 1 bone biopsy) from 2000 to 2004 with available tissue blocks were identified from the University of Pittsburgh Medical Center Presbyterian Hospital, 18 of which had previously performed flow cytometric immunophenotypic studies that included CD38. The patients included 21 men and 8 women aged from 38 to 85 years (median age, 68 years). The routine histological sections and available immunophenotypic studies were reviewed. All 28 cases tested were CD5-positive, and all 22 cases tested were CD23-positive. One additional patient had flow cytometric studies only performed on a subsequent pleural effusion which demonstrated a CD5-positive, CD23negative, FMC7-negative k monoclonal B-cell population with weak CD20 and surface immunoglobulin expression. After confirming the diagnosis of CLL/SLL, cases (except for the spleens and pleural biopsy) were morphologically divided into 1 of 2 categories independently by each of the authors with any discrepancies resolved by consensus. Those with predominantly uniform small- to intermediate-sized lymphocytes and relatively small, well-separated, or nondistinct PCs were considered of btypicalQ CLL/SLL type. This corresponds with the pseudofollicular and probably diffuse subtypes of CLL/SLL as defined by Lennert [23]. Cases were categorized as batypicalQ if the PCs were large and irregular, confluent, or if they had more paraimmunoblasts
L.A. Soma et al. and other large transformed cells than are usually present. This corresponds largely to the tumor-forming subtype as defined by Lennert [23]. Cases demonstrating at least 20% CD38 neoplastic cells as assessed from a retrospective review of flow cytometry reports were considered CD38-positive. The flow cytometric immunophenotypic studies were performed using previously described methods [24]. CD38 assessment was based on a 3-color combination that included CD38-phycoerythrin, CD19-PC5, and CD5-fluorescein isothiocyanate. Immunohistochemical stains were performed on deparaffinized, formalin-fixed tissue sections using antibodies for the following antigens: ZAP-70 (2F3.2, Upstate Biotechnology, Lake Placid, NY; 1:100 dilution after an initial subset of cases were stained at 1:500 dilution), MUM1/IRF4 (monoclonal antibody provided by B. Falini; 1:10 dilution after an initial subset of cases were stained at 1:100 dilution), CD3 (polyclonal, 1:250 dilution; Dako, Carpenteria, Calif), and CD20 (L26, 1:1000 dilution; Dako). CD3 and CD20 immunostains that had been performed using a streptavidinbiotin peroxidase detection system with the Ventana Automated Immunostainer (Ventana Medical Systems, Tucson, Ariz) were available in some cases and were not repeated. The remainder of the immunostains were performed manually using a streptavidin-biotin complex method (Vector Laboratories, Burlingame, Calif) as described previously [24]. The following antigen retrieval techniques were used: microwave antigen retrieval with citrate buffer for the ZAP70 and MUM1/IRF4 and 0.2% pepsin digestion for the CD3. After comparison with CD3 and CD20 stains to assess T/NKand B-cell distribution, and after determining that the T/NK cells were ZAP-70–positive as expected, cases were evaluated for nuclear ZAP-70 staining of the CLL/SLL as follows: positive (staining of majority of cells throughout), weak PC staining only, and negative. The last-named category included both the clearly negative cases plus cases that were indeterminate usually because of weak cytoplasmic staining without definite nuclear positivity. One case did not demonstrate T-cell staining and was not included in the ZAP-70 analyses. Cases were evaluated for nuclear MUM1/ IRF4 staining as follows: positive (majority of small lymphocytes and PCs with moderate to strong positivity), PC positive with additional weaker staining of the surrounding small lymphocytes, and weak/moderate to strong PC staining only. The staining category definitions were determined after an initial review of the cases. Statistical analyses were performed using the Fisher exact test and Prism software (GraphPad Software, San Diego, Calif). The
Fig. 2 ZAP-70 immunohistochemical staining with comparison to CD3 T/NK-cell stain. A, Note the distribution of the CD3-positive cells. B, In comparison with the CD3 stain in A, numerous CLL/SLL cells show nuclear ZAP-70 positivity. C, At higher magnification, note the numerous positive lymphoid cells but negative endothelial cells. D, In this case, only the PCs appear to be weakly ZAP-70–positive in this more dilute stain. E, See the distribution of the CD3-positive T/NK cells mostly around the central reactive germinal center, which can be compared with (F) the nuclear ZAP-70 staining in both the T/NK cells and CLL/SLL cells (CD3 and ZAP-70 immunoperoxidase stains with hematoxylin counterstain, original magnification 10 [A, B], 100 [C], 10 [D], 40 [E, F]).
Chronic lymphocytic leukemia/small lymphocytic lymphoma
155
156
L.A. Soma et al. Table 2 Comparison of MUM1/IRF4 expression to the PC pattern and ZAP-70 and CD38 expression MUM-1– positive
MUM-1 PC–predominanta
Total
Typical PC Atypical PC
5 0
5 16
10 16
ZAP-70–positive ZAP-70–negativeb
2 5
12 9
14 14
CD38-positive CD38-negative
3 1
11 3
14 4
a
The MUM1 PC–predominant category includes cases with PC staining and either absent or weak staining of the remaining lymphocytes. b The ZAP-70–negative category includes the totally negative cases and the indeterminate cases.
MUM1/IRF4 groups with weak or negative inter-PC staining were combined for comparison purposes.
3. Results All evaluable cases had PCs with 10 having a typical pattern (Fig. 1A) and 16 an atypical pattern (Fig. 1B,C). These patterns were correlated with the immunohistological staining patterns (Table 1). ZAP-70 was positive in 14 of 28 evaluable cases, including 3 that, with the ZAP-70 antibody used at a 1:500 dilution, showed apparent weak PC staining only (Fig. 2). No statistical correlations, however, could be documented between staining pattern and morphologic pattern or with any of the other markers tested. MUM1/IRF4 was uniformly expressed throughout in 7 cases, none with atypical PCs (where evaluable). An additional 14 cases had positive PCs plus weaker staining of surrounding lymphocytes and 8 had positive PCs only (Fig. 3). There was no significant correlation between MUM1/IRF4 and ZAP-70 or CD38; however, the difference in PC morphology between those with overall MUM1/IRF4 positivity versus all others was statistically significant ( P = .004) (Table 2). CD38 was positive in 14 of 18 cases with no statistically significant associations with ZAP-70, MUM1/IRF4, or PC pattern.
4. Discussion Although PCs are a unique and an almost invariable microenvironmental feature of CLL/SLL in tissue biopsies, Fig. 3 MUM1/IRF4 immunohistochemical staining. A, Numerous CLL/SLL cells have at least nuclear staining in this case. B, At higher magnification, note the numerous positive cells. C, In contrast, in this case, essentially only the PCs show positive staining (MUM1/IRF4 immunoperoxidase stain with hematoxylin counterstain, original magnification 10 [A, C], 100 [B]).
Chronic lymphocytic leukemia/small lymphocytic lymphoma their relationship to the new paradigm of CLL/SLL being divided into 2 major clinical categories based on a variety of immunophenotypic markers and immunoglobulin heavy chain mutational status has received little attention. For that reason, the immunoarchitectural features of tissue biopsies with CLL/SLL were studied using the ZAP-70 and MUM-1/ IRF4 prognostic markers, and the results correlated with the morphologic features of the PCs as well as with flow cytometrically determined CD38 expression. The observation that PCs may demonstrate antigenic differences compared with the surrounding neoplastic small lymphocytes in CLL/SLL is not a new one. In addition to frequently showing increased expression of proliferationassociated markers such as CD71 (OKT9, transferrin receptor) and Ki-67, PCs in a subset of CLL/SLL cases also show increased expression of HLA-DC (33.1), HLA-DR, CD20 (B1), and CD23 [17-19]. CD23, a low-affinity receptor for immunoglobulin E, is considered a B-cell bactivation markerQ and can lead to entry of resting CLL/ SLL cells into the cell cycle [25]. Higher serum levels of CD23 have also been reported to be an adverse prognostic indicator in CLL/SLL [26]. More recently, it has been reported that expression of survivin, an antiapoptotic protein, in lymph nodes with CLL/SLL is also only detected in PCs [19]. In contrast, other markers may be either downregulated in the PCs or up-regulated in non-PC cells, such as IgD [27], CD9 (BA-2, p24) [17], which modulates cell adhesion and migration [28], and the B-lineage–associated antigen identified by the PI-153 antibody [17]. Decreased CD9 expression in CLL has been associated with more pronounced bone marrow infiltration [28]. Although imperfect, there is a correlation between some of these in vivo changes and the changes induced in CLL/SLL cells exposed in vitro to the mitogen phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) [17]. TPA exposure also leads to inconsistent increased expression of CD38 (OKT10) [17] and to increased cytoplasmic immunoglobulin expression [29]. Two decades ago, these data were used to suggest that CLL/SLL bis representative of a precursor of secretory B cells (plasma cells), which may arise from differentiation pathways distinct from the T cell dependent development pathway related to germinal follicle formationQ [17]. These observations are consistent with the gene profiling studies, suggesting that CLL/SLL of all types is a neoplasm of memory-type B cells [30,31], consistent with the bpreferredQ model of Ferrarini and Chiorazzi [1] that even the bmutatedQ cases may arise from a T cell–independent pathway and consistent with the hypothesis that selective, continued microenvironmental stimulation of the CLL/SLL cells takes place in the PCs. Variations in PC extent and morphology are also well recognized. Although a direct association with prognosis has not been documented [17,21], more extensive PCs in follow-up compared with diagnostic biopsies have been reported [17] as well as a correlation between large numbers of paraimmunoblasts and prolymphocytes, large PC, and/or
157 nuclear irregularities in the neoplastic cells and a diagnosis of batypicalQ CLL/SLL in the blood or bone marrow [20]. Recently, there has been great interest in the assessment of ZAP-70, CD38, and, to a much lesser extent, MUM1/IRF4, both from a clinical and biological perspective in CLL/SLL [3-5,7-15]. ZAP-70, a protein tyrosine kinase [32-35], is currently considered the best phenotypic surrogate of mutational status in CLL/SLL, and positivity is associated with an adverse prognosis [8-11]. We demonstrated that, at least in a minority of cases, there was greater PC ZAP-70 staining than in the surrounding CLL/SLL cells. Carreras et al [11] reported stronger ZAP-70 staining, in general, in the larger cells of the PCs. We did not document an association with PC pattern. MUM1/IRF4 is a transcriptional regulator reported to play a role in gene expression in response to B-cell signaling [36]. Among normal B cells, its expression is associated with a late germinal center/postgerminal center phenotype and mutated IGHV [36]. MUM1/IRF4 expression in peripheral blood lymphocytes is up-regulated by mitogenic stimuli [37]. The role of MUM1/IRF4 expression in CLL/SLL prognostication is not well defined [14,15]. Ito et al [15], who reported MUM1/IRF4 to be an adverse prognostic indicator and whose study included lymph node and bone marrow biopsies, did not show a correlation with mutational status, or like in our series, with CD38 expression. Chang et al [14], who reported that MUM1/IRF4 expression was associated with a better prognosis and whose study included bone marrow biopsies only, did report that all but 1 of 8 nonmutated cases were MUM1/IRF4-negative compared with 7 of 14 mutated cases. Our study demonstrates that PCs uniformly express MUM1/IRF4, whereas the remaining small lymphocytes show more variable staining. Others have also reported preferential MUM1/IRF4 expression in PCs in at least some cases, with some bnegativeQ cases reported as well [15,36]. The constant presence of MUM1/IRF4 PC staining seen in our study supports the important role that this transcriptional regulator must play in PC cells, independent of whether the majority of the CLL/SLL cells are positive or not. It is also consistent with the concepts that B-cell signaling may have a role to play even in the ZAP-70– negative and mutated cases, that PCs largely mimic the phenotypic changes seen in CLL/SLL cells stimulated with TPA, and that PCs represent antigen-stimulated CLL/SLL cells. The significance of the fact that the most extensive MUM1/IRF4 staining was only seen in cases with typicalappearing PCs remains to be determined. Expression of the CD38 activation marker is another well-established adverse prognostic indicator in CLL/SLL [3-5]. Using flow cytometric analysis, neither we nor others have shown a statistically significant correlation between CD38 expression and PC morphology [21]. Given the reported increase in CD38 expression with in vitro activation of CLL/SLL cells [17] and consistent with the finding of greater CD38 expression in lymph nodes than in
158 peripheral blood samples [12,13], CD38 expression might be a feature especially of CLL/SLL cells exposed to the PC microenvironment. Although other explanations are also possible, the observation that the majority of the cases in this study (77%) showed a higher percentage of flow cytometrically evaluated CD38-positive cells in the larger compared with the smaller cell region would be consistent with this possibility (data not shown). This study, therefore, highlights the differential expression of some prognosis-associated antigens in the PCs of CLL/SLL, provides additional evidence that B-cell stimulation with activation and potentially B-cell signaling preferentially take place, at least in part, in the PC, and supports previous observations that phenotypic changes in PCs often mimic findings of mitogenic/immunologic stimulation of CLL/SLL cells. The constant MUM1/IRF4 expression in PCs is also consistent with the prevailing concept that even unmutated CLL/SLL is a neoplasm of memory-type B cells. Finally, the heterogeneity in the staining of CLL/SLL cells based, in part, on microenvironmental factors, is important not only in understanding the biology of this neoplasm but also in evaluating studies looking at the prognostic significance of these markers.
Acknowledgments The authors would like to thank Dr Brunangelo Falini for the gift of the MUM1/IRF4 antibody used in this study and Ms Mary Jo Buffo, Ms Lisa Fitchwell, and Ms Kimberly Fuhrer for their help with the organizational and technical aspects of this study.
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