Immunophenotypic Differentiation Between Neoplastic Plasma Cells in Mature B-Cell Lymphoma vs Plasma Cell Myeloma

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1 Hematopathology / PLASMA CELLS IN LYMPHOMA VS MYELOMA Immunophenotypic Differentiation Between Neoplastic Plasma Cells in Mature B-Cell Lymphoma vs Plasma Cell Myeloma Adam C. Seegmiller, MD, PhD, Yin Xu, MD, PhD, Robert W. McKenna, MD, and Nitin J. Karandikar, MD, PhD Key Words: Plasma cells; Non-Hodgkin lymphoma; Plasma cell myeloma; Flow cytometry DOI: /5EL22BH45PHUPM8P Abstract Some non-hodgkin lymphomas show marked plasmacytic differentiation. In such cases, it may be difficult to differentiate these lymphomas from plasmacytoma or myeloma, especially with limited diagnostic material. However, there may be immunophenotypic differences in the plasma cells in these disorders that distinguish them. This study characterizes the immunophenotypes of neoplastic plasma cells in 41 cases of B-lineage non-hodgkin lymphoma and compares them with those in plasma cell myeloma. We found that plasma cells in lymphoma were significantly more likely to express CD19, CD45, and surface immunoglobulin and less likely to express CD56 than those in myeloma. We further show that CD19 and CD56 expression can be used reliably to distinguish these entities. Myeloma-associated osseous lesions and solitary plasmacytoma of bone showed myeloma-like immunophenotypes. However, some extramedullary plasmacytomas showed lymphoma-like phenotypes, suggesting that, in reality, they may represent non- Hodgkin lymphomas with extensive plasmacytic differentiation. Plasmacytic differentiation is a common feature of marginal zone lymphomas (MZLs) and lymphoplasmacytic lymphomas (LPLs) 1-6 and may sometimes blur the distinction between the 2 entities. 7 Plasmacytic differentiation is less commonly seen in other B-cell lymphomas, including small lymphocytic lymphoma (SLL) 8 and diffuse large B- cell lymphoma (DLBCL). 9 Occasionally, extensive plasmacytic differentiation in a B-cell lymphoma can make it difficult to distinguish it from a primary plasma cell (PC) dyscrasia, such as extraosseous plasmacytoma, especially when the diagnostic material is limited It would be helpful in such cases to have an adjunctive diagnostic testing modality, such as immunophenotypic analysis by flow cytometry (FC), to assist in the resolution of these diagnostic dilemmas. For such testing to be useful, there must be detectable immunophenotypic differences between the neoplastic cells in these 2 diseases. Many studies have demonstrated immunophenotypic differences between normal and neoplastic PCs in PC myeloma. Unlike normal PCs, myeloma cells usually do not express the pan B cell marker CD Although myeloma cells still express CD38 brightly, the levels tend to be slightly less than those seen in normal PCs. 15 In addition, myeloma cells often show strong aberrant expression of the natural-killer cell marker CD56, which is not expressed on normal PCs Furthermore, myeloma cells are much more likely to express CD117 14,17 and CD than their normal counterparts. However, there is some evidence that neoplastic PCs in mature B-cell neoplasms may have an immunophenotype more like normal PCs than like myeloma cells. 16,19 Thus, it is possible that NHLs with plasmacytic differentiation may be distinguished 176 Am J Clin Pathol 2007;127: DOI: /5EL22BH45PHUPM8P

2 Hematopathology / ORIGINAL ARTICLE from PC dyscrasias by the immunophenotype of the neoplastic PCs. In the present study, we compared the immunophenotypic features of neoplastic PCs in mature B-cell lymphomas and PC dyscrasias by FC. We demonstrated significant differences that can reliably distinguish the 2 diagnoses. Materials and Methods Cases The University of Texas Southwestern Medical Center at Dallas FC database was searched for cases of B-cell non- Hodgkin lymphoma (NHL) with plasmacytic differentiation, defined as a lymphoma with 2 separate, discontinuous neoplastic populations detectable by FC: a light chain restricted, CD38(moderately +) B-lymphocyte population, and a distinct CD38(bright +) population with the same light-chain restriction Image 1A. We identified 41 cases meeting these criteria between June 2001 and July As control cases, 41 consecutive cases of bone marrow PC myeloma, ending in July 2005, were identified from the same FC database. In these cases, the diagnosis was confirmed by established World Health Organization clinicopathologic criteria. 20 In addition, the database was searched for solid PC tumors examined during the same period, yielding a total of 13 cases of osseous or extraosseous plasmacytoma, each confirmed by histologic examination. Flow Cytometry Specimen processing, antibody staining, FC, and data analysis were performed as described previously. 21 In all cases, FC panels included fluorochrome-conjugated antibodies targeted against CD19,, CD38, and monoclonal and polyclonal reagents against κ and λ light chains. Panels included CD45 and CD56 in all myeloma cases and in 33 and 18 cases of NHL, respectively. FC was performed using FACSCalibur flow cytometers and data collected using CELLQuest software (BD Biosciences, San Jose, CA). Cluster analysis of events was performed using Painta-Gate software (BD Biosciences). Neoplastic PCs were identified by bright expression of CD38 and restricted membrane and/or intracellular light chain expression (Image 1). These PC clusters were designated positive for a particular antigen if more than 20% of the cells expressed the antigen using an appropriate isotypic control cutoff. Statistical Analysis Frequencies of immunophenotypes in NHL vs PC myeloma were compared by using the Fisher exact test. Results Case Characteristics To identify cases of NHL with plasmacytic differentiation, we searched our institutional FC database to identify cases of NHL that included a separate neoplastic PC population (see the Materials and Methods section). The resulting 41 NHL specimens came from a variety of tissue sources: 20 from bone marrow aspirates, 18 from lymph node or tissue biopsies, and 3 from peripheral blood or body fluids. Of the cases, 21 had a known diagnosis, including 8 DLBCLs, 7 LPLs, 5 MZLs, and 1 SLL. Adequate histologic material was available and examined in 18 of these cases, all of which showed clear plasmacytic differentiation. Two cases were bone marrow specimens with small neoplastic populations detected by FC but not visually apparent on histologic examination. In 1 case, the original histologic material was not available for reexamination. Twenty cases were outside referrals for which no corresponding morphologic information was available. PCs in NHL vs Myeloma Image 1A demonstrates a typical case of NHL with plasmacytic differentiation. The blue events represent the primary neoplastic B-lymphocyte population, defined by restricted κ light-chain expression. In the majority of the cases (36 of 41 cases), these cells were negative for CD5 and CD10, consistent with the diagnosis of LPL, MZL, or DLBCL. In 4 cases, there was at least partial expression of CD5; the single case of SLL fell into this category. In 2 cases (1 MZL and 1 DLBCL), expression of CD10 was detected. The red events in Image 1A represent the corresponding neoplastic PC population, defined by bright expression of CD38 and restricted surface and intracellular κ light-chain restriction. Except for light-chain restriction, the immunophenotype of these cells was similar to that seen in normal PCs: CD19+,, CD45+, and CD56. Image 1B illustrates the immunophenotype of a typical case of PC myeloma. The neoplastic PCs, highlighted in red, are defined by bright expression of CD38 and restricted intracellular λ light chain expression. Unlike normal PCs and the PCs seen in the cases of NHL, these neoplastic PCs were negative for CD19 and CD45 and expressed CD56. These general immunophenotypic differences were relatively consistent throughout the 82 cases of NHL and PC myeloma that were examined Table 1. The most significant difference between the two entities was in CD19 expression. The majority of neoplastic PCs (95%) in NHL expressed CD19, while 90% of PCs in myeloma were CD19 (P <<.001). Use of this marker alone yielded a diagnostic accuracy of 93% in distinguishing these disease entities. Am J Clin Pathol 2007;127: DOI: /5EL22BH45PHUPM8P 177

3 Seegmiller et al / PLASMA CELLS IN LYMPHOMA VS MYELOMA A Side Scatter CD38 CD45 B Side Scatter Forward Scatter CD56 Forward Scatter CD38 CD19 CD19 CD45 CD56 Image 1 A, Non-Hodgkin lymphoma. B, Plasma cell myeloma. Blue, neoplastic B cells; red, neoplastic plasma cells. 178 Am J Clin Pathol 2007;127: DOI: /5EL22BH45PHUPM8P

4 Hematopathology / ORIGINAL ARTICLE Table 1 Expression of Cell Surface Markers on Neoplastic PCs in B- Cell Non-Hodgkin Lymphomas and PC Myelomas Antigen/Disease No. (%) Positive P * CD19 Lymphoma (n = 41) 39 (95) <<.001 Myeloma (n = 41) 4 (10) Lymphoma (n = 41) 13 (32).81 Myeloma (n = 41) 11 (27) CD45 Lymphoma (n = 33) 30 (91) <<.001 Myeloma (n = 41) 17 (41) CD56 Lymphoma (n = 18) 6 (33).01 Myeloma (n = 41) 29 (71) Surface immunoglobulin Lymphoma (n = 41) 31 (76).006 Myeloma (n = 41) 18 (44) PC, plasma cell. * By the Fisher exact test. Expression of CD56 also differed between PCs in NHL and myeloma. CD56 was expressed in 29 (71%) of 41 cases of myeloma, in contrast with only 6 (33%) of 18 cases of NHL. Of note, the expression of CD56 among the CD56+ NHL PCs was significantly dimmer compared with myeloma PCs. The average ± SE mean fluorescence intensity of CD56 on PCs in these 6 cases was 106 ± 18, whereas the average ± SE CD56 mean fluorescence intensity in the 29 CD56+ myelomas was 1,225 ± 178 (P <<.001). By adding dim-to-negative CD56 expression as a secondary diagnostic criterion, 3 of 6 cases misdiagnosed by CD19 expression alone would now be correctly categorized, for a total diagnostic accuracy of 96%. There were also significant differences in the expression of other cell surface markers. Neoplastic PCs in NHL were Table 2 Expression of Cell Surface Markers in Osseous Plasma Cell Tumors more likely to express CD45 and surface immunoglobulin than those in PC myeloma (Table 1). Although these differences were statistically significant, they did not show high practical usefulness for distinguishing between these 2 disease entities (diagnostic accuracies of 73% and 66%, respectively). Moreover, these additional criteria did not enhance the discriminatory power beyond that of CD19 or CD56 expression. There was no significant difference in expression between PCs in NHL and myeloma (Table 1). However, was expressed on PCs in 32% and 27% of NHL and myeloma cases, respectively, distinct from normal PCs, which are typically. 13 Testing Diagnostic Usefulness One scenario in which this analysis could be useful is with small biopsy or fine-needle aspiration specimens of localized PC tumors. To that end, we retrospectively examined FC data from 13 other PC tumors: 6 osseous PC lesions associated with PC myeloma, 3 solitary plasmacytomas of bone, and 4 extraosseous plasmacytomas Table 2. As expected, the osseous lesions associated with myeloma expressed a CD19 myeloma-like immunophenotype. The single case in which PCs expressed CD19 (case 5) showed clear CD56 expression. Similarly, the solitary plasmacytomas of bone showed a myeloma-like phenotype, consistent with their identification as primary PC dyscrasias. In contrast, 2 of 4 extraosseous plasmacytomas (Table 2, cases 10 and 12), expressed an NHL-like immunophenotype, ie, CD19+, CD45+, and CD56. Of note, both of these cases were thyroid lesions. This suggests that these lesions may indeed represent NHLs with extensive plasmacytic differentiation rather than primary extraosseous plasmacytomas. Case No./Site CD38 CD19 CD45 CD56 sig icig Osseous lesions associated with myeloma 1/Ilium b+ + λ+ λ+ 2/Sternum b+ + + λ+ 3/Skull b+ + λ+ 4/Paraspinal b+ + κ+ κ+ 5/Ischium b κ+ κ+ 6/Upper arm b+ + ND κ+ κ+ Solitary plasmacytoma of bone 7/Spine b+ + κ+ 8/Humerus b+ + ND κ+ κ+ 9/Spine b+ ND κ+ Extraosseous plasmacytoma 10/Thyroid b λ+ ND 11/Inguinal lymph node b+ + κ+ κ+ 12/Thyroid b+ + + ND κ+ 13/Orbit b+ + + κ+ κ+ b+, bright positive; icig, intracellular immunoglobulin; ND, not done; sig, surface immunoglobulin; +, positive;, negative. Am J Clin Pathol 2007;127: DOI: /5EL22BH45PHUPM8P 179

5 Seegmiller et al / PLASMA CELLS IN LYMPHOMA VS MYELOMA Discussion Our study demonstrates that neoplastic PCs associated with NHL are immunophenotypically distinct from those of primary PC dyscrasias, such as PC myeloma and plasmacytoma. The former tend to express CD19, CD45, and surface immunoglobulin and express little or no CD56, whereas the latter usually have the reverse immunophenotype. The differences are especially marked for CD19 and CD56, the combination of which can very accurately distinguish these 2 pathologic entities. One possible criticism of the present study is that the cases of NHL with plasmacytic differentiation were identified by FC alone, rather than by histologic examination. Thus, there may be some question as to whether the cells defined as PCs by FC are really PCs or simply an activated subset of the neoplastic B cells. The cells were selected based on bright expression of CD38, a known characteristic of PCs 15 distinguishing them from mature B cells and hematogones that express moderate levels of CD These PCs formed distinct clusters of CD38(bright +) events completely separate from the highest CD38-expressing neoplastic B cells (Image 1A). The validity of this method is strengthened by the fact that morphologic examination confirmed plasmacytic differentiation in 18 of 18 available cases. Although there is extensive literature documenting plasmacytic differentiation in NHL, only a few studies have compared the immunophenotype of the neoplastic PCs with that of the primary neoplastic B-cell population. These studies include a single case series of LPL (San Miguel et al 19 ) and 3 case reports, 11,23,24 for a total of 17 cases. In all of these cases, the neoplastic PCs expressed CD19. Only San Miguel et al 19 examined other markers in cases of LPL. Similar to our study, they found that most neoplastic PCs were CD19+, CD45+, and CD56. It is interesting that they also showed that in 13 of 14 cases, the neoplastic PCs expressed. In the present study, we found expression on PCs in 32% of NHLs (Table 1). Given that normal PCs are generally, 13 this may represent an immunophenotypic aberrancy in differentiated PCs in NHL. In addition to these series, Ely and Knowles 16 examined CD56 expression on 65 NHLs with plasmacytic differentiation by immunohistochemical analysis. They found no discernible CD56 expression by any of these tumors. This largely agrees with the present study because the dim CD56 expression observed in 33% of our cases is not likely to be detected by the less sensitive immunohistochemical method. Recent studies indicate that CD45 expression is higher in proliferating PCs early in PC development. 25 CD45 declines thereafter and is dim or negative in terminally differentiated PCs. Thus, the fact that PCs in NHL are more likely to express CD45 than those in myeloma may suggest differences in differentiation stage. Furthermore, loss of CD45 tends to occur in terminal or more aggressive forms of myeloma. 25 Whether this holds true for PCs in NHL is not known. The immunophenotype of PCs in myeloma has been extensively studied ,26 As in the present study, these reports show that CD19 is rarely expressed in PC myeloma (<1%-10% of cases). Furthermore, the frequency of CD56 expression in these reports was very similar to findings of the present study (71%-79% vs 71%). However, the present study demonstrated more frequent expression of (27% vs 6%- 16%) and CD45 (41% vs 9%). It is unclear whether this minor difference is a function of statistical anomalies or differences in FC materials and/or techniques, such as different antibody clones or conjugated fluorophores. CD45 expression varies considerably in different studies (18%-40%). 12,26 This may be due to variability in the stage, genetics, and biology of the neoplastic cells in each study. 25,26 The differences in and CD45 expression may be important to the degree that they change the primary diagnosis and quantification of PC myeloma. However, because these markers are less important in distinguishing myeloma from NHL with plasmacytic differentiation (see earlier text), these differences have little or no impact on the results and conclusions of the present study. These results have particular application in the diagnosis of extramedullary plasmacytoma. The difficulty of differentiating this entity from NHL is highlighted by multiple case reports in the literature Based on morphologic features, some have suggested that many of these tumors represent extensive plasmacytic differentiation of an MZL or other NHL, rather than primary PC neoplasms. 27 This theory is supported in part by the present study in which 2 of 4 tumors diagnosed as extraosseous plasmacytoma were immunophenotypically consistent with NHL with plasmacytic differentiation (Table 2, cases 10 and 12). However, our results also support the notion that some of these cases may indeed represent true primary PC dyscrasias (plasmacytomas), with phenotypic features similar to myeloma. The present study demonstrated the diagnostic usefulness of flow cytometric immunophenotyping as an adjunctive test that can help discriminate primary PC neoplasms from B-cell NHLs with plasmacytic differentiation. From the Department of Pathology, University of Texas Southwestern Medical Center at Dallas. Address reprint requests to Dr Karandikar: Dept of Pathology, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX References 1. Berger F, Isaacson PG, Piris MA, et al. Lymphoplasmacytic lymphoma/waldenström macroglobulinemia. In: Jaffe ES, Harris NL, Stein H, et al, eds. Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2001: World Health Organization Classification of Tumours. 180 Am J Clin Pathol 2007;127: DOI: /5EL22BH45PHUPM8P

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