Critical Analysis and Diagnostic Usefulness of Limited Immunophenotyping of B-Cell Non-Hodgkin Lymphomas by Flow Cytometry

Hematopathology / FLOW CYTOMETRIC IMMUNOPHENOTYPING IN B-CELL NON-HODGKIN LYMPHOMA Critical Analysis and Diagnostic Usefulness of Limited Immunophenotyping of B-Cell Non-Hodgkin Lymphomas by Flow Cytometry Zahid Kaleem, MD, 1 Glenda White, MT(ASCP), 1 and Robin T. Vollmer, MD 2 Key Words: Immunophenotype; Lymphoma; Flow cytometry; Immunoglobulins Abstract We report the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of various immunophenotypes characteristic of each class of B-cell non-hodgkin lymphoma (NHL) based on analysis of 352 morphologically wellcharacterized B-cell NHLs and 175 benign lymph nodes (LNs) using 2-color flow cytometry. All B-cell NHLs that exhibited a characteristic immunophenotype (except diffuse large B-cell lymphoma) had a high NPV. The immunophenotypes of small lymphocytic lymphoma and mantle cell lymphoma showed high specificity, but only small lymphocytic lymphoma also showed a high PPV. One third of follicular lymphomas coexpressed CD23 and CD10. Diffuse large B-cell NHL showed no consistent immunophenotype. About 90% of all benign LNs expressed no substantial amounts of CD5, CD10, or CD23. Most benign LNs also failed to express substantial amounts of immunoglobulin heavy chains. In contrast, about 90% of NHLs showed expression of 1 or 2 heavy chains. The expression pattern of immunoglobulin light chains was not found helpful in favoring one lymphoma type over another. The usefulness of each immunophenotype for each lymphoma group is of particular diagnostic importance in limited specimens, such as fine-needle aspiration biopsies, small core biopsies, body effusions, extranodal sites, and nodal tissues with various artifacts. The usefulness of flow cytometric immunophenotyping as an adjunct in the diagnosis and classification of B-cell non-hodgkin lymphomas (B-cell NHLs) cannot be overemphasized. It has become a powerful tool for specimens with limited morphologic information, such as from small core and fine-needle aspiration biopsies and evaluation of body effusions. In a few circumstances, however, undue reliance on strict criteria may lead to a wrong diagnosis. Accurate interpretation of complex data not only is a daunting task for the pathologist not familiar with this art but also can be a challenge for the expert. The characteristic immunophenotypes of various lymphomas are well known; the diagnostic usefulness and limitations, however, have received little scrutiny. 1,2 The purpose of the present study was to determine the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of various immunophenotypic profiles characteristic of each class of B-cell NHL to facilitate a correct diagnosis. We also report the incidence of usual and atypical immunophenotypes for each lymphoma group to emphasize the lack of complete faithfulness of any one immunophenotype for a particular lymphoma type. Our results are based on a retrospective analysis of 352 well-characterized B-cell NHLs using 2-color flow cytometry. We contrast our data with a control group comprising 175 benign lymph nodes (LNs). The expression pattern of immunoglobulin heavy chains in our group of lymphomas also provided invaluable insight into the possible mechanisms of immunoglobulin heavy chain class switching and recombinations of the variable [V(D)J] and constant (C H ) regions of the immunoglobulin genes. 136 Am J Clin Pathol 2001;115:136-142 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE Materials and Methods Material Selection All cases were retrieved from the files of Lauren V. Ackerman Laboratory of Surgical Pathology, Barnes-Jewish Hospital/Washington University Medical Center, St Louis, MO, between January 1, 1993, and June 30, 2000, including 352 consecutive B-cell NHLs and 175 consecutive benign/reactive lymph nodes. The cases were classified morphologically according to the standard criteria as defined elsewhere with or without adjunctive immunohistochemical and molecular studies. 1 For the purpose of the present study, all morphologically equivocal or atypical cases were excluded. We wanted to derive the immunophenotypic data only from morphologically unequivocal cases that represent well-defined disease entities so that we could confidently use and extrapolate the immunophenotypic data to correctly identify morphologically atypical cases. This included certain cases in which tissue fixation or other artifacts made an unequivocal morphologic classification impossible, such as the distinction between small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL) in poorly fixed tissue. Also excluded were cases of plasmacytoma/myeloma, T-cell rich B-cell lymphoma, posttransplant B-cell lymphoproliferative disorders, and prolymphocytic and large cell transformations of SLL/chronic lymphocytic lymphoma (CLL). Most of the samples were LNs, but selected cases of extranodal soft tissue (EN), bone marrow (BM), and peripheral blood (PB) also were included. The following cases were included: SLL, 48 (LN, 42; EN, 6); prolymphocytic lymphoma/leukemia de novo (PLL), 2 (LN, 1; PB, 1); lymphoplasmacytoid lymphoma (LpcL) with or without Waldenström macroglobulinemia, 16 (LN, 3; EN, 4; BM, 9); marginal zone B-cell lymphoma (MZL), 36 (LN, 12; spleen, 6; mucosa-associated lymphoid tissue [MALT]-type, 18); MCL, 33 (LN, 28; EN, 5); follicle center cell lymphoma (FCCL), 101 (LN, 90; EN, 11); diffuse large B-cell lymphoma (DLBCL), 80 (LN, 55; EN, 25); small noncleaved cell/burkitt lymphoma (SNCL), 18 (LN, 13; EN, 5); and hairy cell leukemia (HCL), 18 (spleen, 4; BM, 10; PB, 4). All patients with LpcL in the BM had Waldenström macroglobulinemia. Follicular lymphoma included cases with total or partial nodularity. Only de novo diffuse large B- cell NHLs were selected, including cases with CD30 (Ki-1) expression. Morphologic Examination All specimens were obtained and prepared for morphologic examination using standard techniques. Briefly, LNs and extranodal tissues were received fresh and divided appropriately into several parts allocated for routine light microscopy, flow cytometric examination, and possible gene rearrangement studies (most cases). The tissues were fixed in 10% buffered formalin and B-5 fixative and processed routinely, and the sections were stained with H&E for light microscopy. Bone marrow aspirate smears and peripheral blood specimens were air dried and stained with Wright- Giemsa technique. Bone marrow biopsy specimens were fixed in 10% buffered formalin and stained with H&E and Leder stains. Immunophenotypic analysis was performed on formalin-fixed paraffin-embedded tissue. The sections were stained using the avidin-biotin peroxidase complex method. All cases were reviewed and verified independently by 2 pathologists. Flow Cytometry Fresh tissues were transported immediately in RPMI solution to the flow cytometry laboratory. Lymphocytes were disaggregated and released from solid tissue by RPMI injection. Cells were stained with various combinations of the following fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled monoclonal antibodies against the following antigens: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD19, CD20, CD23, CD25, CD103, and HLA-DR; IgG, IgM, IgD, and IgA heavy chains; and kappa and lambda light chains. The typical combinations were as follows: CD19-FITC/CD5-PE, CD23-FITC/CD19-PE, CD10-FITC/CD19-PE, kappa-fitc/cd19-pe, lambda- FITC/CD19-PE, CD3-FITC/CD20-PE, CD11c-FITC/CD19- PE, and CD25-FITC/CD19-PE. In several cases (January 1993-December 1995), CD20 was used in place of CD19. A complete panel including CD5, CD10, and CD23 was run in 319 cases of lymphomas (SLL, 42; PLL, 2; LpcL, 12; MZL, 35; MCL, 25; FCCL, 92; DLBCL, 77; SNCL, 18; HCL, 16) and 68 benign LNs Table 1. Two-color flow cytometric immunophenotyping was performed on a FACScan (Becton-Dickinson, San Jose, CA) or a Coulter XL cytometer (Coulter, Miami, FL) by collecting 10,000 ungated list-mode events, selecting an appropriate lymphocyte gate on the combination of forward and side scatter, and analyzing cells with the most appropriate lymphocyte gate. In cases of more than 1 lymphocyte gate, the gate with the most B cells and/or showing aberrant coexpression of antigens and closely corresponding to the cell size of the lymphoma in question was chosen for analysis. However, with few exceptions, similar data were obtained from additional gates when present. All antibodies were purchased from Becton-Dickinson or Coulter and used according to the manufacturer s guidelines. Appropriate isotypic controls always were run. An antigen was considered positively expressed when at least 25% of the gated cells expressed that antigen. A 25% cutoff level yielded the best combined sensitivity and specificity American Society of Clinical Pathologists Am J Clin Pathol 2001;115:136-142 137

Kaleem et al / FLOW CYTOMETRIC IMMUNOPHENOTYPING IN B-CELL NON-HODGKIN LYMPHOMA and the smallest number of false-positive and false-negative results compared with cutoff levels of 15%, 20%, 30%, and 35% (data not shown). Light-chain clonality was determined when the immunoglobulin kappa/immunoglobulin lambda light-chain ratio was more than 4:1 or less than 0.5:1. Statistical Analysis The sensitivity, specificity, PPV, and NPV were calculated for the most common and the usual immunophenotype for each lymphoma category only in cases in which all 3 antigens (CD5, CD10, and CD23) were run (Table 1). These calculations also included 68 cases of benign lymph nodes. Diffuse large B-cell NHL is a heterogeneous group and does not exhibit any one immunophenotype; the most common is CD5, CD10, CD23, but other immunophenotypes include CD5, CD10+, CD23 ; CD5+, CD10, CD23 ; CD5, CD10+, CD23+; and CD5, CD10, CD23+. For this reason, the sensitivity, specificity, PPV, and NPV were not determined for any immunophenotypes of DLBCL. The sensitivity of an immunophenotype reflects how accurately it identifies the presence of a lymphoma known to have that particular immunophenotype, whereas the specificity of an immunophenotype reflects how accurately it detects the absence of a lymphoma known not to carry that particular immunophenotype. High sensitivity reflects a low false-negative rate, whereas high specificity reflects a low false-positive rate. The predictive value of an immunophenotype reflects how often the immunophenotype is expected to agree with the actual diagnosis. The following simple formulas were used for calculations: Sensitivity = True-Positive Cases/(True-Positive Cases + False-Negative Cases) 100 Specificity = True-Negative Cases/(True-Negative Cases + False-Positive Cases) 100 PPV = True-Positive Cases/(True-Positive Cases + False-Positive Cases) 100 NPV = True-Negative Cases/(True-Negative Cases + False-Negative Cases) 100 Results Overall, the typical immunophenotypes were seen in 205 (84.7%) of 242 lymphomas (included are SLL, PLL, MCL, MZL, LpcL, FCCL, SNCL, and HCL) that displayed a characteristic phenotype (Table 1). Diffuse large B-cell NHL is a heterogeneous group and does not carry a single phenotypic profile. 1,3 The phenotype of SLL/CLL was highly specific and had a high PPV and NPV but was observed in only 90% of the cases. The immunophenotype for MCL, in contrast, had a high specificity and NPV but lacked sensitivity (76%) and PPV (70%). The characteristic phenotypes of LpcL (CD5, CD10, CD23 ), MZL (CD5, CD10, CD23 ), SNCL (CD5, CD10+, CD23 ), and HCL (CD5, CD10, CD23 ) all had a high NPV but a very low PPV and specificity. All combinations of a positive and negative phenotype were seen in one or the other lymphoma except positivity for all 3 antigens (CD5+, CD10+, CD23+). Despite the fact that a substantial proportion of FCCLs may express CD23, several authoritative sources do not Table 1 Immunophenotypes of Various Lymphomas Using 25% Cutoff Level Compared With Benign Nodes * b-ln SLL PLL LpcL MZL MCL FCCL DLBCL SNCL HCL Immunophenotype (68) (42) (2) (12) (35) (25) (92) (77) (18) (16) CD5+, CD10, CD23+ 0 (0) 38 (90) 0 (0) 0 (0) 1 (3) 0 (0) 0 (0) 2 (2) 0 (0) 0 (0) Sensitivity/specificity (%) 90/99 PPV/NPV (%) 93/99 CD5+, CD10, CD23 0 (0) 3 (7) 0 (0) 0 (0) 0 (0) 19 (76) 0 (0) 5 (6) 0 (0) 0 (0) Sensitivity/specificity (%) 76/98 PPV/NPV (%) 70/98 CD5, CD10, CD23 60 (88) 0 (0) 2 (100) 11(92) 31 (88) 5 (20) 9 (10) 33 (43) 4 (22) 12 (75) Sensitivity/specificity (%) 92/58 88/61 75/58 PPV/NPV (%) 7/99 19/99 7/98 CD5, CD10+, CD23 2 (3) 0 (0) 0 (0) 0 (0) 1 (3) 0 (0) 50 (54) 26 (34) 14 (78) 2 (12) Sensitivity/specificity (%) 54/83 78/78 PPV/NPV (%) 52/84 15/98 CD5+, CD10+, CD23+ 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) CD5, CD10, CD23+ 6 (9) 1(2) 0 (0) 1 (8) 2 (6) 0 (0) 5 (5) 7 (9) 0 (0) 2 (12) CD5, CD10+ CD23+ 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 28 (30) 4 (5) 0 (0) 0 (0) CD5+, CD10+, CD23 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (4) 0 (0) 0 (0) 0 (0) 0 (0) CD5, CD11c+, CD25+ 11 (69) b-ln, benign lymph nodes; DLBCL, diffuse large B-cell lymphoma; FCCL, follicle center cell lymphoma; HCL, hairy cell leukemia; LpcL, lymphoplasmacytoid lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone B-cell lymphoma; NPV, negative predictive value; PLL, prolymphocytic lymphoma/leukemia, de novo; PPV, positive predictive value; SLL, small lymphocytic lymphoma; SNCL, small noncleaved/burkitt lymphoma. * Data are given as number (percentage) of cases. The numbers in parentheses with the lymphoma type indicate the total number of cases for that lymphoma type in which all 3 antigens (CD5, CD10, and CD23) were analyzed. Sensitivity and specificity, PPV, and NPV are given when they were calculated. 138 Am J Clin Pathol 2001;115:136-142 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE indicate CD23 expression as a variable feature of FCCL. 4 Noticeably, in our series 28 (30%) of 92 FCCLs expressed CD23 with CD10 and another 5 (5%) expressed only CD23. None of the 16 cases of HCL lacked CD11c, but only 11 (69%) showed expression of CD25. All 10 cases of HCL in which a tartrate-resistant acid phosphatase stain was performed showed a positive reaction, including 5 cases that were negative for CD25. In 2 cases in which this stain was not performed, CD103 was positively expressed by flow cytometry. The immunophenotypic profile for the expression of immunoglobulin heavy chains was characteristic, although not specific, in most cases Table 2. Eleven (12%) of 93 lymphomas expressed no substantial amounts of any heavy chains. In 75 (81%) of 93 cases, only 1 heavy chain was expressed; IgM was the most common (43/75), and IgD the least common (1/75). Concurrent expression of IgM and IgD was the most common pattern among lymphomas that express 2 surface heavy chains. The expression patterns for SLL, LpcL, MZL, MCL, DLBCL, SNCL, and HCL were similar to those reported previously. 1 The one exception, however, was FCCL, which showed isolated expression of IgG in 7 (33%) of 21 cases and had a relative lack of IgM expression. This is in contrast with the reported frequency of IgM and IgD expression. 5 Seventy-one (86%) of 82 heavychain expressing lymphomas showed expression that exceeded 50%. Almost all cases (339/352) expressed either immunoglobulin kappa (199/352) or immunoglobulin lambda (140/352) light chains (Table 2). In contrast with the previously reported observation, 6 immunoglobulin kappa was the more commonly expressed light chain in our group of 33 MCLs. Twelve cases did not express either immunoglobulin kappa or immunoglobulin lambda light chain; and 1 case of DLBCL showed equal amounts of immunoglobulin kappa and lambda light chains. 7 Of 122 cases of lymphomas in which HLA-DR was analyzed, expression was seen in substantial amounts (median, 94%) in 120 cases (98.4%). The median expression in benign LNs, in contrast, was 40% with a range of 15% to 90% and a mean of 42% (SD, 16%). Discussion The characteristic immunophenotypic profiles help substantially in properly classifying various B-cell NHLs, especially when the tissue is limited, as in small needle core biopsy specimens, fine-needle aspiration biopsy specimens, and biopsy specimens with extensive fixation or crush artifact. The usefulness of most phenotypic profiles, however, is limited by lack of complete faithfulness. Among other immunophenotypically atypical cases, we have seen 1 case of pulmonary MALT-type MZL that expressed CD5 and CD23 without CD10 Image 1. This patient in this case had no lymphadenopathy or peripheral lymphocytosis, and the specimens showed all characteristic morphologic features of MALT-type MZL, including the presence of benign germinal centers, a population of centrocyte-like cells, plasma cells, and numerous lymphoepithelial lesions. Proliferation centers were not present. We know of only one other well-documented case of MZL that showed this aberrant immunophenotype. 8 In HCL, we found that CD25 was seen in only two thirds of cases, consistent with previously reported figures (66%-75%), 9,10 whereas CD11c was a more consistent marker, found in all of our 18 cases. We found the CD5+, CD10, CD23+ immunophenotype highly specific for Table 2 Surface Immunoprofile for the Expression of Immunoglobulin (Ig) HCs and LCs in Various Lymphomas and Benign Lymph Nodes * IgM+, IgM+, IgM+, IgG+, No Ig- Ig-kappa Ig-lambda LC IgG+ IgM+ IgD+ IgA+ IgD+ IgG+ IgA+ IgA+ HC LC LC Negative Benign (25) 0 4 0 0 5 0 0 0 16 NA NA NA SLL (18) 1 9 0 0 3 0 0 0 5 23/48 24/48 1/48 LpcL (4) 0 4 0 0 0 0 0 0 NA 12/16 4/16 0/16 MZL (7) 1 5 0 0 1 0 0 0 NA 18/36 18/36 0/36 MCL (17) 0 10 0 1 5 1 0 0 NA 20/33 13/33 0/33 FCCL (21) 7 3 1 2 1 0 1 1 5 58/101 39/101 4/101 DLBCL (15) 1 6 0 1 5 1 0 0 1 45/80 29/80 5/80 SNCL (7) 0 6 0 0 1 0 0 0 NA 9/18 7/18 2/18 HCL (4) 4 0 0 0 0 0 0 0 NA 12/18 6/18 0/18 Total for NHL 14 43 1 4 16 2 1 1 11 197/350 140/350 12/350 b-ln, benign lymph nodes; DLBCL, diffuse large B-cell lymphoma; FCCL, follicle center cell lymphoma; HC, heavy chain; HCL, hairy cell leukemia; LC, light chain; LpcL, lymphoplasmacytoid lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone B-cell lymphoma; NHL, non-hodgkin lymphoma; NPV, negative predictive value; PLL, prolymphocytic lymphoma/leukemia, de novo; PPV, positive predictive value; SLL, small lymphocytic lymphoma; SNCL, small noncleaved/burkitt lymphoma. * The numbers in parentheses following the lymphoma type are the total number of cases in which heavy chains were analyzed. One additional case of DLBCL had expression of Ig-kappa and Ig-lambda LCs in a 1:1 ratio. The 2 cases of PLL both expressing Ig-kappa are not shown. American Society of Clinical Pathologists Am J Clin Pathol 2001;115:136-142 139

Kaleem et al / FLOW CYTOMETRIC IMMUNOPHENOTYPING IN B-CELL NON-HODGKIN LYMPHOMA Image 1 Pulmonary mucosa-associated lymphoid tissue type marginal zone lymphoma showing several lymphoepithelial lesions that expressed CD5 and CD23 but not CD10 (H&E, 100). Image 2 Nodal CD5 mantle cell lymphoma that showed a monotonous proliferation of intermediate-sized cells with irregular nuclei and frequent mitoses (H&E, 400) that showed diffuse nuclear overexpression of cyclin-d1 (not shown) and that lacked bcl-6 expression. CLL/SLL and the CD5+, CD10, CD23 immunophenotype highly specific for MCL. The other phenotypic profiles showed no substantial degree of specificity for any of the lymphoma groups. In most cases of SLL/CLL, almost parallel levels of CD5 and CD23 expression were observed. In a few cases, however, positive expression of CD23 was substantially less than the level of CD5 expression but still greater than 25%. The converse was not observed. In addition to 1 case of pulmonary MALT-type lymphoma, only 2 other cases of de novo DLBCL showed expression of CD5 and CD23; both cases had immunoblastic morphologic features, and the patients had no previous or concurrent evidence of SLL/CLL. Neither case of de novo PLL expressed CD5, CD10, or CD23, and both expressed the immunoglobulin kappa light chain. The negative immunophenotypic profile, CD5, CD10, CD23, was the most common overall. This is not only because it is the characteristic profile for a variety of lymphomas (LpcL, PLL, MZL, DLBCL, and HCL) but also because it can result from lack of CD5 expression in MCL Image 2 and lack of CD10 expression in FCCL Image 3 and SNCL. Cases of FCCL without CD10 expression most likely represent clonal proliferations of a subpopulation of CD10 follicle center cells rather than loss of CD10 expression as a result of malignant transformation. The phenotype CD5, CD10, CD23 is also the most common immunophenotype in benign lymph nodes and not infrequently poses diagnostic problems in distinguishing a benign LN from MZL with nondiagnostic light-chain ratios. Isolated expression of either CD10 or CD23 may, however, be seen in benign lymph nodes. Both of our cases of benign LNs that expressed CD10 were examples of florid follicular hyperplasia. Thus, it is noteworthy that high CD10 expression in B-cell proliferations without any substantial expression of immunoglobulin kappa and lambda light chains represents a florid follicular hyperplasia or a follicular lymphoma. 7 Likewise the 6 cases of benign LNs that showed expression of CD23 (the activation antigen) also showed cortical and paracortical hyperplasia. The same immunophenotype (CD5, CD10, CD23+), however, also may be seen in some lymphomas, including CD5 SLL/CLL and DLBCL and CD10 FCCL and cases of MZL. The important finding is that benign LNs do not express CD5 (>25%) or combined CD10 and CD23 in substantial amounts (>50%). Thus, expression of CD5 is not a feature of benign lymphoid proliferations and suggests involvement by SLL/CLL, MCL, or, rarely, DLBCL. All characteristic immunophenotypic profiles for all lymphomas carry very high (98%) NPVs, ie, absence of the expected immunophenotypic profile for a lymphoma excludes the presence of that particular lymphoma type with a high degree of certainty. The one exception is DLBCL, which does not carry any one immunophenotype because it represents a heterogeneous group of large cell lymphomas rather than a single clinicopathologic entity. 3 We also have found that the profile CD5+, CD10+, CD23 is rare; the only example in our series was that of a case of MCL. The coexpression of CD5 and CD10 is highly unusual because CD5 is 140 Am J Clin Pathol 2001;115:136-142 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE Image 3 Nodal follicular lymphoma that totally lacked CD10 expression showing numerous follicles composed predominantly of small cleaved cells (H&E, 20). expressed by a subpopulation of prefollicular mantle zone cells that lose CD5 as they differentiate into follicular cells and acquire CD10. It is of much diagnostic importance to note that an aberrant immunophenotype due to lack of expression of an antigen was much more common than an aberrant immunophenotype as a result of anomalous expression of an antigen in a lymphoma not usually considered to express that antigen. Thus, in our series, 5 cases of MCL lacked CD5 (CD5, CD10, CD23 ), and none showed anomalous expression of CD23 (CD5+, CD10, CD23+). In none of our 339 light-chain expressing lymphomas was any preferential expression noted of one or the other light chain in any lymphoma type; thus, this expression may not be used to assist in favoring one lymphoma type over another. This is emphasized especially for MCLs, in which immunoglobulin lambda light chain is reportedly more commonly expressed than immunoglobulin kappa light chain; however, our results did not confirm that observation. The diagnostic use of heavy chains can provide valuable information in selected cases. One important use is in excluding the diagnoses of LpcL and SNCL with a reasonable degree of certainty if IgM is not expressed. The expression level of heavy chains can help differentiate a benign from a malignant lymphoproliferative disorder in atypical cases. In none of our 25 cases of benign lymph nodes was the expression greater than 50%. Most cases, in fact, did not express any heavy chains in amounts greater than 25%. In contrast, when expression of a heavy chain was seen in any of the lymphomas, the level usually exceeded 50% and, in many cases, 75%. One of the inferences that we can draw from this observation (because comparable numbers of cells per gate were analyzed in benign and malignant lesions) is that lymphoma cells, except in SLL/CLL, perhaps carry more immunoglobulin molecules per cell than benign lymphocytes. Another interesting finding of potential diagnostic use was the lack of expression of substantial amounts of IgA in benign nodes, whereas expression of IgA (>50%) in isolation or in combination with another heavy chain was seen in 6 cases of lymphoma. This, therefore, suggests that IgA expression in substantial amounts in lymph nodes is suggestive of malignancy since IgA-producing B-lymphocytes normally reside predominantly in epithelium, and only a small fraction of follicle center B cells in benign LNs produce IgA. 11 HLA-DR, which is expressed by all B lymphocytes and only activated T lymphocytes, was expressed in higher levels in our B-cell lymphomas (median, 94%) than in benign nodes (median, 40%) and may help as an adjunct when the question is benign vs malignant. The expression pattern of heavy chains in lymphomas not only is diagnostically helpful but also provides invaluable insight into the possible mechanisms of heavy-chain class switching and recombinations. Unstimulated virgin B cells express IgM with IgD at the cell surface, whereas antigen-stimulated mature B cells express only one type of heavy chain, IgG, IgA, or IgE, as a result of class switching. 11 The linear recombination model implies that during class switching, the V(D)J segment recombines with gamma, alpha, or epsilon gene segments with deletion of the preceding gene segments. 12 The expression of IgM with IgG or IgA in rare lymphomas in our series suggests that recombination may occur during class switching (at least in lymphomas) between V(D)J and C gamma or C alpha without deleting C mu, but with deletion of the C delta segment of heavy-chain genes or, alternatively, formation of a large primary transcript comprising segments of mu, delta, gamma, and alpha genes with subsequent excision of the delta segment during maturation of messenger RNA and splicing of mu with gamma or alpha segments. The expression of both IgG and IgA in another case suggests that during class switching, recombination of V(D)J with C gamma leads to formation of a primary transcript that comprises C gamma and adjacent C alpha segments, with expression of IgG and IgA as a result of differential splicing. Indeed, much of our current knowledge of the normal immune system is derived from studies on hematolymphoid malignant neoplasms. The evaluation of antigen expression must be determined in a substantial population of B lymphocytes. What exactly constitutes a substantial population is a matter of debate and arbitration. We routinely analyze 10,000 cells in total, when ample specimen is available. Caution must be exercised in making a diagnosis of malignant neoplasm in a smaller population of cells (<1,000 B lymphocytes). This American Society of Clinical Pathologists Am J Clin Pathol 2001;115:136-142 141

Kaleem et al / FLOW CYTOMETRIC IMMUNOPHENOTYPING IN B-CELL NON-HODGKIN LYMPHOMA caution must be followed more so in lesions with a negative immunophenotypic profile than in lymphoid proliferations with substantial (>50%) aberrant expression of 1 (CD5) or 2 (CD10 and CD23) antigens. Based on our experience, we suggest that more than 80% of B lymphocytes in a gate comprising at least 1,000 cells may be considered a substantial population. In cases with lower proportions of B cells in a gate, the following guidelines may be used: more than 50% B cells in a gate of 2,000 or more cells; more than 25% B cells in a gate consisting of 4,000 or more cells; and more than 20% B cells in a gate of 6,000 or more cells. We believe that the application of these guidelines and the knowledge of the sensitivity, specificity, PPV, and NPV of each immunophenotype for a particular lymphoma type would be most useful for evaluating fine-needle aspiration biopsy specimens, lymphoid effusions, small core biopsy specimens, extranodal tissues, and morphologically atypical nodal lymphoid proliferations. 9. Stetler-Stevenson M, Medeiros LJ, Jaffe ES. Immunophenotypic methods and findings in the diagnosis of lymphoproliferative diseases. In: Jaffe ES, ed. Surgical Pathology of the Lymph Node and Related Organs. 2nd ed. Philadelphia, PA: Saunders; 1995:22-57. 10. Carey JL, Hanson CA. Flow cytometric analysis of leukemia and lymphoma. In: Keren DF, Hanson CA, Hurtubise PE, eds. Flow Cytometry and Clinical Diagnosis. Chicago, IL: ASCP Press; 1994:197-308. 11. Roitt I, Brostoff J, Male D. Immunology. St Louis, MO: Mosby; 1998:93-106. 12. Harriman W, Volk H, Defanoux N, et al. Immunoglobulin class switch recombinations. Annu Rev Immunol. 1993;11:361-384. From the 1 Lauren V. Ackerman Laboratory of Surgical Pathology, Barnes-Jewish Hospital/Washington University Medical Center, St Louis, MO; and 2 the Department of Pathology, Duke University and VA Medical Center, Durham, NC. Address reprint requests to Dr Kaleem: Washington University School of Medicine, Dept of Pathology, Box 8118, 660 S Euclid Ave, St Louis, MO 63110. References 1. Chan JKC, Banks PM, Cleary ML, et al. A revised European- American classification of lymphoid neoplasms proposed by the international lymphoma study group: a summary version. Am J Clin Pathol. 1995;103:543-560. 2. Morse EE, Yamase HT, Greenberg BR, et al. The role of flow cytometry in the diagnosis of lymphomas: a critical analysis. Ann Clin Lab Sci. 1994;24:6-11. 3. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphomas identified by gene expression profiling. Nature. 2000;403:503-511. 4. Mann RB. Follicular lymphomas. In: Jaffe ES, ed. Surgical Pathology of the Lymph Node and Related Organs. 2nd ed. Philadelphia, PA: Saunders; 1995:252-282. 5. Anderson KC, Bates MP, Slaughenhoupt BL, et al. Expression of human B-cell associated antigens on leukemias and lymphomas: a model for human B-cell differentiation. Blood. 1984;63:1424-1433. 6. Lardelli P, Bookman MA, Sundeen J, et al. Lymphocytic lymphoma of intermediate differentiation: morphologic and immunophenotypic spectrum and clinical correlations. Am J Surg Pathol. 1990;14:752-763. 7. Kaleem Z, Zehnbauer BA, White G, et al. Lack of expression of surface immunoglobulin light-chains in B-cell non- Hodgkin lymphomas. Am J Clin Pathol. 2000;113:399-405. 8. Banks PM, Isaacson PG. MALT lymphomas in 1997: where do we stand? Am J Clin Pathol. 1999;111(suppl 1):S75-S83. 142 Am J Clin Pathol 2001;115:136-142 American Society of Clinical Pathologists