B-Cell Lymphomas With Coexpression of CD5 and CD10

Transcription

1 Hematopathology / B-CELL LYMPHOMAS WITH COEXPRESSION OF CD5 AND CD10 B-Cell Lymphomas With Coexpression of CD5 and CD10 Henry Y. Dong, MD, PhD, Wojciech Gorczyca, MD, PhD, Zach Liu, MD, Patricia Tsang, MD, C. Daniel Wu, MD, Patti Cohen, MD, and James Weisberger, MD Key Words: B-cell lymphoma; Classification; Flow cytometry; Immunohistochemistry; CD5; CD10; bcl-1; bcl-6; Coexpression DOI: /U98ADVKUC26R2RJA Abstract Coexpression of CD5 and CD10 is highly unusual in B-cell lymphomas and may pose a diagnostic challenge. We report 42 cases of B-cell lymphoma with simultaneous expression of CD5 and CD10. They made up approximately 0.4% of all B-cell lymphomas seen during the study period and included the following cases: large B-cell lymphoma (LBCL), 14 (33%); follicular lymphoma (FL), 10 (24%); mantle cell lymphoma (MCL), 9 (21%); chronic lymphocytic leukemia, 4 (10%); acute precursor B-cell lymphoblastic leukemia/lymphoma, 2 (5%); and other low-grade B-cell lymphomas, 3 (7%). All MCLs had overexpression of bcl-1 or the t(11;14) and were CD43+. All FLs had typical histomorphologic features and were bcl-2+ and bcl-6+ but CD43. Of 14 LBCLs, 5 were histologically high-grade. Six (43%) of 14 patients with LBCL died within 10 months of diagnosis of CD5+CD10+ lymphoma (median survival, 4 months), including all 3 patients with stage IV disease and 2 of 5 with histologically high-grade lymphoma. Our findings indicate that coexpression of CD5 and CD10 is rare but occurs in diverse subtypes of B-cell lymphoma. Investigation of bcl-1, bcl-6, and CD43 and morphologic evaluation may resolve the potential confusion in diagnosis and lead to the recognition of the correct lymphoma subtype. Differential expression of CD cluster antigens has been used extensively for delineating stages of B-cell development during the past few decades. It was proposed in the revised European-American lymphoma classification 1 and adopted in the World Health Organization (WHO) classification of hematopoietic malignant neoplasms 2 that the presence of these surface antigens on lymphoma cells may recapitulate that of their normal counterparts. The antigenic profiles then can help define the origin of the neoplastic lymphocytes, providing the foundation for lymphoma classification. As a result, immunophenotyping has become an integral part of the practice of pathology for the diagnosis and classification of lymphoma. Two of the most useful antigens in immunosubtyping B-cell lymphomas are CD5 and CD10. CD5 is characteristically expressed in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and mantle cell lymphoma (MCL). It is a 67-kd signal-transducing glycoprotein initially identified on the T-cell surface and is involved in the regulation of T-cell activation. It also is found on a small subset of normal B cells. Normal CD5+ B cells are defined as B1a cells with a distinct distribution and functional properties, which are thought to originate primarily from fetal liver rather than bone marrow. It also has been documented that CD5 can be up-regulated and expressed during B-cell activation. Normal CD5+ B cells are thought to remain in circulation and may be involved in the production of low-affinity autoreactive antibodies. 3,4 While CLL/SLL may represent a bona fide CD5+ B-cell lymphoproliferative disorder, the relationship of MCL to normal CD5+ B cells is not entirely understood. CD10 is characteristically expressed in acute precursor B- or T-cell lymphoblastic leukemia/lymphoma (ALL) and lymphomas of follicle center cell origin, ie, follicular 218 Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA

2 Hematopathology / ORIGINAL ARTICLE lymphoma (FL), Burkitt lymphoma, and a subset of diffuse large B-cell lymphomas (LBCLs). 1,2 It is a 100-kd transmembrane glycoprotein initially identified as the common acute lymphoblastic leukemia antigen, or CALLA. 5 CD10 is expressed on normal precursor B and T cells and follicle center B cells in the hematopoietic system. Recognition of CD5 or CD10, in conjunction with morphologic and genetic features, may identify specific lymphoma subtypes constituting more than 80% of all B-cell lymphomas other than large cell lymphoma in the United States, 6 such as FL, MCL, CLL/SLL, ALL, and Burkitt lymphoma. Detection of CD5 or CD10 positivity is part of the standard workup for diagnosis of lymphoma by immunohistochemical and flow cytometric (FCM) methods. Given the wide acceptance of fine-needle aspiration (FNA) biopsy in conjunction with FCM in recent years as a useful approach to lymphoma diagnosis, 7-9 these 2 antigens are essential to lymphoma classification when histomorphologic material is unavailable. While CD antigens such as CD5 and CD10 may be specific for stages of B-cell development and can be used conveniently for identifying specific common lymphoma subtypes, such specificity is not absolute on neoplastic cells. They not only might be absent from an entity usually associated with their expression, such as CD5 MCL 10,11 and CD10 FL, but they also can be expressed unexpectedly elsewhere, such as CD5+ marginal zone B-cell lymphoma (MZL) and CD10+ hairy cell leukemia. 18,19 In addition, a few cases of CD5+CD10+ B-cell lymphoma have been reported in the literature These unusual features may obscure the correct diagnosis and mislead management decisions when only a limited workup is performed. Although they are relatively uncommon, they seem to contradict to some degree the general concepts that underpin current B-cell lymphoma classification. We report herein a series of 42 cases of B-cell lymphoma that simultaneously expressed CD5 and CD10. Our data indicate that CD5 and CD10 coexpression may be encountered in diverse subtypes of B-cell lymphoma. However, definitive lymphoma classification can be determined by using the WHO criteria based on a combination of histopathologic features, complete immunophenotype, and genetic features. We emphasize that the inclusion of bcl-1 and bcl-6 in the immunohistochemical panel was especially helpful for characterizing these unusual lymphoma cases. Materials and Methods Case Collection Cases of B-cell lymphoma that were positive for both CD5 and CD10 were obtained from the IMPATH (New York, NY) case files during a 26-month period. The results of immunophenotyping were based on FCM, immunohistochemical analysis, or both. These cases were submitted for hematopathology diagnostic consultation from 40 institutions or hematology-oncology clinics in various clinical settings. FCM Studies The standard antibody panel used for routine FCM studies with fresh tissue or cells included CD45, CD5, CD10, CD11c, CD19, CD20, CD22, CD23, CD38, CD56, kappa, lambda, and T-cell antigens CD2, CD3, CD7, and CD8. Multicolor FCM studies were performed with fresh cell suspensions in accordance with guidelines outlined in the US-Canadian consensus conference on flow cytometry. 25 Briefly, cell suspensions were preincubated in RPMI 1640 medium supplemented with 10% fetal bovine serum to minimize nonspecific binding of antibodies. Erythrocytes in bone marrow and blood specimens were lysed with a 0.008% solution of ammonium chloride. Cells were washed with phosphate-buffered saline and incubated with cocktails of antibodies (Becton Dickinson Immunocytometry Systems, San Jose, CA, exclusively prepared for IMPATH) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC). Contents of the relevant antibody cocktails have been published previously. 10 In some cases, additional staining with PE-conjugated CD5 was performed when results with FITC-conjugated CD5 (same clone) were negative. Data acquisition and analysis were performed on FACSCalibur flow cytometers (Becton Dickinson Immunocytometry Systems) with CellQuest software (Becton Dickinson Immunocytometry Systems). Display of CD45 vs right-angle orthogonal light scatter was used to gate the target lymphoma cell population. In some cases of LBCL, display of the forward vs right-angle light scatter also was used to gate the large cell population for immunophenotypic analysis. The levels of antigen expression were depicted on multiple dual-parameter scattergrams. Positive immunoreactivity was defined as having enhanced fluorescence intensity compared with the negative control and isotype controls in every case. 26 Nonviable cells were excluded by using 7- amino actinomycin D. Immunohistochemical Analysis Formalin-fixed, paraffin-embedded tissue sections were used for immunohistochemical studies in all cases. The standard antibody panel included CD3, CD5, CD10, CD20, CD21, CD23, CD43, CD45, CD79a, bcl-1, bcl-2, bcl-6, kappa, lambda, and Ki-67. The primary antibodies and their staining conditions are listed in Table 1. The immunohistochemical staining was performed using a labeled streptavidin-biotin procedure in TechMate 500 automatic Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA 219

3 Dong et al / B-CELL LYMPHOMAS WITH COEXPRESSION OF CD5 AND CD10 Table 1 Primary Antibodies and Staining Conditions for Immunohistochemical Analysis Antibody Vendor Antibody Clone Dilution Antigen Retrieval * ph CD3 DAKO, Carpinteria, CA Polyclonal 1:2,000 Target retrieval Low CD5 Novocastra, Burlingame, CA 4C7 1:1,000 Target retrieval High CD10 Novocastra 56C6 1:50 Target retrieval Low CD20 DAKO L26 1:2,000 Target retrieval Low CD21 Becton Dickinson, San Jose, CA 1F8 1:1, % Pepsin, 37 C CD23 DAKO MH016 1:50 Target retrieval Low CD43 DAKO L60 1:75,000 Target retrieval Low CD45 DAKO/Zymed, San Francisco, CA Cocktails Target retrieval Low CD79a DAKO HM57 1:800 Target retrieval Low bcl-1 Neomarkers, Fremont, CA DCS-6 1:500 Target retrieval High bcl-2 DAKO 124 1:800 Target retrieval Low bcl-6 DAKO PG-B6p 1:20 Target retrieval High kappa DAKO Polyclonal 1:600,000 Target retrieval Low lambda DAKO Polyclonal 1:400,000 Target retrieval Low Ki-67 DAKO MIB-1 1:2,000 Citrate buffer/steaming * The target retrieval solution was from DAKO. For CD45, cocktails of DAKO clones PD7/26 and 2B11 (1:4,000 dilution) and Zymed clone 1.22/4.14 (1:20,000 dilution) were used. immunostainers (Ventana Medical Systems, Tucson, AZ). Briefly, 4-µm sections were incubated with unconjugated primary antibodies after antigen retrieval, followed by incubation with biotinylated secondary antibodies and streptavidin-conjugated peroxidase (BioGenex, San Ramon, CA). The colorimetric reaction was completed with diaminobenzidine (Sigma, St Louis, MO) as the chromogenic substrate. Antigen retrieval was performed with the heat-induced epitope retrieval method in target retrieval solution (DAKO) at an appropriate ph (Table 1), unless otherwise specified. A negative control with omission of the primary antibody was included in each case. A positive control with known positive tissue was included for each section. For cases initially diagnosed by flow cytometry only, the original tissue blocks were requested for immunohistochemical stains and morphologic evaluation, whenever possible, to confirm the lymphoma classification at the time of the diagnosis. For cases without original tissue blocks, paraffin sections made from the fresh tissue initially submitted to IMPATH for FCM studies were used for immunohistochemical analysis and sometimes for histologic correlation when tissue was adequate. Fluorescence In Situ Hybridization In cases in which only fresh cell suspensions from an FNA biopsy or peripheral blood were available, fluorescence in situ hybridization (FISH) was attempted, whenever possible, to detect the specific chromosome translocation. Interphase FISH was performed using a dual-color, dual-fusion probe set (VYSIS, Downers Grove, IL) for the t(11;14) between the bcl-1 gene and the immunoglobulin heavy chain (IgH) locus or the t(14;18) between bcl-2 and IgH, following the manufacturer s recommendation. Normal cells had 2 red and 2 green signals. Cells positive for chromosomal translocation had 1 red, 1 green, and 2 orange fusion signals. For each case, a total of 200 to 500 nuclei were scored under a fluorescent microscope at the IMPATH cytogenetic laboratory. Lymphoma Classification All lymphoma subtypes were classified by the criteria described in the WHO classification with respect to histomorphologic features, immunophenotype, and genetic features. 2 For CD5+CD10+ B-cell lymphoma of the non large cell type, immunoreactivity with bcl-6 and bcl-2 was required for the diagnosis of FL, and immunoreactivity with bcl-1 was required for MCL. When the specimens were FNA only and no tissue sections were available, detection of the t(11;14) was required in addition to the available immunophenotype for a definitive diagnosis of MCL. Cases of low-grade B-cell lymphoma, not otherwise classified, included those that lacked bcl-1 and bcl-6 and for which tissue was insufficient for complete histologic evaluation. The diagnosis of high-grade B-cell lymphoma required a high proliferation fraction with virtually all neoplastic cells positive for Ki-67, together with the corresponding morphologic features, such as a starry-sky pattern and a high mitotic rate. In cases for which there was only a limited number of fresh cells for FCM studies and no additional material for histologic, immunohistochemical, or cytogenetic evaluation, the CD5+CD10+ lymphoma was categorized by cell size based on the forward light scatter characteristics by FCM studies and cytomorphologic features with cytocentrifuged or touch-imprint samples from the FCM specimen. Some of these cases thus were defined as LBCL without further specification. All cases of biclonal and composite or collision lymphomas that had 2 distinct clonal B-cell populations were carefully excluded. 220 Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA

4 Hematopathology / ORIGINAL ARTICLE Results Clinical Features We identified 42 cases of CD5+CD10+ B-cell lymphoma during the study period. There were equal numbers of men and women (M/F ratio, 1:1), ranging in age from 25 to 103 years (median, 67 years). The anatomic locations most frequently involved by CD5+CD10+ B-cell lymphoma were lymph nodes (20/42), especially in the neck and followed by the groin and axilla; peripheral blood (8/42; in 6 cases only blood specimens were available); and bone marrow (5/42; in 3 cases only bone marrow specimens were available). Various extranodal and extramedullary sites included head and neck (4/42, in soft palate, parotid, lacrimal gland, and oropharynx), soft tissue (4/42, in chest wall, scalp, psoas muscle, and omentum), gastrointestinal tract (3/42, 1 in stomach and 2 in small intestine), spleen (1 case), and breast (1 case). Eight patients (19%) were in leukemic phase with lymphoma cells constituting 30% to 80% of peripheral lymphocytes, including 2 cases each of ALL, CLL/SLL, MCL, and LBCL. In 5 cases, there was a history of B-cell lymphoma, but coexpression of CD5 and CD10 was not described in the pathology reports for those previous lymphomas. One of the lymphomas occurred as a recurrent grade 2 FL in a lymph node at the same location. Another occurred as a high-grade diffuse LBCL, which progressed from a preexisting MZL that was diagnosed 3 years earlier in the parotid gland at the same location. Detailed histories were not available for the other 3 cases. There were 2 cases of CD5+ precursor B-cell ALL. One occurred de novo in a 25-year-old man, and the other manifested as blast crisis of chronic myelogenous leukemia in a 50-year-old man. Table 2 Summary of Clinical Data for Large B-Cell Lymphoma Cases Follow-up data were limited because of the large number of institutions or clinics in various clinical settings involved. Among patients with non large cell lymphomas, 1 patient with grade 2 FL had persistent disease 2 years after chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). One patient with recurrent MCL and the patient with de novo CD5 precursor B-cell ALL died of the disease 2 and 4 months after diagnosis, respectively. Clinical data were available for all patients with LBCL and are summarized in Table 2. Of 14 patients with LBCL, 3 patients had clinical stage IV disease and 5 had histologically high-grade lymphoma. Three patients had recurrent lymphoma, all with high-grade histologic features. Six (43%) of the 14 patients died within 10 months of the current diagnosis (median survival, 4 months). These included all 3 patients with stage IV disease, 2 of 5 with high-grade lymphoma (both occurred as relapse), and 1 with splenic LBCL. Four patients completed treatment and were in complete remission with follow-up ranging from 3 to 10 months (median, 8 months). Two patients (103 and 72 years old) who had localized disease did not receive any treatment and were alive at 6 and 3 months, respectively, after the diagnosis. The other 2 patients were undergoing chemotherapy (CHOP plus rituximab) at the time of manuscript preparation; their outcome has yet to be determined. Immunophenotypic Features The 42 cases of CD5+CD10+ B-cell lymphoma included 14 cases of LBCL (33%), 10 of FL (24%), 9 of MCL (21%), 4 of CLL/SLL (10%), 3 of low-grade B-cell lymphoma, not otherwise classified (7%), and 2 of ALL (5%). Overall, the CD5+CD10+ lymphomas represented approximately 0.4% of all B-cell lymphomas (>10,200 Case No./ Disease Ki-67 Lymphoma Blood Marrow Clinical Follow-up Sex/Age (y) Location (%) History Involvement Involvement Stage Treatment Response (mo) 1/F/70 Inguinal LN 100 No No No III CHOP NR DOD (5) 2/M/89 Bone marrow ND No No Yes IV None DOD (2) 3/M/49 Small bowel 100 No No No Unknown CHOP CR NED (10) 4/M/61 Axillary LN 100 Yes No No Unknown CHOP CR NED (10) 5/M/40 Bone marrow ND No Yes Yes IV CHOP, h-cvad CR (6 mo) DOD (10) 6/F/43 Parotid 100 Yes Unknown Unknown Unknown CHOP, XRT CR NED (6) 7/F/103 Breast 70 No No No Unknown None AWD (6) 8/M/67 Stomach 100 Yes No No Unknown CHOP PR DOD (5) 9/F/82 Spleen 70 No No No Ia CVP, rituximab NR DOD (3) 10/M/30 Neck LN No No No III CHOP, XRT CR NED (3) 11/F/72 Neck LN ND No No No Unknown None AWD (3) 12/M/63 Psoas muscle ND No No No III CHOP, rituximab AWD * (3) 13/F/81 Blood ND No Yes Yes IV Unknown DOD (2) 14/F/86 Soft palate 80 No No No III CHOP, rituximab AWD * (1) AWD, alive with disease; CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone; CR, complete remission; CVP, cyclophosphamide, vincristine, and prednisone; DOD, died of disease; h-cvad, high-dose cyclophosphamide, vincristine, doxorubicin, and dexamethasone; LN, lymph node; ND, not done; NED, no evidence of disease; NR, no response; PR, partial remission; XRT, radiation therapy. * Undergoing treatment; the outcome is undetermined. Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA 221

5 Dong et al / B-CELL LYMPHOMAS WITH COEXPRESSION OF CD5 AND CD10 Table 3 Summary of Immunophenotypic Study Results * Lymphoma No. (%) CD19 CD20 CD5 CD10 bcl-1 bcl-2 bcl-6 CD43 CD23 CD11c kappa lambda LBCL 14 (33) 14/14 14/14 14/14 14/14 0/0/10 7/10 9/12 0/12 3/7 4/9 5/9 FL 10 (24) 10/0/0/0/10 0/0/0/10 0///10 6//10 MCL 9 (21) 9/9 9/9 9/9 9/9 7/9 8/8 0/6 7/7 0/9 4/8 5/9 4/9 CLL/SLL 4 (10) 4/4 4/4 4/4 4/4 0/1 1/1 0/1 1/1 3/4 2/4 3/4 1/4 ALL 2 (5) 2/2 1/2 2/2 2/2 ND ND ND ND 0/2 0/2 0/2 0/2 BCL 3 (7) 3/3 3/3 3/3 3/3 0/3 2/2 0/3 1/3 1/3 2/3 2/3 1/3 Total 42 (100) 42/42 41/42 42/42 42/42 7/33 31/31 17/30 18/33 8/40 13/34 20/37 15/37 ALL, acute precursor B- or T-cell lymphoblastic leukemia/lymphoma; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; FL, follicular lymphoma; LBCL, large B-cell lymphoma; MCL, mantle cell lymphoma; ND, not done. * Data are given as number positive/number tested unless otherwise indicated. The remaining 2 cases had t(11;14) detected by FISH. Both cases were positive for terminal deoxynucleotidyl transferase with either no CD20 or dim CD20 in a subset. Low-grade B-cell lymphoma, not otherwise classified. cases) at our institution. Of all cases of MCL, FL, and CLL/SLL diagnosed at IMPATH during the same period, approximately 4.2%, 0.7%, and 0.3%, respectively, were positive for both CD5 and CD10. The immunophenotypic results for all cases are summarized in Table 3. FCM Results Of 42 cases, 31 (74%) were submitted initially for FCM studies; for 17 of these cases, specimens were available for FCM only. By FCM analysis, the CD5+ and CD10+ staining on neoplastic B cells in each case was demonstrated by an increased fluorescence intensity compared with that on the isotype controls Image 1, Image 2, and Image 3. Multicolor fluorescence staining with CD5 and CD10 in the same tube further confirmed that both CD5 and CD10 were coexpressed on the same cell population (Image 2 and Image 3). Although the expression level of CD5 or CD10 in each case might have varied, the variable fluorescence intensity always showed a coherent single cell population. A discrete population of B cells positive for only CD5 or CD10, which was clearly separable from the double-positive cells, was absent in all cases. The relative intensity of CD5 and CD10 did not seem to correlate with lymphoma subtypes and did not necessarily contribute to lymphoma classification. For example, expression of CD5 could be as strong as the expression of CD10 on cells of FL (Image 1), and CD10 could be as bright as CD5 on MCL (Image 2). However, the intensity of CD10 tended to be more variable in both FL and MCL cases. In 2 cases, the CD10 intensity varied widely from bright to virtually negative within a single cell population (Image 3). In contrast, the intensity of CD5 usually was less variable. In cases in which only FNA specimens were available for FCM studies, additional stains, such as CD43 and bcl-2, often were performed in an attempt to obtain maximal information for lymphoma subtyping. The presence of CD43 usually favored MCL over FL, despite an overexpression of ,000 1, R lgg2a FITC CD45 APC 7AAD FC7 SSC kappa CD10 PE FSC lgg2a APC lambda CD5 FITC Image 1 Flow cytometric studies of a lymph node biopsy specimen with analysis of the CD45+ viable cells shows a monoclonal kappa B-cell population (CD19+) with coexpression of CD5 and CD10 (inset, isotype controls). A diagnosis of follicular lymphoma was established by morphologic examination and positive bcl-6 staining performed subsequently in a tissue section. AAD, amino actinomycin D; APC, allophycocyanin; FITC, fluorescein isothiocyanate; FSC, forward scatter; PE, phycoerythrin; SSC, side scatter. 222 Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA

6 Hematopathology / ORIGINAL ARTICLE kappa PE lambda PE kappa PE lambda PE CD19 FITC CD19 FITC CD10 PE CD10 PE CD23 FITC CD5 FITC Image 2 Flow cytometric studies of a lymph node biopsy specimen show a monoclonal kappa B-cell population with coexpression of CD5 and CD10. (NOTE: CD5 and CD10 staining was performed in the same tube.) CD23 expression was negative. A diagnosis of mantle cell lymphoma was established by morphologic examination and positive bcl- 1/cyclin D1 staining performed subsequently in a tissue section. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin. CD23 FITC CD5 FITC CD5 PE CD10 PE CD10 PE CD5 PE bcl-2 FITC Image 3 Flow cytometric studies of a lymph node fineneedle aspiration biopsy specimen show a monoclonal kappa B cell population with coexpression of CD5, CD10, CD43, and bcl-2. A diagnosis of mantle cell lymphoma was established by subsequent detection of t(11;14) by fluorescence in situ hybridization in the same specimen. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin. CD19 PE CD10 PE CD5 FITC CD43 FITC CD5 FITC bcl-2 in the same clonal B-cell population (Image 3). As shown in Table 3, CD43 was positive in all cases of MCL and negative in all cases of FL. CD11c and CD23 were not helpful for distinguishing MCL from FL. There were 3 cases of CLL/SLL with characteristically low-intensity expression of surface immunoglobulin light chain, CD20, and CD22. The presence of CD23 helped confirm the diagnosis, in conjunction with cytomorphologic examination. There were 2 cases of ALL diagnosed by expression of terminal deoxynucleotidyl transferase and lack of surface light chain. The case of de novo ALL was CD34+. The case derived from chronic myelogenous leukemia also expressed myeloid antigen CD33 and cytoplasmic IgM but lacked expression of CD34. Immunohistochemical Results For 11 (26%) of 42 cases, only paraffin blocks were available for immunohistochemical analysis and morphologic Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA 223

7 Dong et al / B-CELL LYMPHOMAS WITH COEXPRESSION OF CD5 AND CD10 evaluation. By immunoperoxidase staining, coexpression of CD5 and CD10 in these cases was detected on the vast majority of neoplastic cells with at least moderate intensity. For 20 of 31 cases submitted for FCM analysis, tissue sections also were available. Of these cases, 10 (50%) were unequivocally positive for both CD5 and CD10 by immunohistochemical analysis. The remaining 10 cases were positive for either CD5 or CD10 corresponding to the expected lymphoma subtype or positive for both CD5 and CD10, with expression of 1 marker being weak and focal. The overall positivity rates for CD5 and CD10 by immunohistochemical analysis were 70% (14/20) and 80% (16/20), respectively. The 6 cases that were CD5 included FL (5 cases) and MCL (1 case). The 4 cases that were CD10 included MCL (3 cases) and LBCL (1 case). All cases of MCL were CD20+, CD43+, bcl-1+, and bcl-2+ but bcl-6. All cases of FL were CD20+, bcl-2+, and bcl-6+ but CD43 and bcl-1. Among 14 cases of LBCL, 10 with immunohistochemical analysis and histologic evaluation were CD20+ and bcl-2+ but bcl-1. Seven of the 10 had coexpression of bcl-6 in addition to CD5 and CD10, and 6 of the 7 also were CD43+. Three cases lacked both bcl-1 and bcl-6. For the remaining 4 cases, no material was available for immunohistochemical analysis, so they were designated as LBCL without further characterization. Correlation With Histopathologic Findings For 31 (74%) of 42 cases, formalin-fixed, paraffinembedded tissue sections were available for evaluation, including all cases of FL and 7 of 9 cases of MCL. The additional 2 cases of MCL were FNA specimens, for which the diagnosis of MCL was confirmed by FISH detection of the t(11;14). The morphologic features of CD10+ MCL were not distinct from those of ordinary CD10 cases. All cases had the morphologic features of the classic type of MCL. The lymphoma cells typically were composed of monotonous small to medium-sized cells with scant cytoplasm and irregular nuclear contours (centrocytic) in a diffuse or vaguely nodular pattern Image 4A and were bcl-1+ Image 4B. Scattered epithelioid histiocytes also were seen in some of the cases. Features of the blastic variant were not seen in any of these cases. Likewise, the morphologic features of CD5+ FL were not distinctive. All cases with sufficient tissue showed atypical follicular lymphoproliferation characteristic of FL. Most cases also had variable diffuse areas and were associated with sclerosis, a common change seen in FL. All but 1 case was morphologically low grade (grade 1-2 of 3). Only 1 case was grade 3 with a component of diffuse LBCL. Both diffuse and follicular components expressed CD5 and CD10. In 2 cases with cutaneous involvement, there was a nodular lymphoproliferation with cytologic features reminiscent of monocytoid B cells (abundant pale cytoplasm and centrocyte-like nuclei) Image 4C. However, the presence of strong expression of bcl-6 and bcl-2 in addition to CD10 in virtually all neoplastic cells confirmed the diagnosis of FL Image 4D. Histologic features of the so-called floral variant of FL were not seen in this series. The morphologic features of CD5+CD10+ LBCLs varied. Features of highly aggressive or high-grade B-cell lymphoma were seen in 5 of 10 cases. All 5 cases had a high proliferation fraction as indicated by strong Ki-67 nuclear immunoreactivity in virtually all neoplastic cells Image 5F. Among them, 4 of 5 cases were characterized by a diffuse or nodular architecture, a prominent starry-sky pattern, the presence of numerous apoptotic cells, and brisk mitosis. However, the cytomorphologic features of these cells usually were less monotonous than those seen in typical Burkitt lymphoma Image 5A and Image 5B. Immunophenotypically, the neoplastic cells all were positive for CD43 Image 5C, bcl-2 Image 5D, and bcl-6 Image 5E, in addition to CD5 and CD10 Image 5G and Image 5H. The strong expression of bcl-2 in these cases does not favor atypical Burkitt lymphoma according to the WHO classification criteria. The fifth case had predominantly dyshesive anaplastic large cells Image 4E that lacked both bcl-1 and bcl-6. The remaining 5 cases of LBCL had cytologic features of either centroblastic or cleaved large cells; 3 of these cases were positive for bcl-6. Typical immunoblastic features were not seen. For 1 of 4 CLL/SLL cases, tissue sections were available; neoplastic B cells were CD5+ and CD10+ with a typical pattern of proliferation centers or pseudofollicles. The expression of bcl-1 was negative. The histologic features in this case, together with the strong expression of CD43 and bcl-2, confirmed the diagnosis of CLL/SLL despite lack of CD23 by immunohistochemical analysis. FCM studies were not performed in this case. One of the low-grade B-cell lymphomas in the small intestine showed diffuse proliferation of small lymphocytes with a prominent plasmacytic component Image 4F, most likely representing an extranodal MZL of the mucosa-associated lymphoid tissue type with plasmacytic differentiation. Neither bcl-1 nor bcl-6 was positive in this case. Discussion Our results indicate that CD5 and CD10 may be coexpressed on a single clonal population in approximately 0.4% of B-cell lymphomas. It is a rare event but may occur in diverse subtypes of B-cell lymphoma. These cases include, in order of frequency found in the present study, diffuse 224 Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA

8 Hematopathology / ORIGINAL ARTICLE A B C E D F Image 4 Examples of histopathologic features of B-cell lymphoma coexpressing CD5 and CD10. A, Mantle cell lymphoma, classic type (H&E, 400). B, Staining with bcl-1 in the same case ( 400). C, Follicular lymphoma involving skin with a monocytoid appearance (H&E, 400). D, Staining with bcl-6 in the same case ( 400). E, Low-grade B-cell lymphoma with plasmacytoid differentiation negative for both bcl-1 and bcl-6 (H&E, 400). F, Diffuse large B-cell lymphoma with bizarre anaplastic cells negative for both bcl-1 and bcl-6 (H&E, 400). Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA 225

9 Dong et al / B-CELL LYMPHOMAS WITH COEXPRESSION OF CD5 AND CD10 A B C Image 5 An example of large B-cell lymphoma shows diffuse proliferation of large neoplastic cells (A, H&E, 100; B, H&E, 400) positive for CD43 (C, 400), bcl-2 (D, 400), bcl-6 (E, 400), Ki-67 (F, 400), CD5 (G, 400), and CD10 (H, 400). D LBCL (14/42), FL (10/42), MCL (9/42), CLL/SLL (4/42), low-grade B-cell lymphoma, not otherwise classified (3/42), and ALL (2/42). The overall frequency of this unusual phenotype in B-cell lymphomas may be an underestimate, because cases without FCM studies might have been missed owing to the lower detection rate by immunohistochemical analysis alone that we observed. The discrepancy between FCM and immunohistochemical analyses in detecting CD5 and CD10 coexpression may be due to the sensitivity of the methods for detecting CD5, CD10, or both. Despite reports that new monoclonal antibodies for immunohistochemical analysis, such as 4C7 for CD5 27 and 56C6 for CD10, 28 have sensitivities comparable to FCM analysis under optimal conditions, in general the results of immunohistochemical analysis are more prone to suboptimal tissue preservation and adverse effects of various fixatives, which are inevitable in our practice as a referral center and may account for the variation in the immunohistochemical results. The contribution of immunophenotyping is crucial for the diagnosis and classification of lymphoma using the WHO classification criteria, particularly when an FNA or a peripheral blood specimen is the only specimen available for analysis. In these cases, FCM studies often can efficiently resolve the uncertainty of cytomorphologic examination alone in dealing with lymphomas other than large cell lymphomas. 7 However, coexpression of CD5 and CD10 complicates the interpretation of lymphoma immunophenotyping, which until recently has emphasized the value of CD5 and CD10 for defining common subtypes of B-cell lymphoma. All of 226 Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA

10 Hematopathology / ORIGINAL ARTICLE E F G Image 5 (cont) The high proliferation fraction (Ki-67 = 100% of neoplastic cells) suggests a high-grade lymphoma. H our cases, therefore, required additional immunophenotypic and/or genotypic studies for the final lymphoma classification. These included, in particular, detection of bcl-1 and bcl- 6 expression. A few case reports in the literature have described the CD5 and CD10 double-positive immunophenotype in rare cases of CLL/SLL or other low-grade B-cell lymphoma, 20 MCL, 12 rare cases of FL of the floral variant, 21 and occasional cases of high-grade B-cell lymphoma Approximately 10% of FL cases were reported to be CD5+ in a large series, 13 although details were not available and the finding has not been confirmed independently. Owing to the rarity of these cases and lack of bcl-1 and/or bcl-6 studies in most of the previous reports, it is unclear whether the tentative lymphoma classification in all these reports fulfilled the standards of the current WHO criteria. Our study included 42 cases with the analysis of bcl-1 and bcl-6 in addition to a broad panel of antibodies against various CD antigens. Our data further underscore the importance of bcl-1 and bcl-6 as a part of complete immunophenotyping in lymphoma classification, particularly when the expression of CD5, CD10, or both is ambiguous or the histologic and immunophenotypic features seem incompatible. Overexpression of bcl-1 due to the t(11;14), a specific chromosome translocation between the bcl-1 and IgH loci, is considered the hallmark of MCL. 29 Although plasma cell myeloma 30 and hairy cell leukemia 31 also may express bcl-1 at a relatively high frequency, the morphologic features, together with lack of cytoplasmic immunoglobulin or CD25 and CD103 expression, may easily distinguish MCL from the other 2 entities. The transcription factor bcl-6 is Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA 227

11 Dong et al / B-CELL LYMPHOMAS WITH COEXPRESSION OF CD5 AND CD10 expressed consistently only in follicle center cells and their derivatives in the normal hematopoietic system. 32,33 While CD10 is positive in approximately 60% and 90% of FL cases by immunohistochemical 13 and FCM analyses, 12,14 respectively, and it may be present on lymphoma cells other than FL, bcl-6 seems to be a reliable marker for FL. However, it is noteworthy that bcl-6 expression in LBCL may not necessarily indicate follicular origin, for chromosomal translocation or gene amplification involving 3q27 also may lead to bcl-6 overexpression Recent DNA profiling data indicate that bcl-6 may be expressed in either germinal center B- cell like or activated B-cell like LBCLs at a similar frequency as well. 37 In addition to bcl-1 and bcl-6, differential expression of CD43 in B-cell lymphomas may aid in lymphoma classification. It has been well documented in the literature that CD43 expression can be detected in the majority of CD5+ B-cell lymphomas (MCL, CLL/SLL) but in only 1% to 5% of FLs. 38 In our study, all cases of MCL and no cases of FL were CD43+. Burkitt lymphoma, on the other hand, differs from FL by coexpressing CD43 and lacking bcl-2. A subset of LBCLs in the present study was CD10+, CD20+, CD43+, bcl-2+, and bcl-6+. Such an immunophenotype is rather unusual for either FL or Burkitt lymphoma. Hence, our results indicate that CD43 is an additional valuable marker useful for distinguishing MCL from FL and FL from Burkitt lymphoma in practice, even though CD5 and CD10 may be coexpressed in MCL, FL, or Burkitt lymphoma. Although the obscure phenotype resulting from the coexpression of CD5 and CD10 might cast doubts on the diagnostic criteria for immunophenotype in the WHO classification, our results demonstrate that classification of CD5+CD10+ B-cell lymphoma can be established with certainty based on the principles of the WHO classification. Despite the fact that CD5+CD10+ B-cell lymphoma consists of heterogeneous entities, the extended workup required only a few additional stains (bcl-1, bcl-6, and CD43). When only fresh cell suspensions for FCM studies are available, cytogenetic analysis by interphase FISH or karyotyping, whichever is feasible, may contribute to lymphoma classification. In this regard, it is important to obtain a proper specimen in a timely manner to pursue the opportunity. In the present study, 2 of 2 cases of non large cell lymphoma for which only an FNA specimen was available were diagnosed as MCL based on the presence of the t(11;14) detected by interphase FISH. Although detection of the t(14;18) also is helpful for establishing a diagnosis of FL and is academically interesting for studying these cases, the typical histologic features combined with the typical phenotype (CD10+, bcl-6+, bcl-2+, CD43 ) associated with FL was sufficiently diagnostic in practice. Thus, cytogenetic studies for FL were performed rarely in this series. The biologic basis for coexpression of CD5 and CD10 on B cells is unclear. Such an immunophenotype has never been clearly documented for normal B cells at any developmental stage. Because both FL and MCL were described as centrocytic lymphoma in the updated Kiel classification and were thought to be derivatives of B-cell follicles, 39 it has been tempting to speculate that FL and MCL may be related to one another and to B-cell follicle development. Coexpression of CD5 and CD10 may lend further support to the speculation. However, FL and MCL distinguish themselves by the mutually exclusive expression of bcl-1 and bcl-6, even in the presence of both CD5 and CD10. The diversity of B-cell lymphomas that coexpressed CD5 and CD10 in our study further suggests that such coexpression may not be related to follicular ontogeny, particularly in light of the 2 cases of CD5+CD10+ precursor B-cell ALL. CD5 expression has been reported infrequently in nearly all B-cell lymphoma subtypes besides CLL/SLL and MCL, including FL, 21 Burkitt lymphoma, 23 MZL, LBCL, 24,40,41 and hairy cell leukemia. 19,42 Hence, the expression of CD5 in B-cell lymphoma could be an acquired phenomenon under neoplastic conditions. Because other T-cell antigens, such as CD2, CD7, and CD8, also have been seen occasionally in B-cell lymphomas, especially in LBCLs, 43 coexpression of CD5 in neoplastic B cells may simply represent a spectrum of B-cell lymphomas aberrantly expressing T-cell antigens. In fact, 1 case of FL in the present study also expressed strong CD7 detected by both FCM and immunohistochemical analyses. Similarly, CD10 also has been reported in hairy cell leukemia, 18,19 myeloma, 44,45 and, rarely, lymphoplasmacytic lymphoma. 12 Our study provides additional evidence that CD10 may be expressed in lymphoma subtypes other than ALL or those of follicle center B-cell origin. Our results suggest that expression of neither CD5 nor CD10 on neoplastic B cells is absolutely lineage- or developmental stage restricted. It is plausible that coexpression of CD5 and CD10 is due to aberrant antigen expression commonly occurring in a neoplastic process. An analogous situation can be seen with CD27 and CD148. Both initially were defined as antigens specific for normal memory B cells and absent on normal naive B cells. 46,47 However, they are expressed in essentially all types of B-cell lymphomas, including those presumably derived from naive B cells. 48,49 In light of these observations, one ought to be aware that neoplastic B cells may bear unusual features, perhaps as a result of their genetic abnormalities, despite retaining many of the usual features of their normal counterparts. In practice, lymphoma classification should depend on a comprehensive profiling and not be confused by unusual expression of certain specific cell surface molecules, such as in the case of CD5 and CD Am J Clin Pathol 2003;119: DOI: /U98ADVKUC26R2RJA

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