T-Cell-Rich Large-B-Cell Lymphomas Contain Non-Activated CD8+ Cytolytic T Cells, Show Increased Tumor Cell Apoptosis, and Have Lower Bcl-2 Expression Than Diffuse Large-B-Cell Lymphomas
1998; Elsevier BV; Volume: 153; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65685-4
ISSN1525-2191
AutoresRaymond E. Felgar, Katherine R. Steward, John B. Cousar, William R. Macon,
Tópico(s)Immune Cell Function and Interaction
ResumoThe factor(s) responsible for the reduced B cell number and increased T cell infiltrate in T-cell-rich large-B-cell lymphomas (TCRBCLs) have not been well characterized. We studied 18 TCRBCLs and 12 diffuse large-B-cell lymphomas (DLBCLs) to compare the 1) predominant T cell subpopulation(s), 2) expression of cytotoxic granule proteins (TIA-1 and granzyme B), 3) level of tumor cell apoptosis (Apoptag system, Oncor, Gaithersburg, MD), and 4) expression of Ki-67 (Mib-1) and apoptosis-related proteins (fas (CD95), bcl-2, and p53). T cells in TCRBCLs and DLBCLs were predominantly CD8+ T cells expressing αβ T-cell receptors and TIA-1 (16 of 18 TCRBCLs with >50% TIA-1+ small lymphocytes) but lacking granzyme B (16 of 18 TCRBCLs with <25% granzyme B+ small lymphocytes). Scattered apoptotic tumor cells (confirmed with CD20 co-labeling) were present in 15 of 18 TCRBCLs, with 14 of 15 cases having <10% apoptotic cells. No apoptotic cells were seen in 12 of 12 DLBCLs. In 16 of 16 immunoreactive TCRBCLs, 50% bcl-2+ tumor cells. CD95 (fas) expression was also lower, with 3 of 18 (16.7%) TCRBCLs versus 4 of 12 (33%) DLBCLs having >25% CD95+ tumor cells. TCRBCLs and DLBCLs had similar levels of p53 and Ki-67 (Mib-1) expression. Thus, T cells in TCRBCLs are non-activated cytotoxic T lymphocytes (TIA-1+, granzyme B−). Tumor cell apoptosis (perhaps cytotoxic T cell mediated) may partly account for the decreased number of large (neoplastic) B cells in TCRBCLs, but other factors (ie, decreased bcl-2 expression) may also be needed. The factor(s) responsible for the reduced B cell number and increased T cell infiltrate in T-cell-rich large-B-cell lymphomas (TCRBCLs) have not been well characterized. We studied 18 TCRBCLs and 12 diffuse large-B-cell lymphomas (DLBCLs) to compare the 1) predominant T cell subpopulation(s), 2) expression of cytotoxic granule proteins (TIA-1 and granzyme B), 3) level of tumor cell apoptosis (Apoptag system, Oncor, Gaithersburg, MD), and 4) expression of Ki-67 (Mib-1) and apoptosis-related proteins (fas (CD95), bcl-2, and p53). T cells in TCRBCLs and DLBCLs were predominantly CD8+ T cells expressing αβ T-cell receptors and TIA-1 (16 of 18 TCRBCLs with >50% TIA-1+ small lymphocytes) but lacking granzyme B (16 of 18 TCRBCLs with <25% granzyme B+ small lymphocytes). Scattered apoptotic tumor cells (confirmed with CD20 co-labeling) were present in 15 of 18 TCRBCLs, with 14 of 15 cases having <10% apoptotic cells. No apoptotic cells were seen in 12 of 12 DLBCLs. In 16 of 16 immunoreactive TCRBCLs, 50% bcl-2+ tumor cells. CD95 (fas) expression was also lower, with 3 of 18 (16.7%) TCRBCLs versus 4 of 12 (33%) DLBCLs having >25% CD95+ tumor cells. TCRBCLs and DLBCLs had similar levels of p53 and Ki-67 (Mib-1) expression. Thus, T cells in TCRBCLs are non-activated cytotoxic T lymphocytes (TIA-1+, granzyme B−). Tumor cell apoptosis (perhaps cytotoxic T cell mediated) may partly account for the decreased number of large (neoplastic) B cells in TCRBCLs, but other factors (ie, decreased bcl-2 expression) may also be needed. T-cell-rich large-B-cell lymphoma (TCRBCL) is a term used to describe a group of diffuse large-B-cell lymphomas with a prominent infiltrate of small T lymphocytes.1Ramsay AD Smith WJ Isaacson PG T-cell-rich B-cell lymphoma.Am J Surg Pathol. 1988; 12: 433-443Crossref PubMed Scopus (229) Google Scholar, 2Macon WR Williams ME Greer JP Stein RS Collins RD Cousar JB T-cell-rich B-cell lymphomas: a clinicopathologic study of 19 cases.Am J Surg Pathol. 1992; 16: 351-363Crossref PubMed Scopus (109) Google Scholar, 3Krishnan J Wallberg K Frizzera G T-cell-rich large B-cell lymphoma: a study of 30 cases supporting its histologic heterogeneity and lack of clinical distinctiveness.Am J Surg Pathol. 1994; 18: 455-465Crossref PubMed Scopus (105) Google Scholar, 4Baddoura FK Chan WC Masih AS Mitchell D Sun NCJ Weisenburger DD T-cell-rich B-cell lymphoma: a clinicopathologic study of eight cases.Am J Clin Pathol. 1995; 103: 65-75Crossref PubMed Scopus (57) Google Scholar, 5Chittal SM Brousset P Voigt J-J Delsol G Large B-cell lymphoma rich in T-cells and simulating Hodgkin's disease.Histopathology. 1991; 19: 211-220Crossref PubMed Scopus (116) Google Scholar, 6Delabie J Vandenberghe E Kennes C Verhoef G Foschini MP Stul M Cassiman JJ De Wolf-Peeters C Histiocyte-rich B-cell lymphoma: a distinct clinicopathologic entity possibly related to lymphocyte predominant Hodgkin's disease, paragranuloma subtype.Am J Surg Pathol. 1992; 16: 37-48Crossref PubMed Scopus (138) Google Scholar The percentage of T cells needed to classify a lymphoma as a TCRBCL is somewhat controversial, but most authors would accept tumors as TCRBCL if at least 65% to 90% of the lymphoid infiltrate is T cells, with the remainder being large B cells.7Farhi DC T-cell-rich B-cell lymphoma: reflections on changes in hematopathology.Am J Clin Pathol. 1995; 103: 4-5PubMed Google Scholar The B cells have been shown to be a clonal, presumably neoplastic cell population by demonstration of light chain restriction and/or by the presence of immunoglobulin gene rearrangements.2Macon WR Williams ME Greer JP Stein RS Collins RD Cousar JB T-cell-rich B-cell lymphomas: a clinicopathologic study of 19 cases.Am J Surg Pathol. 1992; 16: 351-363Crossref PubMed Scopus (109) Google Scholar Despite the abundance of T cells, most studies indicate the clinical behavior of TCRBCLs is not significantly different from other diffuse large-B-cell lymphomas (DLBCLs), which typically have few tumor-infiltrating lymphocytes (TILs). Previous studies by Chittal et al5Chittal SM Brousset P Voigt J-J Delsol G Large B-cell lymphoma rich in T-cells and simulating Hodgkin's disease.Histopathology. 1991; 19: 211-220Crossref PubMed Scopus (116) Google Scholar and Delabie et al6Delabie J Vandenberghe E Kennes C Verhoef G Foschini MP Stul M Cassiman JJ De Wolf-Peeters C Histiocyte-rich B-cell lymphoma: a distinct clinicopathologic entity possibly related to lymphocyte predominant Hodgkin's disease, paragranuloma subtype.Am J Surg Pathol. 1992; 16: 37-48Crossref PubMed Scopus (138) Google Scholar have indicated that some TCRBCLs may follow a more aggressive clinical course. These studies examined a total of nine and six patients, respectively. Of the combined 15 patients, 6 followed a very aggressive clinical course with short survival (≤18 months). Of these six patients with an aggressive clinical course, it is interesting to note that four had been originally diagnosed and treated as Hodgkin's disease rather than non-Hodgkin's lymphoma. Therefore, the poor results in some of these patients may have been related to inappropriate initial therapy rather than due to an inherently more aggressive biological behavior. More recent studies by Greer et al8Greer JP Macon WR Lamar RE Wolff SN Stein RS Flexner JM Collins RD Cousar JB T-cell-rich B-cell lymphomas: diagnosis and response to therapy of 44 patients.J Clin Oncol. 1995; 13: 1742-1750Crossref PubMed Scopus (57) Google Scholar and Rodriguez et al9Rodriguez J Pugh WC Cabanillas F T-cell-rich B-cell lymphoma.Blood. 1993; 82: 1586-1589PubMed Google Scholar have examined larger series of 44 and 23 patients, respectively, and have shown no significant survival difference between TCRBCLs and DLBCLs. Finally, data examining bone marrow involvement in TCRBCLs10Skinnider BF Connors JM Gascoyne RD Bone marrow involvement in T-cell-rich B-cell lymphoma.Am J Clin Pathol. 1997; 108: 570-578PubMed Google Scholar have shown no significant difference in overall 4-year survival in patients with TCRBCL involving bone marrow compared with a group of control patients with bone marrow involvement by histologically concordant DLBCL when treated with curative intent. The few TCRBCLs analyzed by flow cytometry or frozen-section immunohistochemistry have shown a predominance of CD4+ T cells with a CD4:CD8 ratio compatible with a reactive infiltrate.1Ramsay AD Smith WJ Isaacson PG T-cell-rich B-cell lymphoma.Am J Surg Pathol. 1988; 12: 433-443Crossref PubMed Scopus (229) Google Scholar, 2Macon WR Williams ME Greer JP Stein RS Collins RD Cousar JB T-cell-rich B-cell lymphomas: a clinicopathologic study of 19 cases.Am J Surg Pathol. 1992; 16: 351-363Crossref PubMed Scopus (109) Google Scholar At least one case report with flow cytometric immunophenotyping has suggested that the predominant T cell subpopulation is CD8+.11Kawada H Watanabe S Yoshida M Fukuda R Kobayashi N Masumoto A Ogawa Y Ohbayashi Y Yonekura S Ichikawa Y Flow cytometric analysis of T-cell-rich B-cell lymphoma.Acta Haematol. 1994; 92: 164-166Crossref PubMed Scopus (5) Google Scholar However, no previous studies have fully characterized the T cells based on functional and T cell receptor (TCR) framework antigen expression. Recently, a number of antibodies have been developed to allow CD4 and CD8 subtyping of T cells in fixed, paraffin-embedded tissue sections to assess the type of TCR expressed and to evaluate the presence of cytotoxic-granule-associated proteins such as TIA-1 and granzyme B.12Felgar RE Macon WR Kinney MC Roberts S Pasha T Salhany KE TIA-1 expression in lymphoid neoplasms: identification of subsets with cytotoxic T lymphocyte or natural killer cell differentiation.Am J Pathol. 1997; 150: 1893-1900PubMed Google Scholar, 13Macon WR Salhany KE T-cell subset analysis of peripheral T-cell lymphomas by paraffin immunoperoxidase and correlation of CD4/CD8 results to flow cytometry.Am J Clin Pathol. 1998; 109: 610-617PubMed Google Scholar, 14Oudejans JJ Kummer JA Jiwa M van der Valk P Ossenkoppele GJ Kluin PM Kluin-Nelemans JC Meijer CLM Granzyme B expression in Reed-Sternberg cells of Hodgkin's disease.Am J Pathol. 1996; 148: 233-240PubMed Google Scholar Furthermore, the factor(s) responsible for the increased number of T cells and/or the reduced number of neoplastic B cells in TCRBCLs have not been extensively studied. Previous studies from our group have suggested that interleukin (IL)-4 may play a role in the proliferation of T cells and/or the suppression of B cell growth.15Macon WR Cousar JB Waldron JA Hsu S-M Interleukin-4 may contribute to the abundant T-cell reaction and paucity of neoplastic B cells in T-cell-rich B-cell lymphomas.Am J Pathol. 1992; 141: 1031-1036PubMed Google Scholar However, the role of other proliferation-associated and/or apoptosis-associated proteins has not been investigated. We therefore undertook the present study to compare TCRBCLs and DLBCLs to 1) define the predominant T cell phenotype in TCRBCLs in paraffin-embedded, fixed tissue sections, 2) to assess the expression of cytotoxic T lymphocyte (CTL)-associated proteins, namely TIA-1 and granzyme B, in the T cell infiltrate of TCRBCL and DLBCL, 3) to examine tissue sections for evidence of apoptosis in both TCRBCL and DLBCL using a commercially available terminal deoxynucleotidyl transferase (TdT) end-labeling technique (Apoptag, Oncor, Gaithersburg, MD), and 4) to examine and compare the expression of the proliferation-associated marker Ki-67 (Mib-1) and the apoptosis-associated proteins fas (CD95), bcl-2, and p53 in TCRBCL versus DLBCL. Eighteen TCRBCLs and twelve DLBCLs were evaluated by paraffin immunoperoxidase staining for the antigens described below and for the presence of apoptotic tumor cells using the Apoptag in situdetection kit (Oncor). The clinicopathological features of the cases chosen for study have been previously published.2Macon WR Williams ME Greer JP Stein RS Collins RD Cousar JB T-cell-rich B-cell lymphomas: a clinicopathologic study of 19 cases.Am J Surg Pathol. 1992; 16: 351-363Crossref PubMed Scopus (109) Google Scholar, 15Macon WR Cousar JB Waldron JA Hsu S-M Interleukin-4 may contribute to the abundant T-cell reaction and paucity of neoplastic B cells in T-cell-rich B-cell lymphomas.Am J Pathol. 1992; 141: 1031-1036PubMed Google Scholar As defined in previous publications,2Macon WR Williams ME Greer JP Stein RS Collins RD Cousar JB T-cell-rich B-cell lymphomas: a clinicopathologic study of 19 cases.Am J Surg Pathol. 1992; 16: 351-363Crossref PubMed Scopus (109) Google Scholar, 8Greer JP Macon WR Lamar RE Wolff SN Stein RS Flexner JM Collins RD Cousar JB T-cell-rich B-cell lymphomas: diagnosis and response to therapy of 44 patients.J Clin Oncol. 1995; 13: 1742-1750Crossref PubMed Scopus (57) Google Scholar, 15Macon WR Cousar JB Waldron JA Hsu S-M Interleukin-4 may contribute to the abundant T-cell reaction and paucity of neoplastic B cells in T-cell-rich B-cell lymphomas.Am J Pathol. 1992; 141: 1031-1036PubMed Google Scholar lymphomas were classified as TCRBCLs if the majority (ie, more than 50%) of cells were small T cells by paraffin immunoperoxidase studies for CD3 and/or CD45RO (UCHL-1). One TCRBCL (case 17 in 2Macon WR Williams ME Greer JP Stein RS Collins RD Cousar JB T-cell-rich B-cell lymphomas: a clinicopathologic study of 19 cases.Am J Surg Pathol. 1992; 16: 351-363Crossref PubMed Scopus (109) Google Scholar) and three DLBCLs (see15Macon WR Cousar JB Waldron JA Hsu S-M Interleukin-4 may contribute to the abundant T-cell reaction and paucity of neoplastic B cells in T-cell-rich B-cell lymphomas.Am J Pathol. 1992; 141: 1031-1036PubMed Google Scholar) described previously could not be evaluated in the present study because of insufficient tissue. Paraffin sections (4 μm) from B5-fixed tissues were stained by the streptavidin-biotin complex method and horseradish peroxidase (HRP) reacted with 2′,5′-diaminobenzidine (DAB) according to manufacturer's instructions or previously published modifications12Felgar RE Macon WR Kinney MC Roberts S Pasha T Salhany KE TIA-1 expression in lymphoid neoplasms: identification of subsets with cytotoxic T lymphocyte or natural killer cell differentiation.Am J Pathol. 1997; 150: 1893-1900PubMed Google Scholar, 13Macon WR Salhany KE T-cell subset analysis of peripheral T-cell lymphomas by paraffin immunoperoxidase and correlation of CD4/CD8 results to flow cytometry.Am J Clin Pathol. 1998; 109: 610-617PubMed Google Scholar, 16Shipley WR Hammer RD Lennington WJ Macon WR Paraffin immunohistochemical detection of CD56, a useful marker for neural cell adhesion molecule (NCAM), in normal and neoplastic fixed tissues.Appl Immunohistochem. 1997; 5: 87-93Crossref Scopus (43) Google Scholar for expression of the following antigens: CD20 (L26), CD8, CD3 (polyclonal), p53, and bcl-2, all from Dako, Carpinteria, CA; CD56 (123C3) from Zymed Corp., South San Francisco, CA; CD4 from Vector Laboratories, Burlingame, CA; TCR-αβ (βF-1) and TCR-γδ (TCRδ1), both from Endogen, Woburn, MA; Ki-67 (Mib-1) from Immunotech, Westbrook, ME; and TIA-1 from Coulter Immunology, Hialeah, FL. In addition, immunoperoxidase staining for CD95 (fas) expression was performed using a rabbit polyclonal antiserum (Santa Cruz Biotechnology, Santa Cruz, CA), with microwave epitope retrieval in 10 mmol/L sodium citrate, pH 6.0 (two 5-minute applications). Paraffin-embedded, B5-fixed sections (4 μm) were deparaffinized in 2 g% iodine in xylene (10 minutes at 25°C), followed by three washes (five minutes each at room temperature) in xylene alone. Slides were rehydrated by sequential washes (5 minutes each) in 100% ethanol (two times), 70% ethanol, and phosphate-buffered saline (PBS; 50 mmol/L sodium phosphate, 200 mmol/L sodium chloride, pH 7.4). Endogenous peroxidase activity was inactivated by incubating slides in 3% hydrogen peroxide in PBS (5 minutes at 25°C), followed by three washes in PBS (5 minutes each at 25°C). After blocking nonspecific antibody-binding sites (Protein Blocker, Research Genetics, Huntsville, AL) for 5 minutes at 25°C, slides were incubated with anti-CD95 at a 1:50 dilution in PBS for 60 minutes at 37°C and washed three times in PBS. Staining was then detected using biotinylated goat anti-rabbit antibody (Dako; 60 minutes at 37°C, followed by three washes in PBS), streptavidin-conjugated horseradish peroxidase (HRP; Research Genetics; 30 minutes at 25°C, followed by three washes in PBS), and DAB (Stable DAB, Research Genetics; two applications of 5 minutes each at 25°C). Sections were then washed three times in distilled water, stained with hematoxylin (Autohematoxylin, Research Genetics), and mounted onto coverglasses using standard techniques. Immunoperoxidase staining for human granzyme B expression (1:20 dilution, clone GrB-7; Monosan, Uden, The Netherlands) was performed by the streptavidin-biotin complex method using HRP with DAB on B5-fixed paraffin-embedded sections (4 μm) pretreated by microwave epitope retrieval in 100 mmol/L sodium citrate, according to the manufacturer's directions. In all immunostaining procedures, the adequacy of staining was verified with appropriate positive controls, including tonsil (CD20, CD3, CD4, CD8, βF1, TIA-1, and Mib-1), hepatosplenic γδ T cell lymphoma (granzyme B, TCRδ1), retinoblastoma (CD56), follicular lymphoma (bcl-2), breast carcinoma (p53), and reactive lymph node (CD95). Mercury salts and paraffin were removed from sections (4 μm) of B5-fixed tissues by soaking in 2 g% iodine in xylene for 10 minutes followed by three washes (5 minutes each) in xylene only. Sections were then rehydrated and processed using the Apoptag in situdetection kit (Oncor) according to the manufacturer's instructions, except that tissues were counterstained with Gill's hematoxylin (Fisher Scientific, Fair Lawn, NJ). Positive control sections (from a case of HIV-related lymphadenopathy) were run with each experiment. TCRBCL cases were also double labeled for CD20 expression (L26) followed by biotinylated goat anti-mouse antibody (Dako) and a streptavidin-biotin detection system with alkaline-phosphatase and fast red substrate (Research Genetics) according to the manufacturer's instructions. Antibody staining was evaluated independently and jointly by at least two pathologists (R.E. Felgar, K.R. Steward, and/or W.R. Macon). Staining of the small lymphocytes was scored as follows: 0, no staining; 1+, 1% to 25% small lymphocytes positive; 2+, 26% to 50% small lymphocytes positive; 3+, 51% to 75% small lymphocytes positive; 4+, 76% to 100% small lymphocytes positive. Staining of the large (neoplastic) lymphocytes was also evaluated using the following scale: 0, no staining; 1+, 1% to 10% large cells positive; 2+, 11% to 25% large cells positive; 3+, 26% to 50% large cells positive; 4+, >50% large cells positive. The general demographic features of the patients from whom the TCRBCLs and DLBCLs were obtained for study were similar. The TCRBCL group consisted of 10 males and 8 females with a mean age of 52.2 years (median, 53.5 years); the DLBCL group consisted of 7 males and 5 females with a mean age of 58.7 years (median, 56.5 years). The age range of the TCRBCL group (18 to 92 years), however, was much broader than that seen in the DLBCL group (46 to 74 years). Immunophenotyping data for the small lymphocytes in the TCRBCL group are summarized in Table 1. In 16 of 18 TCRBCLs, more than 50% of the TILs were TIA-1+ (score of 3+ to 4+) with most of them also expressing CD8 and αβ TCRs (βF1+) (see Table 1 and Figure 1). One case (number 4 in Table 1) had a predominance of CD4+ and βF1+ TILs that were also TIA-1+. Staining for granzyme B showed a few cells staining in all cases but a much lower number staining in comparison with TIA-1 (16 of 18 cases with 25% or fewer granzyme B+cells), suggesting a non-activated functional status. In most cases, few lymphocytes stained for CD4, CD56, or γδ TCRs (TCRδ1+). Staining of the DLBCLs showed only a few scattered small lymphocytes with a lower percentage of TIA-1+ cells (9 of 12 cases with <50% small lymphocytes TIA-1+) than in TCRBCLs; the percentage of small lymphocytes in DLBCLs expressing granzyme B was also generally lower in comparison with TIA-1 expression, with 8 of 12 cases showing <25% granzyme B+ cells (see Table 2). In general, most of the TILs in DLBCLs were also CD8+ and βF1+ (see Table 2 and Figure 2). Because most lymphocytes were CD8+ and βF1+, staining for CD56 and TCRδ1 was not evaluated in the DLBCLs.Table 1Immunophenotype of Small T Cells in T-Cell-Rich Large-B-Cell LymphomasCaseAge (Years)SexTIA-1GrBCD4CD8CD56βF1TCRδ1155M3+1+03+1+3+1+266M3+1+02+1+2+1+342F3+1+02–3+1+1+1+418F3+1+4+1+1+4+1+568F3+1+01+1+2+1+674M3+1+04+1+2–3+1+744F3+1+1+2–3+1+3+1+847M2+1+1+2+1+3+1+969M3+1+02–3+1+3+1+1056F4+2+04+1+1+1+1167F3+1+1+1+1+4+1+1257M3+1+2+3+1+4+1+1352M4+1+2+3+1+4+1+1436M3+1+1+4+1+3+1+1536F4+1+02+1+4+1+1618M1+1+1+1+1+2–3+1+1792F4+1+1+3+1+2–3+1+1843M4+3+1–2+4+1+3+1+Patients 10 and 12 represent relapsed lymphomas with a TCRBCL pattern, occurring 8 and 7 years, respectively, after an initial diagnosis of DLBCL. (See also Table 2, Table 4 footnotes.) GrB, granzyme B; M, male; F, female. Results are shown based on the following scoring system: 0, no small cells marking; 1+, 1% to 25% small cells marking; 2+, 26% to 50% small cells marking; 3+, 51% to 75% small cells marking; 4+, 76% to 100% small cells marking. Open table in a new tab Table 2Immunophenotype of Small T Cells in Diffuse Large-B-Cell LymphomasCaseAge (Years)SexTIA-1GrBCD4CD8βF1162M2–3+1+02–3+NR274M3+1+03+3+346M2+2+02+2+473F2+1–2+03+3+558F2+2+1+3+3+657M1+1+0–1+3+3+756F3+2–3+03+3+866M1+003+3+956M3+1+03+3+1052M1+001–2+3+1150F2+1+02+2–3+1254F1+1+02+2+Initial neoplasm in case number 6 was a DLBCL; patient had relapsed lymphoma 7 years later with a TCRBCL pattern (case 12 in Table 1, Table 3). The initial neoplasm in case 7 also was a DLBCL, with a relapse 8 years later with a TCRBCL pattern (case 10 in Table 1, Table 3). See Table 1 for scoring system. GrB, granzyme B; M, male; F, female. Open table in a new tab Figure 2Staining pattern in a typical case of DLBCL (case 2 in Table 2, Table 4). A: Typical H&E appearance of large transformed lymphocytes. B: CD8 staining; C: TIA-1 staining; D: βF1 staining, showing that a few TILs are present that are also CD8+ and βF1+ T cells.E: No apoptotic tumor cells are detectable with the Apoptag assay system. Magnification (oil immersion), ×1000 (A andE) and ×600 (B toD).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Patients 10 and 12 represent relapsed lymphomas with a TCRBCL pattern, occurring 8 and 7 years, respectively, after an initial diagnosis of DLBCL. (See also Table 2, Table 4 footnotes.) GrB, granzyme B; M, male; F, female. Results are shown based on the following scoring system: 0, no small cells marking; 1+, 1% to 25% small cells marking; 2+, 26% to 50% small cells marking; 3+, 51% to 75% small cells marking; 4+, 76% to 100% small cells marking. Initial neoplasm in case number 6 was a DLBCL; patient had relapsed lymphoma 7 years later with a TCRBCL pattern (case 12 in Table 1, Table 3). The initial neoplasm in case 7 also was a DLBCL, with a relapse 8 years later with a TCRBCL pattern (case 10 in Table 1, Table 3). See Table 1 for scoring system. GrB, granzyme B; M, male; F, female. TdT end-labeling studies using the Apoptag in situdetection kit highlighted the presence of apoptotic cells in 15 of 18 cases of TCRBCL (see Table 3 and Figure 1); double-labeling studies using L26 with an immunoalkaline phosphatase detection system showed that these cells were marking as CD20+ B cells (ie, consistent with the neoplastic cell population; see Figure 1.) In general, the number of apoptotic cells in TCRBCLs was low, with 14 of 15 cases having 51% large cells marking. NR, tissue nonreactive with antibody (confirmed on repeat staining); M, male; F, female. Open table in a new tab Table 4Immunophenotype of Large (Neoplastic) B Cells in Diffuse Large-B-Cell LymphomasCaseAge (Years)SexApoptagp53Bcl-2Mib-1 (Ki-67)CD95 (fas)162M02+4+4+0274M01+3+2–3+4+346M01+4+1+1+473F04+01+3+558F01+4+1+0657M02+02+4+756F01+1+1+0866M02+3+3+1+956M01+4+2+01052M01+4+1+01150F04+1+2+3+1254F03+4+2+0Initial neoplasm in case 6 was a DLBCL; patient had relapsed lymphoma 7 years later with a TCRBCL pattern (case 12 in Table 1, Table 3). The initial neoplasm in case 7 also was a DLBCL, with a relapse 8 years later with a TCRBCL pattern (case 10 in Table 1, Table 3). See Table 3 for scoring system. M, male; F, female. Open table in a new tab Patients 10 and 12 represent relapsed lymphomas with a TCRBCL pattern, occurring 8 and 7 years, respectively, after an initial diagnosis of DLBCL. (See also Table 2, Table 4 footnotes.) Results are shown based on the following scoring system: 0, no large cells marking; 1+, 1% to 10% large cells marking; 2+, 11% to 25% large cells marking; 3+, 26% to 50% large cells marking; 4+, >51% large cells marking. NR, tissue nonreactive with antibody (confirmed on repeat staining); M, male; F, female. Initial neoplasm in case 6 was a DLBCL; patient had relapsed lymphoma 7 years later with a TCRBCL pattern (case 12 in Table 1, Table 3). The initial neoplasm in case 7 also was a DLBCL, with a relapse 8 years later with a TCRBCL pattern (case 10 in Table 1, Table 3). See Table 3 for scoring system. M, male; F, female. Data regarding the expression of the proliferation-associated antigen Ki-67 (Mib-1 staining) and apoptosis-related antigens (p53, bcl-2, and CD95/fas) in the tumor (large B) cell population are summarized in Table 3, Table 4. TCRBCLs had a lower percentage of tumor cells expressing bcl-2 (16 of 16 immunoreactive cases had <25% bcl-2+ tumor cells) than DLBCLs (4 of 12 cases with 50% bcl-2+ tumor cells). CD95 (fas) expression was slightly lower in TCRBCLs, as 3 of 18 (16.7%) cases had >25% CD95+ tumor cells as compared with DLBCLs, which had 4 of 12 (33%) cases with >25% CD95+ tumor cells. In general, there were no consistent differences in the percentage of tumor cells expressing Ki-67 (Mib-1+) or p53 between TCRBCLs and DLBCLs. In both TCRBCLs and DLBCLs, 83% of cases (15 of 18 TCRBCLs and 10 of 12 DLBCLs) stained positively for Mib-1 in <25% of tumor cells. There was also comparable p53 staining, as 15 of 18 (83%) TCRBCLs and 9 of 12 (75%) DLBCLs were positive in 50% of the small lymphocytes (score of 3+ or 4+; Table 1), and in an additional 3 cases, CD8+ lymphocytes appeared to be the predominant T cell subset (score of 2+ to 3+; Table 1). Previous studies on peripheral T cell lymphomas by our group13Macon WR Salhany KE T-cell subset analysis of peripheral T-cell lymphomas by paraffin immunoperoxidase and correlation of CD4/CD8 results to flow cytometry.Am J Clin Pathol. 1998; 109: 610-617PubMed Google Scholar have estimated the sensitivity of CD8 immunostaining by this paraffin immunoperoxidase (PIP) technique to be 90% using flow cytometry as the comparative standard. PIP CD4 staining, in contrast, had an estimated sensitivty of 64%. Therefore, it is possible that we have underestimated the true CD4+ T cell subset in the present study. Indeed, in six cases (cases 2, 5, 9, 11, 13, and 16 in Table 1), the total percentage of CD4+and CD8+ cells combin
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