Peripheral T-Cell Lymphoma Complicated by a Proliferation of Large B Cells

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1 Hematopathology / LARGE B-CELL RICH T-CELL LYMPHOMA Peripheral T-Cell Lymphoma Complicated by a Proliferation of Large B Cells John P.T. Higgins, MD, Matthijs van de Rijn, MD, PhD, Carol D. Jones, James L. Zehnder, MD, and Roger A. Warnke, MD Key Words: Epstein-Barr virus; Large B-cell lymphoma; Peripheral T-cell lymphoma; Immunosuppression; Immunohistochemistry Abstract We studied 14 cases that showed a morphologic appearance of peripheral T-cell lymphoma and contained substantial numbers of CD20+ large B cells. In all but 2 cases, the CD20+ large cells showed a mix of kappa and lambda light chain expression. Two cases showed a focal predominance of kappa expression. In situ hybridization using the EBER1 probe for detection of Epstein-Barr virus (EBV) RNA was performed on every case. EBV RNA was present in 10 cases. Of 8 cases with EBV RNA stained by immunohistochemistry for the latent membrane protein of EBV, 6 were positive. Double-labeling immunohistochemistry and in situ hybridization confirmed that EBV was present in the large B cells. Polymerase chain reaction (PCR) analysis showed a clonal rearrangement of the T-cell receptor (TCR)-gamma chain gene in 12 of 13 cases tested. One additional case showed a clonal rearrangement of the TCR-beta chain gene by Southern blot hybridization. PCR analysis showed a clonal immunoglobulin gene rearrangement in 5 cases, a suggestion of a clonal rearrangement in 1, an oligoclonal pattern in 4, and a polyclonal pattern in 4. The finding of large B and T cells may result in a misdiagnosis of a reactive process or of T-cell rich B-cell lymphoma. The presence of EBV in some cases could cause further confusion with the reactive T- and B-immunoblastic proliferation of infectious mononucleosis. Numerous investigators have identified Epstein-Barr virus (EBV) in peripheral T-cell lymphoma The prevalence of EBV varies markedly according to the subtype of T-cell lymphoma: lymphoblastic and low-grade cutaneous T-cell lymphoma are consistently negative; T-lineage anaplastic large cell lymphoma is usually negative; and angioimmunoblastic lymphadenopathy like T-cell lymphoma, Lennert lymphoma, and pleomorphic and immunoblastic T-cell lymphoma may show EBV RNA in numbers approaching 100% of cases. 8,9,15-17 The fraction of positive cells also varies dramatically, from rare cells (<1 per medium-power field) to virtually every cell. 17,18 Early studies suggested that EBV was present exclusively in T-lineage cells, 12 but EBV subsequently has been demonstrated in cells of B and T lineages. 2,7,8,16-19 In 1 study, the EBV-positive cells were frequently negative for B- and T- lineage markers. 11 More recently, Ho et al 19 described 23 cases of EBV-associated peripheral T-cell lymphoma in which molecular studies showed clonal T-cell receptor (TCR)-beta gene rearrangement and a suggestion of clonal immunoglobulin heavy chain gene rearrangement. 19 Doublelabeling studies of these cases showed that the majority of the EBV-infected cells were of B lineage. It has been suggested that the milieu of the T-cell lymphoma facilitates the proliferation of the EBV-harboring B cells as occurs in the setting of immunosuppression related to HIV infection or organ transplantation. 4,7,9,20 We describe a series of lymphomas that histologically and by molecular analysis are peripheral T-cell lymphomas but are accompanied by an infiltrate of large B cells. These large B cells usually showed a mix of kappa and lambda light chain expression. EBV was present in most but not all cases. The finding of large B and T cells can result in misdiagnosis of this T-cell lymphoma as a reactive process or as large B-cell 236 Am J Clin Pathol 2000;114: American Society of Clinical Pathologists

2 Hematopathology / ORIGINAL ARTICLE lymphoma. Furthermore, the presence of EBV in association with a proliferation of T- and B-lineage cells may suggest a diagnosis of infectious mononucleosis. The occurrence of a large B-cell proliferation against a background of peripheral T- cell lymphoma has not been emphasized previously. Materials and Methods Cases Cases were obtained from the hematolymphoid consultation service in the Division of Surgical Pathology, Stanford University Medical Center, Stanford, CA. Cases of peripheral T-cell lymphoma in which large CD20-expressing B cells were discernible in the T-cell areas were included in the series. Initially, cases were required to show molecular evidence of T- cell clonality; however, because molecular studies will fail to identify approximately 20% of cases of peripheral T-cell lymphoma, 21 this requirement was waived for one of the later cases in the series. This case showed compelling histologic and immunophenotypic findings for peripheral T-cell lymphoma. The majority of the cases were referred from pathologists practicing in community settings in northern California. One case, case 11, was reported previously. 22 These cases were all presented as an abstract at the 1999 Annual Meeting of the United States and Canadian Academy of Pathology. 23 Light Microscopy and Immunohistochemistry Histologic sections were prepared from tissue fixed in 10% formalin, embedded in paraffin, and cut at 3 to 4 µm. Sections for light microscopy were stained with H&E. Sections were stained for immunohistochemistry on an automated immunostainer (Ventana ES, Ventana Medical Systems, Tucson, AZ) using the biotin-avidin technique in which diaminobenzidine was used as a chromogen. 24 Monoclonal antibodies used were as follows: L26 (anti-cd20), Ber-H2 (anti-cd30), 1F8 (anti-cd21), and LMP-1 (latent membrane protein; CS1-4, DAKO, Carpinteria, CA). Polyclonal antibodies used were as follows: CD3p, kappa, and lambda (DAKO). Microwave pretreatment was used for L26, Ber-H2, and CD3p. 1F8, kappa, and lambda were pretreated with protease (Ventana Medical Systems). In Situ Hybridization for Epstein-Barr Viral RNA In situ hybridization was performed on sections cut from formalin-fixed paraffin-embedded tissue using methods previously described. 25,26 Slides were deparaffinized in xylene and hydrated through graded alcohols. Deparaffinized tissues then were digested in proteinase-k, 3 µg/ml in a 50-mmol/L concentration of tris(hydroxymethyl)aminomethane (Tris), ph 7.5, for 30 minutes at 37 C, and then washed in water followed by 95% ethanol for 3 minutes and then 100% ethanol for 3 minutes and air dried. The following then were added to each slide in sequence: 12 µl of 2 hybridization buffer (Sigma, St Louis, MO), 10 µl of formamide, 1 µl (20 ng/µl) of biotinylated EBV probe (Operon, Alameda, CA), and 1.5 µl of deionized water. A coverglass was applied, and each slide was incubated for a minimum of 4 hours at 37 C. Slides then were washed in a 50 mmol/l concentration of Tris, a 150-mmol/L concentration of sodium chloride, a 0.1% solution of Triton X-100 (Sigma), ph 7.5, and Tris-buffered saline (TBS) and incubated in streptavidin-alkaline phosphatase for 40 minutes at 4 C, then washed in TBS followed by a Trissaline mixture, ph 9.5. Slides then were incubated in substrate solution (nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt, both in N,N-Dimethyl formamide [Sigma]) for 20 to 40 minutes at room temperature and washed briefly in water. A blue-black reaction product over the nucleus constituted a positive reaction. A known EBVpositive tumor served as a positive control, and an oligo dt probe was used to document preservation of messenger RNA. Double-Labeling Immunohistochemistry and In Situ Hybridization When slides were available, cases that showed staining for Epstein-Barr virus encoded RNA (EBER) were processed for double-labeling immunohistochemistry and in situ hybridization as previously described. 27 Sections were dewaxed and rehydrated in descending alcohol concentrations. Endogenous peroxidase was inactivated with 3% hydrogen peroxide, and slides were microwaved in citrate buffer when appropriate. Primary monoclonal antibodies used were as follows: L26 and mb1 (anti-cd79a) (DAKO) and Leu22 (anti- CD43) (Becton Dickinson, San Jose, CA). The polyclonal antibody was CD3p (DAKO). Microwave pretreatment was performed in citrate buffer for CD3p, mb1, and the control slides in which phosphate-buffered saline (PBS) incubation was substituted for the primary antibody incubation. Slides were incubated with the primary antibody for 30 minutes followed by rinsing twice in PBS. The slides then were incubated with goat antimouse biotinylated secondary antibody for 40 minutes and rinsed twice in TBS. A 40-minute incubation with streptavidin alkaline phosphatase was followed by rinsing in TBS and application of fast red substrate for 25 minutes and TBS, PBS, and water rinses. Slides then were treated with proteinase-k, 3 µg/ml in a 50- mmol/l concentration of Tris, ph 7.5, for 30 minutes at 37 C, rinsed in water, and allowed to air dry. Twenty-five microliters of biotinylated EBER probe at 1 ng/µl in a 40% solution of formamide, 1 hybridization buffer (Sigma) were added under a coverglass, and the slides were incubated overnight at 37 C. The slides then were washed in a 50-mmol/L concentration of American Society of Clinical Pathologists Am J Clin Pathol 2000;114:

3 Higgins et al / LARGE B-CELL RICH T-CELL LYMPHOMA Tris, a 150-mmol/L concentration of sodium chloride, a 0.1% solution of Triton X-100 at ph 7.5, and finally in TBS. A second 40-minute incubation in streptavidin alkaline phosphatase was performed in a Coplin jar. Slides were rinsed twice again in TBS and then a Tris-saline mixture, ph 9.5. Nitroblue tetrazolium substrate was added and incubated for 20 to 40 minutes at room temperature. A final rinse in water was followed by application of a coverglass. TCR-gamma and Immunoglobulin Heavy Chain Polymerase Chain Reaction Analysis DNA from formalin-fixed paraffin-embedded specimens was extracted using a modification of methods previously described. 28 Amplification and heteroduplex analysis for TCR-gamma gene rearrangement were adapted from Chhanabhai et al. 21 The amplification of the TCR-gamma V-J junction was performed using a single set of consensus primers: V1 (5 - TACATCCACTGGTACCTACACCA-3 ) for the V-gamma-I (V-gamma 1-8) and J1/2 (5 -CCCGTCGACTACCTTG- GAAATGTTGTATTCTTC-3 ) for the J-gamma-1 and J- gamma-2 segments. Each reaction mixture consisted of 2U of AmpliTaq Gold polymerase (Perkin-Elmer, Foster City, CA), 10 pmol of each primer, 125-µmol/L concentrations of each deoxynucleoside triphosphate (dntp) in Perkin-Elmer polymerase chain reaction (PCR) buffer II with a 1.5-mmol/L concentration of magnesium chloride and 500 ng of DNA template to a volume of 50 µl. A touch down amplification protocol consisted of 10 minutes of denaturing at 95 C, followed by 20 cycles of 95 C denaturing (40 seconds), 65 C annealing (30 seconds), decreasing by 1 C per cycle, 72 C extension (30 seconds), followed by 15 cycles with the same denaturing and extension times and temperatures and an annealing temperature of 45 C for 30 seconds. The final step consisted a 5-minute extension at 72 C. Touchdown amplification protocols have been shown to improve the specificity and yield of PCR reactions. 29 Ten microliters of each PCR amplicon was loaded on a 2% agarose gel. A band of approximately 300 base pairs indicated that the amplification of the TCR-gamma was successful. Only successfully amplified specimens were analyzed further by heteroduplex electrophoresis. The heteroduplex annealing step was performed as follows: using the thermal cycler, 20 µl of the PCR reaction plus 2 µl of a 0.05-mol/L concentration of EDTA were denatured at 95 C for 5 minutes and then cooled to 37 C over a 30-minute period and held at 4 C until loading onto the mutation detection enhanced (MDE) gel. The 1 MDE gel was prepared using 2 MDE stock solution (FMC Bioproducts, Rockland, ME) in a 54- mmol/l concentration of Tris-borate, a 0.6-mmol/L concentration of EDTA and polymerized with TEMED (BioRad, Hercules, CA) and freshly made 10% ammonium persulfate (Ameresco, Solon, OH). We used a Protean Xii vertical gel system (BioRad) with a gel size of mm. The gel was run in a 54-mmol/L concentration of Tris-borate and a 0.6-mmol/L concentration of EDTA at 80 V for 16 hours. After staining with 1 µg/ml of ethidium bromide, heteroduplex patterns were photographed under UV light. In each experiment, a monoclonal control (Jurkat T-cell lymphoma cell line) and polyclonal control (tonsil) were run in parallel with the test samples. TCR-gamma gene PCR was performed at the National Cancer Institute, National Institutes of Health, Bethesda, MD, on the first of 2 biopsy specimens from case 10 using the method described by McCarthy et al 30 (Mark Raffeld, MD, National Institutes of Health, verbal communication). A final case, case 11, showed extensive DNA degradation, precluding heteroduplex analysis for detection of TCR-gamma gene rearrangement; however, fresh tissue from this case was evaluated successfully by Southern blot hybridization and showed a clonal rearrangement of the TCR-beta gene. Amplification and analysis for immunoglobulin gene rearrangement were adapted from the method described by Lozano et al 31 for frozen tissue. The amplification of the immunoglobulin heavy chain complementarity-determining region 3 was performed using a single set of primers: JH3 (5 - AACTGCAGAGGAGACGGTGACC-3 ), which is a consensus primer of the immunoglobulin heavy chain (IgH) J regions, and FRIII (5 -CTGTCGACACGGCCGTGTAT- TACTG-3 ), which is a consensus primer for the third framework segment of the IgH variable regions. Each reaction mixture consisted of 2.5 U of AmpliTaq Gold polymerase, 50 pmol of each primer, a 500-µmol/L concentration of each dntp in Perkin-Elmer PCR buffer II with a 3-mmol/L concentration of magnesium chloride and 500 ng of DNA template to a volume of 50 µl. A touch down amplification protocol consisted of 10 minutes of denaturing at 95 C, followed by 18 cycles of 95 C denaturing (40 seconds), 60 C annealing (30 seconds), decreasing by 1 C per cycle, followed by 17 cycles with the same denaturing time and temperature and an annealing temperature of 41 C for 30 seconds. The final step consisted of a 5-minute extension at 72 C. Ten microliters of each PCR amplicon was loaded on a 2% agarose gel. A band of approximately 100 to 150 base pairs indicated that the amplification of the IgH was successful. Only successfully amplified specimens were analyzed further by electrophoretic separation of 20 µl of amplicon on a 10% polyacrylamide gel. The gel was prepared on a Mini-Protean ii apparatus (BioRad) and run at 200 V for 1 hour. After staining with 1 µg/ml of ethidium bromide, the gel was photographed under UV light. In each experiment, the following controls were run in parallel with the test samples: a monoclonal control (SUDHL4 lymphoma cell line), a sensitivity control (SUDHL4 DNA diluted to 10% in 238 Am J Clin Pathol 2000;114: American Society of Clinical Pathologists

4 Hematopathology / ORIGINAL ARTICLE polyclonal tonsillar DNA), a polyclonal control (tonsil), and a blank (no template DNA). Results Clinical features and favored diagnoses of the referring pathologists are given in Table 1. One patient, case 2, had AIDS. Three patients had a skin rash. In case 7, this was described as a rash without qualification, while 1 patient, case 5, had generalized pruritus and urticaria that were not responsive to corticosteroid therapy. Another patient, case 11, had violaceous skin nodules. This patient had rheumatoid arthritis for which he was taking gold and prednisone. Three patients had hypergammaglobulinemia. This was polyclonal in 2 cases (6 and 13), and was related to a monoclonal IgG kappa paraprotein in 1 (case 5). One patient (case 13) had anemia and hypercalcemia. Flow cytometry had been performed in 2 cases. In case 12, a population of CD3, CD4+ T cells was revealed. Interestingly, this case showed expression of CD3 by immunohistochemistry. This discrepancy may be related to a cytoplasmic localization of the antigen. Flow cytometry in case 2 revealed a normal distribution of CD4+ and CD8+ T cells without loss of pan T-cell antigen expression. Thirteen of 14 cases were submitted with a favored diagnosis from the referring pathologist. Only 1 case was diagnosed definitively as peripheral T-cell lymphoma. A diagnosis of T-cell lymphoma was considered in an additional 3 cases. Other diagnoses considered for these 3 cases included histiocytic lymphoma, mixed cellularity Hodgkin disease, and T-cell rich B-cell lymphoma. Four cases were identified as lymphoma without determination or prediction of lineage. Two were classified as mixed cellularity Hodgkin disease. Three were considered to represent reactive hyperplasia or reactive immunoblastic proliferations. Two cases, cases 2 and 10, had subsequent biopsy specimens that showed diffuse large B-cell lymphoma. In case 10, this occurred 6 months to 1 year after the initial biopsy. In case 2, the large B-cell lymphoma was discovered 1 year and 10 months after the first biopsy. Histologic Features All 14 cases showed diffuse distortion or effacement of lymph node architecture. A recurring pattern was that of paracortical expansion by a population of atypical cells with vesicular chromatin and medium to large nuclei Image 1. Ten cases showed conspicuous cytoplasmic clearing of the atypical cell population. Twelve cases showed vascular proliferation. Nine cases showed infiltration of the atypical cell population by eosinophils. Six cases contained clusters of epithelioid histiocytes. Residual follicles, when present, showed a reactive appearance with germinal center formation. In short, the histologic appearance was typical of peripheral T-cell lymphoma. In some cases, a distinct population of large cells could be seen against the background of atypical medium to large cells Image 2. Thick-walled vessels and a burned-out lymphocyte-depleted appearance, features of angioimmunoblastic lymphadenopathy, were not striking in any of the cases. Table 1 Clinical Features Case No./ Sex/ Age (y) Biopsy Site Clinical Findings Submitting Diagnosis 1/F/36 Inguinal node Diffuse lymphadenopathy, B symptoms, Non-Hodgkin lymphoma positive Epstein-Barr virus titer 2/M/52 Cervical node AIDS, localized right cervical lymphadenopathy Benign lymphoid hyperplasia 3/M/80 Cervical node Localized left cervical lymphadenopathy Atypical immunoblastic proliferation 4/M/79 Cervical node Localized left cervical lymphadenopathy Lymphoma, not otherwise specified 5/M/64 Axillary node Diffuse lymphadenopathy, urticaria, IgG kappa MCHD paraprotein, polyclonal hypergammaglobulinemia 6/M/71 Inguinal node Diffuse lymphadenopathy, polyclonal Florid polyclonal immunoproliferative reaction hypergammaglobulinemia 7/F/69 Cervical node Diffuse arthralgias, skin rash MCHD 8/F/82 Axillary node Diffuse lymphadenopathy, fever Large cell lymphoma, histiocytic vs T-cell 9/F/51 Cervical node Cervical, mediastinal, and abdominal adenopathy None given 10/M/41 Cervical node Cervical and submental lymphadenopathy Peripheral T-cell lymphoma 11/M/73 Unspecified Rheumatoid arthritis, violaceous skin nodules, Angiocentric lymphoma peripheral node splenomegaly 12/F/77 Inguinal node Diffuse lymphadenopathy Peripheral T-cell lymphoma vs MCHD vs TCRBCL 13/M/76 Cervical node Anemia, hypercalcemia, polyclonal Lymphoma, possible lymphoplasmacytic hypergammaglobulinemia lineage 14/M/68 Cervical node Bilateral cervical adenopathy T-cell lymphoma vs Hodgkin disease IgG, immunoglobulin G; MCHD, mixed cellularity Hodgkin disease; TCRBCL, T-cell rich B-cell lymphoma. American Society of Clinical Pathologists Am J Clin Pathol 2000;114:

5 Higgins et al / LARGE B-CELL RICH T-CELL LYMPHOMA Image 1 (Case 12) Typical morphologic appearance of our cases, showing a paracortical proliferation of cells with cleared cytoplasm admixed with prominent vessels (original magnification 50). Image 2 (Case 12) A distinct population of very large cells with vesicular chromatin and large nucleoli is visible against the atypical cell background (original magnification 100). Table 2 Immunohistochemical and Molecular Findings Light Chain CD21 Staining Case No. Monotypia Pattern LMP-1 EBV In Situ TCR-gamma PCR VDJ PCR 1 No AILD-like Positive Positive Positive Negative 2 * No Normal ND Positive Positive Positive 3 Focal kappa Extensive Negative Negative Positive Suggestive 4 No Normal Negative Negative Positive Negative 5 No ND Positive Positive Positive Positive 6 Focal kappa Normal Negative Negative Positive Oligoclonal 7 No Normal Negative Negative Positive Negative 8 No Normal Negative Positive Positive Positive 9 No Partial AILD pattern Positive Positive Positive Oligoclonal 10 No Normal ND Positive Positive Oligoclonal 11 No Normal Negative Positive ND Negative 12 No AILD-like Positive Positive Positive Positive 13 No Partial AILD pattern Positive Positive Negative Oligoclonal 14 No Normal Positive Positive Positive Positive AILD, angioimmunoblastic lymphadenopathy/dysproteinemia; EBV, Epstein-Barr virus; LMP, latent membrane protein; ND, not done; TCR, T-cell receptor. * This patient subsequently developed diffuse large B-cell lymphoma. Although there was extensive CD21 staining, the pattern correlated with staining for CD20 and was thought to be normal. This patient had a subsequent biopsy that revealed diffuse large B-cell lymphoma. The second biopsy revealed kappa light chain restriction and a clonal immunoglobulin heavy chain gene rearrangement. EBV in situ, TCR-gamma PCR, and Southern blot hybridization were negative on the second biopsy specimen. TCR-beta clonality confirmed by Southern blot hybridization. Immunohistochemistry Results of immunohistochemical, in situ hybridization, and molecular studies are given in Table 2. The atypical interfollicular cells were invariably positive for CD3 Image 3. Although the nuclear size of the T- cell population varied from case to case, at least occasional CD3+ large cells were identified in every case. The B-cell marker L26 stained variable numbers of large cells admixed with the T cells in the interfollicular areas Image 4. Large B cells constituted 5% to 10% of the cell population. In most cases, kappa and lambda light chain staining of the large cells in the interfollicular areas approximated the normal ratio of 2:1 to 3:1 Image 5 and Image 6. In 2 cases (3 and 6), a subtle focal suggestion of a predominance of kappa light chain expression was noted. In case 3, the kappa/lambda ratio was approximately 5:1 to 10:1, while in case 6, this ratio was estimated at 10:1 to 20:1. In a third case (case 10), an initial biopsy revealed a clonal rearrangement of the TCRgamma gene, and in situ hybridization studies revealed the presence of EBV RNA. A subsequent biopsy revealed disappearance of the TCR-gamma clone with emergence of a 240 Am J Clin Pathol 2000;114: American Society of Clinical Pathologists

6 Hematopathology / ORIGINAL ARTICLE Image 3 (Case 12) Immunohistochemical stain for CD3 highlighting medium to large cells. The largest cells are negative (hematoxylin counterstain, original magnification 100). Image 4 (Case 12) Immunohistochemical stain for CD20 highlighting numerous large B cells in the paracortical area (hematoxylin, original magnification 100). Image 5 (Case 12) Immunohistochemical stain for kappa light chain highlighting a proportion of the large cells (hematoxylin counterstain, original magnification 100). Image 6 (Case 12) Immunohistochemical stain for lambda showing a virtually identical pattern to that seen for kappa (hematoxylin counterstain, original magnification 100). proliferation of large B cells with a clonal rearrangement of the immunoglobulin heavy chain gene. This patient s second specimen showed a kappa predominance of greater than 20:1 by immunohistochemistry. The findings in the second specimen warranted a diagnosis of diffuse large B-cell lymphoma. Eight of 13 cases tested showed no increase in CD21 staining or expansion of CD21+ follicular dendritic cell processes into the paracortex, findings that are usually present in angioimmunoblastic T-cell lymphoma. Case 3 showed a striking amount of CD21 staining; however, the distribution of this staining correlated with that seen for CD20 and was thought to be related to follicular hyperplasia. Two cases (9 and 10) showed focal expansion of follicular dendritic cell processes, and 2 cases (1 and 12) showed a well-developed diffuse extension of follicular dendritic cell processes into the paracortex, characteristic of angioimmunoblastic lymphadenopathy. Five of 6 cases tested showed staining of a variable number of large cells for CD30 (data not shown). EBV Studies Of 14 patients, 10 had evidence of EBV in their lymph nodes as detected by in situ hybridization Image 7. In these American Society of Clinical Pathologists Am J Clin Pathol 2000;114:

7 Higgins et al / LARGE B-CELL RICH T-CELL LYMPHOMA Image 7 (Case 12) In situ hybridization for Epstein-Barr virus (EBV) RNA showing numerous medium and large cells with nuclear and nucleolar staining for the EBV-encoded RNA (EBER-1) probe (hematoxylin counterstain, original magnification 100). Image 8 (Case 13) Double-label immunohistochemistry and in situ hybridization. Many of the cells labeled with the Epstein-Barr virus encoded RNA (EBER) probe also express CD20 (arrows). Occasional EBER-labeled cells do not express CD20 (arrowheads) (original magnification 100). cases, the number of positive cells varied substantially. The labeled nuclei included large and medium-sized forms. The portions of the tissue in which EBER-labeled cells were dense were also rich in CD20+ large cells. In 6 of the 10 positive cases, the number of EBER-positive and CD20+ cells were comparable. In 3, the number of CD20+ cells exceeded the number of EBER-positive cells, and in 1 case, the number of EBER-positive cells exceeded the number of CD20+ cells. Discrepant staining in favor of CD20 may relate to less than optimal preservation of RNA. Discrepant staining in favor of EBER may relate to loss of CD20 antigen expression in EBV-infected B cells. This possibility is supported by the fact that staining for CD3 did not account for the EBER-positive, CD20 population. In case 10, EBV was present on an initial biopsy specimen, but was no longer demonstrable on a subsequent specimen. Eight of the cases that showed the presence of EBV by in situ hybridization were examined for LMP-1 expression. In 6 of these cases, rare cells stained for LMP-1. All 5 cases that were negative for EBV by in situ hybridization were also negative for LMP-1, including the second biopsy specimen from the patient whose initial biopsy specimen had been EBV positive (case 10). Double-Labeling Immunohistochemistry and In Situ Hybridization In situ hybridization for EBER was coupled with antibody stains for CD20, CD43, CD3, and CD79 in 6 cases that were known to harbor EBV (cases 1, 9, and 11-14). Interpretable or partially interpretable results were obtained in cases 11, 12, 13, and 14. Case 13 showed expression of CD20 by many EBERlabeled cells Image 8. Many of the double-positive cells were large, while EBER-positive nuclei that lacked CD20 were often medium sized or small. A similar pattern was observed for CD79; however, there were fewer unlabeled nuclei. In contrast, none of the labeled cells expressed CD3 Image 9. Virtually all of the EBER-positive cells were negative for CD43, but rare double-positive cells were observed. Case 11 showed expression of CD20 by much of the EBER-positive population, while CD43 was not expressed by the EBER-positive cells. CD3 and CD79 results were equivocal owing to a lack of discernible EBER staining. Cases 12 and 14 showed weak CD20 expression by the EBER-positive cells, while results for CD43, CD3, and CD79 were equivocal owing to a lack of discernible EBER staining. Despite strong positive staining on slides used for single-label EBV in situ hybridization, cases 1, 9, and 12 showed no discernible EBER labeling on any of the antibody-stained slides or on the PBS controls from the double-labeling study. In these cases, the in situ hybridization failed to work on slides pretreated with microwave heating and on those that were not pretreated. This rate of successful double-labeling is similar to that reported by others. 7,17 In summary, each of 4 interpretable cases showed the presence of EBV in CD20-expressing B cells, while no case showed EBERpositive T cells. 242 Am J Clin Pathol 2000;114: American Society of Clinical Pathologists

8 Hematopathology / ORIGINAL ARTICLE T-gamma PCR Studies In 1 case (case 11), TCR-gamma heteroduplex analysis was not performed because of extensive DNA degradation. However, in this case, a clonal rearrangement of the TCRbeta gene was detected by Southern blot hybridization. Twelve of the remaining 13 cases showed a clonal population of T cells by PCR analysis. One case (case 13), did not show evidence of a clonal TCR-gamma gene rearrangement despite the presence of adequate tissue for evaluation. Frozen tissue was not available for this case, and, thus, Southern blot hybridization could not be performed. For case 10, the initial biopsy revealed evidence of TCR-gamma clonality. This patient s follow-up biopsy did not show clonality by PCR of the TCR-gamma gene or by Southern blot hybridization, using probes for the TCR-beta gene. PCR Studies of the Immunoglobulin Gene Heavy Chain Fourteen cases were tested for clonal VDJ segment recombination by PCR. Four different patterns of PCR products were seen Image 10 (Table 2): 4 cases showed a polyclonal smear of amplicons; 4 cases showed the presence of 3 or more bands and were interpreted as oligoclonal; 1 case showed a single faint band that was suggestive of a single clonal population; and the remaining 5 cases showed 1 or 2 strong bands indicating a definite clonal B-cell population. In addition, in 1 of the cases, the second biopsy from case 10 that showed an oligoclonal pattern by VDJ PCR, Southern blot hybridization confirmed a clonal immunoglobulin gene rearrangement. Discussion Our cases are morphologically peripheral T-cell lymphoma, a diagnosis that was confirmed by molecular studies in 13 of 14 cases. In addition, EBV was present in 10 cases. Double-labeling studies confirmed the presence of EBV in CD20+ B cells in 4 cases. Molecular evidence of clonal or oligoclonal rearrangement of the immunoglobulin heavy chain gene was demonstrated in 10 cases. Because our cases invariably showed the striking presence of large B cells by immunohistochemical studies, the descriptive name large B-cell rich T-cell lymphoma accurately captures the morphologic features of this process. The diagnostic implications of our observations are highlighted by the fact that our cases occasionally were interpreted initially as large B-cell lymphoma or as a reactive immunoblastic proliferation. Our cases bear a resemblance to cases of EBV-positive peripheral T-cell lymphoma previously described in the literature. They are most similar to the cases reported by Ho and Image 9 (Case 13) Double-label immunohistochemistry and in situ hybridization. None of the Epstein-Barr virus encoded RNA positive cells expresses CD3 (original magnification 100). Image 10 IgH FR3-JH polymerase chain reaction products analyzed by polyacrylamide gel electrophoresis (10%) showing a spectrum of gene rearrangement clonality banding patterns. Lane 1 (case 4), polyclonal; lane 2 (case 6), oligoclonal; lane 3 (case 13), oligoclonal with 1 band predominating; lane 4 (case 2), monoclonal; lane 5 (SUDHL4 cell line), monoclonal positive control; lane 6 (10% SUDHL4 in tonsil), sensitivity control; lane 7 (tonsil), polyclonal control; lane 8 (no DNA), negative control. American Society of Clinical Pathologists Am J Clin Pathol 2000;114:

9 Higgins et al / LARGE B-CELL RICH T-CELL LYMPHOMA Table 3 Differential Diagnosis of Large B-Cell Rich T-Cell Lymphoma Atypical Cells Present Molecular Evidence of Clonality EBER-1 and/or Phenotype of Atypical Cells LMP-1 Large Small T Cell B Cell TCRBCL CD20+, CD3, CD30+/ No Yes No/Yes No Yes Classic HD CD15+,CD30+, CD20+/, CD3 No/Yes Yes No No Occasional LBCRTCL CD20+, CD3+, CD30+/ Yes/No Yes Yes Yes Variable Infectious CD3+, CD20+, CD30+ Yes Yes No No Variable mononucleosis EBER, Epstein-Barr virus encoded RNA; HD, Hodgkin disease; LBCRTCL, large B-cell rich T-cell lymphoma; LMP, latent membrane protein; TCRBCL, T-cell rich B-cell lymphoma. colleagues 19 who studied 23 cases of peripheral T-cell lymphoma in which the presence of EBER was revealed by in situ hybridization studies. Double-labeling studies showed that there was a greater number of EBER-containing CD20+ B cells than of EBER-containing CD3+ T cells. All of their cases showed PCR evidence of T-cell clonality, and some of the cases showed a faint band suggesting B-cell clonality. However, we have approached our cases in an entirely different manner from other investigators. While others have studied peripheral T-cell lymphoma for the presence of EBV and then have performed double-labeling and molecular studies to characterize the EBV-positive cell population, we selected cases on the basis solely of the immunohistochemical finding of a population of large B cells in a peripheral T- cell lymphoma. Some of our cases lack EBV entirely, yet a large B-cell proliferation is apparent. While some investigators have noted that in EBV-harboring T-cell lymphomas the EBV is present in B- immunoblasts, 8,17-19 the observation of a large B-cell population has never been emphasized. Thus, our EBV-positive cases might form a subclass of the cases reported by Ho and colleagues 19 because our series does not include cases in which a population of EBVinfected B cells is below the threshold of detection by light microscopy and immunophenotypic studies. Some of our cases may represent angioimmunoblastic T-cell lymphoma. This is supported by the finding of a skin rash in 3 patients and of hypergammaglobulinemia in 3 patients. However, only 2 of our cases showed a well-developed expansion of follicular dendritic cell processes into the paracortical areas. This is said to be a virtually constant feature of angioimmunoblastic T-cell lymphoma. 32 While cytoplasmic clearing of tumor cells and a mild increase in vascularity were features observed in our cases, the marked increase in vascularity and burned-out appearance that define angioimmunoblastic T-cell lymphoma were not present. In all of our cases, the morphologic appearance of the T-cell lymphoma was more in keeping with a diagnosis of peripheral T-cell lymphoma, unspecified than angioimmunoblastic T-cell lymphoma. It is interesting to compare our series with that reported by Loke et al. 33 These authors described 4 T-cell rich lymphomas that initially had been diagnosed as T-cell lymphoma (2 cases of angioimmunoblastic lymphadenopathy like T-cell lymphoma and 2 cases of pleomorphic T-cell lymphoma) on histologic grounds. The cases showed molecular evidence of immunoglobulin heavy chain gene rearrangement without TCR-beta chain gene rearrangement. Of the 4 cases, 2 also showed clonal rearrangement of the immunoglobulin light chain genes. All 4 cases showed evidence of EBV DNA by Southern blot analysis. The authors considered the 2 cases with only immunoglobulin heavy chain gene rearrangements to represent lymphoma of probable B-cell lineage and the 2 cases with heavy and light chain gene rearrangements to represent lymphoma of definite B-cell lineage. The authors describe their cases as having T cells with frequent nuclear pleomorphism in addition to an abundance of high endothelial venules, and admixture of eosinophils, histiocytes, and other cells. These features are similar to those seen in our cases. In the series by Loke et al, 33 then, the importance of molecular studies for excluding the possibility of T-cell lymphoma underlying a proliferation of EBV-infected large B cells is highlighted. Alternatively, these cases may have been identical to ours with the exception that the molecular studies did not disclose the T-cell clone. Such a failure of peripheral T-cell lymphoma to demonstrate clonal rearrangement of the TCR-beta gene has been described previously. 34 The diagnosis of peripheral T-cell lymphoma complicated by a proliferation of large B cells depends on extensive immunophenotypic and molecular evaluation. The light microscopic appearance of our cases was invariably suggestive of peripheral T-cell lymphoma. Immunophenotypic evaluation showed the majority of the atypical cells to mark with the T-lineage marker CD3. However, CD20+ large cells stood out clearly in the interfollicular areas in every case. In a majority of the cases, light chain staining revealed a mix of kappa- and lambda-expressing large cells. Even though such cases sometimes showed molecular evidence of a clonal 244 Am J Clin Pathol 2000;114: American Society of Clinical Pathologists

10 Hematopathology / ORIGINAL ARTICLE rearrangement of the immunoglobulin heavy chain gene, this light chain staining pattern argued against a diagnosis of T- cell rich large B-cell lymphoma. However, in 2 cases, the light chain stains demonstrated at least a suggestion of monotypia of the B-cell population. Molecular studies also were valuable because they often revealed a proliferation of clonal T cells together with clonal, oligoclonal, or polyclonal B-lineage cells. However, it should be stressed that, as is true of our case 13, TCR-gamma PCR performed on paraffinembedded tissue will fail to detect clonality in about 20% of T-cell lymphoma cases. 21 This may be because the TCR genes have not been rearranged, because of small numbers of tumor cells in the tissue sample, or because the primers do not span the rearranged portions of the antigen receptor gene particular to that neoplasm. Thus, it is critical that the high index of suspicion for T-cell lymphoma obtained from the histologic appearance be maintained throughout the workup of the case. We do not know the incidence of large B-cell rich T- cell lymphoma. Our cases were seen primarily in the hematopathology consultation service and, thus, will be influenced by referral bias. We obtained 13 of our cases during a 1-year period (1 case had been reported previously and was several years old). During this time, we evaluated approximately 1,800 cases in consultation. All cases of large B-cell rich T-cell lymphoma probably were recognized, since the same consultants reviewed all of the consultation cases during this time period. Thus, 0.72% of our consultation cases represented large B-cell rich T-cell lymphoma. While referral bias precludes assessment of a true incidence of this process, this calculation may provide some suggestion of how likely large B-cell rich T-cell lymphoma is to be encountered in other consultation services. Neither immunoglobulin heavy chain nor TCR-gamma gene rearrangements are lineage specific, as rare B-cell lymphomas can have TCR rearrangements, and even rarer T-cell lymphomas can have VDJ rearrangements. 35 However, 5 of our cases clearly have distinct clonal T-cell and clonal B-cell populations. First, T-cell processes that show immunoglobulin heavy chain gene rearrangements often represent precursor lymphoblastic disease rather than mature peripheral T-cell lymphomas. 35,36 Likewise, B-cell processes that show TCR-gamma gene rearrangements are also often immature. 37 Second, and more important, the molecular observations are paralleled by the histologic and immunohistochemical findings. Distinct populations of atypical T- and B-lineage cells are discernible in every case. The B-cell population shows a mix of kappa and lambda light chain expression in most cases and the focal finding of a dominant light chain in 2 cases. This correlates perfectly with the VDJ PCR findings of a spectrum of polyclonal, oligoclonal, and monoclonal immunoglobulin heavy chain gene rearrangements. The diagnostic implications of our findings are readily apparent. In 3 of our cases, the referring pathologist had made a provisional morphologic diagnosis of peripheral T-cell lymphoma but been dissuaded from this diagnosis by the finding of scattered large B cells in the interfollicular areas. The presence of a mix of large B and T cells in the interfollicular areas raised the possibility of a reactive immunoblastic proliferation such as occurs in acute mononucleosis. This diagnosis would have been supported by the lack of light chain restriction and by the presence of EBV in many of our cases. However, the morphologic resemblance to acute mononucleosis is only superficial. Several of our cases showed a well-developed vascular pattern and an infiltrate of eosinophils. Furthermore, the spectrum of cytologic appearances of the interfollicular cells and the frequent occurrence of plasmacytoid cells, characteristic of acute mononucleosis, were lacking. Some parallels may be drawn between the B-cell proliferations seen in our cases and those that occur in the setting of HIV and transplantation-associated lymphoma. Our large B-cell proliferations complicating T-cell lymphoma show some of the heterogeneity that others have observed in the HIV-associated lymphomas. 38 Thus, many cases are EBVpositive, while others are not. 15 Some may show small noncleaved cell morphologic features, while others show immunoblastic or large cell morphologic features or show the morphologic features of Hodgkin disease. 39,40 Furthermore, molecular findings may correlate with the morphologic appearance of the neoplasm. 41 These features may prove to have prognostic relevance in our patients as they appear to have in HIV-associated lymphoma. 38 This is corroborated by the finding of a shortened survival in patients with peripheral T-cell lymphoma who show a clonal immunoglobulin heavy chain gene rearrangement, 42 as well as the finding that the presence of EBV is correlated with constitutional symptoms and an aggressive clinical course in T-lineage lymphoma. 16 Two of our patients developed subsequent diffuse large B-cell lymphomas, providing further insight into the nature of this process. From the Department of Pathology, Stanford University Medical Center, Stanford, CA. Supported in part by grant CA34233 from the National Cancer Institute, National Institutes of Health, Bethesda, MD. Presented in part as a lecture at the March 1999 Annual Meeting of the United States and Canadian Academy of Pathology, San Francisco, CA. Address reprint requests to Dr Higgins: Dept of Pathology, Stanford University Medical Center, 300 Pasteur Dr, Stanford, CA. Acknowledgments: We thank Kathleen Jones, Eva Pfendt, Lourdes Villanueva, and Michael Durnen for assistance with American Society of Clinical Pathologists Am J Clin Pathol 2000;114:

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