Composite Angioimmunoblastic T-Cell Lymphoma and Diffuse Large B-Cell Lymphoma A Case Report and Review of the Literature

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1 Hematopathology / COMPOSITE T- AND B-CELL LYMPHOMA Composite Angioimmunoblastic T-Cell Lymphoma and Diffuse Large B-Cell Lymphoma A Case Report and Review of the Literature Yin Xu, MD, PhD, 1 Robert W. McKenna, MD, 1 Mai P. Hoang, MD, 1 Robert H. Collins, MD, 2 and Steven H. Kroft, MD 1 Key Words: Composite lymphoma; Angioimmunoblastic lymphoma; Diffuse large B-cell lymphoma; Epstein-Barr virus DOI: /VD2D98MEMB3FWH34 Abstract We report a rare case of composite angioimmunoblastic T-cell lymphoma (AILT) and diffuse large B-cell lymphoma occurring in a 48-year-old woman with generalized lymphadenopathy and hepatosplenomegaly. The patient initially sought care at a local hospital with a single enlarged left cervical lymph node. Histologic examination of the node was interpreted as an atypical immunoblastic proliferation. She developed generalized lymphadenopathy 10 months later and was referred to our institution for further evaluation. The recent biopsy of the cervical node showed typical features of AILT. Flow cytometric immunophenotyping identified an aberrant CD4+ T-cell population that lacked surface CD3. Polymerase chain reaction analysis of the T-cell receptor gamma gene revealed a clonal rearrangement. In addition to the AILT, the lymph node showed partial involvement by a diffuse large B-cell lymphoma. The B lymphoma cells and admixed immunoblasts and Reed-Sternberg like B cells in the AILT were positive for Epstein-Barr virus (EBV) by in situ hybridization. Our findings raise the possibility that the EBV-associated large B-cell lymphoma is a secondary event in AILT via EBV infection or reactivation followed by clonal expansion of an immortalized EBV-infected B cell clone. Angioimmunoblastic T-cell lymphoma (AILT) is one of the most common subtypes of peripheral T-cell lymphoma. 1 Lymph nodes characteristically show diffusely infiltrating T cells with clear cytoplasm, arborizing vessels, proliferation of follicular dendritic cells, and increased B- immunoblasts that usually contain Epstein-Barr virus (EBV). 2 Occasional Reed-Sternberg (RS)-like cells are also seen. 2 It is histologically difficult to distinguish AILT from angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), a systemic lymphoproliferative disorder of unknown cause originally described by Frizzera et al. 3,4 Supporting data for a diagnosis of AILT include clonal T- cell receptor (TCR) gene rearrangement and an aberrant phenotype of the T cells. 2 In the past, AILT was considered a malignant transformation of AILD, but it currently is thought to most likely arise de novo as a peripheral T-cell lymphoma. 5 Patients often seek care because of generalized lymphadenopathy, hepatosplenomegaly, fever, anemia, and skin rash. 6 The clinical outcome is poor, largely owing to associated immune dysregulation with opportunistic infection. 6,7 Although immunoglobulin gene rearrangement occasionally has been detected in AILT, 8-10 only a few case reports documenting development of B-cell lymphoma in AILD or AILT have been reported in the literature. 9,11-13 An early series demonstrated composite AILD and immunoblastic lymphoma; however, no immunophenotypic or molecular data were available to determine cell lineage and clonality. 14 We describe a patient with composite AILT and EBV-associated diffuse large B-cell lymphoma (DLBCL). 848 Am J Clin Pathol 2002;118: DOI: /VD2D98MEMB3FWH34

2 Hematopathology / CASE REPORT Case Report A 48-year-old woman initially experienced an enlarged left anterior cervical lymph node, which waxed and waned for 2 months. She sought medical attention at a local hospital. Biopsy of the node was interpreted as an atypical immunoblastic proliferation. Flow cytometric findings of the node were reported to lack evidence of non-hodgkin lymphoma. Polymerase chain reaction (PCR) analysis was reported to show a minor monoclonal band in immunoglobulin heavy chain (IgH) analysis and no evidence of clonal TCR gene rearrangement. A clearcut diagnosis of lymphoma was not made at that time. She was treated with a 2-week course of acyclovir, which led to a reduction in size of the node. She sought care because of a sudden onset of marked swelling of the left side of the neck 9 months later. Lymphadenopathy involving bilateral axillary, left supraclavicular, and right inguinal lymph nodes was noted. A computed tomography scan showed an enlarged spleen and enlarged visceral lymph nodes. A repeated lymph node biopsy was interpreted as showing a similar histologic picture to that seen previously. During the following month, the patient then was referred to the University of Texas Southwestern Medical Center, Dallas, for reevaluation of the lymphadenopathy. The physical examination revealed a well-appearing middle-aged woman in no acute distress. Multiple enlarged lymph nodes were easily identified. The spleen was palpable 2 cm below the left costal margin without discrete masses or tenderness. Other physical findings were unremarkable. She denied any symptoms such as fever, night sweats, or weight loss. Her medical history was remarkable for a malignant melanoma, Clark level II, on the back of her left shoulder 4 years earlier, treated with wide local excision. The CBC count showed a hemoglobin of 14.2 g/dl (142 g/l), hematocrit of 41.8% (0.42), a WBC count of 6,100/µL ( /L) with a normal differential count, and a platelet count of 182 /µl ( /L). A rebiopsy of the lymph node on the left side of the neck, which was 6 cm at the time, revealed a composite AILT and DLBCL. The bone marrow was negative for lymphoma involvement. The patient received a 6-cycle cyclophosphamide, doxorubicin [hydroxydaunomycin], vincristine [Oncovin], and prednisone (CHOP) regimen and achieved a complete remission. However, she relapsed a few months later. She has been placed on etoposide, methylprednisolone [Solu-Medrol], cytarabine [high-dose Ara-C], and cisplatin [platinum] (ESHAP) salvage therapy with plans for autologous stem cell transplantation. Materials and Methods Histologic and Immunohistochemical Studies Fresh biopsy tissue was fixed in B-5 fixative, 10% neutral buffered formalin, or both; embedded in paraffin; and sectioned at 3 µm. Sections were stained with H&E for morphologic evaluation. Immunohistochemical analysis was performed on the paraffin sections with the following antibodies and dilutions: CD3 (1:200), CD21 (1F8, 1:50), and CD30 (Ki-1, 1:20) (DAKO, Carpinteria, CA); CD15 (1:10, Becton Dickinson, San Jose, CA); and CD20 (L26, 1:40) and CD45 (1:40) (Signet, Dedham, MA). The immunohistochemical analysis was performed on a TechMate automated immunostainer (Ventana Biotek, Tucson, AZ) with a streptavidin-biotin peroxidase detection system, preceded by microwave antigen retrieval in citrate buffer (BioGenex, San Ramon, CA). In Situ Hybridization In situ hybridization for the detection of EBV-encoded nuclear RNAs (EBERs) was performed on paraffin sections using standard procedures and commercially available reagents (DAKO In Situ Hybridization Detection Systems). 15 Flow Cytometry Tissue processing and antibody staining were performed as previously described. 16 Antibodies against CD2 (55.2), CD3 (SK7), CD4 (SK3), CD5 (L17F12), CD7 (4H9), CD8 (SK1), CD10 (W8E7), CD19 (SJ25C1), CD20 (L27), CD38 (HB7), CD45 (2D1), CD45RO (UCHL-1), and monoclonal kappa (TB28-2) and lambda (I-155-2) immunoglobulins were obtained from Becton Dickinson. Antibodies against FMC-7, CD23 (B6), and polyclonal immunoglobulin kappa (goat) and lambda (goat) were obtained from Coulter- Immunotech (Hialeah, FL). Anti-CD30 (BerH2) was obtained from DAKO. These antibodies were conjugated with fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, or allophycocyanin. Flow cytometric data were acquired using a 4-color FACSCalibur flow cytometry instrument with CELLQuest software (Becton Dickinson). Data analysis was performed using Paint-A-Gate Software (Becton Dickinson). Nonviable cells and debris were excluded based on forward and orthogonal light scatter properties. Positivity for an antigen was defined as at least 10% of lymphoma events beyond a 2% threshold based on the same population in an isotypic control tube. Molecular Analysis DNA was extracted from tissue and purified using the QIAamp Mini Kit (Qiagen, Valencia, CA). Rearrangement of the IgH chain was studied by PCR using primers for 3 framework Am J Clin Pathol 2002;118: DOI: /VD2D98MEMB3FWH34 849

3 Xu et al / COMPOSITE T- AND B-CELL LYMPHOMA regions: FR1 17 and J H, 18 FR2 19 and J H, 18 and FR3 18 and J H. 18 Rearrangement of the TCR gamma chain was examined by PCR using a mixture of consensus V and J region primers 20,21 in a single reaction. PCR was performed according to a previously described method. 22 The products were analyzed by polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). Results The cervical lymph node biopsy performed 10 months after initial diagnosis showed a totally effaced nodal architecture. The lymph node was infiltrated diffusely by small to medium-sized lymphocytes with clear cytoplasm and mildly irregular nuclei intermingled with numerous large transformed cells in a background of prominent arborizing high endothelial venules Image 1A. Immunohistochemical studies revealed that the small to medium-sized lymphocytes were CD3+ T cells and the large lymphocytes were predominantly CD20+, CD30+ B immunoblasts. In situ hybridization for EBER showed strong nuclear positivity in the immunoblasts Image 1B. An irregular expanded meshwork of dendritic cells was prominent throughout the node as highlighted by CD21 immunohistochemical analysis Image 1C. These morphologic features were typical for AILT. An additional feature was the presence of scattered RS-like cells with irregular multilobated nuclei and large eosinophilic nucleoli (Image 1A, inset). These showed membrane staining for CD15, CD30, and CD20 and strong nuclear positivity for EBV by in situ hybridization. Finally, the lymph node showed a single 1-mm focus consisting of sheets of pleomorphic large malignant lymphoid cells Image 2A. These exhibited irregular nuclei, vesicular chromatin, prominent nucleoli, and moderately abundant cytoplasm with a brisk mitotic rate, atypical mitotic figures, and A B C Image 1 Angioimmunoblastic T-cell lymphoma in the cervical lymph node. A, The node architecture was effaced by a diffuse infiltrate of small to medium-sized lymphocytes with clear cytoplasm in a background of arborizing endothelial venules (H&E, 400). Inset: Occasional Reed-Sternberg like cells were identified in the lesion (H&E, 400). B, In situ hybridization for Epstein-Barr virus (EBV)-encoded nuclear RNA 1. Interspersed immunoblasts showed strong nuclear staining for EBV ( 400). C, Irregular expanded meshwork of follicular dendritic cells highlighted by CD21 immunohistochemical analysis ( 20). 850 Am J Clin Pathol 2002;118: DOI: /VD2D98MEMB3FWH34

4 Hematopathology / CASE REPORT A B C Image 2 A, A focus of large cell lymphoma coexistent with angioimmunoblastic T-cell lymphoma (H&E, 100). B, Sheets of pleomorphic large lymphoid cells with atypical mitotic figures and karyorrhexis (H&E, 1,000). C, The large lymphoma cells are of B-cell origin, as indicated by CD20 immunohistochemical analysis ( 400). karyorrhexis Image 2B. The cytologic features of these cells were distinct from those of the B immunoblasts and RS-like cells in the remaining tissue. Immunohistochemical analysis for CD20 confirmed a B-cell lineage of DLBCL Image 2C. The B lymphoma cells also were positive for EBER. Flow cytometric immunophenotyping of the lymph node biopsy identified a population of aberrant small T lymphocytes comprising approximately 27% of total viable cellular events. These were positive for CD2, cytoplasmic CD3, CD4, CD5, CD38, and CD45; partially positive for CD7; and negative for surface CD3, terminal deoxynucleotidyl transferase, and B-cell markers Image 3. Approximately 20% of the T cells in the sample expressed CD10 beyond the threshold for CD10 in the isotypic control (Image 3). PCR analysis of the TCR gamma gene demonstrated a strong clonal band confirming T-cell clonality. PCR analysis of the IgH gene lacked evidence of clonal rearrangement. Sections of the lymph node biopsy performed 10 months earlier were reviewed, although the interpretation was limited by a small amount of material and suboptimal fixation. This showed a largely effaced nodal architecture with a few residual lymphoid follicles. There was a diffuse infiltrate of predominantly small lymphocytes with irregular nuclei, which were identified as T lymphocytes by CD3 immunohistochemical analysis. Numerous transformed large cells were present, which were composed of mixed B and T immunoblasts as indicated by immunohistochemical analysis. A rich vascular network was present in the lesion. Discussion AILD originally was described as a reactive lymphoproliferative disorder of T lymphocytes. 4 The vast majority of the proliferating cells are CD4+ or, less often, CD8+. 2 Am J Clin Pathol 2002;118: DOI: /VD2D98MEMB3FWH34 851

5 Xu et al / COMPOSITE T- AND B-CELL LYMPHOMA 1,024 SSC CD3 Cytoplasmic CD ,024 FSC CD TdT CD8 CD5 CD CD4 CD7 CD10 Image 3 Immunophenotypic analysis of angioimmunoblastic T-cell lymphoma by 4-color flow cytometry. The neoplastic T cells were CD2+, cytoplasmic CD3+, CD4+, CD5+, and partially positive for CD7 and CD10 but CD8 and surface CD3. Red events, neoplastic T lymphocytes; green events, normal T lymphocytes; blue events, CD10 T lymphocytes; black events, T lymphocytes expressing CD10 beyond the isotypic control threshold. FSC, forward scatter; SSC, side scatter; TdT, terminal deoxynucleotidyl transferase. Subsequent molecular and cytogenetic studies of AILD identified clonal TCR gene rearrangement and chromosomal abnormalities in the majority of the cases, 10,23 leading to the conclusion that most cases previously regarded as AILD actually represent AILT, a distinct type of peripheral T-cell lymphoma. On a morphologic basis, the presence of clusters of cells with abundant clear cytoplasm has been used to distinguish AILT from AILD. Distinctive features of AILT are a prominent expanded meshwork of follicular dendritic cells and varying numbers of polyclonal B immunoblasts that, in the majority of cases, contain EBV. The present case demonstrated a morphologically characteristic AILT. Immunophenotyping identified an aberrant CD4+ T-cell population. In addition, approximately 50% of the aberrant T cells expressed CD10 by flow cytometry. CD10 has been reported to be aberrantly expressed in the neoplastic T cells of AILT. 24 Thus, the presence of CD10+ T cells in our case also strongly supported a diagnosis of AILT. Finally, PCR analysis of TCR gene rearrangement confirmed a clonal T- cell population. Clonal IgH gene rearrangements have been found in a minority of cases of AILD. 8-10,23 It has been reported that cases with IgH rearrangements have a higher rate of hemolytic anemia and a higher remission rate with corticosteroid therapy. 23 A trend toward better overall survival also was reported for such cases. 23 In some instances, the B-cell clones appeared and disappeared during the course of the disease. 9 A few cases of AILD and AILT with subsequent development of B-cell lymphoma have been described in the literature. Abruzzo et al 11 reported a case in which a 46-year-old patient with a 2-year history of AILD developed large B-cell immunoblastic lymphoma. Molecular studies revealed clonal IgH rearrangement but a germline configuration of the TCR beta chain gene. At autopsy, the patient had widespread B-cell lymphoma involving multiple organs. The neoplastic B cells were EBER+ by in situ hybridization of EBV. Mazur et al 12 described a 64-year-old patient with a history of AILD for 18 months who developed a primitive malignant lymphoma with blood and marrow involvement. Studies for various types of immunoglobulins and E (erythrocyte) rosette formation 852 Am J Clin Pathol 2002;118: DOI: /VD2D98MEMB3FWH34

6 Hematopathology / CASE REPORT showed absence of these surface markers on lymphoma cells. The lymphoma morphologically resembled Burkitt lymphoma, and a t(8;14)(q24;q32) was identified in all 15 malignant cells examined by karyotypic analysis. The patient died shortly after the diagnosis of B-cell lymphoma. Boros et al 25 reported a case of AILD with development of immunoblastic lymphoma 2 months later. The patient had polyclonal hypergammaglobulinemia at initial diagnosis and developed monoclonal IgM kappa during the course of the disease. Despite chemotherapy, the patient died 1 month after the development of the lymphoma. Recently, Zettl et al 13 reported 2 cases of AILT with subsequent development of DLBCL. In all these cases, the clinical outcome was very poor. The time from the initial diagnosis of AILD to the development of lymphoma ranged from 2 months to 3 years. The temporal relationship of the 2 processes in these cases suggests that there might be a causal relationship between them. Our case was unique in that the diagnostic lymph node contained AILT and a focal large B-cell lymphoma. PCR analysis of the IgH gene performed on fresh biopsied tissue was negative for clonal rearrangement. Molecular studies for B-cell clonality were not performed on the paraffin tissue block because the focus of large B-cell lymphoma was lost in the deeper sections. However, a clonal IgH gene rearrangement was identified by PCR in the original biopsy specimen, although no morphologic evidence of a B-cell neoplasm was present at that time. The coexistence of AILT and DLBCL in the same lymph node is unlikely to be coincidental. B-cell immunoblasts in the AILT and the B-cell lymphoma both contained EBV RNA. We hypothesize that EBV may have had a role in the pathogenesis of the B-cell lymphoma in this case. The immune dysregulation present in AILT may have permitted EBV infection or reactivation, which could have had a role in the proliferation of B-cell immunoblasts and clonal expansion of an immortalized EBV-infected B-cell clone. Therefore, the development of B-cell lymphoma can be a consequence of the disease progression of AILT. Zettl et al 13 also reported 10 AILTs that contained a prominent population of atypical, transformed, large B cells, which formed focally confluent sheets. The authors did not specify the size or distribution of these collections and did not interpret them as DLBCL. These transformed B cells were EBV+ by EBER in situ hybridization or immunohistochemical analysis for EBV latency protein 1 and EBV nuclear antigen 2. The finding of focally concentrated, transformed, EBV+ large B cells suggests the possibility that these are precursor lesions of DLBCL. An interesting finding in the present case was the presence of occasional EBV+ RS-like cells within the AILT. Quintanilla-Martinez et al 26 reported such RS-like cells in 3 peripheral T-cell lymphomas, 2 of which were AILTs. As in the present case, the RS-like cells had immunophenotypic features of classic Hodgkin cells: CD30+, CD15+, and CD20+/. The presence of RS-like cells in AILT might create diagnostic confusion with Hodgkin lymphoma; recognition of cytologic atypia or immunophenotypic aberrancy in the background T cells is critical for making the appropriate diagnosis. Whereas RS cells in classic Hodgkin lymphoma have been documented to represent clonal B-cell populations, the scattered RS cells within the peripheral T-cell lymphomas reported by Quintanilla-Martinez et al 26 were documented to be oligoclonal by single-cell PCR. Clonal EBV+ RS-like cells also have been documented in various small B-cell lymphomas; these may be related clonally to the small B-cell lymphoma or may represent an independent clonal population. 27 Furthermore, the neoplastic cells in overt Hodgkin lymphoma arising in the background of small B-cell lymphomas have been documented to be related clonally to the non-hodgkin lymphoma cells. 28,29 These findings further underscore the likely role of EBV in B- cell lymphomagenesis in the setting of AILT. It is unfortunate that sufficient material was not available to explore the clonal relationship of the EBV+ DLBCL and the EBV+ B immunoblasts and RS-like cells in the present case. Composite lymphoma has been defined as the simultaneous appearance of 2 histologically distinct lymphomas in the same anatomic site. Most are thought to arise from 2 different clones; occasional cases may be related clonally and represent progressive evolutionary stages of the same neoplastic clone Composite lymphoma involving both T and B lineages is an unusual event, and only a few cases have been reported in the literature One case report described a patient with a history of mycosis fungoides who developed chronic lymphocytic leukemia. This manifested in the skin as a composite lymphoma affecting an earlobe. 32 In another case report, a Japanese patient with a history of chronic phase adult T-cell leukemia developed a composite adult T-cell lymphoma and EBV-associated diffuse large B-cell lymphoma. 34 A composite T cell rich large B-cell lymphoma and peripheral T-cell lymphoma also was reported in a 24-year-old patient with a history of nodular lymphocyte predominance Hodgkin lymphoma. 33 Most recently, 3 cases of composite lymphomas were reported consisting of a peripheral T-cell lymphoma, not otherwise specified, and EBV-associated DLBCL. 13 We report a composite AILT and large B-cell lymphoma. We hypothesize that the immune dysregulation characteristically seen in AILT permitted clonal expansion of an EBV+ B-cell clone, with subsequent development of large B-cell lymphoma. From the Departments of 1 Pathology and 2 Medicine, University of Texas Southwestern Medical Center, Dallas. Address reprint requests to Dr Kroft: Dept of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX Am J Clin Pathol 2002;118: DOI: /VD2D98MEMB3FWH34 853

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