EBV Positive Diffuse Large B Cell Lymphoma with Negative Pan-B Cell Markers, Case Report, and Literature Review

Most B cell lymphomas are positive for one or more B cell markers including CD19, CD20, CD79a, or PAX5. However, rare cases of mature B cell lymphoma not expressing any B cell markers have been characterized and recognized as distinct diagnostic entities by current classification guidelines, including plasmablastic lymphoma, primary effusion lymphoma, and ALK-positive large B cell lymphoma. We present a case of pan-B cell marker negative, EBV positive diffuse large B cell lymphoma that is positive for OCT2, BOB1, and clonal immunoglobulin gene rearrangement that does not meet diagnostic criteria for any B cell lymphoma by current 4th and 5th Ed beta version WHO Hematolymphoid Tumors classification. In challenging cases like the one presented, utilizing OCT2 and BOB1 immunohistochemical stains can assist in determining B cell lineage. The WHO tumor classification system should consider adding OCT2 and BOB1 as alternative B cell lineage markers into their corresponding categories.


Introduction
Lineage of hematolymphoid tumors is usually determined by fow cytometry or immunohistochemical staining profles performed on parafn-embedded tissue.CD19, CD20, CD79a, and PAX5 are widely used as markers to determine B cell lineage from the pre-B cell to the plasma cell stage [1][2][3][4].OCT2 is a transcription factor that regulates immunoglobulin gene transcription with its co-activator BOB1 has been shown to be both sensitive and specifc in determining B cell lineage [1,5].
B cell lymphomas that are nonetheless typically negative for traditional pan-B cell markers have been observed and well characterized, including ALK positive large B cell lymphoma, primary efusion lymphoma, plasmablastic lymphoma, HHV8+ large B cell lymphoma, and unclassifable large B cell lymphoma [6][7][8][9], Table 1.
Te current 5th Ed beta version WHO Hematolymphoid Tumors classifcation listed the following essential and desirable diagnostic criteria for EBV positive difuse large B cell lymphoma [11].Essential: (1) Partial or total architectural efacement of afected tissue.(2) Atypical lymphoid infltrate composed of either sheets of large malignant cells or many scattered large, transformed cells of variable morphology including HRS-like cells in a richly cellular reactive background, often accompanied by necrosis.(3) Large cells confrmed to be of B cell lineage (e.g., CD20, PAX5, or CD79a expression).( 4) EBV present in the majority of large B cells.
(5) Absence of in-born or acquired immunodefciency or a history of lymphoma.(6) Exclusion of other EBV-related lymphomas and lymphoproliferative disorders.Desirable: (1) EBV DNA detectable in serum or whole blood (in selected cases).We present a case of pan-B cell marker negative, EBV positive large cell lymphoma with a clonal immunoglobulin gene rearrangement and positivity for OCT2 and BOB1, which successfully determined B cell lineage.
Te patient's clinical condition worsened, leading to hospitalization due to frequent falls.During this admission, bone marrow and inguinal lymph node biopsies were performed.

Materials and Methods
Formalin-fxed (10% neutral, bufered) parafn embedded sections were stained by routine hematoxylin and eosin preparations.Immunohistochemical analysis was performed on Ventana automated machine using Dako antibodies.A panel of stains were performed, including markers for B cells (CD20, CD19, CD79a, and PAX5), T cells (CD2, CD3, CD4, CD5, CD7, CD8, and CD43), follicular dendritic cells (CD21), plasma cells (CD138), cytotoxic Tcells (granzyme B and TIA1), hematopoietic precursors and Hodgkin lymphoma work up (CD45LCA, CD10, CD15, CD30, BCL2, and BCL6), cytokeratin marker (pan-CK), viral stains for HHSV8 and EBV, and proliferative index Ki67.EBV in situ hybridization stain is performed on a Ventana BenchMark Ultra using Ventana ISH iVIEW Blue Plus Detection Kit and EBER1 (Epstein-Barr Virus-Encoded RNA1) DNP probe.Additionally, OCT2 and BOB1 immunohistochemical stains were performed at Mayo Clinic reference laboratory.All immunohistochemical stains follow the manufacturer's protocol, and all stains were interpreted at the University of Kansas Medical Center.TCR gene rearrangement study was performed at Mayo Clinic reference laboratory.Mayo Clinic Laboratories use polymerase chain reaction (PCR) using a multiplex primer method based on the BIOMED-2 strategy to amplify the T cell receptor beta and T cell receptor gamma loci.
Te bone marrow biopsy showed a hypercellular bone marrow and involved by an atypical lymphoid proliferation consisting of large, atypical cells that were positive for CD2, CD30, CD45LCA, and EBV (Figures 1(g) and 1(h)).
Te morphology and immunohistochemical staining pattern were consistent with a diagnosis of EBV positive difuse large B cell lymphoma involving the bone marrow, and the patient started treatment with brentuximab vedotin, cyclophosphamide, doxorubicin, and prednisolone (BV-CHP).Te case was also reviewed by Dr. Elaine Jafe, NIH, USA, and the diagnosis was concurred.

Discussion
Pan-B cell marker negative B cell lymphomas are rare, most commonly observed in ALK positive large B cell lymphoma (ALK + LBCL), plasmablastic lymphoma (PBL), primary efusion lymphoma (PEL), HHV8+ large B cell lymphoma (HHV8+ LBCL), and unclassifable large B cell lymphoma.Te ALK + LBCL, PBL, PEL, and HHV8+ LBCL diagnoses can be made based on clinical presentation, morphology, ALK and HHV8 positivity, and expression of plasma cell associated antigens [1,12,13].A small portion of cases of pan-B cell negative large B cell lymphoma, however, pose a diagnostic challenge and pitfall.Yin et al. [5] investigated the diagnostic utility of OCT2 and BOB1 for cases in which pan-B cell marker expression is negative.Te cases included ALK + LBCL, PBL, PEL, HHV8 + LBCL, and unclassifed large B cell lymphoma.Teir study showed 74% of the cases were positive for OCT2, 85% of their cases were positive for BOB1, and 94% of their cases were positive for at least one of those markers.Importantly, non-B cell neoplasms were all negative for both OCT2 and BOB1.Tey concluded that OCT2 and BOB1 were both sensitive and specifc for determination of B cell lineage.Chu et al. [12] investigated immunohistochemical markers to establish B cell lineage in CD20 negative B cell neoplasms.Te cases included B cell lymphoblastic leukemia/lymphoblastic lymphoma, mature B cell neoplasms that were CD20 negative following rituximab therapy, and CD20 negative DLBCLs.For this discussion, the focus will be on the CD20 negative DLBCL cases.Teir study noted that all cases were negative for CD79a and only one case was positive for PAX5.However, all the cases were positive for either OCT2 or BOB1.Tey concluded that OCT2 and BOB1 may be the most useful for determining B cell lineage in CD20 negative DLBCL cases, such as plasmablastic lymphoma and primary efusion lymphoma.
Case Reports in Hematology Similar to our case, Nakatsuaka et al. [14] presented a case of EBV positive difuse large B cell lymphoma (DLBCL) that was negative for pan-B cell markers but positive for OCT2 and BOB1.Immunoglobulin gene rearrangement testing detected a clonal gene rearrangement.Tey concluded that utilizing OCT2 and BOB1 to determine B cell lineage in cases with unusual immunophenotype profles can lead to the correct diagnosis and subsequent appropriate treatment.
Our case showed scattered, large, atypical cells in a background of lymphocytes, histiocytes, and plasma cells.Case Reports in Hematology lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, EBV positive DLBCL, T cell/histiocyte rich large B cell lymphoma, anaplastic lymphoma kinase (ALK) positive large B lymphoma, and anaplastic large cell lymphoma.Te extensive immunohistochemical panel performed ruled out many of the possible diagnoses.Te large, atypical cells were negative for pan-B cell markers, ALK, HHV8, and CD15, and positive for CD45LCA, which ruled out nodular lymphocyte predominant Hodgkin lymphoma, T cell/histiocyte rich large B cell lymphoma, classic Hodgkin lymphoma, ALK positive B cell lymphoma, and cavitary primary efusion lymphoma.While MUM1 was positive in our case, CD138 was negative, and the tumor cells did not show immunoblastic morphology, so plasmablastic lymphoma was less likely.Te large, atypical cells were positive for CD2, CD30, Oct2, BOB1, and EBV and lacked the morphology of classic "Hallmark cells."A clonal immunoglobulin gene rearrangement was detected, and TCR gene rearrangement was equivocal.Te equivocal TCR result may indicate that the pattern of gene rearrangements is atypical compared to typical polyclonal T cell processes, but not defnitive for a monoclonal T cell population.Tese fndings led to the determination that this tumor was of B cell origin.A diagnosis of EBV positive large B cell lymphoma rather than ALK negative anaplastic large cell lymphoma or T cell lymphomas was rendered (Figure 2).Our case does not ft within a specifc diagnostic category in the 4 th Ed or 5 th Ed beta version of WHO Classifcation of Tumors Hematolymphoid Tumors, and it is important to consider utilizing OCT2 and BOB1 to determine B cell lineage for lymphoma that is negative for pan-B cell markers.We believe these markers should be incorporated into future editions of the WHO Classifcation of Tumors Hematolymphoid Tumors.

Figure 1 :Figure 2 :
Figure 1: Representative sections of lymph node and bone marrow biopsies.Te lymph node biopsy shows a difuse infltrate of large, atypical cells (A).Te large, atypical cells were positive for Oct2 (B), BOB1 (C), EBV (D), CD30 (E), and CD45LCA (F).Representative section of the bone marrow core biopsy (G) and (H) shows infltration of the marrow space by large, atypical cells.