Composite Lymphoma with Follicular Lymphoma Transformation to Clonally Related Epstein–Barr Virus (EBV) Positive Diffuse Large B-Cell Lymphoma and EBV-PositiveClassic Hodgkin Lymphoma

While the Epstein–Barr virus (EBV) is known to drive de novo lymphomagenesis, it may rarely contribute to transformation of indolent lymphoma as well. Some EBV-related lymphomas represent a diagnostic challenge with important prognostic and therapeutic implications. We describe a case of follicular lymphoma (FL) transformation to both EBV + diffuse large B-cell lymphoma (DLBCL) and EBV + classic Hodgkin lymphoma (cHL), the latter of which was only identified retrospectively after selective outgrowth during DLBCL therapy. Finally, we describe successful salvage therapy with brentuximab vedotin plus nivolumab. This is the first known case of composite lymphoma with FL, EBV + DLBCL, and EBV + cHL within a single lymph node. The disease course highlights the importance of careful morphologic examination and comprehensive immunophenotypic characterization of EBV + lymphomas to ensure proper clinical care and underscores the potential for novel therapies currently under investigation. This trial is registered with NCT01671813.


Introduction
Epstein-Barr virus (EBV) exerts a well-established role in the de novo pathogenesis of various aggressive lymphomas [1].However, its function in the transformation of indolent lymphomas is less appreciated.Defnitive classifcation of EBV + lymphomas can be challenging, with disparate treatments being indicated for some diferential diagnostic considerations.Here, we describe the frst known case of a composite lymphoma with both EBV + difuse large B-cell lymphoma (DLBCL) and EBV + classic Hodgkin lymphoma (cHL) arising in a background of follicular lymphoma (FL), with the cHL component only identifed retrospectively following pure EBV + cHL relapse.Furthermore, we demonstrate a clonal relationship between all three lymphoma morphologies and report successful salvage therapy of relapsed cHL with brentuximab vedotin plus nivolumab.

Case Presentation
Te patient is a 54-year-old male who presented to an outside institution with pruritus, fatigue, and cervical lymphadenopathy.On labs, he was found to have anemia, renal insufciency, and hypercalcemia.Excisional biopsy of the left cervical lymph node identifed grade 3A follicular lymphoma (FL) in a follicular pattern.However, a staging positron emission tomography (PET) scan identifed multifocal hypermetabolic lymphadenopathy above and below the diaphragm without mediastinal involvement measuring up to 6.7 cm by 5 cm and with a max intensity of 20.2 SUV, as well as mixed sclerotic and lytic bony lesions with SUV up to 26.8.Due to concern for aggressive disease transformation, an excisional biopsy of a hypermetabolic right inguinal lymph node was performed prior to initiation of therapy.
Te biopsied right inguinal node once again showed areas of grade 3A FL (top parts of Figure 1(A, I)), and fow cytometry detected a clonal B-cell population expressing CD10, CD20, and a lambda light chain.In addition to FL, a region around the periphery of the node, comprising 5-10% of the tissue, showed difuse architecture with central necrosis.Part of this difuse proliferation was composed of sheets of large lymphoid cells with vesicular chromatin and variably prominent nucleoli (Figure 1(A, B)).Tese cells were positive for CD20 (Figure 1(C)), PAX-5 (Figure 1(D)), CD30 (Figure 1(E)), OCT-2 (Figure 1(G)), and EBER (Figure 1(H)) and negative for CD15 (Figure 1(F)).Te large lymphoma cells were also positive for BCL-6 (weak), BCL-2, and MUM1 and negative for CD10 (not shown).Tese fndings were consistent with a composite lymphoma suggestive of FL transformation to EBV + DLBCL.Areas with FL only were negative for EBER but showed scattered CD30 positive cells within the neoplastic follicles.Outside FISH studies were positive for BCL6 gene rearrangement (80%) and a subclonal gain of BCL2 (20%) and negative for BCL2 and MYC rearrangements.
Following the diagnosis of EBV + DLBCL, the patient was treated at the outside institution with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) [2].Interval PETscans revealed partial metabolic response after 2 cycles, mixed response with possible radiologic progression in the right iliac after 4 cycles, and disease progression after 6 cycles (Figure 2(a)).Te patient was subsequently enrolled on a randomized phase 3 clinical trial comparing the CD19targeted antibody-drug conjugate loncastuximab plus rituximab versus rituximab, gemcitabine, and oxaliplatin.By PET, the patient showed a mild improvement after 4 cycles and disease persistence after 5 cycles (Figure 2(b)).At that time, the patient discontinued treatment on the trial less than one year from initial diagnosis.
Te patient subsequently established care at our institution and underwent a right inguinal lymph node excision.Sections showed an enlarged lymph node with architectural efacement by an atypical, vaguely nodular, mixed infltrate composed of moderately numerous Reed-Sternberg (R-S) cells and variants with background small lymphocytes and eosinophils (Figure 1(Q, R Tere was no evidence of FL or DLBCL in this specimen by morphology or fow cytometry.Te patient's plasma was positive for EBV at 25,029 IU/ml. Due to the lack of response to therapy and the discrepant morphology between the diagnostic and persistence/relapse specimens, we performed a careful retrospective examination of the initial EBV + DLBCL transformation.In addition to regions demonstrating monomorphic DLBCL, we identifed a small portion of transformed lymphoma with distinct morphology, background cellularity, and immunophenotype (Figure 1(I, J)).Tis area showed scattered R-S cells and variants in a background of small lymphocytes and scattered eosinophils.Te lymphoma cells were negative for CD20 (Figure 1(K)), OCT-2 (Figure 1(O)), BOB1, and CD79a and positive for PAX-5 (weak; Figure 1(L)), CD30 (Figure 1(M)), CD15 (Figure 1(N)), MUM1, and EBER (Figure 1(P)).Te overall morphology and phenotype of this region were consistent with occult EBV + cHL comprising less than 5% of the involved tissue, resulting in a revised diagnosis of composite lymphoma with both EBV + DLBCL and EBV + cHL arising in a background of FL.
Tis unusual transformation event was suggestive of divergent clonal evolution of FL, but FISH performed on the relapse specimen was negative for the BCL6 gene rearrangement and gain of BCL2.To further evaluate the clonal relatedness between the patient's historical FL, EBV + DLBCL, and EBV + cHL, B-cell receptor gene rearrangement studies were performed to assess for shared clonal peaks.Extracted DNA from the initial FL diagnostic specimen, macrodissected monomorphic EBV + DLBCL, and relapsed EBV + cHL were analyzed for clonal B-cell gene rearrangements using Biomed II primers.All three specimens shared peaks in both ΚA and ΚB tubes (x2) and, in addition, EBV + DLBCL and EBV + cHL shared peaks for IgH FR1 and FR2 (Table 1).No clonal peaks were identifed in the FR1 or FR2 reactions for the initial FL specimen.Tese data confrmed a clonal relationship between all three lymphoma morphologies.
In light of the new diagnosis of EBV + cHL and the fact that-despite its occult nature on previous biopsy-this disease component proved refractory to treatment with anthracycline-based therapy, the patient was started on brentuximab vedotin plus nivolumab [3] with a complete metabolic response after 2 cycles seen on the PET scan (Figure 2(c)).Te patient was subsequently referred for autologous hematopoietic stem cell transplant consolidation.

Discussion
Composite lymphoma, the coexistence of two or more distinct lymphoma subtypes in a single biopsy, is an uncommon fnding.Te distinct components of composite lymphomas can include a wide spectrum of lymphoma entities which may or may not be clonally related on a case-by-case basis.While cHL has been reported as a composite lymphoma with either DLBCL or FL, occasionally in association with EBV [4,5], we believe that this case is the frst report of all three lymphomas arising in a single lymph node.Furthermore, we demonstrate a clonal relationship between the three lymphomas using B-cell receptor gene rearrangement studies.Te combination of shared clonality and the presence of EBV only in the DLBCL and cHL components support a potential role of EBV in FL transformation in this instance.Finally, while the initial therapy was directed to B-cell lymphoma, R-CHOP contains doxorubicin which is essential for the treatment of Hodgkin lymphoma [6].Te patient's refractoriness to frontline chemotherapy led us to believe that subsequent high-dose chemotherapy unlikely to cure the disease, and thus, a choice of brentuximab vedotin and nivolumab was made [3,7], which showed initial efcacy as salvage therapy.

2
Case Reports in Hematology EBV exhibits a known association with lymphoma, especially large B-cell lymphomas, cHL and T/NK cell lymphoproliferative disorders [1].A pathogenic role has been established in lymphomagenesis, particularly in the setting of compromised immunity.Case reports suggest that the virus may occasionally contribute to aggressive disease transformation, not only to de novo pathogenesis [8][9][10].In the case presented herein, we hypothesize that the immunologically tolerogenic microenvironment of FL may have enabled EBV reactivation, as the patient had no other identifed immunosuppressive state, nor had he begun chemotherapy at the time of composite lymphoma diagnosis.Previous studies have shown that EBV + DLBCL may rarely ensue from an antecedent FL with a variable number of EBV + cells [9,10].In our case, no EBER staining was identifed in the initial FL.However, neoplastic follicles did show scattered CD30 positive cells, though the exact identity of the CD30+ cells within neoplastic follicles is uncertain.Interestingly, CD30 positivity has been associated with EBV positivity in FL [10].Transformation of FL to cHL has also been reported, often with a clonal relationship [11], and EBV may play a role in an FL-to-cHL transition in rare instances [9].Although B-cell clonality studies showed clonal relatedness among all components of this composite lymphoma, the cHL relapse did not harbor the BCL6 gene rearrangement or gain of BCL2 as identifed in the composite lymphoma.Tis fnding suggests that EBV may have supplied diferent oncogenic factors to drive cHL transformation.While the overall morphologic and genetic fndings supported EBV-mediated transformation of FL to both DLBCL and cHL, an alternative hypothesis is that EBV reactivation in a common clonally rearranged lymphoma precursor triggered divergent lymphomagenesis.It remains uncertain whether the BCL6 rearrangement and BCL2 gain were present in FL, DLBCL, or both, but the high proportion of FL in the composite lymphoma favors its presence at least in that component.Te factors that enable EBV-driven lymphoma transformation remain to be further elucidated, though data are emerging to this end [5].
From a diagnostic standpoint, EBV + DLBCL can present as a monomorphic form, a polymorphic form and, occasionally, as a gray zone between DLBCL and cHL [12].According to the World Health Organization and International Consensus Classifcation criteria, the latter cases should be diagnosed as EBV + DLBCL [13,14].Appropriate therapy for all morphologic types of EBV + DLBCL is generally considered to be like that for EBV-DLBCL.However, this case showed a distinct component with cHL morphology and immunophenotype, albeit representing a minor portion of the biopsied tissue.Moreover, persistence of only EBV + cHL following multiple lines of B-cell directed therapy (R-CHOP, the trial regimen suspected to be loncastuximab plus rituximab due to total CD19 negativity of relapse biopsy) further supported the distinct biology of the cHL component.
Tis case highlights the importance of careful assessment of EBV + DLBCL, especially the polymorphic form, to exclude cHL due to diferent therapeutic implications.Lymphoma cell morphology, background cellularity, nodal versus extranodal presentation, and comprehensive immunophenotypic assessment of the B-cell program (CD20, PAX5, OCT2, BOB1, and CD79a) are all critical for the diferential diagnosis between polymorphic EBV + DLBCL and EBV + cHL [13].Torough diagnostic evaluation often requires excisional biopsy and occasionally necessitates image guidance with PET/CT to identify FDG-avid sites of involvement.Failure to exhaustively assess all components of a surgical specimen may result in inefective therapy and selective outgrowth of aggressive components of a lymphoma.Furthermore, the eradication of DLBCL and FL components but persistence and subsequent overt relapse as cHL in this case raises the question as to whether the identifcation of composite lymphomas with both DLBCL and cHL should alter initial therapy [15].An ongoing clinical trial of brentuximab vedotin in EBV + DLBCL may support future initial therapy with activity against both lymphomas (https://clinicaltrials.gov/ct2/show/NCT01671813).