EBV-positive Diffuse Large B-cell Lymphoma Research Articles

Clinicopathological and Molecular Characteristics of Rare EBV-associated Diffuse Large B-cell Lymphoma With IRF4 Rearrangement.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) is a rare form of aggressive B-cell lymphoma with limited molecular information reported regarding interferon regulatory factor 4 (IRF4) status. Here, we presented 3 EBV-positive DLBCL cases with IRF4 rearrangement (EBV+DLBCL-IRF4-R) verified by fluorescence in situ hybridization (FISH). Three patients, including 1 male and 2 females (median age: 64y; range: 45 to 68y), had normal immune function. During a median follow-up of 12 months (range: 0 to 24 mo), 2 patients succumbed to the disease, and 1 patient achieved complete response. Three tumors were present in the mediastinum, stomach, and thalamus, respectively. All three tumors exhibited DLBCL morphology and were identified as the non-germinal center B-cell subtype, with EBV-encoded small RNA positivity ranging from 70% to 80%. RNA sequencing was able to identify RHOH and IGH as fusion partners of IRF4 in two cases. No MYC and BCL2 rearrangements were detected in 3 cases by FISH and RNA sequencing. Next-generation sequencing revealed a low mutation burden, and only IRF4 was recurrently mutated in two EBV+DLBCL-IRF4-R cases. Using the LymphGen 2.0 classifier, 1 case was classified as the MCD (including MYD88L265P and CD79B mutations) subtype. We report rare EBV+DLBCL-IRF4-R that may enhance our understanding of the diverse spectrum of large B-cell lymphoma.

EBV-positive diffuse large B-cell lymphoma, not otherwise specified: 2024 update on the diagnosis, risk-stratification, and management.

Epstein Barr virus-positive (EBV+) diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS) is an aggressive B-cell lymphoma associated with EBV infection included in the WHO classification of lymphoid neoplasms since 2016. Although historically associated to poor prognosis, outcomes seem to have improved in the era of chemoimmunotherapy. The diagnosis is established through meticulous pathological evaluation. Detection of EBV-encoded RNA (EBER) is the standard diagnostic method. The ICC 2022 specifies EBV+ DLBCL, NOS as occurring when >80% of malignant cells express EBER, whereas the WHO-HAEM5 emphasizes that the majority of tumor cells should be EBER positive without setting a defined threshold. The differential diagnosis includes plasmablastic lymphoma, DLBCL associated with chronic inflammation, primary effusion lymphoma, among others. The International Prognostic Index (IPI) and the Oyama score can be used for risk-stratification. The Oyama score includes age >70 years and presence of B symptoms. The expression of CD30 and PD-1/PD-L1 are emerging as potential adverse but targetable biomarkers. Patients with EBV+ DLBCL, NOS, should be staged and managed following similar guidelines than patients with EBV-negative DLBCL. EBV+ DLBCL, NOS, however, might have a worse prognosis than EBV-negative DLBCL in the era of chemoimmunotherapy. Therefore, inclusion of patients in clinical trials when available is recommended. There is an opportunity to study and develop targeted therapy in the management of patients with EBV+ DLBCL, NOS.

Trophoblast Cell Surface Antigen 2 (Trop2) Is Expressed in Cases of EBV-Positive Diffuse Large B-Cell Lymphoma Emerging from Angioimmunoblastic T-Cell Lymphoma

Trophoblast Cell Surface Antigen 2 (Trop2) Is Expressed in Cases of EBV-Positive Diffuse Large B-Cell Lymphoma Emerging from Angioimmunoblastic T-Cell Lymphoma

Epstein-Barr virus-positive iris diffuse large B-cell lymphoma detected by metagenomic next-generation sequencing

PurposeEpstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) is a relatively rare subtype of DLBCL. Herein, we report a case of a patient with EBV-positive iris DLBCL after undergoing penetrating keratoplasty and discuss its possible pathogenesis.MethodsA 72-year-old male patient presented to our hospital with progressive blurring of vision in the left eye for the past 4 months. Small white nodular lesions were observed on the iris and retinal surface of the left eye, with a white cloud-like opacity in the vitreous cavity.ResultsThe patient was eventually diagnosed with EBV-positive iris DLBCL after undergoing pathological and metagenomic tests. After injecting methotrexate in the left vitreous cavity and administering systemic and local antiviral treatments, the ocular lesions disappeared.ConclusionEBV infection, drug immunosuppression, and aging-related immune deterioration may play significant roles in the pathogenesis of EBV-positive iris DLBCL.SynopsisEpstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) is a new subtype of DLBCL, which rarely occurs. Herein, we report a case of a patient with EBV-positive iris DLBCL after undergoing penetrating keratoplasty and discuss its possible pathogenesis.

Frequent expression of PD-L1 in BLS-type diffuse large B-cell lymphoma: implications for aggressiveness and immunotherapy

BLS-type diffuse large B-cell lymphoma (DLBCL) denotes an uncommon, aggressive variant of DLBCL presenting initially in bone marrow, liver and spleen without lymphadenopathy or mass lesion. Patients with BLS-type DLBCL present frequently with haemophagocytic syndrome which often leads to early patient demise. Programmed death ligand 1 (PD-L1) plays a negative regulatory role on effector T cells and is an important target of immunotherapy. Assessment of PD-L1 expression in BLS-type DLBCL may carry therapeutic implications and provide mechanistic insights. Standard immunohistochemical analysis for PD-L1 was performed in seven cohorts for this study: (1) DLBCL-not otherwise specified (NOS) (n=201); (2) Epstein-Barr virus (EBV)-positive DLBCL (n=26); (3) thymic (primary mediastinal) DLBCL (n=12); (4) intravascular LBCL (n=3); (5) high-grade B-cell lymphoma, NOS (n=12); (6) BLS-type DLBCL (n=37); and (7) systemic DLBCL involving bone marrow (n=28). We found that PD-L1 was positive in 12.9% of DLBCL-NOS cases, 46.2% of EBV-positive DLBCL, 91.7% of thymic LBCL, none of intravascular LBCL, 8.3% of high-grade B-cell lymphoma-NOS, and 56.8% of BLS-type DLBCL. By comparison, only 14.3% of bone marrow cases involved by systemic DLBCL were positive for PD-L1 (p<0.001). Interestingly, BLS-type DLBCL more frequently showed activated B-cell phenotype (86.5% vs 65.2%, p=0.010), a high Ki-67 proliferative index (97.1% vs 63.3%, p<0.001), MYC overexpression (90.9% vs 56.2%, p=0.023), presence of haemophagocytic syndrome (86.5% vs 4.0%, p<0.001), and poorer overall survival (p<0.001) than DLBCL-NOS. These data suggest that the poor prognosis of BLS-type DLBCL may be explained by both extrinsic tumour microenvironment factors and intrinsic genetic factors of tumour cells, such as PD-L1-associated inactivation of anti-tumour immunity for the former, and MYC pathway activation-related aggressiveness for the latter.

CD30 protects EBV-positive diffuse large B-cell lymphoma cells against mitochondrial dysfunction through BNIP3-mediated mitophagy

Epstein-Barr virus (EBV) positive diffuse large B-cell lymphoma (EBV+ DLBCL) predicts poor prognosis and CD30 expression aggravates the worse consequences. Here, we reported that CD30 positivity was an independent prognostic indicator in EBV+ DLBCL patients in a retrospective cohort study. We harnessed CRISPR/Cas9 editing to engineer the first loss-of-function models of CD30 deficiency to identify that CD30 was critical for EBV+ DLBCL growth and survival. We established a pathway that EBV infection mediated CD30 expression through EBV-encoded latent membrane protein 1 (LMP1), which involved NF-κB signaling. CRISPR CD30 knockout significantly repressed BCL2 interacting protein 3 (BNIP3) expression and co-IP assay indicated a binding between CD30 and BNIP3. Moreover, silencing of CD30 induced mitochondrial dysfunction and suppressed mitophagy, resulting in the accumulation of damaged mitochondria by depressing BNIP3 expression. Additionally, CRISPR BNIP3 knockout caused proliferation defects and increased sensitivity to apoptosis. All the findings reveal a strong relationship between mitophagy and adverse prognosis of EBV+ DLBCL and discover a new regulatory mechanism of BNIP3-mediated mitophagy, which may help develop effective treatment regimens with anti-CD30 antibody brentuximab vedotin to improve the prognosis of CD30+ EBV+ DLBCL patients.

PD-1 Blockade Combined with R-CHOP in Patients with Newly Diagnosed EBV-Positive Diffuse Large B-Cell Lymphoma: A Retrospective Analysis

Background Epstein-Barr virus (EBV) -positive diffuse large B cell lymphoma (EBV+ DLBCL) is a relatively rare aggressive B-cell lymphoma. Most patients present with advanced disease, high incidence of extranodal involvement of 30% to 80% (Keane, Eur J Haematol, 2019), as well as poor prognosis when treated with immunochemotherapy. However, no consensus on standard treatment has been established by now. It was found that the expression of programmed death ligand-1 (PD-L1) was increased in EBV+ DLBCL (Kim, Cancer Res Treat, 2019). In addition, chronic EBV infection results in T-cell exhaustion that can be reversed by PD-1 blockade. Compared with PD-L1-positive EBV-negative DLBCL cell lines, the application of PD-1 blockade in PD-L1-positive EBV+ DLBCL cell lines can significantly restore T cell proliferation and activity(Quan, PLoS One, 2015), suggesting that targeting the PD-L1/PD-1 pathway represents a potential therapy for EBV+ DLBCL. Therefore, we retrospectively analyzed the data of nineteen patients with EBV+ DLBCL who were treated with PD-1 blockade combined with RCHOP in our center, hoping to provide reference for subsequent research. Methods Eligible pts had treatment-naive histologically confirmed diagnosis of EBV+ DLBCL. Additional eligibility criteria included at least one measurable lesion, acceptable haptic and hematological function. Pts with primary CNS lymphoma, primary mediastinal large B-cell lymphoma, secondary CNS involvement were excluded. Additional exclusion criteria included history of immune system disease, uncontrolled infection. Once deemed eligible, pts will receive six cycles of RCHOP combined with PD-1 blockade(21 days per cycle) as induction therapy and subsequently maintenance with PD-1 blockade for 1 year(21 days per cycle). End of treatment(EOT) responses were assessed by PET-CT according to the Lugano Classification 2014. The primary objective was to assess the overall response rate(ORR) at EOT. Results Between May 1, 2019, and Sep 1, 2022, 19 pts were enrolled. Pts' baseline demographics, clinical and pathological characteristics are shown in Table 1. The median age was 64 years old without significant gender difference. The majority of pts were intermediate- to high-risk by IPI. EBV DNA can be detected in blood serum at diagnosis in more than half of all patients. No patient was diagnosed with double expressor lymphoma among 18 specimens that were detected c-Myc and BCL-2 with immumohistochemical staining. EBV-encoded RNA (EBER) was positive(&amp;gt;80% of the tumor cells) in all specimens. PD-L1 positivity(&amp;gt;10% of the tumor cells) or CD30 positivity(&amp;gt;5% of the tumor cells) was also quite common. Besides, no double hit lymphoma was confirmed in patient with Fluorescence situ hybridization test(n=13). Treatment course and efficacy evaluation of all pts are summarized in Figure 1. All patients enrolled in this study had completed 6 cycles of induction therapy. Totally, the ORR at EOT was 94.7%, and all achieved CMR. The ORR of patients with PD-L1 positivity(n=10) was numerically higher than that of patients with negative PD-L1 expression (n=5) (100% vs 80%). EBV DNA detected in blood serum of thirteen patients have decreased to undetectable level after induction therapy. The immune-related adverse events (irAEs) were emphatically analyzed. One patient had recurrent fever after every dose of PD-1 blockade without obvious evidence of infection. Another patient developed grade 3 diarrhea since the second cycle of PD-1 blockade maintenance. Colonoscopy showed mucosal swelling and multiple superficial ulcers in the whole colon, which indicated immune colonitis. Maintenance therapy was therefore discontinued. No irAE was observed in the rest of patients. With a median follow-up time of 13.1m (7.2-38.5m), the median PFS and OS were not reached. Both of 1 year OS and PFS was 89.5%. Overall, 2 pts experienced disease progression (1 at EOT, 1 at 2 months after ASCT), 2 pts died (1 due to progressive disease, 1 due to COVID-19 infection ). Conclusions PD-1 blockade combined with R-CHOP as induction therapy in patients with newly diagnosed EBV+ DLBCL has demonstrated satisfactory response and limited irAEs. Longer follow-up is still warranted to confirm the long-term benefit, especially for patients with maintenance of PD-1 blockade. Still, we believe this therapeutic strategy can be regarded as a successful attempt at improving prognosis for pts with EBV+ DLBCL.

Somatic Mutational Landscape and Transcriptomic Profiles of Lymphoproliferative Disorders with Epstein-Barr Virus Infection in Activated PI3K Delta Syndrome

Introduction: Germline gain-of-function variants in PIK3CD and PIK3R1 cause activated PI3K delta syndrome (APDS) types 1 and 2, respectively. Lymphoproliferative disorder (LPD) and susceptibility to various infections, including Epstein-Barr virus (EBV), are well-known features of APDS. EBV-associated LPDs are frequently observed in patients with APDS, and oligoclonal LPD might transform malignant lymphoma (ML). However, somatic molecular alterations in LPD and ML are not fully elucidated. Here, we report clinical characteristics and somatic mutational landscape and transcriptomic profiles of LPDs with EBV infection in APDS. Methods: LPD and ML in APDS diagnosed during 2011-2022 were recruited all over Japan. Clinical characteristics and outcomes were analyzed. EBV infection status was analyzed by EBER ISH and RNA sequencing (RNA-seq). Whole-exome sequencing (WES), RNA-seq, and single cell RNA-seq (scRNA-seq) were also performed. Results: Five LPD and 5 ML (borderline ML: n = 2, diffuse large B-cell lymphoma (DLBCL): n = 2, classical Hodgkin lymphoma: n = 1) cases were enrolled. Seven cases were APDS1 and 3 cases were APDS2. The median follow-up period was 2.9 (0.5-8.4) years. LPD cases were treated with rituximab monotherapy (n = 1), prednisolone (n = 3), and hematopoietic cell transplantation (HCT) (n = 1). Rituximab-combined chemotherapy was performed in borderline ML and DLBCL (n = 4) or ABVD regimen in Hodgkin lymphoma cases (n = 1). Sirolimus was used in two ML cases. HCT was performed in 9 cases. Two-year overall survival and event-free survival (events: relapse and death) were 78.8% and 70.0% in total LPDs, respectively, and 60% and 40% in ML cases. EBV was negative in 3 cases and positive in 2 cases of LPD cases, and positive in 4 cases, and negative in 1 case of ML cases. The median of 18 (10-48) and 42 (18-189) somatic variants were detected by tumor/germline pair WES in LPD (n = 5) and ML (n = 7, 2 samples were from the same patient at different time points), respectively. The recurrent variants were identified in ZFHX3 (n = 4), TRPM2 (n = 3), BEND3, CDH23, UBR4, ARAF, TET2, FBXO10, MAP4K1, KMT2C, KMT2D and PIK3R1 (n = 2) in LPD and ML cases. In transcriptome analysis, genes associated with lymphocyte activation including B-cell receptor signaling were upregulated in LPD, cell-cell adhesion, cellular response to TNF, and IL-18 signaling pathway were upregulated in ML samples. We performed correlation analysis to elucidate a relationship between EBV infection status and host transcriptomic profiles using RNA-seq data. Interestingly, in addition to cell-cell adhesion and inflammatory machinery, the upregulated molecules of the p53 pathway and negative regulation of the immune system were correlated with transcripts associated with EBV latency, such as EBNA, LMP-1, and LMP-2. In single-cell transcriptome analysis, LPD sample consisted of two large clusters of T cells and B cells with 10 subsets, and EBV-positive DLBCL sample consisted of one independent large cluster with 9 subsets. In LPD sample, components indicative of the normal lymphocyte differentiation process, including the germinal center, were preserved. The molecules involved in lymphocyte activation were upregulated in LPD subsets. In DLBCL sample, the genes associated with mitotic G2-M phases, chromatin organization and RNA metabolism were upregulated in the subsets in which EBV transcripts were more actively transcribed. Discussion and Conclusions: Our retrospective analysis demonstrated that the outcome of LPD and ML in APDS is not satisfactory. scRNA-seq data revealed that cells in LPD still retain the signature of normal lymph node. However, once transformed with EBV, EBV infection may affect epigenetic machineries including chromatin organizer followed by active RNA transcription involved in cell-cell adhesion, inflammatory machineries, molecules of p53 pathway and negative regulation of immune system in the host genome. As the outcome of ML is inferior to LPD, preemptive HCT would be an option for LPD cases.

A case of Epstein-Barr virus-associated primary vitreoretinal lymphoma in an immunosuppressed patient.

To describe a rare case of Epstein-Barr virus (EBV)-positive primary vitreoretinal lymphoma (PVRL) in an immunosuppressed patient. Observational case report. A 64-year-old man under immunosuppressive therapy for rheumatic arthritis was referred for 2-month of blurred vision and decreased visual acuity in the right eye. Only mutton-fat keratic precipitates and mild vitreous opacity were found in the right eye without (sub-)retinal or sub-retinal pigment epithelial lesions. Vitreous biopsy and systemic workup suggested the diagnosis of PVRL of diffuse large B cell lymphoma (DLBCL) subform. Neoplastic cells stained positive for EBV antigens, EBV-encoded small RNA and Epstein-Barr nuclear antigen 2, consistent with EBV-positive DLBCL. Intravitreal methotrexate was effective in improving ocular symptoms. Our case provided evidence on the association of EBV infection with PVRL.

P04 Leg ulceration in a patient with antisynthetase syndrome

Abstract A 42-year-old woman presented with leg ulceration on a background of antisynthetase syndrome and immunosuppression with rituximab, azathioprine and prednisolone. The initial lesion was an erythematous patch on the lower leg, which increased in size with violaceous changes. The area subsequently broke down to form an ulcer. On examination, she had a 7-cm ulcerating lesion with central necrosis. Clinically, the appearance was suspicious of pyoderma gangrenosum; biopsy demonstrated scarring without features of vasculitis, atypia or malignancy. The ulcer did not show improvement as expected after oral and topical steroid treatment, with new fleshy nodular growth. Repeat biopsy showed angiocentric atypical lymphocytes with positive Epstein–Barr virus (EBV)-encoded RNA (EBER) staining. Further incisional biopsy demonstrated large, atypical B lymphocytes with a diffuse sheet-like morphology and a poorly circumscribed base. Immunohistochemistry was positive for CD79a, CD30, CD20, MUM1, BCL6, and EBER. Overall, the findings were consistent with EBV-positive diffuse large B-cell lymphoma (DLBCL). The main differential diagnosis was EBV-positive mucocutaneous ulcer (MCU), another EBV-associated lymphoproliferative disorder, albeit with an indolent course (Dojcinov SD, Venkataraman G, Raffeld M et al. EBV positive mucocutaneous ulcer—a study of 26 cases associated with various sources of immunosuppression. Am J Surg Pathol 2010; 34:405–17). In this case, the diffuse sheet-like morphology and poorly circumscribed base favoured EBV-positive DLBCL. The lesion fully healed with withdrawal of immunosuppression and treatment with rituximab therapy, and involved field radiotherapy. First described in 2003, EBV-positive DLBCL is a rare, classically aggressive lymphoma initially believed to only occur in patients older than 50 years of age (Oyama T, Ichimura K, Suzuki R et al. Senile EBV+ B-cell lymphoproliferative disorders. Am J Surg Pathol 2003; 27:16–26). Since the revision of the World Health Organization classification in 2016, it is also recognized to present in younger patients, with an associated better prognosis. EBV-positive DLBCL occurs in both nodal and extranodal sites. Therefore, cutaneous presentations must complete staging investigations to rule out skin involvement secondary to a nodal lymphoma. Our patient highlights a rare complication of immunosuppression. We illustrate the relevance of considering rare causes of ulceration in patients with autoimmune conditions and immunosuppression and demonstrate the importance of considering malignancy in a nonhealing ulcer. Multiple biopsies may be needed in ulcers that poorly respond to treatment. While EBV-positive DLBCL and EBV-positive MCU can be difficult to differentiate, it is vital for prognostication.

CD30 protects EBV‐positive diffuse large B‐cell lymphoma cells against mitochondrial dysfunction through increasing BNIP3 expression

Introduction: EBV-positive diffuse large B-cell lymphoma (EBV+DLBCL) predicts poor prognosis. Studies have found that CD30 expression was more frequent in EBV+DLBCL patients compared to EBV-negative (EBV-) DLBCL and high CD30 expression was associated with poor survivals in EBV+DLBCL. All these facts suggested a synergistic effect between CD30 expression and EBV infection. Methods: FARAGE and GM12878 are type II and III latency EBV-infected B cell lines while RAJI and DAUDI are type I. SUDHL-2, U2932 and RIVA are EBV-negative DLBCL cell lines. CRISPR/Cas9 editing was employed to engineer the loss-of-function models of genes involved in this study. Results: A total of 451 patients were included and the results showed patients had worse PFS (P = 0.007) and OS (P = 0.007) when CD30 was co-expressed with EBER in DLBCL (Figure 1A). Immunoblot analysis showed that CD30 expression in EBV latency II and III cell lines were higher than EBV latency I and EBV-negative cell lines (Figure 1B). Relevant experiments indicated that the tumor cells with high CD30 expression demonstrated rapid proliferation and were less susceptible to apoptosis (Figure 1C and 1D) and CD30-KO mice showed significantly smaller tumor size compared to CD30 wild type (WT) mice (Figure 1E). A LMP1 overexpression resulted in CD30 expression (Figure 1F). To elucidate how LMP1 regulated CD30, we found LMP1 overexpression increased p-Btk and p-p65 (Figure 1G). Immunoblot analysis detected a decrease in CD30 expression when latency II and III cells were treated with ibrutinib (a BTK inhibitor) and ibrutinib also abolished LMP1-induced p-p65 and p-IκBα (Figure 1H). We performed gene expression analysis following CD30 KO in FARAGE and the results indicated depression of CD30 inhibited BNIP3 expression (Figure 1I). Immunoblot analysis showed that BNIP3 expression in the KO group were significantly lower compared to those in the control group (Figure 1J). IHC staining results revealed a significant decrease of BNIP3 expression in CD30-KO mice (Figure 1K). We found that CD30 KO by CRISPR/Cas9 system led to a significant fall in the MMP (Figure 1L). Moreover, the accumulation of damaged mitochondria was observed in CD30-deficient tumor cells in mice by TEM (Figure 1M). Above results suggested that silencing of CD30 resulted in mitochondrial damage and the inhibition of mitochondrial activity. As BNIP3 was identified as a target gene for CD30, we investigated the effect of BNIP3 in EBV+DLBCL. Collectively, our results validated an important role of BNIP3 in promoting EBV+DLBCL survivals (Figure 1N and 1O). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Molecular Targeted Therapies, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

EBV-Positive Diffuse Large B-Cell Lymphoma in Association with Polyurethane Textured Breast Implants: Case Report and Literature Overview.

Lymphomas associated with breast implants are mostly of the T-cell type which typically present as a late-onset seroma or palpable mass adjacent to the implant. Primary lymphomas of the breast, in the absence of breast implants, are mostly of the B-cell type. However, we present a case of an Epstein-Barr virus-positive diffuse large B-cell lymphoma in a patient with polyurethane textured implants. A 75-year-old woman presented with a sudden onset of swelling in her right breast. Her medical history revealed a unilateral mastectomy at the age of 48 for an invasive ductal adenocarcinoma in her left breast. Reconstruction was performed using bilateral McGhan style 150 implants. Nine years later, she was diagnosed with Baker IV capsular contracture and bilateral rupture, as seen on magnetic resonance imaging. A complete capsulectomy with implant change (Polytech, Replicon® SL HP) and mastopexy on the right was performed. Given her history and the sudden onset of swelling, the situation was concerning. Ultrasound showed a large mass in contact with the implant and an effusion around it. After mastectomy withexplantation and capsulectomy, she was diagnosed with an Epstein-Barr virus-associated diffuse large B-cell lymphoma (DLBCL) of the capsule in association with textured breast implants. We report the first case of polyurethane textured implant association with the rare entity of EBV+ DLBCL. Our aim is to renew awareness of the clinical significance of late periprosthetic seroma and highlight the need for documenting all cases to advance our understanding of breast implant-associated lymphoma. This journal requires that authors assign a level of evidence to each article. For a full description of these evidence-based medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Dynamic changes in circulating EBV-DNA load during treatment have prognostic values in EBV+ DLBCL-NOS: a Chinese cohort study.

EBV-positive diffuse large B-cell lymphoma, not otherwise specified (EBV+ DLBCL-NOS), is an EBV-positive clonal B-cell lymphoid proliferation and circulating EBV-DNA is a great indicator for prognosis among EBV associated disease. In this retrospective study, we report 66 EBV+ DLBCL cases among 2137 DLBCL-NOS cases diagnosed from 2013 to 2021 (prevalence of 6.0%). After a median follow-up of 27months, progression-free survival (PFS) and overall survival (OS) at 2years were 39.5% ± 6.2% and 53.6% ± 6.4%, respectively. Multivariate analysis showed that only the biomarker of the positivity of post treatment EBV-DNA had a borderline correlation with shorter PFS and OS (PFS: P = 0.053; OS: P = 0.065). Patients were divided into three subgroups according to dynamic changes of EBV-DNA status: EBV-DNA persistently negative group, EBV-DNA persistently positive group, and EBV-DNA transformed from positive to negative group; among the three groups, patients of the persistently positive group had worst PFS and OS (P = 0.0527 and P = 0.0139, respectively). Decline in EBV copies correlated significantly with treatment response as well. In conclusion, circulating EBV-DNA level played a vital role in prognostic and monitoring marker for EBV+ DLBCL-NOS.

Diffuse large B-cell lymphoma in 1-year male with founder Slavic RAG1 mutation: case report

Background and AimsLymphomas in RAG1 deficiency are extremely rare, especially in early childhood. Only a few cases of lymphoma in RAG1 deficiency have been previously described in the literature. MethodsSanger sequencing of Slavic founders mutation (RAG1-c.256_257del, p.Lys86ValfsTer33, IL7Rα - c.132C>G, p.Ser44Arg, NBN1 -c.657_661del5, p.Lys219AsnfsTer16 genes) in DNA of died children with malignancy in early age in Belarus. ResultsWe report a case of 14 month-old boy affected by diffuse large B-cell lymphoma diagnosed with RAG1 deficiency. A male patient of Belarus origin was born full-term from non-consanguineous parents. He had a history of recurrent pneumonia since 3 months of age. The patient was hospitalized in 14 month-old with subfebrile fever, wet cough, lymphadenopathy and cyanosis of the nasolabial triangle. Chest x-ray showed bilateral polysegmental pneumonia. Microbiological analysis of sputum found Candida. Ultrasound examination showed hepato-(+3 cm)-spleno- (+3 cm)-megaly and lymphadenopathy of multiple groups of lymph nodes (cervical, inguinal and axillary). Lymph-node, liver, and intestines biopsies were suggestive of EBV-positive Diffuse Large B-cell lymphoma, NOS. He had progressive leukopenia (from 3100 cells/µl to 200 cells/µl). Lymphopenia (400 cells/µl), decreased T-cells (300 cells/µl) and elevated activated T-cells (41%) were detected. B-cell percentage was in normal ranges (33%), but absolute number was slightly decreased (160 cells/µl). TRECs – 0 per 1 million leukocytes, KRECs – 0 per 1 million leukocytes. According to the clinical and immunological phenotype, combined immunodeficiency was suspected.The patient died (age 15 m) one month after the start of the chemotherapy cycle due to sepsis and multiple organ failure without genetic diagnosis. His material was studied post-mortem due to the lymphoma at early age and place of birth (Brest, Western Belarus). Targeted Sanger sequencing revealed a homozygous deletion in RAG1 gene (NM_000448.3) c.256_257del (p.Lys86ValfsTer33). ConclusionsWe described a rare case of EBV-positive diffuse large B-cell lymphoma NOS in a patient with homozygous founder Slavic mutation in the RAG1 gene.

Breast implant associated EBV-positive Diffuse Large B-cell lymphoma: an underrecognized entity?

Breast-implant associated (BIA) lymphoma is an infrequent type of cancer occurring in the fluid and fibrous capsule around a textured breast implant. Recently, both the 2022 WHO 5th edition classification of Haematological tumours (WHO HAEM5) and 2022 International Consensus Classification of Mature Lymphoid Neoplasms (22ICC), recognized breast implant-associated Anaplastic Large Cell Lymphoma (BIA-ALCL) as a definitive entity, defined as a mature CD30-positive T-cell lymphoma, confined by a fibrous capsule, in a breast implant setting. Only few B-cell lymphomas have been reported in the literature to be associated with breast implants. Here we report two EBV-positive Diffuse Large B-cell lymphomas (EBV + DLBCL) in relation to a breast implant, both expressing CD30 as well as EBV latency type 3. Both lesions were considered as DLBCL associated with chronic inflammation (CI-DLBCL), but one presented as a 7 cm solid mass, while the other presented as a fibrin-associated DLBCL (FA-DLBCL) in an HIV patient. Clinically, both are in complete remission 6 months or longer after capsulectomy and graft removal, without additional chemotherapy.Such cases, characterized by large CD30-positive cells, can easily be misdiagnosed as BIA-ALCL if the cell of origin is not further established. Therefore, a diagnostic panel including lineage-specific B-and T-cell markers and EBER in situ hybridization is essential to recognize this rare entity, to understand lymphomagenesis, to predict outcome and to define clinical approach.

TET2 and LILRB1 mutations are frequent in Epstein-Barr virus-positive diffuse large B-cell lymphoma especially in elderly patients.

Diffuse large B-cell lymphoma (DLBCL) harboring Epstein-Barr virus (EBV) primarily occurs in patients who have underlying immunodeficiency or in elderly patients but is also reported in young, immunocompetent patients. The authors investigated the pathologic differences in EBV-positive DLBCL in these three groups of patients. In total, 57 patients with EBV-positive DLBCL were included in the study; of these, 16 patients had associated immunodeficiency, 10 were young (younger than 50years), and 31 were elderly (aged 50years or older). Immunostaining for CD8, CD68, PD-L1, and EBV nuclear antigen 2, and panel-based next-generation sequencing was performed on formalin-fixed, paraffin-embedded blocks. Immunohistochemistry revealed EBV nuclear antigen 2 positivity in 21 of the 49 patients. The degree of CD8-positive and CD68-positive immune cell infiltration and PD-L1 expression did not differ significantly in each group. Extranodal site involvement was more common in young patients (p=.021). In mutational analysis, the genes with the highest mutation frequency were PCLO (n=14), TET2 (n=10), and LILRB1 (n=10). For the TET2 gene, all 10 mutations were found in elderly patients (p=.007). Compared with a validation cohort, both TET2 and LILRB1 showed a higher mutation frequency in EBV-positive patients than in EBV-negative patients. EBV-positive DLBCL occurring in three different age and immune status groups showed similar pathologic characteristics. Notably, a high frequency of TET2 and LILRB1 mutations was characteristic of this disease in elderly patients. Further studies are needed to determine the role of TET2 and LILRB1 mutations in the development of EBV-positive DLBCL along with immune senescence. Epstein-Barr virus-positive diffuse large B-cell lymphoma occurring in three different groups (immunodeficiency-associated, young, and elderly) showed similar pathologic characteristics. The frequency of TET2 and LILRB1 mutations was high in elderly patients with Epstein-Barr virus-positive diffuse large B-cell lymphoma.

Genomic Profiling and Clinicopathological Features in Peruvian Patients with EBV-Positive Diffuse Large B-Cell Lymphomas, NOS

Introduction: Epstein Barr virus-positive (EBV+) diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS) is a newly recognized entity by the World Health Organization. EBV+ DLBCL, NOS is commonly encountered in Latin American countries and carries a dismal prognosis. It has been published studies that have evaluated the genetic profiling in EBV +DLBCL from Asian and Western countries. However, there are lacking of studies from Latin America. This study aimed to characterize and compare the genetic profiling and clinicopathological features in Peruvian EBV+ with EBV- DLBCL patients. Methods: This retrospective cohort study included patients ≥18 years from one institution in Peru from 2010 to 2020. Hematopathologists reviewed pathological samples to confirm the diagnosis of EBV+ DLBCL cases. An EBV+ case was confirmed when the EBER test had a positivity more or equal to 40%. We collected clinicopathological data by reviewing the medical records of the patients. Twenty paraffin-embedded biopsy samples were analyzed and tumor DNA was purified using silica columns (QIAGEN, Valencia, CA, USA). The quantity and quality of the purified DNA were evaluated by spectrophotometry. We compared the genetic profiling with ten EBV+ versus ten EBV- DLBCL samples. Results: Twenty-two candidate genes were selected from previous reports of exome analysis with the diagnosis of DLBC. The median age at diagnosis was similar in both groups (58 years old, range 19-89). There was a male predominance in EBV+ (57%) compared to EBV- DLBCL cases (43%). Sixty-eight percent of patients with EBV+ had advanced disease (Ann arbor stage III-IV), compared to 48% of EBV- DLBCL patients. The non-germinal center was more common in EBV+ (60%) than EBV- cases (40%). All patients received either R-CHOP-like regimens as a first-line treatment. The overall response rate was 91% for EBV- compared to 74% for EBV+ DLBCL. We found mutations in MYC (10%), NOTCH2 (5%), STAT6 (5%) , ANKRD11(5%), TP53 (5%), KMT2A (5%), YY1 (5%), CARD11 (5%), ARID1A (5%) and RHOA (5%) in EBV+ cases, meanwhile, ARID1A (10%) , CCR6 (10%), KMT2A (5%), CARD11(5%), NOTCH2 (5%), TP53 (5%), and ANKRD11(5%) in EBV - DLBCL cases. Conclusions: This is the first Latin American study that have performed the genomic pattern in Peruvian EBV+ in comparison to EBV- DLBCL patients. Overall, our data suggest that Peruvian EBV+ DLBCL have different genetic profiling compared to East and Western countries. Also, we found that EBV+ and EBV- patients share similar drive mutations. Our findings should be validated in a larger prospective cohort of Latin American patients.

Impact of Epstein-Barr virus infection in patients with inflammatory bowel disease.

A high prevalence of Epstein-Barr virus (EBV) infection in patients with inflammatory bowel disease (IBD) has been reported in many case reports and studies; thus, the association between EBV and IBD has gained increasing attention. Patients with IBD are at an increased risk of opportunistic EBV infection owing to the common use of immunomodulators. EBV infection in IBD patients can cause various complications, including superimposed viral colitis, which is associated with chronicity, exacerbation, and poor prognosis of refractory IBD, and can induce progression to lymphoproliferative disorders, such as EBV-positive mucocutaneous ulcer (EBVMCU), lymphomatoid granulomatosis (LYG), hemophagocytic lymphohistiocytosis (HLH) and diffuse large B-cell lymphoma (DLBCL). It has been suggested to screen for EBV before initiating immunosuppressive therapy and monitor the status of EBV infection in patients with IBD, especially those who are EBV-seronegative and have a risk of primary EBV infection. Clinicians should also be careful of misdiagnosing IBD and EBV-associated lymphoproliferative diseases due to similarities in both clinical symptoms and endoscopic manifestations. Withdrawal of immunosuppressants has been shown to be an effective strategy to achieve remission of disease at the time of EBV diagnosis, but antiviral therapy remains controversial. The present review aims to describe the characteristics of the complications caused by EBV infection and generalize the recent research progress on and challenges caused by EBV infection in IBD patients. The literature for writing this review was collected from 'PubMed' research engine. The keywords 'inflammatory bowel disease and Epstein-Barr virus' or 'ulcerative colitis and Epstein-Barr virus' or 'Crohn's disease and Epstein-Barr virus' were used to collect the literature and relevant papers were collected to help writing this review.

S2724 EBV as a Very Unusual Etiology of Mucocutaneous Ulcer of the Neo-Ileum in a Patient With Crohn's Disease

Introduction: A member of the herpes virus family, Epstein Barr virus (EBV) is one of the most common human viruses. It can cause latent infections in humans, leading to serious health complications, including lymphomas and lymphoproliferative disorders. Mucocutaneous ulcers associated with EBV (EBVMCU) are a recently established entity of the lymphoproliferative disorders in 2010. Case Description/Methods: An 82-year-old male with Crohn's disease s/p right hemicolectomy with the formation of neo-ileum seven years earlier, bladder cancer, lung cancer s/p lobectomy in remission for both cancers. He presented to the hospital complaining of bright red blood per rectum. He reports chronic loose stools attributed to a history of Crohn's disease and right hemicolectomy seven years earlier when 30 cm colon was removed, including terminal ileum to the proximal transverse colon. The patient reports that he believed his Crohn's was never symptomatic and was worried about the side effects of biologic Crohn's treatment, which he refused in the past. Colonoscopy revealed extensive ulceration at the neo-ileum with friable mucosa. The histology shows scattered large atypical EBV-positive cells of B cell lineage in inflamed and ulcerated mucosa with granulation tissue. A definitive diagnosis could not be made, and further biopsies were recommended. PET scan and CT scan were unremarkable for metabolically active malignant lesions or lymphadenopathy. Colonoscopy repeated two months later showed the same ulcerated lesion with the same histological features mentioned earlier. (Figure) Discussion: EBVMCUs manifest as shallow sharply circumscribed ulcers on the mucosa or skin and are histologically distinguished by the proliferation of variable-sized, atypical B-lymphocytes, making the site of the ulcers in the neo-ileum very atypical and unique. The lesion usually develops in individuals with impaired immunity, such as those who are elderly, have iatrogenic immunosuppression, have primary immune disorders, or are HIV/AIDS-infected. The lack of a tumor mass favors an EBV-positive mucocutaneous ulcer, a newly recognized entity that can involve the GIT and occurs as an age-related disease and in immunosuppressed patients. In contrast to other EBV-related lymphoproliferative disorders, such as EBV-positive diffuse large B-cell lymphoma, EBVMCU has a good prognosis. A watch-and-wait approach or reduction in immunosuppressants are usually sufficient to cause EBVMCU patients to achieve remission.Figure 1.: Neo-ileum biopsy: The atypical cells are large with ovoid to irregular nuclear membrane, hyperchromatic nuclei, variable prominent nucleoli, and cytoplasmic clearing.

High Predictive Value of the Soluble ZEBRA Antigen (Epstein-Barr Virus Trans-Activator Zta) in Transplant Patients with PTLD.

The ZEBRA (Z EBV replication activator) protein is the major transcription factor of EBV, expressed upon EBV lytic cycle activation. An increasing body of studies have highlighted the critical role of EBV lytic infection as a risk factor for lymphoproliferative disorders, such as post-transplant lymphoproliferative disease (PTLD). We studied 108 transplanted patients (17 PTLD and 91 controls), retrospectively selected from different hospitals in France and in the Netherlands. The majority of PTLD were EBV-positive diffuse large B-cell lymphomas, five patients experienced atypical PTLD forms (EBV-negative lymphomas, Hodgkin’s lymphomas, and T-cell lymphomas). Fourteen patients among the seventeen who developed a pathologically confirmed PTLD were sZEBRA positive (soluble ZEBRA, plasma level above 20 ng/mL, measured by an ELISA test). The specificity and positive predictive value (PPV) of the sZEBRA detection in plasma were 98% and 85%, respectively. Considering a positivity threshold of 20 ng/mL, the sensitivity of the sZEBRA was 82.35% and the specificity was 94.51%. The mean of the sZEBRA values in the PTLD cases were significantly higher than in the controls (p < 0.0001). The relevance of the lytic cycle and, particularly, the role of ZEBRA in lymphomagenesis is a new paradigm pertaining to the prevention and treatment strategies for PTLD. Given the high-specificity and the predictive values of this test, it now appears relevant to investigate the lytic EBV infection in transplanted patients as a prognostic biomarker.