EBER Research Articles

PD-L1 expression in EBV-associated gastric carcinomas

Assessment of the incidence of PD-L1 expression in EBV-associated gastric adenocarcinomas, as well as clarification of the clinical and morphological characteristics and median survival of patients with PD-L1-positive EBV-associated gastric cancer. Samples of surgical material from 127 patients with stomach cancer were studied. Each sample was stained by in situ hybridization using primers for the Epstein-Barr virus-encoded small RNAs (EBER). Expression of PD-L1 was assessed immunohistochemically (PD-L1 SP263, PD-L1 SP142). The results obtained were compared with the main clinical and morphological characteristics of gastric cancer and median survival of patients. The detection rate of PD-L1 SP263 and PD-L1 SP142 in EBV-associated gastric adenocarcinoma in our sample was 100% and 76.9% respectively, thus, PD-L1 expression (SP263, SP142) is significantly more frequently detected in EBV-associated gastric carcinomas. It was found that patients with positive expression of PD-L1 in EBV-associated gastric carcinomas are younger (mean age 56.3 years for SP263 and 55.6 years for SP142), belonging to male gender. In addition, this group is dominated by proximal localization of tumors, ulcerative form of growth, tubular histological type, intermediate subtype according to P. Lauren. These characteristics do not depend on the antibody clone: positive expression of SP142 and SP 263 was detected in the same patients with a few exceptions. The overall median survival of patients with positive PD-L1 status SP263 in EBV-associated gastric carcinomas was 35 months, for patients with positive PD-L1 status SP142 - 25 months. Median survival of SP142 PD-L1 positive patients is higher than overall median survival of PD-L1 negative patients in EBV-associated gastric carcinomas. It was found that PD-L1 status in EBV-associated gastric cancer is not a significant prognostic factor. A single PD-L1 status does not significantly affect the prognosis in patients with gastric cancer, including those in the group of EBV-associated carcinomas, and can only be considered in conjunction with 'classic' clinical and morphological characteristics, primarily with the stage of the tumor process, since they determine the prognostic properties of the tumor.

Study of gastric adenocarcinomas association with the Epstein-Barr virus-encoded small RNAs

Assessment of the incidence of EBV-associated gastric adenocarcinomas in a sample of Russian patients, as well as clarification of the clinical and morphological characteristics and median survival of patients with EBV-associated gastric cancer. We used samples of surgical material from 282 patients with a verified diagnosis of gastric cancer. Each sample was stained by in situ hybridization using primers for the Epstein-Barr virus-encoded small RNAs (EBER). The results obtained were compared with the main clinical and morphological characteristics of gastric cancer. The detection rate of EBV-associated gastric adenocarcinoma in our sample was 9.57%. EBER-positive tumors much more often (p=0.021) belong to the intermediate type according to the P. Lauren classification (66.67%) in comparison with EBER-negative tumors (38.82%). EBER-positive tumors significantly more often (p=0.035) belong to high-grade tumors - 75.00% in comparison with EBER-negative tumors (52.13%). The overall median survival of all patients with EBER-positive tumors (53.5 months) was higher compared to the overall median survival of all patients with EBER-negative tumors - 36.5 months (p=0.5379). The median survival of patients with EBER-positive stage III tumors (30.0 months) was also higher compared to that for patients with EBER-negative tumors - 20.0 months (p=0.5622). It was found that a single EBER status is not a significant prognostic factor (HR=1.0143; CI: 0.9897-1.0196). Separately taken EBER-status is not a significant independent prognostic factor and can be considered only in conjunction with the «classical» clinical and morphological characteristics, primarily with the stage of the tumor process, since it is they that determine the prognostic properties of the tumor.

EBV+ high-grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements: a multi-institutional study.

It is unknown whether Epstein-Barr virus (EBV) infection can occur in high-grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, also known as double- or triple-hit lymphoma (DHL/THL). Here we report 16 cases of EBV+ DHL/THL from screening 846 cases of DHL/THL and obtaining additional EBV+ cases through multi-institutional collaboration: eight MYC and BCL2 DHL, six MYC and BCL6 DHL and two THL. There were eight men and eight women, with a median age of 65 years (range = 32-86). Two patients had a history of follicular lymphoma and one had AIDS. Nine of 14 patients had an International Prognostic Index of ≥3. Half of the cases showed high-grade/Burkitt-like morphology and the other half diffuse large B cell lymphoma morphology. Using immunohistochemistry, the lymphoma cells were positive for MYC (n = 14 of 16), BCL2 (n = 12 of 16), BCL6 (n = 14 of 16), CD10 (n = 13 of 16) and MUM1 (n = six of 14). Using Hans' algorithm, 13 cases were classified as germinal centre B cell (GCB) and three as non-GCB. The lymphomas frequently showed an EBV latency Type I with a median EBV-encoded small RNAs of 80% positive cells (range = 20-100%). After a median follow-up of 36.3 months (range = 2.0-41.6), seven patients died, with a median survival of 15.4 months (range = 3.4-47.3) after diagnosis of EBV+ DHL/THL. Five of six patients with MYC and BCL6 DHL were alive, including four in complete remission. In contrast, only four of 10 patients with MYC and BCL2 DHL or THL were alive, including two in complete remission. The median survival in patients with MYC and BCL6 DHL was unreached and was 21.6 months in patients with MYC and BCL2 DHL or THL. EBV infection in DHL/THL is rare (~1.5%). Cases of EBV+ DHL/THL are largely similar to their EBV- counterparts clinicopathologically. Our findings expand the spectrum of EBV+ B cell lymphomas currently recognised in the World Health Organisation classification and suggest differences between EBV+ MYC and BCL2 DHL versus EBV+MYC and BCL6 DHL that may have therapeutic implications.

High PTX3 expression is associated with a poor prognosis in diffuse large B-cell lymphoma.

Tumor‐associated macrophages (TAMs) are associated with a poor prognosis of diffuse large B‐cell lymphoma (DLBCL). As macrophages are heterogeneous, the immune polarization and their pathological role warrant further study. We characterized the microenvironment of DLBCL by immunohistochemistry in a training set of 132 cases, which included 10 Epstein–Barr virus‐encoded small RNA (EBER)‐positive and five high‐grade B‐cell lymphomas, with gene expression profiling in a representative subset of 37 cases. Diffuse large B‐cell lymphoma had a differential infiltration of TAMs. The high infiltration of CD68 (pan‐macrophages), CD16 (M1‐like), CD163, pentraxin 3 (PTX3), and interleukin (IL)‐10‐positive macrophages (M2c‐like) and low infiltration of FOXP3‐positive regulatory T lymphocytes (Tregs) correlated with poor survival. Activated B cell‐like DLBCL was associated with high CD16, CD163, PTX3, and IL‐10, and EBER‐positive DLBCL with high CD163 and PTX3. Programmed cell death‐ligand 1 positively correlated with CD16, CD163, IL‐10, and RGS1. In a multivariate analysis of overall survival, PTX3 and International Prognostic Index were identified as the most relevant variables. The gene expression analysis showed upregulation of genes involved in innate and adaptive immune responses and macrophage and Toll‐like receptor pathways in high PTX3 cases. The prognostic relevance of PTX3 was confirmed in a validation set of 159 cases. Finally, in a series from Europe and North America (GSE10846, R‐CHOP‐like treatment, n = 233) high gene expression of PTX3 correlated with poor survival, and moderately with CSF1R, CD16, MITF, CD163, MYC, and RGS1. Therefore, the high infiltration of M2c‐like immune regulatory macrophages and low infiltration of FOXP3‐positive Tregs is associated with a poor prognosis in DLBCL, for which PTX3 is a new prognostic biomarker.

Clinicopathological features of syncytial variant nodular sclerosis Hodgkin lymphoma

The syncytial variant of nodular sclerosis Hodgkin lymphoma (SV-NSHL) is uncommon, and its clinicopathological features have not been well analyzed. In this study, we collected 142 cases of SV-NSHL. There were 76 (53.5%) males and 66 (46.5%) females with a median age of 28 years (range, 12-78); 59.9% were younger than 30 years. Patients usually presented with a mediastinal mass (97.6%) and often with bulky (35.7%) and advanced stage disease (3 or 4) (39.7%). Follow-up showed that 61.1% of patients developed relapse after standard therapy. Morphologically, the neoplasms were composed of sheets or large clusters of pleomorphic neoplastic cells with a cohesive appearance that were frequently associated with necrosis. Neutrophils and eosinophils were frequent in the background, whereas small lymphocytes were decreased. Immunohistochemical analysis showed that the following markers were frequently positive: CD30 (100%), MUM1/IRF4 (96%), PAX5 (95%), PD-L1 (91%), and CD15 (80%). CD45 was negative in all cases. Epstein-Barr virus-encoded small RNA (EBER) was detected in 19% of cases. In this cohort, age above 30 years (P=.0430), presence of B-type symptoms (P=.0394), elevated serum lactate dehydrogenase level (P=.0004), and disease relapse (P=.0108) were associated with a poorer overall survival. In contrast, patients with EBER-positive neoplasms had a better survival (P=.0418). Compared with a control group of non-SV-NSHL patients, patients with SV-NSHL were associated with a poorer overall survival (P=.011). These data suggest that SV morphology is associated with a poorer prognosis in patients treated with traditional standard-of-care therapy for classic Hodgkin lymphoma.

A case of Epstein Barr virus-associated primary squamous cell carcinoma of stomach

BackgroundPrimary squamous cell carcinoma (SCC) of stomach is extremely rare. The pathogenesis of SCC of stomach remains unclear. There is only one report that Epstein Barr virus (EBV) infection may be involved in the pathogenesis of SCC arising in the stomach ever before. Here, we report a case of Epstein Barr virus infection-associated primary SCC of stomach in a 70-year-old woman. She was presented to the referring hospital with hematemesis. Initial endoscopy revealed a bleeding gastric ulcer in the upper part of gastric corpus and the coagulation therapy was followed. After a 3-month follow-up, endoscopy revealed a submucosal tumor-like protrusion instead of an ulcer. Computed tomography revealed a mass in the upper part of stomach and swollen lymph nodes along with the lesser curvature and para-aortic lymph node. Biopsy could not confirm the definitive diagnosis. We performed total gastrectomy with para-aortic lymph node sampling. Histological analysis revealed squamous cell carcinoma with EBV infection with lymph node metastases. Tumor cells were positive for EBV-encoded small RNA (EBER) by in situ hybridization. The postoperative course was uneventful and the patient was discharged on day 11 after the operation. CapeOX was started as adjuvant chemotherapy, and the patient remains alive without recurrence 7 months after surgery.ConclusionThis is the first case report of EBV infection-associated primary SCC of the stomach diagnosed by in situ hybridization of EBER. EBV infection may be related to the pathogenesis of primary SCC. Further evidence and studies are required to establish optimal strategy for this rare disease.

Clinical Outcomes of Elderly Patients with Advanced-Stage Classic Hodgkin Lymphoma Who Received an ABVD Regimen: A Multi-Center Retrospective Study in Japan (HORIZON study)

Clinical Outcomes of Elderly Patients with Advanced-Stage Classic Hodgkin Lymphoma Who Received an ABVD Regimen: A Multi-Center Retrospective Study in Japan (HORIZON study)

Detection of Epstein-Barr virus encoded small RNA genes in oral squamous cell carcinoma and non-cancerous oral cavity samples

BackgroundEpstein-Barr Virus (EBV) is a human oncogenic virus that can lead to cancer in lymphoid and epithelial cells and is one of the hypothesized causes of oral cavity lesions including oral squamous cell carcinoma (OSCC), but the etiological association remains undetermined. The present investigation aimed to explore the EBV presence, viral load, and EBV-encoded small RNA (EBER) sequence variation in tissue samples of patients with OSCC and other oral cavity lesions including oral lichen planus (OLP), and oral irritation fibroma (OIF).MethodsIn total, 88 oral cavity samples (23 with OSCC, 29 with OLP, and 36 with OIF diagnosis) were examined by Real-Time PCR technique and some of them were sequenced.ResultsViral EBER sequence was detected in 6 out of the 23 OSCC (31.4%), 6 out of the 29 OLP (20.7%), and 3 out of the 36 OIF cases (8.3%). The mean EBV copy number was higher in OSCC samples (1.2 × 10−2 ± 1.3 × 10−2 copies/cell) compared to OLP (2.2 × 10−3 ± 2.6 × 10−3 copies/cell) and OIF (2.4 × 10−4 ± 2.0 × 10−4 copies/cell) samples, although this difference was not statistically significant (P = 0.318). The EBER gene was amplified and sequenced in 5 OSCC, 3 OLP, and 2 OIF samples with high EBV viral load. One OSCC, two OLP, and two OIF isolates showed different nucleotide variations compared with EBV-WT and AG876 prototype sequences: C6834T, C6870T, C6981T, C7085T, C7085G, and C7094T.ConclusionIn our study the presence of more than one genome copies per tumor cell indicates the possible role of EBV infection in oral cancers. However, more studies should be conducted to clarify the role of EBV in OSCC carcinogenesis.

S1928 Seeing the Forest for the Trees: Not All Colitis Is IBD

Introduction: Gastrointestinal involvement by Epstein-Barr virus (EBV) infection is rare, and almost always occurs in states of immunodeficiency or chronic inflammation. We present a case of EBV colitis in a healthy immunocompetent patient. Case description/methods: A 33-year-old man presented with 3 weeks of bloody diarrhea, fatigue, malaise, and weight loss. On exam, he had tender cervical lymphadenopathy. Labs showed leukocytosis of 19.2 K/UL (64% lymphocytes), hemoglobin of 11.5 g/dL, and elevated CRP of 23.8 mg/L. Liver enzymes were elevated, with ALT of 156 U/L, AST of 125 U/L, and alkaline phosphatase of 220 U/L. Albumin (4 g/dL) and platelets (221 K/UL) were normal. Tests for HIV, viral hepatitis, and stool bacteria, ova, and parasites were negative. Computed tomography showed splenomegaly, enlarged intraabdominal lymph nodes, and diffuse colon wall thickening with hyperemia. Colonoscopy revealed patchy ulcers, friability, and erythema in the colon, with normal intervening mucosa and terminal ileum. He was referred to the Inflammatory Bowel Disease (IBD) service, and initial pathology showed mild active chronic pancolitis. Due to symptom severity, he was started on IV methylprednisolone 20 mg twice daily and rapidly improved in 2 days. Final pathology showed positive EBV-encoded small RNA (EBER) in situ hybridization (50 cells/hpf) without evidence of an underlying lymphoproliferative disorder. Serologies showed acute EBV infection (IgM+, IgG-, PCR viral load 341 IU/mL), and liver biopsy showed EBV hepatitis. Infectious Disease (ID) agreed with supportive therapy. He was discharged on 5 days of prednisone 40 mg daily. On 1-week follow up, he was asymptomatic off steroids. Repeat colonoscopy will be performed in 3 months. Discussion: Acute EBV infection classically presents with lymphadenopathy, splenomegaly, and constitutional symptoms. EBV colitis is exceedingly rare in immunocompetent patients, especially in the absence of lymphoma, with only 3 published case reports. Our case highlights the importance of maintaining a holistic approach to arrive at a unifying diagnosis. EBV in colon biopsies is not always pathologic; serologies are needed to determine active or latent infection. Coordinating with ID is prudent. Management is typically supportive, given the self-limited course; the role of corticosteroids and antivirals remains unclear. Repeat colonoscopy with biopsies to evaluate mucosal healing and to rule out underlying IBD is recommended.Figure 1.: Figure 1A. 4.3 x 3.5 cm mass anterior to a large sigmoid diverticulum with Hounsfield units of 22-36 (red arrow). Hounsfield units of lipomas range from 30 to 70. Figure 1B. 4 cm pedunculated polyp 20 cm from the anal verge. Figure 1C. Microscopy depicting submucosal mass with mature white adipose tissue (red arrow).

Clinicopathologic Analysis of Primary Adrenal Diffuse Large B-Cell Lymphoma: A Reappraisal of 23 Japanese Patients Based on EBV Association and PD-L1 Expression in Tumor Cells.

Primary adrenal diffuse large B-cell lymphoma (PA-DLBCL) is rare. We investigate 23 Japanese patients with PA-DLBCL to understand the clinicopathologic features and biological behavior of this disease. The 17 males and 6 females had a median age of 74 years (range: 40 to 86 y). Tumor cells harbored Epstein-Barr virus-encoded small RNA (EBER) in 9 (39%) samples, including samples from the 2 patients with methotrexate-associated B-cell lymphoproliferative disorder. Programmed cell death ligand 1 (PD-L1) expression was detected in tumor cells of 6 (26%) samples, including 1 EBER+ and 5 EBER- samples. Four (17%) patients exhibited an intravascular proliferating pattern, and all 4 patient samples showed positive staining for PD-L1 in tumor cells. Among those patients, 3 showed intravascular proliferating pattern accompanied by a diffuse extravascular proliferation of tumor cells, and 1 patient was diagnosed with intravascular large B-cell lymphoma. We divided the 23 patients into 3 groups: EBER+ (n=9, 39%), EBER-PD-L1+ (n=5, 22%), and EBER-PD-L1- (n=9, 39%). A comparison of the outcomes among the 3 groups showed significant differences in overall survival (P=0.034). The EBER+ group had the worst prognosis, and the EBER-PD-L1- group had the best prognosis. We also compared the outcomes among the 3 groups that received rituximab-containing chemotherapies. Both the overall survival and progression-free survival were significantly different among these groups (P<0.001 and P=0.002, respectively). In conclusion, we evaluated 3 types of PA-DLBCL and found that each had unique clinical, pathologic, and prognostic features. Our results suggested that immune senescence, iatrogenic immunodeficiency, and immune evasion contribute to the development of PA-DLBCL.

EBV downregulates the m6A “writer” WTAP in EBV-associated gastric carcinoma

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is characterized by the clonal growth of EBV-infected stomach epithelial cells. It has been reported that N6-methyladenosine (m6A) methylation can regulate the splicing, expression, decay and translation of mRNAs. Wilms' tumor 1-associating protein (WTAP) is an m6A “writer” with methyltransferase activity. An m6A RNA methylation quantification kit and immunofluorescence (IF) showed that the m6A total RNA methylation level of the Epstein-Barr virus-negative gastric carcinoma (EBVnGC) cell line (SGC7901) was higher than that in the EBVaGC cell line (GT38). To investigate the underlying mechanism of the downregulated expression of m6A RNA methylation, we analyzed the expression of WTAP. The results showed that the expression of WTAP protein in EBVaGC cell lines was significantly lower than that in EBVnGC cell lines according to western blotting and IF. Through plasmid overexpression and RNA interference technology, we further found that EBV-encoded small RNA1 (EBER1) could downregulate WTAP expression by activating the NF-κB signaling pathway. In addition, WTAP could increase proliferation and inhibit migration in gastric carcinoma cell lines. In summary, EBER1 of EBV potentially regulated WTAP by affecting the NF-κB signaling pathway and WTAP further affected cell proliferation and migration.

Small intestinal follicular lymphoma induced by methotrexate: a case report

BackgroundMethotrexate-associated lymphoproliferative disorder (MTX-LPD) is a rare but critical complication that develops in patients treated with MTX. Although MTX-LPD has been recently reported, the incidence of follicular lymphoma in the intestine is very low.Case presentationA 73-year-old woman who had been receiving MTX for over 10 years visited our hospital complaining of postprandial abdominal pain and nausea. Upper and lower digestive tract endoscopies did not show any abnormal findings. A patency capsule was stagnated at the proximal part of the ileum with a mild dilation on the oral side. An oral balloon endoscopy revealed shallow ulcerative lesions in the jejunum. She was diagnosed with MTX-LPD based on histopathological findings. The symptoms did not improve with the discontinuation of MTX, and the patient required partial resection of the small intestine. The test result for Epstein-Barr virus-encoded small RNA was negative. She was diagnosed with follicular lymphoma based on the histology findings of a surgical specimen. Postoperative positron emission tomography-computed tomography and bone marrow aspiration did not show any findings of lymphoma. On follow-up, no recurrence was noted four years after the surgery.ConclusionsHerein, we report the first case of follicular lymphoma that occurred in the small intestine, negative for Epstein-Barr virus-encoded small RNA. If intestinal symptoms occur during MTX administration, it is important to directly observe by endoscopy and perform histological examination.

Computed tomography radiomics to predict EBER positivity in Epstein-Barr virus-associated gastric adenocarcinomas: a retrospective study.

The relevance of Epstein-Barr virus (EBV) in gastric carcinoma has been represented by the existence of EBV-encoded small RNA (EBER) in the tumor cells and has prognostic significance in gastric cancer, while gastric adenocarcinoma represents the most frequently occurring gastric malignancy. To observe the capacity of radiomic features extracted from contrast-enhanced computed tomography (CE-CT) images to differentiate EBER-positive gastric adenocarcinoma from EBER-negative ones. A total of 54 patients with gastric adenocarcinoma (EBER-positive: 27, EBER-negative: 27) were retrospectively examined. Radiomic imaging features were extracted from all regions of interest (ROI) delineated by two experienced radiologists on late arterial phase CT images. We distinguished related radiomic features through the two-tailed t test and applied them to construct a decision tree model to evaluate whether EBER in situ hybridization positive had appeared. Nine radiomics features were significantly related to EBER in situ hybridization status (P < 0.05), four of which were used to build the decision tree through backward elimination: Correlation_ AllDirection_offset7, Correlation_ angle135_offset7, RunLengthNonuniformity_ AllDirection_offset1_SD, and HighGreyLevelRunEmphasis_ AllDiretion_offset1_SD. The decision tree model consisted of seven decision nodes and six terminal nodes, three of which demonstrated positive EBER in situ hybridization. The specificity, sensitivity, and accuracy of the model were 84%, 80%, and 81.7%, respectively. The area under the curve of the decision tree model was 0.87. Radiomics based on CE-CT could be applied to predict EBER in situ hybridization status preoperatively in patients with gastric adenocarcinoma.

Virome Capture Sequencing in Castleman Disease Identifies Associations with Herpesviridae Family Members but No Novel Viruses

Castleman disease (CD) describes multiple distinct and heterogeneous disorders defined by common lymph node histopathology, including atrophic or hyperplastic germinal centers, prominent follicular dendritic cells, hypervascularization, polyclonal lymphoproliferation, and/or polytypic plasmacytosis. Unicentric CD (UCD) involves a solitary lymph node station that displays CD histopathology, whereas multicentric CD (MCD) involves multiple regions of enlarged nodes. Most UCD patients do not experience systemic symptoms and are successfully treated by removal of the enlarged node. In contrast, MCD involves constitutional symptoms, cytopenia, hepatosplenomegaly, fluid accumulation, cytokine storm-associated multiple organ system dysfunction, and is often fatal. In 1994, Kaposi sarcoma-associated herpesvirus/human herpesvirus-8 (HHV-8) was identified as the etiological driver of MCD in immunocompromised individuals. However, approximately 50% of MCD patients are HHV-8 negative (idiopathic MCD (iMCD)). Its idiopathic nature limits targeted treatment for iMCD patients who do not respond to anti-Interleukin-6 therapy. iMCD is itself heterogeneous, and recent work has identified at least one distinct sub-population of iMCD patients with a severe clinical presentation featuring thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly (iMCD-TAFRO). Several hypotheses have been proposed to explain the pathophysiology of iMCD and UCD, all of which remain untested. One notable hypothesis entertains that iMCD and UCD are in fact driven by viral infection–either HHV-8 that is not detected by current diagnostic methods, a novel virus, or a known but unanticipated virus. In support of the last possibility, there are several case reports of associations with Epstein-Barr virus (EBV), HHV-6 and Hepatitis B virus (HBV); however, neither a systematic study of known viruses, nor a search for novel viruses in CD has been reported.To this end, we employed Virome Capture Sequencing for Vertebrate Viruses (VirCapSeq-VERT),-a RNA-hybrid capture, deep sequencing and bioinformatics pipeline designed for viral discovery. In accordance with the Treaty of Helsinki, we obtained freshly frozen lymph node tissue from patients with iMCD (n=11) and UCD (n=12) as experimental conditions. HHV-8-positive MCD (n=2) was included as a positive control for HHV-8 infection. Lymph nodes from patients with lymphoma without clinical suspicion of HHV-8 infection (n=6) and an additional reactive lymph node were included as a negative control. HHV-8 was only detected in HHV-8-positive MCD and not in iMCD or UCD, consistent with prior clinical testing. Additionally, we did not identify any previously unknown viruses in iMCD or UCD lymph node tissue. Members of the herpesviridae family, including EBV, HHV-6 and HHV-7, were found in all conditions. Interestingly, in iMCD, the identification of herpesviridae family members was associated with relapsing disease and the more clinically severe iMCD-TAFRO variant. Additional numerous associations of unclear significance were made with viruses such as Human Endogenous Retrovirus Type K (HERV-K), transfusion transmitted virus/Torque teno virus (TTV), and Pegivirus.To investigate the significance of the EBV infection, we analyzed the number of EBV positive cells by in situ hybridization on lymph node tissue from a separate cohort of patients with CD and related disorders. The mean number of Epstein-Barr virus-encoded small RNAs (EBER) positive cells/high-powered fields (HPF) across 10 HPF was significantly lower in UCD (2 ± 2.3; n=9) and iMCD (1.7 ± 2.8; n=12) than in HHV-8 positive MCD (106.5 ± 75.6; n=2) and EBV-associated lymphoproliferation (63.5 ± 54.4; n=2) (p < 0.005 for all comparisons). The only iMCD-TAFRO case in this cohort had 4 EBER-positive cells/HPF. Further study is needed with larger cohorts of patients to validate the prognostic and potentially pathogenic significance of these associations, and in particular, the association of EBV and other herpesviridae family members with iMCD-TAFRO. DisclosuresFajgenbaum:Janssen Pharmaceuticals: Research Funding.

Guidelines in Clinical Practice: Appraisal of Quality Metrics in the Management of Newly Diagnosed Diffuse Large B-Cell Lymphoma Based on the American Society of Hematology Practice Improvement Module

Background: The National Comprehensive Cancer Network (NCCN) has issued well-established guidelines for the management and treatment of non-Hodgkin's lymphoma (NHL), in particular for diffuse large B-cell lymphoma (DLBCL). In 2014, the American Society of Hematology established practice improvement module (ASH PIM) incorporating quality metrics in six areas: pathologic diagnosis, staging, hepatitis B testing, use of growth factors, vaccination, and fertility counseling. Currently, such practice improvement modules have allowed physicians to monitor the quality of care in their practice, and reiterating the significance of these clinical guidelines. In an appraisal of quality metrics undertaken at Virginia Mason Medical Center (VMMC), Seattle, Washington, we sought to review four of the ASH PIM metrics and several other metrics to assess adherence to treatment guidelines for Diffuse Large B-Cell Lymphoma (DLBCL) and examine the need for institutional improvement.Methods: A longitudinal cohort of all patients who were diagnosed with DLBCL and received treatment at VMMC between January 1st, 2005 to December 31st, 2016 was identified using VMMC's Tumor Registry. Utilizing the electronic medical records, each patient's chart was reviewed for these quality metrics: pathologic diagnosis, staging of lymphoma, hepatitis and HIV serology testing, echocardiogram (ECHO) prior to starting treatment, clymphoma recurrence risk categorization, chemotherapy education, use of Rituximab therapy, G-CSF use, and post-therapy CT or CT-PET scan. Parameters for pathological diagnosis included assessment of expression of CD20, germinal center vs non-germinal center phenotype based on reporting of MUM1, CD10 and BCL-6, fluorescence in-situ hybridization (FISH) and IHC studies for the presence of traslocation/overexpression of c-Myc, BCL-2 or BCL-6, and EBV assessment by EBV-encoded small RNA1 (EBER1) in-situ hybridization. Staging was evaluated based on imaging in the form of a CT or PET-CT scan prior to treatment initiation and if a bone marrow biopsy (BMB) was also obtained. Serology testing for hepatis B (HBV) and hepatitis C (HCV), and HIV was reviewed. Patients receiving anthracycline-based treatments were assessed for having undergone an ECHOcardiogram prior to initiation of treatment. Post-treatment PET-CT was noted based on the presence of an internal report or outside records.Results: A total of 179 patients were included in this analysis. The median age of this cohort was 67 years (range 24 -91 years) and 54% of these patients were male. Pre-treatment viral serologic screening was performed in 87% of patients for HBV, 78% for HCVFISH f and 92% for HIV. All the specimen were uniformly tested for expression of CD20, and the germinal center vs. non-germinal center phenotype was determined in 97% of patients. c-Myc and BCL2/BCL6 data was available in only 32% of patients. For staging, all patients had imaging (CT/PET-CT), while bone marrow was obtained in only 72% of patients. Post-treatment imaging was available in 97% of cases. Additional data analysis of treatment-related quality metrics is underway, and shall be presented at the ASH annual meeting.Conclusion: Performing regular quality assessments by employing standardized metrics provide opportunities to improve the institutional quality of care for patients with newly diagnosed DLBCL, and allows to build and ensure better adherence to evolving NCCN patient-care guidelines ASH PIM. However, performance on the metrics relying on clinical documentation may not be as high as for those relying on objective testing. Whether such quality metrics affect real-time treatment-decisons and patient outcomes needs further evaluation. DisclosuresNo relevant conflicts of interest to declare.

A Novel Prognostic Model for Primary Gastric Diffuse Large B-Cell Lymphoma in the Rituximab Era, Integrating EBV Harboring on Tumor Cells

Introduction: EBV‐positive (EBV+) diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS) often shows an aggressive clinical course with frequent extranodal disease including gastrointestinal involvement. However, the clinicopathological and prognostic significance of the EBV+ disease among primary gastric DLBCL (gDLBCL) has not been confirmed. Recently targeted therapies using antibodies against programmed cell death 1 (PD-1) and its ligand (PD-L1) have shown great promise in the treatment of relapsed or refractory DLBCL, but there remains a need for reliable methods to identify the candidates for novel immune-therapies.We analyzed the clinicopathological characteristics of gDLBCL and shed the light on the EBV harboring on tumor cells and PD-L1 expression as prognostic indicators for the future clinical trials of PD-1 blockade.Patients and methods: A total of 157 patients previously diagnosed as primary gDLBCL and treated with rituximab-containing chemotherapies were retrospectively examined. All cases were tested for EBV-encoded small RNA (EBER) by in situ hybridization (ISH). The expression of PD-L1 was immunohistochemically evaluated (clone SP142, Spring Bioscience, Pleasanton, CA).Results: Our series consisted of 157 patients: 92 males and 65 females (male: female ratio = 1.4), with a median onset age of 67 years (range, 32-89 years). 66 patients (42%) showed Lugano stage Ⅱ2/ⅡE/Ⅳ, 59 (40%) with high levels of soluble interleukin -2 receptors (sIL-2R >1000U/ml), 45 (29%) with IPI HI/H, and 34 (22%) with B symptoms. 39 patients (38%) had multiple gastric lesions, 19 (12%) with the presence of Bulky mass, and 29 (60%) with Helicobacter pylori infection. 92 patients (61%) had a non-germinal center B-cell immunophenotype (non-GCB). All of 157 patients received multiagent chemotherapy combined with rituximab, with an additional irradiation in 55 (35%). EBV harboring was found on the tumor cells in 12 (7.6%). There was no significant difference in the clinicopathological findings between EBV+ and EBV‐negative (EBV−) subgroups. But, notably, the EBV+ patients had a significantly worse overall survival (OS) than EBV− ones (P=0.004). Multivariate analysis of survival revealed that EBER positivity (P=0.012), the presence of multiple gastric lesions (P=0.014), and IPI HI/H (P=0.017) were adverse independent prognostic factors in gDLBCL. We constructed a prognostic model by combining these prognostic variables as follows: patients with a score of 0 (n=43), no adverse factors; patients with a score of 1 (n=36), one factor; and patients with a score of 2/3 (n=24), two or three factors. The 5-year OS rates for these 3 groups were 100%, 91%, and 43%, respectively. Furthermore, this model significantly stratified gDLBCL patients separately by survival (P <0.001, Figure). Although none of gDLBCL cases expressed PD-L1 on tumor cells, 7 (78%) of 9 cases with EBV+ gDLBCL were positive for PD-L1 in ≥20% of nonmalignant stromal cells in microenvironment (miPD-L1+), which showed a higher frequency than in EBV− ones (78% vs 42%, P=0.13).Conclusions: EBV+ gDLBCL showed a significantly worse prognosis. Our prognostic model appeared to well define the clinical outcome of gDLBCL patients. PD-L1 expression in nonmalignant stromal cells was a common feature of EBV+ gDLBCL. They could be good candidates to benefit from anti-PD-1/PD-L1 therapy. A novel prognostic model by integrating EBV harboring on tumor cells may serve as a risk‐stratification for future clinical trials in which high‐risk patients can be treated with newly developing immune-therapies to improve their outcomes. [Display omitted] DisclosuresShimada:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria.

Self-regressing oral CD30-positive, EBV-negative, T-cell lymphoproliferative lesions. A poorly understood process highlighted by ominous clinicopathologic features and indolent behavior

Intraoral, primary, CD30-positive (CD30+) T-cell lymphoproliferative disorders (TLPDs) are uncommon, and their clinicopathologic presentation and management can vary and may be challenging. Herein, we present a retrospective study of 4 examples of self-regressing primary CD30+ TLPD affecting the gingiva. Archived files were retrospectively reviewed for oral CD30+ TLPDs featuring (1) proper immunohistochemical documentation, (2) Epstein-Barr virus negativity, (3) adequate follow-up information corroborating regression, and (4) no history of hematopoietic malignancy or related-mucocutaneous disease. Three women and 1 man (age range, 55-82 years; mean, 68.3 years) presented with rapidly growing gingival ulcers. Microscopic evaluation revealed diffuse infiltration by sheets of large, atypical cells admixed with lymphocytes and eosinophils, showing angiocentric distribution, focal neurotropism, and muscle infiltration. The lesional cells consistently stained for CD3 and CD30 and were variably immunoreactive against CD2, CD4, CD5, CD7, and CD8, but were negative for ALK1 and EBV-encoded small RNA. TCR-γ gene rearrangement studies revealed a monoclonal T-cell population in 1 case. All lesions showed complete regression 2 to 8 weeks postoperatively (mean follow-up, 4.5 weeks). Notwithstanding their alarming clinicopathologic appearance, there are CD30+ TLPDs confined to the oral cavity that have an indolent course. However, clinical staging is essential to exclude aggressive systemic malignancy.

Lymphoepithelial carcinoma of the parotid gland: a unique example showing p16 immunoreactivity.

Lymphoepithelial carcinoma (LEC) shows characteristic histology of nesting growth of tumor cells with unclear differentiation against the lymphoid stroma background. Although rare in salivary glands, it has previously been recognized as a type of undifferentiated carcinoma but is currently clearly defined as an independent disease separate from undifferentiated carcinoma. We report a case of LEC that developed in the parotid gland and was immunohistochemically positive for p16, which suggested the causative involvement of human papillomavirus (HPV). The patient was a 38-year-old Japanese male aware of mass formation in the left parotid area for 8years. Parotidectomy was performed and there have been no signs of recurrence or metastasis for 18month post-operation. The tumor was histologically typical except for Epstein-Barr virus (EBV)-encoded small RNA (EBER)-negative in situ hybridization (ISH), but p16-positivity by immunohistochemistry, and also frequent contact with extended and expanded pre-existing ductal structures. Although usually strongly associated with EBV infection, the tumor could be regarded to have eventually reached completion as a LEC lesion associated with HPV infection possibly through the pathway shared with squamous cell carcinoma. EBER-ISH remains the most promising index for confirming diagnosis of LEC, but EBV-negative result alone should not prevent diagnosis of LEC.

PLASMA EPSTEIN-BARR VIRUS (EBV) DNA AS A BIOMARKER FOR DIAGNOSIS OF SYRIAN EBV-POSITIVE BURKITT’S LYMPHOMA

Epstein-Barr virus - positive Burkitt’s Lymphoma is defined as the presence of Epstein-Barr virus (EBV) in tumor cells, the standard way to detect (EBV) in Burkitt’s Lymphoma is in-situ hybridization (ISH) of EBV-encoded small RNA (EBERs) in tumor cells. The present study aimed to evaluate plasma Epstein-Barr virus (EBV) DNA as a noninvasive biomarker for diagnosis and prognosis of EBV-positive Burkitt’s lymphoma. The study included 40 newly diagnosed patients with Burkitt’s lymphoma, ranging in age from 4 to 60 years, and 55 sex and age-matched controls. Forty formalin-fixed paraffin-embedded blocks of Burkitt’s lymphoma tissue samples were used to investigate the EBV by in-situ hybridization detection of the EBERs. Plasma EBV DNA was quantified by real-time quantitative polymerase chain reaction (PCR) for all Burkitt lymphoma patients prior to therapy and for control. The results showed that (22/40, 55%) of Burkitt lymphoma were positive for histological EBER, whereas plasma EBV DNA was detectable (range from 1.2×104 to 4.7×106 copies/mL) in all EBV-positive Burkitt lymphoma samples (22/22). EBV DNA was undetectable in all cases of EBV-negative Burkitt lymphomas (18/18) and all healthy control (55/55). It is worth mentioning that our results demonstrated that the EBV DNA load was significantly high in the EBV-positive BL patients suffering poor prognostic state. In conclusion: Plasma EBV-DNA can be used as a noninvasive biomarker for diagnosis and prognosis of EBV-positive Burkitt’s lymphoma.

Case report: histopathology and molecular pathology analysis on enteric tissue of a COVID-19 patient

AimsPatients with COVID-19 can also have enteric symptoms. Here we analyzed the histopathology of intestinal detachment tissue from a patient with COVID-19.MethodsThe enteric tissue was examined by hematoxylin & eosin stain, PAS (Periodic acid–Schiff) staining, Gram staining, Ziehl–Neelsen stain and Grocott’s Methenamine Silver (GMS) Stain. The distribution of CD3, CD4, CK20 and CD68, cytomegalovirus (CMV) and Herpes Simplex Virus (HSV) antigen were determined by immunohistochemistry. In situ hybridization (ISH) of SARS-CoV-2 and Epstein-Barr virus-encoded small RNA (EBER) were also performed.ResultsWe observed mucosal epithelium shedding, intestinal mucosal erosion, focal inflammatory necrosis with hemorrhage, massive neutrophil infiltration, macrophage proliferation accompanied by minor lymphocyte infiltration. Fungal spores and gram positive cocci but not mycobacteria tuberculosis were identified. Immunohistochemistry staining showed abundant CD68+ macrophages but few lymphocytes infiltration. HSV, CMV and EBV were negative. ISH of SARS-CoV-2 RNA showed positive signal which mostly overlapped with CD68 positivity.ConclusionsThe in situ detection of SARS-CoV-2 RNA in intestinal macrophages implicates a possible route for gastrointestinal infection. Further study is needed to further characterize the susceptibility of enteric cells to SARS-CoV-2 infection.