Downregulation of KLF5 by EBER1 via the ERK signaling pathway in EBV-positive nasopharyngeal carcinoma cells: implications for latent EBV infection.

Nasopharyngeal carcinoma (NPC) carcinogenesis and malignant transformation are intimately associated with Epstein-Barr virus (EBV) infection. A zinc-fingered transcription factor known as Krüppel-like factor 5 (KLF5) has been shown to be aberrantly expressed in a number of cancer types. However, little is known about the regulatory pathways and roles of KLF5 in EBV-positive NPC. Our study found that KLF5 expression was significantly lower in EBV-positive NPC than in EBV-negative NPC. Further investigation revealed that EBER1, which is encoded by EBV, down-regulates KLF5 via the extracellular signal-regulated kinase (ERK) signalling pathway. This down-regulation of KLF5 by EBER1 contributes to maintaining latent EBV infection in NPC. Furthermore, we uncovered the biological roles of KLF5 in NPC cells. Specifically, KLF5 may influence the cell cycle, prevent apoptosis, and encourage cell migration and proliferation - all of which have a generally pro-cancer impact. In conclusion, these findings offer novel strategies for EBV-positive NPC patients' antitumour treatment.

Pseudouridylation of Epstein-Barr virus noncoding RNA EBER2 facilitates lytic replication.

Epstein-Barr virus (EBV) expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2, which are preserved in all clinical isolates of EBV, thus underscoring their essential function in the viral life cycle. Recent epitranscriptomics studies have uncovered a vast array of distinct RNA modifications within cellular as well as viral noncoding RNAs that are instrumental in executing their function. Here we show that EBER2 is marked by pseudouridylation, and by using HydraPsiSeq the modification site was mapped to a single nucleotide within the 3' region of EBER2. The writer enzyme was identified to be the snoRNA-dependent pseudouridine synthase Dyskerin, which is the catalytic subunit of H/ACA small nucleolar ribonucleoprotein complexes, and is guided to EBER2 by SNORA22. Similar to other noncoding RNAs for which pseudouridylation has a positive effect on RNA stability, loss of EBER2 pseudouridylation results in a decrease in RNA levels. Furthermore, pseudouridylation of EBER2 is required for the prolific accumulation of progeny viral genomes, suggesting that this single modification in EBER2 is important for efficient viral lytic replication. Taken together, our findings add to the list of RNA modifications that are essential for noncoding RNAs to implement their physiological roles.

Loss of viral genome with altered immune microenvironment during tumour progression of Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) is one of the major drivers of gastric carcinogenesis. EBV infection is established before tumour initiation and is generally maintained throughout tumour development; however, the significance of EBV in tumour maintenance and progression remains to be elucidated. Here, we report eight cases of EBV-associated gastric carcinoma (EBVaGC) with intratumoural heterogenous expression of EBV-encoded small RNA (EBER), a highly expressed latent gene of EBV, and demonstrate clinicopathological characteristics of these rare cases. By performing detailed histological assessment of EBER-positive and -negative components of each case, detection of EBV genome in tumour cells by fluorescence in situ hybridisation, TP73 methylation analysis, whole exome sequencing, and targeted gene panel sequencing, we identified tumours in two patients to be collision tumours of different origins. In the other six patients, some genetic/epigenetic alterations were shared between EBER-positive and -negative components, suggesting that EBV was eliminated from tumour cells during progression. Interestingly, in both tumour types, programmed death ligand 1 and intratumoural infiltration of CD8+ T lymphocytes were lower in EBER-negative than in EBER-positive components, suggesting an immunogenic role of EBV. To the best of our knowledge, this study is the first to demonstrate the detailed histological features and genetic/epigenetic alterations in EBVaGC with heterogenous EBER expression; the loss of EBV may benefit tumour progression and immune evasion and might be clinically important for selecting treatment strategies for such cancers. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Epstein-Barr virus noncoding RNA EBER2 transactivates the UCHL1 deubiquitinase to accelerate cell growth.

The Epstein-Barr virus (EBV) transforms resting B cells and is involved in the development of B cell lymphomas. We report here that the viral noncoding RNA EBER2 accelerates B cell growth by potentiating expression of the UCHL1 deubiquitinase that itself increased expression of the Aurora kinases and of cyclin B1. Importantly, this effect was also visible in Burkitt's lymphoma cells that express none of the virus's known oncogenes. Mechanistically, EBER2 bound the UCHL1 messenger RNA (mRNA), thereby bringing a protein complex that includes PU.1, a UCHL1 transactivator, to the vicinity of its promoter. Although the EBV oncogene LMP1 has been suggested to induce UCHL1, we show here that EBER2 plays a much more important role to reach significant levels of the deubiquitinase in infected cells. However, some viruses that carried a polymorphic LMP1 had an increased ability to achieve full UCHL1 expression. This work identifies a direct cellular target of a viral noncoding RNA that is likely to be central to EBV's oncogenic properties.

Frequent expression of PD-L1 in lymphocyte-rich hepatocellular carcinoma: A report of 4 cases.

BACKGROUND: Programmed death ligand 1 (PD-L1) is an immune checkpoint inhibitor. PD-L1 binds to its receptor programmed death receptor (PD-1) expressed by immune cells and plays a key role in regulating immune responses. Engagement of PD-L1 on cancer cells and PD-1 on immune cells avoid destruction of tumour cells by immune cells. Immunostaining with PD-L1 has been suggested as a biomarker predictive of antiPD-L1 immunotherapy. Lymphocyte-rich hepatocellular carcinoma (LrHCC) is a rare histological HCC subtype which is characterised by neoplastic epithelial cells intermixed with numerous immune cells. METHODS: Here in we investigated immunohistochemical PD-L1 expression in 4 cases of LrHCC. Tumour proportion score (TPS) and immune cell score was recorded. Immunophenotypic characterization of the tumour and inflammatory cells was also done. Epstein-Barr encoding region (EBER) in situ hybridization (ISH) assay as performed in all four tumours. RESULTS: Expression of PD-L1 was demonstrated in tumour epithelial cells and immune cells in all four cases. Incomplete to membranous staining was demonstrated in the tumour cells. Tumour proportion score (TPS) was 1.2-20 %. Immune cells demonstrated membranous and cytoplasmic immunostaining. Immune cell score was ≥1 % to >10 %. CONCLUSION: PD-L1 expression in both tumour and immune cells suggests distinct immunogenic feature and potential role of antiPD-L1 therapies in cases with inoperable disease.

Disparity analysis and prognostic value of pretreatment whole blood Epstein-Barr virus DNA load and Epstein-Barr encoding region status in lymphomas: A retrospective multicenter study in Huaihai Lymphoma Working Group.

Elevated Epstein-Barr virus (EBV) DNA load is common in lymphomas. However, it remains unclear whether the disparity in viral load and its prognostic value in lymphomas are correlated with Epstein-Barr encoding region (EBER) status. In this retrospective multicenter study, we collected the data of pretreatment whole blood EBV DNA (pre-EBV DNA) and EBER status and evaluated their disparity and prognostic values in lymphomas. A total of 454 lymphoma patients from December 2014 to August 2020 were retrospectively retrieved. Mann-Whitney U test, Kruskal-Wallis test and Bonferroni's adjustment were used to explore the disparity of EBV DNA and EBER status in lymphomas. Time-dependent receiver operating characteristic analysis and MaxStat analysis were used to determine optimal cutoff points of pre-EBV DNA load. Univariable and multivariable Cox proportional hazards models were established for the estimation of prognostic factors. The positive rate of EBV DNA in natural killer T-cell lymphoma (NKTL) patients was higher than that in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and Hodgkin lymphoma (HL) patients, and the median positive pre-EBV copy number of NKTL was also higher than that of FL and DLBCL. EBV DNA could clearly distinguish the prognosis of DLBCL, NKTL, HL and peripheral T-cell lymphoma, and the integration of EBER status and EBV DNA could differentiate the prognosis of HL patients. Multivariable results revealed that pre-EBV DNA load had an effect on the prognosis of NKTL, FL and DLBCL. The status of pre-EBV DNA and EBER were disparate. Whole blood pre-EBV DNA predicted the prognosis of lymphomas, and the combination of EBV and EBER status could differentiate the prognosis of HL.

Epstein-Barr virus EBER1 and murine gammaherpesvirus TMER4 share conserved in vivo function to promote B cell egress and dissemination.

The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4) establish life-long latency in circulating B cells. The precise determinants that mediate in vivo gammaherpesvirus latency and tumorigenesis remain unclear. The EBV-encoded RNAs (EBERs) are among the first noncoding RNAs ever identified and have been the subject of decades of studies; however, their biological roles during in vivo infection remain unknown. Herein, we use a series of refined virus mutants to define the active isoform of MHV68 noncoding RNA TMER4 and demonstrate that EBV EBER1 functionally conserves this activity in vivo to promote egress of infected B cells from lymph nodes into peripheral circulation.

Kinase Activity of PAR1b, Which Mediates Nuclear Translocation of the BRCA1 Tumor Suppressor, Is Potentiated by Nucleic Acid-Mediated PAR1b Multimerization.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell-cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

Virus-Mediated Inhibition of Apoptosis in the Context of EBV-Associated Diseases: Molecular Mechanisms and Therapeutic Perspectives.

Epstein-Barr virus (EBV), the representative of the Herpesviridae family, is a pathogen extensively distributed in the human population. One of its most characteristic features is the capability to establish latent infection in the host. The infected cells serve as a sanctuary for the dormant virus, and therefore their desensitization to apoptotic stimuli is part of the viral strategy for long-term survival. For this reason, EBV encodes a set of anti-apoptotic products. They may increase the viability of infected cells and enhance their resistance to chemotherapy, thereby contributing to the development of EBV-associated diseases, including Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), gastric cancer (GC), nasopharyngeal carcinoma (NPC) and several other malignancies. In this paper, we have described the molecular mechanism of anti-apoptotic actions of a set of EBV proteins. Moreover, we have reviewed the pro-survival role of non-coding viral transcripts: EBV-encoded small RNAs (EBERs) and microRNAs (miRNAs), in EBV-carrying malignant cells. The influence of EBV on the expression, activity and/or intracellular distribution of B-cell lymphoma 2 (Bcl-2) protein family members, has been presented. Finally, we have also discussed therapeutic perspectives of targeting viral anti-apoptotic products or their molecular partners.

CARMIL2 Immunodeficiency with Epstein Barr Virus Associated Smooth Muscle Tumor (EBV-SMT). Report of a Case with Comprehensive Review of Literature.

Background: Primary immunodeficiency (PID) having defects related to lymphocyte cytotoxic pathway or T-cell dysfunction are well known for developing opportunistic infections and Epstein-Barr virus (EBV)-associated diseases. CARMIL2 deficiency is a recently described combined immunodeficiency (CID) disorder characterized by defective CD28-mediated T cell co-stimulation, altered cytoskeletal dynamics, susceptibility to various infections and Epstein Barr Virus smooth muscle tumor (EBV-SMT). Case report: We report a homozygous CARMIL2 pathogenic variant presenting with recurrent infections and EBV associated smooth muscle tumor (SMT) in a child. Conclusion: The present study reports that EBV SMT may occur in a child with CARMIL2 deficiency.

[Study of gastric adenocarcinomas association with the Epstein-Barr virus-encoded small RNAs]. / Issledovanie assotsiatsii adenokartsinom zheludka s malymi virusnymi RNK virusa Epshteina­Barr.

OBJECTIVE: 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. MATERIAL AND METHODS: 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. RESULTS: 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). CONCLUSION: Separately taken EBER-status is not a significant independent prognostic factor and can be considered only in conjunction with the «classical¼ clinical and morphological characteristics, primarily with the stage of the tumor process, since it is they that determine the prognostic properties of the tumor.

[PD-L1 expression in EBV-associated gastric carcinomas]. / Ekspressiya PD-L1 v EBV-assotsiirovannom rake zheludka.

OBJECTIVE: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Detection of Epstein-Barr virus encoded RNA in fixed cells and tissues using CRISPR/Cas-mediated RCasFISH.

Identification of Epstein-Barr virus (EBV)-infected cells is critical for the diagnosis and clinical management of EBV-associated diseases. EBV-encoded RNA (EBER) located in the nucleus is a reliable marker due to its high levels of expression and inherent stability in tissue specimens. EBER in situ hybridization has long been the gold standard for detecting tumor-associated latent EBV infection and is valuable in determining the primary site and radiation fields of EBV-related malignancies. However, reliable detection is somewhat restricted by diffused signal and time-consuming procedure of this method, especially when proteins and RNA needed to be labeled simultaneously. Here, we optimized and validated our CRISPR-dCas9 mediated in situ RNA imaging tool-RCasFISH that enabled us to detect EBER rapidly and was compatible with IHC methods in fixed cells and tissue sections. Our approach could provide an attractive alternative for the molecular diagnosis of latent EBV infection.

PD-L1 and IDO1 are potential targets for treatment in patients with primary diffuse large B-cell lymphoma of the CNS.

BACKGROUND: Programmed cell death 1 (PD-1) and its ligands PD-L1 and PD-L2, as well as Indoleamine 2,3-deoxygenase (IDO1) can be expressed both by tumor and microenvironmental cells and are crucial for tumor immune escape. We aimed to evaluate the role of PD-1, its ligands and IDO1 in a cohort of patients with primary diffuse large B-cell lymphoma of the CNS (PCNSL). MATERIAL AND METHODS: Tissue microarrays (TMAs) were constructed in 45 PCNSL cases. RNA extraction from whole tissue sections and RNA sequencing were successfully performed in 33 cases. Immunohistochemical stainings for PD-1, PD-L1/paired box protein 5 (PAX-5), PD-L2/PAX-5 and IDO1, and Epstein-Barr virus encoding RNA (EBER) in situ hybridization were analyzed. RESULTS: High proportions of PD-L1 and PD-L2 positive tumor cells were observed in 11% and 9% of cases, respectively. High proportions of PD-L1 and PD-L2 positive leukocytes were observed in 55% and 51% of cases, respectively. RNA sequencing revealed that gene expression of IDO1 was high in patients with high proportion of PD-L1 positive leukocytes (p = .01). Protein expression of IDO1 in leukocytes was detected in 14/45 cases, in 79% of these cases a high proportion of PD-L1 positive leukocytes was observed. Gene expression of IDO1 was high in EBER-positive cases (p = .0009) and protein expression of IDO1 was detected in five of six EBER-positive cases. CONCLUSION: Our study shows a significant association between gene and protein expression of IDO1 and protein expression of PD-L1 in the tumor microenvironment of PCNSL, possibly of importance for prediction of response to immunotherapies.

EBER in situ hybridization in subcutaneous aluminum granulomas/lymphoid hyperplasia: A diagnostic clue to differentiate injection-associated lymphoid hyperplasia from other forms of pseudolymphomas and cutaneous lymphomas.

BACKGROUND: Subcutaneous vaccination or desensitization may induce persistent nodules at the injection sites. Without the knowledge of prior injection, histopathological work-up may be challenging. OBJECTIVE: Aim of this study was to contribute to the histopathological work-up of unclear subcutaneous nodules, especially their differentiation from cutaneous lymphoma. METHODS: We retrospectively reviewed clinical data and histopathological slides of four patients with subcutaneous nodules, which were suspected to suffer from cutaneous T- or B-cell lymphoma. Sections of these cases and 12 negative controls were stained with hematoxylin and eosin and a standardized immunohistochemical panel of B- and T-cell markers including EBER in situ hybridization as well as electron microscopy. RESULTS: In all cases, large histiocytes with granular cytoplasm compatible with intracellular aluminum hydroxide were present. EBER in situ hybridization revealed positive staining of these granular histiocytes while staining was absent in negative controls. LIMITATIONS: Post hoc completion of medical history revealed that vaccination or specific immunotherapy had been applied before at the biopsy site in only three out of four patients; one patient was lost to follow-up. CONCLUSION: EBER in situ hybridization is an adjunctive tool to differentiate aluminum-induced granuloma/lymphoid hyperplasia from other forms of pseudolymphoma and cutaneous B- or T-cell lymphomas.

Epstein-Barr Virus and Helicobacter pylori co-infection in patients with gastric cancer and duodenale ulcer.

We aimed to detect EBV/Hp (Epstein-Barr Virus/Helicobacter pylori) co-infection by determining the number of copies of EBV/EBER-1 in the gastric biopsy samples of the Hp (+) GC, peptic ulcer (PU), and non-ulcer dyspepsia (NUD) cases. The patient group (PG), with 34 patients (34 GC and 30 PU patients) and a control group with 40 NUD cases were included. All patients and controls were Hp positive. EBV/EBNA-1 IgG were measured by the Anti-EBNA-1 ELISA IgG kit. Determination and quantification of EBV/EBER-1 gene region was performed by qPCR. EBV/EBER-1 positivity was 35.29% (12/34), 6.6% (2/30) and 2.5% (1/40) in GC, PU and 40 NUD cases, respectively. A significant difference was found between the GC and NUD cases (p=0.001). A significant difference was found between the groups for mean EBV/EBER-1 copy numbers (p=0.019). No significant difference was found between GC and the NUD cases (p=0.1455) for EBV/EBNA-1 IgG antibody positivity. EBV/EBER-1 positivity (OR=3.319), and age ≥55 years old (OR=2.331) were found to be a significant in multivariate logistic regression. In conclusion, our data suggest that the GC risk by EBVand Hp co-infection increased 3.3 times.

Prevalence and characteristics of Epstein-Barr virus associated gastric carcinoma in Gansu Province, Northwest China with mRNA expression of glycoprotein BMRF2.

Gansu province is a region with the highest gastric cancer incidence and mortality in Northwest China. Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is an important subtype of gastric cancer which shows specific clinicopathological features such as older-age bias, male predominance, lower lymph-node-metastasis, and a better cancer-related survival comparing to EBV-negative gastric cancers. However, the prevalence of EBVaGC has never been studied in Gansu Province, Northwest China. The present study investigated the incidence, characteristics, and EBV messenger RNA (mRNA) profile of EBVaGC in this area. We have collected 270 stomach samples from gastric cancer patients and analyzed the presence of EBV DNA and EBV-encoded small RNAs (EBERs) by nested polymerase chain reaction (PCR) and in situ hybridization, respectively. The EBV mRNA profiling was investigated by quantitative reverse transcription PCR (qRT-PCR). EBV DNA was detected in 51/95 patients (53.7%), while EBER transcripts were detected in 18/270 patients (6.7%). EBER positivity was significantly associated with older age and less lymph node metastasis, but no obvious association with gender or histological type of tumors. The expression of EBV genes was observed with different patterns, and the mRNA of glycoprotein BMRF2 was detected in EBVaGC. The present study showed unique clinicopathological features and mRNA expression patterns of EBVaGC in Gansu Province, Northwest China, suggesting that geographic variation can contribute to new epidemiological features in EBVaGC. The transcript of glycoprotein BMRF2 was observed consistently in EBVaGC, which may serve as a biomarker and play a role in the pathogenesis of EBVaGC in Gansu Province, Northwest China.

Epstein-Barr virus-associated enteritis with multiple ulcers: The first autopsy case.

Epstein-Barr virus (EBV)-associated enteritis is extremely rare and has not been well characterized. Herein, we present the first autopsy case of EBV-associated enteritis with multiple ulcers in a 73-year-old Japanese male. The patient had abdominal pain and was clinically diagnosed with enteritis. An endoscopic examination revealed multiple ulcers at the terminal ileum. His condition worsened due to serosanguinous bowel discharge and the patient was then admitted to the hospital. Ileocecal and subtotal small intestinal resection was performed for repetitive hemorrhage from ulcers. However, the patient died due to uncontrolled hemorrhage. An autopsy was then performed in order to explore the cause of ulcers in the small intestine. Macroscopic findings revealed multiple ulcers with occasional cobblestone-like appearance of the ileum. Histological analysis revealed marked infiltration of lymphocytes and plasma cells around the ulcer. EBV-encoded RNA in situ hybridization (EBER-ISH) revealed positive inflammatory cells. Cytomegalovirus was immunohistochemically negative. Macroscopic and microscopic findings obtained from autopsy specimens showed no foci of inflammation and EBER-ISH-positive stromal cells in the esophagus, stomach, and colorectum. EBV-associated enteritis can cause uncontrolled repetitive hemorrhage from ulcers and result in critical condition of the patient, which can be used for differential diagnosis.

EBV-encoded RNA1-positive cells in the bone marrow specimens of patients with EBV-negative lymphomas and sarcomas.

Epstein-Barr virus (EBV) infection is associated with pathogenesis of various cancers, including extranodal natural killer/T-cell lymphoma, nasal type (ENKL). ENKL tumor cells are positive for EBV-encoded RNA1 (EBER1), which is the most useful marker to identify ENKL tumor cells in histopathology. Currently, EBER1 in situ hybridization (ISH) is recommended to evaluate bone marrow (BM) involvement of ENKL. However, the actual burden of EBER1-positive cells in normal BM specimens remains unclear. In the present study, we performed EBER1 ISH on 111 BM specimens, which were obtained during an initial staging procedure in patients with EBV-negative cancers and were also negative for BM involvement. One or more EBER1-positive cells per whole specimen were observed in 38 specimens (34%). The number of EBER1-positive cells was distributed as follows: single positive cell, n = 17; two positive cells, n = 13; three positive cells, n = 3; and four positive cells, n = 5. These findings suggest that four or fewer EBER1-positive cells can be observed in BM specimens of patients with non-EBV-related cancers. The clinical implications of a small number of EBER1-positive cells in BM specimens of patients with ENKL should be evaluated in further studies.

Clinical Significance of BCL2, C-MYC, and BCL6 Genetic Abnormalities, Epstein-Barr Virus Infection, CD5 Protein Expression, Germinal Center B Cell/Non-Germinal Center B-Cell Subtypes, Co-expression of MYC/BCL2 Proteins and Co-expression of MYC/BCL2/BCL6 Proteins in Diffuse Large B-Cell Lymphoma: A Clinical and Pathological Correlation Study of 120 Patients.

Background: Clinical significance of germinal center B-cell (GCB) and non-GCB sub-categorization, expression of MYC, BCL2, BCL6, CD5 proteins and Epstein Barr virus encoded RNA (EBER) positivity in diffuse large B-cell lymphoma (DLBCL) remain controversial. Could these biomarkers accurately identify high risk DLBCL patients? Are MYC, BCL2 and BCL6 proteins expression feasible as baseline testing to predict c-Myc, BCL2 or BCL6 gene rearrangements? Aims: To investigate prognostic values of GCB/non-GCB sub-categorization, Double Protein Expression Lymphoma (DPL), Triple Protein Expression Lymphoma (TPL), positivity of CD5 protein and EBER in patients with DLBCL disease. To evaluate correlation between BCL2 , c-Myc and BCL6 gene rearrangements with BCL2, MYC and BCL6 proteins expression. Methods: Diagnostic tissue samples of 120 DLBCL patients between January 2012 to December 2013 from four major hospitals in Malaysia were selected. Samples were subjected to immunohistochemical staining, fluorescent in-situ hybridization (FISH) testing, and central pathological review. Pathological data were correlated with clinical characteristics and treatment outcome. Results: A total of 120 cases were analysed. Mean age of diagnosis was 54.1 years ± 14.6, 64 were males, 56 were females, mean follow up period was 25 months (ranged from 1 to 36 months). Of the 120 cases, 74.2% were non-GCB whereas 25.8% were GCB, 6.7% were EBER positive, 6.7% expressed CD5 protein, 13.3% were DPL and 40% were TPL. The prevalence of c-Myc, BCL2, BCL6 gene rearrangements were 5.8%, 5.8%, and 14.2%, respectively; and 1.6% were Double Hit Lymphoma (DHL). EBER positivity, DPL, TPL, c-Myc gene rearrangement, BCL2 gene rearrangement, extra copies of BCL2 gene and BCL6 gene rearrangement were associated with shorter median overall survival (P<0.05). IPI score was the significant determinants of median overall survival in DPL and TPL (P<0.05). CD5 protein expression and GCB/non-GCB sub-categorization did not affect treatment outcome (P>0.05). Overall, c-Myc, BCL2 and BCL6 gene rearrangements showed weak correlation with expression of MYC, BCL2 and BCL6 proteins (P>0.05). Fluorescent in situ hybridization is the preferred technique for prediction of treatment outcome in DLBCL patients. Conclusion: c-Myc, BCL2, and BCL6 gene rearrangements, EBER expression, DHL, TPL and IPI score are reliable risk stratification tools. MYC, BCL2 and BCL6 proteins expression are not applicable as baseline biomarkers to predict c-Myc, BCL2, and BCL6 gene rearrangements.