Detection Of Epstein-Barr Virus Research Articles

P137 Lesions of Cytomegalovirus and Epstein Barr virus of the proximal gastrointestinal tract in patients with Inflammatory Bowel Disease after COVID-19

BackgroundThe problem of opportunistic infections in IBD remains one of the most pressing issues in the diagnosis and treatment of this pathology. Particular attention among opportunistic infections in IBD is given to cytomegalovirus infection(CMV), the Epstein-Barr virus(EBV). Aim: To assess the frequency of detection of CMV and EBV in biopsies taken from the mucous membranes of the proximal gastrointestinal tract (GIT) in patients with IBD who underwent COVID-19.Methods72 patients with CD(40 w/32 m) and 69 patients with UC(39 m/30 w) who underwent COVID-19, in whom CMV and EBV were not previously detected, were examined. The age of the patients was from 18 to 62 years(39.4±2.7). All patients(p.) underwent a standard endoscopic examination(EE), disease activity was assessed in accordance with the ECCO regulations; the determination of CMV and EBV was carried out by PCR in biopsies of mucous membranes from the proximal GIT.ResultsEE in 27 p.with CD revealed deeper and more widespread ulcerative lesions of the gastric mucosa(CMV+11 p.40.7%/15.3%;EBV+12 p.44.4%/16.7 %;CMV and EBV+4 p.14.8%/5.5%), in 16 p.similar changes were detected in the esophagus(CMV+7 p.43.7%/9.7%,EBV+6 p.37.5%/8.3%;CMV and EBV+3 p.18.7%/4.2%),in 49 p.in the duodenal region(CMV+17 p.34.7%/23.6 %,EBV+23 p.46.9%/31.9%;CMV and EBV+9 p.18.4%/12.5%).EE with UC revealed deeper and more widespread ulcerative lesions of the gastric mucosa in 29 p. (CMV+12 p.41.4%/17.4%;EBV+13 p.44.8%/18.8%; CMV and EBV+4 p.3.8%/5.8%),in 19 p. similar changes were detected in the esophagus(CMV+9 p.47.7%/13.1%,EBV+6 p.31.6%/8.7%;CMV and EBV+4 p.21.1%/5.8%),in 44 p. in the duodenal region(CMV+17 p.38.6%/24.6%,EBV+23 p. 52.3%/33.3%;CMV and EBV+4 p.9.1%/5.8%).Thus, in patients with CD, when determining CMV in biopsy materials by PCR,an isolated positive result(48.6%) was obtained in 35 cases,for EBV(isolated)-41 cases(56.9%), in 16 pat. a combined lesion of CMV and EBV was revealed(22.2%); the localization of lesions was dominated by the duodenal region(68.1%),the stomach (37.5%)and the esophagus(22.2%). In patients with UC, when determining CMV in biopsy materials by PCR,an isolated positive result(55.1%) was obtained in 38 cases,for EBV(isolated)-42 cases(60.7%),in 12 pat. a combined damage to CMV and EBV(17.4%); the localization of lesions was dominated by the duodenal region(63.8%),the stomach(42%) and the esophagus(27.5%).The clinical and endoscopic picture indicates a more severe course of IBD in the presence of herpes viruses detected by PCR examination of biopsies taken from the mucous membranes of the proximal GIT.ConclusionThe findings allow us to recommend the need for more thorough inspection during endoscopy in patients with IBD who have undergone COVID-19.

Factors associated with the presence of the Epstein–Barr virus in the oral cavity of high school students from the city of Cali (Colombia)

IntroductionThe Epstein–Barr virus (EBV) is classified as a group 1 carcinogen. The main route of EBV transmission is oral, through saliva. The present study aimed to determine the frequency of EBV detection in the oral cavity in high school students in the city of Cali (Colombia). Materials and methodsAnalytical cross-sectional study in order to determine the frequency of EBV detection in the oral cavity, the reasons for its prevalence and its association with several factors, in 1565 individuals. The variables analyzed were sociodemographic factors, oral hygiene, oral health, sexual behavior, cigarrete smoking and alcohol intake. The association between the EBV detection and the variables evaluated was done through a generalized linear regression model with logarithmic linkage and Poisson distribution with robust variance. ResultsThe percentage of exposure to EBV in the oral cavity was 38.40% (CI 95%: 36.02–40.84). The frequency of presenting EBV exposure was 22% higher in men and the risk increased according to sexual behaviour. An inverse association with the school grade was found: the eleventh-grade participants had 27% less frequency of exposure to EBV than the lower grades (sixth to eighth). When analyzing the logistic model to study the association between EBV detection and independent variables, the association was overestimated. The overestimation ranged from 27% to 47% depending on the type of variable. ConclusionsThe frequency of EBV detection in the oral cavity of healthy students was similar to that previously described. Factors associated to sexual behavior increased the risk of opportunity to be exposed to EBV.

Kaposi's Sarcoma-Associated Herpesvirus, but Not Epstein-Barr Virus, Co-infection Associates With Coronavirus Disease 2019 Severity and Outcome in South African Patients.

In South Africa, the Coronavirus Disease 2019 (COVID-19) pandemic is occurring against the backdrop of high Human Immunodeficiency Virus (HIV), tuberculosis and non-communicable disease burdens as well as prevalent herpesviruses infections such as Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). As part of an observational study of adults admitted to Groote Schuur Hospital, Cape Town, South Africa during the period June–August 2020 and assessed for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, we measured KSHV serology and KSHV and EBV viral load (VL) in peripheral blood in relation to COVID-19 severity and outcome. A total of 104 patients with PCR-confirmed SARS-CoV-2 infection were included in this study. 61% were men and 39% women with a median age of 53 years (range 21–86). 29.8% (95% CI: 21.7–39.1%) of the cohort was HIV positive and 41.1% (95% CI: 31.6–51.1%) were KSHV seropositive. EBV VL was detectable in 84.4% (95% CI: 76.1–84.4%) of the cohort while KSHV DNA was detected in 20.6% (95% CI: 13.6–29.2%), with dual EBV/KSHV infection in 17.7% (95% CI: 11.1–26.2%). On enrollment, 48 [46.2% (95% CI: 36.8–55.7%)] COVID-19 patients were classified as severe on the WHO ordinal scale reflecting oxygen therapy and supportive care requirements and 30 of these patients [28.8% (95% CI: 20.8–38.0%)] later died. In COVID-19 patients, detectable KSHV VL was associated with death after adjusting for age, sex, HIV status and detectable EBV VL [p = 0.036, adjusted OR = 3.17 (95% CI: 1.08–9.32)]. Furthermore, in HIV negative COVID-19 patients, there was a trend indicating that KSHV VL may be related to COVID-19 disease severity [p = 0.054, unstandardized co-efficient 0.86 (95% CI: –0.015–1.74)] in addition to death [p = 0.008, adjusted OR = 7.34 (95% CI: 1.69–31.49)]. While the design of our study cannot distinguish if disease synergy exists between COVID-19 and KSHV nor if either viral infection is indeed fueling the other, these data point to a potential contribution of KSHV infection to COVID-19 outcome, or SARS-CoV-2 infection to KSHV reactivation, particularly in the South African context of high disease burden, that warrants further investigation.

A nanofluidic preconcentrator integrated with an aluminum-based nanoplasmonic sensor for Epstein-Barr virus detection

Epstein-Barr virus (EBV) positivity is one of the indexes for diagnosing nasopharyngeal carcinoma (NPC). Moreover, systemic inflammatory responses can easily be triggered in patients who are both EBV- and coronavirus disease 2019 (COVID-19)-positive. Development of rapid and highly sensitive EBV screening methods has become important. In this study, a nanofluidic preconcentrator integrated with a nanoslit Fano resonance biosensor was developed to detect latent membrane protein 1 (LMP1) for an EBV diagnosis. Through nanoimprinting and aluminum deposition, the low-cost nanoslit plasmonic sensing chip can be mass-produced. The nanoporous membrane was patterned on a sensing chip as an ion selective channel to concentrate LMP1 proteins. Anti-LMP1 immunoglobulin G was then modified to a sensing chip to immunosense LMP1. The Fano resonant spectrum of the capped nanoslit array produced a transmission peak followed by a dip. We recorded and analyzed the spectrum using four methods, including area, center of mass, peak value, and dip value methods. With preconcentration, a limit of detection (LOD) of 100 pg/ml and a sensing range of 100 pg/ml to 10 µg/ml was achieved using the peak value.

Identifying optimal clinical trial candidates for locoregionally advanced nasopharyngeal carcinoma: Analysis of 9468 real-world cases and validation by two phase 3 multicentre, randomised controlled trial

Background and purposeThis study aims to identify the optimal high-risk candidates for clinical trials in locoregionally advanced nasopharyngeal carcinoma (NPC). Materials and methodsNon-metastatic NPC patients (n = 9,468) were included. Recursive partitioning analyses (RPA) were performed to generate risk stratification. Receiver operating characteristics curve was used to determine the cut-off value of pre-treatment Epstein-Barr virus (EBV) DNA for progression-free survival (PFS). Individual-level data from two clinical trials were used for validation. ResultsAnatomic stratification based on T and N category (eighth edition TNM, TNM-8) classified the N2-3 or T4 as an anatomic high-risk group with 5-year PFS of 69% (95% confidence interval: 68–71%). Prognostic stratification identified patients with pre-treatment EBV DNA ≥4000 copies/mL as a prognostic high-risk group with 5-year PFS of 69% (67–70%). The c-index was significantly higher for anatomic stratification (0.621, p < 0.001) and prognostic stratification (0.585, p < 0.001) compared with existing TNM-8 stage groups (0.562). The validation cohorts based on clinical trials data showed greater PFS benefit than the results of the original trials [Hazard ratio: NCT01245959, 0.64 vs. 0.67; NCT01872962, 0.42 vs. 0.52]. Moreover, detectable post-treatment EBV DNA indicated a high risk of progression with 5-year PFS of 38.7% and was the most adverse independent factor for all endpoints. ConclusionsN2-3 or T4 NPC patients were ideal candidates for multicenter clinical trials in locoregionally advanced NPC. Patients with detectable post-treatment EBV DNA are suitable candidates for adjuvant trials.

Detection of Epstein-Barr virus in gastric adenocarcinoma: qPCR and FISH comparison

EBV-associated gastric cancer accounts for about 10% of all gastric carcinomas worldwide. We aimed to verify the prevalence of EBV in gastric adenocarcinoma samples using FISH and qPCR and comparing the results obtained by both techniques. Gastric cancer samples from 191 cases were analyzed. The FISH assay was performed to detect small EBV RNAs (EBER1) and qPCR was performed to detect the EBV-EBNA-1 gene region. Cohen’s kappa index and the chi-square test were used to compare the methodologies and investigate correlations with the clinical-pathological data of the gastric adenocarcinoma patients. Most of the patients were men, and the average age was 60 years. The intestinal subtype cancer presented more aggressive stages with 90% of patients having a reactive FISH for EBV (EBV+), although the virus infection frequency in epithelial gastric tissue was only 1%. No positive association with clinicopathological features and EBV+ was found by FISH. Using qPCR analysis, the percentage of positive samples was lower (52.4%), and a positive association was found in samples from older patients (> 60 years). Interestingly, 71 qPCR-negative cases were detected by FISH in the presence of non-epithelial cells and in 10 qPCR-positive cases with no evidence of EBV according to FISH. The concordance between the two techniques was low, with only 57.6%. FISH is more informative for associating the gastric carcinoma with EBV positivity in tumor/epithelial cells; however, qPCR can provide relevant information regarding the progression and characteristics of neoplasia.

Clinical Significance of Peripheral Blood EBV-DNA Determination and Genotyping in Lymphoma Patients

To explore the clinical significance of Epstein-Barr virus（EBV） detection and classification in peripheral blood of lymphoma patients. 101 lymphoma patients were enrolled, the clinical characteristics of the patients were collected, including ages, sex, types of lymphoma, Ann Arbor stages, extranodal infiltration and lactate dehyhrogenase. Fluorescent quantitative PCR technology was used to detect the EBV-DNA. Polymerase chain reaction and Agarose gel electrophoresis was used for determination of EB genotyping. The difference between curative effect in EBV-DNA+ and EBV-DNA－ patients, the correlation of adverse factors and EBV infection of the patients were analyzed. 68.3% (69/101) of the patients showed EBV-DNA positive. EBV-positive lymphoma patients showed more adverse prognostic factors than the patients with EBV-negative, which may lead to poorer disease outcome. Among the 46 B-cell non-Hodgkin's lymphoma patients, the overall response rate of EBV-positive patients (60.7%) was lower than EBV-negative patients(88.9%) (P<0.05); For 19 patients with Hodgkin's lymphoma, the overall response rate of EBV-positive patients (46.2%) was lower than EBV-negative patients (100%), the differences were statistically significant (P<0.05). Among 69 patients with EBV-infected lymphoma, 98.6% (68/69) showed type-2 EB virus, and 1.4% (1/69) were type-1 and type-2 mixed infections. Most of EBV-positive in lymphoma patients were EBV type 2, patients with EBV-DNA+ shows poorer efficacy than EBV-DNA－ patients.

Quantitative Off-Target Detection of Epstein-Barr Virus–Derived DNA in Routine Molecular Profiling of Hematopoietic Neoplasms by Panel-Based Hybrid-Capture Next-Generation Sequencing

Epstein-Barr virus (EBV) is associated with hematologic and solid tumors. We utilized a hybridization capture-based next-generation sequencing (NGS) platform targeting 400 genes associated with hematological malignancies to detect and quantify nontargeted viral-derived EBV reads that aligned to the EBV reference contig (NC_007605). We evaluated 5234 samples from 3636 unique patients with hematological neoplasms and found that 100 samples (1.9%) in 93 unique patients had ≥6 EBV reads (range, 6 to 32,325; mean, 827.5; median, 54). Most (n=73, 73%) represented known EBV-associated conditions, and the most common was post-transplant lymphoproliferative disorders (n=21, 29%). Documented EBV viremia was found in 4 of 27 samples with a moderate quantity of EBV reads and conditions not known to be EBV associated, whereas suspected viremia or low-level activation was likely in the remaining 23 samples. A good correlation (Spearman r=0.8; 95% CI, 0.74-0.85) was found between EBV reads by NGS and systematic semiquantitative EBV-encoded RNA in situ hybridization in 162 available samples, particularly at greater EBV involvement. An optimal threshold for significant morphologic EBV involvement was found to be≥10 reads by the receiver operating characteristic analysis (area under the curve, 0.990; 95% CI, 0.9974%-1.000%). Thus, in addition to mutational analysis, hybrid-capture-based NGS panels can detect and quantitate off-target EBV-derived viral DNA, which correlates well with morphology.

Simultaneous occurrence of Epstein-Barr virus (EBV) in periodontal pockets and in oral squamous cell carcinoma: a cross-sectional study.

This study aimed to investigate the detection of Epstein-Barr virus (EBV) in oral squamous cell carcinoma (OSCC) and to verify the concordance of EBV-DNA frequency in subgingival sites and in the OSCC. A cross-sectional study with 30 OSCC patients, aged from 44 to 88years old, was conducted. Samples were collected in subgingival sites and at the OSCC, then submitted to DNA isolation, qPCR, and genotyping. Descriptive statistic was performed to report the frequency of EBV-DNA in all samples, and McNemar test was applied to verify the concordance among the EBV-DNA frequency in both sites. The individuals presented 62years old in average, and the majority were male (66.6%). EBV-DNA was detected in 56.7% OSCC lesions. Among the subgroup of 19 dentate individuals, high concordance (73.7%) in both EBV-DNA detection and the absence in subgingival sites and OSCC was observed, and it was statistically significant (p < 0.05). We report the notable occurrence of EBV-DNA in OSCC; also, the presence of EBV in periodontal sites may contribute to find it in OSCC, although the possible contribution of EBV in the OSCC remains to be investigated. The identification of this easily accessible site of EBV latent infection may help to improve the patient's quality of life by maintenance of oral/periodontal health condition and preventing further possible disorders related to the virus, and also encourages new approaches for investigating EBV, periodontitis, and OSCC.

Establishment and Characterization of a Human Primary Myelofibrosis Cell Line

Aims: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal proliferation of stem cells and myeloid cells. JAK2, CARL, and MPL mutations could be detected in most patients, but 15% of PMF patients could be absent of these three gene mutations. PMF has shorter median survival time and poorer prognosis when compared with closely related MPNs, polycythemia vera (PV) and essential thrombocythemia (ET). Allogenic hematopoietic stem cells (allo-HSCT) is the only curative therapy for PMF patients, however transplantation-related complications and deaths were observed in more than 50% patients. Hydroxyurea and Ruxolitinib are commonly used drugs to improve clinical symptoms of PMF patients, but lack of anti-tumor activities. These drugs could not reverse bone marrow fibrosis or induce cytogenetic remission. Basic research work of PMF is limited as to the lack of human PMF cell line. Here, we aimed to establish and characterize a patient-derived human PMF cell line, which would provide a useful tool for PMF research and screenings for novel drugs.Methods: The bone marrow cells were obtained from a 61-year-old male patient, who was diagnosed as PMF for 2 years. Mono-nuclear cells were isolated and cultured in a continuous culture system. The cells were characterized by different methods, including STR profiling, morphology observation with Giemsa staining, flow cytometry, chromosome analysis, Epstein-Barr virus (EBV) detection, mycoplasma detection, whole-genome-exon sequencing, and cell proliferation assay.Results: The cells have continued to grow in liquid culture for more than 50 passages using the same culture conditions. Doubling time was about 48-72 hours. The STR profiling identified that the cultured cells and the patient's bone marrow cells were from the same origin. The cells are free from EBV and mycoplasma. Giemsa-Wrights staining showed primitive erythrocytes (Figure A), which was consistent with CD71 positive detected by flow cytometry. Chromosome analysis revealed a hyperdiploid karyotype, which was 47, XY,+12[1]/48,idem,+9[9]. Whole-genome-exon sequencing identified mutations of ASXL1, TP53, IKZF1, IDH1, FLT3, and TET1 (Figure B). Now this cell line, designated ZYXY-M2, is collected by China center for type culture collection (CCTCC).Discussion: Established cell lines are invaluable tools in cell biology. Right now, HEL and SET-2 are two cell lines that are most widely used in MPN-related researches. HEL is an erythroleukemia cell line which was derived from a patient with leukemia transformation of Hodgkin's disease, and SET-2 is a megakaryoblastic cell line which was derived from a patient with leukemic transformation of ET. Here, we established a cell line, ZYXY-M2, derived from a PMF patient and it would prove to be a valuable model for the study of PMF. ZYXY-M2 could help to explore the pathogenesis and development of PMF, novel drug screening, and may also help to set up xenograft PMF mouse models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Association between Epstein-Barr virus and periodontitis: A meta-analysis.

PurposePrevious studies have found that Epstein-Barr virus (EBV) is associated with periodontitis, though some controversy remains. This meta-analysis aimed to clarify and update the relationship between EBV and periodontitis as well as clinical parameters.MethodsA comprehensive search was conducted in the PubMed and Scopus databases in December 2020. Original data were extracted according to defined inclusion and exclusion criteria. Outcomes were analyzed, including overall odds ratios (ORs) and 95% confidence intervals (CIs). A random-effects model was used, and publication bias was assessed by Egger’s and Begg’s tests. Sensitivity analysis was used to evaluate the stability of the outcome.ResultsTwenty-six studies were included in the present meta-analysis, involving 1354 periodontitis patients and 819 healthy controls. The included studies mostly showed high quality. The overall quantitative synthesis for the association between EBV and periodontitis was an increased odds ratio when subgingival EBV was detected OR = 7.069, 95% CI = 4.197–11.905, P<0.001). The results of subgroup analysis suggested that the association of EBV with periodontitis was significant in Asian, European, and American populations (P<0.001; P = 0.04; P = 0.003, respectively) but not in African populations (P = 0.29). Subgroup analysis by sample type showed that subgingival plaque (SgP), tissue and gingival crevicular fluid GCF were useful for EBV detection (P<0.001). EBV detection amplification methods included nested PCR, multiplex PCR and PCR (P<0.001; P = 0.05, P<0.001, respectively), but EBV detection by real-time PCR and loop-mediated isothermal amplification presented no significant result (P = 0.06; P = 0.3, respectively). For the clinical parameters of periodontitis, pocket depth (PD) and bleeding of probing (BOP) percentages were higher in the EBV-positive sites than in the EBV-negative sites (MD 0.47 [0.08, 0.85], P = 0.02; MD 19.45 [4.47, 34.43], P = 0.01).ConclusionsA high frequency of EBV detection is associated with an increased risk of periodontitis. The EBV association was particularly significant in all populations except in African populations. Subgigival plaque (SgP), tissue and GCF were not significantly different useful material for detecting EBV in periodontitis. Nested PCR and multiplex PCR are reliable methods for this purpose. In the presence of EBV, PD and BOP are reliable clinical parameters for gingival inflammation. However, some caution in such interpretation is justified due to heterogeneity among studies. A suggested extension could assess the parallel influence of other human herpesviruses.

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.

Continuous polymerase chain reaction microfluidics integrated with a gold-capped nanoslit sensing chip for Epstein-Barr virus detection

We present the first combination of a microfluidic polymerase chain reaction (PCR) with a gold nanoslit-based surface plasmon resonance (SPR) sensor for detecting the DNA sequence of latent membrane protein 1 (LMP1). The PCR microchannel was produced through a laser scribing technique, and the SPR nanoslit chip was manufactured via hot-embossing nanoimprinting lithography. Afterward, the LMP1 DNA probe was adsorbed onto the SPR chip of the integrated device through electrostatic interactions for further detection. The device can complete the analytical procedure in around 36 min, while the traditional machine requires 105 min to achieve similar signals under the same PCR thermal cycles. The calibration curve with serially diluted LMP1 DNA exhibited the accuracy (R2 > 0.99) and sensitivity (limit of detection: ∼10−11 g/mL) of the device. Moreover, extracted DNA from Epstein-Barr virus (EBV)-positive cells were directly detected through the integrated chip. In brief, this all-in-one chip can amplify gene fragments at the front-end and detect them at the back-end, decreasing the time required for the analysis without compromising accuracy or sensitivity. We believe this label-free, real-time, low-cost device has enormous potential for rapid detection of various viruses, such as EBV and COVID-19.

Epstein-Barr Virus Latent Membrane Protein-1 Expression in Nasopharyngeal Carcinoma.

PURPOSENasopharyngeal carcinoma (NPC), a malignant neoplasm of the epithelium covering the nasopharynx, is a rare disease in most parts of the world. Epstein-Barr virus (EBV), the most potent oncogenic virus, coupled with environmental and genetic factors has been identified to play a role in the development of NPC. An array of methods for detecting the virus do exist, from serologic detection of antibodies to DNA amplification. There is paucity of local data on the status of EBV infection in relation to NPC within the region, and this study attempts to shed more light on the subject.METHODSThis was a retrospective cross-sectional laboratory-based study on histologically confirmed, archived tissues from July 2015 to June 2019. Immunohistochemistry expression of latent membrane protein-1 (LMP-1) was used to detect EBV infection in the tissues.RESULTSA total of 71 cases were enrolled in this study. The mean age was 47.87 years ± 16.84 years with a male-to-female ratio of 1.5:1. There was a unimodal distribution of EBV detection, with the peak (26.8%) at 36-45 years. About 45.1% of the 71 samples tested positive for LMP-1, all of which were nonkeratinizing carcinoma. Nonkeratinizing carcinoma was the most common histopathologic subtype (n = 67; 94.4%), with the majority (38 of 67; 56.7%) being undifferentiated and 29 of 67 (43.3%) differentiated. Keratinizing and basaloid subtypes had two cases each, representing 2.8%.CONCLUSIONA significant proportion of NPC, particularly nonkeratinizing histologic subtype, seems to show LMP-1 positivity by immunohistochemistry, which may be adopted in resource-constrained settings to detect EBV infection in these tissue biopsies.

Rapid Internal Control Reference Recombinase-Aided Amplification Assays for EBV and CMV Detection

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 ( P < 0.05), respectively. In comparison with those of qPCR, the sensitivity of the EBV and CMV ICR-RAAs using the DNA by thermal lysis was 72.22% and 80.00%, respectively; the specificity was 100.00%; and the Kappa values were 0.764 and 0.878 ( P < 0. 05), respectively. Thus, rapid and specific detection of EBV and CMV is possible using ICR-RAA assays.

Development and validation of deep learning classifiers to detect Epstein-Barr virus and microsatellite instability status in gastric cancer: a retrospective multicentre cohort study

SummaryBackgroundResponse to immunotherapy in gastric cancer is associated with microsatellite instability (or mismatch repair deficiency) and Epstein-Barr virus (EBV) positivity. We therefore aimed to develop and validate deep learning-based classifiers to detect microsatellite instability and EBV status from routine histology slides.MethodsIn this retrospective, multicentre study, we collected tissue samples from ten cohorts of patients with gastric cancer from seven countries (South Korea, Switzerland, Japan, Italy, Germany, the UK and the USA). We trained a deep learning-based classifier to detect microsatellite instability and EBV positivity from digitised, haematoxylin and eosin stained resection slides without annotating tumour containing regions. The performance of the classifier was assessed by within-cohort cross-validation in all ten cohorts and by external validation, for which we split the cohorts into a five-cohort training dataset and a five-cohort test dataset. We measured the area under the receiver operating curve (AUROC) for detection of microsatellite instability and EBV status. Microsatellite instability and EBV status were determined to be detectable if the lower bound of the 95% CI for the AUROC was above 0·5.FindingsAcross the ten cohorts, our analysis included 2823 patients with known microsatellite instability status and 2685 patients with known EBV status. In the within-cohort cross-validation, the deep learning-based classifier could detect microsatellite instability status in nine of ten cohorts, with AUROCs ranging from 0·597 (95% CI 0·522–0·737) to 0·836 (0·795–0·880) and EBV status in five of eight cohorts, with AUROCs ranging from 0·819 (0·752–0·841) to 0·897 (0·513–0·966). Training a classifier on the pooled training dataset and testing it on the five remaining cohorts resulted in high classification performance with AUROCs ranging from 0·723 (95% CI 0·676–0·794) to 0·863 (0·747–0·969) for detection of microsatellite instability and from 0·672 (0·403–0·989) to 0·859 (0·823–0·919) for detection of EBV status.InterpretationClassifiers became increasingly robust when trained on pooled cohorts. After prospective validation, this deep learning-based tissue classification system could be used as an inexpensive predictive biomarker for immunotherapy in gastric cancer.FundingGerman Cancer Aid and German Federal Ministry of Health.

Abstract 2264: Molecular detection and genotyping of human papillomavirus in presence of some oncogenic viruses among Egyptian breast cancer women

Abstract Introduction High risk human papillomaviruses (HR-HPVs) has a possible role in development and pathogenesis of breast cancer (BC) disease. Other oncogenic viruses showed to be linked to BC such as Epstein Barr virus (EBV), and Human mammary tumor virus (HMTV). Presence of high risk HPV genotypes, EBV or HMTV could affect the severity course of BC. Therefore, the aim of the current study is to explore distribution of HPV with regards to its genotypes and the presence of EBV and HMTV among Egyptian BC women with its correlation to age and hormonal status. Patients &amp; Methods This study was conducted on 40 fresh tissues of BC patients and blood samples from 30 apparently healthy controls. All samples were investigated for detection of HPV, EBV and HMTV DNAs using qualitative PCR. Detection of HPV viral load was performed using quantitative real time PCR. HPV genotypes were determined on 10 purified positive PCR product using Sanger sequencing of HPV L1 region. The obtained sequences were compared to the known HPV DNA sequences at National center for biotechnology information (NCBI) and/or Papillomavirus Episteme (Pave) data bases using their online BLAST and taxonomy tools. Data were correlated with age and hormonal status. Results All the tested 3 oncogenic viruses were not detected into 30 controls. HPV, EBV and HMTV were detected in 20 (50%), 23 (57.5%), 28 (70%) respectively of the 40 BC patients by using PCR assay. HPV median load of the 20 positive samples was 4800 copies/μL. Seven different HPV genotypes were detected included high risk genotypes ((HPV 18 in 3 samples), 70 and 52), low risk genotype (11) detected into only one sample along with 3 other genotypes (102, 114 and 151). One sample showed no significant similarity with any sequence registered in the used databases. Presence of EBV and HMTV DNAs and high risk HPV genotypes was more associated with young age (lower than 50 years), P value = 0.015, 0.001 and 0.05, respectively but were not associated with hormonal status of BC patients. Conclusion HMTV was the most detected viral DNAs among BC patients, High risk HPVs were identified into 10% of studied Egyptian BC patients and were associated with young age. Further larger sample size study is needed to evaluate role of HPV genotypes on pathogenesis and outcome of BC disease. STDF Acknowledgment This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant No.22944. Citation Format: Nasra F Abdel Fattah, Shimaa A Metwally, Samah A Loutfy, Maha A Abo-Shadi, Ahmed B Barakat, Omar A Rabee, Ahmed M Osman, Amany M Helal, Tarek M Hashem, Manar M Moneer. Molecular detection and genotyping of human papillomavirus in presence of some oncogenic viruses among Egyptian breast cancer women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2264.

Ebv-Mir-BART11-3p, Ebv-Mir-BART9 and Ebv-Mir-BART10 Are Present in the Plasma of HIV-Infected Subjects with Lymphoma and Are Potential Lymphoma Biomarkers

Epstein Barr virus (EBV) has been implicated in the lymphomagenesis, particularly of HIV-related lymphomas. EBV-infected cells are able to share miRNAs with other cells via exosomes and certain EBV-miRNAs were found in cells not infected with the virus but from patients with increased levels of EBV copies in plasma such as HIV-infected individuals. Thus, EBV-miRNAs might be detected in the plasma and might be relevant for the diagnosis and follow-up of HIV-related lymphomas.This case-control study included a group of 26 HIV-infected patients with lymphoma that responded to treatment and a control-group of HIV-infected subjetcs without lymphoma matched for age, gender, CD4-lymphocyte counts, plasma HIV viremia and time from HIV infection. We evaluated the presence of 43 EBV-miRNAs in frozen plasma samples from patients with lymphoma at diagnosis (Dx-group, N=26), one year before diagnosis (Pre-group, N=15) and complete response (CR-group, N=26). RNA was extracted from 200µl of plasma using Trizol LS (Ambion, Carlsbad, CA) and cel-miR-39 was used as spike-in. The detection of EBV-miRNAs was assessed by QRT-PCR in a BioMark™ HD System of Fluidigm (San Francisco, CA) using TaqMan® microRNA assays and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA). The following clinical and biological parameters were collected from records: age, gender, ECOG score, extranodal and bulky disease, B symptoms, Ann-Arbor stage, serum lactate dehydrogenase (LDH) and beta2-microglobulin, International Prognostic Index (IPI), HCV and HBV serology, history of opportunistic infection and of AIDS-defining illnesses, onset of combination antiretroviral therapy, CD4-lymphocyte counts, plasma HIV viremia. McNemar's and Wilcoxon tests were used for paired comparisons between groups. P-values of less than .05 were considered statistically significant.Overall, 30 EBV-miRNAs were detected in the Dx-group whereas only 8 EBV-miRNAs where found in the control-group and 19 in the CR-group. EBV-miRNAs were detected more frequently in the Dx-group (17 out of 26) than in the control-group (7 out of 26) (P =.002) and in the CR-group (11 out of 26) (P=.02). The presence of 6 EBV-miRNAs (ebv-miR-BART11-3p, ebv-miR-BART9, ebv-miR-BART10, ebv-miR-BART15, ebv-miR-BART31, and ebv-miR-BART19) could be associated with the lymphoma condition, since they were detected significantly different in the Dx-group and the control-group (Figure 1). Importantly, 3 of those EBV-miRNAs (ebv-miRBART11-3p, ebv-miR-BART9 and ebv-miRBART10) were found to be significantly less frequent in the CR group, reinforcing the idea that they could be derived from lymphoma cells and be used as biomarkers for diagnosis and follow up of HIV-related lymphoma. EBV-miRNAs cannot be used as early biomarkers of lymphoma in HIV-infected patients because none of the 3 EBV-miRNAs associated with lymphoma was significantly found in samples before diagnosis. The detection in plasma of ebv-miR-BART11-3p, ebv-miR-BART10 or ebv-miR-BART9 was not associated with having tumor cells infected with EBV (Figure 1). Only the detection of ebv-miR-BART9 was associated with detectable EBV loads [7/14 (50%) vs. 1/12 (8.3%), P=.036] (Figure 1). The following were the statistically significant associations between the presence of the 3 potential EBV-miRNA lymphoma biomarkers and the clinical-biological features studied. The presence of ebv-miR-BART10 in plasma was associated with IPI>2 [8/14 (57.1%) vs. 1/9 (11.1%), P=.004] and ECOG>2 [8/14 (57.1%) vs. 1/11 (9.1%), P=.011]. Also, more patients with ebv-miR-BART11-3p expression in plasma had ECOG>2 than patients without it [7/13 (53.8%) vs. 1/12 (8.3%), P=.030].Ebv-miR-BART11-3p, ebv-miR-BART9 and ebv-miR-BART10 can be detected in the plasma of patients with HIV-related lymphoma, independently of the presence of EBV in tumor cells or of EBV plasma viremia. They are potential biomarkers of lymphoma to be used in the diagnosis and follow up of HIV-infected subjects with the advantage of being easily determined by QRT-PCR employing noninvasive procedures.This work was supported in part by 2014 SGR225 (GRE) from CERCA Programme/Generalitat de Catalunya, and by funds from Josep Carreras International Foundation, “la Caixa” Foundation, DKMS, and Celgene Spain. [Display omitted] DisclosuresBaptista:DKMS: Research Funding; la Caixa Foundation: Research Funding; Carreras International Foundation: Research Funding; CERCA Programme/Generalitat de Catalunya: Research Funding; Celgene Spain: Research Funding. Sancho:Roche: Honoraria; Laboratorios Servier: Consultancy; Mundipharma: Honoraria; Kern Pharma: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Ribera:Celgene: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Amgen Inc.: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Incyte: Research Funding, Speakers Bureau; ARIAD: Research Funding, Speakers Bureau; Roche: Honoraria. Navarro:Celgene Spain: Research Funding; DKMS: Research Funding; la Caixa Foundation: Research Funding; Josep Carreras International Foundation: Research Funding; CERCA Programme/Generalitat de Catalunya: Research Funding.

EBV Reactivation Incidence and Severity in Multiple Sclerosis Patients Treated with Autologous Haematopoietic Stem Cell Transplant and Comparing with Aplastic Anaemia Treated with ATG—Single Site Experience

INTRODUCTION: Autologous haematopoietic stem cell transplant(HSCT) with anti-thymocyte globulin (ATG) based conditioning is currently being explored for its efficacy and safety in the treatment of Multiple Sclerosis (MS). Despite increasing experience gained, the procedure still carries significant risk of morbidity. Epstein Barr Virus (EBV) infection and its role in pathogenesis of MS remain undetermined. EBV reactivation and post-transplant lymphoproliferative disorders (PTLD) are well described in allogeneic HSCT and uncommon with auto HSCT for other malignant conditions. Similarly patients with Aplastic anaemia (AA) treated with standard immunosuppressive therapy with ATG/ciclosporin also have increased risk of EBV reactivation but reports of PTLD in this group are very rare. The incidence of clinically significant EBV reactivation in MS patients undergoing HSCT is increasingly recognised. This retrospective study reports rates of EBV reactivation, PTLD incidence and associated clinical sequelae in patients with MS undergoing HSCT as compared with AA patients receiving ATG in our centre.PATIENTS AND METHODS: 36 patients with MS had AHSCT between Feb 2012 and 2017. 35 were conditioned using standard protocol of cyclophosphamide and ATG (2.5mg/kg/day for 3 days), and one with BEAM-ATG. A Comparator group of 40 patients with AA consecutively treated with ATG (40mg/kg/day for 4 days) between Sept 2013 and Nov 2016 was selected. Prior exposure to EBV (EBV IgG) was assessed pre-HSCT. EBV levels monitoring was performed on whole blood samples by quantitative polymerase chain reaction (PCR) as per standard centre protocol. Data was collected retrospectively.RESULTS: All patients had previous EBV exposure. All MS patients with relapsing remitting (n=22) and primary/secondary progressive (n=14) disease were previously treated with median 2 (range, 0-6) disease modifying therapies (DMT) and 23 had at least 1 highly active DMTs. Median age of 43yrs (range, 22-64yrs) versus 52yrs (range, 19-68yrs) in AA group, with 35 patients severe/very severe AA and 5 non-severe treated with ATG. Where follow up data was available there was no difference in EBV reactivation rates (80.6% vs 65% P-0.13). The median time to EBV detection was 28 days (IQ range, 19-41d) vs 11 days (IQ range 5-39 d)with a maximal EBV DNA titre at median of 39 days (IQ range 29.5-49.5d) vs 11.5 days(IQ Range 0-43.5) in MS and AA cohort respectively.Patients were stratified into their maximum level of reactivation based on the amount of EBV copy numbers; 0-100,000 copies, 100,000 - 500,000 copies and >500,000 copies. In MS group (Table 1), 8 patients had symptomatic EBV reactivation; defined as persistent fever, lymphadenopathy and/or B symptoms. 10 (34.5%) MS patients had EBV copies>500,000. 3 of these had findings consistent with PTLD on CT scan, however none had histological diagnosis. 3 other MS patients had worsening neurological symptoms with one presenting as pseudo-relapse. All 6 of these patients were treated with anti-CD20 monoclonal therapy (Rituximab; 375mg/m2 weekly for 4 weeks) with rapid resolution of their symptoms. MS patients were more likely to develop high EBV (>500k) levels (p-0.03) and receiver operating characteristics (ROC) curve analysis (Fig 1) confirmed high EBV levels>500,000 correlate with high sensitivity 85.5%, specificity 82.5% (p-0.004) for clinical events requiring treatment. No significant rituximab related adverse events noted in treated group. As expected, no significant symptomatic EBV and no PTLD events were noted in AA cohort and none required any rituximab therapy for the EBV reactivations.Significantly raised paraproteinaemia was observed in 21 (72.4%) of MS cohort compared to 3 (11.5%) in the AA group (p < 0.01). 3 of the MS cohort experienced paraprotein levels greater than 20 g/L and one (IgG 61.1g/L) developed hyper-viscosity requiring plasma exchange and developed neurological symptoms mimicking a MS relapse.CONCLUSION: EBV reactivation was observed to occur commonly in patients with MS with significant symptoms and risk of neurological sequelae and PTLD events in this group, which is unlike the AA cohort treated with comparable ATG doses. Regular EBV monitoring in first 3 months should be mandated in MS patients undergoing HSCT and we recommend cut-off of 500,000 copy numbers as trigger for consideration of pre-emptive rituximab therapy. [Display omitted] DisclosuresPotter:Jazz: Honoraria; Pfizer: Other: Advisory board. Raj:Celgene: Speakers Bureau; Mallinckrodt: Speakers Bureau; MSD: Speakers Bureau; Bloodwise: Research Funding. Pagliuca:Bluebird: Honoraria; Gilead: Honoraria; Astellas: Consultancy, Speakers Bureau; Pfizer: Honoraria; Merck: Honoraria, Research Funding; Jazz: Honoraria; Basilea: Honoraria.

Examining the dynamics of Epstein-Barr virus shedding in the tonsils and the impact of HIV-1 coinfection on daily saliva viral loads.

Epstein-Barr virus (EBV) is transmitted by saliva and is a major cause of cancer, particularly in people living with HIV/AIDS. Here, we describe the frequency and quantity of EBV detection in the saliva of Ugandan adults with and without HIV-1 infection and use these data to develop a novel mathematical model of EBV infection in the tonsils. Eligible cohort participants were not taking antiviral medications, and those with HIV-1 infection had a CD4 count >200 cells/mm3. Over a 4-week period, participants provided daily oral swabs that we analysed for the presence and quantity of EBV. Compared with HIV-1 uninfected participants, HIV-1 coinfected participants had an increased risk of EBV detection in their saliva (IRR = 1.27, 95% CI = 1.10-1.47) and higher viral loads in positive samples. We used these data to develop a stochastic, mechanistic mathematical model that describes the dynamics of EBV, infected cells, and immune response within the tonsillar epithelium to analyse potential factors that may cause EBV infection to be more severe in HIV-1 coinfected participants. The model, fit using Approximate Bayesian Computation, showed high fidelity to daily oral shedding data and matched key summary statistics. When evaluating how model parameters differed among participants with and without HIV-1 coinfection, results suggest HIV-1 coinfected individuals have higher rates of B cell reactivation, which can seed new infection in the tonsils and lower rates of an EBV-specific immune response. Subsequently, both these traits may explain higher and more frequent EBV detection in the saliva of HIV-1 coinfected individuals.