Epstein-Barr Virus Infection Status Research Articles

Infectious mononucleosis complicated by transitory Epstein-Barr virus infection of T and natural killer cells.

Epstein-Barr virus (EBV) typically infects B cells in infectious mononucleosis (IM), but a rare case shows EBV infection in T cells. Seven cases of lymphoproliferative disorder caused by EBV-positive cytotoxic T/natural killer (NK) cell proliferation in the lymph nodes, termed IM with transient EBV infection of T and NK cells (EBV + T/NK cells in IM), are reported here. The purpose of the study is to describe clinicopathological features of EBV + T/natural killer (NK) cells in IM of the lymph node. We retrospectively analysed seven cases of Chinese children and young people adults with EBV + T/NK cells in IM. We used morphological observation, immunohistochemical staining, EB virus in situ hybridisation detection, and analysis of T-cell receptor gene rearrangement. The patients were healthy prior to illness, experiencing sudden onset occurring in all the patients, with high fever as the first symptom, followed by lymphadenopathy and hepatosplenomegaly. Diagnosis occurred < 1.5months of symptom onset. Most lymphocytes in lesions expressed CD3 and Granzyme B or TIA-1 and lacked CD5. CD56 was expressed in numerous cells in 5 of the 7 cases. EBV-encoded RNA (EBER) was detected in medium-to-large-sized cells (50-100 cells per cell/high-power field). T-cell receptor (TCR) gene rearrangement was seen in six cases, with monoclonal rearrangement in four cases. Treatment was conservative treatment but not chemotherapy. Four received anti-HLH therapy and others anti-inflammatory treatment. All patients survived with relapse after long-term clinical observation and follow-up. EBV + T/NK cells in IM can elicit malignant features that mimic T/NK-cell lymphoma pathologically and benign features mimicking IM clinically. These findings indicate that EBV + T/NK cells in IM could serve as valuable diagnosis. Additional clinical information, including age of onset (children and young people), nature of onset (sudden), disease course (short), symptoms (systemic), EBV infection status (acute), and lymph node involvement, is crucial for accurate diagnosis and prognostic evaluation.

A retrospective cohort study of Epstein-Barr virus infection status and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) and the Epstein-Barr virus (EBV) are very closely related. This study estimated the impact of EBV infection status on clinical manifestations and disease remission in patients with SLE. A retrospective study was performed using electronic health records of patients with SLE. The SLE disease activity index (SLEDAI-2K) was used to assess disease activity. VCAIgM or EAIgM positive or EBVDNA copies ≥ 50IU/mL were defined as lytic infection group, EBNA-IgG or VCAIgG-positive and who were negative for both VCAIgM and EAIgM with EBVDNA copies < 50IU/mL were defined as the latent infection group. The endpoint (disease remission) was defined as a decrease in SLEDAI-2K score of ≥ 1 grade or ≥ 4 points from baseline. The association between EBV infection status and disease remission was assessed using propensity score weighting and multivariable Cox regression models. There were 75 patients with SLE in the EBV lytic infection group and 142 patients in the latent infection group. The SLEDAI-2K score was higher in the lytic infection group (10.00 (6.25, 16.00) vs. 8.00 (5.00, 10.00), Z = 3.96, P < 0.001). There was a significant difference in the effect of EBV lytic infection on disease remission compared to latent infection (HR 0.30, 95% CI 0.19-0.49, P < 0.001). Patients with SLE with lytic EBV infection have higher disease activity and take longer to achieve remission. Our study furthers our understanding of the relationship between SLE and EBV infection and may inform better treatment practices in the future.

Tonsillar cytotoxic CD4 T cells are involved in the control of EBV primary infection in children

CD4 T cells play a key role in Epstein Barr virus (EBV) infection, by modulating latent antigen expression, and exhibiting cytotoxic and regulatory properties. Our aim was to evaluate the presence of Granzyme B (GZMB) and Foxp3 CD4 T cells at different EBV infection status and latency profiles. We examined CD4, GZMB, Foxp3, IL10, TGF-β, CD4-GZMB and CD4-Foxp3 expression at the tonsils of pediatric patients with different infective status and EBV latency profiles. CD4+, GZMB+, Foxp3+, CD4-GZMB+ and CD4-Foxp3+ cell counts were higher at the interfollicular region. Higher expression of CD4-GZMB was found in primary infected patients compared to healthy carriers. In patients that expressed latency III antigens, we demonstrated lower CD4+, CD4-GZMB+, CD4-Foxp3+ expression; a negative correlation between the immunoregulatory cytokine IL-10+ and GZMB+ as well as a positive correlation of IL-10+ and CD4+. In patients expressing the lytic protein BMRF1, a positive correlation of TGF-β+ with CD4-GZMB+ and CD4-Foxp3+ was observed. Our findings indicate that CD4-GZMB+ cells are involved in the restriction of primary EBV infection in pediatric patients, which could partially explain the lack of symptoms, whereas both CD4-GZMB+ and CD4-Foxp3+ cells could be involved in the modulation of latency.

Expression of the immune checkpoint molecules PD‑L1 and PD‑1 in EBV‑associated lymphoproliferative disorders: A meta‑analysis.

Epstein-Barr virus (EBV) has been implicated in the development of a wide range of lymphoproliferative disorders. In this process, the role of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) has remained to be clarified. A meta-analysis of 20 studies was performed and risk ratios (RRs) with 95% confidence intervals (CIs) were used to evaluate the association between PD-L1/PD-1 expression and the status of EBV infection. The results showed that the expression level of PD-L1 in tumor cells was significantly higher in EBV+ cases with a pooled RR of 2.26 (95% CI, 1.63-3.14; P<0.01), particularly in subtypes of diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma. Similarly, EBV infection increased the expression of PD-L1 in immune cells with a pooled RR of 2.20 (95% CI, 1.55-3.12; P<0.01). In subtypes of DLBCL and post-transplant lymphoproliferative disorder, the expression of PD-L1 in immune cells is increased in EBV+ cases. Regarding the expression level of PD-1 in tumor-infiltrating lymphocytes (TILs), no significance was found between EBV infection and PD-1 expression, with a pooled RR of 1.10 (95% CI, 0.81-1.48; P>0.05). The present meta-analysis demonstrated that in EBV-associated lymphoproliferative disorders, EBV infection was associated with the expression level of PD-L1 in tumor cells and immune cells but was not associated with the expression of PD-1 in TILs.

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

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

Markers of Epstein-Barr Virus Infection in Association with the Onset and Poor Control of Rheumatoid Arthritis: A Prospective Cohort Study.

Although the connection between Epstein-Barr virus (EBV) and rheumatoid arthritis (RA) has been studied for over 40 years, many questions still need clarification. The study aimed to analyze the possible association between anti-EBV antibody titers, EBV DNA viremia, EBV infection status and EBNA1 (Epstein-Barr nuclear antigen 1-EBNA1) variants and clinical parameters of RA patients. This prospective cohort study included 133 RA patients and 50 healthy controls. Active/recent EBV infection was more prevalent in RA patients than in controls (42% vs. 16%, p < 0.001). RA patients had higher titers of anti-EBV-CA-IgM (capsid antigen-CA) and anti-EBV-EA(D)-IgG (early antigen-EA) antibodies than controls (p = 0.003 and p = 0.023, respectively). Lower levels of anti-EBNA1-IgG and anti-EBV-CA-IgG were observed in RA patients who received methotrexate (anti-EBNA1 IgG p < 0.001; anti-EBV-CA IgG p < 0.001). Based on amino acid residue on position 487, two EBNA1 prototypes were detected: P-Thr and P-Ala. Patients with active/recent EBV infection had a five times more chance of having RA and a nearly six times more chance of getting RA. Also, EBV active/recent infection is twice more likely in newly diagnosed than in methotrexate-treated patients. Further studies are needed to clarify "who is the chicken and who is the egg" in this EBV-RA relationship.

P16INK4a and pRb expression in laryngeal squamous cell carcinoma with and without infection by EBV or different genotypes of HPV: a retrospective study

BackgroundLaryngeal squamous cell carcinoma (LSCC) represents one of the principal tumors of the head and neck. Human papillomavirus (HPV) and Epstein–Barr virus (EBV) are considered risk factors for the development and the clinical prognosis of LSCC. High levels of p16INK4a are suggested as a surrogate marker of HPV or EBV infection in some head and neck tumors but in LSCC is still controversial. Furthermore, pRb expression may be considered an additional biomarker but it has not been clearly defined. This work aimed to compare the expression of pRb and p16INK4a as possible biomarkers in tumor tissues with and without infection by EBV or different genotypes of HPV from patients with LSCC.MethodsTumor samples from 103 patients with LSCC were previously investigated for the presence and genotypes of HPV using the INNO-LiPA line probe assay and for the infection of EBV by qPCR. p16 INK4a and pRb expression was assessed by immunohistochemistry.ResultsOf the 103 tumor samples, expression of p16INK4a was positive in 55 (53.4%) and of this, 32 (56.1%) were positive for HPV whereas 11 (39.3%) were EBV positive but both without a significantly difference (p > 0.05). pRb expression was positive in 78 (75.7%) and a higher frequency of this expression was observed in HPV negative samples (87.0%) (p = 0.021) and in high-risk HPV negative samples (85.2%) (p = 0.010). No difference was observed when comparing pRb expression and EBV infection status (p > 0.05).ConclusionOur results support the suggestion that p16INK4a is not a reliable surrogate marker for identifying HPV or EBV infection in LSCC. On the other hand, most of our samples had pRb expression, which was more frequent in tumors without HPV, suggesting that pRb could indicate HPV negativity. However, more studies with a larger number of cases are required, including controls without LSCC and evaluating other molecular markers to determine the real role of p16INK4a and pRb in LSCC.

Does Epstein-Barr virus-positive gastric cancer establish a significant relationship with the multiple genes related to gastric carcinogenesis?

Gastric cancer has been categorized into molecular subtypes including Epstein-Barr virus (EBV)-positive tumors, which provide clinicopathological and prognostic information. In this study, we investigated the EBV infection status of patients with gastric cancer and its correlation with the clinicopathological characteristics and multiple genes related to gastric carcinogenesis. The data of 460 gastric cancer patients who underwent curative gastrectomy with D2 lymph node dissection between January 2017 and February 2022 were analyzed. The clinicopathological features and prognosis of the patients with EBV-positive gastric cancers were compared with those of EBV-negative gastric cancers. Immunohistochemistry for epidermal growth factor receptor (EGFR), C-erb B2, Ki-67, and p53 was performed. Additionally, in situ hybridization was conducted to detect EBV, and microsatellite instability (MSI) analysis was used to assess the deficiency in mismatch repair (MMR) genes. EBV-positivity and MSI were identified in 10.4% and 37.3% of gastric cancer patients, respectively. EBV positivity was associated with male gender (P = 0.001), proximal location (P = 0.004), poorly differentiated histological type (P = 0.048), moderate to severe lymphoid stroma (P = 0.006), high Ki-67 expression (P = 0.02), and a shorter resection margin. EGFR was more often expressed in EBV-negative gastric cancers (P < 0.001). MSI tumors were associated with older age (P = 0.01), the presence of lymphatic invasion (P = 0.02), less perineural invasion (P = 0.05), and the presence of H. pylori infection (P = 0.05). EBV positive gastric cancer is associated with increased Ki-67 and decreased EGFR expression and a shorter resection margin due to the prominent lymphoid stroma. However, MMR deficiency is not associated with EBV status even though MSI gastric cancer is related to H. pylori status.

Comprehensive single-cell transcriptomic and proteomic analysis reveals NK cell exhaustion and unique tumor cell evolutionary trajectory in non-keratinizing nasopharyngeal carcinoma

BackgroundNonkeratinizing nasopharyngeal carcinoma (NK-NPC) has a strong association with Epstein-Barr virus (EBV) infection. The role of NK cells and the tumor cell evolutionary trajectory in NK-NPC remain unclear. In this study, we aim to investigate the function of NK cell and the evolutionary trajectory of tumor cells in NK-NPC by single-cell transcriptomic analysis, proteomics and immunohistochemistry.MethodsNK-NPC (n = 3) and normal nasopharyngeal mucosa cases (n = 3) were collected for proteomic analysis. Single-cell transcriptomic data of NK-NPC (n = 10) and nasopharyngeal lymphatic hyperplasia (NLH, n = 3) were obtained from Gene Expression Omnibus (GSE162025 and GSE150825). Quality control, dimension reduction and clustering were based on Seurat software (v4.0.2) process and batch effects were removed by harmony (v0.1.1) software. Normal cells of nasopharyngeal mucosa and tumor cells of NK-NPC were identified using copykat software (v1.0.8). Cell-cell interactions were explored using CellChat software (v1.4.0). Tumor cell evolutionary trajectory analysis was performed using SCORPIUS software (v1.0.8). Protein and gene function enrichment analyses were performed using clusterProfiler software (v4.2.2).ResultsA total of 161 differentially expressed proteins were obtained between NK-NPC (n = 3) and normal nasopharyngeal mucosa (n = 3) by proteomics (log2 fold change > 0.5 and P value < 0.05). Most of proteins associated with the nature killer cell mediated cytotoxicity pathway were downregulated in the NK-NPC group. In single cell transcriptomics, we identified three NK cell subsets (NK1-3), among which NK cell exhaustion was identified in the NK3 subset with high ZNF683 expression (a signature of tissue-resident NK cell) in NK-NPC. We demonstrated the presence of this ZNF683 + NK cell subset in NK-NPC but not in NLH. We also performed immunohistochemical experiments with TIGIT and LAG3 to confirm NK cell exhaustion in NK-NPC. Moreover, the trajectory analysis revealed that the evolutionary trajectory of NK-NPC tumor cells was associated with the status of EBV infection (active or latent). The analysis of cell-cell interactions uncovered a complex network of cellular interactions in NK-NPC.ConclusionsThis study revealed that the NK cell exhaustion might be induced by upregulation of inhibitory receptors on the surface of NK cells in NK-NPC. Treatments for the reversal of NK cell exhaustion may be a promising strategy for NK-NPC. Meanwhile, we identified a unique evolutionary trajectory of tumor cells with active status of EBV-infection in NK-NPC for the first time. Our study may provide new immunotherapeutic targets and new sight of evolutionary trajectory involving tumor genesis, development and metastasis in NK-NPC.

Comparative Analysis of Primary and Reactivated EB Virus Infection Associated Hemophagocytic Lymphohistiocytosis

To compare the clinical characteristics of children with hemophagocytic lymphocytosis (HLH) associated with primary Epstein-Barr virus (EBV) infection and EBV reactivation, and explore the effects of different EBV infection status on the clinical indexes and prognosis of HLH. The clinical data of 51 children with EBV associated HLH treated in Henan Children's Hospital from June 2016 to June 2021 were collected. According to the detection results of plasma EBV antibody spectrum, they were divided into EBV primary infection-associated HLH group (18 cases) and EBV reactivation-associated HLH group (33 cases). The clinical features, laboratory indexes and prognosis of the two groups were analyzed and compared. There were no significant differences in age, gender, hepatomegaly, splenomegaly, lymphadenopathy, neutrophil count in peripheral blood, hemoglobin content, platelet count, plasma EBV-DNA load, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, albumin, fibrinogen, triglyceride, ferritin, hemophagocytosis in bone marrow, NK cell activity and sCD25 between the two groups(P>0.05). The central nervous system involvement and CD4/CD8 in EBV reactivation-associated HLH group were significantly higher than those in primary infection-associated HLH group, but the total bilirubin was significantly lower than that in primary infection-associated HLH group (P<0.05). After treatment according to HLH-2004 protocol, the remission rate, 5-year OS rate and 5-year EFS rate of patients in EBV reactivation-associated HLH group were significantly lower than those in EBV primary infection-associated HLH group (P<0.05). EBV reactivation-associated HLH is more likely to cause central nervous system involvement and the prognosis is worser than EBV primary infection-associated HLH, which requires intensive treatment.

Multiancestry genomic and transcriptomic analysis of gastric cancer.

Gastric cancer is among the most common malignancies worldwide, characterized by geographical, epidemiological and histological heterogeneity. Here, we report an extensive, multiancestral landscape of driver events in gastric cancer, involving 1,335 cases. Seventy-seven significantly mutated genes (SMGs) were identified, including ARHGAP5 and TRIM49C. We also identified subtype-specific drivers, including PIGR and SOX9, which were enriched in the diffuse subtype of the disease. SMGs also varied according to Epstein-Barr virus infection status and ancestry. Non-protein-truncating CDH1 mutations, which are characterized by in-frame splicing alterations, targeted localized extracellular domains and uniquely occurred in sporadic diffuse-type cases. In patients with gastric cancer with East Asian ancestry, our data suggested a link between alcohol consumption or metabolism and the development of RHOA mutations. Moreover, mutations with potential roles in immune evasion were identified. Overall, these data provide comprehensive insights into the molecular landscape of gastric cancer across various subtypes and ancestries.

Oral Microbiota Alteration and Roles in Epstein-Barr Virus Reactivation in Nasopharyngeal Carcinoma.

Microbiota has recently emerged as a critical factor associated with multiple malignancies. Nasopharyngeal carcinoma (NPC) is highly associated with Epstein-Barr virus (EBV); the oncovirus resides and is transmitted in the oral cavity. However, the alternation of oral microbiota in NPC patients and its potential link to EBV reactivation and host cell response under the simultaneous existence of EBV and specific bacteria is largely unknown. Here, oral microbiota profiles of 303 NPC patients and controls with detailed clinical information, including serum EBV anti-virus capsid antigen (VCA) IgA level, were conducted. A distinct microbial community with lower diversity and imbalanced composition in NPC patients was observed. Notably, among enriched bacteria in patients, Streptococcus sanguinis was associated with anti-VCA IgA, an indicator of NPC risk and EBV reactivation. By measuring the concentration of its metabolite, hydrogen peroxide (H2O2), in the saliva of clinical patients, we found the detection rate of H2O2 was 2-fold increased compared to healthy controls. Further coculture assay of EBV-positive Akata cells with bacteria in vitro showed that S. sanguinis induced EBV lytic activation by its metabolite, H2O2. Host and EBV whole genome-wide transcriptome sequencing and EBV methylation assays showed that H2O2 triggered the host cell signaling pathways, notably tumor necrosis factor alpha (TNF-α) via NF-κB, and induced the demethylation of the global EBV genome and the expression of EBV lytic-associated genes, which could result in an increase of virus particle release and potential favorable events toward tumorigenesis. In brief, our study identified a characterized oral microbial profile in NPC patients and established a robust link between specific oral microbial alteration and switch of latency to lytic EBV infection status in the oral cavity, which provides novel insights into EBV's productive cycle and might help to further clarify the etiology of NPC. IMPORTANCE EBV is classified as the group I human carcinogen and is associated with multiple cancers, including NPC. The interplays between the microbiota and oncovirus in cancer development remain largely unknown. In this study, we investigate the interactions between resident microbes and EBV coexistence in the oral cavity of NPC patients. We identify a distinct oral microbial feature for NPC patients. Among NPC-enriched bacteria, we illustrated that a specific species, S. sanguinis, associated with elevated anti-IgA VCA in patients, induced EBV lytic activation by its by-product, H2O2, and activated the TNF-α/NF-κB pathway of EBV-positive B cells in vitro, together with increased detection rate of H2O2 in patients' oral cavities, which strengthened the evidence of bacteria-virus-host interaction in physiological circumstances. The effects of imbalanced microbiota on the EBV latent-to-lytic switch in the oral cavity might create the likelihood of EBV infection in epithelial cells at the nasopharynx and help malignant transition and cancer development.

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

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

Clinicopathological features of angioimmunoblastic T-cell lymphoma pattern Ⅰ

Objective: To investigate the clinicopathological features of angioimmunoblastic T-cell lymphoma pattern Ⅰ (AITL Pattern Ⅰ). Methods: The clinicopathological data of 11 AITL Pattern Ⅰ cases that were diagnosed at the Beijing Friendship Hospital Affiliated to Capital Medical University (10 cases) and Beijing Lu Daopei Hospital (1 cases) from January 2019 to October 2021 were retrospectively collected. Immunophenotype, Epstein-Barr virus infection status and T cell receptor (TCR) clonality of the tumor cells were tested, and clinicopathological features of cases were analyzed. Results: Among the 11 AITL Pattern Ⅰ cases, the male to female ratio was 1.2∶1.0. The median age was 59 years (range 47-78 years). Seven cases had B symptoms, while eleven cases presented with systemic lymphadenopathy. According to Ann Arbor system staging, two cases were classified as stage Ⅰ-Ⅱ, and 9 cases as stage Ⅲ-Ⅳ. Hepatosplenomegaly was present in two cases (2/11), three cases (3/11) had skin rash and pruritus, and two cases (2/11) had pleural effusion. Previously, 6 cases (6/11) were diagnosed as reactive hyperplasia, 1 case (1/11) as EBV-associated lymphoproliferative disorder, and 4 cases (4/11) as hyperplasia of lymphoid tissue, which was unable to exclude lymphoma. Histologically, all the 11 cases showed hyperplastic follicles in the paracortical regions with well-formed germinal centers. The hyperplastic follicles showed ill-defined borders and attenuated mantle zones in 7 cases. Mantle zones completely disappeared in 4 cases. The follicles were surrounded by a thin layer of atypical lymphocytes with bright or faintly stained cytoplasm. In 2 cases, the clear cells were located between the germinal centers and the thin residual mantle cell layers, showing a circular growth pattern. The cells were medium in size, with irregular karyotype, coarse chromatin and indistinct nucleoli. Immunohistochemically, CD21 staining showed that the meshworks of follicular dendritic cells(FDC)were mainly confined to the follicles. There was a subtle expansion of the meshworks of FDC in 4 cases with ill-defined borders. The atypical cells surrounding the follicles expressed CD3 (11/11), CD4 (11/11), PD-1 (11/11), CXCL13 (6/11), ICOS (10/11) and CD10 (7/11). PD-1 staining showed a strong perifollicular pattern, and a small number of positive cells were scattered around the high endothelial veins in the interfollicular region. CXCL13, ICOS and CD10 showed similar distribution patterns. EBV-encoded small RNA probe (EBER) in situ hybridization showed that EBER positive B lymphocytes were scattered in the interfollicular region (5-20/HPF) in all cases. T cell receptor gene rearrangement was monoclonal in all cases. Conclusions: Diagnosing AITL Pattern Ⅰ may be challenging and requires comprehensive analysis of clinical manifestations, histological morphology, immunophenotype and gene rearrangement results.

1248P Identification of Epstein-Barr virus (EBV)-associated gastric cancer at RNA-level by evaluating transcriptional status of seven EBV crucial genes

EBV has been an emerging biomarker to inform clinical management of GC, according to the potential mechanisms of EBV infection on GC immune check point blockade (ICB) efficacy. The gold standard for detecting EBV has long been EBV-encoded RNA (EBER) in situ hybridization (ISH). However, with the increasing demand for multi-molecular biomarkers detection recommended by standard treatment guidelines for GC, a single EBV detection has been challenged in clinical practice with limited samples. Herein, an NGS-panel based on RNA for detecting EBV was established and validated in the study. Seven genes including, EBER1, EBER2, and EBNA1, LMP1, LMP2A/B, BZLF1, BARF1 were involved in the NGS panel to evaluate EBV infection status. 42 tumor tissue samples from GC, with half EBV positive and half EBV negative evaluated by ISH were collected, as well as their paired normal tissue sample, for NGS detection. The seven genes expression profile in the enrolled samples were comprehensively analyzed. The raw read counts of the five genes EBNA1, LMP1, LMP2A/B, BZLF1, BARF1 based on RNA sequencing in normal tissue samples were detected as zero. Only 12 normal tissue samples were detected EBER1and/or EBER2, of which the raw read counts did not exceed 10. In ISH-EBV negative tumor samples, raw read counts of the five genes EBNA1, LMP1, LMP2A/B, BZLF1, BARF1 based on RNA were also zero. And, expression signals were detected for EBER1 in 7 samples and EBER2 in 3 samples, and the raw RNA counts did not exceed 17 and 5, respectively. However, in ISH-EBV positive tumor samples, the average number of RNA raw read counts of EBER1 was 7424, with the minimum value was 458; the average number of RNA raw read counts of EBER2 was 3730, with the minimum value was 137. Besides, RNA expression signals of three gene, EBNA1, LMP2A/B, and BARF1, were also been detected with lower raw read counts. Due to the significantly high expression characteristics of EBER1/EBER2 in EBV infected positive samples, the detection of EBV based on RNA by NGS panel is feasible, which provides a basis for the subsequent design of NGS panel including EBV and other molecular biomarkers for GC.

Prevalence of Epstein-Barr Virus Infection and Mismatch Repair Protein Deficiency and the Correlation of Immune Markers in Tibetan Patients with Gastric Cancer.

Background Gastric cancer (GC) is a major cause of cancer-related death in China. Immunotherapies based on PD-1/PD-L1 inhibitors have improved the survival of some patients with GC. Epstein–Barr virus (EBV) infection, mismatch repair (MMR) deficiency, and tumor immune microenvironment (TIME) markers (such as CD3, CD8, and PD-L1) may help to identify specific patients who will respond to PD-1/PD-L1 inhibitors. Considering racial heterogeneity, the pattern of TIME markers in Tibetan patients with GC is still unclear. We aimed to identify the prevalence of EBV infection and the MMR status and their association with immune markers in Tibetan GC to aid in patient selection for immunotherapy. Materials and Methods From 2001 to 2015, we retrospectively collected 120 tissue samples from consecutive Tibetan GC patients and constructed tissue microarrays. EBV infection was assessed by Epstein–Barr-encoded RNA (EBER) in situ hybridization, and MMR protein levels were measured. Immune markers (including CD3 and CD8) in intraepithelial, stromal, and total areas were detected by immunohistochemistry (IHC). PD-L1 expression was assessed by the combined positive score (CPS). We also analyzed the relationships of EBV infection and MMR status with immune markers. Results Of the 120 samples, 11 (9.17%) were EBV positive (+), and 6 (5%) were MMR deficient (dMMR). PD-L1 CPS ≥1% was found in 32.5% (39/120) of Tibetan GC patients. EBV infection was associated with higher numbers of CD3+ T cells (P < 0.05) and CD8+ T cells (P < 0.05) and higher PD-L1 expression (P < 0.05). For the limited number of dMMR patients, no significant relationship was observed between dMMR and TIME markers (P > 0.05). Conclusions In Tibetan GC patients, the rates of EBV infection, dMMR, and positive PD-L1 expression were 9.17%, 5%, and 32.5%, respectively. EBV infection was associated with the numbers of CD3+ T cells and CD8+ T cells and PD-L1 expression within the tumor. These markers may guide the selection of Tibetan GC patients for immunotherapy.

Clinical Significance of Epstein-Barr Virus and Helicobacter pylori Infection in Gastric Carcinoma.

Epstein-Barr virus (EBV) and Helicobacter pylori (HP) coinfection may synergistically induce severe inflammatory responses in the stomach tissue, increasing the risk of developing gastric cancer. We aimed to analyze the effect of EBV and HP coinfection on the clinicopathologic features and prognosis of gastric cancer, as well as to evaluate the role of EBV infection in non-gastric carcinoma with lymphoid stroma (non-GCLS). Overall, 956 patients who underwent surgery for gastric cancer between September 2014 and August 2015 were eligible and divided into groups, according to GCLS morphology, EBV infection, and HP infection. Clinicopathologic characteristics and oncologic outcomes were analyzed retrospectively. EBV and HP coinfection was significantly associated with male sex, proximal location, GCLS morphology, and equivocal p53 expression (p<0.001). Multivariate analysis revealed that EBV infection alone (hazard ratio [HR], 0.362; 95% CI, 0.131 to 0.996; p=0.049) and lower third location (HR, 0.624; 95% CI, 0.413 to 0.943; p=0.025) were inversely correlated with overall survival. During median follow-up period of 72 months, overall survival rate was not significantly different between the EBV and HP coinfection group and others (97.6% vs 86.8%; log-rank p=0.144). In non-GCLS patients (n=920), overall survival rate was not significantly different between the EBV infection group and others (96.9% vs 86.4%; log-rank p=0.126). EBV and HP coinfection is not an independent prognostic factor for gastric cancer. EBV infection status, regardless of HP infection, affects the clinicopathologic features of all types of gastric cancer. However, it does not lead to a significant difference in overall survival of non-GCLS patients.

Clinicopathological features and prognosis of cytotoxic T-cell lymphoma: analysis of 134 cases

Objective: To investigate the clinicopathological features and prognosis of cytotoxic T-cell lymphoma (CTL). Methods: The clinicopathological data of 134 CTL patients in Beijing Friendship Hospital Affiliated to Capital Medical University, the 989 Hospital of PLA Joint Logistics Support force (formerly the 152 Hospital) and the Fourth Hospital of Hebei Medical University from 2008 to 2020 were retrospectively collected. Immunophenotype, Epstein-Barr virus infection status and T cell receptor (TCR) clonality of tumor cells were assessed, and clinicopathological features and prognosis of patients were analyzed. Results: Among the 134 CTL patients, the male to female ratio was 1.7∶1.0, the median age was 49.5 years (range 3-83 years), and 100 cases (74.6%) were under 60 years old. Forty-six point nine percent of the patients (53/113) had B symptoms. Most of the patients presented with systemic superficial lymphadenopathy. According to the Ann Arbor staging system, 36.8% (39/106) of the patients were in stage Ⅰ-Ⅱ, and 63.2% (67/106) in stage Ⅲ-Ⅳ. The rate of extranodal involvement was 51.6% (66/128). Spleen was involved in 24.2% (31/128) of the cases. Morphology showed diffuse growth of abnormal lymphocytes, infiltrating and destroying normal tissue structure. Immunohistochemical staining showed that tumor cells expressed T cell antigens (CD2, CD3, CD5, and CD7), and 72.0% (77/107) of them had decreased or lost expression of one or more antigens. According to the numbers of CD4 and CD8 expression in tumor cells, 70 cases (52.2%) were grouped into CD8+>CD4+group. The expression rates of TIA-1 and granzyme B were 99.2% (119/120) and 79.8% (95/119), respectively. CD20 abnormal expression rate was 27.6% (37/134) and CD56 was negative in all cases. The median Ki-67 proliferative index was 45.0% (range 5%-80%). In situ hybridization of small RNA encoded by Epstein-Barr virus was negative. Clonal TCR gene rearrangement analysis was performed on 49 cases and was positive in all cases. Ninety-one patients were followed up for a median of 36 months (range, 1 to 240 months), and 40 of the 91 patients (44.0%) died. The twenty-three patients were in complete remission (including 13 cases with localized single extranodal mass). The 3-year and 5-year overall survival rates were 53.5% and 49.4%, respectively. Univariate analysis showed that B symptom, spleen involvement, extranodal involvement, clinical stage, CD8+>CD4+phenotype, abnormal expression of CD20 and Ki-67 proliferation index (>60%) were associated with overall survival (P<0.05). The multivariate Cox regression analyses showed that spleen involvement and CD8+>CD4+ phenotype were independent prognostic factors for overall survival in CTL patients. Conclusions: CTL are more commonly found in adult males under 60 years old, often accompanied by B symptom, with a high proportion of extranodal involvement and more CD8 positive phenotypes. Spleen involvement and CD8+>CD4+phenotype are independent predictors of CTL overall survival. Some patients with localized extranodal CTL may have a good prognosis.

P689 Multicenter, cross-sectional, observational study on Epstein–Barr viral infection status and thiopurine use by age group in patients with inflammatory bowel disease in Japan

Abstract Background The Epstein–Barr virus (EBV) infection status in patients with inflammatory bowel disease (IBD), particularly those using thiopurines, may be associated with the risk of lymphoproliferative disorder and hemophagocytic lymphohistiocytosis. This was the first multicenter survey of EBV infection in Japanese patients with IBD (UMIN000033004). Factors related to the EBV infection status were also investigated. Methods This cross-sectional observational study of the EBV infection status in pediatric and adult patients with IBD was conducted at five tertiary institutions in Japan. Serum EBV anti-viral capsid antigen (VCA) IgG, and EBV anti-VCA IgM were measured in 495 patients with IBD□(UC:313 patients, CD:174 patients, IBD-U:8 patients). Patient characteristics (sex, age at diagnosis, age at onset, duration of illness, and disease location), treatments, and blood examination results were obtained from the medical records at each institution. Prior EBV infection was defined as anti-VCA IgM negativity and anti-VCA IgG positivity. Results mong the 495 patients, 56.2% (278/495) were male, and the median age at diagnosis of IBD was 18 years. The median age at the time of the anti-VCA IgG examination was 25 years(range: 0–92 years). Of the 495 patients, 9 were anti-VCA IgM-positive and 354 were anti-VCA IgG-positive (seroprevalence: 72.8%). The proportion of patients with prior EBV infection was 0% for those aged &amp;lt;5 years, &amp;lt;60% for those aged &amp;lt;30 years, and &amp;gt;90% for those aged &amp;gt;30 years. The patients’ current thiopurine use was 37.8% (187/486) for all anti-VCA IgM negative patients and 42.3% (85/201) for anti-VCA IgM negative patients aged &amp;lt;20 years. The proportion of EBV-uninfected patients using thiopurines was 28.4% (52/183) for all patients and 51.8% (44/85) for aged &amp;lt;20 years. Age was significantly associated with anti-VCA IgG seropositivity (p &amp;lt; 0.01, odds ratio: 0.902, 95% confidence interval: 0.880–0.925). No cases of lymphoproliferative disorder, hemophagocytic lymphohistiocytosis, or chronic active EBV infection were reported. Conclusion Approximately 30% of Japanese patients with IBD were EBV-uninfected, including those using thiopurines. Age was a significant factor for anti-VCA IgG seropositivity.

Epstein-Barr virus infection mediated TP53 and Bcl-2 expression in nasopharyngeal carcinoma pathogenesis.

Epstein Barr virus (EBV) stimulates neoplastic transformation of nasopharyngeal epithelial cells through various molecular mechanisms, predominantly affecting inactivation of tumor-suppressor genes and activation of oncogenes. EBV infection is a major risk factor for nasopharyngeal carcinoma (NPC), yet its role in the carcinogenesis is not clear. EBV infection alters the expression of antiapoptotic proteins and tumor suppressor proteins. Therefore, this study investigated the correlation between EBV infection status with B cell lymphoma-2 (Bcl-2) and TP53 protein expression amongst laryngeal and nasopharyngeal cancer cases. This study was performed using 22 nasopharyngeal and 11 laryngeal cancer cases. EBV infection status, TP53 and Bcl-2 protein expression was studied using immunohistochemistry. The majority of the laryngeal cancer cases exhibited a poor prognosis and presented low Bcl-2 expression. A total of 22.7% cases were infected with EBV in the NPC cases. Upregulated TP53 expression was associated with EBV infection in the NPC cohort, and EBV infection was correlated with TP53 upregulation in the patients with NPC, suggesting mutual regulation between TP53 and EBV.