Expression and prognostic significance of the PD-1/PD-L1 pathway in AIDS-related non-Hodgkin lymphoma.

OBJECTIVE: Immune tolerance and evasion play a critical role in virus-driven malignancies. However, the phenotype and clinical significance of programmed cell death 1 (PD-1) and its ligands, PD-L1 and PD-L2, in aggressive acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin lymphoma (AR-NHL) remain poorly understood, particularly in the Epstein-Barr virus (EBV)-positive subset. METHODS: We used in situ hybridization with EBV-encoded RNA (EBER) to assess the EBV status. We performed immunohistochemistry and flow cytometry analysis to evaluate components of the PD-1/PD-L1/L2 pathway in a multi-institutional cohort of 58 patients with AR-NHL and compared EBV-positive and EBV-negative cases. RESULTS: The prevalence of EBV+ in AR-NHL was 56.9% and was associated with a marked increase in the expression of PD-1/PD-L1/PD-L2 in malignant cells. Patients with AR-NHLs who tested positive for both EBER and PD-1 exhibited lower survival rates compared to those negative for these markers (47.4% vs. 93.8%, p = 0.004). Similarly, patients positive for both EBER and PD-L1 also demonstrated poorer survival (56.5% vs. 93.8%, p = 0.043). Importantly, PD-1 tissue-expression demonstrated independent prognostic significance for overall survival in multivariate analysis and was correlated to elevated levels of LDH (r = 0.313, p = 0.031), increased PD-1+ Tregs (p = 0.006), and robust expression of EBER (r = 0.541, p < 0.001) and PD-L1 (r = 0.354, p = 0.014) expression. CONCLUSIONS: These data emphasize the importance of PD-1-mediated immune evasion in the complex landscape of immune oncology in AR-NHL co-infected with EBV, and contribute to the diagnostic classification and possible definition of immunotherapeutic strategies for this unique subgroup.

Effect of pre-emptive rituximab on EBV DNA levels and prevention of post-transplant lymphoproliferative disorder in pediatric kidney transplant recipients: A case series from the pediatric nephrology research consortium.

BACKGROUND: There are scant data on the effect of rituximab on EBV DNA levels and prevention of post-transplant lymphoproliferative disorder (PTLD) in pediatric kidney transplant recipients with EBV DNAemia. METHODS: Kidney transplant recipients with EBV DNAemia treated with rituximab to prevent PTLD between 7/1999 and 7/2019 at five pediatric centers were included. Those with confirmed PTLD at the onset of rituximab were excluded. Primary outcomes included percentage change in EBV DNAemia and occurrence of PTLD post rituximab. RESULTS: Twenty-six pediatric kidney transplant recipients were included. Median age at transplant was 4 years (IQR 2.1-10.3). EBV DNA load monitoring by qPCR was performed at 1-3 month intervals. EBV DNAemia onset occurred at a median of 73 days post-transplant (IQR 52-307), followed by DNAemia peak at a median of 268 days (IQR 112-536). Rituximab was administered at a median of 9 days post peak (IQR 0-118). Rituximab regimens varied; median dose 375 mg/m2 (IQR 375-439) weekly for 1-4 doses per course. Following rituximab, EBV DNA load decreased to <10% of baseline at 120 days in 20/26 patients; however, only 30% achieved complete resolution at last follow-up (median 2094 days post-transplant [IQR 1538-3463]). Two (7%) developed PTLD at 915 and 1713 days post rituximab. All recipients had functioning grafts. One death occurred in a child with PTLD following remission due to unrelated reasons. CONCLUSIONS: In the largest pediatric kidney transplant recipient case series with EBV DNAemia given rituximab to prevent PTLD, rituximab achieved a short-term reduction in DNA load; however, recurrent DNAemia is common.

Role of plasma EBV-DNA load and EBER status on newly diagnosed peripheral T-cell lymphoma.

PURPOSE: To explore the prognostic and therapeutic role of Epstein-Barr Virus (EBV) on peripheral T-cell lymphoma (PTCL). METHODS: Totally 262 newly diagnosed PTCL patients who were hospitalized from January 2014 to December 2022 were retrospectively enrolled. Molecular analysis included 31 eligible patients. EBV-encoded RNA (EBER) presence in tumor tissue and EBV DNA levels in patients at baseline (DNA1) and after 4 cycles of chemotherapy (DNA4) were assessed. RESULTS: Our findings revealed that the EBER-positive cohort exhibited significant differences compared to counterparts in overall survival (OS, P = 0.047) and progression-free survival (PFS, P = 0.009). Both DNA1 and DNA4 were significantly associated with inferior OS. Multivariate analysis demonstrated that DNA4 independently affected PTCL prognosis for OS (hazard ratio = 5.1617; 95% confidence interval 1.1017-24.1831; P = 0.037). Treatment with the cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) plus azacytidine regimen showed a better OS compared to CHOP or CHOP plus etoposide for patients with partially positive EBER and EBER positive statuses (P = 0.192), although the improvement was not statistically significant. This study delineated the genetic paradigm of PTCL, comparing genetic differences by EBV status and found that EBER partially positive plus positive patients were more likely to have DNMT3A (P = 0.002), RHOAG17V (P = 0.023), and TET2 mutations (P = 0.032). CONCLUSION: EBER, DNA1, and DNA4 emerged as sensitive markers for prognosis. CHOP plus azacytidine might present a preferable option for PTCL patients with DNA methylation due to EBV infection.

Aflatoxin B1 and Epstein-Barr virus-induced CCL22 expression stimulates B cell infection.

Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.

Characterization of the brain virome in human immunodeficiency virus infection and substance use disorder.

Viruses can infect the brain in individuals with and without HIV-infection: however, the brain virome is poorly characterized. Metabolic alterations have been identified which predispose people to substance use disorder (SUD), but whether these could be triggered by viral infection of the brain is unknown. We used a target-enrichment, deep sequencing platform and bioinformatic pipeline named "ViroFind", for the unbiased characterization of DNA and RNA viruses in brain samples obtained from the National Neuro-AIDS Tissue Consortium. We analyzed fresh frozen post-mortem prefrontal cortex from 72 individuals without known viral infection of the brain, including 16 HIV+/SUD+, 20 HIV+/SUD-, 16 HIV-/SUD+, and 20 HIV-/SUD-. The average age was 52.3 y and 62.5% were males. We identified sequences from 26 viruses belonging to 11 viral taxa. These included viruses with and without known pathogenic potential or tropism to the nervous system, with sequence coverage ranging from 0.03 to 99.73% of the viral genomes. In SUD+ people, HIV-infection was associated with a higher total number of viruses, and HIV+/SUD+ compared to HIV-/SUD+ individuals had an increased frequency of Adenovirus (68.8 vs 0%; p<0.001) and Epstein-Barr virus (EBV) (43.8 vs 6.3%; p=0.037) as well as an increase in Torque Teno virus (TTV) burden. Conversely, in HIV+ people, SUD was associated with an increase in frequency of Hepatitis C virus, (25 in HIV+/SUD+ vs 0% in HIV+/SUD-; p=0.031). Finally, HIV+/SUD- compared to HIV-/SUD- individuals had an increased frequency of EBV (50 vs 0%; p<0.001) and an increase in TTV viral burden, but a decreased Adenovirus viral burden. These data demonstrate an unexpectedly high variety in the human brain virome, identifying targets for future research into the impact of these taxa on the central nervous system. ViroFind could become a valuable tool for monitoring viral dynamics in various compartments, monitoring outbreaks, and informing vaccine development.

The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation.

Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.

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

OBJECTIVES: 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. METHOD: A retrospective study was performed using electronic health records of patients with SLE. The SLE disease activity index (SLEDAI-2 K) was used to assess disease activity. VCAIgM or EAIgM positive or EBVDNA copies ≥ 50 IU/mL were defined as lytic infection group, EBNA-IgG or VCAIgG-positive and who were negative for both VCAIgM and EAIgM with EBVDNA copies < 50 IU/mL were defined as the latent infection group. The endpoint (disease remission) was defined as a decrease in SLEDAI-2 K 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. RESULTS: There were 75 patients with SLE in the EBV lytic infection group and 142 patients in the latent infection group. The SLEDAI-2 K 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). CONCLUSIONS: 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.

Shared and distinct interactions of type 1 and type 2 Epstein-Barr Nuclear Antigen 2 with the human genome.

BACKGROUND: There are two major genetic types of Epstein-Barr Virus (EBV): type 1 (EBV-1) and type 2 (EBV-2). EBV functions by manipulating gene expression in host B cells, using virus-encoded gene regulatory proteins including Epstein-Barr Nuclear Antigen 2 (EBNA2). While type 1 EBNA2 is known to interact with human transcription factors (hTFs) such as RBPJ, EBF1, and SPI1 (PU.1), type 2 EBNA2 shares only ~ 50% amino acid identity with type 1 and thus may have distinct binding partners, human genome binding locations, and functions. RESULTS: In this study, we examined genome-wide EBNA2 binding in EBV-1 and EBV-2 transformed human B cells to identify shared and unique EBNA2 interactions with the human genome, revealing thousands of type-specific EBNA2 ChIP-seq peaks. Computational predictions based on hTF motifs and subsequent ChIP-seq experiments revealed that both type 1 and 2 EBNA2 co-occupy the genome with SPI1 and AP-1 (BATF and JUNB) hTFs. However, type 1 EBNA2 showed preferential co-occupancy with EBF1, and type 2 EBNA2 preferred RBPJ. These differences in hTF co-occupancy revealed possible mechanisms underlying type-specific gene expression of known EBNA2 human target genes: MYC (shared), CXCR7 (type 1 specific), and CD21 (type 2 specific). Both type 1 and 2 EBNA2 binding events were enriched at systemic lupus erythematosus (SLE) and multiple sclerosis (MS) risk loci, while primary biliary cholangitis (PBC) risk loci were specifically enriched for type 2 peaks. CONCLUSIONS: This study reveals extensive type-specific EBNA2 interactions with the human genome, possible differences in EBNA2 interaction partners, and a possible new role for type 2 EBNA2 in autoimmune disorders. Our results highlight the importance of considering EBV type in the control of human gene expression and disease-related investigations.

The Role of Epstein-Barr Virus (EBV) Infected Gastric Cancer in Increasing microRNA124 (miR-124) Promoter Methylation and Enhancer of Zeste Homolog 2 (EZH2) Gene Expression.

The tumor suppressor microRNAs, miR-21, miR-124, and miR-494, participate in the controlling several cellular processes. To assess target miRNAs promoter methylation levels, we investigated 304 pairs of gastric cancer (GC) tissues and non-tumor tissues. We used a commercial real-time polymerase chain reaction (RT-PCR) for Epstein-Barr virus (EBV) and Helicobacter pylori kit to detect EBV and H. pylori DNA in GC tissues. After finding hypermethylation in the promoter of the miR-124 gene, we evaluated its expression level using quantitative PCR (qPCR). Bioinformatics analysis confirmed miR-124 as a target of enhancer of Zeste homolog 2 (EZH2). Additionally, qPCR confirmed the association between EZH2 and miR-124. EBV and H. pylori DNA were detected in 9.5% and 15.1% of GC patients, respectively. Our findings also revealed significant differences in the miR-124 methylation levels among EBV-infected GC patients, H. pylori infected GC patients, GC patients without EBV and H. pylori infection, and non-tumor tissue. Bioinformatics and qPCR assays suggested an inverse relationship between the expression levels of EZH2 and miR-124 in EBV-infected GC patients. Our data revealed hypermethylation of the miR-124 promoter and significant reduction in its expression in EBV-infected GC tissues. It is possible that miR-124 may target EZH2 by binding to the 3'-UTR of the EZH2 gene, thus potentially contributing to the development of EBV-infected GC.

Plasma Epstein-Barr virus microRNA BART8-3p as a potential biomarker for detection and prognostic prediction in early nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) encoded microRNA BART8-3p (miR-BART8-3p) was significantly associated with the metastasis in nasopharyngeal carcinoma (NPC). To explore the clinical values of plasma miR-BART8-3p in patients with early NPC. We retrospectively analyzed 126 patients with stage I and II NPC. A receiver operating characteristic curve was used to examine the diagnostic performance. Kaplan‒Meier analysis was applied to determine survival differences. Cox regression was used for univariate and multivariate analyses. Compared to healthy subjects, plasma EBV miR-BART8-3p was highly expressed in early NPC patients. The sensitivity, specificity, and area under the curve value of plasma miR-BART8-3p combined with plasma EBV DNA was up to 88.9%, 94.4%, and 0.931. Compared to patients with low expression of miR-BART8-3p, patients with high expression of miR-BART8-3p had poorer 5-year overall survival (OS) (98.9% vs. 91.1%, P = 0.025), locoregional recurrence-free survival (LRRFS) (100% vs. 83.9%, P < 0.001) and distant metastasis-free survival (DMFS) (98.9% vs. 88.0%, P = 0.006). Risk stratification analysis revealed that high-risk patients (with high levels of EBV DNA and miR-BART8-3p) had inferior OS, LRRFS, and DMFS than low-risk patients (without high levels of EBV DNA and miR-BART8-3p). Multivariate analysis verified that the high-risk group was an unfavorable factor for OS, LRRFS, and DMFS. A combination of plasma EBV miR-BART8-3p and EBV DNA could be a potential biomarker for the diagnosis and prognosis in early NPC.

Gastric Cancer in the Era of Epigenetics.

Gastric cancer (GC) remains a significant contributor to cancer-related mortality. Novel high-throughput techniques have enlightened the epigenetic mechanisms governing gene-expression regulation. Epigenetic characteristics contribute to molecular taxonomy and give rise to cancer-specific epigenetic patterns. Helicobacter pylori (Hp) infection has an impact on aberrant DNA methylation either through its pathogenic CagA protein or by inducing chronic inflammation. The hypomethylation of specific repetitive elements generates an epigenetic field effect early in tumorigenesis. Epstein-Barr virus (EBV) infection triggers DNA methylation by dysregulating DNA methyltransferases (DNMT) enzyme activity, while persistent Hp-EBV co-infection leads to aggressive tumor behavior. Distinct histone modifications are also responsible for oncogene upregulation and tumor-suppressor gene silencing in gastric carcinomas. While histone methylation and acetylation processes have been extensively studied, other less prevalent alterations contribute to the development and migration of gastric cancer via a complex network of interactions. Enzymes, such as Nicotinamide N-methyltransferase (NNMT), which is involved in tumor's metabolic reprogramming, interact with methyltransferases and modify gene expression. Non-coding RNA molecules, including long non-coding RNAs, circular RNAs, and miRNAs serve as epigenetic regulators contributing to GC development, metastasis, poor outcomes and therapy resistance. Serum RNA molecules hold the potential to serve as non-invasive biomarkers for diagnostic, prognostic or therapeutic applications. Gastric fluids represent a valuable source to identify potential biomarkers with diagnostic use in terms of liquid biopsy. Ongoing clinical trials are currently evaluating the efficacy of next-generation epigenetic drugs, displaying promising outcomes. Various approaches including multiple miRNA inhibitors or targeted nanoparticles carrying epigenetic drugs are being designed to enhance existing treatment efficacy and overcome treatment resistance.

Role of Peroxiredoxin 1 Induced by Epstein-Barr Virus Infection in Nasopharyngeal Carcinoma.

BACKGROUND/AIM: Nasopharyngeal carcinoma (NPC), a common cancer in Southern China, is associated with Epstein-Barr Virus (EBV) infection. Although many therapies for NPC have been established, the definite role of EBV in NPC remains unclear. Therefore, this work focuses on LMP2A, a latent EBV gene, and investigates whether LMP2A is related to peroxiredoxin 1 (PRDX1) in EBV-positive NPC. MATERIALS AND METHODS: The mRNA and protein expression levels of LMP2A, PRDX1, and beta-catenin were compared in patient samples. To identify molecular mechanisms, EBV-negative NP69 and EBV-positive C666-1 NPC cell lines were used. After making an agar cell block for cell slides, the intensity of LMP2A expression was observed visually. To measure the level of reactive oxygen species, both fluorescence microscope and flow cytometry were used. To investigate the intracellular signaling molecular mechanisms with and without the LMP2A gene, reverse transcription polymerase chain reaction and western blotting were used. RESULTS: Both patient samples and cells of nasopharyngeal carcinoma infected with EBV had increased expression of LMP2A compared with controls, and high ROS levels were identified. Cell viability assay showed that LMP2A promoted cell growth by regulating gene expression. Furthermore, LMP2A induced the expression of PRDX1 and beta-catenin. LMP2A also increased the expression of both cyclin B1 and cyclin D1. CONCLUSION: In NPC cells, PRDX1 and beta-catenin were regulated through LMP2A expression, which reduced cell growth through cell cycle-related gene expression. This study suggests that LMP2A could be a target molecule for inhibiting cancer progression in NPC cells infected with EBV.

Peripheral T cell lymphoma initially presenting in lung biopsies: A diagnostic challenge.

BACKGROUND: Primary or secondary pulmonary involvement by peripheral T cell lymphoma (PTCL) is rare and difficult to diagnose particularly via lung biopsies. METHODS: 22 cases of PTCL diagnosed initially in lung biopsies between January 2006 and November 2020 were retrospectively reviewed followed at Nanjing Drum Tower Hospital and the First Affiliated Hospital of Zhengzhou University, respectively, including clinical manifestations, baseline biochemical indexes, images, histological findings and other available ancillary studies such as immunostaining, Epstein-Barr virus encoded RNA (EBER) in situ hybridization and T-cell receptor rearrangement analysis upon diagnosis. RESULTS: The median age of these patients was 59 years old (range: 29-82 years) at diagnosis. The majority of them complained of fever, cough and fatigue. Computed tomography scans mainly revealed multiple ill-defined nodules/masses of various sizes and densities with or without air bronchogram. Microscopically, most lesions showed lymphoid cells with clear cytoplasm and irregular nuclear contours diffusely infiltrating alveolar septa or alveolar spaces in an inflammatory background. Several cases had a predominance of small neoplastic cells (n = 4) with atypical, irregular nuclei. One case showed a diffuse monotonous pattern of growth. Angioinvasion and necrosis were not uncommon findings. The neoplastic cells in all cases were positive for one or more T-cell markers, and negative for B-cell-lineage antigens and EBER. 19 out of 22 patients had complete follow-up information, and 17 patients were dead at the last follow-up. CONCLUSIONS: Pulmonary involvement by PTCL is rare with dismal outcome. Aggressive clinical course and several clinicopathologic clues, albeit unspecific, may alert the pathologists of the possibilities of pulmonary PTCLs.

A novel multiplex real-time PCR assay for the detection of cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1/2 and strategies for application to blood screening.

A multiplex real-time PCR has been developed to simultaneously detect transfusion-transmissible pathogens cytomegalovirus, Epstein-Barr virus and herpes simplex virus, as well as to provide sample quality testing, for the conserved regions of the cytomegalovirus UL123 gene, the Epstein-Barr virus BKRF1 gene, and the herpes simplex virus 1/2 UL30 gene, tested on 500 blood donors and 320 transfusion recipients. The laboratory sensitivities for all 3 pathogens were 100 copies/µL. Compared to the commercial real-time PCR reference kit, the multiplex real-time PCR assay for the detection of CMV, EBV and HSV presented 100% consistency. In 820 whole blood samples, the multiplex real-time PCR assay identified 34 (4.15%) positive for CMV DNA, 15 (1.83%) positive for EBV DNA, and 6 (0.73%) positive for HSV DNA. For blood transfusions in high-risk groups, whole blood herpes virus test should be included in the spectrum of pathogen testing for blood donors and recipients.

Post cross-linked ROS-responsive poly(ß-amino ester)-plasmid polyplex NPs for gene therapy of EBV-associated nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common tumors in South China and Southeast Asia and is thought to be associated with Epstein-Barr virus (EBV) infection. Downregulation of latent membrane protein 1 (LMP1) encoded by EBV can reduce the expression of NF-κB and PI3K, induce apoptosis, and inhibit the growth of EBV-related NPC. For targeted cleavage of the Lmp1 oncogene via the CRISPR/Cas9 gene editing system, a post cross-linked ROS-responsive poly(ß-amino ester) (PBAE) polymeric vector was developed for the delivery of CRISPR/Cas9 plasmids both in vitro and in vivo. After composition optimization, the resultant polymer-plasmid polyplex nanoparticles (NPs) showed a diameter of ∼230 nm and a zeta potential of 22.3 mV with good stability. Compared with the non-cross-linked system, the cross-linked NPs exhibited efficient and quick cell uptake, higher transfection efficiency in EBV-positive C666-1 cells (53.5% vs. 40.6%), more efficient gene editing ability against the Mucin2 model gene (Muc2) (17.9% vs. 15.4%) and Lmp1 (8.5% vs. 5.6%), and lower intracellular reactive oxygen species (ROS) levels. The NPs achieved good tumor penetration and tumor growth inhibition in the C666-1 xenograft tumor model via Lmp1 cleavage, indicating their potential for gene therapy of EBV-related NPC.

Epstein-Barr Virus BRLF1 Induces PD-L1 Expression in Nasopharyngeal Carcinoma Cells.

Nasopharyngeal carcinoma (NPC) is a specific human malignancy with unique geographic distribution and genetic backgrounds. Although early treatment with radio-chemotherapy has been proven effective for NPC therapy, its therapeutic efficacy substantially diminishes in the late stages of this malignancy. In the tumor microenvironment of NPC, PD-L1 has been demonstrated as a critical factor in impairing T cell activation. As an etiological role for NPC development, it is found that Epstein-Barr virus (EBV) latent proteins upregulated PD-L1 expression. However, whether EBV lytic protein affects PD-L1 expression remains unclear. In this study, through monitoring the mRNA expression pattern of lytic genes and PD-L1 in EBV-positive NPC cell line NA, EBV immediately-early gene BRLF1(Rta) was found to have the potential for PD-L1 activation. Furthermore, we identified that Rta expression enhanced PD-L1 expression in mRNA and protein levels through quantitative real-time polymerase chain reaction and western blotting analysis. The luciferase reporter assay revealed that Rta expression enhanced PD-L1 promoter activity. We also demonstrated that Rta-induced PD-L1 expressions could impair interleukin 2 secretion of T cells, and this mechanism may be through ERK activation. These results displayed the importance of EBV Rta in PD-L1 expression in NPC and may give an alternative target for NPC therapy.

Lysine demethylase LSD1 is associated with stemness in EBV-positive B cell lymphoma.

EBV-infected lymphoma has a poor prognosis and various treatment strategies are being explored. Reports suggesting that B cell lymphoma can be induced by epigenetic regulation have piqued interest in studying mechanisms targeting epigenetic regulation. Here, we set out to identify an epigenetic regulator drug that acts synergistically with doxorubicin in EBV-positive lymphoma. We expressed the major EBV protein, LMP1, in B-cell lymphoma cell lines and used them to screen 100 epigenetic modifiers in combination with doxorubicin. The screening results identified TCP, which is an inhibitor of LSD1. Further analyses revealed that LMP1 increased the activity of LSD1 to enhance stemness ability under doxorubicin treatment, as evidenced by colony-forming and ALDEFLUOR activity assays. Quantseq 3' mRNA sequencing analysis of potential targets regulated by LSD1 in modulating stemness revealed that the LMP1-induced upregulation of CHAC2 was decreased when LSD1 was inhibited by TCP or downregulated by siRNA. We further observed that SOX2 expression was altered in response to CHAC2 expression, suggesting that stemness is regulated. Collectively, these findings suggest that LSD1 inhibitors could serve as promising therapeutic candidates for EBV-positive lymphoma, potentially reducing stemness activity when combined with conventional drugs to offer an effective treatment approach.

Pulmonary Epstein-Barr virus-associated smooth muscle tumor after kidney transplantation: two case reports with review of differential diagnosis.

Pulmonary nodules are a common complication in solid organ transplant recipients, and may have various underlying causes, with Epstein-Barr virus-associated smooth muscle tumor (EBV-SMT) being one of them. Given the rarity of this entity, we describe the diagnosis and therapeutic interventions for post-transplant EBV-SMT in two individuals. Both cases involved female patients who were diagnosed with multiple pulmonary nodules 60 months and 116 months, respectively, after receiving living-related kidney transplantation. Pathological examination revealed a spindle cell tumor, with immunophenotype and EBV in situ hybridization supporting the diagnosis of EBV-SMT. After diagnosis, these two patients underwent intervention by decreasing their intake of immunosuppressants. As of the latest follow-up, the patients' lesion size remained stable, and their overall condition was favorable. We also reviewed literature about the morphological and molecular pathological features of EBV-SMT and highlighted the diagnosis and differential diagnosis of pulmonary spindle cell lesions especially in the setting of immunosuppression.

Clinicopathological and molecular genetic alterations in monomorphic-epitheliotropic intestinal T-cell lymphoma of the small intestine.

BACKGROUND: Small intestinal monomorphic-epitheliotropic intestinal T-cell lymphoma (MEITL) is a rare aggressive T-cell lymphoma originating in the gastrointestinal tract. This study aimed to investigate the clinicopathological features, immunophenotypes, and molecular genetic changes of MEITL. METHODS: The clinicopathological data for three patients with surgically resected MEITL of the small intestine were collected. Next, immunohistochemical labeling, Epstein-Barr virus (EBV) in situ hybridization, assessment of clonal rearrangement of T-cell receptor (TCR) genes, and next-generation sequencing (NGS) were performed. RESULTS: Of the three patients, two were male and one was female, with ages of 61, 67, and 73 years, respectively. Clinical manifestations were predominantly abdominal pain and distension. Histopathology revealed infiltrative growth of small-to-medium-sized lymphocytes with a consistent morphology between the intestinal walls, accompanied by an obvious pro-epithelial phenomenon. The expression of CD3, CD8, CD43, CD56, TIA-1, CD103, H3K36me3, and Bcl-2 was detected, and the Ki-67 proliferation index ranged from 50% to 80%. All three patients tested negative for EBER. However, monoclonal rearrangement of the TCR gene was detected in them. NGS testing showed a JAK3 mutation in all three cases. Further, STAT5B, SETD2, and TP53 mutations were each observed in two cases, and a BCOR mutation was found in one case. All patients were treated with chemotherapy after surgery. Two patients died 7 and 15 month post-operation, and one patient survived for 5 months of follow-up. CONCLUSIONS: Our findings demonstrate that mutations in JAK3 and STAT5B of the JAK/STAT pathway and inactivation of the oncogene SETD2 markedly contribute to the lymphomagenesis of MEITL.