EBV-positive Nasopharyngeal Carcinoma Research Articles

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

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

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

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. 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. 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. 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.

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.

Combination of nivolumab with standard induction chemotherapy in children and adults with EBV-positive nasopharyngeal carcinoma : Protocol of aprospective multicenter phase2trial.

Treatment of Epstein-Barr virus(EBV)-positive nasopharyngeal carcinoma (NPC) with cisplatin/5-fluorouracil (5-FU) induction chemotherapy, followed by radiochemotherapy and subsequent interferon‑β, has yielded high survival rates in children, adolescents, and young adults. Aprevious study has shown that reduction of radiation dose from 59.4 to 54.0 Gy appears to be safe in patients with complete response (CR) to induction chemotherapy. As immune checkpoint-inhibitors have shown activity in NPC, we hypothesize that the addition of nivolumab to standard induction chemotherapy would increase the rate of complete tumor responses, thus allowing for areduced radiation dose in agreater proportion of patients. This is aprospective multicenter phase2 clinical trial including pediatric and adult patients with their first diagnosis of EBV-positive NPC, scheduled to receive nivolumab in addition to standard induction chemotherapy. In cases of non-response to induction therapy (stable or progressive disease), and in patients with initial distant metastasis, treatment with nivolumab will be continued during radiochemotherapy. Primary endpoint is tumor response on magnetic resonance imaging (MRI) and positron emission tomography (PET) after three cycles of induction chemotherapy. Secondary endpoints are event-free (EFS) and overall survival (OS), safety, and correlation of tumor response with programmed cell death ligand 1 (PD-L1) expression. As cure rates in localized EBV-positive NPC today are high with standard multimodal treatment, the focus increasingly shifts toward prevention of late effects, the burden of which is exceptionally high, mainly due to intense radiotherapy. Furthermore, survival in patients with metastatic disease and resistant to conventional chemotherapy remains poor. Primary objective of this study is to investigate whether the addition of nivolumab to standard induction chemotherapy in children and adults with EBV-positive NPC is able to increase the rate of complete responses, thus enabling a reduction in radiation dose in more patients, but also offer patients with high risk of treatment failure the chance to benefit from the addition of nivolumab. EudraCT (European Union Drug Regulating Authorities Clinical Trials Database) No. 2021-006477-32.

Diabetes can increase the prevalence of EBV infection and worsen the prognosis of nasopharyngeal carcinoma

Epstein‒Barr virus (EBV) infection is a primary oncogenic factor of nasopharyngeal carcinoma (NPC) that elicits epithelial-mesenchymal transition (EMT). Although diabetic patients are more susceptible to various infectious diseases, the pathological association with virus-related NPC has not yet been clarified. Herein, we evaluated the influence of diabetes on the clinicopathological changes of 70 patients with NPC. Disease-specific survival (DSS) modified by viral infection was also analysed. The proportion of NPC patients with diabetes was 32.9% (23/70 cases), and 91.3% (21/23 cases) were infected with EBV detected by EBER-I in situ hybridisation. NPC with diabetes showed an effect on EMT evaluated by immunostaining for E-cadherin and vimentin, which was correlated with HbA1c levels. Receiver operating characteristic (ROC) curve analysis determined a HbA1c level of 6.5% as the cut-off value for primary disease death at 2 years [area under the curve (AUC) 0.76; sensitivity 0.64; and specificity 0.81]. High HbA1c levels (≥6.5%) significantly increased the number of lymph node metastases in NPC compared to low HbA1c levels (<6.5%, p<0.01). Diabetic NPC patients had a significantly poorer prognosis than all non-diabetic patients (DSS, 72 months vs not reached, p<0.05). Diabetic EBV-positive NPC patients had a significantly poorer prognosis than non-diabetic EBV-positive patients (DSS, 35 months vs not reached, p<0.01). Multivariate analysis using the Cox proportional hazards model also suggested that HbA1c ≥6.5% was a significant factor in poor prognosis, with a hazard ratio of 6.84 (p<0.05). Collectively, our results revealed for the first time a high prevalence of EBV infection, poor prognosis and the importance of proper glycaemic control in diabetic NPC patients.

Abstract PO-016: SSTR2 use and molecular profile in EBV-positive nasopharyngeal carcinoma: A single institution experience

Abstract Background: Nasopharyngeal carcinoma (NPC) is a rare malignancy endemic in Southeast Asia. EBV infection is recognized as a major risk factor playing important roles in oncogenic pathways. Somatostatin receptor 2 (SSTR2) is a marker used in neuroendocrine tumors and appears to demonstrate high prevalence in EBV+ NPCs. Methods: We identified a total of 16 patients with biopsy-proven WHO type 3 EBV+ (via EBER) NPC in which SSTR2 expression via immunohistochemistry (IHC) was analyzed in either primary or metastatic lesions. Additionally, risk factors (smoking or alcohol), P16 via IHC, PD-L1 via IHC, tumor mutation burden (TMB), microsatellite instability (MSI), genomic alterations via Next Generation Sequencing (NGS), and time to progression (TTP) in months were reported. Results: Mean age of diagnosis was 60 years old, 14 were male, 13 were locally advanced and 37.5% former or heavy smokers. Ten patients were Asian with the rest being Latino (4), European (1) and unknown (1). Fourteen out of sixteen patients (87.5%) had SSTR2 expression either in primary lesions or metastatic lesions. P16 was measured in seven cases, all of them being negative. PDL1 was measured in 11 cases, 9 had high expression (≥ 20 CPS), 2 had expression (1-19 CPS) and 5 was not measured. In 10 cases TMB was evaluated, seven had low TMB and three intermediate TMB. From the TMB intermediate cases, all of them were MSI stable and two high PDL1 expression. From the 10 patients with MSI status all of them were stable. Out of the 16 patients, 11 patients had NGS performed from which 9 patients had clinically significant alterations. 4 out of 9 patients (44.4%) had an 11q13.2-13.3 amplification followed by 33.3% having a 9p21 loss and 22.2% TP53 mutation. Survival, median survival was not reached and median TTP was 23 months with the most common site being the liver. Conclusions: Given the high prevalence of SSTR2 in EBV+ NPCs (87.5%) in our cohort, SSTR2 via IHC may be a good screening tool for EBV + NPCs with potential diagnostic and therapeutic implications. 11q amplifications being present in 44.4% of our cohort might also be of diagnostic significance in NPC. Prognostic and clinical implications warrant further investigation. Citation Format: Juan J. Juarez-Vignon Whaley, Michelle Afkhami, Mykola Onyshchenko, Thomas Gernon, Robert Kang, Ellie Maghami, Sagus Sampath, Arya Amini, Diana Bell, Victoria M. Villaflor. SSTR2 use and molecular profile in EBV-positive nasopharyngeal carcinoma: A single institution experience [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-016.

Preclinical evaluation of the VEGF/Ang2 bispecific nanobody BI 836880 in nasopharyngeal carcinoma models.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is endemic to parts of Asia and overexpression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α are common in NPC. Anti-vascular agents have known clinical activity in patients with recurrent/ metastatic NPC and in this study, we investigated the anti-tumor effect of BI 836880, a humanized bispecific nanobody against VEGF and angiopoietin-2 (Ang2), in preclinical models of EBV-positive and EBV-negative NPC. The efficacy of BI 836880 was also compared with bevacizumab, a recombinant humanized monoclonal antibody against VEGF. We found that BI 836880 could exert growth-inhibitory effect on endothelial cells (HUVEC-C) and the EBV-negative NPC cell line (HK1), but to a lesser extent in the EBV-positive NPC cell lines, C17C and C666-1. In patients-derived xenograft (PDX) models of NPC - Xeno-2117 and Xeno-666, BI 836880 could suppress tumor growth and Ki67, as well as induce tumor necrosis and reduce microvessel density. Moreover, treatment with BI 836880 increased the level of macrophage infiltration in both PDX tumor models of NPC, suggesting that BI 836880 may exert immunomodulatory effect on the NPC immune microenvironment. When compared with bevacizumab, BI 836880 appeared to show at least comparable activity as bevacizumab in terms of its anti-proliferative and anti-angiogenic effects. This study showed that BI 836880 has anti-proliferative, anti-angiogenic and possibly immunomodulatory effect in clinical models of NPC, therefore the dual targeting of VEGF and Ang2 signaling in NPC should be further investigated.

TCR-T cells armored with immune checkpoint blockade in EBV-positive nasopharyngeal carcinoma: The first-in-human phase 1/2 trial.

6047 Background: Nasopharyngeal carcinoma (NPC) refraction and metastasis is common, but therapeutics are limited. As adoptive immunotherapy has emerged as effective against other cancers, engineered T cells bearing a transgenic Epstein-Barr virus (EBV)-specific TCR (TCR-T) represent a viable approach to treat EBV-associated NPC. Given PD-1/PD-L1 induced T cell hypofunction, we herein report preliminary findings of a phase I trial of EBV-targeting TCR-T cells armored with secreted PD-1 blockade for patients who failed in two or more lines of standard therapies. Methods: Patients with advanced NPCs were consented and screened for EBV serotype and HLA haplotype. EBV+/HLA-A*02+ patients were enrolled in a rapid titration setting to escalate the single infusion dose from 5x106 to 1.0x107 and 5.0x107 /kg TCR-T cells. After safety evaluation, the 5.0X107 /kg cohort was expanded for further investigation of combining with IL-2 administration. Fludarabine/cyclophosphamide were administered prior to TCR-T cell transfer as pre-conditioning. Patient monitoring and peripheral blood analysis occurred weekly over the first month and then monthly until disease progression or patient withdrawal. The primary objective was to determine safety and a recommended phase 2 dose (RP2D), while the secondary objective was investigator assessed ORR (RECIST v1.1). Results: One patient per TCR-T dose was treated, and, as no dose-limiting toxicity (DLT) has been observed, dose level 3 (5.0x107 /kg) was expanded with IL-2 administration upon TCR-T cell infusion. As of January 2023, six patients have been treated. One patient (16.7%) exhibited grade 1 CRS (n=6). No grade 4 treatment-related adverse events (TRAEs) have been observed, with leukopenia and fever being the most common AEs. Two patients (33.3%) were assessed as partial response (PR), with one reaching a response duration of 9 months to date. Three patients (50%) were assessed as stable disease (SD). For all patients, pharmacokinetic analysis revealed that levels of TCR-T cells in peripheral blood peak between 3- and 14-days post-infusion, with a maximum duration of 180 days. Conclusions: EBV-targeting TCR-T cells armored with PD-1 blockade are well tolerated. Initial results from this ongoing study indicate that EBV proteins may be safe and effective TCR-T targets to achieve superior outcomes in advanced EBV-positive NPC patients. Clinical trial information: NCT04139057 . [Table: see text]

Clinical efficacy and biomarker analysis of dual PD-1/CTLA-4 blockade in recurrent/metastatic EBV-associated nasopharyngeal carcinoma

Single-agent checkpoint inhibitor (CPI) activity in Epstein-Barr Virus (EBV) related nasopharyngeal carcinoma (NPC) is limited. Dual CPI shows increased activity in solid cancers. In this single-arm phase II trial (NCT03097939), 40 patients with recurrent/metastatic EBV-positive NPC who failed prior chemotherapy receive nivolumab 3 mg/kg every 2 weeks and ipilimumab 1 mg/kg every 6 weeks. Primary outcome of best overall response rate (BOR) and secondary outcomes (progression-free survival [PFS], clinical benefit rate, adverse events, duration of response, time to progression, overall survival [OS]) are reported. The BOR is 38% with median PFS and OS of 5.3 and 19.5 months, respectively. This regimen is well-tolerated and treatment-related adverse events requiring discontinuation are low. Biomarker analysis shows no correlation of outcomes to PD-L1 expression or tumor mutation burden. While the BOR does not meet pre-planned estimates, patients with low plasma EBV-DNA titre (<7800 IU/ml) trend to better response and PFS. Deep immunophenotyping of pre- and on-treatment tumor biopsies demonstrate early activation of the adaptive immune response, with T-cell cytotoxicity seen in responders prior to any clinically evident response. Immune-subpopulation profiling also identifies specific PD-1 and CTLA-4 expressing CD8 subpopulations that predict for response to combined immune checkpoint blockade in NPC.

Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients.

Epstein-Barr virus (EBV) IgA and IgG antibodies in serum from nasopharyngeal carcinoma (NPC) patients are well-established markers for EBV-positive NPC. Luminex-based multiplex serology can analyze antibodies to multiple antigens simultaneously; however, the detection of both IgA and IgG antibodies requires separate measurements. Here we describe the development and validation of a novel duplex multiplex serology assay, which can analyze IgA and IgG antibodies against several antigens simultaneously. Secondary antibody/dye combinations, as well as serum dilution factors, were optimized, and 98 NPC cases matched to 142 controls from the Head and Neck 5000 study (HN5000) were assessed and compared to data previously generated in separate IgA and IgG multiplex assays. EBER in situ hybridization (EBER-ISH) data available for 41 tumors was used to calibrate antigen-specific cut-offs using receiver operating characteristic (ROC) analysis with a prespecified specificity of ≥90%. A directly R-Phycoerythrin-labeled IgG antibody in combination with a biotinylated IgA antibody and streptavidin-BV421 reporter conjugate was able to quantify both IgA and IgG antibodies in a duplex reaction in a 1:1000 serum dilution. The combined assessment of IgA and IgG antibodies in NPC cases and controls from the HN5000 study yielded similar sensitivities as the separate IgA and IgG multiplex assays (all > 90%), and the duplex serological multiplex assay was able to unequivocally define the EBV-positive NPC cases (AUC = 1). In conclusion, the simultaneous detection of IgA and IgG antibodies provides an alternative for the separate IgA/IgG antibody quantification and may present a promising approach for larger NPC screening studies in NPC endemic areas.

Long-Term Outcomes of Nasopharyngeal Carcinoma by Epstein-Barr Virus Status in the Chinese Population: A Multicenter Investigation.

Background: Because the vast majority of nasopharyngeal carcinoma (NPC) in Chinese patients is a direct result of Epstein-Barr virus (EBV) infection, there is a dearth of data for EBV-negative patients in this population. This multicenter study sought to examine the clinical characteristics of EBV-negative patients and compare long-term outcomes with a propensity-matched (1:1.5) EBV-positive cohort. Methods: NPC patients with known EBV status from four hospitals were collated (2013-2021). A logistic regression model was conducted to evaluate the relationship between patient characteristics and EBV status. The Kaplan-Meier method and Cox regression analysis were used to analyze survival data. Results: This study analyzed 48 (40%) EBV-negative and 72 (60%) EBV-positive patients. The median follow-up time was 63.5 months. Most EBV-negative NPC patients (77.1%) were diagnosed in advanced stages with a higher rate (87.5%) of positive lymph node disease, and no significant prognostic factors were discerned in this subpopulation. The EBV-negative disease was more associated with the keratinizing subtype (18.8% vs. 1.4%, p < 0.05). Compared to EBV-negative NPC patients, EBV-positive NPC patients were more likely to develop a local recurrence (9.7% vs. 0%, p = 0.026). There was no statistical difference in mortality (EBV-negative vs. EBV- positive, 8.3% vs. 4.2%, p = 0.34) during the follow-up period. Although the median PFS and median OS were not reached, the 3-year PFS rate was 68.8% vs. 70.8% (EBV-negative vs. EBV-positive, p = 0.06), the 3-year OS rate was 70.8% vs. 76.4% (EBV-negative vs. EBV-positive, p = 0.464), the 5-year PFS rate was 56.3% vs. 50% (EBV-negative vs. EBV-positive, p = 0.451), and the 5-year OS rate was 56.3% vs. 58.3% (EBV-negative vs. EBV-positive, p = 0.051), respectively. These data show that EBV-positive NPC patients seem to have a tendency to gain better survival compared with EBV-negative NPC patients. Conclusions: Most of the EBV-negative patients were in the middle and late stages at the time of diagnosis and were more associated with the keratinizing subtype. EBV status may be associated with prognosis in NPC. EBV positivity seems to be associated with better survival in NPC patients. Still, due to the small cohort of patients and the short observation period for a number of patients, further work is required to corroborate these conclusions.

Abstract 4716: Characterization of 3D genome in nasopharyngeal carcinoma (NPC)

Abstract Background: Epstein-Barr virus (EBV) can be detected in more than 90% of NPC patients in Hong Kong. It has been shown that EBV infection could hijack the human epigenome in B cell lymphoma and EBV+ gastric cancer, however, the role of EBV in epigenetic dysregulation remains largely unknown in NPC. Recently, our epigenomic landscape study demonstrated EBV was associated with changes of chromatin accessibility in NPC, which were largely involved in the binding sites of CTCF, a master regulator of chromosome organization. Therefore, we aim to further characterize the 3D chromatin structure in NPC. Methods: We used the Hi-C (Omni-C Seq) to profile the chromatin interactions in the EBV-positive (EBV+) NPC, EBV-negative (EBV-) NPC, and normal immortalized epithelial (NPE) cell lines. The Omni-C Seq data were integrated with the ATAC-Seq, RNA-Seq, and CUT&amp;RUN-Seq data. The histone markers including H3K4me3, H3K27ac, and H3K27me3, were applied to classify the active and inactive status of the chromatins. The chromatin looping and topologically associated domains (TADs) were defined by the CTCF binding sites. Results: We observed a much larger compartmentalization size in EBV+ NPC with a consistent increase of active to inactive (A to B) compartments, compared to NPE and EBV- NPC. Pathway enrichment analysis showed that these changes potentially influenced immune response, cell differentiation and development, metabolism, ERBB pathway, and calcium signaling transduction. In addition to the changes in compartmentalization, we observed a reduction of CTCF binding signals at the TADs boundaries and the loss of chromatin looping at the 10kb resolution. Consistently, the aggregate peak analysis has shown a significant reduction of the CTCF anchor, indicating the abandonment of architectural CTCF in EBV+ NPC. More importantly, we found EBV tethering to the host chromatins. In the EBV genome, the tethering sites were often located at the regions associated with OriP, EBNA1, and BARTs. In the human genome, the tethering sites were dramatically enriched in the inactive to active (B to A) compartments, which were highly associated with the enhancer regions with H3K27ac. We identified a specific target gene CD74 with EBV tethering at the nearby super-enhancer region. Our results suggest the upregulation of CD74 could be controlled via remodulating the host 3D genome in EBV+ NPC. The spatial transcriptome analysis indicated that the expression of CD74 was highly correlated with HLA-DRA. The spatial analysis further showed that the NPC patients with upregulated HLA-DRA often had de novo distant metastasis after treatment (p=0.007). Conclusions: The EBV tethering to the host genome has a significant impact on host 3D genome organization, affecting chromatin accessibility and gene regulation, providing survival advantages of cancer cell growth and potentially contributing to immune suppression and metastasis. Citation Format: Dittman Lai-Shun Chung, Larry Ka-Yue Chow, Wei Dai. Characterization of 3D genome in nasopharyngeal carcinoma (NPC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4716.

Comparative Analysis of the Humoral Immune Response to the EBV Proteome across EBV-Related Malignancies.

Epstein-Barr virus (EBV) is linked to multiple cancers, including classical Hodgkin lymphoma (cHL), endemic Burkitt lymphoma (eBL), nasopharyngeal carcinoma (NPC), and extranodal natural killer/T-cell lymphoma (NKTCL). Anti-EBV IgG and IgA antibody responses targeting 202 sequences from 86 EBV proteins were measured using the same EBV whole proteome array across four case-control studies investigating EBV-positive cHL, eBL, NPC, and NKTCL (407 cases/620 controls). We grouped EBV-targeted antibodies into pathways by immunoglobulin type (IgA and IgG) and life-cycle stage (latent, immediate early lytic, early lytic, late lytic, and glycoprotein) and evaluated their association with each cancer type. In an additional analysis, we focused on the subset of 46 individual antibodies representing the top candidates for each cancer and compared their associations across the four cancer types using multivariable linear regression models. IgA antibody responses targeting all EBV life-cycle stages were associated with NPC but limited to anti-early lytic stage for cHL. NPC and eBL were associated with IgG antibodies across the viral life cycle; cHL with antibodies in the early lytic, late lytic and glycoprotein stages; and NKTCL with antibodies in the latent, immediate early lytic and early lytic phases. EBNA3A, BBLF1, BDLF4, and BLRF2 IgG antibodies were associated with all cancer types. Our observed similarities and differences across four EBV-associated cancers may inform EBV-related oncogenesis. Understanding the comparative humoral immune response across EBV-related cancers may aid in identifying shared etiologic roles of EBV proteins and inform unique pathogenic processes for each cancer.

Synergistic Association of Hepatitis B Surface Antigen and Plasma Epstein-Barr Virus DNA Load on Distant Metastasis in Patients With Nasopharyngeal Carcinoma

Hepatitis B surface antigen (HBsAg) reportedly increases the risk of distant metastasis among patients with nasopharyngeal carcinoma (NPC). However, the associated potential interaction and changes in hazard ratios (HRs) between HBsAg and different plasma Epstein-Barr (EBV) DNA levels are unknown. Moreover, the potential HBsAg-positive-associated NPC metastatic mechanism remains unclear. To investigate the prognostic value and biological associations of HBsAg and plasma EBV DNA levels on distant metastasis in patients with NPC. Retrospective cohort study performed at Sun Yat-sen University Cancer Center between January 2010 and January 2013. A total of 792 patients with nonmetastatic NPC were enrolled. The median (range) follow-up time was 62.1 (1.4-83.4) months. Of these patients, 17.8% presented with HBsAg positivity. Cytological experiments were performed to evaluate the role of HBsAg in the invasion and migration of EBV-positive NPC cells. Data analysis was performed from July 2020 to April 2021. The primary end point was distant metastasis-free survival. Association rules were used to identify new rules related to distant metastasis. Interaction plots, univariate and multivariate Cox regression analyses, stratification analysis, and quantification using HRs were conducted. Additionally, cell migration and invasion assays, as well as Western blotting, were performed in the cytological validation. Among the 792 patients, 576 (72.7%) were male, with a median (IQR) age of 45 (38-53) years. The HBsAg-positive group exhibited a significant interaction and increased risk of distant metastasis when plasma EBV DNA cutoff levels were 1.5 × 1000 copies/mL or greater. The HR was 9.16 (95% CI, 2.46-34.14) when the plasma EBV DNA load reached 6 × 1000 copies/mL, which was higher than that in patients with stage IV disease (HR, 2.01; 95% CI, 1.13-3.56; P = .02). In cytological experiments, HBsAg promoted epithelial-mesenchymal transition by upregulating vimentin and fibronectin in EBV-positive NPC cells in vitro, thereby promoting invasion and migration of EBV-positive NPC cells. In this cohort study, the observed synergistic association between HBsAg and plasma EBV DNA load represented a novel potential mechanism underlying the increased risk of distant metastasis in patients with NPC. Hence, attention should be paid to patients with NPC with HBsAg positivity, especially when the plasma EBV DNA level is 6 × 1000 copies/mL or greater. Consideration of this synergistic association will contribute to more accurate individualized management.

EBV LMP1-C terminal binding affibody molecule downregulates MEK/ERK/p90RSK pathway and inhibits the proliferation of nasopharyngeal carcinoma cells in mouse tumor xenograft models.

Nasopharyngeal carcinoma (NPC), is an Epstein-Barr virus (EBV) associated malignancy most common in Southern China and Southeast Asia. In southern China, it is one of the major causes of cancer-related death. Despite improvement in radiotherapy and chemotherapy techniques, locoregional recurrence and distant metastasis remains the major causes for failure oftreatment in NPC patients. Therefore, finding new specific drug targets for treatment interventions are urgently needed. Here, we report three potential ZLMP1-C affibody molecules (ZLMP1-C15, ZLMP1-C114 and ZLMP1-C277) that showedspecific binding interactions for recombinant and native EBV LMP1as determined by epitope mapping, co-localization and co-immunoprecipitation assays. The ZLMP1-C affibody molecules exhibited high antitumor effects on EBV-positive NPC cell lines and displayed minimal cytotoxicity towards EBV-negative NPC cell line. Moreover, ZLMP1-C277 showed higher antitumor efficacy than ZLMP1-C15 and ZLMP1-C114 affibody molecules. The ability of ZLMP1-C277 decrease the phosphorylation levels of up-stream activator phospho-Raf-1(Ser338), phospho-MEK1/2(Ser217/Ser221), phospho-ERK1/2(Thr202/Thr204), thereby leading to downstream suppression of phospho-p90RSK(Ser380) and transcription factor c-Fos. Importantly, tumor growth was reduced in tumor-bearing mice treated with ZLMP1-C277 and caused no apparent toxicity. Taken together, our findings provide evidence that ZLMP1-C277 as a promising therapeutic agent in EBV-associated NPC.

Preclinical study of LMP1-RNAi-based anti-tumor therapy in EBV-positive nasopharyngeal carcinoma.

RNA interference (RNAi) treatment has been proven to be an important therapeutic approach in cancer based on downregulation of target-oncogenes, but its clinical efficacy still needs further investigation. LMP1 is usually presented by Epstein-Barr virus (EBV)-positive tumor cells like EBV-associated nasopharyngeal carcinoma (NPC) and acts as an oncogene in tumorigenesis. However, the mechanism of LMP1 as a proto-oncogene in nasopharyngeal carcinoma is still unclear. Two sequence-specific shRNAs 1 and 2 were designed to target the different nucleotide loci of EBV latent antigen LMP1 gene and a series of in vivo and in vitro experiments were performed to investigate the therapeutic effect of sequence-specific shRNAs targeting LMP1 and its related molecular mechanisms in EBV-positive NPC. LMP1-shRNA2 generated a truncated LMP1 mRNA and protein, whereas LMP1-shRNA1 completely blocked LMP1 mRNA and protein expression. Both LMP1-shRNAs inhibited the proliferation and migration of NPC cells overexpressing LMP1 (NPC-LMP1) as well as the NPC-associated myeloid-derived suppressor cell (MDSC) expansion in vitro. However, LMP1-shRNA2 maintained the immunogenicity of NPC-LMP1 cells, which provoked MHC-class I-dependent T cell recognition. LMP1-shRNAs inhibited tumor growth in nude mice but did not reach statistical significance compared to control groups, while the LDH nanoparticle loaded LMP1-shRNAs and the antigen-specific T cells induced by NPC-LMP1 cells treated with LMP1-shRNA2 significantly reduced tumor growth in vivo. LMP1-RNAi-based anti-tumor therapy could be a new hope for the clinical efficacy of RNAi treatment of tumors like NPC.

Abstract 5762: Genomic correlates of response to immune checkpoint blockade in Epstein-Barr virus-associated pulmonary lymphoepithelioma-like carcinoma

Abstract Introduction: Pulmonary lymphoepithelioma-like carcinoma (PLELC) is a rare and distinct subtype of non-small cell lung cancer (NSCLC). The use of immune checkpoint inhibitors (ICIs) for advanced PLELC and tumor genomic features underlying varied treatment responses remains poorly investigated. Methods: The study involved 33 PLELC patients with clinically annotated outcomes to ICIs. Targeted sequencing covering 520 cancer related genes were performed in 22 patients to identify single nucleotide variations (SNVs), copy number variations (CNVs), tumor mutation burden (TMB) and microsatellite instability. Mutation profiles of PLELC were compared with other NSCLC subtypes including lung adenocarcinoma (LUAD) and squamous cell lung carcinoma (LUSC) as well as EBV (Epstein-Barr virus) positive nasopharyngeal carcinoma (NPC) in public databases. Associations between PLELC mutation profiles and treatment responses to ICIs were analyzed. An external dataset of ICI-treated melanoma was used in further analysis of B2M. Results: The objective response rate (ORR), disease control rate (DCR), durable clinical benefit (DCB) rate, and median PFS was 18.2%, 60.6%, 45.5%, and 4.5 months respectively. Genetic alterations were frequently identified in B2M (9 of 22, 41%), CDKN1B (7 of 22, 32%), TP53 (6 of 22, 27%), SGK1 (5 of 22, 23%), NFKBIA (4 of 22, 18%) and TNFAIP3 (4 of 22, 18%). CNVs were comprehensively presented in PLELC while SVNs were less common. In our study, 8 of 9 tumors with B2M mutations and all tumors with CDKN1B, SGK1, TNFAIP3 mutations harbored copy number amplifications. Mutation profiles of PLELC were largely different from those of LUAD and LUSC but were similar to NPC. The median TMB was 2.99 mutations/Mb. All tumors were microsatellite stable (MSS). Patients with higher TMB showed improved DCR (p=0.029), PFS (p=0.056) and overall survival (p=0.038) than those with lower TMB. Patients with lower baseline plasma EBV DNA level also had longer PFS (p&amp;lt;0.001). The presence of B2M amplifications was significant associated with higher TMB level (p=0.022). The PFS of 7 patients with high TMB and amplified B2M were significantly longer than that of other patients (p=0.046). In the external dataset, higher expression of B2M was associated with better ICI responses for melanoma (p&amp;lt;0.05). Pathways involving anti-tumor immune responses such as NK cell-mediated cytotoxicity, antigen processing and presentation were correlated with high B2M expression while Wnt, TGF-beta and MAPK pathways that define poor response to ICI were correlated with low B2M expression. Conclusion: Our study observed comprehensive CNVs in PLELC patients which is a unique molecular feature compared with other NSCLC subtypes and EBV-positive NPC. Higher TMB level and amplifications of B2M in PLELC patients may be used as potential biomarkers to indicate better treatment responses to ICIs. Citation Format: Li-na He, Xuanye Zhang, Xinze Lv, Yuxi Zhou, Min Li, Ting Hou, Wenjie Sun, Li Zhang, ShaoDong Hong. Genomic correlates of response to immune checkpoint blockade in Epstein-Barr virus-associated pulmonary lymphoepithelioma-like carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5762.

MDM2 is a useful diagnostic marker for nasopharyngeal carcinoma

BackgroundThe MDM2 gene appears to be involved in the development of nasopharyngeal carcinoma. The aim of this study was to examine MDM2 expression in a series of nasopharyngeal carcinoma biopsies to explore its potential diagnostic significance. MethodsThe study cohort consisted of 26 nasopharyngeal carcinomas, including 22 EBV positive non-keratinizing squamous cell carcinomas (NKSCC), 1 EBV negative NKSCC and 3 EBV negative keratinizing SCC. For comparison, we selected 48 oropharyngeal carcinomas, including 17 HPV positive SCC (14 non-keratinizing and 3 keratinizing) and 31 HPV negative SCCs (28 keratinizing and 3 non-keratinizing). In addition, we examined MDM2 expression in a group of 26 cervical lymph node metastases, including 5 with EBV positive nasopharyngeal NKSCC and 21 from oropharyngeal carcinoma (18 non keratinizing HPV positive, 1 keratinizing HPV positive, 1 keratinizing HPV negative and 1 non-keratinizing HPV negative). Finally, 2 bone metastases from EBV positive nasopharyngeal NKSCC were also included. A tissue microarray was constructed from formalin-fixed paraffin embedded tumor tissue specimens. Sections were immunostained for MDM2 and in situ hybridization for EBER and CISH analysis for the MDM2 gene were also conducted in all cases. ResultsOverall, MDM2 positivity was detected in 28 of 102 SCCs (27.2 %). MDM2 positivity was significantly more frequent in EBV positive NKSCC (80 %) than in oropharyngeal HPV positive NKSCC (6.1 %) and keratinizing SCCs (9.4 %) (p < 0.001, Pearson chi square). Considering only the primary tumors, 86.4 % of the nasopharyngeal carcinomas were positive, versus 13.5 % of the oropharyngeal carcinomas (p < 0.001, Pearson chi square). Considering the lymph node metastases, 3 of 5 EBV positive carcinomas with nasopharyngeal primary were positive, whereas only one of the HPV positive carcinomas was positive. Finally, both the bone metastases from EBV positive nasopharyngeal carcinoma were positive for MDM2. No amplification of the MDM2 gene was identified by in situ hybridization analysis. ConclusionsOur data indicate that MDM2 could be a valuable diagnostic marker to support the diagnosis of nasopharyngeal EBV positive NKSCC.

Exosomal HMGA2 protein from EBV-positive NPC cells destroys vascular endothelial barriers and induces endothelial-to-mesenchymal transition to promote metastasis

Increased vascular permeability facilitates metastasis. Cancer-secreted exosomes are emerging mediators of cancer-host crosstalk. Epstein-Barr virus (EBV), identified as the first human tumor-associated virus, plays a crucial role in metastatic tumors, especially in nasopharyngeal carcinoma (NPC). To date, whether and how exosomes from EBV-infected NPC cells affect vascular permeability remains unclear. Here, we show that exosomes from EBV-positive NPC cells, but not exosomes from EBV-negative NPC cells, destroy endothelial cell tight junction (TJ) proteins, which are natural barriers against metastasis, and promote endothelial-to-mesenchymal transition (EndMT) in endothelial cells. Proteomic analysis revealed that the level of HMGA2 protein was higher in exosomes derived from EBV-positive NPC cells compared with that in exosomes derived from EBV-negative NPC cells. Depletion of HMGA2 in exosomes derived from EBV-positive NPC cells attenuates endothelial cell dysfunction and tumor cell metastasis. In contrast, exosomes from HMGA2 overexpressing EBV-negative NPC cells promoted these processes. Furthermore, we showed that HMGA2 upregulates the expression of Snail, which contributes to TJ proteins reduction and EndMT in endothelial cells. Moreover, the level of HMGA2 in circulating exosomes is significantly higher in NPC patients with metastasis than in those without metastasis and healthy negative controls, and the level of HMGA2 in tumor cells is associated with TJ and EndMT protein expression in endothelial cells. Collectively, our findings suggest exosomal HMGA2 from EBV-positive NPC cells promotes tumor metastasis by targeting multiple endothelial TJ and promoting EndMT, which highlights secreted HMGA2 as a potential therapeutic target and a predictive marker for NPC metastasis.

EBV miRNAs BART11 and BART17-3p promote immune escape through the enhancer-mediated transcription of PD-L1

Epstein-Barr virus (EBV) is reportedly the first identified human tumor virus, and is closely related to the occurrence and development of nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and several lymphomas. PD-L1 expression is elevated in EBV-positive NPC and GC tissues; however, the specific mechanisms underlying the EBV-dependent promotion of PD-L1 expression to induce immune escape warrant clarification. EBV encodes 44 mature miRNAs. In this study, we find that EBV-miR-BART11 and EBV-miR-BART17-3p upregulate the expression of PD-L1 in EBV-associated NPC and GC. Furthermore, EBV-miR-BART11 targets FOXP1, EBV-miR-BART17-3p targets PBRM1, and FOXP1 and PBRM1 bind to the enhancer region of PD-L1 to inhibit its expression. Therefore, EBV-miR-BART11 and EBV-miR-BART17-3p inhibit FOXP1 and PBRM1, respectively, and enhance the transcription of PD-L1 (CD274, http://www.ncbi.nlm.nih.gov/gene/29126), resulting in the promotion of tumor immune escape, which provides insights into potential targets for EBV-related tumor immunotherapy.