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Previously, we showed that quantification of lymphoma‐associated miRNAs miR‐155‐5p, ‐127‐3p and let‐7a‐5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV‐based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV‐miRNA quantitative real‐time reverse‐transcription PCR (q‐RT‐PCR) assay in an ISO‐13485 certified quality‐management system (QMS). Liposomes encapsulated with a synthetic (nematode‐derived) miRNA spike‐in controlled for EV isolation by automated size‐exclusion chromatography (SEC). Additional miRNA spike‐ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed‐effect modelling with bootstrapping determined the diagnostic performance of the quality‐controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76–0.92) compared to an AUC of 0.87 (CI: 0.80–0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality‐controlled plasma EV‐miRNA assay is technically robust, taking EV‐miRNA as liquid biopsy assay an important step closer to clinical evaluation. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) persists in nasopharyngeal (NPC) and gastric carcinomas (EBVaGC) in a tightly latent form. Cytolytic virus activation (CLVA) therapy employs gemcitabine and valproic acid (GCb+VPA) to reactivate latent EBV into the lytic phase and antiviral valganciclovir to enhance cell death and prevent virus production. CLVA treatment has proven safe in phase-I/II trials with promising clinical responses in patients with recurrent NPC. However, a major challenge is to maximize EBV lytic reactivation by CLVA. Curcumin, a dietary spice used in Asian countries, is known for its antitumor property and therapeutic potential. Novel curcuminoids that were developed to increase efficacy and bioavailability may serve as oral CLVA adjuvants. We investigated the potential of curcumin and its analogs (curcuminoids) to trigger the EBV lytic cycle in EBVaGC and NPC cells. EBV-reactivating effects were measured by immunoblot and immunofluorescence using monoclonal antibodies specific for EBV lytic proteins. Two of the hit compounds (41, EF24) with high lytic inducing activity were further studied for their synergistic or antagonistic effects when combined with GCb+VPA and analyzed by cytotoxicity and mRNA profiling assays to measure the EBV reactivation. Curcuminoid as a single agent significantly induced EBV reactivation in recombinant GC and NPC lines. The drug effects were dose- and time-dependent. Micromolar concentration of curcuminoid EF24 enhanced the CLVA effect in all cell systems except SNU719, a naturally infected EBVaGC cell that carries a more tightly latent viral genome. These findings indicated that EF24 has potential as EBV lytic activator and may serve as an adjuvant in CLVA treatment. [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC) is the most prevalent head and neck cancer in Indonesia, with 100% Epstein-Barr virus (EBV) infection in tumor cells. NPC is rare in the Netherlands. The involvement of EBV in NPC pathogenesis is reflected by early onset aberrant IgA antibody responses to various EBV proteins. Screening for elevated EBV-IgA levels is proposed for NPC risk assessment in endemic countries but is poorly studied in nonendemic regions. This study analyzed the overall diversity (immunoblot) as well as the prevalence and normalized levels of IgA responses to immunodominant peptide epitopes of EBV proteins VCA P18, EBNA 1, and Zebra (Zta) (N-terminus, P 125, P 130, full-length recombinant Zebra) in Indonesian ( n =50) and Dutch ( n =50) patients with NPC. The results confirmed that elevated levels of IgA-VCA P18 and IgA-EBNA 1 were found in both NPC populations, but that IgA-Zta was more variable. IgA-Zta responses were more pronounced in Indonesian NPC cases, reflecting more frequent EBV reactivation overall. IgA-VCA P18 and IgA-EBNA are independent tumor markers and are both necessary for NPC risk assessment. Overall, these results confirmed the diagnostic benefit of combined IgA-VCA P18/-EBNA 1 testing for NPC risk assessment in endemic and nonendemic populations., Competing Interests: The authors have declared that no conflicting interests exist in this study.Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Cell-free microRNA (miRNA) in biofluids released by tumors in either protein or vesicle-bound form, represent promising minimally-invasive cancer biomarkers. However, a highly abundant non-tumor background in human plasma and serum complicates the discovery and detection of tumor-selective circulating miRNAs. We performed small RNA sequencing on serum and plasma RNA from Nasopharyngeal Carcinoma (NPC) patients. Collectively, Epstein Barr virus-encoded miRNAs, more so than endogenous miRNAs, signify presence of NPC. However, RNAseq-based EBV miRNA profiles differ between NPC patients, suggesting inter-tumor heterogeneity or divergent secretory characteristics. We determined with sensitive qRT-PCR assays that EBV miRNAs BART7-3p, BART9-3p and BART13-3p are actively secreted by C666.1 NPC cells bound to extracellular vesicles (EVs) and soluble ribonucleoprotein complexes. Importantly, these miRNAs are expressed in all primary NPC tumor biopsies and readily detected in nasopharyngeal brushings from both early and late-stage NPC patients. Increased levels of BART7-3p, BART9-3p and particularly BART13-3p, distinguish NPC patient sera from healthy controls. Receiver operating characteristic curve analysis using sera from endemic NPC patients, other head and neck cancers and individuals with asymptomatic EBV-infections reveals a superior diagnostic performance of EBV miRNAs over anti-EBNA1 IgA serology and EBV-DNA load (AUC 0.87-0.96 vs 0.86 and 0.66 respectively). The high specificity of circulating EBV-BART13-3p (97%) for NPC detection is in agreement with active secretion from NPC tumor cells. We conclude EV-bound BART13-3p in circulation is a promising, NPC-selective, biomarker that should be considered as part of a screening strategy to identify NPC in endemic regions., (© 2018 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Purpose: Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) and highly prevalent in Indonesia. EBV-DNA load can be used for early diagnosis and may have prognostic value. In this study, EBV-DNA load was evaluated in minimal invasive nasopharyngeal (NP) brushings and whole blood for initial diagnosis and therapy assessment against the standard-of-care diagnosis by biopsy with EBV-RISH and standard EBV-IgA serology., Experimental Design: NP brushings and blood samples were collected from 289 consecutive ENT patients suspected of NPCs and 53 local healthy controls. EBV-DNA load was quantified by real-time PCR and serology by peptide-based EBV-IgA ELISA. Tissue biopsies were examined by routine histochemistry and by EBER RNA in situ hybridization., Results: Repeated NP brushing was well tolerated by patients and revealed high viral load in the 228 NPC cases at diagnosis than 61 non-NPC cancer cases and healthy controls (P < 0.001). The diagnostic value of EBV-DNA load in blood and EBV-IgA serology was inferior to the NP brush results. The level of EBV-DNA load in brushes of patients with NPC was not related to T, N, or M stage, whereas elevated EBV-DNA load in blood correlated with N and M stage. EBV-DNA levels in brushings and whole blood showed a significant reduction at 2 months after treatment (P = 0.001 and P = 0.005, respectively), which was not reflected in EBV-IgA serology., Conclusions: NP brush sampling combined with EBV-DNA load analysis is a minimal invasive and well-tolerated diagnostic procedure, suited for initial diagnosis and follow-up monitoring of NPCs.

Purpose: Nasopharyngeal carcinoma (NPC) is causally linked to Epstein-Barr virus (EBV) infection. Because all tumor cells carry EBV, the virus itself is a potential target for therapy. In these tumor cells, EBV hides in a latent state and expresses only a few non-immunogenic proteins for EBV maintenance and contributes to tumor growth. We developed a cytolytic virus activation (CLVA) therapy for NPC treatment, reactivating latent EBV, triggering immune recognition, and inducing susceptibility to antiviral therapy., Experimental Design: CLVA therapy combines gemcitabine (GCb) and valproic acid (VPA) for virus activation and tumor clearance with (val)ganciclovir (GCV) as the antiviral drug to block virus replication and kill proliferating virus-infected cells. CLVA treatment was optimized and validated in NPC cell lines and subsequently tested in 3 Dutch patients with NPC that was refractory to conventional treatment., Results: In NPC cell lines, both GCb and VPA can induce the lytic cycle of EBV. Their combination resulted in a strong synergistic effect. The addition of GCV resulted in higher cytotoxicity compared with chemotherapy alone, which was not observed in EBV-negative cells. CLVA therapy was analyzed in 3 patients with end-stage NPC. Patients developed increased levels of viral DNA in the circulation originating from apoptotic tumor cells, had disease stabilization, and experienced improved quality of life., Conclusions: Our results in the initial CLVA-treated patients indicate that the therapy had a biological effect and was well tolerated with only moderate transient toxicity. This new virus-specific therapy could open a generic approach for treatment of multiple EBV-associated malignancies., (©2012 AACR.)

Objective: Epstein-Barr virus (EBV)-positive lymphomas in HIV carriers are paralleled by elevated EBV-DNA loads in the circulation. Approximately 20% of asymptomatic HIV carriers also show elevated circulating EBV-DNA loads. We aimed to characterize the nature of this EBV DNA and to determine the transcriptional phenotype of EBV in blood, in relation to serological parameters., Design: A total of 197 random asymptomatic HIV carriers, representing 2% of the Dutch HIV-positive population, were sampled for blood, peripheral blood mononuclear cells and plasma. In addition, 39 EBV-DNA carriers were sampled twice, with a 5-year interval., Methods: EBV-DNA loads were determined by LightCycler-based real-time polymerase chain reaction (PCR). EBV transcription was studied by nucleic acid sequence-based amplification and reverse transcriptase PCR. IgA and IgG antibodies to EBV antigens EBNA1 and VCA-p18 were quantified by synthetic peptide-based enzyme-linked immunosorbent assay., Results: : Elevated EBV-DNA loads were found in whole blood of 19.3% of the tested HIV population, which were persistent in 82%. Plasma samples were EBV-DNA negative and circulating EBV DNA could be attributed to the B-cell compartment. Transcription of only LMP2 and (non-translated) transcripts from the BamHI-A region of the EBV genome was found, whereas EBNA1, LMP1 and lytic EBV transcripts were absent despite high cellular EBV-DNA loads. IgA-reactivity to VCA-p18 was seen in 69%. IgG to VCA-p18 was significantly higher in high EBV-DNA load carriers., Conclusion: Asymptomatic HIV carriers show aberrant EBV persistence in the circulation, characterized by elevated, B-cell-associated EBV-DNA loads. EBV transcription is restricted, arguing for EBV gene shutdown in circulating EBV-carrying B cells. Increased IgA and IgG reactive to VCA-p18 is indicative of increased lytic EBV replication, possibly occurring at mucosal lymphoid sites but not in the circulation.

Nasopharyngeal carcinoma (NPC) is the most prevalent ENT-tumour in Indonesia. We investigated the primary diagnostic value of Epstein-Barr virus (EBV) DNA load and mRNA detection in noninvasive nasopharyngeal (NP) brushings, obtained prospectively from consecutive Indonesian ENT-patients with suspected NPC (N=106) and controls. A subsequent routine NP biopsy was taken for pathological examination and EBER-RISH, yielding 85 confirmed NPC and 21 non-NPC tumour patients. EBV DNA and human DNA load were quantified by real-time PCR. NP brushings from NPC patients contained extremely high EBV DNA loads compared to the 88 non-NPC controls (p<0.0001). Using mean EBV DNA load in controls plus 3 SD as cut-off value, specificity, sensitivity, positive and negative predictive values were 98, 90, 97 and 91%, respectively. Epstein-Barr nuclear antigen 1 (EBNA1) and the carcinoma-specific BARF1 mRNA were detected by nucleic acid sequence based amplification and found in 86 and 74% of NP brushings, confirming NPC tumour cell presence. EBV RNA positivity was even higher in fresh samples stored at -80 degrees C until RNA expression analyses (88% for both EBNA1 and BARF1). EBV RNA-negative NP brushings from proven NPC cases had the lowest EBV DNA loads, indicating erroneous sampling. No EBV mRNA was detected in NP brushings from healthy donors and non-NPC patients. In conclusion, EBV DNA load measurement combined with detection of BARF1 mRNA in simple NP brushings allows noninvasive NPC diagnosis. It reflects carcinoma-specific EBV involvement at the anatomical site of tumour development and reduces the need for invasive biopsies. This procedure may be useful for confirmatory diagnosis in large serological NPC screening programs and has potential as prognostic tool., (Copyright (c) 2006 Wiley-Liss, Inc.)