Epstein-Barr virus mRNA profiles and viral DNA methylation status in nasopharyngeal brushings from nasopharyngeal carcinoma patients reflect tumor origin.

Undifferentiated nasopharyngeal carcinoma (NPC) is 100% associated with Epstein-Barr virus (EBV) as oncogenic driver. NPC is often diagnosed late due to initial vague complaints and obscured location. Prior studies suggest that measurement of EBV DNA load and RNA transcripts in nasopharyngeal (NP) brushings is useful for minimally invasive NPC diagnosis. However, whether these EBV markers relate to local virus replication or reflect tumor origin remains to be demonstrated. To resolve this, we analysed EBV-DNA characteristics and quantified latent and lytic viral RNA transcripts in NP brushings and matching frozen NP-biopsy specimens from patients suspected of having NPC. We observed non-fragmented and Cp-promotor methylated EBV-DNA in both NP brushings and biopsies suggestive of tumor origin. Using quantitative RT-PCR we determined expression levels of 7 critical latent (EBER1, Qp-EBNA1, EBNA2, BART, LMP1, LMP2, BARF1) and 5 lytic (Zta, Rta, TK, PK and VCA-p18) RNA transcripts. Although latent and early lytic RNA transcripts were frequently detected in conjunction with high EBV viral load, in both brushings and biopsies the latent transcripts prevailed and reflected a typical NPC-associated latency-II transcription profile without EBNA2. Late lytic RNA transcripts were rare and detected at low levels mainly in NP brushings, suggestive of abortive viral reactivation rather than complete virus replication. EBV-IgA serology (EBNA1, VCA, Zta) did not correlate to the level of viral reactivation in situ. Overall, viral RNA profiling, DNA fragmentation and methylation analysis in NP brushings and parallel biopsies indicate that NP brush sampling provides a true and robust indicator of NPC tumor presence.

Can Epstein-Barr virus DNA load in nasopharyngeal brushings or whole blood predict recurrent nasopharyngeal carcinoma in a non-endemic region? A prospective nationwide study of the Dutch Head and Neck Oncology Cooperative Group.

This study estimated the value of quantitative measurements of EBV markers in the clinical management of nasopharyngeal carcinoma in a non-endemic area. The aim was to predict prognosis and detect recurrent and residual disease. In 72 patients, EBV DNA load in blood and nasopharyngeal brushes, and IgA VCA-p18 and EBNA1 in plasma were measured at different time points. At diagnosis and post-treatment, a cut-off value was used for detecting disease [positive (PPV) and negative (NPV) predictive value]. The markers were correlated as a continuous variable with tumor stage, disease-free survival (DFS) and overall survival (OS). The Cox hazard ratio model assessed hazard ratios. At diagnosis, the markers were above the COV in 45, 92, 85 and 83 % of the patients, respectively. Post-treatment, DNA load test in blood and brush had the best discriminating power (blood DNA load test: PPV 39 % and NPV 97 %, brush for local disease: PPV 75 % and NPV 99 %). Post-treatment, DNA load in blood was the best predictor for OS and DFS [hazard ratio 3.2 (95 % CI 1.51-3.5) and 2.3 (95 % CI 1.72-5.8)]. Assessing the EBV DNA load in blood has significant prognostic value, although the clinical value is for discussion. The EBV DNA load in the brush might improve early detection of local failures post-treatment.

Aberrant Epstein-Barr virus antibody patterns and chronic lymphocytic leukemia in a Spanish multicentric case-control study.

BACKGROUND: Epstein-Barr virus (EBV)-related malignancies harbour distinct serological responses to EBV antigens. We hypothesized that EBV serological patterns can be useful to identify different stages of chronic lymphocytic leukemia. METHODS: Information on 150 cases with chronic lymphocytic leukemia and 157 frequency-matched (by age, sex and region) population-based controls from a Spanish multicentre case-control study was obtained. EBV immunoglobulin G serostatus was evaluated through a peptide-based ELISA and further by immunoblot analysis to EBV early antigens (EA), nuclear antigen (EBNA1), VCA-p18, VCA-p40 and Zebra. Two independent individuals categorized the serological patterns of the western blot analysis. Patients with very high response and diversity in EBV-specific polypeptides, in particular with clear responses to EA-associated proteins, were categorized as having an abnormal reactive pattern (ab_EBV). Adjusted odds ratios (OR) and 95% confidence interval (CI) were estimated using logistic regression models. RESULTS: Almost all subjects were EBV-IgG positive (>95% of cases and controls) whereas ab_EBV patterns were detected in 23% of cases (N = 34) and 11% of controls (N = 17; OR: 2.44, 95% CI, 1.29 to 4.62; P = 0.006), particularly in intermediate/high risk patients. Although based on small numbers, the association was modified by smoking with a gradual reduction of ab_EBV-related OR for all Rai stages from never smokers to current smokers. CONCLUSIONS: Highly distinct EBV antibody diversity patterns revealed by immunoblot analysis were detected in cases compared to controls, detectable at very early stages of the disease and particularly among non smokers. This study provides further evidence of an abnormal immunological response against EBV in patients with chronic lymphocytic leukemia.

Epstein-Barr virus-targeted therapy in nasopharyngeal carcinoma.

PURPOSE: Despite successful primary treatment of nasopharyngeal carcinoma (NPC), the incidence of distant metastasis remains 25-34 %. Treatment options are limited, and survival is poor. Intratumoural Epstein-Barr virus (EBV) was used as treatment target. In NPC, EBV is present in a latent state, expressing only few non-immunogenic viral products. Gemcitabine and valproic acid can trigger EBV to the lytic state, wherein viral kinases are expressed, making EBV-positive tumour cells susceptible for antiviral therapy with, i.e. valganciclovir, and inducing an EBV-specific immune response. METHODS: This drug combination was applied in eight patients with EBV-positive NPC, refractory to conventional treatment. The primary endpoints were safety, tolerability and clinical response. Secondary endpoint was to get proof of concept based on biomarkers, i.e. pharmacokinetics, EBV-DNA load in whole blood and nasopharyngeal brushes, EBV-RNA profiling for proof of lytic induction, EBV-IgG and EBV-IgA levels and diversity and EBV-specific T cell response. RESULTS: The best observed clinical response was partial in two patients (25 %) and stable disease in three patients (37.5 %). The median survival was 9 months (95 % confidence interval 7-17 months). Effective dose levels were reached. Peaking of EBV-DNA loads in blood and brush proved the biological effect on EBV during most treatment cycles. In one patient, RNA profiling confirmed lytic EBV induction. EBV-IgG and EBV-IgA antibody levels were already high before treatment and did not change during treatment. No changes in EBV-specific T cell response were detected. CONCLUSION: The treatment was safe with manageable side effects, clinical response was observed, and viral activation corroborated.

Epstein-Barr virus serology as a potential screening marker for nasopharyngeal carcinoma among high-risk individuals from multiplex families in Taiwan.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated cancer that is highly treatable when diagnosed early, with 5-year disease-free survival of approximately 90%. However, NPC is typically diagnosed at advanced stages, in which disease-free survival is <50%. There is, therefore, a need for clinical tools to assist in early NPC detection, particularly among high-risk individuals. METHODS: We evaluated the ability of anti-EBV IgA antibodies to detect incident NPC among high-risk Taiwanese individuals. NPC cases (N = 21) and age- and sex-matched controls (N = 84) were selected. Serum collected before NPC diagnosis was tested for ELISA-based IgA antibodies against the following EBV peptides: EBNA1, VCAp18, EAp138, Ead_p47, and VCAp18 + EBNA1 peptide mixture. The sensitivity, specificity, and screening program parameters were calculated. RESULTS: EBNA1 IgA had the best performance characteristics. At an optimized threshold value, EBNA1 IgA measured at baseline identified 80% of the high-risk individuals who developed NPC during follow-up (80% sensitivity). However, approximately 40% of high-risk individuals who did not develop NPC also tested positive (false positives). Application of EBNA1 IgA as a biomarker to detect incident NPC in a previously unscreened, high-risk population revealed that 164 individuals needed to be screened to detect 1 NPC and that 69 individuals tested positive per case detected. CONCLUSIONS: EBNA1 IgA proved to be a sensitive biomarker for identifying incident NPC, but future work is warranted to develop more specific screening tools to decrease the number of false positives. IMPACT: Results from this study could inform decisions about screening biomarkers and referral thresholds for future NPC early-detection program evaluations.

Epstein-Barr virus DNA load in nasopharyngeal brushings and whole blood in nasopharyngeal carcinoma patients before and after treatment.

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.

Characterization of ELISA detection of broad-spectrum anti-Epstein-Barr virus antibodies associated with nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) infection is associated with undifferentiated nasopharyngeal carcinomas (NPC). A distinct seroreactivity pattern to EBV is predictive of subsequent risk of sporadic and familial nasopharyngeal carcinomas. There are currently no accepted screening tools for guiding the clinical management of individuals at high-risk for nasopharyngeal carcinomas, particularly unaffected relatives from nasopharyngeal carcinoma multiplex families. Therefore, the reproducibility of a panel of largely synthetic peptide-based anti-EBV antibody ELISAs was evaluated and their ability to distinguish nasopharyngeal carcinoma cases from controls was explored. IgG and IgA antibodies against 6 different EBV antigens (10 assays, total) were tested on sera from 97 individuals representing the full spectrum of anti-EBV seroprevalence (i.e., healthy individuals with no known EBV seroreactivity, healthy individuals with known EBV seroreactivity, and nasopharyngeal carcinoma cases). Each specimen was tested in triplicate to assess within-batch and across-batch variation, and the triplicate testing was repeated on two separate days. Reproducibility was assessed by the coefficients of variation (CVs) and intraclass correlation coefficients (ICCs). All markers were detectable in 17% or more of samples. For all but one marker, the overall, within-batch, and across-batch CVs were below 15%, and the ICCs were above 70% for all but three markers. Sensitivity of these markers to detect prevalent nasopharyngeal carcinomas ranged from 22% to 100%, and among unaffected controls, most distinguished those with and without known seropositivity. In conclusion, a large number of EBV markers can be measured reliably in serum samples using peptide-based anti-EBV ELISAs.

Cytolytic virus activation therapy for Epstein-Barr virus-driven tumors.

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.

Humoral immune responses to Epstein-Barr virus encoded tumor associated proteins and their putative extracellular domains in nasopharyngeal carcinoma patients and regional controls.

Epstein-Barr virus (EBV) latency proteins EBNA1, LMP1, LMP2, and BARF1 are expressed in tumor cells of nasopharyngeal carcinoma (NPC). IgG and IgA antibody responses to these non-self tumor antigens were analyzed in NPC patients (n=125) and regional controls (n=100) by three approaches, focusing on the putative LMP1, LMP2 extracellular domains. Despite abundant IgG and IgA antibody responses to lytic antigens and EBNA1, patients had low titer (1:25-1:100) IgG to LMP1 (81.2%), LMP2 (95.6%), and BARF1 (84.8%), while immunoblot showed such reactivity in 24.2%, 12.5%, and 12.5% at 1:50 dilution, respectively. Few IgA responses were detected, except for EBNA1. Controls only showed IgG to EBNA1. ELISA using peptides from different domains of LMP1, LMP2, and BARF1 also yielded mostly negative results. When existing, low level IgG to intracellular C-terminus of LMP1 (62.9%) prevailed. Rabbit immunization with peptides representing extracellular (loop) domains yielded loop-specific antibodies serving as positive control. Importantly, these rabbit antibodies stained specifically extracellular domains of LMP1 and LMP2 on viable cells and mediated complement-driven cytolysis. Rabbit anti-LMP1 loop-1 and -3 killed 50.4% and 59.4% of X50/7 and 35.0% and 35.9% of RAJI cells, respectively, and 22% of both lines were lysed by anti-LMP2 loop-2 or -5 antibodies. This demonstrates that (extracellular domains of) EBV-encoded tumor antigens are marginally immunogenic for humoral immune responses. However, peptide-specific immunization may generate such antibodies, which can mediate cell killing via complement activation. This opens options for peptide-based tumor vaccination in patients carrying EBV latency type II tumors such as NPC.

Serological and virological investigation of the role of the herpesviruses EBV, CMV and HHV-6 in post-infective fatigue syndrome.

Multiple previous studies have sought evidence for ongoing, active infection with, or reactivation of, Herpesviruses in patients with chronic fatigue syndrome (CFS), with conflicting results. This study aimed to clarify this by studying 20 patients enrolled in a well-characterized model of the onset and evolution of CFS, the prospective cohort of the Dubbo Infection Outcomes Study (DIOS). The patients selected for examination included five CFS patients with primary Epstein-Barr virus (EBV) infection; five CFS patients with acute viral infection not caused by EBV; and 10 matched controls with prompt resolution of primary EBV infection. Serum samples from three timepoints were assayed using a comprehensive range of serological assays for EBV, HHV-6, and CMV. Viral genomes were assessed using quantitative PCR assays. All patients were seropositive for HHV-6, and 10 were seropositive for CMV at infection baseline (five patients and five controls). Low titer CMV IgM antibodies were found at infection baseline in two of these cases and three control patients. HHV-6 IgG antibody titers were highest at infection baseline but did not differ between the CFS cases and the control patients. There were increases in EBV IgG VCA p18, EBNA-1 IgG, and EA IgG titers over time, but these did not differ between CFS cases and control patients. EBV and HHV6 DNA levels were at control levels in a minority of samples, and CMV was undetectable in all samples. These data do not support the hypothesis of ongoing or reactivated EBV, HHV-6, or CMV infection in the pathogenesis of CFS.