Variable EBV DNA load distributions and heterogeneous EBV mRNA expression patterns in the circulation of solid organ versus stem cell transplant recipients

Epstein-Barr virus (EBV) driven post-transplant lymphoproliferative disease (PTLD) is a heterogeneous and potentially life-threatening condition. Early identification of aberrant EBV activity may prevent progression to B-cell lymphoma. We measured EBV DNA load and RNA profiles in plasma and cellular blood compartments of stem cell transplant (SCT; n = 5), solid organ transplant recipients (SOT; n = 15), and SOT having chronic elevated EBV-DNA load (n = 12). In SCT, EBV DNA was heterogeneously distributed, either in plasma or leukocytes or both. In SOT, EBV DNA load was always cell associated, predominantly in B cells, but occasionally in T cells (CD4 and CD8) or monocytes. All SCT with cell-associated EBV DNA showed BARTs an

Epstein-Barr virus (EBV) serology for predicting distant metastases in a white juvenile patient with nasopharyngeal carcinoma and no clinical response to EBV lytic induction therapy

BACKGROUND: We describe a case of a 16-year-old white girl with Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC). METHODS.: At diagnosis, the patient had characteristic immunoglobulin (Ig)A and IgG responses to EBNA1, viral capsid antigen (VCA)-p18, and early antigens (EAs), with no detectable EBV DNA in her blood. Combined chemotherapy and radiotherapy resulted in complete remission. Eighteen months later, the patient's IgA responses to EBNA1 and p18 and both IgA and IgG anti-EA increased, without apparent recurrence. Five months later, lung metastases were found. She underwent surgical removal of the lung metastases and conventional chemotherapy, but had intraabdominal lymph node metastasis and mediastinal lesions develop. The patient was then treated with a novel treatment consisting of 5-fluorouracil plus valproic acid and subsequent valganciclovir to induce lytic EBV replication. This resulted in the first detectable EBV DNA levels in the blood but did not result in clinical response. RESULTS: The patient's disease progressed, and the patient declined further cancer treatment and died. CONCLUSION: In contrast to EBV DNA load, EBV serology was useful in predicting distant NPC metastasis after initial complete remission in this patient

Curcuminoids as EBV Lytic Activators for Adjuvant Treatment in EBV-Positive Carcinomas

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

Comparison of Quantitative Competitive PCR with LightCycler-Based PCR for Measuring Epstein-Barr Virus DNA Load in Clinical Specimens

The aim of this study was to develop a LightCycler-based real-time PCR assay for monitoring the Epstein-Barr virus (EBV) DNA load in unfractionated whole blood. This assay was compared with quantitative competitive PCR (Q-PCR) for EBV. The LightCycler-based assay was highly sensitive and reproducible when quantifying plasmid DNA in either the presence or absence of healthy donor blood DNA. Amplifying plasmid DNA in DNA backgrounds from different donors slightly increased the variation of quantification, indicating that clinical specimen DNA has an influence on quantification. In most transplant recipients, a good correlation was observed between EBV DNA load dynamics determined by LightCycler and Q-PCR in follow-up samples, although the correlation between absolute values of EBV DNA loads was weak and occasional samples were false negative in the LightCycler assay. In 253 cross-sectional blood samples from patients with Burkitt's lymphoma, infectious mononucleosis, or human immunodeficiency virus infection, a weak but significant correlation between the two methods was found (r(2) = 0.37, P < 0.001). Our results indicate that the clinical specimen DNA background may influence the absolute values of EBV DNA load in LightCycler analyses but that this effect is rare. LightCycler PCR is very well suited for monitoring of EBV DNA load dynamics, and its diagnostic value is comparable to that of Q-PCR. To avoid false negativity or underestimation of viral load, future internal calibration of the LightCycler is recommended. This would also enhance EBV load assay standardization and interinstitute comparisons

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

Cytolytic virus activation therapy and treatment monitoring for Epstein-Barr virus associated nasopharyngeal carcinoma in a mouse tumor model

Undifferentiated nasopharyngeal carcinoma (NPC) is 100% associated with Epstein-Barr virus (EBV). Expression of viral proteins in the tumor cells is highly restricted. EBV reactivation by CytoLytic Virus Activation (CLVA) therapy triggers de novo expression of early viral kinases (PK and TK) and uses antiviral treatment to kill activated cells. The mechanism of tumor elimination by CLVA was analyzed in NPC mouse model using C666.1 cells. Valproic acid (VPA) was combined with gemcitabine (GCb) to stimulate EBV reactivation, followed by antiviral treatment with ganciclovir (GCV). A single cycle of CLVA treatment resulted in specific tumor cell killing as indicated by reduced tumor volume, loss of EBV-positive cells in situ, and paralleled by decreased EBV DNA levels in circulation, which was more pronounced than treatment with GCb alone. In vivo reactivation was confirmed by presence of lytic gene transcripts and proteins in tumors 6 days after GCb/VPA treatment. Virus reactivation was visualized by [124I]-FIAU accumulation in tumors using PET-scan. This studied showed that CLVA therapy is a potent EBV-specific targeting approach for killing tumor cells. The [124I]-FIAU appears valuable as PET tracer for studies on CLVA drug dosage and kinetics in vivo, and may find clinical application in treatment monitoring

Aberrant Epstein-Barr virus persistence in HIV carriers is characterized by anti-Epstein-Barr virus IgA and high cellular viral loads with restricted transcription

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

Vesicle-bound EBV-BART13-3p miRNA in circulation distinguishes nasopharyngeal from other head and neck cancer and asymptomatic EBV-infections

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