Diagnostic Value of Measuring Epstein-Barr Virus (EBV) DNA Load and Carcinoma-Specific Viral mRNA in Relation to Anti-EBV Immunoglobulin A (IgA) and IgG Antibody Levels in Blood of Nasopharyngeal Carcinoma Patients from Indonesia

Abstract

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and is strongly associated with Epstein-Barr virus (EBV).We investigated the primary diagnostic value of circulating EBV DNA and anti-EBV immunoglobulin G (IgG) and IgA levels in Indonesian NPC patients (n ‫؍‬ 149). By a 213-bp Epstein-Barr virus nuclear antigen 1 (EBNA1)-based real-time LightCycler PCR, 72.5% of patients were positive for EBV DNA in whole blood, with 29.5% having levels above a previously determined clinical cutoff value (COV) of 2,000 EBV DNA copies/ml, the upper level in healthy carriers. In a 99-bp LightCycler PCR, 85.9% of patients were positive and 60.4% had levels above the COV. This assay quantified a significantly higher EBV load than the 213-bp PCR assay (P < 0.0001), suggesting that circulating EBV DNA is fragmented. Using data from 11 different studies, we showed a significant inverse correlation between PCR amplicon size and the percentage of patients positive for circulating EBV DNA (Spearman's rho ‫؍‬ ؊0.91; P < 0.0001). EBV DNA loads were unrelated to anti-EBV IgG or IgA levels, as measured by VCA-p18 and EBNA1-specific synthetic peptide-based enzyme-linked immunosorbent assays. The presence of circulating tumor cells was assessed by amplification of BamHI-A rightward frame 1 (BARF1) mRNA, a viral oncogene abundantly expressed in EBV-carrying carcinomas but virtually absent from EBV-associated lymphomas. Despite high EBV DNA loads and the presence of EBNA1 and human U1A small nuclear ribonucleoprotein mRNA, BARF1 mRNA was never detected in blood. We conclude that amplicon size significantly influences EBV DNA load measurement in NPC patients. The circulating EBV DNA load is independent of serological parameters and does not reflect intact tumor cells. The primary diagnostic value of the EBV DNA load for the detection of NPC is limited.

Multipronged attenuation of macrophage-colony stimulating factor signaling by Epstein–Barr virus BARF1

“…A mutation in the BARF1 gene, which will allow the direct testing of BARF1 function in infection, was described recently in the EBV-related rhesus lymphocryptovirus (8). Finally, the BARF1: M-CSF structure provides additional evidence that BARF1 modulates M-CSF-mediated immune responses and may provide a basis for beginning to understand any putative role of BARF1 in the alteration of cellular growth (17,(29)(30)(31)(32).…”

Noninvasive diagnosis of nasopharyngeal carcinoma: Nasopharyngeal brushings reveal high Epstein‐Barr virus DNA load and carcinoma‐specific viral BARF1 mRNA

“…Finally, EBV DNA load in the NP brushing was unrelated to EBV DNA load in the whole blood of patients (r 2 5 0.05; p 5 0.07), as determined by a recently described 99 bp LC-PCR. 22 EBV RNA is abundantly detectable in NP brushings: EBV is transcriptionally active and expresses the carcinoma-specific BARF1 oncogene For a total of 78 out of 85 NP brushings samples (92%) from NPC patients, RNA profiling could be performed using NASBA and RT-PCR. As a control for RNA quality and in order to detect general EBV transcriptional activity, we performed a NASBA assay for EBNA1 mRNA, which is expressed in all EBV-associated malignancies, including NPC.…”

“…22 The circulating EBV DNA is fragmented and is probably derived from apoptosed NPC cells releasing their DNA content into the blood. 22,23 Furthermore, blood samples from NPC patients are BARF1 mRNA-negative, 22 indicating absence of circulating tumour cells. Thus demonstration of an elevated viral DNA burden plus carcinoma-specific viral mRNA expression in NP brushings is preferable in patients suspected for NPC.…”

“…22 EBV DNA in the blood of NPC patients appears highly fragmented, reflecting tumour apoptosis or necrosis. 22,23 Because serology and quantitation of circulating viral DNA only indirectly reflect carcinoma-associated activity of EBV in the nasopharyngeal (NP) region, we further explored the consistent etiological link between NPC and EBV in this study. We hypothesized that NPC may be more directly reflected by elevated viral DNA levels plus carcinoma-specific viral transcriptional activity at the site of the primary tumour.…”

“…11 Quantitation of circulating EBV DNA may be useful for prognostic monitoring in a subset of patients, 9 but because of low or negative EBV DNA values in a significant number of NPC patients, this method is less suited for primary diagnosis. 22 EBV DNA in the blood of NPC patients appears highly fragmented, reflecting tumour apoptosis or necrosis. 22,23 Because serology and quantitation of circulating viral DNA only indirectly reflect carcinoma-associated activity of EBV in the nasopharyngeal (NP) region, we further explored the consistent etiological link between NPC and EBV in this study.…”