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Journal of Clinical Microbiology, July 2005, p. 3066-3073, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3066-3073.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Servi J. C. Stevens,1 Sandra A. W. M. Verkuijlen,1 Bambang Hariwiyanto,2 Harijadi,2 Jajah Fachiroh,2 Dewi K. Paramita,2 I. Bing Tan,3 Sophia M. Haryana,2 and Jaap M. Middeldorp1*

Department of Pathology, Vrije Universiteit Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands,1 Gadjah Mada University, Yogyakarta, Indonesia,2 Department of Otolaryngology-Head & Neck Surgery, Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands3

Received 23 November 2004/ Returned for modification 10 February 2005/ Accepted 29 March 2005

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.

* Corresponding author. Mailing address: Department of Pathology, Vrije Universiteit Medical Centre, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Phone: 31-20-4444070. Fax: 31-20-4442964. E-mail: j.middeldorp{at}vumc.nl.

Journal of Clinical Microbiology, July 2005, p. 3066-3073, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3066-3073.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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