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

Abstract

Nasopharyngeal carcinoma (NPC) is the most prevalent ENTtumour 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 5 106) 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 280°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. ' 2006 Wiley-Liss, Inc.Key words: EBV; NPC; diagnosis; viral load; oncogene; carcinoma Undifferentiated nasopharyngeal carcinoma (NPC WHO type III) is virtually 100% associated with Epstein-Barr virus (EBV) and has a reported high incidence in most of South-East Asia and intermediate incidence in North-African populations and in Inuit. [1][2][3] In Indonesia, with an ethnically diverse population of 225 million people, NPC is the most common ENT tumour with high prevalence among native populations and a yearly overall incidence estimated at 6.2/100,000. 4 Extremely high incidence was recently documented in native populations living on the island of Sulawesi. 5 In Yogyakarta, Central Java, NPC is the most prevalent tumour among man and 4th most prevalent among females, with a male/female ratio of 2.4, constituting respectively 22 and 8% of all diagnosed malignancies. 4 The strong etiological link between EBV and NPC has been known for over 3 decades [1][2][3]6 and is reflected by abnormal anti-EBV antibody profiles, increased circulating EBV DNA levels and by distinct EBV gene expression in the tumour cells. 7-11 Classically, NPC is considered to have a latency type 2 EBV transcription, with expression of EBV-encoded small RNAs 1 and 2 (EBER1/2), BamHI A rightward transcript...

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).…”

Broadening Specificity and Enhancing Cytotoxicity of Adoptive T Cells for Nasopharyngeal Carcinoma Immunotherapy

“…Indeed, T-cell cultures generated with conventional protocols are virtually devoid of BARF1-specific effectors. Considering that BARF1 is expressed by the majority of NPCs from both endemic and nonendemic areas (13)(14)(15)(16), the availability of a protocol able to generate EBV-specific CTLs efficiently targeting BARF1 may also enhance the still unsatisfactory rates of clinical responses obtainable by adoptive immunotherapy in this setting (9,10).…”

“…The oncogenic EBV protein BARF1 is expressed in the majority of NPC cases (13)(14)(15)(16) and may constitute an attractive therapeutic target. In particular, we have previously demonstrated that NPC patients show strong spontaneous CD4…”

Exosomes as the Promising Biomarker for Epstein-Barr Virus (EBV)-Associated Cancers

“…In addition, other pathogenic factors that are enriched in EBV exosomes such as EBV DNAs, EBV mRNAs and EBV miRNAs have also been shown to be potential biomarkers for EBV cancer diagnosis [56][57][58][59] ( Table 2). Using quantitative PCR (qPCR), Yip and colleagues showed that high-EBV DNAs could be detected in plasma/serum of NPC patients at late stages of disease and the viral loads were associated with poor survival or frequent relapse in NPC patients [56,59].…”