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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.

international journal of cancer

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