RESUMEN
The detection of Epstein-Barr virus (EBV) DNA load in nasopharyngeal (NP) brushing samples for diagnosis of nasopharyngeal carcinoma (NPC) has attracted great attention. Further improvements that eliminate the need for clinical settings will greatly extend its application. A total of 250 participants were recruited to obtain NP brushing samples. Brush sampling both with and without the guide of endoscopy was conducted in 38 NPC patients. EBV DNA load, EBV RNA transcript and EBV DNA C promoter methylation status were, respectively, evaluated. Typical latency II transcripts were observed in brushing samples from NPC patients but not controls. Unlike in tissues, multiple lytic gene transcripts were observed not only in NPC patients but also in controls. Apart from EBV RNA transcript, samples from NPC patients also showed higher levels of EBV DNA load and C promoter methylation degree than their controls. Qualitative analysis further showed that EBV DNA C promoter was methylated in all NPC patients but in only 18.4% of the control group. Combined analysis of EBV DNA methylated degree and EBV DNA load increased the sensitivity to 100% in the detection of NPC. Using qualitative methylated type as the criteria, up to 89.5% of samples collected via blind brushing showed consistent results with samples collected via endoscopy-guided brushing from NPC patients. Detection of the methylation status of EBV DNA C promoter in NP brushing samples shows great potential in diagnosing NPC and may provide an appealing alternative for the non-invasive detection and screening of NPC without the need for clinical settings.
Asunto(s)
Metilación de ADN , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/diagnóstico , Neoplasias Nasofaríngeas/diagnóstico , ADN Viral/genética , Infecciones por Virus de Epstein-Barr/complicaciones , Femenino , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/fisiología , Humanos , Masculino , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/virología , Regiones Promotoras Genéticas , Sensibilidad y Especificidad , Proteínas Virales/genética , Latencia del VirusRESUMEN
BACKGROUND: Tissue specimens for nasopharyngeal carcinoma (NPC) research are scarce because of sampling difficulties. Previous studies have suggested non-invasive nasopharyngeal brushing as an effective sampling method for NPC diagnosis. The present study aimed to evaluate the feasibility of nasopharyngeal brushing in the acquisition of NPC nucleic acids for research. METHODS: Nasopharyngeal brushing samples were acquired from 24 healthy individuals and 48 NPC patients. Tissues from 48 NPC and 18 nasopharyngitis patients were collected by endoscopic biopsy. The expression levels of tumor suppressor genes (TSGs) and Epstein-Barr virus (EBV)-encoded microRNAs as well as EBV DNA copy number were measured by quantitative polymerase chain reaction in both types of samples. RESULTS: Among six TSGs examined, the expression levels of two genes were significantly decreased in nasopharyngeal brushing and tissue samples from NPC patients as compared with those from healthy/nasopharyngitis individuals. Four EBV-encoded microRNAs, mir-bart1-5p, mir-bart5, mir-bart6-5p, and mir-bart17-5p, were significantly up-regulated in both NPC brushing and tissue samples compared with those from healthy/nasopharyngitis controls (P < 0.001). EBV DNA was significantly increased in both nasopharyngeal brushing samples (P < 0.001) and tissue samples (P < 0.001) from NPC patients in comparison with those from healthy controls. CONCLUSIONS: Nasopharyngeal brushing can obtain sufficient tumoral materials for the analysis of viral nucleic acid, including EBV-encoded microRNAs and EBV DNA. For the detection of TSG expression, nasopharyngeal brushings was feasible but inferior to tissue samples. This study confirms nasopharyngeal brushing as an applicable sampling method that can aid in nucleic acid-based NPC research.
Asunto(s)
ADN de Neoplasias/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Manejo de Especímenes/métodos , ADN Viral/genética , Endoscopía/métodos , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Estudios de Factibilidad , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Humanos , MicroARNs/genética , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/virología , Nasofaringe/virología , ARN Viral/genética , Reproducibilidad de los ResultadosRESUMEN
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.