Development of a Non-Invasive Method, Multiplex Methylation Specific PCR (MMSP), for Early Diagnosis of Nasopharyngeal Carcinoma

Abstract

Increasing evidence demonstrated that inactivation of tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event during carcinogenesis. Aiming at developing early diagnostic or prognostic tools for various tumors, we took an EBV-associated tumor, nasopharyngeal carcinoma (NPC), as a model and developed a powerful assay based on “multiplex methylation specific-PCR (MMSP)”. The MMSP assay was designed to detect tumor-specific methylation status of several NPC-related genes and was capable of acquiring multiplex information simultaneously through a single PCR reaction with the tiny tumor DNA derived from the direct body fluid close to the primary tumor. In this study, we collected paired nasopharyngeal (NP) swabs and NPC biopsies from 49 NPC patients and twenty noncancerous controls. A panel of markers including two EBV, and two cellular TSG markers were applied in this NPC-specific-MMSP assay. We optimized the working condition of MMSP so that it provides information equal to that from the corresponding separate PCRs. The results showed that MMSP patterns of NPC swab were largely consistent with those of corresponding biopsies and significantly distinguished themselves from those of 20 noncancerous volunteers. Among the 69 samples (49 NPCs and 20 normal controls), the sensitivity of detecting NPC from NP swabs is 98%. The specificity is as high as 100%. In conclusion, being characterized by its noninvasiveness, high reproducibility and informativeness, MMSP assay is a reliable and potential diagnostic tool for NPC. It paves the way for the development of population screening and early diagnosis approaches for various tumor types.

Diagnostic potential of methylated <em>DAPK</em> in brushing samples of nasopharyngeal carcinoma

“…After carefully filtering all the potential papers according to the selection criteria, a total of 17 studies were included in the meta-analysis. Among them, three studies evaluated the association between DAPK methylation and the risk of NPC using blood samples,31–33 and five studies used brushing samples 33–36. Nine other studies assessed the correlation of DAPK methylation and the risk of NPC using NPC and non-tumorous tissue samples 17,26,27,35–40…”

“…Thus, the inactivation of the apoptotic pathway by aberrant methylation has been associated with chemoresistance 67,68. As an important member of the apoptotic pathway, the role of abnormal hypermethylation of DAPK in NPC has been investigated by many researchers 31–36. The detection of DAPK methylation in different NPC patients may be one reason for the inconsistent conclusions about the association between DAPK methylation and the risk of NPC 33,35.…”

Up-regulation of Lamin A/C Expression in Epstein-Barr Virus Immortalized B Cells and Burkitt Lymphoma Cell Lines of Activated B Cell Phenotype

“…Strikingly, overlapping hypomethylated blocks of 1.7 GB were also observed in the epigenomes of unrelated EBV-negative carcinomas, including colon, lung, breast and thyroid carcinomas and Wilms' tumors [54]. This implies that tumor-specific methylation changes may be used as biomarkers in methylation-specific diagnostic PCR assays from the blood for the early detection of occult tumors [63][64][65]. Furthermore, hypermethylation at specific tumor suppressor loci may serve as a predictor for the success of chemotherapy [66,67].…”

Non invasive detection of LMP 1 LMP 2 Epstein Barr Latent membrane protein load in the diagnosis of nasopharyngeal carcinoma in Vietnamese population based on nasopharyngeal brushing samples

“…thus, most of NPC patients are only diagnosed when the tumor has reached an advanced stage [3,7,8]. Thus, the early diagnosis for NPC is essential in achieving a satisfactory therapy effect [10]. As NPC tumor, Epstein-Barr virus (EBV) infection, one of the high-risk factors leading to NPC, has been recognized to be strongly linked to NPC.…”