Comparison of Promoter Hypermethylation Pattern in Salivary Rinses Collected with and without an Exfoliating Brush from Patients with HNSCC

BackgroundSalivary rinses have been recently proposed as a valuable resource for the development of epigenetic biomarkers for detection and monitoring of head and neck squamous cell carcinoma (HNSCC). Both salivary rinses collected with and without an exfoliating brush from patients with HNSCC are used in detection of promoter hypermethylation, yet their correlation of promoter hypermethylation has not been evaluated. This study was to evaluate the concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush from patients with HNSCC. Methodolgy57 paired salivary rinses collected with or without an exfoliating brush from identical HNSCC patients were evaluated for promoter hypermethylation status using Quantitative Methylation-Specific PCR. Target tumor suppressor gene promoter regions were selected based on our previous studies describing a panel for HNSCC screening and surveillance, including P16, CCNA1, DCC, TIMP3, MGMT, DAPK and MINT31. Principal FindingsIn salivary rinses collected with and without brush, frequent methylation was detected in P16 (8.8% vs. 5.2%), CCNA1 (26.3% vs. 22.8%), DCC (33.3% vs. 29.8%), TIMP3 (31.6% vs. 36.8%), MGMT (29.8% vs. 38.6%), DAPK (14.0% vs. 19.2%), and MINT31 (10.5% vs. 8.8%). Spearman's rank correlation coefficient showed a positive correlation between salivary rinses collected with and without brush for P16 (ρ = 0.79), CCNA1 (ρ = 0.61), DCC (ρ = 0.58), TIMP3 (ρ = 0.10), MGMT (ρ = 0.70), DAPK (ρ = 0.51) and MINT31 (ρ = 0.72) (PP16, 82.5% for CCNA1, 78.9% for DCC, 59.7% for TIMP3, 84.2% for MGMT, 84.2% for DAPK, and 94.7% for MINT31. ConclusionsOur study demonstrated strong correlations of gene promoter hypermethylation between salivary rinses collected with and without an exfoliating brush. Salivary rinse collection without using an exfoliating brush may offer a cost effective, rapid, non-invasive, and reliable means for development of epigenetic salivary rinse biomarkers.背景：近年来，唾液冲洗液被认为是开发用于头颈部鳞状细胞癌（head and neck squamous cell carcinoma, HNSCC）检测与监测的表观遗传生物标志物（epigenetic biomarkers）的宝贵样本来源。目前已有研究使用来自头颈部鳞状细胞癌患者的、使用或未使用细胞刷采集的唾液冲洗液来检测启动子高甲基化（promoter hypermethylation），但尚未评估这两种采集方式所得样本的启动子高甲基化相关性。本研究旨在评估头颈部鳞状细胞癌患者的、使用与未使用细胞刷采集的唾液冲洗液之间的启动子高甲基化一致性。研究方法：本研究针对57份来自同一头颈部鳞状细胞癌患者的配对唾液冲洗液样本（分别使用或未使用细胞刷采集），采用定量甲基化特异性PCR（Quantitative Methylation-Specific PCR）检测其启动子高甲基化状态。靶肿瘤抑制基因（tumor suppressor gene）启动子区域的选择基于我们既往用于头颈部鳞状细胞癌筛查与监测的标志物组合研究，所纳入的基因包括P16、CCNA1、DCC、TIMP3、MGMT、DAPK及MINT31。主要研究结果：在使用与未使用细胞刷采集的唾液冲洗液中，各基因均检测到较高频率的甲基化：P16（8.8% vs 5.2%）、CCNA1（26.3% vs 22.8%）、DCC（33.3% vs 29.8%）、TIMP3（31.6% vs 36.8%）、MGMT（29.8% vs 38.6%）、DAPK（14.0% vs 19.2%）以及MINT31（10.5% vs 8.8%）。斯皮尔曼等级相关系数（Spearman's rank correlation coefficient）分析显示，两种采集方式所得样本的启动子高甲基化水平呈正相关：P16（ρ=0.79）、CCNA1（ρ=0.61）、DCC（ρ=0.58）、TIMP3（ρ=0.10）、MGMT（ρ=0.70）、DAPK（ρ=0.51）以及MINT31（ρ=0.72），各基因的样本一致性分别为：CCNA1 82.5%、DCC 78.9%、TIMP3 59.7%、MGMT 84.2%、DAPK 84.2%以及MINT31 94.7%。结论：本研究证实，使用与未使用细胞刷采集的唾液冲洗液之间的基因启动子高甲基化水平具有显著相关性。无需使用细胞刷的唾液冲洗液采集方式，可为表观遗传唾液生物标志物的开发提供一种成本低廉、快速、无创且可靠的手段。