Purpose: Current endoscopy-based screening and surveillance programs have not been proven effective at decreasing esophageal adenocarcinoma (EAC) mortality, creating an unmet need for effective molecular tests for early detection of this highly lethal cancer. We conducted a genome-wide methylation screen to identify novel methylation markers that distinguish EAC and high-grade dysplasia (HGD) from normal squamous epithelium (SQ) or nondysplastic Barrett's esophagus (NDBE). Experimental Design: DNA methylation profiling of samples from SQ, NDBE, HGD, and EAC was performed using HM450 methylation arrays (Illumina) and reduced-representation bisulfate sequencing. Ultrasensitive methylation-specific droplet digital PCR and next-generation sequencing (NGS)-based bisulfite-sequencing assays were developed to detect the methylation level of candidate CpGs in independent esophageal biopsy and endoscopic brushing samples. Results: Five candidate methylation markers were significantly hypermethylated in HGD/EAC samples compared with SQ or NDBE (P < 0.01) in both esophageal biopsy and endoscopic brushing samples. In an independent set of brushing samples used to construct biomarker panels, a four-marker panel (model 1) demonstrated sensitivity of 85.0% and 90.8% for HGD and EACs respectively, with 84.2% and 97.9% specificity for NDBE and SQ respectively. In a validation set of brushing samples, the panel achieved sensitivity of 80% and 82.5% for HGD and EAC respectively, at specificity of 67.6% and 96.3% for NDBE and SQ samples. Conclusions: A novel DNA methylation marker panel differentiates HGD/EAC from SQ/NDBE. DNA-methylation–based molecular assays hold promise for the detection of HGD/EAC using esophageal brushing samples.

中文翻译：

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用于检测食管腺癌和高度不典型增生的新型 DNA 甲基化生物标志物组合

目的：目前基于内窥镜检查的筛查和监测计划尚未被证明可以有效降低食管腺癌 (EAC) 死亡率，因此对有效分子检测以早期发现这种高度致命的癌症的需求未得到满足。我们进行了全基因组甲基化筛选，以确定新的甲基化标记，以区分 EAC 和高度不典型增生 (HGD) 与正常鳞状上皮 (SQ) 或非发育不良巴雷特食管 (NDBE)。实验设计：使用 HM450 甲基化阵列 (Illumina) 和简化代表性硫酸氢盐测序对来自 SQ、NDBE、HGD 和 EAC 的样品进行 DNA 甲基化分析。开发了超灵敏甲基化特异性液滴数字 PCR 和基于下一代测序 (NGS) 的亚硫酸氢盐测序测定法，用于检测独立食管活检和内窥镜刷牙样本中候选 CpG 的甲基化水平。结果：与食管活检和内镜刷牙样本中的 SQ 或 NDBE 相比，HGD/EAC 样本中的 5 个候选甲基化标记物显着高甲基化（P < 0.01）。在用于构建生物标记物组的一组独立刷牙样本中，四标记物组（模型 1）对 HGD 和 EAC 的敏感性分别为 85.0% 和 90.8%，对 NDBE 和 SQ 的特异性分别为 84.2% 和 97.9%。在一组刷牙样本验证中，专家组对 HGD 和 EAC 的敏感性分别为 80% 和 82.5%，特异性为 67.6% 和 96。NDBE 和 SQ 样品为 3%。结论：一种新型 DNA 甲基化标记物组可将 HGD/EAC 与 SQ/NDBE 区分开来。基于 DNA 甲基化的分子检测有望使用食管刷检样本检测 HGD/EAC。