Background and Aims: Early identification of malignant biliary strictures (MBS) is challenging, with up to 20% classified as indeterminants after preliminary testing and tissue sampling with endoscopic retrograde cholangiopancreatography (ERCP). We aimed to evaluate the use of methylated DNA markers (MDM) from biliary brushings to enhance MBS detection in a prospective cohort. Methods: Candidate MDMs were evaluated for their utility in MBS diagnosis through a series of discovery and validation phases. DNA was extracted from biliary brushing samples, quantified, bisulfite-converted, and then subjected to methylation-specific Droplet Digital Polymerase Chain Reaction (ddPCR). Patients were considered to have no malignancy if the sampling was negative and there was no evidence of malignancy after 1 year or definitive negative surgical histopathology. Results: Fourteen candidate MDMs were evaluated in the discovery phase, with top-performing and new markers evaluated in the technical validation phase. The top four MDMs were TWIST1, HOXA1, VSTM2B, and CLEC11A, which individually achieved AUC values of 0.82, 0.81, 0.83, and 0.78, respectively, with sensitivities of 59.4%, 53.1%, 62.5%, and 50.0%, respectively, at high specificities for malignancy of 95.2-95.3% for the final biologic validation phase. When combined as a panel, the AUC was 0.86, achieving 73.4% sensitivity and 92.9% specificity, which outperformed cytology and fluorescent in situ hybridization (FISH). Conclusions: The selected methylated DNA markers demonstrated improved performance characteristics for the detection of MBS compared to cytology and FISH. ​ Therefore, MDMs should be considered viable candidates for inclusion in diagnostic testing algorithms.​

中文翻译：

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甲基化 DNA 标记在恶性胆管狭窄诊断中的应用


背景和目的：恶性胆道狭窄 (MBS) 的早期识别具有挑战性，经过初步测试和内镜逆行胰胆管造影 (ERCP) 组织取样后，高达 20% 的恶性胆道狭窄 (MBS) 被归类为不确定因素。我们的目的是评估胆道刷检中甲基化 DNA 标记 (MDM) 的使用，以增强前瞻性队列中 MBS 的检测。方法：通过一系列发现和验证阶段，评估候选 MDM 在 MBS 诊断中的效用。从胆道刷检样本中提取 DNA，进行定量、亚硫酸氢盐转化，然后进行甲基化特异性微滴数字聚合酶链反应 (ddPCR)。如果采样结果为阴性并且1年后没有恶性肿瘤证据或手术组织病理学结果明确阴性，则认为患者没有恶性肿瘤。结果：在发现阶段评估了 14 个候选 MDM，在技术验证阶段评估了表现最好的标记物和新标记物。排名前四的 MDM 是 TWIST1、HOXA1、VSTM2B 和 CLEC11A，它们各自的 AUC 值分别为 0.82、0.81、0.83 和 0.78，灵敏度分别为 59.4%、53.1%、62.5% 和 50.0%。最终生物验证阶段的恶性肿瘤特异性高达 95.2-95.3%。当组合为一组时，AUC 为 0.86，实现了 73.4% 的敏感性和 92.9% 的特异性，优于细胞学和荧光原位杂交 (FISH)。结论：与细胞学和 FISH 相比，选定的甲基化 DNA 标记在检测 MBS 方面表现出改进的性能特征。 因此，MDM 应被视为包含在诊断测试算法中的可行候选者。