Global Clinical Biomarkers and Companion Diagnostics, Global Early Development, EMD Serono Research and Development Institute, Billerica, MA, USA.
Global Clinical Biomarkers and Companion Diagnostics, Global Early Development, Merck Biopharma, Merck KGaA, Darmstadt, Germany.
BMC Cancer. 2021 Jul 10;21(1):797. doi: 10.1186/s12885-021-08497-x.
MutL Homolog 1 (MLH1) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. As cancer-specific DNA methylation changes in body fluids are limited, it is particularly challenging to develop clinically applicable liquid biopsy methodologies with high sensitivity and specificity. The purpose of this study was to develop a fit-for-purpose methylation sensitive restriction enzyme (MSRE) based digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC.
Primers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to MSRE HpaII digestion. Plasma samples from 20 healthy donors and 28 CRC patients were analyzed with the optimized MSRE procedure using ddPCR.
Using methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Based on the results from the ddPCR assay using 1 ng circulating cell-free DNA (cfDNA) input from healthy donors or CRC samples, ROC curves were generated with an area under the curve (AUC) value of 0.965 (95% CI: 0.94, 0.99). The statistically optimal assay sensitivity and specificity was achieved when 8 positive droplets were used as acceptance criteria (78% sensitivity and 100% specificity, 95% CI: 0.45, 0.95). A tiered-based cutoff (20, 50, 80% percentile based) was applied to distinguish CRC samples with different methylation level.
Our study demonstrated that the liquid biopsy assay for MLH1 promoter methylation detection using purely quantitative ddPCR is a simple and highly sensitive procedure that provides reliable methylation detection in ctDNA. The MSRE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development.
MutL 同系物 1（MLH1）启动子甲基化与微卫星不稳定的结直肠癌（CRC）相关。甲基化状态与癌症发展和进展之间的强相关性导致人们对使用循环肿瘤 DNA（ctDNA）中的甲基化标记物进行早期癌症检测和纵向监测越来越感兴趣。由于体液中癌症特异性 DNA 甲基化变化有限，因此开发具有高灵敏度和特异性的临床适用液体活检方法特别具有挑战性。本研究旨在开发一种适用于目的的甲基化敏感限制性内切酶（MSRE）基于数字液滴 PCR（ddPCR）检测法，以检测晚期 CRC 中 ctDNA 中的 MLH1 启动子甲基化。
设计引物和探针以扩增 MLH1 启动子的 CpG 位点。将甲基化和非甲基化对照基因组 DNA 切成小块以模拟 ctDNA，并进行 MSRE HpaII 消化。使用优化的 MSRE 程序，通过 ddPCR 分析 20 名健康供体和 28 名 CRC 患者的血浆样本。
使用甲基化和非甲基化对照，我们优化了 HpaII 酶消化条件，以确保完全消化并避免假阳性。基于使用 1ng 循环无细胞 DNA（cfDNA）输入的来自健康供体或 CRC 样本的 ddPCR 检测结果，生成了 ROC 曲线，曲线下面积（AUC）值为 0.965（95%CI：0.94，0.99）。当将 8 个阳性液滴用作接受标准时，达到了统计上最佳的检测灵敏度和特异性（78%的灵敏度和 100%的特异性，95%CI：0.45，0.95）。应用分层截止值（基于 20、50、80%分位数）来区分具有不同甲基化水平的 CRC 样本。
我们的研究表明，使用纯定量 ddPCR 检测 MLH1 启动子甲基化的液体活检检测是一种简单且高度敏感的程序，可提供 ctDNA 中可靠的甲基化检测。MSRE-ddPCR 方法还可应用于其他感兴趣的基因，其中甲基化模式可能为未来的临床生物标志物和/或伴随诊断开发提供有临床意义的信息。
