Pathology, Christian Medical College and Hospital Vellore, Vellore, Tamil Nadu, India.
Pathology, Christian Medical College and Hospital Vellore, Vellore, Tamil Nadu, India
J Clin Pathol. 2023 Dec;76(12):860-864. doi: 10.1136/jcp-2023-208953. Epub 2023 Sep 14.
promoter methylation analysis in formalin-fixed paraffin-embedded (FFPE) tissues can be challenging since the DNA obtained is often fragmented. Bisulfite conversion, which is essential to determine methylation status, further degrades DNA. While conventional methylation-specific PCR (MSP) and pyrosequencing assays have long been used to determine the methylation status of , this study was designed to determine the utility of one-tube DNA extraction method coupled with a droplet digital PCR (ddPCR) assay, to study the epigenetic changes in the promoter region of the gene using DNA obtained from FFPE.The FFPE blocks of 30 (n=30) patients with Central Nervous System (CNS) WHO grade 4 tumours, previously tested by MSP (2011-2021) were retrieved; DNA was extracted using one-tube extraction method and bisulfite converted. All converted samples were analyzed for methylation status of the promoter region with a laboratory designed Methylation-Specific ddPCR (MS ddPCR) using degenerate primers and probes that were labelled with FAM or HEX flurocein dye.Of the 30 cases, 20 cases were methylated and 10 cases were unmethylated by MS ddPCR. The results of MS ddPCR were then compared with those obtained by MSP and found to be concordant in 93.3% (28/30) of the cases and discordant in 2 cases. The Cohen's kappa coefficient (κ) was 0.84. The sensitivity, specificity, positive predictive value and negative predictive value of the assay in detecting the methylation status was found to be 95%, 90%, 95% and 90%.The results show that MS ddPCR is a valuable tool to detect the methylation status of MGMT in FFPE with high sensitivity. This method is cost-effective and easy to perform and could be an attractive alternative to the routine method of MSP.
在福尔马林固定石蜡包埋（FFPE）组织中进行启动子甲基化分析具有挑战性，因为获得的 DNA 通常是片段化的。亚硫酸氢盐转化是确定甲基化状态所必需的，它进一步降解 DNA。虽然传统的甲基化特异性 PCR（MSP）和焦磷酸测序检测已长期用于确定 的甲基化状态，但本研究旨在确定一种管 DNA 提取方法与液滴数字 PCR（ddPCR）检测的结合使用，以研究使用从 FFPE 获得的 DNA 研究 基因启动子区域的表观遗传变化。FFPE 块取自 30 名（n=30）中枢神经系统（CNS）WHO 4 级肿瘤患者，这些患者之前通过 MSP（2011-2021）进行了测试；使用单管提取方法和亚硫酸氢盐转化提取 DNA。使用实验室设计的甲基化特异性 ddPCR（MS-ddPCR），使用简并引物和探针，用 FAM 或 HEX 荧光染料标记，对所有转化样本进行 启动子区域的甲基化状态分析。在 30 例中，20 例 MS-ddPCR 为甲基化，10 例为未甲基化。然后将 MS-ddPCR 的结果与 MSP 获得的结果进行比较，发现 93.3%（28/30）的病例结果一致，2 例结果不一致。Cohen's kappa 系数（κ）为 0.84。该检测法检测 MGMT 甲基化状态的敏感性、特异性、阳性预测值和阴性预测值分别为 95%、90%、95%和 90%。结果表明，MS-ddPCR 是一种检测 FFPE 中 MGMT 甲基化状态的灵敏工具。该方法具有成本效益且易于操作，可能是 MSP 常规方法的有吸引力的替代方法。
