da Silva Sara Iolanda Oliveira, Domingos Tabata Alves, Kupper Bruna Elisa Catin, De Brot Louise, Aguiar Junior Samuel, Carraro Dirce Maria, Torrezan Giovana Tardin
Clinical and Functional Genomics, International Research Center/CIPE, A.C.Camargo Cancer Center, São Paulo, Brazil.
Department of Anatomic Pathology, A.C.Camargo Cancer Center, São Paulo, Brazil.
Exp Mol Pathol. 2023 Apr;130:104855. doi: 10.1016/j.yexmp.2023.104855. Epub 2023 Feb 7.
Detecting MLH1 promoter methylation is highly relevant to differentiate between possible Lynch syndrome patients or patients with sporadic causes of MLH1/PMS2 deficiency in colorectal (CRC) and endometrial cancers. Here, we aimed to develop a test for assessing MLH1 promoter methylation based in next generation sequencing (NGS), and to evaluate the concordance of MLH1 methylation and BRAF-V600 mutation status in CRC. For that, we performed a series of experiments with DNA from tumor, saliva and commercial control samples and our in house developed amplicon-based NGS test. In patients' samples, MLH1 methylation above 10% was only observed in tumors with MLH1/PMS2 loss. We confirmed the reproducibility and accuracy of MLH1 promoter analysis performing a serial dilution experiment with completely methylated and unmethylated control DNAs and a comparison between two NGS platforms (Ion Proton and Illumina). In MLH1/PMS2 deficient tumors, the MLH1 methylation status was concordant with the BRAF mutation status in 90% (18/20) of the cases. Our amplicon-based NGS test showed a great sensitivity and specificity for detecting MLH1 methylation in CRC samples, with a high agreement with the evaluation of BRAF mutation. This simple and affordable test could be used as a reflex test to identify patients with sporadic causes of MLH1/PMS2 deficiency in CRC, aiding to genetic test referral and identification of Lynch syndrome patients.
检测MLH1启动子甲基化对于区分结直肠癌（CRC）和子宫内膜癌中可能患有林奇综合征的患者或MLH1/PMS2缺乏的散发性病因患者具有高度相关性。在此，我们旨在开发一种基于下一代测序（NGS）的评估MLH1启动子甲基化的检测方法，并评估CRC中MLH1甲基化与BRAF-V600突变状态的一致性。为此，我们对来自肿瘤、唾液和商业对照样本的DNA以及我们内部开发的基于扩增子的NGS检测方法进行了一系列实验。在患者样本中，仅在MLH1/PMS2缺失的肿瘤中观察到MLH1甲基化高于10%。我们通过使用完全甲基化和未甲基化的对照DNA进行系列稀释实验以及比较两个NGS平台（Ion Proton和Illumina），证实了MLH1启动子分析的可重复性和准确性。在MLH1/PMS2缺陷的肿瘤中，90%（18/20）的病例中MLH1甲基化状态与BRAF突变状态一致。我们基于扩增子的NGS检测方法在检测CRC样本中的MLH1甲基化方面显示出很高的灵敏度和特异性，与BRAF突变评估高度一致。这种简单且经济实惠的检测方法可作为一种补充检测，用于识别CRC中MLH1/PMS2缺乏散发性病因的患者，有助于基因检测转诊和林奇综合征患者的识别。
