Amplicon-based NGS test for assessing MLH1 promoter methylation and its correlation with BRAF mutation in colorectal cancer patients

• Published in: Experimental and molecular pathology Vol. 130; p. 104855
• Main Authors: da Silva, Sara Iolanda Oliveira, Domingos, Tabata Alves, Kupper, Bruna Elisa Catin, De Brot, Louise, Aguiar Junior, Samuel, Carraro, Dirce Maria, Torrezan, Giovana Tardin
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Inc 01-04-2023
• Subjects: BRAF mutation; Colorectal cancer; Colorectal Neoplasms - diagnosis; Colorectal Neoplasms - genetics; Colorectal Neoplasms, Hereditary Nonpolyposis - diagnosis; Colorectal Neoplasms, Hereditary Nonpolyposis - genetics; Colorectal Neoplasms, Hereditary Nonpolyposis - pathology; DNA Methylation - genetics; Endometrial cancer; Germ-Line Mutation; High-Throughput Nucleotide Sequencing; Humans; Lynch syndrome; Microsatellite Instability; Mismatch repair deficiency; Mismatch Repair Endonuclease PMS2 - genetics; MLH1 methylation; Mutation - genetics; MutL Protein Homolog 1 - genetics; Proto-Oncogene Proteins B-raf - genetics; Reproducibility of Results; MLH1 methylation; Mismatch repair deficiency; Endometrial cancer; Colorectal cancer; BRAF mutation; Lynch syndrome
• ISSN: 0014-4800; 1096-0945
• DOI: 10.1016/j.yexmp.2023.104855

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.
•Detecting MLH1 promoter methylation is highly relevant to identify Lynch syndrome patients.•Amplicon based NGS is a simple and low-cost method for detecting MLH1 methylation.•The test showed high agreement with BRAF mutation status in colorectal cancer.