M1929 Utility of MS-Mlpa Methylation Analysis in the Diagnosis of Lynch Syndrome

Abstract

Despite the advances in the selection of patients for genetic testing in Lynch syndrome at least a third of patients with Bethesda criteria and lack of MLH1 expression have sporadic tumours and should not receive genetic testing. The aim of this study is to investigate the role of the methylation analysis using MS-MLPA in the diagnosis of Lynch syndrome. MSMLPA is a novel and methodologically easy technique for quantitative measure of gene methylation. Methods. Sixty colorectal cancer (CRC) specimens with loss of MLH1 expression were selected. DNA from paraffin-embedded tumours was tested for 2 quantitative methylation detection methods: Methylight (Applied biosystems) and MS-MLPA (Methylation Specific-Multiplex Ligation Probe Amplification) (MCR Holland) calculating Methylation Ratio in MLH1. Somatic mutation in BRAF V600E was investigated from tumour DNA. Germline mutations in MLH1 were analyzed by sequentiation. Results: Correlation of quantitative results of the 2 methylation analysis techniques (Methylation and MS-MLPA) was 96.7% (p <0,001). Germline mutations in MLH1 were found in 8 out of 60 patients (13,3%). None of the tumours from these patients with Lynch syndrome showed significant methylation. Forty-two out of 52 (80%) tumours from patients without germline mutation in MLH1 showed significant methylation using Methylight. Using MS-MLPA, methylation was found in 44 out of 52 patients (85%). Both techniques had sensitivity and negative predictive value of 100 % to detect germline mutation patients. Specificity was 80 % for Methylight and 85 % for MS-MLPA, and positive predictive value (PPV) was 44 % for Methylight and 50 % for MS-MLPA. These values were clearly better than the obtained using BRAF V600E mutation, with a specificity of 53,8%. Conclusion: Methylation analysis is a high sensitivity and specific method for detecting sporadic CRC unstable cases. These results highlight the role of MS-MLPA assays as a simple and reliable method for selection of patients for genetic testing in Lynch syndrome.