Biomarkers & Clinical Research

Biography

Biography: Martin Weber

Abstract

Disease-specific alterations of DNA methylation patterns are frequently found in cancer and are currently considered to be suitable biomarkers. These alterations are optimal biomarkers for early detection and disease monitoring when detectable in cell-free DNA. We have developed a multiplexed methyl sensitive restriction enzyme (MSRE) qPCR protocol allowing up to 96 parallel qPCR reactions out of 400µl patient’s plasma. The method was applied on 1) a panel of 35 methylation markers discovered in lung tissue by a targeted DNA methylation micro-array and 2) 63 candidate markers derived form genome wide methylation analyses using Illumina-Methylation450K-Beadchip. To test the feasibility for differential diagnosis via cell free DNA methylation testing, multivariate classification using MSREqPCR data from marker panel 1) enabled differentiation of Lung cancer (n=348; Adeno-Ca: n=100, Squamous cell-Ca: n=100, Small cell-Ca: n=100, and Large cell Ca: n=48) versus cancer-free controls (n=332) with an AUC of 0.81-0.91 depending on the lung cancer entity. Using panel 2) a total of 204 serum and plasma samples (lung cancer, n=33; fibrotic Interstitial lung disease - ILD, n=68; COPD, n=42; healthy, n=61) were tested. ROC analysis revealed an AUC of 0.91 for lung cancer, 0.815 for fibrotic ILD, 0.73 for COPD, and 0.828 for all diseases versus healthy controls. Additionally a set of independent cancer and normal samples could be classified on basis of the top 4 markers (HOXD10, PAX9, PTPRN2 and STAG3) showing an AUC 0.85. Based on 2 discovery studies Lung-cancer biomarkers suitable for minimal invasive diagnostics using multiplexed DNA methylation measurement from 10ng circulating cfDNA have been defined. Both marker-sets and the MSREqPCR approach will be the basis for further validation to improve minimal invasive diagnostics. Moreover, this study confirms that our strategy is suitable for qualifying tissue derived DNA-methylation markers for liquid biopsy testing.