Abstract 726: Ultrasensitive quantification of promoter methylation in cell-free circulating DNA for early detection of lung cancer

Abstract

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. Early detection of lung cancer using Low Dose Computed Tomography (LDCT) screening has been shown to decrease the mortality rate. However, most nodules found are deemed to be benign upon further invasive testing. Thus, complementary minimally-invasive tests are being sought that will help discriminate malignant from benign nodules. Molecular biomarkers are increasingly becoming part of routine clinical practice for the diagnosis, prognosis or prediction of treatment response, with improved disease management and survival outcome. Cell-free circulating DNA (cfDNA) in body fluids, including serum, plasma and urine, has recently emerged as a surrogate for tumor DNA. In addition to providing a minimally-invasive source of tumor DNA, cfDNA reflects molecular alterations and tumor heterogeneity. Epigenetic changes, including DNA methylation, occur early in carcinogenesis. Cancer cells are characterized both by global hypomethylation and hypermethylation of CpG islands in gene promoter regions. Analysis of tumor-specific DNA methylation in cfDNA is a promising strategy for applying epigenetic biomarkers to the detection of cancers at an early-stage. In a prior genome-wide analysis of DNA methylation, we identified a locus methylated de novo in fresh frozen tumor tissues resected from stage I lung cancer patients, that had high discriminatory power to distinguish tumor from non-tumor tissues in multiple patient cohorts. High promoter methylation was also associated with shorter cancer-specific survival. The present study aims at evaluating the diagnostic significance of promoter methylation in cfDNA, and the prognostic value in formalin-fixed paraffin-embedded (FFPE) tissues. We developed a methylation-specific droplet digital PCR (ddPCR) assay to quantify rare methylation events. DNA was subjected to bisulfite treatment to convert unmethylated cytosine residues to uracil. We designed specific primers and probe containing 7 CpGs to only amplify the methylated promoter. Experimental conditions were first optimized using fully methylated and unmethylated control DNAs, DNA extracted from lung cancer cell lines, germline cells and lung tissues (paired tumor and non-tumor). The ddPCR assay has a limit of detection of 30 haploid genomes equivalent of methylated promoter DNA, and a limit of quantification of a single methylated allele present at 0.2% (i.e., 1 methylated copy among 500 unmethylated copies). We demonstrated the assay linearity, reproducibility and specificity for the methylated locus. Differences in methylation levels between tumors and adjacent tissues were also observed. We have thus established a robust and ultrasensitive method for standardized determination of promoter methylation status in cfDNA. We are currently evaluating its potential value for noninvasive diagnosis and prognosis of lung cancer patients. Citation Format: Delphine Lissa. Ultrasensitive quantification of promoter methylation in cell-free circulating DNA for early detection of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 726. doi:10.1158/1538-7445.AM2017-726