Abstract 5604: Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer - A clinical biomarker discovery and validation study

Background: Screening for colorectal cancer (CRC) using fecal occult blood tests (FOBT) reduces CRC mortality, and many CRC screening programs therefore use FOBT. However, FOBT is not the best approach; first, because population acceptance is low due to unpleasantness of fecal sampling; second, because bowel tumors bleed only intermittently, which limits FOBT sensitivity. Development of a novel blood-based screening approach may alleviate these problems. Objective: This study aims to develop and validate novel blood-based biomarker assays to achieve high patient compliance, sensitivity and specificity. Methods and materials: CRC-specific DNA methylation biomarker candidates were identified using a genome-wide discovery strategy based on >4,000 Illumina 450K DNA methylation arrays. We designed digital droplet PCR assays to detect top biomarker candidates in circulating cell-free DNA (cfDNA) isolated from plasma. Initially, sensitivity and specificity of biomarkers were evaluated in validation cohort 1 consisting of plasma collected from 114 symptomatic CRC patients and 86 colonoscopy-confirmed healthy controls. To ensure comparable technical sensitivities for all assays in all samples, we used a fixed cfDNA input of 4,500 copies per ddPCR reaction. Next, we tested markers in validation cohort 2, a selected cohort of 8 mL plasma collected from participants in the Danish national screening program. This cohort comprised 131 asymptomatic CRCs and 869 controls enriched for comorbidities like adenomas, other cancers, diabetes, arthritis, hypertension, inflammatory bowel disease and arteriosclerosis. Results: Our discovery identified 12 DNA methylation biomarkers. Their performance was evaluated in validation cohort 1, and the three best performing markers showed a sensitivity of 89% at a specificity of 99%, which is superior to FOBT. Sensitivity increased with stage, reaching 65%, 85%, 78% and 83% for stage I-IV, respectively. When evaluated in validation cohort 2, the sensitivity was 51% for CRC samples with an input of at least 4,500 copies of cfDNA, and sensitivity was reduced equally for all disease stages compared with results from validation cohort 1. Sensitivity correlated positively with cfDNA input, and it is therefore critical to collect sufficient plasma volumes to achieve high sensitivities in clinical practice. Finally, specificity was 92.4 % even though we enriched for comorbidities in this cohort, and specificity was 95.7% in controls with a clean colon and no comorbidities. Conclusion: Our systematic biomarker discovery and validation study identified a three-gene DNA methylation panel with superior performance in plasma compared to FOBT. Citation Format: Sarah Østrup Jensen, Mai-Britt Worm Ørntoft, Nadia Øgaard, Helle Kristensen, Mads Heilskov Rasmussen, Jesper Bertram Bramsen, Peter Mouritzen, Mogens Rørbæk, Anne-Sofie Kannerup, Søren Laurberg, Hans Jørgen Nielsen, Claus Lindbjerg Andersen. Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer - A clinical biomarker discovery and validation study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5604.