Abstract 3391: Methylation analysis using TriMeth indicate that asymptomatic colorectal cancers (CRCs) release less circulating tumor DNA (ctDNA) compared to symptomatic cancers
Authors: Jensen Sarah Østrup , Øgaard Nadia , Ørntoft Mai-britt Worm , Rasmussen Mads Heilskov , Bramsen Jesper Bertram , Therkildsen Christina , Andersen Claus Lindbjerg 
Published: June 2022

Abstract
Background: Early detection of cancer is key to identify tumours at a time when patients can be offered curatively intended treatments and outcomes are superior. Previous studies have presented sensitive methods for detection of ctDNA in early-stage cancer, but very few have been validated in asymptomatic individuals e.g. a true screening setting. Furthermore, many methods rely on expensive sequencing-based approaches that may not be cost-effective for population-based screening. Recently, we developed TriMeth, a simple CRC-specific ctDNA detection test based on three DNA methylation markers, which showed a sensitivity of 85% (stage I: 80%, stage II: 85%, stage III: 89%, stage IV: 88%) and a specificity of 99% when evaluated in CRC patients and controls (n=434).
Objective: This study aimed to assess the performance of TriMeth cell-free DNA methylation analysis in asymptomatic individuals.
Methods and materials: TriMeth was evaluated in 727 average-risk, asymptomatic individuals from the Danish population-based CRC screening program aged 50-74 years. This cohort included 105 CRC patients (stage I: n=53, stage II: n=24, stage III: n=19, stage IV: n=9) and 622 age and gender-matched controls enriched for comorbidities common to the screening population, such as, diabetes (n=47), arthritis (n=9), hypertension (n=279), arteriosclerosis (n=90), inflammatory bowel disease (n=6), adenomas (n=49), and other cancers (n=91). For comparison, TriMeth was also applied to a cohort of 43 symptomatic CRC patients and 42 cancer-free controls.
Results: In the asymptomatic cohort, the specificity was consistently high (99%) despite the enrichment for controls with comorbidities in this cohort. The sensitivity was 36%, and was reduced for all disease stages compared to symptomatic cancers, though most markedly reduced for stage I CRC (stage I: 9%, stage II: 67%, stage III: 53%, stage IV: 78%). For a subset of asymptomatic individuals (n=91), an additional aliquot of plasma was analyzed, revealing a very high reproducibility of TriMeth (Spearman corr.coeff. r=0.95, p=7.6e-14). However, merging data from the paired samples did not significantly increase sensitivity. TriMeth showed consistently high accuracy in the symptomatic cohort (sensitivity: 84% and specificity: 100%).
Conclusion: TriMeth is highly specific and not affected by comorbidities commonly found in 50-74 years old individuals invited for CRC screening. TriMeth showed high sensitivity for all stages when evaluated in symptomatic CRC patients, but the sensitivity was significantly decreased in asymptomatic cancers, also when stage-stratified. This indicate that asymptomatic cancers, independent of stage, shed less ctDNA than symptomatic cancers and highlights the importance of evaluating methods for early cancer detection in asymptomatic individuals.
Citation Format: Sarah Østrup Jensen, Nadia Øgaard, Mai-britt Worm Ørntoft, Mads Heilskov Rasmussen, Jesper Bertram Bramsen, Christina Therkildsen, Claus Lindbjerg Andersen. Methylation analysis using TriMeth indicate that asymptomatic colorectal cancers (CRCs) release less circulating tumor DNA (ctDNA) compared to symptomatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3391.