Tumor methylation patterns to measure tumor fraction in cell-free DNA.

Abstract

3052 Background: Cell-free DNA (cfDNA) tumor fraction (TF), the proportion of tumor molecules in a cfDNA sample, is a direct measurement of signal for cfDNA cancer applications. The Circulating Cell-free Genome Atlas study (CCGA; NCT02889978) is a prospective, multi-center, observational, case-control study designed to support development of a methylation-based, multi-cancer detection test in which a classifier is trained to distinguish cancer from non-cancer. Here we leveraged CCGA data to examine the relationship between cfDNA containing tumor DNA methylation patterns, TF, and cancer classification performance. Methods: The CCGA classifier was trained on whole-genome bisulfite sequencing (WGBS) and targeted methylation (TM) sequencing data to detect cancer versus non-cancer. 822 samples had biopsy WGBS performed; of those, 231 also had cfDNA targeted methylation (TM) and cfDNA whole-genome sequencing (WGS). Biopsy WGBS identified somatic single nucleotide variants (SNV) and methylation variants (MV; defined as methylation patterns in sequenced DNA fragments observed commonly in biopsy but rarely [ < 1/10,000] in the cfDNA of non-cancer controls [n = 898]). Observed tumor fragment counts (SNV in WGS; MV in TM), were modeled as a Poisson process with rate dependent on TF. TF and classifier limits of detection (LOD) were each assessed using Bayesian logistic regression. Results: Across biopsy samples, a median of 2,635 MV was distributed across the genome, with a median of 86.8% shared with ≥1 participant, and a median of 69.3% targeted by the TM assay. TF LOD from MV was 0.00050 (95% credible interval [CI]: 0.00041 - 0.00061); MV and SNV estimates were concordant (Spearman’s Rho: 0.820). MV TF estimates explained classifier performance (Spearman’s Rho: 0.856) and allowed determination of the classifier LOD (0.00082 [95% CI: 0.00057 - 0.00115]). Conclusions: These data demonstrate the existence of methylation patterns in tumor-derived cfDNA fragments that are rarely found in individuals without cancer; their abundance directly measured TF, and was a major factor influencing classification performance. Finally, the low classifier LOD (~0.1%) motivates further clinical development of a methylation-based assay for cancer detection. Clinical trial information: NCT02889978 .