3049 Background: For multi-cancer detection using cfDNA, TOO determination is critical to enable safe and efficient diagnostic follow-up. Previous array-based studies captured < 2% of genomic CpGs. Here, we report genome-wide fragment-level methylation patterns across 811 cancer cell methylomes representing 21 tumor types (97% of SEER cancer incidence), and define effects of this methylation database on TOO prediction within a machine learning framework. Methods: Genomic DNA from 655 formalin-fixed paraffin-embedded (FFPE) tumor tissues and 156 isolated cells from tumors was subjected to a prototype 30x whole-genome bisulfite sequencing (WGBS) assay, as previously reported in the Circulating Cell-free Genome Atlas (CCGA) study (NCT02889978). Two independent TOO models, one with and one without the methylation database, were fitted on training samples; each was used to predict on the test set. A WGBS classifier was used to detect cancer at 98% specificity; reported TOO results reflect percent agreement between predicted and true TOO among those detected cancers (166 cases: 81 stage I-III, 69 stage IV, 16 non-informative). Results: Genome-wide methylation data generated from this database allowed fragment-level analysis and coverage of ~30 million CpGs across the genome (~60-fold greater than array-based approaches). Incorrect TOO assignments decreased by 35% (20% to 13%) after incorporating methylation database information into TOO classification. Improvement was observed across all cancer types and was consistent in early-stage cancers (stage I-III). Respective performances in breast cancer (n = 23) were 87% vs 96%; in lung cancer (n = 32) were 85% vs 88%; in hepatobiliary (n = 10) were 70% vs 90%; and in pancreatic cancer (n = 17) were 94% vs 100%. Results using an optimized approach informed by these results in a large cohort of CCGA participants will be reported. Conclusions: Incorporating data from a large methylation database improved TOO performance in multiple cancer types. This supports feasibility of this methylation-based approach as an early cancer detection test across cancer types. Clinical trial information: NCT02889978.
Dissociation of chick embryonic tissue for FACS and preparation of isolated cells for genome-wide downstream assays
