Validation of a sensitive, tissue-free blood test for biomarker discovery and tumor burden assessment

Background: Rapid progress in molecular and computational technologies for circulating tumor DNA (ctDNA) analysis is transforming precision oncology, allowing tumor detection, relapse surveillance, and treatment selection without invasive tissue sampling. Among these approaches, methylation profiling of ctDNA offers a sensitive and quantitative measure of tumor burden. However, signal-to-noise ratios varies across cancer types and technologies, underscoring the need for tumor-specific assay optimization and rigorous validation. We previously developed a tissue-free, methylation-based assay and demonstrated its clinical validity in lung, colorectal, and liver cancers. Here we report expanded, larger scale validation across additional tumor types, including pancreatic cancer (PDAC) and bile tract cancer (BTC). Methods: The CanCatch ® Surf classifier was trained and subsequently locked, as previously described. The total analytical performance testing dataset comprises >1,000 contrived and clinical samples (10-30 ng input). In this study, we constructed dilution series of PDAC- and BTC-derived cell-line and cfDNA mixtures spanning 0.001-0.5% tumor allele fraction (TAF). The in-silico titration dataset was generated by digitally blending sequencing reads from PDAC and BTC patients with those from cancer-free donors at defined proportions (0.001-0.5%). Probit regression identified the limit of detection (LoD) as the lowest TAF detected with ≥95% probability; the limit of blank (LoB) was the per-sample positivity rate in age-matched cancer-free donors. Results: The analytical sensitivity for PDAC and BTC reached 0.02% for in silico mixtures, cell-line dilutions and cfDNA titrations, with no false positives among 72 age-matched cancer-free donors (0 %; 95% CI 0-5.0%). Evaluation of 176 pre-treatment plasmas (57 PDAC, 50 BTC, 69 controls) demonstrated a sensitivity of 84.2% for PDAC (stage I 75.0 %, II 86.7%, III 83.3%, IV 100%) and 82.0% for BTC (stage I 71.4%, II 66.7%, III 93.3%, IV 92.3%), both at 98.6 % specificity; larger cohorts are in progress, and updated results will be presented as available. Conclusions: CanCatch ® Surf's non-invasive approach to ctDNA detection in solid tumors has demonstrated performance comparable to traditional tissue-based methods, thus holding promise for broader clinical use in monitoring disease recurrence and assessing treatment effectiveness.