Abstract
BACKGROUND: Blood-based cell-free DNA (cfDNA) methylation testing has emerged as a promising approach for multi-cancer early detection (MCED), holding the potential to improve cancer survival rates. However, traditional bisulfite-based methods often encounter sensitivity limitations in detecting early-stage malignancies or certain cancer types. In the INSPECTOR study, we developed a MCED and cancer signal origin (CSO) system specifically designed for early-stage or hard-to-detect cancers, including those of the lung, breast, colorectum, liver, esophagus, stomach, pancreas, and ovary. METHODS: We established a comprehensive methylation marker discovery database (n = 6,342) by integrating public datasets (n = 4,699) and in-house samples (n = 1,643), all processed using human TET (hTET) enzyme-assisted whole-methylome sequencing (GM-seq). This enabled the design of a targeted panel encompassing 155,362 methylated CpG sites. Leveraging hTET-assisted high-depth next-generation sequencing (NGS), our blood test achieved a median unique depth of 1,093×. Multicenter case-control cohorts, including various pathological subtypes, were used for training, validation, and independent validation of MCED and CSO models, and to verify the clinical feasibility. RESULTS: Clinical validation was conducted across multi-center case-control cohorts, including 1,071 participants in the training set, 581 in the validation set, and 824 in the independent validation set. The MCED assay demonstrated robust performance with a specificity of 99.1% and sensitivity of 83.2% in the training set, 99.0% and 81.8% in the validation set, and comparable results in the independent validation set (99.0% specificity, 81.9% sensitivity). Notably, sensitivity reached 65.5% for stage I cancers, 79.7% for stage II, and 71.3% for stages I-II combined. The sensitivities for different cancer types were as follows: esophageal (79.2%), gastric (76.1%), colorectal (86.2%), pancreatic (66.7%), liver (100.0%), lung (72.9%), breast (88.9%), and ovarian (87.9%). The CSO model exhibited strong accuracy, with top-1 cancer origin prediction rates of 87.9% (validation) and 87.4% (independent validation), rising to 95.1% and 94.5% for top-2 predictions, respectively. For stage I cancers specifically, the top-1 accuracy was 85.5%. CONCLUSIONS: These findings underscore the efficacy of the hTET-assisted cfDNA methylation sequencing system across diverse cancer types, particularly in early stages. Enzyme-assisted NGS test of methylated cfDNA thus enhances the clinical utility of non-invasive blood-based screening.