Clinical validation of red blood cell DNA for early detection of advanced colorectal neoplasia: A single-center prospective observational case-control clinical trial.

3568 Background: Early detection of colorectal cancer (CRC) improves survival, but current screening is limited by poor colonoscopy compliance and low cfDNA sensitivity for early-stage lesions and advanced adenomas (AA). Beyond tumor shedding, solid tumors remotely disrupt bone marrow hematopoiesis, inducing systemic genomic instability in hematopoietic lineages. Leveraging this mechanism, we developed a noninvasive detection strategy using genome-wide profiling of DNA remnants in mature red blood cells (rbcDNA). We previously showed that tumor-induced IL-18/NR4A1 signaling drives locus-specific DNA damage in hematopoietic progenitors, generating rbcDNA signatures distinct from shedding-dependent cfDNA and enabling improved detection of early-stage cancer. Here, we report the first multicenter and prospective clinical validation of this rbcDNA-based assay for the detection of early colorectal neoplasia (NCT05875584). Methods: We enrolled 1,251 colonoscopy-confirmed participants (561 non-advanced neoplasia controls (non-AN), 330 AA, 360 CRC). Peripheral blood (1–2 mL) was collected from each participant, and rbcDNA was isolated and sequenced by low-coverage whole-genome sequencing (~2×), as previously described (PMID: 40341742). Participants were randomly assigned (8:2) to discovery and test cohorts. The discovery cohort was used for CRC- and AA-associated rbcDNA features identification and model development, while the test cohort was used for cutoff optimization. This locked model was externally validated in two independent cohorts. A prospective, observational case-control study was also conducted, in which blood and stool samples were collected simultaneously to compare the rbcDNA classifier with quantitative FIT (qFIT). Among 598 enrolled participants, 585 were included in the final analysis (299 non-AN, 206 AA, and 80 CRC). Results: At a predefined 90% specificity, the rbcDNA classifier achieved sensitivities of 85% for AA and 95% for CRC in the test cohort. Across two external cohorts, the assay consistently achieved 80% and 78% sensitivity for advanced colorectal neoplasia, 91% sensitivity for CRC in both cohorts, and 92% and 91% specificity. In the prospective study, rbcDNA yielded 90% sensitivity for CRC and 60% for AA at 90% specificity, with specificity remaining comparable in participants with non-neoplastic findings and non-advanced adenomas. Compared with qFIT, rbcDNA matched overall CRC sensitivity (90% vs. 88%) but outperformed it for stage I CRC (83% vs. 61%) and advanced adenomas (60% vs. 18%), including >50% of large sessile serrated lesions and tubular adenomas (≥1 cm) versus <15% by qFIT. Conclusions: This first prospective clinical study demonstrates that rbcDNA provides a highly sensitive, non-invasive approach for early colorectal neoplasia detection. Clinical trial information: NCT05875584 .