Detection of Circulating Tumor DNA Shedding from Advanced Precancerous Lesions

Minimally invasive, blood-based tests for the detection of colorectal cancer (CRC) and advanced precancerous lesions (APLs) have emerged as a screening tool to aid in the early detection of cancer. While cell-free (cf)DNA detection of CRC has demonstrated high sensitivity and specificity, APL detection has been less successful. In an effort to understand these limitations, we evaluated the prevalence of APL cfDNA biomarkers from plasma samples. Formalin-fixed, paraffin-embedded (FFPE) or fresh frozen (FF) APL tissue and matched normal blood samples from 61 (FFPE n=27, FF n=34) individuals were obtained through biobanks. Whole-genome sequencing (WGS) data was used to design custom, research-use only, personalized, mPCR-NGS-based circulating tumor (ct)DNA assays. Matched plasma samples collected at the same time as tissue and whole blood collection were evaluated for the presence of circulating APL biomarkers in plasma. Sensitivity was calculated in FFPE and FF for the overall cohorts, by APL subtype, and lesion size. Because the subtype distribution differed from expected prevalence in screening trials (pmids 38477985, 40455622), a distribution was constructed to be representative of the final pivotal study so that a reflective sensitivity estimate could be calculated. Overall at a 90% specificity, APL plasma sensitivity adjusted for subtype incidence was 25% and 37% in matched FFPE and FF tissue samples, respectively. The lower plasma detection rate in FFPE samples could be due to the compromised tissue gDNA integrity and personalized mPCR assay design-ability with FFPE tissue preservation methodology. Median plasma sample variant allele frequencies (VAF) of detected samples was 3.6x10-5 and 1.4x10-5 for FFPE and FF, respectively. APL plasma sensitivity for was calculated for each APL subtype, including high-grade dysplasia (FFPE: 40%, N=10; FF: 38%, N=10), villous growth >25% (FFPE: 25%, N=8; FF: 38%, N=13), tubular adenomas >10 mm (FFPE: 25%, N=4; FF: 33%, N=3), and serrated lesions >10 mm (FFPE: 20%, N=5; FF: 50%, N=8). In general, sensitivity increased as lesion size increased (FFPE: 4-10 mm 20%, 11-20 mm 30%, 21-30 mm 50%, ≥31 mm 50%; FF: 4-10 mm 22%, 11-20 mm 47%, 21-30 mm 67%, ≥31 mm: 33%). In this study, we established a baseline of APL ctDNA shedding using a tissue-informed ctDNA assay that can be used for optimizing assays designed to detect APLs at expected levels in cell-free DNA. Citation Format: Fei Lu, Liliana Cerna, Spenser Alexander, Noura Tbeileh, Esha Atolia, Dina Hafez, Eser Kirkizlar, Matthew Rabinowitz, Alexey Aleshin, Trupti Kawli.. Detection of cfDNA shedding from advanced precancerous lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3856.