Utilization of nano-hmC-seal technology to detect epigenetic signatures of peritoneal metastasis in cell-free DNA (cfDNA) in patients with colorectal and high-grade appendiceal cancer.

e15510 Background: Peritoneal metastases from lower GI tumors are common and cause significant morbidity. Assays of circulating-tumor DNA (ctDNA) to identify peritoneal recurrence after curative-surgical intent are limited due to the low abundance of DNA shed by these tumors, especially mucinous tumors. We demonstrate for the first time an ultra-sensitive test to detect biomarkers modified by the epigenetic mark 5-hydroxymethylcytosine (5hmC) in human cfDNA utilizing nano-hmC seal technology to identify patients with peritoneal metastases (PM) after curative-intent resection. Methods: Patients treated with curative intent resection of colorectal (CRC) and high-grade appendiceal (HGA) cancers underwent prospective plasma collection. Peritoneal disease was documented by direct visual inspection (laparoscopy/laparotomy). Plasma samples were analysed using nano-hmC-seal technique to obtain 5hmC signatures. Genome-wide differential analysis was used to identify significant 5hmC- differentially modified gene body regions (p-value < 0.01 and log-fold change > 10%). Functional annotation analysis was performed to further characterize differentially 5hmC -modified genes noted on multivariate logistic models. Results: Of 64 patients included in the study (CRC = 26; HGA = 38), 47 (73%) were PM+. The median DNA extracted per sample was 23.5 ng (IQR: 15.8-38.3), and no sample failed sequencing criteria. After genome-wide analysis, 39 gene body regions were identified as differentially 5hmC-modified, and 14 genes remained significant after multivariate adjustment. The signatures for PM+ patients were distinguishable from PM- patients using hierarchical clustering and principal component analyses. Functional annotation analysis based on the 14 genes point to alterations in immune and microbiome-related pathways in PM+ group (Table). Conclusions: We report for the first time the detection of peritoneal metastasis in cfDNA from human lower GI cancers using nano-5hmC seal epigenomic technology. This technology could be exploited to detect recurrences in tumors that shed very low levels of cfDNA. This signature is being validated in a prospective clinical trial (NCT 04157322).[Table: see text]