5-hydroxymethylation signatures in plasma circulating cell-free DNA as markers for appendiceal and colorectal peritoneal metastasis.

Abstract

195 Background: Noninvasive tests for peritoneal metastasis (PM) detection lack sensitivity. Genome-wide mapping of 5-hydroxymethylcytosine (5hmC) on nanogram quantities of peripheral plasma circulating cell-free DNA (PcfDNA) was previously shown to differentiate non-metastatic colorectal cancer from healthy subjects. We aimed to examine if patients with colorectal cancer (CRC), high grade appendiceal cancer (HGA) or low grade appendiceal cancer (LGA) with PM have distinct signatures of 5hmC in PcfDNA compared to each other and to patients matched for tumors without PM. Methods: We analyzed plasma samples from a prospectively collected tissue bank. To correlate 5hmC signatures with intraoperative findings, only patients who underwent abdominal surgery in proximity to plasma collection were selected. Key steps of PcfDNA processing included extraction from plasma, nano-hmC-Seal chemical labeling and enrichment of 5hmC-modified fragments, next-generation sequencing, and mapping to the reference human genome. DESeq2 R package was finally used to compare relative 5hmC enrichment and detect distinct 5hmC signatures according to disease histology and PM presence. Results: Plasma samples were collected between 11/2016 – 3/2019 from 46 patients with CRC (n = 21), HGA (n = 17) and LGA (n = 8). Of those, 32 (70%) had PM based on intraoperative findings (median peritoneal cancer index score = 15, range 2-39) and 14 did not have PM. Most samples (91%) were collected on the same day as surgery. An average of 24 million paired-end reads were sequenced for each sample. Four samples (8.7%) were excluded from the analysis due to low sequencing coverage or high duplication level. Unique 5hmC enrichment patterns were found to discriminate with p < 0.05 between CRC PM and HGA PM (n = 616 differentially hydroxymethylated genes (DHMGs)), CRC/HGA PM and LGA PM (n = 1074 DHMGs), and CRC/HGA PM and CRC/HGA patients without PM (n = 1576 DHMGs). Conclusions: Distinct signatures of 5hmC in PcfDNA could differentiate patients with CRC/HGA/LGA PM from each other and from patients with similar tumor histologies without PM. Thus 5hmC signatures in PcfDNA might potentially serve as a sensitive marker of occult PM.