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Patients with ulcerative colitis (UC) are enrolled into surveillance programs for the early detection of colorectal cancer (CRC). However, most patients under surveillance are low-risk and never progress to CRC, while a significant proportion of CRCs in UC form without a preceding confirmed diagnosis of dysplasia. High resolution chromosomal copy-number alteration (CNA) analysis of unselected formalin-fixed paraffin embedded biopsies taken at surveillance colonoscopies using low pass whole genome sequencing (lpWGS) offers an appealing approach to CRC stratification.

We conducted a retrospective case-control study to compare the CNA burden in four unselected non-neoplastic left-sided colorectal biopsies from patients with E2/E3 UC derived 1-5 years prior to HGD/CRC detection (cases), with that of biopsies from patients who subsequently remained HGD/CRC-free for at least 5 years (controls). The two patient groups were matched by age, gender, duration of IBD and PSC status. lpWGS was performed using a standardised pipeline for epithelial enrichment, DNA extraction, library preparation, next generation sequencing and bioinformatic analysis.

476 biopsies, derived from 42 cases and 77 controls, were analysed. Nearly 80% of patients had a detectable CNA in at least one of their biopsies, with the maximal CNA burden in a typical biopsy involving a median 1.1% of that biopsy’s genome. The CNA burden was significantly greater in the rectum compared to the sigmoid colon and descending colon. The most common CNA events were losses of between 1-30 megabases involving the sub-telomeric regions of chromosomes 5-9 and 22, which were found in similar proportion in both case and control biopsies. However, losses extending beyond sub-telomeric regions, as well as copy number gains, were found more frequently in cases biopsies (p<0.0001). The most discriminating CNA event was the presence of such a loss extending beyond subtelomeric regions in any of the patient’s four biopsies, with a high specificity exceeding 0.95 (see Kaplan-Meier plot). ROC analysis demonstrates that lpWGS output has a fair level of accuracy at predicting future HGD/CRC risk (AUC 0.73).

We identified multiple biopsies, predominantly in cases, with a surprisingly marked CNA burden involving over 10% of the genome, highlighting the fluid phenotype-genotype relationship. Non-dysplastic colitic epithelium can bear a significant burden of CNAs and maintain phenotypic stability for years without neoplastic transformation. Remarkably, by analysing the CNA burden of only four random biopsies, derived from less than 0.05% of the colonic surface area, we can significantly discriminate between case and control cohorts.

Protein profiling in patients with inflammatory bowel diseases (IBD) for diagnostic and therapeutic purposes is underexplored. Assessment of interactions between genetics and the plasma proteome could lead to identification of novel disease-associated molecular pathways. In this study, we performed the largest gene-protein association analysis thus far in patients with IBD, taking into account relevant phenotypic covariates and integrating information from multiple biological data layers.

Ninety-two (92) inflammation-related proteins were quantified in plasma of 1,028 patients with IBD (567 Crohn’s disease [CD]; 461 ulcerative colitis [UC]) and 148 healthy individuals to assess proteome-phenotype associations. Both whole-exome sequencing (WES) and global screening array (GSA) data of 919 patients with IBD were included to study associations between over 8 million genetic variants and protein levels (protein quantitative trait loci [pQTL]). Cis-pQTLs were defined within ± 1 Mb of the region of each protein-coding gene center, whereas trans-pQTLs were outside of that region. After adjusting for phenotypic covariates, a step-wise conditional analysis was used to identify all independent pQTLs in CD and UC separately, followed by a meta-analysis. Intestinal mucosal RNA sequencing and fecal metagenomic data were used for complementary analyses.

Thirty-four (34) proteins were differentially abundant between IBD and healthy individuals, of which 24 proteins independent of active inflammation. (Figure 1) Seventy-two (72) proteins were significantly associated to 14 phenotypic factors, including age, sex, medication use, and surgical history. (Figure 2) Fibroblast growth factor-19 (FGF-19) levels were decreased in CD patients with ileal disease or a history of ileocecal resection. Thirteen (13) novel cis-pQTL variants were identified and 10 replicated from previous studies, together affecting 21 different plasma proteins. One trans-pQTL variant of the FUT2 gene (rs602662) and two independent cis-pQTL variants of the CCL25 gene significantly affected plasma C-C motif chemokine ligand 25 (CCL25) levels. (Figure 3) Intestinal gene expression data revealed an overlapping cis-expression (e)QTL-variant (rs3745387) of the CCL25 gene. The FUT2 rs602662 trans-pQTL variant associated significantly with reduced abundances of multiple fecal butyrate-producing bacteria, including the genus Blautia and the species Faecalibacterium prausnitzii.

This study shows that both genotype and multiple disease phenotypes strongly associate with the plasma proteome in patients with IBD and identifies disease-associated pathways that may help to improve disease management in the future.