Mutational Analysis Identifies Therapeutic Biomarkers in Inflammatory Bowel Disease-Associated Colorectal Cancers

Abstract

Purpose: Inflammatory bowel disease-associated colorectal cancers (IBD-CRC) are associated with a higher mortality than sporadic colorectal cancers. The poorly defined molecular pathogenesis of IBD-CRCs limits development of effective prevention, detection, and treatment strategies. We aimed to identify biomarkers using whole-exome sequencing of IBD-CRCs to guide individualized management.Experimental Design: Whole-exome sequencing was performed on 34 formalin-fixed paraffin-embedded primary IBD-CRCs and 31 matched normal lymph nodes. Computational methods were used to identify somatic point mutations, small insertions and deletions, mutational signatures, and somatic copy number alterations. Mismatch repair status was examined.Results: Hypermutation was observed in 27% of IBD-CRCs. All hypermutated cancers were from the proximal colon; all but one of the cancers with hypermutation had defective mismatch repair or somatic mutations in the proofreading domain of DNA POLE Hypermutated IBD-CRCs had increased numbers of predicted neo-epitopes, which could be exploited using immunotherapy. We identified six distinct mutation signatures in IBD-CRCs, three of which corresponded to known mechanisms of mutagenesis. Driver genes were also identified.Conclusions: IBD-CRCs should be evaluated for hypermutation and defective mismatch repair to identify patients with a higher neo-epitope load who may benefit from immunotherapies. Prospective trials are required to determine whether IHC to detect loss of MLH1 expression in dysplastic colonic tissue could identify patients at increased risk of developing IBD-CRC. We identified mutations in genes in IBD-CRCs with hypermutation that might be targeted therapeutically. These approaches would complement and individualize surveillance and treatment programs. Clin Cancer Res; 24(20); 5133-42. ©2018 AACR.

Figure 1. Somatic mutational rates and survival analysis of IBD-CRC.

(A). Mutational frequency in each of the 34 IBD-CRCs ordered by overall mutation rate. There is a clear separation between the 10 hypermutator cancers and the 24 non-hypermutator cancers. With the exception of 15G1, 15G2, and 6J, the hypermutator cancers showed elevated mutation rates of both SNVs and InDels. No InDels were found in the exome of 21M. (B) Kaplan-Meier plot of overall survival stratified by cancer mutator phenotype. Patients with hypermutators cancers had increased survival compared with patients with non-hypermutator cancers (log rank test, P=.04).

Figure 2. Clinical and genetic characteristics of the IBD-CRC.

Top panel: Mutational rate of each cancer case ordered by tumour site and mutation rate. The red line segregates the proximal and distal bowel cancers. Second panel: Tumour site, Dukes’ stage, cancer type, underlying IBD, immunohistochemical testing of MLH1 (MLH1 IHC) and promoter methylation status of MLH1 (MLH1 Pr-M). Third panel: selected genes and somatic nonsense, non-silent and InDel mutations. The colours indicate the predicted effect of the mutation on the protein sequence, and the number indicates the number of mutations with the same predicted effect. Note that in cases where a gene has multiple mutations with different predicted effects, only the effect type with the highest priority will be shown. Further details are in the Supplementary Methods and Materials. The complete list of mutations and their effects is in Supplementary Tables S2A - S2B. Lower panel: Contribution of signatures of mutational processes (A-F) in each cancer case. Each signature A-F corresponds to a signature identified by Alexandrov et al. (Signatures 10, 1, 13/2, 17, 6 and 5, respectively) with cosine similarities ranging from 0.82 to 0.97 (Supplementary Table S3).

Figure 3. Signatures of mutational processes in IBD-CRC.

(A). The somatic mutation spectra of the IBD-CRC and sporadic CRC datasets are represented by each of the six possible substitution types, C>A, C>G, C>T, T>A, T>C, T>G (including their reverse complements) in the context of their immediate 5’ and 3’ flanking bases, giving 96 possible motif combinations. For each substitution type, the 16 motifs are listed in order from left to right by the 5’ flanking base (A, C, G, then T), then by the 3’ flanking base (A, C, G, then T). The IBD-CRC panel shows the mutation spectrum for 34 IBD-associated colorectal cancers; the CRC (TGCA) panel shows the mutation spectrum for 115 colon adenocarcinomas and 267 rectum adenocarcinomas from downloaded from TCGA Data Portal, and the CRC (NHS/HPFS) panel show the mutation spectrum for a cohort of 619 CRCs from Giannakis et al. (14). (B) Six distinct mutation signatures (A-F) were extracted from IBD-CRCs and on comparison with the Alexandrov Signatures 1 to 30 (33) using cosine similarity (Supplementary Table S3), the correspondence is: Signature A and Signature 10 (POLE mutations); Signature B and Signature 1 (age/spontaneous deamination of 5-methylcyotosine); Signature C and Signature 13/2 (AID/APOBEC activation); Signature D and Signature 17 (unknown aetiology); Signature E and Signature 6 (mismatch repair deficiency and microsatellite instability); Signature F and Signature 5 (unknown aetiology; found in all cancer types).

Figure 4. Hotspot microsatellite InDels, driver genes and recurrent somatic copy number alterations in IBD-CRC genomes.

(A). Hotspot InDels and driver genes in IBD-CRC. Somatic SNVs and InDels were used to identify hotspot InDels and driver genes in hypermutator (n=9) and non-hypermutator cancers (n=24). Because hypermutator case 15G1 and 15G2 were related, SNV and InDels were merged for this analysis. (B) Chromosome arm-level somatic copy number alterations. Shown are predicted SCNAs that cover at least 90% of a chromosome arm (left panel), and the frequency of the SCNAs in IBD-CRC (this study), and in sporadic CRC studies by Sheffer et al. (33) and TCGA (25) (right panel). Chromosome arms are listed in descending order of frequency in IBD-CRC. Grey boxes in the frequency columns indicate unknown values. IBD-CRC cases are ordered left to right by overall SNV and InDel mutation rate. Cases to the left of the dashed line are hypermutator cancers.

Figure 5. Predicted HLA Class I neo-epitopes in IBD-CRCs.

The cancers with the highest mutational rates (hypermutators) generated the largest number of predicted HLA Class I neo-epitopes.