Molecular evolution of rectal cancer

Abstract

Colorectal cancer (CRC) is a significant health burden, with rectal cancers accounting for approximately one-third of new diagnoses. With rectal cancers presenting as a distinct subset of CRCs in both their pathology and treatment, this work aimed to use next-generation sequencing to explore the molecular evolution of rectal tumours from adenomas to advanced carcinomas undergoing treatment, before exploring a specific subset of atypical BRAF mutant CRCs which appeared enriched in cancers of the distal colon and rectum. In a cohort of five patients with multi-region whole-genome sequencing (WGS) of large rectal adenomas that had yet to progress despite their size, no clonal molecular alterations associated with invasive carcinoma were found outside of truncal mutations in APC and KRAS. However, subclonal oncogenic mutations in TP53 and MYC were found in one patient (1/5, 20%). All tumours were microsatellite stable, and polygenic clonal evolution was observed between regions. Reconstructed tumour phylogenies suggested linear selective-sweeps operated early in development, followed by a switch towards branched evolution later in development to modulate tumour fitness more subtly. Focus was next shifted towards late-stage rectal cancers undergoing the selection pressure of neoadjuvant chemoradiotherapy. Longitudinally sampled rectal carcinoma tissue, or existing sequencing data, was obtained from the RHYTHM-I and XERXES clinical trials respectively. RHYTHM-I samples underwent WGS (5 patients) and RNA-seq (9 patients), whereas existing WGS data was leveraged for XERXES (6 patients). Somatic copy-number alterations and clonal heterogeneity underwent the largest changes between the pre-treatment and first-dose timepoints, and mutation signatures associated with chemoradiotherapy were observed in some samples. Finally, a combined cohort of sequencing data for 7,478 CRCs was assembled to characterise so-called atypical (class 2 or 3) BRAF-mutant CRCs, due to their apparent enrichment in the distal colon and rectum, which contrasts the canonical BRAF V600E variant (class 1). These classes of BRAF mutation are rarer, and paradoxically have reduced (class 2) or absent (class 3) kinase activity relative to class 1. The dataset consisted of 708 class 1 (9.47%), 33 class 2 (0.44%), and 90 class 3 (1.2%) BRAF mutant CRCs. These tumours were associated with the distal colon and rectum (60.7% class 2 and 59.2% class 3), microsatellite stability (0% and 4.9% MSI), and concomitant Ras pathway mutations (27.3% and 47.7%) – many of which were atypical in CRC themselves. Patients with atypical BRAF mutations had a better prognosis than those with V600E mutations, however concomitant Ras mutation may attenuate this affect. Conditional mouse models of class 3 mutant Braf homologous to BRAF G466V, D594N, and D594G were developed for use in future work further exploring atypical BRAF. In conclusion, this work provides additional insight into the dynamic nature of molecular evolution within rectal cancers throughout development and treatment. Rectal tumours are not constrained to a single model of clonal evolution, shifting from a series of early selective-sweeps towards a more branched model of evolution later in development. For late-stage rectal carcinomas undergoing neoadjuvant chemoradiotherapy, the earliest doses appear to have the greatest effect on tumour heterogeneity, likely through the loss of highly-vulnerable subclones. This study further provides new insights into the distinct subset of atypical BRAF mutant CRCs, particularly in that they appear to amplify existing Ras signalling following a two-mutation mini-driver model of cancer evolution, in addition to exhibiting distinct clinicopathological characteristics that contrast strongly with the canonical view of BRAF mutant CRC.