Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The Braf^V637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

Colorectal cancer is one of the world’s most prevalent and lethal cancers. Mutations of the KRAS gene occur in ~40% of metastatic colorectal cancers. While this cohort has historically been difficult to manage, the last few years have shown exponential growth in the development of selective inhibitors targeting KRAS mutations. Their foremost mechanism of action utilizes the Switch II binding pocket and Cys12 residue of GDP-bound KRAS proteins in G12C mutants, confining them to their inactive state. Sotorasib and Adagrasib, both FDA-approved for the treatment of non-small cell lung cancer (NSCLC), have been pivotal in paving the way for KRAS G12C inhibitors in the clinical setting. Other KRAS inhibitors in development include a multi-targeting KRAS-mutant drug and a G12D mutant drug. Treatment resistance remains an issue with combination treatment regimens including indirect pathway inhibition and immunotherapy providing possible ways to combat this. While KRAS-mutant selective therapy has come a long way, more work is required to make this an effective and viable option for patients with colorectal cancer.

Colorectal cancer is an important cause of morbidity and mortality worldwide. The current treatment landscape includes chemotherapy, targeted therapy, immunotherapy, radiotherapy, and surgery. A key challenge to improving patient outcomes is the significant inter-patient heterogeneity in treatment response. Tumour organoids derived from the patients’ tumours via surgically resected or endoscopically biopsied tissue, have emerged as promising models for personalised medicine. This review synthesises the findings, to date, of studies which have explored the efficacy of ex vivo organoid sensitivity testing for predicting treatment response. Most studies have focused on predicting the response to standard-of-care radiotherapy and chemotherapy options. There is strong evidence to support organoid sensitivity testing of ionising radiation, 5-fluorouracil, and irinotecan, and to a lesser extent, oxaliplatin and TAS-102. Fewer studies have used organoids to identify patients who are likely to benefit from novel treatment options that otherwise remain in clinical trials. This review also summarises recent advancements in organoid culture to include non-epithelial components of the tumour microenvironment, to allow testing of immunotherapy and certain targeted therapy options. Overall, further prospective trials will support the implementation of organoid-based personalised medicine for colorectal cancer patients in the future.

Sessile serrated adenomas (SSAs) are common polyps which give rise to 20–30% of colorectal cancer (CRC). SSAs display clinicopathologic features which present challenges in surveillance, including overrepresentation in young patients, proclivity for the proximal colon and rarity of histologic dysplasia (referred to then as SSAs with dysplasia, SSADs). Once dysplasia develops, there is rapid progression to CRC, even at a small size. There is therefore a clinical need to separate the “advanced” SSAs at high risk of progression to SSAD and cancer from ordinary SSAs. Since SSAs are known to accumulate methylation over time prior to the development of dysplasia, SSAD backgrounds (the remnant SSA present within an SSAD) likely harbour additional methylation events compared with ordinary SSAs. We therefore performed MethyLight and comprehensive methylation array (Illumina MethylationEPIC) on 40 SSAD backgrounds and 40 matched ordinary SSAs, and compared the methylation results with CRC methylation, CRC expression and immunohistochemical data. SSAD backgrounds demonstrated significant hypermethylation of CpG islands compared with ordinary SSAs, and the proportion of hypermethylated probes decreased progressively in the shore, shelf and open sea regions. Hypomethylation occurred in concert with hypermethylation, which showed a reverse pattern, increasing progressively away from the island regions. These methylation changes were also identified in BRAF-mutant hypermethylated CRCs. When compared with CRC expression data, SV2B, MLH1/EPM2AIP1, C16orf62, RCOR3, BAIAP3, OGDHL, HDHD3 and ATP1B2 demonstrated both promoter hypermethylation and decreased expression. Although SSAD backgrounds were histologically indistinguishable from ordinary SSAs, MLH1 methylation was detectable via MethyLight in 62.9% of SSAD backgrounds, and focal immunohistochemical MLH1 loss was seen in 52.5% of SSAD backgrounds. Significant hyper- and hypomethylation events occur during SSA progression well before the development of histologically identifiable changes. Methylation is a heterogeneous process within individual SSAs, as typified by MLH1, where both MLH1 methylation and focal immunohistochemical MLH1 loss can be seen in the absence of dysplasia. This heterogeneity is likely a generalised phenomenon and should be taken into account in future methylation-based studies and the development of clinical methylation panels.

Objective: Sessile serrated lesions (SSLs) are common across the age spectrum, but the BRAF mutant cancers arising occur predominantly in the elderly. Aberrant DNA methylation is uncommon in SSL from young patients. Here, we interrogate the role of ageing and DNA methylation in SSL initiation and progression. Design: We used an inducible model of Braf mutation to direct recombination of the oncogenic Braf V637E allele to the murine intestine. BRAF mutation was activated after periods of ageing, and tissue was assessed for histological, DNA methylation and gene expression changes thereafter. We also investigated DNA methylation alterations in human SSLs. Results: Inducing Braf mutation in aged mice was associated with a 10-fold relative risk of serrated lesions compared with young mice. There were extensive differences in age-associated DNA methylation between animals induced at 9 months versus wean, with relatively little differential Braf-specific methylation. DNA methylation at WNT pathway genes scales with age and Braf mutation accelerated age-associated DNA methylation. In human SSLs, increased epigenetic age was associated with high-risk serrated colorectal neoplasia. Conclusions: SSLs arising in the aged intestine are at a significantly higher risk of spontaneous neoplastic progression. These findings provide support for a new conceptual model for serrated colorectal carcinogenesis, whereby risk of Braf-induced neoplastic transformation is dependent on age and may be related to age-associated molecular alterations that accumulate in the ageing intestine, including DNA methylation. This may have implications for surveillance and chemopreventive strategies targeting the epigenome.

Copy number profiles of paired primary and metastatic colorectal cancers Copy number profiles of paired primary and metastatic colorectal cancers