**5. Conclusions**

*Advances in the Molecular Understanding of Colorectal Cancer*

and intermediate levels of gene hypermethylation [22]. Despite clear enrichment of certain gene mutations within CMS groups, such as high rates of BRAF mutation in CMS1 and KRAS mutations in CMS3, no single genetic aberration was found to be limited to one subtype, and no subtype was defined by a single molecular event. Further integrative genomic analysis did not draw any clear associations either,

Further exploration of gene expression data revealed insight into the underlying biology of the subtypes: CMS1 samples showed strong immune activation and infiltration with CD4+ T helper cells, CD8+ cytotoxic T cells and natural killer (NK) cells along with strong activation of immune evasion pathways. CMS2 showed marked upregulation of Wnt and MYC downstream targets and higher expression of oncogenes EGFR, ERBB2, insulin-like growth factor 2 (IGF-2), insulin receptor substrate 2 (IRS-2) and transcription factor hepatocyte nuclear factor 4α (HNF4α). CMS3 samples showed enrichment for multiple metabolism signatures which are keeping with the described notion that activating KRAS mutations induce prominent metabolic adaptation [23, 24]. CMS4 tumours showed upregulation of genes associated with epithelial-mesenchymal transition (EMT), such as transforming

highlighting the poor genotype–phenotype correlation in this cancer.

growth factor β (TGF-β) and integrins, as well as stromal invasion.

rior survival following relapse was noted in the CMS2 subgroup.

**4.2 Clinical utility of the consensus molecular subtypes**

**4.1 Clinical and prognostic associations of the consensus molecular subtypes**

Further prognostic associations of the CMS subtypes have been explored via retrospective analysis of large clinical trial datasets, as have their association with biological therapies. 392 KRAS wild-type samples from the CALGB 80405 dataset were analysed via a NanoString platform to determine their CMS subtype classification, and this was correlated with survival [25]. It was found that CMS1 tumours treated with bevacizumab had significantly longer OS compared to those treated with cetuximab. CMS2 tumours treated with bevacizumab had a trend towards shorter OS than those treated with cetuximab. A meta-analysis of six randomised trials, including the CRYSTAL and FIRE-3 datasets, also confirmed the improvement in PFS and OS of left-sided tumours (CMS2) treated with anti-EGFR antibodies compared to no significant benefit for right-sided tumours (CMS1) [26]. No survival differences were found for left- or right-sided tumours treated with bevacizumab. This suggests sidedness of the primary tumour that determines efficacy of biological therapies, and this can possibly be explained by the biological differences of tumours from different sides of the bowel: left-sided tumours overexpress the EGFR ligands amphiregulin (AREG) and epiregulin (EREG) and also display amplifications of markers of cetuximab sensitivity, whereas right-sided tumours show reduced expression of EGFR ligands [27].

Much hope was placed upon the CMS classification system allowing stratification of patients for clinical trials to validate the prognostic and predictive value of the subgroups and enable translation into clinical care. Although CMS classification

Associations between CMS subgroups and clinical features and prognosis were also investigated and showed that CMS1 tumours were more common in females, more likely to be right-sided and of higher histopathological grade. Conversely, CMS2 tumours were more likely to be left-sided and present at more advanced stages. CMS4 tumours show the worst OS and RFS even after adjustment for BRAF and KRAS mutations and MSI status. CMS1 tumours display good survival but very poor SAR in keeping with known data of MSI tumours associated with BRAFV600E mutations. CMS2 and CMS3 subgroups display intermediate survival, but a supe-

**12**

Much progress has been made in our understanding of the complex underlying biology of CRC which leads to heterogeneous drug responses and outcomes. Comprehensive integrative molecular analysis has led to the identification of molecularly distinct subgroups within this disease, and the consensus molecular subtypes have enabled some refinement of these subgroups. However, widespread reproducibility and confirmation of the clinical utility of CMS classification still need to be addressed. There are vast amounts of data being generated from molecular classification systems, and this needs to be prospectively integrated into clinical trial design in order to confirm biomarkers of resistance and response as well as to allow rational combinations of therapies to be explored. The ultimate goal is to streamline biomarker and drug codevelopment and recruit patients to innovative clinical trials of targeted agents to which they are more likely to respond based on the underlying molecular makeup of their tumours.
