Recent advances in gene expression analysis have culminated in the publication of a Consensus Molecular Subtyping (CMS) system by the Colorectal Cancer Subtyping Consortium (CRCSC) that stratifies CRC into one of four subtypes based on transcriptional profiling. The CRCSC study reported an association between CMS4 and worse patient outcome, and between CMS1 and survival after relapse. Although many subsequent reports mention the prognostic potential of CMS, no study, to date, has validated the prognostic impact of the subtyping system compared to the routinely used staging for primary CRC.
In a large, single-institution cohort of chemotherapy-naïve, surgically treated colorectal cancers, we have shown that traditional TNM staging outperforms molecular subtyping in prognostication of CRC. Post-surgical staging of this cohort was carried out according to UICC guidelines and staging was similar to that expected of a treatment-naïve cohort. Association of stage with clinical variables found few associations beyond the parameters used to carry out staging, namely lymph-node involvement and distant metastases. As previously reported for other cohorts [18, 19], the association of increasing stage with metastasis was also largely driven by liver metastases.
In addition to histological staging, we carried out consensus molecular subtyping (CMS), based on RNA-sequencing derived gene-expression profiles from tumour tissue. Stratification into CMS yielded similar proportions of CMS1 and CMS3 and unclassified tumours as described by the CRCSC [8]. Our cohort contained a considerably greater proportion of CMS2 tumours at 47%, compared to 37% reported by CRCSC, and fewer CMS4 tumours, 6% compared to 23%. The difference in reported proportions of CMS may be, at least in part, accounted for by the inclusion criteria of surgery with curative intent and the exclusion of patients who received neo-adjuvant chemo- or radiotherapy in this study. A recent report by Trumpi et al. reported that neoadjuvant therapy induces a mesenchymal phenotype in residual tumour cells and, as such, may lead to an increase in the reporting of CMS4 subtypes [20]. These criteria may have excluded many advanced-stage tumours, which were shown to be associated with CMS4 [8]. Intra-tumoural heterogeneity may also affect the classification of CMS4 tumours, as the EMT-associated genes seen in CMS4 tumours may reflect upregulated genes derived from fibroblast and mesenchymal cells present in the stromal background rather than directly from the tumour itself [9, 10, 21, 22], and several studies have suggested that the location and number of tumour biopsies can undermine the accuracy of CMS [23,24,25]; a limitation of this study is the use of a single tumour sample to carry out gene-expression profiling.
Stratification into CMS showed similar associations with clinic-pathological variables as previously reported by CRCSC and other studies. CMS1 tumours were associated with right-side, female, node-negative and poorly-differentiated, with a high proportion of mucinous histology and less likely to be seen in younger patients under 60 years of age. CMS2 tumours made up nearly half of our cohort and were predominantly left-sided tumours found in male patients, and showed a negative association with mucinous type. Patients with CMS3 type tumours were associated with a lower TNM stage. CMS4 tumours were associated with younger age, rectal tumours and presented at an advanced TNM stage with lymph node positivity. Indeed, lymph-node positivity is shown to increase through CMS1, 2, 3 to CMS4.
The established association between increasing tumour stage and poorer outcome was recapitulated in our cohort, in terms of progression-free and overall survival. While post-surgical staging is the mainstay of prognostication in most clinical centres, the potential for refining prognostication using molecular features has been widely investigated, and the combined use of different clinical and molecular markers have shown links with prognosis in CRC, e.g. while BRAF mutations have been associated with poorer outcome [26], the effects of these mutations may be mitigated in MSI tumours [27]. KRAS mutations are also associated with a poorer outcome, but this association is stronger in distal compared to proximal tumours [28].
The original study by Guinney et al. first describing consensus molecular subtyping showed an association between CMS4 and poor overall survival, and between CMS1 and survival after relapse [8]. Several studies have incorporated CMS with other molecular features to in order to refine prognostic groups, and have described poorer outcomes in BRAF-mutated CMS1 MSS tumours, and KRAS-mutated CMS2/3 MSS tumours [29], and favourable outcomes in CMS1 MSI tumours [30]. Although many subsequent publications have emphasised the prognostic importance of CMS, the utility of CMS as a stand-alone prognostic tool in the clinical setting has not been investigated in an independent cohort. Survival analysis showed an association between CMS and both progression-free and overall survival in our cohort, and this was largely due to the difference between CMS4 and the other CMS classes. However, after adjusting for age and sex, CMS4 was not an independent prognostic marker for survival in this study. Including both TNM stage and CMS in models of overall survival shows that TNM significantly explains mortality independently of age and gender, whereas CMS does not. A potential limitation of the study is the relatively low numbers of CMS4 tumours, as discussed above, and that almost half of the tumours in our cohort are CMS2, and this imbalance may affect the power of our study to detect effects specific to CMS1, 3 and 4.
Clinical management of CRC is usually based on histological staging, with stage 1 tumours conservatively managed with surgery and tumours with nodal or distant metastases (Stage 3 and 4) usually treated with adjuvant chemotherapy. Stage 2 tumours remain a conundrum in terms of prognostication, as approximately 20% of patients with Stage 2 CRC die from the disease [31]. Various factors including acute presentation with obstruction and perforation, histological factors such as perineural and perivascular invasion, as well as high grade, have been used as markers of poor prognosis, and as such indicators for adjunctive postoperative chemotherapy. Further stratification using molecular markers, such as BRAF and KRAS mutations, and MSI status [32] have been investigated with regard to their prognostic potential in this tumour group, but have not widely adopted to direct clinical management.
Molecular subtyping is a cornerstone of precision medicine in cancer treatment, and the mutation status of genes in the EGFR pathways, including RAS genes, PIK3CA, PTEN and BRAF have been shown to predict response to EGFR blockade therapy in CRC [33]. MSI status and the effect of the tumour microenvironment, in particular the amount and type of tumour infiltrating lymphocytes, have more recently been proposed as predictors of response to immunotherapy [34]. To date, although CMS1 tumours encompass a large proportion of MSI positive CRC, no targeted treatment options based solely on CMS have been proposed. In the context of metastatic CRC, CMS appears to associate with survival in clinical trials of patients with wild-type KRAS tumours, treated with anti-EGFR or VEGF inhibitors [35, 36]. However, in primary CRC and outside the clinical trials setting, improved stratification of CRC had not yet been demonstrated using CMS. In this study, we have observed that subtyping of TNM-stratified tumours into CMS could improve prognostication for Stage 2 CRC; tumours that were CMS3 subtype had significantly lower overall survival compared to other molecular subtypes. This demonstrates, for the first time, the potential utility of CMS in improving prognostication of CRC in combination with existing methods.
Differential gene expression between Stage 2 patients who died and those who were alive at the end of the follow-up period, identified significant up-regulation of immune-related genes and biologic processes, and tumour-suppressor genes, associated with survival. The importance of the immune microenvironment in tumour progression has been demonstrated in solid tumours, and has been linked to outcome in CRC. Our findings suggest that immune signatures may identify Stage 2 patients with good prognosis, for whom surgery alone may suffice, and conversely a CMS3 signature may identify patients who would benefit from adjuvant chemotherapy/increased surveillance. Further evaluations of the genetic signatures identified in this study, and prospective validation using a more clinically accessible platform e.g. gene panel test, will be necessary to confirm these findings.
