Timothy Stanley Maughan

Purpose: Risk stratification in localized colorectal cancer (CRC) remains inadequate. Transcriptome-based classifiers such as consensus molecular subtyping (CMS) improve this, but are challenging to implement. We determined the performance of a novel image-based method for CRC CMS subtyping (imCMS) using routine pathology images from the SCOT trial and investigated association of imCMS subtypes with clinicopathologic and tumor molecular and immune factors, treatment benefit, and survival. Methods: We applied a novel imCMS classifier to stage II/III CRCs from the SCOT trial (ISRCTN59757862) which examined 3 versus 6 months of adjuvant oxaliplatin-based chemotherapy following curative-intent surgery. We determined correlates of imCMS subgroups and investigated imCMS prognostic and predictive value for CRC recurrence-free interval, overall survival (OS), and survival after CRC recurrence (SAR) by univariable and multivariable Cox regression. Results: A total of 2,816 cases were informative for imCMS classification, of which 664 (24%) were imCMS1 (immune), 987 (35%) imCMS2 (canonical), 433 (15%) imCMS3 (metabolic), 653 (23%) imCMS4 (mesenchymal), and 79 (3%) of mixed subtype. Analysis confirmed expected associations of imCMS1 with deficient DNA mismatch repair (dMMR) and imCMS3 with KRAS mutation and revealed substantially lower intraepithelial CD8 ⁺ and intrastromal FoxP3 ⁺ T-cell infiltrate (CD8 ^IE -FoxP3 ^IS ) in imCMS4 tumors. imCMS classification was strongly prognostic independent of clinicopathologic factors, dMMR, and CD8 ^IE -FoxP3 ^IS in multivariable analysis but did not predict differential benefit from chemotherapy duration or regimen. The combination of imCMS with CD8 ^IE -FoxP3 ^IS groups defined CRC subgroups with substantially different recurrence risk. Analysis of OS and SAR revealed notable discordance in imCMS3 and imCMS4 prognostic value before and after relapse. Conclusion: imCMS classification improves CRC prognostication beyond existing tumor factors and is potentially deliverable in clinical practice. imCMS prediction of therapeutic benefit requires further study.

To describe the steps taken in the creation of a Data Pack containing clinical data from the FOCUS4 randomized controlled trial in colorectal cancer, repre

Supplementary Table 8. Differential gene expression results (post-DESeq2 analysis) for BRAF, KRAS, NRAS, and the EGFR-ligands AREG and EREG across two normal-colorectum RNAseq cohorts. Sorted by adjusted p.values (Benjamini-Hochberg).

Supplementary Figure 1. Study and data availability overview (see Supplementary Table 1).

Supplementary Table 6. Logistic regression model of atypical BRAF mutation versus V600E status with covariates MSI status, age, sex, and tumour location.

Suplementary Table 3a-b. Summarised BRAF mutation characteristics per cohort following the 1-2-3 classification system (Table 3a) and the A-B-C classification system (Table 3b). Table cells with values less than 5 have been censored to facilitate Genomics England reporting restrictions. An uncensored version of Supplementary Table 3 is available within the Genomics England 100,000 Genomes Project Research Environment upon request.

Supplementary Figure 5. Atypical KRAS mutations and additional Ras pathway mutations. A CONSORT-style diagram shows the breakdown of tumours with atypical KRAS mutations by accompanying mutations in KRAS, BRAF, NRAS or NF1.

Supplementary Table 7. Logistic regression models of CMS groups by BRAF mutation class, with covariates: tumour location (left or right colon), MSI status, age, sex, and additional Ras status (Class 1/2/3 left, A-B-C right).

Supplementary Data 1. Sanger sequencing chromatograms presented in Figure 3C-D.

Supplementary Table 1. Details of each cohort included in the study