Molecular subtype assay to reveal anti-EGFR response sub-clones in colorectal cancer (CRC).

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658 Background: We previously stratified CRC into five intrinsic gene expression subtypes (CRCAssigner) and four consensus molecular subtypes (CMS1-4) using expensive and time-consuming microarray/RNAseq platforms. Recently, we developed a low-cost (NanoCRC) assay using nCounter platform (NanoString Technology) and robust gene signatures to classify individual samples into both CRCAssigner and CMS subtypes. Here, we tested the assays using formalin-fixed paraffin-embedded (FFPE) patient samples from Royal Marsden Hospital. Given increased sensitivity to cetuximab in one of our CRCAssigner subtypes transit-amplifying (TA) that represents partly CMS2 subtype, we associated anti-EGFR response to the presence of TA sub-clones. Methods: FFPE from resected and biopsy CRC samples were profiled for our NanoCRC assay. Along with our single-sample subtyping method, we developed a tool to identify sub-clones of TA within individual samples. RAS/BRAF wild-type patients treated with single agent anti-EGFR therapy were statistically associated with sub-clonal molecular profiles, sidedness and single gene expression. Results: NanoCRC assay was highly reproducible with > 0.9 correlation coefficient. Among 76 samples (from 64 patients), all the five CRCAssigner, four CMS subtypes (including the mixed samples) and their associations with RAS/BRAF mutations were successfully identified. Subtype-mutational associations were similar to our previous publications. Among 15 KRAS/BRAF wild-type (wt) patients with single agent anti-EGFR therapy response data, TA sub-clone was identified in 7/9 with clinical benefit and 1/6 with no benefit (p < 0.05). The other sub-clones, sidedness and individual genes within the panels were not significantly associated. This TA sub-clone association was further confirmed using a publicly available cetuximab response data (39 KRAS wt; Khambata-Ford data; p=0.02). Conclusions: Overall, we developed a robust NanoCRC assay to classify CRC FFPE samples in to molecular subtypes. With caveat of small numbers, the presence of TA sub-clones is significantly associated with cetuximab response. Extensive validation is warranted.