Circulating Tumour DNA Sequencing Identifies a Genetic Resistance-Gap in Colorectal Cancers with Acquired Resistance to EGFR-Antibodies and Chemotherapy

SIMPLE SUMMARY: Recent studies have shown the potential of next generation sequencing (NGS) for the identification of genetic variants in tumour DNA that has been released into the bloodstream (ctDNA). However, such variants are often rare in the sample and error correction is required to filter out false calls. In this study we used error corrected ctDNA-sequencing to identify genetic drivers of resistance in metastatic colorectal cancer (mCRC) patients that had become resistant to combined chemotherapy and EGFR-antibody treatment. Our data showed that ctDNA-seq could detect common and novel resistance mechanisms in cases of both primary and acquired resistance. ctDNA-seq could therefore facilitate patient stratification to novel therapies and avoid ineffective treatment with EGFR-antibodies. Furthermore, the data revealed a lack of detectable genetic resistance in a large fraction of the cancer cell population, indicating a need to investigate other, potentially non-genetic, resistance mechanisms. ABSTRACT: Epidermal growth factor receptor antibodies (EGFR-Abs) confer a survival benefit in patients with RAS wild-type metastatic colorectal cancer (mCRC), but resistance invariably occurs. Previous data showed that only a minority of cancer cells harboured known genetic resistance drivers when clinical resistance to single-agent EGFR-Abs had evolved, supporting the activity of non-genetic resistance mechanisms. Here, we used error-corrected ctDNA-sequencing (ctDNA-Seq) of 40 cancer genes to identify drivers of resistance and whether a genetic resistance-gap (a lack of detectable genetic resistance mechanisms in a large fraction of the cancer cell population) also occurs in RAS wild-type mCRCs treated with a combination of EGFR-Abs and chemotherapy. We detected one MAP2K1/MEK1 mutation and one ERBB2 amplification in 2/3 patients with primary resistance and KRAS, NRAS, MAP2K1/MEK1 mutations and ERBB2 aberrations in 6/7 patients with acquired resistance. In vitro testing identified MAP2K1/MEK1 P124S as a novel driver of EGFR-Ab resistance. Mutation subclonality analyses confirmed a genetic resistance-gap in mCRCs treated with EGFR-Abs and chemotherapy, with only 13.42% of cancer cells harboring identifiable resistance drivers. Our results support the utility of ctDNA-Seq to guide treatment allocation for patients with resistance and the importance of investigating further non-canonical EGFR-Ab resistance mechanisms, such as microenvironmentally-mediated resistance. The detection of MAP2K1 mutations could inform trials of MEK-inhibitors in these tumours.