This five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.

Summary Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF mutant resistant cell line models across seven different clinically-relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. In order to identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF mutant colorectal cancer.

A patient with metastatic BRAF -mutated colorectal cancer initially responded to combined EGFR and BRAF inhibition with panitumumab plus vemurafenib. Preexisting cells with increased MET gene copy number in the archival tumor tissue likely underwent clonal expansion during treatment, leading to the emergence of MET amplifi cation in the rebiopsy taken at progression. In BRAF -mutated colorectal cancer cells, ectopic expression of MET conferred resistance to panitumumab and vemurafenib, which was overcome by combining BRAF and MET inhibition. Based on tumor genotyping and functional in vitro data, the patient was treated with the dual ALK-MET inhibitor crizotinib plus vemurafenib, thus switching to dual MET and BRAF blockade, with rapid and marked effectiveness of such strategy. Although acquired resistance is a major limitation to the clinical effi cacy of anticancer agents, the identifi cation of molecular targets emerging during the fi rst treatment may afford the opportunity to design the next line of targeted therapies, maximizing patient benefi t. SIGNIFICANCE:MET amplifi cation is here identifi ed-clinically and preclinically-as a new mechanism of resistance to EGFR and BRAF dual/triple block combinations in BRAF -mutated colorectal cancer. Switching from EGFR to MET inhibition, while maintaining BRAF inhibition, resulted in clinical benefi t after the occurrence of MET-driven acquired resistance. Cancer Discov; 6(9);

Purpose: KRAS mutations confer adverse prognosis to colorectal cancer, and no targeted therapies have shown efficacy in this patient subset. Paracrine, nongenetic events induced by KRAS-mutant tumor cells are expected to result in specific deregulation and/or relocation of tumor microenvironment (TME) proteins, which in principle can be exploited as alternative therapeutic targets. Experimental Design: A multimodal strategy combining ex vivo/in vitro phage display screens with deep-sequencing and bioinformatics was applied to uncover TME-specific targets in KRAS-mutant hepatic metastasis from colorectal cancer. Expression and localization of BCAM and LAMA5 were validated by immunohistochemistry in preclinical models of human hepatic metastasis and in a panel of human specimens (n ¼ 71). The antimetastatic efficacy of two BCAM-mimic peptides was evaluated in mouse models. The role of BCAM in the interaction of KRAS-mutant colo-rectal cancer cells with TME cells was investigated by adhesion assays. Results: BCAM and LAMA5 were identified as molecular targets within both tumor cells and TME of KRAS-mutant hepatic metastasis from colorectal cancer, where they were specifically overexpressed. Two BCAM-mimic peptides inhibited KRAS-mutant hepatic metastasis in preclinical models. Genetic suppression and biochemical inhibition of either BCAM or LAMA5 impaired adhesion of KRAS-mutant colorectal cancer cells specifically to endothelial cells, whereas adhesion to pericytes and hepatocytes was unaffected. Conclusions: These data show that the BCAM/LAMA5 system plays a functional role in the metastatic spreading of KRASmutant colorectal cancer by mediating tumor-TME interactions and as such represents a valuable therapeutic candidate for this large, currently untreatable patient group. Clin Cancer Res; 22(19); 4923-33. Ó2016 AACR.