Background: Colorectal cancer (CRC) has been shown to acquire RAS and EGFR ectodomain mutations as mechanisms of resistance to epidermal growth factor receptor (EGFR) inhibition (anti-EGFR). After anti-EGFR withdrawal, RAS and EGFR mutant clones lack a growth advantage relative to other clones and decay; however, the kinetics of decay remain unclear. We sought to determine the kinetics of acquired RAS/EGFR mutations after discontinuation of anti-EGFR therapy.Patients and methods: We present the post-progression circulating tumor DNA (ctDNA) profiles of 135 patients with RAS/ BRAF wild-type metastatic CRC treated with anti-EGFR who acquired RAS and/or EGFR mutations during therapy. Our validation cohort consisted of an external dataset of 73 patients with a ctDNA profile suggestive of prior anti-EGFR exposure and serial sampling. A separate retrospective cohort of 80 patients was used to evaluate overall response rate and progression free survival during re-challenge therapies.Results: Our analysis showed that RAS and EGFR relative mutant allele frequency decays exponentially (r 2 ¼0.93 for RAS; r 2 ¼0.94 for EGFR) with a cumulative half-life of 4.4 months. We validated our findings using an external dataset of 73 patients with a ctDNA profile suggestive of prior anti-EGFR exposure and serial sampling, confirming exponential decay with an estimated half-life of 4.3 months. A separate retrospective cohort of 80 patients showed that patients had a higher overall response rate during re-challenge therapies after increasing time intervals, as predicted by our model. Conclusion:These results provide scientific support for anti-EGFR re-challenge and guide the optimal timing of re-challenge initiation.

Purpose Liquid biopsies allow the tracking of clonal dynamics and detection of mutations during treatment. Experimental design We evaluated under blinded conditions the ability of cell free DNA (cfDNA) to detect RAS/BRAF mutations in the plasma of 42 metastatic colorectal cancer patients treated on a phase Ib/II trial of FOLFOX and dasatinib, with or without cetuximab. Results Prior to treatment, sequencing of archival tissue detected mutations in 25/42 patients (60%), while the cfDNA assay detected mutations in 37/42 patients (88%). Our cfDNA assay detected mutations with allele frequencies as low as 0.01%. After exposure to treatment, 41/42 patients (98%) had a cfDNA detected RAS/BRAF mutation. Of 21 patients followed with serial measurements who were RAS/BRAF mutant at baseline, 11 (52%) showed additional point mutation following treatment and 3 (14%) no longer had detectable levels of another mutant allele. Of RAS/BRAF wild type tumors at baseline, 4/5 (80%) showed additional point mutations. cfDNA quantitative measurements from this study closely mirrored changes in CEA and CT scan results, highlighting the importance of obtaining quantitative data beyond the mere presence of a mutation. Conclusions Our findings demonstrate the development of new RAS/BRAF mutations in patients regardless of whether they had pre-existing mutations in the pathway, demonstrating a convergent evolutionary pattern.

PURPOSE Atypical, non-V600 BRAF ( aBRAF) mutations represent a rare molecular subtype of metastatic colorectal cancer (mCRC). Preclinical data are used to categorize aBRAF mutations into class II (intermediate to high levels of kinase activity, RAS independent) and III (low kinase activity level, RAS dependent). The clinical impact of these mutations on anti-EGFR treatment efficacy is unknown. PATIENTS AND METHODS Data from 2,084 patients with mCRC at a single institution and from an external cohort of 5,257 circulating tumor DNA (ctDNA) samples were retrospectively analyzed. Overall survival (OS) was calculated using Kaplan-Meier and log-rank tests. Statistical tests were two-sided. RESULTS BRAF mutations were harbored by 257 patients, including 36 with aBRAF mutations: 22 class III, 10 class II, four unclassified. For patients with aBRAF mCRC, median OS was 36.1 months, without a difference between classes, and median OS was 21.0 months for patients with BRAFV600E mCRC. In contrast to right-sided predominance of tumors with BRAFV600E mutation, 53% of patients with aBRAF mCRC had left-sided primary tumors. Concurrent RAS mutations were noted in 33% of patients with aBRAF mCRC, and 67% of patients had microsatellite stable disease. Among patients with aBRAF RAS wild-type mCRC who received anti-EGFR antibodies (monotherapy, n = 1; combination therapy, n = 10), no responses to anti-EGFR therapy were reported, and six patients (four with class III aBRAF mutations, one with class II, and one unclassified) achieved stable disease as best response. Median time receiving therapy was 4 months (range, 1 to 16). In the ctDNA cohort, there was an increased prevalence of aBRAF mutations and subclonal aBRAF mutations ( P < .001 for both) among predicted anti-EGFR exposed compared with nonexposed patients. CONCLUSION Efficacy of anti-EGFR therapy is limited in class II and III aBRAF mCRC. Detection of aBRAF mutations in ctDNA after EGFR inhibition may represent a novel mechanism of resistance.

Innate and acquired resistance to anti-EGFR therapy (EGFRi) is a major limitation in the treatment of metastatic colorectal cancer (mCRC). Although RAS genes are the most commonly mutated innate and acquired oncogenes in cancer, there are a number of other mechanisms which limit the effectiveness of EGFRi. Patients with innate resistance have been found to contain BRAFV600E mutations, and possibly MET, MEK, PIK3CA, PTEN and HER2 alterations. Meanwhile, BRAFV600E mutations may also be involved in acquired resistance to EGFRi, in addition to EGFR ectodomain mutations, MET alterations, and possibly HER2 amplification. In addition, paracrine effects and cell fate mechanisms of resistance are being increasingly described as contributing to acquired resistance. Utilization of circulating tumor DNA has been paramount in monitoring the dynamic nature of acquired resistance, and has helped to guide treatment decisions, particularly in the EGFRi rechallenge setting. Herein, we provide an in-depth review of EGFRi resistance mechanisms and describe the current therapeutic landscape in the hopes of identifying effective rechallenge strategies.

Background Aberrant activation of the intracellular tyrosine kinase Src has been implicated as a mechanism of acquired chemotherapy resistance in metastatic colorectal cancer (mCRC). Here, the oral tyrosine kinase Src inhibitor, dasatinib, was investigated in combination with FOLFOX and cetuximab. Methods We performed a phase IB/II study of 77 patients with previously-treated mCRC. Primary objectives were to determine the MTD, dose-limiting-toxicities, pharmacodynamics, and efficacy. Using a 3+3 design, patients received FOLFOX6 with cetuximab and escalating doses of dasatinib(100, 150, 200mg daily), followed by a 12 patient expansion cohort at 150mg. Phase II studies evaluated FOLFOX plus dasatinib 100mg in KRAS c12/13mut patients, or in combination with cetuximab if KRAS c12/13WT. FAK and paxillin were utilized as surrogate blood biomarkers of Src inhibition, and paired biopsies of liver metastases were obtained in patients in the expansion cohort. Results In Phase IB, the dose-limiting-toxicities were grade 3/4 fatigue(20%), and neutropenia(23%). In Phase II, grade 3/4 fatigue(23%) and pleural effusions(11%) were present. Response rates were 20%(6/30) in the Phase IB escalation and expansion cohort, and 13%(3/24) and 0%(0/23) in the KRAS c12/13WT and mutant cohorts of Phase II, respectively. Median PFS was 4.6, 2.3, and 2.3 months, respectively. There was no evidence of Src inhibition based on surrogate blood biomarkers or paired tumor biopsies. Conclusions The combination of dasatinib plus FOLFOX with or without cetuximab showed only modest clinical activity in refractory CRC. This appears to be primarily due to a failure to fully inhibit Src at the achievable doses of dasatinib.