Summary A main limitation of therapies that selectively target kinase signaling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of EGFR, is effective in a subset of KRAS wild type metastatic colorectal cancers1. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug2. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood3-8. Here, we show for the first time that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance but resistant cells remained sensitive to combinatorial inhibition of EGFR and MEK. Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6/10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab treated patients as early as 10 months prior to radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months prior to radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Colorectal cancer (CRC) is a genetic disease governed by clonal evolution1. Genotyping CRC tissue is employed for therapeutic purposes but this approach has significant limitations. A tissue sample represents a single snapshot in time, is subjected to selection bias due to tumor heterogeneity, and can be difficult to obtain. We exploited circulating DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during therapies with the anti-EGFR antibodies cetuximab or panitumumab. We identified genomic alterations in KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1 in ctDNA of patients with primary or acquired resistance to EGFR blockade. Mutant RAS clones, which rise in blood during EGFR blockade, decline upon withdrawal of anti-EGFR antibodies indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells, which had acquired resistance to cetuximab, reveals that upon antibody withdrawal KRAS clones decay, while the population regains drug sensitivity. ctDNA profiles of patients who benefit from multiple challenging with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results reveal that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of re-challenge therapies based on EGFR blockade.

As for other types of malignancy, colorectal cancer is not a homogeneous disease but actually comprises multiple entities that vary in natural history and molecular pathogenesis. This heterogeneity explains why molecular cancer therapeutics against individual disease driver targets have proven to be effective in only a fraction of cases. One prototypical example is provided by the anti-epidermal growth factor receptor (EGFR) monoclonal antibodies cetuximab and panitumumab, which are approved for the treatment of metastatic colorectal cancer. In unselected patients, the extent of clinical benefit from monotherapy with either drug hovers near the threshold for statistical significance, with response rates of approximately 10% ( 3-5 ).The population of potential responders has been recently enriched thanks to a biomarker-development strategy that is driven by the plausible biological rationale that constitutive activation of signaling pathways parallel to or downstream from EGFR, such as the RAS-RAF axis, should circumvent EGFR inhibition and therefore preclude sensitivity to EGFRtargeted agents ( 6 ). Indeed, the authors of both retrospective and prospective trials have convincingly demonstrated the inefficacy of EGFR-neutralizing antibodies in metastatic colorectal cancer patients with common (codons 12 and 13) KRAS mutations ( 7-12 ).Along this line, in a number of retrospective studies investigators have provided initial evidence that rare KRAS mutations as well as NRAS , BRAF , and (possibly) PIK3CA mutations also are significantly associated with low response rates ( 13-16 ). When considering the cumulative incidence of Only a fraction of patients with metastatic colorectal cancer receive clinical benefit from therapy with anti-epidermal growth factor receptor (EGFR) antibodies, which calls for the identification of novel biomarkers for better personalized medicine. We produced large xenograft cohorts from 85 patient-derived, genetically characterized metastatic colorectal cancer samples ("xenopatients") to discover novel determinants of therapeutic response and new oncoprotein targets. Serially passaged tumors retained the morphologic and genomic features of their original counterparts. A validation trial confirmed the robustness of this approach: xenopatients responded to the anti-EGFR antibody cetuximab with rates and extents analogous to those observed in the clinic and could be prospectively stratified as responders or nonresponders on the basis of several predictive biomarkers. Genotype-response correlations indicated HER2 amplification specifically in a subset of cetuximab-resistant, KRAS/NRAS/BRAF/PIK3CA wild-type cases. Importantly, HER2 amplification was also enriched in clinically nonresponsive KRAS wild-type patients. A proof-of-concept, multiarm study in HER2-amplified xenopatients revealed that the combined inhibition of HER2 and EGFR induced overt, long-lasting tumor regression. Our results suggest promising therapeutic opportunities in cetuximab-resistant patients with metastatic colorectal ca...