The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction–based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.

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.

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EGFR targeted monoclonal antibodies are effective in a subset of metastatic colorectal tumors (mCRC). Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in patients who do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies demonstrate that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived CRC xenografts, MET amplification correlated with resistance to EGFR blockade which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in CRC and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.