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.

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.

How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent therapy is unknown. We studied EGFR blockade in colorectal cancer to assess whether tissue and liquid biopsies can be integrated with radiological imaging to monitor the impact of individual oncogenic alterations on lesion-specific responses. Biopsy of a patient's progressing liver metastasis following prolonged response to cetuximab revealed a K57T MEK1 mutation as a novel mechanism of acquired resistance. This lesion regressed upon treatment with panitumumab and the MEK inhibitor trametinib. In ctDNA, mutant MEK1 levels declined with treatment, but a previously unrecognized KRAS Q61H mutation was also identified that increased despite therapy. This same KRAS mutation was later found in a separate non-responding metastasis. In summary, parallel analyses of tumor biopsies and serial ctDNA monitoring show that lesion-specific radiographic responses to subsequent targeted therapies can be driven by distinct resistance mechanisms arising within separate tumor lesions in the same patient.

The development of molecularly targeted anticancer agents relies on large panels of tumourspecific preclinical models closely recapitulating the molecular heterogeneity observed in patients. Here we describe the mutational and gene expression analyses of 151 colorectal cancer (CRC) cell lines. We find that the whole spectrum of CRC molecular and transcriptional subtypes, previously defined in patients, is represented in this cell line compendium. Transcriptional outlier analysis identifies RAS/BRAF wild-type cells, resistant to EGFR blockade, functionally and pharmacologically addicted to kinase genes including ALK, FGFR2, NTRK1/2 and RET. The same genes are present as expression outliers in CRC patient samples. Genomic rearrangements (translocations) involving the ALK and NTRK1 genes are associated with the overexpression of the corresponding proteins in CRC specimens. The approach described here can be used to pinpoint CRCs with exquisite dependencies to individual kinases for which clinically approved drugs are already available.

Entrectinib is a fi rst-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profi ling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1 , p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confi rmed that either mutation renders the TRKA kinase insensitive to entrectinib. These fi ndings can be immediately exploited to design next-generation TRKA inhibitors. SIGNIFICANCE:We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identifi cation of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the fi rst time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements. Cancer Discov; 6(1);[36][37][38][39][40][41][42][43][44]

Purpose: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models.Experimental Design: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance.Results: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab.Conclusions: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor-antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies.

IntroductionPioneering clinical studies have shown that transplantation of genetically modified hematopoietic stem cells may cure severe genetic diseases such as severe combined immunodeficiencies (SCID), 1,2 chronic granulomatous disease (CGD), 3 and lysosomal storage disorders. 4 Unfortunately, some of these studies showed also the limitations of retroviral gene transfer technology, which may cause severe and sometimes fatal adverse effects. In particular, insertional activation of proto-oncogenes by vectors derived from the Moloney murine leukemia virus (MLV) caused T-cell lymphoproliferative disorders in patients with X-linked SCID 5,6 and premalignant expansion of myeloid progenitors in patients with CGD. 3 Preclinical studies showed that HIV-derived lentiviral vectors are less likely to cause insertional gene activation, 7,8 although they can still interfere with normal gene expression at the posttranscriptional level, as observed in a clinical trial of gene therapy for ␤-thalassemia. 9 The molecular bases of vector-induced genotoxicity and the influence of vector design, transduction protocols, and the patient's genetic background in inducing severe adverse effects are still poorly understood. A better understanding of the interactions between retroviral vectors and the human genome may provide new cues to explain these phenomena and a rational basis for predicting genotoxic risks in gene therapy.A large number of studies have focused on the molecular mechanisms by which mammalian retroviruses choose their integration sites in the target cell genome. After entering a cell, retroviral RNA genomes are reverse transcribed into double-stranded DNA and assembled in preintegration complexes (PICs) containing viral and cellular proteins. PICs translocate to the nucleus and associate with the host cell chromatin, where the viral integrase mediates proviral insertion in genomic DNA. Integration is a nonrandom process, whereby PICs of different viruses recognize components or features of the host cell chromatin in a specific fashion. 10 For HIV and other lentiviruses, the LEDGF/p75 protein has been identified as the main factor tethering PICs to active chromatin, 11 whereas mechanisms underlying integration site selection of other retroviruses remain largely unknown. We recently showed that MLV-derived vectors integrate preferentially in hot spots near genes controlling growth and development of hematopoietic cells and flanked by defined subsets of transcription factor binding sites (TFBSs) and suggested that MLV PICs are tethered to active regulatory regions by basal components of the transcriptional machinery. 12,13 The MLV integrase and long terminal repeat enhancer are the main determinants of the selection of TFBS-rich regions of the genome. 13,14 We used ligation-mediated polymerase chain reaction (LM-PCR) and pyrosequencing to build a genomewide, high-definition map of Ͼ 32 000 MLV integration sites in the genome of human CD34 ϩ hematopoietic progenitor cells (HPCs) and used gene expression profiling, chroma...

Blockade of the epidermal growth factor receptor (EGFR) with the monoclonal antibodies cetuximab or panitumumab is effective in a subset of colorectal cancers (CRCs), but the emergence of resistance limits the efficacy of these therapeutic agents. At relapse, the majority of patients develop RAS mutations, while a subset acquires EGFR extracellular domain (ECD) mutations. Here we find that patients who experience greater and longer responses to EGFR blockade preferentially develop EGFR ECD mutations, while RAS mutations emerge more frequently in patients with smaller tumour shrinkage and shorter progression-free survival. In circulating cell-free tumour DNA of patients treated with anti-EGFR antibodies, RAS mutations emerge earlier than EGFR ECD variants. Subclonal RAS but not EGFR ECD mutations are present in CRC samples obtained before exposure to EGFR blockade. These data indicate that clonal evolution of drug-resistant cells is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies.