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Supplementary Figure S1. MEK1 K57 mutation confers resistance to anti-EGFR antibodies in CRC preclinical models. Supplementary Figure S2. Dual blockade of EGFR and MEK restores sensitivity to CRC cells expressing MEK1 K57N or K57T. Supplementary Figure S3. Longitudinal analysis of founder mutations in patient plasma during panitumumab and trametinib treatment. Supplementary Table S1. 1000 gene sequencing panel. Supplementary Table S2. 40 gene targeted sequencing panel. Supplementary Table S3. Summary of targeted sequencing and ddPCR data on tissue specimens. Supplementary Table S4. Next generation sequencing data from plasma ctDNA. Supplementary Table S5. Summary of serial ctDNA analyses. Supplementary Table S6. Ratio of resistance-associated genetic alterations and founder mutations in serial plasma ctDNA timepoints.

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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 cancer, whose medical treatment in the chemorefractory setting remains an unmet clinical need.Significance: Direct transfer xenografts of tumor surgical specimens conserve the interindividual diversity and the genetic heterogeneity typical of the tumors of origin, combining the flexibility of preclinical analysis with the informative value of population-based studies. Our suite of patient-derived xenografts from metastatic colorectal carcinomas reliably mimicked disease response in humans, prospectively recapitulated biomarker-based case stratification, and identified HER2 as a predictor of resistance to anti-epidermal growth factor receptor antibodies and of response to combination therapies against HER2 and epidermal growth factor receptor in this tumor setting. Cancer Discovery; 1(6); 508–23. ©2011 AACR.Read the Commentary on this article by Ciardiello and Normanno, p. 472This article is highlighted in the In This Issue feature, p. 457

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The file lists Supplementary Materials and Methods and relative References.

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. 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. Cancer Res; 76(15); 4504–15. ©2016 AACR.

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Supplementary Tables S1-S3 - List of drug concentrations to generate resistant cell lines (S1); Drug concentrations applied in the screening depicted in Figure 6 (S2); Primers for gene amplification and sequencing (S3). Supplementary Figure S1. Synergistic activity of targeted therapy combinations in BRAF mutant colorectal cancer cells. Supplementary Figure S2. Amplification of mutant BRAF V600E confers resistance to combined EGFR and MEK targeting in colorectal cancer cells. Supplemental Figure S3. Cytotoxicity induced by ERK inhibition in VACO432 resistant to BRAF combination therapies.

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Purpose: Gene mutations along the Ras pathway (KRAS, NRAS, BRAF, PIK3CA) occur in approximately 50% of colorectal cancers (CRC) and correlate with poor response to anti–EGF receptor (EGFR) therapies. We assessed the effects of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) and phosphoinositide 3-kinase (PI3K)/mTOR inhibitors, which neutralize the major Ras effectors, in patient-derived xenografts from RAS/RAF/PIK3CA-mutant metastatic CRCs (mCRC).Experimental Design: Forty mCRC specimens harboring KRAS, NRAS, BRAF, and/or PIK3CA mutations were implanted in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Each xenograft was expanded into four treatment arms: placebo, the MEK inhibitor AZD6244, the PI3K/mTOR inhibitor, BEZ235, or AZD6244 + BEZ235. Cases initially treated with placebo crossed over to AZD6244, BEZ235, and the anti-EGFR monoclonal antibody cetuximab.Results: At the 3-week evaluation time point, cotreatment of established tumors with AZD6244 + BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to overt tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244 (disease control rates, DCRs: AZD6244, 27.5%; BEZ235, 42.5%). Triple therapy with cetuximab provided further advantage (DCR, 88%). The extent of disease control declined at the 6-week evaluation time point (DCRs: AZD6244, 13.9%; BEZ235, 16.2%; AZD6244 + BEZ235, 34%). Cross-analysis of mice harboring xenografts from the same original tumor and treated with each of the different modalities revealed subgroups with preferential sensitivity to AZD6244 (12.5%), BEZ235 (35%), or AZD6244 + BEZ235 (42.5%); another subgroup (10%) showed equivalent response to any treatment.Conclusions: The prevalent growth-suppressive effects produced by MEK and PI3K/mTOR inhibition suggest that this strategy may retard disease progression in patients. However, data offer cautionary evidence against the occurrence of durable responses. Clin Cancer Res; 18(9); 2515–25. ©2012 AACR.

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Legends

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. 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. Cancer Res; 76(15); 4504–15. ©2016 AACR.

BACKGROUND: Combined MET and BRAF inhibition showed clinical benefit in a patient with rectal cancer carrying BRAF(V600E) and MET amplification. However after 4 months, acquired resistance emerged and the patient deceased shortly after disease progression. The mechanism of resistance to this drug combination is unknown.METHODS: We analysed plasma circulating tumour DNA obtained at progression by exome sequencing and digital PCR. MET gene and mRNA in situ hybridisation analyses in two bioptic specimens obtained at progression were used to confirm the plasma data.RESULTS: We identified in plasma MET gene hyper-amplification as a potential mechanism underlying therapy resistance. Increased MET gene copy and transcript levels were detected in liver and lymph node metastatic biopsies. Finally, transduction of MET in BRAF mutant colorectal cancer cells conferred refractoriness to BRAF and MET inhibition.CONCLUSIONS: We identified in a rectal cancer patient MET gene hyper-amplification as mechanism of resistance to dual BRAF and MET inhibition.

Background The diagnosis of colorectal cancer (CRC) is routinely accomplished through histopathologic examination. Prognostic information and treatment decisions are mainly determined by TNM classification, first defined in 1968. In the last decade, patient-specific CRC genomic landscapes were shown to provide important prognostic and predictive information. Therefore, there is a need for developing next generation sequencing (NGS) and bioinformatic workflows that can be routinely used for the assessment of prognostic and predictive biomarkers. Materials and Methods To foster the application of genomics in the clinical management of CRCs, the IDEA workflow has been built to easily adapt to the availability of patient specimens and the clinical question that is being asked. Initially, IDEA deploys ad-hoc NGS assays to interrogate predefined genomic target sequences (from 600 kb to 30 Mb) with optimal detection sensitivity. Next, sequencing data are processed through an integrated bioinformatic pipeline to assess single nucleotide variants, insertions and deletions, gene copy-number alterations, and chromosomal rearrangements. The overall results are gathered into a user-friendly report. Results We provide evidence that IDEA is capable of identifying clinically relevant molecular alterations. When optimized to analyze circulating tumor DNA, IDEA can be used to monitor response and relapse in the blood of patients with metastatic CRC receiving targeted agents. IDEA detected primary and secondary resistance mechanisms to ERBB2 blockade including sub-clonal RAS and BRAF mutations. Conclusions The IDEA workflow provides a flexible platform to integrate NGS and bioinformatic tools for refined diagnosis and management of patients with advanced CRC.

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., Some colorectal cancer patients respond well to treatment with anti-EGFR antibodies, however response is almost invariably followed by acquired resistance. Here, the authors show that patients with shorter responses acquire RAS mutations, while those relapsing later preferentially develop mutations in the extracellular domain of EGFR.

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show 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 mitogen-activated protein-kinase kinase (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 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before 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 before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Purpose: Gene mutations along the Ras pathway (KRAS, NRAS, BRAF, PIK3CA) occur in approximately 50% of colorectal cancers (CRC) and correlate with poor response to anti–EGF receptor (EGFR) therapies. We assessed the effects of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) and phosphoinositide 3-kinase (PI3K)/mTOR inhibitors, which neutralize the major Ras effectors, in patient-derived xenografts from RAS/RAF/PIK3CA-mutant metastatic CRCs (mCRC). Experimental Design: Forty mCRC specimens harboring KRAS, NRAS, BRAF, and/or PIK3CA mutations were implanted in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Each xenograft was expanded into four treatment arms: placebo, the MEK inhibitor AZD6244, the PI3K/mTOR inhibitor, BEZ235, or AZD6244 + BEZ235. Cases initially treated with placebo crossed over to AZD6244, BEZ235, and the anti-EGFR monoclonal antibody cetuximab. Results: At the 3-week evaluation time point, cotreatment of established tumors with AZD6244 + BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to overt tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244 (disease control rates, DCRs: AZD6244, 27.5%; BEZ235, 42.5%). Triple therapy with cetuximab provided further advantage (DCR, 88%). The extent of disease control declined at the 6-week evaluation time point (DCRs: AZD6244, 13.9%; BEZ235, 16.2%; AZD6244 + BEZ235, 34%). Cross-analysis of mice harboring xenografts from the same original tumor and treated with each of the different modalities revealed subgroups with preferential sensitivity to AZD6244 (12.5%), BEZ235 (35%), or AZD6244 + BEZ235 (42.5%); another subgroup (10%) showed equivalent response to any treatment. Conclusions: The prevalent growth-suppressive effects produced by MEK and PI3K/mTOR inhibition suggest that this strategy may retard disease progression in patients. However, data offer cautionary evidence against the occurrence of durable responses. Clin Cancer Res; 18(9); 2515–25. ©2012 AACR.

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 cancer, whose medical treatment in the chemorefractory setting remains an unmet clinical need. Significance: Direct transfer xenografts of tumor surgical specimens conserve the interindividual diversity and the genetic heterogeneity typical of the tumors of origin, combining the flexibility of preclinical analysis with the informative value of population-based studies. Our suite of patient-derived xenografts from metastatic colorectal carcinomas reliably mimicked disease response in humans, prospectively recapitulated biomarker-based case stratification, and identified HER2 as a predictor of resistance to anti-epidermal growth factor receptor antibodies and of response to combination therapies against HER2 and epidermal growth factor receptor in this tumor setting. Cancer Discovery; 1(6); 508–23. ©2011 AACR. Read the Commentary on this article by Ciardiello and Normanno, p. 472 This article is highlighted in the In This Issue feature, p. 457

The genetic variability among Italian populations of the white-clawed crayfish (Austropotamobius pallipes) was examined to determine their phylogeography and to assess their conservation status as a management unit. A fragment of the mitochondrial DNA COI gene of 107 specimens from ten populations was sequenced, and the phylogenetic relationships were established. Two out of three haplotypes sampled in two French populations from the Rhone basin were shared with Italian populations. Despite a moderate level of genetic variability within the Italian populations of A. pallipes, no genetic structure was revealed. It has been suggested that there have been translocation events throughout the Alpine barrier between the North-Western Italian basins and the Rhone basin. Genetic exchangeability of the French and Italian populations was demonstrated in this study, and a shift of conservation efforts towards the native, congeneric Austropotamobius italicus is recommended.

Colorectal cancers (CRCs) evolve by a reiterative process of genetic diversification and clonal evolution. The molecular profile of CRC is routinely assessed in surgical or bioptic samples. Genotyping of CRC tissue has inherent limitations; a tissue sample represents a single snapshot in time, and it is subjected to spatial selection bias owing to tumor heterogeneity. Repeated tissue samples are difficult to obtain and cannot be used for dynamic monitoring of disease progression and response to therapy. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment with the epidermal growth factor receptor (EGFR)-specific antibodies cetuximab or panitumumab. We identified alterations in ctDNA of patients with primary or acquired resistance to EGFR blockade in the following genes: KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1. Mutated KRAS clones, which emerge in blood during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells that had acquired resistance to cetuximab reveals that upon antibody withdrawal KRAS clones decay, whereas the population regains drug sensitivity. ctDNA profiles of individuals who benefit from multiple challenges with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results indicate that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of rechallenge therapies based on EGFR blockade.

The development of molecularly targeted anticancer agents relies on large panels of tumour-specific 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.

Cetuximab and panitumumab are monoclonal anti-EGFR antibodies (moAbs) currently used for the treatment of advanced RAS wild type colorectal cancers (CRC). Emergence of acquired resistance invariably limits the efficacy of these agents, and the dynamics of clonal evolution during anti-EGFR blockade are poorly understood. At progression, RAS mutations represent the most common genetic alterations, while EGFR extracellular domain (ECD) mutations are acquired by a smaller cohort of patients. We found that the mutation profile correlates with the clinical outcome of patients; in particular those who develop RAS mutations upon EGFR blockade achieve reduced tumor shrinkage and shorter duration of response respect to patients in which EGFR ECD mutations emerge during therapy. We investigated in preclinical models the potential role of RAS and EGFR ECD mutations during the emergence of acquired resistance, by tracking the evolution of clones in a genetically barcoded population of CRC cells chronically treated with cetuximab. We observed that therapeutic (target therapy, chemotherapy) and environmental (reduced nutrient condition) pressures differentially shape the clonal composition of CRC cell populations, leading to the emergence of clones with the highest fitness in presence of the external pressure. In conclusion, a multistep clonal evolution process characterizes the development of drug resistance and is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies. Citation Format: Sabrina Arena, Beth Van Emburgh, Giulia Siravegna, Luca Lazzari, Giovanni Crisafulli, Giorgio Corti, Benedetta Mussolin, Federica Baldi, Michela Buscarino, Alice Bartolini, Emanuele Valtorta, Joana Vidal, Beatriz Bellosillo, Giovanni Germano, Filippo Pietrantonio, Agostino Ponzetti, Joan Albanell, Salvatore Siena, Andrea Sartore-Bianchi, Federica Di Nicolantonio, Clara Montagut, Alberto Bardelli. Emergence of RAS or EGFR mutant clones affects duration of response to EGFR blockade in colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2913. doi:10.1158/1538-7445.AM2017-2913

A metastatic colorectal cancer (mCRC) patient carrying CAD-ALK translocation achieved partial response to an experimental ALK inhibitor and then progressed after 5 months. We studied whether urine cell-free, trans-renal DNA (tr-DNA) could be used to monitor tumor burden and patient’s response. A NGS panel was developed to interrogate 52 common cancer gene rearrangements and 14 frequently mutated genes in cancer patients. A TP53 p.R248W mutation and the CAD-ALK genomic breakpoint (rearrangement) were identified in the tumor tissue and matched plasma circulating tumor DNA (ctDNA) Urine samples were longitudinally obtained from the patient during ALK inhibitor treatment in parallel with blood. To detect the CAD-ALK translocation in urine tr-DNA we designed ultra-short (51 bp amplicon) primer pairs spanning the genomic breakpoint as a unique tumor marker. This approach allowed the non-invasive monitoring of the gene fusion in urine with amounts paralleling tumor burden. Of note, CAD-ALK gene fusion was apparent in urine tr-DNA before radiological confirmation of disease progression. The same strategy was applied to plasma ctDNA and the results were compared. To detect point mutations in urine tr-DNA, we exploited a peptide nucleic acids (PNA)-CLAMP PCR coupled with droplet digital PCR (ddPCR) analysis to specifically suppress amplification of wild-type DNA fragments. Custom PNA probes were designed for TP53 codon 248, and a ddPCR assay was optimized to detect the TP53 p.R248W mutation, which was then identified in all urine tr-DNA samples, with absolute copies correlating with tumor burden throughout ALK inhibitor treatment. In conclusion, we find that urine tr-DNA can be exploited to non-invasively monitor tumor burden by detecting tumor-specific gene fusions as well as point mutations. Citation Format: Giulia Siravegna, Andrea Sartore-Bianchi, Benedetta Mussolin, Laura Palmieri, Federica Tosi, Andrea Cassingena, Luca Novara, Michela Buscarino, Giorgio Corti, Giovanni Crisafulli, Alice Bartolini, Mark Erlander, Federica Di Nicolantonio, Salvatore Siena, Alberto Bardelli. Tracking CAD-ALK gene translocation in urine and plasma of a colorectal cancer patient treated with ALK blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3834. doi:10.1158/1538-7445.AM2017-3834

Colorectal cancers (CRCs) that are sensitive to the anti-epidermal growth factor receptor (EGFR) antibodies cetuximab or panitumumab almost always develop resistance within several months of initiating therapy. We report the emergence of polyclonal KRAS, NRAS, and BRAF mutations in CRC cells with acquired resistance to EGFR blockade. Regardless of the genetic alterations, resistant cells consistently displayed mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) activation, which persisted after EGFR blockade. Inhibition of MEK1/2 alone failed to impair the growth of resistant cells in vitro and in vivo. An RNA interference screen demonstrated that suppression of EGFR, together with silencing of MEK1/2, was required to hamper the proliferation of resistant cells. Indeed, concomitant pharmacological blockade of MEK and EGFR induced prolonged ERK inhibition and severely impaired the growth of resistant tumor cells. Heterogeneous and concomitant mutations in KRAS and NRAS were also detected in plasma samples from patients who developed resistance to anti-EGFR antibodies. A mouse xenotransplant from a CRC patient who responded and subsequently relapsed upon EGFR therapy showed exquisite sensitivity to combinatorial treatment with MEK and EGFR inhibitors. Collectively, these results identify genetically distinct mechanisms that mediate secondary resistance to anti-EGFR therapies, all of which reactivate ERK signaling. These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that MEK inhibitors, in combination with cetuximab or panitumumab, should be tested in CRC patients who become refractory to anti-EGFR therapies.

How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent lines of therapy is unknown. Exposure to therapy may result in selection of sub-clonal cell populations, capable of growing under drug pressures. Therefore, a single-lesion biopsy at disease progression may vastly underrepresent the molecular heterogeneity of resistant tumor clones in an individual patient and may fail to detect the existence of distinct but important resistance mechanisms that could impact clinical response. We identified a colorectal cancer (CRC) patient in whom multiple tumor biopsies were obtained at resistance following prolonged response to with the anti-EGFR antibody cetuximab. Full-exome sequencing of 1000 cancer genes of both primary tumor and progression biopsy revealed a TP53 mutation in all samples and a novel MEK1 p.K57T mutation in one of the progressing liver biopsy. A mutation at the same MEK1 codon was identified in the cetuximab-resistant HCA46 CRC cell line. Biochemical analysis showed constitutive activation of MEK and ERK despite cetuximab treatment. However, the combination of the MEK inhibitor trametinib with either cetuximab or panitumumab restored sensitivity, suggesting a potential therapeutic strategy to overcome resistance to EGFR blockade caused by this mutation. Accordingly, the patient was treated with the combination of panitumumab and trametinib. After 3 months, imaging demonstrated a reduction in size of the biopsied liver metastasis harboring the MEK1 mutation, but revealed that some other lesions had progressed. Plasma collected prior to therapy was analyzed by next-generation sequencing confirming the presence of both TP53 and MEK1 variants, but surprisingly unveiling a previously unrecognized KRAS mutation. ddPCR analysis of longitudinal timepoints of ctDNA unveiled that TP53 mutant levels dropped after initiation of therapy, but rose later during treatment in parallel with CEA tumor marker levels. However, MEK1 mutant levels declined sharply, indicating effective suppression of MEK1 mutant clones by panitumumab and trametinib; while KRAS mutant levels rose, indicating outgrowth of a resistant KRAS-mutant clone. Biopsy of a different liver metastasis that progressed despite panitumumab and trametinib revealed the same KRAS mutation identified in ctDNA. In summary these findings illustrate how distinct acquired resistance mechanisms can arise concomitantly in separate metastases within the same patient, leading to mixed lesion-specific responses to subsequent targeted therapies. Liquid biopsy approaches, in association with single-tumor biopsies, have the potential to detect the presence of simultaneous resistance mechanisms residing in separate metastases in a single patient and to monitor the effects of subsequent targeted therapies. Citation Format: Mariangela Russo, Giulia Siravegna, Lawrence S. Blaszkowsky, Giorgio Corti, Giovanni Crisafulli, Leanne G. Ahronian, Benedetta Mussolin, Eunice L. Kwak, Michela Buscarino, Luca Lazzari, Emanuele Valtorta, Mauro Truini, Nicholas A. Jessop, Hayley E. Robinson, Theodore S. Hong, Mari Mino-Kenudson, Federica Di Nicolantonio, Ashraf Thabet, Andrea Sartore-Bianchi, Salvatore Siena, A John Iafrate, Ryan B. Corcoran, Alberto Bardelli. Tumor heterogeneity and lesion-specific response to targeted therapy in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 878.

Purpose: KRAS mutations represent the main cause of resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs) in metastatic colorectal cancer (mCRC). We evaluated whether highly sensitive methods for KRAS investigation improve the accuracy of predictions of anti-EGFR MoAbs efficacy. Experimental Design: We retrospectively evaluated objective tumor responses in mCRC patients treated with cetuximab or panitumumab. KRAS codons 12 and 13 were examined by direct sequencing, MALDI-TOF MS, mutant-enriched PCR, and engineered mutant-enriched PCR, which have a sensitivity of 20%, 10%, 0.1%, and 0.1%, respectively. In addition, we analyzed KRAS codon 61, BRAF, and PIK3CA by direct sequencing and PTEN expression by immunohistochemistry. Results: In total, 111 patients were considered. Direct sequencing revealed mutations in codons 12 and 13 of KRAS in 43/111 patients (39%) and BRAF mutations in 9/111 (8%), with almost all of these occurring in nonresponder patients. Using highly sensitive methods, we identified up to 13 additional KRAS mutations compared with direct sequencing, all occurring in nonresponders. By analyzing PIK3CA and PTEN, we found that of these 13 patients, 7 did not show any additional alteration in the PI3K pathway. Conclusions: The application of highly sensitive methods for the detection of KRAS mutations significantly improves the identification of mCRC patients resistant to anti-EGFR MoAbs. Clin Cancer Res; 17(14); 4901–14. ©2011 AACR.

How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent lines of therapy is unknown. Exposure to therapy may result in selection of sub-clonal cell populations, capable of growing under drug pressures. Therefore, a single-lesion biopsy at disease progression may vastly underrepresent the molecular heterogeneity of resistant tumor clones in an individual patient and may fail to detect the existence of distinct but important resistance mechanisms that could impact clinical response. To this aim, we identified a colorectal cancer (CRC) patient in whom multiple tumor biopsies were obtained at resistance following prolonged response to with the anti-EGFR antibody cetuximab and irinotecan therapy. Full-exome sequencing of 1000 cancer genes of both primary tumor and progression biopsy revealed a TP53 mutation in all samples and a novel MAP2K1 p.K57T mutation in one of the progressing liver biopsy. Interestingly, a mutation at the same MAP2K1 codon was identified in the cetuximab-resistant HCA46 CRC cell line. Biochemical analysis of preclinical model showed constitutive activation of MEK and ERK despite cetuximab treatment. Exogenous expression of the same mutant MEK1, but not wild-type MEK1, in an independent RAS-WT CRC cell line, LIM1215, conferred resistance to cetuximab or panitumumab. However, the combination of the MEK inhibitor trametinib with either cetuximab or panitumumab restored sensitivity, suggesting a potential therapeutic strategy to overcome resistance to EGFR blockade caused by this mutation. Accordingly, the patient was treated with the combination of panitumumab and trametinib. After 3 months, imaging demonstrated a reduction in size of the biopsied liver metastasis harboring the MAP2K1 mutation, but revealed that some other lesions had progressed. Plasma for circulating DNA (ctDNA) analysis was longitudinally collected during combinatorial treatment. Pre-treatment plasma was analyzed using next-generation sequencing (NGS), confirming the presence of both TP53 and MAP2K1 variants, but surprisingly unveiling an additional KRAS mutation. ddPCR analysis of longitudinal timepopints of ctDNA unveiled that TP53 mutant levels dropped after initiation of therapy, but rose later during treatment with concomitantly/in parallel to CEA ones. However, MAP2K1 mutant levels declined, while KRAS mutant ones rose markedly during therapy, indicating outgrowth of a resistant KRAS-mutant clone. Biopsy of a different liver metastasis' segment that progressed despite panitumumab and trametinib revealed the same KRAS mutation identified in ctDNA. In summary these findings illustrate how individual metastatic lesions can develop distinct resistance mechanisms to targeted agents, leading to striking differences in lesion-specific response to subsequent targeted therapies. As more trials evaluating targeted therapy strategies designed to overcome specific acquired resistance mechanisms enter the clinic, genomic results from single-tumor biopsies should be interpreted with caution. By contrast, liquid biopsy approaches have the potential to detect the presence of simultaneous resistance mechanisms residing in separate metastases in a single patient and to monitor the effects of subsequent targeted therapies. Citation Format: Giulia Siravegna, Mariangela Russo, Lawrence S. Blaszkowsky, Giorgio Corti, Giovanni Crisafulli, Leanne G. Ahronian, Benedetta Mussolin, Eunice L. Kwak, Michela Buscarino, Luca Lazzari, Emanuele Valtorta, Mauro Truini, Nicholas A. Jessop, Hayley E. Robinson, Theodore S. Hong, Mari Mino-Kenudson, Federica Di Nicolantonio, Ashraf Thabet, Andrea Sartore-Bianchi, Salvatore Siena, John Iafrate, Ryan B. Corcoran, Alberto Bardelli. Acquisition of resistance to anti-EGFR therapy drives genomic heterogeneity and lesion-specific responses in colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR01.

Colorectal Cancer (CRC) is characterized by wide genetic, biological and clinical heterogeneity. Oncogenic events, such as KRAS, NRAS and BRAF mutations, are known negative biomarker of response to EGFR targeted therapies. In addition to mutational analysis, transcriptional profiling has been recently exploited to identify distinct CRC molecular subtypes, associated with biological and clinical features such as cell of origin, microsatellite instability, prognosis and response to treatments. Detailed analysis of the relationships between the subtypes and clinical/biological features requires a large panel of preclinical models closely recapitulating the molecular heterogeneity of CRC. To this aim, we assembled a large collection of 151 CRC cell lines. For each line, we: (i) verified genetic identity by short tandem repeat (STR) analysis; (ii) assessed microsatellite instability (MSI) status; (iii) sequenced mutational hotspots in the KRAS, BRAF, NRAS and PIK3CA genes; (iv) performed microarray-based global mRNA expression profiling; (v) evaluated sensitivity to the EGFR-targeting drug cetuximab. STR analysis revealed that some cell lines previously thought to be unrelated are indeed derived from the same individual. This result was confirmed by cell line hierarchical clustering based on mRNA expression profiles. Overall, the mutational landscape of the compendium was concordant with what observed in patients (mutation rates: KRAS = 47%; BRAF = 17%; NRAS = 0.7%; PIK3CA = 18%). Similarly, sensitivity to cetuximab was confined to lines without RAS or BRAF mutations. Gene expression profiling was exploited to assign cell lines to molecular subtypes, according to five different published transcriptional classifiers. We found that all molecular subtypes previously identified in CRC patients were robustly maintained in the lines. Moreover, significant overlaps were detected between individual subtypes across distinct classifiers. Subtype-specific molecular and pharmacological associations previously defined in CRC samples were largely recapitulated in the compendium. In particular, MSI+ cells were significantly enriched in inflammatory and goblet subtypes and less prevalent in the Transit Amplifying (TA) and Stem groups. Cell lines carrying BRAF mutations clustered in the inflammatory subtype, while RAS mutations were equally distributed among all subtypes. Notably, in TA lines without RAS or BRAF mutations, the fraction of cetuximab-sensitive cells was strongly enriched (56%), confirming clinical data and indicating that addiction to the EGFR pathway is an intrinsic feature of this CRC subtype. In conclusion, our results describe a powerful preclinical resource reflecting the molecular and functional heterogeneity of CRC, which can be used to explore multidimensional information of potential clinical relevance. Citation Format: Gabriele Picco, Mariangela Russo, Carlotta Cancelliere, Michela Buscarino, Claudio Isella, Simona Lamba, Barbara Martinoglio, Federica Di Nicolantonio, Alberto Bardelli, Enzo Medico. Colorectal cancer cell lines recapitulate molecular and pharmacological features of clinical samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2015-583

11073 Background: We previously reported that acquired resistance to anti-EGFR antibodies (moAbs) is associated with the emergence of KRAS mutations that can be detected in the blood before disease...

Occurence of acquired resistance constitutes one of the major limitations to tumor treatment. In colorectal cancer, the use of anti-EGFR antibodies such as cetuximab and panitumumab is effective in only 15-20% of patients, till secondary resistance to targeted treatment develops. Shedding light on the molecular basis of resistance represents then a mandatory effort in order to foster new lines of therapy. We recently showed that KRAS mutations are responsible for the emergence of secondary resistance to EGFR therapy in a subset of CRC patients (Misale et al., Nature 2012). Here we investigated the role of additional genetic alterations in the development of resistance to EGFR blockade in CRCs. We initially exploited the “liquid biopsy” approach to measure tumor derived DNA mutations in the blood of patients who initially responded and then became refractory to either cetuximab or panitumumab. We found that multiple KRAS and NRAS alleles often emerge during treatment. To better characterize the role of these genetic alterations in CRC, we generated preclinical models, by chronic treatment of several CRC cell lines with cetuximab or panitumumab until resistant derivatives emerged. The resistant populations were a mixture of clones bearing an heterogeneous panel of genetic alterations in KRAS, NRAS and BRAF at various frequencies. Biochemical pathway analyses revealed that, regardless of the genetic alterations, resistant derivatives consistently displayed MEK and ERK activation, which persisted on EGFR inhibition. Collectively, these results identify genetically distinct mechanisms that mediate secondary resistance to anti-EGFR therapies, all of which converge on the reactivation of ERK signaling. With this work, we provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with anti-EGFR drugs; we propose that MEK inhibitors, in combination with cetuximab or panitumumab, should be considered for the treatment of CRC patients who become refractory to anti-EGFR therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B110. Citation Format: Sabrina Arena, Sandra Misale, Simona Lamba, Giulia Siravegna, Alice Lallo, Sebastijan Hobor, Mariangela Russo, Michela Buscarino, Andrea Sartore-Bianchi, Katia Bencardino, Alessio Amatu, Salvatore Siena, Federica Di Nicolantonio, Alberto Bardelli. Heterogeneous genetic alterations emerge during acquired resistance to anti-EGFR therapy in colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B110.

Monoclonal antibodies targeting EGFR are used in the clinic to treat KRAS wild type metastatic colorectal cancer (CRC). After an initial response, secondary resistance occurs thereby limiting the clinical benefit of these drugs. Relapsed patients have no further therapeutic options, therefore, elucidating the molecular basis of resistance is a prerequisite for the development of further lines of therapy. We took advantage from a panel of CRC cell lines sensitive to EGFR inhibition by treating with cetuximab or panitumumab until resistant derivatives emerged. The resistant populations were heterogeneous mixture of cells bearing different alterations in KRAS, NRAS and BRAF genes at various frequencies. An RNA interference (siRNA) genetic screening unveiled that suppression of MEK1/2 together with silencing of EGFR was effective in impairing the growth of the resistant cells. Combinatorial treatment with pimasertib, a selective allosteric MEK inhibitor, together with cetuximab re-sensitizes anti-EGFR resistant derivatives despite the genetic alterations heterogeneity. Combinatorial treatment was effective in vitro and in vivo both in cell derived xenografts and in an acquired resistant patient derived xenograft (xenopatient). These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that MEK inhibitors, in combination with cetuximab or panitumumab, should be tested in clinical trials in CRC patients who become refractory to anti-EGFR therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C94. Citation Format: Sandra Misale, Sabrina Arena, Simona Lamba, Giulia Siravegna, Alice Lallo, Sebastijan Hobor, Mariangela Russo, Michela Buscarino, Andrea Sartore-Bianchi, Katia Bencardino, Alessio Amatu, Salvatore Siena, Federica Di Nicolantonio, Alberto Bardelli. Heterogeneous mechanisms of acquired resistance to anti-EGFR therapies in colorectal cancer are sensitive to concomitant inhibition of EGFR and MEK. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C94.

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 cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. 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 (Figure 1). 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 (Table 1). 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.

Background Monitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution. Patients and methods We exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying aCAD-ALK translocation during treatment with an ALK inhibitor. Results Using a custom next generation sequencing panel we identified the genomicCAD-ALK rearrangement and aTP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of theCAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient’s clinical course. Detection of theCAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identifiedALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib. Conclusion We find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.