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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.

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

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.

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