Andrea Cassingena

Amplification of the MET Receptor Drives Resistance to Anti-EGFR Therapies in Colorectal Cancer

EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. 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 tumors that 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 show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer 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 colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.

Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients

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.

Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (HERACLES): a proof-of-concept, multicentre, open-label, phase 2 trial

BACKGROUND We previously found that dual HER2 blockade with trastuzumab and lapatinib led to inhibition of tumour growth in patient-derived xenografts of HER2-amplified metastatic colorectal cancer. In this study, we aimed to assess the antitumour activity of trastuzumab and lapatinib in patients with HER2-positive colorectal cancer. METHODS HERACLES was a proof-of-concept, multicentre, open-label, phase 2 trial done at four Italian academic cancer centres. We enrolled adult patients with KRAS exon 2 (codons 12 and 13) wild-type and HER2-positive metastatic colorectal cancer refractory to standard of care (including cetuximab or panitumumab), an Eastern Cooperative Oncology Group performance status of 0 or 1, and at least one measurable lesion. We defined HER2 positivity in tumour samples by use of immunohistochemistry and fluorescence in-situ hybridisation in accordance with our previously validated colorectal cancer-specific diagnostic criteria. Eligible patients received intravenous trastuzumab at 4 mg/kg loading dose followed by 2 mg/kg once per week, and oral lapatinib at 1000 mg per day until evidence of disease progression. The primary endpoint was the proportion of patients achieving an objective response (defined as complete response or partial response), which was assessed by independent central review in the intention-to-treat population. This trial is registered with EudraCT, number 2012-002128-33. FINDINGS Between Aug 27, 2012, and May 15, 2015, we screened 914 patients with KRAS exon 2 (codons 12 and 13) wild-type metastatic colorectal cancer and identified 48 (5%) patients with HER2-positive tumours, although two died before enrolment. Of these patients, 27 were eligible for the trial. All were evaluable for response. At the time of data cutoff on Oct 15, 2015, with a median follow-up of 94 weeks (IQR 51-127), eight (30%, 95% CI 14-50) of 27 patients had achieved an objective response, with one patient (4%, 95% CI -3 to 11) achieving a complete response, and seven (26%, 95% CI 9-43) achieving partial responses; 12 (44%, 95% CI 25-63) patients had stable disease. Six (22%) of 27 patients had grade 3 adverse events, which consisted of fatigue in four patients, skin rash in one patient, and increased bilirubin concentration in one patient. No grade 4 or 5 adverse events were reported. We detected no drug-related serious adverse events. INTERPRETATION The combination of trastuzumab and lapatinib is active and well tolerated in treatment-refractory patients with HER2-positive metastatic colorectal cancer. FUNDING Associazione Italiana Ricerca Cancro (AIRC), Fondazione Oncologia Niguarda Onlus, and Roche.

The genomic landscape of response to EGFR blockade in colorectal cancer

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.

Promoter CpG island hypermethylation of the DNA repair enzyme MGMT predicts clinical response to dacarbazine in a phase II study for metastatic colorectal cancer.

Purpose: O6-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair protein removing mutagenic and cytotoxic adducts from O6-guanine in DNA. Approximately 40% of colorectal cancers (CRC) display MGMT deficiency due to the promoter hypermethylation leading to silencing of the gene. Alkylating agents, such as dacarbazine, exert their antitumor activity by DNA methylation at the O6-guanine site, inducing base pair mismatch; therefore, activity of dacarbazine could be enhanced in CRCs lacking MGMT. We conducted a phase II study with dacarbazine in CRCs who had failed standard therapies (oxaliplatin, irinotecan, fluoropyrimidines, and cetuximab or panitumumab if KRAS wild-type). Experimental Design: All patients had tumor tissue assessed for MGMT as promoter hypermethylation in double-blind for treatment outcome. Patients received dacarbazine 250 mg/m2 intravenously every day for four consecutive days, every 21 days, until progressive disease or intolerable toxicity. We used a Simon two-stage design to determine whether the overall response rate would be 10% or more. Secondary endpoints included association of response, progression-free survival, and disease control rate with MGMT status. Results: Sixty-eight patients were enrolled from May 2011 to March 2012. Patients received a median of three cycles of dacarbazine (range 1–12). Grades 3 and 4 toxicities included: fatigue (41%), nausea/vomiting (29%), constipation (25%), platelet count decrease (19%), and anemia (18%). Overall, two patients (3%) achieved partial response and eight patients (12%) had stable disease. Disease control rate (partial response + stable disease) was significantly associated with MGMT promoter hypermethylation in the corresponding tumors. Conclusion: Objective clinical responses to dacarbazine in patients with metastatic CRC are confined to those tumors harboring epigenetic inactivation of the DNA repair enzyme MGMT. Clin Cancer Res; 19(8); 2265–72. ©2013 AACR.

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

BACKGROUND O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. RESULTS Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). CONCLUSIONS Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

Epigenetic Inactivation of the BRCA1 Interactor SRBC and Resistance to Oxaliplatin in Colorectal Cancer

Background A major problem in cancer chemotherapy is the existence of primary resistance and/or the acquisition of secondary resistance. Many cellular defects contribute to chemoresistance, but epigenetic changes can also be a cause. Methods A DNA methylation microarray was used to identify epigenetic differences in oxaliplatin-sensitive and -resistant colorectal cancer cells. The candidate gene SRBC was validated by single-locus DNA methylation and expression techniques. Transfection and short hairpin experiments were used to assess oxaliplatin sensitivity. Progression-free survival (PFS) and overall survival (OS) in metastasic colorectal cancer patients were explored with Kaplan–Meier and Cox regression analyses. All statistical tests were two-sided. Results We found that oxaliplatin resistance in colorectal cancer cells depends on the DNA methylation–associated inactivation of the BRCA1 interactor SRBC gene. SRBC overexpression or depletion gives rise to sensitivity or resistance to oxaliplatin, respectively. SRBC epigenetic inactivation occurred in primary tumors from a discovery cohort of colorectal cancer patients (29.8%; n = 39 of 131), where it predicted shorter PFS (hazard ratio [HR] = 1.83; 95% confidence interval [CI] = 1.15 to 2.92; log-rank P = .01), particularly in oxaliplatin-treated case subjects for which metastasis surgery was not indicated (HR = 1.96; 95% CI = 1.13 to 3.40; log-rank P = .01). In a validation cohort of unresectable colorectal tumors treated with oxaliplatin (n = 58), SRBC hypermethylation was also associated with shorter PFS (HR = 1.90; 95% CI = 1.01 to 3.60; log-rank P = .045). Conclusions These results provide a basis for future clinical studies to validate SRBC hypermethylation as a predictive marker for oxaliplatin resistance in colorectal cancer.

Integrated molecular dissection of the epidermal growth factor receptor (EFGR) oncogenic pathway to predict response to EGFR-targeted monoclonal antibodies in metastatic colorectal cancer

The introduction of KRAS testing as a diagnostic tool to select patients for epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies for metastatic colorectal cancer is widely regarded as a key advance in the field of personalized cancer medicine. Oncologists are now facing emerging issues in the treatment of metastatic colorectal cancer, including: (i) the identification of additional genetic determinants of primary resistance to EGFR-targeted therapy for further improving selection of patients; (ii) the explanation of rare cases of patients carrying KRAS-mutated tumors who have been reported to respond to either cetuximab or panitumumab and (iii) the discovery of mechanisms of secondary resistance to anti-EGFR antibody therapies. Here we discuss the potential role of comprehensive dissection of the key oncogenic nodes in the EGFR signaling cascade to predict resistance and sensitivity to EGFR monoclonal antibodies in metastatic colorectal cancer. Current data suggest that, together with KRAS mutations, the evaluation of BRAF and PIK3CA/PTEN alterations could also be useful for selecting patients with reduced chance to benefit from EGFR-targeted therapy. Furthermore, measuring EGFR gene copy number also appears relevant to positively identify responders. Up until now, each of these markers has been mainly assessed as a single event, often in retrospective analyses and patients’ series. As these molecular alterations display overlapping patterns of occurrence, this adds considerable complexity to the drawing of an algorithm suitable for clinical decision-making. We suggest that in the near future comprehensive molecular analysis of the entire oncogenic pathway triggered by the EGFR should be performed, thus enhancing the prediction ability of individual markers.

Molecular landscape of acquired resistance to targeted therapy combinations in BRAF mutant colorectal cancer

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.

Therapeutic implications of resistance to molecular therapies in metastatic colorectal cancer

Metastatic colorectal cancer (mCRC) patients carrying KRAS mutated tumors do not benefit from epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies. Indeed, the mutational status of KRAS is currently a validated predictive biomarker employed to select mCRC patients for EGFR targeted drugs. When patients fail standard 5-fluorouracil-, oxaliplatin-, irinotecan- and bevacizumab-based therapies, EGFR-targeted salvage therapy can be prescribed only for those individuals with KRAS wild-type cancer. Thus, clinicians are now facing the urgent issue of better understanding the biology of KRAS mutant disease, in order to devise novel effective therapies in such defined genetic setting. In addition to KRAS, recent data point out that BRAF and PIK3CA exon 20 mutations hamper response to EGFR-targeted treatment in mCRC, potentially excluding from treatment also patients with these molecular alterations in their tumor. This review will focus on current knowledge regarding the molecular landscape of mCRC including and beyond KRAS, and will summarize novel rationally-developed combinatorial regimens that are being evaluated in early clinical trials.