Stefania Napolitano

Increased TGF-α as a Mechanism of Acquired Resistance to the Anti-EGFR Inhibitor Cetuximab through EGFR–MET Interaction and Activation of MET Signaling in Colon Cancer Cells

Purpose: Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by onset of resistance. Experimental Design: We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab. Results: Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET–dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR–MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells. Conclusions: These results suggest that overexpression of TGF-α through induction of EGFR–MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer. Clin Cancer Res; 19(24); 6751–65. ©2013 AACR.

Primary and Acquired Resistance of Colorectal Cancer Cells to Anti-EGFR Antibodies Converge on MEK/ERK Pathway Activation and Can Be Overcome by Combined MEK/EGFR Inhibition

Purpose: The EGFR-independent activation of the RAS/RAF/MEK/MAPK pathway is one of the resistance mechanisms to cetuximab. Experimental Design: We have evaluated, in vitro and in vivo, the effects of BAY 86-9766, a selective MEK1/2 inhibitor, in a panel of human colorectal cancer cell lines with primary or acquired resistance to cetuximab. Results: Among the colorectal cancer cell lines, five with a KRAS mutation (LOVO, HCT116, HCT15, SW620, and SW480) and one with a BRAF mutation (HT29) were resistant to the antiproliferative effects of cetuximab, whereas two cells (GEO and SW48) were highly sensitive. Treatment with BAY 86-9766 determined dose-dependent growth inhibition in all cancer cells, including two human colorectal cancer cells with acquired resistance to cetuximab (GEO-CR and SW48-CR), with the exception of HCT15 cells. Combined treatment with cetuximab and BAY 86-9766 induced a synergistic antiproliferative and apoptotic effects with blockade in the MAPK and AKT pathway in cells with either primary or acquired resistance to cetuximab. The synergistic antiproliferative effects were confirmed using other two selective MEK1/2 inhibitors, selumetinib and pimasertib, in combination with cetuximab. Moreover, inhibition of MEK expression by siRNA restored cetuximab sensitivity in resistant cells. In nude mice bearing established human HCT15, HCT116, SW48-CR, and GEO-CR xenografts, the combined treatment with cetuximab and BAY 86-9766 caused significant tumor growth inhibition and increased mice survival. Conclusion: These results suggest that activation of MEK is involved in both primary and acquired resistance to cetuximab and the inhibition of EGFR and MEK could be a strategy for overcoming anti-EGFR resistance in patients with colorectal cancer. Clin Cancer Res; 20(14); 3775–86. ©2014 AACR.

Mechanisms of resistance to anti-epidermal growth factor receptor inhibitors in metastatic colorectal cancer

The prognosis of patients with metastatic colorectal cancer (mCRC) remain poor despite the impressive improvement of treatments observed over the last 20 years that led to an increase in median overall survival from 6 mo, with the only best supportive care, to approximately 30 mo with the introduction of active chemotherapy drugs and targeted agents. The monoclonal antibodies (moAbs) cetuximab and panitumumab, directed against the epidermal growth factor receptor (EGFR), undoubtedly represent a major step forward in the treatment of mCRC, given the relevant efficacy in terms of progression-free survival, overall survival, response rate, and quality of life observed in several phase III clinical trials among different lines of treatment. However, the anti-EGFR moAbs were shown only to be effective in a subset of patients. For instance, KRAS and NRAS mutations have been identified as biomarkers of resistance to these drugs, improving the selection of patients who might derive a benefit from these treatments. Nevertheless, several other alterations might affect the response to these drugs, and unfortunately, even the responders eventually become resistant by developing secondary (or acquired) resistance in approximately 13-18 mo. Several studies highlighted that the landscape of responsible alterations of both primary and acquired resistance to anti-EGFR drugs biochemically converge into MEK-ERK and PIK3CA-AKT pathways. In this review, we describe the currently known mechanisms of primary and acquired resistance to anti-EGFR moAbs together with the various strategies evaluated to prevent, overcame or revert them.

Primary and Acquired Resistance of Colorectal Cancer to Anti-EGFR Monoclonal Antibody Can Be Overcome by Combined Treatment of Regorafenib with Cetuximab

Purpose: In colorectal cancer, the activation of the intracellular RAS–RAF and PIK3CA–AKT pathways has been implicated in the resistance to anti-EGFR mAbs. We have investigated the role of regorafenib, an oral multikinase inhibitor, in combination with cetuximab, an anti-EGFR mAb, to overcome anti-EGFR resistance. Experimental Design: We have tested, in vitro and in vivo, the effects of regorafenib in a panel of human colorectal cancer cell lines with a KRAS mutation (SW480, SW620, HCT116, LOVO, and HCT15) or with a BRAF mutation (HT29), as models of intrinsic resistance to cetuximab treatment, and in two human colorectal cancer cell lines (GEO and SW48) that are cetuximab-sensitive, as well as in their derived cells with acquired resistance to cetuximab (GEO-CR and SW48-CR). Results: Treatment with regorafenib determined a dose-dependent growth inhibition in all colorectal cancer cell lines. The combined treatment with cetuximab and regorafenib induced synergistic antiproliferative and apoptotic effects in cetuximab-resistant cell lines by blocking MAPK and AKT pathways. Nude mice were injected s.c. with HCT116, HCT15, GEO-CR, and SW48-CR cells. The combined treatment caused significant tumor growth inhibition. Synergistic antitumor activity of regorafenib plus cetuximab was also observed in an orthotopic colorectal cancer model of HCT116 cells. In particular, the combined treatment induced a significant tumor growth inhibition in the primary tumor site (cecum) and completely prevented metastasis formation. Conclusions: The combined treatment with cetuximab and regorafenib could be a strategy to overcome resistance to anti-EGFR therapies in metastatic colorectal cancer patients. Clin Cancer Res; 21(13); 2975–83. ©2015 AACR.

Therapeutic value of EGFR inhibition in CRC and NSCLC: 15 years of clinical evidence

Epidermal growth factor receptor (EGFR) plays a key role in tumour evolution, proliferation and immune evasion, and is one of the most important targets for biological therapy, especially for non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). In the past 15 years, several EGFR antagonists have been approved for the treatment of NSCLC and metastatic CRC (mCRC). To optimise the use of anti-EGFR agents in clinical practice, various clinical and molecular biomarkers have been investigated, thus moving their indication from unselected to selected populations. Nowadays, anti-EGFR drugs represent a gold-standard therapy for metastatic NSCLC harbouring EGFR activating mutation and for RAS wild-type mCRC. Their clinical efficacy is limited by the presence of intrinsic resistance or the onset of acquired resistance. In this review, we provide an overview of the antitumour activity of EGFR inhibitors in NSCLC and CRC and of mechanisms of resistance, focusing on the development of a personalised approach through 15 years of preclinical and clinical research.

Maintenance Treatment with Cetuximab and BAY86-9766 Increases Antitumor Efficacy of Irinotecan plus Cetuximab in Human Colorectal Cancer Xenograft Models

Purpose: The use of cetuximab in the treatment of metastatic colorectal cancer is limited by development of resistance. Experimental Design: We have investigated in three models of highly epidermal growth factor receptor (EGFR)–dependent colorectal cancer xenografts, the effect of maintenance therapy with different kinase inhibitors alone or in combination with cetuximab, after cytotoxic treatment induction with irinotecan plus cetuximab. Results: SW48, LIM 1215, and GEO colorectal cancer cell lines were engrafted into nude mice and treated for 3 weeks with irinotecan and/or cetuximab. The combined treatment induced a significant reduction of tumor size. A subsequent experiment was performed in all three xenograft models in which after an induction treatment with irinotecan plus cetuximab, mice were randomly assigned to one of the following treatments: control, cetuximab, regorafenib, a selective PIK3CA inhibitor (PIK3CAi), a selective MEK inhibitor (MEKi), and/or the combination of each inhibitor with cetuximab. The cetuximab plus MEKi treatment determined the best antitumor activity with suppression of tumor growth. This effect was prolonged for 13 to 15 weeks after cessation of therapy and was accompanied by prolonged survival. Antitumor activity was accompanied by inhibition of the MAPK and MEK pathways. Moreover, in the cetuximab plus MEKi-treated SW48 xenograft group, KRAS mutations as a mechanism of acquired resistance were detected in 25% of cases compared with 75% KRAS mutations in the MEKi-treated group. Conclusions: A possible strategy to prevent and/or overcome resistance to anti-EGFR inhibitors in metastatic colorectal cancer is a maintenance therapy with cetuximab plus MEKi after an initial treatment with irinotecan plus cetuximab. Clin Cancer Res; 21(18); 4153–64. ©2015 AACR.

Clinical outcome and molecular characterisation of chemorefractory metastatic colorectal cancer patients with long-term efficacy of regorafenib treatment

Please click here to see linked paper Background To investigate the potential predictors of response to regorafenib, in chemorefractory metastatic colorectal cancer (mCRC) patients with long-term efficacy from regorafenib treatment. Methods Retrospective, single institution analysis of patients with chemorefractory mCRC treated with regorafenib, in clinical practice setting. 123 patients were treated and stratified into two groups according to number of cycles received (<7 and ≥7). Overall survival (OS), progression-free survival (PFS) and safety were evaluated. 20 tumour samples (10 poor and 10 long responders) were analysed with the OncoMine Comprehensive Assay for 143 genes. Results A good Eastern Cooperative Oncology Group performance status, a lung limited metastatic disease and a long history of metastatic disease were significantly associated with better OS and PFS from treatment with regorafenib. Mutations were mostly found in TP53, KRAS and PIK3CA as well as in NRAS, ERBB2, SMAD4 and PTEN genes. BCL2L1, ERBB2, KRAS, MYC, GAS6 gene amplifications were detected as well as ALK rearrangement. No significant correlation between molecular alterations and response to regorafenib was observed. However, HER2 gene alterations were found in three poor responder patients, suggesting a potential role in regorafenib resistance. Conversely, GAS6 amplification and SMAD4 mutation, detected in two long responder patients, might suggest a role of epithelial–mesenchymal transition phenotype in regorafenib response. Conclusion A subgroup of long responder patients to regorafenib treatment was identified and a comprehensive molecular characterisation was performed; however, further research efforts are essential to confirm our data.

E46Phase III study of regorafenib versus placebo as maintenance therapy in RAS wild type metastatic colorectal cancer (RAVELLO trial)

TPS789 Background: Treatment of metastatic colorectal cancer (mCRC) has improved due to the introduction of more active chemotherapies (CT) and novel targeted agents that have significantly increased response rate (RR), progression free survival (PFS) and overall survival (OS). Recently, CORRECT and CONCUR trials have demonstrated both activity and efficacy of regorafenib, a small multi-kinase inhibitor, as monotherapy in pretreated mCRC. The wide range of action of regorafenib makes it an ideal candidate for monotherapy in earlier disease treatment lines in which different pathways could be involved in the acquisition of resistance. To improve long term efficacy of first line therapy several therapeutic approaches of maintenance treatment have been explored in mCRC. Methods: RAVELLO is an academic randomized, double-blind, placebo-controlled, multi-center, phase III study designed to evaluate efficacy and safety of regorafenib as maintenance treatment after first line therapy. Eligible patients: patholog...

Present and future of metastatic colorectal cancer treatment: A review of new candidate targets

In the last two decades, great efforts have been made in the treatment of metastatic colorectal cancer (mCRC) due to the approval of new target agents for cytotoxic drugs. Unfortunately, a large percentage of patients present with metastasis at the time of diagnosis or relapse after a few months. The complex molecular heterogeneity of this disease is not completely understood; to date, there is a lack of predictive biomarkers that can be used to select subsets of patients who may respond to target drugs. Only the RAS-mutation status is used to predict resistance to anti-epidermal growth factor receptor agents in patients with mCRC. In this review, we describe approved targeted therapies for the management of metastatic mCRC and discuss new candidate targets on the horizon.

Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation

Purpose Cetuximab and panitumumab have an effective therapeutic response in a subset of RAS Wild-Type (WT) metastatic colorectal cancers (mCRCs). Despite molecular-driven selection, all patients do not respond to epidermal growth factor receptor (EGFR) inhibitors and the onset of secondary resistance limits their clinical benefit. Experimental Design We tested, in vitro and in vivo, the effect of SYM004, a 1:1 mixture of two recombinant human-mouse chimeric monoclonal antibodies (mAbs) directed against non-overlapping epitopes of the EGFR, on CRC models with acquired resistance to cetuximab. Results SYM004 showed a potent growth inhibitory effect in CRC cell lines with acquired resistance to cetuximab and MET activation. SYM004 treatment determined a significant induction of apoptosis and a strong inhibition of MET, AKT and MAPK phosphorilation in these resistant models. The data may further suggest SYM004 -driven induced internalization and degradation of the antibody-receptor complex, which prevents cross-interaction between EGFR and MET even in the presence of TGFα. Moreover, in vivo xenograft studies demonstrated that SYM004 has stronger antitumor activity than cetuximab in CRC models. Importantly, in the current work we observed a response to therapy in all cetuximab resistant tumors mice treated with SYM004. More importantly, four out of seven mice continue to respond to SYM004 after 30 weeks of treatment underling the prolonged effect of the drug. Conclusion These results suggest that the treatment with SYM004 could be a strategy to overcome acquired resistance to first generation of anti-EGFR therapies in mCRC as a result of MET activation.PURPOSE Cetuximab and panitumumab have an effective therapeutic response in a subset of RAS Wild-Type (WT) metastatic colorectal cancers (mCRCs). Despite molecular-driven selection, all patients do not respond to epidermal growth factor receptor (EGFR) inhibitors and the onset of secondary resistance limits their clinical benefit. EXPERIMENTAL DESIGN We tested, in vitro and in vivo, the effect of SYM004, a 1:1 mixture of two recombinant human-mouse chimeric monoclonal antibodies (mAbs) directed against non-overlapping epitopes of the EGFR, on CRC models with acquired resistance to cetuximab. RESULTS SYM004 showed a potent growth inhibitory effect in CRC cell lines with acquired resistance to cetuximab and MET activation. SYM004 treatment determined a significant induction of apoptosis and a strong inhibition of MET, AKT and MAPK phosphorilation in these resistant models. The data may further suggest SYM004 -driven induced internalization and degradation of the antibody-receptor complex, which prevents cross-interaction between EGFR and MET even in the presence of TGFα. Moreover, in vivo xenograft studies demonstrated that SYM004 has stronger antitumor activity than cetuximab in CRC models. Importantly, in the current work we observed a response to therapy in all cetuximab resistant tumors mice treated with SYM004. More importantly, four out of seven mice continue to respond to SYM004 after 30 weeks of treatment underling the prolonged effect of the drug. CONCLUSION These results suggest that the treatment with SYM004 could be a strategy to overcome acquired resistance to first generation of anti-EGFR therapies in mCRC as a result of MET activation.