Maria Apicella

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.

Targeted therapies for gastric cancer: failures and hopes from clinical trials

Gastric cancer is the third leading cause of cancer mortality worldwide. As surgery is the only curative treatment strategy and conventional chemotherapy has shown limited efficacy -with a median overall survival of 10 months- new treatments are urgently needed. Trastuzumab and Ramucirumab (targeting HER2 and VEGFR2, respectively) are the only targeted therapies approved so far. Indeed, most Phase III clinical trials evaluating molecular drugs in gastric cancer failed. This review will retrace the relevant clinical trials with molecular therapies performed in gastric cancer patients, discussing the possible reasons for their failure and indicating new perspective for a real improvement of the treatment of this disease.Gastric cancer is the third leading cause of cancer mortality worldwide. As surgery is the only curative treatment strategy and conventional chemotherapy has shown limited efficacy -with a median overall survival of 10 months- new treatments are urgently needed. Trastuzumab and Ramucirumab (targeting HER2 and VEGFR2, respectively) are the only targeted therapies approved so far. Indeed, most Phase III clinical trials evaluating molecular drugs in gastric cancer failed. This review will retrace the relevant clinical trials with molecular therapies performed in gastric cancer patients, discussing the possible reasons for their failure and indicating new perspective for a real improvement of the treatment of this disease.

Dual MET/EGFR therapy leads to complete response and resistance prevention in a MET -amplified gastroesophageal xenopatient cohort

Amplification of the MET oncogene occurs in 2–4% of gastroesophageal cancers and defines a small and aggressive subset of tumors. Although in vitro studies have given very promising results, clinical trials with MET inhibitors have been disappointing, showing few and short lasting responses. The aim of the work was to exploit a MET-amplified patient-derived xenograft model to optimize anti-MET therapeutic strategies in gastroesophageal cancer. We found that despite the high MET amplification level (26 gene copies), in the absence of qualitative or quantitative alterations of EGFR, MET inhibitors induced only tumor growth inhibition, whereas dual MET/EGFR inhibition led to complete tumor regression. Importantly, the combo treatment completely prevented the onset of resistance, which quite rapidly appeared in tumors treated with MET monotherapy. We found that this secondary resistance was due to EGFR activation and could be overcome by dual MET/EGFR inhibition. Similar results were also obtained in a MET-addicted, established gastric cancer cell line. In vitro experiments performed on tumor-derived primary cells confirmed that MET inhibitors were not able to abrogate the activation of downstream transducers and that only the combined MET/EGFR treatment completely shut off the signaling. Previously reported cases, as well as those described here, showed only partial and transient sensitivity to anti-MET therapy. The finding that combined anti-MET/EGFR therapy—even in the absence of EGFR genetic alterations—induced complete and durable response, represents a proof of concept and guarantees further investigations, opening a new perspective of treatment for these patients.

Rituximab Treatment Prevents Lymphoma Onset in Gastric Cancer Patient-Derived Xenografts

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples.

miR‐205 mediates adaptive resistance to MET inhibition via ERRFI1 targeting and raised EGFR signaling

The onset of secondary resistance represents a major limitation to long‐term efficacy of target therapies in cancer patients. Thus, the identification of mechanisms mediating secondary resistance is the key to the rational design of therapeutic strategies for resistant patients. MiRNA profiling combined with RNA‐Seq in MET‐addicted cancer cell lines led us to identify the miR‐205/ERRFI1 (ERBB receptor feedback inhibitor‐1) axis as a novel mediator of resistance to MET tyrosine kinase inhibitors (TKIs). In cells resistant to MET‐TKIs, epigenetically induced miR‐205 expression determined the downregulation of ERRFI1 which, in turn, caused EGFR activation, sustaining resistance to MET‐TKIs. Anti‐miR‐205 transduction reverted crizotinib resistance in vivo, while miR‐205 over‐expression rendered wt cells refractory to TKI treatment. Importantly, in the absence of EGFR genetic alterations, miR‐205/ERRFI1‐driven EGFR activation rendered MET‐TKI‐resistant cells sensitive to combined MET/EGFR inhibition. As a proof of concept of the clinical relevance of this new mechanism of adaptive resistance, we report that a patient with a MET‐amplified lung adenocarcinoma displayed deregulation of the miR‐205/ERRFI1 axis in concomitance with onset of clinical resistance to anti‐MET therapy.

Abstract A04: Combined anti–MET/EGFR treatment results in complete tumor regression and prevents resistance onset in a MET-amplified gastroesophageal xenopatient cohort

Gastric cancer is the world third leading cause of cancer mortality. In spite of the significant therapeutic advances, the overall clinical outcome for patients with advanced gastric cancer is poor, with 5-20% 5-year survival. The only targeted therapy approved so far are trastuzumab, and Ramucirumab which have given unsatisfactory results. Around 50% of gastric tumors bear genetic alterations affecting tyrosine kinase pathways (mainly EGFR, HER3, FGFR2 and MET pathways, besides HER2) but their clinical validation as tumor drivers is missing. The need for new therapeutic options and the possible presence of “druggable” targets prompted us to investigate potential targeted therapies for this disease. Our project aims at identifying and validating targeted therapeutic strategies in gastric cancer, through the generation of a platform of gastric tumor patient-derived xenografts (PDXs), animal models in which tumor surgical specimens are directly transferred into mice. Upon engraftment, the tumor is split and re-implanted in a cohort of mice, allowing the simultaneous testing of different drugs on the same tumor. Thanks to the establishment of a network of 15 Italian centers for samples collection, we generated around 80 gastric PDXs and successfully derived cell lines and organoids from engrafted tumors. Among the tumors collected so far, we found HER2, EGFR, FGFR2, MET and KRAS amplifications. This gastric PDX platform will be exploited for: 1) Validation of candidate oncogenes as relevant targets and identification of efficient therapeutic strategies 2) Identification of novel molecular targets; 3) identification of genetic predictors of response/resistance. In the PDX platform we identified one tumor bearing a high level of MET gene amplification (26 gene copies). We thus performed a preclinical study on a cohort of patient-derived xenografts generated from the MET-amplified gastroesophageal tumor. Despite the high amplification level, MET inhibitors induced only a partial response, while the combined anti-MET/EGFR treatment led to complete tumor regression. Most important, the combo treatment also prevented resistance onset. This data represent the proof of concept that a combined anti-MET/EGFR therapy can be more effective than anti-MET treatment alone in MET-amplified gastroesophageal tumors, in the absence of EGFR genetic lesions. Citation Format: Simona Corso, Maria Apicella, Silvia Menegon, Marilisa Cargnelutti, Maria Stella Scalzo, Rossella Reddavid, Paola Cassoni, Anna Sapino, Maurizio De Giuli, Silvia Giordano. Combined anti–MET/EGFR treatment results in complete tumor regression and prevents resistance onset in a MET-amplified gastroesophageal xenopatient cohort. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A04.

Abstract 622: Gastric cancer in the age of targeted agents: identification and validation of novel therapeutic strategies through the generation of a patient-derived xenografts platform

Gastric cancer is the world third leading cause of cancer mortality. In spite of the significant therapeutic advances, the overall clinical outcome for patients with advanced gastric cancer is poor, with 5-20% 5-year survival. The only targeted therapy approved so far are trastuzumab, and Ramucirumab which have given unsatisfactory results. Around 50% of gastric tumors bear genetic alterations affecting tyrosine kinase pathways (mainly EGFR, HER3, FGFR2 and MET pathways, besides HER2) but their clinical validation as tumor drivers is missing. The need for new therapeutic options and the possible presence of ‘druggable’ targets prompted us to investigate potential targeted therapies for this disease. Our project aims at identifying and validating targeted therapeutic strategies in gastric cancer, through the generation of a platform of gastric tumor patient-derived xenografts (PDXs), animal models in which tumor surgical specimens are directly transferred into mice. Upon engraftment, the tumor is split and re-implanted in a cohort of mice, allowing the simultaneous testing of different drugs on the same tumor. Thanks to the establishment of a network of 15 Italian centers for samples collection, we generated around 80 gastric PDXs and successfully derived cell lines and organoids from engrafted tumors. Among the tumors collected so far, we found HER2, EGFR, FGFR2, MET and KRAS amplifications. This gastric PDX platform will be exploited for: 1) Validation of candidate oncogenes as relevant targets and identification of efficient therapeutic strategies 2) Identification of novel molecular targets; 3) identification of genetic predictors of response/resistance. In the PDX platform we identified one tumor bearing a high level of MET gene amplification (26 gene copies). We thus performed a preclinical study on a cohort of patient-derived xenografts generated from the MET-amplified gastroesophageal tumor. Despite the high amplification level, MET inhibitors induced only a partial response, while the combined anti-MET/EGFR treatment led to complete tumor regression. Most important, the combo treatment also prevented resistance onset. This data represent the proof of concept that a combined anti-MET/EGFR therapy can be more effective than anti-MET treatment alone in MET-amplified gastroesophageal tumors, in the absence of EGFR genetic lesions. Citation Format: Silvia Menegon, Maria Apicella, Cristina Migliore, Tania Capeloa, Marilisa Cargnelutti, Maurizio Degiuli, Anna Sapino, Paola Cassoni, Michele De Simone, Paolo M. Comoglio, Silvia Marsoni, Simona Corso, Silvia Giordano. Gastric cancer in the age of targeted agents: identification and validation of novel therapeutic strategies through the generation of a patient-derived xenografts platform. [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 622.

Abstract B12: Understanding the role of the tumor microenvironment in mediating resistance to anti-MET drugs

Resistance to targeted therapy represents a major challenge to the treatment of cancer patients. Many data support the role of tumor stroma in modulating tumor progression, but little is known about its potential role in drug resistance. This is mostly due to the fact that resistance to targeted therapies has been mainly studied in vitro; thus in vivo tumor models that better mimic human tumors are absolutely needed. Since MET, the tyrosine kinase receptor for Hepatocyte Growth Factor, is frequently implicated in resistance to kinase-targeted therapies and MET Tyrosine kinase inhibitors (TKIs) are currently in advanced phases of clinical trials, our aim was to investigate a possible role of non-cell-autonomous (microenvironment-mediated) mechanisms of resistance to MET-TKIs. We generated in vivo models of MET-addicted tumors resistant to anti-MET treatment, subcutaneously injecting the non–small cell lung carcinoma cell line-EBC-1 and the gastric carcinoma cells GTL16 in NOD-SCID mice and treating them with MET inhibitors (JNJ-38877605 or CRIZOTINIB) until resistance onset. In parallel, untreated tumors were used as control. We analyzed both tumors and cells (tumor cells and murine fibroblasts) isolated and put in culture from WT and resistant (RES) tumors. Through cell viability assays we demonstrated that six out of seven xeno-derived tumor cell lines were not or poorly resistant in vitro to MET inhibitors, suggesting a possible involvement of microenvironment in sustaining resistance. We reasoned that the stroma of the resistant tumors could be qualitatively and/or quantitatively different from that of the wild-type tumors. To support this idea murine-gene arrays showed a significantly different gene expression profile between WT and resistant murine stroma. Gene-annotation enrichment analysis showed that inflammatory pathways and cancer-associated fibroblast (CAF) genes were particularly up-regulated in resistant versus wt tumors. In order to understand whether the stroma may confer resistance to MET TKIs, we started to perform in vitro- and in vivo- co-colture experiments. In vitro experiments suggest that tumor cells co-cultured with fibroblasts were less sensitive to the MET inhibitors. We also performed preliminary in vivo experiments to demonstrate that the stroma of resistant tumors can induce resistance. We injected wild-type EBC1 cells (highly sensitive to MET inhibitors), transduced with the luciferase gene, into pre-existing wild-type or resistant tumors. Then we treated mice with the MET inhibitor JNJ-38877605 and monitored the luciferase signal. At the end of the experiment, by IVIS imaging analysis, we observed a decrease of the EBC1 luciferase signal (that is death of the WT labeled injected cells) in WT tumors treated with the MET inhibitor, but an increased luciferase signal in the resistant tumors (meaning that luciferase-labeled WT cells did not die in the presence of the MET inhibitor when in the contest of resistant tumors). All together our experiments suggest that the microenvironment of resistant tumors can induce resistance to targeted therapies, protecting tumor cells from the effect of the drug. Citation Format: Simona Corso, Maria Apicella, Silvia Giordano. Understanding the role of the tumor microenvironment in mediating resistance to anti-MET drugs. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B12.