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P3.03-019 Molecular Characterization of Malignant Pleural Mesothelioma (MPM) by next Generation Sequencing
January 2017
James Chung-Man Ho,1 Kin-Pong U,2 Shi Xu,1 Paul Ning-Man Cheng,2 Sze Kwan Lam1 1Department of Medicine, The University of Hong Kong, Hong Kong Sar/ Hong Kong Prc, 2Bio-Cancer Treatment International Ltd, Hong Kong/Hong Kong Prc Background: Malignant pleural mesothelioma (MPM) is a global health issue. Pegylated arginase (PEG-BCT-100) has shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and human melanoma. We aimed to study the preclinical anticancer effects of BCT-100 in MPM. Methods: A panel of 5 mesothelioma cell lines (from ATCC) was used to study the in vitro effect of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 (± chemotherapy) were studied using two nude mice xenograft models. Protein expression and arginine concentration were evaluated by Western Blot and ELISA respectively. Cellular location of BCT-100 was detected by immunohistochemistry and immunoflorescence staining. TUNEL assay was used to identify cellular apoptotic events. Results: BCT-100 reduced in vitro cell viability (IC50: 13-24 mU/ml) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. Argininosuccinate synthetase was expressed in H28, H226, H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT100 (60 mg/kg) significantly suppressed tumor growth with increased median survival in both xenograft models. No beneficial effect was observed when combining BCT-100 with pemetrexed or cisplatin. BCT100 decreased serum and intratumoral arginine level. BCT-100 was mainly located in cytosol of tumor cells. Apoptosis (PARP cleavage in 211H xenograft, Bcl-2 downregulation and cleavage of PARP and caspase 3 in H226 xenograft as well as TUNEL-positive staining in both xenografts) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenograft and suppression of cyclin A2, E1, H and CDK4 in H226 xenograft) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms. Conclusion: MPM tumor growth was suppressed by BCT-100 via apoptosis and G1 arrest in vivo. This provides scientific evidence to support further clinical exploration of BCT-100 in treatment of MPM. (Acknowledgment: This research was supported by Hong Kong Pneumoconiosis Compensation Fund Board, HKSAR.) Keywords: malignant pleural mesothelioma, Xenograft, pegylated arginase, apoptosis
Abstracts
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P3.03-019 Molecular Characterization of Malignant Pleural Mesothelioma (MPM) by next Generation Sequencing Topic: Mesothelioma Transitional Susana Cedres,1 Am Martinez De Castro,1 Nuria Pardo,1 Alejandro Navarro,1 Alex Martinez,1 Fabiola Amair,1 Fabricio Racca,1 Eulalia Scheenaard,2 Sergi Recasens,2 Iris De La Fuente,2 Andrea Retter,2 Marta Vilaro,2 Ana Vivancos,2 Enriqueta Felip1 1 Medical Oncology, Vall D0 hebron Institute of Oncology/ vall D0 hebron University Hospital, Barcelona/Spain, 2 Medical Oncology, Vall D0 hebron Institute of Oncology, Barcelona/Spain Background: Malignant pleural mesothelioma (MPM) is a highly aggressive pleural tumor associated with asbestos exposure and with limited survival despite treatment. Chromosomal abnormalities are abundant in MPM and the most frequently mutated genes are BAP1, NF2 and CDKN2A. Expanded molecular profile in MPM may provide targetable molecular aberrations and improve treatment options for these patients (p). Methods: Thirty two MPM patients who progressed to standard chemotherapy underwent genetic tumor profiling in a molecular prescreening program at our institution between 2006 and 2015. Paraffin-embedded biopsies were used for the analysis. Mass detection (MassARRAY, Sequenom) including analysis of mutations in 25 oncogenes was used and since June 2014 multiplexed amplicon sequencing (AmpliSeq, Illumina) was implemented assessing mutations in 59 oncogenes. No patients received systemic treatment prior to obtain the tumor sample. Results: Demographics: male/female (22/10); median age 60.5 (range 32-83 years); histology epithelial/no epithelial (24/8); PS 0/1 (15/17); stage III/IV (14/13). All patients were treated with chemotherapy. Sequenom was performed in 21 p and AmpliSeq in 11 p. Median follow up was 23.3 months and median overall survival (OS) for entire cohort was 30.2 months. The median OS for patients with epitheliod and no-epithelioid histology was 31.5 vs 21.4 months (p¼0.033). Genetic alterations were detected in 5 patients (4 p with AmpliSeq and 1 p with Sequenom). The mutations detected were RNF43/ ZNRF3 (2p), PI3KCA (1p), APC (1p) and P53 (1p). We did not identify significant association between mutations with histology, gender and clinical stage (p>0.05). Median survival for patients with mutations was not reached and for patients without mutations was 30.2 m (p¼0.462).
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Conclusion: This study shows genetic alterations are not frequent in MPM and AmpliSeq detected more genetic alterations than Sequenom. With a limited number of patients, we suggest further investigations about the role of mutations in Wnt pathway in MPM. Keywords: malignant pleural mesothelioma, next generation sequencing
P3.03-020 Cytotoxicology Assessment of MWCNT on MeT-5A Cells Topic: Mesothelioma Transitional Li Ju, Li Jin Zhu, Wei Wu, Min Yu, Jian Lin Lou Occupational Disease Prevention, Zhejiang Academy of Medical Sciences, Hangzhou/China Background: Muti-walled carbon nanotube (MWCNT) is widely used worldwide, but reports already show MWCNT is toxic to experimental animals and cell lines, even causes severe caner, such as mesothelioma. The mechanism of MWCNT toxicities is not very clearly. So, in this study, some toxic effects of MWCNT on MeT-5A cells are evaluated, to offer clues for further study. Methods: Cell survival rate was detected by LDH, and cell cycle/ cell apoptosis were measured by flow cytometry. Cell scratch assay was used to evaluate the cell migration capacity. Results: The cytotoxicity of MWCNT on MeT-5A cells are dose- and time-dependent. Cell cycle are blocked in G1/ G2 phase, cells in S phase are reduced. The same as cell proliferation, the apoptosis of MeT-5A cells is also timedependent. MeT-5A cells display a significant reduction of cell migration after MWCNT exposure for 24h and 48h. Conclusion: The cytotoxicity of MWCNT on MeT-5A cells are dose- and time-dependent and the cellular behavior are perturbed by MWCNT treatments.
P3.03-021 When RON MET TAM in Mesothelioma: All Druggable for One, and One Drug for All? Topic: Mesothelioma Transitional Anne-Marie Baird,1 David Easty,2 Monika Jarzabek,3 Liam Shiels,3 Alex Soltermann,4 Stéphane Raeppel,5 Lauren Mcdonagh,2 Chengguang Wu,4 Chandra Goparju,6 Bryan Stanfill,7 Martin Barr,8
Journal of Thoracic Oncology
Vol. 12 No. 1S
Daisuke Nonaka,9 Bruno Murer,10 Dean Fennell,11 Dearbhaile O’Donnell,12 Luciano Mutti,13 Stephen Finn,14 Sinead Cuffe,15 Harvey Pass,16 Isabelle Schmitt-Opitz,17 Annette Byrne,3 Kenneth O’Byrne,18 Steven Gray8 1St. James’ Hospital & Queensland University of Technology, Dublin/QLD/ Ireland, 2Thoracic Oncology Research Group, St James’ Hospital & Trinity College Dublin, Dublin/Ireland, 3Dept. of Physiology and Medical Physics & Centre for Systems Medicine, Royal College of Surgeons in Ireland, Dublin/ Ireland, 4Dept. of Clinical Pathology, University Hospital Zurich, Zurich/Switzerland, 5Chemrf Laboratories, Montreal/QC/Canada, 6Dept. of Thoracic Surgery, NYU Langone Medical Center, New York/NY/United States of America, 7Csiro, Brisbane/ACT/Australia, 8Thoracic Oncology Research Group, Labmed Directorate, Dublin/ Ireland, 9Dept. of Histopathology, The Christie NHS Foundation Trust, Manchester/United Kingdom, 10 Department of Clinical Pathology, Ospedale Dell’Angelo, Venice/Italy, 11Cancer Research Uk Centre, University of Leicester & Leicester University Hospitals, Leicester/ United Kingdom, 12Dept. of Oncology, Hope Directorate, Dublin/Ireland, 13School of Environment & Life Sciences, University of Salford, Salford/United Kingdom, 14Dept of Histopathology, St James’ Hospital, Dublin/Ireland, 15Dept of Medical Oncology, St James’ Hospital, Dublin/Ireland, 16 Department of Cardiothoracic Surgery, NYU Langone Medical Center, New York/NY/United States of America, 17 Dept. of Thoracic Surgery, University Hospital Zurich, Zurich/Switzerland, 18Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane/Australia Background: Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos. Untreated, MPM has a median survival time of 6 months, and most patients die within 24 months of diagnosis. Therefore an urgent need exists to identify new therapies for treating MPM patients. The potential for therapeutically targeting receptor tyrosine kinase (RTK) signalling networks is emerging as a critical mechanism in ‘oncogene addicted’ cancer, with RTK inhibitors evolving as areas of considerable importance in cancer therapy. Furthermore, RTK hetero-dimerization has emerged as a key element in the development of resistance to cancer therapy. As such TKIs which target several RTKs may have superior efficacy compared with TKIs targeting individual RTKs. We and others have identified c-MET, RON, Axl and Tyro3 as RTKs frequently overexpressed and activated in MPM, making these attractive candidate therapeutic targets. A number of orally bioavailable small molecule inhibitors have been developed which can target these receptors. LCRF0004 specifically targets RON, whereas
James Chung-Man Ho,1 Kin-Pong U,2 Shi Xu,1 Paul Ning-Man Cheng,2 Sze Kwan Lam1 1Department of Medicine, The University of Hong Kong, Hong Kong Sar/ Hong Kong Prc, 2Bio-Cancer Treatment International Ltd, Hong Kong/Hong Kong Prc Background: Malignant pleural mesothelioma (MPM) is a global health issue. Pegylated arginase (PEG-BCT-100) has shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and human melanoma. We aimed to study the preclinical anticancer effects of BCT-100 in MPM. Methods: A panel of 5 mesothelioma cell lines (from ATCC) was used to study the in vitro effect of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 (± chemotherapy) were studied using two nude mice xenograft models. Protein expression and arginine concentration were evaluated by Western Blot and ELISA respectively. Cellular location of BCT-100 was detected by immunohistochemistry and immunoflorescence staining. TUNEL assay was used to identify cellular apoptotic events. Results: BCT-100 reduced in vitro cell viability (IC50: 13-24 mU/ml) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. Argininosuccinate synthetase was expressed in H28, H226, H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT100 (60 mg/kg) significantly suppressed tumor growth with increased median survival in both xenograft models. No beneficial effect was observed when combining BCT-100 with pemetrexed or cisplatin. BCT100 decreased serum and intratumoral arginine level. BCT-100 was mainly located in cytosol of tumor cells. Apoptosis (PARP cleavage in 211H xenograft, Bcl-2 downregulation and cleavage of PARP and caspase 3 in H226 xenograft as well as TUNEL-positive staining in both xenografts) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenograft and suppression of cyclin A2, E1, H and CDK4 in H226 xenograft) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms. Conclusion: MPM tumor growth was suppressed by BCT-100 via apoptosis and G1 arrest in vivo. This provides scientific evidence to support further clinical exploration of BCT-100 in treatment of MPM. (Acknowledgment: This research was supported by Hong Kong Pneumoconiosis Compensation Fund Board, HKSAR.) Keywords: malignant pleural mesothelioma, Xenograft, pegylated arginase, apoptosis
Abstracts
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P3.03-019 Molecular Characterization of Malignant Pleural Mesothelioma (MPM) by next Generation Sequencing Topic: Mesothelioma Transitional Susana Cedres,1 Am Martinez De Castro,1 Nuria Pardo,1 Alejandro Navarro,1 Alex Martinez,1 Fabiola Amair,1 Fabricio Racca,1 Eulalia Scheenaard,2 Sergi Recasens,2 Iris De La Fuente,2 Andrea Retter,2 Marta Vilaro,2 Ana Vivancos,2 Enriqueta Felip1 1 Medical Oncology, Vall D0 hebron Institute of Oncology/ vall D0 hebron University Hospital, Barcelona/Spain, 2 Medical Oncology, Vall D0 hebron Institute of Oncology, Barcelona/Spain Background: Malignant pleural mesothelioma (MPM) is a highly aggressive pleural tumor associated with asbestos exposure and with limited survival despite treatment. Chromosomal abnormalities are abundant in MPM and the most frequently mutated genes are BAP1, NF2 and CDKN2A. Expanded molecular profile in MPM may provide targetable molecular aberrations and improve treatment options for these patients (p). Methods: Thirty two MPM patients who progressed to standard chemotherapy underwent genetic tumor profiling in a molecular prescreening program at our institution between 2006 and 2015. Paraffin-embedded biopsies were used for the analysis. Mass detection (MassARRAY, Sequenom) including analysis of mutations in 25 oncogenes was used and since June 2014 multiplexed amplicon sequencing (AmpliSeq, Illumina) was implemented assessing mutations in 59 oncogenes. No patients received systemic treatment prior to obtain the tumor sample. Results: Demographics: male/female (22/10); median age 60.5 (range 32-83 years); histology epithelial/no epithelial (24/8); PS 0/1 (15/17); stage III/IV (14/13). All patients were treated with chemotherapy. Sequenom was performed in 21 p and AmpliSeq in 11 p. Median follow up was 23.3 months and median overall survival (OS) for entire cohort was 30.2 months. The median OS for patients with epitheliod and no-epithelioid histology was 31.5 vs 21.4 months (p¼0.033). Genetic alterations were detected in 5 patients (4 p with AmpliSeq and 1 p with Sequenom). The mutations detected were RNF43/ ZNRF3 (2p), PI3KCA (1p), APC (1p) and P53 (1p). We did not identify significant association between mutations with histology, gender and clinical stage (p>0.05). Median survival for patients with mutations was not reached and for patients without mutations was 30.2 m (p¼0.462).
S1356
Conclusion: This study shows genetic alterations are not frequent in MPM and AmpliSeq detected more genetic alterations than Sequenom. With a limited number of patients, we suggest further investigations about the role of mutations in Wnt pathway in MPM. Keywords: malignant pleural mesothelioma, next generation sequencing
P3.03-020 Cytotoxicology Assessment of MWCNT on MeT-5A Cells Topic: Mesothelioma Transitional Li Ju, Li Jin Zhu, Wei Wu, Min Yu, Jian Lin Lou Occupational Disease Prevention, Zhejiang Academy of Medical Sciences, Hangzhou/China Background: Muti-walled carbon nanotube (MWCNT) is widely used worldwide, but reports already show MWCNT is toxic to experimental animals and cell lines, even causes severe caner, such as mesothelioma. The mechanism of MWCNT toxicities is not very clearly. So, in this study, some toxic effects of MWCNT on MeT-5A cells are evaluated, to offer clues for further study. Methods: Cell survival rate was detected by LDH, and cell cycle/ cell apoptosis were measured by flow cytometry. Cell scratch assay was used to evaluate the cell migration capacity. Results: The cytotoxicity of MWCNT on MeT-5A cells are dose- and time-dependent. Cell cycle are blocked in G1/ G2 phase, cells in S phase are reduced. The same as cell proliferation, the apoptosis of MeT-5A cells is also timedependent. MeT-5A cells display a significant reduction of cell migration after MWCNT exposure for 24h and 48h. Conclusion: The cytotoxicity of MWCNT on MeT-5A cells are dose- and time-dependent and the cellular behavior are perturbed by MWCNT treatments.
P3.03-021 When RON MET TAM in Mesothelioma: All Druggable for One, and One Drug for All? Topic: Mesothelioma Transitional Anne-Marie Baird,1 David Easty,2 Monika Jarzabek,3 Liam Shiels,3 Alex Soltermann,4 Stéphane Raeppel,5 Lauren Mcdonagh,2 Chengguang Wu,4 Chandra Goparju,6 Bryan Stanfill,7 Martin Barr,8
Journal of Thoracic Oncology
Vol. 12 No. 1S
Daisuke Nonaka,9 Bruno Murer,10 Dean Fennell,11 Dearbhaile O’Donnell,12 Luciano Mutti,13 Stephen Finn,14 Sinead Cuffe,15 Harvey Pass,16 Isabelle Schmitt-Opitz,17 Annette Byrne,3 Kenneth O’Byrne,18 Steven Gray8 1St. James’ Hospital & Queensland University of Technology, Dublin/QLD/ Ireland, 2Thoracic Oncology Research Group, St James’ Hospital & Trinity College Dublin, Dublin/Ireland, 3Dept. of Physiology and Medical Physics & Centre for Systems Medicine, Royal College of Surgeons in Ireland, Dublin/ Ireland, 4Dept. of Clinical Pathology, University Hospital Zurich, Zurich/Switzerland, 5Chemrf Laboratories, Montreal/QC/Canada, 6Dept. of Thoracic Surgery, NYU Langone Medical Center, New York/NY/United States of America, 7Csiro, Brisbane/ACT/Australia, 8Thoracic Oncology Research Group, Labmed Directorate, Dublin/ Ireland, 9Dept. of Histopathology, The Christie NHS Foundation Trust, Manchester/United Kingdom, 10 Department of Clinical Pathology, Ospedale Dell’Angelo, Venice/Italy, 11Cancer Research Uk Centre, University of Leicester & Leicester University Hospitals, Leicester/ United Kingdom, 12Dept. of Oncology, Hope Directorate, Dublin/Ireland, 13School of Environment & Life Sciences, University of Salford, Salford/United Kingdom, 14Dept of Histopathology, St James’ Hospital, Dublin/Ireland, 15Dept of Medical Oncology, St James’ Hospital, Dublin/Ireland, 16 Department of Cardiothoracic Surgery, NYU Langone Medical Center, New York/NY/United States of America, 17 Dept. of Thoracic Surgery, University Hospital Zurich, Zurich/Switzerland, 18Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane/Australia Background: Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos. Untreated, MPM has a median survival time of 6 months, and most patients die within 24 months of diagnosis. Therefore an urgent need exists to identify new therapies for treating MPM patients. The potential for therapeutically targeting receptor tyrosine kinase (RTK) signalling networks is emerging as a critical mechanism in ‘oncogene addicted’ cancer, with RTK inhibitors evolving as areas of considerable importance in cancer therapy. Furthermore, RTK hetero-dimerization has emerged as a key element in the development of resistance to cancer therapy. As such TKIs which target several RTKs may have superior efficacy compared with TKIs targeting individual RTKs. We and others have identified c-MET, RON, Axl and Tyro3 as RTKs frequently overexpressed and activated in MPM, making these attractive candidate therapeutic targets. A number of orally bioavailable small molecule inhibitors have been developed which can target these receptors. LCRF0004 specifically targets RON, whereas