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P2.01-016 Analysis of 5 Differential miRNA Expression in NSCLC Patients
January 2017
Abstracts
S793
Health Network, Toronto/ON/Canada, 4Cancer Clinical Research Unit, University Health Network, Toronto/ON/ Canada Background: Across multiple cancer histologies, many significantly down-regulated genes reside within chromosomal regions with increased number of copies, and vice versa. These “paradoxical genes” have been usually ignored as a noise, but could be a consequence of epigenetic regulatory mechanisms, including microRNA-mediated control of mRNA transcription. Methods: To identify paradoxical genes in lung adenocarcinoma (LUAD) we curated and analyzed gene expression and copy number aberrations across 1,064 LUAD samples, including newly-generated aCGH data from 65 samples. We then analyzed 9 LUAD microRNA expression studies to compile a list of consistently deregulated microRNAs. Finally, using microRNA:gene networks from mirDIP we examined possible association between microRNAs and paradoxical genes. Results: We identified 85 genes whose differential expression consistently contrasts the aberrations of their copy numbers. 70 genes were validated using TCGA-LUAD data. We showed that paradoxical expression of these genes is associated with 19 microRNAs, whose significant deregulation in LUAD has been consistently reported. Importantly, these genes form a clinically significant prognostic signature.
Conclusion: Paradoxical gene expression, caused by microRNA deregulation, is preserved across patient cohorts, and forms a prognostic LUAD signature. Keywords: microRNA, prognostic signature, Copy number aberrations, lung adenocarcinoma
P2.01-016 Analysis of 5 Differential miRNA Expression in NSCLC Patients Topic: Analysis of RNA Marzena A. Lewandowska,1 Lukasz Zolna,2 Aleksandra Chrząstek,2 Janusz Kowalewski2 1 Molecular Oncology and Genetics Departament, The F. Lukaszczyk Oncology Center, Collegium Medicum, Nicolaus Copernicus Univeristy, Bydgoszcz/Poland, 2 Department of Thoracic Surgery and Tumors, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz/Poland Background: There are two main types of lung cancer: non-small cell lung cancer (NSCLC) which represents 8085% cases of lung cancer and small cell lung cancer (SCLC) which is about 10-15% cases of lung cancer. The 5-year survival rate for patients with lung cancer vary depending on the stage of the cancer when it is diagnosed. Unfortunately, most of patients with lung cancer are diagnosed on later stage of disease (stage III and IV). In our research we try to find marker among miRNA that can predict occurring of lung cancer on the earlier stage.
S794
Methods: Isolation of miRNA from plasma was performed by miRCURY RNA Isolation Kit e Biofluids (Exiqon) from NSCLC patients and controls. Synthesis of cDNA and qPCR were carried out using miRCURY LNATM Universal RT microRNA PCR with LNATM enhanced PCR Primers (Exiqon). Statistical calculations were executed on 11 samples as a Pre-eliminary data. Results: Results are shown in the following table.
Conclusion: We assed level of 5 different miRNAs circulating in the blood of NSCLC patients using qPCR. Our initial results show that different miRNA can be used to stratify patients and miRNA. Expression of hsa-miR451a is decreasing in NSCLC versus negative control. Interestingly up-regulated hsa-miR-660-5p was recently described as a prognostic marker in breast cancer but our result preliminary results showed constant decrease in hsa-miR-660-5p expression in all patients’ groups vs controls. The examination on the bigger cohort of patients is necessary to receive a more statistically significant and conclusive data. Keywords: cell-free microRNA, biomarkers, miRNA expression profiling, NSCLC
P2.01-017 Circulating miRNAs in Lung Cancer Are Associated to Pro-Tumorigenic and Immunosuppressive Microenvironment Topic: Analysis of RNA Orazio Fortunato,1 Cristina Borzi,1 Giovanni Centonze,1 Massimo Milione,2 Davide Conte,1 Mattia Boeri,1 Carla Verri,1 Linda Calzolari,1 Francesca Andriani,1 Luca Roz,1 Veronica Huber,3 Agata Cova,3 Chiara Camisaschi,3 Chiara Castelli,3 Licia Rivoltini,3 Claudio Tripodo,4 Ugo Pastorino,5 Gabriella Sozzi1 1Tumor Genomics Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milano/Italy, 2Anatomic Pathology Unit, Department of Pathology and Laboratory Medicine,
Journal of Thoracic Oncology
Vol. 12 No. 1S
Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milan/Italy, 3Unit of Immunotherapy of Human Tumors, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milano/Italy, 4Tumor Immunology Unit Department of Health Science, Human Pathology Section, University of Palermo School of Medicine, Palermo/Italy, 5Thoracic Surgery, Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Palermo/Italy Background: We previously reported the identification of diagnostic miRNA signatures in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. Circulating miRNAs are released into the bloodstream by different mechanisms such as passive leakage from damaged cells or active secretion through extracellular-vesicles or protein complexes. Methods: To evaluate the potential origin and the release of the 24 miRNAs of the diagnostic signature we analyzed their expression by real-time or digital PCR in both cells and conditioned medium (CM) from cancer cell and different cell types of the lung microenvironment. Lung tissues and cell-blocks were analyzed by miRNAs in situ hybridization (ISH). Modulation of miRNAs after in vitro treatments known to induce changes associated with cancer progression, in different cell types was assessed and correlated to changes observed in circulating miRNAs signatures. Results: 24-miRNAs analysis showed higher abundance in specific cellular components such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells or mir-451 and 142-3p in hematopoietic cells. Generally, tumor cells showed lower levels of miRNAs compared to bronchial epithelial cells. MiRNAs specific localization in lung tissue was confirmed by ISH. We observed that mir-451 is specifically expressed in lung interstitial alveolar walls while mir-126 by endothelial cells outside the tumor bulk; miR-145 is characteristic of fibroblast and muscle cells and miR-142-3p of hematopoietic cells, fibroblast and muscle whereas mir-21 is over-expressed in the tumor. The analysis of miRNAs in CM showed that miRNAs secretion is correlated with cellular expression for most cell types (Pearson correlation range: 0.41-0.80). Interestingly, platelets and granulocytes were the components that mostly secreted miRNAs. In vitro experiments showed that endothelial cells under hypoxic condition up-regulate mir-126 and that mir-145 was up-regulated and secreted in lung cancer-associated compared to normal fibroblasts. Interestingly, during conversion of T lymphocytes into T regulatory cells up-regulation of mir-15b, mir-19b and mir-320 was observed whereas
Abstracts
S793
Health Network, Toronto/ON/Canada, 4Cancer Clinical Research Unit, University Health Network, Toronto/ON/ Canada Background: Across multiple cancer histologies, many significantly down-regulated genes reside within chromosomal regions with increased number of copies, and vice versa. These “paradoxical genes” have been usually ignored as a noise, but could be a consequence of epigenetic regulatory mechanisms, including microRNA-mediated control of mRNA transcription. Methods: To identify paradoxical genes in lung adenocarcinoma (LUAD) we curated and analyzed gene expression and copy number aberrations across 1,064 LUAD samples, including newly-generated aCGH data from 65 samples. We then analyzed 9 LUAD microRNA expression studies to compile a list of consistently deregulated microRNAs. Finally, using microRNA:gene networks from mirDIP we examined possible association between microRNAs and paradoxical genes. Results: We identified 85 genes whose differential expression consistently contrasts the aberrations of their copy numbers. 70 genes were validated using TCGA-LUAD data. We showed that paradoxical expression of these genes is associated with 19 microRNAs, whose significant deregulation in LUAD has been consistently reported. Importantly, these genes form a clinically significant prognostic signature.
Conclusion: Paradoxical gene expression, caused by microRNA deregulation, is preserved across patient cohorts, and forms a prognostic LUAD signature. Keywords: microRNA, prognostic signature, Copy number aberrations, lung adenocarcinoma
P2.01-016 Analysis of 5 Differential miRNA Expression in NSCLC Patients Topic: Analysis of RNA Marzena A. Lewandowska,1 Lukasz Zolna,2 Aleksandra Chrząstek,2 Janusz Kowalewski2 1 Molecular Oncology and Genetics Departament, The F. Lukaszczyk Oncology Center, Collegium Medicum, Nicolaus Copernicus Univeristy, Bydgoszcz/Poland, 2 Department of Thoracic Surgery and Tumors, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz/Poland Background: There are two main types of lung cancer: non-small cell lung cancer (NSCLC) which represents 8085% cases of lung cancer and small cell lung cancer (SCLC) which is about 10-15% cases of lung cancer. The 5-year survival rate for patients with lung cancer vary depending on the stage of the cancer when it is diagnosed. Unfortunately, most of patients with lung cancer are diagnosed on later stage of disease (stage III and IV). In our research we try to find marker among miRNA that can predict occurring of lung cancer on the earlier stage.
S794
Methods: Isolation of miRNA from plasma was performed by miRCURY RNA Isolation Kit e Biofluids (Exiqon) from NSCLC patients and controls. Synthesis of cDNA and qPCR were carried out using miRCURY LNATM Universal RT microRNA PCR with LNATM enhanced PCR Primers (Exiqon). Statistical calculations were executed on 11 samples as a Pre-eliminary data. Results: Results are shown in the following table.
Conclusion: We assed level of 5 different miRNAs circulating in the blood of NSCLC patients using qPCR. Our initial results show that different miRNA can be used to stratify patients and miRNA. Expression of hsa-miR451a is decreasing in NSCLC versus negative control. Interestingly up-regulated hsa-miR-660-5p was recently described as a prognostic marker in breast cancer but our result preliminary results showed constant decrease in hsa-miR-660-5p expression in all patients’ groups vs controls. The examination on the bigger cohort of patients is necessary to receive a more statistically significant and conclusive data. Keywords: cell-free microRNA, biomarkers, miRNA expression profiling, NSCLC
P2.01-017 Circulating miRNAs in Lung Cancer Are Associated to Pro-Tumorigenic and Immunosuppressive Microenvironment Topic: Analysis of RNA Orazio Fortunato,1 Cristina Borzi,1 Giovanni Centonze,1 Massimo Milione,2 Davide Conte,1 Mattia Boeri,1 Carla Verri,1 Linda Calzolari,1 Francesca Andriani,1 Luca Roz,1 Veronica Huber,3 Agata Cova,3 Chiara Camisaschi,3 Chiara Castelli,3 Licia Rivoltini,3 Claudio Tripodo,4 Ugo Pastorino,5 Gabriella Sozzi1 1Tumor Genomics Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milano/Italy, 2Anatomic Pathology Unit, Department of Pathology and Laboratory Medicine,
Journal of Thoracic Oncology
Vol. 12 No. 1S
Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milan/Italy, 3Unit of Immunotherapy of Human Tumors, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milano/Italy, 4Tumor Immunology Unit Department of Health Science, Human Pathology Section, University of Palermo School of Medicine, Palermo/Italy, 5Thoracic Surgery, Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Palermo/Italy Background: We previously reported the identification of diagnostic miRNA signatures in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. Circulating miRNAs are released into the bloodstream by different mechanisms such as passive leakage from damaged cells or active secretion through extracellular-vesicles or protein complexes. Methods: To evaluate the potential origin and the release of the 24 miRNAs of the diagnostic signature we analyzed their expression by real-time or digital PCR in both cells and conditioned medium (CM) from cancer cell and different cell types of the lung microenvironment. Lung tissues and cell-blocks were analyzed by miRNAs in situ hybridization (ISH). Modulation of miRNAs after in vitro treatments known to induce changes associated with cancer progression, in different cell types was assessed and correlated to changes observed in circulating miRNAs signatures. Results: 24-miRNAs analysis showed higher abundance in specific cellular components such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells or mir-451 and 142-3p in hematopoietic cells. Generally, tumor cells showed lower levels of miRNAs compared to bronchial epithelial cells. MiRNAs specific localization in lung tissue was confirmed by ISH. We observed that mir-451 is specifically expressed in lung interstitial alveolar walls while mir-126 by endothelial cells outside the tumor bulk; miR-145 is characteristic of fibroblast and muscle cells and miR-142-3p of hematopoietic cells, fibroblast and muscle whereas mir-21 is over-expressed in the tumor. The analysis of miRNAs in CM showed that miRNAs secretion is correlated with cellular expression for most cell types (Pearson correlation range: 0.41-0.80). Interestingly, platelets and granulocytes were the components that mostly secreted miRNAs. In vitro experiments showed that endothelial cells under hypoxic condition up-regulate mir-126 and that mir-145 was up-regulated and secreted in lung cancer-associated compared to normal fibroblasts. Interestingly, during conversion of T lymphocytes into T regulatory cells up-regulation of mir-15b, mir-19b and mir-320 was observed whereas