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P2.02-068 BRG1 and p53 Expression in Resected Stage I – III Non-Small Cell Lung Cancer
November 2017 association with global demethylation suggests that therapeutics targeting methylation such as decitabine may have different effects on LKB1 loss and LKB1 WT tumors, a hypothesis which we are currently testing. We are also continuing to study FOXA1/2/3 and KLF5 to determine the mechanism by which LKB1 regulates its demethylation program. Keywords: epigenetics, LKB1
P2.02-068 BRG1 and p53 Expression in Resected Stage I e III Non-Small Cell Lung Cancer A. Elegbede,1 M. Koebel,2 A. D’Silva,1 M. Dean,3 E. Enwere,3 R. Tudor,1 A. Gibson,1 H. Li,4 S. Otsuka,5 G. Bebb6 1Oncology, University of Calgary, Calgary/CA, 2Pathology, University of Calgary, Calgary/CA, 3Translational Research Lab. Tom Baker Cancer Centre University of Calgary, Calgary/CA, 4University of Calgary, Calgary/CA, 5 Medicine, University of Calgary, Calgary, AB/CA, 6Medicine, University of Calgary and Alberta Health Services, Calgary/CA Background: Evidence suggests a striking significance of BRG1 in nonsmall cell lung cancer (NSCLC) development and prognosis including its ability to modulate NSCLC response to commonly used chemotherapy. The human SW1/SNF (switching defective/ sucrose non-fermenting) family of chromatin remodeling complexes are composed of multisubunit proteins among which are the BRG1 and INI-1. Both BRG1 (SMARCA4) and INI-1 (SMARCB1, BAF47, SNF7) are implicated in different cancers. We aimed at identifying the frequency of the SW1/ SNF protein components loss and the significance in NSCLC. Given that inactivating BRG1 mutations could coexist with TP53 alterations in NSCLC cell lines, we also included the tumor suppressor protein p53 in our analysis. Method: We analyzed the expression of the SW1/SNF subunits (BRG1 and INI-1) and p53 proteins from resected stage I e III NSCLC using the immunohistochemistry. Mouse monoclonal anti-p53 (DO-7, Dako Omnis) and anti-INI-1 (MRQ-27, Cell Marque) antibodies were used for p53 and INI-1 protein detection respectively. BRG1 was detected using the rabbit monoclonal anti-BRG1 antibody (EPNCIR111A, Abcam). Clinical data were obtained from the Glans-Look lung cancer database and patients diagnosed of NSCLC from 2003 to 2006 were included in the study. Result: A total of 150 cases were identified, {Adenocarcinoma n ¼82 (54.7%), Squamous cell carcinoma n ¼ 50 (33.3%), Large cell carcinoma n ¼ 7 (4.7%) and others n ¼ 11 (7.3%: Adenosquamous, Bronchioalveolar and Giant cell carcinomas)}.The BRG1 protein was absent in 6% (9/150) of cases with the overall highest number (3.3%) seen in adenocarcinoma. Whereas normal INI-1 protein was expressed in all samples. Within NSCLC subtypes, the rate of BRG1 loss was highest in the large cell carcinomas at 28.1% (2/7) and lowest in squamous cell subtype at 2% (1/50) while only 6.1% (5/82) of adenocarcinoma showed BRG1 loss. The frequency of aberrant p53 protein expression (including overexpression, cytoplasmic and complete expression loss) was at 63% (95/150) and 6 (2 large cell and 4 adenocarcinoma) of the 95 abnormal p53 cases (6.3%) had BRG1 loss. Conclusion: BRG1 loss seems to be a low occurrence in NSCLC. Although a high rate of BRG1 inactivating mutations were previously reported in NSCLC cell lines, the present result suggests that these mutations may still result in the expression of possibly an abnormal BRG1. BRG1 loss appears to coexist with p53 abnormality in NSCLC. Additional multivariate and outcome analysis will be presented. Keywords: SW1/SNF complex, BRG1, p53, NSCLC
P2.02-069 Targeting Neuropilin-1 in NSCLC M. Barr,1 G. Pidgeon,2 S. Gray,1 K. Gately,1 E. Hams,3 P. Fallon,3 S. Cuffe,1 S. Finn,4 K. O’Byrne5 1Thoracic Oncology Research Group, St James’s Hospital & Trinity College Dublin, Dublin/IE, 2 Surgery, St James’s Hospital & Trinity College Dublin, Dublin/IE,
Abstracts
S2125
3 Medicine, Trinity College Dublin, Dublin/IE, 4Dept of Histopathology, St James’s Hospital & Trinity College Dublin, Dublin/IE, 5Translational Research Institute, Institute of Health and Biomedical Innovation, Brisbane, QLD/AU
Background: Neuropilin-1 (NP1) is expressed by a wide variety of human tumor cell lines and diverse human neoplasms, and is implicated in mediating the effects of VEGF on the proliferation, survival and migration of cancer cells. It is extensively expressed in tumor vasculature, where NP1 over-expression is associated with tumor progression and poor clinical outcome. In this study, we examined the effects of targeting NP1 in NSCLC both in vitro and in vivo. Method: A panel of NSCLC cell lines (H460, H647, A549 and SKMES) were screened for NP1 at the mRNA and protein levels by RT-PCR and Western blotting, respectively. Cellular expression and localization of NP1 was further examined by immunocytochemistry, while a panel of retrospective resected lung tumors and matched normal tissues were stained by immunohistochemistry. The effects of targeting NP1 on cell proliferation (BrdU ELISA), apoptosis (FACS, HCS) and downstream survival signaling pathways (Western Blot) were examined under normoxic and hypoxic (0.1% O2) cell growth using anti-NP1 neutralizing antibodies. Cell survival was assessed in response to treatment of NSCLC cells with a range of chemotherapeutic agents in combination with NP1 neutralizing antibodies. Using a human xenograft model, tumor growth studies were carried out in nude mice following subcutaneous injection of NP1 over-expressing cells relative to empty vector controls. Result: All lung cancer cell lines examined expressed NP1 with the exception of the H460 cell line. Immunocytochemistry analysis confirmed cellular expression and localization of this receptor, particularly in the leading edges of migrating cells, suggesting a possible role in cell migration. In a small cohort of resected NSCLC patients, tumor expression of NP1 was high relative to their matched normal lung tissues in adenocarcinoma, squamous cell and large cell neuroendocrine carcinomas. Cell proliferation and apoptosis were significantly altered in NSCLC cells expressing NP1. While hypoxia induced the expression of NP1, treatment of cells with NP1 neutralizing antibodies reduced hypoxia-mediated cell proliferation and decreased expression of PI3K and MAPK signaling pathways. In a preliminary study, treatment with NP1 neutralizing antibodies sensitized NSCLC cells to the cytotoxic effects of chemotherapy. In vivo, H460 cells overexpressing NP1 significantly increased tumor growth in NOD/SCID mice relative to empty vector controls. Conclusion: These data suggest a role for the Neuropilin-1 receptor in promoting cell survival and tumor growth in NSCLC and may offer potential as a therapeutic biological strategy in lung cancer. Keywords: Cell survival, NSCLC, Neuropilin
P2.02-070 C-MET as a Biomarker in Patients with Surgically Resected Non-Small Cell Lung Cancer G. Tsakonas,1 F.R. Hirsch,2 S. Ekman1 1Dept of Oncology-Pathology, Karolinska Institutet, Stockholm/SE, 2Other, Univ. of Colorado Cancer Center, Aurora, CO/US Background: c-MET protein overexpression has been proposed as potential prognostic as well as predictive biomarker for targeted therapy in non-small cell lung cancer (NSCLC), albeit its role in the clinical setting has not been firmly established yet and no clear cutoff value exists for immunohistochemistry (IHC) score. Method: We designed a retrospective cohort study, consisting of 725 patients with surgically removed non-small cell lung cancer. IHC was conducted in tissue microarrays (TMA) from lung tumors and healthy tissue adjacent to the tumor, using a specific antibody against human c-MET (MET PharmDx). IHC staining was quantified using H-scores
P2.02-068 BRG1 and p53 Expression in Resected Stage I e III Non-Small Cell Lung Cancer A. Elegbede,1 M. Koebel,2 A. D’Silva,1 M. Dean,3 E. Enwere,3 R. Tudor,1 A. Gibson,1 H. Li,4 S. Otsuka,5 G. Bebb6 1Oncology, University of Calgary, Calgary/CA, 2Pathology, University of Calgary, Calgary/CA, 3Translational Research Lab. Tom Baker Cancer Centre University of Calgary, Calgary/CA, 4University of Calgary, Calgary/CA, 5 Medicine, University of Calgary, Calgary, AB/CA, 6Medicine, University of Calgary and Alberta Health Services, Calgary/CA Background: Evidence suggests a striking significance of BRG1 in nonsmall cell lung cancer (NSCLC) development and prognosis including its ability to modulate NSCLC response to commonly used chemotherapy. The human SW1/SNF (switching defective/ sucrose non-fermenting) family of chromatin remodeling complexes are composed of multisubunit proteins among which are the BRG1 and INI-1. Both BRG1 (SMARCA4) and INI-1 (SMARCB1, BAF47, SNF7) are implicated in different cancers. We aimed at identifying the frequency of the SW1/ SNF protein components loss and the significance in NSCLC. Given that inactivating BRG1 mutations could coexist with TP53 alterations in NSCLC cell lines, we also included the tumor suppressor protein p53 in our analysis. Method: We analyzed the expression of the SW1/SNF subunits (BRG1 and INI-1) and p53 proteins from resected stage I e III NSCLC using the immunohistochemistry. Mouse monoclonal anti-p53 (DO-7, Dako Omnis) and anti-INI-1 (MRQ-27, Cell Marque) antibodies were used for p53 and INI-1 protein detection respectively. BRG1 was detected using the rabbit monoclonal anti-BRG1 antibody (EPNCIR111A, Abcam). Clinical data were obtained from the Glans-Look lung cancer database and patients diagnosed of NSCLC from 2003 to 2006 were included in the study. Result: A total of 150 cases were identified, {Adenocarcinoma n ¼82 (54.7%), Squamous cell carcinoma n ¼ 50 (33.3%), Large cell carcinoma n ¼ 7 (4.7%) and others n ¼ 11 (7.3%: Adenosquamous, Bronchioalveolar and Giant cell carcinomas)}.The BRG1 protein was absent in 6% (9/150) of cases with the overall highest number (3.3%) seen in adenocarcinoma. Whereas normal INI-1 protein was expressed in all samples. Within NSCLC subtypes, the rate of BRG1 loss was highest in the large cell carcinomas at 28.1% (2/7) and lowest in squamous cell subtype at 2% (1/50) while only 6.1% (5/82) of adenocarcinoma showed BRG1 loss. The frequency of aberrant p53 protein expression (including overexpression, cytoplasmic and complete expression loss) was at 63% (95/150) and 6 (2 large cell and 4 adenocarcinoma) of the 95 abnormal p53 cases (6.3%) had BRG1 loss. Conclusion: BRG1 loss seems to be a low occurrence in NSCLC. Although a high rate of BRG1 inactivating mutations were previously reported in NSCLC cell lines, the present result suggests that these mutations may still result in the expression of possibly an abnormal BRG1. BRG1 loss appears to coexist with p53 abnormality in NSCLC. Additional multivariate and outcome analysis will be presented. Keywords: SW1/SNF complex, BRG1, p53, NSCLC
P2.02-069 Targeting Neuropilin-1 in NSCLC M. Barr,1 G. Pidgeon,2 S. Gray,1 K. Gately,1 E. Hams,3 P. Fallon,3 S. Cuffe,1 S. Finn,4 K. O’Byrne5 1Thoracic Oncology Research Group, St James’s Hospital & Trinity College Dublin, Dublin/IE, 2 Surgery, St James’s Hospital & Trinity College Dublin, Dublin/IE,
Abstracts
S2125
3 Medicine, Trinity College Dublin, Dublin/IE, 4Dept of Histopathology, St James’s Hospital & Trinity College Dublin, Dublin/IE, 5Translational Research Institute, Institute of Health and Biomedical Innovation, Brisbane, QLD/AU
Background: Neuropilin-1 (NP1) is expressed by a wide variety of human tumor cell lines and diverse human neoplasms, and is implicated in mediating the effects of VEGF on the proliferation, survival and migration of cancer cells. It is extensively expressed in tumor vasculature, where NP1 over-expression is associated with tumor progression and poor clinical outcome. In this study, we examined the effects of targeting NP1 in NSCLC both in vitro and in vivo. Method: A panel of NSCLC cell lines (H460, H647, A549 and SKMES) were screened for NP1 at the mRNA and protein levels by RT-PCR and Western blotting, respectively. Cellular expression and localization of NP1 was further examined by immunocytochemistry, while a panel of retrospective resected lung tumors and matched normal tissues were stained by immunohistochemistry. The effects of targeting NP1 on cell proliferation (BrdU ELISA), apoptosis (FACS, HCS) and downstream survival signaling pathways (Western Blot) were examined under normoxic and hypoxic (0.1% O2) cell growth using anti-NP1 neutralizing antibodies. Cell survival was assessed in response to treatment of NSCLC cells with a range of chemotherapeutic agents in combination with NP1 neutralizing antibodies. Using a human xenograft model, tumor growth studies were carried out in nude mice following subcutaneous injection of NP1 over-expressing cells relative to empty vector controls. Result: All lung cancer cell lines examined expressed NP1 with the exception of the H460 cell line. Immunocytochemistry analysis confirmed cellular expression and localization of this receptor, particularly in the leading edges of migrating cells, suggesting a possible role in cell migration. In a small cohort of resected NSCLC patients, tumor expression of NP1 was high relative to their matched normal lung tissues in adenocarcinoma, squamous cell and large cell neuroendocrine carcinomas. Cell proliferation and apoptosis were significantly altered in NSCLC cells expressing NP1. While hypoxia induced the expression of NP1, treatment of cells with NP1 neutralizing antibodies reduced hypoxia-mediated cell proliferation and decreased expression of PI3K and MAPK signaling pathways. In a preliminary study, treatment with NP1 neutralizing antibodies sensitized NSCLC cells to the cytotoxic effects of chemotherapy. In vivo, H460 cells overexpressing NP1 significantly increased tumor growth in NOD/SCID mice relative to empty vector controls. Conclusion: These data suggest a role for the Neuropilin-1 receptor in promoting cell survival and tumor growth in NSCLC and may offer potential as a therapeutic biological strategy in lung cancer. Keywords: Cell survival, NSCLC, Neuropilin
P2.02-070 C-MET as a Biomarker in Patients with Surgically Resected Non-Small Cell Lung Cancer G. Tsakonas,1 F.R. Hirsch,2 S. Ekman1 1Dept of Oncology-Pathology, Karolinska Institutet, Stockholm/SE, 2Other, Univ. of Colorado Cancer Center, Aurora, CO/US Background: c-MET protein overexpression has been proposed as potential prognostic as well as predictive biomarker for targeted therapy in non-small cell lung cancer (NSCLC), albeit its role in the clinical setting has not been firmly established yet and no clear cutoff value exists for immunohistochemistry (IHC) score. Method: We designed a retrospective cohort study, consisting of 725 patients with surgically removed non-small cell lung cancer. IHC was conducted in tissue microarrays (TMA) from lung tumors and healthy tissue adjacent to the tumor, using a specific antibody against human c-MET (MET PharmDx). IHC staining was quantified using H-scores