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899 A Three-gene Expression Signature Model for Risk Stratification of Patients With Neuroblastoma
S218
european journal of cancer 48, suppl. 5 (2012) S25–S288
899 A Three-gene Expression Signature Model for Risk Stratification of Patients With Neuroblastoma I. Garcia1 , G. Mayol1 , J. Rios2 , N.K.V. Cheung3 , A. Obertheur4 , M. Fischer4 , J.M. Maris5 , G.M. Brodeur5 , J. Mora1 , C. Lavarino1 . 1 Hospital Sant Joan de ´ Oncology, Barcelona, Spain, 2 IDIBAPS Hospital Clinic, Biostatistics, Deu, Barcelona, Spain, 3 Memorial Sloan Kettering Cancer Center, Pediatrics, New York, USA, 4 Children’s Hospital of Cologne, Pediatric Oncology, Cologne, Germany, 5 Children’s Hospital of Philadelphia, Oncology, Pennsylvania, USA Background: Neuroblastoma is an embryonal tumor with contrasting clinical courses. Despite elaborate stratification strategies, precise clinical risk assessment still remains a challenge. The purpose of this study was to develop a PCR-based predictor model to improve clinical risk assessment of neuroblastoma patients. Methods: The model was developed using real-time PCR gene expression data from 96 samples, and tested on separate expression data sets obtained from real-time PCR and microarray studies comprising 362 patients. Results: Based on our prior study of differentially expressed genes in favorable and unfavorable neuroblastoma subgroups, we identified three genes, CHD5, PAFAH1B1 and NME1, strongly associated with patient outcome. The expression pattern of these genes was used to develop a PCRbased single score predictor model. The model discriminated patients into two groups with significantly different clinical outcome (Set 1 5-year overall survival [OS]: 0.93±0.03 vs 0.53±0.06, 5-year event free survival [EFS]: 0.85±0.04 vs 0.042±0.06, both P < 0.001; Set 2 OS: 0.97±0.02 vs 0.61±0.1, P = 0.005, EFS: 0.91±0.8 vs 0.56±0.1, P < 0.001 and Set 3 OS: 0.99±0.01 vs 0.56±0.06, EFS: 0.96±0.02 vs 0.43±0.05, both P < 0.001). Multivariate analysis showed that the model was an independent marker for survival (P < 0.001, for all). In comparison with accepted risk stratification systems, the model robustly classified patients in the total cohort, and in different clinically relevant risk subgroups. Conclusion: We propose for the first time in neuroblastoma, a technically simple PCR-based predictor model that could help refine current risk stratification systems. 900 FoxG1 Confers Resistance to PI3K Inhibitors Through the Repression of FoxO Activity G. Folch Codera1 , M.M. Inda1 , J. Seoane1 . 1 Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain Glioblastoma (GBM), WHO grade IV glioma, is the most frequent and aggressive primary brain tumour of the adulthood and its treatment is nowadays dismal. The PI3K pathway is a key regulator of most cellular processes. Components of the PI3K pathway are frequently mutated in GBM leading to overactivation of the pathway. Several compounds targeting the PI3K pathway are currently being developed and clinically tested. Besides other actions, PI3K inhibitors restore FoxO function by preventing its phosphorylation. The FoxO subfamily of forkhead transcription factors are tumour suppressors that regulate a wide variety of genes involved in key cellular processes such as glucose metabolism, oxidative stress, cell cycle and apoptosis. The forkhead transcription factor FoxG1 plays a critical role in the brain development. Moreover, FoxG1 is the human homologue of the avian viral oncogene Qin. Here we have studied the role of FoxG1 in human glioma as an inhibitor of FoxO activity and the implications of this regulation in treatments with anti-PI3K agents. The immunohistochemical analysis of a tissue microarray of 90 gliomas showed that FoxG1 was expressed in 75% of human gliomas. Furthermore, FoxG1 expression confered poor prognosis in GBM. Using co-immunoprecipitation, we found that FoxG1 bound to FoxO. RT2 -PCR and WB analyses also showed that FoxG1 repressed the FoxOmediated induction of cytostatic and pro-apoptotic genes in GBM cells. Moreover, PI3K inhibitor NVP-BKM120 was found to induce expression of FoxO targets and cell cycle arrest in GBM cells. FoxG1 was found to prevent the PI3K inhibitor-mediated induction of FoxO targets and cell cycle arrest. Finally, in an orthotopic in vivo approach, FoxG1 knock down enhanced the anti-tumoural activity of PI3K inhibitor NVP-BKM120 in GBM. Hence, this study indicates that FoxG1 confers resistance to PI3K inhibitors through the repression of FoxO activity. 901 Nuclear Variant ErbB380kDa Activates Cyclin D1 Expression and Cell Proliferation − a Model for Prostate Cancer Progression? M. El Maassarani1 , G. Fromont2 , D. Fauvin3 , P. Seite4 . 1 University, CNRS FRE 3511, Poitiers, France, 2 University/CHU, Department of Pathology, Poitiers, France, 3 University/CNRS, Poitiers, France, 4 University, CNRS FRE3511, Poitiers, France We have recently described a nuclear variant of the ErbB3 receptor in the nuclei of the H358 lung adenocarcinoma cell line. In vitro, the nuclear
Sunday 8 − Tuesday 10 July 2012
ErbB380kDa protein functions as a transcriptional activator that binds to the Cyclin D1 promoter to activate Cyclin D1 protein expression and increase cell proliferation. ErbB3 has been suggested to play a role in prostate cancer progression, but the mechanisms involved remain unclear. Previous studies have demonstrated a link between ErbB3 activation and androgen receptor (AR) activity. Activation of ErbB3 increases AR transcriptional activity and cell proliferation, likely by suppression of the ErbB3-binding protein (Ebp1). Alternatively, AR activation has been shown to suppress ErbB3 expression in androgen dependant prostate cancer cell lines, leading to increase ErbB3 expression in case of androgen withdrawal. In order to validate our molecular model, we analyzed the expression and cellular localization of ErbB380kDa , Ebp1 and Cyclin D1 in prostate cancer cell lines as well as normal and tumoral prostate tissues. Taken together, our in vitro and in vivo data suggest that ErbB380kD a could be a good indicator of prostate cancer progression from hormone-sensitive to castration-resistant tumors and that the increased proliferation observed in the latter may be in part mediated through activation of Cyclin D1, triggered by nuclear ErbB3.
Sunday 8 − Tuesday 10 July 2012 Poster Session
Experimental / Molecular Therapeutics, Pharmacogenomics 902 BRAFV600E Inhibitor Resistance Mechanisms − Co-treatment With TRAIL and PI3K Inhibitors can Sensitise to Apoptosis Resistant Colon Cancer Cells A. Pintzas1*, E. Oikonomou1 , T. Joyce1 , V. Kosmidou1 , M. Koc2 , V. Sourkova2 , L. Andera2 . 1 Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Greece, 2 Laboratory of Cell Signaling and Apoptosis, Institute of Molecular Genetics, Czech Academy of Sciences, Czech Republic Introduction: Sporadic colorectal cancer is a major cause of death worldwide. 30−40% of colorectal tumours bear K-RAS and up to 15% a BRAF mutations. Notably, K-RAS and BRAF mutant neoplasms overexpress TRAIL receptors, which may sensitise these tumours to TRAIL induced cell death [1]. In colon cancer appearance of oncogenic alterations in BRAF or KRAS mutations, tend to co-exist with those in PIK3CA and mutated PI3K has been shown to interfere with the successful application of MEK inhibitors. Materials and Methods: A panel of colorectal cancer cell lines bearing endogenous BRAF mutations, as well as cell models with either ovrexpressed or silenced BRAFV600E were employed. Treatment in 2D or 3D cell cultures was with BRAF, PI3K, HSP90 inhibitors and rational combinations with TRAIL. Results and Discussion: When PLX4720, a selective BRAFV600E inhibitor, was used to treat colon tumours, resistance was recorded. In an attempt to discover rational therapeutic combinations to re-sensitize oncogene driven tumours to apoptosis, treatment of two genetically different BRAF(V600E) mutant colon cancer cell lines with PLX4720 conferred complete resistance to cell death. Even though p-MAPK/ ERK kinase (MEK) suppression was achieved, TRAIL, an apoptosis inducing agent, was used synergistically in order to achieve cell death by apoptosis in RKO(BRAFV600E/PIK3CAH1047) cells [2]. Pharmacological suppression of the PI3K pathway further enhances the synergistic effect between TRAIL and PLX4720 in RKO cells, indicating the presence of PIK3CA(MT) as the inhibitory factor. We have successfully utilised combinations of two chemically unrelated BRAF(V600E) inhibitors in combination with TRAIL in a BRAF(V600E) mutated background and provided insight for new anti-cancer strategies, where the activated PI3KCA mutant oncogene should be suppressed. We have further analysed the signature of oncogenic BRAFV600E in colon cancer cells and have revealed deregulated cancer cell pathways and functions such as altered expression of DNA repair complex and apoptotic pathway components [3]. These may contribute to oncogenic BRAF cross-talk with key apoptotic pathways and sensitisation/resistance to therapeutics. Further evidence for the potential impact of these findings in personalised therapeutic strategies will be presented. Conclusions: BRAFV600E bearing colon tumour cells can confer resistance to BRAF inhibitors. Co-treatment with PI3K inhibitors and TRAIL can sensitise these cells to apoptosis, thus providing an opportunity for potential personalised treatment protocols to be further tested in the clinic. Reference(s) [1] Oikonomou, E., Kosmidou, V., Katseli, A., Kothonidis, K., Mourtzoukou, D., Kontogeorgos, G., Andera, L., Zografos, G., and Pintzas, A. (2009). TRAIL Receptor Upregulation Correlates to KRAS/ BRAF Mutations in Human Colon Cancer Tumours and Respective Normal Tissue. Int. J. Cancer 125, 2127–2135.
european journal of cancer 48, suppl. 5 (2012) S25–S288
899 A Three-gene Expression Signature Model for Risk Stratification of Patients With Neuroblastoma I. Garcia1 , G. Mayol1 , J. Rios2 , N.K.V. Cheung3 , A. Obertheur4 , M. Fischer4 , J.M. Maris5 , G.M. Brodeur5 , J. Mora1 , C. Lavarino1 . 1 Hospital Sant Joan de ´ Oncology, Barcelona, Spain, 2 IDIBAPS Hospital Clinic, Biostatistics, Deu, Barcelona, Spain, 3 Memorial Sloan Kettering Cancer Center, Pediatrics, New York, USA, 4 Children’s Hospital of Cologne, Pediatric Oncology, Cologne, Germany, 5 Children’s Hospital of Philadelphia, Oncology, Pennsylvania, USA Background: Neuroblastoma is an embryonal tumor with contrasting clinical courses. Despite elaborate stratification strategies, precise clinical risk assessment still remains a challenge. The purpose of this study was to develop a PCR-based predictor model to improve clinical risk assessment of neuroblastoma patients. Methods: The model was developed using real-time PCR gene expression data from 96 samples, and tested on separate expression data sets obtained from real-time PCR and microarray studies comprising 362 patients. Results: Based on our prior study of differentially expressed genes in favorable and unfavorable neuroblastoma subgroups, we identified three genes, CHD5, PAFAH1B1 and NME1, strongly associated with patient outcome. The expression pattern of these genes was used to develop a PCRbased single score predictor model. The model discriminated patients into two groups with significantly different clinical outcome (Set 1 5-year overall survival [OS]: 0.93±0.03 vs 0.53±0.06, 5-year event free survival [EFS]: 0.85±0.04 vs 0.042±0.06, both P < 0.001; Set 2 OS: 0.97±0.02 vs 0.61±0.1, P = 0.005, EFS: 0.91±0.8 vs 0.56±0.1, P < 0.001 and Set 3 OS: 0.99±0.01 vs 0.56±0.06, EFS: 0.96±0.02 vs 0.43±0.05, both P < 0.001). Multivariate analysis showed that the model was an independent marker for survival (P < 0.001, for all). In comparison with accepted risk stratification systems, the model robustly classified patients in the total cohort, and in different clinically relevant risk subgroups. Conclusion: We propose for the first time in neuroblastoma, a technically simple PCR-based predictor model that could help refine current risk stratification systems. 900 FoxG1 Confers Resistance to PI3K Inhibitors Through the Repression of FoxO Activity G. Folch Codera1 , M.M. Inda1 , J. Seoane1 . 1 Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain Glioblastoma (GBM), WHO grade IV glioma, is the most frequent and aggressive primary brain tumour of the adulthood and its treatment is nowadays dismal. The PI3K pathway is a key regulator of most cellular processes. Components of the PI3K pathway are frequently mutated in GBM leading to overactivation of the pathway. Several compounds targeting the PI3K pathway are currently being developed and clinically tested. Besides other actions, PI3K inhibitors restore FoxO function by preventing its phosphorylation. The FoxO subfamily of forkhead transcription factors are tumour suppressors that regulate a wide variety of genes involved in key cellular processes such as glucose metabolism, oxidative stress, cell cycle and apoptosis. The forkhead transcription factor FoxG1 plays a critical role in the brain development. Moreover, FoxG1 is the human homologue of the avian viral oncogene Qin. Here we have studied the role of FoxG1 in human glioma as an inhibitor of FoxO activity and the implications of this regulation in treatments with anti-PI3K agents. The immunohistochemical analysis of a tissue microarray of 90 gliomas showed that FoxG1 was expressed in 75% of human gliomas. Furthermore, FoxG1 expression confered poor prognosis in GBM. Using co-immunoprecipitation, we found that FoxG1 bound to FoxO. RT2 -PCR and WB analyses also showed that FoxG1 repressed the FoxOmediated induction of cytostatic and pro-apoptotic genes in GBM cells. Moreover, PI3K inhibitor NVP-BKM120 was found to induce expression of FoxO targets and cell cycle arrest in GBM cells. FoxG1 was found to prevent the PI3K inhibitor-mediated induction of FoxO targets and cell cycle arrest. Finally, in an orthotopic in vivo approach, FoxG1 knock down enhanced the anti-tumoural activity of PI3K inhibitor NVP-BKM120 in GBM. Hence, this study indicates that FoxG1 confers resistance to PI3K inhibitors through the repression of FoxO activity. 901 Nuclear Variant ErbB380kDa Activates Cyclin D1 Expression and Cell Proliferation − a Model for Prostate Cancer Progression? M. El Maassarani1 , G. Fromont2 , D. Fauvin3 , P. Seite4 . 1 University, CNRS FRE 3511, Poitiers, France, 2 University/CHU, Department of Pathology, Poitiers, France, 3 University/CNRS, Poitiers, France, 4 University, CNRS FRE3511, Poitiers, France We have recently described a nuclear variant of the ErbB3 receptor in the nuclei of the H358 lung adenocarcinoma cell line. In vitro, the nuclear
Sunday 8 − Tuesday 10 July 2012
ErbB380kDa protein functions as a transcriptional activator that binds to the Cyclin D1 promoter to activate Cyclin D1 protein expression and increase cell proliferation. ErbB3 has been suggested to play a role in prostate cancer progression, but the mechanisms involved remain unclear. Previous studies have demonstrated a link between ErbB3 activation and androgen receptor (AR) activity. Activation of ErbB3 increases AR transcriptional activity and cell proliferation, likely by suppression of the ErbB3-binding protein (Ebp1). Alternatively, AR activation has been shown to suppress ErbB3 expression in androgen dependant prostate cancer cell lines, leading to increase ErbB3 expression in case of androgen withdrawal. In order to validate our molecular model, we analyzed the expression and cellular localization of ErbB380kDa , Ebp1 and Cyclin D1 in prostate cancer cell lines as well as normal and tumoral prostate tissues. Taken together, our in vitro and in vivo data suggest that ErbB380kD a could be a good indicator of prostate cancer progression from hormone-sensitive to castration-resistant tumors and that the increased proliferation observed in the latter may be in part mediated through activation of Cyclin D1, triggered by nuclear ErbB3.
Sunday 8 − Tuesday 10 July 2012 Poster Session
Experimental / Molecular Therapeutics, Pharmacogenomics 902 BRAFV600E Inhibitor Resistance Mechanisms − Co-treatment With TRAIL and PI3K Inhibitors can Sensitise to Apoptosis Resistant Colon Cancer Cells A. Pintzas1*, E. Oikonomou1 , T. Joyce1 , V. Kosmidou1 , M. Koc2 , V. Sourkova2 , L. Andera2 . 1 Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Greece, 2 Laboratory of Cell Signaling and Apoptosis, Institute of Molecular Genetics, Czech Academy of Sciences, Czech Republic Introduction: Sporadic colorectal cancer is a major cause of death worldwide. 30−40% of colorectal tumours bear K-RAS and up to 15% a BRAF mutations. Notably, K-RAS and BRAF mutant neoplasms overexpress TRAIL receptors, which may sensitise these tumours to TRAIL induced cell death [1]. In colon cancer appearance of oncogenic alterations in BRAF or KRAS mutations, tend to co-exist with those in PIK3CA and mutated PI3K has been shown to interfere with the successful application of MEK inhibitors. Materials and Methods: A panel of colorectal cancer cell lines bearing endogenous BRAF mutations, as well as cell models with either ovrexpressed or silenced BRAFV600E were employed. Treatment in 2D or 3D cell cultures was with BRAF, PI3K, HSP90 inhibitors and rational combinations with TRAIL. Results and Discussion: When PLX4720, a selective BRAFV600E inhibitor, was used to treat colon tumours, resistance was recorded. In an attempt to discover rational therapeutic combinations to re-sensitize oncogene driven tumours to apoptosis, treatment of two genetically different BRAF(V600E) mutant colon cancer cell lines with PLX4720 conferred complete resistance to cell death. Even though p-MAPK/ ERK kinase (MEK) suppression was achieved, TRAIL, an apoptosis inducing agent, was used synergistically in order to achieve cell death by apoptosis in RKO(BRAFV600E/PIK3CAH1047) cells [2]. Pharmacological suppression of the PI3K pathway further enhances the synergistic effect between TRAIL and PLX4720 in RKO cells, indicating the presence of PIK3CA(MT) as the inhibitory factor. We have successfully utilised combinations of two chemically unrelated BRAF(V600E) inhibitors in combination with TRAIL in a BRAF(V600E) mutated background and provided insight for new anti-cancer strategies, where the activated PI3KCA mutant oncogene should be suppressed. We have further analysed the signature of oncogenic BRAFV600E in colon cancer cells and have revealed deregulated cancer cell pathways and functions such as altered expression of DNA repair complex and apoptotic pathway components [3]. These may contribute to oncogenic BRAF cross-talk with key apoptotic pathways and sensitisation/resistance to therapeutics. Further evidence for the potential impact of these findings in personalised therapeutic strategies will be presented. Conclusions: BRAFV600E bearing colon tumour cells can confer resistance to BRAF inhibitors. Co-treatment with PI3K inhibitors and TRAIL can sensitise these cells to apoptosis, thus providing an opportunity for potential personalised treatment protocols to be further tested in the clinic. Reference(s) [1] Oikonomou, E., Kosmidou, V., Katseli, A., Kothonidis, K., Mourtzoukou, D., Kontogeorgos, G., Andera, L., Zografos, G., and Pintzas, A. (2009). TRAIL Receptor Upregulation Correlates to KRAS/ BRAF Mutations in Human Colon Cancer Tumours and Respective Normal Tissue. Int. J. Cancer 125, 2127–2135.