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573 Sonic Hedgehog Pathway Activation Reveals Hexokinase-2-mediated Aerobic Glycolysis as a Novel Target for Medulloblastoma Therapy
Poster Session – New Molecular Targets 570 POSTER MiR-124 Targets Androgen Receptor and Inhibits Tumorigenesis of Prostate Cancer Cells R. de Vere White1 , X.B. Shi1 . 1 University of California, Comprehensive Cancer Center, Sacramento, USA Background: The human genome may encode >1000 miRNAs which negatively regulate approximately 60% protein-coding genes. Studies have shown miR-124 is significantly decreased in several cancer types and dysregulation of miR-124 may be involved in the pathogenesis of human cancer. Whether miR-124 contributes to the initiation and progression of prostate cancer (CaP) remains poorly understood. This study was meant to explore the role of miR-124 in CaP. Methods: Total RNA was isolated from fresh-frozen prostatic tissues or cell lines using TRIzol reagent. The miR-124 level was measured using qPCR and/or Northern blot analysis. Since the 3 UTR of androgen receptor (AR) mRNA contains a broadly conserved miR-124-binding site, Western blotting and luciferase assay were performed to validate miR-124-mediated regulation of AR. In addition, the effects of miR-124 on growth of CaP cells were evaluated by WST proliferation assay and tumorigenesis in nude mice. Results: Seven prostate cell lines (2 benign, 5 malignant) and 18 matched benign/malignant prostatic tissues were examined for their miR-124 levels. A reduced expression of miR-124 was seen in all malignant cell lines compared to that in benign cell lines, and in 15 of 18 (83%) CaP samples relative to benign matches. Northern blot analysis of seven cell lines and five matched prostate tissues exhibited similar alteration of miR-124. These data provide strong evidence that miR-124 is significantly reduced in CaP cell lines and in a majority of clinical CaP samples. To validate regulation of AR by miR-124, CaP cells were transiently transfected with synthetic miR124 mimic (miR-124m). We observed miR-124-mediated downregulation of the AR protein and its downsteam effectors PSA and miR-125b. To verify that the miR-124 binding site is responsible for regulation by miR124, the AR 3’ UTR was cloned into the pRIM Reporter-luciferase vector and then cotransfected with miR-124m into CaP cells. Transfection of miR124m resulted in 40% reduction of the luciferase activity. We also examined whether clinical CaP samples having low level of miR-124 overexpress the AR using IHC analysis, and found a reverse correlation between miR-124 and AR expression levels in 7 of 8 CaP samples. These data suggest that miR-124 targets the AR and down-regulation of miR-124 results in increased expression of the AR in CaP cells. We also tested and found that treatment with miR-124m induced significant inhibition of cell proliferation compared to treatment with miR-NC control. We also evaluated the effect of miR-124 on tumorigenesis of 22Rv1 CaP cells and observed that tumors derived from miR-124-transfected cells grew substantially slowly, when compared to miR-NC tumors. Conclusions: Loss of miR-124 expression may be a common event in CaP. Since miR-124 directly targets the AR, downregulation of miR-124 reduces negative regulation, which results in increased expression of the AR in clinical CaP tissue. Additionally, downregulation of miR-124 may contribute to pathogenesis of CaP. 571 POSTER Targeting the GLUT1 Transporter as a Novel Therapeutic Approach to Exploit the Dependency of Cancer Cells On the “Warburg Effect” D.A. Chan1 , P. Pearson2 , J.M. Vernier3 , S. Hershenson4 , J. Freddo5 , R. Tabibiazar6 , A.J. Giaccia7 , P.M. O’Connor8 . 1 UCSF, Department of Radiation Oncology, San Francisco California, USA; 2 Ruga Corporation, Preclinical Development, Palo Alto California, USA; 3 Ruga Corporation, Chemistry, Palo Alto California, USA; 4 Ruga Corporation, Pharmaceutics, Palo Alto California, USA; 5 Ruga Corporation, Clinical Development, Palo Alto California, USA; 6 Ruga Corporation, Palo Alto California, USA; 7 Stanford University School of Medicine, Department of Radiation Oncology, Stanford California, USA; 8 Ruga Corporation, Research & Development, Palo Alto California, USA Tumor metabolism is undergoing a renaissance driven in large-part by identification of ‘genetic drivers’ (e.g., VHL, PTEN, RAS or PI3K) that reprogram tumors to become dependent on glycolysis for growth and survival. Within the repertoire of metabolic changes induced during tumor evolution is the selective up-regulation of a highly facilitative glucose transporter, GLUT1 that feeds the insatiable appetite tumors have for glucose. The dependency of tumors on glucose supplied through a GLUT1-dependent pipeline provides a potential Achilles heel for therapeutic intervention. To identify novel agents capable of selectively killing GLUT1-positive cancer cells we performed a synthetic lethal screen comprising of an isogenic pair of kidney cancer cell lines differing in VHL tumor suppressor status. Interrogation with a 130,000 chemical diversity set derived several lead molecules including STF-31 that selectively killed VHL-deficient, GLUT1 positive cells (Chan et al., Sci Transl. Med., 2011). Expanding beyond these earlier studies we demonstrate the exquisite in vitro and in vivo sensitivity of
Friday 9 November 2012 175 a panel of ovarian cancer cell lines to GLUT1 inhibition including the finding of equivalent activity in cisplatin-sensitive and resistant cell backgrounds (A2780 IC50 = 3.0 nM, A2780/CP IC50 = 3.3 nM). Furthermore, in vivo antitumor studies in orthotopic ovarian cancer models revealed a dosedependent inhibition of tumor growth at well-tolerated doses. Furthermore, efficacy correlated with a drug-induced reduction in fluorodeoxyglucoseuptake by PET imaging compared to vehicle controls. These single-agent and on-going drug-combination studies will be presented and provides a data platform from which to pursue clinical studies targeting patients with GLUT1-dependent tumors. Giaccia, A.J. and O’Connor, P.M. are Joint Senior Authors. 572 POSTER Integrated Genomic Analysis Identifies Candidate Genes in Luminal B Breast Cancer L. Addou-Klouche1 , S. Moulessehoul2 , D. Birnbaum3 , M. Chaffanet3 . 1 Institut Paoli-almettes, Molecular Oncology, Marseille, France; 2 ` Algeria; 3 Institut Paoli-Calmettes, UDL-Sba, Biology, Sidi-Bel-Abbes, Molecular Oncology, Marseille, France Background: Breast cancer is a complex and heterogeneous disease. Characterization of genomic alterations such as amplification of oncogenes and loss of tumor suppressor genes (TSG) combined with expression data could identify candidate genes. We focused on luminal B breast cancer molecular subtype whose clinical course is particularly pejorative and for which no targeted therapy exists. Material and Methods: High-Throughput molecular analysis such comparative genomic hybridization on microarrays (aCGH) was used to characterize the regions targeted by chromosomal alterations in breast cancers. In addition, an integrated analysis of genomic and expression profiling from DNA microarrays has contributed to the identification of candidate genes. Results: We demonstrate that the candidate gene L3MBTL4 is targeted by multiple genomic alterations, suggesting its involvement as a potential TSG in luminal B breast cancer molecular subtype. Furthermore, our comparative analyses of integrated profiles of breast cancers identified specific luminal B molecular subtype candidate genes. Conclusion: High-Throughput molecular analysis of breast cancer has already revealed some part of their potential. Such integrated approaches could contribute to better understand the various levels of the molecular changes in the mammary oncogenesis and identify new markers. 573 POSTER Sonic Hedgehog Pathway Activation Reveals Hexokinase-2-mediated Aerobic Glycolysis as a Novel Target for Medulloblastoma Therapy T. Gershon1 , A. Crowther1 , A. Tikunov2 , I. Garcia1 , H. Yuan3 , J. Macdonald2 , M. Deshmukh4 . 1 UNC-Chapel Hill, Neurology, Chapel Hill NC, USA; 2 UNC-Chapel Hill, Biomedical Engineering, Chapel Hill NC, USA; 3 UNC-Chapel Hill, Radiology, Chapel Hill NC, USA; 4 UNC-Chapel Hill, Cell and Developmental Biology, Chapel Hill NC, USA Medulloblastoma is the most common malignant brain tumor in children. Conventional therapy for medulloblastoma fails to cure many patients and leaves many survivors with long-term neuro-cognitive injury. For children with recurrent medulloblastoma, there is no curative therapy. Targeted therapies offer the possibility of reducing treatment toxicity while improving efficacy. The molecular heterogeneity of medulloblastoma, however, may complicate the selection of appropriate targets. To define molecular targets for therapy that may be valid across medulloblastoma subgroups, we probed for distinguishing metabolic features common to medulloblastoma and cerebellar granule neuron progenitors (CGNPs), putative cells of origin from which medulloblastomas arise. We performed an integrated analysis of metabolism and gene expression in CGNPs with and without Sonic Hedgehog (Shh), their endogenous mitogen. Because our analysis highlighted Hexokinase-2 (Hk2) as key metabolic regulator induced by Shh, we studied the effect of conditional genetic Hk2 deletion in CGNP development. We then we crossed Hk2 conditional knockout mice with transgenic SmoM2 mice that develop spontaneous medulloblastoma and determined changes in SmoM2-driven tumorigenesis. We found Shh induced aerobic glycolysis by driving Hk2 expression in CGNPs, both in vitro and in vivo. Hk2 deletion abrogated Shhinduced glycolysis and altered the strict regulation of CGNP differentiation in development. Medulloblastomas retained Hk2 expression typical of CGNPs. While all SmoM2 mice developed medulloblastoma, deletion of Hk2 markedly reduced tumor malignancy and increased survival. While 100% of SmoM2 mice died of medulloblastoma by P20, SmoM2 mice with Hk2 deletion survived a median of 31 days and 30% were longterm survivors. Comparison of pathology of medulloblastoma with and without intact Hk2 demonstrated that Hk2 deletion reduced tumor growth specifically by promoting differentiation.
176 Friday 9 November 2012 Our results demonstrate that aerobic glycolysis is an essential feature of medulloblastoma and reveal a novel link between cellular metabolism and progenitor state. Importantly, we found that aerobic glycolysis is primarily a neuro-developmental program, regulated by Shh, that is coopted to promote neoplastic growth as neural progenitors give rise to medulloblastoma. In Hk2-deleted tumors where aerobic glycolysis was prevented, the consequence was increased differentiation and reduced tumor growth. These findings connect developmental signaling pathways with patterns of metabolism in cancer, while also demonstrating the potential efficacy of metabolic therapy for medulloblastoma through targeting of Hk2. 574 POSTER FGFR1, a Significant Prognostic Factor in Muscle Invasive Bladder Cancer S. Rha1 , S. Lim1 , M. Koh2 , H. Jeong2 , H. Kim1 , S. Lee1 , J. Ahn1 . 1 Yonsei Cancer Center Yonsei University Medical College, Medical Oncology Internal Medicine, Seoul, Korea; 2 Yonsei University Medical College, Pathology, Seoul, Korea Background: The aim of this study is to investigate the value of receptor tyrosine kinase (RTK) expression as prognostic markers and therapeutic targets in muscle invasive urothelial cancer (UC) patients. Among the various methods, we chose immunohistochemical (IHC) stain, one of the most practical and relevant tools frequently used in routine practice. Methods: We retrospectively analyzed the data of 98 patients with muscle invasive UC who underwent radical cystectomy between 2005 and 2010 in Yonsei Cancer Center. Using formalin fixed paraffin embedded tissues of primary tumors, IHC was done for HER2, FGFR1, and FGFR3. Tumors with grade 2 and 3 staining were considered positive for HER2. For FGFR1 or 3, any immunoreactivity against antibody was considered positive. We performed univariate and multivariate analysis to find out the prognostic impact of each molecule and the interaction between them. Results: With a median follow-up duration of 26.6 months (range 1−84 months), the median recurrence free survival (RFS) was 17.7 months (95% CI: 11.8~23.6), and the median overall survival (OS) was 37.3 months (95% CI: 23.7–50.9). There were 41 (41.8%), 44 (44.9%), and 14 (14.2%) patients who have over-expressed HER2, FGFR1, and FGFR3, respectively. In univariate analysis, patients with over-expression of any RTKs showed shorter median RFS (15.8 (95% CI: 10.4–21.2) months vs. not reached; p = 0.008) and OS (26.5 (95% CI: 17.5–35.5) months vs. not reached; p = 0.011) than without any expression of RTKs. When we look into the prognostic value of each RTK, it showed a significantly shorter median RFS (12.9 vs. 37.1 months, p < 0.01) and OS (22.3 months vs. not reached, p = 0.01) in patients with FGFR1 expression. However, there was no significant difference in RFS or OS according to the HER2 and FGFR3 expression status. FGFR1 remained as a significant prognostic factor for OS with hazard ratio of 2.23 (95% CI: 1.27–3.90, p < 0.01). Furthermore, HER2 positivity tended to have adverse effect on survival in FGFR1 negative group. Conclusion: Our result indicates that FGFR1 expression, but not FGFR3 is associated with disease recurrence and poorer overall survival in muscle invasive UC patients after radical cystectomy. It also showed the possibility of the interaction between HER2 and FGFR1 with relation to the prognosis. Given these results based on IHC assay, FGFR1 might be feasible for prognosis prediction and a potential therapeutic target through thorough validation in muscle invasive bladder cancer. 575 POSTER RAIDs: Rational Molecular Assessments and Innovative Drug Selection, an EU Funded Project C. Kurzeder1 , S. Scholl2 , M. Kamal2 , E. Banu3 , G. Kenter4 , A. Mustea5 , C. Ngo6 , M. Popovic7 . 1 Kliniken Essen-Mitte, Gynaekologische Onkologie, ´ Essen, Germany; 2 Institut Curie, Departement d’Oncologie, Paris, France; 3 Saint Constantin Private Hospital, Medical Oncology, Brasov, Romania; 4 University of Amsterdam, Gynaecological Oncology, Amsterdam, The Netherlands; 5 University of Greifswald, Gynaecology, Greifswald, Germany; 6 Institut Curie, Department of Surgical Oncology, Paris, France; 7 Institute of oncology of Vojvodina, Department of Gynaecologic Oncology, Vojvodina, Serbia Background: Cervical cancer (CC) is the second most common malignancy in women worldwide. Although CC is a single diagnostic entity and infection of high-risk HPV is recognized as an important initiating event in tumor genesis, CC exhibits differences in clinical behavior. Stratification of CC into subclasses for progression and response to treatment remains to be defined. At present, the dominant targets under scrutiny for innovative CC treatments are the EGFR/PI3K pathway, proliferation/DNA checkpoint, angiogenesis inhibition, and anti-HPV vaccines. There have been no high
Poster Session – New Molecular Targets resolution genetic investigations such as whole genome/exome sequencing or protein profiling. We are lacking prognostic and predictive biomarkers in CC and there is a growing need for the development of biomarkers to follow up the course of the disease. Material and Methods: ‘RAIDs’ is a multidisciplinary co-operation between academic clinical centers, SMEs and translational research platforms. It will combine New Generation Sequencing (NGN) and Reverse Phase Protein array (RPPA) in a large patient population prior to standard therapy. ‘RAIDs-0’ is a cognitive cohort study intended to define patient stratification for targeted therapies. Subsequently targeted clinical trials are planned using an HPV directed vaccine trial or direct viral targeting in association with standard therapy. In addition, high throughput screening techniques will be performed in CC cell lines to identify new molecules of relevance for CC or CC microenvironment targeting. These molecules will be validated in preclinical mouse models. Results: RAIDs will start in October 2012 in 7 European countries. It aims to define a set of stratification criteria based on molecular profiling. Its results should give insight into dominant genomic and protein signaling pathway alterations, enabling the identification of prognostic and predictive biomarkers for standard or targeted therapy in CC. Immunological data from trials involving vaccine or direct viral targeting will provide information on immune rejection or tolerance of this virally transmitted disease. Conclusions: The RAIDs consortium aims to provide a safer and more efficient therapy for the individual patient. This can be achieved through: a) the acquisition of molecular data for better treatment decisions; b) targeted pilot trials directed at specific alterations; c) the continuous evaluation of standards of care by comparison of care given in multiple centers in Europe. RAIDs should raise awareness in countries with lesser screening practices and improve the quality of life for women with cancer. 576 POSTER GLUT-1, CAIX and HKII Expression in Localized Cervical Carcinoma Patients Treated by Exclusive Radiotherapy and Concomitant Chemoradiotherapy P. Moreno-Acosta1 , S. Schyrly Carrillo1 , O. Oscar Gamboa1 , J. Jineth Acosta2 , J. Joseph Balart Serra3 , N. Nicolas Magne4 , A. Alfredo Romero-Rojas5 . 1 Instituto Nacional de Cancerologia, Bogota´ D.C., Colombia; 2 Universidad Nacional de Colombia, Bogota´ D.C., Colombia; 3 ´ Translacional (IDIBELL) Instituto Catalan ´ de Laboratorio de Investigacion Oncolog´ıa, Barcelona., Spain; 4 5Department of Radiotherapy Institut de ´ ´ Cancerologie de la Loire, Jarez, France; 5 Grupo de Patolog´ıa Oncologica Instituto Nacional de Cancerolog´ıa, Bogota D.C., Bogota D.C., Colombia Objective: The aim of the present study was to determine and describe the proteins expression, CAIX, GLUT-1, HKII, and evaluate a possible association of expression of these with tumor response to exclusive radiotherapy and concomitant chemoradiotherapy in squamous cell carcinomas of the uterine cervix. Methods: This retrospective cohort study included 66 patients in FIGO stages IIB and IIIB for the period from 2001 to 2007. The men age of patients was 47 years (ranging from 26 to 72) years). 22 patients were treated with exclusive radiotherapy and 44 with concomitant chemoradiotherapy. The protein expression of CAIX, GLUT-1 and HKII was evaluated and determined by immunohistochemistry in biopsies taken before treatment. Results: We found a greater increase in the expression of GLUT 1 (74%), followed by CAIX (41%) and HKII (18%). The simultaneous expression or co-expression of GLUT1 and CAIX was found to be significant (p <0.002) compared with GLUT 1 and HKII. By comparing the expression with mediate response to treatment (responses evaluated after three months of terminate treatment) in nonresponders to treatment, we observed a trend of risk of 1.4 times when expressed simultaneously the three proteins. In anemic patients (Hgb <11 g/dl, anemic hypoxia) was observed a trend of risk of 4.3 times of treatment failure. Although no significant association was found between protein expression and survival analysis, we found that overall and free survival rates of disease decreased by 20% when coexpressed GLUT 1 and HKII, and approximately up to 80% when expressed GLUT-1, CAIX and HKII. Conclusions: The increased expression of GLUT 1 on CAIX and HKII reaffirms the concept that tumors of cervical cancer have a high consumption of glucose, a process associated with high energy requirements demanded by cells to maintain the tumor phenotype. Detecting the expression of GLUT 1 can contribute to make clear how the mechanisms of hypoxia lead to invasion. The detection and study before treatment of the expression of CAIX, GLUT 1 and HKII in squamous cell carcinomas of the uterine cervix, considered as biological factors pre-existing, contributes to infer the metabolic and hypoxic state, as also at the rational use of new modalities in radiotherapy, chemotherapy, as bioreductiva chemotherapy and gene therapy in the regulation of hypoxia.
Friday 9 November 2012 175 a panel of ovarian cancer cell lines to GLUT1 inhibition including the finding of equivalent activity in cisplatin-sensitive and resistant cell backgrounds (A2780 IC50 = 3.0 nM, A2780/CP IC50 = 3.3 nM). Furthermore, in vivo antitumor studies in orthotopic ovarian cancer models revealed a dosedependent inhibition of tumor growth at well-tolerated doses. Furthermore, efficacy correlated with a drug-induced reduction in fluorodeoxyglucoseuptake by PET imaging compared to vehicle controls. These single-agent and on-going drug-combination studies will be presented and provides a data platform from which to pursue clinical studies targeting patients with GLUT1-dependent tumors. Giaccia, A.J. and O’Connor, P.M. are Joint Senior Authors. 572 POSTER Integrated Genomic Analysis Identifies Candidate Genes in Luminal B Breast Cancer L. Addou-Klouche1 , S. Moulessehoul2 , D. Birnbaum3 , M. Chaffanet3 . 1 Institut Paoli-almettes, Molecular Oncology, Marseille, France; 2 ` Algeria; 3 Institut Paoli-Calmettes, UDL-Sba, Biology, Sidi-Bel-Abbes, Molecular Oncology, Marseille, France Background: Breast cancer is a complex and heterogeneous disease. Characterization of genomic alterations such as amplification of oncogenes and loss of tumor suppressor genes (TSG) combined with expression data could identify candidate genes. We focused on luminal B breast cancer molecular subtype whose clinical course is particularly pejorative and for which no targeted therapy exists. Material and Methods: High-Throughput molecular analysis such comparative genomic hybridization on microarrays (aCGH) was used to characterize the regions targeted by chromosomal alterations in breast cancers. In addition, an integrated analysis of genomic and expression profiling from DNA microarrays has contributed to the identification of candidate genes. Results: We demonstrate that the candidate gene L3MBTL4 is targeted by multiple genomic alterations, suggesting its involvement as a potential TSG in luminal B breast cancer molecular subtype. Furthermore, our comparative analyses of integrated profiles of breast cancers identified specific luminal B molecular subtype candidate genes. Conclusion: High-Throughput molecular analysis of breast cancer has already revealed some part of their potential. Such integrated approaches could contribute to better understand the various levels of the molecular changes in the mammary oncogenesis and identify new markers. 573 POSTER Sonic Hedgehog Pathway Activation Reveals Hexokinase-2-mediated Aerobic Glycolysis as a Novel Target for Medulloblastoma Therapy T. Gershon1 , A. Crowther1 , A. Tikunov2 , I. Garcia1 , H. Yuan3 , J. Macdonald2 , M. Deshmukh4 . 1 UNC-Chapel Hill, Neurology, Chapel Hill NC, USA; 2 UNC-Chapel Hill, Biomedical Engineering, Chapel Hill NC, USA; 3 UNC-Chapel Hill, Radiology, Chapel Hill NC, USA; 4 UNC-Chapel Hill, Cell and Developmental Biology, Chapel Hill NC, USA Medulloblastoma is the most common malignant brain tumor in children. Conventional therapy for medulloblastoma fails to cure many patients and leaves many survivors with long-term neuro-cognitive injury. For children with recurrent medulloblastoma, there is no curative therapy. Targeted therapies offer the possibility of reducing treatment toxicity while improving efficacy. The molecular heterogeneity of medulloblastoma, however, may complicate the selection of appropriate targets. To define molecular targets for therapy that may be valid across medulloblastoma subgroups, we probed for distinguishing metabolic features common to medulloblastoma and cerebellar granule neuron progenitors (CGNPs), putative cells of origin from which medulloblastomas arise. We performed an integrated analysis of metabolism and gene expression in CGNPs with and without Sonic Hedgehog (Shh), their endogenous mitogen. Because our analysis highlighted Hexokinase-2 (Hk2) as key metabolic regulator induced by Shh, we studied the effect of conditional genetic Hk2 deletion in CGNP development. We then we crossed Hk2 conditional knockout mice with transgenic SmoM2 mice that develop spontaneous medulloblastoma and determined changes in SmoM2-driven tumorigenesis. We found Shh induced aerobic glycolysis by driving Hk2 expression in CGNPs, both in vitro and in vivo. Hk2 deletion abrogated Shhinduced glycolysis and altered the strict regulation of CGNP differentiation in development. Medulloblastomas retained Hk2 expression typical of CGNPs. While all SmoM2 mice developed medulloblastoma, deletion of Hk2 markedly reduced tumor malignancy and increased survival. While 100% of SmoM2 mice died of medulloblastoma by P20, SmoM2 mice with Hk2 deletion survived a median of 31 days and 30% were longterm survivors. Comparison of pathology of medulloblastoma with and without intact Hk2 demonstrated that Hk2 deletion reduced tumor growth specifically by promoting differentiation.
176 Friday 9 November 2012 Our results demonstrate that aerobic glycolysis is an essential feature of medulloblastoma and reveal a novel link between cellular metabolism and progenitor state. Importantly, we found that aerobic glycolysis is primarily a neuro-developmental program, regulated by Shh, that is coopted to promote neoplastic growth as neural progenitors give rise to medulloblastoma. In Hk2-deleted tumors where aerobic glycolysis was prevented, the consequence was increased differentiation and reduced tumor growth. These findings connect developmental signaling pathways with patterns of metabolism in cancer, while also demonstrating the potential efficacy of metabolic therapy for medulloblastoma through targeting of Hk2. 574 POSTER FGFR1, a Significant Prognostic Factor in Muscle Invasive Bladder Cancer S. Rha1 , S. Lim1 , M. Koh2 , H. Jeong2 , H. Kim1 , S. Lee1 , J. Ahn1 . 1 Yonsei Cancer Center Yonsei University Medical College, Medical Oncology Internal Medicine, Seoul, Korea; 2 Yonsei University Medical College, Pathology, Seoul, Korea Background: The aim of this study is to investigate the value of receptor tyrosine kinase (RTK) expression as prognostic markers and therapeutic targets in muscle invasive urothelial cancer (UC) patients. Among the various methods, we chose immunohistochemical (IHC) stain, one of the most practical and relevant tools frequently used in routine practice. Methods: We retrospectively analyzed the data of 98 patients with muscle invasive UC who underwent radical cystectomy between 2005 and 2010 in Yonsei Cancer Center. Using formalin fixed paraffin embedded tissues of primary tumors, IHC was done for HER2, FGFR1, and FGFR3. Tumors with grade 2 and 3 staining were considered positive for HER2. For FGFR1 or 3, any immunoreactivity against antibody was considered positive. We performed univariate and multivariate analysis to find out the prognostic impact of each molecule and the interaction between them. Results: With a median follow-up duration of 26.6 months (range 1−84 months), the median recurrence free survival (RFS) was 17.7 months (95% CI: 11.8~23.6), and the median overall survival (OS) was 37.3 months (95% CI: 23.7–50.9). There were 41 (41.8%), 44 (44.9%), and 14 (14.2%) patients who have over-expressed HER2, FGFR1, and FGFR3, respectively. In univariate analysis, patients with over-expression of any RTKs showed shorter median RFS (15.8 (95% CI: 10.4–21.2) months vs. not reached; p = 0.008) and OS (26.5 (95% CI: 17.5–35.5) months vs. not reached; p = 0.011) than without any expression of RTKs. When we look into the prognostic value of each RTK, it showed a significantly shorter median RFS (12.9 vs. 37.1 months, p < 0.01) and OS (22.3 months vs. not reached, p = 0.01) in patients with FGFR1 expression. However, there was no significant difference in RFS or OS according to the HER2 and FGFR3 expression status. FGFR1 remained as a significant prognostic factor for OS with hazard ratio of 2.23 (95% CI: 1.27–3.90, p < 0.01). Furthermore, HER2 positivity tended to have adverse effect on survival in FGFR1 negative group. Conclusion: Our result indicates that FGFR1 expression, but not FGFR3 is associated with disease recurrence and poorer overall survival in muscle invasive UC patients after radical cystectomy. It also showed the possibility of the interaction between HER2 and FGFR1 with relation to the prognosis. Given these results based on IHC assay, FGFR1 might be feasible for prognosis prediction and a potential therapeutic target through thorough validation in muscle invasive bladder cancer. 575 POSTER RAIDs: Rational Molecular Assessments and Innovative Drug Selection, an EU Funded Project C. Kurzeder1 , S. Scholl2 , M. Kamal2 , E. Banu3 , G. Kenter4 , A. Mustea5 , C. Ngo6 , M. Popovic7 . 1 Kliniken Essen-Mitte, Gynaekologische Onkologie, ´ Essen, Germany; 2 Institut Curie, Departement d’Oncologie, Paris, France; 3 Saint Constantin Private Hospital, Medical Oncology, Brasov, Romania; 4 University of Amsterdam, Gynaecological Oncology, Amsterdam, The Netherlands; 5 University of Greifswald, Gynaecology, Greifswald, Germany; 6 Institut Curie, Department of Surgical Oncology, Paris, France; 7 Institute of oncology of Vojvodina, Department of Gynaecologic Oncology, Vojvodina, Serbia Background: Cervical cancer (CC) is the second most common malignancy in women worldwide. Although CC is a single diagnostic entity and infection of high-risk HPV is recognized as an important initiating event in tumor genesis, CC exhibits differences in clinical behavior. Stratification of CC into subclasses for progression and response to treatment remains to be defined. At present, the dominant targets under scrutiny for innovative CC treatments are the EGFR/PI3K pathway, proliferation/DNA checkpoint, angiogenesis inhibition, and anti-HPV vaccines. There have been no high
Poster Session – New Molecular Targets resolution genetic investigations such as whole genome/exome sequencing or protein profiling. We are lacking prognostic and predictive biomarkers in CC and there is a growing need for the development of biomarkers to follow up the course of the disease. Material and Methods: ‘RAIDs’ is a multidisciplinary co-operation between academic clinical centers, SMEs and translational research platforms. It will combine New Generation Sequencing (NGN) and Reverse Phase Protein array (RPPA) in a large patient population prior to standard therapy. ‘RAIDs-0’ is a cognitive cohort study intended to define patient stratification for targeted therapies. Subsequently targeted clinical trials are planned using an HPV directed vaccine trial or direct viral targeting in association with standard therapy. In addition, high throughput screening techniques will be performed in CC cell lines to identify new molecules of relevance for CC or CC microenvironment targeting. These molecules will be validated in preclinical mouse models. Results: RAIDs will start in October 2012 in 7 European countries. It aims to define a set of stratification criteria based on molecular profiling. Its results should give insight into dominant genomic and protein signaling pathway alterations, enabling the identification of prognostic and predictive biomarkers for standard or targeted therapy in CC. Immunological data from trials involving vaccine or direct viral targeting will provide information on immune rejection or tolerance of this virally transmitted disease. Conclusions: The RAIDs consortium aims to provide a safer and more efficient therapy for the individual patient. This can be achieved through: a) the acquisition of molecular data for better treatment decisions; b) targeted pilot trials directed at specific alterations; c) the continuous evaluation of standards of care by comparison of care given in multiple centers in Europe. RAIDs should raise awareness in countries with lesser screening practices and improve the quality of life for women with cancer. 576 POSTER GLUT-1, CAIX and HKII Expression in Localized Cervical Carcinoma Patients Treated by Exclusive Radiotherapy and Concomitant Chemoradiotherapy P. Moreno-Acosta1 , S. Schyrly Carrillo1 , O. Oscar Gamboa1 , J. Jineth Acosta2 , J. Joseph Balart Serra3 , N. Nicolas Magne4 , A. Alfredo Romero-Rojas5 . 1 Instituto Nacional de Cancerologia, Bogota´ D.C., Colombia; 2 Universidad Nacional de Colombia, Bogota´ D.C., Colombia; 3 ´ Translacional (IDIBELL) Instituto Catalan ´ de Laboratorio de Investigacion Oncolog´ıa, Barcelona., Spain; 4 5Department of Radiotherapy Institut de ´ ´ Cancerologie de la Loire, Jarez, France; 5 Grupo de Patolog´ıa Oncologica Instituto Nacional de Cancerolog´ıa, Bogota D.C., Bogota D.C., Colombia Objective: The aim of the present study was to determine and describe the proteins expression, CAIX, GLUT-1, HKII, and evaluate a possible association of expression of these with tumor response to exclusive radiotherapy and concomitant chemoradiotherapy in squamous cell carcinomas of the uterine cervix. Methods: This retrospective cohort study included 66 patients in FIGO stages IIB and IIIB for the period from 2001 to 2007. The men age of patients was 47 years (ranging from 26 to 72) years). 22 patients were treated with exclusive radiotherapy and 44 with concomitant chemoradiotherapy. The protein expression of CAIX, GLUT-1 and HKII was evaluated and determined by immunohistochemistry in biopsies taken before treatment. Results: We found a greater increase in the expression of GLUT 1 (74%), followed by CAIX (41%) and HKII (18%). The simultaneous expression or co-expression of GLUT1 and CAIX was found to be significant (p <0.002) compared with GLUT 1 and HKII. By comparing the expression with mediate response to treatment (responses evaluated after three months of terminate treatment) in nonresponders to treatment, we observed a trend of risk of 1.4 times when expressed simultaneously the three proteins. In anemic patients (Hgb <11 g/dl, anemic hypoxia) was observed a trend of risk of 4.3 times of treatment failure. Although no significant association was found between protein expression and survival analysis, we found that overall and free survival rates of disease decreased by 20% when coexpressed GLUT 1 and HKII, and approximately up to 80% when expressed GLUT-1, CAIX and HKII. Conclusions: The increased expression of GLUT 1 on CAIX and HKII reaffirms the concept that tumors of cervical cancer have a high consumption of glucose, a process associated with high energy requirements demanded by cells to maintain the tumor phenotype. Detecting the expression of GLUT 1 can contribute to make clear how the mechanisms of hypoxia lead to invasion. The detection and study before treatment of the expression of CAIX, GLUT 1 and HKII in squamous cell carcinomas of the uterine cervix, considered as biological factors pre-existing, contributes to infer the metabolic and hypoxic state, as also at the rational use of new modalities in radiotherapy, chemotherapy, as bioreductiva chemotherapy and gene therapy in the regulation of hypoxia.