- Email: [email protected]
Expression patterns and prognostic value of Cyclin-dependent kinase 5 (Cdk5) in colorectal tumours
S204
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
been observed in the tumor growth kinetics between NSG, hu-NSG, and huNSG-SGM3, suggesting that the human immune cells are not inhibiting tumor growth in this model. Two PDX models and one cancer cell line with high PDL1 levels were used to test response to anti-PD1 therapy in hu-NSG-SGM3 mice. Human lymphocytes in Hu-NSG-SGM3 mice infiltrated the transplanted tumor microenvironment. High PD-1 expression by CD8+ T cells and Tregs were present within the breast cancer microenvironment suggesting anergy and immunosuppression in hu-NSG-SGM3 mice. Treatment with the antiPD-1 receptor antibody pembrolizumab (Keytruda) significantly reduced tumor growth and the PD-1 expression level on lymphocytes. Conclusions: Thus, PDX-bearing hu-NSG-SGM3 mice might serve as a new and improved platform for preclinical immuno-oncology efficacy studies. Conflict of interest: Other Substantive Relationships: The Jackson Laboratory is a not-for-profit biomedical research institution which is partially supported by the sale of mice and related services to the global biomedical research community. 881 Urine biomarker profiling contains structure and predicts prostate cancer hormone therapy relapse H. Curley1 , M. Yazbek-Hanna2 , R. Hurst2 , R. Kumar2 , I. Mills3 , J. Schalken4 , J. Clark2 , D. Brewer2 , C. Cooper2 . 1 UEA, School of Biology, Norwich, United Kingdom, 2 UEA, School of Medicine, Norwich, United Kingdom, 3 Centre for Molecular Medicine Norway, Prostate Cancer, Oslo, Norway, 4 Institute for Molecular Life Sciences Radboud UMC, Urology, Nijmegen, Netherlands Background: Prostate cancer (PCa) is the second most common cancer in men worldwide and the most common in the UK [1]. In 2012 an estimated 307,000 men died from prostate cancer worldwide. Although generally slowgrowing, a subset of cancers progress to fatal disease. At diagnosis, it is often unclear what the correct treatment pathway is and what the response to treatment will be. In this study we have explored the utility of RNA harvested from urine microvesicles with an aim to identify prognostic subtypes of PCa, and an expression signature to predict response to hormone therapy. Material and Methods: A preliminary study was conducted where urine samples were collected at the Norfolk and Norwich University Hospital, post-DRE. Microvesicular RNA was harvested from 0.8mM filtered urine supernatant [2] converted to cDNA and amplified (Nugen Ovation picoSL WTA kit). 50 gene probes were selected from a literature research for prostate cancer diagnostic and prognostic transcripts, and their expression levels analysed using NanoString nCounter technology [3] in 187 samples (of varying clinical stage). Latent Process Decomposition (LPD) [4] was used to interrogate the expression data and standard tools were used for survival analyses. A secondary study has also been performed where urine microvesicle samples were harvested as above from multiple centres and were sent to NanoString with an increased number of probes (167). 497 samples from NNUH were combined with 27 from Dublin, 133 from ICR and 112 from USA, as well as 95 cell (non-microvesicle) samples from NNUH. Data received underwent similar analyses. Results: LPD analysis of the 50-gene expression data identified 4 clusters that had associated clinical structure; clusters 1 and 2 predominantly consisted of advanced and high-risk samples, in contrast, clusters 3 and 4 had mixed clinical content but had higher proportions of intermediate risk and benign samples respectively. A model based on the signature of 4 RNA transcripts was found to be a significant predictor of relapse after hormone therapy (p = 0.002; Cox proportional hazards model, AUC of 0.801). Conclusions: This study demonstrates that RNA in urine microvesicles contains PCa specific transcripts (TMPRSS2/ERG), and biomarker information on PCa disease severity and response to PCa therapy. We are currently analysing the second NanoString project to utilise as validation for the results found in the first analysis. No conflict of interest. 882 Expression patterns and prognostic value of Cyclin-dependent kinase 5 (Cdk5) in colorectal tumours V. Ruiz de Porras1 , S. Cabrero-De las Heras1 , S. Bystrup1 , J.L. Subirats2 , E. Musulen ´ 2 , J.L. Manzano3 , V. Moreno4 , L. Layos3 , C. Buges ´ 3, E. Martinez-Balibrea1 . 1 Health Sciences Research Institute of the “Germans Trias i Pujol” Foundation, Resistance- Chemotherapy and Predictive Biomarkers group, Badalona- Barcelona, Spain, 2 University Hospital Germans Trias i Pujol, Pathology department, Badalona, Spain, 3 University Hospital Germans Trias i Pujol, Medical Oncology Service, Badalona, Spain, 4 Bellvitge Biomedical Research Institute, Colorectal cancer research group, L’Hospitalet de Llobregat, Spain Background: Cdk5 is an atypical cyclin-dependent kinase − because of its activation by binding to p35 and p39 proteins − indispensable for normal brain development and deregulated in neurodegenerative diseases such as Alzheimer’s. Given the great similarity between the cellular and molecular mechanisms orchestrating neuronal migration during development of the nervous system and cancer cell migration during metastasis, it is not surprising
that Cdk5 has been reported to play a role in the latter. Cdk5 is overexpressed in several tumours, and has been associated with a worse prognosis. However, very little is known about a possible implication of Cdk5 in colorectal cancer (CRC). Our aim was to investigate the expression patterns of Cdk5 and p35 in human CRC cell lines and tumour samples for further evaluation of its role as a prognostic marker. Material and Methods: Human HT29, LoVo, HCT116, DLD1, LS513, SW480, HCT15, Caco2, LS1745 and SW1427 CRC cell lines were used in the present study. Cdk5, p35 protein expression was analysed by western blot in cell lines and in 12 frozen stage IV colorectal adenocarcinomas and their corresponding normal tissues. The presence of Cdk5-p35 active complex was studied by immunoprecipitation and kinase activity assay using H1 as target of phosphorylation. Cdk5 and p35 immunostaining was analysed in a tissue microarray containing 35 stage IV CRC cases. Cdk5 mRNA expression was studied in 98-paired adjacent normal and tumour tissues from stage II MSS CRC patients and in 256 stage II and III MSS frozen colorectal tumours. Kaplan meyer curves and log rank test was used to study association of expression data with desease-free survival (DFS) and overall survival (OS) in stage II and III patients. Results: Both Cdk5 and p35 proteins were detected at high levels in 10 CRC cell lines and their functional interaction was confirmed by immunoprecipitation and in vitro kinase assays. We observed a time-dependent increase (from 24 to 96 h) of Cdk5 and p35 protein levels in cell culture. Cdk5 (p = 0.003) and p35 (p = 0.03) were overexpressed in stage IV colorectal adenocarcinomas as compared to their corresponding normal adjacent tissues. Both proteins were also detected by inmunohistochemistry in human colorectal tumours. Higher Cdk5 mRNA levels (p < 0.0001) were found in tumours as compared to the corresponding normal tissues. Interestingly, high Cdk5 tumour levels were significantly associated with a worse DFS and OS (HR = 4.15, p = 0.022 and HR = 8.25, p = 0.049). Conclusion: Cdk5 and p35 were significantly overexpressed in CRC at both, protein and mRNA levels. Overexpression of Cdk5 was associated with a worse prognostic in stage II, III (MSS), suggesting a role of this protein in CRC progression. Further experiments are on-going in order to elucidate the mechanisms by which Cdk5 exerts its tumour promoting functions. No conflict of interest. 883 UM Cure 2020 − A consortium of European experts in uveal melanoma to identify new therapies for patients with metastatic disease N. Dhomen1 , R. Marais1 , M.J. Jager2 , B.E. Snaar-Jagalska3 , S. Coupland4 , B. Romanowska-Dixon5 , M.C. Mione6 , A. Valente7 , B. Ryll8 , R. Ruijtenbeek9 , A. Prestat10 , E. Vinolo11 , S. Roman-Roman12 . 1 CRUK Manchester Institute, Molecular Oncology Group, Manchester, United Kingdom, 2 Leiden University Medical Centre, Laboratory of Ophthalmology, Leiden, Netherlands, 3 Leiden University, Institute of Biology Leiden, Leiden, Netherlands, 4 University of Liverpool, Liverpool Ocular Oncology Research Group, Liverpool, United Kingdom, 5 Jagiellonian University, Department of Ophthalmology and Ocular Oncology, Krakow, Poland, 6 Karlsruhe Institute of Technology, Institute of Toxicology and Genetics, Karlsruhe, Germany, 7 Champalimaud Foundation, Champalimaud Foundation, Lisbon, Portugal, 8 Melanoma Patient Network Europe, MPNE, Uppsala, Sweden, 9 PamGene International BV, Research & Development, ’s-Hertogenbosch, Netherlands, 10 PEP-Therapy, PEP-Therapy, Paris, France, 11 Seeding Science, Seeding Science, Paris, France, 12 Institut Curie, Translational Research Department, Paris, France Introduction: Uveal melanoma (UM) is a rare intraocular tumour with an incidence of 5 cases per million individuals per year. Up to a third of UM patients develop metastases, most often in the liver, and these are invariably fatal. Despite new discoveries in the genetic and molecular background of the primary tumour, little is known about the metastatic disease; moreover, there is no therapy to either prevent or treat UM metastases. UM Cure 2020 has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 667787, and started in January 2016. Materials and Methods: In UM Cure 2020, we will identify and validate at the preclinical level novel therapeutic approaches for the treatment of UM metastases. To achieve our goal, we have established a consortium comprising key European Centres involved in UM patient care, UM clinical, translational and basic research, a patient organisation and two biotech companies with expertise in biomarkers and early clinical development. An ambitious multidisciplinary approach is proposed to move from tissue characterisation to preclinical evaluation of single or combinations of drugs. This approach includes the characterisation of the genetic landscape of metastatic UM and its microenvironment, proteomic studies to address signalling pathway deregulation, and the establishment of novel relevant in vitro and in vivo preclinical models of the metastatic disease. We also aim to validate accurate surrogate endpoint biomarkers to evaluate therapies and detect metastases as early as possible. Underpinning this will be the UM Cure 2020 virtual biobank registry, linking biobanks in the 4 referral centres involved into a harmonised
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
been observed in the tumor growth kinetics between NSG, hu-NSG, and huNSG-SGM3, suggesting that the human immune cells are not inhibiting tumor growth in this model. Two PDX models and one cancer cell line with high PDL1 levels were used to test response to anti-PD1 therapy in hu-NSG-SGM3 mice. Human lymphocytes in Hu-NSG-SGM3 mice infiltrated the transplanted tumor microenvironment. High PD-1 expression by CD8+ T cells and Tregs were present within the breast cancer microenvironment suggesting anergy and immunosuppression in hu-NSG-SGM3 mice. Treatment with the antiPD-1 receptor antibody pembrolizumab (Keytruda) significantly reduced tumor growth and the PD-1 expression level on lymphocytes. Conclusions: Thus, PDX-bearing hu-NSG-SGM3 mice might serve as a new and improved platform for preclinical immuno-oncology efficacy studies. Conflict of interest: Other Substantive Relationships: The Jackson Laboratory is a not-for-profit biomedical research institution which is partially supported by the sale of mice and related services to the global biomedical research community. 881 Urine biomarker profiling contains structure and predicts prostate cancer hormone therapy relapse H. Curley1 , M. Yazbek-Hanna2 , R. Hurst2 , R. Kumar2 , I. Mills3 , J. Schalken4 , J. Clark2 , D. Brewer2 , C. Cooper2 . 1 UEA, School of Biology, Norwich, United Kingdom, 2 UEA, School of Medicine, Norwich, United Kingdom, 3 Centre for Molecular Medicine Norway, Prostate Cancer, Oslo, Norway, 4 Institute for Molecular Life Sciences Radboud UMC, Urology, Nijmegen, Netherlands Background: Prostate cancer (PCa) is the second most common cancer in men worldwide and the most common in the UK [1]. In 2012 an estimated 307,000 men died from prostate cancer worldwide. Although generally slowgrowing, a subset of cancers progress to fatal disease. At diagnosis, it is often unclear what the correct treatment pathway is and what the response to treatment will be. In this study we have explored the utility of RNA harvested from urine microvesicles with an aim to identify prognostic subtypes of PCa, and an expression signature to predict response to hormone therapy. Material and Methods: A preliminary study was conducted where urine samples were collected at the Norfolk and Norwich University Hospital, post-DRE. Microvesicular RNA was harvested from 0.8mM filtered urine supernatant [2] converted to cDNA and amplified (Nugen Ovation picoSL WTA kit). 50 gene probes were selected from a literature research for prostate cancer diagnostic and prognostic transcripts, and their expression levels analysed using NanoString nCounter technology [3] in 187 samples (of varying clinical stage). Latent Process Decomposition (LPD) [4] was used to interrogate the expression data and standard tools were used for survival analyses. A secondary study has also been performed where urine microvesicle samples were harvested as above from multiple centres and were sent to NanoString with an increased number of probes (167). 497 samples from NNUH were combined with 27 from Dublin, 133 from ICR and 112 from USA, as well as 95 cell (non-microvesicle) samples from NNUH. Data received underwent similar analyses. Results: LPD analysis of the 50-gene expression data identified 4 clusters that had associated clinical structure; clusters 1 and 2 predominantly consisted of advanced and high-risk samples, in contrast, clusters 3 and 4 had mixed clinical content but had higher proportions of intermediate risk and benign samples respectively. A model based on the signature of 4 RNA transcripts was found to be a significant predictor of relapse after hormone therapy (p = 0.002; Cox proportional hazards model, AUC of 0.801). Conclusions: This study demonstrates that RNA in urine microvesicles contains PCa specific transcripts (TMPRSS2/ERG), and biomarker information on PCa disease severity and response to PCa therapy. We are currently analysing the second NanoString project to utilise as validation for the results found in the first analysis. No conflict of interest. 882 Expression patterns and prognostic value of Cyclin-dependent kinase 5 (Cdk5) in colorectal tumours V. Ruiz de Porras1 , S. Cabrero-De las Heras1 , S. Bystrup1 , J.L. Subirats2 , E. Musulen ´ 2 , J.L. Manzano3 , V. Moreno4 , L. Layos3 , C. Buges ´ 3, E. Martinez-Balibrea1 . 1 Health Sciences Research Institute of the “Germans Trias i Pujol” Foundation, Resistance- Chemotherapy and Predictive Biomarkers group, Badalona- Barcelona, Spain, 2 University Hospital Germans Trias i Pujol, Pathology department, Badalona, Spain, 3 University Hospital Germans Trias i Pujol, Medical Oncology Service, Badalona, Spain, 4 Bellvitge Biomedical Research Institute, Colorectal cancer research group, L’Hospitalet de Llobregat, Spain Background: Cdk5 is an atypical cyclin-dependent kinase − because of its activation by binding to p35 and p39 proteins − indispensable for normal brain development and deregulated in neurodegenerative diseases such as Alzheimer’s. Given the great similarity between the cellular and molecular mechanisms orchestrating neuronal migration during development of the nervous system and cancer cell migration during metastasis, it is not surprising
that Cdk5 has been reported to play a role in the latter. Cdk5 is overexpressed in several tumours, and has been associated with a worse prognosis. However, very little is known about a possible implication of Cdk5 in colorectal cancer (CRC). Our aim was to investigate the expression patterns of Cdk5 and p35 in human CRC cell lines and tumour samples for further evaluation of its role as a prognostic marker. Material and Methods: Human HT29, LoVo, HCT116, DLD1, LS513, SW480, HCT15, Caco2, LS1745 and SW1427 CRC cell lines were used in the present study. Cdk5, p35 protein expression was analysed by western blot in cell lines and in 12 frozen stage IV colorectal adenocarcinomas and their corresponding normal tissues. The presence of Cdk5-p35 active complex was studied by immunoprecipitation and kinase activity assay using H1 as target of phosphorylation. Cdk5 and p35 immunostaining was analysed in a tissue microarray containing 35 stage IV CRC cases. Cdk5 mRNA expression was studied in 98-paired adjacent normal and tumour tissues from stage II MSS CRC patients and in 256 stage II and III MSS frozen colorectal tumours. Kaplan meyer curves and log rank test was used to study association of expression data with desease-free survival (DFS) and overall survival (OS) in stage II and III patients. Results: Both Cdk5 and p35 proteins were detected at high levels in 10 CRC cell lines and their functional interaction was confirmed by immunoprecipitation and in vitro kinase assays. We observed a time-dependent increase (from 24 to 96 h) of Cdk5 and p35 protein levels in cell culture. Cdk5 (p = 0.003) and p35 (p = 0.03) were overexpressed in stage IV colorectal adenocarcinomas as compared to their corresponding normal adjacent tissues. Both proteins were also detected by inmunohistochemistry in human colorectal tumours. Higher Cdk5 mRNA levels (p < 0.0001) were found in tumours as compared to the corresponding normal tissues. Interestingly, high Cdk5 tumour levels were significantly associated with a worse DFS and OS (HR = 4.15, p = 0.022 and HR = 8.25, p = 0.049). Conclusion: Cdk5 and p35 were significantly overexpressed in CRC at both, protein and mRNA levels. Overexpression of Cdk5 was associated with a worse prognostic in stage II, III (MSS), suggesting a role of this protein in CRC progression. Further experiments are on-going in order to elucidate the mechanisms by which Cdk5 exerts its tumour promoting functions. No conflict of interest. 883 UM Cure 2020 − A consortium of European experts in uveal melanoma to identify new therapies for patients with metastatic disease N. Dhomen1 , R. Marais1 , M.J. Jager2 , B.E. Snaar-Jagalska3 , S. Coupland4 , B. Romanowska-Dixon5 , M.C. Mione6 , A. Valente7 , B. Ryll8 , R. Ruijtenbeek9 , A. Prestat10 , E. Vinolo11 , S. Roman-Roman12 . 1 CRUK Manchester Institute, Molecular Oncology Group, Manchester, United Kingdom, 2 Leiden University Medical Centre, Laboratory of Ophthalmology, Leiden, Netherlands, 3 Leiden University, Institute of Biology Leiden, Leiden, Netherlands, 4 University of Liverpool, Liverpool Ocular Oncology Research Group, Liverpool, United Kingdom, 5 Jagiellonian University, Department of Ophthalmology and Ocular Oncology, Krakow, Poland, 6 Karlsruhe Institute of Technology, Institute of Toxicology and Genetics, Karlsruhe, Germany, 7 Champalimaud Foundation, Champalimaud Foundation, Lisbon, Portugal, 8 Melanoma Patient Network Europe, MPNE, Uppsala, Sweden, 9 PamGene International BV, Research & Development, ’s-Hertogenbosch, Netherlands, 10 PEP-Therapy, PEP-Therapy, Paris, France, 11 Seeding Science, Seeding Science, Paris, France, 12 Institut Curie, Translational Research Department, Paris, France Introduction: Uveal melanoma (UM) is a rare intraocular tumour with an incidence of 5 cases per million individuals per year. Up to a third of UM patients develop metastases, most often in the liver, and these are invariably fatal. Despite new discoveries in the genetic and molecular background of the primary tumour, little is known about the metastatic disease; moreover, there is no therapy to either prevent or treat UM metastases. UM Cure 2020 has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 667787, and started in January 2016. Materials and Methods: In UM Cure 2020, we will identify and validate at the preclinical level novel therapeutic approaches for the treatment of UM metastases. To achieve our goal, we have established a consortium comprising key European Centres involved in UM patient care, UM clinical, translational and basic research, a patient organisation and two biotech companies with expertise in biomarkers and early clinical development. An ambitious multidisciplinary approach is proposed to move from tissue characterisation to preclinical evaluation of single or combinations of drugs. This approach includes the characterisation of the genetic landscape of metastatic UM and its microenvironment, proteomic studies to address signalling pathway deregulation, and the establishment of novel relevant in vitro and in vivo preclinical models of the metastatic disease. We also aim to validate accurate surrogate endpoint biomarkers to evaluate therapies and detect metastases as early as possible. Underpinning this will be the UM Cure 2020 virtual biobank registry, linking biobanks in the 4 referral centres involved into a harmonised