- Email: [email protected]
15 Genetic biomarkers in head and neck squamous cell carcinoma
e32
Abstracts / Oral Oncology 51 (2015) e27–e55
Introduction: Next-generation sequencing (NGS) techniques render genetic investigations of tumor material more effective and will help to understand tumor biology and overcome mechanisms of treatment resistance. Targeted NGS which is feasible also for archival FFPE tumor material represents an attractive approach for the development of personalized cancer treatment. We describe here the establishment of an HNSCC-specific cancer gene panel for targeted NGS. Methods: NGS data available at the cBioPortal reporting on whole exome sequencing of 412 HNSCC samples from three independent patient cohorts (Broad Institute, Science 2011; John Hopkins University, Science 2011; TCGA, provisional) were used. For the definition of relevant genes to be included in a specific HNSCC gene panel, selection criteria such as the relative mutational frequency in HNSCC, the presence of hotspot mutations within the coding region or the occurrence of mutations at recurrent positions were used. In order to consider the biological function of particular genes, genes were mapped to pathways using different databases. Results: Overall, 1710 genes with genetic alterations fulfilling the selection criteria were identified in the HNSCC datasets. Of these, less than 10% of affected genes were altered in more than 5% of patients, speaking for a huge genetic heterogeneity of head and neck cancer. By our selection criteria, a gene set of 901 genes was identified, covering a coding region of 2.94 Mb in total. Of these genes, only 652 could be mapped to specific pathways, resulting in a final coding region length of the selected genes of 2.15 Mb to be targeted by panel NGS. The gene list covered a wide spectrum of already known but also novel unexpected genetic lesions. Conclusion: Application of NGS for comprehensive molecular characterization of HNSCC revealed new insights into HNSCC biology and is expected to lead to the identification of new druggable targets also for this tumor entity. The gene list obtained by our approach can be used for the establishment of an HNSCC-specific panel for targeted NGS. Such a gene panel will help in the assessment the intratumor genetic heterogeneity and the characterization of spontaneous and treatment-induced clonal evolution of tumors and will thereby contribute to the development of personalized medicine in HNSCC. doi:10.1016/j.oraloncology.2015.02.017
15 Genetic biomarkers in head and neck squamous cell carcinoma P. Golusinski a, X. Zhi b, K. Lamperska a, L. Luczewski a, N.J. Schork c, W. Golusinski a, M. Masternak b a
Poznan, Poland Orlando, FL, United States c La Jolla, CA, United States b
Outcome Objectives: Head and neck squamous cell carcinoma (HNSCC) is known as one of the six most common human cancers mainly caused by consumption of tobacco and alcohol. There is also a genetic factor; however, the genetic markers are not yet established. Our objectives were to:
1. Validate the genetic signature of molecular targets expressed by tumors in HNSCC. 2. Determine potential biomarkers for earlier detection, potential therapies and prediction of patients’ survival. Methods: The HNSCC patients were recruited to the study in the Greater Poland Cancer Centre in 2010. Oral cancer and normal epithelium tissue taken at a minimum of 2 cm distal from the
tumors’ margins from 41 patients were used for analysis by Cancer Pathway Finder array and followed with real-rime PCR. Results: Analysis indicated up-regulation of 11 genes including KRT14, ACLY, MCM2, SKP2, STMN1, CDC20, SNAI2, MKI67, SLC2A1, BCL2L11, IGFBP3 (P < 0.05) suggesting altered regulation of cell cycle, cell senescence, metabolism, apoptosis and hypoxia. Five years patient follow up survival analysis indicated that SKP2, KRT14, FOXC2, Acly, PGF, OCLN, CDH2, LDHA, VEGFC, BCL2L11, CA9 genes expression was significantly associated with survival of the patients. Conclusion: Our data indicate that there is significant activation of several cellular pathways in tumor tissue that should be further investigated. Importantly, observed significant association between the expression of SKP2, KRT14, FOXC2, Acly, PGF, OCLN, CDH2, LDHA, VEGFC, BCL2L11, CA9 and survival indicate that the level of the expression of these genes in tumor tissue may predict the survival of the patient. doi:10.1016/j.oraloncology.2015.02.018
Session 4 – Tumor microenvironment (immunology) 17 Impact of polymorphonuclear cells (PMN) in head and neck cancer progression: A mouse model to investigate time dependent functions of PMN K. Moses, S. Lang, S. Brandau Essen, Germany Background: We could previously show that high polymorphonuclear granulocyte (PMN) infiltration in head and neck cancer (HNC) tissues correlated with poor survival of patients with advanced disease. Further in vitro studies showed that human PMN are manipulated by tumor cells to acquire a protumoral phenotype. In order to gain further insight into the mechanisms of PMN-mediated effects during different phases of tumor progression, we characterize PMN expansion, infiltration into tumor tissue and activation by the tumor in a murine in vivo model of HNC. Methods: Murine HNC cell lines were injected subcutaneous and orthotopic (M. myelohyoideus) into immunocompetent mice. Tissues were analyzed for the presence of different cell types by immunohistochemistry. PMN depletion was performed in mice using Gr1 and Ly6G specific antibodies at different time points of tumor progression. Isolated cells were further analyzed by different assays using flow cytometry. Results: Tumors were infiltrated by PMN in both sites of tumor growth. Peripheral granulocytes accumulated in spleen and blood upon tumor progression. Depletion of granulocytes in tumor bearing mice resulted in delayed tumor progression. This effect was highly dependent on the time point of PMN depletion, with early time points resulting in the most prominent growth inhibition. Enhanced infiltration by T cells and reduced angiogenesis was observed in PMN depleted animals of different tumor localizations. Furthermore, we could show immunosuppressive functions of tumor induced PMN in vitro, with immature PMN being the most suppressive subset. Additional depletion of CD8 T cells in PMN depleted animals counteracted the delay in tumor progression. Conclusions: In this model PMN are especially important for the initial steps of tumor formation. Our results suggest that protumoral PMN inhibit an antitumor CD8 T cell response in vivo resulting in accelerated tumor growth. Interference with PMN-mediated tumor progression may offer new treatment options for patients with highly inflammatory tumor growth. doi:10.1016/j.oraloncology.2015.02.019
Abstracts / Oral Oncology 51 (2015) e27–e55
Introduction: Next-generation sequencing (NGS) techniques render genetic investigations of tumor material more effective and will help to understand tumor biology and overcome mechanisms of treatment resistance. Targeted NGS which is feasible also for archival FFPE tumor material represents an attractive approach for the development of personalized cancer treatment. We describe here the establishment of an HNSCC-specific cancer gene panel for targeted NGS. Methods: NGS data available at the cBioPortal reporting on whole exome sequencing of 412 HNSCC samples from three independent patient cohorts (Broad Institute, Science 2011; John Hopkins University, Science 2011; TCGA, provisional) were used. For the definition of relevant genes to be included in a specific HNSCC gene panel, selection criteria such as the relative mutational frequency in HNSCC, the presence of hotspot mutations within the coding region or the occurrence of mutations at recurrent positions were used. In order to consider the biological function of particular genes, genes were mapped to pathways using different databases. Results: Overall, 1710 genes with genetic alterations fulfilling the selection criteria were identified in the HNSCC datasets. Of these, less than 10% of affected genes were altered in more than 5% of patients, speaking for a huge genetic heterogeneity of head and neck cancer. By our selection criteria, a gene set of 901 genes was identified, covering a coding region of 2.94 Mb in total. Of these genes, only 652 could be mapped to specific pathways, resulting in a final coding region length of the selected genes of 2.15 Mb to be targeted by panel NGS. The gene list covered a wide spectrum of already known but also novel unexpected genetic lesions. Conclusion: Application of NGS for comprehensive molecular characterization of HNSCC revealed new insights into HNSCC biology and is expected to lead to the identification of new druggable targets also for this tumor entity. The gene list obtained by our approach can be used for the establishment of an HNSCC-specific panel for targeted NGS. Such a gene panel will help in the assessment the intratumor genetic heterogeneity and the characterization of spontaneous and treatment-induced clonal evolution of tumors and will thereby contribute to the development of personalized medicine in HNSCC. doi:10.1016/j.oraloncology.2015.02.017
15 Genetic biomarkers in head and neck squamous cell carcinoma P. Golusinski a, X. Zhi b, K. Lamperska a, L. Luczewski a, N.J. Schork c, W. Golusinski a, M. Masternak b a
Poznan, Poland Orlando, FL, United States c La Jolla, CA, United States b
Outcome Objectives: Head and neck squamous cell carcinoma (HNSCC) is known as one of the six most common human cancers mainly caused by consumption of tobacco and alcohol. There is also a genetic factor; however, the genetic markers are not yet established. Our objectives were to:
1. Validate the genetic signature of molecular targets expressed by tumors in HNSCC. 2. Determine potential biomarkers for earlier detection, potential therapies and prediction of patients’ survival. Methods: The HNSCC patients were recruited to the study in the Greater Poland Cancer Centre in 2010. Oral cancer and normal epithelium tissue taken at a minimum of 2 cm distal from the
tumors’ margins from 41 patients were used for analysis by Cancer Pathway Finder array and followed with real-rime PCR. Results: Analysis indicated up-regulation of 11 genes including KRT14, ACLY, MCM2, SKP2, STMN1, CDC20, SNAI2, MKI67, SLC2A1, BCL2L11, IGFBP3 (P < 0.05) suggesting altered regulation of cell cycle, cell senescence, metabolism, apoptosis and hypoxia. Five years patient follow up survival analysis indicated that SKP2, KRT14, FOXC2, Acly, PGF, OCLN, CDH2, LDHA, VEGFC, BCL2L11, CA9 genes expression was significantly associated with survival of the patients. Conclusion: Our data indicate that there is significant activation of several cellular pathways in tumor tissue that should be further investigated. Importantly, observed significant association between the expression of SKP2, KRT14, FOXC2, Acly, PGF, OCLN, CDH2, LDHA, VEGFC, BCL2L11, CA9 and survival indicate that the level of the expression of these genes in tumor tissue may predict the survival of the patient. doi:10.1016/j.oraloncology.2015.02.018
Session 4 – Tumor microenvironment (immunology) 17 Impact of polymorphonuclear cells (PMN) in head and neck cancer progression: A mouse model to investigate time dependent functions of PMN K. Moses, S. Lang, S. Brandau Essen, Germany Background: We could previously show that high polymorphonuclear granulocyte (PMN) infiltration in head and neck cancer (HNC) tissues correlated with poor survival of patients with advanced disease. Further in vitro studies showed that human PMN are manipulated by tumor cells to acquire a protumoral phenotype. In order to gain further insight into the mechanisms of PMN-mediated effects during different phases of tumor progression, we characterize PMN expansion, infiltration into tumor tissue and activation by the tumor in a murine in vivo model of HNC. Methods: Murine HNC cell lines were injected subcutaneous and orthotopic (M. myelohyoideus) into immunocompetent mice. Tissues were analyzed for the presence of different cell types by immunohistochemistry. PMN depletion was performed in mice using Gr1 and Ly6G specific antibodies at different time points of tumor progression. Isolated cells were further analyzed by different assays using flow cytometry. Results: Tumors were infiltrated by PMN in both sites of tumor growth. Peripheral granulocytes accumulated in spleen and blood upon tumor progression. Depletion of granulocytes in tumor bearing mice resulted in delayed tumor progression. This effect was highly dependent on the time point of PMN depletion, with early time points resulting in the most prominent growth inhibition. Enhanced infiltration by T cells and reduced angiogenesis was observed in PMN depleted animals of different tumor localizations. Furthermore, we could show immunosuppressive functions of tumor induced PMN in vitro, with immature PMN being the most suppressive subset. Additional depletion of CD8 T cells in PMN depleted animals counteracted the delay in tumor progression. Conclusions: In this model PMN are especially important for the initial steps of tumor formation. Our results suggest that protumoral PMN inhibit an antitumor CD8 T cell response in vivo resulting in accelerated tumor growth. Interference with PMN-mediated tumor progression may offer new treatment options for patients with highly inflammatory tumor growth. doi:10.1016/j.oraloncology.2015.02.019