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Dissecting heterogeneity and molecular mechanisms involved in paranasal sinus cancer
S54
Poster abstracts
Poster Session – Preclinical Models, Wednesday 29 November 2016
into the clinics. Furthermore, preclinical models have to be identified for developing of immunotherapy combinations and elucidating the mechanisms of action of immunotherapies. Determining appropriate regimens requires setting multiple parameters, including dose levels, administration frequencies, durations of treatment for each drug in the combination, and sequence of administration. Those parameters have to be tested before settled trials. Other challenges will include discovering of appropriate biomarkers for efficacy and safety and selecting appropriate tumor type. Material and Methods: We tested several syngeneic models as CT26, MC38, B16F10 and 4T1 towards their respond to PD-L1 checkpoint inhibitor and radiation and measured the infiltration of immune cells. Further to this, we followed two strategies to generate mice with a humanized immune system: human PBMC and human hematopoietic stem cells. These different mouse models harbouring human immune cells are applied to study the efficacy and mode of action of new immunotherapeutic drug and combinations. Results: In a preclinical combination trial in syngeneic mice a PD-L1 inhibitor was combined with local radiation of the tumor. The evaluation of the PD-L1 expression in the tumors revealed that radiation stimulate PD-L1. Furthermore, more infiltrating immune cells were detected. For the humanized mouse models, PBMC were co-inoculated with human breast tumor cells into immunodeficient mice. Increased efficacy of the humanized mAB trastuzumab was seen compared to xenografted tumors without PBMC. On the other hand we reconstituted a human immune system in mice by engrafting human hematopoietic stem cells. At the time when the human immune system was developed, established patient-derived tumors were transplanted on these reconstituted humanized mice. Different tumor entities are growing in these humanized mice either similar to non humanized mice or slightly slower. During the observation period no tumor rejections by the human immune cells were evident, although tumor growth was accompanied by an increase of human T cells in the peripheral blood. Humanized mice bearing various PDX tumors were treated with approved therapeutic checkpoint inhibitors. Ipilimumab as well as nivolumab lead to a slight tumor growth delay and an increased percentage of T cells in the blood and in the tumor. The combination of both inhibitors showed a synergistic effect on tumor growth inhibition. Conclusions: Our syngeneic and humanized mouse models enable appropriate preclinical assessment of immune-based therapeutic antitumor strategies. No conflict of interest. 148 Poster (Board P119) Dissecting heterogeneity and molecular mechanisms involved in paranasal sinus cancer L. De Cecco1 , R. Granata2 , E. Orlandi3 , C. Fallai3 , L. Licitra2 , M. Giannoccaro1 , F. Perrone4 , S. Pilotti4 , P. Quattrone4 , C. Facco5 , F. Sessa5 , M. Turri-Zanoni6 , P. Battaglia6 , P. Castelnuovo6 , P. Antognoni7 , N. Paielli4 , L. Tonella1 , S. Canevari1 , P. Bossi8 . 1 Fondazione IRCCS Istituto Nazionale dei Tumori, Department of Experimental Oncology and Molecular Medicine, Milan, Italy; 2 Fondazione IRCCS Istituto Nazionale dei Tumori, Head and Neck Medical Oncology Department, Milan, Italy; 3 Fondazione IRCCS Istituto Nazionale dei Tumori, Radiotherapy 2 Unit, Milan, Italy; 4 Fondazione IRCCS Istituto Nazionale dei Tumori, Pathology Department, Milan, Italy; 5 ASST Sette Laghi, University of Insubria, Pathology Department, Varese, Italy; 6 ASST Sette Laghi, University of Insubria, Head and Neck Surgery, Varese, Italy; 7 ASST Sette Laghi, University of Insubria, Radiotherapy Department, Varese, Italy; 8 Istituto Nazionale Tumori, Head & Neck Cancer Medical Unit, Milano, Italy Background: Epithelial, non-glandular paranasal sinus cancer is a rare disease, with a global dismal prognosis. There are no recognized targeted treatments and the knowledge of molecular mechanisms involved in the resistance to available therapies (surgery, radiation, chemotherapy) is limited. Dissecting the heterogeneity of paranasal sinus cancers and providing valuable information on the biology of the malignancy is eagerly needed to improve therapeutic approaches. Material and Methods: We selected a retrospective cohort of 53 paranasal sinus cancer cases including the following histologies: (i) sinonasal undifferentiated cancer (SNUC); (ii) sinonasal neuroendocrine cancer (SNEC); (iii) non keratinizing squamous cell cancer (NKSCC). Treatment consisted in different combinations of surgery, radiation and systemic therapies. Whole-transcriptome profiling was performed by microarray analysis using the DASL assay and BeadArray Chips (Illumina). Unsupervised tumour subtype identification was performed applying k-means clustering and 1-Pearson correlation as distance matrix as implemented in the R package ConsensusClusterPlus. We used a 1000 re-sampling interaction model and we tested the existence of 2 < k < 5 clusters. We explored the potential biological processes differentiating the subtypes
through Gene Set Enrichment Analysis (GSEA) that reveals the regulatory relationships among genes providing a systematic understanding of molecular mechanisms. Functional pathway analysis was performed imposing a significant level of FDR <25% and interrogating gene sets belonging to Hallmark Gene Set Collection that represents a collection of “hallmark” gene sets as part of The Molecular Signatures Database obtained by a combination of automated approaches and expert curation. Results: Consensus unsupervised clustering was applied to the geneexpression data matrix and revealed the presence of three stable clusters of samples: (i) ClusterA, n = 28 cases (53%); (ii) ClusterB n = 8 cases (15%); (iii) ClusterC n = 17 cases (32%). ClusterA showed a significant enrichment of proliferation pathways and gene-sets associated with G2/M checkpoint as in progression through the cell division cycle. In addition ClusterA is linked with deregulation of genes of MYC, MTOR signalling. ClusterB was characterized by enrichment in KRAS and inflammation pathways. ClusterC was linked to deregulation of genes of extracellular matrix and WNT signalling. Conclusion: The present preliminary report is the first in-deep molecular analysis of paranasal sinus cancer that proves the capability of transcriptomic profiling to capture molecules and functional pathways deregulated in this disease. The identification of gene-expression patterns associated across the different histologies and to clinical outcome is ongoing. Conflict of interest: This study is supported by Associazione Italiana Ricerca Cancro (AIRC IG 17422 to PB). 149 Poster (Board P120) Spheroid formation assay for in vitro assessment and expansion of circulating cancer stem cells (cCSC) in patients with breast cancer M. Pizon1 , D. Schott1 , U. Pachmann1 , K. Pachmann1 . 1 Transfusion Center Bayreuth, Transfusion Center Bayreuth, Bayreuth, Germany Background: Cells with sphere forming capacity are present in the peripheral blood of breast cancer patients and represent a significant impediment to efficacious treatment due to their putative role in progression, metastasis and chemotherapy resistance. The isolation and identification of cCSC is very difficult, because of their rarity. Here we report that we are routinely able to generate spheroids from the peripheral blood of breast cancer patients. The identification of cCSC could help to develop novel therapeutic strategies specially targeting cancer stem cells. Methods: All white blood cells including circulating epithelial tumor cells from 1 ml blood were cultured under conditions favoring growth of tumorspheres from 72 patients with breast cancer, including a subpopulation of 23 patients with metastatic disease. CETCs were determined using the maintrac® method. Gene expression profiles of single CETCs and tumorspheres of the same patients were analyzed using qRT-PCR. Results: Sphere formation was observed in 79% of patients. The number of tumorspheres was dependend on stage of disease. Furthermore, the most important factor influencing the growth of tumorspheres was the treatment with chemotherapy. Patients treated with chemotherapy had significantly lower numbers of tumorspheres compared to patients without chemotherapy. The spheroids were found to consist of cells with cancer stem cell characteristics such as upregulation of stem cell genes (Sox2, Okt4, Nanog, EpCAM, CD 133,) self-renewal, and high aldehyde dehydrogenase (ALDH) activity. There was no sphere formation in a control group with 50 healthy donors. Conclusion: This study reports on a method for generating and growing tumorspheres from peripheral blood and confirms the hypotheses that cells with cancer stem cells properties are present in the blood of patients with breast cancer. Understanding the biology of tumorspheres may contribute to the identification of novel therapeutic opportunities. No conflict of interest. 150 Poster (Board P121) Evaluation of potential predictive biomarker for cetuximab in a panel of colorectal cancer patient-derived xenografts (PDX) M. Rivera1,2 , J. Hoffmann1 , A. Wulf-Goldenberg1 , P.M. Schlag3 , J. Merk4 , W. Walther2,5 , I. Fichtner1 . 1 Experimental Pharmacology & Oncology ¨ Berlin-Buch GmbH, Project Manager, Berlin, Germany; 2 Max Delbruck Center for Molecular Medicine, Translational Oncology, Berlin, Germany; 3 Charite´ Comprehensive Cancer Center, Charite´ Campus Mitte, Berlin, Germany; 4 Labor Dr. Merk & Kollegen GmbH, Project Management, Ochsenhausen, Germany; 5 Experimental and Clinical Research Center, Charite´ − University Medicine Berlin, Translational Oncology, Berlin, Germany Background: Cetuximab is approved as EGFR targeted therapy for colorectal cancer (CRC) together with activating mutations in the MAPK pathway (KRAS and BRAF) as predictive biomarker. However there is a group of resistant patients for which an improved predictive biomarker is
Poster abstracts
Poster Session – Preclinical Models, Wednesday 29 November 2016
into the clinics. Furthermore, preclinical models have to be identified for developing of immunotherapy combinations and elucidating the mechanisms of action of immunotherapies. Determining appropriate regimens requires setting multiple parameters, including dose levels, administration frequencies, durations of treatment for each drug in the combination, and sequence of administration. Those parameters have to be tested before settled trials. Other challenges will include discovering of appropriate biomarkers for efficacy and safety and selecting appropriate tumor type. Material and Methods: We tested several syngeneic models as CT26, MC38, B16F10 and 4T1 towards their respond to PD-L1 checkpoint inhibitor and radiation and measured the infiltration of immune cells. Further to this, we followed two strategies to generate mice with a humanized immune system: human PBMC and human hematopoietic stem cells. These different mouse models harbouring human immune cells are applied to study the efficacy and mode of action of new immunotherapeutic drug and combinations. Results: In a preclinical combination trial in syngeneic mice a PD-L1 inhibitor was combined with local radiation of the tumor. The evaluation of the PD-L1 expression in the tumors revealed that radiation stimulate PD-L1. Furthermore, more infiltrating immune cells were detected. For the humanized mouse models, PBMC were co-inoculated with human breast tumor cells into immunodeficient mice. Increased efficacy of the humanized mAB trastuzumab was seen compared to xenografted tumors without PBMC. On the other hand we reconstituted a human immune system in mice by engrafting human hematopoietic stem cells. At the time when the human immune system was developed, established patient-derived tumors were transplanted on these reconstituted humanized mice. Different tumor entities are growing in these humanized mice either similar to non humanized mice or slightly slower. During the observation period no tumor rejections by the human immune cells were evident, although tumor growth was accompanied by an increase of human T cells in the peripheral blood. Humanized mice bearing various PDX tumors were treated with approved therapeutic checkpoint inhibitors. Ipilimumab as well as nivolumab lead to a slight tumor growth delay and an increased percentage of T cells in the blood and in the tumor. The combination of both inhibitors showed a synergistic effect on tumor growth inhibition. Conclusions: Our syngeneic and humanized mouse models enable appropriate preclinical assessment of immune-based therapeutic antitumor strategies. No conflict of interest. 148 Poster (Board P119) Dissecting heterogeneity and molecular mechanisms involved in paranasal sinus cancer L. De Cecco1 , R. Granata2 , E. Orlandi3 , C. Fallai3 , L. Licitra2 , M. Giannoccaro1 , F. Perrone4 , S. Pilotti4 , P. Quattrone4 , C. Facco5 , F. Sessa5 , M. Turri-Zanoni6 , P. Battaglia6 , P. Castelnuovo6 , P. Antognoni7 , N. Paielli4 , L. Tonella1 , S. Canevari1 , P. Bossi8 . 1 Fondazione IRCCS Istituto Nazionale dei Tumori, Department of Experimental Oncology and Molecular Medicine, Milan, Italy; 2 Fondazione IRCCS Istituto Nazionale dei Tumori, Head and Neck Medical Oncology Department, Milan, Italy; 3 Fondazione IRCCS Istituto Nazionale dei Tumori, Radiotherapy 2 Unit, Milan, Italy; 4 Fondazione IRCCS Istituto Nazionale dei Tumori, Pathology Department, Milan, Italy; 5 ASST Sette Laghi, University of Insubria, Pathology Department, Varese, Italy; 6 ASST Sette Laghi, University of Insubria, Head and Neck Surgery, Varese, Italy; 7 ASST Sette Laghi, University of Insubria, Radiotherapy Department, Varese, Italy; 8 Istituto Nazionale Tumori, Head & Neck Cancer Medical Unit, Milano, Italy Background: Epithelial, non-glandular paranasal sinus cancer is a rare disease, with a global dismal prognosis. There are no recognized targeted treatments and the knowledge of molecular mechanisms involved in the resistance to available therapies (surgery, radiation, chemotherapy) is limited. Dissecting the heterogeneity of paranasal sinus cancers and providing valuable information on the biology of the malignancy is eagerly needed to improve therapeutic approaches. Material and Methods: We selected a retrospective cohort of 53 paranasal sinus cancer cases including the following histologies: (i) sinonasal undifferentiated cancer (SNUC); (ii) sinonasal neuroendocrine cancer (SNEC); (iii) non keratinizing squamous cell cancer (NKSCC). Treatment consisted in different combinations of surgery, radiation and systemic therapies. Whole-transcriptome profiling was performed by microarray analysis using the DASL assay and BeadArray Chips (Illumina). Unsupervised tumour subtype identification was performed applying k-means clustering and 1-Pearson correlation as distance matrix as implemented in the R package ConsensusClusterPlus. We used a 1000 re-sampling interaction model and we tested the existence of 2 < k < 5 clusters. We explored the potential biological processes differentiating the subtypes
through Gene Set Enrichment Analysis (GSEA) that reveals the regulatory relationships among genes providing a systematic understanding of molecular mechanisms. Functional pathway analysis was performed imposing a significant level of FDR <25% and interrogating gene sets belonging to Hallmark Gene Set Collection that represents a collection of “hallmark” gene sets as part of The Molecular Signatures Database obtained by a combination of automated approaches and expert curation. Results: Consensus unsupervised clustering was applied to the geneexpression data matrix and revealed the presence of three stable clusters of samples: (i) ClusterA, n = 28 cases (53%); (ii) ClusterB n = 8 cases (15%); (iii) ClusterC n = 17 cases (32%). ClusterA showed a significant enrichment of proliferation pathways and gene-sets associated with G2/M checkpoint as in progression through the cell division cycle. In addition ClusterA is linked with deregulation of genes of MYC, MTOR signalling. ClusterB was characterized by enrichment in KRAS and inflammation pathways. ClusterC was linked to deregulation of genes of extracellular matrix and WNT signalling. Conclusion: The present preliminary report is the first in-deep molecular analysis of paranasal sinus cancer that proves the capability of transcriptomic profiling to capture molecules and functional pathways deregulated in this disease. The identification of gene-expression patterns associated across the different histologies and to clinical outcome is ongoing. Conflict of interest: This study is supported by Associazione Italiana Ricerca Cancro (AIRC IG 17422 to PB). 149 Poster (Board P120) Spheroid formation assay for in vitro assessment and expansion of circulating cancer stem cells (cCSC) in patients with breast cancer M. Pizon1 , D. Schott1 , U. Pachmann1 , K. Pachmann1 . 1 Transfusion Center Bayreuth, Transfusion Center Bayreuth, Bayreuth, Germany Background: Cells with sphere forming capacity are present in the peripheral blood of breast cancer patients and represent a significant impediment to efficacious treatment due to their putative role in progression, metastasis and chemotherapy resistance. The isolation and identification of cCSC is very difficult, because of their rarity. Here we report that we are routinely able to generate spheroids from the peripheral blood of breast cancer patients. The identification of cCSC could help to develop novel therapeutic strategies specially targeting cancer stem cells. Methods: All white blood cells including circulating epithelial tumor cells from 1 ml blood were cultured under conditions favoring growth of tumorspheres from 72 patients with breast cancer, including a subpopulation of 23 patients with metastatic disease. CETCs were determined using the maintrac® method. Gene expression profiles of single CETCs and tumorspheres of the same patients were analyzed using qRT-PCR. Results: Sphere formation was observed in 79% of patients. The number of tumorspheres was dependend on stage of disease. Furthermore, the most important factor influencing the growth of tumorspheres was the treatment with chemotherapy. Patients treated with chemotherapy had significantly lower numbers of tumorspheres compared to patients without chemotherapy. The spheroids were found to consist of cells with cancer stem cell characteristics such as upregulation of stem cell genes (Sox2, Okt4, Nanog, EpCAM, CD 133,) self-renewal, and high aldehyde dehydrogenase (ALDH) activity. There was no sphere formation in a control group with 50 healthy donors. Conclusion: This study reports on a method for generating and growing tumorspheres from peripheral blood and confirms the hypotheses that cells with cancer stem cells properties are present in the blood of patients with breast cancer. Understanding the biology of tumorspheres may contribute to the identification of novel therapeutic opportunities. No conflict of interest. 150 Poster (Board P121) Evaluation of potential predictive biomarker for cetuximab in a panel of colorectal cancer patient-derived xenografts (PDX) M. Rivera1,2 , J. Hoffmann1 , A. Wulf-Goldenberg1 , P.M. Schlag3 , J. Merk4 , W. Walther2,5 , I. Fichtner1 . 1 Experimental Pharmacology & Oncology ¨ Berlin-Buch GmbH, Project Manager, Berlin, Germany; 2 Max Delbruck Center for Molecular Medicine, Translational Oncology, Berlin, Germany; 3 Charite´ Comprehensive Cancer Center, Charite´ Campus Mitte, Berlin, Germany; 4 Labor Dr. Merk & Kollegen GmbH, Project Management, Ochsenhausen, Germany; 5 Experimental and Clinical Research Center, Charite´ − University Medicine Berlin, Translational Oncology, Berlin, Germany Background: Cetuximab is approved as EGFR targeted therapy for colorectal cancer (CRC) together with activating mutations in the MAPK pathway (KRAS and BRAF) as predictive biomarker. However there is a group of resistant patients for which an improved predictive biomarker is