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DNA damage responses and chemosensitivity of breast cancer tissue slices ex vivo
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 domains; and (4) provide hypotheses about molecular mechanisms and downstream effects of domain mutations. We make all results available in an interaction web resource (www.mutationaligner.org), which enables discovery and exploration of mutation hotspots identified in protein domains in more than 5000 cancer patient samples across 22 different tumor types. Conclusion: Using cancer genomics datasets from thousands of tumor samples in 22 tumor types, we analyze somatic missense mutations in protein domains and discover new domain mutation hotspots. By associating mutations in infrequently altered genes with mutations in frequently altered paralogous genes that are known to contribute to cancer, this study provides many new clues to the functional role of rare mutations in cancer. No conflict of interest. 143 Characterization of the genomic profile of pseudomyxoma peritonei using amplicon sequencing combined with exome sequencing 2 ¨ , P. Nummela1 , L. Saarinen1 , A. Thiel1 , R. Lehtonen1 , P. Jarvinen 3 H. Jarvinen ¨ ¨ 1. , L.A. Aaltonen1 , A. Lepisto¨ 3 , S. Hautaniemi1 , A. Ristimaki 1 University of Helsinki, Genome-Scale Biology Research Program, Helsinki, Finland, 2 Helsinki University Central Hospital, Dept. of Urology, Helsinki, Finland, 3 Helsinki University Central Hospital, Dept. of Surgery, Helsinki, Finland
Introduction: Pseudomyxoma peritonei (PMP) is a rare but fatal disease in which mucinous tumor cells grow in the peritoneal cavity and secrete voluminous mucinous ascites. The genomic profile of PMP, typically having quite low tumor cell content among the mucus, has started to open by the advent of the powerful technique of next-generation sequencing. Material and Methods: We analyzed 19 formalin-fixed, paraffin-embedded PMP tumor samples with an amplicon sequencing panel covering the mutational hotspot areas of 48 cancer-related genes (average coverage 3580x). Further, to expand the analysis to genes and pathways outside this panel, we sequenced the whole coding exome (average coverage 120x) of nine of these tumors and validated these results with ultra-deep amplicon sequencing. Results and Discussion: KRAS was mutated in all 19 PMP tumors and GNAS in over half of the cases (63%). Other detected mutations, including AKT1, ATM, PIK3CA, SMAD4, and TP53, were mostly detected in 5% and maximally 16% (SMAD4) of the cases. The genetic background of mainly appendix-originating PMP tumors thus seems to resemble more pancreatic intraductal papillary mucinous neoplasms (IPMNs) than colorectal cancers. In addition to GNAS, our exome sequencing approach revealed also seven other genes of the cAMP-protein kinase A (PKA) pathway to be mutated in our PMP tumors (8/9 tumors having this pathway affected), which may account for the highly increased secretion of mucus. In addition, we validated mutations in six genes belonging to the TGF-beta pathway, which may further enhance cell proliferation from that induced by mutated KRAS. Conclusion: Since cAMP-PKA pathway was mutated in 89% of the tumors, inhibition of this pathway might reduce mucus production in most of the patients, thereby suppressing the progression of the disease. No conflict of interest. 144 DNA damage responses and chemosensitivity of breast cancer tissue slices ex vivo D. Van Gent1 , K. Naipal1 , N. Verkaik1 , T. Meijer1 , J. Hoeijmakers1 , C. Van Deurzen2 , R. Kanaar1 , A. Jager3 . 1 Erasmus MC, Molecular Genetics, Rotterdam, Netherlands, 2 Erasmus MC, Pathology, Rotterdam, Netherlands, 3 Erasmus MC, Medical Oncology, Rotterdam, Netherlands Background: Tumor cell characteristics and sensitivities have been studied extensively in two dimensional cell cultures. Much less is known about therapy response in three dimensional growth conditions in the natural tumor microenvironment. Material and Methods: We optimized growth conditions for patient-derived breast tumor slices with a thickness of approximately 30 cell layers. These conditions allowed maintenance of the tumor slices without loss of viability or proliferation over a period of at least 7 days, allowing extensive analysis of tumor cell parameters, such as DNA damage response characteristics and sensitivity to chemotherapy. Results: We used this experimental set up to determine DNA double strand break repair deficiencies in a series of approximately 150 unselected primary breast tumors. We found Homologous Recombination deficiency in 13% of the breast tumors, in many cases associated with triple negative breast cancer. We also showed proof of principle that HR deficient tumor slices were PARP inhibitor and cisplatin sensitive. We are currently adapting the technology for use on biopsy material, for which we will present the first results comparing ex vivo responses and treatment outcome in the patient. In addition to these mechanistic studies, we also investigated whether chemotherapy response could be predicted from ex vivo reaction to chemotherapy. We found a range of sensitivities to treatment with 5-Fluoro-Uracil/Adriamycin/cyclophosphamide
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combinations. As proof of principle, we found one clinically insensitive tumor to be insensitive ex vivo, as well. Conclusion: Breast tumor tissue slices provide a novel opportunity to monitor tumor response to therapy. Under optimal conditions, these slices can be maintained for more than a week without loss of viability, which enables extended experimental protocols. No conflict of interest.
Monday 11 July 2016 Poster Session
Cancer Genomics, Epigenetics and Genome Instability II 145 Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing J. Demeulemeester1,2 , E.K. Møller3,4 , P. Kumar5,6 , N. Silje3,4 , D.C. Wedge7 , K.P. White8 , B. Naume4,9 , V.N. Kristensen3,4,10 , P. Van Loo1,2 , T. Voet5,6 . 1 The Francis Crick Institute, Cancer Genomics, London, United Kingdom, 2 KU Leuven − University of Leuven, Department of Human Genetics − Human Genome Laboratory, Leuven, Belgium, 3 Institute for Cancer Research − Oslo University Hospital, Department of Genetics, Oslo, Norway, 4 Institute for Clinical Medicine − University of Oslo, K.G. Jebsen Center for Breast Cancer Research, Oslo, Norway, 5 KU Leuven − University of Leuven, Department of Human Genetics − Laboratory of Reproductive Genomics, Leuven, Belgium, 6 Wellcome Trust Sanger Institute, Single-cell Genomics Centre, Hinxton, United Kingdom, 7 Wellcome Trust Sanger Institute, Cancer Genome Project, Hinxton, United Kingdom, 8 Institute for Genomics & Systems Biology − University of Chicago, Department of Human Genetics, Chicago, USA, 9 Oslo University Hospital, Department of Oncology, Oslo, Norway, 10 Oslo University Hospital, Department of Pathology, Oslo, Norway Introduction: Single-cell micro-metastases of solid tumors often occur in bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. Unfortunately, the molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer. Material and Method: We sequenced the genomes of 40 single cells isolated by micromanipulation from the bone marrow of six patients using established immunocytochemical markers and morphologic characteristics for epithelial tumor cells. We compared the cells’ DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, and genotyped somatic mutations called on bulk tumor exomes in the singlecell sequences. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enabled ordering of CNA events in molecular pseudo-time. Results and Discussion: CNA landscape analysis revealed that a quarter of the cells are DTCs disseminating from the observed tumor. The remaining cells represented non-aberrant ‘normal’ cells and ‘aberrant cells of unknown origin’ that have CNA profiles discordant from the tumor. Probing somatic mutations confirmed that these cells did not derive from the same lineages as the observed breast cancers. Evolutionary reconstruction pinpointed the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis. Conclusions: Single-cell sequencing of bone-marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells of unknown origin is prominent in bone marrow. Author contributions: JD, EKM and PK contributed equally to this work. KPW, BN, VNK, PVL and TV are joint senior authors. No conflict of interest. 146 The expression of MNT, a MYC antagonist, is autoregulated at the mRNA and protein level ˜ 1 , F. Ourique1 , J. Aresti1 , P.J. Hurlin2 , J. Leon1 . 1 Instituto M.C. Lafita1 , J. Liano de Biomedicina y Biotecnolog´ıa de Cantabria- UC-CSIC, Department of Molecular and Cellular Signalling, Santander, Spain, 2 Shriners Hospitals for Children- Oregon Health & Science University, Department of Cell and Developmental Biology, Portland, USA Background: MNT is a basic-helix-loop-helix-leucine zipper protein from the MXD family. MNT and other MXD proteins are antagonists of MYC, one of the most frequently altered oncoproteins in human cancer. Both MYC and MXD proteins heterodimerize with MAX and bind to E-boxes within regulatory regions of target genes, resulting in the activation (MYC-MAX) or repression (MXD-MAX) of their transcription. MNT expression is ubiquitous and does not fluctuate during cell cycle, so it overlaps with the expression of MYC at critical
Introduction: Pseudomyxoma peritonei (PMP) is a rare but fatal disease in which mucinous tumor cells grow in the peritoneal cavity and secrete voluminous mucinous ascites. The genomic profile of PMP, typically having quite low tumor cell content among the mucus, has started to open by the advent of the powerful technique of next-generation sequencing. Material and Methods: We analyzed 19 formalin-fixed, paraffin-embedded PMP tumor samples with an amplicon sequencing panel covering the mutational hotspot areas of 48 cancer-related genes (average coverage 3580x). Further, to expand the analysis to genes and pathways outside this panel, we sequenced the whole coding exome (average coverage 120x) of nine of these tumors and validated these results with ultra-deep amplicon sequencing. Results and Discussion: KRAS was mutated in all 19 PMP tumors and GNAS in over half of the cases (63%). Other detected mutations, including AKT1, ATM, PIK3CA, SMAD4, and TP53, were mostly detected in 5% and maximally 16% (SMAD4) of the cases. The genetic background of mainly appendix-originating PMP tumors thus seems to resemble more pancreatic intraductal papillary mucinous neoplasms (IPMNs) than colorectal cancers. In addition to GNAS, our exome sequencing approach revealed also seven other genes of the cAMP-protein kinase A (PKA) pathway to be mutated in our PMP tumors (8/9 tumors having this pathway affected), which may account for the highly increased secretion of mucus. In addition, we validated mutations in six genes belonging to the TGF-beta pathway, which may further enhance cell proliferation from that induced by mutated KRAS. Conclusion: Since cAMP-PKA pathway was mutated in 89% of the tumors, inhibition of this pathway might reduce mucus production in most of the patients, thereby suppressing the progression of the disease. No conflict of interest. 144 DNA damage responses and chemosensitivity of breast cancer tissue slices ex vivo D. Van Gent1 , K. Naipal1 , N. Verkaik1 , T. Meijer1 , J. Hoeijmakers1 , C. Van Deurzen2 , R. Kanaar1 , A. Jager3 . 1 Erasmus MC, Molecular Genetics, Rotterdam, Netherlands, 2 Erasmus MC, Pathology, Rotterdam, Netherlands, 3 Erasmus MC, Medical Oncology, Rotterdam, Netherlands Background: Tumor cell characteristics and sensitivities have been studied extensively in two dimensional cell cultures. Much less is known about therapy response in three dimensional growth conditions in the natural tumor microenvironment. Material and Methods: We optimized growth conditions for patient-derived breast tumor slices with a thickness of approximately 30 cell layers. These conditions allowed maintenance of the tumor slices without loss of viability or proliferation over a period of at least 7 days, allowing extensive analysis of tumor cell parameters, such as DNA damage response characteristics and sensitivity to chemotherapy. Results: We used this experimental set up to determine DNA double strand break repair deficiencies in a series of approximately 150 unselected primary breast tumors. We found Homologous Recombination deficiency in 13% of the breast tumors, in many cases associated with triple negative breast cancer. We also showed proof of principle that HR deficient tumor slices were PARP inhibitor and cisplatin sensitive. We are currently adapting the technology for use on biopsy material, for which we will present the first results comparing ex vivo responses and treatment outcome in the patient. In addition to these mechanistic studies, we also investigated whether chemotherapy response could be predicted from ex vivo reaction to chemotherapy. We found a range of sensitivities to treatment with 5-Fluoro-Uracil/Adriamycin/cyclophosphamide
S21
combinations. As proof of principle, we found one clinically insensitive tumor to be insensitive ex vivo, as well. Conclusion: Breast tumor tissue slices provide a novel opportunity to monitor tumor response to therapy. Under optimal conditions, these slices can be maintained for more than a week without loss of viability, which enables extended experimental protocols. No conflict of interest.
Monday 11 July 2016 Poster Session
Cancer Genomics, Epigenetics and Genome Instability II 145 Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing J. Demeulemeester1,2 , E.K. Møller3,4 , P. Kumar5,6 , N. Silje3,4 , D.C. Wedge7 , K.P. White8 , B. Naume4,9 , V.N. Kristensen3,4,10 , P. Van Loo1,2 , T. Voet5,6 . 1 The Francis Crick Institute, Cancer Genomics, London, United Kingdom, 2 KU Leuven − University of Leuven, Department of Human Genetics − Human Genome Laboratory, Leuven, Belgium, 3 Institute for Cancer Research − Oslo University Hospital, Department of Genetics, Oslo, Norway, 4 Institute for Clinical Medicine − University of Oslo, K.G. Jebsen Center for Breast Cancer Research, Oslo, Norway, 5 KU Leuven − University of Leuven, Department of Human Genetics − Laboratory of Reproductive Genomics, Leuven, Belgium, 6 Wellcome Trust Sanger Institute, Single-cell Genomics Centre, Hinxton, United Kingdom, 7 Wellcome Trust Sanger Institute, Cancer Genome Project, Hinxton, United Kingdom, 8 Institute for Genomics & Systems Biology − University of Chicago, Department of Human Genetics, Chicago, USA, 9 Oslo University Hospital, Department of Oncology, Oslo, Norway, 10 Oslo University Hospital, Department of Pathology, Oslo, Norway Introduction: Single-cell micro-metastases of solid tumors often occur in bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. Unfortunately, the molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer. Material and Method: We sequenced the genomes of 40 single cells isolated by micromanipulation from the bone marrow of six patients using established immunocytochemical markers and morphologic characteristics for epithelial tumor cells. We compared the cells’ DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, and genotyped somatic mutations called on bulk tumor exomes in the singlecell sequences. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enabled ordering of CNA events in molecular pseudo-time. Results and Discussion: CNA landscape analysis revealed that a quarter of the cells are DTCs disseminating from the observed tumor. The remaining cells represented non-aberrant ‘normal’ cells and ‘aberrant cells of unknown origin’ that have CNA profiles discordant from the tumor. Probing somatic mutations confirmed that these cells did not derive from the same lineages as the observed breast cancers. Evolutionary reconstruction pinpointed the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis. Conclusions: Single-cell sequencing of bone-marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells of unknown origin is prominent in bone marrow. Author contributions: JD, EKM and PK contributed equally to this work. KPW, BN, VNK, PVL and TV are joint senior authors. No conflict of interest. 146 The expression of MNT, a MYC antagonist, is autoregulated at the mRNA and protein level ˜ 1 , F. Ourique1 , J. Aresti1 , P.J. Hurlin2 , J. Leon1 . 1 Instituto M.C. Lafita1 , J. Liano de Biomedicina y Biotecnolog´ıa de Cantabria- UC-CSIC, Department of Molecular and Cellular Signalling, Santander, Spain, 2 Shriners Hospitals for Children- Oregon Health & Science University, Department of Cell and Developmental Biology, Portland, USA Background: MNT is a basic-helix-loop-helix-leucine zipper protein from the MXD family. MNT and other MXD proteins are antagonists of MYC, one of the most frequently altered oncoproteins in human cancer. Both MYC and MXD proteins heterodimerize with MAX and bind to E-boxes within regulatory regions of target genes, resulting in the activation (MYC-MAX) or repression (MXD-MAX) of their transcription. MNT expression is ubiquitous and does not fluctuate during cell cycle, so it overlaps with the expression of MYC at critical