- Email: [email protected]
The KDM5B demethylase in the normal and malignant mammary gland
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
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172 14q32 miRNA cluster: A hot spot for osteosarcoma risk in a Spanish population
174 Involvement of miR-3117 in pediatric acute lymphoblastic leukemia susceptibility
I. Mart´ın-Guerrero1 , N. Bilbao-Aldaiturriaga1 , J. Uriz2 , A. Garc´ıa-Orad1,3 . University of the Basque Country, Genetics- Physical Anthropology and Animal Physiology, Bilbao, Spain, 2 University Hospital Donostia, Unit of Pediatric Oncohematology, San Sebastian, Spain, 3 BioCruces Health Research Institute, Pediatrics, Barakaldo, Spain
1 A. Gutierrez-Camino1 , S. Varona-Fernandez ´ , N. Garc´ıa de Andoin2 , 5 ˜ , V. Dolzan6 , I. Astigarraga3,7 , A. Navajas3 , A. Sastre4 , A. Carbone´ Baneres I. Mart´ın-Guerrero1 , A. Garc´ıa-Orad1,3 . 1 University of the Basque Country, Genetics- Physical Anthropology and Animal Physiology, Bilbao, Spain, 2 Hospital Donostia, Pediatrics, San Sebastian, Spain, 3 BioCruces Health Research Institute, Pediatrics, Barakaldo, Spain, 4 University Hospital La Paz, Oncohaematology, Madrid, Spain, 5 Hospital Miguel Servet, Pediatrics, Zaragoza, Spain, 6 Institute of Biochemistry, Faculty of Medicine, Ljubljana, Slovenia, 7 University Hospital Cruces, Pediatrics, Bilbao, Spain
1
Background: Recent genome wide association study (GWAS) in osteosarcoma risk showed that several significant results were located in intergenic positions. These results suggest that non-coding regions could play an important role in the risk of osteosarcoma. One of the most studied noncoding molecules are microRNAs (miRNAs), whose deregulation has been associated with osteosarcoma evolution. Therefore, genetic variants affecting miRNA function could contribute to the risk of the disease. To date, only three polymorphisms in miRNAs have been analyzed in relation to the risk of osteosarcoma, and two of them showed significant association. In this context, this study aimed to evaluate the involvement of all genetic variants in premiRNAs in the risk of osteosarcoma. Material and Methods: We selected all SNPs described in miRNAs with a MAF >0.1. A total of 213 SNPs in 206 pre-miRNAs were analyzed in a cohort of patients (n = 74) and their corresponding controls (n = 160) using Goldengate Veracode technology. c2 or Fisher and FDR were used. Results: The most remarkably finding was the association detected between 4 SNPs located at 14q32 miRNA cluster. Interestingly, miRNAs in this cluster have been associated with MYC deregulation. Therefore, genetic variations in these miRNAs could lead to their downregulation, which in turn would lead to the overexpression of MYC. This result supports the idea that this region is a hotspot for the development of the disease. Conclusion: In conclusion, different variants in 14q32 miRNA cluster could be implicated in osteosarcoma susceptibility. Acknowledgement: This project was supported by RETICS (RD12/0036/0060, RD12/0036/0036), UPV/EHU (UFI 11/35) and Basque Government (IT66113). No conflict of interest. 173 Genetic variant in miR-618 in the risk of chronic lymphocytic leukemia I. Mart´ın-Guerrero1 , A. D´ıaz-Navarro1 , A. Gutierrez-Camino1 , A. Garc´ıa-Orad1,2 . 1 University of the Basque Country, Genetics- Physical Anthropology and Animal Physiology, Bilbao, Spain, 2 BioCruces Health Research Institute, Pediatrics, Barakaldo, Spain Background: Recent Genome Wide Association Studies (GWAS) have found new genetic variants associated with chronic lymphocytic leukemia (CLL) susceptibility, several of which were located in non-coding regions. In fact, nowadays it is known that more than 40% of significant variants associated with cancer risk are in non-coding regions, where non-coding RNAs are located. MicroRNAs (miRNAs) are one of the most studied non-coding RNAs that have been involved in the risk of CLL. In a previous study of our group including 46 SNPs in 42 pre-miRNAs, we observed rs11614913 and rs2114358 associated with the risk of CLL. Considering these results, we aimed to determine the involvement in the risk of CLL of all the SNPs described in pre-miRNAs with a MAF >1% in a larger population. Material and Methods: A total of 213 SNPs in 206 miRNAs were genotyped in 164 CLL patients and 237 cancer-free controls using Veracode GoldenGate Technology (Illumina). Results: One polymorphism at miR-618 showed the most significant association under the dominant model (OR = 0.49; 95% CI: 0.29–0.81; p = 0.005). Remarkably, this polymorphism was previously found to be associated with follicular lymphoma, altering the expression of miR-618. In addition, this miRNA was observed, in silico, to regulate genes associated with CLL, such as BCL2, LEF1 or QPCT. Conclusion: These findings suggest that polymorphisms in pre-miRNAs contribute to the risk of CLL. Large-scale studies are needed to validate the current findings. Acknowledgement: This project was supported by RETICS (RD12/0036/0060, RD12/0036/0036), UPV/EHU (UFI11/35) and Basque Government (IT661-13, S-PE13UN079). No conflict of interest.
Background: Recently, several Genome wide associations studies (GWAS) have found genetic variants associated with pediatric acute lymphoblastic leukemia (ALL) risk. The interest of these studies was mainly focused in coding regions. However, nowadays it is known that more than 40% of significant variants associated with cancer risk are situated in non-coding regions, where non-coding RNAs are located. One of the non-coding RNAs more related with cancer are microRNAs (miRNAs), which have been shown to be dysregulated in ALL, suggesting that they may have a role in ALL risk. Therefore, variants in miRNAs that may alter its function could contribute to childhood B-ALL predisposition. The aim of this study was to determine if SNPs in miRNAs are involved in B-ALL susceptibility. Material and Methods: Blood samples of 296 B-cell ALL patients in complete remission and 426 healthy controls of Spanish and a Slovenian cohort were analyzed. We selected all the SNPs described in pre-miRNAs with a MAF >1% (213 SNPs in 206 miRNAs). VeraCode GoldenGate platform was used. Results: Among the most interesting results, a variant in the seed region of miR-3117 was associated with B-ALL in both populations (p = 0.006). Of note is that in silico analyses show that miR-3117 could regulate genes of the MAPK pathway, which is known to promote leukemogenesis. Conclusion: Our results suggest that a SNP in miR-3117 may be involved in B-ALL susceptibility and give new keys to understand its biology. Acknowledgement: This project was supported by RETICS (RD12/0036/0060, RD12/0036/0036), UPV/EHU (UFI 11/35) and Basque Government (IT661-13, S-PE12UN060). No conflict of interest.
175 The KDM5B demethylase in the normal and malignant mammary gland F. Kogera1 , C. Steven1 , B. Spencer-Dene2 , G. Picco1 , V. Tajadura-Ortega1 , Y.H. Chen3 , E. Bennett3 , J. Quist4 , J. Taylor-Papadimitriou1 , J. Burchell1 . 1 KCL, Research Oncology, London, United Kingdom, 2 Cancer Research UKCrick Institute, Experimental Histopathology Lab, London, United Kingdom, 3 University of Copenhagen, Copenhagen Centre for Glycomics, Copenhagen, Denmark, 4 KCL, Breast Cancer Now Unit, London, United Kingdom Background: It is becoming clear that changes occurring in cancer, including breast cancer reflect a dysregulated epigenome. Histone marks play a major role in determining the chromatin state of a cell, and the H3K4me3 mark is associated with active transcription. The KDM5 family of histone demethylases specifically demethylate the H3K4me3 mark and therefore act as transcriptional repressors. KDM5B, a KDM5 family member, was cloned in our lab as being down regulated when a cell over expressing HER2 was treated with Herceptin, and has been found to be widely expressed in breast cancer. Here, we investigate the role of KDM5B in the normal gland and in breast cancer, using breast cancer cell lines and a transgenic mouse model. Materials and Methods: Expression of KDM5B in breast cancers has been documented in samples from Cancer databases. Cell lines derived from different breast cancer subtypes have been analysed for KDM5B expression and the effect of a KO and KD of KDM5B examined. While constitutive KO of KDM5B in the mouse is embryonic lethal, a transgenic strain carrying a demethylase −ve mutant (D ARID domain deleted) is viable. This strain has been used to document an important function for KDM5B in the developing and pregnant mammary gland. Results: High expression of KDM5B was observed in the ER+ and HER2+ breast cancer subtypes. In the DARID mouse mammary gland, a transient delay in mammary tree expansion at mid pregnancy is observed. At this stage, expansion of the luminal progenitor cells is evident and involves the JAK/STAT5 signalling pathway. However, in the DARID mouse, STAT5 activation is dramatically reduced, implicating a role of KDM5B in this activation. We find that, decreased STAT5 activation in the DARID gland is due to increased levels of Caveolin1 (CAV1), an inhibitor of STAT5 activation. Thus in the normal gland KDM5B represses CAV1 expression during development of the luminal cell lineage. The effect of CAV1 on STAT5 activation must be mediated in a paracrine fashion, as CAV1 is not expressed in the luminal cells where STAT5 is functional but is limited to stromal and myoepithelial cells. KDM5B is expressed in these cells as well as in the luminal cells and thus could repress CAV1 expression.
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EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
Conclusions: The importance of CAV1 expression in stromal cells in breast cancer is well documented and high expression is associated with good prognosis. Conversely, expression of CAV1 is indicated to be a marker of triple negative cancers. A CRISPR KO of KDM5B in a HER2+ cell line shows increased CAV1 expression, signifying regulation of CAV1 by KDM5B. We are currently setting up methods to examine the effect of KDM5B on CAV1 expression in breast cancer associated fibroblasts to understand the role of cell phenotype in the interactions between KDM5B and CAV1 in modifying JAK/STAT signalling pathways in breast cancer. No conflict of interest. 177 Comprehensive characterization of matched pre-treatment biopsies and residual disease of chemotherapy treated breast cancer M. Hoogstraat1 , E. Lips2 , L. Mulder2 , P. Nederlof3 , G. Sonke4 , S. Rodenhuis4 , J. Wesseling2 , L.F.A. Wessels1 . 1 Netherlands Cancer Institute, Molecular Carcinogenesis, Amsterdam, Netherlands, 2 Netherlands Cancer Institute, Molecular Pathology, Amsterdam, Netherlands, 3 Netherlands Cancer Institute, Pathology, Amsterdam, Netherlands, 4 Netherlands Cancer Institute, Medical Oncology, Amsterdam, Netherlands Introduction: Neoadjuvant chemotherapy is standard of care for locally advanced breast cancer, unfortunately not all patients benefit from this treatment. Even after decades of research, we still cannot predict which tumors will and which ones will not respond. This may in part be due to tumor heterogeneity, as the sample taken before treatment not necessarily represents the tumor cell population that causes therapy resistance. Methods: To test this hypothesis and to study potential mechanisms of therapy resistance, we collected matched blood, pre-treatment and residual disease samples from 21 breast cancer patients treated with doxorubicin and cyclophosphamide in a neoadjuvant setting. Specifically, tumors were selected with a tumor percentage >50% after chemotherapy to enrich for resistant samples and ensure high quality data. RNA and whole exome sequencing data were generated to characterize somatic mutations, copy number alterations and gene expression profiles, and histopathological characteristics were determined to obtain a comprehensive profile of all tumor samples. Results: Both the comparison of somatic variants and copy number alterations revealed a very diverse image: in several cases, high-level amplifications, large genomic gains or losses, and mutations in known oncogenes or tumor suppressors such as MAP3K1 and RUNX1 were either lost or gained during treatment, while in other cases no such changes were detected. We observed a remarkable number of genetic alterations involved in cell cycle progression and DNA damage checkpoints, including amplification of MDM2, CCND1 and CDK4, and copy number loss or mutations in CDKN1B and ATM. Strikingly, both cases of CDKN1B loss were identified in pre-treatment samples and no longer detectable in the surgery specimen. In contrast, CCND1, CDK4 and MDM2 amplifications were retained, although CCND1 expression decreased significantly in CCND1 amplified tumors. In addition, eighty percent of tumors showed a decreased proliferation rate after chemotherapy, where the high-proliferative ER+ (Luminal B) tumors were most severely affected. This trend was also visible in a validation cohort of 94 ER+ samples, but the prognosis of Luminal B tumors that showed a decrease in proliferation was still significantly worse than that of Luminal A tumors that did not show an altered proliferation rate. Conclusion: Our results confirm that biologically relevant genomic alterations can differ between pre- and post-treatment samples, which greatly impedes biomarker discovery. In addition, our findings emphasize the aggressiveness and chemotherapy insensitivity of CCND1 amplified ER+ breast cancers, and stress the need for better treatment regimens for these patients. In contrast, genomic loss of CDKN1B may be a marker for (partial) sensitivity to chemotherapy. No conflict of interest. 178 Epigenetic control of CD24 expression in colorectal cancer M. Ayub1 , W. Bodmer1 . 1 University of Oxford, Oncology, Oxford, United Kingdom Understanding the mechanism and maintenance of heterogeneity in Colorectal Cancer (CRC) is of paramount interest. By cloning CRC cell lines we have demonstrated that CD24 based heterogeneity is quite common and methylation is directly involved in the evolution and maintenance of heterogeneity in Colorectal Cancer. LS174T, a CRC cell line was cloned using FACS and two clones were found to have differential expression of CD24. They differ in morphology, clonogenicity and growth rate. Later SW480, DLD1, RKO and HCT116 were also found to be heterogeneous with clones of different CD24 level. To understand the regulation of CD24, Bisulfite modified DNA from clones were sequenced. Our study has revealed a region near CD24 promoter region, which is directly methylated. When treated with 5-azacytidine, the CD24 negative clones re-expressed CD24 again. We have also found that CD24 is dynamically regulated and the expressions in clones change towards the expression profile
of their parents over time. This is a clear evidence of convergent evolution in cancer cell lines. CD24 is an important marker of cancer stem cell. So far there has been no clear understanding of the mechanism of its regulation. Here we have reported that CD24 is regulated by direct methylation in Colorectal Cancer (CRC) cell lines. We have also shown that clones with differential expression of CD24 can be isolated and stably maintained from CRC cell lines. No conflict of interest. 179 Digital sorting enables whole-exome and low-pass whole-genome sequencing from low tumor-content formalin-fixed paraffin embedded (FFPE) biopsies C. Forcato1 , J. Laliberte2 , C. Bolognesi1 , C. Schumacher2 , G. Buson1 , C. Mangano1 , P. Tononi1 , G. Medoro1 , T. Harkins2 , N. Manaresi1 . 1 Silicon Biosystems spa, R&D, Bologna, Italy, 2 Swift Biosciences Inc, R&D, Ann Arbor, USA Introduction: Recent data from ongoing basket trials show that for 20% of patients, biopsies are discarded for precision oncology because of too small sample size and/or low-tumor content. Here we describe a workflow for whole-exome (WES) and low-pass whole-genome (WGS) sequencing of pure populations of tumor cells obtained from very low tumor-cellularity FFPE samples using DEPArray™ (Silicon Biosystems) sorting technology. Materials and Method: A FFPE 50 mm thick section from breast infiltrating ductal carcinoma, with 10% tumor cellularity, was dissociated into a cell suspension. Using DEPArray™ digital sorter, 419 cells from 100%-pure tumor and 497 cells from normal stromal subpopulations were recovered based on Keratin/Vimentin immunofluorescence and DNA content. After lysis, Illumina® compatible libraries were prepared from cell-sorted or DNA-extracted samples using Accel-NGS® 2S DNA Library Kit from Swift Biosciences, amplified, enriched using SeqCap EZ MedExome enrichment kit (Roche) and sequenced on a HiSeq 2500 at 29x mean coverage producing 100x2 paired-end reads. An aliquot of pre-capture libraries were used for a low-pass (0.06x mean coverage) WGS on MiSeq to analyze copy number alterations (CNA). Sequences were aligned to hg19 genome and preprocessed using open source software (BWA, Picard, GATK). Variant calls were obtained using samtools mpileup. In lowpass WGS analyses, Control-FREEC algorithm was used to obtain copy number calls, using the mode without control sample. Results and Discussion: Matched stromal/tumor analysis of B-allele frequency of heterozygous SNPs precisely identified Loss-of-Heterozygosity (LOH) regions, as well as Copy-gain regions, both undetectable on unsorted genomic DNA (gDNA). Genetic analyses readily revealed a clinically relevant homozygous (23/23 = 100% reads) TP53:p.L111R somatic mutation in a LOH region in sorted-tumor fraction, missed in unsorted gDNA, where the mutation was not called as present in only 1 read out of 20 (5%). Due to the low tumor cellularity, no copy number alterations were detected in low-pass WGS of unsorted gDNA, showing a flat profile undistinguishable from DEPArray™ purified stromal cells, whereas in sorted tumor cells we detected numerous copy-number alterations in form of gains and losses. Conclusions: DEPArray™ sorting combined with Accel-NGS® 2S library kit allow to obtain whole-exome and low-pass whole-genome data on pure tumor and stroma of FFPE samples, offering a clear picture of tumor-specific variants including LOH and copy-numbers independently of original tumor content. Conflict of interest: Other Substantive Relationships: Employees at Silicon Biosystems spa: C. Forcato, C. Bolognesi, G. Buson, C. Mangano, P. Tononi, G. Medoro, N. Manaresi. Employees at Swift Biosciences Inc: J. Laliberte, C. Schumacher, T. Harkins. 180 An oncogenomics-based in vivo screen identifies novel melanoma tumor-suppressors M. Olvedy1 , J.C. Tisserand2 , F. Luciani1 , B. Boeckx3 , F. Rambow1 , J. Wouters4 , J. Van den Oord4 , D. Lambrechts3 , P. De Sepulveda2 , J.C. Marine1 . 1 VIB, Center for the Biology of Disease, Leuven, Belgium, 2 ´ Institut National de la Sante´ et de la Recherche Medicale, Centre de ´ Recherche en Cancerologie de Marseille, Marseille, France, 3 VIB, Vesalius Research Center, Leuven, Belgium, 4 KU Leuven, Department of Pathology, Leuven, Belgium Introduction: Human cutaneous melanomas are notoriously known as the most aggressive and treatment-resistant cancers. Their complex cytogenetic profiles, due to extremely high mutation burden and frequent aneuploidies, however, hamper the discovery of genes that drive this cancer and thus obstruct the development of novel therapeutics to combat melanoma. There is therefore a pressing need for designing biologically meaningful approaches that would facilitate the identification and validation of the driver lesions. We have developed an innovative screen combining the oncogenomic analysis with cross-species comparison and in vivo validation using a refined mouse model system to discover novel genes driving melanoma. Methods: We collected 59 melanoma lesions from various genetically induced mouse melanoma models driven either by activating mutations in BRaf or
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172 14q32 miRNA cluster: A hot spot for osteosarcoma risk in a Spanish population
174 Involvement of miR-3117 in pediatric acute lymphoblastic leukemia susceptibility
I. Mart´ın-Guerrero1 , N. Bilbao-Aldaiturriaga1 , J. Uriz2 , A. Garc´ıa-Orad1,3 . University of the Basque Country, Genetics- Physical Anthropology and Animal Physiology, Bilbao, Spain, 2 University Hospital Donostia, Unit of Pediatric Oncohematology, San Sebastian, Spain, 3 BioCruces Health Research Institute, Pediatrics, Barakaldo, Spain
1 A. Gutierrez-Camino1 , S. Varona-Fernandez ´ , N. Garc´ıa de Andoin2 , 5 ˜ , V. Dolzan6 , I. Astigarraga3,7 , A. Navajas3 , A. Sastre4 , A. Carbone´ Baneres I. Mart´ın-Guerrero1 , A. Garc´ıa-Orad1,3 . 1 University of the Basque Country, Genetics- Physical Anthropology and Animal Physiology, Bilbao, Spain, 2 Hospital Donostia, Pediatrics, San Sebastian, Spain, 3 BioCruces Health Research Institute, Pediatrics, Barakaldo, Spain, 4 University Hospital La Paz, Oncohaematology, Madrid, Spain, 5 Hospital Miguel Servet, Pediatrics, Zaragoza, Spain, 6 Institute of Biochemistry, Faculty of Medicine, Ljubljana, Slovenia, 7 University Hospital Cruces, Pediatrics, Bilbao, Spain
1
Background: Recent genome wide association study (GWAS) in osteosarcoma risk showed that several significant results were located in intergenic positions. These results suggest that non-coding regions could play an important role in the risk of osteosarcoma. One of the most studied noncoding molecules are microRNAs (miRNAs), whose deregulation has been associated with osteosarcoma evolution. Therefore, genetic variants affecting miRNA function could contribute to the risk of the disease. To date, only three polymorphisms in miRNAs have been analyzed in relation to the risk of osteosarcoma, and two of them showed significant association. In this context, this study aimed to evaluate the involvement of all genetic variants in premiRNAs in the risk of osteosarcoma. Material and Methods: We selected all SNPs described in miRNAs with a MAF >0.1. A total of 213 SNPs in 206 pre-miRNAs were analyzed in a cohort of patients (n = 74) and their corresponding controls (n = 160) using Goldengate Veracode technology. c2 or Fisher and FDR were used. Results: The most remarkably finding was the association detected between 4 SNPs located at 14q32 miRNA cluster. Interestingly, miRNAs in this cluster have been associated with MYC deregulation. Therefore, genetic variations in these miRNAs could lead to their downregulation, which in turn would lead to the overexpression of MYC. This result supports the idea that this region is a hotspot for the development of the disease. Conclusion: In conclusion, different variants in 14q32 miRNA cluster could be implicated in osteosarcoma susceptibility. Acknowledgement: This project was supported by RETICS (RD12/0036/0060, RD12/0036/0036), UPV/EHU (UFI 11/35) and Basque Government (IT66113). No conflict of interest. 173 Genetic variant in miR-618 in the risk of chronic lymphocytic leukemia I. Mart´ın-Guerrero1 , A. D´ıaz-Navarro1 , A. Gutierrez-Camino1 , A. Garc´ıa-Orad1,2 . 1 University of the Basque Country, Genetics- Physical Anthropology and Animal Physiology, Bilbao, Spain, 2 BioCruces Health Research Institute, Pediatrics, Barakaldo, Spain Background: Recent Genome Wide Association Studies (GWAS) have found new genetic variants associated with chronic lymphocytic leukemia (CLL) susceptibility, several of which were located in non-coding regions. In fact, nowadays it is known that more than 40% of significant variants associated with cancer risk are in non-coding regions, where non-coding RNAs are located. MicroRNAs (miRNAs) are one of the most studied non-coding RNAs that have been involved in the risk of CLL. In a previous study of our group including 46 SNPs in 42 pre-miRNAs, we observed rs11614913 and rs2114358 associated with the risk of CLL. Considering these results, we aimed to determine the involvement in the risk of CLL of all the SNPs described in pre-miRNAs with a MAF >1% in a larger population. Material and Methods: A total of 213 SNPs in 206 miRNAs were genotyped in 164 CLL patients and 237 cancer-free controls using Veracode GoldenGate Technology (Illumina). Results: One polymorphism at miR-618 showed the most significant association under the dominant model (OR = 0.49; 95% CI: 0.29–0.81; p = 0.005). Remarkably, this polymorphism was previously found to be associated with follicular lymphoma, altering the expression of miR-618. In addition, this miRNA was observed, in silico, to regulate genes associated with CLL, such as BCL2, LEF1 or QPCT. Conclusion: These findings suggest that polymorphisms in pre-miRNAs contribute to the risk of CLL. Large-scale studies are needed to validate the current findings. Acknowledgement: This project was supported by RETICS (RD12/0036/0060, RD12/0036/0036), UPV/EHU (UFI11/35) and Basque Government (IT661-13, S-PE13UN079). No conflict of interest.
Background: Recently, several Genome wide associations studies (GWAS) have found genetic variants associated with pediatric acute lymphoblastic leukemia (ALL) risk. The interest of these studies was mainly focused in coding regions. However, nowadays it is known that more than 40% of significant variants associated with cancer risk are situated in non-coding regions, where non-coding RNAs are located. One of the non-coding RNAs more related with cancer are microRNAs (miRNAs), which have been shown to be dysregulated in ALL, suggesting that they may have a role in ALL risk. Therefore, variants in miRNAs that may alter its function could contribute to childhood B-ALL predisposition. The aim of this study was to determine if SNPs in miRNAs are involved in B-ALL susceptibility. Material and Methods: Blood samples of 296 B-cell ALL patients in complete remission and 426 healthy controls of Spanish and a Slovenian cohort were analyzed. We selected all the SNPs described in pre-miRNAs with a MAF >1% (213 SNPs in 206 miRNAs). VeraCode GoldenGate platform was used. Results: Among the most interesting results, a variant in the seed region of miR-3117 was associated with B-ALL in both populations (p = 0.006). Of note is that in silico analyses show that miR-3117 could regulate genes of the MAPK pathway, which is known to promote leukemogenesis. Conclusion: Our results suggest that a SNP in miR-3117 may be involved in B-ALL susceptibility and give new keys to understand its biology. Acknowledgement: This project was supported by RETICS (RD12/0036/0060, RD12/0036/0036), UPV/EHU (UFI 11/35) and Basque Government (IT661-13, S-PE12UN060). No conflict of interest.
175 The KDM5B demethylase in the normal and malignant mammary gland F. Kogera1 , C. Steven1 , B. Spencer-Dene2 , G. Picco1 , V. Tajadura-Ortega1 , Y.H. Chen3 , E. Bennett3 , J. Quist4 , J. Taylor-Papadimitriou1 , J. Burchell1 . 1 KCL, Research Oncology, London, United Kingdom, 2 Cancer Research UKCrick Institute, Experimental Histopathology Lab, London, United Kingdom, 3 University of Copenhagen, Copenhagen Centre for Glycomics, Copenhagen, Denmark, 4 KCL, Breast Cancer Now Unit, London, United Kingdom Background: It is becoming clear that changes occurring in cancer, including breast cancer reflect a dysregulated epigenome. Histone marks play a major role in determining the chromatin state of a cell, and the H3K4me3 mark is associated with active transcription. The KDM5 family of histone demethylases specifically demethylate the H3K4me3 mark and therefore act as transcriptional repressors. KDM5B, a KDM5 family member, was cloned in our lab as being down regulated when a cell over expressing HER2 was treated with Herceptin, and has been found to be widely expressed in breast cancer. Here, we investigate the role of KDM5B in the normal gland and in breast cancer, using breast cancer cell lines and a transgenic mouse model. Materials and Methods: Expression of KDM5B in breast cancers has been documented in samples from Cancer databases. Cell lines derived from different breast cancer subtypes have been analysed for KDM5B expression and the effect of a KO and KD of KDM5B examined. While constitutive KO of KDM5B in the mouse is embryonic lethal, a transgenic strain carrying a demethylase −ve mutant (D ARID domain deleted) is viable. This strain has been used to document an important function for KDM5B in the developing and pregnant mammary gland. Results: High expression of KDM5B was observed in the ER+ and HER2+ breast cancer subtypes. In the DARID mouse mammary gland, a transient delay in mammary tree expansion at mid pregnancy is observed. At this stage, expansion of the luminal progenitor cells is evident and involves the JAK/STAT5 signalling pathway. However, in the DARID mouse, STAT5 activation is dramatically reduced, implicating a role of KDM5B in this activation. We find that, decreased STAT5 activation in the DARID gland is due to increased levels of Caveolin1 (CAV1), an inhibitor of STAT5 activation. Thus in the normal gland KDM5B represses CAV1 expression during development of the luminal cell lineage. The effect of CAV1 on STAT5 activation must be mediated in a paracrine fashion, as CAV1 is not expressed in the luminal cells where STAT5 is functional but is limited to stromal and myoepithelial cells. KDM5B is expressed in these cells as well as in the luminal cells and thus could repress CAV1 expression.
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EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
Conclusions: The importance of CAV1 expression in stromal cells in breast cancer is well documented and high expression is associated with good prognosis. Conversely, expression of CAV1 is indicated to be a marker of triple negative cancers. A CRISPR KO of KDM5B in a HER2+ cell line shows increased CAV1 expression, signifying regulation of CAV1 by KDM5B. We are currently setting up methods to examine the effect of KDM5B on CAV1 expression in breast cancer associated fibroblasts to understand the role of cell phenotype in the interactions between KDM5B and CAV1 in modifying JAK/STAT signalling pathways in breast cancer. No conflict of interest. 177 Comprehensive characterization of matched pre-treatment biopsies and residual disease of chemotherapy treated breast cancer M. Hoogstraat1 , E. Lips2 , L. Mulder2 , P. Nederlof3 , G. Sonke4 , S. Rodenhuis4 , J. Wesseling2 , L.F.A. Wessels1 . 1 Netherlands Cancer Institute, Molecular Carcinogenesis, Amsterdam, Netherlands, 2 Netherlands Cancer Institute, Molecular Pathology, Amsterdam, Netherlands, 3 Netherlands Cancer Institute, Pathology, Amsterdam, Netherlands, 4 Netherlands Cancer Institute, Medical Oncology, Amsterdam, Netherlands Introduction: Neoadjuvant chemotherapy is standard of care for locally advanced breast cancer, unfortunately not all patients benefit from this treatment. Even after decades of research, we still cannot predict which tumors will and which ones will not respond. This may in part be due to tumor heterogeneity, as the sample taken before treatment not necessarily represents the tumor cell population that causes therapy resistance. Methods: To test this hypothesis and to study potential mechanisms of therapy resistance, we collected matched blood, pre-treatment and residual disease samples from 21 breast cancer patients treated with doxorubicin and cyclophosphamide in a neoadjuvant setting. Specifically, tumors were selected with a tumor percentage >50% after chemotherapy to enrich for resistant samples and ensure high quality data. RNA and whole exome sequencing data were generated to characterize somatic mutations, copy number alterations and gene expression profiles, and histopathological characteristics were determined to obtain a comprehensive profile of all tumor samples. Results: Both the comparison of somatic variants and copy number alterations revealed a very diverse image: in several cases, high-level amplifications, large genomic gains or losses, and mutations in known oncogenes or tumor suppressors such as MAP3K1 and RUNX1 were either lost or gained during treatment, while in other cases no such changes were detected. We observed a remarkable number of genetic alterations involved in cell cycle progression and DNA damage checkpoints, including amplification of MDM2, CCND1 and CDK4, and copy number loss or mutations in CDKN1B and ATM. Strikingly, both cases of CDKN1B loss were identified in pre-treatment samples and no longer detectable in the surgery specimen. In contrast, CCND1, CDK4 and MDM2 amplifications were retained, although CCND1 expression decreased significantly in CCND1 amplified tumors. In addition, eighty percent of tumors showed a decreased proliferation rate after chemotherapy, where the high-proliferative ER+ (Luminal B) tumors were most severely affected. This trend was also visible in a validation cohort of 94 ER+ samples, but the prognosis of Luminal B tumors that showed a decrease in proliferation was still significantly worse than that of Luminal A tumors that did not show an altered proliferation rate. Conclusion: Our results confirm that biologically relevant genomic alterations can differ between pre- and post-treatment samples, which greatly impedes biomarker discovery. In addition, our findings emphasize the aggressiveness and chemotherapy insensitivity of CCND1 amplified ER+ breast cancers, and stress the need for better treatment regimens for these patients. In contrast, genomic loss of CDKN1B may be a marker for (partial) sensitivity to chemotherapy. No conflict of interest. 178 Epigenetic control of CD24 expression in colorectal cancer M. Ayub1 , W. Bodmer1 . 1 University of Oxford, Oncology, Oxford, United Kingdom Understanding the mechanism and maintenance of heterogeneity in Colorectal Cancer (CRC) is of paramount interest. By cloning CRC cell lines we have demonstrated that CD24 based heterogeneity is quite common and methylation is directly involved in the evolution and maintenance of heterogeneity in Colorectal Cancer. LS174T, a CRC cell line was cloned using FACS and two clones were found to have differential expression of CD24. They differ in morphology, clonogenicity and growth rate. Later SW480, DLD1, RKO and HCT116 were also found to be heterogeneous with clones of different CD24 level. To understand the regulation of CD24, Bisulfite modified DNA from clones were sequenced. Our study has revealed a region near CD24 promoter region, which is directly methylated. When treated with 5-azacytidine, the CD24 negative clones re-expressed CD24 again. We have also found that CD24 is dynamically regulated and the expressions in clones change towards the expression profile
of their parents over time. This is a clear evidence of convergent evolution in cancer cell lines. CD24 is an important marker of cancer stem cell. So far there has been no clear understanding of the mechanism of its regulation. Here we have reported that CD24 is regulated by direct methylation in Colorectal Cancer (CRC) cell lines. We have also shown that clones with differential expression of CD24 can be isolated and stably maintained from CRC cell lines. No conflict of interest. 179 Digital sorting enables whole-exome and low-pass whole-genome sequencing from low tumor-content formalin-fixed paraffin embedded (FFPE) biopsies C. Forcato1 , J. Laliberte2 , C. Bolognesi1 , C. Schumacher2 , G. Buson1 , C. Mangano1 , P. Tononi1 , G. Medoro1 , T. Harkins2 , N. Manaresi1 . 1 Silicon Biosystems spa, R&D, Bologna, Italy, 2 Swift Biosciences Inc, R&D, Ann Arbor, USA Introduction: Recent data from ongoing basket trials show that for 20% of patients, biopsies are discarded for precision oncology because of too small sample size and/or low-tumor content. Here we describe a workflow for whole-exome (WES) and low-pass whole-genome (WGS) sequencing of pure populations of tumor cells obtained from very low tumor-cellularity FFPE samples using DEPArray™ (Silicon Biosystems) sorting technology. Materials and Method: A FFPE 50 mm thick section from breast infiltrating ductal carcinoma, with 10% tumor cellularity, was dissociated into a cell suspension. Using DEPArray™ digital sorter, 419 cells from 100%-pure tumor and 497 cells from normal stromal subpopulations were recovered based on Keratin/Vimentin immunofluorescence and DNA content. After lysis, Illumina® compatible libraries were prepared from cell-sorted or DNA-extracted samples using Accel-NGS® 2S DNA Library Kit from Swift Biosciences, amplified, enriched using SeqCap EZ MedExome enrichment kit (Roche) and sequenced on a HiSeq 2500 at 29x mean coverage producing 100x2 paired-end reads. An aliquot of pre-capture libraries were used for a low-pass (0.06x mean coverage) WGS on MiSeq to analyze copy number alterations (CNA). Sequences were aligned to hg19 genome and preprocessed using open source software (BWA, Picard, GATK). Variant calls were obtained using samtools mpileup. In lowpass WGS analyses, Control-FREEC algorithm was used to obtain copy number calls, using the mode without control sample. Results and Discussion: Matched stromal/tumor analysis of B-allele frequency of heterozygous SNPs precisely identified Loss-of-Heterozygosity (LOH) regions, as well as Copy-gain regions, both undetectable on unsorted genomic DNA (gDNA). Genetic analyses readily revealed a clinically relevant homozygous (23/23 = 100% reads) TP53:p.L111R somatic mutation in a LOH region in sorted-tumor fraction, missed in unsorted gDNA, where the mutation was not called as present in only 1 read out of 20 (5%). Due to the low tumor cellularity, no copy number alterations were detected in low-pass WGS of unsorted gDNA, showing a flat profile undistinguishable from DEPArray™ purified stromal cells, whereas in sorted tumor cells we detected numerous copy-number alterations in form of gains and losses. Conclusions: DEPArray™ sorting combined with Accel-NGS® 2S library kit allow to obtain whole-exome and low-pass whole-genome data on pure tumor and stroma of FFPE samples, offering a clear picture of tumor-specific variants including LOH and copy-numbers independently of original tumor content. Conflict of interest: Other Substantive Relationships: Employees at Silicon Biosystems spa: C. Forcato, C. Bolognesi, G. Buson, C. Mangano, P. Tononi, G. Medoro, N. Manaresi. Employees at Swift Biosciences Inc: J. Laliberte, C. Schumacher, T. Harkins. 180 An oncogenomics-based in vivo screen identifies novel melanoma tumor-suppressors M. Olvedy1 , J.C. Tisserand2 , F. Luciani1 , B. Boeckx3 , F. Rambow1 , J. Wouters4 , J. Van den Oord4 , D. Lambrechts3 , P. De Sepulveda2 , J.C. Marine1 . 1 VIB, Center for the Biology of Disease, Leuven, Belgium, 2 ´ Institut National de la Sante´ et de la Recherche Medicale, Centre de ´ Recherche en Cancerologie de Marseille, Marseille, France, 3 VIB, Vesalius Research Center, Leuven, Belgium, 4 KU Leuven, Department of Pathology, Leuven, Belgium Introduction: Human cutaneous melanomas are notoriously known as the most aggressive and treatment-resistant cancers. Their complex cytogenetic profiles, due to extremely high mutation burden and frequent aneuploidies, however, hamper the discovery of genes that drive this cancer and thus obstruct the development of novel therapeutics to combat melanoma. There is therefore a pressing need for designing biologically meaningful approaches that would facilitate the identification and validation of the driver lesions. We have developed an innovative screen combining the oncogenomic analysis with cross-species comparison and in vivo validation using a refined mouse model system to discover novel genes driving melanoma. Methods: We collected 59 melanoma lesions from various genetically induced mouse melanoma models driven either by activating mutations in BRaf or