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402: Mutational landscape of the mediator complex across human cancers
S96
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
400 Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing E. Heitzer1 , S. Mohan1 , P. Ulz1 , I. Lafer1 , M. Auer1 , S. Lax2 , G. Hoefler3 , T. Bauernhofer4 , J.B. Geigl1 , M.R. Speicher1 . 1 Medical University of Graz, Institute of Human Genetics, Graz, Austria, 2 General Hospital Graz West, Department of Pathology, Graz, Austria, 3 Medical University of Graz, Institute of Pathology, Graz, Austria, 4 Medical University of Graz, Division of Oncology, Graz, Austria Introduction: Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Material and Method: Here we analyzed plasma DNA from 10 patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultrasensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Results and Discussion: Surprisingly we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in 4 patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in 7 patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Conclusion: Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression. No conflict of interest. 401 Chromatin remodelling by the p400 ATPase influences DNA double-strand breaks repair and genetic instability independently of the H2AZ histone variant incorporation Y. Canitrot1 , C. Courilleau2 , C. Chailleux2 , G.C. Taty-Taty2 , M. Quaranta2 , A. Jauneau3 , E. Boutet-Robinet4 , D. Trouche5 . 1 LBCMCP Universite´ P Sabatier, Toulouse, France, 2 LBCMCP Universite´ P Sabatier, CNRS ˆ UMR5088, Toulouse, France, 3 Plateforme Microscopie Imagerie Pole ´ etale, ´ Biotechnologie Veg CNRS FR3450, Castanet Tolosan, France, 4 INRA UMR1131, Toxalim Research Centre in Food Toxicology, Toulouse, France, 5 LBCMCP Universite´ P Sabatier, CNRS UMR5088, Toulouse, France Background: DNA damage signalling and repair take place in a chromatin context. Consequently, chromatin modifying enzymes, including ATPdependent chromatin remodelling enzymes play an important role in the management of DNA damage and particularly for DNA double strand breaks (DSB). Material and Methods: We use siRNA approach to deplete p400 in mammalian cells (the osteosarcoma cell line U2OS and immortalized human fibroblasts). These cell lines express GFP reporter system to evaluate DSB repair by the two main pathways, homologous recombination (HR) and non homologous end joining (NHEJ). We investigated the presence of DNA damage by comet assay and the response to DNA damage was also examined by immunofluorescence against numerous actors of the DNA damage response (gH2AX, 53BP1, rad51). Recruitment of proteins to DSB was studied by chromatin immunoprecipitation and protein–protein interactions were investigated by FRET/FLIM technique. Results: We show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, while p400 is not required for DNA damage signalling, DNA double strand breaks repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair and survival following DNA damage. Strikingly, p400 and Rad51 are present in the same complex and interact and both favour chromatin remodelling around DSBs. The ATPase activity of p400 is necessary to the incorporation of the histone variant H2AZ. However, we did not observe any link between H2A.Z incorporation and the phenotypes observed after p400 depletion. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodelling enzyme involved in chromatin decompaction around DNA double strand breaks. Conclusions: We identified the p400 ATPase as an actor of the regulation of DSB repair by HR and an important brake to the generation of genetic instability. No conflict of interest.
402 Mutational landscape of the mediator complex across human cancers 1 A. Offermann1 , Z. Shaikhibrahim1 , D. Bohm ¨ , M. Deng1 , S. Perner1 . 1 Institute of Pathology University Hospital of Bonn, Department of Prostate Cancer Research, Bonn, Germany Introduction: The Mediator complex is a key regulator of protein codinggenes and an integrative hub for signaling pathways, and contains the four modules head, middle, tail and kinase. Even though altered expression of several Mediator subunits were shown in various cancers, mutations were reported for only a few subunits in some cancers. Material and Method: We utilized deposited exome data of 6,010 patients from The Cancer Genome Atlas and analyzed the mutation status of all 33 Mediator subunits in 20 different human cancers. Results and Discussion: We found a unique mutational landscape, identifying the kinase and tail as hot spots across all cancers. Furthermore, a recurrent mutation in the MED12L kinase subunit was found across six cancer types, whereas two recurrent mutations in the MED15 tail subunit were detected in five different cancers. Conclusion: Our systematic and comprehensive analysis reveals for the first time the mutational landscape of the whole Mediator complex in cancer, and suggests that mutations in specific subunits may constitute a hallmark of cancer. No conflict of interest. 403 Comparative evaluation of EGFR mutation testing of tissue and cytology using direct sequencing, pyrosequencing and peptide nucleic acid clamping in lung adenocarcinoma W. Kim1 , K.W. Min1 , K.Y. Lee2 . 1 Konkuk University School of Medicine, Pathology, Seoul, South Korea, 2 Konkuk University School of Medicine, Internal Medicine, Seoul, South Korea Background: The importance of sensitive methods for the EGFR mutant detection is emphasized. The aim of study is to examine the comparative and concordance analyses among application of direct sequencing, pyrosequencing and peptide nucleic acid (PNA) clamping to detect EGFR gene mutation using archived tissue and cytology specimens. Materials and Methods: Samples from a total of 63 cases, which were diagnosed as adenocarcinoma of the lung at Konkuk university medical center between 2006 and 2012, were collected. Based on the above three methods, the concordance rates of EGFR mutations for in exon 18, 19, 20, 21 were analyzed and validated in comparative tissue and cytology specimens. Results: Comparison of EGFR mutation detection between tissue and cytology had a high concordance rate (ú coefficients: 0.836). The diagnostic performance of pyrosequencing and PNA clamping in tissue (82.5% and 77.5% of sensitivity in tissue, 80% and 72.5% of sensitivity in cytology, respectively) was higher than that of direct sequencing (67.5% of sensitivity in tissue, 67.5% of sensitivity in cytology) as well as cytology. Additionally, among patients who had EGFR wild type by single method, EGFR mutations were detected by other methods. Conclusions: Cytology specimens had a diagnostic performance for the detection of EGFR mutations. For detecting EGFR mutation, pyrosequencing or PNA clamping was more sensitive than direct sequencing. In cases of EGFR mutation negative patients, repeating test using other methods might be considered for improving diagnsotic accuracy. No conflict of interest. 405 Tolerance to mitotic defects contributes to chromosomal instability in post-tetraploid progeny 1 A. Kuznetsova1 , S. Muller ¨ 2 , M. Durrbaum ¨ , Z. Storchova1 . 1 Max Planck Institute for Biochemistry, Maintenance of Genome Stability, Martinsried/Munich, Germany, 2 Ludwig Maximilian University, Institute for Human Genetics, Munich, Germany
Background: Aneuploidy, defined as alterations in both chromosome number and structure, along with chromosomal instability (CIN) are common hallmarks of cancer. Mounting evidence suggests that aneuploidy and CIN can facilitate carcinogenesis in humans. One of the possible routes to aneuploidy and CIN can be via an unstable tetraploid intermediate. However, the mechanisms contributing to CIN observed in post-tetraploid progeny remain elusive. Material and Methods: We recapitulated unstable tetraploid intermediate model using in vitro evolution of tetraploids derived from otherwise chromosomally stable and p53-proficient human cells. Results: The majority of newly formed tetraploid cells undergo p53-dependent cycle arrest after chromosome missegregation already in the first tetraploid mitosis, similarly as reported for aneuploid cells formed after induced chromosome missegregation. However, around 1% of cells escapes the arrest and further proliferates. We found that the cells display increased levels of chromosome missegregation even despite normal centrosome numbers. Analyzed post-tetraploid cell lines share remarkable features with some cancers: for example, near-triploid karyotype, variable frequencies of
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
400 Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing E. Heitzer1 , S. Mohan1 , P. Ulz1 , I. Lafer1 , M. Auer1 , S. Lax2 , G. Hoefler3 , T. Bauernhofer4 , J.B. Geigl1 , M.R. Speicher1 . 1 Medical University of Graz, Institute of Human Genetics, Graz, Austria, 2 General Hospital Graz West, Department of Pathology, Graz, Austria, 3 Medical University of Graz, Institute of Pathology, Graz, Austria, 4 Medical University of Graz, Division of Oncology, Graz, Austria Introduction: Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Material and Method: Here we analyzed plasma DNA from 10 patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultrasensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Results and Discussion: Surprisingly we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in 4 patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in 7 patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Conclusion: Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression. No conflict of interest. 401 Chromatin remodelling by the p400 ATPase influences DNA double-strand breaks repair and genetic instability independently of the H2AZ histone variant incorporation Y. Canitrot1 , C. Courilleau2 , C. Chailleux2 , G.C. Taty-Taty2 , M. Quaranta2 , A. Jauneau3 , E. Boutet-Robinet4 , D. Trouche5 . 1 LBCMCP Universite´ P Sabatier, Toulouse, France, 2 LBCMCP Universite´ P Sabatier, CNRS ˆ UMR5088, Toulouse, France, 3 Plateforme Microscopie Imagerie Pole ´ etale, ´ Biotechnologie Veg CNRS FR3450, Castanet Tolosan, France, 4 INRA UMR1131, Toxalim Research Centre in Food Toxicology, Toulouse, France, 5 LBCMCP Universite´ P Sabatier, CNRS UMR5088, Toulouse, France Background: DNA damage signalling and repair take place in a chromatin context. Consequently, chromatin modifying enzymes, including ATPdependent chromatin remodelling enzymes play an important role in the management of DNA damage and particularly for DNA double strand breaks (DSB). Material and Methods: We use siRNA approach to deplete p400 in mammalian cells (the osteosarcoma cell line U2OS and immortalized human fibroblasts). These cell lines express GFP reporter system to evaluate DSB repair by the two main pathways, homologous recombination (HR) and non homologous end joining (NHEJ). We investigated the presence of DNA damage by comet assay and the response to DNA damage was also examined by immunofluorescence against numerous actors of the DNA damage response (gH2AX, 53BP1, rad51). Recruitment of proteins to DSB was studied by chromatin immunoprecipitation and protein–protein interactions were investigated by FRET/FLIM technique. Results: We show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, while p400 is not required for DNA damage signalling, DNA double strand breaks repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair and survival following DNA damage. Strikingly, p400 and Rad51 are present in the same complex and interact and both favour chromatin remodelling around DSBs. The ATPase activity of p400 is necessary to the incorporation of the histone variant H2AZ. However, we did not observe any link between H2A.Z incorporation and the phenotypes observed after p400 depletion. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodelling enzyme involved in chromatin decompaction around DNA double strand breaks. Conclusions: We identified the p400 ATPase as an actor of the regulation of DSB repair by HR and an important brake to the generation of genetic instability. No conflict of interest.
402 Mutational landscape of the mediator complex across human cancers 1 A. Offermann1 , Z. Shaikhibrahim1 , D. Bohm ¨ , M. Deng1 , S. Perner1 . 1 Institute of Pathology University Hospital of Bonn, Department of Prostate Cancer Research, Bonn, Germany Introduction: The Mediator complex is a key regulator of protein codinggenes and an integrative hub for signaling pathways, and contains the four modules head, middle, tail and kinase. Even though altered expression of several Mediator subunits were shown in various cancers, mutations were reported for only a few subunits in some cancers. Material and Method: We utilized deposited exome data of 6,010 patients from The Cancer Genome Atlas and analyzed the mutation status of all 33 Mediator subunits in 20 different human cancers. Results and Discussion: We found a unique mutational landscape, identifying the kinase and tail as hot spots across all cancers. Furthermore, a recurrent mutation in the MED12L kinase subunit was found across six cancer types, whereas two recurrent mutations in the MED15 tail subunit were detected in five different cancers. Conclusion: Our systematic and comprehensive analysis reveals for the first time the mutational landscape of the whole Mediator complex in cancer, and suggests that mutations in specific subunits may constitute a hallmark of cancer. No conflict of interest. 403 Comparative evaluation of EGFR mutation testing of tissue and cytology using direct sequencing, pyrosequencing and peptide nucleic acid clamping in lung adenocarcinoma W. Kim1 , K.W. Min1 , K.Y. Lee2 . 1 Konkuk University School of Medicine, Pathology, Seoul, South Korea, 2 Konkuk University School of Medicine, Internal Medicine, Seoul, South Korea Background: The importance of sensitive methods for the EGFR mutant detection is emphasized. The aim of study is to examine the comparative and concordance analyses among application of direct sequencing, pyrosequencing and peptide nucleic acid (PNA) clamping to detect EGFR gene mutation using archived tissue and cytology specimens. Materials and Methods: Samples from a total of 63 cases, which were diagnosed as adenocarcinoma of the lung at Konkuk university medical center between 2006 and 2012, were collected. Based on the above three methods, the concordance rates of EGFR mutations for in exon 18, 19, 20, 21 were analyzed and validated in comparative tissue and cytology specimens. Results: Comparison of EGFR mutation detection between tissue and cytology had a high concordance rate (ú coefficients: 0.836). The diagnostic performance of pyrosequencing and PNA clamping in tissue (82.5% and 77.5% of sensitivity in tissue, 80% and 72.5% of sensitivity in cytology, respectively) was higher than that of direct sequencing (67.5% of sensitivity in tissue, 67.5% of sensitivity in cytology) as well as cytology. Additionally, among patients who had EGFR wild type by single method, EGFR mutations were detected by other methods. Conclusions: Cytology specimens had a diagnostic performance for the detection of EGFR mutations. For detecting EGFR mutation, pyrosequencing or PNA clamping was more sensitive than direct sequencing. In cases of EGFR mutation negative patients, repeating test using other methods might be considered for improving diagnsotic accuracy. No conflict of interest. 405 Tolerance to mitotic defects contributes to chromosomal instability in post-tetraploid progeny 1 A. Kuznetsova1 , S. Muller ¨ 2 , M. Durrbaum ¨ , Z. Storchova1 . 1 Max Planck Institute for Biochemistry, Maintenance of Genome Stability, Martinsried/Munich, Germany, 2 Ludwig Maximilian University, Institute for Human Genetics, Munich, Germany
Background: Aneuploidy, defined as alterations in both chromosome number and structure, along with chromosomal instability (CIN) are common hallmarks of cancer. Mounting evidence suggests that aneuploidy and CIN can facilitate carcinogenesis in humans. One of the possible routes to aneuploidy and CIN can be via an unstable tetraploid intermediate. However, the mechanisms contributing to CIN observed in post-tetraploid progeny remain elusive. Material and Methods: We recapitulated unstable tetraploid intermediate model using in vitro evolution of tetraploids derived from otherwise chromosomally stable and p53-proficient human cells. Results: The majority of newly formed tetraploid cells undergo p53-dependent cycle arrest after chromosome missegregation already in the first tetraploid mitosis, similarly as reported for aneuploid cells formed after induced chromosome missegregation. However, around 1% of cells escapes the arrest and further proliferates. We found that the cells display increased levels of chromosome missegregation even despite normal centrosome numbers. Analyzed post-tetraploid cell lines share remarkable features with some cancers: for example, near-triploid karyotype, variable frequencies of