431: Identification of deregulated genes in colorectal cancer metastasis through whole genome and transcriptome sequencing

EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 Conclusions: Our results provide novel evidence that the NuRD complex and PRC2 coordinate to suppress gene expression, which reveals a novel link among epigenetic regulation, mTOR pathway and tumorigenesis. No conflict of interest. 428 Detecting and quantifying low level variants in Sanger sequencing traces E. Schreiber1 , S. Berosik1 , M. Wenz1 . 1 Thermo Fisher, Life Sciences Solutions, South San Francisco, USA Introduction: Automated fluorescent dye-terminator DNA Sequencing using capillary electrophoresis (also known as CE or Sanger sequencing) has been instrumental in the detailed characterization of the human genome and is now widely used as gold standard method for verification of mutation findings, notably in tumor samples. The primary information of the DNA sequencing process is the identification of the nucleotides and of possible sequence variants. A largely unexplored feature of fluorescent Sanger sequencing traces is the quantitative information embedded therein. With the growing need for quantifying somatic mutations in tumor tissue it is desirable to exploit the potential of the quantitative information obtained from sequencing traces. Materials and Methods: To this end, we have developed a software tool that converts a Sanger sequencing trace file into a comma separated values (.csv) file containing numerical data of peak data characteristics that can be explored and analyzed using conventional spreadsheet software. The web-based tool can be accessed at http://apps.lifetechnologies.com/ab1peakreporter. The output file contains the peak height and quality values for each nucleotide and peak height ratios for all 4 bases at any given locus allowing the detection and assessment of subtle changes at any given allele. Results and Discussion: We demonstrate the utility of this tool by analyzing mixed DNA samples with known amounts of spiked in variant alleles from the human TP53 gene ranging from 2.5%, 5%, 7.5%, 10%, 15% and 25% and show that the minor alleles could be readily detected below the 10% level. Conclusion: Enabling high sensitivity detection of minor alleles with a widely available and simple to use technology like Sanger sequencing will be useful for verification of results obtained from next generation sequencing (NGS) platforms. Conflict of interest: Corporate-sponsored research: all authors are employees of Thermo Fisher Scientific. 429 Targeted next-generation sequencing − Identification of Lynch syndrome cases B.A. Talseth-Palmer1 , T.J. Evans1 , A. Spigelman2 , R.J. Scott3 . 1 University of Newcastle and Hunter Medical Research Institute, School of Biomedical Sciences and Pharmacy, Newcastle NSW, Australia, 2 Hunter Family Cancer Service and St Vincent’s Hospital Clinical School and St Vincent’s Hospital Hereditary Cancer Clinic, Hunter New England Health and University of NSW, Newcastle and Sydney NSW, Australia, 3 University of Newcastle and Hunter Medical Research Institute and Hunter Area Pathology Service, School of Biomedical Sciences and Pharmacy and Hunter New England Health, Newcastle NSW, Australia Introduction: Cancer is a major burden to society and in some cases preventable. Hereditary non-polyposis colorectal cancer (HNPCC), also known as Lynch syndrome (LS) is an inherited cancer syndrome that accounts for 2−5% of all colon cancer cases. A germline mutation in DNA mismatch repair (MMR) genes can only be identified in ~50% of probands with a clinical diagnosis of HNPCC. On top of high risk of colorectal cancer (CRC), women diagnosed with LS are also at high risk of developing endometrial cancer (EC). The rate of MSI tumours reported in EC cases is much higher compared to other cancers, illustrating that an abnormal DNA MMR pathway might play a role in EC tumourigenesis. This study aims to search for novel mutations/genes in the MMR pathway in patients with a clinical diagnosis of HNPCC and in sporadic EC cases. Material and Method: Utilising targeted next-generation sequencing (NGS) we screened 22 genes involved in the DNA MMR pathway in 15 HNPCC probands, 13 sporadic EC cases and 2 MMR mutation positive controls (LS cases). In brief, the experiment pipeline consisted of; extracted DNA amplified by ligation-mediated PCR, purified and hybridised to NibleGen human custom array for enrichment before each captured library was loaded on a HiSeq2000 platform and sequencing for each captured library was performed independently to ensure 100x coverage. The bioinformatics analysis consisted of data filtering (removing adaptors contamination and low-quality reads from raw reads), alignments and summary of data production, plus SNP and InDel calling, annotation and statistic utilising commercially available software. Results: Preliminary analysis indicates the presence of an exonic frameshift insertion in LIG1, a gene not previously associated with HNPCC/LS, in 1 HNPCC case and two exonic non-frameshift deletions in MSH3 in 4 HNPCC cases and 5 EC cases. On average we detected 74 and 66 novel SNPs and 73 and 71 novel InDels in the HNPCC and EC cases respectively. All variants

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identified will be followed up with in silico analysis including protein function prediction programs and splice prediction programs where relevant. Reference to appropriate variant databases and comprehensive literature searches will also be performed to ascertain the likely significance of all identified variants. These functions will be incorporated into the bioinformatics pipeline wherever possible to reduce the workload required. Conclusion: This dataset has the potential to discover novel mutations/genes associated with HNPCC, which will expand the criteria for a molecular diagnosis of LS. The discovery of new genetic loci affecting the risk of developing cancer will have major implications for the families of affected individuals, providing enhanced testing and personalised screening programs. No conflict of interest. 431 Identification of deregulated genes in colorectal cancer metastasis through whole genome and transcriptome sequencing C. Hauser1,3 , B. Hutter2 , I. Buchhalter2 , A. Shavinskaya3 , M. Abba1,3 , H. Yazdanparast3 , R. Eils2,4 , B. Brors2 , H. Allgayer1,3 . 1 University of Heidelberg Medical faculty Mannheim, Experimental Surgery, Mannheim, Germany, 2 German cancer research centre (DKFZ), Theoretical Bioinformatics, Heidelberg, Germany, 3 German cancer research centre (DKFZ), Molecular Oncology of Solid Tumors, Heidelberg, Germany, 4 Institute of Pharmacy and Molecular Biotechnology and Bioquant Center, University of Heidelberg, Germany Introduction: Colorectal cancer is the second most common cancer and the second leading cause of cancer related death in men and women in Germany according to the Robert Koch Institute. The main reason for cancer related death is the development of metastasis (Chaffer and Weinberg, Science 2011). Thus, there is an urgent need to increase the knowledge on the main players and pathways leading to metastases. Mutations causing cells to metastasize occur initially in the primary tumor but might only affect a small subpopulation of cells. Materials and Methods: We conducted whole genome and transcriptome sequencing of a colorectal tumor and liver metastasis from the same patient and analysed the occurrence of mutations in the primary tumor and metastasis. The genome sequencing was carried out at a high coverage and on two different platforms, IlluminaHiSeq and Complete Genomics, in order to identify mutations present at a lower frequency in the primary tumor. The data was then analysed for mutations present in both tumor and metastasis as well as mutations with a higher variant allele frequency (VAF) in the metastasis than in the tumor. Results and Conclusion: We identified 16 genes affected by SNVs that had a more than 1.5 fold higher VAF in metastasis than in the tumor and may thus be relevant to the metastatic process. While some of these genes have been described in the context of colorectal or other cancers, several genes are new candidates and most have not yet been associated with the metastatic process. No conflict of interest. 432 Comparison of two library construction strategies for targeted resequencing of BRCA1/2 genes in Bulgarian breast cancer patients on NGS platform D. Dacheva1 , I. Popov1 , R. Dodova1 , T. Goranova1 , A. Mitkova1 , R. Kaneva1 , V. Mitev1 . 1 Medical University − Sofia, Molecular Medicine Center Medical Chemistry and Biochemistry, Sofia, Bulgaria Background: Breast cancer is the most commonly diagnosed malignancy and the most frequent cause of death in women due to cancer. According to the recent estimates 55% to 65% of the women carrying a harmful BRCA1 mutation and around 45% of those with BRCA2 mutation will develop breast cancer by the age of 70. Having 15.8 Kb open reading frame and being highly polymorphic, BRCA1/2 are a representative model for testing the accuracy, sensitivity and specificity of the ION Torrent PGM® Sequencer. Material and Methods: In the current study we included 28 female breast cancer patients divided in two groups. They were tested to compare two methods for library preparation using an in-house primer set for target enrichment in combination with Ion Xpress® Plus gDNA and Amplicon library preparation kit (1st − 16 samples) and commercially available primer pools covering the BRCA1/2 CDS (Ion AmpliSeq® BRCA1/2 Community panel) (2nd − 12 samples). The samples were sequenced with the ION Torrent PGM® Sequencer and all detected variations confirmed with Sanger sequencing on a 3130xl Genetic Analyzer (Applied Biosystems® ). Results and Discussion: We reached 98% sensitivity and 94.5% specificity for library constructed with home-made primers compared to 100% sensitivity and 97.6% specificity for commercially available Ion AmpliSeq® BRCA1 and BRCA2 Community Panel. Median coverage of the regions of interest (ROI) calculated for the 1st group was 94.3% at 1× and 76.1% at 30× and for the 2nd group − 99.5% and 98% respectively. Some of the amplicons in the 1st group were lost, most probably during the size selection step of the