Abstract 812: Whole genome discovery of genetic alterations carried by pancreatic adenocarcinoma.
Authors: Macchini Marina , Astolfi Annalisa , Casadei Riccardo , Indio Valentina , Serra Carla , Vecchiarelli Silvia , Grassi Elisa , Ricci Claudio , D'Ambra Marielda , Taffurelli Giovanni , Santini Donatella , Pezzilli Raffaele , Minni Francesco , Biasco Guido , Di Marco Mariacristina 
Published: August 2014

Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a 5-year survival rate of 4%. Even if some genetic hits leading to PDAC development are known, a deeper knowledge of the genetic landscape of tumor is necessary to identify druggable targets.
Methods: PDAC samples from 14 localized and advanced cases of pancreatic adenocarcinomas were collected by ultrasound-guided biopsy used for DNA and RNA extraction. High resolution copy number analysis was performed on Affymetrix SNP array 6.0 and analyzed with segmentation algorithm against a reference of 270 Ceu HapMap individuals (Partek Genomic Suite). Whole transcriptome massively parallel sequencing was performed at 75x2 bp on a HiScanSQ Illumina platform. An average of 7,3x107 reads per sample were generated, with a mean read depth of 50X. Single nucleotide variants (SNVs) were detected with SNVMix2 and compared with genetic variation databases (dbSNP, 1000genomes, Cosmic). Non-synonimous SNVs were analyzed with SNPs&GO and SIFT to predict disease association.
Results: Whole transcriptome sequencing showed that PDAC samples exhibited a mean of 145 (range: 61-240) non-synonimous SNVs, of which 16 on average are potentially disease-related. 9/14 patients exhibited both macroscopic and cryptic cytogenetic alterations, with a mean of 10 copy number alterations per patient, while 5 patients did not show any copy number gain or loss. Most frequent gains were observed in 18q11.2 involving GATA6 (3/14) and 19q13 targeting AKT2 (3/14) while hotspot deleted regions were found on 18q21 (7/14), 17p13 (6/14), 9p21.3 (6/14), 15q (5/14) and 1q35 (4/14). Merging copy number and RNAseq data we highlighted the major oncogenic hits of PDAC, confirming the prevalence (9/14) of KRAS mutations, in one case also NRAS (G13D), and the central role of the three oncosuppressor CDKN2A (mutated in 3 cases and deleted in 6 cases, either in hetero- or homozygosity), SMAD4 (altered by either point mutation or gene deletion in 8/14 cases), and TP53 (lost in 7/14 samples). We identified novel pathogenic single nucleotide variants and frameshift mutations, and inter- and intra-chromosomal translocation events that give rise to chimeric fusion transcripts. Even if the mutation profile was heterogeneous in different patients the signaling pathways affected were shared, and included KRAS/MAPK, TGFbeta and integrin signaling, proliferation and apoptosis, DNA damage response, and epithelial to mesenchymal transition. New oncogenic alterations emerged, as ARID1A that was inactivated by somatic mutation (G91R and P1425fs) or copy number loss in 6 patients, NOTCH2 and HMGCR, that displayed pathogenic mutations in 15-20% of the analyzed patients.
Conclusions: Next generation sequencing combined with high resolution cytogenetic analysis can improve the understanding of carcinogenesis and highlight new biomarkers for early diagnosis and potential therapeutic targets in PDAC.
Citation Format: Marina Macchini, Annalisa Astolfi, Riccardo Casadei, Valentina Indio, Carla Serra, Silvia Vecchiarelli, Elisa Grassi, Claudio Ricci, Marielda D'Ambra, Giovanni Taffurelli, Donatella Santini, Raffaele Pezzilli, Francesco Minni, Guido Biasco, Mariacristina Di Marco. Whole genome discovery of genetic alterations carried by pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 812. doi:10.1158/1538-7445.AM2013-812