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Context Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with few effective therapies. Although next-generation sequencing (NGS) further clarified the genomic complexity of PDAC, genes or pathways that specifically drive tumour progression or metastasis are not well understood. Objective Our challenge is to implement a whole transcriptome massively parallel sequencing (RNASeq) study to better understand the PDAC molecular biology for diagnosis and prognosis of PDAC. Methods We collected a total of 17 PDAC samples by ultrasound-guided biopsy or by surgical specimen for RNA extraction. Fourteen samples were analyzed by RNASeq, performed at 75x2 bp on a HiScanSQ Illumina platform. To study gene expression profiling related to poor outcome, we first studied differentially expressed genes between “poor” (overall survival 36 months; n=3) prognosis in a total of 6 PDAC sample. Results The relative presence of tumor cells in the sample was evaluated based on the presence of KRAS mutation. A total of 211 genes were differentially expressed. Genes involved in the p53 signalling pathway (CSNK1G1, TGFA), the Wnt/β-catenin (DKK1, WNT7B, WNT10A), insulin-like growth factor system (IGF2) and EGF receptor signalling pathway (EGF) were highly upregulated in “poor” PDAC samples. Interestingly, we found a strong overexpression of CA125/MUC16, recently demonstrated to be involved in PDCA and ovarian cancer invasion by stimulating matrix metalloproteinases 7. Conclusion Components of p53 signalling pathway, Wnt/β-catenin pathways, insulin-like growth factor system and MUC16 might be useful molecular markers in PDCA as their overexpression seems to be related with cancer growth, invasion and prognosis. Further validation of the role of these genes is necessary for translation in clinical practice., JOP. Journal of the Pancreas, Vol 14, N° 5S (2013): September (Suppl.) - p. 528-602

Context We merged clinical history of a locally advanced pancreatic cancer (LAPC) patient with data obtained from a whole transcriptome massively parallel sequencing (RNASeq). Case report A 56-year-old man with histological diagnosis of LAPC. After obtained informed consent, we collected a fragment of pancreatic lesion. Patient received 6 induction cycles treatment with gemcitabine and oxaliplatin (GEMOX) from November 2011, followed by chemoradiotherapy with bi-weekly gemcitabine 50 mg/m2 for 6 weeks. CT-scan demonstrated partial response, so patient received additional 12 cycles of GEMOX, with further response, which currently persist since 17 months (to 3.8 cm vs. 2.5 cm). At the same time, the RNASeq was performed at 75x2 bp on a HiScanSQ (Illumina Inc., San Diego, CA, USA) platform. Single nucleotide variants (SNVs) were detected with SNVMix2 and filtered on dbSNP, 1000 Genomes Project, and Cosmic databases. Non-synonymous SNVs were analyzed with SNPs&GO and PROVEAN. We highlighted the major oncogenic hits of LAPC, confirming KRAS mutations, CDKN2A and SMAD4 deletions. RNASeq analysis showed a somatic mutation p.H672D involving the catalytic domain of hydroxymethylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway, not yet reported neither in the COSMIC nor in the ICGC databases. Furthermore, 4 new genomic rearrangements leading to fusion genes emerged: 2 in-frame gene fusions regulating RAS-MAPK and apoptotic pathways (ANKRD44-GULP1 on chromosome 2; ATXN10-TMEM49; chromosome 22 and 17) and 2 out-of-frame fusions [t(15;3) and t(19;22)] leading to SMAD3-KIAA1143 and LTBP4-SPATS2L, both disrupting genes of the TGFbeta pathway. Conclusion We found a novel somatic alteration involving HMGCR in LAPC. Due to the key role of HMGCR in cellular transformation, we hypothesize a strong potential in the development and outcome of LAPC, whose the optimal treatment remains to be elucidated. Trials that integrate RNASeq data with clinical options are needed., JOP. Journal of the Pancreas, Vol 14, N° 5S (2013): September (Suppl.) - p. 528-602

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.

Context Pancreatic cancer (PC) is characterized by a 5-year survival rate of 4%. The molecular mechanisms involved in the high tumorigenicity of PC are not yet well-known. Methods PC samples from 7 localized and advanced cases of pancreatic adenocarcinomas were collected by ultrasound-guided biopsy. Whole transcriptome RNA libraries were sequenced at 75x2 bp on a HiScanSQ Illumina platform. An average of 4.5x107 reads per sample were generated, with a mean read depth of 40. Sequences were mapped to the human genome (build hg19) and the single nucleotide variants (SNVs) were detected with SNVMix2 tool. The SNVs were compared with genetic variations databanks (dbSNP, 1000genomes, Cosmic) in order to highlight the novel variants. The non-synonimous SNVs were considered at the protein level and analyzed with SNPs&GO, in order to predict whether a variation is disease-related or neutral. Results Pancreatic adenocarcinomas exhibited a mean of 139 (range: 65-252) non-synonimous SNVs, of which 12 on average are potentially disease-related. We found mutations in oncogenes and tumor suppressor genes known to be related to pancreatic tumors, as 4/7 patients exhibited KRAS oncogenic variants (p.G12D, R or V) two of which carried also mutations either in SMAD4 or PIK3CA, one patient showed both TP53 and CDKN2A inactivating mutations and one a mutation in SMARCA4. Novel disease-related SNVs were found in genes regulating cell cycle progression and proliferation, apoptosis, TGFbeta and integrin-signaling, epithelial to mesenchymal transition and nucleotide excision repair. Of interest some genes were mutated in multiple patients, as CBLC that regulates intracellular signalling by multiple tyrosine kinase genes. Conclusions Next generation sequencing analysis, through the identification of the oncogenic alterations carried by tumor cells, can improve the understanding of pancreatic carcinogenesis and highlight new biomarkers for early diagnosis and potential therapeutic targets in pancreatic cancer., JOP. Journal of the Pancreas, Vol 13, N° 5S (2012): September (Suppl.) - p. 548-650