title: Characterization of pancreatic ductal adenocarcinoma using whole transcriptome sequencing and copy number analysis by single-nucleotide polymorphism array.
abstract: The aim of the current study was to implement whole transcriptome massively parallel sequencing (RNASeq) and copy number analysis to investigate the molecular biology of pancreatic ductal adenocarcinoma (PDAC). Samples from 16 patients with PDAC were collected by ultrasound‑guided biopsy or from surgical specimens for DNA and RNA extraction. All samples were analyzed by RNASeq performed at 75x2 base pairs on a HiScanSQ Illumina platform. Single‑nucleotide variants (SNVs) were detected with SNVMix and filtered on dbSNP, 1000 Genomes and Cosmic. Non‑synonymous SNVs were analyzed with SNPs&GO and PROVEAN. A total of 13 samples were analyzed by high resolution copy number analysis on an Affymetrix SNP array 6.0. RNAseq resulted in an average of 264 coding non‑synonymous novel SNVs (ranging from 146‑374) and 16 novel insertions or deletions (In/Dels) (ranging from 6‑24) for each sample, of which a mean of 11.2% were disease‑associated and somatic events, while 34.7% were frameshift somatic In/Dels. From this analysis, alterations in the known oncogenes associated with PDAC were observed, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations (93.7%) and inactivation of cyclin‑dependent kinase inhibitor 2A (CDKN2A) (50%), mothers against decapentaplegic homolog 4 (SMAD4) (50%), and tumor protein 53 (TP53) (56%). One case that was negative for KRAS exhibited a G13D neuroblastoma RAS viral oncogene homolog mutation. In addition, gene fusions were detected in 10 samples for a total of 23 different intra‑ or inter‑chromosomal rearrangements, however, a recurrent fusion transcript remains to be identified. SNP arrays identified macroscopic and cryptic cytogenetic alterations in 85% of patients. Gains were observed in the chromosome arms 6p, 12p, 18q and 19q which contain KRAS, GATA binding protein 6, protein kinase B and cyclin D3. Deletions were identified on chromosome arms 1p, 9p, 6p, 18q, 10q, 15q, 17p, 21q and 19q which involve TP53, CDKN2A/B, SMAD4, runt‑related transcription factor 2, AT‑rich interactive domain‑containing protein 1A, phosphatase and tensin homolog and serine/threonine kinase 11. In conclusion, genetic alterations in PDCA were observed to involve numerous pathways including cell migration, transforming growth factor‑β signaling, apoptosis, cell proliferation and DNA damage repair. However, signaling alterations were not observed in all tumors and key mutations appeared to differ between PDAC cases.
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Mariacristina
authors.initials: M
authors.lastname: Di Marco
authors.affiliation: Interdepartmental Center of Cancer Research, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Astolfi
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Grassi
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Vecchiarelli
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Macchini
authors.affiliation: Interdepartmental Center of Cancer Research, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Indio
authors.affiliation: Department of Medical and Surgical Sciences, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Casadei
authors.affiliation: Department of Medical and Surgical Sciences, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Ricci
authors.affiliation: Department of Medical and Surgical Sciences, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Marielda
authors.initials: M
authors.lastname: D'Ambra
authors.affiliation: Department of Medical and Surgical Sciences, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Taffurelli
authors.affiliation: Department of Digestive Diseases and Internal Medicine, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Serra
authors.affiliation: Liver and Multiorgan Transplant Unit, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Ercolani
authors.affiliation: Pathology Unit, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Donatella
authors.initials: D
authors.lastname: Santini
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Antonia
authors.initials: A
authors.lastname: D'Errico
authors.affiliation: Department of Medical and Surgical Sciences, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Antonio Daniele
authors.initials: AD
authors.lastname: Pinna
authors.affiliation: Department of Medical and Surgical Sciences, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Minni
authors.affiliation: Interdepartmental Center of Cancer Research, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Sandra
authors.initials: S
authors.lastname: Durante
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Laura Raffaella
authors.initials: LR
authors.lastname: Martella
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Sant'Orsola‑Malpighi Hospital, Bologna I‑40100, Italy.
authors.forename: Guido
authors.initials: G
authors.lastname: Biasco
mesh_terms: D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D059467:Transcriptome