title: Whole exome sequencing (WES) on formalin-fixed, paraffin-embedded (FFPE) tumor tissue in gastrointestinal stromal tumors (GIST).
abstract: BACKGROUND
  Next generation sequencing (NGS) technology has been rapidly introduced into basic and translational research in oncology, but the reduced availability of fresh frozen (FF) tumor tissues and the poor quality of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) has significantly impaired this process in the field of solid tumors. To evaluate if data generated from FFPE material can be reliably produced and potentially used in routine clinical settings, we performed whole exome sequencing (WES) from tumor samples of Gastrointestinal stromal tumors (GIST), either extracted FF or FFPE, and from matched normal DNA.
  
  
  METHODS
  We performed whole exome enrichment and sequencing at 100bp in paired end on four GIST samples, either from FFPE or fresh-frozen tissue, and from matched normal DNA.
  
  
  RESULTS
  The integrity of DNA extracted from FFPE was evaluated by a modified RAPD PCR method, thus identifying high quality (HQ) and low quality (LQ) FFPE. DNA library production and exome capture was feasible for both classes of FFPE, despite the smaller yield and insert size of LQ-FFPE. WES produced data of equal quality from FF and FFPE, while only HQ-FFPE yielded an amount of data comparable to FF samples. Bioinformatic analysis showed that the percentage of variants called both in FF and FFPE samples was very high in HQ-FFPE, reaching 94-96 % of the total number of called variants. Classification of somatic variants by nucleotide substitution type showed that HQ-FFPE and FF had similar mutational profiles, while LQ-FFPE samples carried a much higher number of mutations than the FF counterpart, with a significant enrichment of C > T/G > A substitutions. Focusing on potential disease-related variants allowed the discovery of additional somatic variants in GIST samples, apart from the known oncogenic driver mutation, both from sequencing of FF and FFPE material. False positive and false negative calls were present almost exclusively in the analysis of FFPE of low quality. On the whole this study showed that WES is feasible also on FFPE specimens and that it is possible to easily select FFPE samples of high quality that yield sequencing results comparable to the FF counterpart.
  
  
  CONCLUSIONS
  WES on FFPE material may represent an important and innovative source for GIST research and for other solid tumors, amenable of possible application in clinical practice.
authors.affiliation: "Giorgio Prodi" Cancer Research Center, University of Bologna, Bologna, Italy. annalisa.astolfi@unibo.it1.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Astolfi
authors.affiliation: "Giorgio Prodi" Cancer Research Center, University of Bologna, Bologna, Italy. milena.urbini@yahoo.com.
authors.forename: Milena
authors.initials: M
authors.lastname: Urbini
authors.affiliation: "Giorgio Prodi" Cancer Research Center, University of Bologna, Bologna, Italy. valentina.indio2@unibo.it.
authors.forename: Valentina
authors.initials: V
authors.lastname: Indio
authors.affiliation: Department of Specialized, Experimental and Diagnostic Medicine, S. Orsola-Malpighi Hospital, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy. margherita.nannini@unibo.it.
authors.forename: Margherita
authors.initials: M
authors.lastname: Nannini
authors.affiliation: "Giorgio Prodi" Cancer Research Center, University of Bologna, Bologna, Italy. genovese.chiaragiusy@gmail.com.
authors.forename: Chiara Giusy
authors.initials: CG
authors.lastname: Genovese
authors.affiliation: Pathology Unit, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy. donatella.santini@aosp.bo.it.
authors.forename: Donatella
authors.initials: D
authors.lastname: Santini
authors.affiliation: Department of Specialized, Experimental and Diagnostic Medicine, S. Orsola-Malpighi Hospital, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy. maristella.saponara@unibo.it.
authors.forename: Maristella
authors.initials: M
authors.lastname: Saponara
authors.affiliation: Department of Specialized, Experimental and Diagnostic Medicine, S. Orsola-Malpighi Hospital, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy. anna.mandrioli@gmail.com.
authors.forename: Anna
authors.initials: A
authors.lastname: Mandrioli
authors.affiliation: Transplant, General and Emergency Surgery Department, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy. giorgio.ercolani2@unibo.it.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Ercolani
authors.affiliation: Department of Specialized, Experimental and Diagnostic Medicine, S. Orsola-Malpighi Hospital, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy. giovanni.brandi@unibo.it.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Brandi
authors.affiliation: "Giorgio Prodi" Cancer Research Center, University of Bologna, Bologna, Italy. guido.biasco@unibo.it.|Department of Specialized, Experimental and Diagnostic Medicine, S. Orsola-Malpighi Hospital, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy. guido.biasco@unibo.it.
authors.forename: Guido
authors.initials: G
authors.lastname: Biasco
authors.affiliation: "Giorgio Prodi" Cancer Research Center, University of Bologna, Bologna, Italy. maria.pantaleo@unibo.it.|Department of Specialized, Experimental and Diagnostic Medicine, S. Orsola-Malpighi Hospital, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy. maria.pantaleo@unibo.it.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Pantaleo
mesh_terms: D004273:DNA, Neoplasm / Q000235:genetics*; D059472:Exome / Q000235:genetics*; D005557:Formaldehyde / Q000737:chemistry; D046152:Gastrointestinal Stromal Tumors / Q000235:genetics* / Q000473:pathology; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D016612:Paraffin Embedding; D019105:Random Amplified Polymorphic DNA Technique; D016707:Tissue Fixation
chemical_list: D004273:DNA, Neoplasm; D005557:Formaldehyde