Abstract
BackgroundNext 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.MethodsWe 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. ResultsThe 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.ConclusionsWES 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1982-6) contains supplementary material, which is available to authorized users.
Highlights
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
DNA derived from FF samples amplifies a prevalent pool of fragments of around 500 bp, with multiple bands of higher size, while FFPEderived DNA is either of high quality (HQ-FFPE) and shows the 500 bp band as the longest amplifiable fragment (GIST193, GIST174, GIST165), or of low quality (LQ-FFPE), showing only shorter amplified fragments of no more than 300–400 bp (GIST127) (Fig. 1a)
We show that it is possible to achieve results comparable to FF when performing exome sequencing on FFPE tumor samples, Fig. 4 Validation of selected somatic mutations by Sanger sequencing on FF and FFPE tumor DNA and on PB-derived DNA. a platelet-derived growth factor receptor alpha (PDGFRA) p.D842V mutation detected in GIST165 tumor DNA from FF and FFPE samples. b KIT p.L576P mutation present in GIST174 patient, both in FF and FFPE. c SDHA p.S384X detected in patient GIST193 in heterozygosis in the germline, and in homozygosis in tumor DNA. d False negative LATS2 p.Q937X somatic stop-gain mutation
Summary
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. While FFPE specimens are frequently analyzed by amplicon-based or targetedcapture NGS panels [6,7,8,9,10], the possibility to reliably perform whole genome or whole exome sequencing (WES) in archival tumor samples still represents a challenge, both from the technical and bioinformatic point of view [11,12,13,14,15,16]. About 10-15 % of GIST do not exhibit neither KIT or PDGFRA mutations and have been defined as KIT/PDGFRA wild type (WT), which represent an extremely heterogeneous subgroup, characterized by different subsets with distinct molecular hallmarks [22, 23]
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