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.

Fig. 1

Quality control of DNA extracted from FFPE samples. a RAPD PCR performed on FF tumors and FFPE –derived DNA. b-c. KAPA HgDNA quantitative PCR QC kit, summarized as Q-score of the 129 bp vs the control 41 bp amplicon (b) and as the 305 bp vs the 41 bp (c)

Fig. 2

Concordance between FF and FFPE data. All the variants called, including common polymorphism and novel variants mapping on the 37 Mb Exome target region were classified as Shared if called in both FF and FFPE samples, as False Negative (FN) if called only in FF sample, as False Positive (FP) if detected only in FFPE, or ND if not sufficiently covered in either type of sample

Fig. 3

Total number of somatic SNVs detected in FF and FFPE samples, stratified based on the type of nucleotide substitution

Fig. 4

Validation of selected somatic mutations by Sanger sequencing on FF and FFPE tumor DNA and on PB-derived DNA. a 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 (both from FF and FFPE). d False negative LATS2 p.Q937X somatic stop-gain mutation. This mutation is present in both FF and FFPE samples, but fails to be detected by WES of FFPE due to low coverage. e False positive GPR45 p.V203M missense variant. This putative mutation is a present only in FFPE and not in FF sample from GIST127, probably due to cytosine deamination induced by formalin fixation