Whole exome sequencing (WES) on formalin-fixed, paraffin-embedded (FFPE) tumor tissue in gastrointestinal stromal tumors (GIST).

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.

Whole transcriptome sequencing identifies BCOR internal tandem duplication as a common feature of clear cell sarcoma of the kidney.

PURPOSE: Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor that is frequently difficult to distinguish among other childhood renal tumors due to its histological heterogeneity. This work evaluates genetic abnormalities carried by a series of CCSK samples by whole transcriptome sequencing (WTS), to identify molecular biomarkers that could improve the diagnostic process. METHODS: WTS was performed on tumor RNA from 8 patients with CCSK. Bioinformatic analysis, with implementation of a pipeline for detection of intragenic rearrangements, was executed. Sanger sequencing and gene expression were evaluated to validate BCOR internal tandem duplication (ITD). RESULTS: WTS did not identify any shared SNVs, Ins/Del or fusion event. Conversely, analysis of intragenic rearrangements enabled the detection of a breakpoint within BCOR transcript recurrent in all samples. Three different in-frame ITD in exon15 of BCOR, were detected. The presence of the ITD was confirmed on tumor DNA and cDNA, and resulted in overexpression of BCOR. CONCLUSIONS: WTS coupled with specific bioinformatic analysis is able to detect rare genetic events, as intragenic rearrangements. ITD in the last exon of BCOR is recurrent in all CCSK samples analyzed, representing a valuable molecular marker to improve diagnosis of this rare childhood renal tumor.