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Abstract
The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.
Highlights
Whole genome sequencing (WGS) or whole exome sequencing (WES) are excellent tools for the comprehensive and explorative detection of genetic alterations in tumor DNA extracted from cancer cells and tissues [10]
Dilutions (50, 10, 3, and 1%) of DNA extracted from FFPE cell line blocks of the colon cancer cell line LoVo in DNA derived from a FFPE tonsil were used to exemplarily determine the sensitivity and reproducibility of the commercially available amplicon panel cancer hotspot panel v2 (CHPv2)
DNA-aliquots of the four dilutions were provided for Next-Generation Sequencing (NGS) to the three PGM sites
Summary
Whole genome sequencing (WGS) or whole exome sequencing (WES) are excellent tools for the comprehensive and explorative detection of genetic alterations in tumor DNA extracted from cancer cells and tissues [10] These techniques produce overwhelming amounts of data that require extensive bioinformatics analyses [5]. For the diagnostic detection of cancer-associated somatic alterations relevant for therapeutic decisions, targeted ampliconbased Next-Generation Sequencing (NGS) of cancer gene panels has evolved as a promising approach [19]. For this method, short genomic regions are captured or PCRamplified at the initial part of library preparation, and subsequently subjected to NGS. The amount of data created and bioinformatics are limited, while sequencing depth can be very high, allowing for sensitive detection of even subclonal mutations [18, 29]
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