Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics.

Hirsch B1,2, Endris V3,4, Lassmann S3,5, Weichert W3,6, Pfarr N3,6, Schirmacher P3,4, Kovaleva V3,5, Werner M3,5, Bonzheim I3,7, Fend F3,7, Sperveslage J3,7, Kaulich K3,8, Zacher A3,8, Reifenberger G3,8, Köhrer K3,8, Stepanow S3,8, Lerke S3,9, Mayr T3,9, Aust DE3,9, Baretton G3,9, Weidner S3,10, Jung A3,10, Kirchner T3,10, Hansmann ML3,11, Burbat L12,3, von der Wall E12,3, Dietel M12,3, Hummel M12,3.

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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.