Utility of different massive parallel sequencing platforms for mutation profiling in clinical samples and identification of pitfalls using FFPE tissue

In the growing field of personalised medicine, the analysis of numerous potential targets is becoming a challenge in terms of work load, tissue availability, as well as costs. The molecular analysis of non-small cell lung cancer (NSCLC) has shifted from the analysis of the epidermal growth factor receptor (EGFR) mutation status to the analysis of different gene regions, including resistance mutations or translocations. Massive parallel sequencing (MPS) allows rapid comprehensive mutation testing in routine molecular pathological diagnostics even on small formalin-fixed, paraffin-embedded (FFPE) biopsies. In this study, we compared and evaluated currently used MPS platforms for their application in routine pathological diagnostics. We initiated a first round-robin testing of 30 cases diagnosed with NSCLC and a known EGFR gene mutation status. In this study, three pathology institutes from Germany received FFPE tumour sections that had been individually processed. Fragment libraries were prepared by targeted multiplex PCR using institution-specific gene panels. Sequencing was carried out using three MPS systems: MiSeq™, GS Junior and PGM Ion Torrent™. In two institutes, data analysis was performed with the platform-specific software and the Integrative Genomics Viewer. In one institute, data analysis was carried out using an in-house software system. Of 30 samples, 26 were analysed by all institutes. Concerning the EGFR mutation status, concordance was found in 26 out of 26 samples. The analysis of a few samples failed due to poor DNA quality in alternating institutes. We found 100% concordance when comparing the results of the EGFR mutation status. A total of 38 additional mutations were identified in the 26 samples. In two samples, minor variants were found which could not be confirmed by qPCR. Other characteristic variants were identified as fixation artefacts by reanalyzing the respective sample by Sanger sequencing. Overall, the results of this study demonstrated good concordance in the detection of mutations using different MPS platforms. The failure with samples can be traced back to different DNA extraction systems and DNA quality. Unknown or ambiguous variations (transitions) need verification with another method, such as qPCR or Sanger sequencing.