Universität zu Köln

Fassunke, Jana, Haller, Florian, Hebele, Simone, Moskalev, Evgeny A., Penzel, Roland, Pfarr, Nicole, Merkelbach-Bruse, Sabine and Endris, Volker (2015). Utility of different massive parallel sequencing platforms for mutation profiling in clinical samples and identification of pitfalls using FFPE tissue. Int. J. Mol. Med., 36 (5). S. 1233 - 1244. ATHENS: SPANDIDOS PUBL LTD. ISSN 1791-244X

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Abstract

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 (TM), GS Junior and PGM Ion Torrent (TM). 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Fassunke, Jana UNSPECIFIED UNSPECIFIED UNSPECIFIED Haller, Florian UNSPECIFIED UNSPECIFIED UNSPECIFIED Hebele, Simone UNSPECIFIED UNSPECIFIED UNSPECIFIED Moskalev, Evgeny A. UNSPECIFIED UNSPECIFIED UNSPECIFIED Penzel, Roland UNSPECIFIED UNSPECIFIED UNSPECIFIED Pfarr, Nicole UNSPECIFIED UNSPECIFIED UNSPECIFIED Merkelbach-Bruse, Sabine UNSPECIFIED UNSPECIFIED UNSPECIFIED Endris, Volker UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-388020
DOI:	10.3892/ijmm.2015.2339
Journal or Publication Title:	Int. J. Mol. Med.
Volume:	36
Number:	5
Page Range:	S. 1233 - 1244
Date:	2015
Publisher:	SPANDIDOS PUBL LTD
Place of Publication:	ATHENS
ISSN:	1791-244X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language PARAFFIN-EMBEDDED TISSUE; NUCLEIC-ACID EXTRACTION; DNA EXTRACTION; LUNG-CANCER; EGFR; ARTIFACTS; SPECIMENS; TUMORS; KRAS Multiple languages Medicine, Research & Experimental Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/38802