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Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing
Published on: October 18, 2013
Utility of different massive parallel sequencing platforms for mutation profiling in clinical samples and
Jana Fassunke1, Florian Haller2, Simone Hebele2
1Institute of Pathology, University of Cologne, Medical Centre, D-50924 Cologne, Germany.
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™, 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.
Insights
Massive parallel sequencing (MPS) platforms show high concordance for detecting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). DNA quality and extraction methods impact analysis success, highlighting the need for verification of ambiguous variants.
Area of Science:
- Oncology
- Genetics
- Molecular Diagnostics
Background:
- Personalized medicine requires efficient molecular analysis of cancer targets.
- Non-small cell lung cancer (NSCLC) diagnostics increasingly involve comprehensive gene mutation testing.
- Massive parallel sequencing (MPS) offers rapid, broad mutation profiling from limited tissue samples.
Purpose of the Study:
- To evaluate and compare the performance of different MPS platforms for routine NSCLC diagnostics.
- To assess the concordance of EGFR mutation status detection across multiple pathology institutes.
- To identify challenges and best practices for MPS implementation in molecular pathology.
Main Methods:
- A round-robin study involving 30 NSCLC cases with known EGFR mutation status.
- Three pathology institutes utilized FFPE tissue sections for targeted multiplex PCR and MPS.
- Sequencing performed on MiSeq™, GS Junior, and PGM Ion Torrent™ platforms with varied data analysis approaches.
Main Results:
- 100% concordance was achieved in determining the EGFR mutation status across all participating institutes for 26 successfully analyzed samples.
- MPS platforms demonstrated good overall concordance in mutation detection.
- Sample analysis failures were linked to DNA quality issues and extraction variability; minor variants required confirmation via qPCR or Sanger sequencing.
Conclusions:
- MPS platforms are suitable for routine NSCLC molecular diagnostics with high concordance for EGFR mutations.
- Standardization of DNA extraction and quality control is crucial for reliable MPS results.
- Independent verification methods are recommended for ambiguous or low-frequency variants detected by MPS.
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