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L. La Fleur
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-021 - Mutation Profiling by Targeted Next-Generation Sequencing for Diagnostics and Patient Cohort Screening in FFPE NSCLC Samples (ID 920)
09:30 - 09:30 | Author(s): L. La Fleur
- Abstract
Background:
Recent discovery of the landscape of somatic mutations in non-small cell lung cancer (NSCLC), and introduction of new therapeutics have raised the demands for multiplex mutation assays. In exploratory research, mutation profiling has largely been performed on fresh-frozen tissue from surgical specimens. However, for patients with advanced disease the assays need to be adapted to small formalin-fixed paraffin embedded (FFPE) biopsies and cytology preparations. Targeted next-generation sequencing (NGS) techniques are now being developed to address these challenges and have now reached the point where they are more cost efficient than previously used methods, hence there is a need to optimize and validate these techniques to determine if they are robust enough to work in clinical diagnostics.
Methods:
Here we have developed and evaluated Haloplex gene panels in comparison to pyrosequencing and quantitative PCR(qPCR), i.e. the current standard methods for molecular diagnostics of solid tumours in Sweden. The target enrichment was focused on short DNA fragments and included independent capture of complementary strands, “two strand capture”, to address fragmentation and base damage induced by formalin fixation. The panels include all exons of 18-32 genes (for lung cancer and other solid tumors respectively) with known clinical relevance. Seventy-one clinical samples (NSCLC, colorectal carcinoma and melanoma), with known mutational status of hotspots in KRAS, BRAF, NRAS, PIK3CA and EGFR, were selected for analysis. DNA was prepared from FFPE tissues and used for library preparation using the panels and subsequently sequenced on an Illumina MiSeq instrument.
Results:
A complete concordance was seen between the previously defined pyrosequencing and qPCR genotypes and the corresponding variants detected using the gene panels. Both point mutations and smaller indels (<25bp) could be detected by this technique using an in-house bioinformatic pipeline. False positive FFPE-induced mutation artefacts could reliably be identified by the two-strand filter. The technical sensitivity of mutation detection was determined to 2%, and we have decided to use a 5% variant allele frequency threshold for clinical reporting. In addition, clonality and subclonality could be discovered in patients with complex tumour disease (mixed or multiple tumour lesions) by analysis of the mutation patterns. An extended 85 gene panel has also been designed to screen for mutations in NSCLC patient cohorts for clinical molecular research.
Conclusion:
We believe that the established lung cancer gene panels for targeted enrichment and NGS can replace pyrosequencing and qPCR for molecular diagnostics in NSCLC, and will be useful for screening of unselected population-based prospective and retrospective lung cancer patient cohorts in clinical research.
