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B. Gao
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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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P3.04-052 - Next Generation Exome Sequencing of Archival Lung Cancer Resection Specimens (ID 2159)
09:30 - 09:30 | Author(s): B. Gao
- Abstract
Background:
Genetic testing of non-small cell lung cancer has grown rapidly in recent years to accommodate expansion of the number of agents with molecular targets. Whole exome sequencing (WES) has been proposed as a method to comprehensively assess tumor mutation status that could replace current piecemeal approaches to predictive testing. The feasibility of WES for formalin fixed paraffin embedded (FFPE) clinical samples has recently been documented. However, several issues remain to be resolved before this platform can be adopted for routine clinical use. The purpose of the present study is to evaluate tissue coring as a method for obtaining DNA from FFPE tumor tissue, to assess the gene coverage of libraries prepared from FFPE, to determine how best to identify specific validated treatment targets, and to determine mutation load in clinical samples.
Methods:
We extracted DNA from 0.6 mm tissue cores selected both from tumor rich regions of paraffin blocks and normal lung tissue. DNA quality was assessed by Bioanalyzer and Qbit testing. A sequencing library was prepared using the Agilent Sure Select XT5 (v5) library kit. DNA was sequenced using an Illumina Hiseq 2500 ultrahigh throughput sequencing system. We used two flow cells for each of 4 samples to obtain a high level of coverage and to determine the effect of reducing coverage on mutation detection by computational methods. We used the DNA from non-tumoral regions to identify genomic polymorphisms and to then compile lists of mutations that were suspected of have a deleterious effect on the host. As a control, we tested DNA from each tumor by a clinically validated multiplexed panel (Illumina True Site panel). We compared our sequencing results with the TCGA database for the respective tumors.
Results:
DNA yield was 13 and 17 micrograms for the SCC and adenocarcinoma respectively. After shearing to 200 base pairs and library preparation, excellent quality DNA was obtained for sequencing. All of the mutations detected by Miseq analysis were detected by WES. Several mutations identified by WES have not been documented in TCGA. The mutations of the two tumors are sumarized below, including mutation load.WES Mutations SCC Adenocarcinoma Nonsynonomous SNV 247 51 Stopgain SNV 16 1 Fs deletion 10 1 Non-fs substitution 9 7 Fs insertion 2 2 Non-fs deletion 1 3 Non-fs insertion 1 0 Stoploss SNV 1 0 Splice region abnormality 9 0 Not present in TCGA 37 7 Present in TCGA 265 59 Mutations detected by Miseq TP53 (p.G245R) EGFR exon19 del CTNNB1 (p.S45C) Total (Mutation Load) 302 66
Conclusion:
This study confirms that WES is feasible on FFPE tissue and that the two tumors sequenced fall into the two categories, high and low mutation loads. The mutations identified include several that have not previously been reported. All mutations identified by high coverage clinical platforms were also detected by WES. WES may be suitbable for clinical application.
