Mutational Screening in KRAS, BRAF, EGFR, C-KIT and PDGFRain Colorectal Carcinoma (CRC) and Non-Small Cell Lung Cancer (NSCLC) Using Next-Generation Sequencing (NGS)
Tanja Hinrichsen, Oliver Wachter, Barbara Dockhorn-Dworniczak, Hanns-Georg Klein. Center for Human Genetics and Laboratory Medicine Dr. Klein and Dr. Rost, Munich, Bavaria, Germany; Institute of Pathology Kempten, Kempten, Bavaria, Germany
Background: Treatment with monoclonal antibodies (mAB) or „small molecules“ (e.g. tyrosine kinase inhibitor, TKI) depends on the mutational status of certain genes in the tumour tissue of solid tumours. In CRC, the mutational status of KRAS and BRAF correlates with a response to mABs, in NSCLC the mutational status of EGFR and KRAS correlates with a benefit to TKI treatment. Other important genes in gastrointestinal stromal tumour (GIST) are c-KIT and PDGFRa. With NGS, is likely to improve the diagnostics in solid tumours. Discrete templates of the DNA can be amplified and sequenced clonally with a high coverage in a time-saving and cost-efficient manner. Mutations can be detected in a wildtype-background which enables the detection of minorities and is another important step towards personalized medicine.
Design: NGS was applied for a molecular screening of the complete coding region of KRAS, BRAF, EGFR, C-KIT and PDGFRa to investigate for mutations in FFPE-tumour tissue. A set of 48 CRC-FFPE-samples and a set of 48 NSCLC-FFPE-samples were analyzed. DNA was isolated and amplicon preparation (300 bp) was automated with the Fluidigm Access Array System (Fluidigm, South San Francisco, CA). Four Access Arrays were performed and multiplex Identifiers (MIDs) were added manually. After pooling and purification the pooled library was sequenced with GS-FLX (454 Life Sciences, Branford, CT, USA) with an aimed 200xcoverage.
Results: For the NSCLC-set, 544777 reads which passed filter criteria were generated with an average coverage of approximately 118x. First results show mutations in KRAS in the known hotspot regions in a subset of EGFR-negative NSCLC-patients. For the CRC-set, 720509 reads which passed filter criteria were generated with an average coverage of approximately 156x. The analysis of the data is still going on. Data for both runs will be presented.
Conclusions: The combination of the GS FLX and the Access Array System could offer a high-throughput amplicon resequencing solution applicable on DNA from FFPE-tissue. Our data suggest that NGS is a feasible method in routine molecular pathology to examine mutational status of tumour-relevant genes which are the basis for personalized medicine.
Tuesday, March 20, 2012 1:15 PM
Platform Session: Section E, Tuesday Afternoon