B22

As a result of ongoing high throughput mutation screening efforts, a more complete list of genes involved with tumorigenesis and cancer progression has emerged. However, current sequencing technologies lack sensitivity and scalability for more widespread use in analyzing tumor tissues for sequence mutations. For the purposes of simultaneously surveying known sequence mutations in a single assay, we have customized an oligonucleotide-based multiplex high-thoughput SNP genotyping assay to target previously described somatic variants. For validation, 114 unique mutations representing 61 genes were surveyed in 33 unique cancer cell lines. Variants assayed include oncogenic mutations of PIK3CA, KRAS and EGFR, as well as inactivating mutations of p53 and PTEN. 
 The assay successfully identified 94% (107/114) of known mutations. This included the successful identification of 11/11 of ‘hotspot’ mutations of CTNNB1 and p53, as well as deletions in the PTEN gene (950 953delTACT). In order to test the tractability of this platform in detecting mutations in tumor tissues contaminated with normal cells, a set of DNAs from 10 cancer cell lines was diluted with normal wild type DNA. This showed mutation detection was most reduced with <= 20% tumor DNA (1:5 tumor:normal; 60% success at this ratio). In most cases, complex mutations (hotspots) and adjacent repetitive sequences accounted for the reduced success in identifying mutations.
 These results demonstrate a novel high-throughput method (up to 1,536 mutations in a single assay) for the simultaneous screening of somatic mutations in tumor tissue. Small material requirements (250 ng gDNA), and the ability to successfully detect mutations in the presence of normal tissue make this technique suitable for use with frozen and archival tumor specimens. Further, this can aid in identifying previously unknown patterns of co-existing mutations within tumor types, which can aid in predicting drug sensitivity and the design of treatment strategies.

Third AACR International Conference on Molecular Diagnostics in Cancer Therapeutic Development-- Sep 22-25, 2008; Philadelphia, PA