Abstract 4863: Pathway-focused cancer mutation profiling with somatic mutation PCR arrays

Abstract

Abstract Recent advances in sequencing technologies have led to significant increases in the number of known cancer somatic mutations. Translating such information into therapeutic benefits requires molecular characterization of all major human tumor types and the advent of research tools that enable interrogation of multiple mutations in multiple cancer genes simultaneously, with high accuracy and scalability. As an increasing number of important signal transduction pathways, such as the EGFR-RAS-RAF pathway, are implicated in a variety of cancers, the utility of pathway-focused mutation analysis arrays has increased. This study examined the use of real-time PCR somatic mutation detection arrays that target all known functionally important mutation sites within an entire signal transduction pathway. Each array contains 80 to 90 ARMS® primers and 5’ hydrolysis probe-based qPCR assays that allow simultaneous detection of the most frequent and functionally important mutations for multiple genes in a specific pathway. In this study, different cancer sample types (fresh-frozen or FFPE samples from cancer cell lines and tissues) were profiled on qPCR arrays. To establish methodology validity, pyrosequencing was used as a performance benchmark. FFPE tissue sections of lung adenocarcinoma originating from different sources and with dramatically different DNA quality were tested to examine the tolerance of qPCR-based mutation detection to sample quality variation. Finally, data analysis algorithms were developed to maximize the accuracy of mutation/genotype calls. Results indicated that allele-specific PCR amplification combined with probe detection can achieve sufficient analytical specificity and sensitivity for detecting as little as 1% mutant alleles in a background of wild type genomic DNA. In addition, the method can detect mutations in FFPE samples over a wide range of quality (i.e. with differing extents of DNA degradation), and the mutations detected were verified by pyrosequencing analysis. Interestingly, multiple mutations across different genes within the EGFR pathway, and more than one mutation within the EGFR gene, were detected in lung adenocarcinoma samples, indicating either acquisition of multiple mutations by the same cell population or sample heterogeneity. In conclusion, it was demonstrated that somatic mutation profiling in the context of a pathway can be easily accomplished by using qPCR arrays and can be applied to biomarker development for a variety of human cancers. Disclaimer: The applications presented here are for research use only. Not for use in diagnostic procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4863. doi:10.1158/1538-7445.AM2011-4863