Abstract #1642: High throughput SNP analysis from formalin-fixed, paraffin-embedded tumor tissues
Authors: Matthew Sikora, Jacklyn Thibert, Janine Salter, Mitch Dowsett, Michael Johnson, James Rae
Journal: Cancer Research (2009)

Historically, most of the large prospective clinical trials that established the value of adjuvant chemotherapy for cancer did not collect peripheral blood samples for genetic analysis, thereby severely limiting the possibility of retrospective pharmacogenomic studies designed to correlate outcome and toxicity with patient genetics. The majority of these trials did, however, archive formalin-fixed, paraffin-embedded (FFPE) tumor tissue. We and others have previously demonstrated that genotyping for single nucleotide polymorphisms (SNPs) in clinically relevant polymorphic genes can be reliably conducted on DNA extracted from FFPE tumor samples, and that the genotypes obtained agree with those from matched whole blood samples. Although many interesting findings have now been generated using this approach, several factors have limited its wider application. The blocks from historical clinical trials are an extremely valuable and limited resource, and sections used for preparing DNA for genotyping cannot be used for other important uses such as the evaluation of protein marker expression by immunohistochemistry, or array-based determination of mRNA levels. Furthermore, the relatively modest number of genotyping assays that could reliably be conducted on DNA extracted from the few FFPE sections available has also inhibited wider application. We now report technical advances that dramatically increase the number of genotypes that can be generated from a single FFPE sample using standard Taqman-based SNP genotyping assays and, furthermore, demonstrate that FFPE tumor cores, of the type used to generate tumor microarrays, can also be used as a source of DNA for high-order genotyping. We demonstrate that through careful determination of the amount of DNA of sufficient integrity for PCR amplification within the samples generated from FFPE materials, it is possible to significantly decrease the amount of template DNA used in standard Taqman-based SNP genotyping assays. This increases both the PCR amplification efficiency as well as the overall reliability of the final genotype. Using these improved methods, DNA was prepared and analyzed from matched sections and cores. On average, these samples yielded sufficient DNA for over 1,000 SNP determinations from a 5 \#956;m section, or from a 0.6 mm core, a significant increase over the 30-50 genotypes obtained from a standard 5 \#956;m section using previously established methods. The dramatic increase in the number of genotyping assays now possible from a single FFPE section should facilitate the greatly expanded application of pharmacogenetic studies to historical clinical trials. We believe that our demonstration of the use of tumor cores in this context is of particular importance since it has previously been established that the harvesting of tumor cores has minimal impact on the utility of the donor blocks for diagnostic and other purposes.  Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1642.