High-efficiency genotype analysis from formalin-fixed, paraffin-embedded tumor tissues

The Pharmacogenomics Journal (2011) 11, 348–358 & 2011 Macmillan Publishers Limited. All rights reserved 1470-269X/11 www.nature.com/tpj ORIGINAL ARTICLE High-efficiency genotype analysis from formalinfixed, paraffin-embedded tumor tissues MJ Sikora1, JN Thibert2, J Salter3, M Dowsett3, MD Johnson4 and JM Rae1,2 1 Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA; 2 Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI, USA; 3 Academic Department of Biochemistry, Royal Marsden Hospital, London, UK and 4Department of Oncology, Georgetown University Medical Center, Washington, DC, USA Correspondence: Dr JM Rae, Department of Internal Medicine, University of Michigan, 4520B MSRB I, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA. E-mail: Single-nucleotide polymorphisms (SNPs) can be assayed using DNA isolated from archival formalin-fixed, paraffin-embedded (FFPE) samples, making retrospective pharmacogenetic studies possible. In this study, we describe methods that significantly increase the number of SNP determinations possible using FFPE samples. Quantifying the amount of DNA amenable to PCR (amplification-quality DNA, AQ-DNA) allows a significant reduction in the amount of sample required for Taqman-based SNP assays. Optimizing AQ-DNA input increases PCR amplification efficiency and SNP determination accuracy. DNA was extracted from 39 FFPE tumor sections and matched tumor and stromal cores, which were of the type used to generate tissue microarrays. Sections and tumor cores yielded sufficient AQ-DNA for more than 1000 SNP determinations. Seven SNPs were assessed following individual assay optimization for minimal AQ-DNA. Genotypes from tumor cores for single SNPs were 92.3–100% concordant with those obtained from sections. Using these methods, the number of SNP genotypes that can be determined from single FFPE samples is greatly increased expanding the genetic association studies possible from limited archival specimens. The use of tumor cores is of particular importance as the harvesting of tumor cores has minimal impact on the utility of the donor blocks for other purposes. The Pharmacogenomics Journal (2011) 11, 348–358; doi:10.1038/tpj.2010.50; published online 15 June 2010 Keywords: genotyping; paraffin-embedded; tumor samples; genetic polymorphism Introduction Received 11 December 2009; revised 18 March 2010; accepted 27 April 2010; published online 15 June 2010 Until recently, clinical trials in cancer did not routinely collect and store patient DNA samples, thus limiting the ability to conduct pharmacogenetic analysis of many large, landmark clinical trials. Another potential source of patient DNA does, however, exist in the form of formalin-fixed, paraffin-embedded (FFPE) tumor samples that were collected for the majority of clinical trials in oncology. Previously, we and others have demonstrated that DNA can be obtained from these tumor blocks, that single-nucleotide polymorphism (SNP) genotypes can be reliably determined from this DNA, and that the genotypes of SNPs in genes of pharmacogenetic interest (primarily involved in drug metabolism) derived from the tumor samples match those of the germline DNA.1–4 This technological advance demonstrated that FFPE samples could be used for pharmacogenetic analyses of historical prospective clinical trials, thereby allowing the existing wealth of large, carefully conducted clinical trials of chemotherapeutic agents to be mined for associations between inherited gene variants with drug toxicities and clinical outcomes. Maximizing SNP analyses from FFPE tumor samples MJ Sikora et al 349 Using the DNA extracted from FFPE tumor samples for genotype analyses presents significant technical challenges due to the relatively low quantity and poor quality of the template DNA that is extracted from these samples. Standard DNA isolation methods used for FFPE samples typically produce severely sheared and fragmented DNA, which is frequently not optimal for PCR-based genotyping.5–8 All commonly used PCR-based genotyping assays have been designed to work using high-quality DNA isolated from viable cells. Real-time PCR-based Taqman assays, for instance, involve the amplification of DNA segments 80–150 base pairs (bp) in length containing the SNP of interest.9 In addition, 5–10 mm FFPE tissue block sections typically only yield sufficient DNA for 30–50 Taqman-based genotyping reactions (MJS and JNT, unpublished observations). Furthermore, Taqman PCR reactions using DNA extracted from FFPE samples are frequently inefficient, and yield irregular fluorescence output curves, making allelic determination difficult or ambiguous. Common practice when confronted with DNA samples that are difficult to genotype is to increase the amount of template DNA used per reaction. However, we have observed that doing this with FFPE-derived DNA often results in worsening rather than improvement of the PCR reaction. A simple method to determine the optimal amount of a given sample of FFPE-derived DNA for each assay would be valuable. In this report, we describe methods to analyze DNA harvested from FFPE materials that allows the assessment of overall quality and quantification of ‘amplification-quality DNA’ or ‘AQ-DNA’ (DNA fragments large enough for efficient PCR-based analysis). Using materials evaluated in this manner, we describe how minimizing the amount of input DNA in Taqman-based genotyping reactions significantly improves PCR amplification efficiency, increases the accuracy of allelic determinations, and greatly increases the number of genotyping assays that can be performed per sample. We also demonstrate that the FFPE tumor cores of the type used to generate tissue microarrays (TMAs) can be used as a source of DNA for Taqman-based genotyping. The methodological approaches described herein facilitate the improved application of Taqman-based SNP genotyping to FFPE-derived DNA, significantly increasing the number of assays that can be conducted using what is a valuable and limited tissue resource. from 4 to 19 years, with a median age of 15 years. Specific FFPE samples used in each data figure are indicated in Supplementary Table S1. Tumor and stromal areas were marked on a hematoxylin and eosin-stained slide and then transposed onto the associated paraffin block. Cores (0.6 mm) were punched in the marked areas using the Beecher tissue arrayer MTA1 (Beecher Instruments, Sun Prairie, WI, USA) and the extracted cores were transferred to RNAse- and Dnase-free tubes. The needle punch was cleaned with ethanol and allowed to dry fully between each core acquisition to prevent cross contamination. Two 10 mm paraffin-embedded sections were cut from the same paraffin block before core acquisition. A new blade was used for each patient sample to eliminate the potential for cross-contamination. Gloves were worn at all times. DNA extraction from FFPE tissue samples Our pr (...truncated)