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Concordance of metabolic enzyme genotypes assayed from paraffin-embedded, formalin-fixed breast tumors and normal lymphatic tissue.

ABSTRACT: OBJECTIVES:Translational epidemiology studies often use archived tumor specimens to evaluate genetic hypotheses involving cancer outcomes. When the exposure of interest is a germline polymorphism, a key concern is whether the genotype assayed from tumor-derived DNA is representative of the germline. We evaluated the concordance between breast tumor-derived and normal lymph node-derived genotypes for three polymorphic tamoxifen-metabolizing enzymes. METHODS:We assayed paired DNA samples extracted from archived tumor and normal lymph node tissues from 106 breast cancer patients. We used TaqMan assays to determine the genotypes of three enzyme variants hypothesized to modify tamoxifen effectiveness, ie, CYP2D6*4, UGT2B15*2, and UGT1A8*2. We assessed genotype agreement between the two DNA sources by calculating the percent agreement and the weighted kappa statistic. RESULTS:We successfully obtained genotypes for CYP2D6*4, UGT2B15*2, and UGT1A8*2 in 99%, 100%, and 84% of the paired samples, respectively. Genotype concordance was perfect for the CYP2D6*4 and UGT1A8*2 variants (weighted kappa for both = 1.00; 95% confidence interval [CI] 1.00, 1.00). For UGT2B15*2, one pair out of 106 gave a discordant result that persisted over several assay repeats. CONCLUSIONS:We observed strong agreement between DNA from breast tumors and normal lymphatic tissue in the genotyping of polymorphisms in three tamoxifen-metabolizing enzymes. Genotyping DNA extracted from tumor tissue avoids the time-consuming practice of microdissecting adjacent normal tissue when other normal tissue sources are not available. Therefore, the demonstrated reliability of tumor-derived DNA allows resources to be spent instead on increasing sample size or the number of polymorphisms examined.

SUBMITTER: Ahern TP

PROVIDER: S-EPMC2998813 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Concordance of metabolic enzyme genotypes assayed from paraffin-embedded, formalin-fixed breast tumors and normal lymphatic tissue.

Ahern Thomas P TP Christensen Mariann M Cronin-Fenton Deirdre P DP Lunetta Kathryn L KL Rosenberg Carol L CL Sørensen Henrik Toft HT Lash Timothy L TL Hamilton-Dutoit Stephen S

Clinical epidemiology 20101022

<h4>Objectives</h4>Translational epidemiology studies often use archived tumor specimens to evaluate genetic hypotheses involving cancer outcomes. When the exposure of interest is a germline polymorphism, a key concern is whether the genotype assayed from tumor-derived DNA is representative of the germline. We evaluated the concordance between breast tumor-derived and normal lymph node-derived genotypes for three polymorphic tamoxifen-metabolizing enzymes.<h4>Methods</h4>We assayed paired DNA sa ...[more]

PMID: 21152250

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Project description:1. The application of massively parallel sequencing has led to the identification of aberrant druggable pathways and somatic mutations within therapeutically relevant genes in gastro-oesophageal cancer. Given the widespread use of formalin-fixed paraffin-embedded (FFPE) samples in the study of this disease, it would be beneficial, especially for the purposes of biomarker evaluation, to assess the concordance between comprehensive exome-wide sequencing data from archival FFPE samples originating from a prospective clinical study and those derived from fresh-frozen material. 2. We analysed whole-exome sequencing data to define the mutational concordance of 16 matched fresh-frozen and FFPE gastro-oesophageal tumours (N = 32) from a prospective clinical study. We assessed DNA integrity prior to sequencing and then identified coding mutations in genes that have previously been implicated in other cancers. In addition, we calculated the mutant-allele heterogeneity (MATH) for these samples. 3. Although there was increased degradation of DNA in FFPE samples compared with frozen samples, sequencing data from only two FFPE samples failed to reach an adequate mapping quality threshold. Using a filtering threshold of mutant read counts of at least ten and a minimum of 5% variant allele frequency (VAF) we found that there was a high median mutational concordance of 97% (range 80.1-98.68%) between fresh-frozen and FFPE gastro-oesophageal tumour-derived exomes. However, the majority of FFPE tumours had higher mutant-allele heterogeneity (MATH) scores when compared with corresponding frozen tumours (p < 0.001), suggesting that FFPE-based exome sequencing is likely to over-represent tumour heterogeneity in FFPE samples compared to fresh-frozen samples. Furthermore, we identified coding mutations in 120 cancer-related genes, including those associated with chromatin remodelling and Wnt/β-catenin and Receptor Tyrosine Kinase signalling. 4. These data suggest that comprehensive genomic data can be generated from exome sequencing of selected DNA samples extracted from archival FFPE gastro-oesophageal tumour tissues within the context of prospective clinical trials.

Dataset Information

Concordance of metabolic enzyme genotypes assayed from paraffin-embedded, formalin-fixed breast tumors and normal lymphatic tissue.

Publications

Concordance of metabolic enzyme genotypes assayed from paraffin-embedded, formalin-fixed breast tumors and normal lymphatic tissue.

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