Abstract
BackgroundCancer pharmacogenetic studies use archival tumor samples as a DNA source when germline DNA is unavailable. Genotyping DNA from formalin-fixed paraffin embedded tumors (FFPE-T) may be inaccurate due to FFPE storage, genetic aberrations, and/or insufficient DNA extraction. Our objective was to assess the extent and source of genotyping inaccuracy from FFPE-T DNA and demonstrate analytical validity of FFPE-T genotyping of candidate single nucleotide polymorphisms (SNPs) for pharmacogenetic analyses. MethodsCancer pharmacogenetics SNPs were genotyped by Sequenom MassARRAYs in DNA harvested from matched FFPE-T, FFPE lymph node (FFPE-LN), and whole blood leukocyte samples obtained from breast cancer patients. No- and discordant-call rates were calculated for each tissue type and SNP. Analytical validity was defined as any SNP with <5% discordance between FFPE-T and blood and <10% discordance plus no-calls. ResultsMatched samples from 114 patients were genotyped for 247 SNPs. No-call rate in FFPE-T was greater than FFPE-LN and blood (4.3% vs. 3.0% vs. 0.5%, p < 0.001). Discordant-call rate between FFPE-T and blood was very low, but greater than that between FFPE-LN and blood (1.1% vs. 0.3%, p < 0.001). Samples with heterozygous genotypes were more likely to be no- or discordantly-called in either tissue (p < 0.001). Analytical validity of FFPE-T genotyping was demonstrated for 218 (88%) SNPs. ConclusionsNo- and discordant-call rates were below concerning thresholds, confirming that most SNPs can be accurately genotyped from FFPE-T on our Sequenom platform. FFPE-T is a viable DNA source for prospective–retrospective pharmacogenetic analyses of clinical trial cohorts.
Highlights
In cancer there are two relevant genomes, that of the tumor and the patient (McLeod, 2013)
After exclusion of four patients’ samples due to unacceptable genotype call rates in blood (
The no-call rate in FFPE lymph node (FFPE-LN) was approximately 70% of that of formalin-fixed paraffin embedded tumors (FFPE-T), suggesting that most of the non-calling in FFPE-T is attributable to the FFPE process, consistent with what we reported previously for matched CYP2D6 genotyping (Rae et al, 2013)
Summary
In cancer there are two relevant genomes, that of the tumor (somatic) and the patient (germline) (McLeod, 2013). Cancer pharmacogenetic studies use archival tumor samples as a DNA source when germline DNA is unavailable. Our objective was to assess the extent and source of genotyping inaccuracy from FFPE-T DNA and demonstrate analytical validity of FFPE-T genotyping of candidate single nucleotide polymorphisms (SNPs) for pharmacogenetic analyses. Methods: Cancer pharmacogenetics SNPs were genotyped by Sequenom MassARRAYs in DNA harvested from matched FFPE-T, FFPE lymph node (FFPE-LN), and whole blood leukocyte samples obtained from breast cancer patients. Results: Matched samples from 114 patients were genotyped for 247 SNPs. No-call rate in FFPE-T was greater than FFPE-LN and blood (4.3% vs 3.0% vs 0.5%, p < 0.001).
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