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High-quality genotyping data from formalin-fixed, paraffin-embedded tissue on the drug metabolizing enzymes and transporters plus array.

Author(s): Vos HI, van der Straaten T, Coenen MJ, Flucke U, te Loo DM, Guchelaar HJ

Publication: J Mol Diagn, 2015, Vol. 17, Page 4-9

PubMed ID: 25528187 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare Affymetrix DMET genotyping data obtained using FFPE specimens to that obtained using saliva or blood from patients with osteosarcoma. 16 FFPE blocks and matched blood (13) or saliva (3) specimens from patients with osteosarcoma were stored for 3-19 years. DNA was extracted from sections containing normal lung, muscle, lymph node, and/or skin using a modification of the QIAamp DNA Micro Kit, from blood using the QIAamp DNA Blood Midi kit and from saliva using the Oragene DNA kit. Blood and saliva are assumed to have been stored frozen, but conditions of archival storage were not described. Fluids were compared with tissues as a group, but saliva was not compared with blood and tissue types were not compared with each other.

   Summary of Findings:

   While all 4 PCR products (100-400 bp) were amplifiable from blood specimens, the 400 bp product was not amplifiable from FFPE specimens and the 300 bp and 200 bp products were only amplifiable from 2/16 and 10/16 FFPE specimens, respectively. The mean genotyping call rates were 99.4% ± 0.30% for blood/saliva DNA and 98.9% ± 1.0% for FFPE specimens. Importantly, 14/16 FFPE specimens yielded call rates >98%, and the 2 specimens with lower call rates also only allowed for amplification of the 100 bp fragment. For all 1931 markers, the genotyping concordance between matched FFPE and blood/saliva specimens was 97.4%, but when no calls were excluded, the concordance for pairs ranged from 97.46-99.48% with all specimens except 1 having concordance of >98.5%. When rare alleles were excluded, genotype concordance increased slightly for all 16 specimens (98.42-99.89%), and exclusion of 4 markers based on cluster plots further increased concordance. 81.1% of discordant calls were due to no calls or rare allele calls using FFPE specimens. 11.01% of discordant calls were due to a homozygous call using blood or saliva and a heterozygous call using FFPE, and 6.2% of discordant calls were due to the reverse. Only 1.13% and 0.5% of discordant calls were due to full switches (homozygous one allele to homozygous another) and allele deletions, respectively. 13 of the 1931 markers had significant differences in allele frequency between FFPE and blood/saliva specimens, but the authors state 9 of these had unreliable clustering. Importantly, discrepant calls resulted in discrepant haplotype and phenotype results for 7 genes between FFPE and blood/saliva specimens. The genotypes of 13 SNPs were confirmed using Illumina BeadArrays of blood or saliva specimens.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Saliva
   • Tissue - Lymph Node
   • Tissue - Muscle (Skeletal)
   • Tissue - Skin
   • Tissue - Lung

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Sarcoma

   Platform:

   Analyte	Technology Platform
   DNA	DNA microarray
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   PCR Specific	Length of gene fragment	100 bp 200 bp 300 bp 400 bp
   DNA microarray Specific	Technology platform	Illumina BeadArray Affymetrix DMET Plus array
   Biospecimen Acquisition	Biospecimen location	Blood Saliva Lung Muscle Skin Lymph

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