Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.
DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies.
Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore, Singapore.
Shenli
S
Zhang
Department of Medical Oncology, National Cancer Centre, Singapore, Singapore.
Iain B
IB
Tan
Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.
Nur S
NS
Sapari
Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom.
Heike I
HI
Grabsch
Department of Gastrointestinal Oncology, Royal Marsden Hospital, Sutton, United Kingdom.
Alicia
A
Okines
Department of Gastrointestinal Oncology, Royal Marsden Hospital, Sutton, United Kingdom.
Elizabeth C
EC
Smyth
Department of Gastrointestinal Surgery, Kanagawa Cancer Center, Yokohama, Japan.
Toru
T
Aoyama
Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom.
Lindsay C
LC
Hewitt
Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom.
Imran
I
Inam
Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom.
Dan
D
Bottomley
Medical Research Council Clinical Trials Unit, University College London, London, United Kingdom.
Matthew
M
Nankivell
Medical Research Council Clinical Trials Unit, University College London, London, United Kingdom.
Sally P
SP
Stenning
Department of Gastrointestinal Oncology, Royal Marsden Hospital, Sutton, United Kingdom.
David
D
Cunningham
Department of Pathology, Royal Marsden Hospital, Sutton, United Kingdom.
Andrew
A
Wotherspoon
Gastroenterological Center, Yokohama City University Medical Center, Yokohama, Japan.
Akira
A
Tsuburaya
Department of Gastrointestinal Surgery, Kanagawa Cancer Center, Yokohama, Japan.
Takaki
T
Yoshikawa
Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore; Department of Pathology, National University Health System, Singapore, Singapore.
Richie
R
Soong
Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore, Singapore; Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore; Department of Cellular and Molecular Research, National Cancer Centre, Singapore, Singapore; Cancer Therapeutics and Stratified Oncology Group, Genome Institute of Singapore, Singapore, Singapore. Electronic address: gmstanp@duke-nus.edu.sg.
Patrick
P
Tan
D002460:Cell Line; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005557:Formaldehyde / Q000737:chemistry; D018628:Gene Dosage; D005819:Genetic Markers / Q000235:genetics; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010232:Paraffin / Q000737:chemistry; D016612:Paraffin Embedding / Q000379:methods; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D016707:Tissue Fixation / Q000379:methods
D005819:Genetic Markers; D005557:Formaldehyde; D010232:Paraffin; D004247:DNA