title: The efficacy of uracil DNA glycosylase pretreatment in amplicon-based massively parallel sequencing with DNA extracted from archived formalin-fixed paraffin-embedded esophageal cancer tissues.
abstract: Advances in mutation testing for molecular-targeted cancer therapies have led to the increased use of archived formalin-fixed paraffin-embedded (FFPE) tumors. However, DNA extracted from FFPE tumors (FFPE DNA) is problematic for mutation testing, especially for amplicon-based massively parallel sequencing (MPS), owing to DNA fragmentation and artificial C:G > T:A single nucleotide variants (SNVs) caused by deamination of cytosine to uracil. Therefore, to reduce artificial C:G > T:A SNVs in amplicon-based MPS using FFPE DNA, we evaluated the efficacy of uracil DNA glycosylase (UDG) pretreatment, which can eliminate uracil-containing DNA molecules, with 126 archived FFPE esophageal cancer specimens. We also examined the association between the frequency of C:G > T:A SNVs and DNA quality, as assessed by a quantitative PCR (qPCR)-based assay. UDG pretreatment significantly lowered the frequency of C:G > T:A SNVs in highly fragmented DNA (by approximately 60%). This effect was not observed for good- to moderate-quality DNA, suggesting that a predictive assay (i.e., DNA quality assessment) needs to be performed prior to UDG pretreatment. These results suggest that UDG pretreatment is efficacious for mutation testing by amplicon-based MPS with fragmented DNA from FFPE samples.
authors.affiliation: Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Sunto-gun, Japan.
authors.forename: Masakuni
authors.initials: M
authors.lastname: Serizawa
authors.affiliation: Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Sunto-gun, Japan; Division of Gastrointestinal Oncology, Shizuoka Cancer Center, Sunto-gun, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Yokota
authors.affiliation: Department of Gastroenterology and Hematology, Faculty of Medicine, University of Toyama, Toyama, Japan.
authors.forename: Ayumu
authors.initials: A
authors.lastname: Hosokawa
authors.affiliation: Pathology Division, Shizuoka Cancer Center, Sunto-gun, Japan.
authors.forename: Kimihide
authors.initials: K
authors.lastname: Kusafuka
authors.affiliation: Department of Gastroenterology and Hematology, Faculty of Medicine, University of Toyama, Toyama, Japan.
authors.forename: Toshiro
authors.initials: T
authors.lastname: Sugiyama
authors.affiliation: Division of Esophageal Surgery, Shizuoka Cancer Center, Sunto-gun, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Tsubosa
authors.affiliation: Division of Gastrointestinal Oncology, Shizuoka Cancer Center, Sunto-gun, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yasui
authors.affiliation: Pathology Division, Shizuoka Cancer Center, Sunto-gun, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakajima
authors.affiliation: Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Sunto-gun, Japan; Third Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan. Electronic address: ykoh@wakayama-med.ac.jp.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Koh
mesh_terms: D004247:DNA / Q000032:analysis; D004938:Esophageal Neoplasms / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D016707:Tissue Fixation; D051981:Uracil-DNA Glycosidase / Q000378:metabolism*
chemical_list: D004247:DNA; D051981:Uracil-DNA Glycosidase
keywords: Amplicon-based massively parallel sequencing; Cytosine deamination; Formalin-fixed paraffin-embedded samples; Molecular cancer diagnostics; Uracil DNA glycosylase