title: Mutation detection of epidermal growth factor receptor and KRAS genes using the smart amplification process version 2 from formalin-fixed, paraffin-embedded lung cancer tissue.
abstract: Recent evidence indicates that the presence of epidermal growth factor receptor (EGFR) or KRAS mutations in non-small cell lung cancer (NSCLC) can predict the response of the tumor to gefinitib. However, it is difficult to detect these mutations using formalin-fixed, paraffin-embedded (FFPE) tissues because the fixation process and aging can damage the DNA. In this study, we describe our work in adapting the Smart Amplification Process version 2 (SmartAmp2) to detect EGFR or KRAS mutations in DNA extracted from FFPE tissues. We were able to detect these mutations in 37 (97%) of 38 FFPE lung cancer tissue samples within 60 minutes with the SmartAmp2 assay and to confirm the correlation between EGFR mutations in FFPE tissues and gefitinib responsiveness. All mutations had previously been confirmed in the 38 samples using DNA extracted from frozen tissues. Electrophoresis results indicated that PCR analysis was not reliable for DNA extracted from FFPE tissue when primers with a long amplicon (>300 bp) were used. This study confirms that the SmartAmp2 assay is suitable for use with DNA extracted from FFPE as well as frozen tissues.
authors.affiliation: Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Miyamae
authors.forename: Kimihiro
authors.initials: K
authors.lastname: Shimizu
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Mitani
authors.forename: Takuya
authors.initials: T
authors.lastname: Araki
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kawai
authors.forename: Masaru
authors.initials: M
authors.lastname: Baba
authors.forename: Seiichi
authors.initials: S
authors.lastname: Kakegawa
authors.forename: Masayuki
authors.initials: M
authors.lastname: Sugano
authors.forename: Kyoichi
authors.initials: K
authors.lastname: Kaira
authors.forename: Alexander
authors.initials: A
authors.lastname: Lezhava
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
authors.forename: Koujirou
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Izumi
authors.initials: I
authors.lastname: Takeyoshi
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics*; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry*; D000077156:Gefitinib; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016612:Paraffin Embedding / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D011799:Quinazolines / Q000627:therapeutic use; D017422:Sequence Analysis, DNA / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D018631:ras Proteins / Q000235:genetics*
chemical_list: D000970:Antineoplastic Agents; D005404:Fixatives; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D011799:Quinazolines; D005557:Formaldehyde; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D000077156:Gefitinib