Establish of new detection method of gene mutations using cell-free DNA in supernatant fluids

• Project/Area Number: 26460462
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Human pathology
• Research Institution: Kurume University
• Principal Investigator: kawahara akihiko 久留米大学, 大学病院, 医療技術員 (00469347)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2014: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 分子病理学 / 遊離DNA / EGFR遺伝子変異 / 体腔液細胞診 / 病理学 / 分子病理学的検査 / 細胞診 / 上澄みDNA / 液状化細胞診 / 肺癌

Outline of Final Research Achievements

We examined whether it was possible to detect EGFR mutations in cytology cell-free DNA (ccfDNA) from the supernatant fluids of bronchial and effusion cytology samples. Quantity and fragmentation of ccfDNA in the supernatant fluid and cell damage and CC3 expression in the sediment gradually increased in a time dependent manner in the cell lines. In the 74 clinical samples, the detection of EGFR mutations using ccfDNA showed a high sensitivity in samples with malignant cells. In contrast, EGFR mutations were not found in negative samples. The twenty-two patients of EGFR mutations were detected in 63.6% of ccfDNA from supernatant fluids. EGFR mutations were found in 81.3% (13/16) of malignant cell patients, whereas EGFR mutations were not found in the negative samples. Our results suggest that ccfDNA might help to compensate for the lack of adequate DNA in EGFR mutation analysis, and ccfDNA supernatant fluids can be used to detect EGFR mutation in the same way as cancer cell sediments.

Research Products

• [Journal Article] EGFR mutation status in cell-free DNA supernatant of bronchial washings and brushings. (2015)
  • Author(s): Kawahara A, Fukumitsu C, Taira T, Abe H, Takase Y, Mutara K, Yamaguchi T, Azuma K, Ishii H, Takamori S, Akiba J, Hoshono T, Kage M.
  • Journal Title: Cancer Cytopathology Volume: 123 Issue: 10 Pages: 620-628
  • DOI: 10.1002/cncy.21583
  • Peer Reviewed
• [Journal Article] Fixation effect of SurePath preservative fluids using EGFR mutation-specific antibodies for immunocytochemistry. (2014)
  • Author(s): Kawahara A, Taira T, Abe H, Watari K, Murakami Y, Fukumitsu C, Takase Y, Yamaguchi T, Akiba J, Azuma K, Ono M, Kage M
  • Journal Title: Cancer Cytopathology Volume: 122 Issue: 2 Pages: 145-152
  • DOI: 10.1002/cncy.21355
  • Peer Reviewed
• [Journal Article] EML4-ALK-positive lung adenocarcinoma with signet-ring cells (2014)
  • Author(s): Kawahara A, Akiba J, Abe H, Yamaguchi T, Taira T, Azuma K, and Kage M
  • Journal Title: Diagnostic cytopathology Volume: 42 Issue: 5 Pages: 460-463
  • DOI: 10.1002/dc.22936
  • Peer Reviewed
• [Journal Article] Evaluation of immunohistochemistry using two different antibodies and procedures for primary lung adenocarcinoma harboring anaplastic lymphoma kinase rearrangement (2014)
  • Author(s): Akiba J, Kawahara A, Abe H, Azuma K, Yamaguchi T, Taira T, Fukumitsu C, Takase Y, Yasumoto M, Umeno Y, Todoroki K, Kurita T, Yamaguchi R, Kage M, Yano H
  • Journal Title: Oncol Letters Volume: 8 Issue: 5 Pages: 2155-2159
  • DOI: 10.3892/ol.2014.2511
  • Peer Reviewed
• [Journal Article] FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib, a pan-EGFR family kinase inhibitor (2014)
  • Author(s): Azuma K, Kawahara A, Sonoda K, Nakashima K, Tashiro K, Watari K, Izumi H, Kage M, Kuwano M, Ono M, Hoshino T
  • Journal Title: Oncotarget Volume: 5 Pages: 5908-5919
  • Peer Reviewed
• [Journal Article] Humoral immune responses to EGFR-derived peptides predict progression-free and overall survival of non-small cell lung cancer patients receiving gefitinib. (2014)
  • Author(s): Azuma K, Komatsu N, Hattori S, Matsueda S, Kawahara A, Sasada T, Itoh K, Hoshino T.
  • Journal Title: PLoS One. Volume: 9 Issue: 1 Pages: e86667-e86667
  • DOI: 10.1371/journal.pone.0086667
  • Peer Reviewed
• [Presentation] Detection of EGFR mutation status in cell-free DNA supernatant of effusion cytology (2016)
  • Author(s): Kawahara Akihiko, Fukumitsu Chihiro, Taira Tomoki, Abe Hideyuki, Takase Yorihiko, Murata Kazuya, Yamaguchi Tomohiko, Naito Yoshiki, Akiba Jun
  • Organizer: 40th European Congress of Cytology
  • Place of Presentation: イギリス リバプール
  • Year and Date: 2016-10-02
  • Int'l Joint Research
• [Presentation] Epidermal growth factor receptor mutation status in cell-free DNA supernatant of bronchial cytology samples (2016)
  • Author(s): Kawahara Akihiko, Fukumitsu Chihiro, Taira Tomoki, Abe Hideyuki, Takase Yorihiko, Murata Kazuya, Yamaguchi Tomohiko, Akiba Jun, Kage Masayoshi
  • Organizer: The 19th International Congress of Cytology (ICC2016)
  • Place of Presentation: 横浜
  • Year and Date: 2016-05-29
  • Int'l Joint Research
• [Presentation] Detection of activating EGFR mutation status using cytology cell free DNA in supernatant from liquid-based cytology of bronchial washing and bronchial brushing samples. (2015)
  • Author(s): Kawahara Akihiko, Fukumitsu Chihiro, Taira Tomoki, Abe Hideyuki, Takase Yorihiko, Murata Kazuya, Yamaguchi Tomohiko, Tanikawa Ken and Masayoshi Kage.
  • Organizer: 39th European Congress of Cytology.
  • Place of Presentation: Italy, Milan.
  • Year and Date: 2015-09-20
  • Int'l Joint Research
• [Presentation] Fixation effect of SurePath preservative fluids using EGFR mutation-specific antibodies for immunocytochemistry (2014)
  • Author(s): Kawahara Akihiko, Taira Tomoki, Abe Hideyuki, Fukumitsu Chihiro, Takase Yorihiko, Yamaguchi Tomohiko, Akiba Jun, Masayoshi Kage
  • Organizer: 38th European Congress of Cytology
  • Place of Presentation: Switzerland, Geneva
  • Year and Date: 2014-09-27 – 2014-09-30
• [Presentation] LBCの導入効果と分子病理診断への可能性 (2014)
  • Author(s): 河原明彦
  • Organizer: 第28回関東臨床細胞学会
  • Place of Presentation: 静岡県沼津市
  • Year and Date: 2014-09-13
  • Invited
• [Presentation] EGFR遺伝子変異特異的抗体を用いたSurePath細胞保存液の固定効果 (2014)
  • Author(s): 河原明彦，多比良朋希，安倍秀幸，山口知彦，吉田友子，髙瀨頼妃呼，福満千容，谷川 健，秋葉 純，鹿毛政義
  • Organizer: 第55回日本臨床細胞学会総会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2014-06-05 – 2014-06-07