Development of highly sensitive genotyping method by blood circulating tumor DNA enrichment using PIP polyamide

• Project/Area Number: 16K09333
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: Chiba Cancer Center (Research Institute)
• Principal Investigator: Denda Tadamichi 千葉県がんセンター(研究所), 消化器内科, 部長 (90466053)
• Co-Investigator(Kenkyū-buntansha): 渡部 隆義 千葉県がんセンター(研究所), がん研究開発グループ, 研究員 (60526060) ; 奥村 和弘 千葉県がんセンター(研究所), がんゲノムセンター 実験動物研究部, 研究員 (80584680) ; 永瀬 浩喜 千葉県がんセンター(研究所), がん遺伝創薬研究室, 研究所長 (90322073)
• Project Period (FY): 2016-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2018: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: PIポリアミド / KRAS変異 / ctDNA / リキッドバイオプシー / 大腸がん / リッキドバイオプシー / ピロールイミダゾール含有ポリアミド / KRAS / 癌 / トランスレーショナルリサーチ / 遺伝子

Outline of Final Research Achievements

We have developed a novel approach that specifically enriches mutant KRAS alleles using biotinylated PI-polyamides, which can be designed to bind to the minor groove of specific DNA sequences. We investigated the clinical utility of this approach for the detection of KRAS mutations in ctDNA. Biotinylated PI-polyamides were synthesized to target common KRAS codon 12 mutations. A total of 21 matched tumor and serum samples were collected from colorectal cancer (CRC) patients. In this study, results from the digital PCR analysis are compared before and after enrichment assay. KRAS mutations were detected before enrichment assay in 47.6% of patients, whereas they were detected after enrichment assay in 81.0% of patients. Among the 10 patients with nonmetastatic CRC, the detection rate after enrichment assay was significantly higher than that before enrichment assay. This newly developed approach significantly improved the identification of KRAS mutations in ctDNA of the CRCs.

Academic Significance and Societal Importance of the Research Achievements

本研究は、膵臓がんや大腸がんで高頻度に変異を起こすRAS遺伝子の変異を標的とした新規技術である。RAS遺伝子の二本鎖DNAの変異配列に特異的に結合し、二本鎖の状態で変異DNAを集めることができるPIポリアミドを合成し、患者血清由来DNAからRAS変異二本鎖DNAを濃縮することに成功した。これまで血液の中のがんの変異DNAを検出することは困難であったが、本技術を用いることで、二本鎖の状態でRAS変異DNAを抽出し、非常に高い感度でがん細胞由来のDNAを検出でき、ステージ１や２の早期のがんでも診断ができることが明らかとなった。この技術は再発がんや早期がんスクリーニング技術などに応用が可能である。

Research Products

• [Journal Article] Enrichment technique to allow early detection and monitor emergence of KRAS mutation in response to treatment. (2019)
  • Author(s): Kitagawa Y, Okumura K, Watanabe T, Tsukamoto K, Kitano S, Nankinzan R, Suzuki T, Hara T, Soda H, Denda T, Yamaguchi T, Nagase H.
  • Journal Title: Sci Rep. Volume: 9 Issue: 1 Pages: 11346-11346
  • DOI: 10.1038/s41598-019-47700-9
  • Peer Reviewed / Open Access
• [Journal Article] Observational study of first-line chemotherapy including cetuximab in patients with metastatic colorectal cancer: CORAL trial. (2019)
  • Author(s): Muro K, Itabashi M, Hashida H, Masuishi T, Bando H, Denda T, Yamanaka T, Ohashi Y, Sugihara K.
  • Journal Title: Jpn J Clin Oncol. Volume: 49(4) Issue: 4 Pages: 339-346
  • DOI: 10.1093/jjco/hyy189
  • Peer Reviewed / Open Access
• [Journal Article] A multicentre, prospective study of plasma circulating tumour DNA test for detecting RAS mutation in patients with metastatic colorectal cancer. (2019)
  • Author(s): Bando H, Kagawa Y, Kato T, Akagi K, Denda T, Nishina T, Komatsu Y, Oki E, Kudo T, Kumamoto H, Yamanaka T, Yoshino T.
  • Journal Title: Br J Cancer. Volume: 120(10) Issue: 8 Pages: 982-986
  • DOI: 10.1038/s41416-020-0766-1
  • Peer Reviewed / Open Access
• [Journal Article] Exploration of potential prognostic biomarkers in aflibercept plus FOLFIRI in Japanese patients with metastatic colorectal cancer. (2019)
  • Author(s): Hamaguchi T, Denda T, Kudo T, Sugimoto N, Ura T, Yamazaki K, Fujii H, Kajiwara T, Nakajima TE, Takahashi S, Otsu S, Komatsu Y, Nagashima F, Moriwaki T, Esaki T, Sato T, Itabashi M, Oki E, Sasaki T, Chiron M, Yoshino T.
  • Journal Title: Cancer Sci. Volume: 110(11) Issue: 11 Pages: 3565-3572
  • DOI: 10.1111/cas.14198
  • NAID: 120007127702
  • Peer Reviewed / Open Access
• [Journal Article] aCGH Analysis of Predictive Biomarkers for Response to Bevacizumab plus Oxaliplatin- or Irinotecan-Based Chemotherapy in Patients with Metastatic Colorectal Cancer (2019)
  • Author(s): Fujita Y, Taguri M, Yamazaki K, Tsurutani J, Sakai K, Tsushima T, Nagase M, Tamagawa H, Ueda S, Tamura T, Tsuji Y, Murata K, Taira K, Denda T, Moriwaki T, Funai S, Nakajima TE, Muro K, Tsuji A, Yoshida M, Suyama K, Kurimoto T, Sugimoto N, Baba E, Seki N, Sato M, Shimura T, Boku N, Hyodo I, Yamanaka T, Nishio K.
  • Journal Title: Oncologist. Volume: 24 Issue: 3 Pages: 327
  • DOI: 10.1634/theoncologist.2018-0119
  • URL: https://localhost/en/publications/26a05734-e681-4082-8f8b-5e3b4c83aae7
  • Peer Reviewed / Open Access
• [Journal Article] Phase II trial of aflibercept with FOLFIRI as a second-line treatment for Japanese patients with metastatic colorectal cancer. (2019)
  • Author(s): Denda T, Sakai D, Hamaguchi T, Sugimoto N, Ura T, Yamazaki K, Fujii H, Kajiwara T, Nakajima TE, Takahashi S, Otsu S, Komatsu Y, Nagashima F, Moriwaki T, Esaki T, Sato T, Itabashi M, Oki E, Sasaki T, Sunaga Y, Ziti-Ljajic S, Brillac C, Yoshino T.
  • Journal Title: Cancer Sci. Volume: 110 Issue: 3 Pages: 1032-1043
  • DOI: 10.1111/cas.13943
  • NAID: 120007128072
  • Peer Reviewed
• [Journal Article] A phase II trial to evaluate the efficacy of panitumumab combined with fluorouracil-based chemotherapy for metastatic colorectal cancer: the PF trial. (2018)
  • Author(s): Munemoto Y, Kanda M, Oba K, Kim HM, Takemoto H, Denda T, Nagata N, Takano N, Fukunaga M, Kataoka M, Tokunaga Y, Sakamoto J, Mishima H.
  • Journal Title: Cancer Chemother Pharmacol. Volume: 81 Issue: 5 Pages: 829-838
  • DOI: 10.1007/s00280-018-3556-1
  • Peer Reviewed
• [Journal Article] Role of Predictive Value of the Modified Glasgow Prognostic Score for Later-line Chemotherapy in Patients With Metastatic Colorectal Cancer. (2018)
  • Author(s): suchihashi K, Ito M, Moriwaki T, Fukuoka S, Taniguchi H, Takashima A, Kumekawa Y, Kajiwara T, Yamazaki K, Esaki T, Makiyama A, Denda T, Satake H, Suto T, Sugimoto N, Katsumata K, Ishikawa T, Kashiwada T, Oki E, Komatsu Y, Okuyama H, Sakai D, Ueno H, Tamura T, Yamashita K, Kishimoto J, Shimada Y, Baba E
  • Journal Title: Clin Colorectal Cancer. Volume: 17 Issue: 4 Pages: 687-697
  • DOI: 10.1016/j.clcc.2018.07.004
  • Peer Reviewed
• [Journal Article] Relationship Between Thymidine Kinase 1 Expression and Trifluridine/Tipiracil Therapy in Refractory Metastatic Colorectal Cancer: A Pooled Analysis of 2 Randomized Clinical Trials. (2018)
  • Author(s): Yoshino T, Yamazaki K, Shinozaki E, Komatsu Y, Nishina T, Baba H, Tsuji A, Tsuji Y, Yamaguchi K, Sugimoto N, Denda T, Muro K, Takayama T, Esaki T, Hamamoto Y, Moriwaki T, Shimada Y, Goto M, Nakayama N, Fujii H, Tanase T, Ohtsu A
  • Journal Title: Clin Colorectal Cancer. Volume: 17 Issue: 4 Pages: 719-732
  • DOI: 10.1016/j.clcc.2018.07.009
  • Peer Reviewed
• [Journal Article] Tumor Sidedness and Enriched Gene Groups for Efficacy of First-line Cetuximab Treatment in Metastatic Colorectal Cancer. (2018)
  • Author(s): Sunakawa Y, Mogushi K, Lenz HJ, Zhang W, Tsuji A, Takahashi T, Denda T, Shimada K, Kochi M, Nakamura M, Kotaka M, Segawa Y, Tanioka H, Negoro Y, Moran M, Astrow SH, Hsiang J, Stephens C, Fujii M, Ichikawa W.
  • Journal Title: Mol Cancer Ther. Volume: 17 Issue: 12 Pages: 2788-2795
  • DOI: 10.1158/1535-7163.mct-18-0694
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Clinical Validation of Newly Developed Multiplex Kit Using Luminex xMAP Technology for Detecting Simultaneous RAS and BRAF Mutations in Colorectal Cancer: Results of the RASKET-B Study. (2018)
  • Author(s): Taniguchi H, Okamoto W, Muro K, Akagi K, Hara H, Nishina T, Kajiwara T, Denda T, Hironaka S, Kudo T, Satoh T, Yamanaka T, Abe Y, Fukushima Y, Yoshino T.
  • Journal Title: Neoplasia. Volume: 20 Issue: 12 Pages: 1219-1226
  • DOI: 10.1016/j.neo.2018.10.004
  • Peer Reviewed
• [Journal Article] Propensity Score Analysis of Regorafenib Versus Trifluridine/Tipiracil in Patients with Metastatic Colorectal Cancer Refractory to Standard Chemotherapy (REGOTAS): A Japanese Society for Cancer of the Colon and Rectum Multicenter Observational Study. (2018)
  • Author(s): Moriwaki T, Fukuoka S, Taniguchi H, Takashima A, Kumekawa Y, Kajiwara T, Yamazaki K, Esaki T, Makiyama C, Denda T, Satake H, Suto T, Sugimoto N, Enomoto M, Ishikawa T, Kashiwada T, Sugiyama M, Komatsu Y, Okuyama H, Baba E, Sakai D, Watanabe T, Tamura T, Yamashita K, Gosho M, Shimada Y.
  • Journal Title: Oncologist. Volume: 23 Issue: 1 Pages: 7-15
  • DOI: 10.1634/theoncologist.2017-0275
  • NAID: 120007134517
  • Peer Reviewed
• [Journal Article] Early morphological change for predicting outcome in metastatic colorectal cancer after regorafenib. (2017)
  • Author(s): Arai H, Miyakawa K, Denda T, Mizukami T, Horie Y, Izawa N, Hirakawa M, Ogura T, Tsuda T, Sunakawa Y, Nakajima TE.
  • Journal Title: Oncotarget. Volume: 8 Issue: 66 Pages: 110530-110539
  • DOI: 10.18632/oncotarget.22807
  • Peer Reviewed / Open Access
• [Journal Article] The subgroups of the phase III RECOURSE trial of trifluridine/tipiracil (TAS-102) versus placebo with best supportive care in patients with metastatic colorectal cancer. (2017)
  • Author(s): Van Cutsem E, Mayer RJ, Laurent S, Winkler R, Gravalos C, Benavides M, Longo-Munoz F, Portales F, Ciardiello F, Siena S, Yamaguchi K, Muro K, Denda T, Tsuji Y, Makris L, Loehrer P, Lenz HJ, Ohtsu A; RECOURSE Study Group.
  • Journal Title: Eur J Cancer. Volume: 90 Pages: 63-72
  • DOI: 10.1016/j.ejca.2017.10.009
  • Peer Reviewed / Open Access
• [Journal Article] CEA Response and Depth of Response (DpR) to Predict Clinical Outcomes of First-Line Cetuximab Treatment for Metastatic Colorectal Cancer. (2017)
  • Author(s): Sunakawa Y, Tsuji A, Denda T, Segawa Y, Negoro Y, Shimada K, Kochi M, Nakamura M, Kotaka M, Tanioka H, Takagane A, Tani S, Yamaguchi T, Watanabe T, Takeuchi M, Fujii M, Ichikawa W.
  • Journal Title: Target Oncol. Volume: 12 Issue: 6 Pages: 787-794
  • DOI: 10.1007/s11523-017-0527-0
  • Peer Reviewed
• [Journal Article] Cumulative incidence, risk factors and prognostic impact of venous thromboembolism in Japanese patients with advanced gastric cancer. (2017)
  • Author(s): Arai H, Hironaka S, Minashi K, Denda T, Shimokawa M, Yamaguchi T.
  • Journal Title: Jpn J Clin Oncol. Volume: 47 Issue: 10 Pages: 942-948
  • DOI: 10.1093/jjco/hyx107
  • Peer Reviewed
• [Journal Article] Phase II study of S-1 plus leucovorin in patients with metastatic colorectal cancer: Regimen of 1 week on, 1 week off. (2017)
  • Author(s): Li J, Xu R, Xu J, Denda T, Ikejiri K, Shen L, Toh Y, Shimada K, Kato T, Sakai K, Yamamoto M, Mishima H, Wang J, Baba H.
  • Journal Title: Cancer Sci. Volume: 108 Issue: 10 Pages: 2045-2051
  • DOI: 10.1111/cas.13335
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Combined assessment of EGFR-related molecules to predict outcome of 1st-line cetuximab-containing chemotherapy for metastatic colorectal cancer. (2016)
  • Author(s): Sunakawa Y, Yang D, Moran M, Astrow SH, Tsuji A, Stephens C, Zhang W, Cao S, Takahashi T, Denda T, Shimada K, Kochi M, Nakamura M, Kotaka M, Segawa Y, Masuishi T, Takeuchi M, Fujii M, Nakajima T, Ichikawa W, Lenz HJ.
  • Journal Title: Cancer Biol Ther. Volume: 17 Pages: 751-759
  • Peer Reviewed
• [Journal Article] Early Tumor Shrinkage and Depth of Response as Predictors of Favorable Treatment Outcomes in Patients with Metastatic Colorectal Cancer Treated with FOLFOX Plus Cetuximab (JACCRO CC-05). (2016)
  • Author(s): Tsuji A, Sunakawa Y, Ichikawa W, Nakamura M, Kochi M, Denda T, Yamaguchi T, Shimada K, Takagane A, Tani S, Kotaka M, Kuramochi H, Furushima K, Koike J, Yonemura Y, Takeuchi M, Fujii M, Nakajima T.
  • Journal Title: Target Oncol. Volume: 11 Pages: 799-806
  • Peer Reviewed
• [Journal Article] Phase II trial of panitumumab with irinotecan as salvage therapy for patients with advanced or recurrent colorectal cancer (TOPIC study). (2016)
  • Author(s): Nishi T, Hamamoto Y, Nagase M, Denda T, Yamaguchi K, Amagai K, Miyata Y, Yamanaka Y, Yanai K, Ishikawa T, Kuroki Y, Fujii H.
  • Journal Title: Oncol Lett. Volume: 11 Pages: 4049-4054
  • Peer Reviewed
• [Journal Article] Randomized study of FOLFIRI plus either panitumumab or bevacizumab for wild-type KRAS colorectal cancer-WJOG 6210G. (2016)
  • Author(s): Shitara K, Yonesaka K, Denda T, Yamazaki K, Moriwaki T, Tsuda M, Takano T, Okuda H, Nishina T, Sakai K, Nishio K, Tokunaga S, Yamanaka T, Boku N, Hyodo I, Muro K.
  • Journal Title: Cancer Sci. Volume: 107 Issue: 12 Pages: 1843-1850
  • DOI: 10.1111/cas.13098
  • NAID: 120007135107
  • Peer Reviewed
• [Journal Article] Pharmacokinetic dose adjustment of 5-FU in modified FOLFOX7 plus bevacizumab for metastatic colorectal cancer in Japanese patients: a-JUST phase II clinical trial. (2016)
  • Author(s): Denda T, Kanda M, Morita Y, Kim HM, Kashiwada T, Matsuda C, Fujieda S, Nakata K, Murotani K, Oba K, Sakamoto J, Mishima H.
  • Journal Title: Cancer Chemother Pharmacol. Volume: 78 Issue: 7 Pages: 1253-1261
  • DOI: 10.1080/15384047.2016.1178426
  • NAID: 120006220413
  • Peer Reviewed
• [Journal Article] Prognostic Impact of Primary Tumor Location on Clinical Outcomes of Metastatic Colorectal Cancer Treated With Cetuximab Plus Oxaliplatin-Based Chemotherapy: A Subgroup Analysis of the JACCRO CC-05/06 Trials. (2016)
  • Author(s): Sunakawa Y, Ichikawa W, Tsuji A, Denda T, Segawa Y, Negoro Y, Shimada K, Kochi M, Nakamura M, Kotaka M, Tanioka H, Takagane A, Tani S, Yamaguchi T, Watanabe T, Takeuchi M, Fujii M, Nakajima T.
  • Journal Title: Clin Colorectal Cancer. Volume: S1533-0028 Issue: 3 Pages: 30204-3
  • DOI: 10.1016/j.clcc.2016.09.010
  • Peer Reviewed
• [Presentation] 抗血栓薬内服下における早期胃癌に対する内視鏡的粘膜下層剥離術のセカンドルック内視鏡に関する検討 (2019)
  • Author(s): 石垣飛鳥,鈴木拓人,南金山理乃,北川善康,傳田忠道,山口武人
  • Organizer: 第108回日本消化器内視鏡学会関東支部例会
• [Presentation] Whole-exome sequencing in metastatic or locally advanced pancreatic cancer patients with long-term survival (2019)
  • Author(s): 須藤研太郎,横井左奈,小原收,中村和貴,辻本彰子,喜多絵美里,長谷川嘉則,中山学,石井浩,傳田忠道,山口武人
  • Organizer: 第17回日本臨床腫瘍学会学術集会
• [Presentation] ESD 治療により診断、治療し得たDysplasiaを伴う尖圭コンジローマの一例 (2019)
  • Author(s): 橋本佳恵,石垣飛鳥,喜多絵美里,辻本彰子,天沼祐介,須藤研太郎,中村和貴,三梨桂子,傳田忠道,山口武人,鈴木拓人,南金山理乃,北川善康,荒木章伸,伊丹真紀子
  • Organizer: 日本消化器病学会関東支部第356回例会
• [Presentation] TRICOLORE試験（大腸癌第3相試験: mFOLFOX6/CapeOX+Bev vs S-1/CPT-11+Bev）のQOL解析 (2019)
  • Author(s): 佐藤温,高島淳生,傳田忠道,蒲生真紀夫,高畑武功,天貝賢二,倉持英和,近藤仁,的場周一郎,有岡仁,小松嘉人,山口達郎,嶋田顕,森田智視,石岡千加史
  • Organizer: 第57回日本癌治療学会学術集会
• [Presentation] TRICOLORE試験に基づいた転移を有する大腸癌に対する一次治療の費用効果分析 (2019)
  • Author(s): 後藤励,高島淳生,傳田忠道,蒲生真紀夫,倉持英和,天貝賢二,近藤仁,杉山克郎,中村路夫,畑中一映,石岡千加史,小松嘉人,山口達郎,嶋田顕,森田智視
  • Organizer: 第57回日本癌治療学会学術集会
• [Presentation] 結腸直腸癌の原発部位別遺伝子プロファイル解析 GI-Screen 2013-01-CRC 付随研究 (2019)
  • Author(s): 水上拓郎,高橋政樹,砂川優,結城敏志,賀川義規,高島淳生,舛石俊樹,原浩樹,傳田忠道,森脇俊和,塩澤学,沖英次,岡本渉,吉野孝之,中島貴子
  • Organizer: 第57回日本癌治療学会学術集会
• [Presentation] 抗血栓薬内服者における胃ESD後のセカンドルック内視鏡についての検討 (2019)
  • Author(s): 石垣飛鳥,鈴木拓人,南金山理乃,北川善康,傳田忠道,山口武人
  • Organizer: JDDW
• [Presentation] 微小陥凹形態を呈した十二指腸Neuroendocrine tumor の一例 (2019)
  • Author(s): 橋本佳恵,石垣飛鳥,辻本彰子,喜多絵美里,天沼裕介,須藤研太郎,中村和貴,三梨桂子,傳田忠道,石井浩,山口武人,鈴木拓人,南金山理乃,北川善康,川名秀忠,荒木彰伸,伊丹真紀子
  • Organizer: 日本消化器病学会関東支部第357回例会
• [Presentation] PIポリアミドを用いた高感度ctDNA検出技術の開発と臨床応用の検討 (2018)
  • Author(s): 北川善康,傳田忠道,山口武人
  • Organizer: 消化器病学会・消化器内視鏡学会・消化器外科学会・消化器がん検診学会
• [Presentation] 血中微量二本鎖核酸分子を標的とした PI ポリアミドを用いたがん遺伝子変異同定法の開発と臨床応用の検討 (2018)
  • Author(s): 北川善康、奥村和弘、鈴木拓人、渡部隆義、早田浩明、傳田忠道、永瀬浩喜、山口武人
  • Organizer: 日本癌病態治療研究会
• [Presentation] 血中微量二本鎖核酸分子を標的とした PI ポリアミドを用いたがん遺伝子変異同定法の開発と臨床応用の検討 (2017)
  • Author(s): 北川善康、奥村和弘、鈴木拓人、渡部隆義、早田浩明、傳田忠道、永瀬浩喜、山口武人
  • Organizer: 第26回日本癌病態治療研究会
• [Presentation] RAS野生型大腸癌における原発部位とcetuximab併用療法の予後との相関: JACCRO CC-05/06 (2016)
  • Author(s): 砂川 優, 辻 晃仁, 傳田忠道, 嶋田 顕, 東風 貢, 中村 将人, 小高 雅人, 畝川 芳彦, 谷岡 洋亮, 根来 裕二, 山口 達郎, 竹内 正弘, 藤井 雅志, 市川 度, 中島 聰總
  • Organizer: 第54回日本癌治療学会学術集会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2016-10-20
• [Presentation] Cetuximab併用療法耐性大腸癌に対するS-1/CPT-11/bevacizumabの第Ⅱ相試験 (2016)
  • Author(s): 谷岡 洋亮,辻 晃仁, 東風 貢, 高橋 孝夫, 傳田忠道, 小川 雅彰, 渡邉 貴紀, 金 浩敏, 嶋田 顕:
  • Organizer: 第54回日本癌治療学会学術集会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2016-10-20
• [Presentation] 切除不能な大腸癌症例におけるセツキシマブを含む一次治療の観察研究 (CORAL) (2016)
  • Author(s): 傳田忠道,室圭, 板橋道朗, 大橋靖雄, 杉原健一
  • Organizer: 第54回日本癌治療学会学術集会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2016-10-20