詳細

坂井　和子 （サカイ　カズコ）

  • 医学科　特命准教授
Last Updated :2024/04/25

コミュニケーション情報 byコメンテータガイド

  • コメント

    がん分野における遺伝子解析に関する内容。

研究者情報

学位

  • 博士（理学）（お茶の水女子大学）

ホームページURL

J-Global ID

現在の研究分野（キーワード）

    がん分野における遺伝子解析に関する内容。

研究分野

  • ライフサイエンス / 腫瘍診断、治療学

経歴

  • 2012年  近畿大学医学部助教

研究活動情報

論文

  • Akiko Arimura; Kazuko Sakai; Kazuhisa Kaneshiro; Takafumi Morisaki; Saori Hayashi; Kimihisa Mizoguchi; Mai Yamada; Masaya Kai; Mayumi Ono; Kazuto Nishio; Masafumi Nakamura; Makoto Kubo
    Cancers 2024年03月
  • Shinichi Sakamoto; Keisuke Ando; Sangjon Pae; Xue Zhao; Kazuko Sakai; Kodai Sato; Shinpei Saito; Yasutaka Yamada; Junryo Rii; Yusuke Goto; Tomokazu Sazuka; Yusuke Imamura; Naohiko Anzai; Koichiro Akakura; Kazuto Nishio; Tomohiko Ichikawa
    Anticancer research 44 2 639 - 647 2024年02月 
    BACKGROUND/AIM: The prognostic significance of androgen receptor amplification (AR amp) in cell-free DNA (cfDNA) was studied in Japanese patients with castration-resistant prostate cancer (CRPC). PATIENTS AND METHODS: A total of 120 serum samples were obtained from 38 patients with CRPC. Serum cfDNA was purified and the AR copy number was determined. Factors associated with progression-free survival (PFS) and overall survival (OS) were statistically investigated. RESULTS: The number of patients administered enzalutamide (Enza)/abiraterone (Abi)/docetaxel (DTX) was 33/25/11, respectively. The median PSA was 16.5 ng/ml. Thirty patients (79%) had bone metastases and three patients (7.9%) had lung metastases. The median follow-up was 655 days. The median initial AR copy number was 1.27 (1.10-11.50); an AR copy number of 1.27 or higher was defined as an AR-amp. Regarding PFS, the presence of AR-amp, Gleason score (GS), and ALP were significant factors in univariate analysis. In multivariate analysis, AR amplification was an independent prognostic factor (hazard ratio=7.7, p=0.0035). For OS, PSA and AR-amp were significant factors. In multivariate analysis, AR-amp (hazard ratio=4.65, p=0.0188) was the only independent prognostic factor. CONCLUSION: AR-amp was associated with high nadir PSA and low iPSA/PSA ratio. AR-amp was significantly associated with poor prognosis in Japanese patients with CRPC.
  • Terufumi Yoshida; Kazuko Sakai; Masaki Kaibori; Mitsuaki Ishida; Shogo Tanaka; Shoji Kubo; Takuya Nakai; Marco De Velasco; Hideyuki Matsushima; Koji Tsuta; Mitsugu Sekimoto; Kazuto Nishio
    Oncology Letters 27 3 2024年01月
  • Teruaki Takasaki; Yasuyuki Hamabe; Kenta Touchi; Golam Iftakhar Khandakar; Takeshi Ueda; Hitoshi Okada; Kazuko Sakai; Kazuto Nishio; Genzoh Tanabe; Reiko Sugiura
    Oxidative medicine and cellular longevity 2024 7683793 - 7683793 2024年 
    The extracellular signal-regulated kinase (ERK) MAPK pathway is dysregulated in various human cancers and is considered an attractive therapeutic target for cancer. Therefore, several inhibitors of this pathway are being developed, and some are already used in the clinic. We have previously identified an anticancer compound, ACA-28, with a unique property to preferentially induce ERK-dependent apoptosis in melanoma cells. To comprehensively understand the biological cellular impact induced by ACA-28, we performed a global gene expression analysis of human melanoma SK-MEL-28 cells exposed to ACA-28 using a DNA microarray. The transcriptome analysis identified nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcription factor that combats oxidative stress, as the most upregulated genetic pathway after ACA-28 treatment. Consistently, ACA-28 showed properties to increase the levels of reactive oxygen species (ROS) as well as Nrf2 protein, which is normally repressed by proteasomal degradation and activated in response to oxidative stresses. Furthermore, the ROS scavenger N-acetyl cysteine significantly attenuated the anticancer activity of ACA-28. Thus, ACA-28 activates Nrf2 signaling and exerts anticancer activity partly via its ROS-stimulating property. Interestingly, human A549 cancer cells with constitutively high levels of Nrf2 protein showed resistance to ACA-28, as compared with SK-MEL-28. Transient overexpression of Nrf2 also increased the resistance of cells to ACA-28, while knockdown of Nrf2 exerted the opposite effect. Thus, upregulation of Nrf2 signaling protects cancer cells from ACA-28-mediated cell death. Notably, the Nrf2 inhibitor ML385 substantially enhanced the cell death-inducing property of ACA-28 in pancreatic cancer cells, T3M4 and PANC-1. Our data suggest that Nrf2 plays a key role in determining cancer cell susceptibility to ACA-28 and provides a novel strategy for cancer therapy to combine the Nrf2 inhibitor and ACA-28.
  • Satoshi Ikeda; Masahiro Tsuboi; Kazuko Sakai; Toshihiro Misumi; Hiroaki Akamatsu; Hiroyasu Shoda; Noriaki Sakakura; Atsushi Nakamura; Yasuhisa Ohde; Hidetoshi Hayashi; Kyoichi Okishio; Morihito Okada; Ichiro Yoshino; Jiro Okami; Kazuhisa Takahashi; Norihiko Ikeda; Masayuki Tanahashi; Yuichi Tambo; Haruhiro Saito; Shinichi Toyooka; Hidetoshi Inokawa; Toyofumi Chen-Yoshikawa; Toshihide Yokoyama; Tatsuro Okamoto; Noriko Yanagitani; Masahide Oki; Makoto Takahama; Kenji Sawa; Hirohito Tada; Kazuhiko Nakagawa; Tetsuya Mitsudomi; Kazuto Nishio
    Molecular oncology 2023年10月 
    The phase III IMPACT study (UMIN000044738) compared adjuvant gefitinib with cisplatin plus vinorelbine (cis/vin) in completely resected epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). Although the primary endpoint of disease-free survival (DFS) was not met, we searched for molecular predictors of adjuvant gefitinib efficacy. Of 234 patients enrolled in the IMPACT study, 202 patients were analyzed for 409 cancer-related gene mutations and tumor mutation burden using resected lung cancer specimens. Frequent somatic mutations included tumor protein p53 (TP53; 58.4%), CUB and Sushi multiple domains 3 (CSMD3; 11.8%), and NOTCH1 (9.9%). Multivariate analysis showed that NOTCH1 co-mutation was a significant poor prognostic factor for overall survival (OS) in the gefitinib group and cAMP response element binding protein (CREBBP) co-mutation for DFS and OS in the cis/vin group. In patients with NOTCH1 co-mutations, gefitinib group had a shorter OS than cis/vin group (Hazard ratio 5.49, 95% CI 1.07-28.00), with a significant interaction (P for interaction = 0.039). In patients with CREBBP co-mutations, the gefitinib group had a longer DFS than the cis/vin group, with a significant interaction (P for interaction = 0.058). In completely resected EGFR-mutated NSCLC, NOTCH1 and CREBBP mutations might predict poor outcome in patients treated with gefitinib and cis/vin, respectively.
  • 妊孕性温存治療中BRAF融合型神経内分泌癌成分の転移が急速に進行した子宮内膜癌の1例
    加嶋 洋子; 宮川 知保; 高矢 寿光; 坂井 和子; 西尾 和人; 松村 謙臣
    日本癌治療学会学術集会抄録集 61回 P51 - 1 2023年10月
  • Junko Tanizaki; Hiroaki Kuroda; Toshihide Yokoyama; Makoto Takahama; Hiroyasu Shoda; Atsushi Nakamura; Yoshitaka Kitamura; Nobuaki Mamesaya; Yoshihisa Kadota; Kenji Sawa; Kyoichi Okishio; Morihito Okada; Chihiro Suminaka; Kenta Noda; Kazuko Sakai; Yasutaka Chiba; Kazuto Nishio; Kenji Chamoto; Tasuku Honjo; Nobuyuki Yamamoto; Kazuhiko Nakagawa; Hidetoshi Hayashi
    JTO Clinical and Research Reports 100590 - 100590 2023年10月
  • Pten欠損前立腺癌進展における骨髄由来抑制細胞のプロファイリング(Profiling myeloid-derived suppressor cells during mouse prostate cancer progression)
    野澤 昌弘; デベラスコ・マルコ; 倉 由吏恵; 坂井 和子; 安富 正悟; 西本 光寿; 南 高文; 森 康範; 藤田 和利; 吉村 一宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 82回 803 - 803 2023年09月
  • 前立腺癌と大腸癌そして潰瘍性大腸炎の関連性の探索(Systemic inflammation as a link between prostate cancer, colorectal cancer, and ulcerative colitis)
    倉 由吏恵; デベラスコ・マルコ; 坂井 和子; 藤田 和利; 安富 正悟; 森 康範; 南 高文; 野澤 昌弘; 吉村 一宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 82回 1416 - 1416 2023年09月
  • Kazuto Nishio; Kazuko Sakai; Makoto Nishio; Takashi Seto; Carla Visseren-Grul; Michelle Carlsen; Tomoko Matsui; Sotaro Enatsu; Kazuhiko Nakagawa
    Translational lung cancer research 12 8 1702 - 1716 2023年08月 
    BACKGROUND: An exploratory, proof-of-concept, liquid biopsy addendum to examine biomarkers within cell-free DNA (cfDNA) in the RELAY phase 3, randomized, double-blind, placebo-controlled study was conducted. RELAY showed improved progression-free survival (PFS) with ramucirumab (RAM), a human immunoglobulin G1 vascular endothelial growth factor receptor 2 antagonist, plus erlotinib (ERL), a tyrosine kinase inhibitor, compared with placebo (PL) plus ERL. METHODS: Treatment-naïve patients with endothelial growth factor receptor (EGFR)-mutated metastatic non-small cell lung cancer were randomized (1:1) to RAM + ERL or PL + ERL. Plasma samples were collected at baseline, on treatment, and at 30-day post-study treatment discontinuation follow-up. Baseline and treatment-emergent gene alterations and EGFR-activating mutation allele counts were investigated by next-generation sequencing (NGS) and droplet digital polymerase chain reaction (ddPCR), respectively. cfDNA concentration and fragment size were evaluated by real-time polymerase chain reaction and the BioAnalyzer. Patients with a valid baseline plasma sample were included (70 RAM + ERL, 61 PL + ERL). RESULTS: TP53 mutation was the most frequently co-occurring baseline gene alteration (43%). Post-study treatment discontinuation EGFR T790M mutation rates were 54.5% (6/11) and 41.2% (7/17) by ddPCR, and 22.2% (2/9) and 29.4% (5/17) by NGS, in the RAM + ERL and PL + ERL arms, respectively. EGFR-activating mutation allele count decreased at Cycle 4 in both treatment arms and was sustained at follow-up with RAM + ERL. PFS improved for patients with no detectable EGFR-activating mutation at Cycle 4 vs. those with detectable EGFR-activating mutation. Total cfDNA concentration increased from baseline at Cycle 4 and through to follow-up with RAM + ERL. cfDNA fragment size was similar between treatment arms at baseline [mean (standard deviation) base pairs: RAM + ERL, 173.4 (2.6); PL + ERL, 172.9 (3.2)] and was shorter at Cycle 4 with RAM + ERL vs. PL + ERL [169.5 (2.8) vs. 174.1 (3.3), respectively; P<0.0001]. Baseline vs. Cycle 4 paired analysis showed a decrease in cfDNA fragment size for 84% (48/57) and 23% (11/47) of patient samples in the RAM + ERL and PL + ERL arms, respectively. CONCLUSIONS: EGFR-activating mutation allele count was suppressed, total cfDNA concentration increased, and short fragment-sized cfDNA increased with RAM + ERL, suggesting the additional anti-tumor effect of RAM may contribute to the PFS benefit observed in RELAY with RAM + ERL vs. PL + ERL. TRIAL REGISTRATION: ClinicalTrials.gov; identifier: NCT02411448.
  • Naoya Kemmotsu; Kiichiro Ninomiya; Kei Kunimasa; Takamasa Ishino; Joji Nagasaki; Yoshihiro Otani; Hiroyuki Michiue; Eiki Ichihara; Kadoaki Ohashi; Takako Inoue; Motohiro Tamiya; Kazuko Sakai; Youki Ueda; Hiromichi Dansako; Kazuto Nishio; Katsuyuki Kiura; Isao Date; Yosuke Togashi
    International journal of cancer 2023年08月 [査読有り]
     
    Intracranial metastases are common in nonsmall-cell lung cancer (NSCLC) patients, whose prognosis is very poor. In addition, intracranial progression is common during systemic treatments due to the inability to penetrate central nervous system (CNS) barriers, whereas the intracranial effects of cancer immunotherapies remain unclear. We analyzed clinical data to evaluate the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies compared with those treated without PD-1 blockade therapies, and found that the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies was significantly lower than that in patients treated with cytotoxic chemotherapies. In murine models, intracranial rechallenged tumors after initial rejection by PD-1 blockade were suppressed. Accordingly, long-lived memory precursor effector T cells and antigen-specific T cells were increased by PD-1 blockade in intracranial lesions. However, intracranial rechallenged different tumors are not suppressed. Our results indicate that cancer immunotherapies can prevent intracranial progression, maintaining long-term effects intracranially as well as systemically. If intracranial recurrence occurs during the treatment with PD-1 blockade therapies, aggressive local therapies could be worthwhile.
  • Ken Kamata; Mamoru Takenaka; Naoshi Nishida; Akane Hara; Yasuo Otsuka; Hidekazu Tanaka; Shunsuke Omoto; Kosuke Minaga; Kentaro Yamao; Yasutaka Chiba; Kazuko Sakai; Kazuto Nishio; Tomohiro Watanabe; Masatoshi Kudo
    International journal of clinical oncology 28 11 1511 - 1519 2023年08月 
    BACKGROUND: This prospective cohort study evaluated the feasibility of using endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) samples for comprehensive mutational analysis of cancer-related genes using microtissues. METHODS: Fifty patients with suspected pancreatic cancer presenting consecutively at the Kindai University Hospital between January 2018 and January 2019 were enrolled. Cancerous tissues from EUS-FNB were obtained from each tumor and subjected to histological examination and mutational analysis. The primary endpoint was the collection rate of EUS-FNB specimens suitable for comprehensive cancer panels using deep sequencing. Clinical history and genetic variations between the disease control and progressive disease groups of patients on chemotherapy were evaluated as secondary endpoints. RESULTS: The collection rate of EUS-FNB specimens suitable for comprehensive cancer panels using deep sequencing was 93.6%. The cancer panel was sequenced for 25 patients with pancreatic cancer treated initially with systemic chemotherapy. Mutation in p53 and Smad4 were positively and negatively associated, respectively, with disease control at the initial evaluation. The median time to progression in 15 patients with p53 and without Smad4 mutations was 182.0 days; whereas, it was 92.5 days in other 10 patients; this difference was significant (p = 0.020). CONCLUSIONS: Tissue samples from EUS-FNB were suitable for mutational analysis. Pancreatic cancers with p53 and without Smad4 mutations responded better to chemotherapy and had a better prognosis than those others.
  • Koji Haratani; Atsushi Nakamura; Nobuaki Mamesaya; Shigeki Mitsuoka; Yasuto Yoneshima; Ryota Saito; Junko Tanizaki; Yasuhito Fujisaka; Akito Hata; Kosuke Tsuruno; Tomohiro Sakamoto; Shunsuke Teraoka; Masahide Oki; Hiroshi Watanabe; Yuki Sato; Yusuke Nakano; Tomoyuki Otani; Kazuko Sakai; Shuta Tomida; Yasutaka Chiba; Akihiko Ito; Kazuto Nishio; Nobuyuki Yamamoto; Kazuhiko Nakagawa; Hidetoshi Hayashi
    Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 18 10 1334 - 1350 2023年06月 
    INTRODUCTION: The PACIFIC regimen of consolidation therapy with the programmed cell death-ligand 1 inhibitor durvalumab after definitive concurrent chemoradiation therapy has become a standard of care for individuals with unresectable stage III NSCLC. Nevertheless, approximately half of the treated patients experience disease progression within 1 year, with the mechanisms of treatment resistance being poorly understood. We here performed a nationwide prospective biomarker study to explore the resistance mechanisms (WJOG11518L:SUBMARINE). METHODS: A total of 135 patients with unresectable stage III NSCLC who received the PACIFIC regimen were included for comprehensive profiling of the tumor microenvironment by immunohistochemistry, transcriptome analysis, and genomic sequencing of pretreatment tumor tissue and flow cytometric analysis of circulating immune cells. Progression-free survival was compared on the basis of these biomarkers. RESULTS: The importance of preexisting effective adaptive immunity in tumors was revealed for treatment benefit regardless of genomic features. We also identified CD73 expression by cancer cells as a mechanism of resistance to the PACIFIC regimen. Multivariable analysis of immunohistochemistry data with key clinical factors as covariables indicated that low CD8+ tumor-infiltrating lymphocyte density and the high CD73+ cancer cells were independently associated with poor durvalumab outcome (hazard ratios = 4.05 [95% confidence interval: 1.17-14.04] for CD8+ tumor-infiltrating lymphocytes; 4.79 [95% confidence interval: 1.12-20.58] for CD73). In addition, whole-exome sequencing of paired tumor samples suggested that cancer cells eventually escaped immune pressure as a result of neoantigen plasticity. CONCLUSIONS: Our study emphasizes the importance of functional adaptive immunity in stage III NSCLC and implicates CD73 as a promising treatment target, thus providing insight forming a basis for development of a new treatment approach in NSCLC.
  • Kimio Yonesaka; Hidetoshi Hayashi; Atsushi Nakamura; Yuki Sato; Koichi Azuma; Shinya Sakata; Motoko Tachihara; Satoshi Ikeda; Toshihide Yokoyama; Kentaro Ito; Yukihiro Yano; Hirotaka Matsumoto; Haruko Daga; Akito Hata; Kazuko Sakai; Yasutaka Chiba; Kazuto Nishio; Nobuyuki Yamamoto; Kazuhiko Nakagawa
    Clinical lung cancer 2023年06月 
    BACKGROUND: Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has limited treatment options for patients with EGFR-mutated non-small-cell lung cancer (NSCLC). Although osimertinib or afatinib alone induced drug-resistant clones with EGFR secondary mutation in a preclinical model, its combination prevented the appearance of these mutations. We investigated alternating-dose therapy of osimertinib and afatinib in patients with EGFR-mutant NSCLC in a single-arm Phase II trial. METHODS: Treatment-naïve patients with stage IV NSCLC harboring an activating EGFR mutation were enrolled. Alternating cycles of osimertinib (80 mg/day) followed by afatinib (20 mg/day) were administered every 8 weeks. Genomic analysis was performed using circulating tumor DNA obtained before and after the treatment. RESULTS: Among the 46 enrolled patients, the median progression-free survival was 20.2 months. The overall response rate was 69.6%. The median overall survival was not reached. Among the 26 plasma samples obtained after the acquisition of resistance, 3 showed an increased MET gene copy number, and 1 showed BRAF mutation. Meanwhile, no EGFR secondary mutation was detected. CONCLUSION: The efficacy of our treatment was not significantly different from osimertinib alone, as reported previously in untreated advanced NSCLC patients with EGFR mutations. Although the sample size was limited, this treatment may prevent the emergence of EGFR secondary mutations that trigger drug resistance. Further studies are warranted to establish the significance of this treatment. CLINICAL TRIAL REGISTRATION: jRCTs051180009.
  • Toshiaki Takakura; Hiroaki Kanemura; Kazuko Sakai; Kazuto Nishio; Kazuhiko Nakagawa; Hidetoshi Hayashi
    JTO clinical and research reports 4 6 100523 - 100523 2023年06月 
    Resistance to ROS1 tyrosine kinase inhibitors is inevitable, but it has been unclear whether crizotinib might be effective after the development of entrectinib resistance. We here present a case of ROS1-rearranged NSCLC that responded to crizotinib after tumor progression due to MET polysomy during entrectinib treatment. This case suggests that crizotinib is an effective option for patients with MET polysomy, even after disease progression on entrectinib.
  • Kenichi Suda; Kazuko Sakai; Tatsuo Ohira; Takaaki Chikugo; Takao Satou; Jun Matsubayashi; Toshitaka Nagao; Norihiko Ikeda; Yasuhiro Tsutani; Tetsuya Mitsudomi; Kazuto Nishio
    Cancers 15 9 2023年05月 
    BACKGROUND: The Idylla™ EGFR Mutation Test is an ultra-rapid single-gene test that detects epidermal growth factor receptor (EGFR) mutations using formalin-fixed paraffin-embedded specimens. Here, we compared the performance of the Idylla EGFR Mutation Test with the Cobas® EGFR Mutation Test v2. METHODS: Surgically resected NSCLC specimens obtained at two Japanese institutions (N = 170) were examined. The Idylla EGFR Mutation Test and the Cobas EGFR Mutation Test v2 were performed independently and the results were compared. For discordant cases, the Ion AmpliSeq Colon and Lung Cancer Research Panel V2 was performed. RESULTS: After the exclusion of five inadequate/invalid samples, 165 cases were evaluated. EGFR mutation analysis revealed 52 were positive and 107 were negative for EGFR mutation in both assays (overall concordance rate: 96.4%). Analyses of the six discordant cases revealed that the Idylla EGFR Mutation Test was correct in four and the Cobas EGFR Mutation Test v2 was correct in two. In a trial calculation, the combination of the Idylla EGFR Mutation Test followed by a multi-gene panel test will reduce molecular screening expenses if applied to a cohort with EGFR mutation frequency >17.9%. CONCLUSIONS: We demonstrated the accuracy and potential clinical utility of the Idylla EGFR Mutation Test as a molecular screening platform in terms of turnaround time and molecular testing cost if applied to a cohort with a high EGFR mutation incidence (>17.9%).
  • Hanako Sato; Kosuke Murakami; Risa Fujishima; Tomoyuki Otani; Kazuko Sakai; Kazuto Nishio; Noriomi Matsumura
    BMC Women's Health 23 1 2023年04月 
    Abstract Background Uterine angioleiomyoma is benign tumor that composed of smooth muscle cells and thick-walled vessels. It is a very rare condition reported to present as lower abdominal mass, accompanied by dysmenorrhea and hypermenorrhea. However, its clinical presentation is not known. Case presentation We report the case of a 44-year-old Japanese woman who developed severe anemia with disseminated intravascular coagulation without obvious external bleeding. The patient had a huge abdominal mass of over 20 cm in size, which was thought to be a uterine tumor. She received daily blood transfusions and her condition improved rapidly after she underwent hysterectomy. Pathological examination of the tumor revealed spindle-shaped cells with little atypia and mitosis, and numerous large vessels with smooth muscle and thrombus in the vessels. Conclusions Uterine angioleiomyoma was identified as the cause of the coagulation abnormality. CCND2 and AR gene amplification was detected in the tumor. Uterine tumors that present with coagulopathy despite a clinical course suggestive of benign disease should undergo differential diagnosis for uterine angioleiomyoma.
  • Pten欠損前立腺癌マウスモデルにおいてクルクミンモノグルクロニドは腫瘍免疫微小環境を改善する(Curcumin monoglucuronide reprograms the tumor micro-immune environment in mouse Pten-null prostate cancer)
    倉 由吏恵; デベラスコ・マルコ; 坂井 和子; 橋本 士; 西本 光寿; 安富 正悟; 森 康範; 南 高文; 野澤 昌弘; 藤田 和利; 吉村 一宏; 西尾 和人; 植村 天受
    日本泌尿器科学会総会 110回 PP62 - 04 2023年04月
  • Takeshi Fujita; Kazuko Sakai; Natsumi Uehara; Yujiro Hoshi; Anjin Mori; Hajime Koyama; Mitsuo Sato; Kazuya Saito; Yasuhiro Osaki; Kazuto Nishio; Katsumi Doi
    Oncology letters 25 3 121 - 121 2023年03月 
    Vestibular schwannoma (VS) is the most common tumor of the cerebellopontine angle. Despite the increasing diagnosis of sporadic VS over the past decade, the use of traditional microsurgeries to treat VS has decreased. This is likely a result of the adoption of serial imaging as the most common initial evaluation and treatment strategy, especially for small-sized VS. However, the pathobiology of VSs remains unclear, and elucidating the genetic information of tumor tissue may reveal novel insights. The present study performed a comprehensive genomic analysis of all exons in the key tumor suppressor and oncogenes from 10 small (<15 mm) sporadic VS samples. The evaluations identified NF2, SYNE1, IRS2, APC, CIC, SDHC, BRAF, NUMA1, EXT2, HRAS, BCL11B, MAGI1, RNF123, NLRP1, ASXL1, ADAMTS20, TAF1L, XPC, DDB2 and ETS1 as mutated genes. The current study could not draw any new conclusions about the relationship between VS-related hearing loss and gene mutations; however, it did reveal that NF2 was the most frequently mutated gene in small sporadic VS.
  • Masanobu Tsubaki; Tomoya Takeda; Yuuichi Koumoto; Takehiro Usami; Takuya Matsuda; Shiori Seki; Kazuko Sakai; Kazuto Nishio; Shozo Nishida
    Cell proliferation 56 6 e13420  2023年02月 
    The development of BCR::ABL1 tyrosine kinase inhibitors (TKIs), such as dasatinib, has dramatically improved survival in cases of chronic myeloid leukaemia (CML). However, the development of resistance to BCR::ABL1 TKIs is a clinical problem. BCR::ABL1 TKI resistance is known to have BCR::ABL1-dependent or BCR::ABL1-independent mechanisms, but the mechanism of BCR::ABL1 independence is not well understood. In the present study, we investigated the mechanism of BCR::ABL1-independent dasatinib resistance. The expression and activation level of genes or proteins were evaluated using array CGH, real time PCR, or western blot analysis. Gene expression was modulated using siRNA-mediated knockdown. Cell survival was assessed by using trypan blue dye method. We found that dasatinib-resistant K562/DR and KU812/DR cells did not harbour a BCR::ABL1 mutation but had elevated expression and/or activation of MOS, TPL2 and ERK1/2. In addition, MOS siRNA, TPL2 siRNA and trametinib resensitized dasatinib-resistant cells to dasatinib. Moreover, expression levels of MOS in dasatinib non-responder patients with CML were higher than those in dasatinib responders, and the expression of TPL2 tended to increase in dasatinib non-responder patients compared with that in responder patients. Our results indicate that activation of ERK1/2 by elevated MOS and TPL2 expression is involved in dasatinib resistance, and inhibition of these proteins overcomes dasatinib resistance. Therefore, MOS, TPL2 and ERK1/2 inhibitors may be therapeutically useful for treating BCR::ABL1-independent dasatinib-resistant CML.
  • Satoshi Watanabe; Kazuko Sakai; Naoya Matsumoto; Jun Koshio; Akira Ishida; Tetsuya Abe; Daisuke Ishikawa; Tomohiro Tanaka; Ami Aoki; Tomosue Kajiwara; Kenichi Koyama; Satoru Miura; Yuka Goto; Tomoki Sekiya; Ryo Suzuki; Kohei Kushiro; Toshiya Fujisaki; Naohiro Yanagimura; Aya Ohtsubo; Satoshi Shoji; Koichiro Nozaki; Yu Saida; Hirohisa Yoshizawa; Kazuto Nishio; Toshiaki Kikuchi
    Cancers 15 1 2022年12月 
    Anaplastic lymphoma kinase (ALK)-positive lung cancer is a rare cancer that occurs in approximately 5% of non-small-cell lung cancer (NSCLCs) patients. Despite the excellent efficacy of ALK-tyrosine kinase inhibitor in ALK-positive NSCLCs, most patients experience resistance. We conducted a phase II study to investigate the combination of alectinib with bevacizumab in ALK-positive NSCLC patients after failure of alectinib. In this study, ALK-positive nonsquamous NSCLC patients previously treated with alectinib received bevacizumab 15 mg/kg on day 1 every 3 weeks and alectinib 600 mg/day until disease progression. The primary endpoints were progression-free survival (PFS) and the safety of alectinib and bevacizumab. The secondary endpoints included overall survival (OS) and correlation of circulating tumor DNA and plasma proteins with PFS. Of the 12 patients treated, the median PFS was 3.1 months (95% CI 1.2-16.1), and the median OS was 24.1 months (95% CI 8.3-not estimable). The EML4-ALK fusion gene in circulating tumor DNA was significantly correlated with shorter PFS (1.2 months vs. 11.4 months, HR 5.2, p = 0.0153). Two patients experienced grade 3 adverse events; however, none of the patients required dose reduction. Although the primary endpoint was not met, alectinib combined with bevacizumab showed clinical efficacy in ALK-positive patients.
  • Kentaro Tanaka; Kenji Chamoto; Sho Saeki; Ryusuke Hatae; Yuki Ikematsu; Kazuko Sakai; Nobuhisa Ando; Kazuhiro Sonomura; Shinsuke Kojima; Masanori Taketsuna; Young Hak Kim; Hironori Yoshida; Hiroaki Ozasa; Yuichi Sakamori; Tomoko Hirano; Fumihiko Matsuda; Toyohiro Hirai; Kazuto Nishio; Takuro Sakagami; Masanori Fukushima; Yoichi Nakanishi; Tasuku Honjo; Isamu Okamoto
    Science translational medicine 14 675 eabq0021  2022年12月 
    Despite the success of cancer immunotherapies such as programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) inhibitors, patients often develop resistance. New combination therapies with PD-1/PD-L1 inhibitors are needed to overcome this issue. Bezafibrate, a ligand of peroxisome proliferator-activated receptor-γ coactivator 1α/peroxisome proliferator-activated receptor complexes, has shown a synergistic antitumor effect with PD-1 blockade in mice that is mediated by activation of mitochondria in T cells. We have therefore now performed a phase 1 trial (UMIN000017854) of bezafibrate with nivolumab in previously treated patients with advanced non-small cell lung cancer. The primary end point was the percentage of patients who experience dose-limiting toxicity, and this combination regimen was found to be well tolerated. Preplanned comprehensive analysis of plasma metabolites and gene expression in peripheral cytotoxic T cells indicated that bezafibrate promoted T cell function through up-regulation of mitochondrial metabolism including fatty acid oxidation and may thereby have prolonged the duration of response. This combination strategy targeting T cell metabolism thus has the potential to maintain antitumor activity of immune checkpoint inhibitors and warrants further validation.
  • W. Okamoto; K. Sakai; A. Makiyama; Y. Yamamoto; K. Shitara; T. Denda; N. Izawa; Y. Nakano; T. Nishina; T. Esaki; H. Hara; Y. Miura; N. Boku; K. Yamazaki; S. Hironaka; T. Misumi; I. Hyodo; K. Muro; K. Nishio
    ESMO Open 7 6 100592 - 100592 2022年12月
  • Yujiro Hayashi; Kazutoshi Fujita; Kazuko Sakai; Shogo Adomi; Eri Banno; Satoshi Nojima; Eisuke Tomiyama; Makoto Matsushita; Taigo Kato; Koji Hatano; Atsunari Kawashima; Takafumi Minami; Eiichi Morii; Hirotsugu Uemura; Kazuto Nishio; Norio Nonomura
    Scientific reports 12 1 16642 - 16642 2022年10月 
    During tumorigenesis, certain tissues are colonized by mutant clones with oncogenic driver mutations as precancer lesions. These mutations can facilitate clonal expansion and may contribute to malignant transformation. The molecular features of low-grade non-muscle invasive bladder cancer (NMIBC) and high-grade bladder cancer are so distinct that they are thought to follow different evolutionary tumorigenesis pathways. Although NMIBC accounts for most bladder tumors, the somatic mutation patterns in "precancer" urothelium of patients with NMIBC remain unclear. Here, we analyzed specimens of normal urothelium and bladder tumors from patients with low-grade and high-grade NMIBC and investigated the genomic evolution of the cancer. Somatic mutations were analyzed using 50 oncogene-targeted sequences and droplet digital polymerase chain reaction for TERT promoter mutations. Somatic mutations in TERT promoter, FGFR3, and CDKN2A were characteristically identified in the normal urothelium of patients with NMIBC. These mutations, consistently identified in both tumor and normal specimens, likely affect clonal expansion during the malignant transformation of NMIBC. Though larger samples and comprehensive study are warranted to confirm our results, the difference in mutational landscape of the precancerous urothelium of patients with bladder cancer could offer deeper understandings of genomic evolution in bladder tumorigenesis.
  • Naoyuki Iwahashi; Midori Ikezaki; Yoshihiro Komohara; Yukio Fujiwara; Tomoko Noguchi; Kaho Nishioka; Kazuko Sakai; Kazuto Nishio; Mitsuharu Ueda; Yoshito Ihara; Kenji Uchimura; Kazuhiko Ino; Kazuchika Nishitsuji
    PNAS Nexus 1 3 2022年08月 [査読有り]
     
    Abstract Recent studies suggested that aggregates of mutant p53 proteins may propagate and impair normal p53 functioning in recipient cells. Our previous study showed that cancer cell-derived p53 aggregates that cells internalized interfered with p53-dependent apoptosis in recipient cells. However, involvement of p53 aggregate propagation in cancer pathology has not been fully elucidated. Here, we screened patients with high-grade serous ovarian carcinoma, which is characterized by an extremely high frequency of TP53 gene mutations, to show that patients with cytoplasmic p53 deposits have a poor prognosis compared with patients with complete p53 absence or strong nuclear p53 positivity. Cytoplasmic p53 in the patients with poor prognosis consisted of protein aggregates, which suggests that p53 aggregates are oncogenic drivers. Indeed, an inhibitor of p53 aggregation restored cellular apoptosis, a proper p53 function, in p53 aggregate-bearing patient-derived tumor organoids. In cell-based assays, endogenous and exogenous mutant p53 aggregates hindered chemotherapeutic activity of cisplatin, which depends on normal p53 functions. This inhibition was reduced by blocking p53 aggregation or internalization of p53 aggregates. Our study, thus indicates the involvement of p53 aggregate transmission in poor prognosis and in chemotherapy resistance in cancers.
  • Hiroaki Kanemura; Hidetoshi Hayashi; Shuta Tomida; Junko Tanizaki; Shinichiro Suzuki; Yusuke Kawanaka; Asuka Tsuya; Yasushi Fukuda; Hiroyasu Kaneda; Keita Kudo; Takayuki Takahama; Ryosuke Imai; Koji Haratani; Yasutaka Chiba; Tomoyuki Otani; Akihiko Ito; Kazuko Sakai; Kazuto Nishio; Kazuhiko Nakagawa
    JTO clinical and research reports 3 8 100373 - 100373 2022年08月 
    Introduction: Despite a considerable benefit of adding immune checkpoint inhibitors (ICIs) to platinum-based chemotherapy for patients with extensive-stage SCLC (ES-SCLC), a durable response to ICIs occurs in only a small minority of such patients. Methods: A total of 135 patients with ES-SCLC treated with chemotherapy either alone (chemo-cohort, n = 71) or together with an ICI (ICI combo-cohort, n = 64) was included in this retrospective study. Tumors were classified pathologically as inflamed or noninflamed on the basis of programmed death-ligand 1 expression and CD8+ tumor-infiltrating lymphocyte density. Immune-related gene expression profiling was performed, and predicted neoantigen load was determined by whole-exome sequencing. Results: Among patients in the ICI combo-cohort, median progression-free survival was 10.8 and 5.1 months for those with inflamed (n = 7) or noninflamed (n = 56) tumors, respectively (log-rank test p = 0.002; hazard ratio of 0.26). Among the 89 patients with immune-related gene expression profiling data available, inflamed tumors had a higher T cell-inflamed GEP score than did noninflamed tumors (-0.18 versus -0.58, p < 0.001). The 12-month progression-free survival rate was 16.1% and 0% for patients in the ICI combo-cohort harboring tumors with a high (n = 26) or low (n = 18) frameshift neoantigen load, respectively. A high-frameshift neoantigen load was associated with up-regulation of gene signatures related to antigen presentation and costimulatory signaling. A durable clinical benefit of ICI therapy was observed only in patients with inflamed tumors and a high-frameshift neoantigen load. Conclusions: Expression of programmed death-ligand 1, CD8+ T cell infiltration, and a high-frameshift neoantigen load are associated with clinical benefit of ICI therapy in ES-SCLC. Clinical trial registration: UMIN000041056.
  • Yusuke Makutani; Kazuko Sakai; Masahiro Yamada; Toshiaki Wada; Takaaki Chikugo; Takao Satou; Yoko Iwasa; Hidekazu Yamamoto; Marco A de Velasco; Kazuto Nishio; Junichiro Kawamura
    International journal of clinical oncology 27 7 1180 - 1187 2022年04月 
    BACKGROUND: The Biocartis Idylla™ platform is a fully automated, real-time PCR-based diagnostic system. The Idylla™ KRAS and NRAS-BRAF Mutation Tests have been developed for the qualitative detection of mutations in KRAS, NRAS and BRAF genes, facilitating the genomic profiling of patients with colorectal cancer. The aim of the present study was to evaluate clinical performances of these tests in Japan. METHODS: The RAS and BRAF mutation statuses of 253 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues were analyzed using the Investigational Use Only Idylla™ KRAS Mutation Test and the Idylla™ NRAS-BRAF Mutation Test and an in vitro diagnostics (IVD) kit (MEBGEN RASKET™-B kit). RESULTS: The success rate for obtaining a valid mutational data without retest of the Idylla tests was 97.6% (247/253): 111 KRAS mutations (43.8%), 9 NRAS mutations (3.6%), and 36 BRAF V600E mutations (14.2%) were detected using the Idylla tests. Compared with the MEBGEN RASKET-B results, the positive concordance rate was 97.4%, the negative concordance rate was 95.7%, and the overall concordance rate was 95.3% (κ = 0.919, 95% CI 0.871-0.967). The average turnaround time to Idylla™ KRAS and NRAS-BRAF Mutation Test was 5.6 working days (range: 3-11 days). CONCLUSION: This result demonstrates a high concordance between the Idylla™ KRAS and NRAS-BRAF Mutation Tests and an existing IVD kit. In this manner, the Idylla™ mutation tests were validated for the detection of clinically significant KRAS, NRAS, and BRAF mutations in FFPE samples from colorectal cancer patients.
  • Hidetoshi Hayashi; Kimio Yonesaka; Atsushi Nakamura; Daichi Fujimoto; Koichi Azuma; Shinya Sakata; Motoko Tachihara; Satoshi Ikeda; Toshihide Yokoyama; Osamu Hataji; Yukihiro Yano; Katsuya Hirano; Haruko Daga; Hideaki Okada; Yasutaka Chiba; Kazuko Sakai; Kazuto Nishio; Nobuyuki Yamamoto; Kazuhiko Nakagawa
    Lung cancer (Amsterdam, Netherlands) 168 38 - 45 2022年04月 
    INTRODUCTION: Alternation of osimertinib and afatinib is a potential approach to overcome osimertinib resistance and to allow complementation of drug efficacy without compromising safety in patients with epidermal growth factor receptor gene (EGFR)-mutated non-small cell lung cancer (NSCLC). METHODS: Treatment-naive patients with stage IV NSCLC harboring an activating EGFR mutation (L858R or exon-19 deletion) were enrolled. Alternating cycles of osimertinib at 80 mg/day for 8 weeks followed by afatinib at 20 mg/day for 8 weeks were administered. The primary end point was 12-month progression-free survival (PFS) probability. RESULTS: Forty-six patients were enrolled and treated with study therapy. The 12-month PFS probability was 70.2% (60% confidence interval [CI], 63.9-75.6%; 95% CI, 54.2-81.5%), which did not meet the primary end point. After a median follow-up time of 25.7 months, the median PFS was 21.3 months (95% CI, 16.3 months-not reached). The overall response rate was 69.6% (95% CI, 54.2-82.3%). The most common treatment-related adverse events (any grade or grade ≥ 3, respectively) were diarrhea (73.9%, 4.3%), rash acneiform (63.0%, 2.2%), and paronychia (52.2%, 0%). Five cases of pneumonitis, two of grade 2 and thres of grade 3, were apparent, all of which developed during osimertinib treatment. Exploratory evaluation of circulating tumor DNA suggested that coexisting TP53 mutations did not influence PFS for the alternating therapy. CONCLUSIONS: Alternating therapy with osimertinib and afatinib for treatment-naive patients with EGFR- mutated advanced NSCLC did not meet its primary end point, despite the encouraging efficacy and safety profile of this treatment strategy.
  • Hidetoshi Hayashi; Shunichi Sugawara; Yasushi Fukuda; Daichi Fujimoto; Satoru Miura; Keiichi Ota; Yuichi Ozawa; Satoshi Hara; Junko Tanizaki; Koichi Azuma; Shota Omori; Motoko Tachihara; Kazumi Nishino; Akihiro Bessho; Yasutaka Chiba; Koji Haratani; Kazuko Sakai; Kazuto Nishio; Nobuyuki Yamamoto; Kazuhiko Nakagawa
    Clinical cancer research : an official journal of the American Association for Cancer Research 28 5 893 - 902 2022年03月 
    PURPOSE: Although the efficacy of programmed cell death-1 (PD-1) blockade is generally poor for non-small cell lung cancer (NSCLC) with activating mutations of the epidermal growth factor receptor (EGFR) gene, EGFR tyrosine kinase inhibitors (TKIs) may improve the tumor immune microenvironment. We performed a randomized study to assess whether nivolumab improves outcome compared with chemotherapy in such patients previously treated with EGFR-TKIs. PATIENTS AND METHODS: Patients with EGFR-mutated NSCLC who acquired EGFR-TKI resistance not due to a secondary T790M mutation of EGFR were randomized 1:1 to nivolumab (n = 52) or carboplatin-pemetrexed (n = 50). The primary endpoint was progression-free survival (PFS). RESULTS: Median PFS and 1-year PFS probability were 1.7 months and 9.6% for nivolumab versus 5.6 months and 14.0% for carboplatin-pemetrexed [log-rank P < 001; hazard ratio (HR) of 1.92, with a 60% confidence interval (CI) of 1.61-2.29]. Overall survival was 20.7 and 19.9 months [HR, 0.88 (95% CI, 0.53-1.47)], and response rate was 9.6% and 36.0% for nivolumab and carboplatin-pemetrexed, respectively. No subgroup including patients with a high tumor mutation burden showed a substantially longer PFS with nivolumab than with carboplatin-pemetrexed. The T-cell-inflamed gene expression profile score (0.11 vs. -0.17, P = 0.036) and expression of genes related to cytotoxic T lymphocytes or their recruitment were higher in tumors that showed a benefit from nivolumab. CONCLUSIONS: Nivolumab did not confer a longer PFS compared with carboplatin-pemetrexed in the study patients. Gene expression profiling identified some cases with a favorable tumor immune microenvironment that was associated with nivolumab efficacy.
  • Golam Iftakhar Khandakar; Ryosuke Satoh; Teruaki Takasaki; Kana Fujitani; Genzoh Tanabe; Kazuko Sakai; Kazuto Nishio; Reiko Sugiura
    Cells 11 4 2022年02月 
    The mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol-3 kinase (PI3K)/AKT pathways are dysregulated in various human cancers, including pancreatic ductal adenocarcinoma (PDAC), which has a very poor prognosis due to its lack of efficient therapies. We have previously identified ACAGT-007a (GT-7), an anti-cancer compound that kills ERK-active melanoma cells by inducing ERK-dependent apoptosis. Here, we investigated the apoptosis-inducing effect of GT-7 on three PDAC cell lines and its relevance with the MAPK/ERK and PI3K/AKT signaling pathways. GT-7 induced apoptosis in PDAC cells with different KRAS mutations (MIA-Pa-Ca-2 (KRAS G12C), T3M4 (KRAS Q61H), and PANC-1 (KRAS G12D)), being T3M4 most susceptible, followed by MIA-Pa-Ca-2, and PANC-1 was most resistant to apoptosis induction by GT-7. GT-7 stimulated ERK phosphorylation in the three PDAC cells, but only T3M4 displayed ERK-activation-dependent apoptosis. Furthermore, GT-7 induced a marked down-regulation of AKT phosphorylation after a transient peak in T3M4, whereas PANC-1 displayed the strongest and most sustained AKT activation, followed by MIA-Pa-Ca-2, suggesting that sustained AKT phosphorylation as a determinant for the resistance to GT-7-mediated apoptosis. Consistently, a PI3K inhibitor, Wortmannin, abolished AKT phosphorylation and enhanced GT-7-mediated apoptosis in T3M4 and MIA-Pa-Ca-2, but not in PANC-1, which showed residual AKT phosphorylation. This is the first report that ERK stimulation alone or in combination with AKT signaling inhibition can effectively induce apoptosis in PDAC and provides a rationale for a novel concurrent targeting of the PI3K/AKT and ERK pathways.
  • Masaki Kaibori; Kazuko Sakai; Hideyuki Matsushima; Hisashi Kosaka; Kosuke Matsui; Marco A De Velasco; Mitsugu Sekimoto; Kazuto Nishio
    Hepatology international 16 1 135 - 147 2022年02月 
    BACKGROUND/PURPOSE OF THE STUDY: Tumor heterogeneity based on copy number variations is associated with the evolution of cancer and its clinical grade. Clonal composition (CC) represents the number of clones based on the distribution of B-allele frequency (BAF) obtained from a genome-wide single nucleotide polymorphism (SNP) array. A higher CC number represents a high degree of heterogeneity. We hypothesized and evaluated that the CC number in hepatocellular carcinoma (HCC) tissues might be associated with the clinical outcomes of patients. METHODS: Somatic mutation, whole transcriptome, and CC number based on copy number variations of 36 frozen tissue samples of operably resected HCC tissues were analyzed by targeted deep sequencing, transcriptome analysis, and SNP array. RESULTS: The samples were classified into the heterogeneous tumors as poly-CC (n = 26) and the homogeneous tumors as mono-CC (n = 8). The patients with poly-CC had a higher rate of early recurrence and a significantly shorter recurrence-free survival period than the mono-CC patients (7.0 months vs. not reached, p = 0.0084). No differences in pathogenic non-synonymous mutations, such as TP53, were observed between the two groups when targeted deep sequencing was applied. A transcriptome analysis showed that cell cycle-related pathways were enriched in the poly-CC tumors, compared to the mono-CC tumors. Poly-CC HCC is highly proliferative and has a high risk of early recurrence. CONCLUSION: CC is a possible candidate biomarker for predicting the risk of early postoperative recurrence and warrants further investigation.
  • J Tanizaki; K Yonemori; K Akiyoshi; H Minami; H Ueda; Y Takiguchi; Y Miura; Y Segawa; S Takahashi; Y Iwamoto; Y Kidera; K Fukuoka; A Ito; Y Chiba; K Sakai; K Nishio; K Nakagawa; H Hayashi
    Annals of oncology : official journal of the European Society for Medical Oncology 33 2 216 - 226 2022年02月 
    BACKGROUND: Cancer of unknown primary (CUP) has a poor prognosis. Given the recent approval of immune checkpoint inhibitors for several cancer types, we carried out a multicenter phase II study to assess the efficacy of nivolumab for patients with CUP. PATIENTS AND METHODS: Patients with CUP who were previously treated with at least one line of systemic chemotherapy constituted the principal study population. Previously untreated patients with CUP were also enrolled for exploratory analysis. Nivolumab (240 mg/body) was administered every 2 weeks for up to 52 cycles. The primary endpoint was objective response rate in previously treated patients as determined by blinded independent central review according to RECIST version 1.1. RESULTS: Fifty-six patients with CUP were enrolled in the trial. For the 45 previously treated patients, objective response rate was 22.2% [95% confidence interval (CI), 11.2% to 37.1%], with a median progression-free survival and overall survival of 4.0 months (95% CI, 1.9-5.8 months) and 15.9 months (95% CI, 8.4-21.5 months), respectively. Similar clinical benefits were also observed in the 11 previously untreated patients. Better clinical efficacy of nivolumab was apparent for tumors with a higher programmed death-ligand 1 expression level, for those with a higher tumor mutation burden, and for microsatellite instability-high tumors. In contrast, no differences in efficacy were apparent between tumor subgroups based on estimated tissue of origin. Adverse events were consistent with the known safety profile of nivolumab. No treatment-related death was observed. CONCLUSIONS: Our results demonstrate a clinical benefit of nivolumab for patients with CUP, suggesting that nivolumab is a potential additional therapeutic option for CUP.
  • Shinya Rai; Hiroaki Inoue; Kazuko Sakai; Hitoshi Hanamoto; Mitsuhiro Matsuda; Yasuhiro Maeda; Takahiro Haeno; Yosaku Watatani; Takahiro Kumode; Kentaro Serizawa; Yasuhiro Taniguchi; Chikara Hirase; J Luis Espinoza; Yasuyoshi Morita; Hirokazu Tanaka; Takashi Ashida; Yoichi Tatsumi; Kazuto Nishio; Itaru Matsumura
    Cancer science 113 2 660 - 673 2022年02月 
    We previously examined the utility of rituximab-bendamustine (RB) in patients with follicular lymphoma (FL) exhibiting less than optimal responses to 2 cycles of the R-CHOP chemotherapy regimen. The aim of this study was to identify molecular biomarkers that can predict prognosis in RB-treated patients in the context of the prospective cohort. We first analyzed the mutational status of 410 genes in diagnostic tumor specimens by target capture and Sanger sequencing. CREBBP, KMT2D, MEF2B, BCL2, EZH2, and CARD11 were recurrently mutated as reported before, however none was predictive for progression-free survival (PFS) in the RB-treated patients (n = 34). A gene expression analysis by nCounter including 800 genes associated with carcinogenesis and/or the immune response showed that expression levels of CD8+ T-cell markers and half of the genes regulating Th1 and Th2 responses were significantly lower in progression of disease within the 24-mo (POD24) group (n = 8) than in the no POD24 group (n = 31). Collectively, we selected 10 genes (TBX21, CXCR3, CCR4, CD8A, CD8B, GZMM, FLT3LG, CD3E, EOMES, GZMK), and generated an immune infiltration score (IIS) for predicting PFS using principal component analysis, which dichotomized the RB-treated patients into immune IIShigh (n = 19) and IISlow (n = 20) groups. The 3-y PFS rate was significantly lower in the IISlow group than in the IIShigh group (50.0% [95% CI: 27.1-69.2%] vs. 84.2% [95% CI: 58.7-94.6%], P = .0237). Furthermore, the IIS was correlates with absolute lymphocyte counts at diagnosis (r = 0.460, P = .00355). These results suggest that the T-cell-associated immune markers could be useful to predict prognosis in RB-treated FL patients. (UMIN:000 013 795, jRCT:051 180 181).
  • Masato Maekawa; Terumi Taniguchi; Kazuto Nishio; Kazuko Sakai; Kazuyuki Matsushita; Kaname Nakatani; Takayuki Ishige; Makoto Ikejiri; Hiroshi Nishihara; Kuniko Sunami; Yasushi Yatabe; Kanako C Hatanaka; Yutaka Hatanaka; Yoshihiro Yamamoto; Keita Fukuyama; Shinya Oda; Kayoko Saito; Mamoru Yokomura; Yuji Kubo; Hiroko Sato; Yoshinori Tanaka; Misa Fuchioka; Tadashi Yamasaki; Koichiro Matsuda; Kiyotaka Kurachi; Kazuhiro Funai; Satoshi Baba; Moriya Iwaizumi
    Scientific reports 12 1 1494 - 1494 2022年01月 
    To implement precision oncology, analytical validity as well as clinical validity and utility are important. However, proficiency testing (PT) to assess validity has not yet been systematically performed in Japan. To investigate the quality of next-generation sequencing (NGS) platforms and cancer genome testing prevalent in laboratories, we performed pilot PT using patient samples. We prepared genomic DNA from the cancer tissue and peripheral blood of 5 cancer patients and distributed these to 15 laboratories. Most participating laboratories successfully identified the pathogenic variants, except for two closely located KRAS variants and 25 bp delins in EGFR. Conversely, the EGFR L858R variant was successfully identified, and the allele frequency was similar for all the laboratories. A high DNA integrity number led to excellent depth and reliable NGS results. By conducting this pilot study using patient samples, we were able to obtain a glimpse of the current status of cancer genome testing at participating laboratories. To enhance domestic cancer genome testing, it is important to conduct local PT and to involve the parties concerned as organizers and participants.
  • Kimio Yonesaka; Junko Tanizaki; Osamu Maenishi; Koji Haratani; Hisato Kawakami; Kaoru Tanaka; Hidetoshi Hayashi; Kazuko Sakai; Yasutaka Chiba; Asuka Tsuya; Hiroki Goto; Eri Otsuka; Hiroaki Okida; Maki Kobayashi; Ryoto Yoshimoto; Masanori Funabashi; Yuuri Hashimoto; Kenji Hirotani; Takashi Kagari; Kazuto Nishio; Kazuhiko Nakagawa
    Clinical cancer research : an official journal of the American Association for Cancer Research 28 2 390 - 403 2022年01月 
    PURPOSE: EGFR-tyrosine kinase inhibitor (TKI) is a standard first-line therapy for activated EGFR-mutated non-small cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance. Clinical trials of the HER3-targeting antibody-drug conjugate patritumab deruxtecan (HER3-DXd) demonstrated its anticancer activity in EGFR-mutated NSCLC; however, the mechanisms that regulate HER3 expression are unknown. This study was conducted with the aim to clarify the mechanisms underlying HER3 regulation in EGFR-mutated NSCLC tumors and explored the strategy for enhancing the anticancer activity of HER3-DXd in EGFR-mutated NSCLC. EXPERIMENTAL DESIGN: Paired tumor samples were obtained from 48 patients with EGFR-mutated NSCLC treated with EGFR-TKI(s). HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pretreatment to post-EGFR-TKI resistance acquisition. The anticancer efficacy of HER3-DXd and osimertinib was evaluated in EGFR-mutated NSCLC cells. RESULTS: We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared with paired pretreatment samples. RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation, especially in tumors from patients who received continuous EGFR-TKI therapy. An in vitro study also showed that EGFR-TKI increased HER3 expression, repressed AKT phosphorylation in multiple EGFR-mutated cancers, and enhanced the anticancer activity of HER3-DXd. CONCLUSIONS: Our findings help clarify the mechanisms of HER3 regulation in EGFR-mutated NSCLC tumors and highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC.
  • Shinichiro Suzuki; Kimio Yonesaka; Takeshi Teramura; Toshiyuki Takehara; Ryoji Kato; Hitomi Sakai; Koji Haratani; Junko Tanizaki; Hisato Kawakami; Hidetoshi Hayashi; Kazuko Sakai; Kazuto Nishio; Kazuhiko Nakagawa
    Clinical cancer research : an official journal of the American Association for Cancer Research 28 2 428 - 428 2022年01月
  • Koichiro Nozaki; Satoshi Watanabe; Kazuto Nishio; Kazuko Sakai; Toshiaki Kikuchi
    Translational cancer research 11 1 295 - 298 2022年01月 
    Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows great clinical activity in non-small cell lung cancer (NSCLC) patients with EGFR mutations regardless of T790M mutation at first-line chemotherapy. Previous studies demonstrated that there are few patients with initial resistance to osimertinib. Here, we describe a case to report the efficacy of afatinib in an EGFR-mutated NSCLC patient with early progression to first-line osimertinib treatment. A 68-year-old Japanese male was diagnosed with stage IVB lung adenocarcinoma with the EGFR L858R mutation in exon 21. Two months after the start of osimertinib, his tumor progressed at the initial response evaluation. Because he refused to receive cytotoxic chemotherapy, afatinib treatment was initiated. He was administered afatinib, and the tumor shrank. After five months of afatinib treatment, nevertheless the primary tumor was not enlarged, he experienced disease progression with leptomeningeal metastasis and passed away. To elucidate the resistance mechanisms of osimertinib in this patient, we performed next-generation sequencing (NGS) on tumor samples from pleural effusions after osimertinib failure. NGS revealed no specific gene mutations causing resistance to osimertinib except for the EGFR L858R mutation; however, his tumor had a relatively high tumor mutational burden. Afatinib is considered an option for EGFR-mutated patients with early progression to osimertinib.
  • Toshiaki Takahashi; Kazuko Sakai; Hirotsugu Kenmotsu; Kiyotaka Yoh; Haruko Daga; Tatsuo Ohira; Tsuyoshi Ueno; Tadashi Aoki; Hidetoshi Hayashi; Koji Yamazaki; Yukio Hosomi; Toyofumi F. Chen-Yoshikawa; Norihito Okumura; Yuichi Takiguchi; Akimasa Sekine; Tomohiro Haruki; Hiromasa Yamamoto; Yuki Sato; Hiroaki Akamatsu; Takashi Seto; Sho Saeki; Kenji Sugio; Makoto Nishio; Hidetoshi Inokawa; Nobuyuki Yamamoto; Kazuto Nishio; Masahiro Tsuboi
    CANCER SCIENCE 2021年11月 
    The mutation status of tumor tissue DNA (n = 389) of resected stage II-III non-squamous non-small-cell lung cancer (Ns-NSCLC) was analyzed using targeted deep sequencing as an exploratory biomarker study (JIPANG-TR) for the JIPANG study, a randomized phase III study of pemetrexed/cisplatin (Pem/Cis) vs vinorelbine/cisplatin (Vnr/Cis). The TP53 mutation, common EGFR mutations (exon 19 deletion and L858R), and KRAS mutations were frequently detected. The frequency of the EGFR mutation was significant among female patients. Patients with an EGFR mutation-positive status had a significantly shorter recurrence-free survival (RFS) time (24 mo vs not reached) (HR, 1.64; 95% CI, 1.22-2.21; P = .0011 for EGFR mutation status). Multivariable analysis identified both the pathological stage and EGFR mutation status as independent prognostic factors for RFS (HR, 1.78; 95% CI, 1.30-2.44; P = .0003 for disease stage; and HR, 1.57; 95% CI, 1.15-2.16; P = .0050 for EGFR mutation status). This study demonstrated that the EGFR mutation has either a poor prognostic or predictive impact on a poor response to postoperative chemotherapy with platinum doublet chemotherapy for stage II-III Ns-NSCLC patients. This result supports a role for mandatory molecular diagnosis of early-stage Ns-NSCLC for precision oncology and signifies the importance of adjuvant for the 3rd generation tyrosine kinase inhibitor rather than platinum-based chemotherapy. This study is registered with the UMIN Clinical Trial Registry (UMIN 000012237).
  • Yutaka Hatanaka; Takeshi Kuwata; Eiichi Morii; Yae Kanai; Hitoshi Ichikawa; Takashi Kubo; Kanako C Hatanaka; Kazuko Sakai; Kazuto Nishio; Satoshi Fujii; Wataru Okamoto; Takayuki Yoshino; Atsushi Ochiai; Yoshinao Oda
    Pathology international 71 11 725 - 740 2021年10月 
    Clinical cancer genomic testing based on next-generation sequencing can help select genotype-matched therapy and provide diagnostic and prognostic information. Pathological tissue from malignant tumors obtained during routine practice are frequently used for genomic testing. This article is aimed to standardize the proper handling of pathological specimens in practice for genomic medicine based on the findings established in "Guidelines on the handling of pathological tissue samples for genomic medicine (in Japanese)" published by The Japanese Society of Pathology (JSP) in 2018. The two-part practical guidelines are based on empirical data analyses; Part 1 describes the standard preanalytic operating procedures for tissue collection, processing, and storage of formalin-fixed paraffin-embedded (FFPE) samples, while Part 2 describes the assessment and selection of FFPE samples appropriate for genomic testing, typically conducted by a pathologist. The guidelines recommend that FFPE sample blocks be used within 3 years from preparation, and the tumor content should be ≥30% (minimum 20%). The empirical data were obtained from clinical studies performed by the JSP in collaboration with leading Japanese cancer genome research projects. The Japanese Ministry of Health, Labour, and Welfare (MHLW) recommended to comply with the JSP practical guidelines in implementing cancer genomic testing under the national health insurance system in over 200 MHLW-designated core and cooperative cancer genome medicine hospitals in Japan.
  • Yoshinori Imamura; Kaoru Tanaka; Naomi Kiyota; Hidetoshi Hayashi; Ichiro Ota; Akihito Arai; Shigemichi Iwae; Shujiro Minami; Katsunari Yane; Tomoko Yamazaki; Yoshiaki Nagatani; Masanori Toyoda; Takayuki Takahama; Kazuko Sakai; Kazuto Nishio; Naoki Otsuki; Ken-Ichi Nibu; Hironobu Minami
    Medical oncology (Northwood, London, England) 38 11 128 - 128 2021年09月 
    The clinical utility of systemic therapy and genomic profiling in non-squamous-cell head and neck cancer (NSCHNC) has not been fully elucidated. This phase II trial evaluated the efficacy and safety of docetaxel and cisplatin combination in the first-line setting. Eligibility criteria were recurrent and/or metastatic NSCHNC; progressive disease within the last 6 months; no prior systemic therapy; and ECOG performance status of 0-1. Patients received docetaxel (75 mg/m2 on day 1) and cisplatin (75 mg/m2 on day 1), repeated every 21 days for 6 cycles. The primary endpoint was confirmed objective response rate (ORR). The secondary endpoints included progression-free survival (PFS), overall survival (OS), and adverse events. Next-generation sequencing (NGS) was performed using the Ion AmpliSeq Cancer Hotspot Panel v2. Twenty-three patients were enrolled from November 2012 to October 2016, of whom 8 were male. Median age was 57 years. Ninety-six percent of cases were metastatic. Among 22 evaluable patients, confirmed ORR was 45% (95% confidential interval 24-68%). With a median follow-up period of 18.8 months, median PFS and OS were 6.7 and 20.1 months, respectively. Grade 3/4 adverse events included febrile neutropenia (39%) and anemia (22%). No treatment-related deaths were observed. NGS analysis revealed potential treatment targets, including ERBB2, KIT, and ALK. The docetaxel and cisplatin combination regimen can be considered a new treatment option in recurrent and/or metastatic NSCHNC, although primary prophylaxis for febrile neutropenia should be considered. Diverse genomic alterations may lead novel treatment options.This trial was registered with the UMIN Clinical Trials Registry as UMIN000008333 on [September 1st, 2012].
  • Toshio Fujino; Kenichi Suda; Kazuko Sakai; Isao Murakami; Shigeki Shimizu; Shuta Ohara; Takamasa Koga; Akira Hamada; Junichi Soh; Kazuto Nishio; Tetsuya Mitsudomi
    Clinical Lung Cancer 2021年09月
  • Shinichiro Suzuki; Kimio Yonesaka; Takeshi Teramura; Toshiyuki Takehara; Ryoji Kato; Hitomi Sakai; Koji Haratani; Junko Tanizaki; Hisato Kawakami; Hidetoshi Hayashi; Kazuko Sakai; Kazuto Nishio; Kazuhiko Nakagawa
    Clinical cancer research : an official journal of the American Association for Cancer Research 27 20 5697 - 5707 2021年08月 
    PURPOSE: Treatment with KRAS G12C inhibitors such as sotorasib can produce substantial regression of tumors in some patients with non-small cell lung cancer (NSCLC). These patients require alternative treatment after acquiring resistance to the inhibitor. The mechanisms underlying this acquired resistance are unclear. The purpose of this study was to identify the mechanisms underlying acquired sotorasib resistance, and to explore potential treatments for rescuing patients with sotorasib-resistant KRAS G12C NSCLC cells. EXPERIMENTAL DESIGN: Clones of sotorasib-sensitive KRAS G12C NSCLC H23 cells exposed to different concentrations of sotorasib were examined using whole-genomic transcriptome analysis, multiple receptor kinase phosphorylation analysis, and gene copy number evaluation. The underlying mechanisms of resistance were investigated using immunological examination, and a treatment aimed at overcoming resistance was tested in vitro and in vivo Results: Unbiased screening detected subclonal evolution of MET amplification in KRAS G12C NSCLC cells that had developed resistance to sotorasib in vitro MET knockdown using siRNA restored susceptibility to sotorasib in these resistant cells. MET activation by its amplification reinforced RAS cycling from its inactive form to its active form. In addition to RAS-mediated MEK-ERK induction, MET induced AKT activation independently of RAS. Crizotinib, a MET inhibitor, restored sensitivity to sotorasib by eliminating RAS-MEK-ERK as well as AKT signaling. MET/KRAS G12C dual inhibition led to tumor shrinkage in sotorasib-resistant xenograft mice. CONCLUSIONS: MET amplification leads to the development of resistance to KRAS G12C inhibitors in NSCLC. Dual blockade of MET and KRAS G12C could be a treatment option for MET amplified, KRAS G12C-mutated NSCLC.
  • Marco A. De Velasco; Yurie Kura; Naomi Ando; Noriko Sako; Eri Banno; Kazutoshi Fujita; Masahiro Nozawa; Kazuhiro Yoshimura; Kazuko Sakai; Kazuhiro Yoshikawa; Kazuto Nishio; Hirotsugu Uemura
    Cancers 13 16 3975 - 3975 2021年08月 
    Significant improvements with apalutamide, a nonsteroidal antiandrogen used to treat patients suffering from advanced prostate cancer (PCa), have prompted evaluation for additional indications and therapeutic development with other agents; however, persistent androgen receptor (AR) signaling remains problematic. We used autochthonous mouse models of Pten-deficient PCa to examine the context-specific antitumor activity of apalutamide and profile its molecular responses. Overall, apalutamide showed potent antitumor activity in both early-stage and late-stage models of castration-naïve prostate cancer (CNPC). Molecular profiling by Western blot and immunohistochemistry associated persistent surviving cancer cells with upregulated AKT signaling. While apalutamide was ineffective in an early-stage model of castration-resistant prostate cancer (CRPC), it tended to prolong survival in late-stage CRPC. Molecular features associated with surviving cancer cells in CRPC included upregulated aberrant-AR, and phosphorylated S6 and proline-rich Akt substrate of 40 kDa (PRAS40). Strong synergy was observed with the pan-AKT inhibitor GSK690693 and apalutamide in vitro against the CNPC- and CRPC-derived cell lines and tended to improve the antitumor responses in CNPC but not CRPC in vivo. Upregulation of signal transducer and activator of transcription 3 (STAT3) and proviral insertion in murine-1 (PIM-1) were associated with combined apalutamide/GSK690693. Our findings show that apalutamide can attenuate Pten-deficient PCa in a context-specific manner and provides data that can be used to further study and, possibly, develop additional combinations with apalutamide.
  • Esuteru Hirokawa; Satomi Watanabe; Kazuko Sakai; Masayuki Takeda; Chihiro Sato; Takayuki Takahama; Kazuto Nishio; Kazuhiko Nakagawa
    Thoracic cancer 12 16 2283 - 2287 2021年08月 
    Epidermal growth factor receptor (EGFR) kinase domain duplication (KDD) has been identified as an oncogenic driver in 0.05% to 0.14% of non-small cell lung cancer (NSCLC) patients. However, little is known of the efficacy of EGFR tyrosine kinase inhibitors (TKIs) for such patients. Here, we report the case of a 45-year-old Japanese woman with NSCLC positive for EGFR-KDD (duplication of exons 18-25) who developed carcinomatous meningitis and showed a marked response to the EGFR-TKIs erlotinib and osimertinib. As far as we are aware, this is the first report of EGFR-TKI efficacy for carcinomatous meningitis in a NSCLC patient harboring EGFR-KDD.
  • Kazuko Sakai; Marco A De Velasco; Yurie Kura; Kazuto Nishio
    Cancers 13 15 2021年07月 
    Colitis is a risk factor for colorectal cancer (CRC) and can change the dynamics of gut microbiota, leading to dysbiosis and contributing to carcinogenesis. The functional interactions between colitis-associated CRC and microbiota remain unknown. In this study, colitis and CRC were induced in BALB/c mice by the administration of dextran sodium sulfate (DSS) and/or azoxymethane (AOM). Whole transcriptome profiling of normal colon was then performed, and gene set enrichment analysis (GSEA) revealed enriched fatty acid metabolism, oxidative phosphorylation, and PI3K-Akt-mTOR signaling in the tissues from DSS/AOM mice. Additionally, immunohistochemical staining showed increased expression levels of phosphorylated S6 ribosomal protein, a downstream target of the PI3K-Akt-mTOR pathway in the inflamed mucosa of DSS/AOM mice. Fecal microbes were characterized using 16S rDNA gene sequencing. Redundancy analysis demonstrated a significant dissimilarity between the DSS/AOM group and the others. Functional analysis inferred from microbial composition showed enrichments of the sphingolipid signal and lipoarabinomannan biosynthetic pathways. This study provides additional insights into alterations associated with DSS/AOM-induced colitis and associates PI3K-Akt-mTOR, sphingolipid-signaling and lipoarabinomannan biosynthetic pathways in mouse DSS/AOM-induced colitis.
  • Toshiharu Sakurai; Marco A De Velasco; Kazuko Sakai; Tomoyuki Nagai; Hiroki Nishiyama; Kentaro Hashimoto; Hirotsugu Uemura; Hisato Kawakami; Kazuhiko Nakagawa; Hiroyuki Ogata; Kazuto Nishio; Masatoshi Kudo
    Molecular oncology 2021年07月 
    Immune checkpoint inhibitors (ICIs) are widely used to treat various malignancies. Although the gut microbiome is known to influence the efficacy of ICIs on epithelial tumors, the functional interactions between gut taxa and colonic mucosa remain poorly understood. Here we performed transcriptomic profiling and 16S rRNA sequencing to investigate the relationships between mucosal gene expression and microbial composition with ICI responses and gastrointestinal immune-related adverse events (GI irAEs). In responders, genes related to DNA repair and cell cycle signatures were enriched in responders whereas signatures related to innate immune response, NFAT and IFN-γ signaling pathways were enriched in nonresponders. Gut microbial composition revealed an association between moderate GI irAE and favorable response to ICI therapy. Favorable therapeutic responses to ICI and GI irAE treatments were associated with taxa classified as Enterobacteriaceae and were related to ribonucleoprotein complex biogenesis, cytokine-mediated signaling pathway, tRNA metabolic process, and ribonucleoprotein complex assembly in the colon. These findings open new perspectives for improving the efficacy and safety of cancer immunotherapy.
  • Marco A. De Velasco; Kazuko Sakai; Yurie Kura; Eri Banno; Naomi Ando; Noriko Sako; Nobutaka Shimizu; Kazutoshi Fujita; Masahiro Nozawa; Kazuhiro Yoshimura; Kazuto Nishio; Hirotsugu Uemura
    Cancer Research 2021年07月
  • Marco A. De Velasco; Yurie Kura; Noriko Sako; Naomi Ando; Kazuko Sakai; Alwin Schuller; Kazutoshi Fujita; Masahiro Nozawa; Kazuhiro Yoshimura; Kazuto Nishio; Hirotsugu Uemura
    Cancer Research 2021年07月
  • Marco A. De Velasco; Kazuko Sakai; Yurie Kura; Naomi Ando; Noriko Sako; Kazutoshi Fujita; Masahiro Nozawa; Kazuhiro Yoshimura; Kazuto Nishio; Hirotsugu Uemura
    Cancer Research 2021年07月
  • Marco A. De Velasco; Yurie Kura; Kazuko Sakai; Kazutoshi Fujita; Masahiro Nozawa; Kazuhiro Yoshimura; Kazuto Nishio; Hirotsugu Uemura
    Cancer Research 2021年07月
  • Marco A. De Velasco; Yurie Kura; Naomi Ando; Kazuko Sakai; Nobutaka Shimizu; Eri Banno; Masahiro Nozawa; Kazuhiro Fujita; Kazuhiro Yoshimura; Kazuto Nishio; Hirotsugu Uemura
    Cancer Research 2021年07月
  • Atsushi Osoegawa; Masafumi Yamaguchi; Tomomi Nakamura; Ryotaro Morinaga; Kentaro Tanaka; Kosuke Kashiwabara; Takashi Miura; Takayuki Suetsugu; Taishi Harada; Tatsuma Asoh; Kenichi Taguchi; Kazuki Nabeshima; Junji Kishimoto; Kazuko Sakai; Kazuto Nishio; Kenji Sugio
    JTO clinical and research reports 2 7 100191 - 100191 2021年07月 
    Introduction: Although treatment with osimertinib confers survival benefits in patients with lung cancer with the EGFR T790M mutation, the mechanism of acquired resistance to osimertinib remains poorly understood. We conducted a prospective observational study to identify the mechanism on the basis of repeated tissue biopsies. Methods: Patients with EGFR-mutated advanced lung cancer with a T790M mutation detected on a tissue biopsy underwent a rebiopsy after developing acquired resistance to osimertinib. Nucleic acids extracted from the biopsy samples were subjected to targeted resequencing (Oncomine Comprehensive Assay), and circulating cell-free DNA (ccfDNA) was analyzed by CAncer Personalized Profiling by deep Sequencing (AVENIO ctDNA Surveillance Kit). Results: Between November 2016 and March 2020, a total of 87 patients were screened. Among them, 44 developed acquired resistance. Of these, 19 samples from rebiopsies and 12 from preosimertinib biopsies were able to be analyzed by an Oncomine Comprehensive Assay. A ccfDNA analysis was performed in 16 patients. Regarding the mechanisms of acquired resistance, structural change in EGFR, namely, C797S, G796S, or L792V, was the most frequent alteration, being observed in 57.9% of the cases. MET gain was observed in 31.6% of the cases, and gains in cell cycle genes were observed in 26.3% of the cases. In addition, we identified GAS6 gain and an ATM mutation in a patient with small-cell transformation and a BRAF V600E mutation in a patient with oligoprogressive disease. Conclusions: A repeated tissue biopsy and a ccfDNA analysis were useful in analyzing the mechanisms underlying acquired resistance. A long treatment history of EGFR TKIs may result in a high percentage of EGFR structural change.
  • Masahiro Morita; Naoshi Nishida; Kazuko Sakai; Tomoko Aoki; Hirokazu Chishina; Masahiro Takita; Hiroshi Ida; Satoru Hagiwara; Yasunori Minami; Kazuomi Ueshima; Kazuto Nishio; Yukari Kobayashi; Kazuhiro Kakimi; Masatoshi Kudo
    Liver Cancer 10 4 380 - 393 2021年07月 
    Introduction: Although immune checkpoint inhibitors (ICIs) have been considered as promising agents for the treatment of advanced hepatocellular carcinoma (HCC), previous clinical trials revealed that the response to anti-programmed cell death protein 1 (anti-PD-1) monotherapy was as low as 20%. Identifying subgroups that respond well to ICIs is clinically important. Here, we studied the prognostic factors for anti-PD-1 antibody treatment based on the molecular and immunological features of HCC. Methods: Patients who were administered anti-PD1 antibody for advanced HCC at Kindai University Hospital were included. Clinicopathological backgrounds and antitumor responses were examined in 34 cases where tumor tissues before treatment were available. Transcriptome analysis was performed using 40 HCC samples obtained from surgical resection, and immune status was compared between 20 HCCs with activating mutations in β-catenin and those without the mutations using transcriptome-based immunogram. Results: Univariate analysis showed that the disease control rate was significantly better in patients with α-fetoprotein < 400 ng/mL, negative for β-catenin/glutamate synthetase (GS) staining, high combined positive score (CPS) of programmed death-ligand 1 (PD-L1), and increased infiltration of CD8+ cells in tumor tissues. Among them, negative staining of β-catenin/GS, CPS of PD-L1 ≥ 1, and high degree of CD8+ tumor-infiltrating lymphocytes (TILs) were significantly associated with longer survival in both progression-free survival (PFS) and overall survival (OS). The combination of these factors well stratified the survival of the patients on anti-PD-1 antibody in both PFS and OS (p < 0.0001 and p = 0.0048 for PFS and OS, respectively). In addition, the immunogram revealed that tumor-carrying mutations in β-catenin showed downregulation of immune-related genes, especially in those related to priming and activation by dendritic cells, interferon-γresponse, inhibitory molecules, and regulatory T cells. Discussion/Conclusion: The combined score including Wnt/β-catenin activation, CPS of PD-L1, and degree of CD8+ TILs in HCC is informative for predicting the response to ICI in HCC cases. Constitutive activation of β-catenin can induce an immune cold phenotype with downregulation of immune-related genes, and immunohistochemistry-based evaluation is beneficial for identifying the subgroup that shows a good response to ICI.
  • Kosuke Murakami; Akiko Kanto; Kazuko Sakai; Chiho Miyagawa; Hisamitsu Takaya; Hidekatsu Nakai; Yasushi Kotani; Kazuto Nishio; Noriomi Matsumura
    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 34 11 2071 - 2079 2021年06月 
    Recent studies have reported cancer-associated mutations in normal endometrium. Mutations in eutopic endometrium may lead to endometriosis and endometriosis-associated ovarian cancer. We investigated PIK3CA mutations (PIK3CAm) for three hotspots (E542K, E545K, H1047R) in eutopic endometrium in patients with ovarian cancer and endometriosis from formalin-fixed paraffin-embedded specimens by laser-capture microdissection and droplet digital PCR. The presence of PIK3CAm in eutopic endometrial glands with mutant allele frequency ≥ 15% were as follows: ovarian clear cell carcinoma (OCCC) with PIK3CAm in tumors, 20/300 hotspots in 11/14 cases; OCCC without PIK3CAm, 42/78 hotspots in 11/12 cases; high-grade serous ovarian carcinoma, 8/45 hotspots in 3/5 cases; and endometriotic cysts, 5/63 hotspots in 5/6 cases. These rates were more frequent than in noncancer nonendometriosis controls (7/309 hotspots in 5/17 cases). In OCCC without PIK3CAm, 7/12 (58%) cases showed multiple hotspot mutations in the same eutopic endometrial glands. In 3/54 (5.6%) cases, PIK3CAm was found in eutopic endometrial stroma. Multisampling of the OCCC tumors with PIK3CAm showed intratumor heterogeneity in three of eight cases. In two cases, PIK3CAm was detected in the stromal component of the tumor. Homogenous PIK3CAm in the epithelial component of the tumor matched the mutation in eutopic endometrial glands in only one case. Eutopic endometrial glands in ovarian cancer and endometriosis show high frequency of PIK3CAm that is not consistent with tumors, and multiple hotspot mutations are often found in the same glands. While the mutations identified in eutopic endometrium may not be driver mutations in the patient's cancer, these are still driver mutations but this specific clone has not undergone the requisite steps for the development of cancer.
  • Ryoji Kato; Hidetoshi Hayashi; Kazuko Sakai; Shinichiro Suzuki; Koji Haratani; Takayuki Takahama; Junko Tanizaki; Yoshikane Nonagase; Kaoru Tanaka; Takeshi Yoshida; Masayuki Takeda; Kimio Yonesaka; Hiroyasu Kaneda; Kazuto Nishio; Kazuhiko Nakagawa
    International journal of clinical oncology 26 9 1628 - 1639 2021年06月 
    BACKGROUND: We here applied cancer personalized profiling by deep sequencing (CAPP-seq) to analysis of circulating tumor DNA (ctDNA) to identify resistance mechanisms in osimertinib-treated patients with EGFR T790M-positive non-small cell lung cancer (NSCLC). METHODS: The study included patients with EGFR activating mutation-positive advanced NSCLC who were positive for T790M in tumor tissue or plasma after previous treatment with an EGFR tyrosine kinase inhibitor, who received osimertinib at Kindai University Hospital between August 2014 and September 2017, and for whom plasma collected after progression on osimertinib was available. Clinical data were extracted from medical records. Patients with innate resistance to osimertinib were defined as those whose best response was progressive disease or stable disease for < 6 months, whereas patients with a complete or partial response or stable disease for > 6 months were considered as having acquired resistance. RESULTS: We performed CAPP-seq for 20 patients at progression on osimertinib. Distinct patterns of genomic alterations were apparent in patients with innate versus acquired resistance. Mutations in PIK3CA, KRAS, or BRAF and copy number gain for EGFR, ERBB2, or MET were more common in patients with innate resistance than in those with acquired resistance. In addition, one patient who underwent a repeat biopsy was found to harbor the C797S mutation of EGFR after disease progression during osimertinib rechallenge, with this mutation not having been detected at the time of initial progression on osimertinib. CONCLUSIONS: CAPP-seq analysis of ctDNA was able to identify potentially targetable genetic alterations in patients with osimertinib resistance.
  • Kazuto Nishio; Takashi Seto; Makoto Nishio; Martin Reck; Edward B Garon; Kazuko Sakai; Koichi Goto; Terufumi Kato; Yoichi Nakanishi; Toshiaki Takahashi; Nobuyuki Yamamoto; Katsuyuki Kiura; Yuichiro Ohe; Tomohide Tamura; Carla Visseren-Grul; Bente Frimodt-Moller; Rebecca R Hozak; Sameera R Wijayawardana; Annamaria Zimmermann; Gosuke Homma; Sotaro Enatsu; Kazuhiko Nakagawa
    JTO clinical and research reports 2 6 100171 - 100171 2021年06月 
    Introduction: The phase 3 RELAY global study (NCT02411448) revealed significant improvement in progression-free survival (PFS) with ramucirumab plus erlotinib (RAM + ERL) compared with placebo plus ERL (PL + ERL) in untreated EGFR-mutated metastatic NSCLC (hazard ratio [HR] = 0.59 [95% confidence interval (CI): 0.46-0.76, p < 0.0001]). This prespecified analysis evaluates efficacy, safety, and postprogression EGFR T790M rates of RELAY patients enrolled in Japan. Methods: Patients were randomized (1:1) to oral ERL (150 mg/d) plus intravenous RAM (10 mg/kg) or PL every 2 weeks. End points included PFS (primary), safety (secondary), and biomarker analyses (exploratory). Plasma samples collected at baseline and poststudy treatment discontinuation were evaluated for EGFR T790M mutations by next-generation sequencing. Results: The Japanese subset included 211 of 449 (47.0%) RELAY patients (RAM + ERL, n = 106; PL + ERL, n = 105). Median PFS was 19.4 versus 11.2 months for RAM + ERL versus PL + ERL treatment (HR = 0.610 [0.431-0.864]) in the Japanese intent-to-treat population, 16.6 versus 12.5 months (HR = 0.701 [0.424-1.159]) in the EGFR exon 19 deletion subgroup, and 19.4 versus 10.9 months (HR = 0.514 [0.317-0.835]) in the EGFR exon 21 L858R subgroup, respectively. Adverse events of grade 3 or above with RAM + ERL included hypertension (24.8%, all grade 3) and dermatitis acneiform (23.8%). Postprogression treatment-emergent T790M rates were similar between arms (RAM + ERL: 47%, 9 of 19 patients; PL + ERL: 50%, 20 of 40 patients). Conclusions: Clinically meaningful efficacy was observed with RAM + ERL versus PL + ERL in the RELAY Japanese subset, with no new safety concerns. Postprogression T790M rates were similar across treatment arms, indicating the addition of RAM did not affect the ERL-associated EGFR T790M rates at disease progression.
  • Chiho Miyagawa; Hisamitsu Takaya; Kazuko Sakai; Kazuto Nishio; Maho Konishi; Sachiko Minamiguchi; Toshihide Shimada; Noriomi Matsumura
    The oncologist 26 5 356 - 361 2021年05月 
    Recently, several malignant peritoneal mesotheliomas (MPMs), occurring in young women without asbestos exposure and with fusion genes such as anaplastic lymphoma kinase (ALK) and Ewing sarcoma breakpoint region 1, have been reported. In the present case, we encountered MPM with STRN-ALK fusion in a 17-year-old female adolescent. The case did not respond to chemotherapy and is currently in a clinical trial of alectinib. This is the fourth reported case of MPM with STRN-ALK fusion. Of the 45 cancer cases with STRN-ALK fusion in which the fusion partners were examined, all cases except for the current case showed fusion of exon 3 of STRN and exon 20 of ALK. This is the first case with fusion of exon 2 of STRN and exon 20 of ALK. Further advances in cancer genomic medicine may help clarify the clinical significance of this new fusion. KEY POINTS: Malignant peritoneal mesotheliomas (MPMs) can occur in young women without asbestos exposure and show fusion genes that activate anaplastic lymphoma kinase (ALK) by gene rearrangement. ALK rearrangement and the fusion partner can be detected by companion diagnostics and by next generation sequencing. Patients with MPMs with ALK rearrangement may benefit from target therapy.
  • Masayuki Takeda; Takayuki Takahama; Kazuko Sakai; Shigeki Shimizu; Satomi Watanabe; Hisato Kawakami; Kaoru Tanaka; Chihiro Sato; Hidetoshi Hayashi; Yoshikane Nonagase; Kimio Yonesaka; Naoki Takegawa; Tatsuya Okuno; Takeshi Yoshida; Soichi Fumita; Shinichiro Suzuki; Koji Haratani; Kazumasa Saigoh; Akihiko Ito; Tetsuya Mitsudomi; Hisashi Handa; Kazuya Fukuoka; Kazuhiko Nakagawa; Kazuto Nishio
    The oncologist 26 4 e588-e596  2021年04月 
    BACKGROUND: Implementation of personalized medicine requires the accessibility of tumor molecular profiling in order to allow prioritization of appropriate targeted therapies for individual patients. Our aim was to study the role of comprehensive genomic profiling assays that may inform treatment recommendations for patients with solid tumors. MATERIALS AND METHODS: We performed a prospective study to evaluate the feasibility of application of the FoundationOne CDx panel-which detects substitutions, insertions and deletions, and copy number alterations in 324 genes, select gene rearrangements, and genomic signatures including microsatellite instability and tumor mutation burden (TMB)-to patients with advanced or recurrent solid tumors before its approval in Japan. RESULTS: A total of 181 samples were processed for genomic testing between September 2018 and June 2019, with data being successfully obtained for 175 of these samples, yielding a success rate of 96.7%. The median turnaround time was 41 days (range, 21-126 days). The most common known or likely pathogenic variants were TP53 mutations (n = 113), PIK3CA mutations (n = 33), APC mutations (n = 32), and KRAS mutations (n = 29). Among the 153 patients assessed for TMB, the median TMB was 4 mutations/Mb, and tumors with a high TMB (≥10 mutations/Mb) were more prevalent for lung cancer (11/32) than for other solid tumor types (9/121, Fisher's exact test p < .01). No clear trend toward increased efficacy for immune checkpoint inhibitor (ICI) monotherapy or ICI combination chemotherapy in patients with a high programmed cell death-ligand 1 tumor proportion score or a high TMB was apparent. Among the 174 patients found to harbor known or likely pathogenic actionable alterations, 24 individuals (14%) received matched targeted therapy. CONCLUSION: The FoundationOne CDx assay was performed with formalin-fixed, paraffin-embedded tumor specimens with a success rate of >95%. Such testing may inform the matching of patients with cancer with investigational or approved targeted drugs. IMPLICATIONS FOR PRACTICE: This prospective cohort study was initiated to investigate the feasibility and utility of clinical application of FoundationOne CDx. A total of 181 samples were processed for genomic testing between September 2018 and June 2019, with data being successfully obtained for 175 of these samples, yielding a success rate of 96.7%, and 24 individuals (14%) received matched targeted therapy.
  • Shigeki Shimizu; Kazuko Sakai; Takaaki Chikugo; Takao Satou; Naoki Shiraishi; Tetsuya Mitsudomi; Kazuto Nishio
    Oncology letters 21 4 320 - 320 2021年04月 
    Pulmonary sarcomatoid carcinoma (PSC) is classified as poorly differentiated, and non-small cell lung carcinomas that contained a component of sarcoma or sarcoma-like differentiation are rare. The underlying carcinogenetic mechanism governing PSC remains unclear. The current study investigated the underlying carcinogenetic mechanism of PSC based on the hypothesis that it involves the epithelial-mesenchymal transition (EMT) process. Mutation analysis of PSCs, including carcinosarcoma, pleomorphic carcinoma and epithelial carcinoma specimens, was performed using targeted deep sequencing, whole transcriptome analysis and digital spatial profiling (DSP). PSCs exhibit a distinct mutation profile, with TP53, SYNE1 and APC mutations. Therefore, clustering of the gene expression profiles allowed the PSCs to be distinguished from the epithelial carcinomas. Increased gene expression of fibronectin in PSC was an important contributor to differential profiles. Pathway analysis revealed enhanced activity of the integrin-linked kinase (ILK) signaling pathway in the PSCs. DSP analysis using 56 antibodies of marker proteins confirmed significantly higher expression of fibronectin in PSCs. Intratumor heterogeneity of fibronectin expression was observed in sarcoma components. In conclusion, epithelial-mesenchymal transition process mediated by ILK signaling may be associated with carcinogenetic mechanisms of PSC. Overexpression of fibronectin mediated by ILK signaling appears to serve a role in the EMT involved in the PSC transformation process.
  • Kenichi Suda; Kazuko Sakai; Keiko Obata; Shuta Ohara; Toshio Fujino; Takamasa Koga; Akira Hamada; Junichi Soh; Kazuto Nishio; Tetsuya Mitsudomi
    Clinical lung cancer 22 2 e141-e145  2021年03月 
    BACKGROUND: Several clinical and preclinical studies suggest that non-small cell lung cancers (NSCLCs) with EGFR compound mutations were associated with lower efficacies of first-generation EGFR inhibitors than tumors with single EGFR mutation. Some researchers hypothesize that EGFR mutation status is heterogeneous in such tumors and that second-generation EGFR inhibitors may eliminate cancer cells with uncommon EGFR mutations from tumors with EGFR compound mutations. However, this hypothesis is currently unproven; therefore, we performed the current study to determine if tumor cells with EGFR compound mutations are present in heterogeneous or homogeneous manners. PATIENTS AND METHODS: Multiregion analysis was performed for surgically resected primary NSCLC tumors with EGFR compound mutations to examine the intratumor heterogeneity of EGFR compound mutations. In addition, we evaluated the intertumor heterogeneity of EGFR compound mutations using 2 pleural disseminations obtained from a patient with NSCLC at exploratory thoracotomy and 9 primary or metastatic lesions obtained from 2 autopsied NSCLC patients. Digital polymerase chain reaction, target sequencing, or direct sequencing were used to detect EGFR mutations. RESULTS: This study included 5 NSCLC cases; their compound mutations were L858R+S768I, G719X+S768I, G719A+R776H, L858R+E709G, and L858R+I759M. Noncancerous pulmonary tissues from each patient did not harbor EGFR mutations, which revealed that all mutations were somatic. We did not detect any intra- or intertumor heterogeneity in these EGFR compound mutations. CONCLUSION: No intra- or intertumor heterogeneity was observed for EGFR compound mutations. Our results indicate that both EGFR mutations were truncal and selective elimination of cancer cells with uncommon EGFR mutations is unrealistic.
  • Ryoji Kato; Koji Haratani; Hidetoshi Hayashi; Kazuko Sakai; Hitomi Sakai; Hisato Kawakami; Kaoru Tanaka; Masayuki Takeda; Kimio Yonesaka; Kazuto Nishio; Kazuhiko Nakagawa
    British journal of cancer 124 5 914 - 924 2021年03月 
    BACKGROUND: Cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) suppress antitumour immunity, and the tyrosine kinase inhibitor nintedanib has antifibrotic effects. METHODS: We performed a preclinical study to evaluate whether nintedanib might enhance antitumour immunity by targeting CAFs and thereby improve the response to immune checkpoint blockade (ICB). RESULTS: Whereas nintedanib did not suppress the growth of B16-F10 melanoma cells in vitro, it prolonged survival in a syngeneic mouse model of tumour formation by these cells, suggestive of an effect on the TME without direct cytotoxicity. Gene expression profiling indeed showed that nintedanib influenced antitumour immunity and fibrosis. Tumoural infiltration of CD8+ T cells and granzyme B production were increased by nintedanib, and its antitumour activity was attenuated by antibody-mediated depletion of these cells, indicating that nintedanib suppressed tumour growth in a CD8+ T cell-dependent manner. Moreover, nintedanib inhibited the proliferation and activation of fibroblasts. Finally, the combination of nintedanib with ICB showed enhanced antitumour efficacy in B16-F10 tumour-bearing mice. CONCLUSIONS: Our results suggest that nintedanib targeted CAFs and thereby attenuated the immunosuppressive nature of the TME and promoted the intratumoural accumulation and activation of CD8+ T cells, with these effects contributing to enhanced antitumour activity in combination with ICB.
  • Shugo Sakihama; Kazuho Morichika; Rumiko Saito; Megumi Miyara; Takashi Miyagi; Masaki Hayashi; Junnosuke Uchihara; Takeaki Tomoyose; Kazuiku Ohshiro; Shingo Nakayama; Sawako Nakachi; Satoko Morishima; Kazuko Sakai; Kazuto Nishio; Hiroaki Masuzaki; Takuya Fukushima; Kennosuke Karube
    Cancer science 112 3 1300 - 1309 2021年03月 
    Genetic alterations in adult T-cell leukemia/lymphoma (ATLL), a T-cell malignancy associated with HTLV-1, and their clinical impacts, especially from the perspective of viral strains, are not fully elucidated. We employed targeted next-generation sequencing and single nucleotide polymorphism array for 89 patients with ATLL in Okinawa, the southernmost islands in Japan, where the frequency of HTLV-1 tax subgroup-A (HTLV-1-taxA) is notably higher than that in mainland Japan, where most ATLL cases have HTLV-1-taxB, and compared the results with previously reported genomic landscapes of ATLL in mainland Japan and the USA. Okinawan patients exhibited similar mutation profiles to mainland Japanese patients, with frequent alterations in TCR/NF-ĸB (eg, PRKCB, PLCG1, and CARD11) and T-cell trafficking pathways (CCR4 and CCR7), in contrast with North American patients who exhibited a predominance of epigenome-associated gene mutations. Some mutations, especially GATA3 and RHOA, were detected more frequently in Okinawan patients than in mainland Japanese patients. Compared to HTLV-1-taxB, HTLV-1-taxA was significantly dominant in Okinawan patients with these mutations (GATA3, 34.1% vs 14.6%, P = .044; RHOA, 24.4% vs 6.3%, P = .032), suggesting the contribution of viral strains to these mutation frequencies. From a clinical viewpoint, we identified a significant negative impact of biallelic inactivation of PRDM1 (P = .027) in addition to the previously reported PRKCB mutations, indicating the importance of integrated genetic analysis. This study suggests that heterogeneous genetic abnormalities in ATLL depend on the viral strain as well as on the ethnic background. This warrants the need to develop therapeutic interventions considering regional characteristics.
  • Kazuko Sakai; Toshiharu Sakurai; Marco A De Velasco; Tomoyuki Nagai; Takaaki Chikugo; Kazuomi Ueshima; Yurie Kura; Takayuki Takahama; Hidetoshi Hayashi; Kazuhiko Nakagawa; Masatoshi Kudo; Kazuto Nishio
    Frontiers in oncology 11 763468 - 763468 2021年 
    Immune checkpoint inhibitors (ICIs) have become the standard of care for several cancers. However, ICI therapy has also been associated with various immune-related adverse events (irAEs). Clinical manifestations of immune-related colitis resemble those of inflammatory bowel diseases such as ulcerative colitis (UC). The composition of the bowel microflora is thought to influence the development of inflammatory bowel disease and irAE colitis. We profiled the gene expressions and microbe compositions of colonic mucosa from patients with solid cancers receiving anti-PD-L1 antibody treatment; we then compared the expression profiles associated with irAE colitis with those associated with UC. The pathway enrichment analysis revealed functional similarities between inflamed regions of irAE colitis and UC. The common enriched pathways included leukocyte extravasation and immune responses, whereas non-inflamed mucosa from patients with irAE colitis was distinct from patients with UC and was characterized by the recruitment of immune cells. A similarity between the microbiota profiles was also identified. A decreased abundance of Bacteroides species was observed in inflamed regions from both irAE colitis and UC based on a microbiota composition analysis of 16S rDNA sequencing. Pathways associated with molecule transport systems, including fatty acids, were enriched in inflamed and non-inflamed irAE colitis and inflamed UC, similar to Piphillin-inferred KEGG pathways. While UC is characterized by local regions of inflammation, ICI treatment extends to non-inflammatory regions of the colonial mucosa where immune cells are reconstituted. This analysis of the similarity and heterogeneity of irAE colitis and UC provides important information for the management of irAE colitis.
  • Kazuko Sakai; Takayuki Takahama; Mototsugu Shimokawa; Koichi Azuma; Masayuki Takeda; Terufumi Kato; Haruko Daga; Isamu Okamoto; Hiroaki Akamatsu; Shunsuke Teraoka; Akira Ono; Tatsuo Ohira; Toshihide Yokoyama; Nobuyuki Yamamoto; Kazuhiko Nakagawa; Kazuto Nishio
    Molecular oncology 15 1 126 - 137 2021年01月 
    The WJOG8815L phase II clinical study involves patients with non-small cell lung cancer (NSCLC) that harbored the EGFR T790M mutation, which confers resistance to EGFR tyrosine kinase inhibitors (TKIs). The purpose of this study was to assess the predictive value of monitoring EGFR genomic alterations in circulating tumor DNA (ctDNA) from patients with NSCLC that undergo treatment with the third-generation EGFR-TKI osimertinib. Plasma samples of 52 patients harboring the EGFR T790M mutation were obtained pretreatment (Pre), on day 1 of treatment cycle 4 (C4) or cycle 9 (C9), and at diagnosis of disease progression or treatment discontinuation (PD/stop). CtDNA was screened for EGFR-TKI-sensitizing mutations, the EGFR T790M mutation, and other genomic alterations using the cobas EGFR Mutation Test v2 (cobas), droplet digital PCR (ddPCR), and targeted deep sequencing. Analysis of the sensitizing-and T790M-EGFR mutant fractions (MFs) was used to determine tumor mutational burden. Both MFs were found to decrease during treatment, whereas rebound of the sensitizing EGFR MF was observed at PD/stop, suggesting that osimertinib targeted both T790M mutation-positive tumors and tumors with sensitizing EGFR mutations. Significant differences in the response rates and progression-free survival were observed between the sensitizing EGFR MF-high and sensitizing EGFR MF-low groups (cutoff: median) at C4. In conclusion, ctDNA monitoring for sensitizing EGFR mutations at C4 is suitable for predicting the treatment outcomes in NSCLC patients receiving osimertinib (Clinical Trial Registration No.: UMIN000022076).
  • Kazuko Sakai; Masahiro Tsuboi; Hirotsugu Kenmotsu; Takeharu Yamanaka; Toshiaki Takahashi; Koichi Goto; Haruko Daga; Tatsuo Ohira; Tsuyoshi Ueno; Tadashi Aoki; Kazuhiko Nakagawa; Koji Yamazaki; Yukio Hosomi; Koji Kawaguchi; Norihito Okumura; Yuichi Takiguchi; Akimasa Sekine; Tomohiro Haruki; Hiromasa Yamamoto; Yuki Sato; Hiroaki Akamatsu; Takashi Seto; Sho Saeki; Kenji Sugio; Makoto Nishio; Kazunori Okabe; Nobuyuki Yamamoto; Kazuto Nishio
    Cancer science 112 1 388 - 396 2021年01月 
    The JIPANG study is a randomized phase III study of pemetrexed/cisplatin (Pem/Cis) versus vinorelbine/cisplatin (Vnr/Cis) for completely resected stage II-IIIA non-squamous non-small cell lung cancer (Ns-NSCLC). This study did not meet the primary endpoint (recurrence-free survival, RFS) but Pem/Cis had a similar efficacy to Vnr/Cis with a better tolerability. Tumor mutation burden (TMB) is thought to have a predictive value of immune checkpoint inhibitors. However, the relevance of TMB to cytotoxic chemotherapy remains unknown. This exploratory study investigates the relationship between tumor mutation profiles and clinical outcome of Pem/Cis. Formalin-fixed, paraffin-embedded tumor tissues (n = 389) were obtained from the patients. Mutation status of tissue DNA was analyzed by targeted deep sequencing. Epidermal growth factor receptor (EGFR) mutations were detected frequently in Ns-NSCLC (139/374). Patients without any EGFR mutations experienced longer RFS in the Pem/Cis arm versus Vnr/Cis arms. Pem/Cis in patients with high TMB (≥12-16 mut/Mb) tended to have improved survival. In patients with wild-type EGFR, TMB ≥ 12 mut/Mb was significantly associated with improved RFS with Pem/Cis versus Vnr/Cis (not reached vs 52.5 months; hazard ratio (HR) 0.477). It could be proposed that TMB was predictive of RFS benefit with Pem/Cis versus Vnr/Cis in Ns-NSCLC. Further investigation is required to determine whether TMB combined with EGFR mutation status could be used as a predictive biomarker.
  • Tomoya Fukui; Kazuko Sakai; Jiichiro Sasaki; Mikiko Ishihara Kakegawa; Satoshi Igawa; Hisashi Mitsufuji; Masayuki Takeda; Takayuki Takahama; Kazuhiko Nakagawa; Kazuto Nishio; Katsuhiko Naoki
    Cancer biomarkers : section A of Disease markers 31 2 119 - 126 2021年 
    BACKGROUND: The advancement of cancer genomics has allowed for multiplex gene assays using next-generation sequencing (NGS) to be practically implemented, however, a clinical practice system remains to be established. OBJECTIVE: We evaluated the feasibility of clinical sequencing using NGS-based multiplex gene assays between cooperating medical institutions in patients with advanced cancers. METHODS: In this observational study, DNA and RNA samples prepared from existing tumor tissues were subjected to comprehensive genomic profiling using targeted sequencing. RESULTS: From January 2017 to March 2019, 36 samples from 33 patients were assessed. Of all patients, 27 (82%) had lung cancer, with the median age of 50 years (range 38-83). Multiplex gene panel tests were successfully carried out on 35/36 (97%) samples. Potentially actionable gene alterations were identified in 10/30 (33%) samples (3 HER2, 2 KRAS, 2 ALK, 1 PIK3CA, 1 RET, and 1 CDKN2A). In the 6 samples examined for resistant mechanisms, ALK I1171N mutation and MET copy number gain were detected in 2 patients with ALK rearrangement-positive lung cancer. CONCLUSIONS: Clinical sequencing using NGS-based multiplex gene assays between collaborating domestic medical institutions was feasible, with a success rate of > 97%. Overall, clinical sequencing benefits therapeutic decision-making in patients with advanced cancer.
  • Satomi Watanabe; Masayuki Takeda; Tomoyuki Otani; Takeshi Yoshida; Kazuko Sakai; Elizabeth Olek; S Michael Rothenberg; Jennifer Kherani; Pearl P French; Kazuto Nishio; Akihiko Ito; Kazuhiko Nakagawa
    JCO precision oncology 5 2021年
  • Yoriaki Komeda; Toshiharu Sakurai; Kazuko Sakai; Yasuyoshi Morita; Arito Hashimoto; Tomoyuki Nagai; Satoru Hagiwara; Itaru Matsumura; Kazuto Nishio; Masatoshi Kudo
    World journal of clinical cases 8 24 6389 - 6395 2020年12月 
    BACKGROUND: Concomitant ulcerative colitis (UC) and idiopathic thrombocytopenic purpura (ITP) is a rare phenomenon. The management of UC with ITP can be challenging, since a decreased platelet count augments UC. CASE SUMMARY: A 24-year-old man with UC and steroid-resistant ITP experienced UC flare. Although continuous infusion of cyclosporine was initiated, UC did not improve. The administration of tofacitinib subsequently led to the induction of remission. The patient has maintained remission of UC and ITP for over one year on tofacitinib treatment. Whole transcriptomic sequencing was performed for inflamed rectal mucosae obtained before and after the initiation of Janus kinase (JAK) inhibitor, suggesting that distinct molecular signatures seemed to be regulated by JAK inhibitors and other conventional therapies including tumor necrosis factor lockers. CONCLUSION: Tofacitinib should be considered in refractory cases of UC with ITP.
  • Akiko Honobe; Kazuko Sakai; Yosuke Togashi; Takehiro Ohnuma; Tatsuyoshi Kawamura; Kazuto Nishio; Takashi Inozume
    Journal of dermatological science 100 3 217 - 219 2020年12月
  • Hidetoshi Hayashi; Yuichi Takiguchi; Hironobu Minami; Kohei Akiyoshi; Yoshihiko Segawa; Hiroki Ueda; Yasuo Iwamoto; Chihiro Kondoh; Koji Matsumoto; Shin Takahashi; Hisateru Yasui; Toshiyuki Sawa; Yusuke Onozawa; Yasutaka Chiba; Yosuke Togashi; Yoshihiko Fujita; Kazuko Sakai; Shuta Tomida; Kazuto Nishio; Kazuhiko Nakagawa
    JAMA oncology 6 12 1931 - 1938 2020年12月 
    Importance: Although profiling of gene expression and gene alterations by next-generation sequencing (NGS) to predict the primary tumor site and guide molecularly targeted therapy might be expected to improve clinical outcomes for cancer of unknown primary site (CUP), to our knowledge, no clinical trial has previously evaluated this approach. Objective: To assess the clinical use of site-specific treatment, including molecularly targeted therapy based on NGS results, for patients with CUP. Design, Setting, and Participants: This phase 2 clinical trial was conducted at 19 institutions in Japan and enrolled 111 previously untreated patients with the unfavorable subset of CUP between March 2015 and January 2018, with 97 patients being included in the efficacy analysis. Eligibility criteria included a diagnosis of unfavorable CUP after mandatory examinations, including pathological evaluation by immunohistochemistry, chest-abdomen-pelvis computed tomography scans, and a positron emission tomography scan. Interventions: RNA and DNA sequencing for selected genes was performed simultaneously to evaluate gene expression and gene alterations, respectively. A newly established algorithm was applied to predict tumor origin based on these data. Patients received site-specific therapy, including molecularly targeted therapy, according to the predicted site and detected gene alterations. Main Outcomes And Measures: The primary end point was 1-year survival probability. Secondary end points included progression-free survival (PFS), overall survival (OS), objective response rate, safety, efficacy according to predicted site, and frequency of gene alterations. Results: Of 97 participants, 49 (50.5%) were women and the median (range) age was 64 (21-81) years. The cancer types most commonly predicted were lung (21 [21%]), liver (15 [15%]), kidney (15 [15%]), and colorectal (12 [12%]) cancer. The most frequent gene alterations were in TP53 (45 [46.4%]), KRAS (19 [19.6%]), and CDKN2A (18 [18.6%]). The 1-year survival probability, median OS, and median PFS were 53.1% (95% CI, 42.6%-62.5%), 13.7 months (95% CI, 9.3-19.7 months), and 5.2 months (95% CI, 3.3-7.1 months), respectively. Targetable EGFR mutations in tumor specimens were detected in 5 patients with predicted non-small-cell lung cancer (5.2%), 4 of whom were treated with afatinib; 2 of these patients achieved a durable PFS of longer than 6 months. Conclusions and Relevance: This study's findings suggest that site-specific treatment, including molecularly targeted therapy based on profiling gene expression and gene alterations by NGS, can contribute to treating patients with the unfavorable subset of CUP. Trial Registration: UMIN Identifier: UMIN000016794.
  • Tomoko Noguchi; Naoyuki Iwahashi; Kazuko Sakai; Kaho Matsuda; Hitomi Matsukawa; Saori Toujima; Kazuto Nishio; Kazuhiko Ino
    Cancers 12 11 2020年11月 
    Liquid biopsies from circulating tumor DNA (ctDNA) have been employed recently as a non-invasive diagnostic tool for detecting cancer-specific gene mutations. Here, we show the comprehensive gene mutation profiles of ctDNA in 51 patients with different histological subtypes of stage I-IV ovarian cancer, and their association with clinical outcomes. The ctDNA extracted from pre-treatment patients' plasma were analyzed using Cancer Personalized Profiling by Deep Sequencing targeting 197 genes. Of 51 patients, 48 (94%) showed one or more non-synonymous somatic mutations, including TP53 (37.3%), APC (17.6%), KRAS (15.7%), EGFR (13.7%), MET (11.8%), PIK3CA (11.8%), NPAP1 (11.8%), and ALK (9.8%). The most frequently mutated genes were as follows: TP53 in high-grade serous carcinoma (66.7%), APC in clear cell carcinoma (30.8%), PIK3CA in endometrioid carcinoma (40%), and KRAS in mucinous carcinoma (66.7%). Higher cell-free (cf)DNA concentration significantly correlated with worse progression-free survival (PFS) in all patients as well as stage III-IV patients (p = 0.01 and 0.005, respectively). Further, patients with any pathogenic mutations showed significantly worse PFS (p = 0.048). Blood tumor mutational burden detected from ctDNA did not significantly correlate with the histological subtypes or survival. Collectively, clinico-genomic profiles of individual ovarian cancer patients could be identified using ctDNA and may serve as a useful prognostic indicator. These findings suggest that ctDNA-based gene profiling might help in establishing personalized therapeutic strategies.
  • Toshiharu Sakurai; Hiroki Nishiyama; Kazuko Sakai; Marco A De Velasco; Tomoyuki Nagai; Yoriaki Komeda; Hiroshi Kashida; Akiyoshi Okada; Isao Kawai; Kazuto Nishio; Hiroyuki Ogata; Masatoshi Kudo
    Scientific reports 10 1 19186 - 19186 2020年11月 
    Given that sustained remission is the ultimate treatment goal in the management of patients with ulcerative colitis (UC), the decision to stop anti-tumor necrosis factor (anti-TNF) treatment in UC patients is difficult. The aim of this study was to evaluate mucosal microbiota and gene expression profiles associated with long-term remission after discontinuation of anti-TNF therapy. In nine UC patients who received anti-TNF therapy for 6 months, microbiota isolated from uninflamed mucosae and gene expression in inflamed and uninflamed mucosae were investigated at week 0 and at week 24. At treatment initiation, Fusobacterium sp. and Veillonella dispar were over-represented in the relapse group compared with the non-relapse group. After treatment, Dorea sp. and Lachnospira sp. were over-represented in the non-relapse group. In the relapse group only, a significant shift in gut bacterial community composition was found between week 0 and week 24. Gene expression of ALIX (PDCD6IP) and SLC9A3 was significantly higher in the non-relapse group than in the relapse group. Lastly, we used machine learning methods to identify relevant gene signatures associated with sustained remission. Statistical analyses of microbiota and expression profiles revealed differences between UC patients who did or did not keep remission after the discontinuation of TNF inhibitors.Trial registration: UMIN000020785: Evaluation of adalimumab therapy in mesalazine-resistant or -intolerant ulcerative colitis; an observational study (EARLY study).
  • Shuta Ohara; Kenichi Suda; Kazuko Sakai; Masaya Nishino; Masato Chiba; Masaki Shimoji; Toshiki Takemoto; Toshio Fujino; Takamasa Koga; Akira Hamada; Junichi Soh; Kazuto Nishio; Tetsuya Mitsudomi
    Translational lung cancer research 9 5 1915 - 1923 2020年10月 
    Background: Recent studies of advanced lung cancer patients have shown that circulating tumor DNA (ctDNA) analysis is useful for molecular profiling, monitoring tumor burden, and predicting therapeutic efficacies and disease progression. However, the usefulness of ctDNA analysis in surgically resected lung cancers is unclear. Methods: This study included 20 lung cancer patients with clinical stage IIA-IIIA disease. Preoperative and postoperative (3-12 days) plasma samples were collected for ctDNA analysis. Cancer personalized profiling by deep sequencing, which can detect mutations in 197 cancer-related genes, was used for ctDNA detection. The cohort consisted of 18 men and 2 women with a median age of 69 (range, 37-88) years. Sixteen patients (80%) had a history of smoking. Histologically, there were four squamous cell carcinomas, 13 adenocarcinomas, two adenosquamous cell carcinomas, and one small cell carcinoma. Results: At the time of data analysis, the 20 patients had been monitored for a median follow-up of 12 months. Eight patients (40%) were positive for preoperative ctDNA, and this was significantly correlated with tumor size (≥5 vs. <5 cm, P=0.018). Four patients (20%) were positive for postoperative ctDNA, and this was significantly correlated with histological grade (3 vs. 1 or 2, P=0.032). Postoperative positivity for ctDNA also predicted shorter recurrence-free survival (RFS) (P=0.015), while pre- and post-operative carcinoembryonic antigen levels (P=0.150 and P=0.533, respectively) and preoperative positivity for ctDNA (P=0.132) were not correlated with RFS. Conclusions: Detecting ctDNA postoperatively was a poor prognostic factor in surgically resected lung cancer patients that may suggest there is minimal residual disease (MRD).
  • Kenichi Suda; Isao Murakami; Keiko Obata; Kazuko Sakai; Toshio Fujino; Takamasa Koga; Shuta Ohara; Akira Hamada; Junichi Soh; Kazuto Nishio; Tetsuya Mitsudomi
    Lung cancer (Amsterdam, Netherlands) 148 100 - 104 2020年10月 [査読有り]
     
    BACKGROUND: Overcoming acquired resistance against targeted therapies to improve outcomes of lung cancer patients harboring driver mutations is a critical issue. While drug therapy oriented to a resistance mechanism appears attractive, spatial heterogeneity of resistance mechanisms in each patient will diminish treatment efficacy. However, the frequency, clinical backgrounds, clinical implications, and patterns of spatial heterogeneity in resistance mechanisms to EGFR tyrosine kinase inhibitors (TKIs) are largely unknown. PATIENTS AND METHODS: This study included 128 specimens from 24 autopsied patients with lung adenocarcinoma harboring EGFR mutation. Acquired resistance mechanisms reported as relatively frequent in lung cancer, e.g., T790 M and other secondary EGFR mutations, MET and ERBB2 gene amplification, and histological transformation, were retrospectively examined. All patients had received 1st/2nd generation EGFR-TKI and showed acquired resistance to the drug before death. No patient received osimertinib. RESULTS: No resistance mechanism was identified in two patients. T790M mutation was detected in 20 patients (83 %); however, nine of these patients also had lesions without T790M mutation. Among 22 patients whose resistance mechanisms were identified, ten had spatial heterogeneity of resistance mechanisms (45 %), and these patients had significantly shorter time-to-treatment failure compared with those without heterogeneity (median 4.7 months vs. 14.7 months, p = 0.0004). CONCLUSION: We observed significant spatial heterogeneity of acquired resistance mechanisms to EGFR-TKIs in lung adenocarcinoma. Our results also indicate that the incidence of resistance mechanisms may vary based on the biopsied tumor locations.
  • Toshio Shimizu; Kazuto Nishio; Kazuko Sakai; Isamu Okamoto; Kunio Okamoto; Masayuki Takeda; Maiko Morishita; Kazuhiko Nakagawa
    Cancer chemotherapy and pharmacology 86 2 211 - 219 2020年08月 [査読有り]
     
    PURPOSE: This phase I study was conducted to evaluate the safety and pharmacokinetics of YM155, a potent, selective survivin inhibitor, in combination with erlotinib in patients with EGFR TKI refractory advanced non-small cell lung cancer (NSCLC). METHODS: The pimary objectives were to evaluate the safety and tolerability of YM155 at escalating doses (3.6, 4.8, 6.0, and 8.0 mg/m2/days) administered every 3 weeks as continuous intravenous infusion over 168 h in combination with erlotinib at a fixed dose (150 mg, once a day). Secondary objectives were to assess the pharmacokinetics of YM155, antitumor activity, and the relationship between biomarkers and efficacy. The changes in survivin expression in biopsied tumor pre- and post-YM155 administration and serum cytokine levels were also analyzed. RESULTS: Fifteen patients were treated. The most common YM155-related adverse event was the presence of urine microalbumin, whereas grades 3/4 toxicities were rare. One patient who received 4.8  mg/m2/days YM155 developed a dose-limiting grade 2 serum creatinine elevation. YM155 exposure in plasma showed dose proportionality across all dose ranges tested. No pharmacokinetic interaction occurred between YM155 and erlotinib. The serum cytokines IL-8, G-CSF, and MIP-1b showed decreasing trends in patients who achieved progression-free survival of ≥ 12 weeks. Durable stable disease for ≥ 24 weeks was observed in two patients. CONCLUSION: Up to 8.0 mg/m2/days YM155 administered every 3 weeks in combination with erlotinib exhibited a favorable safety profile and moderate clinical efficacy. These results suggest that inhibiting survivin is a potential therapeutic strategy for select patients with EGFR TKI refractory NSCLC. TRIAL REGISTRATION: UMIN000031912 at UMIN Clinical Trials Registry (UMIN-CTR).
  • Hitomi Sakai; Masayuki Takeda; Kazuko Sakai; Kazuto Nishio; Kazuhiko Nakagawa
    Molecular and clinical oncology 13 2 175 - 178 2020年08月 [査読有り]
     
    Anorectal melanoma is a rare disease with a poor prognosis and its response to immunotherapy remains poorly studied. The current study reports a case of recurrent anorectal melanoma in a 60-year-old woman that has exhibited a durable response to ipilimumab for >2 years. Given that the combination of nivolumab and ipilimumab was not approved for use in unresectable or metastatic melanoma at the time of presentation, the patient was initially treated with nivolumab monotherapy and switched to ipilimumab after nivolumab failure. The tumor was microsatellite stable, had an intermediate tumor mutation burden and was negative for programmed cell death-ligand-1 expression. However, the neutrophil-to-lymphocyte ratio in peripheral blood remained at <5 throughout the disease course. Although mucosal melanoma is not caused by ultraviolet radiation and has a lower mutation burden than cutaneous melanoma, the present case responded well to immunotherapy. Further evaluation of potential biomarkers for such patients is required.
  • Naoshi Nishida; Kazuko Sakai; Masahiro Morita; Tomoko Aoki; Masahiro Takita; Satoru Hagiwara; Yoriaki Komeda; Mamoru Takenaka; Yasunori Minami; Hiroshi Ida; Kazuomi Ueshima; Kazuto Nishio; Masatoshi Kudo
    Liver cancer 9 4 426 - 439 2020年08月 [査読有り]
     
    Background and Aim: Immune checkpoint inhibitors are promising agents for the treatment of hepatocellular carcinomas (HCC) refractory to conventional therapies. To enhance the efficacy of this treatment, immunological and molecular characteristics of HCC with programmed cell death ligand 1 (PD-L1) should be explored. Methods: Clinical backgrounds, PD-L1 expression, and the amount of CD8+ tumor-infiltrating mononuclear cells (TIMCs) were analyzed in 154 HCCs. The expression of 3 stem cell markers and co-inhibitory receptors on tumor cells and TIMCs, respectively, were examined by immunohistochemical analysis. Somatic mutations in the 409 cancer-associated genes and TERT promoter were determined; HCCs were classified based on the presence of gene alterations affecting the 8 oncogenic pathways. The results were validated using the dataset from the Cancer Genome Atlas. Results: The expression of PD-L1 in the HCCs was positively correlated with progressive tumor features, the presence of cytokeratin 19 (CK19), Sal-like protein 4 (SALL4), and the mutations of genes involving the phosphatidyl inositol 3-kinase (PI3K)-Akt pathway. Although CD8+ cells were densely infiltrated in PD-L1-positive tumors, these TIMCs frequently expressed multiple co-inhibitory receptors. However, a subset of PD-L1-positive tumors characterized by activating mutations of the PI3K-Akt pathway showed a low degree of TIMCs. Conversely, PD-L1-negative HCCs were associated with mutations in the β-catenin pathway and a small number of TIMCs, although the expression of co-inhibitory receptors was rare. Conclusions: PD-L1-positive HCCs frequently showed an inflamed phenotype with stem cell features; a subset of PD-L1-positive HCCs with mutations in the PI3K-Akt pathway showed a non-inflamed phenotype. In HCCs with dense infiltration of TIMCs, CD8+ cells expressed multiple co-inhibitory receptors, suggesting T cell exhaustion. On the other hand, PD-L1-negative HCCs showed mutations leading to β-catenin activation and exhibited a non-inflamed background. These characteristics should be taken into consideration for developing novel combination therapies using immune checkpoint inhibitors.
  • Tomoko Noguchi; Kazuko Sakai; Naoyuki Iwahashi; Kaho Matsuda; Hitomi Matsukawa; Tamaki Yahata; Saori Toujima; Kazuto Nishio; Kazuhiko Ino
    Oncology letters 19 4 2713 - 2720 2020年04月 [査読有り]
     
    Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) is a novel ultrasensitive next-generation sequencing-based approach that is used to detect circulating tumor DNA (ctDNA). The aim of the present study was to compare the gene mutation profiles and blood tumor mutation burden (bTMB) measured between pre- and post-neoadjuvant chemotherapy (NAC), utilizing CAPP-seq for plasma ctDNA in patients with advanced ovarian cancer. The current study included 10 patients (6 NAC-sensitive and 4 NAC-resistant) clinically diagnosed as having stage III or IV ovarian cancer and were administered NAC between May 2017 and February 2019. The plasma ctDNA samples were collected at pre- and post-NAC, and comprehensive gene mutation analysis was performed using CAPP-seq. In 5 out of 6 NAC-sensitive cases, the variant allele frequency (VAF) of non-synonymous somatic mutations decreased following NAC. In 2 out of the 4 NAC-resistant cases, the VAF of non-synonymous somatic mutations increased, and new somatic mutations emerged following NAC. In regard to TP53 mutation, the rate of TP53 mutation in the NAC-resistant cases was significantly higher compared with NAC-sensitive cases. Finally, the bTMB decreased significantly after NAC treatment in the NAC-sensitive cases, even though there were no significant differences in the pretreatment bTMB levels between the NAC-sensitive and NAC-resistant cases. These results indicated that gene mutation can be profiled and monitored using liquid biopsy-based CAPP-Seq in patients with advanced ovarian cancer with NAC treatment, and TP53 mutation in the ctDNA and bTMB may be novel biomarkers that can be used for patient monitoring during NAC treatment.
  • Takayuki Takahama; Koichi Azuma; Mototsugu Shimokawa; Masayuki Takeda; Hidenobu Ishii; Terufumi Kato; Haruhiro Saito; Haruko Daga; Yuko Tsuboguchi; Isamu Okamoto; Kohei Otsubo; Hiroaki Akamatsu; Shunsuke Teraoka; Toshiaki Takahashi; Akira Ono; Tatsuo Ohira; Toshihide Yokoyama; Kazuko Sakai; Nobuyuki Yamamoto; Kazuto Nishio; Kazuhiko Nakagawa
    Cancer 126 9 1940 - 1948 2020年02月 [査読有り]
     
    BACKGROUND: Liquid biopsy allows the identification of patients whose tumors harbor specific mutations in a minimally invasive manner. No prospective data have been available for the efficacy of osimertinib in patients with non-small cell lung cancer (NSCLC) who develop resistance to first- or second-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and who test positive for the TKI resistance-conferring T790M mutation of EGFR by liquid biopsy. Therefore, a phase 2 study was conducted to assess the efficacy and safety of osimertinib in such patients. METHODS: Eligible patients had advanced or recurrent NSCLC with known TKI-sensitizing mutations of EGFR, had documented disease progression after treatment with at least 1 first- or second-generation EGFR TKI, and were positive for the T790M mutation in plasma according to the Cobas EGFR Mutation Test v2 (Roche Diagnostics) or droplet digital polymerase chain reaction analysis. Patients were treated with osimertinib (80 mg/d) until disease progression. The primary endpoint was the overall response rate (ORR) in patients positive for T790M in plasma by the Cobas assay. RESULTS: Between June 2016 and November 2017, 276 patients were screened for their T790M status with a liquid biopsy. Seventy-four patients were positive for T790M in plasma, and 53 of these individuals were enrolled in the study. The ORR for evaluable patients positive for T790M in plasma by the Cobas assay (n = 49) was 55.1% (95% confidence interval [CI], 40.2%-69.3%). The median progression-free survival for all evaluable patients (n = 52) was 8.3 months (95% CI, 6.9-12.6 months). CONCLUSIONS: The results demonstrate the utility of liquid biopsy for the detection of T790M with the Cobas EGFR Mutation Test v2. Plasma genotyping with this assay is informative for treatment selection in clinical practice when tumor sampling is not feasible.
  • Hisamitsu Takaya; Hidekatsu Nakai; Kazuko Sakai; Kazuto Nishio; Kosuke Murakami; Masaki Mandai; Noriomi Matsumura
    Gynecologic oncology 156 2 415 - 422 2020年02月 [査読有り]
     
    OBJECTIVE: High-grade serous ovarian cancers (HGSOC) are genomically characterized by homologous recombination deficiency (HRD) and TP53 mutations, which lead to intratumor heterogeneity (ITH). This study aimed to reveal the relationship between HRD, ITH and prognosis and analyze their changes during treatment. METHODS: We obtained 573 SNP array and gene expression array data from The Cancer Genome Atlas. SNP array data were processed to calculate the Clonality Index (CI) and loss of heterozygosity (LOH) scores. Gene expression array data were used for classifying molecular subtypes. Additionally, we obtained 33 samples from 20 HGSOC patients, including 4 samples from interval debulking surgery (IDS) and 9 samples from recurrent surgery. RESULTS: We divided HGSOC samples into 2 groups. The high CI group showed a high recurrent risk, and the high LOH group showed a statistically good prognosis. Combining the two factors, the high LOH/low CI group showed a statistically good prognosis. In terms of molecular subtypes, the mesenchymal subtype, which had a poor prognosis, showed a high CI with statisitically significant difference and the immunoreactive subtype, which had a good prognosis, showed a tendency to have a high LOH score. Throughout treatment, the CI decreased to one at the IDS (n = 4) and then increased at recurrence (n = 3). LOH scores greatly decreased in two cases at the IDS. CONCLUSIONS: ITH and HRD were associated with prognosis in HGSOC. ITH decreased after neoadjuvant chemotherapy, suggesting that the chemo-resistant cancer clone remains after chemotherapy.
  • Hidenobu Ishii; Koichi Azuma; Kazuko Sakai; Yoshiko Naito; Norikazu Matsuo; Takaaki Tokito; Kazuhiko Yamada; Tomoaki Hoshino; Kazuto Nishio
    Scientific reports 10 1 691 - 691 2020年01月 [査読有り]
     
    Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) were developed to target the EGFR T790M resistance mutation in non-small cell lung cancer (NSCLC) patients resistant to first- or second-generation EGFR-TKIs. To investigate the efficacy of afatinib treatment for EGFR T790M-positive NSCLC patients showing resistance to osimertinib and alterations in somatic mutations and tumor mutation burden (TMB) in plasma circulating tumor DNA (ctDNA) during afatinib treatment, we conducted a prospective study using Cancer Personalized Profiling by deep Sequencing (CAPP-Seq). Nine NSCLC patients with EGFR T790M mutation who showed resistance to third-generation EGFR-TKIs were enrolled in this study and treated with afatinib. Plasma samples were collected before treatment, 4 weeks after treatment, and at disease progression. The mutation profile and TMB in plasma ctDNA were analyzed by CAPP-Seq. The objective response rate and median progression-free survival associated with afatinib were 0% and 2.0 months, respectively. The C797S mutation-mediated resistance to osimertinib was observed in one patient and following afatinib treatment in two patients; the C797S mutations occurred in the same allele as the T790M mutation. After afatinib treatment, afatinib-sensitive mutant alleles, such as ERBB2, and TMB decreased. We have demonstrated that detection of mutant allele frequency and TMB of ctDNA by CAPP-Seq could help determine the effectiveness of and resistance to afatinib. Although afatinib monotherapy for T790M-positive NSCLC resistant to osimertinib was less effective, the action for multiclonal mutant alleles and TMB might contribute to further treatment strategy.
  • Masayuki Takeda; Kazuko Sakai; Hidetoshi Hayashi; Kaoru Tanaka; Koji Haratani; Takayuki Takahama; Ryoji Kato; Kimio Yonesaka; Kazuto Nishio; Kazuhiko Nakagawa
    Lung cancer (Amsterdam, Netherlands) 139 28 - 34 2020年01月 [査読有り]
     
    OBJECTIVES: The T790M secondary mutation of epidermal growth factor receptor gene (EGFR) is the most common mechanism of acquired resistance to first- or second-generation EGFR tyrosine kinase inhibitors (TKIs). We investigated the association between gene mutation profile in EGFR mutation-positive non-small cell lung cancer (NSCLC) before EGFR-TKI treatment and T790M status after EGFR-TKI treatment. MATERIALS AND METHODS: A total of 57 EGFR mutation-positive NSCLC patients who had undergone a repeat biopsy (tissue or liquid) after failure of treatment with a first- or second-generation EGFR-TKI and who had sufficient tumor tissue available from before treatment for genetic analysis was enrolled. The gene mutation profile of tumor tissue obtained before EGFR-TKI treatment was evaluated by next-generation sequencing with a comprehensive cancer gene panel (409 genes). The number of potentially damaging nonsynonymous mutations was predicted with PolyPhen-2 software. RESULTS: Progression-free survival during EGFR-TKI treatment did not differ significantly between patients who developed T790M-mediated resistance and those who developed T790M-independent resistance. The predicted number of damaging nonsynonymous mutations in pretreatment tumor tissue was significantly lower in patients who developed T790M-mediated resistance than in those with T790M-independent resistance (P =  0.049). CONCLUSIONS: Coexisting mutations in tumor tissue before EGFR-TKI treatment may contribute to the emergence of cell clones responsible for development of T790M-dependent or T790M-independent TKI resistance in patients with EGFR-mutated NSCLC. Multiplex genomic testing of pretreatment tumor tissue may thus provide a means of identifying patients likely to develop T790M-mediated TKI resistance and therefore inform treatment selection.
  • Kimio Yonesaka; Eiji Iwama; Hidetoshi Hayashi; Shinichiro Suzuki; Ryoji Kato; Satomi Watanabe; Takayuki Takahama; Junko Tanizaki; Kaoru Tanaka; Masayuki Takeda; Kazuko Sakai; Koichi Azuma; Yasutaka Chiba; Shinji Atagi; Kazuto Nishio; Isamu Okamoto; Kazuhiko Nakagawa
    Scientific reports 9 1 19501 - 19501 2019年12月 [査読有り]
     
    Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are standard therapy for EGFR-mutant non-small cell lung cancer (NSCLC). Preclinically, HER3 ligand heregulin induces resistance to EGFR-TKIs, whereas the pan-human EGFR family inhibitor afatinib remains effective. Here, we examined whether soluble heregulin levels have clinical implications for EGFR-mutant NSCLC treated with EGFR-TKIs. Soluble heregulin was immunologically measured in plasma from EGFR-mutant NSCLC patients. Cutoff values were determined by 1-year PFS ROC curve. The relationship between soluble heregulin and PFS following EGFR-TKI therapy was analyzed by Cox proportional hazards model. Seventy-three patients were enrolled: 44 were treated with 1st-generation and 29 with 2nd-generation EGFR-TKIs. Soluble heregulin levels varied (range: 274-7,138 pg/mL, median: 739 pg/mL). Among patients treated with 1st-generation EGFR-TKIs, those with high heregulin (n = 20, >800 pg/mL) had a tendency for shorter PFS than those with low heregulin (n = 24, <800 pg/mL), with median PFS of 322 and 671 days, respectively. Cox proportional hazards model also indicated a trend toward resistance against 1st-generation EGFR-TKIs (HR: 1.825, 95% CI: 0.865-4.318) but not against 2nd-generation EGFR-TKIs. Soluble heregulin potentially correlates with resistance to EGFR-TKIs but not 2nd-generation EGFR-TKIs in patients with EGFR-mutant NSCLC.
  • Satomi Watanabe; Tomoyuki Otani; Tsutomu Iwasa; Takayuki Takahama; Masayuki Takeda; Kazuko Sakai; Kazuto Nishio; Akihiko Ito; Kazuhiko Nakagawa
    Clinical breast cancer 19 5 e589-e592  2019年10月 [査読有り]
  • Kimio Yonesaka; Kaoru Tanaka; Mutsukazu Kitano; Hisato Kawakami; Hidetoshi Hayashi; Masayuki Takeda; Kazuko Sakai; Kazuto Nishio; Katsumi Doi; Kazuhiko Nakagawa
    Oncogenesis 8 10 54 - 54 2019年09月 [査読有り]
     
    The anti-epidermal growth factor receptor (EGFR) antibody cetuximab is standard therapy for head and neck squamous cell carcinoma (HNSCC). However, most HNSCC tumors are resistant to it and require alternative treatments. Here, we explored the mechanism of cetuximab resistance and evaluated its clinical relevance in HNSCC. An unbiased comprehensive transcriptome analysis was performed on cetuximab-resistant HNSCC FaDuCR cells. The causative resistance genome was knocked down with siRNA, cell signaling was immunologically analyzed, and drug efficacy was evaluated in vitro and in vivo. The mRNA in situ hybridization (ISH) of the causative genome was performed using 28 excised HNSCC tumors and its relationship with cetuximab efficacy was analyzed. FaDuCR cells were resistant to cetuximab, whereas parental FaDu cells were susceptible to it. FaDuCR cells expressed consistently higher levels of phosphorylated Akt than FaDu cells despite cetuximab exposure. A comprehensive transcriptome analysis revealed that the HER3-ligand heregulin was upregulated in FaDuCR cells compared to FaDu cells. Heregulin knockdown in FaDuCR cells repressed HER3 and Akt phosphorylation and recovered cetuximab anticancer efficacy. In contrast, pan-HER family tyrosine kinase inhibitors such as afatinib decreased HER3 and Akt phosphorylation in FaDuCR cells and inhibited FaDuCR tumor growth. Two of the 28 HNSCC tumor samples presented aberrant heregulin expression comparable to that of FaDuCR cells and were resistant to cetuximab therapy. In HNSCC, heregulin-mediated HER3-Akt activation causes resistance to cetuximab but not to second-generation EGFR-tyrosine kinase inhibitors. Subpopulations with aberrant heregulin-expressing HNSCC might be resistant to cetuximab.
  • Marco A De Velasco; Yurie Kura; Kazuko Sakai; Yuji Hatanaka; Barry R Davies; Hayley Campbell; Stephanie Klein; Youngsoo Kim; A Robert MacLeod; Koichi Sugimoto; Kazuhiro Yoshikawa; Kazuto Nishio; Hirotsugu Uemura
    JCI insight 4 17 2019年09月 [査読有り]
     
    Sustained therapeutic responses from traditional and next-generation antiandrogen therapies remain elusive in clinical practice due to inherent and/or acquired resistance resulting in persistent androgen receptor (AR) activity. Antisense oligonucleotides (ASO) have the ability to block target gene expression and associated protein products and provide an alternate treatment strategy for castration-resistant prostate cancer (CRPC). We demonstrate the efficacy and therapeutic potential of this approach with a Generation-2.5 ASO targeting the mouse AR in genetically engineered models of prostate cancer. Furthermore, reciprocal feedback between AR and PI3K/AKT signaling was circumvented using a combination approach of AR-ASO therapy with the potent pan-AKT inhibitor, AZD5363. This treatment strategy effectively improved treatment responses and prolonged survival in a clinically relevant mouse model of advanced CRPC. Thus, our data provide preclinical evidence to support a combination strategy of next-generation ASOs targeting AR in combination with AKT inhibition as a potentially beneficial treatment approach for CRPC.
  • Iwama E; Sakai K; Hidaka N; Inoue K; Fujii A; Nakagaki N; Ota K; Toyozawa R; Azuma K; Nakatomi K; Harada T; Hisasue J; Sakata S; Shimose T; Kishimoto J; Nakanishi Y; Nishio K; Okamoto I
    Cancer 126 1 219 - 227 2019年09月 [査読有り]
     
    BACKGROUND: The aim of this study was to evaluate the potential of liquid biopsy for prediction of the efficacy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment and for assessment of the changes in genetic alterations during such treatment. METHODS: Plasma samples were prospectively collected from non-small cell lung cancer patients with EGFR-activating mutations during EGFR-TKI treatment until disease progression and were analyzed for such mutations with droplet digital polymerase chain reaction and for other somatic alterations with next-generation sequencing. RESULTS: One hundred patients, including 87 who were EGFR-TKI naïve, were enrolled. Median progression-free survival was significantly shorter for EGFR-TKI-naïve patients with EGFR-activating mutations detected in plasma at baseline than for those without them (7.9 vs 19.0 months; P < .001), with the values being significantly longer for initially positive patients who became negative for these mutations at 12 or 24 weeks than for those who remained positive. An increase in the number of alleles positive for EGFR-activating mutations in plasma during treatment was associated with disease progression, with a hazard ratio of 4.72 (95% CI, 2.07-10.79; P < .001) for EGFR-TKI-naïve patients showing an increase within 36 weeks. For 55 patients with available samples, the total number of somatic alterations (other than activating mutations or T790M of EGFR) in plasma was higher at disease progression than at baseline (33 vs 19; P = 0.04). CONCLUSION: Liquid biopsy shows potential for prediction of EGFR-TKI efficacy and elucidation of clonal tumor evolution during targeted therapy.
  • Haratani K; Hayashi H; Takahama T; Nakamura Y; Tomida S; Yoshida T; Chiba Y; Sawada T; Sakai K; Fujita Y; Togashi Y; Tanizaki J; Kawakami H; Ito A; Nishio K; Nakagawa K
    Journal for immunotherapy of cancer 7 1 251  2019年09月 [査読有り]
  • Kazuko Sakai; Masayuki Takeda; Shigeki Shimizu; Takayuki Takahama; Takeshi Yoshida; Satomi Watanabe; Tsutomu Iwasa; Kimio Yonesaka; Shinichiro Suzuki; Hidetoshi Hayashi; Hisato Kawakami; Yoshikane Nonagase; Kaoru Tanaka; Junji Tsurutani; Kazumasa Saigoh; Akihiko Ito; Tetsuya Mitsudomi; Kazuhiko Nakagawa; Kazuto Nishio
    Scientific reports 9 1 11340 - 11340 2019年08月 [査読有り]
     
    Medical oncologists are challenged to personalize medicine with scientific evidence, drug approvals, and treatment guidelines based on sequencing of clinical samples using next generation sequencer (NGS). Knowledge-based curation systems have the potential to help address this challenge. We report here the results of examining the level of evidence regarding treatment approval and clinical trials between recommendations made by Watson for Genomics (WfG), QIAGEN Clinical Insight Interpret (QCII), and Oncomine knowledge-based reporter (OKR). The tumor samples obtained from the solid cancer patients between May to June 2018 at Kindai University Hospital. The formalin-fixed paraffin-embedded tumor samples (n = 31) were sequenced using Oncomine Comprehensive Assay v3. Variants including copy number alteration and gene fusions identified by the Ion reporter software were used commonly on three curation systems. Curation process of data were provided for 25 solid cancers using three curation systems independently. Concordance and distribution of curated evidence levels of variants were analyzed. As a result of sequencing analysis, nonsynonymous mutation (n = 58), gene fusion (n = 2) or copy number variants (n = 12) were detected in 25 cases, and subsequently subjected to knowledge-based curation systems (WfG, OKR, and QCII). The number of curated information in any systems was 51/72 variants. Concordance of evidence levels was 65.3% between WfG and OKR, 56.9% between WfG and QCII, and 66.7% between OKR and QCII. WfG provided great number of clinical trials for the variants. The annotation of resistance information was also observed. Larger differences were observed in clinical trial matching which could be due to differences in the filtering process among three curation systems. This study demonstrates knowledge-based curation systems (WfG, OKR, and QCII) could be helpful tool for solid cancer treatment decision making. Difference in non-concordant evidence levels was observed between three curation systems, especially in the information of clinical trials. This point will be improved by standardized filtering procedure and enriched database of clinical trials in Japan.
  • Naoyuki Iwahashi; Kazuko Sakai; Tomoko Noguchi; Tamaki Yahata; Hitomi Matsukawa; Saori Toujima; Kazuto Nishio; Kazuhiko Ino
    Scientific reports 9 1 10426 - 10426 2019年07月 [査読有り]
     
    Liquid biopsies of circulating tumor DNA (ctDNA) have recently been used as a non-invasive diagnostic tool for detecting tumor-specific mutations. We present a study of ctDNA liquid biopsies in gynecological cancer using an ultrasensitive next-generation sequencing-based method for ctDNA detection named CAncer Personalized Profiling by deep Sequencing (CAPP-Seq). We performed CAPP-Seq with plasma-ctDNA obtained from 16 patients with gynecological cancer. In all cases, at least one non-synonymous somatic mutation was detected in the ctDNA. In the pre-treatment ctDNA, 4 of 16, 4/16, 5/16, 2/16, 2/16, and 2/16 patients had TP53, KRAS, APC, PIK3CA, BRCA1, and EGFR mutations, respectively. MET gene copy-number gains were detected in the ctDNA of 2 of 16 patients, and FISH analysis of the paired tumor samples confirmed these results. In 2 neoadjuvant chemotherapy-treated ovarian cancer patients, the changes in gene mutation patterns were associated with the treatment response. These findings suggest that CAPP-Seq-based liquid biopsies can be used for the genetic characterization of independent gynecological cancers with high frequency, and might be clinically useful for non-invasive tumor genotyping and therapeutic response monitoring.
  • Kitazono S; Sakai K; Yanagitani N; Ariyasu R; Yoshizawa T; Dotsu Y; Koyama J; Saiki M; Sonoda T; Nishikawa S; Uchibori K; Horiike A; Nishio K; Nishio M
    Cancer science 110 10 3350 - 3357 2019年07月 [査読有り]
     
    Most patients with epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC) will inevitably develop acquired resistance induced by treatment with EGFR tyrosine kinase inhibitors (EGFR-TKI). The mechanisms of resistance to EGFR-TKI are multifactorial, and the detection of these mechanisms is critical for treatment choices in patients who have progressed after EGFR-TKI therapy. We evaluated the feasibility of a molecular barcode method using next-generation sequencing to detect multifactorial resistance mechanisms in circulating tumor DNA and compared the results with those obtained using other technologies. Plasma samples were collected from 25 EGFR mutation-positive NSCLC patients after the development of EGFR-TKI resistance. Somatic mutation profiles of these samples were assessed using two methods of next-generation sequencing and droplet digital PCR (ddPCR). The positive rate for EGFR-sensitizing mutations was 18/25 (72.0%) using ddPCR, 17/25 (68.0%) using amplicon sequencing, and 19/25 (76.0%) using molecular barcode sequencing. Rate of the EGFR T790M resistance mutation among patients with EGFR-sensitizing mutations was shown to be 7/18 (38.9%) using ddPCR, 6/17 (35.3%) using amplicon sequencing, and 8/19 (42.1%) using molecular barcode sequencing. Copy number gain in the MET gene was detected in three cases using ddPCR. PIK3CA, KRAS and TP53 mutations were detected using amplicon sequencing. Molecular barcode sequencing detected PIK3CA, TP53, KRAS, and MAP2K1 mutations. Results of the three assays were comparable; however, in cell-free DNA, molecular barcode sequencing detected mutations causing multifactorial resistance more sensitively than did the other assays.
  • Kazuko Sakai; Tatsuo Ohira; Jun Matsubayashi; Azusa Yoneshige; Akihiko Ito; Tetsuya Mitsudomi; Toshitaka Nagao; Emi Iwamatsu; Jin Katayama; Norihiko Ikeda; Kazuto Nishio
    Cancer science 110 6 2044 - 2049 2019年06月 [査読有り]
     
    Gene fusions play an important role in the carcinogenesis of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for precision medicine. We used formalin-fixed, paraffin-embedded tissue samples of non-small cell lung cancer from 150 EGFR mutation-negative cases and 10 fusion status-known cases and compared the performance of the Oncomine Dx Fusion Transcript Test (ODxFT) with FISH break-apart for the detection of ALK, RET, and ROS1 fusion genes. RNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining, and the ALK fusion gene was independently determined using these assays. Fusion detection analyses were successfully carried out using ODxFT in 150 cases, with only one invalid case. ALK fusion genes were detected at a frequency of 7.3% (11/150) in the lung cancer specimens. Concordance rate between the ODxFT and ALK-FISH analyses was 99.3% (148/149). Sensitivity and specificity were 91.7% and 99.3%, respectively. All the samples with a known fusion status were accurately matched between the two assays. Our results show a high concordance rate between the ODxFT and ALK-FISH analyses. ODxFT was thus validated as an effective method for detecting clinically significant ALK fusion genes in paraffin-embedded tissue samples.
  • Kunimasa K; Nakamura H; Sakai K; Tamiya M; Kimura M; Inoue T; Nishino K; Kuhara H; Nakatsuka SI; Nishio K; Imamura F; Kumagai T
    Lung cancer (Amsterdam, Netherlands) 132 59 - 64 2019年06月 [査読有り]
     
    OBJECTIVES: SMARCA4-deficient thoracic sarcoma(DTS) is a recently identified new entity of thoracic malignancies characterized by inactivation of SMARCA4. Patients with SMARCA4-DTS have a particulary aggresive clinical course and no effective treatments. However, the detailed clinical features of SMARCA4-DTS remain unclear. Here, we report the clinical courses and molecular profiles of two cases of SMARCA4-DTS. MATERIALS AND METHODS: We experienced strikingly similar two patients of SMARCA4-DTS. The clinicopathologic features were reviewed, and detailed immunohistochemical and comprehensive cancer panel analysis with next generation sequencing confirmed the diagnosis. RESULTS: Our cases had many clinical and radiological observations characteristic of SMARCA4-DTS in common. Immunohistochemical staing showed complete loss of SMARCA4 in tumor cells. Loss of function mutations were detected in SMARCA4. We found that severe SREs comprise a new significant clinical feature of SMARCA4-DTS. CONCLUSION: Integrated clinico-radiologic-pathologic-genetic diagnosis is essential for SMARCA4-DTS and physicians should pay attention to severe SREs during the clinical course of this disease.
  • Masayuki Takeda; Kazuko Sakai; Takayuki Takahama; Kazuya Fukuoka; Kazuhiko Nakagawa; Kazuto Nishio
    Cancers 11 6 2019年05月 [査読有り]
     
    : Recent progress in understanding the molecular basis of cancer-including the discovery of cancer-associated genes such as oncogenes and tumor suppressor genes-has suggested that cancer can become a treatable disease. The identification of driver oncogenes such as EGFR, ALK, ROS1, BRAF and HER2 has already been successfully translated into clinical practice for individuals with solid tumor. Next-generation sequencing (NGS) technologies have led to the ability to test for multiple cancer-related genes at once with a small amount of cells and tissues. In Japan, several hospitals have started NGS-based mutational profiling screening in patients with solid tumor in order to guide patients to relevant clinical trials. The Ministry of Health, Labor, and Welfare of Japan has also approved several cancer gene panels for use in clinical practice. However, there is an urgent need to develop a medical curriculum of clinical variant interpretation and reporting. We review recent progress in the implementation of NGS in Japan.
  • Otsubo K; Sakai K; Takeshita M; Harada D; Azuma K; Ota K; Akamatsu H; Goto K; Horiike A; Kurata T; Nakagaki N; Nosaki K; Iwama E; Nakanishi Y; Nishio K; Okamoto I
    The oncologist 24 8 1022 - 1026 2019年04月 [査読有り]
     
    Patients with non-small cell lung cancer (NSCLC) treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) eventually acquire resistance to these drugs. The identification of various resistance mechanisms for determination of subsequent treatment for these patients will require a method for simultaneous detection of multiple genetic alterations with high sensitivity. We performed cancer personalized profiling by deep sequencing (CAPP-Seq) with circulating tumor DNA obtained from patients with NSCLC who acquired resistance to first- or second-generation EGFR-TKIs. Plasma samples from 27 patients were analyzed, and 24 samples underwent CAPP-Seq successfully. Original activating EGFR mutations were detected in 23 patients, with the remaining patient showing MET amplification. With regard to known mechanisms of EGFR-TKI resistance, the T790M mutation of EGFR was detected in 17 of the 24 patients, MET amplification in 9 patients (6 of whom also harbored T790M), ERBB2 amplification in 2 patients (1 of whom also harbored T790M), and EGFR amplification in 4 patients (all of whom harbored T790M). Our results thus show that CAPP-Seq is applicable to clinical samples for the identification of multiple somatic mutations in circulating tumor DNA obtained from patients with NSCLC at the time of disease progression during treatment with first- or second-generation EGFR-TKIs. Patients positive for the T790M mutation of EGFR were also found to constitute a molecularly heterogeneous population. KEY POINTS: CAPP-Seq is applicable to clinical samples for the identification of multiple somatic mutations.The T790M mutation of EGFR is associated with amplification of MET, ERBB2, or EGFR in NSCLC patients resistant to EGFR-TKIs.T790M-positive patients are molecularly heterogeneous, and genetic alterations coexisting with T790M may differ between patients treated with first-generation or second-generation EGFR-TKIs.
  • Yumi Aoyama; Kazuko Sakai; Taiichi Kodaka; Hiroko Tsunemine; Kazuto Nishio; Tomoo Itoh; Daichi Inoue; Takayuki Takahashi
    Journal of clinical and experimental hematopathology : JCEH 59 1 29 - 33 2019年03月 [査読有り]
     
    Myelodysplastic/myeloproliferative neoplasm (MDS/MPN) with ring sideroblasts and thrombocytosis (MDS/MPN with RS-T), which exhibits both an increased number of marrow ring sideroblasts and thrombocytosis, is a rare disorder classified as one of the newly established forms of MDS/MPN in the WHO 2016 classification. A 77-year-old female with marked thrombocytosis of 1,024×109/L was tentatively diagnosed with essential thrombocythemia in 2011, and the thrombocytosis was controlled using hydroxycarbamide and low-dose busulfan. In 2016, the leukocyte count increased to a peak value of 68.8×109/L (86.6% mature neutrophils) during platelet-reduction therapy. Bone marrow aspirate exhibited hypercellularity with ring sideroblasts comprising 41.5% erythroblasts without excess myeloblasts. Cytogenetic examination demonstrated the JAK2 V617F mutation and chromosomal abnormality of 46,XX,del(20)(q1?). Furthermore, dysplastic features of erythroid and granuloid precursors, as well as many large atypical megakaryocytes, were observed. Further genetic examinations revealed the SF3B1 K700E mutation, but not amplification of the JAK2 gene or pathogenic mutations in the 13 other genes examined. A diagnosis of MDS/MPN with RS-T was established and hyperleukocytosis was controlled using a higher dose of hydroxycarbamide. Although the patient maintained a stable disease state, she became RBC transfusion-dependent. Hyperleukocytosis, regardless of chemotherapy, is rare and may be novel in this disorder.
  • Yoshihiko Fujita; Masataka Taguri; Kentaro Yamazaki; Junji Tsurutani; Kazuko Sakai; Takahiro Tsushima; Michitaka Nagase; Hiroshi Tamagawa; Shinya Ueda; Takao Tamura; Yasushi Tsuji; Kohei Murata; Koichi Taira; Tadamichi Denda; Toshikazu Moriwaki; Sadao Funai; Takako Eguchi Nakajima; Kei Muro; Akihito Tsuji; Motoki Yoshida; Koichi Suyama; Takuya Kurimoto; Naotoshi Sugimoto; Eishi Baba; Nobuhiko Seki; Mikio Sato; Takaya Shimura; Narikazu Boku; Ichinosuke Hyodo; Takeharu Yamanaka; Kazuto Nishio
    The oncologist 24 3 327 - 337 2019年03月 [査読有り]
     
    BACKGROUND: The randomized phase III study (WJOG4407G) showed equivalent efficacy between FOLFOX and FOLFIRI in combination with bevacizumab as the first-line treatment for metastatic colorectal cancer (mCRC). We studied whole genome copy number profiles using array-based comparative genomic hybridization (aCGH) analysis of tumor tissue samples obtained in this study. The aim of this study was to identify gene copy number alterations that could aid in selecting either FOLFOX or FOLFIRI in combination with bevacizumab for patients with mCRC. MATERIALS AND METHODS: DNA was purified from 154 pretreatment formalin-fixed paraffin-embedded tissue samples (75 from the FOLFOX arm and 79 from the FOLFIRI arm) of 395 patients enrolled in the WJOG4407G trial and analyzed by aCGH. Genomic regions greater than 1.2-fold were regarded as copy number gain (CNG). RESULTS: Patient characteristics between the treatment arms were well balanced except for tumor laterality (left side; 64% in FOLFOX arm and 80% in FOLFIRI arm, p = .07). FOLFIRI showed a trend toward better response rate (RR), progression-free survival (PFS) and overall survival (OS) than FOLFOX in the patients with CNG of chromosome 8q24.1 (Fisher's exact test, p = .134 for RR; interaction test, p = .102 for PFS and p = .003 for OS) and 8q24.2 (Fisher's exact test, p = .179 for RR; interaction test, p = .144 for PFS and p = .002 for OS). CONCLUSION: Chromosome 8q24.1-q24.2 may contain genes that could potentially serve as predictive markers for selecting either FOLFOX or FOLFIRI in combination with bevacizumab for treatment of patients with mCRC. IMPLICATIONS FOR PRACTICE: Bevacizumab has been used as a standard first-line treatment for patients with metastatic colorectal cancer (mCRC) in combination with either oxaliplatin-based or irinotecan-based chemotherapy. Until now, there has been no predictive marker to choose between the two combination chemotherapies. This array-based comparative genomic hybridization analysis revealed that the difference in therapeutic effect between the two combination chemotherapies is prominent in patients with mCRC with gene copy number gain in chromosome 8p24.1-p24.2. Such patients showed more favorable response and survival when treated with irinotecan-based combination chemotherapy. Overlapping genes commonly found in this region may be predictive biomarkers of the efficacy of the combination chemotherapy with bevacizumab.
  • Hayashi H; Kurata T; Takiguchi Y; Arai M; Takeda K; Akiyoshi K; Matsumoto K; Onoe T; Mukai H; Matsubara N; Minami H; Toyoda M; Onozawa Y; Ono A; Fujita Y; Sakai K; Koh Y; Takeuchi A; Ohashi Y; Nishio K; Nakagawa K
    Journal of clinical oncology : official journal of the American Society of Clinical Oncology 37 7 570 - 579 2019年03月 [査読有り]
     
    PURPOSE Although gene expression profiling is a promising diagnostic technique to determine the tissue of origin for patients with cancer of unknown primary site (CUP), no clinical trial has evaluated yet site-specific therapy directed by this approach compared with empirical chemotherapy. We therefore performed a randomized study to assess whether such site-specific therapy improves outcome compared with empirical chemotherapy in previously untreated patients with CUP. PATIENTS AND METHODS Comprehensive gene expression profiling was performed by microarray analysis, and an established algorithm was applied to predict tumor origin. Patients with CUP were randomly assigned (1:1) to receive standard site-specific therapy or empirical paclitaxel and carboplatin (PC). The primary end point was 1-year survival rate. RESULTS One hundred thirty patients were randomly assigned and had sufficient biopsy tissue for molecular analysis. Efficacy analysis was performed for 50 and 51 patients in the site-specific therapy and empirical PC arms, respectively. Cancer types most commonly predicted were pancreatic (21%), gastric (21%), and lymphoma (20%). The 1-year survival rate was 44.0% and 54.9% for site-specific treatment and empirical PC ( P = .264), respectively. Median overall and progression-free survival were 9.8 and 5.1 months, respectively, for site-specific treatment versus 12.5 and 4.8 months for empirical PC ( P = .896 and .550, respectively). Median overall survival (16.7 v 10.6 months; P = .116) and progression-free survival (5.5 v 3.9 months; P = .018) were better for predicted more-responsive than less-responsive tumor types. CONCLUSION Site-specific treatment that was based on microarray profiling did not result in a significant improvement in 1-year survival compared with empirical PC, although prediction of the original site seemed to be of prognostic value.
  • Yonesaka K; Takegawa N; Watanabe S; Haratani K; Kawakami H; Sakai K; Chiba Y; Maeda N; Kagari T; Hirotani K; Nishio K; Nakagawa K
    Oncogene 38 9 1398 - 1409 2019年02月 [査読有り]
     
    EGFR tyrosine kinase inhibitors (TKIs) are standard therapy for EGFR-mutant non-small cell lung cancer (NSCLC); however, these tumours eventually acquire chemoresistance. U3-1402 is an anti-HER3 antibody-drug conjugate with a novel topoisomerase I inhibitor, DXd. In the current study, we evaluated the anticancer efficacy of U3-1402 in EGFR-mutant NSCLC cells with acquired resistance to EGFR-TKIs. HCC827GR5 and PC9AZDR7 are EGFR-TKI-resistant clones for gefitinib and osimertinib, respectively. U3-1402 alone or in combination with the EGFR-TKI erlotinib demonstrated potent anticancer efficacy in HCC827GR5 cells using an in vitro growth inhibition assay and in vivo xenograft mouse model. U3-1402 induced apoptosis in HCC827GR5 cells accompanying phosphorylation of histone H2A.X, a marker of DNA damage, but did not block HER3/PI3K/AKT signalling. Further, we found using flow cytometry that the cell surface HER3 expression level in HCC827GR5 cells was twice that found in HCC827 cells, indicating internalization of U3-1402 was increased in resistant cells. In addition, administration of U3-1402 notably repressed growth of EGFR-TKI osimertinib-resistant PC9AZDR7 xenograft tumours, and that PC9AZDR7 cells expressed five times greater cell surface HER3 than PC9 cells. Furthermore, using immunofluorescent microscopy, HER3 was observed predominantly in the nucleus of PC9 cells, but was localized in the cytoplasm of PC9AZDR7 cells. This finding indicates that altered trafficking of the HER3-U3-1402 complex may accelerate linker payload cleavage by cytoplasmic lysosomal enzymes, resulting in DNA damage. Our results indicate that administration of U3-1402 alone or in combination with an EGFR-TKI may have potential as a novel therapy for EGFR-TKI-resistant EGFR-mutant NSCLC.
  • Takeda M; Sakai K; Nishio K; Nakagawa K
    OncoTargets and therapy 12 5355 - 5358 2019年 [査読有り]
     
    The discovery of RET rearrangement in non-small cell lung cancer (NSCLC) has prompted development of molecularly targeted therapy for such tumors, with several clinical trials being under way to evaluate the therapeutic effects of multitargeted tyrosine kinase inhibitors. The sensitivity of RET fusion-positive NSCLC to cytotoxic chemotherapy has remained unclear, however. We here report a case of NSCLC positive for the CCDC6-RET fusion gene that benefited from treatment with pemetrexed over a period of 30 months, suggesting that thymidylate synthase-targeted drugs such as pemetrexed may show efficacy for NSCLC harboring RET fusions.
  • Satomi Watanabe; Hidetoshi Hayashi; Koji Haratani; Shigeki Shimizu; Junko Tanizaki; Kazuko Sakai; Hisato Kawakami; Kimio Yonesaka; Junji Tsurutani; Yosuke Togashi; Kazuto Nishio; Akihiko Ito; Kazuhiko Nakagawa
    Cancer science 110 1 52 - 60 2019年01月 [査読有り]
     
    The efficacy of programmed cell death-1 (PD-1) blockade in patients with non-small cell lung cancer (NSCLC) positive for epidermal growth factor receptor (EGFR) gene mutations has been found to be limited, but the underlying mechanisms for this poor response have remained obscure. Given that the recognition by T cells of tumor antigens presented by major histocompatibility complex class I (MHC-I) molecules is essential for an antitumor immune response, we examined the effects of EGFR tyrosine kinase inhibitors (TKIs) on MHC-I expression in NSCLC cell lines. Appropriate EGFR-TKIs increased MHC-I expression at the mRNA and cell surface protein levels in NSCLC cells positive for EGFR mutations including those with the T790M secondary mutation. Trametinib, an inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK, also increased MHC-I expression, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor buparlisib did not, suggesting that the MEK-ERK pathway mediates the down-regulation of MHC-I expression in response to EGFR activation. Immunohistochemical analysis of EGFR-mutated NSCLC specimens obtained before and after EGFR-TKI treatment also revealed down-regulation of phosphorylated forms of EGFR and ERK in association with up-regulation of MHC-I, an increased number of infiltrating CD8+ T cells, and increased PD-1 ligand 1 expression after such treatment. Our results thus suggest that mutational activation of EGFR inhibits MHC-I expression through the MEK-ERK pathway in NSCLC and thereby contributes to the poor response of such tumors to immunotherapy. Further studies are warranted to evaluate the relation between EGFR-MEK-ERK signaling in and the immune response to EGFR-mutated NSCLC. .
  • Sakai H; Takeda M; Sakai K; Nakamura Y; Ito A; Hayashi H; Tanaka K; Nishio K; Nakagawa K
    Lung cancer (Amsterdam, Netherlands) 127 59 - 65 2019年01月 [査読有り]
     
    OBJECTIVES: Immune-checkpoint inhibitors (ICIs) are now an established therapeutic option for advanced non-small cell lung cancer (NSCLC). It has remained unclear, however, whether cytotoxic chemotherapy affects the immune microenvironment in NSCLC wild type for EGFR and ALK. MATERIALS AND METHODS: We retrospectively evaluated changes in programmed cell death 1-ligand 1 (PD-L1) expression, tumor mutation burden (TMB), and CD8+ tumor-infiltrating lymphocyte (TIL) density in NSCLC patients who underwent rebiopsy at the site of recurrence after postoperative platinum-based adjuvant chemotherapy, or in those who underwent rebiopsy after one or more chemotherapeutic regimens at the advanced stage. The PD-L1 tumor proportion score (TPS) and CD8+ TIL density were determined by immunohistochemistry. TMB was estimated by next-generation sequencing with a cancer gene panel (409 genes). RESULTS: Seventeen patients with NSCLC wild type for EGFR and ALK were enrolled. Although PD-L1 TPS tended to be increased in rebiopsy samples compared with initial biopsy tissue, this difference was not significant (P =  0.113). Seven patients showed an increase in PD-L1 TPS, with this change being pronounced in four. Two cases in which PD-L1 TPS increased from 0 to 90% or from 0 to 95% after cytotoxic chemotherapy also showed a durable response to subsequent treatment with an ICI. No substantial correlation between PD-L1 TPS and TMB was apparent either before (R = 0.112) or after (R = 0.101) chemotherapy. A moderate correlation was detected between PD-L1 TPS and CD8+ TIL density before chemotherapy (R = 0.517) and a negligible correlation after (R = 0.0219). CONCLUSION: Cytotoxic chemotherapy may change the biological characteristics of tumors including PD-L1 expression level and TMB.
  • Iwama E; Sakai K; Azuma K; Harada D; Nosaki K; Hotta K; Nishio M; Kurata T; Fukuhara T; Akamatsu H; Goto K; Shimose T; Kishimoto J; Nakanishi Y; Nishio K; Okamoto I
    Cancer science 109 12 3921 - 3933 2018年12月 [査読有り]
     
    Liquid biopsy offers a potential alternative to tissue biopsy for detection of genetic alterations in cancer, and it has been introduced into clinical practice to detect the tyrosine kinase inhibitor (TKI) resistance-conferring T790M mutation of epidermal growth factor receptor (EGFR) in patients with non-small-cell lung cancer (NSCLC). We prospectively collected tumor and plasma samples from 25 NSCLC patients who harbored activating mutations of EGFR and experienced failure of treatment with afatinib. The samples were analyzed by digital PCR (dPCR) and next-generation sequencing (NGS). T790M was detected in plasma with a respective sensitivity and specificity of 83.3% and 70.0% by dPCR and 50.0% and 70.0% by NGS relative to analysis of corresponding tumor samples. Quantitation of T790M based on the ratio of the number of T790M alleles to that of activating mutation alleles (T/A ratio) improved the specificity of plasma analysis to 100% for both dPCR and NGS without a reduction in sensitivity. Although several afatinib resistance mechanisms other than T790M-including copy number gain of NRAS or MET-were identified in tumor samples, the corresponding genetic alterations were not detected in plasma. TP53 mutations were frequently identified in plasma and tumor samples, with most such mutations also having been detected before afatinib treatment. The presence of de novo TP53 mutations was associated with reduced progression-free survival. Quantitation of T790M in plasma is thus a clinically relevant approach to determine the T790M status of tumors. In addition, genetic alterations coexisting with EGFR mutations can affect the efficacy of EGFR-TKI treatment.
  • Masayoshi Ishida; Naoyuki Kawao; Kiyotaka Okada; Kohei Tatsumi; Kazuko Sakai; Kazuto Nishio; Hiroshi Kaji
    Endocrinology 159 11 3775 - 3790 2018年11月 [査読有り]
     
    It is well known that sex differences exist concerning the severity of osteoporosis and bone metabolism, suggesting that factors other than sex hormones might be responsible for sex differences of bone metabolism. We therefore examined sex differences of osteoblast phenotypes of mouse osteoblasts and then performed comparative gene expression analyses using a comprehensive DNA microarray between female and male osteoblasts. Alkaline phosphatase (ALP) activity, mineralization, and the expression of Osterix, ALP, and bone sialoprotein were significantly lower in mouse female osteoblasts compared with male osteoblasts. We identified Serpina3n, a novel serine protease inhibitor, as the gene whose expression has the highest ratio of females to males. A reduction in endogenous levels of Serpina3n by small interfering RNA significantly enhanced the mRNA levels of Runx2, ALP, osteocalcin, and type I collagen (Col1a1) in both male and female osteoblasts. Moreover, Serpina3n overexpression significantly suppressed the mRNA levels of Osterix, ALP, osteocalcin, and Col1a1 in MC3T3-E1 cells. Serpina3n overexpression did not affect Osterix, ALP, and osteocalcin mRNA levels enhanced by bone morphogenetic protein (BMP)-2 in ST2 cells, adipogenic differentiation in ST2 and 3T3-L1 cells, and receptor activator of nuclear factor κB ligand-induced osteoclast formation in RAW264.7 cells, although it significantly suppressed mineralization in ST2 cells differentiated into osteoblasts by BMP-2. In conclusion, we found Serpina3n as the most female osteoblast-dominant gene. Serpina3n exerts a suppression of the osteoblast phenotypes such as Col1a1 expression and ALP activity in differentiated osteoblasts, which might partly explain sex differences of the osteoblast phenotypes in mice.
  • Naoyuki Iwahashi; Kazuko Sakai; Tomoko Noguchi; Tamaki Yahata; Saori Toujima; Kazuto Nishio; Kazuhiko Ino
    Oncology letters 16 5 6431 - 6436 2018年11月 [査読有り]
     
    Liquid biopsies of circulating tumor DNA (ctDNA) can detect molecular alterations, including tumor-specific mutations, and have recently been used as a non-invasive diagnostic, prognostic, and predictive tool. However, this technique is not commonly used in the gynecological field. Gene mutation profiling of liquid biopsy samples was performed using CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), a novel next-generation sequencing-based approach to ultrasensitive ctDNA detection, in order to make it possible to molecularly diagnose metastatic colorectal cancer to the ovary. Liquid biopsy (plasma) samples and formalin-fixed paraffin-embedded tumor samples were obtained from two patients with ovarian tumors, who had a history of surgery for colorectal cancer, and comprehensive gene mutation profiling was conducted using CAPP-Seq. In patient 1, mutations were identified in the same three regions in both the ovarian tumor and preoperative plasma sample (in the KRAS G13D, APC E1306*, and TP53 H193Y genes). In patient 2, mutation was identified in the same one region in all the primary colorectal tumor, the ovarian tumor, and preoperative plasma sample (in APC R216* gene). These mutations are well-known genetic signatures of colorectal cancer, suggesting that the ovarian tumor was metastatic. Tthe gene mutation patterns of colorectal cancer were examined by subjecting liquid biopsy samples from patients with suspected metastatic ovarian tumors to CAPP-Seq. Gene mutation profiling of liquid biopsy samples can contribute to the preoperative differential diagnosis of metastatic ovarian cancer and its subsequent personalized treatment.
  • K. Kunimasa; H. Nakamura; K. Sakai; M. Kimura; T. Inoue; M. Tamiya; K. Nishino; T. Kumagai; S. Nakatsuka; H. Endo; M. Inoue; K. Nishio; F. Imamura
    Annals of Oncology 29 10 2145 - 2147 2018年10月 [査読有り]
  • Sunami K; Takahashi H; Tsuchihara K; Takeda M; Suzuki T; Naito Y; Sakai K; Dosaka-Akita H; Ishioka C; Kodera Y; Muto M; Wakai T; Yamazaki K; Yasui W; Bando H; Fujimoto Y; Fukuoka S; Harano K; Kawazoe A; Kimura G; Koganemaru S; Kogawa T; Kotani D; Kuboki Y; Matsumoto H; Matsumoto S; Mishima S; Nakamura Y; Sawada K; Shingaki S; Shitara K; Umemoto K; Umemura S; Yasuda K; Yoshino T; Yamamoto N; Nishio K; Japanese Society of Medical Oncology; Japan Society of Clinical Oncology; Japanese Cancer Association
    Cancer science 109 9 2980 - 2985 2018年09月 [査読有り]
     
    In Japan, the social (medical) health-care system is on the way to being developed to advance personalized medicine through the implementation of cancer genomic medicine, known as cancer clinical sequencing, which uses a next-generation sequencer. However, no Japanese guidance for cancer genomic testing exists. Gene panel testing can be carried out to help determine patient treatment, confirm diagnosis, and evaluate prognostic predictions of patients with mainly solid cancers for whom no standard treatment is available. This guidance describes how to utilize gene panel testing according to the type of cancer: childhood cancer, rare cancer, carcinoma of unknown primary, and other cancers. The level of evidence classification for unified use in Japan is also detailed. This guidance establishes the basic principles of the quality control of specimens, requirements of medical institutions, informed consent, handling of data during the postanalysis stage, and treatment options based on the evidence level. In Japan, gene panel testing for cancer treatment and diagnosis is recommended to comply with this guidance. This is a collaborative work of the Japanese Society of Medical Oncology, Japan Society of Clinical Oncology, and the Japanese Cancer Association.
  • Cecily P Vaughn; José Luis Costa; Harriet E Feilotter; Rosella Petraroli; Varun Bagai; Anna Maria Rachiglio; Federica Zito Marino; Bastiaan Tops; Henriette M Kurth; Kazuko Sakai; Andrea Mafficini; Roy R L Bastien; Anne Reiman; Delphine Le Corre; Alexander Boag; Susan Crocker; Michel Bihl; Astrid Hirschmann; Aldo Scarpa; José Carlos Machado; Hélène Blons; Orla Sheils; Kelli Bramlett; Marjolijn J L Ligtenberg; Ian A Cree; Nicola Normanno; Kazuto Nishio; Pierre Laurent-Puig
    BMC cancer 18 1 828 - 828 2018年08月 [査読有り]
     
    BACKGROUND: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. METHODS: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. RESULTS: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. CONCLUSION: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.
  • Toshimi Takano; Junji Tsurutani; Masato Takahashi; Takeharu Yamanaka; Kazuko Sakai; Yoshinori Ito; Junya Fukuoka; Hideharu Kimura; Hidetaka Kawabata; Kenji Tamura; Koji Matsumoto; Kenjiro Aogi; Kazuhiko Sato; Kazuto Nishio; Kazuhiko Nakagawa; Toshiaki Saeki
    Breast (Edinburgh, Scotland) 40 67 - 75 2018年08月 [査読有り]
     
    BACKGROUND: For human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC) with progression on trastuzumab-based therapy, continuing trastuzumab beyond progression and switching to lapatinib combined with chemotherapy are both valid options. We conducted an open-label, randomized phase II trial to compare the efficacy of these strategies. PATIENTS AND METHODS: Women with HER2-positive MBC previously treated with trastuzumab and taxanes were randomly assigned to receive trastuzumab plus capecitabine (HX) or lapatinib plus capecitabine (LX). The primary endpoint was progression-free survival (PFS) and the secondary endpoints included overall survival (OS) and the objective response rate (ORR). To explore the predictive value of the differential benefit of anti-HER2 drugs, PIK3CA mutations were assessed using circulating tumor DNA. RESULTS: Eighty-six patients (43 in each arm) were enrolled. The median PFS was 6.1 months in the HX arm and 7.1 months in the LX arm (hazard ratio, 0.81; 90% CI, 0.55-1.21; p = 0.39); the median OS was 31.0 months in the HX arm and was not reached in the LX arm (hazard ratio, 0.58; 95% CI, 0.26-1.31; p = 0.18). The ORR was 40% in the HX arm and 41% in the LX arm. PIK3CA mutations were detected in 23% of the 35 analyzed patients, and in patients without PIK3CA mutations, LX yielded relatively longer PFS and OS than HX. CONCLUSION: In women with HER2-positive MBC previously treated with trastuzumab and taxanes, no significant differences in PFS and OS were observed between patients treated with LX and HX. TRIAL REGISTRATION NUMBER: UMIN000005219.
  • Kimio Yonesaka; Koji Haratani; Shiki Takamura; Hitomi Sakai; Ryoji Kato; Naoki Takegawa; Takayuki Takahama; Kaoru Tanaka; Hidetoshi Hayashi; Masayuki Takeda; Sigeki Kato; Osamu Maenishi; Kazuko Sakai; Yasutaka Chiba; Takafumi Okabe; Keita Kudo; Yoshikazu Hasegawa; Hiroyasu Kaneda; Michiko Yamato; Kenji Hirotani; Masaaki Miyazawa; Kazuto Nishio; Kazuhiko Nakagawa
    Clinical Cancer Research 24 11 2653 - 2664 2018年06月 [査読有り]
  • Naoki Oiso; Kazuko Sakai; Shigeto Yanagihara; Kazuto Nishio; Akira Kawada
    European journal of dermatology : EJD 28 3 414 - 415 2018年06月 [査読有り]
  • Yosuke Makuuchi; Hidetoshi Hayashi; Koji Haratani; Junko Tanizaki; Kaoru Tanaka; Masayuki Takeda; Kazuko Sakai; Shigeki Shimizu; Akihiko Ito; Kazuto Nishio; Kazuhiko Nakagawa
    Oncotarget 9 33 23315 - 23319 2018年05月 [査読有り]
     
    The second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) alectinib and ceritinib are standard treatment options for patients with non-small cell lung cancer (NSCLC) positive for ALK fusion genes. However, almost all patients eventually develop resistance to these drugs. We here report a case of ALK-rearranged NSCLC that developed resistance to alectinib but remained sensitive to ceritinib. The L1196M mutation within the ALK fusion gene was detected after failure of consecutive treatment with crizotinib and alectinib, but no other mechanism underlying acquired resistance to ALK-TKIs was found to be operative. Given the increasing application of ALK-TKIs to the treatment of patients with ALK-rearranged NSCLC, further clinical evaluation is warranted to provide a better understanding of the mechanisms of acquired resistance to these agents and to inform treatment strategies for such tumors harboring secondary mutations.
  • Hitomi Sakai; Junji Tsurutani; Tsutomu Iwasa; Yoshifumi Komoike; Kazuko Sakai; Kazuto Nishio; Kazuhiko Nakagawa
    Breast Cancer 25 5 1 - 9 2018年04月 [査読有り]
     
    Background: Trastuzumab emtansine (T-DM1) is approved for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive advanced breast cancer (ABC), and has high efficacy. However, some patients exhibit primary resistance to T-DM1, and thus methods that can predict resistance in clinical practice are needed. Genomic analysis of circulating tumor DNA (ctDNA) in plasma is a non-invasive and reproducible method. This study aimed to predict primary resistance to T-DM1 by combining genomic analysis of ctDNA and other clinicopathological features of patients with HER2-positive ABC. Methods: The study population comprised 34 patients with HER2-positive ABC who had been treated with T-DM1. Correlations between clinicopathological characteristics of patients and primary resistance to T-DM1 were examined, and HER2 gene copy number and PIK3CA gene mutations were analyzed using plasma ctDNA samples obtained from 16 patients before T-DM1 administration. Results: Among the 34 patients, nine (26.5%) had progressive disease at the first efficacy analysis these patients were considered to have primary resistance to T-DM1. No significant difference was found in the rate of primary resistance to T-DM1 between groups. Among 16 patients whose ctDNA was analyzed, four showed primary resistance to T-DM1. These four patients showed negative HER2 gene amplification in ctDNA and were ER-positive and/or PR-positive by immunohistochemistry. Conclusions: HER2 gene amplification in ctDNA and ER and PR status may predict primary resistance to T-DM1. A liquid biopsy before the initiation of T-DM1 treatment could be a non-invasive way to predict whether a patient would exhibit primary resistance to T-DM1.
  • Masayuki Takeda; Kazuko Sakai; Hidetoshi Hayashi; Kaoru Tanaka; Junko Tanizaki; Takayuki Takahama; Koji Haratani; Kazuto Nishio; Kazuhiko Nakagawa
    Oncotarget 9 30 21132 - 21140 2018年04月 [査読有り]
     
    Unlike common epidermal growth factor receptor gene (EGFR) mutations that confer sensitivity to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC), mutations in exon 20 of either EGFR or the human EGFR2 gene (HER2) are associated with insensitivity to EGFR-TKIs, with treatment options for patients with such mutations being limited. Clinical characteristics, outcome of EGFR-TKI or nivolumab treatment, and the presence of coexisting mutations were reviewed for NSCLC patients with exon-20 mutations of EGFR or HER2 as detected by routine application of an amplicon-based next-generation sequencing panel. Between July 2013 and June 2017, 206 patients with pathologically confirmed lung cancer were screened for genetic alterations including HER2 and EGFR mutations. Ten patients harbored HER2 exon-20 insertions (one of whom also carried an exon-19 deletion of EGFR), and 12 patients harbored EGFR exon-20 mutations. Five of the 13 patients with EGFR mutations were treated with EGFR-TKIs, two of whom manifested a partial response, two stable disease, and one progressive disease. Among the seven patients treated with nivolumab, one patient manifested a partial response, three stable disease, and three progressive disease, with most (86%) of these patients discontinuing treatment as a result of disease progression within 4 months. The H1047R mutation of PIK3CA detected in one patient was the only actionable mutation coexisting with the exon-20 mutations of EGFR or HER2. Potentially actionable mutations thus rarely coexist with exon-20 mutations of EGFR or HER2, and EGFR-TKIs and nivolumab show limited efficacy in patients with such exon-20 mutations.
  • Sugimoto, Kouichi; De Velasco, Marco A.; Kura, Yurie; Sakai, Kazuko; Nozawa, Masahiro; Yoshimura, Kazuhiro; Yoshikawa, Kazuhiro; Nishio, Kazuto; Uemura, Hirotsugu
    CANCER SCIENCE 109 357 - 357 2018年01月 [査読有り]
  • Naoki Oiso; Kazuko Sakai; Tomohiko Narita; Shigeto Yanagihara; Kazuto Nishio; Akira Kawada
    The Journal of dermatology 45 1 e5-e6  2018年01月 [査読有り]
  • Hidetoshi Hayashi; Yasutaka Chiba; Kazuko Sakai; Tomonobu Fujita; Hiroshige Yoshioka; Daisuke Sakai; Chiyoe Kitagawa; Tateaki Naito; Koji Takeda; Isamu Okamoto; Tetsuya Mitsudomi; Yutaka Kawakami; Kazuto Nishio; Shinichiro Nakamura; Nobuyuki Yamamoto; Kazuhiko Nakagawa
    CLINICAL LUNG CANCER 18 6 719 - 723 2017年11月 [査読有り]
     
    Antibodies to programmed cell death-1 (PD-1), such as nivolumab, have shown promising clinical activity in patients with advanced non-small-cell lung cancer (NSCLC), but their efficacy appears to be less pronounced in patients with such tumors harboring epidermal growth factor receptor gene (EGFR) mutations. Recent findings suggest that patients with EGFR mutation-positive NSCLC who develop resistance to tyrosine kinase inhibitors (TKIs) due to mechanisms other than acquisition of the secondary T790M mutation of EGFR are more likely to benefit from nivolumab treatment, possibly as a result of a higher level of expression of the PD-1 ligand PD-L1, than are patients who are T790M-positive. The WJOG8515L study (UMIN ID: 000021133) is a randomized phase II trial to compare nivolumab with the combination of carboplatin and pemetrexed in patients with EGFR mutation-positive nonsquamous NSCLC who have developed resistance to EGFR-TKIs due to mechanisms other than T790M. Eligible patients are those with stage IV or recurrent EGFR mutation-positive NSCLC who experience disease progression after therapy with more than 1 EGFR-TKI, including gefitinib, erlotinib, or afatinib; they must show no evidence of the T790M mutation on analysis of a tumor biopsy specimen obtained after progression on such EGFR-TKI therapy, or, if T790M is detected, they must again experience progression on subsequent treatment with a third-generation EGFR-TKI. The primary endpoint is progression-free survival (PFS), and secondary end points include overall survival (OS), objective response rate, duration of response, safety, and OS and PFS according to PD-L1 expression level. Recruitment started in May 2016 and is ongoing.
  • Yasuhiro Taniguchi; Hirokazu Tanaka; Espinoza J. Luis; Kazuko Sakai; Takahiro Kumode; Keigo Sano; Kentarou Serizawa; Shinya Rai; Yasuyoshi Morita; Hitoshi Hanamoto; Kazuo Tsubaki; Kazuto Nishio; Itaru Matsumura
    INTERNATIONAL JOURNAL OF HEMATOLOGY 106 5 691 - 703 2017年11月 [査読有り]
     
    Myeloproliferative neoplasms (MPNs), including polycythemia vera and essential thrombocythemia, are frequently associated with thrombotic complications. Prevention of thrombotic events is thus a primary aim of the current treatment for these disorders. Although it is known that microparticles (MPs), which are small vesicles released from cell membranes and circulate in the blood, directly contribute to thrombosis via their procoagulant activity, potential associations between plasma levels of MPs and the risk of thrombotic events in MPNs have not been reported. In the present study, we characterized plasma levels of MPs and assessed their potential association with the occurrence of thrombotic events in 59 patients with MPNs. Plasma levels of procoagulant MPs expressing tissue factor (TF+ MPs) were significantly higher in patients suffering thrombotic events than in patients without such events (median/mu l plasma: 33.8 vs 47.2, p = 0.02). Among patients who developed thrombotic events, irrespective of patients' blood counts, TF+ MP were significantly higher in patients without cytoreductive therapy than in those receiving cytoreductive therapy (101.2 vs. 42.5, p < 0.001). These results suggest that elevated levels of TF+ MP may be considered as a novel surrogate marker for thrombotic events in MPN patients. Further studies are needed to clarify the mechanism involved.
  • Kazuto Nishio; Kazuko Sakai
    Japanese Journal of Cancer and Chemotherapy 44 10 813 - 816 2017年10月 [査読有り]
     
    Cancer Clinical Sequencing is a method for making the treatment decision for each cancer patient. Implication of the clinical sequencing is rapidly in progress in Japan. In general, the tumor FFPE samples obtained by biopsy or operatory resection are subjected to amplicon sequencing. The process was divided into pre-analysis, analysis, and post-analysis processes. Quality assurance and control are necessary for all process. In addition, the expert members for clinical sequencing team is essential to make clinical sequencing reports based on the NGS analysis.
  • Kazuko Sakai; Masayo Ukita; Jeanette Schmidt; Longyang Wu; Marco A. De Velasco; Alan Roter; Luis Jevons; Kazuto Nishio; Masaki Mandai
    CANCER LETTERS 405 22 - 28 2017年10月 [査読有り]
     
    Intratumoral heterogeneity of cancer cells remains largely unexplored. Here we investigated the composition of ovarian cancer and its biological relevance. A whole-genome single nucleotide polymorphism array was applied to detect the clonal composition of 24 formalin-fixed, paraffin-embedded samples of human ovarian cancer. Genome-wide segmentation data consisting of the log2 ratio (log2R) and B allele frequency (BAF) were used to calculate an estimate of the clonal composition number (CC number) for each tumor. Somatic mutation profiles of cancer-related genes were also determined for the same 24 samplesby next-generation sequencing. The CC number was estimated successfully for 23 of the 24 cancer samples. The mean +/- SD value for the CC number was 1.7 +/- 1.1 (range of 0-4). A somatic mutation in at least one gene was identified in 22 of the 24 ovarian cancer samples, with the mutations including those in the oncogenes KRAS (29.2%), PIK3CA (12.5%), BRAF (8.3%), FGFR2 (4.2%), andJAK2 (4.2%) as well as those in the tumor suppressor genes TP53 (54.2%), FBXW7 (8.3%), PTEN (4.2%), and RB1 (4.2%). Tumors with one or more oncogenic mutations had a significantly lower CC number than did those without such a mutation (1.0 +/- 0.8 versus 2.3 +/- 0.9, P = 0.0027), suggesting that cancers with driver oncogene mutations are less heterogeneous than those with other mutations. Our results thus reveal a reciprocal relation between oncogenic mutation status and clonal composition in ovarian cancer using the established method for the estimation of the CC number. (C) 2017 The Author(s). Published by Elsevier B.V.
  • Yuichi Murakami; Kahori Sonoda; Hideyuki Abe; Kosuke Watari; Daiki Kusakabe; Koichi Azuma; Akihiko Kawahara; Jun Akiba; Chitose Oneyama; Jonathan A. Pachter; Kazuko Sakai; Kazuto Nishio; Michihiko Kuwano; Mayumi Ono
    ONCOTARGET 8 41 70736 - 70751 2017年09月 [査読有り]
     
    Second- and third-generation inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase activity (EGFR-TKIs) are improving the treatment of patients with non-small cell lung cancer. Here we established two sublines (BR1-8 and BR2-3) resistant to a second-generation inhibitor, afatinib, from the human lung cancer cell line HCC827 that harbors a mutation that activates the tyrosine kinase activity of EGFR. These afatinib-resistant sublines were resistant to first-generation EGFR-TKIs, gefitinib and erlotinib, and a third-generation EGFR-TKI, osimertinib. These resistant sublines showed markedly reduced levels of multiple EGFR family proteins, including the activated mutant EGFR, and complete loss of EGFR amplification as compared with their parental HCC827 cells harboring amplification of EGFR gene. Treatment with the multikinase inhibitor dasatinib or transfection with a SRC small interfering RNA inhibited cell survival and AKT phosphorylation in drug-resistant sublines to a greater extent compared with HCC827 cells. Further, the migration of drug-resistant cells was greater compared with that of HCC827 cells and was inhibited by dasatinib or an FAK inhibitor. These findings indicate that compensatory activation of SRC family kinases (SFKs) and FAK supports the survival and migration of afatinib-resistant cells when the expression of multiple EGFR family proteins was mostly abrogated. Combinations of potent drugs that target SFKs and FAK may overcome the resistance of lung cancer cells to second-generation TKIs.
  • Emiko Udo; Bungo Furusato; Kazuko Sakai; Leah M. Prentice; Tomonori Tanaka; Yuka Kitamura; Tomoshi Tsuchiya; Naoya Yamasaki; Takeshi Nagayasu; Kazuto Nishio; Junya Fukuoka
    DIAGNOSTIC PATHOLOGY 12 1 62  2017年08月 [査読有り]
     
    Background: Ciliated muconodular papillary tumors (CMPTs) are newly recognized rare peripheral lung nodules that are histologically characterized by ciliated columnar, goblet, and basal cells. Although recent studies have shown that CMPTs constitute a neoplastic disease, the complete histogenesis of CMPTs is not fully understood and molecular data are limited. Methods: We reviewed four cases of CMPT and performed immunohistochemical and genomic analyses to establish CMPT profiles. Results: All cases were positive for hepatocyte nuclear factor-4 alpha and mucin 5B and negative for programmed death ligand 1 expression, as determined by immunohistochemistry. The genetic analysis revealed three pathogenic mutations (BRAF V600E, AKT1 E17K, and KRAS G12D), with the KRAS mutation reported here for the first time. Conclusion: Histological and genetic profiles indicate that CMPTs are likely neoplastic and exhibit features similar to mucinous adenocarcinoma. This suggests that some CMPTs may be a precursor lesion of mucinous adenocarcinoma.
  • K. Haratani; H. Hayashi; T. Tanaka; H. Kaneda; Y. Togashi; K. Sakai; K. Hayashi; S. Tomida; Y. Chiba; K. Yonesaka; Y. Nonagase; T. Takahama; J. Tanizaki; K. Tanaka; T. Yoshida; K. Tanimura; M. Takeda; H. Yoshioka; T. Ishida; T. Mitsudomi; K. Nishio; K. Nakagawa
    ANNALS OF ONCOLOGY 28 7 1532 - 1539 2017年07月 [査読有り]
     
    Background: The efficacy of programmed death-1 blockade in epidermal growth factor receptor gene (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) patients with different mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) is unknown. We retrospectively evaluated nivolumab efficacy and immune-related factors in such patients according to their status for the T790M resistance mutation of EGFR. Patients and methods: We identified 25 patients with EGFR mutation-positive NSCLC who were treated with nivolumab after disease progression during EGFR-TKI treatment (cohort A). Programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) density in tumor specimens obtained after acquisition of EGFR-TKI resistance were determined by immunohistochemistry. Whole-exome sequencing of tumor DNA was carried out to identify gene alterations. The relation of T790M status to PD-L1 expression or TIL density was also examined in an independent cohort of 60 patients (cohort B). Results: In cohort A, median progression-free survival (PFS) was 2.1 and 1.3 months for T790M-negative and T790M-positive patients, respectively (P = 0.099; hazard ratio of 0.48 with a 95% confidence interval of 0.20-1.24). Median PFS was 2.1 and 1.3 months for patients with a PD-L1 expression level of >= 1% or <1%, respectively (P = 0.084; hazard ratio of 0.37, 95% confidence interval of 0.10-1.21). PFS tended to increase as the PD-L1 expression level increased with cutoff values of >= 10% and >= 50%. The proportion of tumors with a PD-L1 level of >= 10% or >= 50% was higher among T790M-negative patients than among T790M-positive patients of both cohorts A and B. Nivolumab responders had a significantly higher CD8(+) TIL density and nonsynonymous mutation burden. Conclusion: T790M-negative patients with EGFR mutation-positive NSCLC are more likely to benefit from nivolumab after EGFR-TKI treatment, possibly as a result of a higher PD-L1 expression level, than are T790M-positive patients.
  • Masanobu Tsubaki; Tomoya Takeda; Toshiki Kino; Kazuko Sakai; Tatsuki Itoh; Motohiro Imano; Takashi Nakayama; Kazuto Nishio; Takao Satou; Shozo Nishida
    Oncotarget 8 24 38717 - 38730 2017年06月 [査読有り]
     
    Resistance to the breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitor (TKI) imatinib poses a major problem when treating chronic myeloid leukemia (CML). Imatinib resistance often results from a secondary mutation in BCR-ABL1. However, in the absence of a mutation in BCR-ABL1, the basis of BCR-ABL1-independent resistance must be elucidated. To gain insight into the mechanisms of BCR-ABL1-independent imatinib resistance, we performed an array-based comparative genomic hybridization. We identified various resistance-related genes, and focused on MET. Treatment with a MET inhibitor resensitized K562/IR cells to BCR-ABL1 TKIs. Combined treatment of K562/IR cells with imatinib and a MET inhibitor suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) activation, but did not affect AKT activation. Our findings implicate the MET/ERK and MET/JNK pathways in conferring resistance to imatinib, providing new insights into the mechanisms of BCR-ABL1 TKI resistance in CML.
  • Masayuki Takeda; Kazuko Sakai; Kazuhiko Nakagawa; Kazuto Nishio
    Transl Cancer Res 6 3 633 - 638 2017年06月 [査読有り]
     
    The development of targeted therapies for lung cancer based on the presence of corresponding specific biomarkers has highlighted the need for molecular diagnostic tests capable of the analysis of multiple actionable genetic alterations in a single tumor sample. Amplicon-based next-generation sequencing (NGS)- as opposed to conventional Sanger-based sequencing-has been introduced to facilitate the performance of multiple genomic tests with small amounts of tissue. In Japan, several institutions including the National Cancer Center and university hospitals have initiated NGS-based clinical testing for cancer patients and are able to provide access to investigational drugs or approved targeted agents matched to detected molecular alterations. However, no NGS system has yet been approved for the detection of somatic mutations by the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan. Further development of precision medicine in clinical practice in Japan will require changes to the medical curriculum to support the interpretation and annotation of NGS data. In this review, we introduce NGS-based clinical sequencing projects that are ongoing in Japan-in particular, those focusing on lung cancer-and we discuss issues relating to the integration of NGS into clinical practice.
  • Eri Banno; Yosuke Togashi; Marco A. De Velasco; Takuro Mizukami; Yu Nakamura; Masato Terashima; Kazuko Sakai; Yoshihiko Fujita; Ken Kamata; Masayuki Kitano; Masatoshi Kudo; Kazuto Nishio
    INTERNATIONAL JOURNAL OF ONCOLOGY 50 6 2049 - 2058 2017年06月 [査読有り]
     
    Akt2 is an isoform of Akt, and an association between Akt2 and resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been suggested in pancreatic cancer (PC) in vitro. In this study, we investigated the association between Akt2 expression as evaluated using immunohistochemistry and the outcome of patients with advanced PC who had received treatment with erlotinib (an EGFR-TKI). Although the difference was not significant, patients with high levels of Akt2 expression tended to have a poorer response and a shorter progression-free survival period after treatment with erlotinib plus gemcitabine than those with low expression levels (P=0.16 and 0.19, respectively). In vitro, an Akt2-amplified PC cell line and Akt2-overexpressed cell lines exhibited resistance to anti-EGFR therapies, including erlotinib, but combined treatment with BYL719 (a PI3K inhibitor) cancelled this resistance. Our findings suggest that Akt2 might be associated with the resistance to anti-EGFR therapies, especially the use of erlotinib against PC, and that this resistance can be overcome by combined treatment with a PI3K inhibitor. Akt2 expression could become a predictive biomarker for erlotinib resistance in PC.
  • Masato Chiba; Yosuke Togashi; Eri Bannno; Yoshihisa Kobayashi; Yu Nakamura; Hidetoshi Hayashi; Masato Terashima; Marco A. De Velasco; Kazuko Sakai; Yoshihiko Fujita; Tetsuya Mitsudomi; Kazuto Nishio
    BMC CANCER 17 1 281  2017年04月 [査読有り]
     
    Background: Non-small cell lung cancer (NSCLC) harboring common epidermal growth factor receptor (EGFR) gene mutations (exon 19 deletion or exon 21 L858R) respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The secondary T790 M mutation in exon 20 of the EGFR gene is the most common type of acquired resistance mutation. Several reports have also shown that other secondary mutations (L747S, D761Y and T854A), while uncommon, can induce acquired resistance to first-generation EGFR-TKIs. However, little is known about the anticancer activities of second-or third-generation EGFR-TKIs. Methods: Uncommon secondary mutations were introduced into Ba/F3 cells along with the sensitive EGFR L858R mutation (Ba/F3-L858R/L747S, Ba/F3-L858R/D761Y, and Ba/F3-L858R/T854A), and the sensitivities to various EGFR-TKIs were then investigated. Results: Both the Ba/F3-L858R/L747S and Ba/F3-L858R/D761Y cell lines exhibited weak resistances to first-generation reversible EGFR-TKIs, while the Ba/F3-L858R/T854A cell line exhibited a strong resistance. In contrast, irreversible EGFR-TKIs, especially third-generation EGFR-TKIs, were capable of overcoming these resistances. Western blot analyses demonstrated that gefitinib (first-generation) inhibited the phosphorylation of EGFR to a lesser extent in cells with these secondary mutations than in cells with the sensitive L858R mutation alone. In contrast, afatinib and osimertinib (second-and third-generation) inhibited the phosphorylation of EGFR in cells with these secondary mutations to a similar extent as that seen in cells with the sensitive L858R mutation alone. Conclusions: Our experimental findings suggest that irreversible EGFR-TKIs, especially third-generation EGFR-TKIs, can be effective against uncommon secondary mutations and that switching to third-generation EGFR-TKIs could be a promising treatment strategy for patients with acquired resistance because of these uncommon secondary mutations.
  • Naoki Oiso; Kazuko Sakai; Kazuto Nishio; Akira Kawada
    PIGMENT CELL & MELANOMA RESEARCH 30 2 269 - 272 2017年03月 [査読有り]
  • T. Mizukami; K. Sakai; S. Naruki; T. Taniyama; Y. Horie; N. Izawa; T. Tsuda; T. Fujino; N. Boku; H. Yasuda; T. Fukunaga; T. Eguchi Nakajima; K. Nishio
    ANNALS OF ONCOLOGY 28 2 437 - 438 2017年02月 [査読有り]
  • Mizukami T; Togashi Y; Naruki S; Banno E; Terashima M; de Velasco MA; Sakai K; Yoneshige A; Hayashi H; Fujita Y; Tomida S; Nakajima TE; Fujino T; Boku N; Ito A; Nakagawa K; Nishio K
    Mol Carcinog 56 1 106 - 117 Wiley 2017年01月 [査読有り]
  • E. Iwama; K. Sakai; K. Azuma; T. Harada; D. Harada; K. Nosaki; K. Hotta; F. Ohyanagi; T. Kurata; T. Fukuhara; H. Akamatsu; K. Goto; T. Shimose; J. Kishimoto; Y. Nakanishi; K. Nishio; I. Okamoto
    ANNALS OF ONCOLOGY 28 1 136 - 141 2017年01月 [査読有り]
     
    Background: Analysis of circulating cell-free DNA (cfDNA) is under intensive investigation for its potential to identify tumor somatic mutations. We have now explored the usefulness of such liquid biopsy testing with both the digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) during treatment of patients with the epidermal growth factor receptor (EGFR) inhibitor afatinib. Patients and methods: Eligible patients had advanced lung adenocarcinoma with EGFR activating mutations and were treated with afatinib. Plasma samples were collected before and during (4 and 24 weeks) afatinib treatment as well as at disease progression. Tumor and plasma DNA were analyzed by dPCR and NGS. Results: Thirty-five patients were enrolled. The objective response rate and median progression-free survival (PFS) were 77.1% and 13.8 months, respectively. Tumor and plasma DNA were available for 32 patients. dPCR and NGS detected EGFR activating mutations in 81.3% and 71.9% of baseline cfDNA samples, respectively. In 19 patients treated with afatinib for similar to 24 weeks, the number of EGFR mutant alleles detected in cfDNA by dPCR declined rapidly and markedly after treatment onset, becoming undetectable or detectable at only a low copy number (<10 copies per milliliter) at 4 weeks. Median PFS was slightly longer for patients with undetectable EGFR mutant alleles in cfDNA at 4 weeks than for those in whom such alleles were detectable (14.3 versus 10.0 months). A total of 45 somatic mutations was identified in baseline tumor DNA, and 30 (66.7%) of these mutations were identified in cfDNA by NGS. Allele frequency for somatic mutations in cfDNA determined by NGS changed concordantly during afatinib treatment with the number of EGFR mutant alleles determined by dPCR. Conclusions: Monitoring of cfDNA by dPCR is informative for prediction of afatinib efficacy, whereas that by NGS is reliable and has the potential to identify mechanisms of treatment resistance.
  • Takuro Mizukami; Yosuke Togashi; Saeko Naruki; Eri Banno; Masato Terashima; Marco A. de Velasco; Kazuko Sakai; Azusa Yoneshige; Hidetoshi Hayashi; Yoshihiko Fujita; Shuta Tomida; Takako Eguchi Nakajima; Takashi Fujino; Narikazu Boku; Akihiko Ito; Kazuhiko Nakagawa; Kazuto Nishio
    MOLECULAR CARCINOGENESIS 56 1 106 - 117 2017年01月 [査読有り]
     
    Although fibroblast growth factor (FGF) signals are strongly associated with malignancy, limited information is available regarding the role of the FGF9 signal in colorectal cancer (CRC). In this study, we investigated the frequency of FGF9 amplification in CRC clinical specimens and the association between the FGF9 gene and resistance to anti-EGFR therapies. In clinical samples, an FGF9 copy number gain of 5 copies was observed at a frequency of 8/145 (5.5%) and tended to be related to wild-type KRAS (7/96, 7.3%). Furthermore, FGF9 amplification was not observed in any of the samples from the 15 responders to anti-EGFR therapies but was observed in one sample from the seven non-responders with wild-type KRAS, and two samples from non-responders also had high FGF9 mRNA expression levels. FGF9 amplification was validated using a fluorescence in situ hybridization (FISH) analysis, and FGF9-amplified sections showed readily detectable signals originating from FGF9 protein when examined using immunohistochemistry. In both the in vitro and in vivo experiments using FGF9-overexpressing CRC cell lines, FGF9 overexpression induced strong resistance to anti-EGFR therapies via the enforced FGFR signal, and this resistance was cancelled by the application of an FGFR inhibitor. Considering these results, the FGF9 gene may play an important role in resistance to anti-EGFR therapies in patients with CRC, and such resistance might be overcome by combined treatment with an anti-FGFR inhibitor. These findings strongly encourage the development of FGFR-targeted therapy for CRC patients with FGF9 gene upregulation. (c) 2016 Wiley Periodicals, Inc.
  • Hiromichi Matsuoka; Hiroyasu Kaneda; Kazuko Sakai; Atsuko Koyama; Kazuto Nishio; Kazuhiko Nakagawa
    Clinical Lung Cancer 18 1 e85 - e87 2017年01月 [査読有り]
  • Kohei Shitara; Kimio Yonesaka; Tadamichi Denda; Kentaro Yamazaki; Toshikazu Moriwaki; Masahiro Tsuda; Toshimi Takano; Hiroyuki Okuda; Tomohiro Nishina; Kazuko Sakai; Kazuto Nishio; Shoji Tokunaga; Takeharu Yamanaka; Narikazu Boku; Ichinosuke Hyodo; Kei Muro
    CANCER SCIENCE 107 12 1843 - 1850 2016年12月 [査読有り]
     
    This randomized phase II trial compared panitumumab plus fluorouracil, leucovorin, and irinotecan (FOLFIRI) with bevacizumab plus FOLFIRI as second-line chemotherapy for wild-type (WT) KRAS exon 2 metastatic colorectal cancer (mCRC) and to explore the values of oncogenes in circulating tumor DNA (ctDNA) and serum proteins as predictive biomarkers. Patients with WT KRAS exon 2 mCRC refractory to first-line chemotherapy containing oxaliplatin and bevacizumab were randomly assigned to panitumumab plus FOLFIRI or bevacizumab plus FOLFIRI. Of 121 randomly assigned patients, 117 were eligible. Median overall survival (OS) for panitumumab plus FOLFIRI and bevacizumab plus FOLFIRI were 16.2 and 13.4months [hazard ratio (HR), 1.16; 95% CI, 0.76-1.77], respectively. Progression-free survival (PFS) was also similar (HR, 1.14; 95% CI, 0.78-1.66). KRAS, NRAS, and BRAF status using ctDNA was successfully examined in 109 patients, and mutations were identified in 19 patients (17.4%). Panitumumab plus FOLFIRI showed favorable survival compared with bevacizumab plus FOLFIRI in WT patients and unfavorable survival in those with mutations (P for interaction=0.026 in OS and 0.054 in PFS). OS with bevacizumab plus FOLFIRI was better than panitumumab plus FOLFIRI in patients with high serum vascular endothelial growth factor-A (VEGF-A) levels and worse in those with low levels (P for interaction=0.016). Second-line FOLFIRI plus panitumumab and FOLFIRI plus bevacizumab showed a similar efficacy in patients with WT KRAS exon 2 mCRC. RAS and BRAF mutation in ctDNA could be a negative predictive marker for panitumumab.
  • Masato Chiba; Yosuke Togashi; Shuta Tomida; Hiroshi Mizuuchi; Yu Nakamura; Eri Banno; Hidetoshi Hayashi; Masato Terashima; Marco A. De Velasc; Kazuko Sakai; Yoshihiko Fujita; Tetsuya Mitsudomi; Kazuto Nishio
    INTERNATIONAL JOURNAL OF ONCOLOGY 49 6 2236 - 2244 2016年12月 [査読有り]
     
    Several receptor tyrosine kinases (RTKs) including EGFR, ALK, and MET have been identified as therapeutic targets in non-small cell lung cancer (NSCLC). Among the downstream pathways of RTKs, the MAPK pathway is particularly important for cancer cell proliferation, differentiation, and survival. In this study, the effects of MEK inhibitors (trametinib and PD0325901) in several NSCLC cell lines with driver gene alterations, especially RTK genes, were tested in vitro using an MTT assay, and a wide range of sensitivities was found. In particular, all the EGFR-mutated cell lines were resistant to MEK inhibitors, whereas all the MET-amplified cell lines were sensitive. A bioinformatics technique and western blot analyses showed that the PI3K/AKT pathway is more activated in EGFRmutated NSCLC than in MET-amplified NSCLC, and a PI3K inhibitor enhanced the sensitivity to trametinib in the EGFR-mutated cell lines, suggesting that this pathway is associated with resistance to MEK inhibitors. Although the HCC827 cell line (EGFR mutation) was resistant to MEK inhibitors, the HCC827CNXR cell line, whose driver gene shifts from EGFR to MET, exhibited enhanced sensitivity to MEK inhibitors, indicating the biological importance of the MAPK pathway for MET-amplified NCSLC. Furthermore, a synergistic effect of crizotinib (a MET inhibitor) and trametinib was observed in MET-amplified NCLC cell lines. Our findings indicate that the MAPK pathway is biologically important for MET-amplified NSCLC and strongly encourage the development of combination therapy with a MET inhibitor and a MEK inhibitor against MET-amplified NSCLC.
  • Tatsuo Ohira; Kazuko Sakai; Jun Matsubayashi; Naohiro Kajiwara; Masatoshi Kakihana; Masaru Hagiwara; Masaaki Hibi; Koichi Yoshida; Junichi Maeda; Keishi Ohtani; Toshitaka Nagao; Kazuto Nishio; Norihiko Ikeda
    CANCER SCIENCE 107 11 1660 - 1666 2016年11月 [査読有り]
     
    Next-generation sequencing (NGS) and digital PCR technologies allow analysis of the mutational profile of circulating cell-free DNA (cfDNA) in individuals with advanced lung cancer. We have now evaluated the feasibility of cfDNA sequencing for mutation detection in patients with non-small cell lung cancer at earlier stages. A total of 150 matched tumor and serum samples were collected from non-small cell lung cancer patients at stages IA-IIIA. Amplicon sequencing with DNA extracted from tumor tissue detected frequent mutations in EGFR (37% of patients), TP53 (39%), and KRAS (10%), consistent with previous findings. In contrast, NGS of cfDNA identified only EGFR, TP53, and PIK3CA mutations in three, five, and one patient, respectively, even though adequate amounts of cfDNA were extracted (median of 4936 copies/mL serum). Next-generation sequencing showed a high accuracy (98.8%) compared with droplet digital PCR for cfDNA mutation detection, suggesting that the low frequency of mutations in cfDNA was not due to a low assay sensitivity. Whereas the yield of cfDNA did not differ among tumor stages, the cfDNA mutations were detected in seven patients at stages IIA-IIIA and at T2b or T3. Tumor volume was significantly higher in the cfDNA mutation-positive patients than in the negative patients at stages T2b-T4 (159.1 +/- 58.0 vs. 52.5 +/- 9.9cm(3), P=0.014). Our results thus suggest that tumor volume is a determinant of the feasibility of mutation detection with cfDNA as the analyte.
  • Masaaki Hibi; Hiroyasu Kaneda; Junko Tanizaki; Kazuko Sakai; Yosuke Togashi; Masato Terashima; Marco Antonio De Velasco; Yoshihiko Fujita; Eri Banno; Yu Nakamura; Masayuki Takeda; Akihiko Ito; Tetsuya Mitsudomi; Kazuhiko Nakagawa; Isamu Okamoto; Kazuto Nishio
    CANCER SCIENCE 107 11 1667 - 1676 2016年11月 [査読有り]
     
    Fibroblast growth factor receptor (FGFR) gene alterations are relatively frequent in lung squamous cell carcinoma (LSCC) and are a potential targets for therapy with FGFR inhibitors. However, little is known regarding the clinicopathologic features associated with FGFR alterations. The angiokinase inhibitor nintedanib has shown promising activity in clinical trials for non-small cell lung cancer. We have now applied next-generation sequencing (NGS) to characterize FGFR alterations in LSCC patients as well as examined the antitumor activity of nintedanib in LSCC cell lines positive for FGFR1 copy number gain (CNG). The effects of nintedanib on the proliferation of and FGFR signaling in LSCC cell lines were examined invitro, and its effects on tumor formation were examined invivo. A total of 75 clinical LSCC specimens were screened for FGFR alterations by NGS. Nintedanib inhibited the proliferation of FGFR1 CNG-positive LSCC cell lines in association with attenuation of the FGFR1-ERK signaling pathway invitro and invivo. FGFR1 CNG (10.7%), FGFR1 mutation (2.7%), FGFR2 mutation (2.7%), FGFR4 mutation (5.3%), and FGFR3 fusion (1.3%) were detected in LSCC specimens by NGS. Clinicopathologic features did not differ between LSCC patients positive or negative for FGFR alterations. However, among the 36 patients with disease recurrence after surgery, prognosis was significantly worse for those harboring FGFR alterations. Screening for FGFR alterations by NGS warrants further study as a means to identify patients with LSCC recurrence after surgery who might benefit from nintedanib therapy.
  • Junko Tanizaki; Eri Banno; Yosuke Togashi; Hidetoshi Hayashi; Kazuko Sakai; Masayuki Takeda; Hiroyasu Kaneda; Kazuto Nishio; Kazuhiko Nakagawa
    LUNG CANCER 101 11 - 15 2016年11月 [査読有り]
     
    Comprehensive genomic profiling for non-small cell lung cancer (NSCLC) is likely to identify more patients with rare genetic alterations including uncommon epidermal growth factor receptor gene (EGFR) mutations. It remains unclear how such patients should be treated, however. We here report a case of NSCLC positive for two uncommon mutations of EGFR and a KRAS mutation, including its treatment with the second-generation EGFR tyrosine kinase inhibitor (TKI) afatinib. Tumor specimen obtained by a NSCLC patient with no smoking history was analyzed by next-generation sequencing. Comprehensive genomic profiling revealed that the patient harbored the EGFR mutations G719C and S768I as well as the E49K mutation of KRAS. Treatment with afatinib was clinically effective as confirmed by PET-CT scans of bone metastases and by a marked decrease in the serum concentration of carcinoembryonic antigen. Afatinib was the most effective among seven EGFR-TKIs tested in inhibiting the growth of Ba/F3 cells expressing EGFR(S768I), showing an efficacy similar to that apparent with cells expressing the common EGFR mutant L858R, whereas first- and third-generation EGFR-TKIs were markedly less effective against EGFR(S7681) than against EGFR(L858R). These data suggest that EGFR-TKIs differ in their activity toward cells expressing EGFR(S7681) in vitro. Consistently, afatinib was clinically effective for the treatment of NSCLC harboring G719C and S768I mutations of EGFR. Further studies are warranted to determine the most appropriate EGFR-TKI for treatment of NSCLC harboring uncommon EGFR mutations. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
  • Norikazu Matsuo; Koichi Azuma; Kazuko Sakai; Satoshi Hattori; Akihiko Kawahara; Hidenobu Ishii; Takaaki Tokito; Takashi Kinoshita; Kazuhiko Yamada; Kazuto Nishio; Tomoaki Hoshino
    SCIENTIFIC REPORTS 6 36458  2016年11月 [査読有り]
     
    The most common event responsible for resistance to first-and second-generation (1st and 2nd) epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) is acquisition of T790M mutation. We examined whether T790M is related to clinicopathologic or prognostic factors in patients with relapse of EGFR mutant non-small cell lung cancer (NSCLC) after treatment with 1st or 2nd EGFR-TKIs. We retrospectively reviewed the T790M status and clinical characteristics of 73 patients with advanced or recurrent NSCLC who had been treated with EGFR-TKIs and undergone rebiopsy at Kurume University Hospital between March 2005 and December 2015. T790M mutation was more frequent in patients with EGFR exon 19 deletion mutation (63%, 26/41) than in those with L858R mutation (38%, 12/32) (p = 0.035). The median total duration of 1st or 2nd EGFR-TKI treatment was significantly longer in patients with T790M mutation than in those without (15.3 months vs 8.1 months, p < 0.001). Multivariate analysis revealed that the type of EGFR mutation and the total duration of EGFR-TKI treatment were significantly associated with T790M prevalence. Patients with EGFR exon 19 deletion mutation who receive long-term EGFR-TKI therapy show a high prevalence of T790M mutation. The present data are potentially important for clinical decision-making in NSCLC patients with EGFR mutation.
  • Takayuki Takahama; Kazuko Sakai; Masayuki Takeda; Koichi Azuma; Toyoaki Hida; Masataka Hirabayashi; Tetsuya Oguri; Hiroshi Tanaka; Noriyuki Ebi; Toshiyuki Sawa; Akihiro Bessho; Motoko Tachihara; Hiroaki Akamatsu; Shuji Bandoh; Daisuke Himeji; Tatsuo Ohira; Mototsugu Shimokawa; Yoichi Nakanishi; Kazuhiko Nakagawa; Kazuto Nishio
    ONCOTARGET 7 36 58492 - 58499 2016年09月 [査読有り]
     
    Introduction: Next-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to overcome resistance to earlier generations of such drugs mediated by a secondary T790M mutation of EGFR, but the performance of a second tumor biopsy to assess T790M mutation status can be problematic. Methods: We developed and evaluated liquid biopsy assays for detection of TKI-sensitizing and T790M mutations of EGFR by droplet digital PCR (ddPCR) in EGFR mutation-positive non-small cell lung cancer (NSCLC) patients with acquired EGFR-TKI resistance. Results: A total of 260 patients was enrolled between November 2014 and March 2015 at 29 centers for this West Japan Oncology Group (WJOG 8014LTR) study. Plasma specimens from all subjects as well as tumor tissue or malignant pleural effusion or ascites fluid from 41 patients were collected after the development of EGFR-TKI resistance. All plasma samples were genotyped successfully and the results were reported to physicians within 14 days. TKI-sensitizing and T790M mutations were detected in plasma of 120 (46.2%) and 75 (28.8%) patients, respectively. T790M was detected in 56.7% of patients with plasma positive for TKI-sensitizing mutations. For the 41 patients with paired samples obtained after acquisition of EGFR-TKI resistance, the concordance for mutation detection by ddPCR in plasma compared with tumor tissue or malignant fluid specimens was 78.0% for TKI-sensitizing mutations and 65.9% for T790M. Conclusions: Noninvasive genotyping by ddPCR with cell-free DNA extracted from plasma is a promising approach to the detection of gene mutations during targeted treatment.
  • Yu Nakamura; Yosuke Togashi; Hirokazu Nakahara; Shuta Tomida; Eri Banno; Masato Terashima; Hidetoshi Hayashi; Marco A. de Velasco; Kazuko Sakai; Yoshihiko Fujita; Takatsugu Okegawa; Kikuo Nutahara; Suguru Hamada; Kazuto Nishio
    MOLECULAR CANCER THERAPEUTICS 15 8 1988 - 1997 2016年08月 [査読有り]
     
    The prognosis for patients with advanced esophageal or head-and-neck squamous cell carcinoma (ESCC or HNSCC) remains poor, and the identification of additional oncogenes and their inhibitors is needed. In this study, we evaluated the sensitivities of several ESCC and HNSCC cell lines to HER inhibitors (cetuximab, erlotinib, and afatinib) in vitro and found two cell lines that were hypersensitive to afatinib. Sequence analyses for the afatinib-targeted HER family genes in the two cell lines revealed that one cell line had a previously reported activating EGFR L861Q mutation, whereas the other had an HER4 G1109C mutation of unknown function. No amplification of HER family genes was found in either of the two cell lines. The phosphorylation level of HER4 was elevated in the HER4 G1109C mutation-overexpressed HEK293 cell line, and the mutation had a transforming potential and exhibited tumorigenicity in an NIH3T3 cell line, indicating that this HER4 mutation was an activating oncogenic mutation. Afatinib dramatically reduced the phosphorylation level of EGFR or HER4 and induced apoptosis in the two cell lines. In vivo, tumor growth was also dramatically decreased by afatinib. In a database, the frequencies of HER family gene mutations in ESCC or HNSCC ranged from 0% to 5%. In particular, HER4 mutations have been found relatively frequently in HNSCC. Considering the addiction of cancer cells to activating oncogenic EGFR or HER4 mutations for proliferation, HNSCC or ESCC with such oncogenic mutations might be suitable for targeted therapy with afatinib. (C) 2016 AACR.
  • Eri Banno; Yosuke Togashi; Yu Nakamura; Masato Chiba; Yoshihisa Kobayashi; Hidetoshi Hayashi; Masato Terashima; Marco A. de Velasco; Kazuko Sakai; Yoshihiko Fujita; Tetsuya Mitsudomi; Kazuto Nishio
    CANCER SCIENCE 107 8 1134 - 1140 2016年08月 [査読有り]
     
    Most patients with non-small cell lung cancer (NSCLC) harboring common epidermal growth factor receptor (EGFR) mutations, such as deletions in exon 19 or the L858R mutation in exon 21, respond dramatically to EGFR tyrosine kinase inhibitors (EGFR-TKI), and their sensitivities to various EGFR-TKI have been well characterized. Our previous article showed the in vitro sensitivities of EGFR exon 18 mutations to EGFR-TKI, but little information regarding the sensitivities of other uncommon EGFR mutations is available. First, stable transfectant Ba/F3 cell lines harboring EGFR L858R (Ba/F3-L858R), L861Q (Ba/F3-L861Q) or S768I (Ba/F3-S768I) mutations were created and their drug sensitivities to various EGFR-TKI were examined. Both the Ba/F3-L861Q and Ba/F3-S768I cell lines were less sensitive to erlotinib, compared with the Ba/F3-L858R cell line, but their sensitivities to afatinib were similar to that of the Ba/F3-L858R cell line. The Ba/F3-L861Q cell line was similarly sensitive and the Ba/F3-S768I cell line was less sensitive to osimertinib, compared with the Ba/F3-L858R cell line. The results of western blot analyses were consistent with these sensitivities. Next, similar experiments were also performed using the KYSE270 (L861Q) and KYSE 450 (S768I) cell lines, and their results were compatible with those of the transfectant Ba/F3 cell lines. Our findings suggest that NSCLC harboring the EGFR L861Q mutation might be sensitive to afatinib or osimertinib and that NSCLC harboring the EGFR S768I mutation might be sensitive to afatinib. Overall, afatinib might be the optimal EGFR-TKI against these uncommon EGFR mutations.
  • Masaki Kaibori; Kazuko Sakai; Morihiko Ishizaki; Hideyuki Matsushima; Marco A. De Velasco; Kosuke Matsui; Hiroya Iida; Hiroaki Kitade; A-Hon Kwon; Hiroaki Nagano; Hiroshi Wada; Seiji Haji; Tadashi Tsukamoto; Akishige Kanazawa; Yutaka Takeda; Shigekazu Takemura; Shoji Kubo; Kazuto Nishio
    ONCOTARGET 7 31 49091 - 49098 2016年08月 [査読有り]
     
    The multi-kinase inhibitor sorafenib is clinically approved for the treatment of patients with advanced hepatocellular carcinoma (HCC). We previously reported that fibroblast growth factor 3 and 4 (FGF3/FGF4) amplification is a predictor of a response to sorafenib. This study aims to analyze the relationship between FGF-FGF receptor (FGFR) genetic alterations and the response to sorafenib. Formalin-fixed, paraffin-embedded tissue specimens from HCC patients who had achieved a complete response (CR, N=6) or non-CR (N=39) to sorafenib were collected and were examined for FGF-FGFR gene alterations using next generation sequencing and copy number assay. FGFR mutations were detected in 5 of 45 (11.1%) cases. There was no significant association between FGFR mutation status and the response to sorafenib. We detected no increase in the FGF3/FGF4 copy number in CR cases. An FGF19 copy number gain was detected more frequently among CR cases (2/6, 33.3%) than among non-CR cases (2/39, 5.1%) (P = 0.024, Chi-squared test). In conclusion, a copy number gain for FGF19 may be a predictor of a response to sorafenib, in addition to FGF3/FGF4 amplification.
  • Masato Terashima; Yosuke Togashi; Katsuaki Sato; Hiroshi Mizuuchi; Kazuko Sakai; Kenichi Suda; Yu Nakamura; Eri Banno; Hidetoshi Hayashi; Marco A. De Velasco; Yoshihiko Fujita; Shuta Tomida; Tetsuya Mitsudomi; Kazuto Nishio
    CLINICAL CANCER RESEARCH 22 14 3663 - 3671 2016年07月 [査読有り]
     
    Purpose: This study investigated whether mutations of receptor tyrosine kinase (RTK) genes detected using next-generation sequencing (NGS) are suitable therapeutic targets. Experimental design: Fifty surgically resected non-small cell lung cancer (NSCLC) samples were target resequenced using NGS. We then investigated the functions of the identified RTK gene mutations, including their oncogenic potential, in vitro. Results: Mutations in RTK genes were found in 20 samples (EGFR, 15; ERBB4, 1; ALK, 1; DDR2, 2; FGFR1, 1), mutations in MAPK pathway genes were found in nine samples (KRAS, 7; NRAS, 1; BRAF, 2), and mutations in PI3K pathway genes were found in three samples (PIK3CA, 1; PTEN, 3). Among the mutations in RTKs, the functions of four mutations were unclear (ERBB4 D245G; DDR2 H246R and E655K; FGFR1 A263V). These mutations did not exhibit any transformational activities. Neither the phosphorylation nor the protein expressions of RTKs were changed by the DDR2 H246R, ERBB4 D245G, and FGFR1 A263V mutations, although the expression level of the DDR2 protein harboring the E655K mutation was particularly low. Collagen stimulation decreased cellular proliferation through p38 activation in the DDR2 wild-type-overexpressed cell lines, whereas the growth-suppressive effect was weakened in DDR2 E655K-overex-pressed cell lines. Furthermore, the DDR2 E655K protein strongly bound to ubiquitin ligase E3 (Cbl-b), and the mutant protein expression was increased after treatment with a proteasome inhibitor. Conclusions: Our experimental findings suggest that RTK mutations are not always suitable as therapeutic targets. The DDR2 E655K mutation can play a role in cancer progression by reducing the growth-inhibitory effect of collagen. (C) 2016 AACR.
  • Satomi Watanabe; Masayuki Takeda; Takayuki Takahama; Tsutomu Iwasa; Junji Tsurutani; Junko Tanizaki; Toshio Shimizu; Kazuko Sakai; Yoshitaka Wada; Noritaka Isogai; Kazuto Nishio; Kazuhiko Nakagawa
    INVESTIGATIONAL NEW DRUGS 34 3 394 - 396 2016年06月 [査読有り]
     
    Extramammary Paget's disease is a malignant intraepithelial carcinoma, which constitutes less than 1 % of all vulvar malignancies. Surgical resection is the first treatment of choice and standard chemotherapy has not been established for advanced or recurrent disease. Experimental and clinical studies have identified human epidermal growth receptor 2 as a potential therapeutic target. A 63-year-old male was referred for recurrent extramammary Paget's disease after surgery. Human epidermal growth receptor 2 was shown to be overexpressed and amplified by immunohistochemical analysis and fluorescence in situ hybridization analysis, respectively. After two cycles of trastuzumab monotherapy, all lymph node metastases decreased in size. However, he experienced recurrence in the lymph nodes during the seven courses of trastuzumab. As a subsequent treatment, trastuzumab was administered in combination with docetaxel and pertuzumab; clinical response was sustained for 12 months without significant adverse events.
  • Shimizu Toshio; Nishio Kazuto; Sakai Kazuko; Hayashi Hidetoshi; Okamoto Kunio; Takeda Masayuki; Iwasa Tsutomu; Tanaka Kaoru; Aoyama Koji; Morishita Maiko; Nakagawa Kazuhiko
    JOURNAL OF CLINICAL ONCOLOGY 34 15 2016年05月 [査読有り]
  • M. Takeda; K. Sakai; K. Okamoto; H. Hayashi; K. Tanaka; T. Shimizu; K. Nishio; K. Nakagawa
    ANNALS OF ONCOLOGY 27 4 748 - 750 2016年04月 [査読有り]
     
    RET fusion genes have recently been identified as new "druggable" drivers in 1% to 2% of lung adenocarcinomas, with several clinical trials now being under way to evaluate the therapeutic effects of RET tyrosine kinase inhibitors in patients with RET fusion-positive lung cancer. We here describe a case study of long-term efficacy of docetaxel plus nintedanib (BIBF 1120) that was manifest over 33 months in a female never-smoker with non-small cell lung cancer wild-type for EGFR and ALK. Multiplex genetic testing of lung biopsy specimens revealed a CCDC6-RET fusion gene but no other actionable mutations. Our findings suggest that RET rearrangement as identified by multiplex testing is a potential target for nintedanib therapy.
  • Hiroshi Mizuuchi; Kenichi Suda; Isao Murakami; Kazuko Sakai; Katsuaki Sato; Yoshihisa Kobayashi; Masaki Shimoji; Masato Chiba; Yuichi Sesumi; Kenji Tomizawa; Toshiki Takemoto; Yoshitaka Sekido; Kazuto Nishio; Tetsuya Mitsudomi
    CANCER SCIENCE 107 4 461 - 468 2016年04月 [査読有り]
     
    Mutant selective epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as rociletinib and AZD9291, are effective for tumors with T790M secondary mutation that become refractory to first-generation EGFR-TKI. However, acquired resistance to these prospective drugs is anticipated considering the high adaptability of cancer cells and the mechanisms remain largely obscure. Here, CNX-2006 (tool compound of rociletinib) resistant sublines were established by chronic exposure of HCC827EPR cells harboring exon 19 deletion and T790M to CNX-2006. Through the analyses of these resistant subclones, we identified two resistant mechanisms accompanied by MET amplification. One was bypass signaling by MET amplification in addition to T790M, which was inhibited by the combination of CNX-2006 and MET-TKI. Another was loss of amplified EGFR mutant allele including T790M while acquiring MET amplification. Interestingly, MET-TKI alone was able to overcome this resistance, suggesting that oncogenic dependence completely shifted from EGFR to MET. We propose describing this phenomenon as an "oncogene swap."Furthermore, we analyzed multiple lesions from a patient who died of acquired resistance to gefitinib, then found a clinical example of an oncogene swap in which the EGFR mutation was lost and a MET gene copy was gained. In conclusion, an "oncogene swap" from EGFR to MET is a novel resistant mechanism to the EGFR-TKI. This novel mechanism should be considered in order to avoid futile inhibition of the original oncogene.
  • Katsuaki Sato; Kenichi Suda; Shigeki Shimizu; Kazuko Sakai; Hiroshi Mizuuchi; Kenji Tomizawa; Toshiki Takemoto; Kazuto Nishio; Tetsuya Mitsudomi
    PLOS ONE 11 4 e0154186  2016年04月 [査読有り]
     
    The receptor tyrosine kinase AXL is a member of the Tyro3-Axl-Mer receptor tyrosine kinase subfamily. AXL affects several cellular functions, including growth and migration. AXL aberration is reportedly a marker for poor prognosis and treatment resistance in various cancers. In this study, we analyzed clinical, pathological, and molecular features of AXL expression in lung adenocarcinomas (LADs). We examined 161 LAD specimens from patients who underwent pulmonary resections. When AXL protein expression was quantified (0, 1+, 2+, 3+) according to immunohistochemical staining intensity, results were 0: 35%; 1+: 20%; 2+: 37%; and 3+: 7% for the 161 samples. AXL expression status did not correlate with clinical features, including smoking status and pathological stage. However, patients whose specimens showed strong AXL expression (3+) had markedly poorer prognoses than other groups (P = 0.0033). Strong AXL expression was also significantly associated with downregulation of E-cadherin (P = 0.025) and CD44 (P = 0.0010). In addition, 9 of 12 specimens with strong AXL expression had driver gene mutations (6 with EGFR, 2 with KRAS, 1 with ALK). In conclusion, we found that strong AXL expression in surgically resected LADs was a predictor of poor prognosis. LADs with strong AXL expression were characterized by mesenchymal status, higher expression of stem-cell-like markers, and frequent driver gene mutations.
  • H. Nagai; N. Oiso; S. Tomida; K. Sakai; S. Fujiwara; Y. Nakamachi; S. Kawano; A. Kawada; K. Nishio; C. Nishigori
    BRITISH JOURNAL OF DERMATOLOGY 174 3 633 - 635 2016年03月 [査読有り]
  • K. Yonesaka; K. Hirotani; H. Kawakami; M. Takeda; H. Kaneda; K. Sakai; I. Okamoto; K. Nishio; P. A. Jaenne; K. Nakagawa
    ONCOGENE 35 7 878 - 886 2016年02月 [査読有り]
     
    Human epidermal growth factor receptor (HER) 3 is aberrantly overexpressed and correlates with poor prognosis in non-small cell lung cancer (NSCLC). Patritumab is a monoclonal antibody against HER3 that has shown promising results in early-phase clinical trials, but an optimal target population for the drug has yet to be identified. In the present study, we examined whether heregulin, a HER3 ligand that is also overexpressed in a subset of NSCLC, can be used as a biomarker to predict the antitumorigenic efficacy of patritumab and whether the drug can overcome the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) resistance induced by heregulin. Patritumab sensitivity was associated with heregulin expression, which, when abolished, resulted in the loss of HER3 and AKT activation and growth arrest. Furthermore, heregulin overexpression induced EGFR TKI resistance in NSCLC cells harbouring an activating EGFR mutation, while HER3 and AKT activation was maintained in the presence of erlotinib in heregulin-overexpressing, EGFR-mutant NSCLC cells. Sustained HER3-AKT activation was blocked by combining erlotinib with either anti-HER2 or anti-HER3 antibody. Notably, heregulin was upregulated in tissue samples from an NSCLC patient who had an activating EGFR mutation but was resistant to the TKI gefitinib. These results indicate that patritumab can overcome heregulin-dependent EGFR inhibitor resistance in NSCLC in vitro and in vivo and suggest that it can be used in combination with EGFR TKIs to treat a subset of heregulin-overexpressing NSCLC patients.
  • Naoki Takegawa; Kimio Yonesaka; Kazuko Sakai; Hiroto Ueda; Satomi Watanabe; Yoshikane Nonagase; Tatsuya Okuno; Masayuki Takeda; Osamu Maenishi; Junji Tsurutani; Taroh Satoh; Isamu Okamoto; Kazuto Nishio; Takao Tamura; Kazuhiko Nakagawa
    ONCOTARGET 7 3 3443 - 3450 2016年01月 [査読有り]
     
    Background: Patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS benefit from epidermal growth factor receptor (EGFR)-targeted therapy. However, patients who are treated with anti-EGFR antibodies will eventually develop the resistance to those agents. HER2 amplification is one of the mechanisms conferring resistance to anti-EGFR antibody therapy and could therefore be a potential therapeutic target. The aim of this study was to detect HER2 amplification in circulating tumor DNA (ctDNA) from patients with CRC and acquired resistance to anti-EGFR antibody therapy Results: Our data showed that 22% (4/18) of patients in the cohort exhibited HER2 amplification. One of these patients was found to be positive for HER2 amplification in matched tumor specimens collected after cetuximab therapy, at which point the patient had acquired cetuximab resistance, despite being negative for HER2 amplification prior to therapy. Methods: We analyzed plasma ctDNA using digital polymerase chain reaction (PCR) from 18 patients with CRC, who had been treated with anti-EGFR antibody-based therapy (cetuximab) and subsequently acquired resistant cetuximab. HER2 gene copy number was analyzed using fluorescence in situ hybridization in tumor samples before and after acquisition of resistance to cetuximab-based therapy. Conclusion: Analysis of plasma ctDNA by digital PCR could be useful for detecting HER2 amplification in patients with CRC who were resistant to anti-EGFR antibody therapy.
  • Kenichi Suda; Isao Murakami; Kazuko Sakai; Kenji Tomizawa; Hiroshi Mizuuchi; Katsuaki Sato; Kazuto Nishio; Tetsuya Mitsudomi
    LUNG CANCER 91 36 - 40 2016年01月 [査読有り]
     
    Objectives: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are used as a first line therapy for metastatic lung cancer harboring somatic EGFR mutation. However, acquisition of resistance to these drugs is almost inevitable. T790M (threonine to methionine substitution at codon 790 of the EGFR gene) and MET amplification are well-known resistance mechanisms, and we previously demonstrated that three of six autopsied patients showed inter-tumor heterogeneity in resistance mechanisms by analyzing T790M and MET gene copy number (Suda et al., 2010). To further elucidate the role of heterogeneity in acquired resistance, here we performed further analyses including additional five patients. Materials and methods: We analyzed somatic mutations in 50 cancer-related genes for 26 EGFR-TKI refractory lesions from four autopsied patients using target sequencing. MET and ERBB2 copy numbers were analyzed by real-time PCR. Data for additional one patient was obtained from our recent study (Suda et al., 2015). Relationship between heterogeneity in resistance mechanism(s) and time to treatment failure (TTF) of EGFR-TKI and post-progression survival (PPS) were analyzed. Results and conclusion: We observed heterogeneity of resistance mechanisms in two of four patients analyzed (T790M + MET gene copy number gain, and mutant EGFR loss + unknown). We also identified quantitative heterogeneity in EGER T790M mutation ratio among EGFR-TKI refractory lesions. In analyzing patient outcomes, we found that patients who developed multiple resistance mechanisms had shorter TIT compared with those who developed single resistance mechanism (p = 0.022). PPS after EGFR-TKI treatment failure was compatible between these two groups (p=0.42). These findings further our understanding of acquired resistance mechanisms to EGFR-TKIs, and may lead to better treatment strategies after acquisition of resistance to first generation EGFR-TKIs in lung cancer patients with EGFR mutations. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
  • Tomoyuki Nagai; Tokuzo Arao; Kazuto Nishio; Kazuko Matsumoto; Satoru Hagiwara; Toshiharu Sakurai; Yasunori Minami; Hiroshi Ida; Kazuomi Ueshima; Naoshi Nishida; Kazuko Sakai; Nagahiro Saijo; Kanae Kudo; Hiroyasu Kaneda; Daisuke Tamura; Keiichi Aomatsu; Hideharu Kimura; Yoshihiko Fujita; Seiji Haji; Masatoshi Kudo
    DIGESTIVE DISEASES 34 6 702 - 707 2016年 [査読有り]
     
    Background: Epithelial-mesenchymal transition (EMT) is considered to play a critical role in cancer progression and metastasis. However, the impact of EMT on the prognosis of hepatocellular carcinoma (HCC) is still elusive. In this study, we examined the relationship between the expression of EMT markers and recurrence-free survival (RFS) and overall survival (OS) in HCC patients after hepatic resection. Summary:The mRNA expression of 15 genes related to EMT was assessed by quantitative real-time polymerase chain reaction in cancerous tissues from 72 patients who underwent hepatic resection of HCC between January 2005 and December 2010 at our hospital. The upregulation of TWIST and the downregulation of tight junction protein ZO-1 (TJP1) were significantly associated with shorter RFS as well as OS. Increased levels of TWIST and decreased levels of TJP1 should be predictive markers for poor prognosis in patients with HCC after hepatectomy; those could serve as potential biomarkers for the treatment of HCC. Key Messages: A low level of TJP1 and high level of TWIST expression were prognostic factors predicting HCC after hepatic resection. (C) 2016 S. Karger AG, Basel
  • M. Takeda; K. Sakai; M. Terashima; H. Kaneda; H. Hayashi; K. Tanaka; K. Okamoto; T. Takahama; T. Yoshida; T. Iwasa; T. Shimizu; Y. Nonagase; K. Kudo; S. Tomida; T. Mitsudomi; K. Saigo; A. Ito; K. Nakagawa; K. Nishio
    ANNALS OF ONCOLOGY 26 12 2477 - 2482 2015年12月 [査読有り]
     
    The clinical implementation of genomic profiling for lung cancer with high-throughput, multiplex tests is warranted to allow prioritization of appropriate therapies for individual patients. Multiplex genomic testing can assist physicians in matching patients with approved or experimental targeted treatments. Background: The clinical implementation of genomic profiling for lung cancer with high-throughput, multiplex tests is warranted to allow prioritization of appropriate therapies for individual patients. We have now applied such testing to detect actionable mutations that may inform treatment recommendations in lung cancer. Patients and methods: We prospectively applied amplicon sequencing panels that cover both mutational hotspots in 22 genes related to lung and colon tumorigenesis as well as 72 major variants of ALK, RET, ROS1, and NTRK1 fusion transcripts. We then determined the proportion of patients who received genotype-directed therapy and their overall survival (OS). Results: Tumor specimens from 110 patients with lung cancer recruited between July 2013 and March 2015 were analyzed. The most common genetic alterations were TP53 mutations in 42 patients, followed by EGFR mutations in 25, STK11 mutations in 12, and KRAS mutations in 10. Potentially actionable mutations were identified in 44 patients including 50% of those with adenocarcinoma and 14% of those with squamous cell carcinoma. The OS of patients with advanced or recurrent cancer who had an actionable mutation and received targeted therapy (median OS not achieved) was significantly longer than that of those with no mutation (18.1 months, P = 0.041) or of those with a mutation not so treated (6.1 months, P = 0.0027). Conclusions: Multiplex genomic testing was performed on formalin-fixed, paraffin-embedded tumor specimens with a success rate of >= 95%. Such testing can assist physicians in matching patients with approved or experimental targeted treatments. Clinical trial registration: The University Medical Hospital Information Network (UMIN) Clinical Trials Registry under the identifier UMIN000014782.
  • Yasuo Iwamoto; Tetsuya Mitsudomi; Kazuko Sakai; Takeharu Yamanaka; Hiroshige Yoshioka; Makoto Takahama; Masahiro Yoshimura; Ichiro Yoshino; Masayuki Takeda; Shunichi Sugawara; Tomoya Kawaguchi; Toshiaki Takahashi; Mitsunori Ohta; Yukito Ichinose; Shinji Atagi; Morihito Okada; Hideo Saka; Kazuhiko Nakagawa; Yoichi Nakanishi; Kazuto Nishio
    CLINICAL CANCER RESEARCH 21 23 5245 - 5252 2015年12月 [査読有り]
     
    Purpose: The aims of this study were to evaluate the efficacy and safety of S-1 versus cisplatin (CDDP)+S-1 in patients with completely resected stage II and IIIA non-small cell lung cancer, and to identify predictive biomarkers whose expression in the tumors was significantly associated with patient outcome. Experimental Design: A total of 200 patients were randomly assigned to receive either S-1 (40 mg/m(2) twice per day) for 2 consecutive weeks repeated every 3 weeks for 1 year (S group) or CDDP (60 mg/m2) on day 1 plus oral S-1 (40 mg/m2 twice per day) for 2 consecutive weeks repeated every 3 weeks for four cycles (CS group) within 8 weeks after surgery. The primary endpoints were relapse-free survival (RFS) at 2 years and identification of predictive biomarkers whose expressions have been reported to be associated with CDDP or fluoropyrimidine sensitivity. Results: The RFS rate at 2 years was 65.6% (95% confidence intervals; CI, 55.3-74.0%) in the S group and 58.1% (95% CI, 47.7-67.2%) in the CS group. The only gene with interaction of P < 0.05 was uridine monophosphate synthase (UMPS; P = 0.0348). The benefit that members of the S group had over members of the CS group was higher expression of UMPS. In vitro and in vivo experiments confirmed that overexpression of UMPS enhanced the antitumor effect of fluoropyrimidine. Conclusions: Adjuvant S-1 monotherapy might be preferable to CDDP+S-1 for patients with completely resected NSCLC. UMPS expression may define a patient subset that would benefit from long-term postoperative S-1 monotherapy. (C) 2015 AACR.
  • Yasumasa Yoshioka; Yosuke Togashi; Takaaki Chikugo; Akihiro Kogita; Masataka Taguri; Masato Terashima; Takuro Mizukami; Hidetoshi Hayashi; Kazuko Sakai; Marco A. de Velasco; Shuta Tomida; Yoshihiko Fujita; Tadao Tokoro; Akihiko Ito; Kiyotaka Okuno; Kazuto Nishio
    CANCER 121 24 4359 - 4368 2015年12月 [査読有り]
     
    BACKGROUND: Although colorectal mucinous adenocarcinomas (MCs) are conventionally regarded as exhibiting high-grade differentiation, they can be divided by differentiation into 2 groups according to the glandular appearance: low-grade mucinous adenocarcinoma (low-MC) and high-grade mucinous adenocarcinoma (high-MC). METHODS: Patients with colorectal cancer (CRC) who underwent surgical resection between 2000 and 2012 were enrolled in this study. Among the cases with MC, the clinicopathological and genetic differences between low-MC and high-MC were investigated with next-generation sequencing. RESULTS: A total of 1373 patients with CRC were analyzed. Forty patients (2.9%) had MC, and 13 patients had high-MC. Patients with MC had significantly shorter disease-free survival (DFS) and overall survival (OS) periods than those with nonmucinous carcinoma. When low-MC patients and high-MC patients were compared, those with high-MC had significantly shorter DFS and OS periods than those with low-MC. Multivariate analyses revealed that high-MC was significantly associated with both shorter DFS and shorter OS, but low-MC was not. A genome analysis revealed that low-MC had a considerably larger number of mutations than high-MC, and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and adenomatous polyposis coli mutations were particularly frequently found in low-MC. In contrast, SMAD family member 4 (SMAD4) mutations were frequently found in high-MC. CONCLUSIONS: High-MC is an independent prognostic factor in CRC (but low-MC is not), and it is genetically different from other CRCs, including low-MC. Both the clinicopathological differences and the genetic differences suggest that low-MC and high-MC should be distinguished in clinical settings. (C) 2015 American Cancer Society.
  • Hidenobu Ishii; Koichi Azuma; Kazuko Sakai; Akihiko Kawahara; Kazuhiko Yamada; Takaaki Tokito; Isamu Okamoto; Kazuto Nishio; Tomoaki Hoshino
    ONCOTARGET 6 31 30850 - 30858 2015年10月 [査読有り]
     
    As the development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has become an issue of concern, identification of the mechanisms responsible has become an urgent priority. However, for research purposes, it is not easy to obtain tumor samples from patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC) that has relapsed after treatment with EGFR-TKIs. Here, using digital PCR assay as an alternative and noninvasive method, we examined plasma and tumor samples from patients with relapsed NSCLC to establish the inter-relationships existing among T790M mutation, activating EGFR mutations, HER2 amplification, and MET amplification. Paired samples of tumor and blood were obtained from a total of 18 patients with NSCLC after they had developed resistance to EGFR-TKI treatment, and the mechanisms of resistance were analyzed by digital PCR. Digital PCR analysis of T790M mutation in plasma had a sensitivity of 81.8% and specificity of 85.7%, the overall concordance between plasma and tissue samples being 83.3%. MET gene copy number gain in tumor DNA was observed by digital PCR in three patients, of whom one exhibited positivity for MET amplification by FISH, whereas no patient demonstrated MET and HER2 copy number gain in plasma DNA. Digital PCR analysis of plasma is feasible and accurate for detection of T790M mutation in NSCLC that becomes resistant to treatment with EGFR-TKIs.
  • Takeda Masayuki; Sakai Kazuko; Terashima Masato; Kaneda Hiroyasu; Hayashi Hidetoshi; Tanaka Kaoru; Iwasa Tsutomu; Yoshida Takeshi; Takahama Takayuki; Nishio Kazuto; Nakagawa Kazuhiko
    JOURNAL OF THORACIC ONCOLOGY 10 9 S700 - S701 2015年09月 [査読有り]
  • Kenichi Suda; Isao Murakami; Kazuko Sakai; Hiroshi Mizuuchi; Shigeki Shimizu; Katsuaki Sato; Kenji Tomizawa; Shuta Tomida; Yasushi Yatabe; Kazuto Nishio; Tetsuya Mitsudomi
    SCIENTIFIC REPORTS 5 14447  2015年09月 [査読有り]
     
    Lung cancers often harbour a mutation in the epidermal growth factor receptor (EGFR) gene. Because proliferation and survival of lung cancers with EGFR mutation solely depend on aberrant signalling from the mutated EGFR, these tumours often show dramatic responses to EGFR tyrosine kinase inhibitors (TKIs). However, acquiring resistance to these drugs is almost inevitable, thus a better understanding of the underlying resistance mechanisms is critical. Small cell lung cancer (SCLC) transformation is a relatively rare acquired resistance mechanism that has lately attracted considerable attention. In the present study, through an in-depth analysis of multiple EGFR-TKI refractory lesions obtained from an autopsy case, we observed a complementary relationship between SCLC transformation and EGFR T790M secondary mutation (resistance mutation). We also identified analogies and differences in genetic aberration between a TKI-refractory lesion with SCLC transformation and one with EGFR T790M mutation. In particular, target sequencing revealed a TP53 P151S mutation in all pre-and post-treatment lesions. PTEN M264I mutation was identified only in a TKI-refractory lesion with SCLC transformation, while PIK3CA and RB1 mutations were identified only in pre-treatment primary tumour samples. These results provide the groundwork for understanding acquired resistance to EGFR-TKIs via SCLC transformation.
  • Y. Togashi; H. Mizuuchi; Y. Kobayashi; H. Hayashi; M. Terashima; K. Sakai; E. Banno; T. Mizukami; Y. Nakamura; M. A. de Velasco; Y. Fujita; S. Tomida; T. Mitsudomi; K. Nishio
    ANNALS OF ONCOLOGY 26 8 1800 - 1801 2015年08月 [査読有り]
  • Kazuko Sakai; Haruhiko Takeda; Norihiro Nishijima; Etsuro Orito; Kouji Joko; Yasushi Uchida; Namiki Izumi; Kazuto Nishio; Yukio Osaki
    ONCOTARGET 6 25 21636 - 21644 2015年08月 [査読有り]
     
    The multi-kinase inhibitor sorafenib is now used as standard therapy for advanced hepatocellular carcinoma (HCC). Predictive biomarkers of response to sorafenib are thus necessary. The purpose of this study was to assess the feasibility of using targeted DNA and RNA sequencing to elucidate candidate biomarkers of sorafenib response using fine-needle biopsy, formalin-fixed paraffin-embedded (FFPE) specimens in patients with HCC. Targeted DNA and RNA deep sequencing were feasible for the evaluation of fine-needle biopsy FFPE specimens obtained from 46 patients with HCC treated with sorafenib. Frequent mutations of suppressor genes, such as CTNNB1 (34.8%) and TP53 (26.1%), were detected in the HCC tumors. After excluding these suppressor genes, the average numbers of detected oncogene mutations differed significantly between the non-PD and PD groups (P = 0.0446). This result suggests that the oncogene mutational burden in the tumor might be associated with the clinical response to sorafenib. We have identified candidate gene expression (TGFa, PECAM1, and NRG1) in tumor for the prediction of sorafenib response and PFS by RNA sequencing. Our findings provide new insights into biomarkers for sorafenib therapy and allow us to discuss future therapeutic strategies.
  • Takuro Mizukami; Yosuke Togashi; Shunsuke Sogabe; Eri Banno; Masato Terashima; Marco A. de Velasco; Kazuko Sakai; Yoshihiko Fujita; Shuta Tomida; Takako Eguchi Nakajima; Narikazu Boku; Kazuto Nishio
    INTERNATIONAL JOURNAL OF ONCOLOGY 47 2 499 - 505 2015年08月 [査読有り]
     
    Since the prognosis of unresectable advanced gastric cancer remains poor, novel therapeutic strategies are needed. Somatic MEKI gene mutations have been reported as oncogenic activating mutations in gastric cancer, and MEK inhibitors can be effective against such gastric cancers. In the present study, however, activated EGFR and HER2 signals after treatment with a MEK inhibitor (trametinib) were found in a MEK1-mutated gastric cancer cell line (OCUM-1 cell line) using a phospho-receptor tyrosine kinase array. The phosphorylation of EGFR and HER2 reactivated ERK1/2, which had been inhibited by trametinib, and EGF stimulation led to resistance to trametinib in this cell line. Lapatinib, an EGFR and an HER2 inhibitor, reversed the activation of ERK1/2 by inhibiting the phosphorylation of EGFR and HER2 and cancelled the resistance. The combination of trametinib and lapatinib synergistically inhibited the cell growth of the OCUM-1 cell line and strongly induced apoptosis by inhibiting the activated EGFR and HER2 signals. These results suggest that the EGFR and HER2 signals play a salvage role and are related to resistance to MEK inhibitors in MEK1-mutated gastric cancer. Moreover, combination therapy with trametinib and lapatinib can exhibit a synergistic effect and may contribute to overcoming the resistance to MEK inhibitors.
  • Nishio K; Sakai K; Togashi Y
    Nihon rinsho. Japanese journal of clinical medicine 73 8 1315 - 1322 2015年08月 [査読有り]
  • Kazuko Sakai; Junji Tsurutani; Takeharu Yamanaka; Azusa Yoneshige; Akihiko Ito; Yosuke Togashi; Marco A. De Velasco; Masato Terashima; Yoshihiko Fujita; Shuta Tomida; Takao Tamura; Kazuhiko Nakagawa; Kazuto Nishio
    PLOS ONE 10 5 e0121891  2015年05月 [査読有り]
     
    Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (alpha = 2 x 10(-5)). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.
  • Yosuke Togashi; Hidetoshi Hayashi; Kunio Okamoto; Soichi Fumita; Masato Terashima; Marco A. de Velasco; Kazuko Sakai; Yoshihiko Fujita; Shuta Tomida; Kazuhiko Nakagawa; Kazuto Nishio
    LUNG CANCER 88 1 16 - 23 2015年04月 [査読有り]
     
    Background: Some of patients with non-small cell lung cancer (NSCLC) harboring somatic activating mutations of the epidermal growth factor receptor gene (EGFR mutations) show poor responses to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) treatment. Cigarette smoking is the strongest documented risk factor for the development of lung cancer. Nicotine, while not carcinogenic by itself, has been shown to induce proliferation, angiogenesis, and the epithelial-mesenchymal transition; these effects might be associated with EGFR-TKI resistance. Materials and methods: PC-9 and 11-18 cell lines (EGFR-mutated NSCLC cell lines) were cultured with 1 mu M nicotine for 3 months and were designated as PC-9/N and 11-18/N cell lines, respectively. The sensitivities of these cell lines to EGFR-TKI were then tested in vitro. Moreover, the association between the smoking status and the progression-free survival (PFS) period was investigated in patients with EGFR-mutated NSCLC who were treated with gefitinib. Results: The PC-9/N and 11-18/N cell lines were resistant to EGFR-TKI, compared with controls. The phosphorylation of EGFR in these cell lines was reduced by EGFR-TKI to a smaller extent than that observed in controls, and a higher concentration of EGFR-TKI was capable of further decreasing the phosphorylation. Clinically, smoking history was an independent predictor of a poor PFS period on gefitinib treatment. Conclusions: Chronic nicotine exposure because of cigarette smoking mediates resistance to EGFR-TKI via an EGFR signal. Smoking cessation is of great importance, while resistance may be overcome through the administration of high-dose EGFR-TKI. (c) 2015 Elsevier Ireland Ltd. All rights reserved.
  • Akihiro Kogita; Yosuke Togashi; Hidetoshi Hayashi; Eri Banno; Masato Terashima; Marco A. De Velasco; Kazuko Sakai; Yoshihiko Fujita; Shuta Tomida; Yoshifumi Takeyama; Kiyotaka Okuno; Kazuhiko Nakagawa; Kazuto Nishio
    INTERNATIONAL JOURNAL OF ONCOLOGY 46 3 1025 - 1030 2015年03月 [査読有り]
     
    Non-small cell lung cancer (NSCLC) carrying echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangements is hypersensitive to ALK inhibitors, including crizotinib and alectinib. Crizotinib was initially designed as a MET inhibitor, whereas alectinib is a selective ALK inhibitor. The MET signal, which is inhibited by crizotinib but not by alectinib, is dysregulated in many human cancers. However, the role of the MET signal in ALK-positive NSCLC remains unclear. In this study, we found that hepatocyte growth factor (HGF), ligand of MET, mediated the resistance to alectinib, but not to crizotinib, via the MET signal in ALK-positive NSCLC cell lines (H3122 and H2228 cell lines). In addition, alectinib activated the MET signal even in the absence of HGF and the inhibition of the MET signal enhanced the efficacy of alectinib. These findings suggest that activated MET acts as a salvage signal in ALK-positive NSCLC. This novel role of the MET signal in ALK-positive NSCLC may pave the way for further clinical trials examining MET inhibitors.
  • Akihiro Kogita; Yasumasa Yoshioka; Kazuko Sakai; Yosuke Togashi; Shunsuke Sogabe; Takuya Nakai; Kiyotaka Okuno; Kazuto Nishio
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 458 1 52 - 56 2015年02月 [査読有り]
     
    Intra- and inter-tumor heterogeneity may hinder personalized molecular-target treatment that depends on the somatic mutation profiles. We performed mutation profiling of formalin-fixed paraffin embedded tumors of multi-regional colon cancer and characterized the consequences of intra- and inter-tumor heterogeneity and metastasis using targeted re-sequencing. We performed targeted re-sequencing on multiple spatially separated samples obtained from multi-regional primary colon carcinoma and associated metastatic sites in two patients using nextgeneration sequencing. In Patient 1 with four primary tumors (P1-1, P1-2, P1-3, and P1-4) and one liver metastasis (H1), mutually exclusive pattern of mutations was observed in four primary tumors. Mutations in primary tumors were identified in three regions; KARS (G13D) and APC (R876*) in P1-2, TP53 (A161S) in P1-3, and KRAS (G12D), PIK3CA (Q546R), and ERBB4 (T272A) in P1-4. Similar combinatorial mutations were observed between P1-4 and H1. The ERBB4 (T272A) mutation observed in P1-4, however, disappeared in H1. In Patient 2 with two primary tumors (P2-1 and P2-2) and one liver metastasis (H2), mutually exclusive pattern of mutations were observed in two primary tumors. We identified mutations; KRAS (G12V), SMAD4 (N129K, R445*, and G508D), TP53 (R175H), and FGFR3 (R805W) in P2-1, and NRAS (Q61K) and FBXW7 (R425C) in P2-2. Similar combinatorial mutations were observed between P2-1 and H2. The SMAD4 (N129K and G508D) mutations observed in P2-1, however, were nor detected in H2. These results suggested that different clones existed in primary tumors and metastatic tumor in Patient 1 and 2 likely originated from P1-4 and P2-1, respectively. In conclusion, we detected the muti-clonalities between intra- and inter-tumors based on mutational profiling in multi-regional colon cancer using next-generation sequencing. Primary region from which metastasis originated could be speculated by mutation profile. Characterization of inter- and inter-tumor heterogeneity can lead to underestimation of the tumor genomics landscape and treatment strategy of personal medicine. (C) 2015 Published by Elsevier Inc.
  • Togashi Y; Hayashi H; Terashima M; de Velasco MA; Sakai K; Fujita Y; Tomida S; Nakagawa K; Nishio K
    Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 10 1 93 - 101 2015年01月 [査読有り]
     
    Introduction: Patients with non-small-cell lung cancer (NSCLC) with somatic activating mutations of the epidermal growth factor receptor gene (EGFR mutations) generally respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs). beta-Catenin is a key component of the Wnt/beta-Catenin signal and is an important oncogene that is involved in the pathogenesis and progression of malignant tumors, especially cancer stem cells. Methods and Results: We found that EGFR-mutated NSCLC cell lines exhibited a high expression level of beta-Catenin, compared with cell lines with the wild-type EGFR gene, and XAV939 (a beta-Catenin inhibitor) enhanced the sensitivities to EGFR-TKI in EGFR-mutated NSCLC cell lines. In EGFR-mutated NSCLC cell lines with the acquired resistance threonine-to-methionine mutation in codon 790 (T790M) mutation, XAV939 enhanced the sensitivity of the cells to an irreversible EGFR-TKI but not a reversible EGFR-TKI. The combination of XAV939 and EGFR-TKIs strongly inhibited the beta-Catenin signal and strongly decreased the phosphorylation of EGFR, compared with the use of EGFR-TKIs alone, suggesting an interaction between EGFR and the beta-Catenin signal. The stem cell-like properties of the EGFR-mutated cell line carrying the T790M mutation were inhibited by XAV939 and BIBW2992 (an irreversible EGFR-TKI). Furthermore, the stem cell-like properties were strongly inhibited by a combination of both the agents. A xenograft study demonstrated that beta-Catenin knockdown enhanced the antitumor effect of BIBW2992 in the EGFR-mutated NSCLC cell line carrying the T790M mutation. Conclusion: Our findings indicate that beta-Catenin might be a novel therapeutic target in EGFR-mutated NSCLC carrying the T790M mutation.
  • Kazuko Sakai; Masayuki Takeda; Isamu Okamoto; Kazuhiko Nakagawa; Kazuto Nishio
    ONCOLOGY LETTERS 9 1 405 - 410 2015年01月 [査読有り]
     
    Hepatocyte growth factor (HGF) expression is a poor prognostic factor in various types of cancer. Expression levels of HGF have been reported to be regulated by shorter poly(dA) sequences in the promoter region. In the present study, the poly(dA) mononucleotide tract in various types of human cancer cell lines was examined and compared with the HGF expression levels in those cells. Short deoxyadenosine repeat sequences were detected in five of the 55 cell lines used in the present study. The H69, IM95, CCK-81, Sui73 and H28 cells exhibited a truncated poly(dA) sequence in which the number of poly(dA) repeats was reduced by 5 bp. Two of the cell lines exhibited high HGF expression, determined by reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The CCK-81, Sui73 and H28 cells with shorter poly(dA) sequences exhibited low HGF expression. The cause of the suppression of HGF expression in the CCK-81, Sui73 and H28 cells was clarified by two approaches, suppression by methylation and single nucleotide polymorphisms in the HGF gene. Exposure to 5-Aza-dC, an inhibitor of DNA methyltransferase 1, induced an increased expression of HGF in the CCK-81 cells, but not in the other cells. Single-nucleotide polymorphism (SNP) rs72525097 in intron 1 was detected in the Sui73 and H28 cells. Taken together, it was found that the defect of poly(dA) in the HGF promoter was present in various types of cancer, including lung, stomach, colorectal, pancreas and mesothelioma. The present study proposes the negative regulation mechanisms by methylation and SNP in intron 1 of HGF for HGF expression in cancer cells with short poly(dA).
  • Yosuke Togashi; Akihiro Kogita; Hiroki Sakamoto; Hidetoshi Hayashi; Masato Terashima; Marco A. de Velasco; Kazuko Sakai; Yoshihiko Fujita; Shuta Tomida; Masayuki Kitano; Kiyotaka Okuno; Masatoshi Kudo; Kazuto Nishio
    CANCER LETTERS 356 2 819 - 827 2015年01月 [査読有り]
     
    We previously reported that activin produces a signal with a tumor suppressive role in pancreatic cancer (PC). Here, the association between plasma activin A and survival in patients with advanced PC was investigated. Contrary to our expectations, however, patients with high plasma activin A levels had a significantly shorter survival period than those with low levels (median survival, 314 days vs. 482 days, P = 0.034). The cellular growth of the MIA PaCa-2 cell line was greatly enhanced by activin A via non-SMAD pathways. The cellular growth and colony formation of an INHBA (beta subunit of inhibin)overexpressed cell line were also enhanced. In a xenograft study, INHBA-overexpressed cells tended to result in a larger tumor volume, compared with a control. The bodyweights of mice inoculated with INHBA-overexpressed cells decreased dramatically, and these mice all died at an early stage, suggesting the occurrence of activin-induced cachexia. Our findings indicated that the activin signal can promote cancer progression in a subset of PC and might be involved in cachexia. The activin signal might be a novel target for the treatment of PC. (C) 2014 Elsevier Ireland Ltd. All rights reserved.
  • Kazuko Sakai; Azusa Yoneshige; Akihiko Ito; Yoji Ueda; Satoshi Kondo; Hitoshi Nobumasa; Yoshihiko Fujita; Yosuke Togashi; Masato Terashima; Marco A. De Velasco; Shuta Tomida; Kazuto Nishio
    SPRINGERPLUS 4 7  2015年01月 [査読有り]
     
    We compared the performance of the 3D-Gene (R) mutation assay (3D-Gene (R) KRAS mutation assay kit) with the Scorpion-ARMS (therascreen (R) KRAS RGQ PCR Kit) and Luminex (MEBGEN (TM) KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene (R) mutation assay without an invalid case. The concordance rate between the 3D-Gene (R) mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene (R) mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples.
  • Shunsuke Sogabe; Yosuke Togashi; Hiroaki Kato; Akihiro Kogita; Takuro Mizukami; Yoichi Sakamoto; Eri Banno; Masato Terashima; Hidetoshi Hayashi; Marco A. de Velasco; Kazuko Sakai; Yoshihiko Fujita; Shuta Tomida; Takushi Yasuda; Yoshifumi Takeyama; Kiyotaka Okuno; Kazuto Nishio
    MOLECULAR CANCER THERAPEUTICS 13 12 3098 - 3106 2014年12月 [査読有り]
     
    The prognosis for patients with unresectable advanced or recurrent gastric cancer remains poor. The identification of additional oncogenes with influences similar to those of epidermal growth factor receptor gene mutations, upon which the growth of cancer cells is dependent, is needed. In this study, we evaluated sensitivity to MEK inhibitors (GSK1120212 and PD0325901) in several gastric cancer cell lines in vitro and found three poorly differentiated gastric cancer cell lines that were hypersensitive to the inhibitors. The sequence analyses in these three cell lines revealed that one cell line had a novel MEK1 mutation, while the other two had previously reported KRAS and MEK1 mutations, respectively; the gene statuses of the other resistant cell lines were all wild-type. Experiments using MEK1 expression vectors demonstrated that the MEK1 mutations induced the phosphorylation of ERK1/2 and had a transforming potential, enhancing the tumorigenicity. The MEK inhibitor dramatically reduced the phosphorylation of ERK1/2 and induced apoptosis in the cell lines with MEK1 mutations. In vivo, tumor growth was also dramatically decreased by an inhibitor. One of the 46 gastric cancer clinical samples that were examined had a MEK1 mutation; this tumor had a poorly differentiated histology. Considering the addiction of cancer cells to active MEK1 mutations for proliferation, gastric cancer with such oncogenic MEK1 mutations might be suitable for targeted therapy with MEK inhibitors. (C)2014 AACR.
  • M. Tanioka; K. Sakai; T. Sudo; T. Sakuma; K. Kajimoto; K. Hirokaga; S. Takao; S. Negoro; H. Minami; K. Nakagawa; K. Nishio
    BREAST CANCER RESEARCH AND TREATMENT 147 3 513 - 525 2014年10月 [査読有り]
     
    Several trials have confirmed that the pathological complete response (pCR) rates after neoadjuvant chemotherapy (NAC) are significantly lower in HER2-positive/ER-positive patients than in HER2-positive/ER-negative patients. To understand this phenomenon, we investigated the association between NAC resistance and CCND1, which is frequently overexpressed in ER-positive tumors. Pretreatment formalin-fixed tumor tissues were collected from 75 HER2-positive patients receiving NAC comprised anthracyclines, taxanes, and trastuzumab. Seventeen gene transcripts along with PIK3CA mutations were detected using MassARRAY (Sequenom, San Diego, CA). The gene expression levels were dichotomized according to the median values. The immunohistochemical expression of ER, PTEN, BCL-2, and cyclin D1 was scored. The relationship between the variables was assessed using the Spearman correlation. A logistic regression analysis was performed to detect predictors of pCR, which was defined as no invasive tumor in the breast or axilla. Forty-seven percent of the cases were ER-positive and 52 % (40/63 % in ER-positive/ER-negative) achieved a pCR. Among the ER-positive patients, the CCND1 gene expression level was 2.1 times higher than that in ER-negative patients and was significantly correlated with the expression of cyclin D1 protein. In a univariate analysis, a pCR was associated with high mRNA levels of ESR1, PGR, LMTK3, HER2, IGF1R, INPP4B, PDL-1, BCL-2, and CCND1 (P a parts per thousand currency sign 0.05). In contrast, none of these genes were significantly correlated with a pCR among the ER-negative tumors and only EGFR was significantly correlated with a pCR. PIK3CA mutations or PTEN loss were not associated with a pCR in either group. After excluding ESR1 (r = 0.58), PGR (r = 0.64), and IGF1R (r = 0.59), the expressions of which were correlated with CCND1, a multivariate analysis revealed that CCND1 [P = 0.043; OR, 0.16] and HER2 [P = 0.012; OR, 11.2] retained its predictive value for pCR among ER-positive patients, but not among ER-negative patients. A High Level of CCND1 gene expression is a poor predictor of a pCR and provides a rationale for evaluating CDK4/6 inhibitors in HER2-positive/ER-positive breast cancer patients.
  • Akihiro Kogita; Yosuke Togashi; Hidetoshi Hayashi; Shunsuke Sogabe; Masato Terashima; Marco A. De Velasco; Kazuko Sakai; Yoshihiko Fujita; Shuta Tomida; Yoshifumi Takeyama; Kiyotaka Okuno; Kazuhiko Nakagawa; Kazuto Nishio
    INTERNATIONAL JOURNAL OF ONCOLOGY 45 4 1430 - 1436 2014年10月 [査読有り]
     
    Patients with non-small cell lung cancer (NSCLC) with echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangements generally respond to ALK inhibitors such as crizotinib. However, some patients with EML4-ALK rearrangements respond poorly to crizotinib. Hypoxia is involved in the resistance to chemotherapeutic treatments in several cancers, and we investigated the association between the responses to ALK inhibitors and hypoxia. Sensitivity of the H3122 NSCLC cell line (EML4-ALK rearrangement) to ALK inhibitors (crizotinib or alectinib) was investigated during a normoxic or hypoxic state using an MTT assay. We found that the cell line was resistant to the inhibitors during hypoxia. Hypoxia mediated morphologic changes, including cell scattering and the elongation of the cell shape, that are characteristic of the epithelial-mesenchymal transition (EMT). A migration assay demonstrated that the number of migrating cells increased significantly during hypoxia, compared with during normoxia. Regarding EMT-related molecules, the expressions of slug, vimentin, and fibronectin were increased while that of E-cadherin was decreased by hypoxia. In addition, hypoxia inducible factor 1A-knockdown cancelled the hypoxia-induced EMT and resistance. Our findings indicate that hypoxia induces resistance to ALK inhibitors in NSCLC with an EML4-ALK rearrangement via the EMT.
  • Kazuko Sakai; Shinsuke Kazama; Yuzo Nagai; Koji Murono; Toshiaki Tanaka; Soichiro Ishihara; Eiji Sunami; Shuta Tomida; Kazuto Nishio; Toshiaki Watanabe
    ONCOTARGET 5 20 9641 - 9649 2014年10月 [査読有り]
     
    Neoadjuvant chemoradiotherapy has been introduced in patients with surgically resected rectal cancer and reduced the local recurrence. Heterogeneity exists in rectal cancer, and we hypothesized that there are subclones resistant to chemoradiotherapy within the cancer mass. We performed DNA-targeted sequencing of pre- and post-treatment tumor tissues obtained from 20 rectal cancer patients who received chemoradiotherapy. The variant frequency of the mutant clones was compared between pre- and post-treatment samples of nine non-responder patients. RNA-targeted sequencing of 57 genes related to sensitivity to chemotherapy and radiotherapy was performed for the paired samples. Immunohistochemical analyses of p53 expression were also performed on the paired samples from the nine non-responder patients. DNA-sequencing detected frequent mutations of suppressor genes including TP53, APC and FBXW7 in the post-treatment samples of the nine non-responders. The frequency of TP53 mutations showed significant increases after chemoradiotherapy. RNA-targeted sequencing of 29 tumor tissues demonstrated that decreased expression of three genes and increased expression of four genes were detected in the post-treatment samples. Significantly increased expression of TP53 was observed in the post-treatment samples. Immunohistochemical staining for p53 revealed that increased p53 intensity scores were observed after chemoradiotherapy. These results suggest that the tumors with TP53 mutations tend to accumulate through chemoradiotherapy.
  • Terashima M; Fujita Y; Togashi Y; Sakai K; De Velasco MA; Tomida S; Nishio K
    Oncotarget 5 16 7040 - 7050 2014年08月 [査読有り]
     
    The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cells. To this end, we expressed KIAA1199-MBP fusion protein and performed a pull-down assay. In addition, KIAA1199-overexpressing cancer cell lines were constructed using a retroviral vector and were used for further experiments. A pull-down analysis showed that the glycogen phosphorylase kinase beta-subunit (PHKB) interacted with the C-terminal region of KIAA1199 protein. Furthermore, we observed the interaction of KIAA1199 with glycogen phosphorylase brain form (PYGB) under serum-free conditions. The interaction promoted glycogen breakdown and cancer cell survival. Our findings indicate that KIAA1199 plays an important role in glycogen breakdown and cancer cell survival and that it may represent a novel target for cancer therapy.
  • Masato Terashima; Kazuko Sakai; Yosuke Togashi; Hidetoshi Hayashi; Marco A. De Velasco; Junji Tsurutani; Kazuto Nishio
    SPRINGERPLUS 3 417  2014年08月 [査読有り]
     
    Triple-negative breast cancer (TNBC) is associated with a higher incidence of recurrence and distant metastasis and a poor prognosis, whereas effective treatment strategies remain to be established. Finding an effective treatment for TNBC has become imperative. We examined the effect of the combination of S-1 (or 5-FU in an in vitro study) and eribulin in TNBC cell lines. The in vitro effect of the combination was examined in four TNBC cell lines (MDA-MB-231, MDA-MB-468, BT-549 and MX-1) using a combination index and isobolograms. In addition, we assessed the effect of the combination in an MDA-MB-231 tumor xenograft model. A synergistic effect was observed in three TNBC cell lines (MDA-MB-231, MDA-MB-468, and MX-1), and in an in vivo study, the combination of S-1 and eribulin resulted in significantly higher antitumor effects compared with S-1 or eribulin alone. 5-FU induced epithelial-mesenchymal transition (EMT) change in the TNCB cell line, as supported by the decreased expression of epithelial marker and the increased expression of mesenchymal markers. Meanwhile, TGF-beta induced EMT changes in a TNBC cell line and decreased the sensitivity to 5-FU. This result suggests that 5-FU-induced EMT changes reduce the sensitivity to 5-FU. In contrast, eribulin induced a mesenchymal-epithelial transition (MET) in a TNBC cell line. The EMT phenotype induced by 5-FU was also canceled by eribulin. We demonstrate that the combination of S-1 (5-FU) and eribulin exerts a synergistic effect for TNBC cell lines through MET-induction by eribulin. Therefore, this combination therapy may be a potential treatment option for TNBC.
  • Hyonchol Kim; Hideyuki Terazono; Yoshiyasu Nakamura; Kazuko Sakai; Akihiro Hattori; Masao Odaka; Mathias Girault; Tokuzo Arao; Kazuto Nishio; Yohei Miyagi; Kenji Yasuda
    PLOS ONE 9 8 e104372  2014年08月 [査読有り]
     
    An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as "imaging biomarkers'', with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 mu m(2) and (2) nucleus area larger than 90 mu m(2) were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 mu m(2) were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers-cluster area, nuclei area, nuclei number, and ratio of perimeter-can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.
  • Yosuke Togashi; Hiroki Sakamoto; Hidetoshi Hayashi; Masato Terashima; Marco A. de Velasco; Yoshihiko Fujita; Yasuo Kodera; Kazuko Sakai; Shuta Tomida; Masayuki Kitano; Akihiko Ito; Masatoshi Kudo; Kazuto Nishio
    MOLECULAR CANCER 13 126  2014年05月 [査読有り]
     
    Background: Transforming growth factor, beta (TGFB) signal is considered to be a tumor suppressive pathway based on the frequent genomic deletion of the SMAD4 gene in pancreatic cancer (PC); however; the role of the activin signal, which also belongs to the TGFB superfamily, remains largely unclear. Methods and results: We found a homozygous deletion of the activin A receptor, type IB (ACVR1B) gene in 2 out of 8 PC cell lines using array-comparative genomic hybridization, and the absence of ACVR1B mRNA and protein expression was confirmed in these 2 cell lines. Activin A stimulation inhibited cellular growth and increased the phosphorylation level of SMAD2 and the expression level of p21(CIP1/WAF1) in the Sui66 cell line (wild-type ACVR1B and SMAD4 genes) but not in the Sui68 cell line (homozygous deletion of ACVR1B gene). Stable ACVR1B-knockdown using short hairpin RNA cancelled the effects of activin A on the cellular growth of the PC cell lines. In addition, ACVR1B-knockdown significantly enhanced the cellular growth and colony formation abilities, compared with controls. In a xenograft study, ACVR1B-knockdown resulted in a significantly elevated level of tumorigenesis and a larger tumor volume, compared with the control. Furthermore, in clinical samples, 6 of the 29 PC samples (20.7%) carried a deletion of the ACVR1B gene, while 10 of the 29 samples (34.5%) carried a deletion of the SMAD4 gene. Of note, 5 of the 6 samples with a deletion of the ACVR1B gene also had a deletion of the SMAD4 gene. Conclusion: We identified a homozygous deletion of the ACVR1B gene in PC cell lines and clinical samples and proposed that the deletion of the ACVR1B gene may mediate an aggressive cancer phenotype in PC. Our findings provide novel insight into the role of the activin signal in PC.
  • Isamu Okamoto; Kazuko Sakai; Satoshi Morita; Hiroshige Yoshioka; Hiroyasu Kaneda; Koji Takeda; Tomonori Hirashima; Yoshihito Kogure; Tatsuo Kimura; Toshiaki Takahashi; Shinji Atagi; Takashi Seto; Toshiyuki Sawa; Masashi Yamamoto; Miyako Satouchi; Motoyasu Okuno; Seisuke Nagase; Koichi Takayama; Keisuke Tomii; Tadashi Maeda; Satoshi Oizumi; Shinji Fujii; Yusaku Akashi; Kazumi Nishino; Noriyuki Ebi; Kazuhiko Nakagawa; Yoichi Nakanishi; Kazuto Nishio
    ONCOTARGET 5 8 2293 - 2304 2014年04月 [査読有り]
     
    Archival formalin-fixed, paraffin-embedded (FFPE) tumor specimens were collected from advanced NSCLC patients enrolled in LETS phase III trial comparing first-line S-1/carboplatin with paclitaxel/carboplatin and subjected to multiplex genotyping for 214 somatic hotspot mutations in 26 genes (LungCarta Panel) and 20 major variants of ALK, RET, and ROS1 fusion genes (LungFusion Panel) with the Sequenom MassARRAY platform. MET amplification was evaluated by fluorescence in situ hybridization. A somatic mutation in at least one gene was identified in 48% of non-squamous cell carcinoma and 45% of squamous cell carcinoma specimens, with EGFR (17%), TP53 (11%), STK11 (9.8%), MET (7.6%), and KRAS (6.2%). Mutations in EGFR or KRAS were associated with a longer or shorter median overall survival, respectively. The LungFusion Panel identified ALK fusions in six cases (2.5%), ROS1 fusions in five cases (2.1%), and a RET fusion in one case (0.4%), with these three types of rearrangement being mutually exclusive. Nine (3.9%) of 229 patients were found to be positive for de novo MET amplification. This first multiplex genotyping of NSCLC associated with a phase III trial shows that MassARRAY-based genetic testing for somatic mutations and fusion genes performs well with nucleic acid derived from FFPE specimens of NSCLC tissue.
  • Hisato Kawakami; Isamu Okamoto; Kyoichi Terao; Kazuko Sakai; Minoru Suzuki; Shinya Ueda; Kaoru Tanaka; Kiyoko Kuwata; Yume Morita; Koji Ono; Kazuto Nishio; Yasumasa Nishimura; Katsumi Doi; Kazuhiko Nakagawa
    CANCER MEDICINE 2 6 933 - 941 2013年12月 [査読有り]
     
    Human papillomavirus (HPV) is a major etiologic factor for oropharyngeal squamous cell carcinoma (OPSCC). However, little is known about HPV-related OPSCC in Japan. During the study, formalin-fixed, paraffin-embedded OPSCC specimens from Japanese patients were analyzed for HPV DNA by the polymerase chain reaction (PCR) and for the surrogate marker p16 by immunohistochemistry. For HPV DNA-positive, p16-negative specimens, the methylation status of the p16 gene promoter was examined by methylation-specific PCR. Overall survival was calculated in relation to HPV DNA and p16 status and was subjected to multivariate analysis. OPSCC cell lines were examined for sensitivity to radiation or cisplatin in vitro. The study results showed that tumor specimens from 40 (38%) of the 104 study patients contained HPV DNA, with such positivity being associated with tumors of the tonsils, lymph node metastasis, and nonsmoking. Overall survival was better for OPSCC patients with HPV DNA than for those without it (hazard ratio, 0.214; 95% confidence interval, 0.074-0.614; P = 0.002). Multivariate analysis revealed HPV DNA to be an independent prognostic factor for overall survival (P = 0.015). Expression of p16 was associated with HPV DNA positivity. However, 20% of HPV DNA-positive tumors were negative for p16, with most of these tumors manifesting DNA methylation at the p16 gene promoter. Radiation or cisplatin sensitivity did not differ between OPSCC cell lines positive or negative for HPV DNA. Thus, positivity for HPV DNA identifies a distinct clinical subset of OPSCC with a more favorable outcome in Japanese.
  • Kazuko Sakai; Atsushi Horiike; Darryl L. Irwin; Keita Kudo; Yoshihiko Fujita; Azusa Tanimoto; Toshio Sakatani; Ryota Saito; Kyohei Kaburaki; Noriko Yanagitani; Fumiyoshi Ohyanagi; Makoto Nishio; Kazuto Nishio
    CANCER SCIENCE 104 9 1198 - 1204 2013年09月 [査読有り]
     
    A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR-tyrosine kinase inhibitors (EGFR-TKIs). A non-invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non-small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR-TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression-free survival (PFS) of the patients with the T790M mutation (n=21) was not statistically different from that of the patients without the mutation (n=54, P=0.94). When patients under 65years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M-positive patients (n=11) was significantly shorter than that of the T790M-negative patients (n=29, P=0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR-TKI therapy.
  • Kazuko Sakai; Atsushi Horiike; Darryl L. Irwin; Keita Kudo; Yoshihiko Fujita; Azusa Tanimoto; Toshio Sakatani; Ryota Saito; Kyohei Kaburaki; Noriko Yanagitani; Fumiyoshi Ohyanagi; Makoto Nishio; Kazuto Nishio
    Cancer Science 104 9 1198 - 1204 2013年09月 [査読有り]
     
    A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR-tyrosine kinase inhibitors (EGFR-TKIs). A non-invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non-small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR-TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression-free survival (PFS) of the patients with the T790M mutation (n = 21) was not statistically different from that of the patients without the mutation (n = 54, P = 0.94). When patients under 65 years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M-positive patients (n = 11) was significantly shorter than that of the T790M-negative patients (n = 29, P = 0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR-TKI therapy. © 2013 Japanese Cancer Association.
  • Issei Kurahashi; Yoshihiko Fujita; Tokuzo Arao; Takayasu Kurata; Yasuhiro Koh; Kazuko Sakai; Koji Matsumoto; Maki Tanioka; Koji Takeda; Yuichi Takiguchi; Nobuyuki Yamamoto; Asuka Tsuya; Nobuaki Matsubara; Hirofumi Mukai; Hironobu Minami; Naoko Chayahara; Yasuhiro Yamanaka; Keisuke Miwa; Shin Takahashi; Shunji Takahashi; Kazuhiko Nakagawa; Kazuto Nishio
    PLOS ONE 8 5 63249  2013年05月 [査読有り]
     
    Background: The biological basis for cancer of unknown primary (CUP) at the molecular level remains largely unknown, with no evidence of whether a common biological entity exists. Here, we assessed the possibility of identifying a common diagnostic biomarker for CUP using a microarray gene expression analysis. Methods: Tumor mRNA samples from 60 patients with CUP were analyzed using the Affymetrix U133A Plus 2.0 GeneChip and were normalized by asinh (hyperbolic arc sine) transformation to construct a mean gene-expression profile specific to CUP. A gene-expression profile specific to non-CUP group was constructed using publicly available raw microarray datasets. The t-tests were performed to compare the CUP with non-CUP groups and the top 59 CUP specific genes with the highest fold change were selected (p-value<0.001). Results: Among the 44 genes that were up-regulated in the CUP group, 6 genes for ribosomal proteins were identified. Two of these genes (RPS7 and RPL11) are known to be involved in the Mdm2-p53 pathway. We also identified several genes related to metastasis and apoptosis, suggesting a biological attribute of CUP. Conclusions: The protein products of the up-regulated and down-regulated genes identified in this study may be clinically useful as unique biomarkers for CUP.
  • Tanizaki Junko; Okamoto Isamu; Okabe Takafumi; Sakai Kazuko; Tanaka Kaoru; Hayashi Hidetoshi; Kaneda Hiroyasu; Takezawa Ken; Nishio Kazuto; Nakagawa Kazuhiko
    CANCER RESEARCH 73 8 2013年04月 [査読有り]
  • M. Takeda; I. Okamoto; K. Sakai; H. Kawakami; K. Nishio; K. Nakagawa
    Annals of Oncology 23 11 2931 - 2936 2012年11月 [査読有り]
     
    Background: The EML4-ALK fusion oncogene represents a recently identified molecular target in a subset of patients with non-small-cell lung cancer (NSCLC). Limited data have been available, however, on the outcome of first-line platinum-based chemotherapy in patients with EML4-ALK-positive advanced NSCLC who have not been treated with an ALK kinase inhibitor. Patients and methods: The efficacy of platinum-based chemotherapy was compared between patients with advanced nonsquamous NSCLC who harbor EML4-ALK and those who harbor EGFR mutations and those with neither molecular abnormality. Results: Among 200 patients with advanced nonsquamous NSCLC, 18 (9.0%) were positive for EML4-ALK, 31 (15.5%) harbored EGFR mutations, and 151 (75.5%) were wild type for both abnormalities. Platinum-based combination chemotherapy showed similar efficacies in the EML4-ALK, EGFR mutation, and wild-type cohorts in terms of response rate and progression-free survival, and overall survival in the EML4-ALK cohort closely resembled that in the wild-type cohort. Within the EML4-ALK cohort, patients with variants 1 or 3 of the fusion gene were predominant and did not appear to differ in their sensitivity to the platinum-based regimens. Conclusion: Patients with EML4-ALK-positive advanced NSCLC manifest an aggressive clinical course similar to that of those with wild-type tumors if the effective targeted therapy is not instituted. © The Author 2012.
  • Junko Tanizaki; Isamu Okamoto; Takafumi Okabe; Kazuko Sakai; Kaoru Tanaka; Hidetoshi Hayashi; Hiroyasu Kaneda; Ken Takezawa; Kiyoko Kuwata; Haruka Yamaguchi; Erina Hatashita; Kazuto Nishio; Kazuhiko Nakagawa
    CLINICAL CANCER RESEARCH 18 22 6219 - 6226 2012年11月 [査読有り]
     
    Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib show marked efficacy in patients with non-small cell lung cancer positive for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown. Experimental Design: We established lines of EML4-ALK-positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK-positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristics. Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of epidermal growth factor receptor (EGFR), HER2, and HER3 was upregulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal-regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion. Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK. Clin Cancer Res; 18(22); 6219-26. (C)2012 AACR.
  • Hidetoshi Hayashi; Isamu Okamoto; Hideharu Kimura; Kazuko Sakai; Yasumasa Nishimura; Kazuto Nishio; Kazuhiko Nakagawa
    ANTICANCER RESEARCH 32 10 4533 - 4537 2012年10月 [査読有り]
     
    Background: Little is known about the occurrence and consequences of epidermal growth factor receptor gene (EGFR) mutations and the fusion of the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes in locally advanced non-small cell lung cancer (NSCLC). Patients and Methods: We retrospectively examined 37 patients with locally advanced NSCLC treated with definitive thoracic radiotherapy (TRT). Characteristics were compared among patients classified positive for EGFR mutations, EML4-ALK rearrangement, or patients who were double-negative for these changes. Results: We identified 11 (29.7%) patients with EGFR mutations and 3 (8.1%) with an EML4-ALK rearrangement. Progression-free survival of patients with EGFR mutation-positive NSCLC was similar to the one of those with double-negative disease (13.1 and 18.6 months). The incidence of local recurrence was higher in EGFR mutation positive patients with NSCLC than in double-negative NSCLC. Conclusion: EGFR mutations and the EML4-ALK rearrangements were detected in substantial proportions of patients with locally advanced NSCLC. The efficacy of TRT was limited in patients with EGFR mutations.
  • Yoshihiko Fujita; Rafiqul Islam; Kazuko Sakai; Hiroyasu Kaneda; Kanae Kudo; Daisuke Tamura; Keiichi Aomatsu; Tomoyuki Nagai; Hidekazu Kimura; Kazuko Matsumoto; Marco A. de Velasco; Tokuzo Arao; Tadashi Okawara; Kazuto Nishio
    INVESTIGATIONAL NEW DRUGS 30 5 1878 - 1886 2012年10月 [査読有り]
     
    Resveratrol (3, 4', 5-trihydroxy-trans-stilbene), a natural phytoalexin found in grapes and wine, has anti-proliferative activity on human-derived cancer cells. In our study, we used a conventional condensation reaction between aldehydes and amines to provide a number of aza-resveratrol (3, 4', 5-trihydroxy-trans- aza-stilbene) derivatives in an attempt to screen for compounds with resveratrol's action but with increased potency. Aza-resveratrol and its hydroxylated derivative (3, 4, 4', 5-tetrahydroxy-trans- aza-stilbene) showed a more enhanced anti-proliferative effect than resveratrol in an MCF-7 breast carcinoma cell line. To identify the cellular targets of the aza derivatives of resveratrol, we conjugated the latter aza-stilbene compound with epoxy-activated agarose and performed affinity purification. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, was identified as a major target protein in MCF-7 cell lysates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). The aza-resveratrol and its hydroxylated derivative, but not resveratrol, were also found to be potent inhibitors of MIF tautomerase activity, which may be associated with their inhibitory effects on MIF bioactivity for cell growth.
  • Hiroyasu Kaneda; Isamu Okamoto; Kazuko Sakai; Junko Tanizaki; Masayuki Takeda; Kazuto Nishio; Kazuhiko Nakagawa
    ACTA ONCOLOGICA 51 7 942 - U150 2012年09月 [査読有り]
  • Wataru Okamoto; Isamu Okamoto; Tokuzo Arao; Kiyoko Kuwata; Erina Hatashita; Haruka Yamaguchi; Kazuko Sakai; Kazuyoshi Yanagihara; Kazuto Nishio; Kazuhiko Nakagawa
    MOLECULAR CANCER THERAPEUTICS 11 7 1557 - 1564 2012年07月 [査読有り]
     
    Therapeutic strategies that target the tyrosine kinase MET hold promise for gastric cancer, but the mechanism underlying the antitumor activity of such strategies remains unclear. We examined the antitumor action of the MET tyrosine kinase inhibitor crizotinib (PF-02341066) in gastric cancer cells positive or negative for MET amplification. Inhibition of MET signaling by crizotinib or RNA interference-mediated MET depletion resulted in induction of apoptosis accompanied by inhibition of AKT and extracellular signal-regulated kinase phosphorylation in gastric cancer cells with MET amplification but not in those without it, suggesting that MET signaling is essential for the survival of MET amplification-positive cells. Crizotinib upregulated the expression of BIM, a proapoptotic member of the Bcl-2 family, as well as downregulated that of survivin, X-linked inhibitor of apoptosis protein (XIAP), and c-IAP1, members of the inhibitor of apoptosis protein family, in cells with MET amplification. Forced depletion of BIM inhibited crizotinib-induced apoptosis, suggesting that upregulation of BIM contributes to the proapoptotic effect of crizotinib. Crizotinib also exhibited a marked antitumor effect in gastric cancer xenografts positive for MET amplification, whereas it had little effect on those negative for this genetic change. Crizotinib thus shows a marked antitumor action both in vitro and in vivo specifically in gastric cancer cells positive for MET amplification. Mol Cancer Ther; 11(7); 1557-64. (C)2012 AACR.
  • Kazuko Sakai; Isamu Okamoto; Ken Takezawa; Tomonori Hirashima; Hiroyasu Kaneda; Masayuki Takeda; Kazuko Matsumoto; Hideharu Kimura; Yoshihiko Fujita; Kazuhiko Nakagawa; Tokuzo Arao; Kazuto Nishio
    JOURNAL OF THORACIC ONCOLOGY 7 5 913 - 918 2012年05月 [査読有り]
     
    Introduction: The presence of the transforming fusion gene echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) in non-small-cell lung cancer (NSCLC) is a predictive marker for the efficacy of anaplastic lymphoma kinase inhibitors. However, the currently available assays for the detection of the different variants of EML4-ALK have limitations. Methods: We developed an assay system for the detection of EML4-ALK variants 1, 2, 3a, 3b, 4, 5a, 5b, 6, or 7 transcripts in total RNA obtained from formalin-fixed, paraffin-embedded (FFPE) specimens of NSCLC tissue. The assay is based on region-specific polymerase chain reaction amplification of EML4-ALK complementary DNA followed by specific single-base primer extension and analysis of the extension products by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The assay was validated by fluorescence in situ hybridization and the results confirmed by subcloning and sequencing of polymerase chain reaction products. Results: Evaluation of the analytic sensitivity of the assay with serial dilutions of plasmids containing EML4-ALK complementary DNA sequences revealed it to be capable of the reliable detection of one copy of each plasmid per reaction. The assay also detected EML4-ALK variants 1 or 3 in three FFPE samples of surgically resected NSCLC shown to be positive for anaplastic lymphoma kinase rearrangement by fluorescence in situ hybridization. Furthermore, the assay identified variant 1 of EML4-ALK in 3 of 20 FFPE biopsy samples from patients with advanced NSCLC. All positive samples were confirmed by subcloning and sequencing. Conclusions: Our novel assay is highly sensitive and effective for the detection of EML4-ALK in FFPE specimens.
  • Masayuki Takeda; Isamu Okamoto; Kazuko Sakai; Kaoru Tanaka; Masaaki Terashima; Kazuto Nishio; Kazuhiko Nakagawa
    CLINICAL LUNG CANCER 13 2 157 - 158 2012年03月 [査読有り]
  • J. Tanizaki; I. Okamoto; K. Takezawa; K. Sakai; K. Azuma; K. Kuwata; H. Yamaguchi; E. Hatashita; K. Nishio; P. A. Janne; K. Nakagawa
    BRITISH JOURNAL OF CANCER 106 4 763 - 767 2012年02月 [査読有り]
     
    BACKGROUND: Although most non-small-cell lung cancer (NSCLC) patients with the echinoderm microtubule-associated protein-like 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene - benefit from ALK tyrosine kinase inhibitors (ALK-TKIs), the efficacy of these drugs varies greatly among individuals. METHODS: The antitumour action of ALK-TKIs in EML4-ALK-positive NSCLC cell lines was evaluated from their effects on cell proliferation, signal transduction, and apoptosis. RESULTS: The ALK-TKI TAE684 inhibited cell proliferation and induced apoptosis, in association with inhibition of STAT3 and ERK phosphorylation, in EML4-ALK-positive H3122 cells. TAE684 inhibited STAT3 phosphorylation, but not ERK phosphorylation, and it showed little effect on cell proliferation or apoptosis, in EML4-ALK-positive H2228 cells. The combination of TAE684 and a MEK inhibitor-induced marked apoptosis accompanied by inhibition of STAT3 and ERK pathways in H2228 cells. Such dual interruption of STAT3 and ERK pathways induced downregulation of the antiapoptotic protein survivin and upregulation of the proapoptotic protein BIM. CONCLUSION: Our results indicate that interruption of both STAT3-survivin and ERK-BIM pathways is required for induction of apoptosis in NSCLC harbouring EML4-ALK, providing a rationale for combination therapy with ALK and MEK inhibitors in EML4-ALK-positive NSCLC patients for whom ALK inhibitors alone are ineffective.
  • Kazuyuki Furuta; Tokuzo Arao; Kazuko Sakai; Hideharu Kimura; Tomoyuki Nagai; Daisuke Tamura; Keiichi Aomatsu; Kanae Kudo; Hiroyasu Kaneda; Yoshihiko Fujita; Kazuko Matsumoto; Yasuhide Yamada; Kazuyoshi Yanagihara; Masaru Sekijima; Kazuto Nishio
    CANCER SCIENCE 103 2 221 - 227 2012年02月 [査読有り]
     
    Whole genome-scale integrated analyses of exon array and array-comparative genomic hybridization are expected to enable the identification of unknown genetic features of cancer cells. Here, we evaluated this approach in 22 gastric and colorectal cancer cell lines, focusing on protein kinase genes and genes belonging to the cadherincatenin family. Regarding alternative splicing patterns, several cancer cell lines predominantly expressed isoform 1 of protein kinase A catalytic subunit beta (PRKACB). Paired gastric cancer specimens demonstrated that isoform 1 of PRKACB was a novel cancer-related variant transcript in gastric cancers. In addition, the exon array analysis clearly identified exon 3 or exon 34 skipping in catenin beta 1, a short intron insertion with exon 9 skipping in CDH1, and a deletional transcript of CDH13. These abnormal transcripts were shown to have arisen from small genomic deletions. Meanwhile, an integrated analysis of 11 gastric cancer cell lines revealed that four cell lines amplified fibroblast growth factor receptor 2, with truncated forms observed in two of the cell lines. Gene amplification, and not the truncated form, was found to determine the sensitivity to a fibroblast growth factor receptor inhibitor, indicating that our cell line panel might be useful for cell-based evaluations of specific inhibitors. Using an integrated analysis, we identified several abnormal transcripts and genomic alterations in gastric and colorectal cancer cells. Our approach might enable genetic changes to be identified more efficiently, and the present results warrant further investigation using clinical samples and integrated analyses. (Cancer Sci 2012; 103: 221227)
  • Makoto Nishio; Takeharu Yamanaka; Kazuko Matsumoto; Hideharu Kimura; Kazuko Sakai; Asao Sakai; Takashi Sone; Atsushi Horiike; Fumiaki Koizumi; Kazuo Kasahara; Tatsuo Ohira; Norihiko Ikeda; Nagahiro Saijo; Tokuzo Arao; Kazuto Nishio
    JOURNAL OF THORACIC ONCOLOGY 6 11 1889 - 1894 2011年11月 [査読有り]
     
    Introduction: The epidermal growth factor receptor (EGFR) mutation status is a validated biomarker for the stratification of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) treatment in patients with non-small cell lung cancer (NSCLC); however, its use is limited in patients with wild-type EGFR, and new biomarkers are needed. We hypothesized that the serum concentration of heparan sulfate (HS), which activates oncogenic growth factor receptor signaling through EGFR and non-EGFR signaling pathways, may be a novel glycobiological biomarker for EGFR-TKIs treatment in NSCLC. Methods: The pretreatment serum HS concentrations were determined using enzyme-linked immunosorbent assay in 83 patients with stage IV non-small cell lung adenocarcinoma who received EGFR-TKIs treatment. The relationship between the serum HS concentrations and patient characteristics, tumor response, progression-free survival (PFS), and overall survival (OS) were analyzed. Results: Patient sex, performance status, smoking history, and EGFR mutation status were associated with tumor response. The serum HS concentrations were significantly higher among patients with progressive disease than among those without progressive disease (p = 0.003). Furthermore, the serum HS concentrations were strongly associated with a poor PFS and OS in a univariate Cox analysis (p = 0.0022 and p = 0.0003, respectively). A stratified multivariate Cox model according to the EGFR mutation status showed that higher HS concentrations were significantly associated with a shorter PFS and OS (p = 0.0012 and p = 0.0003). Conclusion: We concluded that a high-serum HS concentration was strongly related to a poor treatment outcome of EGFR-TKIs and may be a promising noninvasive and repeatable glycobiological biomarker in cancer treatment.
  • H. Kaneda; T. Arao; K. Matsumoto; M. A. De Velasco; D. Tamura; K. Aomatsu; K. Kudo; K. Sakai; T. Nagai; Y. Fujita; K. Tanaka; K. Yanagihara; Y. Yamada; I. Okamoto; K. Nakagawa; K. Nishio
    BRITISH JOURNAL OF CANCER 105 8 1210 - 1217 2011年10月 [査読有り]
     
    BACKGROUND: Activin A is a multi-functional cytokine belonging to the transforming growth factor-beta (TGF-beta) superfamily; however, the effect of activin A on angiogenesis remains largely unclear. We found that inhibin beta A subunit (INHBA) mRNA is overexpressed in gastric cancer (GC) specimens and investigated the effect of activin A, a homodimer of INHBA, on angiogenesis in GC. METHODS: Anti-angiogenic effects of activin A via p21 induction were evaluated using human umbilical vein endothelial cells (HUVECs) in vitro and a stable INHBA-introduced GC cell line in vivo. RESULTS: Compared with TGF-beta, activin A potently inhibited the cellular proliferation and tube formation of HUVECs with induction of p21. A promoter assay and a chromatin immunoprecipitation assay revealed that activin A directly regulates p21 transcriptional activity through Smads. Stable p21-knockdown significantly enhanced the cellular proliferation of HUVECs. Notably, stable p21-knockdown exhibited a resistance to activin-mediated growth inhibition in HUVECs, indicating that p21 induction has a key role on activin A-mediated growth inhibition in vascular endothelial cells. Finally, a stable INHBA-introduced GC cell line exhibited a decrease in tumour growth and angiogenesis in vivo. CONCLUSION: Our findings highlight the suppressive role of activin A, unlike TGF-beta, on tumour growth and angiogenesis in GC. British Journal of Cancer (2011) 105, 1210-1217. doi:10.1038/bjc.2011.348 www.bjcancer.com Published online 6 September 2011 (C) 2011 Cancer Research UK
  • J. Tanizaki; I. Okamoto; K. Sakai; K. Nakagawa
    BRITISH JOURNAL OF CANCER 105 6 807 - 813 2011年09月 [査読有り]
     
    BACKGROUND: MET is a receptor tyrosine kinase (RTK) whose gene is amplified in various tumour types. We investigated the roles and mechanisms of RTK heterodimerisation in lung cancer with MET amplification. METHODS: With the use of an RTK array, we identified phosphorylated RTKs in lung cancer cells with MET amplification. We examined the roles and mechanisms of action of these RTKs with immunoprecipitation, annexin V binding, and cell migration assays. RESULTS: We identified epidermal growth factor receptor (EGFR), human EGFR (HER) 2, HER3, and RET in addition to MET as highly phosphorylated RTKs in lung cancer cells with MET amplification. Immunoprecipitation revealed that EGFR, HER2, HER3, and RET each formed a heterodimer exclusively with MET and that these associations were markedly reduced in extent by treatment with a MET kinase inhibitor. RNA interference-mediated depletion of EGFR, HER2, or HER3 induced apoptosis in association with inhibition of AKT and ERK signalling pathways, whereas depletion of HER2 or RET inhibited both cell migration and STAT3 signalling. CONCLUSION: Our data suggest that heterodimers of MET with EGFR, HER2, HER3, or RET have differential roles in tumour development, and they provide new insight into the function of trans-phosphorylated RTKs as heterodimerisation partners of MET in lung cancer with MET amplification. British Journal of Cancer (2011) 105, 807-813. doi: 10.1038/bjc.2011.322 www.bjcancer.com Published online 16 August 2011 (C) 2011 Cancer Research UK
  • Tokuzo Arao; Kazuko Matsumoto; Kazuyuki Furuta; Kanae Kudo; Hiroyasu Kaneda; Tomoyuki Nagai; Kazuko Sakai; Yoshihiko Fujita; Daisuke Tamura; Keiichi Aomatsu; Fumiaki Koizumi; Kazuto Nishio
    ANTICANCER RESEARCH 31 9 2787 - 2796 2011年09月 [査読有り]
     
    Acquired resistance to antiangiogenic drugs has emerged as a potentially important issue in clinical settings; however, the underlying molecular and cellular mechanism of resistance to vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitor (TKI) remains largely unclear. We evaluated the cellular characteristics of human umbilical vein endothelial cell (HUVEC) clones, which are resistant to VEGFR2-TKI (Ki8751) to elucidate this mechanism of resistance to antiangiogenic drugs. Resistant HUVEC clones were 10-fold more resistant to VEGFR2-TKI than the parental cells and they exhibited an almost complete absence of VEGF-mediated cellular proliferation. The mRNA expression analysis revealed that expression of VEGFR1, VEGFR2 and VEGFR3 was lower in resistant clones, while that of several angiogenic ligands was increased. The protein expression of VEGFR2 was markedly down-regulated in two (R5 and R6 clone) out of five resistant clones. Focusing on the R5 clone, VEGF stimulation did not increase the phosphorylation of VEGFR2 or the dimerization of VEGFR2. The inhibition of phospho-AKT by VEGFR2-TKI was also weakened more than 10-fold in the R5 clone. Finally, a microarray analysis revealed that some angiogenesis-associated, and some angiogenesis-specific genes, including platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31, homeobox A9 (HOXA9), and endothelial cell-specific molecule 1 (ESM1), were remarkably down-regulated in all the resistant clones compared with the parental cells. HUVEC clones resistant to VEGFR2-TK1 exhibited down-regulation of VEGFR2, a decreased signal response to VEGF stimulation, and the loss of vascular endothelial markers. These results strongly suggest that an escape from VEGFR2 signaling-dependency is one of the cellular mechanisms of resistance to VEGFR2-TK1 in vascular endothelial cells.
  • K. Takezawa; I. Okamoto; W. Okamoto; M. Takeda; K. Sakai; S. Tsukioka; K. Kuwata; H. Yamaguchi; K. Nishio; K. Nakagawa
    BRITISH JOURNAL OF CANCER 104 10 1594 - 1601 2011年05月 [査読有り]
     
    BACKGROUND: Although a high level of thymidylate synthase (TS) expression in malignant tumours has been suggested to be related to a reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in non-small cell lung cancer (NSCLC). We have now investigated the effect of TS overexpression on pemetrexed sensitivity in NSCLC cells. METHODS: We established NSCLC cell lines that stably overexpress TS and examined the effects of such overexpression on the cytotoxicity of pemetrexed both in vitro and in xenograft models. We further examined the relation between TS expression in tumour specimens from NSCLC patients and the tumour response to pemetrexed by immunohistochemical analysis. RESULTS: The sensitivity of NSCLC cells overexpressing TS to the antiproliferative effect of pemetrexed was markedly reduced compared with that of control cells. The inhibition of DNA synthesis and induction of apoptosis by pemetrexed were also greatly attenuated by forced expression of TS. Furthermore, tumours formed by TS-overexpressing NSCLC cells in nude mice were resistant to the growth-inhibitory effect of pemetrexed observed with control tumours. Finally, the level of TS expression in tumours of non-responding patients was significantly higher than that in those of responders, suggestive of an inverse correlation between TS expression and tumour response to pemetrexed. CONCLUSION: A high level of TS expression confers a reduced sensitivity to pemetrexed. TS expression is thus a potential predictive marker for response to pemetrexed-based chemotherapy in NSCLC patients. British Journal of Cancer (2011) 104, 1594-1601. doi: 10.1038/bjc.2011.129 www.bjcancer.com Published online 12 April 2011 (C) 2011 Cancer Research UK
  • Koichi Azuma; Junji Tsurutani; Kazuko Sakai; Hiroyasu Kaneda; Yasuhito Fujisaka; Masayuki Takeda; Masahiro Watatani; Tokuzo Arao; Taroh Satoh; Isamu Okamoto; Takayasu Kurata; Kazuto Nishio; Kazuhiko Nakagawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 407 1 219 - 224 2011年04月 [査読有り]
     
    Agents that target HER2 have improved the prognosis of patients with HER2-amplified breast cancers. However, patients who initially respond to such targeted therapy eventually develop resistance to the treatment. We have established a line of lapatinib-resistant breast cancer cells (UACC812/LR) by chronic exposure of HER2-amplified and lapatinib-sensitive UACC812 cells to the drug. The mechanism by which UACC812/LR acquired resistance to lapatinib was explored using comprehensive gene hybridization. The FGFR2 gene in UACC812/LR was highly amplified, accompanied by overexpression of FGER2 and reduced expression of HER2, and a cell proliferation assay showed that the IC50 of PD173074, a small-molecule inhibitor of FGFR tyrosine kinase, was 10,000 times lower in UACC812/LR than in the parent cells. PD173074 decreased the phosphorylation of FGFR2 and substantially induced apoptosis in UACC812/LR, but not in the parent cells. FGFR2 appeared to be a pivotal molecule for the survival of UACC812/LR as they became independent of the HER2 pathway, suggesting that a switch of addiction from the HER2 to the FGFR2 pathway enabled cancer cells to become resistant to HER2-targeted therapy. The present study is the first to implicate FGFR in the development of resistance to lapatinib in cancer, and suggests that EGER-targeted therapy might become a promising salvage strategy after lapatinib failure in patients with HER2-positive breast cancer. (C) 2011 Elsevier Inc. All rights reserved.
  • Kanae Kudo; Tokuzo Arao; Kaoru Tanaka; Tomoyuki Nagai; Kazuyuki Furuta; Kazuko Sakai; Hiroyasu Kaneda; Kazuko Matsumoto; Daisuke Tamura; Keiichi Aomatsu; Marco A. De Velasco; Yoshihiko Fujita; Nagahiro Saijo; Masatoshi Kudo; Kazuto Nishio
    CLINICAL CANCER RESEARCH 17 6 1373 - 1381 2011年03月 [査読有り]
     
    Purpose: BIBF 1120 is a potent, orally available triple angiokinase inhibitor that inhibits VEGF receptors (VEGFR) 1, 2, and 3, fibroblast growth factor receptors, and platelet-derived growth factor receptors. This study examined the antitumor effects of BIBF 1120 on hepatocellular carcinoma (HCC) and attempted to identify a pharmacodynamic biomarker for use in early clinical trials. Experimental Design: We evaluated the antitumor and antiangiogenic effects of BIBF 1120 against HCC cell line both in vitro and in vivo. For the pharmacodynamic study, the phosphorylation levels of VEGFR2 in VEGF-stimulated peripheral blood leukocytes (PBL) were evaluated in mice inoculated with HCC cells and treated with BIBF 1120. Results: BIBF 1120 (0.01 mu mol/L) clearly inhibited the VEGFR2 signaling in vitro. The direct growth inhibitory effects of BIBF 1120 on four HCC cell lines were relatively mild in vitro (IC50 values: 2-5 mu mol/L); however, the oral administration of BIBF 1120 (50 or 100 mg/kg/d) significantly inhibited the tumor growth and angiogenesis in a HepG2 xenograft model. A flow cytometric analysis revealed that BIBF 1120 significantly decreased the phosphotyrosine (pTyr) levels of VEGFR2(+)CD45(dim) PBLs and the percentage of VEGFR2(+)pTyr(+) PBLs in vivo; the latter parameter seemed to be a more feasible pharmacodynamic biomarker. Conclusions: We found that BIBF 1120 exhibited potent antitumor and antiangiogenic activity against HCC and identified VEGFR2(+)pTyr(+) PBLs as a feasible and noninvasive pharmacodynamic biomarker in vivo. Clin Cancer Res; 17(6); 1373-81. (C)2010 AACR.
  • Tomoyuki Nagai; Tokuzo Arao; Kazuyuki Furuta; Kazuko Sakai; Kanae Kudo; Hiroyasu Kaneda; Daisuke Tamura; Keiichi Aomatsu; Hideharu Kimura; Yoshihiko Fujita; Kazuko Matsumoto; Nagahiro Saijo; Masatoshi Kudo; Kazuto Nishio
    MOLECULAR CANCER THERAPEUTICS 10 1 169 - 177 2011年01月 [査読有り]
     
    The epithelial mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Sorafenib, a VEGFR inhibitor with activity against RAF kinase, is active against hepatocellular carcinoma (HCC); however, the possible involvement of sorafenib in the EMT remains unclear. Here, we examined the effect of sorafenib on the EMT. Hepatocyte growth factor (HGF) induced EMT-like morphologic changes and the upregulation of SNAI1 and N-cadherin expression. The downregulation of E-cadherin expression in HepG2 and Huh7 HCC cell lines shows that HGF mediates the EMT in HCC. The knockdown of SNAI1 using siRNA canceled the HGF-mediated morphologic changes and cadherin switching, indicating that SNAI1 is required for the HGF-mediated EMT in HCC. Interestingly, sorafenib and the MEK inhibitor U0126 markedly inhibited the HGF-induced morphologic changes, SNAI1 upregulation, and cadherin switching, whereas the PI3 kinase inhibitor wortmannin did not. Collectively, these findings indicate that sorafenib downregulates SNAI1 expression by inhibiting mitogen-activated protein kinase (MAPK) signaling, thereby inhibiting the EMT in HCC cells. In fact, a wound healing and migration assay revealed that sorafenib completely canceled the HGF-mediated cellular migration in HCC cells. In conclusion, we found that sorafenib exerts a potent inhibitory activity against the EMT by inhibiting MAPK signaling and SNAI1 expression in HCC. Our findings may provide a novel insight into the anti-EMT effect of tyrosine kinase inhibitors in cancer cells. Mol Cancer Ther; 10(1); 169-77. (C)2011 AACR.
  • Kazuo Kasahara; Tokuzo Arao; Kazuko Sakai; Kazuko Matsumoto; Asao Sakai; Hideharu Kimura; Takashi Sone; Atsushi Horiike; Makoto Nishio; Tatsuo Ohira; Norihiko Ikeda; Takeharu Yamanaka; Nagahiro Saijo; Kazuto Nishio
    Clinical Cancer Research 16 18 4616 - 4624 2010年09月 
    Purpose: The epidermal growth factor receptor (EGFR) mutation status has emerged as a validated biomarker for predicting the response to treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKI) in patients with non-small cell lung cancer. However, the responses to EGFR-TKIs vary even among patients with EGFR mutations. We studied several other independently active biomarkers for EGFR-TKI treatment. Experimental Design: We retrospectively analyzed the serum concentrations of 13 molecules in a cohort of 95 patients with non-small cell lung adenocarcinoma who received EGFR-TKI treatment at three centers. The pretreatment serum concentrations of amphiregulin, β-cellulin, EGF, EGFR, epiregulin, fibroblast growth factor-basic, heparin-binding EGF-like growth factor, hepatocyte growth factor (HGF), platelet-derived growth factor β polypeptide, placental growth factor, tenascin C, transforming growth factor-β, and vascular endothelial growth factor (VEGF) were measured using enzyme-linked immunosorbent assay and a multiplex immunoassay system. The associations between clinical outcomes and these molecules were evaluated. Results: The concentrations of HGF and VEGF were significantly higher among patients with progressive disease than among those without progressive disease (P < 0.0001). HGF and VEGF were strongly associated with progression-free survival (PFS) and overall survival (OS) in a univariate Cox analysis (all tests for hazard ratio showed P < 0.0001). A stratified multivariate Cox model according to EGFR mutation status (mutant, n = 20 wild-type, n = 23 unknown, n = 52) showed that higher HGF levels were significantly associated with a shorter PFS and OS (P < 0.0001 for both PFS and OS). These observations were also consistent in the subset analyses. Conclusions: Serum HGF was strongly related to the outcome of EGFR-TKI treatment. Our results suggest that the serum HGF level could be used to refine the selection of patients expected to respond to EGFR-TKI treatment, warranting further prospective study. ©2010 AACR.
  • Kazuo Kasahara; Tokuzo Arao; Kazuko Sakai; Kazuko Matsumoto; Asao Sakai; Hideharu Kimura; Takashi Sone; Atsushi Horiike; Makoto Nishio; Tatsuo Ohira; Norihiko Ikeda; Takeharu Yamanaka; Nagahiro Saijo; Kazuto Nishio
    CLINICAL CANCER RESEARCH 16 18 4616 - 4624 2010年09月 [査読有り]
     
    Purpose: The epidermal growth factor receptor (EGFR) mutation status has emerged as a validated biomarker for predicting the response to treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKI) in patients with non-small cell lung cancer. However, the responses to EGFR-TKIs vary even among patients with EGFR mutations. We studied several other independently active biomarkers for EGFR-TKI treatment. Experimental Design: We retrospectively analyzed the serum concentrations of 13 molecules in a cohort of 95 patients with non-small cell lung adenocarcinoma who received EGFR-TKI treatment at three centers. The pretreatment serum concentrations of amphiregulin, beta-cellulin, EGF, EGFR, epiregulin, fibroblast growth factor-basic, heparin-binding EGF-like growth factor, hepatocyte growth factor (HGF), platelet-derived growth factor beta polypeptide, placental growth factor, tenascin C, transforming growth factor-alpha, and vascular endothelial growth factor (VEGF) were measured using enzyme-linked immunosorbent assay and a multiplex immunoassay system. The associations between clinical outcomes and these molecules were evaluated. Results: The concentrations of HGF and VEGF were significantly higher among patients with progressive disease than among those without progressive disease (P < 0.0001). HGF and VEGF were strongly associated with progression-free survival (PFS) and overall survival (OS) in a univariate Cox analysis (all tests for hazard ratio showed P < 0.0001). A stratified multivariate Cox model according to EGFR mutation status (mutant, n = 20; wild-type, n = 23; unknown, n = 52) showed that higher HGF levels were significantly associated with a shorter PFS and OS (P < 0.0001 for both PFS and OS). These observations were also consistent in the subset analyses. Conclusions: Serum HGF was strongly related to the outcome of EGFR-TKI treatment. Our results suggest that the serum HGF level could be used to refine the selection of patients expected to respond to EGFR-TKI treatment, warranting further prospective study. Clin Cancer Res; 16(18); 4616-24. (c) 2010 AACR.
  • Kaneda Hiroyasu; Arao Tokuzo; Tanaka Kaoru; Tamura Daisuke; Aomatsu Keiichi; Kudo Kanae; Sakai Kazuko; De Velasco Marco Antonio; Matsumoto Kazuko; Yoshihiko Fujita; Yamada Yasuhide; Tsurutani Junji; Okamoto Isamu; Nakagawa Kazuhiko; Nishio Kazuto; Nagai Tomoyuki; Furuta Kazuyuki
    CANCER RESEARCH 70 2010年04月 [査読有り]
  • Hiroyasu Kaneda; Tokuzo Arao; Kaoru Tanaka; Daisuke Tamura; Keiichi Aomatsu; Kanae Kudo; Kazuko Sakai; Marco A. De Velasco; Kazuko Matsumoto; Yoshihiko Fujita; Yasuhide Yamada; Junji Tsurutani; Isamu Okamoto; Kazuhiko Nakagawa; Kazuto Nishio
    CANCER RESEARCH 70 5 2053 - 2063 2010年03月 [査読有り]
     
    Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (CRC; 28-fold/colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using small interfering RNA resulted in a decrease in p21(CIP1/WAF1) expression, and a reporter assay and a chromatin immunoprecipitation assay showed that p21 was one of the target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity [ enhanced green fluorescent protein (EGFP): 2/15, FOXQ1: 7/15] and enhanced tumor growth (437 +/- 301 versus 1735 +/- 769 mm(3), P < 0.001) in vivo. Meanwhile, stable p21 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1 overexpression upregulated several genes that have positive roles for tumor growth, including VEGFA, WNT3A, RSPO2, and BCL11A. CD31 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining of the tumor specimens showed that FOXQ1 overexpression mediated the angiogenic and antiapoptotic effect in vivo. In conclusion, FOXQ1 is overexpressed in CRC and enhances tumorigenicity and tumor growth presumably through its angiogenic and antiapoptotic effects. Our findings show that FOXQ1 is a new member of the cancer-related FOX family. Cancer Res; 70(5); 2053-63. (C) 2010 AACR.
  • Kazuko Sakai; Hideyuki Yokote; Kimiko Murakami-Murofushi; Tomohide Tamura; Nagahiro Saijo; Kazuto Nishio
    CANCER SCIENCE 98 9 1498 - 1503 2007年09月 [査読有り]
     
    A humanized anti-HER2 monoclonal antibody pertuzumab (Omnitarg, 2C4), binding to a different HER2 epitope than trastuzumab, is known as an inhibitor of heterodimerization of the HER receptors. Potent antitumor activity against HER2-expressing breast and prostate cancer cell lines has been clarified, but this potential is not clear against lung cancers. The authors investigated the in vitro anti-tumor activity of pertuzumab against eight non-small cell lung cancer cells expressing various members of the HER receptors. A lung cancer 11_18 cell line expressed a large amount of HER2 and HER3, and its cell growth was stimulated by an HER3 ligand, heregulin (HRG)-alpha. Pertuzumab significantly inhibited the HRG-alpha-stimulated cellular growth of the 11_18 cells. Pertuzumab blocked HRG-alpha-stimulated phosphorylation of HER3, mitogen-activated protein kinase (MAPK), and Akt. In contrast, pertuzumab failed to block epidermal growth factor (EGF)-stimulated phosphorylation of EGF receptor (EGFR) and MAPK. Immunoprecipitation showed that pertuzumab inhibited HRG-alpha-stimulated HER2/HER3 heterodimer formation. HRG-alpha-stimulated HER3 phosphorylation was also observed in the PC-9 cells co-overexpressing EGFR, HER2, and HER3, but the cell growth was neither stimulated by HRG-alpha nor inhibited by pertuzumab. The present results suggest that pertuzumab is effective against HRG-alpha-dependent cell growth in lung cancer cells through inhibition of HRG-alpha-stimulated HER2/HER3 signaling.
  • Hideharu Kimura; Kazuko Sakai; Tokuzo Arao; Tatsu Shimoyama; Tomohide Tamura; Kazuto Nishio
    CANCER SCIENCE 98 8 1275 - 1280 2007年08月 [査読有り]
     
    Cetuximab (Erbitux, IMC-C225) is a monoclonal antibody targeted to the epidermal growth factor receptor (EGFR). To clarify the mode of antitumor action of cetuximab, we examined antibody-dependent cellular cytotoxicity (ADCC) activity against several tumor cell lines expressing wild-type or mutant EGFR. ADCC activity and complement-dependent cytolysis activity were analyzed using the CytoTox 96 assay. ADCC activities correlated with the EGFR expression value (R = 0.924). ADCC activities were detected against all tumor cell lines, except K562 cells in a manner dependent on the cellular EGFR expression level, whereas complement-dependent cytolysis activity was not detected in any of the cell lines. The ADCC activity mediated by cetuximab was examined in HEK293 cells transfected with wild-type EGFR (293W) and a deletional mutant of EGFR (293D) in comparison with the mock transfectant (293M). ADCC activity was detected in 293W and 293D cells, in a cetuximab dose-dependent manner, but not in 293M cells (< 10%). These results indicate that ADCC-dependent antitumor activity results from the degree of affinity of cetuximab for the extracellular domain of EGFR, independent of EGFR mutation status. These results suggest ADCC activity to be one of the modes of therapeutic action of cetuximab and to depend on EGFR expression on the tumor cell surface.
  • Y. Basaki; F. Hosoi; Y. Oda; A. Fotovati; Y. Maruyama; S. Oie; M. Ono; H. Izumi; K. Kohno; K. Sakai; T. Shimoyama; K. Nishio; M. Kuwano
    ONCOGENE 26 19 2736 - 2746 2007年04月 [査読有り]
     
    Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.
  • Kazuko Sakai; Hideyuki Yokote; Kimiko Murakami-Murofushi; Tomohide Tamura; Nagahiro Saijo; Kazuto Nishio
    BIOCHEMICAL JOURNAL 397 3 537 - 543 2006年08月 [査読有り]
     
    The existence of an in-frame deletion mutant correlates with the sensitivity of lung cancers to EGFR (epidermal growth factor receptor)-targeted tyrosine kinase inhibitors. We reported previously that the in-frame 15-bp deletional mutation (deIE746-A750 type deletion) was constitutively active in cells. Kinetic parameters are important for characterizing an enzyme; however, it remains unclear whether the kinetic parameters of deletion mutant EGFR are similar to those of wild-type EGFR. We analysed autophosphorylation in response to ATP and inhibition of gefitinib for deletion mutant EGFR and wild-type EGFR. Kinetic studies, examining autophosphorylation, were carried out using EGFR fractions extracted from 293-p Delta 15 and 293-pEGFR cells transfected with deletion mutant EGFR and wild-type EGFR respectively. We demonstrated the difference in activities between unstimulated wild-type (K-m for ATP = 4.0 +/- 0.3 mu M) and mutant EGFR (K-m for XFP = 2.5 +/- 0.2 mu M). There was no difference in K-m values between EGF-stimulated wild-type EGFR (K-m for ATP = 1.9 + 0.1 mu M) and deletion mutant EGFR (K-m for ATP = 2.2 +/- 0.2 mu M). These results suggest that mutant EGFR is active without ligand stimulation. The K-i value for gefitinib of the deletion mutant EGFR was much lower than that of wild-type EGFR. These results suggest that the deletion mutant EGFR has a higher affinity for gefitinib than wild-type EGFR.
  • Kazuko Sakai; Tokuzo Arao; Tatsu Shimoyama; Kimiko Murofushi; Masaru Sekijima; Naoko Kaji; Tomohide Tamura; Nagahiro Saijo; Kazuto Nishio
    FASEB JOURNAL 20 2 311 - 313 2006年02月 [査読有り]
  • K Sakai; K Sakurai; M Sakai; M Hoshino; Y Goto
    PROTEIN SCIENCE 9 9 1719 - 1729 2000年09月 [査読有り]
     
    Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. beta-Lactoglobulin A has a free thiol at Cysl21, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCl at pH 7.5, producing a modified beta-lactoglobulin (TNB-blg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-blg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional H-1-NMR. The CD spectra of TNB-blg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the ct-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-blg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-blg is monomeric while the intact protein is dimeric. Tn contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-blg were monomeric. The unfolding transition of TNB-blg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The H-1-NMR spectrum for TNB-blg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-blg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-blg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.
  • 坂井 和子; 山崎 領; 星野 大; 河田 康志; 後藤 祐児
    生物物理 40 S162  一般社団法人 日本生物物理学会 2000年
  • 柏井 宏之; 坂井 和子; 櫻井 一正; 星野 大; 後藤 祐児
    生物物理 40 S24  一般社団法人 日本生物物理学会 2000年
  • R Yamasaki; M Hoshino; T Wazawa; Y Ishii; T Yanagida; Y Kawata; T Higurashi; K Sakai; J Nagai; Y Goto
    JOURNAL OF MOLECULAR BIOLOGY 292 5 965 - 972 1999年10月 [査読有り]
     
    To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine. As substrate proteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5. The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography. The recovered complexes were then observed by fluorescence microscopy. For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese. The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins. (C) 1999 Academic Press.
  • 坂井 和子; 山崎 領; 星野 大; 河田 康志; 後藤 祐児
    生物物理 39 S132  一般社団法人 日本生物物理学会 1999年

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    日本泌尿器科学会総会 2020年12月 (一社)日本泌尿器科学会総会事務局
  • 前立腺癌特異的Ptenノックアウトマウスモデルを用いたマルチチロシンキナーゼ阻害薬であるTAS-115の免疫調節について
    倉 由吏恵; デベラスコ・マルコ; 坂井 和子; 清水 信貴; 森 康範; 吉村 一宏; 吉川 和宏; 西尾 和人; 植村 天受
    日本泌尿器科学会総会 2020年12月 (一社)日本泌尿器科学会総会事務局
  • リアルタイムPCRは前立腺癌の腫瘍免疫プロファイルと免疫反応性の評価を可能とする
    植村 天受; 倉 由吏恵; 坂井 和子; 清水 信貴; 森 康則; 野澤 昌弘; 吉村 一宏; 吉川 和宏; 西尾 和人; デベラスコ・マルコ
    日本泌尿器科学会総会 2020年12月 (一社)日本泌尿器科学会総会事務局
  • 非扁平非小細胞肺癌に対するPem+CisとVnr+Cisの比較第III相試験における腫瘍変異負荷の意義(JIPANG-TR)
    坂井 和子; 坪井 正博; 釼持 広知; 後藤 功一; 大平 達夫; 中川 和彦; 細見 幸生; 滝口 裕一; 山本 寛斉; 赤松 弘朗; 佐伯 祥; 杉尾 賢二; 山本 信之; 西尾 和人
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • EGFR T790M変異を有する非小細胞肺癌に対するオシメルチニブの獲得耐性メカニズム
    山口 正史; 小副川 敦; 中村 朝美; 森永 亮太郎; 田中 謙太郎; 柏原 光介; 三浦 隆; 末次 隆行; 田口 健一; 鍋島 一樹; 岸本 淳司; 坂井 和子; 西尾 和人; 杉尾 賢二
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • 異種間遺伝子発現解析による免疫プロファイリングへの応用について
    坂野 恵理; 倉 由吏恵; 坂井 和子; 藤田 至彦; 野澤 昌弘; 吉川 和宏; 西尾 和人; デベラスコ・マルコ; 植村 天受
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • 肺肉腫様癌の分子プロファイリング
    竹ヶ原 京志郎; 坂井 和子; 光冨 徹哉; 西尾 和人
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • 第1/2世代EGFRキナーゼ阻害剤に対する獲得耐性機序のinter-tumor heterogeneityとその臨床的意義
    須田 健一; 村上 功; 小畑 慶子; 藤野 智大; 坂井 和子; 宗 淳一; 西尾 和人; 光冨 徹哉
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • アンドロゲン除去療法は前立腺特異的Ptenノックアウトマウスにおいて免疫療法の抗腫瘍効果を増強する
    植村 天受; 倉 由吏恵; 坂井 和子; 野澤 昌弘; 吉川 和宏; 西尾 和人; デベラスコ・マルコ
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • 大腸炎誘発大腸癌と微生物叢の多様性のインタラクトーム解析
    西尾 和人; 坂井 和子; 倉 由吏恵; 竹ヶ原 京志郎; デベラスコ・マルコ
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • 前立腺癌特異的Ptenノックアウトマウスにおけるマイクロバイオームについての検討
    倉 由吏恵; 坂井 和子; 藤田 至彦; 野澤 昌弘; 吉川 和宏; 西尾 和人; デベラスコ・マルコ; 植村 天受
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • Pten欠損前立腺癌におけるJAK1/2標的治療が糞便中のマイクロバイオームに与える影響について
    デベラスコ・マルコ; 倉 由吏恵; 坂井 和子; 野澤 昌弘; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • がん分子標的薬とバイオマーカー
    西尾 和人; 坂井 和子
    日本癌学会総会記事 2020年10月 (一社)日本癌学会
  • 岩間 映二; 坂井 和子; 高濱 隆幸; 下川 元継; 東 公一; 武田 真幸; 加藤 晃史; 駄賀 晴子; 寺岡 俊輔; 高橋 利明; 大平 達夫; 横山 俊秀; 山本 信之; 中川 和彦; 西尾 和人
    肺癌 2020年10月 (NPO)日本肺癌学会
  • 婦人科がんにおけるクリニカルシークエンス 婦人科がんゲノム医療におけるリキッドバイオプシーの展望
    西尾 和人; 野口 智子; 岩橋 尚幸; 坂井 和子; 井箟 一彦
    日本癌治療学会学術集会抄録集 2020年10月 (一社)日本癌治療学会
  • PD-L1高発現の進行NSCLCにおける総腫瘍径とICIによる治療効果の関係性を検討する観察研究  [通常講演]
    鈴木 慎一郎; 加藤 了資; 原谷 浩司; 林 秀敏; 谷崎 潤子; 尾崎 智博; 長谷川 喜一; 大田 隆代; 千葉 康敬; 伊藤 彰彦; 坂井 和子; 西尾 和人; 中川 和彦
    肺癌 2019年11月 (NPO)日本肺癌学会
  • 固形がんに対する腫瘍遺伝子網羅的解析結果に関する観察研究  [通常講演]
    杉本 藍; 福井 朋也; 佐々木 治一郎; 石原 未希子; 日吉 康弘; 井川 聡; 坂井 和子; 武田 真幸; 高濱 隆幸; 中川 和彦; 西尾 和人; 猶木 克彦
    肺癌 2019年11月 (NPO)日本肺癌学会
  • EGFR-TKI治療における前向きリキッドバイオプシー研究  [通常講演]
    大坪 孝平; 岩間 映二; 坂井 和子; 藤井 亜希子; 中垣 憲明; 西尾 和人; 岡本 勇
    肺癌 2019年11月 (NPO)日本肺癌学会
  • Impact of Co-Mutations in EGFR-Mutated NSCLC Before EGFR-TKIs on T790M Mutation Status After TKIs(和訳中)  [通常講演]
    武田 真幸; 坂井 和子; 林 秀敏; 田中 薫; 原谷 浩司; 高濱 隆幸; 加藤 了資; 米阪 仁雄; 西尾 和人; 中川 和彦
    肺癌 2019年11月 (NPO)日本肺癌学会
  • Next-generation sequencing(NGS)を用いた外科切除症例における再発予測因子としての意義  [通常講演]
    小原 秀太; 須田 健一; 坂井 和子; 藤野 智大; 古賀 教将; 西野 将矢; 濱田 顕; 千葉 眞人; 武本 智樹; 宗 淳一; 西尾 和人; 光冨 徹哉
    肺癌 2019年11月 (NPO)日本肺癌学会
  • ALK融合遺伝子陽性非小細胞肺癌におけるALK-TKI耐性機序に関する検討  [通常講演]
    田中 薫; 谷崎 潤子; 野長瀬 祥兼; 原谷 浩司; 酒井 瞳; 加藤 了資; 渡邉 諭美; 吉田 健史; 佐藤 千尋; 林 秀敏; 坂井 和子; 西尾 和人; 中川 和彦
    肺癌 2019年11月 (NPO)日本肺癌学会
  • 実臨床におけるリキッドバイオプシーの役割 CAPP-Seqを用いたLiquid biopsyによるT790M陽性非小細胞肺癌のオシメルチニブ耐性因子の検討  [通常講演]
    加藤 了資; 林 秀敏; 米阪 仁雄; 原谷 浩司; 酒井 瞳; 高濱 隆幸; 岩朝 勤; 田中 薫; 吉田 健史; 武田 真幸; 金田 裕靖; 清水 重喜; 坂井 和子; 伊藤 彰彦; 西尾 和人; 中川 和彦
    肺癌 2019年11月 (NPO)日本肺癌学会
  • 実臨床におけるリキッドバイオプシーの役割 Circulating tumor DNAを用いた分子診断の現状と課題  [通常講演]
    坂井 和子; 西尾 和人
    肺癌 2019年11月 (NPO)日本肺癌学会
  • 近未来の病理診断 Digital Spatial Profiling Technologyを用いての肺原発Carcinosarcomaの検討  [通常講演]
    清水 重喜; 坂井 和子; 白石 直樹; 小原 秀太; 須田 健一; 武本 智樹; 筑後 孝章; 佐藤 隆夫; 光冨 徹哉; 西尾 和人
    肺癌 2019年11月 (NPO)日本肺癌学会
  • 【がんゲノム情報による免疫療法のPrecision Medicine】がんゲノム医療の現状と今後 免疫療法への応用  [通常講演]
    西尾 和人; 坂井 和子
    癌と化学療法 2019年09月 (株)癌と化学療法社
     
    がん遺伝子パネル検査がわが国でも承認され、プレシジョンメディスンが本格的に実装され、その体制作りが進められている。がんの遺伝子変化を解析することにより効果が期待される治療薬の使用が期待されるが、多くは分子標的薬である。また、実際に指定された薬を用いることができる割合は必ずしも多くない。そのなかで免疫チェックポイント阻害薬のバイオマーカーが注目されている。効果予測マーカーとして、PD-L1の発現と独立した腫瘍変異負荷の臨床応用が待ち望まれている。すでにわが国でも臓器を越えコンパニオン診断薬として承認されたMSI検査やdMMR関連検査も、ICIの選択に有用である。これらはがん遺伝子パネル検査でも情報が得られ、免疫療法を含めたより多くの抗悪性腫瘍薬が用いられることが期待される。(著者抄録)
  • リアルタイムPCRを用いた腫瘍免疫プロファイルと免疫反応性の評価について(A real-time PCR-based approach to quantitatively assess tumor immune profiles and immune responses)  [通常講演]
    野澤 昌弘; デベラスコ・マルコ; 倉 由吏恵; 坂井 和子; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2019年09月 日本癌学会
  • イソフラボン摂取はマウス前立腺癌転移モデルにおいて癌の進行を抑制し生存期間を延長させる(Chemopreventive effects of dietary isoflavone in conditional Pten/Trp53-deficient mouse model of prostate cancer)  [通常講演]
    森 康範; デベラスコ・マルコ; 倉 由吏恵; 坂井 和子; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2019年09月 日本癌学会
  • TAS-115マルチキナーゼ阻害薬のマウス前立腺癌モデルにおける免疫調整について(Immunomodulation of the multi-tyrosine kinase inhibitor TAS-115 in a mouse Pten-deficient prostate cancer)  [通常講演]
    倉 由吏恵; デベラスコ・マルコ; 坂井 和子; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2019年09月 日本癌学会
  • アパルタミドによる前立腺腫瘍内の免疫環境の変化(Apalutamide reworks the tumor immune microenvironment of prostate tumors)  [通常講演]
    清水 信貴; デベラスコ・マルコ; 倉 由吏恵; 坂井 和子; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2019年09月 日本癌学会
  • 腫瘍免疫環境プロファイルと抗腫瘍免疫反応(Profiling the tumor immune milieu to assess and predict immune responses)  [通常講演]
    デベラスコ・マルコ; 倉 由吏恵; 森 康範; 清水 信貴; 大關 孝之; 坂井 和子; 野澤 昌弘; 吉村 一宏; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2019年09月 日本癌学会
  • PD-L1発現陰性/TMB Highの肺腺癌に対して化学療法とペムブロリズマブの併用療法を施行した1例  [通常講演]
    金村 宙昌; 林 秀敏; 武田 真幸; 高濱 隆幸; 田中 薫; 中川 和彦; 坂井 和子; 西尾 和人
    肺癌 2019年08月 (NPO)日本肺癌学会
  • 【進化する肝細胞癌の薬物療法-2019 Update(Part 1)】ラムシルマブ 小分子キナーゼ阻害剤と抗体薬の作用機序の違いに基づいた薬効の差異に関する考察  [通常講演]
    西尾 和人; 坂井 和子
    肝・胆・膵 2019年08月 (株)アークメディア
  • dPCR、NGSを用いたアファチニブ耐性機序の探索的研究  [通常講演]
    岩間 映二; 中西 洋一; 岡本 勇; 坂井 和子; 西尾 和人
    肺癌 2019年06月 (NPO)日本肺癌学会
  • 進行卵巣癌のNAC症例におけるCAPP-seqによるliquid biopsyの有用性についての検討  [通常講演]
    野口 智子; 岩橋 尚幸; 八幡 環; 東嶋 左緒里; 馬淵 泰士; 八木 重孝; 南 佐和子; 坂井 和子; 西尾 和人; 井箟 一彦
    日本婦人科腫瘍学会雑誌 2019年06月 (公社)日本婦人科腫瘍学会
  • 婦人科癌liquid biopsyを用いたblood Tumor Mutation Burdenの検討  [通常講演]
    岩橋 尚幸; 野口 智子; 坂井 和子; 八幡 環; 西尾 和人; 井箟 一彦
    日本婦人科腫瘍学会雑誌 2019年06月 (公社)日本婦人科腫瘍学会
  • 【Somatic/germline変異とがん医療】がん遺伝子パネル検査における胚細胞変異の検出と二次的所見への対応  [通常講演]
    西尾 和人; 坂井 和子
    がん分子標的治療 2019年06月 (株)メディカルレビュー社
     
    がん遺伝子パネル検査が実装されるにあたり、二次的所見に対する対応が必要となる。また、パネル検査の実施前においては十分な遺伝カウンセリング、インフォームドコンセントなどが必要とされる。技術的には、腫瘍検体のターゲットシーケンスにおいてシーケンスエラー、SNPとの判別は必ずしも完璧ではないことに留意し、結果の解釈、患者説明の段階を要する。腫瘍サンプルのみでのパネル検査における二次的所見は確定的ではなく、遺伝学的検査の要否をエキスパートパネルでは考慮すべきである。(著者抄録)
  • EGFR-TKIに耐性化したEGFR遺伝子変異陽性非小細胞肺癌の遺伝子プロファイルをCAPP-Seqにて検討する観察研究  [通常講演]
    大坪 孝平; 岩間 映二; 白石 祥理; 米嶋 康臣; 井上 博之; 田中 謙太郎; 中西 洋一; 岡本 勇; 坂井 和子; 西尾 和人
    肺癌 2019年06月 (NPO)日本肺癌学会
  • 当院におけるオシメルチニブの使用成績と血漿中cfDNAの有用性に関する検討  [通常講演]
    加藤 了資; 林 秀敏; 米阪 仁雄; 原谷 浩司; 酒井 瞳; 高濱 隆幸; 谷崎 潤子; 岩朝 勤; 田中 薫; 吉田 健史; 武田 真幸; 金田 裕靖; 中川 和彦; 清水 重喜; 伊藤 彰彦; 坂井 和子; 西尾 和人
    肺癌 2019年04月 (NPO)日本肺癌学会
  • 次世代ゲノムシーケンス解析による検討を行った血管肉腫と腺癌成分を有する肺癌肉腫の一例  [通常講演]
    力武 美保子; 青木 茂久; 有働 恵美子; 坂井 和子; 米満 伸久; 古里 文吾; 西尾 和人; 福岡 順也; 戸田 修二
    日本病理学会会誌 2019年04月 (一社)日本病理学会
  • 進行卵巣癌のNAC症例における新規バイオマーカーとしてliquid biopsyの有用性  [通常講演]
    野口 智子; 岩橋 尚幸; 八幡 環; 東島 左緒里; 井箟 一彦; 坂井 和子; 西尾 和人
    和歌山医学 2019年03月 和歌山医学会
  • EGFR遺伝子変異陽性肺癌治療における大規模前向きリキッドバイオプシー研究  [通常講演]
    岩間 映二; 坂井 和子; 中西 洋一; 西尾 和人; 岡本 勇
    日本内科学会雑誌 2019年02月 (一社)日本内科学会
  • 【入門!!リキッドバイオプシー】リキッドバイオプシーの原理  [通常講演]
    西尾 和人; 坂井 和子
    泌尿器外科 2019年01月 医学図書出版(株)
     
    がん領域ではリキッドバイオプシーは、主に循環血中の腫瘍細胞、エクソソーム、血中循環RNA、循環腫瘍DNAを指す。リキッドバイオプシーの生物学的、生物物理学的特徴を把握する研究により、リキッドバイオプシーを補足し、遺伝子解析を行うことで、低侵襲なバイオマーカーとして用いられるようになった。デジタルPCR、次世代シークエンサー、メチル化解析等の技術の発展により、プレシジョンメディスンに貢献すると期待されている。(著者抄録)
  • 日本臨床腫瘍学会・日本癌治療学会・日本癌学会合同 次世代シークエンサー等を用いた遺伝子パネル検査に基づくがん診療ガイダンス(第1.0版)  [通常講演]
    西尾 和人; 山本 昇; 秋田 弘俊; 石岡 千加史; 小寺 泰弘; 角南 久仁子; 鈴木 達也; 武田 真幸; 土原 一哉; 武藤 学; 安井 弥; 山崎 健太郎; 若井 俊文; 梅村 茂樹; 梅本 久美子; 川添 彬人; 木村 元; 久保木 恭利; 小金丸 茂博; 古川 孝広; 小谷 大輔; 坂井 和子; 澤田 憲太郎; 設樂 紘平; 新垣 清登; 高橋 秀明; 内藤 陽一; 中村 能章; 原野 謙一; 坂東 英明; 福岡 聖大; 藤本 祐未; 松本 慎吾; 松本 寛史; 三島 沙織; 安田 華世; 吉野 孝之; 日本臨床腫瘍学会; 日本癌治療学会; 日本癌学会合同次世代シークエンサー等を用いた遺伝子パネル検査に基づくがん診療ガイダンス作成ワーキンググループ
    臨床病理レビュー 2018年11月 臨床病理刊行会
  • 次世代シークエンサを用いたゲノム解析による肝疾患研究 肝癌外科術後再発進行症例に対するソラフェニブ投与奏効例のバイオマーカーFGF19検討  [通常講演]
    海堀 昌樹; 坂井 和子; 西尾 和人
    肝臓 2018年11月 (一社)日本肝臓学会
  • 【循環癌細胞(CTC)とリキッドバイオプシー】固形癌を対象としたリキッドバイオプシーの臨床応用  [通常講演]
    西尾 和人; 坂井 和子
    臨床検査 2018年11月 (株)医学書院
     
    ＜文献概要＞Point ●循環腫瘍DNA(ctDNA)は,デジタルPCRや次世代シークェンサー(NGS)を用いて分子プロファイリングが可能である.●リキッドバイオプシー(LB)は,治療効果予測,治療経過中のモニタリングツールとして,その臨床的有用性が示されている.●肺癌EGFR遺伝子変異検査の血漿検査が承認され,用いられている.
  • CAPP-seqを用いたLiquid biopsyによる婦人科癌の網羅的遺伝子変異プロファイリング  [通常講演]
    岩橋 尚幸; 坂井 和子; 野口 智子; 八幡 環; 東嶋 左緒里; 西尾 和人; 井箟 一彦
    日本癌治療学会学術集会抄録集 2018年10月 (一社)日本癌治療学会
  • 肺癌個別化医療に向けた取り組み  [通常講演]
    武田 真幸; 坂井 和子; 林 秀敏; 田中 薫; 高濱 隆幸; 原谷 浩司; 福岡 和也; 中川 和彦; 西尾 和人
    日本癌治療学会学術集会抄録集 2018年10月 (一社)日本癌治療学会
  • ALK融合遺伝子陽性非小細胞肺癌におけるALK-TKI耐性機序に関する検討  [通常講演]
    田中 薫; 谷崎 潤子; 野長瀬 祥兼; 原谷 浩司; 酒井 瞳; 林 秀敏; 坂井 和子; 西尾 和人; 中川 和彦
    肺癌 2018年10月 (NPO)日本肺癌学会
  • 重症SRE(skeletal related events)をきたしたSMARCA4 deficient thoracic sarcoma(SMARCA4-DTS)の2例  [通常講演]
    國政 啓; 田宮 基裕; 木村 円花; 井上 貴子; 西野 和美; 熊谷 融; 中村 ハルミ; 中塚 伸一; 今村 文生; 坂井 和子; 西尾 和人
    肺癌 2018年10月 (NPO)日本肺癌学会
  • Digital PCR、次世代シークエンサーを用いたアファチニブ耐性機序の探索的研究  [通常講演]
    堀田 勝幸; 岩間 映二; 坂井 和子; 東 公一; 原田 大二郎; 野崎 要; 中西 洋一; 西尾 和人; 岡本 勇
    肺癌 2018年10月 (NPO)日本肺癌学会
  • 加藤 了資; 林 秀敏; 米阪 仁雄; 原谷 浩司; 酒井 瞳; 高濱 隆幸; 谷崎 潤子; 岩朝 勤; 田中 薫; 吉田 健史; 武田 真幸; 金田 裕靖; 清水 重喜; 坂井 和子; 伊藤 彰彦; 西尾 和人; 中川 和彦
    肺癌 2018年10月 (NPO)日本肺癌学会
  • 武田 真幸; 坂井 和子; 林 秀敏; 高濱 隆幸; 田中 薫; 西尾 和人; 中川 和彦
    肺癌 2018年10月 (NPO)日本肺癌学会
  • ALK融合遺伝子陽性非小細胞肺癌におけるALK-TKI耐性機序に関する検討  [通常講演]
    田中 薫; 谷崎 潤子; 野長瀬 祥兼; 原谷 浩司; 酒井 瞳; 林 秀敏; 中川 和彦; 坂井 和子; 西尾 和人
    肺癌 2018年10月 (NPO)日本肺癌学会
  • 次世代の医療を切り拓くLiquid Biopsy CAPP-seqを用いたリキッドバイオプシーによるクリニカルシークエンシング(Liquid biopsy paves the way for next-generation medicine Clinical sequencing of circulating tumor DNA by CAPP-seq)  [通常講演]
    坂井 和子; 西尾 和人
    日本癌学会総会記事 2018年09月 日本癌学会
  • Ptenノックアウトマウス前立腺癌モデルを用いたアビラテロンによる抗腫瘍免疫効果に関する検討(Effect of abiraterone therapy on anti-tumor immunity in a mouse Pten-deficient prostate cancer model)  [通常講演]
    清水 信貴; デベラスコ・マルコ; 倉 由吏恵; 大關 孝之; 森 康範; 野澤 昌弘; 吉村 一宏; 坂井 和子; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2018年09月 日本癌学会
  • 卵巣高異型度漿液性癌ではクローン数とヘテロ接合性の消失は予後とサブタイプに関連する(Clonality and loss of heterozygosity are associated with prognosis and subtypes in high grade serous ovarian cancer)  [通常講演]
    高矢 寿光; 中井 英勝; 坂井 和子; 西尾 和人; 松村 謙臣
    日本癌学会総会記事 2018年09月 日本癌学会
  • 前立腺癌患者の予後不良に関わる遺伝子群の同定(Identification of a gene set associated with poor clinical outcomes in prostate cancer patients)  [通常講演]
    橋本 士; デベラスコ・マルコ; 倉 由吏恵; 坂井 和子; 清水 信貴; 森 康範; 野澤 昌弘; 吉村 一宏; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2018年09月 日本癌学会
  • 免疫療法開発における遺伝子改変マウス前立腺癌モデルの有用性について(Interrogating genetically engineered mouse models of prostate cancer to aid in immunotherapy development)  [通常講演]
    デベラスコ・マルコ; 倉 由吏恵; 森 康範; 清水 信貴; 大關 孝之; 坂井 和子; 野澤 昌弘; 吉村 一宏; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2018年09月 日本癌学会
  • Ptenノックアウトマウス前立腺癌モデルにおけるマルチキナーゼ阻害薬TAS-115の有効性についての検討(Preclinical evaluation of the multi tyrosine kinase inhibitor TAS-115 in mouse Pten-deficient prostate cancer)  [通常講演]
    野澤 昌弘; デベラスコ・マルコ; 倉 由吏恵; 清水 信貴; 森 康範; 吉村 一宏; 坂井 和子; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2018年09月 日本癌学会
  • Pimキナーゼ阻害薬は去勢抵抗性マウス前立腺癌モデルにおいて腫瘍増殖を抑制し生存期間を延長する(PIM inhibition reduces tumor growth and improves survival in mouse advanced castration-resistant prostate cancer)  [通常講演]
    倉 由吏恵; デベラスコ・マルコ; 森 康範; 清水 信貴; 大關 孝之; 坂井 和子; 野澤 昌弘; 吉村 一宏; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2018年09月 日本癌学会
  • 遺伝子改変Ptenノックアウトマウス前立腺癌モデルにおけるApalutamideの有効性についての検討(Preclinical activity of apalutamide (ARN-509) in genetically engineered mouse models of Pten-deficient prostate cancer)  [通常講演]
    森 康範; デベラスコ・マルコ; 倉 由吏恵; 大關 孝之; 清水 信貴; 野澤 昌弘; 吉村 一宏; 坂井 和子; 吉川 和宏; 西尾 和人; 植村 天受
    日本癌学会総会記事 2018年09月 日本癌学会
  • 【がんのPrecision Medicine(精密医療)-SCRUM-Japanの取り組みを中心に-】がんゲノムに精通した人材育成のための教育プログラム  [通常講演]
    西尾 和人; 坂井 和子
    Progress in Medicine 2018年09月 (株)ライフ・サイエンス
  • 【動き始めたがんゲノム医療 深化と普及のための基礎研究課題】(第2章)actionableパスウェイ 上皮間葉移行とがん幹細胞のシグナルパスウェイ  [通常講演]
    西尾 和人; 坂井 和子
    実験医学 2018年09月 (株)羊土社
     
    がん幹細胞(CSC)と上皮間葉移行(EMT)はともに治療抵抗性等にかかわる。がん細胞はEMTによりCSC化するが、可逆的である。EMT機構を標的にしてCSCを排除するアプローチは魅力的だが、EMT-CSCのメカニズムのさらなる理解が必要である。(著者抄録)
  • Kuniko Sunami; Hideaki Takahashi; Katsuya Tsuchihara; Masayuki Takeda; Tatsuya Suzuki; Yoichi Naito; Kazuko Sakai; Hirotoshi Dosaka-Akita; Chikashi Ishioka; Yasuhiro Kodera; Manabu Muto; Toshifumi Wakai; Kentaro Yamazaki; Wataru Yasui; Hideaki Bando; Yumi Fujimoto; Shota Fukuoka; Kenichi Harano; Akihito Kawazoe; Gen Kimura; Shigehiro Koganemaru; Takahiro Kogawa; Daisuke Kotani; Yasutoshi Kuboki; Hiroshi Matsumoto; Shingo Matsumoto; Saori Mishima; Yoshiaki Nakamura; Kentaro Sawada; Sumito Shingaki; Kohei Shitara; Kumiko Umemoto; Shigeki Umemura; Kayo Yasuda; Takayuki Yoshino; Noboru Yamamoto; Kazuto Nishio
    Cancer science 2018年09月 
    In Japan, the social (medical) health-care system is on the way to being developed to advance personalized medicine through the implementation of cancer genomic medicine, known as "cancer clinical sequencing," which uses a next-generation sequencer. However, no Japanese guidance for cancer genomic testing exists. Gene panel testing can be carried out to help determine patient treatment, confirm diagnosis, and evaluate prognostic predictions of patients with mainly solid cancers for whom no standard treatment is available. This guidance describes how to utilize gene panel testing according to the type of cancer: childhood cancer, rare cancer, carcinoma of unknown primary, and other cancers. The level of evidence classification for unified use in Japan is also detailed. This guidance establishes the basic principles of the quality control of specimens, requirements of medical institutions, informed consent, handling of data during the postanalysis stage, and treatment options based on the evidence level. In Japan, gene panel testing for cancer treatment and diagnosis is recommended to comply with this guidance. This is a collaborative work of the Japanese Society of Medical Oncology, Japan Society of Clinical Oncology, and the Japanese Cancer Association.
  • リキッドバイオプシーの現況  [通常講演]
    西尾 和人; 坂井 和子
    検査と技術 2018年08月 (株)医学書院
  • リキッドバイオプシーの現況  [通常講演]
    西尾 和人; 坂井 和子
    検査と技術 2018年08月 (株)医学書院
  • 骨芽細胞の性差に関与する遺伝子の網羅的解析とSerpina3nの役割  [通常講演]
    石田 昌義; 岡田 清孝; 峯 嘉宏; 河尾 直之; 辰巳 公平; 坂井 和子; 西尾 和人; 高藤 義正; 梶 博史
    日本骨代謝学会学術集会プログラム抄録集 2018年07月 (一社)日本骨代謝学会
  • 【クリニカルシークエンスと病理診断】クリニカルシークエンスの概要  [通常講演]
    西尾 和人; 坂井 和子
    病理と臨床 2018年07月 (株)文光堂
  • 【クリニカルシークエンスと病理診断】 クリニカルシークエンスの概要  [通常講演]
    西尾 和人; 坂井 和子
    病理と臨床 2018年07月 (株)文光堂
  • 最新のがん がんゲノム医療の進展  [通常講演]
    西尾 和人; 武田 真幸; 福岡 和也; 中川 和彦; 坂井 和子
    近畿大学医学雑誌 2018年06月 近畿大学医学会
  • 【Liquid biopsyへの期待と限界】 Cell free DNAの臨床的意義 Circulating tumor DNAを中心に  [通常講演]
    西尾 和人; 坂井 和子
    医学のあゆみ 2018年05月 医歯薬出版(株)
     
    Cell free DNA(cfDNA)は、他のリキッドバイオプシーと同様に効果予測、予後予測、診断、治療モニタリングなどの低侵襲検査として大きな期待が寄せられている。肺癌患者に対してEGFR遺伝子変異の血漿検査が保険償還されるなど、がん疾患領域におけるcfDNAの実臨床への展開が進んでいる。NGSによる低頻度変異アレルの検出技術の進歩は、cfDNAなどのリキッドバイオプシーによるプレシジョンメディシンを可能にすると期待されている。(著者抄録)
  • 澤田貴宏; 中村有貴; 高濱隆幸; 坂井和子; 横山省三; 山上裕機; 西尾和人
    日本がん分子標的治療学会学術集会プログラム・抄録集 2018年04月
  • 高濱隆幸; 高濱隆幸; 武田真幸; 坂井和子; 中川和彦; 西尾和人
    日本がん分子標的治療学会学術集会プログラム・抄録集 2018年04月
  • 【前立腺癌のバイオマーカー】 リキッドバイオプシーの現況  [通常講演]
    西尾 和人; 坂井 和子
    Prostate Journal 2018年04月 医学図書出版(株)
     
    末梢血の腫瘍由来核酸はcirculating tumor DNA、エクソソーム中、あるいはcirculating tumor cell中に存在する。これらリキッドバイオプシーを用いた遺伝子解析により早期診断、治療効果予測、予後予測、治療モニタリングを低侵襲で行うことができると期待されている。近年、超高感度法であるデジタルPCR、次世代シーケンサーの技術改良により、網羅的な遺伝子解析が可能となり、リキッドバイオプシーによる前立腺がんのプレシジョン・メディシンが現実味を帯びてきた。(著者抄録)
  • 【Precision medicineをめざした胆道・膵悪性腫瘍ゲノム医療の最前線】 膵癌・胆嚢癌におけるリキッドバイオプシーを用いたがん遺伝子解析  [通常講演]
    西尾 和人; 坂井 和子
    胆と膵 2018年04月 医学図書出版(株)
     
    末梢血の腫瘍由来核酸はcirculating tumor DNA、エクソソーム中、あるいはcirculating tumor cell中に存在する。これらリキッドバイオプシーを用いた遺伝子解析により早期診断、治療効果予測、予後予測、治療モニタリングを低侵襲で行うことができると期待されている。それらが可能となってきたのは、超高感度法であるデジタルPCRの登場による。現在では次世代シーケンサーの技術改良により、網羅的な遺伝子解析が可能となり、プレシジョンメディスンが膵癌・胆嚢癌に対しても現実味を帯びてきた。(著者抄録)
  • ソラフェニブは肝細胞癌株のHGF誘導性上皮間葉移行を阻害する.  [通常講演]
    永井 知行; 荒尾 徳三; 古田 一行; 金田 裕靖; 工藤 可苗; 青松 圭一; 田村 大介; 坂井 和子; 木村 英晴; 藤田 至彦; 松本 和子; 工藤 正俊; 西條 長宏; 西尾 和人
    日本がん分子標的治療学会第15回学術集会 2011年06月 ホテル日航東京, 東京 日本がん分子標的治療学会第15回学術集会
  • Sorafenib inhibits the hepatocyte growth factor-mediated epithelial mesenchymal transition in hepatocellular carcinoma. ソラフェニブは肝細胞癌株において、HGF起因の上皮間葉移行（Epithelial mesenchymal transition）を阻害する.  [通常講演]
    永井 知行; 荒尾 徳三; 坂井 和子; 工藤 可苗; 金田 裕靖; 田村 大介; 青松 圭一; 木村 英晴; 藤田 至彦; 松本 和子; 西條 長宏; 工藤 正俊; 西尾 和人
    第69回日本癌学会学術総会 2010年09月 大阪国際会議場, 大阪 第69回日本癌学会学術総会
  • 転写因子snail,slugは角膜上皮細胞においてEMTを誘導しTP63発現レベルを減少させる  [通常講演]
    青松 圭一; 荒尾 徳三; 松本 和子; 金田 裕靖; 坂井 和子; 木村 英晴; 藤田 至彦; 下村 嘉一; 西尾 和人
    2010年09月
  • 血清中HGFはEGFRチロシンキナーゼ阻害剤の効果予測因子である  [通常講演]
    坂井 和子; 荒尾 徳三; 木村 英晴; 松本 和子; 西條 長宏; 西尾 和人; 金沢大学医学部附属病院; 金沢大学医学部附属病院; 金沢大学医学部附属病院; 癌研究会有明病院; 呼吸器内科; 癌研究会有明病院; 呼吸器内科; 東京医科大学外科学第一講座; 東京医科大学外科学第一講座; 国立病院機構九州がんセンター; 臨床研究部腫瘍統計学研究室
    分子標的治療学会 2010年06月 東京 分子標的治療学会
  • TGF-βは転写因子SNAI1 SNAI2発現を介して角膜上皮細胞に上皮間葉移行を誘導する  [通常講演]
    青松 圭一; 荒尾 徳三; 杉岡 孝二; 松本 和子; 金田 裕靖; 坂井 和子; 西尾 和人; 下村 嘉一
    The Association for Research in Vision and Ophthalmology 2010年05月 アメリカ The Association for Research in Vision and Ophthalmology
     
    上皮間葉移行（EMT, epithelial-mesenchymal transition）は胎生期の原腸形成や癌細胞の浸潤・転移において重要な現象である。培養ヒト角膜上皮細胞（HCEC）に対して、TGF-βにより誘導される細胞形質の変化とEMT関連分子の発現変化について検討を行った。 TGF-β刺激は、HCECに対して細胞増殖を有意に抑制し、アポトーシスを誘導した。また、濃度・時間依存的にリン酸化smad2 の亢進を認めた。Realtime RT-PCR法ではEMT関連分子の内、VIM, FN1, SNAI1, SNAI2の有意な発現上昇を認めた（p＜0.05）。またHCEC は、EGF刺激による細胞形態変化は弱いのに対して、TGF-β刺激により一部の細胞が細胞形態的にEMT特徴的な変化を示した。以上の結果からヒト角膜上皮細胞においてTGF-βシグナル経路はＥＭＴ関連遺伝子の発現変化を制御し、EMTを誘導することを示した。
  • Expression levels of EGFR-ligands are up-regulated in EGFR tyrosine kinase inhibitor-resistant cell line.  [通常講演]
    坂井 和子; 荒尾 徳三; 松本 和子; 金田 裕靖; 青松 圭一; 藤田 至彦; 西尾 和人; 三菱化学メディエンス株式会社; 国立がん研究センタ; 中央病院
    AACR 2010年04月 米国 AACR
  • 新規血管新生阻害剤BIBF1120の肝細胞癌に対する有用性.  [通常講演]
    工藤 可苗; 荒尾 徳三; 坂井 和子; 永井 知行; 田村 大介; 青松 圭一; デベラスコマルコ; 金田 裕靖; 藤田 至彦; 松本 和子; 工藤 正俊; 西尾 和人
    第68回日本癌学会学術総会 2009年10月 パシフィコ横浜, 神奈川 第68回日本癌学会学術総会
  • 新規HER2抗体Pertuzumabは非小細胞肺癌株のHER3シグナルを阻害する  [通常講演]
    坂井 和子; 荒尾 徳三; 松本 和子; 藤田 至彦; 西條 長宏; 西尾 和人; 国立がんセンター中央病院; 国立がんセンター中央病院
    日本癌学会 2009年10月 横浜 日本癌学会

MISC

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2023年04月 -2027年03月 
    代表者 : 前川 真人; 西尾 和人; 中島 光子; 坂井 和子; 才津 浩智; 山下 計太; 岩泉 守哉; 松下 一之
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2023年04月 -2026年03月 
    代表者 : 西尾 和人; 林 秀敏; デベラスコ マルコ; 坂井 和子
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2022年04月 -2025年03月 
    代表者 : 川村 純一郎; 大東 弘治; 幕谷 悠介; 和田 聡朗; 坂井 和子; 家根 由典; 所 忠男; 吉岡 康多; 上田 和毅; 牛嶋 北斗; 岩本 哲好
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 星田 義彦; 大島 至郎; 大前 陽輔; 西尾 和人; 坂井 和子; 冨田 裕彦
     
    近年のメトトレキサート(MTX)の普及とともに、MTX 関連リンパ増殖性疾患(MTX-LPD)の発生が増加しており、これが日常の関節リウマチ(RA)診療における解決すべき課題の一つとなっている。MTX-LPD は、RA 治療薬である MTX の低容量投与中に発生するリンパ増殖性疾患(LPD)で、時に 『リバーシブルなリンパ腫』という興味深い病態を示す。本疾患は薬剤関連疾患で、その発症には個人差があり、疾患を発症する患者と発症しない患者とに分けられ、これは個人の薬剤の感受性・応答性に依存するものと考えられる。近年個人の個々の薬剤に対する感受性・応答性の差が遺伝的違い即ち遺伝子多型(SNP)の違いによって生じることが報告されていることより、 MTX-LPDの発症や病態に宿主側のSNPが関与することが予想できる。 本研究では文書で同意を得たLPDを発症したRA患者の血液検体を使用し、ジャポニカアレイを用い日本人に適したゲノムワイド関連解析(GWAS)によるSNP解析とRNAトランスクリプトーム解析を実施し、これらを統合的に解析する。本解析によりRA患者におけるMTX-LPD発症およびMTX依存性のreversibleな病態を示す症例の個体間のゲノム配列の違い(遺伝的素因)が解明されることが期待でき、我が国の健康・医療戦略に合致したRA診療における『ゲノム医療の実践』が可能となることが期待できる。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 前川 真人; 西尾 和人; 坂井 和子; 岩泉 守哉; 谷 重喜; 中谷 中; 松下 一之
     
    現在、本邦で行われている次世代シークエンサー（NGS）を用いた遺伝子パネル検査の結果の互換性については不明であるため、その実態を調べるための外部精度評価（EQA）を、ヒトの実試料（がん組織と血球細胞）を調査試料として施行した。実試料を使用するため、倫理委員会の承認を受け、提供者の書面での同意を得て行った。5例（肺がん2例と大腸がん3例）を準備し、15施設が参加した。なお、FFPE試料の場合、がん組織内の多様性による誤差を避けたいこと、純粋にNGSの技能を評価したいことから、がん組織と血球細胞から抽出したDNA溶液を配布した。重要な遺伝子異常は、後からサンガー法によるシークエンス、コンパニオン診断薬で分析、デジタルPCRでアレル頻度の測定を行った。 治療に影響する遺伝子異常、それ以外のがん関連遺伝子異常の報告、遺伝子異常のアレル頻度などについて調べた結果、多くの参加施設で適切に遺伝子異常を指摘できていたが、5例の中に、近接した箇所に変異があった場合にバイオインフォマティクスでフィルタアウトされたり、長い欠落変異を検出できなかったり欠落場所がわずかにずれたりした施設があった。遺伝子異常のアレル頻度は、試料によって、また種類によってバラツキに差があった。ライブラリ作製法の違い、がん細胞のみか正常血球細胞とのペア解析かによっても、遺伝子異常の報告内容やアレル頻度が異なる場合もあった。生殖細胞系列の遺伝子異常は当然ながらペア解析で多かった。 本邦で実試料を用いた技能試験は初めてであり、がんゲノム検査に関する重要な点が見えた意義のあるものとなった。自施設で使用している分析法の利点・欠点を知る良い機会になることを再認識することができた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 坂本 信一; 西尾 和人; 金田 篤志; 今村 有佑; デベラスコ マルコ; 坂井 和子; 川上 英良; 落谷 孝広; 安西 尚彦; 植村 天受
     
    １．前立腺癌細胞において、L型アミノ酸トランスポーター１（LAT1）とヘテロダイマーを構成するSLC3A2/4F2hcの解析を行った。 C4-2細胞をsi4F2hcで処理すると、細胞の成長、移動性、浸潤性の能力が抑制されることがわかり、この効果は細胞周期を通じて起こり、S期が有意に減少し、G0/G1期が有意に増加して細胞周期の停止が示唆された。また、4F2hcの下流分子がSKP2であることがRNA seqによって証明された。si4F2hcは、下流の標的であるSKP2を介して、サイクリン依存性キナーゼインヒビター（P21cip1、P27kip1）のタンパク質発現をアップレギュレートする。4F2hcの高発現は無増悪生存率の独立した予後因子であった（HR 11.54, p=0.0357）。また、4F2hcの高発現は、腫瘍の臨床病期（p=0.0255）およびグリソンスコア（p=0.0035）と関連していた。 ２．我々は、初代PC細胞であるLNCaPと、LNCaPから派生したAR-V7を発現するCRPC細胞であるLNCaP95を比較して、エピゲノムとトランスクリプトームの解析を行った。ChIP-seq解析で同定された399個のAR-V7標的領域のうち、377個はホルモン刺激を受けたARが共通して標的となりうる領域であり、22個はAR-V7が特異的に標的とする領域であった。78個のAR-V7標的遺伝子のうち、74個はAR-V7共通の標的遺伝子、4個はAR-V7特異的な標的遺伝子であり、AR-V7ノックダウンによって最も抑制された遺伝子としてSLC3A2/4F2hcを同定した。SLC3A2/4F2hcは、LAT1の結合分子であった。臨床的なCRPC組織で有意に発現しており、これらをノックダウンすることで、アポトーシスや成長停止を介してLNCaP95の細胞増殖を有意に抑制することがわかった。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2023年03月 
    代表者 : 松村 謙臣; 西尾 和人; 坂井 和子; 宮澤 正顯; 高矢 寿光; 佐藤 隆夫; 万代 昌紀; 村上 隆介
     
    卵巣癌を中心とする婦人科癌について、ゲノム解析や腫瘍免疫の解析を行った。その結果、以下の研究成果が得られた。１）癌腫横断的なDNA相同組み替え修復(HR)経路異常(HRD)の解析によって、HR遺伝子変異は対側アレルの欠失（LOH）を伴う場合に機能的に重要であること、HRDにより生じるゲノムの痕跡(genomic scar）が性別およびTP53変異の有無によって大きく異なること、および、HRDが癌腫横断的にプラチナ製剤やPARP阻害剤感受性に関与することを見出した。本研究成果は現在論文投稿中である。２）卵巣明細胞癌や正所性子宮内膜におけるPIK3CA変異を調べて、PIK3CA変異に関する腫瘍内不均一性があることや、正所性子宮内膜には高頻度にpassenger変異と思われるPIK3CA変異が認められることを見出した。本研究成果も現在論文投稿中である。３）婦人科の癌肉腫における腫瘍免疫の状態について、multi omicsデータに基づいて解析し報告した。４）若年女性において、新たなタイプのALK融合遺伝子を有する悪性中皮腫の１例を発見し報告した。５）増殖の遅い卵巣癌細胞に対してUCN-01が細胞障害活性を有することを見出して報告した。６）内膜症関連卵巣癌の発生起源が正所性子宮内膜由来である可能性を提唱する総説論文を発表した。７）遺伝子変異のパターンが、免疫チェックポイント阻害剤の効果と関連していることを見出した。本研究については、現在、論文作成を準備している。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 武田 真幸; 西尾 和人; 坂井 和子
     
    がん遺伝子パネル検査は、複数のゲノム変異が同時に検出可能な次世代シークエンサーを用いて、個々の人に対するがんのゲノム変異を明らかにし、その特性に応じた最適ながん治療の機会を供与することを目的とする検査である。遺伝子パネルには、薬物療法の有効性、確定診断及び予後予測に係る既知の遺伝子が含まれ、遺伝子変異、欠失、挿入、遺伝子融合、コピー数異常等の情報を一度に明らかにすることが可能である。近畿大学がんクリニカルシーケンスに於いても、知識データベース上に記載の無い遺伝子変異(variant of uncertain significance: VUS)が散見され、それらの遺伝子変異の生物学的意義は不明である。我々は、機能未知遺伝子変異に着目し、その変異が分子標的薬の標的となりうるActionableな変異であるかを証明することで、今後遺伝子パネル検査が保険承認される際 に、薬剤導入の根拠となり得る。 近年の分子生物学の進歩により、がん細胞の悪性形質獲得に関連のある複数の遺伝子が同定されるようになった。「１遺伝子変異１診断薬」の原則では、ドライバー遺伝子数の増加に伴い解析に必要な腫瘍量及び測定時間が増加する為、生検等の微小腫瘍組織からも複数遺伝子を同時に測定可能なマルチ遺伝子診断薬の臨床導入が求められている。近畿大学では、2013年からのNGS解析により機能未知の遺伝子変異が複数同定されており、知識データベースに記載の無い機能未知の遺伝子変異に着目し、その変異導入細胞株を作成し、新規治療標的や薬剤感受性マーカーを探し出し、新しいがん治療の開発に繋げたい。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 坂井 和子; 西尾 和人; 海堀 昌樹
     
    がん細胞は均一な集団ではなく、がんの多様性・不均一性（heterogeneity）が存在することが知られており、がん細胞の不均一性は、薬物療法への治療抵抗性の獲得や再発の原因となり、癌治療における解決すべき課題のひとつであると考えられる。肝細胞がんは、世界規模の全ゲノム解析プロジェクトの成果から、発がんに関わる様々な遺伝子異常が見出されたが、肺腺がんにおけるEGFR遺伝子変異やALK融合遺伝子のように強力なドライバー遺伝子変異は少ないことが明らかになっている。本研究では、肝細胞がんにおける腫瘍内不均一性の生物学的特性を理解することにより、腫瘍内不均一性を有する腫瘍の薬物療法に対する治療抵抗性の克服へつなげるため、腫瘍内不均一性の定量評価を行い、臨床病理学的事象との関連を明らかにすることを目的とする。本年度は、近畿大学医学部及び関西医科大学の倫理委員会に承認された研究実施計画書に基づき収集した肝細胞癌検体の腫瘍内不均一性の定量評価を実施した。腫瘍内不均一性の定量評価のためのゲノムワイドコピー数測定に用いた検体種として、凍結組織検体及びホルマリン固定パラフィン包埋検体を用いたところ、ホルマリン固定パラフィン包埋検体におけるバックグラウンドノイズが高く、評価が困難であったため、凍結組織検体39例の定量結果を用い検討を進めた。同検体における腫瘍内不均一性の多寡に関わる細胞内分子をコピー数変化、遺伝子変異の有無、遺伝子発現プロファイルからの解析を行っている。最終年度は、肝細胞癌の腫瘍内不均一性の多寡と関連する病態を明らかにする。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 林 秀敏; 冨田 秀太; 坂井 和子; 伊藤 彰彦; 高濱 隆幸; 原谷 浩司
     
    もしくはその他の免疫チェックポイント阻害薬併用療法を使用した肺がんを中心とした固形がんの臨床検体を使用した免疫関連の遺伝子発現測定を行い、遺伝子発現情報からの分類を行う（免疫プロファイリング）。免疫プロファイリングによるサブグループと抗PD-1/PD-L1 抗体薬＋細胞障害性抗がん薬の併用療法の有効性についての関連を検討する。肺がん等の治療として、免疫チェックポイント阻害薬と細胞障害性抗がん薬併用療法治療が有用（もしくは無効）と考えられる集団を同定する。 上記計画概要を記した研究実施計画書を近畿大学および複数施設にて倫理委員会にて審査に承認が得られた。承認後に抗PD-1/PD-L1抗体と細胞障害性抗がん薬併用療法を行った進行非小細胞肺癌について臨床情報や実臨床で得られた遺伝子変異情報を収集すると同時にその腫瘍組織検体を収集した。 腫瘍組織検体からRNA/DNAを収集し、nCounter, PanCancer, IO360Panelを用いて腫瘍免疫関連遺伝子発現を評価した。現在21検体について解析が終了した。免疫チェックポイント阻害薬と化学療法の併用療法が一部の免疫関連遺伝子発現と相関していることを見出しており、現在追加検体を測定するように準備中である。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2018年04月 -2022年03月 
    代表者 : 家根 旦有; 西尾 和人; 坂井 和子; 藤井 正人
     
    中咽頭癌はHPV感染の有無によって予後が異なることが報告され、2017年のTNM分類の改定ではHPV関連中咽頭癌は独立した項目として分類されることになった。HPV関連中咽頭癌は予後が良好であることから低侵襲治療を開発する目的で、「中咽頭扁平上皮がんに対する集学的治療の効果とヒト乳頭腫ウイルス感染との相関に関するバイオマーカー研究」のプロトコールを作成し、頭頸部癌基礎研究会の会員に多施設共同研究の参加を募った。その結果、14施設からStageⅢ/Ⅳ中咽頭癌85例のサンプルを回収し、近畿大学医学部ゲノム生物学教室でゲノム解析を行った。 採取されたサンプルから遺伝子解析が可能であった症例は80例であった。HPV検査として、PCRによるHPV-DNA検査、ハイブリッドキャプチャーII法、p16免疫染色法のいずれかに陽性であった症例をHPV陽性とみなした。その結果HPV陽性は56例（70%）、HPV陰性は24例（30%）であった。HPVのgenotypeはHPV16型48例（87.5%）、HPV35型3例（5.4%）、HPV58型2例（3.6%）、HPV31型1例（1.8%）、HPV33型1例（1.8%）であった。 ゲノム解析の方法は次世代シーケンサーであるIonProtonシステムを用い、体細胞遺伝子の解析にはIon AmpliSeq Cancer Hotspot Panel v2を用いて行なった。遺伝子解析の結果は、HPV陽性ではPIK3CAの遺伝子変異が最も多く、56 例中12例（21.4%）、次いでFGFR3 5例（8.9%）、PTEN 4例（7.1%）であった。HPV陰性では24例中p5311例（45.8%）、次いでPIK3CA 2例（8.3%）であった。飲酒・喫煙歴の無い症例は9例で、全例がHPV陽性でPIK3CA変異は3例（33.3%）であった。
  • 日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2017年06月 -2022年03月 
    代表者 : 西尾 和人; 角田 郁生; 岡田 斉; 坂井 和子; 上嶋 一臣; 櫻井 俊治
     
    本研究では炎症に起因する病態における宿主とその微小環境との相互作用は、生体リガンドを介した化学コミュニケーションとして作用すると仮説している。抗がん薬の領域において免疫チェックポイント阻害薬は、その効果及び有害事象に、ヒト-細菌叢との間の化学コミュニケーションの理解が重要と考えている。炎症性腸疾患および炎症が介在する消化器がん、中枢神経炎症疾患である多発性硬化症とアルツハイマー病に焦点をあて、微小環境における宿主と細菌叢との相互作用を解析することにより、疾患の分子機構の解明と治療薬の創出につなげることを目的とした。具体的には、1) 腸内細菌叢のメタゲノム、メタトランスクリプトーム解析技術の向上、2)炎症性腸疾患患者における腸上皮細胞、腸内細菌叢のシーケンシングによる炎症性腸疾患の発がんリスクに対する影響および宿主・微生物間のインタラクションと病態との関連性についての検討、3)免疫チェックポイント阻害薬の投与を受けた肝細胞癌患者の糞便中のメタゲノム、メタトランスクリプトーム解析による免疫チェックポイント阻害薬の副作用及び効果に及ぼす常在細菌叢の影響の検討、4)中枢神経系（CNS）炎症性疾患である多発性硬化症（MS）およびアルツハイマー病の発症に対する腸内細菌叢の影響の検討、を目的とした。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 米阪 仁雄; 坂井 和子; 土井 勝美; 北野 睦三; 田中 薫
     
    これまでに頭頚部癌における抗EGFR抗体治療薬セツキシマブの耐性機序は十分解明されていなかった。本研究で明らかになったのはHER3リガンドであるヘレグリンの過剰発現とそれに伴うHER3シグナルの活性化が同剤への耐性をもたらす事である。特にsiRNAによるヘレグリンの選択的発現阻害は、同剤への耐性頭頚部癌細胞株FaDuCRのHER3活性を抑え、感受性を有意に改善した。さらにpanHER阻害剤であるアファチニブによるHER3活性阻害によっても、セツキシマブ耐性が克服された。そして頭頚部癌の腫瘍組織28検体中2検体でヘレグリンの過剰発現を認め、セツキシマブ治療へ耐性を示した。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 坂井 和子; 西尾 和人; 西尾 誠人
     
    本研究では血中からの融合遺伝子検出としてエキソソームを用いたALK融合遺伝子検出を試みた。ヒト由来肺癌細胞株をヌードマウス皮下移植したモデルを用い、次世代シークエンサーによる融合遺伝子検出の条件検討を行った。その後肺癌患者血漿からエキソソーム単離およびRNA抽出を行ない、PCR増幅によるEML4-ALK検出を試みたが検出が困難であった。そこで、遺伝子変異が既知である患者の血中循環無細胞DNA(cfDNA)を用いて検出可能性を検討した。6例のALK融合遺伝子陽性肺がん患者血漿からcfDNAを抽出し、キャプチャー法を用いて検出を試みた結果、1例でEML4-ALK融合遺伝子の検出が認められた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 家根 旦有; 西尾 和人; 坂井 和子; 藤井 正人; 西尾 和人
     
    頭頸部癌基礎研究会の14施設から収集した中咽頭癌stage III / IV 80例を、次世代シーケンサーを用いて分析した。80例中HPV 陽性は56例（70％）、HPV陰性は24例（30％）であった。 HPV 陽性ではPIK3CAが最も多いゲノム変化であり（12例,21.4％）、次にFGFR3（5例,8,9％）、PTEN（4例,7.1％）であった。 HPV陰性では、TP53（11例,45.8％）が最も多い変異であった。 これらの結果は、日本における中咽頭癌のゲノム変化が他の国の遺伝子変異と比べて相違のないことを示した。今後は、HPVの状況によって異なる治療法を考慮する必要があると思われる。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 清田 尚臣; 大月 直樹; 伊藤 智雄; 平井 千浦子; 西尾 和人; 坂井 和子
     
    唾液腺腺癌の中には唾液腺導管癌と免疫組織学的に類似したアンドロゲン陽性唾液腺腺癌が存在し予後不良であった。また、AR陰性唾液腺腺癌はAR陽性唾液腺腺癌及び唾液腺導管癌とは全く異なる免疫組織学的特徴を有し、遺伝子学的特徴も全く異なる疾患であることが見いだされた。 また、アンドロゲン陽性唾液腺腺癌はHER2陽性例が多く、唾液腺導管癌と共に今後はこうアンドロゲン療法や抗HER2療法の対象として治療開発を行えることが見いだされた。さらには、唾液腺腺癌の中にはflt3やRETのような既存の分子標的薬で治療可能な遺伝子変異が存在することも判明し今後症例数を増やして検討を進める。
  • 日本学術振興会：科学研究費助成事業 若手研究(B)
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 坂井 和子
     
    本研究では次世代シーケンサーによる血漿検体からの体細胞遺伝子変異測定系の有用性を示すことを目的とした。大腸癌に見出されるKRAS, NRAS, BRAF遺伝子変異の検出系を構築し、臨床検体での解析を行った結果、標準法と比べて92%の高い一致率を得た。また、肺癌に特徴的な22遺伝子の遺伝子変異検出パネルを用い、肺癌患者血漿検体を用いて検出を試みた結果、EGFR遺伝子変異を含む複数の遺伝子変異を検出した。検出されたEGFR遺伝子変異は、デジタルPCR法と91%の一致率を示した。以上の結果から、次世代シーケンサーを用いた遺伝子変異検出は、従来の高感度手法と比して同等の検出力を示すことが示唆された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 岡本 勇; 中川 和彦; 西尾 和人; 坂井 和子
     
    本研究ではMassARRAYシステムを用いて、200例の進行非小細胞肺癌症例のパラフィン包埋組織よりEML4-ALK融合遺伝子の検出を試み、全例が測定可能であり、うち18例(9.0%)にEML4-ALK融合遺伝子を検出することに成功した。プラチナ併用化学療法の効果はALK転座陽性例、EGFR遺伝子変異陽性例、野生型、いずれも大きな差は見いだされなかった。 さらにMassARRAYを用いて26遺伝子変異をマルチプレックスに解析し気管支鏡で得られた90％以上の検体で解析が可能であり、微小検体が遺伝子診断に使用できることが示された。
  • 日本学術振興会：科学研究費助成事業 若手研究(B)
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 坂井 和子
     
    本研究では、肺がんにおいてＡＬＫ阻害剤が著効するＥＭＬ４－ＡＬＫ融合遺伝子の非侵襲的検出法の確立を目的として、血清検体におけるＥＭＬ４－ＡＬＫ検出を検討した。がん研有明病院の協力を得て、ＥＭＬ４－ＡＬＫ融合遺伝子陽性の患者血清を採取、収集し、血清検体からのＥＭＬ４－ＡＬＫ融合遺伝子の検出を検討した。ＥＭＬ４－ＡＬＫの複数存在するバリアントの同時検出および検出感度の向上をめざし、血清からの核酸抽出方法や測定条件、鋳型として用いる核酸の種類の検討を行い、検出方法の最適化を行った。この結果、ＥＭＬ４－ＡＬＫ融合遺伝子陽性患者１２例中２例の患者血清からＥＭＬ４－ＡＬＫ融合遺伝子の検出に成功した。

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