Details of a Researcher - NISHIHARA, Hiroshi

Papers - NISHIHARA, Hiroshi

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Clinical utility of comprehensive genomic profiling for advanced pancreatic cancer: insights from real-world data analysis

Eiichiro So, Hideyuki Hayashi, Keitaro Shimozaki, Sara Horie, Shotaro Kishimoto, Akihiko Chida, Yuki Saito, Kai Tsugaru, Kenro Hirata, Shigeki Tanishima, Hiroshi Nishihara, Takanori Kanai, Yasuo Hamamoto

International Journal of Clinical Oncology (Springer Science and Business Media LLC) 2025.02

Accepted, ISSN 1341-9625

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CXCL9 and CXCL13 shape endometrial cancer immune-activated microenvironment via tertiary lymphoid structure formation.

Yoshihiro Nagase, Makoto Kodama, Eriko Aimono, Kohei Nakamura, Reika Takamatsu, Keiko Abe, Takuma Yoshimura, Tatsuyuki Chiyoda, Wataru Yamagami, Hiroshi Nishihara

Cancer science 2025.02

Accepted

Immune checkpoint inhibitor (ICI) therapy has been successfully applied to various cancers; however, not all patients respond to ICI therapy. Tumors with an immune-activated environment are highly responsive to ICIs. To identify the cells and molecules essential to the formation of an immune-activated cancer microenvironment, we focused on the tertiary lymphoid structure (TLS) and performed histological and genomic analyses using endometrial cancer material. In the high immunogenic group, numerous TLSs were observed, and CXCL9 and CXCL13 expression was markedly increased. CXCL9-positive antigen-presenting and CXCL13-positive follicular dendritic cells were distributed in the T- and B-cell zones of TLSs, respectively. A group of molecules whose expression was upregulated along with CXCL9 and CXCL13 expression was strongly associated with cellular immunity. These results suggest that CXCL9-expressing antigen-presenting cells and CXCL13-expressing follicular dendritic cells coordinately shape the immune-activated microenvironment through TLS formation. The current findings will contribute to a better understanding of the mechanisms underlying the activated cancer immune microenvironment, thereby advancing the field of precision cancer medicine.

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Clinical value of preoperative circulating tumor DNA before surgery in patients with esophageal squamous cell carcinoma.

Ryota Kobayashi, Satoru Matsuda, Kohei Nakamura, Hirofumi Kawakubo, Keiso Ho, Yosuke Morimoto, Kazuhiko Hisaoka, Yuki Hoshi, Masashi Takeuchi, Kazumasa Fukuda, Jun Okui, Hiroshi Nishihara, Yuko Kitagawa

European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 51 ( 5 ) 109625 - 109625 2025.01

Accepted

INTRODUCTION: A precise preoperative tumor monitoring method that reflects tumor burden during neoadjuvant treatment is required to guide individualized perioperative treatment strategies for esophageal squamous cell carcinoma (ESCC). This study examined the clinical significance of preoperative circulating tumor DNA (ctDNA) in the plasma of patients undergoing neoadjuvant chemotherapy (NAC) followed by esophagectomy. MATERIALS AND METHODS: Plasma samples were collected longitudinally for ctDNA analysis as well as genomic DNA from primary lesions from patients with histologically confirmed ESCC who received neoadjuvant chemotherapy (NAC) followed by subtotal esophagectomy. Next-generation sequencing was used to identify mutations in both the plasma and primary tumors. We evaluated the relationship between ctDNA alterations and recurrence in patients with locally advanced ESCC. RESULTS: Pretreatment samples from 25 patients (100 %) showed the same mutations in both ctDNA and primary tumors; therefore, they were classified as ctDNA-positive before treatment. In the cohort of 25 patients analyzed, those who tested positive for ctDNA after NAC had a significantly higher risk of recurrence; the 36-month recurrence-free survival rates were 92 % for ctDNA-negative patients and 8 % for ctDNA-positive patients (p < 0.001). CONCLUSIONS: Preoperative ctDNA status may be a promising prognostic biomarker that can be assessed before surgery in patients with ESCC who received NAC. Expanded cohort validation will allow for more personalized multidisciplinary treatment approaches for ESCC tailored to ctDNA analysis.

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Clinical characteristics of gastrointestinal stromal tumors with hypoglycemia.

Akihiko Chida, Kenta Kawasaki, Junko Kuramoto, Hideyuki Hayashi, Toru Kawahara, Satomi Makiuchi, Eiichiro So, Satoko Shimizu, Shotaro Kishimoto, Sara Horie, Yuki Saito, Keitaro Shimozaki, Kai Tsugaru, Kazuhiro Togasaki, Kenro Hirata, Hiroshi Nishihara, Yae Kanai, Takanori Kanai, Yasuo Hamamoto

Oncology letters 28 ( 6 ) 568 - 568 2024.12

Accepted

The development of tyrosine-kinase inhibitors has improved survival rates for patients with gastrointestinal stromal tumors (GISTs). Despite the progress, not all the patients can universally receive the benefit from treatment due to the individual underlying conditions in a real-world setting. The present study focused on the well-known but understudied condition of GIST with hypoglycemia. Hypoglycemia in GIST is characterized by hypoglycemic symptoms such as dizziness, sweating and confusion. It is caused by several factors such as multiple liver metastases, drug adverse effects, postoperative complications and paraneoplastic syndrome [non-islet cell tumor hypoglycemia (NICTH)]. Comprehensive analysis of this condition has been hindered due to its rarity, and has been mostly limited to case reports. In the present study, a single-institution retrospective analysis of GIST with hypoglycemia was conducted to investigate its prevalence and prognosis, and the cause of this condition. The present study identified that the prevalence of hypoglycemic episodes of GIST was 4.1% in all patients with GIST, and recurrent hypoglycemic cases had a poor prognosis. The present study identified 1 case with recurrent hypoglycemia due to NICTH. Since NICTH is a rare hypoglycemic cause and requires further evaluation, an autopsy and genetic sequencing were performed using the available clinical materials. Through this histological and genetic investigation, the histological diversity of NICTH-GIST was revealed and insulin-like growth factor II (IGF-II) amplification was identified. Furthermore, a chronological analysis was performed using multiple resected archived samples from the same case, and revealed that diffuse IGF-II expression may have occurred in the early phase of tumor development. The present study catalogued the characteristics of GIST with hypoglycemia with a focus on NICTH-GIST.

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Characterizing multi-PIK3CA mutations across cancer types: Toward precision oncology.

Kohei Nakamura, Marin Ishikawa, Ryutaro Kawano, Eriko Aimono, Takaaki Mizuno, Sachio Nohara, Shigeki Tanishima, Hideyuki Hayashi, Hiroshi Nishihara

Cancer medicine 13 ( 14 ) e70052 2024.07

Accepted

BACKGROUND: PIK3CA mutations are implicated in various cancers, but the implications of multiple concurrent mutations and their orientations within the gene have not been fully explored. METHODS: In this study, we analyzed multi-PIK3CA mutations across a diverse pan-cancer cohort comprising 3564 tumors. RESULTS: Multi-PIK3CA mutations were present in 10.3% of all PIK3CA-mutant tumors, predominantly occurring in breast and gynecological cancers. Notably, mutations within the helical domain (E542:E545) exclusively occurred in the trans-orientation, contrasting with mutations in the kinase ABD and C2 domains, which mainly appeared in the cis orientation. CONCLUSIONS: The distinct pattern of mutation orientations in PIK3CA suggests variable oncogenic potential, with helical domain mutations in the trans-orientation potentially being less oncogenic. These findings highlight the importance of mutation orientation in the PIK3CA gene as potential biomarkers for targeted therapy. This understanding is crucial for designing clinical trials that leverage PI3K inhibitors, aiming for more effective and precise cancer treatment.

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Clinical and Diagnostic Utility of Genomic Profiling for Digestive Cancers: Real-World Evidence from Japan.

Marin Ishikawa, Kohei Nakamura, Ryutaro Kawano, Hideyuki Hayashi, Tatsuru Ikeda, Makoto Saito, Yo Niida, Jiichiro Sasaki, Hiroyuki Okuda, Satoshi Ishihara, Masatoshi Yamaguchi, Hideaki Shimada, Takeshi Isobe, Yuki Yuza, Akinobu Yoshimura, Hajime Kuroda, Seigo Yukisawa, Takuya Aoki, Kei Takeshita, Shinichi Ueno, Junichi Nakazawa, Yu Sunakawa, Sachio Nohara, Chihiro Okada, Ko Nishimiya, Shigeki Tanishima, Hiroshi Nishihara

Cancers 16 ( 8 ) 2024.04

Accepted

The usefulness of comprehensive genomic profiling (CGP) in the Japanese healthcare insurance system remains underexplored. Therefore, this large-scale study aimed to determine the usefulness of CGP in diagnosing digestive cancers. Patients with various cancer types recruited between March 2020 and October 2022 underwent the FoundationOne® CDx assay at the Keio PleSSision Group (19 hospitals in Japan). A scoring system was developed to identify potentially actionable genomic alterations of biological significance and actionable genomic alterations. The detection rates for potentially actionable genomic alterations, actionable genomic alterations, and alterations equivalent to companion diagnosis (CDx), as well as the signaling pathways associated with these alterations in each digestive cancer, were analyzed. Among the 1587 patients, 547 had digestive cancer. The detection rates of potentially actionable genomic alterations, actionable genomic alterations, and alterations equivalent to CDx were 99.5%, 62.5%, and 11.5%, respectively. APC, KRAS, and CDKN2A alterations were frequently observed in colorectal, pancreatic, and biliary cancers, respectively. Most digestive cancers, except esophageal cancer, were adenocarcinomas. Thus, the classification flowchart for digestive adenocarcinomas proposed in this study may facilitate precise diagnosis. CGP has clinical and diagnostic utility in digestive cancers.

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Clinical-histopathological features and cancer gene analysis of cutaneous epithelioid angiosarcoma: A report of 4 cases.

Keiko Tokuchi, Teruki Yanagi, Suguru Kurosawa, Shinya Kitamura, Takuya Maeda, Che Yuan Hsu, Kodai Miyamoto, Hiroshi Nishihara, Hideyuki Ujiie

JAAD case reports 46 45 - 48 2024.04

Accepted

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A familial case of extramammary Paget disease: Analysis of whole-exome sequencing

Takuya Maeda, Teruki Yanagi, Shinya Kitamura, Hiroshi Nishihara, Yusuke Ono, Yusuke Mizukami, Shinya Tanaka, Hideyuki Ujiie

EJC Skin Cancer 2024.03

Accepted

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14年後に再発したBRAF V600E変異を有するlow-grade gliomaの1例

黒田花音, 小田義崇, 岡本迪成, 村木岳史, 種井善一, 王磊, 津田真寿美, 高阪真路, 西原広史, 田中伸哉

日本病理学会会誌 ((一社)日本病理学会) 113 ( 1 ) 482 - 482 2024.02

Accepted, ISSN 0300-9181

Clinical utility of the Oncomine Dx Target Test multi-CDx system and the possibility of utilizing those original sequence data.

Ayaka Saito, Hideki Terai, Tae-Jung Kim, Katsura Emoto, Ryutaro Kawano, Kohei Nakamura, Hideyuki Hayashi, Hatsuyo Takaoka, Akihiko Ogata, Katsuhito Kinoshita, Fumimaro Ito, Lisa Shigematsu, Masahiko Okada, Takahiro Fukushima, Akifumi Mitsuishi, Taro Shinozaki, Keiko Ohgino, Shinnosuke Ikemura, Hiroyuki Yasuda, Ichiro Kawada, Kenzo Soejima, Hiroshi Nishihara, Koichi Fukunaga

Cancer medicine 13 ( 4 ) e7077 2024.02

Accepted

BACKGROUND: Companion diagnostic tests play a crucial role in guiding treatment decisions for patients with non-small cell lung cancer (NSCLC). The Oncomine Dx Target Test (ODxTT) Multi-CDx System has emerged as a prominent companion diagnostic method. However, its efficacy in detecting driver gene mutations, particularly rare mutations, warrants investigation. AIMS: This study aimed to assess the performance of the ODxTT in detecting driver gene mutations in NSCLC patients. Specifically, we aimed to evaluate its sensitivity in detecting epidermal growth factor receptor (EGFR) mutations, a key determinant of treatment selection in NSCLC. MATERIALS AND METHODS: We conducted a retrospective analysis of NSCLC patients who underwent testing with the ODxTT at Keio University Hospital between May 2020 and March 2022. Patient samples were subjected to both DNA and RNA tests. Driver gene mutation status was assessed, and instances of missed mutations were meticulously examined. RESULTS: Of the 90 patients, five had nucleic acid quality problems, while 85 underwent both DNA and RNA tests. Driver gene mutations were detected in 56/90 (62.2%) patients. Of the 34 patient specimens, driver mutations were not detected using the ODxTT; however, epidermal growth factor receptor (EGFR) mutations were detected using polymerase chain reaction-based testing in two patients, and a KRAS mutation was detected by careful examination of the sequence data obtained using the ODxTT in one patient. For the above three cases, carefully examining the gene sequence information obtained using the ODxTT could identify driver mutations that were not mentioned in the returned test results. Additionally, we confirmed comparable instances of overlook results for EGFR mutations in the dataset from South Korea, implying that this type of oversight could occur in other countries using the ODxTT. EGFR mutation was missed in ODxTT in Japan (6.3%, 2/32), South Korea (2.0%, 1/49), and overall (3.7%, 3/81). CONCLUSION: Even if sufficient tumor samples are obtained, rare EGFR mutations (which are excluded from the ODxTT's genetic mutation list) might not be detected using the current ODxTT system due to the program used for sequence analysis. However, such rare EGFR mutations can still be accurately detected on ODxTT's sequence data using next-generation sequencing.

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Identifying intense inflammatory subtype of esophageal squamous cell carcinoma using clustering approach.

Satoru Matsuda, Shota Hoshino, Tadahiro Goto, Hirofumi Kawakubo, Masashi Takeuchi, Ryota Kobayashi, Kohei Nakamura, Hiroya Takeuchi, Hiroshi Nishihara, Yuko Kitagawa

General thoracic and cardiovascular surgery 2024.01

Accepted

OBJECTIVE: To establish a risk-stratification system for predicting the postoperative recurrence of esophageal squamous cell carcinoma, this study aimed to evaluate the prognostic value of clusters based on blood inflammation and coagulation markers and investigate their correlation with serum cytokines and genetic alteration. METHOD: This single-center, retrospective cohort study enrolled 491 patients with esophageal cancer who underwent subtotal esophagectomy between 2004 and 2012. For cluster exploration, nonhierarchical cluster analysis and k-means were applied using serum C-reactive protein, albumin, fibrinogen, and platelet-lymphocyte ratio as variables. Then, multivariate survival analysis was conducted to investigate the association of clusters with recurrence-free survival. To characterize the clusters, serum interleukin-6, interleukin-8, and genetic alteration in primary tumors, the PleSSision-Rapid panel, which can evaluate 160 representative driver genes, was used. RESULTS: Patients were classified into clusters 1, 2, and 3, which included 24 (5%), 161 (33%), and 306 (62%) patients, respectively. Compared with cluster 3, cluster 1 or 2 had significantly worse recurrence-free survival. Based on the multivariable analysis using cluster, pStage, and age as covariates, cluster was an independent prognostic factor for recurrence-free survival (hazard ratio, 1.55; 95% confidence interval, 1.08-2.21; P = 0.02). The percentage of serum interleukin-6 and interleukin-8 levels was the highest in cluster 1, followed by clusters 2 and 3. In 23 patients with available genomic profiles, no significant difference in representative genomic alterations was observed. CONCLUSIONS: Non-biased clustering using inflammation and coagulation markers identified the intense inflammatory subtype, which had an independent prognostic effect on recurrence-free survival.

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Germline BRCA2 variant with low variant allele frequency detected in tumor-only comprehensive genomic profiling.

Hideyuki Hayashi, Kei Kunimasa, Shigeki Tanishima, Kohei Nakamura, Marin Ishikawa, Yasutaka Kato, Eriko Aimono, Ryutaro Kawano, Hiroshi Nishihara

Cancer science 2023.12

Accepted

Germline BRCA1/2 variants in comprehensive genomic profiling (CGP) often exhibit variant allele frequency (VAF) exceeding 50%. However, when genomic loss occurs at the ipsilateral allele, including the germline variant in tumor cells, the VAF is low. This case report presents a patient with uterine sarcoma with a pathogenic BRCA2 mutation and low VAF in tumor-only CGP, which was later identified as a germline variant. When genomic alterations in BRCA1/2 are identified in tumor-only CGP, the possible germline origin of the variants should be considered, even if their VAF is very low.

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Eribulin inhibits growth of cutaneous squamous cell carcinoma cell lines and a novel patient-derived xenograft

Che Yuan Hsu, Teruki Yanagi, Takuya Maeda, Hiroshi Nishihara, Kodai Miyamoto, Shinya Kitamura, Keiko Tokuchi, Hideyuki Ujiie

Scientific Reports 13 ( 1 ) 2023.12

Accepted

Advanced cutaneous squamous cell carcinoma (cSCC) is treated with chemotherapy and/or radiotherapy, but these typically fail to achieve satisfactory clinical outcomes. There have been no preclinical studies to evaluate the effectiveness of eribulin against cSCC. Here, we examine the effects of eribulin using cSCC cell lines and a novel cSCC patient-derived xenograft (PDX) model. In the cSCC cell lines (A431 and DJM-1 cells), eribulin was found to inhibit tumor cell proliferation in vitro as assessed by cell ATP levels. DNA content analysis by fluorescence-activated cell sorting (FACS) showed that eribulin induced G2/M cell cycle arrest and apoptosis. In xenograft models of cSCC cell lines, the administration of eribulin suppressed tumor growth in vivo. We also developed a cSCC patient-derived xenograft (PDX) which reproduces the histological and genetic characteristics of a primary tumor. Pathogenic mutations in TP53 and ARID2 were detected in the patient’s metastatic tumor and in the PDX tumor. The cSCC-PDX responded well to the administration of eribulin and cisplatin. In conclusion, the present study shows the promising antineoplastic effects of eribulin in cSCC. Also, we established a novel cSCC-PDX model that preserves the patient’s tumor. This PDX could assist researchers who are exploring innovative therapies for cSCC.

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<i>BRCA1/2</i> reversion mutations in a pan‐cancer cohort

Kohei Nakamura, Hideyuki Hayashi, Ryutaro Kawano, Marin Ishikawa, Eriko Aimono, Takaaki Mizuno, Hajime Kuroda, Yasuyuki Kojima, Naoki Niikura, Aya Kawanishi, Kei Takeshita, Shinsuke Suzuki, Shinichi Ueno, Kosuke Okuwaki, Jiichiro Sasaki, Masatoshi Yamaguchi, Kenta Masuda, Tatsuyuki Chiyoda, Wataru Yamagami, Chihiro Okada, Sachio Nohara, Shigeki Tanishima, Hiroshi Nishihara

Cancer Science (Wiley) 2023.12

Accepted, ISSN 1347-9032

Abstract

Tumor sensitivity to platinum (Pt)‐based chemotherapy and poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors is increased by homologous recombination deficiency‐causing mutations; in particular, reversion mutations cause drug resistance by restoring protein function. Treatment response is predicted by breast cancer susceptibility gene 1/2 (BRCA1/2) mutations; however, BRCA1/2 reversion mutations have not been comprehensively studied in pan‐cancer cohorts. We aimed to characterize BRCA1/2 reversion mutations in a large pan‐cancer cohort of Japanese patients by retrospectively analyzing sequencing data for BRCA1/2 pathogenic/likely pathogenic mutations in 3738 patients with 32 cancer types. We identified somatic mutations in tumors or circulating cell‐free DNA that could restore the ORF of adverse alleles, including reversion mutations. We identified 12 (0.32%) patients with somatic BRCA1 (n = 3) and BRCA2 (n = 9) reversion mutations in breast (n = 4), ovarian/fallopian tube/peritoneal (n = 4), pancreatic (n = 2), prostate (n = 1), and gallbladder (n = 1) cancers. We identified 21 reversion events—BRCA1 (n = 3), BRCA2 (n = 18)—including eight pure deletions, one single‐nucleotide variant, six multinucleotide variants, and six deletion–insertions. Seven (33.3%) reversion deletions showed a microhomology length greater than 1 bp, suggesting microhomology‐mediated end‐join repair. Disease course data were obtained for all patients with reversion events: four patients acquired mutations after PARP‐inhibitor treatment failure, two showed somatic reversion mutations after disease progression, following Pt‐based treatment, five showed mutations after both treatments, one patient with pancreatic cancer and BRCA1 reversion mutations had no history of either treatment. Although reversion mutations commonly occur in BRCA‐associated cancers, our findings suggest that reversion mutations due to Pt‐chemotherapy might be correlated with BRCA1/2‐mediated tumorigenesis even in non‐BRCA‐associated histologies.

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Clinical availability and characteristics of multigene panel testing for recurrent/advanced gynecologic cancer.

Shoko Kitazawa, Tatsuyuki Chiyoda, Kohei Nakamura, Kensuke Sakai, Tomoko Yoshihama, Hiroshi Nishio, Yusuke Kobayashi, Takashi Iwata, Kouji Banno, Wataru Yamagami, Hiroshi Nishihara, Daisuke Aoki

International journal of clinical oncology 2023.08

Accepted

BACKGROUND: Japan's health insurance covers multigene panel testing. This study aimed to determine the potential availability and utility of gene panel testing clinically in gynecologic oncology. METHODS: We analyzed the characteristics of patients with gynecologic cancer who underwent gene panel testing using FoundationOne® CDx or OncoGuide™ NCC Oncopanel between November 2019 and October 2022. RESULTS: Out of 102 patients analyzed, 32, 18, 43, 8, and 1 had cervical, endometrial, ovarian cancers, sarcoma, and vaginal cancer, respectively. Druggable gene alteration was found in 70 patients (68.6%; 21 with cervical cancer, 15 with endometrial cancer, 28 with ovarian cancer, 5 with sarcoma, and 1 with other). The most common druggable gene alteration was PIK3CA mutation (n = 21), followed by PTEN mutation (n = 12) and high tumor mutation burden (TMB-H) (n = 11). TMB-H was detected in 5 patients with cervical cancer, 5 with endometrial cancer, and 1 with endometrial stromal sarcoma. Eleven patients (10.8%) received molecularly targeted therapy according to their gene aberrations. Gene panel testing was mostly performed when the second-line treatment was ineffective. Of all 102 patients, 60 did not have recommended treatment, and 15 died or had worsened conditions before obtaining the test results. CONCLUSION: Through multigene panel testing, although many patients had druggable gene alterations, 10.8% of them received the recommended treatment. TMB-H was mainly observed in cervical/endometrial cancer, suggesting its potential as a therapeutic biomarker of immune checkpoint inhibitors. Furthermore, patients' prognosis and performance status should be considered before performing the test.

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Tumor Burden Monitoring with Circulating Tumor DNA During Treatment in Patients with Esophageal Squamous Cell Carcinoma.

Yosuke Morimoto, Satoru Matsuda, Hirofumi Kawakubo, Kohei Nakamura, Ryota Kobayashi, Kazuhiko Hisaoka, Jun Okui, Masashi Takeuchi, Eriko Aimono, Kazumasa Fukuda, Rieko Nakamura, Hideyuki Saya, Hiroshi Nishihara, Yuko Kitagawa

Annals of surgical oncology 30 ( 6 ) 3747 - 3756 2023.06

Accepted

BACKGROUND: To guide appropriate treatment strategy, an accurate tumor monitoring modality that reflects tumor burden during neoadjuvant treatment is required for esophageal squamous cell carcinoma (ESCC). We aimed to investigate the clinical utility of circulating tumor DNA (ctDNA) in plasma in patients who received neoadjuvant chemotherapy (NAC) followed by esophagectomy. PATIENTS AND METHODS: Longitudinally collected plasma samples for ctDNA combined with genomic DNA from primary lesions were obtained from patients with histologically confirmed ESCC who underwent NAC followed by subtotal esophagectomy. Next-generation sequencing was performed to identify mutations from the plasma and the primary tumor. The relationships between changes in ctDNA and the pathological response and recurrence were assessed in patients with locally advanced ESCC. RESULTS: In pretreatment samples from 13 patients, multiple concordant mutations in ctDNA and primary tumors were observed in 11 patients (85%), who were classified as ctDNA positive before treatment. The ctDNA positive rate after NAC correlated with the pathological response (responders, 25%; nonresponders, 100%; p = 0.007). The risk of recurrence increased significantly in patients with positive ctDNA after surgery in analysis of 16 patients; the 1-year recurrence-free survival rates were 90 and 0% in ctDNA-negative and ctDNA-positive groups, respectively (p = 0.0008). In two patients with postoperative recurrence, ctDNA was detected approximately 5.5 months earlier than the diagnosis using radiographical imaging. CONCLUSIONS: ctDNA is a promising biomarker for predicting pathological response and postoperative recurrence in ESCC. To demonstrate the external validity, we are currently preparing a multicenter prospective study.

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ASO Author Reflections: Can Circulating Tumor DNA Guide Individualized Treatment for Patients with Esophageal Squamous Cell Carcinoma?

Annals of surgical oncology 30 ( 6 ) 3757 - 3758 2023.06

Accepted

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ASO Visual Abstract: Tumor Burden Monitoring with Circulating Tumor DNA During Treatment in Patients with Esophageal Squamous Cell Carcinoma.

Annals of surgical oncology 30 ( 6 ) 3759 - 3759 2023.06

Accepted

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Prognostic role of the innate immune signature CD163 and "eat me" signal calreticulin in clear cell renal cell carcinoma.

Tadatsugu Anno, Nobuyuki Tanaka, Kimiharu Takamatsu, Kyohei Hakozaki, Ryohei Kufukihara, Yuto Baba, Toshikazu Takeda, Kazuhiro Matsumoto, Shinya Morita, Takeo Kosaka, Shuji Mikami, Hiroshi Nishihara, Ryuichi Mizuno, Mototsugu Oya

Cancer immunology, immunotherapy : CII 72 ( 6 ) 1779 - 1788 2023.06

Accepted

The effects of the innate immune status on patients with clear cell renal cell carcinoma (ccRCC) currently remain unknown. We herein provided more extensive information about the inner landscape of immunobiology of ccRCC. In total, 260 ccRCC samples from three different cohorts consisting of 213 primary tumors and 47 metastases were obtained. We focused on five representative innate immune signatures, CD68, CD163, the "eat me" signal calreticulin, the "don't eat me" signal CD47, and signal regulatory protein α, and examined the role of each signature by quantitative immunohistochemistry. We then conducted an integrated genome mutation analysis by next-generation sequencing. Among the five markers, high CD163 and low calreticulin expression levels were prognostic in ccRCC. The application of a new risk model based on CD163 and calreticulin levels, named the innate immune risk group (high risk: high-CD163/low calreticulin, intermediate risk: high-CD163/high calreticulin or low CD163/low calreticulin, low risk: low-CD163/high calreticulin), enabled the sequential stratification of patient prognosis and malignancy. Although organ-specific differences were observed, metastases appeared to have a higher innate immune risk, particularly in the lungs, with 50% of ccRCC metastases being classified into the high-risk group according to our risk score. An analysis of genomic alterations based on the innate immune risk group revealed that alterations in the TP53/Cell cycle pathway were highly prevalent in high-risk ccRCC patients according to two innate immune signatures CD163 and calreticulin. The present results provide insights into the immune-genomic biology of ccRCC tumors for innate immunity and will contribute to future therapies focused on the innate immune system in solid cancers.

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Cancer gene analysis of liquid-based cytology specimens using next-generation sequencing: A technical report of bimodal DNA- and RNA-based panel application.

Toshiaki Akahane, Tomomi Isochi-Yamaguchi, Natumi Hashiba-Ohnuki, Nobuyuki Bandoh, Eriko Aimono, Yasutaka Kato, Hiroshi Nishihara, Hajime Kamada, Akihide Tanimoto

Diagnostic cytopathology 51 ( 8 ) 493 - 500 2023.05

Accepted

BACKGROUND: As liquid-based cytology (LBC) specimens harbor high-quality DNA, genomic analysis using LBC specimens is beneficial for integrative diagnosis. This study aimed to clarify the feasibility of LBC specimens for a bimodal application of DNA- and RNA-based next-generation sequencing (NGS) panels. METHODS: LBC specimens were prepared from cultured human cancer HEC59 cells using commercially available fixatives (Cellprep, CytoRich Red, and SurePath solutions), and were subjected to NGS for a feasibility study. Clinical LBC specimens of thyroid and salivary gland tumors were prepared using CytoRich Red solution. After DNA and RNA extraction, NGS analyses were performed in a single run using combined DNA- and RNA-based custom-made cancer panels for the detection of gene mutations and fusions. RESULTS: High-quality DNA and RNA were obtained, and the expected gene mutations and fusions were detected in HEC59 cells using all types of LBC fixatives. Most available clinical cases (18 out of 20) exhibited pathogenic gene mutations (15 cases) and fusion genes (3 cases) using the bimodal DNA- and RNA-based panels. Overall, 18 cases (90%) showed oncogenic mutations or fusion genes of diagnostic values. CONCLUSION: Simultaneous application of bimodal DNA- and RNA-based gene panels was useful in NGS analysis using residual LBC specimens for integrative diagnosis. Residual LBC specimens for genomic analysis, including fusion gene analysis, are particularly useful for obtaining genomic information before surgical resection.

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Diffuse-Type Tenosynovial Giant Cell Tumor Arising in the Temporomandibular Joint Extending to the External Auditory Canal: A Case Report and Literature Review.

Suzuki S, Tsuda H, Bandoh N, Goto T, Uemura A, Aoyama T, Nishio A, Makino S, Yamaguchi T, Aimono E, Nishihara H, Harabuchi Y

Ear, nose, & throat journal 102 ( 5 ) 291 - 296 2023.05

Accepted, ISSN 0145-5613

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Clinical predominance of whole‐exome sequencing to evaluate microsatellite instability status

Reika Takamatsu, Kohei Nakamura, Okihide Suzuki, Chihiro Okada, Ryo Mori, Ryutaro Kawano, Hideyuki Hayashi, Marin Ishikawa, Eriko Aimono, Sachio Nohara, Shigeki Tanishima, Arisa Ueki, Hideyuki Ishida, Hiroshi Nishihara

Cancer Science (Wiley) 2023.04

Accepted, ISSN 1347-9032

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The DNA integrity number and concentration are useful parameters for successful comprehensive genomic profiling test for cancer using formalin-fixed paraffin embedded tissue.

Emmy Yanagita, Hiroshi Yamada, Tetsuro Kobayashi, Eriko Aimono, Kohei Nakamura, Akira Hirasawa, Hiroshi Nishihara

Pathology international 73 ( 5 ) 198 - 206 2023.03

Accepted

The acquisition of high-quality biospecimens and the appropriate handling of these materials are indispensable for successful clinical sequencing. We developed a cancer clinical sequencing system targeting 160 cancer genes: PleSSision-Rapid. Through the PleSSision-Rapid system, we have analyzed DNA quality evaluated by DIN (DNA integrity number) with 1329 formalin-fixed paraffin embedded (FFPE) samples including 477 prospectively collected tissues for genomic test (P) and 852 archival samples after routine pathological diagnosis (A1/A2). As a result, the samples with more than DIN 2.1 was 92.0% (439/477) in prospectively collected sample (P), while it was 85.6% (332/388) and 76.7% (356/464) in two types of archival samples (A1/A2). We performed the PleSSision-Rapid sequence using the samples with over DIN 2.1 and DNA concentration >10 ng/μL with which we were able to construct a DNA library, and the probability of sequence success was almost equivalent during all types of specimen processing, at 90.7% (398/439) in (P), 92.5% (307/332) in (A1) and 90.2% (321/356) in (A2), respectively. Our result indicated the clinical benefit to prepare the prospective collection of FFPE materials for indisputable clinical sequence, and that DIN ≥ 2.1 would be a solid parameter for sample preparation of comprehensive genomic profiling tests.

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がんゲノムプロファイリング検査データに基づく十二指腸腫瘍のゲノム病理学的特徴

石川麻倫, 川野竜太郎, 四十物絵理子, 中村康平, 加藤容崇, 林秀幸, 谷嶋成樹, 西原広史

日本病理学会会誌 ((一社)日本病理学会) 112 ( 1 ) 267 - 267 2023.03

Accepted, ISSN 0300-9181

分子病理診断として運用する院内がん遺伝子検査Rapid-Neoの有用性

川野竜太郎, 柳田絵美衣, 山田寛, 石川麻倫, 中村康平, 林秀幸, 谷嶋成樹, 西原広史

日本病理学会会誌 ((一社)日本病理学会) 112 ( 1 ) 341 - 342 2023.03

Accepted, ISSN 0300-9181

DOCK2は日本人集団におけるCOVID-19の重症化遺伝子で,宿主免疫応答に重要な役割を担う

南宮湖, 枝廣龍哉, 高野友美, 西原広史, 熊ノ郷淳, 小池竜司, 木村彰方, 井元清哉, 宮野悟, 小川誠司, 金井隆典, 岡田随象, 福永興壱

日本呼吸器学会誌 ((一社)日本呼吸器学会) 12 ( 増刊 ) 271 - 271 2023.03

Accepted, ISSN 2186-5876

Japanese nationwide observational multicenter study of tumor BRCA1/2 variant testing in advanced ovarian cancer.

Oda K, Aoki D, Tsuda H, Nishihara H, Aoyama H, Inomata H, Shimada M, Enomoto T

Cancer science 114 ( 1 ) 271 - 280 2023.01

Accepted, ISSN 1347-9032

The association between germline BRCA1 and BRCA2 pathogenic variants (mutations: gBRCAm) and ovarian cancer risk is well established. Germline testing alone cannot detect somatic BRCA1/2 pathogenic variants (sBRCAm), which is calculated based on the proportion of tumor BRCAm (tBRCAm) from tumor samples and gBRCAm. Homologous recombination deficiency (HRD) results mainly from genetic/epigenetic alterations in homologous recombination repair-related genes and can be evaluated by genomic instability status. In Japan, the prevalence of tBRCAm, sBRCAm, and HRD remains unclear. This multicenter, cross-sectional, observational study, CHaRacterIzing the croSs-secTional approach to invEstigate the prevaLence of tissue BRCA1/2 mutations in newLy diagnosEd advanced ovarian cancer patients (CHRISTELLE), evaluated the prevalence of tBRCAm, sBRCAm, and HRD in tumor specimens from newly diagnosed patients with ovarian cancer who underwent gBRCA testing. Of the 205 patients analyzed, 26.8% had a tBRCAm, including tBRCA1m (17.6%) and tBRCA2m (9.3%). The overall prevalence of tBRCAm, gBRCAm, sBRCAm, and HRD-positive status was 26.8%, 21.5%, 6.3%, and 60.0%, respectively. The calculated sBRCAm/tBRCAm ratio was 23.6% (13/55), and the prevalence of gBRCA variant of uncertain significance was 3.9%. These results suggest gBRCA testing alone cannot clearly identify the best course of treatment, highlighting the importance of sBRCA testing in Japan. The present results also suggest that testing for tBRCA and HRD should be encouraged in advanced ovarian cancer patients to drive precision medicine.

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Genomic analysis of aggressive ductal adenocarcinoma of the prostate.

Hiroaki Kobayashi, Takeo Kosaka, Kohei Nakamura, Tokuhiro Kimura, Hiroshi Nishihara, Mototsugu Oya

Cancer medicine 12 ( 7 ) 8445 - 8451 2022.12

Accepted

BACKGROUND: Genomic profile analysis using next-generation sequencing can potentially elucidate the pathogenesis of rare cancers. Ductal adenocarcinoma, a rare subtype of prostate cancer, has an aggressive nature. This is the first study to analyze the genomic profile of ductal adenocarcinoma in an Asian population. METHODS: We identified 12 patients newly diagnosed with ductal adenocarcinoma of the prostate at two hospitals, and nine patients (75.0%) had the pure type. Genomic assessment was performed using either the PleSSision testing platform or FoundationOne CDx. RESULTS: At least one genomic alteration occurred in 11 patients (91.7%), and the most frequently mutated gene was tumor suppressor protein p53 (TP53), which was found in six cases (50.0%). Alterations characteristic of this cohort were found in four cases (33.3%) of retinoblastoma transcriptional corepressor 1 (RB1), which was only observed in the pure type. Compared to previous study results, the frequency of genetic alterations in the phosphoinositide 3-kinase (PI3K) pathway (n = 3; 25.0%) and Wnt-β-catenin pathway (n = 5; 41.7%) was comparable, but no alterations in the DNA damage repair (DDR) pathway were observed. None of the patients presented high tumor mutation burden or microsatellite instability. CONCLUSIONS: We found that the Asian cohort with ductal adenocarcinoma had actionable alterations, and a high frequency of alterations in TP53 and RB1 reflected the aggressive nature of the tumor. Genetic analysis using next-generation sequencing is expected to help elucidate the pathogenesis of ductal adenocarcinoma.

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Derivative Chromosome 3 Loss from t(3;6)(q12;q14) Followed by Differential VHL Mutations Underlie Multifocal ccRCC.

Mizutani K, Yokoi S, Sawada S, Sakamoto I, Kameyama K, Kamei S, Hirade K, Sugiyama S, Matsunaga K, Yamada T, Kato Y, Nishihara H, Ishihara S, Deguchi T

Cancer genomics & proteomics 19 ( 6 ) 740 - 746 2022.11

Accepted, ISSN 1109-6535

Background/Aim: The Von Hippel-Lindau (VHL) gene encodes a protein (pVHL) that plays an important role in proteasome degradation of hypoxia inducible factor α (HIFα) through E3 activation. Accumulation of HIFα by loss of functional pVHL promotes tumorigenesis, thus, VHL has tumor suppressor gene capability in clear cell renal cell carcinoma (ccRCC). VHL is the most frequently mutated gene in ccRCC. The complete loss of VHL is mainly achieved by loss of chromosome 3p, which has a VHL coding region in combination with mutation or hypermethylation of the remaining copy of VHL. Given the risk of constitutional chromosome 3 translocation for RCC, it is important to detect the translocation and understand the mechanism underlying the development of multifocal ccRCC. Case Report: A 67- year-old female patient diagnosed with multifocal RCC underwent robot-assisted partial nephrectomy (RAPN) for three kidney tumors. A cancer gene panel test using next generation sequencing (NGS) detected differential VHL mutations (c.533T>G; p.L178R, c.465_466insTA; p.T157Ifs*3, c.343C>A; p.H115N), while VHL mutation was not detected in peripheral blood DNA. A tendency toward copy number loss of genes on der(3) was also detected in all tumors, but not in the germline one. A karyotype analysis revealed a germline translocation between 3 and 6, t(3;6)(q12;q14). Conclusion: Chromosome 3 translocation and loss of derivative chromosome containing 3p and subsequent somatic differential VHL mutations in this case strongly support the previously proposed three-step model to explain the development of familial conventional ccRCC.

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Characterizing cyclin-dependent kinase 12(CDK12)-altered aggressive prostate cancer: a twelve-case series

Tomohiro Iwasawa, Takeo Kosaka, Yota Yasumizu, Hiroshi Hongo, Yoshinori Yanai, Yuto Baba, Kazuhiro Matsumoto, Kohei Nakamura, Hiroshi Nishihara, Mototsugu Oya

International Journal of Clinical Oncology (Springer Science and Business Media LLC) 27 ( 12 ) 1867 - 1873 2022.10

Accepted, ISSN 1341-9625

Abstract

Background

Prostate cancer harboring cyclin-dependent kinase 12 (CDK12) abnormalities is a hot topic due to its distinctive clinical features, such as sensitivity to immune checkpoint inhibitors. In the last few years, precision medicine using comprehensive genome sequencing has become familiar, and the era of precision oncology has arrived in the field of prostate cancer. This study aimed to present the demographic characteristics of patients with CDK12 alterations.

Methods

In 12 patients with detected CDK12 alterations in our hospital between 2015 and 2021, we evaluated their genomic features and clinical course. CDK12 allelic status was classified into three groups: monoallelic loss, potentially biallelic loss, and biallelic loss based on the genome analyses.

Results

Seven patients already had metastatic cancer at the time of diagnosis, and all 12 patients had Gleason grade ≥ 4. Most cases of biallelic loss or potentially biallelic loss were metastatic cancers at the initial staging, and all these cases were categorized into Gleason grade 5. Two of the 12 patients had BRCA2/RB1 co-loss, and the other two had whole genome duplication. Five patients had a long-term survival of > 6 years, but two patients died within 4 years of diagnosis.

Conclusion

This is the first Japanese prostate cancer case series with CDK12 alterations. CDK12-altered prostate cancer is a heterogeneous disease, and accumulating cases with detailed information leads to precision oncology.

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ASO Author Reflections: Does Elevation of the Prognostic Factor Plasma Fibrinogen Level Reflect the Tumor Microenvironment at the Primary Site?

Hoshino S, Matsuda S, Kawakubo H, Yamaguchi S, Nakamura K, Aimono E, Matsui K, Irino T, Fukuda K, Nakamura R, Okita H, Nishihara H, Takeuchi H, Kitagawa Y

Annals of surgical oncology 29 ( 11 ) 6905 - 6906 2022.10

Accepted, ISSN 1068-9265

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Elevation of the Prognostic Factor Plasma Fibrinogen Reflects the Immunosuppressive Tumor Microenvironment in Esophageal Squamous Cell Carcinoma.

Shota Hoshino, Satoru Matsuda, Hirofumi Kawakubo, Shigeo Yamaguchi, Kohei Nakamura, Eriko Aimono, Kazuaki Matsui, Tomoyuki Irino, Kazumasa Fukuda, Rieko Nakamura, Hajime Okita, Hiroshi Nishihara, Hiroya Takeuchi, Yuko Kitagawa

Annals of surgical oncology 29 ( 11 ) 6894 - 6904 2022.10

Accepted, ISSN 1068-9265

INTRODUCTION: Despite previous reports on the clinical significance of plasma fibrinogen (FNG) levels as a prognostic indicator of ESCC, its underlying mechanism remains unclear. This study aimed to validate the prognostic impact of plasma FNG levels and clarify its relationship with primary tumors in patients with esophageal squamous cell carcinoma (ESCC). METHODS: The prognostic impact of FNG was evaluated in patients with ESCC who underwent esophagectomy between 2000 and 2019. The RNA sequencing of the primary ESCC site, which was from pre-operative biopsy, was performed, followed by immune profile characterization using an immunogram. Those profiles were assessed via the immunohistochemical staining of tumor-associated macrophages (TAMs) and clinical response to nivolumab. RESULTS: Multivariate analysis identified FNG as a significant prognostic factor in ESCC. The immunogram suggested an immunosuppressive tumor environment in the high-FNG group. Immunostaining with the TAM markers CD163 and CD204, revealed that the high-FNG group had significantly higher number of TAMs compared with the low-FNG group. The immunosuppressive characteristics were clinically validated in patients with metastatic ESCC; those who had elevated FNG levels showed poor response to nivolumab. CONCLUSION: This study successfully validated the prognostic impact of plasma FNG levels in an expanded cohort with ESCC. Accordingly, our findings showed that increased plasma FNG reflects an immunosuppressive tumor microenvironment that facilitates tumor progression and poor responses to nivolumab.

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The first Japanese case of intraductal cancer of the prostate with checkpoint kinase 2 mutation

Hiroshi Hongo, Takeo Kosaka, Kohei Nakamura, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

Asian Journal of Urology (Elsevier BV) 9 ( 4 ) 480 - 482 2022.10

Accepted, ISSN 2214-3882

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食道癌におけるクラスタリングを用いた炎症凝固サブタイプの提唱とMolecular profiling

松田諭, 川久保博文, 星野尚大, 後藤匡啓, 小林亮太, 福田和正, 中村理恵子, 中村康平, 西原広史, 北川雄光

日本胸部外科学会定期学術集会 ((一社)日本胸部外科学会) 75回 JP2 - 5 2022.10

Accepted

[Panel Discussion - Problems in the Cancer Genomic Medicine].

Chikashi Ishioka, Manabu Muto, Shinichi Yachida, Hidekazu Shirota, Akira Hirasawa, Kiyoshi Miyagawa, Kazuto Ashizawa, Ichiro Kinoshita, Hiroshi Nishihara, Nariaki Matsuura, Akihiro Sakurai

Gan to kagaku ryoho. Cancer & chemotherapy 49 ( 9 ) 1014 - 1017 2022.09

Accepted, ISSN 0385-0684

About 4 and a half years have passed since"Cancer Genome Medicine"was first mentioned in the Third Phase of the Basic Plan to Promote Cancer Control Programs that started in October 2017. Currently, cancer genomic medicine is being carried out by the cancer gene panel test, which is covered by public insurance, mainly at the 12 Cancer Genome Medicine Core Center Hospital designated nationwide by the Ministry of Health, Labor, and Welfare in Japan. Cancer genomic medicine has come to be positioned as a standard medical treatment. However, there are various challenges in operating an expert panel that professionally examines the results of the gene panel tests and reports treatment recommendations and secondary findings that suggest hereditary tumors. In addition, there is an urgent need to disseminate and educate healthcare professionals and patients about cancer genomic medicine. In this panel discussion on January 14, 2022, 10 panelists discussed how to solve these issues and the prospects for the future.

[Additional Remarks on the Issue of Cancer Genomic Medicine-Focusing on Medical Insurance Issues].

Nishihara H

Gan to kagaku ryoho. Cancer & chemotherapy 49 ( 9 ) 1011 - 1013 2022.09

Accepted, ISSN 0385-0684

Squamous cell carcinoma of the prostate with <scp> <i>SMARCA4</i> </scp> alteration in a Japanese patient

Yuta Kaneko, Takeo Kosaka, Kohei Nakamura, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

IJU Case Reports (Wiley) 5 ( 5 ) 323 - 326 2022.09

Accepted, ISSN 2577-171X

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Cancer genomic medicine in Japan and the roles of pharmacists.

Aomori T, Sakurai H, Nishihara H

Pharmacogenetics and genomics 32 ( 6 ) 242 - 245 2022.08

Accepted, ISSN 1744-6872

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Genomic landscape of treatment-naïve urological cancers using next-generation sequencing-based panel test in the Japanese population.

Mizutani K, Hirade K, Sugiyama S, Kato Y, Nishihara H, Ishihara S

International journal of urology : official journal of the Japanese Urological Association (WILEY) 29 ( 8 ) 909 - 911 2022.08

Accepted, ISSN 0919-8172

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Recommendations related to the analytical equivalence assessment of gene panel testing.

Sumimasa Nagai, Hiroshi Nishihara, Takayoshi Suzuki, Kazuto Nishio, Hiroya Taniguchi, Katsuya Tsuchihara, Kohei Nakamura, Reika Takamatsu, Toshihide Ueno, Hiroyuki Aburatani, Takashi Kohno, Shinji Kohsaka

Cancer science 113 ( 10 ) 3282 - 3290 2022.07

Accepted, ISSN 1347-9032

Advances in cancer genome care over the past few years have included the development of gene panel testing for various biomarkers. This article summarizes issues and provides recommendations related to analytical performance evaluations for new oncology gene panels. The scope of these recommendations includes comprehensive genomic profiling assays related to gene panel testing that uses histological or serum specimens to detect gene mutations. As a research project of the Japan Agency for Medical Research and Development Research on Regulatory Science of Pharmaceuticals and Medical Devices, we convened the working group committee that consisted of more than 30 experts from academia, industry, and government. We have discussed the points that should be considered to allow maximal simplification of assessments using clinical specimens in evaluating accuracy and limit of detection in equivalence and analytical performance for three years. We provide recommendations specific to each type of gene mutation as well as to reference standards or specimens used for evaluations. In addition, in order to facilitate the discussion on the analytical performance of gene panel tests by multidisciplinary tumor boards of hospitals, the present recommendations also describe the items that companies are expected to provide information on in their packaging inserts and reports, and the items that are expected to be discussed by multidisciplinary tumor boards. Our working group document will be important for participants in multidisciplinary tumor boards including medical oncologists and genome scientists, and developers of gene panels not only in Japan but also in other countries.

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Real-world application of next-generation sequencing-based test for surgically resectable colorectal cancer in clinical practice.

Masayo Ogiri, Ryo Seishima, Kohei Nakamura, Eriko Aimono, Shimpei Matsui, Kohei Shigeta, Tatsuyuki Chiyoda, Shigeki Tanishima, Koji Okabayashi, Hiroshi Nishihara, Yuko Kitagawa

Future oncology (London, England) 2022.07

Accepted, ISSN 1479-6694

Aim: To evaluate the significance of next-generation sequencing-based gene panel testing in surgically resectable colorectal cancer by analyzing real-world data. Materials & methods: A total of 107 colorectal cancer patients who underwent curative surgery were included, and correlations between next-generation sequencing data and clinicopathological findings were evaluated. Results: More combination patterns in gene alteration were identified in advanced-stage tumors than in early-stage tumors. The copy number alteration count was significantly lower in right-sided colon tumors and early-stage tumors. Homologous recombination deficiency was more often identified in advanced-stage tumors, and high homologous recombination deficiency status was useful for identifying high-risk stage II tumors. Conclusion: Homologous recombination deficiency was identified as a useful result of gene panel testing with novel utility in clinical practice.

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A retrospective study of parotid gland tumors at a single institution.

Suzuki S, Bandoh N, Goto T, Kubota A, Uemura A, Kono M, Sato R, Takeda R, Sakaue S, Yamaguchi-Isochi T, Nishihara H, Takei H, Harabuchi Y

Oncology letters 24 ( 1 ) 207 - 207 2022.07

Accepted, ISSN 1792-1074

The aim of the present study was to analyze the clinical characteristics, surgical treatments and clinical outcome of patients with parotid gland tumors and to compare the results with those cited in the literature. A retrospective study was conducted in 140 patients (male, n=77; female, n=63) with parotid gland tumors who underwent parotidectomy at Hokuto Hospital Department of Otolaryngology-Head and Neck Surgery (Obihiro, Japan) between April 2007 and December 2021. Of the 140 patients enrolled, 118 (84.3%) patients had benign tumors, including 63 (45%) patients with pleomorphic adenomas and 43 (30.7%) patients with Warthin tumors, and 22 patients (15.7%) had parotid carcinoma. Comparison of the three groups of patients with parotid gland tumors indicated that pack years as an indicator of smoking status were significantly higher in patients with Warthin tumors than in those with parotid carcinomas (P=0.011) or pleomorphic adenoma (P<0.001). Fine-needle aspiration cytology (FNAC) was non-diagnostic for only 6 (4.3%) of 140 patients. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of FNAC by both conventional smear and liquid-based cytology (LBC) for parotid carcinomas were 70, 99, 93.3, 94.4 and 82.9%, respectively. Among the 22 patients with parotid carcinoma, extended total/total and superficial parotidectomy were performed in 10 (45%) and 11 (50%) cases, respectively. Total and selective neck dissection of the area from level II to I, II and III were performed in 6 (24%) and 7 (32%) patients, respectively. Postoperative radiotherapy (50 Gy) was performed in 15 (68%) patients. The overall survival (OS) and disease-free survival (DFS) rates at 5 years were 51.5 and 76.4%, respectively. Univariate analysis revealed that age >65 years was significantly associated with poorer 5-year OS (P<0.001) and DFS (P<0.001). Multivariate analysis revealed that an age of more than 65 years combined with high-grade histologic malignancy was associated with worse DFS (P=0.02; hazard ratio, 3.628; 95% confidence interval, 1.283-9.514). In conclusion, the clinical characteristics and treatment outcomes of parotid gland tumors were consistent with the results of previous reports. Smoking may be closely related to the pathogenesis of Warthin tumors. LBC potentially provides improved accuracy in FNAC.

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Hybridisation chain reaction-based visualisation and screening for lncRNA profiles in clear-cell renal-cell carcinoma.

Ryohei Kufukihara, Nobuyuki Tanaka, Kimiharu Takamatsu, Naoya Niwa, Keishiro Fukumoto, Yota Yasumizu, Toshikazu Takeda, Kazuhiro Matsumoto, Shinya Morita, Takeo Kosaka, Eriko Aimono, Hiroshi Nishihara, Ryuichi Mizuno, Mototsugu Oya

British journal of cancer 127 ( 6 ) 1133 - 1141 2022.06

Accepted, ISSN 0007-0920

BACKGROUND: Analysis of long noncoding RNA (lncRNA) localisation at both the tissue and subcellular levels can provide important insights into the cell types that are important for their function. METHODS: By applying new fluorescent in situ hybridisation technique called hybridisation chain reaction (HCR), we achieved a high-throughput lncRNA visualisation and evaluation of clinical samples. RESULTS: Assessing 1728 pairs of 16 lncRNAs and clear-cell renal-cell carcinoma (ccRCC) specimens, three lncRNAs (TUG1, HOTAIR and CDKN2B-AS1) were associated with ccRCC prognosis. Furthermore, we derived a new lncRNA risk group of ccRCC prognosis by combining the expression levels of these three lncRNAs. Examining genomic alterations underlying this classification revealed prominent features of tumours that could serve as potential biomarkers for targeting lncRNAs. We then derived combination of HCR with expansion microscopy and visualised nanoscale-resolution HCR signals in cell nuclei, uncovering intracellular colocalization of three lncRNA (TUG1, HOTAIR and CDKN2B-AS1) signals such as those located intra- or out of the nucleus or nucleolus in cancer cells. CONCLUSION: LncRNAs are expected to be desirable noncoding targets for cancer diagnosis or treatments. HCR involves plural probes consisting of small DNA oligonucleotides, clinically enabling us to detect cancerous lncRNA signals simply and rapidly at a lower cost.

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Influence of response to prior docetaxel on sensitivity to cabazitaxel in prostate cancer patients with PTEN alterations.

Ken Kamisawa, Takeo Kosaka, Kohei Nakamura, Yota Yasumizu, Hiroshi Hongo, Toshikazu Takeda, Kazuhiro Matsumoto, Hiroshi Nishihara, Mototsugu Oya

Cancer science 113 ( 9 ) 3161 - 3168 2022.06

Accepted, ISSN 1347-9032

The purpose of this study was to investigate factors predicting the sensitivity to cabazitaxel therapy in metastatic castration-resistant prostate cancer (mCRPC) patients with phosphatase and tensin homolog deleted from chromosome 10 (PTEN) alterations. This single-institution, retrospective study included 12 mCRPC patients with PTEN alterations who had received cabazitaxel therapy. Five patients (41%) responded to cabazitaxel therapy with a prostate-specific antigen (PSA) level decline of ≥30% from baseline, and all of them had responded to prior docetaxel therapy with a PSA decline of ≥30%. None of the patients with a poor response to prior docetaxel therapy responded well to cabazitaxel therapy. Of the seven patients who did not respond to cabazitaxel and whose PSA declined from baseline was <30%, five (71%) were also refractory to prior docetaxel therapy. The PSA responses to docetaxel and cabazitaxel were significantly correlated (p = 0.027). Kaplan-Meier analysis revealed that progression-free survival (PFS) for cabazitaxel was significantly shorter for prior docetaxel nonresponders (3.3 versus 9.1 months, p = 0.028). Multivariate analysis revealed that a poor response to prior docetaxel (PSA decline < 30%) (hazard ratio [HR] = 6.382, 95% confidence interval [CI] 1.172-34.750, p = 0.032) and baseline PSA of ≥20 ng/ml (HR = 33.584, 95% CI 2.332-483.671, p = 0.010) were independent prognostic factors for PFS with cabazitaxel therapy. These results demonstrate cross-resistance between docetaxel and cabazitaxel. The response to prior docetaxel therapy can influence the sensitivity to cabazitaxel therapy in mCRPC patients with PTEN alterations.

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A Japanese case of castration-resistant prostate cancer with BRCA2 and RB1 co-loss and TP53 mutation: a case report.

Tomohiro Iwasawa, Takeo Kosaka, Shinya Morita, Shuji Mikami, Kohei Nakamura, Hiroshi Hongo, Hiroshi Nishihara, Mototsugu Oya

BMC medical genomics 15 ( 1 ) 138 - 138 2022.06

Accepted

BACKGROUND: Abnormalities in homologous recombination contribute to the aggressive nature of castration-resistant prostate cancer. Retinoblastoma transcriptional corepressor 1 (RB1) and breast cancer 2 (BRCA2) exist close to each other in the same chromosome, and the significance of their concurrent loss has become a hot topic in the field of cancer research. CASE PRESENTATION: A 61-year-old man presented with a chief complaint of a mass on his head and was diagnosed as multiple bone metastases from prostate cancer. He was treated with standard medication, but he died 2 years 6 months after being diagnosed with prostate cancer. Simultaneous biallelic loss of RB1 and BRCA2 as well as a truncating mutation of tumor protein p53 (TP53) were revealed by genomic analysis. CONCLUSION: To our knowledge, this is the first report of castration-resistant prostate cancer (CRPC) with BRCA2 and RB1 co-loss and TP53 mutation. To establish a treatment strategy for highly malignant cases with such multiple genetic features is important.

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Multiple metastases of androgen indifferent prostate cancer in the urinary tract: two case reports and a literature review.

Tsukasa Masuda, Takeo Kosaka, Kohei Nakamura, Hiroshi Hongo, Kazuyuki Yuge, Hiroshi Nishihara, Mototsugu Oya

BMC medical genomics 15 ( 1 ) 118 - 118 2022.05

Accepted

BACKGROUND: Prostate cancer (PC) is mainly known to metastasize to bone, lung and liver, but isolated metastases of prostate cancer, including ductal carcinoma, in the urinary tract are very rare. We describe two patients with nodular masses in the urinary tract (the anterior urethra or the urinary bladder) that were found on cystoscopy during treatment of castration-resistant prostate cancer. CASE PRESENTATION: In both cases, the pathological diagnosis from transurethral tumor resection showed that they were androgen indifferent prostate cancer (AIPC), including aggressive variant prostate cancer (AVPC) in Case 1 and treatment-induced neuroendocrine differentiation prostate cancer (NEPC) in Case 2. In Case 1, Loss of genetic heterozygosity (LOH) of BRCA2 and gene amplification of KRAS was identified from the urethra polyps. In Case 2, homozygous deletion was observed in PTEN, and LOH without mutation was observed in RB1. CONCLUSION: These are the first reports of two cases of urinary tract metastasis of AIPC.

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The oligodendroglial histological features are not independently predictive of patient prognosis in lower-grade gliomas.

Eriel Sandika Pareira, Makoto Shibuya, Kentaro Ohara, Yu Nakagawa, Tokunori Kanazawa, Dai Kamamoto, Yasutaka Kato, Eri Arai, Eriko Aimono, Kazunari Yoshida, Hiroshi Nishihara, Yae Kanai, Hikaru Sasaki

Brain tumor pathology 39 ( 2 ) 79 - 87 2022.04

Accepted, ISSN 1433-7398

The relevance of oligodendroglial histological features to patient prognoses is controversial. 93 LrGGs resected for about 2 decades were re-assessed based on WHO2007 with special interest to pure oligodendroglial diagnosis (oligodendroglioma or anaplastic oligodendroglioma) and presence of CFO features. Those histological features, patients OS, and tumor chromosomal/genetic characteristics were correlated each other in each of the 3 IDH-1p/19q-based molecular groups. There was significant association between 1p19q status with the oligodendroglial histological diagnosis as well as presence of CFO in the entire cohort. The oligodendroglial diagnosis was associated with longer OS in IDHmut/codel group; however, this association was not significant in the multivariate analyses. In IDHmut/noncodel and IDH-wildtype groups, the oligodendroglial diagnosis was not associated with patient OS. Presence of CFO was not associated with patient OS in any molecular groups. Gain of 8q was associated with the oligodendroglial diagnosis in IDHmut/noncodel group. Neither the oligodendroglial diagnosis nor CFO was predictive for the methylation status of the MGMT gene in any molecular groups. The oligodendroglial histological features are not independently predictive of either patient prognosis or chemotherapeutic response in LrGGs, leaving the possibility of marginal favorable association only in IDHmut/codel tumors.

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TP53 variants in p53 signatures and the clonality of STICs in RRSO samples.

Tomoko Akahane, Kenta Masuda, Akira Hirasawa, Yusuke Kobayashi, Arisa Ueki, Miho Kawaida, Kumiko Misu, Kohei Nakamura, Shimpei Nagai, Tatsuyuki Chiyoda, Wataru Yamagami, Shigenori Hayashi, Fumio Kataoka, Kouji Banno, Kokichi Sugano, Hajime Okita, Kenjiro Kosaki, Hiroshi Nishihara, Daisuke Aoki

Journal of gynecologic oncology 33 ( 4 ) e50 2022.03

Accepted, ISSN 2005-0380

OBJECTIVE: Precursor lesions may be identified in fallopian tube tissue after risk-reducing salpingo-oophorectomy (RRSO) in patients with pathogenic variants of BRCA1/2. Serous tubal intraepithelial carcinoma (STIC) is considered a precursor of high-grade serous carcinoma, whereas the significance of the p53 signature remains unclear. In this study, we investigated the relationship between the p53 signature and the risk of ovarian cancer. METHODS: We analyzed the clinicopathological findings and conducted DNA sequencing for TP53 variants of p53 signatures and STIC lesions isolated using laser capture microdissection in 13 patients with pathogenic variants of BRCA1/2 who underwent RRSO and 17 control patients with the benign gynecologic disease. RESULTS: TP53 pathogenic variants were detected significantly higher in RRSO group than control (p<0.001). No difference in the frequency of p53 signatures were observed between groups (53.8% vs 29.4%; p=0.17). TP53 sequencing and next-generation sequencing analysis in a patient with STIC and occult cancer revealed 2 TP53 mutations causing different p53 staining for STICs and another TP53 mutation shared between STIC and occult cancer. CONCLUSION: The sequence analysis for TP53 revealed 2 types of p53 signatures, one with a risk of progression to STIC and ovarian cancer with pathological variants in TP53 and the other with a low risk of progression without pathological variants in TP53 as seen in control.

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Maelstrom functions in the production of Siwi-piRISC capable of regulating transposons in Bombyx germ cells.

Namba Y, Iwasaki YW, Nishida KM, Nishihara H, Sumiyoshi T, Siomi MC

iScience (Elsevier BV) 25 ( 3 ) 103914 - 103914 2022.03

Accepted, ISSN 2589-0042

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Unique characteristics of tertiary lymphoid structures in kidney clear cell carcinoma: prognostic outcome and comparison with bladder cancer.

Tsukasa Masuda, Nobuyuki Tanaka, Kimiharu Takamatsu, Kyohei Hakozaki, Ryohei Takahashi, Tadatsugu Anno, Ryohei Kufukihara, Kazunori Shojo, Shuji Mikami, Toshiaki Shinojima, Kazuhiro Kakimi, Tatsuhiko Tsunoda, Eriko Aimono, Hiroshi Nishihara, Ryuichi Mizuno, Mototsugu Oya

Journal for immunotherapy of cancer 10 ( 3 ) 2022.03

Accepted

BACKGROUND: The aims of this study were (1) to clarify the impact of tertiary lymphoid structure (TLS) status on the outcome and immunogenomic profile of human clear cell renal cell carcinoma (ccRCC) and (2) to determine phenotypic differences in TLSs between different types of genitourinary cancer, that is, urinary ccRCC and bladder cancer. METHODS: We performed a quantitative immunohistological analysis of ccRCC tissue microarrays and conducted integrated genome mutation analysis by next-generation sequencing and methylation array analysis. Since the tumor immune microenvironment of ccRCC often differs from that of other cancer types, we analyzed the phenotypic differences in TLSs between ccRCC and in-house bladder cancer specimens. RESULTS: Varying distribution patterns of TLSs were observed throughout ccRCC tumors, revealing that the presence of TLSs was related to poor prognosis. An analysis of genomic alterations based on TLS status in ccRCC revealed that alterations in the PI3K-mTOR pathway were highly prevalent in TLS-positive tumors. DNA methylation profiling also revealed distinct differences in methylation signatures among ccRCC samples with different TLS statuses. However, the TLS characteristics of ccRCC and bladder cancer markedly differed: TLSs had the exact opposite prognostic impact on bladder cancer as on ccRCC. The maturity and spatial distribution of TLSs were significantly different between the two cancer types; TLSs were more mature with follicle-like germinal center organization and likely to be observed inside the tumor in bladder cancer. Labeling for CD8, FOXP3, PD-1, and PD-L1 showed marked differences in the diversity of the immune microenvironment surrounding TLSs. The proportions of CD8-, FOXP3-, and PD-L1-positive cells were significantly higher in TLSs in bladder cancer than in TLSs in ccRCC; rather the proportion of PD-1-positive cells was significantly higher in TLSs in ccRCC than in TLSs in bladder cancer. CONCLUSION: The immunobiology of ccRCC is unique, and various cancerous phenomena conflict with that seen in other cancer types; therefore, comparing the TLS characteristics between ccRCC and bladder cancer may help reveal differences in the prognostic impact, maturity and spatial distribution of TLSs and in the immune environment surrounding TLSs between the two cancers.

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[Crosstalk of Cancer Genomic Test and Cancer Immune Therapy].

Nishihara H

Gan to kagaku ryoho. Cancer & chemotherapy 49 ( 3 ) 248 - 254 2022.03

Accepted, ISSN 0385-0684

Sclerosing Polycystic Adenosis Arising in the Parotid Gland Without PI3K Pathway Mutations.

Uemura A, Bandoh N, Goto T, Sato R, Suzuki S, Kubota A, Yamaguchi T, Baba S, Kato Y, Nishihara H, Harabuchi Y, Takei H

Head and neck pathology 16 ( 1 ) 282 - 287 2022.03

Accepted, ISSN 1936-055X

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Castration-resistant prostate cancer patient presenting with whole genome doubling with CDK-12 mutation.

Yuto Baba, Takeo Kosaka, Hiroaki Kobayashi, Kohei Nakamura, Shuji Mikami, Hiroshi Nishihara, Makoto Nakanishi, Mototsugu Oya

BMC medical genomics 15 ( 1 ) 32 - 32 2022.02

Accepted

BACKGROUND: The use of whole-genome sequencing in clinical practice has revealed variable genomic characteristics across cancer types, one of which is whole-genome doubling (WGD), which describes the duplication of a complete set of chromosomes. Yet it is relatively rare in prostate cancer and no such case has ever been reported in Japanese patients. CASE PRESENTATION: A 54-year-old patient with prostatic adenocarcinoma with bone and lymph node metastases was started on androgen-deprivation therapy. As the prostate cancer turned castration-resistant, multimodal therapies including taxane- and platinum-based chemotherapy, androgen-receptor antagonist inhibitors, radiotherapy and radium-233 were introduced. Good controls of serum prostate-specific antigen (PSA) level and bone metastases were achieved for more than 13 years since after the initial treatment. During the treatment, a metastatic lymph node biopsy was performed to confirm the tumor histology, and spinal decompression surgery were performed for spinal compression due to lumber vertebral metastases. The immunohistochemical analysis identified PSA and androgen receptor positive tumor cells in both metastatic lesions, while no variable cancer cells were detected in the prostate on second biopsy. Whole-genome sequencing was performed on the biopsied metastatic lymph node in search for another possible treatment and it revealed that the tumor had WGD and CDK12 mutation. The WGD-positive tumor cells contained large and polymorphic nucleus, presumably reflecting on the ploidy abnormality of the chromosomes. CONCLUSIONS: This report is the first case of a Japanese patient presenting with WGD, who survived more than 13 years with multimodal chemotherapies and radiotherapies.

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Precision cancer genome testing needs proficiency testing involving all stakeholders.

Maekawa M, Taniguchi T, Nishio K, Sakai K, Matsushita K, Nakatani K, Ishige T, Ikejiri M, Nishihara H, Sunami K, Yatabe Y, Hatanaka KC, Hatanaka Y, Yamamoto Y, Fukuyama K, Oda S, Saito K, Yokomura M, Kubo Y, Sato H, Tanaka Y, Fuchioka M, Yamasaki T, Matsuda K, Kurachi K, Funai K, Baba S, Iwaizumi M

Scientific reports 12 ( 1 ) 1494 2022.01

Accepted

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First successful case of platinum-based chemotherapy for neuroendocrine prostate cancer with BRCA2 and PTEN alterations.

Minami Omura, Takeo Kosaka, Eriko Aimono, Kohei Nakamura, Hiroshi Hongo, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

IJU case reports 5 ( 1 ) 41 - 44 2022.01

Accepted

INTRODUCTION: Deoxyribonucleic acid repair gene mutations are now being studied in a variety of solid tumors, with the hope of predicting prognosis, pathogenesis, and treatment outcomes. CASE PRESENTATION: We report the case of a Japanese patient with advanced castration-resistant prostate cancer who exhibited a prominent response to platinum therapy and had coexisting BRCA2 and PTEN mutations according to retrospective multigene panel analysis. CONCLUSION: Through a review of clinical outcomes and genetic/pathologic profiling, the presented case provides insights into future management strategies based on the tumor genetic status.

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SARS-CoV-2 RT-qPCR testing of pooled saliva samples: A case study of 824 asymptomatic individuals and a questionnaire survey in Japan.

Junna Oba, Hiroaki Taniguchi, Masae Sato, Masaki Takanashi, Moe Yokemura, Yasunori Sato, Hiroshi Nishihara

PloS one 17 ( 5 ) e0263700 2022

Accepted

From the beginning of the COVID-19 pandemic, the demand for diagnostic and screening tests has exceeded supply. Although the proportion of vaccinated people has increased in wealthier countries, breakthrough infections have occurred amid the emergence of new variants. Pooled-sample COVID-19 testing using saliva has been proposed as an efficient, inexpensive, and non-invasive method to allow larger-scale testing, especially in a screening setting. In this study, we aimed to evaluate pooled RT-qPCR saliva testing and to compare the results with individual tests. Employees of Philips Japan, Ltd. were recruited to participate in COVID-19 screening from October to December 2020. Asymptomatic individuals (n = 824) submitted self-collected saliva samples. Samples were tested for the presence of SARS-CoV-2 by RT-qPCR in both 10-sample pools and individual tests. We also surveyed participants regarding their thoughts and behaviors after the PCR screening project. Two of the 824 individuals were positive by RT-qPCR. In the pooled testing, one of these two had no measurable Ct value, but showed an amplification trend at the end of the PCR cycle. Both positive individuals developed cold-like symptoms, but neither required hospitalization. Of the 824 participants, 471 responded to our online questionnaire. Overall, while respondents agreed that PCR screening should be performed regularly, the majority were willing to undergo PCR testing only when it was provided for free or at low cost. In conclusion, pooled testing of saliva samples can support frequent large-scale screening that is rapid, efficient, and inexpensive.

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Landscape of prognostic signatures and immunogenomics of the AXL/GAS6 axis in renal cell carcinoma.

Kyohei Hakozaki, Nobuyuki Tanaka, Kimiharu Takamatsu, Ryohei Takahashi, Yota Yasumizu, Shuji Mikami, Toshiaki Shinojima, Kazuhiro Kakimi, Takashi Kamatani, Fuyuki Miya, Tatsuhiko Tsunoda, Eriko Aimono, Hiroshi Nishihara, Ryuichi Mizuno, Mototsugu Oya

British journal of cancer 125 ( 11 ) 1533 - 1543 2021.11

Accepted, ISSN 0007-0920

BACKGROUND: Cabozantinib is an oral tyrosine kinase inhibitor in renal cell carcinoma (RCC), whose targets include oncogenic AXL and unique ligand GAS6. Critical gaps in basic knowledge need to be addressed to devise an exclusive biomarker and candidate when targeting the AXL/GAS6 axis. METHODS: To clarify the effects of the AXL/GAS6 axis on RCC, we herein performed a large-scale immunogenomic analysis and single-cell counts including various metastatic organs and histological subtypes of RCC. We further applied genome-wide mutation analyses and methylation arrays. RESULTS: Varying patterns of AXL and GAS6 expression were observed throughout primary RCC tumours and metastases. Scoring individual AXL/GAS6 levels in the tumour centre and invasive margin, namely, the AXL/GAS6 score, showed a good ability to predict the prognosis of clear cell RCC. Metastasis- and histological subtype-specific differences in the AXL/GAS6 score existed since lung metastasis and the papillary subtype were weakly related to the AXL/GAS6 axis. Cell-by-cell immunohistological assessments clarified an immunosuppressive environment in tumours with high AXL/GAS6 scores. Genomic alterations in the PI3K-mTOR pathway and DNA methylation profiling revealed distinct differences with the AXL/GAS6 score in ccRCC. CONCLUSION: The AXL/GAS6 scoring system could predict the outcome of prognosis and work as a robust biomarker for the immunogenomic state in RCC.

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A first case of ductal adenocarcinoma of the prostate having characteristics of neuroendocrine phenotype with PTEN, RB1 and TP53 alterations.

Hiroaki Kobayashi, Takeo Kosaka, Kohei Nakamura, Kazunori Shojo, Hiroshi Hongo, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

BMC medical genomics 14 ( 1 ) 245 - 245 2021.10

Accepted

BACKGROUND: Ductal adenocarcinoma and neuroendocrine cancer are rare subtypes of prostate cancer with poor prognosis and limited therapeutic options. We present the first case of ductal adenocarcinoma having a neuroendocrine phenotype. CASE PRESENTATION: A 63-year-old man presented with gross hematuria and urinary retention, and his serum prostate-specific antigen level was 4.58 ng/mL. We performed transurethral resection of the prostate, and the diagnosis was ductal adenocarcinoma with a Gleason score of 5 + 4 for acinar adenocarcinoma. Magnetic resonance imaging showed local invasion of left lobe of the prostate and bone metastasis of the left trochanteric section of the femur. Multidisciplinary treatments such as androgen deprivation therapy, chemoradiation therapy, and surgery for metastatic lesions have led to long-term survival. Since next-generation sequencing revealed PTEN and RB1 co-loss and TP53 mutations, we re-evaluated the immunohistochemistry and he was found to be positive for synaptophysin. CONCLUSIONS: This is the first Japanese case of ductal adenocarcinoma with a neuroendocrine phenotype. Genetic analysis may help not only guide the therapeutic strategies, but also sometimes with the diagnosis.

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Concordance analysis of microsatellite instability status between polymerase chain reaction based testing and next generation sequencing for solid tumors.

Keitaro Shimozaki, Hideyuki Hayashi, Shigeki Tanishima, Sara Horie, Akihiko Chida, Kai Tsugaru, Kazuhiro Togasaki, Kenta Kawasaki, Eriko Aimono, Kenro Hirata, Hiroshi Nishihara, Takanori Kanai, Yasuo Hamamoto

Scientific reports 11 ( 1 ) 20003 - 20003 2021.10

Accepted

Various malignancies exhibit high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). The MSI-IVD kit, a polymerase chain reaction (PCR)-based method, was the first tumor-agnostic companion diagnostic to detect MSI status in MSI-H solid tumors. Recently, next-generation sequencing (NGS), which can also detect MSI-H/dMMR, has been made clinically available; however, its real-world concordance with PCR-based testing of MSI-H/dMMR remains to be investigated. The co-primary end points included the positive and negative predictive values of MSI-H/dMMR. A retrospective analysis of 80 patients who had undergone both MSI testing and NGS between July 2015 and March 2021 was conducted. Five patients were confirmed to have MSI-H in both examinations. Among the 75 patients diagnosed as microsatellite stable (MSS) by PCR-based testing, one with pancreatic cancer was diagnosed as having MSI-H after NGS. One patient with pancreatic cancer was diagnosed as having MSS in both tests was found to have a mutation in MLH1 by NGS, which was confirmed as dMMR by IHC staining. NGS had positive and negative predictive values of 100% (5/5) and 98.7% (74/75), respectively, for MSI-H. The concordance between NGS and PCR-based testing was 98.8% (79/80). Thus, NGS can be useful for evaluating MSI/MMR status in clinical practice and can be an important alternative method for detecting MSI-H/dMMR in the future.

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胸壁CIC-NUTM1 sarcomaの1例

岡田真彦, 池村辰之介, 江本桂, 西原広史, 林玲奈, 冨保紗希, 齋藤彩夏, 福島貴大, 篠崎太郎, 光石彬史, 胡谷俊樹, 寺井秀樹, 安田浩之, 川田一郎, 副島研造, 福永興壱

肺癌 ((NPO)日本肺癌学会) 61 ( 6 ) 778 - 778 2021.10

Accepted, ISSN 0386-9628

Profiling the inhibitory receptors LAG-3, TIM-3, and TIGIT in renal cell carcinoma reveals malignancy.

Kimiharu Takamatsu, Nobuyuki Tanaka, Kyohei Hakozaki, Ryohei Takahashi, Yu Teranishi, Tetsushi Murakami, Ryohei Kufukihara, Naoya Niwa, Shuji Mikami, Toshiaki Shinojima, Takashi Sasaki, Yusuke Sato, Haruki Kume, Seishi Ogawa, Kazuhiro Kakimi, Takashi Kamatani, Fuyuki Miya, Tatsuhiko Tsunoda, Eriko Aimono, Hiroshi Nishihara, Kazuaki Sawada, Takeshi Imamura, Ryuichi Mizuno, Mototsugu Oya

Nature communications 12 ( 1 ) 5547 - 5547 2021.09

Accepted

A cutting edge therapy for future immuno-oncology is targeting a new series of inhibitory receptors (IRs): LAG-3, TIM-3, and TIGIT. Both immunogenomic analyses and diagnostic platforms to distinguish candidates and predict good responders to these IR-related agents are vital in clinical pathology. By applying an automated single-cell count for immunolabelled LAG-3, TIM-3, and TIGIT, we reveal that individual IR levels with exclusive domination in each tumour can serve as valid biomarkers for profiling human renal cell carcinoma (RCC). We uncover the immunogenomic landscape associated with individual IR levels in human RCC tumours with metastases in various organs and histological subtypes. We then externally validate our results and devise a workflow with optimal biomarker cut-offs for discriminating the LAG-3, TIM-3, and TIGIT tumour profiles. The discrimination of LAG-3, TIM-3, and TIGIT profiles in tumours may have a broad impact on investigations of immunotherapy responses after targeting a new series of IRs.

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ARID1A mutation/ARID1A loss is associated with a high immunogenic profile in clear cell ovarian cancer.

Yuka Kuroda, Tatsuyuki Chiyoda, Miho Kawaida, Kohei Nakamura, Eriko Aimono, Takuma Yoshimura, Mio Takahashi, Keiko Saotome, Tomoko Yoshihama, Naomi Iwasa, Kensuke Sakai, Wataru Yamagami, Hiroshi Nishihara, Daisuke Aoki

Gynecologic oncology 162 ( 3 ) 679 - 685 2021.09

Accepted, ISSN 0090-8258

OBJECTIVES: ARID1A mutation is frequently found in clear cell ovarian cancer (CCC) and endometrioid ovarian cancer (EC). Anti-PD-1 monotherapy has been found to have limited efficacy in epithelial ovarian cancer; however, anti-PD-1 therapy showed significant clinical benefit in some CCC. We sought to define the relationship of ARID1A mutation/ARID1A expression to the immunogenic profile of different histologic subtypes of ovarian cancer. METHODS: We performed next-generation sequencing of 160 cancer-related genes. Also, we analyzed the immunohistochemical status of ARID1A, PD-L1, and CD8 with survival in different histologic subtypes of ovarian cancer in a total of 103 cases. RESULTS: ARID1A mutation was found in 0% of the high-grade serous ovarian cancer (HGSC) (n = 36), 41.5% of the CCC (n = 41), 45.0% of the EC (n = 20), and 33.3% of the mucinous ovarian cancer (MC) (n = 6) cases. ARID1A loss was found in 19.4% of the HGSC, 75.6% of the CCC, 60.0% of the EC and 0% of the MC cases. ARID1A mutation was found to be associated with high PD-L1 (p < 0.001) or CD8 levels (p < 0.001) in CCC but not in other histologic subtypes. Meanwhile, ARID1A loss was associated with high PD-L1 or CD8 levels in CCC (p < 0.001) and HGSC (p < 0.001) but not in EC and MC. In addition, ARID1A mutation was associated with high tumor mutation burden in CCC (p = 0.006). CONCLUSIONS: ARID1A mutation/ARID1A expression is associated with immune microenvironmental factors in CCC but not in EC. ARID1A status can be a biomarker for selecting candidates for immune checkpoint blockade in CCC.

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Spiradenoma of the breast: a rare diagnostic pitfall in the evaluation of solid-basaloid breast lesions with a dual cell population.

Matsumoto H, Takamatsu R, Abe N, Unesoko M, Zaha H, Ishii A, Nakada N, Nishihara H, Tan PH, Yoshimi N

Virchows Archiv : an international journal of pathology 479 ( 2 ) 401 - 405 2021.08

Accepted, ISSN 0945-6317

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Resection of a desmoid-type fibromatosis with a CTNNB1 p.S45P mutation using a cervico-thoracic approach: A case report and literature review.

Sato R, Bandoh N, Goto T, Uemura A, Inoue N, Otomo Y, Nakano H, Yamaguchi T, Kato Y, Nishihara H, Takei H, Harabuchi Y

Auris, nasus, larynx 48 ( 4 ) 777 - 782 2021.08

Accepted, ISSN 0385-8146

Desmoid-type fibromatosis (DF) is a rare, locally infiltrative, and fibroblastic proliferative disease. DF usually arises from abdominal fascial tissue, but in rare cases, it can occur in extra-abdominal areas. A 73-year-old Japanese male complained of a painless, left anterior neck mass of 3-month duration. Computed tomography revealed the mass measured 9 × 7 × 6 cm and extended to the anterior mediastinum, with invasion of the left clavicle. En bloc resection of the tumor with the left sternoclavicular joint and the medial portion of the left clavicle was performed by cervico-thoracic approach with L-shaped partial sternotomy. Histopathologic examination showed fascicular growth of spindle-shaped cells separated by abundant collagen. Immunohistologic examination revealed nuclear staining of β-catenin and cytoplasmic staining of vimentin. Genetic analysis of 160 cancer-related genes by next-generation sequencing (NGS) demonstrated only a missense mutation in the CTNNB1 gene (c.133T>C, p.S45P). DF extending from the neck to the anterior mediastinum is rare. We report the complete resection of a large-sized DF with the clavicular invasion. A low-frequency CTNNB1 mutation of DF was identified. Genetic analysis with NGS was beneficial for the diagnosis.

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Germline Whole-Gene Deletion of FH Diagnosed from Tumor Profiling.

Arisa Ueki, Kokichi Sugano, Kumiko Misu, Eriko Aimono, Kohei Nakamura, Shigeki Tanishima, Nobuyuki Tanaka, Shuji Mikami, Akira Hirasawa, Miho Ando, Teruhiko Yoshida, Mototsugu Oya, Hiroshi Nishihara, Kenjiro Kosaki

International journal of molecular sciences 22 ( 15 ) 2021.07

Accepted

Hereditary leiomyomatosis and renal cell carcinoma (HL (RCC)) entails cutaneous and uterine leiomyomatosis with aggressive type 2 papillary RCC-like histology. HLRCC is caused by pathogenic variants in the FH gene, which encodes fumarate hydratase (FH). Here, we describe an episode of young-onset RCC caused by a genomic FH deletion that was diagnosed via clinical sequencing. A 35-year-old woman was diagnosed with RCC and multiple metastases: histopathological analyses supported a diagnosis of FH-deficient RCC. Although the patient had neither skin tumors nor a family history of HLRCC, an aggressive clinical course at her age and pathological diagnosis of FH-deficient RCC suggested a germline FH variant. After counseling, the patient provided written informed consent for germline genetic testing. She was simultaneously subjected to paired tumor profiling tests targeting the exome to identify a therapeutic target. Although conventional germline sequencing did not detect FH variants, exome sequencing revealed a heterozygous germline FH deletion. As such, paired tumor profiling, not conventional sequencing, was required to identify this genetic deletion. RCC caused by a germline FH deletion has hitherto not been described in Japan, and the FH deletion detected in this patient was presumed to be of maternal European origin. Although the genotype-phenotype correlation in HLRCC-related tumors is unclear, the patient's family was advised to undergo genetic counseling to consider additional RCC screening.

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Prominent response to platinum-based chemotherapy in a patient with BRCA2 mutant-neuroendocrine prostate cancer and MDM2 amplification.

Tatsuaki Daimon, Takeo Kosaka, Hiroshi Hongo, Eriko Aimono, Kohei Nakamura, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

IJU case reports 4 ( 4 ) 216 - 219 2021.07

Accepted

INTRODUCTION: Genomic profiling provides useful information for diagnosis, treatment, and prognosis, and detection of certain defects, such as DNA repair gene aberrations or microsatellite instability, can possibly lead to optimal treatment, but this testing has not been widely used to inform prostate cancer treatment. CASE PRESENTATION: A 55-year-old man sequentially treated for prostate cancer was diagnosed as neuroendocrine prostate cancer from prostate specimens resected because of urinary retention. Subsequently, he received five cycles of platinum-based chemotherapy in total and responded well. We also performed next-generation sequencing of a sample from the prostate specimen and identified a breast cancer susceptibility gene mutation with Murine Double Minute 2 amplification and loss of heterozygosity in retinoblastoma 1. CONCLUSION: We report a neuroendocrine prostate cancer patient with Murine Double Minute 2 amplification who experienced an aggressive course and for whom platinum-based chemotherapy was effective, and one of the reasons for the good response might be the breast cancer susceptibility gene mutation.

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A first Japanese case of BRCA2 and RB1 co-loss organ-confined prostate cancer successfully treated by radical prostatectomy.

Kazuhiro Matsumoto, Takeo Kosaka, Kohei Nakamura, Hiroshi Nishihara, Mototsugu Oya

International cancer conference journal 10 ( 3 ) 170 - 173 2021.07

Accepted

We report a rare case of localized prostate cancer with BRCA2 and RB1 co-loss, which is usually found at a more advanced stage with a poor prognosis. A 59-year-old male with prostate cancer was referred to our hospital for surgical treatment. He had schizophrenia that was well controlled by medicine. He had no family history of prostate cancer, breast cancer, or ovarian cancer. His initial PSA was 4.5 ng/mL, and Gleason score 3 + 4 adenocarcinoma was detected in one of 12 needle biopsy cores. Imaging studies demonstrated the clinical stage to be cT2aN0M0. Therefore, robot-assisted laparoscopic radical prostatectomy (RALP) with bilateral nerve sparing was performed. Based on histopathological analysis, the Gleason score was 4 + 3 and the pathological stage was pT2N0M0 with a negative surgical margin. Genetic sequencing identified BRCA2 and RB1 co-loss with limited loss of heterogeneity (LOH). At 12 months after surgery, his PSA level remained < 0.01 ng/mL. This case suggests the importance of early detection of prostate cancer and the possibility of cure for prostate cancer with high malignant potential. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13691-021-00469-z.

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RT-PCR Screening Tests for SARS-CoV-2 with Saliva Samples in Asymptomatic People: Strategy to Maintain Social and Economic Activities while Reducing the Risk of Spreading the Virus.

Junna Oba, Hiroaki Taniguchi, Masae Sato, Reika Takamatsu, Satoru Morikawa, Taneaki Nakagawa, Hiromasa Takaishi, Hideyuki Saya, Koichi Matsuo, Hiroshi Nishihara

The Keio journal of medicine 70 ( 2 ) 35 - 43 2021.06

Accepted, ISSN 0022-9717

The year 2020 will be remembered for the coronavirus disease 2019 (COVID-19) pandemic, which continues to affect the whole world. Early and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is fundamental to combat the disease. Among the current diagnostic tests, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) is the most reliable and frequently used method. Herein, we discuss the interpretation of RT-qPCR results relative to viral infectivity. Although nasopharyngeal swab samples are often used for RT-qPCR testing, they require collection by trained medical staff. Saliva samples are emerging as an inexpensive and efficient alternative for large-scale screening. Pooled-sample testing of saliva has been applied for mass screening of SARS-CoV-2 infection. Current policies recommend isolating people with borderline cycle threshold (Ct) values (35<Ct <40), despite these Ct values indicating minimal infection risk. We propose the new concept of a "social cut-off" Ct value and risk stratification based on the correlation of Ct with infectivity. We also describe the experience of RT-qPCR screening of saliva samples at our institution. It is important to implement a scientific approach to minimize viral transmission while allowing economic and social activities to continue.

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Determination of Brain Tumor Recurrence using 11 C‐methionine Positron Emission Tomography after Radiotherapy

Shigeru Yamaguchi, Kenji Hirata, Michinari Okamoto, Eku Shimosegawa, Jun Hatazawa, Ryuichi Hirayama, Naoki Kagawa, Haruhiko Kishima, Noboru Oriuchi, Masazumi Fujii, Kentaro Kobayashi, Hiroyuki Kobayashi, Shunsuke Terasaka, Ken‐ichi Nishijima, Yuji Kuge, Yoichi M Ito, Hiroshi Nishihara, Nagara Tamaki, Tohru Shiga

Cancer Science (Wiley) 112 ( 10 ) 4246 - 4256 2021.06

Accepted, ISSN 1347-9032

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Kimura Disease Presenting with Buccal Mass: A Case Report and Literature Review.

Sato R, Bandoh N, Goto T, Ichikawa H, Uemura A, Suzuki S, Yamaguchi T, Aimono E, Nishihara H, Katada A, Harabuchi Y

Head and neck pathology 15 ( 2 ) 657 - 662 2021.06

Accepted, ISSN 1936-055X

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Germline BRCA2 mutation in a case of aggressive prostate cancer accompanied by spinal bulbar muscular atrophy.

Hiroshi Hongo, Takeo Kosaka, Hideyuki Hayashi, Kohei Nakamura, Hiroshi Nishihara, Shuji Mikami, Himisha Beltran, Mototsugu Oya

Asian journal of andrology 24 ( 1 ) 116 - 118 2021.05

Accepted, ISSN 1008-682X

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Biglycan, tumor endothelial cell secreting proteoglycan, as possible biomarker for lung cancer.

Hirofumi Morimoto, Yasuhiro Hida, Nako Maishi, Hiroshi Nishihara, Yutaka Hatanaka, Cong Li, Yoshihiro Matsuno, Toru Nakamura, Satoshi Hirano, Kyoko Hida

Thoracic cancer 12 ( 9 ) 1347 - 1357 2021.05

Accepted, ISSN 1759-7706

OBJECTIVES: In lung cancer, surgery remains the most curative treatment and limited resection is beneficial for patients with low cardiopulmonary function and low malignancy tumors. However, there are no biomarkers of low malignancy to select candidates for limited resection without compromising the outcome of treatments. Recently we identified biglycan (BGN) as a tumor endothelial cell (TEC) marker that is associated with tumor progression in various cancers. In this study, we analyzed the association between BGN expression in TECs in lung cancer and cancer progression in patients. MATERIALS AND METHODS: First, we performed immunohistochemistry of BGN with resected lung tumor tissues of 155 patients who had undergone thoracic surgery and analyzed the correlation between BGN-positive vessel density in primary lung tumors and clinicopathological factors. Second, we measured the BGN levels in preoperative serum of other 46 patients with lung cancer by ELISA, and analyzed the correlation between BGN expression in tumor tissues and blood BGN levels. RESULTS: High BGN expression in the TECs was significantly associated with T factor, and was a significant negative predictor. BGN levels in preoperative serum of 46 patients with lung cancer was significantly correlated with BGN expression in the TECs. Preoperative serum BGN level was significantly lower in healthy volunteers and less invasive adenocarcinoma than in invasive adenocarcinoma and other lung carcinomas. These results suggest that low BGN level in preoperative serum in patients with lung cancer might indicate low malignancy. CONCLUSIONS: BGN can be a potential biomarker for lung cancer.

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First case of ductal adenocarcinoma of the prostate with MAP3K1 homozygous deletion.

Kazunori Shojo, Takeo Kosaka, Kohei Nakamura, Hiroshi Hongo, Hiroaki Kobayashi, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

IJU case reports 4 ( 3 ) 176 - 179 2021.05

Accepted

Introduction: Ductal adenocarcinoma of the prostate is a rare prostate cancer variant and associated with higher stage and greater risk of mortality. Optimal systemic therapy for metastatic ductal adenocarcinoma is not known. Case presentation: A 67-year-old man presented with ductal adenocarcinoma of the prostate accompanied by multiple lung metastases and advanced bone metastases. We performed channel transurethral resection of the prostate and confirmed the diagnosis of ductal adenocarcinoma of the prostate. DNA sequencing identified a TP53 somatic point mutation (p.Gly245Ser) as the pathogenic variant. Furthermore, a homozygous deletion was observed in mitogen-activated protein kinase kinase kinase 1. The patient received enzalutamide but deceased 5 months after presenting to our institution. Conclusion: To our knowledge, this is the first report of ductal adenocarcinoma of the prostate with a mitogen-activated protein kinase kinase kinase 1 homozygous deletion. Accumulation of whole-exome sequencing data is expected to inform future advances in therapy development.

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Impact of clinical targeted sequencing on endocrine responsiveness in estrogen receptor-positive, HER2-negative metastatic breast cancer.

Kanako Hagio, Toraji Amano, Hideyuki Hayashi, Takashi Takeshita, Tomohiro Oshino, Junko Kikuchi, Yoshihito Ohhara, Ichiro Yabe, Ichiro Kinoshita, Hiroshi Nishihara, Hiroko Yamashita

Scientific reports 11 ( 1 ) 8109 - 8109 2021.04

Accepted

Clinical targeted sequencing allows for the selection of patients expected to have a better treatment response, and reveals mechanisms of resistance to molecular targeted therapies based on actionable gene mutations. We underwent comprehensive genomic testing with either our original in-house CLHURC system or with OncoPrime. Samples from 24 patients with estrogen receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer underwent targeted sequencing between 2016 and 2018. Germline and somatic gene alterations and patients' prognosis were retrospectively analyzed according to the response to endocrine therapy. All of the patients had one or more germline and/or somatic gene alterations. Four patients with primary or secondary endocrine-resistant breast cancer harbored germline pathogenic variants of BRCA1, BRCA2, or PTEN. Among somatic gene alterations, TP53, PIK3CA, AKT1, ESR1, and MYC were the most frequently mutated genes. TP53 gene mutation was more frequently observed in patients with primary endocrine resistance compared to those with secondary endocrine resistance or endocrine-responsive breast cancer. Recurrent breast cancer patients carrying TP53-mutant tumors had significantly worse overall survival compared to those with TP53-wild type tumors. Our 160-gene cancer panel will be useful to identify clinically actionable gene alterations in breast cancer in clinical practice.

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Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation.

Ishino K, Hasuwa H, Yoshimura J, Iwasaki YW, Nishihara H, Seki NM, Hirano T, Tsuchiya M, Ishizaki H, Masuda H, Kuramoto T, Saito K, Sakakibara Y, Toyoda A, Itoh T, Siomi MC, Morishita S, Siomi H

Nucleic acids research 49 ( 5 ) 2700 - 2720 2021.03

Accepted, ISSN 0305-1048

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Estimating copy number using next-generation sequencing to determine ERBB2 amplification status.

Kohei Nakamura, Eriko Aimono, Junna Oba, Hideyuki Hayashi, Shigeki Tanishima, Tetsu Hayashida, Tatsuyuki Chiyoda, Takeo Kosaka, Tomoyuki Hishida, Hirohumi Kawakubo, Minoru Kitago, Koji Okabayashi, Takeru Funakoshi, Hajime Okita, Sadakatsu Ikeda, Hiromasa Takaishi, Hiroshi Nishihara

Medical oncology (Northwood, London, England) 38 ( 4 ) 36 - 36 2021.03

Accepted, ISSN 1357-0560

Assessing Erb-b2 receptor tyrosine kinase 2 (ERBB2) amplification status in breast and gastric cancer is necessary for deciding the best therapeutic strategy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently used for assessing protein levels and gene copy number (CN), respectively. The use of next-generation sequencing (NGS) to measure ERBB2 CN in breast cancer is approved by the United States Federal Drug Administration as a companion diagnostic. However, a CN of less than 8 is evaluated as "equivocal", which means that some ERBB2 amplification cases diagnosed as "HER2 negative" might be false-negative cases. We reviewed the results of gene profiling targeting 160 cancer-related genes in breast (N = 90) and non-breast (N = 19) cancer tissue, and compared the ERBB2 CN results with the IHC/FISH scores. We obtained an estimated CN from the measured CN by factoring in the histological proportion of tumor cells and found that an ERBB2-estimated CN of 3.2 or higher was concordant with the combined IHC/FISH outcome in 98.4% (88/90) of breast cancer cases, while this was not always evident among non-breast cancer cases. Therefore, NGS-estimated ERBB2 CN could be considered a diagnostic test for anti-HER2 therapy after the completion of adequate prospective clinical trials.

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Delta-like 1 homolog (DLK1) as a possible therapeutic target and its application to radioimmunotherapy using 125I-labelled anti-DLK1 antibody in lung cancer models (HOT1801 and FIGHT004)

Hironori Takagi, Songji Zhao, Satoshi Muto, Hiroshi Yokouchi, Hiroshi Nishihara, Toshiyuki Harada, Hikaru Yamaguchi, Hayato Mine, Masayuki Watanabe, Yuki Ozaki, Takuya Inoue, Takumi Yamaura, Mitsuro Fukuhara, Naoyuki Okabe, Yuki Matsumura, Takeo Hasegawa, Jun Osugi, Mika Hoshino, Mitsunori Higuchi, Yutaka Shio, Ryuzo Kanno, Miho Aoki, Chengbo Tan, Saki Shimoyama, Shigeo Yamazaki, Hajime Kikuchi, Jun Sakakibara-Konishi, Satoshi Oizumi, Masao Harada, Kenji Akie, Fumiko Sugaya, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Osamu Honjo, Yoshinori Minami, Masaharu Nishimura, Hirotoshi Dosaka-Akita, Koji Nakamura, Akihiro Inano, Hiroshi Isobe, Hiroyuki Suzuki

Lung Cancer (Elsevier BV) 153 134 - 142 2021.03

Accepted, ISSN 0169-5002

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Genetic Profiling of Malignant Melanoma Arising from an Ovarian Mature Cystic Teratoma: A Case Report

Kohei Nakamura, Eriko Aimono, Reika Takamatsu, Shigeki Tanishima, Tomonari Tohyama, Katsutoshi Sasano, Hiroshi Sakuma, Hiroshi Nishihara

International Journal of Molecular Sciences (MDPI AG) 22 ( 5 ) 2436 - 2436 2021.02

Accepted

Ovarian mature cystic teratomas comprise tissues derived from all three germ layers. In rare cases, malignant tumors arise from ovarian mature cystic teratoma. A variety of tumors can arise from mature cystic teratoma, among which primary malignant melanoma (MM), for which no molecular analyses such as genomic sequencing have been reported to date, is exceedingly rare, thereby limiting possible therapeutic options using precision medicine. We used targeted gene sequencing to analyze the status of 160 cancer-related genes in a patient with MM arising from an ovarian mature cystic teratoma (MM-MCT). KRAS amplification and homozygous deletion in PTEN and RB1 were detected in tumor samples collected from the patient. No KRAS amplification has been previously reported in cutaneous MM, indicating that the carcinogenesis of MM-MCT differs from that of primary cutaneous melanomas. A better understanding of the underlying genetic mechanisms will help clarify the carcinogenesis of MM-MCT. In turn, this will enable treatment with novel targeting agents as well as the initial exploration of gene-based precision oncological therapies, which aim to improve treatment outcomes for patients with this disease.

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Multiple squamous cell carcinomas arising on an epidermal nevus harboring HRAS p.G13R mutation.

Atsushi Narahira, Shinya Kitamura, Takuya Maeda, Hiroshi Nishihara, Teruki Yanagi

The Journal of dermatology 48 ( 2 ) e92-e93 - e93 2021.02

Accepted, ISSN 0385-2407

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Prognostic significance of OX40+ lymphocytes in tumor stroma of surgically resected small-cell lung cancer

Hiroshi Yokouchi, Hiroshi Nishihara, Toshiyuki Harada, Toraji Amano, Takayuki Ohkuri, Shigeo Yamazaki, Hajime Kikuchi, Satoshi Oizumi, Hidetaka Uramoto, Fumihiro Tanaka, Masao Harada, Kenji Akie, Fumiko Sugaya, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Mitsunori Higuchi, Osamu Honjo, Yoshinori Minami, Naomi Watanabe, Masaharu Nishimura, Hiroyuki Suzuki, Hirotoshi Dosaka-Akita, Hiroshi Isobe

OncoImmunology (Informa UK Limited) 10 ( 1 ) 1971430 2021.01

Accepted, ISSN 2162-4011

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【大腸癌診療におけるprecision medicine】ゲノム解析を利用した大腸癌原発巣およびその肝転移巣に対するprecision medicine

川俣太, Patch Ann-Marie, Nones Katia, 本間重紀, 西原広史, 神山俊哉, 武冨紹信, Leggett Barbara, Waddell Nicola, Whitehall Vicki

癌の臨床 ((株)篠原出版新社) 65 ( 4 ) 373 - 382 2021.01

ISSN 0021-4949

ゲノム解析を利用して同一患者の大腸癌原発巣と肝転移巣を比較検討し、化学療法の治療経過や転移過程における時空間的なゲノム変異の解明を試みた。その結果、臨床的に「actionable」な大腸癌ドライバー遺伝子が大腸癌と転移巣間では異なり、原発巣の病理結果から再発・転移巣に対する化学療法を選択しても、再発・転移巣には化学療法が奏功しない可能性が考えられた。また、ドライバー遺伝子の相違は原発巣から転移巣における治療過程でのclonal evolutionによって引き起こされることから、治療経過における転移巣切除や肝生検、リキッドバイオプシーを積極的に行うことで、より正確な真の癌個別化医療が進んでいくものと示唆された。

Pulmonary metastasis secondary to abiraterone-resistant prostate cancer with homozygous deletions of BRCA2: First Japanese case.

Mizuki Izawa, Takeo Kosaka, Kohei Nakamura, Junna Oba, Tomoyuki Hishida, Hiroshi Hongo, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

IJU case reports 4 ( 1 ) 14 - 17 2021.01

Accepted

Introduction: Most metastatic prostate cancers acquire the capacity for androgen-independent growth and become resistant to androgen deprivation therapy. A patient-focused treatment strategy is needed for aggressive castration-resistant prostate cancer. Case presentation: We report the case of a 62-year-old man who presented with prostatic adenocarcinoma who was treated by radiation and combined androgen blockade. After completion of first-line therapy, he was diagnosed with multiple metastatic castration-resistant prostate cancer in the lung. Second-line therapy with abiraterone acetate resulted in partial remission of the lung metastases. Thoracic surgery was performed to remove the single lung metastasis remaining. Next-generation sequencing of the specimens demonstrated homozygous loss of BRCA2. We note in this case a heterogeneous response to abiraterone acetate may be related to the somatic BRCA2 deletions. Conclusions: We present the first Japanese case of a metastatic abiraterone acetate-resistant castration-resistant prostate cancer accompanied by BRCA2 mutation.

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CDK12 and HER2 coamplification in two urothelial carcinomas with rapid and aggressive clinical progression.

Yoshinori Yanai, Takeo Kosaka, Kohei Nakamura, Eriko Aimono, Kazuhiro Matsumoto, Shinya Morita, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

Cancer science 111 ( 12 ) 4652 - 4655 2020.12

Cyclin-dependent kinase 12 (CDK12), one of the key factors associated with DNA damage response pathways, is located on chromosome 17 proximal to Erb-B2 receptor tyrosine kinase 2 (ERBB2). In this report, CDK12 and ERBB2 coamplification was detected by targeted next-generation sequencing in two urothelial carcinomas. The staining intensity of the CDK12 and human epidermal growth factor receptor-2 proteins was associated with the prognosis of each urothelial carcinoma case. Our results suggest that CDK12 coamplification with ERBB2 might be associated with tumor aggressiveness and contribution to cancer pathogenesis. Therapies targeting CDK12 should be developed for these patients.

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CDK12andHER2coamplification in two urothelial carcinomas with rapid and aggressive clinical progression

Yoshinori Yanai, Takeo Kosaka, Kohei Nakamura, Eriko Aimono, Kazuhiro Matsumoto, Shinya Morita, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

CANCER SCIENCE (WILEY) 111 ( 12 ) 4652 - 4655 2020.12

ISSN 1347-9032

Cyclin-dependent kinase 12 (CDK12), one of the key factors associated with DNA damage response pathways, is located on chromosome 17 proximal to Erb-B2 receptor tyrosine kinase 2 (ERBB2). In this report,CDK12andERBB2coamplification was detected by targeted next-generation sequencing in two urothelial carcinomas. The staining intensity of the CDK12 and human epidermal growth factor receptor-2 proteins was associated with the prognosis of each urothelial carcinoma case. Our results suggest thatCDK12coamplification withERBB2might be associated with tumor aggressiveness and contribution to cancer pathogenesis. Therapies targeting CDK12 should be developed for these patients.

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Association of Epstein–Barr virus with regression after withdrawal of immunosuppressive drugs and subsequent progression of iatrogenic immunodeficiency‐associated lymphoproliferative disorders in patients with autoimmune diseases

Katsuya Fujimoto, Kanako C. Hatanaka, Yutaka Hatanaka, Ikumi Kasahara, Satoshi Yamamoto, Takahiro Tsuji, Masanobu Nakata, Yasunari Takakuwa, Yoshihito Haseyama, Yumiko Oyamada, Masakatsu Yonezumi, Hiroaki Suzuki, Hajime Sakai, Hiroko Noguchi, Akio Mori, Hiroshi Nishihara, Takanori Teshima, Yoshihiro Matsuno

Hematological Oncology (Wiley) 38 ( 5 ) 799 - 807 2020.12

ISSN 0278-0232

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Clinical implications of next-generation sequencing-based panel tests for malignant ovarian tumors

Keiko Saotome, Tatsuyuki Chiyoda, Eriko Aimono, Kohei Nakamura, Shigeki Tanishima, Sachio Nohara, Chihiro Okada, Hideyuki Hayashi, Yuka Kuroda, Hiroyuki Nomura, Nobuyuki Susumu, Takashi Iwata, Wataru Yamagami, Fumio Kataoka, Hiroshi Nishihara, Daisuke Aoki

CANCER MEDICINE (WILEY) 9 ( 20 ) 7407 - 7417 2020.10

Accepted, ISSN 2045-7634

Precision medicine based on cancer genomics is being applied in clinical practice. However, patients do not always derive benefits from genomic testing. Here, we performed targeted amplicon exome sequencing-based panel tests, including 160 cancer-related genes (PleSSision-160), on 88 malignant ovarian tumors (high-grade serous carcinoma, 27; endometrioid carcinoma, 15; clear cell carcinoma, 30; mucinous carcinoma, 6; undifferentiated carcinoma, 4; and others, 6 (immature teratoma, 1; carcinosarcoma, 3; squamous cell carcinoma, 1; and mixed, 1)), to assess treatment strategies and useful biomarkers for malignant ovarian tumors. Overall, actionable gene variants were found in 90.9%, and druggable gene variants were found in 40.9% of the cases. ActionableBRCA1andBRCA2variants were found in 4.5% of each of the cases.ERBB2amplification was found in 33.3% of mucinous carcinoma cases. Druggable hypermutation/ultramutation (tumor mutation burden >= 10 SNVs/Mbp) was found in 7.4% of high-grade serous carcinoma, 46.7% of endometrioid carcinoma, 10% of clear cell carcinoma, 0% of mucinous carcinoma, 25% of undifferentiated carcinoma, and 33.3% of the other cancer cases. Copy number alterations were significantly higher in high-grade serous carcinoma (P < .005) than in other histologic subtypes; some clear cell carcinoma showed high copy number alterations that were correlated with advanced stage (P < .05) and worse survival (P < .01). A high count of copy number alteration was associated with worse survival in all malignant ovarian tumors (P < .05). Our study shows that targeted agents can be detected in approximately 40% of malignant ovarian tumors via multigene panel testing, and copy number alteration count can be a useful marker to help assess risks in malignant ovarian tumor patients.

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がん遺伝子パネル検査を受診した患者の全身状態の推移および遺伝子結果から、治療へ還元させるための検査実施時期に関する考察

今井光穂, 持田かおり, 藤倉智花, 柳田絵美衣, 山田寛, 四十物絵理子, 中村康平, 林秀幸, 植木有紗, 西原広史

日本癌学会総会記事 ((一社)日本癌学会) 79回 OJ7 - 3 2020.10

Accepted, ISSN 0546-0476

次世代シーケンサーを用いたゲノム解析による、ERBB2増幅症例での治療推奨におけるコピー数のカットオフ値の標準化について

中村康平, 四十物絵理子, 大場純奈, 植木有紗, 今井光穂, 柳田絵美衣, 山田寛, 林秀幸, 西原広史

日本癌学会総会記事 ((一社)日本癌学会) 79回 OJ7 - 6 2020.10

Accepted, ISSN 0546-0476

Extramammary Paget's disease patient-derived xenografts harboring ERBB2 S310F mutation show sensitivity to HER2-targeted therapies.

Takuya Maeda, Shinya Kitamura, Hiroshi Nishihara, Teruki Yanagi

Oncogene 39 ( 36 ) 5867 - 5875 2020.09

Accepted, ISSN 0950-9232

Although the prognosis of advanced extramammary Paget's disease (EMPD) is poor, there have been no preclinical research models for the development of novel therapeutics. This study aims to establish a preclinical research model for EMPD. We transplanted EMPD tissue into immunodeficient NOD/Scid mice. Histopathological and genetic analyses using a comprehensive cancer panel were performed. For in vivo preclinical treatments, trastuzumab, lapatinib, docetaxel, or eribulin were administered to patient-derived xenograft (PDX) models. Tissue transplanted from the EMPD patient was enlarged in NOD/Scid mice and was transplanted into further generations. Both the transplantation of PDX into nu/nu mice and the reanimation of the cryopreserved xenografted tumors in NOD/Scid mice were successful. We also established an EMPD-PDX-derived primary cell culture. Histopathologically, the xenografted tumors were positive for CK7, which was consistent with the patient's tumors. Genetically, the pathogenic mutation ERBB2 S310F was detected in the patient's tumors (primary intraepidermal lesion, metastatic lymph node) and was observed in the xenografted tumors even after continued passages. The xenografted tumors responded well to trastuzumab and lapatinib therapy. Also, cytotoxic agents (docetaxel and eribulin) were effective against the xenografted tumors. This PDX model (EMPD-PDX-H1) could be a powerful tool for the research and development of EMPD treatments.

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Clinical impact of a cancer genomic profiling test using an in-house comprehensive targeted sequencing system.

Hideyuki Hayashi, Shigeki Tanishima, Kyoko Fujii, Ryo Mori, Chihiro Okada, Emmy Yanagita, Yuka Shibata, Ryosuke Matsuoka, Toraji Amano, Takahiro Yamada, Ichiro Yabe, Ichiro Kinoshita, Yoshito Komatsu, Hirotoshi Dosaka-Akita, Hiroshi Nishihara

Cancer science 111 ( 10 ) 3926 - 3937 2020.08

Accepted, ISSN 1347-9032

Precision medicine is a promising strategy for cancer treatment. In this study, we developed an in-house clinical sequencing system to perform a comprehensive cancer genomic profiling test as a clinical examination and analyzed the utility of this system. Genomic DNA was extracted from tumor tissues and peripheral blood cells collected from 161 patients with different stages and types of cancer. A comprehensive targeted amplicon exome sequencing for 160 cancer-related genes was performed using next-generation sequencing (NGS). The sequencing data were analyzed using an original bioinformatics pipeline, and multiple cancer-specific gene alterations were identified. The success rate of our test was 99% (160/161), while re-biopsy was required for 24% (39/161) of the cases. Potentially actionable and actionable gene alterations were detected in 91% (145/160) and 46% (73/160) of the patients, respectively. The actionable gene alterations were frequently detected in PIK3CA (9%), ERBB2 (8%), and EGFR (4%). High tumor mutation burden (TMB) (≥10 mut/Mb) was observed in 12% (19/160) of the patients. The secondary findings in germline variants considered to be associated with hereditary tumors were detected in 9% (15/160) of the patients. Seventeen patients (11%, 17/160) were treated with genotype-matched therapeutic agents, and the response rate was 47% (8/17). The median turnaround time for physicians was 20 days, and the median survival time after the initial visit was 8.7 months. The results of the present study prove the feasibility of implementing in-house clinical sequencing as a promising laboratory examination technique for precision cancer medicine.

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A phase 2 basket trial of combination therapy with trastuzumab and pertuzumab in patients with solid cancers harboring human epidermal growth factor receptor 2 amplification (JUPITER trial).

Kenta Takahashi, Eri Ishibashi, Toshio Kubo, Yohei Harada, Hideyuki Hayashi, Masayuki Kano, Yasushi Shimizu, Hidekazu Shirota, Yukiko Mori, Manabu Muto, Chikashi Ishioka, Hirotoshi Dosaka-Akita, Hisahiro Matsubara, Hiroshi Nishihara, Naoko Sueoka-Aragane, Shinichi Toyooka, Akihiro Hirakawa, Ukihide Tateishi, Satoshi Miyake, Sadakatsu Ikeda

Medicine 99 ( 32 ) e21457 2020.08

Accepted, ISSN 0025-7974

INTRODUCTION: Human epidermal growth factor receptor 2 (HER2) gene amplification and mutations have emerged as oncogenic drivers and therapeutic targets not limited to breast and gastric cancers, but also in a variety of cancers. However, even if an actionable gene alteration is found, the incidence of HER2 amplification in these cancers is less than 5%. It is too difficult to conduct a conventional randomized, controlled trial in a rare fraction. Therefore, we have designed a organ-agnostic basket study, which covers a variety of solid cancers harboring HER2 amplification, in 1 study protocol. METHODS/DESIGN: This trial is a multicenter, single-arm, basket phase 2 study in Japan. Patients with solid cancers harboring HER2 amplification that have progressed with standard treatment, or rare cancers for which there is no standard treatment, will be eligible. Target cancers include bile duct, urothelial, uterine, ovarian, and other solid cancers where HER2 amplification is detected by comprehensive genomic profiling using next-generation sequencing technology. A total of 38 patients will be treated with combination therapy with trastuzumab and pertuzumab every 3 weeks until disease progression, unmanageable toxicity, death, or patient refusal. The primary endpoint is the objective response rate, and secondary endpoints are progression-free survival, overall survival, and duration of response. DISCUSSION: The aim of this trial is to evaluate the safety and efficacy of combination therapy with trastuzumab and pertuzumab in patients with locally advanced or metastatic, solid cancers harboring HER2 amplification. Instead of focusing on 1 organ type, our trial design uses a basket study focusing on HER2 amplification, regardless of the site or origin of the cancer. The results of our study will advance clinical and scientific knowledge concerning the treatment of locally advanced, rare solid cancers harboring HER2 amplification, using the combination of trastuzumab and pertuzumab. TRIAL REGISTRATION: This trial was registered in Japan Registry of Clinical Trials (jCRT) on February 25, 2019, as jRCT2031180150.

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A Japanese patient with ductal carcinoma of the prostate carrying an adenomatosis polyposis coli gene mutation: a case report.

Kota Umeda, Takeo Kosaka, Kohei Nakamura, Toshikazu Takeda, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

Diagnostic pathology 15 ( 1 ) 102 - 102 2020.08

Accepted

BACKGROUND: Ductal carcinoma of the prostate is a histological subtype with a higher mortality than acinar adenocarcinoma. The number of cases is small and there are no treatment guidelines. We believe that this is the first report of ductal carcinoma of the prostate with an adenomatosis polyposis coli (APC) gene mutation in Japan. CASE PRESENTATION: An 85-year-old man presented with gross hematuria, and a papillary tumor in the prostatic urethra that was diagnosed as ductal carcinoma of the prostate following transurethral resection. Genetic analysis found an APC mutation with loss of heterozygosity. Immunostaining revealed focal nuclear translocation of β-catenin. APC mutations associated with loss of β-catenin degradation in the Wnt signaling pathway and result in over accumulation of β-catenin are thought to increase mortality. In this patient, β-catenin migrated into tumor cell nuclei. CONCLUSION: To the best of our knowledge, this is the first report of ductal carcinoma of the prostate with an APC mutation in Japan. The development of a therapeutic Wnt inhibitor is discussed.

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[Management of Cancer Genomic Board and Preparation of Genomic Report].

Nakamura K, Nishihara H

Gan to kagaku ryoho. Cancer & chemotherapy 47 ( 8 ) 1141 - 1148 2020.08

Accepted, ISSN 0385-0684

Detection of somatic TP53 mutation in surgically resected small-cell lung cancer by targeted exome sequencing: association with longer relapse-free survival

Hiroshi Yokouchi, Hiroshi Nishihara, Toshiyuki Harada, Shigeo Yamazaki, Hajime Kikuchi, Satoshi Oizumi, Hidetaka Uramoto, Fumihiro Tanaka, Masao Harada, Kenji Akie, Fumiko Sugaya, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Mitsunori Higuchi, Osamu Honjo, Yoshinori Minami, Naomi Watanabe, Masaharu Nishimura, Hiroyuki Suzuki, Hirotoshi Dosaka-Akita, Hiroshi Isobe

Heliyon (Elsevier BV) 6 ( 7 ) e04439 - e04439 2020.07

Accepted, ISSN 2405-8440

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Next-generation sequencing in residual liquid-based cytology specimens for cancer genome analysis.

Tomomi Yamaguchi, Toshiaki Akahane, Ohi Harada, Yasutaka Kato, Eriko Aimono, Hidehiro Takei, Takashi Tasaki, Hirotsugu Noguchi, Hiroshi Nishihara, Hajime Kamata, Akihide Tanimoto

Diagnostic cytopathology 48 ( 11 ) 965 - 971 2020.06

Accepted

BACKGROUND: Cancer genome profiling of cytology specimens using next-generation sequencing (NGS) requires adequate and good-quality DNA. Genomic examination of cytology samples was conventionally performed on cell block (CB) or smear specimens than on residual liquid-based cytology (LBC) specimens, which are high-quality DNA sources even after long-term storage. METHODS: We estimated tumor fractions of 37 residual LBC specimens, including 30 fine needle aspiration (FNA) samples from the thyroid (12 papillary thyroid carcinomas and two malignant lymphomas), lymph node (13 metastatic carcinomas and one malignant lymphoma), and breast cancer (one phyllodes tumor and one invasive ductal carcinoma), two pancreatic carcinoma samples, and five liquid (ascites, pleural effusion, and cerebrospinal fluid) samples. The DNA was extracted from all samples and subjected to NGS using a customized cancer gene panel comprising 28 cancer-related genes. RESULTS: NGS analysis revealed somatic mutations corresponding to pathological diagnosis with adequate variant allele frequency (VAF) in 24 LBC specimens, which had significantly higher tumor fraction (72.5% ± 4.9%). Ten cases, including the five fluid samples, had very small tumor fractions (7.5% ± 2.3%) to obtain sufficient VAF. Other two samples had high tumor fractions but showed very low VAF, indicating the presence of fusion genes. The remaining one sample yielded no DNA recovery. CONCLUSION: The residual LBC specimens collected by FNA from the thyroid gland and lymph node were verified to carry high tumor fraction and could serve as an alternate source for molecular testing to screen and diagnose cancers without the use of CB or smears.

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Leukotriene B(4) receptor 1 exacerbates inflammation following myocardial infarction.

Horii Y, Nakaya M, Ohara H, Nishihara H, Watari K, Nagasaka A, Nakaya T, Sugiura Y, Okuno T, Koga T, Tanaka A, Yokomizo T, Kurose H

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 34 ( 6 ) 8749 - 8763 2020.06

Accepted, ISSN 0892-6638

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Genetic profiling of patients with adenoid cystic carcinoma of the Bartholin's glands reveals potential new routes for targeted therapies: a case report.

Kohei Nakamura, Eriko Aimono, Shigeki Tanishima, Hidetaka Nomura, Mitsuho Imai, Hideyuki Hayashi, Hiroshi Nishihara

Diagnostic pathology 15 ( 1 ) 64 - 64 2020.05

Accepted

BACKGROUND: Bartholin gland carcinomas (BGCs) are rare tumor types, for which no molecular analyses including genomic sequencing have been reported to date. Adenoid cystic carcinomas (ACCs) of the Bartholin's glands are an atypical histological type of BGC, and currently nothing is known regarding their genetic profiles or similarity to ACC carcinogenesis in other organs including the salivary glands, thereby limiting possible therapeutic options using precision medicine. CASE PRESENTATION: We used targeted gene sequencing to analyze the occurrence of 160 cancer-related genes in two patients with BG-ACC. KRAS and KDM6A mutations were detected in tumor samples collected from each patient. No KRAS mutations have been previously reported in salivary gland ACCs, indicating that the carcinogenesis of BG-ACC differs from that of the salivary gland ACCs. KDM6A mutations are often reported in salivary gland ACCs and facilitate novel gene-targeted therapy, including the use of BET and HDAC inhibitors. CONCLUSIONS: A better understanding of the underlying genetic mechanisms will help to clarify the carcinogenesis of BG-ACC. In turn, this will enable treatment with novel targeting agents, as well as the initial exploration of gene-based precision oncological therapies, which aim to improve treatment outcomes for patients with this disease.

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Osimertinib Overcomes Alectinib Resistance Caused by Amphiregulin in a Leptomeningeal Carcinomatosis Model of ALK-Rearranged Lung Cancer.

Sachiko Arai, Shinji Takeuchi, Koji Fukuda, Hirokazu Taniguchi, Akihiro Nishiyama, Azusa Tanimoto, Miyako Satouchi, Kaname Yamashita, Koshiro Ohtsubo, Shigeki Nanjo, Toru Kumagai, Ryohei Katayama, Makoto Nishio, Mei-Mei Zheng, Yi-Long Wu, Hiroshi Nishihara, Takushi Yamamoto, Mitsutoshi Nakada, Seiji Yano

Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 15 ( 5 ) 752 - 765 2020.05

Accepted, ISSN 1556-0864

INTRODUCTION: Leptomeningeal carcinomatosis (LMC) occurs frequently in anaplastic lymphoma kinase (ALK)-rearranged NSCLC and develops acquired resistance to ALK tyrosine kinase inhibitors (ALK TKIs). This study aimed to clarify the resistance mechanism to alectinib, a second-generation ALK TKI, in LMC and test a novel therapeutic strategy. METHODS: We induced alectinib resistance in an LMC mouse model with ALK-rearranged NSCLC cell line, A925LPE3, by continuous oral alectinib treatment, established A925L/AR cells. Resistance mechanisms were analyzed using several assays, including Western blot and receptor tyrosine kinase array. We also measured amphiregulin (AREG) concentrations in cerebrospinal fluid from patients with ALK-rearranged NSCLC with alectinib-refractory LMC by enzyme-linked immunosorbent assay. RESULTS: A925L/AR cells were moderately resistant to various ALK TKIs, such as alectinib, crizotinib, ceritinib, and lorlatinib, compared with parental cells in vitro. A925L/AR cells acquired the resistance by EGFR activation resulting from AREG overexpression caused by decreased expression of microRNA-449a. EGFR TKIs and anti-EGFR antibody resensitized A925L/AR cells to alectinib in vitro. In the LMC model with A925L/AR cells, combined treatment with alectinib and EGFR TKIs, such as erlotinib and osimertinib, successfully controlled progression of LMC. Mass spectrometry imaging showed accumulation of the EGFR TKIs in the tumor lesions. Moreover, notably higher AREG levels were detected in cerebrospinal fluid of patients with alectinib-resistant ALK-rearranged NSCLC with LMC (n = 4), compared with patients with EGFR-mutated NSCLC with EGFR TKI-resistant LMC (n = 30), or patients without LMC (n = 24). CONCLUSIONS: These findings indicate the potential of novel therapies targeting both ALK and EGFR for the treatment of ALK TKI-resistant LMC in ALK-rearranged NSCLC.

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チームアプローチで実現するゲノム医療と薬剤師の役割がんゲノム医療における患者対応の経験から

四十物絵理子, 井口亜橘, 持田かおり, 今井光穂, 林秀幸, 西原広史

薬学雑誌 ((公社)日本薬学会) 140 ( 5 ) 667 - 668 2020.05

Accepted, ISSN 0031-6903

癌ゲノム医療の現場において、患者の語りを傾聴し、正しく状況を理解すべき職種として、看護師と並んで薬剤師が挙げられる。癌遺伝子外来での患者対応の実情、遺伝性腫瘍症候群について概説し、ゲノム医療における多職種連携の目指すべき姿について述べた。関与する医療者がそれぞれの立場で患者に必要な情報を伝える必要があり、その中でも薬剤師の関与は重要であると考えられた。

Excessive EP4 Signaling in Smooth Muscle Cells Induces Abdominal Aortic Aneurysm by Amplifying Inflammation.

Taro Hiromi, Utako Yokoyama, Daisuke Kurotaki, Al Mamun, Ryo Ishiwata, Yasuhiro Ichikawa, Hiroshi Nishihara, Masanari Umemura, Takayuki Fujita, Shota Yasuda, Tomoyuki Minami, Motohiko Goda, Keiji Uchida, Shinichi Suzuki, Ichiro Takeuchi, Munetaka Masuda, Richard M Breyer, Tomohiko Tamura, Yoshihiro Ishikawa

Arteriosclerosis, thrombosis, and vascular biology 40 ( 6 ) ATVBAHA120314297 - 1573 2020.04

Accepted, ISSN 1079-5642

OBJECTIVE: Excessive prostaglandin E2 production is a hallmark of abdominal aortic aneurysm (AAA). Enhanced expression of prostaglandin E2 receptor EP4 in vascular smooth muscle cells (VSMCs) has been demonstrated in human AAAs. Although moderate expression of EP4 contributes to vascular homeostasis, the roles of excessive EP4 in vascular pathology remain uncertain. We aimed to investigate whether EP4 overexpression in VSMCs exacerbates AAAs. Approach and Results: We constructed mice with EP4 overexpressed selectively in VSMCs under an SM22α promoter (EP4-Tg). Most EP4-Tg mice died within 2 weeks of Ang II (angiotensin II) infusion due to AAA, while nontransgenic mice given Ang II displayed no overt phenotype. EP4-Tg developed much larger AAAs than nontransgenic mice after periaortic CaCl2 application. In contrast, EP4fl/+;SM22-Cre;ApoE-/- and EP4fl/+;SM22-Cre mice, which are EP4 heterozygous knockout in VSMCs, rarely exhibited AAA after Ang II or CaCl2 treatment, respectively. In Ang II-infused EP4-Tg aorta, Ly6Chi inflammatory monocyte/macrophage infiltration and MMP-9 (matrix metalloprotease-9) activation were enhanced. An unbiased analysis revealed that EP4 stimulation positively regulated the genes binding cytokine receptors in VSMCs, in which IL (interleukin)-6 was the most strongly upregulated. In VSMCs of EP4-Tg and human AAAs, EP4 stimulation caused marked IL-6 production via TAK1 (transforming growth factor-β-activated kinase 1), NF-κB (nuclear factor-kappa B), JNK (c-Jun N-terminal kinase), and p38. Inhibition of IL-6 prevented Ang II-induced AAA formation in EP4-Tg. In addition, EP4 stimulation decreased elastin/collagen cross-linking protein LOX (lysyl oxidase) in both human and mouse VSMCs. CONCLUSIONS: Dysregulated EP4 overexpression in VSMCs promotes inflammatory monocyte/macrophage infiltration and attenuates elastin/collagen fiber formation, leading to AAA exacerbation.

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Intratumoral Genomic Heterogeneity May Hinder Precision Medicine Strategies in Patients with Serous Ovarian Carcinoma.

Kohei Nakamura, Eriko Aimono, Shigeki Tanishima, Mitsuho Imai, Akiko Kawano Nagatsuma, Hideyuki Hayashi, Yuki Yoshimura, Kentaro Nakayama, Satoru Kyo, Hiroshi Nishihara

Diagnostics (Basel, Switzerland) 10 ( 4 ) 2020.04

Accepted, ISSN 2075-4418

Precision medicine, which includes comprehensive genome sequencing, is a potential therapeutic option for treating high-grade serous carcinoma (HGSC). However, HGSC is a heterogeneous tumor at the architectural, cellular, and molecular levels. Intratumoral molecular heterogeneity currently limits the precision of medical strategies based on the gene mutation status. This study was carried out to analyze the presence of 160 cancer-related genetic alterations in three tissue regions with different pathological features in a patient with HGSC. The patient exhibited histological heterogeneous features with different degrees of large atypical cells and desmoplastic reactions. TP53 mutation, ERBB2 and KRAS amplification, and WT1, CDH1, and KDM6A loss were detected as actionable gene alterations. Interestingly, the ERBB2 and KRAS amplification status gradually changed according to the region examined. The difference was consistent with the differences in pathological features. Our results demonstrate the need for sampling of the appropriate tissue region showing progression of pathological features for molecular analysis to solve issues related to tumor heterogeneity prior to developing precision oncology strategies.

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Japan society of clinical oncology/Japanese society of medical oncology-led clinical recommendations on the diagnosis and use of tropomyosin receptor kinase inhibitors in adult and pediatric patients with neurotrophic receptor tyrosine kinase fusion-positive advanced solid tumors, cooperated by the Japanese society of pediatric hematology/oncology.

Yoichi Naito, Saori Mishima, Kiwamu Akagi, Ataru Igarashi, Masafumi Ikeda, Susumu Okano, Shunsuke Kato, Tadao Takano, Katsuya Tsuchihara, Keita Terashima, Hiroshi Nishihara, Hiroyki Nishiyama, Eiso Hiyama, Akira Hirasawa, Hajime Hosoi, Osamu Maeda, Yasushi Yatabe, Wataru Okamoto, Shigeru Ono, Hiroaki Kajiyama, Fumio Nagashima, Yutaka Hatanaka, Mitsuru Miyachi, Yasuhiro Kodera, Takayuki Yoshino, Hiroya Taniguchi

International journal of clinical oncology 25 ( 3 ) 403 - 417 2020.03

Accepted, ISSN 1341-9625

BACKGROUND: The development of novel antitumor agents and accompanying biomarkers has improved survival across several tumor types. Previously, we published provisional clinical opinion for the diagnosis and use of immunotherapy in patients with deficient DNA mismatch repair tumors. Recently, efficacy of tropomyosin receptor kinase inhibitors against neurotrophic receptor tyrosine kinase (NTRK) fusion gene-positive advanced solid tumors have been established as the second tumor-agnostic treatment, making it necessary to develop the guideline prioritized for these patients. METHODS: Clinical questions regarding medical care were formulated for patients with NTRK-positive advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by Japan Society of Clinical Oncology (JSCO) and Japanese Society of Medical Oncology (JSMO) voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and Japanese Society of Pediatric Hematology/Oncology, and the public comments among all Societies' members was done. RESULTS: The current guideline describes 3 clinical questions and 15 recommendations for whom, when, and how NTRK fusion should be tested, and what is recommended for patients with NTRK fusion-positive advanced solid tumors. CONCLUSION: In the NTRK guideline, the committee proposed 15 recommendations for performing NTRK testing properly to select patients who are likely to benefit from tropomyosin receptor kinase inhibitors.

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Japan Society of Clinical Oncology provisional clinical opinion for the diagnosis and use of immunotherapy in patients with deficient DNA mismatch repair tumors, cooperated by Japanese Society of Medical Oncology, First Edition.

Saori Mishima, Hiroya Taniguchi, Kiwamu Akagi, Eishi Baba, Yutaka Fujiwara, Akira Hirasawa, Masafumi Ikeda, Osamu Maeda, Kei Muro, Hiroshi Nishihara, Hiroyki Nishiyama, Tadao Takano, Katsuya Tsuchihara, Yasushi Yatabe, Yasuhiro Kodera, Takayuki Yoshino

International journal of clinical oncology 25 ( 2 ) 217 - 239 2020.02

Accepted, ISSN 1341-9625

BACKGROUND: Novel therapeutic agents have improved survival outcomes in patients with advanced solid tumors. In parallel, the development of predictive biomarkers to identify patients who are likely to benefit from a certain treatment has also contributed to the improvement of survival. Recently, clinical trials have reported the efficacy of immune checkpoint inhibitors in the treatment of mismatch repair-deficient (dMMR) advanced solid tumors. In Japan, a PD-1 inhibitor for dMMR advanced solid tumors, regardless of the primary tumor site, has been approved. However, there are some issues related to administering immune checkpoint inhibitors in the clinical practice setting, making it necessary to develop the guidelines. METHODS: Clinical questions (CQs) regarding medical care were formulated for patients with dMMR advanced solid tumors, and evidence to the CQs was collected by manual search to prepare recommendations. Then, the committee members voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other factors. RESULTS: The current guideline, which we consider a provisional clinical opinion at this point, describes the 11 requirements to be considered in terms of patients for whom dMMR testing is recommended, the timing and methods of dMMR testing, and clinical care systems required to perform dMMR testing properly and to administer immune checkpoint inhibitors safely. CONCLUSION: This provisional clinical opinion proposes the requirements for performing dMMR testing properly to select patients who are likely to benefit from immune checkpoint inhibitors and administering them safely.

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Aggressive prostate cancer with somatic loss of the homologous recombination repair gene FANCA: a case report.

Hiroshi Hongo, Takeo Kosaka, Eriko Aimono, Hiroshi Nishihara, Mototsugu Oya

Diagnostic pathology 15 ( 1 ) 5 - 5 2020.01

Accepted

BACKGROUND: Precision medicine based on genomic analysis of germline or tumor tissue is attracting attention. However, there is no consensus on how to apply the results of genomic analysis to treatment. CASE PRESENTATION: A 59-year-old man diagnosed with metastatic prostate cancer was diagnosed with castration-resistant prostate cancer. Although he was sequentially treated with enzalutamide and abiraterone, bone metastasis progression was identified by skeletal scintigraphy. Therefore, we sequentially performed docetaxel therapy followed by cabazitaxel. After the third cycle of cabazitaxel, his prostate-specific antigen level was stable at < 10 ng/mL, and no radiological progression was detected. The patient's formalin-fixed paraffin-embedded tumor biopsy specimen underwent multiple-gene testing by next-generation sequencing, which identified a FANCA homodeletion. No significant germline mutation was observed. CONCLUSIONS: We describe a case of aggressive, castration-resistant prostate cancer with FANCA homodeletion. Genomic analysis of prostate cancer tissue can be useful to determine optimal treatment of such cancers.

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[Required Human and System Resources for Genomic Medicine].

Eriko Aimono, Hiroshi Nishihara

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 140 ( 5 ) 651 - 655 2020

Accepted, ISSN 0031-6903

Advances in genomic medicine have enabled the development of precise cancer therapies (precision cancer medicine) through multigene testing. Toward this end, we have developed a novel clinical sequencing system called PleSSision (Pathologist edited, Mitsubishi Space Software supervised clinical sequence system for personalized medicine) that performs amplicon exome sequencing targeting 160 cancer genes. Using this system, we have examined more than 600 cases over 3 years, and have identified druggable gene alteration in approximately 60% of the cases. Performing such clinical sequencing requires management of the sample quality and sequencing by pathologists and laboratory technicians; bioinformatics analysis by biomedical scientists; and patient care by nurses and pharmacists, all based on specific skills and knowledge of genomics. In addition, patients diagnosed with a hereditary cancer syndrome based on clinical sequencing results must receive care from a genetic counselor and a medical doctor with expertise in genetics. Recently, poly(ADP-ribose)polymerase (PARP) inhibitors and immune checkpoint inhibitors have been used in the treatment of patients with hereditary cancer syndromes, so collaboration involving other medical staff, especially genomic pharmacists, is also required. In this session, we provide an overview of cancer genomic medicine and emphasize the role that genomic pharmacists play in cancer precision medicine.

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[Learning from My Experience: Outpatient Care for Cancer Multigene Genomic Testing].

Eriko Aimono, Aki Iguchi, Kaori Mochida, Mitsuho Imai, Hideyuki Hayashi, Hiroshi Nishihara

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 140 ( 5 ) 667 - 668 2020

Accepted, ISSN 0031-6903

We established an outpatient service in November 2017 to provide cancer gene profiling test services to cancer patients. To date, we have seen approximately 100 patients. Our staff includes genetic counselors and nurses specialized in genetic medicine. Our experience highlights the importance of healthcare professionals having in-depth knowledge of cancer therapeutic drugs and/or investigational drugs based on cancer genome medicine. Recently, poly(ADP-ribose) polymerase (PARP) inhibitors have been approved for treating breast cancer patients with germline BRCA mutation; thus, in-depth knowledge of genetics and skills for genetic counseling are often considered indispensable in working with cancer patients. However, because the prompt treatment of clear and present cancer is the top priority in clinical settings, providing genetic information at that time, including that of unaffected family members, is of low priority for most patients who are dealing with the severe side effects of anti-cancer therapies. Pharmacists have an essential role to play in cancer therapeutics, talking with patients in order to assess their condition and to clarify the status of their treatment with anticancer agents. Genetic pharmacists should therefore work closely with genetics nurses and genetic counselors in the clinical practice of cancer genomic medicine. In this symposium, I would like to describe our experience caring for patients through our outpatient service, and to discuss the ideal framework for multidisciplinary cooperation to promote cancer genomic medicine.

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Impaired mitochondrial oxidative phosphorylation capacity in epicardial adipose tissue is associated with decreased concentration of adiponectin and severity of coronary atherosclerosis.

Takayuki Nakajima, Takashi Yokota, Yasushige Shingu, Akira Yamada, Yutaka Iba, Kosuke Ujihira, Satoru Wakasa, Tomonori Ooka, Shingo Takada, Ryosuke Shirakawa, Takashi Katayama, Takaaki Furihata, Arata Fukushima, Ryosuke Matsuoka, Hiroshi Nishihara, Flemming Dela, Katsuhiko Nakanishi, Yoshiro Matsui, Shintaro Kinugawa

Scientific reports (Scientific Reports) 9 ( 1 ) 3535 - 3535 2019.12

Research paper (scientific journal), Joint Work, Accepted

Epicardial adipose tissue (EAT), a source of adipokines, is metabolically active, but the role of EAT mitochondria in coronary artery disease (CAD) has not been established. We investigated the association between EAT mitochondrial respiratory capacity, adiponectin concentration in the EAT, and coronary atherosclerosis. EAT samples were obtained from 25 patients who underwent elective cardiac surgery. Based on the coronary angiographycal findings, the patients were divided into two groups; coronary artery disease (CAD; n = 14) and non-CAD (n = 11) groups. The mitochondrial respiratory capacities including oxidative phosphorylation (OXPHOS) capacity with non-fatty acid (complex I and complex I + II-linked) substrates and fatty acids in the EAT were significantly lowered in CAD patients. The EAT mitochondrial OXPHOS capacities had a close and inverse correlation with the severity of coronary artery stenosis evaluated by the Gensini score. Intriguingly, the protein level of adiponectin, an anti-atherogenic adipokine, in the EAT was significantly reduced in CAD patients, and it was positively correlated with the mitochondrial OXPHOS capacities in the EAT and inversely correlated with the Gensini score. Our study showed that impaired mitochondrial OXPHOS capacity in the EAT was closely linked to decreased concentration of adiponectin in the EAT and severity of coronary atherosclerosis.

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A first Japanese case of neuroendocrine prostate cancer accompanied by lung and brain metastasis with somatic and germline BRCA2 mutation.

Takeo Kosaka, Hiroshi Hongo, Eriko Aimono, Kazuhiro Matsumoto, Tetsu Hayashida, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

Pathology international (Pathology International) 69 ( 12 ) 715 - 720 2019.12

Joint Work, Accepted, ISSN 13205463

Germline mutations and copy number changes in DNA damage repair (DDR) genes such as BRCA2 are associated with aggressive forms of prostate cancer (PCa). Although the prevalence of BRCA2 variants in PCa is increasing in Japan, the genomic and biological implications in Japanese patients are unclear. An 81-year-old male presented with prostatic adenocarcinoma with neuroendocrine differentiation accompanied by metastatic lung nodule and brain metastases. Platinum-based doublet chemotherapy combined with etoposide resulted in partial and complete remissions of brain and lung metastases, respectively. Next-generation sequencing of biopsy and peripheral blood samples demonstrated a somatic BRCA2 mutation at c.7008-2A>C and a germline mutation at p.E2877*. The patient's son had been diagnosed with breast cancer 2.5 years ago and was found to have the same germline BRCA2 mutation. BRCA2 mutation increases the risks of aggressive PCa and other cancer types in Japanese males. These forms may be highly responsive to platinum-based chemotherapy.

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Cancer gene profiling explores the possible precision medicine for diffuse-type gastric adenocarcinoma.

Marin Ishikawa, Hideyuki Hayashi, Naoya Sakamoto, Shinya Tanaka, Hiroshi Nishihara

Medical oncology (Northwood, London, England) 37 ( 1 ) 10 - 10 2019.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 1357-0560

Diffuse type gastric cancer (DGC), a pathological subtype, is one of the most common malignant solid tumors, and mortality of this tumor is not negligible, especially in early-onset cancer patients. In fact, affirmative personalized treatments based on gene profile have not been established yet. The aim of this study was to provide the possible genotype-matched treatment for DGC through comprehensive examination of genomic variants and analysis of clinicopathological characteristics. We retrospectively studied 23 formalin-fixed, paraffin-embedded samples of patients diagnosed as DGC between January 2003 and December 2015 at the Department of Cancer Pathology, Hokkaido University Graduate School of Medicine. The cases were divided into two groups: early-onset (< 50 years old) and elderly-onset (≥ 50 years old) DGC groups. We performed targeted genomic sequencing using a 163 cancer-related gene panel. The sequencing data were analyzed using an original bioinformatics pipeline called GenomeJack and were clinicopathologically evaluated. Intestinal metaplasia and atrophy were highly observed in the adjacent non-cancerous mucosa in the elderly-onset DGC group compared with those in the early-onset DGC group. The number of somatic variants was significantly higher in the elderly-onset DGC group than in the early-onset DGC group. Fifteen patients (65.2%) harbored at least one genomic alteration of the potential target for genotype-matched treatment. In addition, one patient with hypermutation phenotype was diagnosed as Lynch syndrome due to MLH1 mutation, suggesting the sensitivity for the treatment with immune checkpoint inhibitors. Not only does our study demonstrated the potential utility of the targeted genomic sequencing approach for making informed therapeutic decisions, but it also sheds light on DGC pathogenesis and progression.

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Establishment of permutation for cancer risk estimation in the urothelium based on genome-wide DNA methylation analysis.

Koji Tsumura, Eri Arai, Ying Tian, Ayako Shibuya, Hiroshi Nishihara, Takuya Yotani, Yuriko Yamada, Yoriko Takahashi, Akiko Miyagi Maeshima, Hiroyuki Fujimoto, Tohru Nakagawa, Haruki Kume, Yukio Homma, Teruhiko Yoshida, Yae Kanai

Carcinogenesis (Carcinogenesis) 40 ( 11 ) 1308 - 1319 2019.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 01433334

The aim of this study was to establish permutation for cancer risk estimation in the urothelium. Twenty-six samples of normal control urothelium obtained from patients without urothelial carcinomas (C), 47 samples of non-cancerous urothelium without noticeable morphological changes obtained from patients with urothelial carcinomas (N), and 46 samples of the corresponding cancerous tissue (T) in the learning cohort and 64 N samples in the validation cohort, i.e. 183 tissue samples in total, were analyzed. Genome-wide DNA methylation analysis was performed using the Infinium HumanMethylation 450K BeadChip, and DNA methylation levels were verified using pyrosequencing and MassARRAY. Amplicon sequencing was performed using the GeneRead DNAseq Targeted Panels V2. Although N samples rarely showed genetic mutations or copy number alterations, they showed DNA methylation alterations at 2502 CpG sites compared to C samples, and such alterations were inherited by or strengthened in T samples, indicating that DNA methylation alterations may participate in field cancerization in the urothelium. Receiver operating characteristic curve analysis confirmed the feasibility of cancer risk estimation to identify urothelium at the precancerous stage by DNA methylation quantification. Cancer risk estimation permutation was established using a combination of two marker CpG loci on the HOXC4, TENM3 and TLR1 genes (sensitivity and specificity 96-100%). Among them, the diagnostic impact of 10 patterns of permutation was successfully validated in the validation cohort (sensitivity and specificity 94-98%). These data suggest that cancer risk estimation using procedures such as urine tests during health checkups might become applicable for clinical use.

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Evolutionary Gain of Dbx1 Expression Drives Subplate Identity in the Cerebral Cortex.

Arai Y, Cwetsch AW, Coppola E, Cipriani S, Nishihara H, Kanki H, Saillour Y, Freret-Hodara B, Dutriaux A, Okada N, Okano H, Dehay C, Nardelli J, Gressens P, Shimogori T, D'Onofrio G, Pierani A

Cell reports 29 ( 3 ) 645 - 658.e5 2019.10

Accepted

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Broad Heterochromatic Domains Open in Gonocyte Development Prior to De Novo DNA Methylation.

Yamanaka S, Nishihara H, Toh H, Eijy Nagai LA, Hashimoto K, Park SJ, Shibuya A, Suzuki AM, Tanaka Y, Nakai K, Carninci P, Sasaki H, Siomi H

Developmental cell 51 ( 1 ) 21 - 34.e5 2019.10

Accepted, ISSN 1534-5807

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Japanese Case of Enzalutamide-Resistant Prostate Cancer Harboring a SPOP Mutation With Scattered Allelic Imbalance: Response to Platinum-Based Therapy.

Keitaro Watanabe, Takeo Kosaka, Eriko Aimono, Hiroshi Hongo, Shuji Mikami, Hiroshi Nishihara, Mototsugu Oya

Clinical genitourinary cancer (Clinical Genitourinary Cancer) 17 ( 5 ) e897 - e902 2019.10

Joint Work, Accepted, ISSN 15587673

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Glomangiopericytoma of the Nasal Cavity with CTNNB1 p.S37C Mutation: A Case Report and Literature Review.

Michihisa Kono, Nobuyuki Bandoh, Ryosuke Matsuoka, Takashi Goto, Toshiaki Akahane, Yasutaka Kato, Hiroshi Nakano, Tomomi Yamaguchi, Yasuaki Harabuchi, Hiroshi Nishihara

Head and neck pathology (Head and Neck Pathology) 13 ( 3 ) 298 - 303 2019.09

Joint Work, Accepted, ISSN 1936055X

Glomangiopericytoma (GPC) is a rare mesenchymal tumor arising from the nasal cavity or paranasal sinuses. GPC was categorized as a borderline and low-malignant-potential tumor by the World Health Organization in 2005 and accounts for less than 0.5% of all sinonasal tumors. We report a case of GPC in a 74-year-old woman with a history of recurrent epistaxis and nasal obstruction. A reddish tumor was seen in the right nasal cavity. Enhanced computed tomography showed a mass lesion occupying the right nasal cavity. The tumor, which originated from the nasal septum in the olfactory fissure area, was resected with 5-mm mucosal margins by endoscopic sinus surgery. Histologic examination revealed a uniform proliferation of oval-to-short spindle-shaped cells beneath the epithelium. Immunohistologic analysis demonstrated the tumor cells were positive for α-smooth muscle actin, β-catenin and Vimentin, and negative for AE1/AE3, Bcl-2, CD34, CD117, Factor VIIIR Ag, S-100 protein, or STAT6. The percentage of Ki-67-positive cells was approximately 5%. Genetic analysis using next-generation sequencing revealed a missense mutation in the CTNNB1 gene (c.110C > G, p.S37C). While other CTNNB1 mutations have been described in GPC; this is the first report of this specific mutation. The mutation was confirmed using Sanger sequencing.

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ヒト膵がん由来膵星状細胞を用いた三次元膵がん線維化モデルの構築ならびに異常ECM改築の機序の解析(Pancreatic stellate cells from human pancreatic cancer show aberrant ECM remodeling in 3D engineered fibrotic tissue)

田中啓祥, 西原広史, 正宗淳, 狩野光伸

日本癌学会総会記事 ((一社)日本癌学会) 78回 E - 3005 2019.09

Accepted, ISSN 0546-0476

バイオバンキングが切り拓く新しいがん研究わが国におけるクリニカルバイオバンクネットワークの構築(Innovative cancer research based on biobanking Clinical biobank network in Japan)

武藤学, 金井雅史, 鶴山竜昭, 松本繁巳, 豊岡伸一, 秋田弘俊, 松原久裕, 西原広史, 末岡榮三朗, 池田貞勝

日本癌学会総会記事 (日本癌学会) 78回 S11 - 4 2019.09

Accepted, ISSN 0546-0476

Effectiveness of a genetic test panel designed for gynecological cancer: an exploratory study.

Koichi Ida, Tsutomu Miyamoto, Shotaro Higuchi, Hodaka Takeuchi, Satoshi Yamada, Motoki Ono, Hiroshi Nishihara, Tanri Shiozawa

Medical oncology (Northwood, London, England) (Medical Oncology) 36 ( 7 ) 62 - 62 2019.07

Joint Work, Accepted, ISSN 13570560

To increase diagnostic efficiency and cost-effectiveness, we performed an exploratory genetic test using a newly designed panel containing 28 actionable and druggable genes, alterations in which are frequently reported in gynecological cancers (TANRE-G, Targeted variants ANalysis RElated to Gynecological cancers). Samples consisted of the formalin-fixed, paraffin-embedded tissue of endometrial (4 cases), cervical (3 cases), and ovarian (4 cases) carcinomas. The sequencing procedure was performed using Ion PGM in our institute with related sequencing kits, and data were analyzed using ClinVar. The present system achieved more than 2500 reads in all tumor samples, and enabled a copy number variation analysis. Results showed that actionable and druggable mutations were detected in 82% (9/11) and 64% (7/11) of cases, respectively, which was similar to other commercially available genetic tests. The amplification of MYC and KRAS was also detected. The analysis cost for each sample was JPY 94,000 (USD 850). These results demonstrate the potential of the TANRE-G panel as an effective tool for examining genetic alterations in gynecological cancers.

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Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis.

Toshiaki Akahane, Tomomi Yamaguchi, Yasutaka Kato, Seiya Yokoyama, Taiji Hamada, Yukari Nishida, Michiyo Higashi, Hiroshi Nishihara, Shinsuke Suzuki, Shinichi Ueno, Akihide Tanimoto

PloS one (PLoS ONE) 14 ( 6 ) e0217724 2019.06

Joint Work, Accepted

In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 103 or 2 × 104 cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 103 cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine.

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Epithelial-to-Mesenchymal Transition Is a Mechanism of ALK Inhibitor Resistance in Lung Cancer Independent of ALK Mutation Status.

Koji Fukuda, Shinji Takeuchi, Sachiko Arai, Ryohei Katayama, Shigeki Nanjo, Azusa Tanimoto, Akihiro Nishiyama, Takayuki Nakagawa, Hirokazu Taniguchi, Takeshi Suzuki, Tadaaki Yamada, Hiroshi Nishihara, Hironori Ninomiya, Yuichi Ishikawa, Satoko Baba, Kengo Takeuchi, Atsushi Horiike, Noriko Yanagitani, Makoto Nishio, Seiji Yano

Cancer research (Cancer Research) 79 ( 7 ) 1658 - 1670 2019.04

Joint Work, Accepted, ISSN 00085472

Mutations in the ALK gene are detectable in approximately 40% of ALK-rearranged lung cancers resistant to ALK inhibitors. Although epithelial-to-mesenchymal transition (EMT) is a mechanism of resistance to various targeted drugs, its involvement in ALK inhibitor resistance is largely unknown. In this study, we report that both ALK-mutant L1196M and EMT were concomitantly detected in a single crizotinib-resistant lesion in a patient with ALK-rearranged lung cancer. Digital PCR analyses combined with microdissection after IHC staining for EMT markers revealed that ALK L1196M was predominantly detected in epithelial-type tumor cells, indicating that mesenchymal phenotype and ALK mutation can coexist as independent mechanisms underlying ALK inhibitor-resistant cancers. Preclinical experiments with crizotinib-resistant lung cancer cells showed that EMT associated with decreased expression of miR-200c and increased expression of ZEB1 caused cross-resistance to new-generation ALK inhibitors alectinib, ceritinib, and lorlatinib. Pretreatment with the histone deacetylase (HDAC) inhibitor quisinostat overcame this resistance by reverting EMT in vitro and in vivo. These findings indicate that HDAC inhibitor pretreatment followed by a new ALK inhibitor may be useful to circumvent resistance constituted by coexistence of resistance mutations and EMT in the heterogeneous tumor. SIGNIFICANCE: These findings show that dual inhibition of HDAC and ALK receptor tyrosine kinase activities provides a means to circumvent crizotinib resistance in lung cancer.

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Pancreatic stellate cells derived from human pancreatic cancer demonstrate aberrant SPARC-dependent ECM remodeling in 3D engineered fibrotic tissue of clinically relevant thickness.

Hiroyoshi Y Tanaka, Kentaro Kitahara, Naoki Sasaki, Natsumi Nakao, Kae Sato, Hirokazu Narita, Hiroshi Shimoda, Michiya Matsusaki, Hiroshi Nishihara, Atsushi Masamune, Mitsunobu R Kano

Biomaterials (Biomaterials) 192 355 - 367 2019.02

Joint Work, Accepted, ISSN 01429612

Desmoplasia is a hallmark of pancreatic cancer and consists of fibrotic cells and secreted extracellular matrix (ECM) components. Various in vitro three-dimensional (3D) models of desmoplasia have been reported, but little is known about the relevant thickness of the engineered fibrotic tissue. We thus measured the thickness of fibrotic tissue in human pancreatic cancer, as defined by the distance from the blood vessel wall to tumor cells. We then generated a 3D fibrosis model with a thickness reaching the clinically observed range using pancreatic stellate cells (PSCs), the main cellular constituent of pancreatic cancer desmoplasia. Using this model, we found that Collagen fiber deposition was increased and Fibronectin fibril orientation drastically remodeled by PSCs, but not normal fibroblasts, in a manner dependent on Transforming Growth Factor (TGF)-β/Rho-Associated Kinase (ROCK) signaling and Matrix Metalloproteinase (MMP) activity. Finally, by targeting Secreted Protein, Acidic and Rich in Cysteine (SPARC) by siRNA, we found that SPARC expression in PSCs was necessary for ECM remodeling. Taken together, we developed a 3D fibrosis model of pancreatic cancer with a clinically relevant thickness and observed aberrant SPARC-dependent ECM remodeling in cancer-derived PSCs.

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Pathways of Progression From Intraductal Papillary Mucinous Neoplasm to Pancreatic Ductal Adenocarcinoma Based on Molecular Features.

Yuko Omori, Yusuke Ono, Mishie Tanino, Hidenori Karasaki, Hiroshi Yamaguchi, Toru Furukawa, Katsuro Enomoto, Jun Ueda, Atsuko Sumi, Jin Katayama, Miho Muraki, Kenzui Taniue, Kuniyuki Takahashi, Yoshiyasu Ambo, Toshiya Shinohara, Hiroshi Nishihara, Junpei Sasajima, Hiroyuki Maguchi, Yusuke Mizukami, Toshikatsu Okumura, Shinya Tanaka

Gastroenterology (Gastroenterology) 156 ( 3 ) 647 - 661.e2 2019.02

Joint Work, Accepted, ISSN 00165085

BACKGROUND & AIMS: Intraductal papillary mucinous neoplasms (IPMNs) are regarded as precursors of pancreatic ductal adenocarcinomas (PDAs), but little is known about the mechanism of progression. This makes it challenging to assess cancer risk in patients with IPMNs. We investigated associations of IPMNs with concurrent PDAs by genetic and histologic analyses. METHODS: We obtained 30 pancreatic tissues with concurrent PDAs and IPMNs, and 168 lesions, including incipient foci, were mapped, microdissected, and analyzed for mutations in 18 pancreatic cancer-associated genes and expression of tumor suppressors. RESULTS: We determined the clonal relatedness of lesions, based on driver mutations shared by PDAs and concurrent IPMNs, and classified the lesions into 3 subtypes. Twelve PDAs contained driver mutations shared by all concurrent IPMNs, which we called the sequential subtype. This subset was characterized by less diversity in incipient foci with frequent GNAS mutations. Eleven PDAs contained some driver mutations that were shared with concurrent IPMNs, which we called the branch-off subtype. In this subtype, PDAs and IPMNs had identical KRAS mutations but different GNAS mutations, although the lesions were adjacent. Whole-exome sequencing and methylation analysis of these lesions indicated clonal origin with later divergence. Ten PDAs had driver mutations not found in concurrent IPMNs, called the de novo subtype. Expression profiles of TP53 and SMAD4 increased our ability to differentiate these subtypes compared with sequencing data alone. The branch-off and de novo subtypes had substantial heterogeneity among early clones, such as differences in KRAS mutations. Patients with PDAs of the branch-off subtype had a longer times of disease-free survival than patients with PDAs of the de novo or the sequential subtypes. CONCLUSIONS: Detailed histologic and genetic analysis of PDAs and concurrent IPMNs identified 3 different pathways by which IPMNs progress to PDAs-we call these the sequential, branch-off, and de novo subtypes. Subtypes might be associated with clinical and pathologic features and be used to select surveillance programs for patients with IPMNs.

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Practical procedures for the integrated diagnosis of astrocytic and oligodendroglial tumors.

Yukihiko Sonoda, Hideaki Yokoo, Shinya Tanaka, Manabu Kinoshita, Mitsutoshi Nakada, Hiroshi Nishihara

Brain tumor pathology (Brain Tumor Pathology) 36 ( 2 ) 56 - 62 2019

Joint Work, Accepted, ISSN 14337398

The publication of the 2016 World Health Organization Classification of Tumors of the Central Nervous System (2016 WHO CNS) represented a major change in the classification of brain tumors. However, many pathologists in Japan cannot diagnose astrocytic or oligodendroglial tumors according to the 2016 WHO CNS due to financial or technical problems. Therefore, the Japan Society of Brain Tumor Pathology established a committee for molecular diagnosis to facilitate the integrated diagnosis of astrocytic and oligodendroglial tumors in Japan. We created three levels of diagnoses: Level 1 was defined as simple histopathological diagnosis using hematoxylin and eosin staining and routine cell lineage-based immunostaining. Level 2 was defined as immunohistochemical diagnosis using immunohistochemical examinations using R132H mutation-specific IDH1, ATRX, and/or p53 antibodies. Level 3 was defined as molecular diagnosis, such as diagnosis based on 1p/19q status or the mutation status of the IDH1 and IDH2 genes. In principle, astrocytic and oligodendroglial tumors should be diagnosed based on the 2016 WHO CNS and/or cIMPACT-NOW criteria; however, the findings obtained through our diagnostic flowchart can be added to the histological diagnosis in parentheses. This classification system would be helpful for pathologists with limited resources.

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Analysis of DLL3 and ASCL1 in Surgically Resected Small Cell Lung Cancer (HOT1702).

Megumi Furuta, Jun Sakakibara-Konishi, Hajime Kikuchi, Hiroshi Yokouchi, Hiroshi Nishihara, Hiroyuki Minemura, Masao Harada, Shigeo Yamazaki, Kenji Akie, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Toshiyuki Harada, Yoshinori Minami, Naomi Watanabe, Satoshi Oizumi, Hiroyuki Suzuki, Masaharu Nishimura, Hirotoshi Dosaka-Akita, Hiroshi Isobe

The oncologist (Oncologist) 24 ( 11 ) e1172 - e1179 2019

Joint Work, Accepted, ISSN 10837159

BACKGROUND: Delta-like protein 3 (DLL3) is a Notch ligand that has an important role in the tumorigenesis of small cell lung cancer (SCLC). Recently, rovalpituzumab tesirine (Rova-T), a DLL3-targeted antibody-drug conjugate, has been developed for treating SCLC. DLL3 is a transcriptional target of the achaete-scute homolog-1 (ASCL1) transcription factor, which is involved in pulmonary neuroendocrine cell development. However, the relationship between DLL3 and/or ASCL1 expression and the clinical features of SCLC remains unknown, especially for early-stage resected SCLC. This study aimed to investigate the expression of DLL3 and ASCL1 in resected SCLC samples using immunohistochemical analysis. MATERIALS AND METHODS: We collected 95 surgically resected SCLC samples, which were formalin fixed and paraffin embedded. Immunohistochemistry staining was performed to investigate the correlation between the expression of either DLL3 or ASCL1 and clinicopathological features of study patients. RESULTS: Seventy-seven (83%) of 93 immunohistochemically evaluable samples were positive for DLL3 (expression in ≥1% of tumor cells), and DLL3-high expression (≥75%) was observed in 44 samples (47%). Sixty-one (64%) of 95 samples were positive for ASCL1 (expression in ≥5% of tumor cells). A positive correlation was observed between DLL3 and ASCL1 expression. DLL3 and ASCL1 expression were not associated with survival in SCLC patients. DLL3 was more prevalent in patients with advanced clinical disease. CONCLUSION: DLL3 and ASCL1 were highly expressed in patients with surgically resected SCLC. DLL3 and ASCL1 may be targets for the treatment of SCLC. IMPLICATIONS FOR PRACTICE: This article examines the relationship between delta-like protein 3 (DLL3) and achaete-scute homolog-1 (ASCL1) protein expression with the clinical features of 95 surgically resected small cell lung cancer (SCLC). DLL3 is attracting attention because rovalpituzumab tesirine (Rova-T), a DLL3-targeted antibody-drug conjugate, was developed recently. DLL3 and ASCL1 were highly expressed in patients with surgically resected SCLC. DLL3 and ASCL1 may be targets for the treatment of early-stage SCLC, including with Rova-T.

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Olaparib monotherapy for BRIP1-mutated high-grade serous endometrial cancer

Kohei Nakamura, Eriko Aimono, Shigeki Tanishima, Mitsuho Imai, Akiko Kawano Nagatsuma, Hideyuki Hayashi, Yuki Yoshimura, Kentaro Nakayama, Satoru Kyo, Hiroshi Nishihara

JCO Precision Oncology (JCO Precision Oncology) 3 283 - 290 2019

Research paper (scientific journal), Joint Work, Accepted

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Mutations in bassoon in individuals with familial and sporadic progressive supranuclear palsy-like syndrome.

Ichiro Yabe, Hiroaki Yaguchi, Yasutaka Kato, Yasuo Miki, Hidehisa Takahashi, Satoshi Tanikawa, Shinichi Shirai, Ikuko Takahashi, Mari Kimura, Yuka Hama, Masaaki Matsushima, Shinsuke Fujioka, Takahiro Kano, Masashi Watanabe, Shin Nakagawa, Yasuyuki Kunieda, Yoshio Ikeda, Masato Hasegawa, Hiroshi Nishihara, Toshihisa Ohtsuka, Shinya Tanaka, Yoshio Tsuboi, Shigetsugu Hatakeyama, Koichi Wakabayashi, Hidenao Sasaki

Scientific reports (Scientific Reports) 8 ( 1 ) 819 - 819 2018.12

Research paper (scientific journal), Joint Work

Clinical diagnosis of progressive supranuclear palsy (PSP) is sometimes difficult because various phenotypes have been identified. Here, we report a mutation in the bassoon (BSN) gene in a family with PSP-like syndrome. Their clinical features resembled not only those of PSP patients but also those of individuals with multiple system atrophy and Alzheimer's disease. The neuropathological findings showed a novel three + four repeat tauopathy with pallido-luysio-nigral degeneration and hippocampal sclerosis. Whole-exome analysis of this family identified a novel missense mutation in BSN. Within the pedigree, the detected BSN mutation was found only in affected individuals. Further genetic analyses were conducted in probands from four other pedigrees with PSP-like syndrome and in 41 sporadic cases. Three missense mutations in BSN that are very rarely listed in databases of healthy subjects were found in four sporadic cases. Western blot analysis of tau following the overexpression of wild-type or mutated BSN revealed the possibility that wild-type BSN reduced tau accumulation, while mutated BSN lost this function. An association between BSN and neurological diseases has not been previously reported. Our results revealed that the neurodegenerative disorder associated with the original proband's pedigree is a novel tauopathy, differing from known dementia and parkinsonism syndromes, including PSP.

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Targeted next-generation sequencing of cancer-related genes in thyroid carcinoma: A single institution's experience.

Nobuyuki Bandoh, Toshiaki Akahane, Takashi Goto, Michihisa Kono, Haruyuki Ichikawa, Takahiro Sawada, Tomomi Yamaguchi, Hiroshi Nakano, Yumiko Kawase, Yasutaka Kato, Hajime Kamada, Yasuaki Harabuchi, Kazuo Shimizu, Hiroshi Nishihara

Oncology letters (Oncology Letters) 16 ( 6 ) 7278 - 7286 2018.12

Joint Work, Accepted, ISSN 17921074

Thyroid carcinoma (TC) has characteristic genetic alterations, including point mutations in proto-oncogenes and chromosomal rearrangements that vary by histologic subtype. Recent developments in next-generation sequencing (NGS) technology enable simultaneous analysis of cancer-associated genes of interest, thus improving diagnostic accuracy and allowing precise personalized treatment for human cancer. A total of 50 patients who underwent thyroidectomy between 2014 and 2016 at Hokuto Hospital were enrolled. Total DNA was extracted from formalin-fixed, paraffin-embedded tissue sections and quantified. Targeted regions of 24 cancer-associated genes were amplified by PCR, barcoded and sequenced using an Illumina MiSeq platform. Subjects included 30 patients with papillary carcinoma (PC), two with PC tall cell variant (TVPC), two with PC follicular variant (FVPC), eight with follicular carcinoma, seven with poorly differentiated carcinoma (PDC), and one with anaplastic carcinoma (AC). The BRAF V600E mutation was present in 25 of 30 (83%) patients with PC, 2 of 2 (100%) patients with TVPC, 6 of 7 (86%) patients of PDC, and one patient with AC. PIK3CA mutations were present in 3 of 30 (delPV104P, A1046T and C420R; 10%) patients with PC and 1 of 7 (H1047R; 14%) patients with PDC. The TP53 mutation was present in 1 of 30 (R306*; 3.3%) patients with PC and 1 of 7 (Q152*; 14%) patients with PDC. The NRAS mutation was present in 1 of 2 (Q61K, 50%) patients with FVPC. Statistical analysis showed that patients without the BRAF V600E mutation had advanced pathologic T and N stages compared with those with the mutation (P=0.047 and P=0.019, respectively). The BRAF V600E mutation was not correlated with overall and disease-free survival in patients with PC. A patient with PC with a mutation in EGFR (K852Q) and the PIK3CA mutation had an aggressive course with multiple bone and lung metastases. Detection of mutations in cancer-associated genes using NGS could enhance the understanding of the clinical behavior of TC.

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Genetic mutation analysis of the malignant transformation of sinonasal inverted papilloma by targeted amplicon sequencing

Yasukawa S, Kano S, Hatakeyama H, Nakamaru Y, Takagi D, Mizumachi T, Suzuki M, Suzuki T, Nakazono A, Tanaka S, Nishihara H, Homma A

International Journal of Clinical Oncology (International Journal of Clinical Oncology) 23 ( 5 ) 835 - 843 2018.10

Research paper (scientific journal), Joint Work, ISSN 13419625

© 2018, Japan Society of Clinical Oncology. Background: The mechanism underlying the malignant transformation of inverted papilloma (IP) has not yet been elucidated. Methods: To clarify the genes responsible for the malignant transformation, we analyzed 10 cases of IP, 8 of IP with dysplasia, and 11 of squamous cell carcinoma (SCC) by targeted amplicon sequencing. Results: The number of mutant genes increased in the order of IP < dysplasia < SCC. Significant differences were observed in the mutation rates of three genes (KRAS, APC and STK11) in particular. TP53 was altered frequently in each group and might be involved in malignant transformation based on to the site of the mutation. A comparison of the genetic variants by region of IP tissue among patients with IP alone, and those with dysplasia or SCC revealed significant differences in the mutation rate of the KRAS gene. Conclusion: Identification of genetic mutations in KRAS is effective for predicting the malignant transformation of IP.

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In vitro 3D blood/lymph-vascularized human stromal tissues for preclinical assays of cancer metastasis.

Akihiro Nishiguchi, Michiya Matsusaki, Mitsunobu R Kano, Hiroshi Nishihara, Daisuke Okano, Yoshiya Asano, Hiroshi Shimoda, Satoko Kishimoto, Soichi Iwai, Mitsuru Akashi

Biomaterials (Biomaterials) 179 144 - 155 2018.10

Joint Work, ISSN 01429612

Tumour models mimicking in vivo three-dimensional (3D) microenvironments are of increasing interest in drug discovery because of the limitations inherent to current models. For example, preclinical assays that rely on monolayer or spheroid cell cultures cannot easily predict 3D cancer behaviours because they have no vasculature. Furthermore, there are major differences in cancer behaviour between human and animal experiments. Here, we show the construction of 3D blood/lymph-vascularized human stromal tissues that can be combined with cancer cells to mimic dynamic metastasis for real-time throughput screening of secreted proteinases. We validated this tool using three human carcinoma cell types that are known to invade blood/lymph vessels and promote angiogenesis. These cell types exhibited characteristic haematogenous/lymphogenous metastasis and tumour angiogenesis properties. Importantly, these carcinoma cells selectively secreted different matrix metalloproteinases depending on their metastasis stage and target vasculature, suggesting the possibility of developing drugs that can target each secreted proteinase. We conclude that the 3D tissue tool will be a powerful throughput system for predicting cancer cell responses and time-dependent secretion of molecules in preclinical assays.

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Genomic testing for pancreatic cancer in clinical practice as real-world evidence.

Hideyuki Hayashi, Shigeki Tanishima, Kyoko Fujii, Ryo Mori, Yasunobu Okamura, Emmy Yanagita, Ryosuke Matsuoka, Toraji Amano, Ichiro Kinoshita, Yoshito Komatsu, Hirotoshi Dosaka-Akita, Hiroshi Nishihara

Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.] (Pancreatology) 18 ( 6 ) 647 - 654 2018.09

Joint Work, Accepted, ISSN 14243903

BACKGROUND: Precision medicine guided by comprehensive genome sequencing represents a potential treatment strategy for pancreatic cancer. However, clinical sequencing for pancreatic cancer entails several practical difficulties. We have launched an in-house clinical sequencing system and started genomic testing for patients with cancer in clinical practice. We have analyzed the clinical utility of this system in pancreatic cancer. METHODS: We retrospectively reviewed 20 patients with pancreatic cancer who visited our division. Genomic DNA was extracted from both tumor tissue and peripheral blood mononuclear cells obtained from the patients. We performed a comprehensive genomic testing using targeted amplicon sequencing for 160 cancer-related genes. The primary endpoints were the detection rates of potential actionable and druggable gene alterations. The secondary endpoints were the detection rate of secondary germline findings, the rate of re-biopsy required for genome sequencing, survival time after the initial visit (post-sequencing survival time), and turnaround time. RESULTS: Although re-biopsy was required for 25% (5/20) of all patients, genomic testing was performed in all patients. Actionable and druggable gene alterations were detected in 100% (20/20) and 35% (7/20) of patients, respectively, whereas secondary germline findings were detected in 5% (1/20) of patients. The median turnaround times for physicians and patients were 20 and 26 days, respectively. The median post-sequencing survival time was 10.3 months. Only 10% (2/20) of all patients were treated with therapeutic agents based on the outcomes of genomic testing. CONCLUSIONS: The clinical application of comprehensive genomic testing for pancreatic cancer was feasible and promising in clinical practice.

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Tumor budding and human chorionic gonadotropin-β expression correlate with unfavorable patient outcome in colorectal carcinoma.

Yuji Konishi, Futoshi Kawamata, Hiroshi Nishihara, Shigenori Homma, Yasutaka Kato, Masumi Tsuda, Shinji Kohsaka, Takahiro Einama, Cheng Liu, Tadashi Yoshida, Akihisa Nagatsu, Mishie Tanino, Shinya Tanaka, Hideki Kawamura, Toshiya Kamiyama, Akinobu Taketomi

Medical oncology (Northwood, London, England) (Medical Oncology) 35 ( 7 ) 104 - 104 2018.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 13570560

Tumor budding is thought to represent a manifestation of epithelial-to-mesenchymal transition (EMT) and it has been correlated with poor patient outcomes in colorectal cancer (CRC). Our group recently demonstrated that human chorionic gonadotropin-β (hCGβ) modulates EMT in CRC. In the current study, based on the likely relationships between tumor budding and hCGβ expression, we examined their clinicopathologic significance in CRC. Twenty-eight of 80 (35.0%) CRC showed tumor budding. Tumor budding significantly correlated with lymph node metastasis (P < 0.01), pathologic stage (P < 0.01), lymphatic invasion (P = 0.044), and vascular invasion (P = 0.013). Thirteen of 80 (16.3%) CRC were hCGβ positive on immunohistochemistry. More tumor buds were present in the hCGβ-positive cases (P < 0.01), and tumor budding was significantly correlated with hCGβ positivity (P < 0.01). Cases with both tumor budding and hCGβ expression had the poorest prognosis compared with all other groups (P < 0.01). In conclusion, tumor budding and hCGβ expression are closely associated with EMT, and they are independent prognostic factors in CRC. They identify patients with an "EMT phenotype" who may respond to targeted molecular therapies.

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がんゲノム医療における臨床検査技師の役割

柳田絵美衣, 西原広史

臨床病理 ((一社)日本臨床検査医学会) 66 ( 5 ) 479 - 489 2018.05

ISSN 0047-1860

「がん遺伝子診断部」を開設し、専門の遺伝外来、診療(医療面接)、患者採血、DNA抽出、ライブラリー調整、シークエンス、ゲノム解析、カンファレンス、遺伝カウンセリング、結果報告をすべてインハウスで行う院内完結型クリニカルシークエンス(CLHURC検査)を開始した。次に腫瘍センター内にゲノム医療ユニットを設置し、CLHURC検査と同様に次世代シークエンサー(NGS)を用いてパネル化したがんドライバー遺伝子160種類を解析する網羅的がん遺伝子パネル検査「PleSSision検査」を実施した。従来までの臨床検査とは異なり、専門としている検査分野のみの知識や技術、経験だけでは、がんゲノム医療に携わることは困難で、他職種の医療従事者や専門家との連携が非常に重要であった。臨床検査技師がゲノム医療における多くの役割を求められることによるデメリットは、新しい分野の検査が導入され、臨床検査技師の役割が増すことで人手や環境、設備が不足し、現場が混乱する可能性が高いことであった。メリットは「専門分野の検査」についての知識や技術、経験以外にも、様々な知識や技術を得ることができることであった。

GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells

Shu Z. Wiley, Krishna Sriram, Wenjing Liang, Sarah E. Chang, Randall French, Thalia McCann, Jason Sicklick, Hiroshi Nishihara, Andrew M. Lowy, Paul A. Insel

FASEB Journal (FASEB) 32 ( 3 ) 1170 - 1183 2018.03

Research paper (scientific journal), Joint Work, ISSN 08926638

Themicroenvironment of pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma (desmoplasia) generated by pancreatic cancer-associated fibroblasts (CAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). Using an unbiased GPCRomic array approach, we identified 82 G-protein-coupled receptors (GPCRs) commonly expressed by CAFs derived from 5 primary PDAC tumors. Comparedwith PSCs and PFs, CAFs have increased expression ofGPR68 (a proton-sensingGPCR), with the results confirmed by immunoblotting, The Cancer Genome Atlas data, and immunohistochemistry of PDAC tumors. Coculture of PSCs with PDAC cells, or incubation with TNF-α, induced GPR68 expression. GPR68 activation (by decreasing the extracellular pH) enhanced IL-6 expression via a cAMP/PKA/cAMP response element binding protein signaling pathway. Knockdown of GPR68 by short interfering RNA diminished low pH-induced production of IL-6 and enhancement of PDAC cell proliferation by CAF conditioned media. CAFs from other gastrointestinal cancers also express GPR68. PDAC cells thus induce expression by CAFs of GPR68, which senses the acidic microenvironment, thereby increasing production of fibrotic markers and IL-6 and promoting PDAC cell proliferation. CAF-expressed GPR68 is a mediator of low-pH-promoted regulation of the tumor microenvironments, in particular to PDAC cell-CAF interaction and may be a novel therapeutic target for pancreatic and perhaps other types of cancers.

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心外膜脂肪組織におけるミトコンドリア機能障害は冠動脈狭窄と関連している(Mitochondrial Dysfunction in Epicardial Adipose Tissue is Associated with Coronary Artery Stenosis)

Nakajima Takayuki, Yokota Takashi, Shingu Yasushige, Yamada Akira, Iba Yutaka, Ujihira Kosuke, Wakasa Satoru, Ooka Tomonori, Takada Shingo, Shirakawa Ryosuke, Furihata Takaaki, Tsuda Masaya, Matsumoto Junichi, Katayama Takashi, Fukushima Arata, Saito Akimichi, Matsuoka Ryosuke, Nishihara Hiroshi, Matsui Yoshiro, Kinugawa Shintaro

日本循環器学会学術集会抄録集 ((一社)日本循環器学会) 82回 YIA - 1 2018.03

Accepted

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses

Yae Kanai, Hiroshi Nishihara, Yohei Miyagi, Tatsuhiro Tsuruyama, Kenichi Taguchi, Hiroto Katoh, Tomoyo Takeuchi, Masahiro Gotoh, Junko Kuramoto, Eri Arai, Hidenori Ojima, Ayako Shibuya, Teruhiko Yoshida, Toshiaki Akahane, Rika Kasajima, Kei-Ichi Morita, Johji Inazawa, Takeshi Sasaki, Masashi Fukayama, Yoshinao Oda

Pathology International (Blackwell Publishing) 68 ( 2 ) 63 - 90 2018.02

Research paper (scientific journal), Joint Work, ISSN 13205463

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed “The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research” based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about −180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine.

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Chorionic Gonadotropin-β Modulates Epithelial-Mesenchymal Transition in Colorectal Carcinoma Metastasis.

Futoshi Kawamata, Hiroshi Nishihara, Shigenori Homma, Yasutaka Kato, Masumi Tsuda, Yuji Konishi, Lei Wang, Shinji Kohsaka, Cheng Liu, Tadashi Yoshida, Mishie Tanino, Shinya Tanaka, Hideki Kawamura, Toshiya Kamiyama, Akinobu Taketomi

The American journal of pathology (American Journal of Pathology) 188 ( 1 ) 204 - 215 2018.01

Joint Work, ISSN 00029440

Ectopic production of free β human chorionic gonadotropin (hCGβ) has been associated with aggressive behavior in non-trophoblastic tumors. hCGβ shares common evolutionary sequences with transforming growth factor-β (TGF-β), which represents a major driving force of epithelial-to-mesenchymal transition (EMT). In this study, we examined the biological roles of hCGβ during EMT and its clinical significance in colorectal cancer (CRC) progression. Eighty CRC specimens and 54 preoperative serum samples were analyzed. hCGβ-overexpressing human CRC cell lines were examined for invasiveness and tumorigenicity, and the expression of EMT-associated genes was investigated. In human CRC, histologic hCGβ positivity [13/80 (16.3%)] was lower than serologic hCGβ positivity [13/54 (24.1%)]. However, it was significantly correlated with several clinicopathological features and unfavorable outcome (P < 0.05). hCGβ-overexpressing cell lines had increased invasiveness, migratory ability, and metastatic potential in mice (P < 0.01). Western blot, PCR, and microarray analyses showed hCGβ altered expression of EMT-related genes, including E-cadherin, phosphorylated SMAD2, SNAIL, and TWIST. hCGβ-induced SNAIL and TWIST overexpression levels were reversible by type I and type II TGF-β receptor inhibitors (P < 0.05). hCGβ thus induces EMT via the TGF-β signaling pathway, and it may represent a molecular target in CRC treatment.

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A clinicopathological analysis of six autopsy cases of sudden unexpected death due to infectious aortitis in patients with aortic tears

Marin Ishikawa, Mishie Ann Tanino, Masaya Miyazaki, Taichi Kimura, Yusuke Ishida, Lei Wang, Masumi Tsuda, Hiroshi Nishihara, Kazuo Nagashima, Shinya Tanaka

Internal Medicine (Japanese Society of Internal Medicine) 57 ( 10 ) 1375 - 1380 2018

Research paper (scientific journal), Joint Work, ISSN 09182918

Objective Cardiovascular disease is a leading cause of sudden unexpected death even in hospitalized patients. Infectious aortitis is a rare disease that has the potential to cause aortic tears and hemorrhage followed by sudden death. The aim of this study was to reveal the clinicopathological features of infectious aortitis that are related to sudden unexpected death. Methods We retrospectively reviewed 1,310 autopsy cases over 15 years and selected the cases involving patients who died suddenly due to aortic tears. We analyzed the clinical information and pathological findings. Results One hundred thirty-three of 1,310 cases (10.2%) were autopsied under the clinical diagnosis of unexpected sudden death. Aortic tears were identified in 33 cases (2.5%) and infectious aortitis was diagnosed in 6 (18.2%) of these cases. All cases involved male patients (middle-aged to elderly) with risk factors for atherosclerosis (i.e., hypertension). The laboratory data showed continuous leukocytosis and C-reactive protein elevation, even during the improvement phase, in patients with pre-existing infectious disease. The autopsy findings revealed three types of aortic tears (aneurysms, dissections and penetrating atherosclerotic ulcers with moderate to severe atherosclerosis), and the infiltration of numerous neutrophils at the site of rupture. Gram-positive bacteria were detected in four cases and Gram-negative bacteria were detected in two cases. Discussion We demonstrated that sudden unexpected death caused by infectious aortitis rarely occurred in hospitalized patients, even in the recovery phase of the preceding infectious disease. We therefore recommend that clinicians pay attention to infectious aortitis in patients with infectious disease, particularly elderly patients with atherosclerotic disease, even those who are in the improvement phase. Conclusion Unexpected sudden death by infectious aortitis in the recovery phase of antecedent infection.

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Pathological Quantification of Carotid Artery Plaque Instability in Patients Undergoing Carotid Endarterectomy.

Takao Konishi, Naohiro Funayama, Tadashi Yamamoto, Tohru Morita, Daisuke Hotta, Ryota Nomura, Yusuke Nakagaki, Takeo Murahashi, Kenji Kamiyama, Tetsuyuki Yoshimoto, Takeshi Aoki, Hiroshi Nishihara, Shinya Tanaka

Circulation journal : official journal of the Japanese Circulation Society (Circulation Journal) 82 ( 1 ) 258 - 266 2018

Joint Work, Accepted, ISSN 13469843

BACKGROUND: Unstable atherosclerotic carotid plaques cause cerebral thromboemboli and ischemic events. However, this instability has not been pathologically quantified, so we sought to quantify it in patients undergoing carotid endarterectomy (CEA).Methods and Results:Carotid plaques were collected during CEA from 67 symptomatic and 15 asymptomatic patients between May 2015 and August 2016. The specimens were stained with hematoxylin-eosin and elastica-Masson. Immunohistochemistry was performed using an endothelial-specific antibody to CD31, CD34 and PDGFRβ. The histopathological characteristics of the plaques were studied. By multiple-variable logistic regression analysis, plaque instability correlated with the presence of plaque rupture [odds ratio (OR), 9.75; P=0.013], minimum fibrous cap thickness (OR per 10 μm 0.70; P=0.025), presence of microcalcifications in the fibrous cap (OR 7.82; P=0.022) and intraplaque microvessels (OR 1.91; P=0.043). Receiver-operating characteristics analyses showed that these factors combined into a single score diagnosed symptomatic carotid plaques in patients with carotid artery stenosis with a high level of accuracy (area under the curve 0.92; 95% confidence interval 0.85-0.99 vs. asymptomatic). CONCLUSIONS: This analysis of carotid plaque instability strongly suggested that the diagnostic scoring of carotid plaque instability improves the understanding and treatment of carotid artery disease in patients undergoing CEA.

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Effectiveness of indocyanine green fluorescence in endoscopic marking in gastric cancer surgery: A feasibility study for replacing India ink

Ono H, Kusano M, Nihei M, Hayashi H, Fukushima H, Kawakami M, Danjo Y, Nagashima K, Shimizu Y, Kawamata F, Honda S, Shimamura T, Nishihara H

Journal of Japanese Society of Gastroenterology (Journal of Japanese Society of Gastroenterology) 115 ( 4 ) 385 - 393 2018

Joint Work, ISSN 04466586

© 2018 Japanese Society of Gastroenterology. All rights reserved. When injected, indocyanine green (ICG) immediately combines with lipoproteins to fluoresce. Here, we studied whether ICG fluorescence is effective for endoscopic marking in gastric cancer surgery using a photodynamic eye (PDE) camera and fluorescent endoscope. An ICG solution was endoscopically injected into the submucosal layer of the gastric tumor 3 days before surgery. We observed the lesions using both a PDE camera and a fluorescent endoscope during laparotomy and laparoscopy, respectively; we also observed the fluorescent luminance and fluorescent size of the resected lesions. We could intraoperatively detect the size of the resected lesions in eight patients with early gastric cancer and six patients with advanced gastric cancer. We believe that the use of ICG fluorescence in endoscopic marking requires additional information, such as the volume of the ICG solution and the timing of the ICG injection.

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Copy number profiles of paired primary and metastatic colorectal cancers.

Futoshi Kawamata, Ann-Marie Patch, Katia Nones, Catherine Bond, Diane McKeone, Sally-Ann Pearson, Shigenori Homma, Cheng Liu, Lochlan Fennell, Troy Dumenil, Gunter Hartel, Nozomi Kobayasi, Hideki Yokoo, Moto Fukai, Hiroshi Nishihara, Toshiya Kamiyama, Matthew E Burge, Christos S Karapetis, Akinobu Taketomi, Barbara Leggett, Nicola Waddell, Vicki Whitehall

Oncotarget (Oncotarget) 9 ( 3 ) 3394 - 3405 2018

Joint Work

Liver metastasis is the major cause of death following a diagnosis of colorectal cancer (CRC). In this study, we compared the copy number profiles of paired primary and liver metastatic CRC to better understand how the genomic structure of primary CRC differs from the metastasis. Paired primary and metastatic tumors from 16 patients and their adjacent normal tissue samples were analyzed using single nucleotide polymorphism arrays. Genome-wide chromosomal copy number alterations were assessed, with particular attention to 188 genes known to be somatically altered in CRC and 24 genes that are clinically actionable in CRC. These data were analyzed with respect to the timing of primary and metastatic tissue resection and with exposure to chemotherapy. The genomic differences between the tumor and paired metastases revealed an average copy number discordance of 22.0%. The pairs of tumor samples collected prior to treatment revealed significantly higher copy number differences compared to post-therapy liver metastases (P = 0.014). Loss of heterozygosity acquired in liver metastases was significantly higher in previously treated liver metastasis samples compared to treatment naive liver metastasis samples (P = 0.003). Amplification of the clinically actionable genes ERBB2, FGFR1, PIK3CA or CDK8 was observed in the metastatic tissue of 4 patients but not in the paired primary CRC. These examples highlight the intra-patient genomic discrepancies that can occur between metastases and the primary tumors from which they arose. We propose that precision medicine strategies may therefore identify different actionable targets in metastatic tissue, compared to primary tumors, due to substantial genomic differences.

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Role of insulin receptor substrates in the progression of hepatocellular carcinoma.

Yoshitaka Sakurai, Naoto Kubota, Iseki Takamoto, Atsushi Obata, Masahiko Iwamoto, Takanori Hayashi, Masakazu Aihara, Tetsuya Kubota, Hiroshi Nishihara, Takashi Kadowaki

Scientific reports (NATURE PUBLISHING GROUP) 7 ( 1 ) 5387 - 5387 2017.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

Several cellular signaling pathways, including insulin/IGF signaling, are known to be activated in hepatocellular carcinoma (HCC). Here, we investigated the roles of insulin receptor substrate (Irs) 1 and Irs2, both of which are the major molecules to be responsible for transducing insulin/IGF signaling in the liver, in the development of HCC by inducing chemical carcinogenesis using diethylnitrosamine (DEN) in mice. The Irs1 mRNA and protein expressions were upregulated in the tumors, along with enhanced insulin signaling. Liver-specific Irs1-knockout (LIrs1KO) mice exhibited suppression of DEN-induced HCC development, accompanied by reduced cancer cell proliferative activity and reduced activation of Akt. Gene expression analyses revealed that the tumors in the DEN-treated LIrs1KO mice showed modest metabolic alterations during hepatocarcinogenesis as well as decreased inflammation and invasion potentials. On the other hand, liver-specific Irs2-knockout (LIrs2KO) mice showed a similar pattern of HCC development to the DEN-treated control wild-type mice. Based on the knowledge that Wnt/β-catenin signaling is activated in HCC, we focused on Wnt/β-catenin signaling and demonstrated that Irs1 expression was induced by Wnt3a stimulation in the primary hepatocytes, associated with insulin-stimulated Akt activation. These data suggest that upregulated Irs1 by Wnt/β-catenin signaling plays a crucial role in the progression of HCC.

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A randomized phase II trial of erlotinib vs. S-1 as a third- or fourth-line therapy for patients with wild-type EGFR non-small cell lung cancer (HOT1002).

Yasuyuki Ikezawa, Hajime Asahina, Satoshi Oizumi, Masahiro Watanabe, Kei Takamura, Yasutaka Kawai, Noriyuki Yamada, Toshiyuki Harada, Ichiro Kinoshita, Yuka Fujita, Eisaku Miyauchi, Takahiro Ogi, Toraji Amano, Megumi Furuta, Jun Sakakibara-Konishi, Hiroshi Nishihara, Hirotoshi Dosaka-Akita, Hiroshi Isobe, Masaharu Nishimura

Cancer chemotherapy and pharmacology (SPRINGER) 80 ( 5 ) 955 - 963 2017.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 03445704

PURPOSE: A high proportion of patients with wild-type EGFR non-small cell lung cancer (NSCLC) receive third-line therapy and beyond, with no prospective randomized trials addressing the issue. This study aimed to select the most suitable regimen as a third- or fourth-line therapy for wild-type EGFR NSCLC. METHODS: This multicenter, randomized phase II study in Japan included patients with recurrent or advanced NSCLC with wild-type or unknown EGFR, who progressed after two or three previous chemotherapies. The patients were randomly assigned to erlotinib (150 mg/day, days 1-21) or S-1 (80-120 mg/day, days 1-14) every 3 weeks until disease progression or unacceptable toxicity. The primary endpoint was disease control rate (DCR). The secondary endpoints included overall survival (OS), progression-free survival (PFS), objective response rate (ORR), toxicity, and quality of life (QOL). RESULTS: From 2011 to 2016, 37 patients were randomly assigned to receive erlotinib (E arm, n = 19) and S-1 (S arm, n = 18). This study was terminated prematurely because of poor patient accrual. DCR/ORR were 42.1%/15.8% in the E arm and 66.7%/16.7% in the S arm. Median PFS/OS were 1.6 months/8.0 months in the E arm and 3.3 months/12.2 months in the S arm. In both groups, the most commonly reported grade 3-4 toxicities were fatigue, anorexia, and nausea. One grade 5 pneumonitis occurred in the S arm. No significant difference was seen in QOL. CONCLUSIONS: S-1 as a third- or fourth-line therapy for wild-type EGFR NSCLC showed numerically better clinical outcomes than erlotinib. CLINICAL TRIAL REGISTRATION NO: UMIN000005308.

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The Anti-tumor Effect of Cabozantinib on Ovarian Clear Cell Carcinoma In Vitro and In Vivo

Makiko Nakatani, Hidemichi Watari, Takashi Mitamura, Lei Wang, Yutaka Hatanaka, Kanako C. Hatanaka, Kohei Honda, Toshiyuki Nomura, Hiroshi Nishihara, Shinya Tanaka, Noriaki Sakuragi

ANTICANCER RESEARCH (INT INST ANTICANCER RESEARCH) 37 ( 11 ) 6125 - 6132 2017.11

Research paper (scientific journal), Joint Work, ISSN 02507005

Background: Several reports have shown that the overexpression of the MET proto-oncogene, receptor tyrosine kinase (MET), was more frequently observed in clear cell carcinoma (CCC) than in non-CCC. We evaluated the antitumor activity of cabozantinib, that targets MET. Materials and Methods: A gene expression analysis of tumors from human ovarian cancers was carried out by transcriptome sequencing. An in vitro 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay (MTT assay) and in vivo experiments were performed to determine the activity of cabozantinib. Results: The MET levels were higher in tumors with CCC than high-grade serous carcinoma (2.2-fold). Cabozantinib inhibited cell viability and phosphorylation of AKT and MAPK under the treatment of hepatocyte growth factor in RMG-I CCC cells. The tumors removed from mice given cabozantinib of 10 mg/kg weighed 70% less than control on day 15, and the immunohistochemical reactivity of phosphorylated MET was reduced compared with control mice. Conclusion: Cabozantinib contributes to tumor reduction, and phosphorylated MET represents an attractive target of CCC.

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Biopsy-proven case of Epstein-Barr virus (EBV)-associated vasculitis of the central nervous system.

Kohei Kano, Takayuki Katayama, Shiori Takeguchi, Asuka Asanome, Kae Takahashi, Tsukasa Saito, Jun Sawada, Masato Saito, Ryogo Anei, Kyousuke Kamada, Naoyuki Miyokawa, Hiroshi Nishihara, Naoyuki Hasebe

Neuropathology : official journal of the Japanese Society of Neuropathology (WILEY) 37 ( 3 ) 259 - 264 2017.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 09196544

A 75-year-old woman was admitted to our hospital with rapidly deteriorating consciousness disturbance. She had a 7-year history of rheumatoid arthritis (RA), which had been treated with methotrexate (MTX) and prednisolone. Brain T2-weighted MRI showed diffuse high-intensity lesions in the cerebral subcortical and deep white matter, bilateral basal ganglia and thalamus. A cerebrospinal fluid examination revealed elevated protein levels and positive Epstein-Barr virus (EBV) DNA. Human immunodeficiency virus was negative. Brain biopsy showed perivascular lymphocytic infiltration in the parenchyma and meninx with EBV-encoded small RNA (EBER). Since this case did not fulfill the criteria for chronic active EBV infection (CAEBV), she was diagnosed with Epstein-Barr virus (EBV)-associated vasculitis of the central nervous system. High-dose methylprednisolone, acyclovir, ganciclovir and foscarnet were not effective. Although EBV is a causative agent of infectious mononucleosis (IM), lymphomas and nasopharyngeal carcinomas, vasculitic pathology of the central nervous system with EBV reactivation in the elderly is rare. Immunosuppressive drugs such as steroids and MTX are widely used to treat autoimmune disorders, but may exacerbate the reactivation of EBV. This is the first case of biopsy-proven EBV-positive/HIV-negative vasculitis during the treatment of RA with MTX and steroids. This case indicates that EBV-associated vasculitis needs to be considered as a differential diagnosis of CNS vasculitis.

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PCTAIRE1/CDK16/PCTK1 is overexpressed in cutaneous squamous cell carcinoma and regulates p27 stability and cell cycle.

Teruki Yanagi, Hiroo Hata, Eri Mizuno, Shinya Kitamura, Keisuke Imafuku, Shinichi Nakazato, Lei Wang, Hiroshi Nishihara, Shinya Tanaka, Hiroshi Shimizu

Journal of dermatological science (ELSEVIER IRELAND LTD) 86 ( 2 ) 149 - 157 2017.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 09231811

BACKGROUND: PCTAIRE1 (also known as cyclin-dependent kinase 16 (Cdk16) and PCTK1) is a Cdk family protein that has been implicated in spermatogenesis. We recently revealed the function of PCTAIRE1 in the tumorigenesis of malignancies, including breast and prostate cancers; however, the tumorigenic function of PCTAIRE1 in cutaneous squamous cell carcinoma (SCC) remains unclear. OBJECTIVE: In this study, we investigated the role of PCTAIRE1 in the tumorigenesis of cutaneous SCCs. METHODS AND RESULTS: In cutaneous/oral SCC A431, DJM-1, HSC-3 cells, PCTAIRE1 gene-knockdown was found to diminish cell proliferation as assessed by cell counting and clonogenic assays. FACS analyses of annexin V-PI staining and DNA content showed PCTAIRE1 knockdown to cause G2/M arrest followed by apoptosis. The depletion of PCTAIRE1 was found to lead to the accumulation of tumor suppressor p27 and down-regulation of c-Myc. In tumor xenografts of A431 cells, the conditional knockdown of PCTAIRE1 restores p27 protein expression and suppresses tumor growth. Clinically, in primary tumors from patients with SCC, PCTAIRE1 is more highly expressed in malignant lesions than in adjacent normal epidermis. Conversely, expression levels of p27 are significantly lower in SCC than in normal epidermis. CONCLUSIONS: Our findings reveal a crucial function for PCTAIRE1 in regulating p27, c-Myc levels and tumor growth in cutaneous SCC cells, suggesting that PCTAIRE1 could be a novel target for skin tumor treatment.

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Regulation of endothelial Fas expression as a mechanism of promotion of vascular integrity by mural cells in tumors

Ryosuke Kamei, Hiroyoshi Y. Tanaka, Takao Kawano, Chiharu Morii, Sayaka Tanaka, Hiroshi Nishihara, Caname Iwata, Mitsunobu R. Kano

CANCER SCIENCE (WILEY) 108 ( 5 ) 1080 - 1088 2017.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 13479032

Angiogenesis is a multi-step process that culminates in vascular maturation whereby nascent vessels stabilize to become functional, and mural cells play an essential role in this process. Recent studies have shown that mural cells in tumors also promote and maintain vascular integrity, with wide-reaching clinical implications including the regulation of tumor growth, metastases, and drug delivery. Various regulatory signaling pathways have been hitherto implicated, but whether regulation of Fas-dependent apoptotic mechanisms is involved has not yet been fully investigated. We first compared endothelial FAS staining in human pancreatic ductal adenocarcinomas and colon carcinomas and show that the latter, characterized by lower mural cell coverage of tumor vasculature, demonstrated higher expression of FAS than the former. Next, in an invitro coculture system of MS-1 and 10T1/2 cells as endothelial and mural cells respectively, we show that mural cells decreased endothelial Fas expression. Then, in an invivo model in which C26 colon carcinoma cells were inoculated together with MS-1 cells alone or with the further addition of 10T1/2 cells, we demonstrate that mural cells prevented hemorrhage. Finally, knockdown of endothelial Fas sufficiently recapitulated the protection against hemorrhage seen with the addition of mural cells. These results together suggest that regulation of endothelial Fas signaling is involved in the promotion of vascular integrity by mural cells in tumors.

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Podoplanin promotes progression of malignant pleural mesothelioma by regulating motility and focus formation

Shinji Takeuchi, Koji Fukuda, Tadaaki Yamada, Sachiko Arai, Satoshi Takagi, Genichiro Ishii, Atsushi Ochiai, Shotaro Iwakiri, Kazumi Itoi, Hisanori Uehara, Hiroshi Nishihara, Naoya Fujita, Seiji Yano

CANCER SCIENCE (WILEY) 108 ( 4 ) 696 - 703 2017.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 13479032

Malignant pleural mesothelioma (MPM) is characterized by dissemination and aggressive growth in the thoracic cavity. Podoplanin (PDPN) is an established diagnostic marker for MPM, but the function of PDPN in MPM is not fully understood. The purpose of this study was to determine the pathogenetic function of PDPN in MPM. Forty-seven of 52 tumors (90%) from Japanese patients with MPM and 3/6 (50%) MPM cell lines tested positive for PDPN. Knocking down PDPN in PDPN-high expressing MPM cells resulted in decreased cell motility. In contrast, overexpression of PDPN in PDPN-low expressing MPM cells enhanced cell motility. PDPN stimulated motility was mediated by activation of the RhoA/ROCK pathway. Moreover, knocking down PDPN with short hairpin (sh) RNA in PDPN-high expressing MPM cells resulted in decreased development of a thoracic tumor in mice with severe combined immune deficiency (SCID). In sharp contrast, transfection of PDPN in PDPN-low expressing MPM cells resulted in an increase in the number of Ki-67-positive proliferating tumor cells and it promoted progression of a thoracic tumor in SCID mice. Interestingly, PDPN promoted focus formation invitro, and a low level of E-cadherin expression and YAP1 activation was observed in PDPN-high MPM tumors. These findings indicate that PDPN is a diagnostic marker as well as a pathogenetic regulator that promotes MPM progression by increasing cell motility and inducing focus formation. Therefore, PDPN might be a pathogenetic determinant of MPM dissemination and aggressive growth and may thus be an ideal therapeutic target.

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THE ADAPTOR PROTEIN CRK-INDUCED ERBB2 EXPRESSION PROMOTES TUMOR PROGRESSION AND METASTASIS OF BLADDER CANCER VIA EXOSOMES

Yoshida Kazuhiko, Tsuda Masumi, Matsumoto Ryuji, Semba Shingo, Kimura Taichi, Tanino Mishie, Nishihara Hiroshi, Kondo Tsunenori, Tanabe Kazunari, Tanaka Shinya

JOURNAL OF UROLOGY 197 ( 4 ) E1175 2017.04

Joint Work, Accepted, ISSN 0022-5347

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[Clinical Biobank: A Novel System to Support Cancer Clinical Sequencing in Japan].

Hiroshi Nishihara

Rinsho byori. The Japanese journal of clinical pathology 65 ( 2 ) 167 - 172 2017.02

Single Work, Accepted, ISSN 0047-1860

The development of genomic medicine has enabled us to perform contemporary clinical sequencing, while the acquisition of high-quality biospecimens and the appropriate handling of these materials are indispensable. We started Clinical Biobank, a novel system for on-demand-type banking, in 2014. During the 22 months from August 2014, we stored around 6,000 biospecimens from 1,700 patients, and most of them were obtained for diverse clinical research. The quality and amount of extracted DNA and RNA from PFPE samples were markedly high; thus, these samples are adequate for targeted amplicon sequencing using MiSeq (Illumina). In addition, we recently established the Division of Clinical Cancer Genetics for personalized cancer medicine. We obtain biospecimens including blood and tissue fixed using the PAXgene Tissue system, and the samples are immediately analyzed for targeted amplicon sequencing using MiSeq. The rest of the samples are stored after adequate procedures in a -80 degree freezer. We obtain DNA and RNA from PAX-fixed paraffin- embedded samples with a high quality and sufficient amounts for clinical sequencing after pathological evalua- tion. For three months, we examined 40 cancer patients and performed targeted exome sequencing. As a result, detection rates of actionable and druggable gene mutations were around 85 and 50%, respectively. We have also established the Clinical Biobank Study Group in Japan, collaborating with several universities and cancer centers. We believe that this novel biospecimen repository system will help to effectively estab- lish cancer clinical sequencing throughout Japan.

Clinicopathological evaluation of Sox10 expression in diffuse-type gastric adenocarcinoma.

Marin Kato, Hiroshi Nishihara, Hideyuki Hayashi, Taichi Kimura, Yusuke Ishida, Lei Wang, Masumi Tsuda, Mishie Ann Tanino, Shinya Tanaka

Medical oncology (Northwood, London, England) (HUMANA PRESS INC) 34 ( 1 ) 8 - 8 2017.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 13570560

Sox10, one of the transcription factors, regulates Wnt/β-catenin signaling in diverse developmental processes in normal tissues. Sox10 is also expressed in variable solid tumors such as breast cancer, salivary tumor, hepatocellular carcinoma, ovarian tumor, nasopharyngeal carcinoma, prostate cancer, and digestive cancer. The role of Sox10 during tumorigenesis is still controversial, especially in digestive cancers; thus, we performed clinicopathological evaluation of Sox10 expression in 41 cases of diffuse-type gastric adenocarcinoma (DGA). We examined the expression of Sox10 by immunohistochemical staining and real-time quantitative reverse transcriptase PCR and evaluated the correlation between Sox10 expression and clinicopathological factors. A low-level expression of Sox10 was significantly associated with high-level venous invasion by immunohistochemical evaluation, while it was significantly associated with high-level lymphatic permeation when analyzed by real-time PCR assay. Survival analysis of 41 cases indicated that high level of vascular permeation was a statistically poor prognostic factor, suggesting that derogation of Sox10 would lead to unfavorable patients' outcome through the acceleration of vascular invasion. In this study, we revealed the clinical benefit of evaluation of Sox10 expression to predict the risk of vascular permeation which yields patients' poor prognosis in DGA.

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Expression of Notch1 and Numb in small cell lung cancer.

Hajime Kikuchi, Jun Sakakibara-Konishi, Megumi Furuta, Hiroshi Yokouchi, Hiroshi Nishihara, Shigeo Yamazaki, Hidetaka Uramoto, Fumihiro Tanaka, Masao Harada, Kenji Akie, Fumiko Sugaya, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Toshiyuki Harada, Mitsunori Higuchi, Osamu Honjo, Yoshinori Minami, Naomi Watanabe, Satoshi Oizumi, Hiroyuki Suzuki, Takashi Ishida, Hirotoshi Dosaka-Akita, Hiroshi Isobe, Mitsuru Munakata, Masaharu Nishimura

Oncotarget (IMPACT JOURNALS LLC) 8 ( 6 ) 10348 - 10358 2017

Research paper (scientific journal), Joint Work, Accepted, ISSN 1949-2553

Notch signaling in tumorigenesis functions as an oncogene or tumor suppressor according to the type of malignancy. Numb represses intracellular Notch signaling. Previous studies have demonstrated that Notch signaling suppresses the proliferation of small cell lung cancer (SCLC) cell lines. However, in SCLC, the association between Notch1 and Numb expression and clinicopathological factors or prognosis has remained unclear. In this study, we evaluated the expression of Notch1 and Numb in SCLC. We immunohistochemically assessed 125 SCLCs that were surgically resected at 16 institutions participating in either the Hokkaido Lung Cancer Clinical Study Group Trial (HOT) or the Fukushima Investigative Group for Healing Thoracic Malignancy (FIGHT) between 2003 and 2013. Correlations between Notch1 or Numb expression and various clinicopathological features were evaluated. Notch1 expression was associated with ECOG performance status. Numb expression was associated with age, sex, and pathological histology (SCLC or Combined SCLC). Analysis of cellular biological expression did not demonstrate a significant correlation between the expression of Notch1 and of Numb. Multivariate Cox regression analysis showed that high Notch1 expression was an independent favorable prognostic factor for SCLC(hazard ratio = 0.503, P = 0.023). High Notch1 expression, but not Numb expression, is associated with favorable prognosis in SCLC.

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The Complete Remission of Acquired Immunodeficiency Syndrome-associated Isolated Central Nervous System Lymphomatoid Granulomatosis: A Case Report and Review of the Literature.

Yasuhiro Kano, Minori Kodaira, Atsuhito Ushiki, Makoto Kosaka, Mitsunori Yamada, Kunihiko Shingu, Hiroshi Nishihara, Masayuki Hanaoka, Yoshiki Sekijima

Internal medicine (Tokyo, Japan) (JAPAN SOC INTERNAL MEDICINE) 56 ( 18 ) 2497 - 2501 2017

Joint Work, Accepted, ISSN 09182918

A 49-year-old man presented with gradually progressive aphasia one month after being diagnosed with acquired immunodeficiency syndrome (AIDS). Brain magnetic resonance imaging showed multiple brain lesions with punctate and linear enhancement. A polymerase chain reaction detected Epstein-Barr virus (EBV) in the patient's cerebrospinal fluid. A diagnosis of isolated central nervous system lymphomatoid granulomatosis (CNS-LYG) was made based on the brain biopsy findings. The complete remission of CNS-LYG was achieved by anti-retroviral therapy (ART) alone. In the present case, the development of AIDS-associated CNS-LYG was considered to have been initiated by the reactivation of EBV in the CNS under immunosuppressive conditions. The patient's condition improved with the reconstitution of the patient's immune system.

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Proposal for a Prospective Registry for Moyamoya Disease in Japan.

Ken Kazumata, Masaki Ito, Haruto Uchino, Hiroshi Nishihara, Kiyohiro Houkin

Neurologia medico-chirurgica (JAPAN NEUROSURGICAL SOC) 57 ( 2 ) 66 - 72 2017

Research paper (scientific journal), Joint Work, Accepted, ISSN 04708105

The number of clinical research papers published worldwide on moyamoya disease (MMD) has increased recently. However, the majority of the literature comprises retrospective single-center studies collecting data on small numbers of patients. Several multi-center studies are ongoing in Japan; however, the current data are insufficient for comprehensively outlining the various characteristics of MMD. To enhance our knowledge on epidemiologic, vascular, and genetic aspects of MMD, a prospective multicenter registry will be established in Japan that will help to streamline clinical research as well as improve clinical treatments and long-term outcomes. Patients with MMD or secondary moyamoya syndrome referred to the participating centers will be invited to the registry. Demographic and physiological parameters, along with neuroimaging data will be collected chronologically. Clinical events, including neurological, medical, and surgical interventions will be recorded. Whole blood samples will be collected. Extra- and intra-cranial vascular tissue, and/or cerebrospinal fluid will also be collected from patients who undergo surgical revascularization. These biospecimens will be stored at the repositories and utilized for genome-wide association studies for identifying genetic variants, as well as tissue-specific proteomic, and/or molecular analyses. Ethics approval will be obtained at all facilities collecting biospecimens. The registry will provide descriptive statistics on functional outcomes, surgical techniques used, medications, and neurological events stratified according to patients' clinical characteristics. We expect this study to provide novel insights in the management of MMD patients and design better therapies.

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Prognostic Value of Eosinophil to Leukocyte Ratio in Patients with ST-Elevation Myocardial Infarction Undergoing Primary Percutaneous Coronary Intervention.

Takao Konishi, Naohiro Funayama, Tadashi Yamamoto, Toru Morita, Daisuke Hotta, Hiroshi Nishihara, Shinya Tanaka

Journal of atherosclerosis and thrombosis (JAPAN ATHEROSCLEROSIS SOC) 24 ( 8 ) 827 - 840 2017

Research paper (scientific journal), Joint Work, Accepted, ISSN 13403478

AIM: Leukocyte profile has been related to clinical outcome in patients with ST-segment elevation (STE) myocardial infarction (MI). However, whether eosinophil to leukocyte ratio (ELR) predicts clinical outcome in patients who have undergone primary percutaneous coronary intervention (PCI) remains unclear. Therefore, we examined the prognostic value of ELR in this patient population. METHODS: We retrospectively analyzed the data of 331 consecutive patients who underwent primary PCI for STEMI between January 2009 and March 2015. All leukocyte types were counted and ELR was calculated for all patients 24 h after hospital admission. The primary study endpoint was major adverse cardiac events (MACEs) within up to one year of follow-up duration. RESULTS: MACEs including cardiac deaths in 9.4% of the patients, MI in 1.5%, and target lesion or vessel revascularization in 10.3%, occurred within one year in 68 patients (20.5%). The mean ELR was significantly lower in patients with MACEs than in patients without MACEs (0.20±0.51 vs.0.49±0.66, respectively; p＜0.001). An ELR ＜0.1 at 24 h was identified as the best cut-off value for mortality prediction. Multivariate analysis identified that an ELR ＜0.1 (odds ratio [OR]=0.38; 95% confidence interval [CI]=0.22-0.67; p＜0.001) and chronic kidney disease (OR=2.38; CI=1.33-4.24; p=0.003) are independent predictors of MACEs. CONCLUSION: In primary PCI patients with STEMI, ELR at 24 h was an independent predictor of MACEs in addition to the usual coronary risk factors and commonly used biomarkers.

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Immunohistochemical profiling of receptor tyrosine kinases, MED12, and TGF-βRII of surgically resected small cell lung cancer, and the potential of c-kit as a prognostic marker.

Hiroshi Yokouchi, Hiroshi Nishihara, Toshiyuki Harada, Takashi Ishida, Shigeo Yamazaki, Hajime Kikuchi, Satoshi Oizumi, Hidetaka Uramoto, Fumihiro Tanaka, Masao Harada, Kenji Akie, Fumiko Sugaya, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Mitsunori Higuchi, Osamu Honjo, Yoshinori Minami, Naomi Watanabe, Aya Goto, Hiroyuki Suzuki, Hirotoshi Dosaka-Akita, Hiroshi Isobe, Masaharu Nishimura, Mitsuru Munakata

Oncotarget (IMPACT JOURNALS LLC) 8 ( 24 ) 39711 - 39726 2017

Research paper (scientific journal), Joint Work, Accepted, ISSN 1949-2553

The limited number of available treatments for patients with small-cell lung cancer (SCLC) has prompted us to further investigate the biology of SCLC by molecular profiling. We collected formalin-fixed paraffin-embedded tumor samples from 127 patients with SCLC, who had undergone surgery at 16 institutions between January 2003 and January 2013, and analyzed the association between disease-specific survival and protein expression of c-kit, c-Met, epidermal growth factor receptor, human EGFR-related 2, vascular endothelial growth factor receptor II, anaplastic lymphoma kinase, mediator complex subunit 12 (MED12), and transforming growth factor beta receptor II (TGF-βRII) by immunohistochemistry (IHC). Of the 125 evaluable samples, all tumors expressed MED12, and 123 samples (98.4%) expressed TGF-βRII. MED12 was highly expressed in the nucleus in 92% of the positive samples while TGF-βRII was highly expressed in the cytoplasm in 55% of the positive samples. High c-kit expression was an independent favorable prognostic marker confirmed by multivariate analysis (hazard ratio: 0.543, 95% confidence interval: 0.310-0.953, p = 0.033). Both the relapse free-survival and overall survival of patients who underwent adjuvant chemotherapy were statistically longer in those with high c-kit expression (n = 38) than those with intermediate, low, or no c-kit expression (n = 19) (not reached vs 11.6 months, p = 0.021; not reached vs 25.9 months, p = 0.028). IHC for c-kit may offer a prognostic marker for early-stage SCLC, and the results for MED12 and TGF-βRII may suggest the biological characteristics of SCLC. Further investigation of the roles of their related molecules in early stage SCLC is required.

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ANCA-Negative Granulomatosis with Polyangiitis Presenting with Hypertrophic Cranial Pachymeningitis, Abducens Nerve Palsy, and Stenosis of the Internal Carotid Artery.

Shohei Harabuchi, Nobuyuki Bandoh, Rika Yasukawa, Michihisa Kono, Takashi Goto, Yasuaki Harabuchi, Hidetoshi Ikeda, Hajime Kamada, Hiroshi Nishihara

Case reports in otolaryngology 2017 9687383 - 9687383 2017

Joint Work, Accepted

We report a rare case of granulomatosis with polyangiitis (GPA) presenting with hypertrophic cranial pachymeningitis (HCP), abducens nerve palsy, and stenosis of the internal carotid artery (ICA). A 59-year-old Japanese man presented with a year history of nasal obstruction and a 2-month history of slight headache. Histopathological examination of the granulomatous mucosa in the ethmoid sinuses resected by endoscopic sinus surgery revealed necrotizing vasculitis with multinucleated giant cells. The patient was diagnosed with the limited form of GPA as a result of the systemic examination. He declined immunosuppressive treatment. Eighteen months after the diagnosis of GPA, he presented with diplopia and severe headache. Though nasal findings indicating GPA were not observed in the nasal cavity, CT scan revealed a lesion of the right sphenoid sinus eroding the bone of the clivus. Gadolinium-enhanced MRI of the brain showed thickening of the dura mater around the right cavernous sinus and clivus. Magnetic resonance angiography and cerebral angiography revealed narrowing at the C5 portion of the ICA. Intravenous methylprednisolone pulse therapy followed by oral prednisolone and cyclophosphamide resolved headache and dramatically improved HCP and stenosis of the ICA.

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A case of giant cell-rich solitary fibrous tumor in the external auditory canal

Sayaka Yuzawa, Satoshi Tanikawa, Isamu Kunibe, Hiroshi Nishihara, Kazuo Nagashima, Shinya Tanaka

PATHOLOGY INTERNATIONAL (WILEY-BLACKWELL) 66 ( 12 ) 701 - 705 2016.12

Research paper (scientific journal), Joint Work, ISSN 13205463

We present a rare case of giant cell-rich solitary fibrous tumor (SFT) arising at the left external auditory canal in a 31-year-old woman. The tumor was well-circumscribed and composed of spindle-shaped cells with abundant collagenous bands. Scattered multinucleate giant cells were observed, some of which lined pseudovascular spaces. Although a focal mild-hypercellular area was observed, mitoses were rare and necrosis was absent. Interstitial mast cells were scattered, especially in the hypercellular area. Immunohistochemically, CD34, vimentin, and Bcl-2 presented diffuse positivity. Moreover, both mononuclear spindle cells and multinucleate cells showed nuclear STAT6 positivity, while NAB2-STAT6 fusion gene could not be detected by reverse transcription polymerase chain reaction using formalin-fixed specimen. These findings suggest the pathological diagnosis of giant cell-rich SFT, previously known as giant cell angiofibroma, which is a rare variant of SFT with multinucleate giant cells and occurs predominantly in orbital region. Although giant cell-rich SFTs of extra-orbital sites have been reported, to our knowledge, this is the first case arising in the external auditory canal. Giant cell-rich SFT should be considered as a differential diagnosis of spindle cell lesion with multinucleate giant cells, and STAT6 immunohistochemistry should be performed to distinguish this rare tumor from other mesenchymal neoplasms.

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Severe mitral regurgitation due to mitral leaflet aneurysm diagnosed by three-dimensional transesophageal echocardiography: a case report

Takao Konishi, Naohiro Funayama, Tadashi Yamamoto, Daisuke Hotta, Kenjiro Kikuchi, Katsumi Ohori, Hiroshi Nishihara, Shinya Tanaka

BMC CARDIOVASCULAR DISORDERS (BIOMED CENTRAL LTD) 16 ( 1 ) 2016.11

Research paper (scientific journal), Joint Work, ISSN 1471-2261

Background: A small mitral valve aneurysm (MVA) presenting as severe mitral regurgitation (MR) is uncommon.
Case presentation: A 47-year-old man with a history of hypertension complained of exertional chest discomfort. A transthoracic echocardiogram (TTE) revealed the presence of MR and prolapse of the posterior leaflet. A 6mm in diameter MVA, not clearly visualized by TTE, was detected on the posterior leaflet on a three-dimensional (3D) transesophageal echocardiography (TEE). The patient underwent uncomplicated triangular resection of P2 and mitral valve annuloplasty, and was discharged from postoperative rehabilitation, 2 weeks after the operation. Histopathology of the excised leaflet showed myxomatous changes without infective vegetation or signs of rheumatic heart disease.
Conclusions: A small, isolated MVA is a cause of severe MR, which might be overlooked and, therefore, managed belatedly. 3D TEE was helpful in imaging its morphologic details.

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A novel treatment strategy for pancreatic cancer based on gene profiles

Hayashi H., Nishihara H.

Japanese Journal of Cancer and Chemotherapy 43 ( 11 ) 1326 - 1331 2016.11

Accepted, ISSN 03850684

Pancreatic cancer has one of the highest rates of mortality among malignancies and the development of promising future therapies is strongly required. Recently, the utility of gene aberrations as biomarkers for determining therapeutic strategies has been demonstrated in several types of cancer. The detection of druggable mutations that aid in the selection of effective molecular targeting drugs is feasible in clinical settings for certain cancers. On the other hand, personalized therapy for pancreatic cancer guided by genomic biomarkers has not yet been realized and suitable molecular targets for the disease have been unclear until now. KRAS, CDKN2A, TP53, and SMAD4 have been recognized as major driver genes in pancreatic carcino-genesis. However, it is considered difficult to develop treatment strategies to target genetic aberrations of these four genes. In recent years, genome sequencing has progressively revealed the molecular biological characteristics of pancreatic cancer, including the discovery of novel potential therapeutic targets and low-frequency druggable genetic aberrations. Gene profile-based novel treatment strategies and subsequent attempts to realize precision medicine for pancreatic cancer are steadily ongoing in an effort to achieve improved treatment outcomes.

Aldo-keto reductase 1C1 induced by interleukin-1β mediates the invasive potential and drug resistance of metastatic bladder cancer cells.

Ryuji Matsumoto, Masumi Tsuda, Kazuhiko Yoshida, Mishie Tanino, Taichi Kimura, Hiroshi Nishihara, Takashige Abe, Nobuo Shinohara, Katsuya Nonomura, Shinya Tanaka

Scientific reports (Scientific Reports) 6 34625 - 34625 2016.10

Research paper (scientific journal), Joint Work

In treating bladder cancer, determining the molecular mechanisms of tumor invasion, metastasis, and drug resistance are urgent to improving long-term patient survival. One of the metabolic enzymes, aldo-keto reductase 1C1 (AKR1C1), plays an essential role in cancer invasion/metastasis and chemoresistance. In orthotopic xenograft models of a human bladder cancer cell line, UM-UC-3, metastatic sublines were established from tumors in the liver, lung, and bone. These cells possessed elevated levels of EMT-associated markers, such as Snail, Slug, or CD44, and exhibited enhanced invasion. By microarray analysis, AKR1C1 was found to be up-regulated in metastatic lesions, which was verified in metastatic human bladder cancer specimens. Decreased invasion caused by AKR1C1 knockdown suggests a novel role of AKR1C1 in cancer invasion, which is probably due to the regulation of Rac1, Src, or Akt. An inflammatory cytokine, interleukin-1β, was found to increase AKR1C1 in bladder cancer cell lines. One particular non-steroidal anti-inflammatory drug, flufenamic acid, antagonized AKR1C1 and decreased the cisplatin-resistance and invasion potential of metastatic sublines. These data uncover the crucial role of AKR1C1 in regulating both metastasis and drug resistance; as a result, AKR1C1 should be a potent molecular target in invasive bladder cancer treatment.

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Genetic landscape of meningioma

Sayaka Yuzawa, Hiroshi Nishihara, Shinya Tanaka

BRAIN TUMOR PATHOLOGY (SPRINGER JAPAN KK) 33 ( 4 ) 237 - 247 2016.10

Joint Work, ISSN 14337398

Meningioma is the most common intracranial tumor, arising from arachnoid cells of the meninges. Monosomy 22 and inactivating mutations of NF2 are well-known genetic alterations of meningiomas. More recently, mutations in TRAF7, AKT1, KLF4, SMO, and PIK3CA were identified by next-generation sequencing. We here reviewed 553 meningiomas for the mutational patterns of the six genes. NF2 aberration was observed in 55 % of meningiomas. Mutations of TRAF7, AKT1, KLF4, PIK3CA, and SMO were identified in 20, 9, 9, 4.5, and 3 % of cases, respectively. Altogether, 80 % of cases harbored at least one of the genetic alterations in these genes. NF2 alterations and mutations of the other genes were mutually exclusive with a few exceptions. Clinicopathologically, tumors with mutations in TRAF7/AKT1 and SMO shared specific features: they were located in the anterior fossa, median middle fossa, or anterior calvarium, and most of them were meningothelial or transitional meningiomas. TRAF7/KLF4 type meningiomas showed different characteristics in that they occurred in the lateral middle fossa and median posterior fossa as well as anterior fossa and median middle fossa, and contained a secretory meningioma component. We also discuss the mutational hotspots of these genes and other genetic/cytogenetic alterations contributing to tumorigenesis or progression of meningiomas.

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EXO1 homozygous deletion suppresses the hydroxyurea sensitivity in anaplastic meningioma with extracranial metastases

Sodai Yoshimura, Takashi Ohta, Kotaro Makita, Shun Yamamuro, Yushi Ochai, Koichiro Sumi, Katsunori Shijo, Atsuo Yoshino, Taku Homma, Masahiko Sugitani, Sayaka Yuzawa, Hiroshi Nishihara, Sinya Tanaka

INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE (E-CENTURY PUBLISHING CORP) 9 ( 9 ) 18618 - 18625 2016.09

Research paper (scientific journal), Joint Work, ISSN 1940-5901

Background: Anaplastic meningioma is exceedingly rare, and its clinicopathological features are distinctive. Distant meningioma metastases have previously been reported to be very rare. The most common site of metastasis is the lung, which accounts for 61% of all meningioma metastases. The standard therapy for meningiomas is total resection and/or radiation therapy. Case Report: A 71-year-old man was admitted with headache and left hemiparesis. Magnetic resonance imaging (MRI) revealed mass lesions at the right frontal convexity and left occipital lobe. Following surgery, pathological examinations demonstrated an anaplastic meningioma. At 3 months after the operation and radiation therapy, lung tumor was removed. The pathological findings for the lung tumor resembled those of the brain tumor. A diagnosis of metastatic lung tumor from anaplastic meningioma was made. At 2 months after the lung surgery, MRI disclosed focal recurrence in the frontal convexity area and progression into the cavernous sinus. Abdominal CT detected a new metastatic lesion in the patient's liver. He underwent adjuvant reirradiation consisting of whole brain radiotherapy. In addition, he received chemotherapy using angiogenesis receptor (bevacizumab). Chemotherapy with hydroxyurea was initiated after 2 courses of the bevacizumab chemotherapy, because his hepatic lesion had become more aggressive. The intracranial mass was reduced by 50% at 8 weeks after initiation of the hydroxyurea chemotherapy. However, there was no significant effect on the liver and lung metastases. Analysis and Conclusion: Extracranial metastases have been estimated to occur in only 0.1% of all meningiomas and most often in association with anaplastic meningiomas. While total resection and/or radiation treatment provide the standard therapy for meningiomas, use of chemotherapy against meningiomas has been limited to distant metastases or based on out of surgical criteria. Several reports have demonstrated that hydroxyurea provides on effective chemotherapy for meningiomas including grade I cases. Hydroxyurea halts meningioma cell growth through arrest of the S-phase of the cell cycle, thus inducing apoptosis. Our analysis revealed EXO1 homozygous deletion at exons 8 and 10 in the patient's extracranial metastatic meningioma. The findings suggested that EXO1 homozygous deletion may be associated with suppression of hydroxyurea sensitivity in anaplastic meningiomas. Hydroxyurea should be a justified therapeutic adaptation in cases of anaplastic meningioma without EOX1 homozygous deletion.

Relationship between left coronary artery bifurcation angle and restenosis after stenting of the proximal left anterior descending artery

Takao Konishi, Tadashi Yamamoto, Naohiro Funayama, Hiroshi Nishihara, Daisuke Hotta

CORONARY ARTERY DISEASE (LIPPINCOTT WILLIAMS & WILKINS) 27 ( 6 ) 449 - 459 2016.09

Research paper (scientific journal), Joint Work, ISSN 09546928

Introduction Restenosis after a percutaneous coronary intervention for proximal left anterior descending (pLAD) coronary artery disease remains a clinical challenge. However, the relationship between the left main trunk (LMT)/LAD bifurcation angle and the pLAD artery restenosis is unclear. This study examined the relationship between the LMT-LAD bifurcation angle and restenosis after stent implantation for pLAD disease.
Methods We analysed the data of 177 consecutive patients who underwent stent implantation for pLAD disease, followed by coronary angiography between December 2008 and September 2013. The LMT-LAD bifurcation angle was measured in the left or the right anterior oblique caudal (CAU) angiographic view.
Results and discussion Out of 177 patients, 12 developed in-stent restenosis and 21 developed in-segment restenosis. The mean angle in patients with in-stent restenosis (52.2 degrees +/- 14.5 degrees) in the left anterior oblique CAU view was significantly larger than that in patients without restenosis (32.0 degrees +/- 18.1 degrees; P<0.001). The LMT-LAD angle in the right anterior oblique CAU view was significantly larger in patients with in-segment restenosis (27.3 degrees+/-14.3 degrees) than in patients without restenosis (17.5 degrees+/-10.1 degrees; P<0.001). Moreover, by multivariate analysis, the LMT-LAD angle was an independent predictor of in-stent and in-segment restenosis, after adjustment for significant confounders such as diabetes, hypertension, dyslipidaemia, final minimum lesion diameter and lesion length.
Conclusion This study suggests that a wide LMT-LAD angle is a predictor of restenosis after stent implantation for pLAD artery disease. Copyright (C) 2016 Wolters Kluwer Health, Inc. All rights reserved.

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Analysis of NAB2-STAT6 Gene Fusion in 17 Cases of Meningeal Solitary Fibrous Tumor/Hemangiopericytoma Review of the Literature

Sayaka Yuzawa, Hiroshi Nishihara, Lei Wang, Masumi Tsuda, Taichi Kimura, Mishie Tanino, Shinya Tanaka

AMERICAN JOURNAL OF SURGICAL PATHOLOGY (LIPPINCOTT WILLIAMS & WILKINS) 40 ( 8 ) 1031 - 1040 2016.08

Joint Work, ISSN 01475185

Solitary fibrous tumor/hemangiopericytoma (SFT/HPC) is a mesenchymal tumor that can affect virtually any region of the body. SFT/HPC of the thoracic cavity and soft tissue has been histologically considered a single biological entity termed SFT; in fact, NAB2-STAT6 gene fusion was recently identified in both diseases. In contrast, meningeal SFT and HPC still need to be investigated in detail with regard to gene fusion variants. The aim of this study was to verify the frequency of NAB2-STAT6 fusion and the relationship between fusion variants and clinicopathologic findings of SFT/HPC, especially meningeal SFT/HPC. We examined the NAB2-STAT6 fusion by reverse transcription polymerase chain reaction with 4 cases of meningeal SFT and 13 cases of meningeal HPC. NAB2-STAT6 fusion transcripts were identified in 12 of 17 cases, including NAB2ex6-STAT6ex17 (4/17, 24%), NAB2ex6-STAT6ex16 and NAB2ex4-STAT6ex2 (3/17, 18%, respectively), and NAB2ex5-STAT6ex16 (2/17, 12%). Three cases showed a pseudopapillary pattern, and 2 of them carried NAB2ex6-STAT6ex17. In addition, our meta-analysis revealed that the major fusion variant in meningeal SFT/HPC was NAB2ex6-STAT6ex16/17 (29/54, 54%), which was also common in soft tissue and intraperitoneum/retroperitoneum but rare in thoracic SFT. Fusion variant significantly correlated with age and histologic diagnosis in meningeal SFT/HPC but not with prognosis. Our results represented that meningeal SFT and HPC were in a single biological spectrum with NAB2-STAT6 gene fusion as was nonmeningeal SFT and further confirmed the organ-specific tumorigenic process and morphologic differences on the basis of fusion variants in meningeal SFT/HPC.

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Identification and analysis of CXCR4-positive synovial sarcoma-initiating cells

T. Kimura, L. Wang, K. Tabu, M. Tsuda, M. Tanino, A. Maekawa, H. Nishihara, H. Hiraga, T. Taga, Y. Oda, S. Tanaka

ONCOGENE (NATURE PUBLISHING GROUP) 35 ( 30 ) 3932 - 3943 2016.07

Research paper (scientific journal), Joint Work, ISSN 09509232

Synovial sarcoma accounts for almost 10% of all soft tissue sarcomas, and its prognosis is poor with 5-year survival rates at 36%. Thus, new treatments and therapeutic targets for synovial sarcoma are required. Tumor-initiating cells have been defined by the ability for self-renewal and multipotent differentiation, and they exhibit higher tumorigenic capacity, chemoresistance and radiation resistance, expecting to be a new therapeutic target. In synovial sarcoma, the presence of such stemness remains largely unclear; thus, we analyzed whether synovial sarcoma possessed tumor-initiating cells and explored specific markers, and we discovered that synovial sarcoma cell lines possessed heterogeneity by way of containing a sphere-forming subpopulation highly expressing NANOG, OCT4 and SOX2. By expression microarray analysis, CXCR4 was identified to be highly expressed in the sphere subpopulation and correlated with stem-cell-associated markers. Inhibition of CXCR4 suppressed the cell proliferation of synovial sarcoma cell lines in vitro. The tumor-initiating ability of CXCR4-positive cells was demonstrated by xenograft propagation assay. CXCR4-positive cells showed higher tumorigenicity than negative ones and possessed both self-renewal and multipotent differentiation ability. Immunohistochemical analysis of 39 specimens of synovial sarcoma patients revealed that CXCR4 strongly correlated with poor prognosis of synovial sarcoma. Thus, we conclude that CXCR4 is the marker of synovial sarcoma-initiating cells, a new biomarker for prognosis and a new potential therapeutic target.

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Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system

Sayaka Yuzawa, Hiroshi Nishihara, Shigeru Yamaguchi, Hiromi Mohri, Lei Wang, Taichi Kimura, Masumi Tsuda, Mishie Tanino, Hiroyuki Kobayashi, Shunsuke Terasaka, Kiyohiro Houkin, Norihiro Sato, Shinya Tanaka

MODERN PATHOLOGY (NATURE PUBLISHING GROUP) 29 ( 7 ) 708 - 716 2016.07

Research paper (scientific journal), Joint Work, ISSN 08933952

Recent genetic analyses using next-generation sequencers have revealed numerous genetic alterations in various tumors including meningioma, which is the most common primary brain tumor. However, their use as routine laboratory examinations in clinical applications for tumor genotyping is not cost effective. To establish a clinical sequencing system for meningioma and investigate the clinical significance of genotype, we retrospectively performed targeted amplicon sequencing on 103 meningiomas and evaluated the association with clinicopathological features. We designed amplicon-sequencing panels targeting eight genes including NF2 (neurofibromin 2), TRAF7, KLF4, AKT1, and SMO. Libraries prepared with genomic DNA extracted from PAXgenefixed paraffin-embedded tissues of 103 meningioma specimens were sequenced using the Illumina MiSeq. NF2 loss in some cases was also confirmed by interphase-fluorescent in situ hybridization. We identified NF2 loss and/or at least one mutation in NF2, TRAF7, KLF4, AKT1, and SMO in 81 out of 103 cases (79%) by targeted amplicon sequencing. On the basis of genetic status, we categorized meningiomas into three genotype groups: NF2 type, TRAKLS type harboring mutation in TRAF7, AKT1, KLF4, and/or SMO, and 'not otherwise classified' type. Genotype significantly correlated with tumor volume, tumor location, and magnetic resonance imaging findings such as adjacent bone change and heterogeneous gadolinium enhancement, as well as histopathological subtypes. In addition, multivariate analysis revealed that genotype was independently associated with risk of recurrence. In conclusion, we established a rapid clinical sequencing system that enables final confirmation of meningioma genotype within 7 days turnaround time. Our method will bring multiple benefits to neuropathologists and neurosurgeons for accurate diagnosis and appropriate postoperative management.

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Clinical impacts of mesothelin expression in gastrointestinal carcinomas.

Takahiro Einama, Futoshi Kawamata, Hirofumi Kamachi, Hiroshi Nishihara, Shigenori Homma, Fumihiko Matsuzawa, Tatsuzo Mizukami, Yuji Konishi, Munenori Tahara, Toshiya Kamiyama, Okio Hino, Akinobu Taketomi, Satoru Todo

World journal of gastrointestinal pathophysiology 7 ( 2 ) 218 - 22 2016.05

Joint Work, Accepted

Mesothelin, C-ERC/mesothelin is a 40-kDa cell surface glycoprotein that is normally present on normal mesothelial cells lining the pleura, peritoneum, and pericardium. Moreover, mesothelin has been shown to be overexpressed in several human cancers, including virtually all mesothelioma and pancreatic cancer, approximately 70% of ovarian cancer and extra bile duct cancer, and 50% of lung adenocarcinomas and gastric cancer. The full-length human mesothelin gene encodes the primary product, a 71-kDa precursor protein. The 71-kDa mesothelin precursor is cleaved into two products, 40-kDa C-terminal fragment that remains membrane-bound via glycosylphosphatidylinositol anchor, and a 31-kDa N-terminal fragment, megakaryocyte potentiating factor, which is secreted into the blood. The biological functions of mesothelin remain largely unknown. However, results of recent studies have suggested that the mesothelin may play a role of cell proliferation and migration. In pancreatic cancer, mesothelin expression was immunohistochemically observed in all cases, but absent in normal pancreas and in chronic pancreatitis. Furthermore, the expression of mesothelin was correlated with an poorer patient outcome in several human cancers. The limited mesothelin expression in normal tissues and high expression in many cancers makes it an attractive candidate for cancer therapy. The present review discusses the expression and function of mesothelin in cancer cells and the utility of mesothelin as a target of cancer therapy.

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Novel signaling collaboration between TGF-beta and adaptor protein Crk facilitates EMT in human lung cancer

Aiman Z. Elmansuri, Mishie A. Tanino, Roshan Mahabir, Lei Wang, Taichi Kimura, Hiroshi Nishihara, Ichiro Kinoshita, Hirotoshi Dosaka-Akita, Masumi Tsuda, Shinya Tanaka

ONCOTARGET (IMPACT JOURNALS LLC) 7 ( 19 ) 27094 - 27107 2016.05

Research paper (scientific journal), Joint Work, ISSN 1949-2553

The signaling adaptor protein Crk has been shown to play an important role in various human cancers. However, its regulatory machinery is not clear. Here, we demonstrated that Crk induced EMT in A549 human lung adenocarcinoma cells through differential regulation of Rac1/Snail and RhoA/Slug, leading to decreased expression of E-cadherin and increased N-cadherin, fibronectin, and MMP2 expression. Cancer cells with mesenchymal features produced TGF-beta and also increased the levels of TGF-beta receptor. TGF-beta increased the endogenous levels of Crk and also augmented Crk-dependent expression of Snail and Slug, and conversely TGF-beta receptor inhibitor suppressed the levels of Snail and Slug. Overexpression of Crk was observed at the invasive front of human lung cancer tissues and was significantly associated with poor prognosis. Thus, TGF-beta and Crk collaborate to form a positive feedback loop to facilitate EMT, which may lead to the malignancy of human cancers possibly being affected by their microenvironment.

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Diagnostic Value of Liquid-Based Cytology With Fine Needle Aspiration Specimens for Cervical Lymphadenopathy

Nobuyuki Bandoh, Takashi Goto, Toshiaki Akahane, Natsumi Ohnuki, Tomomi Yamaguchi, Hajime Kamada, Yasuaki Harabuchi, Shinya Tanaka, Hiroshi Nishihara

DIAGNOSTIC CYTOPATHOLOGY (WILEY-BLACKWELL) 44 ( 3 ) 169 - 176 2016.03

Research paper (scientific journal), Joint Work, ISSN 87551039

Background: Cervical lymphadenopathy is a symptom that is frequently seen among outpatients, and it is important to differentiate malignant lesions from reactive lymphoid hyperplasia. Fine needle aspiration (FNA) cytology has been widely used for the diagnosis of cervical lymphadenopathy. However, some limitations of the diagnostic accuracy using conventional smear (CS) cytology have been pointed out. The diagnostic value of liquid-based cytology (LBC) with FNA specimens has not yet been fully proven.
Methods: Forty-two patients with cervical lymphadenopathy who underwent FNA with CS cytology from 2007 to 2011 and 123 patients who underwent FNA with LBC utilizing LBCPREP2 (TM) from 2011 to 2015 were studied. Diagnostic values were compared between the CS and the LBC groups.
Results: Of the total 165 patients representing the combined CS and LBC groups, 81 (49.1%) were diagnosed as benign lymph node and 84 (50.9%) were malignant diseases including 37 (22.4%) of metastatic carcinoma except for thyroid carcinoma, 30 (18.2%) of metastatic thyroid carcinoma, and 17 (10.3%) of malignant lymphoma. The overall statistical values including sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the CS were 75%, 100%, 100%, 78.9%, and 87.1%, respectively, whereas those values for LBC were 91.2%, 100%, 100%, 90.7%, and 95.3%, respectively. The sensitivity of LBC for malignant diseases tended to be higher than that of CS cytology (p = 0.081).
Conclusion: LBC with FNA specimens from cervical lymphadenopathy is a useful and reliable method for the diagnosis of malignant diseases, especially of metastatic carcinomas, due to its increased sensitivity compared with CS cytology. (C) 2016 Wiley Periodicals, Inc.

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Organ-specific efficacy of HSP90 inhibitor in multiple-organ metastasis model of chemorefractory small cell lung cancer.

Shinji Takeuchi, Koji Fukuda, Sachiko Arai, Shigeki Nanjo, Kenji Kita, Tadaaki Yamada, Eiji Hara, Hiroshi Nishihara, Hisanori Uehara, Seiji Yano

International journal of cancer (International Journal of Cancer) 138 ( 5 ) 1281 - 1289 2016.03

Joint Work, ISSN 00207136

Small-cell lung cancer (SCLC) accounts for nearly 15% of lung cancer cases and exhibits aggressive clinical behavior characterized by rapid growth and metastatic spread to multiple organs. About 70% of patients with SCLC present with extensive disease and distant metastases at diagnosis. HSP90 is a 90-kDa molecular chaperone whose association is required for the stability and function of its numerous "client proteins." Here, we assessed the therapeutic potential of the HSP90 inhibitor 17-DMAG in SCLC. Notably, 17-DMAG hindered the viability of human SCLC cell lines-regardless of their chemosensitivity-via the decreased expression of client proteins, including the proto-oncogene c-Raf (also known as RAF1). In an in vivo imaging model of SCLC multiple-organ metastasis with the human SCLC cell line SBC-5, treatment with 17-DMAG remarkably inhibited the formation of metastatic sites in the liver, but was ineffective in hindering the progression of bone lesions. The latter was likely the result of activation of osteoclasts. IGF-1, which is supposed to be rich in bone environment, preserved c-Raf expression and maintained viability of SBC-5 cells treated with 17-DMAG. Furthermore, the combined use of a bisphosphonate with 17-DMAG significantly attenuated the progression of metastases in both the liver and the bone. These findings suggest that therapeutic effects of HSP90 inhibitors may be organ-specific and should be carefully monitored in SCLC clinical trials.

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A case of cerebral astroblastoma with rhabdoid features: a cytological, histological, and immunohistochemical study

Sayaka Yuzawa, Hiroshi Nishihara, Mishie Tanino, Taichi Kimura, Jun Moriya, Yuuta Kamoshima, Kazuo Nagashima, Shinya Tanaka

BRAIN TUMOR PATHOLOGY (SPRINGER JAPAN KK) 33 ( 1 ) 63 - 70 2016.01

Research paper (scientific journal), Joint Work, ISSN 14337398

Astroblastoma is a rare neuroepithelial neoplasm of unknown origin, usually occurring in children and young adults. Here we report a case of astroblastoma with uncommon features in an 18-year-old female. The tumor was a well-circumscribed cystic and solid mass with marked gadolinium enhancement in the right frontal lobe. Cytological examination showed polarized monopolar cells with diminished cohesiveness. Tumor cells possessed eccentric round to oval nuclei with abundant eosinophilic cytoplasm, sometimes having cytoplasmic processes. Histopathologically, the tumor showed perivascular pseudorosettes with prominent vascular sclerosis. Foam cells were frequently infiltrated around blood vessels and among tumor cells. In some areas, a solid growth pattern of plump tumor cells with abundant inclusion-like eosinophilic cytoplasm showing rhabdoid appearance was observed. The immunohistochemical study revealed strong and diffuse positivity for vimentin and epithelial membrane antigen. Tumor cells were focally positive for glial fibrillary acidic protein and cytokeratin AE1/AE3. Nuclear immunoreactivity for INI1 protein was evident. The Ki-67 labeling index was 10.8 %. This tumor was finally diagnosed as low-grade astroblastoma and the patient had no evidence of recurrence without postoperative radiotherapy or chemotherapy during the last 6 months of follow-up. This report describes novel cytological, histopathological, and immunohistochemical features of the rare tumor.

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Rapid immunocytochemistry based on alternating current electric field using squash smear preparation of central nervous system tumors

Jun Moriya, Mishie Ann Tanino, Tomoko Takenami, Tomoko Endoh, Masana Urushido, Yasutaka Kato, Lei Wang, Taichi Kimura, Masumi Tsuda, Hiroshi Nishihara, Shinya Tanaka

BRAIN TUMOR PATHOLOGY (SPRINGER JAPAN KK) 33 ( 1 ) 13 - 18 2016.01

Research paper (scientific journal), Joint Work, ISSN 14337398

The role of intraoperative pathological diagnosis for central nervous system (CNS) tumors is crucial for neurosurgery when determining the surgical procedure. Especially, treatment of carmustine (BCNU) wafers requires a conclusive diagnosis of high-grade glioma proven by intraoperative diagnosis. Recently, we demonstrated the usefulness of rapid immunohistochemistry (R-IHC) that facilitates antigen-antibody reaction under alternative current (AC) electric field in the intraoperative diagnosis of CNS tumors; however, a higher proportion of water and lipid in the brain parenchyma sometimes leads to freezing artifacts, resulting in poor quality of frozen sections. On the other hand, squash smear preparation of CNS tumors for cytology does not affect the frozen artifacts, and the importance of smear preparation is now being re-recognized as being better than that of the tissue sections. In this study, we established the rapid immunocytochemistry (R-ICC) protocol for squash smears of CNS tumors using AC electric field that takes only 22 min, and demonstrated its usefulness for semi-quantitative Ki-67/MIB-1 labeling index and CD 20 by R-ICC for intraoperative diagnosis. R-ICC by AC electric field may become a substantial tool for compensating R-IHC and will be applied for broad antibodies in the future.

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Intraoperative localization of arteriovenous malformation of a jejunum with combined use of angiographic methods and indocyanine green injection: Report of a new technique.

Hiromi Ono, Mitsuo Kusano, Futoshi Kawamata, Yasushi Danjo, Masato Kawakami, Kimimoto Nagashima, Hiroshi Nishihara

International journal of surgery case reports (International Journal of Surgery Case Reports) 29 137 - 140 2016

Joint Work, ISSN 22102612

INTRODUCTION: The localization of small intestine sources of obscure gastrointestinal bleeding has been a challenge. The use of indocyanine green (ICG) is effective in aiding intraoperative localization if a bleeding lesion is identified on angiography. CASE PRESENTATION: A 95-year-old Japanese man presented with hematochezia. Selective angiography of the superior mesenteric artery (SMA) established an arteriovenous malformation (AVM). ICG injection into the feeding vessel was administered intraoperatively, and the demarcated segment of the jejunum was resected. DISCUSSION: Diluted ICG was injected in the SMA by intraoperative angiography, and the region could be easily and clearly visualized by the ICG fluorescence imaging; small patchy poolings of ICG were recognized. Ultimately, the region was diagnosed as an AVM of the jejunum. To the best of our knowledge, this is the first reported description of this technique. CONCLUSION: Our new technique of combining selective angiography with intraoperative ICG injection and focused enterectomy is a safe, accurate, and cost-effective treatment.

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The infrastructure of the biobank system

Takayoshi Suzuki, Hiromitsu Hatakeyama, Shin-Ichiro Yasukawa, Akihiro Homma, Hiroshi Nishihara, Yoshihiro Matsuno, Satoshi Fukuda

Oto-Rhino-Laryngology Tokyo (Society of Oto-Rhino-Laryngology Tokyo) 59 ( 6 ) 371 - 376 2016

Research paper (scientific journal), Joint Work, ISSN 03869687

With the development of analysis technology, high-quality biological materials have been increasingly important for researches. Cryopreservation of fresh samples is recommended with regard to quality control. We consider that support system which enables closer cooperation with pathologists is essential for collection of tissue samples from specimens, lest the procedure should interfere with the pathological diagnosis. Thus, we manage to set up the platform of Biobank and establish its guideline for the acquisition, maintenance and provision of the quality of biological resources in a secure manner.

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Intraprocedural left ventricular free wall rupture diagnosed by left ventriculogram in a patient with infero-posterior myocardial infarction and severe aortic stenosis

Takao Konishi, Naohiro Funayama, Tadashi Yamamoto, Hiroshi Nishihara, Daisuke Hotta, Kenjiro Kikuchi, Hideo Yokoyama, Katsumi Ohori

BMC CARDIOVASCULAR DISORDERS (BIOMED CENTRAL LTD) 16 ( 1 ) 2016

Research paper (scientific journal), Joint Work, ISSN 1471-2261

Background: Left ventricular wall rupture remains a major lethal complication of acute myocardial infarction and hypertension is a well-known predisposing factor of cardiac rupture after myocardial infarction.
Case presentation: An 87-year-old man was admitted to our hospital, diagnosed as acute myocardial infarction (AMI). The echocardiogram showed 0.67-cm(2) aortic valve, consistent with severe aortic stenosis (AS). A coronary angiography showed a chronic occlusion of the proximal left circumflex artery and a 99 % stenosis and thrombus in the mid right coronary artery. During percutaneous angioplasty of the latter, transient hypotension and bradycardia developed at the time of balloon inflation, and low doses of noradrenaline and etilefrine were intravenously administered as needed. The patient suddenly lost consciousness and developed electro-mechanical dissociation. Cardio-pulmonary resuscitation followed by insertion of an intra-aortic balloon pump (IABP) and percutaneous cardiopulmonary support were initiated. The echocardiogram revealed moderate pericardial effusion, though the site of free wall rupture was not distinctly visible. A left ventriculogram clearly showed an infero-posterior apical wall rupture. Surgical treatment was withheld because of the interim development of brain death.
Conclusions: In this patient, who presented with severe AS, the administration of catecholamine to stabilize the blood pressure probably increased the intraventricular pressures considerably despite apparently normal measurements of the central aortic pressure. IABP, temporary pacemaker, or both are recommended instead of intravenous catecholamines for patients with AMI complicated with significant AS to stabilize hemodynamic function during angioplasty.

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Successful and Safe Retrieval of a Thrombus from the Lumen of a Guiding Catheter during Percutaneous Coronary Intervention

Konishi T, Funayama N, Nishihara H

Journal of Clinical Case Reports 6 2 2016

Joint Work, Accepted

Authentic Vascular and Stromal Structure in Animal Disease Model for Nanomedicine

Hiroshi Nishihara, Mitsunobu R. Kano

Intracellular Delivery III 149 - 160 2016

Joint Work

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Prognostic impact of clinical variables on surgically resected small-cell lung cancer: Results of a retrospective multicenter analysis (FIGHT002A and HOT1301A)

Hiroshi Yokouchi, Takashi Ishida, Shigeo Yamazaki, Hajime Kikuchi, Satoshi Oizumi, Hidetaka Uramoto, Fumihiro Tanaka, Masao Harada, Kenji Akie, Fumiko Sugaya, Yuka Fujita, Tatsuro Fukuhara, Kei Takamura, Tetsuya Kojima, Toshiyuki Harada, Mitsunori Higuchi, Yoshifumi Matsuura, Osamu Honjo, Yoshinori Minami, Naomi Watanabe, Hiroshi Nishihara, Hiroyuki Suzuki, Hirotoshi Dosaka-Akita, Hiroshi Isobe, Masaharu Nishimura, Mitsuru Munakata

LUNG CANCER (ELSEVIER IRELAND LTD) 90 ( 3 ) 548 - 553 2015.12

Research paper (scientific journal), Joint Work, ISSN 01695002

Objectives: Several American and Japanese guidelines recommend surgery for patients with c-stage I small-cell lung cancer (SCLC), whereas the European Society of Medical Oncology (ESMO) guidelines recommend surgery for patients with not only c-stage I but also c-stage II (T2N1) SCLC. In addition, previous studies identified various factors other than clinical stage that are related to survival in these patients. Thus, further validation and examination of the association of clinical stage and other clinical variables with survival are required for establishing practical management of early-stage SCLC.
Patients and methods: We reviewed the clinical courses of 156 SCLC patients who had undergone surgery at 17 institutions between January 2003 and January 2013.
Results: Clinical stages (tumor-node-metastasis [TNM] version 7) of the 156 patients were 98 cases in IA, 14 in IB, 16 in IIA, 7 in IIB, 18 in IIIA, and 3 in IIIB. Median overall survival (OS) was 33.3 months (95% confidence interval: 20.9-45.8). Multivariate analysis revealed that OS was longer in patients either at c-stage II and under, with a maximum tumor diameter of <20 mm, with preoperative diagnosis, without a history or presence of other types of cancer, or who underwent prophylactic cranial irradiation (PCI).
Conclusion: These results indicate that a history or presence of other types of cancer might be a major decisive factor for surgery. Patients with c-stages I and II (c-T2N1) can be considered for surgery, and PCI may be useful in patients undergoing surgery in a practical setting, partly supporting the ESMO guidelines.(1) (c) 2015 Elsevier Ireland Ltd. All rights reserved.

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Infective endocarditis presenting initially with ileus complicated by dehiscence of annuloplasty ring

Takao Konishi, Hiroshi Nishihara, Tadashi Ito, Yoshiaki Tanaka

BMC CARDIOVASCULAR DISORDERS (BIOMED CENTRAL LTD) 15 ( 1 ) 2015.10

Research paper (scientific journal), Joint Work, ISSN 1471-2261

Background: Infective endocarditis (IE) on an annuloplasty ring dehiscence is uncommon after mitral valve repair.
Case Presentation: A 53-year-old man underwent mitral annuloplasty with a 24-mm ring for posterior mitral valve prolapse. He underwent repeat valve repair for recurrent mitral valve regurgitation 4 years later. He was re-hospitalised complaining of vomiting, nausea, general fatigue and left abdominal pain 2 months later, and presented with low-grade fever, leukocytosis and an elevated blood concentration of C-reactive protein. An abdominal computed tomography scan showed multiple embolisms in the liver, kidney and spleen. Transoesophageal echocardiography revealed mitral annuloplasty ring dehiscence and vegetations consistent with IE. The infected annuloplasty ring and vegetations were surgically excised. Blood cultures grew coagulasenegative staphylococcus aureus, consistent with the excised mitral valve histology. The postoperative course was uneventful, without recurrence of IE.
Conclusions: Embolic ileus as initial manifestation of IE is rare and might confuse the diagnosis and delay its management. Gastrointestinal signs and symptoms may be the initial manifestations of systemic embolization from infective endocarditis. Transoesophageal echocardiography effectively identified the presence of vegetations and mitral annuloplasty ring dehiscence.

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Histologically confirmed case of cerebral vasculitis associated with Crohn's disease - a case report

Masayuki Gekka, Taku Sugiyama, Masafumi Nomura, Yasutaka Kato, Hiroshi Nishihara, Katsuyuki Asaoka

BMC NEUROLOGY (BIOMED CENTRAL LTD) 15 ( 1 ) 2015.09

Research paper (scientific journal), Joint Work, ISSN 1471-2377

Background: Extraintestinal manifestations in Crohn's disease (CD) are frequent and well recognized. However, neurological involvement secondary to CD is rare, and there have been few histologically confirmed cases of cerebral vasculitis secondary to CD.
Case presentation: A 58-year-old left-handed man with a history of refractory CD who had fever of over 38 degrees C, progression of CD symptoms, and Gerstmann's syndrome consulted our hospital. Laboratory data showed elevation of C-reactive protein (CRP) and hypoproteinemia. T2-weighted magnetic resonance imaging (MRI) revealed a right parietal high-intensity lesion. Catheter angiography showed segmental multiple narrowing and occlusion in the distal part of the middle cerebral artery and anterior cerebral artery. Angiography also revealed multiple venous occlusions in the affected parietal area. To confirm the diagnosis, the patient underwent open biopsy, and histological examination revealed cerebral vasculitis. The patient was then started on high-dose prednisolone (60 mg/day) in addition to his previous therapy, which included mesalazine, adalimumab, and azathioprine. CRP elevation, hypoproteinemia, and gastrointestinal symptoms immediately improved after starting this treatment. Neurological status improved simultaneously with CD symptom improvement, and follow-up brain MRI revealed a reduction in the size of the right parietal lobe lesion. He returned to normal status and was discharged from our hospital 5 weeks after admission.
Conclusion: This is an important case of histologically confirmed cerebral vasculitis associated with CD. The clinical course of our case clearly illustrates the relevance of the occurrence of cerebral vasculitis and the exacerbation of CD.

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Elevated β-catenin pathway as a novel target for patients with resistance to EGF receptor targeting drugs

Asuka Nakata, Ryo Yoshida, Rui Yamaguchi, Mai Yamauchi, Yoshinori Tamada, Andre Fujita, Teppei Shimamura, Seiya Imoto, Tomoyuki Higuchi, Masaharu Nomura, Tatsuo Kimura, Hiroshi Nokihara, Masahiko Higashiyama, Kazuya Kondoh, Hiroshi Nishihara, Arinobu Tojo, Seiji Yano, Satoru Miyano, Noriko Gotoh

Scientific Reports (Springer Nature) 5 ( 1 ) 2015.08

Research paper (scientific journal), Joint Work

There is a high death rate of lung cancer patients. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in some lung adenocarcinoma patients with EGFR mutations. However, a significant number of patients show primary and acquire resistance to EGFR-TKIs. Although the Akt kinase is commonly activated due to various resistance mechanisms, the key targets of Akt remain unclear. Here, we show that the Akt-β-catenin pathway may be a common resistance mechanism. We analyzed gene expression profiles of gefitinib-resistant PC9M2 cells that were derived from gefitinib-sensitive lung cancer PC9 cells and do not have known resistance mechanisms including EGFR mutation T790M. We found increased expression of Axin, a β-catenin target gene, increased phosphorylation of Akt and GSK3, accumulation of β-catenin in the cytoplasm/nucleus in PC9M2 cells. Both knockdown of β-catenin and treatment with a β-catenin inhibitor at least partially restored gefitinib sensitivity to PC9M2 cells. Lung adenocarcinoma tissues derived from gefitinib-resistant patients displayed a tendency to accumulate β-catenin in the cytoplasm. We provide a rationale for combination therapy

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Tyr724 phosphorylation of ELMO1 by Src is involved in cell spreading and migration via Rac1 activation

Yoshinori Makino, Masumi Tsuda, Yusuke Ohba, Hiroshi Nishihara, Hirofumi Sawa, Kazuo Nagashima, Shinya Tanaka

CELL COMMUNICATION AND SIGNALING (BIOMED CENTRAL LTD) 13 ( 1 ) 2015.07

Research paper (scientific journal), Joint Work, ISSN 1478-811X

Background: The complex of Dock180/ELMO1 that functions as a bipartite guanine nucleotide exchange factor for Rac is essential for diverse physiological and pathological processes of cells such as cell migration, phagocytosis, and invasion of cancer cells. Among the Src-family tyrosine kinases (SFKs), it has been reported that Hck directly phosphorylates ELMO1, regulating phagocytosis by promoting activation of Rac1; however, the involvement of other SFKs in ELMO1 phosphorylation has remained unknown. Here, we identified novel tyrosine (Y) residues of ELMO1 phosphorylated by SFKs, and examined the effects on Rac1 activity, cell adhesion, spreading, and cell motility on extracellular matrix (ECM).
Results: In this study, we unveiled that Src and Fyn can induce tyrosine phosphorylation of ELMO1 in in vivo and in vitro phosphorylation assays. Mutational analyses identified both Y720 and Y724 residues of ELMO1 as Src-mediated phosphorylation sites, preferentially on Y724. Single substitution of Y724 to Phe abrogated Rac1 activation triggered by Src. To elucidate the biological function of pY724, we established NIH3T3 cells stably expressing wild-type ELMO1 or its Y724F mutant together with Dock180. Among them, Y724-deficient cells exhibited a depletion of Rac1 activity with diminished phosphorylation of ELMO1 even upon the ECM-stimulation. It is noteworthy that NIH3T3 cells with ELMO1 Y724F were strikingly defective to promote cell spreading on fibronectin-coated dish, concomitantly exhibiting immature assemblies of actin stress fibers and focal adhesions. Eventually, ELMO1 Y724F significantly impaired cell migration.
Conclusion: These results define that Src-mediated Y724 phosphorylation in ELMO1 plays a critical role for cell spreading via activation of Rac1, leading to promotion of cell migration. As the overexpression and/or hyperactivation of Src have been shown in a wide variety of human cancers, Src-mediated phosphorylation of Y724 in ELMO1 may regulate cancer cell adhesion to the ECM, invasion into surrounding tissues, and subsequent distant metastasis.

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Stent thrombosis caused by metal allergy complicated by protein S deficiency and heparin-induced thrombocytopenia: A case report and review of the literature

Takao Konishi, Tadashi Yamamoto, Naohiro Funayama, Beni Yamaguchi, Seiichiro Sakurai, Hiroshi Nishihara, Koko Yamazaki, Yusuke Kashiwagi, Yasuki Sasa, Mitsuru Gima, Hideichi Tanaka, Daisuke Hotta, Kenjiro Kikuchi

Thrombosis Journal (BioMed Central Ltd.) 13 ( 1 ) 2015.07

Research paper (scientific journal), Joint Work, ISSN 1477-9560

A 43-year-old woman recipient of a bare metal coronary stent during an acute anterior myocardial infarction was repeatedly hospitalized with recurrent stent thrombosis (ST) over the following 3 years. Emergent coronary angiography showed a thrombus in the in-stent segment of the proximal left anterior descending artery. We repeatedly aspirated the thrombus, which immediately reformed multiple times. The discontinuation of heparin and administration of thrombolytics and argatroban, followed by repeated balloon dilatations, ended the formation of new thrombi. The patient was found to be allergic to nickel, protein S deficient and carrier of heparin-induced thrombocytopenia antibody. We discuss this case in the context of a) literature pertaining to acute coronary syndromes in the young, and b) the detailed investigations needed to identify thrombotic risk factors. Steroids may be effective to prevent recurrent ST caused by stent allergy.

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Pathological findings of saccular cerebral aneurysms-impact of subintimal fibrin deposition on aneurysm rupture

Masaaki Hokari, Naoki Nakayama, Hiroshi Nishihara, Kiyohiro Houkin

NEUROSURGICAL REVIEW (SPRINGER) 38 ( 3 ) 531 - 540 2015.07

Research paper (scientific journal), Joint Work, ISSN 03445607

Although several studies have suggested that aneurysmal wall inflammation and laminar thrombus are associated with the rupture of saccular aneurysms, the mechanisms leading to the rupture remain obscure. We performed full exposure of aneurysms before clip application and attempted to keep the fibrin cap on the rupture point. Using these specimens in a nearly original state before surgery, we conducted a pathological analysis and studied the differences between ruptured and unruptured aneurysms to clarify the mechanism of aneurysmal wall degeneration. This study included ruptured (n=28) and unruptured (n=12) saccular aneurysms resected after clipping. All of the ruptured aneurysms were obtained within 24 h of onset. Immunostainings for markers of inflammatory cells (CD68) and classical histological staining techniques were performed. Clinical variables and pathological findings from ruptured and unruptured aneurysms were compared. Patients with ruptured or unruptured aneurysms did not differ by age, gender, size, location, and risk factors, such as hypertension, smoking, and hyperlipidemia. The absence or fragmentation of the internal elastica lamina, the myointimal hyperplasia, and the thinning of the aneurysmal wall were generally observed in both aneurysms. The existence of subintimal fibrin deposition, organized laminar thrombus, intramural hemorrhage, neovascularization, and monocyte infiltration are more frequently observed in ruptured aneurysms. Multivariate logistic regression analysis showed that ruptured aneurysm was associated with presence of subintimal fibrin deposition and monocyte infiltration. These findings suggest that subintimal fibrin deposition and chronic inflammation have a strong impact on degeneration of the aneurysmal wall leading to their rupture, and this finding may be caused by endothelial dysfunction.

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Adaptor protein CRK induces epithelial-mesenchymal transition and metastasis of bladder cancer cells through HGF/c-Met feedback loop.

Ryuji Matsumoto, Masumi Tsuda, Lei Wang, Nako Maishi, Takashige Abe, Taichi Kimura, Mishie Tanino, Hiroshi Nishihara, Kyoko Hida, Yusuke Ohba, Nobuo Shinohara, Katsuya Nonomura, Shinya Tanaka

Cancer science (Cancer Science) 106 ( 6 ) 709 - 717 2015.06

Joint Work, ISSN 13479032

We have previously reported that an adaptor protein CRK, including CRK-I and CRK-II, plays essential roles in the malignant potential of various aggressive human cancers, suggesting the validity of targeting CRK in molecular targeted therapy of a wide range of cancers. Nevertheless, the role of CRK in human bladder cancer with marked invasion, characterized by distant metastasis and poor prognosis, remains obscure. In the present study, immunohistochemistry indicated a striking enhancement of CRK-I/-II, but not CRK-like, in human bladder cancer tissues compared to normal urothelium. We established CRK-knockdown bladder cancer cells using 5637 and UM-UC-3, which showed a significant decline in cell migration, invasion, and proliferation. It is noteworthy that an elimination of CRK conferred suppressed phosphorylation of c-Met and the downstream scaffold protein Gab1 in a hepatocyte growth factor-dependent and -independent manner. In epithelial-mesenchymal transition-related molecules, E-cadherin was upregulated by CRK elimination, whereas N-cadherin, vimentin, and Zeb1 were downregulated. A similar effect was observed following treatment with c-Met inhibitor SU11274. Depletion of CRK significantly decreased cell proliferation of 5637 and UM-UC-3, consistent with reduced activity of ERK. An orthotopic xenograft model with bioluminescent imaging revealed that CRK knockdown significantly attenuated not only tumor volume but also the number of circulating tumor cells, resulted in a complete abrogation of metastasis. Taken together, this evidence uncovered essential roles of CRK in invasive bladder cancer through the hepatocyte growth factor/c-Met/CRK feedback loop for epithelial-mesenchymal transition induction. Thus, CRK might be a potent molecular target in bladder cancer, particularly for preventing metastasis, leading to the resolution of clinically longstanding critical issues.

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Systemic Targeting of Lymph Node Metastasis through the Blood Vascular System by Using Size-Controlled Nano carriers

Horacio Cabral, Jun Makino, Yu Matsumoto, Peng Mi, Hailiang Wu, Takahiro Nomoto, Kazuko Toh, Naoki Yamada, Yuriko Higuchi, Satoshi Konishi, Mitsunobu R. Kano, Hiroshi Nishihara, Yutaka Miura, Nobuhiro Nishiyama, Kazunori Kataoka

ACS NANO (AMER CHEMICAL SOC) 9 ( 5 ) 4957 - 4967 2015.05

Research paper (scientific journal), Joint Work, ISSN 19360851

Occult nodal metastases increase the risk of cancer recurrence, demoting prognosis and quality of life of patients. While targeted drug delivery by using systemically administered nanocarriers can potentially control metastatic disease, lymph node metastases have been mainly dealt by locally injecting nanocarriers, which may not always be applicable. Herein, we demonstrated that sub-50 nm polymeric micelles incorporating platinum anticancer drugs could target lymph node metastases in a syngeneic melanoma model after systemic injection, even after removing the primary tumors, limiting the growth of the metastases. By comparing these micelles with clinically used doxorubicin-loaded liposomes (Doxil) having 80 nm, as well as a 70 nm version of the micelles, we found that the targeting efficiency of the nanocarriers against lymph node metastases was associated with their size-regulated abilities to extravasate from the blood vasculature in metastases and to penetrate within the metastatic mass. These findings indicate the potential of sub-50 nm polymeric micelles for developing effective conservative treatments against lymph node metastasis capable of reducing relapse and improving survival.

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Adult classical glioblastoma with a BRAF V600E mutation

Yoshinobu Takahashi, Toshiaki Akahane, Takahiro Sawada, Hidetoshi Ikeda, Akira Tempaku, Shigeru Yamauchi, Hiroshi Nishihara, Shinya Tanaka, Kazumi Nitta, Wataru Ide, Ikuo Hashimoto, Hajime Kamada

WORLD JOURNAL OF SURGICAL ONCOLOGY (BIOMED CENTRAL LTD) 13 ( 1 ) 2015.03

Research paper (scientific journal), Joint Work, ISSN 1477-7819

The B-Raf proto-oncogene serine/threonine kinase (B-Raf) is a member of the Raf kinase family. The BRAF V600E mutation occurs frequently in certain brain tumors such as pleomorphic xanthoastrocytoma, ganglioglioma, and pilocytic astrocytoma, and less frequently in epithelioid and giant cell glioblastoma. BRAF V600E mutation in these cases has been canonically detected using Sanger sequencing or immunohistochemistry but not with next-generation sequencing (NGS). Moreover, to our knowledge, there is no detailed report of the BRAF V600E mutation in an adult glioblastoma with classical histologic features (c-GBM). Therefore, we performed NGS analysis to determine the mutational status of BRAF of 13 glioblastomas (GBMs) (11 primary and 2 secondary cases) and detected one tumor harboring the BRAF V600E mutation. We report here the detection of the BRAF V600E mutation in a patient with c-GBM and describe the patient's clinical course as well as the results of histopathological analysis.

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In vivo imaging models of bone and brain metastases and pleural carcinomatosis with a novel human EML4-ALK lung cancer cell line.

Shigeki Nanjo, Takayuki Nakagawa, Shinji Takeuchi, Kenji Kita, Koji Fukuda, Mitsutoshi Nakada, Hisanori Uehara, Hiroshi Nishihara, Eiji Hara, Hidetaka Uramoto, Fumihiro Tanaka, Seiji Yano

Cancer science (Cancer Science) 106 ( 3 ) 244 - 252 2015.03

Joint Work, ISSN 13479032

EML4-ALK lung cancer accounts for approximately 3-7% of non-small-cell lung cancer cases. To investigate the molecular mechanism underlying tumor progression and targeted drug sensitivity/resistance in EML4-ALK lung cancer, clinically relevant animal models are indispensable. In this study, we found that the lung adenocarcinoma cell line A925L expresses an EML4-ALK gene fusion (variant 5a, E2:A20) and is sensitive to the ALK inhibitors crizotinib and alectinib. We further established highly tumorigenic A925LPE3 cells, which also have the EML4-ALK gene fusion (variant 5a) and are sensitive to ALK inhibitors. By using A925LPE3 cells with luciferase gene transfection, we established in vivo imaging models for pleural carcinomatosis, bone metastasis, and brain metastasis, all of which are significant clinical concerns of advanced EML4-ALK lung cancer. Interestingly, crizotinib caused tumors to shrink in the pleural carcinomatosis model, but not in bone and brain metastasis models, whereas alectinib showed remarkable efficacy in all three models, indicative of the clinical efficacy of these ALK inhibitors. Our in vivo imaging models of multiple organ sites may provide useful resources to analyze further the pathogenesis of EML4-ALK lung cancer and its response and resistance to ALK inhibitors in various organ microenvironments.

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肺高血圧症における血管病変の形態学的・免疫組織学的変化

谷野美智枝, 辻野一三, 石田雄介, 加藤容崇, 王磊, 木村太一, 西原広史, 田中伸哉

日本病理学会会誌 ((一社)日本病理学会) 104 ( 1 ) 311 - 311 2015.03

ISSN 0300-9181

Gastritis cystica profundaを背景にした重複胃癌の免疫組織学的検討

志藤茜, 杢里花, 湯澤明夏, 石川麻倫, 加藤容崇, 石田雄介, 木村太一, 谷野美智枝, 西原広史, 田中伸哉

日本病理学会会誌 ((一社)日本病理学会) 104 ( 1 ) 513 - 513 2015.03

ISSN 0300-9181

Importance of luminal membrane mesothelin expression in intraductal papillary mucinous neoplasms

Takahiro Einama, Hirofumi Kamachi, Hiroshi Nishihara, Shigenori Homma, Hiromi Kanno, Marin Ishikawa, Futoshi Kawamata, Yuji Konishi, Masanori Sato, Munenori Tahara, Kuniaki Okada, Shunji Muraoka, Toshiya Kamiyama, Akinobu Taketomi, Yoshihiro Matsuno, Hiroyuki Furukawa, Satoru Todo

ONCOLOGY LETTERS (SPANDIDOS PUBL LTD) 9 ( 4 ) 1583 - 1589 2015

Research paper (scientific journal), Joint Work, ISSN 17921074

The present study demonstrated that luminal membrane mesothelin expression is a reliable prognostic factor in gastric cancer. Intraductal papillary mucinous neoplasms (IPMNs) often exhibit a spectrum of dysplasia, ranging between adenoma and carcinoma. Therefore, an immunohistochemical analysis of mesothelin expression in IPMN was performed in the present study, focusing on the localization of mesothelin. IPMNs were classified into two groups, IPMNs associated with invasive carcinoma and low-high (L-H) grade dysplasias. The tumors were classified as mesothelin-positive or -negative and in the mesothelin-positive cases, the localization of mesothelin was evaluated as luminal membrane- or cytoplasmic-positive. Among the 37 IPMNs, mesothelin expression was observed in 21 samples (56.8%), including 46.2% (12 out of 26) of the L-H dysplasia and 81.8% (9 out of 11) of the invasive carcinoma samples (P=0.071). Luminal membrane localization was observed in 10 samples (27%), including 15.4% (4/26) of the L-H dysplasia samples and 54.5% (6 out of 11) of the invasive carcinoma samples (P=0.022). Six patients experienced post-operative recurrence, with five of the recurrent tumors exhibiting mesothelin expression and all six exhibiting luminal membrane localization. It was concluded that immunohistochemical examinations for mesothelin expression and localization are clinically useful for prognostic assessments and decision making regarding further treatment subsequent to surgical procedures in patients with IPMN.

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Inhibition of Eph receptor A4 by 2,5-dimethylpyrrolyl benzoic acid suppresses human pancreatic cancer growing orthotopically in nude mice

Hironobu Takano, Toru Nakamura, Takahiro Tsuchikawa, Toshihiro Kushibiki, Kouji Hontani, Kazuho Inoko, Mizuna Takahashi, Shoki Sato, Hirotake Abe, Shintaro Takeuchi, Nagato Sato, Kei Hiraoka, Hiroshi Nishihara, Toshiaki Shichinohe, Satoshi Hirano

ONCOTARGET (IMPACT JOURNALS LLC) 6 ( 38 ) 41063 - 41076 2015

Research paper (scientific journal), Joint Work, ISSN 1949-2553

Ephrin receptor A4 (EphA4) is overexpressed in human pancreatic adenocarcinoma (PDAC) and activate cell growth. Recent studies have identified small molecules that block EphA4. In this study, we investigated the correlation between EphA4 expression and the prognosis of patients with PDAC. We also examined the cytostatic efficacy of 2,5-dimethylpyrrolyl benzoic acid (compound 1), a small molecule that blocks EphA4, in PDAC cells. Overall survival of patients with EphA4 positivity was significantly shorter than that of patients with EphA4 negativity (P = 0.029). In addition, multivariate analysis revealed that EphA4 expression was an independent prognostic factor in PDAC patients (P = 0.039). Compound 1 showed a cytostatic efficacy in PDAC cells expressing EphA4 in vitro and in vivo. Our study indicated that compound 1 suppressed both EphA4 and Akt phosphorylations, and induced apoptosis in PDAC cells expressing EphA4. In conclusion, compound 1 has a high potential as a therapeutic agent for patients with PDAC.

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Polymeric micelles loaded with platinum anticancer drugs target preangiogenic micrometastatic niches associated with inflammation

Hailiang Wu, Horacio Cabral, Kazuko Toh, Peng Mi, Yi-Chun Chen, Yu Matsumoto, Naoki Yamada, Xueying Liu, Hiroaki Kinoh, Yutaka Miura, Mitsunobu R. Kano, Hiroshi Nishihara, Nobuhiro Nishiyama, Kazunori Kataoka

JOURNAL OF CONTROLLED RELEASE (ELSEVIER) 189 1 - 10 2014.09

Research paper (scientific journal), Joint Work, ISSN 01683659

Nanocarriers have been used for specific delivery of therapeutic agents to solid tumors based on the enhanced permeability and retention in cancerous tissues. Despite metastasis is the main reason of cancer-related death and a priority for nanocarrier-based therapies, the targeting ability of nanocarriers to the metastatic disease is poorly understood, especially for preangiogenic micrometastases as nanocarriers usually use the malignant neovasculature for enhancing their accumulation. Thus, herein, we studied the ability of micellar nanocarriers incorporating (1,2-diaminocyclohexane) platinum(II) (DACHPt) for treating liver metastases of bioluminescent murine colon adenocarcinoma C-26, during overt and preangiogenic metastatic stages. After intravenous injection, DACHPt-loaded micelles (DACHPt/m) effectively inhibited the tumor growth in both metastatic tumor models. While the anticancer activity of the micelles against overt metastases was associated with their selective accumulation in cancerous tissues having neovasculature, the ability of DACHPt/m to target preangiogenic metastases was correlated with the inflammatory microenvironment of the niche. This targeting capability of polymeric micelles to preangiogenic metastasis may provide a novel approach for early diagnosis and treatment of metastases. (C) 2014 Elsevier B.V. All rights reserved.

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SS18-SSX-regulated miR-17 promotes tumor growth of synovial sarcoma by inhibiting p21WAF1/CIP1

Yusuke Minami, Shinji Kohsaka, Masumi Tsuda, Kazuhiro Yachi, Nobuaki Hatori, Mishie Tanino, Taichi Kimura, Hiroshi Nishihara, Akio Minami, Norimasa Iwasaki, Shinya Tanaka

CANCER SCIENCE (WILEY-BLACKWELL) 105 ( 9 ) 1152 - 1159 2014.09

Research paper (scientific journal), Joint Work, ISSN 1347-9032

MicroRNA (miRNA) can function as tumor suppressors or oncogenes, and also as potential specific cancer biomarkers; however, there are few published studies on miRNA in synovial sarcomas, and their function remains unclear. We transfected the OncomiR miRNA Precursor Virus Library into synovial sarcoma Fuji cells followed by a colony formation assay to identify miRNAs to confer an aggressive tumorigenicity, and identified miR-17-5p from the large colonies. MiR-17 was found to be induced by a chimeric oncoprotein SS18-SSX specific for synovial sarcoma, and all examined cases of human synovial sarcoma expressed miR-17, even at high levels in several cases. Overexpression of miR-17 in synovial sarcoma cells, Fuji and HS-SYII, increased colony forming ability in addition to cell growth, but not cell motility and invasion. Tumor volume formed in mice in vivo was significantly increased by miR-17 overexpression with a marked increase of MIB-1 index. According to PicTar and Miranda algorithms, which predicted CDKN1A (p21) as a putative target of miR-17, a luciferase assay was performed and revealed that miR-17 directly targets the 3-UTR of p21 mRNA. Indeed, p21 protein level was remarkably decreased by miR-17 overexpression in a p53-independent manner. It is noteworthy that miR-17 succeeded in suppressing doxorubicin-evoked higher expression of p21 and conferred the drug resistance. Meanwhile, introduction of anti-miR-17 in Fuji and HS-SYII cells significantly decreased cell growth, consistent with rescued expression of p21. Taken together, miR-17 promotes the tumor growth of synovial sarcomas by post-transcriptional suppression of p21, which may be amenable to innovative therapeutic targeting in synovial sarcoma.

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Human pathological basis of blood vessels and stromal tissue for nanotechnology

Hiroshi Nishihara

ADVANCED DRUG DELIVERY REVIEWS (ELSEVIER SCIENCE BV) 74 19 - 27 2014.07

Single Work, ISSN 0169409X

The recent development of nanotechnology has already produced clinically applicable "nanodrugs," which are largely dependent on a novel concept for the drug delivery system. Thus the elucidation of local pharmacokinetics of nanodrugs is indispensable for the further development of nanomedicine; however, the detailed pathophysiology associated with nano-sized materials especially in pathologic lesions has not been well-described. In this review article, the microscopic appearance of vascular pericytes in addition to endothelial cells is discussed in the normal state and also in several pathological conditions which could be the major targets for nanomedicine. Moreover, the role of stromal tissue including myofibroblasts is also focused on, as well as inflammatory cells. Finally, the significance of disease-specific tissue structure in the establishment of personalized nanomedicine is discussed. (C) 2014 Elsevier B.V. All rights reserved.

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Epiregulin enhances tumorigenicity by activating the ERK/MAPK pathway in glioblastoma

Shinji Kohsaka, Kunihiko Hinohara, Lei Wang, Tatsunori Nishimura, Masana Urushido, Kazuhiro Yachi, Masumi Tsuda, Mishie Tanino, Taichi Kimura, Hiroshi Nishihara, Noriko Gotoh, Shinya Tanaka

NEURO-ONCOLOGY (OXFORD UNIV PRESS INC) 16 ( 7 ) 960 - 970 2014.07

Research paper (scientific journal), Joint Work, ISSN 15228517

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors, and the establishment of an effective therapeutic reagent is a pressing priority. Recently, it has been shown that the tumor tissue consists of heterogeneous components and that a highly aggressive population should be the therapeutic target.
Through a single subcutaneous passage of GBM cell lines LN443 and U373 in mice, we have developed highly aggressive variants of these cells named LN443X, U373X1, and U373X2, which showed increased tumor growth, colony-forming potential, sphere-forming potential, and invasion ability. We further investigated using microarray analysis comparing malignant cells with their parental cells and mRNA expression analysis in grades II to IV glioma samples.
Adipocyte enhancer binding protein 1, epiregulin (EREG), and microfibrillar associated protein 5 were identified as candidate genes associated with higher tumor grade and poor prognosis. Immunohistochemical analysis also indicated a correlation of a strong expression of EREG with short overall survival. Furthermore, both EREG stimulation and EREG introduction of GBM cell lines were found to increase phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase and resulted in the promotion of colony formation, sphere formation, and in vivo tumor formation. Gefitinib treatment inhibited phosphorylation of EGFR and extracellular signal-regulated kinase and led to tumor regression in U373-overexpressed EREG.
These results suggested that EREG is one of the molecules involved in glioma malignancy, and EGFR inhibitors may be a candidate therapeutic agent for EREG-overexpressing GBM patients.

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Immunohistochemical evaluation of O-6-methylguanine DNA methyltransferase (MGMT) expression in 117 cases of glioblastoma

Masaya Miyazaki, Hiroshi Nishihara, Shunsuke Terasaka, Hiroyuki Kobayashi, Shigeru Yamaguchi, Tamio Ito, Yuuta Kamoshima, Shin Fujimoto, Sadao Kaneko, Masahito Katoh, Nobuaki Ishii, Hiromi Mohri, Mishie Tanino, Taichi Kimura, Shinya Tanaka

NEUROPATHOLOGY (WILEY-BLACKWELL) 34 ( 3 ) 268 - 276 2014.06

Research paper (scientific journal), Joint Work, ISSN 09196544

Temozolomide (TMZ) is an oral alkylating agent which is widely used in the treatment of glioblastoma (GBM) and is composed of astrocytic and/or oligodendroglial tumors, and the evaluation of O6-methylguanine DNA methyltransferase (MGMT) expression is important to predict the response to TMZ therapy. In this study, we conducted immunohistochemical analysis of 117 cases of Japanese GBM including 19 cases of GBM with oligodendroglioma component (GBMO), using a scoring system for quantitative evaluation of staining intensity and proportion of MGMT, and performed survival analysis of these patients. Immunohistochemically, 55 cases (47%) were positive for MGMT with various intensities and proportions (total score (TS)2), while 62 cases (53%) were negative (TS=0). The distribution of MGMT expression pattern was not affected by any clinicopathological parameters such as the histological subtype (GBM vs. GBMO), age and gender. The survival analysis of these patients revealed that the minimal expression of MGMT (TS2) was a significant unfavorable prognostic factor (P<0.001) as well as resectability (P=0.004). Moreover, multivariate analysis showed that minimal MGMT expression in GBM was the most potent independent predictor for progression free survival (P<0.001) and also overall patient survival (P<0.001). This is the first report employing the scoring system for both staining intensity and proportion to evaluate immunohistochemical MGMT expression in GBM. In addition, our results emphases the clinicopathological values of the immunohistochemical approach for MGMT expression in glioma patients as a routine laboratory examination.

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Prognostic Factors for Survival in Patients with High-Grade Meningioma and Recurrence-Risk Stratification for Application of Radiotherapy

Shigeru Yamaguchi, Shunsuke Terasaka, Hiroyuki Kobayashi, Katsuyuki Asaoka, Hiroaki Motegi, Hiroshi Nishihara, Hiromi Kanno, Rikiya Onimaru, Yoichi M. Ito, Hiroki Shirato, Kiyohiro Houkin

PLOS ONE (PUBLIC LIBRARY SCIENCE) 9 ( 5 ) 2014.05

Research paper (scientific journal), Joint Work, ISSN 1932-6203

Background: Radiotherapy for high-grade meningioma (HGM) is one of the essential treatment options for disease control. However, appropriate irradiation timing remains under debate. The object of this study is to discern which prognostic factors impact recurrence in HGM patients and to propose a risk-stratification system for the application of postoperative radiotherapy.Methods: We retrospectively reviewed 55 adult patients who were diagnosed with Grade II and III intracranial meningioma. Cox regression models were applied to the analysis for impact on early recurrence in HGM patients without postoperative radiotherapy.Results: Grade III malignancy (P = 0.0073) and transformed histology (P = 0.047) proved to be significantly poor prognostic factors of early recurrence by multivariate analysis. The other candidates for recurrence factors were Simpson Grade 3-5 resection, preoperative Karnofsky Performance status, <= 70%, and MIB-1 labeling index >= 15%. According to these prognostic factors, postoperative HGM patients could be stratified into three recurrence-risk groups. The prognoses were significantly different between each group, as the 3-year actual recurrence-free rates were 90% in low-risk group, 31% in intermediate-risk group, and 15% in high-risk group.Conclusion: We propose recurrence-risk stratification for postoperative HGM patients using clinically available factors. Our results suggest that the prognosis for patients with high-risk HGMs is dismal, whereas HGM patients belonging to the low-risk group could have favorable prognoses. This stratification provides us with the criteria necessary to determine whether to apply adjuvant radiotherapy to postoperative HGM patients, and to also help identify potentially curable HGMs without adjuvant radiotherapy.

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Sustained elevation of Snail promotes glial-mesenchymal transition after irradiation in malignant glioma

Roshan Mahabir, Mishie Tanino, Aiman Elmansuri, Lei Wang, Taichi Kimura, Tamio Itoh, Yusuke Ohba, Hiroshi Nishihara, Hiroki Shirato, Masumi Tsuda, Shinya Tanaka

NEURO-ONCOLOGY (OXFORD UNIV PRESS INC) 16 ( 5 ) 671 - 685 2014.05

Research paper (scientific journal), Joint Work, ISSN 15228517

Ionizing irradiation is an effective treatment for malignant glioma (MG); however, a higher rate of recurrence with more aggressive phenotypes is a vital issue. Although epithelial-mesenchymal transition (EMT) is involved in irradiation-induced cancer progression, the role for such phenotypic transition in MG remains unknown.
To investigate the mechanism of irradiation-dependent tumor progression in MG, we performed immunohistochemistry (IHC) and qRT-PCR using primary and recurrent MG specimens, MG cell lines, and primary culture cells of MG. siRNA technique was used for MG cell lines.
In 22 cases of clinically recurrent MG, the expression of the mesenchymal markers vimentin and CD44 was found to be increased by IHC. In paired identical MG of 7 patients, the expression of collagen, MMPs, and YKL-40 were also elevated in the recurrent MGs, suggesting the The Cancer Genome Atlas-based mesenchymal subtype. Among EMT regulators, sustained elevation of Snail was observed in MG cells at 21 days after irradiation. Cells exhibited an upregulation of migration, invasion, numbers of focal adhesion, and MMP-2 production, and all of these mesenchymal features were abrogated by Snail knockdown. Intriguingly, phosphorylation of ERK1/2 and GSK-3 were increased after irradiation in a Snail-dependent manner, and TGF- was elevated in both fibroblasts and macrophages but not in MG cells after irradiation. It was noteworthy that irradiated cells also expressed stemness features such as SOX2 expression and tumor-forming potential in vivo.
We here propose a novel concept of glial-mesenchymal transition after irradiation in which the sustained Snail expression plays an essential role.

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microRNA 31 functions as an endometrial cancer oncogene by suppressing Hippo tumor suppressor pathway

Takashi Mitamura, Hidemichi Watari, Lei Wang, Hiromi Kanno, Makiko Kitagawa, Mohamed Kamel Hassan, Taichi Kimura, Mishie Tanino, Hiroshi Nishihara, Shinya Tanaka, Noriaki Sakuragi

MOLECULAR CANCER (BMC) 13 ( 1 ) 2014.04

Research paper (scientific journal), Joint Work, ISSN 1476-4598

Background: We aimed to investigate whether MIR31 is an oncogene in human endometrial cancer and identify the target molecules associated with the malignant phenotype.Methods: We investigated the growth potentials of MIR31-overexpressing HEC-50B cells in vitro and in vivo. In order to identify the target molecule of MIR31, a luciferase reporter assay was performed, and the corresponding downstream signaling pathway was examined using immunohistochemistry of human endometrial cancer tissues. We also investigated the MIR31 expression in 34 patients according to the postoperative risk of recurrence.Results: The overexpression of MIR31 significantly promoted anchorage-independent growth in vitro and significantly increased the tumor forming potential in vivo. MIR31 significantly suppressed the luciferase activity of mRNA combined with the LATS2 3'-UTR and consequently promoted the translocation of YAP1, a key molecule in the Hippo pathway, into the nucleus. Meanwhile, the nuclear localization of YAP1 increased the transcription of CCND1. Furthermore, the expression levels of MIR31 were significantly increased (10.7-fold) in the patients (n = 27) with a high risk of recurrence compared to that observed in the low-risk patients (n = 7), and this higher expression correlated with a poor survival.Conclusions: MIR31 functions as an oncogene in endometrial cancer by repressing the Hippo pathway. MIR31 is a potential new molecular marker for predicting the risk of recurrence and prognosis of endometrial cancer.

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Differential diagnosis of small cell glioblastoma and anaplastic oligodendroglioma: a case report of an elderly man

Kenta Takahashi, Masumi Tsuda, Hiromi Kanno, Junichi Murata, Roshan Mahabir, Yusuke Ishida, Taichi Kimura, Mishie Tanino, Hiroshi Nishihara, Kazuo Nagashima, Shinya Tanaka

BRAIN TUMOR PATHOLOGY (SPRINGER JAPAN KK) 31 ( 2 ) 118 - 123 2014.04

Research paper (scientific journal), Joint Work, ISSN 14337398

Small cell glioblastoma is a histological subtype of glioblastoma with characteristic features of highly proliferative, monotonous small glial cells with high nuclear cytoplasm ratio. Morphologically, malignant lymphoma or small cell metastatic carcinoma should be carefully discriminated. Some cases are difficult to differentiate from anaplastic oligodendroglioma. In this report, we present a case of small cell glioblastoma of an elderly man. The lack of IDH1/2 mutation was confirmed by immunohistochemistry and direct sequencing. Fluorescence in situ hybridization revealed the lower rates of chromosome 1p and 19q deletion. Microsatellite analysis disclosed partial 10q alteration near the PTEN locus. Not only morphological and immunohistochemical examinations, but also cytogenetical investigations for IDH1/2 mutation, 1p/19q loss, and PTEN alteration, are strongly supportive methods for the differential diagnosis of small cell glioblastoma and anaplastic oligodendroglioma.

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中枢神経系原発びまん性大細胞型B細胞性リンパ腫におけるDNAメチル化に関する解析

畑中佳奈子, 畑中豊, 小林浩之, 菅野宏美, 西原広史, 田中伸哉, 三橋智子, 松野吉宏

日本病理学会会誌 103 ( 1 ) 211 2014.03

Joint Work, ISSN 0300-9181

C-ERC/mesothelin provokes lymphatic invasion of colorectal adenocarcinoma

Futoshi Kawamata, Shigenori Homma, Hirofumi Kamachi, Takahiro Einama, Yasutaka Kato, Masumi Tsuda, Shinya Tanaka, Masahiro Maeda, Kazunori Kajino, Okio Hino, Norihiko Takahashi, Toshiya Kamiyama, Hiroshi Nishihara, Akinobu Taketomi, Satoru Todo

JOURNAL OF GASTROENTEROLOGY (SPRINGER JAPAN KK) 49 ( 1 ) 81 - 92 2014.01

Research paper (scientific journal), Joint Work, ISSN 09441174

Background Lymph node metastasis is a key event of colorectal cancer (CRC) progression. Mesothelin is expressed in various types of malignant tumor and associated with an unfavorable prognosis. The full-length mesothelin (Full-ERC) is cleaved by protease into membrane-bound C-ERC/mesothelin and N-ERC/mesothelin which is secreted into the blood. The aim of this study was to examine the biological role of mesothelin in CRC by clinicopathological analysis and in vitro lymphatic invasion assay.
Methods Ninety-one cases of CRC specimens were immunohistochemically examined and the localization of mesothelin in luminal membrane and/or cytoplasm was also evaluated. Lymphatic invasion assay was also performed using the human CRC cell line, WiDr, which was transfected with Full-, N- and C-ERC/mesothelin expression plasmids (Full-WiDr, N-WiDr and C-WiDr).
Results Immunohistochemically, "luminal membrane positive" of mesothelin was identified in 37.4 %, and correlated with lymphatic permeation and lymph node metastasis, but not with patients' prognosis. Interestingly, among the patients with lymph node metastasis (N = 38), "luminal membrane positive" of mesothelin significantly correlated with unfavorable patients' outcome. In addition, lymphatic invasion assay revealed that Full-WiDr and C-WiDr more significantly invaded human lymphatic endothelial cells than the Mock-WiDr (P < 0.01).
Conclusion The luminal membrane expression of mesothelin was associated with unfavorable prognosis of CRC patients with lymph nodemetastasis. Moreover, this is the first report to prove the biological function of C-ERC/mesothelin associated with lymphatic invasion of cancer in vitro.

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Clinicopathological evaluation of cyclooxygenase-2 expression in meningioma: immunohistochemical analysis of 76 cases of low and high-grade meningioma.

Kato Y, Nishihara H, Mohri H, Kanno H, Kobayashi H, Kimura T, Tanino M, Terasaka S, Tanaka S

Brain tumor pathology (SPRINGER JAPAN KK) 31 ( 1 ) 23 - 30 2014.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 14337398

Tumorigenic activity of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the production of prostaglandins (PGs), has been proved for some types of cancer, including brain tumors. We evaluated expression of COX-2 in meningioma, one of the most common intracranial tumors in adults which accounts for 24-30 % of intracranial tumors. We performed immunostaining for COX-2 in 76 cases of meningioma consisting of 44 cases of low-grade (WHO Grade I) and 32 cases of high-grade (29 cases of Grade II and 3 cases of Grade III) meningioma, and evaluated COX-2 expression levels on the basis of staining intensity and proportion in tumor cells. The expression level of COX-2 in meningioma cells was significantly correlated with WHO grade (P = 0.0153). In addition, COX-2 expression was significantly correlated with MIB-1 labeling index for all 76 cases of meningioma (P = 0.0075), suggesting tumor promotion by COX-2 in meningioma progression. Our results may indicate the therapeutic value of non-steroidal anti-inflammatory drugs against meningioma, especially for patients with elevated proliferation, to regulate the tumorigenic activity of COX-2 in meningioma cells.

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Autophagy in the placenta of women with hypertensive disorders in pregnancy

R. Akaishi, T. Yamada, K. Nakabayashi, H. Nishihara, I. Furuta, T. Kojima, M. Morikawa, T. Yamada, N. Fujita, H. Minakami

PLACENTA (W B SAUNDERS CO LTD) 35 ( 12 ) 974 - 980 2014

Research paper (scientific journal), Joint Work, ISSN 01434004

Introduction: Autophagy has not been studied extensively in the human placenta. This study was performed to determine whether autophagy is increased in the placentas of women with hypertensive disorders in pregnancy compared to normotensive pregnancies.
Methods: LC3-II and p62 protein expression were examined by quantitative Western blotting analysis in 40 placentas from women not experiencing labor pains. The 40 placentas were from 13, 8, and 19 women with preeclampsia, gestational hypertension, and normal pregnancy, respectively. Hypertensive disorders in pregnancy included preeclampsia and gestational hypertension.
Results: LC3-II expression was significantly increased, while that of p62 was significantly reduced in 21 placentas of women with hypertensive disorders compared to those with normal blood pressure irrespective of the presence or absence of fetal growth restriction (FGR). LC3-II expression was also significantly increased in 13 placentas of women with preeclampsia irrespective of the presence or absence of FGR.
Discussion: The results of this study suggested that autophagy is active in the placenta of hypertensive disorders even in the absence of FGR. (C) 2014 Elsevier Ltd. All rights reserved.

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Rapid immunohistochemistry based on alternating current electric field for intraoperative diagnosis of brain tumors

Mishie Tanino, Toshio Sasajima, Hiroshi Nanjo, Shiori Akesaka, Masami Kagaya, Taichi Kimura, Yusuke Ishida, Masaya Oda, Masataka Takahashi, Taku Sugawara, Toshiaki Yoshioka, Hiroshi Nishihara, Yoichi Akagami, Akiteru Goto, Yoshihiro Minamiya, Shinya Tanaka

BRAIN TUMOR PATHOLOGY (SPRINGER JAPAN KK) 32 ( 1 ) 12 - 19 2014

Research paper (scientific journal), Joint Work, ISSN 14337398

Rapid immunohistochemistry (R-IHC) can contribute to the intraoperative diagnosis of central nervous system (CNS) tumors. We have recently developed a new IHC method based on an alternating current electric field to facilitate the antigen-antibody reaction. To ensure the requirement of R-IHC for intraoperative diagnosis, 183 cases of CNS tumors were reviewed regarding the accuracy rate of diagnosis without R-IHC. The diagnostic accuracy was 90.7 % (168/183 cases) in which definitive diagnoses were not provided in 17 cases because of the failure of glioma grading and differential diagnosis of lymphoma and glioma. To establish the clinicopathological application, R-IHC for frozen specimens was compared with standard IHC for permanent specimens. 33 gliomas were analyzed, and the Ki-67/MIB-1 indices of frozen specimens by R-IHC were consistent with the grade and statistically correlated with those of permanent specimens. Thus, R-IHC provided supportive information to determine the grade of glioma. For discrimination between glioma and lymphoma, R-IHC was able to provide clear results of CD20 and Ki-67/MIB-1 in four frozen specimens of CNS lymphoma as well as standard IHC. We conclude that the R-IHC for frozen specimens can provide important information for intraoperative diagnosis of CNS tumors.

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Rectal obstruction due to endometriosis: A case report and review of the Japanese literature

Hiromi Ono, Shohei Honda, Yasushi Danjo, Kenji Nakamura, Mihiro Okabe, Takashi Kimura, Masato Kawakami, Kimimoto Nagashima, Hiroshi Nishihara

International Journal of Surgery Case Reports (Elsevier Ltd) 5 ( 11 ) 845 - 848 2014

Research paper (scientific journal), Joint Work, ISSN 22102612

INTRODUCTION Colorectal obstructive endometriosis is relatively rare in Japan and its differentiation from malignancy is often difficult. We report a case of rectal obstructive endometriosis. PRESENTATION OF CASE A 37-year-old woman was referred to our hospital with a suspected ileus. Her chief symptoms were left lower abdominal pain and vomiting. Colonoscopy showed an intraluminal mass of redness in the upper rectum. A proctectomy was performed because of the bowel obstruction. The rectum was filled with an intraluminal mass measuring 5 cm × 4 cm, and endometriosis was diagnosed pathologically. DISCUSSION A preoperative diagnosis of colorectal obstructive endometriosis is often difficult because of the lack of definite diagnostic, clinical, sonographic, or radiological findings that are characteristic of this disease. Medical treatment is not always effective for colorectal obstructive endometriosis, and surgery is often performed. CONCLUSION Colorectal obstructive endometriosis should be considered as a differential diagnosis in cases of various gastrointestinal symptoms in women who are of reproductive age.

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Erratum to: Rapid immunohistochemistry based on alternating current electric field for intraoperative diagnosis of brain tumors [Brain Tumor Pathol, (2014), DOI:10.1007/s10014-014-0188-y]

Brain Tumor Pathology (Springer-Verlag Tokyo) 32 ( 1 ) 20 - 21 2014

Research paper (scientific journal), Joint Work, Accepted, ISSN 1861-387X

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Ability of the Met kinase inhibitor crizotinib and new generation EGFR inhibitors to overcome resistance to EGFR inhibitors.

Shigeki Nanjo, Tadaaki Yamada, Hiroshi Nishihara, Shinji Takeuchi, Takako Sano, Takayuki Nakagawa, Daisuke Ishikawa, Lu Zhao, Hiromichi Ebi, Kazuo Yasumoto, Kunio Matsumoto, Seiji Yano

PloS one (PLoS ONE) 8 ( 12 ) e84700 2013.12

Joint Work

PURPOSE: Although EGF receptor tyrosine kinase inhibitors (EGFR-TKI) have shown dramatic effects against EGFR mutant lung cancer, patients ultimately develop resistance by multiple mechanisms. We therefore assessed the ability of combined treatment with the Met inhibitor crizotinib and new generation EGFR-TKIs to overcome resistance to first-generation EGFR-TKIs. EXPERIMENTAL DESIGN: Lung cancer cell lines made resistant to EGFR-TKIs by the gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression and mice with tumors induced by these cells were treated with crizotinib and a new generation EGFR-TKI. RESULTS: The new generation EGFR-TKI inhibited the growth of lung cancer cells containing the gatekeeper EGFR-T790M mutation, but did not inhibit the growth of cells with Met amplification or HGF overexpression. In contrast, combined therapy with crizotinib plus afatinib or WZ4002 was effective against all three types of cells, inhibiting EGFR and Met phosphorylation and their downstream molecules. Crizotinib combined with afatinib or WZ4002 potently inhibited the growth of mouse tumors induced by these lung cancer cell lines. However, the combination of high dose crizotinib and afatinib, but not WZ4002, triggered severe adverse events. CONCLUSIONS: Our results suggest that the dual blockade of mutant EGFR and Met by crizotinib and a new generation EGFR-TKI may be promising for overcoming resistance to reversible EGFR-TKIs but careful assessment is warranted clinically.

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NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL

Masaya Miyazaki, Hiroshi Nishihara, Hideki Hasegawa, Masato Tashiro, Lei Wang, Taichi Kimura, Mishie Tanino, Masumi Tsuda, Shinya Tanaka

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (ACADEMIC PRESS INC ELSEVIER SCIENCE) 441 ( 4 ) 953 - 957 2013.11

Research paper (scientific journal), Joint Work, ISSN 0006291X

The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while the physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERR and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated. (C) 2013 Elsevier Inc. All rights reserved.

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Cyclic RGD-Linked Polymeric Micelles for Targeted Delivery of Platinum Anticancer Drugs to Glioblastoma through the Blood-Brain Tumor Barrier

Yutaka Miura, Tomoya Takenaka, Kazuko Toh, Shourong Wu, Hiroshi Nishihara, Mitsunobu R. Kano, Yasushi Ino, Takahiro Nomoto, Yu Matsumoto, Hiroyuki Koyama, Horacio Cabral, Nobuhiro Nishiyama, Kazunori Kataoka

ACS NANO (AMER CHEMICAL SOC) 7 ( 10 ) 8583 - 8592 2013.10

Research paper (scientific journal), Joint Work, ISSN 19360851

Ligand-mediated drug delivery systems have enormous potential for improving the efficacy of cancer treatment. In particular, Arg-Gly-Asp peptides are promising ligand molecules for targeting alpha(gamma),beta(3)/alpha(gamma)beta(5) integrins, which are overexpressed in angiogenic sites and tumors, such as intractable human glioblastoma (U87MG). We here achieved highly efficient drug delivery to U87MG tumors by using a platinum anticancer drug-incorporating polymeric micelle (PM) with cyclic Arg-Gly-Asp (cRGD) ligand molecules. Intravital confocal laser scanning microscopy revealed that the cRGD-linked polymeric micelles (cRGD/m) accumulated rapidly and had high permeability from vessels into the tumor parenchyma compared with the PM having nontargeted ligand, "cyclic-Arg-Ala-Asp" (cRAD). As both cRGD/m- and cRAD-linked polymeric micelles have similar characteristics, including their size, surface charge, and the amount of incorporated drugs, it is likely that the selective and accelerated accumulation of cRGD/m into tumors occurred via an active internalization pathway, possibly transcytosis, thereby producing significant antitumor effects in an orthotopic mouse model of U87MG human glioblastoma.

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Downregulation of miRNA-31 induces taxane resistance in ovarian cancer cells through increase of receptor tyrosine kinase MET

T. Mitamura, H. Watari, L. Wang, H. Kanno, M. K. Hassan, M. Miyazaki, Y. Katoh, T. Kimura, M. Tanino, H. Nishihara, S. Tanaka, N. Sakuragi

Oncogenesis (Oncogenesis) 2 2013.07

Research paper (scientific journal), Joint Work, ISSN 2157-9024

Ovarian cancer is one of the most aggressive female reproductive tract tumors. Paclitaxel (PTX) is widely used for the treatment of ovarian cancer. However, ovarian cancers often acquire chemotherapeutic resistance to this agent. We investigated the mechanism of chemoresistance by analysis of microRNAs using the ovarian cancer cell line KFr13 and its PTX-resistant derivative (KFr13Tx). We found that miR-31 was downregulated in KFr13Tx cells, and that re-introduction of miR31 re-sensitized them to PTX both in vitro and in vivo. miR-31 was found to bind to the 3′-UTR of mRNA of MET, and the decrease in MET correlated to higher sensitivity to PTX. Furthermore, co-treatment of KFr13Tx cells with MET inhibitors sensitized the tumor cells to PTX both in vitro and in vivo. In addition, lower levels of miR31 and higher expression of MET in human ovarian cancer specimens were significantly correlated with PTX chemoresistance and poor prognosis. This study demonstrated miR31-dependent regulation of MET for chemoresistance of ovarian cancer, raising the possibility that combination therapy with a MET inhibitor and PTX will increase PTX efficacy. © 2013 Macmillan Publishers Limited All rights reserved.

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Expression of CD163 prevents apoptosis through the production of granulocyte colony-stimulating factor in meningioma

Hiromi Kanno, Hiroshi Nishihara, Lei Wang, Sayaka Yuzawa, Hiroyuki Kobayashi, Masumi Tsuda, Taichi Kimura, Mishie Tanino, Shunsuke Terasaka, Shinya Tanaka

NEURO-ONCOLOGY (OXFORD UNIV PRESS INC) 15 ( 7 ) 853 - 864 2013.07

Research paper (scientific journal), Joint Work, ISSN 15228517

Background. CD163 is a 130-kDa transmembrane protein expressed in human monocytes and macrophages, and the aberrant expression of CD163 in breast and colorectal cancer associated with patients' poor prognosis was reported. Here, we analyzed the expression of CD163 in meningioma, a common intracranial tumor, and its molecular mechanism in association with meningioma progression.
Methods. First, we performed immunohistochemical analysis using 50 human meningioma specimens. Next, we established CD163-overexpressing human meningioma cell lines and investigated its roles in tumor progression in vitro and in vivo.
Results. Immunohistochemically, 26 of 50 human meningioma specimens (52.0%) were positive for CD163 in tumor cells, including benign grade I (48.5%) and grade II (71.4%) cases. Furthermore, CD163 expression was correlated with histological atypical parameters that directly predict the prognosis of meningioma. CD163-overexpressing meningioma cells showed significant suppression of apoptosis and accelerated tumor growth in nude mice. In addition, unexpected splenomegaly affiliated with the xenograft predicted tumor-derived granulocyte colony-stimulating factor (G-CSF) production, which was confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay.
Conclusions. To our knowledge, this is the first report that demonstrates CD163 expression in meningioma not only by immunohistochemistry but also by reverse-transcription polymerase chain reaction, using primary culture cells, and provides the novel molecular function of CD163 to prevent apoptosis through the production of G-CSF in meningioma.

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Immunohistochemical molecular expression profile of metastatic brain tumor for potent personalized medicine.

Kato Y, Nishihara H, Yuzawa S, Mohri H, Kanno H, Hatanaka Y, Kimura T, Tanino M, Tanaka S

Brain tumor pathology (Brain Tumor Pathology) 30 ( 3 ) 167 - 174 2013.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 14337398

Recent progress in molecule-targeting therapy may yield personalized therapeutic strategies for patients with metastatic brain tumors (MBT), the most frequently encountered intracranial tumors. For this purpose, we investigated the molecular expression profile of MBT to establish the pathological basis for personalized diagnosis. We studied 166 MBT specimens including 70 cases of lung cancer and 34 cases of breast cancer, and performed immunostaining for EGFR, COX-2, and O-6-methylguanine-DNA methyltransferase (MGMT), among others, which could be target molecules for therapeutic agents or enable prediction of drug efficacy. Loss of MGMT expression was observed in approximately 20-40 % of MBT derived from lung, breast, and gastrointestinal cancers, indicating the possibility of treatment of MBT patients with temozolomide. In addition, MBT expressed a variety of receptor tyrosine kinases, for example EGFR and HER2, and signal transduction molecules, for example phospho-mTOR and COX-2, irrespective of tumor origin, enabling individualized medication with molecule-targeting drugs. We also identified alteration of molecular expression profile in 4 MBT cases during recurrence. Our results not only reveal the molecular characteristics of MBT but also suggest the possibility of potent personalized medicine for MBT patients. © 2012 The Japan Society of Brain Tumor Pathology.

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Inhibition of GSH synthesis potentiates temozolomide-induced bystander effect in glioblastoma.

Kohsaka S, Takahashi K, Wang L, Tanino M, Kimura T, Nishihara H, Tanaka S

Cancer letters (ELSEVIER IRELAND LTD) 331 ( 1 ) 68 - 75 2013.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 03043835

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors with poor prognosis. Current standard treatment includes chemotherapy using DNA alkylating agent temozolomide (TMZ) concomitant with surgical resection and/or irradiation. However, GBM patients exhibit various levels of the elevated expression of DNA repair enzyme, due to MGMT causing resistance to TMZ. Determination of the MGMT-positive population of primary tumor is important to evaluate the therapeutic efficacy of TMZ. Here we generated TMZ-resistant GBM cells by introducing MGMT into TMZ-sensitive GBM cell line KMG4, and established a model to assess the TMZ-induced bystander effect on TMZ-resistant cells. By mixing TMZ-resistant and -sensitive cells, GBM tumors with MGMT positivity as 50%, 10%, and 1% were generated in vivo. We could not observe any bystander effect of TMZ-induced cell death in tumor with 50% MGMT positivity. Although the bystander effect was observed within 20 days in the case of tumor with 1% MGMT positivity, final tumor size at day 28 was the same as control without sensitive cells. This bystander effect was observed in vitro using conditioned medium of TMZ-damaged GBM cells, and PCR array analysis indicated that the conditioned medium stimulated stress and toxicity pathway and upregulated anti-oxidants genes expression such as catalase and SOD2 in TMZ-resistant cells. In addition, the reduction of the activity of anti-stress mechanism by using inhibitor of GSH synthesis potentiated TMZ-induced bystander effect. These results suggest that GSH inhibitor might be one of the candidates for combination therapy with TMZ for TMZ-resistant GBM patients. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

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Intracranial mass-forming lesion associated with dural thickening and hypophysitis.

Kanno H, Tanino M, Watanabe K, Ozaki Y, Itoh T, Kimura T, Nishihara H, Itoh T, Narita T, Nagashima K, Tanaka S

Neuropathology : official journal of the Japanese Society of Neuropathology (Neuropathology) 33 ( 2 ) 213 - 216 2013.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 09196544

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脳腫瘍の現代病理学 BRAF異常とグリオーマ

田中伸哉, 谷野美智枝, 津田真寿美, 石田雄介, 木村太一, 西原広史, 長嶋和郎

日本病理学会会誌 ((一社)日本病理学会) 102 ( 1 ) 203 - 203 2013.04

ISSN 0300-9181

術中迅速脳腫瘍診断における電解非接触撹拌技術を搭載した迅速免疫染色装置使用の有用性

谷野美智枝, 明坂詩織, 石田雄介, 木村太一, 西原広史, 田中伸哉

日本病理学会会誌 ((一社)日本病理学会) 102 ( 1 ) 349 - 349 2013.04

ISSN 0300-9181

Relationship between Methyl CpG Binding Protein 2 and JC Viral Proteins

Kenta Takahashi, Yasuko Orba, Taichi Kimura, Lei Wang, Shinji Kohsaka, Masumi Tsuda, Mishie Tanino, Hiroshi Nishihara, Kazuo Nagashima, Hirofumi Sawa, Shinya Tanaka

JAPANESE JOURNAL OF INFECTIOUS DISEASES (NATL INST INFECTIOUS DISEASES) 66 ( 2 ) 126 - 132 2013.03

Research paper (scientific journal), Joint Work, ISSN 13446304

JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopathy (PML). Methyl CpG binding protein 2 (MeCP2) is a transcriptional control nuclear protein that is abundantly expressed in neurons. We previously observed that the MeCP2 protein is expressed in JCV large T antigen (TAg)-expressing glial cells in PML brains. To investigate the relationship between MeCP2 and JCV TAg, we examined the promoter activity and mRNA and protein expression levels of MeCP2 in JCV TAg-expressing cells. We found that JCV TAg enhances the promoter activity of MeCP2, but does not enhance the mRNA and protein levels of MeCP2. These results suggest that post-transcriptional mechanisms may play a role in MeCP2 expression.

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Clinical implications of luminal membrane expression of mesothelin in intraductal papillary mucinous neoplasms

Takahiro Einama, Hirofumi Kamachi, Hiroshi Nishihara, Shigenori Homma, Futoshi Kawamata, Munenori Tahara, Masahiko Taniguchi, Hiroyuki Furukawa, Toshiya Kamiyama, Akinobu Taketomi, Yoshihiro Matsuno, Satoru Todo

JOURNAL OF CLINICAL ONCOLOGY (AMER SOC CLINICAL ONCOLOGY) 31 ( 4 ) 2013.02

Joint Work, Accepted, ISSN 0732-183X

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Expression of O(6) -methylguanine DNA methyltransferase (MGMT) and immunohistochemical analysis of 12 pineal parenchymal tumors.

Kanno H, Nishihara H, Oikawa M, Ozaki Y, Murata J, Sawamura Y, Kato M, Kubota K, Tanino M, Kimura T, Nagashima K, Itoh T, Tanaka S

Neuropathology : official journal of the Japanese Society of Neuropathology (6) 32 ( 6 ) 647 - 653 2012.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 09196544

Pineal parenchymal tumors (PPTs) are rare neoplasms which occupy less than 1% of primary CNS tumors. Because of their rare incidence, previous reports on PPTs are limited in number and the useful molecular markers for deciding histological grading and even selecting chemotherapy are undetermined. In this study, we conducted immunohistochemical analysis of 12 PPT specimens, especially for expression of O6-methylguanine DNA methyltransferase (MGMT) to assess whether temozolomide (TMZ) could serve as a possible alternative therapy for PPTs. We analyzed 12 PPTs, consisting of three pineocytomas, six PPTs of intermediate differentiation (PPTIDs), and three pineoblastomas. Immunohistochemical analysis was performed using antibodies against MGMT, synaptophysin, neurofilament protein (NF), p53, and neuronal nuclear antigen (NeuN). Immunohistochemically, 11 out of 12 cases were positive for MGMT. The mean MIB-1 labeling index was less than 1% in pineocytoma, 3.5% in PPTID, and 10.5% in pineoblastoma. All 12 cases were positive for synaptophysin and 11 cases, except one PPTID case, showed positive for NF. Nuclear staining of NeuN was negative in all cases although cytoplasmic staining of NeuN was observed in five cases. No case was positive for p53. Eleven out of 12 cases of PPTs demonstrated MGMT expression, suggesting chemoresistancy to TMZ treatment. This is the first report showing MGMT expression in PPTs. In addition, MIB-1 labeling index correlated with WHO grade, although the immunoreactivity of synaptophysin, NF, NeuN and p53 did not correlate with the histological grade.

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PDGFRβ expression in tumor stroma of pancreatic adenocarcinoma as a reliable prognostic marker.

Yuzawa S, Kano MR, Einama T, Nishihara H

Medical oncology (Northwood, London, England) (4) 29 ( 4 ) 2824 - 2830 2012.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 13570560

Pancreatic adenocarcinoma is a lethal disease that often develops a desmoplastic reaction in tumor stroma. In general, desmoplasia is thought to promote tumor growth. However, its molecular pathology and prognostic potential have not been fully investigated. Here, we investigate 26 cases of pancreatic ductal adenocarcinoma and examine the clinicopathological association between survival and expression levels of several molecular markers for stromal cells. These include alpha-smooth muscle actin (SMA) and platelet-derived growth factor (PDGF) receptor beta (PDGFR beta). Both are markers of activated fibroblasts or pancreatic stellate cells (PSCs) that play an important role in desmoplasia. The staining patterns of both molecular markers were not uniform, so we categorized them into 3 grades (high, middle, and low) according to intensity. Interestingly, Kaplan-Meier analysis revealed that higher expression of PDGFR beta matched shorter prognosis (p = 0.0287, log-rank test) as well as lymphatic invasion and lymph node metastasis, whereas SMA did not (p = 0.6122). Our results suggest the prognostic potential of cancer stroma via PDGF-B signaling. Regulation of PDGF-B-associated signaling crosstalk between cancer cells and stroma cells, therefore, may indicate a possible therapeutic target for desmoplastic malignant tumors such as pancreatic adenocarcinoma.

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Intracellular localization of mesothelin predicts patient prognosis of extrahepatic bile duct cancer.

Kawamata F, Kamachi H, Einama T, Homma S, Tahara M, Miyazaki M, Tanaka S, Kamiyama T, Nishihara H, Taketomi A, Todo S

International journal of oncology (6) 41 ( 6 ) 2109 - 2118 2012.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 10196439

Mesothelin is expressed in various types of malignant tumors, and we recently reported that the expression of mesothelin was related to unfavorable patient outcome in pancreatic ductal adenocarcinoma and gastric adenocarcinoma. In this study, we examined the clinicopathological significance of mesothelin expression in extrahepatic bile duct cancer (EHBDCA), especially in terms of its association with the staining pattern. Tissue samples from 61 EHBDCA (16 hilar cholangiocarcinoma, 17 upper bile duct adenocarcinoma, 20 middle bile duct adenocarcinoma and 8 distal bile duct adenocarcinoma) were immunohistochemically examined. The expression levels of mesothelin in tumor cells was classified into the localization of mesothelin in luminal membrane and/or cytoplasm, in addition to high and low according to the staining intensity and proportion as a conventional analysis. 'High-level expression' of mesothelin (47.5%) was statistically correlated with liver metastasis (P=0.013) and poorer patient outcome (P=0.022), while 'luminal' membrane positive' of mesothelin (52.5%) was more significantly correlated with liver metastasis (P=0.006), peritoneal metastasis (P=0.024) and unfavorable patient outcome (P=0.017). Moreover, we found that 'cytoplasmic expression' isolated from 'luminal membrane negative' of mesothelin represented the best patient prognosis throughout this study. We describe the expression pattern level of mesothelin, i.e., in luminal membrane or cytoplasm both high and low level, evidently indicate the patient prognosis of EH BDCA, suggesting the pivotal role of mesothelin in cancer promotion depending on its intracellular localization.

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Prognostic implication of histological oligodendroglial tumor component: clinicopathological analysis of 111 cases of malignant gliomas.

Kanno H, Nishihara H, Narita T, Yamaguchi S, Kobayashi H, Tanino M, Kimura T, Terasaka S, Tanaka S

PloS one (7) 7 ( 7 ) e41669 2012.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 1932-6203

The favorable prognosis of high-grade oligodendroglial tumor such as glioblastoma (GBM) with oligodendroglioma component (GBMO) has been suggested; however, the studies which examine the prognostic significance of oligodendroglial tumor were limited. In this study, we performed a histopathology-based reevaluation of 111 cases of high grade gliomas according to the latest World Health Organization (WHO), and compared the clinical outcomes between oligodendroglial tumors and pure astrocytic tumors. The survival analysis revealed that the patients with high grade oligodendroglial tumor including GBMO significantly indicated better prognosis compared to the patients with high grade pure astrocytic tumors (GBM and AA, anaplastic astrocytoma) as expected, and the obtained survival curves were almost identical to those from the patients with conventional Grade III or Grade IV tumors, respectively. Moreover, if the cases of oligodendroglial tumor were histopathologically excluded, the patients with AA exhibited extremely poor prognosis which was similar to that of GBM, suggesting that the histological identification of oligodendroglial tumor component, even partially, prescribe the prognosis of high grade glioma patients. This is the prominent report of retrospective clinicopathological analysis for high-grade gliomas throughout Grade III and IV, especially referring to the prognostic value of histological oligodendroglial tumor component; in addition, our results might offer an alternative aspect for the grading of high-grade astrocytic/oligodendroglial tumors.

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CD133 negatively regulates tumorigenicity via AKT pathway in synovial sarcoma.

Kimura T, Wang L, Tabu K, Nishihara H, Mashita Y, Kikuchi N, Tanino M, Hiraga H, Tanaka S

Cancer investigation (5) 30 ( 5 ) 390 - 397 2012.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 07357907

Synovial sarcoma (SS) is an aggressive tumor that accounts for almost 10% of all soft tissue sarcomas. In this study, we found the expression of CD133 in human SS specimens, thus, we focused on the function of CD133 in SS. Separation of the CD133-positive and -negative subpopulations in SS cell lines clarified that the CD133-negative subpopulation exhibited enhanced growth and hyperphosphorylation of AKT. Treatment of Akt inhibitor suppressed the cell growth of CD133-negative subpopulation to the levels of CD133-positive cells. These results suggest that CD133 has negative effect on the growth of cells through AKT-dependent signalling pathway.

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STAT3 inhibition overcomes temozolomide resistance in glioblastoma by downregulating MGMT expression.

Kohsaka S, Wang L, Yachi K, Mahabir R, Narita T, Itoh T, Tanino M, Kimura T, Nishihara H, Tanaka S

Molecular cancer therapeutics (6) 11 ( 6 ) 1289 - 1299 2012.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 15357163

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors with a poor prognosis. Current standard treatment includes chemotherapy with the DNA-alkylating agent temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide-induced DNA damage due to elevated expression of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT). Here, we show that upregulation of both MGMT and STAT3 was accompanied with acquisition of temozolomide resistance in the GBM cell line U87. Inactivation of STAT3 by inhibitor or short hairpin RNA (shRNA) downregulated MGMT expression in GBM cell lines. MGMT upregulation was not observed by the treatment of interleukin (IL)-6 which is a strong activator of STAT3. Contrarily, forced expressed MGMT could be downregulated by STAT3 inhibitor which was partially rescued by the proteasome inhibitor, MG132, suggesting the STAT3-mediated posttranscriptional regulation of the protein levels of MGMT. Immunohistochemical analysis of 44 malignant glioma specimens showed significant positive correlation between expression levels of MGMT and phosphorylated STAT3 (p-STAT3; P < 0.001, gamma = 0.58). Importantly, the levels of both MGMT and p-STAT3 were increased in the recurrence compared with the primary lesion in paired identical tumors of 12 cases. Finally, we showed that STAT3 inhibitor or STAT3 knockdown potentiated temozolomide efficacy in temozolomide-resistant GBM cell lines. Therefore, STAT3 inhibitor might be one of the candidate reagents for combination therapy with temozolomide for patients with temozolomide-resistant GBM. Mol Cancer Ther; 11(6); 1289-99. (C)2012 AACR.

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Pericyte-coverage of human tumor vasculature and nanoparticle permeability.

Zhang L, Nishihara H, Kano MR

Biological & pharmaceutical bulletin (5) 35 ( 5 ) 761 - 766 2012.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 09186158

Nano drug delivery systems (nanoDDS) are a promising strategy for treatment of human tumors. As indicated by our previous work, the extent of pericyte-coverage of tumor vasculature is important to determining nanoDDS efficacy since intratumoral accumulation of nanoDDS is less in tumor models with pericyte-covered vasculature. Here we investigated the clinical relevance of our previous observations in animal models, by determining pericyte coverage using immunohistochemistry of smooth muscle actin (SMA) with CD34: a vascular endothelial marker, in human tumor tissue samples. The investigation revealed that tumor vasculature coverage by pericytes in pancreatic and diffuse-type gastric cancers was significantly greater than in ovarian, colon, and intestinal-type gastric cancers. The latter group of cancers is easier to treat clinically. These observations are consistent with our previous findings in animal models. On the basis of these findings we believe optimization of nanoDDS delivery should be done depend upon a clear understanding of the effects of pericyte vascular coverage.

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A case of clear cell variant of solid-pseudopapillary tumor of the pancreas in an adult male patient.

Tanino M, Kohsaka S, Kimura T, Tabu K, Nishihara H, Sawa H, Kawami H, Kamada H, Shimizu M, Tanaka S

Annals of diagnostic pathology (2) 16 ( 2 ) 134 - 140 2012.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 10929134

A clear cell variant of solid-pseudopapillary tumor (SPT) of the pancreas was initially reported in 2006 as a tumor that arose in the pancreatic body and tail in young adults; to date, only 4 cases of this entity have been reported. Here, we present the case of a 58-year-old man with clear cell variant of SPT with distinctive clinicopathologic features. The tumor was well demarcated, was 2.6 cm in size and mostly composed of multivacuolated clear cells with solid growth, and exhibited the characteristic immunohistochemical positivity of beta-catenin in the cytoplasm and nuclei of the neoplastic cells. In contrast to classical SPT with nuclear positivity, this case was negative for E-cadherin. Direct DNA sequencing of exon 3 of beta-catenin gene demonstrated a single amino acid substitution (serine to phenylalanine) in codon 37, which is the phosphorylation site by GSK beta and frequently found in classical SPT. Electron microscopy demonstrated enlarged mitochondria and endoplasmic reticulum. Despite the fact that previous cases of clear cell variant of SPT arose mainly in the pancreatic body and tail in female young adults (age, 26-32 years), this case suggested that it is possible for a clear cell variant of SPT to arise in the pancreatic head in a middle-aged man. Because the recognition of the clear cell variant of SPT is important for the appropriate diagnosis of primary pancreatic tumor, the present case with its distinctive characteristics may provide new information for a more profound understanding of the pancreatic SPT. (C) 2012 Elsevier Inc. All rights reserved.

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The impact of extent of resection and histological subtype on the outcome of adult patients with high-grade gliomas.

Yamaguchi S, Kobayashi H, Terasaka S, Ishii N, Ikeda J, Kanno H, Nishihara H, Tanaka S, Houkin K

Japanese journal of clinical oncology (4) 42 ( 4 ) 270 - 277 2012.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 03682811

We reviewed the relationship between extent of resection and survival of patients with high-grade gliomas with special consideration of an oligodendroglial component.A retrospective review was performed on 160 adult patients with histological diagnosis of high-grade gliomas since 2000. All histological slides were categorized as high-grade astrocytomas or oligodendroglial tumors. Extent of resection was assessed by early post-operative magnetic resonance imaging and classified as complete resection, incomplete resection and biopsy. Measured outcomes were overall survival and progression-free survival. The independent association of extent of resection and survival was analyzed by the multivariate proportional hazard model adjusting for prognostic factors.The lesions were classified as high-grade astrocytomas in 93 patients and high-grade oligodendroglial tumors in 67 patients. In high-grade astrocytomas, the median survival after complete resection (n 36), incomplete resection (n 36) and biopsy (n 21) was 23.4, 15.3 and 12.6 months, respectively. Complete resection was independently associated with increased overall survival (P 0.001) and progression-free survival (P 0.002) compared with incomplete resection, while incomplete resection was not associated with survival benefit compared with biopsy by multivariate analysis. On the other hand, in high-grade oligodendroglial tumors, the majority of patients were still alive and there is no significant difference in the survival between complete resection (n 24) and incomplete resection (n 33), while even incomplete resection had a significantly longer overall survival (P 0.001) and progression-free survival (P 0.006) compared with biopsy (n 10).Maximal cytoreduction improves the survival of high-grade gliomas, although our data indicated that the impact of extent of resection in high-grade astrocytomas is different from that in high-grade oligodendroglial tumors.

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CRKL plays a pivotal role in tumorigenesis of head and neck squamous cell carcinoma through the regulation of cell adhesion.

Yanagi H, Wang L, Nishihara H, Kimura T, Tanino M, Yanagi T, Fukuda S, Tanaka S

Biochemical and biophysical research communications (1) 418 ( 1 ) 104 - 109 2012.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

The signaling adapter protein CRK is an indispensable molecule involved in regulating the malignant potential of human cancers. CRK-like (CRKL) is a hematopoietic cell-dominant homologue of CRK that is reported to be phosphorylated by BCR-ABL tyrosine kinase in chronic myelogenous leukemia patients, but its biological function in non-hematopoietic tumors remains unclear. In this study, we explored the tumorigenic role of CRKL in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Immunoprecipitation analysis of HNSCC cell line, HSC-3 cells, showed that the dominant binding partner for C3G was CRKL, not CRK. To clarify the molecular function of CRKL, we established lentiviral shRNA-mediated CRKL-knockdown HNSCC cell lines. In CRKL-knockdown HSC-3 and HSC-4 cells, cell growth and motility were diminished compared to control cells. Cell adhesion assays showed that cell attachment onto both fibronectin- and collagen-coated dishes was significantly suppressed in CRKL-knockdown HSC-3 cells, while no significant change was observed for poly-L-lysine-coated dishes. Immunofluorescence staining revealed that focal adhesion was reduced in CRKL-knockdown HSC-3 cells. With a pull-down assay, CRKL-knockdown HSC-3 cells showed decreased amounts of active Rap1 compared to control cells. Moreover, in an in vivo assay, tumor formation of CRKL-knockdown HSC-3 cells in nude mice was significantly abrogated. Our results indicate that CRKL regulates HNSCC-cell growth, motility, and integrin-dependent cell adhesion, suggesting that CRKL plays a principal role in HNSCC tumorigenicity. (C) 2012 Elsevier Inc. All rights reserved.

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Cyclooxygenase-2 inhibition causes antiangiogenic effects on tumor endothelial and vascular progenitor cells.

Muraki C, Ohga N, Hida Y, Nishihara H, Kato Y, Tsuchiya K, Matsuda K, Totsuka Y, Shindoh M, Hida K

International journal of cancer. Journal international du cancer (1) 130 ( 1 ) 59 - 70 2012.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 00207136

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133(+)/vascular endothelial growth factor receptor-2(+) cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs.

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[A case of central nervous system lymphomatoid granulomatosis successfully treated with corticosteroids].

Shimada S, Ueno H, Yamasaki F, Sugiyama K, Yasutomi H, Sekine M, Ohshita T, Kurashige T, Nakamura T, Nishihara H, Yamawaki T, Kurisu K, Matsumoto M

Brain and nerve = Shinkei kenkyu no shinpo (1) 64 ( 1 ) 85 - 87 2012.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 1881-6096

Tuning-up and driving a redox-active rotor

Kume S., Nishihara H.

Chemical Communications (Chemical Communications) 47 ( 1 ) 415 - 417 2011.12

ISSN 13597345

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Co-expression of mesothelin and CA125 correlates with unfavorable patient outcome in pancreatic ductal adenocarcinoma.

Einama T, Kamachi H, Nishihara H, Homma S, Kanno H, Takahashi K, Sasaki A, Tahara M, Okada K, Muraoka S, Kamiyama T, Matsuno Y, Ozaki M, Todo S

Pancreas (8) 40 ( 8 ) 1276 - 1282 2011.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 08853177

Objectives: Recent studies have shown that the high affinity of mesothelin-CA125 interaction might cause intracavitary tumor metastasis. We examined the clinicopathologic significance and prognostic implication of mesothelin and CA125 expression in pancreatic ductal adenocarcinoma.
Methods: Tissue samples from 66 pancreatic ductal adenocarcinomas were immunohistochemically examined. Proportion and intensity of constituent tumor cells with mesothelin and CA125 expression were analyzed and classified as high-level expression, defined as expression by more than 50% of tumor cells and/or moderate to strong staining, or low-level expression otherwise.
Results: A high level of mesothelin was correlated with a higher histological grade (P = 0.049) and the level of blood vessel permeation (P = 0.0006), whereas a high level of CA125 expression was correlated with a higher recurrence rate (P = 0.015). The expression of mesothelin was strongly correlated with that of CA125 (P = 0.0041). Co-expression of mesothelin and CA125 were associated with an unfavorable patient outcome (P = 0.0062).
Conclusions: This is the first report showing that co-expression of mesothelin and CA125 were in pancreatic ductal adenocarcinoma, and such co-expression is associated with a poor prognosis. Our finding suggests that co-expression of these two factors plays a significant role in the acquisition of aggressive clinical behavior.

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A case of lymphoplasmacyte-rich meningioma of the jugular foramen.

Kanno H, Nishihara H, Hara K, Ozaki Y, Itoh T, Kimura T, Tanino M, Tanaka S

Brain tumor pathology (4) 28 ( 4 ) 341 - 345 2011.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 14337398

Lymphoplasmacyte-rich meningioma (LPRM), the most rare variant of meningiomas, features extensive lymphoplasmacytic infiltrates. Although the jugular foramen (JF) is occasionally involved by several types of tumor, such as paragangliomas and schwannomas, meningioma in the JF is an infrequent disease. Here we present an extremely rare case of LPRM found in the JF. A 55-year-old woman complained of paresis in her right eyelid and palsy in the right side of her lip. Hoarseness and dysphagia also occurred in the following month. Radiologic examinations disclosed a mass lesion in the right JF, and the tumor was operatively removed. Microscopically, the tumor was composed of extensive lymphoplasmacytic infiltration with mild vascular proliferation and scattered sheets of epithelioid cells with plump cytoplasm. Although the obvious whorl formation or psammoma bodies were not observed, by immunochemistry the epithelioid cells were positive for epithelial membrane antigen and also progesterone receptor, indicating a meningothelial cell origin. Considering the histological and radiologic findings, we finally diagnosed the case as LPRM, making this the second reported case of LPRM in the JF.

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A population of BJ fibroblasts escaped from Ras-induced senescence susceptible to transformation.

Kohsaka S, Sasai K, Takahashi K, Akagi T, Tanino M, Kimura T, Nishihara H, Tanaka S

Biochemical and biophysical research communications (4) 410 ( 4 ) 878 - 884 2011.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

Oncogenic stimuli such as H-Ras induce oncogene-induced senescence (OIS) in fibroblasts to protect against transformation. Here we found that a population of the human diploid fibroblasts can escape from OIS induced by H-RasV12. We designated these OIS-escaped cells as OISEC (OIS-escaped cells). OISEC lost the expression of p16 which plays an important role for cell cycle arrest for induction of senescence, but OISEC preserved the p16 expression machinery and exhibited senescence by the treatment with hydrogen peroxide (H(2)O(2)) as stress-induced premature senescence (SIPS). OISEC did not possess anchorage-independent growth potential, and functional disruption of p53 and Rb by SV40 early region encoding large T and small t antigens, induced the aneuploidy phenotype and colony-forming potential of OISEC together with the exhibition of in vivo tumor formation. Finally, we also found that the distinctive feature of OISEC is expression of transcription factors, Oct3/4, SOX2, and Nanog which is closely related to stem-like cell features. This study highlights the presence of a cell population which escaped from OIS, and this OISEC may transform into malignant cancer cells by the additional hits of several genes in vivo. (C) 2011 Elsevier Inc. All rights reserved.

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A uniform bimetallic rhodium/iron nanoparticle catalyst for the hydrogenation of olefins and nitroarenes

Nakamula I, Yamanoi Y, Imaoka T, Yamamoto K, Nishihara H

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition) 50 ( 26 ) 5830 - 5833 2011.06

Joint Work, ISSN 14337851

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High grade meningiomaの治療成績とMib-1 indexの関係の検討

成田拓人, 菅野宏美, 湯澤明夏, 寺坂俊介, 小林浩之, 村田純一, 山口秀, 西原広史, 宝金清博, 田中伸哉

Brain Tumor Pathology (日本脳腫瘍病理学会) 28 ( Suppl. ) 092 - 092 2011.05

ISSN 1433-7398

Case of atypical teratoid/rhabdoid tumor in an adult, with long survival.

Takahashi K, Nishihara H, Katoh M, Yoshinaga T, Mahabir R, Kanno H, Kimura T, Tanino M, Ikeda J, Sawamura Y, Nagashima K, Tanaka S

Brain tumor pathology (1) 28 ( 1 ) 71 - 76 2011.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 14337398

Atypical teratoid/rhabdoid tumor (AT/RT) is a malignant tumor that mostly occurs in early childhood and has poor prognosis despite aggressive therapy. Adult cases are rare and, as far as we are aware, only 30 cases have been reported to date. Here we present the case of a 27-year-old female with left parietal AT/RT with the chief complaint of numbness of the right superior limb. First, the tumor was surgically removed and the diagnosis was grade II glioma. With additional radiotherapy, the clinical course after surgery was favorable. After 6 years, she had an operation for recurrence and the diagnosis was grade III glioma. Temozolomide was prescribed, and a disease-free period of 2 years followed. Surgery was performed for a third time for second recurrence with histology of diffuse growth of rhabdoid cells. Immunohistochemistry was partially positive for vimentin and epithelial membrane antigen. Ki-67 labeling index was extremely high and tumor cells showed no staining of INI1 suggestive of diagnosis of AT/RT. We re-evaluated past specimens and none had immunoreactivity of INI1. Ki-67 labeling index and O-6 methylguanine DNA methyltransferase (MGMT) staining were also re-examined and both increased gradually. She is still alive without recurrence for more than 1 year. As far as we are aware, this is the second longest survival of an adult with AT/RT.

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Clinicopathologic application of lectin histochemistry: bisecting GlcNAc in glioblastoma.

Aoyanagi E, Sasai K, Nodagashira M, Wang L, Nishihara H, Ihara H, Ikeda Y, Tanaka S

Applied immunohistochemistry & molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry (6) 18 ( 6 ) 518 - 525 2010.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 15412016

Glycosylation is one of the most common posttranslational modifications and changes in oligosaccharide structures are associated with many human diseases including a number of cancers. Thus, discovering aberrant glycosylation patterns that serve as markers for brain tumor progression and metastasis represents an attractive strategy to improve clinicopathologic diagnosis and to provide aids to the development of novel therapies. To identify glioblastoma (GBM) cells expressing glycoproteins that contain high levels of the bisecting N-acetylglucosamine (GlcNAc) structures, lectin histochemistry was carried out using erythroagglutinating phytohemagglutinin. Although GBM frequently expressed the bisecting GlcNAc, the lectin reactivity varied among tumor regions within individual specimens. Since detailed histopathologic analysis revealed that oligosaccharides with bisecting GlcNAc structures were preferably expressed in tumor regions with low KI67 immunopositivity, immunodetection of the bisecting GlcNAc could be useful to indicate less proliferative regions in human GBM. Our study highlights the potential use of lectin histochemistry to develop new methods for diagnosis that would improve future antiglioma therapy.

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Intraductal papillary mucinous neoplasms(IPMNs)におけるMesothelinとCA125の発現様式の検討

永生高広, 蒲池浩文, 西原広史, 本間重紀, 佐々木彩実, 田原宗徳, 佐藤正法, 三野和宏, 岡田邦明, 近藤征文, 神山俊哉, 松野吉宏, 藤堂省

日本消化器外科学会雑誌 ((一社)日本消化器外科学会) 43 ( Suppl.2 ) 366 - 366 2010.10

ISSN 0386-9768

バイオマーカーによる診断、その他個別化病理診断の基盤作成胃癌におけるシグナル伝達分子のImmunoprofilingの検討(Diagnosis by biomaker, others Immunoprofiling of signaling molecule for gastric cancer: Pathological basis for Taylor made medicine)

西原広史, 菅野宏美, 石川麻倫, 大場彩音, 田中伸哉

日本癌学会総会記事 ((一社)日本癌学会) 69回 232 - 233 2010.08

ISSN 0546-0476

Self-improvement of keratinocyte differentiation defects during skin maturation in ABCA12-deficient harlequin ichthyosis model mice.

Yanagi T, Akiyama M, Nishihara H, Ishikawa J, Sakai K, Miyamura Y, Naoe A, Kitahara T, Tanaka S, Shimizu H

The American journal of pathology (1) 177 ( 1 ) 106 - 118 2010.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0002-9440

Harlequin ichthyosis (HI) is caused by loss-of-function mutations in the keratinocyte lipid transporter ABCA12. The patients often die in the first 1 or 2 weeks of life, although HI survivors' phenotypes improve within several weeks after birth. In order to clarify the mechanisms of phenotypic recovery, we studied grafted skin and keratinocytes from Abca12-disrupted (Abca12(-/-)) mice showing abnormal lipid transport. Abca12(-1-) neonatal epidermis showed significantly reduced total ceramide amounts and aberrant ceramide composition. Immunofluorescence and immunoblotting of Abca12(-1-) neonatal epidermis revealed defective profilaggrin/filaggrin conversion and reduced protein expression of the differentiation-specific molecules, loricrin, kallikrein 5, and transglutaminase 1, although their mRNA expression was up-regulated. In contrast, Abca12(-1-) skin grafts kept in a dry environment exhibited dramatic improvements in all these abnormalities. Increased transepidermal water loss, a parameter representing barrier defect, was remarkably decreased in grafted Abca12(-1-) skin. Ten-passage sub-cultured Abca12(-/-)keratinocytes showed restoration of intact ceramide distribution, differentiation-specific protein expression and profilaggrin/filaggrin conversion, which were defective in primary-cultures. Using cDNA microarray analysis, lipid transporters including four ATP-binding cassette transporters were up-regulated after sub-culture of Abca12(-/-) keratinocytes compared with primary-culture. These results indicate that disrupted keratinocyte differentiation during the fetal development is involved in the pathomechanism of HI and, during maturation, Abca12(-/-) epidermal keratinocytes regain normal differentiation processes. This restoration may account for the skin phenotype improvement observed in HI survivors. (Am J Pathol 2010, 177:106-118; DOI: 10.2353/ajpath.2010.091120)

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Controlled storage of ferrocene derivatives as redox-active molecules in dendrimers.

Ochi Y, Suzuki M, Imaoka T, Murata M, Nishihara H, Einaga Y, Yamamoto K

Journal of the American Chemical Society 132 ( 14 ) 5061 - 9 2010.04

Accepted, ISSN 0002-7863

Dendritic polyphenylazomethines (DPA) could encapsulate ferroceniums by complexation of the electron-donating skeleton of the DPA imines. Upon addition of ferroceniums to a series of dendritic polyphenylazomethines (DPAGX, where X is the generation number, X = 1-4), the UV-vis spectra showed changes in a manner similar to that observed for the complexation of metal ions with DPAGX. Stepwise shifts in the isosbestic point were consistently observed with the number of imine groups in the first and second layers of the generation-4 dendrimer (DPAG4). DPAG2 and DPAG3 were also found to trap 6 equiv of ferroceniums. To investigate the complexation, UV-vis spectroscopy, 57Fe MoÌssbauer spectroscopy, electrospray ionization-mass spectroscopy (ESI-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were performed. We confirmed that neutral ferrocenes cannot complex with the imine group while ferroceniums can. Utilizing the redox property of ferrocenes, we were able to electrochemically control the encapsulation and release of ferrocenes into the DPA in a manner similar to redox-responsive proteins such as ferritin. In addition to ferrocenes, oligoferrocenes could also be trapped in the DPA. The biferrocene cation(1+) was particularly suitable for electrochemical switching due to its stable mixed valence condition. The terferrocene dication(2+) encapsulated into DPAG4 could be fabricated into a thin film, which exhibited the near-infrared absorption of an intervalence charge-transfer (IV-CT) band, pointing the way toward the use of such systems in material science. © 2010 American Chemical Society.

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DOCK2 regulates cell proliferation through Rac and ERK activation in B cell lymphoma.

Wang L, Nishihara H, Kimura T, Kato Y, Tanino M, Nishio M, Obara M, Endo T, Koike T, Tanaka S

Biochemical and biophysical research communications (1) 395 ( 1 ) 111 - 115 2010.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

DOCK2; a member of the CDM protein family, regulates cell motility and cytokine production through the activation of Rac in mammalian hematopoietic cells and plays a pivotal role in the modulation of the immune system. Here we demonstrated the alternative function of DOCK2 in hematopoietic tumor cells, especially in terms of its association with the tumor progression.
Immunostaining for DOCK2 in 20 cases of human B cell lymphoma tissue specimens including diffuse large B cell lymphoma and follicular lymphoma revealed the prominent expression of DOCK2 in all of the lymphoma cells. DOCK2-knockdown (KD) of the B cell lymphoma cell lines, Ramos and Raji, using the lentiviral shRNA system presented decreased cell proliferation compared to the control cells. Furthermore, the tumor formation of DOCK2-KD Ramos cell in nude mice was significantly abrogated. Western blotting analysis and pull-down assay using GST-PAK-RBD kimeric protein suggested the presence of DOCK2-Rac-ERK pathway regulating the cell proliferation of these lymphoma cells. This is the first report to clarify the prominent role of DOCK2 in hematopoietic malignancy. (C) 2010 Elsevier Inc. All rights reserved.

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Analysis of an alternative human CD133 promoter reveals the implication of Ras/ERK pathway in tumor stem-like hallmarks.

Tabu K, Kimura T, Sasai K, Wang L, Bizen N, Nishihara H, Taga T, Tanaka S

Molecular cancer (BIOMED CENTRAL LTD) 9 39 2010.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 1476-4598

Background: An increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. To reveal the molecular machinery underlying the stem-like features in tumor cells, we analyzed a promoter of CD133 gene using human colon carcinoma Caco-2 and synovial sarcoma Fuji cells, which endogenously express CD133 gene.
Results: A reporter analysis revealed that P5 promoter, located far upstream in a human CD133 gene locus, exhibits the highest activity among the five putative promoters (P1 to P5). Deletion and mutation analysis identified two ETS binding sites in the P5 region as being essential for its promoter activity. Electrophoretic mobility shift assays demonstrated the specific binding between nuclear factors and the ETS binding sequence. Overexpression of dominant-negative forms of Ets2 and Elk1 resulted in the significant decrease of P5 activity. Furthermore, treatment of Fuji cells with a specific MEK/ERK inhibitor, U0126, also markedly decreased CD133 expression, but there was no significant effect in Caco-2 cells, suggesting cell type-specific regulation of CD133 expression. Instead, the side population, another hallmark of TSLCs, was dramatically diminished in Caco-2 cells by U0126. Finally, Ras-mediated oncogenic transformation in normal human astrocytes conferred the stem-like capability to form neurosphere-like colonies with the increase of CD133 mRNA expression.
Conclusions: In conclusion, the Ras/ERK pathway at least in part contributes to the maintenance and the acquisition of stem-like hallmarks, although the extent of its contribution is varied in a cell type-specific manner. These findings could help our comprehensive understanding of tumor stemness, and also improve the development of eradicative therapies against human malignancies.

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Sudden death of a patient with pandemic influenza (A/H1N1pdm) virus infection by acute respiratory distress syndrome.

Takiyama A, Wang L, Tanino M, Kimura T, Kawagishi N, Kunieda Y, Katano H, Nakajima N, Hasegawa H, Takagi T, Nishihara H, Sata T, Tanaka S

Japanese journal of infectious diseases (1) 63 ( 1 ) 72 - 74 2010.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 1344-6304

We describe an autopsy case of a patient with pandemic influenza (A/H1N1pdm) virus infection in Japan, who developed rapidly progressive viral pneumonia exhibiting diffuse alveolar damage. A 41-year-old female visited our hospital with a fever of 38.7 degrees C. She was a public health nurse with no underlying disease and had had contact with a group of elementary school students who had been infected with the influenza (A/H1N1pdm) virus I week earlier. She was prescribed oseltamivir and returned to the hotel where she was staying alone. The next day, she was found dead in her hotel room. At autopsy, both lungs were voluminous and microscopic examination revealed acute-stage, severe diffuse alveolar damage with remarkable mononuclear cell infiltration and hyaline membrane formation in the lungs. CD8-positive T lymphocytes were dominantly observed. Immunohistochemically, influenza A viral protein was confirmed in the damaged type 11 pneumocytes and also in the infiltrated macrophages. Real-time RT-PCR analysis of both pre- and post-mortem pharyngeal swabs confirmed a novel influenza (A/H1N1pdm) virus infection. This is the second autopsy case of influenza (A/H1N1pdm) virus infection in Japan, and the findings indicated that the patient died due to an exceptionally rapid progression of viral pneumonia. This case indicates that patients with influenza (A/H1N1pdm) virus infection should be carefully monitor for acute respiratory distress syndrome.

Treating "intractable" tumours using nanoDDS and TGF-beta inhibitor

Kano M.R, Nishihara H, Kataoka K, Miyazono K

European Cells and Materials 20 ( SUPPL.3 ) 2010

Joint Work, Accepted

A case of central nervous system lymphomatoid granulomatosis; characteristics of PET imaging and pathological findings.

Nishihara H, Nakasato M, Sawa H, Murakami H, Yamamoto D, Moriyama K, Kato N, Hashimoto I, Kamada H, Tanaka S

Journal of neuro-oncology (2) 93 ( 2 ) 275 - 278 2009.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0167-594X

Lymphomatoid granulomatosis (LYG) in the central nervous system (CNS) is an uncommon lymphoproliferative disorder with low grade malignant potential. Here we report a case of CNS-LYG, in particular, its characteristics of radioisotope imaging and pathological findings. A 65-year-old man complained of visual disturbance and homonymous hemianopsia was designated. CT and MRI revealed an edematous, enhanced irregular and nodular lesion in the right occipital and parietal lobes. Although 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) scan showed low uptake in the lesion, Methionine (MET)-PET scan indicated high uptake. Proton magnetic resonance spectroscopy ((1)H-MRS) at 3T revealed a decrease of the peak of the N-acetylaspartate (NAA), suggesting a possible neoplastic lesion. The patient was diagnosed with CNS-LYG based on the surgically removed material showing perivascular infiltration of CD3-positive small T-lymphocytes with granulomatous lesions. The post-operative steroid therapy was effective and the recurrence or exacerbation has not been observed by radiological imaging until now.

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Human synovial sarcoma proto-oncogene Syt is essential for early embryonic development through the regulation of cell migration.

Kimura T, Sakai M, Tabu K, Wang L, Tsunematsu R, Tsuda M, Sawa H, Nagashima K, Nishihara H, Hatakeyama S, Nakayama K, Ladanyi M, Tanaka S, Nakayama KI

Laboratory investigation; a journal of technical methods and pathology (6) 89 ( 6 ) 645 - 656 2009.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0023-6837

SYT-SSX protein, resulted from chromosomal translocation, causes synovial sarcoma, which is a malignant tumor accounting for 10% of soft tissue sarcoma. However, biological functions of SYT (synovial sarcoma translocation), also known as SS18, are largely unclear, whereas it has been proven that Syt-null mice die at early stages of embryonic development. Here, we generated Syt-deficient mice and confirmed the reported phenotypes, including growth retardation, open neural tube and haplo-insufficient lethality, and therefore, there is no doubt that Syt is essential for embryonic development. However, placental defects, described in the earlier report, were rarely seen in our mice and we frequently observed cardiac defect in Syt-deficient mice. As the mechanisms responsible for embryonic lethality seem to be complicate, we performed additional experiments. By using primary cultured embryonic fibroblasts, we showed that Syt(-/-) MEFs deregulate actin organization and suppressed cell migration. These observations suggest that Syt may contribute to the signaling pathway important for various cellular functions in vivo and in vitro, and we propose that Syt-deficient MEFs would be a powerful means to understand the biological roles of SYT in vitro. Laboratory Investigation (2009) 89, 645-656; doi:10.1038/labinvest.2009.25; published online 30 March 2009

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Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration.

Watanabe T, Tsuda M, Makino Y, Konstantinou T, Nishihara H, Majima T, Minami A, Feller SM, Tanaka S

Cell research (5) 19 ( 5 ) 638 - 650 2009.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 1001-0602

Upon growth factor stimulation, the scaffold protein, Gab1, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gab1. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of CrkII, is critical for the induction of Gab1-Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gab1, whereas Crk-SH3(N) interacted with the Gab1 mutant, which lacks the clustered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gab1 was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gab1. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gab1 with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gab1-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gab1-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphorylation of Gab1-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

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Colonic metastasis from primary carcinoma of the lung: Report of a case and review of Japanese literature

Hiromi Ono, Mihiro Okabe, Takashi Kimura, Masato Kawakami, Kenji Nakamura, Yasushi Danjo, Hidero Takasugi, Hiroshi Nishihara

Clinical Journal of Gastroenterology 2 ( 2 ) 89 - 95 2009.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 1865-7257

We report a rare case of colonic metastasis from primary carcinoma of the lung. A 59-year-old man who underwent pulmonary surgery for lung cancer was referred to our hospital in June 2007. The patient complained of abdominal pain, and barium enema examination at another hospital had demonstrated a descending colon tumor. Postoperative histopathological and immunohistochemical findings indicated that the tumor was a colonic metastasis of lung cancer. Three months postoperatively, the cancer had metastasized to the brain, and the patient underwent radiotherapy. He survived for more than 1 year after colonic surgery. Clinically apparent metastases from lung cancer to the colon are rare, and in the 50 Japanese cases retrospectively investigated here, the prognosis was poor. © 2008 Springer.

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CNS lymphomatoid granulomatosis with lymph node and bone marrow involvements.

Takiyama A, Nishihara H, Tateishi U, Kimura T, Wang L, Marukawa K, Itoh T, Hashino S, Nagashima K, Tanaka S

Neuropathology : official journal of the Japanese Society of Neuropathology (6) 28 ( 6 ) 640 - 644 2008.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0919-6544

Lymphomatoid granulomatosis (LYG) in the CNS is an uncommon lymphoproliferative disease with characteristic angiocentric lymphoreticular proliferative and granulomatous lesions exhibiting low-grade malignant potential. Here we report a rare case of CNS-LYG, which disseminated to the lymph node and bone marrow. A 50-year-old man was diagnosed with CNS-LYG based on brain biopsy showing perivascular infiltration of CD3-positive small T-lymphocytes without overt nuclear atypism. Eight months after the initial neurological symptoms, inguinal lymph node swelling was found and histopathologically diagnosed as peripheral T-cell lymphoma. TCR gamma-gene rearrangement study using both paraffin-embedded specimens of brain and inguinal lymph node demonstrated an identical clonal band. Considering the clinical course, we concluded lymph node involvement of CNS-LYG, suggesting the malignant potential of CNS-LYG.

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Harlequin ichthyosis model mouse reveals alveolar collapse and severe fetal skin barrier defects.

Yanagi T, Akiyama M, Nishihara H, Sakai K, Nishie W, Tanaka S, Shimizu H

Human molecular genetics (19) 17 ( 19 ) 3075 - 3083 2008.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0964-6906

Harlequin ichthyosis (HI), which is the most severe genodermatosis, is caused by loss-of-function mutations in ABCA12, a member of the ATP-binding cassette transporter family. To investigate the pathomechanism of HI and the function of the ABCA12 protein, we generated ABCA12-deficient mice (Abca12(-/-)) by targeting Abca12. Abca12(-/-) mice closely reproduce the human HI phenotype, showing marked hyperkeratosis with eclabium and skin fissure. Lamellar granule abnormalities and defective ceramide distribution were remarkable in the epidermis. Skin permeability assay of Abca12(-/-) fetuses revealed severe skin barrier dysfunction after the initiation of keratinization. Surprisingly, the Abca12(-/-) mice also demonstrated lung alveolar collapse immediately after birth. Lamellar bodies in alveolar type II cells of the Abca12(-/-) mice lacked normal lamellar structures. The level of surfactant protein B, an essential component of alveolar surfactant, was reduced in the Abca12(-/-) mice. Fetal therapeutic trials with systemic administration of retinoid or dexamethasone, which are effective for HI and respiratory distress, respectively, to the pregnant mother mice neither improved the skin phenotype nor extended the survival period. Our HI model mice reproduce the human HI skin phenotype soon after the initiation of fetal skin keratinization and provide evidence that ABCA12 plays pivotal roles in lung and skin barrier functions.

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Promoter hypomethylation regulates CD133 expression in human gliomas.

Tabu K, Sasai K, Kimura T, Wang L, Aoyanagi E, Kohsaka S, Tanino M, Nishihara H, Tanaka S

Cell research (10) 18 ( 10 ) 1037 - 1046 2008.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 1001-0602

Brain tumor-initiating cells (BTICs) have been enriched using antibodies against the cell surface protein CD133; however, the biological relevance and the regulatory mechanism of CD133 expression in human gliomas are not yet understood. In this study, we initially demonstrated that CD133 was overexpressed in high-grade human glioblastomas where CD133-positive cells were focally observed as a micro-cluster. In addition, CD133 transcripts with exon 1A, 1B, or 1C were predominantly expressed in glioblastomas. To elucidate the mechanism regulating this aberrant expression of CD133, three proximal promoters (P1, P2, and P3) containing a CpG island were isolated. In U251MG and T98G glioblastoma cells, the P1 region flanking exon 1A exhibited the highest activity among the three promoters, and this activity was significantly inactivated by in vitro methylation. After treatment with the demethylating agent 5-azacytidine and/or the histone deacetylase inhibitor valproic acid, the expression level of CD133 mRNA was significantly restored in glioma cells. Importantly, hypomethylation of CpG sites within the P1, P2, and P3 regions was observed by bisulfite sequencing in human glioblastoma tissues with abundant CD133 mRNA. Taken together, our results indicate that DNA hypomethylation is an important determinant of CD133 expression in glioblastomas, and this epigenetic event may be associated with the development of BTICs expressing CD133.

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Hypopharyngeal squamous cell carcinoma producing both granulocyte colony-stimulating factor and parathyroid hormone-related protein.

Tamura K, Yoshinaga T, Tanino M, Kimura T, Yamada N, Nishimura M, Fukuda S, Nishihara H, Shindoh M, Tanaka S

Pathology international (10) 58 ( 10 ) 652 - 656 2008.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 1320-5463

A 57-year-old woman was admitted to Hokkaido University Hospital because of dysphagia. Laryngoscopy indicated hypopharyngeal tumor histologically diagnosed as squamous cell carcinoma (SCC). A combination of radiotherapy and chemotherapy was performed for 2 months, and the hypopharyngeal lesion completely regressed. After 4 months, fever, anorexia, and malaise appeared, and chest X-ray and CT indicated two large tumors in the right lung. Transbroncheal lung biopsy (TBLB) specimens were diagnosed as SCC. Laboratory data showed high levels of serum granulocyte colony-stimulating factor (G-CSF) and parathyroid hormone-related protein (PTHrP). Subsequently, positron emission tomography (PET) showed multiple metastases to several organs including the liver, spine, skull, and thigh. Two months after readmission, the patient died with no success of chemotherapy. At autopsy, the lung tumor was clearly positive for both G-CSF and PTHrP on immunohistostaining. Retrospectively, examination showed that the primary pharyngeal tumor was focally positive for these two cytokines. Thus, a certain population of hypopharyngeal cancer producing G-CSF and PTHrP, spread to various organs and contributed to the rapid progression and poor prognosis. This case is presented with a discussion of several other cases in the literature.

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Careful exclusion of non-neoplastic brain components is required for an appropriate evaluation of O6-methylguanine-DNA methyltransferase status in glioma: relationship between immunohistochemistry and methylation analysis.

Sasai K, Nodagashira M, Nishihara H, Aoyanagi E, Wang L, Katoh M, Murata J, Ozaki Y, Ito T, Fujimoto S, Kaneko S, Nagashima K, Tanaka S

The American journal of surgical pathology (8) 32 ( 8 ) 1220 - 1227 2008.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0147-5185

Evaluation of O-6-methylguanine-DNA methyltransferase (MGMT) expression is important for antiglioma therapy as many clinical trials have demonstrated that promoter hypermethylation and low level expression of MGMT are associated with an enhanced response to alkylating agents. However, here we report that the current strategies used to evaluate MGMT status in gliomas are unreliable. We observed discordance in the MGMT expression status when immunohistochemical evaluation and polymerase chain reaction-based methylation assessments were used: 73% of gliomas with methylated MGMT promoter had substantial numbers of MGMT-immunopositive tumor cells. Furthermore, when MGMT expression was tested in tumor homogenates using reverse transcription-polymerase chain reaction, 43% of tumors were found positive, in comparison to only 24%, when histologic samples were assayed immunohistochemically. To explain these inconsistencies we undertook a detailed immunohistochemical evaluation of tumor samples and found that some gliomas demonstrated remarkably high expression of MGMT in the entire tumor whereas others contained only a small immunopositive area. Additionally, we found that gliomas contained various types of non-neoplastic cells expressing MGMT, including lymphocytes, vascular endothelial cells, and macrophages/microglias, which contribute to overall MGMT expression detected in tumor homogenates, and thus result in overestimation of tumor NIGMT expression. Therefore, to correctly establish MGMT expression in the tumor, which could be informative of glioma sensitivity to alkylating agents, exclusion of non-neoplastic brain components from analysis is required.

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A case of cerebral ganglioneuronal tumor in the parietal lobe of an adult.

Nishihara H, Ozaki Y, Ito T, Yoshinaga T, Tabu K, Tanino M, Nagashima K, Tanaka S

Brain tumor pathology (1) 25 ( 1 ) 45 - 49 2008

Research paper (scientific journal), Joint Work, Accepted, ISSN 1433-7398

Central nervous system (CNS) neuroblastoma/ganglioneuroblastoma is one of the embryonal tumors with neuronal differentiation found in young adults, but it is most common in children, especially in those below the age of 5 years, whereas extraventricular neurocytoma, a rare neuroepithelial tumor with neuronal differentiation, mostly affects young adults. Here we present a rare case of cerebral ganglioneuronal tumor that occurred in a 32-year-old woman. The patient suffered from tonic convulsion, and computed tomography demonstrated a well-demarcated, round tumor 3.3 cm in size with marked calcification in the right parietal lobe. Histological analysis revealed diffuse infiltration of small, round cells with scattered large ganglion-like cells. Immunohistochemically, the tumor cells did not react with any neuronal molecules, except for chromogranin A in ganglion-like large tumor cells, but electron microscopy demonstrated the presence of synapse-like nerve terminal structures without mature postsynaptic density, suggesting the presence of neoplastic tumor components with neuronal differentiation; thus, this tumor was diagnosed as CNS ganglioneuroblastoma with possible low-grade malignancy because the Mib-1 labeling index was less than 3%-4%. Here we discuss the histological entity of cerebral ganglioneuronal tumors, including extraventricular neurocytoma.

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Signaling adaptor protein Crk is indispensable for malignant feature of glioblastoma cell line KMG4.

Wang L, Tabu K, Kimura T, Tsuda M, Linghu H, Tanino M, Kaneko S, Nishihara H, Tanaka S

Biochemical and biophysical research communications (4) 362 ( 4 ) 976 - 981 2007.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

Signaling adaptor protein Crk has been shown to be involved in pathogenesis of human cancers including brain tumor where Crk was reported to be overexpressed. In this study, we addressed whether Crk is indispensable for malignant phenotype of brain tumor. In 20 surgical specimens of glioma, mRNA of both CrkI and CrkII was found to be elevated in malignant tumor. To define a precise role of Crk, we have established Crk-knockdown cell lines of glioblastoma KMG4 by siRNA, and early phase of cell adhesion to laminin was found to be suppressed. Wound healing assay revealed the decreased cell motility in Crk knockdown cells, and suppression of both anchorage-dependent and -independent growth were demonstrated in these cells. Furthermore, in vivo tumor forming potential was also markedly suppressed. These results suggest that Crk is required for early attachment to laminin, cell motility, and growth of glioblastoma cell line KMG4. (C) 2007 Elsevier Inc. All rights reserved.

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Immunohistochemical and gene rearrangement studies of central nervous system lymphomatoid granulomatosis.

Nishihara H, Tateishi U, Itoh T, Nagashima K, Tanaka S

Neuropathology : official journal of the Japanese Society of Neuropathology (5) 27 ( 5 ) 413 - 418 2007.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0919-6544

Lymphomatoid granulomatosis (LYG) is a rare multisystem disorder with characteristic angiocentric lymphoproliferative features, most frequently involving the lung, skin, and rarely the CNS. LYG has been classified into three subtypes based on the relative proportions of atypical and inflammatory infiltrating cells. Most systemic LYGs have been shown to be EBV-associated, T-cell rich, B-cell proliferative disorders. Here, we present four cases of LYG arising from the CNS and have analyzed them by immunohistochemistry to assess the phenotype of the infiltrate, and by PCR-SSCP (single-strand conformation polymorphism) analysis for immunoglobulin heavy chain (IgH) and T-cell receptor (TcR) gamma gene rearrangements. Three cases revealed perivascular infiltration of T-cell dominant lymphoid cells, two cases showed monoclonal TcR gamma gene rearrangement, while the remaining case had a B-cell immunophenotype and monoclonal IgH gene rearrangement with EBV genome expression. This is the first report of a gene rearrangement study on CNS-LYG. We confirm that some cases of CNS-LYG are derived from T-cell monoclonal lymphoproliferative disease, although this disease should be classified as a borderline malignancy and should be separated from overt malignant lymphoma of CNS.

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Type I interferon prolongs cell cycle progression via p21WAF1/CIP1 induction in human colon cancer cells.

Katayama T, Nakanishi K, Nishihara H, Kamiyama N, Nakagawa T, Kamiyama T, Iseki K, Tanaka S, Todo S

International journal of oncology (3) 31 ( 3 ) 613 - 620 2007.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 1019-6439

Type I interferon (IFN) was originally identified as an immunomodulatory cytokine because of its antiviral activity. Further characterization of its biological effects revealed a prominent role in the direct control of cell growth and potent immunomodulatory and antiangiogenic actions. IFN-alpha and IFN-SS had both been classified as type I IFN, but differences in their antitumor activities were reported. We confirmed the difference in the antiproliferative activities of IFN-alpha 2b and IFN-SS toward HT29 and SW480 cells. IFN treatment was observed to prolong cell cycle progression; in particular, the accumulation of S-phase population was one of the most characteristic changes. The prolongation of S-phase progression and transition into G2/M-phase was suggested to be a crucial action of type I IFN on colon cancer. Additionally, IFN activated the p21 promoter gene and induced p21(WAFl/CIP1) expression. Furthermore, the cell cycle prolongation effect of IFN was suppressed when p21 expression was downregulated. Therefore, we confirmed that p21(WAFl/CIP1) was a crucial target molecule for the effects of IFN on the cell cycle. Additionally, the ability of p21 induction differed between IFN-alpha 2b and IFN-SS and correlated with their inhibitory activities toward cell growth. We conclude that type I IFN prolongs cell cycle progression by p21(WAFl/CIP1) induction in human colon cancer cells.

Crk associates with ERM proteins and promotes cell motility toward hyaluronic acid.

Tsuda M, Makino Y, Iwahara T, Nishihara H, Sawa H, Nagashima K, Hanafusa H, Tanaka S

The Journal of biological chemistry (45) 279 ( 45 ) 46843 - 46850 2004.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

Cell migration is a well organized process regulated by the extracellular matrix-mediated cytoskeletal reorganization. The signaling adaptor protein Crk has been shown to regulate cell motility, but its precise role is still under investigation. Herein, we report that Crk associates with ERM family proteins ( including ezrin, radixin, and moesin), activates RhoA, and promotes cell motility toward hyaluronic acid. The binding of Crk with ERMs was demonstrated both by transient and stable protein expression systems in 293T cells and 3Y1 cells, and it was shown that v-Crk translocated the phosphorylated form of ERMs to microvilli in 3Y1 cells by immunofluorescence and immunoelectron microscopy. This v-Crk-dependent formation of microvilli was suppressed by inhibitors of Rho-associated kinase, and the activity of RhoA was elevated by coexpression of c-Crk-II and ERMs in 3Y1 cells. In concert with the activation of RhoA by Crk, Crk was found to associate with Rho-GDI, which has been shown to bind to ERMs. Furthermore, upon hyaluronic acid treatment, coexpression of c-Crk-II and ERMs enhanced cell motility, whereas the sole expression of c-Crk-II or either of the ERMs decreased the motility of 3Y1 cells. These results suggest that Crk may be involved in regulation of cell motility by a hyaluronic acid-dependent mechanism through an association with ERMs.

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Cyclic AMP promotes cAMP-responsive element-binding protein-dependent induction of cellular inhibitor of apoptosis protein-2 and suppresses apoptosis of colon cancer cells through ERK1/2 and p38 MAPK.

Nishihara H, Hwang M, Kizaka-Kondoh S, Eckmann L, Insel PA

The Journal of biological chemistry (25) 279 ( 25 ) 26176 - 26183 2004.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

We recently reported that cAMP suppresses apoptosis in colon cancer cells and induces cellular inhibitor of apoptosis protein-2 (c-IAP2) via a cAMP-responsive element (CRE), suggesting a mechanism for chemoprevention of colon cancer by non-steroidal anti-inflammatory drugs. In this study, we used T84 human colon cancer cells to define the pathway by which increases in cAMP induce c-IAP2 expression. Treatment with several different cAMP agonists stimulated phosphorylation of CRE-binding protein (CREB) and activated expression of c-IAP2 in a CREB-dependent manner. Studies with pharmacological inhibitors revealed that cAMP-dependent phosphorylation of CREB required activation of ERK1/2 and p38 MAPK but was largely independent of protein kinase A. Immunoblots and transcriptional reporter assays using specific inhibitors, as well as expression of constitutively active forms of MEK1 and MKK3, showed that c-IAP2 induction by cAMP is regulated predominantly through ERK1/2 and p38 MAPK and suggested involvement of p90 ribosomal protein S6 kinase and mitogen and stress response kinase-1 as well. Consistent with those results, we found that cAMP-dependent suppression of apoptosis was blocked by treatment with inhibitors of ERK1/2 and p38 MAPK. We conclude that cAMP can induce c-IAP2 expression in colon cancer cells through CREB phosphorylation and CRE-dependent transcription in a manner that involves activation of ERK1/2 and p38 MAPK. These results emphasize that activation of kinases other than protein kinase A can mediate the actions of agents that increase cAMP, particularly in the regulation of CREB-dependent events.

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Application of laser capture microdissection to cytologic specimens for the detection of immunoglobulin heavy chain gene rearrangement in patients with malignant lymphoma.

Orba Y, Tanaka S, Nishihara H, Kawamura N, Itoh T, Shimizu M, Sawa H, Nagashima K

Cancer (4) 99 ( 4 ) 198 - 204 2003.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0008-543X

BACKGROUND. The demonstration of the monoclonality of immunoglobulin heavy chain (IgH) gene rearrangement is an indispensable method for the diagnosis of B-cell lymphoma as well as histocytochemical analysis. For the detection of IgH gene rearrangement, the extraction of DNA from a homogenous cell population is necessary. Recently, the laser capture microdissection (LCM) technique was shown to isolate specific cells from histopathologic specimens for molecular analysis. However, to the authors' knowledge the applicability of LCM to cytologic specimens has not yet been well established.
METHODS. Using LCM, a homogenous population of B-cell lymphoma cells as both histologic sections and cytologic specimens was captured, and genomic DNA was extracted from the captured cells. IgH gene rearrangement was analyzed by the polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) method.
RESULTS. Genomic DNAs were extracted successfully from ethanol-fixed cytologic specimens, but cells were not captured from air-dried specimens. Using PCR-SSCP analysis, the monoclonality of the IgH gene rearrangement was detected in five cases of tissue sections among nine analyzed cases of malignant lymphoma diagnosed immunohistochemically. However, analysis of the cytologic specimens with LCM demonstrated the monoclonality of the IgH gene rearrangement in seven cases of lymphoma.
CONCLUSIONS. The results of the current study suggest that the novel application of LCM to cytologic specimens occasionally exhibits high sensitivity for the detection of IgH gene rearrangement monoclonality compared with the use of histologic sections.

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Inhibition of apoptosis in normal and transformed intestinal epithelial cells by cAMP through induction of inhibitor of apoptosis protein (IAP)-2.

Nishihara H, Kizaka-Kondoh S, Insel PA, Eckmann L

Proceedings of the National Academy of Sciences of the United States of America (15) 100 ( 15 ) 8921 - 8926 2003.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0027-8424

Cyclooxygenase (COX)-2, a rate-limiting enzyme of prostaglandin (PG) production, is overexpressed in colorectal adenomas and adenocarcinomas, and its inhibition by nonsteroidal antiinflammatory drugs protects against colorectal cancer. Mechanisms of cancer promotion by COX-2 are not fully understood, but signaling through prostaglandin (PG)E-2 receptors is a contributing factor. The major PGE(2) receptors on epithelial cells, EP2 and EP4, increase cAMP production, which promotes growth and inhibits apoptosis in some cell types. Here, we show that cAMP agonists, including PGE2, cholera toxin, and a membrane-permeant cAMP analog, protect normal and transformed intestinal epithelial cells from apoptosis induced by diverse stimuli. This protection is associated with cAMP-mediated, rapid induction of cellular inhibitor of apoptosis protein (c-IAP)-2 and delayed induction of LIVIN, but not of six other members of the IAP family. Concurrently and characteristic of IAP functions, the activity, but not generation, of the cleaved form of the central executioner caspase 3 is inhibited. Induction of c-IAP2 expression by cAMP agonists is accompanied by phosphorylation of cAMP response element binding protein and cAMP response element-dependent activation of transcriptional reporters. Furthermore, inhibition of COX-2 in cells overexpressing the enzyme decreases c-IAP2 expression and promotes apoptosis, both of which are reversible by PGE2 addition, suggesting that COX-2-promoted antiapoptosis is mediated by release of PGE2 and subsequent cAMP-dependent c-IAP2 induction. These results help to explain the cancer chemoprotective effects of nonsteroidal anti inflammatory drugs by defining a mechanism through which cAMP signaling can promote the development of colorectal and possibly other epithelial cancers by means of disruption of normal apoptotic processes.

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CrkL directs ASAP1 to peripheral focal adhesions.

Oda A, Wada I, Miura K, Okawa K, Kadoya T, Kato T, Nishihara H, Maeda M, Tanaka S, Nagashima K, Nishitani C, Matsuno K, Ishino M, Machesky LM, Fujita H, Randazzo P

The Journal of biological chemistry (8) 278 ( 8 ) 6456 - 6460 2003.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1. is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.

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JC virus agnoprotein colocalizes with tubulin.

Endo S, Okada Y, Orba Y, Nishihara H, Tanaka S, Nagashima K, Sawa H

Journal of neurovirology (TAYLOR & FRANCIS INC) 9 Suppl 1 10 - 14 2003

Research paper (scientific journal), Joint Work, Accepted, ISSN 1355-0284

The human polyomavirus JC (JCV) encodes an agnoprotein that consists of 71 amino acid residues, with a molecular weight of approximately 8 kDa, from the late protein coding region. The agnoprotein of JCV shares 50% to 60% homology with those of simian virus 40 (SV40) and BK virus (BKV), and the carboxyl-terminal region of JCV agnoprotein is relatively unique. By using specific antibody to the carboxyl-terminal region of JCV agnoprotein, the authors have demonstrated that JCV agnoprotein expressed in the JCV-infected cells, where it localized predominantly in the perinuclear region of the cytoplasm, and colocalizes with the cellular cytoskeletal protein, tubulin. The results suggest that JCV agnoprotein may play a role in the stability of microtubules and the preservation of JCV infected cells via an interaction with tubulin.

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DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines.

Nishihara H, Maeda M, Oda A, Tsuda M, Sawa H, Nagashima K, Tanaka S

Blood (12) 100 ( 12 ) 3968 - 3974 2002.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-4971

The CDM (ced-5 of Caenorhabditis elegans, DOCK180 [downstream of Crk with molecular weight of 180 kDa] of humans, and myoblast city of Drosophila melanogaster) family of proteins has been shown to play a pivotal role in the integrin-mediated signaling pathway under the regulation of an adaptor molecule c-CT10- related kinase II (c-Crk-II) in adherent cells. Recently, hematopoietic cell-specific CDM protein DOCK2 has been shown to be indispensable for lymphocyte migration. However, the regulatory mechanism for DOCK2 is still unknown because DOCK2 lacks a c-Crk-II binding consensus motif. In this study, we demonstrated that DOCK2 bound to CrkL, which is present exclusively in hematopoietic cells both in vivo and in vitro, and we also found that 2 separate regions of DOCK2 contributed to its binding to Src homology 3 (SH3) domain of CrkL. Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells. We also found that CrkL-induced activation of small guanine triphosphatase (GTPase) Rac1 was significantly inhibited by the DOCK2-dCS mutant in 293T cells. Furthermore, the association of DOCK2 and Vav, the guanine-nucleotide exchanging factor (GEF) for Rac1, was demonstrated in Jurkat cells. Finally, the stable expression of DOCK2-dCS mutant in Jurkat cells was shown to reduce cell attachment. These data suggest the presence of a novel protein complex of CrkL, DOCK2, and Vav to regulate Rac1 in leukemia cell lines. (C) 2002 by The American Society of Hematology.

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Characterization of novel Alu- and tRNA-related SINEs from the tree shrew and evolutionary implications of their origins.

Nishihara H, Terai Y, Okada N

Molecular biology and evolution 19 ( 11 ) 1964 - 72 2002.11

Accepted, ISSN 0737-4038

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Contrast-enhanced dynamic computed tomography for the evaluation of tumor angiogenesis in patients with lung carcinoma.

Tateishi U, Kusumoto M, Nishihara H, Nagashima K, Morikawa T, Moriyama N

Cancer (4) 95 ( 4 ) 835 - 842 2002.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0008-543X

BACKGROUND. The objective of this study was to investigate the role of contrast-enhanced dynamic computed tomography (CT) in the evaluation of tumor angiogenesis in patients with lung carcinoma and to assess its importance in predicting tumor size and lymph node involvement.
METHODS. Dynamic CT scans were evaluated retrospectively in 130 patients with primary lung carcinoma who did not have distant metastasis and who underwent surgical resection with mediastinal lymph node dissection. Histopathologic findings of vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) were correlated with the maximum attenuation values of time-attenuation curves (MAV) on dynamic CT scans.
RESULTS. The MAV in VEGF positive tumors was greater compared with the MAV in VEGF negative tumors (66.1 +/- 4.6 vs. 30.9 +/- 2.7, respectively; P < 0.0001). An association was found between the NUV in VEGF positive tumors and MVD (correlation coefficient = 0.650; P < 0.0001). No difference was found in tumor size with pathologic confirmation and the MAV, VEGF expression, or MVD. The MAV, VEGF expression, and MVD in lung tumors with lymph node involvement were greater compared with the same values in lung tumors without lymph node involvement.
CONCLUSIONS. The MAV of dynamic CT may be a predictor of tumor angiogenesis in patients with lung carcinoma and lymph node involvement. (C) 2002 American Cancer Society.

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DOCK2 mediates T cell receptor-induced activation of Rac2 and IL-2 transcription.

Nishihara H, Maeda M, Tsuda M, Makino Y, Sawa H, Nagashima K, Tanaka S

Biochemical and biophysical research communications (3) 296 ( 3 ) 716 - 720 2002.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

DOCK2, a CDM family protein exclusively found in hematopoietic cells, has been shown to play a role in lymphocyte migration by the regulation of actin cytoskeleton. Although DOCK2 has been shown to induce the activation of Rac1, the regulatory mechanism of Rac2, which is a hematopoietic cell-specific small GTPase, is still unknown. In this study, we examined the role of DOCK2 in the activation of Rac2 in hematopoietic cells. DOCK2 was found to associate with the zeta subunit of the CD3 complex of T cell receptors in Jurkat cells and to activate forced expressed Rac2 in 293T cells. In addition, the stable expression of DOCK2 in Jurkat cells exhibited the elevated activity of endogenous Rac2. Furthermore, the transcriptional activity of interleukin-2 (IL-2) was enhanced in DOCK2-expressing Jurkat cells and the dominant negative form of Rac2 suppressed its elevated IL-2 promoter activity. These results suggest that DOCK2 mediates TCR-dependent activation of Rac2, leading to the regulation of IL-2 promoter activity in T cells. (C) 2002 Elsevier Science (USA). All rights reserved.

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Expression of JC virus agnoprotein in progressive multifocal leukoencephalopathy brain.

Okada Y, Sawa H, Endo S, Orba Y, Umemura T, Nishihara H, Stan AC, Tanaka S, Takahashi H, Nagashima K

Acta neuropathologica (2) 104 ( 2 ) 130 - 136 2002.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0001-6322

To examine the function of JC virus (JCV) agnoprotein, we examined the brains of cases of progressive multifocal leukoencephalopathy (PML), which is caused by JCV infection, using a newly generated antibody. The antibody reacted with 8 kDa protein specific for JCV agnoprotein by Western blotting. In vitro analyses showed that JCV capsid protein VP1 and large T antigen (T-Ag) were localized in the nuclei, but that agnoprotein was mainly detected in the cytoplasm of JCV-infected cells with an occasional nuclear staining. In the PML brain, an immunoreactive signal for agnoprotein was distributed in the perinuclear areas and cytoplasmic processes with occasional punctate staining in demyelinating lesions as well as adjacent myelinated areas. Agnoprotein presented mostly in the infected oligodendrocytes and partly in the astrocytes. Using double immunostaining, agnoprotein was seen to be expressed in the cytoplasmic processes of the cells, the nuclei of which were labeled with VP1 and T-Ag, where virus particles existed. Thus, JCV agnoprotein was mostly expressed in the infected oligodendrocytes and mainly localized in the cytoplasmic processes apart from virus particles in the demyelinated lesions.

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Molecular and immunohistochemical analysis of signaling adaptor protein Crk in human cancers.

Nishihara H, Tanaka S, Tsuda M, Oikawa S, Maeda M, Shimizu M, Shinomiya H, Tanigami A, Sawa H, Nagashima K

Cancer letters (1) 180 ( 1 ) 55 - 61 2002.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0304-3835

Crk is a signaling adaptor protein which is mostly composed of SH2 and SH3 domains. and has been shown to play a pivotal role in cell proliferation. differentiation, and migration. Because Crk was originally isolated as an avian sarcoma virus CT10 encoding oncoprotein v-Crk. we examined a potential role for c-Crk in the carcinogenesis of human cancers. First, to analyze gene mutations of c-Crk, we isolated a human bacterial artificial chromosome clone containing Crk genome and exon/intron structures, However, polymerase chain reaction-single strand conformation polymorphism methods failed to show any genomic mutations in the Crk exon which could be related to carcinogenesis. Second, immunohistochemical analysis of c-Crk-II demonstrated that the levels of c-Crk-II were significantly elevated in most of the tumors, particularly in the colon and lung cancers. Furthermore, immunoblot analysis using human lung cancer cell lines revealed that the expression levels of c-Crk-II were correlated to growth rates of L cells. The elevated expression levels of c-Crk-II might be related to the development of human cancers. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

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Lung tumors evaluated with FDG-PET and dynamic CT: the relationship between vascular density and glucose metabolism.

Tateishi U, Nishihara H, Tsukamoto E, Morikawa T, Tamaki N, Miyasaka K

Journal of computer assisted tomography (2) 26 ( 2 ) 185 - 190 2002.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 0363-8715

Purpose: The aim of the current study was to evaluate the relationship between FDG-PET and dynamic CT in lung tumors.
Method: Forty consecutive patients with pulmonary tumors underwent whole-body FDG-PET and contrast-enhanced dynamic CT. The size of tumors was 2.6 +/- 0.2 cm (mean +/- SD) at the largest diameter. The standardized uptake value (SUV) of FDG-PET, peak attenuation (A(PA)), and relative flow (RF) of dynamic CT were evaluated. All patients underwent surgery. and tissue samples were available to be studied. The intratumoral microvessel densities (MVDs) were counted and compared with the radiologic parameters. The duration between radiologic examinations and surgery was within 2 weeks in all patients.
Results: The mean SUV, A(PA), and RF of lung cancers were significantly higher than those of benign lesions (p < 0.05). The mean A(PA) and RF of lung cancers correlated with mean SUV (A(PA) : r = 0.665, p < 0.0001: RF: r = 0.848, p < 0.05) and mean MVD (A(PA): r = 0.801, p < 0.0001; RF: r = 0.723, p < 0.05). The mean SUV of lung cancers correlated with the mean MVD (r = 0.612, p < 0.001). No correlation was found between the mean A(PA), RF, SUV, and MVD in benign tumors.
Conclusion: The A(PA) and RF of dynamic CT correlated with the SUV of FDG-PET imaging in lung cancer. The A(PA) and RF of dynamic CT as an index of blood pooling may be related to increased glucose metabolism in lung cancer.

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Solitary fibrous tumor of the pleura: MR appearance and enhancement pattern.

Tateishi U, Nishihara H, Morikawa T, Miyasaka K

Journal of computer assisted tomography (2) 26 ( 2 ) 174 - 179 2002.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 0363-8715

Purpose: The aim of the current study was to characterize the MR appearance of solitary fibrous tumor of the pleura (SFTP).
Method: Twenty-two consecutive patients with histologically confirmed SFTP were retrospectively evaluated with MRI.
Results: Tumors demonstrated low signal intensity (n = 18, 82%). iso signal intensity (n = 2, 9%), and high signal intensity (n = 2. 9%) on T1-weighted images. On T2-weighted images, tumors were observed as low signal intensity (n = 10, 45%), high signal intensity (n = 2. 9%). and mixed signal intensity (n = 10, 45%). Solid component in the proliferation of tumor cells corresponded to low signal intensity in 12 tumors (55%) on T2-weighted images. On gadolinium-enhanced T1-weighted images, inhomogeneous enhancement (n = 18. 82%), nodular enhancement (n = 6, 27%), and homogeneous enhancement (n = 4, 18%) were observed. The enhanced lesion within the tumors had dense tumor tissue and dilated microvessels in the pathologic specimen.
Conclusion: SFTP shows variable appearance and enhancement pattern on MRI according to morphologic tumor heterogeneity.

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NM-1, a c-Myc-binding Protein, Is a Candidate for a Tumor Suppressor in Leukemia/Lymphoma and Tongue Cancer

Yuko Fujioka, Takahiro Taira, Yuichi Maeda, Shinya Tanaka, Hiroshi Nishihara, Sanae M. M. Iguchi-Ariga, Kazuo Nagashima, Hiroyoshi Ariga

Journal of Biological Chemistry 276 ( 48 ) 45137 - 45144 2001.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

The c-myc oncogene product (c-Myc) is a transcription factor that dimerizes with Max and recognizes the E-box sequence, and it plays key functions in cell proliferation, differentiation, and apoptosis. We previously showed that MM-1 bound to myc box II within the transactivation domain of c-Myc and repressed the E-box-dependent transcriptional activity of c-Myc. Here we report that MM-1 showed features of a tumor suppressor. In an EST data base search for cDNAs homologous to MM-1, we found a frequent substitution of amino acid 157 of MM-1, from alanine to arginine (A157R), and the substitution was observed more in tumor cells than in normal cells. A survey of the A157R mutation of MM-1 in 57 cultured cancer cells and 90 tissues from cancer patients showed that the A157R was present in about 50-60% of leukemia/lymphoma cells and in more than 75% of squamous cell carcinoma of tongue cancer. Although both the A157R and the wild-type MM-1 bound to c-Myc, only A157R lost the activities to repress both the E-box-dependent transcriptional activity of c-Myc and the myc/ras cooperative transforming activity in rat 3Y1 cells. Furthermore, the wild-type MM-1, but not A157R, arrested the growth of 3Y1 cells. The human MM-1 gene was mapped at chromosome 12q12-12q13, where many chromosome abnormalities in cancer cells have been reported. The results suggest that MM-1 is a novel candidate for a tumor suppressor that controls the transcriptional activity of c-Myc.

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Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication.

Suzuki S, Sawa H, Komagome R, Orba Y, Yamada M, Okada Y, Ishida Y, Nishihara H, Tanaka S, Nagashima K

Virology 286 ( 1 ) 100 - 12 2001.07

ISSN 0042-6822

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HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype.

Tanaka S, Katano H, Tsukamoto K, Jin M, Oikawa S, Nishihara H, Sawa H, Sawada K, Shimizu M, Sata T, Fujioka Y, Nagashima K

Pathology international 51 ( 4 ) 293 - 300 2001.04

ISSN 1320-5463

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Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2 alpha

M Nagai, S Tanaka, M Tsuda, S Endo, H Kato, H Sonobe, A Minami, H Hiraga, H Nishihara, H Sawa, K Nagashima

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA (NATL ACAD SCIENCES) 98 ( 7 ) 3843 - 3848 2001

Research paper (scientific journal), Joint Work, ISSN 0027-8424

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2 alpha, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1-181) of SYT-SSX1 and 50 amino acids (aa 156-205) of hBRM/hSNF2 alpha and we found that the overexpression of this binding region of hBRM/hSNF2 alpha significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription-PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2 alpha in human cancer.

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Clear cell variant of malignant melanoma of the uterine cervix: A case report and review of the literature

Mitsuko Furuya, Michio Shimizu, Hiroshi Nishihara, Tomoo Ito, Noriaki Sakuragi, Hiroshi Ishikura, Takashi Yoshiki

Gynecologic Oncology (Academic Press Inc.) 80 ( 3 ) 409 - 412 2001

Research paper (scientific journal), Joint Work, Accepted, ISSN 0090-8258

Background. A rare variant of malignant melanoma of the uterine cervix mimicking clear cell carcinoma or clear cell sarcoma is described. Case. A 33-year-old Japanese woman was admitted to the hospital complaining of genital discharge and lower back pain. The stage was FIGO IIB and radical hysterectomy and pelvic lymphadenectomy were done. Pathological examination, immunohistochemical studies of melanin granules, and molecular analysis of the EWS/ATF-1 fusion gene were also done. A diffuse proliferation of amelanotic clear cells was detected in the uterine cervix. Tumor cells were positive for HMB 45, Melan-A (MART-1), and S-100 protein and negative for epithelial markers. The EWS/ATF-1 fusion gene was not detected. Conclusion. This is apparently the first report of a case of clear cell melanoma of the uterine cervix. Despite its rarity, this variant of malignant melanoma should be considered when diagnosing clear cell neoplasms of the uterine cervix. © 2001 Academic Press.

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HTLV-1-associated bronchopneumonopathy (HAB): CT-pathological correlation

Ukihide Tateishi, Hiroshi Nishihara, Kazuo Miyasaka

Clinical Radiology (Elsevier) 56 ( 8 ) 664 - 666 2001

Research paper (scientific journal), Joint Work, Accepted, ISSN 0009-9260

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Tumor angiogenesis and dynamic CT in lung adenocarcinoma: Radiologic-Pathologic correlation

Ukihide Tateishi, Hiroshi Nishihara, Satoshi Watanabe, Toshiaki Morikawa, Kazuhiro Abe, Kazuo Miyasaka

Journal of Computer Assisted Tomography 25 ( 1 ) 23 - 27 2001

Research paper (scientific journal), Joint Work, Accepted, ISSN 0363-8715

Purpose: The purpose of this work was to evaluate the correlation of tumor angiogenesis and dynamic CT in lung adenocarcinoma. Method: Thirty-five consecutive patients with lung adenocarcinoma underwent dynamic chest CT. Maximum attenuation of dynamic CT was compared with microvessel densities (MVDs) and vascular endothelial growth factor (VEGF) expression by immunohistochemistry. Results: The mean peak attenuation (APA) of lung adenocarcinoma correlated with MVD (r = 0.689, p &lt
0.0001). VEGF positiveness of lung adenocarcinoma was 63%. There was a significant difference in APA between VEGF-positive and -negative lung adenocarcinomas (39.9 ± 3.9 and 24.3 ± 2.3
p &lt
0.05). The mean MVDs of VEGF-positive adenocarcinomas were significantly higher than those of negative ones (82.5 ± 5.9 and 49.2 ± 7.1
p &lt
0.05). The mean APA of VEGF-positive lung adenocarcinomas correlated positively with MVD (r = 0.707, p &lt
0.0001). Conclusion: The APA value of dynamic CT reflects MVD in lung adenocarcinoma. The APA value of dynamic CT might be an index for VEGF-related tumor angiogenesis.

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High-resolution CT findings of macrophage activation syndrome: a case report.

Tateishi U, Nishihara H, Okano M, Kobayashi R, Kobayashi I, Fujita N

Pediatric radiology 30 ( 10 ) 699 - 701 2000.10

Accepted, ISSN 0301-0449

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Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins

Hiroshi Nishihara, Shin Kobayashi, Yuko Hashimoto, Fumiyo Ohba, Naoki Mochizuki, Takeshi Kurata, Kazuo Nagashima, Michiyuki Matsuda

Biochimica et Biophysica Acta - Molecular Cell Research 1452 ( 2 ) 179 - 187 1999.11

Accepted, ISSN 0167-4889

Human DOCK180, which was originally identified as a major protein bound to the Crk oncogene product, is an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. After DOCK180, at least three putative human proteins that manifest high amino acid sequence similarity to DOCK180 have been registered in the GenBank/EMBL database. We have designated one of them, KIAA0209, as DOCK2 and characterize here. DOCK2 mRNA was expressed mostly in peripheral blood cells, followed by slight expression in the spleen and thymus, whereas DOCK180 was expressed in all tissues tested except in peripheral blood cells. Immunostaining of human cadaver tissues revealed that the expression of DOCK2 was limited to the lymphocytes and macrophages of various organs. DOCK2 bound to and activated Rac1, as did DOCK180
however, DOCK2 did not bind to CrkII, which transduces signals at focal adhesions. Thus, DOCK180 and DOCK2 are regulators of Rac and function in adherent and non-adherent cells, respectively.

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JCウイルスcapsid proteinの機能的領域に対する抗体を用いた免疫組織学的検索と感染抑制実験

西原広史, 小林希, 佐藤真実, 宍戸由紀子, 岡田由紀, 田中伸哉, 澤洋文, 古林与志安, 長嶋和郎

日本病理学会会誌 ((一社)日本病理学会) 88 ( 1 ) 333 - 333 1999.03

Accepted, ISSN 0300-9181

Constitutive association of EGF receptor with the CrkII-23 mutant that inhibits transformation of NRK cells by EGF and TGF-beta

S Ota, S Kizaka-Kondoh, Y Hashimoto, H Nishihara, K Nagashima, T Kurata, H Okayama, M Matsuda

CELLULAR SIGNALLING (ELSEVIER SCIENCE INC) 10 ( 4 ) 283 - 290 1998.04

Accepted, ISSN 0898-6568

Crk belongs to the adapter proteins that participate in many signalling pathways from cell surface receptors. We have characterised the CrkII-23 mutant that inhibits the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. To study the biochemical difference, cDNAs of the wild-type CrkII and the CrkII-23 mutant were introduced stably into NIH 3T3 cells expressing EGF receptor (EGFR). Both CrkII and CrkII-23 were phosphorylated on tyrosine upon EGF simulation with similar time course and dose dependency. Whereas the wild-type CrkII bound to EGFR only after EGF stimulation, CrkII-23 bound to EGFR from before stimulation. Mutation in the Src homology (SH) 2 or amino terminal SH3 domain did not abolish the binding of CrkII-23 to EGFR in the quiescent cells, suggesting that the binding is mediated by a novel mechanism. These CrkII-23-derived mutants, however, did not suppress transformation of NRK cells by EGF and TGF-beta. Hence, both the SH2 and amino-terminal SH3 domains are required to inhibit transformation of NRK cells. These results suggest that persistent signalling from CrkII-23 bound to EGFR suppresses transformation by EGF and TGF-beta in NRK23 cells. (C) 1998 Elsevier Science Inc.

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