Elsevier Ireland Ltd.Acta Medica Okayama0169-50027612012Frequent methylation and oncogenic role of microRNA-34b/c in small-cell lung cancer3238ENNorimitsuTanakaShinichiToyookaJunichiSohTakafumiKuboHiromasaYamamotoYuhoMakiTakayukiMuraokaKazuhikoShienMasashiFurukawaTsuyoshiUenoHiroakiAsanoKazunoriTsukudaKeisukeAoeShinichiroMiyoshiSmall-cell lung cancer (SCLC) is an aggressive tumor with a dismal prognosis among primary lung cancers. MicroRNAs (miRNAs) can act as oncogenes or tumor-suppressor genes in human malignancy. The miR-34 family is comprised of tumor-suppressive miRNAs, and its reduced expression by methylation has been reported in various cancers, including non-small cell lung cancer (NSCLC). In this study, we investigated the alteration and tumor-suppressive impact of miR-34s in SCLC. The methylation of miR-34a and miR-34b/c was observed in 4 (36%) and 7 (64%) of 11 SCLC cell lines, respectively. Among the 27 SCLC clinical specimens, miR-34a and miR-34b/c were methylated in 4(15%) and 18 (67%), respectively. In contrast, 13 (28%) miR-34a methylated cases and 12 (26%) miR-34b/c methylated cases were found in 47 NSCLC primary tumors. The frequency of miR-34b/c methylation was significantly higher in SCLC than in NSCLC (p < 0.001). The expressions of miR-34s were reduced in methylated cell lines and tumors and restored after 5-aza-2'-deoxycytidine treatment, indicating that methylation was responsible for the reduced expression of miR-34s. Because the frequency of methylation was higher in miR-34b/c, we focused on miR-34b/c for a functional analysis. We examined the effect of miR-34b/c introduction on cell proliferation, migration and invasion. The transfection of miR-34b/c to two SCLC cell lines (H1048 and SBC5) resulted in the significant inhibition of cell growth, migration, and invasion, compared with control transfectants. Our results indicate that the aberrant methylation of miR-34b/c plays an important role in the pathogenesis of SCLC, implying that miR-34b/c may be a useful therapeutic target for SCLC.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama1021-335X2912013Impact of GLUT1 and Ki-67 expression on early-stage lung adenocarcinoma diagnosed according to a new international multidisciplinary classification133140ENYuhoMakiJunichiSohKouichiIchimuraKazuhikoShienMasashiFurukawaTakayukiMuraokaNorimitsuTanakaTsuyoshiUenoHiromasaYamamotoHiroakiAsanoKazunoriTsukudaShinichiToyookaShinichiroMiyoshiHigh expression levels of glucose transporter isoform 1 (GLUT1) and Ki-67 are reportedly associated with malignancy-related clinicopathological factors in malignant tumors. Recently, a new histological IASLC/ATS/ERS classification for lung adenocarcinoma was proposed. In this study, we investigated the clinicopathological impact of GLUT1 and Ki-67 expression on early-stage lung adenocarcinoma classified according to the IASLC/ATS/ERS classification. One hundred and five patients with completely resected stage IA lung adenocarcinoma were retrospectively classified into two groups, a 'non-invasive type' (n=31) or an 'invasive type' (n=74), based on the IASLC/ATS/ERS classification. GLUT1 and Ki-67 expression status was evaluated using immunohistochemistry. The epidermal growth factor receptor (EGFR) and KRAS mutation status was determined using PCR-based assays. Positive GLUT1 and Ki-67 expression and EGFR and KRAS mutations were detected in 28 (27%), 33 (31%), 51 (49%) and 5 (8%) cases, respectively. Positive GLUT1 expression was significantly associated with a wild-type EGFR and mutant KRAS status. A multivariate analysis revealed that positive GLUT1 expression was independently associated with the 'invasive type'. In multivariate analyses for overall survival (OS) and disease-free survival (DFS), positive Ki-67 and GLUT1 expression was the only independent factor for a poor OS (P=0.012) and DFS (P=0.040), respectively. In addition, when stratified according to the GLUT1 and Ki-67 status, double-positive cases had the poorest DFS and OS times, compared with the other categories. Positive GLUT1 expression is associated with the invasive character of early-stage lung adenocarcinoma and with early disease relapse. Our results strongly suggest that GLUT1 and Ki-67 play important roles in acquiring biological malignant potential in early-stage lung adenocarcinoma.No potential conflict of interest relevant to this article was reported.Elsevier Ireland Ltd.Acta Medica Okayama0169-50028232013The degree of microRNA-34b/c methylation in serum-circulating DNA is associated with malignant pleural mesothelioma485490ENTakayukiMuraokaJunichiSohShinichiToyookaKeisukeAoeNobukazuFujimotoShinsukeHashidaYuhoMakiNorimitsuTanakaKazuhikoShienMasashiFurukawaHiromasaYamamotoHiroakiAsanoKazunoriTsukudaTakumiKishimotoTakemiOtsukiShinichiroMiyoshiObjectives: Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. microRNA-34b/c (miR-34b/c), which plays an important role in the pathogenesis of MPM, is frequently downregulated by DNA methylation in approximately 90% of MPM cases. In this study, we estimated the degree of miR-34b/c methylation in serum-circulating DNA using a digital methylation specific PCR assay (MSP).
Materials and methods: A real-time MSP assay was performed using the SYBR Green method. The melting temperature (Tm) of each PCR product was examined using a melting curve analysis. For a digital MSP assay, 40 wells were analyzed per sample. A total of 110 serum samples from 48 MPM cases, 21 benign asbestos pleurisy (BAP) cases, and 41 healthy volunteers (HVs) were examined.
Results: Positive range of Tm value for miR-34b/c methylation was defined as 77.71-78.79 degrees C which was the mean 3 standard deviations of 40 wells of a positive control. The number of miR-34b/c methylated wells was counted per sample according to this criterion. The number of miR-34b/c methylated wells in MPM cases was significantly higher than that in BAP cases (P = 0.03) or HVs (P < 0.001). Advanced MPM cases tended to have higher number of miR-34b/c methylated wells than early MPM cases. Receiver-operating characteristic (ROC) curve analysis revealed that three number of miR-34b/c methylated wells per sample was the best cut-off of positivity of MPM with a 67% of sensitivity and a 77% specificity for prediction. The area under the ROC curve was 0.77.
Conclusions: Our digital MSP assay can quantify miR-34b/c methylation in serum-circulating DNA. The degree of miR-34b/c methylation in serum-circulating DNA is associated with MPM, suggesting that this approach might be useful for the establishment of a new detection system for MPM.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6812014Preclinical Evaluation of MicroRNA-34b/c Delivery for Malignant Pleural Mesothelioma2326ENTsuyoshiUenoShinichiToyookaTakuyaFukazawaTakafumiKuboJunichiSohHiroakiAsanoTakayukiMuraokaNorimitsuTanakaYuhoMakiKazuhikoShienMasashiFurukawaMasakiyoSakaguchiHiromasaYamamotoKazunoriTsukudaShinichiroMiyoshiOriginal Article10.18926/AMO/52140The microRNA-34s (miR-34s) have p53 response elements in their 5ʼ-flanking regions and demonstrate tumor-suppressive functions. In malignant pleural mesothelioma (MPM), we previously reported that expression of miR-34b and miR-34c (miR-34b/c) was frequently downregulated by methylation in MPM cell lines and primary tumors. The forced overexpression of miR-34b/c showed significant antitumor effects with the induction of apoptosis in MPM cells. In this study, we examined the in vivo antitumor effects of miR-34b/c using adenovirus vector on MPM. We subcutaneously transplanted NCI-H290, a human MPM cell line, into BALB/C mice and injected adenovirus vector expressing miR-34b/c, luciferase driven by the cytomegalovirus promoter (Ad-miR-34b/c or Ad-Luc), or PBS control into tumors over 5mm in diameter. A statistically significant growth inhibition of the tumor volume was observed in the Ad-miR-34b/c group from day 6 onward compared to the Ad-Luc group. The inhibition
rate of Ad-miR-34b/c, compared to the tumor volume treated with Ad-Luc, was 58.6% on day 10 and 54.7% on day13. Our results indicate that adenovirus-mediated miR-34b/c gene therapy could be useful for the clinical treatment of MPM.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6842014Drug Resistance to EGFR Tyrosine Kinase Inhibitors for Non-small Cell Lung Cancer191200ENKazuhikoShienHiromasaYamamotoJunichiSohShinichiroMiyoshiShinichiToyookaReview10.18926/AMO/52785Non-small cell lung cancer (NSCLC) harboring an activating mutation within the epidermal growth factor receptor (EGFR) was defined as a clinically distinct molecular group. These lesions show oncogene addiction to EGFR and dramatic responses to the EGFR tyrosine kinase inhibitors (TKIs). Several large Phase III trials have shown that EGFR-TKIs improved the progression-free survival of patients with EGFR mutant NSCLC compared to conventional chemotherapy. However, the long-term effectiveness of EGFR-TKIs is usually limited because of acquired drug resistance. To overcome this resistance to EGFR-TKIs, it will be essential to identify the specific mechanisms underlying the resistance. Many investigators have attempted to identify the mechanisms using preclinical models and drug-resistant clinical samples. As a result, several mechanisms have been showed to be responsible for the resistance, but not all of the relevant mechanisms have been uncovered. In this review, we provide an overview of mechanisms underlying drug-resistance to EGFR-TKIs, focusing on results obtained with preclinical models, and we present some possible strategies to overcome the EGFR-TKI resistance.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155812632014平成25年度岡山医学会賞 総合研究奨励賞（結城賞）187190ENHiromasaYamamoto受賞対象論文: Yamamoto H, Higasa K, Sakaguchi M, Shien K, Soh J, Ichimura K, Furukawa M, Hashida S, Tsukuda K, Takigawa N, Matsuo K, Kiura K, Miyoshi S, Matsuda F, Toyooka S：Novel germline mutation in the transmembrane domain of HER2 in familial lung adenocarcinomas. J Natl Cancer Inst (2014) 106, djt338No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X7112017Human RAD 17 Polymorphism at Codon 546 Is Associated with the Risk of Colorectal Cancer5968ENYukikoYasudaDepartment of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAkikoSakaiDepartment of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSachioItoDepartment of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKaoriSasaiDepartment of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHiromasaYamamotoDepartment of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesNagahideMatsubaraDepartment of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMamoruOuchidaDepartment of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHiroshiKatayamaDepartment of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKenjiShimizuDepartment of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOriginal Article10.18926/AMO/54826Human RAD17 acts as an activator of checkpoint signals in response to DNA damage. Here we evaluated the association of hRAD17 Leu546Arg (rs1045051), a missense single nucleotide polymorphism, with the risk of colorectal cancer (CRC) in relation to smoking and alcohol consumption habits in 212 CRC patients and 1,142 cancer-free controls in a case-control study conducted in Japan. The results showed that the hRAD17 Leu/Arg genotype compared to the Leu/Leu genotypes was significantly associated with the protective effect on CRC risk with the adjusted odds ratio (OR) of 0.68 [95% confidence interval (CI): 0.49−0.95, p=0.024], and the males with the Arg/Arg genotype had a greater risk of CRC compared to those with the Leu/Leu and Leu/Arg genotypes (OR=1.87, 95%CI 1.03−3.40, p=0.04). In stratified studies, the protective effect of the Leu/Arg genotype on CRC risk was markedly higher in the light smokers (< 20 pack years) (OR=0.61, 95%CI 0.40−0.94, p=0.024) and the rectal cancer patients (OR=0.49, 95%CI 0.31−0.78, p=0.003). The risk of the Arg/Arg genotype was associated with heavy smoking (≥ 20 pack-years) (OR=2.24, 95%CI 1.09−4.61, p=0.03). These findings suggest that the genetic variant of hRAD17 Leu546Arg polymorphism has a significant effect on CRC susceptibility in Japanese.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6712013Silenced Expression of NFKBIA in Lung Adenocarcinoma Patients with a Never-smoking History1924ENMasashiFurukawaJunichiSohHiromasaYamamotoKouichiIchimuraKazuhikoShienYuhoMakiTakayukiMuraokaNorimitsuTanakaTsuyoshiUenoHiroakiAsanoKazunoriTsukudaShinichiToyookaShinichiroMiyoshiOriginal Article10.18926/AMO/49253Nuclear factor of κ-light polypeptide gene enhancer in B cells inhibitor α (NFKBIA), which is a tumor suppressor gene, was found to be silenced in lung adenocarcinomas. We examined NFKBIA expression, mutations in the EGFR and K-ras genes, and EML4-ALK fusion in 101 resected lung adenocarcinoma samples from never-smokers. NFKBIA expression was evaluated using immunohistochemistry. NFKBIA expression was negative in 16 of the 101 samples (15.8%). EGFR and K-ras mutations and EML4-ALK fusion were detected in 61 (60.5%), 1 (1.0%), and 2 (2.0%) of the 101 samples, respectively, in a completely mutually exclusive manner. Negative NFKBIA expression was observed significantly more frequently among the tumors with none of the three genetic alterations compared to those with such alterations (p＝0.009). In addition, negative NFKBIA expression was significantly more frequent among the EGFR-wild type samples compared to the EGFR-mutant samples (p＝0.013). In conclusion, NFKBIA expression was silenced in adenocarcinomas without EGFR/K-ras mutations or EML4-ALK fusion, suggesting that the silencing of NFKBIA may play an important role in the carcinogenesis of adenocarcinomas independent of EGFR/K-ras mutations or EML4-ALK fusion.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X7042016Study about the Efficacy of Metformin to Immune Function in Cancer Patients327330ENMototsuguWatanabeDepartment of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHiromasaYamamotoDepartment of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesShingoEikawaDepartment of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKazuhikoShienDepartment of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesTadahikoShienDepartment of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesJunichiSohDepartment of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKatsuyukiHottaCenter for Innovative Clinical Medicine, Okayama University HospitalJunWadaDepartment of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesShiroHinotsuCenter for Innovative Clinical Medicine, Okayama University HospitalToshiyoshiFujiwaraDepartment of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKatsuyukiKiuraDepartment of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHiroyoshiDoiharaDepartment of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesShinichiroMiyoshiDepartment of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesHeiichiroUdonoDepartment of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesShinichiToyookaDepartment of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesClinical Study Protocols10.18926/AMO/54514A study to evaluate the effect of metformin on the immune system was commenced in July 2014. Metformin is one of the most commonly prescribed drugs for type 2 diabetes, and previous studies have reported that metformin has an anti-tumor effect. The aim of this study is to evaluate the efficacy of metformin on the immune system in human cancer patients in vivo. The primary outcome parameter will be the rate change in the population of CD8＋ T cells, which produce multiple cytokines. No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X7062016Usefulness of Thoracoscopic Debridement for Chronic Empyema after an Extrapleural Pneumonectomy507510ENHidejiroTorigoeDepartment of Thoracic Surgery, Okayama University HospitalShinichiToyookaDepartment of Thoracic Surgery, Okayama University HospitalHiromasaYamamotoDepartment of Thoracic Surgery, Okayama University HospitalJunichiSohDepartment of Thoracic Surgery, Okayama University HospitalShinichiroMiyoshiDepartment of Thoracic Surgery, Okayama University HospitalCase Report10.18926/AMO/54816We present the case of a 65-year-old Japanese man diagnosed with chronic empyema (without a bronchopleural fistula) that occurred 7 months after he underwent an extrapleural pneumonectomy for right malignant pleural mesothelioma (MPM). Following thoracic drainage and irrigation for 1 month, we performed surgery by a thoracoscopic approach, in light of his general condition. We performed debridement and removal of the Gore-Tex polytetrafluoroethylene (PTFE) patch that had been used for the reconstruction of the diaphragm and the pericardium. The empyema had not relapsed when he died from recurrence of the MPM at 4 months after the thoracoscopic surgery. This patientʼs case suggests that thoracoscopic debridement and patch removal can be a therapeutic option for not only early-stage (exudative or fibrinopurulent) empyema but also late-stage (organized and chronic) empyema without a bronchopleural fistula, particularly for patients in poor general condition.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X7132017Reconstruction of Anterior Chest Wall with Polypropylene Mesh: Two Primary Sternal Chondrosarcoma Cases259262ENShinichiKawanaDepartment of Thoracic Surgery, Okayama University HospitalHiromasaYamamotoDepartment of Thoracic Surgery, Okayama University HospitalYuhoMakiDepartment of Thoracic Surgery, Okayama University HospitalSeiichiroSugimotoDepartment of Thoracic Surgery, Okayama University HospitalShinichiToyookaDepartment of Thoracic Surgery, Okayama University HospitalShinichiroMiyoshiDepartment of Thoracic Surgery, Okayama University HospitalCase Report10.18926/AMO/55210 Primary sternal chondrosarcoma is a rare malignant tumor that is refractory to chemotherapy and radiation. Effective therapy is radical resection of the tumor. We present two patients with primary sternal chondrosarcoma who underwent a radical resection of the lower half of the sternum and bilateral ribs, followed by reconstruction with 2 sheets of polypropylene mesh layered orthogonally. The patients have maintained almost the same pulmonary function as preoperative values, with stability of the chest wall. Although there are various ways to reconstruct the anterior chest wall, reconstruction with polypropylene mesh layered orthogonally is an easy-to-use and sufficient method.No potential conflict of interest relevant to this article was reported.