松下 一之

Segawa S, Nishimura M, Sogawa K, Tsuchida S, Murata S, Watanabe M, Matsushita K, Kamei K, Nomura F, Clinical proteomics, 2015, vol. 12, no. 1, pp. 6

Matsushita K, Itoga S, Nishimura M, Furuta K, Nomura F, Rinsho byori. The Japanese journal of clinical pathology, 2015, vol. 63, no. 3, pp. 347-360

Matsushita K, Kitamura K, Rahmutulla B, Tanaka N, Ishige T, Satoh M, Hoshino T, Miyagi S, Mori T, Itoga S, Shimada H, Tomonaga T, Kito M, Nakajima-Takagi Y, Kubo S, Nakaseko C, Hatano M, Miki T, Matsuo M, Fukuyo M, Kaneda A, Iwama A, Nomura F, Oncotarget, 2015, vol. 6, no. 7, pp. 5102-5117

Seimiya M, Suzuki Y, Yoshida T, Sawabe Y, Matsushita K, Nomura F, Clinica chimica acta; international journal of clinical chemistry, 2015, vol. 441, pp. 44-46

Suzuki Y, Matsushita K, Seimiya M, Yoshida T, Sawabe Y, Ogawa M, Nomura F, Clinica chimica acta; international journal of clinical chemistry, 2015, vol. 451, no. Pt B, pp. 316-322

Suzuki Y, Matsushita K, Seimiya M, Yoshida T, Sawabe Y, Ogawa M, Nomura F, Data in brief, 2015, vol. 5, pp. 1092-1095, 2015

Mizokami D, Araki K, Tanaka N, Suzuki H, Tomifuji M, Yamashita T, Ueda Y, Shimada H, Matsushita K, Shiotani A, PloS one, 2015, vol. 10, no. 1, pp. e0116279

Objectives: Recent studies have demonstrated that, in advanced colorectal carcinoma (CRC) patients, extended RAS (in KRAS exons 2-4 and NRAS exons 2-4) and BRAF mutations are negative predictors for anti-EGFR treatment efficacy and negative prognostic factor, respectively. Thus, high-throughput and cost-effective methods for identification of the mutation status are required. Design and methods: We developed a PCR-high-resolution melting (HRM)-based method for screening extended RAS and BRAF mutations, and relative frequency of mutations in formalin-fixed paraffin-embedded samples of CRC was analyzed. Results: Among 93 CRC samples, 29 harbored mutations in KRAS exon 2, and 9 harbored mutations in BRAF exon 15. Analysis of 55 KRAS exon 2 and BRAF exon 15 wild-type CRC samples identified the following mutations: 1/55 in exon 3 and 2/55 in exon 4 of KRAS; 1/55 in exon 2, 3/55 in exon 3, and 0/55 in exon 4 of NRAS. Conclusions: Our PCR-HRM method will enable rapid determination of the extended RAS and BRAF mutation status prior to anti-EGFR treatment in the clinical setting. (C) 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Rahmutulla B, Matsushita K, Nomura F, World journal of gastroenterology : WJG, 2014, vol. 20, no. 46, pp. 17305-17313

Background: Chronic subdural hematoma (CSDH) is known to have a substantial recurrence rate. Artificial cerebrospinal fluid (ACF) is an effective irrigation solution in general open craniotomy and endoneurosurgery, but no evidence of its use in burr-hole surgery exists. Objective: To identify the potential of ACF irrigation to prevent CSDH recurrence. More specifically, to investigate the perioperative and intraoperative prognostic factors, and to identify controllable ones. Methods: To examine various prognostic factors, 120 consecutive patients with unilateral CSDH treated with burr-hole drainage between September 2007 and March 2013 were analyzed. Intraoperative irrigation was performed with one of two irrigation solutions: normal saline (NS; n = 60) or ACF (n = 60). All patients were followed-up for at least 6 months postoperatively. We also examined the morphological alternations of the hematoma outer membranes after incubation with different solutions. Results: Eleven patients (9.2%) had recurrence. Nine patients (15%) required additional surgery in the NS group, whereas only 2 patients (3.3%) in the ACF group required additional surgery. Among preoperative and intraoperative data, age (, 80 years old, P =.044), thrombocyte (>22.0, P=.037), laterality (right, P=.03), and irrigation solution (ACF, P=.027) were related to smaller recurrence rates by log-rank tests. Only the type of irrigation solution used significantly correlated with recurrence in favor of ACF in both Cox proportional hazards (relative hazard: 0.20, 95% confidence interval (CI): 0.04-0.99; P=.049) and logistic regression models (odds ratio, 0.17, 95% CI: 0.03-0.92; P=.04) using these factors. Histological examinations of the hematoma membranes showed that the membranes incubated with NS were loose and infiltrated by inflammatory cells compared with those incubated with ACF. Conclusion: Irrigation with ACF decreased the rate of CSDH recurrence.

「コンパニオン診断」の基礎知識-概念,関連するガイドライン,情勢など-について概説した.「コンパニオン診断」は患者,個人の層別化とそのための疾患との因果関係が科学的に証明されたバイオマーカー(生体物質)の同定が,薬剤の開発や使用に必須であるという考え方である.個人のゲノム,癌細胞の状態(さまざまな分子メカニズムの変化)を正確に測定する技術のみならず,生体試料の採取,保存方法,試薬類,検査法,検査機器などの標準化,精度管理も「コンパニオン診断」が普遍性を保つためには必要である.(著者抄録)

Objective: Previous histochemical studies in the demyelinating form of Guillain-Barr syndrome (GBS), acute inflammatory demyelinating polyneuropathy (AIDP), have shown complement deposition on the surface of Schwann cells, and therefore unknown epitopes would be present on the outer surface of Schwann cells. Methods: We used a proteomic-based approach to search for the target molecules of AIDP in the extracted proteins from schwannoma cells. Sera were obtained from 40 patients with GBS, 31 controls with inflammatory disease, and 46 normal controls. Results: We found that patients with AIDP after cytomegalovirus (CMV) infection have serum autoantibodies against membrane-organizing extension spike protein (moesin), which is expressed in the Schwann cell processes at the nodes of Ranvier and is crucial for myelination. Of the 40 patients with GBS, 6 had recent CMV infection and 5 of them (83%) had high levels of serum immunoglobulin G antibodies against moesin. The anti-moesin antibodies were found in none of the control subjects with disease including 5 with CMV infection but no neuropathy, and only 2 (4%) of the 46 normal control subjects. Immunocytochemistry showed that moesin was stained at the distal tips of schwannoma cells by sera from the patients with CMV-related AIDP but not by sera from controls. Conclusion: Moesin is a possible immunologic target molecule of pathogenic autoantibodies in patients with CMV-related AIDP. Classification of evidence: This study provides Class II evidence that levels of serum anti-moesin antibodies accurately distinguishes CMV-related AIDP from non-CMV-related AIDP (sensitivity 83%, specificity 93%).

Risk-reducing salpingo-oophorectomy for reducing future cancer risk in women with hereditary breast and ovarian cancer syndrome is rarely performed in Japan; therefore, the cancer preventive effect of risk-reducing salpingo-oophorectomy for hereditary breast and ovarian cancer syndrome among the Japanese population remains unclear. Here, we report the first case of serous tubal intraepithelial carcinoma identified through a risk-reducing salpingo-oophorectomy in a Japanese woman with hereditary breast and ovarian cancer syndrome and who had a deleterious germline mutation of E1214X in BRCA1, but not a BRCA2 mutation. A pre-operative examination revealed multiple uterine leiomyomas but no adnexal mass. Robotic-assisted bilateral salpingo-oophorectomy together with hysterectomy was performed. A pathological examination identified serous tubal intraepithelial carcinoma in the right fallopian tube with no dissemination. Serous tubal intraepithelial carcinoma is implicated as an origin of invasive cancer of the fallopian tube with peritoneal dissemination; prophylactic salpingo-oophorectomy is currently the only method to identify this occult cancer. Our case demonstrated that risk-reducing salpingo-oophorectomy can detect occult cancers, including serous tubal intraepithelial carcinoma, thereby preventing future cancer development in the Japanese hereditary breast and ovarian cancer syndrome population.

The far-upstream element-binding protein-interacting repressor (FIR) is a c-myc transcriptional suppressor. FIR is alternatively spliced to lack the transcriptional repression domain within exon 2 (FIR Delta exon2) in colorectal cancers. FIR and FIR Delta exon2 form homo- or heterodimers that complex with SAP155. SAP155, a subunit of the essential splicing factor 3b subcomplex in the spliceosome, is required for proper P27Kip1 pre-mRNA splicing, and P27Kip1 arrests cells at G1. In contrast, FIR was co-immunoprecipitated with Ku86 and DNA-PKcs. siRNA against Ku86/Ku70 decreased FIR and P27Kip1 expression, whereas siRNA against FIR decreased Ku86/XRCC5 and P27Kip1 expression. Thus the mechanical interaction of FIR/FIR Delta exon2/SAP155 bridges c-myc and P27Kip1 expression, potentially integrates cell-cycle progression and c-myc transcription in cell. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. Because DNA breaks generate the recruitment of Ku86/Ku70 to bind to the broken DNA ends, the possible involvement of FIR and Ku86/Ku70 interaction in the BLM-induced DNA damage repair response was investigated in this study. First, BLM treatment reduced SAP155 expression and increased FIR and FIR Delta exon2 mRNA expression as well as the ratio of FIR Delta exon2:FIR in hepatoblastoma cells (HLE and HLF). Second, FIR or FIR Delta exon2 adenovirus vectors (Ad-FIR or Ad-FIR Delta exon2) increased Ku86/Ku70 and P27Kip1 expression in vitro. Third, BLM decreased P27Kip1 protein expression, whereas increased P27Kip1 and gamma H2AX expression with Ad-FIR Delta exon2. Together, the interaction of FIR/SAP155 modulates FIR splicing and involves in cell-cycle control or cell fate via P27Kip1 and c-myc in BLM-induced DNA damage pathway. This novel function of FIR splicing will contribute to clinical studies of cancer management through elucidating the mechanical interaction of FIR/FIR Delta exon2/SAP155 as a potential target for cancer treatment.

AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR). METHODS: Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR (HA-FIR) expression vector. A fusion gene-deficient, non-transmissible, Sendai virus (SeV) vector encoding FIR cDNA, SeV/dF/FIR, was prepared. SeV/dF/FIR was examined for its gene transduction efficiency, viral dose dependency of antitumor effect and apoptosis induction in HeLa (cervical squamous cell carcinoma) cells and SW480 (colon adenocarcinoma) cells. Antitumor efficacy in a mouse xenograft model was also examined. The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A (SSA), a SAP155 inhibitor, or SAP155 siRNA which induce c-Myc by increasing FIR Delta exon2 in HeLa cells. RESULTS: FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression. Thus, FIR expressing vectors are potentially applicable for cancer therapy. FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2 (FIR Delta exon2), counteracting FIR for c-Myc protein expression. Furthermore, FIR forms a complex with SAP155 and inhibits mutual well-established functions. Thus, both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application. SeV/dF/FIR, a cytoplasmic RNA virus, was successfully prepared and showed highly efficient gene transduction in in vivo experiments. Furthermore, in nude mouse tumor xenograft models, SeV/dF/FIR displayed high antitumor efficiency against human cancer cells. SeV/dF/FIR suppressed SSA-activated c-Myc. SAP155 siRNA, potentially produces FIR Delta exon2, and led to c-Myc overexpression with phosphorylation at Ser62. HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells. A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells. CONCLUSION: SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model, thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment. (C) 2014 Baishideng Publishing Group Co., Limited. All rights reserved.

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. In recent years, studies have shown a definite association between periodontal disease and other inflammatory conditions of the body. High-throughput analysis of proteins has become possible with the development of MS technology. This breakthrough in proteome technology enables comparative studies of comprehensive protein expression and identification of protein. In case of periodontal disease, proteome analysis using 2DE, as well as gel-free methods, has been reported. As a fluid lying in close proximity to periodontal tissue, the gingival crevicular fluid (GCF) is the principal target in the search for biomarkers of periodontal disease, because its protein composition may reflect the disease pathophysiology. Biochemical marker analysis of GCF is effective for objective diagnosis in the early and advanced stages of periodontal disease. Increasing numbers of recent reports have provided evidence that the proteomic approach is a promising tool for the discovery and identification of biochemical markers of periodontal disease. This search is of continuing interest in the field of experimental and clinical periodontal disease research. In this article, we summarize recent comprehensive proteomic studies aimed at discovering and identifying biomarkers of periodontal disease in GCF.

クラスリン重鎖(CHC)の免疫組織化学染色は食道粘膜内腫瘍の病理組織診断に有用か検討した。食道粘膜内腫瘍組織は内視鏡下粘膜切除術(EMR)または内視鏡的粘膜下層剥離術(ESD)で切除した47例のパラフィン包埋標本を用いた。新たな組織診断マーカーであるCHCを食道癌の病理組織診断に応用し、良性病変(炎症およびLIN)と悪性病変(HINおよび上皮内がん)を診断するための有用性について検討し、感度・特異度はそれぞれ75%・95%と高い正診率が得られた。さらに、p53およびKi67の染色性の判断基準を作成し、各種マーカーの正診率を求め比較し、CHC染色の成績はP53またはKi-67と同等またはそれ以上であり、さらにCHCとp53とKi-67の3種類のマーカーを組み合わせた結果、感度・特異度は90%・95%と上昇した。

Background/Aim: Survivin is expressed in the nucleus and/or cytoplasm of various types of malignant tumor cells. Nuclear survivin is indispensable for complete mitosis, while cytoplasmic survivin functions as an apoptosis inhibitor. We examined the difference in the survivin expression among stromal cells of fibroadenoma, and benign and malignant phyllodes tumors. Materials and Methods: Tumor sections were immunohistochemically stained with an anti-human survivin antibody and the labeling index of survivin was calculated. Results: In stromal cells of all tumors, survivin was expressed in the nuclei but not in the cytoplasm. The labeling indices of the stromal cells in five malignant phyllodes tumors (20.5±3.0) were significantly greater than those observed in eight fibroadenomas (1.9±0.6) or nine benign phyllodes tumors (3.0±0.9). Conclusion: In the present study it was shown that stromal cells in malignant phyllodes tumors express nuclear survivin more extensively than stromal cells in benign phyllodes tumors or fibroadenomas.

The majority of human cancer shows chromosomal instability (CIN). Although the precise mechanism remains largely uncertain, proper progression of mitosis is crucial. B-type lamins were suggested to be components of the spindle matrix of mitotic cells and to be involved in mitotic spindle assembly; thus, B-type lamins may contribute to the maintenance of chromosome integrity. Here, using a proteomic approach, we identified lamin B2 as a novel protein involved in CIN. Lamin B2 expression decreased in colorectal cancer cell lines exhibiting CIN, as compared with colorectal cancer cell lines exhibiting microsatellite instability (MIN), which is mutually exclusive to CIN. Importantly, lamin B2 knockdown in MIN-type colorectal cancer cells induced CIN phenotypes such as aneuploidy, chromosome mis-segregation and aberrant spindle assembly, whereas ectopic expression of lamin B2 in CIN-type colorectal cancer cells prevented their CIN phenotypes. Additionally, immunohistochemical analysis showed a lower expression of lamin B2 in cancer tissues extracted from patients with sporadic colorectal cancer (CIN-type) than that from patients with hereditary non-polyposis colorectal cancer HNPCC; MIN type). Intriguingly, mitotic lamin B2 in MIN cancer cells was localized outside the spindle poles and mitotic lamin B2 localization was diminished in CIN cancer cells, suggesting an important role of lamin B2 in proper mitotic spindle formation. The obtained results suggest that lamin B2 maintains chromosome integrity by ensuring proper spindle assembly and that its downregulation causes CIN in colorectal cancer.

Introduction: Although genetic information is essential for molecular targeted therapy for personalized medicine, tissue sampling for genetic analysis remains challenging. We investigated the utility of bronchoscopic sampling in non-small-cell lung cancer (NSCLC) patients compared with conventional histological materials for multiple genetic analyses. Methods: Patients with NSCLC proven by onsite cytological evaluation during bronchoscopic survey were eligible for this study. After conventional needle aspiration biopsy by flexible bronchofiberscopy of primary lesions or convex-probe endobronchial ultrasound of lymph nodes, the used needle was rinsed with saline, and the ultra-microsample (uMS) was used for cytological diagnosis and genetic analysis. Gene mutations and fusion genes were examined by high-resolution melting analysis and direct sequencing. The results from the uMS and those from conventional histological samples were compared. Results: A total of 134 lesions (48 primary and 86 metastatic) were analyzed. Adenocarcinoma (n = 80), squamous-cell carcinoma (n = 43), and NSCLC (n = 11) samples were pathologically confirmed in histological cores; however, malignancies were detected in only 45 (34%) of the corresponding uMS. In 62 samples, genetic disorders, including epidermal growth factor receptor (n = 21), K-ras (n = 11), and BRAF mutations (n = 1); anaplastic lymphoma kinase (n = 5), receptor tyrosine kinase (n = 1), and RET fusion genes (n = 1); and silent mutations (n = 22), were identified. In total, 1474 molecular tests were performed, and 1464 tests (99.3%) were identical for both histological samples and uMS. Conclusion: Bronchoscopic uMS (biopsy needle rinsed fluids) are useful for multiple genetic examinations in NSCLC.

Rahmutulla B, Matsushita K, Satoh M, Seimiya M, Tsuchida S, Kubo S, Shimada H, Ohtsuka M, Miyazaki M, Nomura F, Oncotarget, 2014, vol. 5, no. 9, pp. 2404-2417

Background: Although serum albumin levels (sALB) have been quantified by the dye-binding method with bromocresol green (BCG) or bromocresol purple (BCP) on a routine basis, accurate measurement of sALB with these methods is difficult. The modified BCP method is highly specific to albumin without being affected by sample preservation to enable stable and accurate quantification of albumin concentrations. A change in the albumin measurement method may alter the diagnosis of nephrotic syndrome. Methods: sALB was measured in 295 patients including 98 patients with chronic renal disease by the modified BCP method, BCG method, and immunonephelometry as the gold standard. Results: sALB measured by the modified BCP method was well correlated with levels measured by immunonephelometry. sALB obtained by the BCG method was significantly higher than the levels measured by the modified BCP method (p < 0.001, Student's t-test). This tendency was more evident in patients with chronic renal disease than other patients. When the threshold value of sALB for the diagnosis criteria of nephrotic syndrome (<= 25 g/L) and a high risk of thrombosis (<= 20 g/L) in nephrotic syndrome was based on the BCG method, the revised criteria in the modified BCP method would be <= 20.5 and <= 14.9 g/L, respectively. Conclusions: Overestimation of sALB by the BCG method affected diagnosis of nephrotic syndrome. The method by which sALB is measured should be specified in both clinical and research settings in nephrology.

Background: Although serum albumin levels (sALB) have been quantified by the dye-binding method with bromocresol green (BCG) or bromocresol purple (BCP) on a routine basis, accurate measurement of sALB with these methods is difficult. The modified BCP method is highly specific to albumin without being affected by sample preservation to enable stable and accurate quantification of albumin concentrations. A change in the albumin measurement method may alter the diagnosis of nephrotic syndrome. Methods: sALB was measured in 295 patients including 98 patients with chronic renal disease by the modified BCP method, BCG method, and immunonephelometry as the gold standard. Results: sALB measured by the modified BCP method was well correlated with levels measured by immunonephelometry. sALB obtained by the BCG method was significantly higher than the levels measured by the modified BCP method (p < 0.001, Student's t-test). This tendency was more evident in patients with chronic renal disease than other patients. When the threshold value of sALB for the diagnosis criteria of nephrotic syndrome (<= 25 g/L) and a high risk of thrombosis (<= 20 g/L) in nephrotic syndrome was based on the BCG method, the revised criteria in the modified BCP method would be <= 20.5 and <= 14.9 g/L, respectively. Conclusions: Overestimation of sALB by the BCG method affected diagnosis of nephrotic syndrome. The method by which sALB is measured should be specified in both clinical and research settings in nephrology.

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. Gingival crevicular fluid (GCF) is extracted from the gingival sulcus and pocket. Analysis of biochemical markers in GCF, which predict the progression of periodontal disease, may facilitate disease diagnosis. However, no useful GCF biochemical markers with high sensitivity for detecting periodontal disease have been identified. Thus, the search for biochemical markers of periodontal disease is of continued interest in experimental and clinical periodontal disease research. Using tandem mass tag (TMT) labeling, we analyzed GCF samples from healthy subjects and patients with periodontal disease, and identified a total of 619 GCF proteins based on proteomic analysis. Of these, we focused on two proteins, matrix metalloproteinase (MMP)-9 and neutrophil gelatinase-associated lipocalin (LCN2), which are involved in the progression of periodontal disease. Western blot analysis revealed that the levels of MMP-9 and LCN2 were significantly higher in patients with periodontal disease than in healthy subjects. In addition, ELISA also detected significantly higher levels of LCN2 in patients with periodontal disease than in healthy subjects. Thus, LC-MS/MS analyses of GCF using TMT labeling led to the identification of LCN2, which may be a promising GCF biomarker for the detection of periodontal disease.

Suzuki Y, Seimiya M, Yoshida T, Sawabe Y, Matsushita K, Nomura F, Rinsho byori. The Japanese journal of clinical pathology, 2013, vol. 61, no. 8, pp. 665-670

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which carries a significant risk of progression to cirrhosis and hepatocellular carcinoma. Since NASH is a progressive but reversible condition, it is desirable to distinguish NASH from simple steatosis, and to treat NASH patients at an early stage. To establish appropriate diagnosis and therapy, the pathological mechanisms of the disease should be elucidated; however, these have not been fully clarified for both NASH and simple steatosis. This study aims to reveal the differences between simple steatosis and NASH. METHODS: This study used fatty liver Shionogi (FLS) mice as a NASH model, for comparison with dd Shionogi (DS) mice as a model of simple steatosis. Genome-wide gene expression analysis was performed using Affymetrix GeneChip Mouse Genome 430 2.0 Array, which contains 45101 probe sets for known and predicted genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to investigate gene expression changes and protein localizations. RESULTS: DNA microarray analysis of the liver transcriptomes and qRT-PCR of both types of mice revealed that LCN2, CXCL1 and CXCL9 mRNAs were overexpressed in FLS mouse livers. Immunohistochemistry showed that CXCL1 protein was mainly localized to steatotic hepatocytes. CXCL9 protein-expressing hepatocytes and sinusoidal endothelium were localized in some areas of inflammatory cell infiltration. Most interestingly, hepatocytes expressing LCN2, a kind of adipokine, were localized around almost all inflammatory cell clusters. Furthermore, there was a positive correlation between the number of LCN2-positive hepatocytes in the specimen and the number of inflammatory foci. CONCLUSIONS: Overexpression and distinct localization of LCN2, CXCL1 and CXCL9 in the liver of fatty liver Shionogi mice suggest significant roles of these proteins in the pathogenesis of NASH.