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Central nervous system tumors are classified based on an integrated diagnosis combining histology and molecular characteristics, including IDH1/2 and H3‐K27M mutations, as well as 1p/19q codeletion. Here, we aimed to develop and assess the feasibility of a glioma‐tailored 48‐gene next‐generation sequencing (NGS) panel for integrated glioma diagnosis. We designed a glioma‐tailored 48‐gene NGS panel for detecting 1p/19q codeletion and mutations in IDH1/2, TP53, PTEN, PDGFRA, NF1, RB1, CDKN2A/B, CDK4, and the TERT promoter (TERTp). We analyzed 106 glioma patients (grade II: 19 cases, grade III: 23 cases, grade IV: 64 cases) using this system. The 1p/19q codeletion was detected precisely in oligodendroglial tumors using our NGS panel. In a cohort of 64 grade Ⅳ gliomas, we identified 56 IDH‐wildtype glioblastomas. Within these IDH‐wildtype glioblastomas, 33 samples (58.9%) showed a mutation in TERTp. Notably, PDGFRA mutations and their amplification were more commonly seen in TERTp‐wildtype glioblastomas (43%) than in TERTp‐mutant glioblastomas (6%) (P = .001). Hierarchical molecular classification of IDH‐wildtype glioblastomas revealed 3 distinct groups of IDH‐wildtype glioblastomas. One major cluster was characterized by mutations in PDGFRA, amplification of CDK4 and PDGFRA, homozygous deletion of CDKN2A/B, and absence of TERTp mutations. This cluster was significantly associated with older age (P = .021), higher Ki‐67 score (P = .007), poor prognosis (P = .012), and a periventricular tumor location. We report the development of a glioma‐tailored NGS panel for detecting 1p/19q codeletion and driver gene mutations on a single platform. Our panel identified distinct subtypes of IDH‐ and TERTp‐wildtype glioblastomas with frequent PDGFRA alterations., A glioma‐tailored 48‐gene next‐generation sequencing panel identified distinct subtypes of wildtype IDH and TERT promoter glioblastomas with PDGFRA alterations.

Purkinje cell protein 4/peptide 19 (PCP4/PEP19) is 7.6 kDa peptide originally found in Purkinje cells. PCP4/PEP19 is a differentiation maker of Purkinje cells, where it functions as an antiapoptotic factor. Cerebral neuronal cells also express PCP4/PEP19, which may be related to neuronal cell survival. However, evidence suggests that PCP4/PEP19 may also be involved in neuronal differentiation. Here, we investigated the effects of PCP4/PEP19 expression on neuronal differentiation by analyzing neurite outgrowth, and expression of neuronal differentiation markers in cultured human neuroblastoma M17 cells. When PCP4/PEP19 expression was reduced by siRNA-mediated knockdown, neurite outgrowth was significantly increased. Among many differentiation markers tested, expression of NeuroD1 was increased, while that of Ascl1 was decreased upon PCP4/PEP19 knockdown. Furthermore, luciferase reporter assays revealed that PCP4/PEP19 knockdown upregulated NeuroD1 and downregulated Ascl1 expression, at the transcriptional level. These results suggest a new function of PCP4/PEP19, which suppresses neurite outgrowth and neuronal differentiation through the regulation of NeuroD1 and Ascl1 expression in M17 cells. Furthermore, immunohistochemical studies showed that PCP4/PEP19 localizes in the nuclei of human neuroblastoma cells. Therefore, PCP4/PEP19 may also be an intranuclear negative regulator of neuronal differentiation and may thus be a potential therapeutic target to promote cellular differentiation in human neuroblastoma. PCP4/PEP19 knockdown in neuroblastoma cells induce neurite outgrowth, upregulation of NeuroD1 and downregulation of Ascl1 expression, suggesting that PCP4/PEP19 can suppress neurite outgrowth and neuronal differentiation through the regulation of NeuroD1 and Ascl1. Immunohistochemistry shows nuclear localization of PCP4/PEP19 in neuroblastoma cells. PCP4/PEP19 may therefore be an intranuclear negative regulator of neuronal differentiation.

Background Since short bowel syndrome (SBS) patients face life-threatening conditions, the development of therapeutic strategies to induce intestinal adaptation has been investigated. Ghrelin, a ligand of growth hormone (GH) secretagogue-receptor that stimulates the release of GH and insulin like growth factor-1 (IGF-1), has several pleiotropic effects. We investigated whether ghrelin induces intestinal adaptation in parenterally fed rats with SBS. Methods Sprague-Dawley rats underwent venous catheterization and were divided into 3 groups: those receiving 90% small bowel resection while leaving the proximal jejunum and distal ileum (90% SBR) with TPN (SBS/TPN group), those receiving 90% SBR with TPN + ghrelin (SBS/TPN/ghrelin group), and those receiving sham operation and fed chow (sham group). Ghrelin was administered intravenously at 10 μg/kg/day. On Day 13, the rats were euthanized and the small intestine harvested, and the histology and crypt cell proliferation rates (CCPR), apoptosis, and nutrient transporter protein levels were analyzed and the plasma hormones were measured. Results The villus height and crypt depth of the ileum in the SBS/TPN/ghrelin group were significantly higher than in the SBS/TPN group. The CCPR of the jejunum and the ileum significantly increased by the administration of ghrelin; however, the apoptosis rates did not significantly differ between the SBS/TPN and SBS/TPN/ghrelin groups. Significant differences did not exist in the plasma IGF-1 and nutrient transporter protein levels among three groups. Conclusions The intravenous administration of ghrelin stimulated the morphological intestinal adaptation of the ileum to a greater degree than the jejunum due to the direct effect of ghrelin.

Lung cancer remains a disease of high mortality, despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in lung neoplasms. Our immunohistochemistry (IHC) studies have shown that high MUC4 expression correlates with a poor outcome. We have also shown that the expression of several mucin genes in cancer cell lines is regulated by DNA methylation. We evaluated the expression level of MUC4, mRNA and several DNA hypomethylation factors in lung tissue samples from 33 patients with various lung lesions. The results indicated that the DNA methylation status of MUC4 matched the expression level of mRNA. In addition, the TET1 (Ten-Eleven Translocation) mRNA showed a significant correlation with the status of DNA methylation of MUC4. Furthermore, the treatment of a lung cancer cell line with TET1 siRNA caused a reduction in MUC4 mRNA expression. Thus, we suggest that TET1 mediated DNA hypomethylation plays a key role in the expression of MUC4. This is the first report that TET1 mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer. The analysis of these epigenetic changes may be useful for diagnosing carcinogenic risk.

Aim We have recently established a novel swine model for studies of atherosclerosis using MicrominipigsTM (µMPs) fed a high-fat/high-cholesterol diet (HcD). Using this swine model, we re-evaluated the effects of dietary cholic acid (CA) on serum lipid profile, atherosclerosis and hepatic injuries. Methods The µMPs were fed HcD supplemented with 0.7% CA (HcD＋CA) for eight weeks, and the effect of CA on serum lipoprotein levels, expression of oxidative stress markers, adiposity and lesion formation in the aorta, liver, and other organs was investigated. Results The HcD＋CA-fed group exhibited more visceral adiposity, progression of atherosclerosis and higher serum levels of oxidative stress markers than the HcD-fed group, even though they showed similar serum lipid levels. The liver demonstrated increased lipid accumulation, higher expression of oxidative stress markers, accelerated activation of foamy Kupffer cells and stellate cells, and increased hepatocyte apoptosis, indicating non-alcoholic fatty liver disease (NAFLD). Intriguingly, foamy macrophage mobilization was observed in various organs, including the reticuloendothelial system, pulmonary capillary vessels and skin very often in HcD＋CA-fed µMPs. Conclusion To our knowledge, this is the first large animal model, in which visceral obesity, NAFLD and atherosclerosis are concomitantly induced by dietary manipulation. These data suggest the detrimental effects of CA, potentially through local and systemic activation of oxidative stress-induced signaling to macrophage mobilization, on the acceleration of visceral adiposity, atherosclerosis and NAFLD.

Purpose: Pancreatic cancer remains a disease of high mortality despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic cancers. MUC1 and MUC4 expression are related to the aggressive behavior of human neoplasms and a poor patient outcome. In contrast, MUC2 is a tumor suppressor, and we have previously reported that MUC2 is a favorable prognostic factor in pancreatic neoplasia. This study investigates whether the methylation status of three mucin genes from postoperative tissue specimens from patients with pancreatic neoplasms could serve as a predictive biomarker for outcome after surgery. Experimental Design: We evaluated the methylation status of MUC1, MUC2, and MUC4 promoter regions in pancreatic tissue samples from 191 patients with various pancreatic lesions using methylation-specific electrophoresis. Then, integrating these results and clinicopathologic features, we used support vector machine-, neural network-, and multinomial-based methods to develop a prognostic classifier. Results: Significant differences were identified between the positive- and negative-prediction classifiers of patients in 5-year overall survival (OS) in the cross-validation test. Multivariate analysis revealed that these prognostic classifiers were independent prognostic factors analyzed by not only neoplastic tissues but also nonneoplastic tissues. These classifiers had higher predictive accuracy for OS than tumor size, lymph node metastasis, distant metastasis, and age and can complement the prognostic value of the TNM staging system. Conclusions: Analysis of epigenetic changes in mucin genes may be of diagnostic utility and one of the prognostic predictors for patients with pancreatic ductal adenocarcinoma.

// Takuya Yoshimura 1 , Taiji Hamada 2 , Hiroshi Hijioka 1 , Masakazu Souda 2 , Kazuhito Hatanaka 2 , Takako Yoshioka 3 , Sohsuke Yamada 2 , Masato Tsutsui 4 , Yoshihisa Umekita 5 , Norifumi Nakamura 1 , Akihide Tanimoto 2 1 Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan 2 Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan 3 Department of Pathology, Children’s Cancer Center, National Center for Child Health and Development, Tokyo, Japan 4 Department of Pharmacology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan 5 Division of Organ Pathology, Faculty of Medicine, Tottori University, Yonago, Japan Correspondence to: Akihide Tanimoto, email: akit09@m3.kufm.kagoshima-u.ac.jp Keywords: PCP4/PEP19, breast cancer, EMT, migration, invasion Abbreviations: Purkinje cell protein 4/peptide 19, PCP4/PEP19; epithelial-mesenchymal transition, EMT Received: September 02, 2015 Accepted: January 22, 2016 Published: February 20, 2016 ABSTRACT Purkinje cell protein (PCP) 4/peptide (PEP) 19 is expressed in Purkinje cells where it has a calmodulin-binding, anti-apoptotic function. We recently demonstrated that PCP4/PEP19 is expressed and inhibit apoptosis in human breast cancer cell lines. In the present study we investigated the role of PCP4/PEP19 in cell morphology, adhesion, migration, and invasion in MCF-7 and T47D human breast cancer cell lines. Knockdown of PCP4/PEP19 reduced the formation of filopodia-like cytoplasmic structures and vinculin expression, and enhanced E-cadherin expression. Activities of migration, invasion, and cell adhesion were also decreased after the knockdown of PCP4/PEP19 in MCF-7 and T47D cells. These results suggested that PCP4/PEP19 promotes cancer cell adhesion, migration, and invasion and that PCP4/PEP19 may be a potential target for therapeutic agents in breast cancer treatment which act by inhibiting epithelial-mesenchymal transition and enhancing apoptotic cell death.

Peroxiredoxin 4 (PRDX4) is a member of the peroxiredoxin family of antioxidant enzymes. Previously, we reported that PRDX4 can restrain the initiation and progression of nonalcoholic steatohepatitis by reducing local and systemic reactive oxygen species (ROS) levels. Oxidative stress is recognized as a key factor in hepatocarcinogenesis, and a high ROS level has also been found in hepatocellular carcinoma (HCC). Here, our aim is to investigate roles of PRDX4 in the initiation and progression of HCC.In this study, for hepatocarcinogenesis, wild-type (WT), PRDX4 knockout (PRDX4

The Purkinje cell protein 4/peptide 19 (PCP4/PEP19) is a novel breast cancer cell expressing peptide, originally found in the neural cells as an anti-apoptotic factor, could inhibit cell apoptosis and enhance cell migration and invasion in human breast cancer cell lines. The expression of PCP4/PEP19 is induced by estrogens in estrogen receptor-positive (ER+) MCF-7 cells but also highly expressed in ER- SK-BR-3 cells. In this study, we investigated the effects of PCP4/PEP19 on aromatase gene expression in MCF-7 and SK-BR-3 human breast cancer cells. In SK-BR-3 cells but not in MCF-7 cells, PCP4/PEP19 knockdown by siRNA silencing decreased the aromatase expression in gene transcriptional level. When PCP4/PEP19 was overexpressed by CMV promoter-driven PCP4/PEP19 expressing plasmid transfection, aromatase gene transcription increased in SK-BR-3 cells. This aromatase gene transcription is mainly mediated through promoter region PI.1, which is usually active in the placental tissue but not in the breast cancer tissue. These results indicate a new function of PCP4/PEP19 that would enhance aromatase gene upregulation to supply estrogens in heterogeneous cancer microenvironment.

// Taiji Hamada 1 , Masakazu Souda 1 , Takuya Yoshimura 2 , Shoko Sasaguri 1 , Kazuhito Hatanaka 1 , Takashi Tasaki 1 , Takako Yoshioka 1 , Yasuyo Ohi 3 , Sohsuke Yamada 4 , Masato Tsutsui 5 , Yoshihisa Umekita 6 and Akihide Tanimoto 1 1 Department of Molecular and Cellular Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. 2 Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. 3 Department of Pathology, Sagara Hospital, Social Medical Corporation Hakuaikai, Kagoshima, Japan. 4 Department of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. 5 Department of Pharmacology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan. 6 Division of Organ Pathology, Faculty of Medicine, Tottori University, Yonago, Japan. Correspondence to: Akihide Tanimoto, e-mail: akit09@m3.kufm.kagoshima-u.ac.jp Key words: PCP4/PEP19, breast cancer, apoptosis, CaMKK, Akt Received: April 23, 2014 Accepted: June 23, 2014 Published: July 08, 2014 ABSTRACT The PCP4/PEP19 is a calmodulin-binding anti-apoptotic peptide in neural cells but its potential role in human cancer has largely been unknown. We investigated the expression of PCP4/PEP19 in human breast cancer cell lines MCF-7, SK-BR-3, and MDA-MB-231 cells, and found that estrogen receptor (ER)-positive MCF-7 and ER-negative SK-BR-3 cells expressed PCP4/PEP19. In the MCF-7 cells, cell proliferation was estrogen-dependent, and PCP4/PEP19 expression was induced by estrogen. In both cell lines, PCP4/PEP19 knockdown induced apoptosis and slightly decreased Akt phosphorylation. Knockdown of calcium/calmodulin-dependent protein kinase kinase 1 (CaMKK1), resulting in decreased phospho-Akt Thr308 , enhanced apoptosis in SK-BR-3 but not in MCF-7 cells. CaMKK2 knockdown moderately decreased phospho-Akt Thr308 and increased apoptosis in MCF-7 cells but not in SK-BR-3 cells. These data indicated that PCP4/PEP19 regulates apoptosis but exact mechanism is still unknown. PCP4/PEP19 can therefore potentially serve as independent oncotarget for therapy of PCP4/PEP19-positive breast cancers irrespective of ER expression.

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.

Diethlstilbestrol (DES) is a synthetic estrogen prescribed to several millions of pregnant women worldwide. The risk for breast cancer after age 40 in women prenatally exposed to DES has been reported; however, the precise mechanism of susceptibility to breast cancer remains to be resolved. We investigated the global gene expression profile of terminal end buds (TEBs), the target of carcinogen, in rat mammary glands neonatally exposed to a low- or high-dose DES at postnatal days (PND) 35 and 45, equivalent to the peripubertal stage in humans. In all groups, the number of TEBs gradually increased, peaked at PND35 and decreased at PND49. At PND35 and 49, the low-dose DES-treated group (low-DES group) showed the highest number of TEBs. In the low-DES group at PND35, β-casein, γ-casein and whey acidic protein (WAP) mRNA expression increased 8.2-fold, 26.1-fold and 13.3-fold, respectively, whereas γ-casein and WAP mRNA decreased 17.6-fold and 27.7-fold, respectively, at PND49. The most significant network revealed by Ingenuity Pathway Analysis (IPA) software showed the relevance of NF-κB in low-DES group. The present findings suggest that the deregulation of mammary gland differentiation and development-related genes could be induced and cause the increased number of terminal duct lobular units (TDLUs) in human mammary glands of DES daughters in a critical period of mammary gland development.

Accumulating evidence indicates that oxidative stress plays a critical role in initiating the progression of inflammatory and fibrotic liver diseases, including cholestatic hepatitis. Peroxiredoxin 4 (PRDX4) is a secretory antioxidase that protects against oxidative damage by scavenging reactive oxygen species (ROS) in both the intracellular compartments and extracellular space. In this study, we examined the in vivo net effects of PRDX4 overexpression in a murine model of cholestasis. To induce cholestatic liver injury, we subjected C57BL/6J wild-type (WT) or human PRDX4 (hPRDX4) transgenic (Tg) mice to sham or bile duct ligation (BDL) surgery for seven days. Our results showed that the liver necrosis area was significantly suppressed in Tg BDL mice with a reduction in the severity of liver injuries. Furthermore, PRDX4 overexpression markedly reduced local and systemic oxidative stress generated by BDL. In addition, suppression of inflammatory cell infiltration, reduced proliferation of hepatocytes and intrahepatic bile ducts, and less fibrosis were also found in the liver of Tg BDL mice, along with a reduced mortality rate after BDL surgery. Interestingly, the composition of the hepatic bile acids (BAs) was more beneficial for Tg BDL mice than for WT BDL mice, suggesting that PRDX4 overexpression may affect BA metabolism during cholestasis. These features indicate that PRDX4 plays an important role in protecting against liver injury following BDL and might be a promising therapeutic modality for cholestatic diseases.

Background: Pleomorphic adenoma of the salivary gland (PA) is essentially a benign neoplasm. However, patients with recurrent PA are difficult to manage. There are rare reports on useful immunohistochemical markers to detect a high risk of recurrence when the primary lesions are resected. Aims: To find a new marker to predict the recurrence of PA. Methods: Primary lesions of PA were collected from nine patients showing subsequent recurrence and from 40 patients without recurrence during at least 10 years of follow up of the disease. Paraffin wax embedded tumour samples of the two groups were examined for the expression profiles of MUC1 (differentially glycosylated forms), MUC2, MUC4, MUC5AC, and MUC6 using immunohistochemistry. Several clinicopathological factors were also examined. Results: In univariate analysis of the factors examined, MUC1/DF3 high expression (more than 30% of the neoplastic cells stained) in the primary lesions was seen more frequently in patients with recurrence (four of nine) than in those without recurrence (three of 40; p = 0.011). Larger tumour size (more than 3.0 cm) of the primary PA was also a significant (p = 0.035) risk factor for the recurrence of PA. In multivariate analysis, only high expression of MUC1/DF3 was found to be a significant independent risk factor for the recurrence of PA (p = 0.021). Conclusions: Expression of MUC1/DF3 in PA is a useful marker to predict its recurrence. Those patients with PA showing positive MUC1/DF3 expression should be followed up carefully.

The purpose of this study was to evaluate a telemetry system for examining the cardiovascular system in the conscious common marmoset. Parameters obtained were blood pressure, heart rate, respiratory rate, ECG, body temperature and locomotor activity, and these were continuously recorded on a data recorder via the telemetry system and then processed by a computerized system. Diurnal rhythms of blood pressure, heart rate, body temperature and locomotor activity were observed in this system. We studied the effects of astemizole (antihistamine) and nicardipine (Ca2+ channel blocker) on cardiovascular parameters. Astemizole at 30 mg/kg (p.o.) and at 1 to 3 mg/kg (i.v.), prolonged QT interval and induced ventricular extrasystole. Torsades de pointes occurred in one of three cases at 3 mg/kg (i.v.) and 30 mg/kg (p.o.), while it did not affect the blood pressure, respiratory rate and body temperature. Nicardipine at 30 mg/kg (p.o.) caused sustained hypotension and tachycardia. These results demonstrate the usefulness of the telemetry system using the common marmoset for evaluating the cardiovascular effects of drugs under physiological conditions.

Many patients with invasive ductal carcinoma of the pancreas (IDC) have a poor outcome. MUC4 expression has been implicated as a marker for diagnosis and progression of IDC, but there are no studies of the relation between MUC4 expression and patient prognosis in IDC.To investigate the prognostic significance of MUC4 expression in IDC.The expression profiles of MUC4, ErbB2, p27, and MUC1 were investigated in IDC tissues from 135 patients by means of immunohistochemistry.MUC4 was expressed in 43 of the 135 patients with IDC (31.9%). The survival of 21 patients with high MUC4 expression (20% of neoplastic cells stained) was significantly worse than that of the 114 patients with low MUC4 expression (20% of neoplastic cells stained) (p = 0.0043). Univariate analysis showed that high MUC4 expression (p = 0.0061), large primary tumour status (T2) (p = 0.0436), distant metastasis (p = 0.0383), lymphatic invasion (p = 0.0243), and surgical margins (p = 0.0333) were significant risk factors affecting the outcome of patients with IDC. Backward stepwise multivariate analysis showed that MUC4 expression (p = 0.0121), lymph node metastasis (p = 0.0245), and lymphatic invasion (p = 0.0239) were significant independent risk factors. ErbB2, p27, and MUC1 were not independent risk factors.This study shows that MUC4 expression in IDC is a new independent factor for poor prognosis and predicts the outcome of patients with IDC.