Overproduction of thrombopoietin by BRAFV600E-mutated mouse hepatocytes and contribution of thrombopoietin to hepatocarcinogenesis.

In hepatocarcinogenesis induced by diethylnitrosamine (DEN) in B6C3F1 mice, the BrafV637E mutation, corresponding to the human BRAFV600E mutation, plays a pivotal role. The livers of transgenic mice with a hepatocyte-specific human BRAFV600E mutation weighed 4.5 times more than that of normal mice and consisted entirely of hepatocytes, resembling DEN-induced preneoplastic hepatocytes. However, these transgenic mice spontaneously died 7 wk after birth, therefore this study aimed to clarify the causes of death. In the transgenic mice, the liver showed thrombopoietin (TPO) overexpression, which is associated with eventual megakaryocytosis and thrombocytosis, and activated platelets were deposited in hepatic sinusoids. TPO was also overexpressed in the DEN-induced hepatic tumors, and sinusoidal platelet deposition was observed in the hepatic tumors of humans and mice. Podoplanin was expressed in some of the Kupffer cells in the liver of the transgenic mice, indicating that platelet activation occurred via the interaction of podoplanin with C-type lectin receptor 2 (CLEC-2) on the platelet membrane. Additionally, erythrocyte dyscrasia and glomerulonephropathy/interstitial pneumonia associated with platelet deposition were observed. In the transgenic mice, aspirin (Asp) administration prevented platelet activation, reduced the liver/body weight ratio, decreased the platelet deposition in the liver, kidney, and lung, and prevented erythrocyte dyscrasia and ameliorated the renal/pulmonary changes. Thrombopoietin overproduction by BRAFV600E-mutated hepatocytes may contribute to hepatocyte proliferation via thrombocytosis, platelet activation, and the interaction of platelets with hepatic sinusoidal cells, while hematologic, renal, and pulmonary disorders due to aberrant platelet activation may lead to spontaneous death in the transgenic mice.

Role of the BrafV637E mutation in hepatocarcinogenesis induced by treatment with diethylnitrosamine in neonatal B6C3F1 mice.

The BrafV637E mutation is frequently reported in mouse hepatic tumors, depending on the mouse strain, and corresponds to the human BrafV600E mutation. In this study, we detected the BrafV637E mutation by whole-exome analysis in 4/4 hepatic tumors induced by neonatal treatment with diethylnitrosamine (DEN) in male B6C3F1 mice. We also detected the BrafV637E mutation in 54/63 (85.7%) hepatic lesions, including microscopic foci and grossly visible tumors, by PCR-direct sequencing. Although the mutation was detected in 5/7 (71.4%) hepatic tumors induced by neonatal DEN treatment followed by repeated CCl4 administration, it was not detected in 24 tumors induced by CCl4 treatment without DEN or in eight spontaneous lesions in B6C3F1 mice, suggesting that the mutation is induced by the genotoxic action of DEN. The DEN-induced tumors exhibited hyperphosphorylation of ERK1 and Akt, suggesting that the BrafV637E mutation might activate the MAPK and Akt pathways. Moreover, the DEN-induced tumors overexpressed mRNAs for the oncogene-induced senescence (OIS) markers such as p15Ink4b and p19Arf as well as pro-survival/pro-proliferative cytokines/chemokines such as complement C5/C5a, ICAM-1, IL-1 receptor antagonist and CXCL9, suggesting that the BrafV637E mutation influences the expression of genes involved in either OIS or cellular growth/survival. Liver-specific expression of mutated Braf under control of the albumin enhancer/promoter resulted in an enlarged liver that consisted entirely of small basophilic hepatocytes resembling DEN-induced preneoplastic hepatocytes with ERK1/Akt hyperphosphorylation and C5/C5a overexpression. These results indicate that the BrafV637E mutation induces hepatocytic changes in DEN-induced hepatic tumors. © 2016 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Outcomes of trastuzumab therapy in HER2-positive early breast cancer patients.

BACKGROUND: Previous large trials with trastuzumab (TZM) showed improved outcome in patients with HER2-positive early-stage breast cancer. However, the efficacy and safety of TZM in Japanese patients have not been fully evaluated. We have therefore conducted an observational study in Japan. METHODS: This was a retrospective and a prospective observational study in which data on women with histologically confirmed HER2-positive invasive breast cancer who received TZM for stage I-IIIC disease were collected from 56 institutions that participated in the Japan Breast Cancer Research Group and the efficacy of each treatment regimen analyzed. RESULTS: A total of 2,024 patients treated between July 2009 and June 2011 were initially enrolled in this study; in August 2013, the patient cohort comprised 2,009 patients. Of these, 142 (7.5 %) were aged ≥70 years, 1,097 (58.1 %) had clinically node-negative (cN0) breast cancer, and 883 (47.4 %) were estrogen receptor-positive. Treatment options were neoadjuvant therapy (662 patients) and adjuvant therapy with TZM (1,228 patients). Three-year overall survival (OS) rates in the entire cohort and in the neoadjuvant and adjuvant cohorts, respectively, were 98.9 [95 % confidence interval (CI) 98.2-99.3], 98.3 (95 % CI 96.8-99.1 %), and 99.2 % (95 % CI 98.4-99.6), respectively. Three-year disease-free survival (DFS) rates in the entire cohort and in the neoadjuvant and adjuvant cohorts, respectively were 94.2 (95 % CI 93.0-95.2), 94.8 (95 % CI 93.0-95.9), and 93.1 (95 % CI 90.7-94.9 %), respectively. Multivariate analysis showed that age and nodal status negatively correlated with DFS. Age was the only factor which correlated with OS rate. Adverse events (AEs) associated with TZM and grade 3/4 AEs were reported in 356 (18.8 %) and 14 (0.6 %) patients, respectively. Grade 3/4 cardiac toxicities were reported in 11 patients. CONCLUSION: Based on data from our patient cohort of Japanese women with HER2-positive early-stage breast cancer, the efficacy and safety of systemic therapy with TZM are comparable to data from previously conducted large trials. Progress in anti-HER2 therapy for patients aged ≥70 years who have a poorer prognosis is needed.

Homologous recombination contributes to the repair of acetaldehyde-induced DNA damage.

Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g. in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reactions among biological substances including DNA, RNA, and protein, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we explored mechanisms involved in the repair of acetaldehyde-induced DNA damage by examining the cellular sensitivity to acetaldehyde in the collection of human TK6 mutant deficient in each genome maintenance system. Among the mutants, mismatch repair mutants did not show hypersensitivity to acetaldehyde, while mutants deficient in base and nucleotide excision repair pathways or homologous recombination (HR) exhibited higher sensitivity to acetaldehyde than did wild-type cells. We found that acetaldehyde-induced RAD51 foci representing HR intermediates were prolonged in HR-deficient cells. These results indicate a pivotal role of HR in the repair of acetaldehyde-induced DNA damage. These results suggest that acetaldehyde causes complex DNA damages that require various types of repair pathways. Mutants deficient in the removal of protein adducts from DNA ends such as TDP1-/- and TDP2-/- cells exhibited hypersensitivity to acetaldehyde. Strikingly, the double mutant deficient in both TDP1 and RAD54 showed similar sensitivity to each single mutant. This epistatic relationship between TDP1-/- and RAD54-/- suggests that the protein-DNA adducts generated by acetaldehyde need to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of the genotoxic and mutagenic effects of acetaldehyde.

SOD1 gains pro-oxidant activity upon aberrant oligomerization: change in enzymatic activity by intramolecular disulfide bond cleavage.

Copper-zinc superoxide dismutase (SOD1) has been proposed as one of the causative proteins of amyotrophic lateral sclerosis (ALS). The accumulation of non-native conformers, oligomers, and aggregates of SOD1 in motor neurons is considered responsible for this disease. However, it remains unclear which specific feature of these species induces the onset of ALS. In this study, we showed that disulfide-linked oligomers of denatured SOD1 exhibit pro-oxidant activity. Substituting all the cysteine residues in the free thiol state with serine resulted in the loss of both the propensity to oligomerize and the increase in pro-oxidant activity after denaturation. In contrast, these cysteine mutants oligomerized and acquired the pro-oxidant activity after denaturation in the presence of a reductant that cleaves the intramolecular disulfide bond. These results indicate that one of the toxicities of SOD1 oligomers is the pro-oxidant activity induced by scrambling of the disulfide bonds. Small oligomers such as dimers and trimers exhibit stronger pro-oxidant activity than large oligomers and aggregates, consistent with the trend of the cytotoxicity of oligomers and aggregates reported in previous studies. We propose that the cleavage of the intramolecular disulfide bond accompanied by the oligomerization reduces the substrate specificity of SOD1, leading to the non-native enzymatic activity.

Outcomes of trastuzumab therapy in HER2-positive early breast cancer patients: extended follow-up of JBCRG-cohort study 01.

BACKGROUND: Previous large trials of trastuzumab (TZM) demonstrated improved outcomes in patients with HER2-positive early breast cancer. However, its effectiveness and safety in Japanese patients is not yet clear. Recently, new anti-HER2 agents were developed to improve treatment outcomes, but the patient selection criteria remain controversial. PURPOSE: The aim of this study was to evaluate the long-term effectiveness of TZM therapy as perioperative therapy for HER2-positive operable breast cancer in daily clinical practice and to create a recurrence prediction model for therapeutic selection. METHODS: An observational study was conducted in Japan (UMIN000002737) to observe the prognosis of women (n = 2024) with HER2-positive invasive breast cancer who received TZM for stage I-III C disease between July 2009 and June 2011. Moreover, a recurrence-predicting model was designed to evaluate the risk factors for recurrence. RESULTS: The 5- and 10-year disease-free survival (DFS) rates were 88.9 (95% CI 87.5-90.3%) and 82.4% (95% CI 79.2-85.6%), respectively. The 5- and 10-year overall survival (OS) rates were 96% (95% CI 95.1-96.9%) and 92.7% (95% CI 91.1-94.3%), respectively. Multivariate analysis revealed that the risk factors for recurrence were an age of ≥ 70 years, T2 or larger tumors, clinically detected lymph node metastasis, histological tumor diameter of > 1 cm, histologically detected lymph node metastasis (≥ n2), and the implementation of preoperative treatment. The 5-year recurrence rate under the standard treatment was estimated to be > 10% in patients with a score of 3 or greater on the recurrence-predicting model. CONCLUSION: The recurrence-predicting model designed in this study may improve treatment selection of patients with stage I-III C disease. However, further studies are needed to validate the scores generated by this model.

Survival Outcomes of Retreatment with Trastuzumab and Cytotoxic Chemotherapy for HER2-Positive Recurrent Patients With Breast Cancer Who Had Been Treated with Neo/adjuvant Trastuzumab Plus Multidrug Chemotherapy: A Japanese Multicenter Observational Study.

BACKGROUND: There are little data on the usefulness of trastuzumab (TZM) retreatment as the first-line treatment for patients with HER2 (human epidermal growth factor receptor 2)-positive breast cancer recurrence after perioperative treatment with TZM. AIM: To clarify the outcome and safety of TZM retreatment in patients with recurrent HER2-positive breast cancer. METHOD: An observational study was conducted on patients who relapsed after primary systemic therapy with TZM using the central registration system. The primary end point was progression-free survival (PFS). Secondary end points consisted of the response rate, overall survival (OS), and safety. RESULT: In total, 34 patients were registered between July 2009 and June 2012. The median follow-up time was 23.7 months (2-24 months). The 1- and 2-year PFS rates were 46.9% (95% confidence interval (95% CI): 29.2%-62.9%) and 29.8% (95% CI: 15.0%-46.3%), respectively (median 10.6 months). The median PFS time for patients receiving TZM combined with CTx was 13.9 months. The 1-and 2-year OR rates were 93.9 (95% CI: 77.9%-98.4%) and 84.8% (95% CI: 67.4%-93.4%). Trastuzumab-induced grade 3/4 adverse events were not observed. CONCLUSIONS: This study suggests that the PFS and OS in Japanese patients who relapsed after perioperative TZM therapy improved or were similar to those in previous reports. Differences in patient backgrounds and treatments must be considered when interpreting the results. Trastuzumab should be used combination with CTx and/or HTx for retreatment. Retreatment with TZM is safe.Trial registration: UMIN000002738.

[Results of survey conducted on perioperative chemotherapy and supportive care in primary breast cancer (JBCRG01)].

We carried out a survey of supportive care at institutions that participated in the JBCRG01 study (FEC followed by docetaxel) as neoadjuvant therapy for operable breast cancer. The purpose was to share the information of supportive care for the treatment effect of perioperative intensive chemotherapy among institutions. Appropriate supportive care for nausea, vomiting, edema and febrile neutropenia (FN) is important with respect to the safety of chemotherapy. According to the results of the questionnaire, support from the family and the relationships with doctors, nurses and pharmacists familiar with the chemotherapy were important. The equipment and service for outpatients' cancer chemotherapy center are also important. This multicenter study enhances the exchange of information among institutes. The results of this survey suggest that adequate supportive care makes anthracycline and taxane chemotherapy manageable in the outpatient setting.

Serotonin transporter gene promoter polymorphism and autism: a family-based genetic association study in Japanese population.

Autism is now widely accepted as a biological disorder which, by and large, starts before birth. It has been shown that serotonin (5-HT) is associated with several psychological processes and hyperserotoninemia is observed in some autistic patients. The results of previous reports about family-based association studies between the serotonin transporter (5-HTT) gene promoter polymorphism and autism are controversial. In this study, an analysis using the transmission/disequilibrium test (TDT) between the 5-HTT gene promoter polymorphism and autism in 104 trios, all ethnically Japanese, showed no significant linkage disequilibrium (P=0.17). Recently, it has been reported that some haplotypes at the serotonin transporter locus may be associated with the pathogenesis of autism. Therefore, further investigations by haplotype analyses are necessary to confirm the implications of genetic variants of the serotonin transporter in the etiology of autism.

Physical and biochemical properties of mammalian DNase X proteins: non-AUG translation initiation of porcine and bovine mRNAs for DNase X.

DNase X is the first human DNase protein identified as being homologous with DNase I. In the present study we describe the isolation of several mammalian DNase X cDNAs and the molecular characterization of their coding proteins. A sequence comparison reveals some conserved characteristics: all the mammalian DNase X proteins have an N-terminal signal peptide, a potential N-linked glycosylation site and a C-terminal hydrophobic domain. Human DNase X, ectopically expressed in HeLa S3 cells, is located in the ER (endoplasmic reticulum) and is modified by an N-linked glycosylation at Asn-243. Gene expression analyses show that the high expression level in muscular tissues, a known feature of human DNASE X, is also observed in mouse DNase X. Interestingly, the translation of porcine and bovine DNase X proteins occurs in the absence of an in-frame AUG initiation codon. We show that their mRNAs utilize a conserved CUG triplet for translation initiation.

Interim analysis of a phase II trial of cyclophosphamide, epirubicin and 5-fluorouracil (CEF) followed by docetaxel as preoperative chemotherapy for early stage breast carcinoma.

PURPOSE: A single-arm phase II multicenter trial of the combination of cyclophosphamide (C), epirubicin (E), and 5-fluorouracil (F) followed by docetaxel as neoadjuvant chemotherapy is being conducted by the Japan Breast Cancer Research Group. This report describes an interim analysis of the clinical response and safety of 79 patients who finished preoperative chemotherapy and surgery. PATIENTS AND METHODS: Patients with operable breast cancer received C at 500 mg/m2, E at 100 mg/m2, and F at 500 mg/m2 every 21 days for 4 cycles followed by docetaxel at 75 mg/m2 every 21 days for 4 cycles. RESULTS: Of the 79 patients evaluable for analysis the median age was 46 years (28-59), and 61 patients (77.2%) had T2 tumors. A total of 312 of 316 (98.7%) cycles of CEF and 296 of 312 (94.9%) cycles of docetaxel were administered. Average total cumulative dose was 92% and 95% for CEF and docetaxel, respectively. The rate and grade of edema, neuropathy, arthralgia and myalgia were higher with docetaxel than with CEF. The overall clinical response rate was 70.9%. Breast conserving surgery was performed in 31 of 42 patients (73.8%) with a base-line tumor size of more than 3 cm. CONCLUSIONS: Interim data suggest that CEF followed by docetaxel is an active and tolerable neoadjuvant chemotherapy regimen. A final analysis is planned for 2005.

Granulomatous mastitis diagnosed by core-needle biopsy and successfully treated with corticosteroid therapy: a case report.

A 46-year-old woman presented to our hospital with a rapidly growing lump in her right breast. Fine-needle aspiration (FNA) cytology and core needle biopsy of the mass revealed many epithelioid cells admixed with multinucleated Langhans-type giant cells, neutrophils, lymphocytes, and stromal cells, leading to a diagnosis of granulomatous mastitis. Mammography and ultrasonography provided little information for differentiating between granulomatous mastitis and carcinoma. This patient was successfully treated with low dose and short period of corticosteroid therapy.

Early crisis intervention to patients with acute stress disorder in general hospital.

This report presents 2 patients who were diagnosed to have acute stress disorder (ASD), received early psychiatric intervention (crisis intervention as a short-term psychotherapy), and subsequently had good outcome. Encounter with an event that causes psychological trauma may induce post-traumatic stress disorder (PTSD). However, the 2 patients described here have shown no particular mental symptoms for more than 2 years after the event and are leading normal lives. Psychological debriefing as a group used to be regarded as effective for the prevention of PTSD, but early identification of the stress-related disorder and intensive treatment of individual patients is recently considered to be more necessary. Both of the 2 patients presented here showed good outcome, and early crisis intervention in individual patients is suggested to be effective for the treatment of stress-related disorders and prevention of PTSD.

[Child and adolescent psychiatry its problems and foresight].

Accompanying the fall in birth rate, problems pertaining to the child's mind such as school in attendance, bullying, violence in the school, intrafamilial violence, eating disorders, substance abuse, and child abuse have rocketed and diversified, in addition to affecting increasingly lower age groups. The importance of child and adolescent psychiatry has never been more profound, but our country, without a chair in Child and Adolescent Psychiatry in the medical school framework, and lacking recognition of Child and Adolescent Psychiatry as a clinical department has undoubtedly become an underdeveloped country in terms of child and adolescent psychiatric care. The medical schools have been in the process of review and reorganization these past few years. The range of mental science is wide, and despite being a major discipline constituting one of the two arms of medical science together with somatic medicine, it is regarded as a minor existence in our country. This is the time to re-establish mental science, with areas such as child and adolescent psychiatry, geriatric psychiatry, social psychiatry, and crime psychiatry placed on an equal footing with general psychiatry. Turning our eyes on the world, the children are being robbed of their mental health as refugees, through child labor, starvation, and civil war. The demand of this age is true symbiosis, surpassing differences in race, religion, language, and culture, which is probably the indispensable element in the quest for a happy future for the children of this age.

A randomized, double-blind, placebo-controlled study of atomoxetine in Japanese children and adolescents with attention-deficit/hyperactivity disorder.

OBJECTIVES: Until the recent approval of methylphenidate (MPH), Japan had no approved treatment for attention-deficit/hyperactivity disorder (ADHD). The need still exists for an effective, safe, nonstimulant treatment. This first placebo-controlled Japan study of an ADHD nonstimulant therapy assessed atomoxetine efficacy and safety to determine the optimal dose for controlling ADHD symptoms in children and adolescents. METHODS: A total of 245 Japanese children and adolescents, aged 6-17 years and diagnosed with ADHD, were randomly assigned to receive placebo or one of three atomoxetine doses (0.5, 1.2, and 1.8 mg/kg per day) over 8 weeks. Symptoms were assessed with the Japanese Attention-Deficit/Hyperactivity Disorder Rating Scale-IV-Parent Version: Investigator scored and integrated with teacher reports (ADHD RS-IV-J:I/Sch). Adverse events, vital signs, laboratory tests, and electrocardiograms (ECGs) were obtained for safety analysis. RESULTS: In all, 234 patients completed the study. Atomoxetine at 1.8 mg/kg per day was significantly superior to placebo in reducing ADHD symptoms (p = 0.01; one-sided). Decreased appetite and vomiting were significantly greater in the atomoxetine treatment groups; however, no clinically significant differences were observed. Two patients discontinued due to affect lability and headache. A linear dose-response and vital signs similar to those from other atomoxetine studies were observed. CONCLUSION: Atomoxetine provides an effective and safe nonstimulant option for the treatment of Japanese pediatric patients with ADHD.

Phase II study of preoperative sequential FEC and docetaxel predicts of pathological response and disease free survival.

Purpose This multicenter phase II study examined the impact of pathological effect on survival after preoperative chemotherapy in Japanese women with early stage breast cancer. Patients and methods Prior to surgery, patients received four cycles of FEC (fluorouracil 500 mg/m(2), epirubicin 100 mg/m(2), cyclophosphamide 500 mg/m(2) q3w) followed by four cycles of docetaxel (75 mg/m(2) q3w). Primary endpoint was 3 year disease free survival (DFS) stratified by the absence or presence of Quasi-pCR (QpCR; absence of invasive tumor or only focal residual tumor cells). Secondary endpoints were predictors for QpCR, clinical response, breast conservation rate, and safety. Results Between June 2002 and June 2004, 202 women were enrolled. Among 191 assessable patients, 25% achieved QpCR. With 40 months median follow-up, 3 year DFS was estimated at 91% for all patients. 3 year DFS for patients with QpCR was 98% vs. 89% without QpCR (hazard ratio 0.38 [95% Confidence Interval 0.09-0.84], P = 0.0134). HER2 status and response to FEC were independent predictors of QpCR. The overall clinical response was 75%; 85% of patients achieved breast conservation. Grade 3/4 neutropenia was the most common adverse event, observed in 44% and 35% of patients during FEC and docetaxel, respectively. Treatment related side effects were manageable; there were no treatment related fatalities. Conclusion FEC followed by docetaxel is an active and manageable preoperative regimen for women with early stage breast cancer. QpCR following preoperative chemotherapy predicts favorable DFS. HER2 overexpression and clinical response to FEC predict QpCR.

The selective action of D2 dopamine receptor antisense oligodeoxynucleotide on the expression of the dopamine receptor subtype mRNA in rat striatum.

We have evaluated the effect of the D2 dopamine receptor antisense oligodeoxynucleotide (D2 AS ODN) on the gene expression of all five dopamine receptor subtypes including D1, D2, D3, D4 and D5 dopamine receptor in the rat striatum. The levels of D2 dopamine receptor mRNA are significantly decreased at 6, 12, 24 h after the last injection of three time injections of D2 AS ODN, although D1, D3, D4 and D5 subtype mRNA levels did not significantly reduced at any time. The present study is the first to demonstrate the selective effect of D2 AS ODN on D2 dopamine receptor mRNA among all five dopamine receptor subtypes and the effectiveness of D2 AS ODN without 6-hydroxydopamine.