TERT promotor region rearrangements analyzed in high-risk neuroblastomas by FISH method and whole genome sequencing.

BACKGROUND: Unfavorable neuroblastomas (NBLs) achieve telomere stabilization via telomerase activation through MYCN amplification, TERT promoter region (TERT-PR) rearrangements, or alternative telomere lengthening of telomeres. No well-established methods are available for investigating TERT-PR rearrangements. We examined the relationship between and prognosis by fluorescence in situ hybridization (FISH) upstream and downstream of TERT to establish a simple analysis method. PROCEDURE: TERT-PR rearrangements were analyzed in 3 M MYCN amplified cases and, 11MYCN non-amplified cases (1 MS case, 1 L2 case and 2 M cases less than 18 months, and 1 L2 case and  6 M cases over 18 months old at diagnosis) to determine if MYCN and TERT-PR rearrangement were independent prognostic factors. In total, 14 patients (11 males, 3 females; median age 36.4 months, range 1-122 months) with NBLs were evaluated at Hiroshima University. We identified MYCN amplification, TERT expression, and TERT-PR rearrangements. TERT-PR rearrangement was detected by FISH upstream and downstream of TERT on Chr5.p15.33. For TERT-PR rearranged cases, we characterized the fusion partners by whole genome sequencing. RESULTS: We detected TERT-PR rearrangements in two NBL samples. Both samples were high-risk NBLs and MYCN single NBLs, and their TERT expression levels were extremely higher than in the other samples. Genomic translocation occurred at chromosome 5p15.33 according to whole genome sequencing, agreeing with the FISH results. One case showed translocation of the chr5.p15.33 SLCA6A19 gene to 22q12.3, and another case showed chr5p15.33 to chr5q33.3. CONCLUSIONS: FISH is a useful diagnostic tool for evaluating high-risk NBLs in which TERT-PR rearrangements have occurred.

Outcome and Late Complications of Hepatoblastomas Treated Using the Japanese Study Group for Pediatric Liver Tumor 2 Protocol.

PURPOSE: We report here the outcomes and late effects of the Japanese Study Group for Pediatric Liver Tumors (JPLT)-2 protocol, on the basis of cisplatin-tetrahydropyranyl-adriamycin (CITA) with risk stratification according to the pretreatment extent of disease (PRETEXT) classification for hepatoblastoma (HB). PATIENTS AND METHODS: From 1999 to 2012, 361 patients with untreated HB were enrolled. PRETEXT I/II patients were treated with up-front resection, followed by low-dose CITA (stratum 1) or received low-dose CITA, followed by surgery and postoperative chemotherapy (stratum 2). In the remaining patients, after 2 cycles of CITA, responders received the CITA regimen before resection (stratum 3), and nonresponders were switched to ifosfamide, pirarubicin, etoposide, and carboplatin (ITEC; stratum 4). Intensified chemotherapeutic regimens with autologous hematopoietic stem-cell transplantation (SCT) after resection were an optional treatment for patients with refractory/metastatic disease. RESULTS: The 5-year event-free and overall survival rates of HB patients were 74.2% and 89.9%, respectively, for stratum 1, 84.8% and 90.8%%, respectively, for stratum 2, 71.6% and 85.9%%, respectively, for stratum 3, and 59.1% and 67.3%%, respectively, for stratum 4. The outcomes for CITA responders were significantly better than those for nonresponders, whose outcomes remained poor despite salvage therapy with a second-line ITEC regimen or SCT. The late effects, ototoxicity, cardiotoxicity, and delayed growth, occurred in 61, 18, and 47 patients, respectively. Thirteen secondary malignant neoplasms (SMNs), including 10 leukemia, occurred, correlating with higher exposure to pirarubicin and younger age at diagnosis. CONCLUSION: The JPLT-2 protocol achieved up-front resectability in PRETEXT I/II patients with no annotation factors, and satisfactory survival in patients who were CITA responders in the remaining patients. However, outcomes for CITA nonresponders were unsatisfactory, despite therapy intensification with ITEC regimens and SCT. JPLT-2 had a relatively low incidence of cardiotoxicity but high rates of SMNs.

Inflammatory myofibroblastic tumors of the lung carrying a chimeric A2M-ALK gene: report of 2 infantile cases and review of the differential diagnosis of infantile pulmonary lesions.

We report 2 infantile cases of pulmonary tumor carrying a chimeric A2M-ALK gene. A2M-ALK is a newly identified anaplastic lymphoma kinase (ALK)-related chimeric gene from a tumor diagnosed as fetal lung interstitial tumor (FLIT). FLIT is a recently recognized infantile pulmonary lesion defined as a mass-like lesion that morphologically resembles the fetal lung. Grossly, FLIT characteristically appears as a well-circumscribed spongy mass, whereas the tumors in these patients were solid and firm. Histologically, the tumors showed intrapulmonary lesions composed of densely proliferating polygonal or spindle-shaped mesenchymal cells with diffuse and dense infiltrations of inflammatory cells forming microcystic or micropapillary structures lined by thyroid transcription factor 1-positive pneumocytes, favoring inflammatory myofibroblastic tumor rather than FLIT. The proliferating cells were immunoreactive for ALK, and A2M-ALK was identified in both tumors with reverse-transcription polymerase chain reaction. The dense infiltration of inflammatory cells, immunoreactivity for ALK, and identification of an ALK-related chimeric gene suggested a diagnosis of inflammatory myofibroblastic tumor. Histologically, most reported FLITs show sparse inflammatory infiltrates and a relatively low density of interstitial cells in the septa, although prominent infiltration of inflammatory cells and high cellularity of interstitial cells are seen in some FLITs. The present cases suggest that ALK rearrangements, including the chimeric A2M-ALK gene, may be present in these infantile pulmonary lesions, especially those with inflammatory cell infiltration. We propose that these infantile pulmonary lesions containing a chimeric A2M-ALK gene be categorized as a specific type of inflammatory myofibroblastic tumor that develops exclusively in neonates and infants.

Resectability and tumor response after preoperative chemotherapy in hepatoblastoma treated by the Japanese Study Group for Pediatric Liver Tumor (JPLT)-2 protocol.

BACKGROUND/PURPOSE: We aimed to clarify whether surgical resectability and tumor response after preoperative chemotherapy (preCTx) represented prognostic factors for patients with hepatoblastoma (HBL) in the JPLT-2 study (1999-2012). METHODS: Patients (N=342) with HBL who underwent preCTx were eligible. PRETEXT, CHIC risk stratification (standard [SR], intermediate [IR] and high risk [HR]) at diagnosis, POST-TEXT, and tumor resectability were evaluated by imaging. Tumor response was classified into responders (CR or PR) and nonresponders (NC or PD) according to RECIST criteria. RESULTS: There were 7 PRETEXT I, 106 II, 143 III, and 86 IV, including 71 metastatic HBLs. In POST-TEXT, 12 PRETEXT II, 42 III, and 58 IV were down-staged. The 5-year EFS/OS rates of 198 SR, 73 IR, and 71 HR-HBLs were 82/94%, 49/64%, and 28/34%, respectively. In 198 SR, 154 of 160 responders and 24 of 38 nonresponders survived event-free (P<0.01). In 73 IR, 12 of 24 whose tumors remained unresectable experienced recurrence, 9 of whom were nonresponders (P<0.01). In 71 HR, chemoresponders and tumor resectability after preCTx correlated with favorable outcomes (P<0.05). CONCLUSIONS: Evaluation of response and tumor resectability after preCTx is useful for predicting prognosis in HBLs. To improve outcomes, we should reconsider surgical procedures according to resectability and chemoresponsiveness. LEVEL OF EVIDENCE: Level II.

Telomere biology including TERT rearrangements in neuroblastoma: a useful indicator for surgical treatments.

PURPOSE: Our telomere biology study of neuroblastomas (NBLs) has revealed that unfavorable NBLs acquired telomere stabilization by telomerase activation or ALT (alternative lengthening of telomeres). Recently, genomic rearrangements in a region proximal to the telomerase reverse transcriptase (TERT) gene have been discovered in NBLs. In this study, TERT rearrangements were examined in NBLs along with their relationship to other aspects of telomere biology. METHODS: In 121 NBLs, including 67 cases detected by mass-screening whose telomere length, telomerase activity, ALT with ATRX/DAXX alterations, and MYCN amplification were already known, TERT rearrangements were examined using GeneChip SNP arrays. RESULTS: The 11 ATRX/DAXX mutated ALT cases and 29 cases with high telomerase activity showed poor prognosis. MYCN amplification and TERT rearrangements were independently detected in 16 and 13 cases, respectively, and these alterations were significantly correlated with high telomerase activity. In 81 infant cases, MYCN amplification, TERT rearrangements and ATRX mutations were detected in 3, 4, and 3 cases, respectively. Among them, 6 cases showed progression or recurrences. CONCLUSIONS: Telomere stabilization in NBLs is acquired by telomerase activation through MYCN amplification, TERT rearrangements or by ALT. Since these tumors usually show progression and recurrence, complete resection should be considered, even in infant cases. LEVEL OF EVIDENCE: Prognosis study, level III.

Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger.

The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.

Vascular access for long-term hemodialysis/hemodiafiltration patients.

PURPOSE: We investigated the characteristics of patients who received long-term hemodialysis/hemodiafiltration (HD/HDF) treatment for over 30 years at our group of hospitals and type of vascular access (VA) used. SUBJECTS AND METHODS: As of August 2014, 950 patients were receiving HD/HDF treatment at one of our hospitals. Of those, we investigated 41 (4.3%) undergoing long-term treatment in regard to their characteristics and VA type. The items subjected to analysis were sex, primary illness, age at time of dialysis initiation, present age, duration (years) of HD/HDF, type of arteriovenous fistula (AVF) and arteriovenous graft (AVG), history of surgery and AVF persistence rate. RESULTS: The subjects consisted of 22 men and 19 women, and their mean HD/HDF duration was 33.4 ± 2.8 years. For primary illness, the majority (n = 31) had chronic glomerulonephritis. The age at time of dialysis initiation was 31.7 ± 7.76 years and present age was 64.5 ± 7.65 years. They had received 3.8 VA surgeries. For present VA type, 23 patients (56.0%) had an AVF and 13 (31.7%) an AVG, while 4 AVF patients (9.7%) had a history of AVG use. One patient (2.4%) had a superficialized artery. The mean HD/HDF duration of the 13 AVG patients was 7 years and the longest was 18 years. AVF persistence rate estimated by the Kaplan-Meier method was 75% at 30 years after dialysis initiation. CONCLUSIONS: The present results suggest that the ratio of patients with AVG increased with prolonged HD/HDF treatment. AVG has a higher probability of complications and lower patency as compared to AVF, thus careful management is needed. On the other hand, AVG contributes more to a good long prognosis, as it offers efficient dialysis. In cases of vascular deterioration due to long-term hemodialysis, it is inevitable to change from AVF to AVG, thus the ratio of AVG patients is expected to increase in cases of long-term HD/HDF.

Circulating free DNA as non-invasive diagnostic biomarker for childhood solid tumors.

PURPOSE: Our aims are to determine circulating free DNA (cfDNA) in childhood solid tumor patients who underwent surgical intervention and to analyze any relationships with clinical parameters. METHODS: Fourty-four consenting children admitted with solid tumors between 2010 and 2014 were recruited. CfDNAs isolated from 0.5mL plasma obtained before and 1-30days after surgery were analyzed by next-generation sequencing (NGS: IonTorrent Cancer Hotspot panel) and by gene amplification analysis using a digital PCR (dPCR) platform. RESULTS: Total amounts of cfDNA were 54-825ng and were significantly associated with stage of disease. In cfDNA, 15 mutations or deletions (2 ALK, 2 TP53, 1 WT1, 3 CTNNB1, 1 APC, 1 KIT, 1 RET, 1 CDNK2AT, and 3 SMARCB1) were identified. In 10 neuroblastoma suspected cases, 2 showed high copy numbers of MYCN using dPCR. The positive rate in our cohort was 36%, and all of these aberrations were detected in the original tumors. None of the aberrations were detectable in cfDNA after surgery except for three cases whose tumors remained after surgery. CONCLUSIONS: These data demonstrate the feasibility and potential utility of mutation/deletion/amplification screening in cfDNA using NGS and dPCR for the detection of tumor biomarkers in children with solid tumors. These markers also have the potential utility to evaluate complete resection after surgery.

Mortality and morbidity in primarily resected hepatoblastomas in Japan: Experience of the JPLT (Japanese Study Group for Pediatric Liver Tumor) trials.

BACKGROUND: In the Japanese Study Group for Pediatric Liver Tumor (JPLT) protocols (JPLT-1 and 2) for evaluating the cure rate of risk-stratified hepatoblastoma, primary resection was permitted in PRETEXT I and II cases, followed by postoperative chemotherapy. METHODS: In approximately 500 enrolled cases, resection was performed as the initial treatment in 60 cases, including all 18 PRETEXT I, 30 PRETEXT II, and 12 ruptured cases. The clinical features, surgical procedures, complications, and survival rates were compared in these three groups. RESULTS: All 18 PRETEXT I cases underwent complete resection by lobectomy or segmentectomy (n=14) or nonanatomical partial hepatectomy (NPH) (n=4). The 30 PRETEXT II cases underwent primary resection by right or left lobectomy (n=16), NPH (n=10), or other procedures (n=4). Of these 30 cases, operational death occurred in 1 newborn, and recurrence occurred in 7 cases (14.6%), including 6 NPH cases and 4 older cases (aged >3years). Of the 12 ruptured cases, 7 (58.3%) showed recurrence. Event-free survival rates at 5years in the 3 groups were 88%, 70%, and 32%, respectively. CONCLUSIONS: Primary resection for PRETEXT I or II HB cases should be performed by anatomical resection according to strict surgical guidelines. More intensified chemotherapy is required for primary resected cases whose tumors have ruptured.

Pentraxin 3 is a local inflammatory marker in atrial fibrillation.

Increasing evidence indicates that inflammation contributes to the pathogenesis of atrial fibrillation (AF). Pentraxin 3 (PTX3) is produced abundantly in local inflammatory lesions while C-reactive protein (CRP) is produced mainly in the liver. In this study, we investigated whether a local level of PTX3 might be a sensitive marker for the local inflammation of AF. Blood from the periphery and left atrial appendage (LAA) was sampled from 23 patients with AF undergoing pulmonary vein isolation, and from 10 control subjects with Wolff-Parkinson-White syndrome. We measured peripheral and LAA plasma concentrations of CRP, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and PTX3. Plasma PTX3 concentrations in both locations were higher in patients with AF than in control subjects. PTX3 concentrations were significantly higher in the LAA than the periphery in patients with AF (3.7 ± 1.4 vs 3.3 ± 1.2 ng/ml, P < 0.01), but not in control subjects (2.4 ± 0.5 vs 2.4 ± 0.5 ng/ml, not significant). Patients and controls showed no significant differences in CRP, IL-6, or TNF-α concentrations between the periphery and LAA. Interestingly, there was a significant positive correlation between LAA plasma concentrations of PTX3 and left atrial volume (r = 0.55, P < 0.01). These data demonstrate that local PTX3 production in the left atrium might reflect the local inflammation of AF.

Increase in serum triglyceride was associated with coronary plaque vulnerability in a patient with rheumatoid arthritis.

Rates of morbidity and mortality from cardiovascular disease are high in patients with rheumatoid arthritis (RA); however, the mechanisms and biomarkers that reflect coronary plaque vulnerability have not yet been established. We present a case of acute coronary syndrome (ACS) presumably caused by exacerbation of chronic inflammation of RA, in which an abrupt increase in serum triglyceride was seen on the day of onset of ACS but not during effort angina. This case suggests that RA patients with an abrupt increase in triglyceride need intensive care including anti-platelet and statin therapy for the prevention of coronary plaque rupture. .

Detection of MYCN amplification using blood plasma: noninvasive therapy evaluation and prediction of prognosis in neuroblastoma.

PURPOSE: Amplification of neuroblastoma derived (avian)v-myc myelocytomatosis viral related oncogene (MYCN) is an important risk-stratified indicator in neuroblastoma. To evaluate the feasibility of noninvasive measurement of MYCN amplification, we analyzed MYCN amplification in stored blood plasma samples. METHODS: We used quantitative real-time PCR to determine MYCN copy numbers in plasma-derived DNA of 10 healthy volunteers and 50 neuroblastoma cases. The copy number was calculated as the ratio of copies of MYCN to those of a reference gene. Plasma samples obtained after surgery or neoadjuvant therapy were also analyzed in five cases and four cases, respectively. RESULTS: In 34 neuroblastoma cases, MYCN was nonamplified in both tumor tissue and blood plasma. In 16 neuroblastoma cases, MYCN was amplified in both tumor tissue and blood plasma; 13 of the 16 cases showed poor outcomes. MYCN amplification was undetectable in blood plasma shortly after surgery or neoadjuvant therapy. The correlation coefficient between MYCN copy numbers in tumor tissue and in blood plasma was approximately 0.9. CONCLUSION: We can detect MYCN amplification of tumor tissue noninvasively and quantitatively by measuring the MYCN copy number in blood plasma. Determination of MYCN copy number in plasma may be useful when evaluating surgery and neoadjuvant chemotherapy.

A cisplatin plus pirarubicin-based JPLT2 chemotherapy for hepatoblastoma: experience and future of the Japanese Study Group for Pediatric Liver Tumor (JPLT).

INTRODUCTION: The Japanese Study Group for Pediatric Liver Tumor (JPLT) has conducted cooperative treatment studies on hepatoblastoma (HBL) since 1991. The JPLT2 protocol was launched in 1999 to evaluate the efficacy of cisplatin/pirarubicin (CITA) under risk stratification. European and North American groups showed the improvement of HBL patients by pre- and postoperative chemotherapeutic regimens. Therefore, we evaluated the results of JPLT study and considered the future aspect of JPLT. METHODS: A total of 389 children with malignant hepatic tumors were enrolled in JPLT-2 until 2010. Data from 331 HBL cases were analyzed. RESULTS AND DICUSSION: Of the 331 patients enrolled, their 5-year overall survival and event-free survival rates were 83.3 and 68.0%, respectively. While outcomes of standard-risk cases (tumors involving 3 or fewer sectors of the liver) were excellent, those of high-risk cases (tumors involving 4 sectors of the liver or with distant metastases) remained poor. For 26 high-risk or relapse/refractory HBL cases, high-dose chemotherapy (HDC) with stem cell transplantation (SCT) was carried out. Among them, 6 of 12 relapse or refractory cases died. Compared with other regimens, the CITA regimen achieved similar or superior rates of survival among children with standard-risk HBL, while HDC with SCT was not effective in patients with high-risk HBL. Presently, a global Children's Hepatic Tumor International Consortium (CHIC) project is ongoing, with a focus on international cooperation and risk stratification in the field of rare liver cancers in children. More promising strategies, including liver transplantation and new targeting drugs under global risk stratification, are being proposed.

Androgen receptor promotes sex-independent angiogenesis in response to ischemia and is required for activation of vascular endothelial growth factor receptor signaling.

BACKGROUND: Hypoandrogenemia is associated with an increased risk of ischemic diseases. Because actions of androgens are exerted through androgen receptor (AR) activation, we studied hind-limb ischemia in AR knockout mice to elucidate the role of AR in response to ischemia. METHODS AND RESULTS: Both male and female AR knockout mice exhibited impaired blood flow recovery, more cellular apoptosis, and a higher incidence of autoamputation after ischemia. In ex vivo and in vivo angiogenesis studies, AR-deficient vascular endothelial cells showed reduced angiogenic capability. In ischemic limbs of AR knockout mice, reductions in the phosphorylation of the Akt protein kinase and endothelial nitric oxide synthase were observed despite a robust increase in hypoxia-inducible factor 1α and vascular endothelial cell growth factor (VEGF) gene expression. In in vitro studies, siRNA-mediated ablation of AR in vascular endothelial cells blunted VEGF-stimulated phosphorylation of Akt and endothelial nitric oxide synthase. Immunoprecipitation experiments documented an association between AR and kinase insert domain protein receptor that promoted the recruitment of downstream signaling components. CONCLUSIONS: These results document a physiological role of AR in sex-independent angiogenic potency and provide evidence of novel cross-talk between the androgen/AR signaling and VEGF/kinase insert domain protein receptor signaling pathways.

Eosinophilic myocarditis due to Churg-Strauss syndrome with markedly elevated eosinophil cationic protein.

A 67-year-old woman with asthma visited our hospital with increasing dyspnea and new-onset paresthesia and purpura in her legs. Physical examination showed a wheeze, pretibial edema, and surrounding purpura. Chest X-rays showed cardiac decompensation and an electrocardiogram revealed a new ST-T change. Laboratory data showed leukocytosis, hypereosinophilia (10,450/µL), troponin T(+), elevated BNP, and markedly elevated eosinophil cationic protein (ECP) (> 150 ng/mL). Echocardiography revealed diffuse left ventricular hypokinesis (ejection fraction 30%) with increased wall thickness. Coronary angiography was normal. Cardiac magnetic resonance imaging implied diffuse myocardial edema and subendocardial late gadolinium enhancement. Skin biopsy of purpura showed superfi cial perivascular dermatitis with remarkable eosinophilic infiltrations. No evidence of drug allergies, parasitic infection, or myeloproliferative disorder was detected. Based on these findings, a diagnosis of eosinophilic myocarditis due to Churg-Strauss syndrome was considered. She was administered prednisolone at a dose of 1 mg/kg, cyclophosphamide, and diuretics. Several markers of eosinophilic myocarditis and heart failure gradually improved, including ECP. She was discharged 30 days later with no cardiac event. Eosinophilic myocarditis is characterized by predominantly eosinophilic infi ltration. Eosinophilic granule proteins, such as ECP and major basic protein, play important roles in the pathogenesis of eosinophilic myocarditis. We experienced a rare case of eosinophilic myocarditis due to Churg-Strauss syndrome. Markedly elevated ECP played an important role in the early diagnosis and subsequent reduction in ECP served as a marker of monitoring. In an asthmatic patient with dyspnea, hypereosinophilia, and vasculitis, Churg-Strauss syndrome with eosinophilic myocarditis should be considered.

Clinical feature of anaplastic lymphoma kinase-mutated neuroblastoma.

PURPOSE: Anaplastic lymphoma kinase (ALK) has recently been identified as a gene conferring a predisposition for neuroblastoma. We have analyzed tyrosine kinase domain mutations and amplification/expression of the ALK gene and focused on clinical features of neuroblastoma cases with ALK aberrations. METHODS: The frequency of ALK mutations, copy number gain, and expression were analyzed in 538 neuroblastoma tumors derived from 361 cases, including 161 cases detected by mass screening. These cases were analyzed according to clinicopathologic features including the International Neuroblastoma Staging System and patient outcomes. RESULTS: Three cases (0.8%) had ALK amplification, and 16 cases (5.2%) had missense mutations at positions F1174, F1245, D1249, and R1275. Among them, 7 cases were diagnosed at more than 14 months of age, and 11 cases were infants, including 9 cases detected by mass screening and 1 multiple neuroblastoma with a germline mutation. Of the 11 infants, 3 cases relapsed, and 1 case died of disease. Among cases detected by screening, activated ALK cases showed significantly worse prognosis (P = .002). Of 7 older cases, 5 had MYC amplifications, and 5 died of disease. The expression levels of ALK were up-regulated in cases with unfavorable outcomes. In cases with activated ALK neuroblastoma, survival rates of patients detected by screening were significantly better than those in the clinically detected group (P = .025). CONCLUSIONS: The results of the present study support the hypothesis that activated ALK tumors represent a specific subset of neuroblastomas. These tumors usually develop in infants and may have a high capacity for recurrence.

Wnt signaling and telomerase activation of hepatoblastoma: correlation with chemosensitivity and surgical resectability.

PURPOSE: Recently, it became apparent that telomerase directly modulated Wnt signaling as a cofactor in a ß-catenin transcriptional complex. In this study, we investigated Wnt/ß-catenin signaling and telomerase activation in hepatoblastoma (HBL). METHODS: Tumors derived from 56 HBL cases treated with the Japanese Study Group for Pediatric Liver Tumors (JPLT) Protocol-2 were analyzed for oncogenic mutations (missense mutations and interstitial deletions in the third exon) of the CTNNB1 gene-encoding ß-catenin and for the expression levels of telomerase reverse transcriptase (TERT). RESULTS: Oncogenic mutations of CTNNB1 were detected in 42 cases (75%). The expression levels of TERT were significantly higher in 14 cases without mutation (P < .05) and in 8 cases with metastasis (P < .01). Interestingly, Wnt/ß-catenin target genes were significantly activated in the tumors without mutations (P = .013). In cases with mutations, preoperative chemotherapy was more effective (P = .008), and complete resection rate was higher (P = .034). Consequently, 2 patients with mutations and 4 patients without mutations died of disease (P = .013). High expression of TERT was detected in all tumors of these dead patients. CONCLUSIONS: Wnt/ß-catenin signaling in the HBLs without CTNNB1 mutations was activated by high expression of TERT. The clinical courses in HBLs without CTNNB1 mutations seemed to be unfavorable because of chemoresistance and low rates of resectability.

Activation of peroxisome proliferator-activated receptor α in megakaryocytes reduces platelet-derived growth factor-BB in platelets.

AIM: Platelet-derived growth factor (PDGF)-BB plays a crucial role in atherosclerosis and vascular remodeling by promoting the migration and proliferation of vascular smooth muscle cells. The objective of this study was to clarify the pleiotropic effect of peroxisome proliferator-activated receptor α (PPARα) activators on PDGF-BB expression in megakaryocytes and platelets. METHODS AND RESULTS: The expression of PPARα in a human erythroleukemia (HEL) cells was clearly detected by reverse transcriptase-PCR and immunofluorescence microscopy. The expression level of PPARα in HEL cells was unchanged regardless of differentiation into megakaryocytic cells by treatment with phorbol 12-myristate 13 acetate (TPA). The TPA-induced expression of PDGF-B mRNA and PDGF-BB protein levels in culture media was significantly decreased by treatment with PPARα activators, Wy14643 and fenofibric acid, in a dose-dependent manner. PDGF-BB expression induced by inflammatory cytokines, including interleukin-1ß or interleukin-6, was also significantly suppressed by treatment with PPARα activators. Immunohistochemistry of human bone marrow showed the expression of PPARα in both the nucleus and cytoplasm of megakaryocytes. In addition, PDGF-BB levels in platelets were significantly decreased from 1,800±870 to 1,470±840 pg/10(5) platelets (mean±SD, p<0.05) by treatment with 300 mg fenofibrate once daily for 4 weeks in 13 patients with dyslipidemia. CONCLUSIONS: Activation of PPARα in megakaryocytes reduces PDGF-BB expression in platelets. PPARα activators may exert vasculo-protective action through suppression of PDGF-BB production in a megakaryocyte/platelet pathway.

High plasma aldosterone concentration is a novel risk factor of cognitive impairment in patients with hypertension.

Cognitive impairment leading to dementia is associated with high prevalence of hypertension, decreased quality of life and poor prognosis. Aldosterone is known as a risk factor for cardiovascular and cerebrovascular diseases. In addition, mineral corticoid receptors are abundantly expressed in the hippocampus, which plays a pivotal role in cognitive function; however, it has not been determined whether plasma aldosterone level is associated with cognitive impairment in patients with hypertension. We enrolled 68 patients with essential hypertension and assessed their cardiovascular risk factors, including blood pressure, hyperlipidemia, diabetes mellitus, obesity, smoking, history of cerebral infarction, renal function, parameters of inflammation, oxidative stress and nitric oxide bioavailability, a parameter of cerebral blood flow and carotid plaque by ultrasound examination, plasma renin activity and plasma aldosterone concentration (PAC). The mini-mental state examination (MMSE) was used to evaluate cognitive function. The relevance of cardiovascular risk factors and MMSE score was statistically evaluated. Multiple regression analysis showed that age (P < 0.01), PAC (P < 0.01) and history of cerebral infarction (P < 0.05) were inversely and independently associated with MMSE score. Mineral corticoid receptor antagonists, including spironolactone and eplerenone, increased MMSE score in seven patients with hypertension, but not in the controls. In conclusion, increased PAC is associated with impaired cognitive function and mineral corticoid receptor blockade may protect against not only cardiovascular mortality, but also cognitive impairment in patients with hypertension.

Androgen receptor counteracts Doxorubicin-induced cardiotoxicity in male mice.

Doxorubicin (Dox) has been used as a potent anticancer agent, but serious cardiotoxicity precludes its use in a wide range of patients. We have reported that the androgen-androgen receptor (AR) system plays important roles in cardiac growth and protection from angiotensin II-induced cardiac remodeling. The present study was undertaken to clarify whether the androgen-AR system exerts a cardioprotective effect against Dox-induced cardiotoxicity. Male AR knockout (ARKO) and age-matched littermate male wild-type (WT) mice at 25 wk of age were given ip injections of Dox (20 mg/kg) or a vehicle. The survival rate and left ventricular function in Dox-treated male ARKO mice were reduced compared with those in Dox-treated male WT mice. Electron microscopic study showed prominent vacuole formation of myocardial mitochondria in Dox-treated male ARKO mice. Cardiac oxidative stress and apoptosis of cardiomyocytes were increased more prominently by Dox treatment in male ARKO mice than in male WT mice. In addition, Dox-induced reduction in the expression of cardiac mitochondria transcription factor A (Tfam) and phosphorylation of serine-threonine kinase (Akt) was more pronounced in male ARKO mice than in male WT mice. In cardiac myoblast cells, testosterone up-regulated Akt phosphorylation and Tfam expression and exerted an antiapoptotic effect against Dox-induced cardiotoxicity. Collectively, the results demonstrate that Dox-induced cardiotoxicity is aggravated in male ARKO mice via exacerbation of mitochondrial damage and superoxide generation, leading to enhanced apoptosis of cardiomyocytes. Thus, the androgen-AR system is thought to counteract Dox-induced cardiotoxicity partly through activation of the Akt pathway and up-regulation of Tfam to protect cardiomyocytes from mitochondrial damage and apoptosis.