Search

Abstract Introduction We present a case of a 9‐year‐old girl diagnosed with a myeloproliferative neoplasm (MPN) harboring both monosomy 7 and an ALK/ROS1 fusion gene. Case presentation The neoplasm was resistant to conventional AML chemotherapy and required hematopoietic cell transplantation (HCT) to achieve remission. Discussion MPNs with monosomy 7 and ALK/ROS1 fusions occur in a wide age range of children and adults, and require HCT for long‐term remission. Furthermore, these cases can be responsive to ALK inhibitors. Conclusion This report underscores the potential need to reclassify such MPNs as a distinct entity, which has unique therapeutic implications.

Abstract Previous reports have shown that a gain of the chromosome 9 short arm (9p) is associated with choroid plexus hyperplasia (CPH). Furthermore, CPH can lead to communicating hydrocephalus; however, no cases of CPH with 9p gain requiring choroid plexus resection have been reported. Here, we describe the first case in which a 9p hexasomy/tetrasomy mosaic patient required choroid plexus resection for hydrocephalus. This finding suggested that the 9p copy number is correlated with CPH severity.

Abstract ETV6::Fyn‐related kinase (FRK), which is a Src family tyrosine‐kinase‐related fusion gene and firstly identified in our patient with paediatric high risk B cell precursor acute lymphoblastic leukaemia (B‐ALL), has no evidence of efficacy of tyrosine kinase inhibitor in vivo. We performed functional analysis of ETV6::FRK to establish molecular targeting therapy and determined that dasatinib could abrogate proliferation activity of ETV6::FRK through the repression of FRK‐STAT3/STAT5 pathway in vitro and significantly extended the survival time of the xenografted mice in vivo (p

Abstract Genetic information encoded by DNA is packaged in the nucleus using the chromatin structure. The accessibility of transcriptional elements in DNA is controlled by the dynamic structural changes of chromatin for the appropriate regulation of gene transcription. Chromatin structure is regulated by two general mechanisms, one is histone modification and the other is chromatin remodeling in an ATP‐dependent manner. Switch/sucrose nonfermentable (SWI/SNF) complexes utilize the energy from ATP hydrolysis to mobilize nucleosomes and remodel the chromatin structure, contributing to conformational changes in chromatin. Recently, the inactivation of encoding genes for subunits of the SWI/SNF complexes has been documented in a series of human cancers, accounting for up to almost 20% of all human cancers. For example, human SNF5 (hSNF5), the gene that encodes a subunit of the SWI/SNF complexes, is the sole mutation target that drives malignant rhabdoid tumors (MRT). Despite remarkably simple genomes, the MRT has highly malignant characteristics. As a key to understanding MRT tumorigenesis, it is necessary to fully examine the mechanism of chromatin remodeling by the SWI/SNF complexes. Herein, we review the current understanding of chromatin remodeling by focusing on SWI/SNF complexes. In addition, we describe the molecular mechanisms and influences of hSNF5 deficiency in rhabdoid tumors and the prospects for developing new therapeutic targets to overcome the epigenetic drive of cancer that is caused by abnormal chromatin remodeling.

Abstract Pathogenic variants in the HIBCH gene cause HIBCH deficiency, leading to mitochondrial disorders associated with valine metabolism. Patients typically present with symptoms such as developmental regression/delay, encephalopathy, hypotonia and dystonia. Brain magnetic resonance imaging (MRI) shows bilateral lesions in the basal ganglia with/without brainstem involvement. Here, we report a case of a Japanese patient with Leigh-like syndrome caused by novel HIBCH variants. Long-term follow-up MRI revealed progressive cerebellar atrophy, which expands the phenotypic spectrum of HIBCH deficiency.

Abstract Most cases of rhabdomyosarcoma (RMS) are sporadic and not associated with the Lynch syndrome (LS) spectrum. We report a young adult patient with RMS and a family history of colorectal cancer. Comprehensive cancer genomic profiling (CGP) of his tumor revealed a likely pathogenic variant of MSH2, NM_000251.3:c.1741delA (p.I581Lfs*9), which was also present in his blood sample. The widespread use of CGP may reveal that RMS can be a rare manifestation of LS.

High-risk neuroblastoma (HR-NB) patients remain far from obtaining optimal outcomes, with more than 50% relapse/regrowth rate despite current intensive multimodal therapy. This originated from the activation/proliferation of chemoresistant minimal residual disease (MRD). MRD with a significant prognostic was reported by several quantitative PCR (qPCR) or droplet digital PCR (ddPCR) assays quantitating different sets of NB-associated mRNAs (NB-mRNAs). The 7NB-mRNAs ddPCR assay quantitating CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs was reported to outperform other qPCR assays by a retrospective in-house observational study. In the present study, the Japan Children’s Cancer Group (JCCG) Neuroblastoma Committee conducted a prospective multicenter observational study aimed at evaluating a prognostic value of MRD in bone marrow (BM-MRD) and peripheral blood (PB-MRD) detected by 7NB-mRNAs ddPCR assay. Between August 2018 and August 2022, 7 HR-NB patients who registered for JCCG clinical trials (JN-H-11 and JN-H-15) were enrolled. A total of 19 BM and 19 PB samples were collected, and 4/15 BM and 4/15 PB samples were classified as progressive disease (PD)/non-PD samples. BM-MRD and PB-MRD estimated area under curve (AUC) of 0.767 and 0.800 with a significant accuracy (AUC > 0.7). The present study validated a prognostic value of BM-MRD obtained by a previous study (AUC 0.723) and revealed the significant accuracy of PB-MRD as well as BM-MRD.

Abstract Rhabdomyosarcoma exhibits tumor‐specific energy metabolic changes that include the Warburg effect. Since targeting cancer metabolism is a promising therapeutic approach, we examined the antitumor effects of suppressing lipid metabolism in rhabdomyosarcoma. We suppressed lipid metabolism in rhabdomyosarcoma cells in vitro by administering an inhibitor of malonyl‐CoA decarboxylase, which increases malonyl‐CoA and decreases fatty acid oxidation. Suppression of lipid metabolism in rhabdomyosarcoma cells decreased cell proliferation by inducing cell cycle arrest. Metabolomic analysis showed an increase in glycolysis and inactivation of the pentose phosphate pathway. Immunoblotting analysis revealed upregulated expression of the autophagy marker LC3A/B‐II due to increased phosphorylation of AMP‐activated protein kinase, a nutrient sensor. p21 protein expression level also increased. Inhibition of both lipid metabolism and autophagy suppressed tumor proliferation and increased apoptosis. In vivo studies involved injection of human Rh30 cells into the gastrocnemius muscle of 6‐week‐old female nude mice, which were divided into normal chow and low‐fat diet groups. The mice fed a low‐fat diet for 21 days showed reduced tumor growth compared to normal chow diet‐fed mice. Suppression of lipid metabolism disrupted the equilibrium of the cancer‐specific metabolism in rhabdomyosarcoma, resulting in a tumor growth‐inhibition effect. Therefore, the development of treatments focusing on the lipid dependence of rhabdomyosarcoma is highly promising.

Intraperitoneal venous malformations are uncommon. Therefore, the prognosis of patients has not been determined, and appropriate treatments have not been established. We have reported the case of a neonate with an extensive intraperitoneal venous malformation. She did not have a developmental disorder nor a functional disability; thus, she was observed without treatment. However, the patient died suddenly of obstructive venous return disorder due to thrombosis in a vein draining from the venous malformation, followed by blood pooling in the expanding venous malformation. Extensive intraperitoneal venous malformations can be associated with a lethal prognosis owing to thrombosis. Anticoagulation therapy should be considered proactively for prophylaxis of thrombotic dysfunction.

Abstract B7-H3 (also known as CD276) is associated with aggressive characteristics in various cancers. Meanwhile, in alveolar rhabdomyosarcoma (ARMS), PAX3-FOXO1 fusion protein is associated with increased aggressiveness and poor prognosis. In the present study, we explored the relationship between PAX3-FOXO1 and B7-H3 and the biological roles of B7-H3 in ARMS. Quantitative real time PCR and flow cytometry revealed that PAX3-FOXO1 knockdown downregulated B7-H3 expression in all the selected cell lines (Rh-30, Rh-41, and Rh-28), suggesting that PAX3-FOXO1 positively regulates B7-H3 expression. Gene expression analysis revealed that various genes and pathways involved in chemotaxis, INF-γ production, and myogenic differentiation were commonly affected by the knockdown of PAX3-FOXO1 and B7-H3. Wound healing and transwell migration assays revealed that both PAX3-FOXO1 and B7-H3 were associated with cell migration. Furthermore, knockdown of PAX3-FOXO1 or B7-H3 induced myogenin expression in all cell lines, although myosin heavy chain induction varied depending on the cellular context. Our results indicate that PAX3-FOXO1 regulates B7-H3 expression and that PAX3-FOXO1 and B7-H3 are commonly associated with multiple pathways related to an aggressive phenotype in ARMS, such as cell migration and myogenic differentiation block.

Ephrin type-B receptor 4 (EPHB4), expressed in tumors including rhabdomyosarcoma, is a suitable target for chimeric antigen receptor (CAR)-T cells. Ligand-independent activation of EPHB4 causes cell proliferation and malignant transformation in rhabdomyosarcoma, whereas ligand-dependent stimulation of EPHB4 induces apoptosis in rhabdomyosarcoma. Therefore, we hypothesized that ligand-based, EPHB4-specific CAR-T cells may kill rhabdomyosarcoma cells without stimulating downstream cell proliferation mechanisms. We developed novel CAR-T cells by targeting EPHB4 via EPHRIN B2, a natural ligand of EPHB4. The generation of EPHB4-CAR-T cells via piggyBac (PB) transposon-based gene transfer resulted in sufficient T cell expansion and CAR positivity (78.5% ± 5.9%). PB-EPHB4-CAR-T cells displayed a dominant stem cell memory fraction (59.4% ± 7.2%) as well as low PD-1 expression (0.60% ± 0.21%) after 14 days of expansion. The PB-EPHB4-CAR-T cells inhibited EPHB4-positive tumor cells without activating cell proliferation downstream of EPHB4, even after multiple tumor re-challenges and suppressed tumor growth in xenograft-bearing mice. Therefore, PB-EPHB4-CAR-T cells possess a memory-rich fraction without early T cell exhaustion and show potential as promising therapeutic agents for treating rhabdomyosarcoma and other EPHB4-positive tumors.

Abstract Background Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. There are two subtypes, fusion gene-positive RMS (FP-RMS) and fusion gene-negative RMS (FN-RMS), depending on the presence of a fusion gene, either PAX3-FOXO1 or PAX7-FOXO1. These fusion genes are thought to be oncogenic drivers of FP-RMS. By contrast, the underlying mechanism of FN-RMS has not been thoroughly investigated. It has recently been shown that HMGA2 is specifically positive in pathological tissue from FN-RMS, but the role of HMGA2 in FN-RMS remains to be clarified. Methods In this study, we used FN-RMS cell lines to investigate the function of HMGA2. Gene expression, cell growth, cell cycle, myogenic differentiation, tumor formation in vivo, and cell viability under drug treatment were assessed. Results We found that HMGA2 was highly expressed in FN-RMS cells compared with FP-RMS cells and that knockdown of HMGA2 in FN-RMS cells inhibited cell growth and induced G1 phase accumulation in the cell cycle and myogenic differentiation. Additionally, we showed using both gain-of-function and loss-of-function assays that HMGA2 was required for tumor formation in vivo. Consistent with these findings, the HMGA2 inhibitor netropsin inhibited the cell growth of FN-RMS. Conclusions Our results suggest that HMGA2 has important role in the oncogenicity of FP-RMS and may be a potential therapeutic target in patients with FN-RMS.

Abstract Background Few clinical trials have been reported for patients with intermediate-risk neuroblastoma because of the scarcity of the disease and the variety of clinical and biological characteristics. A multidisciplinary treatment that consists of multidrug chemotherapy and surgery is expected to lead to a good prognosis with few complications. Therefore, a clinical trial for patients with intermediate-risk tumors was designed to establish a standard treatment that reduces complications and achieves good outcomes. Methods We planned a prospective phase 2, single-arm study of the efficacy of image-defined risk factors (IDRF)-based surgical decision and stepwise treatment intensification for patients with intermediate-risk neuroblastomas. For the localized tumor group, IDRF evaluations will be performed after each three-course chemotherapy, and surgery will be performed when appropriate. For patients with metastatic tumors, a total of five chemotherapy courses will be performed, and primary lesions will be removed when the IDRF becomes negative. The primary endpoint is 3-year progression-free survival rate, and the secondary endpoints include 3-year progression-free survival rates and overall survival rates of the localized group and the metastasis group and the incidence of adverse events. From international results, 75% is considered an appropriate 3-year progression-free survival rate. If this trial’s expected 3-year progression-free survival rate of 85% is statistically greater than 75% in the lower limit of the 95.3% confidence interval, with an accuracy 10% (85 ± 10%), both groups require more than 65 patients. Discussion This study is the first clinical trial on the efficacy of IDRF-based surgical decision and stepwise treatment intensification for patients with intermediate-risk neuroblastomas. We expect that this study will contribute to the establishment of a standard treatment for patients with intermediate-risk neuroblastoma. Trial registration UMIN000004700 , jRCTs051180203 ; Registered on December 9, 2010.

Paragangliomas (PGL) are extremely rare tumors, especially in children. While minimally invasive surgical techniques are well established in adult patients, the evidence in children is extremely limited. PGL have a rich blood flow and are located around large vessels; thus, operative exposure and resection are often difficult due to proximity to major vascular structures including renal vessels, vena cava, and aorta. In particular, surgery for interaortocaval large PGL is extremely challenging. In this report, we presented a case of a child with hormonally active interaortocaval large PGL that was resected laparoscopically.A 14-year-old girl was transferred to the emergency room due to acute chest pain and vomiting after falling from a vaulting box. At presentation, her blood pressure was 218/137 mmHg and enhanced computed tomography of her abdomen revealed a 6.5-cm hypervascular tumor with intratumoral hemorrhage in the interaortocaval area. The patient's plasma catecholamine and 24-h urinary catecholamine level were found to be elevated. 131I-MIBG scan showed increased uptake in the tumor. The definitive diagnosis of PGL was made and we conducted elective laparoscopic excision of the retroperitoneal tumor. Careful and meticulous dissection was needed to separate the tumor from the surrounding vessels and tissues using a sealing device. Intraoperative hypertension was controlled using phentolamine, and the surgeon closely communicated with the anesthesiologist intraoperatively. Complete resection of the tumor was achieved laparoscopically. Total blood loss was 175 mL and the operating time was 422 min. Six months later, she remained well with no evidence of recurrence.The laparoscopic approach gave excellent exposure and observation of the tumor's relationship to surrounding structures. Our findings suggest that laparoscopic tumor removal for interaortocaval large PGL is a feasible and safe procedure in children with the improvement of laparoscopic surgical skills and devices.

Abstract Background Late cardiovascular disease-related adverse events are one of the most common causes of premature mortality among long-term survivors of childhood cancer. As it is difficult to reduce the heart dose with traditional anteroposterior–posteroanterior field whole lung irradiation for pulmonary metastasis, improved radiation techniques are highly desirable. We report a case treated with whole lung irradiation using volumetric modulated arc therapy. Case presentation A 3-year-old Japanese girl with pulmonary metastases of Wilms’ tumor received 12 Gy in 8 fractions of whole lung irradiation using volumetric modulated arc therapy. The treatment was well tolerated, and the course was completed as planned without any toxicity. We found statistically significant reduced volumetric modulated arc therapy irradiation doses to organs at risk relative to those of the standard anteroposterior–posteroanterior field technique. The mean heart dose was 8.5 Gy for volumetric modulated arc therapy and 12.3 Gy for the anteroposterior–posteroanterior field. The doses to liver and thyroid were also more favorable with volumetric modulated arc therapy than with the anteroposterior–posteroanterior field technique. We confirmed the dosimetric advantages of volumetric modulated arc therapy over anteroposterior–posteroanterior field in whole lung irradiation in terms of superior normal organ protection. Conclusions Effective heart sparing is possible for whole lung irradiation using volumetric modulated arc therapy. Large-scale studies using standardized procedures should be conducted to validate our results.

Abstract Background There is no established standard chemotherapy for recurrent pediatric solid tumors such as neuroblastoma and sarcoma. Since some of these tumor cells show dysfunctions in homologous recombination repair, the goal is to conduct a phase I study of olaparib, a poly(ADP-ribose) polymerase inhibitor. In this clinical trial, the aims are to evaluate the safety, tolerability, and efficacy of olaparib in pediatric patients with refractory solid tumors and to recommend a dose for phase II trials. Methods In this open-label, multicenter study, olaparib tablets (62.5, 125, and 187.5 mg/m2 b.i.d.) will be administered orally in a standard 3 + 3 dose escalation design. Patients aged 3 to 18 years with recurrent pediatric solid tumors are eligible. Pharmacokinetic and pharmacodynamic analyses will also be performed. Discussion This study aims to extend the indications for olaparib by assessing its safety and efficacy in pediatric refractory solid tumor patients. Trial registration UMIN-CTR (UMIN000025521); Registered on January 4, 2017.

Neuroblastomas (NBs) exhibit broad and divergent clinical behaviors and tumor risk classification at diagnosis is crucial for the selection of an appropriate therapeutic strategy for each patient. The present study aimed to validate the clinical relevance of International Neuroblastoma Risk Group (INRG) prognostic and genomic markers in a Japanese NB cohort using a retrospective analysis. Follow-up data based on 30 common INRG queries in 605 NB cases diagnosed in Japan between 1990 and 2014 were collected and the genome signature of each tumor sample was integrated. As previously indicated, age, tumor stage, MYCN, DNA ploidy, the adrenals as the primary tumor site, serum ferritin and lactate dehydrogenase (LDH) levels, segmental chromosome aberrations, and the number of chromosome breakpoints (BP) correlated with lower survival rates, while the thorax as the primary tumor site and numerical chromosome aberrations correlated with a favorable prognosis. In the patient group with stage 4, MYCN non-amplified tumors (n = 225), one of the challenging subsets for risk stratification, age ≥ 18 months, LDH ≥ 1400 U/L, and BP ≥ 7 correlated with lower overall and event-free survival rates (p < 0.05). The genome subgroup GG-P2s (partial chromosome gain/loss type with 1p/11q losses and 17q gain, n = 30) was strongly associated with a lower overall survival rate (5-year survival rate: 34%, p < 0.05). Therefore, the combination of the tumor genomic pattern (GG-P2s and BP ≥ 7) with age at diagnosis and LDH will be a promising predictor for MYCN-non-amplified high-risk NBs in patient subsets, in accordance with previous findings from the INRG project.

Purpose: Giant mediastinal germ cell tumor (MGCT) requires a well-planned advanced surgical approach. We retrospectively reviewed our surgical strategy for giant MGCT. Methods: Five children (median age, 5 years) with giant MGCT were treated in our institute from 2012 to 2016. Results: The initial diagnosis was made by tumor markers and image inspection in all cases. Benign teratomas (2 girls) and malignancies (3 boys) were treated with upfront surgery and radical tumorectomy after neo-adjuvant chemotherapy, respectively. After detailed 3D-CT, radical tumor excision was performed supported by a skilled pediatric cardiovascular surgeon. The basic approach was as follows: under cardiopulmonary support (CPS) or with CPS on standby, via median sternotomy, the pericardium and phrenic nerve were resected en bloc with the tumor, followed by diaphragmatic plication. Open biopsy was performed via lateral thoracotomy in 1 patient who showed dense adhesion and fistula formation in the lung; lobectomy via hemi-clamshell incision was required. No deaths or severe sequelae occurred in this series. Conclusions: Resectability is the most important predictor of outcomes for MGCTs. Preoperative 3D-CT and CPS can enable complete resection and ensure surgical safety. Well-functioned surgical team is critical success factor in such advanced surgery.

Introduction: Cunninghamella bertholletiae although rarely causing mucormycosis, is responsible for the highest mortality among mucormycetes. The diagnosis of mucormycosis is challenged by the absence of specific biomarkers. Herein, we report a fatal case of C. bertholletiae infection and detection of its DNA in the serum by polymerase chain reaction (PCR). Presentation of case: A 23-year-old male with refractory osteosarcoma was admitted with multiple lung metastases. He was on oral voriconazole prophylaxis after pulmonary aspergillosis. He suffered from fever during temporary neutropenia following chemotherapy and showed several neurological and respiratory symptoms. Despite liposomal-amphotericin B administration, the symptoms rapidly progressed, and he died five days after the onset of neurological symptoms.We retrospectively evaluated the filamentous fungus isolated after his death from gastric juices. Based on the sequence of the internal transcribed spacer (ITS) region we identified the fungal isolate as C. bertholletiae. A 146-bp portion of the D1/D2 region was quantified by quantitative-PCR using DNA extracted from the serum. C. bertholletiae DNA load in the serum was 18.0 copies/μL on the day of onset of neurological symptoms, with the highest (101.0 copies/μL) on the day of his death. Discussion: Detection of circulating DNA of mucormycetes in the blood would greatly enhance the diagnosis of mucormycosis. Rapid diagnosis might alleviate mortality due to mucormycosis. Conclusion: The present case-report suggests that the quantification of C. bertholletiae DNA in the serum could be useful for the diagnosis and evaluation of mucormycosis pathogenesis in patients.

Pediatric malignancies are thought to be risk factors of second malignant neoplasms. However, lung cancer associated with rhabdomyosarcoma is extremely rare. When multiple pulmonary lesions are observed in cancer patients, resection is necessary to distinguish them from metastatic lesions. We report an extremely rare case of synchronous bilateral multiple lung cancers showing ground glass nodules (GGNs) associated with rhabdomyosarcoma in a 15-year-old girl with vulvar rhabdomyosarcoma. When we identify GGNs in adolescents with a history of rhabdomyosarcoma, we should consider lung cancer as a differential diagnosis in addition to pulmonary metastasis usually showing solid nodule. In the present case with tiny lesions, surgical resection using lipiodol-marking helped to ensure both an early diagnosis and curative treatment. Keywords: Synchronous bilateral multiple lung cancers, Adolescent, Rhabdomyosarcoma

Background: Retinoblastomas account for 4% of malignancies in children, 1-2% of which are diffuse infiltrating retinoblastomas. Diffuse anterior retinoblastoma is rare and does not involve the retina. Here, we report on a diffuse anterior retinoblastoma with large sarcoidosis-like nodules on the iris that were responsive to anti-inflammatory therapy. Case: We present a 6-year-old girl who had anterior uveitis with white nodules on the iris and posterior surface of the cornea in her right eye. The nodules initially responded well to anti-inflammatory treatment. However, anterior segment optical coherence tomography (AS-OCT) showed that the nodules gradually grew, shrinking the iris. We then collected the aqueous humor for diagnosis. A biopsy revealed clusters of small cells with a high nuclear-to-cytoplasm ratio with partial rosette formation. Therefore, we diagnosed diffuse anterior retinoblastoma without retinal involvement and performed enucleation of the right eye. The histopathology demonstrated undifferentiated cells similar to those seen on the biopsy, and tumor cells invaded the iris stroma, posterior surface of the cornea, ciliary body, and sclera. After the enucleation, she underwent chemotherapy and remains alive. Conclusion: A differential diagnosis of retinoblastoma should be considered when white nodules refractory to anti-inflammatory therapy occur in the eye, even in the absence of obvious retinal masses. AS-OCT findings are useful in assessing retinoblastoma.

Obesity is a global health concern that promotes chronic low-grade inflammation, leading to insulin resistance, a key factor in many metabolic diseases. Angiotensin 1–7 (Ang 1–7), a component of the renin-angiotensin system (RAS), exhibits anti-inflammatory effects in obesity and related disorders, though its mechanisms remain unclear. In this study, we examined the effect of Ang 1–7 on inflammation of white adipose tissue (WAT) in dietary-induced obese mice. Monocyte chemoattractant protein-1 (MCP-1) produced by white adipocytes and tumour necrosis factor-α (TNF-α) produced by macrophages are pro-inflammatory cytokines and interact to form a pathogenic loop to exacerbate obesity-induced inflammation. We found that Ang 1–7 reduced MCP-1 and TNF-α gene expressions and the number of crown-like structures, which are histological hallmarks of the pro-inflammatory process, in visceral epididymal WAT (eWAT) and reduced circulating MCP-1 and TNF-α levels, accompanied by improvement in insulin resistance, in dietary-induced obese mice. Furthermore, Ang 1–7 reduced MCP-1 and TNF-α secretions in 3T3-L1 white adipocytes and RAW 264.7 macrophages, respectively, which are in vitro experimental models mimicking obesity condition. Our results suggest that Ang 1–7 directly acts on WAT to mitigate obesity-induced inflammation. Thus, this study provides novel insights into the underlying mechanism of anti-obesity effects of Ang 1–7.

We previously developed a method for determining MYCN gene amplification status using cell-free DNA fragments released from cancer cells into the blood of patients with neuroblastoma (NB). Here, we analyzed the relationship between MYCN amplification (MNA) status and neuroblastoma prognosis. We screened serum samples from 151 patients with NB for MNA, using real-time quantitative PCR, and compared the results with MYCN status determined using paired tumor samples. We additionally investigated whether MNA status correlates with patient survival. When a cut-off value of 5 was used, serum-based MNA analysis was found to show good sensitivity (86%) and very high specificity (95%). The sensitivities for stage 1 and 2 might be acceptable, even though it is not as good as for stage 3 and 4 (67% for stage 1 and 2, 92% for stage 3, and 87% for stage 4). MNA status correlated with overall survival in our cohort of 82 patients, with survival data available (p < 0.01). The hazard ratio of MNA status was 4.98 in patients diagnosed at less than 18 months of age (95% confidence interval, 1.00-24.78), and 1.41 (95% confidence interval, 0.63-3.14) for those diagnosed at 18 months of age or older. Serum-based MNA analysis is rapid and non-invasive compared with tumor-based MNA analysis, and has potential to predict tumor MNA status. There is still a room to improve the sensitivity of the test for tumors of stages 1 and 2, nonetheless this assay might help to determine therapeutic strategies prior to tumor biopsy, especially for patients with a life-threatening condition, as well as for patients of less than 18 months of age whose risk-grouping and treatment allocation depends on their MNA status.