A nationwide phase II study of delayed local treatment for children with high-risk neuroblastoma: The Japan Children's Cancer Group Neuroblastoma Committee Trial JN-H-11.

PURPOSE: Survival rates of patients with high-risk neuroblastoma are unacceptable. A time-intensified treatment strategy with delayed local treatment to control systemic diseases has been developed in Japan. We conducted a nationwide, prospective, single-arm clinical trial with delayed local treatment. This study evaluated the safety and efficacy of delayed surgery to increase treatment intensity. PATIENTS AND METHODS: Seventy-five patients with high-risk neuroblastoma were enrolled in this study between May 2011 and September 2015. Delayed local treatment consisted of five courses of induction chemotherapy (cisplatin, pirarubicin, vincristine, and cyclophosphamide) and myeloablative high-dose chemotherapy (melphalan, etoposide, and carboplatin), followed by local tumor extirpation with surgery and irradiation. The primary endpoint was progression-free survival (PFS). The secondary endpoints were overall survival (OS), response rate, adverse events, and surgical complications. RESULTS: Seventy-five patients were enrolled, and 64 were evaluable (stage 3, n = 8; stage 4, n = 56). The estimated 3-year PFS and OS rates (95% confidence interval [CI]) were 44.4% [31.8%-56.3%] and 80.7% [68.5%-88.5%], resspectively. The response rate of INRC after completion of the treatment protocol was 66% (42/64; 95% CI: 53%-77%; 23 CR [complete response], 10 VGPR [very good partial response], and nine PR [partial response]). None of the patients died during the protocol treatment or within 30 days of completion. Grade 4 adverse effects, excluding hematological adverse effects, occurred in 48% of patients [31/64; 95% CI: 36%-61%]. Major Surgical complications were observed in 25% of patients [13/51; 95% CI: 14%-40%]. CONCLUSION: This study indicates that delayed local treatment is feasible and shows promising efficacy, suggesting that this treatment should be considered further in a comparative study of high-risk neuroblastoma.

Pediatric Precision Medicine at the National Cancer Center Japan: Prospective Genomic Study of Pediatric Patients with Cancer as Part of the TOP-GEAR Project.

PURPOSE: This single-center, prospective molecular profiling study characterizes genomic alterations and identifies therapeutic targets in advanced pediatric solid tumors. METHODS: As part of the TOP-GEAR (Trial of Onco-Panel for Gene profiling to Estimate both Adverse events and Response by cancer treatment) project at the National Cancer Center (NCC), Japan, we enrolled pediatric patients with a refractory or recurrent disease during August 2016-December 2021 and performed genomic analysis of matched tumors and blood using originally developed cancer gene panels, NCC Oncopanel (ver. 4.0) and NCC Oncopanel Ped (ver. 1.0). RESULTS: Of 142 patients (age, 1-28 years) enrolled, 128 (90%) were evaluable for genomic analysis; 76 (59%) patients harbored at least one reportable somatic or germline alteration. The tumor samples were collected during the initial diagnosis in 65 (51%) patients, after treatment initiation in 11 (9%) patients, and upon either disease progression or relapse in 52 (41%) patients. The leading altered gene was TP53, followed by MYCN, MYC, CDKN2A, and CDK4. The commonly affected molecular processes were transcription, cell-cycle regulation, epigenetic modifiers, and RAS/mitogen-activated protein kinase signaling. Twelve (9%) patients carried pathogenic germline variants in cancer-predisposing genes. Potentially actionable findings were identified in 40 (31%) patients; to date, 13 (10%) patients have received the recommended therapy on the basis of their genomic profiles. Although four patients had access to targeted therapy through clinical trials, the agents were used in nine patients in an off-label setting. CONCLUSION: The implementation of genomic medicine has furthered our understanding of tumor biology and provided new therapeutic strategies. However, the paucity of proposed agents limits the full potential of actionability, emphasizing the significance of facilitating access to targeted cancer therapies.

ATM depletion induces proteasomal degradation of FANCD2 and sensitizes neuroblastoma cells to PARP inhibitors.

BACKGROUND: Genomic alterations, including loss of function in chromosome band 11q22-23, are frequently observed in neuroblastoma, which is the most common extracranial childhood tumour. In neuroblastoma, ATM, a DNA damage response-associated gene located on 11q22-23, has been linked to tumorigenicity. Genetic changes in ATM are heterozygous in most tumours. However, it is unclear how ATM is associated with tumorigenesis and cancer aggressiveness. METHODS: To elucidate its molecular mechanism of action, we established ATM-inactivated NGP and CHP-134 neuroblastoma cell lines using CRISPR/Cas9 genome editing. The knock out cells were rigorously characterized by analyzing proliferation, colony forming abilities and responses to PARP inhibitor (Olaparib). Western blot analyses were performed to detect different protein expression related to DNA repair pathway. ShRNA lentiviral vectors were used to knockdown ATM expression in SK-N-AS and SK-N-SH neuroblastoma cell lines. ATM knock out cells were stably transfected with FANCD2 expression plasmid to over-expressed the FANCD2. Moreover, knock out cells were treated with proteasome inhibitor MG132 to determine the protein stability of FANCD2. FANCD2, RAD51 and γH2AX protein expressions were determined by Immunofluorescence microscopy. RESULTS: Haploinsufficient ATM resulted in increased proliferation (p < 0.01) and cell survival following PARP inhibitor (olaparib) treatment. However, complete ATM knockout decreased proliferation (p < 0.01) and promoted cell susceptibility to olaparib (p < 0.01). Complete loss of ATM suppressed the expression of DNA repair-associated molecules FANCD2 and RAD51 and induced DNA damage in neuroblastoma cells. A marked downregulation of FANCD2 expression was also observed in shRNA-mediated ATM-knockdown neuroblastoma cells. Inhibitor experiments demonstrated that the degradation of FANCD2 was regulated at the protein level through the ubiquitin-proteasome pathway. Reintroduction of FANCD2 expression is sufficient to reverse decreased proliferation mediated by ATM depletion. CONCLUSIONS: Our study revealed the molecular mechanism underlying ATM heterozygosity in neuroblastomas and elucidated that ATM inactivation enhances the susceptibility of neuroblastoma cells to olaparib treatment. These findings might be useful in the treatment of high-risk NB patients showing ATM zygosity and aggressive cancer progression in future.

Development of an osteosarcoma model with MYCN amplification and TP53 mutation in hiPS cell-derived neural crest cells.

Mesenchymal stem cell- or osteoblast-derived osteosarcoma is the most common malignant bone tumor. Its highly metastatic malignant phenotypes, which are often associated with a poor prognosis, have been correlated with the modulation of TP53- and cell-cycle-related pathways. MYC, which regulates the transcription of cell-cycle modulating genes, is used as a representative prognostic marker for osteosarcoma. Another member of the MYC oncoprotein family, MYCN, is highly expressed in a subset of osteosarcoma, however its roles in osteosarcoma have not been fully elucidated. Here, we attempted to create an in vitro tumorigenesis model using hiPSC-derived neural crest cells, which are precursors of mesenchymal stem cells, by overexpressing MYCN on a heterozygous TP53 hotspot mutation (c.733G>A; p.G245S) background. MYCN-expressing TP53 mutated transformed clones were isolated by soft agar colony formation, and administered subcutaneously into the periadrenal adipose tissue of immunodeficient mice, resulting in the development of chondroblastic osteosarcoma. MYCN suppression decreased the proliferation of MYCN-induced osteosarcoma cells, suggesting MYCN as a potential target for a subset of osteosarcoma treatment. Further, comprehensive analysis of gene expression and exome sequencing of MYCN-induced clones indicated osteosarcoma-specific molecular features, such as the activation of TGF-ß signaling and DNA copy number amplification of GLI1. The model of MYCN-expressing chondroblastic osteosarcoma was developed from hiPSC-derived neural crest cells, providing a useful tool for the development of new tumor models using hiPSC-derived progenitor cells with gene modifications and in vitro transformation.

Polycomb group protein BMI1 protects neuroblastoma cells against DNA damage-induced apoptotic cell death.

The overexpression of BMI1, a polycomb protein, correlates with cancer development and aggressiveness. We previously reported that MYCN-induced BMI1 positively regulated neuroblastoma (NB) cell proliferation via the transcriptional inhibition of tumor suppressors in NB cells. To assess the potential of BMI1 as a new target for NB therapy, we examined the effects of reductions in BMI1 on NB cells. BMI1 knockdown (KD) in NB cells significantly induced their differentiation for up to 7 days. BMI1 depletion significantly induced apoptotic NB cell death for up to 14 days along with the activation of p53, increases in p73, and induction of p53 family downstream molecules and pathways, even in p53 mutant cells. BMI1 depletion in vivo markedly suppressed NB xenograft tumor growth. BMI1 reductions activated ATM and increased γ-H2AX in NB cells. These DNA damage signals and apoptotic cell death were not canceled by the transduction of the polycomb group molecules EZH2 and RING1B. Furthermore, EZH2 and RING1B KD did not induce apoptotic NB cell death to the same extent as BMI1 KD. Collectively, these results suggest the potential of BMI1 as a target of molecular therapy for NB and confirmed, for the first time, the shared role of PcG proteins in the DNA damage response of NB cells.

Comparative epigenomics by machine learning approach for neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the second most common pediatric solid tumor. Because the number of genetic mutations found in tumors are small, even in some patients with unfavorable NB, epigenetic variation is expected to play an important role in NB progression. DNA methylation is a major epigenetic mechanism, and its relationship with NB prognosis has been a concern. One limitation with the analysis of variation in DNA methylation is the lack of a suitable analytical model. Therefore, in this study, we performed a random forest (RF) analysis of the DNA methylome data of NB from multiple databases. RESULTS: RF is a popular machine learning model owing to its simplicity, intuitiveness, and computational cost. RF analysis identified novel intermediate-risk patient groups with characteristic DNA methylation patterns within the low-risk group. Feature selection analysis based on probe annotation revealed that enhancer-annotated regions had strong predictive power, particularly for MYCN-amplified NBs. We developed a gene-based analytical model to identify candidate genes related to disease progression, such as PRDM8 and FAM13A-AS1. RF analysis revealed sufficient predictive power compared to other machine learning models. CONCLUSIONS: RF is a useful tool for DNA methylome analysis in cancer epigenetic studies, and has potential to identify a novel cancer-related genes.

Polycomb EZH1 regulates cell cycle/5-fluorouracil sensitivity of neuroblastoma cells in concert with MYCN.

In the present study, we found that EZH1 depletion in MYCN-amplified neuroblastoma cells resulted in significant cell death as well as xenograft inhibition. EZH1 depletion decreased the level of H3K27me1; the interaction and protein stabilization of MYCN and EZH1 appear to play roles in epigenetic transcriptional regulation. Transcriptome analysis of EZH1-depleted cells resulted in downregulation of the cell cycle progression-related pathway. In particular, Gene Set Enrichment Analysis revealed downregulation of reactome E2F-mediated regulation of DNA replication along with key genes of this process, TYMS, POLA2, and CCNA1. TYMS and POLA2 were transcriptionally activated by MYCN and EZH1-related epigenetic modification. Treatment with the EZH1/2 inhibitor UNC1999 also induced cell death, decreased H3K27 methylation, and reduced the levels of TYMS in neuroblastoma cells. Previous reports indicated neuroblastoma cells are resistant to 5-fluorouracil (5-FU) and TYMS (encoding thymidylate synthetase) has been considered the primary site of action for folate analogues. Intriguingly, UNC1999 treatment significantly sensitized MYCN-amplified neuroblastoma cells to 5-FU treatment, suggesting that EZH inhibition could be an effective strategy for development of a new epigenetic treatment for neuroblastoma.

The PRC2 molecule EED is a target of epigenetic therapy for neuroblastoma.

Epigenetic modifications by polycomb repressive complex (PRC) molecules appear to play a role in the tumorigenesis and aggressiveness of neuroblastoma (NB). Embryonic ectoderm development (EED) is a member of the PRC2 complex that binds to the H3K27me3 mark deposited by EZH2 via propagation on adjacent nucleosomes. We herein investigated the molecular roles of EED in MYCN-amplified NB cells using EED-knockdown (KD) shRNAs, EED-knockout sgRNAs, and the EED small molecule inhibitor EED226. The suppression of EED markedly inhibited NB cell proliferation and flat and soft agar colony formation. A transcriptome analysis using microarrays of EED-KD NB cells indicated the de-repression of cell cycle-regulated and differentiation-related genes. The results of a GSEA analysis suggested that inhibitory cell cycle-regulated gene sets were markedly up-regulated. Furthermore, an epigenetic treatment with the EED inhibitor EED226 and the HDAC inhibitors valproic acid/SAHA effectively suppressed NB cell proliferation and colony formation. This combined epigenetic treatment up-regulated cell cycle-regulated and differentiation-related genes. The ChIP sequencing analysis of histone codes and PRC molecules suggested an epigenetic background for the de-repression of down-regulated genes in MYCN-amplified/PRC2 up-regulated NB.

Infantile Stage M Neuroblastoma With 11q Deletion, Mimicking Stage MS.

A 4-month-old boy with abdominal distension was diagnosed with adrenal neuroblastoma with numerous metastases to the liver and nodules in the skin and muscles. Marked hepatomegaly spontaneously regressed with decreasing tumor marker levels, and the final diagnosis was stage M based on radiologic findings confirming metastasis to the pancreas. The neuroblastoma did not have the MYCN amplification but had an 11q aberration. Chemotherapy was initiated at age 6 months with a successful response. Our case reflects the heterogenous clinical behavior of neuroblastoma and highlights the challenging issue of the difference between stage M and stage MS neuroblastoma in infants.

FGFR2 loss sensitizes MYCN-amplified neuroblastoma CHP134 cells to CHK1 inhibitor-induced apoptosis.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. In this study, one CHK1i-sensitive neuroblastoma cell line, CHP134, was investigated, which characteristically carries MYCN amplification and a chromosome deletion within the 10q region. Among several cancer-related genes in the chromosome 10q region, mRNA expression of fibroblast growth factor receptor 2 (FGFR2) was altered in CHP134 cells and associated with an unfavorable prognosis of patients with neuroblastoma. Induced expression of FGFR2 in CHP134 cells reactivated downstream MEK/ERK signaling and resulted in cells resistant to CHK1i-mediated cell growth inhibition. Consistently, the MEK1/2 inhibitor, trametinib, potentiated CHK1 inhibitor-mediated cell death in these cells. These results suggested that FGFR2 loss might be prone to highly effective CHK1i treatment. In conclusion, extreme cellular dependency of ERK activation may imply a possible application for the MEK1/2 inhibitor, either as a single inhibitor or in combination with CHK1i in MYCN-amplified neuroblastomas.

Loss of p53 suppresses replication stress-induced DNA damage in ATRX-deficient neuroblastoma.

Genetic aberrations are present in the ATRX gene in older high-risk neuroblastoma (NB) patients with very poor clinical outcomes. Its loss-of-function (LoF) facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells and is strongly linked to replication stress (RS) and DNA damage through G-quadruplex (G4) DNA secondary structures. However, limited information is available on ATRX alteration-related NB tumorigenesis. We herein knocked out (KO) ATRX in MYCN-amplified (NGP) and MYCN single copy (SK-N-AS) NB cells with wild-type (wt) and truncated TP53 at the C terminus, respectively, using CRISPR/Cas9 technologies. The loss of ATRX increased DNA damage and G4 formation related to RS in TP53 wt isogenic ATRX KO NGP cells, but not in SK-N-AS clones. A gene set enrichment analysis (GSEA) showed that the gene sets related to DNA double-strand break repair, negative cell cycle regulation, the G2M checkpoint, and p53 pathway activation were enriched in NGP clones. The accumulation of DNA damage activated the ATM/CHK2/p53 pathway, leading to cell cycle arrest in NGP clones. Interestingly, ATRX loss did not induce RS related to DNA damage response (DDR) in TP53-truncated SK-N-AS cells. p53 inactivation abrogated cell cycle arrest and reduced G4 accumulation in NGP clones. The loss of p53 also induced G4 DNA helicases or Fanconi anemia group D2 protein (FANCD2) with ATRX deficiency, suggesting that ATRX maintained genome integrity and p53 deficiency attenuated RS-induced DNA damage in NB cells featuring inactivated ATRX by regulating DNA repair mechanisms and replication fork stability.

CDK4 overexpression is a predictive biomarker for resistance to conventional chemotherapy in patients with osteosarcoma.

Osteosarcoma (OS) is the most common malignant bone tumor, and its sensitivity to preoperative chemotherapy is a significant prognostic factor. The present study aimed to identify potential genomic markers for the prediction of chemosensitivity in patients with OS using a genomic approach. A total of 50 pediatric and adolescent patients diagnosed with high­grade OS were selected. Each pre­therapeutic biopsy sample was subjected to comparative genomic hybridization array analysis and targeted exome sequencing. Although no recurrent gene mutation was observed in chemoresistant tumors, copy number analysis detected recurrent gain of chromosome 12q14.1, which was significantly more frequent (5/21; 24%) in the poor responder cohort than in the good responder cohort (0/29; 0%; P<0.01). Subsequent expression analysis revealed that CDK4 was the only gene in the 12q14.1 gained region with an expression level that was positively associated with copy number gains. In order to elucidate the effect of CDK4 on drug sensitivity, CDK4­overexpressing OS cell lines were treated with cisplatin (CDDP); significant attenuation of CDDP sensitivity, demonstrated by increased cell viability and decreased expression of cleaved caspase­9, was induced by enforced expression of CDK4. In addition, treatment with CDK4/6 inhibitor palbociclib in CDK4­overexpressing U2OS cells facilitated apoptosis and a significant decrease in cell viability in a dose­dependent manner. In conclusion, the results of the present study showed that higher expression and amplification of CDK4 in tumors is a predictive biomarker for resistance to conventional chemotherapy in patients with OS and that palbociclib is a promising drug for this therapeutically challenging cohort.

Retrospective Analysis of INRG Clinical and Genomic Factors for 605 Neuroblastomas in Japan: A Report from the Japan Children's Cancer Group Neuroblastoma Committee (JCCG-JNBSG).

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.

Correction to: Results of a phase II trial for high-risk neuroblastoma treatment protocol JN-H-07: a report from the Japan Childhood Cancer Group Neuroblastoma Committee (JNBSG).

In the October 2018 issue of International Journal of Clinical Oncology.

Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties.

Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumor-promoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.

TAp63 represses transcription of MYCN/NCYM gene and its high levels of expression are associated with favorable outcome in neuroblastoma.

TAp63 is an isoform of p63 gene, a p53 family gene that suppresses tumorigenesis via transcriptional regulation. TAp63 represses transcription of MYC oncogene in glioblastomas; however, its role in another MYC family gene, MYCN, has remained elusive. In this study, we showed that TAp63 repressed transcription of the MYCN gene in human cancer cells. Overexpression of TAp63 in HeLa cells suppressed MYCN expression, whereas knockdown of TAp63 had the opposite effect. By binding to exon 1 of MYCN gene, TAp63 suppressed the promoter activities of MYCN and its cis-antisense gene, NCYM. Other p53 family members, p53 and TAp73, showed lesser ability to suppress MYCN/NCYM promoter activities compared with that of TAp63. All-trans-retinoic acid (ATRA) treatment of MYCN/NCYM-amplified neuroblastoma CHP134 cells induced TAp63 and reduced p53 expressions, accompanied by downregulation of MYCN/NCYM expressions. Meanwhile, TAp63 knockdown inhibited ATRA-induced repression of NCYM gene expression. Blocking the p53 family binding sites by CRISPR-dCas9 system in CHP134 cells induced MYCN/NCYM expression and promoted apoptotic cell death. Expression levels of TAp63 mRNA inversely correlated with those of MYCN/NCYM expression in primary neuroblastomas, which was associated with a favorable prognosis. Collectively, TAp63 repressed MYCN/NCYM bidirectional transcription, contributing to the suppression of neuroblastoma growth.

PPP3CB contributes to poor prognosis through activating nuclear factor of activated T-cells signaling in neuroblastoma.

We previously identified a gain-of-function mutation in PPP3CB in a neuroblastoma (NB) with MYCN amplification. Here we investigated the functional and clinical role of PPP3CB in NB. High PPP3CB expression was an independent indicator predicting poor prognosis of NB. Overexpression of wildtype or mutated PPP3CB (PPP3CBmut) promoted cell growth, but PPP3CB knockdown decreased cell growth in NB cells. Forced expressions of PPP3CB and PPP3CBmut activated NFAT2 and NFAT4 transcription factors and inhibited GSK3ß activity, resulting in the increase in the expressions of c-Myc, MYCN, and ß-catenin, which were downregulated in response to PPP3CB knockdown. Treatment with calcineurin inhibitor cyclosporin A (CsA) or FK506 suppressed cell proliferation and induced apoptotic cell death in both MYCN-amplified and MYCN-non-amplified NB cell lines. Expression of PPP3CB protein was decreased in response to two calcineurin inhibitors. c-Myc, MYCN, and ß-catenin were downregulated at the mRNA and protein levels in CsA or FK506-treated NB cells. Our data indicate that elevated expression of PPP3CB and the expression of its constitutively active mutant contribute to the aggressive behavior of NB tumors and therefore suggest that inhibition of calcineurin activity might have therapeutic potential for high-risk NB.

Results of a phase II trial for high-risk neuroblastoma treatment protocol JN-H-07: a report from the Japan Childhood Cancer Group Neuroblastoma Committee (JNBSG).

BACKGROUND: The Japanese Children's Cancer Group (JCCG) Neuroblastoma Committee (JNBSG) conducted a phase II clinical trial for high-risk neuroblastoma treatment. We report the result of the protocol treatment and associated genomic aberration studies. METHODS: JN-H-07 was a single-arm, late phase II trial for high-risk neuroblastoma treatment with open enrollment from June 2007 to February 2009. Eligible patients underwent five courses of induction chemotherapy followed by high-dose chemotherapy with hematopoietic stem cell rescue. Surgery for the primary tumor was scheduled after three or four courses of induction chemotherapy. Radiotherapy was administered to the primary tumor site and to any bone metastases present at the end of induction chemotherapy. RESULTS: The estimated 3-year progression-free and overall survival rates of the 50 patients enrolled were 36.5 ± 7.0 and 69.5 ± 6.6%, respectively. High-dose chemotherapy caused severe toxicity including three treatment-related deaths. In response to this, the high-dose chemotherapy regimen was modified during the trial by infusing melphalan before administering carboplatin and etoposide. The modified high-dose chemotherapy regimen was less toxic. Univariate analysis revealed that patients younger than 547 days and patients whose tumor showed a whole chromosomal gains / losses pattern had a significantly poor prognosis. Notably, the progression-free survival of cases with MYCN amplification were not inferior to those without MYCN amplification. CONCLUSIONS: The outcome of patients treated with the JN-H-07 protocol showed improvement over the results reported by previous studies conducted in Japan. Molecular and genetic profiling may enable a more precise stratification of the high-risk cohort.

Genomic Amplifications and Distal 6q Loss: Novel Markers for Poor Survival in High-risk Neuroblastoma Patients.

Background: Neuroblastoma is characterized by substantial clinical heterogeneity. Despite intensive treatment, the survival rates of high-risk neuroblastoma patients are still disappointingly low. Somatic chromosomal copy number aberrations have been shown to be associated with patient outcome, particularly in low- and intermediate-risk neuroblastoma patients. To improve outcome prediction in high-risk neuroblastoma, we aimed to design a prognostic classification method based on copy number aberrations. Methods: In an international collaboration, normalized high-resolution DNA copy number data (arrayCGH and SNP arrays) from 556 high-risk neuroblastomas obtained at diagnosis were collected from nine collaborative groups and segmented using the same method. We applied logistic and Cox proportional hazard regression to identify genomic aberrations associated with poor outcome. Results: In this study, we identified two types of copy number aberrations that are associated with extremely poor outcome. Distal 6q losses were detected in 5.9% of patients and were associated with a 10-year survival probability of only 3.4% (95% confidence interval [CI] = 0.5% to 23.3%, two-sided P = .002). Amplifications of regions not encompassing the MYCN locus were detected in 18.1% of patients and were associated with a 10-year survival probability of only 5.8% (95% CI = 1.5% to 22.2%, two-sided P < .001). Conclusions: Using a unique large copy number data set of high-risk neuroblastoma cases, we identified a small subset of high-risk neuroblastoma patients with extremely low survival probability that might be eligible for inclusion in clinical trials of new therapeutics. The amplicons may also nominate alternative treatments that target the amplified genes.

EZH2 regulates neuroblastoma cell differentiation via NTRK1 promoter epigenetic modifications.

The polycomb repressor complex 2 molecule EZH2 is now known to play a role in essential cellular processes, namely, cell fate decisions, cell cycle regulation, senescence, cell differentiation, and cancer development/progression. EZH2 inhibitors have recently been developed; however, their effectiveness and underlying molecular mechanisms in many malignancies have not yet been elucidated in detail. Although the functional role of EZH2 in tumorigenesis in neuroblastoma (NB) has been investigated, mutations of EZH2 have not been reported. A Kaplan-Meier analysis on the event free survival and overall survival of NB patients indicated that the high expression of EZH2 correlated with an unfavorable prognosis. In order to elucidate the functional roles of EZH2 in NB tumorigenesis and its aggressiveness, we knocked down EZH2 in NB cell lines using lentivirus systems. The knockdown of EZH2 significantly induced NB cell differentiation, e.g., neurite extension, and the neuronal differentiation markers, NF68 and GAP43. EZH2 inhibitors also induced NB cell differentiation. We performed a comprehensive transcriptome analysis using Human Gene Expression Microarrays and found that NTRK1 (TrkA) is one of the EZH2-related suppression targets. The depletion of NTRK1 canceled EZH2 knockdown-induced NB cell differentiation. Our integrative methylome, transcriptome, and chromatin immunoprecipitation assays using NB cell lines and clinical samples clarified that the NTRK1 P1 and P2 promoter regions were regulated differently by DNA methylation and EZH2-related histone modifications. The NTRK1 transcript variants 1/2, which were regulated by EZH2-related H3K27me3 modifications at the P1 promoter region, were strongly expressed in favorable, but not unfavorable NB. The depletion and inhibition of EZH2 successfully induced NTRK1 transcripts and functional proteins. Collectively, these results indicate that EZH2 plays important roles in preventing the differentiation of NB cells and also that EZH2-related NTRK1 transcriptional regulation may be the key pathway for NB cell differentiation.