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.

Asymmetric Pericentrosomal CD133 Endosomes Induce the Unequal Autophagic Activity During Cytokinesis in CD133-Positive Human Neuroblastoma Cells.

CD133 is a transmembrane protein that mainly localizes to the plasma membrane in hematopoietic/neural stem cells and cancer stem cells. Although CD133 also localizes to the cytoplasm and is degraded through autophagy, the precise mechanisms responsible for the autophagic degradation of endosomal CD133 currently remain unknown. We demonstrated that endosomal CD133 has unique properties for cell homeostasis. Endosomal CD133 is degraded through p62/SQSTM1-mediated selective autophagy. However, in low basal autophagic cells, such as SK-N-DZ and SH-SY5Y cells, endosomal CD133 accumulates at the pericentrosomal region and conversely suppresses autophagy. Endosomal CD133 also asymmetrically distributes to the pericentrosomal region and induces unequal autophagic activity between 2 daughter cells during cytokinesis in SK-N-DZ and TGW cells. In addition, the asymmetric distribution of pericentrosomal CD133 endosomes and nuclear ß-catenin cooperatively suppresses autophagic activity against p62 in SK-N-DZ cells. Thus, the present study suggests that the asymmetric distribution of pericentrosomal CD133 endosomes induces the symmetry breaking of autophagic activity during cytokinesis in cooperation with nuclear ß-catenin.

Outcome of children with relapsed high-risk neuroblastoma in Japan and analysis of the role of allogeneic hematopoietic stem cell transplantation.

BACKGROUND: In Japan, allogeneic hematopoietic stem cell transplantation is widely performed for recurrent neuroblastomas. This retrospective study aimed to investigate the prognosis of recurrent neuroblastoma in Japan and explore the effectiveness of allogeneic hematopoietic stem cell transplantation. METHODS: Clinical characteristics and data on the treatment of patients with high-risk neuroblastoma who experienced first progression between 2003 and 2010 after attaining complete remission or partial remission were collected from hospitals participating in the Japanese Neuroblastoma Research Group. RESULTS: Data from 61 patients who fulfilled these criteria were collected. The median interval from disease onset to first progression was 19 months (range, 7-65 months), whereas the median observation time of the surviving patients was 18 months (range, 1-69 months). All patients were treated with chemotherapy, where 22 and 3 patients received allogeneic and autologous hematopoietic stem cell transplantation, respectively. Seven patients were alive in second complete remission, and 39 died, including two in complete remission. The 3-year progression-free survival and overall survival rates were 15.3% (SE: 6.1%) and 16.9% (SE: 6.5%), respectively. For patients with allogeneic hematopoietic stem cell transplantation, the 3-year progression-free survival and overall survival were 28.3% (standard error, 12.0%) and 24.3% (standard error, 11.5%), respectively, and for patients without allogeneic hematopoietic stem cell transplantation, the 3-year progression-free survival and overall survival were 6.0% (standard error 5.5%) and 12.0% (standard error 7.6%), respectively. The duration of initial remission (≥ 18 months) and implementation of allogeneic hematopoietic stem cell transplantation were independently predictive of progression-free survival (P = 0.002 and P = 0.017), whereas for overall survival, only allogeneic hematopoietic stem cell transplantation was predictive (P = 0.012). CONCLUSION: Although allogeneic hematopoietic stem cell transplantation contributed to some improvement in prognosis, it was insufficient.

Collagen XI Alpha 1 (COL11A1) Expression in the Tumor Microenvironment Drives Neuroblastoma Dissemination.

BACKGROUND: Neuroblastoma (NB) is among the most common cancers in children. A highly aggressive form of cancer, NB relies on cells in the microenvironment for dissemination particularly cancer associated fibroblast (CAFs). CAFs synthesise the extracellular matrix to create a scaffold for tumor growth thus enabling the carcinogenesis of NB, Collagen, an abundant scaffold protein produced by CAFs, has been implicated in the creation of an optimal tumor microenvironment, however, the expression profile of collagen within NB is not yet known. METHODS: We characterised collagen expression within the tumor-stroma boundary by microarray and confirmed by qRT-PCR and immunohistochemistry. RESULTS: The collagen marker, COL11A1, was also upregulated in NB CD45+ cells and SMA+ CAFs. Furthermore, SMA+ CAFs led to neuroblastoma cell invasion in an in vitro co-culture system which was subsequently attenuated by gene silencing COL11A1. Immunohistochemical staining of clinical tumor samples revealed that high COL11A1 expression in the stroma adjacent to tumour site, significantly associated with advanced cancer stages, age ≥18 months, undifferentiated tumor status, relapse and poor overall survival. CONCLUSION: Collectively, these results suggest that a COL11A1 signature in the NB microenvironment could represent a novel target for therapeutic intervention.

Medical guidelines for Li-Fraumeni syndrome 2019, version 1.1.

Li-Fraumeni syndrome (LFS) is a hereditary tumor that exhibits autosomal dominant inheritance. LFS develops in individuals with a pathogenic germline variant of the cancer-suppressor gene, TP53 (individuals with TP53 pathogenic variant). The number of individuals with TP53 pathogenic variant among the general population is said to be 1 in 500 to 20,000. Meanwhile, it is found in 1.6% (median value, range of 0-6.7%) of patients with pediatric cancer and 0.2% of adult patients with cancer. LFS is diagnosed by the presence of germline TP53 pathogenic variants. However, patients can still be diagnosed with LFS even in the absence of a TP53 pathogenic variant if the familial history of cancers fit the classic LFS diagnostic criteria. It is recommended that TP53 genetic testing be promptly performed if LFS is suspected. Chompret criteria are widely used for the TP53 genetic test. However, as there are a certain number of cases of LFS that do not fit the criteria, if LFS is suspected, TP53 genetic testing should be performed regardless of the criteria. The probability of individuals with TP53 pathogenic variant developing cancer in their lifetime (penetrance) is 75% for men and almost 100% for women. The LFS core tumors (breast cancer, osteosarcoma, soft tissue sarcoma, brain tumor, and adrenocortical cancer) constitute the majority of cases; however, various types of cancers, such as hematological malignancy, epithelial cancer, and pediatric cancers, such as neuroblastoma, can also develop. Furthermore, approximately half of the cases develop simultaneous or metachronous multiple cancers. The types of TP53 pathogenic variants and factors that modify the functions of TP53 have an impact on the clinical presentation, although there are currently no definitive findings. There is currently no cancer preventive agent for individuals with TP53 pathogenic variant. Surgical treatments, such as risk-reducing bilateral mastectomy warrant further investigation. Theoretically, exposure to radiation could induce the onset of secondary cancer; therefore, imaging and treatments that use radiation should be avoided as much as possible. As a method to follow-up LFS, routine cancer surveillance comprising whole-body MRI scan, brain MRI scan, breast MRI scan, and abdominal ultrasonography (US) should be performed immediately after the diagnosis. However, the effectiveness of this surveillance is unknown, and there are problems, such as adverse events associated with a high rate of false positives, overdiagnosis, and sedation used during imaging as well as negative psychological impact. The detection rate of cancer through cancer surveillance is extremely high. Many cases are detected at an early stage, and treatments are low intensity; thus, cancer surveillance could contribute to an improvement in QOL, or at least, a reduction in complications associated with treatment. With the widespread use of genomic medicine, the diagnosis of LFS is unavoidable, and a comprehensive medical care system for LFS is necessary. Therefore, clinical trials that verify the feasibility and effectiveness of the program, comprising LFS registry, genetic counseling, and cancer surveillance, need to be prepared.

De novo evolved gene product NCYM in the pathogenesis and clinical outcome of human neuroblastomas and other cancers.

NCYM is an antisense transcript of MYCN oncogene and promotes tumor progression. NCYM encodes a de novo protein whose open reading frame evolved from noncoding genomic regions in the ancestor of Homininae. Because of its topology, NCYM is always co-amplified with MYCN oncogene, and the mutual regulations between NCYM and MYCN maintain their expressions at high levels in MYCN-amplified tumors. NCYM stabilizes MYCN by inhibiting GSK3ß, whereas MYCN stimulates transcription of both NCYM and MYCN. NCYM mRNA and its noncoding transcript variants MYCNOS have been shown to stimulate MYCN expression via direct binding to MYCN promoter, indicating that both coding and noncoding transcripts of NCYM induce MYCN expression. In contrast to the noncoding functions of NCYM, NCYM protein also promotes calpain-mediated cleavage of c-MYC. The cleaved product called Myc-nick inhibits cell death and promotes cancer cell migration. Furthermore, NCYM-mediated inhibition of GSK3ß results in the stabilization of ß-catenin, which promotes aggressiveness of bladder cancers. These MYCN-independent functions of NCYM showed their clinical significance in MYCN-non-amplified tumors, including adult tumors. This year is the 30th anniversary of the identification of NCYM/MYCNOS gene. On this special occasion, we summarize the current understanding of molecular functions and the clinical significance of NCYM and discuss future directions to achieve therapeutic strategies targeting NCYM.

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.

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.

Tumor suppressor KIF1Bß regulates mitochondrial apoptosis in collaboration with YME1L1.

KIF1Bß, a member of the kinesin superfamily of motor proteins, is a haploinsufficient tumor suppressor mapped to chromosome 1p36.2, which is frequently deleted in neural crest-derived tumors, including neuroblastoma and pheochromocytoma. While KIF1Bß acts downstream of the nerve growth factor (NGF) pathway to induce apoptosis, further molecular functions of this gene product have largely been unexplored. In this study, we report that KIF1Bß destabilizes the morphological structure of mitochondria, which is critical for cell survival and apoptosis. We identified YME1L1, a mitochondrial metalloprotease responsible for the cleavage of the mitochondrial GTPase OPA1, as a physical interacting partner of KIF1Bß. KIF1Bß interacted with YME1L1 through its death-inducing region, as initiated the protease activity of YME1L1 to cleave the long forms of OPA1, resulting in mitochondrial fragmentation. Overexpression of YME1L1 promoted apoptosis, while knockdown of YME1L1 promoted cell growth. High YME1L1 expression was significantly associated with a better prognosis in neuroblastoma. Furthermore, in NGF-deprived PC12 cells, KIF1Bß and YME1L1 were upregulated, accompanied by mitochondrial fragmentation and apoptotic cell death. Small interfering RNA-mediated knockdown of either protein alone, however, remarkably inhibited the NGF depletion-induced apoptosis. Our findings indicate that tumor suppressor KIF1Bß plays an important role in intrinsic mitochondria-mediated apoptosis through the regulation of structural and functional dynamics of mitochondria in collaboration with YME1L1. Dysfunction of the KIF1Bß/YME1L1/OPA1 mechanism may be involved in malignant biological features of neural crest-derived tumors as well as the initiation and progression of neurodegenerative diseases.

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.

Programmed expression of pro-apoptotic BMCC1 during apoptosis, triggered by DNA damage in neuroblastoma cells.

BACKGROUND: The multi-functional BMCC1 (BCH motif-containing molecule at the carboxyl terminal region 1)/PRUNE2 plays a clear role in suppression of tumor activity. In the patients with neuroblastoma (NB), reduced expression of BMCC1 in primary tumor tissues was associated with poor prognosis. By contrast, enforced expression of BMCC1 as well as elevated expression of BMCC1 in response to DNA-damage promotes apoptosis by abrogating Akt-mediated survival pathways. METHODS: We addressed molecular mechanisms underlying changes in regulation of BMCC1 expression during the process of apoptosis, which was promoted by a DNA-damaging drug Cisplatin (CDDP), in NB-derived cells. RESULTS: Elevated expression of BMCC1 was identified as an early response to DNA damage, which is accompanied by phosphorylation of ataxia telangiectasia mutated kinase (ATM) and accumulation of E2F1. Indeed, inhibition of ATM using an ATM inhibitor resulted in a decrease in expression of BMCC1 at mRNA levels. In addition, an E2F-binding sight was required for activation of BMCC1 promoter in response to DNA damage. On the other hand, knockdown of E2F1 yielded abrogated induction of BMCC1 in the cells after treatment with CDDP, suggesting that BMCC1 accumulation was caused by ATM-E2F1-dependent transcription. Finally, we demonstrated that full-length BMCC1 was proteolytically cleaved by apoptosis-activated caspase-9 during advanced stages of apoptosis in SK-N-AS cells. CONCLUSIONS: In this study, we demonstrated the programmed expression of full-length BMCC1 in human NB cells undergoing DNA damage-induced apoptosis. The elucidation of the molecular mechanisms controlling the regulation of BMCC1 during apoptosis initiated by DNA damage provides useful information for understanding drug resistance of tumor cells and spontaneous regression of NB.

Comprehensive evaluation of context dependence of the prognostic impact of MYCN amplification in neuroblastoma: A report from the International Neuroblastoma Risk Group (INRG) project.

BACKGROUND: MYCN amplification (MYCN-A) is an established adverse prognostic factor in neuroblastoma. The extent to which the prognostic impact of MYCN-A depends on other factors has not been fully characterized. PATIENTS AND METHODS: Using the International Neuroblastoma Risk Group database, we constructed Cox models of overall survival (OS) to obtain hazard ratios of the effect of MYCN-A within subgroups defined by other prognostic factors. Cox models assessed the degree to which the prognostic impact of MYCN-A was modulated by each other covariate. We used absolute hazard ratio (HR) differences to construct classification trees to identify subgroups with greatest differential prognostic effect of MYCN-A. RESULTS: In a cohort of 6223 patients with known MYCN status, the OS hazard ratio associated with MYCN-A was 6.3 (95% confidence interval 5.7-7.0, P < .001). Age at diagnosis conferred the largest HR absolute difference for MYCN-A between subgroups (HR absolute difference 16.6; HRs for MYCN-A of 19.6 for <18 months, 3.0 for ≥18 months). MYCN-A remained significantly prognostic of OS after controlling for other factors, abrogating their prognostic strength. Patients whose outcome was most impacted by MYCN status were those who were <18 months, had high mitosis karrhyohexis index (MKI) and low ferritin. CONCLUSION: The prognostic strength of MYCN-A varies depending on which patient subgroup defined by other neuroblastoma risk factors is examined, with greatest strength in patients with otherwise favorable features. MYCN-A has little effect within some subgroups, aiding clinical decision-making if MYCN status cannot be assessed. Subgroups where MYCN-A has large effect may be prioritized for agents targeting Myc family proteins.

Results of a prospective clinical trial JN-L-10 using image-defined risk factors to inform surgical decisions for children with low-risk neuroblastoma disease: A report from the Japan Children's Cancer Group Neuroblastoma Committee.

BACKGROUND: The present study sought to reduce the incidence of treatment complications of low-risk neuroblastoma by using image-defined risk factors (IDRFs) to inform the timing of surgical resection. PROCEDURES: Eligible patients included children (<18 years of age) with stage 1 or 2 disease, children (<365 days of age) with stage 3 disease, and infants with stage 4S disease. In IDRF-negative cases, treatment was completed with surgical resection alone. In IDRF-positive cases, the timing of surgery was determined based on the IDRFs after low-dose chemotherapy with 2-3 of the following four drugs: vincristine, cyclophosphamide, pirarubicin, and carboplatin. The outcome measures were overall survival, progression-free survival, and adverse events. This study was registered with the UMIN Clinical Trials Registry (number 000004355). RESULTS: Of the 60 patients screened between 2010 and 2013, 58 eligible patients were enrolled; 32 were identified as IDRF negative at diagnosis while 26 were identified as IDRF positive and underwent induction chemotherapy. The 3-year overall and progression-free survival rates of the 58 patients were 100% and 82.8% (95% confidence interval: 70.3-90.3), respectively. Neutropenia was the most frequently reported grade 3 or 4 chemotherapy-related form of toxicity (41.7%). With regard to surgical complications, 2.5% of all patients developed pleural effusion and ascites as early complications, while only 2.5% developed renal atrophy as a long-term complication. No fatal toxicities were observed. CONCLUSION: Using IDRFs to inform surgical decision making for the treatment of low-risk neuroblastoma improved prognosis and reduced the incidence of long-term complications.

Co-Inhibition of the DNA Damage Response and CHK1 Enhances Apoptosis of Neuroblastoma Cells.

Checkpoint kinase 1 (CHK1) is a central mediator of the DNA damage response (DDR) at the S and G2/M cell cycle checkpoints, and plays a crucial role in preserving genomic integrity. CHK1 overexpression is thought to contribute to cancer aggressiveness, and several selective inhibitors of this kinase are in clinical development for various cancers, including neuroblastoma (NB). Here, we examined the sensitivity of MYCN-amplified NB cell lines to the CHK1 inhibitor PF-477736 and explored mechanisms to increase its efficacy. PF-477736 treatment of two sensitive NB cell lines, SMS-SAN and CHP134, increased the expression of two pro-apoptotic proteins, BAX and PUMA, providing a mechanism for the effect of the CHK1 inhibitor. In contrast, in NB-39-nu and SK-N-BE cell lines, PF-477736 induced DNA double-strand breaks and activated the ataxia telangiectasia mutated serine/threonine kinase (ATM)-p53-p21 axis of the DDR pathway, which rendered the cells relatively insensitive to the antiproliferative effects of the CHK1 inhibitor. Interestingly, combined treatment with PF-477736 and the ATM inhibitor Ku55933 overcame the insensitivity of NB-39-nu and SK-N-BE cells to CHK1 inhibition and induced mitotic cell death. Similarly, co-treatment with PF-477736 and NU7441, a pharmacological inhibitor of DNA-PK, which is also essential for the DDR pathway, rendered the cells sensitive to CHK1 inhibition. Taken together, our results suggest that synthetic lethality between inhibitors of CHK1 and the DDR drives G2/M checkpoint abrogation and could be a novel potential therapeutic strategy for NB.

Neuroblastoma.

Neuroblastoma is one of the most common solid tumors in children and has a diverse clinical behavior that largely depends on the tumor biology. Neuroblastoma exhibits unique features, such as early age of onset, high frequency of metastatic disease at diagnosis in patients over 1 year of age and the tendency for spontaneous regression of tumors in infants. The high-risk tumors frequently have amplification of the MYCN oncogene as well as segmental chromosome alterations with poor survival. Recent advanced genomic sequencing technology has revealed that mutation of ALK, which is present in ~10% of primary tumors, often causes familial neuroblastoma with germline mutation. However, the frequency of gene mutations is relatively small and other aberrations, such as epigenetic abnormalities, have also been proposed. The risk-stratified therapy was introduced by the Japan Neuroblastoma Study Group (JNBSG), which is now moving to the Neuroblastoma Committee of Japan Children's Cancer Group (JCCG). Several clinical studies have facilitated the reduction of therapy for children with low-risk neuroblastoma disease and the significant improvement of cure rates for patients with intermediate-risk as well as high-risk disease. Therapy for patients with high-risk disease includes intensive induction chemotherapy and myeloablative chemotherapy, followed by the treatment of minimal residual disease using differentiation therapy and immunotherapy. The JCCG aims for better cures and long-term quality of life for children with cancer by facilitating new approaches targeting novel driver proteins, genetic pathways and the tumor microenvironment.

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.

NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3ß resulting in the stabilization of MYCN in human neuroblastomas.

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3ß, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3ß, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3ß inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3ß activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

Unfavorable neuroblastoma prognostic factor NLRR2 inhibits cell differentiation by transcriptional induction through JNK pathway.

The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)-mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA-induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c-Jun, a member of the activator protein-1 (AP-1) family in NB cells. Moreover, the expressions of NLRR2 and c-Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c-Jun reduced NLRR2 expression. We then searched AP-1 binding consensus in the NLRR2 promoter region and confirmed c-Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.

Identification of patient subgroups with markedly disparate rates of MYCN amplification in neuroblastoma: A report from the International Neuroblastoma Risk Group project.

BACKGROUND: MYCN gene amplification (MNA) is a hallmark of aggressive neuroblastoma. This study was performed to determine univariate and multivariate predictors of tumor MNA. METHODS: Data from the International Neuroblastoma Risk Group were analyzed for a subset of 7102 patients with known MYCN status. Chi-square testing and logistic regression were used to identify univariate and multivariate predictors of MYCN status. Recursive partitioning was used to identify groups of patients with maximal differences in rates of MNA. RESULTS: All clinical features (age ≥ 18 months, high ferritin levels, high lactate dehydrogenase [LDH] levels, International Neuroblastoma Staging System stage 4, and adrenal sites) and pathological/biological features (DNA index ≤ 1, high mitosis-karyorrhexis index [MKI], undifferentiated/poorly differentiated grade, unfavorable histology according to the International Neuroblastoma Pathology Classification, and segmental chromosomal aberrations [SCAs]) were significantly associated with MNA. LDH (odds ratio [OR], 8.4; P < .001) and chromosomal 1p loss of heterozygosity (OR, 19.8; P < .001) were the clinical and biological variables, respectively, most strongly associated with MNA. In logistic regression, all variables except chromosome 17q aberration and pooled SCAs were independently predictive of MNA. Recursive partitioning identified subgroups with disparate rates of MNA, including subgroups with 85.7% MNA (patients with high LDH levels who had poorly differentiated adrenal tumors with chromosome 1p deletion) and 0.6% MNA (localized tumors having hyperdiploidy and low MKIs and lacking chromosome 1p aberrations). CONCLUSIONS: MNA is strongly associated with other clinical and biological variables in neuroblastoma. Recursive partitioning has identified subgroups of neuroblastoma patients with highly disparate rates of MNA. These findings can be used to inform investigations of molecular mechanisms of MNA.