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1.  Advances in Risk Classification and Treatment Strategies for Neuroblastoma 
Journal of Clinical Oncology  2015;33(27):3008-3017.
Risk-based treatment approaches for neuroblastoma have been ongoing for decades. However, the criteria used to define risk in various institutional and cooperative groups were disparate, limiting the ability to compare clinical trial results. To mitigate this problem and enhance collaborative research, homogenous pretreatment patient cohorts have been defined by the International Neuroblastoma Risk Group classification system. During the past 30 years, increasingly intensive, multimodality approaches have been developed to treat patients who are classified as high risk, whereas patients with low- or intermediate-risk neuroblastoma have received reduced therapy. This treatment approach has resulted in improved outcome, although survival for high-risk patients remains poor, emphasizing the need for more effective treatments. Increased knowledge regarding the biology and genetic basis of neuroblastoma has led to the discovery of druggable targets and promising, new therapeutic approaches. Collaborative efforts of institutions and international cooperative groups have led to advances in our understanding of neuroblastoma biology, refinements in risk classification, and stratified treatment strategies, resulting in improved outcome. International collaboration will be even more critical when evaluating therapies designed to treat small cohorts of patients with rare actionable mutations.
doi:10.1200/JCO.2014.59.4648
PMCID: PMC4567703  PMID: 26304901
2.  The International Neuroblastoma Risk Group (INRG) Staging System: An INRG Task Force Report 
Journal of Clinical Oncology  2009;27(2):298-303.
Purpose
The International Neuroblastoma Risk Group (INRG) classification system was developed to establish a consensus approach for pretreatment risk stratification. Because the International Neuroblastoma Staging System (INSS) is a postsurgical staging system, a new clinical staging system was required for the INRG pretreatment risk classification system.
Methods
To stage patients before any treatment, the INRG Task Force, consisting of neuroblastoma experts from Australia/New Zealand, China, Europe, Japan, and North America, developed a new INRG staging system (INRGSS) based on clinical criteria and image-defined risk factors (IDRFs). To investigate the impact of IDRFs on outcome, survival analyses were performed on 661 European patients with INSS stages 1, 2, or 3 disease for whom IDRFs were known.
Results
In the INGRSS, locoregional tumors are staged L1 or L2 based on the absence or presence of one or more of 20 IDRFs, respectively. Metastatic tumors are defined as stage M, except for stage MS, in which metastases are confined to the skin, liver, and/or bone marrow in children younger than 18 months of age. Within the 661-patient cohort, IDRFs were present (ie, stage L2) in 21% of patients with stage 1, 45% of patients with stage 2, and 94% of patients with stage 3 disease. Patients with INRGSS stage L2 disease had significantly lower 5-year event-free survival than those with INRGSS stage L1 disease (78% ± 4% v 90% ± 3%; P = .0010).
Conclusion
Use of the new staging (INRGSS) and risk classification (INRG) of neuroblastoma will greatly facilitate the comparison of risk-based clinical trials conducted in different regions of the world.
doi:10.1200/JCO.2008.16.6876
PMCID: PMC2650389  PMID: 19047290
3.  Time to disease progression in children with relapsed or refractory neuroblastoma treated with ABT-751: A report from the Children's Oncology Group (ANBL0621) 
Pediatric blood & cancer  2013;61(6):990-996.
Purpose
ABT-751 is an orally bioavailable sulfonamide that binds the colchicine site of beta-tubulin, thus inhibiting microtubule polymerizaton. A prior phase I study established the recommended dose in children with solid tumors as 200 mg/m2 PO daily × 7 days every 21 days and subjects with neuroblastoma experienced prolonged stable disease. We conducted a phase 2 study in children with progressive neuroblastoma to determine if ABT-751 prolonged the time to progression (TTP) compared to a hypothesized standard based on a historical control population.
Experimental Design
Children and adolescents (n=91) with a median (range) age 7.7 (2.3-21.5) years and progressive neuroblastoma were enrolled and stratified by disease status into disease measureable by CT/MRI (n=47) or disease assessable by 123I-metaiodobenzylguanine scintigraphy (MIBG, n=44). Response was evaluated using RECIST for measureable disease and the Curie scale for MIBG-avid disease.
Results
ABT-751 was well tolerated. Two complete responses and four partial responses were achieved. The median TTP was 42 days (95% CI: 36, 56) in the measureable disease stratum and 45 days (95% CI: 42, 85) in the MIBG-avid disease stratum. These values are similar to TTP in the historical control group (n=136, median TTP 42 days). One-year progression free (PFS) and overall survival (OS) for the combined strata (n=91) were 13%±4%, 48%±5%, respectively.
Conclusions
Although ABT-751 has many characteristics of an ideal maintenance agent for neuroblastoma, the low objective response rate and failure to prolong TTP indicate that ABT-751 is not sufficiently active to warrant further development for neuroblastoma.
doi:10.1002/pbc.24900
PMCID: PMC5127168  PMID: 24347462
neuroblastoma; time to progression; clinical trial; microtubule inhibitor; childhood cancer
4.  Phase II Study of Irinotecan and Temozolomide in Children With Relapsed or Refractory Neuroblastoma: A Children's Oncology Group Study 
Journal of Clinical Oncology  2010;29(2):208-213.
Purpose
This phase II study was conducted to determine the response rate associated with use of irinotecan and temozolomide for children with relapsed/refractory neuroblastoma.
Patients and Methods
Patients with relapsed/refractory neuroblastoma measurable by cross-sectional imaging (stratum 1) or assessable by bone marrow aspirate/biopsy or metaiodobenzylguanidine (MIBG) scan (stratum 2) received irinotecan (10 mg/m2/dose 5 days a week for 2 weeks) and temozolomide (100 mg/m2/dose for 5 days) every 3 weeks. Response was assessed after three and six courses using International Neuroblastoma Response Criteria. Of the first 25 evaluable patients on a given stratum, five or more patients with complete or partial responses were required to conclude that further study would be merited.
Results
Fifty-five eligible patients were enrolled. The objective response rate was 15%. Fourteen patients (50%) on stratum 1 and 15 patients (56%) on stratum 2 had stable disease. Objective responses were observed in three of the first 25 evaluable patients on stratum 1 and five of the first 25 evaluable patients on stratum 2. Less than 6% of patients experienced ≥ grade 3 diarrhea. Although neutropenia was observed, less than 10% of patients developed evidence of infection while neutropenic.
Conclusion
The combination of irinotecan and temozolomide was well tolerated. The objective response rate of 19% in stratum 2 suggests that this combination may be effective for patients with neuroblastoma detectable by MIBG or marrow analysis. Although fewer objective responses were observed in patients with disease measurable by computed tomography/magnetic resonance imaging, patients in both strata seem to have derived clinical benefit from this therapy.
doi:10.1200/JCO.2010.31.7107
PMCID: PMC3058276  PMID: 21115869
5.  Significance of MYCN Amplification in International Neuroblastoma Staging System Stage 1 and 2 Neuroblastoma: A Report From the International Neuroblastoma Risk Group Database 
Journal of Clinical Oncology  2009;27(3):365-370.
Purpose
Treatment of patients with localized neuroblastoma with unfavorable biologic features is controversial. To evaluate the outcome of children with low-stage MYCN-amplified neuroblastoma and develop a rational treatment strategy, data from the International Neuroblastoma Risk Group (INRG) database were analyzed.
Patients and Methods
The database is comprised of 8,800 patients. Of these, 2,660 patients (30%) had low-stage (International Neuroblastoma Staging System stages 1 and 2) neuroblastoma, known MYCN status, and available follow-up data. Eighty-seven of these patients (3%) had MYCN amplified tumors.
Results
Patients with MYCN-amplified, low-stage tumors had less favorable event-free survival (EFS) and overall survival (OS) than did patients with nonamplified tumors (53% ± 8% and 72% ± 7% v 90% ± 1% and 98% ± 1%, respectively). EFS and OS were statistically significantly higher for patients whose tumors were hyperdiploid rather than diploid (EFS, 82% ± 20% v 37% ± 21%; P = .0069; OS, 94% ± 11% v 54% ± 15%; P = .0056, respectively). No other variable had prognostic significance. Initial treatment consisted of surgery alone for 29 (33%) of 87 patients. Details of additional therapy were unknown for 14 patients. Twenty-two patients (25%) underwent surgery and moderate-intensity chemotherapy; another 22 underwent surgery, intensive chemotherapy, and radiation therapy. Nine of the latter 22 underwent stem cell transplantation. Survival in patients who received transplantation did not differ from survival in those who did not receive transplantation.
Conclusion
Among patients with low-stage, MYCN-amplified neuroblastoma, outcomes of patients with hyperdiploid tumors were statistically, significantly better than those with diploid tumors. The data suggest that tumor cell ploidy could potentially be used to identify candidates for reductions in therapy. Further study of MYCN-amplified, low-stage neuroblastoma is warranted.
doi:10.1200/JCO.2008.17.9184
PMCID: PMC2651034  PMID: 19047282
6.  Phase 1 Study of Vorinostat as a Radiation Sensitizer with 131I-Metaiodobenzylguanidine (131I-MIBG) for Patients with Relapsed or Refractory Neuroblastoma 
Purpose
131I-metaiodobenzylguanidine (MIBG) is a radiopharmaceutical with activity in neuroblastoma. Vorinostat is a histone deacetylase inhibitor that has radiosensitizing properties. The goal of this phase 1 study was to determine the maximum tolerated doses of vorinostat and MIBG in combination.
Experimental Design
Patients ≤ 30 years with relapsed/refractory MIBG-avid neuroblastoma were eligible. Patients received oral vorinostat (dose levels 180 and 230 mg/m2) daily Days 1–14. MIBG (dose levels 8, 12, 15, and 18 mCi/kg) was given on Day 3 and peripheral blood stem cells on Day 17. Alternating dose escalation of vorinostat and MIBG was performed using a 3+3 design.
Results
27 patients enrolled to 6 dose levels, with 23 evaluable for dose escalation. No dose-limiting toxicities (DLT) were seen in the first three dose levels. At dose level 4 (15 mCi/kg MIBG/230 mg/m2 vorinostat), 1 of 6 patients had DLT with grade 4 hypokalemia. At dose level 5 (18 mCi/kg MIBG/230 mg/m2 vorinostat), two patients had dose-limiting bleeding (one grade 3 and one grade 5). At dose level 5a (18 mCi/kg MIBG/180 mg/m2 vorinostat), 0 of 6 patients had DLT. The most common toxicities were neutropenia and thrombocytopenia. The response rate was 12% across all dose levels and 17% at dose level 5a. Histone acetylation increased from baseline in peripheral blood mononuclear cells collected on Days 3 and 12–14.
Conclusions
Vorinostat at 180 mg/m2/dose is tolerable with 18 mCi/kg MIBG. A phase 2 trial comparing this regimen to single-agent MIBG is ongoing.
doi:10.1158/1078-0432.CCR-14-3240
PMCID: PMC4470833  PMID: 25695691
131I-MIBG; Vorinostat; Radiation Sensitizer; Neuroblastoma; Relapse; Refractory
7.  Clinical, Biologic, and Prognostic Differences on the Basis of Primary Tumor Site in Neuroblastoma: A Report From the International Neuroblastoma Risk Group Project 
Journal of Clinical Oncology  2014;32(28):3169-3176.
Purpose
Neuroblastoma (NB) is a heterogeneous tumor arising from sympathetic tissues. The impact of primary tumor site in influencing the heterogeneity of NB remains unclear.
Patients and Methods
Children younger than age 21 years diagnosed with NB or ganglioneuroblastoma between 1990 and 2002 and with known primary site were identified from the International Neuroblastoma Risk Group database. Data were compared between sites with respect to clinical and biologic features, as well as event-free survival (EFS) and overall survival (OS).
Results
Among 8,369 children, 47% had adrenal tumors. All evaluated clinical and biologic variables differed statistically between primary sites. The features that were > 10% discrepant between sites were stage 4 disease, MYCN amplification, elevated ferritin, elevated lactate dehydrogenase, and segmental chromosomal aberrations, all of which were more frequent in adrenal versus nonadrenal tumors (P < .001). Adrenal tumors were more likely than nonadrenal tumors (adjusted odds ratio, 2.09; 95% CI, 1.67 to 2.63; P < .001) and thoracic tumors were less likely than nonthoracic tumors (adjusted odds ratio, 0.20; 95% CI, 0.11 to 0.39; P < .001) to have MYCN amplification after controlling for age, stage, and histologic grade. EFS and OS differed significantly according to the primary site (P < .001 for both comparisons). After controlling for age, MYCN status, and stage, patients with adrenal tumors had higher risk for events (hazard ratio, 1.13 compared with nonadrenal tumors; 95% CI, 1.03 to 1.23; P = .008), and patients with thoracic tumors had lower risk for events (HR, 0.79 compared with nonthoracic; 95% CI, 0.67 to 0.92; P = .003).
Conclusion
Clinical and biologic features show important differences by NB primary site, with adrenal and thoracic sites associated with inferior and superior survival, respectively. Future studies will need to investigate the biologic origin of these differences.
doi:10.1200/JCO.2014.56.1621
PMCID: PMC4171360  PMID: 25154816
8.  Validation of a Prognostic Multi-gene Signature in High-risk Neuroblastoma using the High Throughput Digital NanoString nCounter™ System 
Molecular oncology  2014;8(3):669-678.
Microarray-based molecular signatures have not been widely integrated into neuroblastoma diagnostic classification systems due to the complexities of the assay and requirement for high-quality RNA. New digital technologies that accurately quantify gene expression using RNA isolated from formalin-fixed paraffin embedded (FFPE) tissues are now available. In this study, we describe the first use of a high-throughput digital system to assay the expression of genes in an “ultra-high risk” microarray classifier in FFPE high-risk neuroblastoma tumors. Customized probes corresponding to the 42 genes in a published multi-gene neuroblastoma signature were hybridized to RNA isolated from 107 FFPE high-risk neuroblastoma samples using the NanoString nCounter™ Analysis System. For classification of each patient, the Pearson’s correlation coefficient was calculated between the standardized nCounter™ data and the molecular signature from the microarray data. We demonstrate that the nCounter™ 42-gene panel sub-stratified the high-risk cohort into two subsets with statistically significantly different overall survival (p=0.0027) and event-free survival (p=0.028). In contrast, none of the established prognostic risk markers (age, stage, tumor histology, MYCN status, and ploidy) were significantly associated with survival. We conclude that the nCounter™ System can reproducibly quantify expression levels of signature genes in FFPE tumor samples. Validation of this microarray signature in our high-risk patient cohort using a completely different technology emphasizes the prognostic relevance of this classifier. Prospective studies testing the prognostic value of molecular signatures in high-risk neuroblastoma patients using FFPE tumor samples and the nCounter™ System are warranted.
doi:10.1016/j.molonc.2014.01.010
PMCID: PMC4004665  PMID: 24560446
High-risk neuroblastoma; gene signature; molecular classifier; NanoString; nCounter
9.  Comparative pharmacokinetics, safety, and tolerability of two sources of ch14.18 in pediatric patients with high-risk neuroblastoma following myeloablative therapy 
Purpose
Dinutuximab (Unituxin™; ch14.18), a monoclonal antibody against disialoganglioside, improved survival as part of post-consolidation therapy for high-risk neuroblastoma. United Therapeutics Corporation (UTC) assumed ch14.18 production from the National Cancer Institute (NCI); this study evaluates pharmacokinetic comparability, safety, and tolerability of UTC and NCI products.
Methods
In this randomized, two-sequence crossover study, 28 patients aged ≤8 years with high-risk neuroblastoma received equivalent ch14.18-UTC or ch14.18-NCI doses. Despite comparable protein content, nominal doses differed: 17.5 mg/m2/day (ch14.18-UTC) and 25 mg/m2/day (ch14.18-NCI). Patients received one product during therapy cycles 1 and 2, the other during cycles 3–5. Ch14.18 pharmacokinetic profile characterization used population modeling (NONMEM® version 7.2). A two-compartment model with first-order distribution and elimination processes described pharmacokinetic data. Estimated product parameters were normalized to UTC nominal dose. For pharmacokinetic comparability, the final model was used to estimate exposure ratios (UTC/NCI) and associated 90 % confidence intervals (CIs) for area under the curve from time zero to infinity (AUCinf) and maximum concentration (Cmax). All comparisons were based on a standardized single-dose regimen (17.5 mg/m2 over 10 h).
Results
Final-model pharmacokinetic parameters were similar to previously published ch14.18-NCI parameters and comparable for UTC and NCI products. Products’ systemic exposures were comparable, with 90 % CIs around ratios for AUCinf (0.96; 90 % CI 0.88–1.04) and Cmax (1.04; 90 % CI 0.98–1.11) within standard bioequivalence bounds (90 % CI 0.80–1.25). Products’ adverse events were similar and consistent with those previously reported.
Conclusions
Equivalent actual ch14.18-UTC and ch14.18-NCI doses produced comparable exposures, with no notable safety or tolerability differences.
Electronic supplementary material
The online version of this article (doi:10.1007/s00280-015-2955-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s00280-015-2955-9
PMCID: PMC4747995  PMID: 26791869
ch14.18; Dinutuximab; Pharmacokinetics; Safety; Tolerability; Unituxin
10.  Second Malignancies in Patients with Neuroblastoma: The Effects of Risk-Based Therapy 
Pediatric blood & cancer  2014;62(1):128-133.
Background
To investigate the incidence of second malignant neoplasms (SMN) for patients with neuroblastoma, we analyzed patients from the SEER database according to three treatment eras (1: 1973–1989, 2: 1990–1996, 3: 1997–2006) corresponding to the introduction of multi-agent chemotherapy, risk-based treatment, and stem cell transplant.
Procedure
The SEER database was mined for all patients with neuroblastoma or ganglioneuroblastoma. Cumulative incidence of SMN was calculated with death as a competing risk. A poisson regression model was used to estimate incidence rate ratios and 95% confidence intervals to compare the rates of SMN between patients in different Eras.
Results
The analytic cohort included 2,801 patients. Thirty-four patients developed a SMN, accounting for 1.2% of all patients. Of the patients who developed a SMN, 47.1% received radiation for their primary neuroblastoma. Fourteen of the SMN were carcinomas, and 10 were hematologic malignancies, with 6 cases of acute myelogenous leukemia. There was no difference in the incidence of SMN in Era 1 compared to Era 3 (p=0.48). The cumulative incidence of SMN at 30 years for high-risk patients was 10.44% (95% CI 3.98–20.52%) compared to 3.57% (95% CI 1.87–6.12%) for non-high-risk patients (p<0.001).
Conclusions
This study showed no increase in the incidence of SMNs for children treated in the most recent treatment era as compared to earlier Eras. However, as the risk for developing SMN does not plateau, the number of SMNs will likely continue to rise in the cohort of patients treated after 1996. Comprehensive follow-up care for these survivors will be important.
doi:10.1002/pbc.25249
PMCID: PMC4237647  PMID: 25251613
Neuroblastoma; SEER; second malignancies; late effects
11.  The Quinoxaline Antitumor Agent (R+)XK469 Inhibits Neuroblastoma Tumor Growth 
Pediatric blood & cancer  2011;56(1):164-167.
The quinoxaline antitumor agent (R+)XK469 mediates its effects by topoisomerase IIB inhibition. This report describes a 14 year-old with relapsed neuroblastoma who experienced disease stabilization for 14 months while receiving (R+)XK469 monotherapy. Due to this favorable response, laboratory studies were undertaken to determine efficacy in the preclinical setting. (R+)XK469 inhibited proliferation, caused G2 cell cycle arrest of neuroblastoma cells in vitro, and inhibited growth of neuroblastoma xenograft tumors. These preclinical results, coupled with the favorable clinical response, demonstrate that (R+)XK469 and similar anti-tumor agents may be effective in the treatment of high-risk neuroblastoma and warrant further testing.
doi:10.1002/pbc.22639
PMCID: PMC4116189  PMID: 20860039
neuroblastoma; topoisomerase inhibitor; relapse; phase I
12.  Metastatic Neuroblastoma Confined to Distant Lymph Nodes (stage 4N) Predicts Outcome in Patients With Stage 4 Disease: A Study From the International Neuroblastoma Risk Group Database 
Journal of Clinical Oncology  2014;32(12):1228-1235.
Purpose
The presence of distant metastases is one of the most powerful predictors of outcome in patients with neuroblastoma. However, the pattern of metastatic spread is not incorporated into current risk stratification systems. Small case series have suggested that patients with neuroblastoma who have metastatic disease limited to distant lymph nodes (4N disease) may have improved outcomes.
Patients and Methods
We analyzed retrospective data from the International Neuroblastoma Risk Group database for patients diagnosed from 1990 to 2002. 4N patients were compared with the remaining stage 4 patients (non-4N), excluding those with missing metastatic site data.
Results
In all, 2,250 International Neuroblastoma Staging System stage 4 patients with complete data were identified, of whom 146 (6.5%) had 4N disease. For 4N patients, event-free survival (EFS; 5-year, 77% ± 4%) and overall survival (OS; 5-year, 85% ± 3%) were significantly better than EFS (5-year, 35% ± 1%) and OS (5-year, 42% ± 1%) for non-4N stage 4 patients (P < .001). 4N patients were more likely to be younger (P < .001) and have tumors with favorable characteristics, including absence of MYCN amplification (89% v 69%; P < .001). In a multivariable analysis, 4N disease remained a significant predictor of outcome (hazard ratio for non-4N v 4N: 3.40 for EFS and 3.69 for OS). Within subgroups defined by age at diagnosis and tumor MYCN status, 4N disease was significantly associated with improved outcomes.
Conclusion
4N represents a subgroup with better outcome than that of other patients with metastatic disease. These findings suggest that the biology and treatment response of 4N tumors differ from other stage 4 tumors, and less intensive therapy should be considered for this cohort. Future exploration of biologic factors determining the pattern of metastatic spread is warranted.
doi:10.1200/JCO.2013.53.6342
PMCID: PMC4876342  PMID: 24663047
13.  Trans-population Analysis of Genetic Mechanisms of Ethnic Disparities in Neuroblastoma Survival 
Background
Black patients with neuroblastoma have a higher prevalence of high-risk disease and worse outcome than white patients. We sought to investigate the relationship between genetic variation and the disparities in survival observed in neuroblastoma.
Methods
The analytic cohort was composed of 2709 patients. Principal components were used to assign patients to genomic ethnic clusters for survival analyses. Locus-specific ancestry was calculated for use in association analysis. The shorter spans of linkage disequilibrium in African populations may facilitate the fine mapping of causal variants in regions previously implicated by genome-wide association studies conducted primarily in patients of European descent. Thus, we evaluated 13 single nucleotide polymorphisms known to be associated with susceptibility to high-risk neuroblastoma from genome-wide association studies and all variants with highly divergent allele frequencies in reference African and European populations near the known susceptibility loci. All statistical tests were two-sided.
Results
African genomic ancestry was associated with high-risk neuroblastoma (P = .007) and lower event-free survival (P = .04, hazard ratio = 1.4, 95% confidence interval = 1.05 to 1.80). rs1033069 within SPAG16 (sperm associated antigen 16) was determined to have higher risk allele frequency in the African reference population and statistically significant association with high-risk disease in patients of European and African ancestry (P = 6.42×10−5, false discovery rate < 0.0015) in the overall cohort. Multivariable analysis using an additive model demonstrated that the SPAG16 single nucleotide polymorphism contributes to the observed ethnic disparities in high-risk disease and survival.
Conclusions
Our study demonstrates that common genetic variation influences neuroblastoma phenotype and contributes to the ethnic disparities in survival observed and illustrates the value of trans-population mapping.
doi:10.1093/jnci/djs503
PMCID: PMC3691940  PMID: 23243203
14.  Neuroblastoma - Undifferentiated Subtype, Prognostic Significance of Prominent Nucleolar Formation and MYC/MYCN Protein Expression: A Report from the Children’s Oncology Group 
Cancer  2013;119(20):3718-3726.
Purpose
To investigate biological/clinicopathological characteristics of neuroblastoma, undifferentiated subtype (NBUD).
Patients and Methods
157 NBUD cases filed at the Children’s Oncology Group Neuroblastoma Pathology Reference Laboratory were studied, and survival rates of the patients were analyzed with known prognostic factors. Immunostainings for MYCN and MYC protein were performed on 68 tumors.
Results
NBUD cases had a poor prognosis (48.4±5.0% 3-year event-free survival [EFS]; 56.5±5.0% overall survival), and were often associated with high Mitosis-Karyorrhexis Index (MKI, 65%), prominent nucleoli (PN, 83%), ≥18months of age (75%), MYCN amplification (MYCN-A, 83%), diploid pattern (63%), and 1pLOH (loss of heterozygosity, 72%). However, these prognostic indicators, except for MYCN status, had no significant impact on survival. Surprisingly, EFS for patients with MYCN-A tumors (53.4±5.6%) was significantly better (P=0.0248) than for patients with MYCN-Non-Amplified (MYCN-NA) tumors (31.7±11.7%), with MYCN-NA and PN (+) tumors having the worst prognosis (9.3+8.8%, p=0.0045). Immunohistochemically, MYCN expression was found in 42/48 MYCN-A tumors. In contrast, MYC expression was almost exclusively found in the MYCN-NA tumors (9/20) especially when they had PN (8/11). Those patients with only MYC-positive tumors had the worst EFS (N=8, 12.5±11.7%) compared with only MYCN-positive (N=39, 49.9±17.7%) and both negative tumors (N=15, 70.0±17.1%) (P=0.0029). High MKI was often found in only MYCN-positive (30/38) but rarely in only MYC-positive (2/8) tumors.
Conclusions
NBUD represents a unique subtype of neuroblastoma associated with a poor prognosis. In this subtype, MYC protein expression may be a new prognostic factor indicating more aggressive clinical behavior than MYCN amplification and subsequent MYCN protein expression.
doi:10.1002/cncr.28251
PMCID: PMC4554323  PMID: 23901000
Neuroblastoma; undifferentiated subtype; MYCN Amplification; MYC/MYCN expression; prognostic factors; MYC gene expression
15.  Truncated DNMT3B isoform DNMT3B7 suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma 
Cancer research  2012;72(18):4714-4723.
Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive pathophysiology of this disease, but little is known about the basis for such changes. In this study, we examined a role for the DNA methyltransferase DNMT3B, in particular, the truncated isoform DNMT3B7 which is generated frequently in cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B expression in primary tumors. Higher levels of DNMT3B7 were detected in differentiated ganglioneuroblastomas compared to undifferentiated neuroblastomas, suggesting that expression of DNMT3B7 may induce a less aggressive clinical phenotype. To test this hypothesis, we investigated the effects of enforced DNMT3B7 expression in neuroblastoma cells, finding a significant inhibition of cell proliferation in vitro and angiogenesis and tumor growth in vivo. DNMT3B7-positive cells had higher levels of total genomic methylation and a dramatic decrease in expression of the FOS and JUN family members that comprise AP1 transcription factors. Consistent with an established antagonistic relationship between AP1 expression and retinoic acid receptor activity, increased differentiation was seen in the DNMT3B7-expressing neuroblastoma cells following treatment with all-trans retinoic acid (ATRA) compared to controls. Our results indicate that DNMT3B7 modifies the epigenome in neuroblastoma cells to induce changes in gene expression, inhibit tumor growth, and increase sensitivity to ATRA.
doi:10.1158/0008-5472.CAN-12-0886
PMCID: PMC3445765  PMID: 22815530
DNMT3B; Neuroblastoma; DNA Methylation
16.  TET1-Mediated Hydroxymethylation Facilitates Hypoxic Gene Induction in Neuroblastoma 
Cell reports  2014;7(5):1343-1352.
SUMMARY
The ten-eleven-translocation 5-methylcytosine dioxygenase (TET) family of enzymes catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxyme-thylcytosine (5-hmC), a modified cytosine base that facilitates gene expression. Cells respond to hypoxia by inducing a transcriptional program regulated in part by oxygen-dependent dioxygenases that require Fe(II) and α-ketoglutarate. Given that the TET enzymes also require these cofactors, we hypothesized that the TETs regulate the hypoxia-induced transcriptional program. Here, we demonstrate that hypoxia increases global 5-hmC levels, with accumulation of 5-hmC density at canonical hypoxia response genes. A subset of 5-hmC gains colocalize with hypoxia response elements facilitating DNA demethylation and HIF binding. Hypoxia results in transcriptional activation of TET1, and full induction of hypoxia-responsive genes and global 5-hmC increases require TET1. Finally, we show that 5-hmC increases and TET1 upregulation in hypoxia are HIF-1 dependent. These findings establish TET1-mediated 5-hmC changes as an important epigenetic component of the hypoxic response.
doi:10.1016/j.celrep.2014.04.040
PMCID: PMC4516227  PMID: 24835990
17.  Outcome After Surgery Alone or With Restricted Use of Chemotherapy for Patients With Low-Risk Neuroblastoma: Results of Children's Oncology Group Study P9641 
Journal of Clinical Oncology  2012;30(15):1842-1848.
Purpose
The primary objective of Children's Oncology Group study P9641 was to demonstrate that surgery alone would achieve 3-year overall survival (OS) ≥ 95% for patients with asymptomatic International Neuroblastoma Staging System stages 2a and 2b neuroblastoma (NBL). Secondary objectives focused on other low-risk patients with NBL and on those who required chemotherapy according to protocol-defined criteria.
Patients and Methods
Patients underwent maximally safe resection of tumor. Chemotherapy was reserved for patients with, or at risk for, symptomatic disease, with less than 50% tumor resection at diagnosis, or with unresectable progressive disease after surgery alone.
Results
For all 915 eligible patients, 5-year event-free survival (EFS) and OS were 89% ± 1% and 97% ± 1%, respectively. For patients with asymptomatic stage 2a or 2b disease, 5-year EFS and OS were 87% ± 2% and 96% ± 1%, respectively. Among patients with stage 2b disease, EFS and OS were significantly lower for those with unfavorable histology or diploid tumors, and OS was significantly lower for those ≥ 18 months old. For patients with stage 1 and 4s NBL, 5-year OS rates were 99% ± 1% and 91% ± 1%, respectively. Patients who required chemotherapy at diagnosis achieved 5-year OS of 98% ± 1%. Of all patients observed after surgery, 11.1% experienced recurrence or progression of disease.
Conclusion
Excellent survival rates can be achieved in asymptomatic low-risk patients with stages 2a and 2b NBL after surgery alone. Immediate use of chemotherapy may be restricted to a minority of patients with low-risk NBL. Patients with stage 2b disease who are older or have diploid or unfavorable histology tumors fare less well. Future studies will seek to refine risk classification.
doi:10.1200/JCO.2011.37.9990
PMCID: PMC3383182  PMID: 22529259
18.  Integrating Cell-Based and Clinical Genome-Wide Studies to Identify Genetic Variants Contributing to Treatment Failure in Neuroblastoma Patients 
High-risk neuroblastoma is an aggressive malignancy with high rates of treatment failure. We evaluated genetic variants associated with in vitro sensitivity to two derivatives of cyclophosphamide for association with clinical response in a separate replication cohort of neuroblastoma patients (n=2,709). Lymphoblastoid cell lines (LCLs) were exposed to increasing concentrations of 4-hydroperoxycyclophosphamide [4HC n=422] and phosphoramide mustard [PM n=428] to determine sensitivity. Genome-wide association studies (GWAS) were performed to identify single nucleotide polymorphisms (SNPs) associated with 4HC and PM sensitivity. SNPs consistently associated with LCL sensitivity were analyzed for associations with event-free survival in patients. Two linked SNPs, rs9908694 and rs1453560, were found to be associated with PM sensitivity in LCLs across populations and were associated with event-free survival in all patients (P=0.01) and within the high-risk subset (P=0.05). Our study highlights the value of cell-based models to identify candidate variants that may predict response to treatment in patients with cancer.
doi:10.1038/clpt.2014.37
PMCID: PMC4029857  PMID: 24549002
neuroblastoma; pharmacogenomics; cell-based models; IKZF3; ZPBP2; expression quantitative trait loci
19.  Pilot Induction Regimen Incorporating Pharmacokinetically Guided Topotecan for Treatment of Newly Diagnosed High-Risk Neuroblastoma: A Children's Oncology Group Study 
Journal of Clinical Oncology  2011;29(33):4351-4357.
Purpose
To assess the feasibility of adding dose-intensive topotecan and cyclophosphamide to induction therapy for newly diagnosed high-risk neuroblastoma (HRNB).
Patients and Methods
Enrolled patients received two cycles of topotecan (approximately 1.2 mg/m2/d) and cyclophosphamide (400 mg/m2/d) for 5 days followed by four cycles of multiagent chemotherapy (Memorial Sloan-Kettering Cancer Center [MSKCC] regimen). Pharmacokinetically guided topotecan dosing (target systemic exposure with area under the curve of 50 to 70 ng/mL/hr) was performed. Peripheral-blood stem cell (PBSC) harvest and surgical resection of residual primary tumor occurred after cycles 2 and 5, respectively. Patients achieving at least a partial response received myeloablative chemotherapy with PBSC rescue and radiation to the presurgical primary tumor volume. Oral 13-cis-retinoic acid maintenance therapy was administered twice daily for 14 days in six 28-day cycles.
Results
Thirty-one patients were enrolled onto the study. No deaths related to toxicity or dose-limiting toxicities occurred during induction. Mucositis rarely occurred after topotecan cycles (9.7%) in contrast to 30% after MSKCC cycles. Thirty patients underwent PBSC collection with median 31.1 × 106 CD34+ cells/kg (range, 1.8 to 541.8 × 106 CD34+ cells/kg), all negative for tumor contamination by immunocytochemical analysis. Targeted topotecan systemic exposure was achieved in 26 (84%) of 31 patients. At the end of induction, 26 patients (84%) had tumor response and one patient had progressive disease. In the overall cohort, 3-year event-free and overall survival were 37.8% ± 9.4% and 57.1% ± 9.4%, respectively.
Conclusion
This pilot induction regimen was well tolerated with expected and reversible toxicities. These data support investigation of efficacy in a phase III clinical trial for newly diagnosed HRNB.
doi:10.1200/JCO.2010.34.3293
PMCID: PMC3221519  PMID: 22010014
20.  Prognostic Value of the Stage 4S Metastatic Pattern and Tumor Biology in Patients With Metastatic Neuroblastoma Diagnosed Between Birth and 18 Months of Age 
Journal of Clinical Oncology  2011;29(33):4358-4364.
Purpose
Patients with neuroblastoma younger than 12 months of age with a 4S pattern of disease (metastases limited to liver, skin, bone marrow) have better outcomes than infants with stage 4 disease. The new International Neuroblastoma Risk Group (INRG) staging system extends age to 18 months for the 4S pattern. Our aim was to determine which prognostic features could be used for optimal risk classification among patients younger than 18 months with metastatic disease.
Methods
Event-free survival (EFS) and overall survival were analyzed by log-rank tests, Cox models, and survival tree regression for 656 infants with stage 4S neuroblastoma younger than 12 months of age and 1,019 patients with stage 4 disease younger than 18 months of age in the INRG database.
Results
Unfavorable biologic features were more frequent in infants with stage 4 disease than in infants with 4S tumors and higher overall in those age 12 to 18 months (although not different for stage 4 v 4S pattern). EFS was significantly better for infants younger than 12 months with 4S pattern than with stage 4 disease (P < .01) but similar for toddlers age 12 to 18 months with stage 4 versus 4S pattern. Among 717 patients with stage 4S pattern, patients age 12 to 18 months had worse EFS than those age younger than 12 months (P < .01). MYCN, 11q, mitosis-karyorrhexis index (MKI), ploidy, and lactate dehydrogenase were independently statistically significant predictors of EFS and more highly predictive than age or metastatic pattern. MYCN, 11q, MKI, histology, and 1p were combined in a survival tree for improved risk stratification.
Conclusion
Tumor biology is more critical than age or metastatic pattern for prognosis of patients age younger than 18 months with metastatic neuroblastoma and should be considered for risk stratification.
doi:10.1200/JCO.2011.35.9570
PMCID: PMC3221520  PMID: 21969516
21.  Clinical and Biologic Features Predictive of Survival After Relapse of Neuroblastoma: A Report From the International Neuroblastoma Risk Group Project 
Journal of Clinical Oncology  2011;29(24):3286-3292.
Purpose
Survival after neuroblastoma relapse is poor. Understanding the relationship between clinical and biologic features and outcome after relapse may help in selection of optimal therapy. Our aim was to determine which factors were significantly predictive of postrelapse overall survival (OS) in patients with recurrent neuroblastoma—particularly whether time from diagnosis to first relapse (TTFR) was a significant predictor of OS.
Patients and Methods
Patients with first relapse/progression were identified in the International Neuroblastoma Risk Group (INRG) database. Time from study enrollment until first event and OS time starting from first event were calculated. Cox regression models were used to calculate the hazard ratio of increased death risk and perform survival tree regression. TTFR was tested in a multivariable Cox model with other factors.
Results
In the INRG database (N = 8,800), 2,266 patients experienced first progression/relapse. Median time to relapse was 13.2 months (range, 1 day to 11.4 years). Five-year OS from time of first event was 20% (SE, ± 1%). TTFR was statistically significantly associated with OS time in a nonlinear relationship; patients with TTFR of 36 months or longer had the lowest risk of death, followed by patients who relapsed in the period of 0 to less than 6 months or 18 to 36 months. Patients who relapsed between 6 and 18 months after diagnosis had the highest risk of death. TTFR, age, International Neuroblastoma Staging System stage, and MYCN copy number status were independently predictive of postrelapse OS in multivariable analysis.
Conclusion
Age, stage, MYCN status, and TTFR are significant prognostic factors for postrelapse survival and may help in the design of clinical trials evaluating novel agents.
doi:10.1200/JCO.2010.34.3392
PMCID: PMC3158599  PMID: 21768459
22.  Presence Of Cancer-Associated Fibroblasts Inversely Correlates With Schwannian Stroma In Neuroblastoma Tumors 
Stromal cells play a central role in the regulation of tumor angiogenesis. Recent studies have shown that stromal myofibroblasts [cancer-associated fibroblasts] actively promote tumor growth and enhance tumor angiogenesis in many types of adult carcinomas. To evaluate the role cancer-associated fibroblasts play in neuroblastoma angiogenesis and investigate their relationship to stromal Schwann cells, we quantified cancer-associated fibroblasts in 60 primary neuroblastoma tumors and in a novel neuroblastoma xenograft model in which murine Schwann cells were induced to infiltrate into the tumor stroma. Tumor sections were examined for presence of microvascular proliferation, a hallmark of tumor angiogenesis. Cancer-associated fibroblasts were characterized by positive immunostaining for α-smooth muscle actin (α-SMA) and were distinguished from pericytes by staining negatively for high-molecular weight caldesmon. α-SMA positive cells were quantified and their number was defined as high when >1.0% of the area was positive. Associations between high cancer-associated fibroblast number, microvascular proliferation, and established prognosticators were analyzed. High numbers of cancer-associated fibroblasts were associated with Schwannian Stroma-poor histopathology and microvascular proliferation. Thirty-seven (80%) of the 46 Schwannian Stroma-poor tumors had high numbers of cancer-associated fibroblasts in the tumor stroma compared to only 2 (14%) of the 14 Schwannian Stroma-rich/dominant tumors (p<0.001). Thirty-three (89%) of 37 tumors with microvascular proliferation had high numbers of cancer-associated fibroblasts compared to 9 (40%) of 22 tumors without microvascular proliferation (p <0.001). In the xenografts with infiltrating Schwann cells (n=10), the number of cancer-associated fibroblasts/mm2 was ~7-fold less than in the control xenografts without stromal Schwann cells (n=9) (mean of 51 ± 30 vs 368 ± 105, respectively; p<0.001). Thus, cancer-associated fibroblasts were inversely associated with presence of Schwann cells, suggesting that Schwann cells may prevent the activation of fibroblasts. A deeper understanding of the role cancer-associated fibroblasts play in neuroblastoma angiogenesis may guide future development of stroma-directed therapeutic strategies.
doi:10.1038/modpathol.2009.52
PMCID: PMC3347894  PMID: 19407854
Cancer-associated fibroblast; Neuroblastoma; Schwannian Stroma
23.  Racial and Ethnic Disparities in Risk and Survival in Children With Neuroblastoma: A Children's Oncology Group Study 
Journal of Clinical Oncology  2010;29(1):76-82.
Purpose
Although health disparities are well-described for many cancers, little is known about racial and ethnic disparities in neuroblastoma. To evaluate differences in disease presentation and survival by race and ethnicity, data from the Children's Oncology Group (COG) were analyzed.
Patients and Methods
The racial/ethnic differences in clinical and biologic risk factors, and outcome of patients with neuroblastoma enrolled on COG ANBL00B1 between 2001 and 2009 were investigated.
Results
A total of 3,539 patients (white, 72%; black, 12%; Hispanic, 12%; Asian, 4%; and Native American, < 1%) with neuroblastoma were included. The 5-year event-free survival (EFS) rates were 67% for whites (95% CI, 65% to 69%), 69% for Hispanics (95% CI, 63% to 74%), 62% for Asians (95% CI, 51% to 71%), 56% for blacks (95% CI, 50% to 62%), and 37% for Native American (95% CI, 17% to 58%). Blacks (P < .001) and Native Americans (P = .04) had a higher prevalence of high-risk disease than whites, and significantly worse EFS (P = .01 and P = .002, respectively). Adjustment for risk group abrogated these differences. However, closer examination of the EFS among high-risk patients who remained event free for 2 years or longer, revealed a higher prevalence of late-occurring events among blacks compared with whites (hazard ratio, 1.5; 95% CI, 1.0 to 2.3; P = .04).
Conclusion
Black and Native American patients with neuroblastoma have a higher prevalence of high-risk disease, accounting for their worse EFS when compared with whites. The higher prevalence of late-occurring events among blacks with high-risk disease suggests that this population may be more resistant to chemotherapy. Studies focused on delineating the genetic basis for the racial disparities observed in this study are planned.
doi:10.1200/JCO.2010.29.6103
PMCID: PMC3055862  PMID: 21098321
24.  Age-Dependent Prognostic Effect by Mitosis-Karyorrhexis Index in Neuroblastoma: A Report from the Children’s Oncology Group 
Prognostic effects of Mitosis-Karyorrhexis Index (MKI) used in the International Neuroblastoma Pathology Classification (INPC) are age-dependent. A total of 4,282 neuroblastomas reviewed at the Children’s Oncology Group Neuroblastoma Pathology Reference Laboratory (8/1/2001–3/31/2012) included 2,365 low-MKI (L-MKI), 1,068 intermediate-MKI (I-MKI), and 849 high-MKI (H-MKI) tumors. Cox proportional hazards models were fit to determine age cut-offs at which the relative risk of event/death was maximized in each MKI class. Backward-selected Cox models were fit to determine the prognostic strength of the age cut-offs for survival in the presence of other prognostic factors. The age cut-offs used in the INPC for L-MKI tumors (<60 months, n = 2,710, 84.0% ± 1.0% event-free survival [EFS], 93.8 ± 0.7% overall survival [OS] vs ≥60 months, n = 195, 49.8% ± 4.6% EFS, 71.7% ± 4.1% OS; P < 0.0001) and I-MKI tumors (<18 months, n = 568, 83.8% ± 2% EFS, 93.7% ± 1.3% OS vs ≥18 months, n = 500, 51.4% ± 2.9% EFS, 66.7% ± 2.7% OS; P < 0.0001) were within the effective range for distinguishing prognostic groups. As for H-MKI tumors (no cut-off age in the INPC, 51.0% ± 2.2% EFS, 64.4% ± 2.1% OS), a new cut-off of 3–4 months was suggested (<4 months, n = 38, 82.3% ± 8.4% EFS, 81.8% ± 8.5% OS vs ≥4 months, n = 811, 49.6% ± 2.2% EFS, 63.7% ± 2.1% OS, P = 0.0034 and 0.0437, respectively). Multivariate analyses revealed that cut-offs of 60 and 18 months for L-MKI and I-MKI tumors, respectively, were independently prognostic. However, the cut-off of 4 months for H-MKI tumors did not reach statistical significance in the presence of other factors. The age cut-offs for MKI classes (60 months for L-MKI, 18 months for I-MKI, no cut-off for H-MKI) in the current INPC are reasonable and effective for distinguishing prognostic groups with increased risk of event/death for older patients.
doi:10.2350/14-06-1505-OA.1
PMCID: PMC4340697  PMID: 25207821
age cut-off; International Neuroblastoma Pathology Classification; mitosis-karyorrhexis index; neuroblastoma; prognosis
25.  The International Neuroblastoma Risk Group (INRG) Classification System: An INRG Task Force Report 
Journal of Clinical Oncology  2009;27(2):289-297.
Purpose
Because current approaches to risk classification and treatment stratification for children with neuroblastoma (NB) vary greatly throughout the world, it is difficult to directly compare risk-based clinical trials. The International Neuroblastoma Risk Group (INRG) classification system was developed to establish a consensus approach for pretreatment risk stratification.
Patients and Methods
The statistical and clinical significance of 13 potential prognostic factors were analyzed in a cohort of 8,800 children diagnosed with NB between 1990 and 2002 from North America and Australia (Children's Oncology Group), Europe (International Society of Pediatric Oncology Europe Neuroblastoma Group and German Pediatric Oncology and Hematology Group), and Japan. Survival tree regression analyses using event-free survival (EFS) as the primary end point were performed to test the prognostic significance of the 13 factors.
Results
Stage, age, histologic category, grade of tumor differentiation, the status of the MYCN oncogene, chromosome 11q status, and DNA ploidy were the most highly statistically significant and clinically relevant factors. A new staging system (INRG Staging System) based on clinical criteria and tumor imaging was developed for the INRG Classification System. The optimal age cutoff was determined to be between 15 and 19 months, and 18 months was selected for the classification system. Sixteen pretreatment groups were defined on the basis of clinical criteria and statistically significantly different EFS of the cohort stratified by the INRG criteria. Patients with 5-year EFS more than 85%, more than 75% to ≤ 85%, ≥ 50% to ≤ 75%, or less than 50% were classified as very low risk, low risk, intermediate risk, or high risk, respectively.
Conclusion
By defining homogenous pretreatment patient cohorts, the INRG classification system will greatly facilitate the comparison of risk-based clinical trials conducted in different regions of the world and the development of international collaborative studies.
doi:10.1200/JCO.2008.16.6785
PMCID: PMC2650388  PMID: 19047291

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