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1.  Clinical Significance of Tumor-Associated Inflammatory Cells in Metastatic Neuroblastoma 
Journal of Clinical Oncology  2012;30(28):3525-3532.
Purpose
Children diagnosed at age ≥ 18 months with metastatic MYCN-nonamplified neuroblastoma (NBL-NA) are at high risk for disease relapse, whereas those diagnosed at age < 18 months are nearly always cured. In this study, we investigated the hypothesis that expression of genes related to tumor-associated inflammatory cells correlates with the observed differences in survival by age at diagnosis and contributes to a prognostic signature.
Methods
Tumor-associated macrophages (TAMs) in localized and metastatic neuroblastomas (n = 71) were assessed by immunohistochemistry. Expression of 44 genes representing tumor and inflammatory cells was quantified in 133 metastatic NBL-NAs to assess age-dependent expression and to develop a logistic regression model to provide low- and high-risk scores for predicting progression-free survival (PFS). Tumors from high-risk patients enrolled onto two additional studies (n = 91) served as independent validation cohorts.
Results
Metastatic neuroblastomas had higher infiltration of TAMs than locoregional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation-related genes than those in patients diagnosed at age < 18 months. Expression of genes representing TAMs (CD33/CD16/IL6R/IL10/FCGR3) contributed to 25% of the accuracy of a novel 14-gene tumor classification score. PFS at 5 years for children diagnosed at age ≥ 18 months with NBL-NA with a low- versus high-risk score was 47% versus 12%, 57% versus 8%, and 50% versus 20% in three independent clinical trials, respectively.
Conclusion
These data suggest that interactions between tumor and inflammatory cells may contribute to the clinical metastatic neuroblastoma phenotype, improve prognostication, and reveal novel therapeutic targets.
doi:10.1200/JCO.2011.40.9169
PMCID: PMC3675667  PMID: 22927533
2.  microRNA Signature and Expression of Dicer and Drosha Can Predict Prognosis and Delineate Risk Groups in Neuroblastoma 
Cancer research  2010;70(20):7841-7850.
Neuroblastoma is a common childhood tumor and accounts for 15% of pediatric cancer deaths. To investigate the microRNA (miRNA) profile and role of Dicer and Drosha in neuroblastoma, we assessed the expression of 162 human miRNAs, Dicer and Drosha in 66 neuroblastoma tumors by using real-time PCR methods. We found global downregulation of miRNA expression in advanced neuroblastoma and identified 27 miRNAs that can clearly distinguish low- from high-risk patients. Furthermore, expression levels of Dicer or Drosha were low in high-risk neuroblastoma tumors, which accounted for global downregulation of miRNAs in advanced disease and correlated with poor outcome. Notably, for patients with non–MYCN-amplified tumors, low expression of Dicer can serve as a significant and independent predictor of poor outcome (hazard ratio, 9.6; P = 0.045; n = 52). Using plausible neural networks to select a combination of 15 biomarkers that consist of 12 miRNAs' signature, expression levels of Dicer and Drosha, and age at diagnosis, we were able to segregate all patients into four distinct patterns that were highly predictive of clinical outcome. In vitro studies also showed that knockdown of either Dicer or Drosha promoted the growth of neuroblastoma cell lines. Our results reveal that a combination of 15 biomarkers can delineate risk groups of neuroblastoma and serve as a powerful predictor of clinical outcome. Moreover, our findings of growth promotion by silencing Dicer/Drosha implied their potential use as therapeutic targets for neuroblastoma.
doi:10.1158/0008-5472.CAN-10-0970
PMCID: PMC4095771  PMID: 20805302
3.  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
4.  miRNA Expression Profiling Enables Risk Stratification in Archived and Fresh Neuroblastoma Tumor Samples 
Purpose
More accurate assessment of prognosis is important to further improve the choice of risk-related therapy in neuroblastoma (NB) patients. In this study, we aimed to establish and validate a prognostic miRNA signature for children with NB and tested it in both fresh frozen and archived formalin-fixed paraffin-embedded (FFPE) samples.
Experimental Design
Four hundred-thirty human mature miRNAs were profiled in two patient subgroups with maximally divergent clinical courses. Univariate logistic regression analysis was used to select miRNAs correlating with NB patient survival. A 25-miRNA gene signature was built using 51 training samples, tested on 179 test samples, and validated on an independent set of 304 fresh frozen tumor samples and 75 archived FFPE samples.
Results
The 25-miRNA signature significantly discriminates the test patients with respect to progression-free and overall survival (P < 0.0001), both in the overall population and in the cohort of high-risk patients. Multivariate analysis indicates that the miRNA signature is an independent predictor of patient survival after controlling for current risk factors. The results were confirmed in an external validation set. In contrast to a previously published mRNA classifier, the 25-miRNA signature was found to be predictive for patient survival in a set of 75 FFPE neuroblastoma samples.
Conclusions
In this study, we present the largest NB miRNA expression study so far, including more than 500 NB patients. We established and validated a robust miRNA classifier, able to identify a cohort of high-risk NB patients at greater risk for adverse outcome using both fresh frozen and archived material.
doi:10.1158/1078-0432.CCR-11-0610
PMCID: PMC4008338  PMID: 22031095
5.  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
6.  Peripheral Neuroblastic Tumors with Genotype-Phenotype Discordance: A Report from the Children’s Oncology Groupand the International Neuroblastoma Pathology Committee 
Pediatric blood & cancer  2012;60(3):363-370.
Background
Of 4,706 peripheral neuroblastic tumors (pNTs) registered on the Children’s Cancer Group and Children’s Oncology Group Neuroblastoma Study between 1989 and 2010, 51 cases (1.1%) had genotype-phenotype discordance characterized by MYCN amplification (indicating poor prognosis) and Favorable Histology (indicating better prognosis).
Procedure
To distinguish prognostic subgroups in the genotype-phenotype discordant pNTs, two subgroups, “conventional” and “bull’s eye”, were identified based on the nuclear morphology. The “conventional” tumors (35 cases) included: Neuroblastoma, Poorly differentiated subtype (NB-PD, 26 cases) with “salt-and-pepper” nuclei; Neuroblastoma, Differentiating subtype (4 cases); Ganglioneuroblastoma, Intermixed (3 cases); and Ganglioneuroma, Maturing subtype (2 cases). The “bull’s eye” tumors included NB-PD with prominent nucleoli (16 cases). Clinicopathologic characteristics of these two subgroups were analyzed. N-myc protein expression was tested immunohistochemically on available tumors.
Results
No significant difference was found between these two subgroups in the distribution of prognostic factors such as age at diagnosis, clinical stage, histopathology category/subtype, mitosis-karyorrhexis index, ploidy, 1p LOH, and unbalanced 11qLOH. However, prognosis of the patients with “conventional” tumors (5-year EFS 85.7±12.2%; OS 89.3±10.3%) was significantly better than those with “bull’s eye” tumors (EFS 31.3±13.0%; OS 42.9±16.2%) (P=0.0010 and 0.0008, respectively). Immunohistochemically all (11/11) tested “conventional” tumors were negative, and 10/11 tested “bull’s eye” tumors were positive for N-myc protein expression.
Conclusions
Based on the presence or absence of prominent nucleoli (the putative site of RNA synthesis/accumulation leading to N-myc protein expression), two prognostic subgroups, “conventional” with a better prognosis and “bull’s eye” with a poor prognosis, were distinguished among the genotype-phenotype discordant pNTs.
doi:10.1002/pbc.24238
PMCID: PMC3397468  PMID: 22744966
neuroblastoma; International Neuroblastoma Pathology Classification; MYCN; genotype-phenotype correlation; prognosis; immunohistochemistry
7.  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
8.  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
9.  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
10.  A Pilot Study of Tandem High Dose Chemotherapy with Stem Cell Rescue as Consolidation for High Risk Neuroblastoma: Children’s Oncology Group study ANBL00P1 
Bone marrow transplantation  2013;48(7):947-952.
Increasing treatment intensity has improved outcomes for children with neuroblastoma. We performed a pilot study in the Children’s Oncology Group (COG) to assess feasibility and toxicity of a tandem myeloablative regimen without total body irradiation (TBI) supported by autologous CD34 selected peripheral blood stem cells. Forty-one patients with high-risk neuroblastoma were enrolled; eight patients did not receive any myeloablative consolidation procedure, and seven received only one. Two patients out of 41 (4.9%) experienced transplant-related mortality. CD34 selection was discontinued after subjects were enrolled due to serious viral illness. From the time of study enrollment, the overall 3-year event-free survival (EFS) and overall survival (OS) were 44.8±9.6% and 59.2±9.2% (N=41). These results demonstrate that tandem transplantation in the cooperative group setting is feasible and support a randomized comparison of single versus tandem myeloablative consolidation with PBSC support for high-risk neuroblastoma.
doi:10.1038/bmt.2012.276
PMCID: PMC3638062  PMID: 23334272
pediatric; neuroblastoma; tandem transplant; hematopoietic stem cell transplant
11.  Response-Dependent and Reduced Treatment in Lower Risk Hodgkin Lymphoma in Children and Adolescents, Results of P9426: A Report from the Children’s Oncology Group 
Pediatric blood & cancer  2012;59(7):1259-1265.
Background
Hodgkin lymphoma is highly curable but associated with significant late effects. Reduction of total treatment would be anticipated to reduce late effects. This aim of this study was to demonstrate that a reduction in treatment was possible without compromising survival outcomes.
Methods
Protocol P9426, a response-dependent and reduced treatment for low risk Hodgkin lymphoma (stages I, IIA, and IIIA1) was designed in 1994 based on a previous pilot project. Patients were enrolled from 10/15/1996 to 09/19/2000. Patients were randomized to receive or not receive Dexrazoxane and received 2 cycles of chemotherapy consisting of Doxorubicin, Bleomycin, Vincristine, and Etoposide. After 2 cycles, patients were evaluated for response. Those in complete response (CR) received 2550 cGy of involved field radiation therapy (IFRT). Patient with partial response or stable disease, received 2 more cycles of chemotherapy and IFRT at 2550 cGy.
Results
There were 294 patients enrolled, with 255 eligible for analysis. The 8 year event free survival (EFS) between the Dexrazoxane randomized groups did not differ (EFS 86.8 + 3.1% with DRZ, and 85.7 + 3.3% without DRZ (p=0.70). Forty five percent of patients demonstrated CR after two cycles of chemotherapy. There was no difference in EFS by histology, rapidity of response, or number of cycles of chemotherapy. Six of the eight secondary malignancies in this study have been previously reported.
Conclusions
Despite reduced therapy and exclusion of most patients with Lymphocyte Predominant histology, EFS and overall survival are similar to other reported studies. The protocol documents that it is safe and effective to reduce therapy in low risk Hodgkin lymphoma based on early response to chemotherapy with rapid responding patients having the same outcome as slower-responding patients when given 50% of the chemotherapy.
doi:10.1002/pbc.24279
PMCID: PMC3468662  PMID: 22911615
Response-dependent; Hodgkin lymphoma; children and adolescents
12.  Application of the Adult International Germ Cell Classification System to Pediatric Malignant Non-Seminomatous Germ Cell Tumors: A Report From the Children’s Oncology Group 
Pediatric blood & cancer  2008;50(4):10.1002/pbc.21304.
Background
The purpose of this analysis is to explore whether the International Germ Cell Classification Consensus (IGCCC) tumor marker criteria, developed for adult males with metastatic malignant germ cell tumors (MGCT), are prognostic among pediatric patients and whether tumor marker data may be relevant in pediatric risk stratification.
Procedure
The IGCCC was applied to 436 pediatric germ cell patients treated on Pediatric Intergroup Studies from 1990 to 1996. Multivariable Cox proportional hazards model identified prognostic variables; survival rates among IGCCC risk groups were compared using the log-rank test. Concordance and relative performance of IGCCC versus COG risk stratification was evaluated.
Results
Applying the IGCCC, 21% of pediatric patients were good risk (GR), 35% intermediate risk (IR), and 44% poor risk (PR). Only modest concordance between IGCCC and COG stratification systems was noted (49%). Nonetheless, the IGCCC identified a group of PR patients who had significantly worse event-free survival (EFS) versus GR/IR patients (6-year EFS 80% vs. 91%), which was similar to the difference observed using the COG system (6-year EFS 77% vs. 90%). The IGCCC performed well within subgroups for which the IGCCC is not intended (prepubertal, female, and non-metastatic patients).
Conclusions
Applying the IGCCC system to pediatric patients produces a different stratification than does the application of the COG system, although both are prognostic. Development of a de novo pediatric prognostic classification is warranted.
doi:10.1002/pbc.21304
PMCID: PMC3836436  PMID: 18085675
germ cell tumors; International Germ Cell Classification; pediatric germ cell tumors; risk stratification; tumor markers
13.  Clinicopathologic Features and Long-Term Outcomes of NUT Midline Carcinoma 
Purpose
NUT midline carcinoma (NMC) is a poorly differentiated squamous cancer characterized by rearrangement of the NUT gene. Research advances have provided opportunities for targeted therapy in NMC, yet the clinical features of this rare disease have not been systematically characterized. We report on a large population of such patients to identify the disease characteristics and treatments, correlate them with outcome, and to consider clinical recommendations.
Experimental Design
A clinical database was established using retrospective demographic and outcomes data available on all known cases of NMC. Questionnaires were completed by treating physicians. Pathologic, demographic, and clinical variables were assessed for 63 patients, the largest cohort of NMC patients studied to date. Outcome data from 54 patients were available for survival analyses.
Results
The diagnosis of NMC has increased annually since 2007. Since 2009, there has been an observed increase in the age at diagnosis (p<0.05). Geographic distribution of NMC patients has been concentrated in the United States (n=41, 65%). The median overall survival for patients with NMC was 6.7 months. The 2-year progression-free survival (PFS) was 9% with a 95% CI of 1%–17% (1-year PFS 15% (5%–24%)) and 2-year overall survival (OS) was 19% with a 95% CI of 7%–31% (1-year OS: 30% (27%–34%). Multivariate analysis suggested that extent of surgical resection and initial radiotherapy were independent predictors of PFS and OS. Notably, no chemotherapeutic regimen was associated with improved outcome.
Conclusions
NMC portends a poor prognosis among all squamous cell neoplasms and appears to be frequently unrecognized. The finding that conventional chemotherapy has been inadequate indicates a pressing need for the development of targeted therapeutics. Intensive local therapies such as gross total resection and radiotherapy might be associated with enhanced survival.
doi:10.1158/1078-0432.CCR-12-1153
PMCID: PMC3473162  PMID: 22896655
NUT midline carcinoma; outcomes; registry
14.  Phase II Randomized Comparison of Topotecan Plus Cyclophosphamide Versus Topotecan Alone in Children With Recurrent or Refractory Neuroblastoma: A Children's Oncology Group Study 
Journal of Clinical Oncology  2010;28(24):3808-3815.
Purpose
Single-agent topotecan (TOPO) and combination topotecan and cyclophosphamide (TOPO/CTX) were compared in a phase II randomized trial in relapsed/refractory neuroblastoma. Because responders often underwent further therapies, novel statistical methods were required to compare the long-term outcome of the two treatments.
Patients and Methods
Children with refractory/recurrent neuroblastoma (only one prior aggressive chemotherapy regimen) were randomly assigned to daily 5-day topotecan (2 mg/m2) or combination topotecan (0.75 mg/m2) and cyclophosphamide (250 mg/m2). A randomized two-stage group sequential design enrolled 119 eligible patients. Toxicity and response were estimated. Long-term outcome of protocol therapy was assessed using novel methods—causal inference—which allowed adjustment for the confounding effect of off-study therapies.
Results
Seven more responses were observed for TOPO/CTX (complete response [CR] plus partial response [PR], 18 [32%] of 57) than TOPO (CR+PR, 11 [19%] of 59;P = .081); toxicity was similar. At 3 years, progression-free survival (PFS) and overall survival (OS) were 4% ± 2% and 15% ± 4%, respectively. PFS was significantly better for TOPO/CTX (P = .029); there was no difference in OS. Older age at diagnosis and lack of MYCN amplification predicted increased OS (P < .05). Adjusting for randomized treatment effect and subsequent autologous stem-cell transplantation, there was no difference between TOPO and TOPO/CTX in terms of the proportion alive at 2 years.
Conclusion
TOPO/CTX was superior to TOPO in terms of PFS, but there was no OS difference. After adjustment for subsequent therapies, no difference was detected in the proportion alive at 2 years. Causal inference methods for assessing long-term outcomes of phase II therapies after subsequent treatment can elucidate effects of initial therapies.
doi:10.1200/JCO.2009.27.5016
PMCID: PMC2940398  PMID: 20660830
15.  The genetic landscape of high-risk neuroblastoma 
Nature genetics  2013;45(3):279-284.
Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%1. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 cases using a combination of whole exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per megabase (0.48 non-silent), and remarkably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, an additional 7.1% had focal deletions), MYCN (1.7%, a recurrent p.Pro44Leu alteration), and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1, and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies reliant upon frequently altered oncogenic drivers.
doi:10.1038/ng.2529
PMCID: PMC3682833  PMID: 23334666
16.  Clinical Significance of NTRK Family Gene Expression in Neuroblastomas 
Pediatric blood & cancer  2011;59(2):226-232.
Background
Neuroblastomas (NBs) are characterized by clinical heterogeneity, from spontaneous regression to relentless progression. The pattern of NTRK family gene expression contributes to these disparate behaviors. TrkA/NTRK1 is expressed in favorable NBs that regress or differentiate, whereas TrkB/NTRK2 and its ligand BDNF are co-expressed in unfavorable NBs, representing an autocrine survival pathway. We determined the significance of NTRK family gene expression in a large, representative set of primary NBs.
Patients and Methods
We analyzed the expression of the following genes in 814 NBs using quantitative real-time RT-PCR: NTRK1, NTRK2, NTRK3, P75/NGFR, NGF, BDNF, IGFR1 and EGFR. Expression (high vs. low) was dichotomized by median expression value and compared to clinical and biological variables as well as outcome.
Results
High NTRK1 expression was strongly correlated with favorable age, stage, MYCN status, histology, ploidy, risk group and outcome (p<0.0001 for all). However, it did not add significantly to the panel of prognostic variables currently used for cooperative group trials. NTRK2 expression was associated with risk factors but not with outcome. High NGF expression was also associated with most risk factors and weakly with unfavorable outcome.
Conclusions
High expression of NTRK1 is strongly associated with favorable risk factors and outcome in a large, representative population of NB patients. It did not add significantly to the current risk prediction algorithm, but it may contribute to future expression classifiers. Indeed, prospective assessment of NTRK1 and NTRK2 expression will identify tumors that would be candidates for NTRK-targeted therapy, either alone or in combination with conventional agents.
doi:10.1002/pbc.23343
PMCID: PMC3258457  PMID: 21990266
neuroblastoma; TrkA/NTRK1; TrkB/NTRK2; TrkC/NTRK3; P75/NGFR; NGF; BDNF; expression; prognosis
17.  Development of an open-source, flexible framework for complex inter-institutional disparate data sharing and collaboration 
Clinical information, “-omic” datasets, and tissue samples are difficult to harmonize and manage for data mining. We have developed a platform for storing clinical research data while providing access to associated data from other information stores. Data on 34 metrics from 11,000 neuroblastoma patients were instantiated into a database. The Django web framework was used to create a model for rapid development of tools and views with a front-end interface for generating complex queries. Working with Nationwide Children’s Hospital, we can now consume their tissue inventory data through an API. The end-user sees the number of patients who both match their search and have tissue available. Since initial implementation, the current tasks revolve around developing a governance structure and the necessary data use agreements. Efforts now are to (1) update the data with 5000 more patients, and (2) link to genomic data stores, facilitating disparate data acquisition for research studies.
PMCID: PMC3814475  PMID: 24303312
18.  Mechanisms of CHD5 Inactivation in Neuroblastomas 
Clinical Cancer Research  2012;18(6):1588-1597.
Purpose
Neuroblastomas (NBs) have genomic, biological and clinical heterogeneity. High-risk NBs are characterized by several genomic changes, including MYCN amplification and 1p36 deletion. We identified the chromatin-remodeling gene CHD5 as a tumor suppressor gene that maps to 1p36.31. Low or absent CHD5 expression is associated with a 1p36 deletion and an unfavorable outcome, but the mechanisms of CHD5 inactivation in NBs are unknown.
Experimental Design
We examined 1) the CHD5 sequence in 188 high-risk NBs investigated through the TARGET initiative; 2) the methylation status of the CHD5 promoter in 108 NBs with or without 1p36 deletion and/or MYCN amplification; and 3) mRNA expression of CHD5 and MYCN in 814 representative NBs using TaqMan low-density array microfluidic cards.
Results
We found no examples of somatically acquired CHD5 mutations, even in cases with 1p36 deletion, indicating that homozygous genomic inactivation is rare. Methylation of the CHD5 promoter was common in the high-risk tumors, and it was generally associated with both 1p deletion and MYCN amplification. High CHD5 expression was a powerful predictor of favorable outcome, and it showed prognostic value even in multivariable analysis after adjusting for MYCN amplification, 1p36 deletion, and/or 11q deletion.
Conclusions
We conclude that 1) somatically acquired CHD5 mutations are rare in primary NBs, so inactivation probably occurs by deletion and epigenetic silencing; 2) CHD5 expression and promoter methylation are associated with MYCN amplification, suggesting a possible interaction between these two genes; and 3) high CHD5 expression is strongly correlated with favorable clinical/biological features and outcome.
doi:10.1158/1078-0432.CCR-11-2644
PMCID: PMC3306487  PMID: 22294723
Neuroblastoma; CHD5; expression; methylation; mutation
19.  Phase II study of oral capsular 4-hydroxyphenylretinamide (4-HPR/fenretinide) in pediatric patients with refractory or recurrent neuroblastoma: A report from the Children’s Oncology Group NSC #374551; IND# 40294 
Purpose
To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma.
Experimental Design
Patients received 7 days of fenretinide: 2475 mg/m2/day divided TID (<18 years) or 1800 mg/m2/day divided BID (≥18 years) every 21 days for a maximum of 30 courses. Patients with stable or responding disease after course 30 could request additional compassionate courses. Best response by course 8 was evaluated in Stratum 1 (measurable disease on CT/MRI +/− bone marrow and/or MIBG avid sites) and Stratum 2 (bone marrow and/or MIBG avid sites only).
Results
Sixty-two eligible patients, median age 5 years (range 0.6–19.9), were treated in Stratum 1 (n=38) and Stratum 2 (n=24). One partial response (PR) was seen in Stratum 2 (n=24 evaluable). No responses were seen in Stratum 1 (n=35 evaluable). Prolonged stable disease (SD) was seen in 7 patients in Stratum 1 and 6 patients in Stratum 2 for 4–45+ (median 15) courses. Median time to progression was 40 days (range 17–506) for Stratum 1 and 48 days (range 17–892) for Stratum 2. Mean 4-HPR steady state trough plasma concentrations were 7.25 µM (coefficient of variation 40–56%) at day 7 course 1. Toxicities were mild and reversible.
Conclusions
Although neither stratum met protocol criteria for efficacy, 1 PR + 13 prolonged SD occurred in 14/59 (24%) of evaluable patients. Low bioavailability may have limited fenretinide activity. Novel fenretinide formulations with improved bioavailability are currently in pediatric Phase I studies.
doi:10.1158/1078-0432.CCR-11-0995
PMCID: PMC3207022  PMID: 21908574
fenretinide; neuroblastoma; Phase II; ANBL0321
20.  Evaluation of Norepinephrine Transporter Expression and Metaiodobenzylguanidine Avidity in Neuroblastoma: A Report from the Children's Oncology Group 
Purpose. 123I-metaiodobenzylguanidine (MIBG) is used for the diagnostic evaluation of neuroblastoma. We evaluated the relationship between norepinephrine transporter (NET) expression and clinical MIBG uptake. Methods. Quantitative reverse transcription PCR (N = 82) and immunohistochemistry (IHC; N = 61) were performed for neuroblastoma NET mRNA and protein expression and correlated with MIBG avidity on diagnostic scans. The correlation of NET expression with clinical features was also performed. Results. Median NET mRNA expression level for the 19 MIBG avid patients was 12.9% (range 1.6–73.7%) versus 5.9% (range 0.6–110.0%) for the 8 nonavid patients (P = 0.31). Median percent NET protein expression was 50% (range 0–100%) in MIBG avid patients compared to 10% (range 0–80%) in nonavid patients (P = 0.027). MYCN amplified tumors had lower NET protein expression compared to nonamplified tumors (10% versus 50%; P = 0.0002). Conclusions. NET protein expression in neuroblastoma correlates with MIBG avidity. MYCN amplified tumors have lower NET protein expression.
doi:10.1155/2012/250834
PMCID: PMC3463166  PMID: 23050139
21.  Integrative genomics identifies LMO1 as a neuroblastoma oncogene 
Nature  2010;469(7329):216-220.
Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths1,2. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10−16, odds ratio of risk allele = 1.34 (95% confidence interval 1.25–1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.
doi:10.1038/nature09609
PMCID: PMC3320515  PMID: 21124317
22.  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
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.  Antitumor Activity of Hu14.18-IL2 in Patients With Relapsed/Refractory Neuroblastoma: A Children's Oncology Group (COG) Phase II Study 
Journal of Clinical Oncology  2010;28(33):4969-4975.
Purpose
The hu14.18-IL2 fusion protein consists of interleukin-2 molecularly linked to a humanized monoclonal antibody that recognizes the GD2 disialoganglioside expressed on neuroblastoma cells. This phase II study assessed the antitumor activity of hu14.18-IL2 in two strata of patients with recurrent or refractory neuroblastoma.
Patients and Methods
Hu14.18-IL2 was given intravenously (12 mg/m2/daily) for 3 days every 4 weeks for patients with disease measurable by standard radiographic criteria (stratum 1) and for patients with disease evaluable only by [123I]metaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow (BM) histology (stratum 2). Response was established by independent radiology review as well as BM histology and immunocytology, and durability was assessed by repeat evaluation after more than 3 weeks.
Results
Thirty-nine patients were enrolled (36 evaluable). No responses were seen in stratum 1 (n = 13). Of 23 evaluable patients in stratum 2, five patients (21.7%) responded; all had a complete response (CR) of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 nonhematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia. Two patients required dopamine for hypotension, and one patient required ventilatory support for hypoxia. Most toxicities were reversible within a few days of completing a treatment course and were expected based on phase I results.
Conclusion
Patients with disease evaluable only by MIBG and/or BM histology had a 21.7% CR rate to hu14.8-IL2, whereas patients with bulky disease did not respond. Hu14.18-IL2 warrants further testing in children with nonbulky high-risk neuroblastoma.
doi:10.1200/JCO.2009.27.8861
PMCID: PMC3020698  PMID: 20921469
25.  ABCC Multidrug Transporters in Childhood Neuroblastoma: Clinical and Biological Effects Independent of Cytotoxic Drug Efflux 
Background
Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux.
Methods
A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)–driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN1/−, 205 hMYCN+/1 mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan–Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided.
Results
Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the “poor prognosis” expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001).
Conclusion
ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.
doi:10.1093/jnci/djr256
PMCID: PMC3156802  PMID: 21799180

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