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1.  Cortical Processing of Respiratory Occlusion Stimuli in Children with Central Hypoventilation Syndrome 
Rationale: The ability of patients with central hypoventilation syndrome (CHS) to produce and process mechanoreceptor signals is unknown.
Objectives: Children with CHS hypoventilate during sleep, although they generally breathe adequately during wakefulness. Previous studies suggest that they have compromised central integration of afferent stimuli, rather than abnormal sensors or receptors. Cortical integration of afferent mechanical stimuli caused by respiratory loading or upper airway occlusion can be tested by measuring respiratory-related evoked potentials (RREPs). We hypothesized that patients with CHS would have blunted RREP during both wakefulness and sleep.
Methods: RREPs were produced with multiple upper airway occlusions and were obtained during wakefulness, stage 2, slow-wave, and REM sleep. Ten patients with CHS and 20 control subjects participated in the study, which took place at the Children's Hospital of Philadelphia. Each patient was age- and sex-matched to two control subjects. Wakefulness data were collected from 9 patients and 18 control subjects.
Measurements and Main Results: During wakefulness, patients demonstrated reduced Nf and P300 responses compared with control subjects. During non-REM sleep, patients demonstrated a reduced N350 response. In REM sleep, patients had a later P2 response.
Conclusions: CHS patients are able to produce cortical responses to mechanical load stimulation during both wakefulness and sleep; however, central integration of the afferent signal is disrupted during wakefulness, and responses during non-REM are damped relative to control subjects. The finding of differences between patients and control subjects during REM may be due to increased intrinsic excitatory inputs to the respiratory system in this state.
doi:10.1164/rccm.200804-606OC
PMCID: PMC2556457  PMID: 18658113
central hypoventilation syndrome; respiratory-related evoked potentials; wakefulness; sleep
2.  Pediatric Phase I Trial and Pharmacokinetic Study of MLN8237, an Investigational Oral Selective Small-Molecule Inhibitor of Aurora Kinase A: A Children's Oncology Group Phase I Consortium Study 
Purpose
MLN8237, a selective small-molecule inhibitor of Aurora kinase A, has activity in a broad range of preclinical pediatric cancer models. We conducted a phase I trial in children with refractory/recurrent solid tumors to define the maximum-tolerated dose, toxicities, and pharmacokinetic properties of MLN8237.
Experimental Design
MLN8237 was administered orally either once daily or divided twice daily for seven days, every 21 days. Using a rolling-six design, four dose levels (45, 60, 80, and 100 mg/m2/day) were evaluated on the once-daily schedule, and two dose levels (60 and 80 mg/m2/d) on the twice-daily schedule. Pharmacokinetic studies were conducted with the initial dose and trough drug concentrations also measured at the steady state.
Results
Thirty-seven patients were enrolled. On the once-daily dosing schedule, myelosuppression was dose limiting in three of four patients at 100 mg/m2, and one of six patients had dose-limiting mood alteration at 80 mg/m2. At 45 mg/m2, one of six patients experienced dose-limiting mucositis. Mucositis and myelosuppression were dose limiting at 80 mg/m2 on the twice-daily schedule, and one of five patients at 60 mg/m2 on the twice-daily schedule experienced a dose-limiting alkaline phosphatase. Five of 11 patients experienced hand–foot–skin syndrome with twice-daily dosing versus one of 21 after once-daily dosing. There was one partial response and six with prolonged stable disease among 33 evaluable subjects.
Conclusion
The twice-daily dose regimen is well tolerated in adults; however, children experienced a greater frequency of myelosuppression and hand–foot–skin syndrome on this schedule. Children tolerated a higher dose and the recommended pediatric phase II dose is 80 mg/m2/d once daily for seven days.
doi:10.1158/1078-0432.CCR-11-3251
PMCID: PMC4008248  PMID: 22988055
3.  Initial Testing of the MDM2 Inhibitor RG7112 by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2012;60(4):633-641.
Background
RG7112 is a selective inhibitor of p53-MDM2 binding that frees p53 from negative control, activating the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. RG7112 was selected for evaluation by the Pediatric Preclinical Testing Program (PPTP) due to the relatively low incidence of p53 mutations in pediatric cancers compared with adult malignancies.
Procedures
RG7112 and its inactive enantiomer RG7112i were evaluated against the 23 cell lines of the PPTP in vitro panel using 96 hour exposure (1 nM to 10 µM). It was tested against the PPTP in vivo panel focusing on p53 wild-type (WT) xenografts at a dose of 100 mg/kg daily for 14 days followed by 4 weeks of observation. Response outcomes were related to MDM2 and p53 expression datasets (http://pptp.nchresearch.org/data.html).
Results
RG7112 demonstrated cytotoxic activity with a lower median IC50 for p53 WT versus p53 mutant cell lines (approximately 0.4 µM versus > 10 µM, respectively). RG7112 induced tumor growth inhibition meeting criteria for intermediate activity (EFS T/C > 2) in 10 of 26 (38%) solid tumor xenografts. Objective responses included medulloblastoma, alveolar rhabdomyosarcoma, Wilms, rhabdoid and Ewing sarcoma xenografts. For the ALL panel, there was 1 partial response, 5 complete responses and 1 maintained complete response. The ALL xenografts expressed the highest levels of p53 among the PPTP panels.
Conclusions
RG7112 induced tumor regressions in solid tumors from different histotype panels, and exhibited consistent high-level activity against ALL xenografts. This high level of activity supports prioritization of RG7112 for further evaluation.
doi:10.1002/pbc.24235
PMCID: PMC3495996  PMID: 22753001
Preclinical Testing; Developmental Therapeutics; RG7112
4.  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
5.  Replication of GWAS-identified neuroblastoma risk loci strengthens the role of BARD1 and affirms the cumulative effect of genetic variations on disease susceptibility 
Carcinogenesis  2012;34(3):605-611.
Several neuroblastoma (NB) susceptibility loci have been identified within LINC00340, BARD1, LMO1, DUSP12, HSD17B12, DDX4, IL31RA, HACE1 and LIN28B by genome-wide association (GWA) studies including European American individuals. To validate and comprehensively evaluate the impact of the identified NB variants on disease risk and phenotype, we analyzed 16 single nucleotide polymorphisms (SNPs) in an Italian population (370 cases and 809 controls). We assessed their regulatory activity on gene expression in lymphoblastoid (LCLs) and NB cell lines. We evaluated the cumulative effect of the independent loci on NB risk and high-risk phenotype development in Italian and European American (1627 cases and 2575 controls) populations. All NB susceptibility genes replicated in the Italian dataset except for DDX4 and IL31RA, and the most significant SNP was rs6435862 in BARD1 (P = 8.4×10–15). BARD1 showed an additional and independent SNP association (rs7585356). This variant influenced BARD1 mRNA expression in LCLs and NB cell lines. No evidence of epistasis among the NB-associated variants was detected, whereas a cumulative effect of risk variants on NB risk (European Americans: P trend = 6.9×10–30, Italians: P trend = 8.55×1013) and development of high-risk phenotype (European Americans: P trend = 6.9×10–13, Italians: P trend = 2.2×10–1) was observed in a dose-dependent manner. These results provide further evidence that the risk loci identified in GWA studies contribute to NB susceptibility in distinct populations and strengthen the role of BARD1 as major genetic contributor to NB risk. This study shows that even in the absence of interaction the combination of several low-penetrance alleles has potential to distinguish subgroups of patients at different risks of developing NB.
doi:10.1093/carcin/bgs380
PMCID: PMC3716226  PMID: 23222812
6.  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
7.  COMBINATION THERAPY TARGETING THE CHK1 AND WEE1 KINASES DEMONSTRATES THERAPEUTIC EFFICACY IN NEUROBLASTOMA 
Cancer research  2012;73(2):776-784.
Neuroblastoma is uniquely sensitive to single-agent inhibition of the DNA damage checkpoint kinase Chk1, leading us to examine downstream effectors of this pathway and identify mitotic regulator Wee1 as an additional therapeutic target in this disease. Wee1 was overexpressed in both neuroblastoma cell lines and high-risk patient tumors. Genetic or pharmacologic abrogation of Wee1 signaling results in marked cytotoxicity in 10/11 neuroblastoma cell lines with a median IC50 of 300nM for the Wee1-selective small molecule inhibitor MK-1775. Murine tumor lines derived from mice that were either heterozygous or homozygous for MycN were particularly sensitive to single-agent inhibition of Wee1 (IC50s of 160 nM and 62 nM, respectively). Simultaneous pharmacologic inhibition of Chk1 and Wee1 acted in a synergistic fashion to further impede neuroblastoma cell growth in vitro, in a manner greater than the individual inhibitors either alone or combined with chemotherapy. Combination Chk1 and Wee1 inhibition also revealed in vivo efficacy in neuroblastoma xenografts. Taken together, our results demonstrate that neuroblastoma cells depend on Wee1 activity for growth, and that inhibition of this kinase may serve as a therapeutic for neuroblastoma patients.
doi:10.1158/0008-5472.CAN-12-2669
PMCID: PMC3548976  PMID: 23135916
Neuroblastoma; Chk1; Wee1; MK-1775; MK-8776; SCH 900776
8.  Initial Testing (Stage 1) of SGI-1776, a PIM1 Kinase Inhibitor, by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2011;59(4):749-752.
The PIM kinase inhibitor, SGI-1776, was tested against the PPTP in vitro (1.0 nM to 10 μM) and in vivo panels (148 mg/kg daily x 5 days for 3 weeks). SGI-1776 exhibited cytotoxic activity in vitro with a median relative IC50 of 3.1 μM. SGI-1776 induced significant differences in EFS distribution in vivo in 9 of 31 solid tumor xenografts and in 1 of 8 of the evaluable ALL xenografts. SGI-1776 induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity in 1 of 39 evaluable models. In contrast, SGI-1776 induced complete responses of subcutaneous MV4;11 (B myeloid leukemia).
doi:10.1002/pbc.23364
PMCID: PMC3276706  PMID: 22052829
Preclinical Testing; Developmental Therapeutics; kinase inhibitors
9.  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
10.  A Functional Screen Identifies miR-34a as a Candidate Neuroblastoma Tumor Suppressor Gene 
Molecular cancer research : MCR  2008;6(5):735-742.
MicroRNAs are small noncoding RNAs that have critical roles in regulating a number of cellular functions through transcriptional silencing. They have been implicated as oncogenes and tumor suppressor genes (oncomirs) in several human neoplasms. We used an integrated genomics and functional screening strategy to identify potential oncomirs in the pediatric neoplasm neuroblastoma. We first identified microRNAs that map within chromosomal regions that we and others have defined as frequently deleted (1p36, 3p22, and 11q23-24) or gained (17q23) in high-risk neuroblastoma. We then transiently transfected microRNA precursor mimics or inhibitors into a panel of six neuroblastoma cell lines that we characterized for these genomic aberrations. The majority of transfections showed no phenotypic effect, but the miR-34a (1p36) and miR-34c (11q23) mimics showed dramatic growth inhibition in cell lines with 1p36 hemizygous deletion. In contrast, there was no growth inhibition by these mimics in cell lines without 1p36 deletions. Quantitative reverse transcription-PCR showed a perfect correlation of absent miR-34a expression in cell lines with a 1p36 aberration and phenotypic effect after mimetic add-back. Expression of miR-34a was also decreased in primary tumors (n = 54) with 1p36 deletion (P = 0.009), but no mutations were discovered in resequencing of the miR-34a locus in 30 neuroblastoma cell lines. Flow cytometric time series analyses showed that the likely mechanism of miR-34a growth inhibition is through cell cycle arrest followed by apoptosis. BCL2 and MYCN were identified as miR-34a targets and likely mediators of the tumor suppressor phenotypic effect. These data support miR-34a as a tumor suppressor gene in human neuroblastoma.
doi:10.1158/1541-7786.MCR-07-2102
PMCID: PMC3760152  PMID: 18505919
11.  Initial Testing (Stage 1) of the Cyclin Dependent Kinase Inhibitor SCH 727965 (Dinaciclib) by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2012;59(7):1266-1274.
Background
SCH 727965 is a novel drug in clinical development that potently and selectively inhibits CDK1, CDK2, CDK5, and CDK9. The activity of SCH 727965 was evaluated against the PPTP’s in vitro and in vivo panels.
Procedures
SCH 727965 was tested against the PPTP in vitro panel using 96 hour exposure at concentrations ranging from 0.1 nM to 1.0 μM. It was tested against the PPTP in vivo panels at a dose of 40 mg/kg administered intraperitoneally twice weekly for 2 weeks and repeated at Day 21 with a total observation period of 6 weeks.
Results
The median IC50 value for the cell lines was 7.5 nM, with less than 4-fold range between the minimum (3.4 nM) and maximum (11.2 nM) IC50 values. SCH 727965 demonstrated an activity pattern consistent with cytotoxicity for most of the cell lines. Forty-three xenograft models were studied and SCH 727965 induced significant delays in event free survival distribution compared to control in 23 of 36 (64%) evaluable solid tumor xenografts and in 3 of 7 ALL xenografts. SCH 727965 did not induce objective responses in the solid tumor panels and the best response observed was stable disease for one osteosarcoma xenograft. In the leukemia panel, there were two objective responses with a complete response observed in a single xenograft.
Conclusions
SCH 727965 shows an interesting pattern of activity suggesting its potential applicability against selected childhood cancers, particularly leukemias.
doi:10.1002/pbc.24073
PMCID: PMC3349821  PMID: 22315240
Preclinical Testing; Developmental Therapeutics; SCH 727965; Dinaciclib
12.  Targeting MYCN: A Good BET for Improving Neuroblastoma Therapy? 
Cancer discovery  2013;3(3):255-257.
Summary
Direct targeting of oncogenic MYC proteins has been an elusive goal of many cancer drug development efforts. In this issue of Cancer Discovery, Stegmaier and colleagues demonstrate that pharmacologically interfering with the bromodomain and extraterminal (BET) class of proteins potently depletes MYCN in neuroblastoma cells, resulting in cellular cytotoxicity and thus providing a novel approach with a potential impact on a previously undruggable major oncogene.
doi:10.1158/2159-8290.CD-13-0018
PMCID: PMC3709873  PMID: 23475876
13.  Initial Testing (Stage 1) of the Multi-targeted Kinase Inhibitor Sorafenib by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2010;55(6):10.1002/pbc.22712.
Background
Sorafenib is an inhibitor of multiple kinases (e.g., VEGF receptors, PDGFR, FLT3, RET, BRAF, KIT) and is approved by FDA for treatment of two adult cancers. The activity of sorafenib was evaluated against the PPTP's in vitro and in vivo panels.
Procedures
Sorafenib was evaluated against the PPTP in vitro panel using 96 hour exposure at concentrations ranging from 1.0 nM to 10.0 μM. It was tested against the PPTP in vivo panels at a dose of 60 mg/kg administered by oral gavage daily for 5 days per week, repeated for 6 weeks.
Results
In vitro sorafenib demonstrated cytotoxic activity, with a median IC50 value of 4.3 μM. Twenty of 23 cell lines had IC50 values between 1.0 and 10.0 μM. A single cell line (Kasumi-1) with an activating KIT mutation had an IC50 value < 1.0 μM (IC50 = 0.02 μM). In vivo sorafenib induced significant differences in EFS distribution compared to control in 27 of 36 (75%) of the evaluable solid tumor xenografts and in 1 of 8 (12.5%) of the evaluable ALL xenografts. Sorafenib induced tumor growth inhibition meeting criteria for intermediate activity (EFS T/C) in 15 of 34 (44%) evaluable solid tumor xenografts. No xenografts achieved an objective response.
Conclusions
The primary in vitro activity of sorafenib was noted at concentrations above 1 μM, with the exception of a more sensitive cell line with an activating KIT mutation. The primary in vivo effect for sorafenib was tumor growth inhibition, which was observed across multiple histotypes.
doi:10.1002/pbc.22712
PMCID: PMC3823056  PMID: 20672370
Preclinical Testing; Developmental Therapeutics; tyrosine kinases
14.  Initial Testing of the Investigational NEDD8 Activating Enzyme Inhibitor MLN4924 by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2011;59(2):10.1002/pbc.23357.
Background
MLN4924 is an investigational first-in-class small molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an essential component of the NEDD8 conjugation pathway, controlling the activity of a subset of ubiquitin-proteasome system (UPS) E3 ligases, multiprotein complexes that transfer ubiquitin molecules to substrate proteins.
Procedures
MLN4924 was tested against the PPTP in vitro panel using 96 hour exposure time at concentrations ranging from 1.0 nM to 10 μM. It was tested in vivo at a dose of 100 mg/kg [66 mg/kg for the acute lymphoblastic leukemia (ALL) xenografts] administered orally twice daily × 5 days. Treatment duration was 3 weeks.
Results
The median relative IC50 for MLN4924 against the PPTP cell lines was 143 nM, (range 15 nM to 678 nM) with that for the Ewing panel being significantly lower (31 nM). MLN4924 induced significant differences in EFS distribution compared to control in 20 of 34 (59%) evaluable solid tumor xenografts. MLN4924 induced intermediate activity (EFS T/C values > 2) in 9 of the 33 evaluable xenografts (27%), including 4 of 4 glioblastoma xenografts, 2 of 3 Wilms tumor xenografts, 2 of 5 rhabdomyosarcoma xenografts, and 1 of 4 neuroblastoma xenografts. For the ALL panel, 5 of 8 evaluable xenografts showed intermediate activity for the EFS T/C measure. MLN4924 did not induce objective responses in the PPTP solid tumor or ALL panels.
Conclusions
MLN4924 showed potent activity in vitro and in vivo showed tumor growth inhibitory activity against a subset of the PPTP solid tumor and ALL xenografts.
doi:10.1002/pbc.23357
PMCID: PMC3823062  PMID: 22012946
Preclinical Testing; Developmental Therapeutics; MLN4924
15.  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
16.  Replication of neuroblastoma SNP association at the BARD1 locus in African-Americans 
Background
Neuroblastoma (NBL) is an often-fatal pediatric cancer more frequent in European-American than African-American children. African-American children, however, are at higher risk for the more severe form of NBL, and have worse overall survival than European-American children. Genome wide association studies (GWAS) have identified several SNPs associated to NBL in children of European descent. Knowledge of their association to NBL in African-American children is still lacking.
Methods
We genotyped and imputed SNPs located in three gene regions reported to be associated to NBL in children of European descent, and tested them for association in 390 African-American NBL patients compared to 2500 healthy, ethnically matched controls.
Results
SNPs in the BARD1 gene region show a similar pattern of association to NBL in African-American and European-American children. The more restricted extent of linkage disequilibrium in the African-American population suggests a smaller candidate region for the putative causal variants than previously reported. Limited association was observed at the other two gene regions tested, including LMO1 in 11p15 and FLJ22536 in 6p22.
Conclusions
Common BARD1 SNPs affect risk of NBL in African-Americans. The role of other SNPs associated to NBL in children of European descent could not be confirmed, possibly due to different patterns of linkage disequilibrium or limited statistical power to detect association to variants with small effect on disease risk. Extension of GWAS to populations of African descent is important to confirm their results and validity beyond the European populations, and can help to refine the location of the putative causal variants.
doi:10.1158/1055-9965.EPI-11-0830
PMCID: PMC3319325  PMID: 22328350
Neuroblastoma; BARD1; African-Americans; SNPs; genetic association
17.  Testing of the Akt/PKB Inhibitor MK-2206 by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2011;59(3):518-524.
Background
MK-2206 is a small molecule allosteric inhibitor of Akt/PKB that is undergoing clinical trials for treatment of cancer.
Procedures
MK-2206 was tested against the PPTP in vitro panel using a 96 hour exposure (1.0 nM-10 μM), and in vivo using thrice weekly dosing for a planned 4 weeks at its maximum tolerated dose (MTD) of 180 mg/kg.
Results
In vitro, the median relative IC50 value for MK-2206 was 2.2μM. Four cell lines with IC50 values < 200 nM included two ALL cell lines (COG-LL-317 and RS4;11), an AML cell line with an activating KIT mutation (Kasumi-1), and a Ewing sarcoma cell line (CHLA-10). In vivo, MK-2206 induced significant differences in EFS distribution compared to control in 12 of 29 (41%) of the evaluable solid tumor xenografts and in 2 of 8 (25%) of the evaluable ALL xenografts. Significant differences in EFS distribution were most frequently noted in the osteosarcoma panel (6 of 6). A single solid tumor xenograft (OS-31) had a greater than two-fold increase in time to event compared to control animals, with all other solid tumor xenografts showing lesser degrees of tumor growth inhibition. Objective responses were not observed for either the solid tumor or ALL xenografts.
Conclusions
MK-2206 showed its most consistent activity in vitro against ALL cell lines and in vivo against osteosarcoma xenografts. However, no objective responses were observed in solid tumor or ALL xenografts. Further preclinical work evaluating MK-2206 in pediatric models in the combination therapy setting may contribute to its pediatric development.
doi:10.1002/pbc.23412
PMCID: PMC3290691  PMID: 22102563
Preclinical Testing; Developmental Therapeutics; MK-2206
18.  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
19.  Safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large-cell lymphoma: a Children’s Oncology Group phase 1 consortium study 
The lancet oncology  2013;14(6):472-480.
Summary
Background
Various human cancers have ALK gene translocations, amplifications, or oncogenic mutations, such as anaplastic large-cell lymphoma, inflammatory myofibroblastic tumours, non-small-cell lung cancer (NSCLC), and neuroblastoma. Therefore, ALK inhibition could be a useful therapeutic strategy in children. We aimed to determine the safety, recommended phase 2 dose, and antitumour activity of crizotinib in children with refractory solid tumours and anaplastic large-cell lymphoma.
Methods
In this open-label, phase 1 dose-escalation trial, patients older than 12 months and younger than 22 years with measurable or evaluable solid or CNS tumours, or anaplastic large-cell lymphoma, refractory to therapy and for whom there was no known curative treatment were eligible. Crizotinib was given twice daily without interruption. Six dose levels (100, 130, 165, 215, 280, 365 mg/m2 per dose) were assessed in the dose-finding phase of the study (part A1), which is now completed. The primary endpoint was to estimate the maximum tolerated dose, to define the toxic effects of crizotinib, and to characterise the pharmacokinetics of crizotinib in children with refractory cancer. Additionally, patients with confirmed ALK translocations, mutations, or amplification (part A2 of the study) or neuroblastoma (part A3) could enrol at one dose level lower than was currently given in part A1. We assessed ALK genomic status in tumour tissue and used quantitative RT-PCR to measure NPM-ALK fusion transcript in bone marrow and blood samples of patients with anaplastic large-cell lymphoma. All patients who received at least one dose of crizotinib were evaluable for response; patients completing at least one cycle of therapy or experiencing dose limiting toxicity before that were considered fully evaluable for toxicity. This study is registered with ClinicalTrials. gov, NCT00939770.
Findings
79 patients were enrolled in the study from Oct 2, 2009, to May 31, 2012. The median age was 10·1 years (range 1·1–21·4); 43 patients were included in the dose escalation phase (A1), 25 patients in part A2, and 11 patients in part A3. Crizotinib was well tolerated with a recommended phase 2 dose of 280 mg/m2 twice daily. Grade 4 adverse events in cycle 1 were neutropenia (two) and liver enzyme elevation (one). Grade 3 adverse events that occurred in more than one patient in cycle 1 were lymphopenia (two), and neutropenia (eight). The mean steady state peak concentration of crizotinib was 630 ng/mL and the time to reach this peak was 4 h (range 1–6). Objective tumour responses were documented in 14 of 79 patients (nine complete responses, five partial responses); and the anti-tumour activity was enriched in patients with known activating ALK aberrations (eight of nine with anaplastic large-cell lymphoma, one of 11 with neuroblastoma, three of seven with inflammatory myofibroblastic tumour, and one of two with NSCLC).
Interpretation
The findings suggest that a targeted inhibitor of ALK has antitumour activity in childhood malignancies harbouring ALK translocations, particularly anaplastic large-cell lymphoma and inflammatory myofibroblastic tumours, and that further investigation in the subset of neuroblastoma harbouring known ALK oncogenic mutations is warranted.
Funding
Pfizer and National Cancer Institute grant to the Children’s Oncology Group.
doi:10.1016/S1470-2045(13)70095-0
PMCID: PMC3730818  PMID: 23598171
20.  Initial Testing (Stage 1) of AT13387, an HSP90 Inhibitor, by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2011;59(1):185-188.
AT13387, a non-geldanamycin inhibitor of heat-shock protein 90 (HSP90), was tested against the PPTP in vitro panel (1.0 nM to 10 μM) and against the PPTP in vivo panels (40 mg/kg or 60 mg/kg) administered orally twice weekly. In vitro AT13387 showed a median EC50 value of 41 nM and exhibited activity consistent with a cytotoxic effect. In vivo AT13387 induced significant differences in EFS distribution compared to controls in 17% evaluable solid tumor xenografts, but in none of the ALL xenografts. No objective tumor responses were observed. In vivo AT13387 demonstrated only modest single agent activity.
doi:10.1002/pbc.23154
PMCID: PMC3154460  PMID: 21538821
Preclinical Testing; Developmental Therapeutics; HSP90 inhibitors
21.  Initial Testing of the CENP-E Inhibitor GSK923295Aby the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2011;58(6):916-923.
Background
The centromere kinesin motor protein CENP-E plays a crucial role in mitosis, and is an appealing molecular target in cancer. GSK923295A is an allosteric inhibitor of CENP-E that is undergoing clinical evaluation.
Procedures
GSK923295A was evaluated against the 23 cell lines in the Pediatric Preclinical Testing Program (PPTP) in vitro panel using 96 hour exposures to concentrations ranging from 1.0 nM to 10.0 μM. GSK923295A was also tested in vivo against the PPTP acute lymphoblastic leukemia (ALL) and solid tumor xenograft panels using a Day 1–3 and Day 8–10 schedule that was repeated at Day 21. The agent was administered via the intraperitoneal (IP) route at a daily dose of 125 mg/kg.
Results
The median IC50 for all PPTP cell lines was 27 nM, with the median IC50 for the ALL panel being the lowest (18 nM) and for the neuroblastoma panel the highest (39 nM). Excessive toxicity was observed for each of the 8 xenografts of the ALL panel in NOD/SCID mice. Thirty-five solid tumor xenograft models were considered evaluable. GSK923295A induced significant differences in EFS distribution compared to controls in 32 of 35 evaluable solid tumor xenografts tested. Objective responses were noted in 13 of 35 solid tumor xenografts, including 9 with maintained complete responses (MCR), and 3 with complete response (CR).
Conclusions
GSK923295A demonstrated significant antitumor activity against solid tumor models, inducing complete responses in Ewing sarcoma, rhabdoid and rhabdomyosarcoma xenografts. These results suggest that CENP-E may be a valuable therapeutic target in pediatric cancer.
doi:10.1002/pbc.23176
PMCID: PMC3163687  PMID: 21584937
Preclinical Testing; Developmental Therapeutics; GSK923295A
22.  Common Variation at BARD1 Results in the Expression of an Oncogenic Isoform that Influences Neuroblastoma Susceptibility and Oncogenicity 
Cancer Research  2012;72(8):2068-2078.
The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1β. In neuroblastoma cells, silencing of BARD1β showed genotype-specific cytotoxic effects, including decreased substrate-adherent, anchorage-independent, and foci growth. In established murine fibroblasts, overexpression of BARD1β was sufficient for neoplastic transformation. BARD1β stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1β as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1β with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma.
doi:10.1158/0008-5472.CAN-11-3703
PMCID: PMC3328617  PMID: 22350409
genome-wide association; neuroblastoma; BARD1; cancer susceptibility genes; functional genomics; oncogenes; genotype-phenotype correlations
23.  New Strategies in Refractory and Recurrent Neuroblastoma 
Neuroblastoma remains responsible for a disproportionate amount of childhood cancer morbidity and mortality despite recent significant advances in understanding the genetic basis of tumor initiation and progression. About half of newly diagnosed patients can be reliably identified as having tumors of low malignant potential, and these children have cure rates of greater than 95% with little or no cytotoxic therapy. On the other hand, the other half of neuroblastomas typically present in an explosive fashion with widely metastatic disease, and reliable tumor-specific biomarkers have been defined for this phenotype as well. Empiric approaches to high-risk neuroblastoma therapy have relied on dramatic escalation of chemotherapy dose intensity, and recently the incorporation of targeted immunotherapy, but nearly 50% of children with high-risk disease will be refractory to therapy or suffer a relapse, both of which are invariably fatal. Future improvements in high-risk neuroblastoma outcomes will require the identification of disease and patient-specific oncogenic vulnerabilities that can be leveraged therapeutically. Rational development of novel approaches to neuroblastoma therapy requires forward-thinking strategies to unequivocally prove activity in the relapse setting, and ultimately efficacy in curing patients when integrated into frontline treatment plans.
doi:10.1158/1078-0432.CCR-11-1409
PMCID: PMC3660732  PMID: 22427348
24.  ATF4 Regulates MYC-mediated Neuroblastoma Cell Death upon Glutamine Deprivation 
Cancer cell  2012;22(5):631-644.
SUMMARY
Oncogenic Myc alters mitochondrial metabolism, making it dependent on exogenous glutamine (Gln) for cell survival. Accordingly, Gln deprivation selectively induces apoptosis in MYC-overexpressing cells via unknown mechanisms. Using MYCN-amplified neuroblastoma as a model, we identify PUMA, NOXA and TRB3 as executors of Gln-starved cells. Gln depletion in MYC-transformed cells induces apoptosis through ATF4-dependent, but p53-independent, PUMA and NOXA induction. MYC-transformed cells depend on both glutamate-oxaloacetate transaminase and glutamate dehydrogenase to maintain Gln homeostasis and suppress apoptosis. Consequently, either ATF4 agonists or glutaminolysis inhibitors potently induce apoptosis in vitro and inhibit tumor growth in vivo. These results reveal mechanisms whereby Myc sensitizes cells to apoptosis and validate ATF4 agonists and inhibitors of Gln metabolism as potential Myc-selective cancer therapeutics.
doi:10.1016/j.ccr.2012.09.021
PMCID: PMC3510660  PMID: 23153536
25.  Combination Testing (Stage 2) of the anti-IGF-1 Receptor Antibody IMC-A12 with Rapamycin by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2011;58(5):729-735.
Background
IMC-A12, a fully human antibody that blocks ligand binding to the Type 1 insulin-like growth factor receptor, and rapamycin, a selective inhibitor of mTORC1 signaling, have both demonstrated significant antitumor activity against PPTP solid tumor models. Here we have evaluated antitumor activity of each agent individually and in combination against nine tumor models.
Procedures
IMC-A12 was administered twice weekly and rapamycin was administered daily for 5 days per week for a planned 4 weeks. The impact of combining IMC-A12 with rapamycin was evaluated using two measures: 1) the “therapeutic enhancement” measure, and 2) a linear regression model for time-to-event to formally evaluate for sub- and supra-additivity for the combination compared to the agents used alone.
Results
Two osteosarcomas, and 1 Ewing sarcoma of the nine xenografts tested showed therapeutic enhancement. The combination effect was most dramatic for EW5 for which PD2 responses of short duration were observed for both single agents and a prolonged PR response was observed for the combination. Both OS-2 and OS-9 showed significantly longer times to progression with the combination compared to either of the single agents, although objective response criteria were not met.
Conclusions
The combination of IMC-A12 with rapamycin was well tolerated, and induced tumor responses that were superior to either single agent alone in several models. These studies confirm reports using other antibodies that inhibit IGF-1 receptor-mediated signaling that indicate enhanced therapeutic effect for this combination, and extend the range of histotypes to encompass additional tumors expressing IGF-1R where this approach may be effective.
doi:10.1002/pbc.23157
PMCID: PMC3166415  PMID: 21630428
Preclinical Testing; Developmental Therapeutics; Rapamycin; IMC-A12

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