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1.  AZ64 inhibits TrkB and enhances the efficacy of chemotherapy and local radiation in neuroblastoma xenografts 
Neuroblastoma is a common pediatric tumor characterized by clinical heterogeneity. Because it is derived from sympathetic neuroblasts, the NTRK family of neurotrophin receptors plays an integral role in neuroblastoma cell survival, growth, and differentiation. Indeed, high expression of NTRK1 is associated with favorable clinical features and outcome, whereas expression of NTRK2 and its ligand, brain-derived neurotrophic factor (BDNF), are associated with unfavorable features and outcome. AZ64 (Astra Zeneca) is a potent and selective inhibitor of the NTRK tyrosine kinases that blocks phosphorylation at nanomolar concentrations. To determine the preclinical activity of AZ64, we performed intervention trials in a xenograft model with NTRK2-overexpressing neuroblastomas. AZ64 alone significantly inhibited tumor growth compared to vehicle-treated animals (p = 0.0006 for tumor size). Furthermore, the combination of AZ64 with conventional chemotherapeutic agents, irinotecan and temozolomide (irino–temo), showed significantly enhanced anti-tumor efficacy compared to irino–temo alone [(p < 0.0001 for tumor size, p < 0.0005 for event-free survival (EFS)]. We also assessed the combination of AZ64 and local radiation therapy (RT) on a neuroblastoma hindlimb xenograft model, and the efficacy of local RT was significantly increased when animals were treated simultaneously with AZ64 (p < 0.0001 for tumor size, p = 0.0006 for EFS). We conclude that AZ64 can inhibit growth of NTRK-expressing neuroblastomas both in vitro and in vivo. More importantly, it can significantly enhance the efficacy of conventional chemotherapy as well as local RT, presumably by inhibition of the NTRK2/BDNF autocrine survival pathway.
doi:10.1007/s00280-012-1879-x
PMCID: PMC4242714  PMID: 22623209
TrkA; TrkB; AZ64; Neuroblastoma; Inhibition; Signaling; Differentiation
2.  Morbidity in Children and Adolescents Following Surgical Correction of Truncus Arteriosus Communis 
American heart journal  2013;166(3):512-518.
Background
Studies of outcome following operative correction of truncus arteriosus communis (TA) have focused on mortality and rates of re-intervention. We sought to investigate the clinical status of children and adolescents with surgically corrected TA.
Methods and Results
A cross-sectional study of subjects with TA was performed. Subjects underwent concurrent genetic testing, electrocardiogram, cardiac magnetic resonance imaging, cardiopulmonary exercise testing, and completed questionnaires assessing health status and health-related quality of life. Review of their medical history provided retrospective information on cardiac re-intervention and utilization of medical care. Twenty-five subjects with a median age of 11.8 (8.1-18.99) years were enrolled. The prevalence of 22q11.2 deletion was 32%. Incidence of hospitalization, cardiac re-intervention, and non-cardiac operations was highest in the first year of life. Combined catheter-based and operative re-intervention rates were 52% on the conduit and 56% on the pulmonary arteries. Right ventricular ejection fraction and end diastolic volume were normal. Moderate or greater truncal valve insufficiency was seen in 11% of subject, and truncal valve replacement occurred in 8% of subjects. Maximal oxygen consumption (p=0002), maximal work (p<0.0001), and forced vital capacity (p<0.0001) were all lower than normal for age and sex. Physical health status and health-related quality of life were both severely diminished.
Conclusion
Patients with TA demonstrate significant co-morbid disease throughout childhood, significant burden of operative and catheter-based re-intervention, and deficits in exercise performance, functional status, and health-related quality of life.
doi:10.1016/j.ahj.2013.05.023
PMCID: PMC3771390  PMID: 24016501
Truncus arteriosus communis; Cardiac MRI; Cardiopulmonary exercise testing; Quality of life
3.  The TAKE-IT study: aims, design, and methods 
BMC Nephrology  2014;15:139.
Background
Effective interventions to improve immunosuppressive medication adherence among adolescent and young adult kidney transplant recipients are desperately needed. This paper describes the aims, design, and methods of the Teen Adherence in Kidney transplant, Effectiveness of Intervention Trial (TAKE-IT) study.
Design and methods
TAKE-IT is a multicentre, prospective, open-label, parallel arm randomized controlled trial that aims to determine the effectiveness of a clinic-based intervention, including educational, organizational, and behavioural components, in improving immunosuppressive medication adherence among adolescent and young adult kidney transplant recipients. Individuals between 11 and 24 years of age who are at least 3 months post-transplant and followed in one of the eight participating pediatric kidney transplant programs, or their affiliated adult transplant programs are eligible to participate. All participating centers are tertiary care pediatric hospitals in Canada or the United States. Adherence is monitored using an electronic multi-dose pillbox for all participants during a 3-month run-in period, followed by a 12-month intervention interval. The primary outcome is ‘taking adherence’, defined as the proportion of prescribed doses of immunosuppressive medications that were taken, as measured using electronic monitoring.
All participants meet with the study ‘Coach’ at 3 month intervals. The intervention, administered by trained lay personnel, targets common adherence barriers. In addition to forming an Adherence Support Team, intervention participants identify personal barriers to adherence and use Action-focused problem-solving to address them, have their electronic adherence data fed back to them, and have the option to receive email, text message, or visual cue dose reminders. Participants in the control group meet with the coach but do not receive the other components of the intervention. The study aims to have 75 participants in each group complete the study.
Discussion
Since recruitment began in Feb. 2012, 198 adolescents have been approached to participate, of whom 130 have completed a baseline visit. As of March 31, 2014, 125 had been randomized, and 86, 68, 61, and 50 participants had completed 6-month, 9-month, 12-month, and 15-month visits respectively.
Trial registration
Clinicaltrials.gov registration NCT01356277 (May 17, 2011).
doi:10.1186/1471-2369-15-139
PMCID: PMC4236658  PMID: 25176317
Adherence; Randomized trial; Adolescent; Intervention; Kidney transplantation
4.  Treatment of Epstein Barr virus-induced haemophagocytic lymphohistiocytosis with rituximab-containing chemo-immunotherapeutic regimens 
British journal of haematology  2013;162(3):376-382.
Summary
Haemophagocytic lymphohistiocytosis (HLH) is a life threatening complication of Epstein-Barr virus (EBV) infection. The anti-CD20 antibody rituximab depletes B cells, leading to improved outcomes for patients with EBV-associated B-lymphoproliferative disorders. To gather data on the use of rituximab in EBV-HLH, we performed a retrospective investigation involving 42 EBV-HLH patients who had received treatment with rituximab-containing regimens. On average, patients received 3 rituximab infusions (range 1 – 10) at a median dose of 375 mg/m2. In all patients, rituximab was administered with other HLH-directed medications, including steroids, etoposide and/or ciclosporin. Rituximab-containing regimens appeared well tolerated and improved clinical status in 43% of patients. Examination of laboratory data obtained prior to and within 2 – 4 weeks after the first rituximab dose revealed significant reductions in EBV load (median load pre-rituximab: 114,200 copies/ml, median post-rituximab: 225 copies/ml, p=0.0001) and serum ferritin levels (median ferritin pre-rituximab: 4,260 μg/l, median post-rituximab: 1,149 μg/l, p=0.001). Thus, when combined with conventional HLH-directed therapies, rituximab improves symptoms, reduces viral load and diminishes inflammation. These data support the incorporation of rituximab into future prospective clinical trials for patients with EBV-HLH.
doi:10.1111/bjh.12386
PMCID: PMC3776423  PMID: 23692048
Epstein-Barr virus; haemophagocytic lymphohistiocytosis; macrophage activation syndrome; rituximab; x-linked lymphoproliferative disease
5.  Analyzing Genetic Association Studies with an Extended Propensity Score Approach 
Statistical applications in genetics and molecular biology  2012;11(5):10.1515/1544-6115.1790 /j/sagmb.2012.11.issue-5/1544-6115.1790/1544-6115.1790.xml.
Propensity scores are commonly used to address confounding in observational studies. However, they have not been previously adapted to deal with bias in genetic association studies. We propose an extension of our previous method (Zhao et al., 2009) that uses a multilevel propensity score approach and allows one to estimate the effect of a genotype under an additive model and also simultaneously adjusts for confounders such as genetic ancestry and patient and disease characteristics. Using simulation studies, we demonstrate that this extended genetic propensity score (eGPS) can adequately adjust and consistently correct for bias due to confounding in a variety of circumstances. Under all simulation scenarios, the eGPS method yields estimates with bias close to 0 (mean=0.018, standard error=0.01). Our method also preserves statistical properties such as coverage probability, Type I error, and power. We illustrate this approach in a population-based genetic association study of testicular germ cell tumors and KITLG and SPRY4 susceptibility genes. We conclude that our method provides a novel and broadly applicable analytic strategy for obtaining less biased and more valid estimates of genetic associations.
doi:10.1515/1544-6115.1790
PMCID: PMC3518898  PMID: 23104843
population-based genetic association; propensity scores; population stratification; confounding; genetic and non-genetic covariates; susceptibility genes
6.  BH3 Response Profiles From Neuroblastoma Mitochondria Predict Activity of Small Molecule Bcl-2 Family Antagonists 
Cell death and differentiation  2009;17(5):872-882.
Bcl-2 family proteins regulate mitochondrial apoptosis downstream of diverse stressors. Cancer cells frequently deregulate Bcl-2 proteins leading to chemoresistance. We have optimized a platform for solid tumors in which Bcl-2 family resistance patterns are inferred. Functional mitochondria were isolated from neuroblastoma cell lines, exposed to distinct BH3-domain peptides, and assayed for cytochrome c release. Such BH3 profiles revealed three patterns of cytochrome c response. A subset had a dominant NoxaBH3 response implying Mcl1-dependence. These cells were more sensitive to small molecules that antagonize Mcl1 (AT-101) than those that antagonize Bcl-2, Bcl-xL and Bcl-w (ABT-737). A second subset had a dominant BikBH3 response, implying a Bcl-xL/-w dependence, and was exquisitely sensitive to ABT-737 (IC50 <200 nM). Finally, most neuroblastoma cell lines derived at relapse were relatively resistant to pro-death BH3 peptides and Bcl-2 antagonists. Our findings define heterogeneity for apoptosis resistance in neuroblastoma, help triage emerging Bcl-2 antagonists for clinical use, and provide a platform for studies to characterize post-therapy resistance mechanisms for neuroblastoma and other solid tumors.
doi:10.1038/cdd.2009.171
PMCID: PMC3690273  PMID: 19893570
Bcl-2 homology proteins; experimental therapeutics; chemoresistance; BH3 mimetics; neuroblastoma
7.  Mitochondrial Bcl-2 family dynamics define therapy response and resistance in neuroblastoma 
Cancer Research  2012;72(10):2565-2577.
Neuroblastoma is a childhood tumor in which transient therapeutic responses are typically followed by recurrence with lethal chemoresistant disease. In this study, we characterized the apoptotic responses in diverse neuroblastomas using an unbiased mitochondrial functional assay. We defined the apoptotic set-point of neuroblastomas using responses to distinct BH3 death domains providing a BH3 response profile, and directly confirmed survival dependencies. We found that viable neuroblastoma cells and primary tumors are primed for death with tonic sequestration of Bim, a direct activator of apoptosis, by either Bcl-2 or Mcl-1, providing a survival dependency that predicts the activity of Bcl-2 antagonists. The Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-737 showed single agent activity against only Bim:Bcl-2 primed tumor xenografts. Durable complete regressions were achieved in combination with non-curative chemotherapy even for highest-risk molecular subtypes with MYCN amplification and activating ALK mutations. Furthermore, the use of unique isogenic cell lines from patients at diagnosis and at the time of relapse showed that therapy resistance was not mediated by upregulation of Bcl-2 homologues or loss of Bim priming, but by repressed Bak/Bax activation. Together, our findings provide a classification system that identifies tumors with clinical responses to Bcl-2 antagonists, defines Mcl-1 as the principal mediator of Bcl-2 antagonist resistance at diagnosis, and isolates the therapy resistant phenotype to the mitochondria.
doi:10.1158/0008-5472.CAN-11-3603
PMCID: PMC3354953  PMID: 22589275
Bcl-2 homology proteins; mitochondrial profiling; animal models; Bcl-2 antagonist
8.  Germline genetic variation and treatment response on CCG-1891 
Pediatric blood & cancer  2011;58(5):695-700.
Background
Recent studies suggest that polymorphisms in genes encoding enzymes involved in drug detoxification and metabolism may influence disease outcome in pediatric acute lymphoblastic leukemia (ALL). We sought to extend current knowledge by using standard and novel statistical methodology to examine polymorphic variants of genes and relapse risk, toxicity, and drug dose delivery in standard risk ALL.
Procedure
We genotyped and abstracted chemotherapy drug dose data from treatment roadmaps on 557 patients on the Children’s Cancer Group ALL study, CCG-1891. Fourteen common polymorphisms in genes involved in folate metabolism and/or phase I and II drug detoxification were evaluated individually and clique-finding methodology was employed for detection of significant gene-gene interactions.
Results
After controlling for known risk factors, polymorphisms in four genes: GSTP1*B (HR=1.94, p=0.047), MTHFR (HR=1.61, p=0.034), MTRR (HR=1.95, p=0.01), and TS (3R/4R, HR=3.69, p=0.007), were found to significantly increase relapse risk. One gene-gene pair, MTRR A/G and GSTM1 null genotype, significantly increased the risk of relapse after correction for multiple comparisons (p=0.012). Multiple polymorphisms were associated with various toxicities and there was no significant difference in dose of chemotherapy delivered by genotypes.
Conclusions
These data suggest that various polymorphisms play a role in relapse risk and toxicity during childhood ALL therapy and that genotype does not play a role in adjustment of drug dose administered. Additionally, gene-gene interactions may increase the risk of relapse in childhood ALL and the clique method may have utility in further exploring these interactions. childhood ALL therapy.
doi:10.1002/pbc.23192
PMCID: PMC3165089  PMID: 21618417
genotype; acute lymphoblastic leukemia; prognosis; toxicity; gene-gene interactions
9.  Differential inhibitor sensitivity of anaplastic lymphoma kinase variants found in neuroblastoma 
Science Translational Medicine  2011;3(108):108ra114.
Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation of cell lines expressing R1275Q-mutated ALK and a cell line with amplified and overexpressed wild-type ALK. By contrast, cell lines harboring F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition results from an increased ATP-binding affinity (as also seen in acquired resistance to EGFR inhibitors), and should be surmountable with higher doses of crizotinib and/or with higher affinity inhibitors.
doi:10.1126/scitranslmed.3002950
PMCID: PMC3319004  PMID: 22072639
10.  Interobserver reproducibility of electroencephalogram interpretation in critically ill children 
Purpose
Correct outcome prediction after cardiac arrest in children may improve clinical decision making and family counseling. Various investigators have used EEG to predict outcome with varying success, but one limiting issue is the potential lack of reproducibility of EEG interpretation. Therefore, we aimed to evaluate interobserver agreement using standardized terminology in the interpretation of EEG tracings obtained from critically ill children following cardiac arrest.
Methods
3 pediatric neurophysiologists scored 74 EEG samples using standardized categories, terminology, and interpretation rules. Interobserver agreement was evaluated using kappa and intra-class correlation coefficients.
Results
Agreement was substantial for the categories of continuity, burst suppression, sleep architecture, and overall rating. Agreement was moderate for seizure occurrence and inter-ictal epileptiform discharge type. Agreement was fair for inter-ictal epileptiform discharge presence, beta activity, predominant frequency, and fastest frequency. Agreement was slight for maximum voltage and focal slowing presence.
Conclusions
The variability of inter-rater agreement suggests that some EEG features are superior to others for use in a predictive algorithm. Using only reproducible EEG features is needed to ensure the most accurate and consistent predictions. Since even seizure identification had only moderate agreement, studies of non-convulsive seizures in critically ill patients must be conducted and interpreted cautiously.
doi:10.1097/WNP.0b013e3182051123
PMCID: PMC3107383  PMID: 21221016
Electroencephalogram; Interobserver variability; Seizure; Pediatric; Hypoxic Ischemic Encephalopathy; Cardiac Arrest
11.  Lestaurtinib Enhances the Anti-tumor Efficacy of Chemotherapy in Murine Xenograft Models of Neuroblastoma 
Purpose
Neuroblastoma, a common pediatric tumor of the sympathetic nervous system, is characterized by clinical heterogeneity, and the Trk family neurotrophin receptors play an important role in this behavior. Expression of TrkA is associated with favorable clinical features and outcome, whereas TrkB expression is associated with an unfavorable prognosis. We wanted to determine if the Trk-selective inhibitor Lestaurtinib had therapeutic efficacy in a preclinical neuroblastoma model.
Experimental Design
We performed intervention trials of Lestaurtinib alone or in combination with other agents in TrkB-overexpressing neuroblastoma xenograft models.
Results
Lestaurtinib alone significantly inhibited tumor growth compared to vehicle-treated animals (p=0.0004 for tumor size, p=0.011 for EFS). Lestaurtinib also enhanced the anti-tumor efficacy of the combinations of topotecan plus cyclophosphamide (p<0.0001 for size, p<0.0001 for EFS), or irinotecan plus temozolomide (p=0.011 for size; p=0.012 for EFS). There was no additive benefit of combining either 13-cis-retinoic acid or fenretinide with Lestaurtinib compared to Lestaurtinib alone. There was dramatic growth inhibition combining Lestaurtinib with Bevacizumab (p<0.0001), but this combination had substantial systemic toxicity.
Conclusions
We show that Lestaurtinib can inhibit growth of neuroblastoma both in vitro and in vivo, and it can substantially enhance the efficacy of conventional chemotherapy, presumably by inhibition of the Trk/BDNF autocrine survival pathway. It may also enhance the efficacy of selected biological agents, but further testing is required to rule out unanticipated toxicities. Our data support the incorporation of Trk inhibitors like Lestaurtinib in clinical trials of neuroblastoma or other tumors relying on Trk signaling pathways for survival.
doi:10.1158/1078-0432.CCR-09-1531
PMCID: PMC2831131  PMID: 20179224
TrkA; TrkB; Lestaurtinib; CEP-701; neuroblastoma; signaling; differentiation
12.  Biological significance of EPHA2 expression in neuroblastoma 
International journal of oncology  2009;35(4):845-850.
Neuroblastoma is a pediatric solid tumor that exhibits striking clinical bipolarity. Despite extensive efforts to treat unfavorable neuroblastoma, survival rate of children with the disease is among the lowest. Previous studies suggest that EPHA2, a member of the EPH family receptor kinases, can either promote or suppress cancer cell growth depending on cellular contexts. In this study, we investigated the biological significance of EPHA2 in neuroblastoma. It was found that tumorigenic N-type neuroblastoma cell lines expressed low levels of EPHA2, whereas hypo-tumorigenic S-type neuroblastoma cell lines expressed high levels of EPHA2 (p<0.005). Notably, inhibitors of DNA methylation and histone deacetylase enhanced EPHA2 expression in N-type cells, suggesting that EPHA2 is epigenetically silenced in unfavorable neuroblastoma cells. Furthermore, ectopic high-level expression of EPHA2 in N-type neuroblastoma cell lines resulted in significant growth suppression. However, Kaplan-Meier survival analysis showed that high EPHA2 expression was not associated with a good disease outcome of neuroblastoma, indicating that EPHA2 is not a favorable neuroblastoma gene, but a growth suppressive gene for neuroblastoma. Accordingly, EPHA2 expression was markedly augmented in vitro in neuroblastoma cells treated with doxorubicin, which is commonly used for treating unfavorable neuroblastoma. Taken together, EPHA2 is one of the effectors of chemotherapeutic agents (e.g., gene silencing inhibitors and DNA damaging agents). EPHA2 expression may thus serve as a biomarker of drug responsiveness for neuroblastoma during the course of chemotherapy. In addition, pharmaceutical enhancement of EPHA2 by non-cytotoxic agents may offer an effective therapeutic approach in the treatment of children with unfavorable neuroblastoma.
PMCID: PMC2827331  PMID: 19724921
neuroblastoma; favorable neuroblastoma genes; drug responsiveness; biomarkers
13.  ODC1 is a critical determinant of MYCN oncogenesis and a therapeutic target in neuroblastoma 
Cancer research  2008;68(23):9735-9745.
Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have co-ordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the rate-limiting enzyme in polyamine biosynthesis, when compared with lower risk tumors, suggesting this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor α-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically-engineered mice (TH-MYCN GEM) extended tumor latency and survival in homozygous mice, and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments anti-tumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and demonstrates initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors.
doi:10.1158/0008-5472.CAN-07-6866
PMCID: PMC2596661  PMID: 19047152
Embryonal tumors; metabolomics; polyamines; oncogene; experimental therapeutics
14.  Detection of Full-Length and Truncated Neurokinin-1 Receptor mRNA Expression in Human Brain Regions 
Journal of neuroscience methods  2007;168(1):127-133.
We have applied a newly developed SYBR green based real-time RT-PCR assay for quantification of full-length and truncated neurokinin-1 receptor (NK1R) mRNA expression in 9 regions of human brain tissues obtained from 23 subjects who died with no evidence of neurological or neurodegenerative disease. The following brain regions were examined: cingulate cortex, cerebellum, nucleus accumbens, caudate nucleus, putamen, pons, hippocampus, locus coeruleus, and basal ganglia. The SYBR green based-real-time PCR was more sensitive than TaqMan probe based real-time PCR in amplifying both full-length and truncated NK1R mRNA. The real-time RT-PCR assay had excellent specificity and sensitivity, with a dynamic range of detection between 100 and 1000,000 copies of the NK1R cDNA per reaction. The truncated NK1R mRNA levels were more abundant than those of the full-length NK1R in most of the regions examined and there was no significant difference in the truncated NK1R mRNA levels among the nine regions studied. There was, however, a significant difference in the expression of full-length NK1R mRNA levels among the nine regions (P=0.0024), and the putamen region expressed the highest full-length NK1R mRNA. Further studies are needed in order to examine the differences between full-length and truncated NK1R in signal transduction and functional consequences in order to delineate the significance of the copresence of the two forms of NK1R in the human brain.
doi:10.1016/j.jneumeth.2007.10.004
PMCID: PMC2243260  PMID: 18035424
15.  CHD5, a Tumor Suppressor Gene Deleted From 1p36.31 in Neuroblastomas 
Background
Neuroblastomas are characterized by hemizygous 1p deletions, suggesting that a tumor suppressor gene resides in this region. We previously mapped the smallest region of consistent deletion to a 2-Mb region of 1p36.31 that encodes 23 genes. Based on mutation analysis, expression pattern, and putative function, we identified CHD5 as the best tumor suppressor gene candidate.
Methods
We determined the methylation status of the CHD5 gene promoter in NLF and IMR5 (with 1p deletion) and SK-N-SH and SK-N-FI neuroblastoma cell lines using methylation-specific sequencing and measured CHD5 mRNA expression by reverse transcription polymerase chain reaction in cells treated with or without 5-aza-2-deoxycytidine, an inhibitor of DNA methylation. We transfected the cells with CHD5 and antisense (AS) CHD5 DNA to assess the effect of CHD5 overexpression and suppression, respectively, on colony formation in soft agar and growth of xenograft tumors in athymic mice. We also analyzed the association of CDH5 expression with outcomes of 99 neuroblastoma patients. Statistical tests were two-sided.
Results
CHD5 expression was very low or absent in neuroblastoma cell lines. The CHD5 promoter was highly methylated in NLF and IMR5 lines, and CHD5 expression increased after treatment with 5-aza-2-deoxycytidine. Clonogenicity and tumor growth were abrogated in NLF and IMR5 cells overexpressing CHD5 compared with antisense CHD5 (clonogenicity: mean no. of colonies per plate, NLF-CHD5, 43 colonies, 95% confidence interval [CI] = 35 to 51 colonies, vs NLF-CHD5-AS, 74 colonies, 95% CI = 62 to 86 colonies, P < .001; IMR5-CHD5, 11 colonies, 95% CI = 2 to 20 colonies, vs IMR5-CHD5-AS, 39 colonies, 95% CI = 17 to 60 colonies, P = .01; tumor growth, n = 10 mice per group: mean tumor size at 5 weeks, NLF-CHD5, 0.36 cm3, 95% CI = 0.17 to 0.44 cm3, vs NLF-CHD5-AS, 1.65 cm3, 95% CI = 0.83 to 2.46 cm3, P = .002; IMR5-CHD5, 0.28 cm3, 95% CI = 0.18 to 0.38 cm3, vs IMR5-CHD5-AS, 1.15 cm3, 95% CI = 0.43 to 1.87 cm3; P = .01). High CHD5 expression was strongly associated with favorable event-free and overall survival (P < .001), even after correction for MYCN amplification and 1p deletion (P = .027).
Conclusions
CHD5 is the strongest candidate tumor suppressor gene that is deleted from 1p36.31 in neuroblastomas, and inactivation of the second allele may occur by an epigenetic mechanism.
doi:10.1093/jnci/djn176
PMCID: PMC2483574  PMID: 18577749
16.  Disparities in the Reporting and Treatment of Health Conditions in Children: An Analysis of the Medical Expenditure Panel Survey 
Health Services Research  2006;41(2):532-549.
Objectives
To determine whether racial and ethnic disparities in health care use differ for physical and behavioral health conditions.
Data Sources
Secondary analysis of the 1996–1997 Medical Expenditure Panel Survey.
Study Design
Retrospective cohort study of children aged 2–18 years old who were members of participating households. Children were categorized as Hispanic, black, or white. Differences in caregiver-reported behavioral and physical health conditions and services use were compared, and estimates were weighted to reflect the complex sampling scheme.
Principal Findings
Of eligible children weighted to represent over 44 million in each year, 13–15 percent were Hispanic, 14 percent black, and 68–70 percent white. After adjusting for potential confounding, Hispanic and black children were less likely to report externalizing behavioral conditions than white children. Black but not Hispanic children were more likely than white children to report asthma. In addition, Hispanic and black children were less likely to report ambulatory visits, and black children were less likely to report receiving a prescription medication than white children. There were no differences in reported emergency room visits or hospitalizations. Interactions between race and various health conditions, health status, insurance, and income were not significant.
Conclusions
In this nationally representative sample, we identified racial and ethnic disparities in the reporting of health conditions and the use of discretionary health services. Disparities differed between those with behavioral conditions and those with physical conditions. These disparities were not explained by traditional measures including the presence of health conditions, health status, insurance, and family income, and suggest that national surveys such as Medical Expenditure Panel Survey may benefit from the inclusion of additional explanatory measures.
doi:10.1111/j.1475-6773.2005.00484.x
PMCID: PMC1702509  PMID: 16584463
Disparities; mental health disorders; health care services; children
17.  Correlates of Behavioral Care Management Strategies used by Primary Care Pediatric Providers 
Objective
To identify correlates of behavioral management strategies and to test whether children with more severe behavioral problems have care transferred to mental health specialists.
Methods
Secondary analysis of the Child Behavior Study. Children ages 4 to 15 years old were identified with new behavioral problems at non-urgent visits to primary care clinicians. Treatment strategies were categorized into mutually exclusive groups: primary care (psychotropic prescription and/or office-based counseling), mental healthcare (referral for or ongoing specialist mental healthcare), joint care (primary care and mental healthcare) or observation. Child-, family-, clinician-, and practice-level characteristics were assessed for association with management strategies using multivariate methods.
Results
A total of 1377 children from 201 practices in 44 states and Puerto Rico were newly identified with behavioral problems. Behavioral/conduct (41%), attentional/hyperactivity (37%), adjustment (32%), and emotional (22%) problems were most commonly identified. Children with comorbid behavioral problems were more likely to be managed with joint care than other treatment strategies. In addition, clinicians who were male or who had greater mental health orientation were more likely to provide joint care than mental healthcare only.
Conclusions
Clinicians were more likely to manage new behavioral problems jointly with mental health providers than use other strategies if children had coexisting mental health problems or if providers had stronger beliefs about psychosocial aspects of care. These results do not support the hypothesis that children with more severe behavioral problems are transferred to specialists but suggest that primary care and mental healthcare clinicians may benefit from collaborating on treatment plans.
doi:10.1016/j.ambp.2006.12.006
PMCID: PMC1832082  PMID: 17368411
mental health; child; decision making; therapeutics

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