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1.  Outcome of Children With Metastatic Medulloblastoma Treated With Carboplatin During Craniospinal Radiotherapy: A Children's Oncology Group Phase I/II Study 
Journal of Clinical Oncology  2012;30(21):2648-2653.
We evaluated the feasibility of administering carboplatin as a radiosensitizer during craniospinal radiation therapy (CSRT) to patients with high-risk medulloblastomas (MBs) and supratentorial primitive neuroectodermal tumors, and we report the outcome in the subset with metastatic (M+) MB.
Patients and Methods
After surgery, patients received 36 Gy CSRT with boosts to sites of disease. During radiation, patients received 15 to 30 doses of carboplatin (30-45 mg/m2/dose), along with vincristine (VCR) once per week for 6 weeks. Patients on regimen A received 6 months of maintenance chemotherapy (MC) with cyclophosphamide and VCR. Once the recommended phase II dose (RP2D) of carboplatin was determined, cisplatin was added to the MC (regimen B).
In all, 161 eligible patients (median age, 8.7 years; range, 3.1 to 21.6 years) were enrolled. Myelosuppression was dose limiting and 35 mg/m2/dose × 30 was determined to be the RP2D of carboplatin. Twenty-nine (36%) of 81 patients with M+ MB had diffuse anaplasia. Four patients were taken off study within 11 months of completing radiotherapy for presumed metastatic progression and are long-term survivors following palliative chemotherapy. Excluding these four patients, 5-year overall survival ± SE and progression-free survival ± SE for M+ patients treated at the RP2D on regimen A was 82% ± 9% and 71% ± 11% versus 68% ± 10% and 59% ± 10% on regimen B (P = .36). There was no difference in survival by M stage. Anaplasia was a negative predictor of outcome.
The use of carboplatin as a radiosensitizer is a promising strategy for patients with M+ MB. Early progression should be confirmed by biopsy.
PMCID: PMC4559602  PMID: 22665539
2.  Phase I and Pharmacokinetic Trial of PTC299 in Pediatric Patients with Refractory or Recurrent Central Nervous System Tumors: a PBTC Study 
Journal of neuro-oncology  2014;121(1):217-224.
PTC299 is a novel, orally-bioavailable small molecule that selectively inhibits vascular endothelial growth factor receptor protein synthesis at the post-transcriptional level. Based on promising preclinical results, we conducted a pediatric phase I study to estimate the maximum tolerated dose (MTD), describe dose-limiting toxicities (DLT) and characterize the pharmacokinetic profile of PTC299 in children with recurrent CNS tumors.
Patients and Methods
PTC299 was administered orally twice or three times daily, depending on the regimen. Four regimens were evaluated using the rolling 6 design, starting with 1.2 mg/kg/dose twice daily and escalating to 2 mg/kg/dose three times daily. Pharmacokinetic studies were performed during the first two courses.
Twenty-seven children (14 male, median age 11.2, range 5.5–21 years) with recurrent brain tumors were treated; 21 were fully evaluable for toxicity assessment. Therapy was well-tolerated, and the only DLT was grade 3 hyponatremia. Grade three and grade four toxicities were uncommon in subsequent cycles. Median AUC0–Tlast values at the 2 mg/kg were similar to those observed in adults. The study was terminated while patients were being treated at the highest planned dose, due to hepatotoxicity encountered in the ongoing adult phase I studies. No complete or partial responses were observed. Two patients with low-grade gliomas were noted to have minor responses, and at the time of the study’s closure, 5 children with low-grade gliomas had been on therapy for 8 or more courses (range 8–16).
PTC299 was well-tolerated at the highest dose level tested (2 mg/kg/dose TID) in children with recurrent brain tumors and prolonged disease stabilization was seen in children with low-grade gliomas.
PMCID: PMC4330963  PMID: 25407389
pediatric brain tumors; antiangiogenic agents; low-grade gliomas; biologic therapy; gliomas
3.  The impact of molecular analysis on the survival of children with embryonal tumors 
Translational Pediatrics  2016;5(1):5-8.
Embryonal tumors represent a heterogeneous group of malignancies characterized by poorly differentiated cells and generally aggressive behavior. Although advances in survival rates have been made in several of these tumor types, including Wilms’ tumor, retinoblastoma, and medulloblastoma, survival of patients with central nervous system (CNS) embryonal tumors, including primitive neuro-ectodermal tumors (PNETs) and atypical teratoid rhabdoid tumors (AT/RT), are particularly poor. Advancing molecular analysis techniques and the development of gene expression profiles has led to the formulation of different subdivisions within many of the umbrella CNS tumor groups with clinical and prognostic implications. Some subgroups have been identified as having improved survivorships, likely not captured by large scale population data given their small numbers and relatively recent characterization. Importantly, identification of differing molecular pathways has begun to result in targeted therapies which may pave the way for even more surviving patients in the coming years.
PMCID: PMC4729043  PMID: 26835399
Embryonal tumors; pediatrics; molecular profiling
4.  Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma 
Nature Communications  2016;7:11185.
Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly paediatric brain tumours where needle biopsies help guide diagnosis and targeted therapies. To address spatial heterogeneity, here we analyse 134 specimens from various neuroanatomical structures of whole autopsy brains from nine DIPG patients. Evolutionary reconstruction indicates histone 3 (H3) K27M—including H3.2K27M—mutations potentially arise first and are invariably associated with specific, high-fidelity obligate partners throughout the tumour and its spread, from diagnosis to end-stage disease, suggesting mutual need for tumorigenesis. These H3K27M ubiquitously-associated mutations involve alterations in TP53 cell-cycle (TP53/PPM1D) or specific growth factor pathways (ACVR1/PIK3R1). Later oncogenic alterations arise in sub-clones and often affect the PI3K pathway. Our findings are consistent with early tumour spread outside the brainstem including the cerebrum. The spatial and temporal homogeneity of main driver mutations in DIPG implies they will be captured by limited biopsies and emphasizes the need to develop therapies specifically targeting obligate oncohistone partnerships.
Diffuse Intrinsic Pontine Gliomas are diagnosed by sampling a small portion of the tumour. Here, using multiple samples from tumours, the authors analyse the spatial and temporal distribution of driver mutations revealing that H3K27M mutations arise first in tumorigenesis followed by a specific invariable sequence of driver mutations, which are homogeneously distributed across the tumour mass.
PMCID: PMC4823825  PMID: 27048880
5.  Sirolimus for progressive neurofibromatosis type 1–associated plexiform neurofibromas: a Neurofibromatosis Clinical Trials Consortium phase II study 
Neuro-Oncology  2014;17(4):596-603.
Plexiform neurofibromas (PNs) are benign peripheral nerve sheath tumors that arise in one-third of individuals with neurofibromatosis type 1 (NF1). They may cause significant disfigurement, compression of vital structures, neurologic dysfunction, and/or pain. Currently, the only effective management strategy is surgical resection. Converging evidence has demonstrated that the NF1 tumor suppressor protein, neurofibromin, negatively regulates activity in the mammalian Target of Rapamycin pathway.
We employed a 2-strata clinical trial design. Stratum 1 included subjects with inoperable, NF1-associated progressive PN and sought to determine whether sirolimus safely and tolerably increases time to progression (TTP). Volumetric MRI analysis conducted at regular intervals was used to determine TTP relative to baseline imaging.
The estimated median TTP of subjects receiving sirolimus was 15.4 months (95% CI:
14.3–23.7 mo), which was significantly longer than 11.9 months (P < .001), the median TTP of the placebo arm of a previous PN clinical trial with similar eligibility criteria.
This study demonstrated that sirolimus prolongs TTP by almost 4 months in patients with NF1-associated progressive PN. Although the improvement in TTP is modest, given the lack of significant or frequent toxicity and the availability of few other treatment options, the use of sirolimus to slow the growth of progressive PN could be considered in select patients.
PMCID: PMC4483073  PMID: 25314964
neurofibromatosis; NF1; plexiform neurofibroma; rapamycin; sirolimus; mTOR
6.  The Role of NG2 Proteoglycan in Glioma 
Translational Oncology  2016;9(1):57-63.
Neuron glia antigen-2 ((NG2), also known as chondroitin sulphate proteoglycan 4, or melanoma-associated chondroitin sulfate proteoglycan) is a type-1 membrane protein expressed by many central nervous system (CNS) cells during development and differentiation and plays a critical role in proliferation and angiogenesis. ‘NG2’ often references either the protein itself or the highly proliferative and undifferentiated glial cells expressing high levels of NG2 protein. NG2 glia represent the fourth major type of neuroglia in the mammalian nervous system and are classified as oligodendrocyte progenitor cells by virtue of their committed oligodendrocyte generation in developing and adult brain. Here, we discuss NG2 glial cells as well as NG2 protein and its expression and role with regards to CNS neoplasms as well as its potential as a therapeutic target for treating childhood CNS cancers.
PMCID: PMC4800061  PMID: 26947882
7.  Reproducibility of Retinal Nerve Fiber Layer Thickness Measures Using Eye-Tracking with Spectral-Domain Optical Coherence Tomography in Children with Non-Glaucomatous Optic Neuropathy 
American journal of ophthalmology  2014;159(1):71-7.e1.
To determine the intra- and intervisit reproducibility of circumpapillary retinal nerve fiber layer (RNFL) thickness measures using eye-tracking assisted spectral domain optical coherence tomography (SD-OCT) in children with nonglaucomatous optic neuropathy.
Prospective longitudinal study
Circumpapillary RNFL thickness measures were acquired with the Spectralis (Heidelberg Engineering) SD-OCT using the eye-tracking feature at two separate study visits. Children with normal and abnormal vision (visual acuity ≥ 0.2 logMAR above normal and or visual field loss) who demonstrated clinical and radiographic stability were enrolled. Intra- and intervisit reproducibility was calculated for the global average and 9 anatomic sectors by calculating the coefficient of variation and intraclass correlation coefficient.
Forty-two subjects (median age 8.6 years, range 3.9 -18.2 years) met inclusion criteria and contributed 62 study eyes. Both the abnormal and normal vision cohort demonstrated the lowest intravisit coefficient of variation for the global RNFL thickness. Intervisit reproducibility remained good for those with normal and abnormal vision, although small but statistically significant increases in the coefficient of variation were observed for multiple anatomic sectors in both cohorts. The magnitude of visual acuity loss was significantly associated with the global (β = 0.026, P < .01) and temporal sector coefficient of variation (β = 0.099, P < .01).
SD-OCT with eye tracking demonstrates highly reproducible RNFL thickness measures. Subjects with vision loss demonstrate greater intra- and intervisit variability than those with normal vision.
PMCID: PMC4455020  PMID: 25256068
8.  Cytogenetic Prognostication Within Medulloblastoma Subgroups 
Shih, David J.H. | Northcott, Paul A. | Remke, Marc | Korshunov, Andrey | Ramaswamy, Vijay | Kool, Marcel | Luu, Betty | Yao, Yuan | Wang, Xin | Dubuc, Adrian M. | Garzia, Livia | Peacock, John | Mack, Stephen C. | Wu, Xiaochong | Rolider, Adi | Morrissy, A. Sorana | Cavalli, Florence M.G. | Jones, David T.W. | Zitterbart, Karel | Faria, Claudia C. | Schüller, Ulrich | Kren, Leos | Kumabe, Toshihiro | Tominaga, Teiji | Shin Ra, Young | Garami, Miklós | Hauser, Peter | Chan, Jennifer A. | Robinson, Shenandoah | Bognár, László | Klekner, Almos | Saad, Ali G. | Liau, Linda M. | Albrecht, Steffen | Fontebasso, Adam | Cinalli, Giuseppe | De Antonellis, Pasqualino | Zollo, Massimo | Cooper, Michael K. | Thompson, Reid C. | Bailey, Simon | Lindsey, Janet C. | Di Rocco, Concezio | Massimi, Luca | Michiels, Erna M.C. | Scherer, Stephen W. | Phillips, Joanna J. | Gupta, Nalin | Fan, Xing | Muraszko, Karin M. | Vibhakar, Rajeev | Eberhart, Charles G. | Fouladi, Maryam | Lach, Boleslaw | Jung, Shin | Wechsler-Reya, Robert J. | Fèvre-Montange, Michelle | Jouvet, Anne | Jabado, Nada | Pollack, Ian F. | Weiss, William A. | Lee, Ji-Yeoun | Cho, Byung-Kyu | Kim, Seung-Ki | Wang, Kyu-Chang | Leonard, Jeffrey R. | Rubin, Joshua B. | de Torres, Carmen | Lavarino, Cinzia | Mora, Jaume | Cho, Yoon-Jae | Tabori, Uri | Olson, James M. | Gajjar, Amar | Packer, Roger J. | Rutkowski, Stefan | Pomeroy, Scott L. | French, Pim J. | Kloosterhof, Nanne K. | Kros, Johan M. | Van Meir, Erwin G. | Clifford, Steven C. | Bourdeaut, Franck | Delattre, Olivier | Doz, François F. | Hawkins, Cynthia E. | Malkin, David | Grajkowska, Wieslawa A. | Perek-Polnik, Marta | Bouffet, Eric | Rutka, James T. | Pfister, Stefan M. | Taylor, Michael D.
Journal of Clinical Oncology  2014;32(9):886-896.
Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication.
Patients and Methods
Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models.
Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas.
Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.
PMCID: PMC3948094  PMID: 24493713
9.  Phase I Trial of Weekly MK-0752 in Children with Refractory Central Nervous System Malignancies: A Pediatric Brain Tumor Consortium Study 
Amplification and high-levels of NOTCH ligand expression have been identified in several types of pediatric brain tumors. A phase I trial of weekly MK-0752, an oral inhibitor of gamma-secretase, was conducted in children with recurrent central nervous system (CNS) malignancies to estimate the maximum tolerated dose, dose-limiting toxicities (DLT), pharmacokinetics (PK), and pharmacodynamics of weekly MK-0752.
MK-0752 was administered once weekly at 1,000 mg/m2 and 1,400 mg/m2 using a rolling-6 design. PK analysis was performed during the first course. NOTCH and HES expression was assessed by immunohistochemistry and Western blot.
Ten eligible patients were enrolled (median age 8.8 years; range 3.1–19.2) with diagnoses of brain stem glioma (n=3), ependymoma (n=2), anaplastic astrocytoma (n=1), choroid plexus carcinoma (n=2), medulloblastoma (n=1), and primitive neuroectodermal tumor (n=1). Nine were evaluable for toxicity. One DLT of fatigue occurred in the 6 evaluable patients enrolled at 1,000 mg/m2/dose. No DLTs were experienced by 3 patients treated at 1,400 mg/m2/dose. Non-dose-limiting grade 3 toxicities included lymphopenia, neutropenia, and anemia. Median number of treatment courses was 2 (range 1–10). Two patients continued on therapy for at least 6 months. The median (range) Cmax of MK-0752 was 88.2 μg/mL (40.6 to 109 μg/mL) and 60.3 μg/mL (59.2 to 91.9 μg/mL) in patients receiving 1,000 mg/m2/week and 1,400 mg/m2/week, respectively. NOTCH expression was decreased in 6 of 7 patients for whom tissue was available at 24 hours post-MK-0752.
MK-0752 is well-tolerated and exhibits target inhibition at 1,000 mg/m2/week and 1,400 mg/m2/week in children with recurrent CNS malignancies.
PMCID: PMC4681692  PMID: 25930724
Notch; recurrent; brain tumor; pediatric
10.  Phase 2 study of safety and efficacy of nimotuzumab in pediatric patients with progressive diffuse intrinsic pontine glioma 
Neuro-Oncology  2014;16(11):1554-1559.
The prognosis of diffuse intrinsic pontine glioma (DIPG) remains poor, with no drug proven to be effective.
Patients with clinically and radiologically confirmed, centrally reviewed DIPG, who had failed standard first-line therapy were eligible for this multicenter phase II trial. The anti-epidermal growth factor receptor (EGFR) antibody, nimotuzumab (150 mg/m2), was administered intravenously once weekly from weeks 1 to 7 and once every 2 weeks from weeks 8 to 18. Response evaluation was based on clinical and MRI assessments. Patients with partial response (PR) or stable disease (SD) were allowed to continue nimotuzumab.
Forty-four patients received at least one dose of nimotuzumab (male/female, 20/24; median age, 6.0 years; range, 3.0–17.0 years). All had received prior radiotherapy. Treatment was well tolerated. Eighteen children experienced serious adverse events (SAEs). The majority of SAEs were associated with disease progression. Nineteen patients completed 8 weeks (W8) of treatment: There were 2 PRs, 6 SDs, and 11 progressions. Five patients completed 18 weeks (W18) of treatment: 1 of 2 patients with PR at W8 remained in PR at W18, and 3 of 6 children with SD at W8 maintained SD at W18. Time to progression following initiation of nimotuzumab for the 4 patients with SD or better at W18 was 119, 157, 182 and 335 days, respectively. Median survival time was 3.2 months. Two patients lived 663 and 481 days from the start of nimotuzumab.
Modest activity of nimotuzumab in DIPG, which has been shown previously, was confirmed: A small subset of DIPG patients appeared to benefit from anti-EGFR antibody treatment.
PMCID: PMC4201068  PMID: 24847085
children; diffuse intrinsic pontine glioma; epidermal growth factor receptor; nimotuzumab; phase II trial
Neuro-Oncology  2014;16(Suppl 5):v147-v148.
BACKGROUND: This study reports the targeting and real-time optical imaging of brainstem tumors using liposomal nanocarriers. Patients with infiltrating brainstem gliomas (BSG) known as diffuse intrinsic pontine gliomas (DIPGs) have one of the poorest survival rates. Blood brain barrier (BBB) is the main obstacle in the delivery of drugs and contrast agents to DIPGs. OBJECTIVE: Here, we demonstrate the specific delivery of liposomal nanoparticles containing Evans blue (nano-EB) to tumor in a murine model of BSG. METHODS/DESIGN: Mice with brainstem tumors were tail vein injected with nano-EB and in vivo tumor enhancement was monitored by optical fluorescence imaging. RESULTS/DISCUSSION: Necropsy analysis conducted 24 hr post injection showed site-specific delivery of nano-EB to the tumor but not adjacent normal tissue. Immunohistochemical assays confirmed high grade tumor at the site of nano-EB accumulation. These findings demonstrate the feasibility of nano-EB for drug delivery and real-time, sensitive optical imaging of BSGs in vivo. The study shows the specific accumulation of liposomal nano-EB nanocarriers in brainstem tumors in our murine model of brainstem glioma. Liposomes are an ideal drug delivery vehicle able to cross the BBB . Specific accumulation of nano-EB in tumor cells is explained by the enhanced permeability and retention (EPR) property of cancer cells which is believed to be due to the abnormal neovasculature with poorly-aligned defective endothelial cells and structure. This pilot study shows the efficacy of liposomes for targeted delivery and possibility of using nano-EB for in vivo imaging of tumors in a murine model of brain tumors.
PMCID: PMC4218370
Neuro-Oncology  2014;16(Suppl 5):v173.
BACKGROUND: Diffuse intrinsic pontine glioma is one of the least understood pediatric tumors. We have recently identified two molecular subtypes of DIPGs (sonic hedgehog and N-Myc). Despite the existence of in vitro and in vivo models of the disease, molecular characterization of these models and their proximity to representing the disease has not been established. Here, we have done comparative protein profiling of a number of DIPG primary cells as well as protein profiles of a single specimen and specimens derived from its' in vivo and in vitro models. METHODS: Human primary DIPG (n = 4) and normal human cells derived from hindbrain (n = 2) were processed for protein profiling. Total protein was extracted and analyzed using SDS PAGE, and analyzed using Q Exactive mass spectrometer (ThermoFisher, CA, USA). Data and pathway analysis were done using the SequestHT and Ingeniuty Pathway Analysis software respectively. Protein was also extracted from a single human DIPG specimen, and its' in vitro and in vivo derived cells. Protein profiling of this specimens was performed as described above. RESULTS: In this study, we first investigated the protein profiles of four widely used DIPG primary lines, all H3.3K27M mutant. We show the overlap of the protein profile of these cells with established human DIPG subtypes. We further investigated the protein profile of a single DIPG tissue to its in vitro and in vivo models. We describe the fidelity of each model with respect to the molecular entity of the original tumor. The largest number of proteins were shared between primary tumor and murine model (846 proteins, 44%), where the primary tumor and cultured cells shared 30% (721) proteins. CONCLUSIONS: Established human DIPG cells represent tumor biology at the protein level. However, differences in molecular pathway of in vitro and in vivo models representing the disease exist.
PMCID: PMC4218482
Neuro-Oncology  2014;16(Suppl 5):v205.
Pediatric diffuse intrinsic pontine glioma (DIPG) is one of the most difficult cancers to treat. pLys27Met (K27M) driver mutation in the H3F3A (H3.3) and HIST1H3B (H3.1) genes of histone are correlated with a subgroup of DIPGs. Other genomic aberrations include p53 mutations and amplification of signaling pathways including PDGFRα. We have recently reported the involvement of Hedgehog (Hh) pathway in a subset of DIPGs. Modulation of Hh and tyrosine kinase receptors may alter the self-renewal properties of cancer stem cells (CSC). NG2 Proteoglycan positive cells that co-express PDGFRα and Olig-2 are present in adult gliomas, where NG2 contributes to the neoplastic transformation of glioma cells. We examined NG2 expression in frozen brainstem specimens of DIPGs and observed significant NG2 expression in DIPGs [10 of 14 (71 %), fold change = 33, p < 0.05)] as compared to the adjacent normal tissue. NG2 expression was associated with histone 3 K27M mutation [8 of 10 (80 %)]. Two mechanisms of NG2 regulation in DIPG were identified: i) histone 3 binds to NG2 promoter, and ii) miR 129-2 negatively regulates NG2. We detected downregulation of miR129-2 in 85.7% (6 of 7) of DIPG tumors compared to normal tissue (FC = -30.79, n = 7 pairs). We also found overall hypermethylation at 8 CpG loci corresponding to the miR129-2 promoter. NG2 knockdown in vitro (shRNA, miR129-2 or demethylating drugs) retards cellular migration. Luciferase assay in primary mouse glioma cells co-transfected with 3'UTR of NG2 and miR129-2 revealed regulation of NG2 by miR129-2. This was further confirmed in vivo by injection of NG2-dsRed transgenic mice with mir129-2 lentivirus. Orthotopic injection of NG2+ cells results in rapid tumor formation while NG2-KD cells fail to form tumors. Our study offers a potential model for the expansion of tumor stem cells and their self-renewal properties in DIPGs.
PMCID: PMC4218597
14.  Intra- and Inter-visit Reproducibility of Ganglion Cell – Inner Plexiform Layer Measurements Using Handheld Optical Coherence Tomography in Children with Optic Pathway Gliomas 
American journal of ophthalmology  2014;158(5):916-923.e1.
To determine the intra- and inter-visit reproducibility of ganglion cell-inner plexiform layer thickness measures using handheld optical coherence tomography (OCT) in sedated children with optic pathway gliomas and/or Neurofibromatosis type 1 (NF1).
Prospective longitudinal cohort study
Children with sporadic optic pathway gliomas and/or NF1 who had ≥ 2 volumes acquired over the macula using handheld OCT during sedation for a clinically indicated MRI were eligible for the intra-visit cohort. Children with repeat handheld OCT imaging within 6 months were eligible for the inter-visit cohort. Total retinal thickness and ganglion cell-inner plexiform layer thickness were measured using custom designed automated segmentation software. Reproducibility was compared across average and anatomic quadrant by calculating the coefficient of variation (CV) and intraclass correlation coefficient (ICC).
Forty-two subjects (median age 5.4 years, range 0.8–12.7 years) contributed 45 eyes to the intra-visit cohort. Thirty-one subject eyes had normal vision and 14 had abnormal vision (decreased visual acuity and/or visual field). Average and quadrant ganglion cell-inner plexiform layer measures demonstrated CVs ≤ 4.5% with excellent ICCs (> .935). The superior quadrant CV differed between subjects with (4.4%) and without (2.1%) vision loss (P < 0.05). Twenty-five subject eyes were eligible for the inter-visit cohort, demonstrating CVs from 1.6% to 5.2%. Inter-visit ICCs were excellent (.955 – .995).
Handheld OCT imaging in sedated children with optic pathway gliomas produces highly reproducible measures of ganglion cell-inner plexiform layer thickness.
PMCID: PMC4250433  PMID: 25068639
15.  Tumors of the Central Nervous System: Clinical Aspects, Molecular Mechanisms, Unanswered Questions, and Future Research Directions 
Journal of child neurology  2008;23(10):1103-1121.
Central nervous system tumors are the most common solid tumors in children. Many histological subtypes and biological variants exist. The 2007 Neurobiology of Disease in Children Symposium, held in conjunction with the 36th annual meeting of the Child Neurology Society, aimed to define current knowledge in the field and to develop specific aims for future clinical, translational, and fundamental science. Because of advances in structural and metabolic imaging, surgical technique, and combination therapies, the life expectancy of children with some of the most common tumors, such as cerebellar astrocytomas and medulloblastomas, has improved. Other common tumor types, including diffuse pontine gliomas and malignant embryonal tumors, still have a dismal prognosis. As novel therapies are identified for pediatric central nervous system tumors, long-term survival may be associated with considerable disability. A cooperative effort is crucial to early diagnosis and to translating basic research findings into safe, effective new treatments.
PMCID: PMC3674821  PMID: 18952577
16.  Cumulative Cisplatin Dose Is Not Associated With Event-Free or Overall Survival in Children With Newly Diagnosed Average-Risk Medulloblastoma Treated With Cisplatin Based Adjuvant Chemotherapy: Report From the Children's Oncology Group 
Pediatric blood & cancer  2013;61(1):102-106.
Survival rates for children with medulloblastoma have risen over the past decade, in part due to the addition of cisplatin-containing adjuvant chemotherapy. Total dose of cisplatin required for optimal treatment is unknown. The purpose of this study was to evaluate the survival outcomes based on cumulative cisplatin doses (CCD) in children with newly diagnosed average-risk medulloblastoma.
CCD data were reviewed for 363 patients in a prospective study evaluating patients between 3 and 21 years with a newly diagnosed average-risk medulloblastoma and treated with craniospinal radiation and post-radiation cisplatin based adjuvant chemotherapy.
Eight-year event-free survival (EFS) and overall survival (OS) estimates were 78.2±2.6% and 83.9±2.4%, respectively. Only 73 patients received the protocol specified CCD of 600 mg/m2, primarily due to mandated cisplatin toxicity-related dose reductions. The median CCD given to those without relapse or death on treatment was 487.5 mg/m2. CCD, as a time-dependent covariate, was not associated with EFS (P=0.54) or OS (P=0.11). The 343 patients who completed chemotherapy failure-free were categorized into four groups according to CCD (n=10; 75–150 mg/m2), (n=26; 151–300 mg/m2), (n 113; 301–450 mg/m2), and (n=194; 451–600 mg/m2). There were no statistically significant differences in distributions of EFS (P=0.53) or OS (P=0.49) among these four groups.
CCD is not associated with EFS or OS suggesting that lower doses of cisplatin may be incorporated into future medulloblastoma trials, thereby limiting its toxicity profile without affecting survival. If ototoxicity is encountered, more stringent cisplatin dose modification/cessation rules seem warranted.
PMCID: PMC4591537  PMID: 23956184
average-risk medulloblastoma; cumulative cisplatin dose; survival outcomes
17.  Reproducibility of Circumpapillary Retinal Nerve Fiber Layer Measurements Using Handheld Optical Coherence Tomography in Sedated Children 
American journal of ophthalmology  2014;158(4):780-787.e1.
To determine the intra- and intervisit reproducibility of circumpapillary retinal nerve fiber layer (RNFL) measures using handheld optical coherence tomography (OCT) in sedated children.
Prospective cross-sectional and longitudinal study
Children undergoing sedation for a clinically indicated MRI for an optic pathway glioma and or Neurofibromatosis type 1 (NF1) had multiple 6 × 6 mm volumes (isotropic 300×300 or non-isotropic 1000×100 samplings) acquired over the optic nerve. Children with two handheld OCT sessions within 6 months were included in the intervisit cohort. The intra- and inter-visit coefficient of variation (CV) and intraclass correlation coefficient (ICC) were calculated for the average and anatomic quadrant circumpapillary RNFL thickness.
Fifty-nine subjects (mean age 5.1 years, range 0.8–13.0 years) comprised the intravisit cohort and 29 subjects (mean age 5.7 years, range 1.8–12.7 years) contributed to the intervisit cohort. Forty-nine subjects had an optic pathway glioma and 10 subjects had NF1 without an optic pathway glioma. The CV was comparable regardless of imaging with an isotropic and non-isotropic volume in both the intra- and intervisit cohorts. The average circumpapillary RNFL demonstrated the lowest CV and highest ICC compared to the quadrants. For the intervisit cohort, the average ICC was typically higher while the CV was typically lower, but not statistically different compared to the other quadrants.
Circumpapillary RNFL measures acquired with handheld OCT during sedation demonstrate good intra- and intervisit reproducibility. Handheld OCT has the potential to monitor progressive optic neuropathies in young children who have difficulty cooperating with traditional OCT devices.
PMCID: PMC4165732  PMID: 24983792
18.  Proteomic profiling of high risk medulloblastoma reveals functional biology 
Oncotarget  2015;6(16):14584-14595.
Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior.
PMCID: PMC4546489  PMID: 25970789
medulloblastoma; proteomics; cancer; cMYC; glycolysis
19.  Survival and secondary tumors in children with medulloblastoma receiving radiotherapy and adjuvant chemotherapy: results of Children's Oncology Group trial A9961 
Neuro-Oncology  2012;15(1):97-103.
The purpose of the trial was to determine the survival and incidence of secondary tumors in children with medulloblastoma receiving radiotherapy plus chemotherapy. Three hundred seventy-nine eligible patients with nondisseminated medulloblastoma between the ages of 3 and 21 years were treated with 2340 cGy of craniospinal and 5580 cGy of posterior fossa irradiation. Patients were randomized between postradiation cisplatin and vincristine plus either CCNU or cyclophosphamide. Survival, pattern of relapse, and occurrence of secondary tumors were assessed. Five- and 10-year event-free survivals were 81 ± 2% and 75.8 ± 2.3%; overall survivals were 87 ± 1.8% and 81.3 ± 2.1%. Event-free survival was not impacted by chemotherapeutic regimen, sex, race, age at diagnosis, or gender. Seven patients had disease relapse beyond 5 years after diagnosis; relapse was local in 4 patients, local plus supratentorial in 2, and supratentorial alone in 1. Fifteen patients experienced secondary tumors as a first event at a median time of 5.8 years after diagnosis (11 >5 y postdiagnosis). All non-CNS solid secondary tumors (4) occurred in regions that had received radiation. Of the 6 high-grade gliomas, 5 occurred >5 years postdiagnosis. The estimated cumulative 10-year incidence rate of secondary malignancies was 4.2% (1.9%–6.5%). Few patients with medulloblastoma will relapse ≥5 years postdiagnosis; relapse will occur predominantly at the primary tumor site. Patients are at risk for development of secondary tumors, many of which are malignant gliomas. This may become an increasing issue as more children survive.
PMCID: PMC3534419  PMID: 23099653
chemotherapy; medulloblastoma; radiotherapy; secondary tumors
20.  Comparative Multidimensional Molecular Analyses of Pediatric Diffuse Intrinsic Pontine Glioma Reveals Distinct Molecular Subtypes 
Acta neuropathologica  2013;127(6):881-895.
Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Here, we present the first comprehensive protein, mRNA, and methylation profiles of fresh frozen DIPG specimens (n=14), normal brain tissue (n=10), and other pediatric brain tumors (n=17). Protein profiling identified 2,305 unique proteins indicating distinct DIPG protein expression patterns compared to other pediatric brain tumors. Western blot and immunohistochemistry validated upregulation of Clusterin (CLU), Elongation Factor 2 (EF2), and Talin-1 (TLN1) in DIPGs studied. Comparisons to mRNA expression profiles generated from tumor and adjacent normal brain tissue indicated two DIPG subgroups, characterized by upregulation of Myc (N-Myc) or Hedgehog (Hh) signaling. We validated upregulation of PTCH, a membrane receptor in the Hh signaling pathway, in a subgroup of DIPG specimens. DNA methylation analysis indicated global hypomethylation of DIPG compared to adjacent normal tissue specimens, with differential methylation of 24 genes involved in Hh and Myc pathways, correlating with protein and mRNA expression patterns. Sequencing analysis showed c.83A>T mutations in the H3F3A or HIST1H3B gene in 77% of our DIPG cohort. Supervised analysis revealed a unique methylation pattern in mutated specimens compared to the wild type DIPG samples.
This study presents the first comprehensive multidimensional protein, mRNA, and methylation profiling of pediatric brain tumor specimens, detecting the presence of two subgroups within our DIPG cohort. This multidimensional analysis of DIPG provides increased analytical power to more fully explore molecular signatures of DIPGs, with implications for evaluating potential molecular subtypes and biomarker discovery for assessing response to therapy.
PMCID: PMC4028366  PMID: 24297113
Diffuse Intrinsic Pontine Glioma (DIPG); Brainstem Glioma; Proteomics; Histone H3; Myc Oncogene; Hedgehog
21.  Handheld Optical Coherence Tomography During Sedation in Young Children With Optic Pathway Gliomas 
JAMA ophthalmology  2014;132(3):265-271.
Monitoring young children with optic pathway gliomas (OPGs) for visual deterioration can be difficult owing to age-related noncompliance. Optical coherence tomography (OCT) measures of retinal nerve fiber layer (RNFL) thickness have been proposed as a surrogate marker of vision but this technique is also limited by patient cooperation.
To determine whether measures of circumpapillary RNFL thickness, acquired with handheld OCT (HH-OCT) during sedation, can differentiate between young children with and without vision loss from OPGs.
This cross-sectional analysis of a prospective observational study was conducted at a tertiary-care children’s hospital. Children with an OPG (sporadic or secondary to neurofibromatosis type 1) who were cooperative for visual acuity testing, but required sedation to complete magnetic resonance imaging, underwent HH-OCT imaging of the circumpapillary RNFL while sedated.
Area under the curve of the receiver operating characteristic, sensitivity, specificity, positive predictive value, and negative predictive value of the average and quadrant-specific RNFL thicknesses.
Thirty-three children (64 eyes) met inclusion criteria (median age, 4.8 years; range, 1.8–12.6 years). In children with vision loss (abnormal visual acuity and/or visual field), RNFL thickness was decreased in all quadrants compared with the normal-vision group (P < .001 for all comparisons). Using abnormal criteria of less than 5% and less than 1%, the area under the curve was highest for the average RNFL thickness (0.96 and 0.97, respectively) compared with specific anatomic quadrants. The highest discrimination and predictive values were demonstrated for participants with 2 or more quadrants meeting less than 5% (sensitivity = 93.3; specificity = 97.9; positive predictive value = 93.3; and negative predictive value = 97.9) and less than 1% (sensitivity = 93.3; specificity = 100; positive predictive value = 100; and negative predictive value = 98.0) criteria.
Measures of RNFL thickness acquired with HH-OCT during sedation can differentiate between young children with and without vision loss from OPGs. For young children who do not cooperate with vision testing, HH-OCT measures may be a surrogate marker of vision. Longitudinal studies are needed to delineate the temporal relationship between RNFL decline and vision loss.
PMCID: PMC4445404  PMID: 24435762
22.  The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma 
Oncotarget  2015;6(14):12141-12155.
Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to induce gliomagenesis. Here, we investigated NG2 expression in a cohort of DIPG specimens (n= 50). We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2. Treatment with 5-Azacytidine, a methyltransferase inhibitor, results in NG2 downregulation in DIPG primary tumor cells in vitro. NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein, GFAP) markers, indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.
PMCID: PMC4494928  PMID: 25987129
DIPG; NG2; PDGF; histone 3; glioma
23.  A standardized autopsy procurement allows for the comprehensive study of DIPG biology 
Oncotarget  2015;6(14):12740-12747.
Diffuse intrinsic pontine glioma (DIPG) is one of the least understood and most deadly childhood cancers. Historically, there has been a paucity of DIPG specimens for molecular analysis. However, due to the generous participation of DIPG families in programs for postmortem specimen donation, there has been a recent surge in molecular analysis of newly available tumor specimens. Collaborative efforts to share data and tumor specimens have resulted in rapid discoveries in other pediatric brain tumors, such as medulloblastoma, and therefore have the potential to shed light on the biology of DIPG. Given the generous gift of postmortem tissue donation from DIPG patients, there is a need for standardized postmortem specimen accrual to facilitate rapid and effective multi-institutional molecular studies.
We developed and implemented an autopsy protocol for rapid procurement, documenting and storing these specimens. Sixteen autopsies were performed throughout the United States and Canada and processed using a standard protocol and inventory method, including specimen imaging, fixation, snap freezing, orthotopic injection, or preservation. This allowed for comparative clinical and biological studies of rare postmortem DIPG tissue specimens, generation of in vivo and in vitro models of DIPG, and detailed records to facilitate collaborative analysis.
PMCID: PMC4494970  PMID: 25749048
Diffuse Intrinsic Pontine Glioma (DIPG); Brainstem Glioma; Autopsy; Histone 3; Orthotopic Injection
24.  Randomized Study of Two Chemotherapy Regimens for Treatment of Low-Grade Glioma in Young Children: A Report From the Children's Oncology Group 
Journal of Clinical Oncology  2012;30(21):2641-2647.
Surgery is curative therapy for pediatric low-grade gliomas (LGGs) in areas of the brain amenable to complete resection. However, LGGs located in areas where complete resection is not possible can threaten both function and life. The purpose of this study was to compare two chemotherapy regimens for LGGs in children younger than age 10 years for whom radiotherapy was felt by the practitioner to pose a high risk of neurodevelopmental injury.
Patients and Methods
Previously untreated children younger than age 10 years with progressive or residual LGGs were eligible. Children were randomly assigned to receive carboplatin and vincristine (CV) or thioguanine, procarbazine, lomustine, and vincristine (TPCV). Children with neurofibromatosis are reported separately.
Of 274 randomly assigned patients who met eligibility requirements, 137 received CV and 137 received TPCV. The 5-year event-free survival (EFS) and overall survival (OS) rates for all eligible patients were 45% ± 3.2% and 86% ± 2.2%, respectively. The 5-year EFS rates were 39% ± 4% for CV and 52% ± 5% for TPCV (stratified log-rank test P = .10; cure model analysis P = .007). On multivariate analysis, factors independently predictive of worse EFS and OS were younger age and tumor size greater than 3 cm2. Tumor location in the thalamus was also associated with poor OS.
The difference in EFS between the regimens did not reach significance on the basis of the stratified log-rank test. The 5-year EFS was higher for TPCV on the basis of the cure model analysis. Differences in toxicity may influence physician choice of regimens.
PMCID: PMC3413276  PMID: 22665535
25.  Efficacy of bevacizumab plus irinotecan in children with recurrent low-grade gliomas—a Pediatric Brain Tumor Consortium study 
Neuro-Oncology  2013;16(2):310-317.
A phase II study of bevacizumab (BVZ) plus irinotecan (CPT-11) was conducted in children with recurrent low-grade glioma to measure sustained response and/or stable disease lasting ≥6 months and progression-free survival.
Thirty-five evaluable patients received 2 doses (10 mg/kg each) of single-agent BVZ intravenously 2 weeks apart and then BVZ + CPT-11 every 2 weeks until progressive disease, unacceptable toxicity, or a maximum of 2 years of therapy. Correlative studies included neuroimaging and expression of tumor angiogenic markers (vascular endothelial growth factor [VEGF], VEGF receptor 2, hypoxia-inducible factor 2α, and carbonic anhydrase 9).
Thirty-five evaluable patients (median age 8.4 y [range, 0.6–17.6]) received a median of 12 courses of BVZ + CPT-11 (range, 2–26). Twenty-nine of 35 patients (83%) received treatment for at least 6 months. Eight patients progressed on treatment at a median time of 5.4 months (range, 1–17.8). Six patients (17.7%) still in follow-up have had stable disease without receiving additional treatment for a median of 40.1 months (range, 30.6–49.3) from initiating therapy. The 6-month and 2-year progression-free survivals were 85.4% (SE ± 5.96%) and 47.8% (SE ± 9.27%), respectively. The commonest toxicities related to BVZ included grades 1–2 hypertension in 24, grades 1–2 fatigue in 23, grades 1–2 epistaxis in 18, and grades 1–4 proteinuria in 15. The median volume of enhancement decreased significantly between baseline and day 15 (P < .0001) and over the duration of treatment (P < .037).
The combination of BVZ + CPT-11 appears to produce sustained disease control in some children with recurrent low-grade gliomas.
PMCID: PMC3895377  PMID: 24311632
bevacizumab; CPT-11; children; gliomas; recurrent

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