Pediatric Brain Cancer Research Articles

Dabrafenib in pediatric patients with BRAF V600–positive high-grade glioma (HGG).

10505Background: The BRAF V600 mutation constitutively activates the MAPK/ERK pathway and is an oncogenic driver in many tumor types, including pediatric brain cancers. There are limited effective ...

Preface: Invited Issue Editor, Professor James T. Rutka, and the Pediatric Malignant Brain Cancers in the Molecular Era.

Preface: Invited Issue Editor, Professor James T. Rutka, and the Pediatric Malignant Brain Cancers in the Molecular Era.

Diffuse Intrinsic Pontine Glioma : Clinical Features, Molecular Genetics, and Novel Targeted Therapeutics.

Diffuse intrinsic pontine glioma (DIPG) is a deadly paediatric brain cancer. Transient response to radiation, ineffective chemotherapeutic agents and aggressive biology result in rapid progression of symptoms and a dismal prognosis. Increased availability of tumour tissue has enabled the identification of histone gene aberrations, genetic driver mutations and methylation changes, which have resulted in molecular and phenotypic subgrouping. However, many of the underlying mechanisms of DIPG oncogenesis remain unexplained. It is hoped that more representative in vitro and preclinical models–using both xenografted material and genetically engineered mice–will enable the development of novel chemotherapeutic agents and strategies for targeted drug delivery. This review provides a clinical overview of DIPG, the barriers to progress in developing effective treatment, updates on drug development and preclinical models, and an introduction to new technologies aimed at enhancing drug delivery.

Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas.

Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M)1-5 are aggressive and universally fatal pediatric brain cancers 6 . Chimeric antigen receptor (CAR)-expressing T cells have mediated impressive clinical activity in B cell malignancies7-10, and recent results suggest benefit in central nervous system malignancies11-13. Here, we report that patient-derived H3-K27M-mutant glioma cell cultures exhibit uniform, high expression of the disialoganglioside GD2. Anti-GD2 CAR T cells incorporating a 4-1BBz costimulatory domain 14 demonstrated robust antigen-dependent cytokine generation and killing of DMG cells in vitro. In five independent patient-derived H3-K27M+ DMG orthotopic xenograft models, systemic administration of GD2-targeted CAR T cells cleared engrafted tumors except for a small number of residual GD2lo glioma cells. To date, GD2-targeted CAR T cells have been well tolerated in clinical trials15-17. Although GD2-targeted CAR T cell administration was tolerated in the majority of mice bearing orthotopic xenografts, peritumoral neuroinflammation during the acute phase of antitumor activity resulted in hydrocephalus that was lethal in a fraction of animals. Given the precarious neuroanatomical location of midline gliomas, careful monitoring and aggressive neurointensive care management will be required for human translation. With a cautious multidisciplinary clinical approach, GD2-targeted CAR T cell therapy for H3-K27M+ diffuse gliomas of pons, thalamus and spinal cord could prove transformative for these lethal childhood cancers.

EP-1856: Reducing out-of-field organ doses during paediatric brain cancer radiotherapy using lead shielding

EP-1856: Reducing out-of-field organ doses during paediatric brain cancer radiotherapy using lead shielding

Characterization of a novel OTX2-driven stem cell program in Group 3 and Group 4 medulloblastoma.

Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self‐renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof‐of‐principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self‐renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.

Study Suggests PD-1 Inhibitors Against Aggressive Pediatric Brain Cancer

Study Suggests PD-1 Inhibitors Against Aggressive Pediatric Brain Cancer

HDL nanoparticles targeting sonic hedgehog subtype medulloblastoma

Medulloblastoma is the most common paediatric malignant brain cancer and there is a need for new targeted therapeutic approaches to more effectively treat these malignant tumours, which can be divided into four molecular subtypes. Here, we focus on targeting sonic hedgehog (SHH) subtype medulloblastoma, which accounts for approximately 25% of all cases. The SHH subtype relies upon cholesterol signalling for tumour growth and maintenance of tumour-initiating cancer stem cells (CSCs). To target cholesterol signalling, we employed biomimetic high-density lipoprotein nanoparticles (HDL NPs) which bind to the HDL receptor, scavenger receptor type B-1 (SCARB1), depriving cells of natural HDL and their cholesterol cargo. We demonstrate uptake of HDL NPs in SCARB1 expressing medulloblastoma cells and depletion of cholesterol levels in cancer cells. HDL NPs potently blocked proliferation of medulloblastoma cells, as well as hedgehog-driven Ewing sarcoma cells. Furthermore, HDL NPs disrupted colony formation in medulloblastoma and depleted CSC populations in medulloblastoma and Ewing sarcoma. Altogether, our findings provide proof of principle for the development of a novel targeted approach for the treatment of medulloblastoma using HDL NPs. These findings present HDL-mimetic nanoparticles as a promising therapy for sonic hedgehog (SHH) subtype medulloblastoma and possibly other hedgehog-driven cancers.

Жидкостные биопсии в детской нейроонкологии: в преддверии возможностей тераностики

Жидкостные биопсии в детской нейроонкологии: в преддверии возможностей тераностики

PDTM-17. IDENTIFICATION OF HUMAN PEDIATRIC BRAIN TUMORS CELLS IN PATIENT DERIVED ORTHOTOPIC XENOGRAFT MOUSE MODELS BY USING SPECIES-SPECIFIC PTGER2 GENE qPCR

AbstractBACKGROUNDPediatric brain tumors continue to be among the most difficult to cure and are the leading cause of cancer-related deaths in children. Research investigating pediatric brain cancer is still impaired by the lack of cancer models. We established a large panel of PDOX models as well as several paired permanent cell lines using and culture techniques. However, an inherent risk of xenograft models is the contamination of isolates of human tumors with host cells. Therefor strategies to identify the human/mouse ratio are essential. In this study we investigated Prostaglandin Receptor EP2 (PTGER2) as a useful target to identifying mouse cell contamination in PDOX models.METHODSpecies specific primers based on Alcoster et al., 2011 were used to detect PTGER2 in xenograft and cultured cells. RT-qPCR was carried out using a StepOnePlus™ RT-PCR System. Reaction mix contained SYBR Green master mix, respective primers and 5ng of total gDNA in a total reaction volume of 10μL. The qPCR conditions were as follows: 50°C-2min, 95°C-10min, 40 cycles of (95°C-15sec, 61°C-1min). Primers were additionally controlled by analysis of amplified products on a 1.2% agarose gel. FACS was used to isolate human cells according to anti-Human HLA-ABC Ab and mouse-specific Ab cocktail surface staining and subjected to qPCR.RESULTSVarious models of pediatric brain cancer established in our group were investigated for mouse cell contamination. Xenograft as well as cultured cells were routinely analyzed. Our study confirms PTGER2 as a useful target for accurately estimate mouse cell in PDOX models. This enabled us to improve detection and characterization of HLA-ABC/mouse-cocktail double negative population as seen in FACS.CONCLUSIONIn conclusion we have successfully implemented an reliable qRT-PCR approach to determine the human/mouse cell ration in any mixed PDOX cell population.

DDIS-17. POLYNITROXYL ALBUMIN INHIBITS BRAIN TUMORIGENIC CASCADE THROUGH A REACTIVE OXYGEN SPECIES MECHANISM ACROSS THE BLOOD BRAIN BARRIER

This study seeks to develop a safe therapy with potential for a broad spectrum of brain tumors. For this pilot study, we have selected medulloblastoma (MB), the most common malignant pediatric brain cancer that arises from the cerebellum. MB can disseminate through the cerebrospinal fluid to the leptomeninges covering the brain and spinal cord. Leptomeningeal metastasis, a frequent finding at diagnosis and recurrence, is a serious complication for which innovative therapies are urgently needed to improve patient survival while reducing treatment-related toxicity. Polynitroxyl albumin (PNA) has been tested at Sanford Research and shown to reduce intracellular levels of reactive oxygen species (ROS) and inhibition of the tumorigenic cascade: proliferation, progression, and metastasis. This ROS inhibition appears to be responsive in multiple cancers, including triple negative breast cancer and two chemoradiation-resistant head & neck squamous cell carcinoma cell lines. Because intracellular ROS are readily membrane permeable, we have tested efficacy of PNA across the blood brain barrier (BBB) in treating MB. For this we used a metastatic mouse model of the Sonic Hedgehog (SHH) subgroup. PNA was administered i.p. on a thrice weekly schedule; an albumin-only control group was also treated. Survival analysis was obtained, along with histopathology of both brain and spinal cord; primary tumor, leptomeningeal dissemination, and secondary tumor growth were examined. In our preclinical model, a significant decrease in disseminated MB within the brain was detected (P < 0.05), along with secondary tumor growth along the spinal cord (P < 0.05), compared to the control group. Our results compliment PNA studies in other cancer models; PNA works across the BBB to decrease proliferation of tumor cells and inhibit metastatic growth. Follow up studies are warranted to compare the therapeutic index of intra- vs extra-cerebral PNA delivery to generate preclinical efficacy data in support of conjunctive MB clinical therapy.

4SC-202 as a Potential Treatment for the Pediatric Brain Tumor Medulloblastoma.

This project involves an examination of the effect of the small molecule inhibitor 4SC-202 on the growth of the pediatric brain cancer medulloblastoma. The small molecule inhibitor 4SC-202 significantly inhibits the viability of the pediatric desmoplastic cerebellar human medulloblastoma cell line DAOY, with an IC50 = 58.1 nM, but does not affect the viability of noncancerous neural stem cells (NSC). 4SC-202 exposure inhibits hedgehog expression in the DAOY cell line. Furthermore, microarray analysis of human medulloblastoma patient tumors indicate significant upregulation of key targets in the Hedgehog signaling pathway and Protein Tyrosine Kinase (PTK7).

Fitness, body composition and vascular health in adolescent and young adult survivors of paediatric brain cancer and cranial radiotherapy.

Background Survivors of paediatric brain cancer and/or cranial radiotherapy (CRT) are at an increased risk of developing serious comorbidities. Established risk factors for chronic disease include central obesity, endothelial abnormalities and diminished fitness. Objectives Here we characterised anthropometry, body composition, bone mineral density (BMD), heart rate (HR), blood pressure (BP), endothelial function, muscular strength and endurance and aerobic fitness in adolescent and young adult (AYA) survivors. Methods Twenty survivors (10 male, 10 female; 20 ± 2 years) were compared with 19 matched controls. Muscular strength was assessed using three repetition maximum tests, while muscular endurance was determined as number of repetitions performed per minute. Peak oxygen uptake (VO2 peak) was assessed on a treadmill using a modified chronotropic protocol. Anthropometric measurements, HR and BP were taken using standard clinical protocols, while body composition and BMD were determined using dual X-ray absorptiometry (DXA). Endothelial function was measured using the flow mediated dilation technique. Results Survivors demonstrated deficits in muscular strength (latissimus dorsi pull-down, p = 0.020; bicep curl, p = 0.009), muscular endurance (squats, p = 0.012; sit-ups, p = 0.030; push-ups, p = 0.013), minute ventilation at peak exericse (p = 0.002) and VO2peak (L/min, p = 0.002; mL/kg/min, p = 0.008; mL/kg LBM/min, p = 0.010). Additionally, survivors had greater waist-to-hip ratios (p = 0.032), resting HR (p = 0.048) and higher percentage of total body (p = 0.017), central (p = 0.009) and peripheral (p = 0.032) fat. Lean body mass (p = 0.004) and BMD (p = 0.005) were lower in the survivor group. Conclusion AYA survivors of paediatric brain cancer and/or CRT exhibit altered body composition, increased resting HR and reduced BMD, muscular strength, muscular endurance and cardiorespiratory fitness compared to controls.

Abstract 4874: Application of quantitative analysis of mouse cell contamination and purification of human tumor cells in patient derived orthotopic xenograft mouse models of pediatric brain tumors

Abstract Background: Pediatric brain tumors continue to be among the most difficult to cure and are the leading cause of cancer-related deaths in children. Research investigating pediatric brain cancer is still impaired by the lack of cancer models. We established a large panel of orthotopic xenograft models as well as several paired permanent cell lines using in vivo and in vitro culture techniques. However an inherent risk of xenograft models is the contamination of isolates of human tumors with host (mouse) cells. Therefor strategies to correctly and timely identify the human/mouse ratio are essential. In this study we investigated Prostaglandin Receptor EP2 (PTGER2) as a useful target to identifying mouse cell contamination in PDOX models. Method: Species specific primers based on Alcoster et al., 2011 were used to detect PTGER2 in genomic DNA extracts of xenograft and cultured cells. Real-time qPCR was carried out using a StepOnePlus™ Real-Time PCR System. Reaction mix contained SYBR Green select master mix, respective primers and 5 ng of total genomic DNA in a total reaction volume of 10 μL. The qPCR conditions were as follows: 50°C-2 min, 95°C-10 min, 40 cycles of (95°C-15 sec, 61°C-1 min). Primer specificity was controlled in addition by analysis of amplified products on a 1.2% agarose gel. FACS was used to isolate human cells according to anti-Human HLA-ABC Ab and mouse-specific Ab cocktail surface staining and subjected to qPCR. Results: Various models of pediatric brain cancer (PXA, MB, GBM, EPN, ATRT, DIPG, PNET) established in our group were investigated for mouse cell contamination. Cells from xenograft isolates as well as from cultured xenografts were routinely analyzed. Our study confirms PTGER2 as a useful target for accurately estimate mouse cell contamination in brain cancer xenograft models. This further enabled us to improve detection and characterization of HLA-ABC/mouse-cocktail double negative population as seen in FACS. Conclusion: In conclusion we have successfully implemented an easy qRT-PCR approach to determine the human/mouse cell ration in any mixed cell population. Particular for PDOX models it is paramount to have reliable information on mouse cell contamination. Citation Format: Frank K. Braun, Mari Kogiso, Lin Qi, Huiyuan Zhang, Yuchen Du, Holly B. Lindsay, Sibo Zhao, Sarah G. Injac, Xiao-Nan Li. Application of quantitative analysis of mouse cell contamination and purification of human tumor cells in patient derived orthotopic xenograft mouse models of pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4874. doi:10.1158/1538-7445.AM2017-4874

Abstract 810: Establishing patient derived preclinical in vitro and in vivo models of pediatric brain cancers

Abstract The recent surge in understanding genomic aberrations of some of the deadliest childhood brain cancers has highlighted the need for robust preclinical models. Such models will allow for robust drug screening and preclinical evaluation of efficacy, toxicity, and tumor penetrance in vivo. Given the rarity and importance of patient derived specimens, handling and processing methods are perhaps the most critical steps for successful establishment of viable and reproducible in vitro and in vivo models. Since specimen source varies (biopsy, autopsy, or cryo-preserved), processing methods should be refined to allow for optimal extraction of maximum numbers of viable cells from each specimen type. We have developed standardized procedures for handling and processing of tissue samples obtained from biopsy, autopsy, or cryo-preserved specimens as well as necropsy tissue obtained from existing xenograft models. Two processing methods for generating viable cell suspensions are described. The first method, which uses collagenase-DNAse mediated digestion of the tissue is efficient with bulky samples and can be used with tissues obtained at autopsy. The second method uses a commercially available enzymatic dissociation kit optimal for small volume samples such as biopsy, cryo-preserved and mouse necropsy specimens. We show that obtaining viable cell suspension from precious tumor tissue by these methods results in successful generation of pre-clinical in vitro and in vivo models of DIPG, pilocytic astrocytoma and medulloblastoma that represent the exact genetic makeup of the original patient tumor. We further demonstrate intracranial injections of these cells into P2 mice for generating orthotopic xenograft models of brainstem or cortical tumors. Our methods and results allow for rapid establishment of preclinical models using rare and valuable childhood brain tumor specimens. These pre-clinical models serve as valuable tools for understanding the molecular mechanisms of the disease, identifying targetable molecules, and screening of novel therapeutics. Citation Format: Sridevi Yadavilli, Madhuri Kambhampati, Jamila Gittens, Eshini Panditharatna, Mojca Stampar, Lindsay B. Kilburn, Suresh Magge, Roger J. Packer, Javad Nazarian. Establishing patient derived preclinical in vitro and in vivo models of pediatric brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 810. doi:10.1158/1538-7445.AM2017-810

TMOD-09. A PATIENT-DERIVED XENOGRAFT MODEL MIMICKING RECURRENT PEDIATRIC MALIGNANT GLIOMA

Abstract BACKGROUND: Pediatric high grade glioma (pHGG) is a devastating cancer of the central nervous system. The prognosis of this most malignant form of brain tumor remains poor, largely due to the inherent resistance of pHGG to therapy, and inevitably leads to recurrence. Unfortunately, because recurrent glioma, or glioma remaining after conventional therapy, frequently has very different molecular characteristics from the original tumor in addition to a vastly different biological behavior, recurrent pHGG has proven exceedingly difficult to treat, with very few treatment options providing clinically meaningful outcomes. As part of the NCI Pediatric Preclinical Testing Program, several pediatric brain cancer cell lines, including the ones used in this study, were developed and characterized. However, to our knowledge, none have been utilized in development of a rat xenograft model of recurrent pHGG. METHODS: Pediatric glioblastoma cells (SJ-GBM2) were obtained from the Children’s Oncology Group Cell Culture and Xenograft Repository. Irradiated SJGBM2-10gy cells were generated by exposure to a total radiation dose of 10 Gy. Following irradiation, the resultant SJGBM2-10gy cells were tested for cell viability and allowed to reach confluence prior to stereotactical implantation into the right striatum of male athymic rats. RESULTS: Less than 1% of total cells survived radiation. Both irradiated and parental cell lines formed tumors in 100% of the animals following intracranial implantation. Irradiated SJGBM2-10gy cells exhibited a more aggressive tumor growth than the parental SJ-GBM2 cell line, resulting in mortality on average 14 days earlier. Histopathological analysis is underway and more advanced imaging studies are planned. CONCLUSION: To our knowledge this is the first report of an intracranial rat xenograft model mimicking recurrent pHGG using irradiated cell lines. It represents a robust and expedient in vivo model for studying recurrent pHGG biology.

Using Epigenetic Reprogramming to Treat Pediatric Brain Cancer

In this issue of Cancer Cell, Nagaraja etal. dissect the molecular mechanisms underlying therapeutic responses to transcriptional disruptors in the fatal pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). Moreover, they identify super-enhancers mediating these effects, highlighting how normal brain developmental programs can be hijacked in cancer.

High OCT4A levels drive tumorigenicity and metastatic potential of medulloblastoma cells.

Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer.

Investigation of Vitamin D Receptor Gene Polymorphism in Pediatric Patients with Brain Cancer.

Aim:In recent years, it is believed that Vitamin D may play a protective role in some cancer types. Certain regions of the Vitamin D receptor (VDR) gene may show a genetic difference in structure. The most frequent polymorphisms in this gene are in Taq-1, Fok-1, and Bsm-1 regions. Some adult cancer types are associated with VDR gene polymorphism such as; colorectal carcinoma, breast carcinoma, and prostate carcinoma. Reviewing the medical literature, no such study had been done on children so far.Materials and Methods:We investigated the association of the three most common gene polymorphisms (Taq-1, Fok-1, and Bsm-1 regions) in VDR gene in 32 children with brain tumors and forty control healthy volunteers.Results:We could not find any relationship between childhood brain tumors and VDR gene polymorphism in these three regions.Conclusion:The present results suggest that the Taq-1, Fok-1, and Bsm-1 polymorphism in the VDR gene and pediatric brain cancers have no association.

Tapping Big Data to Help Little Patients.

Through a large-scale tumor-sequencing effort for pediatric brain cancer, researchers hope to bring personalized care to young patients.