Pediatric Brain Cancer Research Articles

Abstract 2465: Genomic harmonization of the Data Resource Center for Gabriella Miller Kids First Pediatric Research Program

Abstract Gabriella Miller Kids First Pediatric Research Program (GMKF) is a nation-wide, multi-year initiative focused on the integration of large-scale clinically annotated genomic data for childhood cancers and structural birth defects supported by the NIH Common Fund. Awarded by GMKF, the Kids First Data Resource Center (DRC) is tasked to build infrastructure and workflows for data intaking, harmonization, integration and access authorization to empower collaborative discoveries across the GMKF and other integrated datasets. A key challenge for uniform analyses and empowered discovery of large-scale genomic data relates to the diverse genomic processing workflows and methods employed across the sequencing and bioinformatics community. The DRC genomic harmonization team aims to provide “analysis ready” datasets that are “functionally equivalent” across the Kids First datasets and other large-scale genomic data initiatives in order to accelerate the discovery process. Paired with the cloud-based workspace environments of the DRC, such harmonized dataset provide unprecedented opportunities for shared, reproducible discovery by a diverse, collaborative network of researchers. As such, DRC initial pipelines are developed with BWA-MEM alignment on genome build GRCh38 followed by the GATK best practices for germline variant calling and joint genotyping. Common Workflow Language (CWL) is used as the main workflow specification, while Docker technology has been applied to containerize all the tools used by the workflow. Our current workflows are tasked with data harmonization across a number of different experimental platforms including whole genome sequencing, exome sequencing, and RNA-seq. The data processing is done via CAVATICA, an Amazon Web Services (AWS) based cloud computing platform associated with the Kids First DRC Portal co-developed by Seven Bridges Genomics, where workflows feature scatter-gather parallelization and AWS resource optimization. By utilizing such a framework, the DRC team has harmonized over 10,000 WGS and 1,000 RNA-Seq samples across 12 study cohorts within 8 months. This dataset in its current release includes samples from 40 pediatric brain cancers as well as 8 childhood birth defects with the outcome of delivering 150TB harmonized CRAM and 60TB gVCF. With a highly optimized bioinformatics pipeline powered by an efficient cloud-based execution workflow, The DRC platform processes one genome in about 11 hours with an average compute cost of $15 for whole genome alignment and germline variant calling. Here we present our observed challenges and identified opportunities in the analysis and integration of multi-disease pediatric genomic data on a large scale. Citation Format: Yuankun Zhu, Miguel Brown, Batsal Devkota, Bailey Farrow, Bogdan Gavrilovic, Allison Heath, Kyle Hernandez, Avi Kelman, Parimala Killada, Meen Chul Kim, Daniel Kolbman, Mateusz Koptyra, Milan Kovacevic, Maarten Leerkes, Alex Lubneuski, Michele Mattioni, Pichai Raman, Adam Resnick, Nikola Skundric, Deanne Taylor, Junjun Zhang, Bo Zhang, Phillip B. Storm. Genomic harmonization of the Data Resource Center for Gabriella Miller Kids First Pediatric Research Program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2465.

Abstract 255: Antagonizing Na-K-Cl cotransporter NKCC1 impairs medulloblastoma cell viability

Abstract Medulloblastoma, a common pediatric brain cancer, is comprised of four molecular subgroups: Wnt-activated; Shh-activated; group 3; and group 4. Group 3 subgroup patients have the lowest survival rate and the highest rates of relapse and metastasis [1]. The standard-of-care for medulloblastoma consists of surgical removal of the tumor, radiation, and chemotherapy [1]. However, the standard-of-care poses many risk factors including hearing loss, permanent damage to endocrine and neurocognitive function, and secondary tumors [1]. There is a high demand for new treatment options for medulloblastoma. Interestingly, group 3 medulloblastoma tumors have high expression of GABRA5 and SLC12A2, which code for the α5-subunit of the ligand-gated ionotropic γ-aminobutyric acid type A receptor (GABAAR) and the sodium-potassium-chloride cotransporter isoform-1 (NKCC-1), respectively [2]. Several recent studies have provided evidence that ion channel activity contributes to brain cancer progression [3]. If so, then modulating ion channel(s) function may impair cancer cell viability. We have already shown that use of a positive allosteric modulator of the GABAAR can impair viability of group 3 medulloblastoma cells in vitro and in vivo and with greater specificity and potency than standard-of-care chemotherapeutic [4, 5]. Others have shown that inhibition or down-regulation of NKCC-1 can reduce cell viability, increase apoptosis, and decrease migration and invasion of cells derived from various cancer types [3]. We have examined how modulating NKCC-1 and GABAAR alone and simultaneously impacts the viability of group 3 medulloblastoma cells. We find that there is a synergistic effect in the inhibition of NKCC-1 and positive modulation of GABAAR. We will report on these observations and our model for the mechanism of action.

Potent anti-tumor efficacy of palbociclib in treatment-naïve H3.3K27M-mutant diffuse intrinsic pontine glioma.

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a rare and fatal pediatric brain cancer without cure. Seeking therapeutic strategies is still a major challenge in DIPG research. Previous study has shown that dysregulation of G1/S cell cycle checkpoint was common in DIPG and this dysregulation is even more enriched in the H3.3K27 M mutant subgroup. Here we assess potential anti-tumor efficacy of palbociclib, a specific and cytostatic inhibitor of CDK4/6, on high grade H3.3-K27 M-mutant DIPGs in vitro and in vivo.MethodsWe established patient-derived cell lines from treatment-naïve specimens. All the lines have H3.3K27 M mutation. We used a range of biological in vitro assays to assess the effect of palbociclib on growth of DIPGs. Palbociclib activity was also assayed in vivo against three independent DIPG orthotropic xenografts model.FindingsDysregulation of G1/S cell cycle checkpoint is enriched in these DIPGs. Then, we showed that depletion of CDK4 or CDK6 inhibits DIPG cells growth and blocks G1/S transition. Furthermore, palbociclib effectively repressed DIPG growth in vitro. Transcriptome analysis showed that palbociclib not only blocks G1/S transition, it also blocks other oncogenic targets such as MYC. Finally, palbociclib activity was assayed in vivo against DIPG orthotropic xenografts to demonstrate the high efficiency of blocking tumor growth.InterpretationOur findings thus revealed that palbociclib could be the therapeutic strategy for treatment-naïve DIPG with H3.3K27 M mutation.FundBeijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support, Beijing Municipal Natural Science Foundation, Ministry of Science and Technology of China, and National Natural Science Foundation of China.

Medulloblastoma rendered susceptible to NK-cell attack by TGFß neutralization

Background & Aim Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in patients with high-risk disease, and traditional therapies often cause significant permanent morbidity. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy because they recognize malignant cells without the need for a known target, they are readily available from multiple banks, and they expand to significant numbers. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment, such as Transforming Growth Factor β (TGF-β). Methods, Results & Conclusion To overcome the detrimental effects of TGF-β, we modified CB-derived NK cells to express a dominant negative TGF-β receptor II (DNRII) by retroviral transduction and evaluated their ability to kill a TGF-β-secreting MB cell line (Daoy). We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence ofmedulloblastoma-conditioned, TGF-β-richmedia (decreasing to 14.9±2.1% killing at an E:T ratio of 5:1 from 25.1±2.9%, n=10, p Hence, CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation as a novel therapeutic for the treatment of patients with high-risk medulloblastoma.

Development of a large-scale H3.3K27M-specific TCR-transduced T cell manufacturing method for adoptive cell therapy in HLA-A2+ patients with diffuse midline glioma

Background & Aim Diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG), constitutes up to 20% of pediatric brain cancer and has a median survival of less than one year. Genetic studies have revealed that greater than 70% of DMG cases harbor an amino acid substitution from lysine (K) to methionine (M) at position 27 of histone 3 variant 3 (H3.3). We recently reported that a peptide encompassing the H3.3K27M mutation (amino acid position 26-35) is presented by HLA-A*0201+ human glioma cells and induces a specific cytotoxic responses in CD8+ T lymphocytes. Furthermore, we cloned a high-affinity T cell receptor (TCR) that specifically recognizes the H3.3K27M epitope. Alanine scanning demonstrated that the key amino-acid sequence motif in the epitope for the TCR reactivity is not shared by any known human protein. Based on these data, we plan to develop a phase I study of adoptive transfer therapy using autologous T cells transduced with H3.3K27M-specific TCR. To this end, we have initiated developing processes for manufacturing the TCR-transduced T cells. Methods, Results & Conclusion We optimized the codon usage of full-length cDNA for α- and β-chains of the TCR, and cloned them into a TCR retroviral vector system, which incorporates small interfering (si)RNA targeting constant regions of the endogenous TCR α- and β- chains to avoid mispairing between endogenous and transgene TCR chains (Takara Bio, Inc.). PG13 packaging cells were transfected with siTCR retroviral vector and established as the producer cell line. Using Miltenyi CliniMACS Prodigy system, which allows closed and automated manufacturing of T cell products, we activated human T cells (starting number 1 × 10e8 T cells) with MACS GMP T Cell TransActTM(Miltenyi), and subsequently transduced with the siTCR retroviral vector and expanded the cells in the presence of IL-7 and IL-15. We achieved over 20-fold expansion of the T cells with at least 20% transduction efficiency. Furthermore, the vast majority of TCR-transduced T cells showed central memory phenotype represented by co-expression of CCR7 and CD62L. To further optimize manufacturing conditions, we are currently evaluating other parameters, such as cytokine doses and combinations, as well as starting T cell populations. These data suggest that the Prodigy system may represents a feasible system for manufacturing TCR-transduced T cells for adoptive transfer therapy.

Elevated NLR May Be a Feature of Pediatric Brain Cancer Patients.

Pediatric brain tumors are the most common solid tumor type and the leading cause of cancer-related death in children. The immune system plays an important role in cancer pathogenesis and in the response to immunotherapy treatments. T lymphocytes are key elements for the response of the immune system to cancer cells and have been associated with prognosis of different cancers. Neutrophils on the other hand, which secrete pro-angiogenic and anti-apoptotic factors, enhance the ability of tumor cells to grow and develop into metastases. We conducted a retrospective study of 120 pediatric brain cancer patients and 171 elective pediatric patients hospitalized in Dana Children's Hospital and Sheba Medical Center. Data on age, sex, treatment, lymphocyte, neutrophil, and monocyte count were collected from routinely performed preoperative blood tests. Neutrophil-to-lymphocyte ratio (NLR), and the lymphocyte-to-monocyte ratio (LMR) were calculated and significance was determined by paired T test. p < 0.05 was considered as statistically significant. NLR was significantly higher in the pediatric brain cancer patients. The high NLR in pediatric brain cancer patients is the result of a combination of low lymphocytes and high neutrophils. Both of these factors can have a role in cancer development and propagation and also in response to therapy.

Automated High‐Throughput Drug Discovery in Peptide Hydrogel‐Based 3D Cell Cultures

Automated cell‐based high‐throughput screening (HTS) is a powerful tool in drug discovery and currently, there is a push to combine complex cell culture systems with HTS to provide more clinically applicable results. Despite the increasing recognition that three‐dimensional (3D) models provide more in vivo‐like cell culture environments, there are mechanistic requirements inherent to HTS as well as material limitations that make this integration for high‐throughput drug discovery challenging. We used the peptide‐based hydrogel MAX8 tagged with the RGDS sequence to create a synthetic extracellular scaffold to culture cells in 3D, and show a preliminary implementation of the scaffold for 3D HTS with a pilot drug screen targeting the pediatric brain cancer medulloblastoma. MAX8 undergoes assembly under physiological conditions and ambient temperature into a hydrogel with a well‐defined, nanofibrillar matrix, desired porosity and stiffness to provide a biocompatible cell culture matrix. Due to the physical gel properties such as shear‐thinning and the fast gelation kinetics, cells can be homogenously encapsulated into evenly dispensed gel‐cell constructs using automatic distribution of cell‐peptide mixtures into 384‐well assay plates with standard liquid handling equipment. 2,202 compounds were screened in the 384‐well format against cells encapsulated in the hydrogel as well as cells growing on traditional 2D plastic. 82 compounds passed the first round of screening at a single point of concentration. 16‐point dose response was done on those 82 compounds, of which 17 compounds validated. Three‐dimensional cell‐based HTS could be a powerful screening tool that allows researchers to finely tune the cell microenvironment getting more clinically applicable data as a result. Here, we have shown the successful integration of a peptide‐based hydrogel into the high‐throughput format.Support or Funding InformationThis work was supported by funds from the NIH IDeA program, with grants from the National Institute of General Medical Sciences NIGMS (P20‐GM103464, P30‐GM114736, and U54‐GM104941), the DO Believe Foundation and the Nemours Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

EP-1919 Voxel-based assessment of proton RBE in paediatric brain cancer radiotherapy from multimodal imaging

EP-1919 Voxel-based assessment of proton RBE in paediatric brain cancer radiotherapy from multimodal imaging

Finding Hope and Healing When Cure Is Not Possible

Traditional medical training focused on curing disease may not prepare clinicians to provide comfort and solace to their patients facing life-limiting illness. But dying patients and their families still need healing, and clinicians can actively facilitate it. We explore the clinician's role in the healing journey through the lens of pediatric brain cancer. Specifically, we examine how clinicians can help affected families find their way from “focused hope” (which centers on cure) to “intrinsic hope,” which offers a more realistic and resilient emotional foundation as the child's death approaches and letting go becomes essential. Drawing on their clinical experience and medical knowledge, clinicians can help families comprehend the lessons that their seriously ill child's body has to teach, highlighting the importance of cherishing the present and creating new memories that outlast the disease. Clinicians can avoid the mindset of “nothing more can be done,” emphasizing that there is plenty to do in providing physical, emotional, and spiritual comfort. Clinicians can learn how to be “unconditionally present” for patients and families without immersing themselves in anguish and, eventually, how to help the family find freedom from despair and a full life that still honors the child's memory.

Evaluating out-of-field doses during radiotherapy of paediatric brain tumours using lead shielding and flattening-filter free beams

Paediatric radiotherapy comes at the expense of increased risk of late effects due to out-of-field dose caused not only by the treatment itself but also by image guidance. This study examined how the out-of-field dose to selected radiosensitive organs was affected by applying a 1 mm lead shielding during delivery of volumetric-modulated arc therapy (VMAT) for paediatric brain cancer. The study also investigated how the out-of-field dose to the same organs was affected by the use of flattening-filter free (FFF) beams.Out-of-field doses to the thyroid, breast and testes were measured using thermoluminescence dosimeters inserted in two anthropomorphic phantoms equivalent to a 1-year and 5-year old child. Coplanar VMAT plans were prepared for 6 MV and 6 MV FFF photon beams and delivered using a Varian TrueBeam linear accelerator, with and without lead shielding applied to the phantoms.The measured out-of-field doses were as large as 200,9 cGy for the whole treatment, with associated secondary cancer risk being as large as 1,1%. Shielding of the phantoms was found to decrease the out-of-field dose by up to 24%. The use of 6 MV FFF beams yielded a decrease in the dose to the testes by 21–42% compared to 6 MV, while in one case increasing the dose to the thyroid by 18%. The observation that only doses to organs distant to the primary irradiated volume were significantly decreased for FFF can be explained by an increase in internal scatter caused by the softer energy spectrum of the Varian FFF beam.

Diffuse intrinsic pontine glioma cells are vulnerable to low intensity electric fields delivered by intratumoral modulation therapy

IntroductionDiffuse intrinsic pontine glioma (DIPG) is a high fatality pediatric brain cancer without effective treatment. The field of electrotherapeutics offers new potential for other forms of glioma but the efficacy of this strategy has not been reported for DIPG. This pilot study evaluated the susceptibility of patient-derived DIPG cells to low intensity electric fields delivered using a developing technology called intratumoral modulation therapy (IMT).MethodsDIPG cells from autopsy specimens were treated with a custom-designed, in vitro IMT system. Computer-generated electric field simulation was performed to quantify IMT amplitude and distribution using continuous, low intensity, intermediate frequency stimulation parameters. Treatment groups included sham, IMT, temozolomide (TMZ) chemotherapy and radiation therapy (RT). The impact of single and multi-modality therapy was compared using spectrophotometric and flow cytometry viability analyses.ResultsDIPG cells exhibited robust, consistent susceptibility to IMT fields that significantly reduced cell viability compared to untreated control levels. The ratio of viable:non-viable DIPG cells transformed from ~ 6:1 in sham-treated to ~ 1.5:1 in IMT-treated conditions. The impact of IMT was similar to that of dual modality TMZ–RT therapy and the addition of IMT to this treatment combination dramatically reduced DIPG cell viability to ~ 20% of control values.ConclusionsThis proof-of-concept study provides a novel demonstration of marked DIPG cell susceptibility to low intensity electric fields delivered using IMT. The potent impact as a monotherapy and when integrated into multi-modality treatment platforms justifies further investigations into the potential of IMT as a critically needed biomedical innovation for DIPG.

Maternal stress and pediatric brain cancer: A French study

Background: Despite evidence that pediatric cancer induces a trajectory of parental stress, studies including mothers of children with malignant brain tumors remain scarce. The present work aims to add to the scientific literature by evaluating maternal stress with a French translation of the Pediatric Inventory for Parents (PIP) in a population of mothers whose children have been diagnosed with a brain tumor.Procedure: The mothers of 35 children with malignant brain tumors completed the Pediatric Inventory for Parents (PIP), the State-Trait Anxiety Inventory (STAI) and the Family Assessment Device (FAD) at a mean time since diagnosis of 2.3 years.Results: The PIP showed good internal consistency and correlated significantly with the STAI and the FAD. Maternal stress scores were higher in younger mothers and in mothers of younger children. Emotional functioning was the most affected domain.Conclusions: Emotional stress as assessed by the PIP in mothers of children with malignant brain tumors is considerable, and should be routinely assessed, in order to implement appropriate specific psychosocial interventions when needed.

Building the Foundation for a Promising Pediatric Brain Cancer Treatment

Building the Foundation for a Promising Pediatric Brain Cancer Treatment

Racial and ethnic differences in survival of pediatric patients with brain and central nervous system cancer in the United States.

Brain and central nervous system (CNS) cancer is the leading cause of cancer death among children and adolescents in the United States. Data from earlier studies suggested racial and ethnic differences in survival among pediatric patients with brain tumor. This study examined racial/ethnic difference in survival using national data and considered the effects of demographic and clinical factors. Using National Program of Cancer Registries data, 1-, 3-, and 5-year relative survival (cancer survival in the absence of other causes of death) was calculated for patients with brain and CNS cancer aged<20 years diagnosed during 2001-2008 and followed up through 2013. Racial and ethnic differences in survival were measured by sex, age, economic status, stage, anatomic location, and histology. Adjusted racial and ethnic difference in 5-year cancer specific survival was estimated using multivariable Cox regression analysis. Using data from 11 302 patients, 5-year relative survival was 77.6% for non-Hispanic white patients, 69.8% for non-Hispanic black patients, and 72.9% for Hispanic patients. Differences in relative survival by race/ethnicity existed within all demographic groups. Based on multivariable analysis, non-Hispanic black patients had a higher risk of death at 5 years after diagnosis compared to non-Hispanic white patients (adjusted hazard ratio=1.2, 95% confidence interval, 1.1-1.4). Pediatric brain and CNS cancer survival differed by race/ethnicity, with non-Hispanic black patients having a higher risk of death than non-Hispanic white patients. Future investigation of access to care, social and economic barriers, and host genetic factors might identify reasons for disparities in survival.

P140] Flattening filter-free radiotherapy for paediatric brain cancer: Impact on the dose to selected out-of-field organs

Purpose Removal of the flattening filter has been shown to alter the out-of-field dose during radiotherapy. In this study, the dose to selected out-of-field organs is compared for conventional flattened and flattening filter free (FFF) volumetric-modulated arc therapy (VMAT) of paediatric brain cancer. Methods Out-of-field doses to the thyroid, breast and testes were measured using thermoluminescence dosimeters inserted in two anthropomorphic phantoms resembling a 1-year and 5-year old child. Coplanar VMAT plans were prepared for 6 MV and 6 MV FFF photon beams, for two different PTV sizes (2 cm and 5 cm diameter), simulating post-operative radiotherapy of an ependymoma in the posterior fossa. The phantoms were subject to three VMAT fractions of 1,8 Gy each, delivered using a Varian TrueBeam linear accelerator. Some additional irradiations were performed for the 1-year phantom with the head of the phantom removed, to investigate the contribution from internal scatter to the total out-of-field dose. Results For the testes, measured doses showed a statistically significant decrease (Student’s t-test, significance level p 0,05) of 21–42% for FFF compared with conventional plans, for both phantoms as well as both PTV sizes. Doses to the breast showed a significant decrease of approximately 12% for the 5-year phantom for the small PTV, but other breast doses did not show a significant difference with and without the flattening filter. Doses to the thyroid showed a significant increase of approximately 18% for the 1-year phantom for the small PTV, with other dose differences being insignificant. However, doses to all three organs were significantly reduced (19–37%) when removing the head of the 1-year phantom, minimizing the contribution from internal scatter. Conclusions The results show that FFF VMAT of paediatric brain cancer leads to reduced doses to organs far from the field compared to VMAT with conventional flattened beams, while doses to organs close to the field seem to increase. The latter finding can be explained by an increase in internal scatter, which is possibly caused by the softer energy spectrum of the 6 MV FFF beam for the investigated linear accelerator model compared to conventional 6 MV.

P169] Can lead shielding during paediatric brain radiotherapy reduce the risk of secondary cancer?

Purpose Radiotherapy of paediatric patients comes at the expense of increased secondary cancer risk. This study examines whether the use of a 1 mm lead shielding during volumetric-modulated arc therapy (VMAT) for paediatric brain cancer can reduce the secondary cancer risk for the thyroid and breast. Methods Coplanar 6 MV VMAT plans were prepared for two anthropomorphic phantoms resembling a 1-year and 5-year old girl, for two different PTV sizes (2 cm and 5 cm diameter), simulating post-operative radiotherapy of an ependymoma in the posterior fossa. Out-of-field doses to the thyroid and breast were measured using thermoluminescence dosimeters. The phantoms were subject to three fractions (1,8 Gy each), with and without 1 mm lead shielding of the torso. The measured doses were used to calculate lifetime attributable risks for cancer incidence, for the total treatment dose of 54 Gy, in accordance with the BEIR VII report. 1 Results Applying lead shielding yielded dose reductions of 8–18% for the thyroid and 12–18% for the breast. The results were statistically significant (Student’s t-test, significance level p Conclusions Lead shielding of the phantoms resulted in a systematic decrease in the measured doses to the thyroid and breast, reducing the lifetime attributable risks for cancer incidence. The results hereby suggest that a 1 mm thick lead rubber blanket might be a clinically feasible solution for reducing the risk of secondary cancer in paediatric radiotherapy patients.

Activation of ERBB4 in Glioblastoma Can Contribute to Increased Tumorigenicity and Influence Therapeutic Response.

Glioblastoma (GBM) is often resistant to conventional and targeted therapeutics. ErbB2 Receptor Tyrosine Kinase 4 (ERBB4) is expressed throughout normal brain and is an oncogene in several pediatric brain cancers; therefore, we investigated ERBB4 as a prognostic marker and therapeutic target in GBM. Using RT-qPCR, we quantified mRNA encoding total ERBB4 and known ERBB4 variants in GBM and non-neoplastic normal brain (NNB) samples. Using immunohistochemistry, we characterized the localization of total and phosphorylated ERBB4 (p-ERBB4) and EGFR protein in archived GBM samples and assessed their association with patient survival. Furthermore, we evaluated the effect of ERBB4 phosphorylation on angiogenesis and tumorigenicity in GBM xenograft models. Total ERBB4 mRNA was significantly lower in GBM than NNB samples, with the juxtamembrane JM-a and cytoplasmic CYT-2 variants predominating. ERBB4 protein was ubiquitously expressed in GBM but was not associated with patient survival. However, high p-ERBB4 in 11% of archived GBM samples, independent of p-EGFR, was associated with shorter patient survival (12.0 ± 3.2 months) than was no p-ERBB4 (22.5 ± 9.5 months). Increased ERBB4 activation was also associated with increased proliferation, angiogenesis, tumorigenicity and reduced sensitivity to anti-EGFR treatment in xenograft models. Despite low ERBB4 mRNA in GBM, the functional effects of increased ERBB4 activation identify ERBB4 as a potential prognostic and therapeutic target.

Abstract 2085: Exploring mutation signatures in pediatric cancers

Abstract Background: Mutation signatures have been catalogued across most common cancers, providing insights into their underlying mutational processes and suggesting strategies for categorization and subphenotyping. However, these analyses have been limited to mostly adult cancers, likely due to the relative rarity of pediatric analogs and a corresponding lower mutation burden of these tumors. Here we attempted to compare mutation signatures across several pediatric cancers, given these constraints, with their corresponding adult versions. Data and Methods: From the ICGC data portal, we downloaded clinical and mutation data for pediatric brain (medulloblastoma, glioblastoma, neuroblastoma) and blood-based (acute lymphoblastic leukemia, acute myeloid leukemia) cancers. We also obtained data for related adult cancers, including glioblastoma and acute myeloid leukemia. For each cancer, we characterized mutation patterns using trinucleotides centered at somatic SNVs and then estimated the composition of underlying putative COSMIC mutation signatures. For cancers with low mutation rates (impeding estimates of mutation signatures), we grouped mutations from similar samples based on clinical and molecular statuses to infer mutation signatures. Results: Each of the pediatric cancer types strongly exhibited the common cancer mutation Signature 1 (spontaneous deamination of 5-methylcytosine), as might be expected since it has been described across all adult cancer types. Further, pediatric acute myeloid leukemia and glioblastoma both harbored mutation signatures seen in their adult counterparts in addition to unexpected patterns of defective DNA mismatch repair and DNA double-strand break repair. Interestingly, for pediatric glioblastoma we identified Signatures 3, 8, 15 and 16, which were previously unidentified in adult glioblastoma and have been recently implicated in pediatric medulloblastoma. Conclusion: Our findings may be influenced by the sparsity of mutations in pediatric cancers, highlighting the need for quantifying precision in measures of mutation signatures. This notwithstanding, from our initial observations, pediatric cancers appear to exhibit a more diverse set of mutation signatures than their adult counterparts. Further, pediatric cancers of different histologies, particularly those of brain cancers, may share molecular mechanisms more than currently believed. Citation Format: Francis A. San Lucas, Philip Lupo, Austin Brown, Michael Scheurer, Paul Scheet. Exploring mutation signatures in pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2085.

Abstract 5874: Targeting DDX3 in medulloblastoma by RK-33

Abstract Introduction: Medulloblastoma is the most common form of pediatric brain cancer. Current treatments of surgical resection, craniospinal irradiation, and chemotherapy increase survival but around 40% of patients experience disease recurrence. Classically, four molecular subgroups of medulloblastoma have been identified: Wnt; Sonic hedgehog (SHH); Group 3; and Group 4. More recent work has subtyped up to seven molecular subgroups. The RNA helicase DDX3 is involved in many biological activities such as transcription, mRNA translation, splicing, and nuclear export. About 8% of medulloblastoma cases involve mutations in DDX3, of which 11% involve the pediatric Wnt and SHH subtypes. In addition, coinciding DDX3 and CTNNB1 mutations associated with dysregulated Wnt /β-catenin signaling in Wnt-type medulloblastomas have also been reported. Aims and Methods: The goal of this work was to study whether RK-33, a small molecule inhibitor of DDX3, would be effective against medulloblastoma as a single agent and in combination with radiotherapy. We studied DDX3 expression in medulloblastoma cell lines DAOY and UW228 and human tissue microarrays by immunohistochemistry, western blotting, and qRT-PCR. We targeted DDX3 by siRNA and by RK-33 and observed proliferation and viability changes by MTS assays. Flow cytometric analysis was used to determine effect of RK-33 on cell cycle and apoptosis. TOP/FOP promoter assay was utilized to study effect on TCF promoter activity. We used colony formation assays to study effect of combining radiation and RK-33 treatment on cells. DAOY tumors were generated in nude mice and treated with combination therapy to study effects in vivo. Results: High DDX3 expression was observed in 55% of pediatric and 66% of adult medulloblastomas. DDX3 knockdown resulted in reduced cellular proliferation. Both the medulloblastoma cell lines were sensitive to RK-33, with IC50 values of 2.5 µM and 3.5 µM, respectively and resulted in decreased levels of DDX3 expression, with no observable toxicity. RK-33 resulted in a G1 phase cell cycle arrest before the onset of apoptosis. DDX3 knockdown resulted in a significant decrease of TCF activity and reduced transcription of Axin2, CCND1, MYC, and Survivin. A combination of RK-33 and radiation resulted in a significantly higher inhibitory effect than using single treatment of either RK-33 or radiation. We conclude that inhibiting DDX3 via the small molecule inhibitor RK-33 is effective in reducing medulloblastoma tumors and that the effect is synergistic with radiation treatment. Citation Format: Saritha Tantravedi, Farhad Vesuna, Allison Martin, Michael Lim, Charles G. Eberhart G. Eberhart, Paul J. van Diest, Venu Raman. Targeting DDX3 in medulloblastoma by RK-33 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5874.

RONC-14. REPLICATING CLINICAL RADIATION THERAPY PROTOCOLS IN PRECLINICAL BRAIN TUMOUR MODELS

Surgical resection and radiotherapy, often followed by chemotherapy, have been the mainstay of paediatric brain cancer treatment for decades. For brain cancers with a predilection to spread throughout the brain and spinal cord, such as medulloblastoma, craniospinal irradiation represents the standard-of-care. An unavoidable consequence of this is collateral damage of healthy tissue, resulting in potentially devastating long-term sequelae, including suboptimal cognitive development. Animal models that accurately recapitulate the genotypes and phenotypes of paediatric brain cancers are essential to evaluate potential new approaches to therapy ahead of clinical trial. The XRAD image-guided small animal radiotherapy (SmART) system combines CT imaging with precision irradiation using x-rays. Our aim was to optimise preclinical radiotherapy protocols in mouse models of medulloblastoma, ependymoma and glioblastoma. To evaluate the accuracy and efficacy of the device, radiation was administered to mice with orthotopically-implanted brain tumours. Tissue was harvested, and markers of DNA damage and apoptosis used to evaluate tumour targeting. A transgenic model of SHH-subtype medulloblastoma (NeuroD2::SmoA1) demonstrated exquisite sensitivity to irradiation with significant DNA damage and apoptosis two hours post-treatment. Moreover, reduced tumour size was evident by MRI after five days of radiotherapy. Multiple patient-derived xenograft (PDX) models of Group 3 medulloblastoma also demonstrated radiation sensitivity, whereas minimal anti-tumour effects were observed in ependymoma and glioblastoma xenografts. These data serve as baseline information for future experimental protocols that will evaluate combinatorial regimens of radiotherapy, conventional chemotherapy, and novel anti-cancer agents. These experiments enable rigorous evaluation and selection of viable agents to consider for future clinical evaluation.