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MRI is the current gold standard imaging technique for diagnostic evaluation and monitoring of pediatric CNS tumors, however MRI measures are unable to fully relate to tumor biology and molecular stratification. Circulating in blood and cerebrospinal fluid (CSF), miRNAs are an abundant and stable nucleic acid which can be utilized as a tumor biomarker. Relating miRNA biomarkers and radiological tumor measurements may provide improved diagnostic and monitoring tools for pediatric brain tumors. Using a cohort of 54 pediatric brain tumors including low grade glioma, ependymoma, germ cell tumor, medulloblastoma, atypical teratoid rhabdoid tumor and high-grade glioma we attempted to combine MRI findings and circulating miRNA data. The miRNA expression was profiled in 33 CSF and 52 plasma samples using the HTG EdgeSeq platform. Clinically acquired, multi-parametric MRI scans at time-points close in proximity to liquid biopsy collection were collected retrospectively and used to generate volumetric tumor segmentations. We identified unique miRNA targets significantly correlated with MRI features, clinical findings, and patient outcomes. In both CSF and plasma, miRNA expression was identified to correlate with diagnosis and clinical features including tumor grade and survival status (p < 0.05). In CSF, miRNA expression was correlated with MRI measurements including cystic core volume, non-enhancing tumor volume, leptomeningeal disease, tumor size and location (p < 0.05). Combination of miRNA targets and radiomic tumor measurements improved diagnostic predictions between low- and high-grade tumors. In plasma, miRNA expression was correlated with MRI measurements including cystic core volume, location, and leptomeningeal disease (p < 0.05). These results demonstrate utility of miRNAs as a pediatric brain tumor biomarker which combined with imaging features can improve minimally to non-invasive diagnostics and management of pediatric brain tumors.

Current clinical management of pediatric brain tumor patients involves non-invasive imaging studies to monitor therapeutic response and tumor progression. However, results are often inconclusive and unable to capture biological changes that presage progression on imaging. Non-invasive diagnostics, also termed liquid biopsy, have emerged for detection of cell-free cancer material but there are no such standard, clinically defined biomarkers or methods for pediatric brain tumors. Circulating miRNA presents an attractive biomarker platform given its stability in bio-fluids, selective expression in tumors and release from tumor cells into the extracellular environment. Technology development has permitted high throughput analysis of material obtained from biofluids including plasma and cerebrospinal fluid (CSF). We performed miRNA profiling across a cohort of 54 pediatric brain tumors from different histologies (low grade glioma, ependymoma, germinoma, medulloblastoma, atypical teratoid rhabdoid tumor and high-grade glioma) using CSF (33) and plasma (53) with HTG EdgeSeq platform. CSF and plasma specimens clustered independently of each other providing separate biomarker platforms. Consensus clustering performed on CSF specimens revealed clusters correlated with disease severity (tumor grade). We identified miRNA targets closely correlated with tumor grade (p

PURPOSE: Pediatric high grade glioma (pHGG) remains a fatal disease. Increasing the number of patient derived tumor models and reagents will encourage research in pHGG and support the translation of basic science research discoveries. This work describes a recent multi-institution initiative to provide such a resource. METHODS: pHGG tumors with associated clinical data were prospectively collected and sequenced through the Children’s Brain Tumor Tissue Consortium (CBTTC) and Pediatric Brain Tumor Atlas (PBTA) with data deposited into PedcBioPortal for easy access and visualization. Primary tumor was dissociated and cultured to create both adherent and glioma stem cell lines analyzed by targeted and WGS/RNA sequencing. A tissue microarray (TMA) of primary pHGG tumors was created and examined by immunohistochemistry. RESULTS: The pHGG set included 81 collection events (70 patients, 54% at diagnosis, median age of 11 yrs, 52% female, 43% hemispheric). Analysis of somatic mutations and copy number alterations of known glioma genes were of expected distribution (36% H3.3, 47% TP53, 24% ATRX and 7% BRAF V600E variants). There were rare germline variants in mismatch repair genes with resultant tumor hyper-mutation. At least one established adherent and/or suspension glioma stem cell line was generated from 22 (31%) patients and validated by targeted sequencing for driver pHGG mutations. A subset of lines had WGS and RNAseq profiling which computationally clustered with their parental primary tumors. The lines’ cell biology makeup and in vivo tumor formation was also evaluated. A pHGG TMA (n=77), including 37 (53%) of the sequenced pHGG tumors, showed concordance with mutation status when stained for ATRX, H3.3 K27M and TP53. CONCLUSION: The CBTTC and PBTA have created a freely available integrated resource of characterized pHGG primary tumors, corresponding cell lines and archival fixed tissue to advance translational research for pHGG.

The Children’s Brain Tumor Tissue Consortium (CBTTC) is a multi-institutional collaborative research program dedicated to accelerating pediatric brain tumor research. Tumor specimens collected by CBTTC undergo multidimensional tumor analysis including genomic, transcriptomic and proteomic data generation, providing freely available data for researchers worldwide. Moreover, CBTTC advances pediatric research efforts by generating cell lines and and patient-derived xenografts (PDX) providing these freely available materials for researchers worldwide. High-grade gliomas (HGG) in children is the most prevalent among brain tumors with a dismal prognosis. Preclinical testing has been limited due to lack of pediatric specific HGG in vitro and in vivo models. Here we present a cohort of pediatric HGG cell lines developed within the CBTTC. The cells were derived from 10 subjects from fresh or frozen tumor tissues and generated in different culture conditions. Results include the cells’ basic molecular characteristics and tumor formation in vivo, which presents valuable information for preclinical studies. We also report how our cell culture conditions affect the morphology, growth and genetic makeup of developed tumor cell lines. These results can intersect with tumor molecular profiling to provide a transparent view of cell line utility for cell biology and drug development studies. This project serves as an example of CBTTC’s pipeline for cell line development, and brings important insights for the processes of brain tumor specimen derived tool generation.

INTRODUCTION: Activating BRAF-fusion mutations occur frequently in pediatric low-grade gliomas (PLGGs). We have previously shown targeting of KIAA1549-BRAF fusion by PLX4720, the research analog of vemurafenib, results in paradoxical activation of the mitogen activated protein kinase (MAPK) signal cascade. We hypothesized that BRAF-fusions could be targeted by downstream MEK inhibitors (MEKi), and that combinatorial targeting of BRAF-fusions could also prevent acquired resistance to single agent therapy. METHODS: BRAF-fusions were screened with a selection of clinically relevant MEKi and with PLX8394, a second-generation RAF inhibitor, in vitro in cell culture and soft agar. BRAF V600E and RAS mutant cells were also screened for comparison. MEKi/PLX8394 resistant BRAF-fusion cell lines were generated in vitro and interrogated by sequencing, immunoblotting, and RNA-seq analysis to identify potential co-targets. Combinatorial therapy was then tested in vitro and in vivo to confirm efficacy. RESULTS: BRAF-fusions in PLGGs can be targeted by MEKi and by the pan-RAF inhibitor PLX8394. AZD6244, MEK162, GDC0623, and GDC0973 all inhibited downstream ERK phosphorylation; however, GSK1120212 more robustly inhibited MAPK activation in the BRAF-fusion, BRAF V600E, and RAS mutant cells. Interestingly, the MEKi/PLX8394 resistant BRAF-fusion cells all showed activation of phosphatidylinositol 3-kinase (PI3K) pathway. BRAF-fusion parental cells were screened with RAD001, a targeted mTOR inhibitor, resulting in modest downstream inhibition. Co-targeting of the parental BRAF-fusions with GSK1120212 and RAD001 resulted in robust inhibition of down-stream signaling and in significant tumor inhibition, and prevented acquired resistance to targeted inhibition. CONCLUSION: Our work demonstrates that BRAF-fusion oncogenes can be effectively targeted with GSK1120212 as single agent but even more robustly with GSK1120212 and RAD001 co-targeting. This provides preclinical rationale for treating PLGGs harboring BRAF-fusion oncogenes with combinatorial MEK and mTOR inhibitors in clinical trial.