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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.

BACKGROUND Inverted papilloma (IP) is a sinonasal tumor with a well-known potential for malignant transformation. The purpose of this study was to identify the genes and pathways associated with IP, with progression to carcinoma-in-situ and invasive carcinoma. METHODS To determine genes and molecular pathways that may indicate progression and correlate with histologic changes, we analyzed six IP without dysplasia, five IP with carcinoma-in-situ, and 13 squamous cell carcinoma ex-IP by targeted sequencing. The HTG EdgeSeq Oncology Biomarker Panel coupled with next-generation sequencing was used to evaluate 2560 transcripts associated with solid tumors. RESULTS Progressive upregulation of 11 genes were observed (CALD1, COL1A1, COL3A1, COL4A2, COL5A2, FN1, ITGA5, LGALS1, MMP11, SERPINH1, SPARC) in the order of invasive carcinoma > carcinoma-in-situ > IP without dysplasia. When compared with IP without dysplasia, more genes are differentially expressed in invasive carcinoma than carcinoma-in-situ samples (341 downregulated/333 upregulated vs. 195 downregulated/156 upregulated). Gene set enrichment analysis determined three gene sets in common between the cohorts (epithelial mesenchymal transition, extracellular matrix organization, and coagulation). CONCLUSIONS Progressive upregulation of genes specific to IP malignant degeneration has significant clinical implications. This panel of 11 genes will improve concordance of histologic classification, which can directly impact treatment and patient outcomes. Additionally, future studies on larger tumor sets may observe upregulation in the gene panel that preceded histologic changes, which may be useful for further risk stratification.

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

// Payal Jain 1,2,3 , Amanda Silva 1 , Harry J. Han 2 , Shih-Shan Lang 1,2 , Yuankun Zhu 1,3 , Katie Boucher 1,2,3 , Tiffany E. Smith 1,2,3 , Aesha Vakil 4 , Patrick Diviney 5 , Namrata Choudhari 1,2,3 , Pichai Raman 3,6,7 , Christine M. Busch 8 , Tim Delaney 1,2,3 , Xiaodong Yang 9 , Aleksandra K. Olow 10 , Sabine Mueller 9,11,12 , Daphne Haas-Kogan 13 , Elizabeth Fox 8 , Phillip B. Storm 1,2,3 , Adam C. Resnick 1,2,3,6,* and Angela J. Waanders 3,8,14,* 1 Division of Neurosurgery, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 2 Department of Neurosurgery, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 3 Center for Data Driven Discovery in Biomedicine (D3b), The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 4 The Fred Hutchinson Cancer Research Center, Seattle, WA, USA 5 Division of Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 6 Department of Biomedical and Health Informatics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 7 Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia, PA, USA 8 Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 9 Division of Neurology, University of California, San Francisco, CA, USA 10 Amgen, South San Francisco, CA, USA 11 Department of Neurosurgery, University of California, San Francisco, CA, USA 12 Department of Pediatrics, University of California, San Francisco, CA, USA 13 Department of Radiation Oncology, Harvard Medical School, Boston, MA, USA 14 Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA * These authors are co-senior authors and have contributed equally to this work Correspondence to: Angela J. Waanders, email: waandersA@email.chop.edu Keywords : pediatric low-grade glioma, BRAF-fusions, trametinib and everolimus, MAPK pathway, PI3K/mTOR pathway Received : March 03, 2017 Accepted : August 23, 2017 Published : September 15, 2017 Abstract Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.

Astrocytomas are the most common type of brain tumors in children. Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion genes typifying low-grade astrocytomas and V600E BRAF alterations characterizing distinct or higher-grade tumors. Recently, BRAF-targeted therapies, such as vemurafenib, have shown great promise in treating V600E-dependent melanomas. Like V600E BRAF, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation; however, here we characterized the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. We found that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrated that KIAA1549-BRAF fusion-mediated signaling is diminished with disruption of the BRAF kinase dimer interface. In addition, the KIAA1549-BRAF fusion displays increased binding affinity to kinase suppressor of RAS (KSR), an RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. Our data support the development of targeted treatment paradigms for BRAF-altered pediatric astrocytomas and also demonstrate that therapies must be tailored to the specific mutational context and distinct mechanisms of action of the mutant kinase.

The gene expression landscape for biomarker discovery is still limited for many pediatric brain tumors due to insufficient numbers of biorepository collected fresh frozen specimens. The majority of available tissue are formalin-fixed paraffin embedded (FFPE) pathology diagnostic specimens. These specimens, are often of limited quantity and contain compromised RNA material. A number of emerging commercial platforms are described as supporting quantitative expression analysis for low quantity and poor quality materials. Utilizing available platforms, we designed a study to evaluate RNA and miRNA levels in pediatric brain tumor FFPE specimens with limited/compromised access.Experiments were performed with specimens and/or data obtained from Brain Tumor Tissue Consortium (CBTTC) at Children's Hospital of Philadelphia (CHOP). The mRNA gene expression and miRNA target analysis were performed with FFPE material utilizing two commercial platforms: HTG EdgeSeq and Nanostring. The analysis included 5 specimens of low grade glioma or primitive neuroectodermal tumors for mRNA gene expression and 4 specimens of medulloblastoma for miRNA target analysis. FFPE specimens were processed according to manufacturer's protocols. The results were compared with the RNAseq or miRNA sequencing data derived from corresponding tumors' flash frozen specimens' RNA material. We evaluated requirements of each platform for data generation and established between platform analysis correlations. In performed tests, the HTG platform required lowest amounts of specimen's material. For the majority of analyzed genes (>700 genes), the gene expression profile was relatively similar between all three approaches, however each of the platform presented distinctive distribution profile for normalized data. The RNAseq mean read counts values were correlated highly with NanoString (0.81) and of lesser value with HTG (0.7) platform. The RNAseq presented significantly higher variance distribution than other platforms. The miRNA target analysis (>600 genes) distribution of normalized data revealed significantly lower dynamics for NanoString when compared with miRNAseq or HTG panel data. The miRNA mean read counts were highly correlated with HTG (0.77) and little with NanoString (0.22), while miRNASeq presented the highest variance distribution. In summary, we found a significant level of agreement between all three platforms tested for gene expression data generation. As for miRNA target analysis, the HTG platform presented significantly higher agreement with miRNAseq data. We conclude that tested technologies can support data generation from archived FFPE specimens, however, the platform selection process should involve pre-selection data quality analysis, as well as sample size requirements, gene cohort selection, and pricing evaluations. Citation Format: Mateusz P. Koptyra, Namrata Choudhari, Zhang Zhe, Mariarita Santi, Angela Waanders, Adam Resnick. Empowering rare disease cohort biomarker discovery via comparative assessments of gene expression analysis platforms for FFPE pediatric brain tumor specimens [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 2081.

Chimeric Antigen Receptor (CAR) T-cells made with mRNA offer a transient and safe alternative to viral CARs, mitigating the concern for persistent unwanted side effects from constitutively active T-cells. Previous studies have shown that mRNA CARs are transiently effective, but lack CAR persistence and have struggled to show success across tumor types. We hypothesized the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology including the use of modified uracil and a novel purification method with RNaseIII to prevent double stranding that induces toxicity. Using the established CD19 CAR model in B-cell acute lymphoblastic leukemia, we created mRNA CARs using previously described methods and compared them to mRNA CARs created using modification of uracil and/or purification to remove any aberrant double stranded mRNA. Comparing the modified and purified mRNA CAR T cells to those created using prior methods of making mRNA, both modified and purified mRNA CAR T cells showed a two-fold increase in expression of the CAR on their surface initially, as well as a two-fold improvement in cytotoxic killing of leukemia cells in vitro that persisted for up to five days. Both the modified and purified mRNA CAR T cells also showed reduced expression of negative checkpoint regulators compared to original RNA CAR T cells. However, in vivo studies using a patient-derived xenograft model with a single dose of CAR T cells revealed purified RNA CAR T cells offered the most robust 2-log enhanced suppression of leukemic burden. RNaseIII is a novel purification technique that has not yet been reported in the literature for RNA manufacturing. Our results provide a time efficient purification method that can be easily incorporated into RNA production for use in clinical trials, and poise RNA CARs for increased efficacy as new CAR targets emerge and are being tested. Additional studies are ongoing investigating if the observed improved efficacy will translate to improved cytotoxicity in solid tumor models. Citation Format: Jessica Foster, Namrata Choudhari, Adam Resnick, David Barrett. Novel mRNA purification method with RNaseIII improves efficacy of RNA chimeric antigen receptor T cells [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 5614. doi:10.1158/1538-7445.AM2017-5614

Chimeric Antigen Receptor (CAR) T-cells made with mRNA offer a transient and safe alternative to viral CARs, mitigating the concern for persistent unwanted side effects from constitutively active T-cells. Previous studies have shown that mRNA CARs are transiently effective, but lack CAR persistence and have struggled to show success across tumor types. We hypothesized the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology including the use of modified nucleosides and novel purification methods of the RNA to prevent double stranding that induces toxicity. Using the established CD19 CAR model in B-cell leukemia, we created mRNA CARs using previously described methods and compared them to mRNA CARs created using the modifications of pseudo-uridine or N-methyl pseudo-uridine instead of uridine, and purification to remove any aberrant double stranded mRNA. Comparing the modified and purified mRNA CAR T cells to those created using prior methods of making mRNA, the novel mRNA CAR T cells showed a two-fold increase in expression of the CAR on their surface initially, as well as a two-fold improvement in cytotoxic killing of leukemia cells that persisted for up to five days. The modified and purified mRNA CAR T cells also showed reduced expression of negative checkpoint regulators compared to original RNA CAR T cells. In vivo studies using a leukemia mouse model with a single dose of CAR T cells also showed marked improvement using modified and purified mRNA CAR T cells, with purified RNA showing the most robust 2-log enhanced suppression of leukemic burden. Our results provide a novel technique that can be easily incorporated into RNA production for use in clinical trials, and poise RNA CARs for increased efficacy as new CAR targets emerge and are being tested. Citation Format: Jessica Foster, Namrata Choudhari, David Barrett, Adam Resnick. Novel mRNA manufacturing techniques using modification of nucleosides and purification improves the efficacy of mRNA chimeric antigen receptor T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B083.

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP 3 as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP 3 generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.

Low-grade gliomas are the most common tumors of the central nervous system in children. We recently described a 7q34 duplication resulting in a novel KIAA1549-BRAF fusion, that includes the C-terminus BRAF kinase domain, exclusively in the majority of pediatric low-grade gliomas. BRAF activating mutations have been well described in human malignancies; however, little is known about KIAA1549. In silico analysis of the fusion gene predicts two putative transmembrane domains in the N-terminus segment of KIAA1549. Clones of the fusion were created, including the long and short forms found in pediatric tumors and a truncated version lacking a transmembrane domain. NIH/3T3 stable cell lines for each clone were then generated using Plat-E retrovirus. Assays were also performed with transient over-expression of the clones in HEK/293T cells. Activity of the fusion clones was compared to BRAF (wild-type, kinase-dead, V600E mutant), KIAA1549 (wild-type), and Rasv12 mutant. The KIAA1549-BRAF fusion gene constructs all demonstrated activation of the MAPK pathway with increased phosphylation of both MEK 1/2 and ERK1/2; malignant transformation in soft agar assay; and increased transcriptional output. In vivo studies are ongoing but preliminary results demonstrate the KIAA1549-BRAF fusion is sufficient to cause tumor formation in a xenograft murine model. Overall, our sets of experiments reveal the truncated fusion has kinase activity comparable to the canonical V600E BRAF mutant. Wild-type KIAA1549 had no effect on MEK1/2 and ERK1/2 phosphorylation nor does over-expression cause cell transformation. Not surprising RAF specific and MEK specific inhibition resulted in decreased MEK1/2 phosphorylation in BRAF (all variants) and KIAA1549-BRAF fusion transient over-expression in HEK/293T cells and in IP/BRAF kinase assays. Interestingly, RAF specific inhibition of the KIAA1549-BRAF fusion NIH/3T3 stable cell lines resulted in increased MEK 1/2 phosphorylation at low drug concentrations (0.01-10 micromolar) consistent with recent reports describing paradoxical activation of the MAPK pathway in wild-type BRAF. Drug concentrations of greater than 20 micromolar demonstrated inhibition of all of the constructs. Our data suggest the KIAA1549-BRAF fusion gene has oncogenic potential and is sufficient for tumor formation; however, BRAF kinase specific inhibition can result in paradoxical activation of the MAPK pathway at lower drug concentrations. Targeting the KIAA1549-BRAF fusion in pediatric low-grade gliomas is likely still a rational approach but further preclinical studies are needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3457. doi:10.1158/1538-7445.AM2011-3457