Abstract Unlike many high-grade tumors, low-grade glioma (LGG) has faced challenges in advancing scientific and clinical breakthroughs, affected by the limited capacity to develop representative ...preclinical tumor models. Major barriers to establishing traditional LGG cell culture systems have been cellular senescence and the need to introduce additional genetic modifications to obtain cultured growth. Such LGG models, while extremely valuable, require extensive periods for development and result in clonally selected lines which may not present the complexity of LGG tumor biology. To mitigate such challenges in LGG model development and address the need for three-dimensional tissue cultures, we have successfully generated organoids from fresh tissue specimens obtained from surgical resection. We have demonstrated success of this method on various pediatric brain tumor histologies and have expanded these approaches successfully to LGG. Through such efforts, we have developed 14 LGG tumors’ organoid models as well as other low-grade tumor’s models such as craniopharyngioma, schwannoma, meningioma, optic glioma, and oligodendroglioma. The tissue was processed immediately post-extraction and cultured in media solution on rotating platform. Organoid growth was observed within 1-3 weeks of initiation and tested for growth for up to 3 months. Phenotypic analysis revealed organoid cell composition representing clinical histology. The immune component was preserved in organoids for up to 40 days. MAPK pathway activity, a hallmark marker for KIAA-BRAF fusion positive pilocytic astrocytoma was preserved for up to 4 weeks. Our successful deployment of three-dimensional patient-tissue derived organoids provides for an efficient, simple workflow for the generation of unique and transformative preclinical models for the LGG field. Supported by the Children’s Brain Tumor network’s open science model, 3D organoids will provide investigations with broader resources for LGG tumor biology preclinical testing and support translational studies on behalf of the development of novel clinical trials.
INTRODUCTION:
Liquid biopsy is a non-invasive method of diagnosing and monitoring brain tumor patients. miRNA profiling from cerebrospinal fluid (CSF) and plasma has become an increasingly attractive ...biomarker, however targets have yet to be established in CNS tumors. Low grade glioma (LGG) patients often have excellent outcomes, but some have progressive disease or unresectable lesions. There has been limited work in establishing a clinically approved liquid biopsy assay for such patients.
METHODS:
miRNA profiling of 17 plasma and 12 CSF samples from 10 pediatric LGG and 7 DMG patients was performed using the HTG EdgeSeq platform. Clinical information was obtained through chart review. miRNA expression was evaluated with unsupervised consensus clustering and differential analysis.
RESULTS:
At time of data analysis, 5 LGG had progression or recurrence. The remaining LGG (5) had stable or no evidence of disease. Mean follow-up was 23 months. miRNA consensus clustering revealed that LGG with a recurrence, progression, or malignant transformation clustered with DMG rather than with tumors of similar histology or WHO classification. Pilocytic astrocytoma could be identified by miRNA composition in CSF. In plasma, 103 unique miRNAs were identified with significant differential expression between the progression/recurrence LGG cohort versus those with stable or no evidence of disease (logFC > 1, FDR < 0.05). miR-16-5p, a miRNA correlated with tumor growth and invasion in non-CNS tumors, had higher expression in the more aggressive cohort (p = 0.0022). Over 100 miRNAs correlated with histology, survival, disease state, and presence of leptomeningeal disease (p < 0.05).
CONCLUSIONS:
miRNA profiling of CSF and plasma in patients with LGG may predict disease course to better inform patient care and treatment monitoring.
Abstract
Current management of pediatric brain tumor patients relies on imaging as the only non-invasive tool available for monitoring therapeutic response and tumor progression. Results are often ...inconclusive and unable to capture biological changes that presage progression on imaging. Circulating miRNA provides an attractive non-invasive platform because miRNA’s essential role in regulating gene expression, stability in bio-fluids, selective over-expression in tumors, and subsequent active release from tumors into the extracellular environment. We aimed to establish the prognostic and monitoring value of miRNA biomarkers for pediatric brain tumors. We performed miRNA profiling of CSF (n=33) and plasma (n=53) samples across a cohort of 54 pediatric brain tumors from 6 histologies (low grade glioma, ependymoma, germ cell tumor, medulloblastoma, atypical teratoid rhabdoid tumor and high-grade glioma). We utilized unsupervised consensus clustering, differential expression, and machine learning methods to explore miRNA biomarkers. Plasma miRNA signatures were correlated with WHO Grade while CSF signatures revealed a correlation with leptomeningeal disease and higher disease burden (p<0.05). The expression of circulating miRNA revealed histology-specific signatures for low grade glioma, medulloblastoma and germinoma in plasma and CSF (p<0.05). We identified miRNA signatures of significant translational value: (1) A medulloblastoma specific miRNA in CSF can serve as a potential biomarker for tumor monitoring (medulloblastoma specificity was confirmed in independent tissue-based dataset); (2) Pilocytic astrocytoma can be distinguished from high grade glioma in CSF; (3) Most intriguingly, in plasma, miRNA expression identified pilocytic astrocytoma that had relapsed in several months after initial intervention (p<0.05). These results support circulating miRNA in plasma and CSF as biomarkers of diverse clinical diagnostic and prognostic measures. Further utilization of this approach provides unique platforms to inform liquid biopsy-based management in the clinical setting.
Tumors expressing the ABL oncoproteins (BCR/ABL, TEL/ABL, v-ABL) can avoidapoptosis triggered by DNA damaging agents. The tumor suppressor protein p53 is animportant activator of apoptosis in normal ...cells; conversely its functional loss may causedrug resistance. The ABL oncoprotein - p53 paradigm represents the relationship between anoncogenic tyrosine kinase and a tumor suppressor gene. Here we show that BCR/ABLoncoproteins employ p53 to induce resistance to DNA damage in myeloid leukemia cells.Cells transformed by the ABL oncoproteins displayed accumulation of p53 upon DNAdamage. In contrast, only a modest increase of p53 expression followed by activation ofcaspase-3 were detected in normal cells expressing endogenous c-ABL. Phosphatidylinositol-3 kinase-like protein kinases (ATR and also ATM) -dependent phosphorylation of p53-Ser15residue was associated with the accumulation of p53, and stimulation of p21Waf-1 andGADD45, resulting in G2/M delay in BCR/ABL cells after genotoxic treatment. Inhibition ofp53 by siRNA or by the temperature-sensitive mutation reduced G2/M accumulation anddrug resistance of BCR/ABL cells. In conclusion, accumulation of the p53 proteincontributed to prolonged G2/M checkpoint activation and drug resistance in myeloid cellsexpressing the BCR/ABL oncoproteins.
Abstract
Sodium glucose cotransporters (SGLT) are a class of glucose transporters independent of the conventional glucose transporters (GLUT). Like the GLUT, SGLT2 plays an important role in glucose ...uptake in many solid cancers. Importantly, SGLT2 inhibitors are a new class of anti-diabetic drugs and have recently been investigated as a potential anti-cancer therapy in a variety of solid cancers. In fact, a recent study in adult high-grade gliomas showed that an SGLT-specific PET imaging probe (18F Me-4FDG) is sensitive for visualization of high-grade glioma (HGG) with superior tumor-normal brain uptake relative to 18F-FDG-PET. We aimed to investigate the expression of SGLT2 within pediatric brain tumor subtypes using RNA sequencing data from PedcBioPortal and immunohistochemistry of tissue microarrays (TMAs). Using the RNA sequencing data from 603 patients with pediatric brain tumors, we found that HGGs have the highest SGLT2 expression (Z score=0.13± 1.03) and were significantly higher compared to low-grade gliomas (Z score= -0.12, p=0.049), medulloblastomas (Z score=-0.28, p=0.001), and ependymomas (Z score=-0.30, p=0.002). Atypical teratoid rhabdoid tumors (ATRT) also had significantly higher Z score (0.10±0.67) compared to medulloblastomas (p=0.038), and ependymomas (p=0.049). There was no significant difference between HGGs and ATRTs in SGLT2 expression (p=0.899). Analysis of 4 TMAs including HGGs, medulloblastomas, ependymomas, and ATRTs after SGLT2 immunohistochemistry demonstrated strong membranous staining in a small subset and moderate-weak staining in the majority of HGGs. The majority of ATRTs were positive. The majority of medulloblastomas and ependymomas were negative, noting that the membranous nature of the staining may be a limiting factor in evaluation of tumors with less cytoplasm. In conclusion, our work demonstrates that SGLT2 has heterogeneous expression in pediatric brain tumors and is overexpressed in the majority of ATRTs, and a subset of pediatric HGGs, and may represent a compelling paradigm that integrates metabolic imaging and therapy of these tumors.
Abstract
Brain tumors have become the leading cause of cancer-related death in children. An important hurdle to scientific and clinical progress in the field has been the limited availability of ...preclinical tumor models. Historically, few pediatric brain tumor cell lines have been established and these often poorly recapitulate the phenotypes of the original tumors. In recent years, the Children’s Brain Tumor Network (CBTN) has accelerated the development of patient-derived cell lines and xenografts, offering these resources to the community through open-source access. While these models are extremely valuable, their development process can be lengthy and result in clonally selected lines which presents a challenge for studying complex tumor biology. To address the need for three-dimensional tissue culture, our group in conjunction with CBTN, utilized organoid culture from fresh tissue specimens obtained directly from surgical resection of various pediatric brain tumor histologies. This resulted in the development and banking of over 30 organoid models, which included ependymoma, high-grade glioma, medulloblastoma, atypical teratoid-rhabdoid tumor, diffuse midline glioma, and low-grade glioma diagnoses. Tissue was processed within an hour post extraction and cultured with universal media composition for each diagnosis. Organoid growth was observed within 2-3 weeks of initiation and continued for up to three months before banking. Banked organoids established growth upon return to culture. Phenotypic analysis revealed organoid cell composition that represented clinical histology. Importantly, organoids returned to culture post-banking demonstrated similar cell composition to those in the original culture, indicating their utility for subsequent preclinical testing. Here we provide a simple and efficient workflow for the generation and characterization of three-dimensional tumor organoids generated from fresh surgical pediatric brain tumor tissue. The platform has the potential to accelerate investigations into tumor biology and empower a diverse array of translational studies for the pediatric brain tumor field.
Abstract
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<0.001), tumor dissemination or metastases (p<0.001) and survival (p=0.001). Similarly in plasma, through consensus clustering we identified cohorts with correlation to tumor grade. While distinct from those identified in CSF, specific plasma miRNA also correlated with clinical (tumor grade; p<0.001, dissemination or metastasis; p=0.002) and demographic (gender; p=0.001) features. Independently, histology-specific miRNA signature was also identified for low grade glioma, medulloblastoma and germinoma in plasma and CSF. The results present differential expression of cell-free miRNAs in plasma and CSF as biomarkers associated with diverse clinical diagnostic and prognostic measures. Further utilization of this approach provides unique platforms (plasma and/or CSF) to inform liquid biopsy-based management in the clinical setting.
Abstract
There is mounting evidence that tumor microenvironmental pressure selects for somatic genetic alterations that contribute to the formation of distinct morphological characteristics, captured ...on radiology scans as radio-phenotypes. Such phenotypic variations are a source of heterogeneity in clinical manifestation of tumors of the same histology across the patients, and in part, their heterogeneous responses to therapies. Deeper understanding of the associations between genotype and radio-phenotype in pediatric low-grade glioma (pLGG), the most histologically diverse childhood brain tumor, may facilitate precision diagnostic and therapeutic approaches. Here, we categorize pLGGs into distinct and relatively homogeneous imaging subtypes based on radiomic features and further explore the associations of these imaging subtypes with genotype. From multiparametric MRI scans of 167 pLGGs from the Children’s Brain Tumor Network (CBTN), 881 radiomic features and clinical variables (tumor location, age, and gender) were extracted. After dimensionality reduction using principal component analysis (PCA), K-Means clustering was applied on 19 principal components to group the patients into three imaging subtypes. Using whole transcriptomic data from OpenTargets, differential expression and co-expression of network- and pathway-level and immune-related signaling were compared among these three imaging subtypes. Gene Set Enrichment Analysis (GSEA) revealed differentially higher expression of cell cycle regulatory, extracellular matrix (ECM) remodeling, and cell migratory pathways in imaging subtype1 than subtypes 2 and 3, and upregulation of ECM and immune-related pathways in subtypes 1 and 2 compared to subtype3. Based on Gene Sets Net Correlations Analysis (GSNCA), subtype1 exhibited differential co-regulation of TNF/TNFR1 signaling compared to subtype2, and differential co-regulation of RHOG GTPase and TGFB1pathways when compared against subtype3. Subtype2 showed differential co-regulation in NOTCH1 signaling and transcriptional regulatory pathways. Our proposed multi-disciplinary radiomic-genomic analysis approach elucidates the molecular and biological processes in the genotype of the tumors that are associated with emergence of distinct imaging subtypes in pLGG.
Abstract
The involvement of patient families in clinical research in pediatric brain cancer is an underutilized tool that benefits all those involved. These families are ultimately responsible for ...making decisions on treatment, as well as end-of-life decisions such as post-mortem tissue donation. As demonstrated by organizations such as Gift from a Child, listening to family input on how to best have the conversation surrounding post-mortem tissue donation has led to an increase in total donations, as well as parents stating they are overwhelmingly happy with their decision to donate. Tissue donation is just one example of a large, complex hindrance in the field that had not previously been addressed successfully. Due to the rarity and devastating impact of pediatric brain tumors, the family community is tight-knit and passionate. There is incredibly impactful communication that happens between families influencing decision-making for treatment, funding, and advocacy. Partnering with families allows care teams and researchers both the ability to understand the decision-making process as well as educate and discuss with the families. Surveys of families overwhelmingly show satisfaction with the decision to donate post-mortem and dissatisfaction when they are deprived of the option. Frustration with the lack of treatment has led families to become more proactive in both their child’s treatment and future research. Increasingly, family advocates are becoming more sophisticated in their support of research opportunities. An example of this spearheading the creation of an open access, safe, anonymized data bank with clinical data paired to the patient’s tumor tissue. Family foundations are actively partnering with researchers and industry to make it happen. It is clear that this type of partnership is a strong force for change that benefits researchers, clinicians, and most importantly, patients and their families.
Abstract
Pediatric Central Nervous System (CNS) tumors are the leading cause of cancer-related death among children. Identifying new targeted therapies necessitates the use of pediatric cancer models ...that faithfully recapitulate the patient’s disease. However, the generation and characterization of pediatric cancer models has significantly lagged adult cancers, underscoring the urgent need to develop and characterize pediatric CNS models of disease. Herein, we establish a single-site collection of 233 CNS tumour cell lines, representing 14 distinct brain childhood tumor types. We subjected >200 cell lines to multi-omics analyses (DNA-sequencing, RNA-sequencing, DNA methylation, proteomics, phospho-proteomics), and in parallel performed pharmacological and genetic CRISPR-Cas9 loss of function screens to identify pediatric-specific treatment opportunities and biomarkers. Our work provides insight into specific pathway vulnerabilities in molecularly defined pediatric tumor classes and uncovers biomarker-linked therapeutic opportunities of clinical relevance. Cell line data and resources are provided in an open access portal (vicpcc.org.au/dashboard).
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