ABSTRACT
Pediatric low-grade gliomas (pLGG) comprise 35% of all brain tumors. Despite favorable survival, patients experience significant morbidity from disease and treatments. A deeper understanding of pLGG biology is essential to identify novel, more effective, and less toxic therapies. We utilized single cell RNA sequencing (scRNA-seq), spatial transcriptomics, and cytokine analyses to characterize and understand tumor and immune cell heterogeneity across pLGG. scRNA-seq revealed tumor and immune cells within the tumor microenvironment (TME). Tumor cell subsets revealed a developmental hierarchy with progenitor and mature cell populations. Immune cells included myeloid and lymphocytic cells. There was a significant difference between the prevalence of two major myeloid subclusters between pilocytic astrocytoma (PA) and ganglioglioma (GG). Bulk and single-cell cytokine analyses evaluated the immune cell signaling cascade with distinct immune phenotypes among tumor samples. KIAA1549-BRAF tumors appeared more immunogenic, secreting higher levels of immune cell activators and chemokines, compared to BRAF V600E tumors. Spatial transcriptomics revealed the differential gene expression of these chemokines and their location within the TME. A multi-pronged analysis of pLGG demonstrated the complexity of the pLGG TME and differences between genetic drivers that may influence their response to immunotherapy. Further investigation of immune cell infiltration and tumor-immune interactions is warranted. Key points: There is a developmental hierarchy in neoplastic population comprising of both progenitor-like and mature cell types in both PA and GG.A more immunogenic, immune activating myeloid population is present in PA compared to GG. Functional analysis and spatial transcriptomics show higher levels of immune mobilizing chemokines in KIAA1549-BRAF fusion PA tumor samples compared to BRAF V600E GG samples. Importance of the Study: While scRNA seq provides information on cellular heterogeneity within the tumor microenvironment (TME), it does not provide a complete picture of how these cells are interacting or where they are located. To expand on this, we used a three-pronged approach to better understand the biology of pediatric low-grade glioma (pLGG). By analyzing scRNA-seq, secreted cytokines and spatial orientation of cells within the TME, we strove to gain a more complete picture of the complex interplay between tumor and immune cells within pLGG. Our data revealed a complex heterogeneity in tumor and immune populations and identified an interesting difference in the immune phenotype among different subtypes.
ABSTRACT
BACKGROUND: The mitogen-activated protein kinases/extracelluar signal-regulated kinases pathway is involved in cell growth and proliferation, and mutations in BRAF have made it an oncogene of interest in pediatric cancer. Previous studies found that BRAF mutations as well as KIAA1549-BRAF fusions are common in intracranial low-grade gliomas (LGGs). Fewer studies have tested for the presence of these genetic changes in spinal LGGs. The aim of this study was to better understand the prevalence of BRAF and other genetic aberrations in spinal LGG. METHODS: We retrospectively analyzed 46 spinal gliomas from patients aged 1-25 years from Children's Hospital Colorado (CHCO) and The Hospital for Sick Children (SickKids). CHCO utilized a 67-gene panel that assessed BRAF and additionally screened for other possible genetic abnormalities of interest. At SickKids, BRAF V600E was assessed by droplet digital polymerase chain reaction and immunohistochemistry. BRAF fusions were detected by fluorescence in situ hybridization, reverse transcription polymerase chain reaction, or NanoString platform. Data were correlated with clinical information. RESULTS: Of 31 samples with complete fusion analysis, 13 (42%) harbored KIAA1549-BRAF. All 13 (100%) patients with confirmed KIAA1549-BRAF survived the entirety of the study period (median [interquartile range] follow-up time: 47 months [27-85 months]) and 15 (83.3%) fusion-negative patients survived (follow-up time: 37.5 months [19.8-69.5 months]). Other mutations of interest were also identified in this patient cohort including BRAF V600E , PTPN11, H3F3A, TP53, FGFR1, and CDKN2A deletion. CONCLUSION: KIAA1549-BRAF was seen in higher frequency than BRAF V600E or other genetic aberrations in pediatric spinal LGGs and experienced lower death rates compared to KIAA1549-BRAF negative patients, although this was not statistically significant.
ABSTRACT
BACKGROUND: Atypical teratoid/thabdoid tumor (AT/RT) remains a difficult-to-treat tumor with a 5-year overall survival rate of 15%-45%. Proteasome inhibition has recently been opened as an avenue for cancer treatment with the FDA approval of bortezomib (BTZ) in 2003 and carfilzomib (CFZ) in 2012. The aim of this study was to identify and characterize a pre-approved targeted therapy with potential for clinical trials in AT/RT. METHODS: We performed a drug screen using a panel of 134 FDA-approved drugs in 3 AT/RT cell lines. Follow-on in vitro studies used 6 cell lines and patient-derived short-term cultures to characterize selected drug interactions with AT/RT. In vivo efficacy was evaluated using patient derived xenografts in an intracranial murine model. RESULTS: BTZ and CFZ are highly effective in vitro, producing some of the strongest growth-inhibition responses of the evaluated 134-drug panel. Marizomib (MRZ), a proteasome inhibitor known to pass the blood-brain barrier (BBB), also strongly inhibits AT/RT proteasomes and generates rapid cell death at clinically achievable doses in established cell lines and freshly patient-derived tumor lines. MRZ also significantly extends survival in an intracranial mouse model of AT/RT. CONCLUSIONS: MRZ is a newer proteasome inhibitor that has been shown to cross the BBB and is already in phase II clinical trials for adult high-grade glioma (NCT NCT02330562 and NCT02903069). MRZ strongly inhibits AT/RT cell growth both in vitro and in vivo via a moderately well-characterized mechanism and has direct translational potential for patients with AT/RT.
