Risk factors for glioblastoma are shared by other brain tumor types.

The reported risk factors for glioblastoma (GBM), i.e., ionizing radiation, Li-Fraumeni syndrome, Neurofibromatosis I, and Turcot syndrome, also increase the risk of other brain tumor types. Risk factors for human GBM are associated with different oncogenic mutation profiles. Pedigreed domestic dogs with a shorter nose and flatter face (brachycephalic dogs) display relatively high rates of glioma formation. The genetic profiles of canine gliomas are also idiosyncratic. The association of putatively different mutational patterns in humans and canines with GBM suggests that different oncogenic pathways can result in GBM formation. Strong epidemiological evidence for an association between exposure to chemical carcinogens and an increased risk for development of GBM is currently lacking. Ionizing radiation induces point mutations, frameshift mutations, double-strand breaks, and chromosomal insertions or deletions. Mutational profiles associated with chemical exposures overlap with the broad mutational patterns seen with ionizing radiation. Weak statistical associations between chemical exposures and GBM reported in epidemiology studies are biologically plausible. Molecular approaches comparing reproducible patterns seen in spontaneous GBM with analogous patterns found in GBMs resected from patients with known significant exposures to potentially carcinogenic chemicals can address difficulties presented by traditional exposure assessment.

89Zr-labeled ImmunoPET targeting the cancer stem cell antigen CD133 using fully-human antibody constructs.

BACKGROUND: Cancer stem cells play an important role in driving tumor growth and treatment resistance, which makes them a promising therapeutic target to prevent cancer recurrence. Emerging cancer stem cell-targeted therapies would benefit from companion diagnostic imaging probes to aid in patient selection and monitoring response to therapy. To this end, zirconium-89-radiolabeled immunoPET probes that target the cancer stem cell-antigen CD133 were developed using fully human antibody and antibody scFv-Fc scaffolds. RESULTS: ImmunoPET probes [89Zr]-DFO-RW03IgG (CA = 0.7 ± 0.1), [89Zr]-DFO-RW03IgG (CA = 3.0 ± 0.3), and [89Zr]-DFO-RW03scFv - Fc (CA = 2.9 ± 0.3) were radiolabeled with zirconium-89 (radiochemical yield 42 ± 5%, 97 ± 2%, 86 ± 12%, respectively) and each was isolated in > 97% radiochemical purity with specific activities of 120 ± 30, 270 ± 90, and 200 ± 60 MBq/mg, respectively. In vitro binding assays showed a low-nanomolar binding affinity of 0.6 to 1.1 nM (95% CI) for DFO-RW03IgG (CA = 0.7 ± 0.1), 0.3 to 1.9 nM (95% CI) for DFO-RW03IgG (CA = 3.0 ± 0.3), and 1.5 to 3.3 nM (95% CI) for DFO-RW03scFv - Fc (C/A = 0.3). Biodistribution studies found that [89Zr]-DFO-RW03scFv - Fc (CA = 2.9 ± 0.3) exhibited the highest tumor uptake (23 ± 4, 21 ± 2, and 23 ± 4%ID/g at 24, 48, and 72 h, respectively) and showed low uptake (< 6%ID/g) in all off-target organs at each timepoint (24, 48, and 72 h). Comparatively, [89Zr]-DFO-RW03IgG (CA = 0.7 ± 0.1) and [89Zr]-DFO-RW03IgG (CA = 3.0 ± 0.3) both reached maximum tumor uptake (16 ± 3%ID/g and 16 ± 2%ID/g, respectively) at 96 h p.i. and showed higher liver uptake (10.2 ± 3%ID/g and 15 ± 3%ID/g, respectively) at that timepoint. Region of interest analysis to assess PET images of mice administered [89Zr]-DFO-RW03scFv - Fc (CA = 2.9 ± 0.3) showed that this probe reached a maximum tumor uptake of 22 ± 1%ID/cc at 96 h, providing a tumor-to-liver ratio that exceeded 1:1 at 48 h p.i. Antibody-antigen mediated tumor uptake was demonstrated through biodistribution and PET imaging studies, where for each probe, co-injection of excess unlabeled RW03IgG resulted in > 60% reduced tumor uptake. CONCLUSIONS: Fully human CD133-targeted immunoPET probes [89Zr]-DFO-RW03IgG and [89Zr]-DFO-RW03scFv - Fc accumulate in CD133-expressing tumors to enable their delineation through PET imaging. Having identified [89Zr]-DFO-RW03scFv - Fc (CA = 2.9 ± 0.3) as the most attractive construct for CD133-expressing tumor delineation, the next step is to evaluate this probe using patient-derived tumor models to test its detection limit prior to clinical translation.

Cancer Stem Cells: Current Challenges and Future Perspectives.

Despite major advances in health care including improved diagnostic tools, robust chemotherapeutic regimens, advent of precision, adjuvant and multimodal therapies, there is a major proportion of patients that still go on to experience tumor progression and recurrence. Cancer stem cells (CSCs) are shown to be responsible for tumor persistence and relapse. This subpopulation of cancer cells possess normal stem cell like traits of self-renewal, proliferation, and multilineage differentiation. Currently, they are isolated and enriched based on the cell surface markers that can be detected and sorted through fluorescence and magnetic-based cell sorting. In this chapter, we review the current challenges and limitations often encountered in CSC research, including the identification of universal markers, therapy resistance, and new drug development. Current and future perspectives are discussed to address these challenges including utilization of cutting-edge technologies such as next-generation sequencing to elucidate the genome, epigenome, and transcriptome on a single-cell level and genome-wide CRISPR-Cas9 screens to identify novel pathway-based targeted therapies. Further, we discuss the future of precision medicine and the need for the improvement of clinical trial designs.

National multicentered retrospective review of clinical and intraoperative factors associated with the development of cerebellar mutism after pediatric posterior fossa tumor resection.

BACKGROUND: Cerebellar mutism (CM) is characterized by a significant loss of speech in children following posterior fossa (PF) surgery. The biological origin of CM remains unclear and is the subject of ongoing debate. Significant recovery from CM is less likely than previously described despite rigorous multidisciplinary neuro-rehabilitational efforts. METHODS: A national multi-centered retrospective review of all children undergoing PF resection in four midsized Canadian academic pediatric institutions was undertaken. Patient, tumor and surgical factors associated with the post-operative development of CM were reviewed. Retrospective identification of PF surgery patients including those developing and those that did not (internal control). RESULTS: The study identified 258 patients across the 4 centers between 2010 and 2020 (mean age 6.73 years; 42.2% female). Overall, CM was experienced in 19.5% of patients (N = 50). Amongst children who developed CM histopathology included medulloblastoma (35.7%), pilocytic astrocytoma (32.6%) and ependymoma (17.1%). Intraoperative impression of adherence to the floor of the 4th ventricle was positive in 36.8%. Intraoperative abrupt changes in blood pressure and/or heart rate were identified in 19.4% and 17.8% of cases. The clinical resolution of CM was rated to be complete, significant resolution, slight improvement, no improvement and deterioration in 56.0%, 8.0%, 20.0%, 14.0% and 2.0%, respectively. In the cohort of children who experienced post-operative CM as compared to their no-CM counterpart, proportionally more tumors were felt to be adherent to the floor of the 4th ventricle (56.0% vs 49.5%), intraoperative extent of resection was a GTR (74% vs 68.8%) and changes in heart rate were noted (≥ 20% from baseline) (26.0% vs 15.9%). However, a multiple regression analysis identified only abrupt changes in HR (OR 5.97, CI (1.53, 23.1), p = 0.01) to be significantly associated with the development of post-operative CM. CONCLUSION: As a devastating surgical complication after posterior fossa tumor surgery with variable clinical course, identifying and understanding the operative cues and revising intraoperative plans that optimizes the child's neurooncological and clinical outcome are essential.

Intratumoral Delivery of Chimeric Antigen Receptor T Cells Targeting CD133 Effectively Treats Brain Metastases.

PURPOSE: Brain metastases (BM) are mainly treated palliatively with an expected survival of less than 12 months after diagnosis. In many solid tumors, the human neural stem cell marker glycoprotein CD133 is a marker of a tumor-initiating cell population that contributes to therapy resistance, relapse, and metastasis. EXPERIMENTAL DESIGN: Here, we use a variant of our previously described CD133 binder to generate second-generation CD133-specific chimeric antigen receptor T cells (CAR-T) to demonstrate its specificity and efficacy against multiple patient-derived BM cell lines with variable CD133 antigen expression. RESULTS: Using both lung- and colon-BM patient-derived xenograft models, we show that a CD133-targeting CAR-T cell therapy can evoke significant tumor reduction and survival advantage after a single dose, with complete remission observed in the colon-BM model. CONCLUSIONS: In summary, these data suggest that CD133 plays a critical role in fueling the growth of BM, and immunotherapeutic targeting of this cell population is a feasible strategy to control the outgrowth of BM tumors that are otherwise limited to palliative care. See related commentary by Sloan et al., p. 477.

Protocol for mapping the metabolome and lipidome of medulloblastoma cells using liquid chromatography-mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics and lipidomics have recently been used to show that MYC-amplified group 3 medulloblastoma tumors are driven by metabolic reprogramming. Here, we present a protocol to extract metabolites and lipids from human medulloblastoma brain tumor-initiating cells and normal neural stem cells. We describe untargeted LC-MS methods that can be used to achieve extensive coverage of the polar metabolome and lipidome. Finally, we detail strategies for metabolite identification and data analysis. For complete details on the use and execution of this protocol, please refer to Gwynne et al.1.

Compensatory cross-talk between autophagy and glycolysis regulates senescence and stemness in heterogeneous glioblastoma tumor subpopulations.

Despite tremendous research efforts, successful targeting of aberrant tumor metabolism in clinical practice has remained elusive. Tumor heterogeneity and plasticity may play a role in the clinical failure of metabolism-targeting interventions for treating cancer patients. Moreover, compensatory growth-related processes and adaptive responses exhibited by heterogeneous tumor subpopulations to metabolic inhibitors are poorly understood. Here, by using clinically-relevant patient-derived glioblastoma (GBM) cell models, we explore the cross-talk between glycolysis, autophagy, and senescence in maintaining tumor stemness. We found that stem cell-like GBM tumor subpopulations possessed higher basal levels of glycolytic activity and increased expression of several glycolysis-related enzymes including, GLUT1/SLC2A1, PFKP, ALDOA, GAPDH, ENO1, PKM2, and LDH, compared to their non-stem-like counterparts. Importantly, bioinformatics analysis also revealed that the mRNA expression of glycolytic enzymes positively correlates with stemness markers (CD133/PROM1 and SOX2) in patient GBM tumors. While treatment with glycolysis inhibitors induced senescence in stem cell-like GBM tumor subpopulations, as evidenced by increased ß-galactosidase staining and upregulation of the cell cycle regulators p21Waf1/Cip1/CDKN1A and p16INK4A/CDKN2A, these cells maintained their aggressive stemness features and failed to undergo apoptotic cell death. Using various techniques including autophagy flux and EGFP-MAP1LC3B+ puncta formation analysis, we determined that inhibition of glycolysis led to the induction of autophagy in stem cell-like GBM tumor subpopulations, but not in their non-stem-like counterparts. Similarly, blocking autophagy in stem cell-like GBM tumor subpopulations induced senescence-associated growth arrest without hampering stemness capacity or inducing apoptosis while reciprocally upregulating glycolytic activity. Combinatorial treatment of stem cell-like GBM tumor subpopulations with autophagy and glycolysis inhibitors blocked the induction of senescence while drastically impairing their stemness capacity which drove cells towards apoptotic cell death. These findings identify a novel and complex compensatory interplay between glycolysis, autophagy, and senescence that helps maintain stemness in heterogeneous GBM tumor subpopulations and provides a survival advantage during metabolic stress.

Dynamic profiling of medulloblastoma surfaceome.

Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer. The current standard of care (SOC) involves maximal safe resection and chemoradiotherapy in individuals older than 3 years, often leading to devastating neurocognitive and developmental deficits. Out of the four distinct molecular subgroups, Group 3 and 4 have the poorest patient outcomes due to the aggressive nature of the tumor and propensity to metastasize and recur post therapy. The toxicity of the SOC and lack of response in specific subtypes to the SOC underscores the urgent need for developing and translating novel treatment options including immunotherapies. To identify differentially enriched surface proteins that could be evaluated for potential future immunotherapeutic interventions, we leveraged N-glycocapture surfaceome profiling on Group 3 MB cells from primary tumor, through therapy, to recurrence using our established therapy-adapted patient derived xenograft model. Integrin 𝛼5 (ITGA5) was one of the most differentially enriched targets found at recurrence when compared to engraftment and untreated timepoints. In addition to being enriched at recurrence, shRNA-mediated knockdown and small molecule inhibition of ITGA5 have resulted in marked decrease in proliferation and self-renewal in vitro and demonstrated a survival advantage in vivo. Together, our data highlights the value of dynamic profiling of cells as they evolve through therapy and the identification of ITGA5 as a promising therapeutic target for recurrent Group 3 MB.

Safety and efficacy of lorlatinib against ALK-driven refractory or relapsed neuroblastoma.

We spotlight the promising preliminary findings reported by Goldsmith et al. of a phase 1 first-in-child study showcasing the safety and efficacy of lorlatinib against treatment-refractory or relapsed ALK-driven neuroblastoma.1.

An effective kinase inhibition strategy for metastatic recurrent childhood medulloblastoma.

PURPOSE: Medulloblastomas (MBs) constitute the most common malignant brain tumor in children and adolescents. MYC-amplified Group 3 MBs are characterized by disease recurrence, specifically in the leptomeninges, whereby patients with these metastatic tumors have a mortality rate nearing 100%. Despite limited research on such tumors, studies on MB metastases at diagnosis suggest targeting kinases to be beneficial. METHODS: To identify kinase inhibitors that eradicate cells driving therapy evasion and tumor dissemination, we utilized our established patient-derived xenograft (PDX) mouse-adapted therapy platform that models human MB metastatic recurrences following standard chemoradiotherapy. High-throughput screens of 640 kinase inhibitors were conducted against cells isolated from mouse spines in the PDX model and human fetal neural stem cells to reveal compounds that targeted these treatment-refractory, metastatic cells, whilst sparing healthy cells. Blood-brain barrier permeability assays and additional in vitro experimentation helped select top candidates for in vivo studies. RESULTS: Recurrent Group 3 MB PDX spine cells were therapeutically vulnerable to a selective checkpoint kinase 1 (CHK1) inhibitor and small molecular inhibitor of platelet-derived growth factor receptor beta (PDGFRß). Inhibitor-treated cells showed a significant reduction in MB stem cell properties associated with treatment failure. Mice also demonstrated survival advantage when treated with a CHK1 inhibitor ex vivo. CONCLUSION: We identified CHK1 and PDGFRß inhibitors that effectively target MB cells fueling treatment-refractory metastases. With limited research on effective therapies for Group 3 MB metastatic recurrences, this work highlights promising therapeutic options to treat these aggressive tumors. Additional studies are warranted to investigate these inhibitors' mechanisms and recommended in vivo administration.

Metabolism-based targeting of MYC via MPC-SOD2 axis-mediated oxidation promotes cellular differentiation in group 3 medulloblastoma.

Group 3 medulloblastoma (G3 MB) carries the worst prognosis of all MB subgroups. MYC oncoprotein is elevated in G3 MB tumors; however, the mechanisms that support MYC abundance remain unclear. Using metabolic and mechanistic profiling, we pinpoint a role for mitochondrial metabolism in regulating MYC. Complex-I inhibition decreases MYC abundance in G3 MB, attenuates the expression of MYC-downstream targets, induces differentiation, and prolongs male animal survival. Mechanistically, complex-I inhibition increases inactivating acetylation of antioxidant enzyme SOD2 at K68 and K122, triggering the accumulation of mitochondrial reactive oxygen species that promotes MYC oxidation and degradation in a mitochondrial pyruvate carrier (MPC)-dependent manner. MPC inhibition blocks the acetylation of SOD2 and oxidation of MYC, restoring MYC abundance and self-renewal capacity in G3 MB cells following complex-I inhibition. Identification of this MPC-SOD2 signaling axis reveals a role for metabolism in regulating MYC protein abundance that has clinical implications for treating G3 MB.

External ventricular drainage in the management of pediatric patients with posterior fossa tumors and hydrocephalus: a retrospective cohort study.

PURPOSE: To determine whether intraoperative adjunctive EVD placement in patients with a posterior fossa tumor (PFT) led to improved surgical, radiographic, and clinical outcomes compared to those who did not receive an EVD. METHODS: Patients were grouped as those who underwent routine intraoperative adjunctive EVD insertion and those who did not at time of PFT resection. Patients who pre-operatively required a clinically indicated EVD insertion were excluded. Comparative analyses between both groups were conducted to evaluate clinical, radiological, and pathological outcomes. Odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were computed for post-operative outcomes. RESULTS: Fifty-five selected patients were included, 15 who had an EVD placed at the time of PFT resection surgery, and 40 who did not. Children without an EVD did not experience a higher rate of complications or poorer post-operative outcomes compared to those with an EVD placed during resection surgery. There was no significant difference in the degree of gross total resection (p = 0.129), post-operative CSF leak (p = 1.000), and post-operative hemorrhage (p = 0.554) between those with an EVD and those without. The frequency of new cranial nerve deficits post-operatively was higher in those with an EVD (40%) compared to those without (3%, p = 0.001). There was a trend towards more frequently observed post-operative hydrocephalus in the EVD group (p = 0.057). CONCLUSION: The routine use of EVD as an intraoperative adjunct in clinically stable pediatric patients with posterior fossa tumors and hydrocephalus may not be associated with improved radiological or clinical outcomes.

A single center experience in the management of progressive juvenile pilocytic astrocytoma.

BACKGROUND: Juvenile pilocytic astrocytoma (JPA) typically follows an indolent clinical course. The first-line treatment for most JPAs is surgical resection. However, a gross total resection may not be feasible for deep-seated lesions and/or infiltrative tumors, leading to multimodal treatment approaches that may be complicated by patient age and tumor location. Despite the prevalence of pediatric JPAs, there is no single approach to treating progressive disease. METHODS: We investigated the multifaceted management of progressive JPAs through a retrospective analysis of JPAs treated at a single center over an 18-year period (1998-2016). All cases were categorized according to location, whether supratentorial or infratentorial, and for each case we calculated the number of interventions and the time between interventions. RESULTS: We identified a total of 40 JPAs, (11 supratentorial, 29 infratentorial). Total number of interventions among all supratentorial JPA patients was 21 (average 2 interventions/patient). The total number of interventions among infratentorial JPAs was 40 (average 1.4 interventions/patient). CONCLUSIONS: Treatment of progressive JPA is variable and may require numerous surgeries and adjuvant therapies.

Characterization of an RNA binding protein interactome reveals a context-specific post-transcriptional landscape of MYC-amplified medulloblastoma.

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB.

Cancer-selective metabolic vulnerabilities in MYC-amplified medulloblastoma.

MYC-driven medulloblastoma (MB) is an aggressive pediatric brain tumor characterized by therapy resistance and disease recurrence. Here, we integrated data from unbiased genetic screening and metabolomic profiling to identify multiple cancer-selective metabolic vulnerabilities in MYC-driven MB tumor cells, which are amenable to therapeutic targeting. Among these targets, dihydroorotate dehydrogenase (DHODH), an enzyme that catalyzes de novo pyrimidine biosynthesis, emerged as a favorable candidate for therapeutic targeting. Mechanistically, DHODH inhibition acts on target, leading to uridine metabolite scarcity and hyperlipidemia, accompanied by reduced protein O-GlcNAcylation and c-Myc degradation. Pyrimidine starvation evokes a metabolic stress response that leads to cell-cycle arrest and apoptosis. We further show that an orally available small-molecule DHODH inhibitor demonstrates potent mono-therapeutic efficacy against patient-derived MB xenografts in vivo. The reprogramming of pyrimidine metabolism in MYC-driven medulloblastoma represents an unappreciated therapeutic strategy and a potential new class of treatments with stronger cancer selectivity and fewer neurotoxic sequelae.

Characterization of the minimal residual disease state reveals distinct evolutionary trajectories of human glioblastoma.

Recurrence of solid tumors renders patients vulnerable to advanced, treatment-refractory disease state with mutational and oncogenic landscape distinctive from initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profile barcoded tumor stem cell populations through therapy at tumor initiation, MRD, and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors show distinct patterns of recurrence in which clonal populations exhibit either a pre-existing fitness advantage or an equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing reveals a tumor-intrinsic immunomodulatory signature with prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from patients with GBM. Our results provide insight into the innate and therapy-driven dynamics of human GBM and the prognostic value of interrogating the MRD state in solid cancers.

The proteomic landscape of glioblastoma recurrence reveals novel and targetable immunoregulatory drivers.

Glioblastoma (GBM) is characterized by extensive cellular and genetic heterogeneity. Its initial presentation as primary disease (pGBM) has been subject to exhaustive molecular and cellular profiling. By contrast, our understanding of how GBM evolves to evade the selective pressure of therapy is starkly limited. The proteomic landscape of recurrent GBM (rGBM), which is refractory to most treatments used for pGBM, are poorly known. We, therefore, quantified the transcriptome and proteome of 134 patient-derived pGBM and rGBM samples, including 40 matched pGBM-rGBM pairs. GBM subtypes transition from pGBM to rGBM towards a preferentially mesenchymal state at recurrence, consistent with the increasingly invasive nature of rGBM. We identified immune regulatory/suppressive genes as important drivers of rGBM and in particular 2-5-oligoadenylate synthase 2 (OAS2) as an essential gene in recurrent disease. Our data identify a new class of therapeutic targets that emerge from the adaptive response of pGBM to therapy, emerging specifically in recurrent disease and may provide new therapeutic opportunities absent at pGBM diagnosis.

Reoperation in adult patients with recurrent glioblastoma: A matched cohort analysis.

Background: Despite maximal safe cytoreductive surgery and postoperative adjuvant therapies, glioblastoma (GBM) inevitably recurs and leads to deterioration of neurological status and eventual death. There is no consensus regarding the benefit of repeat resection for enhancing survival or quality of life in patients with recurrent GBM. We aimed to examine if reoperation for GBM recurrence incurs a survival benefit as well as examine its complication profile. Methods: We performed a single-center retrospective chart review on all adult patients who underwent resection of supratentorial GBM between January 1, 2008 and December 1, 2013 at our center. Patients with repeat resection were manually matched for age, sex, tumor location, and Karnofsky Performance Status (KPS) with patients who underwent single resection to compare overall survival (OS), and postoperative morbidity. Results: Of 237 patients operated with GBM, 204 underwent single resection and 33 were selected for repeat surgical resections. In a matched analysis there was no difference in the OS between groups (17.8 ± 17.6 months vs 17 ± 13.5 months, P = .221). In addition, repeat surgical resection had a higher rate of postoperative neurological complications compared to the initial surgery. Conclusions: When compared with matched patients who underwent a single surgical resection, patients undergoing repeat surgical resection did not show significant increase in OS and may have incurred more neurological complications related to the repeat resection. Further studies are required to assess which patients would benefit from repeat surgical resection and optimize timing of the repeat resection in selected patients.