Glioblastoma microenvironment-from biology to therapy.

Glioblastoma (GBM) is the most aggressive primary brain cancer. These tumors exhibit high intertumoral and intratumoral heterogeneity in neoplastic and nonneoplastic compartments, low lymphocyte infiltration, and high abundance of myeloid subsets that together create a highly protumorigenic immunosuppressive microenvironment. Moreover, heterogeneous GBM cells infiltrate adjacent brain tissue, remodeling the neural microenvironment to foster tumor electrochemical coupling with neurons and metabolic coupling with nonneoplastic astrocytes, thereby driving growth. Here, we review heterogeneity in the GBM microenvironment and its role in low-to-high-grade glioma transition, concluding with a discussion of the challenges of therapeutically targeting the tumor microenvironment and outlining future research opportunities.

Rapid autopsy of a patient with recurrent anaplastic ependymoma.

ABSTRACTObjective:Our aim was to outline a procedure for obtaining a rapid autopsy in order to collect high-quality postmortem tissue for genomic analysis. METHODS: This report details a bi-institutional collaborative effort to coordinate a rapid autopsy for a pediatric patient who had died at home. We discuss the scientific rationale for offering a rapid autopsy to caregivers of pediatric patients as well as parental perspectives on broaching the subject of autopsy. We then review the logistics and coordination involved with planning a rapid autopsy and the sequence of events needed to maximize tissue quality. RESULTS: We report the successful coordination of a rapid autopsy for a patient who died in a hospice setting at her out-of-state home. The time interval from death to the start of the rapid autopsy procedure was 4.5 hours, despite the logistical considerations demanded by the location of the patient. Tumor aliquots and nonneoplastic tissues were successfully snap frozen for downstream genomic studies. SIGNIFICANCE OF RESULTS: Physicians should consider trialing a rapid autopsy program at their institution that could be offered to caregivers of pediatric patients. This case report offers a framework to help clinicians develop their own rapid autopsy programs as well as guidelines to help streamline this process for appropriate candidates going forward.

An aesthetic approach to the anterior cranial fossa: the endoscopic transadnexal transorbital roof method.

BACKGROUND: This case report describes a transadnexal transorbital roof approach to the anterior cranial fossa to treat an epidural abscess in a patient who had previously undergone extensive craniofacial reconstruction secondary to fibrous dysplasia. A standard frontal craniotomy or even a supraciliary craniotomy would have been burdensome due to the positioning of the porous polyethylene implant as well as extensive scar tissue and adhesions from prior surgical procedures. METHODS: We performed a retrospective review of the case of a 15-year-old male with an epidural abscess treated at a university hospital. RESULTS: The patient tolerated the procedure well with no postoperative complications. Surgical operative time was approximately 1.5 h with minimal blood loss and the patient was discharged on postoperative day 2. Six weeks after antibiotic therapy (intravenous ampicillin/sulbactam, 2,000 mg q.i.d. for 6 days total), an MRI showed complete resolution of the epidural abscess. At that time the patient had no visual changes, extraocular muscle dysfunction, headaches, periorbital cellulitis, or further seizures and is on no antiepileptic therapy. CONCLUSION: Minimally invasive approaches to treat complex problems in certain circumstances can yield excellent results and limit morbidity encountered by more extensive procedures. In the future, more formal studies with a larger subset of patients will help determine what role these approaches will ultimately have. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

BRAF V600E in a preclinical model of pleomorphic Xanthoastrocytoma: Analysis of the tumor microenvironment and immune cell infiltration dynamics in vivo.

Low-grade glioma (LGG) is the most common brain tumor affecting pediatric patients (pLGG) and BRAF mutations constitute the most frequent genetic alterations. Within the spectrum of pLGGs, approximately 70%-80% of pediatric patients diagnosed with transforming pleomorphic xanthoastrocytoma (PXA) harbor the BRAF V600E mutation. However, the impact of glioma BRAF V600E cell regulation of tumor-infiltrating immune cells and their contribution to tumor progression remains unclear. Moreover, the efficacy of BRAF inhibitors in treating pLGGs is limited compared with their impact on BRAF-mutated melanoma. Here we report a novel immunocompetent RCAS-BRAF V600E murine glioma model. Pathological assessment indicates this model seems to be consistent with diffuse gliomas and morphological features of PXA. Our investigations revealed distinct immune cell signatures associated with increased trafficking and activation within the tumor microenvironment (TME). Intriguingly, immune system activation within the TME also generated a pronounced inflammatory response associated with dysfunctional CD8+ T cells, increased presence of immunosuppressive myeloid cells and regulatory T cells. Further, our data suggests tumor-induced inflammatory processes, such as cytokine storm. These findings suggest a complex interplay between tumor progression and the robust inflammatory response within the TME in preclinical BRAF V600E LGGs, which may significantly influence animal survival.

Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma.

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

Safety and Feasibility of Stereotactic Radiosurgery for Patients with 15 or more Brain Metastases.

Background: Current standard of care treatment for patients with ≥15 brain metastases (BM) is whole brain radiation therapy (WBRT), despite poor neurocognitive outcomes. We analyzed our institutional experience of treating these patients with stereotactic radiosurgery (SRS), with the aim of evaluating safety, cognitive outcomes, and survival metrics. Methods: Patients who received SRS for ≥15 BMs in 1 to 5 fractions from 2014 to 2022 were included. Cognitive outcomes were objectively evaluated using serial Patient-Reported Outcome Measurement Information System (PROMIS) scores. The Kaplan-Meier method was used for survival analysis and log-rank test for intergroup comparisons. Results: Overall, 118 patients underwent 124 courses of LINAC-based SRS. The median number of lesions treated per course was 20 (range, 15-94). Most patients received fractionated SRS to a dose of 24 Gy in 3 fractions (81.5%). At the time of SRS, 19.4% patients had received prior WBRT, and 24.2% had received prior SRS. The rate of any grade radiation necrosis (RN) and grade ≥3 RN were 15.3% and 3.2%, respectively. When evaluating longitudinal PROMIS score trends, 25 of 31 patients had a stable/improved PROMIS score. Patients who did not receive prior brain RT had a longer median survival (7.4 months vs 4.6 months, P = .034). The 12m local control was 97.6%, and the cumulative incidence of distant intracranial failure, with death as a competing event, was 46% (95% CI, 36%, 55%). One year freedom from neurologic death, leptomeningeal disease, and salvage WBRT were 89%, 94.6%, and 84%, respectively. Conclusion: We present here one of the largest studies evaluating SRS for patients with ≥15 BMs. SRS was safe, had favorable cognitive outcomes, and had comparable survival outcomes to contemporary studies evaluating WBRT in this population. Treatment-naïve patients had a median survival of >6 months, long enough to benefit from cognitive sparing with SRS. Our study supports randomized studies comparing SRS and hippocampal avoidance WBRT approaches for these patients.

Homeostatic iron regulatory protein drives glioblastoma growth via tumor cell-intrinsic and sex-specific responses.

Background: Glioblastoma (GBM) displays alterations in iron that drive proliferation and tumor growth. Iron regulation is complex and involves many regulatory mechanisms, including the homeostatic iron regulator (HFE) gene, which encodes the homeostatic iron regulatory protein. While HFE is upregulated in GBM and correlates with poor survival outcomes, the function of HFE in GBM remains unclear. Methods: We interrogated the impact of cell-intrinsic Hfe expression on proliferation and survival of intracranially implanted animals through genetic gain- and loss-of-function approaches in syngeneic mouse glioma models, along with in vivo immune assessments. We also determined the expression of iron-associated genes and their relationship to survival in GBM using public data sets and used transcriptional profiling to identify differentially expressed pathways in control compared to Hfe-knockdown cells. Results: Overexpression of Hfe accelerated GBM proliferation and reduced animal survival, whereas suppression of Hfe induced apoptotic cell death and extended survival, which was more pronounced in females and associated with attenuation of natural killer cells and CD8+ T cell activity. Analysis of iron gene signatures in Hfe-knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Further analysis of differentially expressed pathways revealed oxidative stress as the top pathway upregulated following Hfe loss. Hfe knockdown indeed resulted in enhanced 55Fe uptake and generation of reactive oxygen species. Conclusions: These findings reveal an essential function for HFE in GBM cell growth and survival, as well as a sex-specific interaction with the immune response.

Therapeutic utility of engineered myeloid cells in the tumor microenvironment.

Despite promising results shown in hematologic tumors, immunotherapies for the treatment of solid tumors have mostly failed so far. The immunosuppressive tumor microenvironment and phenotype of tumor infiltrating macrophages are among the more prevalent reasons for this failure. Tumor associated macrophages (TAMs, M2-macrophages) are circulating myeloid cells recruited to the local tumor microenvironment, and together with regulatory T cells (T-regs), are reprogrammed to become immune suppressive. This results in the inactivation or hampered recruitment of cytotoxic CD8 + T and Natural Killer (NK) cells. Recently, attempts have been made to try to leverage specific myeloid functions and properties, including their ability to reach the TME and to mediate the phagocytosis of cancer cells. Additionally, myeloid cells have been used for drug delivery and reprogramming the tumor microenvironment in cancer patients. This approach, together with the advancements in genome editing, paved the way for the development of novel cell-mediated immunotherapies. This article focuses on the latest studies that detail the therapeutic properties of genetically engineered or pharmacologically modulated myeloid cells in cancer preclinical models, limitations, pitfalls, and evaluations of these approaches in patients with cancer.

Myeloid cell heterogeneity in the tumor microenvironment and therapeutic implications for childhood central nervous system (CNS) tumors.

Central nervous system (CNS) tumors are the most common type of solid tumors in children and the leading cause of cancer deaths in ages 0-14. Recent advances in the field of tumor biology and immunology have underscored the disparate nature of these distinct CNS tumor types. In this review, we briefly introduce pediatric CNS tumors and discuss various components of the TME, with a particular focus on myeloid cells. Although most studies regarding myeloid cells have been done on adult CNS tumors and animal models, we discuss the role of myeloid cell heterogeneity in pediatric CNS tumors and describe how these cells may contribute to tumorigenesis and treatment response. In addition, we present studies within the last 5 years that highlight human CNS tumors, the utility of various murine CNS tumor models, and the latest multi-dimensional tools that can be leveraged to investigate myeloid cell infiltration in young adults and children diagnosed with select CNS tumors.

Genetically modified IL2 bone-marrow-derived myeloid cells reprogram the glioma immunosuppressive tumor microenvironment.

Gliomas are one of the leading causes of cancer-related death in the adolescent and young adult (AYA) population. Two-thirds of AYA glioma patients are affected by low-grade gliomas (LGGs), but there are no specific treatments. Malignant progression is supported by the immunosuppressive stromal component of the tumor microenvironment (TME) exacerbated by M2 macrophages and a paucity of cytotoxic T cells. A single intravenous dose of engineered bone-marrow-derived myeloid cells that release interleukin-2 (GEMys-IL2) was used to treat mice with LGGs. Our results demonstrate that GEMys-IL2 crossed the blood-brain barrier, infiltrated the TME, and reprogrammed the immune cell composition and transcriptome. Moreover, GEMys-IL2 extended survival in an LGG immunocompetent mouse model. Here, we report the efficacy of an in vivo approach that demonstrates the potential for a cell-mediated innate immunotherapy designed to enhance the recruitment of activated effector T and natural killer cells within the glioma TME.

Single-cell RNA sequencing reveals immunosuppressive myeloid cell diversity during malignant progression in a murine model of glioma.

Recent studies have shown the importance of the dynamic tumor microenvironment (TME) in high-grade gliomas (HGGs). In particular, myeloid cells are known to mediate immunosuppression in glioma; however, it is still unclear if myeloid cells play a role in low-grade glioma (LGG) malignant progression. Here, we investigate the cellular heterogeneity of the TME using single-cell RNA sequencing in a murine glioma model that recapitulates the malignant progression of LGG to HGG. LGGs show increased infiltrating CD4+ and CD8+ T cells and natural killer (NK) cells in the TME, whereas HGGs abrogate this infiltration. Our study identifies distinct macrophage clusters in the TME that show an immune-activated phenotype in LGG but then evolve to an immunosuppressive state in HGG. We identify CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Targeting these intra-tumoral macrophages in the LGG stage may attenuate their immunosuppressive properties and impair malignant progression.

The MIF promoter SNP rs755622 is associated with immune activation in glioblastoma.

Intratumoral heterogeneity is a defining hallmark of glioblastoma, driving drug resistance and ultimately recurrence. Many somatic drivers of microenvironmental change have been shown to affect this heterogeneity and, ultimately, the treatment response. However, little is known about how germline mutations affect the tumoral microenvironment. Here, we find that the single-nucleotide polymorphism (SNP) rs755622 in the promoter of the cytokine macrophage migration inhibitory factor (MIF) is associated with increased leukocyte infiltration in glioblastoma. Furthermore, we identified an association between rs755622 and lactotransferrin expression, which could also be used as a biomarker for immune-infiltrated tumors. These findings demonstrate that a germline SNP in the promoter region of MIF may affect the immune microenvironment and further reveal a link between lactotransferrin and immune activation.

Pediatric versus adult high grade glioma: Immunotherapeutic and genomic considerations.

High grade gliomas are identified as malignant central nervous tumors that spread rapidly and have a universally poor prognosis. Historically high grade gliomas in the pediatric population have been treated similarly to adult high grade gliomas. For the first time, the most recent classification of central nervous system tumors by World Health Organization has divided adult from pediatric type diffuse high grade gliomas, underscoring the biologic differences between these tumors in different age groups. The objective of our review is to compare high grade gliomas in the adult versus pediatric patient populations, highlighting similarities and differences in epidemiology, etiology, pathogenesis and therapeutic approaches. High grade gliomas in adults versus children have varying clinical presentations, molecular biology background, and response to chemotherapy, as well as unique molecular targets. However, increasing evidence show that they both respond to recently developed immunotherapies. This review summarizes the distinctions and commonalities between the two in disease pathogenesis and response to therapeutic interventions with a focus on immunotherapy.

Utility of multimodality molecular profiling for pediatric patients with central nervous system tumors.

Background: As our molecular understanding of pediatric central nervous system (CNS) tumors evolves, so too do diagnostic criteria, prognostic biomarkers, and clinical management decision making algorithms. Here, we explore the clinical utility of wide-breadth assays, including whole-exome sequencing (WES), RNA sequencing (RNA-seq), and methylation array profiling as an addition to more conventional diagnostic tools for pediatric CNS tumors. Methods: This study comprises an observational, prospective cohort followed at a single academic medical center over 3 years. Paired tumor and normal control specimens from 53 enrolled pediatric patients with CNS tumors underwent WES. A subset of cases also underwent RNA-seq (n = 28) and/or methylation array analysis (n = 27). Results: RNA-seq identified the driver and/or targetable fusions in 7/28 cases, including potentially targetable NTRK fusions, and uncovered possible rationalized treatment options based on outlier gene expression in 23/28 cases. Methylation profiling added diagnostic confidence (8/27 cases) or diagnostic subclassification endorsed by the WHO (10/27 cases). WES detected clinically pertinent tier 1 or tier 2 variants in 36/53 patients. Of these, 16/17 SNVs/INDELs and 10/19 copy number alterations would have been detected by current in-house conventional tests including targeted sequencing panels. Conclusions: Over a heterogeneous set of pediatric tumors, RNA-seq and methylation profiling frequently yielded clinically relevant information orthogonal to conventional methods while WES demonstrated clinically relevant added value primarily via copy number assessment. Longitudinal cohorts comparing targeted molecular pathology workup vs broader genomic approaches including therapeutic selection based on RNA expression data will be necessary to further evaluate the clinical benefits of these modalities in practice.

Whole-Brain Radiation Therapy Versus Stereotactic Radiosurgery for Cerebral Metastases.

Whole-brain radiation therapy (WBRT) was frequently used to treat brain metastases in the past. Stereotactic radiosurgery (SRS) is now generally preferred to WBRT for patients with limited brain metastases. SRS can also be used to treat extensive brain metastases (>10-15 metastases), and clinical trials are currently comparing WBRT with SRS for extensive disease. SRS may allow for an increased risk of radiation necrosis or leptomeningeal disease dissemination after treatment. Preoperative SRS and multifraction radiotherapy decrease the risk of these side effects and may soon become standard of care. Combining SRS with immune checkpoint inhibitors may improve patient outcomes.

Biomarker-Based PET Imaging of Diffuse Intrinsic Pontine Glioma in Mouse Models.

Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a universally poor prognosis. Here, we characterize a positron emission tomography (PET) probe for imaging DIPG in vivo In human histological tissues, the probes target, PARP1, was highly expressed in DIPG compared to normal brain. PET imaging allowed for the sensitive detection of DIPG in a genetically engineered mouse model, and probe uptake correlated to histologically determined tumor infiltration. Imaging with the sister fluorescence agent revealed that uptake was confined to proliferating, PARP1-expressing cells. Comparison with other imaging technologies revealed remarkable accuracy of our biomarker approach. We subsequently demonstrated that serial imaging of DIPG in mouse models enables monitoring of tumor growth, as shown in modeling of tumor progression. Overall, this validated method for quantifying DIPG burden would serve useful in monitoring treatment response in early phase clinical trials. Cancer Res; 77(8); 2112-23. ©2017 AACR.

Malignant Astrocytic Tumor Progression Potentiated by JAK-mediated Recruitment of Myeloid Cells.

Purpose: While the tumor microenvironment has been known to play an integral role in tumor progression, the function of nonresident bone marrow-derived cells (BMDC) remains to be determined in neurologic tumors. Here we identified the contribution of BMDC recruitment in mediating malignant transformation from low- to high-grade gliomas.Experimental Design: We analyzed human blood and tumor samples from patients with low- and high-grade gliomas. A spontaneous platelet-derived growth factor (PDGF) murine glioma model (RCAS) was utilized to recapitulate human disease progression. Levels of CD11b+/GR1+ BMDCs were analyzed at discrete stages of tumor progression. Using bone marrow transplantation, we determined the unique influence of BMDCs in the transition from low- to high-grade glioma. The functional role of these BMDCs was then examined using a JAK 1/2 inhibitor (AZD1480).Results: CD11b+ myeloid cells were significantly increased during tumor progression in peripheral blood and tumors of glioma patients. Increases in CD11b+/GR1+ cells were observed in murine peripheral blood, bone marrow, and tumors during low-grade to high-grade transformation. Transient blockade of CD11b+ cell expansion using a JAK 1/2 Inhibitor (AZD1480) impaired mobilization of these cells and was associated with a reduction in tumor volume, maintenance of a low-grade tumor phenotype, and prolongation in survival.Conclusions: We demonstrate that impaired recruitment of CD11b+ myeloid cells with a JAK1/2 inhibitor inhibits glioma progression in vivo and prolongs survival in a murine glioma model. Clin Cancer Res; 23(12); 3109-19. ©2016 AACR.

A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma.

Tumors are capable of coopting hematopoietic cells to create a suitable microenvironment to support malignant growth. Here, we have demonstrated that upregulation of kinase insert domain receptor (KDR), also known as VEGFR2, in a myeloid cell sublineage is necessary for malignant progression of gliomas in transgenic murine models and is associated with high-grade tumors in patients. KDR expression increased in myeloid cells as myeloid-derived suppressor cells (MDSCs) accumulated, which was associated with the transformation and progression of low-grade fibrillary astrocytoma to high-grade anaplastic gliomas. KDR deficiency in murine BM-derived cells (BMDCs) suppressed the differentiation of myeloid lineages and reduced granulocytic/monocytic populations. The depletion of myeloid-derived KDR compromised its proangiogenic function, which inhibited the angiogenic switch necessary for malignant progression of low-grade to high-grade tumors. We also identified inhibitor of DNA binding protein 2 (ID2) as a key upstream regulator of KDR activation during myeloid differentiation. Deficiency of ID2 in BMDCs led to downregulation of KDR, suppression of proangiogenic myeloid cells, and prevention of low-grade to high-grade transition. Tumor-secreted TGF-ß and granulocyte-macrophage CSF (GM-CSF) enhanced the KDR/ID2 signaling axis in BMDCs. Our results suggest that modulation of KDR/ID2 signaling may restrict tumor-associated myeloid cells and could potentially be a therapeutic strategy for preventing transformation of premalignant gliomas.

Next-Generation Rapid Autopsies Enable Tumor Evolution Tracking and Generation of Preclinical Models.

PURPOSE: Patients with cancer who graciously consent for autopsy represent an invaluable resource for the study of cancer biology. To advance the study of tumor evolution, metastases, and resistance to treatment, we developed a next-generation rapid autopsy program integrated within a broader precision medicine clinical trial that interrogates pre- and postmortem tissue samples for patients of all ages and cancer types. MATERIALS AND METHODS: One hundred twenty-three (22%) of 554 patients who consented to the clinical trial also consented for rapid autopsy. This report comprises the first 15 autopsies, including patients with metastatic carcinoma (n = 10), melanoma (n = 1), and glioma (n = 4). Whole-exome sequencing (WES) was performed on frozen autopsy tumor samples from multiple anatomic sites and on non-neoplastic tissue. RNA sequencing (RNA-Seq) was performed on a subset of frozen samples. Tissue was also used for the development of preclinical models, including tumor organoids and patient-derived xenografts. RESULTS: Three hundred forty-six frozen samples were procured in total. WES was performed on 113 samples and RNA-Seq on 72 samples. Successful cell strain, tumor organoid, and/or patient-derived xenograft development was achieved in four samples, including an inoperable pediatric glioma. WES data were used to assess clonal evolution and molecular heterogeneity of tumors in individual patients. Mutational profiles of primary tumors and metastases yielded candidate mediators of metastatic spread and organotropism including CUL9 and PIGM in metastatic ependymoma and ANKRD52 in metastatic melanoma to the lung. RNA-Seq data identified novel gene fusion candidates. CONCLUSION: A next-generation sequencing-based autopsy program in conjunction with a pre-mortem precision medicine pipeline for diverse tumors affords a valuable window into clonal evolution, metastasis, and alterations underlying treatment. Moreover, such an autopsy program yields robust preclinical models of disease.

Endoscopic diagnosis of an MRI-occult, low-grade glioma with ependymal dissemination.

A 21-year-old man presented with triventricular hydrocephalus due to a tectal mass. He underwent an endoscopic third ventriculostomy, and multiple nodules were identified at the floor of the third ventricle intraoperatively. Surgical pathology of one of these lesions demonstrated that the tissue represented a low-grade astrocytoma. The case highlights the existing potential of neuroendoscopy to reveal neuroimaging-occult lesions, in spite of the significant advances of MRI. Furthermore, the combination of the age of the patient, the nonenhancing MRI appearance, and the multifocality of the lesions constitutes a rare and interesting neoplastic presentation within the brain. The constellation of findings likely represents dissemination of a low-grade tectal glioma via the CSF compartment.