Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known. MethodsFour independent nonoverlapping retrospective cohorts of posterior fossa ependymomas (n = 820) were profiled using genome-wide methylation arrays. Risk stratification models were designed based on known clinical and newly described molecular biomarkers identified by multivariable Cox proportional hazards analyses. ResultsMolecular subgroup is a powerful independent predictor of outcome even when accounting for age or treatment regimen. Incompletely resected EPN_PFA ependymomas have a dismal prognosis, with a 5-year progression-free survival ranging from 26.1% to 56.8% across all four cohorts. Although first-line (adjuvant) radiation is clearly beneficial for completely resected EPN_PFA, a substantial proportion of patients with EPN_PFB can be cured with surgery alone, and patients with relapsed EPN_PFB can often be treated successfully with delayed external-beam irradiation. ConclusionThe most impactful biomarker for posterior fossa ependymoma is molecular subgroup affiliation, independent of other demographic or treatment variables. However, both EPN_PFA and EPN_PFB still benefit from increased extent of resection, with the survival rates being particularly poor for subtotally resected EPN_PFA, even with adjuvant radiation therapy. Patients with EPN_PFB who undergo gross total resection are at lower risk for relapse and should be considered for inclusion in a randomized clinical trial of observation alone with radiation reserved for those who experience recurrence. INTRODUCTIONEpendymoma is the third most common posterior fossa tumor of childhood and a major cause of morbidity and mortality in pediatric oncology, occurring across the entire age spectrum. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] Current therapy for posterior fossa ependymoma in children is aggressive surgical resection followed by involved-field radiation, resulting in 7-year event free-survival of 65%. 12,15 Despite the high mortality rate, trials of cytotoxic chemotherapy have failed to reveal a clear survival benefit for chemotherapy over surgery and radiation alone, although definitive pediatric randomized trials of maintenance chemotherapy are still recruiting through cooperative groups (ClinicalTrials.gov identifiers: NCT01096368 and NCT02265770). 1,5,17 In adults, posterior fossa ependymoma is frequently treated with surgery alone. 18Numerous publications have suggested that the most powerful prognostic factor for posterior fossa ependymoma is the extent of surgical resection or, more appropriately, the amount of residual tumor after surgery. This has entailed an aggressive surgical approach, with some oncologists and surgeons tolerating serious neurologic deficits, including the need for tracheostomies and gastrostom...

Together, these studies elucidate the role that TIMs play in mediating adaptive immune resistance in the GBM microenvironment and provide evidence that they can be manipulated pharmacologically with agents that are clinically available. Development of immune resistance in response to active vaccination in GBM can be reversed with dual administration of CSF-1Ri and PD-1 mAb.

Mutations in isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) are found in a subset of gliomas. Among the many phenotypic differences between mutant and wild-type IDH1/2 gliomas, the most salient is that IDH1/2 mutant glioma patients demonstrate markedly improved survival compared with IDH1/2 wild-type glioma patients. To address the mechanism underlying the superior clinical outcome of IDH1/2 mutant glioma patients, we investigated whether overexpression of the IDH1(R132H) protein could affect response to therapy in the context of an isogenic glioma cell background. Stable clonal U87MG and U373MG cell lines overexpressing IDH1(WT) and IDH1(R132H) were generated, as well as U87MG cell lines overexpressing IDH2(WT) and IDH2(R172K). In vitro experiments were conducted to characterize baseline growth and migration and response to radiation and temozolomide. In addition, reactive oxygen species (ROS) levels were measured under various conditions. U87MG-IDH1(R132H) cells, U373MG-IDH1(R132H) cells, and U87MG-IDH2(R172K) cells demonstrated increased sensitivity to radiation but not to temozolomide. Radiosensitization of U87MG-IDH1(R132H) cells was accompanied by increased apoptosis and accentuated ROS generation, and this effect was abrogated by the presence of the ROS scavenger N-acetyl-cysteine. Interestingly, U87MG-IDH1(R132H) cells also displayed decreased growth at higher cell density and in soft agar, as well as decreased migration. Overexpression of IDH1(R132H) and IDH2(R172K) mutant protein in glioblastoma cells resulted in increased radiation sensitivity and altered ROS metabolism and suppression of growth and migration in vitro. These findings provide insight into possible mechanisms contributing to the improved outcomes observed in patients with IDH1/2 mutant gliomas.

DC vaccination with autologous tumor lysate has demonstrated promising results for the treatment of glioblastoma (GBM) in preclinical and clinical studies. While the vaccine appears capable of inducing T cell infiltration into tumors, the effectiveness of active vaccination in progressively growing tumors is less profound. In parallel, a number of studies have identified negative costimulatory pathways, such as programmed death 1/programmed death ligand 1 (PD-1/PD-L1), as relevant mediators of the intratumoral immune responses. Clinical responses to PD-1 pathway inhibition, however, have also been varied. To evaluate the relevance to established glioma, the effects of PD-1 blockade following DC vaccination were tested in intracranial (i.c.) glioma tumor– bearing mice. Treatment with both DC vaccination and PD-1 mAb blockade resulted in long-term survival, while neither agent alone induced a survival benefit in animals with larger, established tumors. This survival benefit was completely dependent on CD8+ T cells. Additionally, DC vaccine plus PD-1 mAb blockade resulted in the upregulation of integrin homing and immunologic memory markers on tumor-infiltrating lymphocytes (TILs). In clinical samples, DC vaccination in GBM patients was associated with upregulation of PD-1 expression in vivo, while ex vivo blockade of PD-1 on freshly isolated TILs dramatically enhanced autologous tumor cell cytolysis. These findings strongly suggest that the PD-1/PD-L1 pathway plays an important role in the adaptive immune resistance of established GBM in response to antitumor active vaccination and provide us with a rationale for the clinical translation of this combination therapy.

Summary Dendritic cell (DC) vaccination is emerging as a promising therapeutic option for malignant glioma patients. However, the optimal antigen formulation for loading these cells has yet to be established. The objective of this study was to compare the safety, feasibility, and immune responses of malignant glioma patients on two different DC vaccination protocols. 28 patients were treated with autologous tumor lysate (ATL)-pulsed DC vaccination, while 6 patients were treated with glioma-associated antigen (GAA) peptide-pulsed DCs. Safety, toxicity, feasibility and correlative immune monitoring assay results were compared between patients on each trial. Due to HLA subtype restrictions on the GAA-DC trial, 6/15 screened patients were eligible for treatment, while 28/32 patients passed eligibility screening for the ATL-DC trial. Elevated frequencies of activated natural killer (NK) cells were observed in the peripheral blood from GAA-DC patients compared with the ATL-DC patients. In addition, a significant correlation was observed between decreased regulatory T lymphocyte (Treg) ratios (post/pre vaccination) and overall survival (OS; p=0.004) in patients on both trials. In fact, Treg ratios were independently prognostic for OS in these patients, while tumor pathology was not in multivariate analyses. In conclusion, these results suggest that ATL-DC vaccination is associated with wider patient eligibility compared with GAA-DC vaccination. Decreased post/pre-vaccination Treg ratios and decreased frequencies of activated NK cells were associated with prolonged survival in patients from both trials, suggesting that these lymphocyte subsets may be relevant immune monitoring endpoints for immunotherapy protocols in malignant glioma patients.

Contrast-enhanced MRI is typically used to follow treatment response and progression in patients with glioblastoma (GBM). However, differentiating tumor progression from pseudoprogression remains a clinical dilemma largely unmitigated by current advances in imaging techniques. Noninvasive imaging techniques capable of distinguishing these two conditions could play an important role in the clinical management of patients with GBM and other brain malignancies. We hypothesized that PET probes for deoxycytidine kinase (dCK) could be used to differentiate immune inflammatory responses from other sources of contrast-enhancement on MRI. Orthotopic malignant gliomas were established in syngeneic immunocompetent mice and then treated with dendritic cell (DC) vaccination and/or PD-1 mAb blockade. Mice were then imaged with [18F]-FAC PET/CT and MRI with i.v. contrast. The ratio of contrast enhancement on MRI to normalized PET probe uptake, which we term the immunotherapeutic response index, delineated specific regions of immune inflammatory activity. On postmortem examination, FACS-based enumeration of intracranial tumor-infiltrating lymphocytes directly correlated with quantitative [18F]-FAC PET probe uptake. Three patients with GBM undergoing treatment with tumor lysate-pulsed DC vaccination and PD-1 mAb blockade were also imaged before and after therapy using MRI and a clinical PET probe for dCK. Unlike in mice, [18F]-FAC is rapidly catabolized in humans; thus, we used another dCK PET probe, [18F]-clofarabine ([18F]-CFA), that may be more clinically relevant. Enhanced [18F]-CFA PET probe accumulation was identified in tumor and secondary lymphoid organs after immunotherapy. Our findings identify a noninvasive modality capable of imaging the host antitumor immune response against intracranial tumors.

Many animal viruses exhibit proficient growth in transformed cells, a property that has been harnessed for the development of novel therapies against cancer. Despite overwhelming evidence for this phenomenon, understanding of the molecular mechanisms enabling tumor-cell killing is rudimentary for most viruses. We report here that growth and cytotoxicity of the prototype oncolytic poliovirus (PV), PVSRIPO, in glioblastoma multiforme (GBM) is promoted by mitogen-activated protein kinases (MAPKs) converging on the MAPK signal-integrating kinase 1 (Mnk1) and its primary substrate, the eukaryotic initiation factor (eIF) 4E. Inducing Mnk1-catalyzed eIF4E phosphorylation through expression of oncogenic Ras substantially enhanced PVSRIPO translation, replication, and cytotoxicity in resistant cells. This effect was mimicked by expression of constitutively active forms of Mnk1 and correlated with enhanced translation of subgenomic reporter RNAs. Our findings implicate Mnk1 activity in stimulation of PVSRIPO cap-independent translation, an effect that can be synergistically enhanced by inhibition of the phosphoinositide-3 kinase (PI3K).

Primary brain tumors, such as glioblastoma (GBM), are remarkably resistant to immunotherapy, even though pre-clinical models suggest effectiveness. To understand this better in patients, here we take advantage of our recent neoadjuvant treatment paradigm to map the infiltrating immune cell landscape of GBM and how this is altered following PD-1 checkpoint blockade using high dimensional proteomics, single cell transcriptomics, and quantitative multiplex immunofluorescence. Neoadjuvant PD-1 blockade increases T cell infiltration and the proportion of a progenitor exhausted population of T cells found within the tumor. We identify an early activated and clonally expanded CD8+ T cell cluster whose TCR overlaps with a CD8+ PBMC population. Distinct changes are also observed in conventional type 1 dendritic cells that may facilitate T cell recruitment. Macrophages and monocytes still constitute the majority of infiltrating immune cells, even after anti-PD-1 therapy. Interferon-mediated changes in the myeloid population are consistently observed following PD-1 blockade; these also mediate an increase in chemotactic factors that recruit T cells. However, sustained high expression of T-cell-suppressive checkpoints in these myeloid cells continue to prevent the optimal activation of the tumor infiltrating T cells. Therefore, future immunotherapeutic strategies may need to incorporate the targeting of these cells for clinical benefit.