The heterogeneous sensitivity of pediatric brain tumors to different oncolytic viruses is predicted by unique gene expression profiles.

Despite decades of research, the prognosis of high-grade pediatric brain tumors (PBTs) remains dismal; however, recent cases of favorable clinical responses were documented in clinical trials using oncolytic viruses (OVs). In the current study, we employed four different species of OVs: adenovirus Delta24-RGD, herpes simplex virus rQNestin34.5v1, reovirus R124, and the non-virulent Newcastle disease virus rNDV-F0-GFP against three entities of PBTs (high-grade gliomas, atypical teratoid/rhabdoid tumors, and ependymomas) to determine their in vitro efficacy. These four OVs were screened on 14 patient-derived PBT cell cultures and the degree of oncolysis was assessed using an ATP-based assay. Subsequently, the observed viral efficacies were correlated to whole transcriptome data and Gene Ontology analysis was performed. Although no significant tumor type-specific OV efficacy was observed, the analysis revealed the intrinsic biological processes that associated with OV efficacy. The predictive power of the identified expression profiles was further validated in vitro by screening additional PBTs. In summary, our results demonstrate OV susceptibility of multiple patient-derived PBT entities and the ability to predict in vitro responses to OVs using unique expression profiles. Such profiles may hold promise for future OV preselection with effective oncolytic potency in a specific tumor, therewith potentially improving OV responses.

Phantom limb pain, traumatic neuroma, or nerve sheath tumor? Illustrative case.

BACKGROUND: Phantom limb pain and traumatic neuromas are not commonly seen in neurosurgical practice. These conditions can present with similar symptoms; however, management of traumatic neuroma is often surgical, whereas phantom limb pain is treated with conservative measures. OBSERVATIONS: A 77-year-old female patient with a long-standing history of an above-the-knee amputation experienced severe pain in her right posterior buttocks area for several years' duration, attributed to phantom limb pain, which radiated down the stump of her leg and was treated with a variety of conservative measures. A recent exacerbation of her pain led to a prolonged hospitalization with magnetic resonance imaging of her leg stump, revealing a mass in the sciatic notch, at a relative distance from the stump. The anatomical location of the mass on the sciatic nerve in the notch led to a presumed radiological diagnosis of nerve sheath tumor, for which she underwent excision. At surgery, a neuroma of the proximal portion of the transected sciatic nerve that had retracted from the amputated stump to the notch was diagnosed. LESSONS: Traumatic neuromas of transected major nerves after limb amputation should be considered in the differential diagnosis of phantom limb pain.

Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding.

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

Lessons learned from phase 3 trials of immunotherapy for glioblastoma: Time for longitudinal sampling?

Glioblastoma (GBM)'s median overall survival is almost 21 months. Six phase 3 immunotherapy clinical trials have recently been published, yet 5/6 did not meet approval by regulatory bodies. For the sixth, approval is uncertain. Trial failures result from multiple factors, ranging from intrinsic tumor biology to clinical trial design. Understanding the clinical and basic science of these 6 trials is compelled by other immunotherapies reaching the point of advanced phase 3 clinical trial testing. We need to understand more of the science in human GBMs in early trials: the "window of opportunity" design may not be best to understand complex changes brought about by immunotherapeutic perturbations of the GBM microenvironment. The convergence of increased safety of image-guided biopsies with "multi-omics" of small cell numbers now permits longitudinal sampling of tumor and biofluids to dissect the complex temporal changes in the GBM microenvironment as a function of the immunotherapy.

Oncolytic viral therapy: a review and promising future directions.

Oncolytic viral therapy is quickly emerging as a promising subset of immunotherapy, which theoretically can target tumor cells while sparing surrounding healthy cells by harnessing the replication machinery of viruses with tropism for tumor cells, resulting in direct oncolysis, and by transforming immunologically "cold" tumor into areas that elicit the host's immune response. This review provides an overview of oncolytic viral therapy until the present day, starting with the original concept in 1912. The general mechanism of oncolytic viruses (OVs) depends on selectively integrating them into tumor cells based on genetic engineering of viral genomic material, inducing oncolysis and eliciting the host's innate immune response. Moreover, a major component of oncolytic viral therapy has been herpes simplex virus, with talimogene laherparepvec being the only FDA-approved oncolytic viral therapy for the treatment of melanomas. This review explores the characteristics, advantages, disadvantages, and therapeutic uses of several DNA and RNA viral families. A snapshot of the oncolytic viral treatments used in the most recent and advanced clinical trials is also provided. Lastly, the challenges of implementing oncolytic viral therapy are explored, both at a molecular and clinical level, with a highlight of promising future directions. In particular, the lack of an optimal delivery method based on tumor type for oncolytic viral therapy poses a significant obstacle, even in clinical studies. Intrathecal continuous delivery of OVs is a promising prospect, potentially by adapting the novel continuous irrigation and drainage IRRAflow catheter. Further exploration and testing of the IRRAflow catheter should be undertaken.

Inaugural Results of the Individualized Screening Trial of Innovative Glioblastoma Therapy: A Phase II Platform Trial for Newly Diagnosed Glioblastoma Using Bayesian Adaptive Randomization.

PURPOSE: The Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT) is a phase II platform trial that uses response adaptive randomization and genomic profiling to efficiently identify novel therapies for phase III testing. Three initial experimental arms (abemaciclib [a cyclin-dependent kinase [CDK]4/6 inhibitor], neratinib [an epidermal growth factor receptor [EGFR]/human epidermal growth factor receptor 2 inhibitor], and CC-115 [a deoxyribonucleic acid-dependent protein kinase/mammalian target of rapamycin inhibitor]) were simultaneously evaluated against a common control arm. We report the results for each arm and examine the feasibility and conduct of the adaptive platform design. PATIENTS AND METHODS: Patients with newly diagnosed O6-methylguanine-DNA methyltransferase-unmethylated glioblastoma were eligible if they had tumor genotyping to identify prespecified biomarker subpopulations of dominant glioblastoma signaling pathways (EGFR, phosphatidylinositol 3-kinase, and CDK). Initial random assignment was 1:1:1:1 between control (radiation therapy and temozolomide) and the experimental arms. Subsequent Bayesian adaptive randomization was incorporated on the basis of biomarker-specific progression-free survival (PFS) data. The primary end point was overall survival (OS), and one-sided P values are reported. The trial is registered with ClinicalTrials.gov (identifier: NCT02977780). RESULTS: Two hundred thirty-seven patients were treated (71 control; 73 abemaciclib; 81 neratinib; 12 CC-115) in years 2017-2021. Abemaciclib and neratinib were well tolerated, but CC-115 was associated with ≥ grade 3 treatment-related toxicity in 58% of patients. PFS was significantly longer with abemaciclib (hazard ratio [HR], 0.72; 95% CI, 0.49 to 1.06; one-sided P = .046) and neratinib (HR, 0.72; 95% CI, 0.50 to 1.02; one-sided P = .033) relative to the control arm but there was no PFS benefit with CC-115 (one-sided P = .523). None of the experimental therapies demonstrated a significant OS benefit (P > .05). CONCLUSION: The INSIGhT design enabled efficient simultaneous testing of three experimental agents using a shared control arm and adaptive randomization. Two investigational arms had superior PFS compared with the control arm, but none demonstrated an OS benefit. The INSIGhT design may promote improved and more efficient therapeutic discovery in glioblastoma. New arms have been added to the trial.

Intratumoral drug-releasing microdevices allow in situ high-throughput pharmaco phenotyping in patients with gliomas.

The lack of reliable predictive biomarkers to guide effective therapy is a major obstacle to the advancement of therapy for high-grade gliomas, particularly glioblastoma (GBM), one of the few cancers whose prognosis has not improved over the past several decades. With this pilot clinical trial (number NCT04135807), we provide first-in-human evidence that drug-releasing intratumoral microdevices (IMDs) can be safely and effectively used to obtain patient-specific, high-throughput molecular and histopathological drug response profiling. These data can complement other strategies to inform the selection of drugs based on their observed antitumor effect in situ. IMDs are integrated into surgical practice during tumor resection and remain in situ only for the duration of the otherwise standard operation (2 to 3 hours). None of the six enrolled patients experienced adverse events related to the IMD, and the exposed tissue was usable for downstream analysis for 11 out of 12 retrieved specimens. Analysis of the specimens provided preliminary evidence of the robustness of the readout, compatibility with a wide array of techniques for molecular tissue interrogation, and promising similarities with the available observed clinical-radiological responses to temozolomide. From an investigational aspect, the amount of information obtained with IMDs allows characterization of tissue effects of any drugs of interest, within the physiological context of the intact tumor, and without affecting the standard surgical workflow.

Recent oncolytic virotherapy clinical trials outline a roadmap for the treatment of high-grade glioma.

Adult and pediatric high-grade gliomas (HGGs) are aggressive cancers of the central nervous system that confer dismal clinical prognoses. Standard radiation and chemotherapy have demonstrated only limited efficacy in HGGs, motivating the accelerated investigation of novel modalities such as oncolytic virus (OV) therapies. OV centered therapies work through a mixed mechanism centered on oncolysis and the stimulation of an antitumor immune response. Three recent clinical trials utilizing herpes simplex virus-1 and adenovirus-based oncolytic virotherapy demonstrated not only the safety and efficacy of OVs but also novel dosing strategies that augment OV response potential. Considering these recent trials, herein we present a roadmap for future clinical trials of oncolytic immunovirotherapy in both adult and pediatric HGG, as well as persistent roadblocks related to the assessment of OV efficacy within and between trials.

Uncovering transcriptomic landscape alterations of CAN-2409 in in vitro and in vivo glioma models.

Rationale: CAN-2409 is a locally delivered oncolytic therapy, which results in vaccination against the injected tumor. CAN-2409 consists of a non-replicating adenovirus armed with the Herpes virus thymidine kinase, which metabolizes ganciclovir into a phosphorylated nucleotide that is incorporated into the tumor cell's genome, thereby inflicting immunogenic cancer cell death. While CAN-2409's immunological impact has been well characterized, its effects on the tumor cells transcriptome remains unknown. We compared the transcriptomic landscape after treatment of glioblastoma models with CAN-2409 in vitro and in vivo to assess how the interplay with the tumor microenvironment influences CAN-2409-mediated transcriptome alterations. Methods: We performed RNA-Seq with CAN-2409 treated patient-derived glioma stem-like cells and tumors of C57/BL6 mice and compared KEGG pathway usage and differential gene expression focusing on immune cell and cytokine profiles. T-cell -killing assays were performed to assess candidate effectors. Results: PCA analysis showed distinct clustering of control and CAN-2409 samples under both conditions. KEGG pathway analysis revealed significant enrichment for p53 signaling and cell cycle pathway, with similar dynamics for key regulators of both pathways in vitro and in vivo, including MYC, CCNB1, PLK1 and CDC20. Selected alterations (PLK1 and CCNB1) were validated at the protein level. Cytokine expression analysis revealed upregulation of pro-inflammatory IL12a under both conditions; immune cell gene profiling showed reduction of myeloid associated genes. T-cell-killing assays showed increased killing in the presence of IL-12. Conclusion: CAN-2409 significantly alters the transcriptome both in vitro and in vivo. Comparison of pathway enrichment revealed mutual and differential utilization of pathways under both conditions, suggesting a modulating influence on the cell cycle in tumor cells, and of the tumor microenvironment on the transcriptome in vivo. IL-12 synthesis likely depends on interactions with the tumor microenvironment, and it facilitates CAN-2409 cell killing. This dataset provides potential to understand resistance mechanisms and identify potential biomarkers for future studies.

PPRX-1701, a nanoparticle formulation of 6'-bromoindirubin acetoxime, improves delivery and shows efficacy in preclinical GBM models.

Derivatives of the Chinese traditional medicine indirubin have shown potential for the treatment of cancer through a range of mechanisms. This study investigates the impact of 6'-bromoindirubin-3'-acetoxime (BiA) on immunosuppressive mechanisms in glioblastoma (GBM) and evaluates the efficacy of a BiA nanoparticle formulation, PPRX-1701, in immunocompetent mouse GBM models. Transcriptomic studies reveal that BiA downregulates immune-related genes, including indoleamine 2,3-dioxygenase 1 (IDO1), a critical enzyme in the tryptophan-kynurenine-aryl hydrocarbon receptor (Trp-Kyn-AhR) immunosuppressive pathway in tumor cells. BiA blocks interferon-γ (IFNγ)-induced IDO1 protein expression in vitro and enhances T cell-mediated tumor cell killing in GBM stem-like cell co-culture models. PPRX-1701 reaches intracranial murine GBM and significantly improves survival in immunocompetent GBM models in vivo. Our results indicate that BiA improves survival in murine GBM models via effects on important immunotherapeutic targets in GBM and that it can be delivered efficiently via PPRX-1701, a nanoparticle injectable formulation of BiA.

Clinical Importance of the lncRNA NEAT1 in Cancer Patients Treated with Immune Checkpoint Inhibitors.

PURPOSE: mAbs targeting the PD-1/PD-L1 immune checkpoint are powerful tools to improve the survival of patients with cancer. Understanding the molecular basis of clinical response to these treatments is critical to identify patients who can benefit from this immunotherapy. In this study, we investigated long noncoding RNA (lncRNA) expression in patients with cancer treated with anti-PD-1/PD-L1 immunotherapy. EXPERIMENTAL DESIGN: lncRNA expression profile was analyzed in one cohort of patients with melanoma and two independent cohorts of patients with glioblastoma (GBM) undergoing anti-PD-1/PD-L1 immunotherapy. Single-cell RNA-sequencing analyses were performed to evaluate lncRNA expression in tumor cells and tumor-infiltrating immune cells. RESULTS: We identified the lncRNA NEAT1 as commonly upregulated between patients with melanoma with complete therapeutic response and patients with GBM with longer survival following anti-PD-1/PD-L1 treatment. Gene set enrichment analyses revealed that NEAT1 expression was strongly associated with the IFNγ pathways, along with downregulation of cell-cycle-related genes. Single-cell RNA-sequencing analyses revealed NEAT1 expression across multiple cell types within the GBM microenvironment, including tumor cells, macrophages, and T cells. High NEAT1 expression levels in tumor cells correlated with increased infiltrating macrophages and microglia. In these tumor-infiltrating myeloid cells, we found that NEAT1 expression was linked to enrichment in TNFα/NFκB signaling pathway genes. Silencing NEAT1 suppressed M1 macrophage polarization and reduced the expression of TNFα and other inflammatory cytokines. CONCLUSIONS: These findings suggest an association between NEAT1 expression and patient response to anti-PD-1/PD-L1 therapy in melanoma and GBM and have important implications for the role of lncRNAs in the tumor microenvironment.

Reduced time to imaging, length of stay, and hospital charges following implementation of a novel postoperative pathway for craniotomy.

OBJECTIVE: The authors created a postoperative postanesthesia care unit (PACU) pathway to bypass routine intensive care unit (ICU) admissions of patients undergoing routine craniotomies, to improve ICU resource utilization and reduce overall hospital costs and lengths of stay while maintaining quality of care and patient satisfaction. In the present study, the authors evaluated this novel PACU-to-floor clinical pathway for a subset of patients undergoing craniotomy with a case time under 5 hours and blood loss under 500 ml. METHODS: A single-institution retrospective cohort study was performed to compare 202 patients enrolled in the PACU-to-floor pathway and 193 historical controls who would have met pathway inclusion criteria. The pathway cohort consisted of all adult supratentorial brain tumor cases from the second half of January 2021 to the end of January 2022 that met the study inclusion criteria. Control cases were selected from the beginning of January 2020 to halfway through January 2021. The authors also discuss common themes of similar previously published pathways and the logistical and clinical barriers overcome for successful PACU pathway implementation. RESULTS: Pathway enrollees had a median age of 61 years (IQR 49-69 years) and 53% were female. Age, sex, pathology, and American Society of Anesthesiologists physical status distributions were similar between pathway and control patients (p > 0.05). Most of the pathway cases (96%) were performed on weekdays, and 31% had start times before noon. Nineteen percent of pathway patients had 30-day readmissions, most frequently for headache (16%) and syncope (10%), whereas 18% of control patients had 30-day readmissions (p = 0.897). The average time to MRI was 6 hours faster for pathway patients (p < 0.001) and the time to inpatient physical therapy and/or occupational therapy evaluation was 4.1 hours faster (p = 0.046). The average total length of stay was 0.7 days shorter for pathway patients (p = 0.02). A home discharge occurred in 86% of pathway cases compared to 81% of controls (p = 0.225). The average total hospitalization charges were $13,448 lower for pathway patients, representing a 7.4% decrease (p = 0.0012, adjusted model). Seven pathway cases were escalated to the ICU postoperatively because of attending physician preference (2 cases), agitation (1 case), and new postoperative neurological deficits (4 cases), resulting in a 96.5% rate of successful discharge from the pathway. In bypassing the ICU, critical care resource utilization was improved by releasing 0.95 ICU days per patient, or 185 ICU days across the cohort. CONCLUSIONS: The featured PACU-to-floor pathway reduces the stay of postoperative craniotomy patients and does not increase the risk of early hospital readmission.

Dual Immunostimulatory Pathway Agonism through a Synthetic Nanocarrier Triggers Robust Anti-Tumor Immunity in Murine Glioblastoma.

Myeloid cells are abundant, create a highly immunosuppressive environment in glioblastoma (GBM), and thus contribute to poor immunotherapy responses. Based on the hypothesis that small molecules can be used to stimulate myeloid cells to elicit anti-tumor effector functions, a synthetic nanoparticle approach is developed to deliver dual NF-kB pathway-inducing agents into these cells via systemic administration. Synthetic, cyclodextrin-adjuvant nanoconstructs (CANDI) with high affinity for tumor-associated myeloid cells are dually loaded with a TLR7 and 8 (Toll-like receptor, 7 and 8) agonist (R848) and a cIAP (cellular inhibitor of apoptosis protein) inhibitor (LCL-161) to dually activate these myeloid cells. Here CANDI is shown to: i) readily enter the GBM tumor microenvironment (TME) and accumulate at high concentrations, ii) is taken up by tumor-associated myeloid cells, iii) potently synergize payloads compared to monotherapy, iv) activate myeloid cells, v) fosters a "hot" TME with high levels of T effector cells, and vi) controls the growth of murine GBM as mono- and combination therapies with anti-PD1. Multi-pathway targeted myeloid stimulation via the CANDI platform can efficiently drive anti-tumor immunity in GBM.

The emerging field of oncolytic virus-based cancer immunotherapy.

Oncolytic viruses (OVs) provide novel and promising therapeutic options for patients with cancers resistant to traditional therapies. Natural or genetically modified OVs are multifaceted tumor killers. They directly lyse tumor cells while sparing normal cells, and indirectly potentiate antitumor immunity by releasing antigens and activating inflammatory responses in the tumor microenvironment. However, some limitations, such as limited penetration of OVs into tumors, short persistence, and the host antiviral immune response, are impeding the broad translation of oncolytic virotherapy into the clinic. If these challenges can be overcome, combination therapies, such as OVs plus immune checkpoint blockade (ICB), chimeric antigen receptor (CAR) T cells, or CAR natural killer (NK) cells, may provide powerful therapeutic platforms in the clinic.

Antigen presentation deficiency, mesenchymal differentiation, and resistance to immunotherapy in the murine syngeneic CT2A tumor model.

Background: The GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy. Methods: To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics. Results: The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants. Conclusion: Although the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways.

Specific targeting of glioblastoma with an oncolytic virus expressing a cetuximab-CCL5 fusion protein via innate and adaptive immunity.

Chemokines such as C-C motif ligand 5 (CCL5) regulate immune cell trafficking in the tumor microenvironment (TME) and govern tumor development, making them promising targets for cancer therapy. However, short half-lives and toxic off-target effects limit their application. Oncolytic viruses (OVs) have become attractive therapeutic agents. Here, we generate an oncolytic herpes simplex virus type 1 (oHSV) expressing a secretable single-chain variable fragment of the epidermal growth factor receptor (EGFR) antibody cetuximab linked to CCL5 by an Fc knob-into-hole strategy that produces heterodimers (OV-Cmab-CCL5). OV-Cmab-CCL5 permits continuous production of CCL5 in the TME, as it is redirected to EGFR+ glioblastoma (GBM) tumor cells. OV-Cmab-CCL5 infection of GBM significantly enhances the migration and activation of natural killer cells, macrophages and T cells; inhibits tumor EGFR signaling; reduces tumor size; and prolongs survival of GBM-bearing mice. Collectively, our data demonstrate that OV-Cmab-CCL5 offers a promising approach to improve OV therapy for solid tumors.

Variants of the adeno-associated virus serotype 9 with enhanced penetration of the blood-brain barrier in rodents and primates.

The development of gene therapies for the treatment of diseases of the central nervous system has been hindered by the limited availability of adeno-associated viruses (AAVs) that efficiently traverse the blood-brain barrier (BBB). Here, we report the rational design of AAV9 variants displaying cell-penetrating peptides on the viral capsid and the identification of two variants, AAV.CPP.16 and AAV.CPP.21, with improved transduction efficiencies of cells of the central nervous system on systemic delivery (6- to 249-fold across 4 mouse strains and 5-fold in cynomolgus macaques, with respect to the AAV9 parent vector). We also show that the neurotropism of AAV.CPP.16 is retained in young and adult macaques, that this variant displays enhanced transcytosis at the BBB as well as increased efficiency of cellular transduction relative to AAV9, and that it can be used to deliver antitumour payloads in a mouse model of glioblastoma. AAV capsids that can efficiently penetrate the BBB will facilitate the clinical translation of gene therapies aimed at the central nervous system.