Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma.

Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter- and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of antigens. In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface antigens that could be targeted by antibodies and chimeric antigen receptor-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas) and 9166 normal tissue samples (from the Genotype-Tissue Expression project), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative antigens. Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

Continuous irrigation with thrombolytics for intraventricular hemorrhage: case-control study.

Intraventricular hemorrhage (IVH) is a complication of a spontaneous intracerebral hemorrhage. Standard treatment is with external ventricular drain (EVD). Intraventricular thrombolysis may improve mortality but does not improve functional outcomes. We present our initial experience with a novel irrigating EVD (IRRAflow) that automates continuous irrigation with thrombolysis.Single-center case-control study including patients with IVH treated with EVD compared to IRRAflow. We compared standard demographics, treatment, and outcome parameters between groups. We developed a brain phantom injected with a human clot and assessed clot clearance using EVD/IRRAflow approaches with CT imaging.Twenty-one patients were treated with standard EVD and 9 patients with IRRAflow. Demographics were similar between groups. Thirty-three percent of patients with EVD also had at least one dose of t-PA and 89% of patients with IRRAflow received irrigation with t-PA (p = 0.01). Mean drain days were 8.8 for EVD versus 4.1 for IRRAflow (p = 0.02). Days-to-clearance of ventricular outflow was 5.8 for EVD versus 2.5 for IRRAflow (p = 0.02). Overall clearance was not different. Thirty-seven percent of EVD patients achieved good outcome (mRS ≥ 3) at 90 days versus 86% of IRRAflow patients (p = 0.03). Assessing only t-PA, reduction in mean days-to-clearance (p = 0.0004) and ICU days (p = 0.04) was observed. In the benchtop model, the clot treated with IRRAflow and t-PA showed a significant reduction of volume compared to control.Irrigation with IRRAflow and t-PA is feasible and safe for patients with IVH. Improving clot clearance with IRRAflow may result in improved clinical outcomes and should be incorporated into randomized trials.

Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma.

Background: Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter-and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of neoantigens. Results: In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface neoantigens that could be targeted by antibodies and chimeric antigen receptor (CAR)-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas [TCGA]) and 9,166 normal tissue samples (from the Genotype-Tissue Expression project [GTEx]), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN , which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative neoantigens. Conclusions: Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

HummingFlow: novel single twist-drill access for ventricular drainage, irrigation, monitoring, and automated local drug delivery in subarachnoid hemorrhage.

OBJECTIVE: The management of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage (aSAH) remains one of the most important targets for neurocritical care. Advances in monitoring technology have facilitated a more thorough understanding of the pathophysiology and therapeutic approaches, but interventions are generally limited to either systemic therapies or passive CSF drainage. The authors present a novel approach that combines a multimodal monitoring bolt-based system with an irrigating ventricular drain capable of delivering intrathecal medications and describe their early experience in patients with aSAH. METHODS: The authors performed a retrospective review of cases treated with the combined Hummingbird multimodal bolt system and the IRRAflow irrigating ventriculostomy. RESULTS: Nine patients were treated with the combined multimodal bolt system with irrigating ventriculostomy approach. The median number of days to clearance of the third and fourth ventricles was 3 days in patients with obstructive intraventricular hemorrhage. Two patients received intrathecal alteplase for intraventricular hemorrhage clearance, and 2 patients received intrathecal nicardipine as rescue therapy for severe symptomatic angiographic vasospasm. CONCLUSIONS: Combined CSF drainage, irrigation, multimodality monitoring, and automated local drug delivery are feasible using a single twist-drill hole device. Further investigation of irrigation settings and treatment approaches in high-risk cases is warranted.

Multimodal Batch-Wise Change Detection.

We address the problem of detecting distribution changes in a novel batch-wise and multimodal setup. This setup is characterized by a stationary condition where batches are drawn from potentially different modalities among a set of distributions in [Formula: see text] represented in the training set. Existing change detection (CD) algorithms assume that there is a unique-possibly multipeaked-distribution characterizing stationary conditions, and in batch-wise multimodal context exhibit either low detection power or poor control of false positives. We present MultiModal QuantTree (MMQT), a novel CD algorithm that uses a single histogram to model the batch-wise multimodal stationary conditions. During testing, MMQT automatically identifies which modality has generated the incoming batch and detects changes by means of a modality-specific statistic. We leverage the theoretical properties of QuantTree to: 1) automatically estimate the number of modalities in a training set and 2) derive a principled calibration procedure that guarantees false-positive control. Our experiments show that MMQT achieves high detection power and accurate control over false positives in synthetic and real-world multimodal CD problems. Moreover, we show the potential of MMQT in Stream Learning applications, where it proves effective at detecting concept drifts and the emergence of novel classes by solely monitoring the input distribution.

Catastrophic delayed cervical arthroplasty failure: illustrative case.

BACKGROUND: Cervical disc replacement (CDR) is an increasingly used alternative to fusion for symptomatic cervical disc disease. While more studies have suggested favorability of CDR over fusion procedures, limited data exist regarding implant fatigability. Here, the authors present a unique and previously unreported failure of the M6-C prosthesis causing spinal cord injury. OBSERVATIONS: A 49-year-old female with history of cervical degenerative disease and prior C4-7 M6-C arthroplasty presented 9 years later after a minor fall from standing. She endorsed bilateral hand numbness ascending to forearms and shoulders, with dysesthesias and weakness. Imaging showed fractured arthroplasty penetrating the spinal cord. Revision surgery found a ruptured arthroplasty annulus with metal piece piercing the spinal cord. Partial C4 and C5 corpectomy was performed to remove the integrated fins of the arthroplasty and inspect the cord and dura. This was reconstructed with a corpectomy cage and plate. The patient made an excellent recovery, with improvement in her weakness and resolution of her sensory symptoms. LESSONS: Possibility of fatigue-related failures presenting years after implantation have only been infrequently reported but can be catastrophic for patients. The authors encourage further discussions in this area, increased counseling with patients, and recommend a patient registry to better document adverse events.

Continuous Tissue Plasminogen Activator Infusion Using a Minimally Invasive Irrigating Catheter for the Treatment of Intraparenchymal Hemorrhage Within the Basal Ganglia: Case Reports.

BACKGROUND AND IMPORTANCE: Intraparenchymal hemorrhage (IPH) is a debilitating and highly morbid type of stroke with limited effective treatment modalities. Minimally invasive evacuation with tissue plasminogen activator (rt-PA) has demonstrated promise for mortality/functional improvements with adequate clot volume reduction. In this study, we report 2 cases of continuous rt-PA infusion using a closed circuit, dual lumen catheter, and irrigation system (IRRAflow) for IPH treatment. CLINICAL PRESENTATION: A 55-year-old man was admitted for acute onset left hemiparesis; he was found to have right basal ganglia IPH. He was treated with continuous rt-PA irrigation using the IRRAflow device, at a rate of 30 mL/h for 119 hours, with a total volume reduction of 87.8 mL and post-treatment volume of 1.2 mL. At 3-month follow-up, he exhibited a modified Rankin score of 4 and improved hemiparesis. A 39-year-old woman was admitted for acute onset left facial droop, left hemianopsia, and left hemiparesis; she was diagnosed with a right basal ganglia IPH. She was treated with drainage and continuous rt-PA irrigation at 30 mL/h for 24 hours, with a total hematoma volume reduction of 41 mL and with a final post-treatment volume of 9.1 mL. At 3-month follow-up, she exhibited a modified Rankin score of 3 with some improvement in left hemiparesis. CONCLUSION: Continuous rt-PA infusion using a minimally invasive catheter with saline irrigation was feasible and resulted in successful volume reduction in 2 patients with IPH. This technique is similar to the Minimally Invasive Surgery Plus rt-PA for Intracerebral Hemorrhage Evacuation (MISTIE) approach but offers the potential advantages of less breaks in the sterile circuit, continuous intracranial pressure monitoring, and may provide more efficient clot lysis compared with intermittent bolusing.

SynNotch-CAR T cells overcome challenges of specificity, heterogeneity, and persistence in treating glioblastoma.

Treatment of solid cancers with chimeric antigen receptor (CAR) T cells is plagued by the lack of ideal target antigens that are both absolutely tumor specific and homogeneously expressed. We show that multi-antigen prime-and-kill recognition circuits provide flexibility and precision to overcome these challenges in the context of glioblastoma. A synNotch receptor that recognizes a specific priming antigen, such as the heterogeneous but tumor-specific glioblastoma neoantigen epidermal growth factor receptor splice variant III (EGFRvIII) or the central nervous system (CNS) tissue-specific antigen myelin oligodendrocyte glycoprotein (MOG), can be used to locally induce expression of a CAR. This enables thorough but controlled tumor cell killing by targeting antigens that are homogeneous but not absolutely tumor specific. Moreover, synNotch-regulated CAR expression averts tonic signaling and exhaustion, maintaining a higher fraction of the T cells in a naïve/stem cell memory state. In immunodeficient mice bearing intracerebral patient-derived xenografts (PDXs) with heterogeneous expression of EGFRvIII, a single intravenous infusion of EGFRvIII synNotch-CAR T cells demonstrated higher antitumor efficacy and T cell durability than conventional constitutively expressed CAR T cells, without off-tumor killing. T cells transduced with a synNotch-CAR circuit primed by the CNS-specific antigen MOG also exhibited precise and potent control of intracerebral PDX without evidence of priming outside of the brain. In summary, by using circuits that integrate recognition of multiple imperfect but complementary antigens, we improve the specificity, completeness, and persistence of T cells directed against glioblastoma, providing a general recognition strategy applicable to other solid tumors.

Diagnostic Challenges in Primary Sacral Tumors and the Yield of Computed Tomography-Guided Needle Biopsy in the Modern Era.

OBJECTIVE: Primary sacral tumors pose unique challenges because of their complex radiographic appearances, diverse pathologic entities, and dramatically different treatment paradigms based on tumor type. Magnetic resonance imaging and computed tomography (CT) can provide valuable information; however, sacral lesions can possess unique radiographic features and pose diagnostic dilemmas. CT-guided percutaneous needle biopsy is a critical component of the diagnostic workup. However, limited data are available on its efficacy for primary sacral tumors. METHODS: The data from patients with newly diagnosed primary sacral lesions during a 12-year period at our hospital were analyzed. The preoperative magnetic resonance imaging findings, biopsy results, and pathological data for patients who required surgery were analyzed. Unique cases in which the final pathologic result was unexpected from the preoperative imaging findings have been highlighted. RESULTS: Of 38 patients who underwent percutaneous needle biopsy, diagnostic tissue was obtained on the first attempt for 31 (82%). Five of the remaining 7 obtained diagnostic tissue on the second attempt, yielding 95% diagnosis, with only two requiring open biopsies. In 2 patients with diagnostic tissue on CT-guided biopsy, an open biopsy was still recommended because of the clinical scenario. In both patients, the open biopsy results matched those of the CT-guided biopsy. For the 18 patients who required surgery, we found 100% correlation between the percutaneous needle biopsy findings and the final pathological diagnosis. No biopsy-induced complications or extraspinal tumor seeding occurred. CONCLUSIONS: CT-guided biopsy is a safe and effective technique. It represents a critical component of the diagnostic algorithm, given the diverse pathological findings of primary sacral lesions and dramatic differences in treatment.

An oncolytic measles virus-sensitive Group 3 medulloblastoma model in immune-competent mice.

Background: Oncolytic measles virus (MV) is effective in xenograft models of many tumor types in immune-compromised mice. However, no murine cell line exists that is tumorigenic, grows in immune-competent mice, and is killed by MV. The lack of such a model prevents an examination of the effect of the immune system on MV oncotherapy. Methods: Cerebellar stem cells from human CD46-transgenic immunocompetent mice were transduced to express Sendai virus C-protein, murine C-Myc, and Gfi1b proteins. The resultant cells were injected into the brain of NSG mice, and a cell line, called CSCG, was prepared from the resulting tumor. Results: CSCG cells are highly proliferative, and express stem cell markers. These cells are permissive for replication of MV and are killed by the virus in a dose- and time-dependent manner. CSCG cells form aggressive tumors that morphologically resemble medulloblastoma when injected into the brains of immune-competent mice. On the molecular level, CSCG tumors overexpress natriuretic peptide receptor 3 and gamma-aminobutyric acid type A receptor alpha 5, markers of Group 3 medulloblastoma. A single intratumoral injection of MV‒green fluorescent protein resulted in complete tumor regression and prolonged survival of animals compared with treatments with phosphate buffered saline (P = 0.0018) or heat-inactivated MV (P = 0.0027). Conclusions: This immune-competent model provides the first platform to test therapeutic regimens of oncolytic MV for Group 3 medulloblastoma in the presence of anti-measles immunity. The strategy presented here can be used to make MV-sensitive murine models of any human tumor for which the driving mutations are known.

Disparities in health care determine prognosis in newly diagnosed glioblastoma.

OBJECTIVE Glioblastoma (GBM) is an aggressive brain malignancy with a short overall patient survival, yet there remains significant heterogeneity in outcomes. Although access to health care has previously been linked to impact on prognosis in several malignancies, this question remains incompletely answered in GBM. METHODS This study was a retrospective analysis of 354 newly diagnosed patients with GBM who underwent first resection at the authors' institution (2007-2015). RESULTS Of the 354 patients (median age 61 years, and 37.6% were females), 32 (9.0%) had no insurance, whereas 322 (91.0%) had insurance, of whom 131 (40.7%) had Medicare, 45 (14%) had Medicaid, and 146 (45.3%) had private insurance. On average, insured patients survived almost 2-fold longer (p < 0.0001) than those who were uninsured, whereas differences between specific insurance types did not influence survival. The adjusted hazard ratio (HR) for death was higher in uninsured patients (HR 2.27 [95% CI 1.49-3.33], p = 0.0003). Age, mean household income, tumor size at diagnosis, and extent of resection did not differ between insured and uninsured patients, but there was a disparity in primary care physician (PCP) status-none of the uninsured patients had PCPs, whereas 72% of insured patients had PCPs. Postoperative adjuvant treatment rates with temozolomide (TMZ) and radiation therapy (XRT) were significantly less in uninsured (TMZ in 56.3%, XRT in 56.3%) than in insured (TMZ in 75.2%, XRT in 79.2%; p = 0.02 and p = 0.003) patients. Insured patients receiving both agents had better prognosis than uninsured patients receiving the same treatment (9.1 vs 16.34 months; p = 0.025), suggesting that the survival effect in insured patients could only partly be explained by higher treatment rates. Moreover, having a PCP increased survival among the insured cohort (10.7 vs 16.1 months, HR 1.65 [95% CI 1.27-2.15]; p = 0.0001), which could be explained by significant differences in tumor diameter at initial diagnosis between patients with and without PCPs (4.3 vs 4.8 cm, p = 0.003), and a higher rate of clinical trial enrollment, suggesting a critical role of PCPs for a timelier diagnosis of GBM and proactive cancer care management. CONCLUSIONS Access to health care is a strong determinant of prognosis in newly diagnosed patients with GBM. Any type of insurance coverage and having a PCP improved prognosis in this patient cohort. Higher rates of treatment with TMZ plus XRT, clinical trial enrollment, fewer comorbidities, and early diagnosis may explain survival disparities. Lack of health insurance or a PCP are major challenges within the health care system, which, if improved upon, could favorably impact the prognosis of patients with GBM.

Novel and shared neoantigen derived from histone 3 variant H3.3K27M mutation for glioma T cell therapy.

The median overall survival for children with diffuse intrinsic pontine glioma (DIPG) is less than one year. The majority of diffuse midline gliomas, including more than 70% of DIPGs, harbor an amino acid substitution from lysine (K) to methionine (M) at position 27 of histone 3 variant 3 (H3.3). From a CD8+ T cell clone established by stimulation of HLA-A2+ CD8+ T cells with synthetic peptide encompassing the H3.3K27M mutation, complementary DNA for T cell receptor (TCR) α- and ß-chains were cloned into a retroviral vector. TCR-transduced HLA-A2+ T cells efficiently killed HLA-A2+H3.3K27M+ glioma cells in an antigen- and HLA-specific manner. Adoptive transfer of TCR-transduced T cells significantly suppressed the progression of glioma xenografts in mice. Alanine-scanning assays suggested the absence of known human proteins sharing the key amino acid residues required for recognition by the TCR, suggesting that the TCR could be safely used in patients. These data provide us with a strong basis for developing T cell-based therapy targeting this shared neoepitope.

Single-cell profiling of human gliomas reveals macrophage ontogeny as a basis for regional differences in macrophage activation in the tumor microenvironment.

BACKGROUND: Tumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue. RESULTS: We perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells. CONCLUSIONS: We conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.

Isocitrate dehydrogenase mutations suppress STAT1 and CD8+ T cell accumulation in gliomas.

Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are among the first genetic alterations observed during the development of lower-grade glioma (LGG). LGG-associated IDH mutations confer gain-of-function activity by converting α-ketoglutarate to the oncometabolite R-2-hydroxyglutarate (2HG). Clinical samples and gene expression data from The Cancer Genome Atlas (TCGA) demonstrate reduced expression of cytotoxic T lymphocyte-associated genes and IFN-γ-inducible chemokines, including CXCL10, in IDH-mutated (IDH-MUT) tumors compared with IDH-WT tumors. Given these findings, we have investigated the impact of IDH mutations on the immunological milieu in LGG. In immortalized normal human astrocytes (NHAs) and syngeneic mouse glioma models, the introduction of mutant IDH1 or treatment with 2HG reduced levels of CXCL10, which was associated with decreased production of STAT1, a regulator of CXCL10. Expression of mutant IDH1 also suppressed the accumulation of T cells in tumor sites. Reductions in CXCL10 and T cell accumulation were reversed by IDH-C35, a specific inhibitor of mutant IDH1. Furthermore, IDH-C35 enhanced the efficacy of vaccine immunotherapy in mice bearing IDH-MUT gliomas. Our findings demonstrate a mechanism of immune evasion in IDH-MUT gliomas and suggest that specific inhibitors of mutant IDH may improve the efficacy of immunotherapy in patients with IDH-MUT gliomas.

GLUT3 upregulation promotes metabolic reprogramming associated with antiangiogenic therapy resistance.

Clinical trials revealed limited response duration of glioblastomas to VEGF-neutralizing antibody bevacizumab. Thriving in the devascularized microenvironment occurring after antiangiogenic therapy requires tumor cell adaptation to decreased glucose, with 50% less glucose identified in bevacizumab-treated xenografts. Compared with bevacizumab-responsive xenograft cells, resistant cells exhibited increased glucose uptake, glycolysis, 13C NMR pyruvate to lactate conversion, and survival in low glucose. Glucose transporter 3 (GLUT3) was upregulated in bevacizumab-resistant versus sensitive xenografts and patient specimens in a HIF-1α-dependent manner. Resistant versus sensitive cell mitochondria in oxidative phosphorylation-selective conditions produced less ATP. Despite unchanged mitochondrial numbers, normoxic resistant cells had lower mitochondrial membrane potential than sensitive cells, confirming poorer mitochondrial health, but avoided the mitochondrial dysfunction of hypoxic sensitive cells. Thin-layer chromatography revealed increased triglycerides in bevacizumab-resistant versus sensitive xenografts, a change driven by mitochondrial stress. A glycogen synthase kinase-3ß inhibitor suppressing GLUT3 transcription caused greater cell death in bevacizumab-resistant than -responsive cells. Overexpressing GLUT3 in tumor cells recapitulated bevacizumab-resistant cell features: survival and proliferation in low glucose, increased glycolysis, impaired oxidative phosphorylation, and rapid in vivo proliferation only slowed by bevacizumab to that of untreated bevacizumab-responsive tumors. Targeting GLUT3 or the increased glycolysis reliance in resistant tumors could unlock the potential of antiangiogenic treatments.