Abstract The tumor microenvironment (TME) presents a challenging setting for anti-tumor immunity due to its extreme metabolic features: nutrient deprivation, metabolite accumulation, acidity and hypoxia. Immune cells become dysfunctional under these unfavorable conditions resulting in diminished anti-tumor activity. Thus, strategies to address immunosuppressive conditions in the TME are warranted. We utilized metabolic inhibitors to modulate the TME and enhance anti-tumor immune responses. Neuroendocrine tumors (NETs) and chimeric antigen receptor (CAR)-T cells for somatostatin receptor 2 (SSTR2), a potential therapeutic target for NETs, were utilized as our experimental model. SSTR2 CAR-T cells were prepared from CD8+ T cells of healthy donors. To recapitulate T cell dysfunction in TME, CAR-T cells were cultured under unfavorable metabolic situations: acidic, hypoxic and low glucose conditions. The CAR-T cells exhibited dysfunctional phenotypes including decreased proliferation and viability and impaired cytotoxicity and cytokine production capacity. Additionally, exhaustion-related markers were significantly upregulated on CAR-T cells especially under acidic conditions. To modulate metabolism of NETs, we utilized inhibitors targeting lactate dehydrogenase A (LDHA) and mitochondrial complex I. LDH inhibitor NCI-006 reduced glucose consumption and lactate (Lac) production from NETs by inhibiting lactate fermentation, resulting in reduced extracellular acidification. However, NETs exhibited metabolic rewiring to oxidative phosphorylation (OXPHOS) after NCI-006 treatment. In contrast, Complex I inhibitor IACS-010759 treatment induced OXPHOS inhibition and rewiring to aerobic glycolysis. Combined treatment with these inhibitors successfully interrupted metabolic rewiring of NETs by blocking both aerobic glycolysis and OXPHOS. Hyperpolarized magnetic resonance imaging (HP-MRI) with 1-13C pyruvate (Pyr) and electron paramagnetic resonance (EPR) pO2 imaging were conducted to monitor in vivo metabolic activities in NET-derived xenograft tumors. Consistent to our in vitro findings, NCI-006 suppressed Lac production from Pyr, and IACS-010759 treatment resulted in elevated pO2 level in xenograft tumors. However, metabolic rewiring was also observed in xenograft model; NCI-006 treatment exacerbated hypoxia and IACS-010759 promoted Lac production from Pyr. To overcome the metabolic rewiring, we attempted combination treatment with NCI-006 and IACS-010759 on xenograft mice. The combination treatment effectively abrogated metabolic flexibility of NET-derived xenograft tumors. Metabolic stress resulted in CAR-T cell dysfunction, while NCI-006 and IACS-010759 showed potential to improve the extracellular environment of the TME. Further studies are warranted to test the feasibility and efficacy of metabolic inhibitors in combination with CAR-T therapy. Citation Format: Takeshi Ito, Shun Kishimoto, Kazutoshi Yamamoto, Jeffrey Brender, Frank Echtenkamp, Daniel Crooks, Murali Cherukuri Krishna, William Marston Linehan. Modulating the tumor microenvironment (TME) to enhance anti tumor immunity using metabolic inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2903.

Renal oxygenation is essential for maintaining kidney function. Disruptions in oxygen delivery can lead to renal hypoxia, which can exacerbate kidney injury through multiple pathways, including inflammation, oxidative stress, and ischemia-reperfusion injury. Despite the recognized importance of oxygenation in renal pathology, noninvasive and reliable methods for assessing kidney oxygen levels are limited. Current techniques either lack sensitivity or involve invasive procedures, restricting their use in routine monitoring. Therefore, there is a pressing need for innovative approaches to map renal oxygenation, particularly in kidney injury. This study evaluated electron paramagnetic resonance (EPR)-based oxygen imaging using the paramagnetic tracer Ox071 to map kidney oxygen levels in mice with cyclophosphamide-induced kidney injury. Urine partial pressure of oxygen (Po2) was also assessed as a potential surrogate marker. EPR oximetry accurately measured kidney oxygen distribution, revealing a temporary increase in Po2 post-injury. Urine oximetry, however, did not reliably reflect changes in kidney oxygenation. Furthermore, EPR oximetry provided high-resolution spatial mapping of oxygen levels within the kidney, allowing for a detailed understanding of the impact of hypoxia on renal tissue. EPR oximetry is a promising, noninvasive tool for monitoring renal oxygenation, offering high-resolution mapping and longitudinal assessment. Its ability to provide detailed information about oxygen distribution within the kidney makes it a valuable tool for studying the pathophysiology of renal diseases and for developing novel therapeutic strategies.NEW & NOTEWORTHY Quantitative spatially resolved measurement of renal oxygenation has the potential to guide clinical decision making in renal disorders such as acute kidney injury. In this study, we demonstrate the utility of electron paramagnetic resonance imaging to provide noninvasive and quantitative high-resolution mapping of kidney oxygen concentrations.

The success of adoptive cell transfer therapy (ACT) hinges on T cell quality, particularly differentiation state, which governs in vivo persistence and long-term anti-tumor efficacy. CD8⁺ T cells with a stem cell memory (SCM) phenotype exhibit enhanced self-renewal and are associated with durable immune responses. However, inducing such phenotypes without impairing T cell expansion or function remains challenging. Cytokine conditioning and signaling pathway modulation have been explored to steer T cell fate toward less-differentiated states. Here, we investigate the combined effects of IL-21 and Wnt/β-catenin pathway activation—via GSK-3β inhibition—on human CD8⁺ T cell programming, and extend these findings to the generation and functional testing of CD19 CAR T cells. Peripheral blood CD8⁺ T cells from healthy donors were stimulated with anti-CD3/CD28 beads for 48 hours, followed by an 8-day expansion phase in IL-7 and IL-15. IL-21 was added in some conditions, alone or in combination with the GSK-3β inhibitor TWS119 to activate Wnt/β-catenin signaling. Exposure durations were varied to identify the critical window for phenotype modulation and optimize treatment conditions. CD8⁺ T cells were transduced with a CD19 CAR incorporating a 4-1BB costimulatory domain. Functional assessments on day10, included immunophenotyping, proliferation and apoptosis assays, metabolic profiling, and repeated antigen-stimulation cytotoxicity assays using CD19⁺ Nalm6 leukemia cells. IL-21 and TWS119 synergized with IL-7/15 to promote an SCM-like phenotype in CD8⁺ T cells, characterized by elevated expression of memory markers, suppression of effector differentiation, and an IL-2–dominant cytokine profile. However, continuous 10-day exposure impaired T cell quality—marked by reduced expansion, decreased viability, attenuated metabolic activity, and increased apoptosis upon restimulation. In contrast, abbreviated exposure—during days 2-4—mitigated these effects while preserving the memory-enriched phenotype, revealing a critical temporal window for optimal programming. When applied to CAR T-cell manufacturing, short-term IL-21/TWS119 conditioning preserved expansion capacity while increasing the frequency of CD8⁺ CD19 CAR T cells with an SCM-like phenotype and an IL-2–dominant cytokine production. In addition to phenotypic changes, lentiviral mediated CAR transduction was enhanced by IL-21/TWS119 treatment. Following repeated antigen challenge with CD19⁺ Nalm6 leukemia cells, conditioned CAR T cells exhibited superior proliferative capacity and sustained cytotoxic activity, in contrast to conventional CAR T cells. Short-term combinatorial conditioning with IL-21 and GSK-3β inhibition (TWS119) effectively biases human CD8⁺ T cells toward a less-differentiated SCM-like phenotype while avoiding the fitness and viability deficits induced by prolonged exposure. This temporal modulation strategy enhances the durability and anti-tumor potency of CD19 CAR T cells under chronic antigen stress. These findings support transient IL-21/Wnt pathway modulation as a viable enhancement to ACT manufacturing pipelines aimed at improving therapeutic T-cell quality and persistence.

Ovarian cancer (OV) has the highest mortality rate among gynecological cancers. As OV progresses, tumor cells spread outside the ovaries to the peritoneal and abdominal cavities, forming cell clusters that float in the ascitic fluid caused by peritonitis carcinomatosa, leading to further dissemination and metastasis. These cell clusters are enriched with cancer stem cells (CSCs) which are responsible for treatment resistance, recurrence, and metastasis. Therefore, targeting CSCs is a potentially effective approach for treating OV. However, understanding how CSCs acquire treatment resistance and identifying targets against CSCs remains challenging. In this study, we demonstrate that 3D-spheroids of OV cell lines exhibit higher stemness than conventional adherent cells. Metabolomics profiling studies have revealed that 3D-spheroids maintain a high-energy state through increased glucose utilization in the citric acid cycle (TCA), efficient nucleotide phosphorylation, and elevated phosphocreatine as an energy buffer. We also found that nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ production, is highly expressed in OV. Furthermore, the approach based on NAMPT dependence rather than histology found NAMPT to be a potential therapeutic target against CSCs, while also serving as a prognostic indicator in OV. Moreover, we identified a previously unrecognized anti-tumor mechanism whereby disulfiram, an aldehyde dehydrogenase (ALDH) inhibitor, synergistically inhibited mitochondrial function when combined with NAMPT inhibitors - leading to cell cycle arrest in G2/M. Finally, the combination of a NAMPT inhibitor and disulfiram showed significant anti-tumor effects and extended survival in an animal model. Our findings demonstrate the potential of spheroids as a preclinical model for targeting OV CSCs and also indicate that the combination of NAMPT inhibitors and disulfiram is a promising therapeutic strategy to overcome recurrent OV.

Human renal cell carcinomas (RCC) have a variety of pathologies and are known to have alterations in cellular metabolism, although many aspects remain unknown. Fumarate hydratase (FH)-deficient tumors exhibit a shift to aerobic glycolytic system due to several factors including loss and mutation of mitochondrial DNA. We investigated the partial pressure of oxygen, vascular permeability, and blood perfusion in FH-deficient UOK262 xenografts and a type 1 papillary RCC xenograft using EPRI and DCE-MRI. Despite low oxygen consumption rates in vitro, UOK262 xenografts showed a modest median pO 2 and increased hypoxic fraction as compared to a type 1 papillary RCC xenografts.

You have accessJournal of UrologyCME1 Apr 2023PD17-01 THE IMPACT OF LOSS AND MUTATION OF MITOCHONDRIAL DNA ON ONCOMETABOLITE ACCUMULATION IN PATIENT-DERIVED FUMARATE HYDRATASE-DEFICIENT RENAL TUMOR CELL LINES Bhargav Arimilli, Daniel Crooks, Ye Yang, Youfeng Yang, Cathy Vocke, Christopher Ricketts, Mark Ball, Teresa Fan, Andrew Lane, and W. Marston Linehan Bhargav ArimilliBhargav Arimilli More articles by this author , Daniel CrooksDaniel Crooks More articles by this author , Ye YangYe Yang More articles by this author , Youfeng YangYoufeng Yang More articles by this author , Cathy VockeCathy Vocke More articles by this author , Christopher RickettsChristopher Ricketts More articles by this author , Mark BallMark Ball More articles by this author , Teresa FanTeresa Fan More articles by this author , Andrew LaneAndrew Lane More articles by this author , and W. Marston LinehanW. Marston Linehan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant cancer syndrome characterized by mutations in the fumarate hydratase gene (FH). We recently reported the discovery of mutations and loss of mitochondrial DNA (mtDNA) in FH-deficient renal tumors and tumor-derived cell lines. Mitochondrial alterations in FH-deficient tumor-derived cell lines ranged from inactivating mutations in mitochondrial Complex I and Complex III subunits to complete loss of mtDNA in two tumor cell lines. The goal of the present study was to characterize the impact of mtDNA mutations on the oncometabolites fumarate and succinate in FH-deficient renal tumor cell lines. METHODS: We performed stable isotope-resolved metabolomics (SIRM) with 13C5,15N2-glutamine tracer on five FH-deficient patient-derived tumor cell lines, and utilized ion chromatography coupled with ultra-high resolution Orbitrap-based mass spectrometry (UHR-ICMS) and NMR spectroscopy to determine the abundance and isotopic labeling pattern of fumarate and its precursors in polar extracts obtained from the tumor cells. RESULTS: We found that several of the FH-deficient tumor cell lines showed elevated succinate levels, with a 13C labeling pattern that was consistent with oxidative production of succinate from glutamine. In contrast, fumarate 13C labeling patterns in these same cell lines was consistent with reductive carboxylation of glutamine and formation of fumarate from the urea cycle and purine biosynthesis, suggesting a deficiency of succinate dehydrogenase (Complex II) activity in the cells. UHR-ICMS analysis of FH-/- cells labeled with 13C1-glutamine confirmed 13C labeling of the purine biosynthetic intermediate adenylosuccinate via reductive carboxylation of glutamine, giving rise to 13C1-fumarate, and this 13C label was localized to the carboxylate carbon of fumarate using an HMBC NMR experiment. CONCLUSIONS: We have uncovered additional lesions in the TCA cycle in patient-derived HLRCC cell lines, giving rise to accumulation of the oncometabolite succinate. These data highlight the metabolic heterogeneity that is observed in genetically-defined human tumors. Source of Funding: NIH NIH MRSP © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e495 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bhargav Arimilli More articles by this author Daniel Crooks More articles by this author Ye Yang More articles by this author Youfeng Yang More articles by this author Cathy Vocke More articles by this author Christopher Ricketts More articles by this author Mark Ball More articles by this author Teresa Fan More articles by this author Andrew Lane More articles by this author W. Marston Linehan More articles by this author Expand All Advertisement PDF downloadLoading ...

Osimertinib, an EGFR tyrosine kinase inhibitor (TKI), is approved for the treatment of lung adenocarcinoma (LUAD) patients with activating EGFR mutations. However, acquired resistance to osimertinib is common. Resistance results from genomic alterations, but also through perturbations in enzyme activities, and epigenetic modifications. We aimed to identify pathways that mediate drug resistance and target them by identifying potent targeted therapies. Using quantitative proteomics and transcriptomics, we revealed altered metabolic pathways such as oxidative phosphorylation, citric acid cycle, and glutamate pathway in LUAD. Osimertinib‐resistant tumors were dependent on alpha‐ketoglutarate (aKG) pool derived from glutamine but not dependent on citrate, aspartate or fumarate for their survival. Moreover, fumarate, a competitive inhibitor of histone demethylases (HDMs) which use aKG as a cofactor, inhibited the viability of resistant cells. We validated these findings in our clinical trial (NCT02759835). Using RNAseq, we also show that in LUAD patients, oxidative phosphorylation, citric acid cycle, and glutamate pathway genes, including glutaminase (GLS) was significantly over expressed upon resistance when compared to their baseline (pre‐treatment) tumors. Moreover, the osimertinib resistant patient‐derived xenograft tumors were resensitized to osimertinib therapy when treated in combination with telaglenastat, a GLS inhibitor. Given that aKG is essential for HDMs, we investigated the activity of HDMs. Compared to baseline, upon development of resistance patients’ tumors and resistant cells exhibited several over expressed HDMs and decreased histone methylation (H3K27me3), which was restored upon treatment with telaglenastat or transient knock down of GLS, suggesting that reduced intracellular aKG due to the inhibition of GLS could have reduced the activity of HDMs. Osimertinib‐resistant xenograft tumors were susceptible to both HDM inhibitor (GSK J4), and to depletion of KDM6B, an aKG‐dependent demethylase. GSK J4 treatment and depletion of KDM6B increased histone methylation in resistant tumors and decreased their viability, thereby potentially suggesting its role in the maintenance of resistant tumors. We further investigated the role of transcription factors in regulating GLS expression. We found that JUN and RELA were over expressed and activated in resistant tumors. Transient depletion of JUN and RELA reduced GLS expression leading to increased histone methylation and reduced viability of resistant cells. Together, these results suggest that the increased glutaminase expression and activity increase the reliance of cells on aKG dependent HDMs, resulting in the transcriptional reprogramming that may be driving the osimertinib resistance that can be potentially targeted by telaglenastat and GSK‐J4 to overcome resistance.Support or Funding InformationIntramural Research Program, National Institutes of Health. Major Opportunity Grant on Metabolism, Center for Cancer Research, National Cancer Institute.

Dysregulated metabolism is an important marker of many disease states, including cancer. For example, in hereditary leiomyomatosis and renal cell cancer (HLRCC), inactivating mutations in fumarate hydratase (FH) lead to accumulation of high levels of fumarate, a so-called “oncometabolite.” Substantial evidence indicates that fumarate stimulates various oncogenic signaling pathways, necessitating sensitive methods to detect the oncometabolite in order to more rapidly diagnose HLRCC as well as to identify new disease settings in which fumarate may play a signaling role. Here, we report development of novel photoactivatable, fluorogenic chemical probes for detection and profiling of fumarate in biological systems. These chemical probes, diaryl tetrazoles, are by themselves inert towards fumarate. However, upon irradiation with UV light, they release nitrileimines that can form fluorescent cycloadducts with fumarate. We have demonstrated that diaryl tetrazoles can sensitively detect FH activity as well as low micromolar levels of fumarate in complex biological samples. We have also shown that diaryl tetrazoles can be used to monitor changes in intracellular fumarate levels in biological samples by live-cell imaging and flow cytometry. Moreover, these compounds are capable of visualizing differences between patient-derived primary HLRCC tumors lacking FH activity and the adjacent normal kidney, highlighting their potential utility in clinical diagnostics. By offering new insights into fumarate reactivity, our studies provide the chemical basis for novel approaches to therapy and diagnosis in cancers driven by oncometabolite accumulation. Citation Format: Chloe Briney, Sarah Bergholtz, Rhushikesh Kulkarni, Daniel Crooks, Chandrasekhar Mushti, Stephen Lockett, Rolf Swenson, W. Marston Linehan, Jordan Meier. Photoinducible detection of the oncometabolite fumarate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2760.

Giant cell arteritis is the most common primary systemic vasculitis in adults aged ≥50 years and peaks in the eighth decade of life. Common symptoms include headache, scalp tenderness and jaw claudication. Elevated acute phase reactants (erythrocyte sedimentation rate and C-reactive protein) are present in >90% of patients. Visual loss is a well-recognised complication, but approximately 2–4% of giant cell arteritis patients experience stroke, most frequently in the vertebrobasilar territory. We describe a 72-year-old male who developed bilateral vertebral artery occlusion and middle cerebellar peduncle infarction secondary to giant cell arteritis in spite of high-dose steroids.

INTRODUCTION AND OBJECTIVES: Despite the advent of genomic sequencing, the clinical management of bladder cancer has not changed drastically, and little progress has been made towards more precisely targeted bladder cancer therapies. Perturbed metabolic pathways in tumors constitute a potential opportunity for new therapeutic strategies. Many cancer cell lines exhibit the ″Warburg Effect,″ in that they depend on glycolysis more than normal cells. In this study, we targeted the perturbed metabolism in bladder cancer using an inhibitor of lactate dehydrogenase (LDH) developed by our institution's drug development program, with and without the inclusion of metformin. METHODS: Using the Seahorse extra-cellular flux analyzer (Agilent Technologies, Santa Clara, CA), we characterized cell lines based on their extracellular acidification rates (ECAR) and oxygen consumption rates (OCR), to determine their relative reliance on glycolysis or oxidative phosphorylation. The LDH inhibitor was tested in select cell lines for effects on proliferation, invasion, and migration. This inhibitor was also tested in vivo, either as a single agent or in combination with metformin. RESULTS: For the first time, we bioenergetically profiled fourteen bladder cancer cell lines based on their ECAR and OCR preference. Most bladder cancer cell lines depend on oxidative phosphorylation to various degrees (based on high OCR) but a few rely more heavily on glycolysis (based on high ECAR). The cell lines that relied more heavily on glycolysis were more sensitive to LDH inhibition; however, the other cell lines responded to LDH inhibition when made more glycolytic by either hypoxia or cotreatment with metformin. The UMUC3 xenograft demonstrated decreased growth with LDH inhibition. CONCLUSIONS: These data describe for the first time the metabolic phenotype of bladder cancer cell lines and demonstrate that glycolytic tumors are sensitive to LDH inhibitor. In tumors dependent on oxidative phosphorylation, a combination of LDH inhibitor with metformin may overcome LDH inhibitor resistance. Figure. No caption available. Figure. No caption available. Source of Funding: This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Objective Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function. Methods Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell‐based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples. Results Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient‐derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology. Interpretation Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy‐lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774–788

Background: Understanding the factors that drive recurrence and radiosensitivity in brain metastases would improve prediction of outcomes, treatment planning and development of therapeutics. We investigated the expression of known metastasis-inducing proteins in human brain metastases.Methods: Immunohistochemistry on metastases removed at neurosurgery from 138 patients to determine the degree and pattern of expression of the proteins S100A4, S100P, AGR2, osteopontin (OPN) and the DNA repair marker FANCD2. Validation of significant findings in a separate prospective series with the investigation of intra-tumoral heterogeneity using image-guided sampling. Assessment of S100A4 expression in brain metastatic and non-metastatic primary breast carcinomas.Results: There was widespread staining for OPN, S100A4, S100P and AGR2 in human brain metastases. Positive staining for S100A4 was independently associated with a shorter time to intracranial progression after resection in multivariate analysis (hazard ratio for negative over positive staining = 0.17, 95% CI: 0.04-0.74, P = 0.018). S100A4 was expressed at the leading edge of brain metastases in image guided sampling and overexpressed in brain metastatic vs non-brain metastatic primary breast carcinomas. Staining for OPN was associated with a significant increase in survival time after post-operative whole-brain radiotherapy in retrospective (OPN negative 3.43 months, 95% CI: 1.36-5.51 vs OPN positive, 11.20 months 95% CI: 7.68-14.72, Log rank test, P<0.001) and validation populations.Conclusions: Proteins known to be involved in cellular adhesion and migration in vitro, and metastasis in vivo are significantly expressed in human brain metastases and may be useful biomarkers of intracranial progression and radiosensitivity.

Objective: Many different gene families are currently being investigated for their potential role in epilepsy and in the response to antiepileptic drugs. A common research challenge is identifying the members of a gene family that are most significantly dysregulated within the human epileptic focus, before taking them forward for resource-intensive functional studies. Published data about transcriptomic changes within the human epileptic focus remains incomplete. A need exists for an accurate in silico system for the prediction of dysregulated genes within the epileptic focus. We present such a bioinformatic system. We demonstrate the validity of our approach by applying it to the solute carrier (SLC) gene family. There are >400 known SLCs. SLCs have never been systematically studied in epilepsy.Methods: Using our in silico system, we predicted the SLCs likely to be dysregulated in the epileptic focus. We validated our in silico predictions by identifying ex vivo the SLCs dysregulated in epileptic foci, and determining the overlap between our in silico and ex vivo results. For the ex vivo analysis, we used a custom oligonucleotide microarray containing exon probes for all known SLCs to analyze 24 hippocampal samples obtained from surgery for pharmacoresistant mesial temporal lobe epilepsy and 24 hippocampal samples from normal postmortem controls.Results: There was a highly significant (p < 9.99 x 10(-7)) overlap between the genes identified by our in silico and ex vivo strategies. The SLCs identified were either metal ion exchangers or neurotransmitter transporters, which are likely to play a part in epilepsy by influencing neuronal excitability.Significance: The identified SLCs are most likely to mediate pharmacoresistance in epilepsy by enhancing the intrinsic severity of epilepsy, but further functional work will be needed to fully evaluate their role. Our successful in silico strategy can be adapted in order to prioritize genes relevant to epilepsy from other gene families.

Increasingly, biomarkers have been identified that correlate with improved overall and progression-free survival (OS and PFS) in glioblastoma, including MGMT methylation status and mutations in the IDH1 gene. In this study, we investigated the clinical and biological factors associated with long-term survival in glioblastoma patients treated with chemoradiotherapy.

Numerous diverse biological pathways are dysregulated in the epileptic focus. Which of these pathways are most critical in producing the biological abnormalities that lead to epilepsy? Answering this question is key to identifying the primary causes of epilepsy and for discovering new therapeutic strategies with greater efficacy than currently available antiepileptics (AEDs). We have performed the largest genome-wide transcriptomic analysis to date comparing epileptic with normal human hippocampi. We have identified 118 differentially expressed and, for the first time, differentially connected pathways in the epileptic focus. Using network mapping techniques, we have shown that these dysregulated pathways, though seemingly disparate, form a coherent interconnected central network. Using closeness centrality analysis, we have identified that the most influential hub pathways in this network are signalling through G protein-coupled receptors, in particular opioid receptors, and their downstream effectors PKA/CREB and DAG/IP3. Next, we have objectively demonstrated that genetic association of gene sets in independent genome-wide association studies (GWASs) can be used to identify causally relevant gene sets: we show that proven causal epilepsy genes, which cause familial Mendelian epilepsy syndromes, are associated in published sporadic epilepsy GWAS results. Using the same technique, we have shown that central pathways identified (opioid receptor and PKA/CREB and DAG/IP3 signalling pathways) are genetically associated with focal epilepsy and, hence, likely causal. Published functional studies in animal models provide evidence of a role for these pathways in epilepsy. Our work shows that these pathways play a central role in human focal epilepsy and that they are important currently unexploited antiepileptic drug targets.

Study Design Case series and review of the literature. Objective To review the management of giant calcified disks in our large cohort and compare with the existing literature. We discuss our surgical technique. Methods Twenty-nine cases of herniated thoracic disk between 2000 and 2013 were reviewed. Eighteen patients were identified as having giant calcified thoracic disks, defined as diffusely calcified disks occupying at least 40% of the spinal canal. Demographic data was collected in addition to presentation, imaging findings, operative details, and outcomes using the modified Japanese Orthopaedic Association (mJOA) scale. Results Giant calcified thoracic disks (GCTDs) are unique clinical entities that require special neurosurgical consideration owing to significant (≥40%) involvement of the spinal canal and compression of the spinal cord, often leading to myelopathy. The median age at diagnosis was 51.2 years (range 37 to 70) with the mean duration of presenting symptoms being 9.9 months (range 2 weeks to 3 years). Seventeen (94.4%) patients presented with at least one sign of myelopathy (hyperreflexia, hypertonia, bladder or bowel dysfunction) with the remaining 1 (5.6%) patient presenting with symptoms in keeping with radiculopathy. Thoracotomy was performed on 17 (94.4%) patients, and 1 (5.6%) patient had a costotransverse approach. Mean follow-up was 19.8 months (range 7 months to 2 years). mJOA score improved in 15 (83.3%) patients. mJOA scores in the other patients remained stable. Conclusions GCTDs are difficult neurosurgical challenges owing to their size, degree of spinal cord compression, and consistency. We recommend a trench vertebrectomy via a thoracotomy in their surgical management. This procedure safely allows the identification of normal dura on either side of the compressed segment prior to performing a diskectomy. Excellent fusion rates were achieved with insertion of rib head autograft in the trench.