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Surgery is the mainstay of treatment for meningioma, the most common primary intracranial tumor, but improvements in meningioma risk stratification are needed and indications for postoperative radiotherapy are controversial. Here we develop a targeted gene expression biomarker that predicts meningioma outcomes and radiotherapy responses. Using a discovery cohort of 173 meningiomas, we developed a 34-gene expression risk score and performed clinical and analytical validation of this biomarker on independent meningiomas from 12 institutions across 3 continents (N = 1,856), including 103 meningiomas from a prospective clinical trial. The gene expression biomarker improved discrimination of outcomes compared with all other systems tested (N = 9) in the clinical validation cohort for local recurrence (5-year area under the curve (AUC) 0.81) and overall survival (5-year AUC 0.80). The increase in AUC compared with the standard of care, World Health Organization 2021 grade, was 0.11 for local recurrence (95% confidence interval 0.07 to 0.17, P

Low-grade gliomas (LGGs) are the most common brain tumor affecting children. We recently reported an early phase clinical trial of a peptide-based vaccine, which elicited consistent antigen-specific T cell responses in pediatric LGG patients. Additionally, we observed radiologic responses of stable disease (SD), partial response (PR), and near-complete/complete response (CR) following therapy. To identify biomarkers of clinical response in peripheral blood, we performed RNA sequencing on PBMC samples collected at multiple time points. Patients who showed CR demonstrated elevated levels of T cell activation markers, accompanied by a cytotoxic T cell response shortly after treatment initiation. At week 34, patients with CR demonstrated both IFN signaling and Poly-IC:LC adjuvant response patterns. Patients with PR demonstrated a unique, late monocyte response signature. Interestingly, HLA-V expression, before or during therapy, and an early monocytic hematopoietic response were strongly associated with SD. Finally, low IDO1 and PD-L1 expression before treatment and early elevated levels of T cell activation markers were associated with prolonged progression-free survival. Overall, our data support the presence of unique peripheral immune patterns in LGG patients associated with different radiographic responses to our peptide vaccine immunotherapy. Future clinical trials, including our ongoing phase II LGG vaccine immunotherapy, should monitor these response patterns.

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

PurposeMyxopapillary ependymoma (MPE) is a distinct histologic variant of ependymoma arising commonly in the spinal cord. Despite an overall favorable prognosis, distant metastases, subarachnoid dissemination, and late recurrences have been reported. Currently, the only effective treatment for MPE is gross-total resection. We characterized the genomic and transcriptional landscape of spinal ependymomas in an effort to delineate the genetic basis of this disease and identify new leads for therapy.Experimental designGene expression profiling was performed on 35 spinal ependymomas, and copy number profiling was done on an overlapping cohort of 46 spinal ependymomas. Functional validation experiments were performed on tumor lysates consisting of assays measuring pyruvate kinase M activity (PKM), hexokinase activity (HK), and lactate production.ResultsAt a gene expression level, we demonstrate that spinal grade II and MPE are molecularly and biologically distinct. These are supported by specific copy number alterations occurring in each histologic variant. Pathway analysis revealed that MPE are characterized by increased cellular metabolism, associated with upregulation of HIF1α. These findings were validated by Western blot analysis demonstrating increased protein expression of HIF1α, HK2, PDK1, and phosphorylation of PDHE1A. Functional assays were performed on MPE lysates, which demonstrated decreased PKM activity, increased HK activity, and elevated lactate production.ConclusionsOur findings suggest that MPE may be driven by a Warburg metabolic phenotype. The key enzymes promoting the Warburg phenotype: HK2, PKM2, and PDK are targetable by small-molecule inhibitors/activators, and should be considered for evaluation in future clinical trials for MPE.

Low‐grade epilepsy‐associated tumors (LEATs) are a common cause of drug‐resistant epilepsy in children. Herein, we demonstrate the feasibility of using tumor tissue derived from stereoelectroencephalography (sEEG) electrodes upon removal to molecularly characterize tumors and aid in diagnosis. An 18‐year‐old male with focal epilepsy and MRI suggestive of a dysembryoplastic neuroepithelial tumor (DNET) in the left posterior temporal lobe underwent implantation of seven peri‐tumoral sEEG electrodes for peri‐operative language mapping and demarcation of the peri‐tumoral ictal zone prior to DNET resection. Using electrodes that passed through tumor tissue, we show successful isolation of tumor DNA and subsequent analysis using standard methods for tumor classification by DNA, including Glioseq targeted sequencing and DNA methylation array analysis. This study provides preliminary evidence for the feasibility of molecular diagnosis of LEATs or other lesions using a minimally invasive method with microscopic tissue volumes. The implications of sEEG electrodes in tumor characterization are broad but would aid in diagnosis and subsequent targeted therapeutic strategies. [ABSTRACT FROM AUTHOR]

Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in

The multifunctional protein - tissue inhibitor of metalloproteinases-1 (TIMP-1) - has been associated with poor prognosis in several types of cancers including glioblastomas. Glioblastomas are the most common and malignant primary brain tumor known for being highly invasive and resistant to therapy. New treatment strategies are continuously being explored and currently vascular endothelial growth factor (VEGF) inhibitors administered in combination with Irinotecan is the most promising second line treatment. TIMP-1 has been associated with decreased response to chemotherapy in breast and colorectal cancer and especially the family of topoisomerase (TOP) inhibitors, such as Irinotecan, has been suggested to be affected by TIMP-1. In the present study, we investigated whether a high TIMP-1 expression in glioblastoma cells played a role in TOP inhibitor resistance. We established two TIMP-1 over-expressing cell lines and evaluated the sensitivity towards the TOP1 inhibitor SN-38 and the TOP2 inhibitor Epirubicin using a viability and a cytotoxicity assay. In addition, we investigated the invasive features of the cells in a brain slice culture model as well as in an orthotopic xenograft model. The results showed that TIMP-1 over-expressing U87MG cell line sub-clones were significantly more resistant than the controls when exposed to SN-38 and Epirubicin. The same tendency was seen for the TIMP-1 over-expressing A172 sub-clones. No significant differences in invasion patterns were observed for TIMP-1 over-expressing sub-clones when compared to controls. In conclusion, the present study suggests that TIMP-1 over-expression reduces the effect of TOP inhibitors in the glioblastoma cell line U87MG. There was no significant effect of TIMP-1 over-expression on tumor cell invasion. The association found between TIMP-1 cellular levels and the effect of TOP inhibitors needs to be validated in clinical patient material. Pediatric supratentorial high-grade astrocytomas (pHGAs) and diffuse intrinsic pontine gliomas (DIPG) are devastating pediatric malignancies for which no effective therapies exist. Poly-(ADP-Ribose)-Polymerase (PARP) protein expression is found in ∼60% of DIPGs suggesting PARP may be a potential therapeutic target. PARP1/2 were characterized by Western-blotting in normal human astrocytes (NHA), pHGA cell lines (SJG2, SF-188), DIPG cell lines (DIPG-M, DIPG58), and one murine brainstem glioma cell line (mBSG). Cell viability in response to different dosages of Olaparib, Veliparib, or Niraparib was determined using the MTT Assay. PARP activity, apoptosis, and DNA damage was determined by Western blotting against PAR, cleaved PARP, and phosphorylated yH2AX, respectively. Cell cycle phases were analyzed using FACS and western blot for p21. Western blotting demonstrated that, compared with NHAs, PARP1 were highly expressed in SJG2, DIPG-M, and DIPG-58 cells. PARP2 expression was only detected in SJG2 cells. All PARP inhibitors reduced PARP activity as indicated by reduced PAR levels. Olaparib reduced SJG2, mBSG, DIPG58 and DIPGM cell viability at concentrations of 5 or 10uM uM (P < 0.05), Whereas Niraparib induced cytotoxicity at concentrations of 2uM and above (P < 0.05). Olaparib and Niraparib induced DNA damage and apoptosis in SJG2 at doses of 5, 10uM and 2, 5, 10uM, respectively. Niraparib induced G2 arrest in mBSG demonstrated by FACS and increased levels of p21 (P < 0.05). Our data provides in vitro evidence that PARP inhibition may be an effective therapeutic avenue for treatment of pHGA and DIPG. Furthermore while all PARP inhibitors suppress PARP activity not all PARP inhibitors reduce cell viability. Thus not all PARP inhibitors can be expected to be equally efficacious in a clinical trial setting. Toca 511 (vocimagene amiretrorepvec), an amphotropic retroviral replicating vector (RRV), can successfully and safely deliver a functional, optimized yeast cytosine deaminase (CD) gene to tumors in orthotopic glioma models. Within infected cells, CD converts 5-fluorocytosine (5-FC) to the anti-cancer drug 5-FU. The combination of Toca 511 with oral extended release 5-FC (Toca FC), is currently in clinical trials for recurrent High Grade Glioma (HGG, NCT01156584 and NCT01470794). Temozolomide (TMZ), in combination with radiation therapy, is the most commonly used first-line chemotherapy treatment for patients with glioblastoma, the most common and aggressive form of primary brain cancer. A separate study (Takahashi et al., this meeting) addresses the potential radiation synergy with Toca 511/5-FC treatment. A subset of patients with certain genetic alterations does not respond well to TMZ treatment and the overall median survival for patients who respond remains poor, suggesting combinatorial approaches may be necessary to significantly improve patient outcomes. To determine whether Toca 511 and 5-FC therapy is compatible with TMZ, we examined the effect of TMZ in combination with Toca 511 and 5-FC in TMZ-sensitive and resistant glioma lines both in vitro and in vivo. We show that in vitro TMZ delays but does not prevent RRV spread, nor interfere with Toca 511 and 5-FC mediated cell killing in glioma tumor cells, and in vivo there is no significant hematologic effect from the combination of 5-FC and the clinically relevant dose of TMZ. A synergistic long-term survival advantage is observed in mice bearing an orthotopic TMZ-sensitive glioma tumor after Toca 511 administration followed by co-administration of TMZ in combination with 5-FC. These results provide support for the investigation of this novel combination treatment strategy for patients with newly diagnosed glioblastoma. BACKGROUND: LAZ is a 21-aminosteroid that has radioprotective effects against radiation-induced lipid peroxidation. Also antiproliferative effects have been reported against glioblastoma cell lines. DESIGN/METHODS: LAZ PEGylated liposomes (Lipo G) were developed at the University of Houston.. Glioblastoma cell line U87-expressing firefly luciferase reporter gene (100,000 cells in 2 µL) was injected intracranially in each SCID mouse. There were 4 treatment groups (n = 8-9, each): brain model (M) without treatment (control), radiation 2Gy weekly (M + R), Lipo G at 5 mg/kg dose intraperitoneally twice per week (M + L) and radiation with Lipo G (M + R + L). Treatment lasted three weeks. Tumor size was monitored using bioluminescence imaging (BLI), in each mouse. Mice were sacrificed after 3 weeks. Brain was harvested. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. Survival was evaluated using Kaplan Meier analysis at P= 0.05. RESULTS: BLI intensity was 4002.03 ± 1737.67, 2034 ± 737.72, 1387.36 ± 684.53 and 2498.89 ± 2521.32 % for M, M + R, M + L and M + R + L, respectively. Tumor size of the M + L group was reduced by 65% compared to control. There was no significant difference in tumor size of radiated groups compared to control group. MDA brain concentration in M + L and M + R + L groups was significantly less than in M + R group (8.27 ± 0.78 and 10.37 ± 3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M + R, M + R + L and M + L groups, respectively. Mean survival of LAZ treated groups (M + L and M + R + L) was significantly longer than that of the control group. CONCLUSIONS: LAZ liposomal formulations reduced tumor growth by 65%. LAZ also protected brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration by 50%. These provocative data warrant further investigation of LAZ as a radiation protectant and chemotherapeutic agent. Patients with malignant brain tumors have a median survival of approximately one year following diagnosis, regardless of currently available treatments which include surgery followed by radiation and chemotherapy. Improvement in the survival of brain cancer patients requires the design of new therapeutic modalities that take advantage of common phenotypes. One such phenotype is the metabolic dysregulation that is a hallmark of cancer cells. It has therefore been postulated that one approach to treating brain tumors may be by metabolic alteration such as that which occurs through the use of the ketogenic diet (KD). The KD is high-fat, low-carbohydrate diet that induces ketosis and has been utilized for the non-pharmacologic treatment of refractory epilepsy. We and others have shown that this diet enhances survival and potentiates standard therapy in mouse models of malignant gliomas, yet the anti-tumor mechanisms are not fully understood. It has been previously shown that caloric restriction, which induces ketosis, reduces microvessel density in mouse and human brain tumor models, suggesting an anti-angiogenic effect. We now report that in animals fed KetoCal® (KC)(4:1 fat:protein/carbohydrates) ad libitum, peritumoral edema is significantly reduced early in tumor progression when compared to those fed a standard rodent diet. Gene expression profiling demonstrated that KC decreases the expression of the gene encoding vascular endothelial growth factor B (VEGFB) and angiopoetin 1 receptor (TEK). Furthermore, protein analysis showed a reduction of platelet endothelial cell adhesion molecule 1 (PECAM1/CD31) in tumors from animals maintained on KC. Taken together our data suggests that KC alters the angiogenic processes involved in malignant progression of gliomas. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use.

The kynurenine pathway (KP) is the principal route of L-Tryptophan (TRP) catabolism leading to the production of kynurenine (KYN), the neuroprotectants, kynurenic acid (KYNA) and picolinic acid (PIC), and the excitotoxic neurotoxin, quinolinic acid (QUIN). The enzymes indoleamine 2,3-dioxygenase-1 (IDO-1), indoleamine 2, 3-dioxygenase-2 (IDO-2) and tryptophan 2,3-dioxygenase (TDO-2) initiate the first step of the KP. Downstream enzymes include kynureninase (KYNU), 3-hydroxyanthranilate 3,4-dioxygenase (3-HAAO), kynurenine hydroxylase (KMO) and 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD). Kynurenine aminotransferase-I (KAT-I) is one of the enzymes responsible for synthesising KYNA. Mounting evidence directly implicates that IDO-1 induction in various tumours is a crucial mechanism facilitating tumour immune evasion and persistence. However, the involvement of the downstream machinery of the KP in brain tumour progression remains unexplored. A complete characterisation of the KP in brain tumours and the role of the KP in maintaining homeostasis between neuroprotection and neurodegeneration in glioma has not yet been investigated. Here we report the first comprehensive characterisation of the KP in cultured human glioma cells and GBM patient plasma. Our qRT-PCR data revealed that interferon-gamma (IFN-γ) (100 IU/ml) stimulation significantly potentiated the expression of IDO-1 IDO-2, KYNU, 3-HAAO, KMO and significantly down-regulated ACMSD and KAT-I expression in cultured human glioma cells. HPLC analysis revealed that IFN-γ stimulation significantly increased KP activity (KYN/TRP ratio), and significantly lowered the KYNA/KYN neuroprotective ratio in human cultured glioma cells. Our HPLC and GCMS data revealed that KP activation was significantly higher and the concentrations of TRP, KYNA, QUIN and PIC and the KYNA/KYN ratio were significantly lower in GBM patient plasma (n = 18) compared to controls. These results provide further evidence for the involvement of the KP in glioma pathophysiology and highlights a potential role of KP products as novel and highly attractive therapeutic targets to evaluate for the treatment of brain tumours.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Very little is known about aggressive forms of this disease, such as metastatic or recurrent MBs. In order to identify pathways involved in aggressive MB pathophysiology, we performed unbiased, whole genome microarrays on MB tumors at both the human and murine levels. Primary human MBs were compared, transcriptomically, to their patient-matched recurrent or metastatic tumors. Expression profiling was also performed on murine tumors from two spontaneously developing MB mouse models (Ptch+/- and Smo/Smo) that present with differing clinical severities. At both the human and murine levels we identified transforming growth factor-beta (TGF-β) as a potential contributor to MB progression/metastasis. Smad3, a major downstream component of the TGF-β pathway, was also evaluated using immunohistochemistry in malignant human tissues and was shown to correlate with MB metastasis and survival. Similarly, Smad3 expression during development identified a subset of cerebellar neuronal precursors as putative cells of origin for the Smad3-positive MBs. To our knowledge, this is the first study that links TGF-β to MB pathogenesis. Our research suggests that canonical activation of this pathway leads to better prognosis for patients.