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Activation of the epidermal growth factor receptor (EGFR) pathway is strongly implicated in the proliferation and migration of neural/glial progenitors and gliomas. EGFR is silenced after development but becomes aberrantly overexpressed in many low-grade and most high-grade gliomas, often in the absence of gene-amplification/activating mutations. The phenotype and functional characteristics of EGFR-expressing human brain cells are not well defined, as is the relationship of EGFR expression to gliomagenesis. We recently reported on the selective retention of EGFR expression in adult human subventricular zone (SVZ) glia. Notably, we also showed a strikingly similar epigenetic landscape at the wild-type EGFR promoter between germinal matrix/SVZ and EGFR-expressing gliomas. Here we undertook a detailed characterization of EGFR-expressing cells in the developing and adult human brain using warm postmortem material, and in human glioblastomas after fresh resection, hypothesizing that EGFR-positive cells harbor stem cell-like properties in vitro, which may implicate them in gliomagenesis. To better characterize the phenotype of human EGFR-expressing brain cells in vivo, we performed detailed confocal immunofluorescence analysis with markers for glial/neuronal lineage differentiation and proliferation. We found that during neural development, many EGFR-positive cells co-express markers of oligodendrocyte lineage, such as Olig2, similarly to most glioblastomas. We then analyzed the behavior of these cells in vitro. We used our novel, human-based protocol to isolate cells directly from fresh human samples, taking advantage of the inherent binding affinity of EGFR for its natural ligand, EGF. Intriguingly, we found that EGFR-positive, but not EGFR-negative, cells isolated from germinal matrix and from glioblastomas display stem-cell characteristics in culture, including self-renewal and trilineage differentiation. Our findings suggest that EGFR expression confers stem-cell-like properties to human progenitors and glioma cells, hint that its maintenance may play a role in gliomagenesis, and provide a novel isolation platform of functionally defined EGFR+ glioma populations for further molecular analyses.

Angiogenesis mediated by vascular endothelial growth factor (VEGF) is a hallmark of glioblastoma. Moreover, VEGF may exert tumor-intrinsic survival properties mediated both by VEGF receptors 1 and 2. Although various VEGF inhibitors have shown limited clinical activity in glioblastoma, notably determined by responses on neuroimaging and prolongation of progression-free survival (PFS), no VEGF antagonist has so far been demonstrated to unequivocally improve overall survival in any clinical setting in glioblastoma. Based on response rates and improved PFS, although not overall survival (OS), the 149 kDa anti-VEGF-A IgG antibody bevacizumab (Avastin®) is approved in the US and Japan for recurrent glioblastoma and in Japan for newly diagnosed glioblastoma. However, it is not approved in the EU. Here we characterize DLX1008, a 26 kDa anti-VEGF-A single chain antibody fragment which binds with 22 pM and 27 pM affinity to mouse and human VEGF-A, respectively. In vitro, in a tube formation assay with human cerebral microvascular endothelial cells (HCMEC), it was demonstrated that DLX1008 is at least as active as Avastin®. In addition, DLX1008 showed superiority to Avastin® in the inhibition of VEGF-A binding to VEGF-R1 in ELISA by a factor of around 10. In vivo, DLX1008 significantly improved survival in a mouse orthotopic U87 xenograft model compared to vehicle control (p = 0.00026) and significantly inhibited tumor growth in a mouse subcutaneous U87 xenograft model compared to vehicle control (p = 0.0021). In summary, these data warrant further clinical development of DLX1008 in a biomarker-driven approach to glioblastoma. DLX1008 may provide improved clinical benefit in glioblastoma, especially in overall survival, due to higher affinity and smaller size leading to improved tumor penetration.

BACKGROUND: Tumor Treating Fields (TTFields) are an anti-mitotic, electric-physical treatment modality. An interim analysis of the phase 3 trial comparing TTFields with maintenance temozolomide (TTFields/TMZ) showed superior progression free and overall survival compared to temozolomide alone (TMZ alone) in patients with newly diagnosed GBM. METHODS: After completion of radiotherapy with concomitant temozolomide, patients were randomized (2:1) to TTFields/TMZ or TMZ alone. The main trial endpoints were progression-free survival (PFS) and overall survival (OS). Here we report the quality of life (EORTC QLQ C-30 / BN20), Karnofsky performance scores (KPS) and minimental status exam (MMSE) results in the interim analysis of the first 315 patients randomized, with a median follow-up of 38 months (range 18-60). RESULTS (intent-to-treat): 210 patients were randomized to TTFields/TMZ and 105 patients to TMZ alone. Patient characteristics were balanced. Median PFS was 7.1 and 4.0 months [Hazard ratio (HR) 0.62, p = 0.001], respectively and OS from randomization was 19.6 and 16.6 months (HR 0.74, p = 0.034), respectively, both favoring TTFields/TMZ. Global health status improved in patients treated with TTFields/TMZ (change from baseline to 3 months [CFB3] = +24% and change from baseline to 6 months [CFB6] = +13%), whereas a decrease was seen in patients treated with TMZ alone (CFB3 = -7% and CFB6 = - 17%). A full analysis of all domains of the QLQ C-30/BN-20 will be presented. No differences in KPS or MMSE was seen between treatment groups over time. CONCLUSIONS: Maintenance TTFields/TMZ provides a clinically and statistically significant improvement in progression-free and overall survival in newly diagnosed GBM. Quality of life was not adversely affected by the continuous use of TTFields and may be improved in some domains of the EORTC QLQ C-30 and BN20. Cognitive and functional capabilities were not impeded by the addition of TTFields to TMZ therapy.

A population-based analysis of patterns of care and outcome in glioma patients diagnosed 1980-1994 in the Canton of Zurich, Switzerland, has confirmed the overall poor prognosis of glioblastoma. To explore changes in outcome over the last decades, the registry data were re-evaluated for patients diagnosed in the time frame 2005-2009. Patients diagnosed with glioblastoma in the Canton of Zurich from 2005-2009 were identified by the Cancer Registry Zurich. Clinical and epidemiological data, as well as molecular markers were assessed, and analyzed using the Kaplan-Meier method and the Cox proportional hazards model. In the current database, there were 264 patients with glioblastoma, including 256 primary glioblastomas with an annual incidence of 3.98 compared to 3.55 in the former patient group. Median age at diagnosis was 60.0 years recently as opposed to 61.3 years previously. Overall survival (OS) for all glioblastoma patients was 41.7% at 1 year, 22.7% at 2 years and 13.1% at 3 years in the present study while previously the OS was significantly lower, namely 17.7% at 1 year, 3.3% at 2 years and 1.2% at 3 years, respectively. Median OS for primary glioblastomas was 11.0 months for the period ending 2009 versus 4.7 months for the patient population of 1980-1994. In the present study, by treatment, the median OS for best supportive care, radiotherapy alone, temozolomide alone or radiotherapy plus temozolomide was 2.0, 6.0, 6.0 or 17.0 months, respectively. Multivariate analysis revealed age, KPS, extent of resection, adjuvant treatment regimens, year of diagnosis, IDH-1 mutation status and MGMT promoter methylation status significantly associated with survival. The OS of patients in the Canton of Zurich with newly diagnosed glioblastoma has markedly improved from the period of 1980-1994 to 2005-2009.

The efficiency of a healthy brain network (the ‘connectome’) is positively related to physical and cognitive function. We sought to determine whether changes in network efficiency were related to loss of heterozygosity (LOH) at 1p/19q, a favorable prognostic marker in low-grade glioma (LGG). The presurgical diffusion tensor imaging (DTI) of LGG patients (WHO grade II) with known 1p/19q status treated at MGH were retrospectively analyzed. Processing included co-registration with anatomical 3D images, probabilistic tractography with FSL, and binary network construction based on the number of streamlines connecting each parcel from the Automated Anatomical Labeling atlas for which a benchmark healthy connectome (HC) is available. Path length (the average shortest number of steps between each network region) of the whole brain and the hemisphere contralateral to the tumor were compared to the HC by one-sample Wilcoxon signed rank tests. Path lengths of patients with and without LOH were compared using Mann-Whitney U tests. Thirteen patients with images acquired between 2008-2014 were included (8 male, mean age 42 ± 14 years, 8 left-sided tumors, median KPS 90). Four patients showed LOH. At the group level, patients had longer whole-brain path length (2.5 ± 0.3 steps, p = 0.023) and contralateral path length (2.0 ± 0.2 steps, p = 0.033) than the HC (2.3 and 1.9 steps, resp.), indicating a less efficient connectome. Patients with LOH had shorter contralateral path length than those without (2.5 ± 0.3 versus 2.3 ± 0.1 steps, p = 0.011). Age, gender, presurgical tumor volume, and performance status did not influence these results significantly. These results indicate that patients with LGG have decreased connectome efficiency even in the contralateral hemisphere, highlighting the global impact of local tumors. 1p/19q LOH is associated with a more efficient connectome, supporting the more favorable performance status and prognosis in these patients. Connectivity is therefore a promising noninvasive biomarker for prognosis and holds potential for treatment monitoring.

BACKGROUND: Distinguishing radiation necrosis (RTN) from recurrent brain tumor (RBT) by standard MRI morphology poses significant challenges. In addition, histologic features associated with RTN and RBT have not been quantified, leading to diagnostic imprecision at lesion resection. We quantitated morphologic MRI characteristics and histologic features in RTN and RBT in irradiated brain tumor patients undergoing lesion resection of imaging progression. METHODS: Retrospective review of irradiated brain tumor patients with imaging progression identified at lesion resection to be predominant RTN (>80% radiation necrosis) or predominant RBT ( >80% active tumor). Two neuroradiologists blinded to diagnosis quantitated (0, 75%) presurgical MRI features. A profile of histologic findings was similarly scored by one neuropathologist blinded to diagnosis. RESULTS: Study group includes 54 cases for MRI review (30 RBT, 20 RTN) and 38 for histology review (20 RBT and 18 RTN). In a multivariate model of quantitated imaging features, probabilities that RTN and RBT can be estimated in glioma patients are 71% for RBT, 65% for RTN via ROC analysis. T1 hyperintensity, necrosis, spreading wavefront and "Swiss cheese/soap bubble" appearance are moderately correlated with RTN. In brain metastasis, 89% of RTN and 70% of RBT cases are correctly classified. In pathology specimens, significant differences in severity of zonal geographic necrosis in glioma (p = 0.002) and metastasis (p = 0.012) and severity of vascular hyalinization in glioma (p= 0.44) were identified. Fibrinoid and coagulative necrosis and vessel wall thickening were not significantly different in RTN versus RBT. CONCLUSIONS: Quantitative assessment of morphologic MRI abnormalities can assist in distinguishing RTN from RBT, especially in brain metastasis. Quantitative assessment of histologic features identified features significantly correlated with RTN versus RBT. A larger sample size is needed to confirm these findings and is ongoing. Grant support: UL1 RR024989 from NCRR/NIH

Identification of Arterial Input Function (AIF) is one of the crucial steps for quantitative analysis of tissue vascular permeability in Dynamic Contrast Enhanced Magnetic Resonance Imaging studies. In practice, the global AIF is usually identified by experts, which never accounts for dispersion, delay, and partial volume effects. In this pilot study, it is hypothesized that embedded tumors are supplied by their surrounding tissue. This study compares the MR estimates of Pharmacokinetic parameters (PK) in Glioblastoma-Multiforme (GBM) tumors for Peritumoral-derived AIF (PTAIF) as opposed to the use of global AIF. Using the trace of the contrast agent concentration (∝ to relaxivity change, ΔR1) in seventeen treatment-naive GBM patients, a neuroradiologist manually picked the global AIF for each patient. A series of two-dimensional Region of Interests were drawn on the periphery of each tumor in each slice to generate a three-dimensional ROI for encompassing the whole tumor. Then the ΔR1 profile was averaged over the 3D-ROI to generate the PTAIF. For each patient, the global AIF and PTAIF profiles were normalized to the brain white matter. Using the Model-Selection technique and Maximum-Likelihood algorithm, all PK parameters of three Toft-derived models (models 1, 2 and 3), were estimated for two AIFs. The results revealed that PK analysis using PTAIF generates higher plasma volumes in model-1 and model-2 (20% and 60%) with 52% lower plasma volume in model-3 compared to global AIF. PK analysis using PTAIF generates lower transfer constants in models-2 and 3 (54% and 90% for outward-transfer-constant of model-2 and 3 respectively and 13% for backward-transfer-constant of model-3). Also, the extra-vascular-extra-cellular space estimated by the PTAIF compared to the AIF shows significant reduction. The results of this study imply that using Tissue Input Function such as PTAIF compared to the globally selected AIF may highly change the estimates of the PK parameters.

BACKGROUND: RTOG 0933 demonstrated reduced risk of NCF decline in patients with brain metastases treated with HA-WBRT versus historical controls treated with standard WBRT. Hippocampal volume (HV) derived from high resolution MRI (HR-MRI) is an established predictor of NCF in Alzheimer's disease. We assessed associations of HV with NCF of patients enrolled on RTOG 0933. METHODS: HV (-Left, -Right, -Total) was calculated from (1) the submitted treatment structure file and (2) independent processing of baseline HR-MRI with validated software (FreeSurfer, FS). HV was correlated with baseline and 4 month NCF scores (Hopkins Verbal Learning Test-Revised (HVLT-R) Total Recall (TR), Immediate Recognition (IR), and Delayed Recall (DR)) using Pearson correlation. Comparisons between deteriorated and non-deteriorated patients were made using Wilcoxon. RESULTS: 39 of the 42 evaluable patients for the RTOG 0933 primary endpoint had hippocampal contours. All 42 had HR-MRIs, but only 21 were processable by FS due to scan quality. HVs from FS were larger than plan contours (median HV-Total 7.2 vs. 5.5 cc), though were significantly correlated (ρ = 0.68, p = 0.001). Larger FS HV-Total and HV-Right were significantly correlated with improved baseline HVLT-R TR (ρ = 0.46/p = 0.04, ρ = 0.51/p = 0.02, respectively) and DR (ρ = 0.46/p = 0.04, ρ = 0.49/p = 0.03, respectively) but not IR. Plan contour HV was not correlated with baseline HVLT-R though there was a trend for HV-Left with 4 month HVLT-R TR (ρ = 0.29/p = 0.07). There were no significant associations between HV and HVLT deterioration or change from baseline to 4 months. CONCLUSIONS: Baseline HV calculated from automatic segmentation of the hippocampus is significantly associated with baseline NCF. HV is not correlated with deterioration in NCF, though analysis is limited by few events and analyzable patients. The importance of HV will be explored further in two developing NRG trials. This project was supported by grants U10CA21661, U10CA180868, U10CA180822, U10CA37422 and UG1CA189867 from the National Cancer Institute (NCI).

IDH1 mutations in lower grade glioma result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, altered gene expression, and cellular transformation. IDH1 mutations, however, are also linked to mutations in the ATRX gene and the hTERT promoter, suggesting that IDH1 mutation contributes to events that stabilize telomeres and help drive the cellular immortalization and transformation processes. To determine if and how IDH1 mutation influences telomeric stabilization and cellular immortalization, pRb/p53-deficient normal human astrocytes (E6/E7 NHA) were lentivirally infected with constructs encoding WT (IDH1 WT) or R132H mutant IDH 1 (IDH1mut). Following selection, the cells were then monitored for 2HG levels, histone modifications, hTERT promoter mutations, ATRX and telomerase expression, and telomere length as they progressed through their lifespan. Introduction of IDH1mut, but not IDH1 WT, resulted in 2HG production and alterations in histone methylation within 20 population doublings (PD). Neither IDH1mut nor IDH1 WT expression, however, had any direct effect on telomerase expression or telomere length, and cells expressing either IDH1 WT or IDH1mut entered a telomere-induced crisis at PD 70. While cells expressing IDH1 WT failed to reactivate telomerase and escape from crisis, cells expressing IDH1mut emerged from crisis after 51 days, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo. The transformed IDH1mut cells did not exhibit the ALT phenotype or hTERT promoter mutations, but rather exhibited telomerase expression and activity, and shortened but stabilized telomeres. These results show that while IDH1 mutations do not directly contribute to ATRX gene mutation, hTERT promoter mutation, or TERT reactivation, they are permissive for the events that indirectly drive the TERT reactivation that contributes to both the immortalization and transformation of astrocytic cells.

Genomic profiling has rapidly become an essential component of brain tumor classification. This technology can be used to detect genetic alterations that may drive the growth of a tumor, and can be used to identify potential targeted treatment options. Herein we present the results of genomic profiling of brain tumor samples obtained over the past two years at our institution. In our patient population, CDKN2A/B loss is frequently found in glioblastoma patients, suggesting that CDK4/6 inhibitors such as palbociclib may have a potential therapeutic role in adult glioblastoma. Other important mutations such as IDH mutations, EGFRvIII and alterations in the PI3k/akt/mTOR pathway can be detected with genomic profiling and lead to individualized treatment strategies.

Molecular heterogeneity in glioblastoma (GBM) tumors presents major challenges to effective treatments. Well characterized patient-derived xenograft models are critical to support the development of better therapies and biomarkers. We established neurosphere cultures (NS) from twelve freshly resected primary and one recurrent GBM tumor and derived orthotopic xenografts in triplicate. All samples were analyzed using exome, low pass whole genome, RNA sequencing and reverse phase protein array (RPPA). We observed that 85% of mutations present in the tumor samples were transmitted to the NS. A majority of mutations shared by both tumor and NS were captured by xenografts with high cellular frequencies suggesting clonal mutations. Few mutations not present in the tumor samples were detected at low frequency in the NPs and approximately 66% of these were transmitted to the xenografts. A median number of 14 mutations were acquired in xenografts relative to NS, suggesting that the brain environment promotes novel somatic events. Genomic profiling of four matching late passage NS revealed that 15 % of the mutations detected in their early passage progenitors were lost, but as many as 59 novel mutations were acquired in extended culture. Consensus hierarchical gene expression profile clustering revealed that 70% of the tumors formed one cluster, while the NP and matching xenografts clustered together in several groups, including one characterized by EGFR amplification and mutations, another encompassing all the proneural subtype. Consistent with these observations, GBM transcriptional subtypes were conserved between tumor and matched NS in 42% of cases, and between NS and xenografts in 92% of cases. Importantly, signaling pathways significantly altered among sample modalities were identified by RPPA and RNAseq analysis, with implications for drug studies. In a panel of GBMs representative of all subclasses, remarkable conservation of original clonal mutations and evidence for clonal evolution were observed in the matched tumor-NP-xenografts.

BACKGROUND: Individuals with Trisomy 21 have a striking cancer distribution, with a 10- to 20- fold increased risk of acute leukemia compared to a markedly decreased incidence of solid tumors. Medulloblastoma, the most common malignant brain tumor of childhood, is particularly rare in the DS population, with only one published case. As demonstrated in a mouse model of DS, the supernumerary chromosome 21 is associated with cerebellar hypoplasia and a decreased number of external granule cell layer progenitor cells (EGLs). Treatment of these mice with sonic hedgehog (Shh) signaling pathway agonists promotes normalization of EGLs and improved cognitive functioning. PATIENT: We describe a 21 month-old male with DS with desmoplastic/nodular medulloblastoma (DNMB) - a tumor derived from Shh dysregulation and over-activation of EGLs. Immunohistochemistry revealed patchy cytoplasmic GAB-1 staining and patchy strong YAP-1 nuclear and cytoplasmic staining, further placing this DNMB into the new consensus molecular classification Shh subgroup. Additional testing revealed a de novo heterozygous germ line mutation in the PTCH1 gene encoding a tumor suppressor protein in the Shh pathway; both parents tested negative for this mutation. CONCLUSIONS: The reduced presence of EGLs in DS patients offers a plausible explanation for the disparity of medulloblastoma in this population. Conversely, patients with PTCH1 germ line mutations experience Shh pathway overstimulation resulting in Gorlin (Nevoid Basal Cell Carcinoma) Syndrome and an increased incidence of malignant transformation of EGLs leading to medulloblastoma formation. This represents the first documented report of an individual with DS (developmental failure of EGLs, decreased incidence of medulloblastoma) simultaneously carrying PTCH1 germ line mutation (overstimulated EGLs, increased incidence of medulloblastoma). Thus, we observe a highly unusual circumstance in which the PTCH1 mutation appears to ‘trump’ the effects of trisomy 21 in causation of Shh-activated medulloblastoma. Additional genomic studies are ongoing and will be presented.

Glioblastoma (GBM) is highly infiltrated by immune cells of the myeloid lineage; however, the immunosuppressive microenvironment established by GBM favors anti-inflammatory activity in these cells and in turn promotes tumor immune escape. We have recently shown that shRNA-mediated knockdown of the β-galactoside-binding lectin galectin-1 (gal-1) in malignant glioma cells leads to glioma eradication within a week of intracranial engraftment (Baker et al, Cancer Research. 2014; 74:5079-90). NK cells are central to the rejection of gal-1-deficient glioma as immunodepletion of these cells with anti-asialo GM1 or NK1.1 antibodies permitted the growth of these tumors. We now report that immunodepletion of Gr-1+ myeloid cells in RAG1−/- mice also permits the growth of gal-1-deficient glioma, despite the presence of NK cells. This intriguing observation establishes that NK cells require collateral support by Gr-1+ myeloid cells to exert anti-glioma immune surveillance activity. We found that gal-1-deficient mouse GL26-Cit cells are pro-inflammatory as they upregulate expression of chemokines CXCL10/IP-10 and RANTES. Seven-fold more Gr-1+/CD11b+ myeloid cells infiltrated the gal-1-deficient GL26-Cit microenvironment compared to infiltration into control GL26-Cit tumors. Myeloid infiltration was followed by a 10-fold increase in the number of NK cells. Implantation of gal-1-deficient GL26-Cit cells into the brain of B6.Myd88−/- mice failed to recruit peripheral blood mononuclear cells (PBMCs) into the tumor microenvironment 72 hours post-engraftment, thus permitting gal-1-deficient glioma growth. This implied that TLR/Myd88−/- signaling is required to induce the entry of PBMCs into the gal-1-deficient microenvironment to cause tumor eradication. Experiments are now underway to determine the identity of the biomolecular factors produced by gal-1-deficient GL26-Cit glioma cells responsible for initiating Myd88 signaling and the receptors and cells in which Myd88 signaling is necessary for glioma rejection. These studies identify a novel innate immune network necessary and sufficient for early glioma rejection without requiring contributions from the adaptive immune system.

BACKGROUND: While novel targeted therapeutics hold great promise in precisely eliminating sensitive subsets of tumour cells, their selectivity may lead to the proliferation of resistant clones and tumour recurrence. In this respect enhancing the distribution of less-selective, older-generation chemotherapeutics may result in improved tumour control and more favourable side effects profile. To this end we are exploring strategies relying on nanoparticle transport of drugs, intra-cavitary and metronomic intra-CSF drug delivery in order to maximise the cytotoxic effect and minimize the toxicity of chemotherapy in paediatric brain tumours. Human-based 3D in vitro models showing both safety and efficacy were established and a number of drug delivery strategies were tested for improved selectivity against medulloblastoma compared to normal human foetal brain tissue. METHODS: The topoisomerase inhibitors etoposide and teniposide were encapsulated in Poly(ethylene glycol)-block-poly(ɛ-caprolactone) (PEG-PCL 5kDa-5kDa) nanoparticles using the solvent-emulsification method. Comparative toxicity towards normal and tumour human cells was determined by treating normal and tumour spheroids with empty nanoparticles, drug-loaded nanoparticles or free drug. Spheroid volume and metabolism dose-response curves were compared between the different formulations. RESULTS: The more lipophilic drug, teniposide, showed higher drug loading (8%w/w) compared to etoposide (0.4%w/w) in the purified nanoparticles. Whereas both drugs exhibited toxicity towards proliferating cells regardless of whether those were normal or tumour, etoposide had a narrow therapeutic window of 3-10 µM where normal brain tissue showed higher viability compared to medulloblastoma cells. In addition, the teniposide-loaded nanoparticles were 1.5 times less toxic to normal tissue and equally cytotoxic to tumour cells compared to free teniposide. CONCLUSIONS: The tumour-selective therapeutic levels of 3-10 µM identified for etoposide can be useful for intra-CSF prophylaxis against distant metastasis. Although the teniposide-loaded nanoparticles offered an incremental improvement in the therapeutic index of teniposide future formulations with optimised release profile or covalently linked drug may yield better results.

Several preclinical studies indicate that neural stem cells can limit or reverse CNS damage by cell replacement, regeneration or delivery of various factors and therapeutic agents to sites of degeneration or tumor. Allogeneic NSC lines have several advantages over autologous ones, including "off the shelf" usage, greater availability to patients, and cost effectiveness. Genetic modification of NSCs for immortalization and retention of stem cell properties with c-myc or v-myc gene has been shown to be clinically useful in trials for stroke (ReNeuron), and brain tumors (Aboody et al). Here, we propose to express L-myc variants in human fetal brain derived NSCs, to extend NSC lifespan and stem cell characteristics. We have isolated and grown NSCs from freshly dispersed 10 week old human fetal brain tissue and propagated in culture. We then transduced these cells with retroviral vectors containing the L-myc gene. NSCs stably expressing L-myc gene (hNSC.Lmyc) were isolated for further characterization. hNSC.Lmyc cells propagated through passage 15, demonstrating a normal karyotype, expression of stem cell markers (Sox-2, A2B5 and nestin) and low expression of differentiation markers (Olig 2 and vimentin) in vitro. We have also demonstrated that hNSC.Lmyc cells retain their tumor-specific migratory abilities in vitro and in vivo for longer periods of time as compared to untransduced primary NSC pools. We further characterized these hNSC.Lmyc cells for gene expression profiling and genome wide DNA sequencing analysis. Somatic mutation analysis comparing early and late passages of hNSC.Lmyc cells showed no change in the genomic sequences. hNSC.Lmyc cells were able to self renew and differentiate into neurons, oligodendrocytes and astrocytes in vitro. In this study we generated and characterized hNSC.Lmyc line that has potential therapeutic use in numerous central nervous system (CNS) diseases.

There has been no clinical observation about histopathological changes in human gliomas under control of the anti-angiogenic treatment, and the tissue-based observation is likely crucial to understand mechanism of action of these treatment. We collected 4 glioblastomas resected under control of neoadjuvant bevacizumab (Bev). Histopathological investigation was performed by hematoxilyn- eosin staining and immunohistochemistry for CD34, VEGF, VEGFR1/2, HIF-1α, and Nestin as compared to 6 control glioblastomas to assess changes in histological features, microvessel density, expression of VEGF and its receptors, tumor hypoxic condition, and cancer stem cells. Tumor resection under control of Bev was uneventful in all of the 4 cases. Intraoperatively, the tumors were clearly less vasculized than usual. Histopathologically, vascular morphology was quite different from usual glioblastomas with universal disappearance of microvascular proliferation. Microvessel density was significantly decreased in the 4 tumors as compared to control tumors. The expression of VEGF and its receptors was decreased in 2 cases of partial response, suggesting possible correlation with radiologic response. The expression of HIF-1α was similar or somewhat decreased in the 4 tumors. The expression of Nestin was significantly decreased in the 4 tumors as compared to control tumors, but there still remained numerous Nestin-positive cells especially around vessels. We provide the first clinicopathological evidence that the anti-angiogenic therapy induces vascular normalization and decrease of microvessel density in glioblastomas. These in situ observation will help to elucidate the mechanism of resistance to Bev and to optimize therapy.

We have initiated a first-in-human pilot study of intravenous delivery of autologous T cells re-directed to the EGFR variant III mutation by means of a lentiviral vector encoding a chimeric antigen receptor (CAR). Patients with recurrent or incompletely resected glioblastoma (GBM) have their tumors screened for the presence of the EGFRvIII mutation by a next-generation sequencing based assay. Eligible patients undergo leukapheresis for collection of autologous T cells, which are then manufactured to form CART-EGFRvIII T cell products. We report preliminary results on the first six patients we have treated. We targeted a challenging subset of GBM patients, with multi-focal recurrent, residual, progressive disease refractory to standard and experimental therapies, including chemotherapy, radiation, bevacizumab and prior immunotherapy. To date, we have found that infusion of CART-EGFRvIII cells is safe, without evidence of off-tumor toxicity such as cross-reactivity to wild type EGFR. No clinical or laboratory signs of systemic cytokine release syndrome have been observed. One patient developed a seizure and non-convulsive status epilepticus nine days after CART-EGFRvIII infusion. Seizures resolved with anti-epileptic medications, and the patient was treated with steroids and anti-IL6 cytokine therapy. All patients have had significant expansion of CART-EGFRvIII cells as measured by flow cytometry and quantitative PCR in peripheral blood samples, despite the use of steroids in two patients. In one of two patients who had operative resection of presumed residual tumor, pathologic evaluation demonstrated inflammation, T cell infiltration, and EGFRvIII antigen loss. In total, two of six heavily pretreated patients are clinically stable three months post-CART infusion. These findings provide preliminary evidence that CART-EGFRvIII cells are safe, without evidence of off-target toxicity and without evidence of cytokine release syndrome, and that these are active T cell products that expand in the blood.

An anti-EGFRvIII peptide vaccine for treating glioblastoma (Rindopepimut) has shown significant clinical potential, recently receiving Breakthrough Therapy Designation from the FDA. Interestingly, we created this vaccine before MHC binding principles were understood so the sequence was arbitrarily chosen and never optimized for immune response. X-ray diffraction of the EGFRvIII peptide reveals that it has structure, unusual for a 13 amino acid peptide, and the novel glycine at the junction, thought to be essential, is not critical for immune recognition. However, the glycine is at a hinge so we hypothesized this position is important for secondary structure. In our EGFRvIII/NIH-3T3 syngeneic tumor model, we tested substitution of the glycine by all 19 other amino acids, length variations, synthetic amino acids and deletion of the glycine (-Gly) (N = 16 or greater for 24 peptides tested). Several substitutions, including Ala, Val, Pro, show a >58% improved survival rate over Rindopepimut. Most substitutions were similar to Rindopepimut, but some, including His, Cys and -Gly, were comparable to control. All peptides elicited antibodies specific for EGFRvIII with a general trend towards high titer (>1:2x105) in the higher performing vaccines. CTL responses by Elispot revealed that the highest performing vaccines elicited a >42% increase in spots over Rindopepimut (p 19 cleavage products, including recombination to form a larger products at ∼2600Da. Rindopepimut produced ∼14 fragments with lower amounts of the 2600Da, while the poorest vaccines showed almost no cleavage and no 2600Da. Our results show that more robust vaccines against EGFRvIII can be produced, that 3D structure is important, and the vaccine shows unexpected complex processing. Further work is being performed to bring these vaccines to clinical trial.

A precise clinically relevant characterization of glioblastoma (GBM) heterogeneity and underlying genomic variability is urgently needed. To evaluate the true potential of radiogenomics, an emerging field for GBM research, we have tested its scope in a pre-clinical setting. First, glioblastoma stem cells (GSCs) are altered to express decreased levels of Periostin (POSTN), previously identified in a radiogenomic screen as a key pro-invasive gene. Second, orthotopic tumors arising from these GSCs, were scanned and analyzed to identify gene specific MRI texture feature (MRTF) signatures, which can simultaneously predict POSTN status in GBM patients. Doxycycline inducible short hairpin RNA mediated knockdown of POSTN in GSC lines were established. 5X10^6 cells were stereotactically implanted in nude mice frontal cortex and randomized into POSTN knockdown and control groups. Monitored tumor growth longitudinally by MRI (n = 3-4 per group). Region of interest (ROI) methods were applied to the rigid registration images based on axial T1-weighted imaging and corresponding fluid-attenuated inversion recovery sequences. After pre-processing, gray level co-occurrence matrices (GLCMs) were generated in 4 angular directions and 3 invariant measures (mean, range and angular variance) of each texture feature is calculated. A matrix of 3x20 invariant features is generated for each gray level. Mean values of normalized MRTFs from edema T1 post and T2, subgroup tumors arising from independent GSCs (p

BACKGROUND: Despite advances in chemotherapy and surgical approaches, the median overall survival (OS) for patients with GBM remains poor. A “supra-maximal” resection, using the sub-pial technique for low-grade gliomas aims to remove immediately proximate abnormal tissue beyond the zone of enhancement on MRI. The purpose of this study is to evaluate the feasibility and survival benefit of this technique in patients with GBM. METHODS: We retrospectively evaluated 86 consecutive patients operated on for primary GBM using the sub-pial technique with or without BCNU wafer implantation and adjuvant therapy. Presenting clinical, radiological and outcome data were collected. Multivariate Cox proportional hazards regression was used to analyze variables associated with survival. Survival was compared using Kaplan-Meier plots to stratify the impact of extent of resection (EOR) and BCNU. RESULTS: Patient age (mean) was 56 years with a 93.8% median EOR. Median OS for the group was 18.1 months and 54, 16.5 and 13.2 for GTR, NTR and STR respectively. 34.2 and 13.2 months for patients with >95% and

While patients with primary glioblastoma (GBM) have benefited from increased overall survival due to treatment with the alkylating chemotherapy temozolomide (TMZ), the benefit of TMZ for patients with diffuse low-grade gliomas (LGG) remains unknown. We previously demonstrated that among ten TMZ-treated patients with initial LGG (IDH1 mutant, 1p19q intact), treatment induced hypermutation in the recurrent tumors of six patients, all six of whom underwent malignant progression to secondary GBM (sGBM) (Johnson & Mazor, Science, 2014). To further explore the relationships among TMZ treatment, hypermutation and malignant progression, we studied tumor evolution in an expanded cohort of 35 TMZ-treated patients by exome-sequencing of paired IDH1/2-mutant LGGs and their post-TMZ recurrences. This expanded cohort allowed us to contrast patterns of hypermutation between 1p19q intact versus co-deleted subgroups. Hypermutated recurrences were common in both subgroups and consistently underwent malignant progression to the highest grade while acquiring somatic mutations in mismatch repair (MMR) genes. In contrast to the 1p19q intact subgroup in which TP53 mutations were present at diagnosis, the hypermutated recurrences of the 1p19q co-deleted subgroup frequently acquired TMZ-associated mutations in TP53. This suggests that TP53 mutations may cooperate with MMR mutations to convert TMZ-induced cytotoxicity to mutagenicity. Once hypermutated clones arose, we found that they dominated subsequent recurrences. In one case, a hypermutated sGBM was resected, but soon recurred with leptomeningeal spread, at which point hypermutated clones were found in the spinal cord. We examined the clonality of hypermutated tumors through exome-sequencing of multiple spatially distinct samples, from which we estimate that hypermutated recurrent tumors can originate from a single initiating cell. Together, these findings further our understanding of the molecular features and clinical behavior of hypermutated clones, raising concerns about the potent mutagenic activity of TMZ and other alkylating agents in LGG patients.

BACKGROUND: Cediranib is an orally available pan-VEGFR tyrosine kinase inhibitor. Previously phase III study in patients with recurrent glioblastoma did not meet primary end of progressive-free survival (PFS) (J Clin Oncol. 2013 Sep 10; 31(26): 3212-3218) METHODS: We identified 1 patient, 53-year old Caucasian female, who developed tumor progression of left posterior frontal 1.2 x 1.5 cm in February, 2009 following surgery on 10/15/08 and concurrent temozolomide (TMZ) and radiation therapy from 11/8/08 -1/6/09 with one cycle of maintenance TMZ. She was enrolled in a randomized, phase III, placebo-controlled, partially blinded clinical trial of cediranib either as monotherapy or in combination with lomustine (CCNU) versus CCNU. RESULTS: She was randomized to receive a combination therapy with 1st cycle CCNU 190 mg and cederanib 20 mg/day on 4/13/09. Four weeks later, she developed grade 4 thrombocytopenia (17, 000) associated with gum bleeding and dizziness. She received a platelet transfusion. She continued with cediranib alone and 2nd reduced CCNU 100 mg was delayed till 6/16/09, 3rd CCNU on 2/2/2010. She developed hypertension, proteinuria and diarrhea from cediranib in April of 2010 and she continued with reduced dose to 15 mg/day alone without CCNU. However, she developed nephrotic syndrome and poorly controlled hypertension and was taken off this study in May 2010. At 6-week MRI showed 50% tumor regression (PR) and complete response at 24-weeks. With no enhancement seen on MRI on 6/4/15, she has been off therapy and in clinical remission over 5 years with high functional level and good quality of life (KPS 90%). CONCLUSION: This is a case report of successful therapy for recurrent glioblastoma with long term remission despite termination of therapy > 5 years from cediranib and limited CCNU dosage.

Metastatic medulloblastoma is a high-risk disease, because the majority of children dying of medulloblastoma expire from metastases rather than recurrence of the primary tumor. Current dogma dictates that medulloblastoma cells are shed from the primary tumor into the CSF, float through the spinal fluid and establish metastatic colonies on the leptomeningeal surface and spinal cord. Empirical evidence for this mechanism is lacking in the literature. Whole genome sequencing on matched trios of human primary tumor, metastases, and peripheral blood showed somatic mutations from the medulloblastoma at low clonal frequency in the blood samples — consistent with circulating tumor cells. In addition, we identified circulating tumor cells (CTC) in the blood of transgenic mice bearing metastatic medulloblastoma, and in mice intra-cranially implanted with patient derived MB. Surprisingly, when we implanted human or mouse medulloblastoma cells in the flank of immunocompromised mice, the MB cells colonized the leptomeningeal space causing death. As these MB xenografts are not in continuity with the CSF, they must have arisen through hematogenous dissemination. Importantly, flow cytometry shows that MB CTCs express CD34, the cell-surface marker used clinically in children to collect peripheral blood stem cells; suggesting that autologous bone marrow transplants from children with medulloblastoma are likely contaminated with CTCs. Finally we established a parabiosis model where two immunocompromised, sibling mice are surgically joined, and a common blood circulation is established. After parabiosis, one mouse is intra-cranially implanted with MB, and the mice are subsequently separated when the xenografted twin becomes symptomatic. The surviving twin goes on to die from leptomeningeal metastases. Our data challenge the existing paradigm of CSF mediated dissemination of medulloblastoma by demonstrating the capacity of medulloblastoma to spread through the blood system. Identification of a systemic route for MB metastases offers new avenues for the diagnosis and therapy of metastatic medulloblastoma.

BACKGROUND: Therapeutic targeting of the immune checkpoints CTLA-4 and PD-1/PD-L1 have demonstrated tumor regression in clinical trials, and phase II trials are ongoing in glioblastoma. Previous reports have suggested that responses are more frequent in patients with tumors expressing PD-L1; however, this has been disputed. At issue is the validation of PD-L1 biomarker assays and prognostic impact. METHODS: Using immunohistochemical analysis, we measured the incidence of PD-L1 expression in 94 patients with glioblastomas. We determined the total number of PD-L1-expressing cells within the glioblastomas and validated this finding using ex vivo glioblastoma flow cytometry, with further analysis of T cell populations. We evaluated the association between PD-L1 expression and median survival time using the protein expression data sets and mRNA from The Cancer Genome Atlas (TCGA). RESULTS: The median percentage of PD-L1-expressing cells in glioblastoma by cell surface staining is 2.77% (range 0-86.6%, n = 92), similar to the percentage found by ex vivo flow cytometry. The majority of glioblastoma patients (61%) had tumors with at least 1% PD-L1-positive cells, and 38% had at least 5% PD-L1 expression. PD-L1 and PD-1 are commonly expressed on glioblastoma-infiltrating T cells. Expression of PD-L1 is prognostic by multivariate analysis for a negative glioblastoma outcome by both immunohistochemistry (p = 0.018) and mRNA expression analysis of TCGA data (p = 0.009). Expression of PD-1 mRNA is correlated with perforin 1 (P 0.0001), both markers of cytolytic activity, as well as the T cell markers CD3 (P < 0.0001), CD4 (P < 0.0001) and CD8 (P < 0.0001). PD-L1 mRNA expression is correlated with granzyme A (P < 0.0001), CD3 (P = 0.0099) and CD4 (P < 0.001). CONCLUSIONS: The incidence of PD-L1 expression in glioblastoma patients is frequent but is confined to a minority subpopulation-similar to other malignancies that have been profiled for PD-L1 expression. Higher expression of PD-L1 is correlated with worse outcome.

BACKGROUND: Neurofibromatosis type 2 (NF2) is associated with histologically benign but morbid tumors. Patient-reported outcomes (PROs) are important measures of patient function for this neuro-oncologic syndrome which impacts quality of life (QOL) due to multiple chronic tumors. Hence, NF2 serves as a model for developing PROs in neuro-oncology trials. The NFTI-QOL is a validated disease-specific QOL-instrument developed in the United Kingdom which has not been compared against historical clinical trial endpoints in NF2 including total vestibular schwannoma (VS) volume and best word recognition score (WRS). METHODS: An IRB-approved prospective observational study was conducted enrolling adult NF2 patients at Johns Hopkins. Measures included: NFTI-QOL, SF-36; total volume of bilateral VS, best WRS, provider- and patient-reported disease severity (ProvSev, PatSev) by institutionally-derived multi-item (e.g. symptom burden, age-of-onset, fatality-risk) and single-item Likert (mild, moderate, severe) scales. RESULTS: Twenty patients were enrolled; mean age at testing 46 + 21 years and age at diagnosis 32 + 18 years; 35% male. Prior treatment included surgery (50%), radiotherapy (25%), and systemic therapy (40%). Mean best WRS was 71.8 + 37% (n = 20). Two patients had no functional hearing and 10 had unilateral hearing. Mean total VS volume was 5.8 + 7.4 cc (n = 15). Additional lesions included meningioma (75%) and ependymoma (20%). Mean NFTI-QOL score was 8.5 + 5.1 and correlated well with all physical dimensions of the SF-36 (all p < 0.002). NFTI-QOL correlated well with PatSev (r = 0.60, 95%CI 0.21-0.82, p < 0.005) and multi-item ProvSev (r = 0.66, 0.31-0.85, p = 0.002) but not single-item ProvSev (r = 0.33, -0.13-0.68, p = 0.15). A trend toward positive correlation was observed between PatSev and multi-item ProvSev (r = 0.41, -0.04-0.72, p = 0.07). NFTI-QOL scores did not correlate with VS volume (r = 0.10, -0.44-0.58, p = 0.73), or best WRS (r = -0.40, -0.71-0.05, p-0.08). CONCLUSION: NFTI-QOL scores did not correlate with hearing function or tumor volume, but did correlate with patient and provider-reported disease severity underscoring the importance of patient-focused measures in therapeutic outcome assessment in NF2.

BACKGROUND: The addition of trastuzumab to cytotoxic chemotherapy has improved outcomes for patients with HER2 positive breast cancer. Increased survival coupled with limited blood-brain barrier (BBB) penetration of trastuzumab may contribute to the increased incidence of brain metastasis in these patients. Half of these patients die of intracranial disease progression rather than extracranial disease. Therefore, strategies to improve survival must include increased CNS disease control in these patients. Lapatinib crosses the BBB and demonstrates modest activity against intracranial metastases. Based upon preclinical data and results of a phase I study, it's hypothesized that lapatinib plus WBRT can improve the intracranial disease control compared to WBRT alone. METHODS: A randomized phase II trial of WBRT (37.5 Gy/3 weeks) plus or minus concurrent lapatinib (1000 mg qd x 21 during WBRT and for 21 days after) was initiated. Non-CNS penetrating HER2 targeted therapy is permitted throughout the study, but patients not on trastuzumab, pertuzumab or any other breast cancer therapy at study entry are not permitted to begin this therapy while on protocol treatment, but may begin it 24 hours after completion of protocol treatment. Eligibility includes HER2+ breast cancer with at least one measurable, unirradiated parenchymal brain metastasis. The two populations targeted for accrual include patients with 1) newly diagnosed, multiple brain metastases or 2) progressive brain metastases after stereotactic radiosurgery (SRS) or surgical resection of 1-3 metastases. Prior lapatinib is allowed. Patients are stratified by breast-specific graded prognostic assessment; use of non-CNS penetrating HER2 targeted therapy; and prior SRS or surgical resection. The primary endpoint is complete response rate in the brain 12 weeks after WBRT. Secondary endpoints include objective response rate, lesion-specific response rate, CNS progression-free survival, and overall survival. 52 of 143 target accrual have enrolled (6/1/2015). Supported by NCI grants U10CA21661, U10CA180868, U10CA180822, U10CA37422 and UG1CA189867.

Background: Depression in patients with brain tumors is associated with impaired quality of life and shorter survival. Altered metabolism of tryptophan to serotonin and kynurenine metabolites may play a role in tumorassociated depression. Our recent studies with alpha[ 11 C]methyl-L-tryptophan (AMT)-PET in brain tumor patients indicated abnormal tryptophan metabolism not only in the tumor mass but also in normal-appearing contralateral brain. In the present study, we explored if tryptophan metabolism in such brain regions is associated with depression. Methods: Twenty-one patients (mean age: 57 years) with a brain tumor (10 meningiomas, 8 gliomas, and 3 brain metastases) underwent AMT-PET scanning. MRI and AMT-PET images were co-registered, and AMT kinetic parameters, including volume of distribution (VD’, an estimate of net tryptophan transport) and K (unidirectional uptake, related to tryptophan metabolism), were measured in the tumor mass and in unaffected cortical and subcortical regions contralateral to the tumor. Depression scores (based on the Beck Depression Inventory-II [BDI-II]) were correlated with tumor size, grade, type, and AMT-PET variables. Results: The mean BDI-II score was 12 ± 10 (range: 2–33); clinical levels of depression were identified in seven patients (33 %). High BDI-II scores were most strongly associated with high thalamic AMT K values both in the whole group (Spearman’s rho = 0.63, p= 0.004) and in the subgroup of 18 primary brain tumors (r= 0.68, p = 0.004). Frontal and striatal VD’ values were higher in the depressed subgroup than in non-depressed patients (p< 0.05); the group difference was even more robust when moderately/severely depressed patients were compared to patients with no/mild depression (frontal: p= 0.005; striatal: p< 0.001). Tumor size, grade, and tumor type were not related to depression scores. Conclusions: Abnormalities of tryptophan transport and metabolism in the thalamus, striatum, and frontal cortex, measured by PET, are associated with depression in patients with brain tumor. These changes may indicate an imbalance between the serotonin and kynurenine pathways and serve as a molecular imaging marker of brain tumor-associated depression. Trial registration: ClinicalTrials.gov NCT02367469

BACKGROUND: Grade IV astrocytomas, formerly known as glioblastoma multiforme (GBM), are the most common primary brain tumors and have the highest mortality. The therapeutic standard for managing this malignancy remains a combination of surgery, chemotherapy, and radiotherapy; however, there is no cure, nor has there been any significant advancement in the clinical approach to GBM since this protocol was established in 2005. This study aims to better understand the molecular and metabolic characteristics of GBM to better define treatment groups and potentially identify new avenues for therapy. This study utilized continuous, commercially-available GBM cell lines U87, U118, A172, and H4, and examined the concentrations of Bcl-2 family proteins on mitochondria and metabolic activity for each of the cell lines. The measures were correlated to temozolamide (TMZ) sensitivity. RESULTS: Western blot analysis of pro-survival and pro-apoptotic Bcl-2 proteins revealed that U118 and U87 expressed high levels of Bcl-2, while A172 had increased Bcl-xL expression. Interestingly, Bcl-2 and Bcl-xL were not detected in H4 cells. Incidentally, pro-apoptotic BH3-only protein levels were increased in H4 and A172 cells. Metabolic analysis of the cell lines revealed that U118 cells were a glutaminolytic, while U87, A172, and H4 were classified as glycolytic. We found that U118 cells were the most resistant followed by U87, A172, and H4, respectively. CONCLUSIONS: We found that Bcl-2 protein profiling was a useful means to determine therapeutic response and resistance. This was enhanced by addingmetabolic stratification, wherein glycolytic cells were shown to be more sensitive TMZ than glutaminolytic cells. We will expand the number of cell lines in our study and increase the cellular profiling to include mitochondrial dynamics. Using this approach, we will produce a method to define GBM responses and outcomes based on the mitochondrial and metabolic context of individual tumors.

We demonstrate that monoamine oxidase B (MAO-B) levels in glioma are far higher than in control brain, and that levels of MAO-B correlate with tumor grade. Monoamine oxidases are able to oxidize a wide range of N-methyl-1,2,3,6-tetrahydropyridine substrates into their respective N-methyl-pyridinium cations (P + -). We have designed a range of methyl-tetrahydropyridine (MP-) compounds that undergo MAO-B specific oxidation from the neutral pro-Drug/Dye species into mitochondrial targeting lipophilic compounds. Coupling methyl-tetrahydropyridine to the neutral fluorophore Bodipy, via a linker, produces MP-Bodipy. In vitro we show that this compound is rapidly converted into the mitochondria targeting cation P + -Bodipy by MAO-B. The conversion of MP-Bodipy into P + -Bodipy is abolished by preincubation with the MAO-B specific inhibitor, Selegiline, and mitochondrial accumulation of P + -Bodipy can be reversed by using an uncoupling agent. MP-Bodipy is a highly specific probe in glioma xenografts in flank and in intracranial tumor models. This targeting of MAO-B overexpression should facilitate fluorescence-guided surgery allowing neurosurgeons to identify gliomal infiltration boundaries during tumor resection. Using a nitrogen mustard moiety (MUS) in place of the dye gives rise to a MAO-B specific MP-MUS/P + -MUS (pro-Drug/Drug) pair; a gliomal specific chemotherapeutic that targets glioma mitochondria via an attack on mitochondrial DNA. MP-MUS is highly effective in selectively damaging gliomal mitochondria, and leads to an alteration of cancer cell phenotype in the few cells that survive in vitro and in vivo. MP-MUS effectively treats primary gliomal xenografts in both flank and intracranial tumor models. In an intracranial xenograft model, over 50% of all animals are completely tumor free with only three treatments of MP-MUS, without causing any significant toxicity in tissues. The others all have minimal tumor left. A wide range of mitochondrial toxins can be coupled to the MAO-B specific MP-/P + - pair allowing us to optimize our drugs for an individual glioma.

ATNT-26. PHASE I/II STUDY OF DIANHYDROGALACTITOL IN PATIENTS WITH RECURRENT MALIGNANT GLIOMA Kent Shih1,2, Mahish Patel1,3, Nicholas Butowski4, Jeffrey Bacha5, Dennis Brown5, Anne Steino5, Richard Schwartz5, Sarath Kanekal5, Lorena Lopez5, and Howard Burris1,2; Sarah Cannon Research Institute, Nashville, TN, USA; Tennessee Oncology, Nashville, TN, USA; Florida Cancer Specialists & Research Institute, Florida, USA; University of California San Francisco Division of NeuroOncology, San Francisco, CA, USA; DelMar Pharmaceuticals, Vancouver, BC, Canada Glioblastoma multiforme (GBM) is the most common brain cancer. Front-line systemic therapy with temozolomide is often ineffective due to O-methylguanine-DNA-methyltransferase (MGMT)-mediated resistance. Dianhydrogalactitol (VAL-083) is a bi-functional DNA N cross-linking agent that crosses the blood-brain barrier and demonstrated cytotoxic activity independent of MGMT in vitro. The main goal of this clinical trial was to determine an appropriate dose for Phase II/III trials in refractory GBM. METHODS: Open-label, single-arm Phase I/II dose-escalation study in patients with histologically-confirmed GBM, previously treated with radiation and must have failed both temozolomide and bevacizumab, unless contraindicated. The study utilized 3 + 3 dose-escalation design. Patients received VAL-083 on days 1, 2, 3 of a 21-day cycle. RESULTS: 30 GBM patients were enrolled across 8 dose cohorts ranging from 1.5 to 50mg/m/d. No drug-related severe adverse events were reported, and myelosuppression was mild at doses ≤40mg/m/d. Dose limiting toxicities (DLT) consisting of thrombocytopenia were observed at 50mg/m/d. Platelet nadir occurred around day 20, and DLT-related symptoms resolved rapidlyand spontaneously without concomitant treatment. Pharmacokinetic analyses show dosedependent linear systemic exposure with a short 1-2h plasma terminal halflife; Cmax ranged 739-1130ng/mL (5.1-7.7 mM) at 40mg/m/d resulting in calculated CNS concentrations within the IC50 range observed for multiple GBM cell-lines in vitro. Preliminary analysis shows increasing dose-dependent median survival with median OS 1⁄4 9.0 months at doses ≥30mg/m/d vs. 4.4 months at doses ,10mg/m/d. An expansion cohort of up to 14 patients has been initiatedat40mg/m/d.Asmall cohort (n 1⁄4 3) atan interim 45mg/m/d dose will also be studied, and the expansion cohort may be continued at this higher dose if safety data warrant. CONCLUSIONS: VAL-083 dosing appears limited by myelosuppression; however, 40mg/m/d dose exhibited favorable safety profile, with a trend toward improved survival vs. lower doses. Updated safety and efficacy data from the expansion cohort will be presented. ClinicalTrials.gov Identifier NCT01478178. Neuro-Oncology 17:v10–v17, 2015. doi:10.1093/neuonc/nov205.26 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.

Pediatric glioblastoma (GBM) patients have

BACKGROUND: GBM is a deadly brain cancer with an average survival of 12 months. In the past, a treatment-resistant Brain Tumor Initiating Cell (BTIC) compartment has been identified and is thought to drive tumor recurrence. One experimental strategy to target BTICs is the use of oncolytic viruses (OVs). A number of studies have found that OVs may be used to target BTICs in vivo and that some require the innate or acquired immune response for their efficacy. Myxoma Virus (MyxV-WT) is a promising oncolytic virus that is highly effective against glioma cell lines but less effective for BTICs. We hypothesized that MyxV virally encoded anti-apoptotic proteins is a limiting factor for oncolytic efficacy in BTICs. By manipulating MyxV-WT and genetic deletion of the anti-apoptotic gene M11L we will have better therapeutic efficacy and enhanced cell death in BTICs that might be synergistic with conventional Temozolomide therapy. RESULTS: We utilized a panel of patient-derived BTICs and developed syngeneic mBTIC line arising in Trp53+/-/Nf1+/- mice. We compared the efficacy of the MyxV-WT with the MyxV, in which the anti-apoptotic protein M011L has been deleted. We found that MyxV-M011L-KO induced significant amount of intrinsic apoptosis as compared to MyxV-WT, resulting in robust BTICs cell death. Silencing the Bax significantly blocked the induction of apoptosis and cell death induced by MyxV-M011L-KO. Using immunocompetent orthotopic syngeneic glioma model, we found that M011L-KO treatment statistically prolonged survival (38 days) as compared to WT or inactivated virus (30 and 27 days respectively) whereas addition of clinical relevant dose of TMZ resulted in 50% long-term survival rate (>90 days). In contrast, MYXV-M011L-KO treatment in SCID models, failed to provide significant survival advantage in both human and murine BTICs. These results suggest that intact immune response is critical factor regulating oncolytic efficacy of MYXV-M011L-KO in vivo.

Primary central nervous system lymphoma (PCNSL) is a rare malignancy confined to CNS. Despite low tendency of systemic dissemination, its prognosis is poor with median overall survival of 4 years when treated with standard of care high-dose methotrexate-based chemoradiotherapy. Despite majority of PCNSL is diffuse large B-cell lymphoma (DLBCL), its response to therapy and biological behavior differ from the systemic disease, necessitating detailed investigation of its pathogenesis and genetic alterations. Here we performed whole-exome sequencing (WES) on 41 PCNSL and paired normal specimens as well as RNA sequencing. Seven cases were classified as germinal center B-cell-like (GCB) subtype (17%), while 34 cases were non-GCB. Progression-free survival was significantly longer in cases with GCB subtype as in systemic DLBCL (p = 0.027). WES yielded sequencing data with a mean coverage of 158Χ with 98% of the targeted bases covered by ≥20 independent reads. We identified a total of 17,385 somatic mutations, of which 62% were nonsynonymous single nucleotide variations. The predominant nucleotide substitution was a C > T transition (57.8%), especially in the context of the sequence GCG. WES revealed a substantial extent of aberrant somatic hypermutation (aSHM) with a high frequency of nonsynonymous somatic mutations especially in PIM1 (90.2%) and BTG2 (65.9%). Genes mutated other than aSHM include MYD88 (85.4%), most of which being L265P activating mutation. These mutations were not associated with GCB/non-GCB subtypes. Notably, mutations in a number of genes related to the NF-κB signaling pathway were also prominent. Copy number aberration analysis identified gains in chromosomes 1q, 7, 12, and 18q, and a loss of 6q. These results suggest that MYD88 and PIM1 mutations may play an important role in PCNSL development regardless of GCB subtypes, and NF-κB pathway could be the therapeutic targets of this intractable disease.

BACKGROUND: Patients with leptomeningeal metastases (LM) have disease burden involving the membranes surrounding the brain, spinal cord or both, and a short disease trajectory. Limited studies have explored presenting symptoms and the relationship to functional status. The objectives of this study are to evaluate the utility of the MD Anderson Symptom Inventory-brain tumor (MDASI-BT) and the MDASI-spine tumor (MDASI-SP) symptom report instruments in capturing presenting symptoms in this population, and to describe functional status (Karnofsky Performance Score [KPS]) in relation to endorsed symptoms and tumor location. METHODS: Clinical records of 45 patients with LM were reviewed for presenting symptoms. Endorsed symptoms were recorded using the MDASI-BT and MDASI-SP. Both instruments were modified to capture dichotomous data (present/not present). Additional demographic and clinical data were collected (age, gender, tumor location, and KPS). Frequency analysis was used to describe symptom occurrence and KPS. Two-way analysis of variance (ANOVA) was performed to explore differences in KPS for tumor location, and endorsed symptoms. RESULTS: The subjects were primarily female (80%), with a median age of 51 years. The most common location of LM was the brain (42.2%) followed by both brain and spine involvement (37.8%). Twenty-one (46.7%) endorsed symptoms corresponding to both the MDASI-BT and the MDASI-SP. Of those endorsing symptoms corresponding with only one instrument, a greater number of patients reported symptoms corresponding with the MDASI-BT (31%). Pain was the most frequently reported presenting symptom (33%), followed by numbness/tingling (31%). KPS at diagnosis ranged from 50% to 100% (median = 80%). Using ANOVA there was no significant difference in KPS for tumor location and endorsed symptoms. CONCLUSIONS: These findings indicate that, based on frequency analysis, the MDASI-BT shows slightly greater utility in capturing symptoms in LM patients at presentation. KPS was not associated with endorsed symptoms or tumor location at presentation of LM.

The use of immunotherapy, specifically immune checkpoint inhibitors, is a rapidly emerging and evolving treatment modality for many solid tumor types. In this abstract, we describe a case of a 70 year old male with history of PD-L1 expressing metastatic poorly differentiated NSCLC treated with anti-PD-L1 therapy who was subsequently diagnosed with a glioblastoma with evidence of unexpected tumor infiltrating lymphocytes (TILs). The lung cancer was initially treated with combination chemotherapy with excellent response. This regimen was stopped prematurely due to significant treatment-related side effects. He then received the investigational anti-PD-L1 monoclonal antibody, MPDL3280A (Roche), under the Phase II Trial of MPDL3280A for PD-L1 positive locally advanced or metastatic NSCLC (NCT02031458). He experienced partial response without significant treatment-related side effects. Two weeks after completion of his 9th cycle of the study drug, he experienced altered mental status, personality changes, and confusion. A contrast brain MRI demonstrated a multifocal, rim-enhancing, left frontal lobe lesion with corpus callosal involvement. Of note, CT brain imaging prior to anti-PD-L1 study enrollment showed no evidence of abnormal enhancement. Stereotactic biopsy was performed with pathology showing glioblastoma with significant reactive lymphoid component noted (majority of lymphoid cells CD20 positive, scattered lymphocytes CD3 positive). He then underwent near image complete resection with pathology again demonstrating glioblastoma with 20-70% tumor necrosis, with 3 genomic alterations (CDKN2A/B loss; APCI1307K; TERT promoter – 124C > T) identified on Foundation One (Cambridge, MA) Next Generation Sequencing. This is the first reported case of a patient treated with anti-PD-L1 therapy for a high grade malignancy, who then developed a de novo glioblastoma with unusual biopsy findings of CD20 and CD3 positive TILs. Although more correlative studies are currently being performed, the immune implications for the use of immunotherapy for pre-treatment of glioblastomas may correspond to improved treatment response and patient outcomes.

BACKGROUND: The AKT1 mutation was newly described in a subset of meningiomas and inhibitors of this mutation have shown promise in clinical trials in multiple cancer types. We sought to determine the frequency of the AKT1 mutation as well as the expression level of multiple growth factor receptors in a large series of patients with Anaplastic meningiomas (AM) WHO °III. METHODS: Patients with AM °III were tested for the AKT1 (E17K) mutation using PCR technique. Additionally, the expression level of the epidermal growth factor receptor (EGFR), the platelet derived growth factor receptor 1alpha (PDGFR) and the vascular endothelial growth factor receptor (VEGFR) was detected by immunohistochemistry (IHC) using the indirect peroxidase technique on tissue multi arrays (TMA) of paraffin-embedded specimens. Staining was evaluated using a semi-quantitative scoring system. Chi-square test was used for statistical evaluation. RESULTS: We identified 22 AM °III patients with 45 tumors (median follow-up of 8 years). None of the examined 45 tumor samples in this cohort showed an AKT1 mutation (0%). Regarding the IHC, recurrent AM showed increasing proliferation with each recurrence. Overexpression of EGFR was associated with malignancy (p < 0.01) and increased with recurrence (median score 2.3). The median overall survival rate for patients with EGFR overexpression was 2.2 years. In contrast, the overall survival of patients with low EGFR expression was not reached yet (p < 0.05). While PDGFR and VEGFR scores were high in malignant tumors, the difference in comparison with benign meningiomas was not significant. A further finding is a marked survival benefit in our patients cohort in respect to the extent of resection when repeated operations were performed (p = 0.025). CONCLUSIONS: Somatic AKT1 mutations are absent in anaplastic meningiomas WHO grade III. Additionally, our data demonstrate that the overexpression of EGFR in AM might make this receptor valuable as a therapeutic target for their treatment.

One of difficulties in intra-CSF chemotherapy for leptomeningeal carcinomatosis (LMC) is a lack of quantitative measure for the treatment response. Previous studies evaluated CSF profiles to predict treatment response or to correlate with prognosis. However, the results were inconsistent mainly due to small number of patients and heterogeneous treatment. We analyzed 283 patients with LMC from solid cancer, who underwent intraventricular chemotherapy at National Cancer Center from 2003 to 2013. CSF parameters were cytology, cell count and protein level. Control of intracranial pressure (ICP) was reviewed as an objective indicator of symptom improvement. CSF profiles before the treatment were significantly different according to sample sites (p < 0.001) in both cell count (lumbar vs. ventricular is mean 23 ± 44 vs. 8 ± 30) and protein level (mean 124 ± 168 vs. 33 ± 66 mg/dL). Initial lumbar CSF protein level was correlated with development of increased ICP (p = 0.059), hydrocephalus (HCP) (p = 0.049), and cauda equine syndrome (CES) (p< 0.001). But, initial ventricular CSF profiles did not reflect LMC disease activity. Decrease of CSF protein level after treatment was associated with increased ICP control (p = 0.017) while cell count change or cytology conversion is not predictive. Patients with low initial protein level (≤ 50mg/dL), or cell count (≤15) show prolonged survival (p = 0.001 and 0.015, respectively). But, decrease of either protein level or cell count after the treatment and cytology response did not significantly affect the patients OS. We suggest that CSF cell count is too low to reflect disease activity or treatment response especially those from ventricle. Initial CSF protein is in accordance with LMC symptoms and the decrease of protein level after the treatment is predictive of treatment response. We need new quantitative pharmacodynamic markers for LMC treatment to improve efficacy of intraventricular chemotherapy.

BACKGROUND: Atypical teratoid rhabdoid tumor (ATRTs) constitute 20% of brain tumors in young children (

PURPOSE: Despite aggressive treatment with radiation therapy plus concurrent and adjuvant temozolomide (TMZ), the prognosis for glioblastoma remain poor. We investigated the potential of targeting signal transducer and activator of transcription-3 (STAT3) to improve the therapeutic outcome of combined radiotherapy and TMZ in glioblastoma. METHODS AND MATERIALS: We evaluated the preclinical potential of a STAT3 inhibitor, Cpd188 combined with temozolomide and radiation by in vitro clonogenic assays using two established glioblastoma cell lines (U251, U87) and two patients-derived glioblastoma cell lines (GBL12, GBL28) and in vivo studies using nude mice bearing intracranial U251 xenografts. RESULTS: Cpd188 potentiated the radiosensitizing effect of TMZ in U251 glioblastoma cell line which has high levels of p-STAT3 expression and in vitro. Increased radiosensitizing effects of TMZ were associated with the induction of apoptosis and the reversion of epithelial-mesenchymal transition (EMT). Cpd188 delayed in vivo tumor growth both alone and in combination with fractionated radiation and TMZ with a trend toward improved survival rates. Immunohistochemical staining of tumor sections showed that Cpd188 decreased the expression of CD31 (a marker of endothelial proliferation), vascular endothelial growth factor, and hypoxia-inducible factor-1α, suggesting that Cpd188 also has anti-angiogenic effects. We also confirmed the radiosensitizing effect of Cpd188 of GBL28 cell line which was originated from a patient who had a glioblastoma and also was confirmed high level of STAT3 expression and unmethylated MGMT. CONCLUSION: These data indicate that Cpd188 has the potential to improve the therapeutic outcome of combined radiotherapy and TMZ in human glioblastoma, especially in patients whose tumor has a high level of STAT3 expression regardless of MGMT methylation status. Work supported by grant (#2012-0004867 & #2013R1A1A2074531) from National Research Foundation, Korean Ministry of Future Creative Science to In Ah Kim.

BACKGROUND: To assess the efficacy of texture analysis (TA) in differentiating Low Grade Gliomas (LGGs) from Normal Appearing White Matter (NAWM) in a cohort of brain cancer patients using T2-FLAIR MR images. Also the impact of the number of gray-levels employed was investigated. METHODS: T2-FLAIR and post-gadolinium (GD) contrast T1-weighted MR images of 11 TCGA patients (mean age 40.8 years, range 27-59.5 years) were retrospectively analyzed. All patients were diagnosed with LGGs (grades II and III). Four different volumes of interest (VOI)s were segmented for each patient by an experienced radiologist: i) tumor, ii)peritumoral WM (pWM), iii) far ipsi-lateral WM (iWM) and iv) contra-lateral WM(cWM). Following VOI segmentation, TA was performed using FLAIR images on MatLab environment. A complete list of invariant texture features derived from the gray-level co-occurrence matrix (GLCM) was calculated. Because the ideal number of gray levels is unknown, we used various gray levels (8, 16, 32, and 64 levels). To assess the efficacy of the calculated features we constructed a decision tree. The model was validated using a 10-fold cross validation of the training data. The accuracy, sensitivity and specificity were calculated. Impact of the number of gray levels on the performance indicators was investigated. RESULTS: For discrimination of LGG from NAWM, TA achieved 100% accuracy. High accuracy (80-87.5% for different gray levels) was achieved for discrimination of all four VOIs. Off note, the peritumoral WM was identified with 86.7% to 93% accuracy from the two remaining NAWM regions combined. Also, from the performance indicators it is evident that the number of gray levels does not affect the calcification. CONCLUSIONS: TA provided excellent accuracy for discrimination between LGG and NAWM. A major strength of the presented work is that it showed high performance in discriminating among normal WM regions depending on their proximity to the tumor.