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Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy because of its diffuse infiltrative growth pattern. Translational research suffers from the lack of a representative DIPG animal model. Hence, human E98 glioma cells were stereotactically injected into the pons of nude mice. The E98 DIPG tumors presented a strikingly similar histhopathology to autopsy material of a DIPG patient, including diffuse and perivascular growth, brainstem- and supratentorial invasiveness and leptomeningeal growth. Magnetic resonance imaging (MRI) was effectively employed to image the E98 DIPG tumor. [(18) F] 3'-deoxy-3'-[(18) F]fluorothymidine (FLT) positron emission tomography (PET) imaging was applied to assess the subcutaneous (s.c.) E98 tumor proliferation status but no orthotopic DIPG activity could be visualized. Next, E98 cells were cultured in vitro and engineered to express firefly luciferase and mCherry (E98-Fluc-mCherry). These cultured E98-Fluc-mCherry cells developed focal pontine glioma when injected into the pons directly. However, the diffuse E98 DIPG infiltrative phenotype was restored when cells were injected into the pons immediately after an intermediate s.c. passage. The diffuse E98-Fluc-mCherry model was subsequently used to test escalating doses of irradiation, applying the bioluminescent Fluc signal to monitor tumor recurrence over time. Altogether, we here describe an accurate DIPG mouse model that can be of clinical relevance for testing experimental therapeutics in vivo.

Background: Conditionally-replicating adenoviruses (CRAds) infect and replicate in tumor cells, releasing viral progeny upon lysis of the cell. This is a dynamic and inherently exponential process and, thus, the assessment of CRAds should incorporate these dynamics. In vitro experiments are therefore prone to subjective assessment because no validated assay exists that truly appreciates the dynamics of the process. An objective assay could simplify experiments and reduce the number of CRAd variants required to enter a full preclinical evaluation. Methods: We developed a simple and practical mathematical model incorporating easily obtainable parameters of the interaction between replicating viruses and growing tumor cells in vitro and, in the present study, validate this model by fitting the predicted values to experimentally-derived values. Results: From the exponential curves of cellular growth and the viral propagation rate in glioma cells, we derive the four parameters needed in this model and show a robust fit to experimental data. Because the initial infection conditions appear to significantly influence the final outcome of CRAd experiments, these conditions are determined using the same cells and correlated with the expression of the primary adenovirus receptor CAR (coxsackie and adenovirus receptor). Conclusions: The results obtained shed light upon the method of action of CRAds and provide an objective and practical model and assay for determining and predicting CRAd activity in tumor cells. Copyright © 2010 John Wiley & Sons, Ltd.

BackgroundAccurate frameless neuronavigation is highly important in cranial neurosurgery. The accuracy demonstrated in phantom models might not be representative for results in patients. Few studies describe the in vivo quantitative accuracy of neuronavigation in patients. The use of a frameless stereotactic drilling technique for stereoelectroencephalography depth electrode implantation in epilepsy patients, as well as diagnostic biopsies, provides a unique opportunity to assess the accuracy with postoperative imaging of preoperatively planned trajectories.MethodsIn 7 patients with refractory epilepsy, 89 depth electrodes were implanted using a frameless stereotactic drilling technique. Each electrode was planned on a preoperative magnetic resonance and computed tomographic scan, and verified on postoperative computed tomographic scan. After fusion of preoperative and postoperative imaging, the accuracy for each electrode was calculated as the Euclidean distance between the planned and observed position of the electrode tip.ResultsThe median Euclidean distance between planned and observed electrode implantations was 3.5 mm (95% confidence interval, 2.9–3.9 mm) with a range of 1.2–13.7 mm.ConclusionsIn this study, we showed that the in vivo accuracy of our frameless stereotactic drilling technique, suitable for stereoelectroencephalography depth electrode placement and diagnostic brain biopsies, was 3.5 mm.

Multicellular tumor spheroids are used as a model to assess the efficacy of replicating oncolytic adenoviruses. As most assays used to assess cellular viability are unsuitable for oncolytic viruses because of ongoing viral replication, we have used positron emission tomography (PET) to sequentially determine the incorporation of 18F-labeled deoxyglucose ( 18F-DG) as a measure of viability and compared the results to more commonly used assays for measuring the effect of oncolytic therapy. Glioma monolayer cultures and spheroids were infected with wild-type replicating adenovirus and viability was measured by 18F-DG incorporation, WST-1 assay, crystal violet assay, and spheroid volume 2 to 10 days following infection. Results show that volume measurements in adenovirus-infected spheroids are confounded by the cytopathic effect occurring in infected cells. 18F-DG PET provides a useful method to assess small differences in cell number and viability following oncolytic viral therapy in glioma monolayer cultures and spheroids without the need for disintegration of these cultures. Moreover, using 18F-DG PET, repeated sequential measurements of spheroid viability can be made, decreasing the required number of spheroids per experiment. This is a valuable feature when using spheroids derived from limited amounts of patient material. Copyright

Purpose: The integrin-targeted conditionally replicating adenovirus Ad5-Δ24RGD has been shown to possess strong oncolytic activity in experimental tumors and is currently being developed toward phase I clinical evaluation for ovarian cancer and malignant glioma. Previously, we reported that combination therapy of Ad5-Δ24RGD with irradiation led to synergistic antitumor activity in s.c. glioma xenografts. In the current study, the underlying mechanism of action to this synergy was studied and the effects of combined therapy were assessed in an orthotopic glioma model. Experimental Design and Results: Sequencing studies in U-87 monolayers showed that delivery of irradiation before Ad5-Δ24RGD infection led to a greater oncolytic effect than simultaneous delivery or infection before irradiation. This effect was not due to enhanced virus production or release. Experiments using a luciferase-encoding vector revealed a small increase in transgene expression in irradiated cells. In tumor spheroids, combination therapy was more effective than Ad5-Δ24RGD or irradiation alone. Staining of spheroid sections showed improved penetration of virus to the core of irradiated spheroids. Mice bearing intracranial tumors received a combination of Ad5-Δ24RGD with 1 × 5 Gy total body irradiation or with 2 × 6 Gy whole brain irradiation. In contrast to the in vitro data and reported results in s.c. tumors, addition of radiotherapy did not significantly enhance the antitumor effect of Ad5-Δ24RGD. Conclusions: Combined treatment with Ad5-Δ24RGD and irradiation shows enhanced antitumor activity in vitro and in s.c. tumors, but not in an orthotopic glioma model. These differential results underscore the significance of the selected tumor model in assessing the effects of combination therapies with oncolytic adenoviruses.

BACKGROUND: The conditionally replicating adenovirus (CRAd) AdDelta24-p53 replicates selectively in Rb mutant cells, and encodes the p53 suppressor protein. It has shown improved oncolytic potency compared to the parental control AdDelta24. As exogenous p53 has been shown to enhance radiosensitivity, the combination of AdDelta24-p53 and AdDelta24 with radiotherapy was assessed in vitro and in vivo against the therapy resistant gliomas.METHODS: In glioma cells, multicellular spheroids and animal xenografts the efficacy of combination therapy was assessed. P53 phosphorylation, induction of apoptosis and viral replication were determined following single or combination therapies.RESULTS: In vitro, AdDelta24-p53 was more effective against glioma cells than the control AdDelta24. Addition of irradiation equally increased the efficacy of both AdDelta24-p53 and AdDelta24 resulting in improved oncolysis compared to single agent treatment. Radiotherapy did not significantly change the replication kinetics of AdDelta24-p53 or AdDelta24. No detectable increase in p53 phosphorylation was observed but combination of radiotherapy and AdDelta24-p53 caused an increase in the percentage of apoptotic cells. In vivo, combination therapy with either AdDelta24 or AdDelta24-p53 significantly increased the number of mice demonstrating tumor regression (100%) as well as long-term survival (50%). No differences between viruses were noted.CONCLUSIONS: Exogenous p53 expression does not appear to increase the synergistic interaction of CRAds combined with radiotherapy. These results however do indicate that radiotherapy provides the time frame in which AdDelta24 and AdDelta24-p53 can eradicate established tumors that would otherwise escape treatment, and establishes the need to combine these modalities to form an effective anti-cancer treatment.

Organotypic cultures from normal neocortical tissue obtained at epilepsy surgery show a severe injury response. This response involves both neuronal degeneration and the proliferation of reactive cells. A salient feature of the reactive cells is the co‐expression of microglial and astrocytic markers. Surprisingly, the reactive cells also began to express neuronal markers Tubulin βIII and MAP2 adding to the confusion about their origin. Concomitant with their appearance in reactive cells MAP2 and Tubulin βIII expression disappeared from neurons. While NeuN expression decreased significantly, it did not entirely disappear from many neurons. Moreover, it was not observed in reactive cells, showing that NeuN is a reliable marker of neurons.

Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, AdΔ24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus AdΔ24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by AdΔ24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2-activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the AdΔ24TIMP-3-treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87δEGFR) tumors with AdΔ24TIMP-3 and AdΔ24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of AdΔ24 did not significantly affect the antitumor efficacy of this oncolytic agent. ©2005 American Association for Cancer Research. Chemicals / CAS: tissue inhibitor of metalloproteinase 3, 145809-21-8, 164781-40-2; Matrix Metalloproteinase 2, EC 3.4.24.24; Tissue Inhibitor of Metalloproteinase-3

Summary Blood-based liquid biopsies, including tumor-educated blood platelets (TEPs), have emerged as promising biomarker sources for non-invasive detection of cancer. Here we demonstrate that particle-swarm optimization (PSO)-enhanced algorithms enable efficient selection of RNA biomarker panels from platelet RNA-sequencing libraries (n = 779). This resulted in accurate TEP-based detection of early- and late-stage non-small-cell lung cancer (n = 518 late-stage validation cohort, accuracy, 88%; AUC, 0.94; 95% CI, 0.92–0.96; p < 0.001; n = 106 early-stage validation cohort, accuracy, 81%; AUC, 0.89; 95% CI, 0.83–0.95; p, Graphical Abstract, Highlights • Tumor-educated platelet (TEP) RNA profiles allow for blood-based cancer diagnostics • Inflammatory conditions only minimally confound TEP-based cancer detection • Swarm intelligence algorithms enable efficient selection of biomarker gene panels • TEP gene panels enable support vector machine-based classification of lung cancer, Best et al. use particle-swarm optimization algorithms and RNA-seq of tumor-educated platelets from patients to generate RNA sets capable of identifying patients with non-small-cell lung cancer, including those having early stage, from individuals without cancer, including those having inflammatory conditions.

After introduction of the NATOPS standardization system the incidence of mishaps in the United States Navy decreased substantially. NATOPS includes standardized programs as the basis for development of an efficient and sound operational procedure. In order to improve patient safety and analogous to NATOPS we propose a standardization program for parts of surgical procedures

Background Systemic delivery of therapeutic agents remains ineffective against diffuse intrinsic pontine glioma (DIPG), possibly due to an intact blood–brain-barrier (BBB) and to dose-limiting toxicity of systemic chemotherapeutic agents. Convection-enhanced delivery (CED) into the brainstem may provide an effective local delivery alternative for DIPG patients. New method The aim of this study is to develop a method to perform CED into the murine brainstem and to test this method using the chemotherapeutic agent carmustine (BiCNU). To this end, a newly designed murine CED catheter was tested in vitro and in vivo. After determination of safety and distribution, mice bearing VUMC-DIPG-3 and E98FM-DIPG brainstem tumors were treated with carmustine dissolved in DW 5% or carmustine dissolved in 10% ethanol. Results Our results show that CED into the murine brainstem is feasible and well tolerated by mice with and without brainstem tumors. CED of carmustine dissolved in 5% DW increased median survival of mice with VUMC-DIPG-3 and E98FM-DIPG tumors with 35% and 25% respectively. Dissolving carmustine in 10% ethanol further improved survival to 45% in mice with E98FM-DIPG tumors. Comparison with existing methods Since genetically engineered and primary DIPG models are currently only available in mice, murine CED studies have clear advantages over CED studies in other animals. Conclusion CED in the murine brainstem can be performed safely, is well tolerated and can be used to study efficacy of chemotherapeutic agents orthotopically. These results set the foundation for more CED studies in murine DIPG models.

The oncolytic adenovirus Delta24-RGD is currently being tested in phase I trials for the treatment of glioblastoma (GBM). Literature suggests that frequently prescribed anticonvulsants for these patients, phenytoin (PHE), valproic acid (VPA) and levetiracetam (LEV), may interfere with cellular mechanisms of cancer or oncolytic virus activity. We therefore investigated the direct effects of these drugs on Delta24-RGD infection and oncolytic activity.The anticonvulsants PHE, VPA, and LEV were combined with Delta24-RGD treatment in established glioma cell lines as well as on a panel of patient-derived GBM cultures. Effects on infection efficiency were assessed using luciferase-encoding adenoviral vectors. Oncolytic activity was determined by WST-1 assay and viral progeny production was quantified by dilution titration.IC50 values of the anti-epileptic drugs on the four glioma cell lines were far above clinically-relevant concentrations. At therapeutic concentrations, the anti-epileptics generally did not alter the infection efficiency of RGD-modified adenovirus, nor affect progeny production or oncolytic activity of Delta24-RGD. The only exception was found in U373 cells, where VPA slightly antagonised the oncolytic effect of Delta24-RGD (from 29% to 55% viability, p0.01) as well as viral progeny production (60% decrease, p0.01). Oncolysis by Delta24-RGD was not inhibited by the anti-epileptics in any of the patient-derived glioma cultures (n=6). In fact, in one culture a slight enhancement of viral oncolysis by PHE and LEV was found, from 89.7% viability to 76% and 62.4%, respectively (p0.01)Therapeutic levels of valproic acid, phenytoin and levetiracetam do not negatively interfere with the infection efficiency or oncolytic activity of Delta24-RGD in patient-derived GBM cells. Therefore, there is no indication that the choice of anticonvulsant for seizure control in glioma patients should take treatment with Delta24-RGD into account.

Growing skull fracture (GSF) is a rare complication of cranial trauma in children younger than 3 years. It is characterised by the presence of a dural defect due to which herniation of the brain tissue can develop, with cystic transformation and resulting cerebral damage.A 5-month-old baby girl presented at the emergency department following a fall from the staircase. Upon examination she showed a left parietal subgaleal haematoma and right-sided hemiparesis. MRI examination of the brain showed a skull fracture and dural defect with progressive herniation of the brain tissue. Neurosurgical reconstruction was carried out.GSF is a rare complication of cranial trauma in young children as a result of rupture of the dura and separating fracture edges. This can lead to cerebral damage and early recognition is important. GSF needs to be considered in children younger than 3 years with a (pulsating) subgaleal haematoma. The diagnosis is made by MRI scan of the brain and neurosurgical treatment consists of watertight closure of the dura combined with skull reconstruction.

The inevitable clinical recurrence of high grade gliomas after standard treatment is due to the highly diffuse infiltrating parts of these tumors, which remain after surgery and respond poorly to radiation and chemotherapy. It has been proposed to employ the homing capacity of neural stem cells (NSCs) to different types of intracerebral pathology for selective targeting of glioma cells, and delivery of transgenic expressed therapeutics. This approach has been Successful in a number of preclinical experimental studies, however, a major drawback for clinical translation has been the limitation of harvesting and ex vivo expansion of NSCs in patients. Here we demonstrate that adipose derived stem cells (ASCs), which are easily harvested in relatively large quantities in humans, display the same tropism for gliomas as NSCs in vitro and in vivo. Both ipsilateral as well as contralateral injection of these cells in brains of glioma-bearing mice, led to extensive homing to the tumor by the ASCs. The potential of loading these cellular vehicles with transgenes was assessed using adenoviral vectors. ASCs could be infected with adenoviral vectors, albeit at very high MOI. Insertion of the arg-gly-asp (RGD) motif into the adenovirus fiber knob, thereby redirecting primary attachment of the virus to integrins, resulted in a striking 7000-fold increase in infection efficiency. However, in vivo migration of adenovirus-infected ASCs was not observed, most likely due to an inflammatory response to these cells which was not observed with control non-infected ASCs. These results indicate that ASCs are an interesting candidate for further development for cell-based therapy of gliomas, however adenoviruses are not appropriate vectors for delivery of transgenes in this context. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

BACKGROUND: Advanced stage and relapsing neuroblastoma (NB) has a poor prognosis with frequent treatment failures, warranting new treatment options and enhanced local tumor control. Treatment with conditionally replicative adenoviruses (CRAds) has shown effectiveness in various preclinical cancer models, but has not yet been evaluated for local control of NB. Here, we tested the efficacy of the CRAd AdDelta24 and of two AdDelta24 derivatives against NB. Derivative AdDelta24-425S11 infects cells deficient in coxsackie/adenovirus receptor (CAR) via the epidermal growth factor receptor (EGFR). Derivative AdDelta24-p53 expresses the tumor suppressor protein p53 to promote oncolysis.METHODS: Expression of CAR and EGFR, and p53 pathway and DNA damage responses were analyzed in six NB cell lines and two xenografts derived from primary NB using immunohistochemistry, reporter gene transactivation, Western blot and fluorescence-activated cell sorting (FACS) analysis. Efficacy of AdDelta24, AdDelta24-425S11 and AdDelta24-p53 against NB was evaluated in vitro by cell viability analysis and in vivo by monitoring subcutaneous xenograft tumor growth in mice and by histological analysis of treated tumors.RESULTS: Neuroblastoma cell lines were sensitive to oncolysis by AdDelta24, with a higher susceptibility of those with functional p53 and intact DNA damage responses. Compared to AdDelta24, AdDelta24-p53 exhibited enhanced oncolytic potency on all NB cell lines independent of their p53 status and AdDelta24-425S11 was more effective against CAR-low IGR-NB8 cells. Moreover, five daily intratumoral injections of 10(8) plaque-forming units (pfu) of AdDelta24-p53 or AdDelta24-425S11 into subcutaneous IGR-NB8 and IGR-N91 xenografts at an advanced tumor stage yielded significant tumor growth delays (TGD). In contrast, at this dose, AdDelta24 did not cause significant TGD of neuroblastoma xenografts. Injection of AdDelta24-p53 was associated with extensive cell lysis, apoptotic cell death, and fibrous fascicles in the tumors.CONCLUSION: CRAds expressing p53 and targeted towards EGFR appear promising new agents for local control in the treatment of neuroblastoma.

s ingediend voor het Amsterdam Kindersymposium 2013 113 Convection Enhanced Delivery of carmustine in diffuse intrinsic pontine glioma S.E.M. Veldhuijzen van Zanten (1), M.H.A. Jansen (1), D.G. van Vuurden (1,2), E. Hulleman (1,2), T. Lagerweij (2), S. Idema (3), D. P. Noske (2,3), N.I. Wolf (4), N.H. Hendrikse (5), W.P. Vandertop (3), G.J.L. Kaspers (1) (1) Department of Pediatrics, hematology oncology, VU medical center, Amsterdam (2) Neuro-oncology Research Group, VUmc Cancer Center Amsterdam (3) Neurosurgical Center Amsterdam (4) Department of Pediatrics, neurology, VU medical center, Amsterdam (5) Department of Clinical Pharmacology and Pharmacy, VU medical center, Amsterdam INTRODUCTION DIPG patients have a dismal prognosis with a median overall survival of nine months. Typically, surgical resection is not possible, radiotherapy has only temporary eff ect and systemic chemotherapeutics fail. Convection Enhanced Delivery (CED) provides high local drug concentrations and equal drug distribution directly at the tumor site through micro-catheters via a pressure gradient. In a murine CED-model, we injected DIPG with carmustine, a highly-active nitrosourea compound, and found a signifi cantly prolonged survival with no severe toxicity. Based on these preclinical data, and on studies in adults showing the safety of the procedure, we developed a clinical study in children with DIPG. METHODS Patients will be included at time of progression after initial radiotherapy. Catheters will be placed based on iPlan Flow simulations. Two cohorts of three patients will receive escalating concentrations of carmustine up to 50% of the maximum tolerated adult dose. Infusion-volume and infusion-rate will be determined by tumor-volume (maximum 7 ml). Co-infusion of gadolinium enables observation of distribution by MRI. Maximum infusion-duration will be 41 hours, during which a patient will be awake and observed intensively. Primary endpoints of this study are safety and feasibility. CONCLUSION Preclinical data has shown carmustine-CED to be safe and eff ective in DIPG mouse models. With this clinical study we intend to show safety and feasibility of carmustine administration via CED in patients with DIPG. This technique improves treatment options, and hopefully the dismal prognosis of patients in the future. Abstract fi gure: 3-dimensional simulation of CED in a child with DIPG (iPlan Flow BrainLAB AG). The tumor volume is orange-shaped. The red tract shows the safest route for catheter placement. In green, the spherically-shaped distribution volume of the therapeutic agent (based on MRI-DTI images). In this simulation the total infused volume was approximately 3-4 ml and the infusion rate was set to 3.0 μl/min. With expanding infusion volume and time, it is possible to distribute the therapeutic agent over the total tumor volume, resulting in high concentrations of the cytotoxic agent directly at the site of action.fi gure: 3-dimensional simulation of CED in a child with DIPG (iPlan Flow BrainLAB AG). The tumor volume is orange-shaped. The red tract shows the safest route for catheter placement. In green, the spherically-shaped distribution volume of the therapeutic agent (based on MRI-DTI images). In this simulation the total infused volume was approximately 3-4 ml and the infusion rate was set to 3.0 μl/min. With expanding infusion volume and time, it is possible to distribute the therapeutic agent over the total tumor volume, resulting in high concentrations of the cytotoxic agent directly at the site of action.