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Diffuse Midline Glioma (DMG) are fatal pediatric brain tumors with no therapies. We leveraged network-based methodologies to dissect the heterogeneity of DMG tumors and to discover Master Regulator (MR) proteins representing pharmacologically accessible, mechanistic determinants of molecularly distinct cell states. We produced the first DMG regulatory network from 122 publicly available RNAseq profiles with ARACNe (Basso et al. Nat Genet 2005), and inferred sample-specific MR protein activity with VIPER (Alvarez et al. Nat Genet 2016) based on the differential expression of their targets. 7 of the top 25 most active MRs found comprise a well-characterized MR block (MRB2) (Paull et al.Cell 2021), frequently activated across aggressive tumors, and enriched in DMG patient MR signatures (Fisher’s Exact Test p=4.4 × 10−18). A CRISPR/Cas9 KO screen across 3 DMG patient cell lines identified a set of 73/77 essential genes that were enriched in the MR signature of 80% of patient samples (GSEA p=0.000034). FOXM1 emerged as an essential MR, significantly activated across virtually all patients. We then generated RNAseq profiles following perturbation with ~300 oncology drugs in 2 DMG cell lines most representative of patient MR signatures, and used this to identify drugs that invert patient MR activity profiles using the NYS/CA Dept. of Health approved OncoTreat algorithm (Alvarez et al. Nat Genet 2018). OncoTreat predicted sensitivity to HDAC, MEK, CDK, PI3K, and proteosome inhibitors in subsets of patients, overlapping with published DMG drug screens. Importantly, 80% of OncoTreat-predicted drugs (p Further analysis of DMG intra-tumor heterogeneity via protein activity inference across DMG single cells from 6 published scRNAseq profiles identified 6 tumor clusters with unique MR signatures co-existing in virtually all patients representing distinct cellular states (2 astrocyte-, 1 oligodendrocyte-, and 3 oligodendrocyte precursor cell-like states). Targetable MRs and OncoTreat-predicted drugs were distinct between these states. Bulk RNAseq analysis recapitulated predictions seen in the more prevalent OPC-like states, but failed to capture MR and drug predictions for the AC-like states (e.g. JAK1/Ruxolitinib and STAT3/Napabucasin). We are currently validating cell state-specific drug predictions in vivo at single-cell resolution in subcutaneous patient-derived xenograft and orthotopic syngeneic DMG models that we have shown recapitulate patient tumor heterogeneity, including with focused ultrasound-mediated drug delivery. This provides a platform to nominate much-needed novel drugs and drug combinations to treat DMG. Citation Format: Ester Calvo Fernandez, Junqiang Wang, Xu Zhang, Hong-Jian Wei, Hanna E. Minns, Aaron T. Griffin, Lukas Vlahos, Timothy J. Martins, Pamela S. Becker, John Crawford, Robyn D. Gartrell, Luca Szalontay, Stergios Zacharoulis, Zhiguo Zhang, Robert Wechsler-Reya, Cheng-Chia Wu, Andrea Califano, Jovana Pavisic. Network-based inference identifies cell state-specific drugs targeting master regulator vulnerabilities in diffuse midline glioma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4304.

Introduction Side-effects during convection enhanced delivery (CED) are poorly understood. We intended to determine the frequency of side-effects during brain stem infusion and determine risk factors for side-effects persisting longer than 24 h. Methods Children with a radiological diagnosis of brain stem diffuse midline glioma/Diffuse Intrinsic Pontine Glioma were treated on compassionate grounds with awake infusion of carboplatin and sodium valproate into the brain stem using the 4-catheter (2 trans-cerebellar 2 trans-frontal) chronic, intermittent Renishaw Drug Delivery System. We used change in the Pontine Neurological Observation Score (PONScore), a standardised neurological assessment tool, to identify side-effects during infusion. Recovery was determined by retrospective chart review. Results 55 infusions were performed in 8 children (3–11 years). Mean PONScore increased during infusion from 3.3 to 5.7 (p-value > 0.001). One hundred and fifty-seven infusion-related side-effects were identified including headache (33/157) and limb weakness (49/157). Fifty-four side-effects persisted > 24 h. Side-effects that had occurred during a previous infusion and those that occurred during infusion via trans-cerebellar catheters were more likely to be persistent with OR 2.333 (95% CI 1.094–4.976; p-value = 0.028) and 2.155 (1.029–4.513; p-value = 0.042) respectively. If infusion was stopped or titrated at onset rather than continued, the side-effect was less likely to persist > 24 h, OR 0.473 (95% CI 0.177–0.948; p-value = 0.037). Most side-effects developed within the first three millilitre of infusion. Conclusions Side-effects during brainstem infusion are common, can be transient or persist longer than 24 h. Neurological injury during infusion may be time dependent and accumulative rather than volume dependent.

Monitoring neurological side-effects in experimental therapy for diffuse intrinsic pontine glioma (DIPG) can be challenging. We aimed to develop a neurological scale that could be used by non-specialists to quantify neurological changes during experimental treatment of DIPG. We developed the Pontine Observational Neurological Score (PONScore) to measure signs and symptoms of DIPG by adapting validated assessment scales of neurological signs and symptoms in children. We developed a prototype score, taught it to paediatric intensive care nursing staff, who used the Score to assess children receiving awake pontine infusion of chemotherapy for treatment of DIPG. We used their feedback to develop the PONScore. Points are allocated for headache, ophthalmoplegia, facial and tongue weakness, dysarthria, paraesthesia, limb weakness and dysmetria with increasing scores reflecting increasing disability. The PONScore was administered every hour during awake pontine infusion. Correlation and agreement calculations between nursing staff, as non-specialists, and a specialist rater were performed in 30 infusions in 6 children (aged 8–11). Changes in PONScore versus volume of infusion are described in a further 55 infusions in 8 children (aged 3–11). The PONScore demonstrated excellent intra-rater reliability with an intra-class co-efficient of 0.98 (95% CI 0.97–0.99; p-value

Diffuse Midline Glioma (DMG), H3K27M altered, confers a dismal survival of 9-15 months and has a non-inflammatory tumor immune microenvironment (TIME). Radiation therapy (RT) is the mainstay treatment for DMG and has been shown in other cancers to recruit an immune component. However, the effect of RT on the DMG TIME has not been explored. In a syngeneic murine model of pontine DMG (PDGFB+, H3.3K27M, p53−/−), mice were treated with single fraction 15Gy RT or sham control, four mice per group. We performed single cell sequencing after CD45 isolation to evaluate the TIME 4 days post RT and compare to untreated tumor (sham control). Unsupervised clustering of 14,848 CD45+ cells revealed 16 immune cell subsets, most abundantly microglia at 75% of cells, with four subtypes representing a spectrum of homeostatic to activated. Microglia from RT are more concentrated in the activated subtypes with an upregulation of interferon response (i.e. Isg15, Ifit3) compared to untreated tumor with an increase in several interferon pathways using REACTOME. Consistent with RT response, RT treated tumors have increase in cell cycle regulatory genes such as Cdkn1a, across all clusters. In non-resident myeloid cells, compared to untreated tumor, RT is associated with a robust upregulation of interferon response genes in both macrophages (Isg15 Fold Change (FC) 2.30; Ifit1 FC 1.64; Ifit3 FC 2.02; Cxcl10 FC 2.29) and dendritic cells (Isg15 FC 2.67; Ifit1 FC 1.72; Ifit3 FC 2.06; Cxcl10 FC 1.50). We also find differential expression of immune checkpoints in RT-treated versus untreated tumor with decreased expression of Lag3, Tim3 (Havcr2), and Csf1R and increased expression of Cd47, Sirpa and Gitr (Tnfrsf18) post RT. In summary, RT stimulates a pro-inflammatory TIME response and alters immune checkpoints in DMG, highlighting the potential for combining RT and immunotherapy in these tumors.

Diffuse midline glioma (DMG) are fatal pediatric brain tumors with no effective systemic therapies. Molecular profiling demonstrates epigenetic dysregulation and heterogeneity, and novel approaches are needed to identify promising drugs and drug combinations. We used network-based computational analysis of RNA-seq to discover Master Regulator (MR) proteins that represent targetable, mechanistic determinants of distinct DMG cell states. We reverse-engineered the first DMG-specific regulatory network from 122 publicly available DMG RNA-seq profiles with ARACNe. Using this network, we measured sample-specific protein activity based on differential expression of their targets via VIPER. Activity-based clustering identified two clusters showing a trend in survival differences (>1 year, by χ2). The most activated MRs (i.e., TOP2A, CENPF, BUB1B, FOXM1, GTSE1, MKI67, E2F8), relative to normal caudate tissue, were enriched in cell cycle regulation members. The cluster with worse outcomes had significantly higher activity. Targetable MRs activated in subsets of patients () included TOP2A, CHEK1, CDK2, and EZH2. RNA-seq profiles were generated in two DMG cell lines following perturbation with ~400 oncology drugs, and used to identify drugs that invert MR activity profiles with the OncoTreat algorithm. We identified four pharmacotypes, and predicted sensitivity to HDAC, proteosome, EGFR, MEK, and PI3K inhibitors (), amongst others, consistent with published DMG high-throughput drug screens. To dissect intra-tumor heterogeneity, we measured protein activity from published single-cell RNA-seq profiles of 6 DMG patients, using single-cell based regulatory networks. Preliminary activity-based analysis identified 6 cell states representing distinct stages of differentiation/proliferation, including an oligodendrocyte precursor cell-like proliferative state whose MRs overlapped with bulk data (i.e. TOP2A, CENPF, FOXM1, E2F8, ZWINT, CCNA2), suggesting this as a key regulatory module. We are identifying cell state-specific MR-inverter drugs with OncoTreat to ultimately suggest drugs and drug combinations that collapse the transcriptional program and induce tumor kill for validation in MR-matched preclinical DMG models.

Diffuse midline gliomas (DMG) are pediatric tumors with dismal prognosis. When these tumors emerge in the brainstem, there exists no feasible method of surgical resection or systemic intervention, making ionizing radiation the sole therapeutic avenue to date. However, radiotherapy (RT) provides only marginal survival benefit as the topographically diffuse and highly infiltrative tumors spread in areas in which the blood-brain barrier (BBB) is relatively intact. Focused ultrasound (FUS) with intravenous microbubbles provides a compelling solution, transiently and non-invasively opening the BBB to allow drug delivery across the cerebrovasculature. Nonetheless, it remains unclear whether FUS can be safely administered at the brainstem in patients receiving RT. Therefore, the goal of this study was to assess the safety and feasibility of FUS administered concurrent with and adjuvant to a clinical hypofractionated radiation scheme for brainstem DMG. Non-tumor bearing B6 albino mice were randomly assorted into control, RT, FUS, and RT+FUS groups. Mice designated RT+FUS received 39Gy/13fx (hypofractionated RT scheme) to the brainstem with two sessions of FUS approximately 1 week apart. A single-element, spherical-segment FUS transducer driven by a function generator through a power amplifier was used with concomitant microbubble injection to sonicate the brainstem. Magnetic resonance imaging (MRI) was used to confirm BBB opening and cardiopulmonary measures were recorded throughout sonication. Vitals were assessed daily, and all treatment animals underwent Kondziela inverted screen testing and sequential weight lifting to assess brainstem-related strength and motor coordination deficits. In both FUS and RT+FUS mice, MRI confirmed brainstem BBB opening and subsequent closure within 96 hours. Mouse weights were stable, with slight drops (mean=5.5%) following FUS that resolved within three days. No attenuation in cardiorespiratory, strength, and motor coordination measurements was observed from FUS. FUS is a safe and feasible technique for brainstem BBB opening concurrent with and adjuvant to clinical hypofractionated RT.

Background The childhood peak of brain tumors suggests that early-life exposures might have a role in their etiology. Hence, we examined in the Greek National Registry for Childhood Hematological Malignancies and Solid tumors (NARECHEM-ST) whether perinatal and early-life risk factors influence the risk of childhood brain tumors. Methods In a nationwide case-control study, we included 203 cases (0–14 years) with a diagnosis of brain tumor in NARECHEM-ST (2010–2016) and 406 age-, sex-, and center-matched hospital controls. Information was collected via interviews with the guardians and we analyzed the variables of interest in multivariable conditional logistic regression models. Results Instrument-assisted delivery was associated with higher (OR: 7.82, 95%CI: 2.18–28.03), whereas caesarean delivery with lower (OR: 0.67, 95%CI: 0.45-0.99) risk of childhood brain tumors, as compared to spontaneous vaginal delivery. Maternal alcohol consumption during pregnancy (OR: 2.35, 95%CI: 1.45–3.81) and history of living in a farm (OR: 4.98, 2.40–10.32) increased the odds of childhood brain tumors. Conversely, higher birth order was associated with lower risk (OR for 2nd vs. 1st child: 0.60, 95%CI: 0.40-0.89 and OR for 3rd vs. 1st: 0.34, 95%CI: 0.18-0.63). Birth weight, gestational age, parental age, history of infertility, smoking during pregnancy, allergic diseases, and maternal diseases during pregnancy showed no significant associations. Conclusions Perinatal and early-life risk factors, and specifically indicators of brain trauma, exposure to toxic agents and immune system maturation, might be involved in the pathogenesis of childhood brain tumors. Larger studies should aim to replicate our findings and examine associations with tumor subtypes.

Despite major advances in molecular profiling and numerous clinical trials, diffuse midline glioma (DMG) remains a fatal disease with median survival of only ~9 months and no identified effective drugs. To address this challenge, we leveraged network-based methodologies to dissect the heterogeneity of DMG tumors and to discover Master Regulator (MR) proteins representing pharmacologically accessible, mechanistic determinants of molecularly distinct DMG cell states. The study has produced the first DMG-specific regulatory network, reverse-engineered from 122 publicly available pediatric DMG RNA-seq profiles with ARACNe (Basso et al. Nat Genet 2005). Using this network, we measured protein activity for each sample via VIPER (Alvarez et al., Nat Genet 2016). Activity-based clustering identified 2 clusters, characterized by significant overall survival difference (>1 year, p-val=0.02 by χ2 analysis). Protein activity signatures were not significantly associated with tumor location and Histone3 mutation status. The most aberrantly activated MR proteins across all DMG patients (i.e., TOP2A, CENPF, BUB1B, FOXM1, GTSE1, MKI67, and E2F8), relative to normal caudate tissue from GTEx, were highly enriched in cell cycle regulation members, with samples in the worst outcome cluster showing significantly higher activity. Pharmacologically accessible MRs found to be significantly activated in subsets of patients (p-val To dissect DMG intra-tumor heterogeneity, we measured protein activity from published single-cell RNA-seq profiles of 6 DMG patients, using single-cell based regulatory networks. Activity-based analysis of tumor cells identified 8 clusters representing distinct differentiation and proliferation stages—i.e. astrocyte-like, oligodendrocyte-like, and multiple oligodendrocyte precursor cell (OPC)-like subpopulations. Consistent with bulk-based findings, these included an OPC-like-cycling population presenting highly aberrant activity of proliferative MRs (i.e. TOP2A, CENPF, FOXM1, E2F8, ZWINT, and CCNA2), suggesting this as a key DMG regulatory module (Tumor Checkpoint). We are working to define targetable MRs in these subpopulations, and generating RNA-seq profiles of SU-DIPG-VI and SF8628, two DMG cell lines showing protein activity similarity to >95% of patient samples by enrichment analysis (p-val < 10E-5), following perturbation with ~400 oncology drugs. This will allow us to identify drugs capable of inducing tumor demise by inverting the activity of the MRs of each tumor subpopulation, using the NY Dept. of Health approved OncoTreat algorithm (Alvarez et al., Nat Genet 2018). We are currently finalizing and validating both MR and drug predictions to nominate novel, much-needed therapeutic strategies. Citation Format: Ester Calvo Fernández, Junqiang Wang, Aaron T. Griffin, Luca Szalontay, Stergios Zacharoulis, Jovana Pavisic, Andrea Califano. A systems biology approach to defining tumor heterogeneity, prognostic and targetable master regulator protein signatures from bulk and single cell RNA-seq in diffuse midline glioma (DMG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 486.

Diffuse Midline Glioma with H3K27M mutation (DMG) is an aggressive unresectable central nervous system tumor of the brainstem, midline thalamus, or spine. Prognosis is poor and systemic agents have been ineffective partially due to limited permeability of the blood brain barrier (BBB). Non-invasive low intensity focused ultrasound (FUS) can be used for BBB opening (BBBO). Our preclinical studies showed safety and feasibility of targeting and BBBO in the brainstem; however, to our knowledge, FUS-guided BBBO for thalamic DMG has yet to be reported. RNA-seq was performed on mouse syngeneic DMG cells 4423 (PDGFB+, H3.3K27M, p53−/−) and results recapitulated molecular programs seen in human tumors. 4423 cells were injected into the thalamus of male B6 (Cg)-Tyrc-2J/J mice at 1.6mm lateral and 1.8mm posterior to the bregma at a depth of 3.2mm. MRI at 14-21 days post injection confirmed thalamic tumor growth. Histological analysis with Hematoxylin and Eosin stain was consistent with thalamic DMG. Tumor implantation rate was 85% and median survival was 38 days post injection. To test safety and feasibility of BBBO for thalamic tumors, a 1.5 MHz FUS transducer was used with concurrent microbubble injection. BBBO assessed by contrast-enhanced MRI. FUS achieved BBBO targeting the thalamic tumor without increased morbidity or mortality and BBB closure was observed on day 3 post-sonication. Next we tested the tolerance of drug delivery with panobinostat post-FUS at a dose of 20mg/kg weekly. The results are preliminary at this time; however, animals tolerated the combination therapy without morbidity or mortality. Preclinical models are crucial to improve the development of new therapeutic strategies. Establishing this syngeneic thalamic DMG murine model provides the opportunity to test FUS as a non-invasive drug delivery technology for thalamic DMG compared to brainstem DMG, and to re-visit therapeutic agents previously considered ineffective due to limited penetration of the BBB.

Histone deacetylase inhibitors have been found preclinically to be among the most active agents against DMGs, however, they are clinically ineffective with systemic delivery due to blood brain barrier limitations and toxicity. Using a repurposed device (implantable subcutaneous pump connected with a catheter directly implanted into the pons/thalamus) we are performing a phase I, standard 3 + 3 dose escalation study to investigate the safety and feasibility of repeated infusions of MTX110 (Midatech Pharma), a water-soluble formulation of panobinostat, via CED. Eligible patents are between 3 and 18 years of age with newly diagnosed DMG following radiation therapy, without hemorrhage or cyst in the tumor, and having intact organ function. Following tumor biopsy and device implantation, patients receive two 48-hour-infusion pulses 7 days apart of MTX110 (30, 60, or 90 mM). The infusion pump is prefilled with MTX110 (and gadolinium for co-infusion to serve as a surrogate for drug distribution) and administered using the wireless N’Vision clinical programmer at a rate of 0.2 mL/hr. Seven patients (30 mM group, n=3 and 60 mM group, n=4) have been treated with the MTX110 infusate. All but one patient had adequate tumor coverage as measured by co-infused gadolinium on MRI. One patient suffered a severe adverse event related to the infusion and tumor anatomy. Four patients had Grade 2 transient neurological deficits related to biopsy (n=1) and the infusion (n=3). In a follow up period of 12-22 months from diagnosis, progression free survival ranges from 8 to 20 months. With one objective response, 3 patients remain alive (2 without progression, both at 12 months, and 1 with progressive disease, at 22 months post diagnosis). Three patients are expected to be treated at 90 mM level. Using MTX110, we demonstrated the safety and feasibility of repeated drug infusion by CED in DMG patients.

Diffuse midline glioma (DMG) is a pediatric tumor with dismal prognosis. Systemic therapeutic strategies have been unsuccessful to date and radiotherapy (RT) remains the standard of care. A central impediment to systemic therapy is the blood-brain barrier (BBB), which precludes drug delivery to the tumor site. Focused ultrasound (FUS) with intravenous microbubbles can transiently and non-invasively circumvent the BBB to enhance drug delivery. Nevertheless, it remains unclear whether FUS is safe at the brainstem in combination with clinical doses of RT. In this study, we hypothesized that FUS-mediated BBB-opening (BBBO) is safe and feasible with 39 Gy RT. To establish a safety timeline, we administered FUS to the brainstem of nontumor bearing mice concurrent with or adjuvant to radiation; then, we validated our findings in a syngeneic orthotopic xenograft DMG model which received repeated sonication concurrent with RT. Male B6 (Cg)-Tyrc-2J/J albino mice received intracranial injection of 4423 mouse DMG cells (PDGFB+, H3.3K27M, p53−/−) at a location posterior and lateral to the lambda. A clinical RT dose of 39 Gy in 13 fractions was delivered to the brainstem with the Small Animal Radiation Research Platform (SARRP) or the XRAD-320 irradiator. FUS was administered with a 0.5 MHz transducer, and both BBBO and tumor volume were monitored with MRI. FUS-mediated BBBO in nontumor bearing mice receiving RT did not affect cardiorespiratory rate, motor function, and tissue integrity. Moreover, tumor bearing mice tolerated repeated brainstem BBBO concurrent with RT. 39 Gy over 13 fractions offered local control, although disease progression occurred in all animals approximately 3-4 weeks post-RT. Ultimately, repeated FUS-mediated BBB opening concurrent with RT is safe and feasible. In our brainstem DMG model, relapse occurs, making it ideal for future tests of combinatorial RT and FUS-mediated drug delivery.

Background Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain cancer with limited treatment options and poor survival. The delivery of systemic therapies in this disease is severely limited by the blood-brain barrier (BBB). Focused ultrasound combined with intravenous microbubbles (FUS+MB) can effectively open the BBB permitting the entry of drugs across the cerebrovasculature. Etoposide is a chemotherapy frequently used in pediatric oncology with well-established anti-tumor effects but limited efficacy when administered systemically in DIPG. Given that FUS+MB in DIPG is not well studied, our goal was to determine the feasibility of ultrasound-mediated BBB opening and etoposide delivery in a preclinical murine pontine glioma model. Methods A syngeneic, orthotopic model was established by stereotactic injection of PDGF-B+PTEN-/-p53-/- murine glioma cells into the pons of B6 albino mice. Mice were randomly divided into control (n=6) or FUS+MB groups (n=6). A single-element, spherical-segment FUS transducer (center frequency=1.5MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to sonicate the tumor and its margins on post-injection day 14. Immediately after treatment, 5 mg/kg of intraperitoneal etoposide was administered to all 12 mice. All animals underwent cardiac puncture and blood sampling, followed by transcardiac perfusion and brain harvesting. Liquid chromatography-mass spectometry was performed on both serum and tumor tissue to measure etoposide levels. Results Contrast-enhanced MRI demonstrated successful BBB opening in all FUS+MB mice. Compared to control (mean=20.98ng/g), etoposide concentration in the sonicated tumor tissue (mean=164.77ng/g) was nearly eight times greater. Lastly, the mean brain tumor–to-serum ratio was more than fivefold higher in the treated mice (1.50%) compared with the control mice (0.28%) (P Conclusions FUS+MB is hereby shown successful in BBB opening and enhanced delivery of etoposide in a preclinical pontine glioma model.

Purpose Glioblastoma (GBM) is a devastating disease. With the current treatment of surgery followed by chemoradiation, outcomes remain poor, with median survival of only 15 months and a 5-year survival rate of 6.8%. A challenge in treating GBM is the heterogeneous integrity of the blood-brain barrier (BBB), which limits the bioavailability of systemic therapies to the brain. There is a growing interest in enhancing drug delivery by opening the BBB with the use of focused ultrasound (FUS). We hypothesize that an FUS-mediated BBB opening can enhance the delivery of etoposide for a therapeutic benefit in GBM. Methods and Materials A murine glioma cell line (Pdgf+, Pten-/-, P53-/-) was orthotopically injected into B6(Cg)-Tyrc-2J/J mice to establish the syngeneic GBM model for this study. Animals were treated with FUS and microbubbles to open the BBB to enhance the delivery of systemic etoposide. Magnetic resonance (MR) imaging was used to evaluate the BBB opening and tumor progression. Liquid chromatography tandem mass spectrometry was used to measure etoposide concentrations in the intracranial tumors. Results The murine glioma cell line is sensitive to etoposide in vitro. MR imaging and passive cavitation detection demonstrate the safe and successful BBB opening with FUS. The combined treatment of an FUS-mediated BBB opening and etoposide decreased tumor growth by 45% and prolonged median overall survival by 6 days: an approximately 30% increase. The FUS-mediated BBB opening increased the brain tumor-to-serum ratio of etoposide by 3.5-fold and increased the etoposide concentration in brain tumor tissue by 8-fold compared with treatment without ultrasound. Conclusions The current study demonstrates that BBB opening with FUS increases intratumoral delivery of etoposide in the brain, resulting in local control and overall survival benefits.

The failure to develop effective therapies for paediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively map assess the extent to which this was present in these tumours through subclonal genomic analyses, and to determine whether distinct tumour subpopulations may interact to promote tumorigenesis by generating novel subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced cases revealed multiple tumour subclones, spatially and temporally co-existing in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations which we propose co-operate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in

Purpose/Objective(s) Diffuse intrinsic pontine glioma (DIPG) is the most lethal brain tumor in children with no effective treatment options to date. The prognosis remains poor with a median overall survival of less than 1 year. Recent in vitro drug screening studies found the histone deacetylase (HDAC) inhibitor, panobinostat, to be highly effective against DIPG. However, as panobinostat has poor blood-brain barrier (BBB) penetration, phase 1 clinical trials using systemic administration of panobinostat have been unsuccessful. Therefore, improved efficacy will require enhancement of delivery into the brain. Focused ultrasound (FUS), a non-ionizing acoustic form of radiation, has been shown to safely and non-invasively open the BBB, which can increase drug delivery. In this study, we hypothesize that FUS-mediated BBB opening (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG. Materials/Methods A syngeneic DIPG model was established by injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) intracranially. A single-element, spherical-segment FUS transducer (center frequency: 0.5 MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to perform BBBO. We then utilized magnetic resonance imaging to evaluate BBBO and tumor progression. Results We first demonstrated that FUS-mediated BBBO is safe and feasible in mice with DIPG tumors by MRI and passive cavitation detection. Our DIPG cell line was shown to be very sensitive to panobinostat in cell viability assay (IC50 = 0.012 μM). The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival. Conclusion The current results demonstrate that FUS could increase the treatment efficacy of panobinostat in a DIPG animal model due to the improved targeted dose of systemic panobinostat in the DIPG tumors. We believe these data establish a strong preclinical rationale to assess the combination of FUS-mediated BBB opening with panobinostat in pediatric patients with DIPG.

BackgroundDiffuse intrinsic pontine gliomas (DIPG’s) are immunologically inert tumors with a median survival of 9–15 months. Radiation therapy (RT) is the mainstay treatment for DIPG but is associated with immunodepletion of the tumor microenvironment (TME) at high dose ranges. FLASH, or ultra-fast dose rate RT, represents a novel ablative technique that may spare TME immune responses while decreasing tumor burden. Here, we present single-cell immune profiling of DIPG tumors treated with FLASH, conventional dose rate RT (CONV) or no RT (SHAM).MethodsMurine H3.3K27M mutant DIPG cells were stereotactically injected and tumor induction confirmed by magnetic resonance imaging (MRI) 15 days later. DIPG-bearing mice were randomly assigned to one of three treatment groups (n=4/group), FLASH, CONV or SHAM. A fourth group with no tumor (NML) was included as a negative biological control. A modified linear accelerator was used to deliver 15 Gy of electron RT to the brainstem at dose rates of 90 Gy/second and 2 Gy/minute, for the FLASH and CONV groups, respectively. Four days post-RT, mice brainstems were harvested, homogenized, stained for CD45 and tagged with a hashtag antibody specific to each group. CD45+ immune cells were isolated and sequenced using the 10X Genomics chromium single-cell 3’ platform. After processing and alignment of the reads using CellRanger with default parameters, the data was quality checked and filtered before hashtag demultiplexing, unsupervised clustering and downstream analysis was implemented following the Seurat R package. Differential expression evaluated based on the non-parametric Wilcoxon rank sum test. Key genes determine by an adjusted p value of < 0.05 based on bonferroni correction and |avg log2FC| > 0.8.ResultsPreliminary analysis identifies 15 clusters with distinct CD45 immune phenotypes (figure 1). Differential gene expression analysis by hashtag antibody (treatment group) reveals 14 clusters differentially expressing key genes, including 3 clusters upregulated in DIPG compared to NML, and 2 clusters upregulated in irradiated tumors compared to SHAM and NML (figure 2). Notably, analysis demonstrates an individual cluster upregulated in FLASH versus all other groups (p = 3.07E-171). Further deconvolution of specific immune phenotypes represented by each cluster is ongoing.Abstract 91 Figure 1tSNE plot based on clustering of RNA signatures, grouped by RNAAbstract 91 Figure 2tSNE plot based on clustering of RNA signatures, grouped by hashtag antibodyConclusionsOur preliminary analysis shows differential immune responses among DIPG tumors compared to NML. We also find several immune cell subsets that are unique to DIPG treated with CONV or FLASH compared to unirradiated samples. Most notably, we identify a single immune cell subset that is exclusive to FLASH alone, indicating that FLASH elicits a unique immune response in murine DIPG.

Children with relapsed brain tumors are less responsive to treatment. These children often receive therapies without having any robust predictive method of potential benefit. Acute slice culturing(ASC) is a methodology permitting freshly operated tumor to undergo a culturing process preserving the tumor’s micro-environment. With the current study, we investigated the feasibility of obtaining therapeutically meaningful data in a timely manner (3–5 days), performing direct drug testing and single cell sequencing using ASC. Previously, we have combined ex vivo slices of intact, patient-derived Glioblastoma tissue with single-cell RNA-seq for small-scale drug screening and assessment of patient and cell type-specific drug responses. We generated slices from preclinical mouse glioma models and surgical specimens from adult Glioblastoma patients, as well as from children with relapsed Ependymomas, Medulloblastomas, and Gliomas. We demonstrated that these acute slices preserved both the tumor heterogeneity and tumor microenvironment observed in single-cell RNA-seq of cells directly isolated from tumor tissue. Testing drug responses, we then treated tissue slices from the Glioblastoma mouse models and different patients with multiple drugs and combinations. This technique allowed us to identify drug-induced transcriptional responses in specific subpopulations of tumor cells, patient-specific drug sensitivities, and drug effects conserved in both mouse and human tumors. Preliminary data suggests that we can apply this procedure within 5–7 days and provide real-time drug screening/single cell sequencing ASC results to Recurrent/ Progressive pediatric Low-Grade Gliomas, High Grade Gliomas, Ependymomas and Medulloblastomas.

Convection-enhanced delivery (CED, the infusion of drugs under controlled pressure to the brain parenchyma via targeted micro-catheters, allows accurate anatomical targeting and delivery of higher (therapeutic) drug concentrations through clinically relevant volumes of brain tissue or tumor. Histone deacetylase inhibitors have been found in vitro to be the most active agents against Diffuse Midline Gliomas (DMGs) Using a novel device (implantable subcutaneous pump connected with catheter directly implanted into the pons/thalamus) we are performing a Phase I safety study of repeated infusions of MTX110 (MTX110, Midatech) in a dose escalation manner. Eligible patients include 3–18 years of age with newly diagnosed DMGs following radiation therapy without evidence of hemorrhage or cysts with intact organ function. Patients undergo a tumor biopsy and a single catheter (Spetzler lumbar shunt catheter, Integra, Plainsboro, NJ) is placed stereotactically into the geometric center of the tumor. A second catheter is inserted subcutaneously with the distal tubing connected to the infusion pump, (SynchroMed II (Medtronic)), also inserted subcutaneously. The infusion pump is prefilled with MTX110 and administered using wireless N’Vison Clinical programmer into two 24-hour infusions, consisting of 20 hours of drug infusions at 0.2mL/hr. The pulse is completed 7 days later. This is a dose escalation study with the infusate consisting of gadolinium and MTX110 (30, 60, or 90 microM). The study describing the first use in children of this device for direct-to-tumor drug delivery is open to recruitment (January 2020) and the preliminary data will be available for presentation by June 2020.

Diffuse intrinsic pontine glioma (DIPG) remains a fatal disease with no effective drugs to date. Mutation-based precision oncology approaches are limited by lack of targetable mutations and genetic heterogeneity. We leveraged systems biology methodologies to discover common targetable disease drivers—master regulator proteins (MRs)—in DIPG to expand treatment options. Using the metaVIPER algorithm, we interrogated an integrated low grade glioma and GBM gene regulatory network with 31 DIPG-gene expression signatures to identify tumor-specific MRs by differential expression of their transcriptional targets. Unsupervised clustering identified MR signatures of upregulated activity in RRM2/TOP2A in 13 patients, CD3D in 5 patients, and MMP7, TACSTD2, RAC2 and SLC15A1/SLC34A2 in individual patients, all of which can be targeted. Notably, intratumoral administration of etoposide by convection enhanced delivery was effective in murine proneural gliomas in which TOP2 was identified as a MR while RRM2—targetable by drugs such as cladribine—has been shown to be a positive regulator of glioma progression whose knock-down inhibits tumor growth. We also prioritized drugs by their ability to reverse MR-activity signatures using a large drug-perturbation database. Patients clustered by predicted drug sensitivities with distinct groups of tumors predicted to respond to proteasome inhibitors, Thiotepa or Volasertib all of which have early evidence in treating gliomas. We will refine this analysis in a multi-institutional study of >100 patient gene expression profiles to define MR signatures driving known biological/molecular disease subtypes, use DIPG cell lines recapitulating common MR architectures to optimize therapy prioritization, and validate our findings in vivo.

Parents of children diagnosed with brain tumors report high levels of stress at diagnosis and feelings of “being lost” on transition to outpatient follow-up care (Jackson AC, et al, 2007). Ssori is a Japanese form of free-style weaving that encourages people facing life-limiting challenges to discover inner strengths. We report our experience with Saori weaving with brain tumor patients and their families in a pediatric oncology outpatient clinic at a major university medical center. During 2019, we offered weaving sessions twice a week. We had a total of 151 encounters with hematology/oncology patients (age 5–18 years), siblings, or parents. Among these patients there were 20 with primary brain tumor diagnoses. Weaving was offered in the art therapy area of the clinic. After creating a fabric, the weavers had the opportunity to have their work sewn into functional objects, such as pillows, bags, purses, or healing pouches filled with beans that can be heated or cooled for comfort. Brain tumor patients readily engaged in weaving, despite various degrees of neurologic disability including hemiparesis or low vision. In the words of an 8 y/o weaver. “This is so cool. Daddy, can we always come when the weavers are here, so I can weave?” And from a mother: “This is great. She’s focused and busy!” Case studies, including a presentation of Legacy work, will be reported. In conclusion, Saori weaving can be an impactful intervention for childhood brain tumor patients and their families in an outpatient clinic setting.

Diffuse intrinsic pontine glioma (DIPG) is the lethal high-grade brain tumor in children with no effective treatment options to date. Despite excessive clinical trials, the prognosis remains poor, with a median overall survival (mOS) of less than 1 year. Genomic studies of DIPG tissue have identified highly recurrent mutations in genes encoding histone H3 resulting in the substitution of lysine to methionine at position 27 (K27M), which is found in approximately 80% of DIPG. Recent drug screening studies identified the histone deacetylase (HDAC) inhibitors panobinostat as a highly effective drug against DIPG in vitro. However, due to the poor Blood-Brain Barrier (BBB) penetration of systemic administration, to enhance the delivery of panobinostat to improve treatment efficacy is needed. Focused ultrasound (FUS) has been shown to be able to safely and non-invasively open BBB to enhance drug delivery. Hence, in this study, we hypothesize that FUS-mediated BBBO (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG. Herein we established the syngeneic DIPG model by intracranially injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) and used FUS and microbubbles to open BBB and enhance the panobinostat delivery. Magnetic resonance (MR) imaging was utilized to evaluate BBBO and tumor progression. We first demonstrated that FUS-mediated BBB-opening is safe and feasible to mice with DIPG tumors by MR imaging and passive cavitation detection. Moreover, this DIPG cell line is very sensitive to panobinostat in in vitro cytotoxicity assay. The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival. The current results demonstrated FUS could increase the treatment efficacy of panobinostat to DIPG animals may be due to the increase of targeted delivery of systemic panobinostat to DIPG tumors in brainstem.

Background The aetiology of primary central nervous system (CNS) tumours remains largely unknown, but their childhood peak points to perinatal parameters as tentative risk factors. In this meta-analysis, we opted to quantitatively synthesise published evidence on the association between birth anthropometrics and risk of primary CNS tumour. Methods Eligible studies were identified via systematic literature review; random-effects meta-analyses were conducted for the effect of birth weight and size-for-gestational-age on childhood and adult primary CNS tumours; subgroup, sensitivity, meta-regression and dose–response by birth weight category analyses were also performed. Results Forty-one articles, encompassing 53,167 CNS tumour cases, were eligible. Birth weight >4000 g was associated with increased risk of childhood CNS tumour (OR: 1.14, [1.08–1.20]; 22,330 cases). The risk was higher for astrocytoma (OR: 1.22, [1.13–1.31]; 7456 cases) and embryonal tumour (OR: 1.16, [1.04–1.29]; 3574 cases) and non-significant for ependymoma (OR: 1.12, [0.94–1.34]; 1374 cases). Increased odds for a CNS tumour were also noted among large-for-gestational-age children (OR: 1.12, [1.03–1.22]; 10,339 cases), whereas insufficient data for synthesis were identified for other birth anthropometrics. The findings remained robust across subgroup and sensitivity analyses controlling for several sources of bias, whereas no significant heterogeneity or publication bias were documented. The limited available evidence on adults (4 studies) did not reveal significant associations between increasing birth weight (500-g increment) and overall risk CNS tumour (OR: 0.99, [0.98–1.00]; 1091 cases) or glioma (OR: 1.03, [0.98–1.07]; 2052 cases). Conclusions This meta-analysis confirms a sizeable association of high birth weight, with childhood CNS tumour risk, particularly astrocytoma and embryonal tumour, which seems to be independent of gestational age. Further research is needed to explore underlying mechanisms, especially modifiable determinants of infant macrosomia, such as gestational diabetes.

Medulloblastoma (MB) is the most common high-grade primary brain malignancy in children and accounts for 1% of adult brain tumors. Previous studies have compared survival in pediatric and adult MB from the National Cancer Institute Surveillance Epidemiology and End Results (SEER) database finding no difference. However, diagnostic subgroup analyses are limited. We examined survival in children (age 0–19) and adults (20–79) coded as MB in the 2018 SEER database (2000–2016), using Kaplan Meier analysis, log-rank test and Cox proportional hazard ratios (HR) with 95% confidence intervals (CI).). MB in SEER-18 is defined as ICD-O-3 histology codes 9470–9474 (n=1,728). ICD 9473, supratentorial PNET (sPNET, n=97) is biologically distinct so was analyzed separately. 5-year survival for MB, excluding sPNET, was similar in children (n = 1,091, 75.3%) and adults (n= 488, 79.1%) (HR=0.97, CI: 0.79 – 1.17, p=0.50). Subtype analyses showed no survival difference comparing adults and children with desmoplastic nodular MB (n=222, p=0.09), large cell MB (n=73, p=0.46), or MB NOS (n=1330, p=0.10). In contrast, children with sPNET had improved survival (n=65, 72.3%) compared to adults (n=29, 51.7%) (HR = 2.0, CI: 1.10 – 3.92; p=0.02,). In conclusion, 2018 SEER data for MB continue to show no survival difference between adults and children, suggesting adult patients could appropriately be entered on pediatric MB treatment protocols. Further analyses of the 2018 data are ongoing adjusting for sex, race, and treatment. Comparison of adults and children with MB and sPNET will be re-evaluated using the new 2016 World Health Organization classification.

BACKGROUND Drug delivery remains a major obstacle in DIPG, as the blood brain barrier (BBB) limits the penetration of systemic therapies to the brainstem. Focused ultrasound (FUS) is an exciting new technology that, when combined with microbubbles, can open the BBB permitting the entry of drugs across the cerebrovasculature. Given that the utility of FUS in brainstem tumors remains unknown, the purpose of our study was to determine the safety and feasibility of this technique in a murine pontine glioma model. METHODS A syngeneic orthotopic model was established by stereotactic injection of PDGF-B+PTEN-/-p53-/- murine glioma cells (10,000/1ul) into the pons of B6 albino mice. A single-element, spherical-segment FUS transducer (center frequency=1.5MHz) driven by a function generator through a power amplifier (acoustic pressure=0.7MPa) was used with concurrent intravenous microbubble injection (FUS+MB) to sonicate the tumor on post-injection day 14. BBB opening was confirmed with gadolinium-enhanced MRI and Evans blue. Kondziela inverted screen (KIS) testing was completed to measure motor function. Mice were either immediately sacrificed for histopathological assessment or serially monitored for survival. RESULTS In mice treated with FUS (n=11), there was no measured deficit in KIS testing. Additionally, the degree of intra-tumoral hemorrhage and inflammation on H&E in control (n=5) and treated mice (n=5) was similar. Lastly, there was no difference in survival between the groups (control, n=6, median=26 days; FUS, n=6, median=25 days, p>0.05). CONCLUSION FUS+MB is a safe and feasible technique to open the BBB in a preclinical pontine glioma model.

e14529 Background: Medulloblastoma (MB) is a malignant neuroectodermal tumor accounting for 30% of pediatric and only 1% of adult brain tumors. In previous studies comparing survival in pediatric and adult MB from the National Cancer Institute Surveillance Epidemiology and End Results (SEER) database no difference has been found. However, diagnostic subgroup analyses have been limited. Methods: We examined survival in children (age 0-19) and adults (20-79) coded as MB in the 2018 SEER database (2000-2016). We used Kaplan Meier analysis, log-rank test and Cox proportional hazard ratios (HR) with 95% confidence intervals (CI). MB in SEER-18 is defined as ICD-O-3 histology codes 9470–9474 (n = 1,728). ICD 9473, supratentorial PNET (sPNET, n = 97) is biologically distinct and therefore it was analyzed separately. Results: We found that 5-year survival for MB, excluding sPNET, was similar in children (n = 1,091, 75.3%) and adults (n = 488, 79.1%) (HR = 0.97, CI: 0.79 – 1.17, p = 0.50). Furthermore, subtype analyses showed no survival difference comparing adults and children with desmoplastic nodular MB (n = 222, p = 0.09), large cell MB (n = 73, p = 0.46), or MB NOS (n = 1330, p = 0.10). Yet, children with sPNET had improved 5-year survival (n = 65, 72.3%) compared to adults (n = 29, 51.7%) (HR = 2.0, CI: 1.10 – 3.92; p = 0.02,). These findings indicate that while survival in patients with MB is similar across age groups, children with sPNET have improved outcomes. Conclusions: In summary, 2018 SEER data for MB continue to show no survival difference between adults and children, suggesting adult patients could appropriately be entered on pediatric MB treatment protocols. Further analyses of the 2018 data are ongoing adjusting for sex, race, and treatment (chemotherapy or radiation). For sPNET, the apparent improved outcomes for children merit further detailed investigation and will be re-evaluated using the new 2016 World Health Organization classification.

Atypical teratoid rhabdoid tumour (ATRT) is a malignant tumour of the central nervous system with a dismal prognosis. There is no consensus on optimal treatment and different multimodal strategies are currently being used in an attempt to improve outcomes. To evaluate the impact of high-dose chemotherapy followed by autologous stem-cell rescue (HD48 SCR), radiotherapy (RT) at first line, intrathecal chemotherapy (IT) and extent of surgical resection upon recurrence-free survival (RFS) and overall survival (OS). An online database search identified prospective and retrospective studies focused on the treatment of children and adolescents with newly diagnosed ATRT. Clinical, therapeutic and outcome data were extracted and an individual pooled data analysis was conducted. Out of 389 publications, 12 manuscripts were included in our review. Data from 332 patients were analysed. Median age at diagnosis was 37 months (range 1-231). HD-SCR, RT and IT had been administered to 28.6% (58/203), 49.6% (118/238) and 21% (65/310) of the patients, respectively. Gross total resection (GTR) had been achieved in 46.5% (152/327) of the cases. In the multivariate analysis, hazard ratios (95% Confidence Interval) for HD-SCR were: RFS-HR = 0.570 (0.357-0.910) p = 0.019, and OS-HR = 0.388 (0.214-0.704) p = 0.002; and for RT: RFS-HR = 0.551 (0.351-0.866) p = 0.01, and OS-HR = 0.393 (0.216-0.712) p = 0.002. IT and GTR were not significantly associated with improved RFS or OS in the multivariate analysis. In our pooled data review, HD-SCR and RT at first line were associated with improved outcomes in children and adolescents with newly diagnosed ATRT.

BACKGROUND: 5-aminolevulinic acid (5-ALA, Gliolan) use is well documented and established in the resection of adult high-grade gliomas. There is growing interest in its safety and usefulness in the paediatric populations. The main challenge in the latter is the diverse range of histopathological tumour types that can occur. We present the UK’s first case series of 5-ALA use in the paediatric population. METHODS: Parental consent was obtained for the 6 patients (age 1.6–15 years, median 6.5 years) to be given a dose of 20mg/kg of 5-ALA 3 hours pre-operatively. Intra-operatively the tumours were viewed under violet-blue light and the presence of fluorescence was recorded. Fluorescence status was compared with histopathological classification and grade. RESULTS: The case series included: a pilocytic astrocytoma (grade I), a pilomyxoid astrocytoma (II), a glioneuronal tumour (II), a ganglioglioma (II-III), an anaplastic ependymoma (III) and an anaplastic medulloblastoma (IV). Only the grade III ependymoma showed strong fluorescence with 5-ALA. The pilocytic astrocytoma (I) and pilomxyoid astrocytoma (II) showed moderate fluorescence and the ganglioglioma (II-III) showed weak fluorescence. No fluorescence was observed in the glioneuronal tumour (II) or the medulloblastoma (IV). No significant adverse drug reactions were recorded. CONCLUSION: This case series adds to the growing body of evidence demonstrating the safety of 5-ALA in the paediatric population. Our case series highlights the need for larger trials into the usefulness of 5-ALA in the different paediatric brain tumour types. KEYWORDS: 5-aminolevulinic, 5-ALA, Gliolan, Fluoresence, Paediatric, Child, Brain tumour

OBJECTIVE: Tectal plate ‘gliomas’ are uncommon, indolent lesions. Presentation is usually with hydrocephalus and diagnosis is typically made radiologically without histological confirmation. Long term data guiding management is lacking. METHOD: Retrospective review of all paediatric tectal plate lesions in our unit over the last 10 years. RESULTS: Fifteen patients (13 boys, 2 girls; mean age 8.9 years) were identified and included. Most presented with increased intracranial pressure. Fourteen patients (93%) had hydrocephalus at presentation. In 13 patients (87%) there was no progression of the lesion. CSF diversion was performed in 14 patients (93%). The initial intervention was endoscopic third ventriculostomy (ETV) in 12 (80%) and ventriculoperitoneal shunting in 3 (20%). Four patients (33%) were biopsied; 2 (17%) were WHO grade 1 pilocytic astrocytomas and 2 (17%) had non-diagnostic biopsies. Four patients (33%) received adjuvant therapy, two upfront (due to atypical radiological appearances) and 2 due to progression. Of the 4 patients receiving adjuvant therapy, 1 had chemotherapy only (Carboplatin and Vincristine), 1 had chemotherapy followed by proton beam therapy and 2 patients had focal radiation therapy (59.4 CGE). All patients were alive with stable disease at follow up (average 50.5 months; range 0–120 months). CONCLUSION: Our experience is in line with the published literature on the natural history of tectal plate ‘gliomas’ and supports the notion that these are low grade tumours that can be managed with cerebrospinal fluid diversion and surveillance imaging. Adjuvant therapy is reserved for progression and achieves long term control.

New molecularly targeted therapies are needed for childhood ependymoma. Angiogenesis and the PDGFR pathway could be potential therapeutic targets. This study aimed to screen ependymomas for the expression and clinicopathological correlates of angiogenic factors and potential therapeutic targets including VEGFR, endoglin (CD105), CD34, CD31, c-Kit, PDGFR-α and PDGFR-β. Immunohistochemistry for angiogenesis factors and PDGFR-α and β was performed in 24 archival tumor samples from children and adults treated for ependymoma at our institution. CD31 density, CD105 density and pericyte coverage index (PCI) were calculated. These findings were correlated with clinical outcome. VEGFR2 was overexpressed in tumor cells in only one out of 24 cases, but was found overexpressed in the vessels in 6 cases. PDGFR-α and β were found to be over-expressed in the ependymoma tumor cells in seven out of 24 cases (29.2 %). CD31 density, CD105 density and PCI did not correlate with expression of PDGFRs. Overexpression of PDGFR-α and β in tumor cells and overexpression of PDGFR-α in tumor endothelium had prognostic significance and this was maintained in multivariate analysis for overexpression of PDGFR-α in tumor cells (2 year progression free survival was 16.7 ± 15.2 for cases with overexpression of PDGFR-α in the tumor vs. 74.5 ± 15.2 for those with low/no expression, hazard ratio = 5.78, p = 0.04). A number of angiogenic factors are expressed in ependymoma tumor cells and tumor endothelium. Preliminary evidence suggests that the expression of PDGFRs could have a prognostic significance in ependymoma. This data suggests that PDGFRs should be further evaluated as targets using novel PDGFR inhibitors.

Tumor vasculature is a promising therapeutic target in glioblastoma. Imaging tumor blood flow may help assess the efficacy of anti-angiogenic treatments. We determined the clinical usefulness of stable xenon CT performed preoperatively in patients with glioblastoma. This is a prospective cohort study. We determined absolute tumor blood flow before surgery in 38 patients with glioblastoma using stable xenon CT. We also histologically examined tumor specimens obtained from surgery and quantified their vascularity (by CD31 and CD105 immunostain), necrosis (by hematoxylin and eosin stain), and the presence of neuronal processes (by neurofilament immunostain). According to the xenon CT blood flow map, there are 3 types of glioblastoma. Type I glioblastomas have unimodal high blood flow histograms; histologically there is little necrosis and vascular proliferation. Type II glioblastomas have unimodal low blood flow histograms; histologically there is prominent necrosis and vascular proliferation. We propose that in type II glioblastoma, the abnormal vessels induced by hypoxia are inefficient at promoting blood flow. Type III glioblastomas have multimodal blood flow histograms. Histologically there is significant neuronal tissue within the tumor. Patients with type III glioblastomas were more likely to develop a post-surgical deficit, consistent with the inclusion of normal tissue within the tumor. Preoperative measurement of absolute blood flow with stable xenon CT in patients with glioblastoma predicts key biological features of the tumor and may aid surgical planning.

Metronomic chemotherapy, which is defined by the frequent, repetitive administration of chemotherapeutic drugs at relatively low doses, and without prolonged drug-free break, is an emerging strategy to fight cancer. Initially thought to act by targeting tumor angiogenesis, additional mechanisms have been recently unveiled, and metronomic chemotherapy is now considered to represent a form of multitargeted therapy. Despite representing a genuine alternative for advanced and/or high-risk cancer therapy, the development of metronomic approaches in pediatric oncology is still in the early stage. The few numbers of large-scale state-of-the-art clinical trials, issues regarding terminology and the limited understanding of the complex and intertwined mechanisms of action of metronomic treatments have limited progress in this important field of research. On March 18 and 19, 2010, the 2nd International Workshop on Metronomic and Anti-Angiogenic Chemotherapy in Paediatric Oncology was held in Marseille, France, and brought together clinicians, basic scientists, physician-scientists, trainees, and students from all around the world. The main aim of this international meeting was to provide a unique forum to 1) reflect on the major advances that have been made in this field of research since its creation, 2) communicate results from the most recent clinical trials and preclinical studies, 3) discuss the current and future challenges of the field, and 4) set forth a solid framework for future collaborative biologic and clinical studies. The present report documents the main preclinical and clinical data that were presented in the keynote and best abstract sessions and delivers the key messages from the meeting.

Low grade gliomas (LGG) contribute to 50% of all central nervous tumors in children and 15% of all gliomas in adults. Temozolomide (TMZ) is an oral alkylating agent with activity in high and LGG. Various regimens of TMZ are currently in use. We attempted to assess the impact of different TMZ regimens on the treatment of LGG. A systematic review of the literature identified all the studies published in Pubmed, EMBASE and Cochrane databases which met the inclusion criteria. The primary outcome measure was the impact of different TMZ regimens on the 12 month progression-free survival (PFS) rates of patients diagnosed with progressive LGG. Secondary outcome measures looked at the ability of the three regimens to elicit an objective response and the associated toxicity. Statistical pooling and calculation of weighted mean average of each proportion (WMAP) was conducted using a random-effects model. 18 studies (736 patients) were analyzed. PFS at 12 months revealed a WMAP of 0.61 (95% CI 0.44-0.78) for regimen A, 0.59 (0.28-0.89) for regimen B, and 0.91 (95% CI 0.83-0.99) for regimen C (Regimen A--200 mg/m(2)/day for 5 days, repeated every 4 weeks; B--75 mg/m(2)/day for 21 days repeated every 4 weeks; C--75 mg/m(2)/day for 7 weeks with 4 weeks of every 11 weeks). In terms of objective response, WMAP were 0.19 (95% 0.13-0.25), 0.27 (95% CI 0.15-0.39) and 0.21 (95% CI 0.10-0.32) for regimen A, B, C respectively. When analyzing hematological toxicity, WMAPs were 0.14 (95% 0.11-0.18), 0.35 (0.14-0.56) and 0.23 (95% CI 0.03-0.43). The bulk of evidence originates from the standard 5 day/month regimen A but with a lack of comparative studies. Analysis revealed significant heterogeneity. Although there is possibly an indication that metronomic regimens of TMZ result in better PFS and response rate when compared to the conventional standard 5 day regimen, insufficient available data and study heterogeneity preclude any safe conclusions. Well designed randomized controlled clinical trials are needed to establish the efficacy of metronomic regimens of TMZ in LGGs.

BACKGROUND: The optimal management of young children with newly-diagnosed supratentorial ependymomas remains poorly-defined. We report outcomes of young children under 7 years old at diagnosis prospectively treated on three sequential multi-center trials of intensive chemotherapy with intent of avoiding irradiation. METHODS: Between 1991 and 2009, 14 children (median/mean ages of 34 months, range 3–74 months) were enrolled on “Head Start” (HS) trials (3 on HS-I, 3 on HS-II and 8 on HS-III). Induction chemotherapy was identical on HS-I and HS-II; on HS-III, this regimen was supplemented with high-dose systemic methotrexate in each cycle. Consolidation was uniform with a single cycle of marrow-ablative thiotepa/etoposide/carboplatin followed by autologous hematopoietic cell rescue. Irradiation was reserved for children >72 months old or with residual disease following Induction. Tumor pathology underwent central review. RESULTS: Ten children achieved initial gross total resections (GTRx) of tumor and 4 subtotal resections 2 of whom achieved GTRx following chemotherapy. Pathology revealed cellular ependymoma in 6 and anaplastic ependymoma in 8. There was 1 toxic death in Induction, on HS-I. Four children relapsed (3 on HS-II, 1 on HS-III), all locally, 3 expiring of disease despite focal irradiation, 20–72 months from initial diagnosis; the fourth continues recurrence-free after GTRx and focal irradiation. Seven patients survive without irradiation. CONCLUSIONS: Young children with supratentorial ependymomas have a good survival outcome when treated with irradiation-avoiding chemotherapy incorporating high-dose systemic methotrexate. However, it remains unclear if similar results could have been achieved with GTRx alone. The prognostic significance of molecular subtype is under evaluation.

Diffuse intrinsic pontine glioma (DIPG) is a lethal disease of middle childhood with few treatment options. The failure of chemotherapy is in part due to poor blood-brain barrier penetration. Convection enhanced delivery (CED) uses direct intraparenchymal infusion to distribute drug through target brain volumes. Valproic acid and carboplatin are readily compatible with CED and kill DIPG cells as monotherapies and with synergy when combined. These drugs were combined and translated into a two-day CED treatment schedule for treatment of DIPG on compassionate grounds using a long-term, implantable, drug delivery system (undergoing approval, Renishaw Plc). This device utilises a bone-anchored transcutaneous port for intermittent drug administration without the need for repeated surgery. 9 patients who had previously received radiotherapy were selected for treatment. Surgical implantation of the device was well-tolerated. A total of 27 cycles (54 infusions) of sodium valproate and carboplatin were delivered achieving infusion volumes between 3.6–5.6 mL per infusion. The most common acute infusion-related side-effects included headache and mild limb weakness and all patients were discharged within 24 hours of finishing treatment. We describe our preliminary experience as a prelude to a forthcoming clinical trial.

Primary intraspinal peripheral primitive neuroectodermal tumors (pPNETs) are extremely rare tumors with only seven reported cases in the literature. The histopathologic diagnosis of this tumor is complex and has led to a variety of treatment approaches. The distinction between central and peripheral type primary spinal cord PNETs has not always been made in the literature, leading to a paucity of data in this disease. We present here two young patients with primary intraspinal pPNET, their treatment and outcome. The first patient, a 27 year old male, presented with an intradural mass extending from L2 through L5, after multiple relapses, he is currently alive with disease after 72 months, the longest survival yet reported. The second patient, a 16 year old female, presented with an intradural mass at the cauda equina from L2 through L5, and is currently alive with responsive disease at 5 months after initial diagnosis. Here, we discuss the clinical course, the pathology and treatment for this disease and review the literature.

Pseudoprogression (PsP) is a treatment-related phenomenon which hinders response interpretation. Its prevalence and clinical impact have not been evaluated in children/adolescents. We assessed the characteristics, risk factors and prognosis of PsP in children/adolescents and young-adults diagnosed with non-brainstem high grade gliomas (HGG) and diffuse intrinsic pontine gliomas (DIPG). Patients aged 1-21 years diagnosed with HGG or DIPG between 1995 and 2012 who had completed radiotherapy were eligible. PsP was assessed according to study-specific criteria and correlated with first-line treatment, molecular biomarkers and survival. Ninety-one patients (47 HGG, 44 DIPG) were evaluable. Median age: 10 years (range, 2-20). Eleven episodes of PsP were observed in 10 patients (4 HGG, 6 DIPG). Rates of PsP: 8.5 % (HGG); 13.6 % (DIPG). Two episodes of PsP were based on clinical findings alone; nine episodes had concurrent radiological changes: increased size of lesions (n = 5), new focal enhancement (n = 4). Temozolomide, MGMT methylation or H3F3A mutations were not found to be associated with increased occurrence of PsP. For HGG, 1-year progression-free survival (PFS) was 41.9 % no-PsP versus 100 % PsP (p = 0.041); differences in 1-year overall survival (OS) were not significant. For DIPG, differences in 1-year PFS and OS were not statistically significant. Hazard ratio (95 %CI) of PsP for OS was 0.551 (0.168-1.803; p = 0.325) in HGG; and 0.308 (0.107-0.882; p = 0.028) in DIPG. PsP occurred in both pediatric HGG and DIPG patients at a comparable rate to adult HGG. PsP was associated with improved 1-yr PFS in HGG patients. PsP had a protective effect upon OS in DIPG patients.

Overexpression of human epidermal growth factor receptor (HER/EGFR) is associated with various tumors, including ependymomas. To investigate whether EGFR inhibition was of benefit in pediatric patients with recurrent ependymoma, a multi-center, randomized, open-label, phase 2 study of oral erlotinib versus oral etoposide was undertaken. Twenty-five patients were randomized to receive erlotinib 85 mg/m(2) daily or etoposide 50 mg/m(2)/day for 21 consecutive days followed by a 7-day rest period. Courses were repeated every 28 days. In the erlotinib arm, no patient achieved a complete, partial, or minor response, and only 2 (15.4 %) patients showed stable disease as their best response. In the etoposide arm, 2 patients (16.7 %) demonstrated partial responses, 1 (8.3 %) patient demonstrated a minor response, and 2 (16.7 %) showed prolonged stable disease, for a prolonged disease control rate of 41.7 %. Three patients received at least nine cycles of etoposide (range 9-24 cycles) before discontinuing at the request of the physician and/or family. Four patients who failed etoposide in this study received erlotinib in a companion single arm study; none had a response. The futility criteria were met at the second interim analysis, and both studies were discontinued. Pharmacokinetics of erlotinib were similar to previous observations in pediatric patients. Overall, erlotinib was well tolerated and safety was consistent with its established profile in adults. The overall risk-benefit profile does not support the use of erlotinib in pediatric patients with recurrent ependymoma, whereas single-agent etoposide appears to have efficacy in a subset of patients.

Pediatric high grade gliomas represent a heterogeneous group of tumors with poor prognoses despite the use of multimodal treatment. Very little progress has been made over the past decades in identifying efficacious therapeutic modalities against both high grade gliomas and diffuse brainstem gliomas in children. The degree of surgical resection is the most important clinical prognostic factor for children with high grade gliomas, and a complete resection should be attempted whenever feasible. The role of radiation therapy in the treatment of older children with high grade gliomas and diffuse brain stem gliomas is undisputed; however the benefit of using radiation for patients less than 6 years of age (with high grade gliomas) might be questionable. Despite the absence of solid evidence to support its use, chemotherapy is routinely used against these tumors. Currently temozolomide is being investigated due to its activity in adult trials and based on preliminary data regarding recurrent disease. A small subgroup of patients can be successfully treated with high dose chemotherapy followed by autologous stem cell rescue. Early trials using this modality in the past had been associated with high morbidity and mortality. High dose chemotherapy with autologous stem cell rescue in selected patients with minimal residual disease, angiogenesis inhibitors, radiosensitizers and other biological modifiers are being currently investigated in phase I/II trials.

Hepatocellular carcinoma is a leading cause of cancer death worldwide in both adult and pediatric patients. Despite many options, no ideal treatment exists for this highly malignant tumor, and management strategies have varied accordingly. Angiogenesis, the formation of new blood vessels, is an essential component of hepatocellular carcinoma biology. Innovative approaches such as targeting the nontransformed, less resistant, tumor-supporting endothelial cells are currently under investigation in hepatocellular carcinoma. This review will focus on the current knowledge of the pathophysiology of hepatocellular carcinoma angiogenesis, as well as the reported data with angiogenesis inhibitors against hepatocellular carcinoma.

BACKGROUND: Patients with advanced, metastatic sarcoma have a poor prognosis, and the overall benefit from the few standard-of-care therapeutics available is small. The rarity of this tumor, combined with the wide range of subtypes, leads to difficulties in conducting clinical trials. The authors previously reported the outcome of patients with a variety of common solid tumors who received treatment with drug regimens that were first tested in patient-derived xenografts using a proprietary method ("TumorGrafts"). METHODS: Tumors resected from 29 patients with sarcoma were implanted into immunodeficient mice to identify drug targets and drugs for clinical use. The results of drug sensitivity testing in the TumorGrafts were used to personalize cancer treatment. RESULTS: Of 29 implanted tumors, 22 (76%) successfully engrafted, permitting the identification of treatment regimens for these patients. Although 6 patients died before the completion of TumorGraft testing, a correlation between TumorGraft results and clinical outcome was observed in 13 of 16 (81%) of the remaining individuals. No patients progressed during the TumorGraft-predicted therapy. CONCLUSIONS: The current data support the use of the personalized TumorGraft model as an investigational platform for therapeutic decision-making that can guide treatment for rare tumors such as sarcomas. A randomized phase 3 trial versus physician's choice is warranted.

Angiogenesis plays a key role in glioblastoma biology and antiangiogenic agents are under clinical investigation with promising results. However, the angiogenic profiles of patients with glioblastoma and their clinical significance are not well understood. Here we characterize the serum angiogenic profile of patients with glioblastoma, and examine the prognostic significance of individual angiogenic factors. Serum samples from 36 patients with glioblastoma were collected on admission and simultaneously assayed for 48 angiogenic factors using protein microarrays. The data were analyzed using hierarchical cluster analysis. Vessel morphology was assessed histologically after immunostaining for the pan-endothelial marker CD31. Tumor samples were also immunostained for tissue inhibitor of metalloproteinase-1 (TIMP-1). Cluster analysis of the serum angiogenic profiles revealed 2 distinct subtypes of glioblastoma. The 2 subtypes had markedly different tumor microvessel densities. A low serum level of TIMP-1 was associated with significantly longer survival independent of patient age, performance status, or treatment. The serum angiogenic profile in patients with glioblastoma mirrors tumor biology and has prognostic value. Our data suggest the serum TIMP-1 level as an independent predictor of survival.

Introduction Overall prognosis for optic pathway glioma (OPG) in children is excellent. Little is known, however, about the effect of chemotherapy on visual outcome of these patients. Material and methods A systematic review of the literature was carried out to identify all studies published in PubMed, EMBASE and Cochrane Library between 1990 and 2008 reporting visual outcome in children with OPG after chemotherapy. Studies were included when they included 10 or more children with OPG, main treatment was chemotherapy and visual outcome was described. Cochrane guidelines for meta-analysis of non-randomised clinical trials were followed and levels of quality were assigned. Results Eighty-five potentially relevant publications were retrieved for detailed evaluation and only 8 were included. The review revealed 0 randomised controlled trials, 0 non-randomised controlled trials, 3 (37.5%) single-arm trials (1 multi-institutional and 2 single institution trials) and 5 (62.5%) retrospective series. All studies achieved a level of evidence of 4 according to CEBM classification (case-series and poor quality cohort and case-control studies). Only 25 of 174 children experienced improvement in vision after chemotherapy (14.4%), responses ranged from 0% to 45.5%. Vision was stable in 82 children (47.1%, range 27–100%). No study documented the duration of the visual response. Discussion Published studies on childhood low grade gliomas have not shown satisfactorily whether chemotherapy improves outcome of vision in children with OPG. Based on our systematic review it appears that treatment with chemotherapy does not improve resulting vision in the majority of children with OPG. The data available does not allow us to assess whether vision is stabilised sufficiently prior to treatment with radiotherapy.

Replying to: M. R. Dawson et al. , 10.1038/nature08254 (2009) Commenting on ref. 1, Dawson 2 claim that metastasis formation is independent of VEGFR1 activity, contradicting work by us and many others, including the original description of flt1TK-/- (VEGFR1-TK-/-) mice in the metastatic setting3. Contrasting the findings by Dawson et al.2, here we show that VEGFR1 knockdown in myelomonocytic cells eradicates micro- and macrometastases in a non-amputation/resection tumour model.