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Background Children ≤36 months with diffuse intrinsic pontine glioma (DIPG) have increased long-term survival (LTS, overall survival (OS) ≥24 months). Understanding distinguishing characteristics in this population is critical to improving outcomes. Methods Patients ≤36 months at diagnosis enrolled on the International DIPG Registry (IDIPGR) with central imaging confirmation were included. Presentation, clinical course, imaging, pathology and molecular findings were analyzed. Results Among 1183 patients in IDIPGR, 40 were eligible (median age: 29 months). Median OS was 15 months. Twelve patients (30%) were LTS, 3 (7.5%) very long-term survivors ≥5 years. Among 8 untreated patients, median OS was 2 months. Patients enrolled in the registry but excluded from our study by central radiology review or tissue diagnosis had median OS of 7 months. All but 1 LTS received radiation. Among 32 treated patients, 1-, 2-, 3-, and 5-year OS rates were 68.8%, 31.2%, 15.6% and 12.5%, respectively. LTS had longer duration of presenting symptoms (P = .018). No imaging features were predictive of outcome. Tissue and genomic data were available in 18 (45%) and 10 patients, respectively. Among 9 with known H3K27M status, 6 had a mutation. Conclusions Children ≤36 months demonstrated significantly more LTS, with an improved median OS of 15 months; 92% of LTS received radiation. Median OS in untreated children was 2 months, compared to 17 months for treated children. LTS had longer duration of symptoms. Excluded patients demonstrated a lower OS, contradicting the hypothesis that children ≤36 months with DIPG show improved outcomes due to misdiagnosis.

Background Diffuse intrinsic pontine glioma (DIPG) remains a clinico-radiologic diagnosis without routine tissue acquisition. Reliable imaging distinction between DIPG and other pontine tumors with potentially more favorable prognoses and treatment considerations is essential. Methods Cases submitted to the International DIPG registry (IDIPGR) with histopathologic and/or radiologic data were analyzed. Central imaging review was performed on diagnostic brain MRIs (if available) by two neuro-radiologists. Imaging features suggestive of alternative diagnoses included nonpontine origin, Results Among 286 patients with pathology from biopsy and/or autopsy, 23 (8%) had histologic diagnoses inconsistent with DIPG, most commonly nondiffuse low-grade gliomas and embryonal tumors. Among 569 patients with centrally-reviewed diagnostic MRIs, 40 (7%) were classified as non-DIPG, alternative diagnosis suspected. The combined analysis included 151 patients with both histopathology and centrally-reviewed MRI. Of 77 patients with imaging classified as characteristic of DIPG, 76 (99%) had histopathologic diagnoses consistent with DIPG (infiltrating grade II-IV gliomas). Of 57 patients classified as likely DIPG with some unusual imaging features, 55 (96%) had histopathologic diagnoses consistent with DIPG. Of 17 patients with imaging features suggestive of an alternative diagnosis, eight (47%) had histopathologic diagnoses inconsistent with DIPG (remaining patients were excluded due to nonpontine tumor origin). Association between central neuro-imaging review impression and histopathology was significant (p < 0.001), and central neuro-imaging impression was prognostic of overall survival. Conclusions The accuracy and important role of central neuro-imaging review in confirming the diagnosis of DIPG is demonstrated.

Abtract Background This study describes imaging features of diffuse intrinsic pontine glioma (DIPG) and correlates with overall survival (OS) and histone mutation status in the International DIPG Registry (IDIPGR). Methods Four hundred cases submitted to the IDIPGR with a local diagnosis of DIPG and baseline MRI were evaluated by consensus review of 2 neuroradiologists; 43 cases were excluded (inadequate imaging or alternative diagnoses). Agreement between reviewers, association with histone status, and univariable and multivariable analyses relative to OS were assessed. Results On univariable analysis imaging features significantly associated with worse OS included: extrapontine extension, larger size, enhancement, necrosis, diffusion restriction, and distant disease. On central review, 9.5% of patients were considered not to have DIPG. There was moderate mean agreement of MRI features between reviewers. On multivariable analysis, chemotherapy, age, and distant disease were predictors of OS. There was no difference in OS between wild-type and H3 mutated cases. The only imaging feature associated with histone status was the presence of ill-defined signal infiltrating pontine fibers. Conclusions Baseline imaging features are assessed in the IDIPGR. There was a 9.5% discordance in DIPG diagnosis between local and central review, demonstrating need for central imaging confirmation for prospective trials. Although several imaging features were significantly associated with OS (univariable), only age and distant disease were significant on multivariable analyses. There was limited association of imaging features with histone mutation status, although numbers are small and evaluation exploratory.

INTRODUCTION The role of diagnostic biopsy in diffuse intrinsic pontine glioma (DIPG) remains in question. Distinguishing radiographically between DIPG and other pontine tumors with more favorable prognosis and different therapy is critically important. METHODS Cases submitted to the International DIPG registry with histopathologic data were analyzed. Central imaging review was performed by two neuro-radiologists; all cases with imaging features or histopathology suggestive of alternative diagnoses were re-reviewed. Imaging features suggestive of alternative diagnoses included non-pontine origin, RESULTS Among 297 patients with pathology from biopsy and/or autopsy available, 27 (9%) had histologic diagnoses not consistent with DIPG, commonly embryonal tumors (n=9) and pilocytic astrocytomas (n=11). 163 patients had diagnostic MRI available for central neuroimaging review. Among 81 patients classified as characteristic of DIPG, 80 (99%) had histopathology consistent with DIPG (diffuse midline glioma, H3K27M-mutant, glioblastoma, anaplastic astrocytoma, diffuse astrocytoma). Among 63 patients classified as likely DIPG, but with unusual imaging features, 59 (94%) had histopathology consistent with DIPG. 19 patients had imaging features suggestive of another diagnosis, including 13 with non-pontine tumor origin; the remaining 6 all had histopathology not consistent with DIPG. Association between central imaging review and histopathology was significant (p CONCLUSIONS The important role and accuracy of central neuroimaging review in diagnosing or excluding DIPG is demonstrated. In patients with pontine tumors for which DIPG is felt unlikely radiographically, biopsy may be considered to guide diagnosis and treatment.

PURPOSE To review data from DIPG Registry patients recorded to have received a second course of radiation therapy (rRT). METHODS The International DIPG Registry was searched for patients with DIPG who were treated with a known dose of rRT. Doses of rRT, timing from initial diagnosis and primary radiation therapy (pRT), radiographic response to rRT and survival from diagnosis (OS) were evaluated. RESULTS Sixty (11.2%) of 535 Registry patients underwent rRT; dose was provided for 44 patients. Median (range) data from those 44 revealed that rRT was given at 12 (2–65) months from initial diagnosis of DIPG and at 9.6 (1–61) months from completion of pRT at a dose of 26.7 (1.8–74) Gy. After completion of rRT, MRI showed response, progression, stable disease or was not available in 19, 8, 3 and 14 patients, respectively. Median PFS and OS were 11 and 18.1 months, respectively. 475 Registry patients did not undergo rRT; their ages, duration of symptoms, and primary treatment with or without chemotherapy were not significantly different from the rRT cohort. Median PFS and OS for the non-rRT patients were 6.9 and 10 months, respectively. rRT patients were more likely to have had radiographic evidence of tumor necrosis at diagnosis than non-rRT patients. CONCLUSIONS Administration of rRT to patients with DIPG has been inconsistent with respect to timing and dose. Toxicity, response and quality of life data are incomplete, but survival appears to be lengthened with rRT. Prospective clinical trials will elucidate benefits and risks of rRT.

BACKGROUND: Pre-clinical determination of disease-specific activity, effective dosing, safety, pharmacokinetics, and CNS delivery can optimize clinical trial designs. The proteasome inhibitor, marizomib, together with the HDAC inhibitor, panobinostat, is active and synergistic in pre-clinical DIPG studies, with target concentrations of 20 and 100 nM, respectively. The adult maximum tolerated dose (MTD) for marizomib is 0.8 mg/m(2). We evaluated the safety, tolerability and pharmacokinetics in a non-human primate model, predictive of pediatric patients. METHODS: Marizomib was administered (10-minute intravenous infusion) at three dose levels: 0.02 (n=4), 0.04 (n=5), and 0.06 mg/kg (n=1), equivalent to human doses (HED) of 0.4, 0.8, and 1.1 mg/m(2), respectively. Marizomib (dose 0.04 mg/kg) was subsequently administered (n=4) 1-hr post-panobinostat (dose 1 mg/kg, HED 20 mg/m(2), p.o.). Drug concentrations were determined by LC-MS/MS using validated assays and PK parameters calculated via noncompartmental methods. RESULTS: Marizomib +/- panobinostat was tolerable with the exception of one animal (single agent marizomib, dose 0.06 mg/kg, HED= 1.2 mg/m(2)) that expired 12–20 hr post administration; no clear etiology was found at necropsy. Remaining adverse events were Gr 1, 2 with the exception of lymphocytopenia, Gr 3 (n=2). Marizomib demonstrated rapid plasma clearance (1.22–10.25 L/min), short plasma half-life (4.45–8.24 min), non-linear increase in AUC(inf), and no significant difference across dose levels. Conversely, in CSF, there was a trend toward increasing exposure with increasing dose. T(1/2) was longer in CSF than blood (18–25 min vs. 4–7 min, respectively). Comparing marizomib PK before and after panobinostat, t(1/2) and clearance were similar (mean 7.80 vs. 9.49 min, and 6.04 vs. 4.24 L/min, respectively); CSF AUC(inf) increased, 65.69 vs. 121.68 min*nM, respectively. CONCLUSIONS: Marizomib penetrates into the CNS and CSF exposure was higher after panobinostat. This combination warrants clinical evaluation in DIPG; correlation of results with preclinical findings is planned.

The Gift from a Child (GFAC) program was inspired by the dream of one child to donate his brain for research, recognizing the need to study tumor tissue collected at diagnosis, recurrence, and at the time of death. Founded by the Swifty Foundation in 2016, GFAC currently is comprised of five “Centers of Excellence” at institutions with expertise in pediatric neuro-oncology. Partnering with the Children’s Brain Tumor Network, the program’s mandate is twofold: make it possible for families to donate no matter where they live in the United States and make tissue available to scientists globally to empower discovery. In order to overcome barriers that have stifled postmortem collection in the past, GFAC has invested in Tissue Navigators - individuals at each center who coordinate all aspects of donation and communicate with families, medical providers, and laboratory scientists. In 2019 alone, GFAC coordinated 55 autopsy collections from multiple diagnosis. A key metric of the program is also capturing the global sharing and usage of each tissue sample, ensuring that tissue isn’t simply “banked” but is actively being actively used to help unravel tumor biology. To date, tissue has been used for genomic and molecular data generation, preclinical model development including cell lines and PDX models, and for novel drug screening. Together with Children’s Brain Tumor Network, the Gift from a Child program is helping to ensure the most precious gift that a family can make is used to accelerate the path to cures.

The Children’s Brain Tumor Network (formerly known as Children’s Brain Tumor Consortium- CBTTC) is a global organization pioneering a model of open-science medical research to improve treatment and discover cures. Started in 2011, our objective was to utilize a regulatory, agreement, and governance architecture to remove existing research barriers that slowed down the pace of research and collaboration. Our network now includes 17 institutions working together to empower research. As of December 2019, over 3,600 subjects have been enrolled resulting in collection of over 45,000 specimens. Clinical data collection is longitudinal and includes medical history, diagnosis, treatment, pathology slides and reports, radiology imaging and reports, and outcome data. The tissue is collected flash-frozen, in freezing media, and fresh for the generation of pre-clinical models including cell lines. Blood is collected from the subject, with blood or saliva collected from the parents for germline comparison. Additionally, the Children’s Brain Tumor Network- Pediatric Brain Tumor Atlas has generated 952 WGS and RNAseq, 221 proteomics, with annotated clinical data. All of this data, both generated raw and processed data, has been made available broadly to the scientific community via cloud-based platforms, including the Gabriella Miller Kids First Data Resource Portal, Cavatica, and PedCbioportal. As of January 2020, we have 45 approved biospecimen requests and 80 genomic/molecular data requests. In summary, the Children’s Brain Tumor Network’s goal is to accelerate the pace of discovery by providing resources and expanding the network of scientists working towards a cure.

Diffuse intrinsic pontine glioma (DIPG) is a diffusely infiltrating high-grade glioma that has a peak incidence from ages 6-8 and only a 9-12 month median survival. Like adult gliomas, DIPG exhibits a significant expansion of myeloid cells, with microglia/macrophages accounting for ~30% of the tumor cell mass (Caretti & Monje, 2014). In adult glioma, these glioma-associated macrophages have been implicated as supporting angiogenesis and tumor cell invasion, proliferation, and survival (Glass & Synowitz, 2014). However, genomic and epigenomic characterizations of adult and pediatric high-grade gliomas revealed striking differences in the molecular nature of these tumors (Sturm et al., 2014), and traditional chemotherapeutic approaches to adult glioblastoma are not effective in DIPG, suggesting these are unique diseases with distinct pathophysiology. Our data demonstrates that tumor-associated macrophages from primary DIPG samples exhibit gene expression signatures that are associated with pro-tumorigenic functions, but are also significantly less inflammatory than tumor-associated macrophages isolated from adult glioblastoma samples. Moreover, the cytokine secretion profiles of patient-derived DIPG cell cultures are dramatically less inflammatory when compared to patient-derived adult high-grade glioma cell cultures, and are more similar to the profile of human neural precursor cell cultures. This suggests that DIPG is less immunogenic than adult GBM, which may allow the tumor cells to evade detection and infiltrate healthy tissue. We additionally show that DIPG cells express CD200, a ligand that inhibits myeloid cell activation. Knockdown of DIPG expression of CD200 increases primary microglial cell phagocytosis in direct co-culture experiments, and also inhibits DIPG progression in in vivo xenograft models. Thus, CD200 may represent a mechanism by which DIPG evades normal immune responses to tumor growth. By understanding the DIPG-microglial interaction in molecular detail, we will gain insight into the pathophysiology of DIPG that could direct future targeted therapies.

BACKGROUND: Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the leading cause of brain tumor-related deaths in children. There are no effective treatments and median survival remains dismal. Genomics identified a mutation in the majority of DMGs, a lysine to methionine substitution (K27M) in histones 3.1 and 3.3, which causes changes in gene expression that promote gliomagenesis. Panobinostat, a multiple histone deacetylase (HDAC) inhibitor, was one of the most effective agents against patient-derived DIPG cell cultures and xenograft models in previous studies and is presently in clinical trial for DIPG. HDAC inhibition with panobinostat may also exhibit activity against H3K27M+ DMG of the thalamus and spinal cord. METHODS: Patient-derived thalamic and spinal cord H3K27M+ DMG cell cultures were treated with single agent panobinostat at a range of concentrations. Cell viability was evaluated using the CellTiter-Glo assay. Panobinostat was systemically administered to murine models of luciferase-expressing spinal cord H3K27M+ DMG. Response to panobinostat was evaluated with IVIS in vivo imaging. RESULTS: HDAC inhibition with panobinostat significantly decreases cell proliferation with an IC50 of 30 nM and 41 nM in the spinal cord and thalamic glioma patient-derived cell cultures respectively. Panobinostat slowed tumor growth in spinal cord glioma orthotopic xenografts compared to vehicle controls. CONCLUSION: This study suggests that HDAC inhibition with panobinostat may also be beneficial for thalamic and spinal cord H3K27M+ DMG.

BACKGROUND: Children

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor characterized by resistance to therapy. While much progress has been made in mapping the genetic and epigenetic landscapes of DIPG, this newfound knowledge has yet to translate into durable clinical benefit for patients. The recent identification and in vivo characterization of ‘tumor microtubes’, membranous extensions that physically connect neighboring cancer cells via gap junctions in adult astrocytoma (Winkler et al., 2015) suggests that a similar mechanism may operate in pediatric high-grade gliomas (pHGG) such as DIPG, potentially contributing to the observed resistance to therapy. Here, we interrogated primary human DIPG samples, patient-derived DIPG orthotopic xenografts and cell cultures for the presence of putative ‘tumor microtubes’ (TMs). Immunohistochemistry and high-resolution confocal microscopy revealed long filamentous structures connecting DIPG cells that exhibit microanatomical parameters consistent with the TMs described in adult astrocytoma. Neuroligin-3 (NLGN3), a critical microenvironmental determinant of glioma progression, induces expression of several genes implicated in the formation of TMs such as TTYH1 and GAP-43. We find that NLGN3 exposure augments the formation of TM-like structures when applied to DIPG cells in vitro. The role that DIPG cell interconnections via TMs play in DIPG resistance to therapy remains to be tested, but may represent a non-cell-intrinsic mechanism of resistance by which DIPG cells form a cooperative network to adapt and survive therapeutic interventions.

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brainstem tumor with a median survival of approximately 10 months. Despite advances in understanding the molecular origins of DIPG, translations to improvement in clinical outcomes have yet to materialize. To date, there has been little target exploration for immunotherapy applications in DIPG. Here we report substantial preclinical efficacy of chimeric antigen receptor (CAR)-bearing T cells targeting the disialoganglioside GD2 as an immunotherapy regimen in patient-derived orthotopic xenograft models of Histone 3 K27M (H3K27M) mutated DIPG. GD2 is highly and uniformly expressed in patient-derived H3K27M DIPG cultures, and in vitro assays demonstrate substantial target-dependent cytokine generation and target cell killing. H3 WT pediatric high-grade gliomas (HGGs), including one case diagnosed as DIPG, do not express significant levels of GD2, and the H3K27M mutation is associated with increased synthesis of ganglioside pathway synthesis enzymes, suggesting expression of the GD2 antigen is driven by H3K27M-induced transcriptional dysregulation. Single-dose systemic administration of GD2-directed CAR T cells in multiple orthotopic xenograft models of DIPG achieves potent and lasting antitumor efficacy within a 4 week period of administration compared to a CD19-directed CAR control cohort. Universal response was observed across multiple cohorts, and treatment-associated toxicity was transient and tolerated during the period of peak anti-tumor activity. If these results are predictive of human response, GD2-directed CAR T cell therapy could have a transformative impact upon DIPG outcomes.

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric cancer with limited therapeutic options. The majority of cases of DIPG exhibit a mutation in histone-3 (H3K27M) that results in oncogenic transcriptional aberrancies. We show here that DIPG is vulnerable to transcriptional disruption using bromodomain inhibition or CDK7 blockade. Targeting oncogenic transcription through either of these methods synergizes with HDAC inhibition and DIPG cells resistant to HDAC inhibitor therapy retain sensitivity to CDK7 blockade. Identification of super-enhancers in DIPG provides insights toward the cell of origin, highlighting oligodendroglial lineage genes, and reveals unexpected mechanisms mediating tumor viability and invasion, including potassium channel function and EPH receptor signaling. The findings presented demonstrate transcriptional vulnerabilities and elucidate previously unknown mechanisms of DIPG pathobiology.

CD47 is an anti-phagocytic cell surface protein mediating cancer cell evasion of phagocytosis by the innate immune system. Preclinical data suggest that CD47 is appears to be an indispensable means by which many cancer cells, including cancer stem cells, overcome intrinsic expression of their pro-phagocytic “eat me” signals. Blockade of CD47 with mAbs enables phagocytosis of cancer cells in vitro and in vivo. We have developed a novel humanized mAb (huCD47-Ab) that specifically binds CD47 and blocks it from interacting with its ligand, signal regulatory protein–α; (SIRPα), on phagocytic cells, resulting in the phagocytosis and elimination of cancer cells through their “eat me” signals. Normal cells generally do not express eat me signals, and are unaffected by CD47 blocking mAb. We observed expression of CD47 on aggressive pediatric tumors, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), Ewing's sarcoma, neuroblastoma, and several cancers of the central nervous system (CNS), including atypical tetroid rhabdoid tumor (ATRT), primitive neuroectodermal tumor (PNET), medulloblastoma (MB), and pediatric high-grade glioma (pHGG). In-vitro phagocytosis assays, of primary patient samples shows significant engulfment by human peripheral blood derived macrophages upon incubation with the HuCD47-Ab antibody as compared to control. Furthermore we observe potent anti-tumor efficacy and survival benefit in MB, pHGG, ATRT and PNET orthotopic xenografts models establishing the ability of the huCD47 antibody to across the blood brain barrier by the CD47 blocking antibody. Using a novel human neural stem cell/brain tumor co-engraftment orthotopic xenograft model we show specificity of the HuCD47 antibody to only tumor cells and not normal human cells. We further observe potent anti-tumor efficacy in non-CNS tumor (ALL, AML and Osteosarcoma) xenograft models as well. Furthermore we demonstrate that HuCD47-Ab can be administered safely to non-human primates at therapeutic serum levels. Pre-IND and IMPD meetings with the FDA in the US and the MHRA in the UK, have been held, and the clinical trials for adult patients with advanced stage malignancies will start in early 2014. After determining safety in these phase I trials, we initiate protocols for adult CNS tumors as part of expansion cohorts, and subsequently extend the trials to pediatric cancer patients, given manageable toxicity and signs of efficacy in the adult patients. This abstract is also presented as Poster A86. Citation Format: Siddhartha S. Mitra, Sharareh Gholamin, Jens-Peter Volkmer, Abdullah Feroze, Jie Liu, Achal Achrol, Lijuan Wang, Leanne Sayles, Michael Zhang, Kathleen Sakamoto, Michelle Monje-Deisseroth, Yoon-Jae Cho, Alejandro Sweet-Cordero, Ravi Majeti, Samuel Cheshier, Irving Weissman. Overcoming immune evasion in pediatric hematologic and solid tumor malignancies: A preclinical study using a humanized anti-CD47 antibody. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR12.

Introduction: Extensive molecular characterization of Brain tumors done in recent years has proven valuable for tumor classification, risk stratification and outcome prediction for current treatment modalities. However the current standard of care has not improved the prognosis and carries an increased risk of cognitive impairments for children with brain tumors. A characteristic feature of tumor progression and recurrence is its ability to evade the immune system. Our hypothesis is that by modulating the innate immune system we can enhance the ability of macrophages to ‘eat and kill’ brain cancer cells. Recent data suggests that the interaction between the cell surface antigen CD47 and its binding partner SIRPα is a mechanism by which non-solid and solid tumors can evade the innate immune system. To see if anti-CD47 therapy is a potential therapy in malignant pediatric brain tumors we first looked at CD47 expression in freshly isolated patient and postmortem samples from 4 different tumor types; Diffused Intrinsic Pontine Glioma, Medulloblastoma, Ependymoma and ATRTs. We tested the hypothesis that blocking CD47- SIRPα interaction induced phagocytosis in an in-vitro phagocytosis assay. We next established orthotopic xenografts models in immune compromised mice and treated them with anti-CD47 humanized antibody, which is currently being developed for clinical trials in hematopoietic and non-CNS malignancies. Result: CD47 expression was upregulated in all tumor types and was present in >90% of the cells in high grade tumors. Increased CD47 expression was observed in CD15+ and CD133+ putative cancer stem cell population. Blocking the CD47- SIRPα interaction increases tumor phagocytosis by macrophages in-vitro. Systemic treatment with anti-CD47 antibody significantly reduced tumor burden in an orthotopic xenograft setting. Conclusion: Anti-CD47 therapy is a viable and effective treatment modality for pediatric high grade brain tumors. Citation Format: Sharareh Gholamin, Siddhartha S. Mitra, Chase Elliott Richard, Achal Achrol, Doosik Kong, Jun Jae Shin, Michelle Monje-Deisseroth, Yoon-Jae Cho, Irving Weissman, Samuel H. Cheshier. Development of Anti-CD47 therapy for pediatric brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5218. doi:10.1158/1538-7445.AM2013-5218