Overcoming barriers to establishing autopsy procurement programs in pediatric patients with central nervous system tumors: a call to develop regional centers.

BACKGROUND: While autopsy-repository programs with a variety of pediatric central nervous system (CNS) tumor types are a critical resource for preclinical neuro-oncology research, few exist and there is no published guidance on how to develop one. The goal of this prospective Pediatric Brain Tumor Repository (PBTR) study was to develop such a program at Cincinnati Children's Hospital Medical Center (CCHMC) and then publish the quantitative and experiential data as a guide to support the development of similar programs. METHODS: Protocols and infrastructure were established-to educate oncologists and families, establish eligibility, obtain consent, address pre- and post-autopsy logistics (e.g., patient and tissue transportation), process and authenticate tissue samples, and collect and analyze data. RESULTS: Of the 129 pediatric CNS tumor patients at CCHMC who died between 2013 and 2018, 109 were eligible for our study. Of these, 74% (81 of 109) were approached for PBTR donation, and 68% (55 of 81) consented. In the final year of the study, approach and consent rates were 93% and 85%, respectively. Median time from death to autopsy (postmortem interval, PMI) was 10 h (range, 1.5-30). In the outpatient setting, PMI increased with distance (from the hospice/home where the patient died to CCHMC). In all patients, PMI appeared to be lower, when consent was obtained more than 24 h before death. CONCLUSIONS: Procurement of autopsy specimens need not be a barrier in neuro-oncology research. Regional centers, strict timing-of-consent, patient education, and dedicated staff are all needed to minimize PMI and, thereby, increase the value of the procured tissue for an array of basic and translational research applications.

A phase I/II study of ribociclib following radiation therapy in children with newly diagnosed diffuse intrinsic pontine glioma (DIPG).

PURPOSE: Cyclin-dependent kinase-retinoblastoma (CDK-RB) pathway is dysregulated in some diffuse intrinsic pontine gliomas (DIPG). We evaluated safety, feasibility, and early efficacy of the CDK4/6-inhibitor ribociclib, administered following radiotherapy in newly-diagnosed DIPG patients. METHODS: Following radiotherapy, eligible patients received ribociclib in 28-day cycles (350 mg/m2; 21 days on/7 days off). Feasibility endpoints included tolerability for at least 6 courses, and a less than 2-week delay in restarting therapy after 1 dose reduction. Early efficacy was measured by 1-year and median overall survival (OS). Patient/parent-by-proxy reported outcomes measurement information system (PROMIS) assessments were completed prospectively. RESULTS: The study included 10 evaluable patients, 9 DIPG and 1 diffuse midline glioma (DMG)-all 3.7 to 19.8 years of age. The median number of courses was 8 (range 3-14). Three patients required dose reduction for grade-4 neutropenia, and 1 discontinued therapy for hematological toxicity following course 4. The most common grade-3/4 toxicity was myelosuppression. After 2 courses, MRI evaluations in 4 patients revealed increased necrotic volume, associated with new neurological symptoms in 3 patients. The 1-year and median OS for DIPG was 89% and 16.1 months (range 10-30), respectively; the DMG patient died at 6 months post-diagnosis. Five patients donated brain tissue and tumor; 3 were RB+ . CONCLUSIONS: Ribociclib administered following radiotherapy is feasible in DIPG and DMG. Increased tumor necrosis may represent a treatment effect. These data warrant further prospective volumetric analyses of tumors with necrosis. Feasibility and stabilization findings support further investigation of ribociclib in combination therapies. TRIAL REGISTRATION: NCT02607124.

Intracranial growing teratoma syndrome (iGTS): an international case series and review of the literature.

PURPOSE: Intracranial growing teratoma syndrome (iGTS) is a rare phenomenon of paradoxical growth of a germ cell tumor (GCT) during treatment despite normalization of tumor markers. We sought to evaluate the frequency, clinical characteristics and outcome of iGTS in Western countries. METHODS: Pediatric patients from 22 North American and Australian institutions diagnosed with iGTS between 2000 and 2017 were retrospectively evaluated. RESULTS: From a total of 777 cases of central nervous system (CNS) GCT, 39 cases of iGTS were identified for an overall frequency of 5%. Pineal region was a more frequent location for iGTS as compared to cases of GCT without iGTS (p < 0.00001). In patients with an initial tissue diagnosis of GCT, immature teratoma was present in 50%. Serum AFP or ßhCG was detectable in 87% of patients (median values 66 ng/mL and 44 IU/L, respectively). iGTS occurred at a median of 2 months (range 0.5-32) from diagnosis, in the majority of patients. All patients underwent surgical resection, leading to gross total resection in 79%. Following surgery, all patients resumed adjuvant therapy or post treatment follow-up for GCT. At a median follow-up of 5.3 years (range 0.2-11.8), 37 (95%) of patients are alive, including 5 with stable residual mass. CONCLUSION: iGTS occurs in 5% of patients with GCT in Western countries. Tumors of the pineal region and GCT containing immature teratoma appear to be associated with a higher risk of developing iGTS. Complete surgical resection is the mainstay of treatment. Overall survival of patients developing iGTS remains favorable.

Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK.

The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis--two crucial arms of cancer growth--AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40's association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.

A pilot study of bevacizumab-based therapy in patients with newly diagnosed high-grade gliomas and diffuse intrinsic pontine gliomas.

Although bevacizumab has not proven effective in adults with newly diagnosed high-grade gliomas (HGG), feasibility in newly diagnosed children with diffuse intrinsic pontine gliomas (DIPG) or HGG has not been reported in a prospective study. In a safety and feasibility study, children and young adults with newly diagnosed HGG received radiotherapy (RT) with bevacizumab (10 mg/kg: days 22, 36) and temozolomide (75-90 mg/m(2)/day for 42 days) followed by bevacizumab (10 mg/kg, days 1, 15), irinotecan (125 mg/m(2), days 1, 15) and temozolomide (150 mg/m(2)/day days 1-5). DIPG patients did not receive temozolomide. Telomerase activity, quality of life (QOL), and functional outcomes were assessed. Among 27 eligible patients (15 DIPG, 12 HGG), median age 10 years (range 3-29 years), 6 discontinued therapy for toxicity: 2 during RT (grade 4 thrombocytopenia, grade 3 hepatotoxicity) and 4 during maintenance therapy (grade 3: thrombosis, hypertension, skin ulceration, and wound dehiscence). Commonest ≥grade 3 toxicities included lymphopenia, neutropenia and leukopenia. Grade 3 hypertension occurred in 2 patients. No intracranial hemorrhages occurred. For DIPG patients, median overall survival (OS) was 10.4 months. For HGG patients, 3-year progression free survival and OS were 33 % (SE ± 14 %) and 50 % (SE ± 14 %), respectively. All 3 tested tumor samples, demonstrated histone H3.3K27M (n = 2 DIPG) or G34R (n = 1 HGG) mutations. QOL scores improved over the course of therapy. A bevacizumab-based regimen is feasible and tolerable in newly diagnosed children and young adults with HGG and DIPG.

Pten deletion causes mTorc1-dependent ectopic neuroblast differentiation without causing uniform migration defects.

Neuronal precursors, generated throughout life in the subventricular zone, migrate through the rostral migratory stream to the olfactory bulb where they differentiate into interneurons. We found that the PI3K-Akt-mTorc1 pathway is selectively inactivated in migrating neuroblasts in the subventricular zone and rostral migratory stream, and activated when these cells reach the olfactory bulb. Postnatal deletion of Pten caused aberrant activation of the PI3K-Akt-mTorc1 pathway and an enlarged subventricular zone and rostral migratory stream. This expansion was caused by premature termination of migration and differentiation of neuroblasts and was rescued by inhibition of mTorc1. This phenotype is reminiscent of lamination defects caused by Pten deletion in developing brain that were previously described as defective migration. However, live imaging in acute slices showed that Pten deletion did not cause a uniform defect in the mechanics of directional neuroblast migration. Instead, a subpopulation of Pten-null neuroblasts showed minimal movement and altered morphology associated with differentiation, whereas the remainder showed unimpeded directional migration towards the olfactory bulb. Therefore, migration defects of Pten-null neurons might be secondary to ectopic differentiation.

MELK-dependent FOXM1 phosphorylation is essential for proliferation of glioma stem cells.

Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM.

Digital sorting of complex tissues for cell type-specific gene expression profiles.

BACKGROUND: Cellular heterogeneity is present in almost all gene expression profiles. However, transcriptome analysis of tissue specimens often ignores the cellular heterogeneity present in these samples. Standard deconvolution algorithms require prior knowledge of the cell type frequencies within a tissue or their in vitro expression profiles. Furthermore, these algorithms tend to report biased estimations. RESULTS: Here, we describe a Digital Sorting Algorithm (DSA) for extracting cell-type specific gene expression profiles from mixed tissue samples that is unbiased and does not require prior knowledge of cell type frequencies. CONCLUSIONS: The results suggest that DSA is a specific and sensitivity algorithm in gene expression profile deconvolution and will be useful in studying individual cell types of complex tissues.

Prox1 is required for granule cell maturation and intermediate progenitor maintenance during brain neurogenesis.

The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the ectopic expression of Prox1 induces premature differentiation of neural stem cells.

Disseminated Juvenile Xanthogranuloma with a Novel MYH9-FLT3 Fusion Presenting as a Blueberry Muffin Rash in a Neonate.

Juvenile xanthogranuloma (JXG) is a benign proliferative histiocytic disorder of the dendritic cell phenotype. It mostly presents in the pediatric age group as a solitary skin lesion. We describe a rare case of an infant born with disseminated JXG who presented with a blueberry muffin rash at birth. A term infant was noted to have multiple petechiae, purple nodules, and macules (1 mm-2 cm in diameter) and hepatosplenomegaly, at the time of birth. Further investigations revealed thrombocytopenia and direct hyperbilirubinemia and a magnetic resonance imaging showed scattered tiny foci of restricted diffusion in multiple areas of the brain. Patient received multiple platelet transfusions in the first few weeks with gradual improvement in thrombocytopenia. Ultimately, a biopsy of one of the lesions revealed the diagnosis of disseminated JXG with notable atypical features. Somatic mutation analysis showed a novel MYH9-FLT3 fusion, but a bone marrow biopsy was negative. The lesions faded over time, relative to patient's growth and normal neurodevelopment was noted at 18 months of age. JXG should be considered in the differentials of blueberry muffin rash in an infant. Although, JXG is mostly a self-limited condition, congenital disseminated JXG may be associated with significant morbidity and mortality.

Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.

It was previously reported that the ciliary epithelium (CE) of the mammalian eye contains a rare population of cells that could produce clonogenic self-renewing pigmented spheres in culture. Based on their ability to up-regulate genes found in retinal neurons, it was concluded that these sphere-forming cells were retinal stem cells. This conclusion raised the possibility that CE-derived retinal stem cells could help to restore vision in the millions of people worldwide who suffer from blindness associated with retinal degeneration. We report here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells. All of the cells in the CE-derived spheres, including the proliferating cells, had molecular, cellular, and morphological features of differentiated pigmented CE cells. These differentiated cells ectopically expressed nestin when exposed to growth factors and low levels of pan-neuronal markers such as beta-III-tubulin. Although the cells aberrantly expressed neuronal markers, they retained their pigmented CE cell morphology and failed to differentiate into retinal neurons in vitro or in vivo. Our results provide an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitor cell. More importantly, our findings highlight the importance of shifting the focus away from studies on CE-derived spheres for cell-based therapies to restore vision in the degenerating retina and improving techniques for using ES cells or retinal precursor cells.

Severe allergic reactions to thiol-based cytoprotective agents mesna and amifostine in a child with a supratentorial primitive neuroectodermal tumor.

Both 2-mercaptoethane sulfonate sodium (mesna) and amifostine's active metabolite WR-1065 are thiol-based cytoprotective agents that are critical components of high-dose chemotherapy regimens used to treat various cancers in both adults and children. This case report describes a patient with a supratentorial primitive neuroectodermal tumor who developed severe drug reactions to both mesna and amifostine/WR-1065, suggesting that the thiol component of these agents triggered the adverse reactions. This report highlights the clinical presentation of drug-induced hypersensitivity syndrome in the context of pediatric oncology and the supportive care measures that, if implemented rapidly, may diminish the reaction severity and allow successful completion of chemotherapy.

Rapid cell-cycle reentry and cell death after acute inactivation of the retinoblastoma gene product in postnatal cochlear hair cells.

Unlike lower vertebrates, mammals are unable to replace damaged mechanosensory hair cells (HCs) in the cochlea. Recently, ablation of the retinoblastoma protein (Rb) in undifferentiated mouse HC precursors was shown to cause cochlear HC proliferation and the generation of new HCs, raising the hope that inactivation of Rb in postmitotic HCs could trigger cell division and regenerate functional HCs postnatally. Here, we acutely inactivated Rb in nearly all cochlear HCs of newborn mice, using a newly developed HC-specific inducible Cre mouse line. Beginning 48 h after Rb deletion, approximately 40% of HCs were in the S and M phases of the cell cycle, demonstrating an overriding role for Rb in maintaining the quiescent state of postnatal HCs. Unlike Rb-null HC precursors, such HCs failed to undergo cell division and died rapidly. HC clusters were restricted to the less differentiated cochlear regions, consistent with differentiation-dependent roles of Rb. Moreover, outer HCs expressed the maturation marker prestin, suggesting an embryonic time window for Rb-dependent HC specification. We conclude that Rb plays essential and age-dependent roles during HC proliferation and differentiation, and, in contrast to previous hypotheses, cell death after forced cell-cycle reentry presents a major challenge for mammalian HC regeneration from residual postnatal HCs.

AMPK-Regulated Astrocytic Lactate Shuttle Plays a Non-Cell-Autonomous Role in Neuronal Survival.

Lactate is used as an energy source by producer cells or shuttled to neighboring cells and tissues. Both glucose and lactate fulfill the bioenergetic demand of neurons, the latter imported from astrocytes. The contribution of astrocytic lactate to neuronal bioenergetics and the mechanisms of astrocytic lactate production are incompletely understood. Through in vivo1H magnetic resonance spectroscopy, 13C glucose mass spectroscopy, and electroencephalographic and molecular studies, here we show that the energy sensor AMP activated protein kinase (AMPK) regulates neuronal survival in a non-cell-autonomous manner. Ampk-null mice are deficient in brain lactate and are seizure prone. Ampk deletion in astroglia, but not neurons, causes neuronal loss in both mammalian and fly brains. Mechanistically, astrocytic AMPK phosphorylated and destabilized thioredoxin-interacting protein (TXNIP), enabling expression and surface translocation of the glucose transporter GLUT1, glucose uptake, and lactate production. Ampk loss in astrocytes causes TXNIP hyperstability, GLUT1 misregulation, inadequate glucose metabolism, and neuronal loss.

IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

Induced Telomere Damage to Treat Telomerase Expressing Therapy-Resistant Pediatric Brain Tumors.

Brain tumors remain the leading cause of cancer-related deaths in children and often are associated with long-term sequelae among survivors of current therapies. Hence, there is an urgent need to identify actionable targets and to develop more effective therapies. Telomerase and telomeres play important roles in cancer, representing attractive therapeutic targets to treat children with poor-prognosis brain tumors such as diffuse intrinsic pontine glioma (DIPG), high-grade glioma (HGG), and high-risk medulloblastoma. We have previously shown that DIPG, HGG, and medulloblastoma frequently express telomerase activity. Here, we show that the telomerase-dependent incorporation of 6-thio-2'deoxyguanosine (6-thio-dG), a telomerase substrate precursor analogue, into telomeres leads to telomere dysfunction-induced foci (TIF) along with extensive genomic DNA damage, cell growth inhibition, and cell death of primary stem-like cells derived from patients with DIPG, HGG, and medulloblastoma. Importantly, the effect of 6-thio-dG is persistent even after drug withdrawal. Treatment with 6-thio-dG elicits a sequential activation of ATR and ATM pathways and induces G2-M arrest. In vivo treatment of mice bearing medulloblastoma xenografts with 6-thio-dG delays tumor growth and increases in-tumor TIFs and apoptosis. Furthermore, 6-thio-dG crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. Together, our findings suggest that 6-thio-dG is a promising novel approach to treat therapy-resistant telomerase-positive pediatric brain tumors. Mol Cancer Ther; 17(7); 1504-14. ©2018 AACR.

Combined mTOR and MEK inhibition is an effective therapy in a novel mouse model for angiosarcoma.

Angiosarcoma is an aggressive malignancy of vascular origin that occurs de novo or in the context of previous cancer therapy. Despite multi-modal aggressive treatment including surgical resection, chemotherapy, and radiation, five-year overall survival remains poor at 35%. Due to its rarity, little is known about its molecular pathology and clinical trials have been extremely difficult to conduct. Development of animal models for rare diseases like angiosarcoma is critical to improve our understanding of tumorigenesis and to test novel treatment regimens. A genetically engineered mouse model for angiosarcoma was generated by conditional deletion of Trp53, Pten, and Ptpn12 in endothelial cells. Tumors arising from these mice recapitulate the histology and molecular pathology of the human disease including hyperactivation of the PI3K/mTOR and MAPK signaling pathways. Treatment of tumor-bearing mice with mTOR or MEK inhibitors effectively inactivated signaling and resulted in reduced proliferation and elevated apoptosis leading to tumor regression. The effect of treatment on tumor growth was transient and proliferation was restored after a period of dormancy. However, combined inhibition of mTOR and MEK resulted in profound tumor regression which was sustained for the duration of treatment. These results suggest that angiosarcoma may be effectively treated by this drug combination. .

Author Correction: AMP kinase promotes glioblastoma bioenergetics and tumour growth.

In the version of this Article originally published, in ref. 34 the first author's name was spelled incorrectly. The correct reference is: Rodón, L. et al. Active CREB1 promotes a malignant TGFß2 autocrine loop in glioblastoma. Cancer Discov. 10, 1230-1241 (2014). This has now been amended in all online versions of the Article.

AMP kinase promotes glioblastoma bioenergetics and tumour growth.

Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.

Publisher Correction: AMP kinase promotes glioblastoma bioenergetics and tumour growth.

In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.