Incidence and survival characteristics of pediatric ganglioglioma from 2004 to 2018, with focus on infratentorial sites.

Background: Ganglioglioma (GG) is a slow-growing glioneuronal neoplasm, most frequently seen in the supratentorial location in older children and associated with epilepsy syndromes. GG is rare in the infratentorial location, hence we embarked upon analyzing the National Cancer Institute's (NCI) Survival, Epidemiology, and End Results (SEER) database to better evaluate GG outcomes by location in comparison to the broader pediatric low-grade glioma (pLGG) population. Methods: Pediatric patients diagnosed with GG and pLGG from 2004 to 2018 were included in the study. Their demographic, clinical, and survival characteristics were analyzed using SEER*Stat. Results: This study describes the largest cohort of pediatric GG, including 852 cases from year 2004 to 2018, with focus on infratentorial sites. Patients with brainstem GG or those with subtotally resected disease were identified as having higher risk of death. Conclusions: Our analysis highlights brainstem GG as a high-risk, poor-prognostic subgroup and elaborates on the incidence and survival characteristic of this lesser-known subgroup.

Germline BRCA2 pathogenic variants in pediatric ganglioglioma: Case report and review of the literature.

BACKGROUND: BRCA1 and BRCA2 are tumor suppressor genes associated with increased risk of breast and ovarian cancer in adulthood. Patients with germline pathogenic variants in these genes have also been reported to develop brain tumors, although it is unclear whether these syndromes are associated with significant increased risk of brain tumor formation. RESULTS: Here, we report a case of a child with germline BRCA2 pathogenic variant presenting with a symptomatic ganglioglioma. To our knowledge, this is the first such patient to be reported. We discuss prior cases of brain tumors in BRCA1/2 patients and evidence for a potential role for BRCA1/2 pathogenic variants in brain tumor formation. CONCLUSION: BRCA2 germline variants may increase the risk of developing some types of pediatric brain tumors, but further study is needed to determine its effect on low-grade glioma formation.

Dose-dependent seizure control with MEK inhibitor therapy for progressive glioma in a child with neurofibromatosis type 1.

BACKGROUND: Low-grade gliomas (LGGs) occurring in children can result in many different neurologic complications, including seizures. MEK inhibitors are increasingly being used to treat LGG, but their effect on associated neurologic symptoms has not been established. RESULTS: Here, we report a patient with neurofibromatosis type 1 (NF1), medically refractory epilepsy (MRE), and an extensive optic pathway glioma (OPG) who developed dose-dependent seizure control while being treated with selumetinib. Seizure frequency rebounded after dose reduction for cardiac toxicity, then improved, and finally ceased after restarting full dosing, allowing confidence in the cause of improvement. CONCLUSION: Selumetinib may have promise in epilepsy management in other children with NF1 or LGG.

R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells.

BACKGROUND: Loss of the Ras GTPase-activating protein neurofibromin promotes nervous system tumor pathogenesis in patients with neurofibromatosis type 1 (NF1). Neurofibromin loss potentially hyperactivates classic Ras (H-Ras, N-Ras, K-Ras), M-Ras, and R-Ras (R-Ras, R-Ras2/TC21) subfamily proteins. We have shown that classic Ras proteins promote proliferation and survival, but not migration, in malignant peripheral nerve sheath tumor (MPNST) cells. However, it is unclear whether R-Ras, R-Ras2 and M-Ras are expressed and hyperactivated in MPNSTs and, if so, whether they contribute to MPNST pathogenesis. We assessed the expression and activation of these proteins in MPNST cells and inhibited them to determine the effect this had on proliferation, migration, invasion, survival and the phosphoproteome. METHODS: NF1-associated (ST88-14, 90-8, NMS2, NMS-PC, S462, T265-2c) and sporadic (STS-26T, YST-1) MPNST lines were used. Cells were transfected with doxycycline-inducible vectors expressing either a pan-inhibitor of the R-Ras subfamily [dominant negative (DN) R-Ras] or enhanced green fluorescent protein (eGFP). Methodologies used included immunoblotting, immunocytochemistry, PCR, Transwell migration, 3H-thymidine incorporation, calcein cleavage assays and shRNA knockdowns. Proteins in cells with or without DN R-Ras expression were differentially labeled with SILAC and mass spectrometry was used to identify phosphoproteins and determine their relative quantities in the presence and absence of DN R-Ras. Validation of R-Ras and R-Ras2 action and R-Ras regulated networks was performed using genetic and/or pharmacologic approaches. RESULTS: R-Ras2 was uniformly expressed in MPNST cells, with R-Ras present in a major subset. Both proteins were activated in neurofibromin-null MPNST cells. Consistent with classical Ras inhibition, DN R-Ras and R-Ras2 knockdown inhibited proliferation. However, DN R-Ras inhibition impaired migration and invasion but not survival. Mass spectrometry-based phosphoproteomics identified thirteen protein networks distinctly regulated by DN R-Ras, including multiple networks regulating cellular movement and morphology. ROCK1 was a prominent mediator in these networks. DN R-Ras expression and RRAS and RRAS2 knockdown inhibited migration and ROCK1 phosphorylation; ROCK1 inhibition similarly impaired migration and invasion, altered cellular morphology and triggered the accumulation of large intracellular vesicles. CONCLUSIONS: R-Ras proteins function distinctly from classic Ras proteins by regulating distinct signaling pathways that promote MPNST tumorigenesis by mediating migration and invasion. Mutations of the NF1 gene potentially results in the activation of multiple Ras proteins, which are key regulators of many biologic effects. The protein encoded by the NF1 gene, neurofibromin, acts as an inhibitor of both classic Ras and R-Ras proteins; loss of neurofibromin could cause these Ras proteins to become persistently active, leading to the development of cancer. We have previously shown that three related Ras proteins (the classic Ras proteins) are highly activated in malignant peripheral nerve sheath tumor (MPNST) cells with neurofibromin loss and that they drive cancer cell proliferation and survival by activating multiple cellular signaling pathways. Here, we examined the expression, activation and action of R-Ras proteins in MPNST cells that have lost neurofibromin. Both R-Ras and R-Ras2 are expressed in MPNST cells and activated. Inhibition of R-Ras action inhibited proliferation, migration and invasion but not survival. We examined the activation of cytoplasmic signaling pathways in the presence and absence of R-Ras signaling and found that R-Ras proteins regulated 13 signaling pathways distinct from those regulated by classic Ras proteins. Closer study of an R-Ras regulated pathway containing the signaling protein ROCK1 showed that inhibition of either R-Ras, R-Ras2 or ROCK1 similarly impaired cellular migration and invasion and altered cellular morphology. Inhibition of R-Ras/R-Ras2 and ROCK1 signaling also triggered the accumulation of abnormal intracellular vesicles, indicating that these signaling molecules regulate the movement of proteins and other molecules in the cellular interior. Video Abstract.

Temporal, spatial, and genetic constraints contribute to the patterning and penetrance of murine neurofibromatosis-1 optic glioma.

BACKGROUND: Brain tumors are the most common solid tumors of childhood, but little is understood about the factors that influence their development. Pediatric low-grade gliomas in particular display unique temporal and spatial localization associated with different genetic mutations (eg, BRAF genomic alterations, mutations in the neurofibromatosis type 1 [NF1] gene) for reasons that remain unclear. NF1 low-grade gliomas typically arise in the optic pathway of young children as optic pathway gliomas (OPGs), likely from a cell of origin that resides within the third ventricular zone (TVZ). However, the factors that contribute to their distinct temporal patterning and penetrance have not been adequately explored. METHODS: TVZ neuroglial progenitor cells (NPCs) were analyzed over the course of mouse brain development. Progenitors isolated by fluorescence-activated cell sorting (FACS) were assessed for functional and molecular differences. The impact of different germline Nf1 mutations on TVZ NPC properties was analyzed using genetically engineered mice. RESULTS: We identify 3 individual factors that could each contribute to Nf1 optic glioma temporal patterning and penetrance. First, there are 3 functionally and molecularly distinct populations of mouse TVZ NPCs, one of which ("M" cells) exhibits the highest clonogenic incidence, proliferation, and abundance during embryogenesis. Second, TVZ NPC proliferation dramatically decreases after birth. Third, germline Nf1 mutations differentially increase TVZ NPC proliferation during embryogenesis. CONCLUSIONS: The unique temporal patterning and penetrance of Nf1 optic glioma reflects the combined effects of TVZ NPC population composition, time-dependent changes in progenitor proliferation, and the differential impact of the germline Nf1 mutation on TVZ NPC expansion.

Classic Ras Proteins Promote Proliferation and Survival via Distinct Phosphoproteome Alterations in Neurofibromin-Null Malignant Peripheral Nerve Sheath Tumor Cells.

Neurofibromin, the tumor suppressor encoded by the neurofibromatosis type 1 (NF1) gene, potentially suppresses the activation of H-Ras, N-Ras, and K-Ras. However, it is not known whether these classic Ras proteins are hyperactivated in NF1-null nerve sheath tumors, how they contribute to tumorigenesis, and what signaling pathways mediate their effects. Here we show that H-Ras, N-Ras, and K-Ras are coexpressed with their activators (guanine nucleotide exchange factors) in neurofibromin-null malignant peripheral nerve sheath tumor (MPNST) cells, and that all 3 Ras proteins are activated. Dominant negative (DN) H-Ras, a pan-inhibitor of the classic Ras family, inhibited MPNST proliferation and survival, but not migration. However, NF1-null MPNST cells were variably dependent on individual Ras proteins. In some lines, ablation of H-Ras, N-Ras, and/or K-Ras inhibited mitogenesis. In others, ablation of a single Ras protein had no effect on proliferation; in these lines, ablation of a single Ras protein resulted in compensatory increases in the activation and/or expression of other Ras proteins. Using mass spectrometry-based phosphoproteomics, we identified 7 signaling networks affecting morphology, proliferation, and survival that are regulated by DN H-Ras. Thus, neurofibromin loss activates multiple classic Ras proteins that promote proliferation and survival by regulating several distinct signaling cascades.

Improving outcomes for neurofibromatosis 1-associated brain tumors.

Children and adults with neurofibromatosis type 1 (NF1) are predisposed to developing CNS tumors, including optic pathway gliomas (OPGs), brainstem gliomas (BSGs) and high-grade gliomas. Although current first-line treatments for low-grade gliomas (OPGs and BSGs) may prevent further tumor growth, they rarely result in restoration of the associated visual or neurological deficits. The availability of accurate small-animal models of NF1-associated brain tumors has established tractable experimental platforms for the discovery and evaluation of promising therapeutic agents. On the basis of these preclinical studies, biologically targeted agents are now being evaluated in children with NF1-associated low-grade brain tumors. Collectively, these models have also begun to reveal potential neuroprotective and risk assessment strategies for this brain tumor-prone population.

Neuregulin-1 overexpression and Trp53 haploinsufficiency cooperatively promote de novo malignant peripheral nerve sheath tumor pathogenesis.

Malignant peripheral nerve sheath tumors (MPNSTs) are Schwann cell-derived malignancies that arise from plexiform neurofibromas in patients with mutation of the neurofibromin 1 (NF1) gene. We have shown that the growth factor neuregulin-1 (NRG1) also contributes to human neurofibroma and MPNST pathogenesis and that outbred C57BL/6J × SJL/J transgenic mice overexpressing NRG1 in Schwann cells (P0-GGFß3 mice) recapitulate the process of neurofibroma-MPNST progression. However, it is unclear whether NRG1 acts predominantly within NF1-regulated signaling cascades or instead activates other essential cascades that cooperate with NF1 loss to promote tumorigenesis. We now report that tumorigenesis is suppressed in inbred P0-GGFß3 mice on a C57BL/6J background. To determine whether NRG1 overexpression interacts with reduced Nf1 or Trp53 gene dosage to "unmask" tumorigenesis in these animals, we followed cohorts of inbred P0-GGFß3;Nf1+/−, P0-GGFß3;Trp53+/− and control (P0-GGFß3, Nf1+/− and Trp53+/−) mice for 1 year. We found no reduction in survival or tumors in control and P0-GGFß3;Nf1+/− mice. In contrast, P0-GGFß3;Trp53+/− mice died on average at 226 days, with MPNSTs present in 95 % of these mice. MPNSTs in inbred P0-GGFß3;Trp53+/− mice arose de novo from micro-MPNSTs that uniformly develop intraganglionically. These micro-MPNSTs are of lower grade (WHO grade II-III) than the major MPNSTs (WHO grade III-IV); array comparative genomic hybridization showed that lower grade MPNSTs also had fewer genomic abnormalities. Thus, P0-GGFß3;Trp53+/− mice represent a novel model of low- to high-grade MPNST progression. We further conclude that NRG1 promotes peripheral nervous system neoplasia predominantly via its effects on the signaling cascades affected by Nf1 loss.

4-Hydroxytamoxifen induces autophagic death through K-Ras degradation.

Tamoxifen is widely used to treat estrogen receptor-positive breast cancer. Recent findings that tamoxifen and its derivative 4-hydroxytamoxifen (OHT) can exert estrogen receptor-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in estrogen receptor-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNST) where estrogen is not involved. In this study, we gained insights into the estrogen receptor-independent cytotoxic effects of OHT by studying how it kills MPNST cells. Although caspases were activated following OHT treatment, caspase inhibition provided no protection from OHT-induced death. Rather, OHT-induced death in MPNST cells was associated with autophagic induction and attenuated by genetic inhibition of autophagic vacuole formation. Mechanistic investigations revealed that OHT stimulated autophagic degradation of K-Ras, which is critical for survival of MPNST cells. Similarly, we found that OHT induced K-Ras degradation in breast, colon, glioma, and pancreatic cancer cells. Our findings describe a novel mechanism of autophagic death triggered by OHT in tumor cells that may be more broadly useful clinically in cancer treatment.

Malignant peripheral nerve sheath tumor invasion requires aberrantly expressed EGF receptors and is variably enhanced by multiple EGF family ligands.

Aberrant epidermal growth factor receptor (EGFR) expression promotes the pathogenesis of malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1, but the mechanisms by which EGFR expression promotes MPNST pathogenesis are poorly understood. We hypothesized that inappropriately expressed EGFRs promote MPNST invasion and found that these kinases are concentrated in MPNST invadopodia in vitro. Epidermal growth factor receptor knockdown inhibited the migration of unstimulated MPNST cells in vitro, and exogenous EGF further enhanced MPNST migration in a substrate-specific manner, promoting migration on laminin and, to a lesser extent, collagen. In this setting, EGF acts as a chemotactic factor. We also found that the 7 known EGFR ligands (EGF, betacellulin, epiregulin, heparin-binding EGF, transforming growth factor-α [TGF-α], amphiregulin, and epigen) variably enhanced MPNST migration in a concentration-dependent manner, with TGF-α being particularly potent. With the exception of epigen, these factors similarly promoted the migration of nonneoplastic Schwann cells. Although transcripts encoding all 7 EGFR ligands were detected in human MPNST cells and tumor tissues, only TGF-α was consistently overexpressed and was found to colocalize with EGFR in situ. These data indicate that constitutive EGFR activation, potentially driven by autocrine or paracrine TGF-α signaling, promotes the aggressive invasive behavior characteristic of MPNSTs.

Transgenic mice overexpressing neuregulin-1 model neurofibroma-malignant peripheral nerve sheath tumor progression and implicate specific chromosomal copy number variations in tumorigenesis.

Patients with neurofibromatosis type 1 (NF1) develop benign plexiform neurofibromas that frequently progress to become malignant peripheral nerve sheath tumors (MPNSTs). A genetically engineered mouse model that accurately models plexiform neurofibroma-MPNST progression in humans would facilitate identification of somatic mutations driving this process. We previously reported that transgenic mice overexpressing the growth factor neuregulin-1 in Schwann cells (P(0)-GGFß3 mice) develop MPNSTs. To determine whether P(0)-GGFß3 mice accurately model human neurofibroma-MPNST progression, cohorts of these animals were monitored through death and were necropsied; 94% developed multiple neurofibromas, with 70% carrying smaller numbers of MPNSTs. Nascent MPNSTs were identified within neurofibromas, suggesting that these sarcomas arise from neurofibromas. Although neurofibromin expression was maintained, P(0)-GGFß3 MPNSTs exhibited Ras hyperactivation, as in human NF1-associated MPNSTs. P(0)-GGFß3 MPNSTs also exhibited abnormalities in the p16(INK4A)-cyclin D/CDK4-Rb and p19(ARF)-Mdm-p53 pathways, analogous to their human counterparts. Array comparative genomic hybridization (CGH) demonstrated reproducible chromosomal alterations in P(0)-GGFß3 MPNST cells (including universal chromosome 11 gains) and focal gains and losses affecting 39 neoplasia-associated genes (including Pten, Tpd52, Myc, Gli1, Xiap, and Bbc3/PUMA). Array comparative genomic hybridization also identified recurrent focal copy number variations affecting genes not previously linked to neurofibroma or MPNST pathogenesis. We conclude that P(0)-GGFß3 mice represent a robust model of neurofibroma-MPNST progression useful for identifying novel genes driving neurofibroma and MPNST pathogenesis.

Genetically engineered mouse models shed new light on the pathogenesis of neurofibromatosis type I-related neoplasms of the peripheral nervous system.

Neurofibromatosis type 1 (NF1), the most common genetic disorder affecting the human nervous system, is characterized by the development of multiple benign Schwann cell tumors in skin and large peripheral nerves. These neoplasms, which are termed dermal and plexiform neurofibromas respectively, have distinct clinical courses; of particular note, plexiform, but not dermal, neurofibromas often undergo malignant progression to form malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy occurring in NF1 patients. In recent years, a number of genetically engineered mouse models have been created to investigate the molecular mechanisms driving the pathogenesis of these tumors. These models have been designed to address key questions including: (1) whether NF1 loss in the Schwann cell lineage is essential for tumorigenesis; (2) what cell type(s) in the Schwann cell lineage gives rise to dermal neurofibromas, plexiform neurofibromas and MPNSTs; (3) how the tumor microenvironment contributes to neoplasia; (4) what additional mutations contribute to neurofibroma-MPNST progression; (5) what role different neurofibromin-regulated Ras proteins play in this process and (6) how dysregulated growth factor signaling facilitates PNS tumorigenesis. In this review, we summarize the major findings from each of these models and their limitations as well as how discrepancies between these models may be reconciled. We also discuss how information gleaned from these models can be synthesized to into a comprehensive model of tumor formation in peripheral nervous system and consider several of the major questions that remain unanswered about this process.

Tamoxifen inhibits malignant peripheral nerve sheath tumor growth in an estrogen receptor-independent manner.

Few therapeutic options are available for malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1 (NF1). Guided by clinical observations suggesting that some NF1-associated nerve sheath tumors are hormonally responsive, we hypothesized that the selective estrogen receptor (ER) modulator tamoxifen would inhibit MPNST tumorigenesis in vitro and in vivo. To test this hypothesis, we examined tamoxifen effects on MPNST cell proliferation and survival, MPNST xenograft growth, and the mechanism by which tamoxifen impeded these processes. We found that 1-5 µM 4-hydroxy-tamoxifen induced MPNST cell death, whereas 0.01-0.1 µM 4-hydroxy-tamoxifen inhibited mitogenesis. Dermal and plexiform neurofibromas, MPNSTs, and MPNST cell lines expressed ERß and G-protein-coupled ER-1 (GPER); MPNSTs also expressed estrogen biosynthetic enzymes. However, MPNST cells did not secrete 17ß-estradiol, exogenous 17ß-estradiol did not stimulate mitogenesis or rescue 4-hydroxy-tamoxifen effects on MPNST cells, and the steroidal antiestrogen ICI-182,780 did not mimic tamoxifen effects on MPNST cells. Further, ablation of ERß and GPER had no effect on MPNST proliferation, survival, or tamoxifen sensitivity, indicating that tamoxifen acts via an ER-independent mechanism. Consistent with this hypothesis, inhibitors of calmodulin (trifluoperazine, W-7), another known tamoxifen target, recapitulated 4-hydroxy-tamoxifen effects on MPNST cells. Tamoxifen was also effective in vivo, demonstrating potent antitumor activity in mice orthotopically xenografted with human MPNST cells. We conclude that 4-hydroxy-tamoxifen inhibits MPNST cell proliferation and survival via an ER-independent mechanism. The in vivo effectiveness of tamoxifen provides a rationale for clinical trials in cases of MPNSTs.