Pediatric Low-grade Astrocytomas Research Articles

Implications of BRAF V600E mutation in gliomas: Molecular considerations, prognostic value and treatment evolution.

Gliomas are molecularly heterogeneous brain tumors responsible for the most years of life lost by any cancer. High-grade gliomas have a poor prognosis and despite multimodal treatment including surgery, radiotherapy, and chemotherapy, exhibit a high recurrence rate. There is a need for new therapeutic approaches based on precision medicine informed by biomarker assessment and BRAF, a key regulator of MAPK signaling pathway, influencing cell differentiation, proliferation, migration and pro-tumorigenic activity, is emerging as a promising molecular target. V600E, is the most frequent BRAF alteration in gliomas, especially in pediatric low-grade astrocytomas, pleomorphic xanthoastrocytoma, papillary craniopharyngioma, epithelioid glioblastoma and ganglioglioma. The possible application of BRAF-targeted therapy in gliomas is continuously growing and there is preliminary evidence of prolonged disease control obtained by BRAF inhibitors in tumors harboring BRAF V600E mutation. The possibility of introducing targeted therapies into the treatment algorithm represents a paradigm shift for patients with BRAF V600E mutant recurrent high-grade and low-grade glioma and BRAF routine testing should be considered in clinical practice. The focus of this review is to summarize the molecular landscape of BRAF across glioma subtypes and the novel therapeutic strategies for BRAF V600E mutated tumors.

Clinical Relevance of BRAF V600E Mutation Status in Brain Tumors with a Focus on a Novel Management Algorithm.

The possible application of BRAF-targeted therapy in brain tumors is growing continuously. We have analyzed clinical strategies that address BRAF activation in primary brain tumors and verified current recommendations regarding screening for BRAF mutations. There is preliminary evidence for a range of positive responses in certain brain tumor types harboring the BRAF V600E mutation. National Comprehensive Cancer Network Guidelines for central nervous system cancers recommend screening for the BRAF V600E mutation in pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and ganglioglioma. We suggest additional testing in glioblastomas WHO grade IV below the age of 30 years, especially those with epithelioid features, papillary craniopharyngiomas, and pediatric low-grade astrocytomas. BRAF-targeted therapy should be limited to the setting of a clinical trial. If the patient harboring a V600E mutation does not qualify for a trial, multimodality treatment is recommended. Dual inhibition of both RAF and MEK is expected to provide more potent and durable effects than anti-BRAF monotherapy. First-generation RAF inhibitors should be avoided. Gain-of-function mutations of EGFR and KIAA fusions may compromise BRAF-targeted therapy. BRAF alterations that result in MAPK pathway activation are common events in several types of brain tumors. BRAF V600E mutation emerges as a promising molecular target. The proposed algorithm was designed to help oncologists to provide the best therapeutic options for brain tumor patients.

HGG-15. MAPK PATHWAY ACTIVATION IN K27M MUTATED GLIOMAS – ASSOCIATION WITH CLINICAL PARAMETERS AND SURVIVAL

AbstractK27M midline gliomas are defined by point mutations within the chromatin modifiers H3 and are represented as a new separate entity in the new version of the WHO classification for tumors of the central nervous system. These tumors arise in midline structures of the brain, the brain stem and the spinal cord. They occur in children and young adults and are usually associated with a bad clinical outcome. We and others previously reported Mitogen-activated protein kinase (MAPK) pathway activation via BRAF or FGFR1 hotspot mutations in single cases of K27M gliomas. In contrast to K27M mutations, MAPK activation is strongly associated with pediatric low-grade astrocytomas, that show a favorable survival. In the context of K27M glioma, the clinical significance of co-occuring BRAF or FGFR1 mutations remains unclear. We screened a cohort of patients with K27M mutated gliomas for BRAF or FGFR1 mutations. So far, we detected three cases with FGFR1 mutations (K656E and N546K) and two cases with BRAF V600E mutations. These cases showed a favorable survival in comparison to other K27M cases in our series and in comparison to previously reported patient cohorts of K27M glioma. Our results shed light on the frequency and clinical impact of MAPK pathway activation in K27M mutated gliomas with implications for targeted therapies and prognosis of patients.

A brain-penetrant RAF dimer antagonist for the noncanonical BRAF oncoprotein of pediatric low-grade astrocytomas.

Activating mutations or structural rearrangements in BRAF are identified in roughly 75% of all pediatric low-grade astrocytomas (PLGAs). However, first-generation RAF inhibitors approved for adult melanoma have poor blood-brain penetrance and are only effective on tumors that express the canonical BRAFV600E oncoprotein, which functions as a monomer. These drugs (type I antagonists that target the "DFG-in" conformation of the kinase) fail to block signaling via KIAA1549:BRAF, a truncation/fusion BRAF oncoprotein which functions as a dimer and is found in the most common form of PLGA. A panel of small molecule RAF inhibitors (including type II inhibitors, targeting the "DFG-out" conformation of the kinase) was screened for drugs showing efficacy on murine models of PLGA and on authentic human PLGA cells expressing KIAA1549:BRAF. We identify a type II RAF inhibitor that serves as an equipotent antagonist of BRAFV600E, KIAA1549:BRAF, and other noncanonical BRAF oncoproteins that function as dimers. This drug (MLN2480, also known as TAK-580) has good brain penetrance and is active on authentic human PLGA cells in brain organotypic cultures. MLN2480 may be an effective therapeutic for BRAF mutant pediatric astrocytomas.

Prechiasmatic transection of the optic nerve in optic nerve glioma: technical description and surgical outcome.

Optic pathway glioma (OPG) encompasses a spectrum of findings ranging from lesions confined to the optic nerve only, lesions affecting the optic chiasm and hypothalamus, and lesions with diffuse involvement of a large part of the optic pathway and neighboring structures. The majority of pediatric low-grade astrocytomas in the optic/chiasmatic region are typical pilocytic astrocytoma. The rest of them (10%) may be other gliomas such as fibrillary pilomyxoid astrocytoma (grade 2 WHO). The postsurgical local recurrence rate of 55 to 76% has been reported in some histological subtypes such as pilomyxoid astrocytoma (grade 2). Performing a prechiasmatic transection might offer a new surgical option to avoid further tumor growth toward the chiasm in the optic nerve glioma with predominantly orbital manifestations. In this retrospective study, four patients (three children, two without neurofibromatosis type 1 (NF1), and one with NF1 and one adult without NF1) with optic nerve glioma without involvement of the chiasm but blindness, disfiguring proptosis, and pain of the affected eye were included. The surgical approach was performed as a combined approach from pterional extradural and intradural. Without any exceptions, vision of the contralateral eye could be preserved and did not show any deterioration after surgery or during the follow-up time between 17 and 106months. Furthermore, in all patients, gross total tumor resection could be achieved. During follow-up observation in all patients, no further tumor progress or recurrences could be observed. None of the patients were treated postoperatively by radiotherapy or chemotherapy. Prechiasmatic transection of the optic nerve in optic nerve glioma without affecting the chiasm might offer a surgical treatment option to control tumor growth and to preserve vision of the contralateral eye.

343 Brominated Noscapine Inhibits the Growth of Pediatric Low Grade Astrocytomas

343 Brominated Noscapine Inhibits the Growth of Pediatric Low Grade Astrocytomas

Pathological and Molecular Advances in Pediatric Low-Grade Astrocytoma

Pediatric low-grade astrocytomas are the most common brain tumors in children. They can have similar microscopic and clinical features, making accurate diagnosis difficult. For patients whose tumors are in locations that do not permit full resection, or those with an intrinsically aggressive biology, more effective therapies are required. Until recently, little was known about the molecular changes that drive the initiation and growth of pilocytic and other low-grade astrocytomas beyond the association of a minority of cases, primarily in the optic nerve, with neurofibromatosis type 1. Over the past several years, a wide range of studies have implicated the BRAF oncogene and other members of this signaling cascade in the pathobiology of pediatric low-grade astrocytoma. In this review, we attempt to summarize this rapidly developing field and discuss the potential for translating our growing molecular knowledge into improved diagnostic and prognostic biomarkers and new targeted therapies.

Abstract B17: Mechanisms of resistance of BRAF fusions to first generation selective BRAF inhibitors

Abstract Astrocytomas are the most common type of central nervous system tumor in children. We and others have previously reported that activated BRAF mutations, via the KIAA1549-BRAF fusion gene, are a key feature of low-grade astrocytomas. We generated several different cell lines stably expressing various altered BRAF and BRAF-fusion constructs and evaluated first and second-generation BRAF inhibitors including PLX4720 and PLX-PB compounds. In vitro kinase assays demonstrated the KIAA1549-BRAF fusion kinase, like V600E BRAF, undergoes inhibition in the presence of both first and second-generation selective BRAF inhibitors. However, KIAA1549-BRAF fusion expressing cell lines were resistant to PLX4720 and displayed paradoxal activation to first-generation BRAF inhibitor suggesting distinct and cell-context dependent mechanisms for MAPK activation. In contrast, KIAA1549-BRAF-fusion-mediated MAPK activation was abrogated by PB-3, a second-generation, “paradox-breaking,” selective BRAF inhibitor. This suggests that pediatric low-grade astrocytomas harboring the KIAA1549-BRAF fusion may be responsive to second-generation BRAF inhibitors. Our current studies are focused on understanding how KIAA1549-BRAF fusion signaling is mechanistically different than canonical BRAF V600E mutant and how this impacts the differential responses to selective BRAF inhibition.

BRAF-KIAA1549 Fusion Predicts Better Clinical Outcome in Pediatric Low-Grade Astrocytoma

Recent studies have revealed that the majority of pediatric low-grade astrocytomas (PLGA) harbor the BRAF-KIAA1549 (B-K) fusion gene resulting in constitutive activation of the RAS/MAPK pathway. However, the clinical significance of this genetic alteration is yet to be determined. We aimed to test the prognostic role of the B-K fusion in progression of incompletely resected PLGA. We retrospectively identified 70 consecutive patients with incompletely resected "clinically relevant" PLGA. We added 76 tumors diagnosed at our institution between 1985 and 2010 as controls. We examined BRAF alterations by reverse transcriptase PCR, FISH, and single-nucleotide polymorphism array analysis and correlated that with progression-free survival (PFS). Overall, 60% of tumors were B-K fusion positive. All patients with B-K fused PLGA are still alive. Five-year PFS was 61% ± 8% and 18% ± 8% for fusion positive and negative patients, respectively (P = 0.0004). B-K fusion resulted in similarly significant favorable PFS for patients who received chemotherapy. Multivariate analysis revealed that B-K fusion was the most significant favorable prognostic factor in incompletely resected PLGA and was independent of location, pathology, and age. In vitro, BRAF overexpression resulted in growth arrest associated with DNA damage (γH2AX expression). Five-year PFS was 68% ± 15% and 0% for patients with B-K fused and γH2AX-expressing PLGA versus negative tumors (P = 0.001). These data suggest that B-K fusion confers a less aggressive clinical phenotype on PLGA and may explain their tendency to growth arrest. Combined analysis of B-K fusion and γH2AX expression can determine prognosis and may be a powerful tool to tailor therapy for these patients.

Targeting Ras-RAF-ERK and its Interactive Pathways as a Novel Therapy for Malignant Gliomas

Malignant gliomas are the most common and the deadliest brain malignancies in adults. Despite the lack of a complete understanding of the biology of these tumors, significant advances have been made in the past decades. One of the key discoveries made in the area of malignant gliomas is that these tumors can be induced and maintained by aberrant signaling networks. In this context, the Ras pathway has been extensively exploited, from both basic and translational perspectives. Although somatic oncogenic mutations of Ras genes are frequent in several cancer types, early investigations on gliomas revealed disappointing facts that the Ras mutations are nearly absent in malignant gliomas and that the BRAF mutations are present in a very small percentage of gliomas. Therefore, the observed deregulation of the Ras-RAF-ERK signaling pathway in gliomas is attributed to its upstream positive regulators, including, EGFR and PDGFR known to be highly active in the majority of malignant gliomas. In contrast to the initial negative results on the somatic mutations of H-Ras, K-Ras and BRAF, recent breakthrough studies on pediatric low-grade astrocytomas uncovered genetic alterations of the BRAF gene involving copy number gains and rearrangements. The 7q34 rearrangements result in a novel in-frame KIAA1549:BRAF fusion gene that possesses constitutive BRAF kinase activity resembling oncogenic BRAF (V600E). In light of the earlier findings and recent breakthroughs, this review summarizes our current understanding of the Ras-RAF-ERK signaling pathway in gliomas and the outcome of preclinical and clinical studies that evaluated the efficacy of Ras-targeted therapy in malignant gliomas.

Duplication of 7q34 in pediatric low-grade astrocytomas detected by high-density single-nucleotide polymorphism-based genotype arrays results in a novel BRAF fusion gene.

In the present study, DNA from 28 pediatric low-grade astrocytomas was analyzed using Illumina HumanHap550K single-nucleotide polymorphism oligonucleotide arrays. A novel duplication in chromosome band 7q34 was identified in 17 of 22 juvenile pilocytic astrocytomas and three of six fibrillary astrocytomas. The 7q34 duplication spans 2.6 Mb of genomic sequence and contains approximately 20 genes, including two candidate tumor genes, HIPK2 and BRAF. There were no abnormalities in HIPK2, and analysis of two mutation hot-spots in BRAF revealed a V600E mutation in only one tumor without the duplication. Fluorescence in situ hybridization confirmed the 7q34 copy number change and was suggestive of a tandem duplication. Reverse transcription polymerase chain reaction-based sequencing revealed a fusion product between KIAA1549 and BRAF. The predicted fusion product includes the BRAF kinase domain and lacks the auto-inhibitory N-terminus. Western blot analysis revealed phosphorylated mitogen-activated protein kinase (MAPK) protein in tumors with the duplication, consistent with BRAF-induced activation of the pathway. Further studies are required to determine the role of this fusion gene in downstream MAPK signaling and its role in development of pediatric low-grade astrocytomas.

BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas

The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.

A mathematical model for self-limiting brain tumors

It is puzzling that certain brain tumors exhibit arrested exponential growth. We have observed in pediatric low-grade astrocytomas (LGA) at a certain volume ≈100– 150 cm 3 that the tumor ceases to grow. This observation led us to develop a macroscopic mathematical model for LGA growth kinetics that assumes the flow through the surface of the astrocytoma of a triggering agent or “promoter” that is uniformly distributed throughout the tumor, thereby providing relatively homogeneous growth. The model relates the transport of the promoter by the electrochemical potential associated with the tumor and diffusion effects through the surface of the growth and its consumption throughout the tumor volume via a pair of ordinary differential equations. The model assumes a constant growth rate, if the promoter density is above some threshold, and is zero otherwise. We also develop equations for an electrochemical (Nernst) transport mechanism for the promoter, and describes the microscopic basis for the macroscopic evolution to the equilibrium state at a well-defined and universal size. The latter description is unstable to asymmetric perturbations and provides a “star-like” shape for emergent tumors and a spheroidal shape for fully developed ones. The underlying assumption in our hypothesis would also result in tumor growth remission beginning from the periphery and proceeding inwards, a feature that has now been validated clinically.

Cytogenetics in pediatric low-grade astrocytomas.

Cytogenetic analysis in certain tumors is a vital part of classification and assignment of prognosis. Few studies have examined the value of cytogenetic analysis in pediatric brain tumors. This is especially true of low-grade astrocytomas (LGA) of childhood. This study examines the correlation between cytogenetic abnormalities and survival in children with low-grade astrocytomas. The literature on adults with LGA suggest better survival for those whose tumors have normal cytogenetics compared to those with abnormal. We hypothesized this would also be true of children with low-grade astrocytomas. A retrospective study was performed of children presenting between 1980 and 1998 to The Children's Hospital, Denver, who had LGA and on whose tumors informative cytogenetics had obtained. One hundred and forty-nine children were diagnosed with histologically proven LGA. Twenty-nine had successful cytogenetic analysis. One or more chromosomal abnormalities were observed in eight tumors while normal karyotypes were observed in 21 tumors. Actuarial progression-free survival at 5 years was 87.5% for the eight children with abnormal cytogenetics and 43% for those with normal (P=0.56). Overall survival at 5 years was 83% for those with abnormal cytogenetics and 78% for those with normal (P=0.8). The differences in progression-free survival and overall survival between these two groups were not significant. Those children with WHO Grade I tumors had significantly superior progression-free and overall survival than those with Grade II tumors. It appears unlikely that, for children with LGA, those with normal cytogenetics have a better prognosis than those with abnormal. Histologic grade is a better predictor of outcome than cytogenetics.

Low-grade astrocytoma: a decade of experience at St. Jude Children's Research Hospital.

To evaluate the impact of primary tumor site, age at diagnosis, extent of resection, and histology on progression-free survival (PFS) in pediatric low-grade astrocytoma. Medical, pathologic, and imaging information were reviewed for 142 children (ages 2 months to 19 years) with low-grade astrocytoma treated between January 1984 and July 1994. Gross total resection (GTR) was attempted for cerebellar and cerebral hemisphere tumors, with biopsy or less aggressive resection used predominantly for tumors in other sites. Surgery was followed by observation in 107 cases, radiation therapy in 31, and chemotherapy in four. The overall survival rate was 90% +/- 3% (SE) at 4 years. PFS was significantly better for patients with cerebellar and cerebral hemisphere tumors (n = 75) than those with tumors in all other sites (P = .0006). Within the former group, there was no significant difference in PFS for patients in whom GTR was achieved versus those with incomplete resections (4-year estimates, 89% and 77%, respectively). Histology (juvenile pilocytic v astrocytoma not otherwise specified [NOS]) was not related to PFS in an analysis that controlled for tumor site and patient age. Patients younger than 5 years at diagnosis had a significantly poorer PFS than older children, regardless of histology (P < .03) or tumor site (P < .002). Treatment for progressive/recurrent disease was effective in a majority of patients, but appeared more successful in patients with hemispheric than thalamic or hypothalamic tumors. The overall survival in this series of pediatric low-grade astrocytomas is excellent. Age at diagnosis and tumor location, but not histology, had a significant impact on PFS. Efforts to improve treatment outcome should focus on young patients (< 5 years) and on those with central midline tumors. The majority of patients with completely resected hemispheric tumors were monitored without further therapy, which supports attempted GTR of cerebral and cerebellar hemisphere low-grade astrocytoma.

Reduced TSC2 RNA and protein in sporadic astrocytomas and ependymomas

Individuals affected with tuberous sclerosis complex (TSC) develop several benign and malignant tumors at increased frequency, including astrocytomas. Tuberin, the protein product of the tuberous sclerosis complex-2 (TSC2) tumor suppressor gene, has been shown to directly inhibit cell growth and is expressed at high levels in normal central nervous system neurons and astrocytes. To determine whether TSC2 RNA and protein are reduced in astrocytomas from individuals without tuberous sclerosis, reverse transcriptase-polymerase chain reaction and immunoblotting analyses were performed on 49 adult astrocytomas, 10 pediatric astrocytomas, and 13 ependymomas. Eighteen of 40 (45%) high-grade (World Health Organization [WHO] grade III/IV) astrocytomas and 4 of 8 (50%) adult low-grade (WHO grade II) astrocytomas demonstrated reduced or absent TSC2 expression, including 1 giant cell astrocytoma, whereas none of the 10 pediatric low-grade astrocytomas analyzed showed a reduction in TSC2 expression. Reduced or absent tuberin was observed in 2 of 6 (33%) ependymomas analyzed. These data demonstrate, for the first time, that reduced or absent TSC2 expression may represent one of the critical genetic events associated with the development of sporadic adult, but not pediatric, astrocytomas.

Proton magnetic resonance spectroscopic imaging of pediatric low-grade astrocytomas

Despite their uniform histologic appearance, pediatric low-grade astrocytomas (LGA) often exhibit a rather unpredictable clinical course. It is presently unclear whether certain specific genetic, immunologic and/or metabolic features underlie these observed variations. In order to address this question we examined the tumor distribution of choline compounds (Cho), creatine (Cr) and N-acetyl aspartate (NAA) in seven children with midline LGA by means of proton magnetic resonance spectroscopy imaging (H-MRSI). Studies were performed with a 1.5 T GE Signa Scanner equipped with the standard head coil; nominal voxel size was 7.5 x 7.5 x 15 mm. This spatial resolution allowed us to select and independently evaluate multiple regions of interest (ROI) in the tumor as well as in areas of normal brain from the same individual. Normalized values of the observed signal intensities demonstrated a lower NAA and Cr content in the tumors than in the surrounding normal brain. Intratumoral Cho signals were also below normal values in all but one patient. The average Cho:NAA ratio was consistently higher in the tumor than in the normal brain. However, there was a wide variation (up to fourfold) in the Cho:NAA ratios of different ROIs, even within the same tumor. Our results clearly indicate that pediatric LGAs are metabolically heterogeneous, a feature that may be relevant to the understanding of their variable biologic behavior. Inasmuch as unique metabolic patterns were observed in some LGAs, we believe that systematic HMRSI studies of these patients may help define subsets within the group with specific therapeutic requirements.

Recent trends in the radiotherapy of pediatric gliomas.

The management of pediatric gliomas is controversial, and is greatly influenced by the site of origin of the tumor. For example, cerebellar low grade tumors are often cured by surgery alone. This is in contrast to the hypothalamic and optic system tumors which are usually not amenable to complete resection. For the low grade astrocytomas, the usual indications for adjuvant treatment include: recurrent tumors after initial complete resection or symptomatic tumors that have been incompletely excised. In addition, treatment is generally indicated in tumors with growth on follow-up imaging, even in the absence of symptoms. In selecting the optimal treatment, the relative efficacies of surgery, chemotherapy and irradiation must be balanced by the potential complications of therapy. The potential risks of delayed intervention include irreversible neurologic impairment and potential lower probability of tumor control. This chapter reviews recent trends in the radiotherapeutic management of pediatric low-grade and malignant astrocytomas, particularly the new more conformal techniques that hold the promise of reduced toxicity in children requiring irradiation.

P53 gene mutations in pediatric brain tumors.

We investigated the frequency of p53 mutations in 47 pediatric brain tumors of various histologic subtypes that were collected over a period of 5 years. The specimens included 15 primitive neuroectodermal tumors (PNETs), 17 low grade astrocytomas, one anaplastic astrocytoma, three glioblastomas (GBMs), one mixed glial tumor, eight ependymomas, one choroid plexus carcinoma, and one gangliocytoma/ganglioneuroma. Mutations were identified by single strand conformation polymorphism analysis of exons 4-8 and verified by sequencing. Mutations were present in 2 of 3 cases of GBM, but not in 17 low grade astrocytomas (P = 0.02, Fisher's exact test). One GBM demonstrated a germline GGC to AGC transition (gly to ser) at codon 245 with loss of the wild-type allele. A second GBM contained a CGG to TGG transition (arg to trp) at codon 248, also with loss of the wild-type allele, but normal tissue was not available for comparison. In addition, one of 15 PNETs retained heterozygosity but demonstrated a somatic CGT to TGT transition (arg to cys) at codon 273. p53 mutations were absent in other histologic subtypes and in two cases with multiple primary cancers. These data are consistent with earlier findings that p53 mutations are rare in PNETs, which are primarily pediatric tumors. In contrast to adult gliomas, p53 mutations in pediatric gliomas appear restricted to the GBMs. The lack of p53 mutations in pediatric low grade astrocytomas suggests not only histological differences, but also a different molecular pathogenesis in adult and pediatric patients.