Targeted genetic and molecular therapies in neurofibromatosis - A review of present therapeutic options and a glimpse into the future.

Neurofibromatosis type 1, the most common phakomatoses, can present with a host of signs and symptoms, usually involving the skin and the peripheral nervous system. It is characterized by a mutation in the neurofibromatosis type 1 gene on chromosome 17q11.2 that codes for the protein neurofibromin. Neurofibromin acts as a tumor suppressor gene by inhibiting rat sarcoma (Ras) activity and its deficiency leads to increased Ras activity, cellular proliferation and tumor formation. This review was conducted to analyze the various targeted therapies at the genetic and molecular level employed to manage the tumors and other clinical presentations associated with neurofibromatosis type 1. Twenty-eight studies of treatment modalities for the conditions associated with neurofibromatosis and which involved either targeted gene therapy or molecular level therapies, including the latest advances, were included in this review. Mitogen-activated protein kinase kinase inhibition, mammalian target of Rapamycin inhibition and Tyrosine kinase inhibition, represent some of the newer treatment options in this category. Although there are a number of trials for providing therapeutic options at the genetic and molecular level for the various physical and psychological morbidities associated with neurofibromatosis type 1, most of them are in the preclinical stage. Increased clinical trials of the molecules and gene therapies could significantly help in managing the various chronic and sometimes, life-threatening conditions associated with neurofibromatosis 1 and these will probably represent the preferred treatment direction of the future.

Lymphocyte Subpopulations in Patients With Neurofibromatosis Type 1-associated Optic Pathway Gliomas and Plexiform Neurofibromas.

BACKGROUND/AIM: Neurofibromatosis type 1 (NF1) is characterized by the occurrence of multisystem tumors, among which the most characteristic are optic pathway gliomas (OPGs) and plexiform neurofibromas (PNFs). With the development of new anticancer drugs targeting the immune system, it is important to examine the immunological status of patients with NF1. Furthermore, the immune system has been suggested as a probable modulator of NF1-associated phenotypes. The objective of this study was the analysis of lymphocyte subset populations with respect to the presence of PNFs and OPGs. PATIENTS AND METHODS: Fifty-three patients with NF1 diagnosed with OPG/PNF were analyzed for lymphocyte subpopulations. RESULTS: Significantly lower levels of B-cells, T-cells and natural killer (NK) cells were observed in the group of patients with PNFs compared to those with OPG. CONCLUSION: Our observation may indicate a correlation between weakened functioning of the immune system and the formation of PNFs.

Moya-Moya syndrome after cranial radiation for optic glioma with NF1. Case report and literature review of syndromic cases.

INTRODUCTION: Moya-Moya angiopathy is a neurovascular disease that predisposes to ischemic or hemorrhagic strokes. It is generated by a steno-occlusion of the terminal portion of the internal carotid arteries, which induces the development of abnormal neovessels in the deep regions of the brain. Some pathologies such as sickle cell disease, Down syndrome or Graves' disease may be associated with Moya-Moya angiopathy. These syndromic forms harbor several differences compared with idiopathic Moya-Moya disease. CASE REPORT: We report the case of a young patient who presented with a syndromic form of Moya-Moya angiopathy after cranial radiation therapy for an optic glioma associated with type 1 neurofibromatosis treated by combined revascularization. We discuss the particularities of syndromic forms, in their presentation and management based on a review of the literature. CONCLUSION: Many diseases can be associated with Moya-Moya syndrome. Symptomatic patients should undergo surgery, but the risk of postoperative complications appears to be greater than that encountered in patients with non-syndromic Moya-Moya angiopathy.

Optic Nerve Glioma in Two Sisters with Family History of Neurofibromatosis Type 1.

Optic nerve glioma (ONG) is associated in 10% of patients with neurofibromatosis (NF) type 1. To date no consensus has been reached regarding the therapeutic approach and prevention of visual impairment in these patients. Reports in the literature vary from a conservative approach (observation) to the use of single treatment modalities or multimodality protocols of surgical removal, radiotherapy, and/or chemotherapy. We present our experience with two siblings with ONG whose mother carries cutaneous stigmata of NF type 1. The younger sister was diagnosed 3 years after the treatment of the older sibling following recommended imaging for screening. Postoperative follow-up for 11 and 15 years, respectively, demonstrated lack of tumor regrowth and preserved vision in the contralateral eye. We discuss the treatment strategy in pediatric patients with orbital ONG associated with NF type 1.

Optic pathway glioma volume predicts retinal axon degeneration in neurofibromatosis type 1.

OBJECTIVE: To determine whether tumor size is associated with retinal nerve fiber layer (RNFL) thickness, a measure of axonal degeneration and an established biomarker of visual impairment in children with optic pathway gliomas (OPGs) secondary to neurofibromatosis type 1 (NF1). METHODS: Children with NF1-OPGs involving the optic nerve (extension into the chiasm and tracts permitted) who underwent both volumetric MRI analysis and optical coherence tomography (OCT) within 2 weeks of each other were included. Volumetric measurement of the entire anterior visual pathway (AVP; optic nerve, chiasm, and tract) was performed using high-resolution T1-weighted MRI. OCT measured the average RNFL thickness around the optic nerve. Linear regression models evaluated the relationship between RNFL thickness and AVP dimensions and volume. RESULTS: Thirty-eight participants contributed 55 study eyes. The mean age was 5.78 years. Twenty-two participants (58%) were female. RNFL thickness had a significant negative relationship to total AVP volume and total brain volume (p < 0.05, all comparisons). For every 1 mL increase in AVP volume, RNFL thickness declined by approximately 5 microns. A greater AVP volume of OPGs involving the optic nerve and chiasm, but not the tracts, was independently associated with a lower RNFL thickness (p < 0.05). All participants with an optic chiasm volume >1.3 mL demonstrated axonal damage (i.e., RNFL thickness <80 microns). CONCLUSIONS: Greater OPG and AVP volume predicts axonal degeneration, a biomarker of vision loss, in children with NF1-OPGs. MRI volumetric measures may help stratify the risk of visual loss from NF1-OPGs.

Quantitative MRI criteria for optic pathway enlargement in neurofibromatosis type 1.

OBJECTIVE: To determine quantitative size thresholds for enlargement of the optic nerve, chiasm, and tract in children with neurofibromatosis type 1 (NF1). METHODS: Children 0.5-18.6 years of age who underwent high-resolution T1-weighted MRI were eligible for inclusion. This consisted of children with NF1 with or without optic pathway gliomas (OPGs) and a control group who did not have other acquired, systemic, or genetic conditions that could alter their anterior visual pathway (AVP). Maximum and average diameter and volume of AVP structures were calculated from reconstructed MRI images. Values above the 95th percentile from the controls were considered the threshold for defining an abnormally large AVP measure. RESULTS: A total of 186 children (controls = 82; NF1noOPG = 54; NF1+OPG = 50) met inclusion criteria. NF1noOPG and NF1+OPG participants demonstrated greater maximum optic nerve diameter and volume, optic chiasm volume, and total brain volume compared to controls (p < 0.05, all comparisons). Total brain volume, rather than age, predicted optic nerve and chiasm volume in controls (p < 0.05). Applying the 95th percentile threshold to all NF1 participants, the maximum optic nerve diameter (3.9 mm) and AVP volumes resulted in few false-positive errors (specificity >80%, all comparisons). CONCLUSIONS: Quantitative reference values for AVP enlargement will enhance the development of objective diagnostic criteria for OPGs secondary to NF1.

Peri-gestational risk factors for pediatric brain tumors in Neurofibromatosis Type 1.

BACKGROUND: Individuals with Neurofibromatosis Type 1 (NF1) are strongly predisposed to developing pediatric brain tumors (PBTs), especially optic pathway gliomas (OPGs). Although developmental factors have been implicated in the origins of PBTs in both human and animal studies, associations between early-life factors and PBTs have not been evaluated in individuals with NF1. Our objective was to evaluate associations between peri-gestational characteristics and PBTs in this population. METHODS: We conducted a cross-sectional study, ascertaining questionnaire and medical record data for 606 individuals<18years old who enrolled in the NF1 Patient Registry Initiative (NPRI) from 6/9/2011-6/29/2015. One hundred eighty-four individuals had reported PBT diagnoses, including 65 who were identified with OPG diagnoses. Cox proportional hazards regression was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for associations between PBT and OPG diagnoses and peri-gestational characteristics (prematurity, birth weight, parental age, plurality, family history of NF1, assisted reproductive technology, maternal vitamin supplementation, and parental smoking). RESULTS: We observed no significant associations between any of the assessed characteristics and PBTs overall or OPGs with the exception of birth weight. After controlling for potential confounding variables, we observed a significant positive association between birth weight quartile and OPGs with a HR of 3.32 (95% CI 1.39-7.94) for the fourth (≥3915.5g) compared to the first (≤3020g) quartile (p for trend=0.001). CONCLUSIONS: Consistent with results for PBTs in the general population, these results suggest that higher birth weights increase OPG risk in individuals with NF1.

No correlation between NF1 mutation position and risk of optic pathway glioma in 77 unrelated NF1 patients.

Neurofibromatosis type 1 (NF1) is a common monogenic disorder whereby affected individuals are predisposed to developing CNS tumors, including optic pathway gliomas (OPGs, occurring in ~15 to 20 % of cases). So far, no definite genotype-phenotype correlation determining NF1 patients at risk for tumor formation has been described, although enrichment for mutations in the 5' region of the NF1 gene in OPG patients has been suggested. We used whole exome sequencing, targeted sequencing, and copy number analysis to screen 77 unrelated NF1 patients with (n = 41) or without (n = 36; age ≥10 years) optic pathway glioma for germline NF1 alterations. We identified germline NF1 mutations in 69 of 77 patients (90 %), but no genotype-phenotype correlation was observed. Our data using a larger patient cohort did not confirm the previously reported clustering of mutations in the 5' region of the NF1 gene in patients with OPG. Thus, NF1 mutation location should not currently be used as a clinical criterion to assess the risk of developing OPGs.

NF1 germline mutation differentially dictates optic glioma formation and growth in neurofibromatosis-1.

Neurofibromatosis type 1 (NF1) is a common neurogenetic condition characterized by significant clinical heterogeneity. A major barrier to developing precision medicine approaches for NF1 is an incomplete understanding of the factors that underlie its inherent variability. To determine the impact of the germline NF1 gene mutation on the optic gliomas frequently encountered in children with NF1, we developed genetically engineered mice harboring two representative NF1-patient-derived Nf1 gene mutations (c.2542G>C;p.G848R and c.2041C>T;p.R681X). We found that each germline Nf1 gene mutation resulted in different levels of neurofibromin expression. Importantly, only R681X(CKO) but not G848R(CKO), mice develop optic gliomas with increased optic nerve volumes, glial fibrillary acid protein immunoreactivity, proliferation and retinal ganglion cell death, similar to Nf1 conditional knockout mice harboring a neomycin insertion (neo) as the germline Nf1 gene mutation. These differences in optic glioma phenotypes reflect both cell-autonomous and stromal effects of the germline Nf1 gene mutation. In this regard, primary astrocytes harboring the R681X germline Nf1 gene mutation exhibit increased basal astrocyte proliferation (BrdU incorporation) indistinguishable from neo(CKO) astrocytes, whereas astrocytes with the G848R mutation have lower levels of proliferation. Evidence for paracrine effects from the tumor microenvironment were revealed when R681X(CKO) mice were compared with conventional neo(CKO) mice. Relative to neo(CKO) mice, the optic gliomas from R681X(CKO) mice had more microglia infiltration and JNK(Thr183/Tyr185) activation, microglia-produced Ccl5, and glial AKT(Thr308) activation. Collectively, these studies establish that the germline Nf1 gene mutation is a major determinant of optic glioma development and growth through by both tumor cell-intrinsic and stromal effects.

3-D imaging mass spectrometry of protein distributions in mouse Neurofibromatosis 1 (NF1)-associated optic glioma.

Neurofibromatosis type 1 (NF1) is a common neurogenetic disorder, in which affected individuals develop tumors of the nervous system. Children with NF1 are particularly prone to brain tumors (gliomas) involving the optic pathway that can result in impaired vision. Since tumor formation and expansion requires a cooperative tumor microenvironment, it is important to identify the cellular and acellular components associated with glioma development and growth. In this study, we used 3-D matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) to measure the distributions of multiple molecular species throughout optic nerve tissue in mice with and without glioma, and to explore their spatial relationships within the 3-D volume of the optic nerve and chiasm. 3-D IMS studies often involve extensive workflows due to the high volume of sections required to generate high quality 3-D images. Herein, we present a workflow for 3-D data acquisition and volume reconstruction using mouse optic nerve tissue. The resulting 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions. BIOLOGICAL SIGNIFICANCE: The current work addresses a number of challenges in 3-D MALDI IMS, driven by the small size of the mouse optic nerve and the need to maintain consistency across multiple 2-D IMS experiments. The 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions, which could then be targeted for identification and related back to the biology observed in gliomas of the optic nerve.

Risk of optic pathway glioma in children with neurofibromatosis type 1 and optic nerve tortuosity or nerve sheath thickening.

BACKGROUND/AIMS: Optic nerve tortuosity and nerve and sheath thickening are observed on MRI in some patients with neurofibromatosis type 1 (NF-1). This study aimed to determine if tortuosity and thickening are associated with the development of optic pathway glioma (OPG) and subsequent vision loss. METHODS: Children with NF-1 who underwent brain MRI between 1992 and 2005, and had at least 1 year of subsequent visual acuity (VA) follow-up, were identified retrospectively. The baseline MRI was independently reviewed by three neuroradiologists for consensus assessment. Tortuosity was identified using validated operational criteria. Optic nerve and sheath thicknesses and VA at last follow-up were directly measured. RESULTS: Of 132 evaluable children, seven (5%) had tortuosity on baseline MRI. 20 subjects (15%) ultimately developed OPG at a median of 1.9 years (range 7 months-8.0 years) following the baseline MRI. Subjects with tortuosity were significantly more likely to develop OPG than those without tortuosity (57% vs 13%, p=0.01). In subjects who developed OPG, the prevalence of tumour-related vision loss was not significantly different between those with and without baseline tortuosity (14% vs 4%, p=0.28). No difference existed between mean baseline optic nerve (2.3 vs 2.2 mm) or sheath (5.2 vs 5.4 mm) thicknesses comparing subjects who did and did not develop OPG. CONCLUSIONS: Optic nerve tortuosity at baseline is associated with OPG development among patients with NF-1, but does not predispose to aggressive OPG with associated vision loss. Neither nerve nor sheath thickening at baseline is associated with OPG development.

Associations between allergic conditions and pediatric brain tumors in Neurofibromatosis type 1.

Individuals with Neurofibromatosis type 1 (NF1) are at increased risk for pediatric brain tumors (PBTs), especially optic gliomas; however, factors influencing their development are largely unknown. Extensive research suggests that allergic conditions protect against brain tumors, particularly gliomas in individuals without NF1. In this large cross-sectional study, we employed two different data sources to evaluate evidence for the hypothesis that allergic conditions (allergies, asthma, and eczema) may protect against PBT development in individuals with NF1. We used self- and parent/legal guardian reported questionnaire data from participants in the NF1 Patient Registry Initiative (NPRI, n = 1660) born from 1933 to 2014 to ascertain allergic condition and PBT diagnosis histories. Medical records (MRs) of 629 NF1 patients at a large medical center born from 1930 to 2012 were also reviewed for PBT and allergic condition diagnoses to evaluate additional evidence for our hypothesis. We used logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for associations between allergic condition diagnoses and PBTs. Both data sources provided limited to no support for a protective effect of allergies or eczema on PBT development. Non-significant inverse associations between asthma and PBTs were observed (NPRI: OR = 0.80, 95% CI 0.55-1.17; MR: OR = 0.71, 95% CI 0.40-1.28) with stronger associations for optic gliomas specifically. Additionally, a significant inverse association was observed in an NPRI subgroup analysis where the reported asthma diagnosis age was younger than the reported PBT diagnosis age (OR = 0.57; 95% CI 0.36-0.89). Our study supports the hypothesis that asthma protects against PBT development in NF1.

Comparative study of the ophthalmological examinations in neurofibromatosis type 1. Proposal for a new screening algorithm.

PURPOSE: Efforts are made to unify the protocol concerning the ophthalmological screening, monitoring and treatment of Optic Pathway Gliomas (OPGs) in children with neurofibromatosis type 1 (NF1). The aim of this study is to compare the most recent recommendations published in 2007 with the screening strategies in NF1 centres. The integration of these data resulted into a recommendation for an improved screening strategy. METHODS: A literature search on PubMed between 1984 and 2013 was performed. A questionnaire on the ophthalmological screening in NF1 was sent to centres of expertise in the field of NF1. Literature and questionnaire data were analysed. Also, findings of a round table discussion on the ophthalmological screening of NF1 patients at the European Paediatric Ophthalmological Society (EPOS) meeting in 2013 were summarized. RESULTS: In most centres ophthalmological screening in NF1 patients is well organized, but is performed longer and at more regular intervals than is mentioned in the recommendations. Visual acuity testing, fundoscopy and pupillary reflexes are carried out unanimously. CONCLUSIONS: There is no uniformity of the ophthalmological screening in NF1 patients. The present recommendation advises to screen annually until the age of 8. Because OPGs are likely to develop before the age of 6 and children do not usually complain of visual problems, OPGs can be missed or detection can be delayed if screening is only yearly performed at this young age. Based on these arguments, about half of our responders screen more frequently and until a later age. Therefore, we suggest performing a six monthly screening until the age of 6 and a yearly examination from 6 years until adulthood. This examination should include visual acuity assessment, pupillary reflexes and a fundoscopy.

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.

Is it time to change the neurofibromatosis 1 diagnostic criteria?

Neurofibromatosis 1 is a complex inherited neurocutaneous disease that is often difficult to diagnose early because of its age-dependent presentation. The diagnosis is also extremely difficult to communicate to patients and their parents because of the disease's clinical variability, unpredictable evolution, and uncertain prognosis. Since 1988, the year of publication of the last Consensus Conference statement concerning the diagnosis of neurofibromatosis 1, our understanding of the disease has naturally increased and, in addition to the availability of increasingly precise molecular analyses, some new clinical signs have been reported such as anaemic nevi, unidentified bright objects, choroidal hamartomas, and a typical neuropsychological phenotype. We critically review the current diagnostic criteria, and suggest the addition of new signs on the basis of published findings and our own clinical experience. This proposal aims to improve diagnostic power in paediatric age, securing a better and more reliable healthcare transition toward adult age. We finally recommend a new Consensus Conference in order to revise the diagnostic criteria, possibly differentiated by age of presentation.

Functional outcome measures for NF1-associated optic pathway glioma clinical trials.

OBJECTIVE: The goal of the Response Evaluation in Neurofibromatosis and Schwannomatosis Visual Outcomes Committee is to define the best functional outcome measures for future neurofibromatosis type 1 (NF1)-associated optic pathway glioma (OPG) clinical trials. METHODS: The committee considered the components of vision, other ophthalmologic parameters affected by OPG, potential biomarkers of visual function, and quality of life measures to arrive at consensus-based, evidence-driven recommendations for objective and measurable functional endpoints for OPG trials. RESULTS: Visual acuity (VA) assessments using consistent quantitative testing methods are recommended as the main functional outcome measure for NF1-OPG clinical trials. Teller acuity cards are recommended for use as the primary VA endpoint, and HOTV as a secondary endpoint once subjects are old enough to complete it. The optic disc should be assessed for pallor, as this appears to be a contributory variable that may affect the interpretation of VA change over time. Given the importance of capturing patient-reported outcomes in clinical trials, evaluating visual quality of life using the Children's Visual Function Questionnaire as a secondary endpoint is also proposed. CONCLUSIONS: The use of these key functional endpoints will be essential for evaluating the efficacy of future OPG clinical trials.

Role of visual evoked potentials in the assessment and management of optic pathway gliomas in children.

OBJECTIVE: The aim of this study is to investigate the role of pattern reversal visual evoked potentials (pVEPs) in the screening and monitoring of optic pathway gliomas (OPGs) in children with and without neurofibromatosis type 1. METHODS: A review of the English literature published between 1980 and 2012 was performed, with comparison of results of retro- and prospective studies. RESULTS: Pattern reversal VEPs have a high sensitivity (85.7-100 %) for the diagnosis of OPGs, moreover they are safe and cost-effective. Conversely, they have a low specificity (43-83 %) and are not widely available. Besides, pattern reversal VEP results can be unreliable in young children, because of the need for a good cooperation. The studies that were analyzed have drawbacks, including the small sample size, the retrospective design, the differences in gold standard for diagnosis, the different interpretation of small changes in VEP results and the lack of control groups. CONCLUSION: There is still debate about the gold standard for the screening and follow-up of OPGs. The added value of pVEPs to the ophthalmic examination is controversial. Randomized controlled trials or prospective multicentre studies are necessary to assess with sufficient accuracy the sensitivity and specificity of pattern reversal VEPs in the screening for OPGs and its follow-up.