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The aim of this study was to characterize cardiac features of patients with neurofibromatosis 1 (NF1) and large deletions of the NF1 gene region. The study participants were 16 patients with large NF1 deletions and 16 age- and sex-matched NF1 patients without such deletions. All the patients were comprehensively characterized clinically and by echocardiography. Six of 16 NF1 deletion patients but none of 16 non-deletion NF1 patients have major cardiac abnormalities (p = 0.041). Congenital heart defects (CHDs) include mitral insufficiency in two patients and ventricular septal defect, aortic stenosis, and aortic insufficiency in one patient each. Three deletion patients have hypertrophic cardiomyopathy. Two patients have intracardiac tumors. NF1 patients without large deletions have increased left ventricular (LV) diastolic posterior wall thickness (p < 0.001) and increased intraventricular diastolic septal thickness (p = 0.001) compared with a healthy reference population without NF1, suggestive of eccentric LV hypertrophy. CHDs and other cardiovascular anomalies are more frequent among patients with large NF1 deletion and may cause serious clinical complications. Eccentric LV hypertrophy may occur in NF1 patients without whole gene deletions, but the clinical significance of this finding is uncertain. All patients with clinical suspicion for NF1 should be referred to a cardiologist for evaluation and surveillance., (© 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: The aim of this study was to analyze growth rate and identify prognostic factors for progression of postoperative plexiform neurofibromas in patients with neurofibromatosis type 1., Methods: We measured postoperative tumor volume change per year on magnetic resonance imaging. Linear regression models were applied to identify risk factors for tumor progression., Results: Fifty-two patients (mean age: 25.4 years (3.2-64.2 years)) with 56 plexiform neurofibromas were analyzed. Initial median tumor volume was 40.3 ml (SD: 1,552 (0-10,800)). Surgical indications included disfigurement (n = 21), pain (n = 20), and functional deficits (n = 16). Sixteen percent of all cases experienced acute surgical complication, and 13% showed late complication. Eight patients (19%; 6 children and 2 adults) with residual tumor had repeat surgery for tumor progression. Median tumor progression was 0.6% change per year (SD ± 27.4; range: -59.2 to 88.1) and 2.9% from baseline (SD ± 163.9; range -1,001.3 to 81.8). Patients aged 21 years and younger had the highest progression rate (P < 0.01). For every year of age, the mean growth rate decreased by -0.463 mean percent (P = 0.03). With age as a continuous variable, age, the site of the tumor, and depth were the only factors associated with tumor progression. Fourteen plexiform neurofibromas (10 nodular and 4 diffuse) in 13 patients (5 children and 8 adults) were completely resected (by visualization) and did not relapse during observation (mean: 2.9 years; range: 1.1-5.8 years)., Conclusion: Age, tumor type, location, and depth are helpful to estimate the progression of plexiform neurofibromas after surgery. Patients benefit from elective surgery of small and completely removable plexiform neurofibromas.

Purpose: The aim of the study was to evaluate the potential usefulness of intratumoural tracer uptake heterogeneity on (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT as compared to a cut-off maximum standardized uptake value (SUVmax) for characterization of peripheral nerve sheath tumours (PNSTs) in neurofibromatosis type 1 (NF1)., Methods: Fifty patients suffering from NF1 were examined by (18)F-FDG PET/CT. Intralesional tracer uptake was analysed qualitatively and semi-quantitatively by measuring the mean and maximum SUV. Uptake heterogeneity was graded qualitatively using a three-point scale and semi-quantitatively by calculating an SUV-based heterogeneity index (HISUV). Cohen's κ was used to determine inter- and intra-rater agreement. Histopathological evaluation and clinical as well as radiological follow-up examinations served as the reference standards., Results: A highly significant correlation between the degree of intratumoural uptake heterogeneity on (18)F-FDG PET and malignant transformation of PNSTs was observed (p < 0.0001). Semi-quantitative HISUV was significantly higher in malignant PNSTs (MPNSTs) than in benign tumours (p = 0.0002). Both intralesional heterogeneity and SUVmax could be used to identify malignant tumours with a sensitivity of 100 %. Cohen's κ was 0.86 for inter-rater agreement and 0.88 for intra-rater agreement on heterogeneity., Conclusion: MPNSTs in patients with NF1 demonstrate considerable intratumoural uptake heterogeneity on (18)F-FDG PET/CT. Assessment of tumour heterogeneity is highly reproducible. Both tumour heterogeneity and a cut-off SUVmax may be used to sensitively identify malignant PNSTs, but the specificity is higher for the latter. A combination of both methods leads to a non-significant improvement in diagnostic performance.

Objectives: Neurofibromatosis 1 (NF1), NF2, and schwannomatosis are characterized by a predisposition to develop multiple neurofibromas and schwannomas. Currently, there is no blood test to estimate tumor burden in patients with these disorders. We explored whether S100β would act as a biomarker of tumor burden in NF since S100β is a classic immunohistochemical marker of astrocytes, oligodendrocytes and Schwann cells and a small study showed S100β concentrations correlate with the volume of vestibular schwannomas., Design and Methods: We calculated whole-body tumor burden in subjects with NF1, NF2, and schwannomatosis using whole-body MRI (WBMRI) and measured the concentration of S100β in plasma using ELISA. We used chi-square tests and Spearman rank correlations to test the relationship between S100β levels and whole-body tumor burden., Results: 127 consecutive patients were enrolled in the study (69 NF1 patients, 28 NF2 patients, and 30 schwannomatosis patients). The median age was 40years, 43% were male, and median whole-body tumor volume was 26.9mL. There was no relationship between the presence of internal tumors and the presence of detectable S100β in blood for the overall group or for individual diagnoses (p>0.05 by chi-square for all comparisons). Similarly, there was no correlation between whole-body tumor volume and S100β concentration for the overall group or for individual diagnoses (p>0.05 by Spearman for all comparisons)., Conclusions: Plasma S100β is not a useful biomarker for tumor burden in the neurofibromatoses. Further work is needed to identify a reliable biomarker of tumor burden in NF patients., (Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Neurofibromatosis type 1 (NF1) is a hereditary tumor syndrome characterized by the development of benign nerve-sheath tumors, which transform to malignant peripheral nerve-sheath tumors (MPNST) in about 8 to 13% of patients with NF1. MPNST are invasive sarcomas with extremely poor prognosis, and their development may correlate with internal tumor load of patients with NF1. Because early identification of patients with NF1 at risk for developing MPNST should improve their clinical outcome, the aim of this study was to identify serum biomarkers for tumor progression in NF1, and to analyze their correlation with tumor type and internal tumor load., Methods: We selected candidate biomarkers for NF1 by manually mining published data sources, and conducted a systematic screen of 56 candidate serum biomarkers using customized antibody arrays. Serum from 104 patients with NF1 with and without MPNST, and from 41 healthy control subjects, was analyzed. Statistical analysis was performed using the non-parametric Mann-Whitney U-test, followed by Bonferroni correction., Results: Our analysis identified four markers (epidermal growth factor receptor, interferon-γ, interleukin-6, and tumor necrosis factor-α) for which significantly different serum concentrations were seen in patients with NF1 compared with healthy controls. Two markers (insulin-like growth factor binding protein 1 (IGFBP1) and regulated upon activation, normal T-cell expressed and secreted (RANTES)) showed significantly higher concentrations in patients with NF1 and MPNST compared with patients with NF1 without MPNST. A correlation with internal tumor load was found for IGFBP1., Conclusion: Our study identified two serum markers with potential for early detection of patients with NF1 at risk for developing MPNST, and four markers that could distinguish between patients with NF1 and healthy subjects. Such markers may be useful as diagnostic tools to support the diagnosis of NF1 and for timely identification of MPNST. Moreover, the data suggest that there is a systemic increase in inflammatory cytokines independently of tumor load in patients with NF1.

The autosomal dominant disorder neurofibromatosis type 2 (NF2) is a hereditary tumor syndrome caused by inactivation of the NF2 tumor suppressor gene, encoding merlin. Apart from tumors affecting the peripheral and central nervous systems, most NF2 patients develop peripheral neuropathies. This peripheral nerve disease can occur in the absence of nerve-damaging tumors, suggesting an etiology that is independent of gross tumor burden. We discovered that merlin isoform 2 (merlin-iso2) has a specific function in maintaining axonal integrity and propose that reduced axonal NF2 gene dosage leads to NF2-associated polyneuropathy. We identified a merlin-iso2-dependent complex that promotes activation of the GTPase RhoA, enabling downstream Rho-associated kinase to promote neurofilament heavy chain phosphorylation. Merlin-iso2-deficient mice exhibited impaired locomotor capacities, delayed sensory reactions and electrophysiological signs of axonal neuropathy. Sciatic nerves from these mice and sural nerve biopsies from NF2 patients revealed reduced phosphorylation of the neurofilament H subunit, decreased interfilament spacings and irregularly shaped axons.

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary cancers associated with neurofibromatosis type I. MPNSTs lack effective treatment options as they often resist chemotherapies and have high rates of disease recurrence. Aurora kinase A (AURKA) is an emerging target in cancer and an aurora kinase inhibitor (AKI), termed MLN8237, shows promise against MPNST cell lines in vitro and in vivo. Here, we test MLN8237 against two primary human MPNST grown in vivo as xenotransplants and find that treatment results in tumour cells exiting the cell cycle and undergoing endoreduplication, which cumulates in stabilized disease. Targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumour susceptibilities, so we turned to three MPNST cell-lines to further study and modulate the cellular responses to AKI. We find that the sensitivity of cell-lines with amplification of AURKA depends upon the activity of the kinase, which correlates with the expression of the regulatory gene products TPX2 and HMMR/RHAMM. Silencing of HMMR/RHAMM, but not TPX2, augments AURKA activity and sensitizes MPNST cells to AKI. Furthermore, we find that AURKA activity is critical to the propagation and self-renewal of sphere-enriched MPNST cancer stem-like cells. AKI treatment significantly reduces the formation of spheroids, attenuates the self-renewal of spheroid forming cells, and promotes their differentiation. Moreover, silencing of HMMR/RHAMM is sufficient to endow MPNST cells with an ability to form and maintain sphere culture. Collectively, our data indicate that AURKA is a rationale therapeutic target for MPNST and tumour cell responses to AKI, which include differentiation, are modulated by the abundance of HMMR/RHAMM.

Purpose: The aim of this study was to compare the diagnostic performance of ¹⁸F-fluorodeoxyglucose (FDG) PET/CT and whole-body MRI for the detection of malignant peripheral nerve sheath tumors (MPNSTs) in patients with neurofibromatosis type 1, and to evaluate a panel of imaging-based criteria serving that purpose., Patients and Methods: Thirty-one patients were examined by whole-body MRI and ¹⁸F-FDG PET/CT. A panel of imaging-based criteria including tumor region, size, shape, margin definition, contrast enhancement, heterogeneity before and after contrast, intratumoral lobulation, target sign, and mean and maximum standardized uptake values (SUVs) were evaluated. A SUVmax cut-off value of 3.5 was used for lesion analysis. Histopathologic evaluation and/or clinical follow-up served as the reference standard., Results: ¹⁸F-FDG PET/CT had a sensitivity of 100%, whereas MRI had a sensitivity of 66.7%. On PET/CT, tumor size (P<0.005), SUVmax (P<0.0001), SUVmean (P<0.0001), and tracer uptake heterogeneity (P=0.002) were significantly associated with MPNSTs. On MRI, intratumoral lobulation (P<0.02), ill-defined margins (P=0.007), and irregular enhancement on T1-weighted imaging (P<0.001) were significantly associated with MPNSTs., Conclusions: Both PET/CT and whole-body MRI may distinguish benign and malignant PNSTs, but PET/CT has higher sensitivity for that purpose. Imaging-based criteria for identification of MPNSTs on both modalities were identified. False-positive results, requiring biopsy or clinical follow-up, may be reduced by using a combination of MRI and PET derived markers, but only at the price of reduced sensitivity.

A distinct polyneuropathy (PNP) syndrome affects up to 66 % of patients with neurofibromatosis II (NF2). Whether this is primarily a diffuse PNP or due to single, surgically amenable mass lesions has not yet been conclusively demonstrated. We aimed to solve this question by investigating the pathomorphological MR imaging correlate of this rare disorder. Eight patients with NF2-PNP were characterized by clinical examination, electrophysiological studies, and genetic analysis. All patients additionally underwent extended peripheral nerve imaging by a novel protocol of large-coverage high-resolution MRI. Quantitative analyses were performed by separately evaluating cross-sectional images, and by categorizing lesions into non-compressive fascicular microlesions (<2 mm), intermediate lesions (2-5 mm), and compressive macrolesions (>5 mm). The predominant imaging findings were non-compressive fascicular microlesions and intermediate lesions. Proximal-to-distal cumulative lesion burden of these lesions correlated strongly with the severity of clinical symptoms of NF2-PNP. In contrast, compressive macrolesions were not found at all in several symptomatic extremities. We conclude that proximal-to-distal accumulation of non-compressive fascicular lesions instead of compressive mass lesions predominantly underlies the clinical manifestation and severity of NF2-associated PNP. Diagnostic management may now be assisted by large-coverage high-resolution imaging of plexus and peripheral nerves. Additionally, the results underscore the feasibility of this new method, which may open up new diagnostic and investigative possibilities for other disseminated disorders of the peripheral nervous system.

Neurofibromatosis type 2 (NF2) is a hereditary tumor syndrome. The hallmark of NF2 is bilateral vestibular schwannoma. In addition, glioma is one of the diagnostic criteria of NF2. In this retrospective study the clinical presentation and histopathological features of 12 spinal gliomas from NF2 patients were assessed. Ten tumors were previously diagnosed as ependymomas and two as astrocytomas. However, upon re-evaluation both astrocytomas expressed epithelial membrane antigen in a dot-like fashion and in one case it was possible to perform electron microscopy revealing junctional complexes and cilia typical for ependymoma. The findings suggest that NF2-associated spinal gliomas are ependymomas. Based on the fact that NF2-associated gliomas are almost exclusively spinal and that no NF2 mutations have been found in sporadic cerebral gliomas, we suggest that "glioma" in the current diagnostic criteria for NF2 should be specified as "spinal ependymoma"., (© 2012 Japanese Society of Neuropathology.)

Nonallelic homologous recombination (NAHR) is one of the major mechanisms underlying copy number variation in the human genome. Although several disease-associated meiotic NAHR breakpoints have been analyzed in great detail, hotspots for mitotic NAHR are not well characterized. Type-2 NF1 microdeletions, which are predominantly of postzygotic origin, constitute a highly informative model with which to investigate the features of mitotic NAHR. Here, a custom-designed MLPA- and PCR-based approach was used to identify 23 novel NAHR-mediated type-2 NF1 deletions. Breakpoint analysis of these 23 type-2 deletions, together with 17 NAHR-mediated type-2 deletions identified previously, revealed that the breakpoints are nonuniformly distributed within the paralogous SUZ12 and SUZ12P sequences. Further, the analysis of this large group of type-2 deletions revealed breakpoint recurrence within short segments (ranging in size from 57 to 253-bp) as well as the existence of a novel NAHR hotspot of 1.9-kb (termed PRS4). This hotspot harbored 20% (8/40) of the type-2 deletion breakpoints and contains the 253-bp recurrent breakpoint region BR6 in which four independent type-2 deletion breakpoints were identified. Our findings indicate that a combination of an open chromatin conformation and short non-B DNA-forming repeats may predispose to recurrent mitotic NAHR events between SUZ12 and its pseudogene., (© 2012 Wiley Periodicals, Inc.)

Background: Neurofibromatosis type-1 (NF1) is caused by mutations of the NF1 gene at 17q11.2. In 95% of non-founder NF1 patients, NF1 mutations are identifiable by means of a comprehensive mutation analysis. 5-10% of these patients harbour microdeletions encompassing the NF1 gene and its flanking regions. NF1 is characterised by tumours of the peripheral nerve sheaths, the pathognomonic neurofibromas. Considerable inter- and intra-familial variation in expressivity of the disease has been observed which is influenced by genetic modifiers unrelated to the constitutional NF1 mutation. The number of plexiform neurofibromas (PNF) in NF1 patients is a highly heritable genetic trait. Recently, SNP rs2151280 located within the non-coding RNA gene ANRIL at 9p21.3, was identified as being strongly associated with PNF number in a family-based association study. The T-allele of rs2151280, which correlates with reduced ANRIL expression, appears to be associated with higher PNF number. ANRIL directly binds to the SUZ12 protein, an essential component of polycomb repressive complex 2, and is required for SUZ12 occupancy of the CDKN2A/CDKN2B tumour suppressor genes as well as for their epigenetic silencing., Methods: Here, we explored a potential association of PNF number and PNF volume with SNP rs2151280 in 29 patients with constitutional NF1 microdeletions using the exact Cochran-Armitage test for trends and the exact Mann-Whitney-Wilcoxon test. Both the PNF number and total tumour volume in these 29 NF1 patients were assessed by whole-body MRI. The NF1 microdeletions observed in these 29 patients encompassed the NF1 gene as well as its flanking regions, including the SUZ12 gene., Results: In the 29 microdeletion patients investigated, neither the PNF number nor PNF volume was found to be associated with the T-allele of rs2151280., Conclusion: Our findings imply that, at least in patients with NF1 microdeletions, PNF susceptibility is not associated with rs2151280. Although somatic inactivation of the NF1 wild-type allele is considered to be the PNF-initiating event in NF1 patients with intragenic mutations and patients with NF1 microdeletions, both patient groups may differ with regard to tumour progression because of the heterozygous constitutional deletion of SUZ12 present only in patients with NF1 microdeletions.

Background: To examine the natural growth dynamics of internal plexiform neurofibromas (PNs) in patients with neurofibromatosis 1 (NF1)., Methods: Two hundred and one NF1 patients underwent whole body MRI (WBMRI). Tumour burden was estimated volumetrically. Non-parametric Spearman's rho correlation coefficients were used to analyse the relationship of growth rate to tumour volume and age. Chi-squared and Mann-Whitney U tests were used for analysing the association of tumour occurrence with sex or age. Chi-squared tests were used to analyse the association of tumour growth with age group., Results: Seventy-one of 171 patients with serial WBMRI exams had internal PNs (median follow up 2.2 years [1.1 to 4.9 years]). Median whole body tumour volume was 86.4 mL [5.2 to 5878.5 mL]) with a median growth rate of 3.7%/year (-13.4 to 111%/year) that correlated with larger whole body tumour volume (P<0.001) and lower age (P=0.004). No new PNs developed in 273.0 patient-years among patients without tumours. Rate of new tumour development among patients with PNs was 0.6%/year (95% confidence interval 0.02 to 3.4%). Twenty-seven (13.5%) tumours increased significantly and were more frequent among children (P<0.001). Growth rate of tumours was inversely correlated with age (Spearman's rho=-0.330, P<0.001). Seventy-one (35.5%) tumours had smaller volumes on follow up (median -3.4%/year [-0.07% to -35.9%/year])., Conclusion: Children with NF1 and internal PNs are at risk for tumour growth. Most PNs grow slowly or not at all, and some decrease in size. New tumours are infrequent in NF1 patients with PNs and unlikely in patients without PNs.

Aim: A substantial proportion of patients with neurofibromatosis type 1 (NF1) have attention-deficit disorder with or without hyperactivity (AD[H]D). This study explored the influence of AD(H)D symptoms on the intellectual profile of patients with NF1., Method: We retrospectively analysed neuropsychological data from 114 children (66 males, 48 females; age range 6-16y; mean age 9y 3mo [SE 3mo]) with NF1 from an NF1 outpatients department. Assessment included psychiatric diagnosis of AD(H)D (DSM-IV-TR criteria) and intelligence testing (Wechsler Intelligence Scale for Children, German version). Magnetic resonance images were available for all patients, intracranial findings being an exclusion criterion. The effects of AD(H)D symptoms on intelligence and on the cognitive profile were tested by analyses of variance., Results: Patients with AD(H)D symptoms performed significantly worse than those without AD(H)D symptoms on all intelligence measures (main effects for Full-scale, Verbal, and Performance IQ; p<0.005). Subtests typically impaired in patients with NF1 (visuospatial skills and arithmetic) were not specifically influenced by AD(H)D symptoms. There were no differences between AD(H)D subtypes., Interpretation: AD(H)D symptoms have a negative impact on the intellectual development of children with NF1. This impact seems to be of an unspecific nature, with a general attenuation of the cognitive profile., (© The Authors. Developmental Medicine & Child Neurology © 2012 Mac Keith Press.)

Neurofibromatosis type-1 (NF1), resulting from NF1 gene loss of function, is characterized by an increased risk of developing benign and malignant peripheral nerve sheath tumors (MPNSTs). Whereas the cellular heterogeneity of NF1-associated tumors has been well studied, the molecular heterogeneity of MPNSTs is still poorly understood. Mutational heterogeneity within these malignant tumors greatly complicates the study of the underlying mechanisms of tumorigenesis. We have explored this molecular heterogeneity by performing loss of heterozygosity (LOH) analysis of the NF1, TP53, RB1, PTEN, and CDKN2A genes on sections of 10 MPNSTs derived from 10 unrelated NF1 patients. LOH data for the TP53 gene was found to correlate with the results of p53 immunohistochemical analysis in the same tumor sections. Further, approximately 70% of MPNSTs were found to display intra-tumoral molecular heterogeneity as evidenced by differences in the level of LOH between different sections of the same tumor samples. This study constitutes the first systematic analysis of molecular heterogeneity within MPNSTs derived from NF1 patients. Appreciation of the existence of molecular heterogeneity in NF1-associated tumors is important not only for optimizing somatic mutation detection, but also for understanding the mechanisms of NF1 tumorigenesis, a prerequisite for the development of specifically targeted cancer therapeutics.

Patients with large deletions of the NF1 gene and its flanking regions (termed NF1 microdeletions) generally exhibit more severe clinical manifestations of neurofibromatosis type-1 (NF1). Here, we have investigated the clinical phenotype displayed by eight patients harbouring mosaic type-2 NF1 microdeletions. These patients did not exhibit facial dysmorphism, attention deficit hyperactivity disorder, delayed cognitive development and/or learning disabilities, cognitive impairment, congenital heart disease, hyperflexibility of joints, large hands and feet, muscular hypotonia or bone cysts. All these features have previously been reported to be disproportionately associated with germline (i.e. non-mosaic) type-1 NF1 microdeletions as compared with the general NF1 population. Plexiform neurofibromas were also less prevalent in patients with mosaic type-2 NF1 microdeletions as compared with patients carrying constitutional (germline) type-1 NF1 microdeletions. Five of the eight patients with mosaic type-2 deletions investigated here had 20-250 cutaneous neurofibromas, but only one of them exhibited a high load of cutaneous neurofibromas (N > 1,000). By contrast, a previous study indicated a high burden of cutaneous neurofibromas (N > 1,000) in 50% of adult patients with germline type-1 NF1 deletions. Patients with germline type-1 NF1 microdeletions have been reported to have an increased lifetime risk of 16-26% for a malignant peripheral nerve sheath tumour (MPNST). In this study, one of the eight investigated mosaic type-2 microdeletion patients developed an MPNST. We conclude that patients with mosaic type-2 NF1 microdeletions may also be at an increased risk of MPNSTs despite their generally milder disease manifestations as compared with germline type-1 NF1 microdeletions.

Objective: To demonstrate incidental findings and scoliosis on whole-body MRI (WBMRI) in patients with neurofibromatosis type 1 and 2 (NF1 & NF2, respectively), and schwannomatosis., Materials and Methods: Institutional review board approval and written informed consent were obtained for this prospective HIPAA-compliant study. A total of 247 subjects (141 with NF1, 55 with NF2, 51 with schwannomatosis; 132 women (53.5%); mean age, 41 years, range, 18-97 years) underwent WBMRI using coronal STIR (TR/TE: 4190/111 ms, TI: 150 ms) and T1-weighted images (TR/TE: 454/10 ms), 10-mm slice thickness, imaging time ~40 min. Images were reviewed for the presence of incidental findings, outside of nerve sheath tumors. The presence of scoliosis was recorded and curve morphology was assessed and quantified., Results: Incidental findings other than scoliosis were recorded in 104/247 (42%) patients, most often affecting the musculoskeletal system (65/247 patients, 26%). We found 16/247 (6.5%) significant incidental findings likely to affect clinical management, including avascular necrosis of bone in eight patients (five with NF2), eight insufficiency fractures, and four non-neurogenic neoplasms (Hodgkin's lymphoma, liposarcoma, dermoid cyst, large uterine myoma requiring excision). Scoliosis was seen in 50/247 patients (20%), including 8/55 with NF2 (15%) and 11/51 with schwannomatosis (22%)., Conclusions: Incidental findings in the neurofibromatoses frequently involve the skeleton. Given the relatively high incidence of unsuspected osteonecrosis and stress fractures, close attention to the skeleton on WBMRI is advised. In addition, knowledge of common incidental findings can help clinicians prepare patients who undergo WBMRI for potential unexpected findings.

Cancer stem cells (CSCs) are believed to be the regenerative pool of cells responsible for repopulating tumors. Gaining knowledge about the signaling characteristics of CSCs is important for understanding the biology of tumors and developing novel anti-cancer therapies. We have identified a subpopulation of cells positive for CD133 (a CSC marker) from human primary malignant peripheral nerve sheath tumor (MPNST) cells which were absent in non-malignant Schwann cells. CD133 was also found to be expressed in human tissue samples and mouse MPNST cells. CD133+ cells were capable of forming spheres in non-adherent/serum-free conditions. The activation levels of Ras and its downstream effectors such as ERK, JNK, PI3K, p38K, and RalA were significantly increased in this population. Moreover, the CD133+ cells showed enhanced invasiveness which was linked to the increased expression of β-Catenin and Snail, two important proteins involved in the epithelial to mesenchymal transition, and Paxilin, a focal adhesion protein. Among other important characteristics of the CD133+ population, endoplasmic reticulum stress marker IRE1α was decreased, implying the potential sensitivity of CD133+ to the accumulation of unfolded proteins. Apoptotic indicators seemed to be unchanged in CD133+ cells when compared to the wild (unsorted) cells. Finally, in order to test the possibility of targeting CD133+ MPNST cells with Ras pathway pharmacological inhibitors, we exposed these cells to an ERK inhibitor. The wild population was more sensitive to inhibition of proliferation by this inhibitor as compared with the CD133+ cells supporting previous studies observing enhanced chemoresistance of these cells.

We analyzed the histologic features of peripheral nerve sheath tumors occurring in 14 patients with schwannomatosis. Among a total of 31 tumors, 19 tumors (61%) showed schwannoma-like nodules within a neurofibroma-like tumor, corresponding to hybrid neurofibroma/schwannoma. At least 1 hybrid tumor occurred in 10 of 14 (71%) schwannomatosis patients. We then retrieved cases of hybrid tumors without documented relation to schwannomatosis from our database and identified 41 tumors arising in 23 patients. More than half of these patients (14/23) were reported to suffer from multiple peripheral nerve sheath tumors, favoring a tumor syndrome. Indeed, analysis of clinical records revealed the diagnosis of neurofibromatosis type 2 (NF2) in 26% (6/23), neurofibromatosis type 1 (NF1) in 9% (2/23), definite schwannomatosis in 4% (1/23), and possible schwannomatosis in 13% (3/23) of patients with multiple nerve sheath tumors. Our findings suggest that hybrid neurofibroma/schwannoma represents a common tumor type in schwannomatosis and shows a striking association with neurofibromatoses.

Neurofibromatosis Type 1 (NF1) is a frequent tumor suppressor gene disorder characterized by multiple benign tumors and high risk of malignancy. Internal tumor burden is a major disease-associated manifestation and can be most adequately assessed by magnetic resonance imaging of the whole body. Approximately 5% of NF1 patients have constitutional large NF1-deletions that are generally associated with more severe clinical manifestations. Here, we investigated whether these deletion patients also have more and/or larger internal tumors by assessing internal tumors and their total volume (exclusive of cutaneous and subcutaneous) in 38 NF1 deletion patients (including eight mosaic cases) and 114 age- and gender-matched NF1 patients without deletions. The incidence of internal tumors was significantly lower in mosaic deletion patients (1/8 = 13%) but did not differ between the 30 nonmosaic deletion patients and the 90 age- and gender-matched NF1 patients without large deletions used as controls. Neither the number nor the total volume of tumors per patient differed significantly between the latter two groups. However, extremely high tumor burden (>3,000 ml) was significantly more frequent among nonmosaic NF1 deletion patients than among NF1 patients without large deletions (13% vs. 1%, P = 0.014). Thus, as a group, patients with NF1 deletions do not exhibit a significantly higher internal tumor burden than NF1 patients without such deletions. However, deletion patients can frequently have extremely large internal tumors and thus demand special attention., (Copyright © 2012 Wiley Periodicals, Inc.)

Neurofibromatosis type-1 (NF1) is associated with the growth of benign and malignant tumors. Approximately 15% of NF1 patients develop malignant peripheral nerve sheath tumors (MPNSTs), underlining the need to identify specific diagnostic/prognostic biomarkers associated with MPNST development. The Affymetrix Genome-Wide Human single-nucleotide polymorphism (SNP) Array 6.0 was used to perform SNP genotyping and copy number alteration (CNA), loss-of-heterozygosity (LOH), and copy number neutral-LOH (CNN-LOH) analyses of DNA isolated from 15 MPNSTs, five benign plexiform neurofibromas (PNFs), and patient-matched lymphocyte DNAs. MPNSTs exhibited high-level CNN-LOH, with recurrent changes occurring in MPNSTs but not PNFs. CNN-LOH was evident in MPNSTs but occurred less frequently than genomic deletions. CNAs involving the ITGB8, PDGFA, Ras-related C3 botulinum toxin substrate 1 (RAC1) (7p21-p22), PDGFRL (8p22-p21.3), and matrix metallopeptidase 12 (MMP12) (11q22.3) genes were specific to MPNSTs. Pathway analysis revealed the MPNST-specific amplification of seven Rho-GTPase pathway genes and several cytoskeletal remodeling/cell adhesion genes. In knockdown experiments employing short-hairpin RAC1, ROCK2, PTK2, and LIMK1 RNAs to transfect both control and MPNST-derived cell lines, cell adhesion was significantly increased in the MPNST cell lines, whereas wound healing, cell migration, and invasiveness were reduced, consistent with a role for these Rho-GTPase pathway genes in MPNST development and metastasis. These results suggest new targets for therapeutic intervention in relation to MPNSTs., (© 2012 Wiley Periodicals, Inc.)

Type-2 NF1 deletions spanning 1.2 Mb are frequently of postzygotic origin and hence tend to be associated with mosaicism for normal cells and those harboring the deletion (del(+/-) cells). Eleven patients with mosaic type-2 deletions were investigated by FISH and high proportions (94-99%) of del(+/-) cells were detected both in whole blood and in isolated CD3+, CD14+, CD15+, and CD19+ leukocytes. Significantly lower proportions of del(+/-) cells (24-82%) were however noted in urine-derived epithelial cells. A patient harboring an atypical large NF1 deletion with nonrecurrent breakpoints was also found to have a much higher proportion of del(+/-) cells in blood (96%) than in urine (51%). The tissue-specific differences in the proportions of del(+/-) cells as well as the X chromosome inactivation (XCI) patterns observed in these mosaic patients suggest that the majority of the deletions had occurred before or during the preimplantation blastocyst stage before the onset of XCI. We postulate that hematopoietic del(+/-) stem cells present at an early developmental stage are characterized by a selective growth advantage over normal cells lacking the deletion, leading to a high proportion of del(+/-) cells in peripheral blood from the affected patients., (© 2011 Wiley Periodicals, Inc.)

Type 1 neurofibromatosis (NF-1), also known as von Recklinghausen disease, is caused by a disorder of a single gene on chromosome 17 that usually restrains cell division. A sequence that is frequently associated with NF-1 is tumor progression from neurofibromas to malignant peripheral nerve sheath tumors (MPNSTs). The aim of this study was to determine the expression of the neural L1 cell adhesion molecule in dermal-diffuse neurofibromas, plexiform neurofibromas, and MPNSTs of NF-1. We retrospectively analyzed surgically resected primary tumors, including 20 dermal neurofibromas, 23 plexiform neurofibromas, and 17 MPNSTs, by immunohistochemistry in paraffin sections of NF-1 tumors with the use of the L1-specific monoclonal antibody UJ127, which does not cross-react with other members of the L1 family. Immunostainings for CD34 and S100 were included to distinguish and allocate L1-expressing Schwann cells and perineural (specialized) fibroblasts. Our data showed that L1 is highly expressed in all benign NF-1 tumors and in some but not all MPNSTs. Furthermore, we demonstrated a correlation between L1 expression and differentiation grade of MPNSTs. There was a significant trend toward lower or nondetectable expression in the poorly differentiated MPNSTs, in contrast to all other tumor entities so far investigated, in which L1 expression correlated positive with malignancy, except for juvenile but not adult-derived neuroblastomas. Future studies are warranted to elucidate the molecular basis of the varying effects of the degree of L1 expression, receptor, and signal transduction mechanisms in different tumors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nonallelic homologous recombination (NAHR) is the major mechanism underlying recurrent genomic rearrangements, including the large deletions at 17q11.2 that cause neurofibromatosis type 1 (NF1). Here, we identify a novel NAHR hotspot, responsible for type-3 NF1 deletions that span 1.0 Mb. Breakpoint clustering within this 1-kb hotspot, termed PRS3, was noted in 10 of 11 known type-3 NF1 deletions. PRS3 is located within the LRRC37B pseudogene of the NF1-REPb and NF1-REPc low-copy repeats. In contrast to other previously characterized NAHR hotspots, PRS3 has not developed on a preexisting allelic homologous recombination hotspot. Furthermore, the variation pattern of PRS3 and its flanking regions is unusual since only NF1-REPc (and not NF1-REPb) is characterized by a high single nucleotide polymorphism (SNP) frequency, suggestive of unidirectional sequence transfer via nonallelic homologous gene conversion (NAHGC). By contrast, the previously described intense NAHR hotspots within the CMT1A-REPs, and the PRS1 and PRS2 hotspots underlying type-1 NF1 deletions, experience frequent bidirectional sequence transfer. PRS3 within NF1-REPc was also found to be involved in NAHGC with the LRRC37B gene, the progenitor locus of the LRRC37B-P duplicons, as indicated by the presence of shared SNPs between these loci. PRS3 therefore represents a weak (and probably evolutionarily rather young) NAHR hotspot with unique properties., (© 2011 Wiley Periodicals, Inc.)

Purpose: Patients with neurofibromatosis 1 (NF1), NF2, and schwannomatosis are at risk for multiple nerve sheath tumors and premature mortality. Traditional magnetic resonance imaging (MRI) has limited ability to assess disease burden accurately. The aim of this study was to establish an international cohort of patients with quantified whole-body internal tumor burden and to correlate tumor burden with clinical features of disease., Methods: We determined the number, volume, and distribution of internal nerve sheath tumors in patients using whole-body MRI (WBMRI) and three-dimensional computerized volumetry. We quantified the distribution of tumor volume across body regions and used unsupervised cluster analysis to group patients based on tumor distribution. We correlated the presence and volume of internal tumors with disease-related and demographic factors., Results: WBMRI identified 1286 tumors in 145/247 patients (59%). Schwannomatosis patients had the highest prevalence of tumors (P = 0.03), but NF1 patients had the highest median tumor volume (P = 0.02). Tumor volume was unevenly distributed across body regions with overrepresentation of the head/neck and pelvis. Risk factors for internal nerve sheath tumors included decreasing numbers of café-au-lait macules in NF1 patients (P = 0.003) and history of skeletal abnormalities in NF2 patients (P = 0.09). Risk factors for higher tumor volume included female gender (P = 0.05) and increasing subcutaneous neurofibromas (P = 0.03) in NF1 patients, absence of cutaneous schwannomas in NF2 patients (P = 0.06), and increasing age in schwannomatosis patients (p = 0.10)., Conclusion: WBMRI provides a comprehensive phenotype of neurofibromatosis patients, identifies distinct anatomic subgroups, and provides the basis for investigating molecular biomarkers that correlate with unique disease manifestations.

Aim: To evaluate the impact of disease visibility on psychological stress factors in neurofibromatosis type 1 (NF1) and to explore the body image of NF1 patients., Methods: 228 adult NF1 patients participated in this cross-sectional survey. The questionnaire assessed perceived disease visibility and patients' body image. Outcome parameters were depression, distress and quality of life. Mediation models were performed to test if body experience mediated the effect of disease visibility on outcome parameters., Results: Adult NF1 patients had a negative body image, expressed by bodily insecurity/uneasiness and fewer feelings of attractiveness and self-confidence. Compared to the body image of patients with other disfiguring diseases, patients with NF1 felt less attractive, displayed less self-confidence (women: p < 0.001; men: p < 0.001) and were more insecure/uneasy and sexually dissatisfied with their bodies (men: p < 0.001; women: p ≤ 0.12, d = 0.28). The found effect of disease visibility on psychological stress was completely mediated by how patients experienced their bodies., Conclusions: Our study shed light on the importance of how NF1 patients experience and appraise their own bodies. We revealed that body image is an important link between disease visibility and psychological well-being. The body image of patients can be improved with psychotherapeutic interventions., (Copyright © 2012 S. Karger AG, Basel.)

Neurofibromatosis type 2 (NF2) is a tumor suppressor syndrome characterized by bilateral vestibular schwannomas (VS) which often result in deafness despite aggressive management. Meningiomas, ependymomas, and other cranial nerve and peripheral schwannomas are also commonly found in NF2 and collectively lead to major neurologic morbidity and mortality. Traditionally, the overall survival rate in patients with NF2 is estimated to be 38% at 20 years from diagnosis. Hence, there is a desperate need for new, effective therapies. Recent progress in understanding the molecular basis of NF2 related tumors has aided in the identification of potential therapeutic targets and emerging clinical therapies. In June 2010, representatives of the international NF2 research and clinical community convened under the leadership of Drs. D. Gareth Evans (University of Manchester) and Marco Giovannini (House Research Institute) to review the state of NF2 treatment and clinical trials. This manuscript summarizes the expert opinions about current treatments for NF2 associated tumors and recommendations for advancing therapies emerging from that meeting. The development of effective therapies for NF2 associated tumors has the potential for significant clinical advancement not only for patients with NF2 but for thousands of neuro-oncology patients afflicted with these tumors., (Copyright © 2011 Wiley Periodicals, Inc.)

Malignant peripheral nerve sheath tumours (MPNST) are aggressive sarcomas that develop in about 10% of patients with the genetic disease neurofibromatosis type 1 (NF1). Molecular alterations contributing to MPNST formation have only partially been resolved. Here we examined the role of Pten, a key regulator of the Pi3k/Akt/mTOR pathway, in human MPNST and benign neurofibromas. Immunohistochemistry showed that Pten expression was significantly lower in MPNST (n=16) than in neurofibromas (n=16) and normal nervous tissue. To elucidate potential mechanisms for Pten down-regulation or Akt/mTOR activation in MPNST we performed further experiments. Mutation analysis revealed absence of somatic mutations in PTEN (n=31) and PIK3CA (n=38). However, we found frequent PTEN promotor methylation in primary MPNST (11/26) and MPNST cell lines (7/8) but not in benign nerve sheath tumours. PTEN methylation was significantly associated with early metastasis. Moreover, we detected an inverse correlation of Pten-regulating miR-21 and Pten protein levels in MPNST cell lines. The examination of NF1-/- and NF1+/+Schwann cells and fibroblasts showed that Pten expression is not regulated by NF1. To determine the significance of Pten status for treatment with the mTOR inhibitor rapamycin we treated 5 MPNST cell lines with rapamycin. All cell lines were sensitive to rapamycin without a significant correlation to Pten levels. When rapamycin was combined with simvastatin a synergistic anti-proliferative effect was achieved. Taken together we show frequent loss/reduction of Pten expression in MPNST and provide evidence for the involvement of multiple Pten regulating mechanisms.

Introduction: Large deletions of the NF1 gene and its flanking regions are frequently associated with a severe clinical manifestation. Different types of gross NF1 deletion have been identified that are distinguishable both by their size and the number of genes included within the deleted regions. Type-1 NF1 deletions encompass 1.4 Mb and include 14 genes, whereas the much less common type-2 NF1 deletions span 1.2 Mb and contain 13 genes. Genotype-phenotype correlations in patients with large NF1 deletions are likely to be influenced by the nature and number of the genes deleted in addition to the NF1 gene. Whereas the clinical phenotype associated with type-1 NF1 deletions has been well documented, the detailed clinical characterization of patients with non-mosaic type-2 NF1 deletions has not so far been reported., Case Presentation: In the present report we characterized two Caucasian European patients with non-mosaic (germline) type-2 NF1 deletions. Our first patient was a 13-year-old girl with dysmorphic facial features, mild developmental delay, large hands and feet, hyperflexibility of the joints, macrocephaly and T2 hyperintensities in the brain. A whole-body magnetic resonance imaging scan indicated two internal plexiform neurofibromas. Our second patient was an 18-year-old man who exhibited dysmorphic facial features, developmental delay, learning disability, large hands and feet, hyperflexibility of the joints, macrocephaly and a very high subcutaneous and internal tumor load as measured volumetrically on whole-body magnetic resonance imaging scans. At the age of 18 years, he developed a malignant peripheral nerve sheath tumor and died from secondary complications. Both our patients exhibited cardiovascular malformations., Conclusions: Our two patients with non-mosaic type-2 NF1 deletions exhibited clinical features that have been reported in individuals with germline type-1 NF1 deletions. Therefore, a severe disease manifestation is not confined to only patients with type-1 NF1 deletions but may also occur in individuals with type-2 NF1 deletions. Our findings support the concept of an NF1 microdeletion syndrome with severe clinical manifestation that is caused by type-1 as well as type-2 NF1 deletions.

Objectives: To define the frequency and clinical features of plexiform neurofibromas (PN) in children with neurofibromatosis type 1., Study Design: Sixty-five children received whole-body magnetic resonance imaging (MRI) and clinical-neurologic examination. Tumor sizes were calculated volumetrically with the program MedX v3.42. χ(2) test, Fisher exact test, t test, and Spearman rank correlation were used for statistical analysis., Results: Seventy-three tumors were detected in 37 of these 65 children. The mean volume of the tumors was 145.4 mL or 4.8 mL/kg body weight. Eighteen of the 73 PNs caused clinical deficits in 17 children, and the other 56 PNs in 20 children were asymptomatic. Symptomatic tumors were larger than asymptomatic ones (9.6 vs 3.3 mL/kg body weight; P = .01). However, in certain body regions, for example, the head, small tumors also caused clinical deficits. Ten of 18 children ≥11.5 years (median age of the 37 children with PNs) had symptomatic PNs compared with 7 of 19 who were <11.5 years (P = .25)., Conclusion: PNs cause clinical deficits in young children. Early detection and regular MRI monitoring help to estimate growth and possible upcoming complications, and are thus beneficial for optimizing treatment and management., (Copyright © 2011 Mosby, Inc. All rights reserved.)

This study examined the effects of 22 putative splicing mutations in the NF2 gene by means of transcript analysis and information theory based prediction. Fourteen mutations were within the dinucleotide acceptor and donor regions, often referred to as (AG/GT) sequences. Six were outside these dinucleotide regions but within the more broadly defined splicing regions used in the information theory based model. Two others were in introns and outside the broadly defined regions. Transcript analysis revealed exon skipping or activation of one or more cryptic splicing sites for 17 mutations. No alterations were found for the two intronic mutations and for three mutations in the broadly defined splicing regions. Concordance and partial concordance between the calculated predictions and the results of transcript analysis were found for 14 and 6 mutations, respectively. For two mutations, the predicted alteration was not found in the transcripts. Our results demonstrate that the effects of splicing mutations in NF2 are often complex and that information theory based analysis is helpful in elucidating the consequences of these mutations., (Copyright © 2011 Wiley-Liss, Inc.)

Patients suffering from neurofibromatosis type 1 and 2 (NF1 and NF2) are the main risk groups to develop peripheral nerve sheath tumours (PNSTs). In the present study, adhesion molecules CD34 and podoplanin were assessed in regard to their value for tumour classification and as indicators for tumour progression. A total of 103 NF1-, NF2- and schwannomatosis-associated neurofibromas, schwannomas and malignant peripheral nerve sheath tumours (MPNST), as well as 20 sporadic vestibular schwannomas and 9 control tissue samples, were labelled immunohistochemically for detection of podoplanin and CD34. CD34 was shown to be expressed in MPNST and all benign PNSTs except for the compact cellular parts of both, schwannomas and atypical neurofibromas. Podoplanin showed an inverse expression pattern to CD34 labelling mainly the compact parts of schwannoma and atypical neurofibroma. MPNSTs were characterized by strong podoplanin staining at the invasive front. NF1-patients who suffered from atypical neurofibromas did not develop MPNST at a higher frequency than other NF1-patients, although these tumours expressed podoplanin. Ki-67 proliferation indices did not differ significantly between neurofibromas, atypical neurofibromas and schwannomas. In accordance with other studies, CD34 was found to be of limited value for classification and grading of PNST due to its ubiquitous expression. Podoplanin expression in schwannoma and atypical neurofibroma adds to other phenotypic and genotypic similarities between these tumours, like nuclear atypia, regressive changes and euploid polyploidisation. Podoplanin expression in atypical neurofibroma was not associated with tumour progression towards MPNST.

The analysis of monozygotic twins (MZ) concordant for neurofibromatosis type 1 (NF1) has indicated that genetic factors exert a major influence on the clinical variability (e.g. the number of café-au-lait spots and/or neurofibromas) evident in this disease. Here, we report on a pair of monozygotic, dichorionic twins who are phenotypically discordant with respect to NF1. Whereas DNA sequence analysis indicated somatic mosaicism for the NF1 nonsense mutation, c.4108C>T (p.Q1370X), in the affected twin II/1, this lesion was apparently absent in his unaffected brother. The observation of heterozygosity for flanking SNP and microsatellite markers rendered it most unlikely that the observed mosaicism with normal cells was due to mutation reversion brought about either by gene conversion or mitotic recombination. Instead, we conclude that the twinning event, which would have taken place within three days post-fertilization, must have preceded the c.4108C>T mutation which is therefore predicted to have occurred during the blastocyst stage, leading to somatic mosaicism with normal cells lacking the mutation. This is the first reported case of monozygotic twins discordant for NF1 in whom mosaicism for a postzygotic NF1 gene mutation has been observed in the affected but not the unaffected twin., (© 2011 Wiley-Liss, Inc.)

This study aims to examine whether or not cancer stem cells exist in malignant peripheral nerve sheath tumors (MPNST). Cells of established lines, primary cultures and freshly dissected tumors were cultured in serum free conditions supplemented with epidermal and fibroblast growth factors. From one established human MPNST cell line, S462, cells meeting the criteria for cancer stem cells were isolated. Clonal spheres were obtained, which could be passaged multiple times. Enrichment of stem cell-like cells in these spheres was also supported by increased expression of stem cell markers such as CD133, Oct4, Nestin and NGFR, and decreased expression of mature cell markers such as CD90 and NCAM. Furthermore, cells of these clonal S462 spheres differentiated into Schwann cells, smooth muscle/fibroblast and neurons-like cells under specific differentiation-inducing cultural conditions. Finally, subcutaneous injection of the spheres into immunodeficient nude mice led to tumor formation at a higher rate compared to the parental adherent cells (66% versus 10% at 2.5 × 10(5)). These results provide evidence for the existence of cancer stem cell-like cells in malignant peripheral nerve sheath tumors.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder caused by heterozygotic inactivation of the NF1 tumor suppressor gene at 17q11.2. The associated phenotypes are highly variable, and modifying genes have been proposed to explain at least in part the intriguing expressivity. Given that haploinsufficiency of the NF1 gene product neurofibromin is responsible for some of the clinical manifestations, variations in expression of the wildtype NF1 allele might modify the phenotype. We therefore investigated epigenetic molecular modifications that could result in variable expression of the normal NF1 allele. To exclude confounding by DNA sequence variations, we analyzed monozygotic twin pairs with NF1 who presented with several discordant features. We fine-mapped the methylation pattern of a nearly 1 kb NF1 promoter region in lymphocytes of 8 twin pairs. All twin pairs showed significant intra-pair differences in methylation, especially of specific promoter subregions such as 5'UTR, exon 1 and intron 1 (+7 to +622), transcription factor binding sites and promoter elements like NF1HCS. Furthermore, we detected significant intra-pair differences in cytosine methylation for the region from -249 to -234 with regard to discordance for optic glioma with a higher grade of methylation in glioma cases. In conclusion, our findings of epigenetic differences of the NF1 promoter in leukocytes within mono zygotic twin pairs may serve as a proof of principle for other tissues. The results point towards a role of methylation patterns of the normal NF1 allele for expression differences and for modification of the NF1 phenotype.

Purpose: Plexiform neurofibromas (pNF) are Schwann cell tumors found in a third of individuals with neurofibromatosis type 1 (NF1). pNF can undergo transformation to malignant peripheral nerve sheath tumors (MPNST). There are no identified serum biomarkers of pNF tumor burden or transformation to MPNST. Serum biomarkers would be useful to verify NF1 diagnosis, monitor tumor burden, and/or detect transformation., Experimental Design: We used microarray gene expression analysis to define 92 genes that encode putative secreted proteins in neurofibroma Schwann cells, neurofibromas, and MPNST. We validated differential expression by quantitative reverse transcription-PCR, Western blotting, and ELISA assays in cell conditioned medium and control and NF1 patient sera., Results: Of 13 candidate genes evaluated, only adrenomedullin (ADM) was confirmed as differentially expressed and elevated in serum of NF1 patients. ADM protein concentrati on was further elevated in serum of a small sampling of NF1 patients with MPNST. MPNST cell conditioned medium, containing ADM and hepatocyte growth factor, stimulated MPNST migration and endothelial cell proliferation., Conclusions: Thus, microarray analysis identifies potential serum biomarkers for disease, and ADM is a serum biomarker of NF1. ADM serum levels do not seem to correlate with the presence of pNFs but may be a biomarker of transformation to MPNST., (©2010 AACR.)

Nonallelic homologous recombination (NAHR) is responsible for the recurrent rearrangements that give rise to genomic disorders. Although meiotic NAHR has been investigated in multiple contexts, much less is known about mitotic NAHR despite its importance for tumorigenesis. Because type-2 NF1 microdeletions frequently result from mitotic NAHR, they represent a good model in which to investigate the features of mitotic NAHR. We have used microsatellite analysis and SNP arrays to distinguish between the various alternative recombinational possibilities, thereby ascertaining that 17 of 18 type-2 NF1 deletions, with breakpoints in the SUZ12 gene and its highly homologous pseudogene, originated via intrachromosomal recombination. This high proportion of intrachromosomal NAHR causing somatic type-2 NF1 deletions contrasts with the interchromosomal origin of germline type-1 NF1 microdeletions, whose breakpoints are located within the NF1-REPs (low-copy repeats located adjacent to the SUZ12 sequences). Further, meiotic NAHR causing type-1 NF1 deletions occurs within recombination hotspots characterized by high GC-content and DNA duplex stability, whereas the type-2 breakpoints associated with the mitotic NAHR events investigated here do not cluster within hotspots and are located within regions of significantly lower GC-content and DNA stability. Our findings therefore point to fundamental mechanistic differences between the determinants of mitotic and meiotic NAHR., (Hum Mutat 31:1163-1173, 2010. © 2010 Wiley-Liss, Inc.)

Background: Large deletions of the NF1 gene region occur in approximately 5% of patients with neurofibromatosis type-1 (NF1) and are associated with particularly severe manifestations of the disease. However, until now, the genotype-phenotype relationship has not been comprehensively studied in patients harbouring large NF1 gene deletions of comparable extent (giving rise to haploinsufficiency of the same genes)., Method: We have performed the most comprehensive clinical/neuropsychological characterisation so far undertaken in NF1 deletion patients, involving 29 patients with precisely determined type-1 NF1 (1.4 Mb) deletions., Results: Novel clinical features found to be associated with type-1 NF1 deletions included pes cavus (17% of patients), bone cysts (50%), attention deficit (73%), muscular hypotonia (45%) and speech difficulties (48%). Type-1 NF1 deletions were found to be disproportionately associated with facial dysmorphic features (90% of patients), tall stature (46%), large hands and feet (46%), scoliosis (43%), joint hyperflexibility (72%), delayed cognitive development and/or learning disabilities (93%) and mental retardation (IQ<70; 38%), as compared with the general NF1 patient population. Significantly increased frequencies (relative to the general NF1 population) of plexiform neurofibromas (76%), subcutaneous neurofibromas (76%), spinal neurofibromas (64%) and MPNSTs (21%) were also noted in the type-1 deletion patients. Further, 50% of the adult patients exhibited a very high burden of cutaneous neurofibromas (N>or=1000)., Conclusion: These findings emphasise the importance of deletion analysis in NF1 since frequent monitoring of tumour presence and growth could potentiate early surgical intervention thereby improving patient survival.

Background: Glomus tumours are benign painful tumours of the glomus body, a thermoregulatory shunt in the digits. Glomus tumours of the fingers and toes are associated with the monogenic disorder neurofibromatosis type 1 (NF1) and are recently recognised as part of the NF1 phenotype., Methods and Results: A multi-institutional experience with 15 individuals with NF1 and glomus tumours of the fingers or toes is reported. The majority of individuals presented with at least two of the symptoms in the classic triad of localised tenderness, severe paroxysmal pain, and sensitivity to cold. Appearance of the nail and finger or toe is often normal. Women are affected more often than men. Multifocal tumours are common. There is often a delay in diagnosis of many years and clinical suspicion is key to diagnosis, although magnetic resonance imaging may be useful in some scenarios. Surgical extirpation can be curative; however, local tumour recurrence and metachronous tumours are common. Three of our patients developed signs and symptoms of the complex regional pain syndrome., Conclusions: Glomus tumours in NF1 are more common than previously recognised and NF1 patients should be specifically queried about fingertip or toe pain.

Large microdeletions encompassing the neurofibromatosis type-1 (NF1) gene and its flanking regions at 17q11.2 belong to the group of genomic disorders caused by aberrant recombination between segmental duplications. The most common NF1 microdeletions (type-1) span 1.4-Mb and have breakpoints located within NF1-REPs A and C, low-copy repeats (LCRs) containing LRRC37-core duplicons. We have identified a novel type of recurrent NF1 deletion mediated by nonallelic homologous recombination (NAHR) between the highly homologous NF1-REPs B and C. The breakpoints of these approximately 1.0-Mb ("type-3") NF1 deletions were characterized at the DNA sequence level in three unrelated patients. Recombination regions, spanning 275, 180, and 109-bp, respectively, were identified within the LRRC37B-P paralogues of NF1-REPs B and C, and were found to contain sequences capable of non-B DNA formation. Both LCRs contain LRRC37-core duplicons, abundant and highly dynamic sequences in the human genome. NAHR between LRRC37-containing LCRs at 17q21.31 is known to have mediated the 970-kb polymorphic inversions of the MAPT-locus that occurred independently in different primate species, but also underlies the syndromes associated with recurrent 17q21.31 microdeletions and reciprocal microduplications. The novel NF1 microdeletions reported here provide further evidence for the unusually high recombinogenic potential of LRRC37-containing LCRs in the human genome.

Plexiform neurofibromas (PNF), one of the major features of neurofibromatosis type 1 (NF1), are characterized by complex cellular composition and mostly slow but variable growth patterns. In this study, we examined the effect of imatinib mesylate, a receptor tyrosine kinase inhibitor, on PNF-derived Schwann cells and PNF tumour growth in vitro and in vivo. In vitro, PNF-derived primary Schwann cells express platelet-derived growth factors receptors (PDGFR) alpha and beta, both targets of imatinib, and cell viability was reduced by imatinib mesylate, with 50% inhibition concentration (IC(50)) of 10 microM. For in vivo studies, PNF tumour fragments xenografted onto the sciatic nerve of athymic nude mice were first characterized. The tumours persisted for at least 63 days and maintained typical characteristics of PNFs such as complex cellular composition, low proliferation rate and angiogenesis. A transient enlargement of the graft size was due to inflammation by host cells. Treatment with imatinib mesylate at a daily dose of 75 mg/kg for 4 weeks reduced the graft size by an average of 80% (n = 8), significantly different from the original sizes within the group and from sizes of the grafts in 11 untreated mice in the control group (P < 0.001). We demonstrated that grafting human PNF tumour fragments into nude mice provides an adequate in vivo model for drug testing. Our results provide in vivo and in vitro evidence for efficacy of imatinib mesylate for PNF.

Large deletions in the NF1 gene region at 17q11.2 are caused by nonallelic homologous recombination (NAHR). The recurrent type-2 NF1 deletions span 1.2 Mb, with breakpoints in the SUZ12 gene and SUZ12P. Type-2 NF1 deletions occur preferentially during mitosis and are associated with somatic mosaicism. A panel of 16 type-2 NF1 deletions was used as a model system in which to investigate whether extended homozygosity across 17q11.2 might be associated with somatic deletion. Using SNP arrays, a 3.2 Mb interval encompassing the NF1 deletion region was found to harbor runs of homozygosity (ROHs) in different human populations. However, ROHs >or=500 kb directly flanking the NF1 deletion region on both sides were not found to occur disproportionately in NF1 patients harboring type-2 deletions compared to controls. Although low allelic diversity in 17q11.2 is unlikely to be a key factor in promoting NAHR-mediated somatic type-2 deletions, a specific ROH of 588 kb (roh1), located some 525 kb proximal to the deletion interval, was found to occur more frequently (P=0.012) in the type-2 deletion patients compared with controls. We postulate that roh1 may act remotely, via an as yet unknown mechanism, to increase the frequency of somatic recombination between the distally duplicated SUZ12 sequences., ((c) 2010 Wiley-Liss, Inc.)

Unlabelled: Although it is known that neurofibromatosis 1 (NF1) patients suffer from vitamin D deficiency and display decreased bone mineral density (BMD), a systematic clinical and histomorphometrical analysis is absent. Our data demonstrate that NF1 patients display high bone turnover and accumulation of osteoid and that supplementation of vitamin D has a beneficial effect on their BMD., Introduction: Neurofibromatosis 1 results in a wide range of clinical manifestations, including decreased BMD. Although it has been reported that NF1 patients have decreased vitamin D serum levels, the manifestation of the disease at the bone tissue level has rarely been analyzed., Methods: Thus, we performed a clinical evaluation of 14 NF1 patients in comparison to age- and sex-matched control individuals. The analysis included dual X-ray absorptiometry osteodensitometry, laboratory parameters, histomorphometric and quantitative backscattered electron imaging (qBEI) analyses of undecalcified bone biopsies., Results: NF1 patients display significantly lower 25-(OH)-cholecalciferol serum levels and decreased BMD compared to control individuals. Histomorphometric analysis did not only reveal a reduced trabecular bone volume in biopsies from NF1 patients, but also a significantly increased osteoid volume and increased numbers of osteoblasts and osteoclasts. Moreover, qBEI analysis revealed a significant decrease of the calcium content in biopsies from NF1 patients. To address the question whether a normalization of calcium homeostasis improves BMD in NF1 patients, we treated four patients with cholecalciferol for 1 year, which resulted in a significant increase of BMD., Conclusion: Taken together, our data provide the first complete histomorphometric analysis from NF1 patients. Moreover, they suggest that low vitamin D levels significantly contribute to the skeletal defects associated with the disease.

Bilateral vestibular schwannomas are the hallmark of neurofibromatosis type 2 (NF2), and these tumors impair hearing and frequently lead to deafness. Neurosurgical intervention, the only established treatment, often damages the vestibular nerve. We report 2 cases in which treatment with bevacizumab (for 3 months in one case and 6 months in the other) induced regression of progressive vestibular schwannomas by more than 40% and substantially improved hearing in the patient treated for 6 months. Bevacizumab therapy may thus provide an effective treatment for progressive vestibular schwannomas in patients with NF2.

Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.

Background: Patients with neurofibromatosis 1 (NF1) are shorter than expected and often have low bone mineral density (BMD), but the pathogenesis of these bony problems is poorly understood., Methods: We performed an exploratory study of BMD, 18 laboratory measures of bone metabolism, and fracture history in 72 adult NF1 patients., Results: Eight of the 18 clinical biochemical measures of bone health had at least 10% of NF1 patients outside the standard reference range. Serum 25-hydroxy-vitamin D concentrations were low in 56% of the NF1 patients, serum parathyroid hormone (PTH) concentrations were high in 34%, and urine deoxypyridinoline cross-link concentrations were high in 50%. Mean serum 25-hydroxy-vitamin D concentrations were significantly lower in people with NF1 than in season matched controls in both summer (p = 0.008) and winter (p<0.001). 36 (50%) of the 72 people with NF1 studied had BMD consistent with osteopenia, and 14 (19%) had BMD consistent with osteoporosis. High serum PTH concentration, high serum bone tartrate resistant acid phosphatase concentration, and high serum calcium concentration were associated with lower BMD among the NF1 patients. Males were more likely than females to have low BMD. The reported frequency of fractures in individuals with NF1 was much higher than in their unaffected siblings and spouses (p<0.001), and pathological fractures were reported only in NF1 patients., Conclusion: People with NF1 often have a generalised abnormality of bone metabolism. Further studies are needed to determine the biochemical and molecular basis of this abnormality.