Functional interactions between neurofibromatosis tumor suppressors underlie Schwann cell tumor de-differentiation and treatment resistance.

Schwann cell tumors are the most common cancers of the peripheral nervous system and can arise in patients with neurofibromatosis type-1 (NF-1) or neurofibromatosis type-2 (NF-2). Functional interactions between NF1 and NF2 and broader mechanisms underlying malignant transformation of the Schwann lineage are unclear. Here we integrate bulk and single-cell genomics, biochemistry, and pharmacology across human samples, cell lines, and mouse allografts to identify cellular de-differentiation mechanisms driving malignant transformation and treatment resistance. We find DNA methylation groups of Schwann cell tumors can be distinguished by differentiation programs that correlate with response to the MEK inhibitor selumetinib. Functional genomic screening in NF1-mutant tumor cells reveals NF2 loss and PAK activation underlie selumetinib resistance, and we find that concurrent MEK and PAK inhibition is effective in vivo. These data support a de-differentiation paradigm underlying malignant transformation and treatment resistance of Schwann cell tumors and elucidate a functional link between NF1 and NF2.

Status and Recommendations for Incorporating Biomarkers for Cutaneous Neurofibromas Into Clinical Research.

OBJECTIVE: To summarize existing biomarker data for cutaneous neurofibroma (cNF) and to inform the incorporation of biomarkers into clinical trial design for cNFs. METHODS: The cNF working group, a subgroup of the Response Evaluation in Neurofibromatosis and Schwannomatosis (REiNS) consortium, was formed to review and inform clinical trial design for cNFs. Between June 2018 and February 2020, the cNF working group performed a review of existing data on genetic biomarkers for cNFs in the setting of neurofibromatosis type 1. We also reviewed criteria for successful biomarker application in the clinic. The group then held a series of meetings to develop a consensus report. RESULTS: Our systematic literature review of existing data revealed a lack of validated biomarkers for cNFs. In our report, we summarize the existing signaling, genomic, transcriptomic, histopathologic, and proteomic data relevant to cNF. Finally, we make recommendations for incorporating exploratory aims for predictive biomarkers into clinical trials through biobanking samples. CONCLUSION: These recommendations are intended to provide both researchers and clinicians with best practices for clinical trial design to aid in the identification of clinically validated biomarkers for cNF.

Enhancing Neurofibromatosis Clinical Trial Outcome Measures Through Patient Engagement: Lessons From REiNS.

OBJECTIVE: As part of an evaluation of the Response Evaluation in Neurofibromatosis and Schwannomatosis (REiNS) International Collaboration patient representative program, we surveyed REiNS members to (1) identify facilitators and barriers to involving patient representatives and (2) understand whether and how involving patient representatives affected recommendations for clinical trial outcomes. METHODS: We administered an anonymous online survey to all REiNS members. Facilitators and barriers to patient representative involvement were solicited using a modified free listing technique; responses were inductively grouped into higher-order categories and ranked based on saliency score (Smith s). Open-ended questions assessed patient representative expectations for engagement, perceived benefits/costs of patient engagement, and patient representative contributions; responses were analyzed using conventional content analysis. RESULTS: A total of 63/172 (37%) members responded, including 18/30 (60%) patient representatives. Providing sufficient opportunities to meaningfully engage in research tasks and cultivating a respectful, inclusive atmosphere were key facilitators to patient representatives' satisfaction and ability to make an impact. Respondents perceived that patient representatives directly (through their input on research tasks) and indirectly (through effects on other stakeholders' knowledge and communication style) improved the organization's research, leading to selection of more meaningful, relevant, and feasible clinical trial outcome measures. Ongoing challenges to patient engagement include difficulty scheduling meetings and concerns about the level of scientific knowledge patient representatives needed to effectively engage. CONCLUSIONS: Involving patient representatives in REiNS improved perceived quality of neurofibromatosis clinical trial outcome measures. Negotiating sufficient opportunities to engage, fostering an inclusive atmosphere, and navigating time pressures are key to effective patient engagement.

Increased Prostate-Specific Membrane Antigen Uptake in Neurofibromatosis.

A 51-year-old man with 30-year neurofibromatosis and 2-month elevated prostate-specific antigen and back pain underwent a Ga-prostate-specific membrane antigen (PSMA) PET/CT scan for possible prostate cancer. Prostate-specific membrane antigen PET/CT imaging showed no abnormal uptake of the prostate. However, in addition to PSMA uptake in his left lung, thorax, and right ilium, which was confirmed being a lung squamous cell carcinoma by a lung biopsy, widespread uptake was also observed in his skin fibroma lesions. This case demonstrates that benign neurofibromatosis could have uptake of PSMA.

Activation of PLCγ/AKT/IκBα/p65 signaling increases inflammation in mast cells to promote growth of cutaneous neurofibroma.

AIM: Cutaneous neurofibroma (cNF), a hallmark feature of neurofibromatosis type 1 (NF1), results in psychological and physical damage to patients. Considering the important role of mast cells in neurofibroma development, the aim of this study was to elucidate the underlying mechanism of the interaction between cNF cells and mast cells. MAIN METHODS: SW10 cells with Nf1 knocked down were used as a cNF cell model. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays, as well as a mouse xenograft tumor model, were used to assess the cNF tumor growth in vivo and in vitro. ELISAs and IHC were used to examine the inflammatory activity of mast cells. KEY FINDINGS: We demonstrated that cNF cells activated mast cells, which in turn promoted the cNF cell growth, while suppression of the inflammatory activity of cNF-associated mast cells reversed their stimulating effect on the growth of cNF cells. Mechanistic studies revealed that SW10 cells upregulated PLCγ/AKT/IκBα/p65 signaling in mast cells, thereby increasing inflammation. Moreover, PLCγ modulated the AKT/IκBα/p65 signaling activity and played a critical role in the interaction of mast cells and cNF cells. Knockdown of PLCγ in mast cells diminished their cNF cell-induced inflammatory activity and subsequently reduced the cNF cell growth in vivo and in vitro. SIGNIFICANCE: This study revealed a novel interaction between mast cells and cNF cells, suggesting a potential strategy for treating cNF by targeting the newly recognized signaling pathway.

The Secretomes of Painful Versus Nonpainful Human Schwannomatosis Tumor Cells Differentially Influence Sensory Neuron Gene Expression and Sensitivity.

Schwannomatosis is a multiple tumor syndrome in which patients develop benign tumors along peripheral nerves throughout the body. The first symptom with which schwannomatosis patients often present, prior to discovery of tumors, is pain. This pain can be debilitating and is often inadequately alleviated by pharmacological approaches. Schwannomatosis-associated pain can be localized to the area of a tumor, or widespread. Moreover, not all tumors are painful, and the occurrence of pain is often unrelated to tumor size or location. We speculate that some individual tumors, but not others, secrete factors that act on nearby nerves to augment nociception by producing neuronal sensitization or spontaneous neuronal firing. We created cell lines from human SWN tumors with varying degrees of pain. We have found that conditioned medium (CM) collected from painful SWN tumors, but not that from nonpainful SWN tumors, sensitized DRG neurons, causing increased sensitivity to depolarization by KCl, increased response to noxious TRPV1 and TRPA1 agonists and also upregulated the expression of pain-associated genes in DRG cultures. Multiple cytokines were also detected at higher levels in CM from painful tumors. Taken together our data demonstrate a differential ability of painful versus non-painful human schwannomatosis tumor cells to secrete factors that augment sensory neuron responsiveness, and thus identify a potential determinant of pain heterogeneity in schwannomatosis.

From process to progress-2017 International Conference on Neurofibromatosis 1, Neurofibromatosis 2 and Schwannomatosis.

The neurofibromatoses are inherited, tumor suppressor disorders that are characterized by multiple, benign peripheral nerve sheath tumors and other nervous system tumors. Each disease is associated with a distinct genetic mutation and with a different pathogenesis and clinical course. Neurofibromatosis 1 (NF1) is common and epitomized by multiple neurofibromas with widespread complications. NF2 and schwannomatosis are rare diseases that are typified by multiple schwannomas that are particularly painful in people with schwannomatosis. Since 1985, the Children's Tumor Foundation (formerly the National Neurofibromatosis Foundation) has hosted an international Neurofibromatosis Conference, bringing together international participants who are focused on NF research and clinical care. The 2017 Conference, held in Washington, DC, was among the largest gatherings of NF researchers to date and included presentations from clinicians and basic scientists, highlighting new data regarding the molecular and cellular mechanisms underlying each of these diseases as well as results from clinical studies and clinical trials. This article summarizes the findings presented at the meeting and represents the current state-of-the art for NF research.

Cutaneous neurofibromas in the genomics era: current understanding and open questions.

Cutaneous neurofibromas (cNF) are a nearly ubiquitous symptom of neurofibromatosis type 1 (NF1), a disorder with a broad phenotypic spectrum caused by germline mutation of the neurofibromatosis type 1 tumour suppressor gene (NF1). Symptoms of NF1 can include learning disabilities, bone abnormalities and predisposition to tumours such as cNFs, plexiform neurofibromas, malignant peripheral nerve sheath tumours and optic nerve tumours. There are no therapies currently approved for cNFs aside from elective surgery, and the molecular aetiology of cNF remains relatively uncharacterised. Furthermore, whereas the biallelic inactivation of NF1 in neoplastic Schwann cells is critical for cNF formation, it is still unclear which additional genetic, transcriptional, epigenetic, microenvironmental or endocrine changes are important. Significant inroads have been made into cNF understanding, including NF1 genotype-phenotype correlations in NF1 microdeletion patients, the identification of recurring somatic mutations, studies of cNF-invading mast cells and macrophages, and clinical trials of putative therapeutic targets such as mTOR, MEK and c-KIT. Despite these advances, several gaps remain in our knowledge of the associated pathogenesis, which is further hampered by a lack of translationally relevant animal models. Some of these questions may be addressed in part by the adoption of genomic analysis techniques. Understanding the aetiology of cNF at the genomic level may assist in the development of new therapies for cNF, and may also contribute to a greater understanding of NF1/RAS signalling in cancers beyond those associated with NF1. Here, we summarise the present understanding of cNF biology, including the pathogenesis, mutational landscape, contribution of the tumour microenvironment and endocrine signalling, and the historical and current state of clinical trials for cNF. We also highlight open access data resources and potential avenues for future research that leverage recently developed genomics-based methods in cancer research.

Current status and recommendations for biomarkers and biobanking in neurofibromatosis.

OBJECTIVE: Clinically validated biomarkers for neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), and schwannomatosis (SWN) have not been identified to date. The biomarker working group's goals are to (1) define biomarker needs in NF1, NF2, and SWN; (2) summarize existing data on biomarkers in NF1, NF2, and SWN; (3) outline recommendations for sample collection and biomarker development; and (4) standardize sample collection and methodology protocols where possible to promote comparison between studies by publishing standard operating procedures (SOPs). METHODS: The biomarker group reviewed published data on biomarkers in NF1, NF2, and SWN and on biobanking efforts outside these diseases via literature search, defined the need for biomarkers in NF, and developed recommendations in a series of consensus meetings. RESULTS: We describe existing biomarkers in NF and report consensus recommendations for SOP and a minimal clinical dataset to accompany samples derived from patients with NF1, NF2, and SWN in decentralized biobanks. CONCLUSIONS: These recommendations are intended to provide clinicians and researchers with a common set of guidelines to collect and store biospecimens and for establishment of biobanks for NF1, NF2, and SWN.

Diagnosis and outcome of childhood perineurioma.

INTRODUCTION: Intraneural perineurioma is a rare peripheral nerve tumor of childhood and early adulthood. Patients demonstrate progressive muscle weakness and atrophy largely without sensory complaints. CASE: We report two children with perineurioma affecting the radial and femoral nerves. Electromyography (EMG), ultrasound, and 3-T MR imaging were important tools for localizing perineurioma and permitting its differentiation from other nerve lesions. The first patient underwent surgical excision of the perineurioma and a traditional nerve graft. At 10 months post-operative follow-up, she demonstrated no meaningful recovery of muscle strength compared to her pre-operative assessment. EMG did confirm axonal continuity indicating that reinnervation had occurred via the nerve graft. The second patient underwent a two-staged surgical procedure that included an end-to-side nerve transfer. At 18 months post-operative follow-up, she demonstrated mild improvement in muscle strength and EMG evidence of ongoing reinnervation. CONCLUSION: The surgical management of perineurioma remains controversial, and reports of clinical recovery after nerve grafts and nerve transfers vary. Nerve transfers have been reported to provide superior results to traditional nerve grafting in adults with post-traumatic plexus injuries. The modest gain in strength of our patient who underwent a nerve transfer raises the question if this may also apply to patients with perineurioma. Additional studies will be required, which must also take into consideration that features of long-standing neuropathy (i.e., limb length discrepancy) have the potential to reduce the likelihood of reinnervation and clinical recovery.

Multiparametric whole-body anatomic, functional, and metabolic imaging characteristics of peripheral lesions in patients with schwannomatosis.

PURPOSE: To describe the anatomic, functional, and metabolic characteristics of peripheral nerve sheath tumors (PNSTs) in patients with schwannomatosis (SWN) on whole-body magnetic resonance imaging (WB-MRI) (anatomic and functional imaging) and fluorine-18-fluorodeoxyglucose positron emission tomography / computed tomography (FDG-PET/CT) (metabolic imaging). MATERIALS AND METHODS: WB-MRIs at 1.5T and 3.0T performed in 13 SWN subjects using short tau inversion recovery (STIR), T1 -weighted (T1 W), contrast-enhanced T1 W, and diffusion-weighted imaging (DWI) with apparent diffusion coefficient (ADC) mapping and FDG-PET/CT were retrospectively reviewed. Two readers reviewed all imaging for the presence and character of peripheral lesions (size, imaging features, ADC values, and standardized uptake values [SUVmax ]) and ancillary findings. Descriptive statistics are reported. RESULTS: In all, 153 index lesions were characterized in 13 patients on WB-MRI. Lesions were characterized as tumors (97% [149/153]) or cysts (3% [4/153]); 96% (143/149) PNSTs were solitary while 4% (6/149) were plexiform. The median size was 2.3 cm (range 1-24.3 cm). On T1 W, 99% (148/149) tumors were homogeneously isointense; on STIR, 81% (121/149) tumors were heterogeneously hyperintense; on postcontrast T1 W, 81% (100/123) tumors enhanced heterogeneously; on DWI, tumor ADC values (×10(-3) mm(2) /s) were variable (minimum ADC range 0.3-2.2, average ADC range 0.9-2.9). The median SUVmax was 6 (range 2.1-11.7) and 10 (2.7-15.3) on early and delayed imaging, respectively. Malignant degeneration was detected in 1% (1/149) with suspicious anatomic, functional, and metabolic characteristics. Ancillary findings included nerve root thickening (23% [3/13]) and spinal canal lesions (15% [2/13]). CONCLUSION: Although the majority of the PNSTs in SWN are benign and solitary, PNSTs can be plexiform, enlarge over time, and, rarely, undergo malignant degeneration. Due to the high metabolic activity in benign PNSTs by FDG-PET/CT in SWN, WB-MRI with functional sequences maybe a more suitable technique for the assessment of disease burden, tumor characterization, and surveillance. J. MAGN. RESON. IMAGING 2016;44:794-803.

Methylation-based classification of benign and malignant peripheral nerve sheath tumors.

The vast majority of peripheral nerve sheath tumors derive from the Schwann cell lineage and comprise diverse histological entities ranging from benign schwannomas and neurofibromas to high-grade malignant peripheral nerve sheath tumors (MPNST), each with several variants. There is increasing evidence for methylation profiling being able to delineate biologically relevant tumor groups even within the same cellular lineage. Therefore, we used DNA methylation arrays for methylome- and chromosomal profile-based characterization of 171 peripheral nerve sheath tumors. We analyzed 28 conventional high-grade MPNST, three malignant Triton tumors, six low-grade MPNST, four epithelioid MPNST, 33 neurofibromas (15 dermal, 8 intraneural, 10 plexiform), six atypical neurofibromas, 43 schwannomas (including 5 NF2 and 5 schwannomatosis associated cases), 11 cellular schwannomas, 10 melanotic schwannomas, 7 neurofibroma/schwannoma hybrid tumors, 10 nerve sheath myxomas and 10 ganglioneuromas. Schwannomas formed different epigenomic subgroups including a vestibular schwannoma subgroup. Cellular schwannomas were not distinct from conventional schwannomas. Nerve sheath myxomas and neurofibroma/schwannoma hybrid tumors were most similar to schwannomas. Dermal, intraneural and plexiform neurofibromas as well as ganglioneuromas all showed distinct methylation profiles. Atypical neurofibromas and low-grade MPNST were indistinguishable with a common methylation profile and frequent losses of CDKN2A. Epigenomic analysis finds two groups of conventional high-grade MPNST sharing a frequent loss of neurofibromin. The larger of the two groups shows an additional loss of trimethylation of histone H3 at lysine 27 (H3K27me3). The smaller one retains H3K27me3 and is found in spinal locations. Sporadic MPNST with retained neurofibromin expression did not form an epigenetic group and most cases could be reclassified as cellular schwannomas or soft tissue sarcomas. Widespread immunohistochemical loss of H3K27me3 was exclusively seen in MPNST of the main methylation cluster, which defines it as an additional useful marker for the differentiation of cellular schwannoma and MPNST.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients.

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.

Elg1, a central player in genome stability.

ELG1 is a conserved gene uncovered in a number of genetic screens in yeast aimed at identifying factors important in the maintenance of genome stability. Elg1's activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice, acts as a tumor suppressor in mice and humans, exhibits physical interactions with components of the human Fanconi Anemia pathway and may be responsible for some of the phenotypes associated with neurofibromatosis. In this review, we summarize the information available on Elg1-related activities in yeast and mammals, and present models to explain how the different phenotypes observed in the absence of Elg1 activity are related.

Expanding the mutational spectrum of LZTR1 in schwannomatosis.

Schwannomatosis is characterized by the development of multiple non-vestibular, non-intradermal schwannomas. Constitutional inactivating variants in two genes, SMARCB1 and, very recently, LZTR1, have been reported. We performed exome sequencing of 13 schwannomatosis patients from 11 families without SMARCB1 deleterious variants. We identified four individuals with heterozygous loss-of-function variants in LZTR1. Sequencing of the germline of 60 additional patients identified 18 additional heterozygous variants in LZTR1. We identified LZTR1 variants in 43% and 30% of familial (three of the seven families) and sporadic patients, respectively. In addition, we tested LZTR1 protein immunostaining in 22 tumors from nine unrelated patients with and without LZTR1 deleterious variants. Tumors from individuals with LZTR1 variants lost the protein expression in at least a subset of tumor cells, consistent with a tumor suppressor mechanism. In conclusion, our study demonstrates that molecular analysis of LZTR1 may contribute to the molecular characterization of schwannomatosis patients, in addition to NF2 mutational analysis and the detection of chromosome 22 losses in tumor tissue. It will be especially useful in differentiating schwannomatosis from mosaic Neurofibromatosis type 2 (NF2). However, the role of LZTR1 in the pathogenesis of schwannomatosis needs further elucidation.

Heat hyperalgesia and mechanical hypersensitivity induced by calcitonin gene-related peptide in a mouse model of neurofibromatosis.

This study examined whether mice with a deficiency of neurofibromin, a Ras GTPase activating protein, exhibit a nociceptive phenotype and probed a possible contribution by calcitonin gene-related peptide. In the absence of inflammation, Nf1+/- mice (B6.129S6 Nf1/J) and wild type littermates responded comparably to heat or mechanical stimuli, except for a subtle enhanced mechanical sensitivity in female Nf1+/- mice. Nociceptive phenotype was also examined after inflammation induced by capsaicin and formalin, which release endogenous calcitonin gene-related peptide. Intraplantar injection of capsaicin evoked comparable heat hyperalgesia and mechanical hypersensitivity in Nf1+/- and wild type mice of both genders. Formalin injection caused a similar duration of licking in male Nf1+/- and wild type mice. Female Nf1+/- mice licked less than wild type mice, but displayed other nociceptive behaviors. In contrast, intraplantar injection of CGRP caused greater heat hyperalgesia in Nf1+/- mice of both genders compared to wild type mice. Male Nf1+/- mice also exhibited greater mechanical hypersensitivity; however, female Nf1+/- mice exhibited less mechanical hypersensitivity than their wild type littermates. Transcripts for calcitonin gene-related peptide were similar in the dorsal root ganglia of both genotypes and genders. Transcripts for receptor activity-modifying protein-1, which is rate-limiting for the calcitonin gene-related peptide receptor, in the spinal cord were comparable for both genotypes and genders. The increased responsiveness to intraplantar calcitonin gene-related peptide suggests that the peripheral actions of calcitonin gene-related peptide are enhanced as a result of the neurofibromin deficit. The analgesic efficacy of calcitonin gene-related peptide receptor antagonists may therefore merit investigation in neurofibromatosis patients.

[Segmental schwannomatosis in upper-extremity: 5 cases report and literature review].

OBJECTIVE: Multiple schwannomas localized in a single body part not crossing the midline constitute a rare variant of neurofibromatosis, segmental schwannomatosis. We report our experience with 5 cases of segmental schwannomatosis of the upper extremity and review the related literature to improve our skills in diagnosis and differentiation. METHODS: Five patients with segmental schwannomatosis received surgical treatment in our department from 2003 to 2012, of whom 4 were female and the other one male. The mean age was 38 years, ranging from 29 to 48 years. In retrospect, we discussed the clinical appearance, histologic characteristics, genetic data and surgical management. RESULTS: A total of 351 patients with schwannomas were treated in the recent decade. There were 326 patients with solitary schwannoma, accounting for 92.88%, 25 with neurofibromatosis type 2 (NF-2), occupying 7.12% and 5 with segmental schwannomatosis representing 1.42% of the total. Schwannomas are limited in one upper extremity and randomly located at ulnar nerve, median nerve and radial nerve and their branches, with no obvious predisposition. Their family history was negative for cutaneous tumors or central nervous system disease. Neurological examinations did not reveal symptoms related to vestibular nerves or optic nerves, which excluded NF-2 preliminarily. The prior symptom of three cases was pain which could be irradiated to the nerve distribution area. No pain but slight numbness was found in two cases. MRI disclosed multiple masses along the course of the nerves. They were isointense to muscle on T1-weighed images and hyperintense to subcutaneous fat on T2-weighed images. All schwannomas were resected and histological sections exhibited a characteristic feature of schwannoma. Follow-up work of 4.5 years was done to 4 cases and no recurrence or impairment of nerves was found. CONCLUSION: Segmental schwannomatosis is characterized by multiple schwannomas localized in one limb (upper extremity in our cases) without vestibular nerve tumors, most frequently seen in females at the age of 30-60 years. Segmental schwannomatosis is rarely seen in the previous literature. We found around 20 cases in English articles and no cases in domestic articles. In consideration of the clinical appearances of these 5 cases and the genetic research in the related literature, we recommend that segmental schwannomatosis is a distinct form of neurofibromatosis which needs to be more studied. We should also pay more attention to differentiating this disease from other forms of neurofibromatosis.

Rasopathies - dysmorphic syndromes with short stature and risk of malignancy.

OBJECTIVES: The term ´Rasopathies´ represents a group of five neurodevelopmental syndromes (Noonan, LEOPARD, Costello, Cardio-facio-cutaneous, and Neurofibromatose-Noonan syndrome) caused by germline mutation in genes encoding proteins involved in RAS/MAPK (rat sarcoma/mitogen-activated protein kinase) signaling pathway. The RAS/MAPK signaling pathway participates in regulation of cell determination, proliferation, differentiation, migration, and senescence and dysregulation of this pathway can lead to the risk of tumorigenesis. In this review, we aim to summarize the current clinical and molecular genetic knowledge on Rasopathies with special attention for the risk of cancer. We propose also clinical and therapeutic approach for patients with malignancy. METHODS: We are reviewing the clinical and molecular basis of Rasopathies based on recent studies, clinical examination, and molecular diagnostics (mutation analysis of causal genes for Rasopathies) in Slovak pediatric patients. RESULTS: Some clinical features, such as short stature, a specific facial dysmorphology and cardiac abnormalities are common to all of Rasopathy syndromes. However, there are unique signs by which the syndromes can differ from each other, especially multiple lentigo in LEOPARD syndrome, increased risk of malignancy in Costello syndrome, dry hyperkeratotic skin in patients with cardio-facio-cutaneous syndrome, and neurofibromas and cafe-au-lait spots in neurofibromatosis-Noonan syndrome. CONCLUSION: Despite the overlapping clinical features, Rasopathy syndromes exhibit unique fenotypical features and the precise molecular diagnostics may lead to confirmation of each syndrome. The molecular diagnostics may allow the detection of pathogenic mutation associated with tumorigenesis.

Melanocytic differentiation is present in a significant proportion of nonpigmented diffuse neurofibromas: a potential diagnostic pitfall.

Whereas the pigmented (melanotic) variant of diffuse neurofibroma (DNF) with positivity for melanocytic markers is well recognized, expression of melanocytic markers in nonpigmented DNF has not been systematically studied. We analyzed 28 unselected consecutive DNFs for expression of melanocytic markers, including melan A, microphthalmia transcription factor (MITF), and HMB-45 antigen. For comparison, we also analyzed 40 localized skin neurofibromas and 7 intraneural neurofibromas. One case of nonpigmented DNF was analyzed by electron microscopy. Of the 28 DNFs studied by immunohistochemistry, 3 were pigmented and 25 nonpigmented. The 3 pigmented DNFs and 9 of 25 (36%) nonpigmented DNFs expressed melan A, MITF, and HMB-45 antigen. These markers were expressed either focally or more diffusely, typically in a minority of the lesional cells, and usually both in the dermal and subcutaneous portion of the DNF. Melan A was expressed in the largest number of the lesional cells (up to 50%), whereas only a small fraction of the melan A-positive cells (from 5% to 10% in most cases) also expressed HMB-45 antigen. None of the 47 non-DNFs expressed these markers. Ultrastructurally, melanosomes were present in some cells in nonpigmented DNF that expressed the melanocytic markers. Twenty-three of 28 (82%) DNFs, including 10 of 12 (83%) DNFs with melanocytic differentiation, were associated with neurofibromatosis type 1. Expression of melanocytic markers, including melan A, HMB-45 antigen, and MITF in DNF is a potential pitfall in differential diagnosis with melanocytic lesions that may clinically or histopathologically resemble DNF, in particular congenital melanocytic nevus with neurotization and neurofibroma-like melanoma.

Expression of SMARCB1 (INI1) mutations in familial schwannomatosis.

Genetic changes in the SMARCB1 tumor suppressor gene have recently been reported in tumors and blood from families with schwannomatosis. Exon scanning of all nine SMARCB1 exons in genomic DNA from our cohort of families meeting the criteria for 'definite' or 'presumptive' schwannomatosis previously revealed constitutional alterations in 13 of 19 families (68%). Screening of four new familial schwannomatosis probands identified one additional constitutional alteration. We confirmed the presence of mRNA transcripts for two missense alterations, four mutations of conserved splice motifs and two additional mutations, in less conserved sequences, which also affect splicing. Furthermore, we found that transcripts for a rare 3'-untranslated region (c.*82C > T) alteration shared by four unrelated families did not produce splice variants but did show unequal allelic expression, suggesting that the alteration is either causative itself or linked to an unidentified causative mutation. Overexpression studies in cells lacking SMARCB1 suggest that mutant SMARCB1 proteins, like wild-type SMARCB1 protein, retain the ability to suppress cyclin D1 activity. These data, together with the expression of SMARCB1 protein in a proportion of cells from schwannomatosis-related schwannomas, suggest that these tumors develop through a mechanism that is distinct from that of rhabdoid tumors in which SMARCB1 protein is completely absent in tumor cells.