Emerging genotype-phenotype relationships in patients with large NF1 deletions.
ABSTRACT: The most frequent recurring mutations in neurofibromatosis type 1 (NF1) are large deletions encompassing the NF1 gene and its flanking regions (NF1 microdeletions). The majority of these deletions encompass 1.4-Mb and are associated with the loss of 14 protein-coding genes and four microRNA genes. Patients with germline type-1 NF1 microdeletions frequently exhibit dysmorphic facial features, overgrowth/tall-for-age stature, significant delay in cognitive development, large hands and feet, hyperflexibility of joints and muscular hypotonia. Such patients also display significantly more cardiovascular anomalies as compared with patients without large deletions and often exhibit increased numbers of subcutaneous, plexiform and spinal neurofibromas as compared with the general NF1 population. Further, an extremely high burden of internal neurofibromas, characterised by >3000 ml tumour volume, is encountered significantly, more frequently, in non-mosaic NF1 microdeletion patients than in NF1 patients lacking such deletions. NF1 microdeletion patients also have an increased risk of malignant peripheral nerve sheath tumours (MPNSTs); their lifetime MPNST risk is 16-26%, rather higher than that of NF1 patients with intragenic NF1 mutations (8-13%). NF1 microdeletion patients, therefore, represent a high-risk group for the development of MPNSTs, tumours which are very aggressive and difficult to treat. Co-deletion of the SUZ12 gene in addition to NF1 further increases the MPNST risk in NF1 microdeletion patients. Here, we summarise current knowledge about genotype-phenotype relationships in NF1 microdeletion patients and discuss the potential role of the genes located within the NF1 microdeletion interval whose haploinsufficiency may contribute to the more severe clinical phenotype.
Project description:Patients with NF1 microdeletion develop more neurofibromas at a younger age, and have an increased risk of malignant peripheral nerve sheath tumors (MPNSTs). We postulated that the increased risk of malignancy could be due to inactivation, in addition to NF1, of a second tumor suppressor gene located in the typical 1.4-Mb microdeletion found in most of the microdeleted patients. We investigated the expression of NF1, the other 16 protein-coding genes and the 2 microRNAs located in the 1.4-Mb microdeletion by means of real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) in a large series of human dermal and plexiform neurofibromas and MPNSTs. Five genes were significantly upregulated: OMG and SUZ12 in plexiform neurofibromas and ATAD5, EVI2A and C17orf79 in MPNSTs. More interestingly, two genes were significantly downregulated (RNF135 and CENTA2) in tumor Schwann cells from MPNST biopsies and in MPNST cell lines. This study points to the involvement of several genes (particularly RNF135 and CENTA2) in the increased risk of malignancy observed in NF1-microdeleted patients.
Project description:Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive soft tissue sarcomas that can occur sporadically or in the setting of the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome. These tumors carry a dismal overall survival. Previous work in our lab had identified ATRX chromatin remodeler (ATRX), previously termed, Alpha Thalassemia/Mental Retardation Syndrome X Linked as a gene mutated in a subset of MPNSTs. Given the great need for novel biomarkers and therapeutic targets for MPNSTs, we sought to determine the expression of ATRX in a larger subset of sporadic and NF1 associated MPNSTs (NF1-MPNSTs). We performed immunohistochemistry (IHC) on 74 MPNSTs (43 NF1-associated and 31 sporadic), 21 plexiform neurofibromas, and 9 atypical neurofibromas. Using this approach, we have demonstrated that 58% (43/74) of MPNSTs have aberrant ATRX expression (<80% nuclear expression) compared to only 7% (2/30) of benign (plexiform and atypical) neurofibromas. Second, we demonstrated that 65% (28/43) of NF1-MPNSTs displayed aberrant ATRX expression as did 48% (15/31) of sporadic MPNSTs. Finally, we show that aberrant ATRX expression was associated with a significantly decreased overall survival for patients with NF1-MPNST (median OS of 17.9 months for aberrant expression and median OS not met (>120 months) for intact expression, p = 0.0276). In summary, we demonstrate that ATRX is aberrantly expressed in the majority of NF1-MPNSTs, but not plexiform or atypical neurofibromas. Additionally, aberrant ATRX expression is associated with decreased overall survival in NF1-MPNST, but not sporadic MPNST and may serve as a prognostic marker for patients with NF1-MPNST.
Project description:Malignant peripheral nerve sheath tumours (MPNSTs) are highly malignant and resistant. Transformation might implicate up regulation of epidermal growth factor receptor (EGFR). Fifty-two MPNST samples were studied for EGFR, Ki-67, p53, and survivin expression by immunohistochemistry and for EGFR amplification by in situ hybridization. Results were correlated with clinical data. EGFR RNA was also quantified by RT-PCR in 20 other MPNSTs and 14 dermal neurofibromas. Half of the patients had a neurofibromatosis type 1 (NF1). EGFR expression, detected in 86% of MPNSTs, was more frequent in NF1 specimens and closely associated with high-grade and p53-positive areas. MPNSTs expressed more EGFR transcripts than neurofibromas. No amplification of EGFR locus was observed. NF1 status was the only prognostic factor in multivariate analysis, with median survivals of 18 and 43 months for patients with or without NF1. Finally, EGFR might become a new target for MPNSTs treatment, especially in NF1-associated MPNSTs.
Project description:Neurofibromatosis type-1 (NF1) is a complex neurogenetic disorder characterised by the development of benign and malignant tumours of the peripheral nerve sheath (MPNSTs). Whilst biallelic NF1 gene inactivation contributes to benign tumour formation, additional cellular changes in gene structure and/or expression are required to induce malignant transformation. Although few molecular profiling studies have been performed on the process of progression of pre-existing plexiform neurofibromas to MPNSTs, the integrated analysis of copy number alterations (CNAs) and gene expression is likely to be key to understanding the molecular mechanisms underlying NF1-MPNST tumorigenesis. In a pilot study, we employed this approach to identify genes differentially expressed between benign and malignant NF1 tumours.SPP1 (osteopontin) was the most differentially expressed gene (85-fold increase in expression), compared to benign plexiform neurofibromas. Short hairpin RNA (shRNA) knockdown of SPP1 in NF1-MPNST cells reduced tumour spheroid size, wound healing and invasion in four different MPNST cell lines. Seventy-six genes were found to exhibit concordance between CNA and gene expression level.Pathway analysis of these genes suggested that glutathione metabolism and Wnt signalling may be specifically involved in NF1-MPNST development. SPP1 is associated with malignant transformation in NF1-associated MPNSTs and could prove to be an important target for therapeutic intervention.
Project description:PURPOSE:An estimated 5-11% of patients with neurofibromatosis type 1 (NF1) harbour NF1 microdeletions encompassing the NF1 gene and its flanking regions. The purpose of this study was to evaluate the clinical phenotype in children and adolescents with NF1 microdeletions. METHODS:We retrospectively analysed 30 children and adolescents with NF1 microdeletions pertaining to externally visible neurofibromas. The internal tumour load was determined by volumetry of whole-body magnetic resonance imaging (MRI) in 20 children and adolescents with NF1 microdeletions. Furthermore, the prevalence of global developmental delay, autism spectrum disorder and attention deficit hyperactivity disorder (ADHD) were evaluated. RESULTS:Children and adolescents with NF1 microdeletions had significantly more often cutaneous, subcutaneous and externally visible plexiform neurofibromas than age-matched patients with intragenic NF1 mutations. Internal neurofibromas were detected in all 20 children and adolescents with NF1 microdeletions analysed by whole-body MRI. By contrast, only 17 (61%) of 28 age-matched NF1 patients without microdeletions had internal tumours. The total internal tumour load was significantly higher in NF1 microdeletion patients than in NF1 patients without microdeletions. Global developmental delay was observed in 28 (93%) of 30 children with NF1 microdeletions investigated. The mean full-scale intelligence quotient in our patient group was 77.7 which is significantly lower than that of patients with intragenic NF1 mutations. ADHD was diagnosed in 15 (88%) of 17 children and adolescents with NF1 microdeletion. Furthermore, 17 (71%) of the 24 patients investigated had T-scores ? 60 up to 75, indicative of mild to moderate autistic symptoms, which are consequently significantly more frequent in patients with NF1 microdeletions than in the general NF1 population. Also, the mean total T-score was significantly higher in patients with NF1 microdeletions than in the general NF1 population. CONCLUSION:Our findings indicate that already at a very young age, NF1 microdeletions patients frequently exhibit a severe disease manifestation which requires specialized long-term clinical care.
Project description:Malignant peripheral nerve sheath tumours (MPNSTs) are aggressive soft tissue tumours that occur either sporadically or in patients with neurofibromatosis type 1. The malignant transformation of the benign neurofibroma to MPNST is incompletely understood at the molecular level. We have determined the gene expression signature for benign and malignant PNSTs and found that the major trend in malignant transformation from neurofibroma to MPNST consists of the loss of expression of a large number of genes, rather than widespread increase in gene expression. Relatively few genes are expressed at higher levels in MPNSTs and these include genes involved in cell proliferation and genes implicated in tumour metastasis. In addition, a gene expression signature indicating p53 inactivation is seen in the majority of MPNSTs. Subsequent microRNA profiling of benign and malignant PNSTs indicated a relative down-regulation of miR-34a in most MPNSTs compared to neurofibromas. In vitro studies using the cell lines MPNST-14 (NF1 mutant) and MPNST-724 (from a non-NF1 individual) show that exogenous expression of p53 or miR-34a promotes apoptotic cell death. In addition, exogenous expression of p53 in MPNST cells induces miR-34a and other miRNAs. Our data show that p53 inactivation and subsequent loss of expression of miR-34a may significantly contribute to the MPNST development. Collectively, our findings suggest that deregulation of miRNAs has a potential role in the malignant transformation process in peripheral nerve sheath tumours.
Project description:Patients with neurofibromatosis type 1 (NF1) develop benign plexiform neurofibromas that frequently progress to become malignant peripheral nerve sheath tumors (MPNSTs). A genetically engineered mouse model that accurately models plexiform neurofibroma-MPNST progression in humans would facilitate identification of somatic mutations driving this process. We previously reported that transgenic mice overexpressing the growth factor neuregulin-1 in Schwann cells (P(0)-GGF?3 mice) develop MPNSTs. To determine whether P(0)-GGF?3 mice accurately model human neurofibroma-MPNST progression, cohorts of these animals were monitored through death and were necropsied; 94% developed multiple neurofibromas, with 70% carrying smaller numbers of MPNSTs. Nascent MPNSTs were identified within neurofibromas, suggesting that these sarcomas arise from neurofibromas. Although neurofibromin expression was maintained, P(0)-GGF?3 MPNSTs exhibited Ras hyperactivation, as in human NF1-associated MPNSTs. P(0)-GGF?3 MPNSTs also exhibited abnormalities in the p16(INK4A)-cyclin D/CDK4-Rb and p19(ARF)-Mdm-p53 pathways, analogous to their human counterparts. Array comparative genomic hybridization (CGH) demonstrated reproducible chromosomal alterations in P(0)-GGF?3 MPNST cells (including universal chromosome 11 gains) and focal gains and losses affecting 39 neoplasia-associated genes (including Pten, Tpd52, Myc, Gli1, Xiap, and Bbc3/PUMA). Array comparative genomic hybridization also identified recurrent focal copy number variations affecting genes not previously linked to neurofibroma or MPNST pathogenesis. We conclude that P(0)-GGF?3 mice represent a robust model of neurofibroma-MPNST progression useful for identifying novel genes driving neurofibroma and MPNST pathogenesis.
Project description:Background: Because neurofibromatosis type I (NF1) is a cancer predisposition disease, it is important to distinguish between benign and malignant lesions, especially in the craniofacial area. Purpose: The purpose of this study is to improve effectiveness in the diagnostic performance in discriminating malignant from benign craniofacial lesions based on computed tomography (CT) using a Keras-based machine-learning model. Methods: The Keras-based machine learning technique, a neural network package in the Python language, was used to train the diagnostic model on CT datasets. Fifty NF1 patients with benign craniofacial neurofibromas and six NF1 patients with malignant peripheral nerve sheath tumors (MPNSTs) were selected as the training set. Three validation cohorts were used: validation cohort 1 (random selection of 90% of the patients in the training cohort), validation cohort 2 (an independent cohort of 9 NF1 patients with benign craniofacial neurofibromas and 11 NF1 patients with MPNST), and validation cohort 3 (eight NF1 patients with MPNST, not restricted to the craniofacial area). Sensitivity and specificity were tested using validation cohorts 1 and 2, and generalizability was evaluated using validation cohort 3. Results: A total of 59 NF1 patients with benign neurofibroma and 23 NF1 patients with MPNST were included. A Keras-based machine-learning model was successfully established using the training cohort. The accuracy was 96.99 and 100% in validation cohorts 1 and 2, respectively, discriminating NF1-related benign and malignant craniofacial lesions. However, the accuracy of this model was significantly reduced to 51.72% in the identification of MPNSTs in different body regions. Conclusion: The Keras-based machine learning technique showed the potential of robust diagnostic performance in the differentiation of craniofacial MPNSTs and benign neurofibromas in NF1 patients using CT images. However, the model has limited generalizability when applied to other body areas. With more clinical data accumulating in the model, this system may support clinical doctors in the primary screening of true MPNSTs from benign lesions in NF1 patients.
Project description:Neurofibromatosis type 1 (NF1) patients develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). These incurable peripheral nerve tumors result from loss of NF1 tumor suppressor gene function, causing hyperactive Ras signaling. Activated Ras controls numerous downstream effectors, but specific pathways mediating the effects of hyperactive Ras in NF1 tumors are unknown. We performed cross-species transcriptome analyses of mouse and human neurofibromas and MPNSTs and identified global negative feedback of genes that regulate Ras/Raf/MEK/ERK signaling in both species. Nonetheless, ERK activation was sustained in mouse and human neurofibromas and MPNST. We used a highly selective pharmacological inhibitor of MEK, PD0325901, to test whether sustained Ras/Raf/MEK/ERK signaling contributes to neurofibroma growth in a neurofibromatosis mouse model (Nf1(fl/fl);Dhh-Cre) or in NF1 patient MPNST cell xenografts. PD0325901 treatment reduced aberrantly proliferating cells in neurofibroma and MPNST, prolonged survival of mice implanted with human MPNST cells, and shrank neurofibromas in more than 80% of mice tested. Our data demonstrate that deregulated Ras/ERK signaling is critical for the growth of NF1 peripheral nerve tumors and provide a strong rationale for testing MEK inhibitors in NF1 clinical trials.
Project description:Neurofibromatosis Type 1 (NF1) is a common genetic disorder and cancer predisposition syndrome (1:3000 births) caused by mutations in the tumor suppressor gene NF1. NF1 encodes neurofibromin, a negative regulator of the Ras signaling pathway. Individuals with NF1 often develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell linage, some of which progress further to malignant peripheral nerve sheath tumors (MPNSTs). Treatment options for neurofibromas and MPNSTs are extremely limited, relying largely on surgical resection and cytotoxic chemotherapy. Identification of novel therapeutic targets in both benign neurofibromas and MPNSTs is critical for improved patient outcomes and quality of life. Recent clinical trials conducted in patients with NF1 for the treatment of symptomatic plexiform neurofibromas using inhibitors of the mitogen-activated protein kinase (MEK) have shown very promising results. However, MEK inhibitors do not work in all patients and have significant side effects. In addition, preliminary evidence suggests single agent use of MEK inhibitors for MPNST treatment will fail. Here, we describe the preclinical efforts that led to the identification of MEK inhibitors as promising therapeutics for the treatment of NF1-related neoplasia and possible reasons they lack single agent efficacy in the treatment of MPNSTs. In addition, we describe work to find targets other than MEK for treatment of MPNST. These have come from studies of RAS biochemistry, in vitro drug screening, forward genetic screens for Schwann cell tumors, and synthetic lethal screens in cells with oncogenic RAS gene mutations. Lastly, we discuss new approaches to exploit drug screening and synthetic lethality with NF1 loss of function mutations in human Schwann cells using CRISPR/Cas9 technology.