Dermal Neurofibromas Research Articles

Abstract A37: Synthetic lethality as a tool to reveal novel vulnerabilities in NF1-associated tumorigenesis

Abstract Neurofibromatosis Type 1 (NF1) is a common (1:2500 births) genetic disorder and cancer predisposition syndrome, caused by mutations in the tumor suppressor gene NF1. One of the known functions of the protein encoded by NF1 (neurofibromin) is that of a negative regulator of Ras pathway signaling, through its Ras-GAP activity. Among other symptoms, individuals with NF1 often develop benign tumors of the peripheral nervous system (called dermal or plexiform neurofibromas), originating from the Schwann cell linage or their precursors. While these benign tumors can cause significant pain and mobility problems, some (~10%) progress further to malignant peripheral nerve sheath tumors (MPNSTs) and are a leading cause of death among NF1 patients. Both plexiform neurofibromas and MPNSTs arise following loss of the remaining WT NF1 allele within the Schwann cell linage (or their precursors). It is believed the majority of NF1-associated MPNSTs originate from preexisting plexiform neurofibromas. Treatment options for the benign plexiform neurofibromas and MPNSTs are extremely limited, mostly relying on surgical resection and broad-spectrum chemotherapy. Finding new molecular targets for therapeutics effective against both benign tumors and MPNSTs are critical for improved patient outcomes and quality of life. The genetic basis of NF1 syndrome make it a top candidate for using synthetic lethal genetic and therapeutic approaches to uncover unique variabilities in NF1 deficient cells. We have developed a drug discovery pipeline to identify targeted therapeutics for treatment NF1-related neoplasia, including MPNSTs. Using CRISPR/Cas9, we have created an immortalized human Schwann cell line that is deficient for the NF1 gene, which when paired with its isogenic wild-type parent is an outstanding research tool for identifying synthetic lethal interactions. The NF1 deficient cells exhibit increased oncogenic phenotypes, including increased anchorage independent growth under low serum conditions, higher basal levels of Ras-GTP, and a tendency to form tumors in athymic nude mice. These isogenic cell lines are currently being utilized for a number of synthetic lethal screens to identify therapeutics and targets specific to cells lacking NF1. These include: 1. A large-scale screen (~24,000 compounds from focused libraries) for drugs that preferentially kill the NF1 deficient cells. This could identify new targeted therapies for potential use in the clinic. 2. Synthetic lethal genetic screens using genome-wide RNAi and CRISPR/Cas9 approaches to knockdown/out expression of additional genes. The design of our system incorporates two key components that we believe provide the best chance of success. First, our studies utilize the correct, tumor specific, human cell type (Schwann cells). Secondly, we have created isogenic pairs of these cells, differing only in their NF1 status, in which to conduct our synthetic lethal screens. NF1 dependent vulnerabilities identified from this study might also have wider reaching implications and uses, as NF1 is often somatically altered in other cancers. The convergence of genome editing technologies with high throughput drug and synthetic lethal genetic screening provide an exciting opportunity for investigation of NF1 and other genetic cancer predisposition syndromes. Citation Format: Kyle B. Williams, Rory L. Williams, Sue K. Rathe, Jon Hawkinson, David A. Largaespada. Synthetic lethality as a tool to reveal novel vulnerabilities in NF1-associated tumorigenesis [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A37.

KIR2DL5 mutation and loss underlies sporadic dermal neurofibroma pathogenesis and growth.

Dermal neurofibromas (DNFs) are benign peripheral nerve sheath tumors thought to originate from Schwann cell progenitors. These tumors represent one of the hallmarks of the neurofibromatosis type 1 (NF1) tumor predisposition syndrome, where they can number in the thousands. While NF1-DNFs arise due to mutations in the NF1 gene, the vast majority of DNFs occur sporadically (sp-DNFs), where the genetic etiology is currently unknown. Herein, we employed whole-exome sequencing of sp-DNFs to identify a recurrent mutation in the KIR2DL5 gene, which codes for a protein suppressor of natural killer (NK) cell activity. While the function of KIR2DL5 outside of the immune system is unknown, we identified a KIR2DL5N173D mutation in three of nine sp-DNFs, resulting in loss of KIR2DL5 protein expression. In contrast, two of these subjects had unrelated tumors, which retained KIR2DL5 protein expression. Moreover, loss of KIR2DL5 expression was demonstrated in 15 of 45 independently-identified sp-DNFs. Consistent with its potential role as a negative growth regulator important for neurofibroma maintenance, ectopic KIR2DL5N173D expression in normal human Schwann cells resulted in reduced KIR2DL5 expression and increased cell proliferation. Similarly, KIR2DL5 short hairpin RNA knockdown (KD) decreased KIR2DL5 protein levels and increased cell proliferation, as well as correlated with PDGFRβ and downstream RAS/AKT/mTOR hyperactivation. Importantly, inhibition of PDGFRβ or AKT/mTOR activity in KIR2DL5-KD human Schwann cells reduced proliferation to control levels. Collectively, these findings establish KIR2DL5 as a new Schwann cell growth regulator relevant to sp-DNF pathogenesis, which links sporadic and NF1-associated DNFs through RAS pathway hyperactivation.

Head and neck dermal neurofibroma contains distinctive mesenchymal-like tumour stem cells governed by neurotrophin microenvironment

Head and neck dermal neurofibroma contains distinctive mesenchymal-like tumour stem cells governed by neurotrophin microenvironment

Neurofibromatosis type 1.

Neurofibromatosis type 1 is a complex autosomal dominant disorder caused by germline mutations in the NF1 tumour suppressor gene. Nearly all individuals with neurofibromatosis type 1 develop pigmentary lesions (café-au-lait macules, skinfold freckling and Lisch nodules) and dermal neurofibromas. Some individuals develop skeletal abnormalities (scoliosis, tibial pseudarthrosis and orbital dysplasia), brain tumours (optic pathway gliomas and glioblastoma), peripheral nerve tumours (spinal neurofibromas, plexiform neurofibromas and malignant peripheral nerve sheath tumours), learning disabilities, attention deficits, and social and behavioural problems, which can negatively affect quality of life. With the identification of NF1 and the generation of accurate preclinical mouse strains that model some of these clinical features, therapies that target the underlying molecular and cellular pathophysiology for neurofibromatosis type 1 are becoming available. Although no single treatment exists, current clinical management strategies include early detection of disease phenotypes (risk assessment) and biologically targeted therapies. Similarly, new medical and behavioural interventions are emerging to improve the quality of life of patients. Although considerable progress has been made in understanding this condition, numerous challenges remain; a collaborative and interdisciplinary approach is required to manage individuals with neurofibromatosis type1 and to develop effective treatments.

Application of the laser diode with central wavelength 975nm for the therapy of neurofibroma and hemangiomas.

This paper presents a newly developed dermatological laser (with a central wavelength 975 nm) for application in therapies requiring deep penetration of tissue, e.g., cutaneous (dermal) neurofibroma (von Recklinghausen disease) and hemangiomas. This laser can work either in pulses or continues wave mode. Laser radiation is transmitted toward the application region by optical fiber with a diameter of 0.6 mm. The compact design of the laser facilitates its transport and increases the comfort of use.

Abstract 772: The molecular landscape of dermal neurofibromatosis

Abstract Background: Neurofibromatosis type I (NF1) is a genetic disorder that disrupts neurological tissue growth and can lead to a diverse set of symptoms including systematic growth of benign tumors, learning disorders and bone deformities. It is a rare disease occurring in only 1 in 3,000 people worldwide. While the disease has been linked to loss of function in the NF1 gene - a known tumor suppressor - there is a high degree of phenotypic diversity in the NF1 patient population, making it difficult to identify the underlying cause of the disease and treat it effectively. In this work we seek to improve overall knowledge of dermal NF1 through global molecular characterization of the disease. Methods: We have collected four dermal neurofibromas and peripheral blood from each of 11 NF1 patients. We analyzed each sample using (1) Whole genome sequencing (WGS) on the Illumina HiSeq X platform, (2) Illumina OMNI2.5 Arrays (3) RNA-Sequencing on an Illumina HiSeq v4 machine and (4) iTRAQ-labeled proteomics. WGS data for both tumor and blood samples from each patient were used to identify patient-specific germ-line mutations as well as tumor-specific somatic mutations in each sample. Single nucleotide polymorphisms identified by the OMNI Arrays were used to identify copy number alterations in both blood and tumor samples. RNA-Seq data and proteomics data were mapped to transcripts and proteins respectively. Results: Preliminary analysis of this data illustrates a diverse genomic landscape of NF1. Hierarchical clustering of copy number alterations largely show samples clustering by tissue, suggesting that most copy number alterations are somatic and not shared across the germline. However, there are two patients that show germline copy number alterations, including one patient with loss in the NF1 region. WGS analysis suggests similar diversity with each patient possessing a distinct combination of germline and somatic mutations of NF1 and other cancer-related genes. Cluster analysis of the RNA-Seq data shows no patient-specific clusters, suggesting that that each tumor executes a unique transcriptional program. Conclusion: This work represents a first-ever attempt to profile the diversity of dermal neurofibromatosis at a molecular level. Preliminary analysis of the data underscores the complexity of this disease and explains, in part, previous difficulty in identifying effective treatments. Ongoing work includes expanding the analysis to include more patient samples and other types of NF1-derived tumors. As an orphan disease, NF1 has been poorly characterized compared to more common cancers. To rectify this, the Children's Tumor Foundation and Sage Bionetworks are collaborating to make NF1 data available to the public to accelerate research and the drug discovery pipeline. We expect that this data will be a resource for other NF1 researchers to assist in the study of this disease at the molecular level. All data and preliminary results are publicly available at http://www.synapse.org/dermalNF Citation Format: Sara JC Gosline, Pamela Knight, Thomas Yu, Nripesh Prasad, Angela Jones, Shristi Shrestha, Braden Boone, Shawn E. Levy, Andrew J. Link, Allison C. Galassie, Hubert Weinberg, Stephen Friend, Salvatore La Rosa, Justin Guinney, Annette Bakker. The molecular landscape of dermal neurofibromatosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 772.

Phase II trial of the MEK1/2 inhibitor selumetinib (AZD6244) in adults with neurofibromatosis type 1 (NF1) and inoperable plexiform neurofibromas (PNs).

TPS2596Background: PNs in NF1 are a major source of morbidity, causing disfigurement, functional impairment, and pain. Selumetinib is an oral inhibitor of MEK 1/2, which may mediate anti-tumor effects in PNs by inhibiting Ras signaling. Selumetinib is currently undergoing evaluation in adult cancers, pediatric brain tumors and NF1 related PNs. Our phase I trial of selumetinib for children with NF1 and inoperable PNs demonstrated unprecedented activity with objective responses (PN volume decrease ≥ 20% using volumetric MRI analysis) in > 50% of patients (pts) enrolled. However, we have observed slow PN regrowth in several patients who required dose reductions for toxicity. This suggests that a certain selumetinib tissue concentration may be required for target inhibition and anti-tumor activity. Methods: This study will evaluate 1) the objective response rate to selumetinib in pts ≥ 18 years old with NF1 and inoperable, growing or symptomatic PNs, 2) PN and dermal neurofibroma (DN) biopsies prior to and on...

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.

Surgical Management of a Superior Mesenteric Artery Aneurysm Resulting from Neurofibromatosis Type I

Abstract: Arterial involvement in neurofibromatosis is infrequent. Stenotic lesions predominate. Aneurysms are less common. They typically involve the aorta, visceral arteries or carotid-vertebral circulation. They may present as a mass or spontaneous rupture. We present the case of a 26-year-old woman with a Von Recklinghausen disease, operated for a giant aneurysm of the duodenopancreatic arcade. Keywords: Aneurysm; Von Recklinghausen disease; visceral arteries; aneurysmorraph Introduction: Neurofibromatosis type I, or Von Recklinghausen disease was first described by Frederich Von Recklinghausen [1]. It is an autosomal dominantly inheritable disease [2]. It includes multiple skin cafe-au-lait macules, tumors of the nervous system, iris hamartomas, and skeletal deformities such as scoliosis [2,3]. Arterial involvement is rare but may have catastrophic complications. We report an unusual case of a non-complicated superior mesenteric artery aneurysm in a 26-year-old woman with neurofibromatosis type I treated surgically under a median laparotomy. Case Presentation: R.E is a 26-year-old woman with neurofibromatosis type I (Recklinghausen’s disease) diagnosed since 5 years, and blood hypertension due to bilateral stenosis of the renal arteries, treated medically and by percutaneous angioplasty. She was admitted to surgery department complaining of abdominal pain and palpitations. The patient had no history of acute or remote trauma. Her physical examination was remarkable for the cafe-au-lait spotty pigmentation of the skin that is typical of patients with von Recklinghausen’s disease (Figure 1). The patient was hemodynamically stable, with a blood pressure of 144/94 mmHg and a pulse of 88 beats/min. A computed tomography scan revealed an occlusion of the celiac artery, and three non-complicated saccular aneurysms: an aneurysm arising from the duodeno-pancreatic arcade measuring 30 mm (Figure 2), an aneurysm of the splenic artery measuring 6 mm, and an aneurysm of pancreatic collateral of the superior mesenteric artery measuring 11 mm (Figure 3). The patient did not have any symptoms of bowel ischemia. So, a surgical repair under median laparotomy was decided.The largest aneurysm was dissected (Figure 4), was opened, and narrowed by an aneurysmorraphy (Figures 5 and 6). The patient made a full recovery and was discharged 4 days postoperatively. A follow-up CT scan (1 month after the procedure) showed patency of the artery without aneurysmal recurrence (Figure 7). Discussion: Von Recklinghausen disease is an autosomal dominant disorder characterized clinically by cafe-au-lait macules, dermal and plexiform neurofibromas, skeletal abnormalities, optic gliomas, and iris hamartomas [2]. The prognosis of this disease depends essentially on the existence of cerebral tumors, which are responsible for death in 72% of cases [4].Vascular involvement has been described by Reubi [5]. It is rare with a reported prevalence of 0.4% to 6.4% [3]. It includes renovascular hypertension by stenosis of the renal artery, cerebral artery stenosis, aortic coarctation, occlusive, arteriovenous malformations and aneurysmal arterial disease [3].Arterial stenotic lesions have been more commonly described than aneurysms [6].The most frequently sites of aneurysms are the subclavian and vertebral vasculature where they can result in catastrophic hemorrhage [7]. Aneurysmal location of the visceral arteries is exceptional [8]. They involve mainly the splenic artery. Only a few cases of superior mesenteric artery aneurysm have been reported in this disease [9]. Fye, et al. [10] described multiple small aneurysms on the branches of the superior mesenteric artery. Huffman et al [8] reported a patient who underwent ligation of a ruptured superior mesenteric artery aneurysm. The vascular lesions described in our patient reflect the widespread vascular involvement with neurofibromatosis. They include three saccular aneurysms located in the visceral arteries, bilateral stenosis of the renal arteries, and occlusion of the celiac artery. The pathogenesis and natural history of these anomalies remains poorly understood [11]. Most patients have asymptomatic abnormalities of multiple vessels [12]. The clinical manifestations of aneurysm in cases of von Recklinghausen’s disease are characterized by compression, hypertension, and rupture [8]. Noninvasive imaging modalities such as duplex ultrasound scanning, CT scan and magnetic resonance angiography are useful diagnostic tools [9]. Visceral vasculopathy causes ischemic bowel disease; and catastrophic retroperitoneal or abdominal hemorrhage has been attributed to spontaneously rupture arterial aneurysms [13]. So, all aneurysms >1 to 1.5 cm in diameter should be treated [14]. The interventional approaches have proven their value, especially in vascular regions that are difficult to access. We decided not to do the endovascular treatment to our patient because operative treatment of large aneurysms is safe, effective, and durable. Conclusion: Arterial vasculopathy is a well-recognized but uncommon manifestation of neurofibromatosis type 1. Occlusive or aneurysmal disease of arteries of all sizes may occur almost anywhere in the body, and their consequences can be extremely dangerous. So, a general screening of all patients with neurofibromatosis type I for arterial disease should be performed to prevent fatal outcomes of occult aneurysms [15].

Mini Review: Potential Role of Hormones in the Tumors of Neurofibromatosis Type-1

Neurofibromatosis type 1 is an autosomal dominant neurocutaneous disorder caused by mutations in the NF1 gene, and presents a very broad spectrum of clinical manifestations and severity, including dermal and plexiform neurofibromas. In this mini review we briefly address the findings present in the literature related to hormone receptors within these tumor types.

Indicator Exploration for Cancers in Women with Neurofibromatosis Type 1 - A Multi-Centre Retrospective Study

Objective: Neurofibromatosis type 1 (NF1) is a complex hereditary syndrome with multi-systemic involvement and propensity to develop a variety of tumors. Despite the increased risk for malignant neoplasms and shortened life-span, there is no targeted cancer surveillance strategy. Clinical features of NF1 and family history may be associated with occurrence of certain neoplasms and serve as indicators for targeted surveillance. Methods: This multi-centre retrospective study reviewed the records of 423 women with NF1. The associations between neoplasms, clinical features and family history were analyzed. Results: The occurrence of breast cancers is positively associated (p = 0.004) with family history of any cancers, 9.6% (12/125) with family history vs. 2.7% (8/298) without. An association between NF1 clinical phenotypes (i.e. dermal neurofibroma burden) and cancer was not observed. However, the rate of malignant peripheral sheath tumor (MPNST) was significantly higher (p = 0.049) in women with plexiform neurofibroma (PN) than women without, 7.9% (11/139) vs. 3.14% (7/223). Women with learning disabilities have a higher rate (p = 0.019) of central nervous system (CNS) tumors including optic glioma (OPG) than women without, 22.2% (20/90) vs.11.2% (21/187). European Americans (EAs) are significantly more likely (p = 0.002) to develop CNS tumors (21.2%, 41/193) than African Americans (AAs) (6.8%, 6/88). Conclusion: Family history of any cancers, preexisting PN, learning disability and EA ancestry is linked to higher risk of breast cancer, MPNST, and CNS tumors/OPG, respectively.

Soluble AXL: a possible circulating biomarker for neurofibromatosis type 1 related tumor burden.

Neurofibromatosis type 1 (NF1) is the most common tumor predisposition disorder affecting 1/3500 worldwide. Patients are at risk of developing benign (neurofibromas) and malignant peripheral nerve sheath tumors (MPNST). The AXL receptor tyrosine kinase has been implicated in several kinds of cancers, but so far no studies have investigated the role of AXL in NF1 related tumorigenesis. Recently, the soluble fraction from the extracellular domain of AXL (sAXL) has been found in human plasma, and its level was correlated to poor prognosis in patients with renal cancer. Compared to normal human Schwann cells, a significantly high expression level of AXL was found in three of the four MPNST cell lines and two of the three primary MPNST tissues. Similarly, the level of sAXL in conditioned media corresponded to the protein and mRNA levels of AXL in the MPNST cell lines. Furthermore, in two different human MPNST xenograft models, the human sAXL could be detected in the mouse plasma. Its level was proportionate to the size of the xenograft tumors, while no human sAXL was detect prior to the formation of the tumors. Treatment with a newly developed photodynamic therapy, prevented further tumor growth and resulted in drastically reduced the levels of sAXL compared to that of the control group. Finally, the level of sAXL was significantly increased in patients with plexiform tumors compared to patients with only dermal neurofibromas, further supporting the role of sAXL as a marker for NF1 related tumor burden.

Somatic neurofibromatosis type 1 (NF1) inactivation events in cutaneous neurofibromas of a single NF1 patient.

Neurofibromatosis type 1 (NF1) (MIM#162200) is a relatively frequent genetic condition that predisposes to tumor formation. The main types of tumors occurring in NF1 patients are cutaneous and subcutaneous neurofibromas, plexiform neurofibromas, optic pathway gliomas, and malignant peripheral nerve sheath tumors. To search for somatic mutations in cutaneous (dermal) neurofibromas, whole-exome sequencing (WES) was performed on seven spatially separated tumors and two reference tissues (blood and unaffected skin) from a single NF1 patient. Validation of WES findings was done using routine Sanger sequencing or Sequenom IPlex SNP genotyping. Exome sequencing confirmed the existence of a known familial splice-site mutation NM_000267.3:c.3113+1G>A in exon 23 of NF1 gene (HGMD ID CS951480) in blood, unaffected skin, and all tumor samples. In five out of seven analyzed tumors, we additionally detected second-hit mutations in the NF1 gene. Four of them were novel and one was previously observed. Each mutation was distinct, demonstrating the independent origin of each tumor. Only in two of seven tumors we detected an additional somatic mutation that was not associated with NF1. Our study demonstrated that somatic mutations of NF1 are likely the main drivers of cutaneous tumor formation. The study provides evidence for the rareness of single base pair level alterations in the exomes of benign NF1 cutaneous tumors.

Differences of 25-hydroxyvitamin D3 concentrations in children and adults with neurofibromatosis type 1

ObjectivesNeurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder, frequently associated with reduced bone mineral density. Serum 25-hydroxyvitamin D3 concentrations in NF1 adults are lower than in healthy controls in autumn respectively winter and are inversely correlated with the number of dermal neurofibromas. We investigated 25-hydroxyvitamin D3 levels in children and adults with neurofibromatosis type 1 in winter and summer and compared them to healthy controls to get more pathogenic insights in vitamin D3 metabolism in NF1 patients. Design and methodsNF1 patients were clinically examined and serum 25-hydroxyvitamin D3 concentrations were measured in 58 NF1 adults and 46 children in winter as well as in summer and compared to sex-, age- and month-matched controls. Results52 adults suffered from 10 to 5000 dermal neurofibromas, whereas none of the children presented neurofibromas. 25-Hydroxyvitamin D3 increased from winter to summer (mean: 21.0 to 46.5nmol/l) in NF1 adults. This increase was even larger (p=0.0001) than in healthy controls (mean: 50.5 to 60.5nmol/l). However, there were no differences of 25-hydroxyvitamin D3 concentrations in NF1 children and healthy controls both in winter and in summer. ConclusionsOnly adults with NF1 showed lower 25-hydroxyvitamin D3 levels in winter and summer, which are unlikely due to impaired UV-dependent dermal synthesis, but rather might be caused by an accelerated catabolism.

The Activation of the WNT Signaling Pathway Is a Hallmark in Neurofibromatosis Type 1 Tumorigenesis

The hallmark of neurofibromatosis type 1 (NF1) is the onset of dermal or plexiform neurofibromas, mainly composed of Schwann cells. Plexiform neurofibromas can transform into malignant peripheral nerve sheath tumors (MPNST) that are resistant to therapies. The aim of this study was to identify an additional pathway in the NF1 tumorigenesis. We focused our work on Wnt signaling that is highly implicated in cancer, mainly in regulating the proliferation of cancer stem cells. We quantified mRNAs of 89 Wnt pathway genes in 57 NF1-associated tumors including dermal and plexiform neurofibromas and MPNSTs. Expression of two major stem cell marker genes and five major epithelial-mesenchymal transition marker genes was also assessed. The expression of significantly deregulated Wnt genes was then studied in normal human Schwann cells, fibroblasts, endothelial cells, and mast cells and in seven MPNST cell lines. The expression of nine Wnt genes was significantly deregulated in plexiform neurofibromas in comparison with dermal neurofibromas. Twenty Wnt genes showed altered expression in MPNST biopsies and cell lines. Immunohistochemical studies confirmed the Wnt pathway activation in NF1-associated MPNSTs. We then confirmed that the knockdown of NF1 in Schwann cells but not in epithelial cells provoked the activation of Wnt pathway by functional transfection assays. Furthermore, we showed that the protein expression of active β-catenin was increased in NF1-silenced cell lines. Wnt pathway activation was strongly associated to both cancer stem cell reservoir and Schwann-mesenchymal transition. We highlighted the implication of Wnt pathway in NF1-associated tumorigenesis.

MicroRNAome profiling in benign and malignant neurofibromatosis type 1-associated nerve sheath tumors: evidences of PTEN pathway alterations in early NF1 tumorigenesis

BackgroundNeurofibromatosis type 1 (NF1) is a common dominant tumor predisposition syndrome affecting 1 in 3,500 individuals. The hallmarks of NF1 are the development of peripheral nerve sheath tumors either benign (dermal and plexiform neurofibromas) or malignant (MPNSTs).ResultsTo comprehensively characterize the role of microRNAs in NF1 tumorigenesis, we analyzed 377 miRNAs expression in a large panel of dermal and plexiform neurofibromas, and MPNSTs. The most significantly upregulated miRNA in plexiform neurofibromas was miR-486-3p that targets the major tumor suppressor gene, PTEN. We confirmed PTEN downregulation at mRNA level. In plexiform neurofibromas, we also report aberrant expression of four miRNAs involved in the RAS-MAPK pathway (miR-370, miR-143, miR-181a, and miR-145). In MPNSTs, significant deregulated miRNAs were involved in PTEN repression (miR-301a, miR-19a, and miR-106b), RAS-MAPK pathway regulation (Let-7b, miR-195, and miR-10b), mesenchymal transition (miR-200c, let-7b, miR-135a, miR-135b, and miR-9), HOX genes expression (miR-210, miR-196b, miR-10a, miR-10b, and miR-9), and cell cycle progression (miR-195, let-7b, miR-20a, miR-210, miR-129-3p, miR-449a, and miR-106b).ConclusionWe confirmed the implication of PTEN in genesis of plexiform neurofibromas and MPNSTs in NF1. Markedly deregulated miRNAs might have potential diagnostic or prognostic value and could represent novel strategies for effective pharmacological therapies of NF1 tumors.

Probe-Based Quantitative PCR Assay for Detecting Constitutional and Somatic Deletions in the NF1 Gene: Application to Genetic Testing and Tumor Analysis

About 5% of patients with neurofibromatosis type 1 (NF1) bear constitutional microdeletions that encompass NF1 (neurofibromin 1) and neighboring genes. These patients are characterized by the development of a high number of dermal neurofibromas (dNFs), mental retardation, and an increased risk of developing a malignant peripheral nerve sheath tumor (MPNST). Additionally, 10% of somatic second hits identified in dNFs are caused by deletions involving the NF1 gene. To detect constitutional and somatic deletions, we developed a probe-based quantitative PCR (qPCR) assay for interrogating the copy number status of 11 loci distributed along a 2.8-Mb region around the NF1 gene. We developed the qPCR assay with Universal ProbeLibrary technology (Roche) and designed a Microsoft Excel spreadsheet to analyze qPCR data for copy number calculations. The assay fulfilled the essential aspects of the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines and used the qBase relative quantification framework for calculations. The assay was validated with a set of DNA samples with known constitutional or somatic NF1 deletions. The assay showed high diagnostic sensitivity and specificity and distinguished between Type-1, Type-2, and atypical constitutional microdeletions in 14 different samples. It also identified 16 different somatic deletions in dNFs. These results were confirmed by multiplex ligation-dependent probe amplification. The qPCR assay provides a methodology for detecting constitutional NF1 microdeletions that could be incorporated as an additional technique in a genetic-testing setting. It also permits the identification of somatic NF1 deletions in tissues with a high percentage of cells bearing 2 copies of the NF1 gene.

Neurofibromatosis Type 1 Complicated by Atypical Coarctation of the Thoracic Aorta

Neurofibromatosis type 1 (NF1) is a relatively common autosomal dominant genetic disorder with a prevalence of 1 in 3,000 (0.03%) at birth. Clinical features are café-au-lait macules, intertriginous freckling, dermal neurofibroma, iris hamartoma (Lisch nodules), and learning disability. NF1 vasculopathy is a serious but underrecognized complication involving the cerebrovascular and cardiovascular systems. The incidence of hypertension in patients with NF1 is around 1% and is associated mainly with renal artery stenosis in children. Only a few cases of thoracic aortic coarctation in association with hypertension and neurofibromatosis have been reported. Here we describe the case of a 4-year-old girl who presented with NF1 and hypertension due to atypical coarctation of the thoracic aorta. The diagnosis of coarctation of the thoracic aorta at the Th5-to-Th6 level was made following catheterization with a pressure gradient of 40 mmHg. The patient underwent surgery comprising resection of the coarctation of the thoracic aorta and graft interposition. On the basis of our findings, annual assessment of blood pressure is advised for patients with NF1.

Huge solitary neurofibroma of the oral cavity – A case report

Solitary neurofibroma is rare benign non-odontogenic tumour of the oral cavity [1]. The World Health Organisation (WHO) has subdivided neurofibromas into two categories, dermal and plexiform. Dermal neurofibroma arises from single peripheral nerve while plexiform neurofibroma is associated with a multiple nerve bundle. Uncommon variant of neurofibroma is the diffuse neurofibroma which typically involves skin and subcutaneous tissue resulting in an enlargement of affected tissues. We present a case of neurofibroma of the floor of the mouth. A 23-year-old female complained of painless swelling in the left side of the floor of mouth, pouting in the left submandibular region since 3 years. Histological features were characteristics of neurofibroma. We review the clinical and pathological features of the neurofibromas arising from the floor of the mouth. Neurofibromas are immunosensitive for the S-100 proteins, indicating its neural origin. Treatment is surgical with excellent prognosis.

Applying Microsatellite Multiplex PCR Analysis (MMPA) for Determining Allele Copy-Number Status and Percentage of Normal Cells within Tumors

The study of somatic genetic alterations in tumors contributes to the understanding and management of cancer. Genetic alterations, such us copy number or copy neutral changes, generate allelic imbalances (AIs) that can be determined using polymorphic markers. Here we report the development of a simple set of calculations for analyzing microsatellite multiplex PCR data from control-tumor pairs that allows us to obtain accurate information not only regarding the AI status of tumors, but also the percentage of tumor-infiltrating normal cells, the locus copy-number status and the mechanism involved in AI. We validated this new approach by re-analyzing a set of Neurofibromatosis type 1-associated dermal neurofibromas and comparing newly generated data with results obtained for the same tumors in a previous study using MLPA, Paralog Ratio Analysis and SNP-array techniques.Microsatellite multiplex PCR analysis (MMPA) should be particularly useful for analyzing specific regions of the genome containing tumor suppressor genes and also for determining the percentage of infiltrating normal cells within tumors allowing them to be sorted before they are analyzed by more expensive techniques.