Integration of single-nuclei RNA-sequencing, spatial transcriptomics and histochemistry defines the complex microenvironment of NF1-associated plexiform neurofibromas.

Plexiform neurofibroma (PN) is a leading cause of morbidity in children with the genetic condition Neurofibromatosis Type 1 (NF1), often disfiguring or threatening vital structures. During formation of PN, a complex tumor microenvironment (TME) develops, with recruitment of neoplastic and non-neoplastic cell types being critical for growth and progression. Due to the cohesive cellularity of PN, single-cell RNA-sequencing is difficult and may result in a loss of detection of critical cellular subpopulations. To bypass this barrier, we performed single-nuclei RNA-sequencing (snRNA-seq) on 8 frozen PN samples, and integrated this with spatial transcriptomics (ST) in 4 PN samples and immunohistochemistry to provide morphological context to transcriptomic data. SnRNA-seq analysis definitively charted the heterogeneous cellular subpopulations in the PN TME, with the predominant fraction being fibroblast subtypes. PN showed a remarkable amount of inter-sample homogeneity regarding cellular subpopulation proportions despite being resected from a variety of anatomical locations. ST analysis identified distinct cellular subpopulations which were annotated using snRNA-seq data and correlated with histological features. Schwann cell/fibroblast interactions were identified by receptor/ligand interaction analysis demonstrating a high probability of Neurexin 1/Neuroligin 1 (NRXN1/NLGN1) receptor-ligand cross-talk predicted between fibroblasts and non-myelinated Schwann cells (NM-SC) and subtypes, respectively. We observed aberrant expression of NRXN1 and NLGN1 in our PN snRNA-seq data compared to a normal mouse sciatic nerve single-cell RNA-seq dataset. This pathway has never been described in PN and may indicate a clear and direct communication pathway between putative NM-SC cells of origin and surrounding fibroblasts, potentially driving disease progression. SnRNA-seq integrated with spatial transcriptomics advances our understanding of the complex cellular heterogeneity of PN TME and identify potential novel communication pathways that may drive disease progression, a finding that could provide translational therapy options for patients with these devastating tumors of childhood and early adulthood.

Multiple Nf1 Schwann cell populations reprogram the plexiform neurofibroma tumor microenvironment.

To define alterations early in tumor formation, we studied nerve tumors in neurofibromatosis 1 (NF1), a tumor predisposition syndrome. Affected individuals develop neurofibromas, benign tumors driven by NF1 loss in Schwann cells (SCs). By comparing normal nerve cells to plexiform neurofibroma (PN) cells using single-cell and bulk RNA sequencing, we identified changes in 5 SC populations, including a de novo SC progenitor-like (SCP-like) population. Long after Nf1 loss, SC populations developed PN-specific expression of Dcn, Postn, and Cd74, with sustained expression of the injury response gene Postn and showed dramatic expansion of immune and stromal cell populations; in corresponding human PNs, the immune and stromal cells comprised 90% of cells. Comparisons between injury-related and tumor monocytes/macrophages support early monocyte recruitment and aberrant macrophage differentiation. Cross-species analysis verified each SC population and unique conserved patterns of predicted cell-cell communication in each SC population. This analysis identified PROS1-AXL, FGF-FGFR, and MIF-CD74 and its effector pathway NF-κB as deregulated in NF1 SC populations, including SCP-like cells predicted to influence other types of SCs, stromal cells, and/or immune cells in mouse and human. These findings highlight remarkable changes in multiple types of SCs and identify therapeutic targets for PN.

Concomitant variants in NF1, LZTR1 and GNAZ genes probably contribute to the aggressiveness of plexiform neurofibroma and warrant treatment with MEK inhibitor.

Neurofibromatosis 1 (NF1) is caused by germline mutations in the NF1 gene and manifests as proliferation of various tissues, including plexiform neurofibromas. The plexiform neurofibroma phenotype varies from indolent to locally aggressive, suggesting contributions of other modifiers in addition to somatic loss of NF1. In this study, we investigated a life-threatening plexiform neurofibroma in a 9-month-old female infant with NF1. Germline mutations in two RASopathy-associated genes were identified using whole-exome sequencing-a de novo pathogenic variant in the NF1 gene, and a known pathogenic variant in the LZTR1 gene. Somatic analysis of the plexiform neurofibroma revealed NF1 loss of heterozygosity and a variant in GNAZ, a gene encoding a G protein-coupled receptor. Cells expressing mutant GNAZ exhibited increased ERK 1/2 activation compared to those expressing wild-type GNAZ. Taken together, we suggest the variants in NF1, LZRT1 and GNAZ act synergistically in our patient, leading to MAPK pathway activation and contributing to the severity of the patient's plexiform neurofibromatosis. After treatment with the MEK inhibitor, trametinib, a prominent clinical improvement was observed in this patient. This case study contributes to the knowledge of germline and somatic non-NF1 variants affecting the NF1 clinical phenotype and supports use of personalized, targeted therapy.

Population pharmacokinetics and exposure-response of selumetinib and its N-desmethyl metabolite in pediatric patients with neurofibromatosis type 1 and inoperable plexiform neurofibromas.

PURPOSE: Selumetinib (ARRY-142886) is a potent, selective, MEK1/2 inhibitor approved in the US for the treatment of children (≥ 2 years) with neurofibromatosis type 1 (NF1) and symptomatic, inoperable plexiform neurofibromas (PN). We characterized population pharmacokinetics (PK) of selumetinib and its active N-desmethyl metabolite, evaluated exposure-safety/efficacy relationships, and assessed the proposed therapeutic dose of 25 mg/m2 bid based on body surface area (BSA) in this patient population. METHODS: Population PK modeling and covariate analysis (demographics, formulation, liver enzymes, BSA, patients/healthy volunteers) were based on pooled PK data from adult healthy volunteers (n = 391), adult oncology patients (n = 83) and pediatric patients with NF1-PN (n = 68). Longitudinal selumetinib/metabolite exposures were predicted with the final model. Exposure-safety/efficacy analyses were applied to pediatric patients (dose levels: 20, 25, 30 mg/m2 bid). RESULTS: Selumetinib and metabolite concentration-time courses were modeled using a joint compartmental model. Typical selumetinib plasma clearance was 11.6 L/h (95% CI 11.0-12.2 L/ h). Only BSA had a clinically relevant (> 20%) impact on exposure, supporting BSA-based administration in children. Selumetinib and metabolite exposures in responders (≥ 20% PN volume decrease from baseline) and non-responders were largely overlapping, with medians numerically higher in responders. No clear relationships between exposure and safety events were established; exposure was not associated with key adverse events (AEs) including rash acneiform, diarrhea, vomiting, and nausea. CONCLUSION: Findings support continuous selumetinib 25 mg/m2 bid in pediatric patients. Importantly, the updated dosing nomogram ensures that patients will receive a clinically active, yet tolerable, dose regardless of differences in BSA and allows dose reductions, if necessary.

MEK inhibitors in RASopathies.

PURPOSE OF REVIEW: An early understanding of the role of the Ras/Raf/MEK/ERK signalling pathway in regulating cell proliferation has set the stage for the development of several potent and selective MEK inhibitors (MEKi). MEKi represent promising therapies for RAS-driven neoplasias and RASopathies associated with increased Ras/MAPK activity. RECENT FINDINGS: Neurofibromatosis 1 (NF1) is a prototypic RASopathy in which early-phase clinical trials with MEKi have been successful in the treatment of plexiform neurofibromas (pNF) and low-grade gliomas (LGGs). The phase 2 trial (SPRINT) of selumetinib in pNF resulted in at least 20% reduction in the size of pNF from baseline in 71% of patients and was associated with clinically meaningful improvements. On the basis of this trial, selumetinib (Koselugo) received FDA approval for children 2 years of age and older with inoperable, symptomatic pNF. The phase 2 trial of selumetinib in LGG resulted in 40% partial response and 96% of patients had 2 years of progression-free survival. SUMMARY: Given the potential of MEK inhibition as an effective and overall well tolerated medical treatment, the use of targeted agents in the NF1 population is likely to increase considerably. Future work on non-NF1 RASopathies should focus on developing preclinical models and defining endpoints for measurement of efficacy in order to conduct clinical trials.

Early administration of imatinib mesylate reduces plexiform neurofibroma tumor burden with durable results after drug discontinuation in a mouse model of neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by plexiform neurofibromas (pNF), which are thought to be congenital tumors that arise in utero and enlarge throughout life. Genetic studies in murine models delineated an indispensable role for the stem cell factor (SCF)/c-kit pathway in pNF initiation and progression. A subsequent phase 2 clinical trial using imatinib mesylate to inhibit SCF/c-kit demonstrated tumor shrinkage in a subset of preexisting pNF; however, imatinib's role on preventing pNF development has yet to be explored. PROCEDURE: We evaluated the effect of imatinib dosed at 10-100 mg/kg/day for 12 weeks to one-month-old Nf1flox/flox ;PostnCre(+) mice, prior to onset of pNF formation. To determine durability of response, we then monitored for pNF growth at later time points, comparing imatinib- with vehicle-treated mice. We assessed gross and histopathological analysis of tumor burden. RESULTS: Imatinib administered preventatively led to a significant decrease in pNF number, even at doses as low as 10 mg/kg/day. Tumor development continued to be significantly inhibited after cessation of imatinib dosed at 50 and 100 mg/kg/day. In the cohort of treated mice that underwent prolonged follow-up, the size of residual tumors was significantly reduced as compared with age-matched littermates that received vehicle control. CONCLUSIONS: Early administration of imatinib inhibits pNF genesis in vivo, and effects are sustained after discontinuation of therapy. These findings may guide clinical use of imatinib in young NF1 patients prior to the substantial development of pNF.

Integrative Analysis Identifies Candidate Tumor Microenvironment and Intracellular Signaling Pathways that Define Tumor Heterogeneity in NF1.

Neurofibromatosis type 1 (NF1) is a monogenic syndrome that gives rise to numerous symptoms including cognitive impairment, skeletal abnormalities, and growth of benign nerve sheath tumors. Nearly all NF1 patients develop cutaneous neurofibromas (cNFs), which occur on the skin surface, whereas 40-60% of patients develop plexiform neurofibromas (pNFs), which are deeply embedded in the peripheral nerves. Patients with pNFs have a ~10% lifetime chance of these tumors becoming malignant peripheral nerve sheath tumors (MPNSTs). These tumors have a severe prognosis and few treatment options other than surgery. Given the lack of therapeutic options available to patients with these tumors, identification of druggable pathways or other key molecular features could aid ongoing therapeutic discovery studies. In this work, we used statistical and machine learning methods to analyze 77 NF1 tumors with genomic data to characterize key signaling pathways that distinguish these tumors and identify candidates for drug development. We identified subsets of latent gene expression variables that may be important in the identification and etiology of cNFs, pNFs, other neurofibromas, and MPNSTs. Furthermore, we characterized the association between these latent variables and genetic variants, immune deconvolution predictions, and protein activity predictions.

A phase 2 study of trametinib for patients with pediatric glioma or plexiform neurofibroma with refractory tumor and activation of the MAPK/ERK pathway: TRAM-01.

BACKGROUND: Pediatric low-grade gliomas (PLGG) are the most frequent brain tumors in children. Up to 50% will be refractory to conventional chemotherapy. It is now known that the majority of PLGG have activation of the MAPK/ERK pathway. The same pathway is also activated in plexiform neurofibromas (PNs) which are low-grade tumors involving peripheral nerves in patients with neurofibromatosis type 1 (NF1). These lesions are known to be refractory to chemotherapy. Specific MEK inhibitors such as trametinib are now available and have been approved for other cancers harboring mutations in the MAPK/ERK pathway such as melanoma. We have observed significant responses to trametinib in patients with refractory PLGG in our institutions and results from the phase I study are promising. The treatment appears not only efficacious but is also usually well tolerated. We hypothesize that we will observe responses in the majority of refractory PLGG and PN treated with trametinib in this phase 2 study. METHODS: The primary objective is to determine the objective response rate of trametinib as a single agent for treatment of progressing/refractory tumors with MAPK/ERK pathway activation. The TRAM-01 study is a phase II multicentric open-label basket trial including four groups. Group 1 includes NF1 patients with progressing/refractory glioma. Group 2 includes NF1 patients with plexiform neurofibroma. Group 3 includes patients with progressing/refractory glioma with KIAA1549-BRAF fusion. Group 4 includes other patients with progressing/refractory glioma with activation of the MAPK/ERK pathway. Eligible patients for a given study group will receive daily oral trametinib at full dose for a total of 18 cycles of 28 days. A total of 150 patients will be enrolled in seven Canadian centers. Secondary objectives include the assessment of progression-free survival, overall survival, safety and tolerability of trametinib, serum levels of trametinib and evaluation of quality of life during treatment. DISCUSSION: Trametinib will allow us to target directly and specifically the MAPK/ERK pathway. We expect to observe a significant response in most patients. Following our study, trametinib could be integrated into standard treatment of PLGG and PN. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03363217 December 6, 2017.

The evolution and multi-molecular properties of NF1 cutaneous neurofibromas originating from C-fiber sensory endings and terminal Schwann cells at normal sites of sensory terminations in the skin.

In addition to large plexiform neurofibromas (pNF), NF1 patients are frequently disfigured by cutaneous neurofibromas (cNF) and are often afflicted with chronic pain and itch even from seemingly normal skin areas. Both pNFs and cNF consist primarily of benign hyperproliferating nonmyelinating Schwann cells (nSC). While pNF clearly arise within deep nerves and plexuses, the role of cutaneous innervation in the origin of cNF and in chronic itch and pain is unknown. First, we conducted a comprehensive, multi-molecular, immunofluorescence (IF) analyses on 3mm punch biopsies from three separate locations in normal appearing, cNF-free skin in 19 NF1 patients and skin of 16 normal subjects. At least one biopsy in 17 NF1 patients had previously undescribed micro-lesions consisting of a small, dense cluster of nonpeptidergic C-fiber endings and the affiliated nSC consistently adjoining adnexal structures-dermal papillae, hair follicles, sweat glands, sweat ducts, and arterioles-where C-fiber endings normally terminate. Similar micro-lesions were detected in hind paw skin of mice with conditionally-induced SC Nf1-/- mutations. Hypothesizing that these microlesions were pre-cNF origins of cNF, we subsequently analyzed numerous overt, small cNF (s-cNF, 3-6 mm) and discovered that each had an adnexal structure at the epicenter of vastly increased nonpeptidergic C-fiber terminals, accompanied by excessive nSC. The IF and functional genomics assays indicated that neurturin (NTRN) and artemin (ARTN) signaling through cRET kinase and GFRα2 and GFRα3 co-receptors on the aberrant C-fiber endings and nSC may mutually promote the onset of pre-cNF and their evolution to s-cNF. Moreover, TrpA1 and TrpV1 receptors may, respectively, mediate symptoms of chronic itch and pain. These newly discovered molecular characteristics might be targeted to suppress the development of cNF and to treat chronic itch and pain symptoms in NF1 patients.

STAT3 inhibition reduces macrophage number and tumor growth in neurofibroma.

Plexiform neurofibroma, a benign peripheral nerve tumor, is associated with the biallelic loss of function of the NF1 tumor suppressor in Schwann cells. Here, we show that FLLL32, a small molecule inhibitor of JAK2/STAT3 signaling, reduces neurofibroma growth in mice with conditional, biallelic deletion of Nf1 in the Schwann cell lineage. FLLL32 treatment or Stat3 deletion in tumor cells reduced inflammatory cytokine expression and tumor macrophage numbers in neurofibroma. Although STAT3 inhibition downregulated the chemokines CCL2 and CCL12, which can signal through CCR2 to recruit macrophages to peripheral nerves, deletion of Ccr2 did not improve survival or reduce macrophage numbers in neurofibroma-bearing mice. Interestingly, Iba1+; F4/80+;CD11b+ macrophages accounted for ~20-40% of proliferating cells in untreated tumors. FLLL32 suppressed macrophage proliferation, implicating STAT3-dependent, local proliferation in neurofibroma macrophage accumulation, and decreased Schwann cell proliferation and increased Schwann cell death. The functions of STAT3 signaling in neurofibroma Schwann cells and macrophages, and its relevance as a therapeutic target in neurofibroma, merit further investigation.

Spatiotemporal Loss of NF1 in Schwann Cell Lineage Leads to Different Types of Cutaneous Neurofibroma Susceptible to Modification by the Hippo Pathway.

Neurofibromatosis type 1 (NF1) is a cancer predisposition disorder that results from inactivation of the tumor suppressor neurofibromin, a negative regulator of RAS signaling. Patients with NF1 present with a wide range of clinical manifestations, and the tumor with highest prevalence is cutaneous neurofibroma (cNF). Most patients harboring cNF suffer greatly from the burden of those tumors, which have no effective medical treatment. Ironically, none of the numerous NF1 mouse models developed so far recapitulate cNF. Here, we discovered that HOXB7 serves as a lineage marker to trace the developmental origin of cNF neoplastic cells. Ablating Nf1 in the HOXB7 lineage faithfully recapitulates both human cutaneous and plexiform neurofibroma. In addition, we discovered that modulation of the Hippo pathway acts as a "modifier" for neurofibroma tumorigenesis. This mouse model opens the doors for deciphering the evolution of cNF to identify effective therapies, where none exist today. SIGNIFICANCE: This study provides insights into the developmental origin of cNF, the most common tumor in NF1, and generates the first mouse model that faithfully recapitulates both human cutaneous and plexiform neurofibroma. The study also demonstrates that the Hippo pathway can modify neurofibromagenesis, suggesting that dampening the Hippo pathway could be an attractive therapeutic target.This article is highlighted in the In This Issue feature, p. 1.

Malignant Peripheral Nerve Sheath Tumors Show Decreased Global DNA Methylation.

Malignant peripheral nerve sheath tumors (MPNST) are aggressive spindle cell neoplasms that may occur sporadically, often in association with radiation exposure, or in the clinical context of Neurofibromatosis type 1. MPNST are known to harbor genetic alterations affecting the function of polycomb repressive complex 2 (PRC2), resulting in profound changes to global H3K27me3 levels. Recent evidence suggests a link between the polycomb complex and DNA methylation. Given the established epigenetic alterations found in MPNST, we aimed to further explore global methylation changes including 5-methylcystosine (5mC), 5-hydroxymethylcytosine (5hmC), and H3K27me3 levels using previously validated immunolabeling protocols in a representative cohort of 28 peripheral nerve sheath tumors (MPNST [n = 8], localized cutaneous neurofibroma [n = 10], and plexiform neurofibroma [n = 10]). MPNST showed significantly decreased levels of H3K27me3 (p < 0.0002) and 5mC (p = 0.0001) with levels of 5hmC showing borderline statistical significance (p = 0.05) when compared to localized and plexiform neurofibromas. Immunohistochemical findings of decreased H3K27me3 and 5mC further our understanding of global epigenetic alterations observable in MPNST and may provide insight into the basis of tumor progression as well as prognostic and treatment implications in the future.

The primacy of NF1 loss as the driver of tumorigenesis in neurofibromatosis type 1-associated plexiform neurofibromas.

Neurofibromatosis type 1 (NF1) is a common tumor-predisposition disorder due to germline mutations in the tumor suppressor gene NF1. A virtually pathognomonic finding of NF1 is the plexiform neurofibroma (PN), a benign, likely congenital tumor that arises from bi-allelic inactivation of NF1. PN can undergo transformation to a malignant peripheral nerve sheath tumor, an aggressive soft-tissue sarcoma. To better understand the non-NF1 genetic contributions to PN pathogenesis, we performed whole-exome sequencing, RNASeq profiling and genome-wide copy-number determination for 23 low-passage Schwann cell cultures established from surgical PN material with matching germline DNA. All resected tumors were derived from routine debulking surgeries. None of the tumors were considered at risk for malignant transformation at the time; for example, there was no pain or rapid growth. Deep (~500X) NF1 exon sequencing was also conducted on tumor DNA. Non-NF1 somatic mutation verification was performed using the Ampliseq/IonTorrent platform. We identified 100% of the germline NF1 mutations and found somatic NF1 inactivation in 74% of the PN. One individual with three PNs had different NF1 somatic mutations in each tumor. The median number of somatic mutations per sample, including NF1, was one (range 0-8). NF1 was the only gene that was recurrently somatically inactivated in multiple tumors. Gene Set Enrichment Analysis of transcriptome-wide tumor RNA sequencing identified five significant (FDR<0.01) and seven trending (0.01⩽FDR<0.02) gene sets related to DNA replication, telomere maintenance and elongation, cell cycle progression, signal transduction and cell proliferation. We found no recurrent non-NF1 locus copy-number variation in PN. This is the first multi-sample whole-exome and whole-transcriptome sequencing study of NF1-associated PN. Taken together with concurrent copy-number data, our comprehensive genetic analysis reveals the primacy of NF1 loss as the driver of PN tumorigenesis.

Targeted NF1 cancer therapeutics with multiple modes of action: small molecule hormone-like agents resembling the natural anticancer metabolite, 2-methoxyoestradiol.

BACKGROUND: Both the number and size of tumours in NF1 patients increase in response to the rise in steroid hormones seen at puberty and during pregnancy. The size of tumours decreases after delivery, suggesting that hormone-targeting therapy might provide a viable new NF1 treatment approach. Our earlier studies demonstrated that human NF1 tumour cell lines either went through apoptosis or ceased growth in the presence of 2-methoxyoestradiol (2ME2), a naturally occurring anticancer metabolite of 17-ß estradiol. Previous reports of treatment with sulfamoylated steroidal and non-steroidal derivatives of 2ME2 showed promising reductions in tumour burden in hormone-responsive cancers other than NF1. Here we present the first studies indicating that 2ME2 derivatives could also provide an avenue for treating NF1, for which few treatment options are available. METHODS: STX3451, (2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline), a non-steroidal sulphamate analogue of 2ME2, was tested in dose-dependent studies of malignant and benign NF1 human tumour cell lines and cell lines with variable controlled neurofibromin expression. The mechanisms of action of STX3451 were also analysed. RESULTS: We found that STX3451-induced apoptosis in human malignant peripheral nerve sheath tumour (MPNST) cell lines, even in the presence of elevated oestrogen and progesterone. It inhibits both PI3 kinase and mTOR signalling pathways. It disrupts actin- and microtubule-based cytoskeletal structures in cell lines derived from human MPNSTs and in cells derived from benign plexiform neurofibromas. STX3451 selectively kills MPNST-derived cells, but also halts growth of other tumour-derived NF1 cell lines. CONCLUSION: STX3451 provides a new approach for inducing cell death and lowering tumour burden in NF1 and other hormone-responsive cancers with limited treatment options.

TAGLN expression is upregulated in NF1-associated malignant peripheral nerve sheath tumors by hypomethylation in its promoter and subpromoter regions.

Neurofibromatosis type 1 (NF1) caused by NF1 gene mutation is a commonly inherited autosomal dominant disorder. Malignant peripheral nerve sheath tumors (MPNSTs), a type of aggressive sarcoma, are a major cause of mortality in NF1 patients. The malignant transformation of benign plexiform neurofibromas (PNs) to MPNSTs is a marked peculiarity in NF1 patients, yet the pathogenesis remains poorly understood. We found that an actin-associated protein transgelin (SM22) was highly expressed in NF1-deficient MPNST tissues compared to NF1-deficient PN tissues using immunohistological staining and primary cultured MPNST cells in western blot analysis. We further found that this transgelin upregulation was caused by increased transcriptional expression of the TAGLN gene encoding transgelin. Comparison of DNA methylation values in the promoter and subpromoter regions of the TAGLN gene in three types of NF1-deficient primary-cultured cells, derived from an NF1 patient's normal phenotype, a benign PN and MPNST tissues, revealed that the TAGLN gene was hypomethylated in the MPNST cells. Next, to determine the functional role of transgelin in MPNST pathogenesis, we manipulated the TAGLN gene expression and investigated the alteration of the RAS-mitogen-activated protein kinase (MAPK) signaling pathway in the normal-phenotypic and malignant tumor cells. The downregulation of TAGLN expression in NF1-deficient MPNST tumor cells through the treatment of the small interfering RNA resulted in a decrease in the RAS activation (GTP-RAS) and the downstream ERK1/2 activation (phosphorylated ERK1/2), while the overexpression of TAGLN in normal-phenotypic NF1-deficient cells caused an increase in RAS and ERK1/2 activation. These results indicate that upregulation of transgelin caused by hypomethylation of the TAGLN gene is closely involved in tumor progression in NF1.

Secretome survey of human plexiform neurofibroma derived Schwann Cells reveals a secreted form of the RARRES1 protein.

To bring insights into neurofibroma biochemistry, a comprehensive secretome analysis was performed on cultured human primary Schwann cells isolated from surgically resected plexiform neurofibroma and from normal nerve tissue. Using a combination of SDS-PAGE and high precision LC-MS/MS, 907 proteins were confidently identified in the conditioned media of Schwann cell cultures combined. Label free proteome profiling revealed consistent release of high levels of 22 proteins by the four biological replicates of NF1 Schwann cell cultures relative to the two normal Schwann cell cultures. Inversely, 9 proteins displayed decreased levels in the conditioned media of NF1 relative to normal Schwann cells. The proteins with increased levels included proteins involved in cell growth, angiogenesis and complement pathway while proteins with decreased levels included those involved in cell adhesion, plasminogen pathway and extracellular matrix remodeling. Retinoic acid receptor responder protein-1 (RARRES1), previously described as an integral membrane tumor suppressor, was found exclusively secreted by NF1 Schwann cells but not by normal Schwann cells. All-trans retinoic acid modulated secretion of RARRES1 in a dose dependent manner. This study shows altered secretion of key proteins in NF1 derived Schwann cells. The potential implication of these proteins in neurofibroma biology is discussed.

Expression of MiTF may be helpful in differentiating cellular neurothekeoma from plexiform fibrohistiocytic tumor (histiocytoid predominant) in a partial biopsy specimen.

BACKGROUND: Overlapping histopathologic features of cellular neurothekeoma (CNT) and plexiform fibrohistiocytic tumor (PFHT), when both are predominantly composed of histiocytoid cells, make distinction between these entities challenging. Some have suggested that CNT and PFHT are related entities. No prior study has demonstrated a reliable immunohistochemical panel to differentiate these entities. METHODS: Skin biopsies diagnosed as CNT and PFHT, from 2004 to 2010 were retrieved with accompanying pathology reports. Each case was reviewed by at least 2 dermatopathologists and 2 soft tissue pathologists for confirmation of diagnosis. All cases were then evaluated for immunohistochemical expression of PAX2, NKIC3, CD10, and microphthalmia transcription factor (MiTF). RESULTS: Histopathologically, the histiocytoid areas of each tumor shared similar architecture, demonstrating nests and fascicles of histiocytoid to spindled cells, with some separation of nests by collagen bands. Both CNT and PFHT were uniformly positive for NKIC3 and CD10, and both were frequently PAX2 positive. MiTF was strongly and diffusely positive in CNT and was consistently negative in the PFHT. CONCLUSIONS: CNT and PFHT share many histopathologic features and immunohistochemical staining patterns. Of the stains we evaluated, we found that expression of MiTF may be a reliable marker for distinguishing CNT from histiocytoid-predominant PFHT, especially in instances where only a small part of the tumor is sampled for evaluation.

Genetically engineered mouse models shed new light on the pathogenesis of neurofibromatosis type I-related neoplasms of the peripheral nervous system.

Neurofibromatosis type 1 (NF1), the most common genetic disorder affecting the human nervous system, is characterized by the development of multiple benign Schwann cell tumors in skin and large peripheral nerves. These neoplasms, which are termed dermal and plexiform neurofibromas respectively, have distinct clinical courses; of particular note, plexiform, but not dermal, neurofibromas often undergo malignant progression to form malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy occurring in NF1 patients. In recent years, a number of genetically engineered mouse models have been created to investigate the molecular mechanisms driving the pathogenesis of these tumors. These models have been designed to address key questions including: (1) whether NF1 loss in the Schwann cell lineage is essential for tumorigenesis; (2) what cell type(s) in the Schwann cell lineage gives rise to dermal neurofibromas, plexiform neurofibromas and MPNSTs; (3) how the tumor microenvironment contributes to neoplasia; (4) what additional mutations contribute to neurofibroma-MPNST progression; (5) what role different neurofibromin-regulated Ras proteins play in this process and (6) how dysregulated growth factor signaling facilitates PNS tumorigenesis. In this review, we summarize the major findings from each of these models and their limitations as well as how discrepancies between these models may be reconciled. We also discuss how information gleaned from these models can be synthesized to into a comprehensive model of tumor formation in peripheral nervous system and consider several of the major questions that remain unanswered about this process.