Malignant Peripheral Nerve Sheath Tumors Samples Research Articles

Characterizing T-cell dysfunction and exclusion signatures in malignant peripheral nerve sheath tumors reveals susceptibilities to immunotherapy.

Malignant peripheral nerve sheath tumors (MPNSTs) are malignant tumors that arise from peripheral nerves and are the leading cause of mortality in Neurofibromatosis Type 1 (NF1). In this study, we characterized whether transcriptomic signatures of T-cell dysfunction (TCD) and exclusion (TCE) that inversely correlate with response to immune checkpoint blockade (ICB) immunotherapy exist in MPNSTs. MPNST transcriptomes were pooled from Gene Expression Omnibus (GEO). For each sample, a tumor immune dysfunction and exclusion (TIDE) score, TCD and TCE subscores, and cytotoxic T-cell(CTL) level were calculated. In the TIDE predictive algorithm, tumors are predicted to have an ICB response if they are either immunologically hot (CTL-high) without TCD or immunologically cold (CTL-low) without TCE. TIDE scores greater than zero correspond with ICB nonresponse. 73 MPNST samples met inclusion criteria, including 50 NF1-associated MPNSTs (68.5%). The average TIDE score was + 0.41 (SD = 1.16) with 22 (30.1%) predicted ICB responders. 11 samples were CTL-high (15.1%) with an average TCD score of + 0.99 (SD = 0.63). Among 62 CTL-low tumors, 21 were predicted to have ICB response with an average TCE score of + 0.31(SD = 1.20). Age(p = 0.18), sex(p = 0.41), NF1 diagnosis (p = 0.17), and PRC2 loss(p = 0.29) were not associated with ICB responder status. Transcriptomic analysis of TCD and TCE signatures in MPNST samples reveals that a select subset of patients with MPNSTs may benefit from ICB immunotherapy.

DDDR-06. NEUROFIBROMATOSIS TUMOR SUPPRESSORS COOPERATIVELY DRIVE TUMOR DE-DIFFERENTIATION AND MEK INHIBITOR RESISTANCE IN PERIPHERAL NERVOUS SYSTEM TUMORS

Abstract Schwann cell derived tumors comprising schwannomas, neurofibromas, and malignant peripheral nerve sheath tumors (MPNSTs) are the most common cancers of the peripheral nervous system and often arise in patients with neurofibromatosis type-1 (NF-1) or type-2 (NF-2). NF-1 is caused by loss of NF1, a negative regulator of Ras signaling, and NF-2 is caused by loss of NF2, a pleiotropic tumor suppressor with numerous functions including inhibition of PAK signaling. However, whether functional interactions exist between the NF1 and NF2 tumor suppressors remain unclear. More broadly, there are currently no effective molecular therapies for patients with Schwann cell tumors beyond the MEK inhibitor selumetinib to treat neurofibromas in patients with NF-1. Here, we integrate DNA methylation profiling, whole exome sequencing, bulk and single-cell RNA sequencing, biochemistry, and pharmacology across human samples, cell lines, and mouse xenografts to identify cellular de-differentiation as a driver of malignant transformation and selumetinib resistance. Single nuclear RNA-sequencing of human neurofibromas (n = 3) or MPNSTs (n = 3) revealed a total of 13 cell types with increased proliferating, de-differentiated tumor cell populations in MPNST samples. Single cell RNA-sequencing of MPNST mouse xenografts revealed persistence of de-differentiated cell populations in selumetinib treated samples compared to vehicle control, suggesting cellular de-differentiation underlies treatment resistance. A genome-wide CRISPRi screen for mediators of selumetinib response in NF1 deficient neurofibroma cells revealed NF2 loss drives selumetinib resistance. Consistently, NF2 suppression in NF1 deficient neurofibroma cells caused Schwann cell de-differentiation and activation of PAK, a serine threonine kinase. Translationally, a small molecule PAK inhibitor in combination with selumetinib formed an effective therapy in mouse MPNST xenografts. In sum, we elucidate a paradigm of de-differentiation driving malignant transformation and treatment resistance in Schwann cell tumors, uncovering a functional link between the NF1 and NF2 tumor suppressors that sheds light on a novel therapeutic vulnerability.

Cell-free DNA ultra-low-pass whole genome sequencing to distinguish malignant peripheral nerve sheath tumor (MPNST) from its benign precursor lesion: A cross-sectional study.

BackgroundThe leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors.Methods and findingsThis multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity). Healthy controls without NF1 (participants = 16, plasma samples = 16), PN (participants = 23, plasma samples = 23), and MPNST (participants = 14, plasma samples = 46) cohorts showed significant differences in tumor fraction in plasma (P = 0.001) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. No other covariates were significant on multivariate logistic regression. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Greater genomic instability including alterations associated with malignant transformation (focal copy number gains in chromosome arms 1q, 7p, 8q, 9q, and 17q; focal copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arms 6p and 9p) was more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (r = 0.39, P = 0.024). On serial analysis, tumor fraction levels in plasma dynamically correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse. Study limitations include a modest MPNST sample size despite accrual from 2 major referral centers for this rare malignancy, and lack of uniform treatment and imaging protocols representing a real-world cohort.ConclusionsTumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations.

Abstract 3898: Loss of polycomb repressive complex 2 activates HOXB8 and remodels super enhancers in malignant peripheral nerve sheath tumors

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are aggressive and highly metastatic soft tissue sarcomas that lack effective treatment. Loss of the functional polycomb repressive complex 2 (PRC2) through genetic alterations in its core components EED and/or SUZ12 is prevalent in this devastating disease. Consequently, the product of PRC2, trimethylation of histone H3 lysine 27 (H3K27me3), a transcriptional repression marker, was found absent in MPNST samples. However, the epigenetic consequences of PRC2 loss remain unclear. Methods: Using an inducible expression vector, we restored a functional PRC2 in three MPNST cell lines that are PRC2 deficient (PRC2-null). We then profiled the accompanied changes in histone modifications by chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-seq) and transcriptional changes by RNA sequencing (RNAseq). To test the clinical relevance of our findings, we performed single-cell RNAseq (scRNAseq) on one PRC2-null patient MPNST sample. Finally, to target the epigenetic abnormity caused by PRC2 loss, we tested the efficacy of a histone deacetylase inhibitor (HDACi) in treating MPNST cells. Results: We confirmed the tumor suppressor role of a functional PRC2 in MPNSTs by showing that PRC2 restoration caused growth inhibition in PRC2-null MPNST cells but not in those with a wildtype PRC2. Using ChIP-seq, we identified 6135 H3K27me3 peaks, which are associated with 2285 genes, that were commonly gained in all three PRC2-null MPNST cell lines with PRC2 restoration. Results of RNAseq show that more than 50% of these genes were kept silenced even without H3K27me3 and about 10% of the highly expressed genes were re-repressed by PRC2 (fold change &amp;lt; 0.7). Interestingly, among the 145 genes that are transcriptionally regulated by polycomb, 91 were found to be co-occupied by H3K4me3 at their promoters. Since co-existence of H3K27me3 and H3K4me3 at the promoter define a bivalent gene, we further investigated the epigenetic changes associated with PRC2 restoration there. Analysis of ChIP-seq results revealed that, accompanied with gains of H3K27me3, there was loss of acetylated H3K27, a marker of super enhancer (SE), at the bivalent sites. Particularly, we identified a group of oncogenic transcription factors (TFs), including homeobox B8 (HOXB8), that were activated due to the loss of PRC2 and their activation was driven by SEs. Furthermore, the analysis of scRNAseq of our patient MPNST sample confirmed that these essential SE-driven TFs can be used to separate malignant cells from the tumor microenvironment. Finally, in vitro studies show that HDACi was highly effective in treating MPNST cells by de-activating these SE-driven TFs. Conclusion: Herein, we report a novel mechanism underlying the polycomb-regulated transcriptional repression. In MPNSTs, the loss of PRC2 caused activation of a group of oncogenic transcription factors through remodeling the SE landscape, which resulted their vulnerabilities to epigenetic treatments by HDACi. Citation Format: Xiyuan Zhang, Hannah E. Lou, Haiyan Lei, Zhihui Liu, Marielle Yohe, Carol Thiele, Brigitte Widemann, Jack F. Shern. Loss of polycomb repressive complex 2 activates HOXB8 and remodels super enhancers in malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3898.

From Genes to -Omics: The Evolving Molecular Landscape of Malignant Peripheral Nerve Sheath Tumor.

Malignant peripheral nerve sheath tumors (MPNST) are rare, aggressive soft tissue sarcomas that occur with significantly increased incidence in people with the neuro-genetic syndrome neurofibromatosis type I (NF1). These complex karyotype sarcomas are often difficult to resect completely due to the involvement of neurovascular bundles, and are relatively chemotherapy- and radiation-insensitive. The lifetime risk of developing MPNST in the NF1 population has led to great efforts to characterize the genetic changes that drive the development of these tumors and identify mutations that may be used for diagnostic or therapeutic purposes. Advancements in genetic sequencing and genomic technologies have greatly enhanced researchers’ abilities to broadly and deeply investigate aberrations in human MPNST genomes. Here, we review genetic sequencing efforts in human MPNST samples over the past three decades. Particularly for NF1-associated MPNST, these overall sequencing efforts have converged on a set of four common genetic changes that occur in most MPNST, including mutations in neurofibromin 1 (NF1), CDKN2A, TP53, and members of the polycomb repressor complex 2 (PRC2). However, broader genomic studies have also identified recurrent but less prevalent genetic variants in human MPNST that also contribute to the molecular landscape of MPNST and may inform further research. Future studies to further define the molecular landscape of human MPNST should focus on collaborative efforts across multiple institutions in order to maximize information gathered from large numbers of well-annotated MPNST patient samples, both in the NF1 and the sporadic MPNST populations.

HuR/ELAVL1 drives malignant peripheral nerve sheath tumor growth and metastasis.

Cancer cells can develop a strong addiction to discrete molecular regulators, which control the aberrant gene expression programs that drive and maintain the cancer phenotype. Here, we report the identification of the RNA-binding protein HuR/ELAVL1 as a central oncogenic driver for malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive sarcomas that originate from cells of the Schwann cell lineage. HuR was found to be highly elevated and bound to a multitude of cancer-associated transcripts in human MPNST samples. Accordingly, genetic and pharmacological inhibition of HuR had potent cytostatic and cytotoxic effects on tumor growth, and strongly suppressed metastatic capacity in vivo. Importantly, we linked the profound tumorigenic function of HuR to its ability to simultaneously regulate multiple essential oncogenic pathways in MPNST cells, including the Wnt/β-catenin, YAP/TAZ, RB/E2F, and BET pathways, which converge on key transcriptional networks. Given the exceptional dependency of MPNST cells on HuR for survival, proliferation, and dissemination, we propose that HuR represents a promising therapeutic target for MPNST treatment.

Aberrations of TBX2-CHK2-p53 Signaling Pathway and its Role in Malignant Peripheral Nerve Sheath Tumors

Background and Objectives: The dismal outcome of malignant peripheral nerve sheath tumor (MPNST) highlights the necessity of identifying new biomarkers and pathogenesis for this aggressive sarcoma. Therefore, it is necessary to detect the aberrations of the TBX2-CHK2-p53 pathway and investigate its biological role in MPNST. Methods: Genetic aberrations of TBX2, CHK2 and p53 were detected by next generation sequencing (NGS) in 10 MPNST samples. Protein expression of TBX2, CHK2, p53, Ki-67 and cyclin D1 were assessed by immunohistochemistry (IHC) in 63 MPNST samples. Results: Our present data demonstrated that there were gene mutations of TBX2, CHK2 and p53 in MPNST samples. TBX2 expression was correlated with American Joint Committee on Cancer (AJCC) stage, recurrence and metastasis. Correlation analysis found that TBX2 was positively correlated with CHK2 (p=0.045) and CHK2 was positively correlated with p53 (p=0.006). Furthermore, both CHK2 and p53 were positively correlated with Ki-67 (p&lt;0.05), which is related to tumor differentiation, metastasis and prognosis. As for survival analyses, patients with high TBX2, CHK2 and p53 expression exhibited shorter disease-free survival (DFS) and overall survival (OS), respectively (p&lt;0.05) and TBX2 and CHK2 were independent prognostic factors for MPNST patients (p&lt;0.05). Conclusion: There are genetic aberrations of the TBX2-CHK2-p53 signaling pathway in MPNST, which might promote the progression of MPNST by increasing Ki-67 expression. Thus, TBX2 and CHK2 might be useful markers for MPNST.

H3K27 trimethylation loss in malignant peripheral nerve sheath tumor: a systematic review and meta-analysis with diagnostic implications.

Multiple studies have reported the loss of trimethylation at lysine (K) 27 on histone 3 (H3K27me3) in high-grade malignant peripheral nerve sheath tumors (MPNSTs). However, the diagnostic potential of this finding in MPNSTs remains yet to be fully substantiated. Correspondingly, our aim was to pool systematically-identified metadata in the literature and substantiate the incidence of H3K27me3 loss in this setting. Searches of 7 electronic databases from inception to May 2019 were conducted following PRISMA guidelines. Articles were screened against pre-specified criteria. The incidence of loss was then pooled by random-effects meta-analysis of proportions. Nine pertinent studies described a total of 823 high-grade MPNST samples. When pooled, incidence (sensitivity) of complete H3K27me3 loss was estimated to be 53% (95% CI 42-64%). For MPNST subtypes, estimated incidences of complete loss in NF1 subtype was 52% (95% CI 41-62), in sporadic subtype was 53% (95% CI 36-70%), in the epithelioid subtype was 0% (95% CI 0-7%), and radiation-associated subtype was 98% (95% CI 86-100%). Finally, incidence of incomplete loss (specificity) in 1231 MPNST-mimic samples was estimated to be 96% (95% CI 90-99%). Certainty of these outcomes ranged from very low to high. The incidence of complete H3K27me3 loss is substantial in high-grade MPNSTs and is low in MPNST-mimics. Greater cohort study and biological investigation will validate the certainty of these findings as well as elucidate their true molecular and clinical significances.

Abstract 3473: Investigating RAS and RTK signaling in MPNST progression and drug resistance

Abstract Neurofibromatosis, type 1 (NF1) is a tumor predisposition syndrome affecting about 1 in 2,500 live births in the United States. The protein product of the NF1 gene, neurofibromin, loses its function as a negative regulator of the RAS protein, leading to the development of painful and disfiguring benign tumors known as a neurofibromas. Eight to thirteen percent of these neurofibromas will transform into malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are aggressive soft tissue sarcomas that do not respond to the standard sarcoma treatment of chemotherapy and surgical resection. There is a critical need for efficacious targeted therapy to increase the average life span of these patients. Current targeted therapy efforts have shown modest efficacy clinically due to the establishment of compensatory feedback mechanisms that propagate tumor progression and recurrence. These feedback mechanisms are not completely understood in MPNSTs. In lung cancer, inhibition of MEK in the RAS pathway has been shown to activate upstream receptor tyrosine kinases (RTK). Genomic studies of MPNST samples have shown that 30% of tumors have amplification of the RTKs MET and EGFR. Furthermore, in a study of the genomic progression of a NF1 patient pre- and post-treatment, we found amplification of MET and EGFR that increased following treatment. In a study on triple-negative breast cancer cells, it has been shown that MET and EGFR crosstalk may be responsible for the reactivation of the inhibited cancer pathways and drug resistance. Therefore, we hypothesize that coordinated deregulated RAS signaling and MEK inhibition leads to MET and EGFR amplification, expression, activation, and crosstalk, which drives MPNST progression, drug resistance, and tumor recurrence. To test our hypothesis, we will use both in vitro and in vivo experimental methods to pursue the following specific aims: (1) determine the effect of MEK inhibition on establishing compensatory feedback loops that increase RTK activation and downstream signaling, (2) determine the role of MET-EGFR crosstalk signaling in MPNST tumor progression and resistance to kinase inhibition, and (3) determine the efficacy of combination RTK and MEK inhibition in vivo. Citation Format: Candace N. King, Carrie Graveel, Matthew Steensma. Investigating RAS and RTK signaling in MPNST progression and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3473.

Immunohistochemical evaluation of H3K27 trimethylation in malignant peripheral nerve sheath tumors

The histological definitive diagnosis of malignant peripheral nerve sheath tumor (MPNST) is quite difficult because the morphological features are not specific and no useful immunohistochemical marker has been identified. Loss-of-function mutations in EED or SUZ12, which encode the core subunit of polycomb repressive complex 2 (PRC2), were reported in MPNSTs, and the mutations were shown to cause inactivation of PRC2, leading to loss of trimethylation of histone H3 at lysine 27 (H3K27me3). Immunohistochemistry of H3K27me3 is expected to be a specific marker for MPNSTs. We evaluated immunohistochemical expression of H3K27me3 in MPNSTs with heterologous components and metachronous cases of MPNSTs. Among 145 MPNST samples, 50 (34.5%) showed complete loss of staining, and 45 (31.0%) showed partial loss of staining. Regarding the backgrounds of MPNSTs, 43 patients of neurofibromatosis type 1 (NF-1)-associated MPNST demonstrated 19 (44.2%) complete and 12 (27.9%) partial loss of H3K27me3. Among MPNSTs with heterologous component, almost all of MPNSTs with epithelioid differentiation (8/9 samples, 88.9%) retained H3K27me3, and malignant Triton tumors without epithelioid component lacked H3K27me3 at high rate (91.7%). Five of 20 metachronous MPNST cases showed significantly reduced expression of H3K27me3 between primary and later-occurring tumors, but in some cases increased expression of H3K27me3 in the clinical course (such as complete loss to partial loss) was observed. If the tumors are recurrent or metastatic, H3K27me3 expression should be reduced or at least maintained because loss of H3K27me3 is due to genetic mutation of EED or SUZ12. MPNSTs, especially those associated with NF-1, can occur in heterochronous and multiple patterns, and the identification of increased expression of H3K27me3 during a patient’s clinical course can be helpful for determining whether the tumors are heterochronous, multiple or not. As heterochronous and multiple tumors may show lower malignancy compared to recurrent or metastatic tumors, favorable prognosis may be expected when H3K27me3 expression is increased.

Expression and inhibition of BRD4, EZH2 and TOP2A in neurofibromas and malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNST) are rare, highly aggressive sarcomas that can occur spontaneously or from pre-existing plexiform neurofibromas in neurofibromatosis type1 (NF1) patients. MPNSTs have high local recurrence rates, metastasize easily, are generally resistant to therapeutic intervention and frequently fatal for the patient. Novel targeted therapeutic strategies are urgently needed. Standard treatment for patients presenting with advanced disease is doxorubicin based chemotherapy which inhibits the actions of the enzyme topoisomerase IIα (TOP2A). Recent molecular studies using murine models and cell lines identified the bromodomain containing protein 4 (BRD4) and enhancer of zeste homolog 2 (EZH2) as novel targets for MPNST treatment. We investigated the expression and potential use of BRD4, EZH2 and TOP2A as therapeutic targets in human NF1-derived MPNSTs. The transcript levels of BRD4, EZH2 and TOP2A were determined in paired formalin-fixed paraffin-embedded (FFPE) neurofibroma/MPNST samples derived from the same NF1 patient and in a set of plexiform neurofibromas, atypical neurofibromas and MPNST. We further examined the effect on cell viability of genetic or pharmacological inhibition of BRD4, EZH2 and TOP2A in an MPNST cell line panel. Our results indicated that in MPNST samples BRD4 mRNA levels were not upregulated and that MPNST cell lines were relatively insensitive to the bromodomain inhibitor JQ1. We corroborated that EZH2 mRNA expression is increased in MPNST but failed to confirm its reported pivotal role in MPNST pathogenesis as EZH2 knockdown by siRNA did not interfere with cellular proliferation and viability. Finally, the relation between TOP2A levels and sensitivity for doxorubicin was examined, confirming reports that TOP2A mRNA levels were overexpressed in MPNST and showing that MPNST cell lines exhibited relatively high TOP2A protein levels and sensitivity to doxorubicin. We tentatively conclude that the potential for effective therapeutic intervention in MPNST by targeting BRD4, EZH2 and TOP2A individually, may be limited. Clinical studies are necessary to ultimately prove the relevance of BRD4 and EZH2 inhibition as novel therapeutic strategies for MPNST.

Abstract 4433: Deregulated microRNAs in NF1 derived malignant peripheral nerve sheath tumors (MPNST) and their role in carcinogenesis

Abstract Background and aim: MPNSTs are rare and highly aggressive soft tissue sarcomas that can occur spontaneously (sporadic MPNST) or from pre-existing plexiform neurofibromas in neurofibromatosis type 1 (NF1) patients. MPNSTs are usually diagnosed relatively late, metastasize easily, are resistant to therapeutic intervention and frequently fatal for the patient. Diagnostic biomarkers that detect early malignant transformation in the NF1 setting as well identification of putative targets for treatment are urgently needed. In this context we investigated the miRNA expression profiles in NF1-derived MPNST. Materials and Methods: Total RNA was isolated from sections of formalin fixed paraffin embedded (FFPE) tumor samples. miRNA expression was determined using a RT-PCR platform (TaqMan® Low Density Array Human MicroRNA Cards; Applied Biosystems). The expression of selected miRNAs was transiently modulated in neurofibroma and MPNST cell lines by transfection with miRNA mimics (Dharmacon) or LNA™ based anti-miRs (Exiqon) and effects on cell proliferation, invasion/migration and Wnt/β-catenin signaling were studied. Results: In order to identify miRNAs that are specifically deregulated in MPNST we analyzed miRNA expression in a unique set paired tumor samples (n=9) of plexiform neurofibroma and MPNST which were derived from the same NF1 patient. At least 90 miRNAs were found to be differentially expressed (FDR &amp;lt;10%; p&amp;lt;0.025) between neurofibromas and MPNST; the vast majority (82/90) of miRNAs being downregulated in MPNST. Interestingly, and in contrast to reports in the literature, sporadic MPNST samples (n=10) could be easily discriminated from NF1-derived MPNST by their miRNA expression profiles. On the basis of their statistical significance, fold difference and expression level, we selected miRNAs to examine their role in MPNST pathogenesis. MiRNA levels were modulated in MPNST (ST88-14, sNF96.2, 90-8TL) and neurofibroma (Hs53.T) cell lines using miRNA mimics for let-7b, miR-16, miR-26a, miR-29a, miR-139-5p, miR-146a and antisense inhibitors for miR-889, miR-135b. The effects of miRNA expression levels on cellular proliferation, invasion and migration were determined. As Wnt/β-catenin signaling is considered a cancer related driver pathway in MPNST we also investigated whether differentially expressed miRNAs could regulate this pathway focusing on miRNA previously linked to Wnt regulation or predicted to target Wnt signaling components. Using TOP/FOP luciferase reporter assays we analyzed the effects of miR-135b, miR-30a, miR-139-5p and miR-146a. Conclusion: miRNAs specifically deregulated in MPNST may fulfil key oncogenic roles and may be used as biomarker or for therapeutic purposes. Citation Format: Azadeh Amirnasr, Patricia F. van Kuijk, Robert M. Verdijk, Walter Taal, Stefan Sleijfer, Erik A. C. Wiemer. Deregulated microRNAs in NF1 derived malignant peripheral nerve sheath tumors (MPNST) and their role in carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4433. doi:10.1158/1538-7445.AM2017-4433

Hypoxia-inducible factor 1 alpha is a poor prognostic factor and potential therapeutic target in malignant peripheral nerve sheath tumor.

BackgroundMalignant peripheral nerve sheath tumor (MPNST) is a rare soft tissue sarcoma with poor prognosis. Hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the cellular response to hypoxia and regulates the expression of multiple genes involved in tumor progression in various cancers. However, the importance of the expression of HIF-1α in MPNSTs is unclear.MethodsThe expression of HIF-1α was examined immunohistochemically in 82 MPNST specimens. Cell culture assays of human MPNST cells under normoxic and hypoxic conditions were used to evaluate the impact of anti-HIF-1α–specific siRNA inhibition on cell survival. A screening kit was employed to identify small molecules that inhibited HIF-1α.ResultsThe nuclear expression of HIF-1α was positive in 75.6% of MPNST samples (62/82 cases). Positivity for HIF-1α was a significant poor prognostic factor both in univariate (P = 0.048) and multivariate (P ≤ 0.0001) analyses. HIF-1α knockdown abrogated MPNST cell growth, inducing apoptosis. Finally, chetomin, an inhibitor of HIF-1α, effectively inhibited the growth of MPNST cells and induced their apoptosis.ConclusionInhibition of HIF-1α signaling is a potential treatment option for MPNSTs.

Prognostic roles for fibroblast growth factor receptor family members in malignant peripheral nerve sheath tumor.

BackgroundMalignant peripheral nerve sheath tumors (MPNST) are rare, highly malignant, and poorly understood sarcomas. The often poor outcome of MPNST highlights the necessity of identifying prognostic predictors for this aggressive sarcoma. Here, we investigate the role of fibroblast growth factor receptor (FGFR) family members in human MPNSTs.ResultsaCGH and bioinformatics analysis identified frequent amplification of the FGFR1 gene. FISH analysis revealed that 26.9% MPNST samples had amplification of FGFR1, with both focal and polysomy patterns observed. IHC identified that FGFR1 protein expression was positively correlated with FGFR1 gene amplification. High expression of FGFR1 protein was associated with better overall survival (OS) and was an independent prognostic predictor for OS of MPNST patients. Additionally, combined expression of FGFR1 and FGFR2 protein characterized a subtype of MPNST with better OS. FGFR4 protein was expressed 82.3% of MPNST samples, and was associated with poor disease-free survival.Materials and MethodsWe performed microarray-based comparative genomic hybridization (aCGH) profiling of two cohorts of primary MPNST tissue samples including 25 patients treated at The University of Texas MD Anderson Cancer Center and 26 patients from Tianjin Medical University Cancer Institute and Hospital. Fluorescence in situ hybridization (FISH) was used to validate the gene amplification detected by aCGH analysis. Another cohort of 63 formalin-fixed paraffin-embedded MPNST samples (including 52 samples for FISH assay) was obtained to explore FGFR1, 2, 3, and 4 protein expression by immunohistochemical (IHC) analysis.ConclusionsOur integrated genomic and molecular studies provide evidence that FGFRs play different prognostic roles in MPNST.

Whole Exome Sequencing Reveals the Order of Genetic Changes during Malignant Transformation and Metastasis in a Single Patient with NF1-plexiform Neurofibroma.

Malignant peripheral nerve sheath tumors (MPNST) occur at increased frequency in individuals with neurofibromatosis type 1 (NF1), where they likely arise from benign plexiform neurofibroma precursors. While previous studies have used a variety of discovery approaches to discover genes associated with MPNST pathogenesis, it is currently unclear what molecular events are associated with the evolution of MPNST from plexiform neurofibroma. Whole-exome sequencing was performed on biopsy materials representing plexiform neurofibroma (n = 3), MPNST, and metastasis from a single individual with NF1 over a 14-year period. Additional validation cases were used to assess candidate genes involved in malignant progression, while a murine MPNST model was used for functional analysis. There was an increasing proportion of cells with a somatic NF1 gene mutation as the tumors progressed from benign to malignant, suggesting a clonal process in MPNST development. Copy number variations, including loss of one copy of the TP53 gene, were identified in the primary tumor and the metastatic lesion, but not in benign precursor lesions. A limited number of genes with nonsynonymous somatic mutations (βIII-spectrin and ZNF208) were discovered, several of which were validated in additional primary and metastatic MPNST samples. Finally, increased βIII-spectrin expression was observed in the majority of MPNSTs, and shRNA-mediated knockdown reduced murine MPNST growth in vivo. Collectively, the ability to track the molecular evolution of MPNST in a single individual with NF1 offers new insights into the sequence of genetic events important for disease pathogenesis and progression for future mechanistic study.

Methylated RASSF1A in malignant peripheral nerve sheath tumors identifies neurofibromatosis type 1 patients with inferior prognosis.

BackgroundMalignant peripheral nerve sheath tumor (MPNST) is a rare and highly aggressive disease with no evidence of effect from adjuvant therapy. It is further associated with the hereditary syndrome neurofibromatosis type 1 (NF1). Silencing of the tumor suppressor gene RASSF1A through DNA promoter hypermethylation is known to be involved in cancer development, but its impact in MPNSTs remains unsettled.MethodsThe RASSF1A promoter was analyzed by methylation-specific PCR in 113 specimens, including 44 NF1-associated MPNSTs, 47 sporadic MPNSTs, 21 benign neurofibromas, and 1 nonneoplastic nerve sheath control.ResultsRASSF1A methylation was found only in the malignant samples (60%) and identified a subgroup among patients with NF1-associated MPNST with a poor prognosis. These patients had a mean 5-year disease-specific survival of 27.3 months (95% CI: 17.2–37.4) versus 47.4 months (95% CI: 37.5–57.2) for NF1 patients with unmethylated promoters, P = 0.014. In multivariate Cox regression analysis, methylated RASSF1A remained an adverse prognostic factor independent of clinical risk factors, P = .013 (hazard ratio: 5.2; 95% CI: 1.4–19.4).ConclusionA considerable number of MPNST samples display hypermethylation of the RASSF1A gene promoter, and for these tumors, this is the first molecular marker that if validated can characterize a subgroup of patients with inferior prognosis, restricted to individuals with NF1.

Trp53 Haploinsufficiency Modifies EGFR-Driven Peripheral Nerve Sheath Tumorigenesis

Malignant peripheral nerve sheath tumors (MPNSTs) are genetically diverse, aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 1 syndrome. Reduced TP53 gene expression and amplification/overexpression of the epidermal growth factor receptor (EGFR) gene occur in MPNST formation. We focused on determining the cooperativity between reduced TP53 expression and EGFR overexpression for Schwann cell transformation in vitro (immortalized human Schwann cells) and MPNST formation in vivo (transgenic mice). Human gene copy number alteration data, microarray expression data, and TMA analysis indicate that TP53 haploinsufficiency and increased EGFR expression co-occur in human MPNST samples. Concurrent modulation of EGFR and TP53 expression in HSC1λ cells significantly increased proliferation and anchorage-independent growth in vitro. Transgenic mice heterozygous for a Trp53-null allele and overexpressing EGFR in Schwann cells had a significant increase in neurofibroma and grade 3 PNST (MPNST) formation compared with single transgenic controls. Histological analysis of tumors identified a significant increase in pAkt expression in grade 3 PNSTs compared with neurofibromas. Array comparative genome hybridization analysis of grade 3 PNSTs identified recurrent focal regions of chromosomal gains with significant enrichment in genes involved in extracellular signal-regulated kinase 5 signaling. Collectively, altered p53 expression cooperates with overexpression of EGFR in Schwann cells to enhance in vitro oncogenic properties and tumorigenesis and progression in vivo.

RETRACTED ARTICLE: Genomic amplification and high expression of EGFR are key targetable oncogenic events in malignant peripheral nerve sheath tumor

BackgroundThe dismal outcome of malignant peripheral nerve sheath tumor (MPNST) highlights the necessity of finding new therapeutic methods to benefit patients with this aggressive sarcoma. Our purpose was to investigate epidermal growth factor receptor (EGFR) as a potential therapeutic target in MPNSTs.Patients and methodsWe performed a microarray based-comparative genomic hybridization (aCGH) profiling of two cohorts of primary MPNST tissue samples including 25 patients treated at The University of Texas MD Anderson Cancer Center (MD Anderson) and 26 patients from Tianjin Medical University Cancer Institute & Hospital (TMUCIH). Fluorescence in situ hybridization (FISH) method was used to validate the gene amplification detected by aCGH analysis. Another independent cohort of 56 formalin fixed paraffin embedded (FFPE) MPNST samples was obtained to explore EGFR protein expression by immunohistochemical analysis. Cell biology detection and validation were performed on human MPNST cell lines ST88-14 and STS26T.ResultsaCGH and pathway analysis of the 51 MPNSTs identified significant gene amplification events in EGFR pathway, including frequent amplifications of EGFR gene itself, which was subsequently validated by FISH assay. High expression of EGFR protein was associated with poor disease-free and overall survival of human MPNST patients. In human MPNST cell lines ST88-14 and STS26T, inhibition of EGFR by siRNA or Gefitinib led to decreased cell proliferation, migration, and invasion accompanied by attenuation of PI3K/AKT and MAPK pathways.ConclusionThese results suggest that EGFR is a potential therapeutic target for MPNST.

Chemically Induced Rat Schwann Cell Neoplasia as a Model for Early-Stage Human Peripheral Nerve Sheath Tumors: Phenotypic Characteristics and Dysregulated Gene Expression

Most malignant human tumors display a high degree of intratumoral heterogeneity at the time of diagnosis that contributes to treatment failure. This also applies to malignant peripheral nerve sheath tumors (MPNSTs) and aggressive soft tissue sarcomas that arise sporadically or in the context of neurofibromatosis type 1. On average, MPNSTs measure 10 cm in diameter at diagnosis. To explore molecular changes associated with early malignant progression and that may be present in most, if not all, tumor cells, we generated expression profiles of ethylnitrosourea-induced trigeminal MPNSTs in rats. Because these tumors cause increased intracranial pressure, they become detectable when they are comparatively minuscule. Histologic analyses revealed close resemblance to human MPNSTs. Compared with normal trigeminal nerve tissue, 365 genes were markedly upregulated and 310 genes were consistently downregulated in all MPNST samples. The molecular signature characteristic of early-stage MPNSTs included upregulation of proliferation and tissue remodeling-associated genes, downregulation of genes involved in Schwann cell differentiation, and the absence of transcripts associated with neuronal components. The transforming growth factor-β pathway was consistently upregulated in all tumor samples. These data suggest that the signaling pathways underlying early malignant progression of Schwann cells might be targeted to prevent tumor growth and/or to treat more advanced lesions.

Genome‐wide high‐resolution analysis of DNA copy number alterations in NF1‐associated malignant peripheral nerve sheath tumors using 32K BAC array

Neurofibromatosis Type I (NF1) is an autosomal dominant disorder characterized by the development of both benign and malignant tumors. The lifetime risk for developing a malignant peripheral nerve sheath tumor (MPNST) in NF1 patients is approximately 10% with poor survival rates. To date, the molecular basis of MPNST development remains unclear. Here, we report the first genome-wide and high-resolution analysis of DNA copy number alterations in MPNST using the 32K bacterial artificial chromosome microarray on a series of 24 MPNSTs and three neurofibroma samples. In the benign neurofibromas, apart from loss of one copy of the NF1 gene and copy number polymorphisms, no other changes were found. The profiles of malignant samples, however, revealed specific loss of chromosomal regions including 1p35-33, 1p21, 9p21.3, 10q25, 11q22-23, 17q11, and 20p12.2 as well as gain of 1q25, 3p26, 3q13, 5p12, 5q11.2-q14, 5q21-23, 5q31-33, 6p23-p21, 6p12, 6q15, 6q23-q24, 7p22, 7p14-p13, 7q21, 7q36, 8q22-q24, 14q22, and 17q21-q25. Copy number gains were more frequent than deletions in the MPNST samples (62% vs. 38%). The genes resident within common regions of gain were NEDL1 (7p14), AP3B1 (5q14.1), and CUL1 (7q36.1) and these were identified in >63% MPNSTs. The most frequently deleted locus encompassed CDKN2A, CDKN2B, and MTAP genes on 9p21.3 (33% cases). These genes have previously been implicated in other cancer conditions and therefore, should be considered for their therapeutic, prognostic, and diagnostic relevance in NF1 tumorigenesis.