Benign Plexiform Neurofibromas Research Articles

Identification of the Determinants of Plexiform Neurofibroma Morbidity in Pediatric and Young Adult Neurofibromatosis Type 1 Patients: A Pilot Multivariate Approach.

Plexiform neurofibromas (PNs) are histologically benign peripheral nerve sheath tumors associated with neurofibromatosis type 1 (NF1) and often lead to significant morbidity due to growth. Management includes watchful waiting, surgery for partial debulking, and, since recently, systemic treatment with MEK inhibitors. However, due to the scarcity of natural history studies, our understanding of the natural progression of PNs to guide clinicians in deciding in whom and when to intervene is scarce. This study aims to describe the characteristics of NF1 patients with PNs and compare those at high risk for PN progression or experiencing significant morbidity from PN (complex PN) with NF1 patients with PNs of lower complexity. In this retrospective cohort study using clinical data from hospital records of NF1 patients with PNs seen at the Sophia Children's Hospital in the Netherlands between 2012 and 2023, we assessed determinants of clinical phenotypes and PN characteristics predictive of outcomes, including PN complexity and the timing of intervention for PN. We assessed the outcomes using logistic regression analysis and Cox regression. Ninety patients with a median age at last evaluation of 15.7 years and a median follow-up duration of 9.8 years were included. Out of 90 individuals with a benign PN, 37 developed plexiform neurofibroma morbidity during follow-up. Older age was (corrected for pathogenic NF1 variant and PN location) significantly associated with plexiform neurofibroma morbidity. Cox regression revealed that craniofacial and trunk PNs were associated with a higher intervention hazard compared to limb PNs. Our pilot multivariate approach identified older age and the location of the PN to be mostly associated with a higher chance of plexiform neurofibroma morbidity and higher intervention hazard. This may contribute to decisions regarding in whom and when to initiate treatment in NF1 patients with PNs.

BIOM-37. EXPLORING L-TYPE AMINO ACID TRANSPORTER 1 (LAT1) AS A DIAGNOSTIC MARKER AND THERAPEUTIC TARGET IN MALIGNANT PERIPHERAL NERVE SHEATH TUMORS (MPNST)

Abstract OBJECTIVES Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that frequently arise from benign plexiform neurofibromas (PN). Approximately half of MPNST are associated with Neurofibromatosis type 1 (NF1). Despite partial elucidation of the molecular mechanisms underlying MPNST, diagnosis remains challenging, and prognosis is poor. Previously, we found that MPNST cells have elevated βIII-spectrin, a cytoskeletal protein involved in cell morphology and regulation of transporters, including L-type Amino Acid Transporter 1 (LAT1). LAT1 facilitates transport of large neutral amino acids and is upregulated in other cancer types. Our objective is to investigate LAT1 as a diagnostic marker and therapeutic target for MPNST. METHODS Gene and protein expression were determined by qPCR and WES protein analysis, respectively. The effect of shRNA knockdown of LAT1 in MPNST cells was assessed using IncuCyte proliferation assays as well as multiple mouse models. Tumor uptake of [18F] FDOPA, primarily transported by LAT1, in MPNST PDX tumors was assessed using PET/CT imaging. RESULTS LAT1 was expressed in MPNST (n=14) vs. benign PN tissues (n=6). Additionally, in human and murine MPNST cells, shRNA-mediated knockdown of LAT1 suppressed proliferation of MPNST cells in vitro and tumor growth in vivo. In a subcutaneous tumor growth model, intratumor injection of shLAT1 lentivirus reduced tumor volume to approximately 50% of control. Furthermore, uptake of [18F] FDOPA by LAT1 in MPNST PDX tumors was detected by PET/CT imaging. WU-356 PDX tissues exhibited higher LAT1 expression as well as increased uptake of [18F] FDOPA in tumors. Conversely, PET imaging showed significantly lower uptake of [18F] FDOPA in LAT1 knockdown tumors (p<0.05). CONCLUSIONS These results suggest that LAT1 promotes the growth and survival of MPNST cells. In addition, these findings demonstrate the potential use of LAT1 and the radiotracer [18F] FDOPA as a diagnostic biomarker in the management of MPNST.

Clinical features and surgical treatments of neurofibromas associated with neurofibromatosis type 1

To explore the clinical features, surgical treatment, and effectiveness of neurofibromas associated with neurofibromatosis type 1 (NF1). A clinical data of 41 patients with NF1 admitted between December 2018 and April 2024 was retrospectively analyzed. There were 15 males and 26 females, with an average age of 27.5 years (range, 5-61 years). Only one type of neurofibroma existed in 3 patients and the rest of the patients had more than two types of neurofibromas. Fourteen patients had total resection of multiple cutaneous neurofibromas (CNF). Eighteen patients of diffuse neurofibromas underwent total, near-total, or subtotal resection. Among the 13 patients of localized nodular neurofibromas, 9 of benign tumors underwent total sub-capsular resection and 4 of malignant peripheral nerve sheath tumor (MPNST) underwent maginal resection, and only 1 underwent postoperative radiotherapy and chemotherapy. Among the 15 patients of plexiform neurofibromas (PNF), 5 patients underwent both superficial and deep PNF resection, 2 underwent the superficial PNF resection, and 8 underwent the large nodular lesions in the deep PNF resection. There were 8 MPNST, of which 7 cases underwent total sub-capsular resection and large tumor capsule resection under neurophysiological monitoring, and 1 case with the tumor located on the top of the head underwent wide resection and skin grafting. One patient underwent proton knife therapy after surgery, 2 patients did not receive radiotherapy, and the remaining patients received conventional radiotherapy. All patients were followed up after surgery, and the follow-up time was 3-66 months, with an average of 25.0 months. Patients with CNF recovered satisfactorily after surgery, and there was no recurrence during follow-up. Patients with diffuse neurofibromas relieved preoperative symptoms after surgery. Three patients with diffuse neurofibromas located in the head and face recurred during follow-up. The patients with benign localized nodular neurofibromas recovered well after surgery, and only 1 patient had transient regional neuralgia after surgery. Among the patients with MPNST, 2 patients died of recurrence and lung metastasis, while the remaining 2 patients had no recurrence and metastasis during follow-up. All preoperative symptoms disappeared in patients with benign PNF, and no tumor recurrence was observed during follow-up. Two patients with PNF located in the brachial plexus had difficulty in shoulder abduction after surgery, 1 patient with PNF located in vagus developed hoarseness after surgery. Among the 8 patients with MPNST in PNF, 1 died of lung metastases and 1 died of systemic failure. The remaining 6 patients were in stable condition during follow-up, and no tumor recurrence or metastasis was observed. According to the clinical features of neurofibromas in patients with NF1, choosing appropriate surgical approaches can obtain good effectiveness. Because of the difficulty of completely resection, diffuse neurofibromas, especially those located in the head and face, are prone to recurrence after surgery. MPNST has the worst prognosis, high incidence of recurrence/metastasis, and short survival period. Total resection combined with radiotherapy can decrease local recurrence.

Proof of Concept for Genome Profiling of the Neurofibroma/Sarcoma Sequence in Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder characterized by the predisposition to develop tumors such as malignant peripheral nerve sheath tumors (MPNSTs) which represents the primary cause of death for NF1-affected patients. Regardless of the high incidence and mortality, the molecular mechanisms underneath MPNST growth and metastatic progression remain poorly understood. In this proof-of-concept study, we performed somatic whole-exome sequencing (WES) to profile the genomic alterations in four samples from a patient with NF1-associated MPNST, consisting of a benign plexiform neurofibroma, a primary MPNST, and metastases from lung and skin tissues. By comparing genomic patterns, we identified a high level of variability across samples with distinctive genetic changes which allow for the definition of profiles of the early phase with respect to the late metastatic stages. Pathogenic and likely pathogenic variants were abundant in the primary tumor, whereas the metastatic samples exhibited a high level of copy-number variations (CNVs), highlighting a possible genomic instability in the late phases. The most known MPNST-related genes, such as TP53 and SUZ12, were identified in CNVs observed within the primary tumor. Pathway analysis of altered early genes in MPNST pointed to a potential role in cell motility, division and metabolism. Moreover, we employed survival analysis with the TCGA sarcoma genomic dataset on 262 affected patients, in order to corroborate the predictive significance of the identified early and metastatic MPNST driver genes. Specifically, the expression changes related to the mutated genes, such as in RBMX, PNPLA6 and AGAP2, were associated with reduced patient survival, distinguishing them as potential prognostic biomarkers. This study underlines the relevance of integrating genomic results with clinical information for early diagnosis and prognostic understanding of tumor aggressiveness.

Early Detection of Malignant and Premalignant Peripheral Nerve Tumors Using Cell-Free DNA Fragmentomics.

Early detection of neurofibromatosis type 1 (NF1)-associated peripheral nerve sheath tumors (PNST) informs clinical decision-making, enabling early definitive treatment and potentially averting deadly outcomes. In this study, we describe a cell-free DNA (cfDNA) fragmentomic approach that distinguishes nonmalignant, premalignant, and malignant forms of PNST in the cancer predisposition syndrome, NF1. cfDNA was isolated from plasma samples of a novel cohort of 101 patients with NF1 and 21 healthy controls and underwent whole-genome sequencing. We investigated diagnosis-specific signatures of copy-number alterations with in silico size selection as well as fragment profiles. Fragmentomics were analyzed using complementary feature types: bin-wise fragment size ratios, end motifs, and fragment non-negative matrix factorization signatures. The novel cohort of patients with NF1 validated that our previous cfDNA copy-number alteration-based approach identifies malignant PNST (MPNST) but cannot distinguish between benign and premalignant states. Fragmentomic methods were able to differentiate premalignant states including atypical neurofibromas (AN). Fragmentomics also adjudicated AN cases suspicious for MPNST, correctly diagnosing samples noninvasively, which could have informed clinical management. Novel cfDNA fragmentomic signatures distinguish AN from benign plexiform neurofibromas and MPNST, enabling more precise clinical diagnosis and management. This study pioneers the early detection of malignant and premalignant PNST in NF1 and provides a blueprint for decentralizing noninvasive cancer surveillance in hereditary cancer predisposition syndromes.

Interferon-Induced Transmembrane Protein 1 (IFITM1) Is Downregulated in Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheath Tumors.

Neurofibromatosis type 1 (NF1), an autosomal dominant genetic disorder, is caused by mutations in the NF1 gene, which encodes the GTPase-activating protein neurofibromin. The pathogenesis of the tumor progression of benign plexiform neurofibromas (PNs) and malignant peripheral nerve sheath tumors (MPNSTs) remain unclear. Here, we found that interferon-induced transmembrane protein 1 (IFITM1) was downregulated in MPNST tissues compared to those in PN tissues from patients with NF1. Overexpression of IFITM1 in NF1-associated MPNST cells resulted in a significant decrease in Ras activation (GTP-Ras) and downstream extracellular regulatory kinase 1/2 (ERK1/2) phosphorylation, whereas downregulation of IFITM1 via treatment with small interfering RNA in normal Schwann cells had the opposite result, indicating that expression levels of IFITM1 are closely associated with tumor progression in NF1. Treatment of MPNST cells with interferon-gamma (IFN-γ) significantly augmented the expression of IFITM1, thereby leading to a decrease in Ras and ERK1/2 activation. Despite the small number of patient samples, these findings may potentially provide a new target for chemotherapy in patients with NF1-associated MPNSTs. In xenograft mice injected with MPNST cells, IFN-γ treatment successfully suppressed tumor progression with increased IFITM1 expression and decreased Ras and ERK1/2 activation in tumor tissues. Collectively, these results suggest that IFITM1 is closely involved in MPNST pathogenesis and that IFN-γ is a good candidate for the therapeutic treatment of MPNSTs in NF1.

Contemporary Approach to Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheath Tumors.

Most malignant peripheral nerve sheath tumors (MPNSTs) are clinically aggressive high-grade sarcomas, arising in individuals with neurofibromatosis type 1 (NF1) at a significantly elevated estimated lifetime frequency of 8%-13%. In the setting of NF1, MPNSTs arise from malignant transformation of benign plexiform neurofibroma and borderline atypical neurofibromas. Composed of neoplastic cells from the Schwannian lineage, these cancers recur in approximately 50% of individuals, and most patients die within five years of diagnosis, despite surgical resection, radiation, and chemotherapy. Treatment for metastatic disease is limited to cytotoxic chemotherapy and investigational clinical trials. In this article, we review the pathophysiology of this aggressive cancer and current approaches to surveillance and treatment.

PET Imaging of Neurofibromatosis Type 1 with a Fluorine-18 Labeled Tryptophan Radiotracer.

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder. Plexiform neurofibromas (PNFs) are benign tumors commonly formed in patients with NF1. PNFs have a high incidence of developing into malignant peripheral nerve sheath tumors (MPNSTs) with a 5-year survival rate of only 30%. Therefore, the accurate diagnosis and differentiation of MPNSTs from benign PNFs are critical to patient management. We studied a fluorine-18 labeled tryptophan positron emission tomography (PET) radiotracer, 1-(2-[18F]fluoroethyl)-L-tryptophan (L-[18F]FETrp), to detect NF1-associated tumors in an animal model. An ex vivo biodistribution study of L-[18F]FETrp showed a similar tracer distribution and kinetics between the wild-type and triple mutant mice with the highest uptake in the pancreas. Bone uptake was stable. Brain uptake was low during the 90-min uptake period. Static PET imaging at 60 min post-injection showed L-[18F]FETrp had a comparable tumor uptake with [1⁸F]fluorodeoxyglucose (FDG). However, L-[18F]FETrp showed a significantly higher tumor-to-brain ratio than FDG (n = 4, p < 0.05). Sixty-minute-long dynamic PET scans using the two radiotracers showed similar kidney, liver, and lung kinetics. A dysregulated tryptophan metabolism in NF1 mice was further confirmed using immunohistostaining. L-[18F]FETrp is warranted to further investigate differentiating malignant NF1 tumors from benign PNFs. The study may reveal the tryptophan-kynurenine pathway as a therapeutic target for treating NF1.

Perinatal folate levels do not influence tumor latency or multiplicity in a model of NF1 associated plexiform-like neurofibromas

ObjectiveIn epidemiological and experimental research, high folic acid intake has been demonstrated to accelerate tumor development among populations with genetic and/or molecular susceptibility to cancer. Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder predisposing affected individuals to tumorigenesis, including benign plexiform neurofibromas; however, understanding of factors associated with tumor risk in NF1 patients is limited. Therefore, we investigated whether pregestational folic acid intake modified plexiform-like peripheral nerve sheath tumor risk in a transgenic NF1 murine model.ResultsWe observed no significant differences in overall survival according to folate group. Relative to controls (180 days), median survival did not statistically differ in deficient (174 days, P = 0.56) or supplemented (177 days, P = 0.13) folate groups. Dietary folate intake was positively associated with RBC folate levels at weaning, (P = 0.023, 0.0096, and 0.0006 for deficient vs. control, control vs. supplemented, and deficient vs. supplemented groups, respectively). Dorsal root ganglia (DRG), brachial plexi, and sciatic nerves were assessed according to folate group. Mice in the folate deficient group had significantly more enlarged DRG relative to controls (P = 0.044), but no other groups statistically differed. No significant differences for brachial plexi or sciatic nerve enlargement were observed according to folate status.

Malignant Peripheral Nerve Sheath Tumors Activate Distinct Immunosuppressive Pathways Following Radiotherapy and are Associated with Immune Depletion In Vivo

Patients with neurofibromatosis type I, caused by NF1 loss, develop benign plexiform neurofibromas (pNF) in their peripheral nervous system (PNS). Malignant transformation of pNFs into malignant peripheral nerve sheath tumors (MPNSTs) occurs following CDKN2A/B and SUZ12 loss, a process associated with radiotherapy (RT). However, the molecular mechanisms underlying RT responses by different PNS cell types remain unclear. We hypothesized normal peripheral nerve cells, pNFs, and MPNSTs harbor distinct RT responses. Patient derived NF1 WT immortalized peripheral nerve cells (iPNs), NF1 mutant pNF cells, and NF1/CDKN2AB/SUZ12 mutant MPNST cells were used to study RT responses in vitro. CRISPRi was used to assess the functional effects of candidate gene repression. In vitro viability was measured by cell counts. Transcriptomic signatures were measured by bulk RNA-sequencing and integrated with single-cell RNA sequencing (scRNA-seq) data from patient-derived pNF and MPNST resection specimens. Radiation dose response curves revealed pNF cells (IC50 0.61 Gy) were more radiosensitive than MPNST cells (4.15 Gy). WT iPNs, NF1 deficient iPNs, and pNFs cells displayed no difference in cell viability (p = 0.67; t-test) following initiation of 2 Gy x 5 fractions, while MPNST cells were significantly more viable (p = 0.02; t-test). Principal component analysis of bulk RNA-sequencing data at 5 or 14 days following 2 Gy x 5 fractions revealed cell line of origin accounted for the greatest inter-sample variation (64.9% variance), with additional components separating samples based on radiation presence and timing. Using the most variable genes in PCA space to identify markers of RT response, iPNs and pNFs upregulated pro-apoptotic pathways (BAD, DAPK3) at 5 days post-radiation while MPNST cells alone upregulated pro-survival growth factor signaling). At 14 days post radiation, MPNST cells uniquely upregulated TGFβ signaling and interferon response circuits. Incorporation of scRNA-seq data revealed enrichment of growth factor signaling and TGFβ signatures in MPNSTs compared to pNF. Moreover, MPNST harbored significantly fewer immune cells than pNFs (p = 0.008, t-test), suggesting cell-autonomous signaling and crosstalk with the microenvironment are both critical to MPNST radioresistance. Our data indicate additional genetic hits beyond NF1 loss may be required for RT-associated malignant transformation of pNFs and radioresistance in MPNSTs. Analysis of transcriptomic responses to RT suggests that upregulated growth factor signaling and TGFβ-associated immunosuppression are distinct features of MPNST. Future work will focus on CRISPRi screens to unbiasedly nominate functional modifiers of RT response in NF1/CDKN2AB deficient tumors, which may be broadly useful in cancer.

Expression of Ras signaling pathway proteins and developmental factors in peripheral nerve sheath tumors of patients with neurofibromatosis type 1.

To characterize expression of factors relevant for Ras signaling and developmental factors in a large series of peripheral nerve sheath tumors (PNST) obtained from patients with neurofibromatosis type 1 (NF1). Tissue micro-array technique was applied to study 520 PNST of 385 NF1 patients by immunohistochemistry for mTor, Rho, phosphorylated MEK, Pax7, Sox9, and periaxin expression. PNST comprised cutaneous neurofibroma (CNF) (n=114), diffuse neurofibroma (DNF) (n=109), diffuse plexiform neurofibroma (DPNF) (n=108), plexiform neurofibroma (PNF) (n=110), and malignant PNST (MPNST) (n=22). All proteins examined showed highest expression levels/highest frequency of expression in MPNST. Benign PNF with potential for malignant dedifferentiation expressed mTor, phosphorylated MEK, Sox9, and periaxin significantly higher/more frequently than other benign neurofibroma subtypes. In NF1-associated PNST, expression of proteins involved in Ras-signaling and development is upregulated not only in MPNST, but also in benign PNF with the potential for malignant dedifferentiation. The differences in protein expression may provide clues for understanding the therapeutic effects of substances applied for reduction of PNST in NF1.

Abstract 997: Cell-free DNA fragmentomics distinguish between benign, pre-malignant and malignant peripheral nerve sheath tumors in neurofibromatosis type 1

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that, in the setting of neurofibromatosis type 1 (NF1), arise within pre-malignant atypical neurofibroma (AN) and benign plexiform neurofibroma (PN). Early surgical resection improves prognosis, however, early detection by imaging and tissue biopsies is challenging due to tissue heterogeneity. In this multi-institutional study we analyze fragmentomic profiles of plasma cell free DNA (cfDNA) to non-invasively distinguish between NF1 associated PN, AN and MPNST. Accurate classification would inform clinical care: standards of care for PN is observation, AN is narrow-margin resection, and MPNST is wide-margin resection. Methods: We performed whole genome sequencing of plasma cfDNA samples from healthy controls (n = 21), patients with PN (n = 113), AN (n = 39) and MPNST (n = 71). cfDNA fragment profiles were analyzed using two complementary approaches. First, we used unsupervised non-negative matrix factorization (NMF) to obtain global fragment length signatures to infer tumor fragment length distributions. The optimal cutpoint was determined after receiver operating characteristic analysis by Youden’s index in one-versus-one (OVO) disease state comparisons. Additionally, we implemented a bin-wise fragmentomic analysis based on DELFI, training a classifier on the ratios of short (100-150bp) and long (151-220bp) fragments in 5 megabase regions across the genome and arm-level features. Results: NMF accurately distinguished disease states on OVO comparisons: MPNST v AN (acc 0.71), MPNST v PN (acc 0.75), MPNST v healthy (acc 0.84), AN v PN (acc 0.70), AN v healthy (acc 0.80) and PN v healthy (acc 0.87). Accuracies were moderately improved in nearly all conditions with bin-wise fragmentomics: MPNST v AN (acc 0.62), MPNST v PN (acc 0.83), MPNST vs healthy (acc 0.86), AN v PN (acc 0.87), AN vs healthy (acc 0.72) and PN vs healthy (acc 0.88). Strikingly, the two AN with the DELFI scores most closely resembling MPNST were separately identified by independent clinical care teams to have very high-risk features and recent history warranting short-interval follow up. Conclusions: This study demonstrates that the spectrum of benign, pre-malignant and malignant peripheral nerve sheath tumors have distinct, disease state specific fragmentomic signatures. Fragmentomics alone outperformed our previously published copy number based cfDNA classifier in all conditions, most notably in low mutational burden healthy, PN and AN states. Finally, preliminary clinical vignettes suggest that this approach may be able to inform surveillance intervals by identifying higher risk premalignant lesions. Together, this work has the potential to enable earlier detection of clinically actionable AN and early-stage MPNST, thereby improving survival outcomes. Citation Format: Alex C. Pan, Russell Taylor Sundby, Jeffrey J. Szymanski, Paul A. Jones, Peter K. Harris, Aadel A. Chaudhuri, Angela C. Hirbe, Jack F. Shern. Cell-free DNA fragmentomics distinguish between benign, pre-malignant and malignant peripheral nerve sheath tumors in neurofibromatosis type 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 997.

Abstract IA014: Mass spectrometry and RNAseq based discovery of frameshift neoantigens and cryptic neoantigens from malignant peripheral nerve sheath tumors for prophylactic vaccines

Abstract Neurofibromatosis type 1 (NF1) syndrome is an autosomal, dominant tumor predisposition syndrome caused by loss-of-function mutations of NF1 gene encoding neurofibromin. Loss of the non-mutant allele of NF1 in a rare Schwann cell or precursor leads to benign plexiform neurofibromas. The main cause of death among NF1 patients is the malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft tissue sarcoma that most likely develops from plexiform neurofibroma, in particular the so-called “atypical” plexiform neurofibroma. Approximately half of MPNSTs are NF1-associated, and NF1 patients have ~15% lifetime risk of developing this terrible cancer. NF1 patients could greatly benefit from prophylactic vaccination that would prevent the malignant transformation to MPNSTs. We hypothesize that malignant transformation leads to the expression of recurrent alternately processed transcripts, such as transcriptionally-induced chimeras, that could express neoantigens and be used as targets for prophylactic vaccines. Such transcripts can be translated to produce novel peptides downstream of frameshift mutations caused by coding exon read-through into introns, mis-splicing from a coding exon to a non-canonical splice acceptors or splice acceptors in other genes. In most cases, a premature termination codon (PTC) will be rapidly encountered by the ribosome translating such transcripts. Therefore, we furthermore hypothesize that these alternately processed transcripts can express what we call “cryptic” neoantigens when treated with drugs that suppress utilization of premature codons such as Ataluren or gentamicin. In such a way, we could administer a prophylactic vaccine and induce conditionally active immune response that would eliminate nascent tumors only when drug treatment is used. We have developed a PTC suppression read-through reporter which results in presentation of the SIINFEKL peptide on C57BL6/J tumor cells. We developed a bioinformatic algorithm that takes deep RNA-seq data and identifies candidate neoepitopes encoded by non-normally processed or mutant mRNAs called Identifying and Verifying Immunogenic Neoepitopes and Neojunctions (IVINN). We have also achieved successful purification of human HLA Class I complex using an anti-pan HLA antibody column, and bound peptides have been isolated and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). We plan to utilize these approaches for mouse model and human MPNST patient derived xenografts. Citation Format: David A. Largaespada. Mass spectrometry and RNAseq based discovery of frameshift neoantigens and cryptic neoantigens from malignant peripheral nerve sheath tumors for prophylactic vaccines [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA014.

Abstract 1615: Developing a novel model of neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors using induced pluripotent stem cells

Abstract The genetic tumor predisposition syndrome Neurofibromatosis type 1 (NF1) results from the inheritance of a mutant copy of NF1, a RAS-GAP tumor suppressor gene. Subsequent loss of the remaining wild-type NF1 allele in Schwann lineage cells of the peripheral nervous system leads to complete functional loss of the encoded protein, neurofibromin. Schwann cells lacking neurofibromin exhibit hyperactive RAS signaling, and contribute to the formation of benign plexiform neurofibromas (PNFs). Through additional mutations in tumor suppressor genes CDKN2A/CDKN2B, these PNFs may escape senescence and progress to atypical neurofibromatous neoplasms of uncertain biological potential (ANNUBP). Furthermore, loss of function mutations in Polycomb Repressive Complex 2 (PRC2) have been strongly implicated in the progression of ANNUBPs to lethal malignant peripheral nerve sheath tumors (MPNSTs). Although these alterations have been associated with MPNST formation, their temporal dependence during Schwann cell development and contribution to malignant transformation using a human cell model has not been studied. We hypothesize that sequential loss of NF1, CDKN2A, and finally SUZ12 in Schwann cells transforms them into MPNST, and this process is possible to study using a human induced pluripotent stem cell (iPSC) model system. To test our hypothesis, we generated Schwann lineage cells from commercially available iPSCs. Cells were subjected to targeted mutations using CRISPR/Cas9 ribonucleoproteins at different stages of development to mimic potential tumorigenesis. Our preliminary results show that multiple iPSC donor lines can reliably be differentiated to Neural Crest Cells (NCCs), and further to mature Schwann cells. We have edited multiple target loci at high levels (&amp;gt;70%) throughout Schwann cell development. NCCs lacking neurofibromin showed an increase in proliferation and migration. While iPSC-derived Schwann cells (iSCs) lacking NF1 and CDKN2A with or without SUZ12 loss became senescent after several passages. These mutant iSCs were unable to form tumors when subcutaneously injected into the flank of NRG mice, while commonly utilized immortalized human Schwann cell lines lacking NF1 were able to form large subcutaneous tumors. Further refinement of this induced-MPNST (iMPNST) model through targeting of CDKN2B, ATRX, or TP53 may allow for better understanding of the order and timing at which these transforming mutations are sufficient to result in MPNST formation. Additionally, the application of single-cell RNA sequencing technology to this system could uncover potential heterogeneity that could harbor a clone primed for malignant transformation. This model could aid in uncovering novel genetic mutations and pathways which contribute to transformation of benign PNFs to MPNSTs, providing additional targets for future therapeutic intervention. Citation Format: Garrett M. Draper, Daniel Panken, Mahathi Patchava, Wendy Hudson, Kyle B. Williams, David A. Largaespada. Developing a novel model of neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors using induced pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1615.

RETRACTED: A case report of a 4-year-old girl with neurofibromatosis type 1

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors. A diagnostic discordance in the original pathology reports has been noticed and requires further evaluation, therefore the paper will be retracted.

Neurofibromin Deficiency Causes Epidermal Growth Factor Receptor Upregulation through the Activation of Ras/ERK/SP1 Signaling Pathway in Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheet Tumor.

Neurofibromatosis type 1 (NF1) is an autosomal dominant human genetic disorder. The progression of benign plexiform neurofibromas to malignant peripheral nerve sheet tumors (MPNSTs) is a major cause of mortality in patients with NF1. Although elevated epidermal growth factor receptor (EGFR) expression plays a crucial role in the pathogenesis of MPNST, the cause of EGFR overexpression remains unclear. Here, we assessed EGFR expression levels in MPNST tissues of NF1 patients and NF1 patient-derived MPNST cells. We found that the expression of EGFR was upregulated in MPNST tissues and MPNST cells, while the expression of neurofibromin was significantly decreased. Manipulation of NF1 expression by NF1 siRNA treatment or NF1-GAP-related domain overexpression demonstrated that EGFR expression levels were closely and inversely correlated with neurofibromin levels. Notably, knockdown of the NF1 gene by siRNA treatment augmented the nuclear localization of phosphorylated SP1 (pSP1) and enhanced pSP1 binding to the EGFR gene promoter region. Our results suggest that neurofibromin deficiency in NF1-associated MPNSTs enhances the Ras/ERK/SP1 signaling pathway, which in turn may lead to the upregulation of EGFR expression. This study provides insight into the progression of benign tumors and novel therapeutic approaches for treatment of NF1-associated MPNSTs.

Survival and NF1 Analysis in a Cohort of Orthopedics Patients with Malignant Peripheral Nerve Sheath Tumors.

Neurofibromatosis type 1 (NF1) is an autosomal dominant tumor syndrome in which benign plexiform neurofibromas are at risk of transforming into malignant peripheral nerve sheath tumors (MPNSTs), a very rare soft-tissue sarcoma. The prognosis of patients with MPNSTs is poor, with most studies reporting <50% survival at five years. However, studies evaluating MPNSTs are limited and report heterogeneous results. Because no MPNST-specific evidence-based treatment guideline exists, individual institutional experiences are very informative to the field. The main objective of this study was to investigate and report MPNST prognostic clinical and genetic biomarkers from our institution's Orthopedics service experience treating 20 cases from 1992 to 2017. Most patients were treated with resection and adjuvant radiation. Extended follow-up, averaging 11.4 years (ranging 1.1 to 25.1), revealed excellent five-year survival rates: 70% for overall and 60% for metastatic disease. An S100 B immunonegative tumor phenotype was associated with a significantly worse outcome than MPNSTs with positive S100 B stain. In addition, NF1 gene mutation analysis was performed on 27 families with NF1 in which at least one affected family member developed MPNSTs. Of the 27 NF1 germline mutations, five were large deletions spanning (or nearly spanning) the gene (18.5%), substantially more than such deletions in NF1 in general, consistent with increased risk of MPNSTs in such cases.

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.

Chromosome 8 gain is associated with high-grade transformation in MPNST.

One of the most common malignancies affecting adults with Neurofibromatosis type 1 (NF1) is the malignant peripheral nerve sheath tumor (MPNST), an aggressive and often fatal sarcoma that commonly arises from benign plexiform neurofibromas. Despite advances in our understanding of MPNST pathobiology, there are few effective therapeutic options, and no investigational agents have proven successful in clinical trials. To further understand the genomic heterogeneity of MPNST, and to generate a preclinical platform that encompasses this heterogeneity, we developed a collection of NF1-MPNST patient-derived xenografts (PDX). These PDX were compared with the primary tumors from which they were derived using copy number analysis, whole exome sequencing, and RNA sequencing. We identified chromosome 8 gain as a recurrent genomic event in MPNST and validated its occurrence by FISH in the PDX and parental tumors, in a validation cohort, and by single-cell sequencing in the PDX. Finally, we show that chromosome 8 gain is associated with inferior overall survival in soft-tissue sarcomas. These data suggest that chromosome 8 gain is a critical event in MPNST pathogenesis and may account for the aggressive nature and poor outcomes in this sarcoma subtype.

Abstract 1889: Allosteric inhibition of SHP2 prevents adaptive resistance to MEK inhibition in neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors

Abstract Introduction: Neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs) are malignant sarcomas occurring in approximately 15% of NF1 patients and always develop within benign precursor lesions (plexiform neurofibromas). The mortality caused by these tumors is high, approaching 100% in patients with unresectable, metastatic or recurrent disease. Targeted inhibition of the MAP kinase (MAPK) pathway has been attempted in clinical trials using MEK inhibitors (MEKi). While emerging data demonstrate effective tumor growth inhibition in benign plexiform neurofibromas, MEK inhibition blocks MPNST progression only to a limited extent. Combination strategies targeting both the MAPK and PI3K/AKT pathways (horizontal inhibition) have had limited clinical success due to excessive toxicities which precludes delivery of optimal therapeutic concentrations. Thus, novel approaches and complementary treatment strategies that are tolerable and prevent adaptive resistance mechanisms in NF1 MPNST are urgently needed. Methods: We have succeeded in establishing orthotopic in vivo models (patient-derived human xenografts-PDXs and genetically engineered mouse models- GEMMs) via implantation of tumor cells into the sciatic nerve, an approach that is critical, because it preserves the tumor microenvironment. Furthermore, we have optimized MPNST culture conditions, allowing us to establish robust primary cultures of MPNSTs while retaining key biological features of the original tumors. Summary of findings: Using this preclinical testing platform, we demonstrate that - 1. A feedback loop mediated by induction of receptor tyrosine kinase (RTK) signaling becomes hyperactivated upon MEK inhibition (MEKi). This subsequently activates the MAPK pathway to such an extent that MEKi alone is unable to completely block signaling to ERK, thereby maintaining tumor proliferation. 2. SHP2 is a protein tyrosine phosphatase (PTP) that functions as a positive signal transducer, acting between RTKs and RAS and thus, functions as physiological mediator for RAS activation. A treatment paradigm where MEKi is combined with SHP099, an allosteric SHP2 inhibitor to counteract the rebound increase in RTK mediated signaling to RAS-MEK-ERK results in effective attenuation of MEK-ERK signaling. In vivo studies demonstrate that the drug combination exhibits synergistic effects on tumor growth inhibition and significantly prolongs survival. 3. SHP099 disrupts a functional complex (SOS1/GRB2) which is essential to RAS-GTP loading and inhibits RAS-mediated downstream MAP kinase signaling in NF1 MPNST. Conclusions - Our results provide a mechanistic context for SHP2's precise role in the regulation of RAS-GTP and demonstrate that SHP2i can prevent adaptive resistance to MEKi. Given that allosteric SHP2 inhibitors are currently in clinical trials (NCT03114319), our studies can be rapidly translated into a clinical trial to evaluate a combination of SHP2i and MEKi as a novel treatment approach in NF1-MPNSTs. Citation Format: Sameer Farouk Sait, Kwan Tang, Rebecca Brown, Daochun Sun, Xunhua Xie, Benjamin Neel, Luis Parada. Allosteric inhibition of SHP2 prevents adaptive resistance to MEK inhibition in neurofibromatosis type 1 associated 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 1889.