Neurofibromatosis Type 1 Inactivation Research Articles

NFS-22. INVESTIGATING MALIGNANT TRANSFORMATION IN NF1 PEDIATRIC GLIOMAS USING AN IPSCS-DERIVED CEREBRAL ORGANOID MODEL

Abstract BACKGROUND Neurofibromatosis Type 1 (NF1) is an autosomal dominant tumor predisposition syndrome affecting 1 in 3000 children around the world. Although NF1 inactivation is recognized as the cause of low-grade glioma (LGG) development, little is known about subsequent genetic alterations that lead to malignant transformation. One of the limitations in understanding the molecular events underlying the malignant transformation of NF1 gliomas is the lack of appropriate NF1 preclinical models for its comprehensive profiling. OBJECTIVE Brain organoids have emerged as powerful in vitro models for studying the interaction between tumor cells and the tumor microenvironment. In this study, we present a novel cerebral organoid model, developed to investigate the molecular events leading to malignant transformation in NF1 pediatric gliomas. METHODS Induced pluripotent stem cells (iPSCs)-derived cerebral organoids were generated using NF1wt and NF1mut iPSCs, the latter reprogrammed from patients with plexiform neurofibromas (Carlos III Health Institute, Spain). Immunofluorescence analysis was performed at four different developmental stages to confirm their correct cytoarchitectural development and differentiation into heterogeneous cell populations. Furthermore, four mCherry-labelled isogenic glioma cells lines, RES 186 wild type, NF1-/-, CDKN2a-/-,NF1-/-/CDKN2a-/- were co-cultured with the developed cerebral organoids and their migration potential and invasiveness were compared using two-photon microscopy. RESULTS/DISCUSSION NF1mut cerebral organoids display ventricular-like zone at early stages of development (d15-d30), characterized by luminal Sox2+ neural stem cells surrounded by Tuj1+ immature neuron. These organized structures disappear at later stages, while progenitor cells start to differentiate into fully mature neurons and glial cells (d45-d60). Additionally, after 10 days of co-culture, all four RES186 isogenic cell lines infiltrated the organoid structure with different invasion patterns. The established organoid model will provide a platform for evaluating the impact of sequential mutational events on LGG transformation, taking into consideration the role exerted by the tumor microenvironment in supporting tumor cell invasion and migration.

P04.01.B MEK INHIBITORS IN PEDIATRIC RECURRENT SYMPTOMATIC UNRESECTABLE LOW-GRADE GLIOMAS HARBORING MAPK ALTERATION: A SINGLE CENTER EXPERIENCE

Abstract BACKGROUND Treatment of inoperable, recurrent, progressive pediatric low-grade gliomas (pLGGs) remains controversial. Recent studies focused on aberrant activation of the mitogen-activated protein kinase (MAPK) pathway, due to gain of function mutations in RAS or BRAF genes or loss or inactivation of NF1. Selumetinib and trametinib, orally bioavailable and selective MEK1/MEK2 inhibitors (MEKi), act on MAPK pathway. In April 2020 the US Food and Drug Administration and in June 2021 the European Medicines Agency approved selumetinib for inoperable plexiform neurofibromas in Neurofibromatosis type 1 (NF1). We evaluated MEKi efficacy and safety in pLLGs harboring MAPK alteration. MATERIAL AND METHODS From July 2021 to February 2023 eight patients (5 females, 3 males, median age 8 years, range 4-18) were admitted to our hospital for symptomatic, recurrent, inoperable pLGGs (location: 3 hypothalamic-optic chiasm region, 2 posterior fossa, 1 temporo-mesial, 1 quadrigeminal plate, 1 diffuse leptomeningeal; MAPK alteration: 3 NF1 inactivation, 4 KIAA1549/BRAF gene fusion, 1 RNF130/BRAF fusion). MEKi were administered (6 selumetinib, 2 trametinib) after disease progression for failure of previous treatments (6 partial resection, 7 multiple chemotherapies, 2 radiotherapy). MRI assessment was performed every 4 months and radiological response was defined in accordance with RAPNO criteria. RESULTS At first evaluation, 3/8 patients (37,5%) showed a partial radiological response (-64,96% mean volume reduction, range 52,61%-77,19%) and 4/8 (50%) stable disease (0-41.67% volume decrease). Disease progression was observed in a patient heavily pretreated for acute leukemia. One patient with leptomeningeal spreading showed progressive disease at second evaluation. Median time to progression has not yet been reached. Six of 8 patients experienced at least one toxicity. The most frequent toxicities reported were mucocutaneus disorders (rash, mucositis, eczema). Three patients presented creatine phosphokinase (CPK) increase (one grade 2 with rapid normalization after therapy discontinuation). All patients on trametinib had to reduce treatment dose (for skin toxicity or increasing blood pressure). CONCLUSION MEKi, selumetinib and trametinib, represent promising strategies in children with recurrent/progressive LGGs harboring MAPK alterations. In our small series, the treatment was effective and well tolerable in patients with localized disease and without other risk factors. The most common toxicities were CPK elevation and rash. Further studies are needed to identify patients who may benefit most from MEKi treatment and to establish whether such treatments are superior to chemotherapy in front line setting for MAPK altered pLGGs.

Somatic Double Inactivation of NF1 Associated with NF1-Related Pectus Excavatum Deformity

Neurofibromatosis type 1 (NF1) is a neurocutaneous genetic disorder with a broad spectrum of associated signs and symptoms, including skeletal anomalies. The association of NF1 with anterior chest wall deformities has been recently reported, especially the pectus excavatum (PE). Over the years, several authors have suggested loss of heterozygosity (LOH) as the possible pathogenic mechanism underlying the development of the typical NF1 skeletal features. Here, we report a NF1 patient with severe chest deformity and harboring the germline heterozygous pathogenic NF1 variant NM_001042492.3: c.4271delC p.(Ala1424Glufs ∗ 4). Through next-generation sequencing (NGS), we investigated the affected cartilage from the PE deformity and identified the additional frameshift variant NM_001042492.3: c.2953delC p.(Gln985Lysfs ∗ 7), occurring as a somatic NF1 second hit mutation. Exome sequencing confirmed the absence of additional variants of potential pathogenic relevance. Western blot analysis showed the absence of wild-type NF1 protein in the cartilage of the patient, consistent with a somatic double inactivation (SDI) of NF1. Taken together, our findings support the role of SDI in NF1-related PE, widening the spectrum of the pathophysiological mechanisms involved in NF1-related skeletal features.

Genomic Patterns of Malignant Peripheral Nerve Sheath Tumor (MPNST) Evolution Correlate with Clinical Outcome and Are Detectable in Cell-Free DNA.

MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups. This article is highlighted in the In This Issue feature, p. 517.

PATH-07. MULTIPLATFORM MOLECULAR ANALYSES REFINE CLASSIFICATION AND PROGNOSTICATION OF GLIOMAS ARISING IN PATIENTS WITH NEUROFIBROMATOSIS TYPE 1

Abstract BACKGROUND Gliomas arising in patients with neurofibromatosis type 1 (NF1) are heterogeneous, occurring from childhood through adulthood, can be histologically low-grade (LG) or high-grade (HG), and follow an indolent or aggressive clinical course. Comprehensive profiling of genetic alterations beyond NF1 and epigenetic classification of these tumors remain limited. METHODS Next-generation sequencing and DNA methylation profiling was performed on gliomas from 47 NF1 patients and correlated with clinicopathologic features, treatment, and outcomes. RESULTS 30 tumors demonstrated biallelic inactivation of NF1 without additional oncogenic alterations (“molecular LG subgroup”, median age 14 yrs). The remaining 17 tumors harbored additional oncogenic alterations beyond NF1 (“molecular HG subgroup”, median age 28), most frequently CDKN2A homozygous deletion (n=13), ATRX mutation (n=8), PIK3CA or PIK3R1 mutation (n=4), and TP53 mutation (n=3). Survival analysis showed significant differences in time to progression (137 vs 11 mos, p< 0.0001) and median survival time (undefined vs 37 mos, p > 0.0001) for molecular LG versus HG subgroups. DNA methylation profiles of the molecular LG subgroup resolved into a new epigenetic cluster closest to but divergent from the three existing reference classes of sporadic pilocytic astrocytoma. DNA methylation profiles of the molecular HG subgroup demonstrated most tumors epigenetically align with either HGAP or various subclasses of IDH-wildtype GBM. CONCLUSION NF1-associated gliomas stratify into two molecular subgroups. The “molecular LG subgroup” occurs primarily during childhood, harbors biallelic NF1 inactivation only, follows a more indolent clinical course, and has a unique epigenetic signature for which we propose the terminology “pilocytic astrocytoma, arising in the setting of NF1”. The “molecular HG subgroup” occurs primarily during adulthood, harbors additional oncogenic alterations including CDKN2A homozygous deletion and ATRX mutation, follows a more aggressive clinical course, and is epigenetically diverse. These findings highlight recurrently altered pathways in NF1-associated gliomas and help inform targeted therapeutic strategies for this patient population.

Multiplatform molecular analyses refine classification of gliomas arising in patients with neurofibromatosis type 1

Gliomas arising in the setting of neurofibromatosis type 1 (NF1) are heterogeneous, occurring from childhood through adulthood, can be histologically low-grade or high-grade, and follow an indolent or aggressive clinical course. Comprehensive profiling of genetic alterations beyond NF1 inactivation and epigenetic classification of these tumors remain limited. Through next-generation sequencing, copy number analysis, and DNA methylation profiling of gliomas from 47 NF1 patients, we identified 2 molecular subgroups of NF1-associated gliomas. The first harbored biallelic NF1 inactivation only, occurred primarily during childhood, followed a more indolent clinical course, and had a unique epigenetic signature for which we propose the terminology “pilocytic astrocytoma, arising in the setting of NF1”. The second subgroup harbored additional oncogenic alterations including CDKN2A homozygous deletion and ATRX mutation, occurred primarily during adulthood, followed a more aggressive clinical course, and was epigenetically diverse, with most tumors aligning with either high-grade astrocytoma with piloid features or various subclasses of IDH-wildtype glioblastoma. Several patients were treated with small molecule MEK inhibitors that resulted in stable disease or tumor regression when used as a single agent, but only in the context of those tumors with NF1 inactivation lacking additional oncogenic alterations. Together, these findings highlight recurrently altered pathways in NF1-associated gliomas and help inform targeted therapeutic strategies for this patient population.

Case series of congenital pseudarthrosis of the tibia unfulfilling neurofibromatosis type 1 diagnosis: 21% with somatic NF1 haploinsufficiency in the periosteum.

Up to 84% of patients with congenital pseudarthrosis of the tibia (CPT) present with neurofibromatosis type 1 (NF1) (NF1-CPT). However, the etiology of CPT not fulfilling the NIH diagnostic criteria for NF1 (non-NF1-CPT) is not well understood. Here, we collected the periosteum tissue from the pseudarthrosis (PA) site of 43 non-NF1-CPT patients and six patients with NF1-CPT, together with the blood or oral specimen of trios (probands and unaffected parents). Whole-exome plus copy number variation sequencing, multiplex ligation-dependent probe amplification (MLPA), ultra-high amplicon sequencing, and Sanger sequencing were employed to identify pathogenic variants. The result showed that nine tissues of 43 non-NF1-CPT patients (21%) had somatic mono-allelic NF1 inactivation, and five of six NF1-CPT patients (83.3%) had bi-allelic NF1 inactivation in tissues. However, previous literature involving genetic testing did not reveal somatic mosaicism in non-NF1-CPT patients so far. In NF1-CPT patients, when the results from earlier reports and the present study were combined, 66.7% of them showed somatic NF1 inactivation in PA tissues other than germline inactivation. Furthermore, no diagnostic variants from other known genes (GNAS, AKT1, PDGFRB, and NOTCH3) related to skeletal dysplasia were identified in the nine NF1 positive non-NF1-CPT patients and six NF1-CPT patients. In conclusion, we detected evident somatic mono-allelic NF1 inactivation in the non-NF1-CPT. Thus, for pediatric patients without NF1 diagnosis, somatic mutations in NF1 are important.

Identification of a germline CSPG4 variation in a family with neurofibromatosis type 1-like phenotype

Neurofibromatosis type 1 (NF1), an autosomal dominant and multisystem disorder, is generally considered to be caused by NF1 inactivation. However, there are also numerous studies showing that Neurofibromatosis type 1-like phenotype can be caused by the abnormalities in the other genes. Through targeted parallel sequencing, whole-exome sequencing, de novo genomic sequencing, and RNA isoform sequencing, we identified a germline V2097M variation in CSPG4 gene probably increased susceptibility to a NF1-like phenotype family. Besides, a series of in vitro functional studies revealed that this variant promoted cell proliferation by activating the MAPK/ERK signaling pathway via hindering ectodomain cleavage of CSPG4. Our data demonstrate that a germline variation in the CSPG4 gene might be a high risk to cause NF1-like phenotype. To our knowledge, this is the first report of mutations in the CSPG4 gene in human diseases.

LGG-06. COMPREHENSIVE GENOMIC CHARACTERIZATION AND INTEGRATED CLINICAL ANALYSIS OF LOW-GRADE GLIOMAS IN CHILDREN WITH NEUROFIBROMATOSIS TYPE 1

BackgroundLow-grade gliomas (LGGs) arising in children with neurofibromatosis type 1 (NF1) are usually not biopsied. To identify secondary genetic alterations or molecular features that may contribute to pathogenesis and correlate with clinical behavior, we initiated a comprehensive molecular and clinical analysis of pediatric NF1-LGGs. MethodsNF1-LGGs were analysed by whole-genome sequencing (31), targeted gene panel sequencing (9), RNAseq transcriptomal profiling (33) and genome-wide DNA methylation analysis (67). Clinical annotation was available for 48 subjects. ResultsMost LGGs harbored bi-allelic NF1 inactivation as the sole genetic abnormality, but 11% had additional alterations (FGFR1 mutation, n=3; PIK3CA mutation, n=2; homozygous 9p21 deletion, n=2; MYB:QKI fusion, n=1; SETD2 mutation, n=1; EGFR amplification, n=1). FGFR1 mutation conferred additional growth advantage in multiple complementary murine Nf1 models. 88% of NF1-LGGs resembled sporadic pilocytic astrocytoma (PA) by methylation, higher than that based on histology. Non-PA methylation patterns included low-grade glial/glioneuronal tumors, rosette-forming glioneuronal tumors, MYB/MYBL1-altered glioma, and high-grade astrocytoma with piloid features (2 tumors histologically diagnosed as LGG). In total, 18% of samples were classified as non-PA and/or harbored an additional non-NF1 mutation. Non-PA methylation class tumors were more likely to harbor an additional non-NF1 mutation (p=0.005). 7.7% of optic pathway hypothalamic gliomas (OPHGs) had other mutations or were not classified by methylation as PA, compared with 20.6% of NF1-LGGs arising elsewhere. There was no difference based on age for the presence of an additional non-NF1 mutation or non-PA methylation class. ConclusionsGiven the overall low occurrence of non-NF1 mutations or non-PA methylation class tumors in this series, routine clinical biopsy of typically-appearing NF1-LGG may not be indicated, particularly for children with OPHG. Biopsy should be considered for non-OPHG tumors refractory to conventional treatment. As additional agents are developed and treatment strategies evolve, the rationale for biopsy of NF1-LGG may become stronger.

Integrated molecular and clinical analysis of low-grade gliomas in children with neurofibromatosis type 1 (NF1).

Low-grade gliomas (LGGs) are the most common childhood brain tumor in the general population and in individuals with the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Surgical biopsy is rarely performed prior to treatment in the setting of NF1, resulting in a paucity of tumor genomic information. To define the molecular landscape of NF1-associated LGGs (NF1-LGG), we integrated clinical data, histological diagnoses, and multi-level genetic/genomic analyses on 70 individuals from 25 centers worldwide. Whereas, most tumors harbored bi-allelic NF1 inactivation as the only genetic abnormality, 11% had additional mutations. Moreover, tumors classified as non-pilocytic astrocytoma based on DNA methylation analysis were significantly more likely to harbor these additional mutations. The most common secondary alteration was FGFR1 mutation, which conferred an additional growth advantage in multiple complementary experimental murine Nf1 models. Taken together, this comprehensive characterization has important implications for the management of children with NF1-LGG, distinct from their sporadic counterparts.

Neurofibromatosis type 1‐related pseudarthrosis: Beyond the pseudarthrosis site

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting approximately 1 in 2,000 newborns. Up to 5% of NF1 patients suffer from pseudarthrosis of a long bone (NF1-PA). Current treatments are often unsatisfactory, potentially leading to amputation. To gain more insight into the pathogenesis we cultured cells from PA tissue and normal-appearing periosteum of the affected bone for NF1 mutation analysis. PA cells were available from 13 individuals with NF1. Biallelic NF1 inactivation was identified in all investigated PA cells obtained during the first surgery. Three of five cases sampled during a later intervention showed biallelic NF1 inactivation. Also, in three individuals, we examined periosteum-derived cells from normal-appearing periosteum proximal and distal to the PA. We identified the same biallelic NF1 inactivation in the periosteal cells outside the PA region. These results indicate that NF1 inactivation is required but not sufficient for the development of NF1-PA. We observed that late-onset NF1-PA occurs and is not always preceded by congenital bowing. Furthermore, the failure to identify biallelic inactivation in two of five later interventions and one reintervention with a known somatic mutation indicates that NF1-PA can persist after the removal of most NF1 negative cells.

Reprogramming Captures the Genetic and Tumorigenic Properties of Neurofibromatosis Type 1 Plexiform Neurofibromas.

SummaryNeurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(−/−) cells, descendants from the tumor originating cell. These NF1(−/−)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(−/−) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available.

RAS-MPAK Pathway Signaling in Health and Disease

The p21ras (Ras) family of signal switch molecules is an essential component of proliferative responses to many extracellular stimuli including most hematopoietic growth factors and cytokines. Phosphorylation of tyrosine residues on activated cytokine/growth factor receptors triggers multiple signaling pathways including JAK/STAT, Ras and PI3K. Activated Ras proteins further activate downstream effectors such as RAF/MEK/ERK, PI3K and RalGDS. Point mutations of Ras proteins are commonly found in human cancers and are estimated to occur in about 30% of cancer cases. These mutations lock Ras in the constitutively active conformation. In addition to point mutations of RAS, other genetic lesions such as the BCR-ABL fusion, PTPN11 mutations, FLT3 internal tandem duplications, CBL mutations and NF1 inactivation all lead to hyperactive Ras signaling. Besides cancers, RAS and Ras-activating mutations have been found in RASopathies, such as Neurofibromatosis type 1 (NF1), Noonan Syndrome, Costello syndrome, and Cardiofaciocutaneous syndrome (CFC).RAS mutations are highly prevalent in hematologic malignancies including chronic and juvenile myelomonocytic leukemia (CMML and JMML), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) and multiple myeloma (MM). In contrast to epithelial cancers where KRAS mutations are highly prevalent, most mutations found in hematologic malignancies alter NRAS. Human genetic data and results from genetic mouse models support the idea that deregulated Ras signaling is initiating event in myeloproliferative neoplasms (MPNs) and MPN/MDS overlapping diseases. RAS mutations are detected among the initiating mutations in many cases of CMML and JMML, and can persist in patients who have achieved remission either spontaneously or after treatment. Furthermore, mutations of Nras and Kras, as well as leukemia-associated mutations that result in hyperactive Ras such as BCR-ABL, loss of NF1, PTPN11 or CBL mutations all induce MPN or MPN/MDS overlapping disease in mouse models. Although in adult de novo AMLs, human genetic data support a model in which RAS and Ras-activation mutations are likely secondary event, recent large scale next generation sequencing studies show that RAS and Ras-activating mutations are much more commonly found in pediatric than adult AML cases, and pediatric AMLs in general lack “adult pre-leukemic mutation' such as TET2, DNMAT3 or IDH1/2. These results suggest a possibility that RAS mutations can be initiating mutations in pediatric AMLs. In addition, even in AMLs where RAS mutations are secondary event, hyperactive Ras promotes leukemic stem cell self-renewal and is critical for leukemia maintenance, underscoring the importance of targeting Ras for leukemia treatment.Targeting Ras proteins remains to be challenging and significant ongoing effort focuses on developing direct inhibitors of Ras. In recent years, small molecules that bind to Ras proteins to prevent GEF-mediated GDP/GTP exchange, or target the RasG12C mutant allele, showed promising pre-clinical results. Besides, targeting Ras effectors, such as MEK/ERK, individually or in combination with other pathways, remains as a valid approach to halt Ras-induced malignant proliferation. Better understanding of the subcellular localization, feedback regulation and redundancy of Ras-activated signaling network under physiological and pathological contexts will shed light on developing new strategies to effectively attack Ras-driven cancers. DisclosuresNo relevant conflicts of interest to declare.

Developmental dosing with a MEK inhibitor (PD0325901) rescues myopathic features of the muscle-specific but not limb-specific Nf1 knockout mouse

Neurofibromatosis Type 1 (NF1) is a common autosomal dominant genetic disorder While NF1 is primarily associated with predisposition for tumor formation, muscle weakness has emerged as having a significant impact on quality of life. NF1 inactivation is linked with a canonical upregulation Ras-MEK-ERK signaling. This in this study we tested the capacity of the small molecule MEK inhibitor PD0325901 to influence the intramyocellular lipid accumulation associated with NF1 deficiency. Established murine models of tissue specific Nf1 deletion in skeletal muscle (Nf1MyoD−/−) and limb mesenchyme (Nf1Prx1−/−) were tested.Developmental PD0325901 dosing of dams pregnant with Nf1MyoD−/− progeny rescued the phenotype of day 3 pups including body weight and lipid accumulation by Oil Red O staining. In contrast, PD0325901 treatment of 4 week old Nf1Prx1−/− mice for 8 weeks had no impact on body weight, muscle wet weight, activity, or intramyocellular lipid. Examination of day 3 Nf1Prx1−/− pups showed differences between the two tissue-specific knockout strains, with lipid staining greatest in Nf1MyoD−/− mice, and fibrosis higher in Nf1Prx1−/− mice.These data show that a MEK/ERK dependent mechanism underlies NF1 muscle metabolism during development. However, crosstalk from Nf1-deficient non-muscle mesenchymal cells may impact upon muscle metabolism and fibrosis in neonatal and mature myofibers.

Abstract 1456: Identifying early genetic steps in malignant transformation of neurofibromatosis type 1- associated plexiform neurofibromas

Abstract BACKGROUND: Neurofibromatosis type 1 (NF1) is a genetic tumor predisposition disorder caused by germline mutations in tumor suppressor NF1. Plexiform neurofibromas (PN) are benign tumors that arise prenatally or early in childhood and affect 30-50% of NF1 population. Somatic inactivation of second copy of NF1 is believed to be primary genetic event leading to PN initiation. NF1 patients have 8-12% lifetime risk of developing malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft tissue sarcoma, often arising from pre-existing PN and atypical NF (ANF). ANF are pre-malignant tumors that often arise within PN and can transform into MPNST. They are distinct from both PN and MPNST clinically and histologically, thus representing an intermediate step in malignant transformation. Several studies identified deletion of the CDKN2A/2B locus as the most frequent genetic event in ANF, however it is not clear whether other genes or pathways play role in PN transformation into ANF and further into MPNST. In this study, we performed genomic analysis of 16 ANF and 4 MPNST matched with normal DNA obtained from 14 and 4 patients, respectively. METHODS: We performed whole exome sequencing and whole transcriptome RNASeq analyses on Illumina Hi-Seq 2500 platform and copy-number variant (CNV) analysis on Illumina HumanOmniExpressExome-8 SNP-arrays. In addition, we performed deep sequencing of NF1 and validation of select mutations on IonTorrent platform. For select tumors we estimated growth rate and metabolic activity by using volumetric MRI and FDG-PET. RESULTS: We identified inactivation of NF1 in the majority of ANF and all MPNST. We also detected CDKN2A/B locus deletion in the majority of ANF and MPNST (heterozygous in ANF and mostly homozygous in MPNST). We determined that PRC2 genes (EED and SUZ12) were mutated in multiple MPNST but never in ANF. We identified a low number of point mutations and small indels in the genomes of ANF (median 1, range 0-4) and somewhat elevated mutation burden in MPNST (median 23, range 18-31), however none of these mutations were recurrent and none of the mutant genes (other than NF1 and CDKN2A) were present in multiple samples. We found 93 CNV per tumor (median) in ANF that constituted ~2% of their genomes. In comparison, we observed 2,249 CNV (median) in MPNST that comprised ~75% of their genomes. We didn’t detect significant correlation between growth rate or metabolic activity and the degree of genomic instability or mutation burden in the tumors, however the size of the sample set was modest. RNAseq data analysis is pending. CONCLUSIONS: It appears that PN-ANF transition is predominantly if not exclusively driven by heterozygous deletion of the CDKN2A/2B locus. Further progression to MPNST likely involves homozygous loss of CDKN2A/B and complete inactivation of the PRC2 complex. Widespread LOH in MPNST may accelerate inactivation of key gatekeepers. Citation Format: Alexander Pemov, Nancy F. Hansen, Rajesh Patidar, Christine Higham, Eva Dombi, Joseph F. Boland, Settara C. Chandrasekharappa, NIH Intramural Sequencing Center, James C. Mullikin, Margaret Wallace, Javed Khan, Eric Legius, Brigitte Widemann, Douglas R. Stewart. Identifying early genetic steps in malignant transformation of neurofibromatosis type 1- associated plexiform neurofibromas [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 1456. doi:10.1158/1538-7445.AM2017-1456

Confirmation of mutation landscape of NF1-associated malignant peripheral nerve sheath tumors.

The commonest tumors associated with neurofibromatosis type 1 (NF1) are benign peripheral nerve sheath tumors, called neurofibromas. Malignant transformation of neurofibromas into aggressive MPNSTs may occur with a poor patient prognosis. A cooperative role of SUZ12 or EED inactivation, along with NF1, TP53, and CDKN2A loss-of-function, has been proposed to drive progression to MPNSTs. An exome sequencing analysis of eight MPNSTs, one plexiform neurofibroma, and seven cutaneous neurofibromas was undertaken. Biallelic inactivation of the NF1 gene was observed in the plexiform neurofibroma and the MPNSTs, underlining that somatic biallelic NF1 inactivation is likely to be the initiating event for plexiform neurofibroma genesis, although it is unlikely to be sufficient for the subsequent MPNST development. The majority (5/8) of MPNSTs in our analyses demonstrated homozygous or heterozygous deletions of CDKN2A, which may represent an early event following NF1 LOH in the malignant transformation of Schwann cells from plexiform neurofibroma to MPNST. Biallelic somatic alterations of SUZ12 was also found in 4/8 MPNSTs. EED biallelic alterations were detected in 2 of the other four MPNSTs, with one tumor having a homozygous EED deletion. A missense mutation in the chromatin regulator KDM2B was also identified in one MPNST. No TP53 point mutations were found in this study, confirming previous data that TP53 mutations may be relatively rare in NF1-associated MPNSTs. Our study confirms the frequent biallelic inactivation of PRC2 subunits SUZ12 and EED in MPNSTs, and suggests the implication of KDM2B.

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.

Evaluation of somatic mutations in tibial pseudarthrosis samples in neurofibromatosis type 1

BackgroundTibial pseudarthrosis is associated with neurofibromatosis type 1 (NF1) and there is wide clinical variability of the tibial dysplasia in NF1, suggesting the possibility of genetic modifiers. Double inactivation of...

Neurofibromin deficiency-associated transcriptional dysregulation suggests a novel therapy for tibial pseudoarthrosis in NF1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole-exome and whole-transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic biallelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1-associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor-suppressor genes, such as p53. However, gene expression profiling indicates that pseudoarthrosis tissue has a tumor-promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant overexpression of specific cancer-associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin-deficient pseudoarthrosis and promote proper bone remodeling and fracture healing.

Microscopic intraneural perineurial cell proliferations in patients with neurofibromatosis type 1

Benign peripheral nerve sheath tumors (PNSTs) showing more than one line of differentiation (hybrid PNSTs) have been increasingly recognized, mainly due to awareness of their existence and as a consequence of increased use of immunohistochemisty during the last decade. Two recent studies suggested overrepresentation of hybrid tumors among benign PNSTs in patients with neurofibromatosis type 1 (NF-1). This study was performed to assess the presence of perineurial cells in microscopic (early) neurofibromatous lesions and normal-looking peripheral nerves in specimens from 5 patients with NF-1 using markers of perineurial cell differentiation (epithelial membrane antigen, claudin-1, and glucose transporter 1). In 2 patients, multiple normal looking nerve fibers as well as hypertrophied nerves and microscopic tumor nodules showed variable intraneural perineurial cell proliferations that frequently occupied the whole nerve fascicle resulting in multiple microscopic reticular perineurioma-like nodules (microscopic hybrid neurofibromatosis/perineuriomatosis). None of the cases showed the onion skin pattern of intraneural perineurioma. However, other nerve fibers within the same specimens showed normal compact rim of perineurium without any detectable intraneural perineurial cells. Both patients had concurrent multiple larger PNSTs (plexiform neurofibromas, hybrid neurofibroma/perineurioma and lesions with features intermediate between the 2 types). One specimen harboring high-grade malignant PNST and 2 specimens with large solitary neurofibromas displayed no intraneural perineurial cells. These observations suggest that intraneural perineurial proliferations are part of the early lesions in the setting of constitutional NF-1 inactivation and support the concept of pure and hybrid perineuriomatous lesions as novel member of the spectrum of PNSTs in NF-1.