Germline Mutations In NF1 Research Articles

Familial Lipomas Without Classic Neurofibromatosis-1 Caused by a Missense Germline NF1 Mutation

Neurofibromatosis type 1 (NF1; OMIM 162200) is an autosomal dominant disorder caused by a wide variety of germline mutations in the NF1 gene. Nearly all individuals with NF1 exhibit café-au-lait macules before the age of 2,1 but can also develop numerous other medical problems, including autism, cognitive deficits, tumors, and congenital heart defects. Although the clinical variability inherent in NF1 has been attributed to stochastic events, it is possible that the specific germline NF1 mutation might be one factor underlying this heterogeneity. To illustrate this point, we report a family with 2 affected members harboring a missense mutation in the NF1 gene who lack the classic features of NF1.

NFB-06. TREATMENT CHALLENGES IN PEDIATRIC GLIOBLASTOMA MULTIFORME WITH CONCURRENT SOMATIC AND GERMLINE NF1 MUTATIONS WITH GERMLINE MISMATCH REPAIR MUTATIONS: TWO UNIQUE CASES

INTRODUCTIONWe report the first known cases of pediatric glioblastoma (GBM) with prior clinical NF1 diagnoses, one with concurrent germline Lynch syndrome (LS) and NF1, and the other with somatic NF1 mutation and germline constitutional mismatch repair deficiency (CMMRD).METHODSTwo pediatric GBM cases with prior NF1 clinical diagnoses based on neurocutaneous criteria were reviewed. Next generation sequencing and immunohistochemical staining were used for somatic and germline NF1 and MMR gene mutation detection, and for MMR protein expression, respectively.RESULTSSixteen year old male with prior NF1 clinical diagnosis had resection of right frontal GBM revealing somatic mutations of POLE and PMS2, but not NF1. His father had confirmed LS with MSH2 mutation and no neurocutaneous stigmata. Patient’s germline testing revealed both pathogenic MSH2 plus NF1 mutations confirming LS and NF1. Treatment consisted of chemoradiation with temozolomide followed by adjuvant temozolomide with stable disease at 8 cycles. Nineteen year old male with former NF1 clinical diagnosis had 2 GBMs, first in left midbrain biopsied revealing somatic PMS2 and NF1 mutations underwent radiation then 7 cycles of temozolomide, then new left frontal GBM underwent resection followed by radiation and 5 cycles of pembrolizumab stable at 5th cycle.CONCLUSIONChildren with NF1 stigmata and GBM can have concurrent NF1 and LS, or CMMRD with NF1 somatic mutations. Our patients tolerated alkylating agents, despite risk for secondary malignancies as upfront therapy and at recurrence checkpoint inhibitors. Upfront therapy in GBM with mismatch repair syndrome with checkpoint inhibitors should be studied.

LGG-09. CORRELATING GENETIC SIGNATURE OF PILOMYXOID ASTROCYTOMAS AND PILOCYTIC ASTROCYTOMAS WITH QUALITATIVE AND QUANTITATIVE MR IMAGING CHARACTERISTICS

PURPOSEPilomyxoid astrocytomas are predominantly located in supra-chiasmatic region and are more clinically aggressive than pilocytic astrocytomas, although recent WHO 2016 classification placed them into the grade I/II category. In our study, we describe imaging correlation of PMA to their genetic signature.MATERIALS AND METHODSWe identified 12 pediatric patients with pathologically proven PMA, PA, and PA with myxoid features in an IRB approved study. Three of the tumors had whole exome somatic and germline sequencing. Qualitative MRI characteristics of location, size, enhancement, edema, T2 and T1 intensity, and multifocality were assessed.RESULTSAmong the PMA, 3 cases were found to have KIAA1549-BRAF fusion, 1 case BRAF V600E mutation, and 2 cases had wildtype BRAF. The BRAF wildtype tumors had atypical imaging features with intraventricular extension of tumor, involvement of frontal lobe parenchyma and one tumor demonstrating increase in size and development of enhancement at 5 years. Whole exome sequencing of BRAF wildtype tumors identified somatic truncation mutations in NF1 R1534X and R1513X with wildtype germline NF1 and missense mutations in KMT2C and GLTSCR1. Among PAM, one was BRAF wildtype with mutations i PTCH1 M956V and PTPN1 (A72V) and demonstrated atypical features of intratumoral hemorrhage on presentation. Among PA, one was positive for KIAA1549-BRAF, one was BRAF wildtype.CONCLUSIONS BRAF wildtype PMA and PA demonstrate atypical tumor localization and are associated with atypical genetic mutations on whole exome sequencing. On the contrary, presence of KIAA1549-BRAF fusion or BRAF V600E mutation within PMA and PA correlates with classic qualitative imaging characteristics.

Spectrum of Hematological Malignancies in 130 Patients with Germline Predisposition Syndromes - Mayo Clinic Germline Predisposition Study

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

Temporal, spatial, and genetic constraints contribute to the patterning and penetrance of murine neurofibromatosis-1 optic glioma.

Brain tumors are the most common solid tumors of childhood, but little is understood about the factors that influence their development. Pediatric low-grade gliomas in particular display unique temporal and spatial localization associated with different genetic mutations (eg, BRAF genomic alterations, mutations in the neurofibromatosis type 1 [NF1] gene) for reasons that remain unclear. NF1 low-grade gliomas typically arise in the optic pathway of young children as optic pathway gliomas (OPGs), likely from a cell of origin that resides within the third ventricular zone (TVZ). However, the factors that contribute to their distinct temporal patterning and penetrance have not been adequately explored. TVZ neuroglial progenitor cells (NPCs) were analyzed over the course of mouse brain development. Progenitors isolated by fluorescence-activated cell sorting (FACS) were assessed for functional and molecular differences. The impact of different germline Nf1 mutations on TVZ NPC properties was analyzed using genetically engineered mice. We identify 3 individual factors that could each contribute to Nf1 optic glioma temporal patterning and penetrance. First, there are 3 functionally and molecularly distinct populations of mouse TVZ NPCs, one of which ("M" cells) exhibits the highest clonogenic incidence, proliferation, and abundance during embryogenesis. Second, TVZ NPC proliferation dramatically decreases after birth. Third, germline Nf1 mutations differentially increase TVZ NPC proliferation during embryogenesis. The unique temporal patterning and penetrance of Nf1 optic glioma reflects the combined effects of TVZ NPC population composition, time-dependent changes in progenitor proliferation, and the differential impact of the germline Nf1 mutation on TVZ NPC expansion.

Genetics of renovascular hypertension in children.

In most cases of renovascular hypertension in children, the cause is unclear. The aim of this study was to investigate genetic variation as a factor in the development of renovascular hypertension in children. In a cohort of 37 unrelated children from a single tertiary referral center, exome sequencing was performed. We assessed variants in recognized and suspected disease genes and searched for novel ones with a gene-based variant-burden analysis. In the majority of patients, exome sequencing could not identify causative variants. We found a pathogenic variant in a recognized associated disease gene in five patients (three pathogenic variants in NF1, one in ELN and a deletion of chromosome 7q11.23, consistent with Williams syndrome). In two other patients, (likely) pathogenic variants were found in putative renovascular hypertension genes (SMAD6 and GLA), with clinical implications for both. Ten additional patients carried variants of uncertain significance (VUS) in known (n = 4) or putative (n = 6) renovascular hypertension disease genes. Rare variant burden analysis yielded no further candidate genes. Genetic contributors, such as germline mutations in NF1, ELN, 7q11.23del were present in only 5 out of 37 (14%) children with renovascular hypertension. Twelve other children (32%) had potentially causal variants identified, including a pathogenic variant in SMAD6; a vasculopathy gene hitherto unknown to link with renovascular hypertension. Most importantly, our data show that exome sequencing can rarely identify the cause of renovascular hypertension in nonsyndromic children. We suggest that nongenetic factors or somatic genetic variation will play a more important role.

PDCT-02. UNIQUE CONCURRENT ASSOCIATION OF SOMATIC AND GERMLINE NF1 MUTATIONS WITH GERMLINE MISMATCH REPAIR MUTATIONS IN PEDIATRIC GLIOBLASTOMA MULTIFORME

Abstract INTRODUCTION We report the first known cases of pediatric glioblastoma (GBM) with prior clinical NF1 diagnoses, one with concurrent germline Lynch syndrome (LS) and NF1, and the other with somatic NF1 mutation and germline constitutional mismatch repair deficiency (CMMRD). Pediatric GBMs in NF1 are rare but increase with age. LS and CMMRD differ by inheritance: autosomal dominant with heterozygous germline mutation (LS), or biallelic mutation (CMMRD) of one of the 4 (possibly 5) MMRD genes (MLH1, MSH2, MSH6, PMS2, or MSH3) - both can develop GBM. METHODS Two pediatric GBM cases with prior NF1 clinical diagnoses (neurocutaneous stigmata) were reviewed. Next generation sequencing and immunohistochemical staining were used for somatic and germline NF1 and MMR gene mutation detection, and for MMR protein expression, respectively. RESULTS Sixteen-year-old male with prior NF1 clinical diagnosis had headaches for one year preceding diagnosis and resection of right frontal GBM with somatic mutations of POLE and PMS2, but not NF1. His father had confirmed LS with MSH2 mutation and no neurocutaneous stigmata. Patient’s germline testing revealed pathogenic MSH2 and NF1 mutations, confirming LS and NF1. Current treatment is chemoradiation. Nineteen-year-old male with former NF1 clinical diagnosis had 2 GBMs, first in left midbrain diagnosed and biopsied (after 3 months of right hemiparesis) with somatic PMS2 and NF1 mutations, and a second left frontal lobe tumor diagnosed and resected 11 months later, with 2 pathogenic germline mutations of PMS2, inherited one from each parent, confirming CMMRD without NF1. Treatment was focal radiation administered to both sites followed by adjuvant chemotherapy. CONCLUSION Children with NF1 stigmata and GBM may have either concurrent NF1 and LS, or CMMRD with NF1 somatic mutations. Confirmatory germline and somatic MMR gene and NF1 mutation testing in children with GBM and NF1 stigmata should be considered as the specific diagnosis affects management.

85P - Improving detection level of somatic mosaicism in neurofibromatosis type 1

BackgroundNeurofibromatosis type 1 (NF-1) is a tumor predisposition syndrome resulting from mutations in NF1 antioncogene. Differential diagnosis with genetic testing is essential in NF-1 diagnostics due to its clinical variability. Identifying of pathogenic mutations is challenging because of the gene size and structure, especially for low-level somatic mosaicism. Notably, patients with mosaic NF-1 demonstrate no less severe phenotype than those with classic form. Nowadays, NGS and Sanger sequencing are recognized diagnostics methods and they both are not sensitive enough to mosaic genotypes with small fractions of alternative alleles. We believe that this can be resolved with a special approach to sequencing data analysis. MethodsIn order to re-evaluate previously obtained NGS (Ion AmpliSeq technology) results for 275 probands with clinical diagnosis “NF-1” or “NF, unspecified” for whom germline mutations in NF1 and NF2 genes were not identified, we developed an improved data analysis pipeline to search for somatic mutations, which includes: (1) programmatic pool-based division of NGS reads, (2) elimination of out-of-design aligned reads, (3) exclusion of systematic sequencing errors, (4) variant calling with low stringency parameters (alternative allele frequency, AF≥0.05; read depth, DP≥20). Newly detected mutations were verified using Sanger sequencing and heteroduplex analysis. Sanger results were analysed using our in-house SeqBase software, highly sensitive to mosaic variants. ResultsApplication of our NGS data reanalysis algorithm allowed us to detect 12 cases (4.3%) of somatic mosaic mutations among 275 probands otherwise lacking molecular verification of neurofibromatosis diagnosis. The majority of the identified mutations are nonsense or frameshift, with AF ranging from 0.051 to 0.296. Mutations were verified with alternative methods of molecular diagnostics. ConclusionsImproved sequencing data analysis allows to detect NF-1 cases with mosaic genotype in cases unresolved by conventional analysis pipeline. Yet, success rate is strongly dependent upon the fraction of the mutant allele, thus alternative quantitative methods are required for exhaustive NF-1 molecular diagnosis. Legal entity responsible for the studyFederal State Budgetary Institution "Research Centre for Medical Genetics". FundingThe research was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation. DisclosureAll authors have declared no conflicts of interest.

Abstract 1686: Comprehensive genomic and transcriptomic profiling of gastrointestinal stromal tumors

Abstract Most adult gastrointestinal stromal tumors (GIST) are driven by activating KIT or PDGFRA mutations. The remaining 10-15% of cases, often referred to as wildtype (WT) GIST, display either alterations of the succinate dehydrogenase complex (SDH) or RAS pathway mutations. To gain additional insight into the biology of GIST, we performed whole-exome or genome and RNA sequencing in 38 GIST patients (WT, n=15; KIT-mutant, n=21; PDGFRA-mutant, n=2) enrolled in a prospective molecular stratification trial of NCT Heidelberg/Dresden and the German Cancer Consortium (DKTK) designed for younger adults with advanced-stage cancer across histologies and patients with rare tumors (NCT/DKTK MASTER). Of the 15 patients with WT GIST, 3 had pathogenic germline mutations in NF1 and 9 harbored SDH alterations (germline, n=5; somatic, n=4). In the 3 patients with quadruple-negative GIST - defined by the absence of KIT, PDGFRA, SDH, or RAS pathway alterations - we detected novel gene fusions affecting RET, FGFR2, and FGF4, respectively. To delineate biologically relevant subgroups of GIST based on RNA sequencing data from the entire cohort (n=34), we used 3 different clustering methods and 4 different measures of stability and consistency. Despite the underlying clinical and molecular heterogeneity, we identified 3 distinct transcriptional subgroups that were characterized by (i) SDH deficiency, (ii) recurrent somatic RB1 alterations and mutational signatures associated with defective homologous recombination DNA repair, and (iii) elevated PDGFRA expression, respectively. Furthermore, we used random forest analysis to identifiy genes that are significantly (p<0.005) differentially expressed between the 3 subgroups. Interestingly, quadruple-negative cases did not form a separate cluster or clustered within a specific subgroup. Collectively, our data illustrate the molecular heterogeneity of advanced-stage GIST and support comprehensive molecular profiling approaches to capture the entire spectrum of clinically actionable genetic alterations, such as diverse fusion genes affecting kinase signaling pathways in quadruple-negative cases or pathogenic germline mutations in patients with inconspicuous family histories. The finding of two separate transcriptional clusters among patients with SDH-proficient GIST may be reflective of distinct regulatory pathways whose molecular underpinnings and clinical actionability warrant further study. Citation Format: Peter Horak, Matea Hajnic, Laura Gieldon, Mario Hlevnjak, Susan Richter, Barbara Hutter, Johanna Falkenhorst, Sebastian Uhrig, Gregor Warsow, Nagarajan Paramasivam, Stefan Gröschel, Barbara Klink, Simon Kreutzfeldt, Christoph Heining, Christoph E. Heilig, Martina Fröhlich, Stephan Richter, Christian Brandts, Wilko Weichert, Philipp Jost, Olaf Neumann, Marc Zapatka, Albrecht Stenzinger, Alexander Marx, Benedikt Brors, Evelin Schröck, Sebastian Bauer, Peter Hohenberger, Hanno Glimm, Claudia Scholl, Stefan Fröhling. Comprehensive genomic and transcriptomic profiling of gastrointestinal stromal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1686.

Low mutation burden and frequent loss of CDKN2A/B and SMARCA2, but not PRC2, define premalignant neurofibromatosis type 1-associated atypical neurofibromas.

Neurofibromatosis type 1 (NF1) is a tumor-predisposition disorder caused by germline mutations in NF1. NF1 patients have an 8-16% lifetime risk of developing a malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft-tissue sarcoma, often arising from preexisting benign plexiform neurofibromas (PNs) and atypical neurofibromas (ANFs). ANFs are distinct from both PN and MPNST, representing an intermediate step in malignant transformation. In the first comprehensive genomic analysis of ANF originating from multiple patients, we performed tumor/normal whole-exome sequencing (WES) of 16 ANFs. In addition, we conducted WES of 3 MPNSTs, copy-number meta-analysis of 26 ANFs and 28 MPNSTs, and whole transcriptome sequencing analysis of 5 ANFs and 5 MPNSTs. We identified a low number of mutations (median 1, range 0-5) in the exomes of ANFs (only NF1 somatic mutations were recurrent), and frequent deletions of CDKN2A/B (69%) and SMARCA2 (42%). We determined that polycomb repressor complex 2 (PRC2) genes EED and SUZ12 were frequently mutated, deleted, or downregulated in MPNSTs but not in ANFs. Our pilot gene expression study revealed upregulated NRAS, MDM2, CCND1/2/3, and CDK4/6 in ANFs and MPNSTs, and overexpression of EZH2 in MPNSTs only. The PN-ANF transition is primarily driven by the deletion of CDKN2A/B. Further progression from ANF to MPNST likely involves broad chromosomal rearrangements and frequent inactivation of the PRC2 genes, loss of the DNA repair genes, and copy-number increase of signal transduction and cell-cycle and pluripotency self-renewal genes.

Immunophenotypic and Genetic Overlap between JMML and CMML

Introduction:Juvenile myelomonocytic leukemia (JMML) is a rare hematological malignancy of early childhood with characteristics of both myeloproliferative neoplasms and myelodysplastic syndromes. JMML shares pathological features and diagnostic criteria with chronic myelomonocytic leukemia (CMML), a malignancy predominantly affecting the elderly. While 85% of patients with JMML have somatic or germline mutations in RAS pathway genes (NF1, NRAS, KRAS, PTPN11, and CBL), the most frequently mutated genes in CMML include TET2, SRSF2, ASXL1, and RAS and are generally somatic-only. The extent to which histone modification genes (ASXL1, EZH2) or spliceosome machinery genes (SF3B1, SRSF2, U2AF1, ZRSR2) play a role in JMML pathogenesis is unclear. Despite mutational differences, both JMML and CMML manifest as myelomonocytic proliferation with varying amounts of dysplasia in the bone marrow. Clusters of clonally-related CD123+ plasmacytoid dendritic cells (PDCs) have been observed in the bone marrow of patients with CMML but have not been investigated in JMML. Here, we report the mutation profiles and immunophenotypic characteristics of JMML specimens from children treated at our institution.Methods:The pathology archives (1987-2017) at the Children's Hospital of Philadelphia (CHOP) were searched to identify JMML cases (n=21) and included formalin fixed paraffin-embedded diagnostic bone marrow biopsies and splenectomy tissue obtained prior to hematopoietic stem cell transplant. JMML diagnosis was confirmed in all cases by clinicopathological review. Cytogenetic analysis and whole genome SNP array were performed at initial clinical presentation. Genomic DNA and RNA were extracted from JMML patients' bone marrow (n=8) and spleen tissue (n=10) for next-generation sequencing analysis of 118 cancer genes for sequence and copy number variants and 110 genes for known and novel fusions via our custom CHOP Hematologic Cancer Panel.CD123 immunohistochemical (IHC) staining was performed on bone marrow and spleen tissues from children with JMML. Presence of CD123+ PDC clusters was evaluated manually and by digital image analysis. CD123 staining was enumerated using the Aperio Image Scope quantitation of membranous staining v9 with the analysis parameters set such that normal endothelial staining was quantified as 1+, and true CD123 staining cells were quantified as 2+ or 3+. The percentage of CD123+ cells (out of total cellularity) was calculated. Bone marrow from patients with non-JMML myeloid malignancies (n=6) and splenectomy tissue from patients with sickle cell anemia (n=8) were used as controls for the CD123 IHC analysis.Results:We confirmed canonical JMML-associated somatic or germline NF1 (n=3), NRAS (n=4), KRAS (n=2), PTPN11 (n=6), or CBL (n=2) mutations in 16 of the 17 (94%) patients with sequencing data. Interestingly, both PTPN11A72T and NF1R2637* mutations were detected in one patient. In addition, we found potential variants in genes affecting histone modifications (ASXL1, DNMT3A, KDM6A, SETD2), spliceosomal processes (SF3B1, U2AF1), transcription (BCOR, RUNX1, ETV6), or cellular growth (SETBP1, BRAF) in 8/17 patients (47%). While mutations in these genes have been well-characterized in other myeloid disorders, many of these alterations have not been reported to date in children with JMML or are currently of unclear biologic and prognostic significance.We also observed increased clustering of CD123+ PDCs in bone marrow and spleens from patients with JMML compared to IHC staining of control tissues. 2.2 ± 0.42% and 1.8 ± 0.74% of cells expressed CD123 in the spleen and bone marrow specimens, respectively. Control bone marrow and spleen samples did not show significant CD123+ staining.Conclusions:Our study demonstrates frequent variants in histone modification, splicing, and transcription-associated genes in JMML specimens in addition to known pathogenic RAS pathway mutations. We further report histopathologic CD123+ PDC clustering in JMML specimens analogous to that observed in CMML, which may aid in the workup of this often difficult-to-diagnose disease. Our findings of genetic and immunophenotypic overlap between JMML and CMML suggest similarities in pathogenesis despite typical presentation at extremes of age. DisclosuresTasian:Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

NF1 deficiency correlates with estrogen receptor signaling and diminished survival in breast cancer

The key negative regulatory gene of the RAS pathway, NF1, is mutated or deleted in numerous cancer types and is associated with increased cancer risk and drug resistance. Even though women with neurofibromatosis (germline NF1 mutations) have a substantially increased breast cancer risk at a young age and NF1 is commonly mutated in sporadic breast cancers, we have a limited understanding of the role of NF1 in breast cancer. We utilized CRISPR–Cas9 gene editing to create Nf1 rat models to evaluate the effect of Nf1 deficiency on tumorigenesis. The resulting Nf1 indels induced highly penetrant, aggressive mammary adenocarcinomas that express estrogen receptor (ER) and progesterone receptor (PR). We identified distinct Nf1 mRNA and protein isoforms that were altered during tumorigenesis. To evaluate NF1 in human breast cancer, we analyzed genomic changes in a data set of 2000 clinically annotated breast cancers. We found NF1 shallow deletions in 25% of sporadic breast cancers, which correlated with poor clinical outcome. To identify biological networks impacted by NF1 deficiency, we constructed gene co-expression networks using weighted gene correlation network analysis (WGCNA) and identified a network connected to ESR1 (estrogen receptor). Moreover, NF1-deficient cancers correlated with established RAS activation signatures. Estrogen-dependence was verified by estrogen-ablation in Nf1 rats where rapid tumor regression was observed. Additionally, Nf1 deficiency correlated with increased estrogen receptor phosphorylation in mammary adenocarcinomas. These results demonstrate a significant role for NF1 in both NF1-related breast cancer and sporadic breast cancer, and highlight a potential functional link between neurofibromin and the estrogen receptor.

Characterizing temporal genomic heterogeneity in pediatric high-grade gliomas

Pediatric high-grade gliomas (pHGGs) are aggressive neoplasms representing approximately 20% of brain tumors in children. Current therapies offer limited disease control, and patients have a poor prognosis. Empiric use of targeted therapy, especially at progression, is increasingly practiced despite a paucity of data regarding temporal and therapy-driven genomic evolution in pHGGs. To study the genetic landscape of pHGGs at recurrence, we performed whole exome and methylation analyses on matched primary and recurrent pHGGs from 16 patients. Tumor mutational profiles identified three distinct subgroups. Group 1 (n = 7) harbored known hotspot mutations in Histone 3 (H3) (K27M or G34V) or IDH1 (H3/IDH1 mutants) and co-occurring TP53 or ACVR1 mutations in tumor pairs across the disease course. Group 2 (n = 7), H3/IDH1 wildtype tumor pairs, harbored novel mutations in chromatin modifiers (ZMYND11, EP300 n = 2), all associated with TP53 alterations, or had BRAF V600E mutations (n = 2) conserved across tumor pairs. Group 3 included 2 tumors with NF1 germline mutations. Pairs from primary and relapsed pHGG samples clustered within the same DNA methylation subgroup. ATRX mutations were clonal and retained in H3G34V and H3/IDH1 wildtype tumors, while different genetic alterations in this gene were observed at diagnosis and recurrence in IDH1 mutant tumors. Mutations in putative drug targets (EGFR, ERBB2, PDGFRA, PI3K) were not always shared between primary and recurrence samples, indicating evolution during progression. Our findings indicate that specific key driver mutations in pHGGs are conserved at recurrence and are prime targets for therapeutic development and clinical trials (e.g. H3 post-translational modifications, IDH1, BRAF V600E). Other actionable mutations are acquired or lost, indicating that re-biopsy at recurrence will provide better guidance for effective targeted therapy of pHGGs.

Abstract P1-08-01: Regulation of estrogen receptor-α by NF1

Abstract Background. Although great strides have been made in targeting the ER pathway for treating ER+ breast cancer, relapse and death is common and is closely linked to resistance to ER-targeting agents. As a result, the majority of deaths from breast cancer still come from ER+ tumors. To discover drivers for endocrine resistance, we have sequenced tumor DNAs from a cohort of >600 patients treated with 5-year tamoxifen (Tam) monotherapy with a median 10.4 years follow up. Our preliminary data show that the worst outcome mutations (Hazard Ratio of ∼3 for relapse) were mostly those of the Neurofibromatosis type 1 (NF1) gene (encoding Neurofibromin), with nonsense/frame shift mutations creating early stop codons. Germline NF1mutations cause neurofibromatosis type 1, a common inherited disorder that predisposes individuals to both benign and malignant tumors of the nervous system, as well as an increased risk for breast cancer. Analysis of DNA sequencing data has also shown that the NF1 gene is mutated in a wide range of common cancers (e.g., melanoma, lymphoma, and cancers of the lung, breast, and colon). Thus, NF1-deficiency underlies the formation and/or progression of a large number of cancers, so that the development of therapies targeted to NF1-deficient malignancies would have broad impact. These observations support the hypothesis that NF1 gene inactivation is associated with aggressive tumor behaviors, such as endocrine therapy resistance in breast cancer. The key focus of this study is to define how the NF1 protein neurofibromin, regulates endocrine therapy resistance. Although neurofibromin is best known as a negative regulator for Ras, our data show that it may have other functions. Method. Our data suggest that many of the identified nonsense/frame shift create a NF1 null state; thus, we have used gene-silencing to recapitulate the effects of such NF1 mutations on the activities of ER+ breast cancer cells. NF1+ and NF1– ER+ breast cancer cells were grown in defined media to measure how estradiol (E2) and Tam impact their growth, transforming activities, and gene expression. The binding between neurofibromin and components of the ER transcriptional pathway was measured biochemically and using the mammalian two-hybrid system. Results. Our data showed that NF1-silenced cells use Tam as an agonist and can grow with very little E2, and these activities are driven by enhanced recruitment of ER to the ERE, leading to efficient expression of many classic ER-responsive genes. Expressing the NF1-GAP domain does not restore normal responses to Tam and E2 in NF1-silenced cells, suggesting that neurofibromincan regulate ER activity in a Ras-independent manner. To investigate the possibility that neurofibromin can directly regulate ER, we found that it can bind ER; furthermore, neurofibromin was more strongly recruited to the ERE by Tam than by E2. Conclusion. Our data support a model whereby neurofibromin acts like a co-repressor for ER. As such,NF1 loss may result in more aggressive tumor behaviors by activating, not only the Ras pathways, but also the ER transcriptional pathways. Simultaneous activation of two powerful oncogenic pathways by the loss of a single tumor suppressor may explain why neurofibromin is such a potent tumor suppressor lost in a wide range of cancers. Citation Format: Zheng Z-Y, Cakar B, Lavere P, Cao J, Yao J, Singh P, Lei JT, Toonen JA, Haricharan S, Anurag M, Shah K, Kavuri M, Chan DW, Chen X, Gutmann DH, Foulds CE, Ellis MJ, Chang EC. Regulation of estrogen receptor-α by NF1 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-01.

The NF1 gene in tumor syndromes and melanoma

Activation of the RAS/MAPK pathway is critical in melanoma. Melanoma can be grouped into four molecular subtypes based on their main genetic driver: BRAF-mutant, NRAS-mutant, NF1-mutant, and triple wild-type tumors. The NF1 protein, neurofibromin 1, negatively regulates RAS proteins through GTPase activity. Germline mutations in NF1 cause neurofibromatosis type I, a common genetic tumor syndrome caused by dysregulation of the RAS/MAPK pathway, ie, RASopathy. Melanomas with NF1 mutations typically occur on chronically sun-exposed skin or in older individuals, show a high mutation burden, and are wild-type for BRAF and NRAS. Additionally, NF1 mutations characterize certain clinicopathologic melanoma subtypes, specifically desmoplastic melanoma. This review discusses the current knowledge of the NF1 gene and neurofibromin 1 in neurofibromatosis type I and in melanoma.

The mTORC1 Complex Is Significantly Overactivated in SDHX-Mutated Paragangliomas

Aim: We aimed at exploring the activation pattern of the mTOR pathway in sporadic and hereditary pheochromocytomas (PCCs) and paragangliomas (PGLs). Methods: A total of 178 PCCs and 44 PGLs, already characterized for the presence of germline mutations in VHL, RET, NF1, MAX, SDHA, SDHB, SDHC, and SDHD as well as somatic mutations in VHL, RET, H-RAS, and MAX, were included in 5 tissue microarrays and tested using immunohistochemistry for mTOR and Rictor as well as the phosphorylated forms of mTOR, p70S6K, AMPK, AKT, 4EBP1, S6, and Raptor. Results: The positive correlation among most of the molecules investigated proved the functional activation of the mTOR pathway in PCCs/PGLs. Total mTOR, p-S6K and p-S6, and mTORC1-associated molecules p-Raptor and p-AMPK were all significantly overexpressed in PGLs rather than in PCCs, and in the head and neck rather than in abdominal locations. None of the markers, except for the low expression of p-mTOR, was associated with malignancy. Cluster 1 PCCs/PGLs had higher total mTOR, p-Raptor, and p-S6 expression than cluster 2 PCCs/PGLs. In contrast, p-mTOR and mTORC2-associated molecule Rictor were significantly overexpressed in cluster 2 tumors. Within cluster 1, molecules active in the mTORC1 complex were significantly overexpressed in SDHX- as compared to VHL-mutated tumors. Conclusion: In summary, the mTOR pathway is activated in a high proportion of PCCs/PGLs, with a preferential overactivation of the mTORC1 complex in PGLs of the head and neck and/or harboring SDHX mutations.

Whole Exome Sequencing of Growing and Non-Growing Cutaneous Neurofibromas from a Single Patient with Neurofibromatosis Type 1.

The growth behaviors of cutaneous neurofibromas in patients with Neurofibromatosis type 1 are highly variable. The role of the germline NF1 mutation, somatic NF1 mutation and mutations at modifying loci, are poorly understood. We performed whole exome sequencing of three growing and three non-growing neurofibromas from a single individual to assess the role of acquired somatic mutations in neurofibroma growth behavior. 1–11 mutations were identified in each sample, including two deleterious NF1 mutations. No trends were present between the types of somatic mutations identified and growth behavior. Mutations in the HIPPO signaling pathway appeared to be overrepresented.

Comprehensive Genetic Analysis in Cases of Juvenile Myelomonocytic Leukemia for Prognostic Estimation

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm (MPN) that occurs during childhood and has a poor prognosis. Somatic or germline mutations in canonical RAS pathway genes, i.e., PTPN11, NF1, NRAS, KRAS, and CBL, are reported be detected in approximately 85% patients. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for JMML. Although spontaneous remission is occasionally observed in others with supportive therapy, some patients show aggressive disease progression despite HSCT. Recent studies have identified several additional genetic events in an array of genes, including SETBP1 and JAK3, but the relationship between genetic alterations and clinical outcomes remains unclear.Patients and Methods: A total of 131 patients (88 boys, 43 girls) with JMML were enrolled in the study. The median age was 15 months (range, 1-160 months). Eighty-two of 131 patients underwent HSCT, and 36 patients died (disease related, n = 27, transplantation-related complications, n = 16, infection, n = 5, unknown, n = 3). We performed comprehensive genetic analyses of the 131 JMML patients using whole-exome sequencing (n = 68, 52%) or targeted deep sequencing (n = 92, 70%), and assessed the impact of genetic alterations on clinical outcomes in 119 patients, excluding 12 patients with Noonan syndrome-related myeloproliferative disorder (NS/MPD).Results: We identified canonical RAS pathway gene mutations in 115 of 131 patients (88%). Although most RAS pathway mutations were mutually exclusive, coexisting secondary RAS pathway mutations were found in nine patients (8%). In addition, 28 patients harbored secondary genetic alterations in other genes, including SETBP1 (n = 10), JAK3 (n = 12), ASXL1 (n = 6), SH3BP1 (n = 1), RRAS2 (n = 2), and SOS1 (n = 3). In total, 34 of 131 patients harbored secondary genetic mutations. In univariate analysis, patients with secondary genetic mutations showed poorer survival rates than patients without these mutations [5-year transplantation-free survival (TFS) (95% CI) = 8.8% (2.3%-21.1%) vs. 24.1% (15.2%-34.1%), p = 0.007; 5-year overall survival (OS) (95% CI) = 49.6% (32.0%-65.0%) vs. 62.3% (50.8%-71.8%), p = 0.135]. On the basis of the dominant canonical RAS pathway mutations classification, patients with PTPN11 and NF1 mutations were significantly associated with the presence of secondary genetic mutations compared to patients with other RAS pathway gene mutations (PTPN11 (20 of 43, 47%), NF1 (5 of 7, 71%), NRAS (2 of 18, 11%), KRAS (4 of 20, 20%), CBL (1 of 17, 6%), p < 0.001). Consistent with previous reports, patients with PTPN11 and NF1 mutations had inferior survival rates than other JMML patients [5-year TFS (95% CI) = 0% vs. 32.7% (21.5%-44.3%), p < 0.001; 5-year OS (95% CI) = 45.3% (31.1%-58.5%) vs. 68.1% (55.2%-78.0%), p = 0.006]. Multivariate survival analysis identified the RAS pathway mutations (i.e., patients with PTPN11 and NF1 mutations vs. others) [TFS: HR (95% CI) = 3.732 (2.382-5.847), p < 0.001; OS: HR (95% CI) = 1.983 (1.117-3.521), p < 0.019] and low platelet count (<33 × 109/L vs. ≥ 33 × 109/L) [TFS: HR (95% CI) = 1.816 (1.160-2.843), p < 0.001] as independent risk factors for TFS and OS. In subgroup analysis of 50 patients with PTPN11 and NF1 mutations, there were no significant survival differences between patients with (n = 25) or without (n = 25) secondary genetic mutations [5-year TFS (95% CI) = 0% vs. 0%, p = 0.753; 5-year OS (95% CI) = 39.6% (20.8%-57.9%) vs. 51.7% (30.9%-69.1%), p = 0.589].Discussion: Consistent with previous studies, secondary genetic mutations were associated with inferior survival rates, but high correlations were observed in JMML patientswith PTPN11 and NF1 mutations. Our results suggest that comprehensive genetic mutational profiling is essential to estimate prognosis and to stratify JMML patients who require HSCT and/or novel treatment modalities. DisclosuresOgawa:Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding. Kojima:SANOFI: Honoraria, Research Funding.

Abstract 772: The molecular landscape of dermal neurofibromatosis

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

Novel SDHB and TMEM127 Mutations in Patients with Pheochromocytoma/Paraganglioma Syndrome.

Pheochromocytomas (Pheo) and paragangliomas (PGL) are rare tumors, with heterogeneous genetic background. In up to 30% of all, apparently sporadic Pheo/PGL cases germline mutations can be identified in one of the 15 genes representing genetic susceptibility for Pheo/PGL. Malignancy is rare but it frequently associates with SDHB mutations. Our aim was to determine the prevalence of germline SDHx, SDHAF2, MAX and TMEM127 mutations in Hungarian patients with apparently sporadic Pheo/PGLs. Mutation screening of the SDHx, SDHAF2, MAX and TMEM127 genes was performed in 82 Hungarian patients with apparently sporadic Pheo/PGL using PCR and bidirectional Sanger sequencing. Disease-causing germline mutations were identified in 11 patients, of which 4 SDHB and 2 TMEM127 mutations were novel. Earlier development of Pheo/PGL, more malignant phenotype and multiple tumors were observed in genetically positive cases especially in those with SDHB mutations. The presence of bilateral or multiple tumors was the most predictive for identification of a pathogenic mutation. Together with cases harboring germline RET, VHL and NF1 mutations, Hungarian patients with Pheo/PGL exhibit a heterogeneous mutation spectrum, indicating that all of the Pheo/PGL susceptibility genes should be tested. Novel genotype-phenotype associations revealed by our study may contribute to improvement of diagnostic approaches and may help to achieve a better clinical follow up for patients with Pheo/PGL.