Mutations In KLF4 Research Articles

BIOM-60. UNDERSTANDING THE INTERSECTION OF RACE, SEX, AND GENOMIC PROFILES IN MENINGIOMA PATHOPHYSIOLOGY

Abstract BACKGROUND While meningioma incidence and outcomes vary by sex and race, the potential underlying molecular differences possibly contributing to these disparities are less understood. Herein, we explore the clinical and genomic characteristics of meningiomas across groups characterized by age and sex. METHODS We analyzed adult patients with primary meningiomas resected from 2012 to 2023. Patients underwent whole exome sequencing, and demographic data were categorized into four groups based on self-reported race and sex, which included White females, White Males, Black females, and Black males. The primary analyses involved comparisons of tumor characteristics, socioeconomic status (median zip code income, % with high school diploma, health insurance status, distance from hospital), genomics, and recurrence rates. RESULTS The study included 509 primary meningiomas. White females exhibited low-risk genomic profiles characterized by driver mutations in KLF4 (+/- TRAF7) (p=0.010) and POLR2A (p=0.045), with no associated CNVs (p=0.410). This benign molecular profile correlated with lower WHO grades (p=0.013) and a lower incidence of recurrence (p=0.0003). Black females had meningiomas enriched with Hedgehog pathway mutations (p=0.0035) and a predilection for the anterior skull base (p=0.034), leading to an increased risk of recurrence (p=0.026). White males showed an aggressive genomic profile, including chromosome 1p loss (p=0.025) and 10q loss (p=0.067), associated with a high risk of recurrence (p=0.0002). Black males had meningiomas with chromosome 14q loss (p=0.0002), the highest incidence of high-grade meningiomas (p=0.048), and a 4.5-fold increased risk of recurrence (OR 4.5 [1.6-11.8]; p=0.006). Black males also experienced the worst progression-free survival (HR 5.5 [2.5-12.4]; p<0.0001), independent of WHO grade, extent of resection, and socioeconomic status. CONCLUSIONS Each demographic has a distinct molecular profile that correlates with clinical behavior in a race and sex-specific manner. Black males are disproportionately affected by biologically aggressive meningiomas, leading to higher tumor grade and recurrence rates.

Abstract C030: Evolutionary and epistatic analyses reveal genic interactions with KRAS during malignant progression of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) patients face poor prognoses attributable to delayed disease detection, resulting in distant metastases and thereby reducing treatment efficacy. The challenge of detecting PDAC lesions during early stages emanates from an inauspicious trifecta of clinical factors: early-stage malignancies are often asymptomatic, existing biomarkers are insufficiently sensitive, and the physical location of the pancreas stymies reliable screening. A promising strategy towards improving outcomes is the resection of high-risk, pre-malignant lesions deemed likely to progress into carcinomas. Intraductal papillary mucinous neoplasms (IPMNs) are both large enough to be detected during abdominal imaging and can originate PDAC lesions. However, a patient may have many IPMN cysts, many of which will never progress. As pancreatic resection carries with it a high risk of morbidity, distinguishing the the evolutionary trajectories of IPMNs that will and will not progress could inform treatment strategies. Additionally, these trajectories may serve as disruptable targets to manage pre- neoplastic lesions, preventing their progression to PDAC. The vast majority PDAC cases contain mutations in KRAS and GNAS with secondary driver mutations in TP53, SMAD4, CDKN2A, and RNF43. Recent work has compared the frequency of these mutations in PDAC with low-grade and high-grade IPMN lesions, finding that they more common in high than low- grade lesions. Interestingly, unlike KRAS or GNAS, KLF4 mutations are prevalent in low-grade IPMNs, rare in high-grade IPMNs, and almost never observed in full-fledged carcinoma, suggesting distinct evolutionary trajectories possibly mediated by epistatic interactions between KLF4 and KRAS. However, the prevalence of a mutation is not dispositive as to its importance in tumorgenesis. Mutations in TTN, for instance, are frequently observed in PDAC and other cancers, but are widely considered to play a modest role, if any, in these cancers. To understand the contribution of a mutation to the evolution of cancer, we must instead deconvolve selection from baseline mutation rate across genomic sites and among patients to distinguish truly oncogenic mutations from mutations that occur frequently merely due to high underlying mutational rates. Here, we quantify the evolutionary and epistatic interplay between KRAS, KLF4, and other frequent mutations in IPMNs and PDAC during malignant transition. First, we report the cancer effect sizes of single-nucleotide variants in IPMNs at varying dysplastic stages. Then, we infer the synergistic and antagonistic interactions between driver mutations, providing insight into the order in which driver mutations occur. We then corroborate this epistasis-inferred ordering with the ordering of mutations recovered from ancestral state reconstruction derived from time-calibrated phylogenies. Lastly, we probe the mutational processes most responsible for conferring highly oncogenic mutations in IPMNs and PDACs to identify potentially actionable or preventable pathways of disease progression. Metabolism Citation Format: Nic Fisk, Derek Song, Margaret Moore, Cole G Jensen, Vincent L. Cannataro, Mofeed Nagib, Jeffrey D Mandell, Luisa Escobar-Hoyos, John W Kunstman, Jeffrey P Townsend. Evolutionary and epistatic analyses reveal genic interactions with KRAS during malignant progression of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C030.

Genetic characterization and mutational profiling of foramen magnum meningiomas: a multi-institutional study.

Foramen magnum (FM) meningiomas pose significant surgical challenges and have high morbidity and mortality rates. This study aimed to investigate the distribution of clinically actionable mutations in FM meningiomas and identify clinical characteristics associated with specific mutational profiles. The authors conducted targeted next-generation sequencing of 62 FM meningiomas from three international institutions, covering all relevant meningioma genes (AKT1, KLF4, NF2, POLR2A, PIK3CA, SMO, TERT promoter, and TRAF7). Patients with a radiation-induced meningioma or neurofibromatosis type 2 (NF2) were excluded from the study. Additionally, patient and tumor characteristics, including age, sex, radiological features, and tumor location, were retrospectively collected and evaluated. The study cohort consisted of 46 female and 16 male patients. Clinically significant driver mutations were detected in 58 patients (93.5%). The most commonly observed alteration was TRAF7 mutations (26, 41.9%), followed by AKT1E17K mutations (19, 30.6%). Both mutations were significantly associated with an anterolateral tumor location relative to the brainstem (p = 0.0078). NF2 mutations were present in 11 cases (17.7%) and were associated with posterior tumor location, in contrast to tumors with TRAF7 and AKT1E17K mutations. Other common mutations in FM meningiomas included POLR2A mutations (8, 12.9%; 6 POLR2AQ403K and 2 POLR2AH439_L440del), KLF4K409Q mutations (7, 11.3%), and PIK3CA mutations (4, 6.5%; 2 PIK3CAH1047R and 2 PIK3CAE545K). POLR2A and KLF4 mutations exclusively occurred in female patients and showed no significant association with specific tumor locations. All tumors harboring AKT1E17K and POLR2A mutations displayed meningothelial histology. Ten tumors exhibited intratumoral calcification, which was significantly more frequent in NF2-mutant compared with AKT1-mutant FM meningiomas (p = 0.047). These findings provide important insights into the molecular genetics and clinicopathological characteristics of FM meningiomas. The identification of specific genetic alterations associated with tumor location, volume, calcification, histology, and sex at diagnosis may have implications for personalized treatment strategies in the future.

PATH-51. GENETIC CHARACTERIZATION AND MUTATIONAL PROFILING OF FORAMEN MAGNUM MENINGIOMAS: A MULTI-INSTITUTIONAL STUDY

Abstract OBJECTIVE Foramen magnum (FM) meningiomas pose significant surgical challenges and have high morbidity and mortality rates. This study aims to investigate the distribution of clinically actionable mutations in FM meningiomas and identify clinical characteristics associated with specific mutational profiles. METHODS We conducted targeted next-generation sequencing of 62 FM meningiomas from three international institutions, covering all relevant meningioma genes (AKT1, KLF4, NF2, POLR2A, PIK3CA, SMO, and TRAF7). Additionally, we retrospectively collected and evaluated patient and tumor characteristics, including age, sex, radiological features, and tumor location. RESULTS Our cohort consisted of 46 female and 16 male patients. Clinically significant driver mutations were detected in 58 patients (93.5%). The most commonly observed alteration was TRAF7 mutations (n=26, 41.9%), followed by AKT1(E17K) mutations (n=19, 30.6%). Both mutations were significantly associated with an anterolateral tumor location relative to the brainstem (p=0.0078). Tumors harboring these mutations predominantly (89.3%) displayed meningiothelial histology. NF2 mutations were present in 11 cases (17.7%) and were associated with posterior tumor location, in contrast to tumors with TRAF7 and AKT1(E17K) mutations. Other common mutations in FM meningiomas included POLR2A mutations (n=8, 12.9%), KLF4 mutations (n=7, 11.6%), and PIK3CA mutations (n=3, 4.8%). These mutations exclusively occurred in female patients and showed no significant correlation with specific tumor locations. Eight tumors exhibited intratumoral calcification which was significantly more frequent in NF2-mutant compared with AKT1-mutant FM meningiomas (p=0.047). CONCLUSIONS Our findings provide important insights into the molecular genetics and clinicopathological characteristics of FM meningiomas. The identification of specific genetic alterations associated with tumor location, calcification, histology, and sex at diagnosis may have implications for personalized treatment strategies in the future.

Clinical Significance of Molecular Alterations and Systemic Therapy for Meningiomas: Where Do We Stand?

Simple SummaryMeningiomas are the most frequent intracranial tumors and comprise a heterogeneous spectrum of diseases, ranging from small, asymptomatic tumors that do not need treatment to large, symptomatic ones causing seizures or neurological deficits that require surgery and/or radiotherapy. Systemic therapy is reserved for progressive or recurrent meningiomas when surgery and/or radiotherapy options have been exhausted, with only modest activity in terms of disease control and survival. Novel molecular alterations are correlated with grading, location, and prognosis of meningiomas. Moreover, some of these driver alterations regulate meningioma growth and progression and may be targeted by specific drugs that are under investigation in clinical trials. Lastly, the microenvironment surrounding meningiomas may also contribute to regulating tumor growth: in particular, PD-L1 and/or M2 macrophage expression may represent a target for immunotherapy.Meningiomas are common intracranial tumors that can be treated successfully in most cases with surgical resection and/or adjuvant radiotherapy. However, approximately 20% of patients show an aggressive clinical course with tumor recurrence or progressive disease, resulting in significant morbidity and increased mortality. Despite several studies that have investigated different cytotoxic agents in aggressive meningiomas in the past several years, limited evidence of efficacy and clinical benefit has been reported thus far. Novel molecular alterations have been linked to a particular clinicopathological phenotype and have been correlated with grading, location, and prognosis of meningiomas. In this regard, SMO, AKT, and PIK3CA mutations are typical of anterior skull base meningiomas, whereas KLF4 mutations are specific for secretory histology, and BAP1 alterations are common in progressive rhabdoid meningiomas. Alterations in TERT, DMD, and BAP1 correlate with poor outcomes. Moreover, some actionable mutations, including SMO, AKT1, and PIK3CA, regulate meningioma growth and are under investigation in clinical trials. PD-L1 and/or M2 macrophage expression in the microenvironment provides evidence for the investigation of immunotherapy in progressive meningiomas.

A new amplicon-based gene panel for next generation sequencing characterization of meningiomas.

Meningiomas are the most frequent primary intracranial tumors. The considerable variety of histological subtypes has been expanded by the definition of molecular alterations, which can improve both diagnostic accuracy and determination of individual patient's outcome. According to the upcoming WHO classification of brain tumors, the in‐time analysis of frequent molecular events in meningiomas may become mandatory to define meningioma subtypes. We have compiled a custom‐made amplicon‐based next generation sequencing (NGS) meningioma panel covering the most frequent known recurrent mutations in 15 different genes. In an unselected consecutive meningioma cohort (109 patients) analyzed over a period of 12 months, we detected mutations in 11 different genes, with most frequent alterations in NF2 (43%), AKT1 E17K (15%), and TRAF7 (13%). In 39 tumors (36%), two different mutations were detected, with NF2 and SUFU (n = 5) and KLF4 and TRAF7 (n = 5) being the most frequent combinations. No alterations were found in POLR2A, CDKN2A, CDKN2B, and BAP1, and no homozygous CDKN2A/B deletion was detected. NF2 mutations were found in tumors of all WHO grades, whereas mutations in KLF4, TRAF7, and SMO were restricted to WHO grade I meningiomas. In contrast, SMARCE1 and TERT mutations were associated with WHO grade II meningiomas (according to the WHO classification 2016). The distribution of mutations across histological subtypes or tumor localization was in line with the existing literature, with typical combinations like KLF4K409Q/TRAF7 for secretory meningiomas and preferential skull base localization of meningiomas harboring SMO and AKT1 E17K mutations. Thus, we present a custom‐made NGS meningioma panel providing a time and cost‐efficient reliable detection of relevant somatic molecular alterations in meningiomas suitable for daily routine.

MNX1-HNF1B Axis Is Indispensable for Intraductal Papillary Mucinous Neoplasm Lineages

Chromatin architecture governs cell lineages by regulating the specific gene expression; however, its role in the diversity of cancer development remains unknown. Among pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMN) with an associated invasive carcinoma (IPMNinv) arise from 2 distinct precursors, and their fundamental differences remain obscure. Here, we aimed to assess the difference of chromatin architecture regulating the transcriptional signatures or biological features in pancreatic cancers. We established 28 human organoids from distinct subtypes of pancreatic tumors, including IPMN, IPMNinv, and PDAC. We performed exome sequencing (seq), RNA-seq, assay for transposase-accessible chromatin-seq, chromatin immunoprecipitation-seq, high-throughput chromosome conformation capture, and phenotypic analyses with short hairpin RNA or clustered regularly interspaced short palindromic repeats interference. Established organoids successfully reproduced the histology of primary tumors. IPMN and IPMNinv organoids harbored GNAS, RNF43, or KLF4 mutations and showed the distinct expression profiles compared with PDAC. Chromatin accessibility profiles revealed the gain of stomach-specific open regions in IPMN and the pattern of diverse gastrointestinal tissues in IPMNinv. In contrast, PDAC presented an impressive loss of accessible regions compared with normal pancreatic ducts. Transcription factor footprint analysis and functional assays identified that MNX1 and HNF1B were biologically indispensable for IPMN lineages. The upregulation of MNX1 was specifically marked in the human IPMN lineage tissues. The MNX1-HNF1B axis governed a set of genes, including MYC, SOX9, and OLFM4, which are known to be essential for gastrointestinal stem cells. High-throughput chromosome conformation capture analysis suggested the HNF1B target genes to be 3-dimensionally connected in the genome of IPMNinv. Our organoid analyses identified the MNX1-HNF1B axis to be biologically significant in IPMN lineages.

Abstract PO-067: A multi-omics study in patient-derived organoids reveals MNX1-HNF1B axis to be indispensable for intraductal mucinous papillary neoplasm lineages

Abstract Chromatin architecture governs cell lineages by regulating the specific gene expression; however, its role in the diversity of cancer development remains unknown. Among pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) and invasive carcinoma with an associated intraductal papillary mucinous neoplasms (invasive IPMNs) arise from two distinct precursors, and their fundamental differences remain obscure. Here, we hypothesized that chromatin profiles may clarify their intrinsic molecular features. We originally established 28 human organoids from distinct subtypes of pancreatic tumors, including IPMN, invasive IPMN, and PDAC as well as from normal ductal cells and performed exome-seq, RNA-seq, ATAC-seq, ChIP-seq, Hi-C, and phenotypic analyses with shRNA or CRISPR interference. Established organoids successfully reproduced the histology of primary tumors. IPMN and invasive IPMN organoids specifically harbored GNAS, RNF43, and KLF4 mutations and showed the distinct lineage related expression profiles compared to PDAC. In addition, chromatin accessibility profiles well stratified the respective tumor groups. Notably, this analysis supported the histological features of tumor subtypes; gastric IPMN gained the stomach-specific accessible regions and the accessible pattern of invasive IPMN linked to diverse gastrointestinal tissues. In contrast, PDAC was characterized by the significant loss of chromatin accessibility seen in normal pancreatic ductal cells. Footprint analysis of transcription factors (TFs) identified specific TFs that are enriched in each tumor subtype. Of note we found the footprint of HNF1B to be active in IPMN lineages but not in PDAC. To address its biological significance, we analyzed the effects of HNF1B by knockdown (KD) experiments and revealed that HNF1B is biologically indispensable for IPMN lineages. We further identified MNX1 as an upstream regulator of HNF1B expression, another TF expressed in multipotent pancreatic progenitor cells. ChIP experiment revealed the enriched binding of MNX1 on the promoter elements of HNF1B in invasive IPMN. Importantly, KD or CRISPR interference of MNX1 impaired the survival of the organoids from invasive IPMN. Moreover, the correlated and high expression patterns of MNX1 and HNF1B in IPMN lineages were validated in a set of human tissues. To identify the common downstream genes of MNX1 and HNF1B, we analyzed RNA-seq and ATAC-seq after KD of the two genes. We found that MNX1-HNF1B axis governed a set of genes including MYC, SOX9, and OLFM4, which are known to regulate stem cell properties in gastrointestinal epithelium. Lastly, to get a broader view of chromatin architecture in these tumors, we performed Hi-C. We found that HNF1B target genes to be three-dimensionally condensed in the genome of invasive IPMN but not in that of PDAC, supporting the functional importance of those genes in invasive IPMN. Collectively, our organoid analyses unraveled the different lineage related chromatin structures correlating to the specific biological behaviors in pancreatic tumor subtypes. Citation Format: Hiroyuki Kato, Keisuke Tateishi, Keisuke Yamamoto, Dousuke Iwadate, Hiroaki Fujiwara, Takuma Nakatsuka, Koji Miyabayashi, Yotaro Kudo, Ijichi Hideaki, Kazuhiko Koike, Mitsuhiro Fujishiro. A multi-omics study in patient-derived organoids reveals MNX1-HNF1B axis to be indispensable for intraductal mucinous papillary neoplasm lineages [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-067.

Clinical Characteristics and Magnetic Resonance Imaging–Based Prediction of the KLF4K409Q Mutation in Meningioma

Meningioma is the most common primary brain tumor in adults. In recent years, several non-neurofibromin 2 mutations, i.e., AKT1, SMO, TRAF7, and KLF4 mutations, specific for meningioma have been identified. This study aims to analyze the clinical impact and imaging characteristics of the KLF4K409Q mutation in meningioma.Clinical, neuropathologic, and imaging data of 170 patients who underwent meningioma resection between 2013 and 2018 were retrospectively collected and tumors were analyzed for the presence of the KLF4K409Q mutation. We collected imaging characteristics, performed volumetric analysis of tumor size and peritumoral edema (PTBE), and calculated the edema index (EI, i.e., ratio of PTBE to tumor volume). Receiver operating characteristic curve analysis was performed to identify cut-off EI values to predict the mutational status of KLF4.Eighteen (10.6%) of the meningiomas carried the KLF4K409Q mutation; these were significantly associated with a secretory subtype (P < 0.001) and sphenoid wing location (P = 0.029). Smaller tumor size (P = 0.007), an increased PTBE (P = 0.012), and an increased EI (P = 0.001) proved to be significantly associated with the KLF4K409Q mutation. In receiver operating characteristic curve analysis, EI predicted the KLF4K409Q mutation with an area under the curve of 0.728 (P = 0.0016).The KLF4K409Q mutation is associated with a distinct small tumor subtype, prone to substantial PTBE. EI is a reliable parameter to predict the KLF4K409Q mutation in meningioma, thus providing a tool for improvement of pre- and perioperative medical management.

Loss-of-Function Mutations in TRAF7 and KLF4 Cooperatively Activate RAS-Like GTPase Signaling and Promote Meningioma Development

Meningiomas are the most common benign brain tumors. Mutations of the E3 ubiquitin ligase TRAF7 occur in 25% of meningiomas and commonly cooccur with mutations in KLF4, yet the functional link between TRAF7 and KLF4 mutations remains unclear. By generating an in vitro meningioma model derived from primary meningeal cells, we elucidated the cooperative interactions that promote meningioma development. By integrating TRAF7-driven ubiquitinome and proteome alterations in meningeal cells and the TRAF7 interactome, we identified TRAF7 as a proteostatic regulator of RAS-related small GTPases. Meningioma-associated TRAF7 mutations disrupted either its catalytic activity or its interaction with RAS GTPases. TRAF7 loss in meningeal cells altered actin dynamics and promoted anchorage-independent growth by inducing CDC42 and RAS signaling. TRAF deficiency-driven activation of the RAS/MAPK pathway promoted KLF4-dependent transcription that led to upregulation of the tumor-suppressive Semaphorin pathway, a negative regulator of small GTPases. KLF4 loss of function disrupted this negative feedback loop and enhanced mutant TRAF7-mediated cell transformation. Overall, this study provides new mechanistic insights into meningioma development, which could lead to novel treatment strategies. SIGNIFICANCE: The intricate molecular cross-talk between the ubiquitin ligase TRAF7 and the transcription factor KLF4 provides a first step toward the identification of new therapies for patients with meningioma.

Multiregion whole-exome sequencing of intraductal papillary mucinous neoplasms reveals frequent somatic KLF4 mutations predominantly in low-grade regions

ObjectiveIntraductal papillary mucinous neoplasms (IPMNs) are non-invasive precursor lesions that can progress to invasive pancreatic cancer and are classified as low-grade or high-grade based on the morphology of the neoplastic...

New molecular targets in meningiomas: the present and the future.

Meningiomas, the most common primary brain tumor, have historically been managed with surgery and radiation. Traditional chemotherapy has not been effective. Fortunately, recent advances in genetic sequencing have led to an improved understanding of the molecular drivers in meningioma. This article aims to discuss the diagnostic and therapeutic implications of recently discovered genetic alterations in meningiomas. Many of the recently discovered genetic alterations correlate with distinct clinical phenotypes. SMO, AKT and PIK3CA mutations are enriched in the anterior skull base. KLF4 mutations are specific for secretory histology, and BAP1 alterations are common in progressive rhabdoid meningiomas. Alterations in TERT, DMD and BAP1 correlate with poor clinical outcomes. Importantly, the discovery of clinically actionable alterations in a number of genes, including SMO, AKT1 and PIK3CA, has opened up novel potential avenues for therapeutic management of meningiomas. Overexpression of PD-L1 in higher grade meningiomas also provides preclinical support for the investigation of checkpoint blockade. The discovery of genetic alterations has improved our understanding of the natural history and classification of meningiomas. Clinical trials with several novel agents targeting driver mutations are currently accruing patients and they can lead to better treatment strategies.

Frequent AKT1E17K mutations in skull base meningiomas are associated with mTOR and ERK1/2 activation and reduced time to tumor recurrence.

Skull base meningiomas are considered to be difficult for surgical treatment. We wondered whether genetic alterations recently identified in benign non-NF2-mutated World Health Organization (WHO) grade I meningiomas are related to clinical features of skull base meningiomas and whether druggable signaling pathways are activated. We analyzed 93 skull base meningiomas (82 WHO grade I, 11 WHO grade II) for mutations of hot spots or the most relevant exons of AKT1, KLF4/TRAF7, SMO, PI3K, and the TERT promoter. The AKT1E17K mutation was present in 31% of patients and was related to meningothelial histology. AKT1E17K had a negative effect on the time to tumor recurrence. Analyses of activated signaling proteins revealed among AKT1E17K tumors a significantly higher rate of phospho-mammalian target of rapamycin (mTOR) and phospho-p70S6K+ tumors. AKT1E17K tumors with immunoexpression of phospho-extracellular signal-regulated kinase 1 or 2 (ERK1/2) were characterized by significantly shorter time to tumor recurrence compared with AKT1wt tumors expressing phospho-ERK1/2 (P = .046). KLF4 mutations (K409Q) were present in 11.8% of cases, with significant association to the secretory/transitional subtype (P < .001). The presence of the KLF4 K409Q mutation was associated with favorable outcome. One phosphatidylinositol-3 kinase (PI3K) mutation but no SMO or TERT promoter mutation was found. AKT1E17K mutation is frequent in skull base meningiomas, results in activation of the mTOR and ERK1/2 signaling pathways, and has negative impact on tumor recurrence. Patients with skull base meningiomas with AKT1E17K mutation might benefit from additional treatment targeting the mTOR pathway. Generally, the PI3K-Akt-mTOR axis might be a potential target for kinase inhibitors in these tumors.

MNGO-21GENOTYPING OF MENINGIOMA BY TARGETED AMPLICON SEQUENCING USING MiSeq

BACKGROUND: Meningiomas are the most common primary brain tumors, accounting for about 27% of all intracranial tumors. Loss of Neurofibromin 2 (NF2) is found in about half of sporadic meningiomas, and recently, mutations in TRAF7, KLF4, AKT1, and SMO are reported in non-NF2 meningioma. To establish practical genotyping methods, we performed targeted amplicon sequencing with 45 meningioma specimens and evaluated the association with clinicopathological features. METHODS: Frozen samples of 45 meningiomas were fixed with PAXgene Tissue System (QIAGEN) and embedded in paraffin (PFPE). Amplicon sequencing panel targeted NF2, TRAF7, KLF4, AKT1 and SMO were designed by GeneRead Mix-n-Match panel and Custom panel (QIAGEN). Genomic DNA was extracted from PFPE tissues and libraries were prepared using GeneRead DNAseq Targeted Panels V2 (QIAGEN). The libraries were sequenced by the MiSeq (Illumina). The raw read data obtained from amplicon sequencing were processed by GeneRead Panel Variant Calling service for analysis of mutations and copy number alterations (CNA). RESULTS: Thirty three cases (73%) harbored NF2 loss, in which 20 cases (61%) simultaneously carried NF2 mutation. Mutations in TRAF7, KLF4, AKT1, and SMO were observed in 6 (13 %), 4 (9 %), 2 (4 %) and 0 (0%) cases, respectively. Altogether, 40 of 45 case (89 %) carried at least one or more gene alteration including mutations in 5 genes listed above and NF2 loss. Result of genotyping was correlated with histological subtypes as follows. All of nine fibrous meningiomas carried NF2 loss, while all of 4 meningiomas with secretory component carried both TRAF7 and KLF4 mutations. A case of chordoid meningioma harbored only AKT1-E17K mutation. CONCLUSIONS: Here we established the clinical sequence system for meningioma by targeted amplicon sequencing with desktop sequencer in which we demonstrated favorable results for CNAs as well as mutations.

Secretory meningiomas are defined by combined KLF4 K409Q and TRAF7 mutations

Meningiomas are among the most frequent intracranial tumors. The secretory variant of meningioma is characterized by glandular differentiation, formation of intracellular lumina and pseudopsammoma bodies, expression of a distinct pattern of cytokeratins and clinically by pronounced perifocal brain edema. Here we describe whole-exome sequencing analysis of DNA from 16 secretory meningiomas and corresponding constitutional tissues. All secretory meningiomas invariably harbored a mutation in both KLF4 and TRAF7. Validation in an independent cohort of 14 secretory meningiomas by Sanger sequencing or derived cleaved amplified polymorphic sequence (dCAPS) assay detected the same pattern, with KLF4 mutations observed in a total of 30/30 and TRAF7 mutations in 29/30 of these tumors. All KLF4 mutations were identical, affected codon 409 and resulted in a lysine to glutamine exchange (K409Q). KLF4 mutations were not found in 89 non-secretory meningiomas, 267 other intracranial tumors including gliomas, glioneuronal tumors, pituitary adenomas and metastases, 59 peripheral nerve sheath tumors and 52 pancreatic tumors. TRAF7 mutations were restricted to the WD40 domains. While KLF4 mutations were exclusively seen in secretory meningiomas, TRAF7 mutations were also observed in 7/89 (8 %) of non-secretory meningiomas. KLF4 and TRAF7 mutations were mutually exclusive with NF2 mutations. In conclusion, our findings suggest an essential contribution of combined KLF4 K409Q and TRAF7 mutations in the genesis of secretory meningioma and demonstrate a role for TRAF7 alterations in other non-NF2 meningiomas.