Genomic Analysis of Non-NF2 Meningiomas Reveals Mutations in TRAF7, KLF4, AKT1, and SMOKadir Sumerkent Department of Neuroscience, Graduate School of Health Sciences Bahcesehir University

i. Introduction ii. Epidemiology iii. Cause iv. Research v. Findings and Result

Agenda

- Meningiomas, arising from the meninges of the central nervous system, are the most common primary brain tumors, with a prevalence of ~170,000 cases in the United States.

- Meningiomas frequently invade surrounding brain and critical neurovascular structures, often causing neurological deficits and requiring surgical inter- vention.

- They are thought to arise from arachnoidal cap cells, which reside in the arachnoid layer covering the surface of the brain.

Introduction

- Loss of Neurofibromin 2 (merlin, NF2) is found in 40 to 60% of meningiomas, but the genetic architecture of the remainder remains obscure, limiting options for the development of rational therapies.

- Usually bening Although most are histologically classified as benign (grade I), about 10% represent atypical (grade II) or anaplastic (grade III) forms.

- Meningioma was first coined by Harvey Cushing (1922)

Introduction

Merlin (also called Neurofibromin 2 or schwannomin) is a cytoskeletal protein. In humans, it is a tumor suppressor protein involved in Neurofibromatosis type II. Sequence data reveal its similarity to the ERM protein family.

The name "merlin" is an acronym for "Moesin-Ezrin-Radixin-Like Protein".

- Tumor grade is the description of a tutor based on how abnormal the tutor cells and the tutor tissue look under a microscope.

- It is an indicator of how quickly a tutor is likely to grow and spread. If the cells of the tutor and the organisation of the tumor’s tissue are close to those of normal cells and tissue, the tumor is called “well-differentiated”. - These tumors tend to grow and spread at a slower rate than tumors

that are “undifferentiated” or “poorly differentiated”, which have abnormal looking cells and may lack normal tissue structures.

- Tumor grade is not the same as the stage of a cancer. Cancer stage refers to the size and/or extend (reach) of the original (primary) tutor and whether or not cancer cells have spread in the body. - Cancer stage is based on factors such as the location of the primary

tumor, tumor size, regional lymph node involvement (the spread of cancer to nearby lymph nodes), and the number of tumors present.

Tumor grades

- Grading systems differ depending on the cancer. In general, tumors are graded as 1, 2, 3, or 4, depending on the amount of abnormality.

- In Grade 1 tumors, the tumor cells and the organisation of the tumor tissue appear close to normal. These tumors tend to grow and spread slowly. In contrast, the cells and the tissue of Grade 3 and Grade 4 tumors do not look like normal cells and tissue. Grade 3 and Grade 4 tumors tend to grow rapidly and spread faster than tumors with a lower grade. General grading system (unless a grading system for a tumor type is specified)

Tumor grades

- GX: Grade cannot be assessed (undetermined grade)- G1: Well differentiated (low grade)- G2: Moderately differentiated (intermediate grade)- G3: Poorly differentiated (high grade)- G4: Undifferentiated (high grade)

Back to meningiomas

- 2-10 cases per 100.000 individuals.

- Account for approx. %20 of all primary intracranial neoplasms. - Majority are bening, with about 1% - 3% classified

as malignant. - 98% are intracranial but may arise anywhere in

central nervous system. - 2.3% of individuals have

undiagnosed asymptomatic meningioma on autopsy.

- Women affected twice as often men.

- Incidence increases with age.

Epidemiology

- Unknown.Cause

Research

Science, 1 March 2013, Vol. 339, Page: 1077

- Problem Loss of Neurofibromin (NF2, Merlin) is found in 40 to 60% of sporadic meningiomas but the genetic architecture of the remainder remains obscure, limiting options for the development of rational therapies.

- MethodTo comprehensively characterise the genomics of meningioma and to gain further in sight into molecular mechanisms of tumor formation, we performed genome-wide genotyping and exam sequencing of 50 nonirradiated grade I and grade II meningiomas and matched normal DNA. For the meningiomas which matching blood samples were available, the mean number of protein-altering somatic mutations was 7.2 (range 1 to 15), a considerably smaller number compared with malignant tumors.

Research

- Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents.

Genotyping

- Exome sequencing, also known as whole exome sequencing (WES or WXS), is a technique for sequencing all of the expressed genes in a genome (known as the exome).

- It consists of two steps: the first step is to select only the subset of DNA that encodes proteins. These regions are known as exons - humans have about 180,000 exons, constituting about 1% of the human genome, or approximately 30 million base pairs.

Exome sequencing

- The second step is to sequence the exonic DNA using any high-throughput DNA sequencing technology.

- The goal of this approach is to identify genetic variants that alter protein sequences, and to do this at a much lower cost than whole-genome sequencing.

- Since these variants can be responsible for both Mendelian and common polygenic diseases, such as Alzheimer's disease, whole exome sequencing has been applied both in academic research and as a clinical diagnostic.

Exome sequencing

- Method contd. We next searched for genes with significantly more somatic mutations than expected by chance. Besides, NF2, were identified increased mutation burden in TNF receptor-associated factor 7 (TRAF7), Krupple-like factor 4 (KLF4), v-akt murine murine thymoma viral oncogene homolog 1 (AKT1), and Smoothened, frizzled family receptor (SMO) (as a group, refereed to as non-NF2 mutant hereafter). Mutations in these genes were mutually exclusive NF2 mutations.

- In addition, we identified single mutations in genes previously reported to play a role in other neoplasia, including CREBBP, PIK3CA and BRCA1 as well as two SMARCB1 mutations, which coexisted with NF2 loss and have previously been reported in meningiomas.

- We next performed targeted resequencing of these top five genes, along with chromosome 22 copy-number analysis, in an independent set of 250 unradiated meningiomas (204 grade I and 46 high grade).

Research

- Findings- In the combined analsis of 300 meningiomas, we identified coding

mutations in one of these five genes and/or evidence for chromosome 22 loss in 237 (79%).

- NF2 mutations were present in 108 (36%). - TRAF7 mutations, which were always exclusive of NF2 mutations

were observed in nearly one-fourth of the meningiomas examined. - In the transcription factor KLF4, we identified a recurrent K409Q

mutation, which almost always co-occured with TRAF7 mutations and were exclusive of NF2 mutations. KLF4 is expressed in meningiomas.

- The known neoplasia-related recurrent mutation, AKT1E17K was identified in 38 meningiomas. Although the AKT1E17K co-occured with TRAF7 mutations in 25 of 38 tumors, it was exclusive of the KLF4K409Q and NF2 mutations except in one case.

- Finally, in 11 tumors, we identified mutations in SMO, which is expressed in meningiomas.

Research

- Method contd. We next evaluated chromosomal instability. Chromosome 22 loss, observed in 149 tumors, was the most common event and was strongly associated with the presence of coding NF2 mutations. These were also significantly associated with higher grade meningiomas. Higher grade tumors also showed an increased number of large scale chromosomal abnormalities and an increased rate of NF2 mutations and were observed more frequently in males than females. Given these observations pointing to distinct tumor subtypes based on mutation profiles, we examined the mutation spectrum correlated with anatomical distribution and histological subtype. We initially grouped cerebral meningiomas into those originating along the skull base or those present in the cerebral hemispheres.

Research

- Interestingly, tumors with NF2 mutations and/or chromosome 22 loss were predominantly found in the hemispheres with nearly all posterior cerebral (parieto-occipital), cerebellar, or spinal meningiomas being NF2/chr22loss tumors. For meningiomas originating from the skull base, we observed a difference between those originating from medial versus lateral regions. The vast majority of non-NF2 meningiomas were medial, whereas the lateral and posterior skull base meningiomas had NF2/chr22loss. Meningiomas with only the recurrent SMO L412F mutation all located to the medial anterior skull base, near the midline. This is particularly interesting because mutations in hedgehog singling result in holoprosencephaly, the midline failure of embryonic forebrain to divide into two hemispheres.

Research

Genomic architecture of meningiomas.

A) NF2, TRAF7, and SMO coding mutations along with recurrent AKT1 E17K and KL4 k409Q variants reveal meningioma subtypes with mutually exclusive profiles. Analysis for chromosome 22 copy number is also shown.

B) TRAF7 mutations, which are identified in 72 of 300 meningiomas analyzed, are clustered with its WD40 domains. The count of recurrent mutations, which are denoted by diamonds is indicated.

C) The recurrent KLF4 K409Q mutation is located within the first zinc finger domain, which makes direct DNA contact.

Genomic architecture of meningiomas.

D) Circos plot of large-scale genomic abnormalities identified (blue: deletion, red: amplification). Whereas all NF2/chr22loss meningiomas (outer circles, n=41, including n=30 with coding NF2 mutations) show chromosome 22 loss, which is typically associated with further chromosomal abnormalities in grade II tumors (n=11, including n=8 with coding NF2 mutations), genomic stability is a hallmark of grade I non-NF2 tumors (inner circles, n=36).

E) Along the skull base, NF2/chr22loss meningiomas originate from the lateral and posterior regions, whereas the vast majority of anterior and medial meningiomas are non-NF2 mutant.

Genomic architecture of meningiomas.

F) Unsupervised hierarchical clustering of gene expression profiles defines two major bening meningioma sub groups, those with NF2/chr22loss and non-NF2 mutant tumors. Each subgroup reveals differential H3K27ac and gene expression profiles.

Consistent with these clinical observations, unsupervised hierarchical clustering of meningiomas based on gene expression and chromatin immunoprecipitation for H3K27 acetylation followed by sequencing analyses confirmed clustering into NF2/chr22loss versus non-NF2 mutant subgroups and revealed several molecules whose acetylation and expression was specific to a subtype. For these differentially expressed genes, there was a strong correlation between expression and ChIP-seq data. Among the non-NF2 meningiomas, SMO mutants were clearly defined by increased expressions and activation of the Hedgehog pathway.

Research

These results clearly identify meningioma sub groups, distinguishing them based on their mutually exclusive distribution of mutations, distinct potential for chromosomal instability and malignancy, anatomical location, histological appearance, gene expression and H3K27ac profile.

Our results show that the mutational profile of a meningioma can largely be predicted based on its anatomical position, which in turn may predict likely drug response. This may prove relevant for surgically unresectable, recurrent, or invasive meningiomas and could spare patients surgery or irradiation, and independent risk factor for progression of these generally bening tumors.

Results

Genomic Analysis of Non-NF2 Meningiomas Reveals Mutations in TRAF7, KLF4, AKT1, and SMOKadir Sumerkent Department of Neuroscience, Graduate School of Health Sciences Bahcesehir University