This study analyzed the genomic profiles of 300 non-NF2 meningiomas and identified mutations in TRAF7, KLF4, AKT1, and SMO genes in addition to NF2 mutations. These mutations were mutually exclusive and defined distinct meningioma subtypes. Meningiomas with NF2 mutations were located in the cerebral hemispheres, while non-NF2 tumors originated from the skull base. Genomic analyses also revealed differences in chromosomal instability, gene expression, and histone modification between NF2 and non-NF2 subtypes. Identifying these genomic characteristics may help predict tumor behavior and inform targeted therapy approaches.

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

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

3. - 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

4. - 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".

5. - 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

6. - 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)

7. Back to meningiomas

8. - 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

9. - Unknown.
Cause

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

12. - 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.
- Method
To 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

13. - 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

14. - 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

15. - 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

16. - 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

17. - 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

18. - 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

19. - 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

20. 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.

21. 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.

22. 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.

23. 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

24. 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

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