Methylation Profiling of Specific Genes in Ependymomas
Naz KANIT1, Pelin YALCIN2, Serhat ERBAYRAKTAR3, Erdener OZER4
1Department of Molecular Medicine, Dokuz Eylul University Institute of Health Sciences, IZMIR, TURKEY
2Department of Medical Biology, Dokuz Eylul University Institute of Health Sciences, IZMIR, TURKEY
3Department of Neurosurgery, Dokuz Eylul University School of Medicine, IZMIR, TURKEY
4Department of Pathology, Dokuz Eylul University School of Medicine, IZMIR, TURKEY
Keywords: DNA methylation, Ependymoma, Epigenetics, Pyrosequencing
Ependymomas are neuroepithelial tumors of the central nervous system with heterogeneous biology and clinical course. The aim of
the present study is to investigate the relationship between the methylation status and clinicopathological parameters in ependymomas.
Material and Method: DNA methylation status of CDKN2A, RASSF1A, KLF4 and ZIC2 genes were quantitatively analyzed with pyrosequencing
in 44 ependymoma tumor tissues. The relationship of methylation profiles with tumor subtype, histological grade and patient age was statistically
Results: DNA methylation analyses for CDKN2A revealed no difference in methylation levels. Of the 31 included samples for optimal ZIC2
methylation analysis, 10 were hypermethylated (32.3%) and this change was significantly found in the adult spinal ependymomas (p=0.01).
KLF4 hypermethylation was observed in 6 of the overall included 35 samples (17.1%); however, there was no statistically significant relation of
the methylation status with tumor subtype, histological grade or age group. RASSF1A hypermethylation was observed in overall 40 included
samples with varying methylation levels. Higher levels of hypermethylation were significantly related to the grade 3 histology (p=0.01) and
spinal ependymomas (p=0.006). The pediatric cases with grade 3 ependymomas and ependymomas of adulthood showed significantly increased
RASSF1A hypermethylation levels (p<0.001 and p=0.001, respectively).
Conclusion: DNA methylation changes are likely to have biological importance in ependymomas. Both ZIC2 and RASSF1A methylation status
may be useful parameters in the subclassification of these tumors.
Ependymomas are neuroepithelial tumors of the central
nervous system (CNS) located both in the brain and spinal
. They are often seen at pediatric ages and demonstrate
low survival rates 2
. The grading system basically depends
on the histology of the tumor; classical ependymomas
are considered as grade 2, whereas ependymomas with
anaplastic histology are classified as grade 3 tumors.
However. the term “anaplastic ependymoma” is no longer
used in the 2021 WHO classification of CNS tumors. The
current classification divides ependymomas into molecular
groups with the supratentorial, posterior fossa, and spinal
anatomic location. When molecular analysis fails or is
unavailable, the NOS suffix should be used 3
Ependymoma is a very genetically heterogeneous cancer,
and even though a number of whole genome sequencing
and global DNA methylation profiling studies have
focused on the prognosis and predicting positive outcome
for the patients, an epigenetic changes-based risk
classification has not yet been established 4,5. In a recent
landmark study by Pajtler et al. 5 using a global DNA
methylation analysis for 500 cases, three major groups of supratentorial, infratentorial and spinal were defined
according to the location of the tumor where each group
has three subgroups. However, they did not determine
any specific marker for each subgroup. Consequently,
all ependymomas are managed with standard treatment,
which causes serious side effects for many of the patients
3. This situation underlines the need for determination
of molecular parameters, which can be used as prognostic
markers in the precision medicine and potential druggable
Studies on the molecular oncogenesis of ependymomas
are basically based on the changes of DNA methylation
patterns. In the present study, we aimed to investigate
the methylation status of the RASSF1A, CDKN2A, KLF4
and ZIC2 genes and to address the relationship of the
methylation profiling of the selected genes with tumor
subtype, histological grade or age group, as well as to
highlight their significance in precision medicine.
The present study enrolled archival formalin-fixed
paraffin-embedded ependymoma tumor tissues of 44
patients treated in our institution from 2000 to 2020 after obtaining approval in accordance with the regulations of
the local institutional ethical committee. The clinical and
demographic data of the corresponding patients including
tumor site, age and gender were obtained from electronic
records. The inclusion criteria were sufficient formalinfixed
paraffin-embedded (FFPE) archival tumor tissue and
available clinical data including age and tumor localization.
Original hematoxylin and eosin (H&E) stained histological
slides of the tumors were reviewed by an expert pathologist
(E.O.) to confirm the histological diagnosis of ependymoma
and select the optimal tissue block for further molecular
analyses. Ependymomas were graded and classified
according to the 2021 WHO classification.
DNA methylation status of the CDKN2A, RASSF1A,
KLF4 and ZIC2 genes were quantitatively analyzed
with pyrosequencing. Each step for DNA isolation and
bisulfite conversion was performed as instructed in the
manufacturer’s protocol. Ten 5μ sections from each tumor
block were prepared to isolate total genomic DNA using
the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany).
The bisulfite conversion of the isolated genomic DNA
of 20 mL volume for each case was carried out using the
EpiTect Bisulfite kit (Qiagen, Germany) to measure the
DNA methylation. The bisulfite treated and commercially
available control DNAs (EpiTect PCR Control DNA Set,
Qiagen, Germany) were used as templates for polymerase
chain reaction (PCR) (PyroMark PCR Kit, Qiagen,
Germany). The target sequences were amplified using
specific biotinylated primers (Cat. no. PM00139993,
PM00013293, PM00039550 and PM00054131; PyroMark
CpG Assay, Qiagen, Germany). PCR products were
then used to generate single stranded DNAs of the
amplified sections and captured with streptavidin-coated
sepharose beads in corresponding buffers of PyroMark
Q24 Advanced CpG Reagents (Qiagen, Germany). The
remainders were washed in the PyroMark Q24 workstation
(Qiagen, Germany). The single stranded DNAs were
incubated with the pyrosequencing primers and sequenced
in the PyroMark Q24 platform (Qiagen, Germany). The
methylation levels were quantified as T to C percentages
and the average values of each CpG site methylation ratios
were calculated. Average values below 8% were considered
as unmethylated 6,7. The methylation levels of RASSF1A
were grouped into four quartiles as suggested in a previous
study, where methylation levels less than 45.3% were
named as Q1, those between 45.3% and 55.08% as Q2,
those between 55.08% and 61.42% as Q3, and those higher
than 61.42% as Q4 8.
Statistical analysis was performed with the IBM SPSS
Statistics v.22 software in order to display the relation
between the methylation status and clinicopathological
data using the chi-squared test. The probability level of 0.05
or less was chosen to represent statistical significance. All
p-values were two-sided and denoted by p. Fisher’s exact
test was used to calculate p values, as the cell frequencies
were too small for the standard chi-squared test to be
Overall 44 ependymoma tumor tissues were analyzed in the
present study. They were non-recurrent tumors and totally
excised. Twenty patients were female (45.5%) and 24 were
male (54.5%), ranging from 1 to 74 years old (mean: 25.5
±20.9), and including 20 pediatric (45.5%) and 24 adult
(54.5%) cases. Based on tumor classification, there were
three groups: supratentorial ependymoma, NOS (n=8,
18.2%), posterior fossa ependymoma, NOS (n=19, 43.2%),
and spinal ependymoma, NOS (n=17, 38.6%). Grade 2
histology was observed in 56.8% of the cases (n=25) and the
remaining cases showed grade 3 histology (n=19, 43.2%).
The pyrosequencing method was utilized to analyze DNA
methylation levels of the four selected genes (Figure 1)
and the samples with weak signals were excluded. For the
CDKN2A methylation analysis, 18 samples were included
in the study, and hypermethylation was not detected in
any case. Of the 31 included samples for optimal ZIC2
methylation analysis, 10 were hypermethylated (32.3%)
and this change was significantly found in the adult spinal
ependymomas, NOS (p=0.01). KLF4 hypermethylation was
observed in six of the overall included thirty-five samples
(17.1%), however, there was no statistically significant
relation of the methylation status with tumor subtype,
grade or age group. RASSF1A methylation was observed
in overall 40 included samples with varying methylation
levels; therefore, four quartiles representing the mean
methylation values were determined, and demonstrated in
Table I. Higher levels of hypermethylation were significantly
related to the grade 3 histology (p=0.01) and spinal
ependymomas, NOS (p=0.006). The pediatric cases with
grade 3 ependymomas and adults with spinal ependymoma,
NOS showed significantly increased hypermethylation
levels (p<0.001 and p=0.001, respectively).
Click Here to Zoom
|Figure 1: Pyrograms for the target sequences of the CDKN2A, ZIC2, KLF4 and RASSF1A genes.
Click Here to Zoom
|Table I: RASSF1A methylation levels in quartiles and their frequencies related to the histological grade, tumor subtype, and age group.
The methylation of RASSF1A, HIC1 and CDKN2A are
the most common epigenetic changes demonstrated in
. Other genes such as MGMT,
BLU, GSTP1, DAPK, FHIT, MGMT, MCJ, RARB, TIMP3, THBS1, TP73, CASP8, TFRSF10C and TFRSF10D have also
been shown with altered DNA methylation patterns, yet
these changes have not been correlated to clinical setting
. In the present study, we analyzed the gene-specific
methylation profiles of the CDKN2A, RASSF1A, KLF4 and ZIC2 genes in the ependymoma tumor tissues. The
RASSF1A and CDKN2A genes were selected for analysis
in this study since methylations of these genes were
previously reported in ependymomas 1,10
. In a previous
study, lower expression levels of ZIC2 were reported in spinal ependymomas in comparison to intracranial
. We therefore investigated the
relationship of this genetic change with DNA methylation.
On the other hand, we selected the KLF4 gene because it
is widely expressed in neural stem cells, some of which are
shown in the ependymomas, and plays a significant role
in their self-renewal, also involved in reprogramming of
somatic cells to pluripotency 14
. In the present study,
DNA methylation analyses were performed using bisulfitebased
pyrosequencing, which is the gold standard method
. We found specific DNA methylation patterns
exhibiting a relationship with age groups, tumor subtype
and grade 3 histology.
The genetic and epigenetic basis of the oncogenesis
of ependymomas has been under scrutiny for the last
decade. The molecular markers that are associated with
the subgroups of ependymomas or related to the patient
age are not yet well known. To the best of our knowledge,
intracranial and spinal ependymomas are likely to have
different genetic and epigenetic signatures. A metaanalysis
by Lee et al. 16 has revealed that NF2 mutations
were associated with spinal ependymomas whereas HIC1
methylation and EPB41L3 deletions were common in
The 2021 WHO classification of CNS tumors divides
ependymomas into molecular groups with the
supratentorial, posterior fossa, and spinal anatomic
location. It also lists genetically defined subtypes of
supratentorial, posterior fossa and spinal ependymomas.
When molecular analysis fails or is unavailable, the NOS
suffix should be used, as we did in our study. In addition,
according to the current classification, a pathologist can
still grade the ependymomas as either CNS WHO grade 2
or grade 3; however the term “anaplastic ependymoma” is
no longer listed 3.
In the present study, we analyzed the DNA methylation
levels of CDKN2A, which is a tumor suppressor gene,
and widely modified in a range of cancers 17-20. Highgrade
gliomas display decreased CDKN2A expression
levels 21, whereas CDKN2A mutations were observed
in the recurrent meningiomas 22. Although the DNA
methylation analysis revealed unmethylated status for
CDKN2A in all ependymoma cases, there are contrasting
results in the literature. In a study with 123 cases, Rousseou
et al. (10) revealed that 21% of all ependymomas showed
CDKN2A promoter methylations, whereas no methylation
was shown in another study similar to our study 23. We
therefore think that further research is needed in order
to address the importance of promoter methylations of
CDKN2A in ependymomas.
ZIC2 is another gene that has been previously shown to
regulate the development of neural tissues and its mutation
and downregulation have been linked to neurodevelopment
24. Alterations of this gene have been found to have a
crucial function in pediatric medulloblastoma pathogenesis
25, as well as in a broad spectrum of cancers (26-28). In
this study, we showed that the ZIC2 gene is hypermethylated
significantly in adult spinal ependymomas. Kim et al. 13
have reported that ZIC2 expression was downregulated
in the spinal ependymomas; however, the mechanism
underlying its downregulation, which is likely to be due
to DNA methylations, was not specifically analyzed.
Although the number of hypermethylated cases in our
study is limited, the association between ZIC2 methylations
and spinal ependymomas in adulthood may be a potential
mechanism of oncogenesis.
KLF4 encodes a tumor suppressor and its methylation can
lead to silencing of the gene, which has been reported in
variety of cancers such as pancreatic cancer, leukemia,
colorectal cancer, and medulloblastoma 29-32. We
analyzed herein the methylation levels of KLF4 and
17.1% of our ependymoma tissues were hypermethylated.
However, we did not observe a significant relationship of
KLF4 hypermethylation with tumor location, patient age
or histology of the tumor. We think that the methylation
status of KLF4 may play a role in the oncogenesis of
ependymomas but further studies with wider series are
needed to address whether KLF4 hypermethylation can be
an epigenetic signature.
Hypermethylation of the RASSF1A promoter is one of the
most common molecular changes in ependymomas 1. In
this study, varying hypermethylation levels of this gene were
analyzed in terms of quartiles in order to define a cut-off in
which the methylation has a significant relationship with the
prognosis. Higher RASSF1A methylations were observed
in pediatric grade 3 ependymomas, and it may therefore be
related to a worse prognosis. Adult spinal ependymomas
showed significantly increased RASSF1A methylation
levels. These results may explain why ependymomas
are genetically subclassified depending on anatomic
localization. Although epigenetic subclassification of
ependymomas is not yet fully established, we underline the
potential importance of RASSF1A promoter methylations
as a molecular marker for classification.
Current cancer research is mainly focused on discovering
biomarkers, which can potentially be targets for drug
selection or to stratify the patients according to the risk
categories. Although there are some important limitations
in our study such as the limited number of positive cases and insufficient prognostic follow-up data, we think DNA
methylation changes could have a biological significance
in ependymomas. Both ZIC2 and RASSF1A methylation
status may be useful parameters in the subclassification of
ependymomas. In addition, RASSF1A hypermethylation
level may play a biological role in pediatric ependymomas,
also a candidate druggable target changes in grade 3
histology. Because current understanding of ependymomas
is limited and there is much need to discover the epigenetic
setting of the disease, we think that further studies with wider
series will provide sufficient knowledge for the epigenetic
changes-based subclassification of ependymomas.
CONFLICT of INTEREST
None of the authors have any conflict of interest
Concept: NK, EÖ, Design: NK, EÖ, Data collection or
processing: NK, PY, SE, EÖ, Analysis or Interpretation:
NK, PY, Literature search: NK, Writing: NK, EÖ, Approval:
NK, PY, SE, EÖ.
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