2016, Volume 32, Number 2, Page(s) 070-081
Prognostic Value of ALDH1, EZH2 and Ki-67 in Astrocytic Gliomas
Shimaa AHMED1, Hayam RASHED1, Abdelmonem HEGAZY2, Abdel Motaleb MOHAMED3, Wael ELMESALLAMY4
1Department of Pathology, Zagazig University, Faculty of Medicine, ZAGAZİG, EGYPT
2Department of Anatomy and Embryology, Zagazig University, Faculty of Medicine, ZAGAZİG, EGYPT
3Department of Clinical Oncology and Nuclear Medicine, Zagazig University, Faculty of Medicine, ZAGAZİG, EGYPT
4Department of Neurosurgery, Zagazig University, Faculty of Medicine, ZAGAZİG, EGYPT
Keywords: Astrocytoma, Immunohistochemistry, ALDH1, EZH2, Ki-67
Tumor stem cells have been found in a variety of neoplasms and stated to have a role in tumor progression. This study aimed to
evaluate the prognostic significance of biomarkers which are said to be related to these cells, i.e., EZH2, ALDH1 and Ki-67, and their correlation
with each other in astrocytic gliomas.
Material and Method: Formalin-fixed, paraffin-embedded tissue specimens of 40 patients with astrocytic glioma who underwent initial surgery
during the period from December 2011 to May 2014 at Zagazig University Hospitals were enrolled in the study. Consecutive 4-μm thick sections
from formalin-fixed, paraffin-embedded tissue blocks were prepared and stained with hematoxylin and eosin for histopathological evaluation.
Immunohistochemical analysis using ALDH1, EZH2 and Ki-67 antibodies were performed to examine the cases.
Results: A total of forty patients; 22 males and 18 females were studied. The lesions were classified as follows: 14 cases of low-grade astrocytoma
(WHO grade I or II), 11 cases of anaplastic astrocytoma (WHO grade III), and 15 glioblastomas (WHO grade IV). There was a significant
increase in ALDH1 immunoreactivity with increasing the grade of astrocytoma (mean ±SD = 0.2 ±0.4, 0.5 ±0.6, 1.1 ±1.3 and 2.95 ±2.97 in grade I
to IV astrocytic gliomas, respectively). This expression was significantly correlated with overall survival (OS) and progression-free survival (PFS)
(P=0.004). EZH2 expression was also significantly associated with advanced grades (mean ±SD =1.35 ±0.4, 3.1 ±2.6, 7.2 ±3.5 and 9.9 ±4.1, in
grade I to IV astrocytic gliomas, respectively). EZH2 and Ki-67 expressions were found to be correlated with OS and PFS (P < 0.001).
Conclusion: Increased expression of ALDH1, EZH2 and KI67 are found to be associated with unfavourable prognosis in patients with astrocytic
gliomas and may predict therapeutic modalities.
Gliomas represent about 80% of primary malignant tumors
in the central nervous system and rank the first among brain
. CNS neoplasms comprise about 3% of primary
malignant tumors in Egypt. They are the most common
solid tumor in children2
. Astrocytic gliomas represent a
heterogeneous group of malignancies which are classified
into low grade astrocytomas (grade III), anaplastic
astrocytomas (grade III) and glioblastoma (GB) (grade IV)3
. GB is the most common and aggressive primary brain
tumor in humans. 52% of primary brain malignancies and
20% of all primary intracranial tumors in the United States
. Patients with astrocytic gliomas have poor
survival despite the recent advances in management. There
have been only a minimal advance in prognosis especially
in GB with a very short median survival5
Brain tumors are thought to originate from a small
population of undifferentiated cells having characteristics
of normal neural stem cells. These cells have been named
after brain tumor stem cells, responsible for tumor initiation
and therapy resistance in primary brain tumors6.
Aldehyde dehydrogenase 1 (ALDH1) is a detoxifying
enzyme that catalyzes oxidation of intracellular aldehydes,
leading to production of retinoic acid from retinol, which
has a major role in cell proliferation and differentiation and
maintenance of stem cell phenotype. ALDH1 is needed for
resistance to alkylating agent through its detoxifying effect7,8. This biomarker has been detected in stem cells of
both normal and neoplastic tissues9.
Polycomb group (PcG) proteins have a major role in
regulating several physiological and pathological activities,
such as oncogenesis10. PcG protein complexes are grouped into complex 1 (PRC1) and complex 2 (PRC2)11. Enhancer of zeste homolog 2 (EZH2) is the core
member of PRC212. EZH2 acts through activating Ras
and the nuclear factor (NF)-κB pathway to control cell
oncogenesis through regulation of transforming growth
factor -β1 expression and functions13. Also, EZH2
interposes histone methylation and recruits DNA methyl
transferase in the silencing of many genes, related to cell
cycle control and survival14,15. Also, it is considered as
a recent biomarker of unfavorable prognosis in cases with
Ki-67 protein is expressed in all active phases of the cell
cycle. It represents an excellent biomarker applicated in
many neurosurgery centers to predict aggressiveness of
gliomas and patients' outcomes17,18.
The current study aimed to evaluate the prognostic role and
predictive value of ALDH1, EZH2 and Ki-67 in astrocytic
|Patients and Tissue Specimens:
(FFPE) tissue specimens of 40 patients with
astrocytomas who underwent initial surgery during the
period from December 2011 to May 2014 at Zagazig
University Hospitals were enrolled in our study and were
obtained from the archives of the Pathology Department,
Zagazig University. Clinical information and detailed
follow-up data for all patients were taken from medical
files. All examined tumors were located in the cerebral
hemispheres; recurrent tumors were excluded. None of the
patients had received chemotherapy or radiotherapy prior
Follow-up information obtained from the clinical records
ranged from 6 to 36 months. Preoperative Karnofsky
performance status (KPS) score was obtained from the
neurological examination data. The study complied with
the guidelines of Zagazig University ethics committee. MRI
repeated every 34 months was used as standard practice
to detect patients' response. A complete response (CR)
was defined as the complete absence of enhancing or nonenhancing
tumor; partial response (PR) was a shrinkage of
>50% of the tumor area; stable disease (SD) was defined as a
decrease of tumor area from 0 to 50% with stable or reduced
steroid dosage; and minor response (MR) as a reduction of
>25% but <50% of the tumor area. An increase of >25% of
the tumor area was designated as progressive disease (PD).
Histopathologic Examination: Ten tissue specimens were
obtained by stereotactic brain biopsies. Subtotal resection was done in 11 cases. In the remaining 19 cases, gross total
resection was performed. All tissue samples were fixed with
neutral 4% formaldehyde solution. Consecutive 4-μm thick
sections from formalin-fixed, paraffin-embedded tissue
blocks were prepared and stained with hematoxylin and
eosin (H&E) for histopathological classification3.
Immunohistochemical Staining: FFPE tissues were cut
into 4-μm thick sections and transferred to positively
charged slides. Then, sections were subjected to dewaxing,
rehydration, blocking with hydrogen peroxide, and antigen
retrieval (Dako target retrieval solution, citrate buffer
pH 6.0) with microwave. The slides were then incubated
overnight at 2-8 ˚C with primary antibodies: anti-ALDH1
rabbit monoclonal antibody (Abcam, Cambridge, UK;
dilution 1:100); EZH2 (BD Biosciences, CA, 11/EZH2;
dilution 1:100); and Ki-67 antibody (Dako, Glostrup,
Denmark, clone MIB-1; dilution 1:50). Incubation with
secondary antibody and product visualization (Dako)
was performed with diaminobenzidine substrate as the
chromogen. The slides were finally counterstained with
Mayer's hematoxylin and washed once each with distilled
water and PBS.
Specimens of infiltrating duct carcinoma of the breast,
liver and tonsillar tissue were used as positive controls for
EZH2, ALDH1 and Ki-67 antibodies, respectively. Negative
controls, obtained by replacement of primary antibodies by
PBS, were included within each slide batch.
Assessment of Immunohistochemical Staining Results
Evaluation of ALDH1 Immunostaining: The degree of
reactivity was carried out by evaluation of the percentage of
cells stained per section. Sections were graded as ALDH1-
negative (no staining), <2% ALDH1-positive and ≥2%
Evaluation of EZH2 Immunostaining: Positive-nuclear
staining cells (nuclear staining) for EZH2 in ten highpower
microscopic fields were counted and the percentage
of positive cells was calculated. Scoring was as follows: 0
(0%), 1 (110%), 2 (1150%) and 3 (>50%). Also, the
staining intensity was scored as: 0 (negative), 1 (weak), 2
(moderate) and 3 (strong). A final score was obtained for
each case by multiplying the two scores. A final score of
≥4.5 was considered to be high19.
Evaluation of Ki-67 Immunostaining: Ki-67 index was
calculated as the percentage of immunoreactive nuclei per
200 cells in five high power fields. Staining of 010% and
≥10% cell nuclei was considered to be low and high indices,
Statistical Analysis: Continuous variables were expressed
as the mean±SD and median (range), and the categorical
variables were expressed as numbers (percentages).
Continuous variables were checked for normality by using
Shapiro-Wilk test. Mann Whitney U test was used to
compare between two groups of non-normally distributed
variables while the Kruskal-Wallis H test was used for
more than two groups. Percentages of categorical variables
were compared using the Pearson's Chi-square (χ2) test.
Spearman's rank correlation coefficient was calculated
to assess correlations between EZH2, ALDH1 and Ki-67;
(+) sign was indicator for direct relationship and (-) sign
was indicator for inverse relationship. Values near 1 were
indicators for strong relationship and values near 0 were
indicators for weak relationship. Overall Survival (OS)
was calculated as the time from surgery to death or the
most recent follow-up contact (censored). Recurrence free
survival (RFS) was calculated as the time from complete
response to recurrence or date of last known recurrent free
status(censored). Stratification of OS and PFS was done according to all clinicopathological parameters. These
time-to-death and time-to-recurrence distributions were
estimated using the method of Kaplan-Meier plot, and
compared using two-sided exact log-rank test. All tests
were two sided. A P-value <0.05 was considered significant.
All statistics were performed using SPSS 22.0 for Windows
(SPSS Inc., Chicago, IL, USA) & MedCalc Windows
(MedCalc Software bvba 13, Ostend, Belgium).
|Patients and Their Clinicopathological Parameters:
study included forty patients; 22 males and 18 females,
with an age range from 7 to 72 years (mean: 40.2 ±20.2).
The lesions were classified as low-grade [5 pilocytic
astrocytomas (WHO I) and 9 diffuse astrocytomas (WHO
II); and 26 were classified as high-grade gliomas [11
anaplastic astrocytomas (WHO III)] and 15 glioblastomas
(WHO IV)]. The clinicopathological data and the treatment
regimen of the patients are shown in Table I
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|Table I: Clinicopathological features of 40 patients with astrocytic glioma
Association Between Astrocytic Glioma Grade and
Clinicopathological Parameters: WHO grade of astrocytic
gliomas was correlated to age, size, extent of tumor
resection, type of adjuvant treatment, PFS, KPS and OS (P
<0.05), but insignificantly associated with patient sex (P =0.
895) (Table I).
Association Between ALDH1 Expression and
Clinicopathological Parameters: During follow-up period,
26 of the 40 patients had recurrent glioma and 20 patients
had died. ALDH1-positivity >2% was associated with short
PFS and OS significantly (P = 0.001) (Figures 1A-D, 2AD).
ALDH1 was cytoplasmic in distribution (Figure 3AC).
This expression was significantly higher in astrocytic
glioma (mean: 1.5 ±2.2) than non-neoplastic brain tissue
(mean: 0) (P =0.003). There was significant difference
between ALDH1 immunoreactivity and different grades of
astrocytic glioma (P =0.014) (Table II). ALDH1 expression was significantly associated with KPS (P < 0.001), however
it was not associated with age, sex or tumor size and type of
tumor resection (Table III).
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|Figure 1: Kaplan-Meier plot of survival: A) In all studied astrocytic glioma patients (N=40), B) Stratified according to ALDH1 expression,
C) Stratified according to EZH2 expression, D) Stratified according to Ki-67 expression.
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|Figure 2: Kaplan-Meier plot of recurrence: A) In all studied astrocytic glioma patients (N=40), B) Stratified according to ALDH1
expression, C) Stratified according to EZH2 expression, D) Stratified according to Ki-67 expression.
Click Here to Zoom
|Figure 3: A) Low-grade astrocytoma showing low levels of ALDH1
cytoplasmic expression (ALDH1; x200). B) High grade astrocytoma
showing low levels of ALDH1 cytoplasmic expression (ALDH1;
x200). C) High grade astrocytoma showing high levels of ALDH1
cytoplasmic expression (ALDH1; x200).
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|Table II: Relation between different grades of astrocytic glioma&clinicopathological parameters in patients (n=40)
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|Table III: Relation between ALDH1, EZH2 and KI67 with the clinicopathological parameters in patients (n=40)
Association Between EZH2 Expression and
Clinicopathological Parameters: EZH2 were expressed in
the nuclei (Figure 4A-D). This expression was significantly
higher in astrocytic glioma (mean: 6.6 ±4.7) than nonneoplastic
brain tissue (mean: 0.8 ±0.2) (P <0.0001). There
was significant difference between EZH2 immunoreactivity
among different grades of astrocytic glioma (P <0.001)
(Table II). EZH2 expression was significantly associated
with age, size, KPS and type of resection (P =0.05). However,
no significant association between EZH2 expression and
sex was found (P =0.94) (Table III). EZH2 overexpression
was also significantly associated with poor OS and PFS (P
<0.0001) (Figures 1A-D, 2A-D).
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|Figure 4: A) Low-grade astrocytoma showing low levels of EZH2 nuclear expression (EZH2; x400). B) High grade astrocytoma showing
high levels of EZH2 nuclear expression (EZH2; x100). C) Giant cell glioblastoma showing the presence of giant cells (H&E; x400).
D) Giant cell glioblastoma showing high levels of EZH2 nuclear expression (EZH2; x400).
Association Between Ki-67 Expression and
Clinicopathological Parameters : Ki-67 was expressed in
the nuclei (Figure 5A-D). There was significant difference
between Ki-67 index among different grades of astrocytic
glioma. High Ki-67 index was significantly associated with
poor OS and PFS (P <0.0001) (Figures 1A-D, 2A-D).
Click Here to Zoom
|Figure 5: A) Low grade astrocytoma showing low levels of Ki-67 nuclear expression (Ki-67; x400). B) Low grade astrocytoma showing
high levels of Ki-67 nuclear expression (Ki-67; x400). C) High grade astrocytoma showing high levels of Ki-67 nuclear expression (Ki-67;
x100). D) High grade astrocytoma showing high levels of Ki-67 nuclear expression (Ki-67; x400).
Correlation Between ALDH1, EZH2 and Ki-67 in All
Studied Astrocytic Glioma Patients: Significant positive
correlation was found between ALDH1 and EZH2 (r =
+0.598, P <0.0001), as well as between ALDH1, EZH2 &
Ki-67 (P <0.0001) (Tables IV,V).
Relation Between ALDH1, EZH2 and Ki-67 and Response
to Therapy in All Studied Astrocytic Glioma Patients:
There was significant association between ALDH1, EZH2
and Ki-67 and response to therapy. Four patients had no
ALDH1 immunoreactivity, low EZH2 and low Ki-67 index.
However, high expression of ALDH1, EZH2 and Ki-67
were associated with progressive disease in 22 cases. Partial response was achieved in 7 patients, while 7 patients had
stable disease (Table VI).
Click Here to Zoom
|Table VI: Relation between ALDH1,EZH2,Ki-67 and type of response in patients (n=40)
Astrocytic gliomas are the most common central nervous
system neoplasms. They are heterogeneous tumors at
molecular and phenotypical levels21
. Despite the great
advances in therapeutic modalities, survival of patients
with astrocytic gliomas remains poor, especially with GB,
which is a highly aggressive tumor resisting treatment22
. Therefore, better understanding of tumor biology,
molecular basis of tumor invasion and spread and
resistance to conventional therapies may aid to improve
Tumor stem cells (TSCs) comprise a subpopulation of
tumor cells with continuous self-renewal capacity and the
ability to initiate tumor growth. TSCs are also responsible
for tumor recurrence and resistance to recent therapeutic modalities24-26. The identification of these cells seems to
be crucial for understanding the process of carcinogenesis,
and for the development of targeted therapies27.
ALDH1 is a specific stem cell marker in normal brain tissue
and it is suggested to be a novel stem cell marker in GB.
High level of ALDH1 in GB cells may be responsible for the
presence of undifferentiated stem cells27. In the current
study, ALDH1 expression was found to be increased
significantly from grade I to grade II astrocytoma, to
anaplastic astrocytoma and glioblastoma (mean ± SD:
0.2± 0.4, 0.5 ±0.6, 1.1± 1.3 and 2.95±2.97, respectively).
ALDH1 was not detected in low-grade astrocytomas or it
was expressed in <2% of cells. For the high-grade astrocytic
gliomas, 18.2% of grade III and 40% of grade IV tumors had
≥2% of ALDH1-positive cells. This leads to the conclusion
that the level of ALDH1 is strongly correlated with astrocytic
glioma grade and necessarily with the patients' prognosis.
Our results go with those of Liu et al.14, who found
that the percentage of ALDH1-positive cells increased
with tumor grade and correlated with unfavorable clinical
course in patients with astrocytic glioma. All these results
stated that TSCs have a major role in the carcinogenesis of
glioma. Rasper et al.27 stated that high level of ALDH1
was detected in 83.3% of GBs. Moreover, they studied
ALDH1 expression in GB cell lines and cell culture and
searched for the neurosphere formation, finding that high
ALDH1 protein levels improve neurosphere formation.
However, there was deficiency in neurosphere formation
after ALDH1 inhibition and lack of ALDH1 leads to
incomplete maturation of TSCs and improper neurosphere
formation27. ALDH1 is responsible for transformation
of retinol to retinoic acid which leads to up-regulation
apoptosis and differentiation genes in the GB cell line28.
Several studies have displayed that the expression of TSCs
biomarkers is positively correlated to tumor grade and
inversely associated with OS and PFS in glioma cases29. In the current study, patients with ALDH1-positivity
of ≥2% was significantly linked to short PFS and OS as
compared to those with positivity of <2% (P = 0.003). This
is in agreement with the results of Liu et al.14, who found
that over-expression of ALDH1 was significantly correlated
with short PFS and OS (P < 0.001).
EZH2 methylate STAT3, leading to increased STAT3
activity by increasing tyrosine phosphorylation of STAT3,
so they promote carcinogenesis of glioblastoma stem-like
cells30. Transcriptional regulation of c-myc by EZH2
involved in glioblastoma cancer stem cell maintenance and
From the therapeutic point of view, EZH 2-mediated signal
loop, EZH2-CDK4/6-pRb-E2F1, which is involved in
carcinogenesis of glioma, could be inhibited by the candidate
miRNA. MiR-138 is the miRNA that is diminished in GB
samples and potent to suppress EZH2 expression through
inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop32.
In the present study, increased expression of EZH2 protein
was significantly associated with advanced grades. None of
the low-grade astrocytic glioma cases exhibited high EZH2
indices. These results are in accord with those of Wu et al.19, who found that the up-regulations of EZH2 proteins
was significantly associated with increased grades (P <
Zhang et al.33, performed gene expression profiling
of glioma and found that the level of EZH2 mRNA
increased expressively in high grade gliomas compared
to lower grades. Therefore, EZH2 may have a great role
in glioma development. Also, overexpression of EZH2
was investigated in metastatic tumors13. Tumor cell
proliferation and progression, and poor outcome in
prostate, endometrial, breast cancer and melanoma were
found to be related with EZH2 over-expression34,35.
In our study, a significant association was present between
EZH2 over-expression and poor OS and PFS (P < 0.001).
Patient survival declines significantly in those with high
EZH2 expression (9 vs. 11 months for both PFS and
OS). These results go with that of Wu et al.19 who
demonstrated that the high level of EZH2 was greatly
linked to decreased survival (P = 0.006). Also, Zhang et al.33 stated that GB cases expressing higher levels of EZH2
were linked to shorter survival relative to those with lower
The current study revealed a significant difference between
Ki-67 indices among different grades of astrocytic glioma.
These results are in a general agreement with previous
results declaring that Ki-67 index was found to correlate
with tumor grade and clinical behavior36,37. But, a study
performed by Chaloob et al.38 did not support such
significant correlation. The authors attributed this situation
to environmental, racial and geographical differences, in
addition to the differences in sample size and antibodies
used for the detection of Ki-67 antigen in the different
Due to the heterogeneity of astrocytic gliomas, it is likely
that only combinations of markers will able to give
sufficient sensitivity and specificity to be useful for clinical
purposes. In this paper, such a combination with EZH2, ALDH1 and Ki-67 in astrocytic gliomas was studied and
an expressive association was observed among them.
Therefore, over-expression of EZH2 and ALDH1 was
significantly correlated with increased cell proliferation
as assessed by high Ki-67 index. This suggests that tumors
expressing EZH2 and ALDH1 will have unfavourable
prognosis, increased recurrence rate, poor response to
therapy and poor survival.
In conclusion, expression of ALDH1, EZH2 and Ki-67 was
found to be increase significantly with astrocytic glioma
progression and increased expression levels were associated
with short patient survival. Our data suggested that ALDH1,
EZH2 and Ki-67 can be considered as potential biomarkers
to detect astrocytic glioma patients with unfavorable
outcome, can be targeted for future therapy and may aid to
stratify the patients into different risk groups.
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