Introduction
Breast cancer (BC) is the most common cancer type
that affects women in the world population. Approximately
1.3 million women are diagnosed with BC annually
worldwide.[] Clinicopathologic features, such
as tumor size, lymph node (LN) status, hormone receptor
status, and invasion, play important roles in prognosis.
Similar to other types of cancers, BC tumorigenesis
is characterized as a multi-step process in which
each step is thought to correlate genetic and/or nongenetic
factors.
Human epidermal growth factor receptor 2 (HER2;
aka erbB2) and its relatives belong to the HER family of
receptor tyrosine kinases. The HER2 protein is a transmembrane
glycoprotein with a size of 185-kD and belongs
to the HER family of growth factor receptors.[]
The HER2 protein is overexpressed and/or amplified
approximately in 15-20% of the BC and has both prognostic
and predictive implications.[] Topoisomerase
2 alpha (TOP2A) gene encodes a DNA topoisomerase
that controls topologic states of DNA at transcription
and replication.[] TOP2A gene is found on chromosome
17 q12-q21, adjacent to the HER2 gene, and
its aberrations (amplification or deletion) have been
shown usually in HER2-positive breast cancers.[]
Epigenetic abnormalities in neoplastic cells, including
hypermethylation and hypomethylation of DNA,
modified patterns of histones, and remodeled chromatin
arrangement, lead to the modified expression
of numerous fundamental genes. A well-categorized
epigenetic change is hypermethylation of tumor-suppressor
promoters that result in improper transcription
silencing of these genes.[] The tumor suppressor gene
RAS-association domain family member 1 (RASSF1A)
encodes a member of the group of RAS effectors that
modulates cell proliferation, apoptosis, and microtubule
stability. Hypermethylation of RASSF1A was detected
in a significant percentage of several primary
tumors.[] Epigenetic silencing of the RASSF1A is assumed
to be an early cancer biomarker, but this process
is extended from primary to metastatic tumors during
tumor progression.[] The adenomatous polyposis
coli (APC) gene, located in chromosome 5q21, plays
an essential role in the pathogenesis of colorectal cancer,
both in the autosomal dominant inherited familial
APC syndrome and in sporadic colorectal cancer.[] It
has been proposed that the impairment of the APC/β-
catenin pathway may play a role in BC. Lack of APC
expression and upregulation of β-catenin have been
identified in human BC and BC cells.[]
Although BC therapy differs by subtype, there are
standard treatments that are currently administered
based on subtype. The oncogenic issues and signalling
pathways that drive these tumor subtypes are definite,
showing that a better comprehension of their molecular
basis will render possibilities for predicting response to
chemotherapy and implementing novel treatment modalities,
to finally improve patient outcomes. Therefore,
in this study, we aimed to investigate the relationship
between HER2/TOP2A aberrations which in predictive
markers in BC and methylation status of RASSF1A/
APC genes in high-risk patients with BC.
Methods
Case Selection
In this study, formalin-fixed paraffin-embedded
(FFPE) sections of tissue from 60 high-risk BC patients
were obtained in the Department of Pathology, Medical
Faculty, Eskisehir Osmangazi University, Eskisehir,
Turkey. The inclusion criteria of samples were applied
to include the BC patients with (1) tumor size ?2 cm
and/or (2) lymphatic metastases and/or distant metastases
and/or (3) patients under 40 years. Clinical parameters,
such as tumor grade, histopathological type,
the status of estrogen receptor (ER) and progesterone
receptor (PR), were obtained from patient"s case files.
All patients in this series were treated using standard
anthracycline-based adjuvant chemotherapy. Informed
consent was obtained from the patients whose clinical
data could be accessed. The use of FFPE samples for
this research was approved by the clinical studies local
Ethics Committee (Eskisehir Osmangazi University-
Medical Research Ethics 2010/173). This study was
conducted in accord with the Helsinki Declaration.
Fluorescencein Situ Hybridization (FISH) Analysis
Fluorescence insitu hybridization (FISH) analysis was
performed on 4 ?m thick sections of FFPE samples.
Commercially available FISH assays of CEP17, HER2
and TOP2A were done according to the manufacturer"s
protocols (Zytovision, Germany). The kit consisted of
a mixture of spectrum green-labeled HER2, spectrum
red labeled TOP2A gene and spectrum aqua labeled
centromere 17 (CEP17) specific probes. The threecolor
FISH analysis was performed on the slides of the
FFPE tissue samples located in two separate, distinct
microscopic areas. The tumors sections, containing at
least 85% puretumor cells, were selected during the histopathological
analyses of the lesions. In the evaluation
of fluorescence spots specific to HER2/TOP2A/CEP17, absolute and relative numbers (relative to chromosome
17 copy number) of the individual genes were scored
in a hundred randomly selected nuclei per tumor using
an Olympus bx61 fluorescence microscope (Olympus,
Tokyo, Japan) and images were captured using image
analysis system (applied imaging, Newcastle, UK). In
each FISH experiment, known positive and negative
controls were used. In the FISH assessments, HER2/
CEP 17, TOP2A/CEP17 ratios were calculated. HER2/
CEP17 ratio of ?2 and TOP2A/CEP17 ratio of ?1.5
were defined as positive for HER2/TOP2A amplification.
TOP2A was considered deleted when TOP2A/
CEP17 ratio <0.8.[11]
Methylation-Sensitive High-Resolution Melting
(MS-HRM) Analysis
After deparaffinization, genomic DNA was extracted
using MagNA Pure Compact Nucleic Acid Isolation
Kit I (Roche) according to the manufacturer"s instructions.
The quantity and purity of isolated DNA were
evaluated by NanoDrop 1000 spectrophotometer
(Thermo Scientific, DE, USA). Subsequently, genomic
DNA modified using sodium bisulfite to deaminate
selectively unmethylated cytosine residues to uracil,
while 5-methyl cytosine residues were not modified.
The bisulphite modification was performed using the
"EpiTect® Bisulfite Kit" (Qiagen) according to the manufacturer"s
recommendations.
To determine the promoter methylation status of
RASSF1A and APC genes, we used a real-time polymerase
chain reaction (PCR) approach followed by high
resolution melting curve analysis (HRM). PCR and
HRM analysis were consecutively performed on a Light-
Cycler® 480 (Roche Applied Science, Laval, PQ, Canada).
PCR was performed in a 19.5 ?l reaction volume, and 10
?l of BSC DNA templates were added to each well which
contained 10 ?lLightCycler 480 High Resolution Melting
(HRM) Master Mix® (Roche), 2.5 ?l MgCl2 and 3.0 ?l of
each primer. The primer sequences were based on the
previous report [12] as follows:Methylated RASSF1A; F-
5"-GTGTTAACGCGTTGCGTATC-3";R-5"-AACCCCGCGAACTAAAAACGA-
3". RASSF1A unmethylated;
F- 5"-TTTGGTTGGAGTGTGTTAATGTG-3";
R- 5"-CAAACCCCACAAACTAAAAACAA-3"APC
methylated; F-5"-TATTGCGGAGTGCGGGTC-3";
R-5"-TCGACGAACTCCCGACGA-3"; APC unmethylated;
F-5"-GTGTTTTATTGTGGAGTGTGGGTT-3";
R- 5"-CCAATCACAAACTCCCAACAA-3". The amplification
consisted of 10 min at 95°C, followed by 50
cycles of 10 s at 95°C, 15 s at annealing temperature and
25 s at 72 °C.Fluorescence data were collected at 25 acquisitions per second. The LC480-HRM Master Mix®
employed a saturating dye (ResolLight?, Roche), which
facilitated the precise measurement of the melt curves of
the amplicons. The Roche Gene Scanning software was
employed for end-product analysis. This algorithm allowed
the raw melt curves to be normalized for fluorescence
intensity, and a temperature shift was applied to
align the normalized melt curves, which facilitated the
analysis of samples with varying crossing point values.
A difference curve was then derived from the first derivative
of the melt curves. Data for the difference melt
curves were transmitted to Excel (Office 2010; Microsoft
Corp., Redmond, WA, USA). Both peak-height and area-
under-the-curve from the normalized, temperatureshifted,
difference curves were used to create a standard
curve and determine the degree of methylation of each
DNA sample.
Statistical Analysis
All statistical analyses were performed using IBM SPSS
for Windows version 20.0 (SPSS, Chicago, IL, USA).
Comparisons of categorical variables between theclinicopathological
parameters, HER2/TOP2A aberrations
and RASSF1A/APC methylation status were performed
using the Fisher exact test and the Monte Carlo chisquare
test. A two-sided p-value <0.05 was considered
statistically significant. Overall survival was estimated
with the Kaplan-Meier method (Log-rank Test).
Results
A total of 60 cases were included in this study. All
cases were female. The median age of patients was
59.23±1.40 years (range 36 to 81 years). There was no
statistical association between histopathological type,
grade and ER/PR status. The baseline clinicopathological
features of the tumor samples are presented in Table
1. The characteristics of tumor samples according to
HER2 gene status are summarized in Table 2.
Table 1: Baseline characteristics of BC patients
Table 2: Characteristics of tumor samples according to HER2 gene status
HER2/TOP2 aberrations
HER2 gene amplification was observed in 15/60 samples
(25%). All samples with HER2 amplificated had
invasive ductal carcinoma. HER2 amplification was
more frequent in higher-grade tumors (p=0.001) and
PR negativity (p=0.025), and TOP2A aberrations
(p=0.004) (Table 3). TOP2A aberration was found in
18/69 (30.0%) (6.6% deletion and 23.4% amplification).
Although there was not any statistical difference,
the majority of the patients with HER2 and TOP2A aberration
were over the age of 45 years.
Table 3: TOP2A gene aberrations according to APC gene
methylation
When the HER2 gene status was evaluated in 19 patients
who were alive and under follow-up, 17 patients
with normal gene copy number were found to have a
0.82 probability of survival at the 4th year of life and 2
cases with HER2 gene amplification had 0.50 probability
of survival at the 4th year of life. The difference was
not statistically significant (Log-rank=0.139, p=0.399).
When the TOP2A gene status was assessed in 19 patients
who were alive and under follow-up, 15 patients
with normal TOP2A gene had a 0.80 probability of survival
in the 3rd year and four patients with TOP2A gene
amplification had 0.25 probability of survival in the 3rd
year. There was no statistically significant difference
(Log-rank=0.710 p=0.399).
RASSF1A/APC methylation
RASSF1A and APC promoter methylation were observed
in 58/60 samples (96.6%) and 26/60 (43.3%),
respectively. The findings showed that there was no significant difference between RASSF1A/APC methylation
status and histopathological type, grade and
ER/PR status (p>0.05). There was a significant relationship
between APC methylation and TOP2A aberration
(p=0.026). APC gene methylation was significantly
higher in patients with TOP2A aberration (p=0.026)
(Table 3).
When RASSF1A gene methylation of 19 patients
who are alive and under with follow-up was examined,
the findings showed that that 18 tumor samples were
methylated, and 1 sample was unmethylated. As APC
gene methylation was assessed in 19 patients who were
alive and under follow up, samples from 11 patients
were unmethylated and samples from 8 patients were
methylated. There was not any statistically significant
result in life analysis carried out with RASSF1A and
APC gene methylation.
Discussion
BC, a heterogeneous disease representing a wide range
of pathological entities and clinical behaviors, is an important
health problem in all over the world as well as
in Turkey.In the present study, we investigated the correlation
between HER2/TOP2A gene aberrations and
RASSF1A/APC promoter methylation status in tumors
with high-risk BC.
Human epidermal growth factor family consists of
several receptors with tyrosine kinase activity which
has an impact on cell proliferation and survival. The
dimerization of HER family members results in the autophosphorylation
of tyrosine residues in the cytoplasmic
domain and induces cell proliferation and tumorigenesis.[
13] While HER family members do not have a
natural ligand for signalling, several synthetic ligands
have been developed and they are shown to be effective
in drug delivery. Of all-family members, HER2 is a
crucial molecule and expression of HER2 is increased
in several cancer types. HER2 amplification is among
the most common genetic alterations in BC.[] HER2
amplification is an adverse prognostic factor and a
predictive biomarker of response to HER2-targeted
treatment.[] Furthermore, HER2 amplification is
functionally proposed as a driver of genomic instability
and thus may simultaneously cause amplification
and activation of other genes.[] Coamplified genes
found in the smallest region of amplification of HER2
amplicon include MED1, STARD3, GRB7, THRA, and
RARA.[16] TOP2A, located in a separate amplicon
downstream to HER2 amplicon, is often modified in
HER2-amplified tumors.[] Targeted inhibition of
Topoisomerase II alpha enzyme at a molecular level accounts for the cytotoxic effect of the TOP2A inhibitors,
such as the anthracycline class.
In the present study, the findings showed that HER2
gene amplification was 25% and TOP2A gene aberrations
were 30% (6.6% deletion and 23.4% amplification).
Several studies have also reported that TOP2A
aberrations are rare in patients with normal HER2.[]
It was reported that TOP2A aberration was present in
50-80% of the patients with HER2 amplification.[]
In the present study, TOP2A aberrations occurred in 17.7% of HER2 non-amplified cases (13.3% deletion
and 4.4% amplification), while TOP2A aberration was
present in 66.6% (13.3% deletion and 53.3% amplification)
of HER2 amplified cases (p=0.004). These results
support many previous studies reporting a close relationship
between HER2 and TOP2A genes, whereas
HER2/TOP2A co-amplification was reported as 35%
by Press et al.,[] as 39% by Bhargava et al.,[] in
the present study, HER2/TOP2A co-amplification was
found in 13.3% of the patients. This result may be due to
the diversity in methodology and/or established cut-off
values. Moreover, although being statistically insignificant,
we found that HER2 and TOP2A co-amplification
was more common in patients with advanced age.
Epigenetic events are crucial factors in the pathogenesis
of human cancers. Aberrant methylation in the
promoter regions of tumor suppressor genes is associated
with carcinogenesis via transcriptional silencing
of gene expression, resulting in the onset and development
of cancer.[] RASSF1A promoter methylation
provides significant prognostic information in early-stage BC patients.[] Vu et al. and Spitzwieser et al. reported
methylation of RASSF1A in 74.68 %, and 94%of
invasive BC.[,] In another study, Jezkova et al. found
that RASSF1A hypermethylation occurred in 92.2% of
the cases.[] In the present study, we found that RASSF1A
methylation was 96.6%. Our result is consistent
with the research findings of Spitzwieser et al. and Jezkova
et al. The higher ratio of RASSF1A methylation is
attributed to that the high-risk patients were included in
this study and that MS-HRM is such a sensitive analysis
measuring a difference as small as 1/1000.
APC gene inactivation causes dysfunction of
β-catenin protein breakdown, and then, induces Tcf/
Lef and results in abnormal transcription of oncogenes,
including c-myc, c-jun and cyclin D1, eventually leads
to carcinogenesis.[] Methylation in the APC gene has
been examined in various types of carcinomas, such as
BC, gastric, esophagus, pancreatic, and lung cancer.
[] Although numerous studies have been conducted,
the relationship between APC promoter methylation
and BC still remains unclear. He et al. reported that the
APC promoter methylation was associated with cancer
stage, lymph node metastases and ER status in BC.[]
Jin et al.[] and Shinozaki et al.[] reported that
APC methylation was associated with BC (p<0.05);
however, Park et al. and Sturgeon et al. suggested APC
methylation had no correlation with BC.[,] In a meta-
analysis (2483 BC patients and 1218 controls), Zhou
et al. demonstrated that the frequency of APC methylation
was significantly higher in BC cases than controls
under a random effect model.[] It was found that APC
gene promoter methylation was 52.1% in sporadic BC
cases, and there was a significant relationship of APC
hypermethylation with tumor stage and 3-year survival
(p<0.05).[] In the present study, the APC gene methylation
was 43.3%. No association was found between
RASSF1A/APC methylation status and histopathological
type, grade and ER/PR status. However, there was a
significant difference between APC gene methylation
and TOP2A aberrations. The samples with a normal
copy number of TOP2A showed 35.7% APC methylation
while samples with TOP2A aberration represented
61.1% APC methylation (p=0.026) (Table 3).
Our results did not show a statistically significant
relationship between HER2/TOP2A gene aberrations,
RASSF1A/APC gene methylation status and survival.
Conclusion
Our results suggested that APC gene promoter hypermethylation
was associated with TOP2A gene aberrations.
These results suggest that TOP2A aberrations contribute to the epigenetic mechanisms in BC. Our
data can provide a new option for individualized treatment.
Additionally, in this study, it was confirmed that
there was a significant relationship between HER2 amplification
and TOP2A gene aberration.
Acknowledgement: This study was presented at the 4th International
Health Science and Family Medicine Congress, 2019.
Peer-review: Externally peer-reviewed.
Conflict of Interest: None declared.
Ethics Committee Approval: The study has been approved
by the Medical research Ethics Committee of the Medical
Faculty of Eskisehir Osmangazi University.
Financial Support: This study was supported by the Eskisehir
Osmangazi University Scientific Research Foundation,
Project number: 201011037.
Authorship contributions: Concept - A.F.N., O.Ç., S.A.;
Design - S.A., B.D.A., A.F.N.; Supervision - S.A., O.Ç.,
B.D.A.; Materials - E.Ç., O.E.; Data collection &/or processing
- A.F.N., O.E., O.Ç.; Analysis and/or interpretation -
A.F.N., O.E., B.D.A.; Literature search - S.A., C.B., A.F.N.;
Writing - A.F.N., O.Ç., C.B.; Critical review - S.A., O.Ç., E.Ç.
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