15018752330
发表时间:2015-11-24 浏览次数:484次
Introduction
Breast cancer is the most common cancer in women (30% of all cancers among
women in developed countries), but is treatable if early diagnosis and treatment
occur. The incidence of breast cancer has constantly increased since 1940 and
based on WHO reports, there is a 2% annual increase in breast cancer prevalence.
There are no exact statistics on the prevalence of breast cancer among the
Iranian population (the source of our study); estimates show that Iran has
moderate, but increasing, prevalence. Both genetic and environmental
susceptibilities are included in breast cancer etiology, but the exact etiology
has not been definitively identified. Current studies confirm the role of the
immune system on the etiology of breast cancer.
It was shown that cancer
cells provoke immune recognition, but the biologic importance of antitumor
innate and adaptive responses, which are frequently detected in cancer-bearing
hosts, remains incompletely understood. The most considerable antitumor response
is made by human cellular immunity mediated by T-lymphocyte and natural killer
(NK) cells. It follows that variants of genes included in the regulation, and
proliferation of T-lymphocyte and NK cells would be effective in predicting the
risk of breast cancer. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is coded by a
gene on chromosome 2q33. It is a member of the immunoglobulin super family,
which transmits an inhibitory signal to T cells. CTLA-4 binds to B-7 on
antigen-presenting cells, and polymorphism of CTLA-4 gene interferes with
surface activity of B-7, preventing T-lymphocyte from activating.In fact, CTLA-4
prevents immune response and its tumor-killing activity. CTLA-4 gene is
composed of 4 exons and possibly plays a significant role in diseases related to
T cells. More than 100 single-nucleotide polymorphisms are recognized on the
CTLA-4 gene. Among them, AG dysmorphisms, located on +49 of exon 1, could
make amino acid (threonine into alanine) on CTLA-4 protein.
Current studies
show that this polymorphism affects the ability of CTLA-4 to bind to B-7 cells
and to activate T cells. These surveys show that translocation of A allele to G
allele on +49 zone decreases the role of CTLA-4 on T-cell responses, although
there are contradictory reports on the relationship between +49 A to G
polymorphisms and cancer development.
Higher expression of CTLA-4 is seen
in persons with thiamine on zone -318 of CTLA-4 gene promoter or
homogenous adenine on exon 1 of codon +49. There have been several studies on
the relationship between polymorphisms on CTLA-4 gene and autoimmune
diseases such as graves, diabetes mellitus type one, lupus and Hashimoto
thyroiditis and the tendency to develop cancer. Results of some studies show
an inverse relation between polymorphisms of autoimmune diseases and
malignancies on CTLA-4 gene. Alleles discovered in autoimmune diseases
are not seen in malignancies or are related to a good prognosis of cancers. In a
study in Iran, results suggested higher risk of breast cancer among AA and AG
genotypes on +49 zone, but there was no difference between -318 CT and -1666 AG
among case and control groups.
Considering the high prevalence of breast
cancer and also some confirmed evidence of a relationship between CTLA-4 gene polymorphisms and breast cancer, we conducted this study to assess the
relationship between CTLA-4 gene polymorphisms and both incidence and
clinic pathologic features of breast cancer. The results of this study would
help physicians to recognize the prognosis and risk ratio of patients with a
high risk of breast cancer.
Methods
Study subjects
The study group consisted of a total
of 100 Iranian women with breast cancer and 100 healthy cancer-free
control individuals. Informed consent was obtained from each subject,
and each participant was then interviewed to collect detailed
information on demographic characteristics such as sex and age. Some
clinic pathologic features of breast cancer patients, such as tumor
size, lymph node involvement, tumor type, and estrogen receptor (ER),
were also obtained from their medical files [Table 1].
atients were recruited between February 2013 and October 2014 at the Shahid
Sadughi Hospital and Cancer Hospital, Yazd, Iran. Control subjects were
cancer-free individuals, and they were randomly selected from the same regions
and the same time period as the patients were collected. The selection criteria
included no individual history of breast or other cancers.
This study was
approved by the Ethics Committee of Shahid Sadoughi University of Medical
Sciences, Yazd, Iran. A written informed consent was taken from all
patients.
Polymorphism genotyping
Peripheral blood
(5 mL) was collected from subjects after informed consent was obtained. Genomic
DNA was extracted from peripheral blood using the DNA extraction kit (BioFlux,
cat: BSC 06M1, Hangzhou, Bioer Technology Co., Ltd, China).
Genotyping
was performed by polymerase chain reaction (PCR)-restriction fragment length
polymorphism method. The polymorphic region was amplified by PCR using the
following primers: (forward) 5'-CTAAGAGCATCCGCTTGCACCT-3' and (reverse) 5'-
TTGGTGTGATGCACAGAAGCCTTT-3' in a 25 μL reaction solution containing 0.3 μg of
genomic DNA, ×1 PCR buffer, 0.3 mmol/L MgCl 2 , 0.2 mmol/L dNTPs, 2 U
tag DNA polymerase and 0.1 μmol/L of each primer.
The following PCR
program was run: 94 °C for 4 min, 30 cycles of 94 °C for 30 s, 58 °C for 30 s
and 7 °C for 45 s. Final extension was carried out at 72 °C for 5 min. The
lengths of the PCR products were 486 bp (1661AG).
The PCR products were
digested with restriction enzymes Tru1I (MseI) according to the manufacturer's
instructions (Thermo Scientific Fermentas, USA) and analyzed by 2% agarose gel
electrophoresis. The cut site for Tru1I (MseI) was 5'-TTAA-3'.
The
digested fragments in 1661AG were 139 and 347 bp. Presence of the A allele was
recognized by detecting digested 347 and 139 bp fragments on gel, and the G
allele by detecting intact primary 486 bp band [Figure 1]. Comparisons of genotype and allele frequencies in
cases and controls were assessed by Chi-square and t-test using SPSS-16.0
(SPSS Inc., Chicago, USA). Statistical software and statistical significance
were set at P ≤ 0.05. The odds ratio and 95% confidence interval were
also calculated.
Results
A total of 100 women with breast cancer and 100 healthy controls were
enrolled in this study. Mean age of the case group was 48.92 ± 9.85
years and of the control group was 37.92 ± 13.67 years (P < 0.001; t-test).
This study was done in patients who presented at Shahid Sadughi
Hospital and Cancer Hospital, Yazd, Iran. The grade, stage and size of
the tumor in the case group are shown in [Table 1].
About 73.3% of patients had grade one tumors, and about 75% had stages 1
and 2 tumors. Tumor size in more than 60.9% of patients was ≤ 2 mm.
Polymorphisms of CTLA-4 gene are shown in [Figure 2]. There is a significant relationship between groups according to gene polymorphisms (P
= 0.03). Frequency of AA polymorphisms in the case group is higher than
in controls whereas AG polymorphisms are more frequent in the control
group.
[Table 1] shows polymorphisms according to tumor stage. There is no significant difference among study groups according to tumor grade (P = 0.21). Also there was no relationship between polymorphism and tumor stage (P = 0.50) and tumor size (P = 0.50).
Considering the difference in mean age between the two study groups, we analyzed polymorphisms of CTLA-4 gene in all participants according to age groups of under or over 40 years, but found no significant difference (P = 0.30) [Table 1]. We determined that the age difference between the two groups had no confounding effect on the study results.
Analysis of metastasis showed that there is a significant relationship between CTLA-4 gene polymorphism and metastasis. Patients with AA genotype had higher rates of metastasis (P = 0.02) [Table 1].
Analysis also showed no relationship between CTLA-4 gene polymorphisms and ER (P = 0.49), progesterone receptor (P = 0.15) and lymph node involvement (P = 0.53) [Table 1].
Discussion
There is increasing attention to the relationship between several genes'
polymorphisms and polygenetic diseases such as hypertension, diabetes, and
various malignancies. T cells and NK cells have a substantial role in working
against tumors. T-lymphocyte, especially T killer cell, is the most important in
defending cells against tumors. CTLA-4 molecule expresses on T-lymphocyte as an
inhibitor and plays different roles in T-cell activity. It could inhibit
amplification of T cells or even induce apoptosis of activated T cells.
CTLA-4-mediated suppression of tumor immunity has been previously reported.
Several studies have demonstrated the effect of CTLA-4 blockade in enhancing
immunity to tumors. There are some studies on the relationship between
CTLA-4 gene polymorphisms and breast cancer, but results were
contradictory. In order to clarify the role of genetic variants of CTLA-4 gene in immune suppression of patients with cancer, the distribution of
CTLA-4 gene single nucleotide polymorphisms (1661AG), in breast cancer
patients and control subjects, were investigated and their associations were
assessed with prognostic factors. The present case control study was done to
determine possible relationships between AA, AG and GG polymorphisms of
CTLA-4 gene and breast cancer-related factors. Results revealed that the
frequency of AA and GG genotypes in breast cancer patients is higher than in
controls, while AG genotype is more frequent in healthy controls. These results
confirm the findings of two other studies in Iran and China, which found GG
genotype is more frequent in breast cancer patients. However, Erfani et al did not find any difference between their study groups in terms of AG
genotype. Another study in China suggested that AG genotype is more prevalent in
breast cancer patients. Furthermore, Sun et al reported that T
cells with AA genotype are less active than those with GG genotype, and AG is
related to different cancer incidence in humans.
The results of our study
did not find any relation between AA, AG and GG genotypes with respect to tumor
stage, grade, receptors or lymph node involvement. These results are in
agreement with results of Wang et al., who found no significant
relationship between AG genotype and tumor size and lymph node involvement.
Erfani et al. found a relationship between AA genotype and lower
lymph node involvement and higher ER expression, but Ghaderi et al found that AA genotype is related to higher rates of lymph node involvement and
tumor size; these findings are different from our findings. Bi et al in his study reported that CTLA-4 expression is higher in stage 2 than
stage 3 patients. There are also some other studies, which revealed that
CTLA-4 gene polymorphisms are related to higher stages and lymph node
metastasis, which is not consistent with our study. According to age, in our
study and also in the Bi et al., there was no relationship between
CTLA-4 gene polymorphisms and age.
Li et alfound a
relationship between all CTLA-4 gene polymorphisms with estrogen and
progesterone receptors. Also, Erfani et al detected a relationship
between AG genotype and ER expression, which was not consistent with our
findings.
One of the limitations of our study is the failure to take into
account risk factors such as age at menarche, menopausal status, and
environmental factors. It is important to investigate the interaction between
single-nucleotide polymorphisms and these factors on the risk of breast cancer
in a larger sample size in further studies.
Based on our study, there is
a relationship between CTLA-4 gene 1661AG polymorphisms and incidence of
breast cancer, but these polymorphisms are not effective for prognosis.
Considering the controversial reports on this issue, more studies are needed
with larger sample size. Also, a critical review and possible meta-analysis of
present studies are needed to make an exact estimation of the results of current
studies.
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