Breast Keywords; breast cancer; foxp3; gene polymorphism. Introduction

Breast cancer is the most common cancer among Egyptian females accounting for 37.7% of all cancer cases. FoxP3 gene is an essential transcription factor in regulatory T cells, its expression affects the proliferation of T reg cells and alters disease susceptibility.  Several studies with variable results had been carried out on   Foxp3  polymorphisms of the promoter region in different ethnic groups but not in Egyptians. In the present study, we evaluated two promoter polymorphisms to find any association with breast cancer.

           Using SSP and PCR-RFLP to identify genotypes of Foxp3 rs3761548 and for rs3761549 respectively.

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With rs3761548 the most prevalent genotype found was AC genotype followed by CC and AA and for (rs3761549) it was   TT followed by CT & CC. Her2 and CA19-5 were associated with risk of metastasis prediction.

We concluded that no significant association was found between Foxp3 SNPs and risk of breast cancer.

Keywords;  breast cancer; foxp3; gene polymorphism.

 

 

 

 

Introduction

The most common cancer among Egyptian females is Breast cancer accounting for 37.7% of their total. It is also the cause of cancer-related mortality accounting for 29.1% of their total 1.

          Both the innate and adaptive immune systems play a role in preventing relapse in women with breast cancer. The players in this fight are T cells, T regulatory cells, natural killer cells, and their cytokines. The immune system status affects cancer prognosis.  Studies support the hypothesis that the immune system has a role in breast cancer etiology. Immunomodulatory therapy has an important role in breast cancer as a treatment 2.

 

Cytokines produced by T lymphocytes are responsible for a powerful immune response. Solid tumors are associated with T-helper type 2 (Th2) cytokine and T-helper 1 (Th1) cells in inducing inflammation that inhibits tumor growth (3).

 

 Regulatory T cells are cells that inhibit T helper response have CD4+ and CD25 hi and express the transcription factor Foxp3 and are responsible for maintaining self-tolerance so mutations of Foxp3  causes decreased  T reg and increase the risk of autoimmune disease (4).

In cancer, T reg cells may be induced by tumors to inhibit the immune response against tumor antigens. In humans, cancer induces peripheral  T reg to be sensitive to apoptosis (5).

 Experimental studies have shown that T reg cells expressing foxp3, transforming growth factor ? (TGF-?), and IL-10 help tumor to inhibit the normal immune response against it. In addition, studies have shown that T reg cells are increased in the peripheral blood and in the tumor microenvironment in humans with breast adenocarcinomas (6).

       Several studies in Asian population have been conducted to investigate any association of FOXP3 promoter polymorphisms, rs3761549, and rs3761548, with breast cancer however,  no association have yet been found (7,8).

Patients and study design:

This study was approved by the  Mansoura faculty of medicine. The ethical committee (IRB) code number is  R16.07.47,   Participants of the study were breast cancer patients who were recruited to the Mansoura Oncology Cancer Center from 2015 to 2016.

3 ml blood from each patient had been collected in tubes containing EDTA and stored at -20°C until the relevant assays were conducted. Information on cancer diagnosis, disease stage (tumor-node-metastasis), cancer treatments, and estrogen, progesterone and human epidermal growth factor receptor-2 (HER-2) receptors status, also CA 15.3 levels were abstracted from medical charts data seen in tables (1,2&3).

Genotyping: Genomic DNA was extracted from whole blood using DNA purification kit  (Qiagen, Inc., Valencia, CA, USA) and stored at -20°C until further use. Using  SSP  for rs3761548 and PCR-RFLP for rs3761549, FOXp3 gene alleles were amplified by same PCR conditions used in (8).

For rs3761548, a band at 209 base pair (bp) is an A allele,   band at 397 bp for C allele with a general product at 564 bp.

Enzymatic Digestion of rs3761549 PCR product of fox at 388 bp, was 308 and 80 bp for wild-type (CC) and three bands for (CT) genotype.

 

Statistical analysis

 SPSS software version 16 was used to calculate percentages, mean, and SD Chi-square was applied to compare the allele and genotype frequencies between the patients and controls and logistic regression analysis to identify the risk associated with genotypes. A p-value <0.05 was considered significant.   Results This study included 275 women median age was (50.5 ± 5.5  years), while (57%) of our  patients were premenopausal (43%) were postmenopausal, recruited to Mansoura Oncology Center and diagnosed with a primary breast cancer, in addition  123 healthy subjects age  and sex-matched were selected to act as a control group. The genotype distribution of  SNP rs3761548  was 51.6%,  CA , 37.1%, CC, and 11.3%  AA in patient group compared to  60% CA, 32% CC and 7 % AA in control group (figure 1)., while in    (rs3761549)  genotypic distribution of TT , CT, CC were 65.5%,32.7%, 1.8 % respectively  in cases   and    59.5%, 38.8%, and 1.7%  in control group respectively(figure 2). There were no differences observed (see table 4).     Logistic regression analysis was conducted for prediction of metastasis within BC patients, using ER, PR, Her2neo, CA 19-5 and Foxp3 SNPs as covariates. Her2 and CA19-5 were associated with risk of metastasis prediction in univariable and multivariable analyses (see table 5).   Discussion: In this study, no association was found between these two polymorphisms (rs3761548) and  (rs3761549) with breast cancer susceptibility. AC genotype of rs3761548 was found to be the most prevalent genotype followed by CC and AA with no significant association with the tumor stages stage. The Stratified data also revealed no association of the rs3761548 genotypes with the stage of tumor or receptor type. With regard to rs 3761549, the  most prevalent genotype was TT  followed by CT and CC  with no significant association with tumor stages.                       We inferred that Foxp3 rs37161548 and  rs3761549 were not potential polymorphic markers for tumor spreading in studied   Egyptian patients with breast cancer.  In an  Israeli population, three SNPs in Foxp3 gene including rs3761548 were studied using Quantitative  PCR and none of them showed a strong association with breast cancer risk(9).   In a meta-analysis conducted  on  an Asian study the frequency of C and T alleles in rs3761549 were found to be  similar in both patients and controls but  TT genotype was found to be completely absent in this study dissimilar to our study  where we found TT allele to be the highest presented genotype followed by polymorphic CT and lastly the CC group(10). In Indian patients with breast cancer, a study examined Foxp3 (rs3761548) in found a highly significant association with the advanced tumor stage (8). Another study on Indian female patients with breast cancer involved    five foxp3 SNPs included    rs3761548 and rs3761549 found that the frequencies of CC, CT, TT genotypes were similar in cases and controls (1%, 99%, and 0%) and there was a complete absence of mutant homozygous genotype (TT) in their study too. None of the demographic and clinical variables were shown to be influenced by this polymorphism(7.  An Iraqi study looked at the rs3761548 polymorphism in 100  breast cancer case and found an association of  (AA) homozygous genotype with breast cancer development and Concluded that (AA)  individuals had a higher susceptibility for developing breast cancer than individuals with other genotypes (CC, CA). 11 A relatively small number of studies could reduce the statistical power of the analysis., All published studies based on Indians, Iraqi and Israeli populations. Therefore, the results of these studies may be applicable to the specified ethnicity alone.   This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.