Influence of ABCB1 C3435T gene polymorphisms on tumor response to neoadjuvant chemotherapy in locally advanced breast cancer patients from Northeastern Brazil.

Background: Breast cancer (BC) is the most common tumor and the leading cause of cancer-related death among the female population worldwide. To evaluate the association between the ABCB1 C3435T single gene nucleotide polymorphisms (SNPs) with the response to neoadjuvant chemotherapy in women with breast cancer. Methods: This study included 32 female patients who received neoadjuvant chemotherapy. The polymorphisms were genotyped through real-time allele-specific polymerase chain reaction (PCR). The statistical analysis was performed using the Fisher's exact test or Pearson's chi-square test in the Statistical Package for Social Sciences (SPSS) version 20.0 software. Results: The genotypes found for the C3435T polymorphism were in Hardy-Weinberg equilibrium and their genotypic distributions were CC= 10 (31.1%), CT= 14 (43.8%), and TT= 08 (25.0%) with χ2: 0.86 and p-value > 0.05. Allele frequencies were C = 0.54 and T = 0.46. There were no significant statistical differences between genotypes considering the response to neoadjuvant chemotherapy and immunohistochemistry; the presence of the T allele was associated with worsen axillary status response to neoadjuvant chemotherapy. Conclusion: No definite association between the presence of C3435T polymorphism and the response to neoadjuvant chemotherapy was observed. Further studies in Brazil involving larger samples will contribute to validating the results of this study. percentage test. The Fisher's exact or Chi-square association tests were used to evaluate the relationship of the tested genotypes (C3435T polymorphism genotypes) and the response to the neoadjuvant chemotherapy, and the lymph node response after therapy. Analyzes were performed using the Statistical Package for the Social Sciences (SPSS)Ò software, version 20.0 for Windows, adopting a 95% confidence and p-value < 0.05 as significant.

cancer (LABC) represents a heterogeneous group of diseases associated with poor prognoses. According to the TNM staging system from the International Union Against Cancer (UICC)/American Joint Cancer Committee (AJCC), primary BC with extension to the skin, chest wall or inflammatory carcinoma, with or without lymph node involvement, and without metastasis can be included in stage III and considered a LABC [2].
Several studies are being conducted on the pharmacogenomics of antineoplastic chemotherapy drugs to reduce the dose and minimize undesirable effects by individualizing treatment. Neoadjuvant chemotherapy (NAC) is one of the most widely used forms of treatment for its effectiveness in certain potentially lethal neoplasms such as those in the breast, especially in patients with LABC [3].
NAC is a systemic therapy given before surgery to minimize the extent of surgery, reducing tumor size, and facilitating a conservative breast approach [4]. Researches had shown a significant increase in pathological response in primary BC patients when docetaxel was administered as the sequential therapy to doxorubicin and cyclophosphamide in a neoadjuvant scenario [5].
Randomized studies showed similar NAC responses compared to adjuvant therapy, [6] encouraging the use of docetaxel in neoadjuvant settings. NAC followed by surgery, and adjuvant radiotherapy is the standard treatment for LABC patients. [7]. However, the frequency of tumor regression shows rates ranging from 10 to 50%, and up to complete pathological response, however, NAC is ineffective in a large number of patients [8]. The reasons for this heterogeneity may be due to genetic and non-genetic factors [9].
Genetic factors may influence the therapeutic efficacy and interfere with the survival of patients with BC, even those at similar clinical stages. Therefore, the availability of new biomarkers that can more accurately predict prognosis and treatment response is a central issue for improving therapeutic strategies [10]. ABCB1, the multi-drug resistance (MDR) gene, is responsible for reducing intracellular drug levels by the ATP-dependent efflux. The main factors leading to simultaneous resistance to various drugs are drug expulsion after entering the plasma membrane via efflux pumps, reduced uptake, altered target enzymes, altered drug metabolism, increased DNA repair, or failure of regulatory mechanisms of programmed cell death (apoptosis). One of these mechanisms is through host efflux pumps, and among them, the P-glycoprotein (P-gp), [10,11] which is involved in the chemotherapy drug resistance presented by tumor cells [12]. Because the current knowledge in this field is inconsistent and sometimes conflicting, further studies are needed to confirm the clinical importance of single gene nucleotide polymorphisms (SNPs) encoding these multidrug resistance enzymes [13].
This is the first study carried out with the primary objective of evaluating the association between the ABCB1 rs1045642 (C3435T) gene polymorphism and the response to NAC in patients with BC in Northeastern Brazil. This study consisted to evaluate the association between the ABCB1 C3435T SNPs and the response to NAC in women with BC.

Results
The demographic characteristics, risk factors, and tumor characteristics of women with BC are described in Table 1. Data are expressed in average + SD The genotypic distribution showed 10 (31.2%) as homozygous (CC), 08 (25%) as homozygous (TT), and 14 (43.8%) as heterozygous (CT).
No association between the presence of the C allele and response to NAC was observed, nor its association with the lymph node status, hormone receptors, HER-2, and KI-67 (Table 2). *p < 0.05 was considered significant. Table 3 shows that, when assessing the association of the T allele with the NAC response, a significant difference was observed about the lymph node status after NAC, however, without association with tumor size reduction or immunohistochemical markers. No statistical difference was observed in any of the studied variables when the association between the wild homozygote (CC) and the mutated (TT) genotype was evaluated (Table 4).  [18]. In a meta-analysis study, nine articles involving the ABCB1-C3435T gene polymorphism (770 patients) indicated that the polymorphism was not associated with the NAC response in BC patients [16,18]. These results are similar to those in our study where we found no association between the SNP C3435T polymorphism of the MDR-1 gene and tumor response to NAC, hormone receptors, and HER-2 and Ki-67 expression.
This study showed that the presence of the T allele was related to worse axillary status .
The last study that mainly assessed axillary status was conducted in Brazil and showed discordant findings, i.e., no relationship between axillary involvement and the relationship between alleles [17]; this discordance may be justified by the difference in sample and country region where the studies were conducted.
The present study is extremely important in the attempt to search for markers involved with the NAC response in women with BC, which could modify the treatment approach adopted for women with BC, especially within the Northeastern region of Brazil.

Conclusions
The study shows that there was no statistical difference between the presence of C3435T polymorphism genotypes regarding the response to NAC; nevertheless, a worse response of axillary status was observed in the presence of the T allele. Further studies in Brazil involving larger samples will contribute to validating the results of this study.

Study Design
This was an observational and prospective study in women with histopathological diagnosis of BC at clinical stage III, without previous treatment, and submitted to NAC.  [14].
The exclusion criteria were intolerance to NAC, which prevented the completion of treatment, pregnancy, male gender, and metastatic or bilateral disease.