Relationship Between Gene Expression Of TOP2A, XRCC1 And Clinicopathological Features Based On Breast Cancer Molecular Subtyping

Breast cancer (BC) is one of the common malignancies for women worldwide. It is reported that the overall incidence of breast cancer increased by 47% in nearly 10 years. Breast cancer has become one of the malignant tumors with the highest female incidence in China. As well as many malignant tumors, the treatment of breast cancer is a comprehensive therapy based on surgical operation. Although the recurrence and metastasis rate are significantly reduced by the comprehensive treatment including surgery,chemotherapy, radiotherapy, endocrine therapy and targeted therapy, there are still about 30% of recurrence and metastasis rates. There are some rules to follow about local treatment of breast cancer-surgery, radiotherapy and systemic therapy-endocrine therapy, targeted therapy. For the systemic chemotherapy of the breast cancer, effective chemotherapy drugs are plenty, including anthracycline, taxane, platinum etc. How to choose the optimal chemotherapy regimen and reduce unnecessary chemotherapy damage are very important.There are a lot of options and uncertainties when clinicians carry out the chemotherapy. At the time of advocating the precise medical treatment, how to choose the right chemotherapy drugs based on molecular biological characteristics of each breast cancer patient and obtain the best results while minimizing the side effects of the treatment are the main subject of this research. A basic question to solve this research problem is to clarify the susceptibility of the breast cancer to the drug targets. Evidence-based medicine has been shown that anthracycline is the cornerstone of breast cancer chemotherapy,while platinum is considered as the second choice after anthracycline and (or) taxane treatments become failed. This study, based on the latest molecules genotype of St.Gallen Consensus for breast cancer, detect the expression diffidences of TOP2A (anthracycline drug resistance gene), XRCC1 (platinum drug resistance gene) and analyze the clinic pathological parameters, while data provide a reference to the clinical fundamental studies of breast cancer and the individualized precision treatment of the breast cancer patients. Meanwhile, the XRCC1 Arg194Trp polymorphism analysis and protein expression of the current clinical treatment difficult type-triple negative breast cancer are studied to learn more about the occurrence and development of breast cancer. This study was conducted in two parts:Part I:TOP2A expression in different molecular subtypes of breast cancer and its clinical significanceBreast cancer is a group of tumors with significant heterogeneity, and there are some differences of clinical characteristics, treatment effect and prognosis among different molecular subtypes of breast cancer. Although targeted drug herceptin therapy can be used for HER2 over-expression type, but only one third of the patients are drug sensitive. Triple negetive breast cancer is lack of specific treatment with poor prognosis. Study of invasion and metastasis mechanisms of different molecular subtypes of breast cancer will help clinicians develop individualized treatment programs to improve the survival rate of patients with breast cancer. Studies have shown that, TOP2A was associated with breast cancer. Currently, there is few research reports associated with TOP2A expression of different molecular subtypes of breast cancer. Through the study of TOP2A expression of different molecular subtypes of breast cancer and their clinicopathological correlations, we aim at providing the new ideas and invasion and metastasis mechanisms for individualized treatment and meaningful experimental basis for different molecular subtypes of breast cancer, particularly for HER2 over-expression and triple-negative breast cancer.Objective:To investigate the expression, clinical significance, correlations of molecules and protein expression level clinicopathological features of TOP2A in different molecular subtypes of tissues of breast invasive ductal carcinoma patients and paracarcinoma tissues.Methods:Using RT-PCR method to detect the expression difference of TOP2AmRNA in breast cancer tissue and para-carcinoma tissue. Using immunohistochemical method to detect the expression level of TOP2A protein in breast cancer tissues; Immunohistochemical method is also used to detect the expression status of ER, PR and ki-67 of the breast cancer cells. Immunohistochemical and FISH method are used to detect the HER2 status. Based on the latest international consensus of St.Gallen, the breast cancer is divided into four kinds of molecular subtypes, including Luminal A, Luminal B, HER2 over-expression and triple negative type. According to the biological behavior characteristics of different subtypes, we discussed the differences and the relations between TOP2AmRNA and expression in protein level. Further statistical analysis of the relationships between the high expression rate of TOP2A protein and the age, tumor T stage, histological grade, axillary lymph node metastasis, ER, PR, HER-2, P53 and Ki-67 of the patients.Results:The results showed that in resources of 118 cases of breast cancer paired into groups, the expression amount of TOP2A in breast tissues was significantly higher (p <0.05) than adjacent tissues; From the multiple comparisons of the expression level of TOP2AmRNA among the different molecular subtypes in breast cancer, we found TOP2AmRNA expression was more common in LuminalB and HER2 over-expression; the high expression of TOP2A protein was also more common in Luminal B type and HER2 over-expression type, and the protein expression of TOP2A was consistent with the expression of mRNA; mRNA levels and protein levels of TOP2A had an positively correlated relationship and was statistically significant (p 0.001, r=0.704). The high expression of TOP2A protein had no significant correlations with age, PR expression status (p>0.05), and was related to tumor size, axillary lymph node-positive, ER-positive, HER-2 gene amplification, Ki-67 expression (p<0.05).Conclusion:1, This study further validated the TOP2A mRNA had significantly higher expression in breast cancer, TOP2A protein was more common in HER2 over-expression type and triple negative type than in the Lumianl type whose prognosis was relatively good, and confirmed the relationship between TOP2A and the pathogenesis of breast cancer from gene and protein levels.2, In molecular subtypes of breast cancer, the high level expression of TOP2A mRNA and protein was more common in Luminal B type and HER2-positive type, and their results had a positive correlation. Thus we considered using simple and practical immunohistochemical method to detect the TOP2A expression instead of mRAN testing, which was in line with the St.Gallen consensus recommendations of using immunohistochemistry to divide the molecular type of breast cancer. The in-depth research of the two types of breast cancer provided experimental evidence to clarify the mechanism of invasive and metastasis of breast cancer and find new therapeutic targets.3,TOP2A expression was positively related to tumor size, axillary lymph node-positive, ER-positive, HER-2 gene amplification and Ki-67 expression. So, the over-expression of TOP2A genes may contribute to breast cancer progression, and thus was related to the degree of malignancy of breast cancer.4,TOP2A was a resistance gene and the target of detection for anthracycline drugs. In clinical work, detecting TOP2A expression by IHC method was the gist for anthracycline selection. TOP2A was not only the detection target of anthracycline drug efficacy, but also as likely as HER2, may be a new target for breast cancer therapy.Part Ⅱ:Research on polymorphisms and protein expression of XRCC1 gene based on molecular subtypes of breast cancerBreast cancer is one of the most common cancers for women; the occurrence and development of breast cancer are very closely related to genetic factors. Searching for the molecular markers related to the prognosis of breast cancer has important significance for cancer therapy. X-ray repair cross-complementing gene 1 (XRCC1) is an important gene involved in DNA damage repair, and plays a key role in maintaining genome stability. Polymorphic change of XRCC1 Argl94Trp site may affect the normal function of XRCC1 protein, resulting in reduced DNA repair capacity, and thus has an association with a variety of cancer biological behavior susceptibility or chemosensitivity. This study detected gene polymorphism of XRCC1 Arg194Trp site and XRCC1 protein expression to understand its relationship with the molecular subtypes of breast cancer and the clinical and pathological features. According to the literature, XRCC1 is a drug resistance gene for platinum. The purpose of this study is to further investigate the value of XRCC1 in the selection of platinum drugs for breast cancer therapy.Objective:Through the study of Arg 194Trp polymorphism of XRCC1 gene and XRCC1 protein expression of 118 cases of sporadic breast cancer, we explore the expression difference of XRCC1Arg 194Trp and XRCC1 protein expression in breast cancer molecular subtype, its correlation with clinical pathology parameters, and correlation between genetic polymorphisms and protein expression, and further explore its potential values in breast cancer platinum drug selection.Methods:Randomly collect the pathological data of 118 cases of breast cancer without clinical preoperative treatment in Qianfoshan Hospital in Shandong Province from March 2014 to December 2015, and re-read the biopsy. According to the St.Gallen consensus molecular genotype in 2015, pyrosequencing was used to detect the polymorphism changes of XRCC1 Arg194Trp in the peripheral bloods of 118 cases of breast cancer patients. Immunohistochemstry was used to detect the status of the protein expression of XRCC1 in breast cancer tissues; Explore XRCC1 Arg194Trp polymorphism, the polymorphism distribution, expression difference, the relationship between clinicopathological features and XRCC1 protein expression in different breast cancer molecular subtype.Results:1,XRCC1 (Arg194Trp) had three visible genotypes, including CC, CT and TT. The results of the study found that in 118 cases of breast cancer patients, CC genotype accounted for 53.39%(63/118), CT genotype accounted for 36.44%(43/118), and TT genotype accounted for 10.17%(12/118).2, There was no connection (P<0.05) between XRCC1Arg194Trp polymorphism and XRCC1 protein expression.3,XRCC1Arg194TrpCT/TT type was more common in HER2 and triple negative breast cancer, while the CC genotype is more common (P<0.05) in Luminal breast cancer; XRCC1Arg194Trp site polymorphism had no significant statistical differences (p>0.05) between Luminal A and B or HER2 over-expression; XRCC1 Arg 194Trp site polymorphism had statistical differences (p<0.05) between the Luminal A type and triple negative type.4,XRCC1 protein expression had no significant statistical differences (p<0.05) between LuminalA/B type breast cancer and HER2 over-expressing/triple negative type. There was no association between the two groups; XRCC1 protein in type B or type LuminalA had no statistically significant difference (p>0.05) between HER2 over-expression and XRCC1 protein expression, and it was significantly different (p <0.05) between LuminalA type and triple negative.5,The polymorphism distribution of XRCC1 Arg194Trp, low expression of XRCC1 had no significant correlation (p>0.05) with age, tumor size, histological grade, but were significantly correlated (P<0.05) with axillary lymph node metastasis, hormone receptor status, HER2 expansion status, Ki67 expression level.6,The low expression of XRCC1 had no significant correlation (p>0.05) with age, tumor size, histological grade, but were significantly correlated (P<0.05) with axillary lymph node metastasis, hormone receptor status, HER2 expansion status, Ki67 expression level.Conclusion:1, The polymorphism of XRCC1Arg 194Trp don’t affect the expression of XRCC1 protein expression, indicating that the XRCC1 protein level was influenced not only by XRCC1Arg 194Trp sites change, but also affected by the changes of other sites.2, XRCC1Arg 194Trp CT/TT type (heterozygous/homozygous mutant type) is more common in HER2-positive type and triple negative type. Combined with drug susceptibility literature reports, we recommend the choice of platinum drugs.3, The low expression of XRCC1 is more common in triple negative type among the four kinds of molecular subtype for breast cancer. Triple negative breast cancer is the most malignant type with poor therapeutic effect.Thus We recommend immunohisto-chemistry to routinely test XRCC1 protein for triple negative breast cancer, and guide the selection of platinum-based chemotherapy drugs for triple negative breast cancer.4, The gene polymorphism of XRCC1Arg194Trp, the low protein expression of XRCC1 are related to the state of ER, PR, HER2, and Ki67, and may be related to the prognosis of the breast cancer.