The Diagnostic Value of DNA Repair Gene in Breast Cancer Recurrence and Metastasis

Background: DNA repair genes play a vital role in the treatment of many cancers, and DNA repair genes can be used in breast cancer recurrence and metastasis research. We found that the expression of DNA repair genes in breast cancer patients after recurrence and metastasis is abnormal, however, the clinical predictive signicance of DNA repair genes is still elusive. Methods: The nested case-control method was used in patients with breast cancer recurrence and metastasis after surgery (n=109) and patients without recurrence and metastasis after surgery (n=109). The proteins and mRNA of DNA repair genes were detected by immunohistochemistry and Real-time PCR respectively. Results: and ERCC1 (OR=1.181, 95%CI: 1.032~1.353, P<0.05) were risk factors for postoperative recurrence and metastasis of breast cancer. Therefore, we used the ROC curve to study the optimal critical values of PARP1, XRCC4, and ERCC1, combined with the comprehensive judgment of clinical experience, we can conclude that PARP1 (cutoff value = 6, Se = 75.23%, Sp = 79.82%), XRCC4 (cutoff value = 6, Se = 78.9%0, Se = 79.82%), ERCC1 (cutoff value = 3, Se = 89.91%, Sp = 47.71%), suggesting that when the PARP1 score is higher than 6 or the XRCC4 score is higher than 6 or the ERCC1 score is higher than 3, the risk of recurrence and metastasis will increases (OR PARP1 = 14.941, 95% CI PARP1 : 4.004~55.758, P <0.05; OR XRCC4 = 16.740, 95% CI XRCC4 : 6.433 ~ 43.560,


Background
Breast cancer is the most general malignancy in China and either world, and its mortality rate rstly in female malignancy [1]. In recent years, the survival rate of breast cancer has been prominently improved by comprehensive treatment such as surgery and chemotherapy. Nevertheless, approximately one-third of breast cancer patients will present metastases. Recurrence and metastasis are bound up with the prognosis of breast cancer patients and it is also the soprattutto cause of death in breast cancer patients [2]. Studies have found that breast cancer patients' postoperative recurrence and metastasis are related to age, tumor pathological tissue type, clinical analysis, postoperative chemotherapy, and endocrine therapy [3]. At the same time, some people have also studied tolerance to treatment as one of the in uencing factors. However, some tumor cells can pass activating self DNA repair mechanisms to resistance to DNA damage drugs [4][5][6]. So some studies have proposed that DNA repair genes have a relationship with the recurrence and metastasis of breast cancer [7].
More and more studies have found that tumor response to chemotherapy drugs is closely related to the regulation of the DNA repair system [8]. Four major DNA repair pathways are currently known: nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), and double strand break repair (DSBR). In cancer, we found that ERCC1, XPA, XRCC1, PARP1, MSH2, MLH1, 53BP1, XRCC4 are closely related to cancer recurrence and metastasis. The ERCC1 and XPA genes in the NER pathway have con rmed that ERCC1 is associated with recurrence and metastasis in ovarian cancer, testicular germ cell tumors and gastric cancer, and high expression of ERCC1 will lead to an increased risk of recurrence and metastasis [9]. BER as one of the DNA repair mechanisms, PARP1 may be one of the major genes involved in tumor cell recurrence and metastasis [10]. In vitro and in vivo studies have suggested that inhibition of PARP1 can reduce tumor cell repair function, thereby enhancing the therapeutic effect of radiotherapy and chemotherapy on tumors [11,12]. DSBR is the most common but most severe type of DNA damage in eukaryotic cells, and is mainly repaired in mammals through non-homologous end joining (NHEJ). Xiaoyan Li et al. found that 53BP1 affects breast cancer patients' sensitivity to 5-Fu, it will results poor prognosis [13]. Some scholars have found that the research on DNA repair genes such as MLH1, XRCC4, 53BP1, ERCC1 and XPA in breast cancer is mainly expressed in breast cancer tissues and its clinical signi cance [14][15][16]. XRCC1 overexpression can inhibit breast cancer cell proliferation and metastasis [17]. MSH2 mutation may be involved in the occurrence and development of early-onset breast cancer in the family of Lynch syndrome [18]. Among them, PARP1 inhibitors have entered the trial stage of clinical treatment of breast cancer [19]. But no further study of their recurrence and metastasis with breast cancer.
DNA repair requires the role of multiple enzymes and genes. A single gene has a limited role in damage repair. Analyzing only an enzyme or gene is not enough to re ect the complexity of DNA repair. Due to ERCC1, XPA, XRCC1, PARP1, MSH2, MLH1, 53BP1, and XRCC4 are more studied in other cancer. But there are few studies in breast cancer recurrence and metastasis. So in this study, nested case-control study was used to explore the expression levels of major molecules of the DNA repair system ERCC1, XPA, XRCC1, PARP1, MSH2, MLH1, 53BP1, and XRCC4 in patients with recurrent and metastatic breast cancer, in order to provide theoretical support for clinical treatment and prognosis.

Sample
The data come from the follow-up cohort of the Cancer Institute of Southwest Medical University. The cohort was collected and followed up in January 2013 at the Department of Breast Medicine, Southwest Medical University Hospital. Cancer patients have collected approximately 1360 cases. Recurrence and metastasis cases and controls selected in this study were collected from this cohort. Patients with recurrence and metastasis during the follow-up period were included in the recurrence and metastasis case group. At the same time, the control group(non-recurrence) was selected according to the 1: 1 pairing principle in this cohort (n = 109, the matching condition was age ± 3 years, the operation time was consistent, and the treatment plan was consistent). The control group(non-recurrence) was surviving patients in the cohort, and no recurrence and metastasis occurred. Finally, 109 cases and 109 controls were included in January 2018. The pathological data used in this study were from the Department of Pathology, A liated Hospital of Southwest Medical University. The data collected included clinical data, pathological data, and treatment options, as well as para n specimens from patients with breast cancer.
After preliminary diagnosis of breast cancer patients in the a liated hospital of Southwest Medical University, materials were obtained from the Department of Pathology.  Table 1.  Anti-sense 5'-GTGATCTCCTTCTGCATCCTGT-3' 2.3 Immunohistochemical detection of DNA repair gene protein expression in para n-embedded tissues of breast cancer patients Para n sections (3 µm) were dried, depara ned, and rehydrated in graded alcohol to water. Heatmediated antigen retrieval was performed using pressure cooker treatment for 10 min in EDTA buffer (pH 9.0). The slides were incubated for 120 min at 25℃ with primary mouse anti-human monoclonal antibodies to ERCC1, XPA, XRCC1, PARP1, MSH2, MLH1, 53BP1 and XRCC4 (Dako, DK). After washing, the sections were incubated with the second antibody (Envision, HRP rabbit/mouse, Dako, DK) for 30 min at 25℃. Negative controls were obtained by omitting the primary antibody. The slides were visualized by DAB.
Expression of 8 DNA repair protein was determined in the nucleus of tumor cells. Five high-power elds (200×) were randomly selected. The extent of the staining was categorized into ve semi-quantitative classes based on the percentages of positive tumor cells: 0, < 5% positive cells; 1, 6-25% positive cells; 2, 26-50% positive cells; 3, 51-75% positive cells; and 4, > 75% positive cells. Staining intensity was scored as 0, negative; 1, weak; 2, moderate; and 3, intensive. Multiplication of the intensity and the percentage scores gave rise to the nal staining score [20].

Statistical analysis
All data were analyzed using SPSS 22.0 statistical software and MedCalc software, and bilateral P values below 0.05 were considered statistically signi cant. The correlation between DNA repair genes and breast cancer recurrence and metastasis was analyzed by χ 2 test, rank sum test, cox risk model and other statistical methods.

The protein expression of DNA repair genes
Immunohistochemical staining results ( Fig. 1) shows that: DNA repair gene protein positive expression mainly in the cytoplasm, repair gene is highly expressed in the transfer of recurrence group in the breast tissue. The MSH2, MLH1, PARP1, XRCC1, XRCC4, 53BP1, ERCC1, XPA of the recurrence and metastasis group were higher than those of the control group(non recurrence and metastasis group)(P < 0.05), we can concluded that all of them are related to the prognosis of recurrence and metastasis of breast cancer.

Clinicopathologic feature of breast cancer patients
The PR index of the recurrence and metastasis group was lower than that of the control group(non recurrence and metastasis group)(P < 0.05). P53, WHO Grade and Lymph node metastasis of the recurrence and metastasis group was higher than that of the control group(non recurrence and metastasis group) (P < 0.05). There was no signi cant difference in Age, ER, HER2, E-cad, Ki67, TNBC, In ltration and Tumor diameter between the two groups (P > 0.05), as shown in Table 2.

Diagnostic value of DNA repair genes
In the previous study, we found that the protein expression of PARP1, XRCC4 and ERCC1 is related to the recurrence and metastasis of breast cancer(P < 0.05). However, the IHC score is a continuous variable and there is no accurate cut-off value for diagnosis. In order to further understand the role of PARP1, ERCC1 and XRCC4 in the prognosis of breast cancer recurrence and metastasis. Therefore, we used the ROC curve to study the optimal critical values of PARP1, XRCC4, and ERCC1, combined with the comprehensive judgment of clinical experience, we can conclude that PARP1 (cutoff value = 6, Se = 75.23%, Sp = 79.82%), XRCC4 (cutoff value = 6, Se = 78.9%0, Se = 79.82%), ERCC1 (cutoff value = 3, Se = 89.91%, Sp = 47.71%), suggesting that when the PARP1 score is higher than 6 or the XRCC4 score is higher than 6 or the ERCC1 score is higher than 3, the risk of recurrence and metastasis will increases. As shown in Fig. 3 and Table 5. Based on the cutoff value predicted by ROC curve and clinical experience, PARP1, XRCC4 and ERCC1 were divided into the high expression group and the low expression group according to the cutoff value, and the effects of PARP1, XRCC4 and ERCC1 on breast cancer recurrence and metastasis were veri ed again. Among them, the risk of recurrence and metastasis in the PARP1 high expression group was 14.235 times that of the low expression group (OR = 14.235, 95%CI:5.59 ~ 36.245, P < 0.05). The risk of recurrence and metastasis in the XRCC4 high expression group was 16.740 times that of the low expression group(OR = 16.740, 95%CI: 6.433 ~ 43.560, P < 0.05). The risk of recurrence and metastasis in patients with ERCC1 high expression group was 5.285 times that of the low expression group (OR = 5.285, 95%CI :1.843 ~ 15.156, P < 0.05). The risk of recurrence and metastasis in patients with lymph node metastasis ≥ 10 was 9.175 times that without lymph node metastasis (OR = 9.175, 95%CI :2.842 ~ 29.625, P < 0.05). As shown in Table 6.   Fig. 4 and Table 8.

Discussion
Chemotherapy is one of the most important treatments for breast cancer after operation. At present, the survival rate of patients has been effectively improved by referring to ER, PR, HER-2, Ki67, TNBC and other indicators. However, the study found that there are still about 30% recurrence and metastasis rates [21]. It shows that the formulation of treatment plans based on the above pathological indicators may be incomplete, and there are other indicators for guiding treatment that can be excavated. In addition, there is still rise the survival rate of patients for adjustment in the formulation of treatment plans. Nowadays, more and more studies have found that the recurrence and metastasis of tumors are closely related to the DNA repair regulatory system related to drug resistance [22][23][24].
At present, not a few DNA repair genes, such as PARP1, XRCC1, 53BP1, ERCC1, have been found to be associated with the prognosis of breast cancer [25,26]. PARP1 promotes the expression of HIF-1α by activating nuclear factor-κB (NF-κB) and promotes the polarization of macrophages M2, leading to the up-regulation of tumor-related macrophages (TAMs), such as tumor necrosis factor-α (TNFα) and IL-6, thus promoting the proliferation, invasion and metastasis of tumor cells, promoting the formation of tumor microvessels and microlymphatics [27]. And ERCC1 proteins can form heterodimers with DNA repair enzyme de ciency complementary gene (XPF) and perform functions by splicing at the 5'end of the damaged DNA single strand. Overexpression of ERCC1 proteins can lead to rapid repair of damaged DNA stagnating in G2/M, leading to resistance to cisplatin chemotherapeutics [28]. In this study, nested case-control single factor analysis showed that protein levels of PARP1, XRCC1, 53BP1, ERCC1 recurrence and metastasis group was higher than control group. We further included that PARP1 (OR = 1.485, 95% CI:1.279-1.725), ERCC1 (OR = 1.181, 95% CI:1.032-1.353) as independent factors for recurrence and metastasis of breast cancer after operation. The results are similar to those of Sunada, S. et al [29]. It shows that XRCC1 and 53BP1 become ineffective factors after excluding other confounding factors, while the high expression of PARP1 and ERCC1 in the course of chemotherapy will destroy the susceptibility of breast cancer cells, it can be make breast cancer cells less susceptible to chemotherapy, leading to resistance of breast cancer cells and even recurrence and metastasis of chemotherapy failure, which is affecting breast cancer. The independent prognostic factors of metastatic patients are not different from the existing studies.
In addition to the above genes related to breast cancer resistance [30], the prognosis-related DNA repair genes MSH2, MLH1, XRCC4 and XPA [31][32][33], which are frequently studied in the elds of colorectal cancer, lung cancer, prostate cancer and esophageal cancer, were also included in the study and analysis.
Univariate analysis showed that MSH2, MLH1, XRCC4 and XPA in patients with recurrence and metastasis were higher than those without recurrence and metastasis. In binary logistic regression, only XRCC4 (OR = 1.419, 95% CI:1.217 ~ 1.656) was a risk factor for recurrence and metastasis of breast cancer, suggesting that XRCC4 was also associated with recurrence and metastasis of breast cancer after operation. The results of high expression of XRCC4 in esophageal and colorectal cancer suggest poor prognosis. It is possible that XRCC4 itself could form dimers and interact with BRCT domain (BRCA1-terminal, BRCT) at the C end of Ligase IV protein through its α spiral stem ring structure in the central region. Damage repair defects lead to accumulation of genetic damage, and then increase the susceptibility of the body to tumors, thereby increasing the risk of recurrence and metastasis of breast cancer patients.
However, the direct use of IHC score to analyze the recurrence and metastasis of breast cancer after surgery is of little signi cance. In order to further understand the role of PARP1, ERCC1 and XRCC4 in predicting the prognosis, metastasis and recurrence of breast cancer, we also studied the best cut-off value of PARP1, ERCC1 and XRCC4. The results showed that PARP1 (Cut-off Value = 6, Se = 75. 23 . It can be seen that the combined detection of three DNA repair proteins has higher clinical diagnostic value than the single determination. It has been pointed out that PARP1 promotes the expression of HIF-1α by activating nuclear factor κB (NF-κB) and promotes the polarization of macrophages M2, leading to the up-regulation of tumor-related macrophages (TAMs), such as tumor necrosis factor alpha (TNFα) and IL-6, thus promoting the proliferation, invasion and metastasis of tumor cells, promoting the formation of tumor microvessels and microlymphatics [34]. Overexpression of ERCC1 also up-regulates TNF-α, while XRCC4 in NHEJ pathway activates DNA-dependent protein kinase complex (DNA-PK), which is essential for the adhesion of monocytes to TNF-α treated endothelial cells. Other studies have found that in the process of tumorigenesis and development, most patients are accompanied by chronic in ammation, and the chronic protraction of in ammation affects the condition of tumors, leading to recurrence and metastasis of tumors [35]. Tumor necrosis factor-α (TNFα) is closely related to the occurrence of cancer.  The diagnose ROC curve of PARP1, XRCC4 and ERCC1 Page 21/24

Figure 2
The diagnose ROC curve of PARP1, XRCC4 and ERCC1 Figure 3 Shows the effect of breast cancer recurrence and metastasis on the mRNA expression of DNA repair gene. Data are described as Mean (SD), n = 109. Statistical differences are expressed as: *P < 0.05.