Study characteristics affecting the responses to neoadjuvant chemotherapy and the prognosis of patients with bilateral breast cancer: A retrospective analysis

BackgroundBilateral breast cancer (BBC) is defined as breast cancer diagnosed in both breasts in the same patient. Neoadjuvant chemotherapy (NAC) is a well-established approach to evaluate the tumor response to chemotherapeutic agents. The consensus is that different responses in characteristics after NAC can affect prognosis in unilateral breast cancer (UBC), but little is known about the responses of the BBC to NAC. This analysis explored the characteristics that can affect the prognosis of patients with BBC.MethodsThe characteristics of patients diagnosed with BBC (n = 126) was collected and the immunohistochemistry staining was used to detect expression levels of estrogen receptor (ER), progesterone receptor (PR), Ki67, and HER2. A statistical analysis of the differences was performed to identify the factors that affect survival times in all patients with BBC.ResultsA logistic regression indicated that the status of sentinel and axillary lymph node, expression of PR of the right breast tumor, and molecular subtype of the right breast tumor might relate to survival times. Tumor size, status of axillary lymph node, clinical stage, tumor type, histological grade, and molecular subtype of the left breast tumor might have a more profound effect on the survival time than the right breast tumor in the synchronous breast cancer (SBBC) patients. A multivariate analysis of overall survival times in patients with metachronous breast cancer (MBBC) showed that age was the only factor affecting survival time. After NAC treatment in SBBC patients, the Kaplan-Meier survival estimate showed that a decrease in tumor size, clinical stage, Ki67 and P53 levels were positive for a prolonged life span. However, a decrease in ER, PR, and HER2 were negative for prolonged life span. Changes in tumor type and molecular subtype also influenced the survival

SBBC and MBBC usually are defined according to the length of the diagnosis time of tumors in both breasts. This classification cannot specify the exact time interval, which ranges from one month to five years, according to other studies [17,18]. In our study, we used a cut-off time interval of one year to distinguish between the two types of BBC. The regime of NAC treatment that patients with SBBC received was docetaxel, epirubicin, and cyclophosphamide and all treatment strategies were carried out in accordance with the guidelines of Chinese society of clinical oncology 2012.v1. All experimental protocols were approved by Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University.

Immunohistochemistry
The tissue sections of the core needle biopsies taken before NAC and the resection of surgery were evaluated by the pathology department of Chongqing Medical University. The staining intensity in the immunohistochemistry was recorded in terms of percentage for estrogen receptor (ER), progesterone receptor (PR), Ki67, and P53. Samples were defined as ER (+) or PR (+) with at least 1% of nuclei stained, and four grades were classified according to percentages ranging from 0-95%: grade 1 0%, grade 2 1-30%, grade 3 31-60%, and grade 4 61-95%. HER2 status was recorded as grades: -or + was negative, 2+ was doubtful, which required fluorescence in situ hybridization (FISH): positive grades showed a more than 2.2-fold increase, and 3+ was positive.

Follow-up
The followed up was carried out by telephone and records of examination in our hospital. If all the common contact was lost, survival state was confirmed by checking for the patient's identification in the police system. The mean follow-up (original data is provided in Supplementary table 1). It is clear in the table 1 that patients with SBBC more often had an early clinical stage (stage I and II at diagnosis) on the right side (P = 0.007), low histological grade on the left side (P = 0.021), and higher HER2 expression on the left side (P = 0.017) than patients with MBBC. A significantly higher expression of hormone receptor (HR) was evident in patients with SBBC compared with patients with MBBC. This led to the conclusion that more luminal subtypes, especially luminal A, are found in patients with SBBC, and more triple-negative subtypes are found in patients with MBBC. Considering the time interval of the two sides and drug resistance, patients with MBBC usually received two regimens of chemotherapy.

MBBC
At the end of follow-up, 101 patients had survived. The differences in the patients and disease characteristics between the survival and non-survival groups are given in Table 2. Interestingly, some obvious differences were identified in these two groups. For the survival group, statistically different characteristics included a smaller tumor size (left P = 0.074; right P = 0.022), negative sentinel lymph node (left P = 0.000; right P = 0.000) of both sides, an earlier clinical stage at diagnosis (left P = 0.038; right P = 0.028), and lower expressions of ER (left P = 0.037; right P = 0.079) and PR (left P = 0.059; right P = 0.047). On the contrary, in the nonsurvival group, higher expression of ER and PR, larger tumor size, and positive sentinel lymph nodes and axillary lymph nodes could be seen. These data showed that higher expression of ER and PR, larger tumor size, and positive sentinel lymph nodes and axillary lymph nodes were associated with survival. 1.14-3.11; P = 0.014), and molecular subtype (HR = 1.952; 95% CI: 1.19-3.20; P = 0.008), all influenced survival. This result indicated that the tumor in the left side of breast might have a more profound effect on survival state than the right side. To exclude whether differences in the characteristics of the two sides contributed to this result, the characteristics between the two sides were compared, but identified no difference (

Changes of characteristics after NAC in SBBC
In patients with SBBC, 16 received NAC treatment, and only 10 patients survived during the follow-up time. Because changes in the characteristics of the tumor after NAC predict a different prognosis in patients with UBC [10-13], characteristics through core needle biopsy and surgical tissue section were collected (Supplementary table 5 and 6). The changes in characteristics were compared before and after NAC between the survival group and non-survival group (Table 6) and also recorded the response to NAC (Table 7). However, no statistical difference was found.  (Table 8). It is also reminded that changes in tumor type and molecular subtype might influence survival time.
Although the life span differed between groups that showed a decrease or no decrease in all characteristics of tumor in both side breasts, this difference did not reach the traditional statistically significant level.
According to patient response to NAC treatment and RECIST 1. however, this difference did not reach the traditional statistically significant level (P = 0.518; Table 9). The factors affecting survival also indicated differences in the cumulative survival time between these two groups ( Figure 2).

Discussion
In our study, we found that some patients with SBBC had different responses to NAC in the tumor in two sides of breasts, but whether this phenomenon affected prognosis is uncertain. No specific guidelines or consensus about follow-up treatment are available. This study was conducted to summarize the clinical characteristics and prognosis of BBC in China and focused on patients with SBBC who received NAC. This study verified the characteristics and prognosis of BBC in China and provided clinical data to explore follow-up treatment plans for patients with BBC.
The incidence of BBC in our study was 2.51% of all patients with BC. Of the total, 64 were diagnosed with SBBC (the incidence was 0.99%) and 98 were diagnosed with MBBC (the incidence was 1.52% . We also found that patients with SBBC more often had early clinical stage at diagnosis of the right side of the breast, and low histological grade of the left side breast, but higher HER2 expression of the left side breast than patients with MBBC. The reasons for these differences and whether they influence prognosis or treatment strategy require further exploration. The univariate analysis of survival times showed that the survival group had an earlier clinical stage in both sides of the tumor compared with the non-survival group, and a higher expression level of ER and PR was seen in the survival group (Table 2). The multivariate analysis showed that the sentinel lymph node and axillary lymph node influenced survival time (Table 3). In our study, the clinical stage of the lymph node status and positive expression of HR were the most important factors affecting survival time in patients with BBC ( Table 2).
The Cox survival analysis of all characteristics in patients with SBBC showed that different characteristics concentrated on the tumor in the left side of breast. This result indicated that tumor in the left side breast might have a more profound effect on the survival state than those in the right side of breast (Table 4). When comparing the characteristics of both sides, we did not find any difference ( in particular a distinction between the left and right breast, which influenced the prognosis of patients with SBBC, such as differences in the physiology and anatomy of the lymphatic system in the chest area. As for patients with MBBC, three factors (including age, left sentinel lymph node, and right Ki67) had a significant influence on overall survival (Table 1).
We compared changes in tumor characteristics before and after NAC between the survival group and non-survival group in patients with SBBC (Table 6). The Kaplan-Meier survival estimate showed that a decrease in tumor size, clinical stage, Ki67, and P53 may prolong life span, but a decrease of ER, PR, and HER2 may shorten life span (Table 8). We also found that changes in tumor type and molecular subtype may influence life span. Although life span differed between the decrease and nodecrease group, the difference did not reach the traditional statistically significant level. This result showed that these characteristic changes, especially in both sides of the breast in patients with SBBC, may have an impact on survival time. However, because of the limited number of patients with this rare type of cancer, we could not draw a definitive conclusion. If the number of patients expands, we believe we could obtain a clinically significant conclusion. Because of the particularity of SBBC, in our next study, we will set a better self-control, for instance, the tumor in two sides breasts have the same clinical stage, but different molecular subtype, to explore whether differences in response to NAC treatment in both sides of the tumor would have an impact on survival times. This result may guide the treatment plan for patients with this rare type of BBC.          CI confidence interval. Figure 1 The flow chart of reasons for exclusion.

Figure 2
The cumulative survival time between response group and no-response group in SBBC patien

Supplementary Files
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