Higher Locoregional Recurrence in Hormone Receptor-Negative Breast Cancer Patients with Residual Disease after Neoadjuvant Treatment Undergoing Breast Conservation Surgery

Background: Neoadjuvant chemotherapy (NAC) has been the standard treatment for locally advanced breast cancer for the purpose of downstaging or for conversion from mastectomy to breast conservation surgery (BCS). There is still a high locoregional recurrence (LRR) rate after NAC. The aim of this study was to determine predictive factors for locoregional recurrence (LRR) in breast cancer patients after NAC. Materials and Methods: Between 2005 and 2017, 1047 breast cancer patients underwent BCS or mastectomy after NAC in Chang Gung Memorial Hospital, Linkou. We obtained data regarding patient and tumor characteristics, chemotherapy regimens, clinical tumor response, tumor subtypes and pathological complete response (pCR), type of surgery, and recurrence. Results: The median follow-up time was 45.1 months (range 0.1-160.3 months). The mean initial tumor size was 4.89 cm (SD ±2.95 cm). Of the 1047 NAC patients, 232 (22.2%) achieved pCR. The BCS and mastectomy rates were 41% and 59%, respectively. Overall, 240 patients experienced tumor recurrence (22.9%). Thirty-ve cases of LRR (14.3%) were noted following BCS, of which 4.3% achieved pCR. Multivariate analysis indicated that independent factors for the prediction of LRR included hormone receptor negative/human epidermal growth factor receptor 2 positive (HR-/HER2+) subtype, HR-/HER2-subtype, and failure to achieve pCR. Further investigation according to the molecular subtype showed that following BCS, HR-/HER2+ non-pCR group had signicantly increased LRR compared with the HR+/HER2+ pCR group (22.2% vs 6.3%, p<0.05), and the HR-/HER2-non-pCR group had signicantly increased LRR compared with the HR-/HER2-pCR group (0% vs 20.4%, p<0.005). Conclusion: Pathological response after NAC is related to the risk of developing LRR. The LRR rate was higher in


Introduction
Neoadjuvant chemotherapy (NAC) has generally been accepted as the standard treatment for locally advanced breast cancer. With the aim of making non-operable breast cancer operable, NAC increases the number of breast conservative therapy (BCS) candidates by downsizing locally advanced tumor [1,2].
Hence, patients undergoing BCS after NAC might have a more favorable cosmetic outcome and quality of life [3].
Despite the fact that increased use of NAC has facilitated the treatment of breast cancer, long-term optimized outcomes and treatment effects are concerning. In their recently published meta-analysis on NAC and adjuvant chemotherapy, Asselain and colleagues reported that NAC has a higher local recurrence rate than adjuvant chemotherapy in patients with locally advanced tumors [4]. Similar results were reported in a meta-analysis by Mauri in 2005 [5]. The frequency of BCS, de ned as lumpectomy with whole breast irradiation, is higher after the use of NAC compared with adjuvant chemotherapy after surgery.
The long-term survival rate among early breast cancer women without NAC who undergo BCS was the same as that among women who undergo radical mastectomy [6][7]. However, several studies have demonstrated no signi cant difference in long-term survival or local recurrence rates between women who underwent BCS and those who underwent radical mastectomy after NAC [8][9]. Although predictive factors for local recurrence in these NAC-treated patient groups have not yet been clari ed yet, the higher local recurrence risk of NAC than of adjuvant chemotherapy is controversial.
NAC is increasingly used in breast cancer therapy, and clinic-pathological subtypes, such as estrogen receptor positive (ER), progesterone receptor positive (PR), and human epidermal growth factor receptor 2 positive (HER-2), can provide prognostic information regarding the risk of local regional recurrence after NAC. Furthermore, pathological complete remission (pCR) after NAC can signi cantly affect the prognosis of breast cancer [10,11]. We would like to investigate these attributed factors for recurrence reported in retrospective studies on NAC. Furthermore, once the pCR-achieved status is con rmed, the omission of mastectomy is feasible. Patients receiving NAC, in total, have higher BCS rate in literature. We investigated these clinic-pathological attributed factors in association with the operation types after NAC.

Patients And Methods
Patient selection: The study was a retrospective cohort study; therefore, the requirement for patient consent was waived. The study was approved by our institutional review board and was performed in accordance with the ethical standards laid down in the 1964 declaration of Helsinki and all subsequent revisions. Female patients diagnosed with non-metastatic invasive breast cancer who received neoadjuvant chemotherapy (NAC) and underwent mastectomy or breast conserving surgery at Chang Gung Memorial Hospital, Linkou, between 2005 and 2017 were enrolled. Eligible patients met the following criteria: histologically proven unilateral invasive breast carcinoma, normal baseline blood count, normal serum creatinine, alanine aminotransferase, aspirate aminotransferase, alkaline phosphatase, and bilirubin levels, a negative pregnancy test, and a World Health Organization (WHO) performance status <2. Exclusion criteria were in ammatory cancer and the initial presence of distant metastasis. Initial staging was determined through physical examination, mammography, ultrasonography of the breast and axillary lymph nodes, chest radiography, a bone scan, and a whole body computed tomography (CT) scan. Clinical information regarding age, tumor histology, molecular subtype, TMN stage, Scarff-Bloom-Richardson (SBR) grade, NAC regimens, post-NAC response status, operation type, pattern of recurrence, and locoregional recurrence free survival were collected. Chemotherapy regimens included anthracyclinebased and taxane-based regimens and the target therapy for HER2 contained trastuzumab and pertuzumab for partial patients. Dose modi cations were based on nadir blood counts and interval toxicity. Surgery (mastectomy or BCS) and axillary sentinel lymph node biopsies or axillary lymph node dissections were performed 2-4 weeks after NAC was completed. The indication for postoperative radiotherapy included all patients after BCS and the patients receiving mastectomy with initial tumor size >5 cm or residual positive lymph node after NAC. Patients received postoperative adjuvant therapy, including hormone therapy, chemotherapy, and target therapy, according to their clinicopathological factors.
Analysis of estrogen receptor, progesterone receptor, HER2 expression and the de nition of pCR: ER, PR, and HER2 analysis was performed on pretreatment core needle biopsy specimens using immunohistochemical (IHC) staining techniques. For ER and PR, positivity was de ned as expression in >1% of tumor cells. For HER2, IHC staining with a score of 3+ (moderate to strong complete membrane staining observed in 10% of the tumor cells) or a positive uorescence in situ hybridization (FISH) test with IHC staining with a score of 2+ (weak to moderate complete membrane staining in 10% of the tumor cells) was de ned as positive. pCR was de ned as no residual invasive breast cancer in the histopathology specimen of the breast and axillary lymph nodes (ypT0/ypTisN0).
Statistical analysis: Numerical data were compared using a Student's t-test and presented as the mean+standard deviation (SD) and a two-tailed p-value <0.05 was considered to be statistically signi cant. Pearson's chi-square test (c 2 test) was used to compare the differences in the proportions of categorical data. A log-rank test and the Kaplan-Meier method were used for survival analysis. For multivariate analysis of prognostic factors, logistic regression was used. All analyses were conducted using SPSS 22.0 for Windows (SPSS, Chicago, IL).
During the follow up period, the recurrence rate was 22.9% (n = 240), of which 8.6% (n = 90) was locoregional recurrence (LRR) and 16.0% was distal metastasis. By the conclusion of this study, 143 (13.7%) patients had died from breast cancer and 19 (1.8%) patients had died from other causes. Furthermore, on univariate analysis of factors affecting LRR, we noted that clinical T4 stage (T4, p = 0.029), clinical N2 stage (p = 0.001), SBR grade 3 (p = 0.007), negative ER (p = 0.001), negative PR (p = 0.002), HR-subtype (p = 0.037), and non-pCR (p < 0.005) were predictors of LRR (Table 4). Comparing between patients who underwent BCS and those who underwent mastectomy revealed no signi cant difference in the overall recurrent rate of the two groups (p = 0.633). In multivariate analysis, only negative hormone receptor subtypes and non-pCR were found to be predictors of LRR (Table 5) In patients who underwent BCS, a signi cant difference was found in the LRR between the pCR and non-pCR groups (p < 0.05). The LRR of the pCR group was 4.3% (n = 6) compared with 10% (n = 29) in the non-pCR group (Table 6). LRRFS was also found to be signi cantly different between the pCR and non-pCR groups, as shown in Fig. 3 (p = 0.033). Further analysis focusing on the subtypes of the BCS group demonstrated that the HR-/HER2 + non-pCR group had signi cantly increased LRR compared with the HR-/HER2 + pCR group (22.2% vs 6.3%, p < 0.05). Furthermore, the HR-/HER2-non-pCR group had signi cantly increased LRR compared with the HR-/HER2-pCR group (0% vs 20.4%, p < 0.005). (Table 7). Moreover, there was no difference in LRR found with regards to hormone receptor positive disease between the between p-CR and non-pCR groups. In the mastectomy group (n = 615), pCR remained an independent factor for favorable LRR compared with non-pCR patients (p = 0.041), with 3 patients (3.3%) in the pCR group developing LRR compared with 52 patients (9.9%) in non-pCR group (Table 6). Further analysis of molecular subtypes indicated no signi cant difference for all subtypes between the pCR and non-pCR groups following mastectomy ( Table 7). The LRRFS rate in the pCR group following mastectomy showed a favorable outcome; however, this was not signi cant when compared with the non-pCR group (p = 0.060) (Fig. 4). The signi cant difference of LRRFS rate was also shown in the pCR group compared with the non-pCR group in HR-/HER2 + and HR-/HER2-patients who underwent BCS (Fig. 5-6).

Discussion
In total, 232 patients (22.2%) achieved pCR. Multivariate analysis indicated that clinical T stage, clinical N stage, and molecular subtype were independent predictors to pCR. A total of 90 patients (8.6%) developed LRR of which 9 were in the pCR group and 81 were in the non-pCR group. Our study con rmed that pCR after NAC provided better local control in all subtypes of breast cancer. The result was in line with the ndings of our previous report published in 2018, in which no LRR occurred in the pCR group and 31 patients (13.2%) in the non-pCR group with signi cant difference in total 263 patients all receiving neoadjuvant weekly epirubicin and docetaxel regimens [12]. One of the aims of this study was to enroll patients with all different subtypes of the same indication to avoid patient bias. Trastuzumab was included as a neoadjuvant treatment in 2010 in our institution. Our study revealed that 232 patients (22.2%) achieved pCR among the 1047 patients underwent while the BCS rate is 41% and the rest of patients received mastectomy (59%). Overall, 240 patients experienced tumor recurrence (22.9%).
Although CTNeoBC pooled analysis did not validate pCR as a surrogate endpoint for an improved eventfree survival or overall survival in all breast cancer subtypes [13], pCR was a suitable surrogate endpoint for selected patients in aggressive subtypes [14]. A recent comprehensive meta-analysis concluded that pCR followed by NAC was associated with signi cantly better event-free survival (EFS) and overall survival (OS), especially for patients with triple-negative and HER2 + breast cancer. The tumor response effect observed in the pCR group was similar in adjuvant chemotherapy and NAC patients [15]. Moreover, data from the combined analysis of the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-18 and B-27 showed that the residual tumor status was an independent predictor of LRR in all patients at the 10 year follow-up, regardless of surgery type [10]. Another large analysis of the European Organisation for Research and Treatment of Cancer (EORTC) 10994/BIG 1 − 00 study of patients with locally advanced breast cancer receiving NAC showed that pCR was a favorable factor with regards to the prediction of LRR after NAC [16]. Several retrospective series also demonstrated that achieving pCR after NAC can result in better local control following surgery [1][2][17][18]. Therefore, achieving pCR was an important factor not only for distant disease control but also for local control.
The results from the EORTC 10994/BIG 1 − 00 study of patients with locally advanced breast cancer receiving NAC showed that breast cancer subtypes, including HER2 + with or without trastuzumab and triple-negative, are predictive factors for high LRR after NAC [16]. Yang et al. reported that 233 patients with stage II-III disease who were treated with NAC, mastectomy, and post-mastectomy radiotherapy had an 8% LRR rate over 5 years with a median follow-up of 62 months. The authors concluded that patients with triple-negative breast cancer had the highest LRR rate and those with HR + and HER2 + breast cancer had favorable LRR rates, regardless of NAC response [18]. In other several retrospective studies, molecular subtypes including HER2 + and triple-negative also showed poor LRR in BCS patients [1,2]. In our study, HR-/HER2 + and HR-/HER2-subtypes were found independent signi cant factors for the prediction of LRR, regardless of treatment response.
The Early Breast Cancer Trialists' Collaborative Group (EBCTCG) recently reported that NAC was associated with more frequent local recurrence than that of adjuvant chemotherapy. The 15-year local recurrence rate was reported to be 21.4% for NACT compared with 15.9% for adjuvant chemotherapy from a meta-analysis of individual patient data from 10 randomized trials with average 9 years of follow-up. The study group also found that patients who underwent NAC had an increased frequency of breastconserving therapy (65%) versus those treated with adjuvant chemotherapy (49%). The largest difference of LRR appeared in planned mastectomy patients and surgery less commonly used patients. The authors concluded tumors downsized by NAC might have higher local recurrence after BCS than tumors of the same dimensions in women who did not undergo NAC. Furthermore, the majority of patients only received anthracycline-based chemotherapy and nal enrollment of patients occurred in 2002 [4]. Previous reports from the NSABP B27 trial showed that anthracycline-based regimens with the addition of taxane were associated with increased pCR rates and better local control [19]. Moreover, neoadjuvant chemotherapy plus trastuzumab was shown to be a predictor factor for favorable long-term survival but trastuzumab cannot be used before 2002 [20]. In our studies, the majority of patients received anthracycline-based regimens combined with taxane-based chemotherapy and every patient underwent appropriate surgery. The results from the combined analysis of NSABP B-18 and B-27 showed that the 10-year cumulative incidence of LRR was 12.3% for mastectomy patients and 10.3% for lumpectomy plus breast radiotherapy patients, indicating no difference between the mastectomy and BCS groups after NAC [10]. There was no signi cant difference in LRR between the BCS and mastectomy groups in the I-SPY trial, despite the fact that the BCS group on average had lower clinical staging [21]. Importantly, a higher rate of breast conservation would not increase the incidence of LRR, which was also con rmed by the NSABP B-18 and the EORTC studies [22][23]. In another pooled analysis of 5500 women, the mastectomy rate was found to be lower in the NAC group than in the adjuvant chemotherapy group, without hampering local control [24]. In our report, 615 (58.7%) patients chose mastectomy, while 432 (41.3%) received BCS. However, the choice of surgery types did not affect the LRR rate-8.9% in mastectomy patients and 8.1% in BCS patients in the total population. Subgroup analysis revealed 35 cases of LRR (14.3%) following BCS, in which 4.3% achieved pCR group. Further investigation according to the molecular subtype showed that in the BCS group, HR-/HER2 + non-pCR patients had signi cantly increased LRR than HR-/HER2 + pCR patients and that HR-/HER2-non-pCR patients had a signi cantly increased LRR than HR-/HER2-pCR patients. In the mastectomy group, an increasing trend with regards to the risk of LRR in the non-pCR group was observed, but this was not signi cant. Caudle et al. reported that 595 patients with HR-/HER2 + and HR-/HER2-subtypes with a poor response to NAC had worse LRR-free survival after BCS. Furthermore, the authors noted that patients with HR+/HER2-and HR+/HER2 + subtypes had excellent LRR-free survival, regardless of tumor response to neoadjuvant chemotherapy [1]. Another study group from Korea revealed HR-/HER2-subtypes and HER2 + without trastuzumab subtypes predicted higher rates of LRR after NAC and BCT, while A pCR was predictive of improved LRR in HR-/HER2-subtype [2]. Moreover, the I-SPY 1 Trial reported that the 5-year local recurrence risk was 0% for mastectomy and 9% for breast conservation in patients with an excellent response to NAC while the local recurrence rate was 12% for mastectomy and 7% for breast conservation in signi cant residual disease [21]. Another critical point was the resection area of operation after NAC. In patients who undergo NAC in order to achieve breast-tumor downstaging to enable BCS, the tumor site should be marked with a clip before initiating NAC, and resection of the entire volume of breast tissue originally occupied by tumor is not necessary [25]. However, the difference of resection area in uencing local recurrence was still unknown in neoadjuvant setting. Local control appeared to be worse in HR-subtype non-pCR BCS patients after NAC in our study, but the effect on overall survival remains unknown.
Further investigation is needed to determine survival outcomes.

Limitation
One of the limitations of this study is that it was a retrospective study from a single institution, which may result in selection bias. This study is also limited by the short follow-up period. Furthermore, not all patients with the same subtype diagnosis received the same chemotherapy regimens Conclusion Pathological response after NAC is related to the risk of developing LRR. The LRR rate was found to be higher in non-pCR patients after NAC, especially in hormone receptor-negative patients who underwent BCS. Therefore, both the pathological response and molecular subtype should be carefully considered when choosing candidates for BCS after NAC. Some strategies to adequate surgery needed to be suggested in BCS patients after NAC, including careful tumor localization, detailed pathological assessment, evaluation of the resection area, and the margin status.
List Of Abbreviations NAC, Neoadjuvant chemotherapy; BCS, breast conservation surgery; LRR, locoregional recurrence; pCR, pathological complete response; HR, hormone receptor; HER2, human epidermal growth factor receptor 2 Declarations Ethics approval and consent to participate: This study was approved by ethics committee of our institution and institutional review board number was 1711150042.

Consent for publication:
Not applicable.
Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests:
The authors declare that they have no competing interests.

Funding:
This study was supported by research plan in Chang Gung Memorial Hospital, study number CMRPG1K0061 Authors' contributions: Hsu-Huan Chou, Wei-Shan Chung and Rong-Yao Ding analyzed and interpreted the patient data, and were major contributors in writing the manuscript and performed the analysis of statistics; Yung-Feng Lo and Shin-Cheh Chen supervised the manuscript. Other authors collected the clinical data. All authors read and approved the nal manuscript          LRRFS rate in the pCR group compared with the non-pCR group in patients who underwent mastectomy. LRRFS rate in the pCR group compared with the non-pCR group in HR-/HER2+ patients who underwent BCS.