Bene ts of a standardized protocol for axillary management after neoadjuvant chemotherapy in a single center

Marina de Paula Canal (  mpcana738@gmail.com ) ACCamargo Cancer Center https://orcid.org/0000-0002-6052-6245 Caroline gomes de almeida Rocha ACCamargo Cancer Center Cynthia Ap. Bueno de Toledo Osório ACCamargo Cancer Center Almir Bitencourt ACCamargo Cancer Center Marina Sonagli ACCamargo Cancer Center Monique Celeste Tavares ACCamargo Cancer Center Solange Moraes Sanches ACCamargo Cancer Center Fabiana Baroni Alves Makdissi ACCamargo Cancer Center


Introduction
Axillary lymph node status is one of the most important prognostic factors in breast cancer. For locally advanced tumors, neoadjuvant chemotherapy (NACT) [1] shows no difference in overall survival (OS)and speci c-cancer survival(SCS) when compared to adjuvant chemotherapy [2], but it favors higher rates of lumpectomy of the breast and axilla [3]. In addition, the current indication for NACT in breast cancer allows for in vivo evaluation of the tumor for systemic treatment, which has an important prognostic value for certain subtypes, such as triple negative and HER2-positive.
Axillary lymph node dissection (ALND) is related to an increased risk of adverse events, such as lymphedema (14%), limitation of upper limb mobility (28%), and neuropathic pain (31%) [4]. Nevertheless, it remains the gold standard for treatment of axillary lymph node tumors, even after complete clinical / radiological response to NACT [1].
The strategy of target axillary dissection has been adopted as a strategy to reduce the FNR in cN+ patients. In this technique, the target lymph node is marked with a metal clip at the time of biopsy, before NACT and up to ve days before surgery. An additional ultrasound (US) is then performed, during which the clipped lymph node receives radioactive 'seeds' [8] and technetium 99 (Tc99) and blue dye, ensuring accurate SLND. With this technique, FNRs as low as 2% have been observed, and in 77% of cases the marked lymph node corresponded to the SLN [9]. However, target axillary dissection has not been universally adopted because of di culties related to pre-and intra-operative localization of previously marked lymph nodes which had shown complete response to NACT [1].
In view of this, to minimize the FNR in relation to axillary dissection, we have developed a standardized protocol at our institution for clipping positive lymph nodes prior to NACT combined with post-NACT axillary management. The objective of this study is to show that it is possible to prevent ALND in clinically negative patients after NACT, using a protocol that is easily accessible to doctors and patients from other centers.

Materials And Methods
This was a retrospective cohort study of data collected from medical records of patients who underwent NACT in a single center, from January 2014 to December 2018. This study was approved by institutional ethics review board. Data collected included molecular subtype of the tumor, clinical stage (T or N) prior to NACT, type of surgery (mastectomy or lumpectomy), and type of axillary dissection (ALND, SLND, or SLND followed by ALND). Patients were included if they had 1) been diagnosed with invasive breast carcinoma and were undergoing NACT, 2)undergone biopsy or slide review at the institution, and 3) received all treatment (chemotherapy and surgery) at the institution. Patients were excluded if they 1) were male, or 2) had in ammatory, metastatic, recurrent, or bilateral tumors.
Patients were examined before and after the institution of a standardized protocol for axillary treatment after NACT.
There were three possible protocols, depending on the lymph node status of each patient: axilla clinically negative (Fig 1 a); up to two clinically positive lymph nodes prior to NACT, which were clipped (Fig 1 b); up to two clinically positive lymph nodes prior to NACT, which were not clipped (Fig 1 c). In short, ALND was performed in patients with two or more clinically positive lymph nodes prior to NACT, patients with persistent disease after NACT (clinical or radiological), and patients whose initial stage was T4 or in ammatory and had no SLN migration.This was followed by SLND in patients with up to two clinically positive lymph nodes prior to NACT, and patients who had had a complete clinical and radiological response after NACT and SLN migration.
SLN marking was performed on the eve of surgery through injection of Tc99 into the breast, close to the tumor area, with lymphoscintigraphy to evaluate migration. Marking with blue dye was performed during surgery through a subdermal injection into the ipslateral breast in the periareolar or superolateral quadrant, depending on the surgeon's preference. When removing a lymph node marked by Tc99 or blue dye (Fig 2), a portable X-ray (Faxitron ® ) con rmed the presence of the clip for protocol validation (Fig 3).
For statistical analysis, descriptive statistical methods were used and the results of categorical variables were expressed as frequencies and percentages. Correlation analysis was performed using Pearson's chisquare test with Yates correction or Fisher's exact test, when indicated. The level of signi cance adopted was 5% (p≤0.05). 3

. Results
A total of 471 female patients were analyzed, 303 patients before and 165 patients after the implementation of this protocol, 03 patients were excluded due to missing data on medical record, aged 24-87 years, divided according to TNM staging (cT1-cT4, cN0,cN+) and molecular subtype (Luminal A and B HER2negative, Luminal HER2positive and HER2hyperexpressed, and triple negative)( Table 1). Of these patients, 295 underwent mastectomy (simple, modi ed radical, or skin-sparing) and 176 underwent breast-conserving surgeries (quadrantectomy or lumpectomy). ALND was performed in 303 patients, SLND in 156, and SLND followed by ALND in nine.
In the period from 2014 to 2017, prior to the implementation of a standardized protocol for pre-NACT axillary management, 303 patients were included (Fig 4).
Beginning in 2017, a standardized protocol for pre-NACT axillary management was instituted, from which time 165 patients were included (Fig 5).
Sixty-seven of these patients were axillary clinically negative, 49 of whom underwent SLND or SLND followed by ALND, and 18 of whom underwent ALND. The remaining 98 patients, who were axillary clinically positive, were submitted to ne needle aspiration biopsy to test for malignancy. Of the 24 patients who tested negative for malignancy, sixteen underwent SLND and eight underwent ALND. Of the 74 who tested positive for malignancy, 43 underwent ALND, 24 underwent SLND, and 7 underwent SLND followed by ALND Prior to implementation of the standard protocol, 77.2% of patients underwent ALND and 22.8% underwent SLND; after implementation, 59.2% underwent ALND and 40.8% SLND. The increase in SLND after protocol implementation was statistically signi cant (p < 0.05).

.Discussion
In the present study we describe a standardized protocol for post-NACT axillary management which increases the e cacy of target axillary dissection and reduces the false negative rate of SLND.
NACT is an important tool in the treatment of certain breast cancers because it not only reduces tumor burden by initially treating the systemic micrometastatic disease, but has also been shown to increase the rate of conservative surgeries in patients who would not otherwise be candidates [12][13][14] NACT also plays a role in axillary downstaging, improving outcomes of clinically positive patients who underwent chemotherapy prior to SLND. The present study similarly found that, in the ninety-eight patients who presented clinically positive axilla, NACT prevented lymphadenectomy in 46% of cases. This result is nearly identical to that of Mamtani et al., whoreported in a prospective study that 70% of clinically positive patients were eligible for SLND after NACT and 48% were able to avoid ALND [15]. Together these studies demonstrate the role of NACT in reducing the need for ALND among patients with lymph node metastases. Considering the performance of lumpectomy after NACT, Bonadonna and Veronesi reported that NACT reduced large tumors to less than 3 cm in 81% of patients, allowing lumpectomy instead of radical mastectomy in 50-75% of patients for whom mastectomy was initially indicated [13][14]. The results of our study were in agreement, allowing lumpectomy in 52.6% of patients with T2 staging.
Regarding molecular subtypes, we observed complete radiological response in 56% of triple negative tumors, and 54% of HER2 positive. A similar study also observed complete radiological response in 59% of cases [16]. Moreover, patients who were triple negative underwent signi cantly more SLND than those in the Luminal (HER2 negative) subgroup. This study has limitations inherent to its retrospective design. First, the medical records were not standardized, especially regarding axillary status prior to the implementation of institutional protocol in 2017. In addition, the relatively small number of patients in the sample may have limited statistical analyses.
Despite this, the present study demonstrates effectiveness in implementing a standardized protocol for axillary management before and after NACT. The institutional protocol used was created with the objective of reducing the number of false-negative results of SLND and minimizing technical limitations of the target axillary dissection that make its routine implementation unfeasible, such as di culty in locating the clipped lymph node, especially when it has a complete response to treatment [17].
In conclusion, the results of this study showed an increase in the frequency of SLND after implementation of a standardized protocol for management of axilla after NACT in breast cancer patients. Prospective studies, with a larger sample and in other centers, are necessary to validate the proposed technique.

Declarations
Funding -Not applicable

Con icts of interest/Competing interests -None
Availability of data and material -The datasets generated during and/or analysed during the current study are not publicly available due to restrictions by our ethics committee but are available from the corresponding author on reasonable request.   X-ray photo of clipped lymph node. Pathological analysis (frozen section) of lymph nodes after NACT [10} was performed in three parts: 1-Macroscopic examination. In the perioperative examination, SLNs were sliced transversely to a thickness of 2 mm and examined by a pathologist for the presence of any whitish areas of hardened consistency, suggestive of residual lymph node metastasis. All slices of lymph node tissue were xed in 10% buffered formalin and included in one or more para n blocks for the de nitive histological evaluation. 2-Microscopic evaluation. In the microscopic evaluation, a pathologist measured the linear dimension of the largest metastatic focus and described the presence of a possible area of pathological response, characterized by brosis, hemorrhage, accumulations of macrophages and a decrease in the lymph node parenchyma. Additional sections of 4-µm thickness were stained with hematoxylin-eosin and analyzed by a pathologist for the presence of isolated cells, or a group of atypical epithelial cells compatible with residual neoplasia, which would determine ALND. The metal clip area was also described, characterized by foreign body-type gigantocellular reaction and lymphocytic in ltrate around amorphous acidophilic material, compatible with the gel shell present in the clips used. 3-Tumor presence. The presence of axillary nodal tumor deposits of any size, including isolated tumor cells, excluded a complete pathological response. Finally, the number of compromised lymph nodes was counted and classi ed to obtain the rCR index and classi cation [11].