We retrospectively identified 85 patients with stage I–III HER2-positive breast cancer and 41 patients with stage I–III TNBC treated with NAC in the Tokushima University Hospital between January 2010 and December 2019. A total of 14 patients with the following characteristics, 1) HER2-positive breast cancer treated with NAC without trastuzumab, 2) incomplete blood sample data, and 3) NAC discontinued due to adverse events were excluded. The final sample included 71 and 41 patients with HER2-positive and TNBC breast cancer, respectively. The follow-up period ended in September 2020.
Neoadjuvant chemotherapy
Patients with HER2-positive breast cancer were preoperatively treated with a trastuzumab plus taxane regimen. Many patients were also treated with an anthracycline-based combination regimen before receiving taxane. This combination regimen involved four cycles of EC (epirubicine 90 mg/m2, cyclophosphamide 600 mg/m2, q3w). Taxane regimens involved 12 cycles of weekly paclitaxel (80 mg/m2) or four cycles of tri-weekly docetaxel (75 mg/m2) doses. Some patients received four cycles of tri-weekly docetaxel (40 mg/m2) and S-1 (40 mg/m2 orally twice per day on days 1-14) regimen (N-1 Study) prior to receiving trastuzumab and taxane regimen [15]. Trastuzumab was administered either at a dose of 4 mg/kg at loading, followed by 2 mg/kg on a weekly basis, or at a dose of 8 mg/kg at loading, followed by 6 mg/kg every 3 weeks. In a few patients, pertuzumab was used in combination with trastuzumab. Pertuzumab was administered at a dose of 840 mg at loading, followed by 420 mg every 3 weeks. In TNBC, the NAC regimen consisted of four cycles of EC, followed by four cycles of tri-weekly docetaxel (75 mg/m2) or 12 cycles of weekly paclitaxel (80 mg/m2). Some patients received the N-1 Study regimen.
ALC ratio and cut-off value
In HER2-positive breast cancer patients, a routine peripheral blood test was performed, and lymphocyte count estimates were obtained before trastuzumab administration (pre-treatment ALC). Subsequently, routine blood tests were performed before the fourth cycle of trastuzumab and docetaxel regimen or before the tenth cycle of trastuzumab and paclitaxel regimen. Changes to ALC after NAC were calculated (post-treatment ALC). The ALC ratio was defined as the pre-treatment ALC/post-treatment ALC values.
In TNBC patients, the ALC ratio was calculated based on estimates obtained from routine blood tests before taxane administration (pre-treatment ALC) and those obtained before the fourth cycle of docetaxel regimen or before the tenth cycle of paclitaxel regimen.
Evaluation of clinicopathological factors
Data on clinical and pathological characteristics of patients were extracted from their medical records, including age, T, N, stage, estrogen receptor status, HER2 status, and histological therapeutic effect. Parameters such as T, N, and stage were clinically evaluated using pre-treatment imaging findings according to the Union for International Cancer Control TNM classification [16]. Estrogen receptor and HER2 status were determined using biopsy specimens obtained prior to NAC.
Estrogen receptor-positive findings were defined as nuclear staining in ≥10% of cancer cells. HER2 protein expression was classified into the following four groups: 0, 1+, 2+, and 3+. Samples of patients with 2+ expression of HER2 were also tested using in situ hybridization for gene amplification. Protein and in situ hybridization were both performed following the American Society of Clinical Oncology/College of American Pathologists guidelines for HER2 testing [17]. HER2-positivity was defined as HER2 protein expression of 3+ or HER2 gene amplification. Histopathological therapeutic effects were evaluated based on whether pCR was achieved, defined as the disappearance of invasive cancer nests in breast tissue.
Statistical analyses
All statistical analyses were performed with JMP Ver.14 (SAS Institute Inc., Cary, NC, USA). The χ2 test was used to determine significant differences in relationships between the ALC ratio (low vs. high) and clinicopathological factors. The ROC curve analysis was performed to determine the cut-off value of the ALC ratio associated with disease recurrence. The Kaplan–Meier curves were generated to estimate disease-free survival (DFS), and the log rank test was used to compare survival between the groups. P-values of <0.05 were considered indicative of a statistically significant finding. Univariate and multivariate analyses of DFS estimates were performed using the Cox proportional hazards model. Multivariate Cox regression analysis included factors such as the ALC ratio (low vs. high), pathology findings (non-pCR vs. pCR), age (>50 vs. ≤50 years), tumor size (T3-4 vs. T1-2), and nodal status (N1-3 vs. N0).
Ethical statement
The study was conducted in accordance with the Declaration of Helsinki. The institutional review board of the Tokushima University Hospital approved this study (CR 3672) and waived the informed consent requirement due to the retrospective nature of this study.