This study (UMIN000022571) was approved by the independent ethics committee at each study site and conducted in agreement with the Helsinki Declaration and ethical guidelines for medical research on humans. Written consent was obtained from patients before initiating any process in this study. If written consent could not be obtained because a patient had died or could not be reached, samples were sent to a central pathology review office after confirmation by each independent ethics committee that the various provisions based on the Ethical Guidelines for Medical and Health Research Involving Human Subjects had been met.
Previous N·SAS-BC 01 and CUBC studies
Details of the N·SAS-BC 01 and CUBC studies and subsequent analyses have been described previously [
3‐
5]. In brief, patients were enrolled in the N·SAS-BC 01 and CUBC studies from October 1996 to April 2001 and from September 1996 to July 2000, respectively. Both studies included patients with resected stage I–IIIA breast cancer, irrespective of their HR status, but the N·SAS-BC 01 study enrolled high-risk (invasive ductal carcinoma with invasive size ≥ 5 mm and grade 2 or 3, or invasive lobular carcinoma, or metaplastic carcinoma), node-negative, and the CUBC study enrolled node-positive patients (ranged 1–9). Patients in both studies were treated with 6 cycles of either CMF or UFT for 2 years. Patients in the CUBC study also received TAM for 2 years, irrespective of their HR expression status, and patients with ER+, PgR+, or both in the N·SAS-BC 01 study were treated with TAM for 5 years. No patient was treated with trastuzumab in the two studies, and radiation therapy was allowed only in the N·SAS-BC 01 study. Protein expression levels of ER and PgR to assess patient characteristics were determined by enzyme immunoassay at each participating hospital.
Detection and evaluation of clinicopathological factors and prognostic outcomes
The study outcomes were RFS and OS according to HR/HER2 subtypes, and the relationships between clinicopathological factors, including age, tumor size, HR, HER2, Ki67, histological grade and TILs, and updated long-term follow-up prognostic outcomes (RFS and OS) in patients treated with CMF and UFT. In this study, RFS was defined as the period from the date of randomization to the last-confirmed date of no recurrence or of death from any cause, and OS was defined as the period from the date of randomization to the date of death from any cause with a cut-off date of June 30, 2018.
Protein expression levels of ER, PgR, HER2, Ki67 and TILs, as well as nuclear and histological grades, were assessed in the paraffin-embedded sections from each patient and evaluated by central pathological review. ER, PgR, and Ki67 protein expression were detected by immunohistochemistry with monoclonal antibodies SP1, 1E2 (Roche Diagnostics K.K., Japan), and MIB-1 (DAKO Japan Agilent, Japan), respectively. ER and PgR protein expression levels were determined as the sum of the proportion score (0–5) and intensity score (0–3), and ER+ and PgR+ were defined as total scores of 3–8 and ER− and PgR− as total scores of 0–2. Based on the expression results, HR positivity (HR +) was defined as ER+ and/or PgR+, and HR negativity (HR−) as ER− and PgR−.
HER2 expression was detected by semi-quantitative IHC assay (DAKO HercepTest II, Agilent, Japan; score 0–3+) in samples from all patients, with further fluorescence in situ hybridization (FISH) analysis in those with 2+ staining (PathVysion HER-2 DNA Probe Kit; Abbott, Japan). HER2 negativity (HER2−) was defined as an IHC score of 0–2 and negative FISH, and HER2-positive (HER2+) as an IHC score of 2 or 3 and positive FISH results. Nuclear grade (grades 1–3) [
8,
9], and histological grade (grades 1–3) [
10] were evaluated in hematoxylin and eosin-stained sections. Expression of TILs was evaluated according to the criteria of International TILs Working Group 2014 [
11‐
13]. Briefly, using an optical microscope under × 200 and × 400 magnifications, the panel pathologists classified TILs levels into the following three grades: low < 10%, intermediate ≥ 10 to ≤ 40%, and high > 40%.
Statistical methods
Summary statistics, number of patients, mean, standard deviation, minimum, median, and maximum values were obtained for the patients’ baseline characteristics. Between-cohort differences were evaluated using χ2 test and t test. RFS and OS were estimated using Kaplan–Meier analysis, and differences between the two treatment cohorts were tested by the log-rank test. Evaluations of RFS and OS in subgroups were adjusted for clinical characteristics chosen based on the selection criterion set at α = 0.15, namely tumor size (< 3 and ≥ 3 cm) and nodal status (0 and ≥ 1). Hazard ratios and 95% confidence intervals (CIs) were determined using univariate unadjusted Cox proportional hazards model to evaluate the prognostic factors and were summarized in forest plots. Statistical significance was set at a two-sided p < 0.05. Statistical analysis was performed using SAS version 9.4 or higher (SAS Institute, Inc., Cary, NC, USA).