Patients and blood samples
We reviewed the electronic records of breast cancer patients treated at Cancer Hospital, Chinese Academy of Medical Sciences (CAMS) between November 1999 and June 2015. The patient selection criteria were as follows: (1) all the patients were female; (2) each patient had complete clinicopathological data, including patient’s age, tumor size, axillary lymph node status, TNM stage, pathological type, vascular invasion, adjuvant chemotherapy and radiotherapy; (3) the patient was pathologically diagnosed with TNBC; (4) the patient was diagnosed with stage I–III TNBC; and (5) the patient had received anthracycline- and/or taxane-containing regimens as adjuvant chemotherapy. The blood samples of the selected patients were derived from the sample bank which has been built to collect tumor tissues and blood samples from breast cancer patients who were treated in our hospital since 1998. Patients without complete clinical information and sufficient blood samples were excluded.
Breast cancer subtype definition
Estrogen receptor and PR statuses were evaluated based on the immunohistochemical (IHC) results of formalin-fixed, paraffin-embedded, primary breast cancer tissues obtained from patients. ER-positive and PR-positive statuses are defined by ≥ 1% nuclear staining. IHC and/or fluorescence in situ hybridization (FISH) were routinely conducted to determine the HER2 status. Breast cancers are classified as HER2-positive if they are scored as 3+ with uniform membrane staining for HER2 in ≥ 10% tumor cells demonstrated by IHC or HER2 gene amplification demonstrated by FISH [single-probe, average HER2 copy number ≥ 6 signals/cell; dual-probe HER2/chromosome 17 centromere (CEP17) ratio ≥ 2.0 with an average HER2 copy number ≥ 4 signals/cell; dual-probe HER2/chromosome enumeration ratio ≥ 2.0 with an average HER2 copy number < 4 signals/cell; HER2/CEP17 ratio < 2.0 with an average HER2 copy number ≥ 6 signals/cell]. Tumors negative for ER, PR, and HER2 were defined as TNBC.
Treatment
The EC regimen [epirubicin (EPI) 90 mg/m2 or pirarubicin (THP) 40–50 mg/m2 on day 1 and cyclophosphamide (CTX) 600 mg/m2 on day 1, repeated every 21 days for 4 cycles], EC-T regimen [EPI 90 mg/m2 and CTX 600 mg/m2 on day 1, repeated every 14 or 21 days for 4 cycles, followed by docetaxel (DOC) 80 mg/m2 on day 1, repeated every 21 days for 4 cycles or paclitaxel (TAX) 175 mg/m2 on day 1, repeated every 14 or 21 days for 4 cycles], ET regimen (EPI 75 mg/m2 or THP 40–50 mg/m2 on day 1 and DOC 75 mg/m2 or TAX 175 mg/m2 on day 2, repeated every 21 days for 6 cycles), TAC regimen (EPI 75 mg/m2 or THP 40–50 mg/m2, CTX 500 mg/m2, and TAX 175 mg/m2 or DOC 75 mg/m2 on day 1, repeated every 21 days for 6 cycles), and CAF regimen [CTX 500 mg/m2 on day 1, EPI 75 mg/m2 or THP 40–50 mg/m2 or doxorubicin (ADM) 50 mg/m2 on day 1, 5-fluorouracil [5-FU] 500 mg/m2 on days 1 and 8, repeated every 21 days for 6 cycles] were classified as anthracycline-based regimens; the EC-T regimen, TAC regimen, ET regimen, TC regimen (DOC 75 mg/m2 or TAX 175 mg/m2 and CTX 600 mg/m2 on day 1, repeated every 21 days for 4 cycles) and carboplatin-taxane regimen [DOC 75 mg/m2 or TAX 175 mg/m2 on day 1, and carboplatin (CAPE) AUC = 5 mg/mL on day 2, repeated every 21 days for 6 cycles] were classified as taxane-based regimens; the EC-T regimen, ET regimen, and TAC regimen were classified as anthracycline-taxane combinational regimens.
Follow-up
Patients were followed up every 3 months during the first year after surgery, then every 4 months in the second year and every 6 months in years 3–5. After that, patients were followed annually until February 17, 2017. Disease progression was diagnosed based on imaging results [computed tomography (CT), magnetic resonance imaging, or positron emission tomography/computed tomography (PET/CT)] and/or biopsy of the metastatic lesions. DFS was defined as the duration between the date of surgery and the date of the first event (locoregional recurrence or distant metastasis or death from any cause, whichever occurred first). Patients who were recurrence-free and alive at the last follow-up were censored.
Selection of tag SNP in the ATG5 gene
First, we screened the National Center for Biotechnology Information (NCBI) SNP database and selected SNPs located in the promoter region, exon, 5′-untranslated region (UTR), and 3′-UTR of the ATG5 gene. The minor allele frequency of the selected SNPs should be more than 0.05. Second, we searched the PubMed database for articles reporting significant roles of candidate ATG5 SNPs in the development, progression, and chemotherapy resistance of different cancers. Combining these research results, we finally decided to genotype four ATG5 SNPs of potential interest (rs473543, rs28656919, rs3761796, and rs506027).
DNA preparation and genotyping
Genomic DNA was isolated from the whole blood using the blood DNA kit (BioTeKe Corpration, Beijing, China) according to the manufacturer’s protocols. Genotyping was performed with the MassARRAY MALDI-TOF System (Sequenom Inc., San Diego, CA, USA). Primers (forward 5′-ACGTTGGATGAGGTGAAAGGTGATTACTTG-3′ and reverse 5′-ACGTTGGATGGGAAGAGAGAAGGACAAGGG-3′) for polymerase chain reaction or single-base extension were designed using the Assay Designer’s software version 3.0 (Sequenom Inc.) and synthesized by the Beijing Genomics Institute (Beijing, China).
Purified primer extension reaction products were dispensed onto a 384-well Spectro CHIP bioarray using MassARRAY Nanodispenser RS1000 (Sequenom Inc.) and determined by the matrix-assisted laser desorption/ionization time-off light mass spectrometer. Genotype analysis was performed through the MassARRAY Typer software version 4.0 (Sequenom Inc.). Negative controls (without DNA) and duplicate samples were included for quality assurance of genotyping.
Statistical analysis
Chi square test was used to evaluate the Hardy–Weinberg equilibrium. SNPs that were not in Hardy–Weinberg equilibrium were excluded from analysis. The survival probability was calculated using the Kaplan–Meier method. Differences across survival curves were compared by the log-rank test. Using Chi square test, we compared the differences in clinicopathological characteristics between patients with and without recurrence. Univariate and multivariate survival analyses were performed using the Cox proportional hazard regression model. The following variables were examined in the univariate analyses for their relations with DFS: patients’ age, tumor size, axillary lymph node status, TNM stage, pathological type, vascular invasion, adjuvant radiotherapy, and ATG5 rs473543 genotype. Factors with a univariate relevant influence on DFS were then included in the multivariate survival analyses. The statistical analyses were performed with the software package SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). A P value of less than 0.05 was considered statistically significant.