Study subjects
We reviewed the electronic records of breast cancer patients treated at the Cancer Hospital, Chinese Academy of Medical Sciences (Beijing, China) between January 2008 and December 2015. The patient selection criteria were as follows: (1) volunteered to participate in this study; (2) female patients; (3) pathologically diagnosed with stage I–III TNBC; (4) complete clinicopathological data including age, tumor size, TNM stage, pathological type, adjuvant chemotherapy, past medical history, family history, smoking and drinking history; (5) available reports of ECG and/or UCG; and (6) sufficient blood samples for SNP genotyping. Exclusion criteria were as follows: (1) symptomatic heart failure; (2) acute phase of coronary heart disease; and (3) previous history of fatal arrhythmia. We followed all patients until August 6, 2017. This study was approved by the Institutional Review Boards of the Cancer Hospital, Chinese Academy of Medical Sciences (No. CH-BC-019).
Breast cancer subtype definition
Estrogen and progesterone receptor statuses were evaluated based on immunohistochemistry (IHC) of formalin-fixed, paraffin-embedded breast cancer tissue samples obtained from the patients. IHC was performed with anti-estrogen receptor and anti-progesterone receptor antibodies. A positive estrogen or progesterone receptor status was defined by nuclear staining of more than 1% cells based on the guidelines of American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) released in 2010. To determine the human epidermal growth factor 2 (HER2) status, IHC was performed or gene amplification was determined using fluorescence in situ hybridization (FISH). Tumors are classified as HER2-positive if scored as 3+ for HER2 in ≥ 10% tumor cells as demonstrated by IHC or if gene amplification is demonstrated by FISH. Tumors negative for estrogen and progesterone receptors and HER2 were defined as TNBCs.
Exposure dose of chemotherapeutic drugs
All TNBC patients received adjuvant/neoadjuvant treatment. The chemotherapy regimens were as follows: (1) the EC regimen [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 4–6 cycles]; (2) the 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 (DTX) 75 mg/m2 on day 1, repeated every 21 days for 4 cycles, or paclitaxel (PTX) 175 mg/m2 on day 1, repeated every 14 or 21 days for 4 cycles]; (3) the ET regimen (EPI 75 mg/m2 or THP 40–50 mg/m2 on day 1 and DTX 75 mg/m2 or PTX 175 mg/m2 on day 2, repeated every 21 days for 6 cycles); (4) the TAC regimen (EPI 75 mg/m2 or THP 40–50 mg/m2, CTX 500 mg/m2, and PTX 175 mg/m2 or DTX 75 mg/m2 on day 1, repeated every 21 days for 6 cycles); (5) the CAF regimen [CTX 500 mg/m2 on day 1, EPI 75 mg/m2 or THP 40–50 mg/m2 or 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]; (6) the TC regimen (DTX 75 mg/m2 or PTX 175 mg/m2 and CTX 600 mg/m2 on day 1, repeated every 21 days for 4 cycles); (7) the carboplatin-taxane regimen [DTX 75 mg/m2 or PTX 175 mg/m2 on day 1, and carboplatin (CAPE) area under receiver-operating curve (AUC) = 5 mg/mL on day 2, repeated every 21 days for 6 cycles].
The EC and CAF regimens were classified as anthracycline-based regimens; the TC regimen was a taxane-based regimen; the EC-T, ET, and TAC regimens were classified as anthracycline–taxane-based regimens.
Electrocardiography and echocardiography
Standard 12-lead ECG was performed before chemotherapy and after most chemotherapy cycles. As we mainly study early-onset cardiac events, we chose to analyze ECG records after chemotherapy. Abnormal electrocardiogram refers to the presence of new abnormalities after any chemotherapy cycle compared with baseline electrocardiogram, regardless of whether the abnormality disappeared during the subsequent chemotherapy cycle. For patients with abnormal electrocardiograms, UCG was performed according to clinical needs. All ECG and UCG records were sent to Fuwai Hospital and re-interpreted by specialists at the Department of Cardiology. The following parameters were analyzed: heart rate (HR), PR interval, QRS duration, and QT(c) interval. We classified cardiac events by ST-T segment abnormalities, elevated myocardial enzymes, arrhythmia, and QRS pattern or duration abnormalities. To clarify the relationship between treatment duration and ECG abnormalities, we defined early abnormalities as those appearing before four cycles of chemotherapy.
Candidate SNP selection and genotyping
In total, 25 SNPs related to autophagy were selected according to the National Center for Biotechnology Information (NCBI) SNP database (https://www.ncbi.nlm.nih.gov/snp/) and the Catalog of Somatic Mutations in Cancer (COSMIC) database (http://cancer.sanger.ac.uk/cosmic). The final candidate SNPs were ataxia telangiectasia mutated (ATM) (rs1003623, rs227060, rs228589, rs664143, and rs664677), autophagy-related (ATG)5 (rs473543 and rs3761796), ATG7 (rs2594971, rs111595248, and rs4684789), ATG12 (rs1058600 and rs5870670), ATG13 (rs13448 and rs10838611), microtubule-associated protein 1 light chain (MAP1LC)-3A (rs4911429 and rs6088521), MAP1LC-3B (rs9903, rs35227715, rs7865, and rs16944733), caspase 3 (CASP3) (rs1049216, rs12108497, and rs2720376), crystallin alpha B (CRYAB) (rs14133), and stathmin 1 (STMN1) (rs182455).
Genomic DNA was extracted from a 1- to 2-mL blood sample that was collected from each patient upon recruitment using a blood DNA kit (BioTeKe Corporation, Beijing, China). A MassARRAY MALDI-TOF System (Sequenom Inc., San Diego, CA, USA) was used to genotype candidate SNPs according to the protocol. The PCR primers and probes (forward 5′-ACGTTGGATGAGTTTCCTCGCTCCTGTTTC-3′ and reverse 5′-ACGTTGGATGCTCTCTCTCTGGATCTGCTC for ATG13 rs10838611; see other probes in Additional file 1: Table S1) were designed according to Assay design 3.1 (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 a MassARRAY Nanodispenser RS1000 (Sequenom Inc.) and determined using a matrix-assisted laser desorption/ionization time-off light mass spectrometer. Genotype analysis was performed through the MassARRAY Typer software version 4.0 (Sequenom Inc.). Duplicate samples and negative controls (without DNA) were used for quality control of genotyping. Concordance for duplicate samples was 100% for all assays. The group information of each sample was concealed for genotyping analysis.
Statistical analyses
We used paired-sample T tests to compare the differences in HR, PR interval, QRS duration, and QT(c) interval before and after chemotherapy. Chi squared test (Pearson’s χ2 test) or binary logistic regression was employed to determine the relationship among abnormalities on ECG or UCG records, demographic characteristics, and drugs used. Continuous correction of the Chi squared test was used when necessary. The Hardy–Weinberg equilibrium test was performed to validate the genotype distributions of each SNP using the χ2 test. The association between abnormalities on ECG or UCG records and genotype distributions of SNPs was estimated by calculating odds ratios (ORs) and their 95% confidence intervals (95% CIs) with multivariate logistic regression analysis. A P value of less than 0.05 was considered statistically significant, and all statistical tests were two-sided. All analyses were performed with SPSS 19.0 (IBM Inc., Chicago, IL, USA) software.