Study subjects and study design
The prospective observational cohort consisted of breast cancer patients who received adjuvant chemotherapy at the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College between May 2017 and February 2019.
The inclusion criteria of patients in our study were as follows: (1) adult female patients; (2) voluntary participation in this study and voluntary inclusion of their data in this report; (3) pathologically diagnosed with malignant breast cancer; and (4) prepared for using adjuvant chemotherapy. The exclusion criteria were as follows: (1) incomplete basic information of the patient; (2) failed to complete the whole cycle of chemotherapy in our hospital; (3) incomplete routine blood, liver and kidney function, coagulation function, myocardial enzyme (Troponin T), NT-proBNP (or BNP), ECG and UCG tests before and after adjuvant chemotherapy; and (4) failure to detect multiple SNP sites.
This study was approved by the Institutional Review Boards of Cancer Hospital, Chinese Academy of Medical Sciences (No. NCT03537339), and the protocol has been registered on ClinicalTrials.gov with the number NCC201712029.
Adjuvant Treatment
The adjuvant chemotherapy regimens for the enrolled patients were determined by the doctor, and the regimens were as follows:
(1) The AC regimen [epirubicin (EPI) 90 mg/ on day 1 and cyclophosphamide (CTX) 600 mg/ on day 1 and repeated every 21 days for 4-6 cycles];
(2) The AC-T(H) regimen and dose-density AC-T(H) [EPI 90 mg/ and CTX 600 mg/ on day 1, repeated every 14 (dose-density) or 21 days for 4 cycles, followed by paclitaxel (PTX) 175 mg/ on day 1 and repeated every 14 days (and trastuzumab 2 mg/kg on day 1 and repeated every 7 days) for 4 cycles];
(3) The TC regimen [docetaxel (DTX) 75 mg/ and CTX 600 mg/ on day 1, repeated every 21 days for 4-6 cycles];
(4) The TCb(H) regimen [DTX 75 mg/ on day 1 (and trastuzumab 2 mg/ on day 1 and repeated every 7 days), carboplatin area under receiver-operating curve (AUC) = 5 mg/mL on day 2 and repeated every 21 days for 6 cycles];
(5) The AT regimen (EPI 75 mg/ or on day 1 and PTX 175 mg/ on day 2, which was repeated every 21 days for 6 cycles);
(6) The TH regimen (PTX 80 mg/ on day 1 and trastuzumab 2 mg/kg on day 1, which was repeated every 7 days for 12 cycles);
Trastuzumab was used in HER2-positive breast patients, and the dosage and usage of trastuzumab was 4 mg/kg for the first dose and then 2 mg/kg, which was repeated every 7 days for 1 year or 8 mg/kg for the first dose, and then 6 mg/kg, which was repeated every 21 days for 1 year.
The AC, AC-T(H), and AT regimens were classified as anthracycline-containing chemotherapy; the AC-TH, TCb(H), and TH regimens were classified as trastuzumab-containing chemotherapy.
Monitoring the cardiac events and cardiotoxicity
All patients should have at least finish the routine blood, liver and kidney function, coagulation function, D-dimer, troponin T, NT-proBNP (or BNP), standard 12-lead ECG (BEIJING FUKUDA FX-7402) and UCG (GE LOGIQTM E9) tests before and after adjuvant chemotherapy. The paper speed of ECG was 25mm/s. All ECG and UCG records were evaluated by the same radiology team at the National Cancer Center.
The concentration of TnT was detected by an electrochemiluminescence assay, and a troponin T high-sensitivity STAT Elecsys platform (Roche® Diagnostics, Manheim, Germany) was applied. The concentration of NT-proBNP was detected by electrochemiluminescence assay using the proBNP II STAT Elecsys platform (Roche® Diagnostics, Manheim, Germany). The cutoff value was defined as the 99th percentile of a healthy population. TnT or NT-proBNP elevation refers to normal before chemotherapy and higher than normal after chemotherapy. All results are recorded in the hospital electronic system for reference.
According to the examination items, the records of cardiac toxicities was divided into four major points: (1) abnormal ECG: nonspecific ST-T segment anomaly; ST-T segment anomaly; sinus arrhythmia; supraventricular arrhythmia; ventricular arrhythmia; low voltage; poor R wave progression; and others; (2) UCG: 10% reduction in the LVEF (absolute value); (3) elevated TnT levels; (4) elevated BNP or NT-proBNP levels. Cardiac events refer to the occurrence of any one or more of the above. Early-onset is defined as cardiac toxicity during and after adjuvant chemotherapy period. The diagnosis of cardiotoxicity is based on a 10% reduction in LVEF before and after adjuvant chemotherapy.
Candidate SNP selection and genotyping
Thirty SNPs from 25 genes were selected from previously reported research and the National Center for Biotechnology Information (NCBI) SNP database (https://www.ncbi.nlm.nih.gov/snp/). The final list of candidate SNPs are shown in Table 1.
Table 1
The Detection of SNP Site
Function | Gene | SNP site | Success rate of detection |
Drug transport | ABCA1 | rs3887137 | 97.68 |
| rs2235047 | 99.74 |
ABCB4 | rs4148808 | 99.48 |
ABCC1 | rs246221 | 99.48 |
| rs3743527 | 99.74 |
ABCC5 | rs7627754 | 98.71 |
SLC22A1 | rs2282143 | 99.48 |
SLC22A2 | rs316019 | 98.45 |
SLC22A5 | rs2631370 | 50.26 |
| rs2631372 | 97.16 |
SLC22A17 | rs4982753 | 99.23 |
SLC28A1 | rs2290271 | 100.00 |
SLC28A3 | rs7853758 | 99.74 |
| rs885004 | 100.00 |
CNT1 | rs2305364 | 99.23 |
Antioxidation | CAT | rs10836235 | 99.74 |
GSTP1 | rs1695 | 98.45 |
HAS3 | rs2232228 | 99.23 |
drug metabolism | CBR3 | rs1056892 | 100.00 |
NOS3 | rs1799983 | 99.48 |
POR | rs13240755 | 0.00 |
NAD (P) H oxidase multienzyme complex | NCF4 | rs1883112 | 99.48 |
CYBA | rs4673 | 99.48 |
DNA repair | ERCC2 | rs13181 | 97.42 |
PRDM2 | rs7542939 | 100.00 |
Sarcomere structure | CELF4 | rs1786814 | 100.00 |
Genes with unknown functions | GPR35 | rs3749172 | 97.68 |
Receptor tyrosine kinase | ERBB2 | rs1136201 | 99.23 |
rs1058808 | 100.00 |
Cardiac autophagy | ATG13 | rs10838611 | 100.00 |
ABC, ATP Binding Cassette; SLC, Solute Carrier; CNT1, Concentrative Nucleotide Transporter 1; CAT,Catalase; GSTP1, Glutathione S-Transferase Pi 1; HAS3, Hyaluronan Synthase 3; CBR3, Carbonyl Reductase 3; NOS3, Nitric Oxide Synthase 3; POR, Cytochrome P450 Oxidoreductase; NCF4, Neutrophil Cytosolic Factor 4; CYBA, Cytochrome B-245 Alpha Chain; ERCC2, Excision Repair Cross-Complementing; PRDM2, PR/SET Domain 2; CELF4, CUGBP Elav-Like Family Member 4; GPR35, G Protein-Coupled Receptor 35; ERBB2, Erb-B2 Receptor Tyrosine Kinase 2; ATG13, autophagy related 13 |
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. Probes 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 SpectroCHIP bioarray using a MassARRAY Nanodispenser RS1000 (Sequenom Inc.) and determined using a matrix-assisted laser desorption/ionization time-of flight mass spectrometer. Genotype analysis was performed using MassARRAY Typer software version 4.0 (Sequenom Inc.). Duplicate samples and negative controls (without DNA) were used as quality controls for the genotyping. The concordance for duplicate samples was 100% for all assays. The group information of each sample was concealed for genotyping analysis. Sites with a more than a 95% successful detection rate were included in the subsequent analysis.
Statistical Analyses
Continuous variables (such as heart rate, QT interval and LVEF) were expressed as the mean ± SD, and the comparison before and after chemotherapy was performed by
paired-sample t-tests. The Hardy-Weinberg equilibrium test was performed to validate the genotype distributions of each SNP using the χ2 test or Fisher’s exact test. Univariable and multivariable analyses were used to identify the significant independent risk factors between the cardiotoxicity and clinical factors or/and genotype distributions of the SNPs. Variables with a p < 0.25[13] in the univariate analysis were included in the multivariate analysis. These results are presented as adjusted odds ratios (ORs) with 95% confidence intervals (CIs). All statistical tests were two-sided, and p values < 0.05 were considered statistically significant. All analyses above were performed with SPSS software (Version 23, SPSS Inc., IBM, NY, USA).