Our hospital-based study recruited 681 breast cancer cases from Fujian Medical University Union Hospital from July 2000 and October 2014. Patient eligibility criteria in this study were as follows: (i) histopathologically confirmed with invasive breast cancer; (ii) subsequently treated with curative surgical resection and systemic therapy; (iii) availability of complete clinical and follow-up data，(iv) no history of other malignancy and (v) alive at least 1 month after surgery. We ruled out 20 patients, containing 6 patients with breast carcinoma in situ and 14 patients with incomplete clinical information according to this screening criteria. Finally, 661 patients with resected breast cancer were included in the present study for predictive analysis.
Clinicopathological and demographic information was collected from the hospital records, and survival data were obtained from the followed-up database renewed annually. The patients were staged according to the 7th version of the American Joint Commission on Cancer (AJCC) tumor-node-metastasis (TNM) staging system . Estrogen receptor (ER)/progesterone receptor (PR) positivity was determined by IHC analysis of the number of positively stained nuclei (≥ 10%), and hormone receptor (HR) positivity was defined as being either ER+ and/or PR+. Tumors were considered human epidermal growth factor-2 (HER2) positive when cells exhibited strong membrane staining (3+). Expressions of 2+ would require further in situ hybridization testing for HER2 gene amplification, while expressions of 0 or 1+ were regarded as negative. The subtypes were categorized : luminal A (ER+, PR+ ＞20%, HER2-, Ki67＜14% or grade I when Ki67 was unavailable), luminal B (HR+, HER2-, Ki67＞14% or grade II/III when Ki67 was unavailable or HR+, HER2+); HER2 enriched (HR-, HER2+) and triple-negative (HR- and HER2-). The Institutional Ethics Committee approved the study, and all participants consented to the testing at the time of their participation and contributed data.
Blood samples were collected in EDTA anticoagulant tubes and were centrifuged within 30 min. Genomic DNA was extracted from blood samples by using the Whole-Blood DNA Extraction Kit (Bioteke, Beijing, China), according to the manufacturer's protocol, and then the genomic DNA was aliquoted and stored at -80°C for future analysis. The purity of the DNA samples was assessed before mtDNA copy number assessment, and samples without sufficient DNA yield were not included for mtDNA copy number analysis.
MtDNA copy number assessment
Relative mtDNA content was measured by a custom-by-design Multiplex AccuCopy™Kit (Genesky Biotechnologies Inc., Shanghai, China) based on a multiplex fluorescence competitive PCR principle as previously described , which can interrogate copy number variation (CNV) status at multiple genomic loci in the same assay reaction.
The methods below briefly describe the manufacturer's process. A total of 2 target genomic segments within the ND1 gene and 6 reference segments (2, 10, 16, 18, 19, and 20 p) were chosen for the AccuCopy assay. The primers for both target and reference segments were synthesized. The forward primers were fluorescent-labeled at Genesky Biotechnologies (Shanghai, China). The competitive DNAs for the ND1 and 6 reference segments were synthesized in double-strand and provided a mixture from Genesky Biotechnologies (Shanghai, China). These competitive DNAs are almost identical to their homologies in the human reference genome except for 1-2 base pairs deleted. The primers of target segments and reference segments and probes information were provided in Table S1.
The PCR reaction was prepared in 20 μl for each sample, containing a mixture of 2 μl target genomic DNA (5 ng/μl) with 2 μl reference segment DNA, 1 μl Multiplex PCR Fluorescence Primer Mix (AccuCopy™), 10 μl 2 × PCR Master Mix (Genesky Biotechnologies), and 5 μl ddH2O. 10 μl 2 × PCR Master Mix (Genesky Biotechnologies), and 5 μl ddH2O. The program used was an initial denaturation step of 95°C for 10 min followed by the first 11 cycles of denaturation at 94°C for 20 s, annealing at 60°C for 40 s (the annealing temperature was decreased by 0.5°C in each consecutive cycle), and elongation at 72°C for 1.5 min, followed by the second 24 cycles of denaturation at 94°C for 20 s, annealing at 59°C for 30 s, and elongation at 72°C for 1.5 min, and a final extension step at 60°C for 60 min and then at 4 °C forever. The PCR product was diluted in a 1:5 ratio, and 1μl diluted products were mixed with 0.5 μl 500 (Liz) size standard and 8.5 μl Hi-Di formamide (both from Applied Biosystems, Foster City, CA). The mixture was subjected to a denaturation step of 95°C for 5 min and electrophoresed in a 3730XL genetic analyzer (ABI, Carlsbad, CA, USA). Raw data were analyzed by GeneMapper 4.0 (ABI). The height and area data for all specific peaks were exported into a Microsoft Excel file.
The sample/competitive (S/C) peak ratio was calculated for the 2 target and 6 reference segments. The S/C ratio for each target fragment was first normalized based on four reference segments, respectively. The 6 normalized S/C ratios were further normalized to the median value in all samples for each reference segment, respectively, and then averaged. If one of the six normalized S/C ratios deviated ＞25% from the average of the other five, it was excluded from further analysis. The copy number of each target segment was determined by the average S/C ratio times two, given that the copy numbers of four reference segments are two in the diploid genome. Twenty samples were randomly selected as blinded duplicates for quality assessment purposes, and excellent concordance was obtained.
Overall survival (OS) and breast cancer-specific survival (BCSS) were our primary endpoints and defined as the time from cancer diagnosis to the date of mortality for all causes and breast cancer, respectively. Invasive disease-free survival (iDFS) and distant disease-free survival (DDFS) were our secondary endpoints and calculated separately from the date of diagnosis to the date of any recurrence and distant recurrence to follow-up cut-off time . Survival data were analyzed using the Kaplan–Meier method with the log-rank test. The latest follow-up date was 2016.12.31, and the median follow-up duration was 78.8 months (ranging from 4 to 189 months). To adjust for possible confounders and evaluate the form of the relationship, Cox proportional hazards models were fitted using different adjustment parameters: model 1 adjusted for age at diagnosis; model 2 additionally adjusted for hormone receptor status, and HER2 status; model 3 was the same as model 2 plus an adjustment for tumor size, Lymph node involvement, and grade. The Cox proportional hazards model results showed the non-linearity in the effects of mitochondrial copy number on cancer progression. Moreover, nonlinear p-splines were created to evalutate the functional form of the relationship between mtDNA copy number and BC patients’ survival. The results of the splines and the estimates for the quintiles 2, 3, 4 and 5 compared with quintile 1 revealed that a categorical analysis of the data by combining the 2nd, 3rd, 4th and 5th quintile of mtDNA copy number and to set quintile 1 as a reference fits the data better than modelling the data on a continuous scale. The Chi-square test was used to examine differences of categorical variables between subgroups. The student's t-test was used to analyze the difference of normally distributed continuous variables between two groups. The Cox regression model calculated the hazard ratios (HRs) and 95% confidence interval (CI) for each factor in multivariate analyses. The possible mtDNAcn-environment interactions were also evaluated by the Cox proportional hazard regression models. All tests were 2-sided, and P-values < 0.05 were considered statistically significant. SAS 9.4 (SAS Institute Inc., Cary, NC) was used for all statistical analyses.