The promoter methylation status in breast cancer and benign control.
The promoter methylation status of DACT1, PAX5, PLCD1, ZNF545 and TET1was assayed in 32 benign control and 237 breast cancer tissues. Only PAX5 (12.5%) and TET1(6.3%) were methylated in control group. PAX5 and PLCD1 showed exceedingly high methylation rates in the cancer patients and the percentages were 69.2% (164/237) and 54.9% (130/237) respectively, whereas the percentages of DACT1, ZNF545 and TET1 are 33.8% (80/237), 28.7% (68/237) and 18.2% (43/237), respectively (Fig 1A, Table 2). Our findings were validated in the TCGA database. In general, the methylation level of those five genes were significantly higher in breast cancer tissues compared to normal tissues (Fig 1B). From these data, we can find that the methylation level of the five genes in breast cancer tissues were negatively correlated with their expression levels (Fig 1C).
To compared the methylated status of those five genes in breast cancer tissues and plasma，we examined both the breast cancer tissues and paired plasma samples from 33 breast cancer patients. The result suggested that PAX5 had higher methylation frequency (78.8%) than DACT1, PLCD1, ZNF545 and TET1 (15.2%, 9.1%, 3.0% and 6.1% in plasma, respectively), which was similar to methylation status in tissue sample (Fig 1D). The concordance methylated status and gene expression analyze showed that PAX5 presented the highest concordance (81.8%) between tissue and paired plasma DNA when compared with the other four genes (DACT1 36.4%, PLCD1 51.5%, ZNF545 45.5%, TET1 0%) (Table 3). We can find that the concordance rates between tissues and plasma in patients largely differ dependently on the genes methylation.
Association between gene methylation status and clinicopathological features in breast cancer
The correlation between methylation status and patient clinicopathological parameters in tissues was lay out in Table 4. Age-dependent promoter methylation has been reported previously in other tumor suppressor genes in several human cancer types(26). In this study, we reported PAX5, PLCD1 and ZNF545 were hypermethylated in elder (> 50 years) breast cancer patients, with the p value of 0.012, 0.013 and 0.012 respectively. TET1 methylation was statistically associated with tumor size (p=0.001). In addition, we found the presence of associations between frequently methylation of PAX5 with positive hormone receptors (p= 0.006 for estrogen receptor and p = 0.032 for progesterone receptor), two elements of ER and PR regarded as putative risk factors for early relapse in breast cancer, suggesting that the poor patient prognosis was related to the promoter methylation of PAX5. Meanwhile, DACT1 and TET1 had higher methylation rate in the HER2-positive group than HER2-negative group (p = 0.020 for DACT1, p =0.015 for TET1). Besides that, hypomethylation of DACT1 was related to progesterone receptor and p53 positive, the p value was 0.035 for progesterone receptor and 0.009 for p53. And we found that the methylation rate of ZNF545 was statistically lower in 60 patients who were given neoadjuvant chemotherapy than in patients who had mastectomy before systemic treatment (p = 0.040), which suggesting that hypomethylation of ZNF545 may related to patient chemo-response. We found that the methylation status of these five genes in patients’ plasma was not associated with clinicopathological parameters except ZNF545 hypermethylated may associated with tumor size (Table S1)
PAX5, PLCD1 and ZNF545 hypomethylation were associated with better survival
Follow-up data on 194 patients enrolled before January 1, 2015 were included in a survival analysis based on DNA methylation status (Fig 2). In this small cohort with a relatively short follow-up time, the survival curves of the methylated and unmethylated groups were nearly parallel except for that of ZNF545. The test revealed a better survival of patients with methylated ZNF545 (p = 0.0350).
Considering the relatively small sample amount and short follow-up time of this study, two online databases, Kaplan–Meier Plotter and bc-GenExMiner V4.0, were employed to adduce indirect evidences to elucidate the value of the five genes on prognostic prediction in breast cancer. From bc-GenExMiner V4.0, high expression of PAX5 (HR:0.87, 95% CI: 0.78-0.98, p=0.0232), PLCD1 (HR:0.81, 95% CI: 0.72-0.91, p=0.0003) and ZNF545 (HR=0.84, 95% CI: 0.70-0.99, p=0.0407) presented better MR-free survival, whereas DACT1 expression showed no significant difference with metastatic relapse in breast cancer (HR: 1.10, 95% CI: 0.98-1.24, p=0.1120). Low expression of TET1 might plays an important role in metastatic relapse (HR:1.26, 95% CI: 1.06-1.48, p=0.0073) (Fig 3A). Analysis of RFS from Kaplan–Meier Plotter indicated that high expression of all five genes predicted showed longer RFS under individual cutoff (Fig 3B).
PAX5, PLCD1 and TET1 had statistically significance for diagnosis in breast cancer
To distinguish breast cancer from breast benign disease, we performed ROC curve analysis and calculated area under the curve (AUC), sensitivity and specificity of each gene (Table 5). According to the results, DACT1, PAX5 and PLCD1 had statistically significance for diagnosis in breast cancer. PAX5 and PLCD1 had both high sensitivity (69.20% and 54.85%, respectively) and high specificity (87.50% and 100.00%, respectively). DACT1 had low sensitivity (33.76%) but high specificity (100.00%). Next, we conducted univariate logistic regression analysis to analyze the methylation status of five genes to predict breast cancer in 237 cancer patients and 32 benign disease patients. Patients with methylation of PAX5 have a higher risk of breast cancer (OR=15.726, 95% CI=5.323-46.463, p<0.001) (Table 6).
Information on methylation level of these 5 genes of 72 pairs as case-control was obtained from TCGA database. The risk of breast cancer with DACT1, PAX5, PLCD1, ZNF454 and TET1 hypermethylation were 1.182, 20.598, 8.412, 3.130 and 12.250 times higher than that with hypomethylation, respectively, and all p values, except DACH1, were less than 0.05 (Table 7). As a result of the multiple linear regression analysis of gene methylation level and breast cancer (Table 8), the higher the methylation level of PAX5 and TET1 was correlated with higher risk of breast cancer, with the coefficients of 0.466 and 0.292, respectively.