Methylation of NBPF1 as a novel marker for the detection of plasma cell-free DNA of breast cancer patients

Background: Recent studies revealed that tumor-specific gene methylation can be detected in the circulating cell-free DNA (cfDNA) of cancer patients; therefore, methylated cfDNA is considered a promising biomarker. Human neuroblastoma breakpoint family member 1 (NBPF1) was originally identified in a neuroblastoma (NB) patient. The present study is the first to evaluate the presence of NBPF1 gene methylation in cfDNA in plasma of breast cancer patients.

Methods: Differentially methylated cfDNA was screened using bisulfite sequencing with a next-generation sequencer (BS-seq) among 25 breast cancer patients, 25 patients with a benign breast disease and 25 healthy female volunteers. Then, five specific methylation sites in the NBPF1 gene were verified in 11 breast cancer samples, and two further sites in the NBPF1 promoter were detected in 52 breast cancer patients (stages I-III), 31 patients with benign breast disease and 30 healthy controls by using methylation-specific PCR (MSP). Furthermore, the association between the methylation statuses of NBPF1 and the clinicopathological characteristics of breast cancer patients was analyzed.

Results: BS-seq demonstrated that the NBPF1 methylation levels in breast cancer patients were higher than those in patients with benign breast disease and healthy controls. The MSP results showed that the methylation rates of two sites in the NBPF1 promoter were 67.1% and 61.4% in breast cancer patients, 48.2% and 59.6% in patients with benign breast disease, and 40.9% and 48.1% in healthy controls, respectively. The methylation rates of one site were significantly different among the three groups (p < .05), with the highest rate in breast cancer patients. Moreover, there was no statistically significant correlation between the NBPF1 promoter methylation and the major clinicopathological features of the patients.

Conclusions: These results indicate that hypermethylation of the NBPF1 promoter occurs in a significant proportion of breast tumors and that NBPF1-methylated cfDNA may serve as a potential tumor marker for breast cancer.