DNA Methylation Status Of Cyclin D2 And HIN-1 Gene In Intraductal Proliferative Lesions Of Breast And Invasive Breast Cancer

[Objective] Breast cancer is one of the most common malignant tumors. The incidence of breast cancer has been increasing in recent years. It is crucial for breast cancer to diagnose and treat in early stages. Breast intraductal hyperplasia lesions are closely associated with breast cancer, the risk to breast cancer development of which increase in several different rates. DNA methylation is a hot topic in breast cancer research recently. Many researches have confirmed that DNA hypermethylation is closely associated with tumorigenesis, however, as gene methylation is tumor-specific, there is no ideal DNA methylation profiles to use in clinical diagnosis of breast cancer. In addition, little is known about DNA hypermethylation from intradutcal hyperplasia lesions to breast cancer . The purpose of this study was to investigate two candidate tumor suppressor genes--cyclin D2 and HIN-1 genes methylation states in usual ductal hyperplasia(UDH), atypical ductal hyperplasia(ADH), ductal carcinoma in situ (DCIS) as well as invasive breast cancer, and to increase our understanding of what the role of the cyclin D2 and HIN-1 gene methylation play in breast tumorigenesis and progression. We hope our research data can provide a theoretical basis for the selection of gene methylation profiles, as well as for the early diagnosis , prevention and treatment of the breast cancer.[Methods] Forty-four cases of intraductal proliferative lesions (12 UDH, 8 ADH, 24 DCIS) of breast ,14 cases of invasive breast cancer and 6 cases of normal breast tissues were collected from Qi Lu Hospital of Shandong University from 2002～2005. The samples were fixed in 10% formalin and paraffin embedded. All archival haematoxylin and eosin-stained sections were reviewed to confirm the diagnosis. The CpG island methylation status of cyclin D2 and HIN-1 gene were analyzed using methylation-specific polymerase chain reaction(MSP). All the data were analyzed by SPSS 13.0 software for windows.[Results] The positive rate of hypermethylation in cyclin D2 gene was 25.0%(3/12) of UDH, 37.5% (3/8) of ADH, 66.7% (16/24) of DCIS and 78.6 % (11/14) in IBC respectively. The positive rates of hypermethylation in HIN-1 gene was 16.7%(2/12) of UDH, 37.5%(3/8) of ADH, 62.5%( 15/24)of DCIS and 85.7% (12/14) of IBC. By contrast, there was no hypermethylation in both cyclin D2 and HIN-1 gene in normal breast tissue. The significant difference of the methylation rate of cyclin D2 gene was found between normal tissue and IBC, UDH and IBC, normal tissue and DCIS, UDH and DCIS (P＜0.05) . There was no significant difference between all other groups (P>0.05). The statistic results of methylation rate in HIN-1 gene were the same to cyclin D2 gene. The rate of methylation increased from normal breast tissue to UDH to ADH to DCIS and to IBC both in cyclin D2 and HIN-1 gene. The statistic results showed that there were liner correlations. In addition, at least one gene methylated in 25%(3/12) of UDH, 50.0%(4/8) of ADH, 87.5% of (21/24) DCIS, 92.9% (13/14) of IBC. The hypermethylation levels in DCIS and IBC were much higher than that of nomal tissue, UDH and ADH, and the differences were significant (P＜0.05) . From UDH, ADH, DCIS to IBC, the methylation rates increased, and statistic result showed there was a liner correlation too. At the same time, Our study found that there was a positive correlation between methylation of cyclin D2 gene and that of HIN-1 gene.[Conclusion] 1. Neither of cyclin D2 and HIN-1 gene are methylated in normal tissue, but both are methylated in DCIS and IBC with high levels. It indicates that the methylation of cyclin D2 and HIN-1 gene plays a role in breast tumorigenesis. 2. That some extent levels of methylation of cyclin D2 and HIN-1 gene in UDH and ADH suggestes the methylation of two genes is a early event in breast tumorigenesis, its accumulation may promote malignant transformation. 3. The positive correlation of cyclin D2 gene methylation with that of HIN-1 indicates that detection the mathylation of cyclin D2 and HIN-1 gene together can provide a profile to diagnose breast cancer in early stage. In addition, hypermethylation in breast intraductal hyperplasia lesions may be biomarkers of cancer risk and targets for cancer chemoprevention.