Quantitation of CDH1 promoter methylation in formalin-fixed paraffin-embedded tissues of breast cancer patients using differential high resolution melting analysis


1 Department of Pharmaceutical Biotechnology, Isfahan University of Medical Sciences, Isfahan, Iran

2 Cancer, Petroleum and Environmental Pollutants Research Centre, Ahvaz Jundishapur University of Medical sciences, Ahvaz, Iran

3 Department of Pharmacology and Toxicology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

4 Applied Physiology Research Centre, Isfahan University of Medical Sciences, Isfahan, Iran


Background: E-cadherin (CDH1) plays an important role in cell–cell adhesion of epithelial tissues. Loss of E-cadherin expression can lead to loss of tissue integrity, metastasis, and cancer progression. Also loss of E-cadherin expression might be related to aberrant promoter methylation of the CDH1 gene. Many studies have been performed on CDH1 promoter methylation, especially in breast cancer. Although most of the studies have used qualitative methods for methylation analysis, this study is designed to quantitatively investigate CDH1 promoter methylation in breast cancer and its correlation with patients' clinicopathological features.
Materials and Methods: Using differential high resolution melting analysis (D-HRMA), the methylation level of the CDH1 gene promoter was quantified in 98 breast cancer formalin-fixed paraffin-embedded (FFPE) tissues and also 10 fresh frozen normal breast tissues.

Results: All samples were detected to be methylated at the CDH1 promoter region. About 74.5% of the breast cancer samples were hypermethylated with an average methylation level of around 60%, while 25.5% of the patients were methylated with the mean methylation level of about 33%, and 90% of the normal samples had a mean methylation level of about 18%. Statistical analyses represented a significant correlation between CDH1 promoter methylation and cancer progression hallmarks, such as, clinical stage, nodal involvement, tumor size, and histological grade.
Conclusion: In summary, quantitation of CDH1 promoter methylation can serve as a diagnostic and prognostic tool in breast cancer. Also D-HRMA can be used as a fast and reliable method for quantitation of promoter methylation.


Caldeira JR, Prando EC, Quevedo FC, Neto FA, Rainho CA, Rogatto SR. CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer. BMC Cancer 2006;6:48.  Back to cited text no. 1
Lombaerts M, van Wezel T, Philippo K, Dierssen JW, Zimmerman RM, Oosting J, et al. E-cadherin transcriptional downregulation by promoter methylation but not mutation is related to epithelial-to-mesenchymal transition in breast cancer cell lines. Brit J Cancer 2006;94:661-71.  Back to cited text no. 2
Sunami E, Shinozaki M, Sim MS, Nguyen SL, Vu AT, Giuliano AE, et al. Estrogen receptor and HER2/neu status affect epigenetic differences of tumor-related genes in primary breast tumors. Breast Cancer Res 2008;10:R46.  Back to cited text no. 3
Luczak MW, Jagodziński PP. The role of DNA methylation in cancer development. Folia Histochem Cytobiol 2006;44:143-54.  Back to cited text no. 4
Miyamoto K, Ushijima T. Diagnostic and therapeutic applications of epigenetics. Jpn J Clin Oncol 2005;35:293-301.  Back to cited text no. 5
Kristensen LS, Mikeska T, Krypuy M, Dobrovic A. Sensitive melting analysis after real time-methylation specific PCR (SMART-MSP): High-throughput and probe-free quantitative DNA methylation detection. Nucleic Acids Res 2008;36:e42.  Back to cited text no. 6
Kajabova V, Smolkova B, Zmetakova I, Sebova K, Krivulcik T, Bella V, et al. RASSF1A promoter methylation levels positively correlate with estrogen receptor expression in breast cancer patients. Transl Oncol 2013;6:297-304.  Back to cited text no. 7
Cho YH, Shen J, Gammon MD, Zhang YJ, Wang Q, Gonzalez K, et al. Prognostic significance of gene-specific promoter hypermethylation in breast cancer patients. Breast Cancer Res Treat 2012;131:197-205.  Back to cited text no. 8
Cho YH, Yazici H, Wu HC, Terry MB, Gonzalez K, Qu M, et al. Aberrant promoter hypermethylation and genomic hypomethylation in tumor, adjacent normal tissues and blood from breast cancer patients. Anticancer Res 2010;30:2489-96.  Back to cited text no. 9
Sebova K, Zmetakova I, Bella V, Kajo K, Stankovicova I, Kajabova V, et al. RASSF1A and CDH1 hypermethylation as potential epimarkers in breast cancer. Cancer Biomark 2012;10:13-26.  Back to cited text no. 10
Wojdacz TK. Methylation-sensitive high-resolution melting in the context of legislative requirements for validation of analytical procedures for diagnostic applications. Expert Rev Mol Diagn 2012;12:39-47.  Back to cited text no. 11
Liu W, Guan M, Su B, Li J, Ma W, Liu C, et al. Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation-sensitive high resolution melting (MS-HRM) analysis: Application in colorectal cancer. Clin Chim Acta 2010;411:940-6.  Back to cited text no. 12
Balic M, Pichler M, Strutz J, Heitzer E, Ausch C, Samonigg H, et al. High quality assessment of DNA methylation in archival tissues from colorectal cancer patients using quantitative high-resolution melting analysis. J Mol Diagn 2009;11:102-8.  Back to cited text no. 13
Malentacchi F, Forni G, Vinci S, Orlando C. Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol. Nucleic Acids Res 2009;37:e86.  Back to cited text no. 14
Naghitorabi M, Mohammadi Asl J, Mir Mohammad Sadeghi H, Rabbani M, Jafarian-Dehkordi A, Javanmard HS. Quantitative evaluation of DNMT3B promoter methylation in breast cancer patients using differential high resolution melting analysis. Res Pharm Sci 2013;8:167-75.  Back to cited text no. 15
Rasti M, Entezam M, Monabati A. Hypermethylation of E-Cadherin and Estrogen Receptor-α gene promoter and its association with clinicopathological features of breast cancer in Iranian patients. Iran J Med Sci 2009;34:186-90.  Back to cited text no. 16
Parrella P, Poeta ML, Gallo AP, Prencipe M, Scintu M, Apicella A, et al. Nonrandom distribution of aberrant promoter methylation of cancer-related genes in sporadic breast tumors. Clin Cancer Res 2004;10:5349-54.  Back to cited text no. 17
Li S, Rong M, Iacopetta B. DNA hypermethylation in breast cancer and its association with clinicopathological features. Cancer Lett 2006;237:272-80.  Back to cited text no. 18
Zhao L, Wang L, Jin F, Ma W, Ren J, Wen X, et al. Silencing of estrogen receptor alpha (ERalpha) gene by promoter hypermethylation is a frequent event in Chinese women with sporadic breast cancer. Breast Cancer Res Treat 2008;117:253-9.  Back to cited text no. 19
Zou D, Yoon HS, Perez D, Weeks RJ, Guilford P, Humar B. Epigenetic silencing in non-neoplastic epithelia identifies E-cadherin (CDH1) as a target for chemoprevention of lobular neoplasia. J Pathol 2009;218:265-72.  Back to cited text no. 20
Jerónimo C, Costa I, Martins MC, Monteiro P, Lisboa S, Palmeira C, et al. Detection of gene promoter hypermethylation in fine needle washings from breast lesions. Clin Cancer Res 2003;9:3413-7.  Back to cited text no. 21
Jung SP, Kim S, Nam SJ, Kim I, Bae JW. The role of the CDH1 promoter hypermethylation in the axillary lymph node metastasis and prognosis. J Breast Cancer 2013;16:16-22.  Back to cited text no. 22
Swift-Scanlan T, Vang R, Blackford A, Fackler MJ, Sukumar S. Methylated genes in breast cancer: Associations with clinical and histopathological features in a familial breast cancer cohort. Cancer Biol Ther 2011;11:853-65.  Back to cited text no. 23
Hoque MO, Prencipe M, Poeta ML, Barbano R, Valori VM, Copetti M, et al. Changes in CpG islands promoter methylation patterns during ductal breast carcinoma progression. Cancer Epidemiol Biomarkers Prev 2009;18:2694-700.  Back to cited text no. 24