title: Methylation in promoter regions of PITX2 and RASSF1A genes in association with clinicopathological features in breast cancer patients.
abstract: Breast cancer is a heterogeneous disease with very different responses to therapy and different length of survival. In many cases, however, the determination of the stage and histopathological characteristics of breast cancer is insufficient to predict prognosis and response to treatment for the vast heterogeneity of the disease. To understand the molecular signature of subtypes of breast cancer, we attempted to identify the methylation status of key tumour suppressor gene Ras association (RalGDS/AF-6) domain family member 1 isoform a (RASSF1A) and a member of the paired-like homeodomain transcription factor family which functions in left-right asymmetry development (PITX2) and to correlate results with known clinicopathological features of breast cancer. Formalin-fixed, paraffin-embedded (FFPE) tissues of breast carcinomas (n = 149) were used for DNA extraction. DNA was modified by bisulphite conversion. Detection of the methylation level of the genes mentioned above was performed by methylation-sensitive high-resolution melting assay (MS-HRM). Based on MS-HRM results for RASSF1A and PITX2, we subdivided the samples into four groups according to methylation level (≤50 % methylated, >50 % methylated, 100 % methylated and completely unmethylated alleles). All degrees of methylation status for both genes underwent analysis of dependence with known clinicopathological features, and we found significant associations. In 134 of 149 (89.9 %) primary breast carcinomas, the RASSF1A promoter was methylated. Total hypermethylation of PITX2 was observed in 60 of 135 (44.4 %) breast cancer cases. RASSF1A hypermethylation had significant association with increased age (p < 0.05), tumour grade (p < 0.0001) and stage (p < 0.0001) in the 100 % methylated group. There was significant association of PITX2 hypermethylation with tumour grade (p < 0.0001) and stage (p < 0.0001). Association between the methylation level of both investigated genes and tumour type was significant for ductal invasive carcinoma cases only (p < 0.0001). This study shows different levels of heterogeneous methylation acquired by MS-HRM assay of the promoter region of RASSF1A and PITX2 and its relationship with clinicopathological features of 149 breast cancer patients. We noticed that immunohistopathological subtypes of breast cancer contain distinct promoter methylation patterns. All these data suggest that hypermethylation of the CpG island promoters of RASSF1A and PITX2 might play an essential role in the very early stages of breast cancer pathogenesis.
authors.affiliation: Department of Oncology JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4C, 036 01, Martin, Slovakia. jezkova@jfmed.uniba.sk.|Clinic of Gynaecology and Obstetrics, Jessenius Faculty of Medicine, Martin University Hospital, Kollarova 2, 036 01, Martin, Slovakia. jezkova@jfmed.uniba.sk.
authors.forename: Eva
authors.initials: E
authors.lastname: Jezkova
authors.affiliation: St. Elizabeth Cancer Institute Hospital, Heydukova 10, 812 50, Bratislava, Slovakia.
authors.forename: Karol
authors.initials: K
authors.lastname: Kajo
authors.affiliation: Department of Oncology JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4C, 036 01, Martin, Slovakia.|Clinic of Gynaecology and Obstetrics, Jessenius Faculty of Medicine, Martin University Hospital, Kollarova 2, 036 01, Martin, Slovakia.
authors.forename: Pavol
authors.initials: P
authors.lastname: Zubor
authors.affiliation: Department of Oncology JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4C, 036 01, Martin, Slovakia.
authors.forename: Marian
authors.initials: M
authors.lastname: Grendar
authors.affiliation: Department of Oncology JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4C, 036 01, Martin, Slovakia.
authors.forename: Bibiana
authors.initials: B
authors.lastname: Malicherova
authors.affiliation: Department of Molecular Medicine JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4C, 036 01, Martin, Slovakia.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mendelova
authors.affiliation: Clinic of Gynaecology and Obstetrics, Jessenius Faculty of Medicine, Martin University Hospital, Kollarova 2, 036 01, Martin, Slovakia.
authors.forename: Karol
authors.initials: K
authors.lastname: Dokus
authors.affiliation: Department of Oncology JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4C, 036 01, Martin, Slovakia.
authors.forename: Zora
authors.initials: Z
authors.lastname: Lasabova
authors.affiliation: Department of Pathological Anatomy, Jessenius Faculty of Medicine, Martin University Hospital, Kollarova 2, 036 01, Martin, Slovakia.
authors.forename: Lukas
authors.initials: L
authors.lastname: Plank
authors.affiliation: Clinic of Gynaecology and Obstetrics, Jessenius Faculty of Medicine, Martin University Hospital, Kollarova 2, 036 01, Martin, Slovakia.
authors.forename: Jan
authors.initials: J
authors.lastname: Danko
keywords: Breast cancer; DNA methylation; High-resolution melting assay; PITX2; RASSF1A