Assessment of DNA methylation status in early stages of breast cancer development

Abstract

Background: Molecular pathways determining the malignant potential of premalignant breast lesions remain unknown. In this study, alterations in DNA methylation levels were monitored during benign, premalignant and malignant stages of ductal breast cancer development.

Methods: To study epigenetic events during breast cancer development, four genomic biomarkers (Methylated-IN-Tumour (MINT)17, MINT31, RARβ2 and RASSF1A) shown to represent DNA hypermethylation in tumours were selected. Laser capture microdissection was employed to isolate DNA from breast lesions, including normal breast epithelia (n=52), ductal hyperplasia (n=23), atypical ductal hyperplasia (n=31), ductal carcinoma in situ (DCIS, n=95) and AJCC stage I invasive ductal carcinoma (IDC, n=34). Methylation Index (MI) for each biomarker was calculated based on methylated and unmethylated copy numbers measured by Absolute Quantitative Assessment Of Methylated Alleles (AQAMA). Trends in MI by developmental stage were analysed.

Results: Methylation levels increased significantly during the progressive stages of breast cancer development; P-values are 0.0012, 0.0003, 0.012, <0.0001 and <0.0001 for MINT17, MINT31, RARβ2, RASSF1A and combined biomarkers, respectively. In both DCIS and IDC, hypermethylation was associated with unfavourable characteristics.

Conclusion: DNA hypermethylation of selected biomarkers occurs early in breast cancer development, and may present a predictor of malignant potential.

Figure 1

Laser capture microdissection of ductal carcinoma in situ. All tissue types assayed in this study were carefully microdissected by LCM from deparaffinized, hematoxylin-stained slide sections of ∼8 μm thickness. A representative slide example of capturing DCIS tissue is shown after LCM in (A), and the microdissected tissues LCM on-cap in (B).

Figure 2

Linearity of LCM surface area and total copy numbers. A series of incremental surface areas of 10⁵, 3 × 10⁵, 10⁶, 3 × 10⁶ and 10⁷ μm² was captured. Tissues were subjected to in situ on-cap bisulfite modification followed by proteinase K treatment. Tissue lysate was used for AQAMA PCR for copy number measurement. Data of MINT17 copy numbers are shown. The Pearson R² was 0.97 for correlation between increasing copy numbers with increasing surface area (P=0.002). X-axis indicates the respective surface areas assayed; the numbers represent captured μm in multiples of ten. This figure also demonstrates adequate PCR efficiency for captured and processed sample DNA, yielding an efficient amplification slope (Pearson R² 0.97) in a serial dilution series.

Figure 3

Methylation Index by tissue type. Boxplots showing Methylation Index of MINT17, MINT31, RARß2 and RASSF1A vs tissue type (normal breast epithelia (NL), ductal hyperplasia (DH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal cancer (IDC)). Boxplots show quartiles, median and outliers.