LINE1 and Alu repetitive element DNA methylation in tumors and white blood cells from epithelial ovarian cancer patients

Abstract

Objective: We determined whether DNA methylation of repetitive elements (RE) is altered in epithelial ovarian cancer (EOC) patient tumors and white blood cells (WBC), compared to normal tissue controls.

Methods: Two different quantitative measures of RE methylation (LINE1 and Alu bisulfite pyrosequencing) were used in normal and tumor tissues from EOC cases and controls. Tissues analyzed included: i) EOC, ii) normal ovarian surface epithelia (OSE), iii) normal fallopian tube surface epithelia (FTE), iv) WBC from EOC patients, obtained before and after treatment, and v) WBC from demographically-matched controls.

Results: REs were significantly hypomethylated in EOC compared to OSE and FTE, and LINE1 and Alu methylation showed a significant direct association in these tissues. In contrast, WBC RE methylation was significantly higher in EOC cases compared to controls. RE methylation in patient-matched EOC tumors and pre-treatment WBC did not correlate.

Conclusions: EOC shows robust RE hypomethylation compared to normal tissues from which the disease arises. In contrast, RE are generally hypermethylated in EOC patient WBC compared to controls. EOC tumor and WBC methylation did not correlate in matched patients, suggesting that RE methylation is independently controlled in tumor and normal tissues. Despite the significant differences observed over the population, the range of RE methylation in patient and control WBC overlapped, limiting their specific utility as an EOC biomarker. However, our data demonstrate that DNA methylation is deranged in normal tissues from EOC patients, supporting further investigation of WBC DNA methylation biomarkers suitable for EOC risk assessment.

Keywords: Alu; DNA methylation; Epithelial ovarian cancer; LINE1; Repetitive elements.

Figure 1. LINE1 and Alu RE methylation in human epithelial ovarian cancer (EOC), normal ovarian surface epithelia (OSE), and normal fallopian tube epithelia (FTE)

LINE1 and Alu methylation were determined using bisulfite pyrosequencing. A) LINE1 and B) Alu methylation in EOC, normal tissues (OSE + FTE), OSE, and FTE samples. Number of samples per group is shown below the y-axis labels. Box and whiskers plots are shown (bars, mean; box, 25-75 percentile; whiskers, 2.5-97.5 percentile). Mann-Whitney two-tailed P-values are shown. NS = not significant. C) Comparison of methylation in patient-matched OSE and FTE tissues. Spearman correlation test results (for LINE1 + Alu) are shown. D) Comparison of LINE1 and Alu methylation in EOC. A linear regression line, and Spearman correlation test results are shown.

Figure 2. LINE1 and Alu RE methylation in WBC from EOC patients and controls

RE methylation was determined using bisulfite pyrosequencing. Post-treatment case and control leukocytes were obtained from the HOPE study. Pre-treatment case samples (PBMC) were also available, for a sub-set of HOPE cases. A) LINE1 methylation in controls (leukocytes), all cases (PBMC + leukocytes), pre-treatment cases (PBMC), and post-treatment cases (leukocytes). B) Alu methylation in controls (leukocytes), all cases (PBMC + leukocytes), pre-treatment cases (PBMC), and post-treatment cases (leukocytes). Box and whiskers plots are presented as described in Fig. 1. Number of samples per group is shown below the y-axis labels. Mann-Whitney two-tailed P-values are shown.

Figure 3. Correlation of LINE1 and Alu methylation in WBC from EOC patients and controls

RE methylation was determined using bisulfite pyrosequencing. A) Association between LINE1 and Alu methylation in controls (leukocytes). B) Association between LINE1 and Alu methylation in all cases (PBMC + leukocytes). C Association between LINE1 and Alu methylation in pre-treatment cases (PBMC). D) Association between LINE1 and Alu methylation in post-treatment cases (leukocytes). In all panels, Spearman correlation test results are shown, and linear regression lines are plotted for statistically significant correlations. NS = not significant.

Figure 4. Correlation of RE methylation in patient-matched tumors and pre-treatment PBMC

RE methylation was determined using bisulfite pyrosequencing. A) LINE1 and B) Alu methylation were compared in patient-matched tumors and pre-treatment PBMC samples. Spearman correlation test results are shown. NS = not significant.