Integrative Analysis of Gene Expression and Promoter Methylation to Differentiate High-Grade Serous Ovarian Cancer from Benign Tumors

Abstract

Background: Ovarian cancer (OC) is the third most common and second most lethal onco-gynecological disease in the world, with high-grade serous ovarian cancer (HGSOC) making up the majority of OC cases worldwide. The current serological biomarkers used for OC diagnosis are lacking sensitivity and specificity, thus new biomarkers are greatly needed. Recently, the chromatin remodeling complex gene ARID1A, Notch and Wnt pathway gene expression, as well as HOX-related gene promoter methylation have been linked with promoting OC. Methods: In this pilot study, 10 gene expression biomarkers and 4 promoter methylation biomarkers were examined as potential diagnostic and prognostic indicators of OC in 65 fresh-frozen gynecologic tumor tissues. Results: Out of 10 genes analyzed, the expression of eight biomarkers was significantly reduced in OC cases compared to benign, and HOX-related gene promoter methylation significantly increased in OC tumors. Out of 14 biomarkers, CTNNB1 showed the best single biomarker separation of HGSOC from benign cases (AUC = 0.97), while a combination of the seven Notch pathway-related gene expressions (NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, JAG2, and HES1) demonstrated the best separation of HGSOC from the benign cases (AUC = 1). Conclusions: The combination of multiple gene expression or gene promoter methylation biomarkers shows great promise for the development of an effective biomarker-based diagnostic approach for OC.

Keywords: diagnostic model; gene expression; gene promoter methylation; high-grade serous ovarian carcinoma.

Figure 1

A heatmap of promoter methylation and relative gene expression in gynecologic tumors. ARID1A met—ARID1A promoter methylation, OC—ovarian cancer, HGSOC—high-grade serous ovarian cancer, other—other malignant gynecologic tumors, G1—grade 1, G3—grade 3, CA125 increase—CA125 serum concentration > 35 U/mL, NA—data not available.

Figure 2

HOX-related (ALX4, HOPX, CDX2) and ARID1A gene promoter methylation in high-grade ovarian cancer (HGSOC, n = 42), non-HGOSC ovarian cancer (Other, n = 14) and benign (n = 9) cases.

Figure 3

Boxplots depicting gene expression in three gynecologic tumor groups: benign (n = 9, blue), high-grade serous ovarian cancer (HGSOC, n = 42, dark pink), and non-HGSOC gynecologic tumors (Other, n = 14, light pink).

Figure 4

ROC plot and its metrics for the gene expression and promoter methylation biomarkers separation of the HGSOC (n = 42) and benign cases (n = 9). ALX4 and CDX2 gene promoter methylation biomarker curves overlap. AUC—area under the curve, npv—negative predictive value, tpr—true positive rate, fpr—false positive rate.

Figure 5

Receiver operator characteristic (ROC) plot and its metrics for the gene expression, promoter methylation biomarker, and serum CA125 biomarker separation of the HGSOC (n = 42) and benign cases (n = 9). Gene expression biomarker combination (all 10 genes), Mixed biomarker combination (all 14 biomarkers), and Notch gene expression biomarker combination (NOTCH1-4, HES1, DLL1, JAG2) curves overlap. HOX gene promoter methylation biomarker combination includes three genes (HOPX, ALX4, CDX2), serum CA125 threshold concentration considered 35 U/mL.