Detection of endometrial cancer via molecular analysis of DNA collected with vaginal tampons

Abstract

Objective: We demonstrate the feasibility of detecting EC by combining minimally-invasive specimen collection techniques with sensitive molecular testing.

Methods: Prior to hysterectomy for EC or benign indications, women collected vaginal pool samples with intravaginal tampons and underwent endometrial brushing. Specimens underwent pyrosequencing for DNA methylation of genes reported to be hypermethylated in gynecologic cancers and recently identified markers discovered by profiling over 200 ECs. Methylation was evaluated individually across CpGs and averaged across genes. Differences between EC and benign endometrium (BE) were assessed using two-sample t-tests and area under the curve (AUC).

Results: Thirty-eight ECs and 28 BEs were included. We evaluated 97 CpGs within 12 genes, including previously reported markers (RASSF1, HSP2A, HOXA9, CDH13, HAAO, and GTF2A1) and those identified in discovery work (ASCL2, HTR1B, NPY, HS3ST2, MME, ADCYAP1, and additional CDH13 CpG sites). Mean methylation was higher in tampon specimens from EC v. BE for 9 of 12 genes (ADCYAP1, ASCL2, CDH13, HS3ST2, HTR1B, MME, HAAO, HOXA9, and RASSF1) (all p<0.05). Among these genes, relative hypermethylation was observed in EC v. BE across CpGs. Endometrial brush and tampon results were similar. Within tampon specimens, AUC was highest for HTR1B (0.82), RASSF1 (0.75), and HOXA9 (0.74). This is the first report of HOXA9 hypermethylation in EC.

Conclusion: DNA hypermethylation in EC tissues can also be identified in vaginal pool DNA collected via intravaginal tampon. Identification of additional EC biomarkers and refined collection methods are needed to develop an early detection tool for EC.

Keywords: Early detection; Endometrial cancer; Methylation; Tampon; Tao brush.

Copyright © 2015 Elsevier Inc. All rights reserved.

Figures

Figure 1

Graphical representation of Spearman correlation coefficients across genes and CpGs. Methylation among CpGs within each gene is highly correlated, with the exception of GTF2A1. GTF2A1 was included as a negative gynecologic cancer control as it is hypermethylated in ovarian cancer but has not been shown to be hypermethylated in endometrial cancer.

Figure 2

Jit plots of absolute methylation percentages as determined in tampon and Tao biospecimens from women with endometrial cancer (case) and benign endometrium (control) for (a) HTR1B, (b) HOXA9, and (c) RASSF1 show consistently higher methylation within biospecimens collected from women with endometrial cancer. Further discovery efforts are ongoing to identify additional methylated genes in EC and other molecular markers that further separate EC from BE.

Figure 3

Receiver operating characteristic (ROC) curve analyses with areas under the curve (AUC) are shown for discrimination between endometrial cancer and benign endometrium using methylation markers. (a) Six novel candidate genes identified via prior profiling and CDH13 (additional CpGs identified on prior profiling) and (b) five genes identified from the gynecologic cancer literature. HAAO and RASSF1 have been published as hypermethylated in endometrial cancer. HSP2A and GTF2A1 have not been shown to be hypermethylated in endometrial cancer, but are methylated in ovarian cancer. HOXA9 hypermethylation in endometrial cancer is a novel finding in this study. Colored lines show ROC curves for individual genes.