Analysis of methylation markers for differentiation of samples from leiomyoma and sarcoma patients

Purpose Pre-operative differentiation of uterine tumors into leiomyoma (LM) and sarcoma (S) is difficult, but may be realized by DNA methylation markers detected in cell-free DNA (cfDNA) of blood or peritoneal washings.

Methods Methylation markers were derived from literature (n=4) or EOC-related projects of our lab (n=3). Quantitative methylation-specific PCR (MSP) assays were established and validated using unmethylated and CpG-Methyltransferase (M.SssI)-methylated DNA. Methylation levels were quantified relative to a bisulfit-specific control region and the 100% methylated control DNA. Analyzed samples consist of bisulfite-treated gDNA from fresh-frozen tissue (LM n= 19; S n=19; normal endometrial tissue n=10) and cfDNA from serum (LM n=28; S=28) or peritoneal washings (LM n=30; S n=30) partially derived from the same patient.

Results Tissue-based analyses resulted in quantitative differences and identified five markers with significantly higher methylation level in leiomyoma or sarcoma tissue compared to normal endometrial tissue (p< 0.05, Mann-Whitney test). In addition, two of these markers were differentially methylated between leiomyoma and sarcoma samples (p< 0.05, Mann-Whitney test). These markers could not differentiate between patients with leiomyoma or sarcoma if analyzed in cfDNA of blood or peritoneal washings (p>0.05, Mann-Whitney test). However, two other markers showed significantly different methylation levels in cfDNA isolated from blood or peritoneal washings.

Conclusion Uterine leiomyoma and sarcoma tissues exhibit epigenetic differences potentially useful as biomarkers. However, tissue-specific differences may not cause significantly changed methylation level in cfDNA. Thus, markers for cfDNA analyses should be identified in the specific target samples.

Publication History

Article published online:
07 October 2020