Epigenetic Analysis of Circulating Tumor Cells
Technical Report,01 Aug 2017,31 Jul 2019
University of Wisconsin Madison United States
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Despite advances in the field, metastatic prostate cancer prognosis remains poor. Although genomic biomarkers are being assessed for clinical relevance, epigenetic modifications have been found to be much more common, some found in more than 90 of prostate cancer tumors. Two of these modifications are GSTP1 and PRAC DNA hypermethylation. Circulating tumor cells CTCs provide a minimally invasive way to monitor patient disease, however they are a rare cell population and current methods of epigenetic analysis are not sensitive enough to assay DNA methylation from such a population. We have developed an assay that can sensitively and specifically enrich for methylated DNA from rare cell populations and have optimized this assay using cell line models to enrich for methylated GSTP1 from as little as one cell. We have tested this assay in patient biopsies, either flow sorted by tumor compartment or unsorted populations, and found GSTP1 methylation in 100 of patient tissue tested, but not in white blood cells WBCs. We also chose 7 patients with castration resistance prostate cancer CRPC as a pilot study for GSTP1 methylation analysis in CTCs. 5 out of 7 71 CRPC CTC samples had GSTP1 methylation detectable above background. In order to measure GSTP1 and PRAC methylation from the same cells, we have made changes to this assay to allow for pre-amplification of target genes prior to qRT-PCR. We have tested this new version of the assay and ensured that the performance is similar to the original version. We have also tested a PRAC primer on cell line models and WBC DNA and found that WBCs may harbor some PRAC methylation, which will need to be addressed prior to use in patient samples. We have collected DNA from patients that will be used to assess GSTP1 methylation and may also be used to assess PRAC methylation if the primer can be optimized to exclude WBC methylation.
- Medicine and Medical Research