A tissue biopsy-based epigenetic multiplex PCR assay for prostate cancer detection

Abstract

Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1 , APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1 , APC and RASSF1 genes, using methylation specific PCR (MSP). The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE) samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.

Clinical Validation of an Epigenetic Assay to Predict Negative Histopathological Results in Repeat Prostate Biopsies

“…However, in the event of limited excess tissue a minimum of 20 μm from formalin fixed, paraffin embedded tissue cores were epigenetically profiled based on the 3 genes GSTP1 , APC and RASSF1 as previously described. 21 After completion of all analyses with the epigenetic assay clinical and patient characteristics were verified and updated with data from subsequent biopsies as necessary.…”

Tissue-based biomarkers in prostate cancer

“…[69, 70] This concept is based on the idea of a field-change in histologically normal tissue adjacent to a focus of adenocarcinoma. [71-73] The assay is used to decide on the need for repeat biopsy in a patient with a previously negative biopsy.…”

Current and Future Applications of Genetic Prostate Cancer Screening In the Urologic Clinic

“…Hypermethylation (which is an increase in the epigenetic methylation of cytosine and adenosine residues in DNA) of GSTP1 and APC is expressed more commonly in prostate tumor tissue than benign tissue. The analysis of the GSTPI and APC genes on the remaining prostate tissue from a previously negative biopsy helps reduce unnecessary repeat biopsies, and also identifies high-risk patients (Trock et al, 2012;Truong et al, 2013;van Neste et al, 2012). The prognostic evaluation by testing DNA methylation remains in the development phase, but still can serve as a tool to distinguish between aggressive and nonaggressive tumors.…”