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

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.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.In the USA, over 70% of the annually ~1,000,000 performed biopsies for prostate cancer suspicion result in a cancer-free diagnosis [1-4], with screening programs typically based on serum PSA levels [5,6]. Due to the limited performance of this marker in terms of suboptimal sensitivity and specificity, there have been recommendations against the utilization of serum PSA levels for prostate cancer screening [7-9]. Several alternatives are actively being explored for prostate cancer screening and detection. This report emphasizes one particular biomarker strategy,i.e. DNA methylation of key gene promoter regions. DNA hypermethylation is an epigenetic change that locks genes in a silent, non-expressed state [10]. Such changes occur both