Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings.

Androgens are a major driver of prostate cancer (PCa) and continue to be a critical treatment target for advanced disease, which includes castration therapy and antiandrogens. However, resistance to these therapies leading to metastatic castration-resistant prostate cancer (mCRPC), and the emergence of treatment-induced neuroendocrine disease (tNEPC) remains an ongoing challenge. Instability of the DNA methylome is well established as a major hallmark of PCa development and progression. Therefore, investigating the dynamics of the methylation changes going from the castration sensitive to the tNEPC state would provide insights into novel mechanisms of resistance. Using an established xenograft model of CRPC, genome-wide methylation analysis was performed on cell lines representing various stages of PCa progression. We confirmed extensive methylation changes with the development of CRPC and tNEPC using this model. This included key genes and pathways associated with cellular differentiation and neurodevelopment. Combined analysis of methylation and gene expression changes further highlighted genes that could potentially serve as therapeutic targets. Furthermore, tNEPC-related methylation signals from this model were detectable in circulating cell free DNA (cfDNA) from mCRPC patients undergoing androgen-targeting therapies and were associated with a faster time to clinical progression. These potential biomarkers could help with identifying patients with aggressive disease.