Use of Droplet Digital PCR for Consistent Detection of TMPRSS2:ERG Gene Fusion Transcripts Initiated In Vitro

Abstract

Gene fusions are hybrid genes that arise from chromosomal rearrangements linking two independent genes. The most common gene fusion in prostate cancer involves the 5 androgen-regulated TMPRSS2 promoter fused with the 3 ETS transcription factor ERG. TMPRSS2:ERG (T:E) gene fusions occur in about half of all prostate cancers and are considered an early event in oncogenesis. Investigations into the mechanism behind T:E gene fusion initiation using in vitro systems are hindered by the technical limitations posed by fluorescence in situ hybridization and suboptimal sensitivity of reverse transcription quantitative PCR. The objective of this study was to develop a reliable, user-friendly method of detecting low abundance T:E gene fusion transcripts as a read-out for putative T:E gene fusions generated in cells. We identified droplet digital PCR (ddPCR) as a sensitive method for detecting rare T:E gene fusion transcripts and observed consistent detection in reactions containing a single T:E gene fragment, or 1 fusion positive cell per 10,000 fusion negative cells. Next, we evaluated dihydrotestosterone (DHT), genotoxic insults (irradiation, etoposide), and inflammatory agents tumor necrosis factor-alpha (TNF) and hydrogen peroxide (H2O2) as initiators of T:E fusions in a fusion negative prostate cell line (LNCaP). Consistent with prior studies, we identified DHT combined with etoposide as potent, synergistic initiators of T:E gene fusion transcript expression in LNCaP cells. We did not detect T:E gene fusion transcripts after LNCaP exposure to irradiation, TNF, or H2O2. We determined that TNF and H2O2 exposure led to global downregulation of androgen receptor signaling, which may have limited the formation or expression of treatment-initiated genomic T:E fusions. Therefore, one limitation of the ddPCR assay is the requirement for T:E fusion mRNA expression. Our proposed method significantly improves the feasibility of testing novel initiators of the T:E gene fusion and can be applied to additional studies investigating mechanisms of gene fusion initiation in prostate and other cancers.