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

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 (RT-qPCR). 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 assessed the sensitivity of droplet digital PCR (ddPCR) by quantifying T:E gene fusion transcripts using gene fragment- or cell-based standard curves. Next, we evaluated dihydrotestosterone (DHT), genotoxic insults (irradiation, etoposide), and inflammatory agents tumor necrosis factor-alpha (TNF-α) and hydrogen peroxide (H₂O₂) as initiators of T:E fusions in a fusion-negative prostate cell line (LNCaP). Finally, we performed RT-qPCR to measure the expression of androgen receptor (AR), AR-regulated genes (TMPRSS2, NKX3.1) and the downstream activation target of TNF-α, NF-κB.

We identified 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. Consistent with prior studies, the ddPCR assay 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-α, or H₂O₂. We determined that TNF-α and H₂O₂ exposure led to global downregulation of AR 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.