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

Area of Science:

• Oncology
• Molecular Biology
• Genetics

Background:

• Gene fusions, such as TMPRSS2:ERG (T:E), are common in prostate cancer, arising from chromosomal rearrangements.
• T:E gene fusions are early events in prostate cancer oncogenesis, occurring in about half of all cases.
• Current methods like FISH and RT-qPCR have limitations in detecting low-abundance T:E gene fusion transcripts.

Purpose of the Study:

• To develop a sensitive and user-friendly method for detecting T:E gene fusion transcripts.
• To use this method to identify potential initiators of T:E gene fusions in prostate cancer cells.

Main Methods:

• Assessed the sensitivity of droplet digital PCR (ddPCR) for quantifying T:E gene fusion transcripts.
• Utilized gene fragment- and cell-based standard curves to evaluate ddPCR sensitivity.
• Exposed fusion-negative LNCaP prostate cancer cells to dihydrotestosterone (DHT), genotoxic agents (irradiation, etoposide), and inflammatory agents (TNF-α, H2O2) to test for T:E fusion initiation.
• Measured androgen receptor (AR) signaling and downstream targets via RT-qPCR.

Main Results:

• ddPCR demonstrated high sensitivity, detecting T:E gene fusion transcripts from single fusion-positive cells.
• DHT combined with etoposide synergistically initiated T:E gene fusion transcript expression in LNCaP cells.
• Irradiation, TNF-α, and H2O2 did not induce detectable T:E gene fusion transcripts; these agents downregulated AR signaling.

Conclusions:

• The developed ddPCR assay is a reliable and sensitive tool for detecting T:E gene fusion transcripts.
• This method enhances the feasibility of investigating novel initiators of gene fusions.
• The approach is applicable to further studies on gene fusion mechanisms in various cancers.