High-Speed Biosensing Strategy for Non-Invasive Profiling of Multiple Cancer Fusion Genes in Urine: Beyond the Abstract

Genomic rearrangements such as the TMPRSS2:ERG fusion gene variants in prostate cancer (PCa) are powerful biomarkers useful for cancer diagnosis, subtyping and predicting prognosis. Fusion genes are a result of chromosomal rearrangements such as translocations, deletions, or inversions. This can result in abnormal gene expression and in turn, leads to tumour initiation and development. With regards to cancer diagnosis and prognosis, fusion genes are emerging as biomarkers of immense value due to their specific presence in tumor tissue only.

The most prevalent group of gene fusions affecting approximately 50% of all PCa cases, is the fusion between TMPRSS2 and ERG genes. Additionally, TMPRSS2:ERG variants are detectable in urine to provide non-invasive PCa diagnostic sampling as an attractive surrogate for needle biopsies. Therefore, rapid and simple assays for identifying multiple urinary TMPRSS2:ERG variants are potentially useful to aid in early cancer detection, immediate patient risk stratification, and prompt personalized treatment. Yet, current methodologies for multiplexed gene fusion detection are limited by laborious, time-consuming protocols and high equipment/reagent costs. Thus, the development of simpler and faster alternatives is still needed.

In this work, we describe a rapid, isothermal ligation-based assay for profiling of multiple TMPRSS2:ERG messenger RNAs (mRNA). The proposed technique is simple and quick to execute; and exploits the specificity of DNA ligase and the speed of isothermal amplification to simultaneously detect multiple fusion gene RNAs within a short sample-to-answer timeframe of 60 min. This approach involves rapid ligation of adjacent probes specific to fusion junctions of different TMPRSS2:ERG variants followed by an isothermal amplification of ligation products for real-time fluorescence readout.

As a proof-of-concept, we demonstrated simultaneous parallel detection of 3 mRNA targets, including the two most common TMPRSS2:ERG fusion variants in PCa and a housekeeping mRNA as loading control. From only 30 ng of starting total RNA sample, we successfully showed non-invasive detection of multiple gene fusion biomarkers in patient urine samples. All patient outcomes were subsequently validated by a PCR-based gold standard approach, thereby underscoring the promise of an accurate and reliable clinical screening technique for non-invasive PCa diagnosis.

Though large-scale population-based clinical studies are still ongoing, previous pilot studies have shown TMPRSS2:ERG fusion variants to be present in about half of all PCa cases. Hence, successful TMPRSS2:ERG profiling in clinical samples may provide quick and accurate diagnosis for clinically-significant PCa and enable targeted therapies for TMPRSS2:ERG inhibition. Our proposed method is potentially a simpler, faster and more cost-effective alternative for detecting multiple TMPRSS2:ERG variants and thus identifying TMPRSS2:ERG-positive PCa subsets for appropriate clinical actions. The successful detection of multiple TMPRSS2:ERG variants in urine samples suggested a possibility for non-invasive PCa screening in the future.

Written by: Kevin M. Koo¹, Eugene J.H. Wee^1*, and Matt Trau^{1, 2*}

¹Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, QLD 4072, Australia
²School of Chemistry and Molecular Biosciences, The University of Queensland, QLD 4072, Australia

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