Background: The challenge for prostate cancer (PCa) diagnosis is to improve the ability to distinguish indolent from aggressive PCa. Increased urinary levels of Distal-less Homeobox 1 (DLX1) and Homeobox C6 (HOXC6) mRNA have been associated with high-grade PCa (Gleason Score ≥7). Reverse transcriptase PCR (RT-PCR)-assays for the quantification of DLX1 and HOXC6 mRNA levels were developed, and clinical validation showed that these urinary markers can significantly improve accuracy for detection of high-grade PCa at biopsy. The aim of this study was to validate the analytical performance of these mRNA-based assays, including specimen stability and analytical sensitivity, specificity, precision, repeatability and reproducibility.
Results: A stabilization buffer, developed for urine preservation under standard pre-processing conditions, makes sample shipment from clinics to laboratories easily feasible. The mRNA in urine samples, preserved in this stabilization buffer, is stable at room temperature up to 5 days from collection, resulting in 100% evaluable rate. The long term stability of the mRNAs in the buffer was shown by similar clinical performances using RNA values obtained immediately after urine collection (area under curve (AUC) 0.72(95%CI: 0.61-0.83)) and after 1-year storage (AUC 0.71 (95%CI: 0.60-0.81)). Test performance was not compromised by most common inhibitors and bacterial strains found in urine. However, an inhibitory effect of hemoglobin was observed.
All precision, reproducibility, instrument and inter-laboratory variation data obtained for the mRNA-based assays met the pre-specified acceptance criteria of a standard deviation less than or equal to 0.5 crossing point. The analysis of 99 whole urine samples at two laboratories indicated a very strong positive correlation (r=0.997, P<0.001). The test outcome in terms of absolute difference in likelihood for high-grade PCa upon biopsy was less than 2% between the two sites.
Conclusions: This study illustrates the robustness of the mRNA assays, enabling testing in clinical routine laboratories and molecular pathology laboratories where the here described automated RNA extraction and PCR platforms are available.