Abstract
BackgroundThe 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.MethodsAnalytical validation of the RT-PCR assays for DLX1, HOXC6 and KLK3 was performed using in vitro transcribed (IVT) specimens of the target genes, covering all aspects of the analytical method including assay sensitivity, specificity, linearity, precision, repeatability and reproducibility using pre-specified acceptance criteria. To confirm that the mRNA assays are sufficient robust for use in clinical routine laboratories, the performance characteristics were verified in an independent laboratory using post-DRE collected urine samples from 101 men scheduled for prostate biopsy.ResultsA 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.ConclusionsThis 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.
Highlights
The challenge for prostate cancer (PCa) diagnosis is to improve the ability to distinguish indolent from aggressive PCa
Clinical sample collection Voided urine samples were collected from a cohort of 99 men scheduled for prostate biopsies due to elevated prostate-specific antigen (PSA) levels, an abnormal digital rectal exam (DRE) or a family history of PCa
Analytical specificity Basic Local Alignment Search Tool (BLAST) searches revealed that the target sequences of Distal-less Homeobox 1 (DLX1), Homeobox C6 (HOXC6) and Kallikrein 3 (KLK3) showed no homology to the DNA of bacteria, viruses, other organisms or other human DNA or RNA sequences
Summary
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. Autopsy on men aged 80 years and older, who died from other causes, revealed that almost 70% had PCa at time of death [4]. This implies that the majority of PCa’s are clinically insignificant and will have an indolent disease course. The most important challenge is to improve the ability to identify aggressive PCa at the time of diagnosis and in an early stage of the disease
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