Analytical Validation of MyProstateScore 2.0—Active Surveillance: A Urinary-Based Clinical RT-PCR Prostate Cancer Assay

Background/Objectives: Active surveillance (AS) is recommended for men with low-risk prostate cancer to minimize overtreatment while monitoring for disease progression. However, current surveillance strategies rely heavily on repeat biopsies, which are invasive and associated with morbidity. MyProstateScore 2.0—Active Surveillance (MPS2-AS) is a urine-based biomarker test developed to predict progression to Grade Group ≥ 2 (GG ≥ 2) and Grade Group ≥ 3 (GG ≥ 3) prostate cancers in men on AS. The objective of this study was to analytically validate the reproducibility and robustness of MPS2-AS analyte detection and risk score calculation across key laboratory variables. Methods: Analytical precision was evaluated using pooled urine specimens processed using the MPS2-AS laboratory workflow. Eight pooled urine samples were tested in a within-laboratory design across five days, with two runs per day, and two replicates per run. Additional reproducibility studies assessed variability across three QuantStudio™ 12K Flex Real-Time PCR Systems and three OpenArray™ chip lots. Ten RNA biomarkers were quantified by RT-PCR and used to calculate the MPS2-AS GG1-2 and GG1-3 risk scores. Variance components were estimated using hierarchical ANOVA. Results: The MPS2-AS analyte measurements demonstrated high precision across within-laboratory testing, with standard deviations ranging from 0.00 to 0.60 and coefficients of variation (%CV) from 0.00 to 4.01%. The reproducibility across qPCR instruments and OpenArray chip lots showed similar robustness, with analyte %CVs of ≤4.57% and ≤4.10%, respectively. These stable analyte measurements translated to reproducible model outputs, with %CV ≤ 10.69% for the GG1-2 risk score and ≤7.20% for the GG1-3 risk score across all tested conditions. No systematic bias was observed between runs, days, instruments, or reagent lots. Conclusions: MPS2-AS demonstrates strong analytical precision and reproducibility for quantifying urinary biomarkers and generating GG1-2 and GG1-3 risk scores. These results support the reliability of MPS2-AS for clinical laboratory implementation and its use as a non-invasive tool to inform biopsy decisions in men with Grade Group 1 prostate cancer undergoing active surveillance.