Discovery of PCA3
In 1999, DD3 (differential display code 3) [now referred to as the PCA3 gene], a non-coding mRNA located on chromosome 9q21-22 with unknown function, was identified as being highly overexpressed (median 66-fold) in more than 95% of malignant prostate tissue compared with benign or normal prostatic tissue.24 Bussemakers and colleagues were first to identify PCA3 using Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR).
They noted a 10- to 100-fold overexpression of PCA3 in 53 of 56 radical prostatectomy specimens compared with non-neoplastic prostate tissues. In 2004, Tinzl et al. described the second-generation PCA3 test known as uPM3 diagnostic test.25 They reported on the outperformance of PCA3 in comparison to PSA, with sensitivity and specificity of 82% and 76% vs. 87% and 16%, respectively. Today, the most current third-generation commercial platform is referred to as the Progensa PCA3 assay (approved in 2012),26 which is marketed by Gen-Probe.
Similar to other urinary markers, PCA3 is collected using a commercial kit after a firm and thorough DRE. Once the first urine catch sample (20-30 mL) is collected, the specimen is placed on ice (to maintain a target temperature between 2° to 8°C) and then shipped to the designated testing sites. The samples can be stored at this temperature for up to 14 days.
The PCA3 assay is run by selecting the RNAs of interest (PCA3 and PSA) from the rest of the RNAs within the urine sample. This extraction is performed using magnetic beads coated with complementary oligonucleotide sequences. Once desired RNAs are isolated, they are amplified by PCR. Chemoluminescent-labeled probes are then used to perform the hybridization protection assay. PCA3 and PSA RNAs are quantified in separate tubes and then PCA3 score is calculated as the PCA3/PSA ratio. The Progensa PCA3 assay final output is the PCA3 score, calculated using the formula: PCA3 Score = (PCA3 mRNA)/(PSA mRNA) × 100026 (Figure 1). This provides a continuous value (ranging from 0-100) that correlates with probability of PCa detection on subsequent transrectal PBx.