There are several blood and urine markers available alone or in combination to further risk stratify patients with a mildly elevated PSA, typically between 2.5 and 10 ng/mL. The intent is to improve upon the poor specificity of PSA and avoid the risks associated with unnecessary biopsies, including the risk of overdiagnosis of GG1 prostate cancer, in patients with a low probability of harboring GG2+ disease. Naturally, with avoidance of biopsies comes the risk of delaying the diagnosis of clinically significant prostate cancer (“false negatives”). Tests that report the likelihood of any prostate cancer, rather than reporting GG2+ prostate cancer are less valuable in terms of ameliorating overdiagnosis of low-grade prostate cancer. Importantly, such biomarkers should not be used in situations in which, based on available clinical and laboratory data, the risk of GG2+ prostate cancer is so low or so high the result of adjunctive biomarkers would not influence the decision of whether to proceed with further testing (e.g., MRI and/or biopsy). For example, in patients with a prostate nodule, a PSA > 10 ng/mL, a strong family history of high-grade prostate cancer, or other significant risk factors, it is unlikely an adjunctive biomarker would change the decision to proceed with biopsy. In contrast, in a patient with a mildly elevated PSA, a very low PSA density (based on available imaging-based volume measurement), no other risk factors, and a desire to avoid biopsy, ongoing screening rather than further testing is preferable. Perhaps the most widely available adjunctive test is percent free PSA. Lower percent free PSA is associated with greater likelihood of identifying prostate cancer on biopsy.171-176 Additionally, it improves upon the prediction of GG2+, primarily in validation studies of multiplex tests that include percent free PSA. For example, in the study validating the use of the 4KscoreTM, exclusion of percent free PSA from the model reduced the AUC from 0.821 (95% CI: 0.790 to 0.852) to 0.699 (95% CI: 0.664 to 0.735).177 Similarly, percent free PSA improves prediction of GG2+ prostate cancer compared to total PSA (AUC 0.661 versus 0.551) in a study demonstrating the value of prostate health index (PHI)TM.178 Numerous studies have shown that higher PSA density (serum PSA [ng/mL] divided by imaging measures of prostate volume [cc]) is associated with the risk of identifying clinically significant prostate cancer on biopsy.179-181 Various thresholds have been proposed, with lower thresholds (e.g., PSA density ≥ 0.07) having higher sensitivity, but lower specificity, than higher thresholds (e.g., PSA density ≥ 0.15). Thus, PSA density is an important component of disease risk assessment when imaging is available for volume measurement. However, the Panel recognizes the continuous nature of risk associated with the spectrum of PSA density values and cautions against use of threshold values in isolation for management decision-making. It is debatable which of the newer biomarkers (alone or in combination) is best, and comparative studies are sparse. A list of available tests for an initial biopsy cohort is summarized in Table 6. With this update, Table 6 now includes additional references describing studies of previously included biomarkers for biopsy-naïve patients.182 In general, the tests are calibrated such that avoiding biopsy in the setting of a sub-threshold test reduces biopsies by about one third, resulting in delayed detection or non-detection of 5% to 10% of clinically significant prostate cancers.183 A meta-analysis of studies that met criteria for inclusion in the evidence base for this Guideline showed that use of secondary biomarkers would reduce the number of biopsies by 35% (95% CI: 26% to 44%; p<0.0001),179, 184-195 and 9% (95% CI: 6% to 11%; p<0.0001)179, 181, 184-189, 191-193, 196 of clinically significant prostate cancers would not be detected. A modeling study evaluating several of the tests in the reflex setting (to refer patients with PSA between 4 to 10 ng/mL to biopsy at pre-specified cutoffs) projected that if patients were screened annually the tests would minimally impact life years or quality-adjusted life years compared with all patients with PSA > 4 ng/mL undergoing biopsy.197 Given their generally significant impact on biopsy reduction and their projected minimal impact on life expectancy, such tests may be of value among patients with modestly elevated PSA tests, especially in patients with a prior negative biopsy in whom PSA alone is not recommended as the sole trigger for re-biopsy. Considerations in selecting a test include test performance characteristics (such as NPV), availability, and familiarity. As in the PSA screening setting, the use of SDM is highly recommended given the uncertainty involved. This statement applies to both initial and repeat biopsy situations. <img src="images/Guidelines/Guideline%20Images/2026%20EDPC/EDPC%202026-%20Table%206.jpg" alt="TABLE 6: Available Biomarker Assays" title="TABLE 6: Available Biomarker Assays" width="700" height="1399" class="blockImg" caption="false" /> (Abbreviations: DRE, digital rectal exam; fPSA, free PSA; iPSA, intact PSA; mRNA, messenger ribonucleic acid; PSA, prostate-specific antigen; SNP, single nucleotide polymorphism.)    In addition to the biomarkers in Table 6, PRSs that are based on SNPs measured in saliva or blood are genetic tests used to predict a person’s risk of developing prostate cancer. Various combinations of SNPs have been mathematically integrated to produce several commercially available options. There is little evidence to mandate which SNP panel or PRS to use and where to threshold risk to create strata with different screening frequencies. The endpoint of the majority of studies on PRS has mainly focused on any detection of prostate cancer, not clinically significant or metastatic/lethal prostate cancer. Few PRS scores have been shown to discriminate between aggressive and indolent prostate cancer risk.234 Calculating a PRS based on genotypes of 66 known prostate cancer loci for 4,967 patients in the ERSPC, the rate of overdiagnosis (e.g., detection of GG1) of screen-detected cancers was 42%, with 58% of these found in the lower PRS risk group and 37% in those with higher PRS risk.235 Adding SNPs to STHLM-3 added 1% to the AUC (from 0.75 to 0.76) for GG2+ (Gleason Score ≥ 7) after the clinical information and protein biomarkers.219 The ongoing large-scale BARCODE-1 trial invited individuals to obtain prostate cancer screening using PRS and recently published the primary outcome. The participation rate was low (22%), which limits generalizability.3, 236 Of over 40,000 patients invited, 6,393 participated and had a PRS calculated, of whom 745 (12%) were in the top 10% of PRS (i.e., elevated risk). Among those, about half (n=468) underwent an MRI and prostate biopsy, irrespective of PSA, and 40% (187/468) had prostate cancer, half of which were considered clinically significant. However, whether PRS testing without PSA improves risk stratification of early detection strategies and favorably balances the risks of unnecessary biopsy, overdiagnosis and detection of clinically significant prostate cancer as compared to other currently available strategies, is unclear.3 It is also important to note that PRS has largely been developed from European-ancestry genome-wide association studies, and their performance across other ancestries remains variable, underscoring the need for careful cross-ancestry calibration and multi-ancestry research. At present, PRS should not independently dictate prostate imaging or biopsy decisions outside of structured screening programs, as their clinical utility in isolation remains limited. Ongoing research efforts focus on the role of PRS in distinguishing aggressive versus indolent prostate cancers.