Background The benefits of prostate cancer screening with serum prostate-specific antigen (PSA) have been largely offset by the high rate of negative prostate biopsies and overdiagnosis of indolent cancers. These outcomes result from the limited diagnostic accuracy of PSA for clinically significant prostate cancer and are considered a major obstacle to realizing the benefits of population-wide screening for prostate cancer.

Methods We analyzed RNAseq data from a prostate cancer compendium to identify novel transcripts associated with cancer and high-grade cancer. Predefined nomination criteria were applied to 58,724 gene targets, yielding 54 differentially expressed transcripts. We designed a custom multiplex qPCR panel for non-invasive detection of candidate transcripts in urine. The panel was applied to a development cohort of men with elevated PSA (3 to 10 ng per milliliter) that underwent prospective, standardized urine collection and prostate biopsy at the University of Michigan. Elastic net modeling was used to derive the optimal model for clinically significant (grade group 2 or higher) prostate cancer, the 18-transcript MyProstateScore 2.0 (MPS2) test. The calibrated, locked MPS2 model was assessed in a blinded, external National Cancer Institute (NCI) – Early Detection Research Network (EDRN) validation cohort and compared to serum PSA, the Prostate Cancer Prevention Trial risk calculator (PCPTrc), and the MyProstateScore (MPS) test. The original MPS assay measures urinary expression of two cancer-associated markers (PCA3, TMPRSS2:ERG) and is endorsed by National Comprehensive Cancer Network guidelines for consideration prior to biopsy in the study population.

Results We performed multiplex urinary testing of 1,623 clinical specimens in total, representing the largest such cohort to our knowledge. The prospective NCI-EDRN validation population included 743 men undergoing per-protocol urine collection and prostate biopsy. The median age was 62 years, median PSA was 5.6 ng per milliliter, and 151 men (20%) had clinically significant prostate cancer on biopsy. The area under the receiver-operating characteristic curve (AUC) for clinically significant prostate cancer was 0.597 (95% Confidence Interval [CI], 0.547 to 0.646) for PSA, 0.659 (95% CI, 0.611 to 0.707) for the PCPTrc, and 0.737 (95% CI, 0.694 to 0.780) for MPS, as compared to 0.818 (95% CI, 0.781 to 0.855) for the optimal MPS2 model (MPS2+). Under a clinically applicable testing approach providing 95% sensitivity for clinically significant cancer, the specificity (equivalent to the percentage of unnecessary biopsies avoided after pre-biopsy testing) was 11% for PSA, 20% for the PCPTrc, and 23% for MPS, as compared to 41% for MPS2+. In all sub-populations, MPS2 testing provided negative predictive value (NPV) of 95% to 99% for clinically significant cancer.

Conclusions In a large, external validation population referred for prostate biopsy, the novel MPS2 assay provided exceptional sensitivity and NPV to rule out clinically significant prostate cancer. These data support the use of MPS2 as a highly accurate secondary test to reduce the harms associated with PSA screening and preserve its long-term benefits.

LynxDx has obtained an exclusive license from the University of Michigan to commercialize MPS2. J.J.T. and A.M.C. are co-founders and scientific advisors to LynxDx. Y. Zhang., L.X., J.S., and Y.S.N. are scientific advisors to LynxDx. LynxDx was not involved in the planning or funding of this study and did not approve its content.

This work was funded by the Michigan-Vanderbilt EDRN Biomarker Characterization Center (U2C CA271854), the EDRN DMCC (U24 CA086368), Michigan Prostate SPORE (P50 CA186786), NCI Outstanding Investigator Award (A.M.C., R35 CA231996), Prostate Cancer Foundation Young Investigator Award (J.T., L.X., 20YOUN11), Prostate Cancer Foundation, Howard Hughes Medical Institute, and the American Cancer Society.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Institutional Review Board of The University of Michigan gave ethical approval for this work.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present work are contained in the manuscript.