Novel non-invasive urine-based gene expression assay discriminates between low- and high-risk prostate cancer before biopsy

Over the past five years, prostate-specific antigen (PSA)-based screening programs have come under scrutiny given concerns about the accuracy of PSA and its downstream effects on diagnosis and treatment.

Specifically, PSA is not cancer-specific and thus, several benign conditions are associated with elevated serum PSA levels. Even though several adjuncts to conventional PSA have been proposed (e.g., PSA density/velocity, PSA doubling time, percentage of free PSA, and several isoforms), there is no optimal threshold value to distinguish between prostate cancer and benign conditions (1). Furthermore, evidence is scarce showing that those PSA ‘modifiers’ provide additional accuracy relative to serum PSA alone (2).

Moreover, the extensive use of PSA screening has led to a significant increase of diagnostic prostate biopsies and higher detection rates of clinically insignificant tumors, which will likely remain indolent over time (3). Hence, influential public health guideline panels, such as the United States Preventive Services Task Force (USPSTF), have recommended against PSA-based screening in all men (4) to minimize the risks of overtreatment of low-grade tumors, as well as associated health care costs and psychological burden to the patient (5,6). Nevertheless, those recommendations have potentially significant consequences for patients harboring intermediate- to high-risk disease, as diagnoses might be delayed up to a certain point where potentially curative treatment is no longer possible. Given the established survival benefit of surgery or radiation therapy in patients with high-grade prostate cancer [Gleason scores (GS) ≥7 or locally advanced clinical stages] (7,8), the ideal prostate cancer early detection tool would be able to (I) identify patients with high-grade tumors to initiate diagnostic and treatment pathways and (II) avoid unnecessary biopsies and overtreatment in men with low-grade or without malignant cancer.

The perfect biomarker for general use in prostate cancer management needs to meet certain strict criteria. In addition to the required high sensitivity and specificity, the ability to differentiate benign from malignant, as well as indolent from aggressive tumors, the ideal marker has to be an inexpensive, easily accessible, and ideally non-invasive test. The concept of urinary prostate cancer biomarkers is not novel. To date, two urinary markers have been identified and adopted into clinical prediction tools to improve prostate cancer diagnosis and risk assessment.

Prostate cancer antigen 3 (PCA3) was initially described in 1999 as a prostate specific messenger ribonucleic acid (mRNA), which was overexpressed up to 66-fold in more than 95% of prostate cancers (9). In several follow-up studies, PCA3 demonstrated superior predictive abilities compared to serum PSA. In 2006, the PCA3 assay was translated into the commercially available Progensa™ PCA3 test (10). PCA3 was included into a predictive nomogram (11), which was externally validated in 2010 (12). Importantly, de la Taille et al. showed that PCA3 was superior in predicting initial biopsy outcome, compared to total PSA, PSA density, and %free PSA (13). Thus, in 2012, the US Food and Drug Administration (FDA) approved the test as a decision tool for the repeat biopsy setting, given that the likelihood of harboring prostate cancer increases, as the PCA3 score is higher (14,15). Specifically, in a cohort of 127 patients with a suspicious digital rectal exam (DRE), and/or persistently elevated PSA levels, and previous suspicious histology on the initial biopsy, Auprich et al. confirmed that PCA3 was the best predictor of prostate cancer at first repeat biopsy, compared to total PSA alone (16). Nevertheless, its role to distinguish indolent from aggressive tumors remains equivocal. In a retrospective study of 305 patients who underwent radical prostatectomy, the PCA3-score was not an independent predictor of extraprostatic extension, seminal vesicle invasion or high-grade disease (GS ≥7) (17).

Second, another important group of genes that are differentially expressed was identified in 2005: the ETS family (v-ets erythroblastosis virus E26 oncogene; ERG and ETS variant gene 1; ETV1). Tomlins et al. showed that these genes were overexpressed in approximately 57% of prostate cancer cases (18), and that this overexpression was most likely driven by an androgen-regulated fusion with the Transmembrane Protease, Serine 2 (TMPRSS2) (18). Following this, TMPRSS2-ERG fusion transcripts were shown to be detectable in urine samples (19). A meta-analysis of 61 studies evaluating men with fusion-positive prostate cancers did not find TMPRSS2-ERG to be a strong predictive marker of disease outcome after radical prostatectomy, as the fusion status was not associated with risk of GS ≥7 vs. GS ≤6 or GS =7 vs. GS ≤6 (20). These, along with results from other studies, suggest that TMPRSS2-ERG fusion may be able to predict tumor stage, however its association with GS or cancer-specific mortality remains unclear.

These two urine-based prostate cancer early detection biomarkers—PCA3 and TMPRSS2-ERG—along with serum PSA were subsequently combined into another urine test, the Mi-Prostate Score (MiPS) (21,22). MiPS + PSA outperformed both PCA3 + PSA and PSA alone for prediction of high-grade prostate cancer defined as GS ≥7 (22). Of note, both Progensa™ and MiPS require pre-collection DRE, which might be perceived as an invasive intervention and thus, do not meet the stringent definition of a ‘perfect’ detection tool.

Against this backdrop, a study by McKiernan et al. in JAMA Oncology found promising results for a novel urine-based gene expression assay to predict high-grade prostate cancer at initial biopsy (23). The authors used an exosome-derived gene expression signature, which included PCA3 and ERG RNA. While the underlying genes are not novel per se, McKiernan et al. were the first to isolate exosomal RNA without previous prostate examination, derive a molecular prostate cancer signature, and prospectively validate the predictive accuracy of this diagnostic tool. Exosomes are miniscule tissue-derived vesicles, which can be secreted by different cell types, including tumor cells, and carry proteins and RNAs that are representative of their tissue origin (24). In this study, 255 patients with serum PSA levels of 2–10 ng/mL were examined to assess the prognostic accuracy of the ExoDx Prostate IntelliScore urine exosome assay. The derived score was then validated in an intended-use population of 519 patients from 22 facilities in the United States. Patients were considered eligible if they had no history of prostate cancer or biopsy, were 50 years or older, and referred for initial prostate biopsy due to a suspicious DRE finding and/or serum PSA levels from 2–10 ng/mL. When estimating the area under receiver operating characteristic curve (AUC) for discrimination of GS ≥7 vs. GS <7 or benign disease, the novel urine exosome gene expression assay in combination with standard of care (PSA, age, race, and family history of prostate cancer) was superior to standard of care alone (AUC: 0.73 vs. 0.63; P<0.001) (23). Similar results were found when the target population was extended to include patients with a serum PSA level of 10–20 ng/mL.

This new tool relies on previously established genomic markers, but is solely first-catch urine-based. While the MiPS does incorporate previously established genes and serum PSA, the ExoDx Prostate IntelliScore urine exosome assay is different as it does not require a DRE (23).

Notably, the predictive accuracy of this novel exosome-derived test is no better than MiPS, which was previously introduced in 2015 (22) and relies on TMPRSS2-ERG, PCA3, as well as clinical variables included in the Prostate Cancer Prevention Trial risk calculator (PSA, family history, outcome of DRE, and prior biopsy) (25). While the AUC for MiPS was 0.779 for predicting high-grade cancer at biopsy in the validation cohort (22), McKiernan et al. reported an AUC of 0.73 in the external validation of 519 patients to discriminate between GS ≥7 vs. GS <7 or benign disease (23). However, it is convincing that the novel test can be conducted by any health professional without precise knowledge of the performance of an adequate DRE and not only by urologists or physicians. Also, patients could be spared another DRE, which may raise compliance and eventually facilitate the clinical workflow, indeed.

Despite these advantages, patients were considered eligible for the novel exosome-derived test if they presented with an elevated serum PSA ranging from 2–10 ng/mL (2–20 ng/mL in subanalyses) and/or a suspicious DRE. Whether a patient with suspicious DRE should undergo this test is a complex manner. Given that a substantial proportion of prostate cancers detected by DRE at PSA levels ≤4 ng/mL are associated with clinically highly aggressive tumors (26), it is debatable if a patient who presents with a suspicious DRE should undergo this test, as it is unlikely to change clinical decision-making.

Novel tumor targets are anxiously needed, and the combination of biomarker templates seems to be a promising approach to improve the prediction of prostate cancer and prostate cancer aggressiveness at biopsy. However, adequate internal and external validation of these markers are necessary. Specifically, prospective validation in randomly invited population-based cohorts is the gold standard to test the predictive accuracy of those novel markers. As such, the Stockholm 3 study group recently validated a new predefined model in a screening cohort of 113,082 men to identify high-risk prostate cancer (GS ≥7) with better accuracy than PSA alone (27). The model included a combination of several plasma protein biomarkers and performed significantly better than PSA alone (27). Nevertheless, regarding exosomes, further research is eagerly awaited. If researchers are able to gain higher yields of exosomes from urine samples, this may help in finding new bladder, prostate, or renal cancer-specific miRNA and mRNA biomarkers.

Acknowledgments

Funding: None.

Footnote

Provenance: This is a Guest Perspective commissioned by Section Editor Peng Zhang (Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Shanghai, China).

Conflicts of Interest: QD Trinh is supported by an unrestricted educational grant from the Vattikuti Urology Institute, a Clay Hamlin Young Investigator Award from the Prostate Cancer Foundation and a Genentech BioOncology Career Development Award from the Conquer Cancer Foundation of the American Society of Clinical Oncology. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Thompson IM, Pauler DK, Goodman PJ, et al. Prevalence of prostate cancer among men with a prostate-specific antigen level < or =4.0 ng per milliliter. N Engl J Med 2004;350:2239-46. [Crossref] [PubMed]
2. Vickers AJ, Savage C, O'Brien MF, et al. Systematic review of pretreatment prostate-specific antigen velocity and doubling time as predictors for prostate cancer. J Clin Oncol 2009;27:398-403. [Crossref] [PubMed]
3. Berman DM, Epstein JI. When is prostate cancer really cancer? Urol Clin North Am 2014;41:339-46. [Crossref] [PubMed]
4. Moyer VAU.S. Preventive Services Task Force. Screening for prostate cancer: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med 2012;157:120-34. [Crossref] [PubMed]
5. Roobol MJ. Is prostate cancer screening bad or good? Summary of a debate at the innovation in urology meeting, September 17-19, 2010, Milan, Italy. Eur Urol 2011;59:359-62. [Crossref] [PubMed]
6. Heijnsdijk EA, der Kinderen A, Wever EM, et al. Overdetection, overtreatment and costs in prostate-specific antigen screening for prostate cancer. Br J Cancer 2009;101:1833-8. [Crossref] [PubMed]
7. Bill-Axelson A, Holmberg L, Garmo H, et al. Radical prostatectomy or watchful waiting in early prostate cancer. N Engl J Med 2014;370:932-42. [Crossref] [PubMed]
8. Wilt TJ, Brawer MK, Jones KM, et al. Radical prostatectomy versus observation for localized prostate cancer. N Engl J Med 2012;367:203-13. [Crossref] [PubMed]
9. Bussemakers MJ, van Bokhoven A, Verhaegh GW, et al. DD3: a new prostate-specific gene, highly overexpressed in prostate cancer. Cancer Res 1999;59:5975-9. [PubMed]
10. Groskopf J, Aubin SM, Deras IL, et al. APTIMA PCA3 molecular urine test: development of a method to aid in the diagnosis of prostate cancer. Clin Chem 2006;52:1089-95. [Crossref] [PubMed]
11. Chun FK, de la Taille A, van Poppel H, et al. Prostate cancer gene 3 (PCA3): development and internal validation of a novel biopsy nomogram. Eur Urol 2009;56:659-67. [Crossref] [PubMed]
12. Auprich M, Haese A, Walz J, et al. External validation of urinary PCA3-based nomograms to individually predict prostate biopsy outcome. Eur Urol 2010;58:727-32. [Crossref] [PubMed]
13. de la Taille A, Irani J, Graefen M, et al. Clinical evaluation of the PCA3 assay in guiding initial biopsy decisions. J Urol 2011;185:2119-25. [Crossref] [PubMed]
14. Deras IL, Aubin SM, Blase A, et al. PCA3: a molecular urine assay for predicting prostate biopsy outcome. J Urol 2008;179:1587-92. [Crossref] [PubMed]
15. Ochiai A, Okihara K, Kamoi K, et al. Clinical utility of the prostate cancer gene 3 (PCA3) urine assay in Japanese men undergoing prostate biopsy. BJU Int 2013;111:928-33. [Crossref] [PubMed]
16. Auprich M, Augustin H, Budäus L, et al. A comparative performance analysis of total prostate-specific antigen, percentage free prostate-specific antigen, prostate-specific antigen velocity and urinary prostate cancer gene 3 in the first, second and third repeat prostate biopsy. BJU Int 2012;109:1627-35. [Crossref] [PubMed]
17. Auprich M, Chun FK, Ward JF, et al. Critical assessment of preoperative urinary prostate cancer antigen 3 on the accuracy of prostate cancer staging. Eur Urol 2011;59:96-105. [Crossref] [PubMed]
18. Tomlins SA, Rhodes DR, Perner S, et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 2005;310:644-8. [Crossref] [PubMed]
19. Laxman B, Tomlins SA, Mehra R, et al. Noninvasive detection of TMPRSS2:ERG fusion transcripts in the urine of men with prostate cancer. Neoplasia 2006;8:885-8. [Crossref] [PubMed]
20. Pettersson A, Graff RE, Bauer SR, et al. The TMPRSS2:ERG rearrangement, ERG expression, and prostate cancer outcomes: a cohort study and meta-analysis. Cancer Epidemiol Biomarkers Prev 2012;21:1497-509. [Crossref] [PubMed]
21. Chevli KK, Duff M, Walter P, et al. Urinary PCA3 as a predictor of prostate cancer in a cohort of 3,073 men undergoing initial prostate biopsy. J Urol 2014;191:1743-8. [Crossref] [PubMed]
22. Tomlins SA, Day JR, Lonigro RJ, et al. Urine TMPRSS2:ERG Plus PCA3 for Individualized Prostate Cancer Risk Assessment. Eur Urol 2016;70:45-53. [Crossref] [PubMed]
23. McKiernan J, Donovan MJ, O'Neill V, et al. A Novel Urine Exosome Gene Expression Assay to Predict High-grade Prostate Cancer at Initial Biopsy. JAMA Oncol 2016;2:882-9. [Crossref] [PubMed]
24. Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002;2:569-79. [PubMed]
25. Thompson IM, Ankerst DP, Chi C, et al. Assessing prostate cancer risk: results from the Prostate Cancer Prevention Trial. J Natl Cancer Inst 2006;98:529-34. [Crossref] [PubMed]
26. Okotie OT, Roehl KA, Han M, et al. Characteristics of prostate cancer detected by digital rectal examination only. Urology 2007;70:1117-20. [Crossref] [PubMed]
27. Grönberg H, Adolfsson J, Aly M, et al. Prostate cancer screening in men aged 50-69 years (STHLM3): a prospective population-based diagnostic study. Lancet Oncol 2015;16:1667-76. [Crossref] [PubMed]