Urine Biomarkers Show Promise for Prostate Cancer

Prostate cancer is the most common non-cutaneous malignancy in men. Traditionally, prostate specific antigen (PSA) is used to screen for disease, whereas a prostate biopsy is required for diagnosis. However, the specificity for PSA is only 20–45 percent,¹ leading to a high number of unnecessary prostate biopsies. Many low-risk prostate cancers are suitable for surveillance alone, so there is an urgent need for minimally invasive prostate cancer biomarkers to accurately distinguish low-risk from aggressive disease.

Urine biomarkers are an attractive option as prostate cancer cells are shed into urine, making screening minimally invasive. Urine biomarkers also assess the entirety of the prostate, unlike prostate biopsies, which represent a small fraction of the cancer. Accurate urinary biomarkers further minimize unnecessary prostate biopsies while accurately determining which prostate cancers require aggressive treatment. 

Urine biomarkers

Although urine cytology was used to diagnose malignancies as early as the 1970s,² it was unable to reliably distinguish prostate from urothelial or renal cancers. Using multiplex immunofluorescence cytology of urine precipitate to identify prostate cancer-associated targets, such as AMACR and Nkx3.1, allowed confirmation of prostate cancer, albeit with a poor sensitivity of 36 percent.¹ Researchers have since identified more specific DNA, mRNA, and protein biomarkers in urine to detect prostate cancers. 

DNA

Candidate biomarkers were first identified in prostate cancer tissue and subsequently evaluated in urine. For example, classic DNA hypermethylation changes in prostate cancer tissues were identified in post-prostate massage urine samples using methylation specific quantitative PCR (qPCR).³ More recently, whole genome sequencing and analysis of copy number variations of urine cell-free DNA has shown benefits in predicting treatment outcomes in patients with aggressive prostate cancer.⁴

mRNA

A breakthrough occurred in 1999 when prostate cancer antigen-3 (PCA3), a noncoding RNA sequence, was detected in prostate cancer tissue.⁵ Unlike PSA, which is present in extra-prostatic tissues, PCA3 is specific to the prostate. PCA3 overexpression induces cell proliferation and invasive phenotype. Developed by DiagnoCure, Inc., PCA3 is the only Food and Drug Administration (FDA) approved prostate cancer urinary biomarker. It is was approved in 2012 for men with elevated PSA and a negative prostate biopsy. A meta-analysis including more than 12,000 patients indicates that PCA3 has a 65 percent sensitivity and 73 percent specificity for cancer detection.⁶

In 2005, researchers looking for prominent prostate cancer oncogene mutations used fluorescence in situ hybridization to identify a recurrent fusion abnormality between two erythroblast transformation specific (ETS) transcription factors: TMPRSS2 and ERG.⁷ TMPRSS2-ERG represents about 85 percent of ETS fusion anomalies and occurs in 30–55 percent of localized prostate cancers. Semi-quantitative RT-PCR for TMPRSS2-ERG provides high specificity (93 percent) for diagnosing cancer but low sensitivity (24–37 percent).⁸

"Urine biomarkers are an attractive option as prostate cancer cells are shed into urine, making screening minimally invasive."

Proteins

Post-prostate massage urine contains proteins that can be analyzed using array and proteomic technology.¹ One promising candidate is engrailed-2 (EN2), a transcription factor important for early mammalian development,⁹ that is quantified via enzyme linked immunosorbent assays (ELISA). In a longitudinal study of 184 men, the test correlated with cancer volume, and the diagnostic sensitivity and specificity were 66 percent and 88 percent, respectively.¹⁰ Currently, a lateral flow immunoassay-based EN2 test is being developed for point-of-care testing.⁹

Multiplexed urinary biomarkers

Combining urinary biomarkers can improve clinical utility and provide a more robust evaluation of the heterogeneity innate to prostate cancers. Testing PCA3 and TMPRSS2-ERG fusion improves sensitivity up to 73 percent (from 65 percent) while maintaining the high specificity of TMPRSS2-ERG fusion alone⁸ This dual approach has been shown to decrease unnecessary prostate biopsies by up to 42 percent.¹ Importantly, PCA3 and TMPRSS2-ERG fusion abnormalities are not as accurate in African American men—a group with increased prostate cancer risk.

Another available test, SelectMDx, uses RT-qPCR to quantify DLX1 and HOXC6 mRNA, along with age, a clinical prostate exam, and PSA levels. SelectMDx consistently outperforms the PCA3 test¹¹ and identifies high-risk prostate cancers. 

Future prostate cancer biomarkers

The use of advanced technologies such as NGS and machine learning have led to a wide array of prostate cancer urine biomarkers. Currently, multiplex panels in development combine classes of biomarkers such as proteins and nucleic acids and utilize a broader spectrum of known mutational anomalies and available biomarkers. Challenges remain to validate and normalize the numerous available biomarkers while improving sensitivity and specificity. 

References

1. Fujita, Kazutoshi, and Norio Nonomura. “Urinary biomarkers of prostate cancer.” International Journal of Urology 25.9 (2018):770–79.
2. Garret, M, and Jassie, M. “Cytologic examination of post prostatic massage specimens as an aid in diagnosis of carcinoma of the prostate.” Acta Cytologica 20 (1976): 126–31.
3. Zhao, Fang, et al. “A urine-based DNA methylation assay, ProCUrE, to identify clinically significant prostate cancer.” Clinical Epigenetics 10.1 (2018). 
4. Xia, Yun, et al. “Copy number variations in urine cell free DNA as biomarkers in advanced prostate cancer.” Oncotarget 7.24 (2016): 35818–31. 
5. Bussemakers, MJ, et al. “DD3: a new prostate-specific gene, highly overexpressed in prostate cancer.” Cancer Research 59.23 (1999): 5975-79
6. Cui, Yong, et al. “Evaluation of prostate cancer antigen 3 for detecting prostate cancer: A systematic review and meta-analysis.” Scientific Reports 6.1(2016). 
7. Tomlins, S. A. “Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer.” Science 310. 5748 (2005): 644–48.
8. Sanguedolce, Francesca, et al. “Urine TMPRSS2: ERG fusion transcript as a biomarker for prostate cancer: Literature review.” Clinical Genitourinary Cancer 14.2 (2016): 117–21. 
9. Connell, Shea, et al. “Integration of urinary EN2 protein & cell-free rna data in the development of a multivariable risk model for the detection of prostate cancer prior to biopsy.” Cancers 13.9 (2021): 2102. 
10. Morgan, Richard, et al. “Engrailed-2 (EN2): A tumor specific urinary biomarker for the early diagnosis of prostate cancer.” Clinical Cancer Research 17.5 (2011): 1090–98.
11. Hendriks, Rianne J., et al. “A urinary biomarker-based risk score correlates with multiparametric MRI for prostate cancer detection.” The Prostate 77.14 (2017): 1401–07.