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Researchers at the Johns Hopkins Kimmel Cancer Center, Johns Hopkins All Children’s Hospital and four other institutions have devised a novel method to test for prostate cancer using biomarkers present in urine, funded in part by the National Institutes of Health. This approach could significantly reduce the need for invasive, often painful biopsies, they say.
By analyzing urine samples from prostate cancer patients before and after prostate-removal surgery, as well as from healthy individuals, researchers identified a panel of three biomarkers — TTC3, H4C5 and EPCAM — that robustly detected the presence of prostate cancer. These biomarkers were detectable in patients prior to surgery but were nearly absent post-surgery, confirming that they originated in prostate tissue.
Researchers tested the three-biomarker panel in a development and validation group. The test had an area under the curve (AUC) of 0.92 (1.0 is a perfect performance). It accurately identified prostate cancer 91% of the time and accurately ruled out people without prostate cancer 84% of the time in the validation study. It also determined that the panel could better than PCA3 distinguish patients with prostate cancer from those with BPH.
The panel maintained diagnostic accuracy in 78.6% (development study) and 85.7% (validation study) of prostate-specific antigen (PSA)-negative prostate cancer cases and distinguished prostate cancer from benign prostate conditions with an AUC of 0.89. These results were published Sept. 2 in eBioMedicine.
TTC3 (tetratricopeptide repeat domain 3) plays a role in asymmetric cell division in cancer cells, H4C5 (H4 clustered histone 5) plays a role in modulating the structure of chromatin (a complex of DNA and proteins found in cells), and EPCAM (epithelial cell adhesion molecule) is a protein highly overexpressed in many human cancers that originate in the epithelial tissue lining the surface of organs and structures throughout the body.
Prostate cancer, one of the leading causes of death in men in the United States, is typically detected by blood tests to measure PSA, a protein produced by cancerous and noncancerous tissue in the prostate. In most men, a PSA level above 4.0 nanograms per milliliter is considered abnormal and may result in a recommendation for prostate biopsy, in which multiple samples of tissue are collected through small needles.
However, the PSA test is not very specific, meaning prostate biopsies are often needed to confirm a diagnosis of cancer, says senior study author Ranjan Perera, Ph.D., director of the Center for RNA Biology at Johns Hopkins All Children’s Hospital in St. Petersburg, Florida, and a professor of oncology and neurosurgery at the Johns Hopkins University School of Medicine. In many cases, these biopsies are negative and can result in unintended complications, Perera says. PSA tests also can lead to unnecessary treatment for very low-grade prostate cancers that are very unlikely to grow and spread over a short period of time.
“This new biomarker panel offers a promising, sensitive and specific, noninvasive diagnostic test for prostate cancer,” says Perera. “It has the potential to accurately detect prostate cancer, reduce unnecessary biopsies, improve diagnostic accuracy in PSA-negative patients, and serve as the foundation for both laboratory-developed and in vitro diagnostic assays.”
The panel was found to be able to detect prostate cancer even when PSA was in the normal range and could distinguish prostate cancer from conditions like prostatitis (inflammation of the prostate) and an enlarged prostate, a condition known as benign prostatic hyperplasia (BPH).
“There is a real need for non-PSA-based biomarkers for prostate cancer, and urine is quite easy to collect in the clinic,” says study co-author Christian Pavlovich, M.D., the Bernard L. Schwartz Distinguished Professor of Urologic Oncology at Johns Hopkins and program director for the Prostate Cancer Active Surveillance Program. “Most urologists feel that an accurate urinary biomarker would be a valuable addition to our current diagnostic armamentarium.”
During the study, investigators studied biomarkers in urine samples from healthy individuals as well as from patients with biopsy-proven prostate cancer undergoing prostate-removal surgeries at The Johns Hopkins Hospital, Johns Hopkins Bayview Medical Center or AdventHealth Global Robotics Institute in Celebration, Florida. They studied 341 urine specimens (107 from healthy individuals, 136 from patients with prostate cancer before surgery and 98 after surgery) during the development of their urine test and an additional 1,055 specimens (162 from healthy individuals, 484 from patients with prostate cancer before surgery and 409 after surgery) to validate the test.
During the performance evaluation phase of testing, the scientists also studied samples from patients with BPH or prostatitis, and healthy controls, from The Johns Hopkins Hospital from 2022–25.
Investigators extracted RNA from prostate cells shed in 50-ml urine samples and analyzed them using RNA sequencing and also real-time quantitative polymerase chain reaction (qPCR) to study gene expression. They also used immunohistochemistry to study biomarkers in samples from cancerous prostate tissue and healthy adjacent tissue, and statistical analyses to compare biomarkers found in the urine and tissue samples.
From an initial 815 prostate-specific genes identified in urine from men with prostate cancers, the investigators prioritized the top 50 genes, then the top nine, and from there selected the three top performers: TTC3, H4C5 and EPCAM for further analysis.
Overall, expression levels of the three biomarkers were significantly higher in urine samples from individuals with prostate cancers than in urine from the healthy controls. The expression of each biomarker diminished to low or undetectable levels in samples taken after surgery. A greater proportion of patients with prostate cancer tested positive for the three biomarkers than for PCA3, another biomarker associated with prostate cancers, in both the development study and the validation study.
“This test has the potential to help physicians improve diagnostic accuracy of prostate cancer, reducing unnecessary interventions while allowing early treatment for those who need it,” says study co-author Vipul Patel, M.D., director of urologic oncology at AdventHealth Cancer Institute in Celebration, Florida. Patel also is medical director of global robotics for AdventHealth’s Global Robotics Institute, and founder of the International Prostate Cancer Foundation. “On behalf of physicians and patient globally, I advocate for further study and progress for these biomarkers.”
Investigators are considering how the biomarker panel could be used alone or combined with a PSA test to make a “super PSA,” Perera says. The next steps for the research are to have an independent trial of the test at another institution and to further develop the test for laboratory use in clinical settings, he says. The investigators have filed a patent, and Johns Hopkins Technology Ventures is helping the team to spin off a company.
The study co-authors were Menglang Yuan, Kandarp Joshi, Yohei Sanada, Bongyong Lee, Rudramani Pokhrel, Alexandra Miller, Ernest K. Amankwah, Ignacio Gonzalez-Gomez, Naren Nimmagadda, Ezra Baraban, Anant Jaiswal and Chetan Bettegowda of Johns Hopkins. Additional co-authors were from Charles University in Prague; the University of Kansas; Orlando Health Medical Group Urology-Winter Park in Orlando, Florida; and AdventHealth Cancer Institute.
The work was supported by the International Prostate Cancer Foundation, the Johns Hopkins Kimmel Cancer Center (NIH grant # P30CA006973), the Bankhead-Coley Cancer Research Program (grant # 24B16) to Perera, and by the Maryland Innovation Initiative Grant to Pavlovich and Perera.
Bettegowda is a consultant for Haystack Oncology, Privo Technologies and Bionaut Labs. He is a co-founder of OrisDx and Belay Diagnostics
END
Johns Hopkins investigators create new urine-based test to ID prostate cancers
2025-09-03
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