The distinction between reading DNA and reading RNA is central to why this test works so well. DNA mutations reveal that a cancer exists, but they say little about what the cancer is actually doing inside the body. RNA, by contrast, reflects which genes a cell is actively using at a given moment, offering a dynamic snapshot of tumor behavior. Maximilian Diehn, MD, PhD, the Jack, Lulu, and Sam Willson Professor and a professor of radiation oncology at Stanford, explained the difference in practical terms: measuring tumor DNA in urine can tell clinicians whether a cancer is present, he said, but analyzing RNA can tell them not only whether a cancer is present but also what it is doing. That added layer of information is what allows the test to move beyond diagnosis and into the realm of treatment prediction.

The clinical need for better tools is substantial. Roughly 85,000 people in the United States are diagnosed with bladder cancer each year, and the disease is notoriously prone to recurrence and expensive to monitor. Diagnosis and surveillance currently depend on cystoscopy, a procedure in which an endoscope is threaded into the bladder to visually inspect for suspicious tissue. Cystoscopy can miss up to 30 percent of cancer cases, and patients with a history of non-muscle-invasive bladder cancer may need to undergo the procedure as often as every three months for the rest of their lives. The physical burden, the anxiety, and the cumulative cost of this surveillance regimen make a reliable noninvasive alternative one of the most sought-after goals in urologic oncology.

Diehn and Ash Alizadeh, MD, PhD, the Moghadam Family Professor, have collaborated on liquid biopsy technology for more than a decade. In 2014, the pair developed CAPP-Seq, short for cancer personalized profiling by deep sequencing, a method capable of detecting trace amounts of tumor DNA in the blood of lung cancer patients. The technique was later shown to classify lymphoma subtypes and flag emerging drug resistance. The researchers wondered whether a similar approach applied to urine could enhance the diagnosis, monitoring, and treatment of bladder cancer, but the bladder presented unique challenges that made a straightforward adaptation impossible.

The central problem is something researchers call the field effect. Normal bladder lining cells often accumulate their own mutations that overlap with those found in a patient’s actual tumor, a consequence of the same environmental exposures, such as smoking, that drive bladder cancer in the first place. A DNA-based test can mistake these bystander mutations for cancer, and correcting for the error typically requires first sequencing a patient’s tumor and normal tissue to learn which mutations to track and which to disregard. RNA-based testing sidesteps this obstacle entirely. Because RNA messages reflect overall gene activity rather than individual mutations, the test does not require matched tumor samples. Alizadeh noted that for DNA-based, tumor-informed approaches, clinicians need to know exactly what a tumor’s mutations look like to track disease, whereas RNA offers a biological signature of disease rather than an exact likeness, and urine provides a noninvasive way to detect and track it.

Designing a urine-based RNA test was nonetheless technically demanding, because RNA molecules in urine are fragmented and relatively rare. The research team, led by graduate student Kevin J. Liu, adapted a genetic sequencing method originally designed for RNA in blood plasma and designed a custom gene panel to identify RNA messages that are rare in healthy urine but abundant when a patient has a genitourinary tumor. The resulting assay, named uRARE-seq, which stands for urine random priming and affinity capture of cell-free RNA fragments for enrichment analysis by sequencing, was applied to samples from patients with bladder, kidney, and prostate cancer. It closely tracked the biology of the tumors and could even distinguish low-grade from high-grade bladder cancer, an important distinction that guides how aggressively a tumor must be treated.

The performance figures are striking. In a study of 683 urine samples from patients and healthy volunteers, uRARE-seq correctly identified 95 percent of people with localized bladder cancer and correctly cleared 90 percent of those who did not have the disease. That sensitivity and specificity outperformed both standard urine cytology, in which urine is examined under a microscope to identify cancerous cells, and an existing DNA-based urine test. The test also proved capable of identifying residual disease left behind after surgery and after BCG immunotherapy, giving clinicians a way to assess treatment response without repeated invasive procedures.

Perhaps the most consequential finding concerns treatment prediction. The researchers discovered that patients who subsequently responded to BCG therapy had pre-treatment urine RNA messages rich in genes involved in T-cell and immune-signaling activity, while those who failed to respond showed molecular signs of rapidly dividing tumor cells. Leveraging this insight, the team developed a biomarker that predicted with high accuracy whether a patient’s tumor was more likely to respond to immunotherapy or to chemotherapy, which is currently used as a second-line treatment when BCG fails or is unavailable. Joseph Liao, MD, the Kathryn Simmons Stamey Professor and chair of the department of urology, shares senior authorship of the study with Diehn and Alizadeh, and emphasized the stakes involved in getting these treatment decisions right.

The supply dynamics of BCG make this predictive capability especially valuable. BCG, a weakened strain of the bacterium used in tuberculosis vaccines, is instilled directly into the bladder through a catheter once a week for six weeks, with maintenance courses that may continue for up to three years. It works by stimulating the patient’s immune system to destroy lingering cancer cells, but it does not work for everyone, since some tumors simply lack the underlying immune activity needed to mount an effective response. Critically, BCG is manufactured by a single global supplier, and shortages are common, forcing urologists to ration doses among patients. A validated version of the test could allow doctors to reserve BCG for those most likely to benefit while moving other patients to chemotherapy sooner, sparing them months of ineffective therapy.

The researchers plan to validate uRARE-seq in larger, prospective trials at multiple centers, with the aim of bringing personalized treatment to bladder cancer patients. Liao pointed out that there are currently no biomarkers at all recommended or approved for surveillance in bladder cancer, and that the existing treatments are incredibly taxing, both physically and financially; guiding those choices, he said, has the potential to help both patients and clinicians. Researchers from Resero Bio, a Stanford spinout based in San Carlos formed to commercialize the technology, and the VA Palo Alto Health Care System contributed to the study. The work was funded by the National Institutes of Health, the Stanford ACED Canary Fellowship, the Tobacco-Related Disease Research Program, and the Virginia and D.K. Ludwig Fund for Cancer Research, among other sources, and several authors hold ownership interests in Resero Bio or related patents and consulting roles.

Subject of Research: Urine cell-free RNA sequencing for bladder cancer detection and treatment response prediction

Article Title: Urine test may improve bladder cancer diagnosis and guide treatment decisions

Article References: Urine test may improve bladder cancer diagnosis and guide treatment decisions. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: bladder cancer, liquid biopsy, urine test, cell-free RNA, uRARE-seq, BCG immunotherapy, chemotherapy, biomarker, cystoscopy, Nature Medicine, Stanford Medicine, precision oncology