A simple urine test devised by researchers at Stanford Medicine and the VA Palo Alto Health Care System can diagnose people with bladder cancer more accurately than current standard methods.

The test also allows physicians to not only assess the cancer’s response to treatment but to predict whether immunotherapy or chemotherapy is likely to be most effective for each patient.

The test is unique in that it measures RNA messages shed by tumour cells into urine, rather than hunting for cancer-related mutations in DNA as most blood and urine liquid biopsies do.

Because RNA reflects which genes a cancer cell is actively using, it captures a tumour’s behaviour in addition to its presence.

“Measuring tumour DNA in urine can tell us whether a cancer is present,” said Maximilian Diehn, MD, PhD, the Jack, Lulu, and Sam Willson Professor and a professor of radiation oncology.

“Analysing RNA can tell us not only whether a cancer is present — but also what it’s doing.”

Diehn shares senior authorship of the study, published in Nature Medicine, with Joseph Liao, MD, the Kathryn Simmons Stamey Professor and chair of the department of urology, and Ash Alizadeh, MD, PhD, the Moghadam Family Professor.

Graduate student Kevin J. Liu is the lead author of the research.

From blood to bladder

About 85,000 people in the United States are diagnosed each year with bladder cancer, which is prone to recurrence and expensive to monitor.

Diagnosis and surveillance currently rely on cystoscopy, in which an endoscope is threaded into the bladder to look for suspicious tissue — a procedure that can miss up to 30% of cancer cases.

High-risk patients with a history of non-muscle-invasive bladder cancer may need to undergo the surveillance procedure every three months.

After surgery to remove a tumour, many patients are treated with a weakened strain of the bacterium used in tuberculosis vaccines called Bacillus Calmette-Guérin, or BCG.

BCG is instilled directly into the bladder through a catheter once a week for six weeks and may be repeated as maintenance for up to three years.

BCG stimulates the patient’s immune system to destroy any lingering cancer cells.

But the treatment doesn’t work for everyone — some tumours simply lack the underlying immune activity needed to spark an effective attack — and there’s been no reliable way to tell in advance who will benefit.

For more than a decade, Diehn and Alizadeh have collaborated on liquid biopsy technology, which involves testing blood and other types of fluids.

In 2014, the pair developed CAPP-Seq (cancer personalised profiling by deep sequencing), a method for detecting trace amounts of tumour DNA in the blood of lung cancer patients and later showed it could also classify lymphoma subtypes and flag emerging drug resistance.

They wondered whether a similar technique used on urine could enhance the diagnosis, monitoring and treatment of bladder cancer.

But the bladder presented some unique challenges.

Normal bladder lining cells often accumulate their own mutations that overlap with those in a patient’s actual tumour.

A DNA test can mistake these bystander mutations — known as the field effect — for cancer, and correcting for them often requires first sequencing a patient’s tumour and normal tissue to learn which mutations to track and which to disregard.

In contrast, because RNA messages reflect overall gene activity rather than individual mutations, RNA tests don’t require matched tumour samples.

“For DNA-based, tumour-informed approaches, you need to know exactly what the tumour’s mutations look like to track disease,” Alizadeh said.

“RNA doesn’t have that problem. You’re looking for a biological signature of disease, not an exact likeness, and urine is a noninvasive way to accurately detect and track bladder cancer.”

Designing a urine-based test focused on RNA rather than DNA, however, was difficult because RNA molecules in urine are fragmented and relatively rare.

The researchers 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 genitourinary tumours.

Applied to samples from patients with bladder, kidney and prostate cancer, the resulting test, called uRARE-seq (for urine random priming and affinity capture of cell-free RNA fragments for enrichment analysis by sequencing), closely tracked the biology of the tumours and could distinguish low-grade from high-grade bladder cancer.

High-accuracy approach

In a study of 683 urine samples from patients and healthy volunteers, the test correctly identified 95% of people with localised bladder cancer and gave a clean bill of health to 90% of those who did not, outperforming both standard urine cytology (in which urine is examined through a microscope to identify cancerous cells) and a DNA-based urine test.

It also identified residual disease left behind after surgery and BCG immunotherapy.

The researchers found that patients who subsequently responded to BCG had pre-treatment urine RNA messages rich in genes involved in T-cell and immune-signalling activity; those who didn’t instead showed signs of rapidly dividing tumour cells.

Leveraging this finding, they developed a biomarker that predicted with high accuracy whether tumours were more likely to respond to immunotherapy versus chemotherapy, which is currently a second-line treatment if BCG is not successful or is unavailable.

Identifying who is most likely to respond to BCG treatment is critical.

BCG is manufactured by a single global supplier, and shortages are common, forcing urologists to ration it among patients.

A validated version of the test could let doctors reserve BCG for those patients who are most likely to benefit from it and move others to chemotherapy sooner.

The researchers plan to validate uRARE-seq in larger, prospective trials at multiple centres, with the aim of bringing personalised treatment to bladder cancer patients.

“There are currently no biomarkers at all that are recommended or approved for surveillance. These treatments are incredibly taxing — physically and financially,” Liao said.

“If we can guide those choices, we have the potential to help both patients and clinicians.”

Source: Stanford Medicine