Bladder cancer currently represents the second most common urological malignancy after prostate cancer, and incidence is expected to increase in the coming decades. While a subset of patients already presents with advanced muscle-invasive bladder cancer, the vast majority are diagnosed with non-muscle-invasive bladder cancer (NMIBC).
The natural history of NMIBC, with frequent recurrences over a long period of time, typically requires repeated follow-up visits for cystoscopic assessment, making bladder cancer one of the most expensive cancers to treat.
The approach to surveillance of NMIBC, in particular, is quite challenging. For many decades, diagnosis and monitoring has relied on two major pillars: cystoscopy and urine cytology.
Unmet needs
Despite technological improvements cystoscopy still misses or misinterprets a significant number of urothelial lesions. Moreover, as an invasive procedure it adds to patient morbidity.
As for urine cytology, and notwithstanding high specificity for detecting high-grade urothelial cancers, its sensitivity is relatively low, missing a significant number of clinically-relevant tumors. Moreover, the reporting system is focused on identifying high-grade cancers and does not reliably detect low-grade lesions, which may still require treatment. Interobserver agreement also remains a concern, and the use of grey zone categories such as “atypical urothelial cells” is challenging to translate into clinical action, since it may represent a vast array of histology results – from benign lesions to high grade urothelial cancer.
Finally, urothelial carcinoma has a tendency for multifocality, and diagnosing an upper tract lesion in voided urine samples can be challenging, since cells are more often degenerated.
Given the limitations of both cystoscopy and cytology, there is an urgent need for more accurate means of diagnosing and monitoring NMIBC patients. Less invasive, more personalized, and more cost-effective tests are required, ultimately leading to better risk stratification, optimal treatment selection, and improved patient outcomes.
Urine: the promising biofluid
Taking advantage of its intimate contact with the urothelium lining of the bladder (and upper urinary tract), urine represents a logical and promising biofluid, carrying valuable diagnostic information about each patient’s bladder cancer at a specific time point. This makes urine the most useful source of liquid biopsy biomarkers for diagnosing and monitoring NMIBC patients.
Among the several analytes within a liquid biopsy sample, aberrations in DNA methylation of tumor DNA – including circulating tumor DNA – are particularly attractive. Silencing of gene expression (namely of tumor suppressor genes) through promoter methylation is part of the epigenetic mechanisms facilitating tumor initiation and progression. Such epigenetic changes occur early, frequently preceding morphological changes that draw clinical attention. This places DNA methylation-based tests as optimal candidates for screening or early diagnosis of cancer, as well as for non-invasive detection of minimal residual disease during follow-up.
One PCR-based assay – that detects aberrations in the DNA methylation pattern of a panel of 15 biomarkers – has a CE-IVD mark and is approved for clinical use by the FDA for non-invasive monitoring of NMIBC patients. The test is convenient and non-invasive (using voided urine samples), requiring a low amount of DNA. Strengths of the workflow include same-day results, standardization of protocol and workflow, and automatic software analysis.
Alongside a quantitative score, the test offers a qualitative result denoting high or low probability of bladder cancer, facilitating clinical decision making. Validation studies show an overall sensitivity and specificity of 74 percent and 84 percent, respectively, or 91 percent and 81 percent for high grade urothelial cancers. What’s more, recent reports have also demonstrated the test’s ability to detect upper tract urothelial cancers.
Follow-up testing
Although cystoscopy cannot be completely replaced, the high negative predictive value – 98 percent for high grade urothelial cancers – of the DNA methylation test makes it a useful complement to the follow-up of bladder cancer patients, working as a “rule-out test”. Results in several centers have validated the strategy of alternating cystoscopy with DNA methylation testing, which led to a decrease in the frequency of cystoscopies, thereby reducing patient burden and boosting cost-effectiveness.
Moreover, studies have shown that patients with positive DNA methylation results have a higher risk of recurrence during follow-up, even before a cystoscopic change can be detected. This anticipatory positive result can help guide the selection of patients for more intense follow-up regimens. Further studies are required to assess the performance of the test in additional indications and diverse populations.
DNA methylation testing in practice
In conclusion, DNA methylation testing constitutes a promising route to precision medicine for NMIBC. And what about the role of pathologists in this odyssey? Pathologists are at the core of precision medicine, having a critical role in the implementation and reporting of any biomarker test. The laboratory’s input to liquid biopsy processing, pre-analytical variables, laboratory workflow, biobanking, and quality control is crucial.
At our Department of Pathology of the Portuguese Oncology Institute of Porto, we have implemented NMIBC DNA methylation testing since January 2025. The pipeline is entirely performed at the Molecular Pathology lab, which received certification for performing this test. This requires a multidisciplinary team of fully trained professionals, careful planning and organization, and constant communication with the urology clinic.
Liquid biopsy biomarker tests are moving towards clinical implementation and changing the paradigm – offering the power of precision oncology information in a non-invasive way. The field of bladder cancer is no exception, where harnessing the power of urine biomarkers may make the difference in personalized patient management.
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