Diagnostic potential of urinary PENK methylation for bladder cancer in patients with hematuria: insights from a prospective and multi-institutional study

Introduction

In bladder cancer (BCa), oncological outcomes are closely associated with tumor stage and grade, with muscle-invasive BCa and high-grade disease carrying poor prognosis (1). Accurate detection of both the presence of BCa and its oncological characteristics is therefore essential for patient management. However, the current gold-standard diagnostic methods—cystoscopy and urine cytology—have limitations. Cystoscopy is invasive and often causes patient discomfort, whereas urine cytology exhibits low sensitivity (2,3). Moreover, these methods provide limited accuracy in assessing the depth of BCa invasion into the bladder wall. Such challenges underscore the ongoing need for noninvasive, reliable methods for BCa detection.

Extensive research has explored potential diagnostic biomarkers for BCa to address these limitations. In particular, liquid biopsy has received increasing attention since the early 2010s, with urine representing an optimal sample owing to its noninvasive collection and direct contact with BCa, which may provide abundant molecular and genomic information (4). Several urinary biomarkers have been investigated, and some are commercially available, including BTA (Polymedco, Cortlandt Manor, NY, USA), NMP22 (Matritech, Newton, MA, USA), UroVysion (Abbott Molecular, Des Plaines, IL, USA), and Immuno-Cyt (Scimedx, Dover, NJ, USA). Nevertheless, even Food and Drug Administration-approved urinary biomarkers demonstrate limited diagnostic performance, with low sensitivity ranging from 58% to 78% (5). Although gene-panel-based approaches have been developed to overcome the limitations of single-target methods, optimal gene combinations remain unclear (4).

In this context, it should be noted that recent evidence suggests that a single DNA target, urinary PENK methylation, may enable precise BCa detection (6-10). The PENK gene encodes an endogenous opioid polypeptide hormone, which is cleaved into enkephalin; hypermethylation of the PENK promoter may inactivate tumor-suppressive pathways in multiple cancers (10). While studies of urinary PENK methylation have shown promising diagnostic accuracy, particularly for high-grade or advanced BCa, most were limited by single-center design or small sample sizes (6-10). Jeong et al. (11) subsequently reported the diagnostic utility of urinary PENK methylation in a large, prospective, multicenter study, published in JAMA Oncology. This commentary reviews these updated data and discusses their implications for clinical practice and the future development of effective BCa diagnostic methods.

Study design and methodological strengths

The study design has several notable strengths. First, the cohort was prospectively enrolled across 10 institutions in the Republic of Korea. Second, the sample size was carefully calculated based on pilot data, considering a BCa detection rate of 13.7% and a dropout rate of 10%, ensuring that the final cohort met the required number of participants. More than 1,000 participants (n=1,099) remained for the final analysis after excluding individuals with other malignancies in the urinary tract, allowing straightforward interpretation of the diagnostic accuracy of urinary PENK methylation for BCa. Third, the study focused on patients presenting with hematuria, a common but frequently etiologically unclear symptom in patients with BCa, thereby providing clinically relevant insights into the potential utility of urinary PENK methylation in resolving this diagnostic dilemma (12-14). Fourth, comparative analyses of diagnostic accuracy among PENK methylation, NMP22, and urine cytology were prespecified, offering an objective assessment of the clinical performance of the urinary PENK methylation test. Methodologically, adherence to the Standards for Reporting of Diagnostic Accuracy guidelines enhanced transparency and rigor. Despite the multicenter design, all pathological specimens were centrally reviewed by a pathologist blinded to urinary test results, minimizing reference standard bias and interobserver variability in grading and staging. Blinding of clinicians and pathologists to DNA methylation results further strengthened the internal validity of the study.

Diagnostic performance

The primary finding is the robust diagnostic potential of the urinary PENK methylation test in detecting and excluding BCa, with a sensitivity of 78.1% [95% confidence interval (CI): 72.6–83.6%] and specificity of 88.8% (95% CI: 86.7–90.8%) (Table 1). Notably, sensitivity increased to 89.2% (95% CI: 86.4–93.8%) for high-grade or invasive BCa, with a specificity of 87.8% (95% CI: 85.6–89.9%). Subgroup analyses demonstrated consistently high sensitivity across clinical categories, including age, sex, smoking status, and type of hematuria, supporting the robustness of the test. Receiver operating characteristic (ROC) curves indicated excellent performance for high-grade or invasive BCa (area under the ROC curve =0.95) and overall BCa (area under the ROC curve =0.88).

Compared with NMP22 and urine cytology, urinary PENK methylation substantially outperformed both tests in sensitivity for overall BCa [NMP22: 44.1% (95% CI: 37.5–50.8%); urine cytology: 32.3% (95% CI: 26.0–38.5%)] and high-grade or invasive BCa [NMP22: 51.5% (95% CI: 44.0–59.0%); urine cytology: 39.7% (95% CI: 32.4–46.9%)], although its specificity was slightly lower for overall BCa [NMP22: 91.9% (95% CI: 90.1–93.8%); urine cytology: 99.5% (95% CI: 99.1–100%)] and high-grade or invasive BCa [NMP22: 91.6% (95% CI: 89.8–93.5%); urine cytology: 99.5% (95% CI: 99.0–100%)]. Combining urinary PENK methylation with either NMP22 or urine cytology for high-grade or invasive BCa improved sensitivities to 91.8% (95% CI: 87.7–95.9%) and 90.2% (95% CI: 85.8–94.6%), respectively, with specificities of 90.2% (95% CI: 85.8–94.6%) and 87.6% (95% CI: 85.3–89.6%), respectively. Of the final cohort of 1,099 patients, 219 (19.9%) were diagnosed with BCa, and 176 (16.0%) had high-grade or invasive BCa. For high-grade or invasive BCa, the positive predictive value was 61.3% (95% CI: 55.4–67.3%), and the negative predictive value was 97.6% (95% CI: 96.6–98.7%).

Clinical implications

The findings from NMP22 and urine cytology indicate that the clinical advantage of urinary PENK methylation lies primarily in its high sensitivity for detecting BCa, particularly high-grade and invasive BCa. This feature corresponds to a low false-negative rate and contributes to high negative predictive values of 94.2% for overall BCa and 97.6% for clinically significant BCa. Although predictive values are prevalence-dependent (positive predictive value decreases and negative predictive value increases in lower-risk populations), the negative predictive values observed in this study are consistently high, aligning with a recent, relatively small-scale study evaluating urinary PENK methylation in patients with hematuria (97.7% in Korean and American populations) (9). These results suggest that the urinary PENK methylation test provides reliable exclusion of BCa in patients with hematuria, acting as a ‘rule-out’ tool. Notably, sensitivity remained high across subgroups defined by age and smoking status, despite evidence that DNA methylation may be influenced by these factors, supporting the robustness of the test in excluding disease (15).

Conversely, the relatively lower specificity and suboptimal positive predictive value of 61.3% for high-grade or invasive BCa indicate a non-negligible rate of false-positive results, limiting the role of urinary PENK methylation as a ‘rule-in’ tool. In clinical practice, this limitation may result in unnecessary cystoscopy, increased patient anxiety, and higher healthcare costs. However, combining urinary PENK methylation with urine cytology may mitigate this issue. According to the study protocol, the combined test (i.e., PENK methylation test and urine cytology) was considered positive when at least one of the two tests was positive. In this approach, cases that are positive by urinary PENK methylation but negative by urine cytology are still classified as positive. The two tests exhibit complementary diagnostic characteristics: urinary PENK demonstrates high sensitivity and negative predictive value, whereas urine cytology offers high specificity and positive predictive value (Table 1). Therefore, interpreting the combined results by considering negative results from PENK methylation alongside positive urine cytology may allow clinicians to reduce unnecessary cystoscopy while maintaining diagnostic accuracy for BCa.

Overall, in patients presenting with hematuria, urinary PENK methylation may be advantageous as an initial screening tool, prioritizing the detection of aggressive disease. Further studies are warranted to validate the clinical utility of this noninvasive combined approach, considering oncological outcomes, cost-effectiveness, and patient-centered factors, including emotional well-being.

Population characteristics and generalizability

However, this study has several limitations. First, all samples were obtained from patients enrolled in the Republic of Korea. Therefore, the diagnostic performance of urinary PENK methylation, particularly its high sensitivity, requires validation in other racial and ethnic populations. Second, the present study focused exclusively on patients presenting with hematuria. Because microscopic or gross hematuria is a common symptom in BCa, the restriction to this population may have inflated the positive predictive value relative to the general population, highlighting the need for larger-scale studies including patients irrespective of hematuria. Third, attrition warrants attention: approximately 12% of enrolled participants were excluded from the final analysis owing to invalid samples, lack of cystoscopy, or protocol deviations. Although not uncommon in prospective diagnostic studies, this attrition may affect real-world feasibility. Fourth, the study excluded patients with coexisting urinary malignancies to enhance result validity. However, PENK does not bind plasma proteins and is exclusively filtered in the glomerulus, suggesting potential as an alternative marker for renal function (16). Consequently, urinary PENK is not specific to the urinary tract, consistent with a recent prospective study reporting positive urinary PENK methylation in patients with cervical (25.0%), colorectal (10.5%), esophageal (4.8%), liver (5.0%), and kidney (5.3%) cancers (10). These potential false-positive findings emphasize that urinary PENK methylation is highly reliable in avoiding false negatives, supporting its utility as an initial BCa screening tool.

Future direction

Over the past decade, multigene assays have demonstrated favorable diagnostic performance for BCa by capturing its heterogeneous genetic background. Regarding DNA methylation, several tests, including AssureMDx, EpiCheck, UroMark, UriFind, and BladMetrix, have been developed (17). Most demonstrate impressive sensitivities, particularly for high-grade disease exceeding 90%, with specificity varying widely from approximately 75% to 95% (17,18). For instance, a prospective, blinded study for high-risk non-muscle invasive BCa surveillance using EpiCheck reported a sensitivity of 100% (95% CI: 64.6–100%) and specificity of 91% (95% CI: 72.2–98.4%) (18). However, as highlighted in a recent meta-analysis, multigene panels also face challenges of high cost and lack of standardization in selecting optimal gene combinations, contrasting with single-marker assays such as PENK methylation (19). Consequently, balancing simplicity with molecular coverage remains an active area of investigation. Comparative effectiveness studies are needed to determine the optimal trade-off among performance, cost, and feasibility. Furthermore, integrating PENK methylation testing into multivariable risk models incorporating clinical and imaging data may enhance overall diagnostic accuracy.

Conclusions

Jeong et al. (11) conducted a multicenter, large-scale study providing compelling evidence that urinary PENK methylation testing demonstrates acceptable diagnostic accuracy for detecting BCa in patients with hematuria. Specifically, the urinary PENK methylation test exhibited high sensitivity for high-grade or invasive BCa and outperformed established urinary biomarkers, including NMP22 and urine cytology. Nonetheless, concerns regarding its suboptimal positive predictive value temper enthusiasm for immediate widespread adoption. Further large-scale validation in ethnically diverse cohorts is essential to confirm the diagnostic potential of urinary PENK methylation testing.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Translational Andrology and Urology. The article has undergone external peer review.

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0092/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0092/coif). The authors have no conflicts of interest to declare.

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