Integrated Methylation and Copy Number Analysis for Noninvasive Bladder Cancer Detection in Urine

Irene J Beijert; Birgit M M Wever; Norbert Moldovan; Yara van den Burgt; Annick Nouwens; Ymke van der Pol; Anouk E Hentschel; Judith Bosschieter; Birgit Lissenberg-Witte; Paul C Kauer; R Jeroen A van Moorselaar; Florent Mouliere; Jakko A Nieuwenhuijzen; Renske D M Steenbergen

doi:10.1200/PO-25-00882

Integrated Methylation and Copy Number Analysis for Noninvasive Bladder Cancer Detection in Urine

Irene J Beijert
, Birgit M M Wever
, Norbert Moldovan
, Yara van den Burgt
, Annick Nouwens
, Ymke van der Pol
, Anouk E Hentschel
, Judith Bosschieter
, Birgit Lissenberg-Witte
, Paul C Kauer
, R Jeroen A van Moorselaar
, Florent Mouliere
, Jakko A Nieuwenhuijzen
, Renske D M Steenbergen^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

PURPOSE: The molecular analysis of urine cell-free DNA offers a noninvasive tool to advanced bladder cancer (BC) management. Assessment of somatic copy number aberration (SCNA) and DNA methylation analysis have emerged as promising approaches for BC detection. Here, we developed an integrated analysis to assess both SCNA and targeted methylation changes from the same template molecules, which we named the integrated sequencing-based copy number and methylation analysis in urine (iSECURE) method.

MATERIALS AND METHODS: Urine samples of 30 patients with primary BC, 28 patients with recurrent BC, and 31 hematuria controls were collected at home. Copy number profiling and tumor fraction (TF) estimation were performed by shallow whole-genome enzymatic methyl sequencing. Methylation sequencing libraries were also used for the measurement of a previously validated three-gene methylation marker panel ( GALR1/HAND2/NRN1) by the quantitative methylation-specific polymerase chain reaction. Diagnostic accuracy was evaluated by logistic regression analysis using Youden's index for sensitivity and specificity and validated using leave-one-out cross-validation (LOOCV).

RESULTS: TF and methylation levels were significantly higher in patients with BC compared with controls ( P < .0001). The individual assays yielded an AUC of 0.91 at 79% sensitivity and 97% specificity for the methylation marker panel and 0.81 at 74% sensitivity and 87% specificity for copy number profiling. The iSECURE workflow yielded a higher diagnostic accuracy with an AUC of 0.94 at 88% sensitivity (90% for primary tumors and 86% for recurrent tumors) and 94% specificity. LOOCV demonstrated an AUC of 0.91 at 84% sensitivity and 90% specificity.

CONCLUSION: We developed a single workflow for copy number profiling and targeted methylation analysis, requiring less input DNA and hands-on time. The high accuracy of iSECURE in home-collected urine samples for detecting both primary and recurrent BC underscores its clinical potential.

Original language	English
Article number	e2500882
Journal	JCO Precision Oncology
Volume	10
Issue number	8
DOIs	https://doi.org/10.1200/PO-25-00882
Publication status	Published - Aug 2026
Externally published	Yes

Keywords

Humans
DNA Methylation
Urinary Bladder Neoplasms/urine
Female
DNA Copy Number Variations
Male
Aged
Middle Aged
Biomarkers, Tumor/urine
Sensitivity and Specificity

Access to Document

10.1200/PO-25-00882

Cite this

Beijert, I. J., Wever, B. M. M., Moldovan, N., van den Burgt, Y., Nouwens, A., van der Pol, Y., Hentschel, A. E., Bosschieter, J., Lissenberg-Witte, B., Kauer, P. C., van Moorselaar, R. J. A., Mouliere, F., Nieuwenhuijzen, J. A., & Steenbergen, R. D. M. (2026). Integrated Methylation and Copy Number Analysis for Noninvasive Bladder Cancer Detection in Urine. JCO Precision Oncology, 10(8), Article e2500882. https://doi.org/10.1200/PO-25-00882

@article{8f1e330a0d6141dba19173742a755641,

title = "Integrated Methylation and Copy Number Analysis for Noninvasive Bladder Cancer Detection in Urine",

abstract = "PURPOSE: The molecular analysis of urine cell-free DNA offers a noninvasive tool to advanced bladder cancer (BC) management. Assessment of somatic copy number aberration (SCNA) and DNA methylation analysis have emerged as promising approaches for BC detection. Here, we developed an integrated analysis to assess both SCNA and targeted methylation changes from the same template molecules, which we named the integrated sequencing-based copy number and methylation analysis in urine (iSECURE) method.MATERIALS AND METHODS: Urine samples of 30 patients with primary BC, 28 patients with recurrent BC, and 31 hematuria controls were collected at home. Copy number profiling and tumor fraction (TF) estimation were performed by shallow whole-genome enzymatic methyl sequencing. Methylation sequencing libraries were also used for the measurement of a previously validated three-gene methylation marker panel ( GALR1/HAND2/NRN1) by the quantitative methylation-specific polymerase chain reaction. Diagnostic accuracy was evaluated by logistic regression analysis using Youden's index for sensitivity and specificity and validated using leave-one-out cross-validation (LOOCV). RESULTS: TF and methylation levels were significantly higher in patients with BC compared with controls ( P < .0001). The individual assays yielded an AUC of 0.91 at 79\% sensitivity and 97\% specificity for the methylation marker panel and 0.81 at 74\% sensitivity and 87\% specificity for copy number profiling. The iSECURE workflow yielded a higher diagnostic accuracy with an AUC of 0.94 at 88\% sensitivity (90\% for primary tumors and 86\% for recurrent tumors) and 94\% specificity. LOOCV demonstrated an AUC of 0.91 at 84\% sensitivity and 90\% specificity. CONCLUSION: We developed a single workflow for copy number profiling and targeted methylation analysis, requiring less input DNA and hands-on time. The high accuracy of iSECURE in home-collected urine samples for detecting both primary and recurrent BC underscores its clinical potential.",

keywords = "Humans, DNA Methylation, Urinary Bladder Neoplasms/urine, Female, DNA Copy Number Variations, Male, Aged, Middle Aged, Biomarkers, Tumor/urine, Sensitivity and Specificity",

author = "Beijert, \{Irene J\} and Wever, \{Birgit M M\} and Norbert Moldovan and \{van den Burgt\}, Yara and Annick Nouwens and \{van der Pol\}, Ymke and Hentschel, \{Anouk E\} and Judith Bosschieter and Birgit Lissenberg-Witte and Kauer, \{Paul C\} and \{van Moorselaar\}, \{R Jeroen A\} and Florent Mouliere and Nieuwenhuijzen, \{Jakko A\} and Steenbergen, \{Renske D M\}",

year = "2026",

month = aug,

doi = "10.1200/PO-25-00882",

language = "English",

volume = "10",

journal = "JCO Precision Oncology",

issn = "2473-4284",

publisher = "Lippincott Williams \& Wilkins",

number = "8",

}

Beijert, IJ, Wever, BMM, Moldovan, N, van den Burgt, Y, Nouwens, A, van der Pol, Y, Hentschel, AE, Bosschieter, J, Lissenberg-Witte, B, Kauer, PC, van Moorselaar, RJA, Mouliere, F, Nieuwenhuijzen, JA & Steenbergen, RDM 2026, 'Integrated Methylation and Copy Number Analysis for Noninvasive Bladder Cancer Detection in Urine', JCO Precision Oncology, vol. 10, no. 8, e2500882. https://doi.org/10.1200/PO-25-00882

TY - JOUR

T1 - Integrated Methylation and Copy Number Analysis for Noninvasive Bladder Cancer Detection in Urine

AU - Beijert, Irene J

AU - Wever, Birgit M M

AU - Moldovan, Norbert

AU - van den Burgt, Yara

AU - Nouwens, Annick

AU - van der Pol, Ymke

AU - Hentschel, Anouk E

AU - Bosschieter, Judith

AU - Lissenberg-Witte, Birgit

AU - Kauer, Paul C

AU - van Moorselaar, R Jeroen A

AU - Mouliere, Florent

AU - Nieuwenhuijzen, Jakko A

AU - Steenbergen, Renske D M

PY - 2026/8

Y1 - 2026/8

N2 - PURPOSE: The molecular analysis of urine cell-free DNA offers a noninvasive tool to advanced bladder cancer (BC) management. Assessment of somatic copy number aberration (SCNA) and DNA methylation analysis have emerged as promising approaches for BC detection. Here, we developed an integrated analysis to assess both SCNA and targeted methylation changes from the same template molecules, which we named the integrated sequencing-based copy number and methylation analysis in urine (iSECURE) method.MATERIALS AND METHODS: Urine samples of 30 patients with primary BC, 28 patients with recurrent BC, and 31 hematuria controls were collected at home. Copy number profiling and tumor fraction (TF) estimation were performed by shallow whole-genome enzymatic methyl sequencing. Methylation sequencing libraries were also used for the measurement of a previously validated three-gene methylation marker panel ( GALR1/HAND2/NRN1) by the quantitative methylation-specific polymerase chain reaction. Diagnostic accuracy was evaluated by logistic regression analysis using Youden's index for sensitivity and specificity and validated using leave-one-out cross-validation (LOOCV). RESULTS: TF and methylation levels were significantly higher in patients with BC compared with controls ( P < .0001). The individual assays yielded an AUC of 0.91 at 79% sensitivity and 97% specificity for the methylation marker panel and 0.81 at 74% sensitivity and 87% specificity for copy number profiling. The iSECURE workflow yielded a higher diagnostic accuracy with an AUC of 0.94 at 88% sensitivity (90% for primary tumors and 86% for recurrent tumors) and 94% specificity. LOOCV demonstrated an AUC of 0.91 at 84% sensitivity and 90% specificity. CONCLUSION: We developed a single workflow for copy number profiling and targeted methylation analysis, requiring less input DNA and hands-on time. The high accuracy of iSECURE in home-collected urine samples for detecting both primary and recurrent BC underscores its clinical potential.

AB - PURPOSE: The molecular analysis of urine cell-free DNA offers a noninvasive tool to advanced bladder cancer (BC) management. Assessment of somatic copy number aberration (SCNA) and DNA methylation analysis have emerged as promising approaches for BC detection. Here, we developed an integrated analysis to assess both SCNA and targeted methylation changes from the same template molecules, which we named the integrated sequencing-based copy number and methylation analysis in urine (iSECURE) method.MATERIALS AND METHODS: Urine samples of 30 patients with primary BC, 28 patients with recurrent BC, and 31 hematuria controls were collected at home. Copy number profiling and tumor fraction (TF) estimation were performed by shallow whole-genome enzymatic methyl sequencing. Methylation sequencing libraries were also used for the measurement of a previously validated three-gene methylation marker panel ( GALR1/HAND2/NRN1) by the quantitative methylation-specific polymerase chain reaction. Diagnostic accuracy was evaluated by logistic regression analysis using Youden's index for sensitivity and specificity and validated using leave-one-out cross-validation (LOOCV). RESULTS: TF and methylation levels were significantly higher in patients with BC compared with controls ( P < .0001). The individual assays yielded an AUC of 0.91 at 79% sensitivity and 97% specificity for the methylation marker panel and 0.81 at 74% sensitivity and 87% specificity for copy number profiling. The iSECURE workflow yielded a higher diagnostic accuracy with an AUC of 0.94 at 88% sensitivity (90% for primary tumors and 86% for recurrent tumors) and 94% specificity. LOOCV demonstrated an AUC of 0.91 at 84% sensitivity and 90% specificity. CONCLUSION: We developed a single workflow for copy number profiling and targeted methylation analysis, requiring less input DNA and hands-on time. The high accuracy of iSECURE in home-collected urine samples for detecting both primary and recurrent BC underscores its clinical potential.

KW - Humans

KW - DNA Methylation

KW - Urinary Bladder Neoplasms/urine

KW - Female

KW - DNA Copy Number Variations

KW - Male

KW - Aged

KW - Middle Aged

KW - Biomarkers, Tumor/urine

KW - Sensitivity and Specificity

U2 - 10.1200/PO-25-00882

DO - 10.1200/PO-25-00882

M3 - Article

C2 - 42585615

SN - 2473-4284

VL - 10

JO - JCO Precision Oncology

JF - JCO Precision Oncology

IS - 8

M1 - e2500882

ER -

Integrated Methylation and Copy Number Analysis for Noninvasive Bladder Cancer Detection in Urine

Abstract

Keywords

Access to Document

Fingerprint

Cite this