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Comparative Analysis of Urine Fractions for Optimal Bladder Cancer Detection Using DNA Methylation Markers.

ABSTRACT: DNA methylation analysis of full void urine and urine pellet seems promising for bladder cancer (BC) detection and surveillance. Urinary cell-free DNA from urine supernatant is now gaining interest for other molecular tests in BC. This study aims to evaluate which urine fraction is preferred for BC diagnosis using methylation markers: full void urine, urine pellet or supernatant. Methylation levels of nine markers were determined in the three urine fractions and correlated with their respective tumor tissues in BC patients and compared to controls. For all markers and marker panel GHSR/MAL, diagnostic performance was determined by calculating the area under the curve (AUC) of the respective receiver operating characteristic curves. For most of the markers, there was a significant correlation between the methylation levels in each of the urine fractions and the matched tumor tissues. Urine pellet was the most representative fraction. Generally, AUCs for BC diagnosis were comparable among the fractions. The highest AUC was obtained for GHSR/MAL in urine pellet: AUC 0.87 (95% confidence interval: 0.73-1.00), corresponding to a sensitivity of 78.6% and a specificity of 91.7%. Our results demonstrate that cellular and cell-free DNA in urine can be used for BC diagnosis by urinary methylation analysis. Based on our comparative analysis and for practical reasons, we recommend the use of urine pellet.

SUBMITTER: Hentschel AE

PROVIDER: S-EPMC7226114 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Comparative Analysis of Urine Fractions for Optimal Bladder Cancer Detection Using DNA Methylation Markers.

Hentschel Anouk E AE Nieuwenhuijzen Jakko A JA Bosschieter Judith J Splunter Annina P van APV Lissenberg-Witte Birgit I BI Voorn J Patrick van der JPV Segerink Loes I LI Moorselaar R Jeroen A van RJAV Steenbergen Renske D M RDM

Cancers 20200402 4

DNA methylation analysis of full void urine and urine pellet seems promising for bladder cancer (BC) detection and surveillance. Urinary cell-free DNA from urine supernatant is now gaining interest for other molecular tests in BC. This study aims to evaluate which urine fraction is preferred for BC diagnosis using methylation markers: full void urine, urine pellet or supernatant. Methylation levels of nine markers were determined in the three urine fractions and correlated with their respective ...[more]

PMID: 32252299

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Project description:BackgroundThe development of accurate urinary biomarkers for non-invasive and cost-effective detection of primary and recurrent bladder tumours is recognized as one of the major clinical needs in bladder cancer diagnostics. The purposes of this study were (1) to validate the results of a previous technical comparison by determining the diagnostic performance of nine methylation markers in urine pellet compared to full void urine, and (2) to validate the diagnostic performance of the optimal marker panel GHSR/MAL from a previous exploratory study in a preclinical setting.MethodsUrine samples of 108 patients with bladder cancer and 100 age- and gender-matched controls were prospectively collected for methylation analysis. Urinary methylation levels of the markers FAM19A4, GHSR, MAL, miR-129, miR-935, PHACTR3, PRDM14, SST and ZIC1 were determined with quantitative methylation-specific PCR in urine pellet. Area under the curves (AUCs) were determined for individual markers and the marker panel GHSR/MAL. The diagnostic performance of the marker panel GHSR/MAL was evaluated in the total study population and in different subgroups of patients with bladder cancer using the Chi-square test. The diagnostic accuracy was assessed by leave-one-out cross-validation.ResultsAll nine urinary methylation markers (FAM19A4, GHSR, MAL, miR-129, miR-935, PHACTR3, PRDM14, SST and ZIC1) showed significantly higher methylation levels in bladder cancer patients than in controls (p < 0.001). Area under the curves (AUCs) of the nine methylation markers tested in urine pellet were similar to AUCs in full void urine of an independent previous cohort. GHSR/MAL reached an AUC of 0.89 (95% confidence interval [CI] 0.84-0.94), at 80% sensitivity and 93% specificity. Sensitivity of GHSR/MAL increased with higher tumour grades, higher tumour stages, in primary vs. recurrent tumours, and in males vs. females.ConclusionsThis technical validation supports the robustness of DNA methylation analysis in urine pellet and full void urine for the non-invasive detection of bladder cancer. Subsequent preclinical validation confirmed the diagnostic potential of GHSR/MAL. These findings underline the diagnostic potential of the marker panel GHSR/MAL for future bladder cancer diagnostics.

Project description:BackgroundBladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. Previous studies have identified several tumor-related genes that are hypermethylated in bladder cancer; however the DNA methylation profile of bladder cancer in Taiwan is not fully understood.MethodsIn this study, we compared the DNA methylation profile of multiple tumor suppressor genes (APC, DAPK, E-cadherin, hMLH1, IRF8, p14, p15, RASSF1A, SFRP1 and SOCS-1) in bladder cancer patients from different Chinese sub-populations including Taiwan (104 cases), Hong Kong (82 cases) and China (24 cases) by MSP. Two normal human urothelium were also included as control. To investigate the diagnostic potential of using DNA methylation in non-invasive detection of bladder cancer, degree of methylation of DAPK, IRF8, p14, RASSF1A and SFRP1 was also accessed by quantitative MSP in urine samples from thirty bladder cancer patients and nineteen non-cancer controls.ResultsThere were distinct DNA methylation epigenotypes among the different sub-populations. Further, samples from Taiwan and China demonstrated a bimodal distribution suggesting that CpG island methylator phentotype (CIMP) is presented in bladder cancer. Moreover, the number of methylated genes in samples from Taiwan and Hong Kong were significantly correlated with histological grade (P < 0.01) and pathological stage (P < 0.01). Regarding the samples from Taiwan, methylation of SFRP1, IRF8, APC and RASSF1A were significantly associated with increased tumor grade, stage. Methylation of RASSF1A was associated with tumor recurrence. Patients with methylation of APC or RASSF1A were also significantly associated with shorter recurrence-free survival. For methylation detection in voided urine samples of cancer patients, the sensitivity and specificity of using any of the methylated genes (IRF8, p14 or sFRP1) by qMSP was 86.7% and 94.7%.ConclusionsOur results indicate that there are distinct methylation epigenotypes among different Chinese sub-populations. These profiles demonstrate gradual increases with cancer progression. Finally, detection of gene methylation in voided urine with these distinct DNA methylation markers is more sensitive than urine cytology.

Project description:Background:Current standard methods used to detect and monitor bladder cancer (BC) are invasive or have low sensitivity. This study aimed to develop a urine methylation biomarker classifier for BC monitoring and validate this classifier in patients in follow-up for bladder cancer (PFBC). Methods:Voided urine samples (N?=?725) from BC patients, controls, and PFBC were prospectively collected in four centers. Finally, 626 urine samples were available for analysis. DNA was extracted from the urinary cells and bisulfite modificated, and methylation status was analyzed using pyrosequencing. Cytology was available from a subset of patients (N?=?399). In the discovery phase, seven selected genes from the literature (CDH13, CFTR, NID2, SALL3, TMEFF2, TWIST1, and VIM2) were studied in 111 BC and 57 control samples. This training set was used to develop a gene classifier by logistic regression and was validated in 458 PFBC samples (173 with recurrence). Results:A three-gene methylation classifier containing CFTR, SALL3, and TWIST1 was developed in the training set (AUC 0.874). The classifier achieved an AUC of 0.741 in the validation series. Cytology results were available for 308 samples from the validation set. Cytology achieved AUC 0.696 whereas the classifier in this subset of patients reached an AUC 0.768. Combining the methylation classifier with cytology results achieved an AUC 0.86 in the validation set, with a sensitivity of 96%, a specificity of 40%, and a positive and negative predictive value of 56 and 92%, respectively. Conclusions:The combination of the three-gene methylation classifier and cytology results has high sensitivity and high negative predictive value in a real clinical scenario (PFBC). The proposed classifier is a useful test for predicting BC recurrence and decrease the number of cystoscopies in the follow-up of BC patients. If only patients with a positive combined classifier result would be cystoscopied, 36% of all cystoscopies can be prevented.

Project description:BackgroundEarly screening for prostate cancer (PCA) remains controversial because of overdiagnosis and overtreatment of clinically insignificant cancers. Even though a number of diagnostic tests have been developed to improve on PSA testing, there remains a need for a more informative non-invasive test for PCA. The objective of this study is to identify a panel of DNA methylation markers suitable for a non-invasive diagnostic test from urine DNA collected following a digital rectal exam (DRE) and/or from first morning void (FV). A secondary objective is to determine if the cumulative methylation is indicative of biopsy findings.MethodsDRE and FV urine samples were prospectively collected from 94 patients and analyzed using 24 methylation-specific quantitative PCR assays derived from 19 CpG islands. The methylation of individual markers and various combinations of markers was compared to biopsy results. A methylation threshold for cancer classification was determined using a target specificity of 70%. The average methylation and the number of positive markers were also compared to the result of the biopsy, and the area under the receiver operating characteristic curves (AUCs) were calculated.ResultsMethylation of all 19 markers was detected in FV and DRE DNAs. Combining the methylation of two or more markers improved on individual marker results. Using 6of19 methylated markers as the threshold for cancer classification yielded a specificity of 71% (95% CI, 0.57-0.86) from both DRE and FV and a sensitivity of 89% (95% CI, 0.79-0.97) from DRE and 94% (95% CI, 0.84-1.0) from FV. The negative predictive value at the 6of19 threshold was ≥ 90 for both DNA types.ConclusionsPCA-specific methylation was detected in both FV and DRE DNA. There was no significant difference in diagnostic accuracy at the 6of19 threshold between DRE and FV urine DNA. The results support the development of a non-invasive diagnostic test to reduce unnecessary biopsies in men with elevated PSA. The test can also provide patients with personalized recommendations based on their own methylation profile.

Dataset Information

Comparative Analysis of Urine Fractions for Optimal Bladder Cancer Detection Using DNA Methylation Markers.

Publications

Comparative Analysis of Urine Fractions for Optimal Bladder Cancer Detection Using DNA Methylation Markers.

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