Identification and Validation of the Methylated TWIST1 and NID2 Genes through Real-Time Methylation-Specific Polymerase Chain Reaction Assays for the Noninvasive Detection of Primary Bladder Cancer in Urine Samples

Renard, Isabelle; Joniau, Steven; Cleynenbreugel, Ben Van; Collette, C.; Naômé, C.; Vlassenbroeck, Ilse; Hubert, N.; Leval, Jean de; Straub, Josef; Criekinge, Wim Van; Hamida, Wissem; Hellel, Majed; Thomas, Alexandre; Leval, Laurence de; Bierau, Katja; Waltregny, David

doi:10.1016/j.eururo.2009.07.041

Cited by 147 publications

(91 citation statements)

References 25 publications

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“…From a list of 108 genes previously reported hypermethylated in bladder cancer, 89 genes were present on the Infinium array and 32 showed methylation (Supplementary Table S11). There was a high degree of agreement (70%) between the genes reported by Renard and colleagues and our findings (32). Similar concordance was observed between the study by Wolff and colleagues using Illuminas Golden Gate technology and the Infinium array (40).…”

Section: Discussionsupporting

confidence: 80%

“…The combination of TWIST and NID2 was reported to have a sensitivity and specificity of 90% and 93%, respectively (32). Recently, 3 other novel markers (GDF15, TMEFF2, and VIM) have been reported to have a sensitivity of 94% and a specificity of 90% to 100%.…”

Section: Discussionmentioning

confidence: 99%

“…In bladder cancer, methylation of single gene has been identified and a possible function as stage marker, or as a urinary marker, has been tested (13,19,(28)(29)(30)(31)(32)(33)(34)(35). However, few studies have used a more global array-based approach.…”

Section: Introductionmentioning

confidence: 99%

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Comprehensive Genome Methylation Analysis in Bladder Cancer: Identification and Validation of Novel Methylated Genes and Application of These as Urinary Tumor Markers

Reinert

Modin

Castaño

et al. 2011

Clinical Cancer Research

183

161

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Purpose: Epigenetic alterations are common and can now be addressed in a parallel fashion. We investigated the methylation in bladder cancer with respect to location in genome, consistency, variation in metachronous tumors, impact on transcripts, chromosomal location, and usefulness as urinary markers.Experimental Design: A microarray assay was utilized to analyze methylation in 56 samples. Independent validation was conducted in 63 samples by a PCR-based method and bisulfite sequencing. The methylation levels in 174 urine specimens were quantified. Transcript levels were analyzed using expression microarrays and pathways were analyzed using dedicated software.Results: Global methylation patterns were established within and outside CpG islands. We validated methylation of the eight tumor markers genes ZNF154 (P < 0.0001), HOXA9 (P < 0.0001), POU4F2 (P < 0.0001), EOMES (P ¼ 0.0005), ACOT11 (P ¼ 0.0001), PCDHGA12 (P ¼ 0.0001), CA3 (P ¼ 0.0002), and PTGDR (P ¼ 0.0110), the candidate marker of disease progression TBX4 (P < 0.04), and other genes with stage-specific methylation. The methylation of metachronous tumors was stable and targeted to certain pathways. The correlation to expression was not stringent. Chromosome 21 showed most differential methylation (P < 0.0001) and specifically hypomethylation of keratins, which together with keratin-like proteins were epigenetically regulated. In DNA from voided urine, we detected differential methylation of ZNF154 (P < 0.0001), POU4F2 (P < 0.0001), HOXA9 (P < 0.0001), and EOMES (P < 0.0001), achieving 84% sensitivity and 96% specificity.Conclusions: We initiated a detailed mapping of the methylome in metachronous bladder cancer. Novel genes with tumor, chromosome, as well as pathway-specific differential methylation in bladder cancer were identified. The methylated genes were promising cancer markers for early detection of bladder cancer. Clin Cancer Res; 17(17); 5582-92. Ó2011 AACR.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

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Comprehensive Genome Methylation Analysis in Bladder Cancer: Identification and Validation of Novel Methylated Genes and Application of These as Urinary Tumor Markers

Reinert

Modin

Castaño

et al. 2011

Clinical Cancer Research

183

161

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“…Given the comparable sensitivities of urine testing and cystoscopy, we suggest that a subsequent study should be carried out investigating a modified surveillance protocol consisting of the urine test followed by cystoscopy, only when the urine test is positive, for patients in the low/ intermediate risk bladder cancer group. A number of recent studies have reported high sensitivities with DNA methylation biomarkers for the detection of bladder cancer in voided urine (36)(37)(38)(39)(40)(41)(42)(43). Three of these studies used methylation specific PCR (MSP) and reported sensitivities in the range of 85 to 94% at a specificity in the range of 93 to 100%.…”

Section: Discussionmentioning

confidence: 99%

A 3-Plex Methylation Assay Combined with the FGFR3 Mutation Assay Sensitively Detects Recurrent Bladder Cancer in Voided Urine

Kandimalla

Masius

Beukers

et al. 2013

Clinical Cancer Research

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Purpose: DNA methylation is associated with bladder cancer and these modifications could serve as useful biomarkers. FGFR3 mutations are present in 60% to 70% of non-muscle invasive bladder cancer (NMIBC). Low-grade bladder cancer recurs in more than 50% of patients. The aim of this study is to determine the sensitivity and specificity of a urine assay for the diagnosis of recurrences in patients with a previous primary NMIBC G1/G2 by using cystoscopy as the reference standard.Experimental Design: We selected eight CpG islands (CGI) methylated in bladder cancer from our earlier genome-wide study. Sensitivity of the CGIs for recurrences detection was investigated on a test set of 101 preTUR urines. Specificity was determined on 70 urines from healthy males aged more than 50 years. A 3-plex assay for the best combination was developed and validated on an independent set of 95 preTUR, recurrence free, and nonmalignant urines (n ¼ 130).Results: The 3-plex assay identified recurrent bladder cancer in voided urine with a sensitivity of 74% in the validation set. In combination with the FGFR3 mutation assay, a sensitivity of 79% was reached (specificity of 77%). Sensitivity of FGFR3 and cytology was 52% and 57%, respectively.Conclusion: The combination of methylation and FGFR3 assays efficiently detects recurrent bladder cancer without the need for stratification of patients regarding methylation/mutation status of the primary tumor. We conclude that the sensitivity of this combination is in the same range as cystoscopy and paves the way for a subsequent study that investigates a modified surveillance protocol consisting of the urine test followed by cystoscopy only when the urine test is positive.

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“…It is thought that this protein limits the growth of many cell types by regulating gene expression (Soprano et al, 2004). In the study conducted by Renard et al (2010), it was demonstrated that 2 genes (TWIST1 and NID2) were frequently methylated in urine samples collected from bladder cancer patients, including those with early-stage and low-grade diseases, with a specificity of 93% and sensitivity of 90%, which was an improvement from the cytological method of detection (48%). Besides the identification of DNA hypermethylation at a single locus, some authors have demonstrated that several genes may be analyzed together to generate a profile of hypermethylated genes.…”

Section: Candidate Epigenetic Biomarkers In the Diagnosis Of Bladder mentioning

confidence: 91%

Epigenetic Biomarkers in Bladder Cancer

Zimbardi¹,

Reis²,

Prando³

et al. 2012

Bladder Cancer - From Basic Science to Robotic Surgery

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Identification and Validation of the Methylated TWIST1 and NID2 Genes through Real-Time Methylation-Specific Polymerase Chain Reaction Assays for the Noninvasive Detection of Primary Bladder Cancer in Urine Samples

Cited by 147 publications

References 25 publications

Comprehensive Genome Methylation Analysis in Bladder Cancer: Identification and Validation of Novel Methylated Genes and Application of These as Urinary Tumor Markers

Comprehensive Genome Methylation Analysis in Bladder Cancer: Identification and Validation of Novel Methylated Genes and Application of These as Urinary Tumor Markers

A 3-Plex Methylation Assay Combined with the FGFR3 Mutation Assay Sensitively Detects Recurrent Bladder Cancer in Voided Urine

Epigenetic Biomarkers in Bladder Cancer

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