Elsevier

European Urology

Volume 58, Issue 1, July 2010, Pages 96-104
European Urology

Bladder Cancer
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

https://doi.org/10.1016/j.eururo.2009.07.041Get rights and content

Abstract

Background

Accumulating evidence suggests that DNA methylation markers could serve as sensitive and specific cancer biomarkers.

Objective

To determine whether a panel of methylated genes would have the potential to identify primary bladder cancer (BCa) in voided urine samples.

Design, setting, and participants

A pharmacologic unmasking reexpression analysis in BCa cell lines was initially undertaken to unveil candidate methylated genes, which were then evaluated in methylation-specific polymerase chain reaction (MSP) assays performed on DNA extracted from noncancerous and cancerous bladder tissues. The most frequently methylated genes in cancerous tissues, with 100% specificity, were retained for subsequent MSP analysis in DNA extracted from urine samples to build and validate a panel of potential methylated gene markers. Urine samples were prospectively collected at three urologic centres from patients with histologically proven BCa and processed for use in real-time MSP and cytologic analysis. Patients with nonmalignant urologic disorders were included as controls.

Measurements

A urine sample was classified as valid when ≥10 copies of the gene encoding ß-actin were measured in the urine sediment genomic DNA. Sensitivity, specificity, and predictive values of the MSP and cytology tests were assessed and compared.

Results and limitations

MSP assays performed on 466 of the 496 (94%) valid urine samples identified two genes, TWIST1 and NID2, that were frequently methylated in urine samples collected from BCa patients, including those with early-stage and low-grade disease. The sensitivity of this two-gene panel (90%) was significantly better than that of cytology (48%), with comparable specificity (93% and 96%, respectively). The positive predictive value and negative predictive value of the two-gene panel was 86% and 95%, respectively.

Conclusions

Detection of the methylated TWIST1 and NID2 genes in urine sediments using MSP provides a highly (≥90%) sensitive and specific, noninvasive approach for detecting primary BCa.

Trial registration

BlCa-001 study – EudraCt 2006-003303-40.

Introduction

Early detection of bladder cancer (BCa) correlates with an increased likelihood for bladder preservation and improved overall survival [1]. In most patients, the bladder tumour will primarily appear as a papillary non–muscle-invasive urothelial carcinoma (UC); up to 70% of these tumours will recur, and 15% will progress in stage and grade [2]. Therefore, patients diagnosed with early-stage BCa undergo frequent monitoring, currently based on cystoscopy and cytology, resulting in BCa becoming one of the most costly of all cancers to manage [3]. Noninvasive detection of early-stage BCa is essential for reducing the cost, morbidity, and mortality of this common malignancy. There is much interest in trying to identify urine-based markers for the diagnosis of this disease. Indeed, urine cytology is highly specific (≈94%) but lacks sensitivity (≈35%), especially for low-grade UC [4], [5], while cystoscopy is highly sensitive but is invasive, costly, and often associated with discomfort [6].

Cancer results from interactions between the environment and genetic and epigenetic factors. Although genetic mutations are often the subject of investigation, such genetic alterations account for only a small percentage of most cancers. Epigenetics indeed represent the most frequent alteration of DNA that can lead to the development and progression of cancer. More specifically, DNA hypermethylation, the most studied epigenetic mechanism, occurs when DNA becomes methylated at CpG-rich regions located in the gene promoter regions, leading to gene inactivation. DNA methylation of critical genes, such as tumour-suppressor genes, is a frequent and early event in neoplastic development [7].

Molecular tests that can determine the methylation status of specific genes can be readily used for the detection of cancer in clinical specimens such as tissue biopsies or body fluids. DNA is an ideal target for molecular detection because it is stable and can be amplified using polymerase chain reaction (PCR)–based techniques that are widely available in clinical laboratories. This technique makes it possible to work with small amounts of DNA from early neoplastic lesions or small cancers and achieve very sensitive detection of cancer cells in a background of largely more abundant normal cells. Critical tumour-suppressor genes are rarely methylated in normal cells, so that aberrant methylation of these genes is now recognised as a hallmark of neoplastic development [8], [9].

The most referenced method for determining the methylation state of critical genes is methylation-specific PCR (MSP), which has been widely used to study the feasibility of detecting hypermethylated genes originating from cancer cells in tissues and body fluids such as serum, urine, stool, and saliva. Numerous reports have shown the advantages of this method, which include increased sensitivity, specificity, and reproducibility over conventional methods based on cellular analysis in use today [10], [11].

A large number of studies have identified methylated genes linked to BCa [12], [13], [14], [15], [16]. The goal of this research was to develop a urine-based DNA methylation assay with high sensitivity, specificity, and reproducibility while using the fewest markers possible for cost-effective diagnostic utility. Herein, we describe the identification process and application of methylation-based DNA markers for the sensitive and specific detection of early-stage BCa.

Section snippets

Bladder tissues

Retrospective formalin-fixed, paraffin-embedded samples of human bladder UC of various grades and stages and nontumoural bladder tissues from cystectomy specimens from patients with neurourologic disorders (without BCa) were used in the study. Eleven 5-μm–thick slices of each sample were serially cut from each paraffin block. One section was routinely haematoxylin and eosin stained for histologic confirmation. The other 10 slices were deparaffinised, processed for DNA extraction, and then

Results

Fig. 1 details the three-step methylated gene marker identification, selection, and validation strategy used in this work.

Discussion

Intense work is being done in the field of bladder tumour markers with the goal of identifying BCa earlier, both at the initial diagnosis and at recurrence of known tumour, without the need for cystoscopic evaluation. Numerous urine-based markers and techniques have been investigated, and some of them are commercially available (eg, based on the detection of soluble antigens or chromosomal aberrations) and have been found to have better sensitivity than cytology but frequently with lower

Conclusions

By applying a three-step marker identification, selection, and validation strategy, we found that the detection in voided urine samples of a set of two methylated genes, TWIST1 and NID2, is highly (≥90%) sensitive and specific for the presence of primary BCa. Collectively, our data showed that these two methylated gene markers are highly suited for the urine-based, noninvasive detection of this disease, including low-grade and low-stage tumours.

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