Bladder CancerIdentification 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
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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