Detection of Bladder Cancer in Urine by a Tumor Suppressor Gene Hypermethylation Panel

Abstract

Purpose: Bladder cancer is potentially curable in the majority of cases; however, the prognosis for patients with advanced disease at presentation remains poor. Current noninvasive tests such as cytology lack sufficient sensitivity to detect low-grade, low-stage tumors. Silencing of tumor suppressor genes, such as p16 INK4a , VHL, and the mismatch repair gene hMLH1, has established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancers. It is also a promising new target for molecular detection in bodily fluids including urine, a readily accessible fluid known to contain bladder cancer cells. Methylation-specific PCR (MSP) can determine the presence or absence of methylation of a gene locus at a sensitivity level of up to 1 methylated allele in 1000 unmethylated alleles, appropriate for identifying cancer cell DNA in a bodily fluid.Experimental Design: We first determined the frequency of hypermethylation of the Rb tumor suppressor gene by bisulfite sequencing and of the p16 INK4a , p14 ARF , APC, and RASSF1A tumor suppressor genes by MSP in 45 bladder cancers. We then designed a panel optimal for diagnostic coverage composed of the APC, RASSF1A, and p14 ARF tumor suppressor genes. This panel was tested for detection of hypermethylation in matched sediment DNA from urine specimens obtained before surgery from the same 45 bladder cancer patients (2 Tis, 16 Ta, 10 T1, and 17 T2-4) as well as normal and benign control DNAs.Results: Hypermethylation of at least one of three suppressor genes (APC, RASSF1A, and p14 ARF ) was found in all 45 tumor DNAs (100% diagnostic coverage). We detected gene hypermethylation in the matched urine DNA from 39 of 45 patients (87% sensitivity), including 16 cases that had negative cytology. No hypermethylation of APC, RASSF1A, or p14 ARF was observed in normal transitional cell DNAs or in urine DNAs from normal healthy individuals and patients with inflammatory urinary disease (cystitis). Furthermore, an unmethylated gene in the tumor DNA was always found to be unmethylated in the matched urine DNA (100% specificity).Conclusions: Promoter hypermethylation of tumor suppressor genes is common in bladder cancer and was found in all grades and stages of tumors examined. Hypermethylation was detected in the urine DNA from 39 of 45 (87%) patients, including cases of early-stage disease amenable to cure. MSP may enhance early detection of bladder cancer using a noninvasive urine test.

Methylational urinalysis: a prospective study of bladder cancer patients and age stratified benign controls

“…Most molecular biomarkers analysed to date, lack sufficient specificity or sensitivity for their widespread use, and require extensive genetic analysis preventing their routine clinical use (Molinie et al, 2001). A promising biomarker recently investigated is aberrant DNA promoter hypermethylation (Dulaimi et al, 2004). This epigenetic marker has potential benefits over previously analysed genetic investigations, as it occurs widely throughout cancerous cells (thus only several loci need analysis) and always affects the same genomic loci (promoter regions) allowing minimal analysis.…”

“…Silencing of tumour suppressor genes (TSG) associated with aberrant hypermethylation has been shown to be a molecular pathway for human cancer development (Herman and Baylin, 2003). As promoter hypermethylation occurs frequently in UC (Catto et al, 2005), several authors have investigated its detection in exfoliated urinary cells as a novel diagnostic test (Maruyama et al, 2001;Chan et al, 2002;Dulaimi et al, 2004;Friedrich et al, 2004). Recently, Dulaimi et al (2004) found that analysis of hypermethylation using a panel of TSG's yielded superior results to cytology in the detection of bladder cancer.…”

“…As promoter hypermethylation occurs frequently in UC (Catto et al, 2005), several authors have investigated its detection in exfoliated urinary cells as a novel diagnostic test (Maruyama et al, 2001;Chan et al, 2002;Dulaimi et al, 2004;Friedrich et al, 2004). Recently, Dulaimi et al (2004) found that analysis of hypermethylation using a panel of TSG's yielded superior results to cytology in the detection of bladder cancer. The authors concluded that methylation analysis had a role as a noninvasive urine test for the early detection of bladder cancer.…”

“…In the 17 normal control urine samples, no aberrant methylation was detected except for RARb in four cases (23.5%). Dulaimi et al (2004) analysed urine DNA from 45 UC patients and 12 normal healthy individuals, for methylation of a TSG panel consisting of APC, RASSF1a and p14. Methylation was detected in 39 out of 45 patients (87% sensitivity; 100% specificity), including 16 cases with negative cytology.…”

Charakterisierung von DNA-Methylierungs-Biomarkern für das Harnblasenkarzinom

“…Klinisch bedeutsam ist trotz des überwiegend gu ten Verlaufs der papillären Tumoren eine hohe Rezidivrate (50-70%), was vor allem auf multifokale Tumorherde zurückzu führen ist. Problematisch ist hierbei ins besondere, dass 10-20% dieser Tumoren zu (Muskel ) invasiven Stadien rekurrie ren können [3].…”