Urine Sediment DNA Research Articles

Quantitative Methylation-Specific Polymerase Chain Reaction Gene Patterns in Urine Sediment Distinguish Prostate Cancer Patients From Control Subjects

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of prostate cancers and is a promising marker for cancer detection. We sought to develop a test for prostate cancer based on a quantitative methylation-specific polymerase chain reaction (QMSP) of multiple genes in urine sediment DNA. We tested urine sediment DNA for aberrant methylation of nine gene promoters (p16INK4a, p14(ARF), MGMT, GSTP1, RARbeta2, CDH1 [E-cadherin], TIMP3, Rassf1A, and APC) from 52 patients with prostate cancer and 21 matched primary tumors by quantitative fluorogenic real-time polymerase chain reaction. We also analyzed urine sediments from 91 age-matched individuals without any history of genitourinary malignancy as controls. Promoter hypermethylation of at least one of the genes studied was detected in urine samples from all 52 prostate cancer patients. Urine samples from the 91 controls without evidence of genitourinary cancer revealed no methylation of the p16, ARF, MGMT, and GSTP1 gene promoters, whereas methylation of RARbeta2, TIMP3, CDH1, Rassf1A, and APC was detected at low levels. Overall, methylation found in urine samples matched the methylation status in the primary tumor. A combination of only four genes (p16, ARF, MGMT, and GSTP1) would theoretically allow us to detect 87% of prostate cancers with 100% specificity. Our data support further development of the noninvasive QMSP assay in urine DNA for early detection and surveillance of prostate cancer.

Detection of Methylated Apoptosis-Associated Genes in Urine Sediments of Bladder Cancer Patients

There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments. We analyzed the methylation status of the 5' regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients. The 5' regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P < or = 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P < or = 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P < or = 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P < or = 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals. Our results indicate that methylation of the 5' region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.

Identification of fibroblast growth factor receptor 3 mutations in urine sediment DNA samples complements cytology in bladder tumor detection.

Mutations in fibroblast growth factor 3 receptor (FGFR3) are frequent events in low-grade bladder tumors. To assess the potential utility of the detection of FGFR3 mutations in a screening modality, the authors analyzed urine sediment DNA samples from 192 patients in a retrospective study. Urine sediment DNA samples from 192 patients were prepared. Seventy-two patients had undergone transurethral resection (TURBT group) of mainly Ta lesions and 120 patients had undergone cystectomy (cystectomy group). The majority of patients in the cystectomy group had more advanced tumors compared with patients in the TURBT group. DNA preparations were screened for FGFR3 mutations in exons 7, 10, and 15 using single-strand conformation polymorphism (SSCP) and DNA sequencing. Using SSCP, 67% of patients in the TURBT group and 28% in the cystectomy group displayed FGFR3 mutations. Comparative analysis of cytology results and FGFR3 mutational analysis were performed in 122 cases. Within the TURBT group, FGFR3 mutation analysis outperformed cytology. FGFR3 mutation analysis identified change in 68% of urine sediment DNA samples whereas cytology recorded the presence of tumor cells in 32% of the DNA samples. In the cystectomy group, cytology outperformed FGFR3 mutation analysis. Cytology recorded tumor detection in 90% of patients, while SSCP identified mutational change in 24%. Combining FGFR3 mutation results with cytology in both groups correctly identified tumor presence in 105 of 122 (86%) of patients. The greater sensitivity of FGFR3 mutation detection over cytology in identifying the presence of low-grade, superficial bladder tumors represents a potential new tool to complement standard cytology in screening patients for bladder tumors and recurrent disease.

Identification of H-ras Mutations in Urine Sediments Complements Cytology in the Detection of Bladder Tumors

Urinary cytology has long been used as a noninvasive screen for the detection of urinary tract cancer but is limited by the generation of false positive and false negative results. More recently, molecular changes associated with urothelial neoplastic progression have been identified in DNA from urine sediments, demonstrating an alternative approach for identifying neoplastic change in the bladder. The purpose of this prospective study was to determine the value of detection of H-ras (also known as HRAS) mutations in urine sediment DNA as a clinical indicator of tumor presence, recurrence, and/or progression. Urine sediments were collected from 100 patients presenting with bladder tumors, with follow-up samples collected from 19 patients. DNA extracted from urine sediments was analyzed for changes in exon 1 of the H-ras gene, using single-strand conformation polymorphism (SSCP) analysis. A representative number of aberrant H-ras/SSCP migrating bands were excised and sequenced to confirm the presence of a mutation. Human bladder specimens were obtained from patients (93 of the 100 patients initially and 18 of the 19 patients studied by follow-up) and histologically evaluated for tumor content and grade. Mutations in exon 1 of the H-ras gene were detected in urine sediments from 44% (44 of 100) of the patients; concordant results were obtained by cytologic analysis, where 33% (31 of 93) of the patients displayed positive cytology. Analysis of the distribution of abnormalities with tumor grade revealed greater detection of low-grade (1-2) lesions using ras analysis (47%) compared with cytology (16%). In contrast, cytology was more effective in identifying the presence of carcinoma in situ. Combined results from these two approaches substantially increased the sensitivity of tumor detection, resulting in the identification of tumors in 60% of patients. Identification of H-ras mutations in DNA from urine sediments facilitates the detection of low-grade bladder tumors and, in combination with cytology, increases the overall tumor detection from 33% to 60%. Preliminary results in patient follow-up suggest that detection of H-ras mutations may have some clinical utility in detecting the presence of abnormal cells in the absence of an overt lesion following cystoscopy or positive cytology.