ABSTRACT
OBJECTIVE: To test DNA methylation profiling in detection of urothelial carcinoma in urine. STUDY DESIGN: Thirty-three bladder specimens were analyzed for the DNA p16INK4a, RASSF1, APC, GSTP, E-Cad and CyclinD2 genes to determine if there is a difference in gene methylation between benign and malignant cases. Urine samples were analyzed in a feasibility study. Finally, methylation profiles of urine samples were obtained and compared with follow-up biopsy diagnoses. RESULTS: We found methylated genes in 18% benign, 37% urothelial carcinoma in situ and 93% infiltrating urothelial carcinoma cases (p = 0.001). Methylation profiles from the 18 urine samples revealed a significantly higher prevalence of methylated genes in carcinoma cases than benign cases (100% vs. 50%, p = 0.025). We analyzed methylation profiles in 37 cytologically atypical urine samples with malignant or benign diagnosis on surgical follow-up andfound that only APC (55% in malignant vs. 0% in benign, p=0.025) and CyclinD2 were differentially methylated (35% in malignant vs. 0% in benign, p=0.2) while p14ARF, p16INK4a, RASSF1, GSTP and E-Cad had similar methylation profiles. CONCLUSION: These results suggest that methylation of p14ARF, p16INK4a, RASSF1, GSTP and E-Cad genes may not accurately identify carcinoma, but methylated APC and CyclinD2 might be useful biomarkers for urothelial carcinoma in urine.
Subject(s)
Carcinoma/diagnosis , Carcinoma/genetics , DNA Fingerprinting/methods , DNA Methylation , Urinary Bladder Neoplasms/diagnosis , Urinary Bladder Neoplasms/genetics , Urothelium/pathology , Aged , Anaphase-Promoting Complex-Cyclosome , Cadherins/genetics , Cadherins/metabolism , Carcinoma/urine , Cyclin D2 , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclins/genetics , Cyclins/metabolism , DNA/genetics , DNA/urine , DNA Fingerprinting/trends , Diagnostic Errors/prevention & control , False Negative Reactions , Feasibility Studies , Genetic Markers/genetics , Humans , Middle Aged , Predictive Value of Tests , Reproducibility of Results , Tumor Suppressor Protein p14ARF/genetics , Tumor Suppressor Protein p14ARF/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitin-Protein Ligase Complexes/metabolism , Urinary Bladder Neoplasms/urine , Urothelium/metabolismABSTRACT
Methylation of tumor suppressor genes has been implicated in breast cancer development. However, methylation profiles of different breast lesions, subtypes of carcinoma in particular, have not been examined in detail. In this study, we use methylation-specific PCR (MSP) to generate gene methylation profiles of different breast lesions and to test the clinical utility of such profiles. We examined the methylation status of three genes, RARbeta2, RASSF1A, and cyclin D2, on 102 samples of breast tissue, from benign (n = 36), to in situ carcinoma (n = 21), to invasive carcinoma (n = 45). We found that almost all cases of invasive carcinoma (96%) contained at least one methylated gene from our panel, whereas gene methylation was less common among benign lesions (42%) and in situ carcinoma (76%). Of the three genes, cyclin D2 methylation was most specific for malignancy because only 1 of 35 benign cases was methylated at this gene (1 case was not informative). The major histologic subtypes of invasive carcinoma show similar methylation profiles in the genes examined. We next performed MSP analysis on archival breast fine-needle aspiration (FNA) biopsy samples and corresponding surgical biopsy specimens and found a high concordance between the two types of specimens. We then analyzed 17 breast FNA biopsy samples with an indeterminate diagnosis. In this setting, MSP had a high specificity (100%) and modest sensitivity (67%) for identifying malignancy.
