MULTIFOCAL TRANSITIONAL CELL CARCINOMA OF THE BLADDER AND UPPER URINARY TRACT: MOLECULAR SCREENING OF CLONAL ORIGIN BY CHARACTERIZING CD44 ALTERNATIVE SPLICING PATTERNS

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Abstract
CD44 is a widely expressed cell surface adhesion molecule in which various isoforms arise from alternative RNA splicing mechanism during cancer initiation. We assessed whether multifocal transitional cell carcinoma of the urothelium is due to field change and/or intraluminal seeding and implantation. In a series of 24 patients with synchronous and/or metachronous multiple urothelial cancers we performed reverse transcription-polymerase chain reaction analysis using a set of primers capable of amplifying all CD44 splice variant isoforms. After polymerase chain reaction products were electrophoresed band intensities with areas corresponding to the major isoforms (that is CD44s, CD44v10 and CD44v8–10) were quantified, and CD44v10-to-CD44s and CD44v8–10-to-CD44s ratios were calculated. Moreover, p53 gene mutations in exons 4 to 11 were screened by direct DNA sequencing. Of these 24 cases 18 showed similar CD44v10-to-CD44s and CD44v8–10-to-CD44s ratios in among multiple urothelial cancers in each case. However, in the remaining 6 cases these ratios were quite different among multiple cancer lesions. Furthermore, different types of p53 mutation were detected among multiple cancer lesions in only 2 of 24 cases, which also indicated different patterns of CD44 alternative splicing. These findings suggest that at least some multiple transitional cell carcinomas of the urothelium seem to be of independent origin based on the analysis of alternative RNA splicing of CD44. Moreover, this hypothesis was further supported by the evaluation of p53 gene mutation.