Detection of p53 gene alteration in renal-cell cancer by micropreparation techniques of tumor specimens.

Abstract

Alterations in the p53 tumor-suppressor gene have been identified in a variety of human malignancies, including renal-cell cancer. A technique for the isolation of tumor areas from tissue specimens to analyze formalin-fixed and paraffin-embedded tumors and try to avoid a disturbance of the results due to genetic background signal by the presence of tumor-infiltrating lymphocytes, was established. The presence of lymphocytes within the tumor areas investigated was determined by immunohistochemical staining for CD3, a lymphatic surface antigen. Following the isolation of about 100-200 tumor cells, PCR-directed molecular genetic analysis was performed. A highly informative allelotyping approach for the detection of loss of heterozygosity (LOH), determining BstU1- and VNTR-polymorphisms, a 100-bp marker directly localized in intron 1 of the p53 gene, as well as screening for mutations by single-strand conformation polymorphism analysis (SSCP) in exons 5-8, were used. Out of 44 renal-cancer specimens, 33 (75%) were informative for PCR-directed RFLP-analysis. Allelic loss at the p53 gene locus was observed in 10 of 33 cases (33%). No correlation between p53 gene alteration and T-stage, histological grade or histological differentiation could be observed. Alterations in the p53 gene, as detected by a molecular genetic as well as an immunohistochemical approach, were correlated to overall survival. During univariate analysis histological grade, lymphnode status and the presence of distant metastases could be identified as prognostic parameters for overall survival. During multivariate analysis none of the factors investigated remained an independent prognosticator for survival. Summarizing these results, it seems unlikely that p53 gene alterations will serve as an important new factor for the clinical prognosis of patients with renal-cell cancer.