[Our experience in using fluorescence in situ hybridization FISH-Uro Vysion in diagnostics of urothelial carcinoma]. / Nase zkusenosti s metodou fluorescencní in situ hybridizace pri detekci uroteliálního karcinomu.

UNLABELLED: Urothelial carcinoma is a disease at high risk of recurrence after the initial therapy (70-80%) and with the tendency to progression accomplishing the recurrence (30%). Long lasting monitoring of patients with urothelial carcinoma is necessary. Cystoscopy and cytology are currently the primary modalities used to detect and monitor urothelial carcinoma. However, cytology has relatively poor sensitivity especially in well differentiated tumors. Cystoscopy is an invasive and relatively expensive method. Therefore, methods improving detection of urothelial carcinoma from urine specimens are employed. Uro Vysion (Vysis) fluorescence in situ hybridization (FISH) for improved detection of urothelial carcinoma was evaluated. MATERIALS AND METHODS: Bladder tumor progression is accompanied by increased chromosomal instability and aneuploidy of chromosomes 3, 7, 17 and loss of locus 9p21. A total of 124 patients were analyzed at Dpts. of Urology and Pathology, Faculty Hospital in Brno. Cytologically analyzed urine specimens were tested by FISH and simultaneously cystoscopy was employed including biopsy for histological examination. RESULTS: FISH analysis was positive in 35 cases, including 5 cases with negative biopsy and cytology. Negative FISH result was detected in 24 cases where the malignant status was determined. The sensitivity of FISH in our series was 58.9% and the specificity 88.1%. CONCLUSIONS: FISH is a relatively simple, speedy and non invasive diagnostic method. It detects the symptoms of malignity on the molecular level, which leads to earlier diagnosis and therapy and, hence, to potential extended survival. FISH makes it possible to take decision in cases of atypical or unclear cytological finding. The FISH method using the Uro Vysion kit appears as a prospective non invasive method capable of early UK detection, with a higher sensitivity than the standard cytology of urine.

Conformational changes in p53 analysed using new antibodies to the core DNA binding domain of the protein.

The p53 protein contains a protease resistant core section that binds to DNA in a sequence specific manner and whose crystal structure has been determined. This core is flanked at the N-terminus by the transcriptional transactivation domain and at the C-terminus by sequences involved in the oligomerisation of the protein. Extensive immunochemical analysis of p53 has shown that dominant antigenic sites lie within these N- and C-terminal domains while few antibodies to the central core have been identified. One of these, PAb240, has been extensively characterised as its epitope is cryptic in the native DNA binding core structure but is exposed by denaturation. This epitope is also exposed on many p53 proteins that contain point mutations in the core domain suggesting that these mutations may have a common affect on the structure of the core. To investigate this further we have generated several new antibodies to novel sites on p53 and mapped their epitopes using synthetic peptides. We find that antibodies to two other discrete sites in the core can also, like PAb240, recognize cryptic epitopes and distinguish mutant from wild-type conformations implying that the point mutations found in p53 in human tumours have widespread effects on the folding pattern of the DNA binding domain.

p53 protein overexpression associates with growth patterns rather than with metastasizing in operable breast cancer.

We have analyzed p53 protein expression in 121 primary breast cancer biopsies by immunohistochemistry using the monoclonal antibody DO-1 and polyclonal serum CM-1. p53 protein overexpression has correlated in our study with mitotic activity (p=0.001), nuclear atypia (p=0.002), less favorable histological type of tumor and in a lesser extent with tumor size. The inverse, but highly significant, correlation (p=0.007) has been observed with lymph node involvement. There was also a trend for higher p53 positivity among DNA aneuploid tumors as compared with DNA diploid cases, but this was not significant. Our study suggests that p53, at least in some patients, may not be directly involved in the process of metastatic progression in breast cancer. Preliminary data would suggest that the detection of p53 protein overexpression could be a useful additional prognostic parameter in breast cancer.