In situ detection of global DNA hypomethylation in exfoliative urine cytology of patients with suspected bladder cancer.

Global DNA hypomethylation is a common phenomenon in bladder cancer. Therefore we investigated whether it is possible to detect and assess global DNA hypomethylation in bladder cancer using a specific monoclonal antibody for 5-methyl-cytosine. Cytospins from exfoliative urine cytology specimens of patients with bladder cancer or a history of bladder cancer, control patients with benign urological diseases and of young healthy volunteers were analyzed. Urothelial carcinoma (UC) cells showed various degrees of nuclear destaining indicating global DNA hypomethylation whereas all specimens from healthy volunteers showed granular nuclear staining indicating regular methylation of repeated DNA sequences. Lowest 5-methylcytosine immunostaining scores were observed in carcinoma cells and a statistically significant difference was observed between urothelial cells of healthy controls or patients with benign disease compared to bladder cancer patients (p<0.01, p<0.05, respectively). In UC cases even morphologically normal urothelial cells often displayed evident hypomethylation. Likewise, in patients with a history of UC, but no cystoscopic evidence of recurrence, morphologically non-malignant urothelial cells presented with some degree of demethylation. Our results strongly support the hypothesis of early global demethylation in bladder cancer. Immunocytochemical staining with the 5-methylcytosine antibody allows simultaneous individual assessment of nuclear morphology and methylation status of a given sample.

Verrucous carcinoma of the buccal mucosa: histopathological, cytological and DNA-cytometric features.

We describe a patient with an exophytic oral lesion diagnosed as verrucous carcinoma. The lesion existed without metastases, at least 5 years. Local excisions led to recurrences and continuous expansion. Scalpel biopsies for histopathological and polymerase chain reaction examination were obtained from characteristic regions of the lesion. Brush biopsies for exfoliative cytology (EC) were taken, in order to screen the mucosal area covered by the lesion. After Feulgen restaining of the smears, nuclear DNA contents were measured using a TV image analysis system. An exophytic lesion of the buccal mucosa was diagnosed as low-grade malignant through histopathology and EC combined with DNA-image cytometry (peritetraploid DNA-aneuploidy). Due to almost normal microscopic appearance of the epithelium of verrucous carcinoma, thorough cytological/DNA-cytometric and histological examinations are needed. Brush biopsies of such neoplastic oral lesions showing DNA-aneuploidy with peritetraploid stemlines should be used for diagnosis and follow-up examination of these patients.

Cytologic and DNA-cytometric examination of oral lesions in lichen planus.

BACKGROUND: The aim of the present study was to evaluate the diagnostic value of exfoliative cytology (EC) and DNA image cytometry applied to oral lesions of lichen planus (LP; n = 56), in order to detect or exclude malignant transformation. METHODS: Brush and excisional biopsies were obtained from 56 patients. In cases of oral LP in which brush biopsies were suspicious for tumor cells, nuclear DNA contents were measured, using a TV Image Analysis System. RESULTS: In 50 patients EC yielded tumor cell-negative, doubtful in four cases and suspicious results obtained in two cases. DNA image cytometry revealed DNA-aneuploidy only in the two suspicious cases. The comparison between cytologic/DNA-cytometric diagnosis and biopsy histology resulted in a total agreement (LP without dysplasia: 54 and squamous cell carcinoma in LP: two cases). CONCLUSIONS: In conclusion, cytology with DNA-cytometry is a highly sensitive, specific, and non-invasive method, which can be used for periodical follow up of oral LP lesions in order to early detect or exclude malignancy.

Expression of BUB1 protein in gastric cancer correlates with the histological subtype, but not with DNA ploidy or microsatellite instability.

During mitosis, the spindle checkpoint delays the onset of anaphase until all chromosomes have attached properly to the mitotic spindle, preventing chromosome missegregation. BUB (budding uninhibited by benzimidazole) 1 is one of the key components of this checkpoint. BUB1 mutations are rare in cancer tissues and no mutations have been identified in gastric cancer. In mice, immunodepletion of BUB1 abolished the spindle checkpoint. Thus, aberrant expression of BUB1 protein could impair mitotic checkpoint function, resulting in aneuploidy, a common phenomenon in gastric cancer. In the present study, an antibody was generated against BUB1 and its expression was studied in gastric cancer tissue sections (n = 80) by immunohistochemistry. Nuclear BUB1 expression was found in all gastric cancer cases. The proportion of tumour cells expressing BUB1 was significantly greater in diffuse-type than in intestinal-type gastric carcinoma (p < 0.001). No correlation was found between BUB1 expression and deoxyribonucleic acid (DNA) ploidy, microsatellite instability or any other histopathological parameters investigated. To the authors' knowledge, this is the first study of BUB1 protein expression in gastric cancer tissues. Different BUB1 protein expression levels in intestinal- and diffuse-type gastric cancer may provide further evidence of a potential link between different genetic pathways and morphological phenotype in gastric carcinogenesis. However, further studies are needed to establish whether there is an association between BUB1 protein expression level and mitotic spindle checkpoint function in gastric cancer.

Overexpression of the mitotic checkpoint genes BUB1, BUBR1, and BUB3 in gastric cancer--association with tumour cell proliferation.

The mitotic spindle assembly checkpoint modulates the timing of anaphase initiation in response to improper alignment of chromosomes at the metaphase plate. The BUB gene family encodes proteins which are part of a large multi-protein kinetochore complex and which are believed to be key components of the checkpoint regulatory pathway. Failure of this surveillance system can lead to genomic instability and could be responsible for the increased incidence of aneuploidy in gastric cancer. Since mutations of BUB genes have not been identified in gastric cancer to date, altered BUB expression levels may significantly impair mitotic checkpoint function. To explore this possibility, the expression levels of BUB1, BUBR1, and BUB3 were determined in 43 gastric carcinomas and corresponding normal gastric mucosa by reverse transcription-polymerase chain reaction (RT-PCR). Gene expression levels were compared with histopathological parameters and DNA ploidy, as well as with proliferative activity, measured by Ki-67 mRNA expression. To the authors' knowledge, this is the first study to investigate the expression levels of mitotic checkpoint genes together with DNA ploidy in gastric cancer. BUB1 was overexpressed in 84%, BUBR1 in 68%, and BUB3 in 79% of gastric cancers. This study also revealed that all three genes were simultaneously overexpressed in 61% of the tumours and that there was a statistically significant positive correlation between overexpression of BUB1, BUBR1 or BUB3 and Ki-67 expression (p < 0.001). Eighty-one per cent of the tumours were classified as aneuploid. However, no correlation was found between ploidy and BUB transcript expression levels. These results suggest that inactivation of the mitotic checkpoint genes BUB1, BUBR1, and BUB3 by epigenetic silencing does not seem to play a role in gastric carcinogenesis. The strong correlation of BUB expression level and tumour cell proliferation suggests that BUB overexpression is a proliferation-dependent phenomenon in gastric cancer. However, overexpression due to lack of normal BUB protein function or due to a yet unknown additional BUB function has to be considered.