IADR Abstract Archives
Cytopathological Analysis of Genetic Damage and Proliferation During Oral Carcinogenesis
Objectives: The objective of this study is to assess the cytopathology as a tool to detect DNA damage and cell proliferation activity during oral carcinogenesis
Methods: Cytological brushes were collected from 4 groups of patients: control (n=25); patients exposed to carcinogens from alcohol and smoking but without any oral lesion (n=26); patients with oral leukoplakia (n=18); patients with oral squamous cell carcinomas (n=20). Collected cells were released in a buffer solution. Part of the cells was applied on glass slides and silver stained for AgNOR (argyrophilic nucleolar organizer region) analysis of cell proliferation. For each sample the AgNORs were quantified and the mean and percentage of cells with more than 4 AgNORs per nuclei were calculated. The remaining cells were dispensed on a FTA card for DNA extraction. Patient’s blood was used as control for Loss of heterozigosity (LOH) analysis. PCR was performed for IFNA amplification. IFNA microsatellite marker is used to detect LOH on 9p21 loci, where the tumor suppressor gene CDKN2A is located. All experiments were performed by a blind and calibrated observer. Kruskal-wallis and Mann-Whitney tests were selected for statistical analysis.
Results: Increased mean AgNOR was observed in the Leukoplakia group compared to the control Group (3,4 versus 2,44, respectively, p=0,01, Kruskal-wallis test). The patients exposed to carcinogens and with leukoplakia showed higher levels of mutations in 9p21 (66,7% and 69,6%, respectively) when compared to the control and carcinoma groups (34,8% and 38,1%, respectively). No significant difference on proliferation activity was observed comparing patients with and without mutations on 9p21 (p= 0.357, Mann-Whitney test).
Conclusions: The collection of samples by cytological brushes is useful to detect alterations on cell proliferation and genetic damage in patients exposed to carcinogens and with oral leukoplakias.
Methods: Cytological brushes were collected from 4 groups of patients: control (n=25); patients exposed to carcinogens from alcohol and smoking but without any oral lesion (n=26); patients with oral leukoplakia (n=18); patients with oral squamous cell carcinomas (n=20). Collected cells were released in a buffer solution. Part of the cells was applied on glass slides and silver stained for AgNOR (argyrophilic nucleolar organizer region) analysis of cell proliferation. For each sample the AgNORs were quantified and the mean and percentage of cells with more than 4 AgNORs per nuclei were calculated. The remaining cells were dispensed on a FTA card for DNA extraction. Patient’s blood was used as control for Loss of heterozigosity (LOH) analysis. PCR was performed for IFNA amplification. IFNA microsatellite marker is used to detect LOH on 9p21 loci, where the tumor suppressor gene CDKN2A is located. All experiments were performed by a blind and calibrated observer. Kruskal-wallis and Mann-Whitney tests were selected for statistical analysis.
Results: Increased mean AgNOR was observed in the Leukoplakia group compared to the control Group (3,4 versus 2,44, respectively, p=0,01, Kruskal-wallis test). The patients exposed to carcinogens and with leukoplakia showed higher levels of mutations in 9p21 (66,7% and 69,6%, respectively) when compared to the control and carcinoma groups (34,8% and 38,1%, respectively). No significant difference on proliferation activity was observed comparing patients with and without mutations on 9p21 (p= 0.357, Mann-Whitney test).
Conclusions: The collection of samples by cytological brushes is useful to detect alterations on cell proliferation and genetic damage in patients exposed to carcinogens and with oral leukoplakias.