Use of correction procedures in ploidy analysis of oral carcinomas

Ploidy analysis is an aid in the diagnosis and evaluation of prognosis of tumors. Image analysis is a relatively simple technique to assess ploidy that can be carried out with accessible equipment. However, it requires the use of accurate technical procedures to avoid methodological errors that may bias the measurements. We previously developed two procedures that are simple to apply in routine work and serve to correct the errors derived from the measurement of large nuclei that are not fully contained in the thickness of the section and those derived from non-specific background readings. In the present study we applied these corrections to the retrospective ploidy analysis of a series of 67 patients with oral carcinoma with a follow-up time of 18 months. Thirty-four patients were alive at the end of the study, 33 were deceased. The ploidy values and the malignancy indices corresponding to the deceased and live patients with TNM stage III and IV carcinomas at the time of biopsy were significantly different. There were no significant differences in ploidy values between live and deceased patients with TNM stage I and II at the time of biopsy. The corrections improved the sensitivity of the method and thus the statistical significance of the data. These data suggest that the method proposed may be of use to estimate lesion evolution, in particular in patients with advanced oral squamous cell carcinomas.

Experimental study to test the potential tumor promotion effect of a tooth bleaching agent

Carbamide Peroxide is routinely employed as a whitener for tooth enamel. Oral mucosa protection is recommended to avoid inflammatory reactions. Experimental work has demonstrated its irritative effect on gastric mucosa when swallowed. The activity of certain oxidizing agents as tumoral promoters has been demonstrated and associated to their capacity to induce hyperplasia. Within this context it seemed of interest to assess the possible action of carbamide peroxide as a tumoral promoter in oral mucosa with or without a precancerous condition. Its action was tested in 2 models which are highly sensitive to chemical cancerization: a) Dorsum skin or SENCAR mice treated with carbamide peroxide daily or twice a week with or without prior initiation with dimethylbenz(a)anthracene (DMBA). Control mice were submitted to the standard carcinogenesis protocol, i.e. initiation with DMBA and promotion with 12-O-tetradecanoyl phorbol acetate (TPA). b) Hamster cheek pouch submitted to topical application of carbamide peroxide 3 times a week with or without prior initiation with DMBA, hamster cheek pouch submitted to repeated topical application of DMBA as a complete carcinogen: application twice a week in the control group and identical treatment + 1 weekly application of carbamide peroxide to evaluate its capacity to enhance the process. The effects were assessed between 1 and 14 weeks of treatment at different intervals for the different experimental protocols. The control cases exhibited hyperplasia and tumor induction in keeping with the known sequence for both carcinogenesis models. None of the cases revealed a promoter or enhancer capacity of carbamide peroxide. These results indicate the lack of risk involved in the application of carbamide peroxide even in oral mucosa with a precancerous condition due to the action of initiation agents such as tobacco and alcohol.

Early detection of alterations associated to oral cancer

The model of hamster cheek pouch carcinogenesis closely mimics the development of human oral cancer. The study of the interaction between chemical carcinogens and radiation in the process of oral carcinogenesis is of interest given that the oral cavity is frequently exposed to chemical carcinogens such as alcohol and tobacco and is the route of entry of therapeutic radiation. In this context, markers of incipient alterations associated to a process of malignant transformation would contribute to early diagnosis and follow-up. The aim of the present study was to assess the early changes produced by carcinogenic agents applied separately or combined in a two-stage carcinogenesis protocol in hamster cheek pouch. The cheek pouch of the hamsters was treated with a single dose of radiation (20 Gy) or 7,12-dimethylbenz(a)anthracene (DMBA) as initiating agents and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoting agent for 1 or 2 weeks. The end-points chosen to identify early alterations were hyperplastic foci and silver-stained nucleolar organizer regions (Ag NOR). The data show that both markers are useful in the detection of early alterations compatible with a process of malignant transformation.

Nucleolar organizer regions in a model of cell hyperactivity and regression

Nucleolar organizer regions stained with colloidal silver techniques (AgNOR) evidence sites of active rRNA transcription. It has been proved that AgNOR undergo a rise in number and variations in size and shape in conditions which traditionally involve enhanced cell proliferation and rRNA transcription. AgNOR have been described as a marker of malignant transformation in multiple entities. Our laboratory has previously described their value as markers of radioinduced damage. The finding, at light microscopy level, that silver staining persisted at later post-irradiation times when cells are characteristically inactive, prompted the present study to correlate findings at light microscopy level with the ultrastructural analysis of nucleoli and their AgNOR in a model of irradiated skin. We herein attempt to explain the biological significance of AgNOR variations in the different phases of radioinduced response (which involves cellular hyperactivity followed by regressive features). Ten Wistar rats were submitted to local irradiation of the left leg (the shielded right leg was used as control) with 50 Gy x rays and killed 15 days post- irradiation. Silver staining was performed on ultrathin sections. In the basal layer of control epithelium silver affinity was established for fibrillar centers (FC) and fibrillar dense components (DFC). During the phase of radioinduced hyperplasia (1-3 days post-exposure) basal cells exhibit large reticular nucleoli, with irregular contours and silver staining on DFC. In the regressive phase (4-5 days post-irradiation) silver staining persists despite the halt in transcriptional activity, associated to homogeneous and compact nucleoli. These findings suggest caution in the interpretation of silver staining patterns.

Simultaneous PAGE, immunoblotting, and immunohistochemical analysis of differentiation associated keratins in lesions of the oral mucosa

The expression of differentiation associated high PM Keratin polypeptides of the oral mucosa lesions were studied by immunohistochemical and immunoblotting techniques applied to adjacent sections of each biopsy specimen. The material studied included specimens of leukoplakia, verrucous carcinoma, squamous cell carcinoma, adenocarcinoma and keratoacanthoma. Little or no expression of 65-67 Kd keratins was evident in squamous cell carcinoma and adenocarcinoma. Hyperkeratotic (both benign and dysplastic) lesions such as verrucous carcinoma, leukoplakia, and keratoacanthoma, showed great variations in the intensity of 65-67 bands and a very irregular immunohistochemical staining pattern. Increased amounts of horny substance was usually accompanied by absence of, or decreased expression of 65-67 Kd keratins, thus indicating a change in the polypeptide composition of the horny layer in pathological conditions of the oral epithelium.