Quantification of cell cycle markers in oral precancer
The overall aims of the studies were to obtain objective measures of oral precancerous lesions based upon studies of the cell cycle and to investigate these parameters as possible prognostic indicators with regard to malignant transformation of these lesions. The majority of the precancerous lesions in the present study were dysplastic. These are the lesions which cause the greatest concern clinically with regard to malignant transformation. The first part of the study investigated the S-phase, growth fraction and the relationship of these to each other and with the degree of dysplasia with the aim of achieving objective measurements of the dysplasia and prognostic information. This was achieved by the use of BrdU for labelling of cells in the S-phase and anti-Ki67 antibody as a marker of cells in the growth fraction. The BrdU labelling index was demonstrated to provide an objective assessment of the dysplastic lesions when compared to the semi-objective method of Smith and Pindborg (1969). The ratio of the S-phase to the growth fraction was higher in those lesions which progressed to malignancy and was cited as a possible prognostic indicator. A number of methodological problems were identified from this first part of the study and these were investigated further by the development of techniques in Chapter 3. Firstly, a method was developed to enable the BrdU labelled tissue to be formalin fixed and then allow other cell cycle associated markers to be studied on sequential sections of the same tissue block. Secondly, numerous antigen retrieval techniques were carried out in order to optimise the immunohistochemical staining of Ki67 and subsequently other antibodies utilised in later parts of the study. Normal oral epithelium, derived post-mortem, was studied in Chapter 4 to investigate the apparent underestimation of proliferating cells identified by anti-Ki67 antibody in Chapter 2. It was apparent that, as in dysplastic epithelia, Ki67 significantly underestimated progenitor cells of the morphologically identified progenitor compartment. Ki67 did not identify all of those cells which would have been expected to be in the cell cycle it was originally claimed to do.