Prognosis in bladder cancer. A study of cytometric, morphometric and immunohistochemical techniques

Bladder cancer affects about 2,000 people in Sweden every year. For adequate treatment, prognostic information is essential. Most of the prognostic factors applied to date still lack satisfactory predictive value. This investigation was conducted on 230 bladder tumours with a known prognosis in order to improve prognostication methods. A new modified technique for the measurement of the DNA content in formalin-fixed paraffin-embedded bladder tumour tissue was developed, by introducing a new enzyme and density gradient centrifugation before performing flow cytometry. The results were comparable with those obtained by analysing fresh or frozen tissue. DNA ploidy was found to correlate positively to stage, histological grade and progression. A methenamine-silver staining technique, which facilitates the identification of mitoses, was developed and applied for determining mitotic frequency and density. The results proved to yield prognostic markers superior to subjective grading. Intermediate grade tumours could be separated into two prognostically highly different groups. Reduced PCNA antigenicity in archival, formalin-fixed, paraffin-embedded bladder tumour tissue was observed. Antigen expression could be retrieved by heating the slides in distilled water in a microwave oven. The application of a citrate buffer could further improve the staining, which correlated to prognosis but had no independent prognostic value. Image analysis was applied in an attempt to develop computer-based grading systems. Analysis of Feulgen-stained tissue sections was performed by an object-based method, digitizing, segmenting and analyzing the images automatically. Furthermore, a texture analysis method, which estimated grey-scale co-occurrence probability matrices on digitized images, was developed; both types of grading were found to correlate significantly to subjective grading.