The long term objectives of this research are to develop a system of biomarkers that can be used by the urologist as an adjunct to cystoscopy in monitoring for recurrence and to aid in the therapeutic management decision. The potential for recurrence and for progression of recurrent disease lies in the phenotype of cells of the bladder field, while the potential for metastasis and invasion lies within the genome of the tumor itself. A technique for determining profiles of markers on tumor, the adjacent field from which it arose and from distant fields within the bladder has been developed using """"""""touch preps"""""""" of tissue obtained at surgery and will be used to identify markers within the bladder field. These measurements will directly establish the profile of markers existing at surgery. Markers will be measured using quantitative fluorescence image analysis, which can measure multiple markers on single cells in relation to morphology. The markers to be measured are as follows. (1) Morphology, is a documented marker for undifferentiated tumors and serves as the reference point for integration of biochemical markers. (2) DNA ploidy, including stem cell ploidy and the presence of cells with greater than 5C DNA predict both aggressive tumors and recurrence, (3) A tumor-related antigen, p3OO detected by the M344 antibody of Y. Fradet is expressed by low-grade tumor cells and premalignant lesions, (4) F- and G-actin are early differentiation-related markers that show a high correlation with bladder cancer risk (p less than 0.001), (5) pl85, the product of the neu oncogene and (6) epidermal growth factor receptor (EGFR) participate in growth control. (7) The product of the p53, the product of the p53 tumor suppressor gene regulates cell division and differentiation. (8) Metastasis markers such as collagenase IV seem to be products of metastatic cells. (9) Deletions or duplications of chromosomes 1, 7, 9, 11 and 17 represent events resulting from genetic instability. Whenever evidence of p53 mutation is seen and the amount of tumor tissue permits, the p53 locus will be selectively amplified by polymerase chain reaction and sequenced. Patients will also be studied longitudinally with marker measurements in bladder wash and voided urine samples to correlate indirect exfoliative cytology with direct field measurements and to determine how the above markers correlate with recurrence. Two models will be used. The stratified risk study involves measuring the marker in a cross section of patients and controls with a graded risk of developing disease and with other urologic conditions to rapidly evaluate how markers fall within the time frame of development of cancer, determine the correlations among markers and their sensitivity and specificity to various endpoints. Markers will also be further validated in longitudinal studies with continued followup with endpoints of survival, recurrence, occurrence and response to therapy to firmly establish those that can be used as biochemical intermediate end-point markers for clinical end-points. The data will be analyzed to develop classification schemas that can improve patient management by predicting occurrence, recurrence, progression and response to therapy.
