In this large population-based study of bladder cancer tumor heterogeneity, we examined changes in cell cycling as a function of tumor grade, stage, and patient characteristics including cigarette smoking to elucidate the specific role of aberrant cell cycling in urothelial carcinomas. To our knowledge, this is the largest study of well-characterized incident cases of bladder cancer that analyzed a comprehensive set of key cell-cycle proteins using novel automated scoring methods.
Our results show that aberrant protein expression of p53, p63, p16, and Ki-67, but not Rb or cyclin D1, correlates with tumor stage and grade. Of the cell-cycle proteins examined in this study, p53 showed the most heterogenic expression pattern with respect to tumor stage and grade, especially when stratified for different staining intensities using novel digital image analysis techniques. Whereas overall expression of p53 was not associated with tumor stage/grade as described in previous smaller studies (20
), we showed a significant relationship with staining intensity. One smaller study by Kelsey and colleagues suggested that the histologic character of p53 staining, especially the intensity, is more indicative of bladder cancer aggressiveness than the percentage of p53 positive nuclei (19
). Applying novel, automated, digital image analysis, we were able to stratify p53 expression for staining intensity and showed that weak p53 expression was significantly higher in low-stage/grade tumors (P
< 0.0001), whereas strong p53 and moderate-to-strong expression were higher in high-stage/grade tumors (P
< 0.0001 and P
= 0.01, respectively). These findings underscore the need for the development of p53 analysis protocols that take into account p53 levels and intensity.
Other cell-cycle molecules associated with stage and grade were p63, p16, and Ki-67. We found an inverse association between p63 expression and tumor stage/ grade similar to the findings of previous studies (23
). Although the exact targets of p63 are unknown, it is thought to regulate the cell cycle through p53-dependent pathways (27
). Several studies have suggested a role for tumor suppressor genes Rb and p16 in bladder cancer progression, by showing that functional inactivation of Rb and p16 is associated with progression of bladder cancer to more aggressive disease(12
), where as expression of p16 is associated with a better outcome in low-grade/ stage bladder cancers (13
). Consistent with these data, we found that p16 expression significantly decreased with increasing tumor stage/grade. However, we identified no significant association between Rb expression and any of the histopathologic factors tested. Previous univariate studies have suggested that cyclin D1 may be used as an inverse indicator for the level of invasiveness in bladder cancer, but multivariate analysis showed that cyclin D1 alone was not an independent prognostic marker (15
). Our results are in agreement with these previous studies in that cyclin D1 expression decreased withtumorstage/gradeinunivariate(datanotshown)but not multivariate models. The cell-cycle molecule with the highest RR associated with high tumor stage/grade analyzed in our study was Ki-67, a known predictor of tumor stage and grade in bladder cancer (16
). Together, these results show significant aberration of cell cycling in the progression of urothelial carcinoma.
Cigarette smoking is a well-established risk factor for bladder cancer and resulted in a 2- to 3-fold increased risk compared with nonsmokers (18
). However, it remains unclear to date whether smoking modifies cell cycling. A number of previous studies have examined the association between smoking and p53 protein alterations, but results have been inconsistent. Two studies found direct relationships between p53 nuclear accumulation and smoking status and smoking intensity (32
), whereas more recent studies have found no association (9
). Such inconsistencies may be attributed to insufficient power and to difficulty in differentiating staining intensities, which was improved by our use of an automated image analysis approach. Taking advantage of our large study population with detailed, historical smoking information, we found that smoking, perhaps the most important bladder cancer risk factor in Western populations, did not modify the expression of any of the key cell-cycle molecules examined. We observed that protein expression was virtually similar between smokers and never-smokers and across additional exposure indices presented in this analysis. These results indicate that smoking does not modify protein expression of key cell-cycle molecules that are documented to play important roles in bladder cancer progression. Therefore, the increased risk of bladder cancer in smokers identified in epidemiologic studies could not be accounted for by aberrant expression of cell-cycle proteins alone. p53 alterations observed in other studies may be induced by a combination of several endogenous and exogenous carcinogens and/or other molecular targets may be affected by the carcinogens in the cigarette smoke.
There were significantly more cases excluded from VT than from ME (P < 0.001), largely due to differences in tissue retrieval rates. However, this difference in the proportion of cases from the 2 states did not bias the distribution of demographic and tumor characteristics or the smoking variables (). Although differences by region were not observed for p16 and Rb protein expression in the univariate analysis, they were found in the multivariant model adjusted for age, gender, race, smoking status, and stage/grade. This finding may be due to additional differences in case characteristics that we were unable to evaluate in this study, such as occupational and environmental exposures or level of education.
In summary, the findings from our large and well-characterized population-based study of histologically confirmed urothelial carcinomas showed significant heterogeneity in protein expression of cell-cycle proteins that are predictive of disease progression. Further studies are needed to assess the molecular mechanisms in bladder cancer associated with various environmental exposures and to further elucidate their role in bladder tumorigenesis.