Detection And Value Of Ki-67, CK20, P53, CyclinD1and Chromosomes3,7,17and9p21in Bladder Neoplasms

ObjectiveTo evaluate the role of immunohistochemistry and fluorescence in situ hybridization (FISH) in the differential diagnosis between bladder inverted papilloma and low grade noninvasive urothelial carcinoma (UC) with an inverted growth pattern. And then, aberrations of chromosomes3,7,17and9p21and abnormal expression of Ki-67and CyclinD1were detected in superficial UC and muscle invasive UC separately to evaluate their relationship with clinicopathologic parameters and prognosis.Materials and methodsPart174cases of urothelial lesions with endophitic growth pattern were obtained from the pathology department of Fudan University Shanghai Cancer Center and Huadong Hospital Affiliated to Fudan University including36cases of inverted papilloma and38cases of low grade noninvasive UC with an inverted growth pattern. Histological features were obtained under light microscopy. And then tissue microarrays were made. Expression of Ki-67, CK20, p53and CyclinD1was detected by immunohistochemistry. Gains of chromosomes3,7and17and homozygous loss of9p21were detected by UroVysion FISH. Differences of expression of proteins and aberrations of chromosomes between inverted papilloma and low grade noninvasive UC with an inverted growth pattern were analyzed by SPSS16.0.Part2250cases of UC were obtained from Fudan University Shanghai Cancer Center, including126cases of superficial UC (53cases of Ta and73cases of T1) and124cases of muscle invasive UC (T2-T4). Clinicopathologic and prognostic information was collected.10cases of nontumorous urothelium were used as negative controls. Gains of chromosomes3,7and17and homozygous loss of9p21were detected by UroVysion FISH. Expression of Ki-67and CyclinDl was detected by immunohistochemistry. Relationship was analyzed between aberrations of chromosomes or abnormal expression of proteins and clinicopathologic variables and prognosis in superficial UC and muscle invasive UC separately.ResultsPart11. Morphologic analysis:The inverted papilloma was mainly composed of anastomosing cords which were thin and congruous in width or nests which were small and regular. Sometimes cysts could be seen in the inferior of nests. In36cases of inverted papilloma, peripheral palisading of nuclei could be seen in31cases (86.1%) and streaming in the inferior of cords or nests could be seen in28cases (77.8%). The mitotic figure was seldom seen, only in the basal layer if there was any. The number of mitotic figure of all the cases with inverted papilloma in this study was less than1/10HPF. Cords of low grade noninvasive UC with an inverted growth pattern were usually thick and irregular and nests were usually big and inordinate. Definite fibrovascular cores could be found in17of38cases (44.7%). Mitotic figures which were common in superficial or middle layers were easy to be found,20out of38cases (52.6%)>1/10HPF in this study.2. Results of immunohistochemistry:1) Expression of Ki-67was significantly lower in inverted papilloma in which only1of36cases (2.8%) was positive than that in UC with an inverted growth pattern in which18of38cases (47.4%) were positive (P<0.001). Expression of CK20was significantly lower in inverted papilloma in which2of36cases (5.6%) were positive than that in UC with an inverted growth pattern in which14of38cases (36.8%) were positive (P=0.001). Expression of P53was lower in inverted papilloma in which10of36cases (27.8%) were positive than that in UC with an inverted growth pattern in which16of38cases (42.1%) were positive, but the difference between them was not statistically significant (P=0.230). Expression of CyclinDl was a little lower in inverted papilloma in which25of36cases (69.4%) were positive than that in UC with an inverted growth pattern in which28of38cases (73.7%) were positive, but the difference between them was not statistically significant (P=0.798).2) When Ki-67was combined with CK20for differencial diagnosis, there were3of36cases (8.3%) with one marker positive in inverted papilloma, and there were13of38cases (34.2%) with neither markers positive in UC with an inverted growth pattern. 3. Results of FISH:cases demonstrating a gain of at least2of chromosomes3,7and17or a homozygous loss of9p21were defined as UroVysion FISH positive. It was positive in24of38cases (63.2%) of UC with an inverted growth pattern while negative in all the cases of inverted papilloma.9of13(69.2%) cases which were negative for both Ki-67and CK20were positive of UroVysion FISH. When Ki-67and CK20were combined with UroVysion FISH, the sensitivity and specificity could reach89.5%and97%respectively.Part21. Association of expression of Ki-67and CyclinD1with pathologic stage and grade1) High expression of Ki-67could be seen in73of125cases (58.4%) of superficial UC and92of124cases (74.2%) of muscle invasive UC. It was significantly associated with high grade in both superficial UC and muscle invasive UC (P<0.001and P<0.001, respectively). Expression of Ki-67showed no significant association with stage in both superficial UC and muscle invasive UC (P=1.000and P=0.443, respectively).2) High expression of CyclinDl which could be seen in69of124cases (55.6%) was significantly associated with low grade in superficial UC (P<0.001). High expression of CyclinDl which could be seen in33of124cases (26.6%) showed no significant association with grade in muscle invasive UC (P=0.3). High expression of CyclinDl was significantly associated with lower stage in low grade UC (P=0.029). The difference was not significant in high grade UC (P=0.068).3) Correlation between Ki-67and CyclinDlThere was a significant inverse relationship between Ki-67and CyclinDl in superficial UC (P=0.003, r=-0.265). No significant relationship was determined in muscle invasive UC(P=0.485).2. Association of expression of Ki-67and CyclinDl with prognosis1) High expression of Ki-67and low expression of CyclinD1were significantly associated with worse disease-free survival in superficial UC (P=0.02and P<0.001, respectively). 2) Neither Ki-67nor CyclinDl showed any association with prognosis in muscle invasive UC (P=0.069and P=0.441, respectively).3. Association of gains of chromosomes3,7and17and homozygous loss of9p21with pathologic stage and grade1)24/60(40%),14/60(23.3%),13/60(21.7%) and18/60(30%) of low grade UC demonstrated gains of chromosomes3,7,17and homozygous loss of9p21, respectively. None of them was associated with pathologic stage (P=1.000, P=0.427, P=0.099and P=0.571, respectively).2)99/119(83.2%),98/119(82.4%),95/119(79.8%) and49/119(41.2%) of high grade UC demonstrated gains of chromosomes3,7,17and homozygous loss of9p21, respectively. Gains of chromosome7and homozygous loss of9p21were associated with higher pathologic stage (P=0.027and P=0.026, respectively). Chromosomes3and7showed no significant association with stage (P=0.322and P=0.108).3)60/101(59.4%),50/101(49.5%),50/101(49.5%) and31/101(30.7%) of superficial UC demonstrated gains of chromosomes3,7,17and homozygous loss of9p21, respectively. Gains of chromosomes3,7and17were associated with higher grade (P<0.001, P<0.001and P<0.001, respectively) while loss of9p21showed no significant association with grade (P=0.831).4)63/78(80.8%),62/78(79.5%),58/78(74.4%)^36/78(46.2%) of muscle invasive UC demonstrated gains of chromosomes3,7,17and homozygous loss of9p21, respectively. Gains of chromosomes3,7and17were associated with higher grade (P<0.001, P<0.001and P<0.001, respectively) while loss of9p21showed no significant association with stage (P=0.097).4. Association of gains of chromosomes3,7and17and homozygous loss of9p21with prognosis1) In superficial UC, Gains of chromosomes7and17were significantly associated with worse disease-free survival (P=0.043and P=0.016, respectively). Aberrations of chromosome3and9p21showed no prognostic significance (P=0.610and P=0.241, respectively). 2) In muscle invasive UC, Gains of chromosome17and homozygous loss of9p21were significantly associated with worse disease-free survival (P=0.013and P=0.042, respectively). Gains of chromosomes3and7showed no prognostic significance (P=0.188and P=0.116, respectively).5. Prediction of disease-free survival1) In superficial UC, high grade, multiple tumors, gains of chromosomes7and17, high expression of Ki-67and low expression of CyclinD1were associated with worse disease-free survival in univariate analysis. Multiple tumors and low expression of CyclinD1were independent predictors in multivariate analyses (P=0.036and P=0.013, respectively).2) In muscle invasive UC, high grade, high stage, gains of chromosomes17and homozygous loss of9p21were associated with worse disease-free survival in univariate analysis. Only high stage was independent in multivariate analyses (P=0.009). Patients with a stage of T3-T4had a worse prognosis than those with T2.Conclusions1. Irregular cords or nests, easily found mitotic figures and fibrovascular cores are features of low grade noninvasive UC with an inverted growth pattern.2. Immunohistochemical evaluation of Ki-67and CK20may be useful in the differential diagnosis of inverted papilloma and low grade noninvasive UC with an inverted growth pattern.3. UroVysion FISH is useful in the diagnosis of inverted papilloma and low grade noninvasive UC with an inverted growth pattern with high sensitivity and specificity. Most UC with an inverted growth pattern that are negative of both Ki-67and CK20can be detected positively by UroVysion FISH.4. Gains of chromosomes3,7and17are associated with high grade in both superficial UC and muscle invasive UC. Gains of chromosome7and homozygous loss of9p21are associated with high pathologic stage in high grade UC.5. High expression of Ki-67is associated with high grade in both superficial UC and muscle invasive UC.6. Low expression of CyclinDl is associated with high grade in superficial UC. Low expression of CyclinDl is associated with high stage in low grade UC. 7. The expressin of CyclinD1is inversely correlated with Ki-67in superficial UC.8. In superficial UC, high grade, multiple tumors, gains of chromosomes7and17, high expression of Ki-67and low expression of CyclinDl are associated with worse disease-free survival in univariate analysis. Multiple tumors and low expression of CyclinD1are independent predictors in multivariate analyses.9. In muscle invasive UC, high grade, high stage, gains of chromosomes17and homozygous loss of9p21are associated with worse disease-free survival in univariate analysis. Only high stage is independent in multivariate analyses.