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Expression of p27Kip1 in bladder cancers: immunohistochemical study and prognostic value in a series of 95 cases
Cancer Letters 186 (2002) 115–120 www.elsevier.com/locate/canlet
Expression of p27 Kip1 in bladder cancers: immunohistochemical study and prognostic value in a series of 95 cases Laetitia Lacoste-Collin, Anne Gomez-Brouchet, Ghislaine Escourrou, Marie-Bernadette Delisle, Thierry Levade, Emmanuelle Uro-Coste* Department of Pathology, and INSERM U466 unit, CHU Rangueil, 31403 Toulouse Cedex 4, France Received 10 April 2002; accepted 21 May 2002
Abstract Prognostic value of p27 Kip1 immunohistochemical expression was evaluated in a series of 95 bladder carcinomas. Low p27 Kip1 expression was correlated with higher tumor grade (P ¼ 0:01) and stage (P ¼ 0:009), associated with poor overall survival (P ¼ 0:01) and, for superficial cancers, with disease-free survival (P ¼ 0:05). Thirty-five cases exhibited a heterogeneous expression related in some instances to tumoral architecture. Seventeen cases showed a cytoplasmic reactivity related to low nuclear expression (P ¼ 0:057). Loss of p27 Kip1 expression is a pejorative event in bladder tumors and inhibition of p27 Kip1 degradation could offer new therapeutic ways. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bladder cancer; p27 Kip1; Prognostic value; Heterogeneous immunostaining
1. Introduction Transitional cell carcinoma of the bladder is the second most prevalent cancer among middle-aged and elderly men [1]. Superficial bladder cancers, which are localized in the mucosal layer (pTa) or invade the lamina propria (pT1), have mostly a good prognosis but present a high risk of relapse (50–70%). Moreover, about 30% of these patients develop tumors that invade the muscularis propria (pT2) or beyond, and present a poorer prognosis [2]. Although pathological grade and stage are of clinical help in bladder cancer therapeutic management, reliable prognostic markers are still lacking. It has been reported that reduced expression of p27 Kip1 predicts
* Corresponding author. Tel.: 133-5-6132-3387; fax: 133-56132-2127. E-mail address: [email protected] (E. Uro-Coste).
poor survival in breast, colon, prostate, and lung cancers [3–6]. p27 Kip1 is a cdk inhibitor that regulates progression from G1 into phase S by binding to the cyclin E-cdk2 complex and inhibiting this kinase [7]. Mutations of the human p27 Kip1 gene is a rare event in human primary tumors, including bladder cancer [8]. The decreased expression of p27 Kip1 is due to posttranscriptional events and has been associated with increased proteasome-mediated degradation in colon and lung cancer [4,6]. Few independent studies have suggested a prognostic value of p27 Kip1 expression in primary bladder cancer but these findings are not uniformly observed [9–13]. In lymphoma and brain tumors, a strong inverse correlation between p27 Kip1 and Ki67 staining has been observed, but in most epithelial tumors p27 Kip1 levels do not appear to correlate with cell proliferation [14]. Decrease of p27 Kip1 levels might confer an anchorage-independent growth
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00319-1
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L. Lacoste-Collin et al. / Cancer Letters 186 (2002) 115–120
to tumor cells, rather than just restoring cell cycle progression [15]. This present study aimed at re-evaluating p27 Kip1 prognostic value in bladder cancers, and investigating characteristics of its immunohistochemical expression.
disease. Tissue specimens were obtained from the Department of Pathology, Rangueil Hospital. Histological grading and staging were assessed according to WHO and Tumor-Node-Metastasis (TNM) classifications, respectively [16,17]. Other clinical and pathological parameters as tumoral size, number of tumors and nodal status, were also recorded and are indicated in Table 1.
2. Materials and methods 2.2. Immunohistochemistry 2.1. Patient characteristics and tissue samples A cohort of 95 patients who underwent routine surgery for bladder cancer at the Division of Urology, Rangueil Hospital (Toulouse, France) between January 1989 and December 1990 was investigated for this study. All of the patients underwent transurethral resection for bladder tumor. The series included 73 men (77%) and 22 women (23%), with a mean age at diagnosis of 69 years (range 41–93) and a mean follow-up of 52 months (range 1–140). The followup of patients was conducted according to standard clinical practice. Nineteen patients presented local relapses, 18 patients developed distant metastasis and 21 patients died of their malignant bladder
Immunohistochemical analysis was performed on routinely processed, Dubosq fixative, paraffinembedded tissues using a streptavidin–biotin immunoperoxidase technique. Five-micrometer representative tissue sections were used. Sections were deparaffinized and rehydrated. Antigenic retrieval was processed by submerging in an EDTA buffer (10 mM; pH 8) and microwaving (25 min at 800 W). Sections were then treated to block endogenous peroxidase activity and nonspecific sites with H2O2 (1.5%; 5 min) and bovine serum (PBS buffer, 20 min), respectively. The primary monoclonal antip27 Kip1 antibody (1:800, Transduction Laboratories, clone 57) was incubated for 2 h at room temperature.
Table 1 Clinical and pathological data Parameter
Tumoral size # 3 cm . 3 cm Number of tumors Single Multiple Pathological T-stage pTa and pT1 pT2 and pT3 Tumor grade I and II III Nodal status No N1 P27 Kip1 cytoplasmic staining No Yes a
n.s., not significant.
Total cases
Mean percentage of p27 Kip1 positive nuclei (^SD)
61 (64%) 34 (36%)
41.9 (^22.1) 41.8 (^22.4)
68 (71.5%) 27 (28.5%)
39 (^20.8) 47.5 (^23.5)
56 (59%) 39 (41%)
46.8 (^22.35) 34.9 (^20.16)
48 (50.5%) 47 (49.5%)
47.3 (^23.3) 36.4 (^19.6)
22 (23.7%) 7 (7.5%)
37.8 (^19.1) 35.4 (^18.4)
78 (82.1%) 17 (17.9%)
42.8 (^23.2) 37.8 (^16.5)
P value a
n.s.
n.s.
0.009
0.01
n.s.
0.057
L. Lacoste-Collin et al. / Cancer Letters 186 (2002) 115–120
After washing, immunostaining was performed using a streptavidin–biotin immunoperoxidase and diaminobenzidine kit (Histostainplus kit, Zymed). Sections were then counterstained with 1% modified Harris hematoxylin. As a negative control, a duplicate section of each tissue sample was immunostained in the absence of the primary antibody. A lymph node with reactive follicles served as a positive control. A strong nuclear staining of lymphocytes and normal urothelium provided an internal positive control for preservation of the p27 Kip1 immunogenicity in most of examined sections. Within tumor cells, the staining reaction for p27 Kip1 was in some cases localized both in the nucleus and the cytoplasm, but the nuclear staining was in most of the cases exclusive. Cell counts were performed at 400£ magnification using a 10 £ 10 counting grid via an eyepiece by two independent observers (L.L.C and E.U.C). Ten different representative fields were counted by each observer, and an index of nuclear p27 Kip1 expression was calculated as a percentage (number of positively immunostained nuclei/total number of tumor nuclei). The presence of cytoplasmic reactivity was noticed. 2.3. Statistical analysis The association between p27 Kip1 expression and other variables shown in Table 1 was analyzed using contingence table methods and tested for significance using the Pearson’s x 2 test. Survival curves were calculated using the Kaplan–Meier method, and the log-rank test was used for the analysis. Patients who died of other cause than bladder cancer during the follow-up were considered as censored data in the survival analysis. Various cut-off points of p27 Kip1 nuclear expression were analyzed. Multivariate analysis was performed using the stepwise Cox regression model. All results were considered statistically significant when the P was less than 0.05. 3. Results 3.1. Characteristics of p27 Kip1 immunostaining Strong expression of p27 Kip1 was consistently noted in nuclei of normal urothelium and there was a consistent but variable reduction in the number of p27 Kip1 stained nuclei in tumoral tissue. p27 Kip1 labeling index
117
ranged from 1 to 91% (Fig. 1A, B). Although most cases showed homogeneous immunostaining, 35 cases (37%) exhibited a heterogeneous expression of p27 Kip1 which was, in some instances, related to tumoral architecture. Five cases of superficial papillary carcinoma displayed high expression of p27 Kip1 in superficial cell layers and low expression in basal cell layers. Nine cases of invasive carcinoma showed a loss of expression of p27 Kip1 in the deepest component compared to the surface of the tumor (Fig. 1C). In most cases (82%), staining was confined to the nucleus. Nevertheless, in 17 cases a cytoplasmic reactivity was also noted (Fig. 1D). This cytoplasmic staining tended to be related to low nuclear expression (P ¼ 0:057) (Table 1). 3.2. Expression of p27 Kip1 correlates with tumor grade and tumor stage The data collected in Table 1 show that low p27 Kip1 expression was strongly associated with increased stage (P ¼ 0:009) when tumors were separated in two groups: superficial (pTa and pT1) versus invasive (pT2 and pT3) tumors. Likewise, low p27 Kip1 expression was strongly correlated with high grade (P ¼ 0:01) when comparing well and moderately differentiated tumors (grades I and II) and poorly differentiated tumors (grade III). On the other hand, there was no relationship between p27 Kip1 expression and other prognostic factors like tumoral size and the number of tumoral localizations. 3.3. Prognostic significance of p27 Kip1 expression The Kaplan–Meier curve of disease-free survival showed that low expression of p27 Kip1 ð#50% of stained nuclei) indicated a higher risk of relapse (P ¼ 0:05) for the patients presenting superficial bladder carcinoma (Fig. 2A). In terms of overall survival for all stages of tumors, patients presenting less than 60% of stained nuclei had a worse prognosis (P ¼ 0:001) (Fig. 2B). In multivariate analysis p27 Kip1 expression appeared not to be an independent prognostic factor. 4. Discussion We have found that a reduced expression of p27 Kip1
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Fig. 1. p27 Kip1 immunostaining in four representative cases of transitional bladder carcinoma. (A) More than 90% of neoplastic cells show a strong nuclear staining (high expression) ( £ 160). (B) Less than 1% of nuclei are stained (low expression). A strong nuclear staining of lymphocytes (arrow) provided a useful internal positive control for preservation of the p27 Kip1 immunogenicity ( £ 400). (C) High expression in superficial papillary tumoral component lay above a loss of expression in the deeper component. Arrow indicates positive lymphocytes identified at higher magnification ( £ 160). (D) In some areas, a distinct cytoplasmic staining is observed without nuclear expression (arrow). Immunostaining restricted to nuclei is observed in reactive lymphocytes (arrowhead) ( £ 400).
is correlated with tumor stage (P ¼ 0:009), grade (P ¼ 0:01), disease-free survival in superficial bladder cancers (P ¼ 0:05) and overall survival in all stages (P ¼ 0:01). These results are in agreement with previous data on the correlation between p27 Kip1 expression and tumor stage and grade in bladder cancers [9,10,12,13]. We have also observed that a low expression level of p27 Kip1 is a good indicator of relapse in superficial tumors and of bad prognosis in superficial and invasive tumors. Nevertheless, in our series p27 Kip1 is not an independent prognostic factor and brings no benefit as compared to grade and stage. However, it may have some interest in specific tumor
subgroups, then it would be interesting to evaluate the prognostic role of p27 Kip1 in a larger cohort of superficial tumors, especially pT1 grade III, a group with varying patient outcome and a challenging therapeutic management. Recently, Sgambato et al. [18] reported that the simultaneous presence of low cyclin-D1, low p27 Kip1 and high Ki67 expression defined a ‘high-risk’ group of patient with papillary superficial cancer. The heterogeneity of p27 Kip1 immunostaining has already been reported for several types of tumors [3,19]. In bladder cancer, heterogeneous immunostaining has been observed in one study [10]. This heterogeneous labeling seems to be prominent in
L. Lacoste-Collin et al. / Cancer Letters 186 (2002) 115–120
bladder tumors. We have observed a heterogeneous staining in 56% of the invasive cases. Some tumors expressed lower p27 Kip1 levels in undifferentiated areas, but regardless to the cytological appearance of tumor cells, some areas in the deepest part of the tumor lost p27 Kip1 expression (23% of invasive carcinoma). These findings suggest that the loss of p27 Kip1 expression is involved in the potential of tumor inva-
Fig. 2. Survival curves. Kaplan–Meier curves for disease-free survival of superficial tumors (A) and overall survival (B) in 95 patients who underwent surgery for primary bladder carcinomas, stratified according to p27 Kip1 expression.
119
sion. Indeed, activation of the cell cycle is not the main mechanism by which p27 Kip1 acts in tumor cells, as expression of p27 kip1 and proliferation rate are not correlated in most epithelial tumors [3,4,14]. In fact, the loss of p27 Kip1 expression may provide the cells the ability to grow without any anchorage. In a murine mammary carcinoma cell line, overexpression of E-cadherin leads to increased p27 Kip1 levels that inhibit cell proliferation and decrease invasion potential [15]. Moreover, Thomas et al. have observed a reduction of p27 Kip1 expression in metastasis of colorectal carcinoma compared to primitive tumors [20]. These findings may indicate that loss of p27 Kip1 expression confers to tumoral epithelial cells the ability to survive without anchorage and provide the tumor cells the opportunity to invade any tissue and to develop metastases. In our tumor series, most of tumors displayed a p27 Kip1 expression restricted to nuclei. However, in 18% of cases, we have observed a cytoplasmic staining which tends to be related with a loss of nuclear expression (P ¼ 0:057). These observations suggest that the decrease of nuclear p27 Kip1 expression is due to cytoplasmic translocation. Indeed, cytoplasmic sequestration of p27 Kip1 by various proteins has been proposed. These proteins include c-myc and the viral oncoprotein E7 of human papillomavirus [21,22]. Moreover, the overexpression of p38 in mammalian cells causes the translocation of p27 Kip1 from the nucleus to the cytoplasm, decreasing the amount of p27 Kip1 in the cell by accelerating its proteasomedependent degradation [23]. Two independent studies reported the presence of cytoplasmic staining in bladder cancer [9,10]. We have not observed a prognostic value for cytoplasmic expression, whereas in other types of tumors, such as colorectal cancer, cytoplasmic expression of p27 Kip1 has been claimed to be related to poor prognosis [24]. In bladder cancers, p27 Kip1 appears to have a prognostic value as it has been demonstrated in others tumors, but is not an independent factor in our study. Nevertheless, it would be interesting to study the role of p27 Kip1 in a larger series of stage pT1 and grade III tumors that represent the most challenging bladder tumor to treat. The heterogeneous immunohistochemical expression of p27 Kip1 observed in the different parts of an invasive tumor suggests a potential role for this protein in maintaining cell prolifera-
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tion in a dependent cell contact-manner. The loss of expression of this protein may allow the cells to proliferate without control of adhesion molecules. Acknowledgements We would like to thank Dr. Jean-Paul Charlet for statistical analysis, and Mr. J.C. Thiers and Mr. M. Mathieu for technical assistance. References [1] R.T. Greenlee, T. Murray, S. Bolden, P.A. Wingo, Cancer statistics, 1999, CA Cancer J. Clin. 50 (2000) 7–33. [2] C.R. Hayter, L.F. Paszat, P.A. Groome, K. Schulze, W.J. Mackillop, The management and outcome of bladder carcinoma in Ontario, 1982–1994, Cancer 89 (2000) 142–151. [3] C. Catzavelos, N. Bhattacharya, Y.C. Ung, J.A. Wilson, L. Roncari, C. Sandhu, Decreased levels of the cell-cycle inhibitor p27/kip1 protein: prognostic implications in primary breast cancer, Nat. Med. 3 (1999) 227–230. [4] M. Loda, B. Cukor, S.W. Tam, P. Lavin, M. Fiorentino, G.F. Draetta, Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinoma, Nat. Med. 3 (1997) 231–234. [5] J. Tsihlias, L. Kapusta, G. DeBoer, I. Morava-Protzner, I. Zbieranowski, N. Bhattacharya, Loss of cyclin-dependent kinase inhibitor p27/kip1 is a novel prognostic factor in localized human prostate adenocarcinoma, Cancer Res. 58 (1998) 542–548. [6] V. Esposito, A. Baldi, A. De Luca, A.M. Groger, M. Loda, G.G. Giordano, Prognostic role of the cyclin-dependent kinase inhibitor p27 in non-small cell lung cancer, Cancer Res. 57 (1997) 3381–3385. [7] C.J. Sherr, Mammalians G1 cyclins, Cell 73 (1993) 1059– 1065. [8] J. Serth, M. Kuczyk, S. Machtens, C. Bokemeyer, R. Herrmann, J. Hartmann, Analysis of the cyclin-dependent kinase inhibitor p27/kip1 in muscle invasive bladder cancer, Oncol. Rep. 6 (1999) 229–233. [9] P. Korkolopoulou, P. Christodoulou, A.E. Konstantinidou, E. Thomas-Tsagli, P. Kapralos, P. Davaris, Cell cycle regulators in bladder cancer: a multivariate survival study with emphasis on p27/kip1, Hum. Pathol. 31 (2000) 751–760. [10] A. Sgambato, M. Migaldi, B. Faraglia, L. Garagnani, G. Romano, C. De Gaetani, Loss of p27/kip1 expression correlates with tumor grade and with reduced disease-free survival in primary superficial bladder cancers, Cancer Res. 59 (1999) 3245–3250.
[11] M.A. Kuczyk, S. Machtens, C. Bokemeyer, K. Hradil, I. Macheel, J. Hartmann, Prognostic value of p27/kip1 and p21/WAF/Cip protein expression in muscle invasive bladder cancer, Oncol. Rep. 6 (1999) 687–689. [12] K.H. Franke, M. Miklosi, P. Goebell, S. Clasen, C. Steinhoff, A.G. Anastasiadis, Cyclin-dependent kinase inhibitor p27/ kip1 is expressed preferentially in early stages of urothelial carcinomas, Urology 56 (2000) 689–695. [13] J.J. Del Pizzo, A. Borkowski, S.C. Jacobs, N. Kyprianou, Loss of cell cycle regulators p27/kip1 and cyclin E in transitional cell carcinoma of the bladder correlates with tumor grade and patient survival, Am. J. Pathol. 155 (1999) 1129–1136. [14] J. Slingerland, M. Pagano, Regulation of the Cdk inhibitor p27 and its deregulation in cancer, J. Cell Physiol. 183 (2000) 10– 17. [15] B. Saint-Croix, C. Sheehan, J.W. Rak, V.A. Florenes, J.M. Slingerland, R.S. Kerbel, E-cadherin-dependent growth suppression is mediated by cyclin-dependent kinase inhibitor p27/kip1, J. Cell Biol. 142 (1998) 557–571. [16] F.K. Mostofi, C.J. Davis Jr, I.A. Sesterhenn, WHO Histological Typing of Urinary Bladder Tumors, Springer, Berlin, 1999. [17] P. Hermanek, R.V.P. Hutter, L.H. Sobin, G. Wagner, Ch. Wittekind, TNM Atlas. Illustrated Guide to the TNM/pTNM Classification of Malignant Tumours, Springer, Berlin, 1997. [18] A. Sgambato, M. Migaldi, B. Faraglia, G. De Aloysio, P. Ferrari, R. Ardito, C. De Gaetani, G. Capelli, A. Cittadini, G.P. Trentini, Cyclin D1 expression in papillary superficial bladder cancer: its association with other cell cycle-associated proteins, cell proliferation and clinical outcome, Int. J. Cancer. 97 (2002) 671–678. [19] Y. Kudo, T. Takata, W. Yasui, I. Ogawa, M. Miyauchi, T. Takekoshi, Reduced expression of cyclin-dependent kinase inhibitor p27/kip1 is an indicator of malignant behavior in oral squamous cell carcinoma, Cancer 83 (1998) 2447–2455. [20] G.V. Thomas, K. Szigeti, M. Murphy, G. Draetta, M. Pagano, M. Loda, Down-regulation of p27 is associated with development of colorectal adenocarcinoma metastases, Am. J. Pathol. 153 (1998) 681–687. [21] J. Vlach, S. Hennecke, K. Alevizopoulos, D. Conti, I.B. Amat, Growth arrest by the cyclin-dependent kinase inhibitor p27/ kip1 is abrogated by c-myc, EMBO J. 15 (1996) 6595–6604. [22] K. Zerfass-Thome, W. Zwerschke, B. Mannhardt, R. Tindle, J.W. Botz, P. Jansen-Du¨ rr, Inactivation of the cdk inhibitor p27/kip1 by the human papillomavirus type 16 E7 oncoprotein, Oncogene 13 (1996) 2323–2330. [23] K. Tomoda, Y. Kubota, J.Y. Kato, Degradation of the cyclindependent-kinase inhibitor p27/kip1 is instigated by Jab1, Nature 398 (1999) 160–164. [24] K. Gunther, A. Jung, U. Volker, M. Meyer, T. Brabletz, K.E. Matzel, p27/kip1 expression in rectal cancer correlates with disease-free survival, J. Surg. Res. 92 (2000) 78–84.
Expression of p27 Kip1 in bladder cancers: immunohistochemical study and prognostic value in a series of 95 cases Laetitia Lacoste-Collin, Anne Gomez-Brouchet, Ghislaine Escourrou, Marie-Bernadette Delisle, Thierry Levade, Emmanuelle Uro-Coste* Department of Pathology, and INSERM U466 unit, CHU Rangueil, 31403 Toulouse Cedex 4, France Received 10 April 2002; accepted 21 May 2002
Abstract Prognostic value of p27 Kip1 immunohistochemical expression was evaluated in a series of 95 bladder carcinomas. Low p27 Kip1 expression was correlated with higher tumor grade (P ¼ 0:01) and stage (P ¼ 0:009), associated with poor overall survival (P ¼ 0:01) and, for superficial cancers, with disease-free survival (P ¼ 0:05). Thirty-five cases exhibited a heterogeneous expression related in some instances to tumoral architecture. Seventeen cases showed a cytoplasmic reactivity related to low nuclear expression (P ¼ 0:057). Loss of p27 Kip1 expression is a pejorative event in bladder tumors and inhibition of p27 Kip1 degradation could offer new therapeutic ways. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bladder cancer; p27 Kip1; Prognostic value; Heterogeneous immunostaining
1. Introduction Transitional cell carcinoma of the bladder is the second most prevalent cancer among middle-aged and elderly men [1]. Superficial bladder cancers, which are localized in the mucosal layer (pTa) or invade the lamina propria (pT1), have mostly a good prognosis but present a high risk of relapse (50–70%). Moreover, about 30% of these patients develop tumors that invade the muscularis propria (pT2) or beyond, and present a poorer prognosis [2]. Although pathological grade and stage are of clinical help in bladder cancer therapeutic management, reliable prognostic markers are still lacking. It has been reported that reduced expression of p27 Kip1 predicts
* Corresponding author. Tel.: 133-5-6132-3387; fax: 133-56132-2127. E-mail address: [email protected] (E. Uro-Coste).
poor survival in breast, colon, prostate, and lung cancers [3–6]. p27 Kip1 is a cdk inhibitor that regulates progression from G1 into phase S by binding to the cyclin E-cdk2 complex and inhibiting this kinase [7]. Mutations of the human p27 Kip1 gene is a rare event in human primary tumors, including bladder cancer [8]. The decreased expression of p27 Kip1 is due to posttranscriptional events and has been associated with increased proteasome-mediated degradation in colon and lung cancer [4,6]. Few independent studies have suggested a prognostic value of p27 Kip1 expression in primary bladder cancer but these findings are not uniformly observed [9–13]. In lymphoma and brain tumors, a strong inverse correlation between p27 Kip1 and Ki67 staining has been observed, but in most epithelial tumors p27 Kip1 levels do not appear to correlate with cell proliferation [14]. Decrease of p27 Kip1 levels might confer an anchorage-independent growth
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00319-1
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to tumor cells, rather than just restoring cell cycle progression [15]. This present study aimed at re-evaluating p27 Kip1 prognostic value in bladder cancers, and investigating characteristics of its immunohistochemical expression.
disease. Tissue specimens were obtained from the Department of Pathology, Rangueil Hospital. Histological grading and staging were assessed according to WHO and Tumor-Node-Metastasis (TNM) classifications, respectively [16,17]. Other clinical and pathological parameters as tumoral size, number of tumors and nodal status, were also recorded and are indicated in Table 1.
2. Materials and methods 2.2. Immunohistochemistry 2.1. Patient characteristics and tissue samples A cohort of 95 patients who underwent routine surgery for bladder cancer at the Division of Urology, Rangueil Hospital (Toulouse, France) between January 1989 and December 1990 was investigated for this study. All of the patients underwent transurethral resection for bladder tumor. The series included 73 men (77%) and 22 women (23%), with a mean age at diagnosis of 69 years (range 41–93) and a mean follow-up of 52 months (range 1–140). The followup of patients was conducted according to standard clinical practice. Nineteen patients presented local relapses, 18 patients developed distant metastasis and 21 patients died of their malignant bladder
Immunohistochemical analysis was performed on routinely processed, Dubosq fixative, paraffinembedded tissues using a streptavidin–biotin immunoperoxidase technique. Five-micrometer representative tissue sections were used. Sections were deparaffinized and rehydrated. Antigenic retrieval was processed by submerging in an EDTA buffer (10 mM; pH 8) and microwaving (25 min at 800 W). Sections were then treated to block endogenous peroxidase activity and nonspecific sites with H2O2 (1.5%; 5 min) and bovine serum (PBS buffer, 20 min), respectively. The primary monoclonal antip27 Kip1 antibody (1:800, Transduction Laboratories, clone 57) was incubated for 2 h at room temperature.
Table 1 Clinical and pathological data Parameter
Tumoral size # 3 cm . 3 cm Number of tumors Single Multiple Pathological T-stage pTa and pT1 pT2 and pT3 Tumor grade I and II III Nodal status No N1 P27 Kip1 cytoplasmic staining No Yes a
n.s., not significant.
Total cases
Mean percentage of p27 Kip1 positive nuclei (^SD)
61 (64%) 34 (36%)
41.9 (^22.1) 41.8 (^22.4)
68 (71.5%) 27 (28.5%)
39 (^20.8) 47.5 (^23.5)
56 (59%) 39 (41%)
46.8 (^22.35) 34.9 (^20.16)
48 (50.5%) 47 (49.5%)
47.3 (^23.3) 36.4 (^19.6)
22 (23.7%) 7 (7.5%)
37.8 (^19.1) 35.4 (^18.4)
78 (82.1%) 17 (17.9%)
42.8 (^23.2) 37.8 (^16.5)
P value a
n.s.
n.s.
0.009
0.01
n.s.
0.057
L. Lacoste-Collin et al. / Cancer Letters 186 (2002) 115–120
After washing, immunostaining was performed using a streptavidin–biotin immunoperoxidase and diaminobenzidine kit (Histostainplus kit, Zymed). Sections were then counterstained with 1% modified Harris hematoxylin. As a negative control, a duplicate section of each tissue sample was immunostained in the absence of the primary antibody. A lymph node with reactive follicles served as a positive control. A strong nuclear staining of lymphocytes and normal urothelium provided an internal positive control for preservation of the p27 Kip1 immunogenicity in most of examined sections. Within tumor cells, the staining reaction for p27 Kip1 was in some cases localized both in the nucleus and the cytoplasm, but the nuclear staining was in most of the cases exclusive. Cell counts were performed at 400£ magnification using a 10 £ 10 counting grid via an eyepiece by two independent observers (L.L.C and E.U.C). Ten different representative fields were counted by each observer, and an index of nuclear p27 Kip1 expression was calculated as a percentage (number of positively immunostained nuclei/total number of tumor nuclei). The presence of cytoplasmic reactivity was noticed. 2.3. Statistical analysis The association between p27 Kip1 expression and other variables shown in Table 1 was analyzed using contingence table methods and tested for significance using the Pearson’s x 2 test. Survival curves were calculated using the Kaplan–Meier method, and the log-rank test was used for the analysis. Patients who died of other cause than bladder cancer during the follow-up were considered as censored data in the survival analysis. Various cut-off points of p27 Kip1 nuclear expression were analyzed. Multivariate analysis was performed using the stepwise Cox regression model. All results were considered statistically significant when the P was less than 0.05. 3. Results 3.1. Characteristics of p27 Kip1 immunostaining Strong expression of p27 Kip1 was consistently noted in nuclei of normal urothelium and there was a consistent but variable reduction in the number of p27 Kip1 stained nuclei in tumoral tissue. p27 Kip1 labeling index
117
ranged from 1 to 91% (Fig. 1A, B). Although most cases showed homogeneous immunostaining, 35 cases (37%) exhibited a heterogeneous expression of p27 Kip1 which was, in some instances, related to tumoral architecture. Five cases of superficial papillary carcinoma displayed high expression of p27 Kip1 in superficial cell layers and low expression in basal cell layers. Nine cases of invasive carcinoma showed a loss of expression of p27 Kip1 in the deepest component compared to the surface of the tumor (Fig. 1C). In most cases (82%), staining was confined to the nucleus. Nevertheless, in 17 cases a cytoplasmic reactivity was also noted (Fig. 1D). This cytoplasmic staining tended to be related to low nuclear expression (P ¼ 0:057) (Table 1). 3.2. Expression of p27 Kip1 correlates with tumor grade and tumor stage The data collected in Table 1 show that low p27 Kip1 expression was strongly associated with increased stage (P ¼ 0:009) when tumors were separated in two groups: superficial (pTa and pT1) versus invasive (pT2 and pT3) tumors. Likewise, low p27 Kip1 expression was strongly correlated with high grade (P ¼ 0:01) when comparing well and moderately differentiated tumors (grades I and II) and poorly differentiated tumors (grade III). On the other hand, there was no relationship between p27 Kip1 expression and other prognostic factors like tumoral size and the number of tumoral localizations. 3.3. Prognostic significance of p27 Kip1 expression The Kaplan–Meier curve of disease-free survival showed that low expression of p27 Kip1 ð#50% of stained nuclei) indicated a higher risk of relapse (P ¼ 0:05) for the patients presenting superficial bladder carcinoma (Fig. 2A). In terms of overall survival for all stages of tumors, patients presenting less than 60% of stained nuclei had a worse prognosis (P ¼ 0:001) (Fig. 2B). In multivariate analysis p27 Kip1 expression appeared not to be an independent prognostic factor. 4. Discussion We have found that a reduced expression of p27 Kip1
118
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Fig. 1. p27 Kip1 immunostaining in four representative cases of transitional bladder carcinoma. (A) More than 90% of neoplastic cells show a strong nuclear staining (high expression) ( £ 160). (B) Less than 1% of nuclei are stained (low expression). A strong nuclear staining of lymphocytes (arrow) provided a useful internal positive control for preservation of the p27 Kip1 immunogenicity ( £ 400). (C) High expression in superficial papillary tumoral component lay above a loss of expression in the deeper component. Arrow indicates positive lymphocytes identified at higher magnification ( £ 160). (D) In some areas, a distinct cytoplasmic staining is observed without nuclear expression (arrow). Immunostaining restricted to nuclei is observed in reactive lymphocytes (arrowhead) ( £ 400).
is correlated with tumor stage (P ¼ 0:009), grade (P ¼ 0:01), disease-free survival in superficial bladder cancers (P ¼ 0:05) and overall survival in all stages (P ¼ 0:01). These results are in agreement with previous data on the correlation between p27 Kip1 expression and tumor stage and grade in bladder cancers [9,10,12,13]. We have also observed that a low expression level of p27 Kip1 is a good indicator of relapse in superficial tumors and of bad prognosis in superficial and invasive tumors. Nevertheless, in our series p27 Kip1 is not an independent prognostic factor and brings no benefit as compared to grade and stage. However, it may have some interest in specific tumor
subgroups, then it would be interesting to evaluate the prognostic role of p27 Kip1 in a larger cohort of superficial tumors, especially pT1 grade III, a group with varying patient outcome and a challenging therapeutic management. Recently, Sgambato et al. [18] reported that the simultaneous presence of low cyclin-D1, low p27 Kip1 and high Ki67 expression defined a ‘high-risk’ group of patient with papillary superficial cancer. The heterogeneity of p27 Kip1 immunostaining has already been reported for several types of tumors [3,19]. In bladder cancer, heterogeneous immunostaining has been observed in one study [10]. This heterogeneous labeling seems to be prominent in
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bladder tumors. We have observed a heterogeneous staining in 56% of the invasive cases. Some tumors expressed lower p27 Kip1 levels in undifferentiated areas, but regardless to the cytological appearance of tumor cells, some areas in the deepest part of the tumor lost p27 Kip1 expression (23% of invasive carcinoma). These findings suggest that the loss of p27 Kip1 expression is involved in the potential of tumor inva-
Fig. 2. Survival curves. Kaplan–Meier curves for disease-free survival of superficial tumors (A) and overall survival (B) in 95 patients who underwent surgery for primary bladder carcinomas, stratified according to p27 Kip1 expression.
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sion. Indeed, activation of the cell cycle is not the main mechanism by which p27 Kip1 acts in tumor cells, as expression of p27 kip1 and proliferation rate are not correlated in most epithelial tumors [3,4,14]. In fact, the loss of p27 Kip1 expression may provide the cells the ability to grow without any anchorage. In a murine mammary carcinoma cell line, overexpression of E-cadherin leads to increased p27 Kip1 levels that inhibit cell proliferation and decrease invasion potential [15]. Moreover, Thomas et al. have observed a reduction of p27 Kip1 expression in metastasis of colorectal carcinoma compared to primitive tumors [20]. These findings may indicate that loss of p27 Kip1 expression confers to tumoral epithelial cells the ability to survive without anchorage and provide the tumor cells the opportunity to invade any tissue and to develop metastases. In our tumor series, most of tumors displayed a p27 Kip1 expression restricted to nuclei. However, in 18% of cases, we have observed a cytoplasmic staining which tends to be related with a loss of nuclear expression (P ¼ 0:057). These observations suggest that the decrease of nuclear p27 Kip1 expression is due to cytoplasmic translocation. Indeed, cytoplasmic sequestration of p27 Kip1 by various proteins has been proposed. These proteins include c-myc and the viral oncoprotein E7 of human papillomavirus [21,22]. Moreover, the overexpression of p38 in mammalian cells causes the translocation of p27 Kip1 from the nucleus to the cytoplasm, decreasing the amount of p27 Kip1 in the cell by accelerating its proteasomedependent degradation [23]. Two independent studies reported the presence of cytoplasmic staining in bladder cancer [9,10]. We have not observed a prognostic value for cytoplasmic expression, whereas in other types of tumors, such as colorectal cancer, cytoplasmic expression of p27 Kip1 has been claimed to be related to poor prognosis [24]. In bladder cancers, p27 Kip1 appears to have a prognostic value as it has been demonstrated in others tumors, but is not an independent factor in our study. Nevertheless, it would be interesting to study the role of p27 Kip1 in a larger series of stage pT1 and grade III tumors that represent the most challenging bladder tumor to treat. The heterogeneous immunohistochemical expression of p27 Kip1 observed in the different parts of an invasive tumor suggests a potential role for this protein in maintaining cell prolifera-
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