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p16INK4a Expression and Clinicopathologic Parameters in Renal Cell Carcinoma
european urology 51 (2007) 732–738
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Kidney Cancer
p16INK4a Expression and Clinicopathologic Parameters in Renal Cell Carcinoma Stephen O. Ikuerowo a, Markus A. Kuczyk b, Reinhard von Wasielewski c, Olayiwola B. Shittu d, Udo Jonas a, Stefan Machtens a, Ju¨rgen Serth a,* a
Department of Urology, Medizinische Hochschule Hannover, Hannover, Germany Department of Urology, Karl-Eberhards University, Tu¨bingen, Germany c Department of Pathology, Medizinische Hochschule Hannover, Hannover, Germany d Division of Urology, University College Hospital and University of Ibadan, Ibadan, Nigeria b
Article info
Abstract
Article history: Accepted August 8, 2006 Published online ahead of print on August 23, 2006
Objectives: The tumour suppressor gene p16INK4a is a cyclin-dependent kinase inhibitor, for which inactivation attributable to promoter hypermethylation or homozygous deletion has been described in malignancies. Little is known about p16INK4a protein levels in renal cell carcinoma (RCC) and its association with clinicopathologic parameters or disease progression. Methods: The expression of the p16INK4a gene was analysed with the use of immunohistochemistry and tissue microarrays (TMA). Tissue cores were obtained from the primary tumour itself, the tumoural invasion front, and histologically benign peritumoural tissue of 397 nephrectomies. For statistical analysis, sections were classified into four groups according to the relative amount of positively stained cells: negative (0%), low (1–10%), intermediate (11–50%), and high positivity (>50%). Follow-up data were analyzed for 198 patients (follow-up period: 2–240 mo; median: 138 mo). Results: Absent or low expression of p16INK4a was observed in 82% of tumour samples. No statistically significant association was found between protein levels detected in tumour, invasion front, or normal renal tissues and any of the clinicopathologic variables. Survival analysis by Kaplan-Meier revealed a significant association between high expression (>50%) of p16INK4a in tumours and better disease-specific survival ( p = 0.03, log-rank test). Cox regression analysis showed that p16INK4a expression is an independent covariate in disease-specific survival ( p < 0.01). Conclusions: The absence of p16INK4a expression in most tumour cells indicates that p16INK4a could be involved in the tumourigenesis of RCC. Immunohistochemically detected positivity for p16INK4a is a positive prognosticator for specific survival in both uni- and multivariate analyses.
Keywords: p16INK4a Renal cell cancer Prognosis Tissue microarray analysis
# 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Medizinische Hochschule Hannover, Department of Urology, OE6247, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Tel. +49 511 5328927; Fax: +49 511 5322926. E-mail address: [email protected] (J. Serth). 0302-2838/$ – see back matter # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2006.08.010
european urology 51 (2007) 732–738
1.
Introduction
Renal cell carcinoma (RCC) represents the most prevalent malignancy of the kidney and accounts for 3% of all adult cancers in the Western world [1]. Surgical removal of organ-confined tumours provides a chance for cure, but approximately 30% of treated patients will develop metastases. RCC is a largely chemo- and radiotherapy-resistant tumour, and partial or complete response to immunobiologic therapy is observed only in a fraction of patients [2–4]. The TNM staging system is widely used for prognosis of RCC patients, and additional parameters such as Fuhrman grade, tumour size, necrosis, vascular invasion, and clinical presentation have been suggested to improve the predictability of RCC [5–7]. Cell cycle progression and cell proliferation are controlled by proteins that frequently have been described to be deregulated in various malignant diseases [8]. The tumour suppressor p16INK4a is a G1/S phase cell cycle inhibitor, which arrests cell replication by binding to the cyclin D1-CDK4/CDK6 complex and inhibition of retinoblastoma (RB) protein phosphorylation. As a result, p16INK4a promotes the formation of a RB-E2F–repressive complex that, in turn, blocks the G1/S progression of the cell cycle [9]. Moreover, it has been suggested that both cyclin D1 overexpression and p16INK4a protein alteration give rise to persistent hyperphosphorylation of RB [10]. Recently, overexpression of cyclin D1 has been described in RCC [11]. Although inactivation of the p16INK4a gene in cell lines derived from RCC attributable to promoter hypermethylation, homozygous deletion or, very rarely, point mutation has been reported, the association of p16INK4a protein levels and clinicopathologic features or prognosis of patients with RCC has not been investigated so far [12]. Therefore, the aim of the present study was to analyse the expression of p16INK4a in RCCs and to assess its prognostic significance in a sufficient number of patients. 2.
Methods
2.1.
Patients and tissue microarrays
For the setup of tissue microarrays, 397 patients who had radical nephrectomy for treatment of RCC between 1981 and 1998 at the Medizinische Hochschule Hannover, Hannover, Germany, were included. The mean age of the patients was 60 yr (range: 31–83). Tissue samples selected by the pathologist were obtained from the primary tumour, the respective invasion front, and the peritumoural, histologically normal kidney, and arranged on tissue microarray blocks (TMAs) as
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described previously [13]. The follow-up data for 238 patients were available and included in the analysis. At the time of the final follow-up update, 112 patients were still surviving; 86 patients had died as a result of the disease and 40 patients of other causes. For statistical analysis, cases with cancerindependent deaths were excluded; thus, 198 patients were considered for the follow-up group (median: 138 mo, range: 2–240 mo). The clinical and histopathologic characteristics as well as the immunohistochemical positivity of the follow-up group of patients strongly resembled the entire group of all nephrectomies (Table 1). Clinical staging and nuclear grading were performed according to the Union International Contre le Cancer (UICC) 1997 TNM classification system [14] and Fuhrman et al. [15], respectively. The TNM 2002 classification, which provides additional subclassification of the T1 group, could not be applied because of a lack of complete information on the tumour size. Histologic classification of tumours according to the Heidelberg system [16] resulted in 363 (91%) conventional, 28 (7%) papillary, and 3 (1%) chromophobic RCCs.
2.2.
Immunohistochemistry
The TMAs were immunohistochemically analysed for p16INK4a protein expression with the use of a mouse antihuman p16 monoclonal antibody (BD Biosciences, San Diego, CA, USA) as described previously [17], except that signal amplification using tyramide was omitted. The anti-p16 antibody was incubated overnight (dilution: 1:60) in a moist chamber at room temperature. Two independent investigators without knowledge of clinicopathologic and follow-up data classified the slides on the basis of the percentage of cells displaying nuclear immunoreactivity detected either in tumour cells (tumour and invasion front specimens) or in tubular cells (peritumoural renal parenchyma). After a preliminary survey of stained specimens, specific cutoff values were chosen to allow robust and efficient classification of stained specimens. Respective tissue specimens were grouped into four classes: negative (0%), low- (1–10%), intermediate- (11–50%), and high-level expression (>50%). No significant interobserver differences were detected, and investigators were blinded against each other.
2.3.
Statistical analysis
Statistical analyses were performed with the use of the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, USA). The association between p16INK4a expression and clinicopathologic characteristics was determined with the use of the chi-square test; p values 0.05 were considered statistically significant. Cases with cancer-independent deaths were not included in the study. Censored cases in Kaplan-Meier plots indicate that patients were still alive at the time of the last follow-up examination. Diseasespecific survival was statistically analysed for a follow-up period of 12 yr (144 mo) with the use of the Kaplan-Meier method and the log-rank test. The Cox proportional hazard model was used to test for the independency of prognostic parameters.
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Table 1 – Clinicopathologic data and correlation with positivity for p16INK4a Variable
All patients (N [%])
Disease-specific follow-up group (N [%])
p value (chi-square test): correlation with p16INK4a expression
Total
397
198
pT category pT1 (n = x) pT2 (n = x) pT3 (n = x) pT4 (n = x)
10 (2.5) 200 (50.4) 169 (42.6) 18 (4.5)
6 (3.0) 106 (53.5) 81 (41.0) 5 (2.5)
Lymph node (pN) Negative Positive Unknown
203 (51.1) 48 (12.1) 146 (36.8)
102 (51.5) 25 (12.6) 71 (35.9)
0.733
Systemic metastasis No Yes Unknown
295 (74.3) 76 (19.1) 26 (6.6)
145 (73.2) 38 (19.2) 15 (7.6)
0.291
TNM stage I II III IV
10 (2.5) 178 (44.8) 127 (32.0) 82 (20.7)
5 (2.5) 85 (43.0) 65 (32.8) 43 (21.7)
Tumour grade G1 G2 G3 G4 Unknown
66 (16.6) 239 (60.2) 33 (8.3) 1 (0.3) 58 (14.6)
34 (17.2) 133 (67.2) 15 (7.6) 0 (0.0) 16 (8.0)
Histologic types Conventional Papillary Chromophobic Unclassified
363 (91.4) 28 (7.0) 3 (0.8) 3 (0.8)
177 (89.4) 18 (9.1) 2 (1.0) 1 (0.5)
Age 60 yr <60 yr
206 (48.0) 191 (52.0)
104 (47.5) 94 (52.5)
0.979
Gender Male Female
238 (60.0) 159 (40.0)
127 (64.0) 71 (36.0)
0.340
Immunopositivity in tumours for p16INK4a Negative (0%) 247 (62) Low (1–10%) 77 (19) Intermediate (11–50%) 47 (12) High (>50%) 26 (7)
3.
Results
3.1.
Immunohistochemistry for p16INK4a expression
After evaluation of 397 immunostained tumour specimens, 247 (62%) exhibited no positivity for p16INK4a, 77 (19%) demonstrated low positivity, 47 (12%) had intermediate positivity, and in 26 (7%) high positivity was observed (Fig. 1, for examples of p16INK4a staining). Signals of p16INK4a were predominantly detected in the nuclei of tumour cells. Infrequent cytoplasmic expression was to a
124 (62) 35 (18) 28 (14) 11 (6)
–
0.107
0.270
0.149
0.098
–
great extent accompanied by additional staining signals in the nuclei. A comparison of p16INK4a positivity in tumour, invasive margin, and peritumoural tissue demonstrated low or missing positivity (0–10% positively stained cells) in 324 (82%) tumour, 265 (67%) invasion front, and 319 (86%) peritumoural specimens. In invasive front tissue a significantly higher positivity was observed when compared with signals obtained from tumoural samples ( p < 0.001). No significant difference in positivity was found for tumour and peritumoural tissue samples.
european urology 51 (2007) 732–738
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Fig. 1 – Immunohistochemical analysis of p16INK4a expression in RCC: (A) low level (<50% of immunopositive nuclei) and (B) high level expression (>50% of immunopositive nuclei) in RCC. Only signals in nuclei of tumour cells were considered for evaluation.
3.2. Association between p16INK4a expression and clinicopathologic features
No statistically significant association was observed between positivity of p16INK4a protein in the primary tumour, the invasion front, or the peritumoural benign renal parenchyma and any of the clinicopathologic factors. The factors investigated include age, gender, tumour localization (right or left), histologic type of the tumour, pT stage, lymph node status, systemic metastasis, and the TNM stage. Table 1 presents a summary of statistical analyses. 3.3.
Survival analyses
Comparative survival analyses of the patient group demonstrating high expression of p16INK4a (>50% positively stained cells) and those exhibiting negative (0%) or low (1–10%) expression demonstrated a statistically significant improved survival for the high positivity group of patients ( p = 0.019 and p = 0.033, log-rank test, Fig. 2A). Borderline significance was obtained for the comparison of high (>50% positively stained cells) and intermediate (11–50%) expression groups ( p = 0.057, Fig. 2A). Comparison of the high-expression group (>50% positively stained cells) with the combined group of
Fig. 2 – Kaplan-Meier analysis of disease-specific survival of patients. (A) Comparative survival analysis of immunohistochemically classified tumours demonstrating negative (0%), low- (1–10%), intermediate(11–50%), and high-level positivity (>50%). (B) Comparison of tumours using a 50% cutoff value for immunopositivity.
zero, low, and intermediate positivity tumours (0–50% positively stained cells) showed a significant better survival for patients exhibiting high expression of p16INK4a ( p = 0.03, log-rank test, Fig. 2B). To assess whether the immunohistochemically detected positivity for p16INK4a in RCC is a statistically independent predictor of disease-specific survival, Cox regression analysis was performed including pT stage, lymph node status, systemic metastasis, and histologic grade of the tumour as statistical covariables. Expression of p16INK4a was found to be an independent variable for the prediction of the disease-specific survival ( p = 0.008). In addition, the pT stage, lymph node involvement, systemic metastasis, and the histologic grade of the tumour were also found to be independent variables in the regression model for prediction of the prognosis (Table 2).
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Table 2 – Results of multivariate analysis of diseasespecific survival Covariate pT stage Lymph node status Systemic metastasis Fuhrmann histologic grade p16INK4a expression
4.
p value 0.000 0.000 0.000 0.035 0.008
Discussion
Inactivation of the p16INK4a gene by hypermethylation of the promoter region, homozygous deletion, and mutation has frequently been identified in various tumour cell lines including those obtained from RCC, suggesting that functional inactivation of p16INK4a is a common event in tumourigenesis [18–20]. Loss of p16INK4a protein function is supposed to induce persistent hyperphosphorylation of the retinoblastoma protein, resulting in an evasion of cell-cycle arrest and promotion of the neoplastic process. To our knowledge the present study is the first to investigate the association between immunohistochemically detected expression of p16INK4a and the clinicopathologic characteristics in RCC. Complete or almost complete loss of p16INK4a was frequently observed in our large group of RCC specimens, which strongly suggested that low levels of p16INK4a participate in the pathogenesis of RCC. Previous studies have also demonstrated low positivity (<5% positively stained cells) of p16INK4a in up to 72% of RCCs [21] or complete loss of p16INK4a expression in 62% of colorectal carcinomas [22]. Although it is debatable whether results of different immunohistochemical studies can be compared on a quantitative basis, it is apparent that our results are similar in tendency. The reason for low protein levels as observed in RCC is not clear so far. Hypermethylation of the p16INK4a promoter and homozygous deletion account only for a fraction of tumours exhibiting inactivation of p16INK4a [12]. Moreover, hyperphosphorylation of the retinoblastoma protein has so far not been characterized in RCC in detail. Positivity for p16INK4a was significantly increased in invasive margin tissue when compared with tumoural samples. Upregulation of p16INK4A has been described in invasive front tissue of basal cell carcinomas, which is accompanied by low proliferative activity of tumour cells [23]. Recently we found that expression of the mitotic marker protein cyclin B1 is decreased in invasion front tissue [17]. Thus, the higher positivity for p16INK4A observed in invasive margin tissue of RCC could possibly indicate the presence of a similar mechanism in RCC.
High-level positivity for p16INK4a was detected in 7% of RCCs. Taking into account that high immunopositivity could not be detected previously in RCC [21], higher levels of p16INK4a protein overall seem more likely to be a rare event in RCC. The question arises whether disease progression in these rare tumours is different from the majority of tumours, which are detected with low positivity for p16INK4a. Interestingly, our study demonstrated that patients showing high positivity for p16INK4a have a significantly improved disease-specific survival. Similar prognostic relevance of immunohistochemically detected p16INK4a expression has also been described previously in other malignant diseases including non-small cell lung cancer, melanoma, and oro- and nasopharyngeal, hepatocellular, and colorectal carcinoma [24–26]. In prostate cancer [27,28] and neuroblastoma [29], overexpression of p16INK4a was found to be associated with poor survival. Other investigators found no prognostic significance of p16INK4a in head and neck squamous cell carcinoma [30,31]. In RCC, however, our findings clearly indicate that immunohistochemically detected positivity of p16INK4a identifies a subset of patients exhibiting a significantly improved clinical outcome. Interestingly, the multivariate survival analysis also demonstrated that immunopositivity of p16INK4a is an independent variable for statistical prediction of the prognosis of RCC patients. Provided that the increased p16INK4a protein levels, as detected by immunohistochemical analysis, correspond with elevated concentrations of functionally active proteins, our data could give evidence that patients suffering from RCC would benefit from artificially increased levels of p16INK4a as recently intended both by gene therapeutic approaches and pharmaceutical trials [32–34].
5.
Conclusions
Our study demonstrated a significant reduction of immunopositivity of p16INK4a in large parts of RCCs, therefore indicating a possible involvement of the gene in tumourigenesis of RCC. Immunohistochemically detected positivity of p16INK4a is a positive prognostic factor in both uni- and multivariate analyses for specific survival.
Acknowledgements We thank Mrs Hepke and Mrs Kuhls for the technical assistance. We thank Mr Vaske for statistical advice.
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References [1] Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1999. CA Cancer J Clin 1999;49:8–31. [2] Motzer RJ, Russo P, Nanus DM, Berg WJ. Renal cell carcinoma. Curr Probl Cancer 1997;21:185–232. [3] Patel PH, Chaganti RS, Motzer RJ. Targeted therapy for metastatic renal cell carcinoma. Br J Cancer 2006;94:614–9. [4] Negrier S, Escudier B, Gomez F, et al. Prognostic factors of survival and rapid progression in 782 patients with metastatic renal carcinomas treated by cytokines: a report from the Groupe Francais d’Immunotherapie. Ann Oncol 2002;13:1460–8. [5] Kattan MW, Reuter V, Motzer RJ, Katz J, Russo P. A postoperative prognostic nomogram for renal cell carcinoma. J Urol 2001;166:63–7. [6] Sorbellini M, Kattan MW, Snyder ME, et al. A postoperative prognostic nomogram predicting recurrence for patients with conventional clear cell renal cell carcinoma. J Urol 2005;173:48–51. [7] Ficarra V, Novara G, Galfano A, Artibani W. Neoplasm staging and organ-confined renal cell carcinoma: a systematic review. Eur Urol 2004;46:559–64. [8] Sherr CJ. The Pezcoller lecture: cancer cell cycles revisited. Cancer Res 2000;60:3689–95. [9] Sherr CJ. The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol 2001;2:731–7. [10] Beasley MB, Lantuejoul S, Abbondanzo S, et al. The P16/ cyclin D1/Rb pathway in neuroendocrine tumors of the lung. Hum Pathol 2003;34:136–42. [11] Hedberg Y, Ljungberg B, Roos G, Landberg G. Expression of cyclin D1, D3, E, and p27 in human renal cell carcinoma analysed by tissue microarray. Br J Cancer 2003;88:1417–23. [12] Sanz-Casla MT, Maestro ML, del Barco V, et al. Loss of heterozygosity and methylation of p16 in renal cell carcinoma. Urol Res 2003;31:159–62. [13] Mengel M, Kreipe H, von Wasielewski R. Rapid and largescale transition of new tumor biomarkers to clinical biopsy material by innovative tissue microarray systems. Appl Immunohistochem Mol Morphol 2003;11:261–8. [14] Guinan P, Sobin LH, Algaba F, et al. TNM staging of renal cell carcinoma: Workgroup No. 3. Union International Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997;80:992–3. [15] Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982;6:655–63. [16] Kovacs G, Akhtar M, Beckwith BJ, et al. The Heidelberg classification of renal cell tumours. J Pathol 1997;183:131–3. [17] Ikuerowo SO, Kuczyk MA, Mengel M, et al. Alteration of subcellular and cellular expression patterns of cyclin B1 in renal cell carcinoma is significantly related to clinical progression and survival of patients. Int J Cancer 2006;119:867–74. [18] Liggett Jr WH, Sidransky D. Role of the p16 tumor suppressor gene in cancer. J Clin Oncol 1998;16:1197–206. [19] Kawada Y, Nakamura M, Ishida E, et al. Aberrations of the p14(ARF) and p16(INK4a) genes in renal cell carcinomas. Jpn J Cancer Res 2001;92:1293–9.
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[20] Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994;264:436–40. [21] Hedberg Y, Ljungberg B, Roos G, Landberg G. Retinoblastoma protein in human renal cell carcinoma in relation to alterations in G1/S regulatory proteins. Int J Cancer 2004; 109:189–93. [22] Cui X, Shirai Y, Wakai T, Yokoyama N, Hirano S, Hatakeyama K. Aberrant expression of pRb and p16(INK4), alone or in combination, indicates poor outcome after resection in patients with colorectal carcinoma. Hum Pathol 2004;35:1189–95. [23] Svensson S, Nilsson K, Ringberg A, Landberg G. Invade or proliferate? Two contrasting events in malignant behavior governed by p16(INK4a) and an intact Rb pathway illustrated by a model system of basal cell carcinoma. Cancer Res 2003;63:1737–42. [24] Weinberger PM, Yu Z, Haffty BG, et al. Prognostic significance of p16 protein levels in oropharyngeal squamous cell cancer. Clin Cancer Res 2004;10:5684–91. [25] Klussmann JP, Gultekin E, Weissenborn SJ, et al. Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus. Am J Pathol 2003;162:747–53. [26] Zhao P, Hu YC, Talbot IC. Expressing patterns of p16 and CDK4 correlated to prognosis in colorectal carcinoma. World J Gastroenterol 2003;9:2202–6. [27] Lee CT, Capodieci P, Osman I, et al. Overexpression of the cyclin-dependent kinase inhibitor p16 is associated with tumor recurrence in human prostate cancer. Clin Cancer Res 1999;5:977–83. [28] Henshall SM, Quinn DI, Lee CS, et al. Overexpression of the cell cycle inhibitor p16INK4A in high-grade prostatic intraepithelial neoplasia predicts early relapse in prostate cancer patients. Clin Cancer Res 2001;7:544–50. [29] Omura-Minamisawa M, Diccianni MB, Chang RC, et al. p16/p14(ARF) cell cycle regulatory pathways in primary neuroblastoma: p16 expression is associated with advanced stage disease. Clin Cancer Res 2001; 7:3481–90. [30] Yuen PW, Man M, Lam KY, Kwong YL. Clinicopathological significance of p16 gene expression in the surgical treatment of head and neck squamous cell carcinomas. J Clin Pathol 2002;55:58–60. [31] Geisler SA, Olshan AF, Weissler MC, et al. p16 and p53 Protein expression as prognostic indicators of survival and disease recurrence from head and neck cancer. Clin Cancer Res 2002;8:3445–53. [32] Wang L, Qi X, Sun Y, Liang L, Ju D. Adenovirus-mediated combined P16 gene and GM-CSF gene therapy for the treatment of established tumor and induction of antitumor immunity. Cancer Gene Ther 2002;9:819–24. [33] Stadler WM, Vogelzang NJ, Amato R, et al. Flavopiridol, a novel cyclin-dependent kinase inhibitor, in metastatic renal cancer: a University of Chicago Phase II Consortium study. J Clin Oncol 2000;18:371–5. [34] Aklilu M, Kindler HL, Donehower RC, Mani S, Vokes EE. Phase II study of flavopiridol in patients with advanced colorectal cancer. Ann Oncol 2003;14:1270–3.
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Editorial Comment Sergio Bracarda, SC Medical Oncology, Ospedale S. Maria della Misericordia, Azienda Ospedaliera-Universitaria di Perugia, Perugia, Italy [email protected] Recent advances in the management of renal cell carcinoma (RCC), such as antiangiogenic treatments, mostly depend on an increased level of knowledge of the molecular machinery of the disease: a complex network of pathways regulating (or disregulating) proliferation, apoptosis, invasion, and much more of the tumoural cell processes [1]. p16INK4a, a product of the INK4a/ARF locus, is a tumour-suppressor molecule acting as a cyclindependent kinase inhibitor (CDKI), previously mainly known as a mediator of cellular senescence [2]. More recently, the strict interaction of p16INK4a with other tumour-suppressor proteins (eg, pRB, retinoblastoma gene protein, and p53) in cell-cycle control has been better understood (eg, its frequent deregulation in cancer) [3]. Previously published studies about p16INK4, however, have depicted a heterogeneous scenario about its role and significance in different cancer types. Ikuerowo and colleagues present the results of a large retrospective
study evaluating the possible role of p16INK4a in RCC tumourigenesis and prognosis. Interestingly, only cases with a high positivity level of p16INK4a showed a statistically significant improved survival, an inverse situation with respect to what is known in breast carcinoma, while any statistically significant relationship was found between p16INK4a status and classic prognostic factors (age, sex, stage, histologic type). Even if this study cannot be considered conclusive for the argument, it shows the possibility of use of new prognostic factors to better define the natural history of ‘‘apparently’’ localized RCC and to hypothesise innovative, targeted treatment approaches for this frequently lethal disease. References [1] Linehan WM, Vaselli J, Srinivasan R, et al. Genetic basis of cancer of the kidney: disease-specific approaches to therapy. Clin Cancer Res 2004;10:6282S–9S. [2] Satyanarayana A, Rudolph KL. p16 and ARF: activation of teenage proteins in old age. J Clin Invest 2004;114:1237–40. [3] Ohtani N, Yamakoshi K, Takahashi A, Hara E. The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression. J Med Invest 2004; 51:146–53.
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Kidney Cancer
p16INK4a Expression and Clinicopathologic Parameters in Renal Cell Carcinoma Stephen O. Ikuerowo a, Markus A. Kuczyk b, Reinhard von Wasielewski c, Olayiwola B. Shittu d, Udo Jonas a, Stefan Machtens a, Ju¨rgen Serth a,* a
Department of Urology, Medizinische Hochschule Hannover, Hannover, Germany Department of Urology, Karl-Eberhards University, Tu¨bingen, Germany c Department of Pathology, Medizinische Hochschule Hannover, Hannover, Germany d Division of Urology, University College Hospital and University of Ibadan, Ibadan, Nigeria b
Article info
Abstract
Article history: Accepted August 8, 2006 Published online ahead of print on August 23, 2006
Objectives: The tumour suppressor gene p16INK4a is a cyclin-dependent kinase inhibitor, for which inactivation attributable to promoter hypermethylation or homozygous deletion has been described in malignancies. Little is known about p16INK4a protein levels in renal cell carcinoma (RCC) and its association with clinicopathologic parameters or disease progression. Methods: The expression of the p16INK4a gene was analysed with the use of immunohistochemistry and tissue microarrays (TMA). Tissue cores were obtained from the primary tumour itself, the tumoural invasion front, and histologically benign peritumoural tissue of 397 nephrectomies. For statistical analysis, sections were classified into four groups according to the relative amount of positively stained cells: negative (0%), low (1–10%), intermediate (11–50%), and high positivity (>50%). Follow-up data were analyzed for 198 patients (follow-up period: 2–240 mo; median: 138 mo). Results: Absent or low expression of p16INK4a was observed in 82% of tumour samples. No statistically significant association was found between protein levels detected in tumour, invasion front, or normal renal tissues and any of the clinicopathologic variables. Survival analysis by Kaplan-Meier revealed a significant association between high expression (>50%) of p16INK4a in tumours and better disease-specific survival ( p = 0.03, log-rank test). Cox regression analysis showed that p16INK4a expression is an independent covariate in disease-specific survival ( p < 0.01). Conclusions: The absence of p16INK4a expression in most tumour cells indicates that p16INK4a could be involved in the tumourigenesis of RCC. Immunohistochemically detected positivity for p16INK4a is a positive prognosticator for specific survival in both uni- and multivariate analyses.
Keywords: p16INK4a Renal cell cancer Prognosis Tissue microarray analysis
# 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Medizinische Hochschule Hannover, Department of Urology, OE6247, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Tel. +49 511 5328927; Fax: +49 511 5322926. E-mail address: [email protected] (J. Serth). 0302-2838/$ – see back matter # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2006.08.010
european urology 51 (2007) 732–738
1.
Introduction
Renal cell carcinoma (RCC) represents the most prevalent malignancy of the kidney and accounts for 3% of all adult cancers in the Western world [1]. Surgical removal of organ-confined tumours provides a chance for cure, but approximately 30% of treated patients will develop metastases. RCC is a largely chemo- and radiotherapy-resistant tumour, and partial or complete response to immunobiologic therapy is observed only in a fraction of patients [2–4]. The TNM staging system is widely used for prognosis of RCC patients, and additional parameters such as Fuhrman grade, tumour size, necrosis, vascular invasion, and clinical presentation have been suggested to improve the predictability of RCC [5–7]. Cell cycle progression and cell proliferation are controlled by proteins that frequently have been described to be deregulated in various malignant diseases [8]. The tumour suppressor p16INK4a is a G1/S phase cell cycle inhibitor, which arrests cell replication by binding to the cyclin D1-CDK4/CDK6 complex and inhibition of retinoblastoma (RB) protein phosphorylation. As a result, p16INK4a promotes the formation of a RB-E2F–repressive complex that, in turn, blocks the G1/S progression of the cell cycle [9]. Moreover, it has been suggested that both cyclin D1 overexpression and p16INK4a protein alteration give rise to persistent hyperphosphorylation of RB [10]. Recently, overexpression of cyclin D1 has been described in RCC [11]. Although inactivation of the p16INK4a gene in cell lines derived from RCC attributable to promoter hypermethylation, homozygous deletion or, very rarely, point mutation has been reported, the association of p16INK4a protein levels and clinicopathologic features or prognosis of patients with RCC has not been investigated so far [12]. Therefore, the aim of the present study was to analyse the expression of p16INK4a in RCCs and to assess its prognostic significance in a sufficient number of patients. 2.
Methods
2.1.
Patients and tissue microarrays
For the setup of tissue microarrays, 397 patients who had radical nephrectomy for treatment of RCC between 1981 and 1998 at the Medizinische Hochschule Hannover, Hannover, Germany, were included. The mean age of the patients was 60 yr (range: 31–83). Tissue samples selected by the pathologist were obtained from the primary tumour, the respective invasion front, and the peritumoural, histologically normal kidney, and arranged on tissue microarray blocks (TMAs) as
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described previously [13]. The follow-up data for 238 patients were available and included in the analysis. At the time of the final follow-up update, 112 patients were still surviving; 86 patients had died as a result of the disease and 40 patients of other causes. For statistical analysis, cases with cancerindependent deaths were excluded; thus, 198 patients were considered for the follow-up group (median: 138 mo, range: 2–240 mo). The clinical and histopathologic characteristics as well as the immunohistochemical positivity of the follow-up group of patients strongly resembled the entire group of all nephrectomies (Table 1). Clinical staging and nuclear grading were performed according to the Union International Contre le Cancer (UICC) 1997 TNM classification system [14] and Fuhrman et al. [15], respectively. The TNM 2002 classification, which provides additional subclassification of the T1 group, could not be applied because of a lack of complete information on the tumour size. Histologic classification of tumours according to the Heidelberg system [16] resulted in 363 (91%) conventional, 28 (7%) papillary, and 3 (1%) chromophobic RCCs.
2.2.
Immunohistochemistry
The TMAs were immunohistochemically analysed for p16INK4a protein expression with the use of a mouse antihuman p16 monoclonal antibody (BD Biosciences, San Diego, CA, USA) as described previously [17], except that signal amplification using tyramide was omitted. The anti-p16 antibody was incubated overnight (dilution: 1:60) in a moist chamber at room temperature. Two independent investigators without knowledge of clinicopathologic and follow-up data classified the slides on the basis of the percentage of cells displaying nuclear immunoreactivity detected either in tumour cells (tumour and invasion front specimens) or in tubular cells (peritumoural renal parenchyma). After a preliminary survey of stained specimens, specific cutoff values were chosen to allow robust and efficient classification of stained specimens. Respective tissue specimens were grouped into four classes: negative (0%), low- (1–10%), intermediate- (11–50%), and high-level expression (>50%). No significant interobserver differences were detected, and investigators were blinded against each other.
2.3.
Statistical analysis
Statistical analyses were performed with the use of the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, USA). The association between p16INK4a expression and clinicopathologic characteristics was determined with the use of the chi-square test; p values 0.05 were considered statistically significant. Cases with cancer-independent deaths were not included in the study. Censored cases in Kaplan-Meier plots indicate that patients were still alive at the time of the last follow-up examination. Diseasespecific survival was statistically analysed for a follow-up period of 12 yr (144 mo) with the use of the Kaplan-Meier method and the log-rank test. The Cox proportional hazard model was used to test for the independency of prognostic parameters.
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Table 1 – Clinicopathologic data and correlation with positivity for p16INK4a Variable
All patients (N [%])
Disease-specific follow-up group (N [%])
p value (chi-square test): correlation with p16INK4a expression
Total
397
198
pT category pT1 (n = x) pT2 (n = x) pT3 (n = x) pT4 (n = x)
10 (2.5) 200 (50.4) 169 (42.6) 18 (4.5)
6 (3.0) 106 (53.5) 81 (41.0) 5 (2.5)
Lymph node (pN) Negative Positive Unknown
203 (51.1) 48 (12.1) 146 (36.8)
102 (51.5) 25 (12.6) 71 (35.9)
0.733
Systemic metastasis No Yes Unknown
295 (74.3) 76 (19.1) 26 (6.6)
145 (73.2) 38 (19.2) 15 (7.6)
0.291
TNM stage I II III IV
10 (2.5) 178 (44.8) 127 (32.0) 82 (20.7)
5 (2.5) 85 (43.0) 65 (32.8) 43 (21.7)
Tumour grade G1 G2 G3 G4 Unknown
66 (16.6) 239 (60.2) 33 (8.3) 1 (0.3) 58 (14.6)
34 (17.2) 133 (67.2) 15 (7.6) 0 (0.0) 16 (8.0)
Histologic types Conventional Papillary Chromophobic Unclassified
363 (91.4) 28 (7.0) 3 (0.8) 3 (0.8)
177 (89.4) 18 (9.1) 2 (1.0) 1 (0.5)
Age 60 yr <60 yr
206 (48.0) 191 (52.0)
104 (47.5) 94 (52.5)
0.979
Gender Male Female
238 (60.0) 159 (40.0)
127 (64.0) 71 (36.0)
0.340
Immunopositivity in tumours for p16INK4a Negative (0%) 247 (62) Low (1–10%) 77 (19) Intermediate (11–50%) 47 (12) High (>50%) 26 (7)
3.
Results
3.1.
Immunohistochemistry for p16INK4a expression
After evaluation of 397 immunostained tumour specimens, 247 (62%) exhibited no positivity for p16INK4a, 77 (19%) demonstrated low positivity, 47 (12%) had intermediate positivity, and in 26 (7%) high positivity was observed (Fig. 1, for examples of p16INK4a staining). Signals of p16INK4a were predominantly detected in the nuclei of tumour cells. Infrequent cytoplasmic expression was to a
124 (62) 35 (18) 28 (14) 11 (6)
–
0.107
0.270
0.149
0.098
–
great extent accompanied by additional staining signals in the nuclei. A comparison of p16INK4a positivity in tumour, invasive margin, and peritumoural tissue demonstrated low or missing positivity (0–10% positively stained cells) in 324 (82%) tumour, 265 (67%) invasion front, and 319 (86%) peritumoural specimens. In invasive front tissue a significantly higher positivity was observed when compared with signals obtained from tumoural samples ( p < 0.001). No significant difference in positivity was found for tumour and peritumoural tissue samples.
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Fig. 1 – Immunohistochemical analysis of p16INK4a expression in RCC: (A) low level (<50% of immunopositive nuclei) and (B) high level expression (>50% of immunopositive nuclei) in RCC. Only signals in nuclei of tumour cells were considered for evaluation.
3.2. Association between p16INK4a expression and clinicopathologic features
No statistically significant association was observed between positivity of p16INK4a protein in the primary tumour, the invasion front, or the peritumoural benign renal parenchyma and any of the clinicopathologic factors. The factors investigated include age, gender, tumour localization (right or left), histologic type of the tumour, pT stage, lymph node status, systemic metastasis, and the TNM stage. Table 1 presents a summary of statistical analyses. 3.3.
Survival analyses
Comparative survival analyses of the patient group demonstrating high expression of p16INK4a (>50% positively stained cells) and those exhibiting negative (0%) or low (1–10%) expression demonstrated a statistically significant improved survival for the high positivity group of patients ( p = 0.019 and p = 0.033, log-rank test, Fig. 2A). Borderline significance was obtained for the comparison of high (>50% positively stained cells) and intermediate (11–50%) expression groups ( p = 0.057, Fig. 2A). Comparison of the high-expression group (>50% positively stained cells) with the combined group of
Fig. 2 – Kaplan-Meier analysis of disease-specific survival of patients. (A) Comparative survival analysis of immunohistochemically classified tumours demonstrating negative (0%), low- (1–10%), intermediate(11–50%), and high-level positivity (>50%). (B) Comparison of tumours using a 50% cutoff value for immunopositivity.
zero, low, and intermediate positivity tumours (0–50% positively stained cells) showed a significant better survival for patients exhibiting high expression of p16INK4a ( p = 0.03, log-rank test, Fig. 2B). To assess whether the immunohistochemically detected positivity for p16INK4a in RCC is a statistically independent predictor of disease-specific survival, Cox regression analysis was performed including pT stage, lymph node status, systemic metastasis, and histologic grade of the tumour as statistical covariables. Expression of p16INK4a was found to be an independent variable for the prediction of the disease-specific survival ( p = 0.008). In addition, the pT stage, lymph node involvement, systemic metastasis, and the histologic grade of the tumour were also found to be independent variables in the regression model for prediction of the prognosis (Table 2).
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Table 2 – Results of multivariate analysis of diseasespecific survival Covariate pT stage Lymph node status Systemic metastasis Fuhrmann histologic grade p16INK4a expression
4.
p value 0.000 0.000 0.000 0.035 0.008
Discussion
Inactivation of the p16INK4a gene by hypermethylation of the promoter region, homozygous deletion, and mutation has frequently been identified in various tumour cell lines including those obtained from RCC, suggesting that functional inactivation of p16INK4a is a common event in tumourigenesis [18–20]. Loss of p16INK4a protein function is supposed to induce persistent hyperphosphorylation of the retinoblastoma protein, resulting in an evasion of cell-cycle arrest and promotion of the neoplastic process. To our knowledge the present study is the first to investigate the association between immunohistochemically detected expression of p16INK4a and the clinicopathologic characteristics in RCC. Complete or almost complete loss of p16INK4a was frequently observed in our large group of RCC specimens, which strongly suggested that low levels of p16INK4a participate in the pathogenesis of RCC. Previous studies have also demonstrated low positivity (<5% positively stained cells) of p16INK4a in up to 72% of RCCs [21] or complete loss of p16INK4a expression in 62% of colorectal carcinomas [22]. Although it is debatable whether results of different immunohistochemical studies can be compared on a quantitative basis, it is apparent that our results are similar in tendency. The reason for low protein levels as observed in RCC is not clear so far. Hypermethylation of the p16INK4a promoter and homozygous deletion account only for a fraction of tumours exhibiting inactivation of p16INK4a [12]. Moreover, hyperphosphorylation of the retinoblastoma protein has so far not been characterized in RCC in detail. Positivity for p16INK4a was significantly increased in invasive margin tissue when compared with tumoural samples. Upregulation of p16INK4A has been described in invasive front tissue of basal cell carcinomas, which is accompanied by low proliferative activity of tumour cells [23]. Recently we found that expression of the mitotic marker protein cyclin B1 is decreased in invasion front tissue [17]. Thus, the higher positivity for p16INK4A observed in invasive margin tissue of RCC could possibly indicate the presence of a similar mechanism in RCC.
High-level positivity for p16INK4a was detected in 7% of RCCs. Taking into account that high immunopositivity could not be detected previously in RCC [21], higher levels of p16INK4a protein overall seem more likely to be a rare event in RCC. The question arises whether disease progression in these rare tumours is different from the majority of tumours, which are detected with low positivity for p16INK4a. Interestingly, our study demonstrated that patients showing high positivity for p16INK4a have a significantly improved disease-specific survival. Similar prognostic relevance of immunohistochemically detected p16INK4a expression has also been described previously in other malignant diseases including non-small cell lung cancer, melanoma, and oro- and nasopharyngeal, hepatocellular, and colorectal carcinoma [24–26]. In prostate cancer [27,28] and neuroblastoma [29], overexpression of p16INK4a was found to be associated with poor survival. Other investigators found no prognostic significance of p16INK4a in head and neck squamous cell carcinoma [30,31]. In RCC, however, our findings clearly indicate that immunohistochemically detected positivity of p16INK4a identifies a subset of patients exhibiting a significantly improved clinical outcome. Interestingly, the multivariate survival analysis also demonstrated that immunopositivity of p16INK4a is an independent variable for statistical prediction of the prognosis of RCC patients. Provided that the increased p16INK4a protein levels, as detected by immunohistochemical analysis, correspond with elevated concentrations of functionally active proteins, our data could give evidence that patients suffering from RCC would benefit from artificially increased levels of p16INK4a as recently intended both by gene therapeutic approaches and pharmaceutical trials [32–34].
5.
Conclusions
Our study demonstrated a significant reduction of immunopositivity of p16INK4a in large parts of RCCs, therefore indicating a possible involvement of the gene in tumourigenesis of RCC. Immunohistochemically detected positivity of p16INK4a is a positive prognostic factor in both uni- and multivariate analyses for specific survival.
Acknowledgements We thank Mrs Hepke and Mrs Kuhls for the technical assistance. We thank Mr Vaske for statistical advice.
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[20] Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994;264:436–40. [21] Hedberg Y, Ljungberg B, Roos G, Landberg G. Retinoblastoma protein in human renal cell carcinoma in relation to alterations in G1/S regulatory proteins. Int J Cancer 2004; 109:189–93. [22] Cui X, Shirai Y, Wakai T, Yokoyama N, Hirano S, Hatakeyama K. Aberrant expression of pRb and p16(INK4), alone or in combination, indicates poor outcome after resection in patients with colorectal carcinoma. Hum Pathol 2004;35:1189–95. [23] Svensson S, Nilsson K, Ringberg A, Landberg G. Invade or proliferate? Two contrasting events in malignant behavior governed by p16(INK4a) and an intact Rb pathway illustrated by a model system of basal cell carcinoma. Cancer Res 2003;63:1737–42. [24] Weinberger PM, Yu Z, Haffty BG, et al. Prognostic significance of p16 protein levels in oropharyngeal squamous cell cancer. Clin Cancer Res 2004;10:5684–91. [25] Klussmann JP, Gultekin E, Weissenborn SJ, et al. Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus. Am J Pathol 2003;162:747–53. [26] Zhao P, Hu YC, Talbot IC. Expressing patterns of p16 and CDK4 correlated to prognosis in colorectal carcinoma. World J Gastroenterol 2003;9:2202–6. [27] Lee CT, Capodieci P, Osman I, et al. Overexpression of the cyclin-dependent kinase inhibitor p16 is associated with tumor recurrence in human prostate cancer. Clin Cancer Res 1999;5:977–83. [28] Henshall SM, Quinn DI, Lee CS, et al. Overexpression of the cell cycle inhibitor p16INK4A in high-grade prostatic intraepithelial neoplasia predicts early relapse in prostate cancer patients. Clin Cancer Res 2001;7:544–50. [29] Omura-Minamisawa M, Diccianni MB, Chang RC, et al. p16/p14(ARF) cell cycle regulatory pathways in primary neuroblastoma: p16 expression is associated with advanced stage disease. Clin Cancer Res 2001; 7:3481–90. [30] Yuen PW, Man M, Lam KY, Kwong YL. Clinicopathological significance of p16 gene expression in the surgical treatment of head and neck squamous cell carcinomas. J Clin Pathol 2002;55:58–60. [31] Geisler SA, Olshan AF, Weissler MC, et al. p16 and p53 Protein expression as prognostic indicators of survival and disease recurrence from head and neck cancer. Clin Cancer Res 2002;8:3445–53. [32] Wang L, Qi X, Sun Y, Liang L, Ju D. Adenovirus-mediated combined P16 gene and GM-CSF gene therapy for the treatment of established tumor and induction of antitumor immunity. Cancer Gene Ther 2002;9:819–24. [33] Stadler WM, Vogelzang NJ, Amato R, et al. Flavopiridol, a novel cyclin-dependent kinase inhibitor, in metastatic renal cancer: a University of Chicago Phase II Consortium study. J Clin Oncol 2000;18:371–5. [34] Aklilu M, Kindler HL, Donehower RC, Mani S, Vokes EE. Phase II study of flavopiridol in patients with advanced colorectal cancer. Ann Oncol 2003;14:1270–3.
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Editorial Comment Sergio Bracarda, SC Medical Oncology, Ospedale S. Maria della Misericordia, Azienda Ospedaliera-Universitaria di Perugia, Perugia, Italy [email protected] Recent advances in the management of renal cell carcinoma (RCC), such as antiangiogenic treatments, mostly depend on an increased level of knowledge of the molecular machinery of the disease: a complex network of pathways regulating (or disregulating) proliferation, apoptosis, invasion, and much more of the tumoural cell processes [1]. p16INK4a, a product of the INK4a/ARF locus, is a tumour-suppressor molecule acting as a cyclindependent kinase inhibitor (CDKI), previously mainly known as a mediator of cellular senescence [2]. More recently, the strict interaction of p16INK4a with other tumour-suppressor proteins (eg, pRB, retinoblastoma gene protein, and p53) in cell-cycle control has been better understood (eg, its frequent deregulation in cancer) [3]. Previously published studies about p16INK4, however, have depicted a heterogeneous scenario about its role and significance in different cancer types. Ikuerowo and colleagues present the results of a large retrospective
study evaluating the possible role of p16INK4a in RCC tumourigenesis and prognosis. Interestingly, only cases with a high positivity level of p16INK4a showed a statistically significant improved survival, an inverse situation with respect to what is known in breast carcinoma, while any statistically significant relationship was found between p16INK4a status and classic prognostic factors (age, sex, stage, histologic type). Even if this study cannot be considered conclusive for the argument, it shows the possibility of use of new prognostic factors to better define the natural history of ‘‘apparently’’ localized RCC and to hypothesise innovative, targeted treatment approaches for this frequently lethal disease. References [1] Linehan WM, Vaselli J, Srinivasan R, et al. Genetic basis of cancer of the kidney: disease-specific approaches to therapy. Clin Cancer Res 2004;10:6282S–9S. [2] Satyanarayana A, Rudolph KL. p16 and ARF: activation of teenage proteins in old age. J Clin Invest 2004;114:1237–40. [3] Ohtani N, Yamakoshi K, Takahashi A, Hara E. The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression. J Med Invest 2004; 51:146–53.