Evaluation of cell proliferation, apoptosis, and angiogenesis in transitional cell carcinoma of the renal pelvis and ureter

BASIC SCIENCE

EVALUATION OF CELL PROLIFERATION, APOPTOSIS, AND ANGIOGENESIS IN TRANSITIONAL CELL CARCINOMA OF THE RENAL PELVIS AND URETER XIANGHUA ZHANG, CHUIZE KONG,

AND

IKUMASA TAKENAKA

ABSTRACT Objectives. To investigate cell proliferation, apoptosis, and angiogenesis and their roles in transitional cell carcinoma (TCC) of the renal pelvis and ureter. Methods. Formalin-fixed and paraffin-embedded tissue blocks from 42 patients with TCC of the renal pelvis and ureter were studied. Cell proliferation was assessed by Ki-67 immunostaining, and the proliferation index (PI) was expressed as a percentage of Ki-67-positive cells. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of TUNEL positive cells. Angiogenesis was evaluated by CD31 immunostaining, and microvessel density (MVD) was expressed as the average of the microvessel count. Results. The PI ranged from 5.9% to 48.0% (median 20.03%), AI from 1.0% to 4.2% (median 2.26%), and MVD from 16.0 to 146.0 (median 56.88) in TCC of the renal pelvis and ureter. Statistical analysis revealed close associations of both PI and MVD with tumor stage and of AI with tumor grade. Our study demonstrated a strong relationship between PI and MVD, but did not show associations of AI with PI or MVD in TCC of the renal pelvis and ureter. Conclusions. It is suggested that the high activity of tumor cell proliferation with rich neovascularization may be related to the high malignant potential of the cancer, and evaluation of cell proliferation combined with angiogenesis may be useful in predicting the progression of the renal pelvic and ureteral TCC. UROLOGY 57: 981–985, 2001. © 2001, Elsevier Science Inc.

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t is known that the growth of a tumor is determined by cell proliferation and cell loss (apoptosis). Angiogenesis, the growth of new blood vessels, has a critical role in tumor growth and metastasis. Recent studies revealed important roles for cell proliferation, apoptosis, and angiogenesis in the tumor progression and the prediction of patient survival in a lot of cancers.1–3 In addition to the increased proliferation with decreased apoptosis, both increased proliferation and apoptosis were demonstrated in a number of cancers, even though they reflect two opposite phenomena.1,2 To provide a much greater blood supply and tissue support, angiogenesis has been related to the tumor formation, progression, and prognosis in a vaFrom the Department of Urology, Kagawa Medical University, Kagawa, Japan Reprint requests: Xianghua Zhang, M.D., Department of Urology, Kagawa Medical University, 1750-1 Kita-gun, Miki-cho, Kagawa 761-0793, Japan Submitted: October 10, 2000, accepted (with revisions): December 11, 2000 © 2001, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED

riety of malignancies, including bladder cancer.3–5 Carcinomas of the renal pelvis and ureter are rare, accounting for only 4% of all urothelial cancers. Most renal pelvic and ureteral cancers are transitional cell carcinomas (TCCs). Little is known concerning the roles of tumor cell proliferation, apoptosis, and angiogenesis in TCC of the renal pelvis and ureter. In the current study, we investigated cell proliferation activity by Ki-67 immunostaining, the frequency of apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL), and the degree of angiogenesis by CD31 immunostaining in a total of 42 cases of renal pelvic and ureteral TCC. Furthermore, we analyzed correlations among cell proliferation, apoptosis, and angiogenesis and their relationships to the established clinicopathologic factors of the tumor. MATERIAL AND METHODS Formalin-fixed and paraffin-embedded tissue blocks from 42 patients (median age 54 years, range 38 to 78) with TCC of 0090-4295/01/$20.00 PII S0090-4295(00)01130-4 981

TABLE I. Summarized results of proliferation index, apoptotic index, and microvessel density in transitional cell carcinoma of the renal pelvis and ureter Characteristics Total Grade 1 2 3 Stage T1 T2 T3-4

Patients (n)

PI (%)

AI (%)

MVD

42

20.03

2.26

56.88

8 22 12

15.63 20.10 23.30

1.71* 2.18 2.79*

48.38 53.86 68.50

14 17 11

11.42† 20.55 30.18†

2.24 2.08 2.57

32.28‡ 58.12 86.27‡

KEY: PI ⫽ proliferation index; AI ⫽ apoptotic index; MVD ⫽ microvessel density. PI and AI expressed as percentage of Ki-67 positive cells or apoptotic cells per 100 tumor cells, respectively; MVD expressed as average of microvessel count. * Statistically significant difference between AI and tumor grade (P ⫽ 0.0008). † Statistically significant difference between PI and tumor stage (P ⫽ 0.0002). ‡ Statistically significant difference between MVD and tumor stage (P ⬍0.0001).

the renal pelvis and ureter were studied. There were 25 men and 17 women and 23 left-sided and 19 right-sided tumors. None of the patients investigated in this study underwent treatment before surgery. The tissue blocks were cut into 5-␮m-thick sections and placed on Poly-L-lysine-treated glass slides. Representative sections were stained with routine hematoxylin-eosin and evaluated. Histologic grading and staging were performed in accordance with the guidelines of the International Union Against Cancer.6

Ki-67 AND CD31 IMMUNOSTAINING After the deparaffinization, hydration, and blocking steps, the sections were incubated with the following mouse monoclonal antibodies for 1 to 2 hours at room temperature: antiKi-67 antigen (1/50 dilution; Zymed Laboratories, San Francisco, Calif) and anti-CD31 (1/10 dilution; Quartett GmbH, Berlin, Germany). The sections were next incubated with biotinylated goat anti-mouse IgG (1/200 dilution, Vector Laboratories) for 30 minutes. After incubation in an avidin-biotinperoxidase complex for 30 minutes, the samples were exposed to diaminobenzidine tetrahydrochloride solution, and counterstained with hematoxylin. To obtain the specificity controls, the primary antibody was replaced by Tris-buffered saline, and all other steps were followed unchanged.

CELL PROLIFERATION AND MICROVESSEL DENSITY ASSESSMENT The proliferation index (PI) was obtained as the ratio of Ki-67-positive cells relative to the total number of counted tumor cells and calculated from observations of at least 1000 cells in each section. The microvessel density (MVD) was determined in the five most intense areas of vascularization (hotspot) of each section. All discrete clusters or single cells stained for CD31 were counted as a vessel. Individual microvessel counts were made under light microscopy with a 200-fold magnification (corresponding to an area of 0.28 mm2) within the designated neovascular hotspot. The average counts were recorded as the MVD for each case.

IN SITU DETECTION OF APOPTOSIS

For the detection of apoptotic cells, in 5-␮m-thick sections of formalin-fixed and paraffin-embedded tissue, the TUNEL 982

FIGURE 1. Regression analysis of the Spearman correlation coefficient (r) for the correlation between PI and MVD in TCC of the renal pelvis and ureter. A significant positive relationship was noted between PI and MVD (r ⫽ 0.804; P ⬍0.0001).

method was used according to the procedures included in the Apop Tag In Situ Apoptosis Detection Kit (Intergen, NY). In brief, after the deparaffinization and blocking steps, incubation with 100 ␮g/mL proteinase K (Sigma) was performed for 15 minutes at room temperature. After prehybridization treatment, the sections were exposed to TdT with digoxigenin-11dUTP and deoxyadenosine triphosphate (dATP), with incubation in a moist chamber for 60 minutes at 37°C and then to anti-digoxigenin-11-dUTP labeling for 30 minutes at room temperature, followed by exposure to 0.05% diaminobenzidine tetrahydrochloride. Counterstaining was achieved with 0.5% methyl green solution. For negative controls, TdT was omitted. As positive controls, DNase I-treated specimens were used. The apoptotic index (AI) was obtained as the ratio of TUNEL-positive cells relative to the total number of counted tumor cells and calculated from observations of at least 1000 cells in each section.

STATISTICAL ANALYSIS The Student t test and the Kruskal-Wallis test were used to analyze the continuous variables in each data set. Correlations between PI, AI, and MVD were checked by the Spearman rank correlation coefficient. A P value less than 0.05 was considered to indicate a statistically significant difference.

RESULTS PROLIFERATION INDEX Positive staining of Ki-67 was detected in the nuclei of the renal pelvic and ureteral TCC cells. The PI ranged from 5.9% to 48.0% (median 20.03%) and was significantly related to tumor stage (P ⫽ 0.0002) (Table I). APOPTOTIC INDEX TUNEL signals were also detected in the nuclei of the renal pelvic and ureteral TCC cells. The AI ranged from 1.0% to 4.2% (median 2.26%) and was closely related to tumor grade (P ⫽ 0.0008). UROLOGY 57 (5), 2001

FIGURE 2. Observations of frequent Ki-67-positive tumor cells, apoptotic cells by TUNEL staining, and CD31positive endothelial cells within the same tumor area in serial sections of a case of TCC of the renal pelvis and ureter. (A) Ki-67 immunostaining. Original magnification ⫻60. (B) TUNEL staining. Arrow indicates area of higher magnification in Fig. C. Original magnification ⫻60. (C) Higher magnification of Fig. B. Original magnification ⫻120. (D) CD31 immunostaining. Original magnification ⫻60.

MICROVESSEL DENSITY Positive staining of CD31 was detected in the membrane of the endothelial cells in the tumor stroma. The MVD ranged from 16.0 to 146.0 (median 56.88) and was significantly associated with tumor stage (P ⬍0.0001). CORRELATIONS AMONG PI, AI, AND MVD Regression analysis showed a strong correlation between PI and MVD in TCC of the renal pelvis and ureter (P ⬍0.0001) (Fig. 1). However, the AI was associated with neither the PI nor MVD in the cancer cells. Morphologically, both Ki-67-positive tumor cells and CD31-positive endothelial cells were commonly detected within the same tumor area, with or without occurrence of frequent TUNEL-positive tumor cells, in the serial sections of several cases (Fig. 2). COMMENT Previous studies demonstrated significant associations of cell proliferation, as detected by Ki-67 UROLOGY 57 (5), 2001

staining, with tumor grade, stage, recurrence, and prognosis in TCC of the bladder.7–9 Furthermore, the incidence of apoptosis was closely related to tumor grade, stage, and even prognosis in bladder10 and renal pelvic and ureteral cancers11 using the TUNEL method. The present study showed close relationships between the PI and tumor stage and between the AI and tumor grade. These data, together with the results presented here, indicate that the degrees of cell proliferation and apoptosis may be implicated in the pathogenesis of renal pelvic and ureteral TCC. Continuously growing numbers of clinicopathologic studies have suggested that highly angiogenic tumors are indeed associated with tumor progression and with shorter patient survival in melanoma and breast, lung, and prostate cancers.4,5,12,13 The degree of angiogenesis correlated significantly with tumor stage and the presence of vascular invasion in bladder TCC.14 Furthermore, tumor angiogenesis has been demonstrated to be an independent indicator of disease recurrence and overall 983

survival in invasive TCC of the bladder.3 Our study also showed a close correlation between MVD and tumor stage. This reveals that quantitative study of MVD may be used as a staging predictor in TCC of the renal pelvis and ureter. Cell proliferation, apoptosis, and angiogenesis are essential for carcinogenesis. However, few data are currently available regarding the relation between cell proliferation, apoptosis, and angiogenesis in clinical cancer research.15–17 In the study of urothelial cancers, the correlations among the three parameters have not been extensively investigated. To our knowledge, this is the first report to investigate the situations of cell proliferation, apoptosis, and angiogenesis in TCC of the renal pelvis and ureter. Regarding the balance between cell proliferation and cell death (apoptosis), recent studies have shown that increased cell proliferation may be accompanied by either decreased apoptosis or increased apoptosis in a number of cancers.1,2 Cell proliferation and apoptosis were closely related to the tumor grade or/and tumor stage in bladder TCC,10,18 and the incidence of apoptosis was particularly related to the mitotic index in TCC of both the bladder and the renal pelvis and ureter.10,11 Although our study demonstrated close associations of an increased PI with tumor stage and an increased AI with tumor grade, apoptotic cell death was not related to cell proliferation in the cancer. This indicates that the increment of AI may not always parallel the increasing PI in the tumor progression of renal pelvic and ureteral TCC. More recently, the degree of angiogenesis has been associated with cell proliferation in prostate, oral, and head and neck cancer12,19,20; other data, however, failed to show such a correlation in lung carcinoma.21 In addition to the close associations of both PI and MVD with tumor stage, the present study demonstrated a significant relationship between PI and MVD in TCC of the renal pelvis and ureter. It is suggested that the higher activity of cell proliferation with rich neovascularization may be related to a higher malignant potential of the cancer. This finding also supports the hypothesis that tumor growth is dependent on the formation of new blood microvessels, even in TCC of the renal pelvis and ureter. On the other hand, evidence suggests that the progressive inhibition of apoptosis and the induction of angiogenesis may contribute to tumor initiation, growth, and metastasis in the pathogenesis of breast cancer.22 An inverse relationship between the degree of angiogenesis and the incidence of apoptosis was also demonstrated in colon, prostate, and gastric carcinomas.17,23,24 The present study failed to show any correlations between MVD and AI in TCC of the renal pelvis and ureter. This reveals that the occurrence of ap984

optosis may not be suppressed by neovascularization in TCC of the renal pelvis and ureter. CONCLUSIONS We demonstrated a strong relationship between cell proliferation and angiogenesis in TCC of the renal pelvis and ureter. In addition to a close association of apoptosis with tumor grade, both cell proliferation and angiogenesis were significantly related to tumor stage. It is suggested that the evaluation of cell proliferation combined with angiogenesis may be useful in predicting the progression of TCC of the renal pelvis and ureter. REFERENCES 1. Koivisto P, Visakorpi T, Rantala I, et al: Increased cell proliferation activity and decreased cell death are associated with the emergence of hormone-refractory recurrent prostate cancer. J Pathol 183: 51–56, 1997. 2. Ito Y, Matsuura N, Sakon M, et al: Both cell proliferation and apoptosis significantly predict shortened disease-free survival in hepatocellular carcinoma. Br J Cancer 81: 747– 751, 1999. 3. Bochner BH, Cote RJ, Weidner N, et al: Angiogenesis in bladder cancer: relationship between microvessel density and prognosis. J Natl Cancer Inst 87: 1603–1612, 1995. 4. Barnhill RL, Fandrey K, Levy MA, et al: Angiogenesis and tumor progression of melanoma: quantification of vascularity in melanocytic nevi and cutaneous malignant melanoma. Lab Invest 67: 331–337, 1992. 5. Toi M, Kashitani J, and Tominaga T: Tumor angiogenesis is an independent prognostic indicator in primary breast carcinoma. Int J Cancer 55: 371–374, 1993. 6. International Union Against Cancer: TNM Classification of Malignant Tumors, 4th ed. New York, Springer-Verlag, 1987. 7. Osen I, Fossa SD, Majak B, et al: Prognostic factors in muscle-invasive bladder cancer treated with radiotherapy: an immunohistochemical study. Br J Urol 81: 862– 869, 1998. 8. Asakura T, Takano Y, Iki M, et al: Prognostic value of Ki-67 for recurrence and progression of superficial bladder cancer. J Urol 158: 385–388, 1997. 9. Suwa Y, Takano Y, Iki M, et al: Prognostic significance of Ki-67 expression in transitional cell bladder carcinoma after radical cystectomy. Pathol Res Pract 193: 551–556, 1997. 10. Lipponen PK, and Aaltomaa S: Apoptosis in bladder cancer as related to standard prognostic factors and prognosis. J Pathol 173: 333–339, 1994. 11. Masuda M, Takano Y, Iki M, et al: Apoptosis in transitional cell carcinoma of the renal pelvis and ureter: association with proliferative activity, bcl-2 expression and prognosis. J Urol 158: 750 –753, 1997. 12. Brawer MYK, Deering RE, Brown M, et al: Predictors of pathologic stage in prostatic carcinoma: the role of neovascularity. Cancer 73: 678 – 687, 1994. 13. Macchiarini P, Fontanini G, Hardin MJ, et al: Relation of neovascularisation to metastasis of non-small-cell lung cancer. Lancet 340: 145–146, 1992. 14. Philp EA, Stephenson TJ, and Reed MW: Prognostic significance of angiogenesis in transitional cell carcinoma of the human urinary bladder. Br J Urol 77: 352–357, 1996. 15. Vindigni C, Miracco C, Spina D, et al: Cell proliferation, cell death and angiogenesis in early and advanced gastric cancer of intestinal type. Int J Cancer 74: 637– 641, 1997. 16. Vermeulen PB, Verhoeven D, Hubens G, et al: MiUROLOGY 57 (5), 2001

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