Cyclin E and Ki67 (MIB1) as markers of proliferative activity in human prostate cancers

Urologic Oncology 6 (2001) 249–253

Original article

Cyclin E and Ki67 (MIB1) as markers of proliferative activity in human prostate cancers: an immunohistochemical study K. Gyftopoulos, M.D., Ph.D.a,*, P. Perimenisa, P. Ravazoulab, G.A. Barbaliasa a Department of Urology, School of Medicine, University of Patras, 26110, Rio, Patras, Greece Department of Pathology, School of Medicine, University of Patras, 26110, Rio, Patras, Greece Received 18 April 2000; received in revised form 12 December 2000; accepted 5 January 2001

b

Abstract Recent evidence has implicated cyclins in the evolution and progression of various malignancies. We immunohistochemically evaluated the expression of cyclin E, a G1 cyclin, along with Ki67-assessed proliferative activity in prostatic carcinoma. Formalin-fixed, paraffin embedded tissue sections from eighty-four previously untreated prostate carcinomas were distributed according to tumor grade and examined for the expression of cyclin E. Results were compared with Ki67 expression as well as tumor grade. Cyclin E was detected in 22 of 84 cases (26.2%) and its expression varied substantially (mean index varied from 4,7–9,0). Ki67 staining was present in 35.7% of cases. Although no correlation with tumor grade was found for either cyclin E or Ki67, a statistically significant correlation was present between the expression of the two biomarkers (Fisher’s test, p0.0001). We examined the proliferative activity of prostatic carcinomas of different tumor grades with markers for Ki67 and cyclin E. Our findings indicate that reactivities with these markers are strongly correlated with each other but their expression seems independent of tumor grade. Further studies on the clinical implications of the combined variable of cyclin E and Ki67 expression might disclose a useful prognostic indicator of survival or tumor relapse in patients with prostate carcinoma. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Prostate cancer; Cyclins; Tumor grade

1. Introduction Normal prostate growth and development depends upon a particular pattern of cell division, with some cell types dividing much more often than others and all divisions occurring in a timely and appropriate fashion [1]. Proliferating cells progress through an orderly sequence of phases that constitute the cell cycle, which is typically divided into four phases: G1, S, G2 and M phase. The regulatory control of the progression of cells through the various stages of cell division is mediated by a group of protein complexes that typically consist of a regulatory subunit called cyclin and a catalytic subunit named cyclin dependent kinase (CDK). Cyclins are divided in two main families: the G1 cyclins (C, D1–3 and E) and the mitotic cyclins (A and B). G1 cyclins are important for the passage of cells through the G1 phase and their entry into the S phase. A growing body of evidence indicates the important role of cyclins in the evolution and progression of various malignancies [2–4]. Several studies assess cyclins as markers of cell proliferation and cell cycle status in several neoplasms, along with the

* Corresponding author. 11 Alkinoou St., 26442 Patras, Greece. Tel: 30-61-425728; fax: 30-61-425728.

widely used Ki67- proliferative activity assessment [5,6]. Ki67, which is most commonly detected by use of the MIB1 antibody, is a 395-kDa antigen encoded by a single gene on chromosome 10 whose expression is tightly associated with the entry of cells into the cell cycle. However, Ki67 is expressed in cells at all stages of the cell cycle, while in contrast cyclin E (as well as other G1 cyclins) is expressed at the G1-S transition, indicating that the cell is beyond the restriction point at late G1. To date studies addressing the cellular distribution of cyclins in human prostatic carcinoma are very limited, although interesting results are reported in several neoplasms [5,7,8]. In the current study we immunohistochemically evaluated the expression of cyclin E and Ki67 in human prostatic carcinoma and examined their possible interrelation along with correlation with tumor grade. 2. Materials and methods 2.1. Tissue samples Human prostatic tissues were obtained from transurethral resection and retropubic or radical prostatectomy, from patients operated in the Department of Urology during the years 1994–1997. Eighty-four cases of primary prostate carcinoma were analyzed from men between the ages of 52 and

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85 years (mean 72 yrs) who had not undergone any hormonal treatment. Tumors were graded according to the Gleason grading system [9] and further divided into 3 subgroups (GRADE I: Gleason score 2–4, n24, GRADE II: G-score 5–7, n41, GRADE III: G-score 8–10, n19). Tissues were fixed in formalin and embedded in paraffin blocks. Sections were cut at 4 m and stained with hematoxylin-eosin for histological diagnosis, with additional sections cut for immunostaining. 3. Immunohistochemistry The commercially available monoclonal antibodies for cyclin E (MCP515, Ylem, Italia) and Ki67 (Dako A/S, Denmark) were used. The immunostaining was performed according to the Streptavidin–Biotin Complex Peroxidase method using the KWIK™ kit (IMMUNON™, Pittsburgh, PA). Specimens of breast carcinoma, known to express cyclin E and Ki67 were used as positive controls. Primary antibody was replaced with non-immune sera for negative control immunostaining. After deparaffinization and rehydration sections were treated with the Microwave antigen unmasking buffer technique [10]. Slides were immersed in a coplin jar with sodium citrate buffer PH6 and heated 3 times for 5 min in a microwave oven (700W). After each 5 min interval the buffer was inspected for vaporization loss and the oven was reactivated for 5 min. Once the 3 cycles were completed, the slides were allowed to cool down at room temperature for 20 min and were rinsed in TBS PH7.6 for 10 min. Incubation with IMMUNON™ protein blocking agent was then performed for 20 min. Sections were then incubated with primary antibody (Dilutions: cyclin E 1:50, Ki67 1:80) and left overnight in moist chambers at 4C. Slides were then washed in TBS for 35 min, first washing in

TBSTWEEN 0.1% and then Kwik™ Biotinylated Universal Secondary Ab Reagent was applied for 20 min. Slides were then washed again in the previously described manner and Kwik™ Streptavidin Peroxidase Reagent was applied for 20 min. Further washing with TBS was followed by incubating the sections with Chromogen-Substrate solution (DAB) for 5 min. The sections were then rinsed in running tap water for 5 min and counterstained with 50% Mayer hematoxylin solution for 1 min. After dehydration slides were cleared with xylene and mounted with Entellan (Merck, Darmstadt, Germany). 3.1. Analysis of staining Morphological assessment of immunostained tissue and manual counting of immunolabeled tumor cells were performed with the aid of an occular grid. Glandular epithelium nuclei were assessed in 10 representative high power fields (40) per case and a Labeling Index (LI) was calculated as the percentage of tumor cell nuclei that exhibited positive staining. Only tumor cells with unequivocal nuclear positivity, irrespective of intensity of staining, were counted as positive. Positivity was considered at a minimal threshold of 5% positive cells. Overall variations in the percentages of nuclear staining for cyclin E and Ki67 and the relationship with tumor grade was evaluated by analysis of variance (ANOVA). The relationship between cyclin E and Ki67 expression was examined by Fisher’s Exact Test. A p value less than 0.05 was considered to be significant. 4. Results Cyclin E staining was present in 22 of 84 cases (26.2%). In general, cyclin E reactivity was limited to the tumor cell nuclei, although occasionally faint cytoplasmic staining was observed. The nuclear staining pattern was diffuse, with

Fig. 1. Strong nuclear reactivity for cyclin E in prostate carcinoma (magnification 200).

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Fig. 2. Strong nuclear reactivity for cyclin E in prostate carcinoma (magnification 200).

positive nuclei sporadically distributed (Figs. 1,2). Nuclear reactivity for cyclin E varied from 0 to 60% of cells. When the immunohistochemical expression was analyzed for each grade group by analysis of variance, no statistically significant differences were observed (p0.05), mean values: 8.2, 9.0 and 4.7 for group grade I, II and III respectively). Ki67 reactivity was strong, localized in the nuclei of tumor cells but limited at 30 of 84 cases (35.7%). Immunoreactive nuclei were randomly scattered across the tumor areas (Fig. 3). The range of LI for Ki67 varied from 0 to 50%. Mean values in each grade group were 8.4, 11.2 and 8.9 for group grade I, II and III respectively. Analysis of variance

between grade groups did not reveal any statistically significant difference (p0.05). Taking into account recent evidence that suggests that Gleason 7 prostate cancer should be considered a unique grade category [11,12], we further divided grade II group into grade 5–6 and grade 7 tumors (n31 and n10 respectively). Analysis of variance of cyclin E expression between the newly formed four groups failed to yield any statistically significant results (p0.05). Similar results were obtained when Ki67 positivity was analyzed. A significant percentage (70%) of Ki67 positive tumors stained positively for cyclin E (21/30). Correlation between

Fig. 3. Nuclear immunostaining for Ki67 in prostate carcinoma (magnification 200).

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cyclin E and Ki67 expression was examined by analyzing positive and negative samples using the Fisher’s Exact Test. Interestingly, a strong correlation was present (p0.0001). 5. Discussion Cyclin E is a highly conserved 45 kDa nuclear protein that was first identified by virtue of its ability to rescue G1cyclin defective budding yeast [13]. It is a major regulator of the progression of cells into S-phase that appears in cells as they cross the restriction point in late G1 phase. Overexpression of cyclin E has been observed in a variety of tumors, including breast, gastric, endometrial, renal pelvis and ureteral carcinoma, thyroidal, hepatocellular carcinoma and melanocytic lesions [4,7,14–20]. Most studies suggest that given the central role of cyclin E in cell division, this may contribute to the malignant phenotype. It is also noteworthy that this overexpression appears to be tumor specific and not merely a secondary event due to increased proliferation of tumor cells. On the other hand, cyclin E expression, unlike cyclin A, is difficult to detect in proliferating cells of normal tissues. These remarkable findings make possible that cyclin E might be an oncogene, although no direct evidence exists up to date [16,21,22]. Studies of cyclin E expression in prostatic carcinoma are limited and results are controversial, mainly due to the extremely limited immunoreactivity of prostatic tumor cells, a fact that suggests that prostatic carcinoma is a tumor of low proliferative activity [23]. In the present study the expression of cyclin E in prostatic tumor cells was limited but the overall expression (26.2%) can not be ignored. In a previous study cyclin E positivity was quite limited (14.2%, 4/28 cases) but the reported focal variation in expression correlates well with our findings, suggesting that this type of variation in expression of cell cycle regulators indicates genetic instability of the tumor [23]. It would be interesting to observe the varying patterns of cyclin E expression in correlation with tumor grade. Although we did not formerly evaluate the staining patterns according to the distribution patterns as previously described (positive, diffuse positive, focal positive, regional positive) [7], repeated evaluation of the sections failed to reveal any pronounced differences between grade groups. Additionally, when the LI for cyclin E was compared in the three grade groups, analysis of variance failed to yield any statistically significant difference. Similar findings have been reported in renal cell carcinoma [8]. The mechanisms underlying cyclin E overexpression have not yet been defined in detail. Reports from studies in tumors other than prostate cancer implicate gene amplification in the phenomenon of cyclin E overexpression in certain tumors [19,24]. Another alternative may include increased mRNA stability, as reported in colorectal carcinomas [22]. Alterations in the transcriptional level upstream the signal transducing pathways could be involved independently of gene amplification. Additionally, abnormal expression of p53 seems to induce the expression of cy-

clin E, although the exact mechanism is obscure [25]. Nevertheless, no matter the mechanism, it is accepted that tumors overexpressing cyclin E may be of high malignant potential. Ki67 has been extensively used as an indicator of the proliferative state in cells, since it is expressed only in cycling cells and bears a short half-life. The estimation of the proliferative activity has prognostic implications in many neoplasias [26–28]. Although studies are limited, the expression of Ki67 in prostate carcinoma is considered an independent prognostic factor of survival, clinical tumor recurrence and PSA-detected relapse [29,30–32]. Recent advances in antigen retrieval techniques allow for measurements of Ki67 in formalin fixed, paraffin embedded tissues. In the present study, Ki67 immunoreactivity was detected in 35.7% of cases. The pattern of immunoreactivity and the number of immunoreactive tumor cells correlate well with the findings in other studies. The limited proportion of tumor cells that exhibit positive staining for Ki67 indicates that proliferative activity is low in prostate cancer. Still, controversy exists on whether Ki67 expression is related to tumor grade. Although Ki67 index and tumor grade correlation has been reported, few other studies suggest that no correlation exists between proliferation index and Gleason score [33]. In our study, analysis of variance failed to yield any statistically significant results between different grade groups. The fact that both cyclin E and Ki67 are markers of cell proliferation and cell cycle in the same complex pathway of cell division makes an interrelation between these two factors possible. Previous studies in breast tumors and gastric adenocarcinomas revealed a strong correlation between cyclin E and Ki67 [15,34,35]. Still, reports on renal cell and prostatic carcinoma failed to show such a positive correlation [8,23]. In the present study, however, a statistically significant correlation was present between cyclin E and Ki67. The observed difference might in part be explained by the smaller number of cases included in the afore mentioned studies. Nevertheless, although cyclin E overexpression seems to be a true tumor cell abnormality and not merely induced by increased proliferation, the possibility still exists that tumors demonstrating high cyclin E expression also feature higher fractions of proliferating cells than tumors with a lower protein expression [15,35,36]. This could be supported by the findings of Western blot analysis of cyclin E alteration and high proliferative activity (estimated by proliferating cell nuclear antigen) in whole cell lysates from surgical samples of prostate cancer patients [19]. Further studies are necessary in order to delineate the mechanisms responsible for cyclin E overexpression. It is of great interest that in the present study the immunohistochemical determination of cyclin E expression in formalin-fixed, paraffinembedded tissue was confirmed. To our knowledge, only one immunohistochemical study of cyclin E expression in prostate cancer is available in the literature to date [23]. Certainly, both markers will have to be further evaluated be-

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fore we conclude to any possible independent predictive role of cyclin E and Ki67 determination in clinical outcome, i.e. survival and tumor recurrence. Still, many issues can be addressed using the above described method in large retrospective studies of archival material from patients with known clinical outcome.

[15]

[16] [17]

6. Conclusions Cyclin E and Ki67 are important molecules of the cell proliferation machinery that are expressed in prostate carcinoma, as determined by immunohistochemical methods. Cyclin E expression seems to be independent from tumor grade but a statistically significant correlation with proliferation index was present. Although Ki67 is generally accepted to be overexpressed in relation with tumor grade, we were unable to show any significant correlation. Further studies are necessary to determine whether these cell cycle antibodies could represent new independent biomarkers in the prediction of prognosis and better treatment planning for patients with prostate carcinoma.

[18]

[19]

[20]

[21]

[22] [23] [24]

References [1] Kirby RS, Christmas TJ, Brawer M. Anatomical and pathological considerations. In: Kirby RS, Christmas TJ, Brawer M, editors. Prostate cancer. London: Mosby, 1996, pp 33–36. [2] Keyomarsi K, Herliczek TW. The role of cyclin E in cell proliferation, development and cancer. Prog Cell Cycle Res 1997;3:171–91. [3] Marches R, Scheuermann RH, Uhr JW. Cancer dormancy: role of cyclin-dependent kinase inhibitors in induction of cell cycle arrest mediated via membrane IgM. Cancer Res. 1998;58:691–7. [4] Steeg PS, Zhou Q. Cyclins and breast cancer. Breast Cancer Res Treat 1998;52:17–28. [5] Prayson RA. Cyclin D1 and MIB1 immunohistochemistry in ependymomas. Am J Clin Pathol 1998;110:629–34. [6] Lai R, Medeiros LJ, Wilson CS, et al. Expression of the cell-cyclerelated proteins E2F–1, p53, mdm-2, p21waf-1 and Ki67 in multiple myeloma: correlation with cyclin D1 immunoreactivity. Mod Pathol 1998;11:642–7. [7] Li S, Shiozawa T, Nakayama K, Nikaido T, Fujii S. Stepwise abnormality of sex steroid hormone receptors, tumor supressor gene products (p53 and Rb) and cyclin E in uterine endometrioid carcinoma. Cancer 1996;77:321–9. [8] Renshaw AA, Loughlin KR, Dutta A. Cyclin A and MIB1 (Ki67) as markers of proliferative activity in primary renal neoplasms. Mod Pathol 1998;11:963–6. [9] Gleason DF. Classification of prostatic carcinomas. Cancer Chemother Rep 1966;50:125–36. [10] Loda M, Fogt F, French F, et al. Androgen receptor immunohistochemistry on paraffin-embedded tissue. Mod Pathol 1994;7:388–91. [11] Twfilli MV, Gheiler EL, Tiguert R, et al. Should Gleason score 7 prostate cancer be considered a unique grade category? Urology 1999;53:372–7. [12] Green GA, Hanlon AL, Al-Saleem T, Hanks GE. A Gleason score of 7 predicts a worse outcome for prostate carcinoma patients treated with radiotherapy. Cancer 1998;83:971–6. [13] Koff A, Cross F, Fisher A, et al. Human cyclin E, a new cyclin that interacts with two members of the CDC2 family. Cell 1991;66:1217–28. [14] Tran TA, Ross JS, Carlson JA, Mihm MC. Mitotic cyclins and cyclin-

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32] [33]

[34]

[35]

[36]

253

dependent kinases in melanocytic lesions. Hum Pathol 1998;29: 1085–90. Nielsen NH, Arnerlöv C, Emdin SO, Landberg G. Cyclin E overexpression, a negative prognostic factor in breast cancer with strong correlation to oestrogen receptor status. Br J Cancer 1996;74:874–80. Sakaguchi T, Watanabe A, Sawade H, et al. Prognostic value of cyclin E and p53 expression in gastric carcinoma. Cancer 1998;82:1238–43. Wang S, Wuu J, Savas L, Patwardhan N, Khan A. The role of cell cycle regulatory proteins, cyclin D1, cyclin E and p27 in thyroid carcinogenesis. Hum Pathol 1998;29:1304–9. Furihata M, Ohtsuki Y, Sonobe H, et al. Prognostic significance of cyclin E and p53 protein overexpression in carcinoma of the renal pelvis and ureter. Br J Cancer 1998;77:783–8. Keyomarsi K, O’Leary N, Molnar G, Lees E, Fingert HJ, Pardee AB. Cyclin E, a potential prognostic marker for breast cancer. Cancer Res 1994;54:380–5. Peng SY, Chou SP, Hsu HC. Association of downregulation of cyclin D1 and overexpression of cyclin E with p53 mutation, high tumor grade and poor prognosis in hepatocellular carcinoma. J Hepatol 1998;29:281–9. Buckley MF, Sweeney KJE, Hamilton JA, et al. Expression and amplification of cyclin genes in human breast cancer. Oncogene 1993;8: 2127–33. Leach FS, Elledge SJ, Sherr CJ, et al. Amplification of cyclin genes in colorectal carcinomas. Cancer REs 1993;53:1986–9. Mashal RD, Lester S, Corless C, et al. Expression of cell cycle regulated proteins in prostate cancer. Cancer Res 1996;56:4259–63. Akama Y, Yasui W, Yokozaki H, et al. Frequent amplification of the cyclin E gene in human gastric carcinomas. Jpn J Cancer Res 1995; 86:617–21. Bonder SM, Minna JD, Jensen SM, et al. Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation. Oncogene 1992;7:43–49. Gerdes J. Ki67 and other proliferation markers useful for immunohistological diagnosis and prognostic evaluations in human malignancies. Sem Cancer Biol 1990;1:199. Scott RJ, Hall PA, Haldane JS, et al. A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction. J Pathol 1991;165:173–7. Quinn CM, Wright NA. The clinical assessment of proliferation and growth in human tumors: evaluation of methods and applications as prognostic variables. J Pathol 1990;160:93. Bubendorf L, Sauter G, Moch H, et al. Ki67 labelling index: an independent predictor of progression in prostate cancer treated by radical prostatectomy. J Pathol 1996;178:437. Bettencourt MC, Bauer JJ, Sesterhenn IA, Mostofi FK, McLeod DG, Moul JW. Ki67 expression is a prognostic marker of prostate cancer recurrence after radical prostatectomy. J Urol 1996;156:1064. Stattin P, Damber JE, Karlberg L, Bergh A. Cell proliferation assessed by Ki67 immunoreactivity on formalin fixed tissues is a predictive factor for survival in prostate cancer. J Urol 1997;157:219–22. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182:311–22. Vartanian RK, Weidner N. Endothelial cell proliferation in prostatic carcinoma and prostatic hyperplasia: correlation with Gleason’s score, microvessel density and epithelial cell proliferation. Lab Invest 1995;73:844–50. Yasui W, Akama Y, Kuniyasu H, et al. Expression of cyclin E in human gastric adenomas and adenocarcinomas: correlation with proliferative activity and p53 status. J Exp Ther Oncol 1996;1:88–94. Scott KA, Walker RA. Lack of cyclin E immunoreactivity in non-malignant breast and association with proliferation in breast cancer. Br J Cancer 1997;76:1288–92. Dutta A, Chandra R, Leiteer LM, Lesters S. Cyclins as markers of tumor proliferation: Immunocytochemical studies in breast cancer. Proc Natl Acad Sci USA 1995;92:5386–90.