Minichromosome maintenance proteins 2 and 5 expression in muscle-invasive urothelial cancer: a multivariate survival study including proliferation markers and cell cycle regulators

Human Pathology (2005) 36, 899 – 907

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Minichromosome maintenance proteins 2 and 5 expression in muscle-invasive urothelial cancer: a multivariate survival study including proliferation markers and cell cycle regulators Penelope Korkolopoulou MD, PhDa,*, Nikolaos Givalos MD, PhDa, Angelica Saetta BSc, PhDa, Athina Goudopoulou BSc, PhDa, Hariklia Gakiopoulou MD, PhDa, Irene Thymara MD, PhDa, Euphemia Thomas-Tsagli MD, PhDb, Efstratios Patsouris MD, PhDa a

Department of Pathology, Athens University, Medical School, GR 115 27 Athens, Greece Department of Pathology, Asklepeion Voula Hospital, GR 166 73 Athens, Greece

b

Received 7 June 2005; accepted 14 June 2005

Keywords: Minichromosome maintenance proteins; Muscle-invasive urothelial cancer; Cell cycle regulators

Summary Evaluation of cell cycle regulators has gained special interest in the effort to increase the amount of prognostic information in malignant tumors. Minichromosome maintenance proteins (MCMs) drive the formation of prereplicative complexes, which is the first key event during G1 phase. Therefore, altered MCM expression may be a hallmark of cell cycle deregulation, which is supposed to be the most essential mechanism in the development and progression of bladder cancer. Our aim was to investigate the value of MCMs as proliferation markers and prognostic indicators in detrusor muscle–invasive urothelial bladder carcinomas. We analyzed immunohistochemically the expression of MCM-2 and MCM-5 in 65 patients with detrusor muscle–invasive urothelial bladder carcinomas in relation with clinicopathologic parameters, patients’ overall and disease-free survival, and the expression of the conventional proliferation index Ki-67 and other cell cycle modulators (p53, pRb, p21WAF1, and p27Kip1). The levels of MCM-2 and MCM-5 were significantly higher in high-grade ( P b .0001), advanced-stage ( P = .001), and nonpapillary tumors ( P b .0001). The expression of MCM-2 and MCM-5 significantly associated with the conventional proliferation index Ki-67 ( P = .0001 for each protein). The expression of MCM-2 or MCM-5 positively correlated with p53 labeling index ( P = .014 and P = .009, respectively). Also, median p21WAF1 labeling index was higher in MCM-5 high expressors ( P = .028). Finally, both MCM-2 and MCM-5 associated significantly with adverse patients’ outcome in both univariate ( P = .0072 and P = .0074, respectively) and multivariate ( P = .0001) analysis. In conclusion, MCM-2 and MCM-5 appear to be reliable proliferation indexes and useful prognostic markers in patients with muscle-invasive urothelial bladder carcinomas. D 2005 Elsevier Inc. All rights reserved.

T Corresponding author. E-mail address: [email protected] (P. Korkolopoulou). 0046-8177/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2005.06.008

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1. Introduction Evaluation of cell cycle regulators has gained special interest in the effort to increase the amount of prognostic information in malignant tumors [1]. Minichromosome maintenance proteins (MCMs) drive the formation of prereplicative complexes (PRCs), which is the first key event during G1 phase [2]. Therefore, altered MCM expression may be a hallmark of cell cycle deregulation, which is supposed to be the most essential mechanism in the development and progression of bladder cancer [1]. In all eukaryotic cells, initiation of DNA synthesis is a complex multistep process tightly coupled to progression through the cell cycle [3]. In the first step, during the G1 phase, key replication initiation factors, including the origin recognition complex, the Cdc6 protein and MCM-2 to MCM-7 are recruited into PRCs at future replication origins, establishing the competence of particular chromatin regions for initiation of DNA synthesis [2,4-6]. The MCMs are highly conserved proteins presumed to act as an enzymatically active helicase [4]. In the second step, unwinding of replication origins and establishment of bidirectional replication forks take place to control entry into the S phase [4]. As the DNA is replicated, MCMs gradually dissociate from chromatin, ensuring that each region of DNA is replicated only once during a single cell cycle because replicated DNA lacks functional PRCs [4]. As the cells leave the cell cycle to enter the quiescent, differentiated, and senescent states, the Cdc6 and MCM components of the replication initiation pathway are down-regulated [7,8]. If the MCMs play a critical role in initiation of DNA synthesis, their expression is expected to correlate with cell proliferation when DNA replication must precede each cell division [2]. Concerning bladder cancer, although there are undoubtedly many aspects of its molecular etiology that remain undefined, it is conceivable that perturbation in cell cycle regulation constitutes the most essential and irrevocable feature of bladder tumorigenesis, upon which all oncogenic influences must ultimately converge [1]. Therefore, the hypothesis tested in the present study was whether immunohistochemical expression of MCMs could provide an accurate means of determining cellular proliferation in bladder carcinomas, in comparison with the expression of conventional proliferation indexes and cell cycle regulators. Our study was focused on detrusor muscle–invasive urothelial bladder carcinomas expected to demonstrate a higher proliferation rate. Moreover, gaining insight into the molecular determinants of the biologic behavior of detrusor muscle–invasive bladder cancer is imperative to identify groups of patients with a particularly poor prognosis. Among the 6 best-known MCM subtypes (MCM-2 to MCM-7) and the recently identified MCM-10 nuclear factor, MCM-2 immunohistochemical expression has been reported to bear some prognostic relevance in stage T1 bladder carcinomas [9], whereas detection of MCM-5 has been detected as a valuable diagnostic tool in urine specimens [10]. Given the

P. Korkolopoulou et al. previous data, MCM-2 and MCM-5 were at first place chosen to be investigated in the subgroup of detrusor muscle– invasive urothelial bladder carcinomas with regard to their potential prognostic value. Prognostic information arising from these 2 proteins was compared with that provided by the conventional clinicopathologic parameters as well as G1/S modulators to test the effectiveness of a potential future use in the routine evaluation of patients with cancer.

2. Material and methods 2.1. Patients This is a retrospective study of 65 consecutive patients with primary detrusor muscle–invasive (T2-T4) N0 M0 urothelial carcinomas (UCs), who presented to Asklepeion Voula Hospital in Athens between 1985 and 1995, for whom paraffin-embedded tissue and clinical information were available. There were 58 men and 7 women with a median age of 74 years (range, 39-93 years). By the time this study was undertaken, 25 patients had died of their disease after a median survival of 22 months (range, 6-92 months). The median follow-up for the remaining 40 patients was 39 months (range, 3-73 months). Thirty-five patients relapsed after a median period of 10 months (range, 2-58 months), whereas the recurrence-free survival for nonrelapsing patients was 30 months (range, 12-60 months). According to our standard clinical protocols between 1985 and 1995, T2 to T4 carcinomas were treated with maximal transurethral resection (TUR) followed by systemic chemotherapy and radiation (n = 46 T2 cases) or cystectomy (all T3-T4 cases and 3 T2 cases). None of the patients had received chemotherapy or radiation before TUR. The staging of tumors was reviewed to conform to Union Internationale Contre le Cancer standards of 2002 [11] and was based on the combination of clinical and histological data, that is, cystoscopy, intravenous urography, computed tomography, ultrasonography, and histological examination, to determine the presence of invasion into or beyond the detrusor muscle. The histological samples consisted of TUR material. In all cases, detrusor muscle was included in the specimens and invasion was diagnosed or confirmed on histology. None of the other investigations diagnosed an invasion of detrusor muscle not seen on histology. All cases were reviewed and assigned a histological grade according to the World Health Organization classification of urothelial neoplasms [12]. In each case, the tumor configuration (papillary status) was also recorded. There were 22 papillary and 43 nonpapillary UCs. The clinicopathologic characteristics of our cases are shown in Table 1.

2.2. Processing of specimens and immunohistochemistry Tissues were fixed immediately after removal in 10% buffered formalin and processed to paraffin wax. Four-

MCM-2 and MCM-5 expression in urothelial cancer

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Table 1 Clinicopathologic characteristics of patients with muscle invasive urothelial bladder carcinomas No. of patients Age (y) (median, range) Sex (male/female) Configuration (papillary/nonpapillary) Histological grade (low/high) Stage (T2/T3/T4)

65 74, 39-93 58:7 22:43 25:40 49:9:7

micrometer serial sections were cut from each specimen on superfrost plus glass slides and left to dry overnight at 378C. Staining for MCM-2 was successful in 60 cases and for MCM-5 in 50 cases. In addition, all cases were stained for Ki-67 and p53. p27Kip1 and p21WAF1 immunostaining was available in 59 cases, whereas pRb staining was available in 64 cases. The following monoclonal antibodies were used: (1) MCA 1859 for MCM-2 (clone CRCT2.1/D1.9H5) (Serotec Ltd, Oxford, UK; diluted 1:500, overnight), (2) MCA 1860 for MCM-5 (clone CRCT5.1/A2.7A3) (Serotec Ltd; diluted 1:200, overnight), (3) MIB-1 for Ki-67 (YLEM, Rome, Italy; purchased prediluted), (4) DO-1 for p53 (Oncogene Science, Uniondale, NY; diluted 1:80, 1 hour), (5) 4D10 for p21WAF1 (Novocastra, Burlingame, Conn; diluted 1:20, overnight), (6) 1B4 for p27Kip1 (Novocastra; diluted 1:60, overnight), and (7) PMG3-245 for pRb (Pharmingen BD, Heidelberg, Germany; diluted 1:400, 18 hours). For MCM-2, MCM-5, Ki-67, p27Kip1, and p21Cip1 detection, antigen retrieval with microwaving for 30 minutes, at 750 W, in citrate buffer of pH 6.0, was required. For p53 detection, sections were treated with target unmasking fluid (Kreatech, Amsterdam, The Netherlands) at 908C for 15 minutes. For pRb, a high-temperature antigenunmasking technique was used, as previously described [13]. The standard 3-step streptavidin-peroxidase technique was used with DAKO Envision kit (DAKO, Carpinteria, Calif). Known positive controls (a normal tonsil for MCM-2, MCM-5, Ki-67, and p27Kip1; a colorectal carcinoma for

p53; a previously irradiated piece of skin for p21WAF1; and a breast carcinoma for pRb) as well as negative controls (sections in which the primary antibody was substituted by nonimmune mouse serum) were also stained in each run. Staining for all antibodies was assessed blindly (ie, without any knowledge of the clinical data) by 2 observers. Whenever a difference of greater than 5% between the 2 assessments was observed, slides were reviewed jointly and a consensus was reached. Nuclei from about 1000 tumor cells from systematically randomized fields (original magnification 40) throughout the entire section were counted, and the labeling index (LI) was calculated as the percentage of labeled nuclei of the total number of tumor cells counted. Whenever heterogenous staining was encountered, counting was performed in areas of highest density of labeled cells, identified at medium magnification (original magnification 20), because it has been proposed that these tumor areas are most likely to be of biologic significance [14 -16]. All clearly identifiable nuclear staining beyond background was recorded as positive for MCM-2, MCM-5, Ki-67, and

Fig. 1 MCM-5 immunohistochemical expression in numerous neoplastic nuclei of a high-grade UC (streptavidin-peroxidase, original magnification 200).

Fig. 3 Distribution of MCM-2 immunohistochemical expression mainly in the basal layers of a low-grade UC (streptavidinperoxidase, original magnification 100).

Fig. 2 MCM-5 immunohistochemical expression in a few nuclei of a low-grade UC (streptavidin-peroxidase, original magnification 200).

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P. Korkolopoulou et al. distribution. Therefore, the relationship between p27Kip1 LI or pRb LI and grade or 2-tiered stage was tested with Student t test. The Mann-Whitney U test was used to compare central tendencies in the distribution of the remaining numerical variables between grades and papillary status categories. Associations between numerical parameters and stage were assessed with Kruskal-Wallis analysis of variance. Spearman rank correlation coefficient was calculated to determine associations between numerical variables. The prognostic effect of various parameters (ie, age, sex, grade, T category, type of treatment, Ki-67 LI, MCM-2 LI, MCM-5 LI, and p53, p27Kip1, p21WAF1, and pRb expression) on clinical outcome (ie, death of disease or recurrence) was tested by plotting survival curves according to KaplanMeier method and comparing groups using the log-rank test, as well as by multivariate analysis. Patients dying of other causes during the follow-up period were treated as censored data. Disease-free survival was defined as time from diagnosis until documentation of recurrent disease with cystoscopy and biopsy. In univariate analysis, the continuous variables (age, MCM-2 LI, MCM-5 LI, KI-67, p21WAF1, p27Kip1, and pRb) were categorized on the basis of the median value. p53 expression was categorized as positive versus negative, using 1% as cutoff value [17]. To avoid any bdata-drivenQ categorization, continuous variables were entered in multivariate analysis as continuous variables. Given that MCM-2 and MCM-5 were strongly interrelated, their significance as independent prognosticators was tested in separate multivariate analyses.

Fig. 4 Numerous MCM-2–positive nuclei in a high-grade UC (streptavidin-peroxidase, original magnification 400).

p21WAF1, but for p53 and p27Kip1, the threshold of positivity was raised to 1% [17-19]. pRb expression was considered to be aberrant if staining was seen in less than 50% of neoplastic population [13]. No endothelial or lymphoid cells were included in the counts although they expressed Ki-67, MCM-2, MCM-5, and p27Kip1.

2.3. Statistical analysis The normality of distributions was tested with the Kolmogorov-Smirnov test. Logarithmic transformation of p27Kip1 LI and pRb LI was necessary to obtain normal

Table 2 MCM-2, MCM-5, Ki-67, proliferating cell nuclear antigen, p53, p27Kip1, p21WAF1, pRb, and p16 immunohistochemical expression according to histological grade, stage, and tumor configuration Grade Low MCM-2 Median P MCM-5 Median P Ki-67 Median P p53 Median P p27Kip1 Median P p21WAF1 Median P pRb Median P

Stage High

T2

Configuration T3-T4

Papillary

Nonpapillary

35 (5-70)

9 (2-30) b.0001

30 (5-70)

29.5 (10-70)

12 (2-20) b.0001

25 (5-70)

(range) (%)

10 (2-60) b.0001

30 (5-70)

15 (2-70) .001

(range) (%)

15 (2-50) b.0001

23 (10-70)

16.5 (2-45) .001

(range) (%)

10 (0.5-30) .001

19 (2-60)

12 (5-45) .11

20 (1-60)

8.5 (2-12) b.0001

20 (1-60)

(range) (%)

>0.0001 (0-60) .072

0.0001 (0-70) .001

5.5 (0-60)

0.0001 (0-8) .002

0.2 (0-70)

(range) (%)

65 (0-100) .132

45 (0.5-99)

45 (0-100) .041

4 (5-99)

61 (0-100) .28

45 (5-99)

(range) (%)

0.2 (0-60) .55

0.5 (0-90)

0.2 (0-90) .430

0.5 (0-10)

0.4 (0-60) .91

0.1 (0-90)

(range) (%)

9 (0.5-85) .74

10 (0-95)

8 (0-85) .73

12.5 (0-95)

10 (2-85) .13

8 (0-95)

0.150 (0-70)

MCM-2 and MCM-5 expression in urothelial cancer

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Statistical analysis was performed using the SPSS for Windows Software (SPSS Inc, Chicago, Ill). A P value of .05 or less was considered indicative of a statistically significant difference.

3. Results 3.1. Immunostaining of MCM-2 and MCM-5 Immunoreactivity for MCM-2 and MCM-5 was seen in all cases ranging from 2% to 70%, with a median of 18% and 20%, respectively. The pattern of staining was mostly nuclear, although a faint cytoplasmic staining was seen in some cases, which was disregarded as nonspecific (Figs. 1- 4). Positive cells were randomly distributed, except for papillary areas in which MCM-2 and MCM-5 preferentially stained the basal cell layers (Fig. 3). When analyzing the relationship between MCM-2 or MCM-5 expression and grade or T category, a significant difference was registered, inasmuch as high-grade and T3 to T4 tumors displayed a higher expression level than the remaining cases (Mann-Whitney U test, P b .0001 for either MCM-2 or MCM-5 and tumor grade, and P = .001 for either MCM-2 or MCM-5 and stage, respectively) (Table 2) (Figs. 1-4). A strong positive correlation also emerged between MCM-2 and MCM-5 expression (Spearman q = 0.862, P = .0001).

3.2. Relationship of MCM-2 LI and MCM-5 LI with proliferation rate To investigate the relation between MCM-2 or MCM-5 expression and tumor’s proliferative potential, we stained our cases for Ki-67. As expected [17], Ki-67 LI increased with increasing grade (Mann-Whitney U test, P = .001).

Fig. 5 Kaplan-Meier overall survival curves according to the immunohistochemical expression of MCM-2 categorized on the basis of the median value (18%). Increased (z18%) MCM-2 expression is significantly associated with poorer overall survival.

Fig. 6 Kaplan-Meier overall survival curves according to the immunohistochemical expression of MCM-5 categorized on the basis of the median value (20%). Increased (z20%) MCM-5 expression is significantly associated with poorer overall survival.

However, the association between Ki-67 LI and stage did not reach statistical significance (Table 2). A statistically significant strong positive correlation was established between MCM-2 or MCM-5 expression level and Ki-67 LI (Spearman q = 0.742 and P = .0001, and q = 0.773 and P = .0001, respectively).

3.3. Expression of p27Kip1, p21WAF1, p53, and pRb Positivity for p27Kip1 was seen in all cases but one and, for p21WAF1, in 50.8% of cases. p53 accumulation was recorded in 50.8% of cases. Altered pRb expression was recorded in 50% of cases. Inflammatory and endothelial cells as well as nonneoplastic urothelium expressed strongly p27Kip1 and pRb but were negative for p53 and p21WAF1.

Fig. 7 Kaplan-Meier overall survival curves according to the immunohistochemical expression of p27Kip1 categorized on the basis of the median value (45%).

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3.5. Survival analysis

Fig. 8 Kaplan-Meier disease-free survival curves according to the immunohistochemical expression of p21WAF1 categorized on the basis of the median value (0.3%).

The pattern of staining for all 4 was nuclear, often with a granular quality. Cells in mitosis were uniformly negative. Significantly higher p53 LI and lower p27Kip1 LI were recorded in T3 to T4 tumors as opposed to T2 tumors (Mann-Whitney U test, 0.001, and t test, 0.041, respectively) (Table 2). The relationship between p53 LI and grade was of marginal significance (Mann-Whitney U test, P = .072) (Table 2).

3.4. Correlations between MCM-2 or MCM-5 expression and p21WAF1, p27Kip1, p53, or pRb expression The expression of MCM-2 or MCM-5 positively correlated with p53 LI although the correlation was rather weak (Spearman q = 0.317, P = .014, and q = 0.366, P = .009, respectively). Also, median p21WAF1 LI was higher in high (z20%) MCM-5 expressors as compared with that in low MCM-5 expressors (Mann-Whitney U test, P = .028).

Table 3

In univariate analysis, the parameters adversely affecting survival were high grade ( P = .0098), advanced T category ( P b .0001), increased (z18%) MCM-2 expression ( P = .0072), increased (z20%) MCM-5 expression ( P = .0074), and increased (z15%) Ki-67 expression ( P = .0023), whereas decreased p27Kip1 expression attained a marginal significance in this regard ( P = .0782) (Figs. 5-7). Accordingly, the only parameters adversely affecting disease-free survival were absence of p21WAF1 expression ( P = .0047) (Fig. 8) and advanced stage ( P = .0303). In multivariate Cox proportional hazard analysis of overall survival, MCM-2 expression emerged as an independent predictor of adverse outcome ( P = .007) along with advanced stage (Table 3 [A1]). MCM-5 failed to emerge as a significant prognosticator ( P = .067) when compared with conventional clinicopathologic parameters (Table 3 [B1]). When the other cell cycle regulators were also introduced in the model, MCM-2 emerged as the only independent predictor of overall survival ( P b .0001) (Table 3 [A2]), whereas MCM-5 demonstrated a strong independent association with overall survival ( P b .0001) along with p53 overexpression ( P = .001) (Table 3 [B2]). As far as disease-free survival is concerned, multivariate analysis demonstrated patients’ age and p21WAF1 immunohistochemical expression as the only independent prognostic factors ( P = .021 and P = .044, respectively) (Table 3 [C1]). When the other cell cycle regulators were removed from the model, T category emerged as the only significant parameter independently affecting disease-free survival ( P = .015) (Table 3 [C2]). Neither MCM-2 nor MCM-5 was a significant parameter in this regard.

4. Discussion In the present study, we have clearly demonstrated the nuclear expression of MCM-2 and MCM-5 in muscleinvasive UCs. A faint cytoplasmic staining was also seen in

Cox proportional hazard estimation of overall and disease-free survival in muscle-invasive UCs

Covariate Overall survival A1. MCM-2 Stage B1. Stage A2. MCM-2 B2. p53 MCM-5 Disease-free survival C1. Age p21WAF1 C2. Stage

Coefficient

SE

P

Relative risk

95% confidence limits for relative risk Lower

Upper

1.031 0.650 1.124 1.948 0.057 0.100

0.382 0.285 0.364 0.517 0.018 0.026

.007 .023 .002 .0001 .001 .0001

2.804 1.916 3.078 7.012 1.058 1.105

1.327 1.095 1.507 2.547 1.023 1.050

5.925 3.352 6.288 19.299 1.095 1.162

0.061 1.093 1.062

0.027 0.542 0.438

.021 .044 .015

1.063 0.335 2.891

1.009 0.116 1.225

1.120 0.969 6.821

MCM-2 and MCM-5 expression in urothelial cancer some cases, which although disregarded as nonspecific, has also been reported in other studies [20-22]. The possible functional role of nonnuclear MCM expression merits further investigation. The significant association between MCM-2 and MCM-5 expression reflects the similar kinetics of these proteins, being up-regulated to accomplish their proliferative task during cancer progression and downregulated during withdrawal of mammalian cells from the cell cycle [7,21]. Recently, several groups have reported that MCM immunoreactivity is a specific marker for proliferating cells [21,23-25]. In contrast to a previous study in stage T1 UCs, where a tendentious correlation was found between MCM-2 and Ki-67 antigen expression [9], in the present study, a statistically significant association was established in muscle-invasive UCs between MCM-2 or MCM-5 expression and Ki-67 LI, mean values of MCM-2 and MCM-5 being higher than those observed for Ki-67, obviously because Ki-67 fails to label cells in early G1 [26-28]. This pattern has also been described in other neoplasms or premalignant conditions [7,8,14-16,26]. Therefore, antibodies to MCMs offer a potentially more accurate means of determining the proliferative fraction within a tumor than the conventional proliferative indexes [7]. Other advantages of MCMs are that they do not detect cells undergoing DNA repair and they are not down-regulated in proliferating cells by nutritional deprivation, which may be operating regionally in solid tumors [26]; their function has been well characterized in several in vitro systems [2] representing a point of convergence for numerous signaling pathways involved in cell growth [7]; and their levels remain constant during the cell cycle, decreasing markedly in cells with a lower proliferation rate [29]. As far as the relationships of MCMs with the other cell cycle modulators are concerned, weak positive correlations emerged between MCM-2 or MCM-5 and p21WAF1 as well as p53. Given the major function of p21WAF1 as an inhibitor of cell cycle progression, its association, although weak, with MCMs seems rather contradictory. However, the elevation of p21WAF1 levels in tumors, characterized by higher proliferative potential, may indicate a tendency of these neoplasms to develop a feedback control mechanism of growth inhibition through p21WAF1 induction [18]. Moreover, as we have already suggested in a previous study in bladder carcinomas [18], elevated p21WAF1 is not sufficient to arrest cells at the G1 checkpoint, probably because p21WAF1 complexes still have kinase activity due to altered stoichiometric regulation [18]. Given that p53 immunoreactivity in UCs has been regarded as a surrogate marker for, although not a proof of, gene mutation or inactivation, the observed correlation between MCM-5 and p53 might be indicative of the positive effect exerted by mutant-type p53 on cell cycle progression. In the present study MCM-2 and MCM-5 expression increased with increasing tumor grade and advancing stage. In a previous study in stage T1 urothelial bladder

905 carcinomas, a significant association was also found between MCM-2 expression and tumor grade inasmuch as high-grade tumors displayed a higher MCM-2 immunoreactivity [9]. Significant associations between MCM overexpression and high grade have also been described in prostate [20] and renal carcinomas [15] as well as in oligodendrogliomas [14]. Moreover, in vitro studies have clearly shown a dramatic decrease in the levels of MCM-2 messenger RNA during the differentiation of human myeloblast HL60 cells [30], which supports our findings. In a variety of dysplastic conditions, MCMs were more highly expressed than Ki-67 [31]. It has been demonstrated that deregulated expression of these proteins is characteristic of dysplasia in cervical squamous neoplasia [8] and also in urothelial neoplasia of the bladder [10]. Therefore, unlike other markers whose up-regulation in high-grade and advanced-stage tumors implies a late event in tumor cancer progression, MCM up-regulation in dysplastic states as well as in higher grade tumors suggests an early event that either is accentuated during dedifferentiation or, from the beginning, provides high-grade cells with a proliferative benefit, enabling them to evolve into highgrade carcinomas. A main scope of our study was to investigate the prognostic relevance of MCM-2 and MCM-5 expression in muscle-invasive UCs. In both univariate and multivariate survival analysis, overexpression of each protein associated significantly with poor overall patients’ survival. The confirmation of the adverse prognostic effects of a notorious group of universally established prognostic factors (grade, stage, and configuration) proves that our cohort was quite representative and that survival analysis was valid. We report for the first time in muscle-invasive urothelial bladder carcinoma the adverse prognostic significance of MCM-2 overexpression, in keeping with previous studies in other malignant neoplasms [14,15,20,32,33]. However, various cutoffs have been used in the different studies to determine MCM-2 overexpression. These cutoffs ranged from 10% and 11.5% [14,20] to 50% [32]. We applied in our study a cutoff of 18% to 20% to compare survival groups, in keeping with that used by Krqger et al [9] in stage T1 UCs to examine progression-free survival of patients. Indeed, in the latter study, MCM-2 overexpression, categorized at a cutoff of 20%, was the only parameter with significant predictive value as far as progressionfree survival was concerned. However, the variation of cutoffs with prognostic relevance found in the different neoplasms implies the need of individualization, according to tumor and/or tissue type, before possible incorporation of MCMs into the routine evaluation of patients with cancer. In contrast to MCM-2 expression whose adverse prognostic significance has been demonstrated in other types of neoplasms, MCM-5 prognostic significance in a neoplastic condition has not been examined so far. Its expression has been investigated mainly in the context of

906 dysplastic and neoplastic conditions as a diagnostic marker [8,10,22,34]. According to our results, MCM-5 expression may additionally prove to be useful as a predictive factor of decreased overall survival in patients with muscle-invasive urothelial bladder carcinomas when compared with other cell cycle regulators. As far as the prognostic value of other cell cycle modulators is concerned, of note is that Ki-67 expression demonstrated a statistically significant correlation with decreased overall survival in univariate but not in multivariate analysis. In multivariate analysis, p53 expression emerged as an independent factor, adversely influencing overall survival, in keeping with previous reports on invasive UCs [35]. As far as disease-free survival is concerned, multivariate analysis demonstrated patients’ age and p21WAF1 immunohistochemical expression as the only independent parameters influencing patients’ disease-free survival, strengthening the results of our previous study [18]. In conclusion, in the present study, we have investigated for the first time MCM-2 and MCM-5 expression in muscleinvasive bladder carcinoma in relation with clinicopathologic parameters, cell cycle modulators, and patients’ survival. Both proteins associated significantly with highgrade and advanced-stage carcinomas as well as with Ki-67 proliferative index. However, a higher proportion of proliferative cells was detected using MCM antibodies, probably reflecting cells in early G1 and suggesting that MCMs offer a more accurate means of determining cells in cycle. Moreover, MCM-2 and MCM-5, but not Ki-67, emerged as independent predictors of poor overall survival, implying that these proteins may prove to be useful prognostic markers in patients with muscle-invasive urothelial bladder carcinomas. Finally, the role of MCMs in muscle-invasive urothelial cancer may not be limited to that of a prognostic marker but may direct avenues of research and therapeutic interventions.

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[9]

[10]

[11] [12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

References [20] [1] Zlotta AR, Schulman CC. Biological markers in superficial bladder tumours and their prognostic significance. Urol Clin North Am 2000; 27:179-189, xi-xii. [2] Tye BK. MCM proteins in DNA replication. Annu Rev Biochem 1999;68:649 - 86. [3] Ritzi M, Knippers R. Initiation of genome replication: assembly and disassembly of replication-competent chromatin. Gene 2000;245: 13 - 20. [4] Labib K, Diffley JF. Is the MCM2-7 complex the eukaryotic DNA replication fork helicase? Curr Opin Genet Dev 2001;11:64 - 70. [5] Lei M, Tye BK. Initiating DNA synthesis: from recruiting to activating the MCM complex. J Cell Sci 2001;114:1447 - 54. [6] Yu Z, Feng D, Liang C. Pairwise interactions of the six human MCM protein subunits. J Mol Biol 2004;340:1197 - 206. [7] Stoeber K, Tlsty TD, Happerfield L, Thomas GA, Romanov S, Bohrow L, et al. DNA replication licensing and human cell proliferation. J Cell Sci 2001;114:2027 - 41. [8] Williams GH, Romanowski P, Morris L, Madine M, Mills AD, Stoeber K, et al. Improved cervical smear assessment using antibodies

[21]

[22]

[23]

[24]

against proteins that regulate DNA replication. Proc Natl Acad Sci U S A 1998;95:14932 - 7. Krqger S, Thorns C, Stfcker W, Mqller-Kunert E, Bfhle A, Feller A. Prognostic value of MCM2 immunoreactivity in stage T1 transitional cell carcinoma of the bladder. Eur Urol 2003;43:138 - 45. Stoeber K, Halsall I, Freeman A, Swinn R, Doble A, Morris L, et al. Immunoassay for urothelial cancers that detects DNA replication protein MCM5 in urine. Lancet 1999;354:1524 - 5. Greene FL, Page DL, Fleming ID, Fritz AG, Balch CM, Haller DG, et al. AJCC cancer staging manual. New York7 Springer-Verlag; 2002. Eble N, Sauter G, Epstein JI, Sesterhenn A. The World Health Organization classification of tumours. Pathology and genetics. Tumours of the urinary system and male genital organs. Lyon7 AIRC Press; 2004. p. 90. Korkolopoulou P, Christodoulou P, Lazaris A, Thomas-Tsagli E, Kapralos P, Papanikolaou A, et al. Prognostic implications of aberrations in p16/pRb pathway in urothelial bladder carcinomas: a multivariate analysis including p53 expression and proliferation markers. Eur Urol 2001;39:167 - 77. Wharton SB, Chan KK, Anderson JR, Stoeber K, Williams GH. Replicative Mcm2 protein as a novel proliferation marker in oligodendrogliomas and its relationship to Ki67 labeling index, histological grade and prognosis. Neuropathol Appl Neurobiol 2001;27:305 - 13. Rodins K, Cheale K, Coleman N, Fox SB. Minichromosome maintenance protein 2 expression in normal kidney and renal cell carcinomas: relationship to tumor dormancy and potential clinical utility. Clin Cancer Res 2002;8:1075 - 81. Hunt DPJ, Freeman A, Morris LS, Burnet NG, Bird K, Davies TW, et al. Early recurrence of benign meningioma correlates with expression of mini-chromosome maintenance–2 protein. Br J Neurosurg 1993;58:51 - 6. Korkolopoulou P, Christodoulou P, Konstantinidou A-E, ThomasTsagli E, Kapralos P, Davaris P. Cell cycle regulators in bladder cancer: a multivariate survival study with emphasis on p27Kip1. Hum Pathol 2000;31:751 - 60. Korkolopoulou P, Konstantinidou A-E, Thomas-Tsagli E, Christodoulou P, Kapralos P, Davaris P. WAF1/p21 expression is an independent prognostic indicator in superficial and invasive bladder cancer. Appl Immunohistochem Mol Morphol 2000; 8:285 - 92. Korkolopoulou P, Christodoulou P, Kapralos P, et al. The role of p53, MDM2 and c-erbB2 oncoproteins, epidermal growth factor receptor and proliferation markers in the prognosis of urinary bladder cancer. Pathol Res Pract 1998;193:767 - 75. Meng MV, Grossfeld GD, Williams GH, Dilworth S, Stoeber K, Mulley TW, et al. Minichromosome maintenance protein 2 expression in prostate: characterization and association with outcome after therapy for cancer. Clin Cancer Res 2001;7:2712 - 8. Heidebrecht HJ, Buck F, Endl E, Kruse ML, Adam-Klages S, Andersen K, et al. Ki-Mcm6, a new monoclonal antibody specific to Mcm6: comparison of the distribution profile of Mcm6 and the Ki67 antigen. Lab Invest 2001;81:1163 - 5. Going JJ, Keith WN, Neilson L, Stoeber K, Stuart RC, Williams GH. Aberrant expression of minichromosome maintenance proteins 2 and 5, and Ki-67 in dysplastic squamous oesophageal epithelium and Barrett’s mucosa. Gut 2002;50:373 - 7. Kodan I, Shomori K, Osaki M, Kuratate I, Ryoke K, Ito H. Expression of minichromosome maintenance 2 (MCM2), Ki-67 and cell cycle– related molecules, and apoptosis in the normal-dysplasia-carcinoma sequence of the oral mucosa. Pathobiology 2001;69:150 - 8. Todorov IT, Werness BA, Wang HQ, Buddharaju LN, Todorova PD, Slocum HK, et al. HsMCM2/BM28: a novel proliferation marker for human tumors and normal tissues. Lab Invest 1998; 78:73 - 8.

MCM-2 and MCM-5 expression in urothelial cancer [25] Freeman A, Morris LS, Mills AD, et al. Minichromosome maintenance proteins as biological markers of dysplasia and malignancy. Clin Cancer Res 1999;5:2121 - 32. [26] Osaki M, Osaki M, Yamashita H, Shomori K, Yoshida H, Ito H. Expression of minichromosome maintenance–2 in human malignant fibrous histiocytomas: correlations with Ki-67 and p53 expression, and apoptosis. Int J Mol Med 2002;10:161 - 8. [27] Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol 2000;182:311 - 22. [28] MacCallum DE, Hall PE. The location of pKi67 in the outer dense fibrillary compartment of the nucleolus points to a role in ribosome biogenesis during the cell division cycle. J Pathol 2000;190:537 - 44. [29] Todorov IT, Pepperkok R, Philipova RN, Kearsey SE, Ansorge W, Werner D. A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division. J Cell Sci 1994;107:253 - 65. [30] Philipova RN, Vassilev AP, Kaneva RP, et al. Expression of the nuclear protein mitotin in differentiating in vitro HL60 cells. Biol Cell 1991;72:47 - 50.

907 [31] Alison MR, Hunt T, Forbes SJ. Minichromosome maintenance (MCM) proteins may be pre-cancer markers. Gut 2002;50:290 - 1. [32] Gonzalez MA, Pinder SE, Callagy G, Vowler SL, Morris LS, Bird K, et al. Minichromosome maintenance protein 2 is a strong independent prognostic marker in breast cancer. J Clin Oncol 2003; 21:4306 - 13. [33] Ramnath N, Hernandez FJ, Tan D-F, Huberman JA, Natarajan N, Beck AF, et al. MCM2 is an independent predictor of survival in patients with non–small-cell lung cancer. J Clin Oncol 2001;22: 4259 - 66. [34] Williams GH, Swinn R, Prevost AT, De Clive–Lowe P, Halsall I, Going JJ, et al. Diagnosis of oesophageal cancer by detection of minichromosome maintenance 5 protein in gastric aspirates. Br J Cancer 2004;91:714 - 9. [35] Esrig D, Elmajian D, Groshen S, Freeman JA, Stein JP, Chen S-C, et al. Accumulation of nuclear p53 and tumor progression in bladder cancer. N Engl J Med 1994;331:1259 - 64.