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Clinical Cancer Research Vol. 12, 2752-2758, May 1, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Biological Characteristics in Bladder Cancer Depend on the Type of Genetic Instability

Yoshiaki Yamamoto1,2, Hideyasu Matsuyama1, Shigeto Kawauchi2, Tomoko Furuya2, Xiu Ping Liu2, Kenzo Ikemoto2, Atsunori Oga2, Katsusuke Naito1 and Kohsuke Sasaki2

Authors' Affiliations: Departments of 1 Urology and 2 Pathology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan

Requests for reprints: Kohsuke Sasaki, Department of Urology, Yamaguchi University School of Medicine, Ube 755-8505, Yamaguchi, Japan. Phone: 81-836-22-2222; Fax: 81-836-22-2223; E-mail: kohsuke{at}yamaguchi-u.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Malignant tumors show an inherent genetic instability that can be classified as microsatellite instability (MSI) or chromosomal instability (CIN). To elucidate the differences in biological characteristics of bladder cancer between the two types of genetic instability, the expression of the mismatch repair (MMR) proteins, Aurora-A and p53 proteins, the number of centrosomes, numerical aberrations of chromosomes and 20q13, and DNA ploidy were examined in 100 human urothelial carcinomas of the bladder.

Experimental Design: Expressions of the MLH1, MSH2, Aurora-A, and p53 proteins and the numbers of centrosomes were immunohistochemically assessed. Numerical aberrations of chromosomes 7, 9, 17, and 20q13 spots were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed by laser scanning cytometry.

Results: The expression levels of the MMR related-proteins decreased in 9 of 100 tumors. Tumors with low MLH1 or MSH2 expression (designated as MSI cancers) were not linked with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, 20q13 gain, DNA aneuploidy, and disease progression. MSI cancers showed a favorable prognosis. CIN cancers (49 cases), defined as tumors with a large intercellular variation in centromere copy numbers, were associated more frequently with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, and 20q13 gain than the others (51 cases). Tumors with disease progression were included in the CIN cancer group.

Conclusions: The present observations suggest that there are differences in the biological characteristics of the two types of genetic instability.

Urothelial carcinoma of the bladder is the second most common malignancy of the genitourinary tract (1). Approximately three quarters of all bladder cancers are low-grade and noninvasive superficial tumors. These cancers generally show a favorable prognosis with respect to mortality; however, tumor recurrence and disease progression are found in 50% to 70% and 10% to 20% of tumors, respectively (2). These tumors frequently show an unfavorable disease course. Unfortunately, there are few reliable markers to distinguish these tumors from other kinds of tumors.

With tumor progression, genetic aberrations accumulate successively in tumor cells due to the inherent genetic instability of malignant tumors, including urothelial carcinomas. Genetic instability is generally categorized into microsatellite instability (MSI) and chromosomal instability (CIN; ref. 3). Defects in mismatch repair (MMR) genes, including hMLH1 and hMSH2, lead directly to the development of MSI. Immunohistochemical determination of MLH1 and MSH2 has been used as a surrogate for MSI determination in colorectal cancer (4–6). MSI accounts for as many as 10% to 20% of sporadic colorectal carcinomas but the frequency of MSI in bladder cancer is controversial (7–14).

The hypothesis that CIN results from abnormalities of genes implicated in mitosis is widely accepted. Centrosome amplification (15–17), overexpression of STK15/BTAK/Aurora-A kinase located on 20q13 (18–20), and p53 mutation and cyclin E overexpression (21) are closely linked with CIN (22) in human malignancies including bladder cancer (23–25). However, in respect to the relationship between genetic instability and the characteristics of urothelial carcinomas, there is much room for argument.

To elucidate the differences in biological characteristics between the two types of genetic instability in bladder cancers, we examined the status of MMR protein expression and abnormalities of CIN-related molecular markers, and compared them with clinicopathologic variables in 100 cases of bladder cancer.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and tissue specimens. Small tissue specimen were obtained from 100 consecutive cases of urothelial carcinoma of the bladder [78 men and 22 women; average age, 69.6 years (range, 33-95 years)] by transurethral resection of the bladder cancer (TURBT, 91 tumors), radical cystectomy (4 tumors), and transurethral bladder biopsy (5 tumors) between 1996 and 2005. Tumor tissue specimens were frozen and stored in a freezer at –80°C until use. The study was approved by the institutional ethical committee at the Yamaguchi University School of Medicine (registration 69), and written informed consent was obtained from each patient. Tumors were graded and staged according to WHO criteria (26) and tumor-node-metastasis classification (27), respectively. The pathologic grade was 1, 2, and 3 for 5, 54, and 41 tumors, respectively. The pathologic stage was pTa, pT1, pT2a, pT2b, pT3a, and pT4 for 21, 63, 8, 3, 4, and 1 tumor, respectively. None of the patients had any history of chemotherapy/radiation therapy before surgery. Patients who underwent bladder preservation surgery underwent a cystoscopic examination as well as urine cytology every 3 months. Those who had undergone cystectomy were examined by routine chest plain radiography and a computerized tomography scan every 6 months and by bone scintigraphy every year. Disease progression was defined as a recurrent tumor having a more advanced stage or higher grade than the primary tumor or when a distant metastasis was radiographically detected. Patient prognosis could be evaluated in 87 pT2 or less tumors that had curative surgery. Tumor recurrence and disease progression were detected in 45 (51.7%) and 20 (23.0%) tumors, respectively, with a mean follow-up period of 41.6 months that ranged from 6 to 94 months. Touch preparations were generated by gently touching each specimen to a glass slide, followed by air-drying at room temperature for 30 minutes, and then processed for immunofluorescence experiments and fluorescence in situ hybridization analysis.

Immunohistochemical staining. Formalin-fixed paraffin-embedded tissue specimens were used for immunohistochemical staining. Specimens were pretreated with microwave irradiation for 14 minutes in 0.01 mol/L citrate-buffered saline (pH 6.0) for antigen retrieval. Endogenous peroxidase activity was blocked by incubation with a 0.3% H2O2 solution for 10 minutes. Tissue sections were then incubated with mouse monoclonal antibodies against MLH1 (PharMingen, San Diego, CA), MSH2 (Oncogene, San Diego, CA), p53 (Dako Corporation, Carpentaria, CA), and a rabbit polyclonal antibody against Aurora-A (Calbiochem, Darmstadt, Germany) at 4°C overnight. Streptavidin-biotin complex and horseradish peroxidase were applied and reaction products were visualized using the HISTOFINE SAB-PO (M) or (R) Immunohistochemical Staining Kit (Nichirei, Tokyo, Japan) according to the instructions of the manufacturer. Sections were counterstained with Mayer's hematoxylin. A colon cancer specimen from previous studies, which showed a positive nuclear reaction for MLH1 and MSH2, was used as a positive control. Two observers (Y.Y. and S.K.) independently examined immunostained specimens in a blinded manner.

A tumor in which <30% of the nuclei were immunohistochemically positive for MLH1 or MSH2 (low expression) was categorized as a MSI cancer (8). Immunohistochemical determination of MLH1 and MSH2 has been used as a surrogate for MSI determination in colorectal cancer (4–6). Although the nature of MSI was not genetically confirmed in this study, bladder cancers with low expressions of MLH1 or/and MSH2 were called "MSI cancers" for convenience in this article. If >80% of the nuclei were positively stained, it was considered to be a normal expression (tumors that did not have a low expression were considered to have a normal expression).

Increased immunoreactivity of p53 was defined as >20% of the nuclei positively stained for p53 (28). Overexpression of Aurora-A was defined as >20% of cells counted showing strong cytoplasmic expression of Aurora-A (19).

Immunofluorescence staining. For immunostaining of centrosomes, cells were fixed with 10% formalin/methanol for 20 minutes at 25°C, washed with PBS, and permeabilized with 0.5% Triton X in PBS for 5 minutes at 25°C, followed by a 1-hour incubation with blocking solution (10% normal goat serum in PBS). Cells were then subjected to immunostaining of centrosomes using a mouse monoclonal anti-{gamma}-tubulin antibody (Sigma, St. Louis, MO) as previously described (29). The antibody-antigen complexes were detected with an Alexa 488– or Alexa 568–conjugated goat anti-mouse immunoglobulin G antibody (Molecular Probes, Eugene, OR) for 1 hour at 37°C. The number of centrosome signals in each cell was determined by manually counting >100 cells in each specimen under an epifluorescence microscope (Olympus, Tokyo, Japan) at x1,000 magnification. Centrosome amplification–positive cases were defined as those having >5% of cells with three or more centrosomes per cell as previously described (25).

Fluorescence in situ hybridization. To determine the copy number of 20q13 and the variant fractions for CIN, multicolor fluorescence in situ hybridization techniques were applied to detect specific regions at 20q13 and centromeric regions for chromosomes 7, 9, and 17. Commercially purchased 20q13 probes (LSI ZNF217, 20q13.2 amplicon) labeled with Spectrum Orange (Vysis, Downers Grove, IL) were cohybridized with centromeric probes specific for chromosome 20 (D20Z1) labeled with fluorescein (Q Biogene, Amsterdam, the Netherlands). Numerical aberrations of chromosomes 7, 9, and 17 were detected using CEP 7 (D7Z1), CEP 9, and CEP 17 (Vysis), respectively. The fixation, hybridization, and posthybridization procedures were done according to protocols recommended by the suppliers. More than 100 nuclei were counted under an epifluorescence microscope equipped with triple bandpass filter sets (DAPI/Spectrum Green/Spectrum Orange) in combination with a single bandpass filter (Spectrum Aqua, Vysis).

Tumors were classified as 20q13 gain when >60% of cells had more than five signals per cell for 20q13 (19).

The variant fraction was defined as the fraction of cells of which the chromosome number was different from the modal chromosome number (22). CIN was tentatively defined as >25% of the average variant fractions of chromosomes 7, 9, and 17, with reference to Lengauer et al. (22).

Measurement of nuclear DNA using a laser scanning cytometer. The nuclear DNA content was measured using a laser scanning cytometer (LSC 101; Olympus) as previously described (30, 31). A DNA histogram was generated and DNA ploidy determined. The DNA index was calculated according to published principles (32). Diploid and aneuploid tumors with DNA indices of ≤1.2 and >1.2 were classified as diploid and aneuploid cancers, respectively (33).

Statistical analysis. Statistical analysis was done using JMP 4.0 statistical software (SAS Institute, Cary, NC). A contingency table with either {chi}2 or Fisher's exact probability test was applied for the univariate analysis. The probability of survival was calculated by the Kaplan-Meier method with statistical differences evaluated by the log-rank test. The Cox proportional hazard model was applied for multivariate analysis with a step-down procedure until all the factors remained significant. For all statistical tests, P < 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Of 100 tumors, 49 (49.0%) that showed large intercellular variations in the number of centromeric signals were classified as CIN cancers. Centrosome amplification positivity was detected in 64 (64.0%) of the 100 tumors. Aurora-A was overexpressed in 65 (65.0%) tumors whereas p53 immunoreactivity was increased in 56 (56.0%). The copy number of 20q13 increased in 42 (42.0%) tumors. DNA aneuploidy was detected in 47 (47.0%).

Relationship of MMR protein expression to molecular variables in bladder cancers. MLH1 and MSH2 protein expression decreased in 6 (6.0%) and 5 (5.0%) tumors, respectively (Fig. 1A-D ), and two tumors showed a decrease in expression for both proteins (Table 1 ). In total, 9 (9.0%) tumors were classified as MSI cancers. These tumors were associated with no centrosome amplification (Fig. 1E and F), Aurora-A overexpression (Fig. 1G and H), increased p53 immunoreactivity, 20q13 gain (Fig. 1I and J), DNA aneuploidy, and features of CIN (Table 1). Six patients with a history of upper urinary tract tumor had normal expression levels of MMR protein. Disease progression was not found in MSI cancers.


Figure 1
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  		Fig. 1. Immunohistochemical staining of MLH1, MSH2, and Aurora-A, and representative tumors with and without centrosome amplification and 20q13 gain in bladder cancers. Immunohistochemical staining revealed that positive and negative staining for MLH1 (A and B, respectively) and MSH2 (C and D, respectively) are observed in tumor cell nuclei. E, representative tumor with centrosome amplification positive. Note the four signals for centrosomes (red). F, representative tumor with centrosome amplification negative. Note the two signals for centrosomes (red). Representative examples of strong (G) and minimal (H) expression of Aurora-A. I, representative tumor with 20q13 gain. Note the 20q13 gain (red) with polysomy of chromosomes 20 (green). J, representative tumor without 20q13 gain. Note two signals for 20q13 (red) with disomy of chromosomes 20 (green).

 

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  		Table 1. Patients with a decrease in MLH1 or MSH2 expression and other molecular markers

 
Relationship of CIN to other variables in bladder cancers. There were statistical differences in the frequency of centrosome amplification (P < 0.0001), Aurora-A overexpression (P < 0.0001), increased p53 immunoreactivity (P = 0.0027), 20q13 gain (P < 0.0001), tumor grade (P < 0.0001), and tumor stage (P = 0.0058) between the CIN+ and CIN– groups (Table 2 ). The relationship between DNA ploidy and other variables was significant (Table 2).


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  		Table 2. Association of several variables with CIN and DNA ploidy

 
Relationship of 20q13 gain (DNA sequence copy number gain at 20q13) and centrosome amplification to clinicopathologic variables. All tumors with 20q13 gain (group 1) exhibited centrosome amplification and Aurora-A overexpression. There was a significant difference in the frequency of CIN between groups 1, 2 (tumors with centrosome amplification but no 20q13 gain), and 3 (tumors with no 20q13 gain and centrosome amplification; 95.2%, 31.8%, and 5.6%; P < 0.0001). Tumors in group 1 had higher frequencies of DNA aneuploidy, multiple tumors, positive urinary cytology, high tumor grade, and advanced tumor stage than those in other groups (Table 3 ). Tumors in group 3 had a significantly higher recurrence-free and progression-free survival rate than those in groups 1 and 2 [P = 0.0345 and 0.0025, respectively (log-rank test)], as shown in Fig. 2 .


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  		Table 3. Grouping stratified by 20q13 gain, centrosome amplification, and clinicopathologic variables

 

Figure 2
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  		Fig. 2. Recurrence-free and progression-free survival stratified by concurrent 20q13 gain and centrosome amplification. A, Kaplan-Meier plot of recurrence-free survival curves stratified by 20q13 gain positivity (group 1), 20q13 gain negativity and centrosome amplification positivity (group 2), and 20q13 gain negativity and centrosome amplification negativity (group 3). B, Kaplan-Meier plot of progression-free survival curves stratified by groups 1, 2, and 3.

 
Relationship of molecular variables to disease progression. The progression in tumors with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, or 20q13 gain was significantly faster than that in those tumors with none of these variables [P = 0.0005, P = 0.0105, P = 0.0171, and P = 0.0375, respectively (log-rank test)].

A univariate analysis of the Cox proportional hazard model revealed that the low MMR protein expression was a predictor of long progression-free survival in bladder cancer (P = 0.0224; Table 4 ). Centrosome amplification was the most reliable predictor for disease progression in multivariate analysis (hazard ratio, 3.562; 95% confidence interval, 1.591-15.188; P = 0.0005; Table 4).


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  		Table 4. Cox proportional hazard model for disease progression in bladder cancer

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Genetic instability in tumors is roughly divided into two types, MSI and CIN. MSI is caused by defects in MMR, and CIN by abnormalities in chromosome segregation (22). In general, malignant tumors show one or both types (22). In this study, approximately half the bladder cancers were classified as CIN. Of 100 tumors, 9.0% presented with a decrease in MMR protein expression and were classified as MSI. Our data are considered reliable as the cohort examined consisted of 100 cases of bladder cancer. Although MSI is not only represented by a low expression level of MMR protein (8), a decrease in MMR protein expression is almost equivalent to MSI in urothelial carcinoma of the bladder (13) and the upper urinary tract (34), as well as in colorectal cancer (4, 5). In this study, we used only an immunohistochemical method to evaluate MSI. This method alone may provide insufficient information for determination of MSI (5, 35); however, the expression status of MLH1 and MSH2 was used as a surrogate marker for MSI determination in colorectal cancers as noted above (4–6). The frequency of MSI estimated by genetic analysis, although generally low, is controversial in bladder cancer (7–10). The frequency of MSI in the present study seems compatible with that reported elsewhere.

Interestingly, all nine bladder cancers with low MMR protein expression were characterized by the absence of centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, 20q13 gain, DNA aneuploidy, and CIN features. In tumors with MSI, p53 mutation was rare (10). Patients with MSI colorectal cancer showed a better prognosis than those with microsatellite stable cancer (36, 37). However, the relationship of MSI to prognosis is still in dispute in bladder cancer (8, 13, 14). In superficial bladder cancers, as well as in colon cancers, the decrease in MMR protein expression was associated with a lower recurrence rate (8). This is supported by the data from the present study. In this study, all nine tumors with low MMR-related protein expression were associated with no disease progression and, in addition, it was a significant predictor of progression-free survival in univariate analysis. These data, together with previous reports, suggest that in bladder cancers, the low expression of MMR-related proteins means lower malignant potential of disease progression compared with others.

In contrast, all tumors with 20q13 gain were accompanied by both centrosome amplification and Aurora-A overexpression. The 20q13 gain was observed in only 3 (7.1%) pTa bladder cancers whereas it was detected in 39 (92.9%) tumors with invasion into the lamina propria and muscle layer. The difference in molecular and clinicopathologic variables was distinct between tumors with and without 20q13 gain. These observations support the hypothesis suggested in previous reports that higher malignant behavior is associated with CIN resulting from 20q13 gain (38), Aurora-A overexpression (18–20), and centrosome amplification (17) in bladder cancer.

Centrosome amplification was reported as an early event in rat mammary carcinogenesis (39) and it was detected even in precancerous lesions of several human cancers (16). There were no differences in prognosis between group 1 (20q13 gain) and group 2 (centrosome amplification but no 20q13 gain) tumors. However, the prognosis of these two groups was statistically worse than that of tumors without 20q13 gain and centrosome amplification (group 3). These findings suggest that centrosome amplification, leading to CIN, confers highly malignant characteristics for tumors, consequently leading to disease progression in bladder cancer.

In conclusion, bladder cancers are classified into two distinct types according to genetic instability: MSI cancers that show relatively favorable prognosis and CIN cancers that show highly malignant behavior. Of the CIN-related molecular markers investigated in this study, centrosome amplification was the strongest predictor for disease progression.


   Acknowledgments
 
We thank Takae Okada for assistance in preparing the materials for immunostaining, Drs. Masaru Okuda and Kenji Kawamura for their technical advice on immunostaining, and Dr. Hiroaki Matsumoto for collecting follow-up data.


   Footnotes
 
Grant support: Grant-in-Aid for Scientific Research (B) grant 15390492 from the Japan Society for the Promotion of Science.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Y. Yamamoto and H. Matsuyama contributed equally to this work.

Received 4/19/05; revised 10/ 7/05; accepted 2/ 8/06.


   References
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 Materials and Methods
 Results
 Discussion
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Clin. Cancer Res., March 15, 2007; 13(6): 1703 - 1712.
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