Clinical Cancer Research Vol. 12, 7004-7008, December 1, 2006
© 2006 American Association for Cancer Research
Imaging, Diagnosis, Prognosis |
Chromosome 20q13.2 Gain May Predict Intravesical Recurrence after Nephroureterectomy in Upper Urinary Tract Urothelial Tumors
Jumpei Akao1,
Hideyasu Matsuyama1,
Yoshiaki Yamamoto1,
Kohsuke Sasaki2 and
Katsusuke Naito1
Authors' Affiliations: Departments of 1 Urology and 2 Pathology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan
Requests for reprints: Katsusuke Naito, Department of Urology, Yamaguchi University School of Medicine, Ube 755-8505, Yamaguchi, Japan. Phone: 81-836-22-2275; Fax: 81-836-22-2276; E-mail: katsunai{at}po.cc.yamaguchi-u.ac.jp.
 |
Abstract
|
|---|
Purpose: Amplification or gain of copy number of chromosome 20q13.2 has been implicated as a causal factor for chromosome instability. We investigated the impact of chromosomal instability and its causative molecular markers, 20q13.2 gain and centrosome amplification, on patient outcome in upper urinary tract transitional cell carcinoma (UUT-TCC).
Experimental Design: The number of centrosomes was assessed by immunohistochemistry. Numerical aberrations of chromosomes 7, 9, and 17 that allowed the estimation of chromosomal instability and 20q13.2 gain were evaluated by fluorescence in situ hybridization in 96 frozen specimens from UUT-TCC and compared with clinicopathologic background and patient outcome.
Results: Chromosomal instability, 20q13.2 gain, and centrosome amplification were detected in 62 of 96 (64.6%), 61 of 96 (63.5%), and 45 of 90 (50.0%) tumors, respectively. 20q13.2 Gain was significantly associated with tumor stage (P = 0.042), chromosomal instability (P < 0.0001), and centrosome amplification (P < 0.0001). Kaplan-Meier analysis showed that 20q13.2 gain was strongly associated with intravesical recurrence-free survival in all patients (P = 0.0050), as well as in patients with grade 2 tumors (P = 0.0011, log-rank test). On multivariate analysis, 20q13.2 gain was found to be the sole independent prognostic factor predicting subsequent intravesical recurrence (hazard ratio, 1.65; 95% confidence interval, 1.03–2.90; P = 0.036).
Conclusions: 20q13.2 gain was strongly associated with a reduced time to intravesical recurrence in all patients. Our data suggest that 20q13.2 gain may be a predictive marker of intravesical recurrence in patients with UUT-TCC.
Upper urinary tract transitional cell carcinoma (UUT-TCC) is a relatively rare cancer that occurs in 
5% of all patients with urothelial cancer (1). The standard treatment for localized UUT-TCC is the surgical removal of the entire affected upper urinary tract (kidney and ureter). In spite of curative surgery, recurrence occurs in two forms: recurrence in the bladder, and local recurrence or distant metastases. The intravesical recurrence rate after surgery has been reported to range from 16% to 50% (2–8). Significant risk for developing intravesical recurrences necessitates the use of routine cystoscopic surveillance. Although stage and grade are associated with local recurrence as well as distant metastasis (9), only a few predictors have been reported for intravesical recurrence after surgery (7, 8). The identification of such prognostic markers for subsequent intravesical recurrence would be beneficial for selection of an individualized strategy for posttreatment surveillance. Two theories have been proposed to explain intravesical recurrence (10). One theory is the "field-effect" theory, in which the entire urothelium is exposed to common carcinogenic insults and multifocal tumors subsequently arise from independent clones of transformed cells (11). The second is the monoclonal theory, in which an initial clone of tumor cells is able to spread via intraluminal dispersion or intraepithelial migration, resulting in recurrence (12). One may predict subsequent intravesical recurrence by exploring the primary tumor after nephroureterectomy, provided that the de novo bladder tumor would be derived from a progenitor cell of the monoclonal origin.
Genetic aberrations consecutively accumulate in tumor cells due to genetic instability inherent to malignant tumors, leading to tumor progression. Genetic instability is generally categorized into microsatellite instability and chromosomal instability in a variety of malignancies, including colon, bladder, and upper urinary tract (13–15). Centrosome amplification, which leads to the formation of multipolar spindles and unequal segregation of chromosomes, is both common and a major factor in chromosomal instability (16–18).
Our previous study has shown that centrosome amplification predicts progression and tumor recurrence in bladder cancer (18), whereas the role of chromosomal instability and its causative molecular event for patient prognosis has not yet been elucidated in UUT-TCC. These findings prompted us to explore whether 20q13.2 gain, chromosomal instability, and centrosome amplification also play an important role in tumor recurrence and progression in UUT-TCC. To the best of our knowledge, this is the first study that shows that 20q13.2 gain could predict subsequent intravesical recurrence in UUT-TCC.
|
Materials and Methods
|
|---|
Patients and tissue specimens. Tissue specimens were obtained from 96 UUT-TCC (65 men and 31 women; median age, 71 years; age range, 48-93 years) by total nephroureterectomy between 1992 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 no. 69), and written informed consent was obtained from each patient. Pathologic grades were classified as grades 1, 2, and 3 in 4, 55, and 37 tumors, respectively. The pathologic stage was Ta, T1, T2, T3, and T4 in 4, 27, 23, 37, and 5 tumors, respectively. The tumor staging system was based on the American Joint Commission on Cancer staging system (19). Tumor locations were renal pelvis and ureter for 55 and 41 tumors, respectively. Patients had no history of neoadjuvant chemotherapy or radiation therapy, or bladder cancer. Postoperative checkups were done every 3 months and consisted of a cystoscopic examination as well as urine cytology for the first 2 years after surgery and every 6 months thereafter. Furthermore, routine chest plain radiography and a computerized tomography scan were done every 6 months, and bone scintigraphy was done every year. Tumor recurrence was defined as a new tumor detected by cystoscopy or radiographic examinations. Disease progression was defined as cases with the occurrence of a recurrent tumor in the bladder (intravesical recurrence) or with either local recurrence or a distant metastasis. For adjuvant therapy, cisplatin-based chemotherapy and radiation therapy were done in 26 and 19 cases, respectively. At a median follow-up of 30 months, intravesical recurrence, local recurrence or distant metastases, and cause-specific death were found in 28, 19, and 17 patients, respectively. The median time to intravesical recurrence, local recurrence or distant metastases, and cause-specific death were 11, 8, and 15 months, respectively.
Tissue preparations of the specimens were processed using the touch preparation technique by gently touching each specimen to a glass slide, followed by air-drying at room temperature for 30 minutes.
Immunofluorescence. 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-100 in PBS for 5 minutes at 25°C. Cells were then incubated with a blocking solution (10% normal goat serum in PBS) for 1 hour and subjected to immunostaining.
For immunostaining of centrosomes, a mouse monoclonal anti-
-tubulin antibody (Sigma, St. Louis, MO) was applied as described previously (18). The antibody-antigen complexes were detected with an Alexa 488– or Alexa 568–conjugated goat anti-mouse IgG antibody (Molecular Probes, Leiden, The Netherlands) for 1 hour at 37°C.
The number of centrosome signals in each cell was determined by observing >100 cells in each sample under an epifluorescence microscope (Olympus, Tokyo, Japan) at x1,000 magnification. Centrosome amplification was defined as >5% of cells having three or more centrosomes per cell (18).
Immunohistochemical staining. Formalin-fixed, paraffin-embedded tissue specimens were used for immunohistochemical staining using a rabbit polyclonal antibody against Aurora-A (Calbiochem, Darmstadt, Germany). Two observers (J.A. and Y.Y.) independently examined immunostained specimens in a blinded manner. The intensity of immunohistochemical staining was scored on a three-point scale as follows: –, no detectable expression; +, weak to moderate expression; ++, strong expression. Overexpression of Aurora-A was defined as >20% of cells counted showing strong cytoplasmic expression of Aurora-A (20).
Fluorescence in situ hybridization. To determine the gain of 20q13 and the variant fractions for chromosomal instability, the multicolor fluorescence in situ hybridization technique was applied to detect specific regions at 20q13.2 and centromeric regions of chromosomes 7, 9, and 17, respectively. Commercially purchased 20q13 probes (LSI ZNF217, 20q13.2 amplicon) labeled with Spectrum Orange (Vysis, Downers Grove, IL) were hybridized. 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 supplier. More than 100 nuclei were counted under an epifluorescence microscope.
Cases were classified as 20q13 gain when over 40% of cells had three or more signals per cell for 20q13 (21).
The fraction of cells whose chromosome number was different from the modal chromosome number was calculated and defined as the variant fraction, an index of chromosomal instability. Chromosomal instability was tentatively defined as over 25% of the average variant fractions of chromosomes 7, 9, and 17 (18, 22).
Statistical analysis. Statistical analysis was done using the JMP 4.0 statistical software (SAS Institute, Cary, NC). The probability of intravesical recurrence was calculated by the Kaplan-Meier method, and statistical differences were evaluated by the log-rank test. A contingency table with either the 
2 test or Fisher's test was applied for univariate analysis. The Cox proportional hazard model was applied for multivariate analysis. For all statistical tests, a P value of <0.05 was considered significant.
|
Results
|
|---|
Overview. 20q13.2 Gain, centrosome amplification, and chromosomal instability were detected in 61 of 96 (63.5%), 45 of 90 (50.0%), and 62 of 96 (64.6%) tumors, respectively. Figure 1
depicts representative cases with and without 20q13.2 gain; numerical aberrations of chromosomes 7, 9, and 17; and centrosome amplification (Fig. 1). Centrosome amplification could not be evaluated in six tumors due to weak spot signals.
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig. 1. Representative tumors with and without 20q13 gain (A and B); numerical aberrations of chromosomes 7, 9, and 17 (C and D); and centrosome amplification (E and F) in UUT-TCC. A and B, cells with and without 20q13 gain as detected by fluorescence in situ hybridization using a 20q13 fluorescent-tagged probe. Nuclei with three or more signals per cell with 20q13 gain (A), and two signals per cell without gain (B). C and D, cells with and without numerical chromosomal aberrations of chromosomes 7, 9, and 17 as detected by fluorescence in situ hybridization using centromere-specific probes as described in the text. Four signals of red (chromosome 7) and aqua blue (chromosome 17) per nucleus showing numerical chromosomal aberrations (C). Two signals of red, green (chromosome 9), and aqua blue (chromosome 17) showing a normal copy number of chromosomes (D). E and F, cells with and without centrosome amplification as detected by -tubulin antibody staining. Centrosomes are located at the cytoplasm of the cells, with three signals showing centrosome amplification (E) and two signals showing no centrosome amplification (F).
|
|
Relationship among chromosomal instability, centrosome amplification, gain of chromosome 20q13.2, and pathologic data. Chromosomal instability was significantly associated with centrosome amplification (P = 0.0044), 20q13.2 gain (P < 0.0001), and tumor grade (P = 0.029). Centrosome amplification was significantly associated with chromosomal instability (P = 0.0044), 20q13.2 gain (P < 0.0001), and tumor grade (P = 0.047). There was a significant difference in the frequency of chromosomal instability (P < 0.0001), centrosome amplification (P < 0.0001), and higher tumor stage (P = 0.042) between patients with 20q13.2 gain and those without (Table 1
).
View this table:
[in this window]
[in a new window]
|
Table 1. Relationship between chromosomal instability, centrosome amplification, gain of chromosome 20q13.2, and pathologic data
|
|
Relationship among chromosomal instability, centrosome amplification, gain of chromosome 20q13.2, and patient prognosis. There was no association between chromosomal instability or centrosome amplification and patient prognosis, including intravesical recurrence, local recurrence or distant metastasis, and cause-specific death. On the other hand, 20q13.2 gain was significantly associated with intravesical recurrence (P = 0.019, Fisher's test; Table 2
).
View this table:
[in this window]
[in a new window]
|
Table 2. Relationship between chromosomal instability, centrosome amplification, gain of chromosome 20q13.2, and patient prognosis
|
|
Relationship between 20q13.2 gain and intravesical recurrence. Patients without 20q13.2 gain had a significantly higher intravesical recurrence-free survival than those with 20q13.2 gain (P = 0.0050, log-rank test; Fig. 2A
). For the subgroup of patients with grade 2 tumors, patients without 20q13.2 gain had a significantly higher intravesical recurrence-free survival than those with 20q13.2 gain (P = 0.0011, log-rank test; Fig. 2B). When the interval from nephroureterectomy to intravesical recurrence was categorized as early (<18 months) and late (18 months or later) recurrence, patients with 20q13.2 gain had a significantly higher frequency of early recurrence than that of late recurrence (Table 3
).
View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig. 2. Kaplan-Meier plot of intravesical recurrence stratified by chromosome 20q13 gain in patients with upper urinary tract urothelial cancer. Kaplan-Meier analysis showed that patients with 20q13.2 gain had a significantly shorter time to intravesical recurrence than those without a 20q13.2 gain, in all tumors (A) and in grade 2 tumors (B). P = 0.005 (A) and P = 0.0011 (B), log-rank test. 20q13.2 gain was defined as cases where >40% of nuclei showed three or more signals of 20q13.2 per cell.
|
|
Multivariate analysis revealed that 20q13.2 gain was the strongest predictor for intravesical recurrence after nephoureterectomy (hazard ratio, 1.65; 95% confidence interval, 1.03-2.90; P = 0.036; Table 4
).
Relationship between 20q13.2 gain and Aurora-A overexpression. Overexpression of Aurora-A was studied in 18 selected cases that showed intravesical recurrence, and the positivity was compared with 20q13.2 gain. The correspondence rate between Aurora-A overexpression and 20q13.2 gain was significant at 88.9% (P = 0.056, Fisher's exact test; Table 5
).
|
Discussion
|
|---|
20q13.2 Amplification has been implicated as a causal factor for chromosome instability in urothelial cancer cell lines (23). Chromosomal aneuploidy generated by Aurora-A/serine-threonine protein kinase located in the vicinity of 20q13.2, or other agents inducing chromosomal instability provides a mechanism for genetic changes and tumor progression (24). Our results support this association of 20q13.2 gain with chromosomal instability and centrosome amplification. We have previously reported a close association of Aurora-A expression and 20q13.2 gain in bladder cancer (18). Preliminary data suggest a close association between Aurora-A expression and 20q13.2 gain in this study. These data suggest that 20q13.2 gain reflects the overexpression of Aurora-A in urothelial cancer.
20q13.2 Gain has been identified as an independent prognostic marker that indicate reduced survival in breast and ovarian cancer (25, 26). Intravesical recurrence after UUT-TCC reportedly occurred in 16% to 50% cases (2–8), which is in agreement with our results. The risk factors for intravesical recurrence have a reported history of bladder cancer and tumor multiplicity (6, 8). Our findings provide important new information with respect to 20q13.2 gain and the prediction of intravesical recurrence after nephroureterectomy. A close association between 20q13.2 gain and intravesical recurrence may be explained by the implantation of seeding cells. Hinotsu et al. (27) proposed two patterns of tumor recurrence of superficial bladder carcinoma: early-phase recurrence, defined by a first peak of estimated hazard risk with <500 days after transurethral resection of the bladder tumor, and a late-phase recurrence. Early recurrence is likely to be due to the implantation of cancer cells during transurethral resection of the bladder tumor. This report may justify our cutoff of 18 months, which discriminates early recurrence from late recurrence, and that early intravesical recurrence may result from the implantation of seeding cells arising from UUT-TCC.
In this study, we showed that 20q13 gain was the only independent predictor for intravesical recurrence. This knowledge may enable us to modify current surveillance protocols to provide optimal, cost-effective follow-up for intravesical recurrence and to develop novel adjuvant strategies to reduce the rate of intravesical recurrence. With respect to disease progression, 20q13.2 gain did not reach significance as a prognostic factor. The small number of patients in this study and the short follow-up period may partially explain this discrepancy.
In conclusion, 20q13.2 gain proved to be an independent prognostic factor for intravesical recurrence, and the detection of 20q13.2 gain may provide crucial prognostic information regarding tumor recurrence. A larger cohort and longer follow-up period may confirm our preliminary data and elucidate the relationship between 20q13.2 gain and disease progression in UUT-TCC.
|
Footnotes
|
|---|
Grant support: Grant-in-Aid for Scientific Research (B) (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.
Received 4/ 4/06;
revised 9/ 7/06;
accepted 9/21/06.
|
References
|
|---|
- Messing EM, Catalona W. Urothelial tumors of the renal pelvis and ureter. Campbell's urology. 7th ed. Philadelphia: WB Saunders; 1998. p. 2383–410.
- Hatch TR, Hefty TR, Barry JM. Time-related recurrence rates in patients with upper tract transitional cell carcinoma. J Urol 1988;140:40–1.[Medline]
- Sakamoto N, Naito S, Kotoh S, et al. Recurrence of bladder tumors following surgery for transitional cell carcinoma of the upper urinary tract. Eur Urol 1991;20:136–9.[Medline]
- Hisataki T, Miyao N, Masumori N, et al. Risk factors for the development of bladder cancer after upper tract urothelial cancer. Urology 2000;55:663–7.[CrossRef][Medline]
- Koga F, Nagamatsu H, Ishimaru H, Mizuo T, Yoshida K. Risk factors for the development of bladder transitional cell carcinoma following surgery for transitional cell carcinoma of the upper urinary tract. Urol Int 2001;67:135–41.[CrossRef][Medline]
- Kang CH, Yu TJ, Hsieh HH, et al. The development of bladder tumors and contralateral upper urinary tract tumors after primary transitional cell carcinoma of the upper urinary tract. Cancer 2003;98:1620–6.[CrossRef][Medline]
- Matsui Y, Utsunomiya N, Ichioka K, et al. Risk factors for subsequent development of bladder cancer after primary transitional cell carcinoma of the upper urinary tract. Urology 2005;65:279–83.[CrossRef][Medline]
- Raman JD, Ng CK, Boorjian SA, Vaughan ED, Jr., Sosa RE, Scherr DS. Bladder cancer after managing upper urinary tract transitional cell carcinoma: predictive factors and pathology. BJU Int 2005;96:1031–5.[CrossRef][Medline]
- Huben RP, Mounzer AM, Murphy GP. Tumor grade and stage as prognostic variables in upper tract urothelial tumors. Cancer 1988;62:2016–20.[CrossRef][Medline]
- Jones TD, Wang M, Eble JN, et al. Molecular evidence supporting field effect in urothelial carcinogenesis. Clin Cancer Res 2005;11:6512–9.[Abstract/Free Full Text]
- Harris AL, Neal DE. Bladder cancer-field versus clonal origin. N Engl J Med 1992;326:759–61.[Medline]
- Hafner C, Knuechel R, Stoehr R, Hartmann A. Clonality of multifocal urothelial carcinomas: 10 years of molecular genetic studies. Int J Cancer 2002;101:1–6.[CrossRef][Medline]
- Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature 1998;396:643–9.[CrossRef][Medline]
- Gryfe R, Kim H, Hsieh ET, et al. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 2000;342:69–77.[Abstract/Free Full Text]
- Amira N, Rivet J, Soliman H, et al. Microsatellite instability in urothelial carcinoma of the upper urinary tract. J Urol 2003;170:1151–4.[CrossRef][Medline]
- Lingle WL, Barrett SL, Negron VC, et al. Centrosome amplification drives chromosomal instability in breast tumor development. Proc Natl Acad Sci U S A 2002;99:1978–83.[Abstract/Free Full Text]
- Pihan GA, Wallace J, Zhou Y, Doxsey SJ. Centrosome abnormalities and chromosome instability occur together in pre-invasive carcinomas. Cancer Res 2003;63:1398–404.[Abstract/Free Full Text]
- Yamamoto Y, Matsuyama H, Furuya T, et al. Centrosome hyperamplification predicts progression and tumor recurrence in bladder cancer. Clin Cancer Res 2004;10:6449–55.[Abstract/Free Full Text]
- Greene FL, Page DL, Fleming ID, et al. AJCC cancer staging manual. 6th ed. New York: Springer-Verlag; 2002. p. 329–34.
- Sen S, Zhou H, Zhang RD, et al. Amplification/overexpression of a mitotic kinase gene in human bladder cancer. J Natl Cancer Inst 2002;94:1320–9.[Abstract/Free Full Text]
- Aust DE, Muders M, Kohler A, et al. Prognostic relevance of 20q13 gains in sporadic colorectal cancers: a FISH analysis. Scand J Gastroenterol 2004;39:766–72.[CrossRef][Medline]
- Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in colorectal cancers. Nature 1997;386:623–7.[CrossRef][Medline]
- Savelieva E, Belair CD, Newton MA, et al. 20q gain associates with immortalization: 20q13.2 amplification correlates with genome instability in human papillomavirus 16 E7 transformed human uroepithelial cells. Oncogene 1997;14:551–60.[CrossRef][Medline]
- Zhou H, Kuang J, Zhong L, et al. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet 1998;20:189–93.[CrossRef][Medline]
- Tanner MM, Tirkkonen M, Kallioniemi A, et al. Amplification of chromosomal region 20q13 in invasive breast cancer: prognostic implications. Clin Cancer Res 1995;1:1455–61.[Abstract]
- Diebold J, Mosinger K, Peiro G, et al. 20q13 and cyclin D1 in ovarian carcinomas. Analysis by fluorescence in situ hybridization. J Pathol 2000;190:564–71.[CrossRef][Medline]
- Hinotsu S, Akaza H, Ohashi Y, Kotake T. Intravesical chemotherapy for maximum prophylaxis of new early phase superficial bladder carcinoma treated by transurethral resection: a combined analysis of trials by the Japanese Urological Cancer Research Group using smoothed hazard function. Cancer 1999;86:1818–26.[CrossRef][Medline]
Top
Abstract
Materials and Methods
