Chromosomal aberrations in early-stage bilharzial bladder cancer

Cancer Genetics and Cytogenetics 132 (2002) 41–45

Chromosomal aberrations in early-stage bilharzial bladder cancer Magdy Sayed Alya, Hussein Mostafa Khaledb,* a Faculty of Science, Cairo University (Beni-Suef Branch), Cairo, Egypt Medical Oncology Department, National Cancer Institute, Kasr El Aini Street Cairo University, Cairo 11796, Egypt Received 19 February 2001; received in revised form 4 June 2001; accepted 5 June 2001

b

Abstract

Bilharzial bladder cancer is one of the most common types of malignancy in both men and women in several developing countries including Egypt. It has several unique clinical, epidemiological, and histological characteristics, suggesting that it is an entity distinct from bladder cancer seen in Western countries. Genetic alterations in bilharzial-related bladder cancer have been studied infrequently, especially in the advanced stages of disease, that is, T3 and T4 classifications. The objective of this study was to extend establishing the baseline cytogenetic profile of this type of malignancy to early T1 and T2 classifications. For this purpose, fluorescence in situ hybridization was applied to interphase nuclei of frozen-stored samples with biotinylated repetitive DNA probes specific for all chromosomes to detect numerical chromosome changes in 35 patients presenting with relatively early-stage pT1 and pT2 disease. Eleven cases had squamous cell carcinoma (SCC) and 24 had transitional cell carcinoma. Six of 24 transitional cell carcinomas had diploid chromosome counts with all the probes. Numerical chromosome aberrations were detected in 18 cases (75%). In 12 cases, a loss of chromosome 9 was observed. In three cases, an additional loss of chromosome 17 was detected. One case demonstrated a loss of chromosome 10, whereas another two cases showed a gain of chromosome 7, next to a loss of chromosome 9. Loss of chromosome Y was observed in nine of the 27 male cases studied (33.3%), in which only one case showed an abnormality whereas four cases were detected next to loss of chromosome 9, and one case showed gain of chromosome 7. Five cases showed loss of chromosome 19 whereas gain of chromosome 4 was detected in two cases. Two of 11 samples of SCC had normal diploid chromosome counts with all the probes used. In four of 11 cases (36.4%) underrepresentation of chromosome 9, compared with the other chromosomes, was detected. An additional loss of chromosome 17 and gain of chromosome 7, next to loss of chromosome 9, was detected in three cases. One case showed loss of chromosome 17 as the only numerical aberration. Loss of the Y chromosome was detected in three cases of which one case had gain of chromosome 7 and one case had loss of chromosome 19. No correlation was found between any of the clinicopathologic parameters examined in this study and the presence or absence of any numerical chromosomal aberrations except for the significant association between schistosomal history and loss of Y chromosome (P0.007). © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction In several developing nations in Africa and in the Middle East—most predominantly Egypt—bladder cancer is one of the most common types of malignancy in both men and women [1]. Bladder cancer in Egypt and other developing countries has several unique clinical, epidemiological, and histological characteristics that suggest that it is an entity distinct * Corresponding author. Tel: 2012-2151040; Fax: 202-3644720. E-mail address: [email protected] (H.M. Khaled).

from bladder cancer in Western countries. One of the differences is that patients generally present at a more advanced stage whereas only 18.5% of cases present with pT1 and pT2 classification of disease [2]. Primary chromosome aberrations are directly related to cancer development and frequently are found as the sole abnormality, often associated with particular types. Secondary abnormalities may be fortuitous or may determine the biological behavior of the tumor. Primary abnormalities would be expected to occur early in tumor development, whereas secondary changes would be more common in the later stages.

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In transitional cell carcinoma (TCC), primary abnormalities involve loss of genetic material (suppressor gene or genes) on chromosome 9 for papillary tumors versus mutation of p53 (chromosome 17) for carcinoma in situ [3]. Additional genetic changes are seen in bladder cancer evolution, including loss of genetic material on chromosomes 1, 11, and 13, duplication of chromosomes 1, 7, and 18, and overexpression of Her2-neu (17q). In contrast, genetic changes in bilharzial-related bladder cancers have been studied infrequently [4,5]. Jones et al. [6] demonstrated high frequency of chromosome 9p21 deletions in squamous cell bladder tumors, which includes the p16 gene (p16 inhibitor of cyclin-dependent kinase 4). In contrast, transitional cell bladder cancers rarely show this mutation, more frequently demonstrating deletion of all chromosome 9. In addition, whereas the frequency of p53 mutations appears to be similar in both, the type and positions of the mutations are reportedly different. The objective of this study is to extend establishing the baseline cytogenetic profile of this type of malignancy to early T1 and T2 classifications. For this purpose, fluorescence in situ hybridization was applied to interphase nuclei of frozen-stored samples with biotinylated repetitive DNA probes specific for all chromosomes to detect numerical chromosome changes in 35 patients presenting with relatively early pT1 and pT2 disease classifications. These findings will help to further verify whether bilharzial bladder cancer is cytogenetically different from bladder cancer present in other places in the world. 2. Materials and methods 2.1. Patients and samples Touch prints of frozen-stored tumor samples from 35 patients with bladder cancer subjected to transurethral resection at the National Cancer Institute of Cairo in Egypt were evaluated in this study. Demographic data of this group of patients including age, gender, bilharzial infestation, pathological type, grade, and classification of the tumor were collected from the hospital charts of the patients. 2.2. Slide preparation Frozen-stored tissues were thawed slowly in cold phosphate-buffered saline (PBS) at 4C and lightly touched on precleaned slides. After air-drying, the slides were incubated in 75 mM KCl for 20 min at 37C and fixed in freshly prepared Carnoy’s solution (three volumes of methanol and one volume of acetic acid). They were air-dried at room temperature and stored at 20C until subsequent analysis. 2.3. Probes and probe labeling The DNA probes specific for chromosomes 2, 3, 4, 8, 12, 13 ⁄ 21, 14, 15, 16, 17, 18, and 19 were obtained commercially as biotinylated inserts (Oncor, Gaithersburg, MD,

USA). The repetitive satellite DNA probes specific for chromosomes 1, 5, 6, 7, 9, 10, 11, 20, X, and Y were labeled with biotin-16-dUTP (Boehringer Mannheim, Indianapolis, IN, USA) by nick translation using a BRL kit. 2.4. Fluorescence in situ hybridization (FISH) Frozen slides were thawed at room temperature, dehydrated in ethanol, and air-dried. Each slide was treated with RNase (100 g/ml) in 2 standard saline citrate (SSC) for 60 minutes at 37C to remove endogenous RNA. Slides were rinsed in 2 SSC and dehydrated in ethanol. Hybridization was performed with a modification of the procedure described by Pinkel et al. [7]. Briefly, 20 l of hybridization mixture containing 1 ng/l biotin-labeled DNA probe, 0.5 g/l salmon sperm DNA, 60% formamide, and 10% dextran sulfate in 2 SSC were applied to each slide, covered with a coverslip, and sealed with rubber cement. Probe and target DNA were denatured simultaneously at 75C for 8 min. Hybridization took place in a moist chamber at 37C overnight. Posthybridization washes were performed in 65% formamide for 20 min and twice in 2 SSC (pH 7.0) for 10 min each at 43C. Signals were detected with fluorescein isothiocyanate–conjugated avidin and amplified with biotinylated goat anti-avidin antibody followed by another layer of avidin (avidin and antiavidin, Vector Laboratories, in a final concentration of 25 g/ml in 4 SSC, pH 7.6, with 15% nonfat dry milk, 0.2% Tween 20). Nuclei were allowed to counterstain in 1 g/ml propidium iodide (PI), with 0.2% diazobicyclo-(2-2-2)-octane (DABCO) as an antifading agent to preserve the fluorescence during prolonged microscopy. To allow a proper evaluation of fluorescence in situ hybridization (FISH) signals, the previously published criteria [8] were adapted. Briefly, 350–450 intact and nonoverlapping interphase nuclei that had signals with more or less the same homogenous fluorescence intensity were counted, and the number of bright fluorescing spots per nucleus were scored for each probe. Minor hybridization spots were not counted. Spots in a paired arrangement (split spots) were counted as one signal. The percentages of nuclei, which had one, two, three, four, or more signals per one nucleus, were calculated for each specimen. Statistical analysis was performed using Fisher’s exact test, and the Mantel-Haenszel rank test for trend.

3. Results Thirty-five Egyptian patients with bladder cancer were included in this study. Their median age was 56 years (range 20–82 years). They were 27 males and eight females with a male–female ratio of 3.3:1. Eleven cases had squamous cell carcinoma (SCC) and 24 had TCC. Grade I tumors were diagnosed in 11 cases, whereas 21 and 3 cases had grade II and III lesions, respectively. Data on pathologic disease stage were available for 34 cases. P1 lesions were encountered in 24 and P2 for 10 cases.

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Table 1 Clinical data of early-stage bilharzial bladder tumors Patient no.

Age

Sex

Bilharziasis

Pathological type

Pathological grade

Tumor classification

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

63 43 42 65 82 50 50 44 65 55 43 50 50 68 20 63 48 72 65 68 72 65 77 45 62 50 20 70 45 44 45 57 45 65 57

M M M M M F M M M M M M M M M M M M M M M M F F M F M M F M F M F F M

N N N Y N N N N Y N N N N N N N Y N N N Y Y N Y Y N Y Y — Y — Y N N —

T T S T T T T T T T T T T T T T T T T T S S T S T T S T S S S S T S S

2 1 2 3 2 2 1 1 2 2 1 1 2 1 2 2 2 2 2 1 2 2 3 2 1 2 2 3 1 2 1 2 2 1 2

1 1 2 2 1 1 1 2 1 1 1 2 2 1 1 1 1 1 2 1 1 1 2 2 1 2 1 — 1 1 1 1 2 1 1

Abbreviations: S, squamous cell carcinoma; T, transitional cell carcinoma.

Information about schistosomal bladder infection was available for 32 of 35 (91.4%) patients. Eleven of 32 (31.4%) patients had schistosomal bladder infection, and 21 of 32 (65.6%) did not. The clinicopathologic data are summarized in Table 1. 3.1. FISH Approximately 90–94% of control lymphocyte nuclei showed two spots (disomy) for all probes specific to autosomal chromosomes, and 93% showed one spot for probes specific to both chromosomes X and Y. All probes also were hybridized to normal bladder urothelial cells of eight cases and counted as a control. The average percentages of nuclei with two signals (disomy) for the autosomal chromosomes were greater than 90%. The total percentages of nuclei with more than three (tetrasomy and other aneusomies) were quite low (3%). The average percentages of nuclei with single signals of these chromosomes were less than 6%. The average percentages of nuclei with single signals of chromosomes X and Y exceeds 93%.

Cutoff values were determined based on the mean 3 standard deviation. If the percentage of nuclei containing one signal was greater than 11%, the tumor was considered as having a monosomic population. The cutoff values for three (trisomy), four (tetrasomy), and more than four (hypertetrasomy) signals were 7%, 6%, and 5%, respectively. For chromosomes X and Y, the cutoff values for two signals and three or more signals were 4% and 2%, respectively. A tumor was considered to have a cell population without signals for chromosome Y (loss of chromosome Y) if the percentage of nuclei without any signals was greater than 10% in two different trials. Fluorescence in situ hybridization was successful in all cases studied. Most of the probes displayed a diploid spot distribution. Table 2 summarizes the FISH results for each of the 35 patients with numerical chromosomal aberrations. Six of 24 TCC cases had diploid chromosome count with all the probes used. Numerical chromosome aberrations were detected in 18 cases (75%). In 12 cases, a loss of chromosome 9 was observed. In three cases, an additional loss of chromosome 17 was detected. One case demonstrated a loss

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of chromosome 10, whereas another two cases showed a gain of chromosome 7, next to a loss of chromosome 9. Two of 11 samples of SCC had normal diploid chromosome count with all the probes used. In four of 11 cases (36.4%), underrepresentation of chromosome 9, compared with the other chromosomes, were detected. An additional loss of chromosome 17 and gain of chromosome 7, next to loss of chromosome 9, was detected in three cases. One case showed loss of chromosome 17 as the only numerical aberration. Loss of the Y chromosome was detected in three cases of which one case was with gain of chromosome 7 and one case with loss of chromosome 19. Loss of chromosome Y was observed in nine of the 27 male cases studied (33.3%), from which one case as the only abnormality observed whereas four cases were detected next to loss of chromosome 9, and one case with gain of chromosome 7. Five cases showed loss of chromosome 19 whereas gain of chromosome 4 was detected in two cases.

Table 2 Numerical chromosomal aberrations of early-stage bilharzial bladder carcinoma patients Chromosome no. Case no.

4

7

9

10

11

17

19

Y

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

4 — — — — — — — — — — 4 — — — — — — — — — — — — — — — — — — — — — 4 —

— — 7 — — — 7 — 7 — — — 7 — — — — — — — — 7 — — 7 — — — — — — — — 7 7

— — 9 — 9 — 9 9 — 9 — — 9 — 9 — 9 9 — 9 — — — — — 9 — — 9 — — — — 9 9

— — — — — — — — — — — 10 — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — 11 — — — — — — — — — — — — — — — — — —

— — 17 — — 17 — — — — — — — 17 — — — 17 — 17 — — — 17 — 17 — — — — — — — 17 17

19 — — — — — — — — 19 — — 19 — — — — — 19 — — — — — — — — — — 19 — — — — —

— — — — — — — Y Y — — — — — Y — Y Y Y — — Y — — Y — Y Y — Y — Y — — —

No aberrations were found in the other studied chromosomes.

3.2. Clinicopathologic correlation None of the clinicopathologic parameters, including schistosomal history, gender and age of the patients, pathologic grade and subtype, and the P classification of disease, had a statistically significant correlation with the presence or absence of any of the numerical chromosomal aberrations observed in this study. Only the presence of schistosomal history was significantly correlated with the loss of Y chromosome (Table 3).

4. Discussion Most studies on the molecular genetics of bladder cancer have focused on transitional cell carcinoma, and few studies have been published on genetic changes of schistosomalrelated bladder cancer in which many cases present with squamous cell carcinoma [9]. The authors previously published a baseline cytogenetic profile of schisosomal-related bladder cancer in Egypt for cases presenting with relatively late tumor stages, that is, T3–4 lesions [4,5]. To our knowledge, this is the first study to report on FISH chromosomal analysis of early T1–2 schistosomal-related bladder cancer cases. Distinct genotypic patterns associated with early and late stages of nonschistsomal bladder cancer were reported previously with two genetic pathways characterizing the evolution of superficial bladder tumors to invasive disease. Chromosome 9 deletion was observed in all superficial papillary tumors (Ta) and almost all tumors invading the lamina propria (T1). However, 3p, 5q, and 17p deletions were absent in the Ta tumors but were identified in invasive bladder cancer [10]. In our material, significant numerical aberrations were observed in T1 and T2 cases and included losses of chromosomes 17, 19, and Y and gains of chromosomes 4 and 7. Loss of chromosome 19 and gain of chromosome 4 were not observed previously in T3, and T4 cases [4]. This represents a different cytogenetic profile from that reported for TCC elsewhere. Unlike T3–4 lesions in which chromosomal anomalies, especially losses of chromosome 9 were associated with a younger age group of patients as well as with a lower grade tumors [4], no significant correlations were found between any of the numerical chromosomal aberrations and the different clinicopathologic indices of T1–2 cases examined in the current study. The only exception to this is the significant association found between loss of Y chromosome and the presence of positive schistosomal infestation. This also was observed in our previous study [4,5] in which schistosomal history was present in all the six cases with T3a–b lesions and with loss of Y chromosome. Thus, the long-term established clinical finding of male preponderance among bilharzial bladder cancer cases and the observed high frequency of loss of Y chromosome in early and late cases of this unique type of bladder carcinoma indicate the presence of a possible tumor suppressor gene on the Y chro-

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Table 3 Correlation between clinicopathologic indices and patients with numerical chromosomal aberrations Numerical chromosomal aberrations Clinicopathologic characteristic Age 60 years (20 cases) 60 years (15 cases) Gender M (27 cases) F (8 cases) Schistosomal history Yes (11 cases) No (21 cases) Pathologic type SCC (11 cases) TCC (24 cases) Pathologic grade Grade I (11 cases) Grade II and III (24 cases) Pathologic P classification T1 (24 cases) T2 (10 cases)

4

7

9

10

11

17

19

Y

1 2

4 4

10 4

1 0

1 0

5 5

3 2

2 1

7 1

11 3

1 0

1 0

6 4

5 0

12 NA

0 3

3 4

2 10

0 1

1 0

2 7

1 4

8/10 4/16 (P0.007)*

1 2

4 4

4 10

0 1

0 1

4 6

1 4

4/7 8/20

2 1

3 5

5 9

1 0

0 1

3 7

0 5

2/8 10/19

2 1

5 2

9 4

0 1

1 0

6 3

3 2

9/20 2/6

6/14 6/13

*P value was corrected for multiple statistical comparisons.

mosome. On the basis of these interesting findings and to complete the cytogenetic profile of bilharzial bladder cancer cases, it is now important to extend FISH analysis to include even earlier cases with Cis and Ta lesions associated with bilharziasis. Such cases may be difficult to assess because of the infrequent presentation of patients with Cis and Ta schistosomal-related bladder carcinoma being less than 1% in most series. Therefore, in conclusion, it may be suggested from this and our previous studies that schistosomal-related bladder cancer has a different cytogenetic profile from that observed for the classic TCC of the bladder. The role of Y chromosome in schistosomal bladder development and progression needs further study both on the macroscopic and molecular levels.

References [1] El-Bolkainy MN, Mokhtar NM, Ghoneim MA, Hussein MH. The impact of schistosomiasis on the pathology of bladder carcinoma. Cancer 1981;48:2643–8. [2] El-Sebai I. Bilharziasis and bladder cancer. Cancer 1977;27:100–6.

[3] Spruck CH III, Ohnseseit PE, Gonzalez-Zuluetra M, Esrig D, Miyao N, Tsai YC, Lerner SP, Schmutte C, Yang AS, Cote R. Two molecular pathways to transitional cell carcinoma of the bladder. Cancer Res 1994;54:784–8. [4] Aly MS, Khaled HM. Chromosomal aberrations in bilharzial bladder cancer as detected by fluorescence in situ hybridization. Cancer Genet Cytogenet 1999;114:62–7. [5] Khaled HM, Aly MS, Magrath IT. Loss of Y chromosome aberrations in bilharzial bladder cancer. Cancer Genet Cytogenet 2000;117:32–6. [6] Jones PA, Buckley JD, Henderson BE, Ross RK, Pike MC. From gene to carcinogen: a rapidly evolving field in molecular epidemiology. Cancer Res 1991;51:3617–20. [7] Pinkel D, Straume T, Gray J. Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 1986;83:2934–8. [8] Van Dekken H, Pizzolo JG, Kelsen DP, Melamed MR. Targeted cytogenetic analysis of gastric tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes. Cytogenet Cell Genet 1990; 54:103–7. [9] Ghoneim M, El-Mekresh MM, El-Baz MA, El-Attar IA, Ashamallah A. Radical cystectomy for carcinoma of the bladder, critical evaluation of the results in 1026 cases. J Urol 1997;158:393–9. [10] Dalbagni G, Presti J, Reuter V, Fair WR, Cordon-Cardo C. Genetic alterations in bladder cancer. Lancet 1993;342:469–72.