Carcinogenesis Advance Access originally published online on November 6, 2003
Carcinogenesis, Vol. 25, No. 3, 343-347,
March 2004
Carcinogenesis vol.25 no.3 © Oxford University Press 2004; all rights reserved.
MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION |
A partial allelotyping of urothelial carcinoma of bladder in the Chinese
Jianjun Zhang1,5,*,
Shan Zheng2,*,
Yanning Gao1,
Jimmy A. Rotolo3,
Zejun Xiao4,
Changling Li4 and
Shujun Cheng1,6
1 Department of Chemical Etiology and Carcinogenesis, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China, 2 Department of Pathology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China, 3 Department of Pharmacology, Joan and Sanford I. Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA and 4 Department of Urology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China
 |
Abstract
|
|---|
The cancer spectrum and genetic pathways underlying tumorigenesis
vary among different ethnic populations due to genetic background
and/or environment discrepancies. We have implied in our previous
study that the genetic alterations found in bladder cancers
of Chinese patients differ from those of Caucasians. We performed
the present study to explore the genetic pathways of the urothelial
carcinoma (UC) in Chinese patients. We carried out a partial
allelotyping of Chinese UC on chromosome arms commonly deleted
in Caucasian UC, and compared the allelo-typing between Chinese
and Caucasian UC. Forty-five Chinese UC specimens were allelotyped
using 30 microsatellite markers on 18 chromosome arms. The most
frequent regions of loss of heterozygosity found included 9q
(54.1%), 17p (51.2%), 9p (48.8%), 18q (42.2%), 3p (41.9%), 16q
(33.5%) and 11p (30.0%). Compared with UC from the UK and US,
the LOH frequencies on most chromosome arms in this study are
higher, with statistically significant differences on 3p, 16q
and 18q. Our results suggest that both consensus and different
alterations exist between Chinese and Caucasian UC, indicating
that genetic alterations of cancer can vary between different
ethnic populations due to genetic and/or etiological discrepancies.
Abbreviations: LOH, loss of heterozygosity; SCC, squamous cell carcinoma; UC, urothelial carcinoma
|
Introduction
|
|---|
Bladder cancer is the sixth most common malignancy world wide
(
1), the sixth most common cancer in men and the tenth most
common cancer in women in China (
2). Urothelial carcinoma (UC)
is the predominant bladder cancer throughout Western countries
and China. Somatic genetic alterations in UC specimens collected
in the UK and the US have been well characterized (
3). In these
studies, loss of heterozygosity (LOH) in several specific regions
of genome identified the probable location of tumor suppressors
that may play a role in the development of UC (
3).
Cancer types and genetic pathways initiating tumorigenesis in the bladder vary among different ethnic populations due to genetic background and/or environment exposure discrepancies. As an example, in Egypt and other regions of the Middle East where the trematode Schistosoma haematobium is endemic, bladder carcinoma is predominantly squamous cell carcinoma (SCC) and the genetic abnormalities are different from that of UC (4). Additionally, schistosomiasis associated UC showed different genetic alterations from that collected in the UK and the US (4). We have implied in our previous study that genetic alterations of bladder cancer in Chinese may be different from that of Caucasian patients (5). However, the molecular events underlying tumor development in the bladder cancer of Chinese are poorly understood.
In order to better understand the genetic pathways of UC in Chinese patients, we screened 45 Chinese patients with UC for LOH on 18 chromosome arms commonly deleted in UC of Caucasian patients, and compared the results from the two populations.
|
Materials and methods
|
|---|
Tissues
Samples from 45 patients with sporadic UC were obtained from
the Department of Urology, Cancer Hospital, Chinese Academy
of Medical Sciences. Age, sex, tumor stage, grade and smoking
history of each patient are listed in
Table I. Tumor stage and
grade were classified according to the tumor-node-metastasis
system and WHO criteria (
6). Surgical specimens were obtained
by transurethral resection of bladder tumors, radical cystectomy
or nephron- ureterectomy. No medications were taken before surgery
in these patients. In each case, freshly excised tumor samples
were frozen immediately in liquid nitrogen for 5 min and stored
at -80°C until DNA extraction. Histopathological examination
was performed on representative sections of each tumor by a
pathologist (S.Z.) before DNA extraction, and only samples that
were comprised of >75% tumor cells were included in this
study. Peripheral blood samples were collected before the patients
underwent operations. Lymphocytes were isolated by addition
of citrate (final concentration 0.2%) to 5 ml peripheral blood
followed by dilution with lymphocyte isolating solution (Institute
of Haematology, CAMS, Tianjin, China), and centrifugation at
room temperature (800–1000
g for 10 min). The layer containing
leukocytes was collected and washed twice with phosphate-buffered
saline. Cell pellets were stored at -80°C until DNA extraction.
DNA preparation
Tumor tissues were pulverized in the presence of liquid nitrogen.
Pulverized tumors and peripheral leukocytes were digested with
50 µg/ml proteinase K in 1% (w/v) sodium dodecyl sulfate
for 8–14 h at 48°C, and twice extracted with phenol–chloroform.
DNA was precipitated by addition of 2 vol of 95% ethanol and
1/3 vol of 3 M sodium acetate.
Microsatellite analysis
DNA from each sample was analyzed using 30 microsatellite markers (Research Genetics, Huntsville, AL) listed in Table II, as described previously (5). Briefly, the forward primer of each marker pair was end-labeled with [
-32P]dATP (New England Nuclear, Boston, MA). In each polymerase chain reaction (PCR), 20–100 ng of DNA was subjected to 35 cycles, in which each cycle consisted of: 95°C for 30 s (desaturating temperature), 52–62°C for 1 min (varying annealing temperature), and 72°C for 1 min (extension temperature). There was a single final extension at 72°C for 10 min. PCR products were separated by electrophoresis on a denaturing 7% urea–polyacrylamide–formamide gel, and the bands were detected by autoradiography.
Informative cases (i.e. cases occurring when the length of two
alleles vary and can be separated by polyacrylamide gel electrophoresis)
were scored as a LOH when a reduction of at least 50% in one
allele compared with the normal (germ-line) allele occurred
(
Figure 1). Initially, LOH was screened visually for loss of
one allele or the presence of microsatellite shifts, and cases
with a ‘partial loss’ were evaluated further by
using the public domain of NIH Imagine Program (developed at
the NIH and available on the Internet at
http://rsb.info.nih.gov/nih-image).
All results were confirmed by at least two independent observers.
Results were verified by independent PCR with increased DNA
templates (100 ng/PCR) in cases with LOH or PCR failure.
View larger version (103K):
[in this window]
[in a new window]
|
Fig. 1. Microsatellite analysis in tumor tissue (T) compared with peripheral blood leukocytes DNA (N). Representative autoradiograms are shown. Heterozygosity or lost alleles are suggested with arrows.
|
|
Allelotyping data from the UK and the US UC for comparison
We used frequently cited data from the UK (
7–
9) and the
US (
10,
11) consisting of 533 UC in these studies. Among the
cases for which tumor stage or grade data were available, the
grade distribution consisted of G1 = 29% (70/242), G2 = 41%
(99/242) and G3 = 30% (73/242) and there are 27% (136/499) pTa,
5% (27/499) pTa–pT1, 29% (145/499) pT1 cases and 38% (191/499)
pT2 UC.
Statistic analysis
2 tests were used to evaluate the relationship between LOH of given microsatellite marker/chromosome arm and sex, smoking history, tumor stage or grade. In addition, 2 tests were employed to compare the frequencies of LOH on each chromosome arm between Caucasian patients and Chinese patients, and to compare the stage distribution between Caucasian patients and Chinese patients. 2 tests were performed using the software package Epi Info version 5.0 (Center for Disease Control, Epidemiology Program Office, Atlanta, GA).
|
Results
|
|---|
Thirty microsatellite markers on 18 chromosome arms were used
in this study. The markers were selected on the basis of their
close proximity either to tumor suppressor genes implicated
in the development of UC (i.e. P16 at 9p21, RB1 at 13q13) or
to minimum regions of deletion mapped in bladder or other tumors.
We intended to use the same microsatellite markers as used for
the UK and US UC patients (
7–
10). However, as we have
identified previously (
5), some of the markers gave very low
rate of heterozygosity or unclear distinct patterns among this
group of patients and were excluded. Two or three markers were
used on 3p, 4q, 9p, 9q, 11q, 17p and 18q to increase the numbers
of informative chromosome arms. Informative results were obtained
for 89.4% (724/810) of chromosome arms examined. LOH results
of each tumor were shown in
Figure 2. The highest frequency
of LOH detected is 9q (54.1%), followed by 17p (51.2%), 9p (48.8%),
18q (42.2%), 3p (41.9%), 16q (33.5%) and 11p (30.0%), as shown
in
Table II. No correlation between LOH of any microsatellite
marker/chromosome arm with sex, smoking history, tumor stage
or grade were found, perhaps due to the relatively small case
number. A partial allelotyping for this series of tumors is
shown in
Figure 3. There is considerable variation in the frequency
of LOH in individual tumors. In one case, a pTa–G2 tumor
(Case 25,
Figure 2) showed retention of heterozygosity at all
informative loci examined, whereas a pT4–G3 tumor (Case
19,
Figure 2) showed LOH on 10 chromosome arms. In general,
high-grade late-stage tumors are prone to show LOH on multiple
arms. On chromosome arms where two or more markers were used,
the same retention/LOH of the markers was observed in most cases.
However, some cases showed different retention/LOH of the markers
on the same chromosome arm, suggesting there may be more than
one tumor suppressor on the same arm involved in the development
of bladder cancer.
View larger version (112K):
[in this window]
[in a new window]
|
Fig. 2. Patterns of LOH of each microsatellite marker (A) and chromosome arm (B) in 45 bladder tumors. Status of each microsatellite marker or chromosome arm is indicated by shading as LOH (black), retention of heterozygosity (white) and non-informative (gray).
|
|
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3. Comparison of partial allelotyping for Chinese UC and UK/US UC (data from Brewster et al., 1994; Knowles et al., 1994; Shaw et al., 1995; Chang et al., 1995 and Presti et al., 1991; refs 7–11). Open bar: LOH frequency in Chinese UC; closed bar: LOH frequency in Caucasian UC.
|
|
As shown in
Table III and
Figure 3, we compared LOH frequencies
of chromosome arms between Chinese bladder tumors and Caucasian
bladder tumors from UK (
7–
9) and the US (
10,
11). The
P value for difference of grade or stage distribution between
Caucasian and Chinese UC is 0.9906 and 0.4165, respectively.
Generally, the LOH frequencies on most chromosome arms in our
group are higher than that of Caucasian patients (
7–
11).
However, only the differences on 3p, 16q and 18q are statistically
significant.
|
Discussion
|
|---|
Tumor incidences, tumor types and genetic abnormalities may
be different among different geographic and/or ethnic populations.
Different risk factor exposure is believed to contribute to
the differences (
12,
13). However, since environment–genetic
interactions play very important roles in the development of
tumors, genetic predisposition is now taken as another explanation
for the differences. A good example is that the mutation pattern
of p53 differs considerably in breast cancers among different
ethnic populations (
14–
17). We implied in our previous
studies that the pattern of LOH in bladder cancer in Chinese
might be different from that of Caucasian patients (
5). Here,
we assessed LOH in chromosomal regions known to be involved
in the development of UC in the UK (
7–
9) and the US (
10,
11).
In comparing the LOH frequencies in our study and previous studies
of UC in Western countries, our results suggest that there are
both consensus and different alterations between Chinese and
Caucasian UC. Indeed, the Chinese tumors showed significant
LOH in all chromosomal regions identified previously in Caucasian
UC.
The frequency of LOH on 17p almost certainly reflects the known high frequency of involvement of TP53 in these tumors. The involvement of TP53 is associated with high tumor grade and stage, which has been well documented (3). However, in our study, the frequency of LOH of TP53-linked microsatellite marker TP53 is lower than that of D17S695 on 17p13.3 (Table II), suggesting that there are other tumor suppressor genes on 17p besides TP53 involved in the development of bladder cancer.
LOH frequencies at 1p, 2p, 4q, 5p, 5q, 11q, 13q, 14q, 17q, 20p are found relatively low (
20%), similar to those of UC in Caucasian patients (see Table III and Figure 3). The most striking differences between the Chinese bladder tumors and all Caucasian tumors studied previously are in the relative frequencies of involvement of 3p, 16q and 18q (Table III; P<0.01). Frequencies of LOH on these chromosome arms in Chinese bladder tumors are >20% higher than those in Caucasian UC and statistically significant. These results suggest that tumor suppressors on chromosome arms 3p, 16q and 18q could play more important roles in the development of UC in Chinese. The target tumor suppressor genes of these deletions have not yet been identified.
In summary, we demonstrate in this study that Chinese bladder UC share genetic alterations with Caucasian bladder tumors and also possess different genetic abnormalities, which may be contributed by different genetic background and/or environment exposure. None of the tumors in this study are occupation or infection related. Thus, the different genetic abnormalities demonstrated in this study may be attributed to other etiological factors because of differences in dietary habits, social behavior or the genetic background discrepancy between Caucasian and Chinese patients. It is probable that the genetic and etiological discrepancies may exist in other malignancies and diseases and must be considered when exploring genetic alterations of diseases.
|
Notes
|
|---|
5 Present address: Laboratory of Signal Transduction, Memorial
Sloan-Kettering Cancer Center, 430 East 67th Street, New York,
NY 10021, USA
6 To whom correspondence should be addressed Email: chengsj{at}pubem.cicams.ac.cn
* These authors contributed equally to this work.
|
Acknowledgments
|
|---|
Supported by the National Natural Foundation of China (30170432)
and Beijing Natural Foundation (7012027). We are indebted to
Dr Jin Jen of John Hopkins Oncology Center, Baltimore, USA,
Dr Nan Hu of NIH, USA and Dr Li Mao of MD Anderson Cancer Center,
University of Texas, Houston, USA for their stimulating suggestions
and valuable technical assistance.
|
References
|
|---|
- Wingo,P.A., Tong,T. and Bolden,S. (1995) Cancer statistics. CA. Cancer J. Clin., 45, 8–30.
- Zhou,R.X. (1994) Epidemiology of bladder tumors. In Zhou,R.X (ed.) Bladder Surgery. People's Health Publishing House, Beijing, pp. 145–147.
- Knowles,M.A. (1998) Molecular genetics of bladder cancer: pathways of development and progression. Cancer Surv., 31, 49–76.[ISI][Medline]
- Shaw,M.E., Elder,P.A., Abbas,A. and Knowles,M.A. (1999) Partial allelotype of schistosomiasis-associated bladder cancer. Int. J. Cancer, 80, 656–661.[CrossRef][ISI][Medline]
- Zhang,J., Fan,Z., Gao,Y., Xiao,Z., Li,C., An,Q. and Cheng,S. (2001) Detecting bladder cancer in the Chinese by microsatellite analysis: ethnic and etiologic considerations. J. Natl Cancer Inst., 93, 45–50.[Abstract/Free Full Text]
- Mostofi,F.K. (1999) Histological typing of urinary bladder tumors. In Mostofi,F.K., Davis,C.J. and Sesterhenn,I.A. (eds) In collabration with Sobin,L.H. and pathologists in 10 counties. Histological Typing of Urinary Bladder Tumors, 2nd Edn. Springer, Berlin, pp. 9–17.
- Brewster,S.F., Gingell,J.C., Browne,S. and Brown,K.W. (1994) Loss of heterozygosity on chromosome 18q is associated with muscle-invasive transitional cell carcinoma of the bladder. Br. J. Cancer, 70, 697–700.[ISI][Medline]
- Knowles,M,A,, Elder,P.A., Williamson,M., Cairns,J.P., Shaw,M.E. and Law,M.G. (1994) Allelotype of human bladder cancer. Cancer Res., 54, 531–538.[Abstract/Free Full Text]
- Shaw,M.E. and Knowles,M.A. (1995) Deletion mapping of chromosome 11 in carcinoma of the bladder. Genes Chromosomes Cancer, 13, 1–8.[ISI][Medline]
- Chang,W.Y., Cairns,P., Schoenberg,M.P., Polascik,T.J. and Sidransky,D. (1995) Novel suppressor loci on chromosome 14q in primary bladder cancer. Cancer Res., 55, 3246–3249.[Abstract/Free Full Text]
- Presti,J.C., Jr, Reuter,V.E., Galan,T., Fair,W.R. and Cordon-Cardo,C. (1991) Molecular genetic alterations in superficial and locally advanced human bladder cancer. Cancer Res., 51, 5405–5409.[Abstract/Free Full Text]
- Ng,I.O., Srivastava,G., Chung,L.P., Tsang,S.W. and Ng,M.M. (1994) Overexpression and point mutations of p53 tumor suppressor gene in hepatocellular carcinomas in Hong Kong Chinese people. Cancer, 74, 30–37.[CrossRef][ISI][Medline]
- Wong,N., Lai,P., Pang,E. et al. (2000) Genomic aberrations in human hepatocellular carcinomas of differing etiologies. Clin Cancer Res., 6, 4000–4009.[Abstract/Free Full Text]
- Sommer,S.S., Cunningham,J., McGovern,R.M., Saitoh,S., Schroeder,J.J., Wold,L.E. and Kovach,J.S. (1992) Pattern of p53 gene mutations in breast cancers of women of the midwestern United States. J. Natl Cancer Inst., 84, 246–252.[Abstract/Free Full Text]
- Hill,K.A. and Sommer,S.S. (2002) p53 as a mutagen test in breast cancer. Environ. Mol. Mutagen, 39, 216–227.[CrossRef][ISI][Medline]
- Blaszyk,H., Vaughn,C.B., Hartmann,A., McGovern,R.M., Schroeder,J.J., Cunningham,J., Schaid,D., Sommer,S.S. and Kovach,J.S. (1994) Novel pattern of p53 gene mutations in an American black cohort with high mortality from breast cancer. Lancet, 343, 1195–1197.[CrossRef][ISI][Medline]
- Hartmann,A., Rosanelli,G., Blaszyk,H., Cunningham,J.M., McGovern,R.M., Schroeder,J.J., Schaid,D.J., Kovach,J.S. and Sommer,S.S. (1995) Novel pattern of P53 mutation in breast cancers from Austrian women. J. Clin. Invest., 95, 686–689.[ISI][Medline]
Received February 27, 2003;
revised September 24, 2003;
accepted October 28, 2003.
CiteULike 
Connotea 
Del.icio.us What's this?
Top
Abstract
Introduction