microsatellite instability in bladder cancer...

[CANCER RESEARCH 53, 5620-5623. December l. 1993]

Advances in Brief

Microsatellite instability in Bladder Cancer I

Mirella Gonzalez-Zulueta, 2 J. Michael Ruppert, Kaori Tokino, Yvonne C. Tsai, Charles H. Spruck III, Noriomi Miyao, Peter W. Nichols, Gregers G. Hermann, Thomas Horn, Kenneth Steven, Ian C. Summerhayes, David Sidransky, and Peter A. Jones 3 Urologic Cancer Research Laboratory, Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, California 90033 [M. G-Z., Y C. Z, C. H. S., N. M., P. W. N., P. A. J.]; Department of Ototaryngology-Head and Neck Surgery, the Johns Hopkins University; Baltimore, Maryland 21205 [J. M. R., K. T., D. S.]; Department of Urology, Herlev Hospital, University of Copenhagen, Herlev, Denmark [G. G. H., T. H., 1(. S.]; and Laboratory of Cancer Biology and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115 [1. C. S.]

A b s t r a c t

Somatic instability at microsatellite repeats was detected in 6 of 200 transitional cell carcinomas of the bladder. Instabilities were apparent as changes in (GT). repeat lengths on human chromosome 9 for four tumors and as alterations in a (CAG). repeat in the androgen receptor gene on the X chromosome for three tumors. Single locus alterations were detected in three tumors, while three other tumors revealed changes in two or more loci. In one tumor we found microsatellite instability in all five loci analyzed on chromosome 9. The alterations detected were either minor 2-base pair changes or larger (>2 base pairs) alterations in repeat length. All six tumors were low stage (Ta-T1), suggesting that these alterations can occur early in bladder tumorigenesis.

I n t r o d u c t i o n

Microsatel l i te markers play an impor tant role in the analysis of L O H 4 in cancer. Microsatel l i tes are t andem iterations o f s imple di-,

tri-, or te t ranucleot ide repeats, and their usefulness can be attr ibuted to

abundancy (1), hypervariabi l i ty (2), fairly even genom ic distr ibution

(3), and ease of detect ion by the PCR.

Microsatel l i tes have been reported to be unstable in some inheri ted

diseases and in some types of cancer. This instability consists of

expans ion or contract ion of D N A within repeat e lements (4). Expan-

sion of a t r inucleot ide repeat is responsible for fragile X syndrome (5),

spinobulbar muscu la r a t rophy (6), myo ton ic dys t rophy (7), Hunt ing-

ton ' s disease (8), and spinocerebel lar ataxia type 1 (9). Dinucleot ide

repeat alterations have recently been l inked to predisposi t ion to colo-

rectal cancer (10, 11), in which changes in the microsatel l i te repeats were shown to be variable, ranging f rom 2-base pair changes to larger

alterations (11), and attr ibuted to RERs (10). Moreover , genet ic in-

stability is present in some inheri ted syndromes (such as xeroderma

p igmen tosum, ataxia-telangiectasia, and B l o o m ' s syndrome) in which

there is an associated predisposi t ion to cancer (12).

We have recently used microsateUites located on h u m a n ch romo-

somes 9 and 17p to de te rmine the sequence of molecular defects ocurr ing in a series o f 200 T C C of the bladder s (13, 14). Recent

ev idence suggests that L OH of c h r o m o s o m e 9 is an early event in the

generat ion of papil lary T C C but not in ca rc inoma in situ of the

Received 8/30/93; accepted 10/18/93. 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.

1 Supported by USPFIS Grant R35 CA49758 from the National Cancer Institute, and a Collaborative Research agreement with Oncor, Inc., Gaithersburg, MD.

2 Recipient of a fellowship from The Government of Navarra, Spain. 3 To whom requests for reprints should be addressed, at the Kenneth Norris Jr. Com-

prehensive Cancer Center, University of Southern California, 1441 East Lake Ave. Los Angeles, CA 90033-0800.

4 The abbreviations used are: LOH, loss of heterozygosity; PCR, polymerase chain reaction; TCC, transitional cell carcinoma; RER, replication error.

5 C. H. Spruck III, P. E Ohneseit, M. Gonzalez-Zulueta, D. Esrig, N. Miyao, Y. C. Tsai, S. P. Lerner, A. S. Yang, R. Cote, L. Dubeau, P. W. Nichols, G. G. Hermann, T. Horn, K. Steven, D. G. Skinner, and P. A. Jones. Two molecular pathways to transitional cell carcinoma of the bladder, submitted for publication.

bladder. 5 Allel ic losses of c h r o m o s o m e 17p and p53 muta t ions were

c o m m o n l y seen in carc inoma in situ and in invasive tumors (15). 5 The

purpose o f the current report is to show that microsatel l i te changes

were apparent in some of these tumors , inc luding four tumors wi th

d inucleot ide repeat al terations in c h r o m o s o m e 9 and three tumors wi th

changes in a t r inucleot ide repeat in the androgen receptor gene located

on the X ch romosome . The cases we report were very interest ing since

one of them showed genet ic alterations at all loci examined in chro-

m o s o m e 9 and all were low stage tumors , sugges t ing that the ge nomic

instabili ty g iv ing rise to these microsatel l i te changes migh t occur as an

early event in b ladder tumorigenesis .

Mater ia l s a n d M e t h o d s

The current results were obtained from the analysis of 200 transitional cell carcinomas of the bladder, 154 of which were previously reported cases (13). 5 TCC specimens were obtained from hospitals in Los Angeles County, CA (n = 90), from the Herlev Hospital in Copenhagen, Denmark (n = 64), and from the Johns Hopkins tumor bank, Baltimore (n = 46). Of these, 112 were fresh- frozen and 88 were paraffin-embedded tissues. Tumors were graded according to the criteria of Bergkvist et al. (16) and staged according to the tumor-nodes- metastasis staging system (17). High molecular weight DNA was prepared from fresh-frozen tumor specimens and matching blood samples by proteinase K digestion and phenol/chloroform extraction as described (18). DNA from archival paraffin-embedded specimens was isolated by microdissecting tumor and normal tissues from hematoxylin and eosin-stained frozen sections as described (19).

Tumor DNA was examined for genetic alterations at seven separate micro- satellites, five localized in chromosome 9 (D9S59, D9S63, D9S64, D9S146, D9S156), one in chromosome 17p (D17S513), and one in the X chromosome (androgen receptor gene locus). Loci D9S59, D9S63, and D9S64 were analyzed for 156 tumors; loci D9S146 and D9S156 were analyzed for 49 tumors; locus D17S513 was analyzed for 90 tumors; the androgen receptor gene locus was analyzed for 25 tumors. The dinucleotide repeat polymorphism, (GT),, at loci on chromosomes 9 and 17p was analyzed by PCR amplification followed by electrophoresis on denaturing 8% polyacrylamide gels as described (20, 21). The sequences of primers used are: locus D9S59, 5'-TTA CAC TAT ACC AAG ACT CC-3' and 5'-AAG GGA ATT CAT CCC CTG CT-3'; locus D9S63, 5'-TFA TAA TGC CGG TCAACC Tr-3' and 5'-CCG GAA G'rT ACT CTA GTC TA-3'; locus D9S64, 5'-GAA GGG CTC T I T ATI" AAC TGA T-3' and 5'-AAC CTG GGC GAC ACA GCAA-3'; locus D9S146, 5'-TGC AAT CAA ATT CCC AGC-3' and 5'-GAG GTG ACA TCT GGA AqT-3'; locus D9S156, 5 '-ATC ACT T I T AAC TGA GGA GG-3' and 5'-AGA TGG TGG TGA ATA GAG GG-3'; locus D17S513, 5'-TTC ACT TGT GGG CTG CTG TC-3' and 5'-TAA GAA AGG CTC CCA CAA GCA-3'. The trinncleotide repeat polymorphism, (CAG),, in the androgen receptor gene (22) was analyzed by PCR, performed in a final volume of 25 txl, containing 50 ng of genomic DNA, 1/xM concentrations of each oligonucleotide primer (5'-GTG CGC GAA GTG ATC CAG AA-3' and 5'-TCT GGG ACG CAA CCT CTC TC-3'), 200/xM of the nonradioactive deoxynucleotides, 2/xCi of [a-32P]dCTP, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgC12, 0.01% gelatin, and 1 unit of Taq DNA polymerase (Boehringer Mannhein Biochemicals, Indianapolis, IN). Twenty- four cycles of 94~ for 1 min, 60~ for 1 min, and 72~ for 1.5 rain were performed with the initial denaturation step and final elongation step length-

5620

Research. on November 30, 2018. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

M I C R O S A T E L L 1 T E I N S T A B I L I T Y IN B L A D D E R C A N C E R

ened to 2 and 3 min, respectively. PCR products were resolved on 5% polyacrylamide-7 M urea gels for 2.5 h at 60 W.

Resul ts N Ta T1

We have recently utilized microsatellite repeat polymorphisms to

investigate allelic deletions of chromosomes 9 in 200 TCCs and 17p

in 90 TCCs. The microsatellite banding patterns observed for 194 of

these cases showed either no changes between normal and tumor DNA

or loss of an allele in the tumor DNA. Fig. l a shows examples of the

same banding pattern being present in DNA microdisected from a

paraffin-embedded superficial papillary grade II tumor and adjacent

normal tissue for two (GT)n repeat polymorphisms at loci D9S59 and

D9S64 (Fig. la , Lanes 1, 2, 5, and 6); an example of LOH, detected

for the same tumor at locus D9S156 is shown in Fig. la , Lane 10. However, differences between normal and tumor DNA banding pat-

terns were observed in the same specimen at loci D9S63 and D9S146 (Fig. l a , Lanes 3, 4, and 7, 8). These differences consisted of shifts in

the electrophoretic mobilities of (GT)n dinucleotide repeat fragments

reflecting a minor 2-base pair expansion of the repeat at locus D9S63 and a >2-base pair expansion at locus D9S146. These alterations were not due to polymerase errors during PCR amplification since results

were reproducible in replicate assays, and in mixing reactions in

which tumor DNA was added to normal DNA from other patient.

Fig. lb shows the results obtained for patient B, whose lamina

propria invasive grade III tumor DNA, obtained from fresh-frozen

N T N T N T N T N T

W

t i

1 2 3 4 5 6 7 8 9 10

D 9 S 5 9 D 9 S 6 3 D 9 S 6 4 D 9 S 1 4 6 D 9 S 1 5 6

N T

~ J

N T N T N T N T

ot 1 2 3 4 5 6 7 8 9 10

D 9 S 5 9 D g s 6 3 D g S 6 4 D 9 S I 4 6 D 9 S I 5 6

Fig. 1. Dinucleotide repeat polymorphisms in normal (N) and tumor (T) tissue from patients with transitional cell carcinoma of the bladder. The microsateUite markers are located at loci D9S59, D9S63, D9S64, D9S146, and D9S156 in chromosome 9. Each normal allele is represented by a major band surrounded by several other lighter bands. (a) (GT)n repeat polymorphisms showing microsatellite abnormalities at loci D9S63 and D9S146. I_~ci D9S59 and D9S64 in Lanes 1, 2, and 5, 6, respectively, showed the same banding pattern for normal and tumor DNA. Locus D9S156 revealed LOH in tumor DNA in lane 10. (b) (GT), repeat polymorphisms in patient B. The lamina propria invasive grade III tumor DNA showed alterations in all five loci examined in chromosome 9. Lanes 2 and 8 show a 2-base pair shift at loci D9S59 and D9S146, respectively. Lanes 4, 6, and 10 contain tumor DNA presenting larger alterations of the allele sizes at loci D9S63, D9S64, and D9S156.

i ....

i;

- 320 bp

- 299 bp

- 275 bp

N T i

Fig. 2. Trinucleotide repeat polymorphisms in the androgen receptor gene in the X chromosome in TCC patients. (a) (CAG)n repeat polymorphism in patient C. Allele sizes are indicated in base pairs. Two alleles of 320 and 299 base pairs are present in the normal DNA (N). A new, truncated allele of 275 base pairs appears in the superficial grade III tumor DNA (Ta) and in the lamina propria invasive grade III tumor DNA (T1). (b) (CAG)n repeat polymorphism in patient D. The tumor DNA (T) shows a major expansion (>2 base pairs) within the trinucleotide repeat. This specimen also showed alterations in (GT)n repeats on chromosome 9.

tissue, showed changes in banding patterns at all five loci analyzed on

chromosome 9 (Fig. lb, Lanes 2, 4, 6, 8, 10). The alterations detected

in this tumor, similarly to the changes observed in the tumor from

patient A, were of two types: a single 2-base pair shift was observed

at loci D9S59 and D9S146 (Fig. lb, Lanes 2, and 8), and larger

alterations (>2 base pairs) were detected at loci D9S63, D9S64, and D9S156 (Fig. lb , Lanes 4, 6, and 10). To rule out the possibility of

specimen contamination or sample switching, we obtained and ana-

lyzed paraffin-embedded tumor and normal material from patient B.

When results from the paraffin-embedded material were compared

with those obtained from the fresh-frozen tumor the same alteration

was detected in DNA from both sources of tumor tissue. Fig. 2 shows the results of the analysis of a trinucleotide repeat

polymorphism in the androgen receptor gene obtained for patients C and D. The tumors from these patients revealed changes in the (CAG)n repeat at the androgen receptor gene locus. Fig. 2a shows that the two tumor specimens from patient C, superficial (Ta) grade III and lamina

5621



MICROSATELLITE INSTABILITY 1N BLADDER CANCER

Table 1 Bladder TCCs showing microsatellite alterations

Tumor Tumor No. of loci altered/no, of loci Patient stage grade examined Names of loci altered Microsatellite alteration

A Ta II 2/7 D9S63, D9S146 Minor and major expansions of (GT)n repeat

B Tt Ill 5/7 Minor and major expansions and contractions of (GT)n repeat

C Ta III 1/7 Major truncation of (CAG)n repeat T1 III 1/7 Major truncation of (CAG)n repeat

D Ta II 5/7 Minor and major contractions of (GT)n repeat; major expansion of (CAG)n repeat

E T1 Iit 1/7 D9S156 Major expansion of (GT)n repeat

D9S59, D9S63, D9S64, D9S146, D9SI56

Androgen receptor gene in X chromosome Androgen receptor gene in X chromosome

D9S63, D9S64, D9S146, D9S156, androgen receptor gene

propria invasive (T1) grade III, contained a new shortened 275-base pair allele that was not present in the normal tissue DNA. These results for patient C were confirmed in tumor DNA obtained from paraffin-embedded material. The new fragment represented a deletion within the trinucleotide repeat element. Sequencing of each of the three alleles revealed that the first two, present in the normal DNA, contained 24 and 17 CAG repeats, respectively, while the third, new allele contained only 9 CAG repeats. In contrast, an expansion within the trinucleotide repeat element is shown in Fig. 2b for the tumor obtained from patient D.

Table 1 summarizes the stages and grades as well as the genetic alterations detected in the tumors in which microsatellite changes were found. All six RER + tumors were low stage (Ta-T1) , grades II-III, with three tumors (from patients C and E) showing alterations in only one of the seven loci analyzed, while three other tumors (from patients A, B, and D) revealed alterations in more than one locus.

In contrast to the results obtained for chromosome 9 and the androgen receptor gene in the X chromosome, the analysis of locus D17S513 in chromosome 17p did not reveal any microsatellite alteration in 90 TCCs analyzed.

Discussion

Our data show that genomic instability as measured by changes in microsatellite repeats occurs in TCC of the bladder. Since alterations were detected in low stage TCCs, including two low grade tumors, genomic instability might be an early event in bladder tumorigenesis. The low number of bladder tumors in which we observed microsatellite changes could be due to the fact that only seven markers were analyzed, five of them on the same chromosome. Thus, our results may reflect only a small part of a genome-wide instability in bladder cancer. However, further studies with a larger number of microsatellite markers would be important to verify this interpretation.

The tumor obtained from patient B was particularly interesting in that it contained microsatellite changes in all five loci analyzed on chromosome 9. This specimen was obtained from a patient who had a history of a kidney TCC and a ureteral TCC resected 7 and 3 years, respectively, before diagnosis of the tumor analyzed in our study, suggesting an association between somatic instability in chromosome 9 and susceptibility to multiple primary tumors.

The data obtained for patient C were informative in relation to the timing of the instability in tumorigenesis since the Ta tumor showed LOH for chromosome 9 whereas the T1 tumor, which was excised 10 months later, had retention for this chromosome (13). Since both tumors contained the new truncated trinucteotide repeat fragment in the androgen receptor gene, it is likely that they were derived from the same transformed cell and that the allelic loss for chromosome 9 occurred after the instability, resulting in a faster growing tumor which was detected earlier. The development of a shortened CAG

5622

repeat allele is intriguing and may be a manifestation of RERs and genomic instability associated with transformation. RER during tumor development may also result in expansions of trinucleotide repeats, such as those which occurred in the tumor from patient D. In contrast to tumors from patient C, the tumor from patient D contained an expansion of the (CAG)n repeat in the androgen receptor gene, and also showed expansions and deletions at (GT), repeats on chromosome 9.

Genetic alterations similar to the ones detected in our study have recently been reported for hereditary nonpolyposis colorectal cancer and one associated ovarian carcinoma (10, 11). Microsatellite changes have not only been linked to familial predisposition to colon cancer, in which a tendency to this type of alteration could be inherited, but have also been detected in sporadic colon carcinomas (10, 11). The changes in repeat lengths that we detected in bladder carcinomas were of two types: major alterations (>2 base pairs) and minor alterations (2-base pair change) in the repeat fragment size, similar to the changes observed in colon cancer (11). The tumors in which we detected microsatellite alterations could be grouped according to the number of loci affected: 3 tumors showed DNA alterations at multiple loci; and 3 tumors showed alterations at only 1 locus.

Our findings that microsatellite instabilities are present as an ap- parently early event in the development of bladder cancer, for which a hereditary predisposition has never been described, suggest that this kind of instability might be common to sporadic human cancers, and that RER-containing tumors might not be unique to hereditary nonpolyposis colorectai cancer families. Thus, it will be interesting to search for microsatellite alterations in other tumor types to investigate the hypothesis that sporadic tumors can acquire this genotype during tumor development and progression.

References

1. Beckman, J., and Weber, J. L. Survey of human and rat microsateilites. Genomics, 12: 627-631, 1992.

2. Litt, M., and Luty, J. A. A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Gellet., 44: 397--401, 1989.

3. Stallings, R. L., Torney, D. C., Hildebrand, C. E., Longmire, J. L., Deaven, L. L., Jett, J. H., Doggett, N. A., and Moysis, R. K. Physical mapping of human chromosomes by repetitive sequence fingerprinting. Proc. Natl. Acad. Sci. USA, 87: 6218-6222, 1990.

4. Nelson, D. L, and Warren, S. T. Trinucleotide repeat instability: when and where? Nat. Genet., 4: t07-108, 1993.

5. Kremer, E. J., Prilchard, M., Lynch, M., Yu, S., Holman, K., Baker, E., Warren, S. T., Schlessinger, D., Sutherland, G. R., and Richards, R. I. Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n. Science (Washington DC), 252: 1711-1714, 1991.

6. Biancalana, V., Serville, E, Pommier, J., Julien, J., Hanauer, A., and Mandel, J. L. Moderate instability of the trinucleotide repeat in spino bulbar muscular atrophy. Hum. Mol. Genet., 1: 255-258, 1992.

7. Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., and Barcelo, J. Myotonic dystrophy mutation: an unstable



MICROSATELLITE INSTABILITY IN BLADDER CANCER

CTG repeat in the 3' untranslated region of the gene. Science (Washington DC), 255: 1253-1255, 1992.

8. The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell, 72: 371-383, 1993.

9. Orr, H. T., Chung, M., Banff, S., Kwiatkowski Jr., T. J., Servadio, A., Beaudet, A. L., McCall, A. E., Duvick, L. A., Ranum, L. P. W., and Zoghbi, H. Y. Expansion of an unstable trinucleotide CAG repeat in spino cerebellar ataxia type 1. Nat. Genet., 4: 221-226, 1993.

10. Aaltonen, L. A., Peltomaki, P., Leach, F. S., Sistonen, P., Pylkkanen, L., Mecklin, J-P., Jarvinen, H., Powell, S. M., Jen, J., Hamilton, S. R., Petersen, G. M., Kinzler, K. W., Vogelstein, B., and de la Chapelle, A. Clues to the pathogenesis of familial colorectal cancer. Science (Washington DC), 260: 812-816, 1993.

11. Thibodeau, S. N., Bren, G., and Schaid, D. Microsatellite instability in cancer of the proximal colon. Science (Washington DC), 260: 816--819, 1993.

12. German, J., Bloom, D., and Passarge, E. Bloom's syndrome. XII. Report from the Registry for 1987. Clin. Genet., 35: 57--69, 1989.

13. Miyao, N., Tsai, Y. C., Lerner, S. P., Olumi, A. E, Spruck. C. H., III, Gonzalez- Zulueta, M., Nichols, P. W., Skinner, D. G., and Jones, P. A. The role of chromosome 9 in human bladder cancer. Cancer Res., 53: 4066-4070, 1993.

14. Sidransky, D., Frost, P., Von-Eschenbach, A., Oyasu, R., Preisinger, A. C., and Vogelstein, B. Clonal origin of bladder cancer. N. Engl. J. Med. 326: 737-740, 1992.

15. Sidransky, D., Von-Eschenbach, A., Tsai, Y. C., Jones, P. A., Summerhayes, I., Mar- shall, E, Paul, M., Green, P., Hamilton, S. R., Frost, P., and Vogelstein, B. Identifi-

cation of p53 mutations in bladder cancers and urine samples. Science (Washington DC), 252: 706--709, 1991.

16. Bergkvist, A., Ljungquist, A., and Moberger, G. Classification of bladder tumors based on the cellular pattern. Acta Chir. Scand., 130: 371-378, 1965.

17. Bears, O. H., Henson, D. E., Hutter, R. V. P., and Myers, M. H. (eds.). American Joint Committee on Cancer. Manual for Staging of Cancer, Ed. 3, pp. 193-198. Philadel- phia: J. B. Lippincott Co., 1988.

18. Tsai, Y. C., Nichols, P. W., Hiti, A. L., Williams, Z., Skinner, D. G., and Jones, P. A. Allelic losses of chromosome 9, 11, and 17 in human bladder cancer. Cancer Res., 50: 44--47, 1990.

19. Spruck, C. H., III, Rideout, W. M., III, Olumi, A. E, Ohneseit, P. E, Yang, A. S., Tsai, Y. C., Nichols, P. W., Horn, T., Hermann, G. G., Steven, K., Ross, R. K., Yu, M. C., and Jones, P. A. Distinct pattern of p53 mutations in bladder cancer: relationship to tobacco usage. Cancer Res., 53: 1162-1166, 1993.

20. Kwiatkowski, D. J., Henske, E. P., Weimer, K., Ozelius, L., Gusella, J. F., and Haines, J. Construction of a GT polymorphism map of human 9q. Genomics, 12: 229-240, 1992.

21. Oliphant, A. R., Wright, E. C., Swensen, J., Grvis, N. A., Goldar, D., and Skolnick, M. H. Dinucleotide repeat polymorphism at the D17S513 locus. Nucleic Acids Res., 19: 4794, 1992.

22. Cutler Allen, R., Zoghbi, H. Y., Moseley, A. B., Rosenblatt, H. M., and Belmont, W. Methylation of HpalI and HhaI sites near the polymorphic CAG repeat in the human androgen receptor gene correlates with X chromosome inactivation. Am. J. Hum. Genet., 51: 1229-1239, 1992.

5623



1993;53:5620-5623. Cancer Res Mirella Gonzalez-Zulueta, J. Michael Ruppert, Kaori Tokino, et al. Microsatellite Instability in Bladder Cancer

Updated version

http://cancerres.aacrjournals.org/content/53/23/5620

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/53/23/5620To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



microsatellite instability in bladder cancer...

Documents