MULTIFOCAL TRANSITIONAL CELL CARCINOMA OF THE BLADDER AND UPPER URINARY TRACT: MOLECULAR SCREENING OF CLONAL ORIGIN BY CHARACTERIZING CD44 ALTERNATIVE SPLICING PATTERNS

0022-5347/04/1723-1127/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 172, 1127–1129, September 2004 Printed in U.S.A.

DOI: 10.1097/01.ju.0000129541.23460.48

MULTIFOCAL TRANSITIONAL CELL CARCINOMA OF THE BLADDER AND UPPER URINARY TRACT: MOLECULAR SCREENING OF CLONAL ORIGIN BY CHARACTERIZING CD44 ALTERNATIVE SPLICING PATTERNS HIDEAKI MIYAKE,* ISAO HARA, SADAO KAMIDONO

AND

HIROSHI ETO

From the Departments of Urology, Hyogo Medical Center for Adults, Akashi and Kobe University School of Medicine, Kobe, Japan

ABSTRACT

Purpose: CD44 is a widely expressed cell surface adhesion molecule in which various isoforms arise from alternative RNA splicing mechanism during cancer initiation. We assessed whether multifocal transitional cell carcinoma of the urothelium is due to field change and/or intraluminal seeding and implantation. Materials and Methods: In a series of 24 patients with synchronous and/or metachronous multiple urothelial cancers we performed reverse transcription-polymerase chain reaction analysis using a set of primers capable of amplifying all CD44 splice variant isoforms. After polymerase chain reaction products were electrophoresed band intensities with areas corresponding to the major isoforms (that is CD44s, CD44v10 and CD44v8 –10) were quantified, and CD44v10to-CD44s and CD44v8 –10-to-CD44s ratios were calculated. Moreover, p53 gene mutations in exons 4 to 11 were screened by direct DNA sequencing. Results: Of these 24 cases 18 showed similar CD44v10-to-CD44s and CD44v8 –10-to-CD44s ratios in among multiple urothelial cancers in each case. However, in the remaining 6 cases these ratios were quite different among multiple cancer lesions. Furthermore, different types of p53 mutation were detected among multiple cancer lesions in only 2 of 24 cases, which also indicated different patterns of CD44 alternative splicing. Conclusions: These findings suggest that at least some multiple transitional cell carcinomas of the urothelium seem to be of independent origin based on the analysis of alternative RNA splicing of CD44. Moreover, this hypothesis was further supported by the evaluation of p53 gene mutation. KEY WORDS: urothelium; carcinoma, transitional cell; genes, p53; cell adhesion molecules

One of the most important characteristics of transitional cell carcinomas (TCCs) of the bladder and upper urinary tract is the high incidence of synchronous and/or metachronous occurrence, that is patients often present with multiple tumors and more than 60% have recurrence after surgical resection.1 Traditionally such a multifocal nature of TCC has been explained by 2 hypotheses.2 One is the field change hypothesis, in which the entire urothelium is exposed to common carcinogenic insults and multifocal tumors subsequently arise from independent clones of transformed cells.3 The alternative seeding hypothesis predicts that an initial clone of tumor cells is able to spread via intraluminal dispersion or intraepithelial migration, resulting in multifocal and/or recurrent growth.4 A number of genetic abnormalities have been shown to be associated with TCC, such as loss of chromosome 9 and mutation of p53 gene, which occurs in approximately 50% of high grade TCCs.5, 6 Recent molecular genetic studies supported the seeding hypothesis by analyzing the clonality of synchronous and/or metachronous TCCs with respect to chromosome 9 loss and p53 mutation.2, 7 However, these studies focused mainly on high grade invasive tumor. Furthermore, detailed investigation of multifocal urothelial cancers, including superficial disease, suggested the development of multifocal tumors from plural clones.6 Considering these findings, the precise mechanism of multifocal development in urothelial cancers seems to require further elucidation. CD44 is a widely distributed cell surface adhesion molecule that has a crucial role in various biological phenomena, such as

lymphocyte homing and activation, cell motility, cell-matrix interaction. and the regulation of tumor cell growth and metastasis.8 Recent studies demonstrated that CD44 contains at least 20 exons, of which 10 can be alternatively spliced in different combinations to make up numerous variant isoforms.9 Several human malignant tumors have been reported to over express CD44 variant isoforms and, thus, widespread attention has been focused on CD44 variant isoforms as candidate markers that may provide clinical information, including tumor spread, metastatic potential and prognosis.10⫺13 Furthermore, various kinds of malignant tumors, such as urothelial cancer, have been demonstrated to show multiple complexed CD44 mRNA transcript patterns.14, 15 These findings suggest that characterizing the expression profile of CD44 variant isoforms in multiple urothelial cancers may help clarify the clonal origin of multiple urothelial tumors. Therefore, in the current study using a series of 24 cases of synchronous and/or metachronous multiple urothelial cancers we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis using a set of primers capable of amplifying all CD44 splice variant isoforms. Band intensities with areas corresponding to the major isoforms (that is CD44s, CD44v10 and CD44v8 –10) were then quantified. Moreover, p53 gene mutations in exons 4 to 11 were screened by direct DNA sequencing. MATERIALS AND METHODS

Patient characteristics and sample collection. Between Accepted for publication March 26, 2004. * Requests for reprints: Department of Urology, Hyogo Medical January 1998 and December, 2002 a total of 52 distinct urothelial tumors of the bladder, ureter and renal pelvis were Center for Adults, 13–70, Kitaohji-cho, Akashi 673-8558, Japan. 1127

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CD44 AND CLONAL ORIGIN OF UROTHELIAL CANCER

obtained from 24 patients who underwent surgical treatment at our institutions. Each tissue specimen was immediately snap frozen and stored at ⫺80C until assessed. It was routinely confirmed by cryostat sectioning before analysis that carcinoma tissues contained more than 90% cancer cells. RT-PCR for CD44. Total RNA was isolated using the method described by Chomczynski and Sacchi.16 cDNA was prepared from 1 ␮g total RNA by random priming using a First-Strand cDNA Synthesis kit (Promega Corp., Madison, Wisconsin) and 200 U Moloney murine leukemia virus RT (Life Technologies, Inc., Gaithersburg, Maryland). After incubation of the reaction mixture at 37C for 90 minutes the cDNA was resuspended in 0.3 M sodium acetate and precipitated with 2.5 volumes of ethanol. Synthesized cDNA, and a 1:1 mixture of Taq Start Antibody (Clontech Laboratories, Inc., Palo Alto, California) and recombinant Taq DNA polymerase (5 U/␮l, Takara, Kusatu, Japan) were added to the standard PCR reaction mixture containing 50 ␮g of each primer, 0.2 mM of each deoxynucleoside triphosphate, 10 mM tris-HCl (pH 8.3), 1.5 mM MgCl2 and 50 mM KCl. PCR reactions were performed with a Perkin Elmer Cetus Gene Amp PCR system 2400 (Perkin Elmer, Norwalk, Connecticut) in a 15 ␮l reaction volume. Samples were heated to 94C for 5 minutes, followed by 35 cycles with each cycle consisting of 3 phases, that is 94C for 30 seconds, 63C for 30 seconds and 72C for 30 seconds. To verify the successful completion of cDNA synthesis for each sample glyceraldehyde-3dehydrogenase cDNA was amplified with the primers, S, 5⬘AAGGCTGGGGCTCAT-TTGCAG-3⬘ and AS, 5⬘- CCAAATTCGTTGTCATACCAGG-3⬘. For analysis of CD44 splice variants expressed in tissue specimens we used the S primer, S1 (5⬘- TCCCAGACGAAGACAGTCCCTGGAT-3⬘), and the AS primer, AS1 (5⬘- CACT-GGGGTGGAATGTGTCTTGGTC-3⬘), thereby, immediately flanking the insertion site for the variant exons in the CD44 open reading frame. Direct DNA sequencing of PCR products was performed with a 373S DNA sequencer (Applied Biosystems, Foster City, California), using standard protocols. PCR products were electrophoresed on 2% agarose gel and visualized by ethidium bromide staining followed by ultraviolet transillumination. The resulting images were density scanned (Atto Densitograph, model AE6905, Macintosh version, Atto, Tokyo, Japan). Band intensities with areas corresponding to the amplimers of CD44v8 – 10, CD44v10 and CD44s were then quantified using the National Institutes of Health image 1.60 program (Wayne Rasband Analytics, National Institutes of Health, Bethesda, Maryland), and CD44v8 –10-to-CD44s and CD44v10-toCD44s ratios were calculated. Measurements were performed by one of us (IH) while blinded to the sample source or the results of any other laboratory investigations. All samples were assessed by 2 independent PCR reactions. Detection of p53 mutations. Mutations in the p53 gene were detected by direct DNA sequencing of PCR products with a 373S DNA sequencer (Applied Biosystems), using standard protocols. Exons 4 to 11 of the p53 gene were individually amplified by PCR reactions. Exons 4 to 11 of the p53 gene were analyzed because previous reports have shown that these exons harbor the majority of inactivating mutations in a diverse variety of human tumors.17

multiple TCCs of the urothelium 18 showed similar CD44v10to-CD44s and CD44v8 –10-to-CD44s ratios among multiple tumors in each case. In contrast, typical results of CD44v10-toCD44s and/or CD44v8 –10-to-CD44s ratios were markedly different among multiple tumors in the remaining 6 cases, that is cases 2, 7, 9, 16, 19 and 22 (see figure). Table 1 lists detailed data on these 6 cases. We subsequently investigated mutation of the p53 gene of multiple urothelial cancers in all 24 cases and p53 gene mutation was detected in 5, that is cases 4, 7, 11, 14 and 19. In 2 of these 5 cases (cases 7 and 19) different types of p53 mutation among multiple tumors were observed, which also showed different patterns of CD44 alternative splicing (table 2). DISCUSSION

One of the most interesting characteristics of urothelial cancer is multiple synchronous and/or metachronous development in heterotopic urothelium. This observation has given rise to the field defect hypothesis, which proposes that the entire urothelium from renal pelvis to urethra may be susceptible to malignant transformation due to a carcinogenic insult.3 On the other hand, several recent clinical and experimental findings support the alternative explanation that the multifocal development of urothelial cancers is due to seeding and/or intraepithelial spreading of the original cancer cells.2, 4, 7 However, it remains controversial whether heterotopic urothelial cancer recurrence can be entirely explained by the seeding hypothesis for certain reasons. 1) Molecular genetic methods for investigating clonal origin are limited, including X chromosome inactivation, mutation analysis of a specific gene and microsatellite analysis. 2) Most tumors evaluated in such studies have been high grade, invasive urothelial cancers. 3) Tumor samples have been obtained from a relatively small number of patients in previous studies. 4) In some other types of malignancy the proposed mechanism of multiplicity remains contradictory among reported studies.18 Therefore, to address the mechanism involved in the multiple development of urothelial cancer it may be required to analyze a significant number of multiple tumors using novel molecular genetic technique in addition to previously reported methods. Recently several investigators isolated a number of different isoforms of CD44 that have been characterized by cDNA sequencing and appear to arise by alternative splicing, and some of this variation has been shown to produce functional roles in the molecule.19 For example, the introduction of a specific CD44 variant isoform resulted in changes in the malignant potential of cultured human cancer cell lines.20 Furthermore, over expression of variant CD44 isoforms has been shown to be associated with disease progression in various human malignant tumors, including urothelial cancer.10⫺15 These findings suggest that analyzing the patterns of CD44 variant isoforms in multiple urothelial cancers may confer important information concerning their clonal origin.

RESULTS

RT-PCR using a set of primers that are capable of amplifying all CD44 splice variant isoforms was initially performed to analyze CD44 alternative splicing patterns among multiple urothelial cancers. In this series we defined different expression of CD44 variant isoforms when the value of CD44v10-to-CD44s and/or CD44v8 –10-to-CD44s ratios in 1 tumor tissue was more than double compared with those in other tumor tissue after considering the effect of possible contamination of normal tissue on the ratios. Of 24 cases of synchronous and/or metachronous

RT-PCR amplification using S1 and AS1 primer sets on cDNAs from surgical specimens of urothelial cancers. PCR products were electrophoresed on 2% agarose gel and visualized by ethidium bromide staining and ultraviolet transillumination.

CD44 AND CLONAL ORIGIN OF UROTHELIAL CANCER TABLE 1. Differential expression of CD44 variant isoforms among multifocal urothelial cancers Tumor No. Pt 2: 1 2 Pt 7: 1 2 3 Pt 9: 1 2 Pt 16: 1 2 Pt 19: 1 2 Pt 22: 1 2 3

Date

Grade

CD44v10/CD44S

CD44v8-10/CD44S

2/98 2/98

1 1

0.02 0.09

0.11 0.02

6/98 6/98 2/99

2 2 3

0.11 0.13 0.08

0.04 1.22 0.74

8/99 8/99

2 2

0.32 0.04

0.07 0.08

3/00 3/00

1 2

0.12 0.10

0.17 0.89

9/01 9/01

3 3

0.12 0.02

0.03 0.39

3/02 7/02 7/02

2 2 3

0.12 0.05 0.08

1.02 2.13 0.68

CONCLUSIONS

These findings suggest that at least some multiple TCCs of the urothelium seem to be of independent origin based on the analysis of alternative RNA splicing of CD44. This hypothesis was further supported by the evaluation of p53 gene mutational analysis. Clarification of the clonal origin of multifocal urotheTABLE 2. p53 mutations in multifocal urothelial cancers Pt 4: 1 2 Pt 7: 1 2 3 Pt 11: 1 2 Pt 14: 1 2 Pt 19: 1 2

lial cancers would have a significant impact on the design of an optimal treatment schedule, and the prevention of recurrence and disease progression. Accordingly further investigation is required in a larger number of multiple urothelial cancer samples using plural methods simultaneously. REFERENCES

In this series 24 cases of multiple urothelial cancers were examined by RT-PCR targeting CD44 alternative splicing as well as direct sequencing of the p53 gene to address the mechanism of the development of multiple urothelial tumors. Of these 24 cases 18 showed similar patterns of CD44 alternative splicing, while CD44v10-to-CD44s and/or CD44v8 – 10-to-CD44s ratios were significantly different among multiple tumors in the remaining 6 (cases 2, 7, 9, 16, 19 and 22). In this study we did not analyze normal adjacent tissue since our previous studies have demonstrated different alternative splicing patterns of CD44 in normal urothelial tissue compared to urothelial cancer lesions.12 As for mutation analysis of the p53 gene, a mutated p53 gene was detected in 5 cases (cases 4, 7, 11, 14 and 19), of which 2 (cases 7 and 19) showed different types of p53 mutation among multiple tumors. Based on these results multiple tumors in 6 cases (cases 2, 7, 9, 16, 19 and 22) could be considered to arise from independent clones. However, the results of these 2 methods were not consistent in 4 of the 6 cases (cases 2, 9, 16 and 22), while the remaining 2 (cases 7 and 19) showed different patterns of CD44 alternative splicing as well as different mutations of the p53 gene. Considering these results, at least 2 of the 24 cases of multiple urothelial cancers could be explained by the field defect hypothesis.

Tumor No.

1129

Date

Grade

Mutation

Codon

4/98 4/98

3 3

AGA-TGA AGA-TGA

65 65

6/98 6/98 2/99

2 2 3

Not found AAG-AAC CGT-CAT

132 273

12/99 12/99

2 3

CGT-CAT CGT-CAT

273 273

1/00 1/00

3 3

AGA-AAA AGA-AAA

280 280

9/01 9/01

3 3

AGA-AAA AGA-TGA

280 65

1. Jones, P. A. and Droller, M. J.: Pathways of development and progression in bladder cancer: new correlations between clinical observations and molecular mechanisms. Semin Urol, 11: 177, 1993 2. Xu, X., Stower, M. J., Reid, I. N., Garner, R. C. and Burns, P. A.: Molecular screening of multifocal transitional cell carcinoma of the bladder using p53 mutations as biomarkers. Clin Cancer Res, 2: 1795, 1996 3. Harris, A. L. and Neal, D. E.: Bladder cancer—field versus clonal origin. N. Engl J Med, 326: 759, 1992 4. Amar, A. D. and Das, S.: Upper urinary tract transitional cell carcinoma in patients with bladder carcinoma and associated vesicoureteral reflux. J Urol, 133: 468, 1985 5. Presti, J. C., Jr., Reuter, V. E., Galan, T., Fair, W. R. and Cordon-Cardo, C.: Molecular genetic alterations in superficial and locally advanced human bladder cancer. Cancer Res, 51: 5405, 1991 6. Takahashi, T., Habuchi, T., Kakehi, Y., Mitsumori, K., Akao, T., Terachi, T. et al: Clonal and chronological genetic analysis of multifocal cancer of the bladder and upper urinary tract. Cancer Res, 58: 5835, 1998 7. Habuchi, T., Takahashi, R., Yamada, H., Kakehi, Y., Sugiyama, T. and Yoshida, O.: Metachronous multifocal development of urothelial cancers by intraluminal seeding. Lancet, 342: 1087, 1993 8. Bartolazzi, A., Peach, R., Aruffo, A. and Stamenkovic, I.: Interaction between CD44 and hyarulonate is directory implicated in the regulation of tumor development. J Exp Med, 180: 53, 1994 9. Tanabe, K. K., Nishi, T. and Saya, H.: Novel variants of CD44 arising from alternative splicing: changes in the CD44 alternative splicing pattern of MCF-7 breast carcinoma cells treated with hyaluronidase. Mol Carcinog, 7: 212, 1993 10. Tanabe, K. K., Ellis, L. M. and Saya, H.: Expression of CD44R1 adhesion molecule in colon carcinomas and metastases. Lancet, 341: 725, 1993 11. Hara, I., Miyake, H., Yamanaka, K., Hara, S., Arakawa, S. and Kamidono, S.: Expression of CD44 adhesion molecule in nonpapillary renal cell carcinomas and normal kidneys. Urology, 54: 562, 1999 12. Miyake, H., Okamoto, I., Hara, I., Gohji, K., Yamanaka, K., Arakawa, S. et al: Highly specific and sensitive detection of malignancy in urine samples from patients with urothelial cancer by CD44v8 –10/CD44v10 competitive RT-PCR. Int J Cancer, 79: 560, 1998 13. Miyake, H., Hara, I., Yamanaka, K., Gohji, K., Arakawa, S. and Kamidono, S.: Expression patterns of CD44 adhesion molecule in testicular germ cell tumors and normal testes. Am J Pathol, 152: 1157, 1998 14. Miyake, H., Eto, H., Arakawa, S., Kamidono, S. and Hara, I.: Over expression of CD44v8 –10 in urinary exfoliated cells as an independent prognostic predictor in patients with urothelial cancer. J Urol, 167: 1282, 2002 15. Nagabhushan, M., Pretlow, T. G., Guo, Y. J., Amini, S. B., Pretlow, T. P. and Sy, M. S.: Altered expression of CD44 in human prostate cancer during progression. Am J Clin Pathol, 106: 647, 1996 16. Chomczynski, P. and Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem, 162: 156, 1987 17. Vogelstein, B. and Kinzler, K. W.: p53 function and dysfunction. Cell, 70: 523, 1992 18. Bedi, G. C., Westra, W. H., Gabrielson, E., Koch, W. and Sidransky, D.: Multiple head and neck tumors: evidence for a common clonal origin. Cancer Res, 56: 2484, 1996 19. Screaton, G. R., Bell, M. V., Jackson, D. G., Cornelis, F. B., Gerth, U. and Bell, J. I.: Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternative spliced exons. Proc Natl Acad Sci USA, 89: 12160, 1992 20. Miyake, H., Hara, I., Okamoto, I., Gohji, K., Yamanaka, K., Arakawa, S. et al: Interaction between CD44 and hyaluronic acid regulates human prostate cancer development. J Urol, 160: 1562, 1998