Evaluation of Epidermal Growth Factor Receptor DNA Amplification and mRNA Expression in Bladder Cancer

0022-534 7/92/14 71-0274$03.00/0 THE JOURNAL OF UROLOGY Copyright© 1992 by AMERICAN UROLOGICAL ASSOCIATION, INC.

Vol. 147, 274-277, January 1992

Printed in U.S.A.

EVALUATION OF EPIDERMAL GROWTH FACTOR RECEPTOR DNA AMPLIFICATION AND mRNA EXPRESSION IN BLADDER CANCER DAVID P. WOOD, JR., WILLIAM R. FAIR

AND

R. S. K. CHAGANTI

From the Laboratory of Cancer Genetics, Sloan-Kettering Institute, Urology Service, Department of Surgery and Department of Pathology, Memorial Hospital, Memorial Sloan-Kettering Cancer Center, New York, New York

ABSTRACT

The epidermal growth factor receptor has been implicated in the malignant transformation of cells because the v-erbB oncogene is a truncated form of the epidermal growth fac±or rereptor. DNA amplification and/or mRNA overexpression of the epidermal growth factor receptor is associated with the malignant potential of several human epithelial tumors. To determine the frequency of epidermal growth factor receptor DNA amplification and mRNA overexpression in bladder cancer, we evaluated 12 bladder tumors for DNA amplification and another 14 bladder tumors for mRNA overexpression. By Southern hybridization, we found no evidence of DNA amplification or gene rearrangements in 12 bladder tumors. Five of 14 bladder tumors overexpressed epidermal growth factor receptor mRNA four to 15 fold compared to normal urothelium from the same bladder by Northern analysis. These findings suggest that epidermal growth factor receptor amplification and/or gene rearrangement occurs infrequently in bladder cancer. Epidermal growth factor receptor mRNA overexpression, however, is found in 36% of bladder tumors and may play a role in bladder tumorogenesis. KEY WORDS:

epidermal growth factor receptor; neoplasms, bladder

Epidermal Growth Factor Receptor is a transmembrane protein that is present in a variety of tissues including urothelium, kidney and breast. 1-:1 Epidermal growth factor receptor is activated by Epidermal Growth Factor, a peptide that is present in urine and by Transforming Growth Factor alpha (TGF-a) which is associated with cellular proliferation. 4 - 8 Investigators, using immunohistochemical staining with monoclonal antibodies directed against the epidermal growth factor receptor, have found increased staining in 40 to 95% of bladder tumors. 9 ·rn Recently, epidermal growth factor receptor immunoreactivity has been correlated with a poor prognosis with high stage tumors staining more frequently than low stage tumors. 11 It is unclear which step of protein synthesis is responsible for epidermal growth factor receptor overproduction in bladder tumors. One study evaluated 29 bladder tumors for epidermal growth factor receptor DNA amplification and found that one tumor had evidence of amplification. 12 We studied the epidermal growth factor receptor mRNA levels in bladder tumors to determine if mRNA overexpression plays a role in epidermal growth factor receptor overproduction. We evaluated 26 bladder tumors for epidermal growth factor receptor DNA amplification or mRNA overexpression to better define the mechanism of epidermal growth factor receptor expression in bladder cancer. MATERIALS AND METHODS

Tissue procurement. Bladder cancer from a group of 12 patients was available for Southern analysis to identify DNA amplification. Additionally, bladder tumor tissue with corresponding grossly normal urothelium from the same bladder was available for mRNA analysis from another 14 patients undergoing radical cystectomy. The normal urothelium was dissected from the underlying muscle by a pathologist, This normal urothelial specimen was taken far from the primary tumor. No patient had histologic evidence of multi-focal carcinoma in situ in the bladder specimen. The most-epithelial-containing porAccepted for publication July 9, 1991. *Requests for reprints: Division of Urology, Chandler Medical Center, University of Kentucky, 800 Rose St., Lexington KY 40536. Supported by NIH Grant CA-05826. 274

tion of the bladder tumor was used for analysis to decrease the amount of normal stromal and vascular tissue in the specimen. Adequate tissue was not available for both DNA and mRNA analysis on the same specimen, All patients underwent surgery at Memorial-Sloan-Kettering Cancer Center. Tissue was snap frozen in liquid nitrogen and stored at -SOC. All bladder tumors studied were transitional cell carcinomas. Frozen tissue was not available for immunohistochemical staining with the epidermal growth factor receptor monoclonal antibody. Southern analysis. High molecular weight DNA was extracted from the tumor as previously described. 13 Ten µg. of DNA was digested with EcoRI restriction endonuclease, electrophoresed on a 0.8% agarose gel and then transferred to nylon membrane using a semi-automatic system (Probe Tech3, Oncor). Random primers were used to radioactively label the probes to a high specific activity. 14 The filters were hybridized and washed as previously described. 13 After washing, the filters were exposed to Kodak XAR film at -SOC for 1 to 3 days, The epidermal growth factor receptor probe was purchased from ATCC (American Type Culture Collection, Rockville, MD) and the JH probe was a gift from J. Ravetch. 15 The JH probe was assumed to be a single copy gene and used to adjust for unequal DNA loading, The signal intensity from each tumor was quantitated on an Ultroscan XL Laser Densitometer (LKB). The epidermal growth factor receptor signal level was divided by the JH level to compensate for differences in DNA loading. The corrected epidermal growth factor receptor level was then compared to the corrected epidermal growth factor receptor signal from normal placenta to determine the level of epidermal growth factor receptor amplification in each tumor, Northern analysis. RNA was isolated using the guanidinium isothiocyanate method followed by a cesium chloride gradient centrifugation.13 Twenty µg. of total RNA was electrophoresed on a 3% formaldehyde/I% agarose gel for 18 to 20 hours. The RNA was then transferred onto Zetaprobe membrane (Biorad). The filter was prehybridized in 50% formamide/lM NaCl/10% Dextran Sulphate/I% SDS (Sodium dodecyl sulfate) for 15 minutes. Two X 106 cpm./ml. of probe and 100 µg./ml. of sheared salmon sperm was added to the prehybridization buffer

275

EPIDERMAL GROWTH FACTOR RECEPTOR EXPRESSION KN BLADDER CANCER

and incubated at 42C for 16 to 18 hours. The filters were then washed in 2X SSC (lx SSC = 0.15M sodium chloride and 0.015M sodium citrate) at room temperature twice for five minutes followed by two washes in 2x SSC and 1% SDS at 60C for 30 minutes each. The two final washes were in O.lx SSC at room temperature for 30 minutes each. The filters were exposed to Kodak XAR film at -SOC for one to four days. To control for differences in the amount of mRNA in each lane, each blot was hybridized with a gamma actin probe (gift of P. Gunning 16 ) using the same hybridization conditions as for the epidermal growth factor receptor probe. The level of mRNA expression in the tumor and normal tissues was determined for both probes using a laser densitometer. The epidermal growth factor receptor level was divided by the gamma actin level for each specimen to adjust for differences in the amount of mRN A loaded in each lane. We then compared the corrected epidermal growth factor receptor mRNA level in the tumor to that in normal urothelium to determine the level ofmRNA overexpression.

Pt # 10 Pl 8 5 4 Pl 1 - 23 Kb 9.4 Kb

~

- 6.6 Kb - 4.4 Kb

RESULTS

Twenty-six bladder tumors were analyzed for either epidermal growth factor receptor DNA amplification or mRNA overexpression. Twelve tumors had DNA analysis and 14 tumors had mRNA analysis (tables 1 and 2). Follow up data are too short to comment on the effect epidermal growth factor receptor expression may have on survival. None of the 12 bladder tumors analyzed by Southern analysis exhibited epidermal growth factor receptor DNA amplification or gene rearrangements (figure 1). Normal urothelium and tumor tissue expressed the expected 10kb epidermal growth factor receptor mRNA band on Northern analysis (figure 2). TABLE 1.

Bladder tumors evaluated by Southern analysis for DNA amplification

Patient Number

Pathologic

DNA Amplifi-

Stage*

cation**

Tl T2 T3 T4 T5 T6 T7 TS T9 TlO Tll Tl2

P3aNlMO P3bNOMO PISNOMO P4aNOMO P3aNOMO PISNOMO PlNOMO P3bNOMO P3aN2MO P3bNOMO PaNOMO PlNOMO

Tl3 Tl4 Tl5 Tl6 Tl7 Tl8 Tl9 T20 T21 T22 T23 T24 T25 T26

Pathologic Stage* P3bNOMO P4bNOMO P3bN!MO P3aNOMO PlNOMO PlNOMO P3bNlMO PaNOMO P3bNOMO P3bNOMO P3aNOMO P3bNOMO P3bNlMO P3bNlMO

- 13.5 Kb

FIG. 1. Southern analysis of DNA from bladder tumors hybridized with epidermal growth factor receptor probe. Germline pattern is seen for all tumors with no evidence of DNA amplification. P-placental DNA as control.

PU

22 .---,

17 .---,

20

113

,---,

16

24

.---,

.---,

.---,

T N T N T N T N T N T N

-10 Kb

Acti11

- 2.1 Kb

mRNA

FIG. 2. Northern analysis evaluating bladder tumors for epidermal growth factor receptor mRNA expression. Tumors from patient #16, #17, #18, #20 and #24 have 12, 15, 10, 5 and 4-fold mRNA overexpression respectively.

2. Bladder tumors evaluated for EGFR mRNA expression by Northern Analysis Patient Number

JH -

EGFR mRNA

* Staging according to the TNM system for bladder cancer by the American Joint Committee on Cancer. ** _ X - number of times DNA amplified. TABLE

2.3 Kb 2.1 Kb

mRNA** overexpression

12X 15X lOX 5X

4X

* Staging according to the TNM system for bladder cancer by the American Joint Committee on Cancer. ** _ x - number of times EGFR mRNA overexpressed.

There were no abnormal size transcripts to suggest a truncated form of the epidermal growth factor receptor mRNA. Five of the 14 tumors analyzed for epidermal growth factor receptor mRNA expression had four to 15 times higher epidermal growth factor receptor mRNA levels in the tumor compared to the corresponding normal urothelium from the same bladder (table 2, figure 2). Two of the tumors with epidermal growth factor receptor mRNA overexpression were muscle invading tumors and the other three were superficial tumors. Because all of the tumors were from radical cystectomy specimens, muscle invading tumors had been present in the bladder prior to surgery. Therefore, the superficial tumors in this cohort of patients are not representative of primary superficial bladder tumors and a correlation between epidermal growth factor receptor mRNA expression and tumor stage is not possible. DISCUSSION

The epidermal growth factor receptor has been implicated in the malignant transformation of cells because the v-erbB oncogene, which produces erythroblastosis and sarcomas in chickens, is a truncated form of the epidermal growth factor receptor.

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WOOD, FAIR AND CHAGANTI

Subsequently, many normal and malignant cells were found to express the epidermal growth factor receptor. 6 Epidermal growth factor and TGF-a bind to epidermal growth factor receptor and activates the receptor to phosphorylate proteins via tyrosine kinase. 17 This phosphorylation may stimulate intracellular or transmembrane signal transduction and increase cell cycle activity or produce malignant transformation of the cell. Urine contains high levels of epidermal growth factor and this has stimulated interest in determining the level of epidermal growth factor receptor expression in urological tumors.4 Several investigators have found elevated epidermal growth factor receptor expression in renal cell carcinoma and bladder cancer thus supporting the idea that the epidermal growth factor receptor may be important in the development or growth oftumors of the kidney ifod bladder. 2 ; 9 ' 18 Published reports have determined epidermal growth factor receptor expression in bladder cancer using monoclonal antibodies to the receptor. By immunohistochemical staining, 40 to 95% of all bladder tumors express epidermal growth factor receptor. 1· 9 • rn, 18 In particular, epidermal growth factor receptor staining was significantly associated with decreased patient survival, tumor progression, and tumor recurrence. Muscle invasive tumors had a 56 to 80% incidence of epidermal growth factor receptor staining compared to 15 to 44 % of superficial tumors. 11 Another study, using Scatchard analysis to better determine the amount of epidermal growth factor receptor in the cell, found that bladder tumors had more epidermal growth factor receptor present than normal urothelium but, except for a few cases, the difference was only two to threefold. 18 Conversely, Messing 10 found that superficial and invasive tumors express epidermal growth factor receptor equally with no significant difference in the staining pattern between the two groups. The differences in these studies using immunohistochemistry is probably related to the different antibodies used and other methodologic considerations.rn Because of these findings, it is important to determine if epidermal growth factor receptor DNA amplification or mRNA overexpression play a role in causing bladder tumors to overproduce the epidermal growth factor receptor. Epidermal growth factor receptor DNA amplification has been found in several tumors including gliomas, and squamous cell carcinomas. 19 • 20 DNA amplification, however, is not essential for epidermal growth factor receptor overproduction. Renal cell carcinoma, which overexpresses epidermal growth factor receptor, does not exhibit evidence of concomitant gene amplification.21 One study has evaluated epidermal growth factor receptor DNA amplification in bladder tumors. They found one of 29 tumors had eightfold DNA amplification which correlated with an increased epidermal growth factor receptor immunoreactivity in that tumor. 12 We found no evidence of epidermal growth factor receptor DNA amplification or gene rearrangement in 12 bladder tumors. These results suggest that DNA amplification plays a minor role in epidermal growth factor receptor overexpression in bladder tumors and that mRNA overexpression or protein overproduction are the main causes. We found 36% of bladder tumors overexpressed epidermal growth factor receptor mRNA four to 15 times compared to normal urothelium from the same bladder. Our level of epidermal growth factor receptor overexpression is lower than that reported by Neal et al. 11 , who used immunohistochemistry. Several reasons could explain this discrepancy. First, tumor heterogeneity, necrosis and infiltration by normal stromal and inflammatory cells can dilute the number of cells that express high levels of epidermal growth factor receptor. Northern blot analysis measures the mRNA level of a given gene in a sample of tissue and unlike immunohistochemistry, cannot differentiate between tumor and nontumor cells. It may be that some tumor cells express high levels of epidermal growth factor

receptor that can be identified by immunohistochemistry but are diluted by nonexpressive cells on Northern analysis. Secondly, Northern analysis may not be sensitive enough to detect small changes in epidermal growth factor receptor mRNA expression that may translate into an overproduction of the receptor which can be seen with immunohistochemistry. Finally, we compared the epidermal growth factor receptor mRNA content in the tumor to normal appearing urothelium from the same bladder. Messing10 found that normal appearing urothelium from bladders that contained a preexisting bladder tumor overexpressed the epidermal growth factor receptor compared to specimens taken from bladders without a tumor present. This finding suggests that a "urothelial field change" has occurred which makes the urothelium more susceptible to tumor formation. However, not all bladders form recurrent tumors thus the significance of this staining pattern is unclear. Because we did not have normal urothelium from noncancer bearing bladders, we do not know the epidermal growth factor receptor mRNA level in completely normal urothelium. In summary, our results better define the expression of the epidermal growth factor receptor in bladder cancer. DNA amplification infrequently occurs in bladder cancer but some tumors overexpress epidermal growth factor receptor mRNA compared to adjacent normal urothelium; the significance of this overexpression is unknown. The correlation between epidermal growth factor receptor mRNA expression and immunohistochemical staining is unclear and a study evaluating mRNA levels and epidermal growth factor receptor staining on the same tumor should be done to answer this question. REFERENCES L Messing, E. M., Hanson, P., Ulrich, P. and Erturk, E,: Epidermal growth factor-interactions with normal and malignant urothelium: in vivo and in situ studies. J. Urol., 138: 1329, 1987. 2. Mydlo, J. H., Michaeli, J., Cordon-Cardo, C., Goldenberg, A. 8., Heston, W. D. W. and Fair, W.R.: Expression of transforming growth factor a and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue, Cancer Res., 49: 3407, 1989. 3. Fitspatrick, S. L., Brightwell, J., Wittliff, J. L., Barrows, G. H. and Schultz, G. 8.: Epidermal growth factor binding by breast tumor biopsies and relationship to estrogen and progestin receptor levels. Cancer Res., 44: 3448, 1984. 4. Lau, J. L. T., Fowler, Jr., J, E. and Ghosh, L.: Epidermal growth factor in the normal and neoplastic kidney and bladder. J. Urol., 139: 170, 1988. 5. Gregory, H.: Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature, 257: 325, 1975. 6. Downdard, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwlee, P., Ullrich, A., Schlessinger, J. and Waterfield, M. D.: Close similarity of epidermal growth factor receptor and verb-B oncogene protein sequences. Nature, 307: 521, 1984. 7. Derynck, R., Roberts, A. B., Winkler, M. E., Chen, E. Y. and Goedde!, D. V.: Human transforming growth factor a: precursor structure and expression in E. coli. Cell, 38: 287, 1984. 8. Rosenthal, A., Lindquist, P. B., Bringman, T. S., Goedde!, D, V. and Derynck, R.: Expression in rat fibroblasts of a human transforming growth factor a results in transformation. Cell, 46: 301, 1986, 9. Neal, D. E., Bennett, M. K., Hall, R. R., Marsh, C., Abel, P. D., Sainbury, J. R. C. and Harris, A. L.: Epidermal growth factor receptors in human bladder cancer: comparison of invasive and superficial tumors. Lancet, l: 366, 1985. 10. Messing, E. M.: Clinical implications of the expression of epidermal growth factor receptors in human transitional cell carcinoma. Cancer Res,, 50: 2530, 1990. 11. Neal, D. E., Sharples, L., Smith, K, Fennelly, J., Hall, R. R. and Harris, A L.: The epidermal growth factor receptor and the prognosis of bladder cancer. Cancer, 65: 1619, 1990. 12. Berger, M. S., Greenfield, C., Gullick, W. J., Haley, J., Downward, J., Neal, D. E., Harris, A. L. and Waterfield, M. D.: Evaluation of epidermal growth factor receptors in bladder tumors. Br. J. Cancer, 56: 533, 1987. 13. Maniatis, T., Fritsch, E. F. and Sambrook, J. (eds): Molecular

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