0022-5347 /91/1453-064 7$03.00/0 Vol. 145, 647-653, March 1991
THE JOURNAL OF UROLOGY Copyright© 1991 by AMERICAN UROLOGICAL ASSOCIATION, INC.
Printed in U.S.A.
EXPERIMENTAL INTRAVESICAL THERAPY FOR SUPERFICIAL TRANSITIONAL CELL CARCINOMA IN A RAT BLADDER TUMOR MODEL GARY D. STEINBERG,* CHARLES B. BRENDLER, ROBERT A. SQUIRE
AND
JOHN T. ISAACS
From the James Buchanan Brady Urological Institute, Department of Urology Oncology and Comparative Medicine, Johns Hopkins Hospital and Johns Hopkins University School of Medicine, Baltimore, Maryland
ABSTRACT
A rodent bladder cancer model that is induced by intravesical instillation of N-methyl-Nnitrosourea (MNU) was characterized. Cohorts of four to five week old female Fisher 344 rats received four biweekly 1.5 mg. doses of intravesical MNU and were sacrificed at various intervals. By week 13 all animals had flat atypia and/or papillary transitional cell tumors, and 67% of the lesions were moderately (grade II) or poorly differentiated (grade III). By week 20, 83% had gross muscle invasive tumors that eventually killed the host. A cohort of 40 MNU treated animals was subsequently treated commencing at week 17 after initiation of MNU with one of three intravesical six week regimens: 1) saline; 2) BCG (Tice strain); or 3) recombinant human tumor necrosis factor (RTNF) plus adriamycin. There was no difference in animal survival or tumor growth in any group of animals commencing therapy at week 17. A second cohort of 107 animals commenced therapy at 13 weeks after initiation of MNU with one of five intravesical six week regimens: 1) intravesical BCG (Tice strain); 2) adriamycin; 3) recombinant human tumor necrosis factor (RTNF); 4) RTNF plus adriamycin; or 5) BCG plus adriamycin. BCG, RTNF or adriamycin alone had no effect on tumor growth; however, BCG plus adriamycin and RTNF plus adriamycin commencing at week 13 significantly inhibited tumor growth and progression. In conclusion, this autochthonous intravesical rodent transitional cell carcinoma model appears useful for the following reasons: 1) it closely resembles human transitional cell carcinoma histologically and biologically in that all animals develop neoplastic changes in-situ that progress to muscle invasion and kill the host; 2) as with human bladder cancer these tumors do not respond to intravesical therapy if treated when tumor burden is large; however, tumor growth is inhibited when treated early; and 3) this model appears appropriate for screening and developing new intravesical treatments for superficial bladder cancer. KEY WORDS:
bladder cancer, adriamycin, recombinant human tumor necrosis factor, bacillus Calmette-Guerin
Bladder cancer is the second most common urologic malignancy in the US with 49,000 new cases and 10,000 deaths per year. 1 Smoking and occupational exposures to various chemicals especially in the dye and petroleum industries are reported to be etiologic factors in approximately 50% of all cases. 2 • 3 Approximately 70% of patients diagnosed each year have superficial (Ta, Tis, T 1 ) disease but these patients are at risk for recurrent tumors and disease progression. 3 - 6 Many of these patients are therefore candidates for intravesical therapy; however, the efficacy of available intravesical agents is only 30 to 70% and all have potentially serious side effects. 4 • 7- 13 In the past, investigators have used serially passaged in-vitro and transplantable bladder cancer animal models to study the effects oftherapy. 14- 22 These models differ from human bladder cancer because of inherent problems of phenotypic and genotypic drift which occur with both continuous cell and animal passage and alter the original biochemical and histological characteristics of the tumor. In addition, autochthonous animal models of bladder cancer induced by the oral administration of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and N-[4-(5nitro-2-furyl)-2-thiazolyl] formamide (FANFT) 16- 18• 23- 25 or intravesical MNU 26- 29 are problematic because of the frequent development of squamous cell carcinomas along with transiAccepted for publication October 19, 1990. * Requests for reprints: Department of Urology, Marburg 145, Johns Hopkins Hospital, Baltimore, MD 21205. Supported by HHS Grant NIDDK DK07552, U.S. Public Health Service Grant RR00130, and the generous contributions of Mr. and Mrs. Merrill Bank, Mr. and Mrs. Seymour Kulick and Mr. and Mrs. Jack Stollof. 647
tional cell carcinomas of the bladder. Using a modification of a model introduced in the 1970s by Hicks et al., 26 • 27 we have developed a model of autochthonous rat transitional cell carcinoma (TCC) 30 that is useful for the following reasons: 1) the tumors arise only from urinary bladder epithelium; 2) the histology of the invasive tumors which grossly develop is transitional cell with occasional focal squamous metaplasia; 3) no leukemias, breast cancers, lymphomas or other tumors are induced; 4) similar to human TCC, 30- 32 the karyotypes are diploid with no consistent pattern of chromosomal aberrations; and 5) similar to human TCC, increased expression or mutation of ras p21 protein does not appear to be necessary for malignant transformation. 33 •34 This model appears ideally suited for the study of new intravesical treatments for superficial TCC. A previous study from our laboratory using this model demonstrated that at 13 weeks after carcinogen exposure 45% of female Fisher 344 rats had superficial TCC, and that at 17 weeks 32% had superficial TCC and 20% had muscle invasive TCC. 30 Therefore, to assess the effect of intravesical therapy and the importance of timing of therapy cohorts of animals began intravesical therapy at 13 and 17 weeks after initiation of MNU with immunotherapeutic agents (BCG and RTNF) individually and in combination with a chemotherapeutic agent (adriamycin). These therapeutic agents were chosen because: 1) BCG is currently believed to be one of the most effective intravesical agents against superficial TCC; 9 • 11- 13 2) recent work in our laboratory by Alexander et al. 35 demonstrated the syn ergistic efficacy of the combination of RTNF and chemotherapeutic agents directed against the enzyme DNA topoisomerase
648
STEINBERG AND ASSOCIATES
II in vitro. This enzyme is responsible for unwinding the DNA double helix during replication, and is thus an important target for chemotherapeutic drugs; 36 3) adriamycin is a DNA topoisomerase II inhibitor35- 38 and is also used intravesically to treat superficial TCC; 39 4) previously, Alexander et al. 21 demonstrated the in-vivo synergistic efficacy of the systemic combination of RTNF and adriamycin in the treatment of MBT-2 bladder cancer cells when implanted subcutaneously in the hind leg of C3H/HE mice; and 5) intravesical RTNF is being tested in phase I trials by the Eastern Cooperative Oncology Group (ECOG). The results of intravesical therapy of this carcinogen induced autochthonous model form the basis of this report. MATERIALS AND METHODS
We modified the methods used in the model introduced by Hicks et al. 26 • 27 by using a different strain of inbred rats and providing continuous antibiotics in their drinking water. 30 Multiple cohorts of four to five week old female Fisher 344 rats (Harlan Sprague Inc., Indianapolis, Ind.) were used for this study, The animals were anesthetized with intraperitoneal nembutal. One gram of N-methyl-N-nitrosourea (MNU) (Sigma, St. Louis, MO.), was dissolved in 100 cc of normal saline. The animals received 0.15 cc (1.5 mg.) of this solution via a 22 gauge teflon angiocath (Travenol Laboratories, Chicago, 11.) intravesically under a continuous flow hood within 45 minutes after preparation of the MNU solution, every other week (week 0, 2, 4, and 6) for a total of four doses. The animals remained anesthetized for approximately 2 hours after catheterization. Routine precautions were followed when working with this carcinogen. MNU was administered under a continuous flow hood, and the excess MNU and animal bedding were incinerated. All animals received water medicated with a combination of trimethoprim sulfamethoxazole, neomycin, and polymyxin B, formulated as described previously40 and Purina rat chow ad libitum. Animals were maintained on a 12 hour lightdark cycle, and were sacrificed at various intervals after they received their last dose of MNU. Approximately 24% (62 of 258) of all animals entered into MNU dosing schedules did not survive all 4 doses. The causes of death were usually related to complete ulceration of the urinary bladder or urosepsis secondary to urethral stricture formation and urinary obstruction. Approximately 5% of animals formed bladder or ureteral calculi, Tissue preparation. The animals were sacrificed by CO 2 gas overdose and necropsy was performed. The urinary bladder and urethra were excised in toto. The liver and lungs were inspected for metastases and, if suspicious, were fixed in formalin or Bouin's solution respectively. The kidneys and ureters were dissected and inspected for the presence of pyonephrosis, stones, or upper tract tumors secondary to reflux. The bladders were inflated with 10% buffered formalin, and a ligature was placed around the urethra. The bladders were fixed for at least 24 hours and embedded in paraffin. Sections were cut transversely through the midportion of the bladder, and nine 5micron sections were taken from each half to adequately sample the entire bladder; these sections were stained with hematoxylin and eosin (H&E). Light microscopy. All sections from each bladder specimen were reviewed under low power. The section that appeared to have the greatest amount of change from normal rat bladder was selected for histologic grading. Typically the histologic changes were relatively uniform in any one specimen, but some areas of the bladder were occasionally more affected than other areas. The tumors were staged by the Union Internationale Cancer Control (UICC) and the American Joint Committee for Cancer Staging tumor, node and metastasis (TNM) system for human bladder cancer as described previously18• 24 • 41 - 44 and categorized into either: 1) hyperplasia, flat or papillary atypia,
with mild to moderate dysplasia; 2) superficial transitional cell carcinoma (TCC) which includes stages stage P. (papillary exophytic tumors with fibrovascular cores and nuclear pleomorphism of the epithelial cells with no evidence of invasion); Stage P;, (tumors confined to the mucosa with full mucosal thickness of marked atypia/dysplasia (carcinoma in-situ)); and stage P 1 (tumors that demonstrate evidence of lamina propria invasion); or 3) bladder wall muscle invasive transitional cell carcinomas (stages P 2 and P 3 ). The tumors were also categorized by histologic grade using conventional criteria. Grade 1 lesions demonstrate minimal nuclear pleomorphism, while grade 2 and 3 lesions show increasing amounts of nuclear pleomorphism, loss of polarity, increased cellularity, prominence of nucleoli, presence of mitoses and formation of giant cells. Therapeutic trials. A cohort of 40 animals that received four doses of MNU was divided into four treatment groups. Group 1 received no further treatment. Groups 2-4 were treated weekly for six weeks intravesically with 0.2 cc of either saline (group 2); BCG (Tice strain 5 ± 3 X 108 colony forming units/ ampule dissolved in 60 cc normal saline) (group 3); or recombinant human tumor necrosis factor (RTNF) (7 x 105 units/ ml. of saline) (Cetus Corp., Emeryville, CA) and adriamycin (two mg./ml.) (Adria Inc., Dublin, OH) (1:1 mixture) (group 4). The dosages of the intravesical agents were at the same concentrations as used for intravesical therapy in humans. Treatment began 17 weeks after initiation of MNU. The animals were followed until dead or premorbid for a maximum of 42 weeks. A second cohort of 107 animals that received four doses of MNU was divided into six treatment groups. Group 1 received no further treatment. Groups 2-6 were treated weekly for six weeks intravesically with 0.2 cc of intravesical: RTNF (group 2); adriamycin (group 3); BCG (group 4); BCG and adriamycin (1:1 mixture) (group 5); or RTNF and adriamycin (group 6). 100
Histopathological Findings in Female Fisher 344 Rat Bladders Treated with 4 Doses of N-methyl-N-nitrosourea (MNU)
90 Legend BO
70
-
D
hyperplasia or atypical dysplasia
rn
superficial TCC Pa, Pis, P1
•
60
C
muscle invasive TCC
~
, P3
Q)
~
50
Q)
a..
40
30
20
10
0
Weeks ofter Initiation of the Carcinogen 8-10 weeks 12-13 (N=IO) (N=22)
14-16 (N=21)
17-18 (N=IO)
20-24 (N=l2)
FIG. 1. Pathological stages of bladder tumors in female Fisher 344 rat bladders treated with four doses of intravesical MNU and sacrificed at various time intervals.
INTRAVESICAL THERAPY FOR SUPERFICIAL TRANSITIONAL CELL CARCINOMA
100
649
Tumor Grode of Rat Bladders Treated with 4 Doses of lntrovesical N-methyl-N-Nitrosourea (MNU)
90 Legend
80
70
D
grade I
~
grade II
II
grade Ill
60
C:
(I) (.) b.
50
(I)
0...
40
30
20
10
Weeks after Initiation of the Carcinogen MNU weeks 10-12 (N=l5)
weeks 13-15 (N=33)
weeks
weeks
16-18 (N=IO)
20-24 (N=l2)
FIG. 2. Pathological grades of bladder tumors in female Fisher 344 rat bladders treated with four doses of intravesical MNU and sacrificed at various time intervals.
FIG. 3. Fisher 344 female rat bladder treated with four doses of MNU with hyperplasia.
The dosages of intravesical agents were the same as above. Treatment began 13 weeks after initiation of MNU. Ten animals from each group were sacrificed at 25 weeks (6 weeks after the last treatment), and histology was evaluated blindly without knowledge of treatment given. RESULTS
Histopathologic progression of MNU treated bladders. All rat bladders treated with intravesical MNU develop progressive neoplastic changes (fig. 1), and the tumors become progressively less differentiated with time (fig. 2). These lesions progress from hyperplasia (fig. 3), atypia (fig. 4), carcinoma in-situ (fig. 5), and papillary TCC (fig. 6) to large bulky muscle invasive tumors (fig. 7) that completely fill the bladder lumen, obstruct the ureters and kill the animal. These large tumors weigh as much as one to two grams (the normal rat bladder weighs approximately 50 mg.). No animal in this study or in our previous study 30 developed squamous cell carcinoma either alone or in combination with transitional cell carcinoma as was described in previous studies of MNU induced bladder can cer. 11- 18, 26 - 28 This may be due to the continuous antibiotics in the animals' drinking water or the use of a strain of rats that is less prone to endogenous stone formation and thus chronic inflammation and squamous metaplasia. 30 Treatment results. The cohort of animals that began treatment at 17 weeks after the initiation of MNU had no apparent response to therapy when assessed by the TNM staging system (table 1). By week 40, 78% of the RTNF/adriamycin animals, 100% of the BCG animals, 88% of the saline animals and 75% of the control animals had bulky, muscle-invasive tumors.
FIG. 4. Fisher 344 female rat bladder treated with four doses of MNU with hyperplasia and mild to moderate cellular atypia.
There was no increase in survival in any treatment group versus the untreated control group. In the second cohort of animals that began treatment at 13 weeks after initiation of MNU, intravesical BCG, RTNF or adriamycin alone had no significant effect on tumor growth. However, the combinations of RTNF with adriamycin and BCG with adriamycin inhibited the growth and progression of tumors significantly (table 2 and fig. 8). Although few of the animals were cured by histological criteria, the tumors in the
650
STEINBERG AND ASSOCIATES
Fm. 5. Fisher 344 female rat bladder treated with four doses of MNU with areas of marked atypia and dysplasia involving full mucosal thickness (carcinoma-in-situ).
Fm. 7. Fisher 344 female rat bladder treated with four doses of MNU with muscle invasive TCC.
had flat carcinoma in situ only and the other had no evidence of disease. The overall mortality of treatment in all groups was 25% and did not vary significantly between treatment groups. These mortalities may be due to either catheterization, the intravesical agent used or possibly the MNU itself. DISCUSSION
Fm. 6. Fisher 344 female rat bladder treated with four doses of MNU with superficial papillary transitional cell carcinoma and mild to moderate cellular atypia.
BCG, RTNF, adriamycin and control groups weighed 1.5 to 2.0 gm. and filled the bladder, while the tumors in the RTNF / adriamycin and BCG/adriamycin groups were small, nonobstructing tumors. Of four additional RTNF/ adriamycin animals sacrificed at 31 weeks, only one had a bulky bladder tumor that filled the lumen, one had small papillary and sessile tumors, and two had no obvious gross tumors. Histopathological examination of the latter two bladders revealed that one
MBT-2 is presently the most frequently used animal model to study bladder cancer. 14• 15• 19- 22 MBT-2 is a transplantable murine serially passaged cell line that was established from a transitional cell carcinoma of the bladder induced by the oral administration of N-[ 4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT). 14-18 This model has been studied extensively and has been used to screen various intravesical and systemic chemotherapeutic agents. However, there are problems in using this model to study superficial TCC of the bladder: 1) it is a tumor serially passaged for over 10 years with resultant genetic drift; 2) it is usually transplanted subcutaneously (the reliability of implantation in the bladder is limited45- 47 ) and thus, one cannot assess the efficacy or adverse effects of intravesical therapy on the host urothelium; 3) it has a very fast in-vivo growth rate (doubling time is less than three days) and thus, it is difficult to study stage progression; 4) by day 34 after implantation the tumor contains highly anaplastic sarcomatous elements; and 5) the tumor is aneuploid with abnormal chromosomes, the development of which may have structurally occurred during serial passage. 16- 18 Few attempts to study intravesical therapy of autochthonous bladder tumors in rodents have been performed to date. Previously Soloway et al. 19 reported on the use of various chemotherapeutic agents instilled intravesically in mice fed F ANFT for 38 weeks and sacrificed at 46 weeks. They found that intravescial cis-platinum, mitomycin C, and thiotepa all reduced tumor incidence but there were no signficant differences in mean and median tumor weights between treated and control groups. In addition, few of the control animals developed invasive disease by 46 weeks, and the histology of these tumors was not described. Lee et al. 20• 22 instilled single cell suspensions of MBT-2 cells into c3H/HE mice bladders and treated these animals with either intravesical RTNF alone or a combination of intravesical BCG and systemic interleukin -II. Intravesical RTNF did not suppress tumor growth; however, the combination of intravesical BCG plus systemic interleukin II did. Bahnson and Ratliff48 instilled MBT-2 cells intravesically into C3H/ HE mice and subsequently treated them with alpha difluoromethylornithine (DFMO) in the drinking water alone and in
INTRAVESICAL THERAPY FOR SUPERFICIAL TRANSITIONAL CELL CARCINOMA TABLE
651
1. Results of intravesical therapy commenced at 17 weeks after the intravesical initiation of the carcinogen MNU in animals followed until
death or 42 weeks Treatment Group
Percent that Survived Therapy
Percent that Died from Bulky Bladder Cancer
Percent with Bulky Tumor at Sacrifice or Death
100
38
75
80
75
88
50
40
100
82
56
78
Control N=8 Saline N= 10 BCG N= 10 RTNF / adriamycin N= 11
Histopathological findings in female Fisher 344 rat bladders treated with 4 doses of intravesical MNU, commenced therapy at 13 weeks and sacrificed at 25 weeks after initiation of the carcinogen
TABLE 2.
Treatment Group
Percent with Hyperplasia, Percent with Flat or Papillary Superficial Atypia/Dysplasia TCC 40 30 50 20 70 60
0 10 10 0 20 30
Control RTNF Adriamycin BCG BCG/Adriamycin RTNF / Adriamycin
Percent with Muscle Invasion 60 50 40 80 10* 10*
* p = 0.04 (in comparison with percent of control animals with muscle invasive TCC).
Results of Treotment Begun at Week 13
90 80 70 +-_
C
Q)
~
Q)
Q.
60
50
40 30
20 10 Control
0
Hyplosia/atypia
RTNF
Adriomycin
BCG
~ Superficial TCC
BCG/Adrlomycin RTNF/Adriomycin
11111 Muscle Invasive TCC
FIG. 8. Histopathological findings as evaluated by TNM system in female Fisher 344 rat bladders treated with four doses of intravesical MNU, commenced therapy at 13 weeks and sacrificed at 25 weeks. combination with intravesical alpha 1 and beta interferon. DFMO alone significantly delayed the outgrowth of tumors without any additive effect when interferon was added. However, these studies did not assess tumor growth but rather inhibition ofMBT-2 cell implantation into the urinary bladder. In this study a model of autochthonous rat TCC of the bladder was developed based upon the original findings of Hicks et al. 26 This model is useful for studying intravesical therapy of superficial TCC for the following reasons: 1) the tumors arise only from bladder urothelium; 2) all animals develop progressive neoplastic changes in-situ within four months after initiation of the carcinogen; 3) these lesions progress through various early stages of neoplasia to bladder wall muscle-invasion and kill the host; 4) the histology of the tumors is transitional cell; and 5) the neoplastic lesions develop at discrete times and in high frequency (fig. 1), thereby allowing treatment to be initiated at known stages of disease. The toxicity and incomplete efficacy of the commonly used intravesical agents has prompted a search for other treatments for superficial TCC of the bladder. Although adriamycin is an
effective agent and intravesical RTNF is in phase I trial, the combination of RTNF and adriamycin has not been tested in humans. Previously Alexander et al. 21 demonstrated the efficacy of the systemic combination of RTNF and adriamycin in the treatment of MBT-2 bladder cancer in-vitro and in-vivo. Therefore, this autochthonous model ofTCC was used to assess the efficacy of combined intravesical therapy (i.e. biological response modifiers such as RTNF and BCG with adriamycin) in inhibiting the growth and progression of these tumors. In this study, BCG, RTNF, and adriamycin had little effect on tumor growth. However, both BCG and RTNF, in combination with adriamycin, significantly reduced tumor growth and delayed tumor progression. Although few animals were cured, most had significantly reduced tumor volumes. At present, BCG is considered one of the most effective single intravesical agents for therapy of superficial TCC. 4 • 49 Although its mechanism for action is unknown, BCG may act through a T-cell dependent mechanism. 5° Following intravesical BCG therapy, Haaff et al. 51 demonstrated the presence of IL-2 in the urine, and Bohle et al. 52 found both IL-1 and TNF in the urine. It is also known that BCG stimulates the endogenous production of TNF. 53 IL-2 is produced predominantly by T-helper cells which activate natural killer T-cells, 54 and TNF is produced predominantly by macrophages. 53 It is possible, therefore, that BCG and TNF act through a similar mechanism in combination with adriamycin to produce a synergistic tumoricidal effect. This may also explain the similar cytotoxic action on tumor cells of both BCG and TNF. Presently the mechanism of action of enhanced tumor cytotoxicity from the combination of biologic response modifiers such as RTNF and BCG and DNA topoisomerase II targeted chemotherapeutic agents is unknown. However, recent evidence suggests that the combination may allow easier access of TNF or TNF-induced nuclear endonucleases to DNA resulting in DNA fragmentation and cell death. 21 • 35• 37• 38 Future studies will be necessary to determine the optimal combinations, timing, and sequence of biologic response modifiers and chemotherapeutic agents targeted at DNA topoisomerase II. This model should be useful in determining this information, and this may lead to more effective treatments for superficial transitional cell carcinoma of the bladder in man. Acknowledgments. The authors would like to thank John Lamb for his excellent technical assistance. Recombinant Human Tumor Necrosis Factor was generously supplied to us by Dr. Abla Creasey of the Cetus Corporation (Emeryville, CA) and adriamycin was supplied to us by Dr. Vernon Verhoef of Adria Laboratories (Dublin, OH). REFERENCES
Silverberg, E., Boring, C. C. and Squires, T. S.: Cancer Statistics 1990. Ca., 40: 1, 1990. 2. Thompson, I. M. and Fair, W. R.: The epidemiology of bladder cancer. AUA update series vol 8, lesson 27, 1989. 3. Thompson, I. M., Peek, M. and Rodriguez, F. R.: The impact of cigarette smoking on stage, grade and number of recurrences of transitional cell carcinomas of the bladder. J. Urol., 137: 401, 1987. 1.
652
STEINBERG AND ASSOCIATES
4. Herr, H. W., Laudone, V. P. and Whitmore, W. F. Jr.: An overview of intravesical therapy for superficial bladder tumors. J. Urol., 138: 1363, 1987. 5. Rubben, H., Lutzeyer, W., Fischer, N., Deutz, F., LaGrange, W., Giani, G. and Members of the Registry for Urinary Tract Tumors: Natural history and treatment of low and high risk superficial bladder tumors. J. Urol., 139: 283, 1988. 6. Heney, N. M., Ahmed, S., Flanagan, M. J., Frable, W., Corder, M. P., Haferman, M. D., Hawkins, I. R. and The National Bladder Cancer Collaborative Group A: Superficial bladder cancer: progression and recurrence. J. Urol., 130: 1083, 1983. 7. Soloway, M. S.: The management of superficial bladder cancer. Cancer, 45: 1856, 1980. 8. Soloway, M. S.: Selecting initial therapy for bladder cancer. Cancer, 60: 502, 1987. 9. Catalona, W. J., Hudson, M.A., Gillen, D. P., Andriole, G. L. and Ratliff, T. L.: Risks and benefits of repeated courses of intravesical bacillus Calmette-Guerin therapy for superficial bladder cancer. J. Urol., 137: 220, 1987. 10. Heney, N. M., Koontz, W.W., Barton, B., Soloway, M., Trump, D. L., Hazra, T. and Weinstein, R. S., For the National Bladder Cancer Group: Intravesical thiotepa versus mitomycin C in patients with Ta, Tl, and Tis transitional cell carcinoma of the bladder: a phase III prospective randomized study. J. Urol., 140: 1390, 1988. 11. Herr, H. W., Baldalment, R. A., Amato, D. A., Laudone, V. P., Fair, W.R. and Whitmore, W. F., Jr.: Superficial bladder cancer treated with bacillus Calmette-Guerin: a multivariate analysis of factors affecting tumor progression. J. Urol., 141: 22, 1989. 12. Kavoussi, L. R., Torrence, R. J., Gillen, D. P., Hudson, M. A., Haaff, E. 0., Dresner, S. M., Ratliff, T. L. and Catalona, W. J.: Results of 6 weekly intravesical bacillus Calmette-Guerin instillations on the treatment of superficial bladder tumors. J. Urol., 139: 935, 1988. 13. Lamm, D. L., Stogdill, V. D., Stogdill, B. J. and Crispen, R. G.: Complications of bacillus Calmette-Guerin immunotherapy in 1,278 patients with bladder cancer. J. Urol., 135: 272, 1986. 14. Soloway, M. S.: Intravescial and systemic chemotherapy of murine bladder cancer: Cancer Res., 37: 2918, 1977. 15. Soloway, M. S. and Murphy, W. M.: Experimental chemotherapy of bladder cancer-systemic and intravesical. Seminars in Oncology, 6: 166, 1979. 16. Murphy, G. P., Sandberg, A. A., Pontes, J.E., Ochi, H., Yoshida, M. and Williams, P. D.: A murine model for bladder cancer. In: Bladder Cancer Part A: Pathology, Diagnosis and Surgery, New York: Alan R. Liss, Inc, pp. 177-200, 1984. 17. Raghavan, D., Debruyne, F., Herr, H., Jocham, D., Kakizoe, T., Okajima, E., Sandberg, A. and Tannock, I.: Experimental models of bladder cancer: a critical review. In: Developments in Bladder Cancer, New York: Alan R. Liss, Inc, pp. 171-208, 1986. 18. Cohen, S. M.: Pathology of experimental bladder cancer in rodents. In: The Pathology of Bladder Cancer, Edited by G. T. Bryan and S. M. Cohen. Boca Raton: CRC Press, vol I, pp. 1-40, 1983. 19. Soloway, M. S., Nissenkorn, I., McCallum, L. W. and Murphy, W. M.: Single and sequential combination intravesical chemotherapy of murine bladder cancer. Urology, 19: 169, 1982. 20. Lee, K. E., O'Donnell, R. W., Schoen, S. and Cockett, A. T. K.: Effect of intravesical administration of tumor necrosis serum and human recombinant tumor necrosis factor on a murine bladder tumor. J. Urol., 138: 430, 1987. 21. Alexander, R. B., Isaacs, J. T. and Coffey, D. S.: Tumor necrosis factor enhances the in vitro and in vivo efficacy of chemotherapeutic drugs targeted at DNA topoisomerase II in the treatment of murine bladder cancer. J. Urol., 138: 427, 1987. 22. Lee, K. E., Weiss, G. H., O'Donnell, R. W. and Cockett, A. T. K.: Reduction of bladder cancer growth in mice treated with intravesical Bacillus Calmette-Guerin and systemic interleukin 2. J. Urol., 137: 1270, 1987. 23. Drago, J. R.: The noble rat bladder cancer model: FANFT-induced tumors. Cancer, 53: 1093, 1984. 24. Fukushima, S., Hirose, M., Tsuda, H., Shirai, T., Hirao, K., Arai, M. and Ito, N.: Histological classification of urinary bladder cancers in rats induced by N-butyl-N-(4-hydroxy-butyl) nitrosamine. Gann, 67: 81, 1976. 25. Herman, C., Vegt, P., Debruyne, F., Vooijs, G. and Ramaekers, F.: Squamous and transitional elements in rat bladder carcinomas
26. 27. 28. 29. 30.
31. 32.
33.
34.
35.
36. 37. 38.
39. 40.
41.
42.
43.
44. 45. 46.
47.
induced by N-butyl-N-(4-hydroxyl-butyl) nitrosamine (BBN). M.J. Pathol., 120: 419, 1985. Hicks, R. M. and Wakefield, J. ST. J.: Rapid induction of bladder cancer in rats with N-methyl-N-nitrosourea I. Histology. Chem. Biol. Interactions, 5: 139, 1972. Hicks, R. M. and Wakefield, J. ST. J.: Membrane changes during urothelial hyperplasia and neoplasia. Cancer Res., 36: 2502, 1976. Severs, N. J., Barnes, S. H., Wright, R. and Hicks, R. M.: Induction of bladder cancer in rats by fractionated intravesicular doses of N-methyl-N-nitrosourea. Br. J. Cancer, 45: 337, 1982. Cox, R. and Irving, C. C.: Effect of N-methyl-N-nitrosourea on the DNA of Rat Bladder epithelium. Cancer Res., 36: 4114, 1976. Steinberg, G. D., Brendler, C. B., Ichikawa, T., Squire, R. A. and Isaacs, J. T .: The characterization of an N-methyl-N -nitrosourea (MNU) induced rat bladder cancer model. Cancer Res., 50: 6668, 1990. Vanni, R., Scarpa, R. M., Nieddu, M. and Usai, E.: Cytogenetic investigation on 30 bladder carcinomas. Cancer Genet. Cytogenet., 30: 35, 1988. Gibas, Z., Prout, G. R., Pontes, J.E., Connolly, J. G. and Sandberg, A. A.: A possible specific chromosome change in transitional cell carcinoma of the bladder. Cancer Genet. Cytogenet., 19: 229, 1986. Meyers, F. J., Gumerlock, P.H., Kokoris, S. P., De Vere White, R. W. and McCormick, F.: Human bladder and colon carcinomas contain activated ras p21: specific detection of twelfth codon mutants. Cancer, 63: 2177, 1989. Feinberg, A. P., Vogelstein, B., Droller, M. J., Baylin, S. B. and Nelkin, B. D.: Mutation affecting the 12th amino acid of the cHa-ras oncogene product occurs infrequently in human cancer. Science, 220: 1175, 1983. Alexander, R. B., Nelson, W. G. and Coffey, D. S.: Tumor necrosis factor: synergistic enhancement of in vitro cytotoxicity from chemotherapeutic drugs targeted at DNA topoisomerase II. Cancer Res., 47: 2403, 1987. Zwelling, L. A.: DNA topoisomerase II as a target of antineoplastic drug therapy. Cancer and Metastasis Reviews, 4: 263, 1985. Tewey, K. M., Rowe, T. C., Yang, L., Hilligan, B. D. and Liu, L. F.: Adriamycin-induced DNA damage mediated by DNA topoisomerase II. Science, 226: 466, 1984. Krosnick, J. A., Mule, J. J., McIntosh, J.E. and Rosenberg, S. A.: Augmentation of a:ntotumor efficacy by the combination of recombinant tumor necrosis factor and chemotherapeutic agents in vivo. Cancer Res., 49: 3729, 1989. Garnick, M. B., Schade, D., Israel, M., Maxwell, B. and Richie, J. P.: Intravesical doxorubicin for prophylaxis in management of recurrent superficial bladder carcinoma. J. Urol., 131: 43, 1984. Henry, J. M. and Isaacs, J. T.: Relationship between tumor size and the curability of metastatic prostatic cancer by surgery alone or in combination with adjuvant chemotherapy. J. Urol., 139: 1119, 1988. Squire, R. A., Sporn, M. B., Brown, C. C., Smith, J.M., Wenk, M. L. and Springer, S.: Histopathological evaluation of the inhibition of rat bladder carcinogenesis by 13-cis-retinoic acid. Cancer Res., 37: 2930, 1977. Squire, R. A.: Classification and differential diagnosis of neoplasms, urinary bladder, rat. Monographs on pathology of laboratory animals: In: Urinary System. Edited by T. C. Jones, U. Mohr and R. D. Hunt. Berlin: Springer-Verlag, pp. 311-317, 1986. Pauli, B. U., Coon, J. S. and Weinstein, R. S.: Transitional cell carcinoma, bladder, rat. Monographs on pathology of laboratory animals: In: Urinary System. Edited by T. C. Jones U. Mohr and R. D. Hunt. Berlin: Springer-Verlag, pp. 322-331, 1986. American Joint Committee for Cancer Staging, Beahrs, 0. H., Myers, M. H., (eds.): Manual for Staging of Cancer. 2nd. Ed., Philadelphia: JB Lippincott, pp. 171, 1983. Soloway, M. S. and Masters, S.: Urothelial susceptibility to tumor cell implantation: influence of cauterization. Cancer, 46: 1158, 1980. Shapiro, A., Kelley, D. R., Oakley, D. M., Catalona, W. J. and Ratliff, T. L.: Technical factors affecting the reproducibility of intravesical mouse bladder tumor implantation during therapy with bacillus Calmette-Guerin. Cancer Res., 44: 3051, 1984. Soloway, M. S., Nissenkorn, I. and McCallum, L.: Urothelial sus-
INTRAVESICAL THERAPY FOR SUPERFICIAL TRANSITIONAL CELL CARCINOlV!A
48.
49.
50.
51.
ceptibility to tumor cell implantation: comparison of cauterization with N-methyl-N-nitrosourea. Urology, 21: 159, 1983. Bahnson, R. R. and Ratliff, T. L.: Inhibition of mouse bladder tumor proliferation by alpha difluoromethylornithine and interferon in vitro and in vivo. J. Urol., 139: 1367, 1988. Morales, A.: Long term results and complications of intracavitary bacillus Calmette-Guerin therapy for bladder cancer. J. Urol., 132: 457, 1984. Ratliff, T. L., Gillen, D. and Catalona, W. J.: Requirement of a thymus-dependent immune response for ECG-mediated antitumor activity. J. Urol., 137: 155, 1987. Haaff, E. 0., Catalona, W. J. and Ratliff, T. L.: Detection of
653
interieukin-2 in the urine of patients with superficial bladder tumors after treatment with intravesical BCG. J. Urol., 136: 970, 1986. 52. Bohle, A., Gerdes, J., Nowc, C., Ulmer, A. F., Musehold, F., Hofsetter, A. G. and Flad, H. D.: Effects of local BCG therapy in patients with bladder carcinoma on phenotypes and function of mononuclear cells. In: International Society Of Urology Reports: Immunotherapy Of Urological Tumors. Edited by J. B. DeKernion. New York: Churchill Livingstone, pp. 83-106, 1990. 53. Old, L. J.: Tumor necrosis factor. Science, 230: 630, 1985. 54. Shalaby, M. R., Pennica, D. and Palladino, M. A.: An overview of the history and biologic properties of tumor necrosis factors. Springer Seminars in Imnmnopathology, 9: 33, 1986.
THE JOURNAL OF UROLOGY Copyright© 1991 by AMERICAN UROLOGICAL ASSOCIATION, INC.
Printed in U.S.A.
EXPERIMENTAL INTRAVESICAL THERAPY FOR SUPERFICIAL TRANSITIONAL CELL CARCINOMA IN A RAT BLADDER TUMOR MODEL GARY D. STEINBERG,* CHARLES B. BRENDLER, ROBERT A. SQUIRE
AND
JOHN T. ISAACS
From the James Buchanan Brady Urological Institute, Department of Urology Oncology and Comparative Medicine, Johns Hopkins Hospital and Johns Hopkins University School of Medicine, Baltimore, Maryland
ABSTRACT
A rodent bladder cancer model that is induced by intravesical instillation of N-methyl-Nnitrosourea (MNU) was characterized. Cohorts of four to five week old female Fisher 344 rats received four biweekly 1.5 mg. doses of intravesical MNU and were sacrificed at various intervals. By week 13 all animals had flat atypia and/or papillary transitional cell tumors, and 67% of the lesions were moderately (grade II) or poorly differentiated (grade III). By week 20, 83% had gross muscle invasive tumors that eventually killed the host. A cohort of 40 MNU treated animals was subsequently treated commencing at week 17 after initiation of MNU with one of three intravesical six week regimens: 1) saline; 2) BCG (Tice strain); or 3) recombinant human tumor necrosis factor (RTNF) plus adriamycin. There was no difference in animal survival or tumor growth in any group of animals commencing therapy at week 17. A second cohort of 107 animals commenced therapy at 13 weeks after initiation of MNU with one of five intravesical six week regimens: 1) intravesical BCG (Tice strain); 2) adriamycin; 3) recombinant human tumor necrosis factor (RTNF); 4) RTNF plus adriamycin; or 5) BCG plus adriamycin. BCG, RTNF or adriamycin alone had no effect on tumor growth; however, BCG plus adriamycin and RTNF plus adriamycin commencing at week 13 significantly inhibited tumor growth and progression. In conclusion, this autochthonous intravesical rodent transitional cell carcinoma model appears useful for the following reasons: 1) it closely resembles human transitional cell carcinoma histologically and biologically in that all animals develop neoplastic changes in-situ that progress to muscle invasion and kill the host; 2) as with human bladder cancer these tumors do not respond to intravesical therapy if treated when tumor burden is large; however, tumor growth is inhibited when treated early; and 3) this model appears appropriate for screening and developing new intravesical treatments for superficial bladder cancer. KEY WORDS:
bladder cancer, adriamycin, recombinant human tumor necrosis factor, bacillus Calmette-Guerin
Bladder cancer is the second most common urologic malignancy in the US with 49,000 new cases and 10,000 deaths per year. 1 Smoking and occupational exposures to various chemicals especially in the dye and petroleum industries are reported to be etiologic factors in approximately 50% of all cases. 2 • 3 Approximately 70% of patients diagnosed each year have superficial (Ta, Tis, T 1 ) disease but these patients are at risk for recurrent tumors and disease progression. 3 - 6 Many of these patients are therefore candidates for intravesical therapy; however, the efficacy of available intravesical agents is only 30 to 70% and all have potentially serious side effects. 4 • 7- 13 In the past, investigators have used serially passaged in-vitro and transplantable bladder cancer animal models to study the effects oftherapy. 14- 22 These models differ from human bladder cancer because of inherent problems of phenotypic and genotypic drift which occur with both continuous cell and animal passage and alter the original biochemical and histological characteristics of the tumor. In addition, autochthonous animal models of bladder cancer induced by the oral administration of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and N-[4-(5nitro-2-furyl)-2-thiazolyl] formamide (FANFT) 16- 18• 23- 25 or intravesical MNU 26- 29 are problematic because of the frequent development of squamous cell carcinomas along with transiAccepted for publication October 19, 1990. * Requests for reprints: Department of Urology, Marburg 145, Johns Hopkins Hospital, Baltimore, MD 21205. Supported by HHS Grant NIDDK DK07552, U.S. Public Health Service Grant RR00130, and the generous contributions of Mr. and Mrs. Merrill Bank, Mr. and Mrs. Seymour Kulick and Mr. and Mrs. Jack Stollof. 647
tional cell carcinomas of the bladder. Using a modification of a model introduced in the 1970s by Hicks et al., 26 • 27 we have developed a model of autochthonous rat transitional cell carcinoma (TCC) 30 that is useful for the following reasons: 1) the tumors arise only from urinary bladder epithelium; 2) the histology of the invasive tumors which grossly develop is transitional cell with occasional focal squamous metaplasia; 3) no leukemias, breast cancers, lymphomas or other tumors are induced; 4) similar to human TCC, 30- 32 the karyotypes are diploid with no consistent pattern of chromosomal aberrations; and 5) similar to human TCC, increased expression or mutation of ras p21 protein does not appear to be necessary for malignant transformation. 33 •34 This model appears ideally suited for the study of new intravesical treatments for superficial TCC. A previous study from our laboratory using this model demonstrated that at 13 weeks after carcinogen exposure 45% of female Fisher 344 rats had superficial TCC, and that at 17 weeks 32% had superficial TCC and 20% had muscle invasive TCC. 30 Therefore, to assess the effect of intravesical therapy and the importance of timing of therapy cohorts of animals began intravesical therapy at 13 and 17 weeks after initiation of MNU with immunotherapeutic agents (BCG and RTNF) individually and in combination with a chemotherapeutic agent (adriamycin). These therapeutic agents were chosen because: 1) BCG is currently believed to be one of the most effective intravesical agents against superficial TCC; 9 • 11- 13 2) recent work in our laboratory by Alexander et al. 35 demonstrated the syn ergistic efficacy of the combination of RTNF and chemotherapeutic agents directed against the enzyme DNA topoisomerase
648
STEINBERG AND ASSOCIATES
II in vitro. This enzyme is responsible for unwinding the DNA double helix during replication, and is thus an important target for chemotherapeutic drugs; 36 3) adriamycin is a DNA topoisomerase II inhibitor35- 38 and is also used intravesically to treat superficial TCC; 39 4) previously, Alexander et al. 21 demonstrated the in-vivo synergistic efficacy of the systemic combination of RTNF and adriamycin in the treatment of MBT-2 bladder cancer cells when implanted subcutaneously in the hind leg of C3H/HE mice; and 5) intravesical RTNF is being tested in phase I trials by the Eastern Cooperative Oncology Group (ECOG). The results of intravesical therapy of this carcinogen induced autochthonous model form the basis of this report. MATERIALS AND METHODS
We modified the methods used in the model introduced by Hicks et al. 26 • 27 by using a different strain of inbred rats and providing continuous antibiotics in their drinking water. 30 Multiple cohorts of four to five week old female Fisher 344 rats (Harlan Sprague Inc., Indianapolis, Ind.) were used for this study, The animals were anesthetized with intraperitoneal nembutal. One gram of N-methyl-N-nitrosourea (MNU) (Sigma, St. Louis, MO.), was dissolved in 100 cc of normal saline. The animals received 0.15 cc (1.5 mg.) of this solution via a 22 gauge teflon angiocath (Travenol Laboratories, Chicago, 11.) intravesically under a continuous flow hood within 45 minutes after preparation of the MNU solution, every other week (week 0, 2, 4, and 6) for a total of four doses. The animals remained anesthetized for approximately 2 hours after catheterization. Routine precautions were followed when working with this carcinogen. MNU was administered under a continuous flow hood, and the excess MNU and animal bedding were incinerated. All animals received water medicated with a combination of trimethoprim sulfamethoxazole, neomycin, and polymyxin B, formulated as described previously40 and Purina rat chow ad libitum. Animals were maintained on a 12 hour lightdark cycle, and were sacrificed at various intervals after they received their last dose of MNU. Approximately 24% (62 of 258) of all animals entered into MNU dosing schedules did not survive all 4 doses. The causes of death were usually related to complete ulceration of the urinary bladder or urosepsis secondary to urethral stricture formation and urinary obstruction. Approximately 5% of animals formed bladder or ureteral calculi, Tissue preparation. The animals were sacrificed by CO 2 gas overdose and necropsy was performed. The urinary bladder and urethra were excised in toto. The liver and lungs were inspected for metastases and, if suspicious, were fixed in formalin or Bouin's solution respectively. The kidneys and ureters were dissected and inspected for the presence of pyonephrosis, stones, or upper tract tumors secondary to reflux. The bladders were inflated with 10% buffered formalin, and a ligature was placed around the urethra. The bladders were fixed for at least 24 hours and embedded in paraffin. Sections were cut transversely through the midportion of the bladder, and nine 5micron sections were taken from each half to adequately sample the entire bladder; these sections were stained with hematoxylin and eosin (H&E). Light microscopy. All sections from each bladder specimen were reviewed under low power. The section that appeared to have the greatest amount of change from normal rat bladder was selected for histologic grading. Typically the histologic changes were relatively uniform in any one specimen, but some areas of the bladder were occasionally more affected than other areas. The tumors were staged by the Union Internationale Cancer Control (UICC) and the American Joint Committee for Cancer Staging tumor, node and metastasis (TNM) system for human bladder cancer as described previously18• 24 • 41 - 44 and categorized into either: 1) hyperplasia, flat or papillary atypia,
with mild to moderate dysplasia; 2) superficial transitional cell carcinoma (TCC) which includes stages stage P. (papillary exophytic tumors with fibrovascular cores and nuclear pleomorphism of the epithelial cells with no evidence of invasion); Stage P;, (tumors confined to the mucosa with full mucosal thickness of marked atypia/dysplasia (carcinoma in-situ)); and stage P 1 (tumors that demonstrate evidence of lamina propria invasion); or 3) bladder wall muscle invasive transitional cell carcinomas (stages P 2 and P 3 ). The tumors were also categorized by histologic grade using conventional criteria. Grade 1 lesions demonstrate minimal nuclear pleomorphism, while grade 2 and 3 lesions show increasing amounts of nuclear pleomorphism, loss of polarity, increased cellularity, prominence of nucleoli, presence of mitoses and formation of giant cells. Therapeutic trials. A cohort of 40 animals that received four doses of MNU was divided into four treatment groups. Group 1 received no further treatment. Groups 2-4 were treated weekly for six weeks intravesically with 0.2 cc of either saline (group 2); BCG (Tice strain 5 ± 3 X 108 colony forming units/ ampule dissolved in 60 cc normal saline) (group 3); or recombinant human tumor necrosis factor (RTNF) (7 x 105 units/ ml. of saline) (Cetus Corp., Emeryville, CA) and adriamycin (two mg./ml.) (Adria Inc., Dublin, OH) (1:1 mixture) (group 4). The dosages of the intravesical agents were at the same concentrations as used for intravesical therapy in humans. Treatment began 17 weeks after initiation of MNU. The animals were followed until dead or premorbid for a maximum of 42 weeks. A second cohort of 107 animals that received four doses of MNU was divided into six treatment groups. Group 1 received no further treatment. Groups 2-6 were treated weekly for six weeks intravesically with 0.2 cc of intravesical: RTNF (group 2); adriamycin (group 3); BCG (group 4); BCG and adriamycin (1:1 mixture) (group 5); or RTNF and adriamycin (group 6). 100
Histopathological Findings in Female Fisher 344 Rat Bladders Treated with 4 Doses of N-methyl-N-nitrosourea (MNU)
90 Legend BO
70
-
D
hyperplasia or atypical dysplasia
rn
superficial TCC Pa, Pis, P1
•
60
C
muscle invasive TCC
~
, P3
Q)
~
50
Q)
a..
40
30
20
10
0
Weeks ofter Initiation of the Carcinogen 8-10 weeks 12-13 (N=IO) (N=22)
14-16 (N=21)
17-18 (N=IO)
20-24 (N=l2)
FIG. 1. Pathological stages of bladder tumors in female Fisher 344 rat bladders treated with four doses of intravesical MNU and sacrificed at various time intervals.
INTRAVESICAL THERAPY FOR SUPERFICIAL TRANSITIONAL CELL CARCINOMA
100
649
Tumor Grode of Rat Bladders Treated with 4 Doses of lntrovesical N-methyl-N-Nitrosourea (MNU)
90 Legend
80
70
D
grade I
~
grade II
II
grade Ill
60
C:
(I) (.) b.
50
(I)
0...
40
30
20
10
Weeks after Initiation of the Carcinogen MNU weeks 10-12 (N=l5)
weeks 13-15 (N=33)
weeks
weeks
16-18 (N=IO)
20-24 (N=l2)
FIG. 2. Pathological grades of bladder tumors in female Fisher 344 rat bladders treated with four doses of intravesical MNU and sacrificed at various time intervals.
FIG. 3. Fisher 344 female rat bladder treated with four doses of MNU with hyperplasia.
The dosages of intravesical agents were the same as above. Treatment began 13 weeks after initiation of MNU. Ten animals from each group were sacrificed at 25 weeks (6 weeks after the last treatment), and histology was evaluated blindly without knowledge of treatment given. RESULTS
Histopathologic progression of MNU treated bladders. All rat bladders treated with intravesical MNU develop progressive neoplastic changes (fig. 1), and the tumors become progressively less differentiated with time (fig. 2). These lesions progress from hyperplasia (fig. 3), atypia (fig. 4), carcinoma in-situ (fig. 5), and papillary TCC (fig. 6) to large bulky muscle invasive tumors (fig. 7) that completely fill the bladder lumen, obstruct the ureters and kill the animal. These large tumors weigh as much as one to two grams (the normal rat bladder weighs approximately 50 mg.). No animal in this study or in our previous study 30 developed squamous cell carcinoma either alone or in combination with transitional cell carcinoma as was described in previous studies of MNU induced bladder can cer. 11- 18, 26 - 28 This may be due to the continuous antibiotics in the animals' drinking water or the use of a strain of rats that is less prone to endogenous stone formation and thus chronic inflammation and squamous metaplasia. 30 Treatment results. The cohort of animals that began treatment at 17 weeks after the initiation of MNU had no apparent response to therapy when assessed by the TNM staging system (table 1). By week 40, 78% of the RTNF/adriamycin animals, 100% of the BCG animals, 88% of the saline animals and 75% of the control animals had bulky, muscle-invasive tumors.
FIG. 4. Fisher 344 female rat bladder treated with four doses of MNU with hyperplasia and mild to moderate cellular atypia.
There was no increase in survival in any treatment group versus the untreated control group. In the second cohort of animals that began treatment at 13 weeks after initiation of MNU, intravesical BCG, RTNF or adriamycin alone had no significant effect on tumor growth. However, the combinations of RTNF with adriamycin and BCG with adriamycin inhibited the growth and progression of tumors significantly (table 2 and fig. 8). Although few of the animals were cured by histological criteria, the tumors in the
650
STEINBERG AND ASSOCIATES
Fm. 5. Fisher 344 female rat bladder treated with four doses of MNU with areas of marked atypia and dysplasia involving full mucosal thickness (carcinoma-in-situ).
Fm. 7. Fisher 344 female rat bladder treated with four doses of MNU with muscle invasive TCC.
had flat carcinoma in situ only and the other had no evidence of disease. The overall mortality of treatment in all groups was 25% and did not vary significantly between treatment groups. These mortalities may be due to either catheterization, the intravesical agent used or possibly the MNU itself. DISCUSSION
Fm. 6. Fisher 344 female rat bladder treated with four doses of MNU with superficial papillary transitional cell carcinoma and mild to moderate cellular atypia.
BCG, RTNF, adriamycin and control groups weighed 1.5 to 2.0 gm. and filled the bladder, while the tumors in the RTNF / adriamycin and BCG/adriamycin groups were small, nonobstructing tumors. Of four additional RTNF/ adriamycin animals sacrificed at 31 weeks, only one had a bulky bladder tumor that filled the lumen, one had small papillary and sessile tumors, and two had no obvious gross tumors. Histopathological examination of the latter two bladders revealed that one
MBT-2 is presently the most frequently used animal model to study bladder cancer. 14• 15• 19- 22 MBT-2 is a transplantable murine serially passaged cell line that was established from a transitional cell carcinoma of the bladder induced by the oral administration of N-[ 4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT). 14-18 This model has been studied extensively and has been used to screen various intravesical and systemic chemotherapeutic agents. However, there are problems in using this model to study superficial TCC of the bladder: 1) it is a tumor serially passaged for over 10 years with resultant genetic drift; 2) it is usually transplanted subcutaneously (the reliability of implantation in the bladder is limited45- 47 ) and thus, one cannot assess the efficacy or adverse effects of intravesical therapy on the host urothelium; 3) it has a very fast in-vivo growth rate (doubling time is less than three days) and thus, it is difficult to study stage progression; 4) by day 34 after implantation the tumor contains highly anaplastic sarcomatous elements; and 5) the tumor is aneuploid with abnormal chromosomes, the development of which may have structurally occurred during serial passage. 16- 18 Few attempts to study intravesical therapy of autochthonous bladder tumors in rodents have been performed to date. Previously Soloway et al. 19 reported on the use of various chemotherapeutic agents instilled intravesically in mice fed F ANFT for 38 weeks and sacrificed at 46 weeks. They found that intravescial cis-platinum, mitomycin C, and thiotepa all reduced tumor incidence but there were no signficant differences in mean and median tumor weights between treated and control groups. In addition, few of the control animals developed invasive disease by 46 weeks, and the histology of these tumors was not described. Lee et al. 20• 22 instilled single cell suspensions of MBT-2 cells into c3H/HE mice bladders and treated these animals with either intravesical RTNF alone or a combination of intravesical BCG and systemic interleukin -II. Intravesical RTNF did not suppress tumor growth; however, the combination of intravesical BCG plus systemic interleukin II did. Bahnson and Ratliff48 instilled MBT-2 cells intravesically into C3H/ HE mice and subsequently treated them with alpha difluoromethylornithine (DFMO) in the drinking water alone and in
INTRAVESICAL THERAPY FOR SUPERFICIAL TRANSITIONAL CELL CARCINOMA TABLE
651
1. Results of intravesical therapy commenced at 17 weeks after the intravesical initiation of the carcinogen MNU in animals followed until
death or 42 weeks Treatment Group
Percent that Survived Therapy
Percent that Died from Bulky Bladder Cancer
Percent with Bulky Tumor at Sacrifice or Death
100
38
75
80
75
88
50
40
100
82
56
78
Control N=8 Saline N= 10 BCG N= 10 RTNF / adriamycin N= 11
Histopathological findings in female Fisher 344 rat bladders treated with 4 doses of intravesical MNU, commenced therapy at 13 weeks and sacrificed at 25 weeks after initiation of the carcinogen
TABLE 2.
Treatment Group
Percent with Hyperplasia, Percent with Flat or Papillary Superficial Atypia/Dysplasia TCC 40 30 50 20 70 60
0 10 10 0 20 30
Control RTNF Adriamycin BCG BCG/Adriamycin RTNF / Adriamycin
Percent with Muscle Invasion 60 50 40 80 10* 10*
* p = 0.04 (in comparison with percent of control animals with muscle invasive TCC).
Results of Treotment Begun at Week 13
90 80 70 +-_
C
Q)
~
Q)
Q.
60
50
40 30
20 10 Control
0
Hyplosia/atypia
RTNF
Adriomycin
BCG
~ Superficial TCC
BCG/Adrlomycin RTNF/Adriomycin
11111 Muscle Invasive TCC
FIG. 8. Histopathological findings as evaluated by TNM system in female Fisher 344 rat bladders treated with four doses of intravesical MNU, commenced therapy at 13 weeks and sacrificed at 25 weeks. combination with intravesical alpha 1 and beta interferon. DFMO alone significantly delayed the outgrowth of tumors without any additive effect when interferon was added. However, these studies did not assess tumor growth but rather inhibition ofMBT-2 cell implantation into the urinary bladder. In this study a model of autochthonous rat TCC of the bladder was developed based upon the original findings of Hicks et al. 26 This model is useful for studying intravesical therapy of superficial TCC for the following reasons: 1) the tumors arise only from bladder urothelium; 2) all animals develop progressive neoplastic changes in-situ within four months after initiation of the carcinogen; 3) these lesions progress through various early stages of neoplasia to bladder wall muscle-invasion and kill the host; 4) the histology of the tumors is transitional cell; and 5) the neoplastic lesions develop at discrete times and in high frequency (fig. 1), thereby allowing treatment to be initiated at known stages of disease. The toxicity and incomplete efficacy of the commonly used intravesical agents has prompted a search for other treatments for superficial TCC of the bladder. Although adriamycin is an
effective agent and intravesical RTNF is in phase I trial, the combination of RTNF and adriamycin has not been tested in humans. Previously Alexander et al. 21 demonstrated the efficacy of the systemic combination of RTNF and adriamycin in the treatment of MBT-2 bladder cancer in-vitro and in-vivo. Therefore, this autochthonous model ofTCC was used to assess the efficacy of combined intravesical therapy (i.e. biological response modifiers such as RTNF and BCG with adriamycin) in inhibiting the growth and progression of these tumors. In this study, BCG, RTNF, and adriamycin had little effect on tumor growth. However, both BCG and RTNF, in combination with adriamycin, significantly reduced tumor growth and delayed tumor progression. Although few animals were cured, most had significantly reduced tumor volumes. At present, BCG is considered one of the most effective single intravesical agents for therapy of superficial TCC. 4 • 49 Although its mechanism for action is unknown, BCG may act through a T-cell dependent mechanism. 5° Following intravesical BCG therapy, Haaff et al. 51 demonstrated the presence of IL-2 in the urine, and Bohle et al. 52 found both IL-1 and TNF in the urine. It is also known that BCG stimulates the endogenous production of TNF. 53 IL-2 is produced predominantly by T-helper cells which activate natural killer T-cells, 54 and TNF is produced predominantly by macrophages. 53 It is possible, therefore, that BCG and TNF act through a similar mechanism in combination with adriamycin to produce a synergistic tumoricidal effect. This may also explain the similar cytotoxic action on tumor cells of both BCG and TNF. Presently the mechanism of action of enhanced tumor cytotoxicity from the combination of biologic response modifiers such as RTNF and BCG and DNA topoisomerase II targeted chemotherapeutic agents is unknown. However, recent evidence suggests that the combination may allow easier access of TNF or TNF-induced nuclear endonucleases to DNA resulting in DNA fragmentation and cell death. 21 • 35• 37• 38 Future studies will be necessary to determine the optimal combinations, timing, and sequence of biologic response modifiers and chemotherapeutic agents targeted at DNA topoisomerase II. This model should be useful in determining this information, and this may lead to more effective treatments for superficial transitional cell carcinoma of the bladder in man. Acknowledgments. The authors would like to thank John Lamb for his excellent technical assistance. Recombinant Human Tumor Necrosis Factor was generously supplied to us by Dr. Abla Creasey of the Cetus Corporation (Emeryville, CA) and adriamycin was supplied to us by Dr. Vernon Verhoef of Adria Laboratories (Dublin, OH). REFERENCES
Silverberg, E., Boring, C. C. and Squires, T. S.: Cancer Statistics 1990. Ca., 40: 1, 1990. 2. Thompson, I. M. and Fair, W. R.: The epidemiology of bladder cancer. AUA update series vol 8, lesson 27, 1989. 3. Thompson, I. M., Peek, M. and Rodriguez, F. R.: The impact of cigarette smoking on stage, grade and number of recurrences of transitional cell carcinomas of the bladder. J. Urol., 137: 401, 1987. 1.
652
STEINBERG AND ASSOCIATES
4. Herr, H. W., Laudone, V. P. and Whitmore, W. F. Jr.: An overview of intravesical therapy for superficial bladder tumors. J. Urol., 138: 1363, 1987. 5. Rubben, H., Lutzeyer, W., Fischer, N., Deutz, F., LaGrange, W., Giani, G. and Members of the Registry for Urinary Tract Tumors: Natural history and treatment of low and high risk superficial bladder tumors. J. Urol., 139: 283, 1988. 6. Heney, N. M., Ahmed, S., Flanagan, M. J., Frable, W., Corder, M. P., Haferman, M. D., Hawkins, I. R. and The National Bladder Cancer Collaborative Group A: Superficial bladder cancer: progression and recurrence. J. Urol., 130: 1083, 1983. 7. Soloway, M. S.: The management of superficial bladder cancer. Cancer, 45: 1856, 1980. 8. Soloway, M. S.: Selecting initial therapy for bladder cancer. Cancer, 60: 502, 1987. 9. Catalona, W. J., Hudson, M.A., Gillen, D. P., Andriole, G. L. and Ratliff, T. L.: Risks and benefits of repeated courses of intravesical bacillus Calmette-Guerin therapy for superficial bladder cancer. J. Urol., 137: 220, 1987. 10. Heney, N. M., Koontz, W.W., Barton, B., Soloway, M., Trump, D. L., Hazra, T. and Weinstein, R. S., For the National Bladder Cancer Group: Intravesical thiotepa versus mitomycin C in patients with Ta, Tl, and Tis transitional cell carcinoma of the bladder: a phase III prospective randomized study. J. Urol., 140: 1390, 1988. 11. Herr, H. W., Baldalment, R. A., Amato, D. A., Laudone, V. P., Fair, W.R. and Whitmore, W. F., Jr.: Superficial bladder cancer treated with bacillus Calmette-Guerin: a multivariate analysis of factors affecting tumor progression. J. Urol., 141: 22, 1989. 12. Kavoussi, L. R., Torrence, R. J., Gillen, D. P., Hudson, M. A., Haaff, E. 0., Dresner, S. M., Ratliff, T. L. and Catalona, W. J.: Results of 6 weekly intravesical bacillus Calmette-Guerin instillations on the treatment of superficial bladder tumors. J. Urol., 139: 935, 1988. 13. Lamm, D. L., Stogdill, V. D., Stogdill, B. J. and Crispen, R. G.: Complications of bacillus Calmette-Guerin immunotherapy in 1,278 patients with bladder cancer. J. Urol., 135: 272, 1986. 14. Soloway, M. S.: Intravescial and systemic chemotherapy of murine bladder cancer: Cancer Res., 37: 2918, 1977. 15. Soloway, M. S. and Murphy, W. M.: Experimental chemotherapy of bladder cancer-systemic and intravesical. Seminars in Oncology, 6: 166, 1979. 16. Murphy, G. P., Sandberg, A. A., Pontes, J.E., Ochi, H., Yoshida, M. and Williams, P. D.: A murine model for bladder cancer. In: Bladder Cancer Part A: Pathology, Diagnosis and Surgery, New York: Alan R. Liss, Inc, pp. 177-200, 1984. 17. Raghavan, D., Debruyne, F., Herr, H., Jocham, D., Kakizoe, T., Okajima, E., Sandberg, A. and Tannock, I.: Experimental models of bladder cancer: a critical review. In: Developments in Bladder Cancer, New York: Alan R. Liss, Inc, pp. 171-208, 1986. 18. Cohen, S. M.: Pathology of experimental bladder cancer in rodents. In: The Pathology of Bladder Cancer, Edited by G. T. Bryan and S. M. Cohen. Boca Raton: CRC Press, vol I, pp. 1-40, 1983. 19. Soloway, M. S., Nissenkorn, I., McCallum, L. W. and Murphy, W. M.: Single and sequential combination intravesical chemotherapy of murine bladder cancer. Urology, 19: 169, 1982. 20. Lee, K. E., O'Donnell, R. W., Schoen, S. and Cockett, A. T. K.: Effect of intravesical administration of tumor necrosis serum and human recombinant tumor necrosis factor on a murine bladder tumor. J. Urol., 138: 430, 1987. 21. Alexander, R. B., Isaacs, J. T. and Coffey, D. S.: Tumor necrosis factor enhances the in vitro and in vivo efficacy of chemotherapeutic drugs targeted at DNA topoisomerase II in the treatment of murine bladder cancer. J. Urol., 138: 427, 1987. 22. Lee, K. E., Weiss, G. H., O'Donnell, R. W. and Cockett, A. T. K.: Reduction of bladder cancer growth in mice treated with intravesical Bacillus Calmette-Guerin and systemic interleukin 2. J. Urol., 137: 1270, 1987. 23. Drago, J. R.: The noble rat bladder cancer model: FANFT-induced tumors. Cancer, 53: 1093, 1984. 24. Fukushima, S., Hirose, M., Tsuda, H., Shirai, T., Hirao, K., Arai, M. and Ito, N.: Histological classification of urinary bladder cancers in rats induced by N-butyl-N-(4-hydroxy-butyl) nitrosamine. Gann, 67: 81, 1976. 25. Herman, C., Vegt, P., Debruyne, F., Vooijs, G. and Ramaekers, F.: Squamous and transitional elements in rat bladder carcinomas
26. 27. 28. 29. 30.
31. 32.
33.
34.
35.
36. 37. 38.
39. 40.
41.
42.
43.
44. 45. 46.
47.
induced by N-butyl-N-(4-hydroxyl-butyl) nitrosamine (BBN). M.J. Pathol., 120: 419, 1985. Hicks, R. M. and Wakefield, J. ST. J.: Rapid induction of bladder cancer in rats with N-methyl-N-nitrosourea I. Histology. Chem. Biol. Interactions, 5: 139, 1972. Hicks, R. M. and Wakefield, J. ST. J.: Membrane changes during urothelial hyperplasia and neoplasia. Cancer Res., 36: 2502, 1976. Severs, N. J., Barnes, S. H., Wright, R. and Hicks, R. M.: Induction of bladder cancer in rats by fractionated intravesicular doses of N-methyl-N-nitrosourea. Br. J. Cancer, 45: 337, 1982. Cox, R. and Irving, C. C.: Effect of N-methyl-N-nitrosourea on the DNA of Rat Bladder epithelium. Cancer Res., 36: 4114, 1976. Steinberg, G. D., Brendler, C. B., Ichikawa, T., Squire, R. A. and Isaacs, J. T .: The characterization of an N-methyl-N -nitrosourea (MNU) induced rat bladder cancer model. Cancer Res., 50: 6668, 1990. Vanni, R., Scarpa, R. M., Nieddu, M. and Usai, E.: Cytogenetic investigation on 30 bladder carcinomas. Cancer Genet. Cytogenet., 30: 35, 1988. Gibas, Z., Prout, G. R., Pontes, J.E., Connolly, J. G. and Sandberg, A. A.: A possible specific chromosome change in transitional cell carcinoma of the bladder. Cancer Genet. Cytogenet., 19: 229, 1986. Meyers, F. J., Gumerlock, P.H., Kokoris, S. P., De Vere White, R. W. and McCormick, F.: Human bladder and colon carcinomas contain activated ras p21: specific detection of twelfth codon mutants. Cancer, 63: 2177, 1989. Feinberg, A. P., Vogelstein, B., Droller, M. J., Baylin, S. B. and Nelkin, B. D.: Mutation affecting the 12th amino acid of the cHa-ras oncogene product occurs infrequently in human cancer. Science, 220: 1175, 1983. Alexander, R. B., Nelson, W. G. and Coffey, D. S.: Tumor necrosis factor: synergistic enhancement of in vitro cytotoxicity from chemotherapeutic drugs targeted at DNA topoisomerase II. Cancer Res., 47: 2403, 1987. Zwelling, L. A.: DNA topoisomerase II as a target of antineoplastic drug therapy. Cancer and Metastasis Reviews, 4: 263, 1985. Tewey, K. M., Rowe, T. C., Yang, L., Hilligan, B. D. and Liu, L. F.: Adriamycin-induced DNA damage mediated by DNA topoisomerase II. Science, 226: 466, 1984. Krosnick, J. A., Mule, J. J., McIntosh, J.E. and Rosenberg, S. A.: Augmentation of a:ntotumor efficacy by the combination of recombinant tumor necrosis factor and chemotherapeutic agents in vivo. Cancer Res., 49: 3729, 1989. Garnick, M. B., Schade, D., Israel, M., Maxwell, B. and Richie, J. P.: Intravesical doxorubicin for prophylaxis in management of recurrent superficial bladder carcinoma. J. Urol., 131: 43, 1984. Henry, J. M. and Isaacs, J. T.: Relationship between tumor size and the curability of metastatic prostatic cancer by surgery alone or in combination with adjuvant chemotherapy. J. Urol., 139: 1119, 1988. Squire, R. A., Sporn, M. B., Brown, C. C., Smith, J.M., Wenk, M. L. and Springer, S.: Histopathological evaluation of the inhibition of rat bladder carcinogenesis by 13-cis-retinoic acid. Cancer Res., 37: 2930, 1977. Squire, R. A.: Classification and differential diagnosis of neoplasms, urinary bladder, rat. Monographs on pathology of laboratory animals: In: Urinary System. Edited by T. C. Jones, U. Mohr and R. D. Hunt. Berlin: Springer-Verlag, pp. 311-317, 1986. Pauli, B. U., Coon, J. S. and Weinstein, R. S.: Transitional cell carcinoma, bladder, rat. Monographs on pathology of laboratory animals: In: Urinary System. Edited by T. C. Jones U. Mohr and R. D. Hunt. Berlin: Springer-Verlag, pp. 322-331, 1986. American Joint Committee for Cancer Staging, Beahrs, 0. H., Myers, M. H., (eds.): Manual for Staging of Cancer. 2nd. Ed., Philadelphia: JB Lippincott, pp. 171, 1983. Soloway, M. S. and Masters, S.: Urothelial susceptibility to tumor cell implantation: influence of cauterization. Cancer, 46: 1158, 1980. Shapiro, A., Kelley, D. R., Oakley, D. M., Catalona, W. J. and Ratliff, T. L.: Technical factors affecting the reproducibility of intravesical mouse bladder tumor implantation during therapy with bacillus Calmette-Guerin. Cancer Res., 44: 3051, 1984. Soloway, M. S., Nissenkorn, I. and McCallum, L.: Urothelial sus-
INTRAVESICAL THERAPY FOR SUPERFICIAL TRANSITIONAL CELL CARCINOlV!A
48.
49.
50.
51.
ceptibility to tumor cell implantation: comparison of cauterization with N-methyl-N-nitrosourea. Urology, 21: 159, 1983. Bahnson, R. R. and Ratliff, T. L.: Inhibition of mouse bladder tumor proliferation by alpha difluoromethylornithine and interferon in vitro and in vivo. J. Urol., 139: 1367, 1988. Morales, A.: Long term results and complications of intracavitary bacillus Calmette-Guerin therapy for bladder cancer. J. Urol., 132: 457, 1984. Ratliff, T. L., Gillen, D. and Catalona, W. J.: Requirement of a thymus-dependent immune response for ECG-mediated antitumor activity. J. Urol., 137: 155, 1987. Haaff, E. 0., Catalona, W. J. and Ratliff, T. L.: Detection of
653
interieukin-2 in the urine of patients with superficial bladder tumors after treatment with intravesical BCG. J. Urol., 136: 970, 1986. 52. Bohle, A., Gerdes, J., Nowc, C., Ulmer, A. F., Musehold, F., Hofsetter, A. G. and Flad, H. D.: Effects of local BCG therapy in patients with bladder carcinoma on phenotypes and function of mononuclear cells. In: International Society Of Urology Reports: Immunotherapy Of Urological Tumors. Edited by J. B. DeKernion. New York: Churchill Livingstone, pp. 83-106, 1990. 53. Old, L. J.: Tumor necrosis factor. Science, 230: 630, 1985. 54. Shalaby, M. R., Pennica, D. and Palladino, M. A.: An overview of the history and biologic properties of tumor necrosis factors. Springer Seminars in Imnmnopathology, 9: 33, 1986.
