Intraluminal Interleukin 2 and Bacillus Calmette-Guerin for Treatment of Bladder Cancer: A Preliminary Report

0022-5347 /87 /1372-0216$02.00/0 Vol. 137, February

THE JOURNAL OF UROLOGY

Copyright© 1987 by The Williams & Wilkins Co.

Printed in U.S.A.

INTRALUMINAL INTERLEUKIN 2 AND BACILLUS CALMETTEGUERIN FOR TREATMENT OF BLADDER CANCER: A PRELIMINARY REPORT PAUL A. MERGUERIAN,* LAURENCE DONAHUE

AND

A. T. K. COCKETT

From the Department of Urology, University of Rochester Medical Center, Rochester, New York

ABSTRACT

The authors treated 13 patients who had undergone resection of transitional cell carcinoma of the bladder with a combination of 3,500 units of interleukin 2 and half the recommended dose of bacillus Calmette-Guerin. Of the patients 11 (85 per cent) remained free of tumor for a mean of 13 months (range 6 to 24 months). These results are comparable to those with bacillus CalmetteGuerin therapy alone. However, a longer followup with a large number of patients is needed to assess the efficacy of this modality compared to conventional bacillus Calmette-Guerin therapy. Side effects after treatment were minor, self limiting (fever, hematuria and bladder irritability) and lasted for 24 hours. Reduction of cell-mediated immune responses during tumor growth is associated with a concomitant decrease in the functional capacity of T-lymphocytes. 1 The proliferative immune responses are controlled by a series of soluble growth factors or activating agents designated as lymphokines. 2 Interleukin 2, a lymphokine, is a protein of low molecular weight produced by T-lymphocytes. 3 Recent investigations would indicate that the slowdown or cessation of cytotoxic activity against tumor cells may be owing to the absence of interleukin 2. 4 Interleukin 2 has been shown to stimulate cytotoxic T-cell precursor proliferation with the final generation of specific cytotoxic Tcells. 5-7 Bacillus Calmette-Guerin (BCG) has been demonstrated to be effective for the intravesical therapy of superficial bladder carcinoma. The rationale for use of BCG is based partly on the hypothesis that placing an immune stimulant in contact with superficial bladder cells bearing antigenic properties would stimulate a host response to the tumor cells, thus, producing a cytotoxic response and lessening the chance of neoplastic transformation. 8-12 We investigated the efficacy of combined therapy with BCG and interleukin 2 in the treatment of bladder carcinoma. The rationale for this mode of therapy is that when BCG is placed intravesically it will produce an acute inflammatory response and will enhance the local proliferation of T-cells. Interleukin 2 then will generate cytotoxic T-cells and, thus, enhance tumor cell lysis.

cells per ml., and stimulated with the mitogen phytohemagglutinin A for 8 hours. After incubation the cells were separated from the solution, washed several times, suspended in a culture medium and incubated for 18 hours, after which they were separated from the suspension that contained the T-cell growth factor. The supernatant then was filtered and concentrated with ultrafilters YM-10 and YM-30.:j: The final product was 50 to 250 times more concentrated. Interleukin 2 then was assayed. Serial 2-fold dilutions of a medium control, a commercially available standard interleukin 2 preparation§ (defined as 500 units per ml.) and our test samples were incubated with 104 HT-2 cells for 24 hours at 37C (6 per cent carbon dioxide at 95 per cent humidity) followed by an 18-hour pulse with tritiated (3 H) thymidine (18 to 25 Ci./ mM., 2 µCi./20 µl. per well). After the incubation period cells were harvested with a multiple automated sample harvester and uptake of 3H-thymidine was measured by liquid scintillation counting. The reciprocal of the dilution giving 50 per cent of the maximum 3H-thymidine incorporation tentatively defined the number of units of interleukin 2 in each sample. The actual number of units was defined by the equation: standardized units of interleukin 2 in test batch

=

MATERIALS AND METHODS

We treated 13 patients with transitional cell carcinoma of the bladder: 4 had stage A, 3 stage Bl and 4 stage B2 disease, and 2 had carcinoma in situ. Of these patients 4 had recurrences despite previous therapy with BCG alone, irradiation therapy and/or pig cell immunotherapy. The treatment protocol consisted of intravesical instillation of 60 mg. BCG.t Each sample of BCG was sent to our microbiology laboratory for colony counts and it was found that the average instillation was only 3 X 107 colony-forming units. This treatment was combined with the instillation of 3,500 units interleukin 2. The buffy coat of human blood was layered with the Ficoll-Hypaque solution. Peripheral mononuclear cells then were collected above the polymeric solution and were removed. The cells were washed and suspended at a density of 3 X 106 Accepted for publication September 2, 1986. * Requests for reprints: Department of Urology, Box 656, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, New York 14642.

t Institut Armand Frappier, Quebec, Canada.

500 units per ml. in defined standard tentatively defined units of interleukin 2 in standard x tentatively defined units of interleukin 2 in test batch.

Weekly intravesical instillations of BCG and interleukin 2 were given for 6 consecutive weeks. The treatment then was continued at monthly intervals for 1 year. Cystoscopy with cytopathological studies and bladder biopsies were performed before treatment, 1 month after completing the 6-week course of therapy and at 3-month intervals. A bladder computerized tomography scan or magnetic resonance imaging of the bony pelvic area was performed at 6-month intervals. The final confirmation of the presence or absence of tumor was made by cystoscopy, flow cytometry and random bladder biopsies. RESULTS

Of the 13 patients who received therapy 11 (85 per cent) remained free of tumor for a mean of 13 months (range 6 to 24 months). One patient had a partial response with tumor regres-

+Amicon Co. Lexington, Massachusetts.

§ Biological Resource Branch, NCI-FCRE, Frederick, Maryland.

216

--

·

• ___ --·;,,_-·.;c----_:-c.• c.-. .......... ---,

.217 response occurred after a second dose of BCG s.nd interleukin 2. This event the rate to 93 per cent. One a was started who had recurrences before treatn1ent 4 remained free of tumor for a mean of 13 months. After BCG and interleukin 2 treatment all bladder -·-"'-·--

2, 2, Skin test with ,,~,.,,,~~protein derivative was monitored before admission to the protocol and at 6-month intervals. All patients had negative purified derivative skin tests before treatment. The skin test converted to positive in only 2 patients. Complications were tolerable, self-limiting and included mild hematuria, frequency, and mild pyrexia lasting for 24 hours. Our population also consisted of 7 patients with infiltrative carcinoma and the outcome of these cases is summarized. CASE HISTORIES

Case 1. J. a 52-year-old woman, was diagnosed as having grade II, stage B2 papillary transitional cell carcinoma in 1979. The received cell immunotherapy and irradiation. In 1982 she received alone. superficial tumors recurred between 1982 and 1985. In July 1984 the was placed on our ---h,~~ of BCG and interleukin 2 recurred thereafter. We decided to give the week course of combined but the tumors recurred, In 1985 a third course of combined BCG and interleukin 2 was given. The patient completed the 6-week subsequently, complete remission occourse of therapy studies and random nu,n,:i..,, were curred. The last 3

man, was diagnosed as stage B2 transitional cell carcinoma in

-84

FIG. 2. A, bladder reveals chronic cystitis with proliferation of mononuclear cells. E, reduced from X40. B, bladder biopsy shows cystitis with large eosinophilic infiltrate. H & E, reduced from xlOO.

-78 -72 -66 -60 -54 -48 -42 -36 -30 -24 -18 -12 MONTHS PRIOR TO TREATMENT

-6

0

6

12

18

Months Post Treatment c BCG

24

+

ll2

FIG. 1. Effect of BCG and interleukin-2 on recurrence of bladder carcinoma. Each line represents separate patient. Dots represent positive biopsies for transitional cell carcinoma. IR, irradiation patients JB, AB, SM, EC, AC, WC and RW had infiltrating tumors. PC, pig cell immunotherapy given. B, intravesical BCG therapy started. UD, urinary diversion performed. PCy, partial cystectomy performed. BIL, intravesical BCG and interleukin-2 therapy started.

218

MERGUERIAN, DONAHUE AND COCKETT

The patient refused cystectomy and he was placed on our protocol. Treatment was started in November, and multiple biopsies and cystoscopic studies revealed chronic cystitis with no evidence of tumor recurrence. Case 3. S. M., a 68-year-old man, had grade III, stage B2 papillary transitional cell carcinoma in July 1984. The patient was a poor risk for an operation with severe atherosclerotic cardiovascular disease and a recent myocardial infarction, and he was placed on our protocol. After treatment several random biopsies and cystoscopic studies revealed cystitis with a prominent eosinophilic component and no tumor. Case 4. E. C., a 75-year-old man, had stage A papillary transitional cell carcinoma in 1970, with no recurrence until stage Bl multiple transitional cell carcinoma developed in June 1982. The patient refused cystectomy and underwent repeated cystoscopic resections of recurrent bladder tumors. In June 1984 the patient was started on our protocol, and repeat cystoscopic studies and random biopsies revealed chronic cystitis with no evidence of tumor. Case 5. A. C., a 65-year-old man, had grade II, stage B papillary transitional cell carcinoma of the bladder in June 1984 and July 1985. The patient refused radical cystectomy and partial cystectomy was performed. The resection margins contained tumor cells. He was placed on our protocol, and subsequent cystoscopic studies and random bladder biopsies showed chronic cystitis with no evidence of tumor recurrence. Case 6. W. C., a 63-year-old man who was a heavy cigarette smoker, had grade III, stage B2 transitional cell carcinoma in August 1983. The patient received 4,000 rad and underwent ilea! loop urinary diversion. He was placed on our protocol with intravesical instillation of BCG and interleukin 2, and he remains free of tumor. Case 7. R. W., a 70-year-old man, had grade HI, stage B transitional cell carcinoma in February 1984. He refused cystectomy and was placed on our protocol. Repeat cystoscopic studies and random bladder biopsies revealed chronic cystitis. DISCUSSION

The effector cell mechanism of white blood cells against tumor cells has many actions. Killer T-cells, activated macrophages, polymorphonuclear leukocytes, K-cells with antibody and N -cells all are possible effectors. Antibody itself with complement and lymphotoxin produced by T-cells also is reported to be cytotoxic in vitro. On the other hand, suppressor T-cells, antigen antibody complexes and toxic substances produced by tumor cells will inhibit or block these effector mechanisms.13 In 1976 Morales and associates reported a marked decrease in the rate of recurrent superficial bladder cancer in 9 patients treated with intravesical and intradermal BCG. 8 Since then, many reports have demonstrated the effectiveness of live BCG in decreasing the rate of recurrent superficial bladder carcinoma and carcinoma in situ to O to 30 per cent, compared to a high (50 per cent) recurrence rate in many patients. 14' 15 Three different effector mechanisms to explain the effects of BCG have been proposed: 1) an immediate inflammatory response caused by live BCG in which the effector cells, macrophages, are activated directly by BCG without T-cell participation, 2) a tuberculin type response, in which the macrophages ingesting BCG stimulate the BCG sensitized T-cells to produce lymphokines and 3) induction of tumor-specific immunity, in which ECG-stimulated proliferating T-cells interact with macrophages that have ingested BCG and tumor cell debris. This may be an important mechanism for the effective induction of tumor-specific killer T-cells. 16 Interleukin 2 or T-cell growth factor represents one element in a cascade of lymphokines released during an immune response. Recently, Rosenstein 17, Mule 18· 19 and Rosenberg20 and their associates defined the lymphokine activated killer cell system. Lymphokine activated killer cells are generated in vitro

by incubating fresh peripheral blood lymphocytes of tumorbearing patients and normal individuals or splenocytes with interleukin 2. Autologous and allogeneic fresh noncultured tumors of varied histology are lysed by lymphokine activated killer cells while sparing normal tissue. In vitro studies have shown that the binding of antigen to an antigen-specific T-cell receptor provides the first signal for T-cell proliferation and induces the expression of interleukin 2 receptors on effector T-cells. The binding of interleukin 2 to the expressed interleukin 2 receptors provides the second signal for T-cell proliferation and induces T-cell mitosis. 3 Thus, the addition of exogenous interleukin 2 to antigen-activated T-cells in vitro and in vivo stimulates a proliferation or numerical expansion of functionally specific T-cells. Interleukin 2 is released by T-cells in response to antigen, presented in the context of proteins of the major histocompatibility complex. In vitro interleukin 2 is produced by stimulating lymphoid cells with lectins, which can substitute for antigen in the accessory cell signal. 3 We presented a preliminary report on 10 patients. 21 To our knowledge treatment with intravesicular instillation of BCG and allogenic interleukin 2 has not been reported previously. Pizza and associates demonstrated tumor regression in 3 of 6 patients with injections of interleukin 2 into the tumor site. 22 We demonstrate clearly in 13 cases that low dose BCG, 3 x 107 as compared to 1010 colony-forming units, combined with interleukin 2 is efficient in preventing recurrent bladder tumors. Our recurrence rate of 20 per cent is comparable to that of intravesical BCG alone. Owing to our short followup the duration of remission is unknown. It has been reported that patients receiving intravesical therapy and whose purified protein derivative skin test converted to positive had a lower recurrence rate than those whose skin test did not convert. 21 In our series the purified protein derivative skin test converted to positive in only 20 per cent of the patients. Owing to this fact and that our recurrence rate is low we believe that interleukin 2 may have a significant role in preventing recurrent bladder tumors. The side effects of combined treatment were minimal and tolerable, including frequency of urination, hematuria, slight dysuria and slight pyrexia that usually lasted 24 hours. We conclude that low dose (10 7 colony-forming units) BCG combined with interleukin 2 administered intravesically is as effective and associated with less side effects than 1010 colonyforming units of BCG for the prophylactic treatment of patients with bladder cancer. It still is too early to predict the length of remission. Further studies are warranted to determine the optimal response and to determine the efficacy of this treatment modality compared to other treatment modalities that include interleukin 2 alone and low dose BCG alone. We currently are conducting a randomized study to compare the effectiveness of half dose (60 mg.) BCG alone compared to the combination of half dose (60 mg.) BCG and interleukin 2. We recognize the need to determine if a half dose of BCG is as effective as a full dose (120 mg.), and if the combination of half dose BCG and interleukin 2 is more effective than the half dose of BCG alone. REFERENCES l. Ting, C. C., Rodrigues, D., Ting, R. C., Wive!, N. and Collins, M.

J.: Suppression of T cell-mediated immunity by tumor cells: immunogenicity versus immunosuppression and preliminary characterization of the suppressive factors. Int. J. Cancer, 24: 644, 1979. 2. Dumonde, D. C., Hamblin, A. S., Kasp-Grochowska, E., Pulley, M. and Wolstencroft, R. A.: Lymphokines and the lymphoendothelial system-an illustration of immunoregulatory integration. In: Immunoregulation. Edited by N. Fabris, E. Garaci, J. Hadden and N. A. Mitchison. New York: Plenum Press, p. 177, 1983. 3. Robb, R. J.: Interleukin 2: the molecule and its function. Immunol. Today, 5: 203, 1984.

219 4. Burger, iJ.,. Elgert, K. l_J. and Farran, L.; Interleukin 2 (1L-2) activity during turnor- growth: IL-2 p=-oduction kinetics absorption of and responses to exogenous IL-2. Cell. ImmunoL 84: 1

1

f),

Ci.

7.

8.

9.

10.

11. 12.

1:1.

228, 1984. Donohue, ,J. I-I., Rosenstein, M., Chang, A. E., Lotze, NL eJ., Robb,

R. ,J. and Rosenberg, S. A.: The administration of interleukin 2 enhances the of sensitized murine to cure a disseminated syngeneric lymphoma. J. 132: 2123, 1984. Yu, A., Watts, H., ,Jaffe, N. and Parkman, R.: Concomitant presence of tumor-specific cytotoxic and inhibitor lymphocytes in patients with osteogenic sarcoma. New Engl. J. Med., 297: 121, 1977. Cheever, M. A., Thompson, ,J. A., Kern, D. E. and Greenberg, P. D.: Interleukin 2 (IL 2) administered in vivo: influence of IL 2 route and timing on T cell growth. J. Immunol., 134: 3895, 1985. Morales, A., Eidinger, D. and Bruce, A. W.: Intracavitary bacillus Calmette-Guerin in the treatment of superficial bladder tumors. ,J. Urol., 116: 180, 1976. Morales, A.: Treatment of carcinoma in situ of the bladder with BCG, a phase II triaL Cancer Immunol. Immunother., 9: 69, 1980. Lamm, D. L., Thor, D. E., Harris, S. C., Reyna, J. A., Stogdill, V. D. and Radwin, H. M.: Bacillus Calmette-Guerin immunotherapy of superficial bladder cancer. ,J. Urol., 124: 38, 1980. Lamm, D. L., Thor, D. E., Stogdill, V. D. and Radwin, H. M.: Bladder cancer immunotherapy. ,J. Urol., 128: 931, 1982. Morales, A.: Long-term results and complications of intracavitary bacillus Calmette-Guerin therapy for bladder cancer. J. Urol., 132: 457, 1984. Fujimoto, S., Greene, M. I. and Sehon, A. H.: Regulation of the immune response to tumor antigens. I. Immunosuppressor cells

1n. ,rnnc.ns'R host. J. IrDmunol., 11£: 791, 1976. 14. Williams, J. L., J. C. and Saunders, N.: T 1 bladder tumours. Brit. J. Urol., 49: 663, 1977. 15. Torti, F. M. and Lum, B. L.: The biology and treatment of superficial bladder cancer. J. Clin. Oncol., 2: 505, 1984. 16. Tokunaga, T., Kataoka, T., Nakamura R. rvL, Yamamoto, S. and Akagawa, K S.: Mode of antitumor action of BCG. GANN, Monogr. Cancer Res., 21: 59, 1978. 17. Rosenstein, M., Yron, I., Kaufmann, Y. and Rosenberg, S. A.: Lymphokine-activated killer cells: lysis of fresh syngeneic natural killer resistant murine tumor cells by lymphocytes cultured in interleukin 2. Cancer Res., 44: 1946, 1984. 18. Mule, J. J., Shu, S., Schwarz, S. L. and Rosenberg, S. A.: Adoptive immunotherapy of established pulmonary metastases with LAK cells and recombinant interleukin-2. Science, 225: 1487, 1984. 19. Mule, J. J., Shu, S. and Rosenberg, S. A.: The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo. ,J. Immunol., 135: 646, 1985. 20. Rosenberg, S. A., Lotze, M. T., Muul, L. M., Leitman, 8., Chang, A. E., Ettinghausen, S. E., Matory, Y. L., Skibber, J. M., Shiloni, E., Yetto, J. T., Seipp, C. A., Simpson, C. and Reichert, C. M.: Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. New Engl. J. Med., 313: 1485, 1985. 21. Merguerian, P. A. and Cockett, A. T. K.: Intracavitary bacillus Calmette-Guerin and interleukin-2 for bladder cancer: a preliminary report. Surg. Forum, 36: 657, 1985. 22. Pizza, G., Severini, G., Menniti, D., De Vinci, C. and Corrado, F.: Tumour regression after intralesional injection of interleukin 2 (IL-2) in bladder cancer. Preliminary report. Int. J. Cancer, 34: 359, 1984. 1