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Levamisole in the Treatment of Non-invasive and Invasive Bladder Cancer: A Preliminary Report
0022-534 7/78/1193-034 7$02. 00/0
VoL 119, March Printed in UB A
THE JoURNAIL OF UROILOGY
Copyright © 1978 by The Williams & Wilkins Co.
LEVAMISOLE IN THE TREATMENT OF NON-INVASIVE AND INVASIVE BLADDER CANCER: A PRELIMINARY REPORT ROBERT B. SMITH,* JEAN DEKERNION, BARBARA BARON, DONALD G. SKINNER AND JOSEPH J. KAUFMAN From the Department of Surgery/Urology, University of California Medical Center and Wadsworth Veterans Administration Hospital, Los Angeles, California
ABSTRACT
We have done a double-blind, randomized, controlled study on 37 patients with transitional cell carcinoma in which levamisole was used as an immune adjuvant in addition to the standard therapy for non-invasive and invasive bladder cancer. Levamisole is administered easily and is well tolerated, especially when compared to other immune adjuvants, such as bacillus CalmetteGuerin or Corynebacterium parvum. Recall antigens, dinitrochlorobenzene reactivity and total lymphocyte count demonstrated little correlation to the initial stage of disease. Monocyte chemotaxis was increased significantly in patients receiving levamisole. Since our study is ongoing no data exist as yet to make any statement regarding the efficacy of levamisole in the treatment of bladder cancer. Levamisole, t an imidazo-thiazole derivative, was developed and has been used successfully for many years as an anthelmintic. In 1971 Renoux and Renoux found that this agent might have immunostirnulative effects. 1 Many studies were instituted that confirmed this observation, especially in immunodeficient patients. 2' 3 Levamisole affects particularly Tcell and phagocyte function (cell-mediated arm of the immune system) in the compromised host. 4 , 5 Clinical trials suggest that the agent seems to be most effective in patients who have minimal tumor burden and who are immunosuppressed secondary to such modalities as chemotherapy or radiation therapy. Studies on the use of levamisole as an adjuvant to surgery in resectable lung cancer patients have demonstrated a significant increase in the survival and interval free of disease over control patients. 6' 7 Rojas and associates demonstrated a similar significant prolongation of the interval free of disease and survival in patients with stage C breast cancer (inoperable) who were treated with extensive radiation therfollowed with levamisole. 8 has been little information regarding the effect of levamisole on patients with large tumor burdens, although patients who do not seem to respond to chemotherapy seem to benefit little from levamisole, On the other hand, the addition of levamisole to an effective chemotherapeutic agent may potentiate the effect. Hortobagyi and associates reported an increase in the response rate and duration of remission in patients with metastatic breast cancer receiving concomitant levamisole and chemotherapy compared to patients receiving chemotherapy alone. 9 Hall and associates showed an increased interval free of disease in patients with refractory malignant melanoma who were treated with actinomycin D plus levamisole compared to those treated with actinomycin D alone. 10 Patients with minimum residual bladder cancer who have undergone curative operations and radiation therapy are candidates for immunostimulation for several reasons. 1) The radiation therapy causes immunodepression for a variable interval. 2) There is a 30 per cent 5-year survival expectancy in such patients (patients with muscle invasion), which can be Accepted for publication May 27, 1977. Read at annual meeting of American Urological Association, Chicago, Illinois, April 24-28, 1977. *Requests for reprints: Division of Urology, 66-118 CHS, UCLA Medical Center, Los Angeles, California 90024. t Janssen Research and Development, Inc., New Brunswick, New Jersey. 347
attributed to the presence of microfoci of tumor at the time of definitive therapy. 3) There have been no proved effective chemotherapeutic agents for the treatment of advanced bladder cancer and, therefore, the prevention of disseminated and local recurrence is mandatory. Patients with stage A recurrent malignant bladder papilfo.. mas also are ideally suited for adjuvant imrnunotherapy. Only about 30 to 40 per cent of these patients will respond to adjuvant thio-tepa therapy. Furthermore, as the number of recurrences increases the risk of incurring invasive or ana", plastic carcinoma increases markedly. Between 30 and 50 per cent of such patients can be expected to require radical operations and radiation therapy eventually. Although clinical trials with levarnisole are in the prelimi, nary stages this drug appears to be an appropriate agent for the treatment of these 2 types of bladder tumors. The drug is relatively innocuous and, therefore, can be justified as adjuvant therapy, even in patients with non-invasive cancer. The high incidence of recurrence in both these stages of bladder cancer further justifies the need for adjuvant therapy. Therefore, our study was designed to evaluate whether levamisole will reduce the rate of recurrence of invasive bladder cancer after definitive surgery and radiation therapy and whether it will decrease the recurrence rate and lengthen the interval free of disease in patients with recurrent stage A bladder carcinoma. MATERIALS AND METHODS
Patients with transitional cell carcinoma of the bladder were admitted to the study and subdivided into the following groups: 1) patients with stage A non-invasive tumors and 2) patients with invasive bladder tumors, treated either with partial or total cystectomy, who have minimal residual disease. In addition to our usual therapy the patients were randomized blindly to receive either levamisole or an identical placebo. Patients with diffuse carcinoma in situ also were admitted to the study as a separate group and placed on levamisole. Since the natural history of carcinoma in situ is so predictable and since np effective chemotherapeutic or radiation therapy program has been devised successfully we did not think a placebo group was necessary in this patient population. However, there is no information as to whether these patients represent good candidates for adjuvant immunotherapy.
348
SMITH AND ASSOCIATES
Patients older than 80 years who had a second malignancy or a life expectancy of less than 3 years, irrespective of their bladder tumor, were excluded. In addition, patients with known metastatic disease or known residual local tumor after definitive therapy were not considered candidates for the study. Patients received 2.5 mg./kg. of either a placebo or levamisole daily for 2 consecutive days every week (rounded off to the highest 50 mg. increment). The medication was started prior to surgery or radiation therapy. Treatment continued on a blind basis for a minimum of 1 year Qr a maximum of 2 years, or until tumor recurrence or metastasis or drug toxicity is noted. The randomization code is broken at that time. Complete blood count, platelet count, absolute lymphocyte, count, chest x-ray, creatinine and SMA-12 are performed every 3 months. Liver and bone scans are performed yearly or more often if clinically indicated. Excretory urograms are performed at 6-month intervals for 2 years and thereafter yearly. Skin tests for delayed cutaneous hypersensitivity and in vitro tests of immune response are repeated every 8 weeks. RESULTS
Our report is preliminary, representing 210 patient months on therapy. A total of 36 patients have been admitted to the study since its inception 12 months ago. Patient entry continues to be active. Of the 36 patients 15 with non-invasive transitional cell carcinoma, 15 with invasive carcinoma and 6 with carcinoma in situ are being studied. Twenty-seven patients continue on the protocol, 5 have been terminated from the study because of recurrent disease, 2 were removed from the study because of side effects and 3 were protocol deviants. Two of 6 levamisole patients and 1 of 9 placebo patients in the non-invasive group had tumor recurrence. No patients in the invasive group (9 stage B tumors, no stage C and 3 stage Dl tumors) have had evidence of recurrent disease to date. However, followup is short, the longest patient being only 12 months post-cystectomy. In the carcinoma in situ group a patient who was given initially a placebo experienced recurrence and was converted to levamisole. Only 1 of 6 levamisole patients has had recurrence thus far. Side effects. Two patients on levamisole experienced side effects of sufficient severity to require their removal from the study. One patient experienced severe neutropenia 4½ months after the initiation of therapy (200 mg. daily for 2 consecutive days each week). Two months before the neutropenic episode a rash was noted and the dosage level was progressively decreased to 100 mg. daily with no diminution in the rash. The drug was discontinued 4½ months after the initiation of therapy when the white blood cell count was noted to be 861/mm. 3 , with 3 per cent segmented neutrophils and 1 per cent band. Forty-eight hours later the white blood count bottomed out at 477/mm. 3 (3 per cent segmented neutrophils). The patient was isolated. During the subsequent 3 weeks the white blood count increased to 1,000/mm. 3 and 5 days later was 3,900/mm. 3 when the patient was discharged from the hospital. A week later the white blood count normalized and was 8,700/mm. 3 with a normal differential. An antineutrophil antibody has been identified, which is cytotoxic to the patient's neutrophils as well as to the neutrophils of random donors. The other patient who had to be removed from the study experienced a severe flu-like syndrome 2 weeks after the initiation of therapy, having lethargy, headache, nausea, vomiting and fever. Symptoms disappeared when the levamisole was withdrawn and the patient has not been rechallenged with the medication. In addition, mild transient side effects have occurred in other patients but were not severe enough to warrant cessation of therapy. One patient experienced mild fever, 1 patient insomnia, 2 patients transient diarrhea, 1 patient euphoria and 1 patient a mild bitter metallic taste.
Interestingly, 2 patients on a placebo experienced side effects. One patient had mild lethargy and another was removed from the placebo because of a severe flu-like syndrome. This patient has been placed on levamisole without problems. Immunologic data. All patients had recall antigens applied, which were not found to be useful. Dinitrochlorobenzene (DNCB) reactivity was investigated in all patients and, thus far, has been somewhat disappointing in regard to the relationship to stage and grade of tumor (table 1). Little difference between tumor stages was seen in the incidence of initial DNCB reactivity: 46. 7 per cent positive in the non-invasive group and 33.3 per cent in the invasive group. Patients in the levamisole and placebo groups noted an increase in DNCB reactivity after therapy (table 2). The increase in reactivity was more marked in the non-invasive group. It is interesting to note that 2 of the 3 patients in the non-invasive group and 1 of the 2 in the carcinoma in situ group who have had recurrences were responsive when rechallenged with DNCB. The correlation of absolute lymphocyte counts has been disappointing in relation to stage and grade of the tumor and response to therapy. No definite pattern could be found in regard to the increase in the absolute lymphocyte count and the response to levamisole therapy or in relation to patients with recurrent disease (table 3). The only immune parameter with any positive correlation to levamisole administration was monocyte chemotaxis. Monocyte chemotaxis was performed on these patients by Dr. D. Nathanson, University of California at Los Angeles. Fourteen study patients were examined in a blind fashion. All 10 levamisole patients tested had an increase in chemotaxis, whereas none of 4 placebo control or 5 non-age-matched controls had increased chemotaxis. An actual diminution in the number of active T-cells was found in patients treated with levamisole. As yet, we have been unable to explain the significance of this finding. DISCUSSION
The number of patients and length of followup is obviously too small to permit any statements to be made in regard to TABLE
Non-invasive Invasive Carcinoma in situ
* Positive =
1. Initial DNCB reactivity Negative Positive* No. Pts.
No. Pts. (%)
8 10 3
7 (46. 7) 5 (33.5) 3 (50)
1.0 cm. or greater erythema or induration with 50 µ,g. DNCB. TABLE
2. DNCB response after therapy Negative
Positive*
No. Pts.
No. Pts. (%)
Non-invasive:
2
Levamisole Placebo Invasive: Levamisole Placebo Carcinoma in situ (all levamisole)
1 1
12 (85. 7) 7 (87.5) 5 (83.3) 7 (58.3) 4 (67) 3 (50) 5 (83.3)
5
2 3 1
* Positive = 1.0 cm. or greater erythema or induration with 50 µ,g. DNCB. TABLE
Non-invasive: Levamisole Placebo Invasive: Levamisole Placebo Carcinoma in situ (all levamisole)
3. Absolute lymphocyte count Pre-Therapy
Post-Therapy
2,027 2,089 1,985 1,631 1,575 1,700 1,587
2,064 1,936 2,193 1,670 1,636 1,743 1,732
LEVAMISOLE IN TREATMENT OF NON-INVASIVE AND INVASIVE BLADDER CANCER
the efficacy of levamisole as an immune adjuvant in the treatment of bladder cancer. We have found the drug to be administered easily and tolerated well with few side effects, especially when compared to other methods of immunotherapy, such as baccillus Calmette-Guerin (BCG) and Corynebacterium parvum. Patient acceptance is vastly superior than with other immunotherapeutic agents with which we have had experience. We have been unable to correlate DNCB reactivity with tumor stage of bladder cancer, although other investigators have been successful in doing so. 11- 13 Catalona and associates showed an excellent correlation between DNCB reactivity and tumor stage (stages O to Bl, 72 per cent; stages B2 and 15 per cent)." They also demonstrated a strong correlation between the decrease in DNCB reactivity and tumor recurrence. Schellhammer and associates similarly demonstrated a 71 per cent positive rate in superficial tumors (stages O to Bl) and a 35 per cent positive response in invasive tumors. 12 Our data suggest that DNCB reactivity is depressed severely in all stages of disease (table 1). Yet to be determined is the significance of apparent improvement in DNCB reactivity in patients who have undergone definitive therapy for their bladder tumors, whether they received levamisole or a placebo. This improvement may be related specifically to decreasing the tumor burden rather than to any active immunostimulative event. The patients treated with levamisole seem to have a higher DNCB response rate (16 of 20 patients, 80 per cent) than those in the placebo group (8 of 12 patients, 67 per cent). These numbers are obviously too small to permit statistical evaluation. One disquieting fact noted in our study is that 3 of 5 patients who already have had recurrences while in this study were DNCB-reactive. The absolute peripheral lymphocyte count has been said by Morales and Eidinger to correlate well with the stage and progression of renal cell carcinoma. 14 Amin and Lich demonstrated a positive relationship between lymphopenia and poor survival in patients with bladder cancer. 15 Zacharski and Linman found that 22.4 per cent of cancer patients had lymphopenia compared to only 6 per cent of controls (lymphopenia being defined as less than 1,000 lymphocytes per mm. 3).'" Our series showed only 2 of31 patients to be lymphopenic (6.5 per cent), which is identical to the control population of Zacharski and Linman. The stage of the tumor bore no relationship to the absolute lymphocyte count in our series (table 3). No increase was noted after treatment in our series. However, an increase was not expected, since most patients were not lymphopenic initially. Of the 2 lymphopenic patients 1 has had an increase in absolute lymphocyte count with levamisole therapy (from 828 to 1,562). In addition, 4 of 5 patients who had recurrences had normal lymphocyte counts. We believe that an absolute lymphocyte count is of little use as a parameter to follow the immune response to bladder cancer. By far the most consistent immune parameter showing a positive correlation with the administration of levamisole is the monocyte chemotaxis. Hausman and Brosman demonstrated defective monocyte function in patients with bladder cancer but could find no correlation between tumor stage and the degree of chemotactic defect. 17 The significance of in-
349
creased monocyte chemotaxis in the patient treated with levamisole is unknown currently. Our efforts with levamisole continue. It is our hope that an increased survival and interval free of disease will be seen, as apparently has been seen in other tumors. REFERENCES
1. Renoux, G. and Renoux, M.: Effet immunostimulant d'un imidothiazole dans !'immunisation des souris contre !'infection par Brucella abortus. C.R. Acad. Sci., 272: 349, 1971. 2. Hirshaut, Y., Pinsky, C., Marquardt, H. and Oettgen, H.F." Effects of levamisole in delayed hypersensitivity reactions in cancer patients (abstract). Proc. Amer. Ass. Cancer Res., 14; 109, 1973. 3. Tripodi, D., Parks, L. C. and Brugmans, J.: Drug-induced restoration of cutaneous delayed hypersensitivity in anergic patients with cancer. New Engl. J. Med., 289: 354, 1973. 4. Biniaminov, M. and Ramot, B.: In-vitro restoration by levamisole of thymus-derived lymphocyte function in Hodgkin's disease (letter). Lancet, 1: 464, 1975. 5. Renoux, G., Renoux, M. and Palat, A.: Influence of levamisole on T-cell reactivity and in survival of intractable cancer patients. Proc. Fogarty International Center. Washington, D. C.: U. S. Government Printing Office, No. 28, 1975. 6. Study Group for Bronchogenic Carcinoma: Immunopotentiation with levamisole in resectable bronchogenic carcinoma: a double-blind controlled trial. Brit. Med. J., 3: 461, 1975. 7. Amery, W. K.: A double-blind levamisole trial in resectable lung cancer. Ann. N. Y. Acad. Sci., 277: 260, 1976. 8. Rojas, A. F., Mickiewicz, E., Feierstein, J. N., Glait, H. and Olivari, A. J.: Levamisole in advanced human breast cancer. Lancet, l: 211, 1976. 9. Hortobagyi, G. N., Gutterman, J. U., Blumenschein, G. R., Tashima, T. K., Buzdar, A. U. and Hersh, E. M.: Levamiso!e in the treatment of breast cancer. Read at 3rd conference of Modulation of Host Immunoresistance in the Prevention or Treatment of Induced Neoplasia. Bethesda: National Institutes of Health, December 13-15, 1976. 10. Hall, S. W., Benjamin, R. S., Heilbrum, L., Lewinski, U,, Gutterman, J. U. and Mavilgit, G.: Chemo-immunotherapy of refractory malignant melanoma with actinomycin-D and levamisole. Read at 3rd conference of Modulation of Host Immunoresistance in the Prevention or Treatment of Induced Neoplasia. Bethesda: National Institutes of Health, December 13-15, 1976. 11. Catalona, W. J., Smolev, J. K. and Harty, J. I.: Prognostic value of host immunocompetence in urologic cancer patients. J. Urol., 114: 922, 1975. 12. Schellhammer, P. F., Bracken, R. B., Bean, M.A., Pinsky, Co M, and Whitmore, W. F.: Immune evaluation with skin testing. A study of testicular, prostatic, and bladder neoplasms. Cancer, 38: 149, 1976. 13. Olsson, C. A., Rao, C. N., Menzoian, J. 0. and Byrd, W. E.: Immunologic unreactivity in bladder cancer patients. J. Uro!., 107: 607, 1972. 14. Morales, A. and Eidinger, D.: Immune reactivity in renal cancer: a sequential study. J. Urol., 115: 510, 1976. 15. Amin, M. and Lich, R.: Lymphocytes and bladder cancer. J. Urol., 111: 165, 1974. 16. Zacharski, L. R. and Linman, J. W.: Lymphocytopenia: its causes and significance. Mayo Clin. Proc., 46: 168, 1971. 17. Hausman, M. S. and Brosman, S. A.: Abnormal monocyte function in bladder cancer patients. J. Urol., 115: 537, 1976.
VoL 119, March Printed in UB A
THE JoURNAIL OF UROILOGY
Copyright © 1978 by The Williams & Wilkins Co.
LEVAMISOLE IN THE TREATMENT OF NON-INVASIVE AND INVASIVE BLADDER CANCER: A PRELIMINARY REPORT ROBERT B. SMITH,* JEAN DEKERNION, BARBARA BARON, DONALD G. SKINNER AND JOSEPH J. KAUFMAN From the Department of Surgery/Urology, University of California Medical Center and Wadsworth Veterans Administration Hospital, Los Angeles, California
ABSTRACT
We have done a double-blind, randomized, controlled study on 37 patients with transitional cell carcinoma in which levamisole was used as an immune adjuvant in addition to the standard therapy for non-invasive and invasive bladder cancer. Levamisole is administered easily and is well tolerated, especially when compared to other immune adjuvants, such as bacillus CalmetteGuerin or Corynebacterium parvum. Recall antigens, dinitrochlorobenzene reactivity and total lymphocyte count demonstrated little correlation to the initial stage of disease. Monocyte chemotaxis was increased significantly in patients receiving levamisole. Since our study is ongoing no data exist as yet to make any statement regarding the efficacy of levamisole in the treatment of bladder cancer. Levamisole, t an imidazo-thiazole derivative, was developed and has been used successfully for many years as an anthelmintic. In 1971 Renoux and Renoux found that this agent might have immunostirnulative effects. 1 Many studies were instituted that confirmed this observation, especially in immunodeficient patients. 2' 3 Levamisole affects particularly Tcell and phagocyte function (cell-mediated arm of the immune system) in the compromised host. 4 , 5 Clinical trials suggest that the agent seems to be most effective in patients who have minimal tumor burden and who are immunosuppressed secondary to such modalities as chemotherapy or radiation therapy. Studies on the use of levamisole as an adjuvant to surgery in resectable lung cancer patients have demonstrated a significant increase in the survival and interval free of disease over control patients. 6' 7 Rojas and associates demonstrated a similar significant prolongation of the interval free of disease and survival in patients with stage C breast cancer (inoperable) who were treated with extensive radiation therfollowed with levamisole. 8 has been little information regarding the effect of levamisole on patients with large tumor burdens, although patients who do not seem to respond to chemotherapy seem to benefit little from levamisole, On the other hand, the addition of levamisole to an effective chemotherapeutic agent may potentiate the effect. Hortobagyi and associates reported an increase in the response rate and duration of remission in patients with metastatic breast cancer receiving concomitant levamisole and chemotherapy compared to patients receiving chemotherapy alone. 9 Hall and associates showed an increased interval free of disease in patients with refractory malignant melanoma who were treated with actinomycin D plus levamisole compared to those treated with actinomycin D alone. 10 Patients with minimum residual bladder cancer who have undergone curative operations and radiation therapy are candidates for immunostimulation for several reasons. 1) The radiation therapy causes immunodepression for a variable interval. 2) There is a 30 per cent 5-year survival expectancy in such patients (patients with muscle invasion), which can be Accepted for publication May 27, 1977. Read at annual meeting of American Urological Association, Chicago, Illinois, April 24-28, 1977. *Requests for reprints: Division of Urology, 66-118 CHS, UCLA Medical Center, Los Angeles, California 90024. t Janssen Research and Development, Inc., New Brunswick, New Jersey. 347
attributed to the presence of microfoci of tumor at the time of definitive therapy. 3) There have been no proved effective chemotherapeutic agents for the treatment of advanced bladder cancer and, therefore, the prevention of disseminated and local recurrence is mandatory. Patients with stage A recurrent malignant bladder papilfo.. mas also are ideally suited for adjuvant imrnunotherapy. Only about 30 to 40 per cent of these patients will respond to adjuvant thio-tepa therapy. Furthermore, as the number of recurrences increases the risk of incurring invasive or ana", plastic carcinoma increases markedly. Between 30 and 50 per cent of such patients can be expected to require radical operations and radiation therapy eventually. Although clinical trials with levarnisole are in the prelimi, nary stages this drug appears to be an appropriate agent for the treatment of these 2 types of bladder tumors. The drug is relatively innocuous and, therefore, can be justified as adjuvant therapy, even in patients with non-invasive cancer. The high incidence of recurrence in both these stages of bladder cancer further justifies the need for adjuvant therapy. Therefore, our study was designed to evaluate whether levamisole will reduce the rate of recurrence of invasive bladder cancer after definitive surgery and radiation therapy and whether it will decrease the recurrence rate and lengthen the interval free of disease in patients with recurrent stage A bladder carcinoma. MATERIALS AND METHODS
Patients with transitional cell carcinoma of the bladder were admitted to the study and subdivided into the following groups: 1) patients with stage A non-invasive tumors and 2) patients with invasive bladder tumors, treated either with partial or total cystectomy, who have minimal residual disease. In addition to our usual therapy the patients were randomized blindly to receive either levamisole or an identical placebo. Patients with diffuse carcinoma in situ also were admitted to the study as a separate group and placed on levamisole. Since the natural history of carcinoma in situ is so predictable and since np effective chemotherapeutic or radiation therapy program has been devised successfully we did not think a placebo group was necessary in this patient population. However, there is no information as to whether these patients represent good candidates for adjuvant immunotherapy.
348
SMITH AND ASSOCIATES
Patients older than 80 years who had a second malignancy or a life expectancy of less than 3 years, irrespective of their bladder tumor, were excluded. In addition, patients with known metastatic disease or known residual local tumor after definitive therapy were not considered candidates for the study. Patients received 2.5 mg./kg. of either a placebo or levamisole daily for 2 consecutive days every week (rounded off to the highest 50 mg. increment). The medication was started prior to surgery or radiation therapy. Treatment continued on a blind basis for a minimum of 1 year Qr a maximum of 2 years, or until tumor recurrence or metastasis or drug toxicity is noted. The randomization code is broken at that time. Complete blood count, platelet count, absolute lymphocyte, count, chest x-ray, creatinine and SMA-12 are performed every 3 months. Liver and bone scans are performed yearly or more often if clinically indicated. Excretory urograms are performed at 6-month intervals for 2 years and thereafter yearly. Skin tests for delayed cutaneous hypersensitivity and in vitro tests of immune response are repeated every 8 weeks. RESULTS
Our report is preliminary, representing 210 patient months on therapy. A total of 36 patients have been admitted to the study since its inception 12 months ago. Patient entry continues to be active. Of the 36 patients 15 with non-invasive transitional cell carcinoma, 15 with invasive carcinoma and 6 with carcinoma in situ are being studied. Twenty-seven patients continue on the protocol, 5 have been terminated from the study because of recurrent disease, 2 were removed from the study because of side effects and 3 were protocol deviants. Two of 6 levamisole patients and 1 of 9 placebo patients in the non-invasive group had tumor recurrence. No patients in the invasive group (9 stage B tumors, no stage C and 3 stage Dl tumors) have had evidence of recurrent disease to date. However, followup is short, the longest patient being only 12 months post-cystectomy. In the carcinoma in situ group a patient who was given initially a placebo experienced recurrence and was converted to levamisole. Only 1 of 6 levamisole patients has had recurrence thus far. Side effects. Two patients on levamisole experienced side effects of sufficient severity to require their removal from the study. One patient experienced severe neutropenia 4½ months after the initiation of therapy (200 mg. daily for 2 consecutive days each week). Two months before the neutropenic episode a rash was noted and the dosage level was progressively decreased to 100 mg. daily with no diminution in the rash. The drug was discontinued 4½ months after the initiation of therapy when the white blood cell count was noted to be 861/mm. 3 , with 3 per cent segmented neutrophils and 1 per cent band. Forty-eight hours later the white blood count bottomed out at 477/mm. 3 (3 per cent segmented neutrophils). The patient was isolated. During the subsequent 3 weeks the white blood count increased to 1,000/mm. 3 and 5 days later was 3,900/mm. 3 when the patient was discharged from the hospital. A week later the white blood count normalized and was 8,700/mm. 3 with a normal differential. An antineutrophil antibody has been identified, which is cytotoxic to the patient's neutrophils as well as to the neutrophils of random donors. The other patient who had to be removed from the study experienced a severe flu-like syndrome 2 weeks after the initiation of therapy, having lethargy, headache, nausea, vomiting and fever. Symptoms disappeared when the levamisole was withdrawn and the patient has not been rechallenged with the medication. In addition, mild transient side effects have occurred in other patients but were not severe enough to warrant cessation of therapy. One patient experienced mild fever, 1 patient insomnia, 2 patients transient diarrhea, 1 patient euphoria and 1 patient a mild bitter metallic taste.
Interestingly, 2 patients on a placebo experienced side effects. One patient had mild lethargy and another was removed from the placebo because of a severe flu-like syndrome. This patient has been placed on levamisole without problems. Immunologic data. All patients had recall antigens applied, which were not found to be useful. Dinitrochlorobenzene (DNCB) reactivity was investigated in all patients and, thus far, has been somewhat disappointing in regard to the relationship to stage and grade of tumor (table 1). Little difference between tumor stages was seen in the incidence of initial DNCB reactivity: 46. 7 per cent positive in the non-invasive group and 33.3 per cent in the invasive group. Patients in the levamisole and placebo groups noted an increase in DNCB reactivity after therapy (table 2). The increase in reactivity was more marked in the non-invasive group. It is interesting to note that 2 of the 3 patients in the non-invasive group and 1 of the 2 in the carcinoma in situ group who have had recurrences were responsive when rechallenged with DNCB. The correlation of absolute lymphocyte counts has been disappointing in relation to stage and grade of the tumor and response to therapy. No definite pattern could be found in regard to the increase in the absolute lymphocyte count and the response to levamisole therapy or in relation to patients with recurrent disease (table 3). The only immune parameter with any positive correlation to levamisole administration was monocyte chemotaxis. Monocyte chemotaxis was performed on these patients by Dr. D. Nathanson, University of California at Los Angeles. Fourteen study patients were examined in a blind fashion. All 10 levamisole patients tested had an increase in chemotaxis, whereas none of 4 placebo control or 5 non-age-matched controls had increased chemotaxis. An actual diminution in the number of active T-cells was found in patients treated with levamisole. As yet, we have been unable to explain the significance of this finding. DISCUSSION
The number of patients and length of followup is obviously too small to permit any statements to be made in regard to TABLE
Non-invasive Invasive Carcinoma in situ
* Positive =
1. Initial DNCB reactivity Negative Positive* No. Pts.
No. Pts. (%)
8 10 3
7 (46. 7) 5 (33.5) 3 (50)
1.0 cm. or greater erythema or induration with 50 µ,g. DNCB. TABLE
2. DNCB response after therapy Negative
Positive*
No. Pts.
No. Pts. (%)
Non-invasive:
2
Levamisole Placebo Invasive: Levamisole Placebo Carcinoma in situ (all levamisole)
1 1
12 (85. 7) 7 (87.5) 5 (83.3) 7 (58.3) 4 (67) 3 (50) 5 (83.3)
5
2 3 1
* Positive = 1.0 cm. or greater erythema or induration with 50 µ,g. DNCB. TABLE
Non-invasive: Levamisole Placebo Invasive: Levamisole Placebo Carcinoma in situ (all levamisole)
3. Absolute lymphocyte count Pre-Therapy
Post-Therapy
2,027 2,089 1,985 1,631 1,575 1,700 1,587
2,064 1,936 2,193 1,670 1,636 1,743 1,732
LEVAMISOLE IN TREATMENT OF NON-INVASIVE AND INVASIVE BLADDER CANCER
the efficacy of levamisole as an immune adjuvant in the treatment of bladder cancer. We have found the drug to be administered easily and tolerated well with few side effects, especially when compared to other methods of immunotherapy, such as baccillus Calmette-Guerin (BCG) and Corynebacterium parvum. Patient acceptance is vastly superior than with other immunotherapeutic agents with which we have had experience. We have been unable to correlate DNCB reactivity with tumor stage of bladder cancer, although other investigators have been successful in doing so. 11- 13 Catalona and associates showed an excellent correlation between DNCB reactivity and tumor stage (stages O to Bl, 72 per cent; stages B2 and 15 per cent)." They also demonstrated a strong correlation between the decrease in DNCB reactivity and tumor recurrence. Schellhammer and associates similarly demonstrated a 71 per cent positive rate in superficial tumors (stages O to Bl) and a 35 per cent positive response in invasive tumors. 12 Our data suggest that DNCB reactivity is depressed severely in all stages of disease (table 1). Yet to be determined is the significance of apparent improvement in DNCB reactivity in patients who have undergone definitive therapy for their bladder tumors, whether they received levamisole or a placebo. This improvement may be related specifically to decreasing the tumor burden rather than to any active immunostimulative event. The patients treated with levamisole seem to have a higher DNCB response rate (16 of 20 patients, 80 per cent) than those in the placebo group (8 of 12 patients, 67 per cent). These numbers are obviously too small to permit statistical evaluation. One disquieting fact noted in our study is that 3 of 5 patients who already have had recurrences while in this study were DNCB-reactive. The absolute peripheral lymphocyte count has been said by Morales and Eidinger to correlate well with the stage and progression of renal cell carcinoma. 14 Amin and Lich demonstrated a positive relationship between lymphopenia and poor survival in patients with bladder cancer. 15 Zacharski and Linman found that 22.4 per cent of cancer patients had lymphopenia compared to only 6 per cent of controls (lymphopenia being defined as less than 1,000 lymphocytes per mm. 3).'" Our series showed only 2 of31 patients to be lymphopenic (6.5 per cent), which is identical to the control population of Zacharski and Linman. The stage of the tumor bore no relationship to the absolute lymphocyte count in our series (table 3). No increase was noted after treatment in our series. However, an increase was not expected, since most patients were not lymphopenic initially. Of the 2 lymphopenic patients 1 has had an increase in absolute lymphocyte count with levamisole therapy (from 828 to 1,562). In addition, 4 of 5 patients who had recurrences had normal lymphocyte counts. We believe that an absolute lymphocyte count is of little use as a parameter to follow the immune response to bladder cancer. By far the most consistent immune parameter showing a positive correlation with the administration of levamisole is the monocyte chemotaxis. Hausman and Brosman demonstrated defective monocyte function in patients with bladder cancer but could find no correlation between tumor stage and the degree of chemotactic defect. 17 The significance of in-
349
creased monocyte chemotaxis in the patient treated with levamisole is unknown currently. Our efforts with levamisole continue. It is our hope that an increased survival and interval free of disease will be seen, as apparently has been seen in other tumors. REFERENCES
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