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Clinical Implications of Transplantability of Induced Bladder Tumors to Intact Transitional Epithelium in Dogs
THE JOURNAL OF UROLOGY
Vol. 75, No. 4, April 1956 Printed in U.S.A.
CLINICAL IMPLICATIONS OF TRANSPLANTABILITY OF INDUCED BLADDER TUMORS TO INTACT TRANSITIONAL EPITHELIUM IN DOGS DONALD F. McDONALD
AND
THEODORE THORSON
From the Division of Urology, Department of Surgery, and the Department of Pathology, University of Washington School of lVIedicine, Seattle, Wash.
In the clinical literature two opposing theories concerning the pathogenesis of recurrent transitional papillary carcinomas are extant. Albarran and Imbert 1 in 1903 and Casper 2 in 1908 voiced the opinion that tumor implants might be responsible for the recurrence of tumors in the ureter in cases of papillary carcinoma of the renal pelvis and in the urinary bladder respectively. This idea was at variance with that expressed in 1890 by Hansemann, 3 who suggested that independent foci of neoplasia developed about the primary tumor site. Since these early papers had not settled the matter, many others wrote of their views. In Kaplan, McDonald and Thompson's 4 paper this controversy was well presented, with reference to papillary carcinoma of the ureter metastases. From their own cases, these authors found that "lesions which involve the ureter are most frequently located at the anatomic points of narrowing and lesions which involve the bladder are most frequently ipsilateral to the tumor in the renal pelvis." Nonetheless, they concluded that independent multicentric origin best explained the recurrence of these tumors at new locations. It seemed that an experimental approach to the problem, since it had not previously been attempted, might clarify the situation. Because we were working with beta-naphthylamine induced papillary tumors of the urinary bladder, we considered their use. However, the dogs at our disposal being of heterogeneous hereditary origin seemed to promise little hope of successful transplantation when compared with rat and mouse inbred strain tumor transplantation experiments. Upon further investigation, we found that newborn puppies were best. Mann 5 reported that only 3 per cent of transplants of a mammary carcinoma grew. Allam, Lombard, Stubbs and Shirer 6 reported unsuccessful attempts to transplant 13 spontaneous canine tumors in purebred beagles and mixed-bred puppies when innoculated subcutaneously, intramuscularly, subarachnoidally, Read at annual meeting, American Urological Association, Los Angeles, Calif., May 16-19, 1955. This investigation was supported in part by a research grant from the National Institutes of Health, U.S. Public Health Service and the John and Mary R. Markle Foundation. 1 Albarran, J. and Imbert, L.: Les Tumeurs du Rein. Paris: Masson et Cie, pp. 452-459, 1903. 2 Casper, L.: Die Recidive der Harnblasenpapillome. Berliner Klin. Wochnschr., 45: 303-308, 1908. 3 Hansemann, D.: Ueber Asymetrische Zelltheilung in Epithelkrebsan und deren Biologische Bedeutung. Virchows Arch. f. Path. Anat., 119: 299-326, 1890. 4 Kaplan, J. H., McDonald, J. R. and Thompson, G. J.: Multicentric origin of papillary tumors of the urinary tract. J. Urol., 66: 792-804, 1951. 5 Mann, F. C.: Attempts to obtain a transplantable tumor in the higher species of animals. Cancer Res., 4: 331-347, 1919. 6 Allam, M. W., Lombard, L. S., Stubbs, E. L. and Shirer, J. F.: Transplantation of a thyroid carcinoma within the canine species. Cancer Res., 14: 734-737, 1954. 690
BLADDER 'J'UMOHS
6H.l
Fra. I. Portion of bcta-naphthylamine-induced dog bladder tumor used for impbnta.1-ion into closed dog bla,dder pouche8.
intracerebrally, intraocularly, intratesticularly and intraperitoneally. found that when puppies three to twelve weeks old were pretreated with :300 total body irradiation and cortisone, a spontaneom;]y occurring canine thyroid carcinoma could be transplanted 100 per cent succe8sfully. The odd8 that our induced bladder tumors would grow when transplanted to intact transitional epithelium in adult untreated mongrel dogs did not seem good. Hughes's7 transplantable mouse bladder tumor was considered. The size of the animal was unsatisfactory for the type of surgical manipulation necessary to the experiment. METHODS
Test animals ,vere operated upon to produce closed bladder pouches not in contact with urine. 8 Papillary tumors obtained from the bladders of animals that had received beta-naphthylamine up to six months prior to use were transplanted to the established bladder pouches two weeks after healing, Histological sectimrn of a portion of each tumor fragment implanted were made. Care was exercised to elo:oe the bladder pouch so that no raw surface to which the tumor could attad1 would be present. Because of anticipated low incidence of sucC'essfol transplants, no untransplanted cont,rol animals were employed. The tumor recipient dogs were sacrificed at three to six month intervals to determine ,Yhether the tumor had survived and grown. No treatment \Yas to the dogs before or after tumor implantation. 7 Hughes, B.: The induction of t,ransplantablc bladder carcinoma in inbred strains or mice. J. Urol. 62: 833-837, 1949, 8 J\IcDonald, D. F. and Lund, R. R.: The role of the urine in vesical carcinoma: I. Confirmation of the 11rogenous theory of pathogenesis. J. Urol., 71: 560-570, 1954.
692
D. F. McDONALD AND T. THORSON
Frn. 2. A, successful tumor transplant to intact transitional epithelial lined bladder pouch 3 months after transplantation. B, papillary carcinoma transplant growing in isolated bladder pouch 3 months after transplant. RESULTS
In three of 7 dogs the pouch contained grossly recognizable papillary protruding tumors which were observed to be present all over the interior surface of the pouch. Microscopically, the transplanted tumors resembled their progenitors. The heavy submucosal infiltrate, with plasma cells and lymphocytes, the hy-
r
BLADDER TUMORS
693
Fm. 3. Submucosal invasion of transplanted experimental papillary carcinoma in isolated bladder pouch 6 months after transplant.
peremia, the atypicism, altered staining properties and numerous mitoses, were reminiscent of the tumors observed in beta-naphthylamine treated dog bladders. Figure 1 is a biopsy of one of the tumor fragments implanted. Figures 2 and 3 represent the tumors seen in the isolated bladder pouches. According to the classification of Ash 9 these were regarded as grade 1 papillary carcinomas. Two of three animals sacrificed at 3 months showed successful tumor transplants. Only one of the four additional animals sacrificed at 6 months was found to have a successful transplant. A total of three successful tumor homotransplants in seven recipient dog bladder pouches was thus observed. DISCUSSION
The fact that transplanted bladder tumors grew under the unfavorable conditions of our experiment lends strong support to the implantation theory. The donors of the tumor fragments had not received beta-naphthylamine for a period of 6 months prior to the time that the tumor fragments were removed and transplanted to the bladder pouches of the recipient animals. In animals given 600 mg. of beta-naphthylamine daily, orally, the earliest reported tumors were at 8 months. It would seem very unlikely that the tiny amount of carcinogen present in the tumor fragment might produce a tumor in such a short time as 3 months. If we were to make clinical inferences from this experiment, they would be that some form of treatment directed at destroying tumor fragments which could be potential implantation metastases, be routinely used. This is not a new idea. In 1908 Casper2 advised treatment of the tumor base with concentrated resorcinol 9
Ash, J.E.: Epithelial tumors of the bladder. J. Urol., 44: 135-145, 1940.
694
D. F. McDONALD AND T. THORSON
and irrigation of the bladder postoperatively with 5 per cent resorcinol. Beer10 reported that recurrences were reduced from 25 per cent to 15 per cent by soaking the bladder and incision with strong alcohol for 5 minutes before sewing up. W ade11 wrote of filling the bladder with silver nitrate, 1 : 1000 solution, preoperatively and bathing the wound and bladder with the same solution before closing. Kirwin12 presented the idea of treating the bladder with 50 per cent phenol in glycerine for 3 minutes, followed by 95 per cent alcohol. Richter13 used trichloroacetic acid treatment before excision of the tumor. He reported no recurrences in nineteen cases. Franksson14 found 37 per cent of 119 recurrent tumors in an area near the primary tumor site, located in bladder incisional scars or around the bladder neck. He believed that these were due to implants. The vesical neck recurrences in patients who had had transurethral resection led him to postulate that damage to the mucosa of the vesical neck predisposed to tumor implantation. He expressed a preference for trichloroacetic acid treatment to prevent implantation of tumor fragments, but did not present a convincingly large group of patients. Semple 15 and Duckworth16 have reported the use of podophyllin suspensions in treatment of bladder tumors. They believe that podophyllin can selectively destroy superficial tumors. Podophyllin treatment has not been tried as a means of preventing implantations. Only a large series of clinical cases in which alternate patients are treated by the methods described can lead to the proper conclusion as to their value. SUMMARY AND CONCLUSIONS
Beta-naphthylamine-induced dog bladder papillary transitional tumors have been successfully homotransplanted to intact bladder epithelium in other mongrel dogs in three of seven attempts. The literature relating to the clinical problem of recurrent bladder tumors and methods used to prevent implantation recurrences has been briefly reviewed. The experimental and clinical observations cited imply that implantation of tumor fragments can occur and that something can and should be done about it. 10 Beer, E.: Physical agents in the treatment of bladder tumors. Am. J. Surg., 44: 113132, 1928. 11 Wade, H.: The treatment of malignant tumors of the urinary bladder. Surg., Gynec. and Obst., 52: 312-323, 1931. 12 Kirwin, T. J.: Papillomatosis of the bladder: New conceptions of etiology and treatment. J. Urol., 49: 1-13, 1943. 13 Richter, S.: Fortsatta Erfarenheter over Kemoelektrokoagulation for Blasen Tumorer. Nord. Med., 24: 2195-2196, 1944. 14 Franksson, C.: Tumours of the urinary bladder. Acta. Chir. Scand., Suppl. 151, Stockholm, 1950. 15 Semple, J.E.: Papillomata of bladder treated with podophyllin. Brit. Med. J., 1: 1235, 1948. 16 Duckworth, D. A.: Treatment of papillomatosis with podophyllin. J. Urol., 64: 740, 1950.
Vol. 75, No. 4, April 1956 Printed in U.S.A.
CLINICAL IMPLICATIONS OF TRANSPLANTABILITY OF INDUCED BLADDER TUMORS TO INTACT TRANSITIONAL EPITHELIUM IN DOGS DONALD F. McDONALD
AND
THEODORE THORSON
From the Division of Urology, Department of Surgery, and the Department of Pathology, University of Washington School of lVIedicine, Seattle, Wash.
In the clinical literature two opposing theories concerning the pathogenesis of recurrent transitional papillary carcinomas are extant. Albarran and Imbert 1 in 1903 and Casper 2 in 1908 voiced the opinion that tumor implants might be responsible for the recurrence of tumors in the ureter in cases of papillary carcinoma of the renal pelvis and in the urinary bladder respectively. This idea was at variance with that expressed in 1890 by Hansemann, 3 who suggested that independent foci of neoplasia developed about the primary tumor site. Since these early papers had not settled the matter, many others wrote of their views. In Kaplan, McDonald and Thompson's 4 paper this controversy was well presented, with reference to papillary carcinoma of the ureter metastases. From their own cases, these authors found that "lesions which involve the ureter are most frequently located at the anatomic points of narrowing and lesions which involve the bladder are most frequently ipsilateral to the tumor in the renal pelvis." Nonetheless, they concluded that independent multicentric origin best explained the recurrence of these tumors at new locations. It seemed that an experimental approach to the problem, since it had not previously been attempted, might clarify the situation. Because we were working with beta-naphthylamine induced papillary tumors of the urinary bladder, we considered their use. However, the dogs at our disposal being of heterogeneous hereditary origin seemed to promise little hope of successful transplantation when compared with rat and mouse inbred strain tumor transplantation experiments. Upon further investigation, we found that newborn puppies were best. Mann 5 reported that only 3 per cent of transplants of a mammary carcinoma grew. Allam, Lombard, Stubbs and Shirer 6 reported unsuccessful attempts to transplant 13 spontaneous canine tumors in purebred beagles and mixed-bred puppies when innoculated subcutaneously, intramuscularly, subarachnoidally, Read at annual meeting, American Urological Association, Los Angeles, Calif., May 16-19, 1955. This investigation was supported in part by a research grant from the National Institutes of Health, U.S. Public Health Service and the John and Mary R. Markle Foundation. 1 Albarran, J. and Imbert, L.: Les Tumeurs du Rein. Paris: Masson et Cie, pp. 452-459, 1903. 2 Casper, L.: Die Recidive der Harnblasenpapillome. Berliner Klin. Wochnschr., 45: 303-308, 1908. 3 Hansemann, D.: Ueber Asymetrische Zelltheilung in Epithelkrebsan und deren Biologische Bedeutung. Virchows Arch. f. Path. Anat., 119: 299-326, 1890. 4 Kaplan, J. H., McDonald, J. R. and Thompson, G. J.: Multicentric origin of papillary tumors of the urinary tract. J. Urol., 66: 792-804, 1951. 5 Mann, F. C.: Attempts to obtain a transplantable tumor in the higher species of animals. Cancer Res., 4: 331-347, 1919. 6 Allam, M. W., Lombard, L. S., Stubbs, E. L. and Shirer, J. F.: Transplantation of a thyroid carcinoma within the canine species. Cancer Res., 14: 734-737, 1954. 690
BLADDER 'J'UMOHS
6H.l
Fra. I. Portion of bcta-naphthylamine-induced dog bladder tumor used for impbnta.1-ion into closed dog bla,dder pouche8.
intracerebrally, intraocularly, intratesticularly and intraperitoneally. found that when puppies three to twelve weeks old were pretreated with :300 total body irradiation and cortisone, a spontaneom;]y occurring canine thyroid carcinoma could be transplanted 100 per cent succe8sfully. The odd8 that our induced bladder tumors would grow when transplanted to intact transitional epithelium in adult untreated mongrel dogs did not seem good. Hughes's7 transplantable mouse bladder tumor was considered. The size of the animal was unsatisfactory for the type of surgical manipulation necessary to the experiment. METHODS
Test animals ,vere operated upon to produce closed bladder pouches not in contact with urine. 8 Papillary tumors obtained from the bladders of animals that had received beta-naphthylamine up to six months prior to use were transplanted to the established bladder pouches two weeks after healing, Histological sectimrn of a portion of each tumor fragment implanted were made. Care was exercised to elo:oe the bladder pouch so that no raw surface to which the tumor could attad1 would be present. Because of anticipated low incidence of sucC'essfol transplants, no untransplanted cont,rol animals were employed. The tumor recipient dogs were sacrificed at three to six month intervals to determine ,Yhether the tumor had survived and grown. No treatment \Yas to the dogs before or after tumor implantation. 7 Hughes, B.: The induction of t,ransplantablc bladder carcinoma in inbred strains or mice. J. Urol. 62: 833-837, 1949, 8 J\IcDonald, D. F. and Lund, R. R.: The role of the urine in vesical carcinoma: I. Confirmation of the 11rogenous theory of pathogenesis. J. Urol., 71: 560-570, 1954.
692
D. F. McDONALD AND T. THORSON
Frn. 2. A, successful tumor transplant to intact transitional epithelial lined bladder pouch 3 months after transplantation. B, papillary carcinoma transplant growing in isolated bladder pouch 3 months after transplant. RESULTS
In three of 7 dogs the pouch contained grossly recognizable papillary protruding tumors which were observed to be present all over the interior surface of the pouch. Microscopically, the transplanted tumors resembled their progenitors. The heavy submucosal infiltrate, with plasma cells and lymphocytes, the hy-
r
BLADDER TUMORS
693
Fm. 3. Submucosal invasion of transplanted experimental papillary carcinoma in isolated bladder pouch 6 months after transplant.
peremia, the atypicism, altered staining properties and numerous mitoses, were reminiscent of the tumors observed in beta-naphthylamine treated dog bladders. Figure 1 is a biopsy of one of the tumor fragments implanted. Figures 2 and 3 represent the tumors seen in the isolated bladder pouches. According to the classification of Ash 9 these were regarded as grade 1 papillary carcinomas. Two of three animals sacrificed at 3 months showed successful tumor transplants. Only one of the four additional animals sacrificed at 6 months was found to have a successful transplant. A total of three successful tumor homotransplants in seven recipient dog bladder pouches was thus observed. DISCUSSION
The fact that transplanted bladder tumors grew under the unfavorable conditions of our experiment lends strong support to the implantation theory. The donors of the tumor fragments had not received beta-naphthylamine for a period of 6 months prior to the time that the tumor fragments were removed and transplanted to the bladder pouches of the recipient animals. In animals given 600 mg. of beta-naphthylamine daily, orally, the earliest reported tumors were at 8 months. It would seem very unlikely that the tiny amount of carcinogen present in the tumor fragment might produce a tumor in such a short time as 3 months. If we were to make clinical inferences from this experiment, they would be that some form of treatment directed at destroying tumor fragments which could be potential implantation metastases, be routinely used. This is not a new idea. In 1908 Casper2 advised treatment of the tumor base with concentrated resorcinol 9
Ash, J.E.: Epithelial tumors of the bladder. J. Urol., 44: 135-145, 1940.
694
D. F. McDONALD AND T. THORSON
and irrigation of the bladder postoperatively with 5 per cent resorcinol. Beer10 reported that recurrences were reduced from 25 per cent to 15 per cent by soaking the bladder and incision with strong alcohol for 5 minutes before sewing up. W ade11 wrote of filling the bladder with silver nitrate, 1 : 1000 solution, preoperatively and bathing the wound and bladder with the same solution before closing. Kirwin12 presented the idea of treating the bladder with 50 per cent phenol in glycerine for 3 minutes, followed by 95 per cent alcohol. Richter13 used trichloroacetic acid treatment before excision of the tumor. He reported no recurrences in nineteen cases. Franksson14 found 37 per cent of 119 recurrent tumors in an area near the primary tumor site, located in bladder incisional scars or around the bladder neck. He believed that these were due to implants. The vesical neck recurrences in patients who had had transurethral resection led him to postulate that damage to the mucosa of the vesical neck predisposed to tumor implantation. He expressed a preference for trichloroacetic acid treatment to prevent implantation of tumor fragments, but did not present a convincingly large group of patients. Semple 15 and Duckworth16 have reported the use of podophyllin suspensions in treatment of bladder tumors. They believe that podophyllin can selectively destroy superficial tumors. Podophyllin treatment has not been tried as a means of preventing implantations. Only a large series of clinical cases in which alternate patients are treated by the methods described can lead to the proper conclusion as to their value. SUMMARY AND CONCLUSIONS
Beta-naphthylamine-induced dog bladder papillary transitional tumors have been successfully homotransplanted to intact bladder epithelium in other mongrel dogs in three of seven attempts. The literature relating to the clinical problem of recurrent bladder tumors and methods used to prevent implantation recurrences has been briefly reviewed. The experimental and clinical observations cited imply that implantation of tumor fragments can occur and that something can and should be done about it. 10 Beer, E.: Physical agents in the treatment of bladder tumors. Am. J. Surg., 44: 113132, 1928. 11 Wade, H.: The treatment of malignant tumors of the urinary bladder. Surg., Gynec. and Obst., 52: 312-323, 1931. 12 Kirwin, T. J.: Papillomatosis of the bladder: New conceptions of etiology and treatment. J. Urol., 49: 1-13, 1943. 13 Richter, S.: Fortsatta Erfarenheter over Kemoelektrokoagulation for Blasen Tumorer. Nord. Med., 24: 2195-2196, 1944. 14 Franksson, C.: Tumours of the urinary bladder. Acta. Chir. Scand., Suppl. 151, Stockholm, 1950. 15 Semple, J.E.: Papillomata of bladder treated with podophyllin. Brit. Med. J., 1: 1235, 1948. 16 Duckworth, D. A.: Treatment of papillomatosis with podophyllin. J. Urol., 64: 740, 1950.