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Pathological Changes Associated With Topical Chemotherapy for Superficial Bladder Cancer
0022-5347 /81/1264-0461$02.00/0 Vol. 126, October
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright© 1981 by The Williams & Wilkins Co.
PATHOLOGICAL CHANGES ASSOCIATED WITH TOPICAL CHEMOTHERAPY FOR SUPERFICIAL BLADDER CANCER WILLIAM M. MURPHY, MARKS. SOLOWAY
AND
PAMELA J. FINEBAUM
From the Department of Pathology, Baptist Memorial Hospital and Department of Urology, University of Tennessee Center for Health Sciences, Memphis, Tennessee
ABSTRACT
With use of previous observations in experimental animals as a basis for comparison the cytologic and histologic changes in human patients who underwent topical chemotherapy for superficial bladder cancer were documented. Despite the potential for inhibition of deoxyribonucleic acid replication these drugs apparently act in vivo as toxic substances, causing increased exfoliation with denudation of papillary and/o:r flat urothelium. Multinucleation was common but confined to superficial cells. Atypical cells, such as those observed after systemic chemotherapy with cyclophosphamide, rarely were present and could be distinguished readily from neoplastic elements. Although topical chemotherapy may suppress tumor growth and progression it apparently does not eradicate the neoplastic process. During the last 5 years we have been interested in the morphologic effects of topical chemotherapy for superficial carcinoma of the bladder. To avoid the multiple variables inherent in the evaluation of patients we examined first the changes in experimental animals, in which conditions of tumorigenesis and treatment could be controlled. 1- 4 From these studies various observations were made. 1) The morphologic changes, cytologic and histologic, of triethylenethiophosphoramide (thio-tepa) and mitomycin C on mammalian bladders are similar. 2) Both of these alkylating agents appear to act in vivo primarily as toxic substances rather than metabolic inhibitors of deoxyribonucleic acid replication, causing increased exfoliation of preserved cells with secondary denudation of urothelium. 3) Multinucleated cells and cells with atypical nuclei are unusual after topical chemotherapy. Although more prominent in treated animals these changes are not specific for drug effect and can be seen after catheterization with instillation of saline alone. They almost always occur in superficial (umbrella) cells. More importantly, these changes can be distinguished easily from malignant tumor cells. 4) Neither thio-tepa nor mitomycin C prevents eventual tumor growth in F ANFTinduced murine bladder cancer, although they may delay recurrence and inhibit progression to higher grade and/or stage neoplasms. With this experience in an experimental system as a basis for comparison we herein determine whether similar observations can be documented in humans. MATERIALS AND METHODS
The patients included in this study had documented superficial bladder carcinoma, had been treated with topical thiotepa and/or mitomycin C, and had had sufficient followup with endoscopic, histologic and cytologic examinations to determine the pathologic changes associated with chemotherapy. Of the 35 patients who have been treated at our institutions 20 met these criteria. Most of the remainder have been treated too recently for adequate followup and a few had insufficient pathologic studies for inclusion in this evaluation. There were 13 men and 7 women, ranging in age at initiation of therapy from 45 to 87 years (median 60 years). Previous treatment included only transurethral resection of the bladder in 16 individuals, while 4 patients had had radiation therapy to the bladder 3 to 16 years before chemotherapy. Ten patients received only thio-tepa and 4 received only mitomycin C. Of the remaining 6 patients 4 were treated with thio-tepa followed by mitomycin C and 2 Accepted for publication November 26, 1980.
received mitomycin C followed by thio-tepa. Thio-tepa was given in 30 to 60 mg. doses instilled each week for the first 4 weeks and monthly thereafter. The total dose varied from 210 to 1,625 mg., depending upon treatment response and patient status. Mitomycin C was administered in 40 mg. doses each week for 8 weeks. Patients were evaluated at 12 weeks and continued on a monthly schedule if a complete response was recorded cystoscopically. These individuals received between 240 and 800 mg. of the drug. Patients were followed at regular intervals with cystoscopy, urinary cytology studies and bladder biopsies. Duration of observation after treatment varied from 7 to 62 months, with a mean of 22.8 months. Tumors documented during followup were termed recurrences, although they may well have been new occurrences or even persistent primary neoplasms. With few exceptions, either cystoscopic urine or voided urine plus bladder washings were obtained at each followup visit. Specimens were processed fresh within 4 hours of collection onto Millipore filters for cytologic evaluation. The cytologic specimens were evaluated for total number of cells on the filters, percentage of degeneration, and the number of multinucleated and atypical cells, as well as the presence of malignant or premalignant elements. For this analysis 268 specimens were examined: 86 had been obtained before and 182 after the initiation of topical chemotherapy (table 1). An average of 4 urinary samples per patient (range O to 18) was submitted before treatment was begun. During followup an average of 9 cytology studies per patient was examined. Biopsies were performed when any abnormality was seen cystoscopically and/or cytology studies indicated that a tumor was present. The specimens were processed according to stand-ard histologic techniques. Sections were examined for diagnostic interpretation, denudation, inflammation, edema and fibrosis. Denudation was semiquantitated by determining the percentage of epithelial surfaces not covered by urothelial cells for each specimen and combining the numbers to calculate total denudation for all biopsies received at a single followup visit. In this way a single value could be expressed even when multiple biopsies were performed. Inflammation, edema and fibrosis were recorded on a semiquantitative scale where O indicates feature absent, ± indicates feature identified only after thorough examination, + indicates feature identified easily and ++ indicates feature prominent or of marked degree. There were 277 samples available for this analysis: 115 obtained before and 162 collected after the initiation of therapy (table 1). Thus, comparisons for drug effect could be made among specimens
461
462
MURPHY, SOLOWAY AND FINEBAUM
obtained before and after chemotherapy in the same patient. For the purpose of this study all pathologic specimens, whether obtained before, during or after treatment, were re-evaluated without knowledge of clinical findings, treatment status or correlative pathologic information. RESULTS
As expected, there was considerable variation in the pathologic findings among specimens. Generally, however, the variation from one sample to another in any 1 individual was less than that of specimens from different patients. Edema and inflammation were constant features of the histologic specimens (inflammation was almost always rated ± to + and edema was judged + on the semiquantitative scale used) and did not distinguish samples collected before therapy from those obtained after the initiation of treatment. Fibrosis rarely was observed and was judged ± when recorded. Over-all, the degree of denudation averaged 14 per cent in biopsies collected before treatment and 37 per cent in specimens obtained during therapy. In 11 of the 18 cases suitable for this comparison the variation between specimens obtained before and during therapy was relatively small (table 2). However, great differences occurred in 7 cases (fig. 1). Among these 7 individuals denudation of samples obtained before treatment ranged from O to 25 per cent compared to 45 to 80 per cent among biopsies collected afterwards. However, there was no relationship between percentage epithelial denudation and total dosage of drug received. Since all patients were treated at the same intervals correlations between amount of denudation and frequency of drug exposure could not be made. Correlations between the animal model and humans were somewhat less positive for the cytologic samples. Specimens obtained before and during treatment were available in 14 of 20 cases. Of these 14 patients differences in cellular preservation TABLE
among samples collected from the same patients occurred in 9. In 7 of these 9 patients a greater percentage of preserved elements were counted in samples obtained after initiation of drug therapy (table 2). The number of exfoliated cells, expressed as a function of cells appearing on consistently prepared membrane filters, was more variable. On the whole, these patient data confirm observations made in the animal model. Nevertheless, they should be interpreted with caution, since they reflect averaging of cell counts on numerous samples and cannot be controlled totally for factors related to multiple instrumentations, surgical procedures, nonspecific inflammation and variable host responses of a genetically heterogeneous species such as man. As in the experimental system cytologic changes that might be interpreted as drug effect were unusual and distinguished easily from the morphologic changes of neoplasia. Multinucleated cells were observed in 69 specimens from 14 patients. These cells occurred in pre-therapy and post-therapy samples, and were confined to superficial (umbrella) cells (fig. 2). More importantly, cells with large, atypical nuclei were identified in only 10 specimens from 6 individuals. Of the thousands of urothelial cells examined only 17 such elements were observed and all appeared in samples obtained after treatment. In 15 of 17 samples the host cell could be identified by its large size and low nuclear:cytoplasmic ratio as a superficial cell, so that morphological changes that might be confused with malignant tumor cells were present in only 2 instances (fig. 3). Although these cells lacked the features of malignancy they exhibited no morphologic characteristics that would enable one to determine whether they were secondary to instrumentation, nonspecific inflammation or reaction to the drugs.
1. Specimens examined before and after initiation of topical
chemotherapy Histology
Total No. specimens Mean No. specimens per pt. (range)
Cytology
Before
After
Before
After
115 6 (1-28)
162 8 (0-35)*
86 4 (0-18)
182 9 (1-32)
• One patient had no biopsies during followup because cystoscopy performed 3 times and cytology studies performed 6 times were unequivocally negative. TABLE
2. Histologic and cytologic changes among patients treated
with topical chemotherapy Before Therapy More denudation More preservation More exfoliation
After Therapy
.No Change
7 7
11
2 3
4
7
0
5
FIG. 1. Denuded urothelium after thio-tepa administration. Few remaining epithelial cells are degenerated partially. Biopsies such as this are common after topical chemotherapy, making histologic evaluation of therapeutic effectiveness difficult. H & E, reduced from X320.
FIG. 2. Multinucleated cells occurring after thio-tepa administration. Similar cells can be observed after other types of stimuli and are not specific for drug effect. A, reduced from X320. B, reduced from X512.
463
TOPICAL CHEMOTHERAPY FOR SUPERFICIAL BLADDER CANCER
•
.
B.
•
•
•• •
• 1-
•
'.\
\
•
II
-·
. ••
•
.
..
ti
• •.
Ill
• .
Ill
•
"
Fm. 3. Atypical urothelial cells after mitomycin C administration. A, despite coarse chromatin and prominent nucleoli abundant cytoplasm and low nuclear:cytoplasmic ratio mark these elements as superficial and benign. B, cell is more difficult to classify. Fortunately, such cells are rare after topical chemotherapy. Reduced from X512.
Fm. 4. Urinary cytology studies reveal cells of low and high grades of malignancy. A, low grade disease. Note large nuclei, high nuclear:cytoplasmic ratio, evenly distributed but finely granular chromatin and nuclear notching. B, high grade disease. Similar changes occur but chromatin is coarse and distributed irregularly. Nucleoli are prominent. Reduced from X512.
In this study cytology was a valuable procedure in the examination of patients with superficial bladder cancer (fig. 4). During followup biopsies were positive for tumor in 14 individuals. Cytology studies had been performed before biopsy in 13. In 7 of the 13 patients (53.8 per cent) a positive cytology study preceded the first positive biopsy, in 3 (23.1 per cent) it was coincident and in only 3 it was negative. Tumor cells appeared in the urine an average of 14 months (range 1 to 25 months) before the histologic documentation. One patient had a positive cytology study before a second recurrence. If this is considered the figures are even more impressive, with a cytologic diagnosis preceding histologic confirmation in 8 of 13 cases (61.5 per cent) and negative cytology studies in only 2 (15.4 per cent). Conversely, cytology studies were positive in 14 of 20 cases during followup. In 11 of these 14 patients tumor had been documented histologically by this writing. Two of the 3 individuals with negative biopsies had grade III tumors initially and all had urothelial abnormalities endoscopically. During the treatment interval 10 of the 20 patients had histologically documented.recurrences (table 3). Of the remaining 10 individuals 4 had positive biopsies >6 months after topical chemotherapy had been discontinued owing to persistent tumor, documented cytologically and/or endoscopically. In 3 additional cases neoplastic cells were observed in urinary specimens during therapy when biopsies contained either denuded or reactive/reparative epithelium. In 2 of these 3 cases tumors appeared cystoscopically, while equivocal endoscopic findings were recorded in the third. Only 3 patients remained free of tumor throughout the study interval, although at any single moment as many as 50 per cent had no evidence of disease (table 4). The total dosage of thio-tepa and/or mitomycin C received at the time recurrence was documented is
TABLE
3. Recurrence after topical chemotherapy
Biopsy pos. Biopsy pos. * Cytology pos., biopsy neg. Cytology neg., biopsy neg.
No. Pts.
Total Dose (mg.)
Interval to Recurrence (mos.)
10 4 3 3
240-1,625 240-640 320-990 210-740
1-44 28-59 4-20 8-41t
* Positive biopsy >6 months after treatment stopped. Three of 4 patients had positive cytology studies during treatment. t No recurrence for duration of followup.
TABLE 4. Patient response to chemotherapy-results of multiple examinations during followup compared to single examination at last visit
No disease: Alive Dead Tumor: Alive Dead Totals
During Followup
Last Visit
2 1
8 2
14
8 2 20
-1. 20
shown in table 3. Recurrences were noted in some cases after only 240 mg. thio-tepa (210 mg. mitomycin C), while in others new tumors were recognized only after much larger total dosages of drugs. There was no consistent relationship between total dosage of drugs and reappearance of tumors. Similar results were observed when the interval to recurrence was considered. Only 1 patient had a new tumor of a higher cytologic grade than the original neoplasm.
464
MURPHY, SOLOWAY AND FINEBAUM DISCUSSION
Thio-tepa and mitomycin C are the most widely used topical chemotherapeutic drugs for the treatment of superficial bladder cancer.5- 10 Both are alkylating agents that act in vitro by interfering with deoxyribonucleic acid synthesis. Previous studies in experimental animals have indicated that these chemicals are similar in their effects on mammalian urothelium. 4 The morphologic changes produced apparently are toxic rather than metabolic and are manifested initially by increased exfoliation of well preserved cells with denudation of the bladder epithelium. After 48 hours denudation persists but fewer, more degenerated elements appear in the urine. Cells with nuclear changes that cannot be attributed to the neoplastic process occur rarely and, when present, they are confined virtually to superficial (umbrella) cells. 2 The data reported herein indicate that similar morphologic changes can be observed in humans. Inflammation and edema were constant histological changes but the severity of this host response was not related to type of drug, dosage or duration of treatment. Although denudation often was extensive fibrosis rarely was observed, indicating that scarring and hematuria from nonre-epithelialized mucosa are not predictable sequelae of long-term therapy. Cytologic examination of urinary samples is a valuable adjunct to cystoscopy and biopsy during followup of patients treated with topical chemotherapeutic agents. The denuding effect of the drugs makes recurrence difficult to detect cystoscopically and to document histologically. Urinary cytology may include cells from a large area of urothelium and reveal tumors not sufficiently large for endoscopic sampling and histologic diagnosis. Positive cytology studies often precede positive biopsies by many months and can provide important information concerning the function of topical chemotherapeutic drugs. 11 Most previous reports regarding the clinical benefits of topical chemotherapy have not included regular examination of urinary samples. 10 In this study had regular followup with urinary cytology studies not been done chemotherapy would have seemed to render 50 per cent of patients free of tumor throughout the treatment interval. In fact, only 15 per cent of the subjects were free of tumor during the entire followup interval. N eoplasia was recognized only in the urinary cytology studies during intravesical chemotherapy in 6 individuals and antedated a positive biopsy during the study interval in 3. Interestingly, in these 3 patients topical chemotherapy had been discontinued, perhaps allowing a previously denuded epithelium to develop clinically recognizable lesions. In humans, as in experimental animals, topical chemotherapy with thio-tepa and/or mitomycin C apparently does not prevent the eventual growth of urothelial carcinoma. 3 Recurrent tumors, documented endoscopically, cytologically or histologically, were identified in 17 of 20 patients at some time during the interval of observation (table 4). Four individuals had histologic confirmation of tumors long after topical therapy had been discontinued but the drugs were stopped because of positive urinary cytology in 3. Although our observations in humans and animals indicate that neither thio-tepa nor mitomycin C completely eradicates superficial cancers it would be unjustifiable to conclude that topical chemotherapy provides no clinical benefit. The ability of these chemicals to suppress tumor de-
velopment, inhibit progression and delay recurrence is an important subject not evaluable by the design of this study. Beneficial effects in these areas are supported by clinical reports, 5-10 previous experimental data3 and the facts that only 1 of our patients had a tumor of higher cytologic grade and that at any single moment (for example at the last followup visit) a significant proportion of patients were alive with no evidence of disease (table 4). REFERENCES 1. Murphy, W. M., Soloway, M. S. and Lin, C. J.: Morphologic effects of thio-TEPA on mouse urothelium. Acta Cytol., 21: 701, 1977.
2. Murphy, W. M., Soloway, M. S. and Lin, C. J.: Morphologic effects of thio-TEPA on mammalian urothelium. Changes in abnormal cells. Acta Cytol., 22: 550, 1978. 3. Murphy, W. M. and Soloway, M. S.: The effect of thio-tepa on developing and established mammalian bladder tumors. Cancer, 45: 870, 1980. 4. Murphy, W. M., Soloway, M. S. and Crabtree, W. N.: The morphologic effects of mitomycin C in mammalian urinary bladder. Cancer, 47: 2567, 1981. 5. Veenema, R. J., Dean, A. L., Jr., Roberts, M., Fingerhut, B., Chowhury, B. K. and Tarassoly, H.: Bladder carcinoma treated by direct instillation of thio-tepa. J. Urol., 88: 60, 1962. 6. Drew, J. E. and Marshall, V. F.: The effects of topical thiotepa on the recurrence rate of superficial bladder cancers. J. Urol., 99: 740, 1968. 7. Byar, D. and Blackard, C.: Comparisons of placebo, pyridoxine, and topical thiotepa in preventing recurrence of stage I bladder cancer. Urology, 10: 556, 1977. 8. Mishina, T., Oda, K., Murata, S., Ooe, H., Mori, Y. and Takahashi, T.: Mitomycin C bladder instillation therapy for bladder tumors. J. Urol., 114: 217, 1975. 9. Bracken, R. B. and Johnson, D.: Treatment of multiple superficial tumors of the urinary bladder with intravesical mitomycin C. In: Mitomycin C: Current Status and New Developments. Edited by S. K. Carter and S. T. Crooke. New York: Academic Press, pp. 205-211, 1979. 10. Soloway, M. S.: Rationale for intensive intravesical chemotherapy for superficial bladder cancer. J. Urol., 123: 461, 1980. 11. Esposti, P. L., Moberger, G. and Zajicek, J.: The cytologic diagnosis of transitional cell tumors of the urinary bladder and its histologic basis. A study of 567 cases of urinary-tract disorder including 170 untreated and 182 irradiated bladder tumors. Acta Cytol., 14: 145, 1970. EDITORIAL COMMENT These authors extended earlier studies of the morphologic effects of topical chemotherapy on the urothelium in animals to humans with superficial bladder cancer. Although specific morphologic changes associated with the topical application of thio-tepa and mitomycin C were not delineated the results from humans showed a higher percentage of exfoliation of urothelium with denudation of stroma and multinucleation of superficial cells in specimens taken after than before initiation of therapy. This study supports the value of sequential cytologic studies of urine as a procedure to follow the course of vesical cancer after topical chemotherapy. Additionally, the findings tend to dispell the concern of many that fibrosis results from multiple bladder biopsies repeated at frequent intervals. Marvin A. Jackson Department of Pathology Howard University College of Medicine Washington, D. C.
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright© 1981 by The Williams & Wilkins Co.
PATHOLOGICAL CHANGES ASSOCIATED WITH TOPICAL CHEMOTHERAPY FOR SUPERFICIAL BLADDER CANCER WILLIAM M. MURPHY, MARKS. SOLOWAY
AND
PAMELA J. FINEBAUM
From the Department of Pathology, Baptist Memorial Hospital and Department of Urology, University of Tennessee Center for Health Sciences, Memphis, Tennessee
ABSTRACT
With use of previous observations in experimental animals as a basis for comparison the cytologic and histologic changes in human patients who underwent topical chemotherapy for superficial bladder cancer were documented. Despite the potential for inhibition of deoxyribonucleic acid replication these drugs apparently act in vivo as toxic substances, causing increased exfoliation with denudation of papillary and/o:r flat urothelium. Multinucleation was common but confined to superficial cells. Atypical cells, such as those observed after systemic chemotherapy with cyclophosphamide, rarely were present and could be distinguished readily from neoplastic elements. Although topical chemotherapy may suppress tumor growth and progression it apparently does not eradicate the neoplastic process. During the last 5 years we have been interested in the morphologic effects of topical chemotherapy for superficial carcinoma of the bladder. To avoid the multiple variables inherent in the evaluation of patients we examined first the changes in experimental animals, in which conditions of tumorigenesis and treatment could be controlled. 1- 4 From these studies various observations were made. 1) The morphologic changes, cytologic and histologic, of triethylenethiophosphoramide (thio-tepa) and mitomycin C on mammalian bladders are similar. 2) Both of these alkylating agents appear to act in vivo primarily as toxic substances rather than metabolic inhibitors of deoxyribonucleic acid replication, causing increased exfoliation of preserved cells with secondary denudation of urothelium. 3) Multinucleated cells and cells with atypical nuclei are unusual after topical chemotherapy. Although more prominent in treated animals these changes are not specific for drug effect and can be seen after catheterization with instillation of saline alone. They almost always occur in superficial (umbrella) cells. More importantly, these changes can be distinguished easily from malignant tumor cells. 4) Neither thio-tepa nor mitomycin C prevents eventual tumor growth in F ANFTinduced murine bladder cancer, although they may delay recurrence and inhibit progression to higher grade and/or stage neoplasms. With this experience in an experimental system as a basis for comparison we herein determine whether similar observations can be documented in humans. MATERIALS AND METHODS
The patients included in this study had documented superficial bladder carcinoma, had been treated with topical thiotepa and/or mitomycin C, and had had sufficient followup with endoscopic, histologic and cytologic examinations to determine the pathologic changes associated with chemotherapy. Of the 35 patients who have been treated at our institutions 20 met these criteria. Most of the remainder have been treated too recently for adequate followup and a few had insufficient pathologic studies for inclusion in this evaluation. There were 13 men and 7 women, ranging in age at initiation of therapy from 45 to 87 years (median 60 years). Previous treatment included only transurethral resection of the bladder in 16 individuals, while 4 patients had had radiation therapy to the bladder 3 to 16 years before chemotherapy. Ten patients received only thio-tepa and 4 received only mitomycin C. Of the remaining 6 patients 4 were treated with thio-tepa followed by mitomycin C and 2 Accepted for publication November 26, 1980.
received mitomycin C followed by thio-tepa. Thio-tepa was given in 30 to 60 mg. doses instilled each week for the first 4 weeks and monthly thereafter. The total dose varied from 210 to 1,625 mg., depending upon treatment response and patient status. Mitomycin C was administered in 40 mg. doses each week for 8 weeks. Patients were evaluated at 12 weeks and continued on a monthly schedule if a complete response was recorded cystoscopically. These individuals received between 240 and 800 mg. of the drug. Patients were followed at regular intervals with cystoscopy, urinary cytology studies and bladder biopsies. Duration of observation after treatment varied from 7 to 62 months, with a mean of 22.8 months. Tumors documented during followup were termed recurrences, although they may well have been new occurrences or even persistent primary neoplasms. With few exceptions, either cystoscopic urine or voided urine plus bladder washings were obtained at each followup visit. Specimens were processed fresh within 4 hours of collection onto Millipore filters for cytologic evaluation. The cytologic specimens were evaluated for total number of cells on the filters, percentage of degeneration, and the number of multinucleated and atypical cells, as well as the presence of malignant or premalignant elements. For this analysis 268 specimens were examined: 86 had been obtained before and 182 after the initiation of topical chemotherapy (table 1). An average of 4 urinary samples per patient (range O to 18) was submitted before treatment was begun. During followup an average of 9 cytology studies per patient was examined. Biopsies were performed when any abnormality was seen cystoscopically and/or cytology studies indicated that a tumor was present. The specimens were processed according to stand-ard histologic techniques. Sections were examined for diagnostic interpretation, denudation, inflammation, edema and fibrosis. Denudation was semiquantitated by determining the percentage of epithelial surfaces not covered by urothelial cells for each specimen and combining the numbers to calculate total denudation for all biopsies received at a single followup visit. In this way a single value could be expressed even when multiple biopsies were performed. Inflammation, edema and fibrosis were recorded on a semiquantitative scale where O indicates feature absent, ± indicates feature identified only after thorough examination, + indicates feature identified easily and ++ indicates feature prominent or of marked degree. There were 277 samples available for this analysis: 115 obtained before and 162 collected after the initiation of therapy (table 1). Thus, comparisons for drug effect could be made among specimens
461
462
MURPHY, SOLOWAY AND FINEBAUM
obtained before and after chemotherapy in the same patient. For the purpose of this study all pathologic specimens, whether obtained before, during or after treatment, were re-evaluated without knowledge of clinical findings, treatment status or correlative pathologic information. RESULTS
As expected, there was considerable variation in the pathologic findings among specimens. Generally, however, the variation from one sample to another in any 1 individual was less than that of specimens from different patients. Edema and inflammation were constant features of the histologic specimens (inflammation was almost always rated ± to + and edema was judged + on the semiquantitative scale used) and did not distinguish samples collected before therapy from those obtained after the initiation of treatment. Fibrosis rarely was observed and was judged ± when recorded. Over-all, the degree of denudation averaged 14 per cent in biopsies collected before treatment and 37 per cent in specimens obtained during therapy. In 11 of the 18 cases suitable for this comparison the variation between specimens obtained before and during therapy was relatively small (table 2). However, great differences occurred in 7 cases (fig. 1). Among these 7 individuals denudation of samples obtained before treatment ranged from O to 25 per cent compared to 45 to 80 per cent among biopsies collected afterwards. However, there was no relationship between percentage epithelial denudation and total dosage of drug received. Since all patients were treated at the same intervals correlations between amount of denudation and frequency of drug exposure could not be made. Correlations between the animal model and humans were somewhat less positive for the cytologic samples. Specimens obtained before and during treatment were available in 14 of 20 cases. Of these 14 patients differences in cellular preservation TABLE
among samples collected from the same patients occurred in 9. In 7 of these 9 patients a greater percentage of preserved elements were counted in samples obtained after initiation of drug therapy (table 2). The number of exfoliated cells, expressed as a function of cells appearing on consistently prepared membrane filters, was more variable. On the whole, these patient data confirm observations made in the animal model. Nevertheless, they should be interpreted with caution, since they reflect averaging of cell counts on numerous samples and cannot be controlled totally for factors related to multiple instrumentations, surgical procedures, nonspecific inflammation and variable host responses of a genetically heterogeneous species such as man. As in the experimental system cytologic changes that might be interpreted as drug effect were unusual and distinguished easily from the morphologic changes of neoplasia. Multinucleated cells were observed in 69 specimens from 14 patients. These cells occurred in pre-therapy and post-therapy samples, and were confined to superficial (umbrella) cells (fig. 2). More importantly, cells with large, atypical nuclei were identified in only 10 specimens from 6 individuals. Of the thousands of urothelial cells examined only 17 such elements were observed and all appeared in samples obtained after treatment. In 15 of 17 samples the host cell could be identified by its large size and low nuclear:cytoplasmic ratio as a superficial cell, so that morphological changes that might be confused with malignant tumor cells were present in only 2 instances (fig. 3). Although these cells lacked the features of malignancy they exhibited no morphologic characteristics that would enable one to determine whether they were secondary to instrumentation, nonspecific inflammation or reaction to the drugs.
1. Specimens examined before and after initiation of topical
chemotherapy Histology
Total No. specimens Mean No. specimens per pt. (range)
Cytology
Before
After
Before
After
115 6 (1-28)
162 8 (0-35)*
86 4 (0-18)
182 9 (1-32)
• One patient had no biopsies during followup because cystoscopy performed 3 times and cytology studies performed 6 times were unequivocally negative. TABLE
2. Histologic and cytologic changes among patients treated
with topical chemotherapy Before Therapy More denudation More preservation More exfoliation
After Therapy
.No Change
7 7
11
2 3
4
7
0
5
FIG. 1. Denuded urothelium after thio-tepa administration. Few remaining epithelial cells are degenerated partially. Biopsies such as this are common after topical chemotherapy, making histologic evaluation of therapeutic effectiveness difficult. H & E, reduced from X320.
FIG. 2. Multinucleated cells occurring after thio-tepa administration. Similar cells can be observed after other types of stimuli and are not specific for drug effect. A, reduced from X320. B, reduced from X512.
463
TOPICAL CHEMOTHERAPY FOR SUPERFICIAL BLADDER CANCER
•
.
B.
•
•
•• •
• 1-
•
'.\
\
•
II
-·
. ••
•
.
..
ti
• •.
Ill
• .
Ill
•
"
Fm. 3. Atypical urothelial cells after mitomycin C administration. A, despite coarse chromatin and prominent nucleoli abundant cytoplasm and low nuclear:cytoplasmic ratio mark these elements as superficial and benign. B, cell is more difficult to classify. Fortunately, such cells are rare after topical chemotherapy. Reduced from X512.
Fm. 4. Urinary cytology studies reveal cells of low and high grades of malignancy. A, low grade disease. Note large nuclei, high nuclear:cytoplasmic ratio, evenly distributed but finely granular chromatin and nuclear notching. B, high grade disease. Similar changes occur but chromatin is coarse and distributed irregularly. Nucleoli are prominent. Reduced from X512.
In this study cytology was a valuable procedure in the examination of patients with superficial bladder cancer (fig. 4). During followup biopsies were positive for tumor in 14 individuals. Cytology studies had been performed before biopsy in 13. In 7 of the 13 patients (53.8 per cent) a positive cytology study preceded the first positive biopsy, in 3 (23.1 per cent) it was coincident and in only 3 it was negative. Tumor cells appeared in the urine an average of 14 months (range 1 to 25 months) before the histologic documentation. One patient had a positive cytology study before a second recurrence. If this is considered the figures are even more impressive, with a cytologic diagnosis preceding histologic confirmation in 8 of 13 cases (61.5 per cent) and negative cytology studies in only 2 (15.4 per cent). Conversely, cytology studies were positive in 14 of 20 cases during followup. In 11 of these 14 patients tumor had been documented histologically by this writing. Two of the 3 individuals with negative biopsies had grade III tumors initially and all had urothelial abnormalities endoscopically. During the treatment interval 10 of the 20 patients had histologically documented.recurrences (table 3). Of the remaining 10 individuals 4 had positive biopsies >6 months after topical chemotherapy had been discontinued owing to persistent tumor, documented cytologically and/or endoscopically. In 3 additional cases neoplastic cells were observed in urinary specimens during therapy when biopsies contained either denuded or reactive/reparative epithelium. In 2 of these 3 cases tumors appeared cystoscopically, while equivocal endoscopic findings were recorded in the third. Only 3 patients remained free of tumor throughout the study interval, although at any single moment as many as 50 per cent had no evidence of disease (table 4). The total dosage of thio-tepa and/or mitomycin C received at the time recurrence was documented is
TABLE
3. Recurrence after topical chemotherapy
Biopsy pos. Biopsy pos. * Cytology pos., biopsy neg. Cytology neg., biopsy neg.
No. Pts.
Total Dose (mg.)
Interval to Recurrence (mos.)
10 4 3 3
240-1,625 240-640 320-990 210-740
1-44 28-59 4-20 8-41t
* Positive biopsy >6 months after treatment stopped. Three of 4 patients had positive cytology studies during treatment. t No recurrence for duration of followup.
TABLE 4. Patient response to chemotherapy-results of multiple examinations during followup compared to single examination at last visit
No disease: Alive Dead Tumor: Alive Dead Totals
During Followup
Last Visit
2 1
8 2
14
8 2 20
-1. 20
shown in table 3. Recurrences were noted in some cases after only 240 mg. thio-tepa (210 mg. mitomycin C), while in others new tumors were recognized only after much larger total dosages of drugs. There was no consistent relationship between total dosage of drugs and reappearance of tumors. Similar results were observed when the interval to recurrence was considered. Only 1 patient had a new tumor of a higher cytologic grade than the original neoplasm.
464
MURPHY, SOLOWAY AND FINEBAUM DISCUSSION
Thio-tepa and mitomycin C are the most widely used topical chemotherapeutic drugs for the treatment of superficial bladder cancer.5- 10 Both are alkylating agents that act in vitro by interfering with deoxyribonucleic acid synthesis. Previous studies in experimental animals have indicated that these chemicals are similar in their effects on mammalian urothelium. 4 The morphologic changes produced apparently are toxic rather than metabolic and are manifested initially by increased exfoliation of well preserved cells with denudation of the bladder epithelium. After 48 hours denudation persists but fewer, more degenerated elements appear in the urine. Cells with nuclear changes that cannot be attributed to the neoplastic process occur rarely and, when present, they are confined virtually to superficial (umbrella) cells. 2 The data reported herein indicate that similar morphologic changes can be observed in humans. Inflammation and edema were constant histological changes but the severity of this host response was not related to type of drug, dosage or duration of treatment. Although denudation often was extensive fibrosis rarely was observed, indicating that scarring and hematuria from nonre-epithelialized mucosa are not predictable sequelae of long-term therapy. Cytologic examination of urinary samples is a valuable adjunct to cystoscopy and biopsy during followup of patients treated with topical chemotherapeutic agents. The denuding effect of the drugs makes recurrence difficult to detect cystoscopically and to document histologically. Urinary cytology may include cells from a large area of urothelium and reveal tumors not sufficiently large for endoscopic sampling and histologic diagnosis. Positive cytology studies often precede positive biopsies by many months and can provide important information concerning the function of topical chemotherapeutic drugs. 11 Most previous reports regarding the clinical benefits of topical chemotherapy have not included regular examination of urinary samples. 10 In this study had regular followup with urinary cytology studies not been done chemotherapy would have seemed to render 50 per cent of patients free of tumor throughout the treatment interval. In fact, only 15 per cent of the subjects were free of tumor during the entire followup interval. N eoplasia was recognized only in the urinary cytology studies during intravesical chemotherapy in 6 individuals and antedated a positive biopsy during the study interval in 3. Interestingly, in these 3 patients topical chemotherapy had been discontinued, perhaps allowing a previously denuded epithelium to develop clinically recognizable lesions. In humans, as in experimental animals, topical chemotherapy with thio-tepa and/or mitomycin C apparently does not prevent the eventual growth of urothelial carcinoma. 3 Recurrent tumors, documented endoscopically, cytologically or histologically, were identified in 17 of 20 patients at some time during the interval of observation (table 4). Four individuals had histologic confirmation of tumors long after topical therapy had been discontinued but the drugs were stopped because of positive urinary cytology in 3. Although our observations in humans and animals indicate that neither thio-tepa nor mitomycin C completely eradicates superficial cancers it would be unjustifiable to conclude that topical chemotherapy provides no clinical benefit. The ability of these chemicals to suppress tumor de-
velopment, inhibit progression and delay recurrence is an important subject not evaluable by the design of this study. Beneficial effects in these areas are supported by clinical reports, 5-10 previous experimental data3 and the facts that only 1 of our patients had a tumor of higher cytologic grade and that at any single moment (for example at the last followup visit) a significant proportion of patients were alive with no evidence of disease (table 4). REFERENCES 1. Murphy, W. M., Soloway, M. S. and Lin, C. J.: Morphologic effects of thio-TEPA on mouse urothelium. Acta Cytol., 21: 701, 1977.
2. Murphy, W. M., Soloway, M. S. and Lin, C. J.: Morphologic effects of thio-TEPA on mammalian urothelium. Changes in abnormal cells. Acta Cytol., 22: 550, 1978. 3. Murphy, W. M. and Soloway, M. S.: The effect of thio-tepa on developing and established mammalian bladder tumors. Cancer, 45: 870, 1980. 4. Murphy, W. M., Soloway, M. S. and Crabtree, W. N.: The morphologic effects of mitomycin C in mammalian urinary bladder. Cancer, 47: 2567, 1981. 5. Veenema, R. J., Dean, A. L., Jr., Roberts, M., Fingerhut, B., Chowhury, B. K. and Tarassoly, H.: Bladder carcinoma treated by direct instillation of thio-tepa. J. Urol., 88: 60, 1962. 6. Drew, J. E. and Marshall, V. F.: The effects of topical thiotepa on the recurrence rate of superficial bladder cancers. J. Urol., 99: 740, 1968. 7. Byar, D. and Blackard, C.: Comparisons of placebo, pyridoxine, and topical thiotepa in preventing recurrence of stage I bladder cancer. Urology, 10: 556, 1977. 8. Mishina, T., Oda, K., Murata, S., Ooe, H., Mori, Y. and Takahashi, T.: Mitomycin C bladder instillation therapy for bladder tumors. J. Urol., 114: 217, 1975. 9. Bracken, R. B. and Johnson, D.: Treatment of multiple superficial tumors of the urinary bladder with intravesical mitomycin C. In: Mitomycin C: Current Status and New Developments. Edited by S. K. Carter and S. T. Crooke. New York: Academic Press, pp. 205-211, 1979. 10. Soloway, M. S.: Rationale for intensive intravesical chemotherapy for superficial bladder cancer. J. Urol., 123: 461, 1980. 11. Esposti, P. L., Moberger, G. and Zajicek, J.: The cytologic diagnosis of transitional cell tumors of the urinary bladder and its histologic basis. A study of 567 cases of urinary-tract disorder including 170 untreated and 182 irradiated bladder tumors. Acta Cytol., 14: 145, 1970. EDITORIAL COMMENT These authors extended earlier studies of the morphologic effects of topical chemotherapy on the urothelium in animals to humans with superficial bladder cancer. Although specific morphologic changes associated with the topical application of thio-tepa and mitomycin C were not delineated the results from humans showed a higher percentage of exfoliation of urothelium with denudation of stroma and multinucleation of superficial cells in specimens taken after than before initiation of therapy. This study supports the value of sequential cytologic studies of urine as a procedure to follow the course of vesical cancer after topical chemotherapy. Additionally, the findings tend to dispell the concern of many that fibrosis results from multiple bladder biopsies repeated at frequent intervals. Marvin A. Jackson Department of Pathology Howard University College of Medicine Washington, D. C.