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DOES TOPICAL INSTILLATION THERAPY INFLUENCE CHROMOSOMAL ABERRATIONS IN SUPERFICIAL BLADDER CANCER?
0022-5347/98/1591-0265$03.00/0 THE JOURNAL OF UROLOGY Copyright 0 1998 by AMERICAN UROLOGICAL ASS~CIATION, INC.
Vol. 159,266-269. January 1998 Printed in U S A .
DOES TOPICAL INSTILLATION THERAPY INFLUENCE CHROMOSOMAL ABERRATIONS IN SUPERFICIAL BLADDER CANCER? ARMIN PYCHA,* CHRISTINE MIAN, JOHANN HOFBAUER, ANDREA HAITEL, HELENE WIENER AND MICHAEL MARBERGER From the Department of Urology and Department of Pathology, University of Vienna, Vienna, Austria
ABSTRACT
Purpose: Patients with a high risk for superficial bladder cancer are treated by topical immunoor chemotherapy after transurethral resection to reduce the chance of recurrence and/or progression. The aim of this study was to analyse if cytogenetical abnormalities, which are known to be constantly related to bladder cancer, are modified or eliminated by topical immuno- or chemotherapy. Materials and Methods: Using fluorescence in situ hybridization (FISH), the influence of topical instillation therapy with Bacillus Calmette-Guerin (BCG) and Mitomycin C (MMC) on numerical aberrations of chromosomes 7,9and 17 was investigated in 25 patients with transitional cell cancer (TCC) of the bladder. Data were compared with histological and clinical outcome. Fifteen TCC patients with similar histological criteria without instillation therapy served as controls. Median follow-up was 30 2 2 months. Results: After BCG treatment 10 of 15 patients (66.6%)developed recurrent and 2/15(13.3%) progressive disease. Three of 15 patients (20.0%)had no evidence of disease. Numerical aberrations did not change in 8 of the 15 BCG patients (53.3%) and changed to a more aggressive pattern in 40.0% (6/15).Five of 10 MMC treated patients (50.0%) developed a recurrent tumor, 2/10 (20.0%) progressed and 3/10(30.0%)had no evidence of disease. Four of 10 (40.0%)of these patients showed stable and 5/10 (50.0%)progressive chromosomal patterns. Only one patient in each group with primary chromosomal alterations changed to a regular diploid chromosomal pattern after therapy according to a complete clinical remission. Conclusion: Even though topical immuno- and chemotherapy may be useful to delay recurrence and progression, chromosomal patterns remain basically unstable. KEYWORDS: bladder cancer, intravesical chemotherapy, BCG therapy, chromosomal aberrations, FISH
%
Patients with a high risk of recurrent and progressive superficial transitional cell cancer of the bladder are in general treated with an additional topical immuno- or chemotherapeutical instillation after transurethral resection (TUR),since topical therapy is known to reduce recurrenceand progression rates.1-6 Oncogenesis of bladder cancer is described as a multiple step genetic alteration.7 It is also known that numerical aberrations of chromosomes 7, 9 and 17 are involved in the history of superficial and invasive cancer.7-11 If chromosomal alterations are postulated as the cause of malignant changes, successful therapy should theoretically lead to the disappearance or at least modification of chromosomal aberration patterns. The aim of this study was to determine if cytogenetical abnormalities known to be constantly related to bladder cancer are modified or eliminated by topical immuno- or chemotherapy. MATERIALS A N D METHODS
Forty patients (16 female, 24 male, average 71 years, range 39 to 85 years) with histologically confirmed transitional cell cancer (TCC) of the bladder were included in this study. At study entry all patients underwent TUR. Six patients showed first manifestation of multifocal pTa tumours, fifteen patients had recurrent but solitary pTa tumours, and 19
patients had stage pT1 tumours. Tumours were graded G1 in 10 cases, G2 in 23 cases and G3 in 7 cases (tables 1and 2). Prior to TUR bladder washings were obtained in all cases. During follow-up 25 patients had either topical instillations with MMC or they were under posttherapeutical follow up after BCG or MMC. Fifteen patients were treated with BCG, type R W M , (BCG-S, Medac, Hamburg, Germany) 2 X 10' colonies in 50 ml. solution per instillation weekly for a period of six weeks. Ten patients received 20 mg. MMC (Kyowa, Tokio, Japan) in 20 ml. solution per instillation every two weeks for 6 to 18months.12 The remaining 15 patients had no instillation therapy and formed the control group. All patients were followed for a median duration of 30 months (range 2832 months) with cystoscopy, voided urine and barbotage cytology every three months. In case of suspicious cystoscopy biopsies were taken. During follow-up chromosomal analysis was performed with every control visit. Table 2 gives data at first analysis, at 15 months and at 30 2 2 months. Bladder washings and touch preparations (imprints) of specimens were obtained at TUR in every patient. Barbotage specimens were collected immediately prior to TUR or at every follow-up cystoscopy by irrigation of the bladder with 50 ml. saline. Bladder washings were spun twice for 5 minutes at 350 g (Hettich Universal centrifuge, Germany), using a sedimentation conus the second time. At least one glass slide was air dried and stained according to the May Griinwald staining. The other slides were wet fixed (spray fixation, Merck, Germany), partly stained according to the Papanicolaou method and partly kept for further evalua-
Accepted for publication July 15, 1997. *Requests for re rints: Department of Urology, University of Vienna, Wahringer 8urt.d 18-20, 1090 Vienna, AusFa. supported by Grant 1389, BiirgermelsterfondeWen. 265
266
4 1
TOPICAL INSTILLATION THERAPY IN SUPERFICIAL BLADDER CANCER
TABLE1. Histology, pre- and posttherapeutic numerical chromosomal aberrations (NCA), kind of therapy, and clinical outcome in imprints and bladder washings of 25 patients with TCC of the bladder. Followup time: 30 2 2 months Retherapeutic NCA Patient 1 2 3 4 5
6 7 8
9 10 11 12 13 14 15
16 17 18
19 20 21 22
23 24 25 NED =
TCC pTaGl multifoc. pTaGl m. pTaG2 multifoc. pTaG2 rec. pTaG2 rec. pTaG2 rec. pTlG2 pTlG2 pTlG2 pTlG2 pT1GZ pTlG2 pTlG3 pTlG3 pTlG3 pTaGl rec. pTaGl rec. pTaGl rec. pTaGZ multifoc. pTaG2 rec. pTaG2 rec. pTlG2 pTlG2 pTlG3 oTlG3 no evidence of disease.
t?
none
-9
+ 17
+7
-9
none
+7 none +7 none +7 none none none +7 +7 none +7
-9 -9 none
+7
none
none none none
-9
+17 none none none none +17 none none none +17 none none none none none none none none none none none + 17 +i7
-9
none -9 -9 -9 -9 none -9 -9
-9 -9 -9
+7
none none none +7 +7 +7
Kind of Therapy
$17
#9
-9 -9
-9 -9 -9
none
BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG MMC MMC MMC MMC MMC MMC MMC MMC MMC MMC
Posttherapeutic NCA
Clinical Outcome
t7
x9
$17
none +7 +7 none +7
-9 none
+17 +17 + 17 none none none + 17 + 17 none none none +17 none + 17 none +17 none none + 17 none none none +17 + 17 +17
-9
-9 none -9 none none -9 -9 -9 none none none none -9 -9 -9 -9 -9 none -9 none -9 none
+7 +7 none none +7 +7 +7 none +7 +7 none +7 none none +7 none none +7 +7 +7
recurrence recurrence cystitis recurrence recurrence progression progression recurrence recurrence recurrence recurrence recurrence NED cystitis recurrence progression recurrence recurrence recurrence cystitis NED cystitis progression recurrence recurrence
TABLE2. Histology, numerical chromosomal abemtwns (NCA) at first analysis and during followup time and clinical outcome in imprints and bladder washings of 15 patients (control group) with TCC of the bladder. Followup time: 30 2 2 months
1 2 3 4
5 6
7 8
9 10
11 12 13 14 15
TCC pTaGl rec. pTaGl rec. pTaGl rec. pTaG2 rec. pTaG2 rec. pTaGZ rec. pTaGZ rec. pTaG2 multifoc. pTaG2 multifoc. pTaG2 multifoc. pTlGl pTlGl pTlG2 pTlG2 pTlG3
NCA 30
NCA 15 Months
NCA a t 1. Analysis Patient
17
#9
#17
t7
#9
117
+7 none none +7 +7
-9
none
+17 none none none none none none none +17 none none +17 none +17 none
+7 none none +7 +7
none -9 -9 -9 none -9 -9 -9 none -9
+ 17 none none none + 17 none none none +17 none none
-9 -9 -9
+7
-9
none
-9
+7
-9
+7 none none
-9
+7 +7
-9 -9 none -9
none
-9
+7
none
+7 none +7 +7 none none +7 +7 none +7
tions. All stained slides were evaluated by routine exfoliative cytology and had identical cytological reports from both staining methods used. Imprint specimens were gathered immediately after TUR. Before placing biopsy material into the fixation fluid (37% formaldehyde), the resected material was rolled on or softly premed against a glass slide. Intraoperative imprints were spray fixed immediately aRer collection. To control the content of cells, one slide was stained according to the Papanicolaou method, the others were used for the detection of numerical chromosomal aberrations. TCC was staged and graded according to the UICC classiikation13 (table 1and 2). Fluorescence in situ hybridization analysis (FISH).Using FISH,specimens were evaluated for numerical aberrations on chromosome 7.9 and 17.The protocol followed in principle the method developed by pinkel et al.,14 slightly modified to shorten procedure time. Before denaturation slides were washed in 50% ethanol and PBS. Denaturation of the specimens (Formamid (Sigma, GemanyY20 x Saline Sodium Citrate (SSC)(Vysis Inc., MA, USA),75C for 5 minutes) and the directly labeled DNA target (Vysis Inc., MA, USA)was followed by hybridization. Hybridization was performed in 30 minutes at 42C. Subsequently slides were washed twice in
-9
none
+ 17
-9 -9
none +17 none
none
-?: 2
#7
#9
+7
none
none none +7 +7 +7
-9
+I +7 +7 none none
+I +7 none +7
Months
-9 -9 none -9 -9 -9 none -9 -9 none -9 -9
none
#17 +17 none none none + 17 none none +17 +17 none none +17 none + 17 none -
Clinical Outcome progression no recurrence recurrence recurrence progression recurrence recurrence progression recurrence no recurrence recurrence recurrence progression recurrence recurrence
SSC solutions of different concentrations. Counterstaining was achieved by application of 10 pl. diamidinophenylindole (DAPI) I1 Wysis Inc., MA, USA) per slide. Spots of two hundred cells were evaluated using 600-fold magnification and images were captured by a Smart Capture System (Vysis Inc., USA) mounted on a fluorescence microscope (Olympus, GerIIlany). As a control for the reliability of the method, lymphocytes of healthy probands were included in every hybridization unit, assuring diploid cells for the most part. According to previously published data8 the cutoff level for true aneusomy was established at 20% for all analysed chromosomes. Data were analysed statistically with the McNemar Chi-square test. RESULTS
Table 1 gives an overview of the histological diagnoses, patterns of analysed chromosomes, the type of topical treatment, the clinical outcome and the chromosomal patterns of the 25 analyzed patients. After treatment 10 patients (66.6%)out of the BCG treated group (group I) developed a recurrent tumour of the same
TOPICAL INSTILLATION THERAPY IN SUPERFICIAL, BLADDER CANCER
stage and grade as the primary one. Two patients (13.3%) developed progressive tumours, and 3 patients (20.0%) showed no evidence of disease, endoscopically, cytologically and histologically. Out of the mitomycin C treated group (group 11) five patients (50.0%) developed recurrent disease. Two patients (20.0%)progressed and 3 patients (30.0%)had no evidence of disease. Recurrence was never observed within the first 5 months after TUR, so that residual primary tumour can be excluded.15 Before treatment 5 patients out of group I showed a singular monosomy 9 (33.3%), 3 patients a singular trisomy 7 (20.0%), and one patient (6.6%)the combination of monosomy 9, trisomy 7 and 17. Six patients (40.0%) showed a mixed chromosomal pattern, i.e. monosomy 9 and trisomy 7 or 17,or aberrations of chromosome 7 and 17 only. Out of group I1 six patients (60.0%) had a single monosomy 9, one patient (10.0%)had aberrations in all analysed chromosomes and 3 patients (30.0%)had a mixed chromosomal aberrations pattern. After treatment 8 patients out of group 1(53.3%) showed the same numerical chromosomal aberrations as before therapy (stable pattern) and six patients (40.0%) changed to aberrations of chromosome 7 and 17 (progressive pattern). Out of group I1 four patients (40.0%) showed a stable chromosomal pattern and five patients (50%) a progressive chromosomal pattern. Only one patient (6.6%) of group I and one patient (10.0%) of group I1 had a regular diploid chromosomal pattern more than 2.5 years after therapy. In these patients the findings correlated with complete clinical remission. The other four patients without clinical and cytological evidence of disease (2 of every group) had a histologically verified unspecific cystitis caused by the BCGtherapy, but nevertheless chromosomal analysis showed persistent or progressive genetic alterations. Statistically no difference was seen for numerical aberrations of chromosomes 7 and 9 before and after therapy. In contrast the finding of trisomy 17 increased significantly after instillation (p = 0.04124).Nevertheless no differences were seen between the two kinds of therapy. At first chromosomal analysis 5 of the 15 patients (33.3%) of the control group showed a single monosomy 9, 2 patients (13.3%) a single trisomy 7 and 4 patients (26.6%) both a monosomy 9 and trisomy 7.Two patients showed aberrations of all three analyzed chromosomes and two patients a mixed chromosomal pattern. Subsequent genetic analysis during follow-up time showed a stable chromosomal pattern in 10 cases (66.6%) and a progressive chromosomal pattern in 33.3%. From the clinical point of view 2 of these 15 patients (13.3%) remained free of recurrence, nine patients (60.0%) had recurrent disease and four patients (26.6%) showed progression (table 2). The chromosomal patterns, mainly persistent or progressive genetic alterations, of the 15 patients of the control group were similar when compared with patients who underwent instillation therapy. No statistically significant difference was seen between the two groups.
267
bladder provoking an infection which induces an immunologcal response that initiates antitumour activity.2.16 T-cell :ompetence is therefore required. MMC has also been viably used for topical instillation after nesection of superficial bladder tumours. The antibiotic MMC s an alkylating agent with antitumour activity.17 It seems to )e tolerated with fewer side-effects than BCG.6 Cytotoxic :hernotherapy destroys rapidly dividing cells and is effective lnly when such cells are present at the time of administraLion.18 Previously published data suggest that BCG is more sfficient in reducing tumour recurrence than intravesical :hemotherapy.l-4.19 In addition chemotherapy appears to be more effective in low grade tumours, whereas BCG seems to be optimally effective in higher-grade tumours.' When considering the pattern of chromosomal aberrations, the patients of group I appeared to have more aggressive tumours than those in group I1 (figs. 1-3). In spite of this, no significant differences in outcome were noted between BCG and MMC in this study. No significant differences in the behaviour of chromosomal patterns in treated patients versus untreated patients were seen during follow-up. Chromosomal aberrations in both groups were already present at the first genetic analysis after TUR and was observed in both patients undergoing instillation therapy and in patients who underwent only TUR during the complete follow-up time, respectively. It seems that deterioration of the chromosomal pattern in bladder cancer is not caused by topical therapy. Reversion to diploid status and loss of chromosomal abnormalities were observed in only two patients, while another 4 patients endoscopically and histologically free of disease showed persisting genetic alterations. Chromosomal anoma-
MONOSOMY 9
DISCUSSION
Previous publications showed that monosomy 9 is an early genetic alteration in the oncogenesis of bladder cancer.7 It appears mostly as single genetic change in superficial tumourslo which recur often but progress rarely and in general have a good prognosis. Aberrations of chromosomes 7 and 17 are related to a more aggressive tumour behaviour.8-11 Tumours with numerical aberrations of these two chromosomes have a substantially higher recurrence and progression rate. A multitude of studied-6.12 describe the benefit of intravesical therapy with BCG in the treatment of carcinoma in situ, Ta and T1 tumours. The profound immunostimulatory effect of BCG led investigators to evaluate its role in the treatment of tumours. Viable bacteria are introduced into the
FIG.1. Distribution of monosomy 9 in 25 andysed patients prim to topical instillation therapy.
268
TOPICAL INSTILLATION THERAPY IN SUPERFICIAL BLADDER CANCER
TRISOMY 7
started, there must not necessarily be a phenotypic expression at the same time. "his could explain absence of recurrent disease, given the resistance of chromosomal changes. Likewise, while multistep acquisition of an aggressive genotype is generally considered the most acceptable theory of biological tumour progression, there are instances where a monoclonal origin has strongly been suggested." The large number of structural and numerical chromosomal changes present in bladder cancer enables cancer cells to biologically behave in different directions. Because of these changes it is conceivable that either cell may be or become resistant to the usual forms of therapy. Even though topical immuno- and chemotherapy may be considered useful to achieve clinical remission, numerical aberrations of the chromosomes 7,9 and 17 remained persistent in 91.7% of all analyzed cases of this study. Moreover, it is possible that aberrations of other chromosomes documented in TCC11 and not evaluated in this study may reflect a change post therapy. Complete remission has been reported in approximately 50% with a follow-up of 3-5 years: and approximately 30%with a follow-up of 10 year^.^.^ Nevertheless the significant enhancement of trisomy 17 despite topical instillation therapy shows that cytogenetical deterioration and therefore progression of disease cannot be avoided. REFERENCES
1. Lamm, D. L.:BCG in perspective: advances in the treatment of superficial bladder cancer. Eur. Urol., 27 (Suppl. 1):2, 1995. 2. RatlifF, T. L.,Ritchey, J. K., Yuan, J. J. J., Andriole, G. L. and Catalona, W. J.: T-cell subsets required for intravesical BCG immunotherapy for bladder cancer. J. Urol., 1 5 0 1018,1993. FIG. 2. Distribution of trisomy 7 in 25 andysed patients prior to 3. Soloway, M. S.:Managing superficial bladder cancer: an overtopical instillation therapy. view. Urol. (Suppl.), 40:5, 1992. 4. Vegt, P. D. J., Witjes, J. A., Witjes, W. P. J., Doesburg, W. H., Debruyne, F. M. J. and van der Meijden, A. P. M.: A randomized study of intravesical mitomycin C, Bacillus CalmetteGuerin Tice and Bacillus Calmette-GuerinRIVM treatment in pTa-pT1 papillary carcinoma and carcinoma in situ of the bladder. J. Urol., 153: 929,1995. 5. Nadler, R. B., Catalona, W. J., Hudson, M. A. and Ratliff, T. L.: Durability of the tumor-free response for intravesical Bacillus Calmette-Guerin therapy. J. Urol., 152 367, 1994. 6. Herr, H. W., Wartinger, D. D., Fair,W. R. and Oettgen, H. F.: Bacillus Calmette-Guerin therapy for superficial bladder cancer: a 10-year followup. J. Urol., 141: 1020,1992. 7. Spruck, C. H., 111, Ohneseit, P. F., Gonzales-Zulueta,M., Esrig, D., Miyao, N., Tsai Lerner, S. P., Schmiitte, C., Yang, A. S., Cote, R., Dubeau, L., Nichols, P. W., Hermann, G. G., Steven, K., Horn, T., Skinner, D. G. and Jones, P. A.: T w o molecular oathwavs to transitional cell carcinoma of the bladder. Cancer .kes., S& 784,1994. 8. Waldman. F. M.. Caroll, P. R., Kerschmann. R., Cohen, M. B., Field, F. G. A d May&, B. H.: Centromekc copy number of chromosome 7 is strongly correlated with tumor grade and labeling index in human bladder cancer. Cancer Res., 51: 3807,1991. 9. O l d , A. F., Tsai, Y.C., Nichols, P. W., Skinner, D. G., Cain, D. R., Bender, L. I. and Jones, P. A: Allelic loss of chromosome 17p distinguishes high grade from low grade transitional cell carcinomas of the bladder. Cancer Res., 50: 7081,1990. 10. Pycha, A., Mian, C., Haitel, A., Hofbauer, J., Wiener, H. and Marberger, M.: Fluorescence in situ hybridization identifies more aggressive types of primarily noninvasive (pTa) bladder cancer. J. Urol., 151: 2116, 1997. 11. Sandberg, A.A. and Berger, C. S.: Review of chromosome studies in urological tumors. 11. Cytogenetics and molecular genetics of bladder cancer. J. Urol., 151: 545,1994. 12. Smak, R., Wiener, H., Foeger, A., Susani, M., Zhang, Z. F., FIG. 3. Distribution of trisomy 17 in 26 analysed patients prior to Maier, U. and Marberger, M.: Cytomorphometry in the monitopical inetillation therapy. toring of bladder cancer under intravesical chemotherapy. Eur. Urol., 29: 391,1996. 13. Hermanek, P. and Sobin, L. H.: UICC TNM Classification of lies in general lead to oncogenetic changes involving genes Malignant Tumours. Berlin, Heidelberg, New York, London, that control or affect cellular growth and difYerentiation.11 Paris,Tokyo, Honkong, Barcelona, Budapest. Fourth Edition, Even though the carcinogene& procese may have already 2nd revision, 1992.
TRISOMY 17
TOPICAL INSTILLATION THERAPY IN SUPERFICIAL BLADDER CANCER
14. Pinkel, D.,Straume, T. and Gray, J. W.: Cytogenetic analysis using quantitative, high sensitivity fluorescence hybridization. Proc. Natl. Acad. Sci., 83: 2934,1986. 15. Flamm, J. and Steiner, R.: Stellenwert der differenzierten transurethralen Resektion beim primiiren oberflachlichen Blasenkarzinom. Urologe A, 3 0 111, 1991. 16. Ratliff, T. L.:Role of the immune response in BCG for bladder cancer. Eur. Urol., 21 (suppl. 2): 17, 1992.
269
17. Murphy, W. M., Soloway, M. S. and Crabtree, W. N.: The morphologic effects of mitomycin in mammalian urinary bladder. Cancer, 47:2567,1981. 18. Akaza, H.: BCG treatment of existing Ta, T1 tumours or carcinoma in situ of the bladder. Eur. Urol., 27 (suppl. 1):9,1995. 19. Van der Meijden, A. P. M.: Practical approaches to the prevention and treatment of adverse reactions to BCG. Eur. Urol., 27 (suppl. 1): 23, 1995.
Vol. 159,266-269. January 1998 Printed in U S A .
DOES TOPICAL INSTILLATION THERAPY INFLUENCE CHROMOSOMAL ABERRATIONS IN SUPERFICIAL BLADDER CANCER? ARMIN PYCHA,* CHRISTINE MIAN, JOHANN HOFBAUER, ANDREA HAITEL, HELENE WIENER AND MICHAEL MARBERGER From the Department of Urology and Department of Pathology, University of Vienna, Vienna, Austria
ABSTRACT
Purpose: Patients with a high risk for superficial bladder cancer are treated by topical immunoor chemotherapy after transurethral resection to reduce the chance of recurrence and/or progression. The aim of this study was to analyse if cytogenetical abnormalities, which are known to be constantly related to bladder cancer, are modified or eliminated by topical immuno- or chemotherapy. Materials and Methods: Using fluorescence in situ hybridization (FISH), the influence of topical instillation therapy with Bacillus Calmette-Guerin (BCG) and Mitomycin C (MMC) on numerical aberrations of chromosomes 7,9and 17 was investigated in 25 patients with transitional cell cancer (TCC) of the bladder. Data were compared with histological and clinical outcome. Fifteen TCC patients with similar histological criteria without instillation therapy served as controls. Median follow-up was 30 2 2 months. Results: After BCG treatment 10 of 15 patients (66.6%)developed recurrent and 2/15(13.3%) progressive disease. Three of 15 patients (20.0%)had no evidence of disease. Numerical aberrations did not change in 8 of the 15 BCG patients (53.3%) and changed to a more aggressive pattern in 40.0% (6/15).Five of 10 MMC treated patients (50.0%) developed a recurrent tumor, 2/10 (20.0%) progressed and 3/10(30.0%)had no evidence of disease. Four of 10 (40.0%)of these patients showed stable and 5/10 (50.0%)progressive chromosomal patterns. Only one patient in each group with primary chromosomal alterations changed to a regular diploid chromosomal pattern after therapy according to a complete clinical remission. Conclusion: Even though topical immuno- and chemotherapy may be useful to delay recurrence and progression, chromosomal patterns remain basically unstable. KEYWORDS: bladder cancer, intravesical chemotherapy, BCG therapy, chromosomal aberrations, FISH
%
Patients with a high risk of recurrent and progressive superficial transitional cell cancer of the bladder are in general treated with an additional topical immuno- or chemotherapeutical instillation after transurethral resection (TUR),since topical therapy is known to reduce recurrenceand progression rates.1-6 Oncogenesis of bladder cancer is described as a multiple step genetic alteration.7 It is also known that numerical aberrations of chromosomes 7, 9 and 17 are involved in the history of superficial and invasive cancer.7-11 If chromosomal alterations are postulated as the cause of malignant changes, successful therapy should theoretically lead to the disappearance or at least modification of chromosomal aberration patterns. The aim of this study was to determine if cytogenetical abnormalities known to be constantly related to bladder cancer are modified or eliminated by topical immuno- or chemotherapy. MATERIALS A N D METHODS
Forty patients (16 female, 24 male, average 71 years, range 39 to 85 years) with histologically confirmed transitional cell cancer (TCC) of the bladder were included in this study. At study entry all patients underwent TUR. Six patients showed first manifestation of multifocal pTa tumours, fifteen patients had recurrent but solitary pTa tumours, and 19
patients had stage pT1 tumours. Tumours were graded G1 in 10 cases, G2 in 23 cases and G3 in 7 cases (tables 1and 2). Prior to TUR bladder washings were obtained in all cases. During follow-up 25 patients had either topical instillations with MMC or they were under posttherapeutical follow up after BCG or MMC. Fifteen patients were treated with BCG, type R W M , (BCG-S, Medac, Hamburg, Germany) 2 X 10' colonies in 50 ml. solution per instillation weekly for a period of six weeks. Ten patients received 20 mg. MMC (Kyowa, Tokio, Japan) in 20 ml. solution per instillation every two weeks for 6 to 18months.12 The remaining 15 patients had no instillation therapy and formed the control group. All patients were followed for a median duration of 30 months (range 2832 months) with cystoscopy, voided urine and barbotage cytology every three months. In case of suspicious cystoscopy biopsies were taken. During follow-up chromosomal analysis was performed with every control visit. Table 2 gives data at first analysis, at 15 months and at 30 2 2 months. Bladder washings and touch preparations (imprints) of specimens were obtained at TUR in every patient. Barbotage specimens were collected immediately prior to TUR or at every follow-up cystoscopy by irrigation of the bladder with 50 ml. saline. Bladder washings were spun twice for 5 minutes at 350 g (Hettich Universal centrifuge, Germany), using a sedimentation conus the second time. At least one glass slide was air dried and stained according to the May Griinwald staining. The other slides were wet fixed (spray fixation, Merck, Germany), partly stained according to the Papanicolaou method and partly kept for further evalua-
Accepted for publication July 15, 1997. *Requests for re rints: Department of Urology, University of Vienna, Wahringer 8urt.d 18-20, 1090 Vienna, AusFa. supported by Grant 1389, BiirgermelsterfondeWen. 265
266
4 1
TOPICAL INSTILLATION THERAPY IN SUPERFICIAL BLADDER CANCER
TABLE1. Histology, pre- and posttherapeutic numerical chromosomal aberrations (NCA), kind of therapy, and clinical outcome in imprints and bladder washings of 25 patients with TCC of the bladder. Followup time: 30 2 2 months Retherapeutic NCA Patient 1 2 3 4 5
6 7 8
9 10 11 12 13 14 15
16 17 18
19 20 21 22
23 24 25 NED =
TCC pTaGl multifoc. pTaGl m. pTaG2 multifoc. pTaG2 rec. pTaG2 rec. pTaG2 rec. pTlG2 pTlG2 pTlG2 pTlG2 pT1GZ pTlG2 pTlG3 pTlG3 pTlG3 pTaGl rec. pTaGl rec. pTaGl rec. pTaGZ multifoc. pTaG2 rec. pTaG2 rec. pTlG2 pTlG2 pTlG3 oTlG3 no evidence of disease.
t?
none
-9
+ 17
+7
-9
none
+7 none +7 none +7 none none none +7 +7 none +7
-9 -9 none
+7
none
none none none
-9
+17 none none none none +17 none none none +17 none none none none none none none none none none none + 17 +i7
-9
none -9 -9 -9 -9 none -9 -9
-9 -9 -9
+7
none none none +7 +7 +7
Kind of Therapy
$17
#9
-9 -9
-9 -9 -9
none
BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG BCG MMC MMC MMC MMC MMC MMC MMC MMC MMC MMC
Posttherapeutic NCA
Clinical Outcome
t7
x9
$17
none +7 +7 none +7
-9 none
+17 +17 + 17 none none none + 17 + 17 none none none +17 none + 17 none +17 none none + 17 none none none +17 + 17 +17
-9
-9 none -9 none none -9 -9 -9 none none none none -9 -9 -9 -9 -9 none -9 none -9 none
+7 +7 none none +7 +7 +7 none +7 +7 none +7 none none +7 none none +7 +7 +7
recurrence recurrence cystitis recurrence recurrence progression progression recurrence recurrence recurrence recurrence recurrence NED cystitis recurrence progression recurrence recurrence recurrence cystitis NED cystitis progression recurrence recurrence
TABLE2. Histology, numerical chromosomal abemtwns (NCA) at first analysis and during followup time and clinical outcome in imprints and bladder washings of 15 patients (control group) with TCC of the bladder. Followup time: 30 2 2 months
1 2 3 4
5 6
7 8
9 10
11 12 13 14 15
TCC pTaGl rec. pTaGl rec. pTaGl rec. pTaG2 rec. pTaG2 rec. pTaGZ rec. pTaGZ rec. pTaG2 multifoc. pTaG2 multifoc. pTaG2 multifoc. pTlGl pTlGl pTlG2 pTlG2 pTlG3
NCA 30
NCA 15 Months
NCA a t 1. Analysis Patient
17
#9
#17
t7
#9
117
+7 none none +7 +7
-9
none
+17 none none none none none none none +17 none none +17 none +17 none
+7 none none +7 +7
none -9 -9 -9 none -9 -9 -9 none -9
+ 17 none none none + 17 none none none +17 none none
-9 -9 -9
+7
-9
none
-9
+7
-9
+7 none none
-9
+7 +7
-9 -9 none -9
none
-9
+7
none
+7 none +7 +7 none none +7 +7 none +7
tions. All stained slides were evaluated by routine exfoliative cytology and had identical cytological reports from both staining methods used. Imprint specimens were gathered immediately after TUR. Before placing biopsy material into the fixation fluid (37% formaldehyde), the resected material was rolled on or softly premed against a glass slide. Intraoperative imprints were spray fixed immediately aRer collection. To control the content of cells, one slide was stained according to the Papanicolaou method, the others were used for the detection of numerical chromosomal aberrations. TCC was staged and graded according to the UICC classiikation13 (table 1and 2). Fluorescence in situ hybridization analysis (FISH).Using FISH,specimens were evaluated for numerical aberrations on chromosome 7.9 and 17.The protocol followed in principle the method developed by pinkel et al.,14 slightly modified to shorten procedure time. Before denaturation slides were washed in 50% ethanol and PBS. Denaturation of the specimens (Formamid (Sigma, GemanyY20 x Saline Sodium Citrate (SSC)(Vysis Inc., MA, USA),75C for 5 minutes) and the directly labeled DNA target (Vysis Inc., MA, USA)was followed by hybridization. Hybridization was performed in 30 minutes at 42C. Subsequently slides were washed twice in
-9
none
+ 17
-9 -9
none +17 none
none
-?: 2
#7
#9
+7
none
none none +7 +7 +7
-9
+I +7 +7 none none
+I +7 none +7
Months
-9 -9 none -9 -9 -9 none -9 -9 none -9 -9
none
#17 +17 none none none + 17 none none +17 +17 none none +17 none + 17 none -
Clinical Outcome progression no recurrence recurrence recurrence progression recurrence recurrence progression recurrence no recurrence recurrence recurrence progression recurrence recurrence
SSC solutions of different concentrations. Counterstaining was achieved by application of 10 pl. diamidinophenylindole (DAPI) I1 Wysis Inc., MA, USA) per slide. Spots of two hundred cells were evaluated using 600-fold magnification and images were captured by a Smart Capture System (Vysis Inc., USA) mounted on a fluorescence microscope (Olympus, GerIIlany). As a control for the reliability of the method, lymphocytes of healthy probands were included in every hybridization unit, assuring diploid cells for the most part. According to previously published data8 the cutoff level for true aneusomy was established at 20% for all analysed chromosomes. Data were analysed statistically with the McNemar Chi-square test. RESULTS
Table 1 gives an overview of the histological diagnoses, patterns of analysed chromosomes, the type of topical treatment, the clinical outcome and the chromosomal patterns of the 25 analyzed patients. After treatment 10 patients (66.6%)out of the BCG treated group (group I) developed a recurrent tumour of the same
TOPICAL INSTILLATION THERAPY IN SUPERFICIAL, BLADDER CANCER
stage and grade as the primary one. Two patients (13.3%) developed progressive tumours, and 3 patients (20.0%) showed no evidence of disease, endoscopically, cytologically and histologically. Out of the mitomycin C treated group (group 11) five patients (50.0%) developed recurrent disease. Two patients (20.0%)progressed and 3 patients (30.0%)had no evidence of disease. Recurrence was never observed within the first 5 months after TUR, so that residual primary tumour can be excluded.15 Before treatment 5 patients out of group I showed a singular monosomy 9 (33.3%), 3 patients a singular trisomy 7 (20.0%), and one patient (6.6%)the combination of monosomy 9, trisomy 7 and 17. Six patients (40.0%) showed a mixed chromosomal pattern, i.e. monosomy 9 and trisomy 7 or 17,or aberrations of chromosome 7 and 17 only. Out of group I1 six patients (60.0%) had a single monosomy 9, one patient (10.0%)had aberrations in all analysed chromosomes and 3 patients (30.0%)had a mixed chromosomal aberrations pattern. After treatment 8 patients out of group 1(53.3%) showed the same numerical chromosomal aberrations as before therapy (stable pattern) and six patients (40.0%) changed to aberrations of chromosome 7 and 17 (progressive pattern). Out of group I1 four patients (40.0%) showed a stable chromosomal pattern and five patients (50%) a progressive chromosomal pattern. Only one patient (6.6%) of group I and one patient (10.0%) of group I1 had a regular diploid chromosomal pattern more than 2.5 years after therapy. In these patients the findings correlated with complete clinical remission. The other four patients without clinical and cytological evidence of disease (2 of every group) had a histologically verified unspecific cystitis caused by the BCGtherapy, but nevertheless chromosomal analysis showed persistent or progressive genetic alterations. Statistically no difference was seen for numerical aberrations of chromosomes 7 and 9 before and after therapy. In contrast the finding of trisomy 17 increased significantly after instillation (p = 0.04124).Nevertheless no differences were seen between the two kinds of therapy. At first chromosomal analysis 5 of the 15 patients (33.3%) of the control group showed a single monosomy 9, 2 patients (13.3%) a single trisomy 7 and 4 patients (26.6%) both a monosomy 9 and trisomy 7.Two patients showed aberrations of all three analyzed chromosomes and two patients a mixed chromosomal pattern. Subsequent genetic analysis during follow-up time showed a stable chromosomal pattern in 10 cases (66.6%) and a progressive chromosomal pattern in 33.3%. From the clinical point of view 2 of these 15 patients (13.3%) remained free of recurrence, nine patients (60.0%) had recurrent disease and four patients (26.6%) showed progression (table 2). The chromosomal patterns, mainly persistent or progressive genetic alterations, of the 15 patients of the control group were similar when compared with patients who underwent instillation therapy. No statistically significant difference was seen between the two groups.
267
bladder provoking an infection which induces an immunologcal response that initiates antitumour activity.2.16 T-cell :ompetence is therefore required. MMC has also been viably used for topical instillation after nesection of superficial bladder tumours. The antibiotic MMC s an alkylating agent with antitumour activity.17 It seems to )e tolerated with fewer side-effects than BCG.6 Cytotoxic :hernotherapy destroys rapidly dividing cells and is effective lnly when such cells are present at the time of administraLion.18 Previously published data suggest that BCG is more sfficient in reducing tumour recurrence than intravesical :hemotherapy.l-4.19 In addition chemotherapy appears to be more effective in low grade tumours, whereas BCG seems to be optimally effective in higher-grade tumours.' When considering the pattern of chromosomal aberrations, the patients of group I appeared to have more aggressive tumours than those in group I1 (figs. 1-3). In spite of this, no significant differences in outcome were noted between BCG and MMC in this study. No significant differences in the behaviour of chromosomal patterns in treated patients versus untreated patients were seen during follow-up. Chromosomal aberrations in both groups were already present at the first genetic analysis after TUR and was observed in both patients undergoing instillation therapy and in patients who underwent only TUR during the complete follow-up time, respectively. It seems that deterioration of the chromosomal pattern in bladder cancer is not caused by topical therapy. Reversion to diploid status and loss of chromosomal abnormalities were observed in only two patients, while another 4 patients endoscopically and histologically free of disease showed persisting genetic alterations. Chromosomal anoma-
MONOSOMY 9
DISCUSSION
Previous publications showed that monosomy 9 is an early genetic alteration in the oncogenesis of bladder cancer.7 It appears mostly as single genetic change in superficial tumourslo which recur often but progress rarely and in general have a good prognosis. Aberrations of chromosomes 7 and 17 are related to a more aggressive tumour behaviour.8-11 Tumours with numerical aberrations of these two chromosomes have a substantially higher recurrence and progression rate. A multitude of studied-6.12 describe the benefit of intravesical therapy with BCG in the treatment of carcinoma in situ, Ta and T1 tumours. The profound immunostimulatory effect of BCG led investigators to evaluate its role in the treatment of tumours. Viable bacteria are introduced into the
FIG.1. Distribution of monosomy 9 in 25 andysed patients prim to topical instillation therapy.
268
TOPICAL INSTILLATION THERAPY IN SUPERFICIAL BLADDER CANCER
TRISOMY 7
started, there must not necessarily be a phenotypic expression at the same time. "his could explain absence of recurrent disease, given the resistance of chromosomal changes. Likewise, while multistep acquisition of an aggressive genotype is generally considered the most acceptable theory of biological tumour progression, there are instances where a monoclonal origin has strongly been suggested." The large number of structural and numerical chromosomal changes present in bladder cancer enables cancer cells to biologically behave in different directions. Because of these changes it is conceivable that either cell may be or become resistant to the usual forms of therapy. Even though topical immuno- and chemotherapy may be considered useful to achieve clinical remission, numerical aberrations of the chromosomes 7,9 and 17 remained persistent in 91.7% of all analyzed cases of this study. Moreover, it is possible that aberrations of other chromosomes documented in TCC11 and not evaluated in this study may reflect a change post therapy. Complete remission has been reported in approximately 50% with a follow-up of 3-5 years: and approximately 30%with a follow-up of 10 year^.^.^ Nevertheless the significant enhancement of trisomy 17 despite topical instillation therapy shows that cytogenetical deterioration and therefore progression of disease cannot be avoided. REFERENCES
1. Lamm, D. L.:BCG in perspective: advances in the treatment of superficial bladder cancer. Eur. Urol., 27 (Suppl. 1):2, 1995. 2. RatlifF, T. L.,Ritchey, J. K., Yuan, J. J. J., Andriole, G. L. and Catalona, W. J.: T-cell subsets required for intravesical BCG immunotherapy for bladder cancer. J. Urol., 1 5 0 1018,1993. FIG. 2. Distribution of trisomy 7 in 25 andysed patients prior to 3. Soloway, M. S.:Managing superficial bladder cancer: an overtopical instillation therapy. view. Urol. (Suppl.), 40:5, 1992. 4. Vegt, P. D. J., Witjes, J. A., Witjes, W. P. J., Doesburg, W. H., Debruyne, F. M. J. and van der Meijden, A. P. M.: A randomized study of intravesical mitomycin C, Bacillus CalmetteGuerin Tice and Bacillus Calmette-GuerinRIVM treatment in pTa-pT1 papillary carcinoma and carcinoma in situ of the bladder. J. Urol., 153: 929,1995. 5. Nadler, R. B., Catalona, W. J., Hudson, M. A. and Ratliff, T. L.: Durability of the tumor-free response for intravesical Bacillus Calmette-Guerin therapy. J. Urol., 152 367, 1994. 6. Herr, H. W., Wartinger, D. D., Fair,W. R. and Oettgen, H. F.: Bacillus Calmette-Guerin therapy for superficial bladder cancer: a 10-year followup. J. Urol., 141: 1020,1992. 7. Spruck, C. H., 111, Ohneseit, P. F., Gonzales-Zulueta,M., Esrig, D., Miyao, N., Tsai Lerner, S. P., Schmiitte, C., Yang, A. S., Cote, R., Dubeau, L., Nichols, P. W., Hermann, G. G., Steven, K., Horn, T., Skinner, D. G. and Jones, P. A.: T w o molecular oathwavs to transitional cell carcinoma of the bladder. Cancer .kes., S& 784,1994. 8. Waldman. F. M.. Caroll, P. R., Kerschmann. R., Cohen, M. B., Field, F. G. A d May&, B. H.: Centromekc copy number of chromosome 7 is strongly correlated with tumor grade and labeling index in human bladder cancer. Cancer Res., 51: 3807,1991. 9. O l d , A. F., Tsai, Y.C., Nichols, P. W., Skinner, D. G., Cain, D. R., Bender, L. I. and Jones, P. A: Allelic loss of chromosome 17p distinguishes high grade from low grade transitional cell carcinomas of the bladder. Cancer Res., 50: 7081,1990. 10. Pycha, A., Mian, C., Haitel, A., Hofbauer, J., Wiener, H. and Marberger, M.: Fluorescence in situ hybridization identifies more aggressive types of primarily noninvasive (pTa) bladder cancer. J. Urol., 151: 2116, 1997. 11. Sandberg, A.A. and Berger, C. S.: Review of chromosome studies in urological tumors. 11. Cytogenetics and molecular genetics of bladder cancer. J. Urol., 151: 545,1994. 12. Smak, R., Wiener, H., Foeger, A., Susani, M., Zhang, Z. F., FIG. 3. Distribution of trisomy 17 in 26 analysed patients prior to Maier, U. and Marberger, M.: Cytomorphometry in the monitopical inetillation therapy. toring of bladder cancer under intravesical chemotherapy. Eur. Urol., 29: 391,1996. 13. Hermanek, P. and Sobin, L. H.: UICC TNM Classification of lies in general lead to oncogenetic changes involving genes Malignant Tumours. Berlin, Heidelberg, New York, London, that control or affect cellular growth and difYerentiation.11 Paris,Tokyo, Honkong, Barcelona, Budapest. Fourth Edition, Even though the carcinogene& procese may have already 2nd revision, 1992.
TRISOMY 17
TOPICAL INSTILLATION THERAPY IN SUPERFICIAL BLADDER CANCER
14. Pinkel, D.,Straume, T. and Gray, J. W.: Cytogenetic analysis using quantitative, high sensitivity fluorescence hybridization. Proc. Natl. Acad. Sci., 83: 2934,1986. 15. Flamm, J. and Steiner, R.: Stellenwert der differenzierten transurethralen Resektion beim primiiren oberflachlichen Blasenkarzinom. Urologe A, 3 0 111, 1991. 16. Ratliff, T. L.:Role of the immune response in BCG for bladder cancer. Eur. Urol., 21 (suppl. 2): 17, 1992.
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17. Murphy, W. M., Soloway, M. S. and Crabtree, W. N.: The morphologic effects of mitomycin in mammalian urinary bladder. Cancer, 47:2567,1981. 18. Akaza, H.: BCG treatment of existing Ta, T1 tumours or carcinoma in situ of the bladder. Eur. Urol., 27 (suppl. 1):9,1995. 19. Van der Meijden, A. P. M.: Practical approaches to the prevention and treatment of adverse reactions to BCG. Eur. Urol., 27 (suppl. 1): 23, 1995.