- Email: [email protected]
470A prospective comparison using fluorescence in situ hybridization and cytology
469
470
A N E W AND R E L I A B L E C U L T U R E S Y S T E M F O R S U P E R F I C I A L L O W GRADE BLADDER TRANSITIONAL CARCINOMA
A P R O S P E C T I V E C O M P A R I S O N U S I N G F L U O R E S C E N C E IN S I T U H Y B R I D I Z A T I O N AND C Y T O L O G Y
Seifert H.H., Prior A., Cronauer M., Mtiller M., Ackermann R., Schulz W. Capponi G. 1, Casazza S. 2, Campodonico E 1, Bandelloni R. 2, Maffezzini M. 1 Heinrich Heine Universit~t Dfisseldorf, Urologische Klinik, Diisseldorf, Germany INTRODUCTION & OBJECTIVES: There are few studies about primary cultures of low grade superficial bladder transitional carcinomas. Most investigators used short term cultures without a passage of the cultured cells. Often the malignant origin of the cultured cells remains unclear. We established a new and reliable culturing system for superficial bladder cancers and discuss tbe suitability of this in vitro system for superficial bladder cancer. MATERIAL & M E T H O D S : Representative fresh tissue specimens were obtained from transurethral resections or radical cystectomies from patients with transitional carcinomas of the bladder. Mean ages of the patients was 74 (range 52-95). Pathological examination revealed 22 pTa, 8 pT1, 16 pT2 tumours (UICC, 1997). The fresh specimens were immediately referred to room temperature transport medium which contained a protein kinase inhibitor. Under stereomicroscopic control specimens were freed from thermic necrosis and damaged tissue. Then the tissues were stored overnight at 4 C in a 0 . 1 % EDTA solution containing hepes and aprotinine. Next day the specimens the turnout cells were separated from the stroma with fine scissors. After centrifugation the cells were plated in 250 ml culture flakes. We used a medium with only 1% serum additionally containing EGF, penicillin, streptomycin, BPE, nonessential aminoacids and 1% supernatant of human bladder fibroblast cultures. After subconfluence the primary cultures were passaged. Malignancy was demonstrated by DNA fluorescopy. RESULTS: Between November 2002 and April 2004 46 primary bladder cancer cultures were performed. The overall success rate was 60.5 %. The malignant origin of the cultured cells was clearly demonstrated by DNA fluorescopy. Interestingly 84.2 % of papillary superficial low grade bladder transitional carcinomas have been cultured successfully compared to only 23.1% of advanced stage tumours respectively. A similar result was seen for G3 tumours with only 28.6 % of successful cultures. Cultures from G1 and G2 grow similarly with a success rate of 85.7 % and 72.3 % respectively. The age of the tissue donor does not influence culture success. Up to 3 passages are possible with superficial low grade TCC cultures. In 24 % of all successful primary cultures a passage was not possible. CONCLUSIONS: We describe a new and reliable culture system which is highly successful in culturing low grade transitional cell carcinoma of the bladder. Up to three passages of the primary tumour cell cultures have been possible demonstrating the viability of the cultured tumour cells. Therefore this culture system can widely be used for functional in vitro experiments simulating superficial bladder cancer.
lGalliera HospitaI, Department of Urology, Genoa, Italy, 2Galliera Hospital, Department of Pathology, Genoa, Italy I N T R O D U C T I O N & O B J E C T I V E S : Transitional cell carcinoma (TCC) of the bladder is currently diagnosed by cystoscopy. Urinary cytology represents the most common tool to enhance cancer detection either for primary diagnosis or during follow-up. Fluorescence in situ hybridization (FISH - Urovision Vysis) can identify cells with chromosomal alterations obtained from voided urine. We compared the sensitivity of urinary cytology and FISH for the detection of bladder TCC. M A T E R I A L & M E T H O D S : From May 2003 till May 2004, 89 urine specimens were collected from 89 patients. 65 of 89 patients had a previous history of bladder TCC, while 24 patients were investigated for hematuria-dysuria. All urine specimens were analyzed with cytology and FISH. FISH was performed using probes to centromeres of 3,7,17 and locus 9p21. Patients were subjected to TUR or mapping of the mucosa for positive or equivocal cystoscopy, and positive cytology or FISH. RESULTS: TCC was present in 48 of 89 patients after TUR. The overall TCC detection was 68.8 % for cytology (33/48 patients), and 83.3 % for FISH (40/48 patients). STAGE FISH CYTOLOGY Ta 6/12 3/i2 T1 19/21 15/21 T2-3 7/7 7/7 Cis 8/8 8/8 GRADE Low 11/17 6/17 High 29/31 27/31 FISH were positive in all positive cytologyes. 4 patients with positive FISH were negative after TUR, whereas only 1 patient has positive cytology with negative TUR. C O N C L U S I O N S : FISH analysis showed a superior detection rate for bladder TCC than conventional cytology, especially for low stage and grade tumours.
471
472
DECREASED EXPRESSION OF INSULIN LIKE GROWTH FACTOR R E C E P T O R (IGF-IR) IS AN I N D E P E N D E N T P R O G N O S T I C F A C T O R IN UPPER URINARY TRACT TRANSITIONAL CELL CARCINOMA
E F F E C T O F H Y P E R T H E R M I A ON T H E C Y T O T O X I C I T Y O F F O U R CHEMOTHERAPEUTIC AGENTS CURRENTLY USED FOR THE TREATMENT OF TRANSITIONAL CELL CARCINOMA OF THE B L A D D E R - AN IN V I T R O S T U D Y
Zigeuner R?, Tannapfel A. 2, Rehak E 3, Langner C. 4 Van der Heijden A., Moonen P., Verhaegh G., Jansen C., Sehalken J., Witjes J.A. LMedical University Graz, Urology, Graz, Austria, 2University of Leipzig, Pathology, Leipzig, Germany, 3Medical University Graz, Biomedical Engineering and Computing, Graz, Austria, 4Medical University Graz, Pathology, Graz, Austria INTRODUCTION & OBJECTIVES: The role of the insulin-like growth factor receptor (IGF-IR) has been well documented for many turnouts. Regarding transitional cell carcinoma (TCC), systematic data are currently lacking. MATERIAL & M E T H O D S : 53 upper urinary tract TCCs and ten cases of nonneoplastic urothelium were chosen for analysis. TCCs included 22 (42%) pT1, 9 (17%) pY2, 22 (42%) pT3, 28 (53%) low grade and 25 (47%) high grade cases, respectively. Immunohistochemical evaluation was performed using a tissue microarray technique. Analysis included semiquantitative assessment of immunostaining (staining of <50% vs. >50% of tumour cells) and staining intensity (weak, moderate, strong). Results were associated with pT-stage, grade, and prognosis. A multivariate analysis including pT-stage, grade, angioinvasion and immunostaining was performed. RESULTS: Normal urothelium showed a strong distinct membranous immunostaining of almost all urothelial cells, stroma and renal parenchyma lacked immunoreactivity. In TCCs, IGF-IR immunoreactivity was noted in all tumours, high expression was noted in 46/53 (87%). A statistically significant decrease of staining intensity was observed with increasing pT-stage (strong intensity: 18/31 [58%] pT1/2 vs. 3/22 [14%] pT3 tumours; p=0.001) and tumour dedifferention (strong intensity: 16/28 [57%] low grade vs. 5/25 [20%] high grade mmours). Semiquantitative expression was associated only with grade (p=0.03), not with pT-stage. 5/7 (71%) and 13/32 (41%) patients with tumours showing low expression and low to moderate intensity, respectively, compared to 9/46 (20%) and 1/21 (5%) patients showing high expression and high intensity developed metastatic disease (p<0.00l and p-0.004, respectively). In multivariate analysis, stage pT3, angioinvasion, and low IGF-IR immunostaining were independent prognostic factors for metastatic disease. C O N C L U S I O N S : In contrast to most human tissues, IGF-IR is strongly expressed in normal urothelium and all TCCs. A decreased expression could be observed with tumour dedifferentiation. Decreased IGF-IR expression is an independent prognostic factor in upper tract TCCs.
European Urology Supplements 4 (2005) No. 3, pp. 120
UMC Nijmegen, Urology,Nijmegen, The Netherlands INTRODUCTION & OBJECTIVES: Hyperthermiacombined with chemotherapy is not a novel cancer treatment. However, the working mechanism of this combination therapy is not fully understood. The current in vitro study investigates the differences in cytotoxicity of mitomycin-C (MMC), epirubicin, gemcitabine and the novel agent EOquinT M at 37°C or 43°C. EOqninT M is a bioreductive alkylating indoloquthone.Although strueturally related to MMC, EOquinT M exhibits a distinct antitumouractivity and there are differences in biochemical activation. MATERIAL & METHODS: The human transitional cell carcinoma (TCC) cell lines used were RT4, RT112, 253J and T24. The cells were seeded in 96-well microtiter plates. After 24 hours cells were treated for 60 minutes with increasing concentrations of MMC, epirubicin, gemcitabine and EOquinT M at a temperature of 37°C or 43°C. After treatment cells were rinsed three times and left for 24 hours in the incubator at 37°C. The influenceof chemotherapyand temperature on cell survival was determined by an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbrmnide)assay. RESULTS: Decreased cell proliferation with increasing concentrations of chemotherapeutic agents was demonstrated. EOquinT M proved to be the most potent agent at both temperatures.Dependingon the type of cell line used, the concentrationEO9 to reach LDso at 37°C was 6 to 78 times lower than the concentration MMC. When hyperthermia was given simultaneously with EOquinTM, the combined potency increased and the EOquinT M dose used to reach LDsodecreased by 21% to 56%. Hyperthermia alone did not demonstratedecreased cell proliferation. However,a synergisticeffect on the decreased cell proliferationwas demonstrated in all ceil lines and ehemotherapeuticagents used, although each with a maximum at a different chemotherapyconcentration and to a different extent. Synergismwas most obvimts in cell lines treated with low dose epirubicin. The differences in LDso as result of applied hyperthermia is expressed in percents and calculated by the equation: A LDsoEag~nt]= (LDsoNgcm~t43oc]/ LDsoE~g...... 3v°c])x 100% (Table 1). Table 1. Treatment
LD50 RTll2
LD50 T24
LD50 253J
LD50 RT4
A LD50 [MMC] A LD50 [EOquinTM] A LD50 [epirubicin] A LD50 [gemcitabine]
85% 79% 13% 67%
17% 60% 5% 69%
71% 44% 21% 85%
86% 47% 23% 78%
CONCLUSIONS: Synergism with hyperthermia and chemotherapy was clearly demonstrated for Epirubicin, EOquinT M , MMC and to a lesser extent gemcitabine. Hyperthermiaalone did not cause decreased cell proliferation. Synergismwas most prominentwith low drug doses, and the most potent drug used in this in vitro study was EOquinT M .
470
A N E W AND R E L I A B L E C U L T U R E S Y S T E M F O R S U P E R F I C I A L L O W GRADE BLADDER TRANSITIONAL CARCINOMA
A P R O S P E C T I V E C O M P A R I S O N U S I N G F L U O R E S C E N C E IN S I T U H Y B R I D I Z A T I O N AND C Y T O L O G Y
Seifert H.H., Prior A., Cronauer M., Mtiller M., Ackermann R., Schulz W. Capponi G. 1, Casazza S. 2, Campodonico E 1, Bandelloni R. 2, Maffezzini M. 1 Heinrich Heine Universit~t Dfisseldorf, Urologische Klinik, Diisseldorf, Germany INTRODUCTION & OBJECTIVES: There are few studies about primary cultures of low grade superficial bladder transitional carcinomas. Most investigators used short term cultures without a passage of the cultured cells. Often the malignant origin of the cultured cells remains unclear. We established a new and reliable culturing system for superficial bladder cancers and discuss tbe suitability of this in vitro system for superficial bladder cancer. MATERIAL & M E T H O D S : Representative fresh tissue specimens were obtained from transurethral resections or radical cystectomies from patients with transitional carcinomas of the bladder. Mean ages of the patients was 74 (range 52-95). Pathological examination revealed 22 pTa, 8 pT1, 16 pT2 tumours (UICC, 1997). The fresh specimens were immediately referred to room temperature transport medium which contained a protein kinase inhibitor. Under stereomicroscopic control specimens were freed from thermic necrosis and damaged tissue. Then the tissues were stored overnight at 4 C in a 0 . 1 % EDTA solution containing hepes and aprotinine. Next day the specimens the turnout cells were separated from the stroma with fine scissors. After centrifugation the cells were plated in 250 ml culture flakes. We used a medium with only 1% serum additionally containing EGF, penicillin, streptomycin, BPE, nonessential aminoacids and 1% supernatant of human bladder fibroblast cultures. After subconfluence the primary cultures were passaged. Malignancy was demonstrated by DNA fluorescopy. RESULTS: Between November 2002 and April 2004 46 primary bladder cancer cultures were performed. The overall success rate was 60.5 %. The malignant origin of the cultured cells was clearly demonstrated by DNA fluorescopy. Interestingly 84.2 % of papillary superficial low grade bladder transitional carcinomas have been cultured successfully compared to only 23.1% of advanced stage tumours respectively. A similar result was seen for G3 tumours with only 28.6 % of successful cultures. Cultures from G1 and G2 grow similarly with a success rate of 85.7 % and 72.3 % respectively. The age of the tissue donor does not influence culture success. Up to 3 passages are possible with superficial low grade TCC cultures. In 24 % of all successful primary cultures a passage was not possible. CONCLUSIONS: We describe a new and reliable culture system which is highly successful in culturing low grade transitional cell carcinoma of the bladder. Up to three passages of the primary tumour cell cultures have been possible demonstrating the viability of the cultured tumour cells. Therefore this culture system can widely be used for functional in vitro experiments simulating superficial bladder cancer.
lGalliera HospitaI, Department of Urology, Genoa, Italy, 2Galliera Hospital, Department of Pathology, Genoa, Italy I N T R O D U C T I O N & O B J E C T I V E S : Transitional cell carcinoma (TCC) of the bladder is currently diagnosed by cystoscopy. Urinary cytology represents the most common tool to enhance cancer detection either for primary diagnosis or during follow-up. Fluorescence in situ hybridization (FISH - Urovision Vysis) can identify cells with chromosomal alterations obtained from voided urine. We compared the sensitivity of urinary cytology and FISH for the detection of bladder TCC. M A T E R I A L & M E T H O D S : From May 2003 till May 2004, 89 urine specimens were collected from 89 patients. 65 of 89 patients had a previous history of bladder TCC, while 24 patients were investigated for hematuria-dysuria. All urine specimens were analyzed with cytology and FISH. FISH was performed using probes to centromeres of 3,7,17 and locus 9p21. Patients were subjected to TUR or mapping of the mucosa for positive or equivocal cystoscopy, and positive cytology or FISH. RESULTS: TCC was present in 48 of 89 patients after TUR. The overall TCC detection was 68.8 % for cytology (33/48 patients), and 83.3 % for FISH (40/48 patients). STAGE FISH CYTOLOGY Ta 6/12 3/i2 T1 19/21 15/21 T2-3 7/7 7/7 Cis 8/8 8/8 GRADE Low 11/17 6/17 High 29/31 27/31 FISH were positive in all positive cytologyes. 4 patients with positive FISH were negative after TUR, whereas only 1 patient has positive cytology with negative TUR. C O N C L U S I O N S : FISH analysis showed a superior detection rate for bladder TCC than conventional cytology, especially for low stage and grade tumours.
471
472
DECREASED EXPRESSION OF INSULIN LIKE GROWTH FACTOR R E C E P T O R (IGF-IR) IS AN I N D E P E N D E N T P R O G N O S T I C F A C T O R IN UPPER URINARY TRACT TRANSITIONAL CELL CARCINOMA
E F F E C T O F H Y P E R T H E R M I A ON T H E C Y T O T O X I C I T Y O F F O U R CHEMOTHERAPEUTIC AGENTS CURRENTLY USED FOR THE TREATMENT OF TRANSITIONAL CELL CARCINOMA OF THE B L A D D E R - AN IN V I T R O S T U D Y
Zigeuner R?, Tannapfel A. 2, Rehak E 3, Langner C. 4 Van der Heijden A., Moonen P., Verhaegh G., Jansen C., Sehalken J., Witjes J.A. LMedical University Graz, Urology, Graz, Austria, 2University of Leipzig, Pathology, Leipzig, Germany, 3Medical University Graz, Biomedical Engineering and Computing, Graz, Austria, 4Medical University Graz, Pathology, Graz, Austria INTRODUCTION & OBJECTIVES: The role of the insulin-like growth factor receptor (IGF-IR) has been well documented for many turnouts. Regarding transitional cell carcinoma (TCC), systematic data are currently lacking. MATERIAL & M E T H O D S : 53 upper urinary tract TCCs and ten cases of nonneoplastic urothelium were chosen for analysis. TCCs included 22 (42%) pT1, 9 (17%) pY2, 22 (42%) pT3, 28 (53%) low grade and 25 (47%) high grade cases, respectively. Immunohistochemical evaluation was performed using a tissue microarray technique. Analysis included semiquantitative assessment of immunostaining (staining of <50% vs. >50% of tumour cells) and staining intensity (weak, moderate, strong). Results were associated with pT-stage, grade, and prognosis. A multivariate analysis including pT-stage, grade, angioinvasion and immunostaining was performed. RESULTS: Normal urothelium showed a strong distinct membranous immunostaining of almost all urothelial cells, stroma and renal parenchyma lacked immunoreactivity. In TCCs, IGF-IR immunoreactivity was noted in all tumours, high expression was noted in 46/53 (87%). A statistically significant decrease of staining intensity was observed with increasing pT-stage (strong intensity: 18/31 [58%] pT1/2 vs. 3/22 [14%] pT3 tumours; p=0.001) and tumour dedifferention (strong intensity: 16/28 [57%] low grade vs. 5/25 [20%] high grade mmours). Semiquantitative expression was associated only with grade (p=0.03), not with pT-stage. 5/7 (71%) and 13/32 (41%) patients with tumours showing low expression and low to moderate intensity, respectively, compared to 9/46 (20%) and 1/21 (5%) patients showing high expression and high intensity developed metastatic disease (p<0.00l and p-0.004, respectively). In multivariate analysis, stage pT3, angioinvasion, and low IGF-IR immunostaining were independent prognostic factors for metastatic disease. C O N C L U S I O N S : In contrast to most human tissues, IGF-IR is strongly expressed in normal urothelium and all TCCs. A decreased expression could be observed with tumour dedifferentiation. Decreased IGF-IR expression is an independent prognostic factor in upper tract TCCs.
European Urology Supplements 4 (2005) No. 3, pp. 120
UMC Nijmegen, Urology,Nijmegen, The Netherlands INTRODUCTION & OBJECTIVES: Hyperthermiacombined with chemotherapy is not a novel cancer treatment. However, the working mechanism of this combination therapy is not fully understood. The current in vitro study investigates the differences in cytotoxicity of mitomycin-C (MMC), epirubicin, gemcitabine and the novel agent EOquinT M at 37°C or 43°C. EOqninT M is a bioreductive alkylating indoloquthone.Although strueturally related to MMC, EOquinT M exhibits a distinct antitumouractivity and there are differences in biochemical activation. MATERIAL & METHODS: The human transitional cell carcinoma (TCC) cell lines used were RT4, RT112, 253J and T24. The cells were seeded in 96-well microtiter plates. After 24 hours cells were treated for 60 minutes with increasing concentrations of MMC, epirubicin, gemcitabine and EOquinT M at a temperature of 37°C or 43°C. After treatment cells were rinsed three times and left for 24 hours in the incubator at 37°C. The influenceof chemotherapyand temperature on cell survival was determined by an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbrmnide)assay. RESULTS: Decreased cell proliferation with increasing concentrations of chemotherapeutic agents was demonstrated. EOquinT M proved to be the most potent agent at both temperatures.Dependingon the type of cell line used, the concentrationEO9 to reach LDso at 37°C was 6 to 78 times lower than the concentration MMC. When hyperthermia was given simultaneously with EOquinTM, the combined potency increased and the EOquinT M dose used to reach LDsodecreased by 21% to 56%. Hyperthermia alone did not demonstratedecreased cell proliferation. However,a synergisticeffect on the decreased cell proliferationwas demonstrated in all ceil lines and ehemotherapeuticagents used, although each with a maximum at a different chemotherapyconcentration and to a different extent. Synergismwas most obvimts in cell lines treated with low dose epirubicin. The differences in LDso as result of applied hyperthermia is expressed in percents and calculated by the equation: A LDsoEag~nt]= (LDsoNgcm~t43oc]/ LDsoE~g...... 3v°c])x 100% (Table 1). Table 1. Treatment
LD50 RTll2
LD50 T24
LD50 253J
LD50 RT4
A LD50 [MMC] A LD50 [EOquinTM] A LD50 [epirubicin] A LD50 [gemcitabine]
85% 79% 13% 67%
17% 60% 5% 69%
71% 44% 21% 85%
86% 47% 23% 78%
CONCLUSIONS: Synergism with hyperthermia and chemotherapy was clearly demonstrated for Epirubicin, EOquinT M , MMC and to a lesser extent gemcitabine. Hyperthermiaalone did not cause decreased cell proliferation. Synergismwas most prominentwith low drug doses, and the most potent drug used in this in vitro study was EOquinT M .