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A comparative phase III study of hexvix® fluorescence and standard cystoscopy in the detection of carcinoma in situ (CIS) of the bladder
437
438
A COMPARATIVE PHASE HI STUDY OF HEXVIXB FLUORESCENCE AND STANDARD CYSTOSCOPY IN THE DETECTION OF CARCINOMA IN SITU (CIS) OF THE BLADDER
DO IMMUNOHISTOCHEMICAL PREDICTION OF A NEURAL BLADDER CANCER
Marberuer M.‘, Witjes F.‘, Schmeller K.‘, Ronald D.‘, Susani M.’
Planr B.‘, Deix T.‘, Draxlcr V.‘, Haitel A.‘, Petsch M.‘, Caspers H.P.‘, Marberger M.’
1NTRODUCTION & OBJECTIVES: Neural networks have been investigated in some medical disciplines including the detection of prostate cancer in patients with low PSA-levels. In superficial bladder cancer a marker such as PSA is missing. Recently a variety of molecular markers have been tested for their ability to predict the outcome in patients with bladder cancer. Only a few give truly independent prognostic information. To explore the potential role of an artificial neural network (ANN) in predicting therapy outcome of patients with superficial bladder cancer, immunohistochemical markers were used in combination with ctinico-pathological factors.
INTRODUCTION & OBJECTIVES: Fluorescence cystoscopy has been reported to improve the detection of CIS lesions but this has not been verified in large multicenter studes. In this study, recruiting patients from 19 centres in 8 European countries, we compared the detection of CIS using hexyl aminolevulinate (HAL) fluorescence and standard white light cystoscopy. MATERIAL & METHODS: Patients suspected of having high-risk bladder cancer received instillation of 50 ml HAL 8 mM for one hour before cystoscopy. The bladder was inspected in white followed by blue light, and biopsies/ resected material of tumours and suspicious areas were assessed by central pathologist. Cystoscopy was performed with D-light (Karl Storz, Germany) allowing for both white light and blue light (375-440 nm) bladder inspection.
MATERIAL & METHODS: In 150 patients with superficial bladder cancer we investigated EGF receptor, ~53, c-erb-B2 and MDM-2 as immunohistochemical parameters and clinico-pathological factors. There were 87 Ta, 62 T 1, 23 Carcinoma in situ, 47 with BCG chemotherapy. Data were analysed using a backpropagation network with one neural layer and 45 bidden nodes. The input neural data consisted of immunohistochemical markers and tumour stage, malignancy grade, presence of a carcinoma in situ, size (tumour >3 cm) and number of resected tumours and mformation on the therapy applied (chemotherapy or immunotherapy). Explored output parameters were recurrence and progression.
RESULTS: Of 211 evaluable patients (80% male), 83 (39%) had CIS. In 17 (21%) patients HAL detected CIS lesions not seen under white light inspection. In 54% of CIS patients HAL detected additional CIS lesions compared to standard cystoscopy, and overall HAL detected significantly more CIS lesions (97%) compared to standard cystoscopy (58%). Of 92 Ta patients, HAL detected additional Ta lesions in 20 patients. Only negligible side effects were reported. including blood biochemistry and hematology. Lesion detection rate (lesions detected blue or white/detected
I CIS
1Dvsulasia
I Ta
in blue+white)
1T2
IT1
I
blue light
167 (97%)
64 (94%)
365 (97%)
79 (96%)
29 (100%)
white light total
101 (58%) 172
36 (53%) 68
329 (88%) 376
72 (88%) 82
28 (97%) 29
CONCLUSIONS: HAL fluorescence detection of CIS of the bladder.
cystoscopy
significantly
improved
early
INCREASE OUTCOME FOR PATIENTS WITH
‘Golzheim Clinic, Urology. Dusseldorf. Germany. ‘University of Vienna, Urology. Vienna, Austria, ‘University of Vienna, Pathology, Vienna, Austria
University of Vienna, Urology, Vienna, Austria. ‘University Medical Center, Urology, Nijmegen, The Netherlands, ‘St. Johanns Spital, Urology, Salrburg. Austria, ‘University of Vienna, Pathology, Vienna, Austria
I
MARKERS NETWORK
KESULTS: The overall accuracy of the neural network in predicting recurrence by use of EGF receptor, ~53, c-erb-B2 and MDM-2 was 84% with 83% sensitivity and specificity of 85%. When employing each marker as the sole predictor of recurrence the overall accuracy decreased compared to utilizing all 4 immunohistochemical markers. In high grade (G3) Tl and Ta tumours the ANN analysis became 78% sensitive and 93% specific with an overall accuracy of 85%. The performance of the ANN on stage progression by use of EGF receptor, ~53, c-erb-B2 and MDM-2 was 74%. The receiver operating curve (ROC) showed an area under the curve of 76%. CONCLUSIONS: Predicting therapy outcome with clinicopathological parameters or immunohistochemical markers as the sole predtctor by use of an ANN is unsatisfactory. Utilizing both immunohistochemical and clinicopathological parameters increases the prognostic accuracy of an ANN for bladder cancer. An ANN is a useful adjunct and provides the clinician with additional information on predicting outcome and the right choice of therapy according to the risk for recurrence and progression.
440
439 HEMATOLOGIC ERYTHROCYTES USING A FLOW METHOD
STUDY OF URINARY AND BLOOD TO IDENTIFY THE SOURCE OF HEMATURIA CYTOMETER-BASED LASER LIGHT-SCATTERING :
Anrulo J.C., Guil M.. Gonzalez J., Lopez-Rub10 M.. Sanchez-Chapado
M
Hospital Principe de Asturias, Department of Urology, Alcala de Henares. Spain INTRODUCTION & OBJECTIVES: A new noninvasive and accurate method to locate the source of hematuria, based on the comparative volumetric analysis of blood and urinary red blood cells using the Technicon H3 system, has been developed (J. Ural 1999; 162: 119). MATERIAL & METHODS: Comparative study between blood and urine implies the comparison between erythrocytes in peripheral blood diluted in urine supematant after centrifugation and urinary erythrocytes in patients with hematuria. Technicon H3 system allows independent measure of cell volume (MCV), hemoglobin concentration (MCHM), red cell distribution width (RDW) and hemoglobin distribution width (HDW). This study has been performed in a series of 148 patients with hematuria in which a glomerular (n=106) or nonglomerutar (n=42) source have been blindly identified, with the intention to determine which is the hematologic study (urinary or comparative) that better discriminates a non-glomerular source. Quantitative comparison was performed using Student’s t-test. Sensitivity, specificity. accuracy and positive and negative predictive values of urinary and comparative analysis were performed for each parameter. ROC curve analysis was also performed. RESULTS: Among the parameters studied, mean values of comparative MCV (p<.OOOl), urinary MCV (p<.OOOl), urinary MCHM (p=.OO7), and urinary HDW (p=.O18) differ according to the source of hematuria. ROC curve analysis reveal area under the curve is 98% for comparative MCV (SE ,009) 84% for urinary MCV (SE .038), 69% for urinary MCHM (SE .046), and 63% for urinary HDW (SE .54). Statistically significant differences that favour comparative MCV (2=4) can be established. With a cut-off value of 13 fl, comparative MCV reaches 91% sensitivity, 92% specificity, 97% positive predictive value, 80% negative predictive value, and 91% accuracy to detect a glomemlar source. CONCLUSIONS: Comparative volumetric analysis (i.e., the difference between MCV of erythrocytes in peripheral blood diluted in urine supernatant after centrifugation and MCV of urine erythrocytes) determined by flow cytometer-based laser light scattering method is definitely the best automatic hematologic study to be incorporated in urologists’ practice to evaluate patients with asymptomatic hematuria. European Urology Supplements 2 (2003) No. 1, pp. 112
QUANTITATIVE DETECTION OF THE TELOMERASE SUBUNITS HTR AND HTERT FOR DIAGNOSIS OF BLADDER CANCER Weikert S.. Miitler M., Krause H., Miller K Ilniversitatsklinikum
Benjamin Franklin. Dept. of Urology, Berlin. (iermany
INTRODUCTION & OBJECTIVES: Telomerasc is an enzyme found predominantly in malignant ceils. This opens new diagnostic perspectivca. However, the standard method of detecting tclomerase activity, the so-called TRAP assay, is not suitable for the diagnostic examination of urine samples. An alternative consists in the detection of the telomerase subunits hTR and hTERT using the reverse-transcriptase polymerase chain reaction (RT-PCR) method. Studies have shown that hTR and hTERT can be detected in urine samples with high sensitivity and specificity. Nevertheless. a method for quantitative evaluation would be desirable, particularly for hTR. To date, however, no prospective investigation in a large patient collective has been published. MATEFUAL & METHODS: Urine samples were obtained from 179 patients with confirmed urothelial carcinoma of the urinary bladder, 186 patients with benign urologic disease (urolithiasis, benign prostatic hypertrophy, urinary tract infections), and 100 healthy controls were collected prospectively and examined without knowledge of the diagnosis. Following extraction of RNA, a highly sensitive quantitative RT-PCR (LightCycler) was used to identify the mRNA of the catalytic subunit of telomerase, hTERT, or the RNA of the telomerase RNA, hTR. RESULTS: In urine samples dertved from patients with &firmed bladder carcinoma, hTERT was detected with a sensitivity of 55.2%. Because of the low expression of hTERT, pTa tumours loss than 1 cm in diameter were often not detected. Specificity in healthy controls was 89.2% and 85% in the entire collective. The ROC curves for quantitative RT-PCR detection of hTR yielded a sensitivity of 77% at a cut-off level of 750 copies. Specificity of hTR in healthy controls was 95.3% and 72. I % for the entire collective. False positive results were encountered particularly often in patients with urolithiasis. CONCLUSIONS: Due to the low expression of hTERT, small tumours are often not detected. Despite its very good specificity, the low sensitivity associated with the detection of hTERT in urine samples does not currently fultil diagnostic expectations. A further refinement in the quantitative RT-PCR is possible and necessary. Much more promising is the quantitative detection of hTR, particularlY in regard to the healthy control collective. Inflammatory processes in the urinary tract do not lead to false positive results.
438
A COMPARATIVE PHASE HI STUDY OF HEXVIXB FLUORESCENCE AND STANDARD CYSTOSCOPY IN THE DETECTION OF CARCINOMA IN SITU (CIS) OF THE BLADDER
DO IMMUNOHISTOCHEMICAL PREDICTION OF A NEURAL BLADDER CANCER
Marberuer M.‘, Witjes F.‘, Schmeller K.‘, Ronald D.‘, Susani M.’
Planr B.‘, Deix T.‘, Draxlcr V.‘, Haitel A.‘, Petsch M.‘, Caspers H.P.‘, Marberger M.’
1NTRODUCTION & OBJECTIVES: Neural networks have been investigated in some medical disciplines including the detection of prostate cancer in patients with low PSA-levels. In superficial bladder cancer a marker such as PSA is missing. Recently a variety of molecular markers have been tested for their ability to predict the outcome in patients with bladder cancer. Only a few give truly independent prognostic information. To explore the potential role of an artificial neural network (ANN) in predicting therapy outcome of patients with superficial bladder cancer, immunohistochemical markers were used in combination with ctinico-pathological factors.
INTRODUCTION & OBJECTIVES: Fluorescence cystoscopy has been reported to improve the detection of CIS lesions but this has not been verified in large multicenter studes. In this study, recruiting patients from 19 centres in 8 European countries, we compared the detection of CIS using hexyl aminolevulinate (HAL) fluorescence and standard white light cystoscopy. MATERIAL & METHODS: Patients suspected of having high-risk bladder cancer received instillation of 50 ml HAL 8 mM for one hour before cystoscopy. The bladder was inspected in white followed by blue light, and biopsies/ resected material of tumours and suspicious areas were assessed by central pathologist. Cystoscopy was performed with D-light (Karl Storz, Germany) allowing for both white light and blue light (375-440 nm) bladder inspection.
MATERIAL & METHODS: In 150 patients with superficial bladder cancer we investigated EGF receptor, ~53, c-erb-B2 and MDM-2 as immunohistochemical parameters and clinico-pathological factors. There were 87 Ta, 62 T 1, 23 Carcinoma in situ, 47 with BCG chemotherapy. Data were analysed using a backpropagation network with one neural layer and 45 bidden nodes. The input neural data consisted of immunohistochemical markers and tumour stage, malignancy grade, presence of a carcinoma in situ, size (tumour >3 cm) and number of resected tumours and mformation on the therapy applied (chemotherapy or immunotherapy). Explored output parameters were recurrence and progression.
RESULTS: Of 211 evaluable patients (80% male), 83 (39%) had CIS. In 17 (21%) patients HAL detected CIS lesions not seen under white light inspection. In 54% of CIS patients HAL detected additional CIS lesions compared to standard cystoscopy, and overall HAL detected significantly more CIS lesions (97%) compared to standard cystoscopy (58%). Of 92 Ta patients, HAL detected additional Ta lesions in 20 patients. Only negligible side effects were reported. including blood biochemistry and hematology. Lesion detection rate (lesions detected blue or white/detected
I CIS
1Dvsulasia
I Ta
in blue+white)
1T2
IT1
I
blue light
167 (97%)
64 (94%)
365 (97%)
79 (96%)
29 (100%)
white light total
101 (58%) 172
36 (53%) 68
329 (88%) 376
72 (88%) 82
28 (97%) 29
CONCLUSIONS: HAL fluorescence detection of CIS of the bladder.
cystoscopy
significantly
improved
early
INCREASE OUTCOME FOR PATIENTS WITH
‘Golzheim Clinic, Urology. Dusseldorf. Germany. ‘University of Vienna, Urology. Vienna, Austria, ‘University of Vienna, Pathology, Vienna, Austria
University of Vienna, Urology, Vienna, Austria. ‘University Medical Center, Urology, Nijmegen, The Netherlands, ‘St. Johanns Spital, Urology, Salrburg. Austria, ‘University of Vienna, Pathology, Vienna, Austria
I
MARKERS NETWORK
KESULTS: The overall accuracy of the neural network in predicting recurrence by use of EGF receptor, ~53, c-erb-B2 and MDM-2 was 84% with 83% sensitivity and specificity of 85%. When employing each marker as the sole predictor of recurrence the overall accuracy decreased compared to utilizing all 4 immunohistochemical markers. In high grade (G3) Tl and Ta tumours the ANN analysis became 78% sensitive and 93% specific with an overall accuracy of 85%. The performance of the ANN on stage progression by use of EGF receptor, ~53, c-erb-B2 and MDM-2 was 74%. The receiver operating curve (ROC) showed an area under the curve of 76%. CONCLUSIONS: Predicting therapy outcome with clinicopathological parameters or immunohistochemical markers as the sole predtctor by use of an ANN is unsatisfactory. Utilizing both immunohistochemical and clinicopathological parameters increases the prognostic accuracy of an ANN for bladder cancer. An ANN is a useful adjunct and provides the clinician with additional information on predicting outcome and the right choice of therapy according to the risk for recurrence and progression.
440
439 HEMATOLOGIC ERYTHROCYTES USING A FLOW METHOD
STUDY OF URINARY AND BLOOD TO IDENTIFY THE SOURCE OF HEMATURIA CYTOMETER-BASED LASER LIGHT-SCATTERING :
Anrulo J.C., Guil M.. Gonzalez J., Lopez-Rub10 M.. Sanchez-Chapado
M
Hospital Principe de Asturias, Department of Urology, Alcala de Henares. Spain INTRODUCTION & OBJECTIVES: A new noninvasive and accurate method to locate the source of hematuria, based on the comparative volumetric analysis of blood and urinary red blood cells using the Technicon H3 system, has been developed (J. Ural 1999; 162: 119). MATERIAL & METHODS: Comparative study between blood and urine implies the comparison between erythrocytes in peripheral blood diluted in urine supematant after centrifugation and urinary erythrocytes in patients with hematuria. Technicon H3 system allows independent measure of cell volume (MCV), hemoglobin concentration (MCHM), red cell distribution width (RDW) and hemoglobin distribution width (HDW). This study has been performed in a series of 148 patients with hematuria in which a glomerular (n=106) or nonglomerutar (n=42) source have been blindly identified, with the intention to determine which is the hematologic study (urinary or comparative) that better discriminates a non-glomerular source. Quantitative comparison was performed using Student’s t-test. Sensitivity, specificity. accuracy and positive and negative predictive values of urinary and comparative analysis were performed for each parameter. ROC curve analysis was also performed. RESULTS: Among the parameters studied, mean values of comparative MCV (p<.OOOl), urinary MCV (p<.OOOl), urinary MCHM (p=.OO7), and urinary HDW (p=.O18) differ according to the source of hematuria. ROC curve analysis reveal area under the curve is 98% for comparative MCV (SE ,009) 84% for urinary MCV (SE .038), 69% for urinary MCHM (SE .046), and 63% for urinary HDW (SE .54). Statistically significant differences that favour comparative MCV (2=4) can be established. With a cut-off value of 13 fl, comparative MCV reaches 91% sensitivity, 92% specificity, 97% positive predictive value, 80% negative predictive value, and 91% accuracy to detect a glomemlar source. CONCLUSIONS: Comparative volumetric analysis (i.e., the difference between MCV of erythrocytes in peripheral blood diluted in urine supernatant after centrifugation and MCV of urine erythrocytes) determined by flow cytometer-based laser light scattering method is definitely the best automatic hematologic study to be incorporated in urologists’ practice to evaluate patients with asymptomatic hematuria. European Urology Supplements 2 (2003) No. 1, pp. 112
QUANTITATIVE DETECTION OF THE TELOMERASE SUBUNITS HTR AND HTERT FOR DIAGNOSIS OF BLADDER CANCER Weikert S.. Miitler M., Krause H., Miller K Ilniversitatsklinikum
Benjamin Franklin. Dept. of Urology, Berlin. (iermany
INTRODUCTION & OBJECTIVES: Telomerasc is an enzyme found predominantly in malignant ceils. This opens new diagnostic perspectivca. However, the standard method of detecting tclomerase activity, the so-called TRAP assay, is not suitable for the diagnostic examination of urine samples. An alternative consists in the detection of the telomerase subunits hTR and hTERT using the reverse-transcriptase polymerase chain reaction (RT-PCR) method. Studies have shown that hTR and hTERT can be detected in urine samples with high sensitivity and specificity. Nevertheless. a method for quantitative evaluation would be desirable, particularly for hTR. To date, however, no prospective investigation in a large patient collective has been published. MATEFUAL & METHODS: Urine samples were obtained from 179 patients with confirmed urothelial carcinoma of the urinary bladder, 186 patients with benign urologic disease (urolithiasis, benign prostatic hypertrophy, urinary tract infections), and 100 healthy controls were collected prospectively and examined without knowledge of the diagnosis. Following extraction of RNA, a highly sensitive quantitative RT-PCR (LightCycler) was used to identify the mRNA of the catalytic subunit of telomerase, hTERT, or the RNA of the telomerase RNA, hTR. RESULTS: In urine samples dertved from patients with &firmed bladder carcinoma, hTERT was detected with a sensitivity of 55.2%. Because of the low expression of hTERT, pTa tumours loss than 1 cm in diameter were often not detected. Specificity in healthy controls was 89.2% and 85% in the entire collective. The ROC curves for quantitative RT-PCR detection of hTR yielded a sensitivity of 77% at a cut-off level of 750 copies. Specificity of hTR in healthy controls was 95.3% and 72. I % for the entire collective. False positive results were encountered particularly often in patients with urolithiasis. CONCLUSIONS: Due to the low expression of hTERT, small tumours are often not detected. Despite its very good specificity, the low sensitivity associated with the detection of hTERT in urine samples does not currently fultil diagnostic expectations. A further refinement in the quantitative RT-PCR is possible and necessary. Much more promising is the quantitative detection of hTR, particularlY in regard to the healthy control collective. Inflammatory processes in the urinary tract do not lead to false positive results.