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Quantitative detection of circulating renal cancer cells in peripheral blood samples: Experience of a two-center study
705 TWO BROTHERS AFFECTED BY RENAL CELL SAME TIME AND WITH UNUSUAL CHROMOSOME
CARClNOMA PATTERNS
AT
706 ALTERED P27 SURVIVAL OF DEMONSTRATED
KIPl EXPRESSION PREDICTS LONG-TERM PATIENTS WITH RENAL CELL CANCER AS BY TISSUE MICROARRAY ANALYSIS
Raffaele D.‘, Patrizia E.‘, Albino G.‘, Mattace Raso D.‘, Pozza M.‘, Gallucci M.’ IENEA, Cellular Toxicology, Rome, Italy, ‘Regina Elena Cancer Urology, Rome, Italy, ‘Universiti La Sapienza, Urology, Rome, Italy
Institute,
INTRODUCTION & OBJECTIVES: Renal Cell Carcinoma (RCC) is the most malignant tumour arising in the kidney and occurs mostly in sporadic form. Familiar RCC is rare and the tumours tend to be bilateral and to appear at an earlier age than in non-familiar cases. We investigated two brothers affected by RCC at the same time, yielding information on the carcinogenic process. MATERIAL & METHODS: We have studied two brothers affected by clear RCC. The brothers presented with hematuria and were diagnosed at the same time. Patient 1, a 47-year-old male, underwent unilateral total nephrectomy for a solid mass of 6 cm at the high pole of the left kidney. This tumour has been classified as pT2 NO MO RCC. Patient 2, a 44-year-old male, underwent unilateral total nephrectomy for a solid mass of 5 cm between the two poles of the left kidney. This case was also classified pT2 NO MO. Three specimens from the primary tumour and three samples from peritumoural normal kidney were taken under sterile conditions for analysis in both cases. Cytogenetic analysis was carried out on one of the normal and one of the tumour samples. and on blood samples. We used flow cytometry (FCM) to evaluate nuclear DNA content and performed cytogenetic analysis. We also carried out fluorescence in situ hybridisation (FISH) with a panel of centromeric probes for chromosomes 3, 7. 8, 9, 12. 17, 20, and Y in interphase cells. RESULTS: Flow cytometry analysis revealed diploid histograms in the tumour samples of patient 1, while an aneuploid cell and “non-malignant” subpopulation was found in the tumour and “non-malignant” samples of patient 2. Tumour samples from the two brothers were studied by FISH and a common numerical chromosome aberration was found: trisomy of chromosome 3 and 7 and monosomy and trisomy of chromosome 9 and 17. Moreover in normal samples from both brothers we found monosomy 9, and in normal sample from patient 1 we found monosomy 17. Cytogenetic analysis revealed trisomy 3 in some cells growth from normal kidney tissue of each brother. CONCLUSIONS: The identification of the same chromosome alterations both brothers appears to provide evidence of an unusual process carcinogenesis, probably due to a common genetic basis.
in of
Kuczyk M.‘, Von der Heyde E.‘, Kobierski Jonas U.’>-Serth J.’
.‘, Merseburger
A.‘, Mengel M.?,
‘Universitiy Medical School Hannover, Urology, Hannover, Germany, ‘Universitiy Medical School Hannover, Pathology, Hannover, Germany INTRODUCTION & OBJECTIVES: It has been suggested that in a variety of human malignancies the loss of the expression of the cdk inhibitor, p27 Kipl is associated with a poor clinical prognosis. For renal cell cancer the prognostic value of p27 expression has been recently indicated. In the present study the high through-put tool of tissue microarray technique has been utilized to determine the prognostic value of p27 expression in a significant number of renal cell cancer specimens. MATERIAL & METHODS: The expression of the p27 gene was investigated in 1260 tissue specimens obtained from 420 patients. For each patient the tissue microarray slides contained a tissue core obtained from histopathologically normal renal tissue, the primary tumour itself as well as from the invasion front. For immunostaining the monoclonal anti - p27 antibody (G173-524, Pharmingen) was applied. Follow - up data were available for 251 patients. Statistical evaluation was done by univariate (Log rank test) and multivariate (step wise logistic regression model) analysis. RESULTS: After a median follow - up of 138 (36-240) months, 88 of 251 patients (35%) had died from tumour progression. To perform the overall survival analysis a cut - off value of 5% for the relative amount of nuclei exhibiting a positive immunohistochemical staining reaction was applied. Decreased expression of the p27 protein within tissue cores obtained from the invasion front was significantly correlated with the clinical prognosis of the patients (univariate analysis, p=O.Ol). In a multivariate stepwise logistic regression analysis, histological grade (p
The results of our study demonstrate that low p27 expression prognostic factor in patients with renal cell carcinoma.
707
708
QUANTITATIVE DETECTION OF CIRCULATING RENAL CANCER BLOOD SAMPLES: EXPERIENCE OF A CELLS IN PERIPHERAL TWO-CENTER STUDY
REVERSE TRANSCRIPTION PCR ASSAY FOR G250 TO DETECT CIRCULATING TUMOUR CELLS IN RENAL CELL CARCINOMA (RCC): RESULTS OF A PROSPECTIVE RANDOMISED TRIAL
Iv$$$
Heidenrelch A., Schrader A., Ohlmann C., Konrad L., Miiller-Rau A
z!+ar”th
U.‘. Schmtdt U:. Bliimke Ms.‘. Fussel S.‘, Bartel F ‘, Melchior A ‘, Lmne
‘Technical Umversity Dresden, Department of Urology, Dresden. Germany. ‘Umversity HalleWittenberg, Institute “f Pathology, Halle/Saalc. Germany, ‘University Halle-Wittenberg, Chmc of Urology, Halle/Saale. Germany INTRODUCTION & OBJECTIVES: Little is known about the tum”ur biological determinants, the time c”urse and the r”ute of dissemination of renal cell carcinoma (RCC). Moreover, the clmical staging does not address the true biological significance of the disease in many cases. The purpose of this study was to demonstrate the efficacy of an enrichment protocol for the detection of circulating turnour cells in the bloodstream of RCC patients based on an optimwed autoMACS CD45 leukocyte depletion strategy (I, 2). The major results of the comparative prospective study in tw” urological centres will be presented and summanzed. MATERIAL & METHODS: At least eight mL ven”us blood samples (BS) where collected prior or after nephrectomy. Mononuclear ceils were isolated by ficoll gradient separation Afterwards, leukocytes were depleted by a standardized and vahdated CD45 autoMACS depletion protocol (2). Cytokeratin (CK) immunocytochemistry was performed using a pan-CK or a CK-8118 antibody. Cytospin slides were evaluated by light microscopy, and tumour cells were counted by a pathologist wth long-term experience in patho-morphological assessment of disseminated tum”ur cells. The numbers of abnormal cells with no, weak or distinct CK expression per BS were calculaled. RESULTS: WC Investigated 279 BS from I53 RCC patients (50 females, 103 males) from our two urologic clinics during the past three years. Turnour cells were identified in X9 BS (32%) from 56 patients (37%). Applying two different immunocytochemical staimng protocols (panCK vs. CK8/18 antibody) the equal rates of positive stained tum”ur cells were observed. However, we also detected many turnour cells (for about half of all cases) without any or with only a very weak CK expression. A.correlation between tum”ur cell number and turnour grading, and an increase of the number of patients having circulating tllmour cells with an advanced turnour stage was observed. Additionally, preliminary results of long-term reevaluation and the follow-up data of s”me of the patients indicate that the detection of circulating tum”ur cells in the blood before and/or immediately after surgery is assoaated often with a relative early tllmour relapse. CONCLUSIONS: The apphed aut”MACS technique resulted in a high rate of depletion of leukocytes and guaranteed an efficient enrichment of tum”ur cells. This allows the search for turnour cells in up to 16 mL whole BS on a single slide. The similar results for both study centres indicate the reproducibihty of the applied isolation methodology (I ,2) and the potential efficacy for future multlcenter studies. Beside the potential impact of this BS-based tumour cell detection method for a molecular tum”ur staging and a molecular determination of prognosis the value of the strategy for monitoring localized and advanced RCC under vari”us treatment options is an attractive field of future studies. References: (I) Bilkenroth et al. Int J Cancer 92: 577-5X2. 2001; (2) Meyc et al. Int J Oncol 21: 521.530, 2002
Phdipps
University, Department of Urology, Marburg, Germany
INTRODUCTION & OBJECTIVES: Radical nephrectomy represents the standard surgical approach to patients with RCC. The retroperitoneal approach (RP) or the transperitoneal route (TP) with early hgation the renal artery to avoid tumour cell dissemination are 2 competing procedures. The impact of the surgical approach on turnour cell shedding, however, has never been evaluated. Recently, G250 has been identified as specific RCC-associated antigen. Purpose of the study was to evaluate the perioperative G250 expression m peripheral blood of patients undergoing radical nephrectomy by the RP or TP route. MATERIAL & METHODS: 68 consecutwe patients wth RCC were prospectively random&d to radical Nx via the RP (n=37) or the TP (n=3 I) approach. RP-Nx included early mobilization of the kidney and secondary ligature of the RA whereas in TP-Nx the RA was ligated prior t” mobihzation. In all pts, blood specimens were collected preoperatively, mtraoperatively (skin incision, skin closure), on postop day I and at hospital discharge. RNA from serum was extracted by TRlzol; following cDNA synthesis PCR for G250 expression was performed with specific primers. RT-PCR for GAPDH served as internal positive control. RESULTS: There were no sigmficant differences between the groups with regard to age, gender or tumour stage. GAPDH expression was positive in all RCC’s and benign renal turnours. Although there was a difference in the frequency of periop. turnour cell shedding. no differences were observed at time of discharge (Table I). Intraop. 16% and 29% of initially G250 negative RCC’s produced positive RTPCR signals during the RP and TP approach, rap. At time of hospital discharge RTPCR was still positwe in IO pts. 8 with a pT3b or pTxNl-2MI RCC and 2 with a DT~ tumour. Preoperative
lntraoperative
Postoperative
Hospital discharge
TP-Nw
50%
57.8%
52.6%
25%
RP-Nx
42%
45%
25%
27.6%
CONCLUSIONS: For the first time, the impact of the surgical approach on tumour cell shedding was evaluated in RCC. Based on our results, the RP approach has no negative impact on postop. turnour cell sheddmg. Although there was no significant correlatton to turnour stage, all metastatic RCC exhibited positive G250 signals at time of discharge. Currently, the role of G250-specific RTPCR for early detection of relapse 1s investigated during f”llow-up.
European
Urology Supplements
2 (2003) No. 1,
pp. 179
CARClNOMA PATTERNS
AT
706 ALTERED P27 SURVIVAL OF DEMONSTRATED
KIPl EXPRESSION PREDICTS LONG-TERM PATIENTS WITH RENAL CELL CANCER AS BY TISSUE MICROARRAY ANALYSIS
Raffaele D.‘, Patrizia E.‘, Albino G.‘, Mattace Raso D.‘, Pozza M.‘, Gallucci M.’ IENEA, Cellular Toxicology, Rome, Italy, ‘Regina Elena Cancer Urology, Rome, Italy, ‘Universiti La Sapienza, Urology, Rome, Italy
Institute,
INTRODUCTION & OBJECTIVES: Renal Cell Carcinoma (RCC) is the most malignant tumour arising in the kidney and occurs mostly in sporadic form. Familiar RCC is rare and the tumours tend to be bilateral and to appear at an earlier age than in non-familiar cases. We investigated two brothers affected by RCC at the same time, yielding information on the carcinogenic process. MATERIAL & METHODS: We have studied two brothers affected by clear RCC. The brothers presented with hematuria and were diagnosed at the same time. Patient 1, a 47-year-old male, underwent unilateral total nephrectomy for a solid mass of 6 cm at the high pole of the left kidney. This tumour has been classified as pT2 NO MO RCC. Patient 2, a 44-year-old male, underwent unilateral total nephrectomy for a solid mass of 5 cm between the two poles of the left kidney. This case was also classified pT2 NO MO. Three specimens from the primary tumour and three samples from peritumoural normal kidney were taken under sterile conditions for analysis in both cases. Cytogenetic analysis was carried out on one of the normal and one of the tumour samples. and on blood samples. We used flow cytometry (FCM) to evaluate nuclear DNA content and performed cytogenetic analysis. We also carried out fluorescence in situ hybridisation (FISH) with a panel of centromeric probes for chromosomes 3, 7. 8, 9, 12. 17, 20, and Y in interphase cells. RESULTS: Flow cytometry analysis revealed diploid histograms in the tumour samples of patient 1, while an aneuploid cell and “non-malignant” subpopulation was found in the tumour and “non-malignant” samples of patient 2. Tumour samples from the two brothers were studied by FISH and a common numerical chromosome aberration was found: trisomy of chromosome 3 and 7 and monosomy and trisomy of chromosome 9 and 17. Moreover in normal samples from both brothers we found monosomy 9, and in normal sample from patient 1 we found monosomy 17. Cytogenetic analysis revealed trisomy 3 in some cells growth from normal kidney tissue of each brother. CONCLUSIONS: The identification of the same chromosome alterations both brothers appears to provide evidence of an unusual process carcinogenesis, probably due to a common genetic basis.
in of
Kuczyk M.‘, Von der Heyde E.‘, Kobierski Jonas U.’>-Serth J.’
.‘, Merseburger
A.‘, Mengel M.?,
‘Universitiy Medical School Hannover, Urology, Hannover, Germany, ‘Universitiy Medical School Hannover, Pathology, Hannover, Germany INTRODUCTION & OBJECTIVES: It has been suggested that in a variety of human malignancies the loss of the expression of the cdk inhibitor, p27 Kipl is associated with a poor clinical prognosis. For renal cell cancer the prognostic value of p27 expression has been recently indicated. In the present study the high through-put tool of tissue microarray technique has been utilized to determine the prognostic value of p27 expression in a significant number of renal cell cancer specimens. MATERIAL & METHODS: The expression of the p27 gene was investigated in 1260 tissue specimens obtained from 420 patients. For each patient the tissue microarray slides contained a tissue core obtained from histopathologically normal renal tissue, the primary tumour itself as well as from the invasion front. For immunostaining the monoclonal anti - p27 antibody (G173-524, Pharmingen) was applied. Follow - up data were available for 251 patients. Statistical evaluation was done by univariate (Log rank test) and multivariate (step wise logistic regression model) analysis. RESULTS: After a median follow - up of 138 (36-240) months, 88 of 251 patients (35%) had died from tumour progression. To perform the overall survival analysis a cut - off value of 5% for the relative amount of nuclei exhibiting a positive immunohistochemical staining reaction was applied. Decreased expression of the p27 protein within tissue cores obtained from the invasion front was significantly correlated with the clinical prognosis of the patients (univariate analysis, p=O.Ol). In a multivariate stepwise logistic regression analysis, histological grade (p
The results of our study demonstrate that low p27 expression prognostic factor in patients with renal cell carcinoma.
707
708
QUANTITATIVE DETECTION OF CIRCULATING RENAL CANCER BLOOD SAMPLES: EXPERIENCE OF A CELLS IN PERIPHERAL TWO-CENTER STUDY
REVERSE TRANSCRIPTION PCR ASSAY FOR G250 TO DETECT CIRCULATING TUMOUR CELLS IN RENAL CELL CARCINOMA (RCC): RESULTS OF A PROSPECTIVE RANDOMISED TRIAL
Iv$$$
Heidenrelch A., Schrader A., Ohlmann C., Konrad L., Miiller-Rau A
z!+ar”th
U.‘. Schmtdt U:. Bliimke Ms.‘. Fussel S.‘, Bartel F ‘, Melchior A ‘, Lmne
‘Technical Umversity Dresden, Department of Urology, Dresden. Germany. ‘Umversity HalleWittenberg, Institute “f Pathology, Halle/Saalc. Germany, ‘University Halle-Wittenberg, Chmc of Urology, Halle/Saale. Germany INTRODUCTION & OBJECTIVES: Little is known about the tum”ur biological determinants, the time c”urse and the r”ute of dissemination of renal cell carcinoma (RCC). Moreover, the clmical staging does not address the true biological significance of the disease in many cases. The purpose of this study was to demonstrate the efficacy of an enrichment protocol for the detection of circulating turnour cells in the bloodstream of RCC patients based on an optimwed autoMACS CD45 leukocyte depletion strategy (I, 2). The major results of the comparative prospective study in tw” urological centres will be presented and summanzed. MATERIAL & METHODS: At least eight mL ven”us blood samples (BS) where collected prior or after nephrectomy. Mononuclear ceils were isolated by ficoll gradient separation Afterwards, leukocytes were depleted by a standardized and vahdated CD45 autoMACS depletion protocol (2). Cytokeratin (CK) immunocytochemistry was performed using a pan-CK or a CK-8118 antibody. Cytospin slides were evaluated by light microscopy, and tumour cells were counted by a pathologist wth long-term experience in patho-morphological assessment of disseminated tum”ur cells. The numbers of abnormal cells with no, weak or distinct CK expression per BS were calculaled. RESULTS: WC Investigated 279 BS from I53 RCC patients (50 females, 103 males) from our two urologic clinics during the past three years. Turnour cells were identified in X9 BS (32%) from 56 patients (37%). Applying two different immunocytochemical staimng protocols (panCK vs. CK8/18 antibody) the equal rates of positive stained tum”ur cells were observed. However, we also detected many turnour cells (for about half of all cases) without any or with only a very weak CK expression. A.correlation between tum”ur cell number and turnour grading, and an increase of the number of patients having circulating tllmour cells with an advanced turnour stage was observed. Additionally, preliminary results of long-term reevaluation and the follow-up data of s”me of the patients indicate that the detection of circulating tum”ur cells in the blood before and/or immediately after surgery is assoaated often with a relative early tllmour relapse. CONCLUSIONS: The apphed aut”MACS technique resulted in a high rate of depletion of leukocytes and guaranteed an efficient enrichment of tum”ur cells. This allows the search for turnour cells in up to 16 mL whole BS on a single slide. The similar results for both study centres indicate the reproducibihty of the applied isolation methodology (I ,2) and the potential efficacy for future multlcenter studies. Beside the potential impact of this BS-based tumour cell detection method for a molecular tum”ur staging and a molecular determination of prognosis the value of the strategy for monitoring localized and advanced RCC under vari”us treatment options is an attractive field of future studies. References: (I) Bilkenroth et al. Int J Cancer 92: 577-5X2. 2001; (2) Meyc et al. Int J Oncol 21: 521.530, 2002
Phdipps
University, Department of Urology, Marburg, Germany
INTRODUCTION & OBJECTIVES: Radical nephrectomy represents the standard surgical approach to patients with RCC. The retroperitoneal approach (RP) or the transperitoneal route (TP) with early hgation the renal artery to avoid tumour cell dissemination are 2 competing procedures. The impact of the surgical approach on turnour cell shedding, however, has never been evaluated. Recently, G250 has been identified as specific RCC-associated antigen. Purpose of the study was to evaluate the perioperative G250 expression m peripheral blood of patients undergoing radical nephrectomy by the RP or TP route. MATERIAL & METHODS: 68 consecutwe patients wth RCC were prospectively random&d to radical Nx via the RP (n=37) or the TP (n=3 I) approach. RP-Nx included early mobilization of the kidney and secondary ligature of the RA whereas in TP-Nx the RA was ligated prior t” mobihzation. In all pts, blood specimens were collected preoperatively, mtraoperatively (skin incision, skin closure), on postop day I and at hospital discharge. RNA from serum was extracted by TRlzol; following cDNA synthesis PCR for G250 expression was performed with specific primers. RT-PCR for GAPDH served as internal positive control. RESULTS: There were no sigmficant differences between the groups with regard to age, gender or tumour stage. GAPDH expression was positive in all RCC’s and benign renal turnours. Although there was a difference in the frequency of periop. turnour cell shedding. no differences were observed at time of discharge (Table I). Intraop. 16% and 29% of initially G250 negative RCC’s produced positive RTPCR signals during the RP and TP approach, rap. At time of hospital discharge RTPCR was still positwe in IO pts. 8 with a pT3b or pTxNl-2MI RCC and 2 with a DT~ tumour. Preoperative
lntraoperative
Postoperative
Hospital discharge
TP-Nw
50%
57.8%
52.6%
25%
RP-Nx
42%
45%
25%
27.6%
CONCLUSIONS: For the first time, the impact of the surgical approach on tumour cell shedding was evaluated in RCC. Based on our results, the RP approach has no negative impact on postop. turnour cell sheddmg. Although there was no significant correlatton to turnour stage, all metastatic RCC exhibited positive G250 signals at time of discharge. Currently, the role of G250-specific RTPCR for early detection of relapse 1s investigated during f”llow-up.
European
Urology Supplements
2 (2003) No. 1,
pp. 179