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IL6 And TGF-betai modulate the invasion of primary renal carcinoma cultures into an artificial stroma
554
553 PROGNOSTIC SIGNIFICANCE IN RENAL CELL CARCINOMA Tasca Andrea’,
Meneghi Agostino’.
OF VASCULAR
LYMPHOCYTE SUBPOPULATIONS: PROGNOSTIC VALUE OF PATIENTS WITH RENAL CELL CARCINOMA UNDER THERAPY WITH IL-2 AND IFN-ALPHA
MICROEMBOLISM
Dante Stefania’
‘Department of Pathology, S. Bortolo Hospital, Urology. S. Bortolo Hospital, Vicenra, Italy
Vicenza, Italy, *Department
of
INTRODUCTION & OBJECTIVES: The prognosis of Renal Cell Carcinoma (RCC) is related to the pathological stage and to the instrinsic ability of the disease to metastasise. Aim of the study is to evaluate the microvascular anatomy of the tumour and to correlate it with the metastatic potential. MATERIALS & METHODS: Eighty-three cases of RCC, operated from February 1996 and December 1998 were retrospectively evaluated. Histopathologic findings were classified according to UICC and AJCC 1997 in renal cell carcinoma, papillary carcinoma and cromophobic carcinoma. Nuclear grading was according to Fuhrman 4 classes classification. Pathologic stages were pTI-2=43, pT3=40 cases respectively. Primary antibody CD 3 I (Biogenix) I:50 dilution was utiliaed to study the tumour vascular network. RESULTS: The vascular network of all RCC was similar with thin capillary vessels of constant diameter. Small cellular clusters of 5-6 cells were surrounded by these small vessels in low-grade turnouts. This capillary network around neoplastic cells was interrupted and focal or diffuse necrosis was present in higher grade RCC. Neoplastic cells were present inside these capillaries in 17% and 68% of pTl-2 of pT3 patients, respectively. The percentage of patients with progressive disease or dead because of metaatatic disease was 6% and 32% in the two groups. In all these patients a neoplastic microembolism was evident. CONCLUSION: A separate connection between the neoplastic capillary network and the vessels of supportive structures around the tumour and between the latter and normal intrarenal vessels is a suggestive hypothesis coming from this paper. The neoplaatic vascular network seems not to have a direct connection with normal renal vessels: a filter should be present at the tumour psudocapsule. According to this hypothesis neoplastic cells circulate inside the intratumoural capillary network only, without gaining access to normal intrarenal and systemic circulation. When the neoplastic growth infiltrates the surrounding structures, a direct access to normal intrarenal vascular network i\ reached, with the possibility of direct anastomosis between neoplastic and normal vessels and subsequent easier metastatic spread. This could be the theoretic support to the therapeutic success in conservative treatment of small RCC in which the integrity of tumour psudocapaule is still present.
Hillie Felix Paolo’
‘, Schneider Thomas’, Zacharias Mario’, Langner Jiirgen’, Fornara
‘Department of Urology, Martin-Luther-University, HalIe/.%, Germany. ‘Department of Immunology, Martin-Luther-University, Halle/Sa. Germany INTRODUCTION & OBJECTIVES: The immune-therapy of the metastatic renal cell carcinoma with interferon alone or in combination with Interleukin-2 represents the therapy of choice at present. Despite intensive efforts, to improve the therapy regarding its effectiveness, the curation could be lifted only unimportantly on over 30%. On reason of the restrictions of the quality of life through the side effects of the cytokines (general condition, fever. accomplishment-decrease), it always participates the goal to decide particularly patients without outlook on a therapy-success. MATERIALS & METHODS: Immun-phaenotyping in the flow-cytometry was overseen 161 immune-therapy-cycles with 95 patients at the department of urology of the Martin-Luther-University Hall-Wittenberg and was analysed. The Lymphocyte subpopulations, total-lymphocytes, total-T-cells, CD4+-T-cells, CD&T-cells, CD2S+-T-cells, HLA-DR+-T-cells, NK-cells and CD57+-cells became as parameter of the immunological activation. By means of the statistical methods (PEARSON, TTest, ANOVA). the curve-courses as well as the values of the individual weeks were examined in reference for the groups complete remission (CR), partial remission (PR). stable disease (SD) and progress (P). RESULTS: In the course of the immune-therapy, an increase of all measured subpopulations was to be proved. The typical curve-course was discontinuous with two maxima in week 3 and 5. In the comparison of the groups I (CR+PR+SD) and 111 (P). CD4+/CDX+-T-cells could for the courses of the CD4+-T-cells as share of the total-T-lymphocytes of the quotient and are determined significantly different courses for the share of NK-cells in the total-lymphocytes. The statements of the literature to the prognostic meaning of CD2S+-T-cells and HLA-DR+-T-cells could not be confirmed lacking statistical significance on reason. With the analysis of individual subpopulations in the individual weeks, 3 and 5 could the increase of NK-cells as share of the total-lymphocytes in week, the increase of the CD57+-cells as share of the NK-cells in week 3 as well as the increase of the CD4+-T-Lymphozyten as share of the T-lymphocytes also in week 3 as significantly differently between the groups I and III is secured. CONCLUSION: From clinical view. the ability of the immune system participates with the analysis of the NK-cells. CD57+-cells, T-cells and CD4+-T-cell5 in the third week to the activation (CD4+-T-cells) and to the tumour-resistance, NK-cells.
556
555 ANALYSIS OF T IMMUNOTHERAPY CELL CARCINOMA
HELPER TYPE IN PATIENTS (MRCC)
1 AND 2 CELLS WITH METASTATIC
DURING RENAL
IL6 AND TGF-BETA1 MODULATE THE INVASION OF PRIMARY RENAL CARCINOMA CULTURES INTO AN ARTIFICIAL STROMA Steiner
Heinzer Hans, Huland Edith, Stephan Miriam, Huland Hartwig Department
of Urology, University
Clinic Hamburg.
Hamburg,
Germany
INTRODUCTION & OBJECTIVES: T helper type I (Thl) and type 2 (Th2) play important immunoregulatory roles, which are categorised as Thl and Th2 on the basis of cytokine production. Th I produce interferon (IFN)-g, while Th2 secrete interleukin (IL)-4 and IL- IO. Recent studies have demonstrated that elevated Th2 cytokines are associated with poor prognosis in cancer patients. The role of Thl and Th2 cells and their alteration during immunotherapy for progressive mRCC is unclear. MATERIALS & METHODS: Peripheral blood lymphocytes (PBL) were obtained from I5 patients before and during IL-2 based immunotherapy. Levels of IFN-g, IL-4, IL-IO and IL-12 were measured by ELISA in a culture of stimulated PBL. For quantitative analysis of IL-4 and IFN-g mRNA competitive RT-PCR was used. The results of the cytokine measurement were correlated to the response to immunotherapy. RESULTS: 7115 patients responded to immunotherapy with atabilisation or partial response. During immunotherapy responding patients showed on the protein level a decrease of Th2 cytokines IL-4 (median 13.2 vs. 9.5 rig/ml). ILIO (median 544.5 vs. 240.5 @ml) and Thl cytokine IFN-g (median I41 vs.43.7 ngiml) compared to levels before treatment. IL-12 values were unchanged (median 2 vs. 2.9 ngiml). Expression of mRNA showed also a decrease of IL-4 mRNA (median copies per cell 794 vs. 112) and IFN-g mRNA (median copies per cell I I9 VS. 39). In contrast in patients with progressive disease Th2 cytokines increased. detected by ELISA (IL-4: I .4 vs. 8.3. IL-IO: 249 v\. 268 rig/ml) and RT-PCR on the RNA level (IL-4 mRNA: IS0 vs. 209). CONCLUSION: Analysis of T helper types showed a Th2-dominant subtype in mRCC patients by RT-PCR. Although the trend was not significant in this small group of patients, we found a predominantly depression of Th2 cells during immunotherapy. In contrast patient\ with propresjive disease showed increasing Th2 cell numbers associated with poor prognosis. Analysis of T helper types might be helpful in monitoring immunotherapiec and identifying patients under risk not responding to therapy.
Thomas’,Junker Udo’, Nuernberger Steffi”, Schubert Joerg’
‘Urology. Friedrich-Schiller-University, Jena. Friedrich-Schiller-University, Jena. Germany
Germany,
‘Immunology,
INTRODUCTION & OBJECTIVES: To investigate a possible role of the cytokines IL-6 and TGF-betal in the process of invasion of renal cell carcinoma (RCC) into an artificial stroma. MATERIALS & METHODS: Primary cultures of RCC (n=15) and control tissue (n=4) were derived immediately after surgical removal of tumours and/or metastases. After testing for cytokeratin (exclusion of fibroblasts), CellTracker”” (Molecular Probes)-labelled RCC were plated onto a cushion of fibroblasts in collagen-I. During IO-14 days cultured cells were re-fed every second day with medium containing IL-6 (IO ngiml), TGF-beta1 (IO @ml) or anti-IL6 (2 mkgiml, neutralises up to 40 ngiml IL6). By serial sectioning and immunocytochemistry expression of proteases and pattern of invasion was assessed. A parameter derived from the area/circumference ratio (Q= I for circle, larger for all other forms) of the tumour cell compartment was used to determine invasive growth. RESULTS: Control tissue renal epithelium forms a straight layer atop the \tromu or forma a lump of cells surrounded by the collagenifibroblast mix. No invasion is observed. RCC cells behave variably. Typically, in non-stimulated cultures, a compact block of proliferating tumour and stroma cells is observed. No invasion occurs (Q=l). However, in certain tumours IL6 leads to a diffuse invasion of single tumour cells (Q=S.6), while TGF-betal yields both proliferation of tumour and stroma cells and invasion of string-like tumour cell aggregates (Q=3.3). lmmunhistochemically strong expression of cathepsin B and L was detected in the borderline between tumour cells and stroma fibroblasts, whereas MMP-2 and -9 were expressed weaker and diffuse. CONCLUSION: The cytokines IL6 and TGF-beta1 modulate the invasive behaviour of RCC. This is of special interest because of the detected elevated serum/plasma levels of TGF-betal in kidney cancer patients. European
Urology
Supplements
1 (2002) No. 1, pp. 141
553 PROGNOSTIC SIGNIFICANCE IN RENAL CELL CARCINOMA Tasca Andrea’,
Meneghi Agostino’.
OF VASCULAR
LYMPHOCYTE SUBPOPULATIONS: PROGNOSTIC VALUE OF PATIENTS WITH RENAL CELL CARCINOMA UNDER THERAPY WITH IL-2 AND IFN-ALPHA
MICROEMBOLISM
Dante Stefania’
‘Department of Pathology, S. Bortolo Hospital, Urology. S. Bortolo Hospital, Vicenra, Italy
Vicenza, Italy, *Department
of
INTRODUCTION & OBJECTIVES: The prognosis of Renal Cell Carcinoma (RCC) is related to the pathological stage and to the instrinsic ability of the disease to metastasise. Aim of the study is to evaluate the microvascular anatomy of the tumour and to correlate it with the metastatic potential. MATERIALS & METHODS: Eighty-three cases of RCC, operated from February 1996 and December 1998 were retrospectively evaluated. Histopathologic findings were classified according to UICC and AJCC 1997 in renal cell carcinoma, papillary carcinoma and cromophobic carcinoma. Nuclear grading was according to Fuhrman 4 classes classification. Pathologic stages were pTI-2=43, pT3=40 cases respectively. Primary antibody CD 3 I (Biogenix) I:50 dilution was utiliaed to study the tumour vascular network. RESULTS: The vascular network of all RCC was similar with thin capillary vessels of constant diameter. Small cellular clusters of 5-6 cells were surrounded by these small vessels in low-grade turnouts. This capillary network around neoplastic cells was interrupted and focal or diffuse necrosis was present in higher grade RCC. Neoplastic cells were present inside these capillaries in 17% and 68% of pTl-2 of pT3 patients, respectively. The percentage of patients with progressive disease or dead because of metaatatic disease was 6% and 32% in the two groups. In all these patients a neoplastic microembolism was evident. CONCLUSION: A separate connection between the neoplastic capillary network and the vessels of supportive structures around the tumour and between the latter and normal intrarenal vessels is a suggestive hypothesis coming from this paper. The neoplaatic vascular network seems not to have a direct connection with normal renal vessels: a filter should be present at the tumour psudocapsule. According to this hypothesis neoplastic cells circulate inside the intratumoural capillary network only, without gaining access to normal intrarenal and systemic circulation. When the neoplastic growth infiltrates the surrounding structures, a direct access to normal intrarenal vascular network i\ reached, with the possibility of direct anastomosis between neoplastic and normal vessels and subsequent easier metastatic spread. This could be the theoretic support to the therapeutic success in conservative treatment of small RCC in which the integrity of tumour psudocapaule is still present.
Hillie Felix Paolo’
‘, Schneider Thomas’, Zacharias Mario’, Langner Jiirgen’, Fornara
‘Department of Urology, Martin-Luther-University, HalIe/.%, Germany. ‘Department of Immunology, Martin-Luther-University, Halle/Sa. Germany INTRODUCTION & OBJECTIVES: The immune-therapy of the metastatic renal cell carcinoma with interferon alone or in combination with Interleukin-2 represents the therapy of choice at present. Despite intensive efforts, to improve the therapy regarding its effectiveness, the curation could be lifted only unimportantly on over 30%. On reason of the restrictions of the quality of life through the side effects of the cytokines (general condition, fever. accomplishment-decrease), it always participates the goal to decide particularly patients without outlook on a therapy-success. MATERIALS & METHODS: Immun-phaenotyping in the flow-cytometry was overseen 161 immune-therapy-cycles with 95 patients at the department of urology of the Martin-Luther-University Hall-Wittenberg and was analysed. The Lymphocyte subpopulations, total-lymphocytes, total-T-cells, CD4+-T-cells, CD&T-cells, CD2S+-T-cells, HLA-DR+-T-cells, NK-cells and CD57+-cells became as parameter of the immunological activation. By means of the statistical methods (PEARSON, TTest, ANOVA). the curve-courses as well as the values of the individual weeks were examined in reference for the groups complete remission (CR), partial remission (PR). stable disease (SD) and progress (P). RESULTS: In the course of the immune-therapy, an increase of all measured subpopulations was to be proved. The typical curve-course was discontinuous with two maxima in week 3 and 5. In the comparison of the groups I (CR+PR+SD) and 111 (P). CD4+/CDX+-T-cells could for the courses of the CD4+-T-cells as share of the total-T-lymphocytes of the quotient and are determined significantly different courses for the share of NK-cells in the total-lymphocytes. The statements of the literature to the prognostic meaning of CD2S+-T-cells and HLA-DR+-T-cells could not be confirmed lacking statistical significance on reason. With the analysis of individual subpopulations in the individual weeks, 3 and 5 could the increase of NK-cells as share of the total-lymphocytes in week, the increase of the CD57+-cells as share of the NK-cells in week 3 as well as the increase of the CD4+-T-Lymphozyten as share of the T-lymphocytes also in week 3 as significantly differently between the groups I and III is secured. CONCLUSION: From clinical view. the ability of the immune system participates with the analysis of the NK-cells. CD57+-cells, T-cells and CD4+-T-cell5 in the third week to the activation (CD4+-T-cells) and to the tumour-resistance, NK-cells.
556
555 ANALYSIS OF T IMMUNOTHERAPY CELL CARCINOMA
HELPER TYPE IN PATIENTS (MRCC)
1 AND 2 CELLS WITH METASTATIC
DURING RENAL
IL6 AND TGF-BETA1 MODULATE THE INVASION OF PRIMARY RENAL CARCINOMA CULTURES INTO AN ARTIFICIAL STROMA Steiner
Heinzer Hans, Huland Edith, Stephan Miriam, Huland Hartwig Department
of Urology, University
Clinic Hamburg.
Hamburg,
Germany
INTRODUCTION & OBJECTIVES: T helper type I (Thl) and type 2 (Th2) play important immunoregulatory roles, which are categorised as Thl and Th2 on the basis of cytokine production. Th I produce interferon (IFN)-g, while Th2 secrete interleukin (IL)-4 and IL- IO. Recent studies have demonstrated that elevated Th2 cytokines are associated with poor prognosis in cancer patients. The role of Thl and Th2 cells and their alteration during immunotherapy for progressive mRCC is unclear. MATERIALS & METHODS: Peripheral blood lymphocytes (PBL) were obtained from I5 patients before and during IL-2 based immunotherapy. Levels of IFN-g, IL-4, IL-IO and IL-12 were measured by ELISA in a culture of stimulated PBL. For quantitative analysis of IL-4 and IFN-g mRNA competitive RT-PCR was used. The results of the cytokine measurement were correlated to the response to immunotherapy. RESULTS: 7115 patients responded to immunotherapy with atabilisation or partial response. During immunotherapy responding patients showed on the protein level a decrease of Th2 cytokines IL-4 (median 13.2 vs. 9.5 rig/ml). ILIO (median 544.5 vs. 240.5 @ml) and Thl cytokine IFN-g (median I41 vs.43.7 ngiml) compared to levels before treatment. IL-12 values were unchanged (median 2 vs. 2.9 ngiml). Expression of mRNA showed also a decrease of IL-4 mRNA (median copies per cell 794 vs. 112) and IFN-g mRNA (median copies per cell I I9 VS. 39). In contrast in patients with progressive disease Th2 cytokines increased. detected by ELISA (IL-4: I .4 vs. 8.3. IL-IO: 249 v\. 268 rig/ml) and RT-PCR on the RNA level (IL-4 mRNA: IS0 vs. 209). CONCLUSION: Analysis of T helper types showed a Th2-dominant subtype in mRCC patients by RT-PCR. Although the trend was not significant in this small group of patients, we found a predominantly depression of Th2 cells during immunotherapy. In contrast patient\ with propresjive disease showed increasing Th2 cell numbers associated with poor prognosis. Analysis of T helper types might be helpful in monitoring immunotherapiec and identifying patients under risk not responding to therapy.
Thomas’,Junker Udo’, Nuernberger Steffi”, Schubert Joerg’
‘Urology. Friedrich-Schiller-University, Jena. Friedrich-Schiller-University, Jena. Germany
Germany,
‘Immunology,
INTRODUCTION & OBJECTIVES: To investigate a possible role of the cytokines IL-6 and TGF-betal in the process of invasion of renal cell carcinoma (RCC) into an artificial stroma. MATERIALS & METHODS: Primary cultures of RCC (n=15) and control tissue (n=4) were derived immediately after surgical removal of tumours and/or metastases. After testing for cytokeratin (exclusion of fibroblasts), CellTracker”” (Molecular Probes)-labelled RCC were plated onto a cushion of fibroblasts in collagen-I. During IO-14 days cultured cells were re-fed every second day with medium containing IL-6 (IO ngiml), TGF-beta1 (IO @ml) or anti-IL6 (2 mkgiml, neutralises up to 40 ngiml IL6). By serial sectioning and immunocytochemistry expression of proteases and pattern of invasion was assessed. A parameter derived from the area/circumference ratio (Q= I for circle, larger for all other forms) of the tumour cell compartment was used to determine invasive growth. RESULTS: Control tissue renal epithelium forms a straight layer atop the \tromu or forma a lump of cells surrounded by the collagenifibroblast mix. No invasion is observed. RCC cells behave variably. Typically, in non-stimulated cultures, a compact block of proliferating tumour and stroma cells is observed. No invasion occurs (Q=l). However, in certain tumours IL6 leads to a diffuse invasion of single tumour cells (Q=S.6), while TGF-betal yields both proliferation of tumour and stroma cells and invasion of string-like tumour cell aggregates (Q=3.3). lmmunhistochemically strong expression of cathepsin B and L was detected in the borderline between tumour cells and stroma fibroblasts, whereas MMP-2 and -9 were expressed weaker and diffuse. CONCLUSION: The cytokines IL6 and TGF-beta1 modulate the invasive behaviour of RCC. This is of special interest because of the detected elevated serum/plasma levels of TGF-betal in kidney cancer patients. European
Urology
Supplements
1 (2002) No. 1, pp. 141