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Transitional Cell Carcinoma (TCC) of the Renal Pelvis: Nuclear DNA Ploidy by Flow Cytometry
Accepted 473
474
HEPATIC DYSFUNCTION SYNDROME(HDS) ASSOCIATED WITH RENAL CELL CARCINOMA(RCC): PRELIMINARY CHARACTERIZATION STUDIES. Harold
TRANSITIONAL CELL CARCINOMA (TCC) OF THE RENAL PELVIS: NUCLEAR DNA PLOIDY BY FLOW CYTOMETRY. Michael L. Blute*, Kenichi Tsushima,* George M. Farrow,* Michael M. Lieber, Rochester, MN (Presentation to be made by Dr. Blute) A flow cytometric method for determining the nuclear DNA ploidy pattern of cells from formalin-fixed paraffin-embedded tissue has been described by Hedley et al (J Histochem Cytochem 31: 1322, 1983). This method a 11 ows 1 aboratory analysis of tumor specimens from groups of patients treated many years previously whose clinical outcome is now known. The Hedley method was carried out on tumor specimens from 111 patients with TCC of the renal pelvis; nuclei were stained with propidium iodide using the Vindelov method (Cytometry 3:323, 1983) and run on a FACS IV Flow Cytometer. Patients underwent surgery between 1960 and 1975. DNA histograms were classified as DNA diploid (or normal), DNA tetraploid (with a significant increase in the 4C peak), or DNA aneuploid. Results and clinical outcome were: DNA Histogram Pattern No. Dead from Metastatic TCC DNA diploid 38 5 (13%) DNA tetraploid 54 21 (39%)* (*p<0.02) DNA aneuploid 19 15 (79%)** (**p<0.001) There was an excellent correlation between histolog1c grade, DNA ploidy pattern, and clinical outcome. No patient with a grade l tumor (n=28) had a DNA aneuploid pattern and none died from TCC. Thirty of 31 patients with grade 3-4 tumors had an abnormal DNA pattern (DNA tetraploid or aneuploid); 26 (84%) died from metastatic TCC. For grade 2 TCC, patients with tumors with DNA aneuploid patterns did markedly worse than patients with tumors with DNA diploid patterns. With well-differentiated and poorlydifferentiated TCC of the renal pelvis, DNA ploidy pattern simply correlated well with histologic grade assigned by an experienced pathologist. For grade 2 TCC, DNA ploidy analysis identified a prognostically important group of DNA abnormal tumors with poor outcome not detectable by light microscopic grading.
N.Keer*,William Coukos*,Julia Sensibar*,Livia Arvay*, Roger Kroes*,James M.Kozlowski*, and Chung Lee*, Chicago, IL. (Presentation to be made by Dr.Coukos) HDS, seen in about 10% of RCC patients, is presumed to result from a hepatotoxin of RCC origin. Since the etiology
of HOS is unknown, a cell line(BA1119) was established from a patient with RCC and clinical HDS in an attempt to iden.;--
tify the putative hepatotoxin. BA1119, when injected s.c, into nude mice resulted in growing tumors which killed the hosts within 10 weeks with no evidence of gross metastasis. The tumor bearing animals exhibited liver inflammation, massive splenomegaly, elevated serum LDH and immunoglobulins. These findings are typical in clinical HDS. The observed splenomegaly could be reproduced by injecting the conditioned media of BA1119 cells into nude miceJ i.p. for 6 weeks. The use of unconditioned mediaJ conditioned media from a non-HDS producing RCC cell lineJ or BA1119 conditioned media heated for 1 hour at 60°C failed to produce splenomegaly in age and sex matched animals. These results suggest that the substance responsible for the splenomegaly in nude mice is a proteinacious material secreted by the BA1119 cells. To further characterize this protein, the protein fraction of
conditioned media of BA1119 cells was subjected to 2-dimensional gel electrophoresis. A non-HDS producing cell line (769P) was used as a control. A unique group of proteins with molecular weights ranging from 85-90 kilodaltons and isoelectric points from 7-8 was associated with the BA1119 cells but not the 769P cells. These results indicate that
1) the BA! 119 cell line retains the ability to produce a paraneoplastic syndrome in nude mice which resembles the
clinical HOS, 2)the BA1119 cell line secretes a heat-labile substance which causes splenomegaly in the nude mouse, and 3)BA1119 cells synthesize at least one group of proteins different from a RCC cell line not producing HDS. Therefore, BA1119 can serve as an experimental model for the
study of HOS in RCC.
415
476
INTERLEUKIN 2 (IL-2) IN THE MANAGEMENT OF PATIENTS WITH RENAL CELL CARCINOMA. George P. Hemstreet, *Robert P. Whitehead, *Laurie B. Owen-Schaub and *Lawrence E. DeBault, Oklahoma City, OK (Presentation to be made by Dr. Hemstreet) Interleukin 2 is a biological response modifier with activity in patients with renal cell carcinoma. Cells of non-T-cell origin are activated by IL-2 to become cytotoxic lymphocytes with T-cell markers and recognize and kill transformed cells (Cell.Immunol.102:1986). The optimal administration of IL-2 to maximize response and reduce toxicity is unknown. In a Phase I-II study, 14 patients werg a~ministered subcutaneous IL-2 at doses of.5-4 X 10 u/M. Toxicity consisted of erythema at the injection site, eosinophilia, and a febrile response were manageable on outpatient basis. No complete response or partial response occurred, but stabilization occurred
2
in three patients. In 4 gati nts treated to date with intravenous IL-2, 4-8 XlO u/M, IV bolus 5 days every other week, one partial response of pulmonary nodules was documented. Toxicity was tolerable with minimal fluid retention and no patients required intubation for management of pulmonary edema and the febrile response was
controlled with 50mg indomethacin. Using in vitro studies with activated LAK we have demonstrated a 75 to BO-fold enrichment of LAK progenitors three days after activation. By flow cytometry, 40% of cells are in the S/G 2/M phase by day 4 and after irradiation 50% decrease is seen within 8-9 hours. The initial generation and maintenance of LAK effector function are dependent on cellular proliferation and have provided the basis for our cur 11 nt regimen, IV IL-2, .lmg/kg/day X 2.5 days with 2 XlO LAK cells X 6 weeks. (Supported by Cetus Corporation and VA Grant 11134-0132).
RENAL
PELVIC
TUMOURS - PERCUTANEOUS INVESTIGATION AND C.R. J. Woodhouse, M.J.Kellett* and H.J.G.Bloom*. The Institute of Urology and the Royal Marsden Hospital, London, UK. (Presentation to be made by Mr. Woodhouse). 10 patients with a radio-lucent filling defect in the renal pelvis have been investigated and treated. 7 had a previous history of bladder transitional cell carcinoma (TCC). Ureteroscopy was impossible in 9. In 7 patients pelvicalyceal TCC was cauterised or resected through a percutaneous track such as would be used for stone removal. In 4 of 7 the contra-lateral kidney had been removed. After surgery an Iridium wire (192lr) was placed in the nephrostomy delivering 4500cGy at 4mm radius over 3-4 days, to prevent track implantation. All tumours were non-invasive, 3 being G1 and 4 G2. One operation was complicated by bleeding but transfusion was not required. Follow-up is 10-48 months. No track implantation has occurred. 1 patient has died from uraemia following massive local recurrence in a solitary kidney. 3 patients have had urothelial recurrence of TCC remote from the operated kidney. In 1 patient the renal pelvis was ruptured during surgery. It healed rapidly and 192Ir wire was implanted a week later. Spontaneous rupture of the renal pelvis occurred at 6 weeks and nephroureterectomy was performed after 2 months of internal drainage failed to produce healing. In another 3 patients with a past history of bladder TCC, percutaneous nephroscopy was used to investigate a radiolucent pelvic lesion suspicious of tumour. In 1 a uric acid stone was found and removed. In 2 no lesion was found. All remain well on follow-up at 12-38 months. Percutaneous surgery and local radiotherapy can be used in the management of pelvi-calyceal TCC. It is particularly valuable in low grade, low stage lesions in solitary kidneys or unfit patients. Radiotherapy should be withheld if there is a renal pelvic leak at surgery.
Til EATMENT.
222A
474
HEPATIC DYSFUNCTION SYNDROME(HDS) ASSOCIATED WITH RENAL CELL CARCINOMA(RCC): PRELIMINARY CHARACTERIZATION STUDIES. Harold
TRANSITIONAL CELL CARCINOMA (TCC) OF THE RENAL PELVIS: NUCLEAR DNA PLOIDY BY FLOW CYTOMETRY. Michael L. Blute*, Kenichi Tsushima,* George M. Farrow,* Michael M. Lieber, Rochester, MN (Presentation to be made by Dr. Blute) A flow cytometric method for determining the nuclear DNA ploidy pattern of cells from formalin-fixed paraffin-embedded tissue has been described by Hedley et al (J Histochem Cytochem 31: 1322, 1983). This method a 11 ows 1 aboratory analysis of tumor specimens from groups of patients treated many years previously whose clinical outcome is now known. The Hedley method was carried out on tumor specimens from 111 patients with TCC of the renal pelvis; nuclei were stained with propidium iodide using the Vindelov method (Cytometry 3:323, 1983) and run on a FACS IV Flow Cytometer. Patients underwent surgery between 1960 and 1975. DNA histograms were classified as DNA diploid (or normal), DNA tetraploid (with a significant increase in the 4C peak), or DNA aneuploid. Results and clinical outcome were: DNA Histogram Pattern No. Dead from Metastatic TCC DNA diploid 38 5 (13%) DNA tetraploid 54 21 (39%)* (*p<0.02) DNA aneuploid 19 15 (79%)** (**p<0.001) There was an excellent correlation between histolog1c grade, DNA ploidy pattern, and clinical outcome. No patient with a grade l tumor (n=28) had a DNA aneuploid pattern and none died from TCC. Thirty of 31 patients with grade 3-4 tumors had an abnormal DNA pattern (DNA tetraploid or aneuploid); 26 (84%) died from metastatic TCC. For grade 2 TCC, patients with tumors with DNA aneuploid patterns did markedly worse than patients with tumors with DNA diploid patterns. With well-differentiated and poorlydifferentiated TCC of the renal pelvis, DNA ploidy pattern simply correlated well with histologic grade assigned by an experienced pathologist. For grade 2 TCC, DNA ploidy analysis identified a prognostically important group of DNA abnormal tumors with poor outcome not detectable by light microscopic grading.
N.Keer*,William Coukos*,Julia Sensibar*,Livia Arvay*, Roger Kroes*,James M.Kozlowski*, and Chung Lee*, Chicago, IL. (Presentation to be made by Dr.Coukos) HDS, seen in about 10% of RCC patients, is presumed to result from a hepatotoxin of RCC origin. Since the etiology
of HOS is unknown, a cell line(BA1119) was established from a patient with RCC and clinical HDS in an attempt to iden.;--
tify the putative hepatotoxin. BA1119, when injected s.c, into nude mice resulted in growing tumors which killed the hosts within 10 weeks with no evidence of gross metastasis. The tumor bearing animals exhibited liver inflammation, massive splenomegaly, elevated serum LDH and immunoglobulins. These findings are typical in clinical HDS. The observed splenomegaly could be reproduced by injecting the conditioned media of BA1119 cells into nude miceJ i.p. for 6 weeks. The use of unconditioned mediaJ conditioned media from a non-HDS producing RCC cell lineJ or BA1119 conditioned media heated for 1 hour at 60°C failed to produce splenomegaly in age and sex matched animals. These results suggest that the substance responsible for the splenomegaly in nude mice is a proteinacious material secreted by the BA1119 cells. To further characterize this protein, the protein fraction of
conditioned media of BA1119 cells was subjected to 2-dimensional gel electrophoresis. A non-HDS producing cell line (769P) was used as a control. A unique group of proteins with molecular weights ranging from 85-90 kilodaltons and isoelectric points from 7-8 was associated with the BA1119 cells but not the 769P cells. These results indicate that
1) the BA! 119 cell line retains the ability to produce a paraneoplastic syndrome in nude mice which resembles the
clinical HOS, 2)the BA1119 cell line secretes a heat-labile substance which causes splenomegaly in the nude mouse, and 3)BA1119 cells synthesize at least one group of proteins different from a RCC cell line not producing HDS. Therefore, BA1119 can serve as an experimental model for the
study of HOS in RCC.
415
476
INTERLEUKIN 2 (IL-2) IN THE MANAGEMENT OF PATIENTS WITH RENAL CELL CARCINOMA. George P. Hemstreet, *Robert P. Whitehead, *Laurie B. Owen-Schaub and *Lawrence E. DeBault, Oklahoma City, OK (Presentation to be made by Dr. Hemstreet) Interleukin 2 is a biological response modifier with activity in patients with renal cell carcinoma. Cells of non-T-cell origin are activated by IL-2 to become cytotoxic lymphocytes with T-cell markers and recognize and kill transformed cells (Cell.Immunol.102:1986). The optimal administration of IL-2 to maximize response and reduce toxicity is unknown. In a Phase I-II study, 14 patients werg a~ministered subcutaneous IL-2 at doses of.5-4 X 10 u/M. Toxicity consisted of erythema at the injection site, eosinophilia, and a febrile response were manageable on outpatient basis. No complete response or partial response occurred, but stabilization occurred
2
in three patients. In 4 gati nts treated to date with intravenous IL-2, 4-8 XlO u/M, IV bolus 5 days every other week, one partial response of pulmonary nodules was documented. Toxicity was tolerable with minimal fluid retention and no patients required intubation for management of pulmonary edema and the febrile response was
controlled with 50mg indomethacin. Using in vitro studies with activated LAK we have demonstrated a 75 to BO-fold enrichment of LAK progenitors three days after activation. By flow cytometry, 40% of cells are in the S/G 2/M phase by day 4 and after irradiation 50% decrease is seen within 8-9 hours. The initial generation and maintenance of LAK effector function are dependent on cellular proliferation and have provided the basis for our cur 11 nt regimen, IV IL-2, .lmg/kg/day X 2.5 days with 2 XlO LAK cells X 6 weeks. (Supported by Cetus Corporation and VA Grant 11134-0132).
RENAL
PELVIC
TUMOURS - PERCUTANEOUS INVESTIGATION AND C.R. J. Woodhouse, M.J.Kellett* and H.J.G.Bloom*. The Institute of Urology and the Royal Marsden Hospital, London, UK. (Presentation to be made by Mr. Woodhouse). 10 patients with a radio-lucent filling defect in the renal pelvis have been investigated and treated. 7 had a previous history of bladder transitional cell carcinoma (TCC). Ureteroscopy was impossible in 9. In 7 patients pelvicalyceal TCC was cauterised or resected through a percutaneous track such as would be used for stone removal. In 4 of 7 the contra-lateral kidney had been removed. After surgery an Iridium wire (192lr) was placed in the nephrostomy delivering 4500cGy at 4mm radius over 3-4 days, to prevent track implantation. All tumours were non-invasive, 3 being G1 and 4 G2. One operation was complicated by bleeding but transfusion was not required. Follow-up is 10-48 months. No track implantation has occurred. 1 patient has died from uraemia following massive local recurrence in a solitary kidney. 3 patients have had urothelial recurrence of TCC remote from the operated kidney. In 1 patient the renal pelvis was ruptured during surgery. It healed rapidly and 192Ir wire was implanted a week later. Spontaneous rupture of the renal pelvis occurred at 6 weeks and nephroureterectomy was performed after 2 months of internal drainage failed to produce healing. In another 3 patients with a past history of bladder TCC, percutaneous nephroscopy was used to investigate a radiolucent pelvic lesion suspicious of tumour. In 1 a uric acid stone was found and removed. In 2 no lesion was found. All remain well on follow-up at 12-38 months. Percutaneous surgery and local radiotherapy can be used in the management of pelvi-calyceal TCC. It is particularly valuable in low grade, low stage lesions in solitary kidneys or unfit patients. Radiotherapy should be withheld if there is a renal pelvic leak at surgery.
Til EATMENT.
222A