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The Isolation and Expansion of Lymphocytes Infiltrating Human Renal Tumors Using Recombinant Interleukin-2; Possible Application for Clinical Use
Accepted 469
470
EFFECT OF TUMOR NECROSIS FACTOR AND INTERFERON GAMMA ON HUMAN RENAL CELL CARCINOMA GROWTH. Loui• R. Kavoussi* and M'Liss A. Hudson•, St. Louis, MO. (Presented by Louis R. Kavoussi) factg~o\~~~~ainde~~~~~,e~g~ifti~;a•~lVN~f fi~~gr b~:~r~~~~n to effectively inhibit the growth of a variety of tumor types. Tumor cells vary in their sensitivity to TNF with some being highly susceptable while others are totally resistant. Recent studies have &hewn that IFNy enhances the antitumor effects of TNF. Pretreatment of cell lines with IFNy either enhances sensitivity or induces sansitivity for resistant lines. Little evidence is available concerning the effects of TNF either alone or in combination with IFNy on growth of kidney tumor cells. The human kidney tumor cell l1nesl 786-0 and CAKI, were tested for susceptibility to TNF and FNy (both are purified recombinant products provided by Biogen, Inc.), Cells were cultured in 96 well plates and treated with varying concentrations of each a9ent alone or combinations of both agents. In the combination studies the cell lines were pretreated for various time intervals with IFNy, washed and tested for TNF activitr, TNF alone inhibited 786-0 eel proliferation by a maximum of 67'l. at 1 ug/ml. CAKI cells were less sensitive to TNF with a maximum inhibition of 40% at 1 ug/ml. Pretreatment of 786-0 cells with IFNy for 24 hours prior to addition of TNF had little affect on the cytolytic activity of TNF, Inhibition was observed to be 67% for TNF alone (1 ug/ml) and 71X with IFNy pretruhent (250 u/ml l. In contrast, the TNF activity against the less sensitive cell line, CAKI, was si9nificantly (P < .005) enhanced by IFNy pretreatment (40% inhibition alone1 59% inhibition after lFNy pretreatment). The enhanced effect was uniformly observed over a TNF dose range of 0.001 to 1 ug/ml. These data demonstrate that kidney tumor cell lines are sensitive to the cytolytic effects of TNF. Moreover 1 the data show that IFNy enhances the effectiveness of TNt for less sensitive kidney tumor cell lines suggesting that combination therapy may induce a more uniform antitumor affect.
THE IMPACT OF INCIDENTALLY-NOTED RENAL CELL CARCINOMA - IS THERE A PLAC~ FOR ROUTINE SCREENING? Ian Thompson*, Michael Peek, and Francisco Rodriguez, San Antonio, TX (Presentation to be made by Dr. Thompson) Two hundred and twelve cases of renal cell carcinoma treated at Brooke Army Medical Center between 1946 and 1985 were reviewed. During these four 10-year time periods, the number of cases identified serendipitously during studies performed for non-urologic complaints increased from 6% for 1946-1955 to 25% for 1976-1985. Although the overall number of localized tumors at the time of diagnosis was 32~ 82% of incidentally-noted tumors were localized. When stage distributions were compared among the four 10-year periods, the only cause for an overall increase in the number of localized tumors was due to those which were noted incidentally. Forty percent of patients continue to present with stigmata of metastatic disease. Life table analysis comparing incidentally-noted tumors with the remainder show that patients with tumors noted incidentally have a lesser chance of dying of their disease (15 vs 75%) and if they do die of disease, the mean time to progression is longer (52 vs 18 months) and the mean time to death is longer (71 vs 21 months). When the four 10-year periods were compared, deleting those patients with incidentally-noted tumors, the percent of patients dying of their disease has not changed in 40 years (approximately 75%). Additionally, again comparing these periods, a shorter time to progression and death is noted in patients with renal cell carcinoma in recent years. These data suggest that the only significant advance which has occurred to improve the survival of patients with renal cell carcinoma has been the increased number of tumors noted incidental to other examinations. The implication of these data is that routine morphologic renal examination with modalities such as ultrasound may be a viable means of improving the therapy of this tumor. Low prevalence of renal tumors would dictate that screening examinations be performed during ultrasound examinations for other causes.
471
472
TUMOR-TARGETED CIIEMOTJIERAPY WI'l'H LIPID CONTRAST MEDIIJN AND MACRO MOLECULAR ANTICANCER DlUJG( SMANCS ) FOR RENAL CELL CARCINOMAS. *Mikio Kobayashi,*Shiro St1gihara,*lliroshi Maeda,*Toshimitsu Konno and llidetoshi
THE ISOLATION AND EXPANSION OF' LYMPHOCYTES INFILTRATING HUMAN RENAL TUMORS USING RECOMBINANT INTERLEUKIN-2; POSSIBLE APPLICATlON FOR CLINICAL USE *Arie Belldegrun and *Steven A. Rosenberg, Bethesda, Maryland (Presentation to be made by Dr, Belldegrun) We have developed a technique for isolating from fresh human renal cell cancers, tumor infiltrating lymphocytes (TIL) that can be significantly expanded in interleukin-2 (IL-2) to numbers sufficient for use in human therapy. Our studies in a variety of tumor models suggest that the adoptive transfer of TIL is from 50 to 100 times more effective than therapy with lyrnphokine activated killer (LAK) cells in mediating the in vivo destruction of established pulmonary and hepatic metastases. Tumor cell suspensions obtained from 34 consecutive fresh human renal cell cancer (RCC) specimens were separated using enzymatic techniques. The total cell recovery was 2.5 x 109 + 1.5 (mean+ SEM) cells per tumor (range: 1 x 108 to 5 x-109 cells): The percentage of tumor cells and lymphocytes in the suspension was 41 + 3.8% (range: 6 to 75%) and 56 + 3.8% (range: 20 to 85%) respectively, Activated TIL within these tumors expand in IL-2 and by 10 to 14 days after initiation of culture a 5 to 15 fold increase in the number of lymphocytes could be achieved with elimination of all autologous tumor cells. TIL continued to expand in cuJture resulting in 50,716 ~ 25, 142 fold increase in the total number of lymphocytes. The majority of TIL were T cytotoxic/suppressor cells (Leu 2+ Leu 4+). Fresh as well as cryopreserved renal 'I'll could lyse almost all autologous and some allogeneic tumor targets in 4-hour chromium release assays. The ability to obtain expanded cytotoxic TIL from cryopreserved tumor suspensions opens the possibility of immunotherapy based on reinfusing the patient with his own TIL once distant metastases appear. These experiments provide a basis for the use of human TIL in the treatment of patients with metastatic RCC. Clinical trials to test this therapeutic approach will soon begin.
Yamanakh.,Maebashi and KUlllamoto,Japan
( Presentation
to be made by Dr. ko bayashi ) 25 patients with renal cell carcinoma were treated with a lypophilic macromolecular drug 1 Poly( stylene-co -malcic acid )-conjugated neocarcinostatin( SMANCS ) dissolved in lipid contrast medium Lipiodol( SMANCS/ LIPIODOL ) • The drug was injected mainly by catheterizing renal artery and the other feeding artery under x-ray monitoring. The procedure of selective arterial administration of 5-20 mg/ml of SMANCS/LIPIODOL was simple to perfonn and was required once every 2-3
;;eeks, Total dose of SMANCS fpr each patient varied from 5 to 35 mg. Doth SMANCS and Lipiodol accmnulated more selectively in ttunor than in any Other tissues and remained in the neovasculature and extra-capillary
space for a long time.
C'l' pattern of the remaining
oil contrast medium in the tumor was characterized by
the high density area being localized mainly in the periphery of the tumor around the central necrosis. When arterial infusion of only SMANCS/LIPIODOL was given,the depos;ted Lipiodol decreased more quickly within two weeks but when the oil contrast me<.li\un with higher viscosity was used, it persistently remained
and
more slowly disappeared. The degree of oil contrast medium remaining in the tumor was found to correlate well with the runount of the agents administered and the anti-cancer effect. Severe side effects were not observed such as myelo-suppression,unendurable pain, nausea and vomitting etc.
221A
470
EFFECT OF TUMOR NECROSIS FACTOR AND INTERFERON GAMMA ON HUMAN RENAL CELL CARCINOMA GROWTH. Loui• R. Kavoussi* and M'Liss A. Hudson•, St. Louis, MO. (Presented by Louis R. Kavoussi) factg~o\~~~~ainde~~~~~,e~g~ifti~;a•~lVN~f fi~~gr b~:~r~~~~n to effectively inhibit the growth of a variety of tumor types. Tumor cells vary in their sensitivity to TNF with some being highly susceptable while others are totally resistant. Recent studies have &hewn that IFNy enhances the antitumor effects of TNF. Pretreatment of cell lines with IFNy either enhances sensitivity or induces sansitivity for resistant lines. Little evidence is available concerning the effects of TNF either alone or in combination with IFNy on growth of kidney tumor cells. The human kidney tumor cell l1nesl 786-0 and CAKI, were tested for susceptibility to TNF and FNy (both are purified recombinant products provided by Biogen, Inc.), Cells were cultured in 96 well plates and treated with varying concentrations of each a9ent alone or combinations of both agents. In the combination studies the cell lines were pretreated for various time intervals with IFNy, washed and tested for TNF activitr, TNF alone inhibited 786-0 eel proliferation by a maximum of 67'l. at 1 ug/ml. CAKI cells were less sensitive to TNF with a maximum inhibition of 40% at 1 ug/ml. Pretreatment of 786-0 cells with IFNy for 24 hours prior to addition of TNF had little affect on the cytolytic activity of TNF, Inhibition was observed to be 67% for TNF alone (1 ug/ml) and 71X with IFNy pretruhent (250 u/ml l. In contrast, the TNF activity against the less sensitive cell line, CAKI, was si9nificantly (P < .005) enhanced by IFNy pretreatment (40% inhibition alone1 59% inhibition after lFNy pretreatment). The enhanced effect was uniformly observed over a TNF dose range of 0.001 to 1 ug/ml. These data demonstrate that kidney tumor cell lines are sensitive to the cytolytic effects of TNF. Moreover 1 the data show that IFNy enhances the effectiveness of TNt for less sensitive kidney tumor cell lines suggesting that combination therapy may induce a more uniform antitumor affect.
THE IMPACT OF INCIDENTALLY-NOTED RENAL CELL CARCINOMA - IS THERE A PLAC~ FOR ROUTINE SCREENING? Ian Thompson*, Michael Peek, and Francisco Rodriguez, San Antonio, TX (Presentation to be made by Dr. Thompson) Two hundred and twelve cases of renal cell carcinoma treated at Brooke Army Medical Center between 1946 and 1985 were reviewed. During these four 10-year time periods, the number of cases identified serendipitously during studies performed for non-urologic complaints increased from 6% for 1946-1955 to 25% for 1976-1985. Although the overall number of localized tumors at the time of diagnosis was 32~ 82% of incidentally-noted tumors were localized. When stage distributions were compared among the four 10-year periods, the only cause for an overall increase in the number of localized tumors was due to those which were noted incidentally. Forty percent of patients continue to present with stigmata of metastatic disease. Life table analysis comparing incidentally-noted tumors with the remainder show that patients with tumors noted incidentally have a lesser chance of dying of their disease (15 vs 75%) and if they do die of disease, the mean time to progression is longer (52 vs 18 months) and the mean time to death is longer (71 vs 21 months). When the four 10-year periods were compared, deleting those patients with incidentally-noted tumors, the percent of patients dying of their disease has not changed in 40 years (approximately 75%). Additionally, again comparing these periods, a shorter time to progression and death is noted in patients with renal cell carcinoma in recent years. These data suggest that the only significant advance which has occurred to improve the survival of patients with renal cell carcinoma has been the increased number of tumors noted incidental to other examinations. The implication of these data is that routine morphologic renal examination with modalities such as ultrasound may be a viable means of improving the therapy of this tumor. Low prevalence of renal tumors would dictate that screening examinations be performed during ultrasound examinations for other causes.
471
472
TUMOR-TARGETED CIIEMOTJIERAPY WI'l'H LIPID CONTRAST MEDIIJN AND MACRO MOLECULAR ANTICANCER DlUJG( SMANCS ) FOR RENAL CELL CARCINOMAS. *Mikio Kobayashi,*Shiro St1gihara,*lliroshi Maeda,*Toshimitsu Konno and llidetoshi
THE ISOLATION AND EXPANSION OF' LYMPHOCYTES INFILTRATING HUMAN RENAL TUMORS USING RECOMBINANT INTERLEUKIN-2; POSSIBLE APPLICATlON FOR CLINICAL USE *Arie Belldegrun and *Steven A. Rosenberg, Bethesda, Maryland (Presentation to be made by Dr, Belldegrun) We have developed a technique for isolating from fresh human renal cell cancers, tumor infiltrating lymphocytes (TIL) that can be significantly expanded in interleukin-2 (IL-2) to numbers sufficient for use in human therapy. Our studies in a variety of tumor models suggest that the adoptive transfer of TIL is from 50 to 100 times more effective than therapy with lyrnphokine activated killer (LAK) cells in mediating the in vivo destruction of established pulmonary and hepatic metastases. Tumor cell suspensions obtained from 34 consecutive fresh human renal cell cancer (RCC) specimens were separated using enzymatic techniques. The total cell recovery was 2.5 x 109 + 1.5 (mean+ SEM) cells per tumor (range: 1 x 108 to 5 x-109 cells): The percentage of tumor cells and lymphocytes in the suspension was 41 + 3.8% (range: 6 to 75%) and 56 + 3.8% (range: 20 to 85%) respectively, Activated TIL within these tumors expand in IL-2 and by 10 to 14 days after initiation of culture a 5 to 15 fold increase in the number of lymphocytes could be achieved with elimination of all autologous tumor cells. TIL continued to expand in cuJture resulting in 50,716 ~ 25, 142 fold increase in the total number of lymphocytes. The majority of TIL were T cytotoxic/suppressor cells (Leu 2+ Leu 4+). Fresh as well as cryopreserved renal 'I'll could lyse almost all autologous and some allogeneic tumor targets in 4-hour chromium release assays. The ability to obtain expanded cytotoxic TIL from cryopreserved tumor suspensions opens the possibility of immunotherapy based on reinfusing the patient with his own TIL once distant metastases appear. These experiments provide a basis for the use of human TIL in the treatment of patients with metastatic RCC. Clinical trials to test this therapeutic approach will soon begin.
Yamanakh.,Maebashi and KUlllamoto,Japan
( Presentation
to be made by Dr. ko bayashi ) 25 patients with renal cell carcinoma were treated with a lypophilic macromolecular drug 1 Poly( stylene-co -malcic acid )-conjugated neocarcinostatin( SMANCS ) dissolved in lipid contrast medium Lipiodol( SMANCS/ LIPIODOL ) • The drug was injected mainly by catheterizing renal artery and the other feeding artery under x-ray monitoring. The procedure of selective arterial administration of 5-20 mg/ml of SMANCS/LIPIODOL was simple to perfonn and was required once every 2-3
;;eeks, Total dose of SMANCS fpr each patient varied from 5 to 35 mg. Doth SMANCS and Lipiodol accmnulated more selectively in ttunor than in any Other tissues and remained in the neovasculature and extra-capillary
space for a long time.
C'l' pattern of the remaining
oil contrast medium in the tumor was characterized by
the high density area being localized mainly in the periphery of the tumor around the central necrosis. When arterial infusion of only SMANCS/LIPIODOL was given,the depos;ted Lipiodol decreased more quickly within two weeks but when the oil contrast me<.li\un with higher viscosity was used, it persistently remained
and
more slowly disappeared. The degree of oil contrast medium remaining in the tumor was found to correlate well with the runount of the agents administered and the anti-cancer effect. Severe side effects were not observed such as myelo-suppression,unendurable pain, nausea and vomitting etc.
221A