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Phase II trial of interleukin-1 alpha plus indomethacin in melanoma patients
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A SIMPLE METHOD OF DETECTING mRNA FOR IL-1 p FROM UNFRACTIONATED WHOLE BLOOD WITH RT-PCR S. Shim& G. Wakabavashi, T. Tsuzuki, T. Miura, M. Ueda, Dept. of Surgery, Keio University School of Medicine., Tokyo, Japan. We describe here a simple method to detect mRNA from unfractionated whole blood using RT-PCR. Using this method, IL-1 /3 gene expression can be detected by RT-PCR from OSml blood sample. m Total RNA is extracted from blood sample by 4M guauidinium isothiocyanate mixture, 0.2M sodium acetate, phenol and chloroform. About 1Opg of total RNA is obtained from 0.5ml whole blood by this procedure. mRNA is reverse transcribed, and IL-1 /3 mRNA selectively amplified by synthetic primers with PCR. The peripheral blood and sscites were obtained from a cancer patient administered with OK-432 intmperitoneally. In both blood and ascites of this patient, IL-l p mRNA was detected after irmaperitoneal injection of OK-432. IL-1 /3 mRNA was shown the most overexpression in the peripheral blood at 36 hours titer OK-432 administration. IL-l,0 mRNA was detected earlier in the ascites than in the peripheral blood. IL- 1 p mRNA was undetectable in normal control blood. Comment Using this method, we could save the time of fractiona& and the amount of blood. Thus, this method may be. applicable to monitoring cytokine gene expression in cancer patient under immunochemotherapy.
ANTIPROLIFERATIVE EFFECT OF TNF, IL-l AND INFs ON LYMPHOBLASTIC LEUKEMIC CELL LINES. Q.Trubiani*. S.Miscia. A.Cataldi. M.R.Panara* and R.Di Primio. 1st. Citomorfologia CNR and 1st. Morfologia Um.Nor. Univ. Chieti, 66100 Chieti, Italy. Tumor necrosis factor (rTNF-alfa), Interleukin1 (IL-l) and Interferon alfa and gamma (INFs) are cytokines synthetized by immune system cells under stimulation of various agents and they show a pleiotropic biological effects. Therefore, we have examined the changes induced on cell proliferation by these cytokines using undifferentiated Terminal Transferase (TdT) positive pre-B (KM-3) and pre-T (RPMI-8402) cells lines and differentiated TdT negative like B cells @AMOS-l) and T cells (MOLTThe cytokines tested do not affect cell 3). proliferation on TdT positive cells while in more differentiated cells it is possible to observe an anti-proliferativeeffectdeterminedby3H-thymidine incorporation. These results suggest that the different effect of cytokines could be related to the differentiation stage acquired by lymphoid cells.
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PHASE II TRIAL OF INTERLELIKIN-1 ALPHA PLUS INDOMETHACIN IN J. W. Smith II, -W. Urba, -9 J. Janik MELANOMA PATIENTS. W. Sharfman, J. Oppenheim, & D. Long". BRMP, DCT, NCI; PRIlDynCorp, NCI-FCRDC, Frederick, MD 21701. After defining the maximum-tolerated dose of IL-1 plus indomethacin from our phase I trial, we designed a phase II trial in melanoma pts. based on preclinical data suggesting anti-tumor activity of the combination in that disease. IL-l was given IV "ver 15 mins. ""ce daily x 7 days at 0.1 mcg/kg with indomethacin 50 mg every 8 hrs. and repeated "rice after a one week rest. 32 pts. were stratified on the basis of their predominant site of metastatic disease - 14 visceral and 18 non-visceral. The median age was 48 yrs. (range 32-77 yrs.), male:female was 24:8, and their Karnofsky performance status More than half the pts. had no prior was 80% or greater. therapy. Of the 13 evaluable pts. with visceral metastases, one pt. has had stable disease and the rest progressed. Of the 17 evaluable pts. with non-visceral disease, one had a PR (80% decrease in tumor) that has lasted 9+ months. Five other pts. had individual metastatic lesions shrink significantly, but overall did not meet the criteria for a PR. Toxicity consisted of grade 2 or less chills, fever, headache, nausea, vomiting & myalgia and grade 3 hypotension (requiring pressors) in 25% of the pts. with the first &/or second doses of IL-I. One 77 yr. old pt. sustained a non-fatal myocardial infarction. We conclude that IL-1 alpha plus indomethacin has a low level of anti-tumor activity against melanoma. Efforts to enhance the regimen by adding etoposide are planned.
LOCAL LOCAL Cancer
IL-2 GIVES SYSTEMIC IFN gamma INDUCTION. Institute, Buffalo,
The purpose of this direct local therapeutic peritumor injections of interferon gamma (IFNg),
associated
systemic
THERAPEUTIC EFFECT Jan Vaage. Roswell NY, USA 14263. study was to compare effects of multiple interleukin-2 (IL-2) and also to compare
therapeutic
effects
VIA Park the and the
on distant
untreated tumors resulting from the peritumor therapeutic injections. The therapeutic effects were tested against intramammary implants of an immunogenic, syngeneic C3H mammary carcinoma. Peritumor IL-2 and IFNg had nearly equal local and systemic therapeutic effects. In a comparison of the therapeutic effects of IL-2 and IFNg injected systemically, only the IFNg injections resulted in IL-2 and IFNg did a significant number of cures. not have additive effect when used in combination, suggesting that they have connected, rather than separate, paths of action in the anti-tumor immune response. The injection of anti-IFNg monoclonal antibody abrogated the systemic therapeutic effect of peritumor IL-2, indicating that the systemic therapeutic effect was the result of IFNg induction at the IL-2 injection site.
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SUBCUTANEOUS (s.c.) ADMINISTRATION OF RECOMBINANT INTERLEUKlN 2 (r-IL2) IN UClENSlVE SMALL CELL LUNG CANCER (SCLC) IN PARTIAL REMISSION AFlER INDUCTlON CHEMOTHERAPY. S.. Biddi. S.
A SIMPLE METHOD FOR MEASURING CYTOKINE PRODUCTION IN VITRO. I van der Ven-Jonaekriia. PNM Demacker. JWM van der Meer. Dept of Internal Medicine, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen,The Netherlands. The production of cytokines, as IL-1 and TNF, plays an important role in the acute phase reaction following au infection.To study whether the cytokine production is qualitatively or quantitatively related to the kind and duration of au infection, we have evaluated the simple method described by Nerad et al (Cytokine 1:139, 1989). In this procedure blood drawn into EDTA is incubated under sterile conditions with LPS and the cytokines released in plasma are determined by specific RIA’s. J&& : compared to Concanavalin A, LPS was a superior stimulant, which resulted in a dose dependent production of IL-1R and TNF in the range 0.01-100 pg/ml. At these concentrations the amounts of both cytokines produced correlated highly (r=O.98). At 10 pglml LPS, Indomethacin (0.5 pglml) had no influence. The maximal production of both cytokines at 10 pglml was already attained after incubation for 6 hour at 37 “C. Intra-individual variation determined in four healthy volunteers ranged from 10 to 49 76 (n=4). Conclusion : This method provides a tool for studying serious infectious diseases, when laboratory facilities are limited.
lstiuio Nazionale Tumori,
Milano, Italy. From 911990 to 411991, 7 pts with extensive SCLC in partial remission after inductiin chemotherapy (Epirubicin + lfosfamide + Etoposide every 21 days foe4 cycles) were treated with r-112at 9 InilliOfl IU/sqm S.C.hvice daily for two days, fcllowed by 3 million IUisqm twice daily for 3 days. Alter 2 days rest, pts. received r-1123 million IU/sqm S.C.tv& daily, 5 days per week for 3 weeks. This was a treatment cycle and was repeatedfcu 3 times with a 3 week rest alter each. We observed 1 pt. with partial remission, 2 pt. with pogressive disease and 4 pts. with stable disease. Treatment was well tolerated and was partally carried out on an oulpatient basis. All pts had grade 2 fever. grade 1 hypotension, myalgia, rhinitis. Eosinophilia (range: 155O/mm3 to 165QOimm3)was detected in all pts, which was greater during the third week of treatment.Prior to and during treatment, ceil surface markers and soluble IL2 receptofs (sll-2R) were analysed. In all patients, increased expression 01 CD25 as well as NK cell-associated CD16-CD56 cell surface ant&ens was observed at the end of each cycle. Mean levels of slL-2R were significanllyhigher during treatmentcompared to basal values @SE: 4957@6 vs 696@34 U/ml; px):OOl) and the highest levels were reached on the second and third we& of each cycle. Moreover the ikwease in slL-2R during the first cycle was greater in nonresponder than in responder patients, although the diHerencewas not statistically significant(&SE: 5866&g vs 4261&6). Investigation on maintainance immunotherapy in patients with extensive SCLC is justified given their bad prognosis even after a complete remission. Preliminaryresults suggest that S.C.low-dose r-112is feasible even on an outpatient basis and has biologicalas well as clinical activity, which remain to be further elucidated.
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
/ 495
271
274
A SIMPLE METHOD OF DETECTING mRNA FOR IL-1 p FROM UNFRACTIONATED WHOLE BLOOD WITH RT-PCR S. Shim& G. Wakabavashi, T. Tsuzuki, T. Miura, M. Ueda, Dept. of Surgery, Keio University School of Medicine., Tokyo, Japan. We describe here a simple method to detect mRNA from unfractionated whole blood using RT-PCR. Using this method, IL-1 /3 gene expression can be detected by RT-PCR from OSml blood sample. m Total RNA is extracted from blood sample by 4M guauidinium isothiocyanate mixture, 0.2M sodium acetate, phenol and chloroform. About 1Opg of total RNA is obtained from 0.5ml whole blood by this procedure. mRNA is reverse transcribed, and IL-1 /3 mRNA selectively amplified by synthetic primers with PCR. The peripheral blood and sscites were obtained from a cancer patient administered with OK-432 intmperitoneally. In both blood and ascites of this patient, IL-l p mRNA was detected after irmaperitoneal injection of OK-432. IL-1 /3 mRNA was shown the most overexpression in the peripheral blood at 36 hours titer OK-432 administration. IL-l,0 mRNA was detected earlier in the ascites than in the peripheral blood. IL- 1 p mRNA was undetectable in normal control blood. Comment Using this method, we could save the time of fractiona& and the amount of blood. Thus, this method may be. applicable to monitoring cytokine gene expression in cancer patient under immunochemotherapy.
ANTIPROLIFERATIVE EFFECT OF TNF, IL-l AND INFs ON LYMPHOBLASTIC LEUKEMIC CELL LINES. Q.Trubiani*. S.Miscia. A.Cataldi. M.R.Panara* and R.Di Primio. 1st. Citomorfologia CNR and 1st. Morfologia Um.Nor. Univ. Chieti, 66100 Chieti, Italy. Tumor necrosis factor (rTNF-alfa), Interleukin1 (IL-l) and Interferon alfa and gamma (INFs) are cytokines synthetized by immune system cells under stimulation of various agents and they show a pleiotropic biological effects. Therefore, we have examined the changes induced on cell proliferation by these cytokines using undifferentiated Terminal Transferase (TdT) positive pre-B (KM-3) and pre-T (RPMI-8402) cells lines and differentiated TdT negative like B cells @AMOS-l) and T cells (MOLTThe cytokines tested do not affect cell 3). proliferation on TdT positive cells while in more differentiated cells it is possible to observe an anti-proliferativeeffectdeterminedby3H-thymidine incorporation. These results suggest that the different effect of cytokines could be related to the differentiation stage acquired by lymphoid cells.
272
275
PHASE II TRIAL OF INTERLELIKIN-1 ALPHA PLUS INDOMETHACIN IN J. W. Smith II, -W. Urba, -9 J. Janik MELANOMA PATIENTS. W. Sharfman, J. Oppenheim, & D. Long". BRMP, DCT, NCI; PRIlDynCorp, NCI-FCRDC, Frederick, MD 21701. After defining the maximum-tolerated dose of IL-1 plus indomethacin from our phase I trial, we designed a phase II trial in melanoma pts. based on preclinical data suggesting anti-tumor activity of the combination in that disease. IL-l was given IV "ver 15 mins. ""ce daily x 7 days at 0.1 mcg/kg with indomethacin 50 mg every 8 hrs. and repeated "rice after a one week rest. 32 pts. were stratified on the basis of their predominant site of metastatic disease - 14 visceral and 18 non-visceral. The median age was 48 yrs. (range 32-77 yrs.), male:female was 24:8, and their Karnofsky performance status More than half the pts. had no prior was 80% or greater. therapy. Of the 13 evaluable pts. with visceral metastases, one pt. has had stable disease and the rest progressed. Of the 17 evaluable pts. with non-visceral disease, one had a PR (80% decrease in tumor) that has lasted 9+ months. Five other pts. had individual metastatic lesions shrink significantly, but overall did not meet the criteria for a PR. Toxicity consisted of grade 2 or less chills, fever, headache, nausea, vomiting & myalgia and grade 3 hypotension (requiring pressors) in 25% of the pts. with the first &/or second doses of IL-I. One 77 yr. old pt. sustained a non-fatal myocardial infarction. We conclude that IL-1 alpha plus indomethacin has a low level of anti-tumor activity against melanoma. Efforts to enhance the regimen by adding etoposide are planned.
LOCAL LOCAL Cancer
IL-2 GIVES SYSTEMIC IFN gamma INDUCTION. Institute, Buffalo,
The purpose of this direct local therapeutic peritumor injections of interferon gamma (IFNg),
associated
systemic
THERAPEUTIC EFFECT Jan Vaage. Roswell NY, USA 14263. study was to compare effects of multiple interleukin-2 (IL-2) and also to compare
therapeutic
effects
VIA Park the and the
on distant
untreated tumors resulting from the peritumor therapeutic injections. The therapeutic effects were tested against intramammary implants of an immunogenic, syngeneic C3H mammary carcinoma. Peritumor IL-2 and IFNg had nearly equal local and systemic therapeutic effects. In a comparison of the therapeutic effects of IL-2 and IFNg injected systemically, only the IFNg injections resulted in IL-2 and IFNg did a significant number of cures. not have additive effect when used in combination, suggesting that they have connected, rather than separate, paths of action in the anti-tumor immune response. The injection of anti-IFNg monoclonal antibody abrogated the systemic therapeutic effect of peritumor IL-2, indicating that the systemic therapeutic effect was the result of IFNg induction at the IL-2 injection site.
273
276
SUBCUTANEOUS (s.c.) ADMINISTRATION OF RECOMBINANT INTERLEUKlN 2 (r-IL2) IN UClENSlVE SMALL CELL LUNG CANCER (SCLC) IN PARTIAL REMISSION AFlER INDUCTlON CHEMOTHERAPY. S.. Biddi. S.
A SIMPLE METHOD FOR MEASURING CYTOKINE PRODUCTION IN VITRO. I van der Ven-Jonaekriia. PNM Demacker. JWM van der Meer. Dept of Internal Medicine, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen,The Netherlands. The production of cytokines, as IL-1 and TNF, plays an important role in the acute phase reaction following au infection.To study whether the cytokine production is qualitatively or quantitatively related to the kind and duration of au infection, we have evaluated the simple method described by Nerad et al (Cytokine 1:139, 1989). In this procedure blood drawn into EDTA is incubated under sterile conditions with LPS and the cytokines released in plasma are determined by specific RIA’s. J&& : compared to Concanavalin A, LPS was a superior stimulant, which resulted in a dose dependent production of IL-1R and TNF in the range 0.01-100 pg/ml. At these concentrations the amounts of both cytokines produced correlated highly (r=O.98). At 10 pglml LPS, Indomethacin (0.5 pglml) had no influence. The maximal production of both cytokines at 10 pglml was already attained after incubation for 6 hour at 37 “C. Intra-individual variation determined in four healthy volunteers ranged from 10 to 49 76 (n=4). Conclusion : This method provides a tool for studying serious infectious diseases, when laboratory facilities are limited.
lstiuio Nazionale Tumori,
Milano, Italy. From 911990 to 411991, 7 pts with extensive SCLC in partial remission after inductiin chemotherapy (Epirubicin + lfosfamide + Etoposide every 21 days foe4 cycles) were treated with r-112at 9 InilliOfl IU/sqm S.C.hvice daily for two days, fcllowed by 3 million IUisqm twice daily for 3 days. Alter 2 days rest, pts. received r-1123 million IU/sqm S.C.tv& daily, 5 days per week for 3 weeks. This was a treatment cycle and was repeatedfcu 3 times with a 3 week rest alter each. We observed 1 pt. with partial remission, 2 pt. with pogressive disease and 4 pts. with stable disease. Treatment was well tolerated and was partally carried out on an oulpatient basis. All pts had grade 2 fever. grade 1 hypotension, myalgia, rhinitis. Eosinophilia (range: 155O/mm3 to 165QOimm3)was detected in all pts, which was greater during the third week of treatment.Prior to and during treatment, ceil surface markers and soluble IL2 receptofs (sll-2R) were analysed. In all patients, increased expression 01 CD25 as well as NK cell-associated CD16-CD56 cell surface ant&ens was observed at the end of each cycle. Mean levels of slL-2R were significanllyhigher during treatmentcompared to basal values @SE: 4957@6 vs 696@34 U/ml; px):OOl) and the highest levels were reached on the second and third we& of each cycle. Moreover the ikwease in slL-2R during the first cycle was greater in nonresponder than in responder patients, although the diHerencewas not statistically significant(&SE: 5866&g vs 4261&6). Investigation on maintainance immunotherapy in patients with extensive SCLC is justified given their bad prognosis even after a complete remission. Preliminaryresults suggest that S.C.low-dose r-112is feasible even on an outpatient basis and has biologicalas well as clinical activity, which remain to be further elucidated.