Immunology Letters, 33 (1992) 127-134
0165 - 2478 / 92 / $ 5.00 © 1992 ElsevierSciencePublishers B.V. All rights reserved IMLET 01807
Chemo-immunotherapy in patients with metastatic melanoma using sequential treatment with dacarbazine and recombinant human interleukin-2: evaluation of hematologic and immunologic parameters and correlation with clinical response Rut Isacson a, Eli Kedar b, Vivian Barak a, Zulma Gazit b, Oded Yurim c, Inna Kalichman a, Hannah Ben-Bassat d, Shoshana Biran a, Michael Schlesinger e, Christopher R. Franks f, G e r d a J. R o e s t f, Peter A. P a l m e r f a n d Eitan Shiloni c aDepartment of Oncology, bLautenberg Center for General and Tumor Immunology, eDepartment of Surgery B, dDepartment of Experimental Surgery, eDepartment of Experimental Medicine, Hadassah University Hospital, Jerusalem, Israel; and fEuroCetus B.V., Amsterdam, The Netherlands
(Received 13 January 1992;revision received28 April 1992; accepted 29 April 1992)
1. Summary We have treated 18 patients with metastatic malignant melanoma (MM) with high-dose IL-2 administered by continuous iv infusion in combination with dacarbazine (DTIC), and correlated the clinical response with various hematologic and immunologic parameters. Two regimens differing in the sequence of treatment were employed, and 1-6 treatment cycles were given, depending on patient response. Two patients had a complete response (CR, 4 6 + m , 14m), two patients a partial response (PR, 16m,6m), one a minimal response and four had a stable disease lasting 2-7 months, thus the response rate (CR + PR) was 22%. None of the following parameters, tested prior to initiation of the therapy Key words: Melanoma; Dacarbazine; Interleukin-2 Correspondence to: Dr. Rut Isacson, Department of Ontology, Hadassah UniversityHospital, Jerusalem 91020, Israel. Abbreviations: MM, malignant melanoma; NK, natural killer;
LAK, lymphokine-activated killer; IL-2, interleukin-2; iv, intravenous; TNF~t, tumor necrosis factor ct; WBC, white blood cells; PBMNL, peripheral blood mononuclear leukocytes.
and 1-2 days after termination of each course of IL-2, correlated with the clinical response: WBC counts (total and differential), levels of blood CD4 and CD8 T cells, N K cells, monocytes and B cells, production of IL-1 and IL-1 inhibitor by monocytes, responsiveness to 3 mitogens, N K / L A K cell activity, and serum levels of IL-l~t, IL2, soluble IL-2 receptor, and TNF~. The only prognostic parameter was the greater increase in the level of IL-2 receptor (Tac)-bearing lymphocytes in the responding patients after 1-3 cycles of IL-2. \The data suggests that non-specific immune parameters have no prognostic value for patients undergoing IL-2-based immunotherapy.
2.
Introduction
Metastatic malignant melanoma (MM) carries a dismal prognosis. Dacarbazine (DTIC) is the most effective chemotherapeutic agent in M M with a response rate of 15-25%; most of these responses are, however, partial and of a short duration [1]. Considering the failure of current chemotherapy to cure or significantly prolong survival of patients with disseminated MM, the search for new therapeutic strategies is mandatory. Immuno127
therapy with interleukin-2 (IL-2), alone or together with IL-2-activated autologous lymphocytes (lymphokine-activated killer (LAK) cells), gave a response rate of approx. 20%, with 5-10% of the patients achieving a complete response in some studies [2-4]. The synergistic therapeutic effects obtained in experimental tumor systems with several chemotherapeutic agents in combination with IL-2 [5-9], stimulated the application of similar regimens in clinical trials. In this phase II study, which is part of a European multicenter study, supported by EuroCetus, D T I C and recombinant human IL-2 (given by continuous iv infusion) were sequentially administered to 18 M M patients. Preliminary reports on clinical responses and toxicity in some of the patients included in our study were recently published [10-12]. We present here the analysis of the hematologic and immunologic parameters tested prior, during, and post-therapy. We report our results separately, because such a broad analysis has not been performed by the other centers included in this study.
repeated at 5 weeks. Maintenance therapy was scheduled 3 weeks after the completion of induction treatment, consisting of IL-2 18 x 106 IU/ m2/24 h for 5 days, alternating with DTIC 850 mg/m 2 i.v. every 3 weeks for 18 weeks. A maximum of 6 cycles of IL-2 were scheduled.
3.2.2. Protocol H (13patients) DTIC 850 mg/m 2 was given by short i.v. infusion on day 1; IL-2 18 × 106 IU/m2/24 h was administered by continuous i.v. infusion for 5 consecutive days (days 4-9). A maximum of 6 treatment cycles with DTIC and IL-2 were scheduled: the first two at 13-day intervals and the next four at 20-day intervals. Clinical response was evaluated every 2 cycles of IL-2. Treatment was discontinued upon disease progression. Further details regarding treatment are given in references [10-12]. The IL-2 ( > 9 7 % pure, 18 x 106 IU/mg) was kindly supplied by EuroCetus B.V., Amsterdam, The Netherlands. 3.3.
3.
Blood samples
Materials and Methods
Eighteen previously untreated patients (11 males, 7 females) with a median age of 40 years (range: 18-64) and a Karnofsky status >/80% were treated. All patients had histologically proven disseminated melanoma with measurable disease (Table 1). Patients were selected according to the criteria defined previously [10-12]. Treatment was given between February 1988 and April 1990. All 18 patients were evaluable for analysis.
Blood (15-25 ml) was drawn with and without heparin immediately prior (baseline), during IL-2 infusion, and 1 or 2 days after each course of IL2. Blood samples were also taken from some patients 4-6 days after IL-2 and 2-4 days after DTIC. Serum aliquots were stored at - 7 0 ° C . Peripheral blood mononuclear leukocytes (PBMNL) were separated by a Ficoll-Hypaque gradient. For cryopreservation, cell aliquots were suspended in 10% DMSO, 20% fetal calf serum and 70% RPMI 1640 medium and stored at - 180°C for 2 weeks up to 12 months.
3.2.
3.4.
3.1.
Patient population
Treatment
Two different regimens of DTIC and IL-2 were employed.
3.2.1. Protocol I (5patients) Two induction cycles of IL-2 18 × 10 6 IU (equivalent to 3 × 106 Cetus Units)/m2/24 h were given by continuous i.v. infusion on days 1-5 and 12-16. DTIC 850 mg/m 2 was given by short i.v. infusion (20-30 min) on day 26. This cycle was 128
Assessment of hematologic parameters and immune functions in vitro
White blood cell (WBC) counts were performed on fresh samples using Coulter counting. Differential counts were done both by Coulter and on Giemsa stained smears. Cell markers, immune functions and cytokine serum levels were assayed on fresh or frozen samples, or on both (see Results). Except for N K / L A K activity which was considerably reduced by cryopreservation
TABLE 1 Patient characteristics Patient No. a
Age
Sex
Disease sites
IL-2 cycles given
D T I C cycles % of IL-2 given scheduled dose given
Response duration (months) b
1 2 3 4 5
37 50 64 49 37
M M M M F
Lung, liver, L N e Liver, spleen Lung, liver, L N SC d nodules Liver, L N
2 4 2 6 2
2 1 5 1
100 100 100 90 100
M R (5) P R (6) P C R (46 + ) P
6 7 8 9 10 11 12 13 14 15
34 42 41 22 39 18 33 55 40 42
M F M F M F F F M
6 4 2 2 4 2 6 2 2
6 4 2 2 4 2 6 2 2
80 70 85 90 70 100 85 90 100
S (7) S (2) P P
16 17 18
38 24 50
LN, SC nodules Lung Lung, liver LN Spleen Lung, liver, SC nodules Mediastinal mass SC nodules LN Adrenal, lung, SC nodules Lung, liver LN Liver
2 6 1 6
2 6 1 4
95 85 100 60
M M M F
CR (14) P
s (5) P P P P R (16) P
s (7)
apatients Nos. 1-5-were treated according to protocol I, patients Nos. 6-18 according to protocol II (see Materials and Methods). bCR, completeresponse; PR, partial response; MR, minimal response; S, stable; P, progression. ¢LN, lymph nodes. dSC, subcutaneous. (see Results), no significant differences in the other parameters tested were noted in preliminary experiments between fresh and cryopreserved samples. Using cryopreserved samples permitted several assays to be run in parallel simultaneously on samples taken at different times during treatment, thereby avoiding day-to-day variations in experimental conditions. Each frozen sample was tested in 2-4 separate experiments and the results represent the means. PBMNL phenotype was tested by standard immunofluorescence techniques and FACS analysis, using the following monoclonal antibodies (Becton-Dickinson, Mountain view, CA): Leu4 (CD3) for total T cells; Leu2 (CD8), Leu3 (CD4), B4 (CD19) for B cells; Ia (HLA-DR), Leu M3 (CD14) for monocytes; Leul 1 (CD16) for FCy receptor-bearing cells; and Tac (CD25) for IL-2 receptor-bearing cells. Results are also expressed in stimulation index = peak level during treatment/ baseline level. Levels of cytokines (IL-I~, IL-2, TNFct) and
soluble IL-2 receptor in patient sera were assayed by enzyme-linked immunosorbent assays (ELISA) (T Cell Sciences, Cambridge, MA). IL-2 was also determined by a bioassay, using a murine T cell line (CTLL-2) in a 48 h [3H]thymidine uptake assay, with results similar to those obtained by ELISA. In vitro secretion of IL-0t and IL-1 inhibitor by freshly obtained blood monocytes during a 24 h incubation was tested as described previously [13]. Results are expressed in stimulation index (see above). Response to the mitogens phytohemagglutinin (PHA), concanavalin A (con A) and pokeweed mitogen (PWM) was tested as described previously [14]. Cytotoxic activity of PBMNL was assayed at 1:1-100:1 effector/target cell ratios in a 4 h 51Cr-release assay against the NK-sensitive target K562 cells and the NK-resistant, LAK-sensitive target SW 48 human colorectal carcinoma cell line, as previously described [15]. Cells of several patients were tested in parallel against the LAKsensitive A375 human melanoma cell line, with re129
suits similar to those obtained with the SW48 cell line. Results are expressed in mean lytic unit (LU)/106 cells, where 1 LU = number of effector cells required to lyse 20% target cells, using 3000 labeled target cells/well. 3.5.
Statistical analysis
Differences between the responding and nonresponding patient groups for each parameter were tested for significance (P
0165 - 2478 / 92 / $ 5.00 © 1992 ElsevierSciencePublishers B.V. All rights reserved IMLET 01807
Chemo-immunotherapy in patients with metastatic melanoma using sequential treatment with dacarbazine and recombinant human interleukin-2: evaluation of hematologic and immunologic parameters and correlation with clinical response Rut Isacson a, Eli Kedar b, Vivian Barak a, Zulma Gazit b, Oded Yurim c, Inna Kalichman a, Hannah Ben-Bassat d, Shoshana Biran a, Michael Schlesinger e, Christopher R. Franks f, G e r d a J. R o e s t f, Peter A. P a l m e r f a n d Eitan Shiloni c aDepartment of Oncology, bLautenberg Center for General and Tumor Immunology, eDepartment of Surgery B, dDepartment of Experimental Surgery, eDepartment of Experimental Medicine, Hadassah University Hospital, Jerusalem, Israel; and fEuroCetus B.V., Amsterdam, The Netherlands
(Received 13 January 1992;revision received28 April 1992; accepted 29 April 1992)
1. Summary We have treated 18 patients with metastatic malignant melanoma (MM) with high-dose IL-2 administered by continuous iv infusion in combination with dacarbazine (DTIC), and correlated the clinical response with various hematologic and immunologic parameters. Two regimens differing in the sequence of treatment were employed, and 1-6 treatment cycles were given, depending on patient response. Two patients had a complete response (CR, 4 6 + m , 14m), two patients a partial response (PR, 16m,6m), one a minimal response and four had a stable disease lasting 2-7 months, thus the response rate (CR + PR) was 22%. None of the following parameters, tested prior to initiation of the therapy Key words: Melanoma; Dacarbazine; Interleukin-2 Correspondence to: Dr. Rut Isacson, Department of Ontology, Hadassah UniversityHospital, Jerusalem 91020, Israel. Abbreviations: MM, malignant melanoma; NK, natural killer;
LAK, lymphokine-activated killer; IL-2, interleukin-2; iv, intravenous; TNF~t, tumor necrosis factor ct; WBC, white blood cells; PBMNL, peripheral blood mononuclear leukocytes.
and 1-2 days after termination of each course of IL-2, correlated with the clinical response: WBC counts (total and differential), levels of blood CD4 and CD8 T cells, N K cells, monocytes and B cells, production of IL-1 and IL-1 inhibitor by monocytes, responsiveness to 3 mitogens, N K / L A K cell activity, and serum levels of IL-l~t, IL2, soluble IL-2 receptor, and TNF~. The only prognostic parameter was the greater increase in the level of IL-2 receptor (Tac)-bearing lymphocytes in the responding patients after 1-3 cycles of IL-2. \The data suggests that non-specific immune parameters have no prognostic value for patients undergoing IL-2-based immunotherapy.
2.
Introduction
Metastatic malignant melanoma (MM) carries a dismal prognosis. Dacarbazine (DTIC) is the most effective chemotherapeutic agent in M M with a response rate of 15-25%; most of these responses are, however, partial and of a short duration [1]. Considering the failure of current chemotherapy to cure or significantly prolong survival of patients with disseminated MM, the search for new therapeutic strategies is mandatory. Immuno127
therapy with interleukin-2 (IL-2), alone or together with IL-2-activated autologous lymphocytes (lymphokine-activated killer (LAK) cells), gave a response rate of approx. 20%, with 5-10% of the patients achieving a complete response in some studies [2-4]. The synergistic therapeutic effects obtained in experimental tumor systems with several chemotherapeutic agents in combination with IL-2 [5-9], stimulated the application of similar regimens in clinical trials. In this phase II study, which is part of a European multicenter study, supported by EuroCetus, D T I C and recombinant human IL-2 (given by continuous iv infusion) were sequentially administered to 18 M M patients. Preliminary reports on clinical responses and toxicity in some of the patients included in our study were recently published [10-12]. We present here the analysis of the hematologic and immunologic parameters tested prior, during, and post-therapy. We report our results separately, because such a broad analysis has not been performed by the other centers included in this study.
repeated at 5 weeks. Maintenance therapy was scheduled 3 weeks after the completion of induction treatment, consisting of IL-2 18 x 106 IU/ m2/24 h for 5 days, alternating with DTIC 850 mg/m 2 i.v. every 3 weeks for 18 weeks. A maximum of 6 cycles of IL-2 were scheduled.
3.2.2. Protocol H (13patients) DTIC 850 mg/m 2 was given by short i.v. infusion on day 1; IL-2 18 × 106 IU/m2/24 h was administered by continuous i.v. infusion for 5 consecutive days (days 4-9). A maximum of 6 treatment cycles with DTIC and IL-2 were scheduled: the first two at 13-day intervals and the next four at 20-day intervals. Clinical response was evaluated every 2 cycles of IL-2. Treatment was discontinued upon disease progression. Further details regarding treatment are given in references [10-12]. The IL-2 ( > 9 7 % pure, 18 x 106 IU/mg) was kindly supplied by EuroCetus B.V., Amsterdam, The Netherlands. 3.3.
3.
Blood samples
Materials and Methods
Eighteen previously untreated patients (11 males, 7 females) with a median age of 40 years (range: 18-64) and a Karnofsky status >/80% were treated. All patients had histologically proven disseminated melanoma with measurable disease (Table 1). Patients were selected according to the criteria defined previously [10-12]. Treatment was given between February 1988 and April 1990. All 18 patients were evaluable for analysis.
Blood (15-25 ml) was drawn with and without heparin immediately prior (baseline), during IL-2 infusion, and 1 or 2 days after each course of IL2. Blood samples were also taken from some patients 4-6 days after IL-2 and 2-4 days after DTIC. Serum aliquots were stored at - 7 0 ° C . Peripheral blood mononuclear leukocytes (PBMNL) were separated by a Ficoll-Hypaque gradient. For cryopreservation, cell aliquots were suspended in 10% DMSO, 20% fetal calf serum and 70% RPMI 1640 medium and stored at - 180°C for 2 weeks up to 12 months.
3.2.
3.4.
3.1.
Patient population
Treatment
Two different regimens of DTIC and IL-2 were employed.
3.2.1. Protocol I (5patients) Two induction cycles of IL-2 18 × 10 6 IU (equivalent to 3 × 106 Cetus Units)/m2/24 h were given by continuous i.v. infusion on days 1-5 and 12-16. DTIC 850 mg/m 2 was given by short i.v. infusion (20-30 min) on day 26. This cycle was 128
Assessment of hematologic parameters and immune functions in vitro
White blood cell (WBC) counts were performed on fresh samples using Coulter counting. Differential counts were done both by Coulter and on Giemsa stained smears. Cell markers, immune functions and cytokine serum levels were assayed on fresh or frozen samples, or on both (see Results). Except for N K / L A K activity which was considerably reduced by cryopreservation
TABLE 1 Patient characteristics Patient No. a
Age
Sex
Disease sites
IL-2 cycles given
D T I C cycles % of IL-2 given scheduled dose given
Response duration (months) b
1 2 3 4 5
37 50 64 49 37
M M M M F
Lung, liver, L N e Liver, spleen Lung, liver, L N SC d nodules Liver, L N
2 4 2 6 2
2 1 5 1
100 100 100 90 100
M R (5) P R (6) P C R (46 + ) P
6 7 8 9 10 11 12 13 14 15
34 42 41 22 39 18 33 55 40 42
M F M F M F F F M
6 4 2 2 4 2 6 2 2
6 4 2 2 4 2 6 2 2
80 70 85 90 70 100 85 90 100
S (7) S (2) P P
16 17 18
38 24 50
LN, SC nodules Lung Lung, liver LN Spleen Lung, liver, SC nodules Mediastinal mass SC nodules LN Adrenal, lung, SC nodules Lung, liver LN Liver
2 6 1 6
2 6 1 4
95 85 100 60
M M M F
CR (14) P
s (5) P P P P R (16) P
s (7)
apatients Nos. 1-5-were treated according to protocol I, patients Nos. 6-18 according to protocol II (see Materials and Methods). bCR, completeresponse; PR, partial response; MR, minimal response; S, stable; P, progression. ¢LN, lymph nodes. dSC, subcutaneous. (see Results), no significant differences in the other parameters tested were noted in preliminary experiments between fresh and cryopreserved samples. Using cryopreserved samples permitted several assays to be run in parallel simultaneously on samples taken at different times during treatment, thereby avoiding day-to-day variations in experimental conditions. Each frozen sample was tested in 2-4 separate experiments and the results represent the means. PBMNL phenotype was tested by standard immunofluorescence techniques and FACS analysis, using the following monoclonal antibodies (Becton-Dickinson, Mountain view, CA): Leu4 (CD3) for total T cells; Leu2 (CD8), Leu3 (CD4), B4 (CD19) for B cells; Ia (HLA-DR), Leu M3 (CD14) for monocytes; Leul 1 (CD16) for FCy receptor-bearing cells; and Tac (CD25) for IL-2 receptor-bearing cells. Results are also expressed in stimulation index = peak level during treatment/ baseline level. Levels of cytokines (IL-I~, IL-2, TNFct) and
soluble IL-2 receptor in patient sera were assayed by enzyme-linked immunosorbent assays (ELISA) (T Cell Sciences, Cambridge, MA). IL-2 was also determined by a bioassay, using a murine T cell line (CTLL-2) in a 48 h [3H]thymidine uptake assay, with results similar to those obtained by ELISA. In vitro secretion of IL-0t and IL-1 inhibitor by freshly obtained blood monocytes during a 24 h incubation was tested as described previously [13]. Results are expressed in stimulation index (see above). Response to the mitogens phytohemagglutinin (PHA), concanavalin A (con A) and pokeweed mitogen (PWM) was tested as described previously [14]. Cytotoxic activity of PBMNL was assayed at 1:1-100:1 effector/target cell ratios in a 4 h 51Cr-release assay against the NK-sensitive target K562 cells and the NK-resistant, LAK-sensitive target SW 48 human colorectal carcinoma cell line, as previously described [15]. Cells of several patients were tested in parallel against the LAKsensitive A375 human melanoma cell line, with re129
suits similar to those obtained with the SW48 cell line. Results are expressed in mean lytic unit (LU)/106 cells, where 1 LU = number of effector cells required to lyse 20% target cells, using 3000 labeled target cells/well. 3.5.
Statistical analysis
Differences between the responding and nonresponding patient groups for each parameter were tested for significance (P
