The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine)... more The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine) is a potent enhancer of the cytotoxic activity of the anti-pan-T lymphocyte (CD5) T101 monoclonal antibody conjugated to purified ricin A-chain (T101-immunotoxin; T101-IT). We also demonstrate that T101-IT in the presence of amantadine does not induce immunotoxin-mediated cytotoxicity in nontarget cells such as human marrow hematopoietic progenitor cells. These results provide further knowledge for the improvement of ex vivo purification of human bone marrow from normal or leukemic T cells prior to allogeneic or autologous stem cell transplantation, respectively. Furthermore, since amantadine has long been employed safely in human therapy, its use in conjunction with immunotoxins might be exploited in vivo.
The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine)... more The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine) is a potent enhancer of the cytotoxic activity of the anti-pan-T lymphocyte (CD5) T101 monoclonal antibody conjugated to purified ricin A-chain (T101-immunotoxin; T101-IT). We also demonstrate that T101-IT in the presence of amantadine does not induce immunotoxin-mediated cytotoxicity in nontarget cells such as human marrow hematopoietic progenitor cells. These results provide further knowledge for the improvement of ex vivo purification of human bone marrow from normal or leukemic T cells prior to allogeneic or autologous stem cell transplantation, respectively. Furthermore, since amantadine has long been employed safely in human therapy, its use in conjunction with immunotoxins might be exploited in vivo.
Two patients (aged 52 and 44) received high-dose sequential chemotherapy followed by myeloablativ... more Two patients (aged 52 and 44) received high-dose sequential chemotherapy followed by myeloablative therapy and autotransplantation as first line treatment of a stage III multiple myeloma with high plasmacell labelling index. Autograft was performed with peripheral blood cells collected after high-dose etoposide followed by rhGM-CSF continuous infusion. During this period very high numbers of circulating hemopoietic progenitors were detected in both patients. Prompt, complete and durable hematological recovery was observed after autograft with peripheral blood stem cells. Thus, we conclude that massively released progenitors after high-dose chemotherapy and rhGM-CSF include stem cells capable of marrow reconstitution.
A patient with T-cell acute lymphoblastic leukemia (T-ALL) in second remission was treated with h... more A patient with T-cell acute lymphoblastic leukemia (T-ALL) in second remission was treated with high doses of chemotherapy and radiotherapy, followed by transplantation of autologous bone marrow purged ex-vivo with an anti-CD5-saporin immunotoxin (OKT1-SAP). Prior to transplantation the bone marrow graft had been considered in complete remission, as assessed by morphology and immunophenotyping. Twenty-two days after transplantation, the disease relapsed in the bone marrow with the same phenotype as at the onset. Retrospective analysis of the transplanted marrow cells by a recently developed high sensitivity and specificity assay (HSS assay), involving immunologic fractionation and T-cell receptor rearrangement analysis, revealed a graft contamination of approximately 0.5% malignant T-cells. This finding, together with the early post-transplant leukemic relapse, strongly suggests that the bone marrow was the source of the leukemic cells. The data are discussed for their implications on residual leukemia detection by gene rearrangement studies.
Although effective in achieving durable remission in selected tumors incurable by conventional-do... more Although effective in achieving durable remission in selected tumors incurable by conventional-dose chemotherapy, high-dose regimens requiring bone marrow transplantation remain too toxic for widespread use. With the aim to improve the therapeutic index of high-dose therapy, we have developed a novel program whereby several non-cross-resistant agents (including total body irradiation at myeloablative dose) were delivered sequentially rather than concurrently. The regimen has been tested in 25 patients with Hodgkin's disease refractory to primary chemotherapy (MOPP and ABVD) or relapsed within 12 months after first complete remission. The efficacy of the sequential regimen (72% complete response rate, 49% event-free survival and freedom from progression at four years, with a median observation of 3.5 years for patients remaining alive) compares favorably with the activity so far reported for the most effective high-dose regimens. Toxicity was low; no toxic deaths occurred and only three life-threatening adverse events were documented after autografting. The excellent tolerability of the final myeloablative course most likely reflects the speed and completeness of haematologic recovery that followed the use of peripheral blood progenitors as the sole or additional source of myeloid stem cells. The reduction of haematologic toxicity was further emphasized when rhG-CSF was employed to both shorten post-chemotherapy neutropenic intervals and to collect large amounts of peripheral blood stem cells. We expect that the use of growth factors will have a major impact on the therapeutic index of high-dose regimens. This, in turn, will confirm their safety as primary treatment in selected high-risk subsets.
It is still not known whether hematopoiesis reconstituted by autografting with the peripheral blo... more It is still not known whether hematopoiesis reconstituted by autografting with the peripheral blood hematopoietic progenitors (CPCs) after myeloablative high dose radiotherapy and/or chemotherapy is durable and capable of coping with the increased demand imposed by boost radiotherapy, surgery, or infection. The durability of hematopoiesis was evaluated in 34 consecutive cancer patients treated with myeloablative total body irradiation (n = 17, median follow-up 3 years, range 3-49 months) and/or alkylating-agent chemotherapy (n = 17, median follow-up 8 months, range 6-41 months) and autografted with CPCs because bone marrow autografting was contraindicated. CPCs (> or = 8 x 10(6) CD34 + cells/kg) had been collected during mobilization into the circulation in response to previous anticancer therapy and hematopoietic growth factor(s). Following brief temporary pancytopenia, all patients achieved normal and durable hematopoiesis. The newly reconstituted hematopoietic system was capable of reacting favorably to stressful and debilitating events such as surgery, radiotherapy, or varicella-zoster infection. No secondary irreversible failure of blood cell production occurred. The documentation of the durability of normal hematopoiesis following myeloablative cancer therapy and autografting with mobilized CPCs implies that the latter procedure, rather than being solely an alternative to bone marrow autografting, represents an advantageous tool of choice permitting substantial amelioration of the therapeutic index of high-dose cancer therapy.
Seventy-seven (68 operable breast cancer with > 9 metastatic axillary nodes and 9 inflamma... more Seventy-seven (68 operable breast cancer with > 9 metastatic axillary nodes and 9 inflammatory breast cancer) entered this study. During hematopoietic recovery after cancer therapy with high-dose cyclophosphamide (7 g/m2; HD-CTX) circulating hematopoietic progenitors were collected by leukapheresis (LK) in all patients and then cryopreserved for autologous transplantation. Following HD-CTX, 70 patients were treated with hematopoietic growth factor(s) for 14 days: 38 with rhGM-CSF (group a), 16 with rhIL-3 (group b), 11 with sequential rhIL-3 and rhGM-CSF (group c), 5 with sequential rhIL-3 and rhG-CSF (group d). Seven control patients (group e) did not receive any growth factor. Leukaphereses, carried out over 2-4 consecutive days per patient, were started earlier in group c and in group d patients (mean day: +12 after HD-CTX). The sequential administration of rhIL-3 and rhG-CSF (group d) resulted in clearly higher yield of CFU-GM and CD34+ cells per leukapheresis (65.9 x 10(4)/Kg versus 20.9 x 10(6)/Kg, respectively) if compared with other groups of treatment.
To assess the toxicity, efficacy, and applicability of high-dose therapy with bone marrow and/or ... more To assess the toxicity, efficacy, and applicability of high-dose therapy with bone marrow and/or peripheral-blood autotransplantation in high-risk, previously untreated patients with multiple myeloma. Thirteen consecutive patients with high-labeling index (LI) multiple myeloma received a novel high-dose sequential (HDS) regimen consisting in the high-dose administration of cyclophosphamide (7 g/m2) followed by vincristine (1.4 mg/m2) plus methotrexate (8 g/m2 with leucovorin rescue), etoposide (2 g/m2) and, finally, total-body irradiation (TBI; 10 Gy) plus melphalan (120 mg/m2) with autografting of peripheral-blood hematopoietic progenitor cells. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 5 micrograms/kg/d) was continuously infused after cyclophosphamide and etoposide both to accelerate hematopoietic recovery and to expand/mobilize the hematopoietic progenitor-cell pool. Among 13 patients, 12 completed the program; 10 (or 77%) achieved a complete response and five are alive and disease-free after a median follow-up duration of 36 months (range, 24 to 52). The durations of both freedom from progression (FFP; median, 38 months) and overall survival (OS; median, 41 months) were significantly superior in the 13 HDS-treated patients as compared with 19 well-matched historical controls. HDS emerges as a highly effective, well-tolerated, and widely accessible regimen capable of imparting a survival benefit to patients with high-LI multiple myeloma. Larger studies using this or similar programs in standard-risk myeloma are clearly warranted.
We report that blood cell autografts, collected by single leukapheresis in cancer patients (n = 1... more We report that blood cell autografts, collected by single leukapheresis in cancer patients (n = 11) at the time of mobilization of hematopoietic progenitors into peripheral blood following anticancer therapy with high-dose cyclophosphamide (HD-CTX) plus interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF/filgrastim), comprise 1.98 +/- 0.39 x 10(5)/kg (mean +/- SE) CD34+ progenitors of dendritic cells (DCs). This number corresponds to 140-fold more progenitors than in a control autograft collected in the steady state. DCs derived from mobilized CD34+ cells, morphologically and immunophenotypically undistinguishable from skin Langerhans cells and DCs from bone marrow and cord blood CD34+ cells, are shown to be powerful stimulators of allogeneic T cell proliferation in primary MLR and of autologous HLA-DR-restricted CD4+ T cell proliferation in response to presentation of xenogenic antigens. We show that the GM-CSF-plus-TNF-alpha-dependent ex vivo generation of DCs from mobilized CD34+ cells is 2.5-fold enhanced by flk-2/flt-3 ligand or c-kit ligand (stem cell factor) and five-fold enhanced by a combination of these growth factors. In addition, the optimal serum for the generation of DCs is autologous HD-CTX recovery-phase serum rather than fetal calf serum (FCS) or steady-state human serum, which are clinically inadequate and ineffective, respectively. In practice, the stimulation of CD34+ cells in a blood cell autograft (15.75 +/- 2.46 x 10(6)/kg) provided by the above four growth factors should permit ex vivo generation of approximately 40 x 10(9) DCs in an adult patient. These new findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.
We compared hematologic and clinical effects of granulocyte-macrophage colony-stimulating factor ... more We compared hematologic and clinical effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) after treatment with high-dose cyclophosphamide (HD-CTX, 7 g/m2), given as the first phase of a high-dose sequential chemotherapy program that includes a myeloablative therapy with mobilized progenitor cell autografting. Forty-nine consecutive patients with non-Hodgkin's lymphoma, Hodgkin's disease, or poor-prognosis breast cancer received GM-CSF (n = 27) or G-CSF (n = 22) after HD-CTX in two consecutive, nonrandomized studies. Cytokines were administered in continuous intravenous (i.v.) infusion for 14 to 15 days at a median dose of 5.5 and 10 micrograms/kg/d, respectively, starting 24 hours after HD-CTX. Neutrophil recovery was faster with G-CSF administration (11.5 v 13.2 days; P = .01), whereas platelet counts recovered more rapidly with GM-CSF (13.7 v 16.6 days; P = .01). Prophylactic platelet transfusions were administered more frequently to patients treated with G-CSF than with GM-CSF (66% v 22% of the patients; P = .02). No clinically significant difference was observed between the two groups concerning days of absolute neutropenia or neutropenic fever. Both cytokines reduced the time to eligibility for subsequent chemotherapy administration compared with historical controls not given cytokine (14 to 16 v 20 days). Both cytokines increased circulation of hematopoietic progenitors. Most side effects were World Health Organization (WHO) median grade 1 to 2, were more frequent during GM-CSF than during G-CSF treatment, and were reversible by simple supportive measures and/or by dose reduction or suspension of the cytokine. Permanent suspension of cytokine administration was never required in either group. GM-CSF or G-CSF administration after HD-CTX reduces hematologic toxicity of high-dose chemotherapy and induces circulation of large amounts of hematopoietic progenitors suitable for autografting in cancer patients.
Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-... more Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-specific T cells in melanoma patients. However, it is not known whether autologous DCs, differentiated by two of the most efficient protocols (from CD34+ progenitors or from monocytes), are equally effective as professional antigen-presenting cells (APCs) when the patients have a low frequency of peptide-specific precursors. To this end, a limiting dilution assay was applied to evaluate the frequency of antigen-specific CTL precursors (CTLps) in peripheral blood of HLA-A*0201+ melanoma patients. Then, from two melanoma patients showing low frequency of CTLps to melanoma antigen-A/melanoma antigen recognized by T cell (Melan-A/Mart-1)(27-35) peptide, autologous DCs were differentiated from granulocyte colony-stimulating factor-mobilized CD34+ progenitors or from monocytes. CD34+- and monocyte-derived DCs were characterized by a similar proportion of CD1a+ cells expressing HLA class II antigens and CD54, CD80, and CD86 molecules. Both types of DC presented Melan-A/Mart-1(27-35) and tyrosinase(369-377) peptides to melanoma-specific CTL clones and were equally effective as peptide-pulsed APCs in the activation of influenza A matrix(58-66)-specific CTLs from high-frequency precursors (1294/10(6) and 1789/10(6) lymphocytes in the two patients). However, efficient activation of Melan-A/Mart-1(27-35)-specific CTLs from low-frequency precursors (158/10(6) and 77/10(6) lymphocytes) of the two patients was markedly dependent on the use of peptide-loaded CD34+-derived DCs. These results suggest that CD34+- and monocyte-derived DCs are not functionally equivalent APCs for the activation of low-frequency peptide-specific CTLps.
Although T-cell-defined tumor antigens have recently been identified in several tumors, human neo... more Although T-cell-defined tumor antigens have recently been identified in several tumors, human neoplastic cells are considered to be poorly immunogenic. The development of clinical approaches to the immunotherapy of human tumors thus requires the identification of effective adjuvants. Dendritic cells (DC) are a specialized system of antigen-presenting cells (APC) that could be utilized as natural adjuvants to elicit antitumor immune responses. In an attempt to overcome the problem of the low frequency of mature DC in peripheral blood, several methods have been applied for the ex vivo generation of human DC by culturing mobilized CD34+ cells or monocytes with combinations of cytokines. As shown in murine models as well as in the human system, after loading with peptides or tumor lysates or infection with recombinant viral vectors, DC expressing tumor antigens are able to elicit specific antitumor T cells and to mediate tumor regression. These initial results suggest that this new approach may lead to effective antitumor responses even in heavily pretreated patients bearing advanced cancers, but further clinical trials are required to validate the efficacy of vaccination with tumor-antigen-loaded DC.
To assess the efficacy, toxicity, and applicability of high-dose therapy administered as adjuvant... more To assess the efficacy, toxicity, and applicability of high-dose therapy administered as adjuvant initial treatment to women with breast cancer with extensive nodal involvement. Sixty-seven patients with stage II to III breast cancer involving > or = 10 axillary nodes received a novel high-dose sequential (HDS) regimen, including the high-dose administration of three non-cross-resistant drugs (cyclophosphamide, methotrexate, and melphalan) given within the shortest interval of time as possible with hematologic and nonhematologic toxicity. Sixty-three patients completed the program as planned, one patient died of acute toxicity, and three patients were switched to standard-dose adjuvant therapy. After a median follow-up duration of 48.5 months and a lead follow-up of 78 months, actuarial relapse-free survival for all 67 registered patients is 57% and overall survival is 70%, respectively. Comparison with a historical control group of 58 consecutive patients showed a significantly superior rate of freedom from relapse for the HDS-treated group (57% v 41%, respectively), in particular when two subgroups of patients, more homogeneous for their number of involved nodes, were compared (65% v 42%). Overall, treatment was of short duration (median, 70 days), required a median of 32 days of hospital stay, and was associated with only a few severe side effects (the most distressing being oral mucositis after melphalan therapy). HDS therapy emerges as an effective and applicable regimen, whose major toxicity was occasional. Final assessment of its value in a randomized, multicenter trial is presently underway.
The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine)... more The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine) is a potent enhancer of the cytotoxic activity of the anti-pan-T lymphocyte (CD5) T101 monoclonal antibody conjugated to purified ricin A-chain (T101-immunotoxin; T101-IT). We also demonstrate that T101-IT in the presence of amantadine does not induce immunotoxin-mediated cytotoxicity in nontarget cells such as human marrow hematopoietic progenitor cells. These results provide further knowledge for the improvement of ex vivo purification of human bone marrow from normal or leukemic T cells prior to allogeneic or autologous stem cell transplantation, respectively. Furthermore, since amantadine has long been employed safely in human therapy, its use in conjunction with immunotoxins might be exploited in vivo.
The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine)... more The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine) is a potent enhancer of the cytotoxic activity of the anti-pan-T lymphocyte (CD5) T101 monoclonal antibody conjugated to purified ricin A-chain (T101-immunotoxin; T101-IT). We also demonstrate that T101-IT in the presence of amantadine does not induce immunotoxin-mediated cytotoxicity in nontarget cells such as human marrow hematopoietic progenitor cells. These results provide further knowledge for the improvement of ex vivo purification of human bone marrow from normal or leukemic T cells prior to allogeneic or autologous stem cell transplantation, respectively. Furthermore, since amantadine has long been employed safely in human therapy, its use in conjunction with immunotoxins might be exploited in vivo.
Two patients (aged 52 and 44) received high-dose sequential chemotherapy followed by myeloablativ... more Two patients (aged 52 and 44) received high-dose sequential chemotherapy followed by myeloablative therapy and autotransplantation as first line treatment of a stage III multiple myeloma with high plasmacell labelling index. Autograft was performed with peripheral blood cells collected after high-dose etoposide followed by rhGM-CSF continuous infusion. During this period very high numbers of circulating hemopoietic progenitors were detected in both patients. Prompt, complete and durable hematological recovery was observed after autograft with peripheral blood stem cells. Thus, we conclude that massively released progenitors after high-dose chemotherapy and rhGM-CSF include stem cells capable of marrow reconstitution.
A patient with T-cell acute lymphoblastic leukemia (T-ALL) in second remission was treated with h... more A patient with T-cell acute lymphoblastic leukemia (T-ALL) in second remission was treated with high doses of chemotherapy and radiotherapy, followed by transplantation of autologous bone marrow purged ex-vivo with an anti-CD5-saporin immunotoxin (OKT1-SAP). Prior to transplantation the bone marrow graft had been considered in complete remission, as assessed by morphology and immunophenotyping. Twenty-two days after transplantation, the disease relapsed in the bone marrow with the same phenotype as at the onset. Retrospective analysis of the transplanted marrow cells by a recently developed high sensitivity and specificity assay (HSS assay), involving immunologic fractionation and T-cell receptor rearrangement analysis, revealed a graft contamination of approximately 0.5% malignant T-cells. This finding, together with the early post-transplant leukemic relapse, strongly suggests that the bone marrow was the source of the leukemic cells. The data are discussed for their implications on residual leukemia detection by gene rearrangement studies.
Although effective in achieving durable remission in selected tumors incurable by conventional-do... more Although effective in achieving durable remission in selected tumors incurable by conventional-dose chemotherapy, high-dose regimens requiring bone marrow transplantation remain too toxic for widespread use. With the aim to improve the therapeutic index of high-dose therapy, we have developed a novel program whereby several non-cross-resistant agents (including total body irradiation at myeloablative dose) were delivered sequentially rather than concurrently. The regimen has been tested in 25 patients with Hodgkin's disease refractory to primary chemotherapy (MOPP and ABVD) or relapsed within 12 months after first complete remission. The efficacy of the sequential regimen (72% complete response rate, 49% event-free survival and freedom from progression at four years, with a median observation of 3.5 years for patients remaining alive) compares favorably with the activity so far reported for the most effective high-dose regimens. Toxicity was low; no toxic deaths occurred and only three life-threatening adverse events were documented after autografting. The excellent tolerability of the final myeloablative course most likely reflects the speed and completeness of haematologic recovery that followed the use of peripheral blood progenitors as the sole or additional source of myeloid stem cells. The reduction of haematologic toxicity was further emphasized when rhG-CSF was employed to both shorten post-chemotherapy neutropenic intervals and to collect large amounts of peripheral blood stem cells. We expect that the use of growth factors will have a major impact on the therapeutic index of high-dose regimens. This, in turn, will confirm their safety as primary treatment in selected high-risk subsets.
It is still not known whether hematopoiesis reconstituted by autografting with the peripheral blo... more It is still not known whether hematopoiesis reconstituted by autografting with the peripheral blood hematopoietic progenitors (CPCs) after myeloablative high dose radiotherapy and/or chemotherapy is durable and capable of coping with the increased demand imposed by boost radiotherapy, surgery, or infection. The durability of hematopoiesis was evaluated in 34 consecutive cancer patients treated with myeloablative total body irradiation (n = 17, median follow-up 3 years, range 3-49 months) and/or alkylating-agent chemotherapy (n = 17, median follow-up 8 months, range 6-41 months) and autografted with CPCs because bone marrow autografting was contraindicated. CPCs (> or = 8 x 10(6) CD34 + cells/kg) had been collected during mobilization into the circulation in response to previous anticancer therapy and hematopoietic growth factor(s). Following brief temporary pancytopenia, all patients achieved normal and durable hematopoiesis. The newly reconstituted hematopoietic system was capable of reacting favorably to stressful and debilitating events such as surgery, radiotherapy, or varicella-zoster infection. No secondary irreversible failure of blood cell production occurred. The documentation of the durability of normal hematopoiesis following myeloablative cancer therapy and autografting with mobilized CPCs implies that the latter procedure, rather than being solely an alternative to bone marrow autografting, represents an advantageous tool of choice permitting substantial amelioration of the therapeutic index of high-dose cancer therapy.
Seventy-seven (68 operable breast cancer with > 9 metastatic axillary nodes and 9 inflamma... more Seventy-seven (68 operable breast cancer with > 9 metastatic axillary nodes and 9 inflammatory breast cancer) entered this study. During hematopoietic recovery after cancer therapy with high-dose cyclophosphamide (7 g/m2; HD-CTX) circulating hematopoietic progenitors were collected by leukapheresis (LK) in all patients and then cryopreserved for autologous transplantation. Following HD-CTX, 70 patients were treated with hematopoietic growth factor(s) for 14 days: 38 with rhGM-CSF (group a), 16 with rhIL-3 (group b), 11 with sequential rhIL-3 and rhGM-CSF (group c), 5 with sequential rhIL-3 and rhG-CSF (group d). Seven control patients (group e) did not receive any growth factor. Leukaphereses, carried out over 2-4 consecutive days per patient, were started earlier in group c and in group d patients (mean day: +12 after HD-CTX). The sequential administration of rhIL-3 and rhG-CSF (group d) resulted in clearly higher yield of CFU-GM and CD34+ cells per leukapheresis (65.9 x 10(4)/Kg versus 20.9 x 10(6)/Kg, respectively) if compared with other groups of treatment.
To assess the toxicity, efficacy, and applicability of high-dose therapy with bone marrow and/or ... more To assess the toxicity, efficacy, and applicability of high-dose therapy with bone marrow and/or peripheral-blood autotransplantation in high-risk, previously untreated patients with multiple myeloma. Thirteen consecutive patients with high-labeling index (LI) multiple myeloma received a novel high-dose sequential (HDS) regimen consisting in the high-dose administration of cyclophosphamide (7 g/m2) followed by vincristine (1.4 mg/m2) plus methotrexate (8 g/m2 with leucovorin rescue), etoposide (2 g/m2) and, finally, total-body irradiation (TBI; 10 Gy) plus melphalan (120 mg/m2) with autografting of peripheral-blood hematopoietic progenitor cells. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 5 micrograms/kg/d) was continuously infused after cyclophosphamide and etoposide both to accelerate hematopoietic recovery and to expand/mobilize the hematopoietic progenitor-cell pool. Among 13 patients, 12 completed the program; 10 (or 77%) achieved a complete response and five are alive and disease-free after a median follow-up duration of 36 months (range, 24 to 52). The durations of both freedom from progression (FFP; median, 38 months) and overall survival (OS; median, 41 months) were significantly superior in the 13 HDS-treated patients as compared with 19 well-matched historical controls. HDS emerges as a highly effective, well-tolerated, and widely accessible regimen capable of imparting a survival benefit to patients with high-LI multiple myeloma. Larger studies using this or similar programs in standard-risk myeloma are clearly warranted.
We report that blood cell autografts, collected by single leukapheresis in cancer patients (n = 1... more We report that blood cell autografts, collected by single leukapheresis in cancer patients (n = 11) at the time of mobilization of hematopoietic progenitors into peripheral blood following anticancer therapy with high-dose cyclophosphamide (HD-CTX) plus interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF/filgrastim), comprise 1.98 +/- 0.39 x 10(5)/kg (mean +/- SE) CD34+ progenitors of dendritic cells (DCs). This number corresponds to 140-fold more progenitors than in a control autograft collected in the steady state. DCs derived from mobilized CD34+ cells, morphologically and immunophenotypically undistinguishable from skin Langerhans cells and DCs from bone marrow and cord blood CD34+ cells, are shown to be powerful stimulators of allogeneic T cell proliferation in primary MLR and of autologous HLA-DR-restricted CD4+ T cell proliferation in response to presentation of xenogenic antigens. We show that the GM-CSF-plus-TNF-alpha-dependent ex vivo generation of DCs from mobilized CD34+ cells is 2.5-fold enhanced by flk-2/flt-3 ligand or c-kit ligand (stem cell factor) and five-fold enhanced by a combination of these growth factors. In addition, the optimal serum for the generation of DCs is autologous HD-CTX recovery-phase serum rather than fetal calf serum (FCS) or steady-state human serum, which are clinically inadequate and ineffective, respectively. In practice, the stimulation of CD34+ cells in a blood cell autograft (15.75 +/- 2.46 x 10(6)/kg) provided by the above four growth factors should permit ex vivo generation of approximately 40 x 10(9) DCs in an adult patient. These new findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.
We compared hematologic and clinical effects of granulocyte-macrophage colony-stimulating factor ... more We compared hematologic and clinical effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) after treatment with high-dose cyclophosphamide (HD-CTX, 7 g/m2), given as the first phase of a high-dose sequential chemotherapy program that includes a myeloablative therapy with mobilized progenitor cell autografting. Forty-nine consecutive patients with non-Hodgkin's lymphoma, Hodgkin's disease, or poor-prognosis breast cancer received GM-CSF (n = 27) or G-CSF (n = 22) after HD-CTX in two consecutive, nonrandomized studies. Cytokines were administered in continuous intravenous (i.v.) infusion for 14 to 15 days at a median dose of 5.5 and 10 micrograms/kg/d, respectively, starting 24 hours after HD-CTX. Neutrophil recovery was faster with G-CSF administration (11.5 v 13.2 days; P = .01), whereas platelet counts recovered more rapidly with GM-CSF (13.7 v 16.6 days; P = .01). Prophylactic platelet transfusions were administered more frequently to patients treated with G-CSF than with GM-CSF (66% v 22% of the patients; P = .02). No clinically significant difference was observed between the two groups concerning days of absolute neutropenia or neutropenic fever. Both cytokines reduced the time to eligibility for subsequent chemotherapy administration compared with historical controls not given cytokine (14 to 16 v 20 days). Both cytokines increased circulation of hematopoietic progenitors. Most side effects were World Health Organization (WHO) median grade 1 to 2, were more frequent during GM-CSF than during G-CSF treatment, and were reversible by simple supportive measures and/or by dose reduction or suspension of the cytokine. Permanent suspension of cytokine administration was never required in either group. GM-CSF or G-CSF administration after HD-CTX reduces hematologic toxicity of high-dose chemotherapy and induces circulation of large amounts of hematopoietic progenitors suitable for autografting in cancer patients.
Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-... more Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-specific T cells in melanoma patients. However, it is not known whether autologous DCs, differentiated by two of the most efficient protocols (from CD34+ progenitors or from monocytes), are equally effective as professional antigen-presenting cells (APCs) when the patients have a low frequency of peptide-specific precursors. To this end, a limiting dilution assay was applied to evaluate the frequency of antigen-specific CTL precursors (CTLps) in peripheral blood of HLA-A*0201+ melanoma patients. Then, from two melanoma patients showing low frequency of CTLps to melanoma antigen-A/melanoma antigen recognized by T cell (Melan-A/Mart-1)(27-35) peptide, autologous DCs were differentiated from granulocyte colony-stimulating factor-mobilized CD34+ progenitors or from monocytes. CD34+- and monocyte-derived DCs were characterized by a similar proportion of CD1a+ cells expressing HLA class II antigens and CD54, CD80, and CD86 molecules. Both types of DC presented Melan-A/Mart-1(27-35) and tyrosinase(369-377) peptides to melanoma-specific CTL clones and were equally effective as peptide-pulsed APCs in the activation of influenza A matrix(58-66)-specific CTLs from high-frequency precursors (1294/10(6) and 1789/10(6) lymphocytes in the two patients). However, efficient activation of Melan-A/Mart-1(27-35)-specific CTLs from low-frequency precursors (158/10(6) and 77/10(6) lymphocytes) of the two patients was markedly dependent on the use of peptide-loaded CD34+-derived DCs. These results suggest that CD34+- and monocyte-derived DCs are not functionally equivalent APCs for the activation of low-frequency peptide-specific CTLps.
Although T-cell-defined tumor antigens have recently been identified in several tumors, human neo... more Although T-cell-defined tumor antigens have recently been identified in several tumors, human neoplastic cells are considered to be poorly immunogenic. The development of clinical approaches to the immunotherapy of human tumors thus requires the identification of effective adjuvants. Dendritic cells (DC) are a specialized system of antigen-presenting cells (APC) that could be utilized as natural adjuvants to elicit antitumor immune responses. In an attempt to overcome the problem of the low frequency of mature DC in peripheral blood, several methods have been applied for the ex vivo generation of human DC by culturing mobilized CD34+ cells or monocytes with combinations of cytokines. As shown in murine models as well as in the human system, after loading with peptides or tumor lysates or infection with recombinant viral vectors, DC expressing tumor antigens are able to elicit specific antitumor T cells and to mediate tumor regression. These initial results suggest that this new approach may lead to effective antitumor responses even in heavily pretreated patients bearing advanced cancers, but further clinical trials are required to validate the efficacy of vaccination with tumor-antigen-loaded DC.
To assess the efficacy, toxicity, and applicability of high-dose therapy administered as adjuvant... more To assess the efficacy, toxicity, and applicability of high-dose therapy administered as adjuvant initial treatment to women with breast cancer with extensive nodal involvement. Sixty-seven patients with stage II to III breast cancer involving > or = 10 axillary nodes received a novel high-dose sequential (HDS) regimen, including the high-dose administration of three non-cross-resistant drugs (cyclophosphamide, methotrexate, and melphalan) given within the shortest interval of time as possible with hematologic and nonhematologic toxicity. Sixty-three patients completed the program as planned, one patient died of acute toxicity, and three patients were switched to standard-dose adjuvant therapy. After a median follow-up duration of 48.5 months and a lead follow-up of 78 months, actuarial relapse-free survival for all 67 registered patients is 57% and overall survival is 70%, respectively. Comparison with a historical control group of 58 consecutive patients showed a significantly superior rate of freedom from relapse for the HDS-treated group (57% v 41%, respectively), in particular when two subgroups of patients, more homogeneous for their number of involved nodes, were compared (65% v 42%). Overall, treatment was of short duration (median, 70 days), required a median of 32 days of hospital stay, and was associated with only a few severe side effects (the most distressing being oral mucositis after melphalan therapy). HDS therapy emerges as an effective and applicable regimen, whose major toxicity was occasional. Final assessment of its value in a randomized, multicenter trial is presently underway.
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