Peripheral blood mobilization of different lymphocyte and dendritic cell subsets with the use of intermediate doses of G-CSF in patients with non-Hodgkin's lymphoma and multiple myeloma.

Between June 2003 and November 2004, we collected mobilized peripheral blood units from 29 patients with non-Hodgkin's lymphoma and multiple myeloma for autologous peripheral blood stem cell transplantation. They received granulocyte colony-stimulating factor (G-CSF) (16 micro g/kg/day) for a total of 5 days. Immediately before and 3 h after the fourth and fifth dose of G-CSF, we performed flow cytometry analysis to quantify: T cells (CD3+CD4+, CD3+CD8+), B cells (CD19+), NK cells (CD3-CD16+CD56+), NKT cells (CD3+CD16+CD56+), type 1 dendritic cells (DC1) (lin-HLA-DR+CD11c+), type 2 dendritic cells (DC2) (lin-HLA-DR+CD123+), regulatory T cells (Tregs) (CD4+CD25+), and activated T cells (CD3+HLA-DR+). All cell subsets were mobilized after G-CSF treatment with the exception of B, NK, and NKT lymphocytes. The median number of Treg cells before and after G-CSF was statistically different (29+/-14.9x10(6)/l vs 70.1+/-46.1x10(6)/l, P<0.02). DCs were mobilized significantly with a 5.9-fold increase in DC2 (15.1+/-30.3x10(6)/l vs 89.8+/-81.0x10(6)/l, P<0.02) and a 2.6-fold increase for DC1 (41+/-42.5x10(6)/l vs 109.5+/-58.0x10(6)/l, P<0.04). Patients received a mean of 3.1+/-1.2x10(7)/kg NK cells, 1.3+/-0.9x10(7)/kg NKT cells, 0.41+/-0.29x10(7)/kg DC1, 0.2+/-0.22x10(7)/kg DC2, and 1.8+/-1.9x10(7)/kg Tregs. In conclusion, intermediate doses of G-CSF induce mobilization of different lymphocyte subsets, with the exception of B, NK, and NKT cells. The mobilization of certain suppressive populations (DC2 and Treg) could be in theory deleterious, at least in patients with cancer.

Clinical relevance of NK, NKT, and dendritic cell dose in patients receiving G-CSF-mobilized peripheral blood allogeneic stem cell transplantation.

To analyze the relationship between the cellular composition of peripheral blood allografts and clinical outcome, we performed a prospective study in 45 adult patients who underwent allogeneic peripheral blood hematopoietic stem cell transplantation (HSCT) from a histocompatibility leukocyte antigen identical sibling donor for different hematological malignancies. The dose of CD34+, CD3+, CD4+, CD8+, and CD19+ lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, type 1 and type 2 dendritic cells (DC1 and DC2), as well as regulatory T (Treg) lymphocytes was analyzed. All patients were conditioned with busulphan and cyclophosphamide (BuCy2) +/- VP-16 and received a short course of methotrexate and cyclosporin-A as graft-versus-host disease (GVHD) prophylaxis. Acute GVHD (aGVHD) was present in 9 of 43 (21%) patients, and chronic GVHD (cGVHD) developed in 18 of 39 (46%) patients. There was a significantly higher incidence of aGVHD in patients receiving more than 6x10(6)/kg CD34+ cells. In univariate analysis, variables associated with better survival were as follows: a dose of less than 1.5x10(7)/kg NKT cells and less than 1.7x10(6)/kg DC2 for disease-free survival (DFS), and a dose of less than 3x10(7)/kg NK cells, less than 1.5x10(7)/kg NKT cells, less than 3x10(6)/kg DC1, and less than 1.7x10(6)/kg DC2 for overall survival (OS). In the Cox regression analysis, the dose of NKT cells was the only variable associated with better DFS, while the doses of NK, NKT, and CD34+ cells (less than 8x10(6)/kg) were associated with better OS. In conclusion, different circulating cell populations, other than CD34+ cells, are also of relevance in predicting the clinical outcome after allogeneic peripheral blood HSCT.

CD4+ CD25+ lymphocyte and dendritic cell mobilization with intermediate doses of recombinant human granulocyte colony-stimulating factor in healthy donors.

We prospectively conducted a quantitative and phenotypic analysis of T, B, natural killer (NK), NKT, type 1 and 2 dendritic cells (DC), and regulatory T cells, before and after mobilization with intermediate doses of granulocyte colony-stimulating factor (G-CSF) (16 microg/kg per day). Between November, 2003, and December, 2004, we collected stem cells from 25 HLA identical sibling donors for allogeneic hematopoietic stem cell transplantation. Before mobilization and 3 h after the fourth and fifth doses of G-CSF, blood samples were taken for blood counts and flow cytometry. The median number of regulatory T cells before and after G-CSF was statistically different (69 +/- 41 x 10(6)/L versus 161 +/- 159 x 10(6)/L, p < 0.01). We observed a 1.7-fold increase in NK and NKT cells (p < 0.009 and p < 0.02, respectively). DC were mobilized with a 11.5-fold increase in type 2 (p < 0.004) and a 8.5-fold increase in type 1 DC (p < 0.003). The patients received a mean of: 2.2 x 10(7)/kg +/- 1.4 x 10(7)/kg of NK cells, 0.95 x 10(7)/kg +/- 0.81 x 107/kg of NKT cells, 0.43 x 107/kg +/- 0.53 x 10(7)/kg of type 1 DC, 0.3 v 10(7)/kg +/- 0.45 x 10(7)/kg of type 2 DC and 1.4 x 10(7)/kg +/- 1.2 x 10(7)/kg of regulatory T cells. Using intermediate doses of G-CSF, we have demonstrated the mobilization of different lymphocyte subsets, in particular regulatory T cells and DC, which can be expanded later and used in the treatment of cancer and autoimmune diseases.

Donor lymphocyte infusions for relapse of chronic myeloid leukemia after allogeneic stem cell transplantation: prognostic significance of the dose of CD3(+) and CD4(+) lymphocytes.

Between December 1993 and November 2001, 30 patients with chronic myeloid leukemia who relapsed after stem cell transplantation were studied. Seventeen patients were not treated before donor lymphocyte infusion (DLI), eight patients received interferon-alpha (IFN-alpha), and five underwent chemotherapy. The method of DLI was the bulk dose regimen. The median time between DLIs was 6 weeks. The median number of infusions was three; the median time from transplant to relapse was 17 months and from relapse to DLI 2 months. Eleven patients (37%) were in molecular/cytogenetic relapse, 14 (47%) in chronic phase, and five (16%) in accelerated or blastic phase. Seventeen patients (57%) developed acute graft-versus-host disease (GVHD). Chronic GVHD was observed in 15 of 24 (62%) patients. Four (13%) patients developed cytopenia after a median of 30 days. Nineteen (63%) patients achieved response, 15 of them developed GVHD. The response rate according to the disease phase was molecular or cytogenetic relapse: 91%, chronic phase: 57%, and accelerated or blastic phase: 20%. The median time to response was 6 months. Patients treated with IFN-alpha or no treatment as well as those who were in molecular/cytogenetic relapse and those who received a CD3(+) cell dose <1 x 10(8)/kg and CD4(+) <8 x 10(7)/kg had better survival. We conclude that patients who receive lower doses of lymphocytes have better survival. In some patients IFN-alpha seems to be a good choice to potentiate the graft-versus-leukemia (GVL) effect.

Intermediate doses of melphalan and dexamethasone are better than vincristine, adriamycin, and dexamethasone (VAD) and polychemotherapy for the treatment of primary plasma cell leukemia.

Primary plasma cell leukemia (PPCL) is a rare form of disease accounting for 1-2 percent of myelomas. Between September 1990 and November 2000, among 540 patients with myeloma studied, 24 fulfilled the criteria of PPCL (4.4 percent). We found high frequencies of female patients (62 percent), Bence Jones proteinuria (79 percent), anemia (88 percent), bleeding (54 percent), confusional syndrome (42 percent), weight loss (71 percent), hepatomegaly (25 percent), splenomegaly (21 percent), leukocytosis (62 percent), and thrombocytopenia (71 percent). High serum levels of creatinine, calcium, lactate dehydrogenase (LDH), and beta(2)-microglobulin were detected in 50 percent, 37 percent, 58 percent, and 71 percent, respectively. Four patients were treated with vincristine, melphalan, cyclophosphamide, prednisone, and adriamycin (VMCPA), 12 with vincristine, adriamycin, and dexamethasone (VAD), and 8 with M-80 (oral melphalan 80 mg/m(2) plus dexamethasone 40 mg/m(2)). There was a trend toward lower values of Karnofsky score (P=0.07) and higher values of LDH (P=0.2) in the VAD group. Other clinical characteristics were comparable among the three groups. Complete plus partial responses were achieved in one and six patients treated with VMCPA and M-80, respectively. All patients treated with VAD failed to respond to treatment. Patients receiving the M-80 regimen experienced higher platelet toxicity (P=0.05), vomiting (P<0.0003), and mucositis. Also, the need for red blood cell transfusions was higher in the M-80 group. Median overall survival was 60 days. Overall survival was better in patients achieving complete or partial response. In conclusion, our study illustrates that intermediate doses of melphalan plus dexamethasone are an effective chemotherapy regimen for this aggressive disease. Response to treatment is the only prognostic factor for survival in these patients.

Allogeneic bone marrow transplantation for chronic myeloid leukemia: a single center experience.

BACKGROUND: Bone marrow transplantation (BMT) is the therapy of choice for patients with chronic myeloid leukemia (CML) who have a human leukocyte antigen (HLA)-identical donor and are under 50 years of age. METHODS: Here, 45 patients with CML were treated with busulfan (Bu) 16 mg/kg and cyclophosphamide (Cy) 120 mg/kg before allogeneic BMT from an HLA-identical sibling 27 (60%) or a 1-antigen mismatch donor 18 (40%). Eighteen patients (40%) were in the early chronic phase (CP) and 27 (60%) in late CP. We used cyclosporin-A (CsA) in 20 patients and cyclosporin-A-methotrexate (CsA-MTX) in 25 for graft-vs.-host disease (GVHD) prophylaxis. RESULTS: We observed a high incidence of acute and chronic GVHD (69% and 67%, respectively). A multivariate analysis identified differences in the sex of the donor and the recipient (p = 0.03) and grade III-IV acute GVHD (p = 0.0001) as significant adverse influences on disease-free survival. Age, sex, chronic GVHD, disease phase, one antigen-mismatch and use of CsA or CsA-MTX had no statistical significance. The 3-year probabilities of relapse, disease-free survival, and overall survival were 11%, 55%, and 60%, respectively. Transplant-related mortality occurred in 31% of the cases. The high frequency of GVHD is explained by HLA determination by serological typing, differences in sex between the donor and recipient, and a high proportion (40%) of 1 antigen-mismatch donors. CONCLUSIONS: BMT is a procedure feasible for patients with CML in early and late chronic phase and even in those with an HLA non-identical donor. Strategies directed to decrease acute GVHD could improve the outcome of these patients.

Prospective randomized clinical trial comparing high-dose ifosfamide + GM-CSF vs high-dose cyclophosphamide + GM-CSF for blood progenitor cell mobilization.

Between August 1994 and June 1999, 56 patients were prospectively randomized to receive ifosfamide 10 g/m2 + GM-CSF 5 microg/kg/day (IFO+GM-CSF n = 28) and cyclophosphamide 4 g/m2 + GM-CSF 5 microg/kg/day (CY+GM-CSF n = 28). Both groups were comparable for age, gender, diagnosis, disease stage and previous chemotherapy. The IFO+GM-CSF group demonstrated a shorter median interval between therapy and apheresis (10 days (8-14) vs 13 days (8-25) P = 0.002), median number of doses of GM-CSF (9 (7-13) vs 15 (9-31) P = 0.001), median of days with aplasia (0.5 (0-10) vs 6 (0-21) P = 0.001), median days with fever (0 (0-6) vs 3 (0-9) P = 0.006) and median of days using i.v. antibiotics (0 (0-11) vs 7.5 (0-19) P = 0.002). The median MNC yield was similar in both groups. The CD34+ cell yield was better in the CY+GM-CSF group (3.14 (0.9-11.8) vs 5.33 (0. 08-32)) but not at significant levels (P = 0.1). White blood cell hematopoietic recovery was more rapid in the CY+GM-CSF group (16 (10-22) vs 13 (10-24) P = 0.02). Platelet engraftment was similar in both groups. Costs of mobilization and transplantation were almost the same: $28 570 ($18 527-$47 028) and $30 020 ($17 281-$67 591), respectively (P = 0.9). There were no differences in disease-free survival and overall survival between both groups. Mild and transient non-hematological toxicity (hemorrhagic cystitis, decrease in serum creatinine clearance and CNS dysfunction) was seen most frequently in the IFO+GM-CSF group.