Prophylactic growth factor-primed donor lymphocyte infusion using cells reserved at the time of transplantation after allogeneic peripheral blood stem cell transplantation in patients with high-risk hematologic malignancies

BACKGROUND: Standard allogeneic bone marrow transplantation (BMT) offers only a small chance of cure for most adult patients with advanced hematologic malignancies. The authors postulated that allogeneic peripheral blood stem cell transplantation (PBSCT) followed by prophylactic growth factor-primed donor lymphocyte infusion (DLI) with cells reserved at harvest would maximize the graft-versus-tumor effects in patients with hematologic malignancies who had a high risk of recurrence. METHODS: Seventeen patients with hematologic malignancies who had a high risk of recurrence were allocated on an intent-to-treat basis to allogeneic PBSCT from human leukocyte antigen-matched sibling donors followed by prophylactic growth factor-primed DLI of cells reserved at harvest for transplantation. RESULTS: The median age was 37 years (range, 19-56 years). All donors underwent two or more apheresis procedures. The median numbers of mononuclear cells (MNCs), CD34 positive (CD34+) cells, and CD3+ cells, respectively, that were collected for 17 donors were 9.0 x 10(8) MNCs/kg (range, 4.9-14.4 x 10(8) MNCs/kg), 13.0 x 10(6) CD34+ cells/kg (range, 2.4-75.2 x 10(6) CD34+ cells/kg), and 5.8 x 10(8) CD3+ cells/kg (range, 3.3-9.9 x 10(8) CD3+ cells/kg) for a mean number of 2.35 apheresis procedures (range, 2.0-4.0 procedures). The median numbers of MNCs and CD3+ cells that were cryopreserved were 2.1 x 10(8) MNCs/kg (range, 0.0-4.4 x 10(8) MNCs/kg) and 1.4 x 10(8) CD3+ cells/kg (range, 0.0-3.5 x 10(8) CD3+ cells/kg). Seven of 17 patients received additional PBSCs, with a median of 5.0 x 10(7) CD3+ cells/kg (range, 3.0-9.9 CD3+ cells/kg) between Day 41 and Day 120. The reasons for inability to administer additional PBSCs in 10 patients included early death (n = 4 patients), severe graft-versus-host disease (GVHD) (n = 3 patients), disease recurrence (n = 2 patients), and harvest failure (n = 1 patient). Of seven patients, two patients died of recurrence, and one died of cytomegalovirus pneumonitis. The surviving four patients were free of disease when last assessed (median follow-up, 597 days) but were suffering from chronic GVHD (one patient had limited GVHD, and three patients had extensive GVHD). CONCLUSIONS: The authors suggest that allogeneic PBSCT with prophylactic growth factor-primed DLI may be a potentially curative strategy for the treatment of hematologic malignancies in patients with a high risk of recurrence. Their approach may offer the additional advantage of collecting enough cells at harvest for the potential use of DLI, which is easy, convenient for donors, and cost effective.     

Circulating CD34+ counts and apheresis planning

The success of peripheral blood progenitor cell (PBPC) transplantation depends upon harvesting adequate numbers of cells and accurate prediction of when to commence apheresis. Although peripheral white cell count (WBC) is commonly used to identify when to initiate apheresis it does not uniformly predict the CD34+ content of the apheresis product nor the number of exchange procedures required. We investigated whether the peripheral blood CD34+ count would not only predict harvest yield but whether it would also predict the number of apheresis procedures needed to generate at least 2 x 10(6)/kg CD34+ cells. CD34+ counts were performed over an 8-month period on the peripheral blood and PBPC harvests of all patients undergoing leucopheresis. Regression analysis showed a highly significant correlation between peripheral blood CD34+ count and yield of CD34+ cells in the apheresis product. The regression plot with WBC was weaker. We have shown that a peripheral CD34+ count > or = 62 x 10(6)/l is required to confidently achieve an adequate harvest in one apheresis, two aphereses are needed if the initial count is > or = 40 x 10(6)/l. Therefore peripheral blood CD34+ counts not only are able to determine the threshold at which to commence apheresis but are useful in predicting apheresis requirements and planning demands on the apheresis service.     

High-versus standard-dose filgrastim (rhG-CSF) for mobilization of peripheral-blood progenitor cells from allogeneic donors and CD34(+) immunoselection.

PURPOSE: The efficacy of a high- versus a standard-dose filgrastim (recombinant human granulocyte colony-stimulating factor, or rhG-CSF) regimen to mobilize peripheral-blood progenitor cells (PBPCs) for allogeneic transplantation was investigated in 75 healthy donors. PATIENTS AND METHODS: From December 1994 to December 1997, 75 consecutive donors (median age, 38 years; range, 17 to 67 years) were assigned to two different schedules of rhG-CSF for PBPC mobilization. Fifty donors received 24 microg rhG-CSF/kg body weight (BW) divided into two daily subcutaneous injections (two doses of 12 microg, group A), whereas 25 were treated with 10 microg rhG-CSF once daily (group B). Apheresis was started on day 4 in group A and on day 5 in group B. Target CD34(+) cell numbers in apheresis products were >/= 4 x 10(6)/kg recipient BW. RESULTS: Cytokine priming and collection of PBPCs were equally well tolerated in both groups. Significantly higher CD34(+) cell numbers in group A with 3. 7 x 10(6)/kg recipient BW/apheresis (0.47 x 10(6)/L apheresis) compared with 2 x 10(6)/kg recipient BW/apheresis (0.25 x 10(6)/L apharesis) in group B were obtained (P <.05). Using standard aphereses (median, 9 L), two doses of 12 microg rhG-CSF/kg allowed collection of >/= 4 x 10(6)/kg CD34(+) cells with two aphereses (range, one to three) in group A versus three aphereses (range, one to six) in group B (P <.015). Donor age, sex, and BW influenced the collection of CD34(+) cell numbers: in particular, significantly higher apheresis results were obtained in donors younger than 40 years compared with donors older than 40 years of age (P <.05). In 65 CD34(+) selection procedures using avidin-biotin immunoabsorption columns (Ceprate SC System, CellPro, Bothell, WA), a median CD34(+) purity of 53%, CD34(+) recovery of 40%, and the collection of 2 x 10(6)/kg CD34(+) cells/selection were achieved. In group A with higher CD34(+) cells/kg/apheresis, CD34(+) purity, recovery, and cell yields were 60%, 45%, and 2.3 x 10(6)/kg/selection, respectively, as compared with 48%, 31%, and 0.7 x 10(6)/kg in group B (P <.05). CONCLUSION: Our results demonstrate that twice daily rhG-CSF (two doses of 12 microg/kg BM) compared with once daily rhG-CSF (10 microg/kg BW), in addition to being well tolerated, significantly improves PBPC mobilization, allows the collection of higher numbers of CD34(+) cells with one or two standard aphereses, and facilitates subsequent selection procedures in healthy allogeneic donors.     

The graft content of donor T cells expressing gamma delta TCR+ and CD4+foxp3+ predicts the risk of acute graft versus host disease after transplantation of allogeneic peripheral blood stem cells from unrelated donors.

PURPOSE: Recently, high numbers of regulatory T cells within the stem cell graft were described to be associated with less graft-versus-host disease (GVHD) after related peripheral blood stem cell transplantation (PBSCT). Studies in mice also suggest a distinct role of gamma delta TCR(+) T cells in mediating GVHD. Therefore, the aim of this study was to define the yet-unknown role of regulatory and gamma delta TCR(+) T cells in human PBSCT from unrelated donors. EXPERIMENTAL DESIGN: The frequency of both T-cell subsets within the graft was analyzed in 63 patients receiving unrelated allogeneic PBSCT. The respective amounts were quantified by flow cytometry and PCR and further correlated with clinical outcome. RESULTS: The grafts contained a median of 11.2 x 10(6)/kg CD4(+)foxp3(+) and 9.8 x 10(6)/kg gamma delta TCR(+) T cells, respectively. Patients receiving more CD4(+)foxp3(+) cells had a lower cumulative incidence of acute GVHD II-IV (44% versus 65%, P=0.03). Interestingly, in patients who received higher concentrations of donor gamma delta TCR(+) T cells, acute GVHD II-IV was more frequent (66% versus 40%, P=0.02). In multivariate analysis, only the graft concentration of gamma delta TCR(+) T cells (P=0.002) and a positive cytomegalovirus status of the recipient (P = 0.03) were significantly associated with the occurrence of acute GVHD II-IV. CONCLUSION: Graft composition of T-cell subsets seems to affect the outcome of patients receiving allogeneic PBSCT from unrelated donors. Therefore, selective manipulation or add-back of particular subsets might be a promising strategy to reduce the incidence of GVHD.     

Successful treatment of metastatic renal cell carcinoma with a nonmyeloablative allogeneic peripheral-blood progenitor-cell transplant: evidence for a graft-versus-tumor effect.

PURPOSE: A 50-year-old man developed progressive pulmonary metastasis resistant to interferon alfa-2b treatment 7 months after he underwent left nephrectomy for stage III renal cell carcinoma. We performed a nonmyeloablative allogeneic peripheral-blood stem-cell transplant in this patient to exploit a possible graft-versus-tumor effect from allogeneic lymphocytes. MATERIALS AND METHODS: The conditioning regimen consisted of fludarabine and cyclophosphamide followed by a T-cell replete, granulocyte-colony stimulating-factor-mobilized peripheral-blood stem-cell transplant from his HLA-identical brother. Cyclosporine was administered from days -4 to +45 to prevent graft rejection and acute graft-versus-host disease (GVHD). RESULTS: Serial polymerase chain reaction analysis of hematopoietic lineage-specific minisatellites initiallyshowed mixed chimerism in CD14(+) and CD15(+) myeloid cells, CD3(+) T cells, and CD34(+) progenitor cells, with rapid conversion to 100% donor T-cell chimerism by day +60 and 100% donor myeloid cells by day +100. Serial computed tomography scans of the chest showed stable disease at day +30, slight regression of pulmonary lesions at day +63, and complete disappearance of all pulmonary metastatic disease by day +110. Mild transient acute GVHD disease of the skin occurred on day +60 and limited chronic GVHD of the skin occurred by day +200. CONCLUSION: The complete regression of metastatic disease, which has now been maintained for more than 1 year, is compatible with a graft-versus-tumor effect.     

CD34 positive selection as prophylaxis against graft versus host disease in allogeneic peripheral blood stem cell transplantation

We report our experience of CD34 positive selection as a means of graft T-cell depletion (TCD) in 14 consecutive HLA-identical Peripheral blood stem cells (PBSC) allografts as prophylaxis against graft versus host disease (GVHDp). CD34 positive selection was performed by immunomagnetic separation achieving a median CD34 and T-cell dose of 4.17 (range 1.4-8.50) x 10(6)/kg and 1.89 (range 0.92-13.18) x 10(4)/kg, respectively, in the graft. This represents 4-log depletion of T-cells. The median time to achieve a neutrophil count of 0.5 x 10(9)/l was 15 days and to achieve a platelet count of 50 x 10(9)/l was 20 days. Only four patients developed acute GVHD at a median of 41 days but this was exclusively mild grade I cutaneous disease and settled with oral steroids. Four patients, all of whom had AML, relapsed or progressed after transplant at a median of 161 (range 109-311) days. One of these had been transplanted in early relapse (9% blasts) whilst another was in second remission. The remaining 10 patients are alive and well. The median progression free survival for the whole population is 69% at 686 days. We conclude that CD34 positive selection by immunomagnetic separation in HLA-identical PBSC allografting achieves significant TCD with clinically trivial acute GVHD, prompt engraftment and an acceptable disease relapse risk.     

Allogeneic peripheral blood stem cell transplantation in acute non-lymphoblastic leukemia.

Unmodified allogeneic peripheral blood stem cell transplantation (alloPBSCT) was performed in 20 consecutive acute non-lymphoblastic leukemia (ANLL) patients from their HLA-identical siblings. There were 11 males and 9 females. Median age was 34 years (range 17-43). Donors were primed with 2.5-15 micrograms/kg/day s.c. granulocyte-colony stimulating factor (G-CSF, Neupogen, Roche). Conditioning regimen was Bu (16 mg/kg) + Cy (120 mg/kg) in 19 patients and high dose Ara-C (3 gr/m2 twice daily for 3 days) for one patient who relapsed after bone marrow transplantation. Eighteen patients were in CR1. CsA + short-term MTX (n = 19) or CsA alone (n = 1) were used for graft versus host disease (GVHD) prophylaxis. The median number of apheresis procedures for each patient was 2 (2-4). A median of 6.5 (3.2-38.2) x 10(8)/kg MNC or 9.4 (2.2-12.4) x 10(6)/kg CD34+ cells were given. Median days to reach granulocyte of > 0.5 x 10(9)/l and platelet of > 50 x 10(9)/l were 12 (10-14) and 15 (11-35) respectively. Day 100 transplant-related mortality was 20 per cent (4/20). Grade 2 to 4 AGVHD was seen in 8 out of 17 (47%) evaluable patients. Severe AGVHD occurred in 3 out of 17 (18%). Clinical CGVHD of all grades developed in 12 out of 17 (70%) evaluable patients. The mean disease-free survival and overall survival were 17 (range: 8-33 months) and 18 months (range: 10-34 months), respectively. In conclusion, alloPBSCT in ANLL is associated with a faster engraftment, no greater incidence of AGVHD, but increased risk of CGVHD.     

Feasibility of ESHAP + G-CSF as peripheral blood hematopoietic progenitor cell mobilisation regimen in resistant and relapsed lymphoma: a single-center study of 22 patients.

The purpose was study the feasibility of ESHAP + G-CSF for peripheral blood hematopoietic progenitor cell (PBPC) mobilisation in resistant/relapsed Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL). Twenty-two consecutive patients with HD (8) and N-HL (14) received ESHAP chemotherapy and G-CSF (5 microg/Kg/d). When a minimum number of 10,000 peripheral blood CD34+ cells/mL was observed patients underwent leukapheresis until a CD34+ cell dose > or = 2.5x10(6)/Kg was collected or the PBPC peak was lost. Blood cells kinetics and toxicity were analysed. Data concerning the day of first apheresis, number of procedures per patient, and cellular yield of the aphereses were recorded. Correlation between the CD34+ cell content in the apheresis product and the two diagnosis groups was attempted. Twelve patients (54%) developed short-lived severe neutropenia (<0.5x10(9)/L). Thrombocytopenia (<25x10(9)/L) had a median duration of 1 day. Fever appeared in 4 patients and CN Staph bacteriemia in 2 cases. Bleeding events did not supervene and no deaths occurred. Aphereses started at day +15 (median) and the median number of apheresis/patient was 2. Seventeen patients underwent 1 or 2 leukaphereses. Thirteen patients (59%) achieved the CD34+ cell target in the first apheresis. NHL patients obtained statistically significant better CD34+ cell collections than HD. Only 2 HD patients failed to mobilise, 1 previously treated with high-dose therapy and autologous bone marrow transplantation. ESHAP + G-CSF has been shown to be feasible for PBPC mobilisation in resistant/relapsed lymphoma. Toxicity was low and CD34+ cell yield high, especially in N-HL. This mobilisation regimen should be further explored in a larger patient population.     

Higher doses of CD34+ peripheral blood stem cells are associated with increased mortality from chronic graft-versus-host disease after allogeneic HLA-identical sibling transplantation.

Allogeneic peripheral blood stem cell transplantation (PBSCT) has emerged as an alternative to bone marrow transplantation. PBSCT can be associated with a higher incidence of chronic graft-versus-host disease (cGVHD). In this study, we investigated whether there was a correlation between the composition of PBSC grafts (CD34+ and CD3+ cells) and hematological recovery, GVHD, relapse, and relapse-free survival (RFS) after myeloablative HLA-identical sibling PBSCT. The evolution of 100 acute or chronic leukemia patients was analyzed. Neither hematological recovery, acute or cGVHD, nor relapse, was significantly associated with CD3+ cell dose. Increasing CD34+ stem cells was associated with faster neutrophil (P=0.03) and platelet (P=0.007) recovery. Moreover, 47 of the 78 patients evaluable for cGVHD (60%; 95% CI, 49-71%) developed extensive cGVHD. The probability of extensive cGVHD at 4 years was 34% (95% CI, 21-47%) in patients receiving a 'low' CD34+ cell dose (<8.3 x 10(6)/kg), as compared to 62% (95% CI, 48-76%) in patients receiving a 'high' CD34+ cell dose (>8.3 x 10(6)/kg) (P=0.01). At a median follow-up of 59 months, this has not translated into a difference in relapse. In patients evaluable for cGVHD, RFS was significantly higher in patients receiving a 'low' CD34+ cell dose as compared to those receiving a 'high' CD34+ cell dose (P=0.04). This difference was mainly because of a significantly higher cGVHD-associated mortality (P=0.01). Efforts to accelerate engraftment by increasing CD34+ cell dose must be counterbalanced with the risk of detrimental cGVHD.     

Less intense conditioning with fludarabine, cyclophosphamide, idarubicin and etoposide (FCIE) followed by allogeneic unselected peripheral blood stem cell transplantation in elderly patients with leukemia.

The objective of this study was to assess toxicity and feasibility of achieving engraftment of allogeneic blood progenitor cells following nonmyeloablative conditioning according to the FCIE protocol (fludarabine 25 mg/m(2)/day, days -7 to -3; cyclophosphamide 200 mg/m(2)/day, days -7 to -3; idarubicin 12 mg/m(2)/day, days -7 to -5; etoposide 250 mg/m(2)/day, days -4 to -3) in elderly patients with leukemia. Eleven patients were included in the study: six patients with acute myeloid leukemia (AML) in complete remission (CR); three patients with refractory or relapsed AML; one patient with chronic myeloid leukemia; one patient with acute lymphoblastic leukemia. The median age of the patients was 62 years. All patients received blood progenitor cells from an HLA-identical sibling with 8.8 x 10(6) CD34(+) cells/kg (median; range 4.7 to 26.2 x 10(6)/kg) and 5.5 x 10(8) CD3(+)cells/kg (median; range 4.5 to 7.9 x 10(8)/kg). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and three courses of methotrexate. The median duration of white blood cell counts <1 x 10(9)/l was 17 days and of platelet counts <50 x 10(9)/l 20 days. In two patients acute GVHD grade I occurred. Nine of 10 patients analyzed developed mixed chimerism. Of seven patients transplanted in CR, three remained in CR 19 to 31 months after transplantation. Three patients with refractory leukemia did not achieve CR, while the patient with relapsed AML achieved a 3rd CR. After a median follow-up time of 22 months, chronic GVHD was mild and limited. The data from this pilot study in elderly patients with leukemia show that the combination of primarily immunosuppressive (FC) and antileukemic (IE) drugs for nonmyeloablative conditioning has moderate nonhematological toxicity and allows engraftment of allogeneic blood progenitor cells.     

CD6+ donor marrow T-cell depletion as the sole form of graft-versus-host disease prophylaxis in patients undergoing allogeneic bone marrow transplant from unrelated donors.

PURPOSE: The role of donor marrow T-cell depletion (TCD) in preventing graft-versus-host disease (GVHD) after transplantation of unrelated allogeneic marrow remains undefined. Because different TCD methodologies differ in the degree and specificity with which T cells are removed, it is likely that transplant outcomes would depend on which technique is used. Herein, we report results in the first 48 recipients of unrelated marrow using CD6+ TCD as the sole form of GVHD prophylaxis. PATIENTS AND METHODS: Median age of patients was 46 years (20 to 58 years). Donors were matched at A/B HLA loci. Ablation consisted of cyclophosphamide and fractionated total-body irradiation (TBI; 14 Gy). To facilitate engraftment, patients also received 7.5 Gy (22 patients) [corrected] or 4.5 Gy (26 patients) [corrected] of total lymphoid irradiation (TLI) before admission. No additional immune suppressive prophylaxis was administered. Granulocyte colony-stimulating factor was administered daily from day +1 to engraftment. RESULTS: All 48 patients demonstrated neutrophil engraftment. An absolute neutrophil count of 500 x 10(6)/L was achieved at a median of 12 days (range, 9 to 23 days). There were no cases of late graft failure. The number of CD34+ cells infused/kg was associated with speed of platelet and neutrophil recovery. The dose of TLI did not influence engraftment. Grades 2-4 acute GVHD occurred in 42% of patients (95% confidence interval [CI], 0.28 to 0.57). Mortality at day 100 was 19%. There have been only five relapses. Estimated 2-year survival was 44% (95% CI, 0.28 to 0.59) for the entire group, 58% for patients less than 50 years of age. In multivariable analysis, age less than 50 years (P =.002), cytomegalovirus seronegative status (P =.04), and early disease status at bone marrow transplant (P =.05) were associated with superior survival. CONCLUSION: CD6+ TCD does not impede engraftment of unrelated bone marrow after low-dose TLI, cyclophosphamide, and TBI. CD6+ TCD as the sole form of GVHD prophylaxis results in an incidence of GVHD that compares favorably with many adult studies of unrelated transplantation using unmanipulated marrow and immune-suppressive medications, especially in light of the median age of our patients (46 years). Although event-free survival in patients less than 50 years of age is very encouraging, older patients experience frequent transplantation-related complications despite TCD.     

Antitumor activity mediated by double-negative T cells

Allogeneic lymphocytes are potent mediators of leukemia and lymphoma remission. The goal of this study was to determine whether single MHC class I locus-mismatched lymphocytes could generate an antilymphoma activity in the absence of graft-versus-host-disease (GVHD) and to understand the underlying mechanisms. Immunoincompetent Scid or lethally irradiated mice were challenged i.v. with a lethal dose of A20 lymphoma cells together with an infusion of single MHC class I locus mismatched splenocytes. Mice that were challenged with A20 cells alone succumbed to lymphoma between 34 and 50 days after infusion. In contrast, >75% of mice that were coinfused with single class I MHC locus mismatched splenocytes survived indefinitely (n = 20) in the absence of GVHD. Interestingly, the number of CD3(+)CD4(-)CD8(-) double-negative (DN) T cells increased 15-fold in mice that did not develop lymphoma. Both DN T cells isolated from the spleens of lymphoma-free mice and DN T cells cloned from na?ve mice were cytotoxic to A20 lymphoma cells in vitro. When DN T cell clones were infused into na?ve mice i.v. together with A20 lymphoma cells, 86% of recipient mice were protected from lymphoma onset and did not develop GVHD (n = 22). To assess whether the systemic injection of DN T cells can also suppress local tumor development, A20 cells were infused i.m., and at the same time DN T cell clones were infused either i.v. or i.m. Results indicated that DN T cells infused systemically (i.v.) could not prevent local tumor outgrowth, but DN T cells coinfused locally (i.m.) prevented local tumor development in 91% of animals (n = 11). Furthermore, we demonstrate that primary DN T cells were also able to prevent tumor growth in 75% of mice when infused together with A20 cells i.m. (n = 12). Together, these results demonstrate that an antilymphoma activity can be generated in mice without causing GVHD. Furthermore, DN T cells can suppress lymphoma cells in vivo and in vitro, suggesting that DN T cells could be used as a novel strategy for the treatment of lymphoma.     

外周血造血干细胞移植治疗恶性血液病53例

背景与目的:造血干细胞移植使恶性血液病的预后得到很大的改观,外周血造血干细胞移植(transplantation of peripheral blood stem cells,PBSCT)逐渐取代了骨髓移植(bone marrow transplantation,BMT)而成为造血干细胞移植的主要方式.本研究观察了自体或异基因外周血造血干细胞移植对53例恶性血液病患者的治疗效果.方法:53例恶性血液病患者于2003年7月～2006年5月在郑州大学第一附属医院血液科接受PBSCT治疗,中位年龄37岁.采用G-CSF或化疗联合G-CSF动员外周血干细胞.自体移植患者接受CD34 细胞的中位数为3.0×106/kg,异基因移植患者接受CD34 细胞的中位数为6.2×106/kg;自体移植采用MAC预处理方案,异基因移植采用改良的Bu/Cy预处理方案;移植物抗宿主病(graft versus host disease,GVHD)的预防采用MTX、环孢菌素A联合骁悉,1例1个点不合患者加用抗淋巴细胞球蛋白.结果:移植后中性粒细胞≥0.5×109/L、血小板≥20×109/L的天数在自体移植中分别为13天和19天,在异基因移植中分别为12天和15天;异基因移植中Ⅰ～Ⅲ度急性GVHD(aGVHD)的发生率为31.4%,慢性GVHD(cGVHD)的发生率为71.4%,复发率在自体移植和异基因移植患者中分别为38.9%和5.7%,700天无病生存率在自体移植和异基因移植患者中分别为57.9%和69.5%.结论:自体和异基因外周血造血干细胞移植均能很快地重建造血,是治疗恶性血液病的重要手段之一.     

Mobilization of peripheral blood progenitor cells with a combination of cyclophosphamide, r-metHuSCF and filgrastim in patients with breast cancer previously treated with chemotherapy.

The objective of our study was to determine the effect of adding r-metHuSCF to Filgrastim and cyclophosphamide for mobilization of peripheral blood progenitor cells (PBPC), on collection of CD34(+) cells and engraftment after autologous stem cell transplant. Twenty-three patients with previously treated stage II-IV breast cancer received cyclophosphamide (3 g/m(2)), Filgrastim 5 microg/kg daily and r-metHuSCF 20 microg/kg daily. Two PBPC collections were performed on consecutive days starting the day the WBC count was above 7.5 x 10(3)/microl. Collection was performed between days +9 and +12 and the median number of CD34(+) cells collected was 9.9 x 10(6)/kg (1.1-53.1) and 6.6 x 10(6)/kg (1.4-33.8) for the first and second apheresis, respectively. Despite being previously treated patients, the target CD34(+) cell dose required for SCT was obtained in all patients. SCT was associated with rapid neutrophil and platelet engraftment and a highly significant correlation was observed between the number of CD34(+) cells infused and engraftment. Treatment with SCF plus filgrastim was well tolerated, with mild to moderate local skin rash being the most frequently reported adverse event. In conclusion, addition of r-metHuSCF induces mobilization of a large number of CD34(+) cells which results in shortening of time to engraftment and hospitalization.     

单一剂量G-CSF用于异基因外周血造血干细胞动员和移植后造血恢复的临床研究

背景与目的:应用粒系集落刺激因子(granulocytecolony-stimulatingfactor,G-CSF)等动员剂从健康供者中动员和获取造血干细胞用于异基因外周血造血干细胞移植(allogeneicperipheralbloodstemcelltransplantation,allo-PBSCT)已在临床广泛开展,但在其作用机理、给药剂量和给药方案等方面还存在许多问题有待进一步研究。本研究旨在探讨单一剂量的G-CSF(250μg/d)用于allo-PBSCT的外周血干细胞(peripheralbloodstemcell,PBSC)动员以及移植后造血恢复的安全性和有效     

High doses of transplanted CD34+ cells are associated with rapid T-cell engraftment and lessened risk of graft rejection, but not more graft-versus-host disease after nonmyeloablative conditioning and unrelated hematopoietic cell transplantation.

This report examines the impact of graft composition on outcomes in 130 patients with hematological malignancies given unrelated donor granulocyte-colony-stimulating-factor-mobilized peripheral blood mononuclear cells (G-PBMC) (n = 116) or marrow (n = 14) transplantation after nonmyeloablative conditioning with 90 mg/m(2) fludarabine and 2 Gy TBI. The median number of CD34(+) cells transplanted was 6.5 x 10(6)/kg. Higher numbers of grafted CD14(+) (P = 0.0008), CD3(+) (P = 0.0007), CD4(+) (P = 0.001), CD8(+) (P = 0.004), CD3(-)CD56(+) (P = 0.003), and CD34(+) (P = 0.0001) cells were associated with higher levels of day 28 donor T-cell chimerism. Higher numbers of CD14(+) (P = 0.01) and CD34(+) (P = 0.0003) cells were associated with rapid achievement of complete donor T-cell chimerism, while high numbers of CD8(+) (P = 0.005) and CD34(+) (P = 0.01) cells were associated with low probabilities of graft rejection. When analyses were restricted to G-PBMC recipients, higher numbers of grafted CD34(+) cells were associated with higher levels of day 28 donor T-cell chimerism (P = 0.01), rapid achievement of complete donor T-cell chimerism (P = 0.02), and a trend for lower risk for graft rejection (P = 0.14). There were no associations between any cell subsets and acute or chronic GVHD nor relapse/progression. These data suggest more rapid engraftment of donor T cells and reduced rejection rates could be achieved by increasing the doses of CD34(+) cells in unrelated grafts administered after nonmyeloablative conditioning.     

Allogeneic Peripheral Blood Stem Cell Transplantation: Is there an increased risk of graft vs host disease in leukemia patients?

Abstract

Fifteen patients with hematological malignancies [9 acute nonlymphoblastic leukemia (ANLL), four chronic myelogenous leukemia (CML), two acute lymphoblastic leukemia (ALL)] received allogeneic peripheral blood stem cell transplantation (alloPBSCT) from HLAidentical sibling donors. Donors received 2.5-15 μg/kg/day of recombinant human granulocyte colony stimulating factor (rhG-CSF) for 5-10 days. Administration of rhG-CSF was well tolerated except for mild to moderate bone pain occurring in all the donors which was relieved by oral paracetamol. A total of 40 leukaphereses were performed for the 15 donors using the bilateral antecubital veins. None of the donors needed central venous line insertion. The median number of apheresis procedures for each patient was 3 (2-3). A median of 7.7 (4-38.2) × 10⁸/kg mononuclear cells, 35 (2.4-90.0)x 10⁶/kg CD34+ cells, 1.85 (0.45-4.8) × 10⁸/kg CD3 and 0.3 (0.16-1.01) × 10⁸/kg natural killer cells were given without any manipulation. Cyclosporin A (CsA) plus short-course methotrexate (MTX) (12 patients) and CsA alone (3 patients) were used for graft versus host disease (GVHD) prophylaxis. Median granulocyte and platelet engraftments were done on days 11 (10-31) and 16 (11-54) respectively. Grades II-IV GVHD occurred in 62% of the patients and grades III-IV in 15%. Twelve patients are still alive with full engraftment and disease-free. In conclusion, alloPBSCT is an alternative to allogeneic bone marrow transplantation, because of the ease of collection and rapid hematological recovery. However, there is a trend for increased acute GVHD in our leukemia patients compared to allogeneic bone marrow.     

The role of thiotepa in allogeneic stem cell transplantation in patients with leukemia

Graft versus host disease (GVHD) and recurrence of basic disease are major obstacles to a successful allogeneic bone marrow transplantation (BMT) outcome. One of the possibilities of maintaining the therapeutic potential of marrow allografting in the absence of GVHD is to intensify the conditioning regimen administered pre-T-cell depleted BMT in order to compensate for the loss of GVH related graft versus leukemia (GVL) effect. In order to do so we used a preparative regimen consisting of three alkylating agents-Busulfan (BU), Thiotepa (TTP) and Cyclophosphamide (CY)-for T-cell depleted allogeneic stem cell transplantation (SCT) instead of the standard BU-CY protocol. The effect of this intensified regimen was investigated in 30 consecutive leukemia patients who underwent T-cell depleted SCT from HLA identical siblings. Sixteen of the patients were males and 14 females, of median age 24 (5-43) years. Fourteen patients had acute myelogenous leukemia (AML), ten acute lymphoblastic leukemia (ALL), four chronic myelogenous leukemia (CML) and two myelodysplastic syndrome. The conditioning regimen consisted of BU 4 mg/kg x 4 days (-8 to -5), TTP 5 mg/kg x 2 days (-4 and -3), and CY 60 mg/kg x 2 days (-2 and -1). Engraftment was normal, with WBC >1.0x10(9)/l at day +18 (10-32), ANC >0.5x10(9)/l at day +21 (9-33) and platelets >25x10(9)/l at day +30 (14-69). Regimen related toxicity (RRT) was moderate and transplant related complications comparable to other conventional conditioning protocols. Overall survival and disease free survival (DFS) at 60 months follow up was 50%. Only three patients (10%), with ALL, relapsed and subsequently died. From the current data it would appear that TTP does not significantly improve BMT outcome in patients with leukemia, when compared to the standard BU-CY conditioning. However, our results with the BU-TTP-CY combination followed by T-cell depleted allogeneic SCT could provide the basis for a prospective randomized study comparing this protocol with the standard BU-CY regimen.     

Synergistic enhancement of antitumor immunity with adoptively transferred tumor-specific CD4+ and CD8+ T cells and intratumoral lymphotactin transgene expression

The lack of efficient T-cell infiltration of tumors is a major obstacle to successful adoptive T-cell therapy. We have shown that transplanted SP2/0 myeloma tumors that have been engineered to express lymphotactin (Lptn) invariably regress under the influence of infiltrating XCR1+T cells and neutrophils. Herein, we characterize these T cells and investigate their therapeutic efficacy, either alone or with Lptn gene therapy. After stimulation with SP2/0 cells, these T cells were CD25+FasL+L-selectin-, expressed XCR-1, and were chemoattracted by Lptn in vitro. They comprised 66% CD4+ Th1 and 33% CD8+ Tc1 cells, both of which expressed significant amounts of IFN-gamma, perforin, and tumor necrosis factor-alpha, but not interleukin-4. The CD4+ Th1 and CD8+ Tc1 cells, which were inhibited and stimulated, respectively, for proliferation with Lptn signaling, displayed 38 and 84% specific killing, respectively, for Ia(d)/H-2K(d)-expressing SP2/0 tumor cells (E:T ratio, 100). In vivo, combined intratumoral Lptn gene transfer and adoptive immunotherapy with these CD4+ and CD8+ T cells eradicated well-established SP2/0 tumors in six of eight mice, and dramatically slowed tumor growth in the other two mice. Cell tracking using labeled T cells confirmed that these cells infiltrated better into the Lptn-expressing tumors than non-Lptn-expressing ones. Control or Lptn adenoviral treatments by themselves did not alter the lethal outcome for tumor-bearing mice, nor did T-cell therapy by itself, although the latter two treatments did slow its time frame. Combined Lptn gene transfer and adoptive CD4+ or CD8+ cell transfers were not nearly as efficacious as the combined Lptn gene and unfractionated T-cell transfers. Taken together, our data provide solid evidence of a potent synergy between adoptive CD4+ and CD8+ T-cell therapy and Lptn gene transfer into tumor tissues, which culminated in the eradication of well-established tumor masses.     

Notch induces human T-cell receptor γδ+ thymocytes to differentiate along a parallel, highly proliferative and bipotent CD4 CD8 double-positive pathway

In wild-type mice, T-cell receptor (TCR) γδ(+) cells differentiate along a CD4 CD8 double-negative (DN) pathway whereas TCRαβ(+) cells differentiate along the double-positive (DP) pathway. In the human postnatal thymus (PNT), DN, DP and single-positive (SP) TCRγδ(+) populations are present. Here, the precursor-progeny relationship of the various PNT TCRγδ(+) populations was studied and the role of the DP TCRγδ(+) population during T-cell differentiation was elucidated. We demonstrate that human TCRγδ(+) cells differentiate along two pathways downstream from an immature CD1(+) DN TCRγδ(+) precursor: a Notch-independent DN pathway generating mature DN and CD8αα SP TCRγδ(+) cells, and a Notch-dependent, highly proliferative DP pathway generating immature CD4 SP and subsequently DP TCRγδ(+) populations. DP TCRγδ(+) cells are actively rearranging the TCRα locus, and differentiate to TCR(-) DP cells, to CD8αβ SP TCRγδ(+) cells and to TCRαβ(+) cells. Finally, we show that the γδ subset of T-cell acute lymphoblastic leukemias (T-ALL) consists mainly of CD4 SP or DP phenotypes carrying significantly more activating Notch mutations than DN T-ALL. The latter suggests that activating Notch mutations in TCRγδ(+) thymocytes induce proliferation and differentiation along the DP pathway in vivo.