Transplantation Of Allogeneic CD34 Research Articles

Transplantation of allogeneic CD34+-selected cells followed by early T-cell add-backs: favorable results in acute and chronic myeloid leukemia

The aim of this study was to investigate preservation of anti-leukemic activity and protection from opportunistic infections after transplantation of allogeneic + cells in patients with hematologic malignancies and bad prognosis. Methods Thirty-three patients [median age 42 years, range 23-55 years, diagnosis AML/myelodysplastic syndrome (MDS) 14, ALL nine, CML seven and multiple myeloma (MM) three] received myeloablative conditioning followed by infusion of selected CD34+ cells from matched unrelated donors (31) or HLA-identical siblings (two). Early donor lymphocyte infusions (DLI; 0.5 and 1.0 x 10(6) CD3+ cells/kg) were given while patients were on immunosuppressive therapy. Ninety-seven per cent of patients engrafted and 24 of 29 patients surviving more than 30 days received at least one pre-emptive DLI. Three patients (10%) developed acute (a)GvHD (two grade I-II, one grade III-IV) spontaneously, and 16 patients (67%) developed aGvHD after DLI (12 grade I-II, four grade III-IV). Eight of 24 evaluable patients developed chronic (c)GvHD (33%, six limited, two extensive). After a median follow-up of 590 days (range 138-1610 days) 18 patients were alive (55%), 16 in complete remission (CR), one in hematologic and one in molecular relapse. Seven patients died after relapse (21%) and eight died from transplantation-related causes (24%). Patients with myeloid malignancies had a significantly better survival than patients with ALL or MM (74%+/-10 vs. 30%+/-13, P<0.05). Early pre-emptive low-dose DLI following transplantation of selected CD34+ cells from unrelated donors after myeloablative conditioning is feasible and effective without undue toxicity, especially in patients with myeloid malignancies.

Allogeneic Transplantation of CD34+ Selected Hematopoietic Cells—Clinical Problems and Current Challenges

Since G-CSF mobilized peripheral blood has become the major source of hematopoietic cells (PBSC) for allogeneic transplantation of patients with h`ematological malignancies, new technologies of T-cell depletion were developed to reduce the risk of graft-versus-host disease. CD34+ selection by immunomagnetic separation has been shown to be the most effective method to achieve a 3 – 4 log depletion of T-cells while preserving the high number of hematopoietic progenitors in the graft. The high number of CD34+ cells contained in the positive fraction have allowed to facilitate engraftment from sibling donors matched for only one haplotype. In addition, studies in pediatric recipients have shown that high numbers of hematopoiteic progenitors infused can lead to timely recovery of T- and B-cells in the first year after transplantation. Positive results have also been reported in patients with chronic myelogenous leukemia who had received CD34+ selected PBSC from HLA-identical sibling donors and subsequent infusions of donor T-cells to establish graft-versus-leukemia effects. On the other hand, studies performed in adults with advanced leukemia receiving CD34+ selected PBSC from haploidentical or unrelated donors demonstrated an increased risk for graft-rejection, relapse and late opportunistic infections. Future efforts will have to concentrate on the restoration of cellular immunity by either adaptively transferring antigen specific effector cells or by studying the use of cytokines like interleukin-7. Although these question have not been resolved yet, allogeneic transplantation of highly purified CD34+ PBSC has become an therapeutic option for patients who are at high-risk for severe GvHD

Serial quantification of lymphoid and myeloid mixed chimerism using multiplex PCR amplification of short tandem repeat-markers predicts graft rejection and relapse, respectively, after allogeneic transplantation of CD34+ selected cells from peripheral blood.

We used multiplex amplification of nine microsatellite sequences (PCR-STR) to analyse chimerism in pure populations of T cells and neutrophils from peripheral blood from 40 patients submitted to an allogeneic transplant, 22 having received a T-cell depleted (TCD) peripheral blood graft by means of CD34(+) selection (allo-PBT/CD34(+)), and 18, an unmodified graft (allo-SCT; 13 allogeneic bone marrow transplants and five allo-PBT). T-cell mixed chimerism (TcMC) was observed in 16 of the 22 (72.3%) patients receiving an allo-PBT/CD34(+), but in only one of the 18 (5.5%) patients receiving an allo-SCT (P=0.0001). TcMC was transient (n=6), stable (n=7), and associated with poor haematopoietic engraftment (n=4). All patients with TcMC who developed graft failure had more than 30% of host T cells. Myeloid MC (MyMC) was observed in four (19%) allo-PBT/CD34(+) patients and in three (17%) allo-SCT patients (P=NS). Five out of seven (71%) patients with MyMC relapsed, all of them diagnosed with myeloid malignancies, as compared with two of the 20 (10%) patients with complete donor chimerism (P&<0.0001). In conclusion, TcMC appears in a significant number of allo-PBT/CD34(+) patients and may be associated with poor engraftment when the percentage of host T cells is >30%; likewise, MyMC appears in a small percentage of recipients of both allo-PBT/CD34(+) and allo-SCT patients, and is associated with leukaemia relapse in myeloid malignancies.

Changing phenotypes of hematopoietic stem cells.

Identification of the surface phenotypes of hematopoietic stem cells is an important subject in both clinical stem cell transplantation and basic research in hematopoiesis. Transplantation of pure populations of stem cells should eliminate the occurrence of graft-vs-host disease (GVHD) in allogeneic transplantation and may reduce the recurrence rate of malignancies in autologous transplantation. Availability of highly enriched populations of stem cells should facilitate investigations in a number of areas such as in vitro expansion, cryogenic storage, and gene therapy using stem cells. An ultimate goal is to develop a method to quantitate the number of living stem cells based on their surface characteristics. During the last two decades, investigators identified a number of surface molecules, which, in combinations, were thought to provide exact definition of murine and human stem cells. Recent studies of murine stem cells, however, clearly demonstrated that expression of the surface antigens of stem cells is under the influence of developmental stages and the kinetic state of the stem cells. This review summarizes the concept of the changing phenotypes of hematopoietic stem cells.

Allogeneic transplantation of CD34+selected cells from peripheral blood from human leukocyte antigen–identical siblings: detrimental effect of a high number of donor CD34+ cells?

Clinical results after T-cell–depleted allografts might be improved by modifying the graft content of progenitor and accessory cells. Although the association of the number of donor T cells with the clinical outcome has been studied extensively, the optimum number of progenitor cells that should be administered to patients is unknown. The characteristics of 84 consecutive human leukocyte antigen (HLA)–identical sibling transplants of granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood progenitor cells depleted of T cells by CD34+ positive selection (allo-PBT/CD34+) were analyzed for their effect on clinical outcome. After a median follow-up of 24 months (range, 1-70 months), 50 patients remain alive (59.5%) and 34 have died (21 [25%] as a result of the transplant and 13 [15.5%] due to disease relapse). The median number of CD34+ cells administered to the patients was 3.9 × 106/kg (range, 1.2-14.3 × 106/kg). A number of CD34+ cells in the inoculum of 1 × 106/kg to 3 × 106/kg was associated with increased survival: 21 of 28 (75%) patients are alive, as compared with 29 of 56 (52%) patients receiving more than 3 × 106/kg (actuarial probability 75% vs. 42%, respectively; P = .01). In the multivariate analysis, the independent prognostic variables for survival were CD34+cell dose 1 × 106/kg to 3 × 106/kg (RR = 4.8; P = .0008), sex-pairing match (RR = 3.2;P = .002), and early stage of disease (RR = 2.8;P = .007). From these results it appears that, in allo-PBT/CD34+ from HLA-identical siblings, a number of CD34+ cells in the inoculum between 1 × 106/kg to 3 × 106/kg is an important factor for better survival, and that higher CD34+ cell doses might be associated with a poorer outcome.

Allogeneic transplantation of CD34+selected cells from peripheral blood from human leukocyte antigen–identical siblings: detrimental effect of a high number of donor CD34+ cells?

AbstractClinical results after T-cell–depleted allografts might be improved by modifying the graft content of progenitor and accessory cells. Although the association of the number of donor T cells with the clinical outcome has been studied extensively, the optimum number of progenitor cells that should be administered to patients is unknown. The characteristics of 84 consecutive human leukocyte antigen (HLA)–identical sibling transplants of granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood progenitor cells depleted of T cells by CD34+ positive selection (allo-PBT/CD34+) were analyzed for their effect on clinical outcome. After a median follow-up of 24 months (range, 1-70 months), 50 patients remain alive (59.5%) and 34 have died (21 [25%] as a result of the transplant and 13 [15.5%] due to disease relapse). The median number of CD34+ cells administered to the patients was 3.9 × 106/kg (range, 1.2-14.3 × 106/kg). A number of CD34+ cells in the inoculum of 1 × 106/kg to 3 × 106/kg was associated with increased survival: 21 of 28 (75%) patients are alive, as compared with 29 of 56 (52%) patients receiving more than 3 × 106/kg (actuarial probability 75% vs. 42%, respectively; P = .01). In the multivariate analysis, the independent prognostic variables for survival were CD34+cell dose 1 × 106/kg to 3 × 106/kg (RR = 4.8; P = .0008), sex-pairing match (RR = 3.2;P = .002), and early stage of disease (RR = 2.8;P = .007). From these results it appears that, in allo-PBT/CD34+ from HLA-identical siblings, a number of CD34+ cells in the inoculum between 1 × 106/kg to 3 × 106/kg is an important factor for better survival, and that higher CD34+ cell doses might be associated with a poorer outcome.

The number of donor CD3+ cells is the most important factor for graft failure after allogeneic transplantation of CD34+ selected cells from peripheral blood from HLA-identical siblings

AbstractThis study analyzed the characteristics of 257 HLA-identical sibling transplants of granulocyte colony-stimulating factor–mobilized peripheral blood progenitor cells depleted of T cells by CD34+ positive selection (allo-PBT/CD34+) for their effect on the incidence of graft failure. Twenty-four patients developed graft failure (actuarial probability, 11%; 95% confidence interval, 7.1-14.9). Prognostic factors considered were sex and age of donor and recipient, donor-recipient blood group compatibility, diagnosis, disease status at transplant, conditioning regimen, cytomegalovirus serology, number of CD34+ and CD3+ cells infused, and cryopreservation. The major factor associated with graft failure was the number of CD3+ cells in the inoculum. Twenty-three of 155 patients receiving a T-cell dose in the graft less than or equal to 0.2 × 106/kg experienced graft failure, compared with only one of 102 patients receiving more than 0.2 × 106/kg (actuarial probability 18% vs 1%, respectively; P = .0001). The actuarial probability of graft failure progressively increased as the number of CD3+ cells in the graft decreased, which was determined by grouping the number of CD3+ cells in quartiles (log-rankP = .03; log-rank for trend P = .003). In the multivariate analysis by the proportional hazard method, 2 covariates entered into regression at a significant level: CD3+ cells less than or equal to 0.2 × 106/kg (risk ratio = 17;P < .0001), and patients with chronic myelogenous leukemia (CML) conditioned with busulphan-based regimens (risk ratio = 4.8; P = .001). From these results it appears that the number of CD3+ cells in the inoculum—with a threshold of 0.2 × 106/kg or less—is the most critical factor in maintaining a sustained engraftment in allo-PBT/CD34+ from HLA-identical siblings. In addition, for patients with CML receiving 0.2 × 106/kg or less CD3+ cells, total body irradiation might be better than busulphan-based regimens.

Transplantation of allogeneic CD34+ peripheral blood stem cells in patients with advanced hematologic malignancy

Sixteen patients with advanced hematologic malignancies were transplanted with HLA-identical allogeneic peripheral blood stem cells (PBSCs) that were selected for CD34+ cells by an avidin-biotin immunoadsorption technique. The median age of patients was 48 years (range, 37 to 67). Patients received 12.0 or 13.2 Gy of total body irradiation followed by 120 mg/kg of cyclophosphamide. Normal donors received 16 mg/kg of granulocyte-colony stimulating factor on days 1 to 6 followed by PBSC harvests on days 4 to 7. PBSC harvests were processed each day on a single avidin-blotin column containing an antibody to the CD34 antigen and processed cells were infused without cryopreservation daily for 4 consecutive days. Prophylaxis against graft-versus-host disease (GVHD) consisted of cyclosporine alone for 5 patients and CSA plus methotrexate for 11 patients. A median of 18.64 (6.74 to 34.97) x 10(8) CD34+ cells/kg patient body weight were collected from each donor. A median of 8.96 (2.62 to 17.34) x 10(8) CD34+ cells/kg patient body weight were recovered after avidin-biotin adsorption which represented a median CD34+ cell yield of 53% (18% to 77%) with a median purity of 62% (34% to 82%). There was a reduction in CD3+ cells from a median of 557.26 (227.73 to 677.77) x 106/kg to 0.73 x 10(4)/kg (0.40 to 3.65), in CD4+ cells from 351.72 (194.47 to 520.11) x 10(6)/kg to 0.40 (0.15 to 1.03) x 10(4)/kg and in CD8+ cells from 169.74 (53.34 to 325.83) x 10(6)/ kg to 0.32 (0.12 to 2.71) x 10(4)/kg representing a median 2.8 (2.19 to 3.14) log reduction in T cells. One patient died of infection on day 3 posttransplant and was unevaluable for recovery of neutrophils. The median day to recovery of 500 neutrophils/mL was 15 (8 to 26) in the remaining 15 patients. Six of 16 patients falled to achieve a platelet count of 20,000/mL before death on days 3 to 97 of transplant-related complications. The median day to achieving platelets of 20,000 mL in the remaining 10 patients was 11 (7 to 31). Eight of 16 patients (50%) died between 3 and 97 days posttransplant, 7 of transplant-related causes, and 1 of progressive disease. Grade 2-4 acute GVHD occurred in 12 out of 14 (86%) and grades 3-4 in 6 out of 14 (43%) evaluable patients. Six of 8 evaluable patients developed clinical chronic GVHD and 1 developed subclinical chronic GVHD. Bone marrow and/or peripheral blood chimerism studies in 12 evaluable patients showed 97% to 100% donor type in 11 patients with 1 patient in relapse showing 40% donor cells 60 to 90 days posttransplant. Four of 16 patients (25%) are alive and disease-free 312 to 576 days after transplant. There were no episodes of graft failure or rejection. This study shows that allogeneic transplantation using CD34+ selected PBSC results in prompt and sustained engraftment. CD34+ selection, as employed in this preliminary study, however, resulted in an apparently higher rate of acute and chronic GVHD. However, The sample size is quite small and precludes a more definitive conclusion regarding GVHD.

Stem cells as vehicles for gene therapy: novel strategy for HIV infection.

Restoration of bone marrow and immune function by means of allogeneic bone marrow transplantation has been attempted in AIDS patients but has not been successful as the donor-derived cells, or their progeny, inevitably became infected. A hairpin ribozyme that specifically cleaves HIV-1 RNA has been developed by F. Wong-Staal et al. and has been demonstrated to confer resistance against HIV-1 infection. Allogeneic transplantation of CD34+ cells or their pluripotent subsets, transduced by vectors bearing this ribozyme gene, can protect the stem cells and their progeny from HIV-1 infection and eventually restores immune function. We have provided evidence that long-term repopulating stem cells can be mobilized into peripheral blood by growth factors. The combination of G-CSF and GM-CSF seems to yield a high frequency of pluripotent stem cells with a CD34+ subset profile that is similar to placental and umbilical cord blood (PUCB). We have then demonstrated a highly efficient transduction of CD34+ cells from PUCB and mobilized leukapheresis products by retroviral vectors bearing the ribozyme gene. Expression of the ribozyme gene, as shown by reverse transcriptase-polymerase chain reaction, was of similar magnitude (70%-90% of cells that grow into colonies). Challenge of the progeny macrophages from such transduced CD34+ cells with monocyte-trophic strains of HIV-1 showed that they were resistant to infection. Thus allogeneic transplantation of CD34+ cells or their pluripotent subsets, transduced with ribozyme gene, can be a promising strategy for the treatment of HIV infection.