Transplantation of highly purified CD34+ progenitor cells from alternative donors in children with refractory severe aplastic anaemia

Without transplantation from a human leucocyte antigen-identical family donor, refractory severe aplastic anaemia (SAA) has an unfavourable prognosis. Conventional transplantation from a matched unrelated donor carries a high rate of mortality. We transplanted large numbers of highly purified CD34+ cells from matched unrelated (n = 4), mismatched unrelated (n = 4) and mismatched related (n = 1) donors into nine children with refractory SAA. The grafts consisted of granulocyte colony-stimulating factor-mobilized peripheral positively selected CD34+ cells. A median of 15.1 x 106/kg CD34+ stem cells and 11 x 103/kg CD3+ T-lymphocytes were infused. No additional pharmacological graft versus host disease (GVHD) prophylaxis was given. At a median follow-up of 47 (range 37-72) months, eight patients (89%) were in complete remission with >90% donor chimaerism and no evidence of GVHD. One patient died on day +238 as a consequence of GVHD. The use of highly purified mobilized CD34+ stem cells warrants further clinical exploration in children with refractory SAA.     

Autografting with CD34+ peripheral blood stem cells: retained engraftment capability and reduced tumour cell content

The efficacy of an immunomagnetic purging method and the Isolex 300 devices were assessed for selecting CD34+ cells from leukapheresis products of 29 patients with non-Hodgkin's lymphoma (NHL), 39 with multiple myeloma and 34 with breast cancer. The mean purity of the CD34+ cell population was 93.6% and the mean recovery was 67.7%. Following enzymatic cleavage by chymopapain the expression of Thy-1 and Leu-8 was significantly reduced without affecting haematological recovery. The population of selected CD34+ cells of 4/8 patients with follicular lymphoma became PCR-negative. A 2.5 log reduction of tumour cells could be achieved in four patients with multiple myeloma as shown by a quantitative PCR assay. There were no tumour cells detectable in any of the 19 CD34+ cell preparations of patients with breast cancer. In 64 patients who received 94 cycles of high-dose therapy, a mean number of 4.7x 10(6) CD34+ cells/kg were autografted. The time needed for platelet reconstitution was different when a comparison was made with 156 patients, who had received unmanipulated leukapheresis products (10 v 12 d, P = 0.006). No significant differences with regard to neutrophil recovery were noted. Five patients had a graft failure. Two of them died (on day 78 and 88 following PBSCT), and three patients were rescued with unmanipulated back-up transplants. In conclusion, the immunomagnetic selection of CD34+ cells provides autografts with reduced tumour cell content and an engraftment ability similar to that of unmanipulated autografts.     

Autologous CD34+ and CD133+ stem cells transplantation in patients with end stage liver disease

AIM:To assess the utility of an autologous CD34 + and CD133 + stem cells infusion as a possible therapeutic modality in patients with end-stage liver diseases.METHODS:One hundred and forty patients with endstage liver diseases were randomized into two groups.Group 1,comprising 90 patients,received granulocyte colony stimulating factor for five days followed by autologous CD34 + and CD133 + stem cell infusion in the portal vein.Group 2,comprising 50 patients,received regular liver treatment only and served a...     

Allogeneic transplantation using CD34+ selected peripheral blood progenitor cells combined with non‐mobilized donor T cells for refractory severe aplastic anaemia

Allogeneic haematopoietic stem cell transplantation is curative for severe aplastic anaemia (SAA ) unresponsive to immunosuppressive therapy. To reduce chronic graft‐versus‐host disease (GVHD ), which occurs more frequently after peripheral blood stem cell (PBSC ) transplantation compared to bone‐marrow transplantation (BMT ), and to prevent graft rejection, we developed a novel partial T‐cell depleted transplant that infuses high numbers of granulocyte colony‐stimulating factor‐mobilized CD 34⁺ selected PBSC s combined with a BMT ‐equivalent dose of non‐mobilized donor T‐cells. Fifteen patients with refractory SAA received cyclophosphamide, anti‐thymocyte globulin and fludarabine conditioning, and were transplanted with a median 8 × 10⁶ CD 34⁺ cells/kg and 2 × 10⁷ non‐mobilized CD 3⁺ T‐cells/kg from human leucocyte antigen‐matched sibling donors. All achieved sustained engraftment with only two developing acute and two developing chronic GVHD . With a 3·5‐year median follow‐up, 86% of patients survived and were transfusion‐independent. When compared to a retrospective cohort of 56 bone‐marrow failure patients that received the identical transplant preparative regimen and GVHD prophylaxis with the exception that the allograft contained unmanipulated PBSC s, partial T‐cell depleted transplant recipients had delayed donor T‐cell chimerism and relative reduction of 75% in the incidence of acute grade II ‐IV GVHD (13% vs. 52%; P  =  0·010) and of 82% in chronic GVHD (13% vs. 72%; P  =  0·0004). In multivariate analysis, partial T‐cell depleted transplants remained significantly associated with a reduced risk of GVHD . In conclusion, for patients with refractory SAA , this novel transplant strategy achieves excellent engraftment and survival when compared to unmanipulated PBSC transplants and dramatically reduces the incidence of both acute and chronic GVHD .     

Transplantation of HLA-mismatched CD34+ selected cells in patients with advanced malignancies: severe immunodeficiency and related complications

This trial was designed to test the use of CD34⁺ selected haemopoietic stem cells (HSC) in HLA-mismatched donor–recipient pairs, following intensive conditioning with thiotepa, antilymphocyte globulin (ALG), cyclophosphamide and single-dose total-body irradiation (sTBI). 10 patients aged 16–50 with advanced malignancies and a two- or three-antigen mismatched family donor entered this study. Donor marrow and G-CSF primed peripheral blood cells were processed separately on CD34 columns (Ceprate). The median number of infused CD34⁺ cells were 5.66 × 10⁶/kg, with 0.55 × 10⁶/kg CD3⁺ cells. Nine patients received cyclosporin for graft-versus-host disease (GvHD) prophylaxis. Median neutrophil counts on day 21 were 2 × 10⁹/l with a median platelet count of 60 × 10⁹/l, but CD4 counts remained extremely depressed throughout the study. Acute GvHD was scored as grade 0–I in two patients, as grade II in seven, and grade III in one. Eight patients died at a median interval of 72 d from HSCT (range 20–144) due to cytomegalovirus (CMV) associated interstitial pneumonitis (IP) ( n  = 5), renal failure ( n  = 1), GvHD ( n  = 1) and Aspergillus meningitis ( n  = 1). Two patients are alive 365–495 d post transplant, one in remission and one in relapse.
This study suggests that large numbers of positively selected mismatched HSC can rapidly engraft after intensive conditioning regimen: however, profound post-transplant immunodeficiency leads to a high risk of lethal infectious complications.     

A prospective analysis of the pattern of immune reconstitution in a paediatric cohort following transplantation of positively selected human leucocyte antigen-disparate haematopoietic stem cells from parental donors

Transplantation of haematopoietic stem cells from human leucocyte antigen (HLA)-disparate parental donors presents a promising new approach for the treatment of patients lacking a HLA-matched donor. Success against major obstacles such as graft-versus-host disease (GvHD) and graft rejection has recently been demonstrated, so that immune reconstitution is one of the prime factors that determines the long-term prognosis following transplantation. Twenty children transplanted with megadoses of highly purified CD34(+) haematopoietic stem cells after rigorous T-cell depletion were prospectively monitored for their immune reconstitution during the first post-transplant year. Natural killer (NK) cells showed a marked increase on d +30. T and B cells began to reconstitute on d +72 and +68 respectively. During extended follow-up, their numbers and proliferative capacity upon mitogen stimulation continually increased. Early reconstituting T cells were predominantly of a primed, activated phenotype with severely skewed T-cell receptor (TCR)-repertoire complexity. Naive T cells emerged 6 months post transplantation, paralleled by an increase in TCR-repertoire diversity. All patients self-maintained sufficient immunoglobulin levels after d +200. This study demonstrates that paediatric recipients of highly purified, haploidentical stem cells are able to reconstitute functioning T-, B- and NK-cell compartments within the first post-transplant year. This, together with the absence of significant GvHD, provides a strong indication for this approach to be considered in children who lack a HLA-matched donor.     

CD34~+ CD38~-细胞对异基因造血干细胞移植的影响研究

目的观察CD34+CD38-细胞对异基因造血干细胞移植术后造血重建和移植物抗宿主病(GVHD)的影响。方法分析2004年1月至2009年12月河南省人民医院血液科全相合异基因外周血干细胞造血干细胞移植78例,CD34+、CD34+CD38-细胞输入量与血缘全相合异基因外周血造血干细胞移植术后造血重建及GVHD发生率间的相关性。结果粒细胞、血小板恢复时间与CD34+CD38-细胞输入量呈负相关(r分别为-0.521、-0.448,P<0.01),与CD34+细胞输入量也呈负相关(r分别为-0.405、-0.371,P<0.05)。急性GVHD、慢性GVHD的发生与CD34+、CD34+CD38-、CD3+、CD4+、CD8+细胞输入量无相关性。结论输入高数量的CD34+CD38-细胞有利于移植术后的粒细胞、血小板快速恢复;对于预测术后造血恢复,CD34+CD38-细胞亚群输入量可能优于CD34+细胞总数。     

CD34+/CD90+ cells infused best predict late haematopoietic reconstitution following autologous peripheral blood stem cell transplantation

This study aimed to identify which graft product subset of cells might be the most predictive of late haematopoietic recovery (three to 12 months) following autologous peripheral blood stem cell transplantation (PBSCT). The relationships between the numbers of reinfused CD34+ cells and their immature subsets such as CD34+/CD90+, CD34+/AC133+, CD34+/CD38- and CD34+/HLA-DR- cells, and haemoglobin, white blood cell (WBC) and platelet counts at 3, 6, 9 and 12 months after PBSCT, were studied in 25 patients with haematological and solid malignancies. The total CD34+ cell number, as well as CD34+/CD90+ and CD34+/AC133+ cell numbers, correlated with platelet counts at 3, 6, 9 and 12 months after PBSCT, but the CD34+/CD90+ cells infused best predicted platelet recovery during the first 12 months after PBSCT (P < 0.0238 at any time-point). The CD34+/AC133+ cell dose also correlated with WBC counts at 3 months post PBSCT. In addition, all patients receiving more than 80 x 10(4) CD34+/CD90+ cells/kg showed platelet counts greater than 100 x 10(9)/l at all points after PBSCT, suggesting that this value of the CD34+/CD90+ cells infused was a threshold dose for durable haematopoietic engraftment after PBSCT.     

Immature granulocyte fraction in the peripheral blood is a practical indicator for mobilization of CD34(+) cells

We propose a simple parameter that improves prediction of the number of CD34(+) cells in blood cells collected by apheresis for autologous peripheral blood stem cell (PBSC) transplantation following administration of granulocyte colony-stimulating factor. The percentage of immature granulocytes including myeloblasts, promyelocytes, myelocytes, and metamyelocytes (LSI for left-shift index) immediately prior to the start of each apheresis correlated with the number of CD34(+) cells in PBSC collections (r = 0.79, P < 0.0001, Y = 0.227X - 0.99, R(2) = 0.623) much better than did the white blood cell count (r = 0.07), currently the most commonly used predictor in deciding the initiation of apheresis. We then used receiver operating characteristic (ROC) curves to determine a cutoff point for LSI to prevent unnecessary apheresis. At LSI > 7.5, sensitivity and specificity of cutoff points in the probability of obtaining >1.0 x 10(6) CD34(+) cells/kg BW were 93.3% and 94.3% (95% CI, 91.4-100.0%), respectively. When LSI reaches 15.25, nearly 100% of apheresis will attain the target CD34(+) cell dose. These findings indicate that LSI is a useful and simple method for predicting the yield of CD34(+) cells before the start of PBSC collection and avoiding unnecessary apheresis.     

Clinical applications of CD34+ cell-selected peripheral blood stem cells

Peripheral blood stem cells (PBSC) are increasingly used for stem cell transplantation after high dose chemotherapy. CD34+ cell selection has also been done for use in autologous transplantation studies Bone marrow (BM) may contain tumor cells at the time of harvesting, and on re-infusion, these cells could contribute to a subsequent relapse. Similarly, tumor cell contamination of PBSC collections has been found in a number of studies. Therefore, purging contaminating tumor cells may prevent cases of relapse. As most tumor cell types do not express CD34 antigen, one of the most widespread applications of CD34+ cell selection is likely to be in tumor cell purging. Similarly, CD34+ cell selection has aided allogeneic transplantation studies. Acute graft-versus-host disease (aGVHD) is a major cause of morbidity and mortality in cases of allogeneic transplantation. As aGVHD is mediated by donor T cells, removal of T cells from the graft by CD34+ cell selection may ensure prophylaxis against aGVHD. Further, high-dose immunosuppression followed by CD34+ cell-selected stem cell rescue is theoretically reasonable as a therapeutic tool for patients with autoimmune disease resistant to conventional therapy. However, patients given T cell-depleted transplantation have an increased risk of opportunistic infection as well as malignancies related to immunosuppression; therefore, close monitoring is warranted. We describe here clinical applications of CD34+ cell-selected PBSC for a variety of diseases, with special emphasis on the efficacy as well as drawbacks of this novel technique.     

Transplantation of CD3/CD19 depleted allografts from haploidentical family donors in paediatric leukaemia

Transplantation of T‐ and B‐cell depleted allografts from haploidentical family donors was evaluated within a prospective phase II trial in children with acute lymphoblastic leukaemia, acute myeloid leukaemia and advanced myelodysplastic syndrome (n = 46). 20 patients had active disease; 19 patients received a second or third stem cell transplantation (SCT). Toxicity‐reduced conditioning regimens consisted of fludarabine or clofarabine (in active disease only), thiotepa, melphalan and serotherapy. Graft manipulation was carried out with immunomagnetic microbeads. Primary engraftment occurred in 88%, with a median time to reach >1·0 × 10⁹/l leucocytes, >20 × 10⁹/l platelets and >0·1 × 10⁹/l T‐cells of 10, 11 and 50 days, respectively. After retransplantation, engraftment occurred in 100%. Acute graft‐versus‐host disease (GvHD) grade II and III‐IV occurred in 20% and 7%, chronic GvHD occurred in 21%. Both conditioning regimens had comparable toxicity. Transplant‐related mortality (TRM) was 8% at one year and 20% at 5 years. Event‐free survival at 3 years was: 25% (whole group), 46% (first, second or third complete remission [CR], first SCT) vs. 8% (active disease, first SCT) and 20% (second or third SCT, any disease status). This approach allows first or subsequent haploidentical SCTs to be performed with low TRM. Patients in CR may benefit from SCT, whereas the results in patients with active disease were poor.     

CD34+-enriched peripheral blood progenitor cells from unrelated donors for allografting of adult patients: high risk of graft failure,infection and relapse despite donor lymphocyte add-back

Fifty-one adults with haematological malignancies were transplanted with CD34+-selected peripheral blood progenitor cells (PBPC) from unrelated donors. The conditioning protocol contained total body irradiation (n = 17) or combinations of busulphan and other alkylating agents (n = 34). Antithymocyte globulin was infused in all patients. The median number of CD3+ T cells infused with the graft after purification with the Isolex 300 system in the first cohort of 18 patients was 2.1 x 10(5)/kg. Prophylactic donor lymphocyte infusion (DLI) containing 1 x 10(5) CD3+ T cells was performed on d 21 in the following 33 patients who had received PBPC purified by the CliniMACS system. Early graft failure occurred in 8/51 patients (16%). After a median follow-up of 31 months (range 8-60), the probability of disease-free survival (DFS) was 36% for the whole group. Reasons for death were opportunistic infections (n = 15), graft-versus-host disease (GvHD, n = 7) and relapse (n = 4). Pre-transplant factors with significant impact on DFS were cytomegalovirus status and risk category of underlying disease. The occurrence of graft failure or GvHD was associated with poor outcome. Recipients of CD34+-selected PBPC from unrelated donors are at high risk of infectious complications, relapse and graft failure which cannot be prevented by early reinfusion of unmodified donor lymphocytes.     

Influence of the different CD34+ and CD34- cell subsets infused on clinical outcome after non-myeloablative allogeneic peripheral blood transplantation from human leucocyte antigen-identical sibling donors

Currently, no information is available regarding the influence of the different CD34+ cell subsets infused on the haematopoietic recovery, following non-myeloablative allogeneic peripheral blood stem cell transplantation (allo-PBSCT). We have explored, in a group of 13 patients receiving non-myeloablative allo-PBSCT from human leucocyte antigen-identical sibling donors, the influence of the total dose of CD34+ haematopoietic progenitor cells (HPC) infused, compared with that of the different CD34+ HPC and CD34- leucocyte subsets in the leukapheresis samples, on both engraftment and clinical outcome. The overall numbers of total CD34+ HPC (P = 0.002) and myelomonocytic-committed CD34+ HPC infused (P = 0.0002) were strongly associated with neutrophil recovery (> 1 x 109 neutrophils/l), the latter being the only independent parameter influencing neutrophil recovery. Regarding long-term engraftment, only the number of immature CD34+ HPC infused/kg correlated with the duration of hospitalization in the first 2 years after discharge (r = -0.75, P = 0.005). Both the overall amount of CD34+ HPC and the number of myelomonocytic CD34+ HPC infused showed a significant influence on the risk of graft-versus-host disease (GVHD). Thus, the overall probability of GVHD was 100%vs 25% for patients receiving >/= 5 x 106 CD34+ HPC or >/= 3.5 x 106 of myelomonocytic-committed CD34+ HPC vs lower doses (P = 0.013). None of the other CD34+ and CD34- cell subsets analysed correlated with development of GVHD. In summary, our results suggest that in non-myeloablative allo-PBSCT, high numbers of CD34+ HPC, especially the myelomonocytic-committed CD34+ progenitors, lead to rapid neutrophil engraftment. However, they also strongly impair clinical outcome by increasing the incidence of GVHD.     

Duration of filgrastim mobilization and apheresis yield of CD34+ progenitor cells and lymphoid subsets in normal donors for allogeneic transplantation

Seventy-seven normal donors underwent leukapheresis for peripheral blood progenitor cell collection beginning on day 4 ( n  = 45) or day 5 ( n  = 32) of filgrastim mobilization (12 μg/kg/d). The two groups were comparable for age, weight, blood volumes processed during leukapheresis and target CD34⁺ cell dose to be collected. The day 5 schedule allowed a more consistent achievement of the target cell dose with one apheresis ( P  = 0.005) and resulted in the initial collection of a significantly larger number of CD34⁺ cells ( P  = 0.009). There was no statistically significant difference in the leukapheresis yield of lymphoid subsets and natural killer cells.     

Determining post-thaw CD34+ cell dose of cryopreserved haematopoietic progenitor cells demonstrates high recovery and confirms their integrity

Background  The acceptable dose of haematopoietic progenitor cells (HPCs) for transplantation is generally based on the number of CD34+ cells determined prior to cryopreservation. Commonly, cryopreservation is associated with total nucleated cell viability loss. Because HPCs have been shown to be more resistant to cryopreservation damage than nucleated cells overall, low viability may not reflect the quality and integrity of the thawed product.
Methods  Peripheral blood HPC products from 45 mobilized allogeneic and autologous donors were harvested by continuous flow blood separation and cryopreserved in 7·5% dimethyl sulfoxide. The number of viable CD34+ cells was determined by flow cytometry. Viability was measured by trypan blue (TB) uptake and 7-aminoactinomycin D (7-AAD) flow cytometry.
Results  Post-thaw HPC products were analysed for viability, CD34+ cell recovery and engraftment capability. The average post-thaw viable CD34+ cell recovery was 86·4%, while the average post-thaw viability, measured by TB or 7-AAD, was 74·0% and 57·0%, respectively. Most of the cells that did not survive cryopreservation were of the granulocyte series. All of the donors who underwent transplantation engrafted, mostly within 14 days.
Conclusions  Our data show that most CD34+ cells survive cryopreservation, regardless of the overall post-thaw total nucleated cell viability. Measuring the number of viable CD34 cells post-thaw might be of importance, and in cases of low viability can confirm the quality of the product issued.     

Viable CD34+/CD133+ blood progenitor cell dose as a predictor of haematopoietic engraftment in multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation

Both CD34 (cluster of differentiation 34) and the more recently described CD133 are markers of primitive stem cells with haematopoietic repopulating ability. Most transplanting centres use a minimum number of CD34+ cells as the requirement for a transplant and consider this a predictor of haematopoietic engraftment. However, transplanted CD34+ cell dose does not always give a close correlation with time to engraftment nor explain delayed engraftment in some patients. We have retrospectively evaluated the potential of measuring viable CD133+ cell numbers in the autograft as an alternative predictor of haematological engraftment after autologous stem-cell transplantation in a cohort of patients with multiple myeloma (MM). We found an average 32% loss of viability of CD34+ cells in the post-thaw sample compared with the fresh sample. Of the original estimated CD34+ cell numbers transplanted per kg, 43% of the thawed samples were double positive for CD34+/CD133+. In this patient group, the CD34+/CD133+ subset gave the closest statistical correlation with time to neutrophil engraftment (p < 0.05), particularly for patients given above median (1.8 × 10⁶/kg) dose of the double-positive cells. The CD34+/CD133+ population was the only parameter to give a significant correlation with white cell engraftment in this patient cohort (p < 0.05). There was no significant correlation between CD34+, viable CD34+ or viable CD34+/CD133+ cells/kilogram with platelet engraftment. Determination of viable CD34+/CD133+ progenitor cell dose in the autograft may be a useful tool to predict neutrophil recovery after autologous transplantation than conventional assessment of CD34+ numbers. These results warrant further investigation of the role of CD133 in haematopoietic engraftment.     

CD34+ cell dose predicts relapse and survival after T-cell-depleted HLA-identical haematopoietic stem cell transplantation (HSCT) for haematological malignancies

Seventy-eight patients with haematological malignancies, received T-cell-depleted stem cell transplants and cyclosporin followed by delayed add-back of donor lymphocytes to prevent leukaemia relapse. The source of stem cells was bone marrow in 50 patients and granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood in 28 patients. In univariate analysis, only the CD34+ cell dose (but not the stem cell source or the T lymphocyte dose) and disease status were predictive for transplant-related mortality, relapse and survival. Patients receiving >/= 3 x 106 CD34+ cells/kg had an overall actuarial survival of 68% compared with 52%, 35% and 10%, respectively, for cell doses of 2-2.99, 1-1.99 and < 1 x 106/kg. Multivariate analysis of risk factors for relapse identified disease risk and CD34+ cell dose as the only factors. Relapse was 62.5% in 38 patients at high risk of relapse vs. 25% for 40 patients at intermediate or low risk. CD34+ cell doses of >/= 3 x 106/kg were associated with a 13.5% relapse vs. 48% for recipients of lower doses. This favourable effect of CD34+ cell dose on relapse was apparent in both high- and intermediate- plus low-risk groups. Our results support the potential benefit of a high stem cell dose in lowering transplant-related mortality (TRM) and in reducing relapse after allogeneic marrow or blood stem cell transplants.     

CD133+肺癌细胞的干细胞特性研究

目的 收获CD133+肺腺癌细胞并评估其肿瘤干细胞特性.方法 从13例新鲜肺腺癌组织中收获CD133+和CD133-肺腺癌细胞,并进行Transwell侵袭实验、裸鼠成瘤实验等对比研究,观察CD133+和CD133-肺腺癌细胞的差异.结果 13例中有10例发现CD133表达,其阳性表达率最高为13.12%,CD133+和CD133-细胞在侵袭性、致瘤能力上有明显区别.结论 CD133在人肺腺癌组织细胞中广泛表达,CD133+较CD133-人肺腺癌细胞具有更强的侵袭性和致瘤能力.