Quantitative Expression Analysis of WT1 Main Isoforms in AML

Abstract Abstract 1469 The Wilms Tumor 1 (WT1) gene was first described as a tumour suppressor gene, but its accurate role in leukemia development has not been completely elucidated. Some authors support the role of WT1 as a prognostic marker in acute myeloid leukemia (AML) based on the assessment of its expression at the mRNA level. However, the prognostic value of the main isoforms of WT1 has been less studied. The aim of this study was to develop a specific quantitative assay to estimate the ratio of expression of the four major WT1 isoforms (A, 5-/KTS-; B, 5+/KTS-; C, 5-/KTS+; D, 5+/KTS+) and to evaluate their prognostic impact. WT1 expression was analyzed in bone marrow samples from 108 patients with AML at diagnosis (65 male/46 female, median age: 61 yr, range: 17 – 91). Likewise, peripheral blood samples of 20 healthy donors and 6 samples of cord blood CD34+ cell selection were analyzed as normal controls. We performed a new method to quantify the ratios of the four major isoforms of WT1. Briefly, to amplify all isoforms within a PCR reaction, specific WT1 primers flanking exon 4 to exon 10 were used in cDNA samples, followed by capillary electrophoresis with laser-induced fluorescence analysis on an ABIPRISM 310 DNA Analyzer (Applied Biosystems, Foster City, CA) and lastly analyzed with the Gene Mapper 4.2 software (Applied Biosystems). The amount of each isoform was calculated by the area under the curve. Subsequent comparisons of isoform ratios were made by standardized calculation of percentage. All values are given as the mean of duplicate PCRs. In parallel, RQ-PCR for total WT1 detection was performed as previously described by Barragan et al. (Haematologica 2004; 89: 926–933). GUS gene was used as housekeeping gene. Eighteen patients (17%) did not express WT1, while 90 patients (83%) overexpressed WT1 above background levels. The median value of each WT1 isoform was: 18% (range: 2 – 73) for A isoform; 16% (range: 7 – 63) for B isoform; 24% (range: 2 – 52) for C isoform; and 33% (range: 3 – 55) for D isoform. None of healthy donors had detectable WT1 levels in peripheral blood. All samples of CD34+ cells expressed the four isoforms of WT1: 21% (range: 2 – 26) for A isoform; 16% (range: 1 – 64) for B isoform; 24% (range: 1 – 47) for C isoform; and 36% (range: 25 – 44) for D isoform. These data reveal that, in our series, the most predominant isoform was +5/+KTS, both in AML and in cord blood CD34+ cell selection samples. There were no significant differences when comparing the proportion of each isoform between the cord blood CD34+ cell selection samples and the cohort of AML patients. There was not significant correlation between the overexpression of total WT1 with the ratio of each isoform, and we were unable to demonstrate that the overexpression of WT1 is due to a particular isoform overexpression. A significant lower event-free survival (EFS) was observed in those patients overexpressing total WT1, taking a cut-off value of 3000 WT1 copies/ GUS copies × 104 (75th percentile, P =.001). However, when the same cut-off as well as the median value for each one of the isoforms was used, we found no significant differences in EFS and in overall survival. To sum up, none of the isoforms were correlated with overexpression of total WT1 or survival. We were unable to find differences between the expression of each isoform of WT1 in CD34+ cells from normal cord blood and in AML patients. Further studies including larger controls need to be carried out. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Recombinant Tpo Stimulates Maturation of Megakaryocytes Derived from Adult Peripheral Blood- but Not Cord Blood-CD34+ Cells.

Blood ◽

10.1182/blood.v104.11.1191.1191 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1191-1191

Author(s):

Karen M. Pastos ◽

William B. Slayton ◽

Lisa M. Rimsza ◽

Martha C. Sola

Keyword(s):

Cord Blood ◽

Peripheral Blood ◽

Developmental Differences ◽

Ploidy Levels ◽

Cd34 Cells ◽

Cord Blood Cd34 ◽

Adult Bone ◽

Adult Peripheral Blood ◽

Cells Cultured

Abstract Umbilical cord blood (CB) is a valuable source of stem cells for transplantation. However, platelet engraftment is slow, taking approximately 70 days for CB transplants versus 20 days for mobilized adult peripheral blood (PB) transplants. This time is not significantly shortened by the administration of recombinant thrombopoietin (rTpo). The cause for the delay in platelet engraftment following CB transplant is unknown. We hypothesized that developmental differences in size and ploidy of neonatal versus adult megakaryocytes (MKs) contribute to this delay. To mimic these two types of transplant in vitro, we compared CB to PB CD34+ cells cultured in adult bone marrow stromal-conditioned media (CM) or unconditioned media (UCM) for 14 days. Increasing doses of rTpo were added to the CM, and the resulting MK maturation was compared with that of UCM with maximal rTpo concentration. MK number and ploidy were determined by flow cytometry using CD41-FITC and propidium iodide, respectively. Increased ploidy levels were expressed as percentage of MKs with a ploidy ≥ 8N. Results represent an average of three independent experiments. Figure Figure As seen in the figure, PB-derived MKs reached highest ploidy levels in the presence of UCM + 100 ng/ml rTpo. When cultured in CM, they exhibited lower ploidy levels, regardless of Tpo concentration. In contrast, CB-derived MKs exhibited higher ploidy levels in response to CM with either 0 or 0.1 ng/ml (physiologic concentration) of rTpo, as compared to higher rTpo concentrations or UCM + 100 ng/ml rTpo. MK numbers increased in response to rTpo in a dose-response manner, regardless of the source of the MKs (data not shown). These results indicate that intrinsic differences between CB- and PB-derived megakaryocytes exist, and that maturation is regulated differently in neonatal versus adult MKs. While Tpo is a potent stimulator of MK maturation in PB-derived MKs, it appears to inhibit this process in CB-derived MKs. These differences may be relevant to understanding the delayed platelet engraftment following CB transplants.

Download Full-text

Co-Transplantation of Autologous Umbilical Cord Matrix Mesenchymal Stem Cells Improves Engraftment of Umbilical Cord Blood in NOD/SCID Mice.

Blood ◽

10.1182/blood.v108.11.2569.2569 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2569-2569

Author(s):

Robb Friedman ◽

Monica Betancur ◽

Hande Tuncer ◽

Laurent Boissel ◽

Curtis Cetrulo ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Peripheral Blood ◽

Scid Mice ◽

Umbilical Cord Matrix ◽

Cd34 Cells ◽

Cord Blood Cd34

Abstract Umbilical cord blood (UCB) is a viable source of hematopoietic stem cells for transplantation of children and adults undergoing treatment for hematological malignancies. However only 4% of adults 70kg and over have a UCB unit available which contains the widely accepted minimum cell dose of 1.5x107 mononuclear cells per kilogram. Co-transplantation of hematopoietic stem cells with mesenchymal stem cells may enhance engraftment and therefore decrease transplant-related morbidity and mortality from delayed leukocyte recovery associated with a low pre-transplant cell dose. Umbilical cord matrix (UCM) cells, found in the Wharton’s Jelly, were easily and reliably extracted from minced pieces of cord by culture in RPMI + 20% fetal bovine serum at 37° and 5% humidified CO2. UCM expand best in 20% FBS but can also be expanded in human serum, autologous serum, and X-VIVO10. Small (1–3mm) minced pieces of umbilical cord can be cyropreserved at the time of delivery in 10% DMSO solution. UCM cells exhibit a fibroblast morphology and express markers common to mesenchymal stem cells: CD73 (SH3), CD105 (SH2), CD 29, CD44, CD49b, CD117, CD166, STRO-1 and HLA-DR. UCM are negative for CD14, CD 19, CD34, and CD45. Morphology and cell surface marker expression is stable after greater than fifteen passages. UCM cells grown in culture were shown to produce more GM-CSF and G-CSF than similar numbers of adult bone marrow mesenchymal stem cells, GM-CSF 178 pg/mL versus 77 pg/mL and G-CSF 82.6 pg/mL versus 7.9 pg/mL. NOD/SCID mice treated with anti-NK 1.1 antibodies and irradiated with 350 cGy were injected with suboptimal (1x104) numbers of cord blood CD34+ cells with and without 1x106 autologous UCM cells, extracted from the same umbilical cord as the cord blood CD34+ cells. Bone marrow was harvested at six weeks post transplant from both femurs and tibias and peripheral blood obtained via cardiac puncture. The percentage of human CD45+ cells in the bone marrow and the peripheral blood was assessed by flow cytometry. NOD/SCID mice transplanted with 1x104 cord blood CD34+ cells alone had 3.0% human CD45+ cell engraftment in the bone marrow and 3.6% human CD45+ cells in the peripheral blood, while NOD/SCID mice transplanted with 1x104 CD34+ cells and 1x106 UCM cells had an average of 27.3% human CD45+ cell engraftment in the bone marrow and 3.9% human CD45+ cells in the peripheral blood. These results indicate a trend towards improved engraftment in vivo with co-transplantation of suboptimal numbers of umbilical cord blood CD34+ cells and autologous umbilical cord matrix cells versus transplantation of suboptimal numbers of umbilical cord CD34+ cells alone.

Download Full-text

Defective Peripheral Blood Endothelial Cell Progenitors in Patients with Scleroderma and Psoriatic Arthritis

Blood ◽

10.1182/blood.v112.11.4724.4724 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4724-4724

Author(s):

Taxiarchis V Kourelis ◽

Akrivi D Manola ◽

Despoina Adamidou ◽

Lazaros Sakkas ◽

Eyagelia Mperou ◽

...

Keyword(s):

Psoriatic Arthritis ◽

Cord Blood ◽

Peripheral Blood ◽

Culture Medium ◽

Autologous Bone Marrow ◽

Autologous Serum ◽

Cd34 Cells ◽

Cord Blood Cd34 ◽

And Function

Abstract Vasculogenesis is known to be defective in patients with scleroderma (SS) and psoriatic arthritis (PA) with vessel loss in the former and hypertrophic blood vessels in the latter in affected areas. We studied the number and function of peripheral blood endothelial progenitors (PBEP) in patients with SS and PA to elucidate the mechanism of EC dysfunction. Materials and Methods: Eleven patients with SS, 13 patients with PA and 7 healthy individuals were studied. We measured CD133+/CD146+ cells in peripheral blood (PB) by immunofluorescence. We performed cell cultures of isolated CD34+ cells in endocult medium and examined the endothelial colonies (EPC). We also performed cocultures of CD34+ and autologous bone marrow stromal cells (BMSC) in double chambers. We also performed cocultures of BMSC from patients and normal endothelial cells from cord blood. Results: The number of CD133+/CD146+ cell in PB was increased the 2 groups of patients compared to controls. The number of EC colonies in endocult did not differ in the 3 groups. The presence of autologous serum within the culture medium reduced the number of colonies in 3 patients with SS. The number and the size of EC colonies from SS patients in vitro were significantly reduced (p<0.01) after co-cultures of autologous BMSC with CD34+ in culture plates with insert while they were increased (p<0.01) from patients with PA. The same was true when cord blood CD34+ cells were cultured in endocult medium in the presence of BMSC of SS and PA patients. Conclusion: EC progenitors from patients with SS and PA are increased in PB and develop normal EC colonies in vitro. They developed decreased colonies in SS and increased colonies in PA when cultured together with with autologous BMSC. This means that possible cell-cell or humoral interactions between EC and some cellular component within BMSC affect the survival and differentiation of EC.

Download Full-text

First Reported Use of a Biosimilar G-CSF in Allogeneic Stem Cell Mobilisation

Blood ◽

10.1182/blood.v118.21.4571.4571 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4571-4571

Author(s):

Nabih Azar ◽

Sylvain Choquet ◽

Alice Garnier ◽

Damien Roos-Weil ◽

Véronique Leblond

Keyword(s):

Body Weight ◽

Stem Cell ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Median Number ◽

Large Hospital ◽

Originator Product ◽

Healthy Donors ◽

Stem Cell Mobilisation ◽

Cd34 Cells

Abstract Abstract 4571 OBJECTIVES: Biosimilar granulocyte colony-stimulating factor (G-CSF) has been approved on the basis of comparable quality, safety and efficacy as the originator product. Approval of biosimilar G-CSF is in the same indications as the originator, including autologous and allogeneic peripheral blood stem cell (PBSC) mobilisation, for which it is being used throughout our large hospital group in Paris, France. Concerns have been raised by professional bodies over use of biosimilar G-CSF in allogeneic transplants. To our knowledge, this is the first reported use of biosimilar G-CSF in healthy donors for allogeneic transplantation. METHODS: Healthy related donors received biosimilar G-CSF (EP-2006, Sandoz Biopharmaceuticals) 10 μg/kg/day for PBSC mobilisation. G-CSF was administered on days 1–4 with the first leukapheresis performed on day 5. If the required number of CD34+ cells per recipient body weight (4 × 106 CD34+ cells/kg) was not collected, G-CSF administration was continued and a second PBSC collection was performed on day 6. Further G-CSF administration and a third leukapheresis was done on day 7 if required. RESULTS: Twelve healthy donors (7 male, 5 female; mean ± SD age 61.0 ± 7.8 years, range 51–73) received biosimilar G-CSF for PBSC mobilisation. Median donor body weight was 82 kg (range 55–115 kg). Median CD34+ cell count on day 5 was 75/ml (range 23–140). A single leukapheresis after 4 injections of biosimilar G-CSF was sufficient to collect the CD34+ cell dose required in six donors with peripheral blood (PB) CD34+ counts ranging from 80–140 ml. Six donors underwent a second leukapheresis after 5 days of biosimilar G-CSF, one of whom (a 73-year old female) underwent a third leukaphereses on day 7 (PB CD34+ at first apheresis 23 ml). Median number of CD34+ cells per recipient bodyweight was 5.5 × 106 cells/kg (range 2.6–8.9). Five donors reported bone pain (4 mild, 1 moderate severity) which was effectively treated with paracetamol. No other adverse events were reported. Blood measurements were normal in all donors when assessed one week after the end of mobilisation. These findings are consistent with our previous use of originator G-CSF (Neupogen®) in allogeneic stem cell mobilisation. CONCLUSION: These preliminary findings suggest biosimilar G-CSF is effective and well-tolerated in allogeneic stem cell mobilisation. Further studies are required to assess longer-term safety outcomes. The use of biosimilar G-CSF may provide important cost-savings when compared with the originator product. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Quantitative Expression of Adhesion Molecules on Granulocyte Colony-Stimulating Factor-Mobilized Peripheral Blood, Bone Marrow, and Cord Blood CD34+Cells

Journal of Hematotherapy & Stem Cell Research ◽

10.1089/152581601317210908 ◽

2001 ◽

Vol 10 (6) ◽

pp. 807-814 ◽

Cited By ~ 19

Author(s):

C. Gigant ◽

V. Latger-Cannard ◽

D. Bensoussan ◽

P. Feugier ◽

P. Bordigoni ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Quantitative Expression ◽

Cd34 Cells ◽

Cord Blood Cd34 ◽

Stimulating Factor ◽

Mobilized Peripheral Blood

Download Full-text

Pro- and Antiapoptotic Proteins Expression on Bone Marrow and Peripheral Blood CD34+Cells in Acute Leukemia (AL) Patients

Blood ◽

10.1182/blood.v118.21.4996.4996 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4996-4996

Author(s):

Elena E. Khodunova ◽

Elena N Parovichnikova ◽

Irina V. Galtzeva ◽

Sergey M. Kulikov ◽

Valeri G Savchenko

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Expression Level ◽

Leukemia Cells ◽

Control Group ◽

Healthy Donors ◽

Cd34 Cells ◽

Significant Difference ◽

Blast Cells

Abstract Abstract 4996 It was shown that drug resistance, poor-risk cytogenetics and poor prognosis in AL is associated with high level of Bcl-2 expression and low Bax/Bcl-2 ratio (<0,3). Fas-antigen (CD95) as a protein triggering the extrinsic apoptotic pathway is differently expressed on hematopoietic precursors. More immature CD34+/CD38- AML blast cells have lower expression of Fas/Fas-L and lower Fas-induced apoptosis than CD34+/CD38+cells. CD34+/CD38− leukemia precursors also have a reduced sensitivity to daunorubicin in vitro and increased expression of multidrug resistance genes (mrp/lrp). CD34+ leukemia cells have not yet been properly characterized regarding the expression of angiotensin converting enzyme (ACE) which regulatory influence on hematopoiesis is now beeing extensively investigated. ACE expression on blast cells is high, but it's still unknown how CD34+ACE+ leukemia cells behave after chemotherapy. Recent publications indicate that CD34+ACE+ hematopoietic precursors transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts. We have studied the dynamics of Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in peripheral blood (PB) and bone marrow (BM) in AL pts during treatment. PB and BM samples were collected before and on +36 day after chemotherapy. The antigens were detected by flow cytometry using monoclonal antibodies. We calculated 10 000 cells in each sample. 19 pts were included in the study: 10 - AML and 9 - ALL. The control group comprised 8 healthy donors. At time of diagnosis there were 40±5,7% of CD34+ cells in BM and 26±4,9% - in PB. There was no significant difference between AML and ALL. CD34+ cells in BM and PB of healthy donors constituted 1,6% and 0,27%, respectively. After induction therapy (+36 day) CD34+ cells decreased in BM to 6,1%±3,3 (p=0,0001), in PB to 3,7%± 2,7 (p=0,0008) in all pts. The data on antigens expression on CD34+ cells of BM and PB are presented in table 1 CD34+/Bcl-2+ CD34+/Bax+ CD34+/CD95+ CD34+/ACE+ BM PB BM PB BM PB BM PB AML pts n=10 0 day 38±11,6* 41±14 24,4±7,9 29,2±7,6* 16,4±8,5 23,2±7,8 21,7±9,5 20,8±8,7* 36 day 13,5±3,4** 23,7±5** 46,2±11,5 50,3±11 19,9±5,5 36,4±10 34±6,6 35±9,2** ALL pts n=9 0 day 36±11 33,7±12 46,2±9,4 37,4±3,7* 3,4±1,1* 7,1±2,5* 41±10,9 33,2±9,7* 36 day 18,4±5,8 26±8,9 38±11,8 40,5±10 26,2±9,1** 40,9±9,2** 34±10 62,8±10** Donors n=8 11,7±1,6 26,1±5,9 22,8±4 67,8±6,7 13,4±3,2 47,7±11,6 28±5,3 68,2±10,2 * − p<0.05 compare with donors ** − p<0.05 compare with day 0 CD34/Bcl-2 expression in BM in AML pts is significantly higher (p=0,04) at the diagnosis comparing with donors. CD34/Bcl-2 expression in PB in AML pts and in BM and PB in ALL pts is higher too, but not significantly. This expression level decreased substantially in BM and PB in AML pts on +36 day comparing with day 0 (p<0,05). We did not found significant changes in ALL pts. CD34/Bax expression in PB is significantly lower (p=0,003) both in AML and ALL pts in comparison with donors. In AML, not in ALL, chemotherapy caused augmentation of Bax expression in CD34+ BM and PB cells on +36 day. BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 and Bax, demonstrating much higher level of pro- and antiapoptotic markers in PB cells. On the contrast CD34+ leukemia cells in BM and PB had similar characteristics regarding CD34/Bcl-2 and CD34/Bax expression. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar. CD95 expression on CD34+ BM and PB before treatment is significantly lower (p=0,01, p=0,008) in ALL pts in comparison with donors, and this expression level increased after chemotherapy (p<0,05). CD34/CD95 expression in AML pts is similar with donors, and we didn't find changes after treatment. CD34/ACE coexpression in BM cells of leukemia pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AML and ALL pts was much lower (p<0,05) than in donors and substantially increased on the day 36. So, our data demonstrate that Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in AL pts and donors significantly differs. The chemotherapy provokes critical changes in CD34/CD95 expression in BM and PB in ALL pts, CD34/Bcl-2 expression in AML pts and ÑÂ34/ACE expression in PB in all AL pts. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Natural killer cells differentiated in vitro from cord blood CD34 + cells are more advantageous for use as an immunotherapy than peripheral blood and cord blood natural killer cells

Cytotherapy ◽

10.1016/j.jcyt.2017.03.068 ◽

2017 ◽

Vol 19 (6) ◽

pp. 710-720 ◽

Cited By ~ 3

Author(s):

Anna Domogala ◽

Michael Blundell ◽

Adrian Thrasher ◽

Mark W. Lowdell ◽

J. Alejandro Madrigal ◽

...

Keyword(s):

Natural Killer Cells ◽

Cord Blood ◽

Natural Killer ◽

Peripheral Blood ◽

Killer Cells ◽

Cd34 Cells ◽

Cord Blood Cd34

Download Full-text

On Demand Plerixafor As a Rescue Strategy in Healthy Donors to Achieve Adequate Stem Cells - a Pilot Study from South India

Blood ◽

10.1182/blood-2019-127045 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5640-5640

Author(s):

Kishore Kumar ◽

Chezhian Subash

Keyword(s):

Stem Cells ◽

Pilot Study ◽

Side Effects ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

High Dose ◽

Healthy Donors ◽

Cd34 Cells ◽

Significant Difference ◽

Control Study

Aims & Objectives: The aim of the study is to check the safety and feasibility of using Plerixafor in healthy donors who have failed to mobilise > 2 x 10 6/kg of CD34 cells on day 1 of aphresis. The indication at present remains off label. Patients/Materials & Methods: Seventeen healthy donors, all more than 18 years of age were enrolled in this study after a proper written informed consent who failed to mobilise adequately on day 1 collection. The dose of Plerixafor used was 0.24mg/kg body weight of donor, 10-11 hours before the aphresis procedure. The GCSF was given from day -5 and the last dose was given 4 hours before the second day aphresis. CD34 was done in pre-aphresis peripheral blood and in product bag and compared with day 1 data. Results: The median donor bw was 54 kg (IQR, 42 kg to 69 kg) and the median recipient bw was 58 kg (IQR, 44 kg to 76 kg).The median CD34+ count in peripheral blood was 11.2/µl on day 4 after G-CSF alone and 24.7.0/µl on day 5 after G-CSF plus Plerixafor. The use of Plerixafor increased the number of circulating CD34 cells in peripheral blood by 2.2 fold.There were no major side effects except for the manageable bone pains which can be attributed to GCSF also. There were no differences in the engraftment statistics and rates of GVHD in comparison to historical cases. Though this is not a randomised control study to compare with second day high dose GCSF cases, the amount of increase in stem cells were statistically significant comparing with historical controls(p< 0.05). After a median followup of six months, no adverse effects were noted in donors. Discussion & Conclusion: Plerixafor is well tolerated in healthy donors and can be used safely in situations of poor mobilisers or when there is a significant difference in weight of donor to receipient. Its a small pilot study. We need a proper randomised control study and a longer follow up to look infor the side effects on healthy donor. Disclosures No relevant conflicts of interest to declare.

Download Full-text