PRMT1 Mediated FLT3 Methylation As a Therapeutic Vulnerability in FLT3-ITD+ AML

Abstract The current view is that treatment failures of AML patients are due to persistence of leukemia stem cells (LSCs). The presence of FMS-like tyrosine kinase-3 (FLT3) Internal tandem duplication (ITD) is associated with poor prognosis. But, FLT3 tyrosine kinase inhibitors (TKI) demonstrate transient clinical activity in FLT3-ITD+ AML patients. Persistent FLT3-ITD+ AML LSC represent a source of relapse. There is a pressing need to target LSC and improve outcomes for FLT3-ITD+ AML patients. PRMT1, the predominant arginine methyltransferase, has been implicated in pathogenesis of AML rare subtype (i.e., acute megakaryoblastic leukemia). Herein, we show that PRMT1 protein expression is significantly increased in LSC-enriched AML CD34+CD38- cells relative to the counterparts of normal peripheral blood stem cells (PBSC) (AML n=9, normal n=8, p=0.0004,). Following PRMT1-knockdown (KD), AML CD34+ cells (n=15) demonstrated varying degrees of apoptosis while the survival of normal cells were not affected. Interestingly, we observed significant apoptosis-induction in a subset of samples bearing FLT3-ITD mutation (6 out of total 15) upon PRMT1-KD (ShCtrl 13.9±3.6%, ShPRMT1 32.5±4.6%, p<0.001). PRMT1-KD induced apoptosis was also more evident in cord blood (CB) CD34+ cells expressing FLT3-ITD (ShCtrl 19.4±1.3%, ShPRMT1 45.2±2.5%, p<0.001) relative to that of FLT3-WT (ShCtrl 27.47±1.8%, ShPRMT1 36.9±1.6%, p=0.0167). In 293T cells ectopically overexpressing FLT3-WT or FLT3-ITD, co-immunoprecipitation (co-IP) indicated greater interaction between PRMT1 and FLT3-ITD. Through RNA-Seq profiling two AML lines (MV4-11, OCI-AML3) plus one FLT3-ITD transduced CB CD34+ cells with PRMT1-KD, we obtained a differentially-regulated gene set as a "PRMT1 signature". This signature was enriched in FLT3-ITD+ AML relative to FLT3-WT AML according to ssGSEA analysis using two AML datasets (GSE14468, GSE10358), indicating PRMT1 may cooperate with FLT3-ITD regulating AML maintenance. Given that PRMT1 directly interacts with FLT3-ITD, we next asked whether PRMT1 catalyzes FLT3-ITD protein methylation. Through mass-spectrometry analysis of a FLT3-ITD+ AML specimen and in vitro methylation assay, we identified that PRMT1 catalyzes FLT3-ITD arginine (R) methylation (Me) at two conserved residues, 972 and 973. Using in-house R972/973 Me antibody, we validated the expression of FLT3 R-Me in FLT3-ITD AML speciemens (7 out of 7). To test R-Me function, we transduced MLL-AF9 (MA9) overexpressing murine c-Kit+ cells with methylation-deficient FLT3-ITD (R972/973K, arginine [R] to lysine [K]) construct, and found that MA9 cells expressing R972/973K underwent more apoptosis than that of WT FLT3-ITD (WT FLT3-ITD 9.7±1.1%, R972/973K 23.7±2.1%, p=0.003). The double transformed cells were further transplanted into recipients for leukemia development. Mice receiving MA9 cells expressing R972/973K exhibited longer survival (median survival: WT FLT3-ITD 36 days, R972/973K 50 days, p=0.002, n=6). Mechanistically, expression of R972/973K did not affect the total tyrosine phosphorylation level of FLT3-ITD. Additionally, FLT3-ITD R-Me expression persisted after a TKI (AC220) treatment. These facts indicated that FLT3-ITD R-Me function is independent of FLT3-ITD kinase activity. We then used a FLT3-ITD+ patient derived xenograft (PDX) model to assess the effects of TKI and PRMT1 inhibition. Following engraftment >1% in peripheral blood, we divided mice (n=24) into 4 groups and treated each with vehicle, MS023 (a type I PRMT inhibitor, ACS Chem Biol. 2016;11:772-781) (160 mg/kg/i.p), AC220 (10 mg/kg/i.g) or combination for 4 weeks. MS023 treatment downregulating FLT3 R-Me levels enhanced elimination of FLT3-ITD AML cells by AC220 treatment (AC220 24.6±13.4% vs combination 7.6±6.5%, p=0.02, n=6). At 16 weeks post-secondary BMT, significant AML burden in single drug treated transplants was observed, but less AML cells were detected in combination-treated transplants (AC220 52.3%, vs combination 25.4%, p<0.001, n=6). MS023 had little effect on long-term in vivo engraftment of CD34+ from a human CB specimen (vehicle 76.3±5.9%, MS023 72.2±3.4%, p=0.21, n=5). In summary, our study demonstrated PRMT1 overexpression contributes to AML stem/progenitor cell survival possibly through FLT3-ITD methylation, supporting further exploration into how PRMT1-mediated FLT3 methylation governs LSC survival. Disclosures Khaled: Alexion: Consultancy, Speakers Bureau; Daiichi: Consultancy; Juno: Other: Travel Funding.

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

Blood ◽

10.1182/blood.v88.11.4132.4132 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4132-4138 ◽

Cited By ~ 5

Author(s):

WI Bensinger ◽

CD Buckner ◽

K Shannon-Dorcy ◽

S Rowley ◽

FR Appelbaum ◽

...

Keyword(s):

Stem Cells ◽

Body Weight ◽

Peripheral Blood ◽

Hematologic Malignancy ◽

Chronic Gvhd ◽

Allogeneic Transplantation ◽

Hematologic Malignancies ◽

Peripheral Blood Stem Cells ◽

Cd34 Cells ◽

Blood Stem Cells

Abstract 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.

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Mobilization of Allogeneic Peripheral Blood Stem Cells in Family Donors with Single-Dose Pegfilgrastim.

Blood ◽

10.1182/blood.v114.22.2148.2148 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2148-2148

Author(s):

Vladan Vucinic ◽

Nadezda Basara ◽

Runa Stiegler ◽

Kristina Bartsch ◽

Constanze Kliem ◽

...

Keyword(s):

Stem Cells ◽

Body Weight ◽

Single Dose ◽

Peripheral Blood ◽

Peripheral Blood Stem Cells ◽

Weight Range ◽

Healthy Donors ◽

Cd34 Cells ◽

Blood Stem Cells ◽

Pbsc Mobilization

Abstract Abstract 2148 Poster Board II-125 Introduction: The standard procedure for obtaining peripheral blood stem cells (PBSC) is donor mobilization with G-CSF. Pegfilgrastim is a covalently bound conjugate of filgrastim and monomethoxypolyethylene glycol with longer half-life elimination due to decreased plasma clearance and could represent an alternative approach for PBSC mobilization in healthy donors. Design and Methods: From July 2006 till August 2009 28 related healthy donors (50% male, 50% female) were treated with single dose of 12 mg pegfilgrastim for mobilization of allogeneic PBSC. The harvests were performed as large-volume, continuous-flow collections using a Cobe Spectra blood cell separator on day 4 and if necessary on day 5 of the mobilization regimen. In case of inadequate CD34+ counts (less than 4×106/kg body weight of recipient on day 5), stimulation was continued with filgrastim. In addition, the serum level of filgrastim was determined twice daily. Results: We present the results of 27 donors (the results of the 28th donor are still pending). In all 27 cases the harvests were successful. In 22 out of 27 donors (82%) only a single apheresis was needed to reach the target. Two of the donors required additional treatment with non-pegylated filgrastim. The maximal concentration of circulating CD34+ cells was achieved on day 4 (median 74.3/μl; range 24.6-136.6). The median yield of CD34+ cells was 5.9×106/kg of the recipients body weight (range 3-14.5), and the median CD3+ count was 9.1×108/kg of the recipient body weight (range 1.4-6.2). Serum filgrastim level peak was on day 2 of the mobilization regimen with a median level of 226 ng/ml (range 35 to 1123 ng/ml), thus preceding the increase of CD34+ cells in blood. The main adverse events were WHO grade 1 and included headaches, bone pain and transient elevations of alkaline phosphatase and lactate dehydrogenase. Conclusion: PBSC mobilization with a single dose of pegfilgrastim is feasible for healthy donors. The graft composition was comparable to that obtained with the conventional regimen of short-term G-CSF. Long-term follow-up of healthy donors treated with pegfilgrastim should be further investigated. Disclosures: No relevant conflicts of interest to declare.

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Ex-Vivo Expanded Peripheral Blood Stem Cells (EVEC) Compared with Un Manipulated Peripheral Blood Stem Cells (PBSC) Autologous Transplantation for Multiple Myeloma: A Pair Match Analysis.

Blood ◽

10.1182/blood.v114.22.502.502 ◽

2009 ◽

Vol 114 (22) ◽

pp. 502-502 ◽

Cited By ~ 3

Author(s):

Noel-Jean Milpied ◽

Gerald Marit ◽

Bernard Dazey ◽

Jean-Michel Boiron ◽

Zoran Ivanovic ◽

...

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Peripheral Blood ◽

Ex Vivo ◽

High Dose ◽

Peripheral Blood Stem Cells ◽

Cd34 Cells ◽

Blood Stem Cells

Abstract Abstract 502 Autologous stem cell transplantation with PBSC after high-dose chemotherapy remains standard therapy for patients with symptomatic Multiple Myeloma (MM). Strategies to minimize complications could significantly reduce the morbidity of that procedure. One possibility could be to shorten the duration of induced neutropenia through the injection of an ex-vivo expanded graft. Nineteen patients (pts) received EVEC after high-dose Melphalan (HDM) (200 mg/m2) as the only graft. The ex-vivo expanded procedure has been described elsewhere (Boiron et al. Transfusion 2006 and Ivanovic et al. Transfusion 2006). Briefly, thawed peripheral blood CD 34+ cells collected after G-CSF mobilisation and selected with immunomagnetic devices were incubated for 10 days in a serum free medium (Maco Biotech HP01) with Stem Cell Factor (Amgen), G-CSF (Amgen) and TPO (Amgen: 7 pts; Cellgenix:12 pts). The expanded cells were then thoroughly washed and injected 48h after the HDM injection. The ex-vivo expansion lead to a median fold of 5,4 for CD34+ cells (1,3-11,8); 118 for CD33+ (1-703880); 3386 for CD14+ (4-101075); 28,5 for CD13+ (10-703880) and 13 for CFUs (6-21). The median N° of CD34+ cells injected was 14×10e6/kg (5,3-48). The results of these transplants were compared to those achieved in 38 pts who received unmanipulated PBSC after HDM. Pts and controls were matched for age, sex, stage of the disease, first line chemotherapy ( VAD or VD) status of the disease at time of transplant, year of transplant, time between diagnosis and transplant, CD34+ mobilisation technique (HD cytoxan + G-CSF or G-CSF alone) and the median N° of total nucleated cells and of CD34+ collected. The results are summarized on the table: There was no secondary neutropenia in the patients who received EVEC. With a median FU of the entire cohort of 30 m, the median OS for pts who received their first transplant with EVEC and with PBSC is 69 m and not reached respectively (p=NS), the median PFS is 18 m and 27 m (p = NS) and the median time to progression is 14 m and 15 m (p=NS). Conclusion: EVEC is feasible, safe and reduce significantly the morbidity of autologous stem cell transplantation after HDM for multiple myeloma. Disclosures: Milpied: Amgen France: Honoraria.

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Seizures During Infusion of Autologous Peripheral Blood Stem Cells Are Related to High White Blood Cell Concentration in the Peripheral Blood Stem Cell Product and Can Be Prevented by Dilution of Product with Autologous Plasma

Blood ◽

10.1182/blood.v118.21.4386.4386 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4386-4386

Author(s):

Carlos Bachier ◽

Grant Potter ◽

Joshua Potter ◽

Charles F. LeMaistre ◽

Paul Shaughnessy ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Blood Cell ◽

White Blood Cell ◽

Peripheral Blood ◽

Cell Transplant ◽

Autologous Plasma ◽

Peripheral Blood Stem Cells ◽

Cd34 Cells ◽

Blood Stem Cells

Abstract Abstract 4386 Seizures are rare during infusion of autologous peripheral blood stem cells (PBSC). We retrospectively analyzed 159 adult patients (pts.) collected consecutively between January 2006 and July 2009. Pts. were collected on either COBE Spectra (COBE) (n=85) or Fresenius AS 104 (Fresenius) (n=74) cell separators and mobilized with granulocyte colony stimulating factor (G-CSF) alone (n=47), G-CSF and Plerixafor (n=26), or G-CSF and chemotherapy (n=66). Pts. characteristics did not differ between the COBE and Fresenius cohorts, but there were differences in PBSC product (Table). Pts. collected with COBE had higher white blood cell (WBC) and total nucleated count (TNC) but lower mononuclear cell (MNC) percentage and cell viability than pts. collected with the Fresenius. Absolute CD34+ cells in the PBSC product, CD34+ cells / kg and total CD34+ cells / kg infused at transplant were not significantly different. CD34+ yields (calculated as the ratio of CD34+ cells /μl of the PBSC product to the patient's peripheral blood CD34+ cells / μl taken on the day of collection) were significantly higher on the COBE than Fresenius. No serious adverse events occurred during PBSC infusion except 3 of 159 pts. developed seizures during infusion of PBSC; all collected on the COBE and all three had product WBC > 590 × 103/μl (compared to a median of 163.3 × 103/ μl for all other products)(Figure). Evaluation of pts. did not identify abnormalities in imaging studies, cerebrospinal fluid analysis, electrolytes, or past history which might explain etiology of seizures. No significant difference in WBC or platelet engraftment was observed in pts. collected with COBE or Fresenius. We then prospectively correlated WBC counts midway and at the end of PBSC collections. Fourteen pts. had 15 apheresis using the Fresenius. Mid- and post-WBC concentrations were 64 +/− 23 × 103/μl and 69 +/− 20 × 103/μl, respectively. Fifty-one pts. had 66 apheresis using COBE, with WBC counts obtained midway and at the end of collection of 287 +/− 150 × 103/μl and 273 +/− 144 × 103/μl, respectively. Mid-WBC accurately correlated with WBC at the end of the collection in both the COBE and Fresenius cohorts (r2 = 0.940 and r2 = 0.904, respectively). Using this information, we prospectively evaluated 65 pts. who underwent 80 PBSC collections in anticipation of an autologous (n=44) or allogeneic (n=7) stem cell transplant between June 2009 and January 2010. Collections for these pts. were performed using the COBE (n=66) or the Fresenius (n=15). Mid-WBC were obtained and products with mid-collection WBC concentration > 450 × 103/uL (n=29) had additional autologous plasma collected at the time of collection for final product dilution to < 450 × 103/uL prior to cryopreservation. Pts weight, volume of PBSC product and CD34+ cells/kg infused did not differ between the pts who received diluted PBSC product and those who did not. There were also no differences in either ANC (12 ± 1.3 days vs. 11.5 ± 1.3 days, dilution vs. non-dilution, p = 0.760) or in platelet engraftment (18 ± 3.7 days vs. 16 ± 2.7 days, dilution vs. non-dilution, p = 0.561). No serious adverse infusion effects were observed in either group. In conclusion, high number of WBC in COBE collections is a possible cause of PBSC infusion related seizures. No seizures were observed after dilution of PBSC with high WBC concentration.TIENT AND PRODUCT CHARACTERISTICSCOBE (±SD)Fresenius (±SD)Number of Products165180Number of Patients8574Age at collection56 ± 1456 ± 15Weight at Collection (kg)82.7 ± 17.979.5 ± 15.9Collections / Patient2 ± 12 ± 1Blood Volume Processed at end of Collection (L)18.0 ± 2.418.1 ± 2.7(*)Product Volume (ml)241 ± 56.8402 ± 72.0Peripheral WBC (103/ μl)36.6 ± 18.933.3 ± 24.5(*)Product WBC(103/ μl)163.3 ± 136.055.8 ± 29.3(*)TNC (1010)3.51 ± 1.861.95 ± 1.19(*)MNC (1010)2.36 ± 1.191.60 ± 0.09(*)MNC (%)75.0 ± 23.385.0 ± 10.8Volume prior to freezing(ml)100 ± 54100 ± 32(*)Post Freeze Viability (%)70 ± 1475 ± 10Peripheral CD34+/ μl24.0 ± 43.825.3 ± 79.1(*)Product CD34+/μl726.7 ± 1325.9264.63 ± 781.0(*)Product / Peripheral CD34+24.87 ± 10.9010.91 ± 6.64Absolute Product CD34+ cells (108)1.77 ± 3.521.14 ± 3.35Product CD34+/kg (106)2.02 ± 4.671.39 ± 4.15Total CD34+ cells infused (106 / kg)3.85 ± 3.203.85 ± 2.24(*) = p values < 0.05 Disclosures: No relevant conflicts of interest to declare.

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Collection of Peripheral Blood Stem Cells in 118 Small Children Weighing 20 Kg or less: Experience of a Single Centre

Blood ◽

10.1182/blood.v128.22.3378.3378 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3378-3378

Author(s):

Jianyun Wen ◽

Yuelin He ◽

Libai Chen ◽

Jing Du ◽

Zhiyong Peng ◽

...

Keyword(s):

Stem Cells ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Thalassemia Major ◽

Median Number ◽

Peripheral Blood Stem Cells ◽

Hematopoietic Stem ◽

Small Children ◽

Cd34 Cells ◽

Blood Stem Cells

Abstract Background: Peripheral blood stem cells (PBSC) are increasingly used as a source of stem cells for either autologous or allogeneic hematopoietic transplantation in children.Although technically similar to adult procedures, PBSC harvest may be difficult in young children, especially in the very small children. Aim: In this study, we aimed to evaluatethe safety and efficacy of harvesting peripheral blood hematopoietic stem cells in very small children,and to provide a guideline. Methods: Between Jan 2013 to Mar 2016, we evaluated 118 children weighing 20 kg or less, with the smallest patient weighing 11 kg. The patients had a median age of 59 months and included 72 children with thalassemia major and 46 young donors. The granulocyte-colony stimulating factor (G-CSF) analogs were used at a dose of 10 mg/kg/day administered subcutaneously once a day and receiving oral calcium for five days before harvesting. Blood was withdrawn at a mean rate of 30-40 ml/min through a temporaryfemoral vein catheter (12 or 14 guage) to ensure adequate blood flow and returned through a larger catheter in a peripheral vein.Total nucleated cells(TNC) and CD34+ cells were estimated in the peripheral blood before collection of the apheresis product. Results: We collected sufficient products from all the children with one to three apheresis procedures. No serious complication was detected in all children and all aphereses were completed within 4 hours.The volume of blood per kilogram processed for each apheresis ranged from 55 to 160ml (median, 85ml). The median number of TNC and CD34+ cells collected were 12×108/kg and 15×106/kg per apheresis, respectively. Conclusions:We conclude that collection of PBSC is a safe and practical procedure in children, even in very small children. Disclosures No relevant conflicts of interest to declare.

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Mobilization and collection of peripheral blood CD34+ cells from patients with Fanconi anemia

Blood ◽

10.1182/blood.v98.10.2917 ◽

2001 ◽

Vol 98 (10) ◽

pp. 2917-2921 ◽

Cited By ~ 37

Author(s):

James M. Croop ◽

Ryan Cooper ◽

Christine Fernandez ◽

Vicki Graves ◽

Susan Kreissman ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Fanconi Anemia ◽

Peripheral Blood ◽

Bone Marrow Failure ◽

Peripheral Blood Stem Cells ◽

Target Weight ◽

Peripheral Blood Cd34 ◽

Cd34 Cells ◽

Blood Stem Cells

Abstract A potential therapeutic option for patients with Fanconi anemia is collection of peripheral blood stem cells prior to the development of severe pancytopenia. These hematopoietic cells potentially could be infused when symptomatic bone marrow failure develops, as autologous rescue after chemotherapy in the event of leukemic transformation, or as targets for gene therapy. Eight patients with Fanconi anemia were mobilized with 10 μg/kg per day of granulocyte colony-stimulating factor (median, 10 ± 4 days) to determine the feasibility of collecting peripheral blood stem cells for future use. Six patients achieved a peripheral blood CD34+ count of ≥ 6/μL and underwent apheresis. The collection goal was 2 × 106 CD34+ cells/kg based on a predicted weight 5 years from the date of collection. A mean of 2.6 ± 0.9 × 106 CD34+ cells/kg of the weight at the time of collection were collected, which corresponded to 1.9 ± 0.4 × 106 CD34+cells/kg of the target weight. The collections required a mean of 4 ± 3 days (range, 2-8 days) of apheresis. Six of the 8 subjects had ≥ 1 × 106 CD34+ cells/kg cryopreserved based on both actual and target weights, and 4 subjects had ≥ 2 × 106 CD34+ cells/kg cryopreserved based on the target weight. These results suggest that some patients with Fanconi anemia can have adequate numbers of CD34+ cells mobilized and collected from the peripheral blood prior to the onset of severe bone marrow failure, but they may require an extended mobilization and multiple days of collection.

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Subsets of CD34+ and Engraftment Kinetics in Allogeneic Peripheral Stem Cell Transplantation.

Blood ◽

10.1182/blood.v108.11.2954.2954 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2954-2954

Author(s):

Domenico Pastore ◽

Anna Mestice ◽

Paola Carluccio ◽

Tommasina Perrone ◽

Manuela Leo ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Peripheral Blood ◽

Conditioning Regimen ◽

The Other ◽

Peripheral Blood Stem Cells ◽

Gvhd Prophylaxis ◽

Cd34 Cells

Abstract Engraftment kinetics in allogeneic peripheral blood stem cell transplantation (alloPBSCT) depend on the number and efficiency of the stem cells in the graft, the conditioning regimen and GvHD prophylaxis. Currently, stem cell evaluation is performed by counting CD34+ cells; however, CD34+ cells are a heterogeneous population including the early uncommitted fraction as well as different subsets committed to one or the other lineage; hence, defining the CD34+ subset most predictive of engraftment and its threshold value would be of the utmost importance. This study aimed to identify which graft product subset of CD34+ cells might be the most predictive of early hematopoietic recovery following alloPBSCT. The relationships between the number of “mature” subsets of CD34+ cells (CD34+/CD33+, CD34+/CD38+, CD34+/DR+ and CD34+/CD133) and “immature” subsets of CD34+ cells (CD34+/CD33−, CD34+/CD38−, CD34+/DR− and CD34+/CD133+) and early neutrophil and platelet engraftment were studied in a homogeneous series (for disease, pre-transplant chemotherapy, conditioning regimen GvHD prophylaxis) of 30 acute myeloid leukemia (AML) patients after alloPBSCT from HLA-identical siblings. All patients received the BU-CY regimen consisting of busulfan 4 mg/kg/day for 4 consecutive days followed by cyclophosphamide 60 mg/kg/day for 2 consecutive days; GvHD prophylaxis included cyclosporin and methotrexate. The CD34+ dose infused ranged from 2.9 to 8.8 × 106/Kg (median 4.6); the percentage of immature CD34+ cells was 36% for CD34+/CD33−, 60% for CD34+/CD38−, 5% for CD34+/DR− and 70% for CD34+/CD133+; this translates into a median dose of 1.6 × 106/Kg (range 0.3–5) for CD34+/CD33−, 2.6 × 106/Kg (range 0.1–6.2) for CD34+/CD38−, 0.4 × 106/Kg (range 0.1–2.3) for CD34+/DR− and 0.95 ×106/Kg (range 0.6–2.3) × 106/Kg for CD34+/CD133+. Median time to achieve engraftment of neutrophils and platelets was 13 days (range 10–16) and 15 days (range 13–19), respectively. In our experience the total CD34+/CD133+ cell number was inversely correlated with the days required for recovery of 0.5 × 109/L neutrophils (r = −0.76, p<0.05) and 100 × 109/L platelets (r = −0.71, p<0.05); this correlation was better than the total CD34+ cells dose and neutrophil (r = −0.71, p<0.05) and platelets engraftment (r = −0.68, p = 0.06). No correlation was found between the other CD34+ subsets and neutrophil and platelets engraftment. With regard to the threshold dose for early neutrophil engraftment, all 14 patients who received more than 1 × 106/Kg of CD34+/CD133+ had a neutrophil count higher than 1.0 × 109/L at 12 days. We suggest that a high number of CD34+/CD133+ peripheral blood stem cells may be associated with faster neutrophils and platelets recovery; these findings may help to predict the repopulating capacity of PBSC in patients after allogeneic PBSCT, especially when a relatively low number of CD34+ cells is infused.

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Outpatient Hematopoietic Grafting in Patients with Multiple Sclerosis Employing Autologous Non-Cryopreserved Peripheral Blood Stem Cells: A Feasibility Study

Blood ◽

10.1182/blood.v126.23.5489.5489 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5489-5489

Author(s):

Guillermo J. Ruiz-Arguelles ◽

Andrés León-Peña ◽

Emilio Medina-Ceballos ◽

Alejandro Ruiz-Arguelles ◽

Manuel A Ruiz-Delgado ◽

...

Keyword(s):

Multiple Sclerosis ◽

Stem Cells ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

High Dose ◽

Outpatient Basis ◽

Peripheral Blood Stem Cells ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Blood Stem Cells

Abstract Background: Multiple sclerosis (MS) is a chronic, inflammatory, debilitating disease that causes destruction of central nervous system (CNS) myelin, with varying degrees of axonal damage. With the goalofd re-setting the immune system, autologous hematopoietic stem cell transplantations (HSCT) have been done in patients with MS since 1996 and more than 700 HSCTs have been performed around the world. The risk of transplant related mortality in HSCT for MS has declined over the past years. Material and methods: Consecutive patients with MS were autografted in a single center using: Hematopoietic stem cells (HSC) were mobilized with cyclophosphamide (Cy), 3 gr/m2 and G-CSF, the procedure was conducted on outpatient basis employing peripheral blood non-frozen HSC and conditioning with high-dose Cy (100 mg/Kg) and post-transplant G-CSF and rituximab. Antibiotics, antimycotics and antivirals were given orally. Results: Thirteen patients with MS were prospectively accrued in the study. There were 7 females and 6 males. Median age was 48 years, range 24 to 65. The expanded disability status scale (EDSS) score of these patients had a median of 5 points (range 1 to 6). All the autografts were started on an outpatient basis and two persons were admitted to the hospital during the procedure (persistent nausea/vomiting and neutropenic fever); they stayed in the hospital for 48 hours. In order to obtain a minimum of 1 x106 viable CD34+ cells/Kg, one to four apheresis were done (median 1). The total number of viable CD34+ cells infused to the patients ranged between 1 and 9.6x106 (median 3.1). Patients recovered above 0.5 x109/L absolute granulocytes on median day 9 (range 6 to 12). No individuals needed transfusions of red blood cells nor platelets transfusions. There were no transplant-related deaths and the 23-month overall survival of the autografted patients is 100%. Median cost of the procedure was 30 000 USD. In 8 persons the EDSS was assessed three months after the graft; it diminished from a median of 4.5 to a median of 2.5. In 5 patients, the three months re-assessment of the EDSS has not been possible as a result of the time elapsed after the autograft. Discussion: These data indicate that it is possible to conduct autotrasplants for patients with MS employing a simplification of the conventional procedures by means of non-frozen peripheral blood stem cells and outpatient conduction. Additional information is needed to asses the efficacy of these procedures in the treatment of patients with MS. Disclosures No relevant conflicts of interest to declare.

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