Intra-Bone Marrow Transplantation of CD34+ Enriched Grafts Following a Non-Myeloablative Conditioning Allows Successful Engraftment In DLA-Identical Canine Litters.

Abstract Abstract 3711 Introduction: Successful engraftment following transplantation of hematopoietic stem cells (HSCT) depends mainly on pre- and posttransplant immunosuppression, graft type and composition as well as on the HSC numbers infused. Whereas some of the aforementioned parameters can be influenced in the clinical setting, the latter one is more difficult to address. HSCTs of grafts with limited HSC numbers are accompanied by increased graft failure rates, longer cytopenias and increased morbidity. Current concepts to overcome low HSC numbers include the combination of two unrelated grafts, expansion techniques, modification of the graft composition or the site of graft infusion. In preliminary rodent studies intra-bone marrow (IBM) compared to intravenous (IV) HSCT led to faster engraftment which might be explained by closer location of the HSC to the stem cell niches. Aims: To investigate the feasibility and efficiency of IBM-HSCT following a non-myeloablative conditioning regimen in a dog-leukocyte antigen (DLA) identical canine HSCT model. Method: DLA-identical siblings were used as donor/recipient pairs for HSCTs. Recipients received a single dose of 2 Gy total body irradiation before HSCT (day 0). Pre- and postgrafting immunosuppression consisted of CSA (d-1 to d+35) and MMF (d0 to d+27). Two IBM-HSCT cohorts were investigated and data compared to IV controls (CON). BM-grafts of the respective donors were infused unmodified IV (CON, n=9) or IBM after HSC enrichment using a buffy coat followed by ficoll density centrifugation (IBM-I, n=6; 5ml total volume) or IBM after HSC enrichment using buffy coat centrifugation only (IBM-II, n=6; 25 ml total volume). In the CON group the graft was infused in the cubital vein. In the IBM-groups the grafts were infused through a BM aspiration needle into the BM of the left humerus and femur over a period of 5 minutes. In 4 IBM animals graft migration analyses were performed using technecium99 marking. Chimerism and BM cellularity were determined at injection and opposite sides. Analyses of chimerism were performed via polymorphic nucleotide repeat analyses weekly. BM cellularity was determined biweekly. Complete blood count was performed daily. Result: Infusion of grafts directly into the BM was feasible: both volumes (5ml, 25ml) could be infused without any leakage at the injection sites. Tc99-marked BM cells stayed predominately at the injection site for the first 24 hours. All animals engrafted. Mean TNC numbers infused were 2.6 ×108/kg (range: 1.6–11.4; CON), 1.6 ×108/kg (range: 1–2.4; IBM-I), 3.7 ×108/kg (range: 2.1–5.8; IBM-II) (IBM-I vs CON: p=0.08, IBM-II vs CON: p=0.9, IBM-I vs II: p<0.02,). Mean CD34+ numbers infused were 0.6 ×106/kg (range: 0.3–2.2; CON), 2.5 ×106/kg (range: 0.3–6.4; IBM-I), 4.3 × 106/kg (range: 1.3–6.5; IBM-II) (IBM-I vs CON: p=0.06, IBM-II vs CON: p=0.01, IBM-I vs II: p=0.4). PBMC chimerisms at d+14, d+28 and d+56 were 25% (range: 8–46), 36% (range: 5–63), 28% (range: 4–54) (CON), 12% (range: 6–18), 23% (range: 14–39), 18% (range: 6–64) (IBM-I) and 40% (range: 23–60), 61% (range: 31–84), 45% (range: 25–70) (IBM-II) (IBM-I vs CON: p=0.03, p=0.05, p=0.1, IBM-II vs CON: p=n.s. (all), IBM-I vs II: p=0.06, p=0.04, p=0.1). Recovery of hematopoiesis occurred significantly slower in both IBM-BMT groups compared to CON (p<0.002): mean leukopenia (<1.0 gpt/l) durations were 4 days (range: 0–15; IBM-I), 0.5 days (range: 0–2, IBM-II) versus 0 days (CON); mean thrombocytopenia durations (<50Gpt/l) were 19 days (range: 2–49, IBM-I), 10 days (range: 8–16, IBM-II) versus 4 days (range: 0–6, CON). However, if only grafts with <2.0 ×106 CD34+/kg were analysed differences concerning leukocytes recovery times diminished between IBM and the IV groups, whereas they persisted in regards to thrombocytopenia. Analyses of cellularity and chimerism within a HSCT recipient (injection vs opposite site) revealed higher initial cellularities and significantly higher BM donor chimerisms up to day +56, both in favour of the injection site (p<0.03). Conclusion: Infusion of HSC grafts up to volumes of 25ml directly into the BM is feasible and allows successful donor engraftment following non-myeloablative conditioning. Duration of cytopenias following IBM-HSCT is still significant, perhaps due to the loss of precursor cells during graft preparation. Further studies are warranted to determine optimal graft preparation and IBM application techniques. Disclosures: No relevant conflicts of interest to declare.

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Antibody-Based Depletion of Hematopoietic Stem Cells Empties Niches for Efficient Transplantation.

Blood ◽

10.1182/blood.v110.11.lb2.lb2 ◽

2007 ◽

Vol 110 (11) ◽

pp. LB2-LB2

Author(s):

Agnieszka Czechowicz ◽

Daniel L. Kraft ◽

Deepta Bhattacharya ◽

Irving L. Weissman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Conditioning Regimen ◽

Myeloablative Conditioning ◽

Hematopoietic Stem ◽

Exact Function ◽

Potential Applications ◽

Conditioning Regimens

Abstract Hematopoietic stem cells (HSCs) are used therapeutically in bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) to correct hematolymphoid abnormalities. Upon intravenous transplantation, HSCs can home to specialized bone marrow niches, self-renew and differentiate and thus generate a new, complete hematolymphoid system. Unfortunately BMT has had limited applications, due to the risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy, that are deemed necessary for HSC engraftment. Elimination of these toxic conditioning regimens could expand the potential applications of BMT to include many non-malignant hematologic disorders, a wide variety of autoimmune disorders such as diabetes and multiple sclerosis, as well as in the facilitation of organ transplantation. The exact function of these traditional myeloablative conditioning regimens is not clear. To elucidate the barriers of HSC engraftment, we transplanted 50–1000 purified HSCs (Ckit+Lin−Sca1+CD34+CD150−) into immunodeficient, Rag2−/− or Rag2−/−gc−/− recipient mice and show that HSC engraftment levels rarely exceed 0.5% following transplantation without toxic conditioning, indicating that the immune system is not the only barrier to engraftment. Additionally, we did not observe a significant increase in HSC engraftment when HSC doses of >250 cells were transplanted. Even when up to 18000 HSC were transplanted, we did not see a linear increase in HSC engraftment, indicating that the increased doses of HSCs transplant inefficiently. We believe this is due to the naturally low frequency of available HSC niches, which we postulate may result from the physiologic migration of HSCs into circulation. Conversely, separation of the graft into small fractions and the subsequent time-delayed transplantation of these doses did result in increased engraftment due to the natural physiologic creation of new available HSC niches. When 1800 HSC were transplanted daily for seven days, the engraftment was 6.1-fold higher than transplantation of 12800 HSC in a single bolus. Here, we provide evidence that, aside from immune barriers, donor HSC engraftment is restricted by occupancy of appropriate niches by host HSCs. Through elimination of host HSCs we are able to increase available HSC niches for engraftment. We have developed a novel system where HSCs can be eliminated by targeting C-kit, a cell surface antigen that is highly expressed on the surface of HSCs. Cultivation of HSCs with ACK2, a depleting antibody specific for c-kit, prevented stem-cell factor (SCF) dependent HSC proliferation in vitro and resulted in cell death. Administration of ACK2 to mice led to the rapid and transient removal of >98% of endogenous HSCs in vivo thus resulting in equal numbers of available niches for engraftment. Following ACK2 clearance from serum, transplantation of these animals with donor HSCs led to chimerism levels of up to 90%, representing a 180-fold increase as compared to unconditioned animals. This non-myeloablative conditioning regimen had few side effects, other than temporary loss of coat color. The HSCs in even untransplanted animals rapidly recovered and animals remained healthy and fertile. This work redefines the way we approach BMT/HSCT, and places great emphasis on the necessity to create available HSC niches prior to transplantation. Extrapolation of these methods to humans may enable efficient yet mild conditioning regimens for transplantation, thus expanding the potential applications of BMT/HSCT.

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Impact of Infusion Rate on the Early Engraftment Following Allogeneic Intra Bone Marrow Infusion of Hematopoietic Stem Cells in a Canine DLA-Identical Reduced Intensity Transplantation Model

Blood ◽

10.1182/blood.v126.23.5406.5406 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5406-5406

Author(s):

Stephanie Schaefer ◽

Juliane Werner ◽

Sandra Lange ◽

Katja Neumann ◽

Christoph Machka ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Infusion Rate ◽

Conflicts Of Interest ◽

Conditioning Regimen ◽

Hematopoietic Stem ◽

Time Points ◽

The Impact ◽

Reduced Intensity

Abstract Introduction: Direct intra bonemarrow (IBM) infusion of hematopoietic stem cells (HSC) is assumed to improve the homing efficiency and to accelerate the early engraftment in comparison to the conventional intravenous application of HSC. Especially for transplantation of low cell numbers i.e. "weak grafts" that is generally associated with delayed engraftment. The direct infusion of HSC in close proximity to the HSC niche by intra bone marrow transplantation (IBMT) might be a promising way. Whether the HSC infusion rate might influence the homing process and therefore the outcome after IBMT is so far unknown. Aims: Herein, we analyzed in a canine DLA-identical littermate model the impact of different graft infusion rates on the hematopoietic recovery as well as on the engraftment kinetics after IBMT following reduced intensity conditioning. Methods: Recipient dogs received IBMT following a 4.5 Gy total body irradiation (TBI). From day (d) -1 until d+35 Cyclosporin A (15mg/kg) was administered orally twice a day as immunosuppression. For IBM transfusion the graft volume was reduced by buffy coat centrifugation and dogs obtained 2x25 ml simultaneously into the humerus and femur. The infusion rate of the graft was 25ml/10 min in group 1 (IBM10, n = 8) and 25 ml/60 min in group 2 (IBM60, n = 7). A 28 day follow-up is currently available for twelve dogs (IBM10 n = 7; IBM60 n = 5). The development of the peripheral blood mononuclear cell (PBMC) and granulocyte chimerism was tested weekly. Blood count, kidney and liver enzymes were monitored routinely. Results: All animals engrafted. One dog of the IBM10 group died at d+15 (infection) and was therefore not included into analysis. The median number of infused total nucleated cells were in IBM10 4.1*108/kg (range 2.3-6.0*108/kg) and in IBM60 3.2*108/kg (range 1.8-4.4*108/kg; p=0.4). The infused CD34+ numbers were median 3.2*106/kg (range: 1.2-10.0*106/kg; IBM10) and 3.6*106/kg (range: 1.5-6.8*106/kg; IBM60; p=0.7). Time of leukocyte recovery was median d+11 after IBMT in both groups (range: d+4 to d+11, IBM10; d+8 to d+14, IBM60; p= 0.5). Median leukocytes nadirs amounted to 0.2*109/l for IBM10 and 0.3*109/l for IBM60 (p= 0.08). The median duration of leukopenia (<1*109/l) were similar (6d, range: 4-11d, IBM10; 3-9d, IBM60) (p= 0.6). Median platelet nadir was 0*109/l for both cohorts (range: 0.0-7.0*109/l, IBM10; 0.0-1.0*109/l, IBM60). The period of thrombocytopenia (≤20.0*109/l) was significantly prolonged in the IBM60 group (median 10d, range) compared to 5d (range: 3-12d) in the IBM10 group (p=0.05). Donor PBMC chimerisms at d+7, d+14 and d+28 were median 22% (range: 8-34%), 50% (range: 29-53%) and 67% (range: 47-73%) in IBM10. The results of PBMC chimerism for IBM60 were 11% (range: 5-34%), 42% (range: 20-42%) and 59% (range: 44-66%) at these time points (p = n.s.). Donor granulocyte chimerisms of median 33% (range: 11-83%), 100% (range: 58-100%) and 100% (range: 82-100%) were detected at d+7, d+14 and d+28 after HSCT in IBM10, respectively. The granulocyte chimerism in IBM60 amounted to 34% (range: 3-87%), 96% (range: 94-100%) and 98% (range: 96-100%) at the above mentioned time points p=n.s. for all time points). Conclusion: Our data suggest that early granulocyte and PBMC engraftment is not influenced by modification of the HSC infusion rate. However, the period of thrombocytopenia seems to be prolonged following a 60 minutes application. Therefore, longer infusion times in an IBMT setting seem not to be beneficial following toxicity reduced conditioning regimen. Disclosures No relevant conflicts of interest to declare.

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Impact of Cytopenia Following Unmanipulated Haploidentical Hematopoietic Stem Cell Transplantation Using Post-Transplant Cyclophoshamide

Blood ◽

10.1182/blood.v128.22.2281.2281 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2281-2281

Author(s):

Villetard Ferdinand ◽

Stefania Bramanti ◽

Samia Harbi ◽

Sabine Fürst ◽

Catherine Faucher ◽

...

Keyword(s):

Bone Marrow ◽

Cell Transplantation ◽

Disease Risk ◽

Conflicts Of Interest ◽

Risk Index ◽

Conditioning Regimen ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Cell Dose ◽

The Impact

Abstract Introduction Allogeneic transplantation from a haploidentical donor (HaploSCT) is an alternative strategy in the treatment of hematologic malignancies in absence of HLA-identical donor. Recent studies reported similar outcome after HaploSCT compared to HLA-identical transplantation in different settings (Bashey, JCO 2013; Wang, Blood 2015; Gosh, JCO 2016). Although survivals seemed promising after HaploSCT, hematopoietic recovery following such a mismatched transplantation could represent a limitation. Thus, our series aims to evaluate hematological recovery after HaploSCT using a post transplantation cyclophosphamide (PT-Cy) platform. Methods This retrospective monocentric study included consecutive patients with following criteria: adults with hematological malignancies; bone marrow or peripheral blood T-replete HaploSCT from 2011 to 2015; non-myeloablative (Baltimore approach) or reduced intensity conditioning (busulfan-based) regimen; PT-Cy as part of GVHD prophylaxis. Patients with primary graft failure were excluded. Absolute neutrophil count (ANC), red cells (RCT) or platelet transfusion (PT) requirements on day 30 (D30) and day 100 (D100) were analyzed among disease-free patients. We first separately evaluated the rate of patients with significant cytopenia in each lineage (defined by ANC < 1 G/L, RCT need, PT need) and searched for impact of pre-transplantation factors on cytopenia (multivariate analyses by binary logistic regression). Then, we evaluated outcome by D30- and D100-landmark analyses according to cytopenia. Results One hundred and forty six patients with a median age of 56 years (range: 19-73) were analyzed: 142 and 117 were evaluable at D30 (4 early deaths) and D100 (17 deaths, 11 relapses), respectively. At D30, 20% of patients had ANC<1G/L, 67% needed RCT and 63% needed PT. Corresponding values at D100 were 20%, 42% and 28%, respectively (Figure 1). At D30: the use of PBSC (HR 9.5, p=0.002) was significantly associated with ANC>1G/L at D30; the use of NMAC Baltimore schema (HR 0.3, p=0.012) and CD34+ cell dose > median (HR 0.4, p=0.041) decreased PT needs while hematopoietic cell transplantation comorbidity index (HCT-CI)≥3 (HR 3.3, p=0.004) was associated with PT needs; no factor was found to significantly influence RCT. At D100: Age>60 years (HR 2.4, p=0.045), female to male HaploSCT (HR 3.3, p=0.020) and HCT-CI≥3 (HR 3.7, p=0.006) were significantly associated with higher risk of RCT need; female to male HaploSCT (HR 3.6, p=0.015) and HCT-CI≥3 (HR 6.9, p=0.001) were associated with PT needs; no factor was found to significantly influence ANC. With a median follow up of 25 months (range: 5-55), cox multivariate model with adjustment by age (continuous), disease risk index (low/intermediate vs high/very high), HCT-CI (0-2 vs ≥3), conditioning regimen (baltimore vs. busulfan-based) and graft source (bone marrow vs PBSC) showed that ANC<1 G/L was strongly associated with higher NRM (HR 2.9, p=0.011) and shorter OS (HR 3.4, p<0.001), overcoming the impact of RCT and PT needs (Figure 2A and 2B). In contrast, D100 analysis showed that PT need was the most determinant factor of increased NRM (HR 13.7, p=0.013) and poor OS (HR 7.3, p=0.003), while both D100 ANC and RCT needs did not impact outcome (Figure 2C and 2D). Discussion We found that cytopenia remain a concern after HaploSCT, leading to increased NRM and OS. The absence of ANC>1G/L at D30 as well as the need of PT at D100 may be considered as a strong post transplantation factor predicting poor outcome. Some pre-transplantation factors of cytopenia have been identified, such as CD34+ cell dose, sex mismatch and graft source. Among them, some may help for donor selection while the optimal donor for HaploSCT is still unknown. Moreover, better neutrophil recovery at D30 is achieved with the use of PBSC. CD34+ optimal cell dose in this setting remains also to be determined. In addition, post transplantation events such GVHD and/or infections should be evaluate to explore their interactions with such cytopenia, aiming to develop early therapeutic interventions. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Eltrombopag Combined with G-CSF and Cyclosporine Could Effect for Severe Acquired Aplastic Anemia

Blood ◽

10.1182/blood-2018-99-115779 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5110-5110 ◽

Cited By ~ 1

Author(s):

Bingyi Wu ◽

Jiahui Cai ◽

Li Yingshi ◽

Dong Ruihong

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Median Time ◽

Antithymocyte Globulin ◽

Complete Blood Count ◽

Conflicts Of Interest ◽

Fungal Infections ◽

Young Patients ◽

Severe Adverse Effect ◽

Hematopoietic Stem

Abstract Backgroud Acquired aplastic anemia (AA) is a potential life-threatening hematopoietic stem cell (HSC) disorder resulting in cytopenia. The first line therapy for AA is HSC transplantation for young patients who have suitable donors and immunosuppressive therapy (IST) with antithymocyte globulin and cyclosporine for the remaining patients. However, about 30% of patients are refractory to IST or relapse after IST. IST with antithymocyte globulin and cyclosporine result in severe complication and mortality infection. To reduce the mortality infection and increase the response of IST for AA is still problem. Eltrombopag, a thrombopoietin mimetic, demonstrated efficacy in restoring trilineage hematopoiesis, has recently emerged as an encouraging and promising agent for patients with refractory AA. To explore the effect of eltrombopag for severe acquired aplastic anemia, we treated seven severe AA patients with eltrombopag combined with cyclosporine and G-CSF. Herein we report initial results of the eltrombopag combined with cyclosporine and G-CSF for severe AA. Methods The diagnostic of AA patient consisted of a complete blood count, a bone marrow biopsy, bone marrow karyotype analysis and assessment of a paroxysmal nocturnal hemoglobinuria (PNH) clone. Patients with SAA aged ≥18 years old who without suitable donors received eltrombopag 75mg/d, cyclosporine 6mg/kg by oral, and G-CSF 300ug/d by subcutaneous injection from diagnosis. Red blood was infused to maintained HB more than 60g/L. Platelet were infused to maintained PLT more than 20x109/L. G-CSF was administered until neutrophil count more than 1.0x109/L. Vale concentration of cyclosporine were maintained more than 100ug/ml in blood plasm and maintained two years. Eltrombopag was taper down when platelet was more than 100x109/L. Eltrombopag was given at least three months. Antibacterial was administered when patient was high fever. Posaconazole were given for fungal infections prophylaxsis. Hematologic improvements were assessed by the National Institutes of Health (NIH) response criteria for AA. Results The median age of 7 patients with SAA was 44 years old (range 19-68 yr). Full hematologic improvements were achieved in 3 patients. All patients achieved platelet and RBC infusion independence. The median time from the first eltrombopag therapy to platelet infusion independence was 35 days (range 33-46d). The median time from the first eltrombopag therapy to RBC infusion independence was 40 days (range 30-50d). Median 6 units (1200ml) (range 3-10U, 600ml -2000ml) RBC and 7 units (2.5x109/unit) platelet were infused. With median 8 months follow-up (3-12 months),3 patients are still full hematologic improvements and 4 platelet and RBC infusion independence. No severe fugual infection was observed in this group patients. ALT slightly elevate in one patient. No other severe adverse effect was observed. Conclusions Treatment of SAA patients with G-CSF、cyclosporine combined with eltrombopag is feasible and effect. Our results deserve further research and confirmation in larger samples. Disclosures No relevant conflicts of interest to declare.

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Are Outcomes After Myeloablative Conditioning Regimen in Double Cord Blood Transplantation (UCBT) Better Than Single UCBT for Adults with Acute Leukemia in Remission?.

Blood ◽

10.1182/blood.v120.21.3083.3083 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3083-3083

Author(s):

Annalisa Ruggeri ◽

Henrique Bittencourt ◽

Guillermo Sanz ◽

Alessandro Rambaldi ◽

Ibrahim Yakoub-Agha ◽

...

Keyword(s):

Multivariate Analysis ◽

Acute Leukemia ◽

Conflicts Of Interest ◽

Cord Blood Transplantation ◽

Chronic Gvhd ◽

Conditioning Regimen ◽

Myeloablative Conditioning ◽

Hematopoietic Stem ◽

Grade Ii ◽

Group 2

Abstract Abstract 3083 Allogeneic hematopoietic stem cell transplantation (HSCT) is indicated for patients (pts) with acute leukemia (AL) with poor-risk cytogenetics or refractoriness to chemotherapy. For adults requiring HSCT urgently, such as pts in first complete remission (CR1), a single (s) or double unit (d) UCBT is a valid stem cells source. In the sUCBT setting, type of conditioning regimen seems to be associated with better outcome (Sanz BMT 2012). With the aim to compare single vs double UCBT after myeloablative conditioning regimen (MAC) in a homogeneous series of pts, we analyzed 239 adults (>18years) with AL in CR1. Pts were transplanted with sUCBT (n=156) or dUCBT (n=83) from 2005–2011 in EBMT centers for ALL (n=101) and AML (n=138). Type of MAC was statistically associated with outcomes therefore pts were analyzed in 3 different groups: Group 1: pts receiving sUCBT with TBI-based+Cy (+Flu) (n=68) (performed in 42 transplant centers (TC)), Group 2: pts receiving sUCBT with Bu+Flu+Thiotepa (n=88) (performed in 23 TC) and Group 3: pts receiving dUCBT with Cy+TBI+Flu (n=83) (performed in 47 TC). No statistical differences were found among the 3 groups for pts and disease characteristics (diagnosis, risk, gender, weight, CMV status, year of UCBT and time from diagnosis to UCBT) however pts in group2 were older than in group1 and 3 (median age 38 vs 33 vs 31 years) (p=0.03). Cytogenetic at diagnosis was available for 176 pts, 39% of pts were classified in the intermediate risk and 56% in unfavorable risk group. Forty-two pts had t(9;22) and 26 FLT3/ITD mutation. No differences on cytogenetic were found among the 3 groups. Thirty one percent of CB units were identical to recipient or had 1 HLA disparity (antigen level typing for HLA-A and B and allelic level for DRB1) while 69% had 2–3 HLA disparities. There was no difference on HLA disparities among the 3 groups. Median infused TNC was 2.9×107/kg for group1, 3×107/kg for group2, and 3.7×107/kg for group3 (p=0.01) and median CD34 was 1.2×105/kg, 1.6×105/kg and 1.5×105/kg, respectively (p=0.32). ATG was part of conditioning regimen in 73% of pts. The use of ATG was different in the 3 groups (70%, 90% and 40% for group1, 2 and 3, respectively p<0.001). GVHD prophylaxis consisted either of CSA±MMF or CSA±steroids in 46% and 22% of pts, respectively. All groups had the same median follow-up time: 24 (range 3–74) months. For group1, group2 and group3, the cumulative incidence (CI) of 60 days neutrophil recovery was 82%, 89% and 87% (p=0.15), with median time of 27, 21 and 24 days, respectively (p<0.001). Chimerism analysis performed at day 100 showed full donor chimerism in 87% of pts (data available for 80% of pts who engrafted). No differences in chimerism status were found between the 3 groups (p=0.47). At day 100, CI of acute GVHD (grade II-IV) was 30% vs 20% vs 45% for group1, group2 and group3, respectively (p=0.001). Pts receiving a dUCBT who developed aGvHD (n=38), experienced mainly grade II aGvHD with skin involvement (grade II (n=25), grade III (n=10), grade IV (n=3)). CI of chronic GvHD at 1 year was 29%, with no differences in the incidence among the groups. At 1 year, CI of TRM was 44% for group1, 33% for group2 and 36% for group3 (p=0.46). In multivariate analysis, two factors were associated with higher TRM: diagnosis of ALL (p=0.048) and age>35 years (p=0.049). One-Hundred-six pts died and the causes of death were infection (n=38), GvHD (n=18), other transplant-related events (n=31) or relapse (n=18). CI of 2y relapse was 25% for group1, 18% for group2 and 16% for group3 (p=0.22). No factors were found to be associated with increase relapse incidence in multivariate analysis. The 2y probability of leukemia-free-survival (LFS) was 31% for group1 (sUCBT-TBI based), 48% for group2 (sUCBT-BuFluTT), and 47% for group3 (dUCBT) (p=0.03). No center effect was found for LFS. In multivariate analysis, use of sUCBT using TBI based MAC (HR=0.9, p=0.003), diagnosis of ALL (HR=0.69, p=0.04) and age>35years (HR=1.4, p=0.04) were independently associated with decreased LFS. In this retrospective based registry analysis, in the myeloablative setting for adults with AL in CR1, outcomes (TRM, RI and LFS) after dUCBT were not statistically different from sUCBT using iv-BuFluTT. However, compared to sUCBT using TBI-based MAC, dUCBT was associated with lower RI and better LFS rates. In the MAC setting, the combination of conditioning regimens and type of graft (single vs. double) may have different impact UCBT outcomes. Disclosures: No relevant conflicts of interest to declare.

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Busulfan-Cyclophosphamide Worsens Peripheral Gvhd While Fludarabine-Busulfan Results in Wide Bone Marrow Involvement in a Murine Model of Acute Gvhd

Blood ◽

10.1182/blood.v128.22.5791.5791 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5791-5791

Author(s):

Xin He ◽

Xiaojun Xu ◽

Yongbin Ye ◽

Qifa Liu

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Bone Marrow Involvement ◽

Conditioning Regimen ◽

Clinical Manifestations ◽

Major Complication ◽

Clinical Situation ◽

Hematopoietic Stem ◽

The Impact

Abstract Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and A proper conditioning regimen is vital to stop the development of aGVHD. To provide a platform for the study of aGVHD and evaluate the impact of different conditioning regimens, we established a murine aGVHD model that simulates the clinical situation and can be conditioned with Busulfan-Cyclophosphamide (BU-Cy) and Fludarabine-BU (Flu-BU). In our study, BALB/c mice were conditioned with BU-Cy or Flu-BU and transplanted with 2×107 bone marrow cells and 2×107splenocytes from either allogeneic (C57BL/6) or syngeneic (BALB/c) donors. The allogeneic recipients conditioned with BU-Cy had shorter survival (P<0.05) and more severe hepatic and intestinal clinical manifestations and pathological changes associated with increased INF-γ expression and diminished IL-4 expression in serum compared to allogeneic recipients conditioned with Flu-BU. Meanwhile increased donor-derived T-cell infiltration and impaired bone marrow B-cell development could be seen in the aGVHD mice conditioned with Flu-BU. Our study showed that the conditioning regimen with BU-Cy resulted in more severe peripheral aGVHD, while the Flu-BU regimen was associated with aGVHD with wide bone marrow involvement. Disclosures No relevant conflicts of interest to declare.

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KIT Blockade Is Sufficient to Sustain Donor Hematopoietic Stem Cell Engraftment in Fanconi Anemia Mice

Blood ◽

10.1182/blood.v126.23.1206.1206 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1206-1206

Author(s):

Shanmuganathan Chandrakasan ◽

Rajeswari Jayavaradhan ◽

Ernst John ◽

Archana Shrestha ◽

Phillip Dexheimer ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Fanconi Anemia ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Conditioning Regimen ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Increased Risk

Abstract Background: Fanconi anemia (FA) is the most common cause of inherited bone marrow failure (BMF). Currently, the only curative option for the BMF in FA is an allogenic hematopoietic stem cell transplant (HSCT). However, due to the underlying DNA repair defect, FA patients poorly tolerate alkylating chemotherapy or irradiation based conditioning, which is necessary for donor engraftment. However, this results in significant short and long term morbidity/mortality and augments the inherent increased risk of malignancies in FA patients. To overcome the adverse effects associated with alkylating conditioning agents, alternate experimental approaches exploiting the inherent hematopoietic stem cell (HSC) defect in FA are of utmost clinical necessity. Objective: To develop a safe KIT blocking antibody (KIT-Ab) based HSCT conditioning regimen for FA that does not involve chemotherapy or irradiation. Method: High purity KIT-Ab was made from the ACK2 hybridoma and its specificity to KIT binding was validated using mast cell assay. Baseline peripheral blood cells and the bone marrow hematopoietic stem and progenitor cell (HSPC) compartment (Lin-Kit+Sca+ and Lin-Kit+Sca+CD150+CD48- cells) of FANCA-/- and FANCD2-/- murine models were analyzed. Mechanistic studies using sorted FA bone marrow HSPC were performed ex vivo. This was followed by definitive primary and secondary transplants experiments following injection of KIT-Ab. Results: Several features of FA hematopoietic stem/progenitor cells (HSPC) suggested their susceptibility to KIT-Ab blockade-mediated killing: (a) Expression of KIT was significantly lower in FANCA-/- HSPC, while expression of its ligand was higher in bone marrow stroma; (b) Moreover, genes associated with apoptosis/senescence, stress and inflammatory signaling that were upregulated in WT-HSPC following KIT-Ab blockade, were upregulated in FANCA-/- HSPC at baseline; (c) Furthermore, FANCA-/- HSPC demonstrated increased susceptibility to KIT-Ab mediated apoptosis and had a reduced proliferative capacity. In-vivo studies following ACK2 injection showed a marked reduction of colony-forming units (CFU-C) from both FANCA-/- and FANCD2-/- mice one week following injection, when compared to WT mice (48% and 76% decrease in CFU-C, respectively). Based on these findings, we evaluated the role of ACK2 as a sole HSCT conditioning regimen in FANCA-/- and FANCD2-/- mice. Indeed, definitive HSCT in both FANCA-/- and FANCD2-/- mice using KIT-Ab based conditioning resulted in donor HSC engraftment with multi-lineage chimerism, which progressively increased to 22-24% by 4-months, and was sustained in secondary transplants. Overall, we show that KIT-blockade alone is an adequate non-genotoxic HSPC-targeted conditioning in FA mice, and its clinical translation could circumvent the extensive transplant-related morbidity/mortality in this disease. Disclosures No relevant conflicts of interest to declare.

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Practice of allogeneic hematopoietic stem cell transplantation for infantile autosomal recessive osteopetrosis

Pediatric Hematology/Oncology and Immunopathology ◽

10.24287/1726-1708-2019-18-2-43-52 ◽

2019 ◽

Vol 18 (2) ◽

pp. 43-52

Author(s):

A. E. Burya ◽

K. I. Kirgizov ◽

E. A. Pristanskova ◽

M. B. Melnikova ◽

V. V. Palm ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Autosomal Recessive ◽

Conditioning Regimen ◽

Unrelated Donor ◽

Post Transplantation ◽

Myeloablative Conditioning ◽

Peripheral Blood Stem Cells ◽

Hematopoietic Stem ◽

Blood Stem Cells

Generalized osteopetrosis is a rare hereditary disease characterized by impairment of skeleton bones formation, bone marrow dysfunction, neurologic deficiency and blindness. The main treatment for osteopetrosis is an allogeneic hematopoietic stem cell transplantation (allo-HSCT). To review and analyze experience of Department of bone marrow transplantation of RDKB (BMT RDKB) of allo-HSCT for patients with autosomal recessive generalized osteopetrosis; to evaluate tolerability and efficacy of the conditioning regiment administered. Between 2010 to 2018 years, 7 patients (2-male, 5-female) with autosomal recessive generalized osteopetrosis underwent allo-HSCT in tDepartment of bone marrow transplantation of RDKB. Median age at the moment of HSCT was 5,5 years (1–11 years). Before the transplantation myeloablative conditioning regimen was used: treosulfan, fludarabine and melphalan for 5 patients, treosulfan, fludarabine and thiotepa for 1 patient and treosulfan with fludarabine for 1 patient. In case of unrelated allo-HSCT antithymocyte globulin was added to the conditioning regimen. Bone marrow from matched (HLA- 10/10) unrelated donor was used for 4 patients, peripheral blood stem cells from matched unrelated donor was used for 1 patient, two grafts of unrelated umbilical cord blood (HLA 8/10 and 9/10) for 1 patient and peripheral blood stem cells from matched (HLA 10/10) from related donor – for 1 patient. For “graft-versus-host” disease (GVHD) prophylaxis either cyclosporine A/tacrolimus and methotrexate/ mofetil mycophenolate was used. White blood cell recovery had been achieved for 6 from 7 patients on +13 to +22 day (median +17 day). Quick autoreconstitution of hemopoesis was observed for the recipient of umbilical cord blood who got one myeloablative drug. The following early post transplantation complications were registered: oropharyngeal mucositis up to II degree in 6 patients, neutropenic enterocolitis up to II degree in 4 patients, up to III degree in 3 patients, sepsis in 3 patients. The GVHD symptoms occurred in 2 cases: skin form of II degree in one patient and skin form of II degree and intestinal form of IV degree in another patient. One patient with neurodegenerative form of osteopetrosis died with increase of hypertensive-hydrocephalus syndrome, cerebral edema with downward cerebellar herniation. During 5-6 months after allo-HSCT the 5 successfully transplanted patients experienced poor graft function but then reduction of extramedullary hemopoesis occurred and full engraftment was achieved. Hypercalcemia was reported in 2–5 months after allo-HSCT and was treated by administration of bisphosphonates. Visual impairment persisted after allo-HSCT. After 4–6 months after transplantation axis skeleton growth occurred for all 5 successfully transplanted patients, skull deformation reduced and no new zones of nerve’s compression were observed. No patients had any developmental delays after the treatment. Allo-HSCT is an effective systemic treatment of autosomal recessive generalized osteopetrosis. However because serious neurodegenerative condition cannot be reversed by allo-HSCT, such treatment may not be recommended for patients with heavy CNS impairment. Myeloablative conditioning regimen with two alkylating agents provides allogeneic reconstitution of hemopoesis. In post transplantation period, measures for hypercalcemia control are necessary. Early diagnostic of autosomal recessive generalized osteopetrosis can help to evaluate feasibility of allo-HSCT and to start treatment on time thus provide chance for long-term rehabilitation and prevention of serious disability. The study was approved by the Independent Ethics Committee of Russian Children's Clinical Hospital.

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Successful Bone Marrow Transplantation Using an Immunomyelosuppressive Conditioning in Patients with Severe Congenital Neutropenia: The Results of a Single-Institute

Blood ◽

10.1182/blood-2019-126653 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1035-1035

Author(s):

Risa Matsumura ◽

Shiho Nishimura ◽

Yoko Mizoguchi ◽

Mizuka Miki ◽

Maki Taniguchi ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Genetic Disorder ◽

Chronic Gvhd ◽

Conditioning Regimen ◽

Mixed Chimerism ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Hematopoietic Stem ◽

Engraftment Failure

Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by recurrent bacterial infections from early infancy due to severe chronic neutropenia. Majority of SCN patients have benefitted by the treatment with granulocyte colony-stimulating factor (G-CSF). However, patients on long-term G-CSF therapy have a relative risk of developing myelodysplastic syndrome/acute myeloid leukemia. The only curable treatment for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning regimens have been applied to the treatment for SCN patients prior to malignant transformation. However, the optimal conditioning of HSCT for SCN patients has not been established. In this study, we conducted bone marrow cell transplantations (BMT) in 16 patients with SCN using an immunomyelosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital. A total of 17 BMT procedures were performed in 16 patients with SCN from 2008 to 2019. Five of 16 patients had experienced the engraftment failure of initial HSCT and 4 of them were referred to our hospital for re-transplantation. Fifteen of 16 patients had a heterozygous mutation in the ELANE gene. Bone marrow cells (BM) were obtained from 6 HLA-matched related, 3 HLA-matched unrelated, and 8 HLA-mismatched unrelated (7/8 antigens) donors, respectively. Conditioning regimen consisted of fludarabine, cyclophosphamide, melphalan, total body irradiation (3.6 Gy) with or without antithymocyte globulin. Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was observed within post-transplant 24 days in all patients. Two patients developed graft failure on day 40 and day 90, respectively, after the temporal engraftment. However, both patients were rescued by second BMT from different HLA-matched unrelated donors receiving the same conditioning regimen. Four patients who received BMT from HLA-matched related donors developed stable mixed chimerism without neutropenia in peripheral blood for 3 to 10 years. Although one patient who received donor lymphocyte infusion due to mixed chimerism developed grade II acute GVHD and limited chronic GVHD, the others did not develop severe GVHD. All patients are alive for 6 months to 11 years after BMT with no signs of severe infections or transplantation-related morbidity. Similar conditioning regimen has been applied to BMT for 35 patients with chronic granulomatous disease (CGD) in our hospital. In that study 4 male adulthood patients with CGD already fathered each child by their wives through spontaneous pregnancy, implying the successful preservation of patients' fertility. Collectively, our results demonstrate that BMT with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. The excellent results in our cohort suggest that indications for proceeding to HSCT could be extended to patients without malignant transformation.The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. Disclosures No relevant conflicts of interest to declare.

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HHV-6 Reactivation Lowers the Risk of Acute Graft-Versus-Host Disease but Enhances the Risk of Infections Due to Activation of CD4+FoxP3+ Lymphocytes.

Blood ◽

10.1182/blood.v114.22.4653.4653 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4653-4653

Author(s):

Andrzej Lange ◽

Emilia Jaskula ◽

Sylwia Madej ◽

Dorota Dlubek

Keyword(s):

Conflicts Of Interest ◽

Survival Rates ◽

Conditioning Regimen ◽

Cd4 Cells ◽

Myeloablative Conditioning ◽

Hematopoietic Stem ◽

Post Transplant ◽

Unrelated Donors ◽

Cmv Reactivation ◽

Cd4 Lymphocytes

Abstract Abstract 4653 CMV, EBV, and HHV-6 reactivation was monitored for 20 days to 4 years after allogeneic hematopoietic stem cell transplantation (alloHSCT) with respect to hematological and immunological reconstitution as well as fate in 118 patients (17 children and 101 adults) who received alloHSCT (100 peripheral blood progenitor cell (PBPC) and 18 bone marrow transplants from 53 siblings and 65 unrelated donors, 64 patients receiving reduced intensive conditioning (RIC) and 54 myeloablative conditioning). All patients on RIC and those transplanted from unrelated donors received anti-thymocyte globulin (ATG). Herpes virus DNA copies were measured by quantitative PCR (primers and probes designed according to Kimura 1999, Machida 2000, and Locatelli 2000). This study focused on HHV-6. It was found that:Multivariate analysis showed that alternative donor (OR=3.06, p=0.038), myeloablative conditioning regimen (OR=0.22, p=0.004), and an HLA-matched (10/10 at allele level) graft (OR=0.14, p=0.011) were risk factors of HHV-6 reactivation. Interestingly, they were different from those of CMV reactivation (acute GvHD grade >=I, OR=2.74, p=0.024).HHV-6 reactivation was found earlier post-transplant than CMV reactivation (19 vs. 47 days, median values, p=0.014) and rather before clinically apparent aGvHD manifestation, in contrast to CMV and EBV infections (percentages of reactivation events prior to aGvHD were 71, 14, and 14 for HHV-6, CMV and EBV, respectively).HHV-6 was associated with an increase in CD4+ lymphocytes (p=0.028, Wilcoxon matched pairs test), in contrast to CMV infection, in which CD4+ cells were rather low.HHV-6 reactivation noted during 100 days post-transplant was associated with a higher fraction of FoxP3+ in CD4+ cells compared with those of counterparts (13% vs. 8.5%, median values, p=0.008).Encephalitis was frequent in HHV-6 (8/14 vs. 26/104, p=0.023) and EBV (9/14 vs. 22/104, p=0.002) positive patients. HHV-6 preceded EBV (14/26 vs. 28/92, p=0.037) but not CMV (9/36 vs. 33/82, p=0.145) reactivation before 100 days after HSCT.HHV-6 positivity in the 100 days post-HSCT was associated with a higher mortality rate early post-transplant (6-month survival rates of HHV-6-positive and -negative cases: 55% vs. 80%, respectively). In conclusion: HHV-6 reactivation affects patients early post-transplant and associates with an increase in CD4+ lymphocytes in blood which frequently have FoxP3 generation potential. Risk of aGvHD is consequently lower, but there is a higher incidence of infections, including EBV, encephalitis, and sepsis, and survival is poor. Disclosures: No relevant conflicts of interest to declare.

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