The mammalian target of rapamycin inhibitor everolimus (RAD001) is a successfully used immunosuppressant in solid-organ transplantation. Several studies have already used RAD001 in combination with calcineurin inhibitors after hematopoietic stem cell transplantation (HSCT). We investigated calcineurin inhibitor-free pre- and post-transplantation immunosuppression of RAD001 combined with mycophenolate mofetil (MMF) in a nonmyeloablative HSCT setting. After nonmyeloablative conditioning with 2 Gy total body irradiation, 8 dogs received HSCT from dog leukocyte antigen-identical siblings. Immunosuppressives were given at doses of 1.5 mg RAD001 twice daily from day -1 to +49, then tapered until day +56, and 20 mg/kg MMF from day 0 to +28, then tapered until day +42. An historical cyclosporin A (CsA)/MMF regimen was used in the control group. All dogs engrafted. Median platelet nadir amounted in all dogs to 0 × 10(9)/L (median, day +10; duration <50 × 10(9)/L, 22 days) and median leukocyte nadir was 1.0 × 10(9)/L (range, .1 to 2.5 × 10(9)/L; median, day +13). Eventually, 5 of 8 (63%) animals rejected their grafts. Two dogs died of infections on day +19 and +25. Pharmacokinetics of RAD001 and MMF showed median trough levels of 19.1 (range, 10.5 to 43.2) μg/L and .3 (.1 to 1.3) mg/L, respectively. The median area under the curve was 325 (range, 178 to 593) μg/L × hour for RAD001 and 29.6 (range, 7.9 to 40.5) ng/L × hour for MMF. All dogs developed clinically mucosal viral infections during the clinical course. Compared with the control group, the level of toxicities for RAD001/MMF increased in all qualities. Combined immunosuppression of RAD001 and MMF after nonmyeloablative HSCT is associated with significant toxicities, including a prolonged platelet recovery time as well as increased infections compared to the CsA/MMF regimen.

Introduction: Successful engraftment following hematopoietic stem cell transplantation (HSCT) depends on factors like immunosuppression, graft composition and number of infused HSC. Whereas the immunosuppression as well as the type and composition of the graft are influenceable low numbers of available HSCs i.e. “weak grafts” remain a clinical challenge. Weak grafts are accompanied by increased graft failure rates and longer cytopenias associated with increased morbidity. Intra bone marrow (IBM) infusion of HSC might be an approach to overcome these problems. Studies in rodents demonstrated faster engraftment with an IBM HSCT approach compared to intravenous (IV) HSCT following myeloablative conditioning. Studies of IBM HSCT following non-myeloablative or reduced intensity conditioning (RIC) are missing. Aims: Exploring the feasibility and efficiency of IBM allogeneic HSCT in comparison to IV HSCT in dog leukocyte antigen (DLA) identical canine littermates using a RIC regimen. Methods: DLA-identical siblings were used as donor/recipient pairs for HSCT. Recipient dogs were conditioned with 4.5 Gy total body irradiation before HSCT (d0) and received 15 mg/kg Cyclosporin A BID as pre- and postgrafting immunosuppression (d-1 to d+35). BM grafts were harvested at d0. In the control group (CON, n=7) unmodified BM was transplanted IV. In the IBM group (n=7) BM harvests were centrifuged and buffy coat of the BM was then transfused simultaneously into the recipient humeri and femura (50 ml, 10 min). 10 dogs are currently evaluable. Chimerism of the peripheral blood mononuclear cells (PBMC) and granulocytes (G) were tested weekly until week 8 and afterwards in larger intervals. Blood cell counts and clinical toxicities such as weight loss were monitored. Results: Infusion of BM directly into the bone was feasible. All animals engrafted. Median number of infused total nucleated cells was 4.0*108/kg (range 2.3-6.0*108/kg, IBM) and 3.3*108/kg (range 1.9-5.0*108/kg, CON, IBM vs CON: p=0.4). Median CD34+ numbers infused were 3.1*106/kg (range:1.2-10.0*106/kg, IBM) and 3.9*106/kg (range: 1.0-7.2*106/kg, CON; IBM vs CON: p= 0.8). Hematopoietic recovery in the IBM and CON groups were similar. Leukocytes recovery (>1.0*109/l) occurred at median d+11 (range: d+10 - d+16, IBM) and d+10 (range: d+9-d+12, CON; IBM vs CON: p=0.3). Median leukocytes nadirs amounted to 0.23*109/l (IBM) and 0.28*109/l (CON; IBM vs CON: p=0.3) and median duration of leukopenia (<1.0*109/l) were 6 days (range: 5.0–11.0, IBM) and 4 days (range: 3.0–6.0, CON; IBM vs CON: p=0.1). Median platelet nadir after IBMT was 10.0*109/l (range: 0.0 - 25.0*109/l) and 6.0*109/l (range: 3.0-15.0*109/l, CON; IBM vs CON: p=0.8). Period of thrombocytopenia (≤50.0*109/l) lasted for 12 days in both groups (p=0.7). Chimerism analyses showed an early and fast increase in donor chimerism in both groups. The PBMC donor chimerism at d+14, d+28 and d+56 were 46% (range: 30-53%), 57% (range: 40-73%), 64% (range: 60-83%) for IBM. Results in CON were 37% (range: 17-93%), 60% (range: 49-100%), 57% (range: 40-100%) (IBM vs CON, p=n.s. (all time points)). The G chimerism values at that specific points were 95% (range: 53-100%), 100% (range: 53-100%), 96% (range: 88-100%) for IBM and 100% (range: 93-100%), 99% (range: 92-100%), 98% (range: 93-100%) for CON (IBM vs CON, p=n.s. (all time points)). Primary goal of the study was the feasibility of the IBM approach. Ethics regulations did not allow to use weak grafts (≤2.0*106/kg) intentionally. However, 4 animals received weak grafts (CON n=2, 1.0 and 2.0*106/kg; IBM n=2, 1.2 and 1.3 *106/kg). Of interest, comparing data of these dogs showed that durations of leukopenia were similar (median 10 days, both groups), but duration of thrombocytopenia were different (median 8 days, IBM vs 22 days, CON). Additionally, long term donor chimerism was higher in the IBM (median 80% PBMC, 100% G) vs CON (median 61% PBMC, 42% G). Conclusion: First, IBM HSCT is a feasible and effective method to deliver HSC directly into the bone marrow following RIC in a canine HSCT model. Second, our preliminary data suggest that IBM HSCT reveals advantageous engraftment differences in regards to platelet recovery and donor chimerism kinetics compared to the IV HSCT when grafts with low HSC numbers were infused. Follow up data of this study and future studies will have to clarify these observations further. Disclosures No relevant conflicts of interest to declare.

The canine hematopoietic stem cell transplantation (HSCT) model has become accepted in recent decades as a good preclinical model for the development of new transplantation strategies. Information on factors associated with outcome after allogeneic HSCT are a prerequisite for designing new risk-adapted transplantation protocols. Here we report a retrospective analysis aimed at identifying risk factors for allograft rejection in the canine HSCT model. A total of 75 dog leukocyte antigen-identical sibling HSCTs were performed since 2003 on 10 different protocols. Conditioning consisted of total body irradiation at 1.0 Gy (n = 20), 2.0 Gy (n = 40), or 4.5 Gy (n = 15). Bone marrow was infused either intravenously (n = 54) or intraosseously (n = 21). Cyclosporin A alone or different combinations of cyclosporine A, mycophenolate mofetil, and everolimus were used for immunosuppression. A median cell dose of 3.5 (range, 1.0 to 11.8) total nucleated cells (TNCs)/kg was infused. Cox analyses were used to assess the influence of age, weight, radiation dose, donor/recipient sex, type of immunosuppression, and cell dose (TNCs, CD34(+) cells) on allograft rejection. Initial engraftment occurred in all dogs. Forty-two dogs (56%) experienced graft rejection at median of 11 weeks (range, 6 to 56 weeks) after HSCT. Univariate analyses revealed radiation dose, type of immunosuppression, TNC dose, recipient weight, and recipient age as factors influencing long-term engraftment. In multivariate analysis, low radiation dose (P < .001) and low TNC cell count (P = .044) were identified as significant independent risk factors for graft rejection. Peripheral blood mononuclear cell chimerism ≥30% (P = .008) and granulocyte chimerism ≥70% (P = .023) at 4 weeks after HSCT were independent predictors of stable engraftment. In summary, these data indicate that even in low-dose total body irradiation-based regimens, the irradiation dose is important for engraftment. The level of blood chimerism at 4 weeks post-HSCT was predictive of long-term engraftment in the canine HSCT model.

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.

No abstract