Good Engraftment Research Articles

Polyurethane foam for skin graft fixation in clinical-relevant ovine burn wound model for wound repair and regeneration research.

The availability of clinical-relevant large animal models for research in wound healing study is limited. Although a few reports described the wound dressing fixation method using polyurethane foam in patients, no animal studies were conducted to investigate efficacy of the polyurethane foam in grafted burn wounds. In the present study, we report a simple fixation method of grafted burned skin using polyurethane foam dressing (Allevyn Non-Adhesive, smith & nephew, UK) in a clinically relevant ovine grafted burn wound model. The dressing was removed at postoperative day 7 after skin graft. The grafted skin was completely engrafted without any complications. This method was safe and easy to perform and associated with good engraftment without any complications. We believe that the polyurethane foam fixation method may be successfully used in clinical practice as well as in preclinical studies for grafted burn wound repair and regeneration research.

Administration of Plerixafor in Poor Mobilizers Allows Mobilization of Haematopoietic Progenitors: Clonogenic Potential and Engraftment Analysis

BACKGROUND Plerixafor (PLX) blocks the binding of stromal cell-derived factor to CXCR4, resulting in hematopoietic stem cell (HSC) release from the bone marrow. It has been successfully used as a mobilizing agent in poor mobilizers (PM). The best strategy is probably the "pre-emptive" (on demand) use, as it allows an "on time" identification of PMs, preventing collection failure and need for further mobilization. Usually a cut-off of < 10/ μL CD34+ cell count at leukocyte recovery is used for PLXa dministration, while a minimum level is not defined, as mobilization may be obtained even with very low values. An important issue that has not been extensively explored so far is represented by the biological characteristics of the yields collected with PLX, especially clonogenicity, and its clinical counterpart, represented by the engraftment times observed after transplant. In this report we retrospectively analysed our data on the use of PLX in PMs, evaluated its efficacy and focused on the clonogenicity of collected stem cells and engraftment post auto transplant (ASCT). METHODS We collected data on PM patients mobilized with PLX between 2011 and 2019:Clinical data: mobilization and collection performance, transplant rate and engraftment times (days to PMN > 500/uL).Biological data: Clonogenicity of HSC collected after PLX, measured by hematopoietic progenitor semi-solid cultures according to Stem Cell Technology. These clinical and biological data were then compared to those obtained in patients mobilized without PLX and in allogeneic donors who received G-CSF in the same period. RESULTS From January 2011 to June 2019 PLX was used in 73 mobilization cycles, performed in 64 patients. Patients characteristics are described in Table 1. 105 doses of PLX were given during 73 mobilization cycles (1,44 doses/cycle) and 93 collections performed (1,27/ cycle). Circulating CD34+ pre PLX administration were in median 6,81/µl (range: 0,27-21), while after treatment 26,20/µl (range: 4,8-155,4). Using a cut-off of ≥ 10/ μL CD34+, a successful mobilization was achieved in 67/73 cycles (success rate 91,7%). Overall 59 out of 64 patients achieved the collection target (92,2%) at any time and 54/64 have been transplanted (84,4%), the other 10 not yet due to mobilization failure (n=2), insufficient yield (n=3), clinical unfitness (n=2) or because too early (n=3). A median of 4 HPC bags were reinfused (range: 2-12) and median time to WBC engraftment was 10 days (8-21). Engrafment times are in line with those of MM and NHL patients mobilized without PLX in the same period of time (median: 10, range: 8-12) at our Institution. Interestingly, 33 out of 35 pts mobilized with PLX (94,2%), compared to 442/449 (98,4%) engrafted in ≤11 days. We analyzed the cellular composition of yields and clonogenicity of HSC collected in 3 different groups:Group A: PLX mobilization (n=93)Group B: chemo + G-CSF without PLX (n=755)Group C: G-CSF mobilization in allogeneic donors (n=206) We failed to demonstrate a statistical difference between clonogenicity in group A and B (p=0,691), while clonogenicity was slightly but significantly higher in group C compared to group A and B (p< 0,001). Data are shown in tables 2,3 and 4. CONCLUSION In our cohort of patients, administration of PLX in PM resulted in successful mobilization of HPCs with good clonogenicity and engraftment potential. The use of PLX allowed an high proportion of patients to undergo ASCT. Disclosures No relevant conflicts of interest to declare.

Ptcy + ATG Compared to Ptcy Alone As Gvhd Prophylaxis for Peripheral Blood Stem Cells Allotransplant Is Associated with Significant Lower Incidence of Acute Gvhd without Increasing Relapse or Death By Infection

Backgound: High-dose post-transplant cyclophosphamide (PTCY) is currently used for GVHD prophylaxis both in the matched and the haplo transplant settings. However, peripheral blood stem cells (PBSC) as source of graft are significantly associated with a higher incidence of GVHD after transplant with PTCY compared to the use of bone marrow, especially with haplodonors. New strategies are needed to decrease GVHD incidence in this setting. Methods: This retrospective study included 115 adults (males n=68, median age: 60 yo (range: 23-71) allotransplanted with PTCY or PTCY+ATG between March 2014 and March 2019 in a single institution. Three conditionings were used: the Baltimore regimen (n=40, period: 11/2013-05/2017) consisting of Fludarabine 30 mg/m²/day (d), d -6 to -2, cyclophosphamide 14.5 mg/kg day -6, low dose total body irradiation 2 Grays d-1; the Clo-Baltimore regimen (n=37, period: 03/2014-04/2017), consisting of Clofarabine 30 mg/m²/d, d -6 to -2, cyclophosphamide 14.5 mg/kg d -6, low dose total body irradiation 2 Grays d-1; and the CloB2A1 regimen (n=38, period: 05/2017-03/2019) consisting of Clofarabine 30 mg/m²/d, d -6 to -2, busulfan 3,4 mg/kg d -3 and d-2, ATG 2,5 mg/kg d-1. The three groups shared most characteristics (gender, disease risk index, type of donor [matched vs haplo], ABO compatibility, donor/recipient CMV status, previous allograft, median age of the donor, median CD34+ and CD45+ graft cells counts). Baltimore patients were younger (median 56 years vs Clo-Baltimore: 61 years and CloB2A1: 60 years, p=0.02) and mostly transplanted for lymphoid diseases (65% vs 8% vs 19%, p<0.001). The median number of CD3+ T cells infused was significantly higher for CloB2A1 patients (27.5 107/kg vs Baltimore 22.65 vs Clo-Baltimore 20.8, p=0.008). All patients received PBSC as stem cell source at d 0 and PTCY 50 mg/kg/d, d +3 and +4 with cyclosporine and mycophenolate mofetyl as GVHD prophylaxis. All patients provided informed consent for data collection before the graft. Results: The median follow-up was 42 (29-64), 33 (7-63) and 15 (3-25) months for the Baltimore, Clo-Baltimore and CloB2A1 groups, respectively. Primary graft failure was significantly higher in the CloB2A1 group (at day+60: 22% (6 haplo, 1 10/10 and 1 9/10) vs Baltimore 5.2% (1 haplo, 1 10/10) vs Clo-Baltimore 0% (p=0.003). Similar 18-month survivals were observed for the three groups (Baltimore/Clo-Baltimore/CloB2A1): OS: 65% (51-81) vs 67.1% (53-84) vs 51.2% (35-73), p=0.27; DFS: 60% (46-77) vs 56.7% (42-75) vs 37.8% (23-62) p=0.20); GRFS (50% (36-68) vs 37.8% (25-57) vs 36.7% (22-60), p=0.63). The incidences of relapse and death as well as the causes of deaths (no more death by infection in the CLOB2A1 sub-group) were also comparable. Conversely, considering engrafted patients, the incidence of grade 2-4 acute GVHD was significantly lower in the CloB2A1 sub-group (19% vs Baltimore: 50% vs Clo-Baltimore: 51.4%, p=0.009) and despite higher numbers of CD3+ T cells infused in this group. The incidence of grade 3-4 acute GVHD and chronic GVHD (considering engrafted patients alive at day+100) was lower also in the CloB2A1 sub-group (6.2% vs 16.6% vs 13.5%, p=0.32 and 16% vs 29.4% vs 31%, p=0.51). Considering only haplotransplants, 18-month OS (p=0.95), DFS (p=0.98) and GRFS (p=0.39) were similar between the three groups (Baltimore: 57.1%, 50% and 35.7%; Clo-Baltimore: 60%, 53.5%, 35.7%; CloB2A1: 57.9%, 48.3%, 46.6%). The incidence of grade 2-4 acute GVHD remained significantly lower in the CloB2A1 haplo sub-group (p=0.01). Considering only matched transplants, 18-month survivals were significantly lower for the CLoB2A1 sub-group (OS: 44.4% vs Baltimore 83.3% vs Clo-Baltimore: 88.8%, p=0.05; DFS: 33% vs 83.3% vs 66.6%, p=0.008; GRFS: 22.2% vs 75% vs 44.4%, p=0.008). Conclusion: The CloB2A1 + PTCY regimen does not appear to be a good platform, neither for haplotransplant nor for matched transplant since 1) the incidence of primary graft failure is relatively high for the former and 2) survivals are lower for the latter. However, this study shows that the addition of one day of ATG to PTCY decreases the incidence of GVHD after transplant without increasing the incidence of relapse or death by infection. The use of ATG +PTCY should be tested in patients receiving PBSC haplo-transplant but with other conditioning regimens allowing for a good engraftment, such as Baltimore-based regimens. These data have to be validated prospectively. Disclosures Chevallier: Incyte: Consultancy, Honoraria; Daiichi Sankyo: Honoraria; Jazz Pharmaceuticals: Honoraria. Peterlin:Daiichi-Sankyo: Consultancy; AbbVie Inc: Consultancy; Astellas: Consultancy; Jazz Pharma: Consultancy. Le Gouill:Janssen-cilag: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Roche Genentech: Consultancy, Honoraria; Gilead-Kite: Consultancy, Honoraria; Servier: Consultancy; Novartis: Consultancy; Takeda: Consultancy; loxo: Consultancy, Honoraria. Moreau:Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

The Bruton’s tyrosine kinase inhibitor acalabrutinib protects from anaphylaxis in a humanized mouse model

There is an unmet need for therapies that can prevent anaphylaxis. Acalabrutinib is a well-tolerated, FDA approved drug for leukemia that targets Bruton’s tyrosine kinase (BTK), an enzyme also thought to be essential for IgE receptor signaling in human mast cells and basophils. We tested the hypothesis that acalabrutinib administration would prevent anaphylaxis in a humanized mouse model. NSG-SGM3 mice were engrafted with human CD34+ cells at age 3-4 weeks. 16 weeks after engraftment, passive systemic anaphylaxis (PSA) was induced by intravenous sensitization with human anti-NP-IgE and challenge 24 hours later with intravenous injection of NP-BSA. Mice were pre-treated with acalabrutinib or vehicle control via oral gavage prior to challenge. Anaphylactic responses were assessed using core body temperature and clinical scoring. NSG-SGM3 mice engrafted with human CD34+ cells showed good engraftment of human mast cells in addition to excellent PSA responses. Increasing the challenge dose of NP-BSA from 5 to 500 μg resulted in increased severity of anaphylaxis, from mild to fatal responses. Two doses of 15 mg/kg acalabrutinib administered 16 and 4 hours prior to challenge completely mitigated, and 1.5 mg/kg partially mitigated, responses to moderate PSA compared to mice gavaged with vehicle. NSG-SGM3 humanized mice represent a unique model for the study of anaphylaxis where the severity of response can be controlled by varying the amount of allergen used for challenge. Furthermore, pretreatment with just two doses of acalabrutinib completely prevented PSA in these mice, suggesting that BTK inhibitors may be able to prevent IgE-mediated anaphylaxis in humans.

Allogreffe : nouveautés et perspectives

The recent advances in the field of allogeneic stem cell transplantation (allo- SCT) have emerged, including the development of transplants from haplo-iden- tical family donors. Indeed, recently, haplo-identical transplants are expansively increasing thanks to the use of high-doses of post-transplant cyclophosphamide which promotes good engraftment while reducing the risk of severe graft-versus-host disease (GVHD). In addition, the improved knowledge of the physiology of GVHD and the emergence of new anti-infectious treatments have reduced the risk of post-allograft complications. Unfortunately, the main cause of mortality after allo-SCT is still represented by the relapse of the hematological malignancy; As a consequence, prophylactic or pre-emptive therapeutic strategies to avoid relapse have been developped these recent years with very encouraging results. Lastly, we discuss the development of CAR T-cells, a new T-cell immunotherapy that can challenge the place of allo- SCT in the close future.

A patient derived xenograft model of cervical cancer and cervical dysplasia.

AimTo develop a patient derived xenograft (PDX) model of cervical cancer and cervical dysplasia using the subrenal capsule.MethodsCervical cancer (12 Squamous Cell Carcinoma, 1 Adenocarcinoma, 1 Adenosquamous Carcinoma), 7 cervical dysplasia biopsy and normal cervical tissues were transplanted beneath the renal capsule of immunocompromised NOD/SCID/gamma mice. Resulting tumours were harvested and portions serially transplanted into new recipient mice for up to three in vivo passages. Parent and xenograft tumours were examined by immunohistochemistry for p16INK41, HPV, and CD-45. Single cell suspensions of mixed mouse and human, or human only cell populations were also transplanted.ResultsThe overall engraftment rate for the primary cervical cancer PDX model was 71.4 ±12.5% (n = 14). Tumours maintained morphological, histoarchitecture and immunohistochemical features of the parent tumour, and demonstrated invasiveness into local tissues. Single cell suspensions did not produce tumour growth in this model. Mean length of time (32.4 +/- 3.5 weeks) for the transplanted tissue to generate a tumour in the animal was similar between successive transplantations. Three of four xenografted cervical dysplasia tissues generated microscopic cystic structures resembling dysplastic cervical tissue. Normal cervical tissue (4 of 5 xenografted) also developed microscopic cervical tissue grafts.ConclusionThe subrenal capsule can be used for a PDX model of human cervical cancer with a good engraftment rate and the ability to model in vivo characteristics of cervical cancer. For the first time we have demonstrated that cervical dysplasia and normal cervical tissue generated microscopic tissues in a PDX model.

The impact of donor and recipient KIR genes and KIR ligands on the occurrence of acute graft-versus-host disease and graft survival after HLA-identical sibling hematopoietic stem cell transplantation

After allogeneic hematopoietic stem cell transplantation (allo-HSCT), donor natural killer (NK) cells trigger alloreactions against potential recipient cells by their killer immunoglobulin-like receptors (KIRs). This study investigated whether KIR/HLA genotypes and KIR haplotypes of donors and recipients exhibit a critical function in the prevalence of acute graft-versus-host disease (aGVHD) and persistence of the graft after HLA-identical sibling allo-HSCT for patients with hematological malignancies. We studied KIR and HLA genotypes in 115 related donors and recipients (56 patients with AML and 59 patients with ALL) who had received allo-HSCT from HLA-matched sibling donors. We evaluated 17 KIR genes and some alleles, including their ligands, using the PCR-SSP assay. KIR gene frequency results between donors and recipients showed that donors had more activating KIR than their recipients. Chi-square comparison of KIR genotype frequencies in donors versus recipients revealed a significant difference (P < 0.001). We found a survival association between the donor lacking and the recipient having group B KIR haplotypes, although this was not statistically significant. This study suggests that we could exploit NK cell alloreactivity as a part of the optimization of donor selection and potential immunotherapeutic regimens to help facilitate good engraftment and reduce the risk of aGVHD incidence after allo-HSCT.

Modern approaches to the technical aspects of split-skin grafting

Split-skin grafting takes the leading place when closing extensive soft tissue defects. Such defects can be the result of both an acute process (pyoinflammatory soft tissue diseases, surgical interventions) and chronic disorders in the skin and underlying tissues (trophic ulcers of venous and arterial genesis, fistulas, decubitus, complications of the diabetic foot syndrome).&#x0D; The main criterion for assessing the result of split-skin grafting is the percentage of engraftment of the skin graft. There are several classifications that characterize the degree of closure of the recipient wound (Petrova VI, Rysmana BV, Gostishcheva VK). According to most authors, the successful outcome of split-skin grafting depends on several groups of factors. Systemic factors include the content of the total blood protein, hemoglobin, which should not be below acceptable standards. To the local - the readiness of the recipient wound to skin plasty. Microcirculation in the recipient bed plays an important role. The opinions of the researchers about the bacterial contamination of the wound and its effect on the processes of engrafting the flap are opposite. Some believe that a good engraftment of an autodermotransplant is possible even in the presence of a wounded pathogenic microflora in the wound. In modern works the negative influence of microorganisms on the results of split-skin grafting has been proved. Moreover, the upper limit of bacterial contamination of wounds is determined, the excess of which inevitably leads to unsatisfactory results of the transplantation of the skin.&#x0D; The method of postoperative donor wound management is a separate issue in plastic and reconstructive surgery. The review examines the criteria for the readiness of a wound for split-skin grafting, various types of preparation of the wound bed for plastic closure, surgical techniques for performing split-skin grafting, and options for closing the donor wound. &#x0D;

In Vivo Evaluation of Short-Term Performance of New Three-Layer Collagen-Based Vascular Graft Designed for Low-Flow Peripheral Vascular Reconstructions.

Aim The aim of this study was to evaluate short-term patency of the new prosthetic graft and its structural changes after explantation. Methods The study team developed a three-layer conduit composed of a scaffold made from polyester coated with collagen from the inner and outer side with an internal diameter of 6 mm. The conduit was implanted as a bilateral bypass to the carotid artery in 7 sheep and stenosis was created in selected animals. After a period of 161 days, the explants were evaluated as gross and microscopic specimens. Results The initial flow rate (median ± IQR) in grafts with and without artificial stenosis was 120 ± 79 ml/min and 255 ± 255 ml/min, respectively. Graft occlusion occurred after 99 days in one of 13 conduits (patency rate: 92%). Wall-adherent thrombi occurred only in sharp curvatures in two grafts. Microscopic evaluation showed good engraftment and preserved structure in seven conduits; inflammatory changes with foci of bleeding, necrosis, and disintegration in four conduits; and narrowing of the graft due to thickening of the wall with multifocal separation of the outer layer in two conduits. Conclusions This study demonstrates good short-term patency rates of a newly designed three-layer vascular graft even in low-flow conditions in a sheep model.

Remissions of Acute Myeloid Leukemia and Blastic Plasmacytoid Dendritic Cell Neoplasm Following Treatment with CD123-Specific CAR T Cells: A First-in-Human Clinical Trial

The current treatment of relapsed or refractory AML is associated with low rates of complete response (CR) and considerable complications. As a result, only a minority of patients (pts) proceed to allogeneic hematopoietic stem cell transplantation (alloHSCT) with curative intent. Furthermore, outcomes for AML pts with disease relapse after alloHSCT are dismal. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and incurable blood cancer with a median survival of <18 months and no standard of care. Thus, there are clear unmet therapeutic needs in both these conditions. CD123 is overexpressed on AML blasts and leukemic stem cell (LSC)-enriched cell subpopulations compared to normal hematopoietic stem cells and myeloid progenitors. High levels of CD123 expression is also one of the diagnostic hallmarks for BPDCN. All these features make CD123 an attractive target for T cell based adoptive immunotherapy.We have previously demonstrated preclinically the anti-AML activity of CD123-chimeric antigen receptor (CAR) T cell therapy (Mardiros, Blood 2013). The CD123CAR contains an anti-CD123 single-chain variable fragment, an optimized IgG4 CH2CH3 linker, a CD28 co-stimulatory domain, and a CD3 zeta signaling domain and is used to engineer T cells for patients enrolled on a single center, first-in-human phase I dose escalation clinical trial open at our Institution (NCT02159495). The primary objective is to test the safety and activity of escalating doses of CD123CAR T cells for patients with relapsed or refractory AML (cohort 1) and BPDCN (cohort 2). Donor-derived or autologous T cells from leukapheresed peripheral blood mononuclear cells were lentivirally transduced with the CD123CAR vector. Prior to T cell infusion, all patients undergo a lymphodepleting regimen including fludarabine 25-30 mg/m2 daily for 3 days and cyclophosphamide 300 mg/m2 daily for 3 days. Pts receive a single dose of CD123CAR T cells with an option for a second infusion if they continue to meet safety and eligibility criteria and still have CD123+ disease. To date, 14 patients have been enrolled and 7 treated (6 AML, 1 BPDCN).All 6 patients in the AML cohort had refractory AML following alloHSCT, and a median of 4 (range: 4-7) prior lines of therapy. Of the 2 pts treated at dose level (DL) 1 (50M CAR+ T), 1 achieved a morphologic leukemic-free state, which lasted 2 months. She received a second infusion 3 months later with subsequent blast reduction from 77.9% to 0.9% (flow cytometry) after 35 days. Of the 4 pts on DL 2 (200M CAR+ T), 1 achieved CR and became transfusion independent. She proceeded to a second alloHSCT on day 70. Restaging on day +161 post-transplant showed she has remained in MRD-negative CR with good engraftment and 100% donor chimerism. Another pt with CR prior to treatment remained in CR at day 28 and has proceeded to a second alloHSCT. The remaining 2 patients had reductions in blast counts, but did not achieve remission. All toxicities were reversible and manageable: cytokine release syndrome (CRS; 4 grade 1, 1 grade 2); 1 adenoviral pneumonia requiring intubation; and 1 grade 3 rash due to drug hypersensitivity. There were no dose limiting toxicities and no treatment-related cytopenias. One pt with prior alloHSCT had mild recurrent cutaneous GVHD, which occurred after the completion of CAR T treatment. Correlative studies including T cell persistence and CD123 expression are underway and will be reported.In the BPDCN cohort, 1 pt has been treated so far, a 74-year-old man with a persistent bulky subcutaneous mass after clinical trial treatment with a CD123 antibody-drug conjugate. Following lymphodepletion, he received a single dose of 100M autologous CD123CAR T cells and continues to be in CR at 60 days post-infusion. Skin biopsies at the tumor site on days 14 and 28 showed no evidence of disease. Restaging work-up at day 28 revealed disease-free bone marrow, no new masses by CT scan, normalized blood counts, and complete resolution of clinical symptoms. The pt tolerated the treatment well with no CRS or neurologic toxicity.In this first-in-human clinical trial of CD123CAR T cell therapy, we have demonstrated the feasibility and safety of targeting CD123. We have also observed promising anti-leukemic activity in both AML and BPDCN. Importantly, no myeloablative effects have been observed, supporting further testing of this immunotherapeutic strategy in both transplant eligible and ineligible patients. DisclosuresStein:Stemline: Consultancy; Amgen: Consultancy, Speakers Bureau.

Brentuximab for Refactory Hodgkin’s Lymphoma: A Case Report

Hodgkin's lymphoma (HL) continues to be an intriguing, and fascinating lymphoma [1]. HL is expected to cure the majority of patients. Brentuximab vedotin is a CD30 antibody. We survey efficacy of brentuximab in patient with HL who was resistant to treatment. A 43 year old male referred with cough and right supraclavicular lymphadenopathy. His disease was HL, nodular sclerosis type (stage IIB and grade II) with megakaryocytosis in bone marrow aspiration (BMB). He was treated with 6 cycles of induction chemotherapy including adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) and achieved a complete remission by imaging assessment. Then he received ifosfamide, carboplatin, etoposide (ICE) plus brentuximab vedotin (1.8 mg/kg every 3 weeks) regimen for 6 courses. At the end of this time he been eligible for autologous bone marrow transplantation (BMT). Bone marrow was done and after 13 days with good engraftment the patient discharge hospital. Therapists should be try with a specific target when they need a new and potent treatment like BMT for an active disease. Brentuximab vendotin is a new drug in policy of recurrence that it used for a few of cases in Iran.

The Addition of Low-Dose Total Body Irradiation to Fludarabine and Melphalan Conditioning in Haplocord Transplantation for High-Risk Hematological Malignancies.

Preliminary evidence indicates that the addition of low-dose total body irradiation (TBI) (2-4 Gy) to reduced intensity conditioning may reduce the rate of relapse in allogeneic stem cell transplants. In very high-risk patients receiving combination haploidentical single-unit cord blood transplants, we have added 4 Gy TBI to the widely used fludarabine, melphalan conditioning regimen, in hopes of reducing relapse and decreasing graft rejection. We retrospectively reviewed the posttransplant outcomes of patients who underwent haplocord stem cell transplant between May 2013 and March 2015 and who received fludarabine 30 mg/m day (D)-7 to -3, melphalan 140 mg/m D-2, and 2 Gy TBI D-4 and -3. All 25 patients achieved primary neutrophil engraftment after a median of 12 days. The median time to platelet engraftment was 27 days. The cumulative incidence of nonrelapse mortality was 16% by D+100 and 33% by 1 year. The cumulative incidence of grade III to IV acute graft-versus-host disease was 36% by D+100. The CIR was 13% by D+100 and 29% by 1 year. The estimated 1-year overall survival and progression-free survival were 40% and 37%, respectively. In a subgroup analysis, we compared the outcome of 13 acute myeloid leukemia patients receiving this conditioning regimen with age and disease risk index-matched acute myeloid leukemia patients receiving fludarabine-melphalan without TBI. The TBI group had lower incidence of relapse at 1 year (15% vs 54%, P = 0.05). Overall, combination fludarabine-melphalan with low-dose TBI after haplocord stem cell transplant assures good engraftment and leads to acceptable toxicity and disease control in the setting of high risk, heavily pretreated patients. These findings warrant further investigation at a larger-scale, prospective level.

The Changing Landscape of Treatment in Pediatric Aplastic Anemia; A Single Institution's Experience

Introduction: Aplastic anemia (AA) is a rare syndrome of bone marrow failure characterized by peripheral blood pancytopenia and marrow aplasia. We report the clinical course and therapeutic approach of children with AA, who were treated in our Department within the last 4 years. Methods: Fifteen children (9 males/ 6 females) of mean age 7.64 years (range: 2 to 15 years) were diagnosed in our unit with AA since 2012. Diagnosis was established by bone marrow aspirate and biopsy and a normal bone marrow karyotype. Evaluation for underlying bone marrow failure syndromes, including Fanconi anemia, Shwachman-Diamond syndrome and paroxysmal nocturnal hemoglobinuria was performed in all cases. Results: Four children were identified with Fanconi anemia, both by cytogenetic and molecular analysis. Eleven children were diagnosed with acquired aplastic anemia (AAA); one probably after treatment with NSAIDs, one patient presented after influenza virus infection, while two patients presented also with transaminasemia of unknown etiology. First line therapy was hematopoietic stem-cell transplantation (HSCT), should an appropriate graft be available. In this respect, three patients (N: 3/4 ) with Fanconi anemia and one patient (N:1/11) with AAA were transplanted from a fully-matched sibling donor. One patient with AAA received autologous cord blood. The remaining ten patients (N: 10/15) received standard immunosuppressive therapy (antithymocyte globulin, cyclosporine-A and methylprednisolone). Eight of the 9 evaluable patients responded to therapy. Six of these patients also received treatment with eltrombopag, an oral thrombopoietin-receptor agonist, for at least six months. Eltrombopag was provided as off-label compassionate use and after having received approval from regulatory authorities. In these 6 patients, treatment with eltrombopag was well tolerated with no additive toxicity. Five patients showed progressive improvement of hematological values during the treatment with eltrombopag. Two patients with AAA, did not respond to immunosuppressive therapy, and subsequently underwent MUD-HSCT. One succumbed due to severe autoimmune hemolytic anemia, while the second has showed good engraftment, but with short post-BMT follow up time. Of note is, that one patient with Fanconi anemia showed full hematological recovery after immunosuppressive treatment. This patient, who was found to be homozygous for a FANC-E mutation, did not have any clinical stigmata. It is speculated that the unusual response to therapy may be due to its very mild clinical phenotype. Conclusions: Survival rates in severe AA have remarkably improved in the last decades due to allo-HSCT, immunosuppressive therapy and intense supportive care. This small series of children with AA underlines the option of new effective modalities. The use of autologous umbilical cord blood should be considered as an alternative first line therapy. Eltrombopag seems to improve platelet count and result in a tri-lineage response, in a manner similar to the one observed in adults with AA. Finally, the efficacy of immunosuppressive treatment in a patient with Fanconi anemia has not been previously described and warrants further evaluation. DisclosuresKattamis:Novartis: Honoraria, Research Funding; ApoPharma: Honoraria.

Sequential intravital imaging reveals in vivo dynamics of pancreatic tissue transplanted under the kidney capsule in mice.

Aims/hypothesisDynamic processes in pancreatic tissue are difficult to study. We aimed to develop an intravital imaging method to longitudinally examine engraftment, vascularisation, expansion and differentiation in mature islets or embryonic pancreases transplanted under the kidney capsule.MethodsIsolated pancreatic islets from adult mice and murine embryonic day (E)12.5 pancreases containing fluorescent biomarkers were transplanted under the kidney capsule of immunodeficient recipient mice. Human islet cells were dispersed, transduced with a lentivirus expressing a fluorescent label and reaggregated before transplantation. Graft-containing kidneys were positioned subcutaneously and an imaging window was fitted into the skin on top of the kidney. Intravital imaging using multiphoton microscopy was performed for up to 2 weeks. Volumes of fluorescently labelled cells were determined as a measure of development and survival.ResultsTransplanted islets and embryonic pancreases showed good engraftment and remained viable. Engraftment and vascularisation could be longitudinally examined in murine and human islet cells. Murine islet beta cell volume was unchanged over time. Transplanted embryonic pancreases increased to up to 6.1 times of their original volume and beta cell volume increased 90 times during 2 weeks.Conclusions/interpretationThis method allows for repeated intravital imaging of grafts containing various sources of pancreatic tissue transplanted under the kidney capsule. Using fluorescent markers, dynamic information concerning engraftment or differentiation can be visualised and measured.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-4049-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Enhanced engraftment, proliferation, and therapeutic potential in heart using optimized human iPSC-derived cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (CMs) are a promising tool for cardiac cell therapy. Although transplantation of induced pluripotent stem cell (iPSC)-derived CMs have been reported in several animal models, the treatment effect was limited, probably due to poor optimization of the injected cells. To optimize graft cells for cardiac reconstruction, we compared the engraftment efficiency of intramyocardially-injected undifferentiated-iPSCs, day4 mesodermal cells, and day8, day20, and day30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day20 CMs was significantly higher compared to other cells. Transplantation of day20 CMs into the infarcted hearts of immunodeficient mice showed good engraftment, and echocardiography showed significant functional improvement by cell therapy. Moreover, the imaging signal and ratio of Ki67-positive CMs at 3 months post injection indicated engrafted CMs proliferated in the host heart. Although this graft growth reached a plateau at 3 months, histological analysis confirmed progressive maturation from 3 to 6 months. These results suggested that day20 CMs had very high engraftment, proliferation, and therapeutic potential in host mouse hearts. They also demonstrate this model can be used to track the fate of transplanted cells over a long time.

Hematopoietic stem cell transplantation in patients with Hurler syndrome – myeloablative conditioning efficiency

Background. Allogenic hematopoietic stem cell transplantation (allo-HSCT) for patients with mucopolysaccharidosis is the radical method of therapy nowadays. Myeloablative conditioning regimens allow to achieve good engraftment and do not compromise clinical status of a patient. Aim – to show effectiveness of allo-HSCT with myeloablative conditioning for patients with Hurler syndrome (HS). Patients and methods . We analyzed 23 allo-HSCT at 22 patients with HS which were performed in 2002–2015. HSCT from unrelated donors (10/10 and 9/10) were performed in 18 cases. Transplant: bone marrow – 73.9 % (n = 17); peripheral blood stem cells – 17.4 % (n = 4); cord blood (CB) – 8.7 % (n = 2). Age median – 1.86 y.o. (10 months – 3.8 years). Conditioning regimen: Busulphan/Treosulphan + Fludarabine + Thiotepa/Melphalan and Antithymocyte Immunoglobulin +/– Rituximab (in case of HSCT from unrelated donor). Results. 19 patients are alive now. 15 patients have complete donor’s chimerism now. All patients have good answer on general disease (best answer correlated with minimal interval between diagnosis and HSCT). Primary graft failure was in one case of CB transplantation. Graft rejection was diagnosed in 3 cases. Causes of death were infection complications in all cases. Results improved during last 5 years – good infection control and therapy of infection and immune complications. Median of follow-up is 48.62 (8–130) months, overall survival 83.2 %, event-free survival – 71.5 %. Conclusion. Our results suggested that allo-HSCT with myeloablative conditioning (with Melphalan inclusion) for the patients with HS is effective way of therapy to stop neurodegeneration processes. Optimization of prevention and treatment of infection complications and graftversus-host disease allows to improve significantly results of HSCT.

The characteristics of human cranial bone marrow mesenchymal stem cells

Recently, cell-based therapy has attracted attention for treatment of central nervous system (CNS) disorders. Bone marrow-derived mesenchymal stem cells (BMSCs) are considered to have good engraftment potential. Therefore, more efficient and less invasive methods to obtain donor cells are required. Here, we established human BMSCs from cranial bone waste (cBMSCs) obtained following routine neurosurgical procedures.cBMSCs and cells obtained from the iliac crest (iBMSCs, standard BMSCs) showed expression of cell surface markers associated with mesenchymal stem cells and multipotency traits such as differentiation into osteogenic and adipogenic lineages. cBMSCs showed higher expression of the neural crest-associated mRNAs p75, Slug, and Snail than iBMSCs. Neurogenic induced cells from cBMSCs expressed the neural markers nestin, Pax6, neurofilament (NF)-L, and NF-M as seen with RT-PCR, and NF-M protein as seen with western blotting at higher levels than cells from iBMSCs. Immunostaining showed a significantly greater proportion of NF-M-positive cells in the population of induced cBMSCs compared with the population of iBMSCs. Thus, cBMSCs showed a greater tendency to differentiate into neuron-like cells than iBMSCs.

Defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous S32I mutation in IκBα

Patients with ectodermal dysplasia with immunodeficiency (ED-ID) caused by mutations in the inhibitor of NF-κB α (IκBα) are susceptible to severe recurrent infections, despite normal T and B cell numbers and intact in vitro lymphocyte function. Moreover, the outcome of hematopoietic stem cell transplantation (HSCT) in these patients is poor despite good engraftment. Mice heterozygous for the IκBα S32I mutation found in patients exhibited typical features of ED-ID. Strikingly, the mice lacked lymph nodes, Peyer's patches, splenic marginal zones, and follicular dendritic cells and failed to develop contact hypersensitivity (CHS) or form germinal centers (GCs), all features not previously recognized in patients and typical of defective noncanonical NF-κB signaling. Lymphotoxin β receptor (LTβR)-driven induction of chemokines and adhesion molecules mediated by both canonical and noncanonical NF-κB pathways was impaired, and levels of p100 were markedly diminished in the mutant. IκBα mutant → Rag2(-/-), but not WT→IκBα mutant, bone marrow chimeras formed proper lymphoid organs and developed CHS and GCs. Defective architectural cell function explains the immunodeficiency and poor outcome of HSCT in patients with IκBα deficiency and suggests that correction of this niche is critical for reconstituting their immune function.

Intrabone Injection of T-Cell Depleted Peripheral Blood Stem Cells from HLA-Haploidentical Donors to Reduce the Risk of Graft Rejection in Children

▪Background. Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective treatment option for patients with malignant and non-malignant hematologic disorders lacking an HLA-compatible donor. Strategies for T-cell depletion (TCD) of the graft, such as positive selection of CD34+ cells, offer the potential to prevent acute and chronic graft-versus-host disease (GVHD). The risk of graft rejection associated with the extensive depletion of both T lymphocytes and accessory cells can be overcome by infusing a very high number (megadose) of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) (exceeding 10x106/kg recipient body weight) to overcome the HLA barrier (Aversa F. et al. Blood 1994). Moreover, the infusion of a megadose of CD34+ cells (higher than 20x106/kg and 12.4x106/kg, respectively) has been shown to result in faster immunological recovery and improved leukemia-free survival probability in children (Handgretinger R. et al. Bone Marrow Transplant 2001; Klingebiel T. et al. Blood 2010).Nevertheless, in the case of donors considered “poor mobilizers” (10-30% of cases), the threshold dose of CD34+ cells needed to ensure the inoculum of a megadose of stem cells might not be achieved. In the setting of cord blood (CB) transplantation, one of the strategies aimed at overcoming the problem of low cellularity is represented by the intrabone injection of CB stem cells, with good engraftment rates even in adult patients. We explored the same strategy in the context of T-cell depleted haplo-HSCT and low graft cellularity due to poor donor mobilization, ensuing in inadequate dose of CD34+cells available after positive selection TCD.Patients and methods. From September 2009 to April 2013, 11 pediatric patients affected by malignant or non-malignant hematological disorders (5 acute lymphoblastic leukemias, 1 acute myeloid leukemia, 1 myelodysplastic syndrome, 2 dyskeratosis congenita, 1 Fanconi anemia) received a T-cell depleted CD34+positively selected PBSC allograft from an HLA-haploidentical related donor.Due to the failure to achieve a target cell dose higher than 12x106 purified CD34+ cells/kg, part of the stem cell inoculum was infused as intrabone injection. The procedure was carried out at the patient bedside by multiple intrabone injections in the superior-posterior iliac crests under sedoanalgesia, as previously described (Frassoni F. et al. Lancet Oncol 2008). The median dose of CD34+ cells infused was 9x106/kg (range, 5-12) while the median number of CD3+ lymphocytes was 0.7x104/kg recipient body weight (range, 0.3-11). About one third of the stem cell inoculum, corresponding to a total volume of 20-40 ml, was given intrabone, while the remaining stem cell portion was infused intravenously.Results.No complication occurred during, or immediately after, the intrabone injection. Nine out of the 11 patients achieved a complete donor engraftment, while graft rejection occurred in 2 patients. The median time for neutrophil engraftment was 13.5 days (range, 12-20), while the median time for platelet recovery was 14 days (range, 13-24). One patient developed grade II acute GVHD and only 1 case of limited chronic GVHD was observed. No transplant-related deaths were observed.Conclusions. Our data suggest that, in the haplo-HSCT setting, the intrabone injection of positively selected CD34+ cells, can be safely used in cases of low graft cellularity due to poor donor mobilization, with the aim of minimizing the risk of graft rejection or poor engraftment. Our preliminary data need to be confirmed in larger series of patients and compared with those obtained with conventional intravenous administration of comparable dose of CD34+ cells. DisclosuresNo relevant conflicts of interest to declare.

The Outcomes of Second Allogeneic Stem Cell Transplantation for Disease Relapse after T Cell Depleted Allogeneic Stem Cell Transplantation: A Single Center Experience-University of Chicago

The outcome of disease relapse after allogeneic stem cell transplantation (allo-SCT) remains very poor. Second SCT has achieved long term survival in select pts. We retrospectively analyzed the outcomes of 65 pts who underwent 2rd SCT for disease relapse at University of Chicago between September 2002 and September 2013. All except 4 pts received T cell depleted (TCD) SCT as 1st SCT mainly with fludarabine (flu)/clofarabine-melphalan (mel) -alemtuzumab; flu-busulfan-alemtuzumab for MRD or MUD, and flu-mel-ATG for haplo-cord SCT. The majority of pts had AML (n=46) and high risk MDS (n=5). The median age at 2nd allo-SCT was 47ys (11-73). 33 pts, 21 pts and 11 pts received MRD, MUD and Haplo-Cord SCT respectively for 2nd SCT. 13 pts (20%) achieved CR before 2nd SCT. 71% pts received TCD conditioning for 2nd SCT. 98% (n=64) and 72% (n=47) pts achieved neutrophil and platelet engraftment at a median time of 11 and 18 days, respectively, following the 2nd SCT. With a median fellow up of 23 (5.5-140) months for survivors after 2nd SCT, the estimated 1 year PFS was 29.2% and 1 year OS was 33.8%. The day 100 treatment related mortality (TRM) rate was 28%, and the cumulative incidence of aGVHD and cGVHD were 23% and 8%, respectively. Not surprisingly, the majority of pts died from disease relapse (22/47, 47%), followed by infection (11/47, 23%) and GVHD (6/47, 13%). In the univariate analysis, pts with remission duration after 1st SCT >=12 months or CR status before 2nd SCT had significantly better PFS (P=0.001 and 0.009) and OS (P=0.001 and 0.013). The differences of 1 year PFS and OS stratified by these two variables were very striking. The 1 years PFS for pts in CR was 62% compared to 21% for pts not in CR; and 1 year PFS for pts with remission duration >=12 months after 1st SCT was 57% comparing around 14% for pts <12 months. On the other hand, donor type (MRD vs. MUD vs. haplo-cord), conditioning (RIC vs. myeloablative) had no influence on PFS and OS. While age (<=60 vs. >60) had no influence on 1 year PFS (P=0.083), younger pts had better 3 year PFS (P=0.026); Alemtuzumab use (n=37 vs. n=16 without alemtuzumab) in the 2nd SCT conditioning for related donors significantly worsened 3 years PFS (44% vs. 8%; P=0.027) but not 1 year PFS, and it had no effect on OS. A scoring system using age (<=60 vs. >60), disease status before 2nd SCT (CR vs. non-CR), and remission duration after 1st SCT (<6, 6-12 and >=12 months) was generated to attempt to classify the pts into different risk categories. The groups of 0 (n=5), 1-2 (n=34), 3-4 (n=26) could separate the 1 year PFS (80% vs. 32.3% vs. 15.4%) and OS (100% vs. 35.3 vs. 19.2%) nicely. Although the numbers are small, pts with a score of 0 (<=60 yrs, in CR, and >=12 months remission duration after 1st SCT) had excellent outcomes with 1 year PFS of 80%, and 1 year OS of 100%. In conclusion, 2nd allo-SCT is a viable option for disease relapse after TCD allo-SCT with acceptable GVHD and good engraftment for those entering transplant in remission and/or remission duration >=12 months after 1st SCT. Abstract 2509. TablePatient Outcomes after 2nd SCTAll pts CR not in CR Remission duration >=12 months Remission duration 6-12 monthsRemission duration <6 months score of 0 score of 1-2 score of >=3 Pts number (n)651352231527534261 yearPFS29.2%61.5%21.2%56.5%13.3%14.8%80%32.3%15.4%3 year PFS21.5%53.8%13.5%43.5%6.7%11.1%80%20.6%11.5%1 year OS33.8%69.2%25%60.9%20%18.5%100%35.3%19.2%3 year OS23.1%46.2%17.3%43.5%6.7%14.8%80%20.6%15.4% DisclosuresArtz:Miltenyi: Research Funding. Stock:Sigma-Tau: Membership on an entity’s Board of Directors or advisory committees, Research Funding.