Single-dose Total Body Irradiation Research Articles

TCRαβ +/CD19 + - Depleted HLA-Haploidentical Hematopoietic Stem Cell Traansplantation in Pediatric Patient with Fanconi Anemia

Background Allogeneic hematopoietic stem cell transplantation (HSCT) currently represents the only proven treatment able to rescue bone marrow failure and prevent the occurrence of clonal evolution in patients with Fanconi Anemia (FA). If HSCT from HLA-identical related donor (MRD) is reported to be associated with better outcomes, this is not available for most patients. On the other hand, the identification of a fully matched unrelated donor (MUD) may be unsuccessful, or require times incompatible with a prompt treatment of the haematological condition. For patients who lack a MRD or MUD, the preferred donor choice is debated. HLA partially matched haploidentical donor (haplo) is virtually immediately available for all patients and haplo-HSCT has been reported over the last few years as a promising alternative for those patients. Following the favorable results already reported from our group of a cohort of pediatric and young adult FA patients underwent haplo-HSCT, based on the selective depletion of TCRαβ + and CD19 + cells graft manipulation, here we present the outcome of an enlarged cohort of patients who received HSCT in 3 different centers. Methods Patients diagnosed with FA and eligible for an allogeneic transplantation, but lacking a MRD or MUD, and with a suitable HLA-haploidentical donor were eligible to this study. The conditioning regimen included intravenous fludarabine, cyclophosphamide and single-dose total body irradiation for most patients. Pretransplant anti-T-lymphocyte globulin was administered in all patient to control in vivo bidirectional donor-recipient alloreactivity. Apheresis and graft manipulation were performed as previously reported (Li Pira, Biol Blood Marrow Transplant 2016). Results Thirty-five patients (19 males and 16 females) underwent haplo-HSCT between September 2011 and April 2023 in 3 different centers (Bambino Gesù Children's Hospital, Rome, Italy; Policlinico San Matteo, Pavia, Italy; Tokyo; National Center for Child Health and Development, Children's Cancer Center, Tokyo, Japan). Twenty-four patients have been previously reported (Strocchio, Blood Adv 2021). Details on patients and donor characteristics, graft composition and conditioning regimen are listed in table 1. One patient with clonal evolution and 1 with AML received fludarabine and AraC 3 weeks before the start of the conditioning. Primary engraftment was achieved in 33 patients (94.3%), with median time for neutrophil and platelet recovery of 12 days (range, 9-20 days) and 10 days (range, 7-14 days), respectively. Primary graft failure occurred in 2 patients and both were successfully transplanted from the other haploidentical parent. The monitoring of chimerism analysis confirmed a stable engraftment of donor hemopoiesis in all engrafted patients. Within 33 evaluable patients, 9 (27.3%) experienced grade 1-2 aGvHD (7 with stage I-II skin-only involvement, 1 with stage I lower gastrointestinal involvement) at a median time of 35.5 days (range, 16-67 days) after HSCT. One patient developed late-onset skin only grade 2 aGvHD at day +211 after HSCT. One patient (3%) experienced grade IV aGvHD (liver and gastrointestinal involvement) at day + 100 from HSCT, 4 weeks after the infusion of adenoviral-specific donor T cells; he is currently receiving ruxolitinib and the complete resolution of GvHD was obtained. Only one patient (3%), developed cGvHD with mild skin involvement. Thirteen patients (39.4%) experienced one or more viral infections or reactivations (6 cytomegalovirus, 4 herpesvirus-6, 2 adenovirus, 1 parainfluenzae); the specific antiviral treatment was administered with a complete resolution of infection in all cases. With a median follow up of 5.5 years (range, 0.6-11.4 years) all the patients are alive and with resolution of hematological complications. Except for the already reported vulvar intraepithelial neoplasia, no additional posttransplant malignancy was observed Conclusions Our data obtained from enlarged cohort of patients previously reported, confirm excellent results in terms of survival and a good rate of engraftment also in a multicenter setting. The low incidence of acute and chronic GvHD, a particularly detrimental complication in patients with DNA repair disorder, makes this approach attractive in this patient population. In FA patients lacking a standard donor, TCRab +/CD19 + haplo-HSCT is confirmed to be a promising strategy.

Total-Body Irradiation Alters White Matter Volume and Microstructural Integrity in Rhesus Macaques

Long-term survivors of brain irradiation can suffer from irreversible injury and cognitive impairment. T1-weighted and diffusion tensor magnetic resonance imaging are used to evaluate brain volumes and white matter (WM) microstructure in neurodevelopmental and neurodegenerative conditions. The goal of this study was to evaluate the long-term effects of single-dose total-body irradiation (TBI) or TBI with 5% partial-body sparing on brain volumetrics and WM integrity in macaques. We utilized MRI scans from a cohort of male rhesus macaques (age range: 3.6-22.8 years), to compare global and regional brain volumes and WM diffusion in survivors of TBI (T1w: n=137, DTI: n=121, dose range: 3.5-10 Gy) against unirradiated controls (T1w: n=48, DTI: n=38). In all ROIs, radiation affected the age-related changes in fractional anisotropy (FA); in general, FA increased across age in both groups, but to a lesser extent in the irradiated group (interaction p-values <0.01). Depending on the ROI, mean diffusivity (MD) decreased or remained the same across age in unirradiated animals, while it increased or did not change in irradiated animals. The increases in MD were driven by changes in radial diffusivity (RD) which followed similar trends across age. Axial diffusivity (AD) did not differ by irradiation status. Age-related changes in relative volumes in controls reflected normal trends in humans with increasing WM and decreasing gray matter (GM) until middle age. Cerebrospinal fluid (CSF) volume did not differ across age in controls. WM volume was lower and CSF volume was higher in young, irradiated macaques. WM volume was similar between groups and CSF volume reduced in older irradiated macaques. GM volume was unaffected by radiation. TBI results in delayed WM expansion and long-term disruption of WM integrity. Diffusion changes suggest that myelin injury in WM is a hallmark of late-delayed radiation induced brain injury.

CytoRADx: A High-Throughput, Standardized Biodosimetry Diagnostic System Based on the Cytokinesis-Block Micronucleus Assay.

In a large-scale catastrophe, such as a nuclear detonation in a major city, it will be crucial to accurately diagnose large numbers of people to direct scarce medical resources to those in greatest need. Currently no FDA-cleared tests are available to diagnose radiation exposures, which can lead to complex, life-threatening injuries. To address this gap, we have achieved substantial advancements in radiation biodosimetry through refinement and adaptation of the cytokinesis-block micronucleus (CBMN) assay as a high throughput, quantitative diagnostic test. The classical CBMN approach, which quantifies micronuclei (MN) resulting from DNA damage, suffers from considerable time and expert labor requirements, in addition to a lack of universal methodology across laboratories. We have developed the CytoRADx™ System to address these drawbacks by implementing a standardized reagent kit, optimized assay protocol, fully automated microscopy and image analysis, and integrated dose prediction. These enhancements allow the CytoRADx System to obtain high-throughput, standardized results without specialized labor or laboratory-specific calibration curves. The CytoRADx System has been optimized for use with both humans and non-human primates (NHP) to quantify radiation dose-dependent formation of micronuclei in lymphocytes, observed using whole blood samples. Cell nuclei and resulting MN are fluorescently stained and preserved on durable microscope slides using materials provided in the kit. Up to 1,000 slides per day are subsequently scanned using the commercially based RADxScan™ Imager with customized software, which automatically quantifies the cellular features and calculates the radiation dose. Using less than 1 mL of blood, irradiated ex vivo, our system has demonstrated accurate and precise measurement of exposures from 0 to 8 Gy (90% of results within 1 Gy of delivered dose). These results were obtained from 636 human samples (24 distinct donors) and 445 NHP samples (30 distinct subjects). The system demonstrated comparable results during in vivo studies, including an investigation of 43 NHPs receiving single-dose total-body irradiation. System performance is repeatable across laboratories, operators, and instruments. Results are also statistically similar across diverse populations, considering various demographics, common medications, medical conditions, and acute injuries associated with radiological disasters. Dose calculations are stable over time as well, providing reproducible results for at least 28 days postirradiation, and for blood specimens collected and stored at room temperature for at least 72 h. The CytoRADx System provides significant advancements in the field of biodosimetry that will enable accurate diagnoses across diverse populations in large-scale emergency scenarios. In addition, our technological enhancements to the well-established CBMN assay provide a pathway for future diagnostic applications, such as toxicology and oncology.

TNFAIP8 (TIPE) Deficiency Exacerbates Acute Graft Versus-Host Disease in Murine Model

Background: Acute graft-versus-host disease (GVHD) occurs after allogeneic hematopoietic cell transplant (alloHCT) and presents a donor immune cell response against host tissues associated with significant morbidity and mortality. Up to 60% of alloHCT recipients will develop acute GVHD. Damage to the gastrointestinal tract from the conditioning is a key step in its pathogenesis by allowing for systemic translocation of lipopolysaccharide (LPS) and other danger signals, hereby promoting host APC activation. Tumor necrosis factor (TNF)–induced protein 8 (TNFAIP8) family is a newly identified group of proteins consisting of TNFAIP8 (TIPE), TIPE1 (TNFAIP8L1, TNF–induced protein 8-like 1), TIPE2 (TNFAIP8L2), and TIPE3 (TNFAIP8L3). TIPE acts as a negative mediator of apoptosis via inhibition of caspase-3 activation and also induces proliferation. TIPE signaling is involved in activation of NF-kappa-B and AP-1 pathways. TIPE-deficient mice have been shown to develop more severe colitis than wild type mice due to TIPE deficiency in non-hematopoietic cells along with higher levels of TNF and IL-6. Given the potential roles of TIPE in regulating these processes and considering the importance of these cytokines and of epithelial injury in the pathophysiology of GVHD, we hypothesized, that TIPE deficiency of allogeneic HCT recipients results in worsened GVHD.Methods: 9 - 12 week old naive C57BL/6 or TIPE knockout C57BL/6 mice were conditioned with 1000cGy single dose TBI, followed by transplantation of 10 million bone marrow cells and 20 million splenocytes from either syngeneic C57BL/6 or allogeneic BALB/c donors. Animals were monitored for clinical GVHD and survival and analyzed at week 6 after HCT for serum cytokines, T cell expansion, whole spleen cell chimerism and cell proliferation marker.Results: Allogeneic wild type recipients demonstrated significantly better survival when compared to allogeneic TIPE knockout recipients by week 6 (66.7% vs 40.0%, p =0.024). All syngeneic animals survived. Clinical GVHD scores were significantly higher in TIPE-deficient allogeneic recipients at Day +28 (p=0.0166), +35 (p=0.0349), +42 (p=0.0241) compared to allogeneic wild type recipients. Day +42 serum cytokine analysis revealed increased IL-17A (p=0.0027), TNF (p=0.0396), IL-6 (p=0.0064) levels in TIPE-deficient allogeneic recipients compared to allogeneic control. This was associated with increased splenic CD4+ (p=0.04) but not CD8+ (p= 0.0898) cell expansion when compared to allogeneic control. TIPE-deficient and allogeneic control mice showed no differences in splenic donor T cell chimerism (84.4 versus 89.4, p=0.178), indicating comparable donor T cell engraftment. Ki-67 expression was significantly decreased in epithelial cells of small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls, and a similar nonsignificant trend was seen in the colon as well. Active caspase-3 expression was significantly increased in the small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls and a similar nonsignificant trend was seen in the colon as well. This observation was associated with significantly increased GI tract pathology scores of TIPE-deficient allogeneic recipients compared to allogeneic control. No differences were observed in pathology of lung and liver between TIPE-deficient allogeneic recipients and allogeneic control. Histopathologic analysis of T cell infiltration in small intestine and colon by immunohistochemistry for CD3 antibody revealed no difference between allogeneic groups, suggesting a role of TIPE-deficient nonhematopoietic cells in GVHD pathophysiology.Conclusion TIPE deficiency in allogeneic C57BL/6 recipients resulted in increased mortality after transplant, which was associated with significant increase in pro-inflammatory cytokines expression and decreased proliferation and increased apoptosis of intestinal epithelial cells, suggesting not only exaggerated inflammatory damage but also decreased epithelial regeneration. Therefore, TIPE seems to be a TNF receptor family member with rather protective function in GVHD gut pathophysiology and overexpression may be a potential future target to prevent or treat this complication after allo HCT. DisclosuresNo relevant conflicts of interest to declare.

Final Height of Patients Who Underwent Bone Marrow Transplantation for Hematological Disorders During Childhood: A Study by the Working Party for Late Effects-EBMT

AbstractFew data are available on the long-term effect of bone marrow transplantation (BMT) on growth. This study examines those factors that play a role in the final height outcome of patients who underwent BMT during childhood. Data on 181 of 230 patients with aplastic anemia, leukemias, and lymphomas who had BMT before puberty (mean age, 9.8 ± 2.6 years) and who had reached their final height were analyzed. An overall decrease in final height standard deviation score (SDS) value was found compared with the height at BMT (P < 107) and with the genetic height (P < 107). Girls did better than boys, and the younger in age the person was at time of BMT, the greater the loss in height. Previous cranial irradiation + single-dose total body irradiation (TBI) caused the greatest negative effect on final height achievement (P < 104). Fractionation of TBI reduces this effect significantly and conditioning with busulfan and cyclophosphamide seems to eliminate it. The type of transplantation, graft-versus-host disease, growth hormone, or steroid treatment did not influence final height. Irradiation, male gender and young age at BMT were found to be major factors for long-term height loss. Nevertheless, the majority of patients (140/181) have reached adult height within the normal range of the general population.

An Amino Acid–Based Oral Rehydration Solution Regulates Radiation-Induced Intestinal Barrier Disruption in Mice

Radiotherapy inadvertently affects gastrointestinal (GI) epithelial cells, causing intestinal barrier disruption and increased permeability. We examined the effect of amino acid-based oral rehydration solution (AA-ORS) on radiation-induced changes of intestinal barrier function and epithelial tight junctions (TJs) in a randomized experimental study using a total-body irradiation (TBI) mouse model. Eight-week-old male Swiss mice received a single-dose TBI (0, 1, 3, or 5Gy), and subsequent gastric gavage with AA-ORS (threonine, valine, serine, tyrosine, and aspartic acid) or saline for 2 or 6 d. Intestinal barrier function of mouse ileum was characterized by electrophysiological analysis of conductance, anion selectivity, and paracellular permeability [fluorescein isothiocyanate (FITC)-dextran]. Ultrastructural changes of TJs were evaluated by transmission electron microscopy. Membrane protein and mRNA expression of claudin-1, -2, -3, -5, and -7, occludin, and E-cadherin were analyzed with western blot, qPCR, and immunohistochemistry. Nonparametric tests were used to compare treatment-dose differences for each time point. Saline-treated mice had a higher conductance at doses as low as 3Gy, and as early as 2 d post-TBI compared with 0Gy (P<0.001). Paracellular permeability and dilution potential were increased 6 d after 5Gy TBI (P<0.001). Conductance decreased with AA-ORS after 2 d in 3-Gy and 5-Gy mice (P<0.05 and P<0.001), and on day 6 after 5Gy TBI (P<0.001). Anion selectivity and FITC permeability decreased from 0.73±0.02 to 0.61±0.03 pCl/pNa (P<0.01) and from 2.7±0.1×105 to 2.1±0.1×105 RFU (P<0.001) in 5-Gy mice treated with AA-ORS for 6 d compared with saline. Irradiation-induced ultrastructural changes of TJs characterized by decreased electron density and gap formation improved with AA-ORS. Reduced claudin-1, -3, and -7 membrane expression after TBI recovered with AA-ORS within 6d, whereas claudin-2 decreased indicating restitution of TJ proteins. Radiation-induced functional and structural disruption of the intestinal barrier in mice is reversed by AA-ORS rendering AA-ORS a potential treatment option in prospective clinical trials in patients with gastrointestinal barrier dysfunction.

TNFAIP8 Deficiency Exacerbates Acute Graft Versus Host Disease in a Murine Model of Allogeneic Hematopoietic Cell Transplantation.

Gastrointestinal acute graft-versus-host disease (GVHD) occurring after allogeneic hematopoietic cell transplant is an allo-reactive T cell and inflammatory cytokine driven organ injury with epithelial apoptosis as 1 of its hallmark findings and is associated with significant mortality. Tumor necrosis factor (TNF)-alpha-induced protein 8 (TNFAIP8 or TIPE) acts as a negative mediator of apoptosis via inhibition of caspase-3 activation, promotes cell proliferation and Tipe deficiency is associated with increased inflammation. To evaluate the role of TIPE in acute GVHD, naive C57BL/6 and Tipe C57BL/6 mice were conditioned with 1000 cGy single dose total body irradiation, followed by transplantation of 10 million bone marrow cells and 20 million splenocytes from either syngeneic C57BL/6 or allogeneic BALB/c donors. Allo TIPE-deficient mice developed exacerbated gut GVHD compared with allo controls and had significantly decreased survival (6 wk overall survival: 85% versus 37%; P < 0.05), higher clinical GVHD scores, more profound weight loss, increased serum proinflammatory cytokines (interleukin-17A, TNF, interleukin-6, and interferon-γ). T-cell infiltration into the ileum was increased; epithelial proliferation was decreased along with significantly higher levels of chemokines KC and monokine induced by gamma interferon. Using bone marrow chimeric experiments, TIPE was found to have a role in both hematopoietic and nonhematopoietic cells. Absence of TIPE results in excessive inflammation and tissue injury after allo-HCT, supporting that TIPE confers immune homeostasis and has tissue-protective function during the development of gut GVHD and may be a potential future target to prevent or treat this complication after allogeneic HCT.

Busulfan Pharmacokinetics and Precision Dosing: Are Patients with Fanconi Anemia Different?

It is well known that pharmacokinetics (PK)-guided busulfan (BU) dosing increases engraftment rates and lowers hepatotoxicity in patients undergoing hematopoietic cell transplantation (HCT). However, there are no published PK data in patients with Fanconi anemia (FA), who are known to have baseline DNA repair defect and related inherent sensitivity to chemotherapy. In our prospective, multi-institutional study of alternative donor HCT for FA using chemotherapy-only conditioning, we replaced the single dose of total-body irradiation with BU at initial doses of 0.8 to 1.0 mg/kg and 0.6 to 0.8 mg/kg given i.v. every 12 hours for 4 doses. Patients received the first dose of i.v. busulfan on day -8, and blood levels for PK were obtained. PK samples were drawn following completion of infusion. BU PK levels were collected at 2 hours, 2 hours and 15 minutes, and 4, 5, 6, and 8 hours from the start of infusion. The remaining 3 doses of BU were given on days -7 and -6. Thirty-seven patients with available BU PK data with a median age of 9.2 years (range, 4.3 to 44 years) are included in the final analyses. The overall BU PK profile in patients with FA is similar to non-FA patients after considering their body weight. In our cohort, a strong correlation between BU clearance and weight supports current practice of per kilogram dosing. However, not surprisingly, we show that the disease (ie, host) sensitivity related to FA is the main determinant of total dose of BU that can be safely administered to patients in this high-risk population. On the basis of our results, we propose an optimal BU concentration at steady-state level of ≤350 ng/mL (equivalent to total cumulative exposure of 16.4 mg*h/L for 4 doses over 2 days) for patients with FA undergoing HCT. To our knowledge, this is the first and largest report of prospective BU PK in patients with FA undergoing HCT, providing an optimal BU target cutoff to achieve stable donor engraftment while avoiding excessive toxicity.

How Adoptive Immunotherapy with Conventional T and Regulatory T Cells Exerts a Gvl Effect without GvHD, after Haploidentical Hematopoietic Transplantation

▪Post-transplant relapse is still a major cause of treatment failure in high-risk acute leukemia (AL) patients. Attempts to manipulate donor T cell alloreactivity to spare normal tissues while killing leukemic cells have been largely unsuccessful. In the search for strategies to separate the GvL effect from GVHD, we investigated the role of a thymic-derived CD4+CD25+ FoxP3+ regulatory T-cell subpopulation (Tregs) that physiologically helps maintain immunological self-tolerance and immune homeostasis. Evidence from murine bone marrow (BM) transplantation across major histocompatibility barriers showed co-infusion of conventional T lymphocytes (Tcons) with Tregs suppressed lethal GVHD without impairing Tcon activity against malignant diseases. In 69 high-risk AL patients who had received an HLA haploidentical T cell-depleted hematopoietic transplant and no post-transplant immunosuppressive GvHD prophylaxis, adoptive immunotherapy with donor Tregs (2 × 106/kg) and Tcons (1 × 106/kg) protected patients from GvHD (Di Ianni et al., Blood 2011) and largely prevented post-transplant leukemia relapse. In fact, only 5% of patients relapsed (Martelli et al., Blood 2014). However, also because such patients had advanced stage disease, TRM was still in the range of that of T cell-depleted haplo transplants, (i.e. 40%, Aversa et al., JCO 2005). A current cohort of AL patients was conditioned with total body irradiation (TBI) (8Gy as single dose TBI or 13.5 Gy as fractionated TBI), cyclophosphamide (30mg/kg), thiotepa (8mg/kg) and fludarabine (200mg/m2). To date, 24 high-risk AL patients (7 ALL, 17 AML) have been enrolled. Twenty-two of the 24 patients engrafted, one AML patient relapsed, 4/22 developed aGvHD (4/4 are alive) and one developed cGvHD, TRM was exceptionally low (Fig. 1). At a median follow up of 2 years, 19 patients are alive. Consequently, probabilities of leukemia-free and leukemia/cGVHD-free survivals are good (Fig. 1). Hypotheses have been put forward to explain the ability of Tregs to prevent of GvHD while preserving the GvL effect mediated by Tcons. However, the mechanism is still unknown. In humans, naïve CD45RA+ Tregs express CXCR4 BM homing receptor and preferentially localize to the BM while memory CD45RO+ Tregs display lower CXCR4 expression and home to the periphery (Booth et al., J Immunol. 2010). Since Tregs that are recovered from peripheral blood and are used for adoptive immunotherapy are CD45RO+ and largely CxCR4 negative, we hypothesized that GvL without GvHD might be due to unopposed Tcon alloreactivity in the BM combined with regulated T cell alloreactivity at the periphery. In order to verify such hypothesis, we infused NSG mice with human leukemia and human Tregs and Tcons. Mice that received leukemia and haploidentical Tcons (without Tregs) cleared leukemia but died of GvHD. T cells harvested from their BM, spleen and liver were predominantly CD8+ and displayed alloreactivity against leukemia. Mice that received leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. T cells harvested from spleen and liver were composed of CD8+ T cells (40%) and CD4+T cells (60%). Purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction. Purified CD8+ T cells displayed no alloreactivity against leukemia. In contrast, T cells harvested from BM were still predominantly CD8+ and still displayed alloreactivity against leukemia suggesting Tcons had retained their alloantigen recognition. Finally, in mice treated with Tregs alone, T cells were recovered only in the spleen and liver, they displayed a CD4+ phenotype and inhibited mixed lymphocyte reaction. No T cells were found in the BM. In contrast, when we infused human purified CD45RA+ naïve Tregs (that display the CXCR4 BM homing receptor), mice died of leukemia progression despite the co-infusion of large doses of Tcons. In the BM we isolated CD8+ T cells that displayed no ability to kill leukemia and CD4+ T cells that displayed regulatory function. Thus, naïve Tregs homed to the BM and blocked the GvL effect of T cons. When CXCR4 was blocked with an anti-CXCR4 antibody, naïve Tregs could not home to the BM and T cons killed leukemia. In conclusion, Treg-Tcon adoptive immunotherapy confines the graft-versus-host alloreaction to the hematopoietic system and, consequently, allows a GvL effect without GvHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Post-Transplant Cyclophosphamide as Sole Graft-versus-Host Disease Prophylaxis Is Feasible in Patients Undergoing Peripheral Blood Stem Cell Transplantation for Severe Aplastic Anemia Using Matched Sibling Donors

High-dose cyclophosphamide (PTCY) after allogeneic hematopoietic cell transplantation (HSCT) has been shown to be effective in preventing graft-versus-host disease (GVHD) after HLA-matched bone marrow transplantation. We performed a phase II study of PTCY given at 50 mg/kg i.v. on days 3 and 4 as the sole GVHD prophylaxis after HSCT for severe aplastic anemia (SAA) in patients receiving granulocyte colony-stimulating factor–mobilized peripheral blood stem cell (PBSC) grafts from HLA-matched related donors after conditioning with fludarabine, CY, and single-dose total body irradiation. Thirty patients with a median age of 29 years (range, 16 to 49) were enrolled in this study. Engraftment was seen in 27 patients (90%) at a median of 16 days (range, 12 to 21) post-HSCT. None of the patients developed veno-occlusive disease of the liver or hemorrhagic cystitis. Grades II to IV acute GVHD was seen in 22% of patients with grades III to IV GVHD in 11.1%. The 2-year cumulative incidence of chronic GVHD was 22.7%. Fourteen patients (46.6%) did not require any further immunosuppression after receiving PTCY. Comparing with 2 historical cohorts of 30 patients each who received cyclosporine and methotrexate (MTX; at 15 mg/m2 [MTX15] and 10 mg/m2 [MTX10]), the incidence of grades II to IV acute GVHD was lower, albeit not significantly, with the use of PTCY (PTCY, 22.2%, vs MTX15, 37.1%, vs MTX10, 53.8%; P = .056), whereas rates of chronic GVHD were significantly reduced (PTCY, 22.7%, vs MTX15, 63.6%, vs MTX10, 76.2%; P = .013). Viral infections including cytomegalovirus were significantly higher with the use of PTCY (60%) compared with cyclosporine and MTX (MTX15, 23.3%, vs MTX10, 33.3%; P = .008). Overall survival was similar between the 3 groups. We conclude that PTCY as the sole GVHD prophylaxis is associated with low rates of acute and chronic GVHD in patients undergoing PBSC transplant for SAA using HLA-matched donors. This trial is registered at CTRI/2010/091/001480.

A Comparison of Fractionated Myeloablative Total Body Irradiation Schedules Combined with Chemotherapy As Conditioning for Allograft Bone Marrow Transplantation in Patients with Acute Leukemia: The Sarasin Study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT)

IntroductionTotal-body irradiation (TBI) has an historical established role in preparative regimens used before allogeneic transplant in both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The most popular myeloablative conditioning consists of 12Gy TBI administered in 6 fractions (2Gy twice daily for 3 days) in combination with cyclophosphamide. This schedule of treatment delivery is, however, time-consuming. With limited availability of irradiation equipment, many departments in the world limit the number of patients receiving TBI due to non-medical reasons. Therefore, one of the possible solutions is to reduce the number of fractions for a similar effective dose. The aim of the SARASIN study was to analyze the impact of fractionation on outcome of patients undergoing allotransplant for ALL and AML.Patients and methodsWe retrospectively compared myeloablative TBI regimens of 3126 patients registered in the EBMT database transplanted between 2000 and 2014 for ALL (n=1783) or AML (n=1343). Pre-transplant chemotherapy consisted mainly of cyclophosphamide (Cy) in 92% and 97% of ALL and AML patients, respectively. TBI was delivered as either 12Gy in 6 fractions (group 1; ALL, n=1362 and AML, n=857), or single dose TBI (STBI) (group 2; ALL, n=54 and AML, n=79), or 9-12Gy in 2 fractions (group 3; ALL, n=173 and AML, n=256), or 12Gy in 3-4 fractions (group 4; ALL, n=194 and AML, n=151). The majority (70%-79%)* of ALL and AML (57%-79%) patients were grafted in 1stcomplete remission (CR1). The rate of transplants from unrelated donors was higher in ALL (24%-50%) as compared to AML (20%-37%) of the patients, with similar rates of non-in vitro T-cell depletion that ranged from 25% to 96% in the 4 TBI groups, respectively. Graft versus host disease (GvHD) prevention consisted mainly (75% to 89%) of cyclosporine and methotrexate.ResultsThe median follow-up was 61 (1-87) months and 85 (1-192) months in the ALL and AML patients, respectively. At 5 years, leukemia free survival (LFS), overall survival (OS), relapse incidence (RI) and non-relapse mortality (NRM) were 46.5% (44.1% - 49%), 50.4% (47.9% - 52.9%), 29% (26.7% - 31.1%), 24.5% (22.5% - 26.6%) in ALL and 45.7% (43% - 48.5%), 48% (45.3% - 51%), 30.4% (28% - 33%) and 23.8% (21.5% - 26.2%), respectively. LFS at 5y in AML and ALL patients were respectively: 48% and 45%, 32% and 45%, 45% and 53%, 42% and 50% in the 4 TBI groups (p=0.082 for AML and p=0.32 for ALL). Additionally, for both AML and ALL, no statistical significance was found between the 4 TBI groups for OS (p=0.82 in ALL; p=0.11 in AML), RI (p=0.29 in ALL; p=0.23 in AML) and for NRM (p=0.58 in ALL; p=0.12 in AML). In multivariate analyses of TBI schedules, comparing the different schedules to the standard 12Gy in 6 fractions (group 1 vs group 2; group 1 vs group 3; group 1 vs group 4), fractionation was not found as independent prognostic factor neither in ALL nor in AML patients for LFS, OS, RI or NRM.ConclusionThe SARASIN study showed that using a TBI dose of 12Gy as pre allogeneic transplantation, fractionation has no impact on relapse or survival neither in ALL, nor in AML patients. Furthermore, the reduction of the number of fractions even in this rather high total body radiation dose level is not associated with increased risk of NRM. Altogether, our data suggests that 12Gy could be delivered safely in less than 6 fractions. Late effects analyses are ongoing. Our findings are not only of considerable practical importance for radiotherapy departments but moreover it may lead to increase TBI availability as pre transplantation conditioning for leukemic patients undergoing allogeneic transplantation. DisclosuresMichallet:Astellas Pharma: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria.

HLA-Haploidentical TCR αβ/CD19-Depleted Hematopoietic Stem Cell Transplantation in Children with Fanconi Anemia

Introduction. Allogeneic HSCT currently represents the only consolidated curative approach for Fanconi anemia (FA) patients, with best results observed in the HLA-identical sibling setting. For patients lacking an HLA-matched related or unrelated donor, haploidentical HSCT virtually assures the opportunity for nearly all patients to benefit from HSCT, offering the advantage of immediate accessibility to the transplant procedure. In order to overcome the limitation of delayed immune recovery, historically associated with this type of allograft, in the last few years we developed a novel method of ex vivo graft manipulation, consisting of the negative depletion of T-cell receptor (TCR) αβ+ T-lymphocytes and CD19 B-cells from peripheral blood stem cells (PBSC) grafts (ClinicalTrial.gov identifier: NCT01810120) (Bertaina el al., Blood 2014). Here we report our analysis in a subgroup of FA patients given TCRαβ/CD19-depleted haploidentical HSCT at our Institution.Patients and methods. Ten consecutive FA patients (6 girls and 4 boys) underwent a TCRαβ/CD19-depleted HSCT from an HLA-haploidentical relative between September 2011 and July 2015. Median age at diagnosis was 6.6 (range 2.7-22.0) years and median age at time of transplantation was 8.1 (range 4.4-22.2) years. The conditioning regimen included Cyclophosphamide 300 mg/m2/day and Fludarabine 30 mg/m2/day for 4 consecutive days (days -6 to -3), with 200 cGy single-dose TBI. Pretransplantation Fresenius® ATG was administered at a dose of 4 mg/kg/day for 3 consecutive days (days -5 to -3) in order to prevent both graft failure and graft-versus-host disease (GVHD). All patients received Rituximab 200 mg/m2 to reduce the risk of Epstein-Barr virus-related post-transplant lymphoproliferative disorders. Selective removal of TCRαβ+ and B-cells was performed on G-CSF-mobilized donor PBSC through labeling with biotinylated anti-TCRαβ antibodies and anti-CD19 antibodies, followed by incubation with anti-biotin antibodies conjugated to paramagnetic beads (CliniMACS; Miltenyi Biotec, Bergisch Gladbach, Germany). No immunosuppressive therapy was administered as post-transplantation prophylaxis against GVHD.Results. The TCRαβ/CD19-depletedgrafts contained a median of 20.40 x106/kg (range 15.80-33.40) CD34+ cells, 5.60 x106/kg (range 1.78-69.60) CD3+ lymphocytes, 0.021 x106/kg (range 0.002-0.043) TCRαβ+ lymphocytes, 5.60 x106/kg (range 1.78-69.60) TCRγδ+ lymphocytes, 0.036 x106/kg (range 0.013-0.079) CD20+ lymphocytes, and 45.30 x106/kg (range 16.2-177.0) NK cells. Engraftment with sustained full donor chimerism was achieved in 9 out of 10 patients, the cumulative incidence of graft rejection being 10% (95% CI, 0-26.8). The patient who rejected his first allograft achieved a complete engraftment after a second HSCT from one-antigen mismatched unrelated donor. No secondary graft failures were observed. The median time for neutrophil and platelet engraftment was 12 days (range, 9-15) and 9 days (range, 8-12), respectively. No patient experienced acute or chronic GVHD in the follow-up period. No transplant-related deaths occurred in our cohort. With a median follow-up of 28 months (range 13.2-39.1), the Kaplan-Meier estimates of OS and DFS were both 100%, while the EFS probability was 90% (95% CI, 47.3-98.5).Discussion. These data suggest that haploidentical HSCT after removal of TCRαβ+ and CD19+ lymphocytes is able to guarantee engraftment with excellent OS and DFS in patients affected by FA. Moreover, given the very low incidence of both acute and chronic GVHD, which has been shown to contribute to the increased risk of developing late post-transplantation malignancies in FA patients, this approach can be considered a very attractive option for FA patients in need of an allograft and lacking an HLA-identical family donor. DisclosuresNo relevant conflicts of interest to declare.

The Total Body Irradiation Schedule Affects Acute Leukemia Relapse After Matched T Cell–Depleted Hematopoietic Stem Cell Transplantation

We sought to determine whether the total body irradiation (TBI) schedule affected outcome in patients with acute leukemia in complete remission who received Tcell-depleted allogeneic hematopoietic stem cell transplantation from HLA identical siblings. The study recruited 55 patients (median age, 48years; age range, 20-66years; 30 men and 25 women; 34 with acute myeloid leukemia and 21 with acute lymphoid leukemia). Hyperfractionated TBI (HTBI) (1.2Gy thrice daily for 4days [for a total dose of 14.4Gy] from day -12 to day -9) was administered to 29 patients. Single-dose TBI (STBI) (8Gy, at a median dose rate of 10.7cGy/min on day -9) was given to 26 patients. All patients achieved primary, sustained engraftment with full donor-type chimerism. At 10years, the overall cumulative incidence of transplant-related mortality was 11% (SE, ±0.1%). It was 7% (SE, ±0.2%) after HTBI and 15% (SE, ±0.5%) after STBI (P=.3). The overall cumulative incidence of relapse was 33% (SE, ±0.5). It was 13% (SE, ±0.5%) after HTBI and 46% (SE, ±1%) after STBI (P=.02). The overall probability of disease-free survival (DFS) was 59% (SE, ±7%). It was 67% (SE, ±0.84%) after HTBI and 37% (SE, ±1.4%) after STBI (P=.01). Multivariate analyses showed the TBI schedule was the only risk factor that significantly affected relapse and DFS (P=.01 and P=.03, respectively). In patients with acute leukemia, HTBI is more efficacious than STBI in eradicating minimal residual disease after HLA-matched Tcell-depleted hematopoietic stem cell transplantation, thus affecting DFS.

Simvastatin mitigates increases in risk factors for and the occurrence of cardiac disease following 10Gy total body irradiation.

The ability of simvastatin to mitigate the increases in risk factors for and the occurrence of cardiac disease after 10 Gy total body irradiation (TBI) was determined. This radiation dose is relevant to conditioning for stem cell transplantation and threats from radiological terrorism. Male rats received single dose TBI of 10 Gy. Age-matched, sham-irradiated rats served as controls. Lipid profile, heart and liver morphology and cardiac mechanical function were determined for up to 120 days after irradiation. TBI resulted in a sustained increase in total- and LDL-cholesterol (low-density lipoprotein-cholesterol), and triglycerides. Simvastatin (10 mg/kg body weight/day) administered continuously from 9 days after irradiation mitigated TBI-induced increases in total- and LDL-cholesterol and triglycerides, as well as liver injury. TBI resulted in cellular peri-arterial fibrosis, whereas control hearts had less collagen and fibrosis. Simvastatin mitigated these morphological injuries. TBI resulted in cardiac mechanical dysfunction. Simvastatin mitigated cardiac mechanical dysfunction 20–120 days following TBI. To determine whether simvastatin affects the ability of the heart to withstand stress after TBI, injury from myocardial ischemia/reperfusion was determined in vitro. TBI increased the severity of an induced myocardial infarction at 20 and 80 days after irradiation. Simvastatin mitigated the severity of this myocardial infarction at 20 and 80 days following TBI. It is concluded simvastatin mitigated the increases in risk factors for cardiac disease and the extent of cardiac disease following TBI. This statin may be developed as a medical countermeasure for the mitigation of radiation-induced cardiac disease.

Mechanisms of graft-versus-leukemia effects after allogeneic stem cell transplantation: effects on the leukemia stem cell?

Total body irradiation (TBI) was originally thought to be capable of eradicating leukemia, but with time it became apparent that we cannot eradicate this disease with TBI.1 Today we know that graft-versus-leukemia effects are more important than previously thought. Strong evidence came from the depletion of T cells from the graft for the prevention of graft-versus-host disease (GVHD): the rate of relapses increased particularly in chronic myelogenous leukemia (CML),2 and in some patients the graft was even rejected. However, some patients with refractory acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were treated with intensive chemotherapy and a single dose of TBI and are alive and in remission today more than 30 years later. Therefore, the question arises whether immune therapy with lymphocytes from the stem cell donor can eradicate leukemia stem cells without the help of high-dose TBI or stem-cell toxic chemotherapy. The best clinical evidence for leukemia stem cells is provided by the occurrence of late relapses presumably arising from radio- and chemo-resistant, quiescent stem cells. Late relapses after allogeneic stem cell transplantation were observed in patients treated for CML3 and less frequently in those treated for AML and ALL.4 In these patients stem cells of leukemia may have been recruited from a quiescent state and may have given rise to open relapse.

Beneficiary Effect of Autologous Hematopoietic Cell Transplantation in Idiopathic Ulcerative Dermatitis C57BL/6 Mice

Idiopathic ulcerative dermatitis (IUD) is a distinctive skin disease in C57BL/6 mice. The pathogenesis of this disease is still unknown but it has been suggested that scratching character of this strain and immune complex induced vasculitis play a role. There is no established treatment for idiopathic ulcerative dermatitis. Naive C57BL/6 with lesions suggestive of IUD with ulceration, necrosis and bleeding were identified and confirmed clinically by institutional veterinarian. Mice with severe lesions were euthanized and skin sample biopsies confirmed IUD by pathology. Mice with mild to moderate ulceration were distributed into two groups, of which one was treated with autologous hematopoietic cell transplantion (HCT) and the remaining were kept as observation control. For HCT, recipients received 950cGy single dose total body irradiation followed by injection of bone marrow cells from IUD-free C57BL/6 mice. Animals were monitored daily until the end of week 4, when autologous transplant recipients showed improved skin condition without ulceration. Compared to observation controls, autologous transplant seemed to accelerate the healing process, suggesting that inducing temporary immune-incompetency targeting auto-reactive B and T lymphocytes seems beneficial in this disease. Suggesting that dysregulation of cellular immune responses is contributory to IUD, our findings further imply potential value for autologous HCT related immunosuppression in the treatment of chronic autoimmune dermatological diseases.

The Prolyl Hydroxylase Inhibitor Dimethyl Oxalyl Glycine Decreases the Severity of Acute Lung Injury After Allogeneic Hematopoietic Cell Transplantation.

AbstractAbstract 3006 Background:Allogeneic hematopoietic cell transplantation (HCT) is increasingly utilized in the treatment of hematologic malignant and non-malignant diseases. Therapy-related toxicity and graft versus host disease (GVHD) remain major challenges, with acute lung injury being associated with significant morbidity and mortality. Prolyl hydroxylase inhibitors (PHI) interfere with signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection resulted in the hypothesis, that the PHI DMOG (Dimethyl oxalyl glycine) may reduce lung injury evolving after murine allogeneic HCT. Methods:14 week old BALB/c were conditioned with 750 cGy single dose TBI, followed by transplantation of 5×10E6 bone marrow cells and 4×10E6 splenocytes from either syngeneic BALB/c or allogeneic C57BL/6 donors. DMOG in PBS was given to both syngeneic and allogeneic recipients at 8mg per mice ip on alternate days for the first two weeks post-transplant and then twice a week till day 50, while syngeneic and allogeneic controls received PBS only. Animals were monitored for clinical GVHD and survival. At day 50 post HCT, gut, liver and lung pathology as well as BAL fluid T cell numbers were assessed by using a scoring system quantifying severity and extent of parenchymal and peribronchial/perivascular infiltrates and flow cytometry, respectively. In addition, T cell expansion was analyzed both in vivo in the spleen on day +7 and in vitro by mixed lymphocyte reaction (MLR). Results:Allogeneic HCT resulted in severe lung pathology in PBS treated animals (mean 118.8±47.19), but was significantly reduced when DMOG was given (36.0±17.99; p < 0.05), and absent in both PBS and DMOG treated syngeneic controls. Consistently, BAL CD4+ and CD8+ T cells in DMOG recipients were decreased by 53.7% and 68.4%, respectively, when compared to PBS controls. Decreased pulmonary injury was associated with a trend towards better overall survival by day 50 (allogeneic DMOG: 59.2% vs allogeneic PBS 40.7%) and all syngeneic recipients survived. GVHD related injury to liver and GI tract at time of analysis was not different between allogeneic groups. Alloreactive proliferative T cell responses in vitro were suppressed in the presence of DMOG at a concentration of 1mM (cpm after 96h: mean 29316±875.9 vs 41735±3085; p < 0.01). In vivo, splenic T cell expansion of CD4+ but not CD8+ T cells was reduced at day +7 as well (p < 0.05). Interestingly, when compared to PBS treated controls reduced mortality of DMOG treated allogeneic HCT recipients could already be seen soon after HCT (day +10: 26.0% vs 11.1%) in association with decreased CD4+ T cell proliferation, while lung injury in this model this early is rather marginal. Conclusion:DMOG treatment decreases the severity of acute noninfectious lung injury and improves overall survival after allogeneic HCT. While impaired early T cell responses likely contribute to improvement of both endpoints, altered recruitment of CD8+ T cells to the lung seems to uniquely affect the development of pulmonary but not gastrointestinal and hepatic injury at later time points. Current studies are ongoing to delineate the effects of DMOG on T cell proliferation and recruitment to different target organs, and to define the role of this novel approach in the prevention and treatment of HCT related complications. Disclosures:No relevant conflicts of interest to declare.

Prognostic Impact of Iron Overload During Follow-up After Allogeneic Stem Cell Transplantation

Abstract 347▪▪This icon denotes a clinically relevant abstract Introduction:Iron overload is frequent in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) due to multiple red blood cell transfusions in the pre- and post-transplant period. Serum ferritin is a routine marker for iron overload. Along with transferrin saturation and soluble transferrin receptor, it was recently shown to strongly impact on survival after HSCT. Reduction of iron overload – e.g. by chelation - might improve the outcome after allogeneic transplantation. To analyze whether patients might benefit from post-transplant interventions aimed at reducing iron overload, we evaluated the impact of iron parameters on survival in a cohort of patients in which complete iron parameters were assessed before and at multiple time-points after transplantation. Methods:We studied 153 consecutive patients undergoing unmanipulated T-cell replete allogeneic transplantation at our center between 2005 and 2009. Among 90 males and 63 females with a median age of 46.5 years (range 18 – 70 years), underlying diseases were AML/MDS (n=87), ALL (n=25), lymphoma/myeloma (n=25), CML/MPN (n=12) and non-malignant (n=4). Donors were HLA-identical (n=78) or one-antigen mismatched (n=2) siblings, or unrelated volunteer donors (n=73). Patients with lymphoma received conditioning with BEAM, fludarabine and single dose total body irradiation (TBI). All other patients were treated with cyclophosphamide and busulfan, or cyclophosphamide and TBI +/− etoposide. Graft-vs-host disease prophylaxis was with Cylosporin A plus either methotrexate or mycophenolate. Serum iron parameters including ferritin, transferrin saturation, transferrin, iron and soluble transferrin receptor (sTfR) were determined before HSCT as well as 3, 6, 12, 24, 36 and 60 months post-transplant. Patients were categorized into groups with high or low iron parameters according to values above or below median at each time-point. Predictors of transplant outcome were further evaluated in multivariable Cox models using disease and stage as covariates. Results:Of the 153 patients, 83 were alive at last contact with a median follow-up of 3.4 years. Ferritin was strongly elevated before HSCT (median 1344 ng/ml, range 16–6507 ng/ml), peaked 3 months post-transplant (median 2508 ng/ml, range 71–9756 ng/ml) and continuously decreased to reach values within the normal range at 5 years (median 242.5 ng/ml, range 28–984ng/ml). Transferrin saturation and iron analogously peaked in the early post-transplant period and subsequently lowered, while transferrin and sTfR increased after an early post-transplant nadir up to 5 years. The post-transplant course of all iron parameters is shown in Fig 1. As demonstrated previously, survival analysis showed a poor survival of patients with a pre-transplant ferritin value above the median (hazard ratio 2.4, p=0.001). Time-stratified landmark survival analysis showed that iron overload (as assessed by ferritin levels above the median at the respective time-point) had a detrimental effect on survival in all periods analyzed (0–6 months p=0.01; 6–12 months p<0.001; 1–2 years p=0.07; and 2–5 years p=0.02. Figure 2). Interestingly, no patient with a ferritin level below the median died more than six months post-transplant. Excess mortality in patients with high ferritin was due to increased transplant related mortality. After adjustment for disease and stage, elevated ferritin values retained their prognostic significance. Analysis of the other iron parameters showed similar trends, but their prognostic value was lower than that of ferritin. [Display omitted] [Display omitted] Conclusions:Iron overload before HSCT as well as during the post-transplant follow-up confers an increased risk of death. Iron parameters significantly impact on survival during all post-transplant intervals analyzed. Thus, our data suggest that patients may benefit from interventions to reduce iron overload after HSCT. Disclosures:No relevant conflicts of interest to declare.

Infusion of Mismatched Ex Vivo Expanded and Cryopreserved Murine Hematopoietic Progenitors Rescues Mice After Lethal Radiation Exposure,

Abstract 4006 Introduction:Despite the large body of research designed to assess the effects of radiation on hematopoiesis, specific therapies aimed at improving the survival of individuals accidentally exposed to higher doses of whole body irradiation are lacking. The ideal therapeutic agent would be an immediately available, easily distributable single agent therapy able to generate at least temporary in vivo hematopoietic recovery, especially of the myeloid lineage, and facilitate return of autologous hematopoiesis. We have previously demonstrated that murine lin−/sca-1+/c-kit+ (LSK) cells which have been ex vivo expanded in the presence of an immobilized Notch ligand form consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG1 (Delta1ext-IgG) can engraft across MHC barriers. We now show that the same product is also capable of reducing radiation toxicity and improving overall survival. Methods: LSK cells were isolated by flow cytometry from whole bone marrow (BM) of Ly5a mice (H-2b, CD45.1) and placed in culture in the presence of immobilized Notch ligand and Iscove’s medium supplemented with cytokines mSCF, hFlt-3 ligand, hIL-6, and hIL-11. After 14 days, expanded cells were harvested and cryopreserved in 90% FBS + 10% DMSO. On the day of transplant, frozen cells were thawed, washed, and resuspended in PBS + 1% FBS. To determine the ability of the expanded cells to contribute to in vivo hematopoietic recovery and provide radioprotection in a MHC mismatched model, Balb-c recipient mice (H-2d, CD45.2) were treated with single dose TBI at 7, 7.5 or 8 Gy followed by infusion of either a PBS + 1% FBS solution (PBS) or 5 × 106expanded cells. The results presented herein represent the combined data from 3 consecutive experiments, including a total of 150 mice for the generation of survival curves and an additional 54 mice to assess donor engraftment. All experiments were approved by the Animal Care and Use Committee (IACUC), animals were observed for a minimum of 30 days to assess survival. Moribund animals that met specific criteria established by the IACUC were euthanized and counted as an experimental death. Separately, mice were sacrificed at 1, 2, and 3 weeks after infusion and peripheral blood and BM collected to determine the degree of donor hematopoiesis by flow cytometry. PB was also collected at day 45 to evaluate donor chimerism and total white counts (WBC) recovery. Results: At a TBI dose of 8 Gy, 97% of mice that received PBS died in contrast to only 52% mortality in the group treated with 5 × 106expanded cells (p<0.0001). Similarly, at a dose of 7.5 Gy, the survival rate at 30 days was 25% and 70% for mice that received PBS and expanded cells, respectively (p=0.0004). When the TBI doses was reduced to 7 Gy, all mice infused with expanded cells survived and only one died in the PBS group (p=0.22) (Figure 1). Of note, no further deaths were observed after 30 days and mice that received 8 Gy recovered WBC by day 45. Evaluation of donor derived hematopoiesis demonstrated in vivo persistence of the mismatched expanded cells through day 28, with peak engraftment occurring before day 14. The percent of donor cells in the peripheral blood one week following infusion ranged between 50 to 95% in all mice that received expanded cells, with higher donor engraftment correlating with increased dose of TBI. The level of donor engraftment then decreased over time, but in the surviving mice remained persistent to levels ranging from 10 to 30%. Importantly, no signs of graft versus host disease (GVHD) were observed despite the full-mismatch between the recipient and donor product. Conclusions: Ex vivo expanded and cryopreserved murine hematopoietic progenitors can be infused safely and effectively to provide temporary donor derived hematopoiesis and rescue mice after lethal acute ionizing radiation. Importantly, such cells can be ex vivo expanded, cryopreserved, and banked for future treatment of neutropenia as a result of accidental radiological exposures or therapies. Furthermore, in vivo transplantation studies demonstrate that short-term engraftment without manifestations of GVHD can be attained across major H-2 barriers, supporting the use of this product as a universal or third party donor product. Studies are on-going aimed at determining an optimal range of cell doses and extending the window after radiation exposure when the cryopreserved, expanded products can be effective. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Xerostomia in children and adolescents after stem cell transplantation conditioned with total body irradiation or busulfan

To study salivary secretion rates and symptoms of xerostomia in children and adolescents conditioned with either radiation therapy or with chemotherapy only in the setting of hematopoietic stem cell transplantation (HSCT). Thirty patients conditioned with 10 Gy single dose TBI (sTBI) and cyclophosphamide (Cy) 60 mg/kg for two days and 35 conditioned busulfan (Bu) and Cy as part of the preparative regimen were included in the study. All patients were treated before 13 years of age, and had survived 2-16 years after HSCT. All patients were interviewed according to a standard questionnaire on symptoms of xerostomia and the unstimulated and stimulated whole salivary secretion rate was determined. The stimulated salivary secretion rates were 0.8±0.5 ml/min in sTBI/Cy group compared to 1.1±0.6 ml/min in the Bu/Cy group (p=0.01). Dysfunction of either unstimulated or stimulated salivary secretion rates were found in 18/30 (60%) in sTBI/Cy group and 9/35 (26%) in Bu/Cy group (p<0.01). There were no differences regarding the number of xerostomia related symptoms in children conditioned with either sTBI/Cy or Bu/Cy. Both unstimulated and stimulated salivary secretion rates were inversely correlated to the total number of complaints of xerostomia. This study shows that children exhibit xerostomia after HSCT irrespective of conditioning with busulfan or sTBI. It is of importance that salivary function is evaluated and that both salivary function as well as the subjective feeling of mouth dryness is evaluated.