Bone Marrow Transplantation Procedures Research Articles

Incidence, Clinical Characteristics and Risk Factors for Poor Mobilization of Hematopoietic Stem Cells in a Colombian Bone Marrow Transplantation Unit: A Retrospective Cohort Study

Background: mobilized peripheral hematopoietic stem cells (PHSC) are the main cell source for autologous bone marrow transplantation. Failure to mobilize an appropriate number of PHSC can jeopardize autologous bone marrow transplantation procedure. Objective: our aim was to describe the clinical and demographics characteristics, the incidence of mobilization failure and the prevalence of risks factors for poor mobilization in patients in whom PHSC mobilization were attempted for autologous bone marrow transplantation between January 1st 2017 and 31 December 2022. A univariate and multivariate analysis (binary logistic regression) were done to determinate the association between risk factors and mobilization failure. Results: 129 patients were included , the patients median age was 54 years. Indications for autologous bone marrow transplantation were multiple myeloma in 50,4 %, lymphoma in 46,5 % and systemic sclerosis in 2,3 % of patients respectively. Mobilization was attempted with Filgrastim in 71,1 %, Filgrastim plus Plerixafor in 26,6 % and chemotherapy (Cyclophosphamide) plus Filgrastim in 2,3 % of patients respectively. Mobilization failure occurred in 9,3 % of patients (12 patients) occurring in 11 % of patients who received Filgrastim, in 5,6 % who received Filgrastim plus Plerixafor and in none of patients who received chemotherapy plus Filgrastim. No difference in mobilization failure was found between patients who received Filgrastim monotherapy versus those who received Filgrastim plus Plerixafor ( χ 2 p=0,388, relative risk: 1,8, confidence interval 95% 0,43 - 8,1). Patients who received Filgrastim plus Plerixafor had been treated more frequently (four of more cycles of Lenalidomide) compared with patients who received Filgrastim alone (63,3 % versus 18,7 %, χ 2 p < 0,001, RR=4,01, CI 95 %; 2,32-6,87).The median age of patients was superior in patients who received Filgrastim plus Plerixafor compared with those who received Filgrastim (58 vs 51 years, U Mann Whitney p = 0,02). The median number of treatment lines tended to be superior in patients receiving Filgrastim plus Plerixafor compared to those receiving Filgrastim (2 lines versus 1 line of therapy, U Mann Withney p= 0,054). Patients who had been treated with intensive chemotherapy regimens like DHAP, ESHAP or Hyper CVAD (p=0,004, RR: 4,3, CI 1,5-12,3)) and patients older than 60 years (p=0,018, RR: 3,4 (1,2-9,85)) were significant more prone to mobilization failure in bivariate and multivariate analysis. Conclusion: mobilization failure occurred in 9,3 % of patients , Filgrastim monotherapy was more frequently used, no difference in mobilization failure was found between those who received Filgrastim versus Filgrastim plus Plerixafor, history of treatment with more than 4 cycles of lenalidomide and older age were more frequently in patients who received Filgrastim plus Plerixafor. History of a treatment intensive chemotherapy regimen and being older than 60 years were consistently associated with mobilization failure in univariate and multivariate analysis.

Assessing the Efficacy of Alkylating Agent Regimens in the Treatment of Infantile Malignant Osteopetrosis: Cyclophosphamide, Busulfan, or Thiotepa.

Infantile malignant osteopetrosis is a debilitating disease that requires total bone marrow irradiation and transplant procedures for patients to survive. The major complication of this procedure is graft vs host disease (GVHD), followed by infections and end organ toxicity. Therefore, current research efforts into treatment mainly aim to reduce GVHD while limiting infections and organ toxicity. Different regimens of alkylating agents have been used to try to reduce GVHD. The most common regimen is cyclophosphamide (Cy) with busulfan (Bu), followed by Cy with Bu and thiotepa (Thio). This meta-analysis aimed to evaluate the efficacy of different treatments by comparing mortality and morbidity causes and rates across groups. The mean one-year survival rate for the Cy, Bu, Thio regimen studies in the human leukocyte antigen (HLA) unmatched group (45.01%) was statistically lower than the one-year survival rate for the studies using just a Cy, Bu regimen (70.8%) in the HLA unmatched studies (p<0.00142). The one-year survival in the studies which had HLA-matched donors was 80.56%, which is statistically higher (p<0.001) than the one-year survival in the HLA-unmatched studies (53.96%), indicating a benefit of finding HLA-matched donors. It seems that price and availability could be a factor in the widespread use of Cy.

Etiologies of hearing loss in Fanconi Anemia

ObjectivesWe aim to describe types of hearing loss associated with Fanconi anemia patients who underwent a bone marrow transplant (BMT) to identify possible etiologies of hearing loss. Additionally, we hope to investigate hearing loss early in life as a potential predictor of needing a BMT surgery. Fanconi anemia is a rare autosomal recessive disease that is the most common inherited bone marrow failure syndrome, characterized by bone marrow failure and multiple congenital anomalies, including hearing loss. This is the largest study to date reviewing types of hearing loss in patients with Fanconi anemia, specifically in those who have undergone BMTs. MethodsA retrospective chart review of patients diagnosed with Fanconi anemia at a single institution, tertiary, referral-based children's hospital with a bone marrow transplant team specializing in Fanconi anemia was conducted from 4/19/1976 to 10/19/2015. History, physical examination, audiometry, and imaging findings were reviewed in patients with and without history of bone marrow transplant. Patient hearing levels, as measured by pure tone audiometry at 500 Hz, 1, 2, and 4 kHz, were evaluated. Patients were grouped by transplant status and results and were assessed to determine type and degree of hearing loss. Statistical analysis was performed to compare the likelihood of bone marrow transplant procedures in Fanconi anemia patients with normal and abnormal hearing. ResultsThere were 252 patients with Fanconi anemia identified via diagnosis search in institutional electronic medical records using CPT codes and cross referencing with the Fanconi Anemia database, 58 of whom had available audiometric data. Of the 58 Fanconi anemia patients with available audiograms, 21 (36%) had abnormal audiograms; 37 patients had normal audiograms. Twenty out of 21 (95%) patients who had abnormal audiograms had undergone bone marrow transplants. Thirty-one of 37 (84%) patients with normal audiograms had received bone marrow transplants. Statistical analysis showed that patients with hearing loss were more likely to require a BMT in the future (OR = 3.87, p = 0.05). Of the patients with abnormal audiograms and a bone marrow transplant (n = 20), 14 (70%) had conductive hearing loss, 5 (25%) had mixed hearing loss, and 1 patient (5%) had sensorineural hearing loss. 13 of 20 patients (65%) had bilateral hearing loss and eight of 20 (40%) had unilateral hearing loss. Of those patients with conductive hearing loss (n = 15), the most common etiologies were Eustachian tube dysfunction (47%), external auditory canal stenosis (33%), and abnormal middle ear anatomy (13%). ConclusionsHearing loss is a common finding in Fanconi anemia patients who have undergone BMTs with conductive hearing loss being the most common audiologic manifestation in our cohort of patients. This demonstrates the necessity of frequent hearing screenings in this population and close collaboration with audiology throughout patient care. Our study indicates that hearing status early in life may be a predictor of needing a bone marrow transplant in the future. Further studies should explore the long-term impact of BMT surgery on hearing status.

Assessing the Presence of Hematopoietic Stem and Progenitor Cells in Mouse Spleen.

Transplantation of hematopoietic material into recipient mice is an assay routinely used to determine the presence and function of hematopoietic stem and progenitor cells (HSPCs) in vivo . The principle of the method is to transplant donor cells being tested for HSPCs into a recipient mouse following bone marrow ablation and testing for reconstitution of hematopoiesis. Congenic mouse strains where donor and recipient differ by a distinct cell surface antigen (commonly CD45.1 versus CD45.2) are used to distinguish between cells derived from the donor and any residual recipient cells. Typically, the transplantation is performed using bone marrow cells, which are enriched for HSPCs. Here, we describe an analogous procedure using hematopoietic material from spleen, allowing detection of functional progenitors and/or stem cells in the spleen that can occur under certain pathologies. Key to the success of this procedure is the prior removal of mature T cells from the donor sample, to minimize graft versus host reactions. As such, this protocol is highly analogous to standard bone marrow transplant procedures, differing mainly only in the source of stem cells (spleen rather than bone marrow) and the recommendation for T cell depletion to avoid potential immune incompatibilities. Graphical abstract: Schematic overview for assessment of stem cells in spleen by transplantation. Single cell suspensions from spleens are depleted of potentially pathogenic mature T lymphocytes by magnetic bead immunoselection using biotinylated antibodies against CD4 and CD8, followed by streptavidin magnetic beads, which are subsequently removed by using a magnet (MojoSort, Biolegend). Successful T cell depletion is then evaluated by Fluorescence Activated Cell Sorting (FACS). T-cell depleted cell suspension is injected intravenously through the retro-orbital sinus into lethally irradiated recipients. Recipients are analyzed for successful engraftment by FACS analysis for the presence of donor-derived mature hematopoietic lineages in the peripheral blood. A second serial transplantation can be used to document the presence of long-term reconstituting stem cells in the periphery of the original donor mice.

Erythroid Chimerism Measurement As a Predictive Tool of Bone Marrow Transplantation Outcome in Sickle Cell Disease

Allogeneic bone marrow transplantation (BMT) is the only curative treatment available for sickle cell disease (SCD). The effectiveness of the treatment is primarily assessed by the chimerism rate, which is the fraction of cells derived from the donor's hematopoietic stem cells (HSCs) relative to the recipient's cells. New less toxic conditioning agents, described as reduced, nonmyeloablative (as opposed to myeloablative conditioning agents) are now used for BMT with sibling donors. Moreover, most sibling donors are sickle cell trait carriers (heterozygous A/S genotype). In the absence of a test performed on erythroid cells, the rate of chimerism is currently assessed either on different subpopulations of myeloid cells by molecular biology techniques or on hemoglobin measurement by chromatography (HPLC). However, the percentage of myeloid chimerism is rarely representative of the percentage of erythroid chimerism in these patients and HPLC cannot allow analysis at the single cell level.An easy-to-implement method for accurate measurement of fetal hemoglobin (HbF) content in individualized red blood cells (RBCs) has recently been developed (Hebert, et. al, AJH 2020). This method relies on flow cytometry for cell individualization and intracellular labeling of HbF with a fluorescent monoclonal antibody for quantification. Based on the same approach, we propose the use of monoclonal antibodies directed against adult hemoglobins (HbA0 and HbA2) or hemoglobin S (HbS). This strategy allows to discriminate, from a blood sample, the different RBC subpopulations coming from either the recipient or the donor, or from the transfusion associated with this procedure, thus allowing us to precisely determine the rates of erythroid chimerism in patients.As a proof of concept, we show here the evaluation of the percentage of erythroid chimerism during a longitudinal follow-up performed on 3 patients with SCD (P1 - 18 months, P2 - 4 months, and P3 - 4 months). The 3 patients received allogeneic BMT from a sibling A/S donor following nonmyeloablative conditioning consisting of the administration of an anti-CD52 antibody associated with radiotherapy (300 cGy TBI - total-body irradiation). We observed for P1 and P2 donor erythroid chimerism >90% and >95%, 1 and 2 months after BMT, respectively (measured after exclusion of transfused RBCs). These results are in accordance with the presence of most RBCs HbA- and HbS-positive, as determined by flow cytometry. In addition, the analysis of the reticulocyte (retic) subpopulations (CD71-positive cells) of these patients provides precise information on the RBC production of the bone marrow following transplantation. Retic donor chimerism levels were >99% for both P1 and P2, 1 month after BMT procedure. P1 and P2 showed stable donor reticulocyte and donor erythroid chimerism levels during the follow-up. On the contrary, P3 presented a different response with 43%, 41% and 5% donor erythroid chimerism 1, 2 and 3 months after BMT, respectively. Retrospectively, this bad response in P3 would have been predicted by low donor reticulocyte chimerism levels (5%, 6% and 0.5% at 1, 2 and 3 months after BMT, respectively). Our erythroid specific chimerism results were also compared to the assessment of chimerism performed on either total white blood cells (WBCs) or lineage specific subpopulations after sorting. Over 18 months after BMT in P1, the correlation between retic chimerism and total WBCs or T lymphocytes (CD3-positive cells) chimerism were not statistically significant (P = 1 and P = 0.833, respectively - Spearman test) (Figure 1).This new tool to monitor BMT outcome to treat SCD shows great precision and potentially a stronger predictive capability than total WBCs or lineage specific chimerism assessed by molecular biology. Our results highlight the importance of analyzing reticulocyte population to better assess BMT efficacy and predict the outcome a few days after the transplant. Its use could also be considered in the context of the various gene therapy strategies currently under development using modified β-globin gene addition or direct mutation correction through CRISPR/Cas9 and homologous recombination system. [Display omitted] DisclosuresBencheikh: Innovhem: Current Employment. Bartolucci: Innovhem: Current holder of individual stocks in a privately-held company.

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

An Emerging Multi-Drug Resistant Yeast Like Fungus Candida auris Infections in ICUs

The incidence of fungal infections in recent years is increasing rapidly and there is an emergence of newer fungal pathogens and anti-fungal drug resistance due to multiple predisposing reasons such as prolonged and indiscriminate use of antibiotic therapy, immunosuppressive corticosteroid therapy, aggressive use of ant-cancer drugs, bone marrow and organ transplantation procedures, and underlying conditions like immunodeficiency diseases (E.g., AIDS) and metabolic disorders such as diabetes mellitus.

Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma

AbstractThe fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5′-monophosphate–activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

Atypical non-progressive semantic impairment following allogeneic bone marrow transplantation in a patient with Waldenström’s macroglobulinemia: A case report

Objective: A case report of a 74-year-old male presenting with an atypical multimodal semantic impairment. The patient was diagnosed with Waldenström macroglobulinemia (WM) for which he received allogeneic bone marrow transplantation (BMT) due to disease progression. Following BMT, he developed a sudden onset of semantic difficulties that have remained unchanged for eight years. No other cognitive functions have been affected and his activities of daily living remain fully preserved.Method: The patient was assessed at our neuropsychology unit with six neuropsychological evaluations over an 8-years follow-up period following BMT. Additional semantic tests were administered during the last three evaluations. Four MRI scans (at age 62, 66, 69 and 74) and 18F-FDG PET (at age 74) were obtained.Results: The patient presents a multimodal semantic impairment, including naming impairment, visual agnosia, prosopoanomia, associative prosopagnosia, topographical disorientation and impaired retrograde memory for public events. MRI scans and 18F-FDG PET revealed bilateral symmetrical atrophy (temporal > frontal) and inferior bilateral temporal lobe hypometabolism, respectively. Neuroradiological examination was unremarkable prior to BMT.Conclusion: Clinical diagnosis remains a challenge given the focal and stable nature of his deficits. We hypothesize that the BMT procedure might have resulted in the temporal lobe damage and subsequent semantic impairment. We recommend obtaining a thorough neuropsychological evaluation of patients who receive allogenic BMT, both prior to and following transplant.

The Critical Role of Cell Homing in Cytotherapeutics and Regenerative Medicine

AbstractRecent advances in cell science and regenerative therapies mimic the therapeutic effects of patients’ own cells when they home to and accumulate at sites of injury. Inspired by stem cell trafficking under in vivo conditions, the facilitation of stem cell homing and the driving of endogenously mobilized mesenchymal stem cells (MSCs) for advanced therapeutics have shown indisputable success in several preclinical studies. In fact, stem cell homing is also relevant for cell transplantation. Ensuring that a sufficient number of transplanted cells arrive at the targeted region is a prerequisite for treatments to be successful, particularly when culture‐expanded cells are injected intravenously. In this progress report, the authors discuss the important role of cell homing following i) the delivery of ex vivo‐manipulated cellular materials, ii) the transplantation of bone marrow in a bone marrow transplant procedure, and iii) endogenous cell mobilization/recruitment in response to injury and disease. Considering a paradigm shift from in vitro tissue engineering to in situ tissue regeneration, current endeavors and future potential in the topics of chemokine‐guided cell homing and the design of cell‐comfortable scaffolds that combine both biochemical and biophysical cues for immunomodulation and in situ tissue regeneration are highlighted.

Experience of Children Undergoing During and After Bone Marrow Transplantation Procedure

Backgrounds Bone marrow transplantation (BMT) involves a lenghty hospital admission in an isolated environment to prevent infection and treatment with high immunosupression followed infusions of donor bone marrow. Thus, BMT has profound effect on the lives of children. There are many studies about parents/caregivers’ experiences regarding own children’s bone marrow transplantation/stem cell transplantation procedures. However, you can find limited studies about children’s experiences in literature. Aim The aim of this study was to identify children’s experience undergoing during and after BMT prosedures. Methods Using a qualitative descriptive desing, seven children, six boys and one girl between 7 and 15 years, were interviewed. Subjects’ responses were taped-recorded, transcribed verbatim and analysed by using content analysis. Finding Themes emerged from in four categories; (i) changing life style (family life, school life, amusement, activities), (ii) isolation, (iii) feeling and reactions (fear of painful procedure, frightening procedure), (iv) medical treatment and side effect. Conclusion Pediatric nurses in transplantation unit are in a exellent position to offer support and guidance to children undergoing BMT should encourage them to explore experience during BMT procedure.

BMT decreases HFD-induced weight gain associated with decreased preadipocyte number and insulin secretion.

Experimental bone marrow transplantation (BMT) in mice is commonly used to assess the role of immune cell-specific genes in various pathophysiological settings. The application of BMT in obesity research is hampered by the significant reduction in high-fat diet (HFD)-induced obesity. We set out to characterize metabolic tissues that may be affected by the BMT procedure and impair the HFD-induced response. Male C57BL/6 mice underwent syngeneic BMT using lethal irradiation. After a recovery period of 8 weeks they were fed a low-fat diet (LFD) or HFD for 16 weeks. HFD-induced obesity was reduced in mice after BMT as compared to HFD-fed control mice, characterized by both a reduced fat (-33%; p<0.01) and lean (-11%; p<0.01) mass, while food intake and energy expenditure were unaffected. As compared to control mice, BMT-treated mice had a reduced mature adipocyte volume (approx. -45%; p<0.05) and reduced numbers of preadipocytes (-38%; p<0.05) and macrophages (-62%; p<0.05) in subcutaneous, gonadal and visceral white adipose tissue. In BMT-treated mice, pancreas weight (-46%; p<0.01) was disproportionally decreased. This was associated with reduced plasma insulin (-68%; p<0.05) and C-peptide (-37%; p<0.01) levels and a delayed glucose clearance in BMT-treated mice on HFD as compared to control mice. In conclusion, the reduction in HFD-induced obesity after BMT in mice is at least partly due to alterations in the adipose tissue cell pool composition as well as to a decreased pancreatic secretion of the anabolic hormone insulin. These effects should be considered when interpreting results of experimental BMT in metabolic studies.

Aplastic Anemia in the Context of Hemolytic Paroxysmal Nocturnal Hemoglobinuria: Feasibility of Antibody-Based Intensive Immunosuppression during Eculizumab Treatment

Paroxysmal nocturnal hemoglobinuria (PNH) may present as hemolytic (classical PNH) or aplastic (AA/PNH syndrome) PNH. While classical PNH patients requires anti-complement treatment (eculizumab), the treatment of AA/PNH patients should target their bone marrow failure (BMF) by immunosuppression (IST), or even bone marrow transplantation (BMT). However, in a few patients clinically meaningful AA and hemolysis may be concomitant, eventually justifying both IST and eculizumab. To date there is no standard treatment for this rare condition. Here we investigated the prevalence of this condition based on a large cohort of PNH patients seen at our reference centers at St. Louis Hospital (Paris) and Federico II University (Naples), looking for patients who have received "intensive IST" in combination with eculizumab. Amongst a total of 145 PNH patients seen between 2007 and 2016, we have identified 6 patients who have received intensive IST during eculizumab treatment ; thus, roughly the prevalence of severe BMF associated with hemolysis is around 5%. All the 6 patients have been initially classified as AA/PNH syndrome at time of eculizumab starting, but did not require specific treatment before; they were all on eculizumab treatment at the standard dose of 900 mg fortnightly, with adequate control of intravascular hemolysis. Five of the patients fulfilled the criteria of severe AA, the latter had very severe isolated neutropenia, and was diagnosed with an immune-mediated agranulocytosis. Since no patient had a HLA-matched related donor for BMT, all the patients received intensive IST according to different institutional regimens; eculizumab was not discontinued to minimize the risk of rebound intravascular hemolysis and thrombotic complications. Three patients (2 AA and 1 agranulocytosis) received standard IST with horse-antithymocyte globulin (h-ATG, 40 mg/kg for 4 consecutive days) combined cyclosporine A (CsA). The 3 other AA patients received alemtuzumab (3-10-30-30 mg subcutaneously in 4 consecutive days) and CsA within the prospective trial NCT00895739; one of these patients a few months later also received a second IST course with rabbit-ATG (3.5 mg/kg for 5 consecutive days) and CsA. All the patients completed the scheduled treatment without any side effect, including infusion-related reactions. Lymphocyte depletion was observed in all patients irrespective of sustained therapeutic complement blockade, with lymphocyte count dropping <100/μL in all cases and lasting for several days (or even months in the case of alemtuzumab). All patients remained on eculizumab treatment, even if one AA patient required an increase of the dose up to 1200 mg because of pharmacokinetic breakthrough; in 2 patients 50% hemolytic complement (CH50) was systematically monitored during the treatment course, without showing any significant change from status at pre-IST treatment. All the patients were available for response assessment at 6 months; globally, 3 out of 6 showed a hematological response. The agranulocytosis patient achieved a partial response (PR) requiring G-CSF chronic treatment. Among the 5 AA patients, we observed 2 CR (1 with h-ATG and 1 with alemtuzumab) and 1 PR (after alemtuzumab); this latter was converted into a CR after a second IST course with r-ATG. One of the CR relapsed at 3 years showing clonal evolution toward a myelodysplastic syndrome, and finally died. All the other patients are alive, keeping their hematological response; of the 3 non-responders, 1 was rescued with an unrelated transplantation, and 2 remain on eculizumab treatment (one has developed thromboembolic complications).This is the first systematic description of the management of severe BMF in PNH patients on anti-complement treatment; we first observed that this rare challenging condition may pertain to about 5% of PNH patients. Then we demonstrated that intensive IST, based on either polyclonal or monoclonal anti-lymphocyte antibodies can be safely delivered even in concomitance of eculizumab. Pharmacological lymphocyte depletion was achieved irrespective of terminal complement inhibition. Clinical efficacy was in the expected range as in broader AA populations, with an overall reponse rate of 50%. Based on these data, intensive IST delivered on top of eculizumab should be considered in PNH patients who develop severe AA during eculizumab treatment lacking a low-risk bone marrow transplant procedure. DisclosuresPeffault de Latour:Amgen: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding. Risitano:Alnylam: Research Funding; Novartis: Research Funding; Alexion Pharmaceuticals: Other: lecture fees, Research Funding; Rapharma: Research Funding.

Haplo-Identical Bone Marrow Transplant Protocol using Reduced Intensity Conditioning for Fundeni Clinical Institute

Abstract Purpose: Even if the romanian population is ethnically compact caucasian-type population, many of the patients referred for bone marrow transplantation lack a suitable donor. In order to expand the donor pool and the accesibility to transplant for those who have indications it is necessary to perform haplo-identical bone marrow transplant procedure in Fundeni Clinical Institute. Since 2009 Romania established a National Volunteer Stem Cell Donor Registry (RNDVCSH), the goal was to enlarge the possibility to find HLA-matched unrelated donors (MUD) for patients. This approach offered transplant for only up to 60%patients referred for transplantation in 2014, even we chose one HLA-mismatch donors. The haploidentical transplant protocol proposed for our institution is based on Sidney Kimmel Comprehensive Cancer Center protocol from Johns Hopkins University School of Medicine. The major milestones of this protocol include: patient eligibility, donor selection criterias, evaluation of the haplo donor, the conditioning regimen plan and additional supportive care, the bone marrow harvest, prophylaxis of graft versus host disease, assessment during and after the transplant. Donor must be HLA-haploidentical first-degree relatives of the patient with signed consent. The patient, parents and children are typed at the allelic level for HLA-A, -B,-C,-DRB1 and -DQB1. They will perform also de HLA-antibody search using cross-match test in complement-dependent cytotoxicity. The conditioning regimen is composed by Fludarabine 30 mg/m2/day (from day -6 to day -2) combined with Cyclophosphamide 14,5 mg/kgIBW/day (from day -6 to day-5) and TBI at 2 Gyîn day -1. In case of lacking TBI procedure at 2 Gy dose in day -1, it could be replaced by two dose of Busulfan iv in day -3 and day-2 (dose=3,2 mg/kgIBW/day) for those with acute and chronic leukemias. The donor will have general anesthesia,the target yield of marrow is 4 x 10^8 total nucleated cells/kg recipient using his IBW. The GVHD prophylaxis consisted of post-transplant Cyclophosphamide (PTCy) of 50mg/kgIBW/day in IV administration in day +3 and +4, followed by tacrolimus and mycophenolatemofetil (MMF) beginning day +5. The MMF will be stopped at day+35, the tacrolimus will continue till 6 months after the transplantation. Conclusion: One of the most important factors affecting transplantation outcome is proper timing.Therefore, donor availability is an crucial issue. Haploidentical related donors are available for almost all patients, so the use of those donors is a viable alternative.

Isolation, Characterization and Application of BCR-ABL-ve Mesenchymal Stem Cells Derived from Peripheral Blood of Chronic Myeloid Leukemia (CML) in Enhancing Potentiality of Bone Marrow Transplantation in CML Patients

Chronic Myeloid leukemia (CML) is a disorder causing uncontrolled growth of myeloproliferative blast cells. Tyrosine kinase inhibitors are not really useful in advanced stages and have their own side effects, reactions and resistance as well. Till date, CML can be fully cured only by bone marrow transplantation (BMT) which has its own limitations and thus need further studies to increase better survival of CML patients by this procedure. Our lab at Jaslok Hospital has been working on studying and characterizing mesenchymal stem cells in various types of hematological malignancies. We came up with a concept of isolating and characterizing mesenchymal stem cells (MSCs) from peripheral blood of a CML patient which can be used in combination with Bone Marrow Transplantation in the same CML patient. Our study has shown that the stem cells derived from peripheral blood of CML patient were found to be BCR/ABL -ve . We designated this cell line as a “BCR/ABL -ve ” cell line. Molecular characterization of these cells further confirmed their Mesenchymal phenotypes with distinct expression of CD105, CD13 and CD73 genes. Interestingly these cells also expressed pluripotency genes such as OCT4 and NANOG and cytokines i.e. IL6 and TNFα. We further confirmed the normal phenotype of BCR/ABL -ve MSCs by localizing expression of H-Ras, Rb, P53, P16, P21, and EGFR and Ki67 cancer related proteins in these cells by immunofluorescence Microscopy and by in vitro transformation assay. Thus, we suggest that these normal BCR/ABL -ve MSCs derived from CML patient’s peripheral blood can be used in addition to BMT procedure for a better recovery of CML disease in near future.

Reducing RIP140 expression in macrophage alters ATM infiltration, facilitates white adipose tissue browning, and prevents high-fat diet-induced insulin resistance.

Adipose tissue macrophage (ATM) recruitment and activation play a critical role in obesity-induced inflammation and insulin resistance (IR). The mechanism regulating ATM activation and infiltration remains unclear. In this study, we found receptor interacting protein 140 (RIP140) can regulate the dynamics of ATM that contribute to adipose tissue remodeling. A high-fat diet (HFD) elevates RIP140 expression in macrophages. We generated mice with RIP140 knockdown in macrophages using transgenic and bone marrow transplantation procedures to blunt HFD-induced elevation in RIP140. We detected significant white adipose tissue (WAT) browning and improved systemic insulin sensitivity in these mice, particularly under an HFD feeding. These mice have decreased circulating monocyte population and altered ATM profile in WAT (a dramatic reduction in inflammatory classically activated macrophages [M1] and expansion in alternatively activated macrophages [M2]), which could improve HFD-induced IR. These studies suggest that reducing RIP140 expression in monocytes/macrophages can be a new therapeutic strategy in treating HFD-induced and inflammation-related diseases.

Visual compatibility of defibrotide with selected drugs during simulated Y-site administration.

The visual compatibility of a solution of defibrotide (the only drug recommended for treatment and prophylaxis of hepatic venoocclusive disease) with solutions of various drugs commonly administered in bone marrow transplant procedures was studied. Solutions of 43 drug products in concentrations typically used in clinical practice were evaluated in 1:1 mixtures with defibrotide solution in glass tubes kept at room temperature. The evaluated products included antiinfectious, corticoid, sedative, analgesic, and cardiovascular agents widely used for hematopoietic stem cell transplantation and other marrow transplant procedures; in most cases, test solutions were prepared via dilution in or reconstitution with sterile water, 0.9% sodium chloride injection, or 5% dextrose injection. The mixtures were visually observed immediately after manual mixing and at specified time points (60, 150, and 240 minutes). Visual compatibility was defined as the absence of color change, haze, fibers, particles, gas generation, and precipitate formation. The effect of mixing order on visual compatibility was ascertained. Of the 43 tested drug solutions, 36 were found to be visually compatible with the defibrotide solution over the entire four-hour study period. Solutions of 7 drugs (amikacin, furosemide, midazolam, mycophenolate mofetil, nicardipine, tobramycin, and vancomycin) were visually incompatible with defibrotide solution. In some cases, evidence of incompatibility was observed intermittently or was dependent on mixing order. Defibrotide solution was found to be visually compatible with solutions of 36 i.v. products that are likely to be coadministered with the drug in a bone marrow transplant unit. Seven drug solutions were visually incompatible with defibrotide solution.

In vivo EPR tooth dosimetry for triage after a radiation event involving large populations.

The management of radiation injuries following a catastrophic event where large numbers of people may have been exposed to life-threatening doses of ionizing radiation will rely critically on the availability and use of suitable biodosimetry methods. In vivo electron paramagnetic resonance (EPR) tooth dosimetry has a number of valuable and unique characteristics and capabilities that may help enable effective triage. We have produced a prototype of a deployable EPR tooth dosimeter and tested it in several in vitro and in vivo studies to characterize the performance and utility at the state of the art. This report focuses on recent advances in the technology, which strengthen the evidence that in vivo EPR tooth dosimetry can provide practical, accurate, and rapid measurements in the context of its intended use to help triage victims in the event of an improvised nuclear device. These advances provide evidence that the signal is stable, accurate to within 0.5 Gy, and can be successfully carried out in vivo. The stability over time of the radiation-induced EPR signal from whole teeth was measured to confirm its long-term stability and better characterize signal behavior in the hours following irradiation. Dosimetry measurements were taken for five pairs of natural human upper central incisors mounted within a simple anatomic mouth model that demonstrates the ability to achieve 0.5 Gy standard error of inverse dose prediction. An assessment of the use of intact upper incisors for dose estimation and screening was performed with volunteer subjects who have not been exposed to significant levels of ionizing radiation and patients who have undergone total body irradiation as part of bone marrow transplant procedures. Based on these and previous evaluations of the performance and use of the in vivo tooth dosimetry system, it is concluded that this system could be a very valuable resource to aid in the management of a massive radiological event.

Successful Salvage Treatment With Clofarabine and Cytarabine In Relapsed/Refractory Acute Myeloid Leukemia

ObjectivesRelapsed/refractory AML patients have a poor prognosis; allogeneic hematopoietic stem cell transplantation (HSCT) is the only chance in this setting to achieve long-term disease-free survival (1). It was previously established the activity of clofarabine plus cytarabine in AML relapse (clofarabine dosed once daily for 5 days with 40 mg/m2 followed 4 hours later by ara-C at 1 g/m2 per day)(2).However, modifications of this combination in AML therapy of relapsed/refractory patients warrant further evaluation. Therefore, our goal was to determine the efficacy and safety of clofarabine at lower dosage followed by cytarabine (Ara-C) in adult patients with relapsed or refractory acute myeloid leukemia (AML) and to evaluate the capacity of this regimen as a bridge for HSCT. MethodsPatients aged 18-65 years with refractory/relapsed AML were treated at the dose of clofarabine 30 mg/mq on days 1-5 and cytarabine 1000 mg/mq gg on days 1-5. We evaluated the complete remission rate (CRR), duration of remission (DOR) and overall survival (OS). Minimal residual disease (MRD) by molecular targeting was considered in all patients. ResultsTwenty-five (25) patients aged 29-64 years (median 47), who were fit for allogenetic HCT, received one cycle of 30 minutes infusion of clofarabine 30 mg/mq, followed 4 hours later by 3 hours infusion of intermediate dose cytarabine 1000 mg/mq days 1-5. Only in the first three patients this schedule was followed by gentuzumab. Nine (36%) patients had refractory disease (seven after one induction regimen, one after two previous regimes, one after a prior hematopoietic stem cell transplant (HSCT); 16 (64%) patients were in their first (12 patients) or second relapse (4 patients); among the 12 patients in first relapse, 5 were from an allogeneic stem cell transplant. Fourteen patients (56%) achieved a complete remission (CR), seven (28%) was refractory and 4 (16%) died of treatment related mortality. Eleven (44%) patients underwent (9 in CR) to allogeneic transplants or DLI infusion (3 patients refractory, and 8 patients relapsed), only one patient underwent to autologous transplant. One patient, who was relapsed after prior HSCT, obtained a CR but he developed acute graft vs host disease after therapy and died in molecular CR*. Among all patients underwent HSCT after Clofa/Ara-c salvage, six patients (50%) are still alive and in complete remission, six patients (50%) died because of HSCT complications or AML relapse. The complete remission rate (CRR) was (56,00 %), the median Overall Survival was 5 months for all patients (range 1-38 M), 11 Months for those underwent to tranplantation and 1,5 Months for non transplanted group. Treatment was complicated by neutropenic fever (n=17), grade III-IV mucositis (n=2) , skin rush (n=4) grade II- III, hepatic transaminase elevations (n=2). Two (n=5) patient died before their disease status could be evaluated. ConclusionsThese preliminary results suggest that combination treatment with clofarabine 30 mg/mq and ARA-C 1000 mg/mq is effective in this particularly poor prognosis category of patients, resulting in an ORR very favorably, representing a potential “bridge” toward bone marrow transplant procedures (among the 14 patients who achieved a CR, twelve (85%) proceeded to HSCT, and six are still alive). The safety profile is acceptable in this relapsed/refractory population, and our results are very similar to previous regimes using higher clofarabine dosages. More studies with this combination in adults are warranted. References1 Estey E. Treatment of relapsed and refractory acute myeloid leukemia. Leukemia. 2000;14:476-479. 2. Faderl S et al, “Results of a pase 1-2 study of clofarabine in combination with cytarabine (ara-C)”Blood 2005 [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Hospital Demand for Blood Components in Ontario Correlates with Selected Diagnostic and Procedural Activities

AbstractAbstract 4272 Introduction:Demand for blood components in Canada is a significant and growing contributor to health care spending, but the drivers of this demand remain poorly characterized. While most institutions do not routinely monitor the transfusion requirements of individual diagnoses and procedures, an institution's overall demand for blood components and the types of clinical activities it provides are both well documented. Development of an institution-level inferential model may allow for more accurate demand forecasting for blood components. Methods:Hospital shipments of all blood components from Canadian Blood Services (red blood cells, platelets, plasma and cryoprecipitate) in the province of Ontario during the period 2006–2009 were merged with institution-level administrative data containing hospital characteristics and selected diagnostic and procedural codes as obtained from inpatient and ambulatory clinical databases maintained by the Canadian Institute for Health Information. Simple linear correlation and several nested multivariable linear regression models were fitted and compared. Results:From 2006–2009, our sample included 137 healthcare facilities, representing approximately 1.4 million units of RBCs, 400 000 units of plasma, 166 000 doses of platelets (defined as either one pool of whole blood-derived platelets or a single apheresis platelet), and 20 000 units of unpooled cryoprecipitate. Institutional demand for these blood components correlated with both hospital-level characteristics and diagnostic and procedural activity data. Institutional demand for red blood cells correlated most strongly with visits for cirrhosis and chemotherapy. Demand for plasma correlated most strongly with visits for cirrhosis and cardiac surgery procedures. Demand for platelets correlated most strongly with bone marrow and solid organ transplant procedures. Demand for cryoprecipitate correlated most strongly with visits for valvular heart disease and cardiac procedures. The strength of these correlations generally remained stable over the four years of analysis, during which time demand for red blood cells and platelets increased, demand for plasma decreased, and demand for cryoprecipitate remained stable. Conclusions:In the largest province of Canada, institutional demand for blood components correlate strongly with disease burden measured by diagnostic and procedural codes contained within administrative data. Disclosures:Pendergrast:Canadian Blood Services: Research Funding. Zagorski:Canadian Blood Services: Research Funding.