Nonmalignant Hematologic Disorders Research Articles

Comparison of Bone Marrow Versus Peripheral Blood in Haploidentical Transplantation Using Post-Transplant Cyclophosphamide- a Retrospective Analysis

Comparison of Bone Marrow Versus Peripheral Blood in Haploidentical Transplantation Using Post-Transplant Cyclophosphamide- a Retrospective Analysis

Fertility Preservation in Childhood Cancer: Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment.

Simple SummarySubstantial strides have been made in treating childhood cancers; however, as a result of chemotherapy and radiotherapy, young males experience long-term side effects, including impaired fertility. Whilst prepubertal testicular tissue can be cryopreserved prior to gonadotoxic treatments, it remains to be determined how to generate mature gametes from the immature human testis tissue. Development of immature germ cells into sperm is a complex process, which is supported by mature Sertoli cells and testosterone produced from Leydig cells. We used an established testicular xenotransplantation model to investigate the effect of puberty hormones, known as gonadotrophins, on functional maturation of the spermatogonial stem cell (SSC) niche. Limited testosterone production and partial maturation of Sertoli cells occurred in prepubertal testis grafts, suggesting that longer periods of grafting and/or identification of additional factors are required to develop testicular transplantation as a model for fertility preservation in male survivors of childhood cancer.Survivors of childhood cancer are at risk for long-term treatment-induced health sequelae, including gonadotoxicity and iatrogenic infertility. At present, for prepubertal boys there are no viable clinical options to preserve future reproductive potential. We investigated the effect of a pubertal induction regimen with gonadotrophins on prepubertal human testis xenograft development. Human testis tissue was obtained from patients with cancer and non-malignant haematological disorders (n = 6; aged 1–14 years) who underwent testis tissue cryopreservation for fertility preservation. Fresh and frozen-thawed testis fragments were transplanted subcutaneously or intratesticularly into immunocompromised mice. Graft-bearing mice received injections of vehicle or exogenous gonadotrophins, human chorionic gonadotrophin (hCG, 20 IU), and follicle-stimulating hormone (FSH, 12.5 IU) three times a week for 12 weeks. The gross morphology of vehicle and gonadotrophin-exposed grafts was similar for both transplantation sites. Exposure of prepubertal human testis tissue xenografts to exogenous gonadotrophins resulted in limited endocrine function of grafts, as demonstrated by the occasional expression of the steroidogenic cholesterol side-chain cleavage enzyme (CYP11A1). Plasma testosterone concentrations (0.13 vs. 0.25 ng/mL; p = 0.594) and seminal vesicle weights (10.02 vs. 13.93 mg; p = 0.431) in gonadotrophin-exposed recipient mice were comparable to vehicle-exposed controls. Regardless of the transplantation site and treatment, initiation and maintenance of androgen receptor (AR) expression were observed in Sertoli cells, indicating commitment towards a more differentiated status. However, neither exogenous gonadotrophins (in castrated host mice) nor endogenous testosterone (in intact host mice) were sufficient to repress the expression of markers associated with immature Sertoli cells, such as anti-Müllerian hormone (AMH) and Ki67, or to induce the redistribution of junctional proteins (connexin 43, CX43; claudin 11, CLDN11) to areas adjacent to the basement membrane. Spermatogonia did not progress developmentally but remained the most advanced germ cell type in testis xenografts. Overall, these findings demonstrate that exogenous gonadotrophins promote partial activation and maturation of the somatic environment in prepubertal testis xenografts. However, alternative hormone regimens or additional factors for pubertal induction are required to complete the functional maturation of the spermatogonial stem cell (SSC) niche.

Hematopoietic stem cells from pluripotent stem cells: Clinical potential, challenges, and future perspectives.

The generation of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) is an active and promising area of research; however, generating engraftable HSCs remains a major obstacle. Ex vivo HSC derivation from renewable sources such as iPSCs offers an experimental tool for studying developmental hematopoiesis, disease modeling, and drug discovery, and yields tremendous therapeutic potential for malignant and nonmalignant hematological disorders. Although initial attempts mostly recapitulated yolk sac primitive/definitive hematopoiesis with inability to engraft, recent advances suggest the feasibility of engraftable HSC derivation from iPSCs utilizing ectopic transcription factor expression. Strategic development for de novo HSC generation includes further investigations of HSC ontogeny, and elucidation of critical signaling pathways, epigenetic modulations, HSC and iPSC microenvironment, and cell‐cell interactions that contribute to stem cell biology and function.

Prognostic impact of Epstein-Barr virus serostatus in patients with nonmalignant hematological disorders undergoing allogeneic hematopoietic cell transplantation: the study of Infectious Diseases Working Party of the European Society for Blood and Marrow Transplantation

AbstractBackgroundIn patients with acute leukemia, lymphoma and chronic malignancies, donor and/or recipient Epstein-Barr virus (EBV) seropositive status increases the risk of development of chronic graft-versus-host disease (cGVHD) after allo-hematopoietic cell transplantation (allo-HCT), while it has no influence on other transplant outcomes. No data are available on the impact of EBV serostatus on transplant outcomes in patients with nonmalignant hematological disorders.ObjectiveWe analyzed the influence of the recipient's (R) and donor's (D) EBV serostatus on transplant outcomes (overall survival (OS); relapse-free survival (RFS); relapse incidence (RI); nonrelapse mortality (NRM); acute graft-versus-host disease (aGVHD); cGVHD) in patients with nonmalignant hematological disorders undergoing allo-HCT.Patients and MethodsA total of 2,355 allo-HCTs performed between 1997 and 2016 for acquired bone marrow failure or hemoglobinopathies were included in this retrospective Registry megafile Infectious Diseases Working Party of the European Society of Blood and Marrow Transplantation (IDWP-EBMT) study.ResultsDemographics: The median age of recipient was 17.7 years (range: 0–77), and 50.8% were children. 79.0% of recipients and 75.4% of donors were EBV-seropositive. 67.8% had HCT from a matched family donor, 4.6% from a mismatched family donor, and 27.6% from an unrelated donor (UD). T-cell depletion was performed in vivo and ex vivo in 82.2% and 6.6% of patients, respectively. Conditioning regimen was myeloablative in 63.7% and reduced intensity conditioning (RIC) in 36.3% of patients. The median follow-up was 4.7 years. Transplant outcomes: EBV-seropositive recipients in comparison with EBV-seronegative recipients had lower OS (85.4% vs. 88.4%, p = 0.035) and higher NRM (10.0% vs. 6.4%, p = 0.018). No other significant differences were found for: RI, RFS, and aGVHD or cGVHD with respect to EBV pretransplant serostatus donor and/or recipient. Multivariate analysis: A trend toward higher risk of development of cGVHD (HR = 1.31; p = 0.081) and better survival (HR = 0.78; p = 0.087) in allo-HCT from EBV-seropositive donors was found. Allo-HCT in EBV-seropositive recipients had a trend toward lower risk of development of cGVHD (HR = 0.75; p = 0.065). When four subgroups (R−/D−, R−/D+, R+/D−, R+/D+ EBV serology) were analyzed, the EBV serostatus had no significant impact on OS, RFS, RI, NRM and development of aGVHD or cGVHD.ConclusionsAllo-HCT from EBV-seropositive versus EBV-seronegative donors are at 31% higher risk of cGVHD in patients with nonmalignant hematological disorders undergoing allo-HCT; however this difference is nonsignificant in multivariate analysis.

Human CD34+ hematopoietic stem cells culture in humanized culture medium for cell therapy

IntroductionHematopoietic stem cell transplantation (HSCT) is a proper treatment for many cancers, including malignant and non-malignant hematologic and immune disorders. However, due to the small quantity of HSCTs, their number should be increased ex vivo. Cell proliferation increase by animal serum can transmit certain diseases and immune problems to the recipient; therefore, replacement of culture media with media of human origin seems to be crucial. Acellular Wharton's jelly (AWJ) can be considered as an alternative medium in this regard. This study aimed to investigate the effect of AWJ substitution for fetal bovine serum (FBS) on the expression of myeloid and lymphoid specific markers and CD34+ cells proliferation pattern following co-culture with mesenchymal stem cells. Materials and methodsIn this study, CD34+ umbilical cord blood HSCs were co-cultured with MSCs in the presence of 10% FBS or AWJ, stem cell factor (SCF), thrombopoietin (TPO), and FLT3-L and the proliferation pattern and expression of myeloid as well as lymphoid lineage markers were then evaluated. ResultsThe expression of myeloid and lymphoid specific markers was increased and decreased in AWJ and FBS containing culture medium, respectively. ConclusionThis study provided evidence for the differentiation of UCB-derived CD34+ cells toward myeloid lineage and lack of their differentiation toward lymphoid lineage in AWJ containing culture medium.

The Use of Post-transplantation Cyclophosphamide in Peripheral Blood HLA-matched Stem Cell Transplantation as Graft-versus-host Disease Prophylaxis in Patients With Malignant or Non-malignant Hematologic Disorders: A Single-center Experience of 52 Patients

IntroductionStudies addressing the utilization of post-transplant cyclophosphamide (CY) as graft-versus-host disease (GVHD) prophylaxis in allogeneic hemopoietic stem cell transplantation from matched sibling donors are limited and with controversial results. Chronic GVHD incidence necessitating systemic treatment is around 35% in peripheral blood stem cell transplantation (PBSCT) from human leukocyte antigen-matched sibling donors. Patients and MethodsIn this study, high-dose CY was added to PBSCT aiming to reduce the incidence of GVHD to reach a lower figure compared with standard GVHD prophylaxis. Fifty-two patients with either benign or malignant hematologic disorders who underwent stem cell transplantation at Nasser Institute Hospital in Egypt from November 2017 to October 2018 were enrolled in this study. Fifty patients had fully human leukocyte antigen-matched siblings, whereas the remaining 2 patients had 1 locus class I mismatched donors. Pre-transplant conditioning regimen was fludarabine and busulfan (FLU/BU) in malignant cases (73.1%) and FLU/CY in benign hematologic disorders (26.9%) and 1 patient with hypocellular myelodysplastic syndrome. For GVHD prophylaxis, CY was given at a dose of 50 mg/kg/day on days 3 and 4 post-transplantation, and cyclosporine (CSA) starting day 5 in 96.1% of patients. For the 1-locus mismatched patients, both CSA and mycophenolate mofetil were administered starting day 5. ResultsThe 1-year incidence of acute GVHD (aGVHD) was 15.3% and for chronic GVHD (cGVHD) was 13.4%. Historical data of GVHD prophylaxis at our center using CSA and methotrexate showed an incidence of 37% for aGVHD and 33.9% for cGVHD. ConclusionsPost-transplant CY GVHD prophylaxis led to significantly less aGVHD (P = .03) and cGVHD (P = .04).

Incidence and time course of neutropenia in patients treated with rituximab‐based therapy for non‐malignant immune‐mediated hematologic diseases

Incidence and time course of neutropenia in patients treated with rituximab‐based therapy for non‐malignant immune‐mediated hematologic diseases

Improving Marrow Collections By Use of Five Side Hole Needle

Authors Harvey, E., Abhyankar, S., Merkel, D, Shune, L. Objectives Marrow harvesting is used to treat both malignant and nonmalignant hematologic disorders. We describe here our method of decreasing both anesthesia and collection times and maintaining or improving the total nucleated cell dose of the product. Method We analyzed 227 marrow harvest between October 2010 and August 2018. From the period of October 2010 through July 2018, we utilized the standard aspirate/biopsy needle. Total number of harvests during this time was 167. Starting in August of 2018, we switched to a 5-side hole marrow harvest needle. Total number of harvest utilizing this needle was 60. Counts were checked midway during all harvests and again close to estimated completion to verify stopping point. Results Anesthesia time for donors decreased 27% from an average of 83 minutes to 61 minutes (44 minimum- 96 maximum). Total harvest time was decreased by 32%, from an average of 44 minutes with the single hole needles to 30 minutes with the five side hole needs. (11 minimum – 66 maximum) (32% decrease). Total nucleated cell dose averaged 4.91 × 108/kg with the use of the single hole needs (n=167) and with the 5-side hole needles the average was 5.13 × 10 8 (N=60) Conclusion Changing from the single hole needle to the five (5) side hole needle improved the efficiency of the marrow harvest from both the collection and an anesthesia timepoints. By decreasing the anesthesia and collection times, we minimize the overall effects of the collection procedure on the donor. Traditional single hole beveled needle on left, 5 side hole needle on right. Close-ups of 5 Side Hole Needle

Acute Kidney Injury and CKD Associated with Hematopoietic Stem Cell Transplantation.

Hematopoietic stem cell transplantation is a life-saving therapy for many patients with cancer, as well as patients with some nonmalignant hematologic disorders, such as aplastic anemia, sickle cell disease, and certain congenital immune deficiencies. Kidney injury directly associated with stem cell transplantation includes a wide range of structural and functional abnormalities, which may be vascular (hypertension, thrombotic microangiopathy), glomerular (albuminuria, nephrotic glomerulopathies), and/or tubulointerstitial. AKI occurs commonly after stem cell transplant, affecting 10%-73% of patients. The cause is often multifactorial and can include sepsis, nephrotoxic medications, marrow infusion syndrome, hepatic sinusoidal obstruction syndrome, thrombotic microangiopathy, infections, and graft versus host disease. The risk of post-transplant kidney injury varies depending on patient characteristics, type of transplant (allogeneic versus autologous), and choice of chemotherapeutic conditioning regimen (myeloablative versus nonmyeloablative). Importantly, AKI is associated with substantial morbidity, including the need for KRT in approximately 5% of patients and the development of CKD in up to 60% of transplant recipients. AKI has been associated universally with higher all-cause and nonrelapse mortality regardless of transplant type, and studies have consistently shown extremely high (>80%) mortality rates in those patients requiring acute dialysis. Accordingly, prevention, early recognition, and prompt treatment of kidney injury are essential to improving kidney and patient outcomes after hematopoietic stem cell transplantation, and for realizing the full potential of this therapy.

Preventing infectious complications when treating non-malignant immune-mediated hematologic disorders.

Immunosuppressants, targeted antibody therapies, and surgical splenectomy are amongst the treatment choices for immune-mediated non-malignant hematologic disorders, with infection being the most common non-hematological adverse event from these therapies. Corticosteroids are associated with a length-of-treatment and dose-dependent risk for infection, including opportunistic infections. Screening and antimicrobial prophylaxis against tuberculosis, Strongyloides stercoralis, and Pneumocystis jirovecii pneumonia, are indicated in selected patients on steroids and with certain risk factors for infection. Rituximab is associated with hepatitis B virus reactivation. All patients planned to be started on rituximab should be screened for hepatitis B surface antigen and total core antibody, with antiviral prophylaxis given depending on test results. In eculizumab treated patients, immunization against meningococcal serogroups ACWY and B is recommended. In addition, some guidelines suggest antibiotic prophylaxis for the duration of eculizumab treatment. In splenectomized patients, counseling and immunization are cornerstones of infection prevention. Several federal and society guidelines about immunizations and prophylactic antimicrobial therapies for patients treated with various immunosuppressive agents exist and are summarized in this manuscript in a clinical-focused table. In addition, management suggestions are made where no formal guidelines exist.

Adherence to Infectious Disease Screening and Immunization Guidelines When Treating Non-Malignant Immune-Mediated Hematologic Disorders

Background : The use of immunosuppressive therapies in the management of non-malignant immune-mediated hematologic disorders (NMIHD) leads to an increased risk of infections. Preventive strategies, such as immunization and infectious disease (ID) screening, reduce the burden of infectious complications. However, lack of compliance to available guidelines among healthcare providers (HCP) represent a major concern. The present study was performed to assess adherence by HCP to national guidelines and recommendations for the prevention of infectious complications in patients (pts) treated for a NMIHD with the goal to identify areas where interventions can be implemented to improve awareness. Methods : We retrospectively analyzed pts diagnosed with AIHA, acquired coagulation factor deficiency (ACFD), TTP, ITP, aHUS, PNH, and antiphospholipid syndrome (APS) between January 2016 and July 2018 at an academic medical center (University of North Carolina). Pts had to have received either rituximab (RTX), eculizumab, or splenectomy. Adherence to existing strategies was assessed based on available recommendations from recognized national entities (e.g. CDC, ACIP), and clinical societies (e.g. ASH) (summarized in Table 1). Immunizations considered for analysis included: pneumococcal, meningococcal and Haemophilus influenzae vaccines ('triple vaccine' if received all together). We considered 'adherence to an immunization recommendation' when, depending on the diagnosis and treatment employed, pts received all recommended vaccines at least 2 weeks prior to therapy or 4 weeks if splenectomy. Adherence to ID screening in pts receiving RTX therapy was defined as pts screened for both hepatitis B surface antigen and total core antibodies. Influenza virus and herpes zoster (HZ) immunization rates were also analyzed. Results : 269 pts from six UNC-affiliated hospitals were included. Pts clinical characteristics are summarized in Table 2. Overall, only 6% of all evaluable pts were appropriately immunized as recommended by national guidelines (5%, 8%, and 8%, for RTX, eculizumab, and splenectomy, respectively) (Table 3). Immunization rates are summarized in Figure 1. In the eculizumab group, although 92% of HCP recommended and prescribed meningococcal immunization, only 38% (n=5/13) received both vaccines (54% either, 8% none). Likewise, although 89% (n=47/53) of pts undergoing splenectomy had received a 'triple vaccine', only 11% of pts received guideline recommended vaccines (both pneumococcal and meningococcal vaccines) and only 8% were appropriately vaccinated by receiving all indicated vaccines at least 4 weeks prior to their scheduled surgery. 68% of pts treated with RTX were appropriately screened for hepatitis B, with lower rates found in young women (screening rate: 77% age >50 vs. 58% ≤50 years, p<0.01; 83% male vs. 58% female, p<0.01) diagnosed with TTP or APS (ITP 74%, AIHA 79%, ACFD 77% vs. TTP 46%, APS 55%, p=0.01). Lastly, 36% of pts received annual influenza virus vaccine, and 11% of eligible pts the HZ vaccine. Conclusion : To the best of our knowledge, there have been no studies conducted in the U.S. assessing adherence to infectious prevention guidelines among different NMIHD. We found that in pts with NMIHD undergoing immunotherapy or splenectomy, many pts received either incomplete recommended immunizations or the immunization was administered too close in proximity to the immunosuppressive therapy or splenectomy, both resulting in impaired immunologic response to vaccines. In addition, only two thirds of pts receiving RTX had pretreatment screening for hepatitis B infection at odds with national recommendations of 100%. Lastly, low immunization rates against influenza virus and HZ were found. Based on these results, we created infographics (supplemental figure), an "easy-to-use" pocket brochure summarizing recommendations (Table 1), and we are building immunization reminders when electronically prescribing immunotherapy, with the aim of improving HCP awareness in our health care system. Although we are unsure about other institution's compliance rate, we suspect that non-compliance to immunization is a systemic problem across the U.S. We will disseminate this data hoping to replicate this study at other academic institutions. There is an urgent need for the development of strategies to improve HCP awareness and adherence to infectious prevention guidelines! Disclosures No relevant conflicts of interest to declare.

The Road to Day 0: Identification of Barriers from HLA Typing to Transplantation in the Era of Haploidentical Grafts and Post-Transplant Cyclophosphamide

Introduction: Allogeneic hematopoietic stem cell transplantation (alloHSCT) offers an effective, potentially life saving treatment for a broad range of malignant and nonmalignant hematologic disorders. To some degree, an adequate performance status, comorbidity and disease control, and an available donor must be present in order to be considered as a suitable candidate. In addition, sociodemographic factors such as insurance coverage, high out-of-pocket costs and transplant center availability might impair access to alloHCT, particularly in low and middle-income countries (LMIC). Currently, in the era of haploidentical grafts donor availability has been largely resolved and is no longer the biggest barrier for reaching transplantation in LMIC, becoming the most common form of alloHSCT in our center. On the other hand, most outcomes-based publications in the field of alloHSCT are focused only on the transplanted, not on an "intent-to-transplant" basis. The current reasons for not being transplanted once a patient is considered for the procedure are unknown. Therefore, as early adopters of haploidentical transplantation we aimed to evaluate patients that were not grafted. Objetive: To determine barriers to alloHSCT and patient outcomes by studying the population that was HLA typed and/or had a consult in our center, regardless of the event of transplantation Methods: All adult patients who were HLA-typed and considered potentially eligible for transplantation in our center were included. Since 2015 haploidentical donors have become the immediate next best option when a matched sibling donor is unavailable in our center, due to a lack of access and increased costs of unrelated donors and umbilical cord blood. Related alloHSCT have been largely subsidized by the government, reducing transplant costs to a minimum for uninsured patients. We performed a retrospective review of patient records from January 2016- December 2018, regardless of baseline diagnosis, donor source and conditioning regimen. We documented demographic variables, identified the reasons for not performing the alloHCT, and performed a landmark analysis from the time of HLA typing in comparison to patients that did reach transplantation. Univariate and multivariate analyses were performed to identify covariates associated to not reaching transplantation. Results: One hundred and thirty five patients were HLA-typed and considered eligible to proceed to alloHCT. Forty-seven (34.8%) were not transplanted. In six cases, alloHSCT was considered unnecessary. The proportion of patients who received transplantation was similar regardless of the availability of a MSD vs haploidentical donor (68.2 vs 66.7% repectively, p=0.53). Transplanted patients had a significantly lower disease risk index (DRI) and more frequently had private healthcare. Other baseline characteristics between transplanted and not transplanted cohorts are presented in Table 1. The reasons for not proceeding with transplantation were disease status (refractory disease or relapse prior to conditioning) in 15 patients (36.6%), followed by procedure rejection by patient/family and/or treatment abandonment in 12 (29.2%), 5 (12.2%) did not have a suitable donor, 4 (9.7%) due to economic difficulties, and 4 (9.7%) developed important comorbidities. A high DRI and lack of insurance coverage were associated with a higher risk for not getting a transplant in both univariate and multivariate analyses (Table 1). As expected, the outcome of patients that were not transplanted was dismal overall and particularly in high/very high DRI patients (Figure 1, Panel A-D). Conclusion: In the era of haploidentical transplantation, the biggest barrier in our limited-resource context is disease activity/relapse, followed by patient rejection/treatment abandonment, and not the availability of a donor. We must perform transplants promptly and increase education for patients and family in order to overcome these limitations. Disclosures Gomez-Almaguer: Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Teva: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

Leptin Receptor As a Functional Marker for Long-Term Repopulating Hematopoietic Stem Cells

Hematopoietic cell transplantation is an invaluable life-saving regimen for patients affected by malignant and non-malignant hematological disorders. However, successful clinical outcomes depend on the abilities of hematopoietic stem (HSCs) and progenitor cells (HPCs) to home to the bone marrow (BM) and then reconstitute a healthy new blood system. Leptin (Lep), a metabolic hormone well-characterized for its regulations of appetite and body weight by acting on the hypothalamus neurons, has a WSXWS motif of the type I cytokine receptor family and has reported hematopoietic effects (Cioffi et al., Nat Med 1996, Bennett et al., Curr Biol 1996, Umemoto et al., Blood 1997, Gainsford et al. Proc Natl Acad Sci USA 1996, Claycombe et al., Proc Natl Acad Sci USA 2008). These studies were however mostly limited to in vitro assays. Recent work demonstrated that Lep receptor(r)+ stromal cells were indispensable for maintenance of HSC/HPC (Comazzetto et al., Cell Stem Cell 2019, Himburg et al., Cell Stem Cell 2018, Zhou et al., Nat Cell Biol 2017). Yet, whether Lepr expression on HSC/HPC has effects on their in vivo functions remain largely unknown. We hypothesized that environmental factors that affect metabolism of HSCs and HPCs, such as those modulated by Lep/Lepr interactions, may be involved in HSC/HPC regulation and the engraftment of these cells. Using flow cytometry analysis, we first assessed expression levels of Lepr on HSCs and HPCs. While only a low percentage of mouse BM HSC/HPC expressed Lepr, both the percentages of Lepr+HSCs (28.5% Lepr+LT-HSC and 17.2% Lepr+ST-HSC) and mean fluorescence intensity (MFI) of surface Lepr on these cells are significantly higher than that of Lepr+HPCs such as CMP, GMP and CLP (3.8%, 1.5%, 0.7% Lepr+ respectively). Despite the fact that HPCs express a lower level of Lepr, intact Lep/Lepr signaling was critical for their functions. This was illustrated by in vitro colony assay of cells taken from Lepr knockout (-/-) mouse BM in which significantly fewer absolute numbers per femur of HPC-derived colonies (CFU-GM, CFU-GEMM, BFU-E) formed compared to WT controls, and these progenitors were in a slow or non-cycling state. To evaluate how Lepr expression affects in vivo HSC/HPC functions, equal numbers of BM C57BL/6 (WT; CD45.2+) Lepr - Lineage-Sca1+cKit+ (LSK) vs. Lepr+LSK cells were sorted and each transplanted with competitive BoyJ (CD45.1+) cells into lethally irradiated CD45.2+/CD45.1+ F1 recipients. A consistently higher engraftment capacity of Lepr+LSK cells was manifested in comparison to Lepr - LSK cells as noted in peripheral blood (PB) at months 1-6 chimerism post-transplant (91% vs 1.1% at month 6). Lepr+HSCs and Lepr+MPPs expressed similar levels of surface CXCR4 in comparison to corresponding Lepr - populations, suggesting that homing differences may not explain increased engraftment of Lepr+ LSK. At month 6, Lepr+LSK, but not Lepr - cells, demonstrated a significant myeloid-biased engraftment (0.24 vs 0.03 respectively for myeloid/lymphoid ratios). This is consistent with the phenotypic finding that compared to Lepr -LSK cells, Lepr+LSK cells contained a significantly lowered percentage of MPP4 progenitor cells (3.6% vs 36%), which have been demonstrated as a lymphoid-biased subset of MPPs (Pietras et al., Cell Stem Cell 2015). In addition, Lepr+LSK cells contained three-fold fewer progenitors as determined by in vitro colony assays. These findings demonstrated that Lepr+LSK cells were enriched for long-term hematopoietic repopulating HSCs, while its counterpart Lepr -LSK cells contained mostly HPCs. The data also suggested that absence of Lepr expression may play a role in fate-decision skewing HSCs towards MPP4 production. For beginning efforts at mechanistic insight, we hypothesized that Lepr+ HSCs and Lepr+MPP may be different than Lepr - cells in mitochondrial activity. Compared to Lepr - cells, Lepr+HSC and Lepr+MPP cells interestingly possessed more robust mitochondrial metabolism, as demonstrated by their mitochondria having significantly higher membrane potential (measured by JC-1 assay). In summary, Lep/Lepr signaling appears to be a functional ligand-receptor axis for maintaining HSC/HPC homeostasis and differentiation cell bias. Moreover, Lepr expression may serve as a functional marker for long-term repopulating HSCs, which has potential translational possibilities, as Lepr is highly conserved between mice and humans. Disclosures No relevant conflicts of interest to declare.

Transplantation of gene-modified haematopoietic stem cells: Application and clinical considerations.

Autologous and allogeneic haematopoietic stem cell (HSC) transplantation has been performed in patients with various malignant and non-malignant haematological disorders for more than 50 years. Ex vivo gene modification of HSCs for autologous transplantation opens up new therapeutic avenues for genetic and infectious diseases. Major advances have been made over the last three decades with respect to gene modification of HSCs and transplantation strategies, ultimately culminating in the approval of two such therapies in Europe (Strimvelis for a rare primary immune deficiency, and LentiGlobin for beta-thalassaemia). Newer gene-modifying technologies and treatment regimens have also recently come to the fore, which hold great promise for the development of safer and more effective treatments. We provide an overview of the current state of gene-modified HSC therapies, highlighting success stories, limitations and important considerations for achieving successful translation of these therapies to the clinic.

DISTRIBUTION OF NON-MALIGNANT HEMATOLOGICAL DISORDERS BY SEX, AGE GROUPS AND TYPE OF DISEASE BASED ON BONE MARROW ASPIRATION IN POPULATION OF KHYBER PAKHTUNKHWA, PAKISTAN

Background: The distribution of non-malignant hematological disorder varies across globe by sex, age groups and type of disease. The objectives of this study were to determine distribution of non-malignant hematological disorders by sex, age groups and type of disease based on bone marrow aspiration in population of Khyber Pakhtunkhwa, Pakistan.
 Materials and Methods: This cross-sectional study was conducted in Department of Pathology, Khyber Teaching Hospital, Peshawar, Pakistan from Jan.-Dec. 2014. 116 cases with non-malignant hematogical disorders were selected. Sex, age groups and type of disease were analyzed by count and percentage with confidence intervals for proportions. Chi-square goodness-of-fit test was used to testify two hypotheses.
 Results: Out of 116 cases, 63(54.31%, 80%CI 48.38-60.24) were men and 53(45.69%, 80% CI 39.76-51.62) women. Age groups wise; 31(26.72%, 80%CI 21.46-31.99) were child (≤17 years), 57(49.14%, 80%CI 43.19-55.09) young adult (18-35 years), 19(16.38%, 80%CI 11.98-20.78) middle-aged adults (36-55 years) and nine (7.76%, 80%CI 4.57-10.94) older adults (≥56 years). Frequency of type of disease was; hemolytic anemia 17(14.66%, 80%CI 10.45-18.86), megaloblastic anemia 16(13.79%, 80%CI 9.69-17.9), bone marrow hypoplasia 14(12.07%, 80%CI 8.19-15.95), iron deficiency anemia 13(11.21%, 80%CI 7.45-14.96), mixed deficiency anemia 9(7.76%, 80%CI 4.57-10.94), ITP 9(7.76%, 80%CI 4.57-10.94), aplastic anemia 8(6.89%, 80%CI 3.88-9.91), bi-liniage cytopenia 7(6.03%, 80%CI 3.2-8.87), erythroid hyperplasia 3(2.59%, 80%CI .7-4.47) and malaria 2(1.72%, 80%CI 0.18-3.27). In 18(15.52%, 80%CI 11.21-19.83) cases the bone marrow was diluted. Null hypothesis for distribution of sex (p=.3532) was accepted while that for age groups was rejected (p=

DISTRIBUTION OF NON-MALIGNANT HEMATOLOGICAL DISORDERS BY SEX, AGE GROUPS AND TYPE OF DISEASE BASED ON BONE MARROW ASPIRATION IN POPULATION OF KHYBER PAKHTUNKHWA, PAKISTAN

Background: The distribution of non-malignant hematological disorder varies across globe by sex, age groups and type of disease. The objectives of this study were to determine distribution of non-malignant hematological disorders by sex, age groups and type of disease based on bone marrow aspiration in population of Khyber Pakhtunkhwa, Pakistan. Materials and Methods: This cross-sectional study was conducted in Department of Pathology, Khyber Teaching Hospital, Peshawar, Pakistan from Jan.-Dec. 2014. 116 cases with non-malignant hematogical disorders were selected. Sex, age groups and type of disease were analyzed by count and percentage with confidence intervals for proportions. Chi-square goodness-of-fit test was used to testify two hypotheses. Results: Out of 116 cases, 63(54.31%, 80%CI 48.38-60.24) were men and 53(45.69%, 80% CI 39.76-51.62) women. Age groups wise; 31(26.72%, 80%CI 21.46-31.99) were child (≤17 years), 57(49.14%, 80%CI 43.19-55.09) young adult (18-35 years), 19(16.38%, 80%CI 11.98-20.78) middle-aged adults (36-55 years) and nine (7.76%, 80%CI 4.57-10.94) older adults (≥56 years). Frequency of type of disease was; hemolytic anemia 17(14.66%, 80%CI 10.45-18.86), megaloblastic anemia 16(13.79%, 80%CI 9.69-17.9), bone marrow hypoplasia 14(12.07%, 80%CI 8.19-15.95), iron deficiency anemia 13(11.21%, 80%CI 7.45-14.96), mixed deficiency anemia 9(7.76%, 80%CI 4.57-10.94), ITP 9(7.76%, 80%CI 4.57-10.94), aplastic anemia 8(6.89%, 80%CI 3.88-9.91), bi-liniage cytopenia 7(6.03%, 80%CI 3.2-8.87), erythroid hyperplasia 3(2.59%, 80%CI .7-4.47) and malaria 2(1.72%, 80%CI 0.18-3.27). In 18(15.52%, 80%CI 11.21-19.83) cases the bone marrow was diluted. Null hypothesis for distribution of sex (p=.3532) was accepted while that for age groups was rejected (p=

Incidence, Risk Factors, and Outcomes of Primary Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for both malignant and nonmalignant hematologic disorders. However, primary poor graft function (PGF) is a serious early complication of allo-HSCT that leads to a poor outcome. Little is known about the characteristics, incidence, and risk factors of primary PGF occurring after allo-HSCT. Here we performed a 1:4 ratio nested case-control study in 830 patients who underwent allo-HSCT between April 2013 and November 2018 at our center. Twenty-four patients (14 males and 10 females; average age, 35.79 years; range, 17 to 53 years) developed primary PGF. On univariate and multivariate analyses, a CD34+ cell dose <5 × 106/kg (P = .003), a serum ferritin (SF) level >2000 ng/mL (P = .008), and splenomegaly (P = .039) were identified as 3 independent risk factors for primary PGF. After a median follow-up of 7.5 months (range, 1 to 48 months), only 5 patients (20.8%) survived. The survival rate of patients with primary PGF was significantly lower than that of patients with good graft function (GGF) (1-year overall survival, 25.0% versus 90.6%; P < .001). Cox regression analysis suggested that PGF and high SF level were strongly associated with rapid death in these patients. In conclusion, allo-HSCT recipients with a low CD34+ cell dose in their graft and exhibiting a high SF level and splenomegaly should be monitored for the development of primary PGF after allo-HSCT, and effective therapies need to be explored.

Comparison of Myeloablative Versus Reduced-Intensity Conditioning Regimens for Allogeneic Hematopoietic Stem Cell Transplantation in Acute Myeloid Leukemia: A Cohort Study

Objective:Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment modality for a variety of malignant and non-malignant hematologic disorders. Myeloablative conditioning (MAC) and reduced-intensity conditioning (RIC) regimens could have different clinical outcomes. This purpose of this study was to assess the long-term outcome of MAC versus RIC regimens in patients with acute myeloid leukemia (AML) undergoing allogeneic HSCT.Materials and Methods:We retrospectively compared long-term outcomes with MAC and RIC regimens in patients with AML who underwent allo-HSCT at our tertiary transplantation center.Results:We analyzed survival outcomes after MAC-HSCT versus RICHSCT among 107 adult patients with AML diagnosed from 2001 through 2017. Of those, 44 patients underwent a MAC regimen, whereas 63 patients received a RIC regimen. The median follow-up time was 37 months (range: 6-210) for the entire group. The 3-year overall survival (OS) for RIC and MAC patients was 67% and 60%, respectively (p>0.05). The 3-year progression-free survival (PFS) for RIC and MAC patients was 88% and 77%. In multivariate analysis, the type of conditioning regimen (RIC vs. MAC) did not influence PFS (p=0.24). Acute graft-versus-host disease (GVHD) was seen in five of the RIC patients and 9 of the MAC patients. Chronic GVHD was seen in 16 of the RIC patients and 6 of the MAC patients. There was no significant difference between the two groups in terms of acute GVHD (p=0.089), but there was a significant difference between the two groups in terms of chronic GVHD (p=0.03).Conclusion:This retrospective analysis confirmed that MAC and RIC regimens had a consistently equivalent rate of OS and PFS in AML patients who underwent allo-HSCT. The choice of MAC versus RIC conditioning regimen might be decided on the basis of patient and disease characteristics.

Umbilical cord blood mesenchymal stem cells application in hematopoietic stem cells expansion on nanofiber three-dimensional scaffold.

Umbilical cord blood (UCB) hematopoietic stem cells (HSCs) transplantation (HSCTs) is considered as a therapeutic strategy for malignant and nonmalignant hematologic disorders. Nevertheless, the low number of HSCs obtained from each unit of UCB can be a major challenge for using these cells in adults. In addition, UCB is a rich source of mesenchymal stem cells (MSCs) creating hopes for nonaggressive and painless treatment in tissue engineering compared with bone marrow MSCs. This study was designed to evaluate the effects of UCB-MSCs application in UCB-HSCs expansion on the nanoscaffold that mimics the cell's natural niche. To achieve this goal, after flow cytometry confirmation of isolated HSCs from UCB, they were expanded on three-dimensional (3D) poly-l-lactic acid (PLLA) scaffolds fabricated by electrospinning and two-dimensional (2D)-culture systems, such as (1) HSCs-MSCs culturing on the scaffold, (2) HSCs culturing on the scaffold, (3) HSCs-MSCs culturing on 2D, and (4) HSCs culturing on 2D. After 7 days, real-time polymerase chain reaction (PCR) was performed to evaluate the CXCR4 gene expression in the mentioned groups. Moreover, for the next validation, the number of total HSCs, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay, scanning electron microscopy imaging, and colony-forming unit assay were evaluated as well. The results of the study indicated that UCB-MSCs interaction with HSCs in 3D-culture systems led to the highest expansion of UCB-HSCs on day 7. Flow cytometry results showed the highest purity of HSCs cocultured with MSCs. Real-time PCR showed a significant increase in gene expression of CXCR4 in the mentioned group. The highest viability and clonogenicity were detected in the mentioned group too. Considered together, our results suggest that UCB-HSCs and MSCs coculturing on PLLA scaffold could provide a proper microenvironment that efficiently promotes UCB-HSCs expansion and UCB-MSCs can also be considered as a promising candidate for UCB-HSCTs.

Predictors of Donor/Host (D/H) Lineage Specific Chimerism Trends in Pediatric Patients Following Allogeneic Hematopoietic Cell Transplantation

The outcome of allogeneic HCT is dependent on several pre-transplant variables. We hypothesize that these pre-transplant factors may influence the donor/host chimerism post HCT, which in its turn may affect the outcome. We performed an analysis to study lineage specific D/H chimerism patterns of total blood leukocytes, myeloid cells/neutrophils, B-cells, NK-cells and T-cells. All consecutive patients who underwent an allogeneic HCT between 01/2010 to 06/2015 on the Pediatric BMT Service at MSKCC were reviewed. Chimerism analyses were performed by short tandem repeat (STR) polymorphism analysis at the American Red Cross Blood Services (Philadelphia, PA). Lineage specific donor chimerism post HCT was studied including donor chimerism trend, and factors with potential impact on chimerism including: age, disease, graft source, and T-cell depletion (TCD). Preliminary analyzes performed on this cohort included Wilcoxon Rank Test and cox proportional hazard analyses. 140 patients with a median age of 11.5 years were included. They included 97 patients with hematologic malignancies, 27 patients with non-malignant hematologic disorders and 16 patients with immunologic disorders. Cytoreduction included TBI in 48 patients or chemotherapy only in 98 patients. 105 patients received T-cell depleted grafts, 28 patients unmodified marrow or peripheral blood grafts, and 7 patients unrelated cord blood grafts. No patients received donor leukocyte infusions (DLI). Observational studies revealed that full donor chimerism of myeloid cells, B-cells and NK-cells, but not T-cells occurred early post-transplant. In Wilcoxon Rank Test, there was no difference in the percentage of total donor leukocytes at 3 months vs 12 months post HSCT (n=30), while the median of donor T-cell chimerism was 51% at 3 months and 91% at 12 months post HSCT (p The studies of the impact of different factors on chimerism were performed, including age, disease, and type of graft. Loss of donor total leukocytes chimerism between 3 and 12 months post-transplant was significantly more pronounced for patients This preliminary analysis of lineage specific chimerism post-transplant showed that donor T-cells may take one year to fully recover post-transplant, mostly following T-cell depleted grafts, without intervention. Cord blood grafts were associated with high donor chimerism throughout the post-transplant period. Lastly, factors associated with loss of donor chimerism post-transplant were younger age and non-malignant disorders. More in-depth analyses are being performed.