Journal articles: 'HSC criteria'

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Hyatt, Sarah A., Wei Wang, Bryce A. Kerlin, and Sarah H. O’Brien. "Applying Diagnostic Criteria for Type 1 von Willebrand Disease to a Pediatric Population." Blood 110, no. 11 (November 16, 2007): 2138. http://dx.doi.org/10.1182/blood.v110.11.2138.2138.

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Abstract Background: Although type 1 von Willebrand disease (VWD) is the most common bleeding disorder seen by pediatric hematologists, making a definitive diagnosis continues to be a challenge in clinical practice. Both the International Society on Thrombosis and Haemostasis (ISTH) and the Hospital for Sick Children in Toronto (HSC) have proposed diagnostic criteria for type 1 VWD. These include abnormal laboratory values, significant mucocutaneous bleeding, and/or a positive family history. Most recently, the ISTH published updated recommendations, which differed only in the requirement of more abnormal laboratory results (VWF:Ag 5–20 IU/ml). We applied ISTH and HSC criteria, as well as updated ISTH criteria, to a large population of pediatric patients diagnosed with type 1 VWD. We hypothesized that a substantial number of patients would not meet either HSC or ISTH diagnostic criteria. Methods: We performed a retrospective medical record review of all type 1 VWD patients at our Hemostasis and Thrombosis Center. We evaluated each record for bleeding history, family history, and laboratory values. Frequencies of fit for HSC, ISTH and updated ISTH criteria were calculated. Mean VWF:Ag, VWF:RCo, and bleeding scores (Rodeghiero et al, J Thromb Haemost, 2006) were compared across populations meeting each proposed criteria. Results: Of 201 patients, 33.9% met the HSC definition of “definitive” type 1 VWD, 4.5% met ISTH definition, and 0% met updated ISTH definition. An additional 56.2% (HSC), 15.4% (ISTH), and 6% (updated ISTH) met definitions of “possible” type 1 VWD. For each proposed definition, criteria for significant mucocutaneous bleeding were most likely to be met, while criteria for abnormal laboratory values were least likely. In fact, 74% of patients had significant bleeding as defined by the HSC (56% as defined by ISTH). We did find significant clinical and laboratory differences between patients labeled as definite, possible, and normal by ISTH and HSC criteria. For example, patients meeting criteria for definite disease by HSC criteria had a mean bleeding score of 3.5 and mean VWF:Ag of 31 IU/ml, compared to 2.6 and 47 IU/ml in patients labeled as possible, and 2.2 and 68 IU/ml in patients labeled as normal (p=0.001 bleeding score, <0.001 mean VWF:Ag). Regardless of whether they met any set of criteria, most patients (94%) received some type of medical intervention (pre-operative or therapeutic desmopressin or VWF replacement). Discussion: We found that the majority of our pediatric type 1 VWD patients did not meet the original ISTH definition of definite or even possible type 1 VWD, thus confirming in a larger population the findings of HSC investigators (Dean et al, Thromb Haemost, 2000). In addition, we have demonstrated that the new ISTH criteria are even more inappropriate for clinical practice in a pediatric population, with 0% of patients meeting criteria for definite disease. Therefore, these criteria failed to identify a substantial number of children and adolescents who presented to medical attention, had significant mucocutaneous bleeding, and required therapeutic interventions. The new ISTH criteria may be an excellent scientific tool for identifying a narrow, severely affected population of patients likely to have autosomal dominant VWD mutations. However, they do not appear to have clinical validity in the pediatric setting.

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Shepherd, Bryan E., Hans-Peter Kiem, Cynthia E. Dunbar, Andre Larochelle, Robert E. Donahue, Ruth Seggewiss, Peter M. Lansdorp, Peter Guttorp, and Janis L. Abkowitz. "Estimating the Replication Rate of Hematopoietic Stem Cells in Non-Human Primates: A Test of Hayflick’s Hypothesis." Blood 106, no. 11 (November 16, 2005): 1710. http://dx.doi.org/10.1182/blood.v106.11.1710.1710.

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Abstract Because hematopoietic stem cells (HSC) cannot be directly observed, stochastic simulations in conjunction with competitive repopulation experiments have been used to estimate the replication rate of feline (1 rep per 8–10 wks) and murine (1 rep per 2.5 wks) HSC (model description and estimates in Nat Med2:190, 1996; Blood96:3399, 2001). These results could suggest that the HSC replication rate decreases with animal size and longevity, perhaps implying that the number of lifetime replications per HSC is limited and conserved in mammals. To test this, we analyzed retroviral vector gene marking and granulocyte telomere length data from baboons and rhesus macaques. Rhesus macaques are approximately the same size (weight 8 kg) as cats and have a similar lifespan (15–20 years); their hematopoietic demand, defined as the number of blood cells required per lifetime, is comparable. In contrast, baboons are larger (15 kg), live for 30 years, and have a hematopoietic demand more similar to humans. We simulated HSC dynamics for virtual baboons and macaques using specific HSC replication rates and then determined if observed data could be a random draw from 1000 simulated datasets. Compatibility of simulated and observed gene marking studies was determined by 3 formal criteria computed for both observed and simulated data: 1) the time after transplantation until the percent of marked cells stabilizes (measured using a change point model), 2) the presence or absence of drift after stabilization (drift is defined as a post-stabilization slope significantly different from 0 and estimated as at least 2% per 100 days), and 3) the amount of residual variation after stabilization (variation about a smoothed fit to the data -- transforming using the variance-stabilization transformation, arcsine of square root). Binomial probabilities were computed to evaluate Criterion 2 and Kolmogorov-Smirnov goodness of fit tests were used for evaluating Criteria 1 and 3, with p-values < 0.05 defining incompatibility. Mice and cat HSC parameters did not yield simulated data compatible with the observed baboon data. Rather, the analyses required that baboon HSC replicated less frequently, approximately once per 27–77 weeks (with 300 transplanted HSC (the estimated number infused); the range was robust to other modeling assumptions). In contrast, cat HSC parameters yielded simulated data compatible with the observed macaque data if 100–500 HSC were transplanted, whereas mice HSC parameters trended towards incompatibility (incompatible for 100 and 500 transplanted HSC; nearly incompatible for 300 transplanted HSC, p=0.07). Next, granulocyte telomere length data were simulated (methods in Exp Hematol32:1040; 2004) and preliminary data yielded best estimates for the replication rate of HSC in macaques and baboons of once per 18 weeks and once per 24 weeks, respectively. Our results (derived from 2 independent experimental approaches) demonstrate that the replication rate of macaque HSC is slower than mouse, whereas the replication rate of baboon HSC is slower than both cat and mouse; argue that the rate of HSC replication inversely correlates with longevity; and support Hayflick’s hypothesis that cells can only undergo a relatively constant finite number of lifetime replications. This apparent evolutionary constraint may extend to human HSC behavior.

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Agarwal, Sachin, Ravi Kant, and Ravi Shankar. "Humanitarian supply chain management frameworks." Benchmarking: An International Journal 26, no. 6 (August 5, 2019): 1749–80. http://dx.doi.org/10.1108/bij-08-2018-0245.

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Purpose The purpose of this paper is to examine and compare extant framework in humanitarian supply chain management (HSCM) and to propose a framework on humanitarian supply chain (HSC) performance measurement based on the content, context and process. Design/methodology/approach The structured keywords, namely humanitarian supply chain (HSC), humanitarian logistic (HL), humanitarian relief chain (HRC) and humanitarian chain (HC) as an exact phrase were searched in the title, abstract and keywords in the academic database. A total of 66 peer-reviewed articles were selected for analysis purpose that reports framework from the reviewed literature. These selected frameworks are categorized in dimensions, namely framework novelty, framework source, recognize elements/constructs of framework, comparative analysis of the framework and in-depth study of HSCM performance measurement. Findings The analysis reveals that the majority of these developed frameworks are novel and academic based. Case study is most prominent research methodology in the development of HSCM framework. Lack of coordination among humanitarian stakeholders is the major challenge in the empirical implementation of framework. This study proposes future research trend toward a unified HSCM framework that will facilitate to uncover the coherent set of elements/constructs in the field of HSCM. Research limitations/implications This study considers peer-reviewed articles published in English language, and excludes conference papers, working articles, technical data/reports and book chapters. Practical implications This study categorizes new dimension for framework analysis and proposed an HSC performance measurement framework which gives new insights to the academicians, practitioners and policy makers for future work. Social implications This examination gives the establishment to facilitate investigation of viable, efficient and effective HSCM, and detail opportunities for practices. Originality/value This study critically analyzes 66 frameworks under the different criteria to identify research gap and trends. Furthermore, this study proposes the HSC performance measurement framework.

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Szmigielska-Kaplon, Anna, Janusz Szemraj, Krzysztof Jamroziak, Agnieszka Pluta, Marta Robak, Anna Krawczynska, Katarzyna Hamara, Katarzyna Szmigielska, Tadeusz Robak, and Agnieszka Wierzbowska. "Polymorphism Of CD44 Influences Efficacy Of CD34+Cells Mobilization In Patients With Hematological Malignancies." Blood 122, no. 21 (November 15, 2013): 3270. http://dx.doi.org/10.1182/blood.v122.21.3270.3270.

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Abstract Background In the last decade peripheral blood was the main source of hematopoietic stem cells (HSC) for autologous (auto-SCT) and allogeneic (allo-SCT) transplantation. The exact mechanisms of HSC mobilization are still not clear and efficacy of the procedure is hardly predictable. Numerous clinical factors including age, number of previous intensive regimens, radiotherapy and type of disease can influence efficacy of CD34+ cell mobilization for auto-SCT in patients with hematologic malignancies. Recently it has been stated that ligand- receptor interactions of adhesion molecules such as SDF1/CXCR4, VLA4/VCAM-1 or CD44/osteopontin play important role in homing of HSC in hematopoietic niche. There is evidence that disruption of the ligand-receptor complex leads to egress of HSC to peripheral blood. Influence of single nucleotide polymorphisms in CD44, VCAM-1 and CXCR4 on efficacy of HSC mobilization was evaluated in healthy donors, but not in patients with hematological disorders. The aim of the present study was evaluation of constitutive polymorphism of genes encoding cytokines and receptors present in HSC niche and their impact on efficacy of mobilization of HSC in patients with hematological malignancies. Patients and Methods 110 (60 females and 50 males) were enrolled to the study. The median age of the patients was 55 (range 22-69) years. All of the patients evaluated were eligible for autologous HSC mobilization and transplantation. The group consisted of patients with multiple myeloma (74), non-Hodgkin lymphoma (19), Hodgkin lymphoma (15) or acute myeloid leukemia (2). The mobilization procedures comprised chemotherapy and then G-CSF at a dose of 10µg/kg daily. ‘Poor mobilizers’ group was defined according to GITMO criteria: patients with peak CD34+ in peripheral blood < 20/μL or total yield <2x 106 CD34+/kg in maximum 3 aphereses. Genotyping was performed using standard PCR-based assays. Three subgroups were established for the genotype in each polymorphism (homozygous more frequent, heterozygous and homozygous less frequent). Results The group of patients (N=108) who achieved minimal threshold for collections (CD34+ at least 10/µl) proceeded to aphereses. Median total yield of CD34+ in this group of patients was 5,6 x 106//kg, while median number of cells collected during first apheresis was 3,3x 106//kg. Median number of days of G-CSF treatment before first apheresis was 10. Fifteen patients fulfilled the criteria for ‘poor mobilizer’. The group of ‘poor mobilizers’ had higher frequency of TT allele in rs13347 (CD44) gene (CC+ CT vs TT p=0,047), the difference was even more pronounced in patients with multiple myeloma (N=72, p=0,027). TT homozygous genotype resulted in reduced CD44 mRNA expression at the time of apheresis in comparison with carries of C allele (median=40 in TT group, median=77 in CT and median=85 in CC group, p<0,001). Patients with TT allele had lower total yield CD34+/kg than the group with allele C (Median=3,7x 106//kg vs. 5,8x 106//kg, p=0.019) and lower number of CD34+cells gathered during first apheresis (0,95x 106//kg vs. 3,3x 106//kg, p=0.04). Multivariate logistic regression analysis including age, sex, diagnosis (multliple myeloma vs. others), number of previous treatment lines and CD44 polymorhism (TT vs CT+CC) revealed TT allele was the only factor associated with 5 fold higher risk of poor mobilization (p=0,037). Polymorphic variants of CXCR4 and VCAM-1 did not influence significantly efficacy of HSC mobilization in our group of patients. In conclusion, our results indicate that among investigated SNPs, only CD44 rs13347 has impact on efficacy HSC mobilization in patients with hematologic malignancies. CD44 SNPs analysis may be helpful for predicting of ‘poor mobilizers’ population that may benefit from newer modalities using adhesion molecules inhibitors. Disclosures: No relevant conflicts of interest to declare.

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McKinney-Freeman, Shannon, Hu Li, Matthew Curran, Sabine Loewer, Catherine Spina, Olaia Naveiras, James J. Collins, and George Q. Daley. "A Systems Biology Approach to Study the Acquisition of Adult Repopulating Potential During Hematopoietic Stem Cell Ontogeny." Blood 114, no. 22 (November 20, 2009): 1479. http://dx.doi.org/10.1182/blood.v114.22.1479.1479.

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Abstract Abstract 1479 Poster Board I-502 Hematopoietic progenitors and stem cells emerge at distinct times and anatomical locations during ontogeny. The first definitive hematopoietic progenitors appear in the yolk sac (YS) around day 8.5 of development (E8.5) while the first cells to meet the functional criteria of hematopoietic stem cell (HSC; cells capable of life-long restoration of the hematopoietic compartment of a recipient whose own hematopoietic system has been destroyed by chemicals or irradiation) can be detected in the aorta-gonads-mesonephros (AGM) at E10 of development. HSC can later be detected in large numbers in the E12.5 placenta, E14.5 fetal liver (FL) and adult bone marrow (BM). Induction of ectopic Cdx4 and HoxB4 expression during murine embryonic stem cell (ESC) differentiation followed by OP9 co-culture yields cells with in vivo hematopoietic repopulating potential (ESC-HSC). We have recently defined the phenotype of ESC-HSC relative to that of HSCs that arise in the embryo during development. Using cell fractionation followed by transplantation into irradiated Rag-2-/-γc-/- mice, we found that ESC-HSC display cell surface markers representative of both embryonic and adult HSC compartments: they express high levels of CD41, are heterogeneous for CD45, express CD150, and lack CD34 expression. We also found that CD150 is a developmentally regulated molecule on the surface of HSC, absent on the earliest HSC compartments of the AGM and placenta, but present on FL and bone marrow HSC. Although hematopoietic cells of the early YS do not display HSC potential when transplanted into the periphery of adult recipients, these cells can acquire this potential if infected with the homeobox gene HoxB4 and cultured on OP9 stroma, similarly to ESC. These data suggest that activation of specific molecular pathways can induce adult repopulating potential. To identify the critical genetic networks regulating the acquisition of adult hematopoietic repopulating potential, we are exploiting prospective purification via flow cytometry and microarray technology to assess the global gene expression profiles of HSC and progenitors from throughout murine ontogeny and embryonic stem cell differentiation. By comparing populations that lack adult repopulating potential (c-kit+CD41+CD34+ E9 YS cells, c-kit+CD41+CD45- ESC-derived cells and VE-cadherin+CD41- ESC-derived cells) with those that possess this activity (VE-cadherin+CD45+ E11.5 AGM, c-kit+CD34medCD45+ E12.5 placenta, Lin-c-kit+Sca-1+CD150+CD48- E14.5 FL, Lin-c-kit+Sca-1+CD150+CD34- BM, and CD41highCD45-CD34- cells from HoxB4 infected ESC expanded on OP9 stroma) we can use a novel gene network inference algorithm dubbed “mode-of-action by network identification” (MNI) to elucidate the key mediators for this functionality. The MNI algorithm predicts the key mediators under a particular perturbation by using a microarray compendium to infer a gene regulatory network that assesses how gene transcripts change in relation to one another and the net external influence. A large compendium with diverse experimental scenarios helps MNI to reverse engineer a comprehensive network model. We are currently using around 11,000 murine gene expression data sets acquired using 430 2.0 Affymetrix array chips to infer a regulatory network model, through which our HSC and progenitors microarray data will be filtered to identify the mode of action of the specific profiles accordingly. By comparing repopulating and non-repopulating populations from mouse ontogeny and ESC, we hope to identify the critical mediators and gene networks regulating the acquisition of adult repopulating potential during development. Disclosures: Daley: iPierian: Consultancy, Equity Ownership; Epizyme: Consultancy; Solasia: Consultancy; MPM Capital: Consultancy.

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Hawary, Amr M., Hazel E. Warburton, Richard J. Brough, Gerald N. Collins, Stephen C. Brown, Patrick H. O'Reilly, and Adebanji AB Adeyoju. "The ‘2-Week Wait’ Rule for Referrals for Suspected Urological Cancers – Urgent Need for Refinement of Criteria." Annals of The Royal College of Surgeons of England 90, no. 6 (September 2008): 517–22. http://dx.doi.org/10.1308/003588408x301082.

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INTRODUCTION All NHS-suspected cancers should be seen within 2 weeks of referral and are referred under government guidelines (Health Service Circular 205; HSC 205). This policy will be subject to review in 2009. Review is vital to allow the appropriate detection of malignancy without overburdening the premium clinic slots with the healthy. PATIENTS AND METHODS A total of 170 consecutive patients were referred from January–June 2005. Referral details, patient information, events and time to diagnosis were recorded. RESULTS Of these 170 patients, 143 were suitable for analysis. Forty-three patients (30%) were referred with frank haematuria, of whom 30% had bladder cancer. Nine percent of patients (n = 13) had microscopic haematuria none of whom had cancer. A quarter of the patients (n = 35) were referred with suspected testis cancer but none had cancer. Forty-one patients were referred with serum prostate-specific antigen (PSA) elevation; 18 cancers were detected in this group. Ten men had PSA values greater than 50 ng/ml. Only two cancers were suitable for radical prostatectomy. No cancer was found in patients less than 50 years of age. CONCLUSIONS A high cancer incidence was found (27.9%), the majority of which was bladder cancer or advanced prostate cancer. Out of the 143 patients, no malignancy was diagnosed in any patient less than 50 years of age, no malignancy was diagnosed in any of the microscopic haematuria group and there was no cancer diagnosed in the group of patients referred with scrotal swellings. We suggest that some guidelines are leading to referral of patients with low cancer risk. When the HSC 205 is revised in 2009, we hope studies such as ours are taken into consideration in order to improve resource utilisation.

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Park, Christopher Y., Yulei Wang, Susan Prohaska, Diane Tseng, and Irving L. Weissman. "MicroRNA Profiling of Human Acute Myeloid Leukemia and Normal Hematopoietic Stem/Progenitor Cells Reveals a Leukemia Stem Cell Signature." Blood 110, no. 11 (November 16, 2007): 779. http://dx.doi.org/10.1182/blood.v110.11.779.779.

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Abstract While numerous studies have contributed important insights into the molecular origins of human acute myeloid leukemia (AML), many may not accurately reflect molecular pathways critical to AML development or maintenance because they ignore the inherent heterogeneity among AML blasts. One subset of blasts - leukemia stem cells (LSCs) - exhibits the unique ability to self-renew and to engraft disease in immunodeficient mouse hosts, suggesting that their elimination is critical to developing curative therapies. In addition, there is little information regarding the role of microRNAs (miRNAs) in regulating gene expression or biologic function in AML. In order to assess the potential contribution of miRNAs to AML LSC biology, we have evaluated the expression profile of 315 mature miRNAs in FACS-purified AML LSC and compared it to both non-LSC blasts as well as normal human bone marrow (BM) derived hematopoietic stem cells (HSC) and committed progenitors using a multiplexed TaqMan-based real-time PCR strategy. SAM analysis with stringent criteria (at least 25% samples with Ct <30, FDR <1%) reveals that AML LSC and non-LSC blasts are more similar to one another than to normal HSC or committed progenitors. Among the BM populations tested, AML LSC and non-LSC populations are most similar to the granulocyte-macrophage progenitor (GMP). A set of miRNAs distinguishes AML LSC and non-LSC from normal HSC and committed progenitors, including 35 miRNAs that are under-expressed and 33 miRNAs that are over-expressed in both AML fractions versus the normal populations; many of these differentially expressed miRNAs show a range of expression exceeding 3 orders of magnitude. Supervised clustering analysis of AML LSC and non-LSC blasts reveals an LSC signature composed of 89 miRNAs, with nearly all differentially expressed miRNAs (86/89) exhibiting lower expression levels in AML LSC than non-LSC blasts. Finally, supervised clustering identifies a “stem-cell” signature composed of 17 miRNAs that are over-expressed in AML LSC and HSC versus committed progenitors. This group of miRNAs does not include miRNAs previously described as being highly expressed in embryonic stem cells. Together, these studies represent the first direct comparison of miRNA expression in a human cancer stem cell to its normal counterpart, thereby identifying miRNAs that may regulate AML LSC and/or normal HSC/committed progenitor function. Initial functional studies in vivo using LNA knockdown strategies indicate that a subset of miRNAs highly expressed in HSC and LSC is important in regulating normal HSC function. We are currently expanding these studies to test the role of these miRNAs in maintaining engrafted AMLs in the xenotransplant setting.

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To, L. Bik, Jean-Pierre Levesque, and Kirsten E. Herbert. "How I treat patients who mobilize hematopoietic stem cells poorly." Blood 118, no. 17 (October 27, 2011): 4530–40. http://dx.doi.org/10.1182/blood-2011-06-318220.

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Abstract Transplantation with 2-5 × 106 mobilized CD34+cells/kg body weight lowers transplantation costs and mortality. Mobilization is most commonly performed with recombinant human G-CSF with or without chemotherapy, but a proportion of patients/donors fail to mobilize sufficient cells. BM disease, prior treatment, and age are factors influencing mobilization, but genetics also contributes. Mobilization may fail because of the changes affecting the HSC/progenitor cell/BM niche integrity and chemotaxis. Poor mobilization affects patient outcome and increases resource use. Until recently increasing G-CSF dose and adding SCF have been used in poor mobilizers with limited success. However, plerixafor through its rapid direct blockage of the CXCR4/CXCL12 chemotaxis pathway and synergy with G-CSF and chemotherapy has become a new and important agent for mobilization. Its efficacy in upfront and failed mobilizers is well established. To maximize HSC harvest in poor mobilizers the clinician needs to optimize current mobilization protocols and to integrate novel agents such as plerixafor. These include when to mobilize in relation to chemotherapy, how to schedule and perform apheresis, how to identify poor mobilizers, and what are the criteria for preemptive and immediate salvage use of plerixafor.

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Yang, Liping, David Bryder, Jörgen Adolfsson, Jens Nygren, Robert Månsson, Mikael Sigvardsson, and Sten Eirik W. Jacobsen. "Identification of Lin–Sca1+kit+CD34+Flt3– short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients." Blood 105, no. 7 (April 1, 2005): 2717–23. http://dx.doi.org/10.1182/blood-2004-06-2159.

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AbstractIn clinical bone marrow transplantation, the severe cytopenias induced by bone marrow ablation translate into high risks of developing fatal infections and bleedings, until transplanted hematopoietic stem and progenitor cells have replaced sufficient myeloerythroid offspring. Although adult long-term hematopoietic stem cells (LT-HSCs) are absolutely required and at the single-cell level sufficient for sustained reconstitution of all blood cell lineages, they have been suggested to be less efficient at rapidly reconstituting the hematopoietic system and rescuing myeloablated recipients. Such a function has been proposed to rather be mediated by less well-defined short-term hematopoietic stem cells (ST-HSCs). Herein, we demonstrate that Lin–Sca1+kithiCD34+ short-term reconstituting cells contain 2 phenotypically and functionally distinct subpopulations: Lin–Sca1+kithiCD34+flt3– cells fulfilling all criteria of ST-HSCs, capable of rapidly reconstituting myelopoiesis, rescuing myeloablated mice, and generating Lin–Sca1+kithiCD34+flt3+ cells, responsible primarily for rapid lymphoid reconstitution. Representing the first commitment steps from Lin–Sca1+kithi CD34–flt3– LT-HSCs, their identification will greatly facilitate delineation of regulatory pathways controlling HSC fate decisions and identification of human ST-HSCs responsible for rapid reconstitution following HSC transplantations.

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Bourin, Philippe, Anne Huynh, Christian Recher, Christian Berthou, Laurent Garderet, Lofti Benbouker, Anne-Marie Perry, et al. "Stem Cell Factor (SCF) for Hematopoietic Stem Cell (HSC) Mobilization: Results of the Randomized IFM 99-01 Trial." Blood 104, no. 11 (November 16, 2004): 2921. http://dx.doi.org/10.1182/blood.v104.11.2921.2921.

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Abstract Introduction : In multiple myeloma, the usual mobilization protocol is toxic because of the use of cyclophosphamide. Several studies showed the interest of SCF. Objective : To compare two mobilization protocols: endoxan 4g/m2 + G-CSF 5μg/kg/j (arm A) versus G-CSF 10μg/kg/j + SCF 25μg/kg/j (arm B) in a prospective, open and, randomized trial. Patients and methods : the studied criteria were the quality of the cell collections (objective > 5.106 CD34+ cells into 2 cytapheresis), the toxicity of the mobilization and graft phases, as well as the post-graft hematopoietic reconstitution. Multiple myeloma patients, less than 65 years old, with 0 or 1 risk factor (ß2microglobulin > 3 mg/L or chromosome 13 deletion) and who have a response ≥ 50% after 3 cures of VAD were included. After a fourth cure of VAD each patient was planed to receive a tandem transplant (IFM 99-02 trial). Results : 150 patients (pts) were included and 138 were eligible (arm A = 67 pts, arm B = 71 pts). Pts and disease characteristics were similar in each arm. The objective of HSC collection was obtained with 92% pts in arm A and 81% pts in arm B (non significant). The total number of CD34+ cells collected were similar: 16.106 CD34+/kg (arm A) versus 15.106 CD34+/kg (arm B). Toxicity of HSC mobilization procedure was significantly different: duration of neutropenia < 500/mm3 (8 days in arm A versus 0 days in arm B, p<0.00001), duration of thrombopenia < 50 000/mm3 (5 days in arm A versus 0 days in arm B, p<0.0001), use of antibiotherapy (43% pts in arm A versus 9% pts in arm B, p<0.00001). 31% pts receiving SCF had local erythema at injection point. One pts experienced a grade 3 allergy in arm B. Hematopoietic reconstitution after first graft (high dose Melphalan 140 mg/m2, G-CSF at day 7) was not significantly different in either arm: duration of neutropenia < 500/mm3 (8 days in arm A versus 10 days in arm B), duration of thrombopenia < 50 000/mm3 (7.5 days in arm A versus 9 days in arm B), number of red blood cells units transfusions (1.5 versus 1.5). The 36 months overall survival probability was not significantly different with HDC mobilization (64%) versus SCF + G-CSF mobilization (87%). Conclusion : In myeloma patients, HSC mobilization with SCF + G-CSF is as effective as HDC + G-CSF, and gives very significant lower toxicities.

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Milone, G., M. Poidomani, S. Coppoletta, E. Mauro, E. Marturano, F. Crispi, A. Spadaro, A. Di Marco, and S. Leotta. "Defibrotide in Prevention of Liver Toxicity in Patients at High Risk of VOD after HSC Transplantation." Blood 112, no. 11 (November 16, 2008): 3275. http://dx.doi.org/10.1182/blood.v112.11.3275.3275.

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Abstract Defibrotide has been proposed as preventive treatment of Veno Occlusive Disease (VOD), data on its use are, however, limited and its effectiveness not yet demonstrated. We have administered Defibrotide as prevention of VOD in pts treated with HSC transplantation because of haematological malignancies, all patients were at high VOD risk because of hyper-ferritinemia (>800 ng/ml) or because not in CR of their underlying disease at time of transplant or being overweight (Actual BW>20% of Ideal BW) or because Hepatitis virus B or C sero-positive. 120 pts were treated with Defibrotide, 77 pts received allogeneic HSC tx and 43 autologous HSC tx, 105 received a myeloablative conditioning (55 pts it was based on busulfan) and 15 pts received a RIC. 48% of patients were affected with Acute leukemia, 23% with Lymphomas, 17% with Multiple Myeloma, 12% by other malignancies. Defibrotide was administered i.v. at dosage of 600 mg/d. from the day of conditioning was started to day +25 together with heparin at low dose (100 IU/Kg c.i.). RESULTS: after prophylaxis with Defibrotide a bilirubin value above 2.5 mg/dl during the first 30 days was observed in 16/120 pts (13%), 7/120 pts (5%) reached Seattle’s VOD criteria but had spontaneous resolution of liver toxicity, only 1 patient (0.8%) had a severe VOD and MOF. Overall survival at 3 years was 60% and it was 54% in allo-transplanted patients. We compared results with those obtained in a group of 78 pts treated by allogeneic or autologous transplants and who received low dose heparin only and not Defibrotide because they were considered at low risk of VOD. Percentage of pts who developed a bilirubin level > 2.5 mg/dl (p=0.12) and percentage of patients that reached criteria for VOD were not different (p= 0.08) in these two groups, numbers of red blood cell transfusions were comparable (p=0.07). When all the 198 studied patients were analyzed using logistic regression for factors important in the development of a bilirubinemia above 2.5 mg/dl we found to be important: use of MTX as prophylaxis of GVHD (P=0.004; Odds ratio 5,153), allogeneic transplant (P=0.007; Odds ratio 7,127) and baseline value of bilirubin (P=0.02; Odds Ratio 4,690). Conclusions: Use of Defibrotide in prophylaxis of VOD after HSC transplantation is safe and when employed in patients at high risk of VOD, it leads to an incidence of severe VOD below 1% with a frequency of liver toxicity equivalent to that found in patients having low risk features. To conclude on efficacy of Defibrotide in comparison to low dose heparin alone, a large randomised comparison is warranted.

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Kabir, Mohammed Humayun. "Necessity of initiating Rating Scale for more reliable assessment of writing skill at HSC level: a case study." IIUC Studies 6 (October 19, 2012): 35–52. http://dx.doi.org/10.3329/iiucs.v6i0.12247.

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In this essay, firstly, I will discuss importance of rating scales, relationship between assessment criteria and operations and conditions and effectiveness of rating scales while assessing writing. Secondly, I will examine a selection of scales and subsequently there will be an estimation of those in meeting objectives of the tests and of the course. Finally, I will recommend a suitable rating scale to test English First paper and English Second Paper writing skill at HSC level in Bangladesh evaluating the existing one where I will mention steps to be taken to reduce inter and intra- rating fluctuation in scoring. DOI: http://dx.doi.org/10.3329/iiucs.v6i0.12247 IIUC Studies Vol.6 2010: 35-52

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Magennis, Tina, and Jennifer Mitchell. "University Entry Scores as a Predictor of Academic Performance in a Health Information Management Program." Health Information Management 28, no. 2 (June 1998): 57–61. http://dx.doi.org/10.1177/183335839802800208.

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The university entry scores for school leavers admitted to the first year of the Bachelor of Applied Science (Health Information Management) degree at the University of Sydney in 1996 were examined to determine whether the Tertiary Entrance Rank (TER) was a good predictor of academic performance, as measured by grade point average (GPA). The study also examined Higher School Certificate (HSC) results in English and mathematics, and preference selection for the health information management (HIM) course to determine whether any of these had predictive validity. The results showed that TER, HSC English and mathematics scores and preference for the course were all poor predictors of academic performance in the student's first year. Low TER was not associated with low GPA and low scores in English and mathematics were not associated with low GPA. There was no significant difference between the performance of those students who listed the HIM course as their first preference and those who did not. These results suggest that there may be no need to establish a minimum entry level for admission to the HIM course, or for prerequisites in English and mathematics. It may be that multiple criteria are required to predict academic success in this course.

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Mokhtari, Saloomeh, Evan Joseph Colletti, Chad Sanada, Zanetta S. Lamar, Paul J. Simmons, Anthony Atala, Diane S. Krause, Esmail D. Zanjani, Christopher D. Porada, and Graca Almeida-Porada. "A Human Bone Marrow-Derived Stromal Cell Population with Hemogenic Potential." Blood 126, no. 23 (December 3, 2015): 1201. http://dx.doi.org/10.1182/blood.v126.23.1201.1201.

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Abstract During ontogeny, definitive hematopoietic stem/progenitor cells (HSC) are thought to arise from vascular endothelial cells, through an endothelial-to-hematopoietic transition, a natural process that occurs in unique, specialized embryonic hemogenic endothelial cells. Developmental studies, and experiments using pluripotent stem cells in an effort to recapitulate this process and thereby gain a better understanding of the emergence of definitive hematopoiesis, have collectively led to the prevailing view that the hemogenic endothelium constitutes a transient population of cells within the embryo that rapidly disappears during development and is absent in the adult. Herein, we provide the first evidence that at early time points of gestation, prior to the establishment of hematopoiesis, a unique subpopulation of Stro-1+ cells present within the inner part of the developing human bone marrow co-expresses APLNR, a marker of angiogenic mesoderm. Moreover, these Stro-1+APLNR+ cells express multiple other markers described for hemogenic endothelium, and subsequently contribute to the vasculature, cartilage, and bone. Importantly, we also show that cells expressing these same markers of primitive mesoderm/hemogenic endothelium persist at low frequency within the adult marrow. These adult-derived cells can be extensively expanded in vitro without loss of potential, but lack hematopoietic colony-forming potential in vitro. However, upon transplantation into a fetal microenvironment, clonally-derived populations of these adult Stro1+ isolated stromal progenitors (SIPs) not only contribute to the vasculature and nascent BM niches, but also efficiently generate, at a clonal level, hematopoietic stem cells (HSC) that are capable of robust, multilineage hematopoietic reconstitution, with generation of both myeloid and lymphoid cells upon serial transplantation. In conclusion, our studies have thus uncovered the latent potential of a highly expandable population of seemingly vestigial adult human somatic cells, whose ontogenic history includes a phenotype identical to that described for hemogenic endothelium. We have also shown that, if provided with the appropriate/necessary inductive factors, these unique adult cells are capable of giving rise to hematopoietic cells that fulfill the gold standard criteria for bona fide HSC. Therefore, these cells could potentially be more amenable to reprogramming technologies, to produce HSC that could be used to treat/cure a broad variety of blood diseases. Disclosures No relevant conflicts of interest to declare.

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Meignin, Véronique, Jean Soulier, Frédéric Brau, Marc Lemann, Eliane Gluckman, Anne Janin, and Gérard Socié. "Little evidence of donor-derived epithelial cells in early digestive acute graft-versus-host disease." Blood 103, no. 1 (January 1, 2004): 360–62. http://dx.doi.org/10.1182/blood-2003-06-1843.

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Abstract Donor origin of epithelial intestinal cells has been studied in animals and humans after transplantation and has been used as evidence of hematopoietic stem cell (HSC) plasticity. However, in the human gastrointestinal tract, no study used X- or Y-chromosome detection by fluorescence in situ hybridization (FISH) coupled with immunologic stainings to characterize cell types on the same tissue section. Here, we combined these techniques on the same section of duodenal epithelium in 6 patients with acute graft-versus-host disease. Donor-derived lymphoid cells were detected in the epithelium and the lamina propria, as expected. However, using our stringent criteria, no donor-derived cells could be proven to be epithelial.

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16

Yong, Agnes S. M., Keyvan Keyvanfar, Rhoda Eniafe, Bipin N. Savani, Katayoun Rezvani, Aarthi Shenoy, Eleftheria K. Koklanaris, et al. "The Level of Minimal Residual Disease in Primitive Progenitor Cells from CML Patients after Allogeneic Stem Cell Transplantation Is Higher Than after Treatment with Tyrosine Kinase Inhibitors." Blood 112, no. 11 (November 16, 2008): 1107. http://dx.doi.org/10.1182/blood.v112.11.1107.1107.

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Abstract The advent of imatinib, and subsequently, other tyrosine kinase inhibitors (TKIs) has relegated allogeneic stem cell transplantation (SCT) to second-or third-line therapy for chronic phase-chronic myeloid leukemia (CML). A significant shortcoming of TKIs in the large majority of good-responder patients is the persistent detection of BCR-ABL transcripts despite achievement of complete cytogenetic response (CCyR) or major molecular response (greater than 3-log reduction of BCR-ABL levels below a standardized baseline in minimal residual disease (MRD) measurements from total mononuclear cells). The presence of this MRD, and the demonstration that primitive CD34+ cells from patients in CCyR still harbor BCR-ABL (Bhatia, et al, Blood 2003) strengthens the opinion that TKIs do not eradicate all CML cells. Conversely, SCT has apparently cured some CML patients with more than 20 years follow-up. Of note, MRD is also quite frequently found in CML patients more than 5 years post-SCT, and donor lymphocyte infusions (DLI) are usually given to patients who satisfy criteria for molecular relapse. We compared the level of BCR-ABL transcripts in primitive hematopoietic cells from patients who were treated with T-depleted SCT with scheduled T-cell addback from D+45 to D+100 post-SCT (n=34) with levels in those taking TKIs (n=4). CD34+ progenitor cells were isolated from leukapheresis collections at D+120 post-SCT (n=13), peripheral blood from D+60 – 66 months post-SCT (n=19), bone marrow aspirates in patients on long-term follow-up (4–10 years) post-SCT (n=8) or 15 – 18 months post-TKI treatment alone (n=4); serial post-SCT samples were available in 6 patients. Using fluorescence activated cell sorting, hematopoietic stem cells (HSC, CD34+CD38-Lin-CD90+), common myeloid progenitors (CMP, CD34+CD38+Lin-IL3Rα+CD45RA-) and granulocyte-macrophage progenitors (GMP, CD34+CD38+Lin-IL3Rα+CD45RA+) were collected. BCR-ABL expression in primitive CD34+ subpopulations and total leukocytes from peripheral blood (PB) was measured using real-time quantitative polymerase chain reaction, with the sensitivity to detect one BCR-ABL-positive cell in 106 normal cells. A median of 112 x 106 mononuclear cells (range 16 – 582 x 106) were available per patient sample, with a median of 3 x 106 CD34+ cells (range 1 – 90 x 106) sorted. There was no difference between the number of HSC, CMP or GMP CD34+ cells collected between MRD negative or MRD positive-post-SCT patients, or TKI-treated patients. All patients with negative MRD in PB (n=12 post-SCT, n=1 post-TKI) did not have detectable BCR-ABL transcripts in primitive CD34+ subpopulations. Furthermore, in PB MRD-positive patients, 3/21 (14%) post-SCT and 2/2 (100%) post-TKI did not have detectable BCR-ABL transcripts in HSC, CMP or GMP populations. 18/21 (86%) PB MRD-positive patients who were post-SCT had detectable BCR-ABL transcripts in at least one primitive CD34+ subpopulation. A rise in BCR-ABL levels in GMP populations tended to herald impending relapse. In post-SCT patients on long-term follow-up with persistent PB MRD positivity not fulfilling criteria for molecular relapse, the highest BCR-ABL levels were in HSC populations. In comparable patients with a major molecular response, post-SCT patients appeared to harbor a greater amount of residual leukemia cells in CD34+ subpopulations than TKI-treated patients. Our observations suggest that although SCT is a curative treatment for CML, the graft-versus-leukemia effect may eliminate only more mature leukemic CD34+ subpopulations in some patients who have enduring positive MRD post-SCT, with persistence of the most primitive leukemic HSCs, which are presumably constrained in patients who do not fulfill criteria for relapse. Conversely, TKI appears to reduce the number of BCR-ABL-positive primitive CD34+ subpopulations, especially GMPs and CMPs, more efficiently. Our data support TKI-treatment as an adjunct to DLI to treat CML relapse post-SCT, and concurrent vaccination strategies which are able to target surface proteins on HSC to eradicate disease.

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Teresa, Fabrício Barreto, and Lilian Casatti. "Development of habitat suitability criteria for Neotropical stream fishes and an assessment of their transferability to streams with different conservation status." Neotropical Ichthyology 11, no. 2 (June 2013): 395–402. http://dx.doi.org/10.1590/s1679-62252013005000009.

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We assessed the preference of 10 fish species for depth and velocity conditions in forested streams from southeastern Brazil using habitat suitability criteria (HSC curves). We also tested whether preference patterns observed in forested streams can be transferred to deforested streams. We used data from fish sampled in 62 five-meter sites in three forested streams to construct preference curves. Astyanax altiparanae, A. fasciatus, Knodus moenkhausii, and Piabina argentea showed a preference for deep slow habitats, whereas Aspidoras fuscoguttatus, Characidium zebra, Cetopsorhamdia iheringi, Pseudopimelodus pulcher, and Hypostomus nigromaculatus showed an opposite pattern: preference for shallow fast habitats. Hypostomus ancistroides showed a multimodal pattern of preference for depth and velocity. To evaluate whether patterns observed in forested streams may be transferred to deforested streams, we sampled 64 five-meters sites in three deforested streams using the same methodology. The preference for velocity was more consistent than for depth, as success in the transferability criterion was 86% and 29% of species, respectively. This indicates that velocity is a good predictor of species abundance in streams, regardless of their condition

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18

Reed, William, Renée Smith, Florinna Dekovic, Joanna Y. Lee, Julie D. Saba, Elizabeth Trachtenberg, Joanna Epstein, Steffany Haaz, Mark C. Walters, and Bertram H. Lubin. "Comprehensive banking of sibling donor cord blood for children with malignant and nonmalignant disease." Blood 101, no. 1 (January 1, 2003): 351–57. http://dx.doi.org/10.1182/blood-2002-02-0394.

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Abstract Banking of cord blood (CB) for unrelated hematopoietic stem cell (HSC) transplantation is well established. However, directed-donor banking of CB for siblings in a current good tissue practices (cGTP) environment has not previously been investigated. Families were eligible for the present study if they were caring for a child with a disorder treatable by HSC transplantation and expecting the birth of a full sibling. We devised standard operating procedures and policies to address eligibility, donor recruitment, donor and recipient evaluation, CB collection, shipping, graft characterization, storage, and release of CB from quarantine. Many of these policies are distinctly different from those established for unrelated-donor CB banks. We enrolled 540 families from 42 states. Collections occurred at several hundred different hospitals. No family was deferred on the basis of health history or infectious disease testing, but departures from standard donor suitability criteria were documented. Disease categories for sibling recipients included malignancy, sickle cell anemia, thalassemia major, nonmalignant hematological conditions, and metabolic errors. Mean CB volume (including anticoagulant) was 103.1 mL; mean nucleated cell count was 8.9 × 108. Cell dose exceeded 1.5 × 107 nucleated cells per kilogram for 90% of banked units. Seventeen units (3.4%) have been transplanted. Sixteen of the 17 CB allograft recipients had stable engraftment of donor cells. Remote-site collection of sibling donor CB can be accomplished with a high success rate and in a cGTP-guided environment. The cellular products have been used successfully for transplantation; their number and characteristics should be adequate to support the first prospective clinical investigations of sibling CB transplantation.

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Rimmer, Emily K., Donald S. Houston, and Kristine Roland. "Adequacy of Bone Marrow Trephine Biopsy in a Tertiary Care Center." Blood 112, no. 11 (November 16, 2008): 4702. http://dx.doi.org/10.1182/blood.v112.11.4702.4702.

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Abstract OBJECTIVES: To review the bone marrow trephine biopsies analyzed at the Health Sciences Centre in Winnipeg, Canada in 2007 and evaluate them in terms of published criteria for adequacy. METHODS: The Health Sciences Centre (HSC) is the largest tertiary care centre in the province of Manitoba, and houses the only leukemia treatment/bone marrow transplant ward. It also provides pathology services to the adjacent clinics of the provincial cancer agency. One thousand and twelve (1012) bone marrow aspirates and biopsies were identified from January 1 – December 31, 2007 through pathology records. Bone marrow biopsies performed on children (age <18 years) and specimens referred from other centers were excluded. A total of 770 bone marrow aspirates and biopsies were identified as meeting the inclusion criteria and 67 were unavailable for evaluation. 703 biopsies were included in the final analysis. Data was collected on location of procedure (HSC ward vs. outpatient cancer clinic), operator and indication of biopsy. The total length of each biopsy and length of interpretable bone marrow were measured. The bone marrow biopsies were compared to published criteria for adequacy: 16 mm total length prior to processing (11 mm after processing), and 8 mm of interpretable marrow after processing. RESULTS: Using 8 mm of interpretable marrow as the criterion of adequacy, the overall adequacy rate was 67% (472/703). There was a significant difference in the percentage of adequate biopsies between operators. Hematologists obtained an adequacy rate of 81% (220/272), Registered Clinical Assistants (RCA) 63% (169/268), oncologists 56% (5/9), residents 50% (71/141), and medical students 54% (7/13), p<0.001). The mean overall length of biopsy after processing was 14.5 mm (SD 5.3 mm), with 10 mm (SD 5.4 mm) of interpretable bone marrow. Hematologists obtained samples with a mean length of interpretable bone marrow of 12.4 mm (SD 5.8 mm), RCA 8.6 mm (SD 3.9 mm), oncologists 10 mm (SD 4.1 mm), residents 8.4 mm (SD 5.7 mm), and medical students 8.2 mm (SD 4.5 mm). There is a significant difference in length of core biopsies obtained by different operators. Hematologists get longer biopsies than residents (p≪0.01), RCA (p≪0.01) and medical students (p<0.05). When looking specifically at those cases (n = 294) in which the requisition submitted at the time of the procedure stated an indication for which an adequate biopsy is crucial (detection of infiltration, diagnosis and staging of lymphoma) or when there was an inadequate/dry aspirate (n = 74), there was no significant difference in percentage of adequate biopsies when compared to the overall adequacy rate (70% vs. 67%, p=NS). When the biopsy was performed at the outpatient cancer clinic, the adequacy rate was 72.5% (342/472), whereas biopsies performed on HSC wards had an adequacy rate of 56.4% (133/236). CONCLUSIONS: A large proportion of bone marrow biopsies performed during the study period were of inadequate size. There was a significant difference in quality of bone marrow biopsies obtained by different operators, and among biopsies performed at different locations. The proportion of adequate samples was no better in cases where the aspirate was dry or where the suspected diagnosis should have mandated collection of an adequate sample. A multidimensional intervention including education and procedural changes will be implemented in order to improve the quality of bone marrow biopsies performed.

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Nishi, Katsuyuki, Taro Sakamaki, Kevin Shuolong Kao, Kay Sadaoka, Momo Fujii, Akifumi Takaori-Kondo, and Masanori Miyanishi. "Age-Associated Myeloid Biased Hematopoiesis Depends on Relative Decrease of Short-Term Hematopoietic Stem Cell." Blood 134, Supplement_1 (November 13, 2019): 2481. http://dx.doi.org/10.1182/blood-2019-122355.

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Aging in the human hematopoietic system is known to be associated with reduced self-renewal capacity, myeloid biased hematopoiesis, and increased incidence of hematological disorders. Amongst these changes, myeloid-biased hematopoiesis leads to decreased adaptive immune system function and increased propensity for myeloid malignancies in aged individuals. Recent reports have supported the concept that myeloid-biased alteration is most likely due to cell intrinsic alterations in the hematopoietic stem cell (HSC) compartment and a clonal shift toward myeloid-biased HSCs. Recently, we demonstrated that Hoxb5 specifically marks HSCs with long-term self-renewal capacity (LT-HSC) within the bone marrow of young mice. Additionally, we further demonstrated that around 80% of the cells included within the immunophenotypic HSC fraction (defined by the cell surface markers: Lineage-c-Kit+Sca-1+Flk2-CD150+CD34-/lo) (hereafter referred to as pHSCs) are not LT-HSCs (Chen JY et al., Nature 2016). In this study, using our LT-HSC-specific reporter mouse model, we sought to understand the mechanisms underlying the myeloid-biased alteration with age. First, to verify that Hoxb5 can be used as a LT-HSC-specific marker in aged mice, we utilized a transplantation assay in which 10 cells of Hoxb5+ or Hoxb5- pHSCs isolated from 2-year-aged mice were transplanted with supporting bone marrow cells into lethally irradiated mice. Any mice showing long-term (≧16week) granulocyte reconstitution (≧1% generation in peripheral blood) were considered as a positive for long-term hematopoiesis. Refer to this criteria, continuous hematopoiesis was observed only within the Hoxb5+pHSC recipients, indicating that Hoxb5 marks LT-HSCs within the pHSC compartment throughout the mouse lifespan. Interestingly, the lineage output derived from transplanted aged-Hoxb5+-HSCs was not skewed towards the myeloid lineage when compared to their young counterparts (the frequency of myeloid lineage contribution in donor cells: Aged vs Young = 42.6% vs 50.5%, n.s.). Our findings suggest that the balance between myeloid and lymphoid lineage output of the LT-HSC (Hoxb5+ pHSC) persists throughout life. To further confirm this hypothesis, we co-transplanted 10 cells of 2-year-aged Hoxb5+ pHSC with 10 cells of EGFP-overexpressed young Hoxb5+ pHSC along with supporting bone marrow cells into the same recipients. At 12 weeks posttransplant, peripheral blood (PB) analysis demonstrated that the frequency of myeloid output in both aged or young donor cells was nearly equivalent (Aged vs Young = 48.0% vs 43.2%, n.s.). According to previous reports, myeloid-related genes are known to be enriched in HSC compartment with age. However, given our previous findings demonstrating that ~80% of the pHSC compartment consists of non-LT-HSCs, we conducted RNA-seq analysis for bulk pHSCs (a mixture of Hoxb5+ and Hoxb5- pHSCs) and pure Hoxb5+ pHSCs isolated from young and 2-year-old mice respectively. Gene Set Enrichment Analysis showed that the previously defined geneset: preGM (granulocyte/macrophage progenitors)-specific genes (Pronk, C. J. et al., Cell Stem Cell 2007) are more highly expressed in aged pHSCs than in young pHSCs (NES = 1.25, q=0.067), whereas no significant gene enrichment of preGM-specific genes in aged Hoxb5+ pHSCs was observed compared to young Hoxb5+pHSCs (NES=-1.02, q=0.333). Given that the frequency of the Hoxb5- pHSC (which possesses only transient self-renewal and give rise to mainly lymphoid cell) in the pHSC compartment is decreased in aged mice (Aged vs Young = 43.3% vs 73.6%, p<0.0001), we hypothesized that the myeloid biased phenotypic change in the HSC compartment with age depends on a relative decrease of Hoxb5- pHSCs. To verify this, we transplanted young Hoxb5+ pHSCs and Hoxb5- pHSCs in a 5:5 ratio (the ratio between Hoxb5+ and Hoxb5- pHSCs in aged mice) or 2:8 ratio (the ratio in young mice). PB analysis at 16 weeks post-transplant demonstrated that recipient mice transplanted with Hoxb5+ pHSCs and Hoxb5- pHSCs in the 5:5 ratio have a greater myeloid lineage output than recipients of the 2:8 ratio (the frequency of myeloid lineage contribution in donor cells: 5:5 ratio vs 2:8 ratio = 31% vs 7%, p<0.05). In conclusion, the present study demonstrates that myeloid biased hematopoiesis in the aged setting results from the alteration of the ratio between Hoxb5+ and Hoxb5- pHSCs rather than cell-intrinsic change. Disclosures Takaori-Kondo: Ono: Research Funding; Takeda: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Janssen: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

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Mahfouz, Reda Z., Englehaupt Rickki, Joyce A. Juersivich, Kathleen Cooper, Lisa Durkin, Tomas Radivoyevitch, Ramon V. Tiu, et al. "Non-Cytotoxic Differentiation Therapy Based On Mechanism of Disease Produces Complete Remission in Myelodysplastic Syndromes (MDS) with High Risk Cytogenetics." Blood 120, no. 21 (November 16, 2012): 1696. http://dx.doi.org/10.1182/blood.v120.21.1696.1696.

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Abstract Abstract 1696 The standard treatment concept and approach in MDS aims for apoptosis-induction in expanding malignant clones in the hope that this will permit recovery by functionally normal hematopoietic stem cells (HSC). This concept is fundamentally undermined, however, by frequent genetic deletion in MDS/AML cells of key apoptosis genes. Hence, therapy selects for relapse with the most apoptosis-resistant malignant subclones (e.g., p53-null) while destroying crucial normal HSC (which have intact apoptosis-pathways). Observations regarding biology of sustained proliferation (MYC activity) in MDS/AML suggested an alternative approach: key late-differentiation MYC-antagonist genes (e.g., CEBPE) are repressed epigenetically even though lineage-specifying transcription factors (TF) (e.g., CEBPA) that drive expression of these genes are highly expressed. The basis for this paradox has been exposed: MDS/AML associated genetic abnormalities (e.g., RUNX1 mutation) favor recruitment of corepressors (e.g., DNMT1) instead of coactivators to lineage-specifying TF, thereby repressing differentiation target genes. Thus, in pre-clinical MDS/AML models, depleting DNMT1 by non-cytotoxic methods triggers MYC-antagonist expression and irreversible cell cycle exit by p53/p16-independent differentiation pathways (CEBPE-p27). The same treatment spares normal HSC, which unlike AML stem cells, do not express high levels of lineage-specifying TF. To translate these observations, this NIH-supported clinical trial (NCT01165996) incorporated the following novelty: (i) Lower dose of the cytidine analogue decitabine (DAC, Eisai) than in any previous trials (3.5–7 mg/m2) since hemoglobinopathy clinical trials demonstrated that these low doses deplete DNMT1 in normal HSC without cytotoxicity/apoptosis; (ii) SC instead of IV administration to avoid high peak levels that cause apoptosis; (iii) Use of the lack of toxicity to administer drug frequently 1–3X/week, to catch more MDS cells in S-phase via greater exposure times and distribution than in previous MDS trials (FDA-approved schedules only treat MDS cells entering S-phase in the first few days of multi-week cycles); (iv) Scientific correlates to measure non-cytotoxic, epigenetic-differentiation mechanism of action. Sample size = 25 as planned, 10/25 (40%) were relapsed/refractory after 5-azacytidine and/or lenalidomide, median age = 72y (range 46–85y), median disease duration = 900d (range 60–4680d). Anti-emetics were not needed, and cytotoxic side-effects e.g., hair loss did not occur. Neutropenic fever (NF) occurred in 11 subjects (5 had NF and 8 had ANC<500 prior to therapy). There was one treatment-associated fatality from sepsis. Though non-cytotoxic, suppression of clonal hematopoiesis is intended: nadir preceded complete remission (CR) 4/25 (16%) or hematologic improvement (HI) 7/25 (28%) (CR + HI = 47%) and 10/25 stable disease (SD) (CR+HI+SD = 84%) (IWG criteria). In transfusion dependent patients durations of transfusion independence for platelets (n=5) were median 343d (range 186–707d), 3/5 ongoing; for RBC (n=10) median 399d (range 290–696d), 5/10 ongoing. Complete cytogenetic remissions (CyR) occurred in 6/12 (50%) and partial CyR in 2/12 (17%) (overall CyR = 67%). Consistent with a p53-independent mechanism, CyR occurred even in poor risk cytogenetics, and increasing DAC frequency salvaged loss of response. Higher marrow cellularity was the best predictor of CR/HI, and CyR was substantially higher than CR/HI, underscoring that despite efficacious clone suppression, marrow HSC reserve is crucial to relief of cytopenia. Non-cytotoxic epigenetic-differentiation mechanism was confirmed by marrow IHC and FCM measurement of DNMT1, g-H2AX, p27/CDKN1B, MYC, and KI67 (3rd party quantification). Differentiation therapy transformed the outlook for M3 AML. Not surprisingly, a worthy goal has been to extend this paradigm. Biology of sustained MYC activity in MDS/AML guided logical repurposing of DAC for non-cytotoxic differentiation therapy even in MDS/AML with complex cytogenetics. Tolerance permits ready application despite age or comorbidities. Treatment exposure time and surrogates of normal HSC reserve are more important predictors of CR/HI than disease-mutations, and some patients may require separate measures (e.g., cytokines) to boost normal HSC diminished by age and previous insults. Disclosures: Reu: Celgene: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Saunthararajah:Cleveland Clinic Innovation: patent application for oral THU-decitabine., patent application for oral THU-decitabine. Patents & Royalties. Off Label Use: lower dose of decitabine, subcutaneous administration, metronomic administration.

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Miharada, Kenichi, Valgardur Sigurdsson, and Stefan Karlsson. "Developmental Pluripotency Associated 5 (Dppa5) Regulates Hematopoietic Stem Cell Reconstitution Capacity by Modulating Cellular Metabolism and ER Stress." Blood 120, no. 21 (November 16, 2012): 847. http://dx.doi.org/10.1182/blood.v120.21.847.847.

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Abstract Abstract 847 Expansion of hematopoietic stem cells (HSCs) will be required to achieve safe and effective transplantation for treatment of malignant blood disorders in adults. Due to regulatory constraints in the bone marrow (BM) stem cell niche, HSCs are quiescent under steady state conditions. Identification of regulators with a potential to expand HSC requires model systems with dividing stem cells, for example embryonic stem (ES) cells. To discover novel regulators that can increase the number of HSCs, we designed a screening protocol to identify possible candidates by comparing gene expression in undifferentiated and differentiated ES cells. We developed several key criteria to select candidates from microarray data and identified four extrinsic soluble factor candidates. One of these candidates, Cripto, was characterized as a critical regulator of HSC in the endosteal hypoxic niche by inducing higher glycolytic activity as an intermediary of the master regulator of hypoxic responses, HIF-1α (Miharada et al, Cell Stem Cell, 2011). Same strategy identified ten intrinsic factors. After initial selection, a second screening of the selected genes using a lentiviral over-expression approach in CD34−CD48−KSL cells identified three potential candidate genes. One of these candidates, Dppa5 (developmental pluripotency associated 5), generated more than 100-fold expansion of phenotypic HSCs upon enforced expression after two weeks culture in vitro. Overexpression of Dppa5 robustly increased the reconstitution level in lethally irradiated mice (15-fold higher than control, n=7, p<0.001) without loosing multi-lineage potential after 14 days culture in vitro, while this enhancement was not seen in the short-term (2 days) culture. It was noteworthy that the enhanced reconstitution by Dppa5 was significantly increased in the secondary recipients, whereas control vector transduced cells failed to reconstitute (n=14, p<0.001). Quantitative real time PCR (qRT-PCR) analysis revealed that the expression of Dppa5 mRNA is significantly higher in long-term HSCs than other progenitor populations. Conversely, down-regulation of Dppa5 using shRNA resulted in a significant severe reduction of reconstitution of the transplanted Dppa5 knockdown HSCs (12-fold reduction, n=6, p<0.001). In order to study what factors are affected by Dppa5 overexpression, two-dimensional gel electrophoresis (2D-DIGE) was performed. The findings showed that the levels of proteins involved in DNA synthesis and protein translation were increased. In contrast, decreased proteins included glycolysis related enzymes and ER stress response proteins. Surprisingly, all decreased glycolysis proteins (pyruvate kinase M2 (Pkm2), phosphoglycerate kinase 1 (Pgk1), and protease serine 1) have been observed as increased proteins when hematopoietic cells were cultured with recombinant Cripto. These proteins, as well as Cripto, are regulated by HIF-1α. Importantly, loss of Pkm2 has been shown to lead to a competitive repopulative advantage following bone marrow transplantation by modulating HSC metabolism (Wang et al., Abstract ISSCR 2012). These reported findings are consistent with our results. Moreover, GRP78 (a cell surface receptor for Cripto), GRP94, Calreticulin, and Pdia3 were decreased upon Dppa5 overexpression: These molecules are key components of ER stress chaperones and also known to be under the control of HIF-1α. Since these findings strongly indicate alteration in HSC metabolism, we measured cellular mitochondrial activity. MitoTracker (MT) staining showed higher intensity of MT in the Dppa5 over-expressed HSC population in the engrafted mice compared with the control (1.36-fold increase, n=4, p<0.05) suggesting that oxidative phosphorylation is enhanced. Molecular analysis has demonstrated that Dppa5 binds to multiple target RNA sequences and regulates the expression of several targets. UV-crosslinking immunoprecipitation (UV-CLIP) followed by qRT-PCR demonstrated binding of Dppa5 to the RNA sequence of GRP94 in hematopoietic cell lines overexpressing Dppa5, in addition to the previously reported genes (Mapk6, Cdc42, and Cyclin F). Thus, Dppa5 is a novel critical regulator of HSC and our preliminary findings indicate that its mechanism is governed by metabolism switching and decreased ER stress responses through GRP94 as an important bridging factor. Disclosures: No relevant conflicts of interest to declare.

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Leonard, Alexis, Naoya Uchida, David F. Stroncek, Sandhya R. Panch, Kamille West, Eoghan George Molloy, Tom Hughes, et al. "Safe and Efficient Peripheral Blood Stem Cell Collection in Patients with Sickle Cell Disease Using Plerixafor." Blood 134, Supplement_1 (November 13, 2019): 1964. http://dx.doi.org/10.1182/blood-2019-126091.

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Background: Hematopoietic stem cell (HSC) gene therapy is potentially curative for sickle cell disease (SCD) provided that a sufficient quantity of long-term engrafting CD34+ HSCs can be safely collected and infused. In patients with SCD, peripheral blood (PB) mobilization with granulocyte colony stimulating factor is contraindicated, and steady-state bone marrow (BM) harvesting is associated with suboptimal HSC quality and yield. Hence, agents that promote safe and effective peripheral HSC mobilization in SCD are required. Methods: This phase I multicenter study investigated the safety and efficacy of plerixafor mobilization (240 µg/kg) followed by apheresis, processing, and HSC enrichment in 15 adult subjects with SCD (NCT03226691). Hydroxyurea treatment was stopped at least two weeks prior to mobilization and all participants who were not receiving long-term transfusion therapy received prophylactic red blood cell exchange prior to plerixafor administration to achieve a target sickle Hb (HbS) <30%. The primary endpoint was efficient collection of PB HSCs (target 2.0x106 CD34+ cells/kg, minimum 1.5x106) after plerixafor mobilization without serious adverse events (SAEs). Collection products were assessed for total WBC, CD34+, CD3+, and CD19+ composition by flow cytometry. Spearman's correlation test was used to assess the relationship between baseline CD34/µL, total CD34+ cells/kg collected, and total blood volume processed. Findings: Fifteen participants with SCD (HbSS n=13, HbSC n=1, HbSβ+ n=1) who met inclusion criteria were enrolled at St. Jude Research Children's Hospital (n=3) or NIH (n=12) between July 2017 and February 2019. All participants except one successfully met the minimum target CD34+ cells/kg yield with 2 or fewer mobilization and apheresis procedures, with almost half the participants (n=7) achieving ≥5.0x106 CD34+ cells/kg. The total WBC count increased by an average of 3.2-fold over baseline (range 1.7-5.0) to an average peak WBC count of 26.5x103/µL (range 14.1-47.4), with counts returning to baseline within 1-2 days. Median total WBC, CD34+, CD19+, and CD3+ cells/kg in the final apheresis product after one (n=12) or two (n=3) collection procedures were 0.9 x109 WBC/kg (range 0.4-1.7), 4.5x106 CD34+ cells/kg (range 0.9-12.0), 3.4x108 CD19+ cells/kg (range 0.5-4.9), and 3.6x108 CD3+ cells/kg (range 1.7-7.3), respectively. There was a strong positive correlation between baseline CD34/µL and total CD34+ cells/kg (rs = 0.7776, 95% CI 0.446-0.9215, p = 0.001). Participants with the lowest pre-apheresis CD34 cell count generally underwent higher blood volume processing (rs = - 0.1443, 95% CI -0.6075 to 0.3921, p = 0.59) in an effort to achieve target yields. However, higher blood volume processing did not translate to higher total CD34+ cells/kg collection yields (rs = - 0.2104, 95% CI -0.6488 to 0.3328, p = 0.43). A median of 97% of positively selected CD34+ cells were CD34high (range 73.6-99.4%) compared to 1.3% CD34low (range 0.09-24.4%) phenotype suggesting enrichment for long-term engrafting HSCs. Seven grade III AEs (two non-pain and five pain related) and one grade IV AE (non-pain - hemolysis) occurred and each resolved with symptomatic treatment. Plerixafor mobilization was overall safe and in most cases generated CD34+ cell yields that were sufficient for both genetic modification and back-up allogeneic transplantation (median cell yield 4.2x106 CD34+ cells/kg). Interpretation: This first multi-institutional phase I study suggests the efficiency and safety of plerixafor mobilization and apheresis collection in 15 adult subjects with SCD. The primary endpoint to obtain a target of 2.0x106 CD34+ cells/kg from the PB was exceeded without SAEs. Importantly, immunophenotyping data suggest that for individuals with SCD, the plerixafor mobilized cell product is more enriched for long-term engrafting HSCs compared to CD34+ cells isolated from BM. Given the risks of general anesthesia and the low quality and yield of CD34+ cells harvested from the BM of individuals with SCD, plerixafor mobilization represents a safe and potentially superior alternative for HSC isolation. Disclosures Hankins: Novartis: Research Funding; ASPHO: Honoraria; NHLBI: Research Funding; LYNKS Foundation: Research Funding; NHLBI: Honoraria; National Committee for Quality Assurance: Consultancy; Global Blood Therapeutics: Research Funding; Bluebird Bio: Consultancy. Sharma:Vertex Pharmaceuticals: Other: Study PI; Doris Duke Foundation: Research Funding. Weiss:GlaxoSmithKline: Consultancy; Rubius INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Cellarity INC: Consultancy.

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Lawrence, H. Jeffrey, Christina M. Ferrell, Sheri T. Dorsam, Hideaki Ohta, R. Keith Humphries, Mika K. Derynck, Chris Haqq, and Corey Largman. "Activation of Stem-Cell Specific Genes by HOXA9 and HOXA10 Homeodomain Proteins in CD34+ Human Cord Blood Cells." Blood 104, no. 11 (November 16, 2004): 3227. http://dx.doi.org/10.1182/blood.v104.11.3227.3227.

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Abstract There is growing evidence for a role of HOX homeodomain (HD) proteins in normal hematopoiesis. Several HOX genes, including HOXA9 and HOXA10, are expressed in primitive hematopoietic cells, and that expression is down-regulated as cells mature, implying a role in early hematopoietic differentiation. In addition, retrovirally enforced over-expression of either of these two HD proteins results in dramatic expansion of hematopoietic stem cells (HSC). Our laboratory recently published a description of the HOXA9 transcriptome in human leukemic cell lines, using a transient over-expression strategy (Dorsam et al. Blood 2004). In that study we observed changes in the mRNA levels of a large number of genes within 24 hours of introduction of a HOXA9 expression vector in these cells. The modulated targets represented a wide variety of functional groups, including oncogenes, cell-cycle proteins, enzymes, membrane proteins and other transcription factors. Interestingly, a number of these putative targets are known to be part of the transcriptome of normal HSC. This previous study raises questions as to whether the HOXA9 targets identified in aneuploid immortalized myeloid cell lines would match the gene targets in normal hematopoietic cells. To answer this question and to compare target genes of two closely related HD proteins, human CD34+ umbilical cord blood cells were transduced with MSCV-based vectors expressing either HOXA9 or HOXA10, and RNA isolated from these cells was amplified and analyzed with cDNA microarrays. Statistical analysis using the Significance Analysis of Microarrays (SAM) algorithm revealed a common signature of several hundred modulated genes, demonstrating that the transcriptomes of HOXA9 and HOXA10 largely overlap in this cellular context. In a second step, CLUSTER analysis was performed, introducing threshold values to increase the likelihood of the identified genes being truly regulated and of biological significance. Using these more stringent criteria, 115 genes were modulated by one or both of the over-expressed genes. Several genes that were up-regulated by both HOX proteins were validated by quantitative real-time RT-PCR. HOXA9 and HOXA10 showed positive regulation of genes in the Wnt pathway, including Wnt 10b and two Wnt receptors Frizzled 1 and Frizzled 5, a important pathway for hematopoietic stem cell (HSC) self renewal. Other validated targets included Ets-related gene (ERG),, alcohol dehydrogenase 1 (ALDH1) and very long chain acyl-CoA synthetase (VLCS-H1), all of which are known to be expressed in normal CD34+ cells. One down-regulated gene in this survey is CYBB, a respiratory burst oxidase component pg91(phox), which is expressed in myeloid cells, and has previously been shown to be repressed by HOXA10. GenMAPP pathway analysis indicated that HOXA9 and HOXA10 repressed expression of several genes involved in heme biosynthesis and three globin genes, indicating a general suppression of the erythroid maturation program. HOXA9 and HOXA10 both appear to activate many HSC-specific genes while repressing genes involved in hematopoietic differentiation.

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Yang, Yan chun, Ya Gao, Ying Xu, Yintian Zhang, Dongmao Zhu, Zhiping Fan, Qifa Liu, and Baohong Ping. "Clinical Characteristics and Outcomes of 82 Patients with Mixed-Phenotype Acute Leukemia." Blood 132, Supplement 1 (November 29, 2018): 1124. http://dx.doi.org/10.1182/blood-2018-99-114348.

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Abstract Objectives: Mixed-phenotype acute leukemia(MPAL) is a rare disease and comprises 2% to 5% of all acute leukemia. Outcomes for MPAL are worse than both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).The complex phenotype exhibited by this type of leukemia resulted in a myriad of treatment approaches.In our study, we retrospective analysis 82 patients in clinical trail, treatment strategy and prognosis. Method: eighty-two patients diagnosed with MPAL at Nan fang hospital from 2006 to 2017 using either EGIL or 2008 WHO criteria were analyzed. Comparison the treatment effect and outcomes between different therapy types. Result: eighty-two patients, including 60 males and 22 females with a median age of 29 years (range, 2 months-72 years), were studied. 61 patients (77%) were older than 18years, 73patients met the criteria for MPAL via EGIL, 68via WHO2008, and 59of these were reported to satisfy both definitions. fifty one of these cases (62.2%) had a B/myeloid phenotype, Twenty four of these cases (29.3%) had a T/myeloid phenotype. The other cases (8.5%) showed immunophenotypic evidence of a B, T, and myeloid lineage in one blast population. Among the 82 cases, 57 cases with successful cytogenetic studies, 20(35.1%) had normal karyotypes and 37patients(64.9%) had abnormal karyotypes. Twelve patients (21.05%) translocation between chromosomes 9 and 22, five (8.8%) patients had 11q23/MLL translocations. Twelve patients (21.05%) had a complex karyotype and eight patients (14%) had other karyotype. The rarity of this disease and the fact that patients with MPAL are excluded from most AML and ALL clinical trials further complicates guidance about therapy. Of the 82 cases, 75 patients underwent the complete first course of treatment and complete remission rate was 49.8%. treatment approaches utilizing therapy for acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and so-called "hybrid" therapy mixing elements of both are 51.9%, 16.7% and 66.7% respectively (P=0.003).OS and EFS with hybrid induction therapy and ALL like induction were not significantly different than those with AML induction by either definition (P>0.05). A total of 60 patients received consolidation treatment, 21 patients received the chemotherapy while 39 patients received stem cell transplant (HSC). The total Median EFS was 21months, in Chemotherapy group and HSC group, the Median EFS was 6 months and 40 months respectively, The 3-year EFS was 26.1% and 55.6% respectively (P=0.038). The total Median OS was 21 months, Median OS showed a significant survival benefit for starting with chemotherapy as compared to HSC( 12 months and 43 months respectively (P=0.001)), The 3-year OS was 19.1% and 57.7% respectively. Conclusion: In this study, ALL like induction therapy or "hybrid" therapy was associated with a more than three-fold greater CR rate than AML therapy. SCT therapy showed a trend for an association with higher OS and EFS for MPAL . Key words: MPAL, Immunophenotype,Treatment strategy Funding Key Sci-Tech Research Projects of Guangdong Province (2014A02021102). Disclosures Fan: National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding.

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Olnes, Matthew J., Phillip Scheinberg, Katherine Calvo, Yong Tang, Susan Soto, Xingmin Feng, Ronan G. Desmond, Jay N. Lozier, Neal S. Young, and Cynthia E. Dunbar. "Eltrombopag Can Stimulate Trilineage Hematopoiesis with Transfusion Independence in Patients with Refractory Severe Aplastic Anemia: Results From a Phase II Trial." Blood 118, no. 21 (November 18, 2011): 54. http://dx.doi.org/10.1182/blood.v118.21.54.54.

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Abstract Abstract 54 Severe aplastic anemia (SAA) is characterized by trilineage marrow hypoplasia and a paucity of hematopoietic stem cell (HSC) progenitors. SAA is treated with immunosuppressive therapy (IST) or allogeneic HSC transplantation (HSCT), with a successful outcome after either treatment in a majority of patients. However, 20–40% of patients without a suitable donor for HSCT and a suboptimal response to IST may have persistent severe thrombocytopenia. Thrombopoietin (TPO) is the principal regulator of platelet production, and it exerts its effects through binding the megakaryocyte progenitor TPO receptor mpl, which stimulates production of mature megakaryocytes and platelets. Several lines of evidence support the concept that signaling through mpl also influences expansion and maintenance of primitive HSCs and multi-potent progenitor cells. Eltrombopag, is a small molecule TPO receptor agonist that stimulates mpl, and increases platelet counts in patients with chronic immune thrombocytopenic purpura (ITP). It is approved for the treatment of chronic ITP (Promacta®). We conducted a non-randomized, pilot phase II study of eltrombopag in SAA patients who remained severely thrombocytopenic at least six months after one or more rounds of IST (clinical trials.gov identifier NCT00922883). Consecutive patients fulfilling the inclusion criteria received eltrombopag 50mg daily with dose escalation every two weeks to a maximum dose of 150mg daily. Primary end points assessed after three months of treatment were changes in peripheral blood counts (platelets, hemoglobin, absolute neutrophil counts), with hematologic response criteria defined a priori for each lineage. Secondary endpoints included the incidence of bleeding events, and health related quality of life. Patients who achieved hematologic responses were maintained on eltrombopag through an extended access protocol. We completed our planned accrual of twenty five patients, and 22 are evaluable for response to date. Median age was 45 years old (range 18–77 years), and the median time from the last course of IST was 13 months (range 6–54 months). Median follow up time was 9 months (range 1–24 months). Nine of twenty two patients (41%) achieved hematologic responses: seven of twenty-two patients (32%) achieved platelet responses with transfusion independence for eight weeks or greater; six patients had improved hemoglobin levels after starting treatment (mean hemoglobin increase of 3.8 g/dL) and 4 patients who were previously dependent on red blood cell transfusions have achieved transfusion-independence. Five neutropenic patients had increased neutrophil counts after treatment with eltrombopag (mean increase 660 cells/uL). Plasma TPO levels did not predict for hematologic response to eltrombopag. Serial bone marrow biopsies performed on patients with hematologic responses demonstrated normalization of trilineage hematopoiesis and cellularity in three of four responders receiving a year or more of therapy, with no increase in reticulin fibrosis (Figure 1). These results represent the first evidence that TPO stimulation can expand the HSC pool in humans, with clinically meaningful trilineage hematologic improvements in patients with SAA, resulting in transfusion-independence and improved quality of life with a simple daily oral regimen. Updated response data on the full 25 patients will be presented at the Society's meeting. Disclosures: Off Label Use: Eltrombopag for severe aplastic anemia.

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Yang, Liping, Jörgen Aolfsson, Robert Månsson, David Bryder, Ole-Johan Borge, Lina Thoren, Ewa Sitnicka, Yutaka Sasaki, Mikael Sigvardsson, and Sten Eirik W. Jacobsen. "A Novel Road Map for Blood Lineage Commitment and Development: Identification of a Lympho-Myeloid Stem/Progenitor Cell Lacking Erythro-Megakaryocytic Potential." Blood 104, no. 11 (November 16, 2004): 2790. http://dx.doi.org/10.1182/blood.v104.11.2790.2790.

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Abstract The recent identification of common myeloid and lymphoid progenitors (CMPs and CLPs, respectively) lends support to the classical and currently prevailing model for hematopoietic commitment and blood lineage development, suggesting that the first and decisive lineage commitment step of adult hematopoietic stem cells (HSCs) results in an immediate and complete separation of myelopoiesis and lymphopoiesis. Virtually all of multipotent (lympho-myeloid) stem and progenitor cells in adult mouse bone marrow (BM) reside in the small Lin−Sca1+kithi (LSK) compartment. We now present data demonstrating that the LSK compartment in adult BM, can be separated into three phenotypically and functionally distinct HSC subsets, based on differential expression of CD34 and flt3. Long-term HSCs (with extensive self-renewing potential) reside in the LSKCD34−flt3− fraction. The LSKCD34+ short-term HSC compartment consists of LSKCD34+flt3− cells fulfilling all criteria of short-term HSCs, being highly enriched in CFU-S activity, and capable of rapidly reconstituting myelo-erythropoiesis and thereby rescuing myeloablated mice. The short-term LSKCD34+flt3− HSCs give upon transplantation rise to LSKCD34+flt3+ cells, that although sustaining a combined lymphoid (B and T cell) and myeloid (granulocyte and monocyte) developmental potential at the single cell level, loose their ability to adapt megakaryocytic and erythroid fates in vitro as well as in vivo. These evidence for the existence of LSKCD34+flt3+ cells with granulocyte, monocyte, B and T cell, but not megakaryocyte and erythroid development potentials, are not compatible with the first lineage commitment step of HSCs resulting in strict separation of common lymphoid and common myeloid differentiation pathways. Based on the present findings we rather propose an alternative road map for blood lineage development in which LSKCD34+flt3− short-term HSCs genereate megakaryocyte-erythroid progenitors and LSKCD34+flt3+ cells upon asymmetric cell divisions.

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Moratiel, Rubén, Raquel Bravo, Antonio Saa, Ana M. Tarquis, and Javier Almorox. "Estimation of evapotranspiration by the Food and Agricultural Organization of the United Nations (FAO) Penman–Monteith temperature (PMT) and Hargreaves–Samani (HS) models under temporal and spatial criteria – a case study in Duero basin (Spain)." Natural Hazards and Earth System Sciences 20, no. 3 (March 27, 2020): 859–75. http://dx.doi.org/10.5194/nhess-20-859-2020.

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Abstract. The evapotranspiration-based scheduling method is the most common method for irrigation programming in agriculture. There is no doubt that the estimation of the reference evapotranspiration (ETo) is a key factor in irrigated agriculture. However, the high cost and maintenance of agrometeorological stations and high number of sensors required to estimate it make it non-plausible, especially in rural areas. For this reason, the estimation of ETo using air temperature, in places where wind speed, solar radiation and air humidity data are not readily available, is particularly attractive. A daily data record of 49 stations distributed over Duero basin (Spain), for the period 2000–2018, was used for estimation of ETo based on seven models against Penman–Monteith (PM) FAO 56 (FAO – Food and Agricultural Organization of the United Nations) from a temporal (annual or seasonal) and spatial perspective. Two Hargreaves–Samani (HS) models, with and without calibration, and five Penman–Monteith temperature (PMT) models were used in this study. The results show that the models' performance changes considerably, depending on whether the scale is annual or seasonal. The performance of the seven models was acceptable from an annual perspective (R2>0.91, NSE > 0.88, MAE < 0.52 and RMSE < 0.69 mm d−1; NSE – Nash–Sutcliffe model efficiency; MAE – mean absolute error; RMSE – root-mean-square error). For winter, no model showed good performance. In the rest of the seasons, the models with the best performance were the following three models: PMTCUH (Penman–Monteith temperature with calibration of Hargreaves empirical coefficient – kRS, average monthly value of wind speed, and average monthly value of maximum and minimum relative humidity), HSC (Hargreaves–Samani with calibration of kRS) and PMTOUH (Penman–Monteith temperature without calibration of kRS, average monthly value of wind speed and average monthly value of maximum and minimum relative humidity). The HSC model presents a calibration of the Hargreaves empirical coefficient (kRS). In the PMTCUH model, kRS was calibrated and average monthly values were used for wind speed and maximum and minimum relative humidity. Finally, the PMTOUH model is like the PMTCUH model except that kRS was not calibrated. These results are very useful for adopting appropriate measures for efficient water management, especially in the intensive agriculture in semi-arid zones, under the limitation of agrometeorological data.

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Kelly, Patrick, B. Balcik, K. Bohn, R. Mueller, I. Jurickova, T. Schuesler, L. Reeves, et al. "Early Results of the Clinical Collection and Genetic Correction of Fanconi Complement Type A Hematopoietic Stem Cells." Blood 106, no. 11 (November 16, 2005): 1283. http://dx.doi.org/10.1182/blood.v106.11.1283.1283.

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Abstract Fanconi anemia (FA) is a genetic syndrome characterized by almost uniform development of aplastic anemia. Current therapies for patients lacking HLA-identical sibling hematopoietic stem cell (HSC) donors have shown high morbidity and mortality in clinical trials. Genetic correction of FA HSC using viral vectors has been demonstrated in animal models. However, harvesting of sufficient CD34+ cells at the time that HSC therapy is clinically indicated is difficult due to the severe bone marrow hypoplasia that accompanies pancytopenia. We have opened two phase I clinical trials that seek to determine if potentially useful numbers of CD34+ cells can be collected early in the course of the disease (collection study) and if these cells, once corrected, can engraft without cytoreduction and demonstrate proliferative advantage in vivo over un-corrected cells (gene transfer study). These studies are being conducted with approval by the FDA/NIH-RAC/Institutional IRB and monitored by an independent DSMB. To date, 4 FA patients have undergone a 20 ml/kg bone marrow harvest (BMH) with an average of 1.3x106 CD34+ cells/kg (range 0.3–2.9x106 CD34+ cells/kg) collected, suggesting that collection of adequate numbers of cells will be challenging, even early in the disease. In the gene transfer study, 3 FA patients with genotype A (FAA) have enrolled, meeting eligibility criteria of FAA, no evidence of malignancy and a minimum of 1x105 viable CD34+ cells/kg for ex vivo culture and gene transfer. BMHs from the 3 patients (2 fresh and one previously cryopreserved) were CD34+ cell selected using the CliniMACS device. Despite collection before significant pancytopenia, an average of only 5x105 CD34+ cells/kg (range 1.5–10x105 CD34+ cells/kg) was purified from these 3 cases representing ~10% of the expected yield from normal individuals. These cells underwent ex vivo gene transfer using cytokine prestimulation in serum-free medium followed 2 exposures to a GALV-MSCV-FANCA vector. Transduction efficiency of the final products determined by real-time PCR analysis of CD34+-derived progenitors averaged 48% (range 40–62%). Equivalent efficiency of correction of mitomycin C hypersensitivity in progenitor cells confirmed this analysis at the functional level. Nucleated cell recovery after ex vivo manipulation was 82–110% of input nucleated cells using freshly harvested bone marrow derived CD34+ cells (N=2). However, despite good CD34+ cell recovery and viability after CD34+ selection, only 6% of input nucleated cells were recovered utilizing CD34+ cells purified from the previously cryopreserved bone marrow and these cells were not re-infused. In the two patients who did receive gene corrected cells, the total cell dose re-infused was 2.5–3.5x105 nucleated cells/kg, reflecting the low number of initial CD34+ cells placed in culture. One patient is now 6 months post re-infusion with no evidence of gene marking observed in her PB or BM. The second patient had detectable FAA vector sequences in her PB early post-infusion (+4 weeks) but had none detected +8 weeks. The data suggest that while gene transfer efficiency in the clinical setting has been significantly improved, collection and expansion (either in vitro or in vivo) of adequate number of HSC may be critical to the success of genetic correction attempts in FA.

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Pina, Cristina, and Tariq Enver. "Transcriptional Programming in Quiescent Human Hematopoietic Stem Cells - Implications for Regulation of Lineage Commitment Decisions." Blood 106, no. 11 (November 16, 2005): 1723. http://dx.doi.org/10.1182/blood.v106.11.1723.1723.

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Abstract Human cord blood-derived CD133+G0 cells are a primitive population highly enriched in hematopoietic stem cells (HSC). We used this population to investigate the molecular characteristics of primitive human HSC, and, in particular, to unveil how different cell fates of quiescence, self-renewal and lineage commitment and differentiation are regulated at the molecular level. We isolated cord blood CD133+ cells in the G0 and G1 compartments of the cell cycle on the basis of low or high RNA content, respectively, as detected by Pyronin Y staining. More than 98% of the CD133+G0 cells were Ki67-negative, and at least 90% did not express CD38, thus confirming the quiescent and primitive status of the cells. Consistent with earlier findings showing that CD133+G0 cells have the highest reported frequency of LTC-IC (1), our quiescent population presented a significantly higher frequency of LTC-IC when compared to CD133+G1 cells. We further showed that CD133+G0 cells had significantly higher colony-forming capacity in progenitor assays and a higher CFU-Mix content. Initial RT-PCR analysis revealed that while both compartments express the erythroid marker beta-globin, myeloid MPO and lymphoid IL7R can only be observed in CD133+G1 cells. This suggests a hierarchy of commitment decisions in relation to cell cycle that places the erythroid signature upstream of myelo-lymphoid differentiation and may be in agreement with revised models of the hematopoietic differentiation tree recently proposed in mouse (2). This hypothesis is currently being assessed at the single-cell level. We next compared the overall gene expression programmes of CD133+G0 and G1 populations using global profiling. Consistent with the sorting criteria, CD133+G1-enriched transcripts have a comparatively higher frequency of cell cycle, protein synthesis and RNA processing, and metabolism-associated genes, which underlines the robustness of the data. The CD133+G1 population is associated with a lymphoid signature, including immunoglobulin heavy and light chains, and the SLAM family member CD48, which is consistent with the revised hierarchy of commitment decisions discussed above. Functional categories comparatively over-represented amongst CD133+G0-enriched genes include transcriptional regulation and signal transduction, suggesting that primitive quiescent HSC are ready to respond to a variety of cues that modulate alternative fate decisions. Since the transcriptional profile of a given population of cells may not reflect the transcriptional profile of each individual cell, we are currently analyzing the expression patterns of CD133+G0-enriched transcription factors (TF) at the single-cell level. This approach may help define subpopulations of cells with unique molecular signatures and suggest functional subcompartments within an otherwise heterogeneous population of primitive hematopoietic cells.

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Guthrie, Iric R., Mark D. Ehrhart, Jose R. Bucheli, and Mark R. Burge. "2141." Journal of Clinical and Translational Science 1, S1 (September 2017): 69. http://dx.doi.org/10.1017/cts.2017.246.

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OBJECTIVES/SPECIFIC AIMS: Thionamides are anti-thyroid drugs (ATD) that are commonly used to treat autonomous thyrotoxicosis. Although efficacious, these medications carry a risk of neutropenia or agranulocytosis in a small but finite proportion of the patients who receive them. Some risk factors for thionamide-induced neutropenia have been identified, including body mass index (BMI) and dose, but the role of race and ethnicity in the pathogenesis of this potentially life-threatening side effect is not known. We hypothesize that there will be no effect of race or ethnicity on the change in absolute neutrophil count (ANC) following initiation of thionamide therapy among adult patients with thyrotoxicosis. METHODS/STUDY POPULATION: Data from the electronic medical record at UNM HSC were obtained using a standard database query for the years 2000–2016. Inclusion criteria were the prescription of an ATD, an ANC recorded within 30 days of initiating ATD therapy (pre-ATD), and an ANC recorded between 75 and 365 days after starting an ANC (post-ATD). Patients taking other agents known to cause neutropenia and agranulocytosis, such as clozapine, allopurinol, or chemotherapy, were excluded. Patients were assigned to racial and ethnic groups as follows: Hispanic, non-Hispanic Caucasian (NHC), native American, Black, and Asian. The post-ATD ANC was defined as the nadir ANC observed after the ATD was started. “Delta ANC” was defined as [(post-ATD ANC)−(pre-ATD ANC)]. ANOVA analysis with Bonferroni-adjusted post-hoc testing was performed to examine differences in the mean changes of ANC across ethnic groups. RESULTS/ANTICIPATED RESULTS: In total, 123 adult patients met inclusion and exclusion criteria and were included in the analysis. No significant difference was found between any of the racial groups with regard to age, sex, BMI, pre-ATD ANC, or the pre-ATD to post-ATD ANC interval. The native American group showed a significantly greater post-ATD ANC (not shown) and Delta-ANC as compared with the other groups. Delta ANC Hispanic=−1.4±3.3, Caucasian=−0.6±3.3, Black=−0.9±4.1, Asian=−3.8±4.8, native American=3.6±5.1 (all units per mm3; p<0.001). DISCUSSION/SIGNIFICANCE OF IMPACT: In this cohort of New Mexicans with thyrotoxicosis, native American race was protective against thionamide-induced neutropenia.

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Hossain, Mohammad Delowar, Rubayet Ferdush, and Sajedar Rahman. "Unusual presentation of patients with obsessive compulsive disorder." Bangladesh Journal of Psychiatry 31, no. 2 (February 6, 2020): 48–50. http://dx.doi.org/10.3329/bjpsy.v31i2.45375.

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Obsessive compulsive disorder (OCD) is a common psychiatric disorder in clinical practice. But few studies have been done in Bangladesh. The aim of the study was to find out the unusual presentation of OCD patients other than common presentation. This was a cross sectional study done at private chamber and clinic in Dhaka, Bangladesh during the period of February, 2018 to January, 2019. Hundred twenty five patients fulfilling the inclusion and exclusion criteria were selected consecutively. After taking written consent a predetermined questionnaire was filled for each patient through face-to-face interview. The results showed that, most of the patients were from 21-30 years of age group (34.4%) with female preponderance (60%). Majority of the patients were married (60%), completed up to HSC level (44.8%) and students (38.4%). Among the patients the unusual presentations of OCD were new experience headache (56%), decreased sexual desire (16%), cough (9.5%), pain (8%), vomiting (8%), fit like behaviour (4%), increased frequency of micturition (2.4%) and deliberate self harm (DSH) (1.6%). The research findings will help us for the assessment, diagnosis and treatment plan. Therefore misdiagnosis and patient sufferings will be reduced. Bang J Psychiatry December 2017; 31(2): 48-50

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Voloshin, Sergei, Andrey Garifullin, Sergey Linnikov, Anastasiya Kuzyaeva, Vasily Shuvaev, Valentina Balashova, Georgiy Rysev, Alexander Chechetkin, and Zanna Chebukina. "The Comparison of Results of Autologous Transplantation Using Non-Cryopreserved and Cryopreserved Hematopoietic Stem Cells (HSC)." Blood 136, Supplement 1 (November 5, 2020): 5–6. http://dx.doi.org/10.1182/blood-2020-142279.

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Background: Bone marrow (peripheral blood stem cells (PBSCs)) autologous transplantation is the standard care for transplant-eligible patients with multiple myeloma. This treatment option is somewhat limited due to the high consumption of economic resources and the access to Cryobank. We performed a retrospective analysis of multiple myeloma patients who underwent autologous transplantation using non-cryopreserved and сryopreserved grafts at our institution from March 2016 to April 2020. Aim: Compare the results of autologous transplantation using non-cryopreserved and cryopreserved hematopoietic stem cells (HSC). Methods: 78 patients with MM were included in the study (male/female ratio 1.3:1). All patients got the standard immunochemotherapy programs. They had remission (≥partial response) till the auto-HSCT. Patients were divided in two groups depending on the technique of HSC storage: non-CRYO (n=35) and CRYO (n=43). Cryopreservation is a standard method of storage of HSC suspension. In our work we used the native HSC suspension which was saved from +4 °C to +6 °C during 72 - 120 hours. An effectivity and safety were evaluated in such parameters as the number of CD34+ and 7AAD- cells, colony-forming ability (CFA). All of these were made after apheresis and before reinfusion of HSC. Also, we compared the duration of hematopoiesis's recovery, the number of platelet transfusions, the length of hospitalization after auto-HSCT. Additionally, the effectiveness of therapy was assessed according to the IMWG response criteria and the level of residual tumor load before SCT and on day +100. Results : There were no differences in the total number of CD34 + cells x 106/kg, or in the level of 7AAD- cells, or in the total CFA. However, there was a significant difference in the percentage of loss of CD34+ cells from the moment of apheresis to the moment of reinfusion. We suppose it was caused adverse effects by temperature changes in CRYO group. In both groups, there were no severe infusion reactions on day 0. The adverse events (nausea, vomiting, tachycardia, increased total bilirubin and indicator liver enzymes) were absent in the non-CRYO group. But 29/43 (67.4%) patients had such symptoms in the CRYO group on day 0. The results are presented in the comparison table (image 1) of the evaluated parameters. All patients had full recovery of hematopoiesis till discharge from the hospital. Neutrophil recovery was achieved at 11th day (range 9-14) and platelets at 12th day (range 8-19) in the non-CRYO group, and 10th day (range 8-14) and 12th day (range 8 -20) in the CRYO group, respectively. The frequency of achieving a partial response before autoHSCT was 37% (13/35), a very good partial response - 40% (14/35), a complete response - 23% (8/35) in the non-CRYO group and 72% (31/43), 14% (6/43) and 14% (6/43) in the CRIO group, respectively. HSCT was improved the efficiency of treatment as well as the frequency of complete and MRD-negative responses in both groups. Partial response after autoHSCT was achieved in 23% (8/35) patients, very good partial response in 40% (14/35), complete response in 37% (13/35) in the non-CRYO group compared to the CRYO group (47% (20/43), 21% (9/43) and 32% (14/43), respectively). The MRD status was assessed before and after autoHSCT in 48 patients. The frequency of MRD-negative response before autoHSCT was 8.7% (2/23), after transplantation - 21.7% (5/23) in the non-CRYO group and 4% (1/25) and 12% (3/25) in the CRYO group, respectively. AutoHSCT was increased the "depth" of the response in 25 patients. However, there were no significant differences between the same-type categories in the non-CRYO and CRYO groups (p>.05). AutoHSCT led to decrease of tumor load (TL). The average TL value was 0.55% before HSCT and 0.018% after HSCT (p=.036) in the non-CRYO group and 2.05 and 0.37 (p=.003) in the CRYO group, respectively. The mean TL after HSCT in the non-CRYO (0.018%) was smaller than mean TL in the CRYO (0.37%) groups (p <.05). Conclusion: The method of storage of PBSCs without cryopreserved is equal to traditional method controlled freezing with Dimethyl sulfoxide and can be used in hospitals which have no a Cryobank in their composition. Table Disclosures Shuvaev: Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

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Dou, Diana R., Arazin Minasian, Maria I. Sierra, Pamela Saarikoski, Jian Xu, Stuart H. Orkin, Sacha L. Prashad, et al. "Inability to Express HOXA Cluster and BCL11A Genes Compromises Self-Renewal and Multipotency of hESC-Derived Hematopoietic Cells." Blood 120, no. 21 (November 16, 2012): 1190. http://dx.doi.org/10.1182/blood.v120.21.1190.1190.

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Abstract Abstract 1190 The inability to derive functional hematopoietic stem cells (HSCs) in vitro from pluripotent cells prevents widespread utilization of HSCs in the clinic; however, the molecular defects compromising the in vitro generated hematopoietic stem/progenitor cells (HSPCs) are unknown. Using a two-step differentiation method in which human embryonic stem cells (hESCs) were first differentiated into embryo bodies (EBs) and then CD34+ cells from hEBs were co-cultured on OP9M2 bone marrow mesenchymal stem cell (MSC) stroma (hEB-OP9), we were able to derive HSPCs expressing the HSC immunophenotype (CD34+CD38−CD90+CD45+) (hereafter termed CD90+HSPCs). Colony forming and stroma co-culture assays demonstrated that the hEB-OP9 CD90+HSPCs were able to differentiate into myelo-erythroid lineages and T-cells. However, when comparing CD90+HSPCs from hEB-OP9 to those from fetal liver (FL)—an in vivo source of HSCs—the former remained severely functionally limited in their proliferative potential and ability to differentiate into B-cells. To identify the basis of the proliferative and differentiation defects, we performed microarray analysis to define gene expression differences between CD90+HSPCs derived from hEB-OP9, FL, early 3–5 week placenta (PL) and an earlier stage of hESC differentiation (hEB). This analysis revealed establishment of the general hematopoietic transcription factor network (e.g. SCL, RUNX1, CMYB, ETV6, HOXB4, MYB), demonstrating the successful differentiation and identification of hematopoietic cells using our two-step culturing techniques and immunophenotype criteria. Moreover, evaluation of Spearman coefficients confirmed CD90+HSPCs isolated from hEB-OP9 culture were brought into closer resemblance of the hFL CD90+HSPCs as compared to to the developmentally immature hEB and hPL CD90+HSPCs. Encouragingly, hEB-OP9 CD90+HSPCs displayed downregulation of expression of genes related to hemogenic endothelium development associated with hEB and hPL while genes critical in HSPC function, including DNA repair and chromatin modification, were upregulated to levels comparable to hFL-HSPCs. However, a subgroup of FL HSPC genes could not be induced in hEB-OP9 HSPCs, including the HOXA cluster genes and BCL11A—implicated in HSC self-renewal and B-cell formation, respectively. Interestingly, absence of HOXA genes and BCL11A and poor proliferative potential were also observed in HSPCs from early placenta, suggesting these defects are not in vitro artifacts but instead reflect an inability of hEB-OP9 HSPCs to complete developmental maturation. To validate the necessity of HOXA genes and BCL11A in proliferation potential and multipotency, we next utilized shRNAs to target MLL—the upstream regulator of the HOXA cluster—, individual HOXA genes, or BCL11A in FL-HSPCs to test whether knockdown was sufficient to recapitulate the defects observed in hESC-derived HSPCs. Knockdown of HOXA7 resulted in the loss of CD34+ cells while HOXA9 shRNA-treated cells displayed a loss of more differentiated CD38hi cells. MLL knockdown depleted both CD38+ and CD34+ populations. BCL11A silencing resulted in the loss of B-cells. These studies identify HOXA genes and BCL11A as developmentally regulated genes essential for generating self-renewing, multipotent HSCs from pluripotent cells. Disclosures: No relevant conflicts of interest to declare.

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Chim, Chor Sang James, and Raymond Liang. "A Staged Approach to the Use of Bortezomib/Thalidomide/Dexamethasone (VTD) in Multiple Myeloma Patients with Suboptimal Initial Cytoreduction Led to a High Complete Remission Rate." Blood 112, no. 11 (November 16, 2008): 5202. http://dx.doi.org/10.1182/blood.v112.11.5202.5202.

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Abstract Autologous hematopoietic stem cell transplantation (AHSCT) and bortezomib are the two most important advances in the treatment of multiple myeloma (MM) in the last decade. Autologous HSC harvest is preferably performed at complete remission (CR) or after maximal reduction of myeloma tumor load. Owing to the high cost of bortezomib, we devised a total therapy incorporating VAD (vincristine, adriamycin, dexamethasone), followed by VTD in those with inadequate cytoreduction (i.e. <75% reduction in paraprotein) to maximize eradication of myeloma cells in the marrow prior to HSC harvest. Those achieving >75% paraprotein reduction proceeded to AHSCT directly. Those with measurable disease after the first AHSCT proceeded to second AHSCT. Response was based on the EBMT criteria (Blade et al, 1998). There were 16 patients with a median age of 50 years (33–64). Majority had advanced stage disease by International staging system. After VAD, 6 patients had >75% response (>90% response, n=3; 75%–90% response, n=3) and hence proceeded to first AHSCT directly. The other 10 patients had VTD salvage therapy, which upgraded response in 9 cases. After VAD and VTD treatments, there were 2, 6,7 and 1 patients achieving CR, >90% response, 75%–90% response and no response respectively. Thirteen patients proceeded to the first AHSCT, which rendered upgrading of response in 9 patients (69%) with CR/near CR in 7 cases (54%) and >90% response in 4 cases (31%). The latter 4 patients proceeded to second AHSCT, resulting in CR/near CR in 9 cases (70%) and >90% in 2 cases (15%). Of the 11 patients with >90% response after first and/or second AHSCT, 5 patients had maintained response with continual CR in 4 cases, whereas 6 patients relapsed (with asymptomatic disease in 4 cases and fatality in 2 cases). Therefore, this strategy of employing bortezomib only in those with insufficient cytoreduction achieved a high CR/near CR rate comparable to that with upfront bortezomib use. However, the frequent relapse implied that the quality of CR is equally important. Moreover, the aggressive fatal relapse in CR patients demonstrated a biologically aggressive subgroup in which CR might be inadequate.

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Ma, Dongdong, Jing Zhang, Hui-feng Lin, Joseph Italiano, and Robert I. Handin. "The identification and characterization of zebrafish hematopoietic stem cells." Blood 118, no. 2 (July 14, 2011): 289–97. http://dx.doi.org/10.1182/blood-2010-12-327403.

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Abstract HSCs are defined by their ability to self-renew and maintain hematopoiesis throughout the lifespan of an organism. The optical clarity of their embryos and the ease of genetic manipulation make the zebrafish (Danio rerio) an excellent model for studying hematopoiesis. Using flow cytometry, we identified 2 populations of CD41-GFP+ cells (GFPhi and GFPlo) in the whole kidney marrow of Tg(CD41:GFP) zebrafish. Past studies in humans and mice have shown that CD41 is transiently expressed in the earliest hematopoietic progenitors and is then silenced, reappearing in the platelet/thrombocyte lineage. We have transplanted flow-sorted GFPhi and GFPlo cells into irradiated adult zebrafish and assessed long-term hematopoietic engraftment. Transplantation of GFPhi cells did not reconstitute hematopoiesis. In contrast, we observed multilineage hematopoiesis up to 68 weeks after primary and secondary transplantation of GFPlo cells. We detected the CD41-GFP transgene in all major hematopoietic lineages and CD41-GFP+ cells in histologic sections of kidneys from transplant recipients. These studies show that CD41-GFPlo cells fulfill generally accepted criteria for HSCs. The identification of fluorescent zebrafish HSCs, coupled with our ability to transplant them into irradiated adult recipients, provide a valuable new tool to track HSC homing, proliferation, and differentiation into hematopoietic cells.

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Crawford, P., F. Kirkpatrick, O. Galway, and K. Watson. "576 ESTABLISHING VIRTUAL MULTIDISCIPLINARY ROUND IN BELFAST TRUST NURSING HOMES: PHARMACIST, NURSING & HEALTHCARE TEAM COLLABORATION." Age and Ageing 50, Supplement_2 (June 2021): ii8—ii13. http://dx.doi.org/10.1093/ageing/afab116.23.

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Abstract Introduction During the first covid surge, 25% of Belfast HSC Trust (BHSCT) care homes were affected, rising to 44% by surge 3, resulting in limited face to face access for healthcare professionals. Nursing home residents required medicine reviews post-covid infection to optimise medicines and reduce pill burden. Method The Care Home Nursing Support Team (CHNST), consultant pharmacist for older people and the lead care home pharmacist medicines optimisation older people (MOOP), rapidly established a multidisciplinary virtual round. Four main steps included: An SOP was established to ensure consistent pathway for nursing home inclusion criteria and team roles. The inclusion group included residents who were furthest from their baseline including weight loss, swallowing difficulties, decreased mobility, altered sitting balance and polypharmacy. The pharmacist developed a proforma template for completion by the nursing home staff to gather key information ahead of the round to improve efficiency eg swallow, renal function, pain, falls risk. The care home resident was included on video link by ipad following careful consent processes. Benefits included enhanced assessment of frailty, mobility, dexterity and adherence. Results Conclusion The multidisciplinary care home rounds provided an efficient means to collaborate with other professionals, while providing holistic & patient-focussed care. Plans are underway for development of an NI MOOP care home pathway.

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Takahashi, Tsutomu, Ritsuro Suzuki, Hiroyasu Ogawa, Takahiro Fukuda, Kazuteru Ohashi, Shuichi Taniguchi, Yoshinobu Kanda, Hiromasa Abe, Yoshihisa Kodera, and Junji Suzumiya. "The Safety of Hematopoietic Stem Cell Harvest from Elderly Family Donor in Japan." Blood 126, no. 23 (December 3, 2015): 1897. http://dx.doi.org/10.1182/blood.v126.23.1897.1897.

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Abstract [Background and Objectives] The number of patients received allogeneic hematopoietic stem cell (HSC) transplantation is increasing year by year, particularly for elderly patients. Related donor is preferable than the unrelated, but the safety of elderly donors has not been clarified. For this purpose, the complications of elderly HSC donor were retrospectively analyzed in comparison with younger donors. [Materials and Methods] From September 2006 to December 2014, a total of 7,896 related HSC donations was reported to the Japan society for hematopoietic cell transplantation (JSHCT) registration system by 391 harvest teams. The day 30 check reports after harvest were available for 6,911 (87.5%) donations. Donors under 18 years old were excluded, and 6,297 donations were analyzed in the present study. For donor age analysis those ranging from 18 to 30 years were regarded as reference. The primary endpoint was the incidence of early severe adverse events (eSAEs) during the harvest or within the 30-day period. Statistical analyses were conducted using Fisher's exact test to compare the donor demographics and to identify the relationships between the incidence of eSAEs. Logistic regression analyses were used to analyze the factors influencing eSAEs. Data analyses were conducted using EZR software, version 1.23 (Saitama Medical Center, Jichi Medical University). This study was approved by the ethical committee of JSHCT and Shimane University. [Results] Median age of donors were 42 years old (range: 18-80). There were 3,232 male and 3,002 female donors. A total of 2,009 donations were planned to collect bone marrow (BM) and 4,288 donations were planned to collect peripheral blood stem cell (PBSC). Nine of the planned BM harvests and 64 of the planned PBSC collections were cancelled because of the various reasons including adverse reactions or poor stem cell mobilization. Three other donations planned to collect BM were changed to PBSC, and four donations planned to collect PBSC were changed to BM, vice versa. These altered cases were included to the analysis as an intention-to-harvest basis. Out of 6,297 HSC donations, 63 donors (1.0%) were reported by the harvest teams to have experienced eSAEs. SAEs were de\x{fb01}ned as follows: death, events dangerous to life, prolongation of hospitalization, permanent failure, disease or abnormality inherited to offspring and other important medical events. The details of eSAEs were pain (8), infection (8), allergy (6), blood access related (5), neuropathy (4), thrombocytopenia (4), liver dysfunction (2), gout (2), tetany (2), epidural hematoma (2), pulmonary embolism (1), and others (19). None died due to eSAEs and 81 percent of donors recovered from eSAEs in an average of 17 days. In comparison with reference (18 to 30 years) the relative risk of eSAEs was 0.74 for donors aged 31-35 years, 0.37 for 36-40 years, 0.62 for 41-45 years, 1.04 for 46-50 years, 0.77 for 51-55 years, 1.27 for 56-60 years, and 2.66 for 61-65 years. Those aged 61-65 years only had significantly elevated risk of eSAE by univariate analysis (P = 0.02). The incidence of eSAE in each age group are shown in the Table. Univariate logistic regression analysis also showed female sex (1.3% vs 0.7%, 95% CI 1.16-3.55, P = 0.01) and current health conditions (1.9% vs 0.9%, 95% CI 0.99-4.12, P = 0.04) were risk factors affecting eSAEs other than age. Donation type (bone marrow or peripheral blood), laboratory abnormality at health screening and JSHCT donor insurance eligibility criteria did not affect the eSAEs. Multivariate analysis revealed that age category of 61-65 years (Table) and female sex (OR 2.03, 95% CI 1.21-3.43, P = 0.01) were independent predictive factors. [Conclusion] The safety of elderly family HSC donors was significantly inferior to younger donors. Elderly family donors above age 61 should be selected carefully. These findings are useful for informed consent at donations of elderly family donors and consideration the upper limit of age of unrelated volunteer donors. Table 1. Relative risk of eSAE adjusted by sex Adjusted N incidence OR 95% CI P-value Total 1.0% 18-30 years 16/1493 1.1% 31-35 years 6/758 0.8% 0.75 0.29-1.91 0.54 36-40 years 3/756 0.4% 0.38 0.11-1.30 0.12 41-45 years 5/745 0.7% 0.62 0.23-1.71 0.36 46-50 years 8/720 1.1% 1.04 0.44-2.44 0.93 51-55 years 6/722 0.8% 0.77 0.30-1.98 0.59 56-60 years 10/739 1.4% 1.26 0.57-2.79 0.57 61-65 years 9/321 2.8% 2.66 1.16-6.06 0.02 66 years - 0/43 0.0% - - - Disclosures No relevant conflicts of interest to declare.

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Papapetrou, Eirini P., Michel Sadelain, and Frederic Bushman. "Genomic Safe Harbors in Human iPS Cells." Blood 118, no. 21 (November 18, 2011): SCI—47—SCI—47. http://dx.doi.org/10.1182/blood.v118.21.sci-47.sci-47.

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Abstract Abstract SCI-47 Current hematopoietic stem cell (HSC) gene therapy relies on randomly integrated retroviral vectors and is hampered by the risk of insertional oncogenesis often leading to leukemia. This risk would be minimized if therapeutic transgenes could be inserted in selected sites of the genome that permit appropriate function without disruption or dysregulation of endogenous genes — referred to as “genomic safe harbors.” The advent of induced pluripotent stem (iPS) cell technology offered unprecedented opportunities for the genetic engineering of human cells. iPS cells, unlike HSC, can be extensively cultured in vitro, enabling the selection and study of unique sites of transgene integration for the first time in a relevant setting. We proposed a definition of safe harbor sites, based on their topology in the genome with relation to coding genes and other genomic landmarks, using five criteria: (i) distance of at least 50 kb from the 5’ end of any gene, (ii) distance of at least 300 kb from any cancer-related gene, (iii) distance of at least 300 kb from any microRNA, (iv) location outside a transcription unit, and (v) location outside ultraconserved regions of the human genome (1). To test them, we developed a strategy to select iPS cell clones harboring a single copy of a randomly integrating vector at sites that meet our safe harbor criteria. In a recent proof-of-principle study, using a model for genetic correction of β-thalassemia major, we demonstrated that erythroid progeny of patient-specific iPS cell clones harboring a lentivirally encoded β-globin transgene in a safe harbor site express therapeutic levels of β-globin without perturbing neighboring genes. This approach, entailing the prospective screening and selection of integration sites, based on combined bioinformatics and functional analyses, provides a robust and dependable strategy for the genetic engineering of human iPS cells. iPS cell-derived cell products used in regenerative medicine will need to be genetically engineered to correct a genetic disease or permit in vivo cell tracking or the elimination of residual undifferentiated cells or progeny gone astray. Our strategy should be broadly applicable to introducing reporter, suicide, or therapeutic genes in a clinically relevant manner. We are currently exploiting this strategy to express a conditional HSV-tk suicide gene for purging of iPS cell progeny from teratoma-initiating cells. With the emergence of improved technologies for homologous recombination into human cells, targeted gene addition may soon become a realistic option if predefined validated safe harbor sites in the human genome are identified. We are utilizing our selection strategy in iPS cells (using lentiviral vectors with reporter cassettes that can be exchanged using Cre recombinase-mediated cassette exchange) as a platform for the de novo discovery and characterization of putative universal safe harbor sites that can be broadly used for the genetic engineering of multiple human cell types. Disclosures: No relevant conflicts of interest to declare.

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Dal Cortivo, Liliane, RITA Creidy, Aurélie Gabrion, Marianne Leruez-Ville, Sebastien Heritier, Fabien Touzot, Guilhem Cros, et al. "Anti CMV and/or Anti Adenovirus IFN-g-Positive CD4+ CD8+ T Lymphocytes for Treatment of Viral Infections After Allogeneic HSC Transplantation: First Results." Blood 120, no. 21 (November 16, 2012): 1906. http://dx.doi.org/10.1182/blood.v120.21.1906.1906.

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Abstract Abstract 1906 Reactivation of latent viruses such as cytomegalovirus (CMV) and adenovirus (AdV) is responsible for infections which may be life-threatening in HSCT recipients. In the post-transplantation period, severity and frequency of these infections depend on (a) the degree of donor-recipient HLA incompatibility and (b) the intensity of immunosuppressive therapy used to prevent immunological complications. Antiviral drugs may be partially effective, often toxic and cannot always control those viral infections.T cell immunity plays a major role in the control of viral infections. It has been demonstrated that the transfer of donor T lymphocytes specifically directed against viral antigens is capable of preventing, controlling and clearing viral infection (Feuchtinger T et al., 2004 and 2010). The present project aimed the evaluation of specific, cell-based immunity against CMV and AdV by injection of IFN-g-positive CD4+and CD8+ donor T lymphocytes isolated ex vivo after stimulation with viral peptides. Methods: Our protocol was designed for pediatric or adult patients treated by allogeneic HSCT and matching the following inclusion criteria: (1) biological and/or clinical symptoms of CMV and/or AdV infection 2) no response or contraindication to conventional antiviral treatment and (3) no or low grade pre-existing aGvHD at inclusion (≤ grade II) controlled by corticoids (<1 mg/kg). Antiviral treatments are allowed during the inclusion period. Donor IFN-g-positive T lymphocytes are isolated with the CliniMACS Cytokine Capture System (Miltenyi Biotech) after incubation with viral peptide pools. Primary evaluation criterion is the efficacy of the treatment on CMV viral load 21 days after the first injection. In the event of a negative or partial response and the absence of aGvHD, a second injection may be scheduled. Secondary evaluation criteria are (1) the occurrence of de novo aGvHD or aggravation of existing aGvHD, (2) the evolution of clinical symptoms potentially related to the infection, (3) the demonstration of biological in vivo expansion of injected T lymphocytes (as evidenced by the IFN-g secretion capacity and specific tetramer assays) and (4) for AdV infection, evaluation of efficacy (viral load, in vivo expansion of transfused lymphocytes, clinical symptoms) and the safety (occurrence of aGvHD) of this immunotherapy. Results: From September 2010 to July 2012, 9 patients were included: 3 male adults (46–54 years, 1 CLL, 1 CML and 1 AA, 2 geno- and 1 pheno-identical transplantation) and 6 children (age: 7–25 months, sex ratio F/M: 4/2, 4 FLH, 1 SCID and 1AA, 4 haplo, 1 geno- and 1 pheno-id transplantation). 4/9 patients were treated for CMV, 3/9 for AdV and 2/9 for CMV and AdV reactivation. 5/9 patients received 2 cytotoxic T lymphocytes (CTL) injections. Mean number of CD3 IFN-g positive cells injected was 4206/kg (1167–6000/kg) with 55% and 69% of CD4 and CD8 anti CMV-T cells and 56% and 61% of CD4 and CD8 anti AdV T cells respectively. Mean delay of first immunotherapy was 109 days (28–270) after transplantation. 2/9 patients were not evaluable due to early death (<21 days post injection) and 1/9 patient died of graft failure 43 days after CTL injection without efficacy on infectious evolution. 6 patients are still alive: 4 with complete, 1 with partial remission of virus replication and 1 recently included, is still under evaluation. An in vivo expansion of transfused CTL was observed (mean expansion was 33 and 35 fold for CD8-IFN-g and CD4-IFN-g positive cells respectively 42 days after injection) in parallel with the decrease of viral load in all alive patients. No aGvHD was detected in the 5/6 evaluated patients. One of 6 presenting cGvH at inclusion need increase of corticotherapy 3 months after second injection of CTL One patient presenting with CMV retinitis received 2 CTL injections without worsening of retina lesions which healed. Conclusion: The CliniMACS Cytokine Capture System allows the isolation of virus-specific T cells in a brief delay (24 hours) with a satisfactory enrichment of both CD4 and CD8 T cells. First results show efficacy of virus-specific T cells injection on viral load without signs of aGvHD in 5/6 evaluable patients. More patients need to be included in this trial in order to confirm these encouraging results. Disclosures: Cambouris: Miltenyi Biotec: Employment.

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Willis, J. P., M. Oguri, M. E. Ramos-Ceja, F. Gastaldello, M. Sereno, C. Adami, S. Alis, et al. "Understanding X-ray and optical selection of galaxy clusters: a comparison of the XXL and CAMIRA cluster catalogues obtained in the common XXL-HSC SSP area." Monthly Notices of the Royal Astronomical Society 503, no. 4 (March 26, 2021): 5624–37. http://dx.doi.org/10.1093/mnras/stab873.

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ABSTRACT Large samples of galaxy clusters provide knowledge of both astrophysics in the most massive virialized environments and the properties of the cosmological model that defines our Universe. However, an important issue that affects the interpretation of galaxy cluster samples is the role played by the selection waveband and the potential for this to introduce a bias in the physical properties of clusters thus selected. We aim to investigate waveband-dependent selection effects in the identification of galaxy clusters by comparing the X-ray MultiMirror (XMM) Ultimate Extra-galactic Survey (XXL) and Subaru Hyper Suprime-Cam (HSC) CAMIRA cluster samples identified from a common 22.6 deg2 sky area. We compare 150 XXL and 270 CAMIRA clusters in a common parameter space defined by X-ray aperture brightness and optical richness. We find that 71/150 XXL clusters are matched to the location of a CAMIRA cluster, the majority of which (67/71) display richness values N > 15 that exceed the CAMIRA catalogue richness threshold. We find that 67/270 CAMIRA clusters are matched to the location of an XXL cluster (defined within XXL as an extended X-ray source). Of the unmatched CAMIRA clusters, the majority display low X-ray fluxes consistent with the lack of an XXL counterpart. However, a significant fraction (64/107) CAMIRA clusters that display high X-ray fluxes are not associated with an extended source in the XXL catalogue. We demonstrate that this disparity arises from a variety of effects including the morphological criteria employed to identify X-ray clusters and the properties of the XMM PSF.

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Binato, Renata, Nathalia Oliveira, Luis Fernando Bouzas, and Eliana Abdelhay. "AML Mesenchymal Stem Cells Signature." Blood 124, no. 21 (December 6, 2014): 2932. http://dx.doi.org/10.1182/blood.v124.21.2932.2932.

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Abstract Background: Acute myeloid leukemia(AML) is a heterogeneous hematological disease characterized by proliferation and accumulation of myeloid precursors in the bone marrow, decrease of apoptosis level and differentiation arrest of these cells. The French, American and British group of leukemia experts (FAB) divided acute myeloid leukemias into eight subtypes based on the characteristics and behavior of leukocytes and the in cell differentiation blockage. Although several studies in the area, events related to the beginning of disease as well as its progression is still unknown. It is believed that malignant transformation in normal Hematopoietic Stem Cells (HSC) can give rise a Leukemic Stem Cell (LSC) and this transformation could be related to changes in Mesenchymal stromal cells (MSC) signaling. In this context, the aim of this work is to analyze the role of MSCs in the leukemic transformation of hematopoietic stem cell. To address this hypothesis we used ChipArray approaches to evaluate the gene expression profile of MSC from AML patients compared to MSC from Healthy donors (HD). Methods: For this purpose, bone marrow(BM) samples from HD and patients diagnosed with AML previously classified into subtypes were placed in culture. We used 7 samples from different subtypes of AML (four from subtype M2, two from M3 and one from M4/M5) and compared with two pools of HD. After 3rd passage, total RNA from MSCs cultures were obtained using RNeasy Mini Kit according to the manufacturer’s instructions (QIAGEN). After that, RNA was processed according to GeneChip whole transcription (WT) sense target-labeling assay (Affymetrix) and hybridized to GeneChip human gene 1.0 ST array (Affymetrix) washed and stained according to the manufacturer’s protocols. Data were analyzed using Partek® software and differentially expressed genes with ³ 2 fold-change was used as criteria to define overexpression or downregulation.To confirm the array assay we performed real-time PCR with a large number of patients with different subtypes. Pathway analysis and related processes was obtained using the Ingenuity Pathways Analysis (IPA) and MetaCoreTM softwares. Results: The results obtained in ChipArray assay showed that there is a common molecular signature to all MSCs AML patients compared to donors. Fifty five genes differentially expressed were found. To confirm the results obtained in ChipArray real-time PCR with some genes was performed with more patients (CCL2, SPON2, MUC12 - overexpressed and MMP16, BMP4, CLDN1, SDC2 and OPN downregulate). The results confirmed the commom molecular signature in all AML patients instead of subtypes.These genes are involved in processes of cellular adhesion, differentiation of osteoclast and Wnt pathway. We further carried out another ChipArray assay with another samples. At this time, an unsupervised analysis with the 55 differentially expressed genes was done to verify if the samples agrouping in clusters that separate AML patients from HD. The results confirmed that this molecular signature is capable to distinguish patients from HD. Changes in MSC signaling could be related to malignant transformation of HSC and some of these genes found in ChipArray are secreted proteins that could affect HSC. To verify if these secreted proteins are reallly differentially expressed in AML patiens we performed Western blot assay with plasma samples from AML patients and HD. The secreted proteins seleted were CCL2 and BMP4 (overexpressed and downregulated in AML patients, respectively). The results confirmed the increase of CCL2 protein and decrease of BMP4 protein in AML patients plasma. In Acute Lymphoblastic Leukemia (ALL), CCL2 was able to increase the adhesion of ALL cells to MSC and to promote survival and proliferation of MSC.BMP4 has been described as an important component produced by the hematopoietic microenvironment that regulates both HSC number and function. Moreover, BMP4 has also implicated BMP4 in homing and engraftment of human hematopoietic stem/progenitor cells. Conclusions: With these results, we could start to elucidate the participation of AML-MSCs in the leukaemogenic process. Altogether, both CCL2 and BMP4 produced and secreted by MSC from BM microenvironment seems to be important for the biology of AML cells. Financial support: CNPq, FAPERJ, INCT. Figure 1 Hierarchical Clustering of the 55 differentially expressed genes. Figure 1. Hierarchical Clustering of the 55 differentially expressed genes. Disclosures No relevant conflicts of interest to declare.

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Cupri, Alessandra, Salvatore Leotta, Uros Markovic, Maria Grazia Camuglia, Giulio Antonio Milone, Angelo Curto Pelle, Valerio Leotta, et al. "Isavuconazole Prophylaxis during Early Phases of Allogeneic HSC Transplantation Is Not Associated to an Increase Need of Cyclosporin-a Dose Modification." Blood 134, Supplement_1 (November 13, 2019): 3271. http://dx.doi.org/10.1182/blood-2019-127120.

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Background: Isavuconazole is an effective treatment of invasive fungal infections (IFI). Since its broad spectrum of activity and favorable toxicity, it could also be used as a prophylaxis. Experience in use of isavuconazole as prophylaxis of IFI is, however, limited. In allogeneic transplant setting, its feasibility should also be assessed in regard of changes of Cyclosporine (CYA) metabolism. Aim: To evaluate feasibility of the administration of Isavuconazole as short term prophylaxis during early phase of allogeneic transplantation and to study its influence on CYA trough blood levels and CYA dosing. Methods: In a prospective study from July 2017 to April 2019, we treated 34 HSC transplantation using Isavuconazole (ISA) prophylaxis, ISA was administered at dosage of 200 mg /day i.v. (with a loading-dose of 200 mg x 3 /days for the first 48 hours) from day + 2 after HSCT infusion and until day + 30. The patients were selected using the criteria: any underlying diagnosis; age 18-70; any type of allogeneic donor; GVHD prophylaxis including CYA i.v.. Blood trough levels of CYA were measured bi-weekly by ELISA. Blood CYA level was maintained between 100 - 300 mcg/ml by dose adjustment. The need for CYA dose modification, IFI rate and patient outcome (TRM, OS) was studied and compared to an historical control group who received prophylaxis using Fluconazole during day +2 to day +30 (n 29). Results: Groups receiving Isavuconazole or Fluconazole were not significanly different in diagnosis, age, sex, disease status at transplant, donor type, stem cell-source, GVHD-prophylaxis and conditioning regimen. No patients in the Fluco-group had a previous invasive fungal infection (IFI), while 2 patients had a previously IFI in the Isa-group. All patients reached engraftment, neutrophils engraftment was reached in a mean of 19 days and 18 days in the Isa and Fluco-group, respectively. No IFI occurred in the fluco-group, while 1 proven IFI (Blastoschyzomyces Capitatus blood-stream infection) occurred in the Isa-group. Mucositis occurred in 56 patients (88%). Mucositis grade III-IV grade of was registered in 62% and 61%, in the Isa- and Fluco-Group respectively. Isavuconazole was well tolerated, without significant toxicity, no patient needed dose-reduction or suspension. Mean values of trough CyA blood-levels were compared at for 1st, 2nd, 3rd and 4th week and no difference was found between the two groups (Mann Whitney U test p value: 0.2, 0.5, 0.8, 0.9 respectively for 1st, 2nd, 3rd and 4th week), Fig 1. Mean number of CYA-dose adjustments was 1.3 /patient in Isa-group and 1.2/patient in Fluco-group. Difference was not significant (p = 0.6, Mann-Whitney U-test). Overall survival (OS) at 1 year was 64 % in Fluco-group (95% CI: 44-79%) and 66% in Isa-group (95% CI: 44-81%) (log-rank, p value: 0.9). Non-relapse mortality (NRM) at 1 year was 16.3% (95% CI: 5.7-31%) in Isa-group and 14.2% (95% CI:4.3-29%) in Fluco-group (Gray test, p: 0.61). Conclusions: In this prospective study, short term prophylaxis using Isavuconazole, after allogeneic HSC transplant, was found to be feasible. No significant difference with standard fluconazole prophylaxis was found in trough CYA blood levels and in the need of cyclosporin dose modification. Further studies are needed extending the lenght of Isavuconazole prophylaxis. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Diercks, Deborah B., Ezra A. Amsterdam, David F. Gaieski, Diane Gurney, W. Frank Peacock, and Gregg C. Fonarow. "Discharge Criteria." Critical Pathways in Cardiology: A Journal of Evidence-Based Medicine 7, no. 2 (June 2008): 111–15. http://dx.doi.org/10.1097/hpc.0b013e318177dfcc.

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Baek, Kyong Oh, Jeong Min Lee, Tae Geom Ku, and Young Do Kim. "Evaluation of By-Pass Fishway Operation for Attraction Efficiency Based on GPS Drifter Field Experiments." Water 13, no. 16 (August 22, 2021): 2302. http://dx.doi.org/10.3390/w13162302.

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The attraction efficiency of a by-pass fishway installed at Gangjeong-Goryeong Weir on the Nakdong River in South Korea was evaluated according to flow rate variation of the main channel. Optimal flow rate that achieved the maximum value of attraction efficiency at the fishway entrance was also determined. The weir can adjust the flow rate of the main channel and the fishway by operating sluice gates. The weighted usable area (WUA) calculated on the basis of habitat suitability criteria (HSC) for target species using River2D (a two-dimensional physical habitat model) was regarded as an indicator of attraction efficiency. The simulated velocity field by River2D was validated by virtue of measured data acquired from GPS drifter field experiments. Additionally, monthly fish monitoring data obtained with a fish trap served as supporting data to confirm the validity of the estimated attraction efficiency. The monitoring results revealed that the fishway attraction efficiency was the highest during the spawning season (from April to June). The target fish used the fishway most frequently in April. However, many other fish species used the fishway in June. Simulation results revealed that the flow rate of the main channel at the weir should be maintained at 190 m3/s in order to most effectively attract the target fish species into the fishway entrance. Managing the optimal flow rate by operating the sluice gate is especially important during the spawning season of the target fish to facilitate upstream and downstream migration.

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Avni, Batia Ronit, Joycelynne Palmer, Tracey Stiller, and Stephen J. Forman. "Eosinophilia As a Biomarker Predicting the Occurrence of Chronic Graft Versus Host Disease,." Blood 118, no. 21 (November 18, 2011): 4084. http://dx.doi.org/10.1182/blood.v118.21.4084.4084.

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Abstract Abstract 4084 Introduction: Chronic graft-versus-host disease (cGVHD) is a major complication after allogeneic hematopoietic stem cell transplantation (HSCT) known to impair quality of life and functional status and adversely affects long-term survival. Given its impact on outcomes post HSCT, biological markers predicting cGVHD appearance could be useful. Eosinophilia has been reported to occur in the setting of both acute and cGVHD. Even though many physicians refer to eosinophilia as a predictor of GVHD, it is unclear whether eosinophilia can serve as a biomarker predicting occurrence of cGVHD in the general HSCT population. Objectives: In order to evaluate whether eosinophilia can predict the development of cGVHD, we have chosen to study a cohort of HSCT patients who developed eosinophilia while being tapered off their immunosuppressive drugs. Methods: From 01/2000 to 12/2009, a matched series of 112 patients who underwent allogeneic HSCT at City of Hope was identified. Of those, 56 patients presented with eosinophilia (>0.5×10^9 /L on two consecutive visits within 4 weeks, not related to relapse or documented allergy, drug reaction or parasitic infection) after day 120, while being tapered from their immunosuppressive drugs (case group). A control group (with no eosinophilia present on two consecutive visits within 4 weeks after day 120) was matched for the following criteria: age (±10 years), transplant year (± 2 years), HSC source (peripheral blood versus bone marrow), donor type (related vs. unrelated), female donor to male recipient, occurrence of acute GVHD and type of GVHD prophylaxis(tacrolimus/sirolimus vs. other). Results: A total of 112 patients were included in the analysis (Table no. 1). The most prevalent indication for HSCT was acute leukemia. Fifty seven percent of patients underwent myeloablative HSCT. There was no statistically significant difference between the case and control group for the matching criteria: age (p=0.88), HSC source, donor type, female donor to male recipient, occurrence of acute GVHD and type of GVHD prophylaxis (p=1). There was also no statistically significant difference between the groups for the different disease risk category (i.e. low risk, intermediate risk, high risk and non malignant) (p=0.4581). In the group of patients presenting with eosinophilia, 89% (50 out of 56) developed cGVHD, as opposed to 59% (33 out of 56) in the control group (p=0.0002, Chi square test). In this setting, eosinophilia was found to have a positive predictive value of 89%. In a multivariate logistic regression model, looking at the mentioned above known cGVHD risk factors, patients with eosinophilia were nearly 7 times more likely to develop cGVHD compared to those without eosinophila (p=0.0003, 95% CI 2.41–20.13). The median time from the development of eosinophilia to first GVHD sign was 20 days. Eighty four percent of patients both in the eosinophilia and the control groups presented with extensive GVHD grading, with skin, mouth and liver being the most prevalent sites for cGVHD occurrence. With a median follow up of 51 month for the entire cohort (range 5.5–132m) 80% of patients were alive in the eosinophilia group vs. 87% in the control group. Conclusions: The development of eosinophilia in the setting of tapering or discontinuation of immunosuppression may serve as a good predictor of cGVHD. Eosinophilia in this setting may be an indicator for early intervention. Disclosures: No relevant conflicts of interest to declare.

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Mannelli, Francesco, Sara Bencini, Benedetta Peruzzi, Paola Guglielmelli, Annalisa Pacilli, Giada Rotunno, Francesca Gesullo, et al. "Multi-Lineage Dysplasia Assessment By Immunophenotype in Myeloproliferative Neoplasms (MPN): Correlation with Disease' Variant, Clinical Features and Molecular Genetics." Blood 134, Supplement_1 (November 13, 2019): 1668. http://dx.doi.org/10.1182/blood-2019-125843.

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Background. MPN are clonal disorders of hematopoietic stem cell (HSC) that include polycythemia vera (PV), essential thrombocytemia (ET) and myelofibrosis (MF), either secondary to ET-PV, or primary (PMF), including pre-fibrotic (pre-PMF) and overt. In MF, multi-lineage dysplasia (MLD) of varying degree can be detected by morphologic analysis of bone marrow (BM), but appraisal of MLD is intrinsically operator-dependent and lacks quantitative information. Multi-parameter flow cytometry (MFC) is used to study dysplasia by investigating aberrant antigen expression profiles during hematopoietic cells' differentiation and maturation, allowing: i) analysis of a much greater cell number; ii) standardization of data by referring to controls; iii) definition of scores to estimate MLD extent. Aim. The aim of the study was to assess MLD in MPN by MFC and correlate with disease' variant, clinical and biological features. Methods. Patients: pts were diagnosed according to 2016 WHO criteria. Driver and high molecular risk mutations (HMR) were determined. MFC: Different BM cell compartments were analyzed with a panel of 28 monoclonal antibodies (MoAb) by 2 acquisition steps: Step 1) HSC and progenitor (GMP and MEP) cells up to >5 x 106 cells/sample were studied by Lin/CD34/CD38/CD90/CD45/CD49f/CD45RA/CD135 MoAb. Step 2) Precursor and mature compartments (B-, neutrophilic, monocytic, erythroid, plasmacytoid dendritic cells, basophils) were identified within CD34+ and/or CD117+ subset. Uncommitted CD38+ precursors were obtained by subtraction. CD117-neg, maturing cells were identified by specific features, ie CD34, CD45 and scatter. MLD was assessed by 2 phenotypic scores: i) one validated by ELN in MDS (Ogata score) includes 4 parameters; ii) the second one, adapted from Matarraz et al (2010; Salamanca score), 63 parameters. Normal phenotypic profile was defined as the interval between mean value (M) ± 2SD per parameter in healthy donors' BM samples. Each parameter scored 0.5, 1 or 2 for values between M ± 2 SD and M ± 3 SD, M ± 3 SD and M ± 4 SD, and over M ± 4 SD of the normal profile, respectively. Results. 51 consecutive MPN pts at diagnosis were enrolled, including 30 MF (7 pre-PMF, 14 overt PMF, 9 post-PV/ET MF); 13 ET, 5 PV, 1 MPN-unclassifiable and 2 MDS/MPN. Median age was 59y (range 24-83). 19 (63.3%) MF patients had JAK2V617F, 9 (30.0%) CALR, 2 (6.7%) MPLW515L mutation. 11 pts (36.7%) harbored at least 1 HMR mutation. According to MIPSS, 6 (20.0%), 11 (36.7%) and 10 (33.3%) were low, intermediate and high risk. By comparing MF pts to controls and ET-PV, altered proportion of immature cell subsets was observed. Specifically, HSC and uncommitted progenitors in MF were increased of 3.8 and 1.5-fold, respectively whereas GMP and downstream precursors were significantly reduced 2 to >6-fold. No significant differences emerged comparing mature compartments, except for an increase of basophils and a decrease of pDC in MF subset (Table 1). When considering MLD, MF patients ranked higher scores for both the Ogata and Salamanca score. Furthermore, Salamanca score values had a trend to correlate with MIPSS risk categories, with median value of 7.75, 13.25 and 15.5 in low, intermediate and high category (KW test P=0.06). MF cases were separated according to Salamanca score ≥ or < to the median value of 13.5: pts with higher score were featured by significant differences in hemoglobin (11.9 vs 13.7 g/dL; P=.015), platelet count (330 vs 558 x109/L; P=.004), LDH (380 vs 275 U/L; P=.034), circulating CD34+ cells (60.4 vs 5.3/mL; P=.007). We also compared prePMF and ET pts, two categories with challenging differential diagnosis: prePMF displayed expansion of HSC (0.015) compared to ET (0.0056; P=0.028) and reduced proportions of downstream precursors, similar to what observed in MF vs non-MF comparison. Conversely, MLD scores were similar: Ogata 1.0 vs 1.0, Salamanca 9.0 vs 7.5 for ET and prePMF, respectively. Conclusions. The assessment of MLD by MFC correlated with MPN variants according to WHO; MF was associated with higher deviation from normal phenotypic profile. MLD scores delineated clinical phenotype and were consistent with MIPSS risk stratification. With the limits of number of pts analyzed, these preliminary data also support a biologic continuum of pre-PMF and MF, the first being characterized by abnormal expansion of immature cell compartments compared to ET but minimal signs of dysplastic maturation unlike overt MF. Disclosures Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Styczynski, Jan, Myriam Labopin, Nabila Elarouci, Adriana Balduzzi, Lidia Gil, Karoline Ehlert, Franca Fagioli, et al. "Pediatric Sibling Donor Complications of Hematopoietic Stem Cell Collection: EBMT Pediatric Diseases Working Party Study." Blood 114, no. 22 (November 20, 2009): 806. http://dx.doi.org/10.1182/blood.v114.22.806.806.

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Abstract Abstract 806 Objective: The analysis of donor safety and early side effects related to hematopoietic stem cells (HSC) collection from bone marrow (BM) or peripheral blood (PB) in pediatric HLA-identical sibling donors. Methods: From 2005 to 2009, data regarding pediatric (<18 years) sibling donors undergoing HSC collection from 38 participating EBMT Centers were registered on specific forms sent to the PDWP Office. Data were centrally analyzed as of July 2009. A multiple logistic regression was performed to express an estimate of the relative risk of BM versus PB donation for the following variables: pain, blood transfusion, prolonged hospital stay, and need for iron supplementation. Results: A total number of 410 pediatric sibling donors with median age of 9 (range; 0.3-18) years were enrolled into the study; including 12% aged <4 yrs and 64% with body weight <40 kg. Children donated BM in 272 (66%), and PB in 138 (34%) cases. The criteria used by transplant centers for PB-HSC collection in children were: discrepancy between donor and recipient body weight, parents decision, undergoing clinical trial (pediatric centers), or transplant protocol (adult centers). Consents for donation were given by parents (49%), court (23%), ethical committee (28%) or social board (0.4%). All BM and 56% of PB donors had general anesthesia (PB donors for central catheter placement), for a median 90 (range; 30-300) and 30 (range 8-285) minutes, respectively. One (0.2%) serious adverse event (SAE) was reported (pneumothorax+hydrothorax) after catheter placement for PB collection. Tachycardia, decrease of blood pressure, sore throat or vomiting after anesthesia occurred in 5-11% cases. Autologous blood transfusion was done in 26% of BM and 7% of PB donors, at median age of 12 years (range 2.8-18). Erythropoietin was administered in 23 BM donors. BM was collected in median volume 18.8 (range; 2-49) ml/kg of donor weight and no severe complications of BM collection were observed. G-CSF priming was performed in all PB donors with median G-CSF dose of 10 (range; 5-20) mcg/kg/day. Median WBC count before collection was 46 (range; 24-101) G/L. Muscle/bone pain, fever, headache, abdominal or back pain, nausea or vomiting related to G-SCF were reported only in 6% of PB donors. Median number of apheresis was 1 (range; 1-3), which lasted for a median of 4 (range; 2-8) hrs. 21% donors experienced hypocalcemia. Thrombocytopenia <70G/L occurred in 3% of PB donors, however platelet transfusion was not required in any case. Blood allotransfusion was done in 23% of BM and 2.6% of PB donors. Iron was supplemented in 73% of BM and 31% of PB donors (p<0.001). Pain after collection occurred in 64% of BM and 15% of PB donors (p<0.001) and persisted for median of 1 day (range; 1-21 for BM and 1-3 for PB donors, respectively). No infections or thrombotic events were reported. Median number of nights spent in hospital before collection was 1 (range; 0-14) for BM and 3 (range; 0-7) for PB donors; while after collection 1 (range; 0-7) for BM and 0 (range; 0-6) for PB donors (p<0.001). Donors felt helpful, happy and proud in most cases, irrespectively of stem cell source. They felt responsible for recipient in 58% of BM and 53% of PB of donors (ns). Willingness to donate again was expressed by 86% and 71% of the BM and PB of donors, respectively (p=0.007). In multiple logistic regression analysis following complications and risk factors were determined: pain (BM collection, OR=12.3, p<0.001; age >4 yrs, OR=9.9, p<0.001), blood allotransfusion (BM collection, OR=22.7, p<0.001; body weight <40 kg, OR=4, p=0.004), prolonged hospital stay (BM collection, OR=3.5, p<0.001), and need for iron supplementation (BM collection, OR=6.9, p<0.001). Conclusions: BM or PB HSC collection in pediatric sibling donors is safe, however there is a risk of mild, short-term and easy to prevent or control early side effects. The risk of SAE in healthy pediatric donors exists, although it is small. Donors and parents must be informed about the risk of possible complication. There is a need of donor outcome and follow-up registry. Disclosures: No relevant conflicts of interest to declare.

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Pu, Jeffrey J., Hetty E. Carraway, Rong Hu, Galina Mukhina, and Robert A. Brodsky. "Relevance of Minor PIG-A Mutations In Acquired Aplastic Anemia and Myelodysplastic Syndromes." Blood 116, no. 21 (November 19, 2010): 4433. http://dx.doi.org/10.1182/blood.v116.21.4433.4433.

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Abstract Abstract 4433 Introduction: Aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndromes (MDS) are acquired bone marrow failure diseases. About 15% AA patients eventually transform to classical PNH; however, MDS patients almost never evolve to classical PNH. PNH originates from a multipotent HSC that acquires a PIG-A mutation. PIG-A gene mutations lead to absence of glycosylphosphatidylinositol-anchor proteins (GPI-AP). In PNH, matching mutations can be found in myeloid, erythroid and lymphoid lineages. Roughly 1 in 50,000 (0.002%) colony-forming-cells (CFC) from healthy controls harbor PIG-A mutations. However, PIG-A mutations in healthy controls arise from progenitors rather than multipotent HSC; and are often transient. More than 50% AA patients and 10~30% MDS patients were reported to have small populations (0.01 to 10%) of PNH granulocytes. The objective of this study is to determine the origin of PIG-A mutations in patients with AA and MDS. Method: A prospective study of 3 AA patients with small PNH granulocyte populations and 17 MDS patients was performed. AA and MDS were diagnosed as per clinical criteria and WHO classification (2008) respectively. The median age for AA patients was 49 (range: 28~66). The median age for MDS patients was 66 (range: 48~79). Two AA patients were managed with supportive care, and 1 received HiCy. Five MDS patients were under observation; 3 each received Aranesp, Revlimid, 5-azacytidine, and MS-275/GM-CSF respectively. PNH granulocyte size was measured by flow cytometry. Bone marrow CD34+ cells were isolated via Ficoll-Paque separation and magnetic selection. Proaerolysin is a toxin that selectively kills GPI-AP intact cells but not PNH cells. PNH CFC assay was performed using proaerolysin-containing methylcellulose culture. T cell enrichment culture was performed using peripheral mononuclear cells, RPMI1640 medium with 10% AB serum, micro-beads coated with antibodies against CD2, CD3, and CD28, rIL-2, in the presence of 1nM proaerolysin. DNA was extracted from individual proaerolysin resistant CFC and T cell colonies and was PCR amplified and sequenced for PIG-A mutation analysis. PIG-A mutations were identified using forward/reverses primers. Result: Of 3 AA patients bearing a small PNH granulocyte population (range: 3.2~3.8%), median granulocyte PIG-A mutation frequency was 1.07% (range: 0.88~1.33%), median bone marrow cellularity was 10% (range: 5~20%), and median CD34+ cell was 0.05% (range: 0.019~0.09%). All AA patients had normal karyotypes. PIG-A mutation analysis on 2 of them showed that PIG-A mutations occurred at multipotent HSC level: 1 had a single base substitution mutation at bp549, switching a C to T in both CFU-Gs and T cells; and the other one had a 3 bps (GTC from bp776 to 778) deletion mutation in cells from both lineages. The third patient had a substitution mutation at bp1005 in CFCs, switching an A to T. Her T cell PIG-A mutation analysis is pending. All mutations were confirmed in 3 independent colonies from each lineage. Of 17 MDS patients, 14 had hypercellular bone marrow, 2 had normal cellularity, and 1 had hypocellularity. Seven had normal karyotypes, 9 had abnormal karyotypes. Only 3 patients (17.6%) had detectable PNH granulocyte populations (range: 0.1~3.2%). Studies on the patient bearing a 3.2% PNH granulocytes indicated that PIG-A mutation occurred at progenitor level and was transient. Proaerolysin-resistant CFC assay only grew 1 proaerolysin-resistant CFU-G in 10 plates with a granulocyte PIG-A mutation frequency of 0.03%. DNA sequence analysis of CFC from that colony demonstrated a PIG-A gene single base T deletion mutation at bp 210. However, no PNH T cells were detected via either flow cytometry or T cell enrichment culture. After 4 months of supportive care, he no longer had detectable PNH cells via either flow cytometry or proaerolysin-resistant CFC colony assay. Another patient, who initially bore a 1.5% PNH granulocyte population, also had his PNH granulocyte population disappeared after 2 months of therapy with 5-azacytidine. The 3rd patient is still on surveillance. No PNH T cells were detected in last 2 patients either. Conclusion: Similar to classical PNH, PIG-A mutations in AA are clonal and arise from multipotent HSC. However, PIG-A mutations in MDS arise from progenitor cells, which likely explains why PNH populations in MDS patients are transient and seldom evolve into classical PNH. Disclosures: No relevant conflicts of interest to declare.

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Schuurhuis, Gerrit J., Bijan Moshaver, Alexander N. Snel, Gert J. Ossenkoppele, and Sonja Zweegman. "Combination of CD34/CD38 Immunophenotypes and Side Population (SP) Reveals the Putative Leukemia Stem Cell/Leukemia Initiating Cell In Acute Myeloid Leukemia." Blood 116, no. 21 (November 19, 2010): 1582. http://dx.doi.org/10.1182/blood.v116.21.1582.1582.

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Abstract Abstract 1582 Although the originally defined CD34+CD38- leukemia stem cell (LSC)/leukemia initiating cell (LIC) in acute myeloid leukemia (AML) still serves as a lead in studies on the characterization of LSC/LIC, in more immuno-compromised mouse models, other stem cell immunophenotypes, i.e. CD34-CD38- and CD34-CD38+ turn out to have LSC/LIC activity (Taussig et al, Blood 2010; 115: 1976). Two functional LSC phenotypes offer the ability not only to restrict the LSC/LIC compartment to a lower frequency compartment, but also to include such CD34- negative immunophenotypes. These concern high activity of aldehyde dehydrogenase (ALDH) or high efflux of Hoechst 44432 (resulting in side population, SP). The present study deals with the possible overlap between CD34/CD38 and SP defined stem cell compartments. Based on our previous results (van der Pol, Haematologica 2003; 88: 983), we defined truly CD34 negative AML as AML with a very small CD34+ population only (usually≤1%), with particular scatter properties, and proven to be of normal origin. Using expression of cell surface markers present on LSC/LIC, but absent on HSC (van Rhenen et al, Leukemia 2007; 21: 1700; Blood 2007; 110: 2659), the LSC/LIC compartment in this type of leukemia (5 cases studied) was shown to be present in the CD34-CD38+ compartment, while the HSC was found in the CD34+CD38- compartment. When determining aberrant cell surface markers within the SP, the only malignant cells present in the SP population were CD34-CD38+. To assess the primitive character of the putative LSC/LIC sub-populations, cell sorting experiments were performed in which all aberrant marker positive SP cells along with marker positive non-SP cells (n=2) and all aberrant marker negative SP cells and marker negative non-SP cells (n=2), were assessed in the liquid culture stem cell assay. SP marker positive cells had 280- and 725-fold more stem cell activity compared to non-SP cells (14,500 versus 20 and 8,400 versus 30, respectively), while for marker negative cells only the SP fraction contained stem cell activity (8,000 and 8,200 colonies per million input cells). Even though the frequency of SP cells is far below that of non-SP cells, the absolute number of colonies was higher in SP than in non-SP cells. In CD34 positive leukemia, in contrast to CD34 negative AML, the SP fraction was largely filled with cells with all combinations of CD34 and CD38, which all fulfill the criteria (marker positivity) for malignancy. Liquid culture assay results were: for marker positive cells, colonies were formed only in the SP compartment in 2/3 cases studied (6,500 and 4,400 colonies per million input cells). In the remaining case colony formation was 195-fold higher in SP versus non-SP (4,100 versus 21 colonies per million input cells). For marker negative cells, in all three cases only the SP fraction produced colonies (5,300, 1,4500 and 90 per million input cells). Malignant character of marker positive cells was confirmed using FISH analysis, immediately after cell sorting prior to cell culturing, and at the end of the liquid culture on plucked colonies. These results show that both in CD34 negative and CD34 positive leukemia the SP fraction contains most primitive cells and has much higher clonogenic potency compared to non-SP cells. In CD34 negative AML, SP HSC are CD34+CD38- and SP LSC/LIC are CD34-CD38+. In CD34 positive AML, SP HSC are CD34+CD38- and SP LSC/LIC may be CD34+CD38-, but also CD34+CD38+, CD34-CD38+ and CD34-CD38-. The absence of CD34 negative engraftment, seen in earlier studies, likely results from relatively high immune competence of the NOD/SCID mice in those studies. The results suggest that CD34 negative leukemia may contain more immune sensitive LSC/LIC. Above that, CD34 negative AML is characterized by lower multidrug resistance (abstract Schuurhuis, Kelder et al, subm for this meeting). From these characteristics favorable outcome for the particular class of patients, defined as truly CD34 negative, can be predicted. This was indeed the case in our large study (394 patients): median overall survival not reached (>41 months) for CD34 negative patients (n=85) and 19 months for CD34 positive patients (n=309) (p=0.006). Our results warrant to consider CD34 negative patients as a completely different entity of AML patients, in terms of stem cell characteristics, which may evoke different therapeutic strategies compared to CD34 positive patients. Disclosures: No relevant conflicts of interest to declare.

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Journal articles on the topic 'HSC criteria'

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