Bone Marrow Regeneration Research Articles

The importance of immunophenotyping by flow cytometry in distinction between hematogones and B lymphoblasts

Hematogones are normal B-lineage lymphoid precursors in the bone marrow. B lymphoblasts are immature neoplastic cells present in patients with precursor B-cell acute lymphoblastic leukemia (B-ALL). Hematogones and B lymphoblasts share characteristics, such as morphological similarity often indistinct and expression of the same antigens in immunophenotypic analysis. Increased numbers of hematogones in patients with B-ALL during regeneration of bone marrow after treatment for leukemia, in cases of disease relapse or marrow transplantation, may be subject to questions about the nature and prognosis of this immature cell. This article presents information about the morphological and immunophenotypic characteristics of B lymphoid precursors and verifies the relevance of immunophenotyping by flow cytometry (FC) in the distinction between those cells. This differentiation is essential to establish a correct prognosis and assist in medical decision about the most appropriate therapeutic scheme.

The Anti-Proliferative Effect of 5-Fluorouracil on Tumor Is Highly Associated with the Renewal of Peripheral White Blood Cells

The efficacy of chemotherapy is thought to be direct killing of tumor cells, but documented studies have been shown that immunity plays a role in its effectiveness. In a pilot study to observe the bone marrow suppression and regeneration in tumor bearing mice induced by single dose injection of 5-fluorouracil (5-FU), we unexpectedly found that tumors grew fast as bone marrow mononuclear cells (BMC) and peripheral white blood cells (PWBC) were decreased quickly during myelosuppression meanwhile significantly slow as repopulating of BMC and PWBC during bone marrow regeneration after 5-FU treatment, no matter whether in low or high dose administration, but the higher the dose was, the lower of the nadir of BMC and PWBC were reached to, as well as the much more powerful duration and strength of the repopulated BMC and PWBC, suggested that the immunity might be a predominant drive in 5-FU chemotherapy. Due to the fact that BMC is the source of PWBC which is its final maturational and functional form, it could be proposed that the anti-proliferative effect of 5-FU on tumor is highly associated with the renewal of PWBC.

Lin-Sca-1+c-KitlowCD48+CD71+ Cells Are the Engine of Bone Marrow Regeneration

The hematopoietic tissue is the most suitable tissue for studies into the biology of regeneration. We examined bone marrow regeneration starting from a very low number of repopulating cells.Mice were irradiated by a sublethal dose of 6 Gy and recovery of hematopoiesis was examined in its three basic compartments: stem and progenitor cells, precursors of blood cells, and peripheral blood between days 8 – 90 following irradiation. The irradiation reduced existing pools of progenitors and stem cells to ̴0.1%. We focused on the pools of stem cells and progenitors that were evaluated by several assays.A novel assay based on engraftment of a defined number of transplanted competitors revealed delayed filling of the niches for long-term repopulating cells (LTRCs; stem cells) as compared to filling of the niches for short-term repopulating cells (STRCs; progenitors) by endogenous STRCs and LTRCs generated by cells surviving irradiation. This indicated a very slow recovery of the pool of stem cells, lagging after recovery of the pool of progenitors. This was confirmed by a traditional competitive transplantation assay, as well as by another novel assay that measured the capacity of bone marrow to establish pools of stem cells and progenitors in lethally irradiated mice. Direct assays of progenitors by the spleen colony (CFU-S) and in vitro CFC-GM assays indicated a low frequency of the clonogenic cells in regenerating bone marrow. Productive hematopoiesis was re-established about 10 days after irradiation in presence of very low pools of progenitors and stem cells.Next we examined numbers of immature hematopoietic cells by flow cytometry focused at lineage negative (Lin-), Sca-1 positive (Sca-1+), c-Kit+ cells (LSK cells), characterized further by means of the CD150, CD48 and CD71 antigenic markers. In absolute terms, only the population of LSK CD150+CD48+ recovered and overshot normal values after day 12 following irradiation. In relative terms, the LSK CD48+ cells (CD150+ and CD150-) represented more than 99% of all LSK cells two weeks after irradiation. Also in relative terms, the LSK CD150+CD48- cells, which are highly enriched for hematopoietic stem cells in normal bone marrow, recovered by day 30 and became relatively abundant later on. Another striking change consisted in initial lack and later severe scarcity of cells with the phenotype of LSK CD150-CD48-. These cells, serving as multipotent progenitors in normal bone marrow thus remained severely depleted in regenerating bone marrow during the whole follow-up period. LSK cells were mostly negative for CD71 (transferrin receptor) in normal bone marrow but became highly positive for this marker between days 10 – 20 after irradiation.In conclusion, our semi-quantitative evaluation of regenerating hematopoiesis reveals its highly peculiar features. Those include an early recovery of blood cell production, followed by significant expansion of the Lin-Sca-1+c-KitlowCD48+CD71+ cells in presence of a highly reduced pool of hematopoietic stem cells. DisclosuresNo relevant conflicts of interest to declare.

Value of Day 21 Flow Cytometry Minimal Residual Disease Assessment in Childhood Acute Lymphoblastic Leukemia (ALL) for Early Identification of Good Responders

The efficacy of induction chemotherapy in childhood acute lymphoblastic leukemia (ALL) is usually evaluated on day 35. However, at this stage, many patients have already begun to recover and present with a regenerative bone marrow (BM) where hematogones may make the identification of residual blast cells problematic both in morphology and in flow cytometry (FCM). In the FRALLE (French Acute Lymphoblastic Leukemia) trials, evaluation is proposed on days 8, 21 and 35. Here we evaluated whether FCM performed on day 21 (D21), when hematogones are still absent, would prove informative.The cohort reported here was constituted of 45 children aged between 1 and 20 years old (median 6) treated for ALL according to the FRALLE recommendations since 2006.There were 81% B-ALL, 17% T-ALL and 2% of mixed phenotype acute leukemia (MPAL, T/My). At diagnosis, the mean percentage of BM blasts was 50%. Classification according to the European Group for Immunophenotyping of Leukemia (EGIL) was 3 B-I, 21 B-II, 11 B-III, 2 B-IV and 1 T-I, 2 T-II and 4 T-III. Extensive immunophenotyping at diagnosis identified a median of 3 leukemia associated immunophenotypes (LAIP, range 1-5), defined as discriminant from hematogones.Corticosensitivity was defined on a complete blood count (CBC) as less than 1 G/L of blast cells on day 8. Chemosensitivity was assessed on a bone marrow aspiration at day 21, both morphologically (< 5% blasts) and in FCM (MRD0). Molecular biology (according to Biomed2) was performed on BM samples collected on days 35 (MRD1) and 70 (MRD2). Follow-up median time was 59 months (3-276).Corticosensitivity was observed for 39/43 patients (one had received corticosteroids for a tonsillitis before being referred and diagnosed with ALL and another one had less than 1 G/L of blasts at diagnosis). Five/44 patients were identified as chemoresistant by morphology on D21 (one aplastic sample). Enough cells were available for minimal residual disease (MRD) by FCM in 43 patients, on bone marrow collected on EDTA. As a mean, 586 328 total nucleated cells were acquired in FCM (range 9 616 - 1 751 000) thereby providing good sensitivity. Multiparameter FCM in 6 to 8 colors was performed on a single tube, customized according to each patient’s LAIP. Five MRD thresholds were defined as follows : level 1, >10-2 detected blasts; level 2, 10-3- <10-2detected blasts; level 3, 10-4- <10-3detected blasts; level 4, 10-5- <10-4 detected blasts or no event detected; level 5, <10-5detected blasts or no event detected. The table below indicates the partition of patients according to these MRD levels on D21. Table 1LevelPatient numbersDetectable MRDAbsence of MRD166028803660415695818Event-free survival (EFS; Kaplan Meier Log rank test) was statistically significant (p=0.023) when comparing patients with level 4 or 5 MRD to the others. Level 3 patients had an intermediate EFS and OS.In an independent cohort of 79 patients with evaluable FMC MRD on day 21 from a different center, the same good prognosis value of MRD lower than 10-4at that time point was confirmed for EFS (p=0.03).Childhood ALL has become of relatively good prognosis with the progress in therapies. However, it is noteworthy that half the patients had detectable MRD at levels 1, 2 or 3, which probably prompted the clinicians to intensify treatment. However, below level 3, i.e.<10-4, FCM MRD on D21 is of excellent prognosis, even if detectable.In summary, this work demonstrates the feasibility of FCM evaluation on D21, with the advantage of not being complicated by the presence of hematogones. Moreover, interpretation remains delicate in morphology at this time point where BM smears may be very poor. FCM thereby provides precious early information on chemosensitivity with a single patient-adapted antibody combination. DisclosuresNo relevant conflicts of interest to declare.

Calcium Calmodulin Dependent Kinase Kinase 2 Regulates Hematopoietic Stem Cell Regeneration and Quiescence

Extracellular free calcium (Ca2+) is the most abundant signaling molecule in the hematopoietic stem cell (HSC) niche, and intracellular calcium is an important second messenger controlling critical functions in HSCs. Accordingly, Ca2+ is a master regulator of HSC biology, and nodes of the Ca2+ signaling network are emerging as critical components of the molecular machinery governing HSC regeneration and quiescence. Ca2+ controls many cell functions by forming a complex with Calmodulin (CaM), which binds and activates a family of Calcium/Calmodulin dependent kinase (CaMK) that includes CaMKI, CaMKII, CaMKIV, CaMKK1 and CaMKK2. We have previously investigated the function of CaMKK2 in blood cells and established this protein as a critical component of the calcium signaling pathway that regulates granulopoiesis and macrophage activation. Here, we investigated the function of CaMKK2 in the regenerative response of HSCs. To determine the expression of CaMKK2 under homeostatic conditions, we examined transgenic reporter mice and identified robust reporter activity in primitive long-term hematopoietic stem cells, but reduced signal in the majority of lineage positive blood and bone marrow cells. To establish whether CaMKK2 has a functional role in controlling hematopoiesis and HSC self-renewal, we examined hematopoietic regeneration in mice deficient for CaMKK2. We demonstrate that Camkk2 -/- mice have accelerated bone marrow and peripheral blood regeneration following hematopoietic injury. Camkk2 -/- HSCs showed increased proliferative capacity in vivo and mobilized into the peripheral blood to establish extra-medullary hematopoiesis during regeneration. To identify the proximal target of CaMKK2, we assessed the phosphorylation status of CaMKI, CaMKIV and adenosine monophosphate activated protein kinase (AMPK), which are known targets of CaMKK2. Our results indicate Camkk2 -/- stem and progenitor cells (HSPC) fail to accumulate phospho-AMPK in response to ionizing radiation. Since AMPK is a negative regulator of the mammalian target of rapamycin (mTOR) pathway, we also analyzed the levels of phospho-ribosomal 6 protein (rpS6), which is a distal target of the mTOR cascade that positively regulates cell size and proliferation. We found that irradiated Camkk2 -/- HSPCs accumulate higher levels of phospho-rpS6 compared to wild type cells. This finding implicates CaMKK2 as a critical component of a negative regulatory circuit that links calcium signaling to AMPK activation and cell proliferation in HSPC. Cumulatively, our data demonstrate that CaMKK2 regulates HSC function and suggests CaMKK2 inhibition is a novel approach to stem cell expansion for clinical therapy. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Association of Hematological Nadirs and Survival in a Non-Human Primate Model of Hematopoietic Syndrome of Acute Radiation Syndrome

Recombinant human interleukin-12 (rHuIL-12, HemaMaxTM) is being developed for mitigation of HSARS under the FDA Animal Rule using a NHP model of HSARS for proof of efficacy and clinical studies in healthy human subjects to demonstrate safety. We have shown previously that a single injection of rHuIL-12 administered 24-25 hours after lethal total body irradiaton (TBI), in the absence of antibiotics, fluids or blood products, resulted in improved survival while filgrastim (G-CSF) did not provide any survival benefit in our NHP HSARS model (Basile et al 2012, Gluzman-Poltorak et al 2014, Gluzman-Poltorak et al 2014).The mechanism by which IL-12 mitigates HSARS following TBI appears to involve multiple effects of IL-12 on hematopoieses. In our previous studies, animals treated with rHuIL-12 showed statistically significant reductions in the occurrence of severe neutropenia and severe thrombocytopenia, as well as attenuated nadirs for lymphocytes, neutrophils, platelets, and reticulocytes.To characterize further relationship between survival and hematological nadirs (lymphocytes, neutrophils, platelets, RBCs and reticulocytes) in the rhesus model of HSARS and to better understand the effects of rHuIL-12 versus G-CSF on blood cell nadirs, we undertook a meta-analysis analysis across three studies in irradiated rhesus monkeys.Animals used in this analysis were irradiated (700 cGy) and treated with a single subcutaneous injection of vehicle (n=64) or rHuIL-12 50-500ng/kg (n=108) 24-25 hours after irradiation, or daily subcutaneous injections of G-CSF at 10μg/kg/d for 18 days starting 24-25 hours after irradiation (n=26). Males and females were equal in each group.Lymphocytes, neutrophils and platelets were significantly lower in decedents versus monkeys that survived to day 60 overall and this was true in each treatment group (p<0.001, Wilcoxon rank-sum test). The mean RBC nadir was significantly higher in survivors compared to non-survivors in the rHuIL-12 group (p = 0.008), but not in the control group or the G-CSF group.Lymphocytes nadir appears to be the strongest and most consistent predictor of death followed by neutrophils and platelets (Spearman’s rank correlation). RBCs and reticulocytes are less informative in terms of predicting survival status.In addition, the receiver operating characteristic (ROC) curve area under the curve (AUC) by nadir value was assessed. The operating characteristics for lymphocytes, neutrophils and platelets allow for relatively large positive predictive values (PPV) of death with relatively high sensitivity. A cutoff value for lymphocytes nadir of 0.08x109/L (values less than or equal predicting death and higher values predicting life) allows for largest PPVs (97.2% and 92.5%) with 76.1% and 62.7% sensitivities for control and rHuIL-12 treatments, respectively. A cutoff value for neutrophils nadir of 0.03x109/L permits for 84% and 71.1% PPVs with sensitivities of 91.3% and 91.5 % for control and rHuIL-12 treatments, respectively. A cutoff value for platelets nadir of 9x109/L permits for 84.1% and 76.8% PPVs with sensitivities of 80.4% and 72.9 % for control and rHuIL-12 treatments, respectively. RBCs and reticulocytes were found less informative.To conclude, in the rhesus model of HSARS we have observed that an augmented hematological nadirs generally predicts an increased potential for survival as this effect reflects early bone marrow regeneration. The nadir for lymphocytes appears to be the strongest and most consistent predictor of death. Decrease of lymphocyte counts has been established as a best marker of bone marrow damage in a large database of human victims of acute radiation (METREPOL, Fliedner et al 2001). Thus, the correlation of our results with the human data supports the validity of our animal model as an accurate representation of human HSARS and its ability to predict effectiveness in humans exposed to lethal radiation. These data also suggest that the significant increase in early bone marrow regeneration seen in our studies, resulting in increases in nadir values for all major blood cell types, may be the main mechanism of action by which rHuIL-12 mitigates the lethality of HSARS.This project has been entirely funded with Federal funds from BARDA/ASPR/DHHS under Contract No. HHSO100201100037C. DisclosuresGluzman Poltorak:Neumedicines Inc.: Employment, Equity Ownership. Vladimir:Neumedicines Inc.: Consultancy, Employment, Equity Ownership. Basile:Neumedicines Inc.: Employment, Equity Ownership, Neumedicines Inc. Patents & Royalties.

Bone marrow regeneration promoted by biophysically sorted osteoprogenitors from mesenchymal stromal cells.

Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as "cell factories" that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies.

Endocan, a potential prognostic and diagnostic biomarker of acute leukemia.

Recent evidence indicated that endocan may be a potential cell marker and a new target for cancers including acute leukemia since the serum endocan level in patients with acute leukemia was associated with the status of the disease, i.e., endocan was higly expressed in untreated acute leukemia, but decreased after chemotherapy and increased again during bone marrow regeneration. The present study showed that there was high level expression of endocan in cytoplasm of bone marrow blasts of patients with acute myeloid leukemia or acute lymphoblastic leukemia. The expression level of endocan was significantly decreased when the patients underwent remission after chemotherapy and re-bounces back when the acute leukemia relapsed. No obvious change in expression of endocan was observed before and after chemotherapy if the patients showed no remission after chemotherapy. (N-(4-Hydroxyphenyl) retinamide), a potent anti-angiogenic agent, could not only down-regulate the expression of vascular epithelial growth factor, but also decrease endocan transcription and expression in NB4 cells, a human acute promyelocytic leukemia cell line. These observations suggest that endocan could act as a predictor for the severity and the prognosis of acute leukemia. The findings could be used as the basis for future targeted therapy directed against bone marrow angiogenesis in acute leukemia treatment.

Adipose‐derived stromal cells grown on a hydroxyapatite scaffold can support hematopoiesis in regenerated bone marrow in vivo

Osteoblastic cells are a key component of the bone marrow (BM) stem cell niche and help regulate hematopoietic stem cells (HSCs). We have previously demonstrated that adipose-derived stromal cells (ADSCs) can differentiate into both osteogenic and chondrogenic cells in vitro. The current study examined whether the anatomical architecture of the BM could be regenerated in vivo by using ADSCs cultured on a hydroxyapatite (HA) scaffold. ADSCs from GFP transgenic mice were cultured in vitro on an HA scaffold. The scaffold with the attached cells was implanted subcutaneously onto the backs of C57/BL6 (Ly5.2) recipient mice. Lineage-negative (Lin-) Ly5.1 BM cells transduced with a lentiviral vector containing the luciferase (Luc) gene were intravenously administered to the recipient mice after lethal irradiation. Eight weeks after BM transplantation, the scaffolds were removed from the first recipient mice and subcutaneously implanted into lethally irradiated second recipient mice. The biodistribution and kinetics of Luc(+) Ly5.1 cells were monitored by bioluminescence imaging and flow cytometry. Luc(+) hematopoietic cells were present in the scaffolds of the secondary implanted mice for at least 8 months. Subcutaneous injection of G-CSF resulted in wide distribution of bioluminescence signals from the original scaffolds to the whole body. Therefore, BM regenerated using ADSCs grown on an HA scaffold can support HSC populations in vivo, suggesting that a functional BM niche is reconstituted. These results may have a significant impact on the development of therapeutic strategies for various hematopoietic diseases.

Randomized comparison of single dose of recombinant human IL-12 versus placebo for restoration of hematopoiesis and improved survival in rhesus monkeys exposed to lethal radiation

BackgroundThe hematopoietic syndrome of the acute radiation syndrome (HSARS) is a life-threatening condition in humans exposed to total body irradiation (TBI); no drugs are approved for treating this condition. Recombinant human interleukin-12 (rHuIL-12) is being developed for HSARS mitigation under the FDA Animal Rule, where efficacy is proven in an appropriate animal model and safety is demonstrated in humans.MethodsIn this blinded study, rhesus monkeys (9 animals/sex/dose group) were randomized to receive a single subcutaneous injection of placebo (group 1) or rHuIL-12 at doses of 50, 100, 250, or 500 ng/kg (groups 2–5, respectively), without antibiotics, fluids or blood transfusions, 24–25 hours after TBI (700 cGy).ResultsSurvival rates at Day 60 were 11%, 33%, 39%, 39%, and 50% for groups 1–5, respectively (log rank p < 0.05 for each dose vs. control). rHuIL-12 also significantly reduced the incidences of severe neutropenia, severe thrombocytopenia, and sepsis (positive hemoculture). Additionally, bone marrow regeneration following TBI was significantly greater in monkeys treated with rHuIL-12 than in controls.ConclusionsData from this study demonstrate that a single injection of rHuIL-12 delivered one day after TBI can significantly increase survival and reduce radiation-induced hematopoietic toxicity and infections. These data significantly advance development of rHuIL-12 toward approval under the Animal Rule as an effective stand-alone medical countermeasure against the lethal effects of radiation exposure.

Megakaryocytes Contribute to the Bone Marrow-Matrix Environment by Expressing Fibronectin, Type IV Collagen, and Laminin

Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, type IV collagen, and laminin are the most abundant around bone marrow sinusoids and constitute a pericellular matrix surrounding megakaryocytes. Most importantly, we report, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vivo. In vitro, fibronectin induced a threefold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, megakaryocyte ploidy level in fibronectin-treated cultures was significantly reduced. Stimulation with type IV collagen resulted in a 1.4-fold increase in megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in megakaryocytes derived from fetal liver progenitor cells. In vivo, megakaryocyte expression of fibronectin and basement membrane components was upregulated during bone marrow reconstitution upon 5-fluorouracil induced myelosuppression, while only type IV collagen resulted upregulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact megakaryocyte behavior differently during their differentiation and highlights a new role for megakaryocyte as ECM-producing cells for the establishment of cell niches during bone marrow regeneration.

ProHNPs are specific markers of normal myelopoiesis

Pro human neutrophil peptides (proHNP)s are proforms of α-defensins produced by precursors of human neutrophils. They are secreted to bone marrow plasma in large amounts by myelocytes. We hypothesized that the plasma concentration of proHNPs might serve as a specific marker of myelopoietic activity, heralding the onset of normal myelopoiesis before reappearance of neutrophils, in the setting of bone marrow regeneration. To investigate this, plasma levels of proHNPs were measured by enzyme-linked immunosorbent assay in blood samples collected from patients undergoing allogeneic (n=11) or autologous (n=16) stem cell transplantations (SCTs) and patients receiving chemotherapy for acute leukemia (n=14). To compare proHNPs with previously suggested myeloid markers, myeloperoxidase (MPO), lysozyme and neutrophil gelatinase-associated lipocalin (NGAL) were also assayed. In all but one patient, chemotherapy led to the complete disappearance of ProHNPs from plasma. It reappeared in plasma on average 6.3 days before reappearance of neutrophils in the allogeneic setting, whereas this was reduced to an average of 2.8 days in the autologous SCT patients who received granulocyte colony-stimulating factor. Patients with acute myeloid leukemia (n=19) had significantly lower levels of plasma proHNPs than healthy controls, indicating that proHNPs are not produced by leukemic blasts. We conclude that plasma concentration of proHNPs is a clinically useful marker of normal myelopoiesis.

Assessment Of Minimal Residual Disease In Acute Myeloblastic Leukemia In Multiparameter Flow Cytometry

Acute myeloid leukemias (AML) represent a vast and complex group of diseases where numerous molecular lesions have been and keep being described. From the immunophenotypic point of view, probably because of the variety of cells in the myeloid lineage, a rather large variety also exists. The detection of minimal residual disease (MRD) in such conditions therefore meets the challenge of tracking the proper anomaly and correctly separate leukemic cells from their normal counterparts. In an oligocentric project initiated in France in late 2006, ten cytometry platforms and six molecular biology laboratories collaborated to detect MRD concomitantly in flow and with molecular techniques. The flow cytometry part of this work is reported here.A total of 307 consecutive patients were tested at diagnosis with a comprehensive common panel allowing for the detection of immature markers and potential leukemia-associated immunophenotypic patterns. Follow-up samples were planned to be obtained after induction and at the end of treatment, with an optional control before the second consolidation. In fine, 274 patients had at least one point of follow up. All samples were tested in a technique of whole bone marrow lysis no wash, avoiding any cell loss. The flow cytometry panel comprised ten five-colors tubes, all containing CD45 as gating marker. A newly developed strategy was devised to analyse MRD data. The approach of the GTLLF (Arnoulet, Cytometry part B, 2011) was first applied to properly identify mature polymorphonuclears, monocytes and lymphocytes, allowing for a negative Boolean selection of immature cells in the region dubbed “bermudes” by this group. Focusing on this area, each combination of the four markers tested together with CD45 was then displayed in a total of six biparametric histograms. For each of them, on the diagnosis sample, quadrant gates were constructed so that the lower left one contained no blast cells. A Boolean operation was then designed to exclude all these six areas, thereby combining the positive blasts present in the three other parts of each quadrant. The resulting population was visualized on a CD45/side scatter biparametric histogram to check that the cells appeared as a focused cluster at a precise position. The same strategy was then applied for each patient's consecutive samples, always checking whether any cells identified with this protocol displayed the scattered pattern of cells engaged in maturation (no MRD) or constituted a focused population without maturation (positive MRD). The amount of MRD was then calculated taking into account as denominator the whole population of nucleated cells in the sample (excluding debris on a live gate). As internal control a specific feature of the Kaluza software was used to merge samples obtained for a given patient in order to display on the same worksheet the diagnosis and follow up samples using the principal component separation provided by the “radar” tool of this software. This original method proved to be easily applicable and provide a consistent help for MRD interpretation.All patients could be assessed for MRD with only two of the ten tubes used. These contained the following combinations : CD15, CD13, CD33, CD34, CD45 and CD7, CD117, CD33, CD34, Cd45. At diagnosis, any combination of expression of CD13, CD33, CD117 and CD34 could be observed, the percentage of positive cases for each of these antigens being respectively 86%, 89%, 81% and 58%. As a whole, 93% of the follow-up samples (MRD) tested contained less than 5% of cells with an immunophenotype comparable to that of diagnosis. This figure was 77% for less than 1% and 43% for less than 0.1%. The strategy devised for files analysis was easily applicable for all patients except those with myelomonocytic leukemia. For some of them, separation of the blasts from the monocytic compartment could be problematic in regenerating bone marrow samples.In conclusion, the flow cytometry part of this multicenter study allowed to establish that the combination of CD45 with CD13, CD33, CD117 and CD34 with the additional information provided by CD5 and CD7 represents a quasi-universal panel, now easy to implement on instruments with 8 or 10 detectors, for the detection of MRD in multiparameter in flow cytometry. Moreover, a powerful strategy of listmodes analysis was developed allowing for the direct comparison of several samples from the same patients and/or of a given sample and normal (control) bone marrow. Disclosures:No relevant conflicts of interest to declare.

Enhancing bone marrow regeneration by SALL4 protein

Hematopoietic stem cells (HSCs) are widely used in transplantation therapy to treat a variety of blood diseases. The success of hematopoietic recovery is of high importance and closely related to the patient’s morbidity and mortality after Hematopoietic stem cell transplantation (HSCT). We have previously shown that SALL4 is a potent stimulator for the expansion of human hematopoietic stem/progenitor cells in vitro. In these studies, we demonstrated that systemic administration with TAT-SALL4B resulted in expediting auto-reconstitution and inducing a 30-fold expansion of endogenous HSCs/HPCs in mice exposed to a high dose of irradiation. Most importantly, TAT-SALL4B treatment markedly prevented death in mice receiving lethal irradiation. Our studies also showed that TAT-SALL4B treatment was able to enhance both the short-term and long-term engraftment of human cord blood (CB) cells in NOD/SCID mice and the mechanism was likely related to the in vivo expansion of donor cells in a recipient. This robust expansion was required for the association of SALL4B with DNA methyltransferase complex, an epigenetic regulator critical in maintaining HSC pools and in normal lineage progression. Our results may provide a useful strategy to enhance hematopoietic recovery and reconstitution in cord blood transplantation with a recombinant TAT-SALL4B fusion protein.

Immunophenotypic profile of nucleated erythroid progenitors during maturation in regenerating bone marrow

This study introduces an in-depth flow cytometric method for the analysis of nucleated erythroid progenitors during bone marrow regeneration. Initial immunophenotypic analysis with the conventional erythroid-associated markers CD36, CD71 and CD235a was supplemented with the analysis of additional markers, including CD105, CD117, CD45, CD38 and cell-scattered light characteristics. Our data show that the expression of CD105 and CD117 is critical for the distinction between four phenotypically different developmental stages of nucleated erythroid progenitors: pro-erythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts and orthochromatophilic erythroblasts. CD105 antigen expression was specifically associated with pro-erythroblasts and basophilic erythroblasts, whereas CD117 was expressed at the earliest pro-erythroblast stage. Both antigens were progressively lost throughout the course of differentiation. These data allow for the identification of aberrant erythroid development in acute erythroid leukemia or myelodysplastic syndrome.

No significant prognostic value of normal precursor B‐cell regeneration in paediatric acute myeloid leukaemia after induction treatment

B-cell precursors (BCP) regeneration in bone marrow (BM) after induction chemotherapy is prognostic for good treatment response in adult acute myeloid leukaemia (AML). We detected BCP regeneration in 81% of 59 paediatric AML patients at first complete remission; this compared to 46% in an adult study. BCP regeneration did not correlate with outcome or minimal residual disease levels. In 36 healthy BM controls, BCP levels were significantly higher in children as compared to adults. Therefore, BCP regeneration does not reflect good response to treatment in paediatric AML, possibly due to the relatively high base-line levels of BCP in children.

CD56 Expression in Normal Immature Granulocytes after Allogeneic Hematopoietic Stem Cell Transplantation

Bone marrow mononuclear cells from 93 patients with hematological malignancies after allogeneic hematopoietic stem cell transplantation (AHSCT) were analyzed using flow cytometry (FCM). The disease was acute myeloblastic leukemia (50 patients), acute lymphoblastic leukemia, and others. Conditioning was myeloablative (80 patients) or reduced intensity. The stem cell source was bone marrow (75 patients), peripheral blood stem cells, or cord blood. After AHSCT, granulocyte colony-stimulating factor was given to all patients. All patients showed engraftment of the donor cells. FCM was conducted on a median of 22 days after AHSCT. The gate was set around a granulocytic region consisting of immature granulocytes. The positivity rates of CD13, CD14, CD15, CD33, CD34, CD56, and HLA-DR in the cells were 59.9 ± 27.4%, 5.8 ± 8.8%, 98.3 ± 1.9%, 92.3 ± 12.4%, 2.6 ± 5.8%, 24.3 ± 16.7%, and 9.1 ± 6.6%, respectively. The greatest value of CD56 positivity was 73.1%. On the basis of CD56 expression, cases of more than 24% CD56 positivity were assigned to the CD56-high group (39 patients), while the rest were assigned to the CD56-low/negative group. There were no significant differences between the two groups in terms of disease status, sex, donor, hematopoietic stem cells, days of FCM analysis, or peripheral blood cell counts around the days of performing FCM. These results indicate that CD56 can be expressed in normal immature granulocytes at a variety of expression levels in regenerative bone marrow. Attention should be paid when evaluating aberrant antigen expression of CD56 in granulocytes.

SpecificDioscoreaPhytoextracts Enhance Potency of TCL-Loaded DC-Based Cancer Vaccines

Dioscorea tuber phytoextracts can confer immunomodulatory activities ex vivo and improve regeneration of bone marrow cells in vivo. In present study, we evaluated specific Dioscorea phytoextracts for use ex vivo as a bone-marrow-derived dendritic cell- (DC-) based vaccine adjuvant for cancer immunotherapy. Fractionated Dioscorea extracts (DsII) were assayed for their effect on maturation and functions of DC ex vivo and antimelanoma activity of DC-based vaccine in vivo. The phytoextract from 50–75% ethanol-precipitated fraction of Dioscorea alata var. purpurea Tainung no. 5 tuber, designated as DsII-TN5, showed a strong augmentation of tumor cell lysate- (TCL-) loaded DC-mediated activation of T-cell proliferation. DsII-TN5 stimulated the expression of CD40, CD80, CD86, and IL-1β in TCL-loaded DCs and downregulated the expression of TGF-β1. DC vaccines prepared by a specific schema (TCL (2 h) + LPS (22 h)) showed the strongest antitumor activity. DsII-TN5 as a DC vaccine adjuvant showed strong antimelanoma activity and reduced myeloid-derived suppressor cell (MDSC) population in tested mice. DsII-TN5 can also activate DCs to enhance Th1- and Th17-related cytokine expressions. Biochemical analysis showed that DsII-TN5 consists mainly of polysaccharides containing a high level (53%) of mannose residues. We suggest that DsII-TN5 may have potential for future application as a potent, cost-effective adjuvant for DC-based cancer vaccines.

Mesenchymal stem cells combined with barrier domes enhance vertical bone formation

To enhance vertical bone formation in a rat calvarium following combination of guided bone regeneration (GBR) and transplantation of bone marrow derived mesenchymal stem cells (bmMSC). Gold domes (7mm radius, 5mm height) were filled with 5×10(5) bmMSC or osteogenic transformed bmMSC (otMSC) that were isolated from tibia of inbred rats and mixed with βTCP. Domes filled with βTCP served as control. Rats were sacrificed after 3months. New bone formation was analysed by histology and histomorphometry. In all rats hard tissue filled the space under the dome. In the lower part of the specimens the newly formed mature bone was continuous with the original calvaria, whereas the upper (distal) part of the augmented tissue contained residual scaffold surrounded by connective tissue. Histomorphometric analysis revealed that cell transplantation doubled vertical bone height: (bmMSC 4.13±0.5mm, otMSC 4.14±0.3mm βTCP 2.29±0.22mm, p≤0.001). Bone area fraction (%) was significantly increased following transplantation of otMSC (47.2±2.5%) when compared with bmMSC (37.3±3.35%) and with βTCP (31.09±2.7%) (p≤0.031 versus bmMSC, p≤0.0004 versus control) In a rat calvaria model transplantation of both otMSC and bmMSC, when combined with GBR significantly enhanced bone formation.

TGFβ Restores Hematopoietic Homeostasis After Chemotherapy.

AbstractAbstract 2344 Background:Hematopoietic stem cells (HSCs) are required for lifelong blood cell production and, to prevent exhaustion, the majority of HSCs are deeply quiescent during steady-state hematopoiesis. Paracrine factors produced by specialized bone marrow niche cells maintain HSC quiescence. Yet, evolution demands a rapid hematopoietic response to stressors such as infection, bleeding or toxin exposure. These triggers set off a remarkable adaptation in hematopoiesis that sacrifices HSPC quiescence, and the protection it affords, to meet an urgent need for new blood cell production. A great deal is known about how the HSPCs are mobilized during these periods of stress but mechanisms driving the return to steady-state hematopoiesis after stress have not been identified. Method/Results:Because TGFβ is one of few negative regulators of hematopoiesis, we measured the levels of active TGFβ and assessed the effects of its downstream signaling during hematopoietic recovery after chemotherapy. During late hematopoietic regeneration, as hematopoietic homeostasis is restored, the levels of active TGFβ spike in whole bone marrow and downstream signaling (as reported by Smad2 phosphorylation) increases in hematopoietic stem and progenitor cells (HSPCs). To assess how TGFβ signaling alters hematopoietic regeneration, we administered myelosuppressive chemotherapy to mice and then, during recovery, treated cohorts with either an antibody that specifically neutralizes active TGFβ (1D11), an isotype control antibody (13C4) or nothing at all. We found that TGFβ blockade accelerated blood count and bone marrow regeneration, and promoted HSCP self-renewal/proliferation by delaying the return of HSPCs to quiescence (G0). In contrast, TGFβ blockade during homeostasis elevates neither blood counts nor bone marrow cellularity and fails to induce HSPCs to emerge from quiescence. Conclusions:The de facto paradigm is that homeostasis is passively reestablished as stress mediators normalize. Our data strongly suggest that this paradigm is incorrect. Rather than being a passive process, it appears that steady-state hematopoiesis is actively reimposed. TGFβ pathway activation marks regenerating HSPCs returning to quiescence and this context-depending signaling helps reestablish homeostasis during recovery from chemotherapy. Together, our data demonstrate that myelosuppression does not drive hematopoiesis using only a cytokine-fueled gas pedal but also taps an active braking mechanism once sufficient recovery has been attained. More directly, our data strongly suggests that TGFb pathway inhibitors could be used to promote multi-lineage hematopoietic reconstitution. This finding significantly extends prior work in the field by opening the door to new, potentially superior, ways to modulate the hematopoietic adaptation to stress. Disclosures:No relevant conflicts of interest to declare.