Changes In Peripheral Blood Counts Research Articles

Longitudinal trends of peripheral blood counts in polycythaemia vera and essential thrombocythemia patients in the UK.

There is sparse evidence of how well haematological targets are met in practice for essential thrombocythemia (ET) and polycythaemia vera (PV) patients. Patient data was collected between 2008 and 2020 from two UK NHS Trusts for ET and PV patients. Longitudinal changes in peripheral blood counts, including the proportion of patients meeting peripheral blood count remission, was modelled. Relative risk of cardiovascular‐related events for patients achieving remission within 3‐months was estimated. A total of 620 ET and 429 PV patients were analysed. For high‐risk patients, haematological parameters decreased in the first months of observation then stabilised within normal reference ranges until year 5. Total time spent in peripheral blood count remission was 39.2% for ET and 29.1% for PV. A lower proportion of ET patients reached target platelet counts (48.3%) compared to WBC (79.1%), whilst PV patients were less likely to reach target haematocrit levels (56.9%) compared to platelets (77.3%) or WBC (74.6%). There was no statistically significant association between reaching target blood counts within 3‐months and cardiovascular risk. Complete haematological remission remains a challenging target in managing PV and ET, however this study was unable to show statistically‐significant evidence that this was associated with increased risk of cardiovascular events.

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

Peripheral blood cytopenias in the aging general population and risk of incident hematological disease and mortality

AbstractPeripheral blood cytopenias may precede the development of hematological malignancies and frequently pose clinical challenges in the older population. The natural course of (mild) cytopenias during aging and their association with hematological disorders in community-dwelling individuals are not well studied. Within the population-based Lifelines cohort (n = 167729), we studied changes in peripheral blood counts, occurrence of cytopenias, and associated hematological outcomes in the context of aging. Development of hematological malignancies and (cause-specific) mortality were evaluated by linkage to nationwide registries. Anemia and thrombocytopenia emerged with older age, in line with a general age-related decline in these blood counts. For neutropenia, no increase in prevalence with older age was observed. Using standard reference limits to define cytopenias, anemia (hazard ratio [HR], 1.84; 95% confidence interval [CI], 1.59-2.12), thrombocytopenia (HR, 1.58; 95% CI, 1.32-1.89), and, especially the concomitant presence of anemia and thrombocytopenia (HR, 4.75; 95% CI, 2.98-7.55) were associated with inferior overall survival. Only a minor proportion of deaths was explained by diagnosed hematological malignancies, with the majority attributable to other causes. Neutropenia, either isolated (HR, 0.88; 95% CI, 0.73-1.06) or combined with another cytopenia, did not affect overall survival. For individuals aged ≥60 years, 5-year cumulative incidence of hematological malignancies was 0.60% (95% CI, 0.50-0.70), with higher incidences among those with anemia (P < .001) or thrombocytopenia (P < .001) but not neutropenia (P = .201). Highest cumulative incidences of diagnoses and mortality from hematological malignancies were observed in individuals with >1 cytopenia. We conclude that anemia and thrombocytopenia, but not neutropenia, are associated with inferior overall survival of community-dwelling individuals. Hematological malignancies develop in a small fraction of these cases.

Gamma Tocotrienol Protects Mice From Targeted Thoracic Radiation Injury.

Radiation injury will result in multiorgan dysfuntion leading to multiorgan failure. In addition to many factors such as radiation dose, dose rate, the severity of the injury will also depend on organ systems which are exposed. Here, we report the protective property of gamma tocotrienol (GT3) in total as well as partial body irradiation (PBI) model in C3H/HeN male mice. We have carried out PBI by targeting thoracic region (lung-PBI) using Small Animal Radiation Research Platform, an X-ray irradiator with capabilities of an image guided irradiation with a variable collimator with minimized exposure to non-targeted tissues and organs. Precise and accurate irradiation of lungs was carried out at either 14 or 16 Gy at an approximate dose rate of 2.6 Gy/min. Though a low throughput model, it is amenable to change the field size on the spot. No damage to other non-targeted organs was observed in histopathological evaluation. There was no significant change in peripheral blood counts of irradiated mice in comparison to naïve mice. Femoral bone marrow cells had no damage in irradiated mice. As expected, damage to the targeted tissue was observed in the histopathological evaluation and non-targeted tissue was found normal. Regeneration and increase of cellularity and megakaryocytes on GT3 treatment was compared to significant loss of cellularity in saline group. Peak alveolitis was observed on day 14 post-PBI and protection from alveolitis by GT3 was noted. In irradiated lung tissue, thirty proteins were found to be differentially expressed but modulated by GT3 to reverse the effects of irradiation. We propose that possible mode of action of GT3 could be Angiopoietin 2-Tie2 pathway leading to AKT/ERK pathways resulting in disruption in cell survival/angiogenesis.

Mutant U2AF1S34F and U2AF1Q157P Induce Distinct RNA Splicing and Hematopoietic Phenotypes In Vivo

Myelodysplastic syndromes (MDS) are the most common myeloid malignancies among the elderly. Patients present with bone marrow (BM) failure manifested by low peripheral blood (PB) counts and are at increased risk of developing acute myeloid leukemia. Mutations of U2AF1, a gene that encodes a spliceosome protein, are identified in 11% of MDS patients. The two most common U2AF1 mutants, S34F and Q157P, alter the splicing of two distinct sets of pre-mRNA targets in vitro and each co-occur with unique gene mutations in MDS patients, suggesting these mutants may affect MDS pathogenesis differently. In mice, U2AF1S34F expression leads to altered splicing, reduced B-cell counts, and features of MDS. Similar studies have not been performed for U2AF1Q157P. To study the impact of U2AF1Q157P expression on splicing and hematopoiesis in vivo, we created a doxycycline (DOX)-inducible ("Tet-On") transgenic mouse that expresses mutant U2AF1Q157P and is isogenic to our previously reported U2AF1S34F and U2AF1WT transgenic mice. First, we confirmed DOX-inducible expression of the U2AF1Q157P transgene in BM by RT-PCR-seq. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P, we performed non-competitive BM transplants into lethally irradiated congenic recipient mice. Donor BM from U2AF1WT or U2AF1S34F mice was also transplanted for comparison. Six weeks after transplant, mice were maintained on DOX chow to induce U2AF1 transgene (U2AF1WT, U2AF1S34F, or U2AF1Q157P) expression (n = 10 mice per genotype). After six weeks on DOX, there were no significant changes in PB counts for U2AF1Q157P mice compared to U2AF1WT controls. In contrast, white blood cell (WBC) and B-cell counts were significantly reduced in U2AF1S34F mice, as reported previously. Assessment of the BM revealed increased numbers (per five leg bones) of hematopoietic stem and progenitor cells (LSK [Lin− Sca-1+ c-kit+] and LK [Lin− Sca-1− c-kit+]) in U2AF1S34F mice (1.33×105 LSK and 7.13×105 LK cells) compared to U2AF1WT (1.04×105 LSK and 5.69×105 LK cells; p &amp;lt; 0.05 for LSK and LK), as reported previously. In contrast, there was no change in LSK cells (1.03×105, p = 0.9668) and a non-significant increase in LK cells (6.84×105, p = 0.0547) in U2AF1Q157P mice compared to U2AF1WT. Both U2AF1S34F and U2AF1Q157P mice shared a significant increase in the number of common myeloid progenitors (CMP) compared to U2AF1WT (2.43×105 and 2.39×105 vs. 1.66×105 cells; p &amp;lt; 0.001 and p &amp;lt; 0.01, respectively), although CFU-C interrogated by methylcellulose assay were significantly increased only for U2AF1S34F mice. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P on stem cell function, we mixed equal numbers of whole BM test cells (CD45.2+; U2AF1Q157P or U2AF1WT) with congenic control wild-type BM competitor cells (CD45.1+/CD45.2+) and transplanted them into lethally irradiated congenic recipient mice (CD45.1+/CD45.2+ ; n = 6 per genotype). As in non-competitive transplants, DOX chow was administered six weeks after transplant. After six weeks on DOX, we observed a relative multi-lineage competitive disadvantage by analysis of peripheral blood chimerism (%CD45.2+ WBC) for U2AF1Q157P test compared to U2AF1WT test cells (49.5% vs. 71.7%, respectively, p &amp;lt; 0.001). In addition, stem and progenitor cells were all significantly reduced in the BM of U2AF1Q157P competitive transplant mice compared to U2AF1WT after 18 weeks of DOX (LSK, 36.1% vs. 92.2%, respectively, p &amp;lt; 0.001; LK, 53.1% vs. 92.0%, p &amp;lt; 0.001). Lastly, using a Nanostring array, we identified consensus 3' splice sites of cassette exons that were increased or decreased in RNA from c-kit enriched mutant (U2AF1S34F or U2AF1Q157P) BM cells relative to U2AF1WT (FDR &amp;lt; 0.1). As expected, we observed altered consensus 3' splice sites at the −3 position (for U2AF1S34F) and +1 position (for U2AF1Q157P) of differentially spliced exons, indicating altered but different pre-mRNA splicing induced by either U2AF1 mutant. In aggregate, hematopoietic expression of U2AF1Q157P causes a multi-lineage competitive disadvantage of BM stem cells and expanded myeloid progenitors in the non-competitive transplant setting, like U2AF1S34F. However, PB counts and lineage distribution are not affected, indicating that the two common U2AF1 mutants, Q157P and S34F, are associated with different hematopoietic phenotypes and alterations to splicing, and may have different roles in MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

Radiation therapy and neutropenia

Acute radiation injury typically manifests as cytopenias: anemia, neutropenia, and thrombocytopenia; however, the measurement of peripheral blood counts belies the complex mechanism and timing of RT effects on hematopoietic stem cell (HSC) populations. There are several potential mechanisms for RT effects on the bone marrow, which are not mutually exclusive and may be complimentary. These include direct damage to HSC and reduction in their number and function, changes to the surrounding bone marrow stroma and microenvironment, and injury to ancillary cell populations that serve to regulate the hematopoietic process (eg, colony-stimulating factor [CSF] or erythropoietin-secreting cells). These effects are most likely with large volume irradiation of the pelvis or spine, as the primary site of functional bone marrow is the pelvis and vertebrae, which account for approximately 60% of total volume. Most of the data for detailed hematopoietic effects of RT are derived from large single fraction total body radiation exposures or from extended field administrations that are no longer used in routine practice. Even very low RT doses (as low as 0.3 Gy) can lead to measurable changes in peripheral blood counts (primarily lymphocytes at these doses) owing to the exquisite radiosensitive nature of these cells. With larger doses, lymphopenia typically occurs almost immediately due to radiation-induced apoptosis in interphase, followed by granulocytopenia, then thrombocytopenia, and ultimately anemia. Granulocyte counts may actually have an initial transient rise owing to mobilization from the periphery into the circulation, followed by a subsequent rapid decline. After an intermediate dose of total body radiation, initial decline in cell counts occurs approximately in 1, 4, 8, and 12 days for lymphocytes, granulocytes, platelets, and red blood cells, respectively. Clinical cytopenia occurs in approximately 1 day, 1 week, 2-3 weeks, and 2-3 months, respectively. These differences between cell lineages are owing to inherent radiosensitivity differences, variations in cell population reserves and mobilization, time required for hematopoietic cell replacement, and the life span of the differentiated peripheral cell (with red blood cells have the longest life cycle).

Alemtuzumab Is Safe and Associated With High Response Rates In Selected Patients With Myelodysplastic Syndrome

▪In a subgroup of patients with myelodysplastic syndrome (MDS) an immune mechanism is believed to cause bone marrow failure. In these individuals immunosuppressive treatment (IST) can restore marrow function. We previously reported a 68% response rate in a phase I/II study of alemtuzumab in 31 patients with low to int-2 risk MDS (Sloand etal JCO Dec 10, 2010:5166-5173). Patients were enrolled according to their predicted probability of response based on an algorithm that incorporated HLA-DR15 positivity, age and transfusion burden (Blood 2003 Oct 15; 102(8):3025-7). We now report updated efficacy data on our cohort of 40 patients and long term follow up data on responders. The first 37 patients received 10mg of alemtuzumab for 10 days intravenously. Alemtuzumab was administered subcutaneously in the subsequent 3 patients. Primary endpoints were changes in peripheral blood counts. Secondary endpoints included improvement in the transfusion requirements (in transfusion- dependent patients), duration of response, and late effects of treatment, relapse and survival. Thirty nine patients were evaluable. One patient discontinued treatment after one dose because of severe hypotension and was excluded from analysis. Median follow-up time was 25 months (3-64 months). Median age was 56 years (23-71 years). The overall response rate (ORR) was 64% (25/39) with 21% of patients having a complete response (8/39). Twenty of 29 (69%) int-1 patients; 4 of 7 (57 %) int-2 patients and 1 of 3 (33%) low risk patients responded following alemtuzumab treatment. Median time to response was 3 months with median duration of response 9 months (range 1-69 months). Eight of 11 patients who relapsed regained their response with addition of cyclosporine and one patient was lost to follow up. The median duration of response in patients rescued with cyclosporine is 37 months (1-42 months). The median duration of response to immunosuppressive treatment (alemtuzumab +/- cyclosporine) is 22.2 months (range 3-69 months). All responding patients who were previously transfusion dependent became transfusion independent after alemtuzumab treatment. One of three patients who received subcutaneous alemtuzumab administration responded. The median survival for all patients was 34 months (range 1-72 months) (figure 1a) with median survival of 36 months (6-72 months) in responding group compared to 16 months (1-56 months) in non-responding group (p<0.001) (figure 1b). Five of twelve patients with abnormal cytogenetics at the start of treatment had a complete cytogenetic remission at one year. One patient with monosomy 7 and another with del 13 were in cytogenetic remission for 4.5 years and 4 years respectively. Decline in their blood counts correlated with re-emergence of initial clone. Three patients with del 13, 5q- and del 5 continue to remain in cytogenetic remission at 4, 2 and 1 years respectively. The absolute neutrophil count at baseline was significantly associated with overall survival on univariate analysis. On multivariate analysis, factors significantly associated with overall survival were male sex, bone marrow hypocellularity/normocellularity, prior immunosuppressive treatment, baseline absolute neutrophil count and platelet count. All patients became lymphopenic after alemtuzumab administration and most patients continued to be lymphopenic at 3 years. Baseline absolute lymphocyte count, ratio of CD4/CD8 cell count, absolute CD4 and NK cell counts and kinetics of lymphocyte recovery were not predictive for response. Alemtuzumab was generally well tolerated with most patients having manageable infusion reactions with the first dose. Sixteen of the 39 patients became transiently positive for EBV DNA and 6 of 29 CMV seropositive patients had reactivation but no patient developed clinically significant viral disease. Alemtuzumab is well tolerated and is effective in a subset of patients with MDS. Long term responses with cytogenetic remissions are possible. Patients who relapse can be rescued with cyclosporine. Immunosuppresion should be considered in patients likely to respond based on a simple algorithm and alemtuzumab is an alternative to ATG based regimens. [Display omitted] [Display omitted] Disclosures:Off Label Use: Alemtuzumab use as immunosuppresive agent in myelodysplastic syndrome.

Resistive exercises, with or without whole body vibration, prevent vertebral marrow fat accumulation during 60 days of head-down tilt bed rest in men

Fat accumulates in the bone marrow of lumbar vertebrae with bed rest. Exercise with or without whole body vibration may counter this effect. Our objectives were to measure 1) the vertebral fat fraction (VFF) of men subjected to bed rest who performed resistive exercises with (RVE, n = 7) or without whole body vibration(RE, n = 8) or no exercise (CTR, n = 9) using three MRI techniques; and 2) changes in peripheral blood counts. Twenty-four healthy men (age: 20-45 yr) underwent -6° head-down tilt (HDT) bed rest for 60 days. MRI was performed using three techniques (fat saturation, proton spectroscopy, and in and out of phase) to measure the fat fraction of L(3), L(4), and/or L(5) at baseline, mid-HDT, and end-HDT. Erythrocytes and leukocytes were counted at HDT days 19, 33, 47, 54, and 60. The mean absolute VFF was increased in the CTR group at mid-HDT and end-HDT (+3.9 ± 1.3 and +3.6 ± 1.2%, respectively, both P < 0.05). The RE group had a smaller VFF change than the CTR group at mid-HDT (-0.9 ± 1.2 vs. +3.9 ± 1.3%, P < 0.05). The RVE group had a smaller VFF change than the CTR group at end-HDT (-2.6 ± 1.9 vs. +3.5 ± 1.2%, P < 0.05). Erythrocyte counts were increased in all groups at HDT day 19 and HDT day 33 and in the RE group at HDT day 54 (all P < 0.05). Bed rest for 60 days at -6° HDT increased lumbar VFF in men beyond natural involution. RVE and RE regimens effectively prevented VFF accumulation. Higher erythrocyte counts were not altered by RVE or RE. Whole body vibration, along with RE administered to people with prolonged immobility, may prevent fat accumulation in their bone marrow.

Radiation Disasters: Role of the BMT Team

Bone marrow transplant (BMT) teams do not generally consider themselves to be emergency responders. But the bone marrow is the most radiosensitive organ in the body, and early changes in peripheral blood counts remain the best indicator of major total-body radiation exposures. Following a mass casualty incident, such as that occasioned by a nuclear detonation, BMT teams should expect that they will be called upon for their expertise in managing severe myelosuppression. Numerous resources, including the Radiation Injury Treatment Network, are available to assist BMT teams in planning for such a role.

Long-Term In Vivo Studies in Mice Unexpectedly Reveal That Prolonged Growth Hormone (GH) and Insulin/Insulin-Like Growth Factor-1 (IGF-1) Signaling Have a Negative Effect on Normal Bone Marrow Hematopoiesis – Implications for GH-Based “rejuvenation” Therapies,

Abstract 3422 Background:Growth hormone (GH) has been proposed as a rejuvenation factor that delays aging in older patients. The long-term results of such therapy, however, have recently become controversial. It is known that GH action is mediated by release of insulin-like growth factor-1 (IGF-1, also known as somatomedin C) from the liver. IGF-1 is an important factor affecting proliferation of several types of cells, including malignant hematopoietic blasts. A role for the GH-IGF-1 axis in normal hematopoiesis still remains controversial and some stimulatory effects to the clonogenicity of normal hematopoiteic stem/progenitor cells (HSPCs) have been described. We have previously reported that IGF-1 does not directly stimulate proliferation of normal clonogenic hematopoietic progenitors (J Clin Invest. 1994;94:320); however, it may promote erythroid differentiation and hemoglobinization of erythroblasts (Leukemia 1998;12:371). Aim of the study: Since long-term studies on the effect of GH-IGF-1 signaling on normal hematopoiesis in vivo have not been performed yet, we investigated the influence of these factors on the hematopoietic system in appropriate mouse models that express low or high levels of plasma GH and IGF-1. Experimental approach: We employed two mutant mouse strains that have low circulating plasma levels of IGF-1 due to a GH-receptor mutation (Laron dwarf mice) or to a defect in GH synthesis in the pituitary gland (Ames dwarf mice). We also employed transgenic mice that express the bovine GH gene (bGH), which results in a constitutively high level of circulating plasma IGF-1. In some experiments, normal wild type or Laron and Ames dwarf mice, with low circulating plasma IGF-1 levels, were injected for a prolonged period of time with recombinant GH or IGF-1. All these animals were analyzed by FACS for the presence of Sca-1+Lin–CD45+ HSPCs in bone marrow (BM) and the clonogenic growth of progenitors from all hematopoietic lineages monitored in standard methylcellulose (CFU-GM and BFU-E) and plasma clot (CFU-Meg) cultures. We also analyzed changes in peripheral blood count by Hemavet. The mice employed in our studies were at different ages (2 months, 6 months, and 2 years), and both female and male mice were analyzed. Results: We observed that Laron and Ames dwarf mice, with low circulating plasma levels of IGF-1, at the age of 1–2 years had ∼3–5 times more Sca-1+Lin–CD45+ in the BM than normal control littermates of the same age. Similarly, these mice also had ∼3–5 times more CFU-GM, BFU-E, and CFU-Meg in BM. The number of Sca-1+Lin–CD45+ cells and clonogenic progenitors from all lineages declined ∼5 fold in these animals (at 2 months and 8 months of age) after 4 weeks of treatment by daily injection of GH or IGF-1. However, interestingly, if this treatment was ceased the number of these cells showed over time a tendency to normalize. In another set of experiments, we evaluated the number of HSPCs in BM of 6-month- and 1-year-old mice that express the bovine GH transgene. We found that these mice have an ∼6-fold and ∼12-fold lower number in the BM of Sca-1+Lin–CD45+ and clonogenic progenitors, respectively, than control transgenic littermates expressing empty vector. Overall, a decrease in the number of HSPCs was accompanied by microcytic anemia. Conclusions: Our data, obtained from several in vivo animal models, show that prolonged exposure to GH-IGF-1 signaling depletes the number of HSPCs in the BM over time. This observation is highly relevant to GH rejuvenation therapies currently employed in humans. Our data indicate that this may lead to the premature depletion of HSPCs, and we also envision that prolonged stimulation of HSPCs by GH-IGF-1 may result in development of leukemias. In support of this notion, Laron dwarf mice and Laron dwarf patients do not develop hematological malignancies, while leukemias are often seen in bGH transgenic mice and in acromegalic patients. Disclosures:No relevant conflicts of interest to declare.

Eltrombopag Can Stimulate Trilineage Hematopoiesis with Transfusion Independence in Patients with Refractory Severe Aplastic Anemia: Results From a Phase II Trial

Abstract 54▪▪This icon denotes a clinically relevant abstractSevere 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. [Display omitted] Disclosures:Off Label Use: Eltrombopag for severe aplastic anemia.

The Bone Marrow Hematopoietic Microenvironment Is Functionally Impaired in Iron-Overloaded Mice,

Abstract 3173 Background and Purpose:Deferasirox (DFX) is an oral iron chelator that enables effective chelation by once daily administration.Since the introduction of DFX, iron chelation therapy (ICT) for transfusional iron overload has attracted increased attention. It is known that excess iron increases oxidative stress and affects various organs, such as the liver, heart and endocrine glands, negatively. Sufficient ICT can remove excess iron and improve organ dysfunction in iron-overloaded patients, and accumulating data has indicated that efficient ICT improves the survival of transfusion-dependent patients with myelodysplastic syndromes (MDS). Recently, we experienced a case of MDS with transfusional iron overload in which the hematopoietic data improved unexpectedly after administration of DFX without any other specific treatments (Okabe H et al. Rinsho Ketsueki, 2009). An increasing number of similar cases has been reported. This clinical observation indicates that iron overload could also affect the hematopoietic system unfavorably, via, as yet, unknown mechanisms. Methods and Results:We generated iron-overloaded mice to investigate how iron overload affects hematopoiesis in vivo. C57BL6 mice were injected with a total of 200 mg of iron dextran, intraperitoneally over 4 weeks. The iron-overloaded mice showed pigmented skin and hepatosplenomegaly, and histological examination showed excess iron deposition in the bone marrow, liver, spleen and heart. The serum and organ iron concentrations in these mice markedly increased. However, the iron-overloaded mice did not show any significant changes in peripheral blood counts or the proportion of immature hematopoietic cells in the bone marrow. To further examine the effects of excess iron on the biological functions of hematopoietic stem and progenitor cells (HSPCs), we performed bone marrow transplantation (BMT) assays. First, to assess the hematopoietic reconstitutional capacity of the HSPCs of iron-overloaded mice, we transplanted bone marrow cells (1×106 cells) from iron-overloaded mice or normal mice into lethally irradiated normal recipient mice along with the same number of normal competitor cells. We found no significant difference in hematopoietic reconstitution between the iron-overloaded donor cells and the normal donor cells, suggesting that the hematopoietic reconstitutional capacity of HSPCs in iron-overloaded mice is not significantly affected by iron. In contrast, when we transplanted bone marrow cells from normal mice (2×106 cells) into iron-overloaded recipients, hematopoietic recovery was significantly delayed, in particular platelet counts (at 2 weeks after BMT, normal recipients vs. iron-overloaded recipients, 63.4±9.4 vs. 18.7±4.7×104/μl, respectively, p<0.001). This indicates that excess iron disturbs the function of the bone marrow microenvironment and delays hematopoietic reconstitution. Microarray and quantitative RT-PCR analysis of non-hematopoietic bone marrow cells (CD45-/Ter119-) from the iron-overloaded mice demonstrated significant reductions in CXCL12, VCAM-1, Kit-ligand and IGF-1, which are important regulators of hematopoiesis. In addition, in the iron-overloaded mice, the serum concentration of erythropoietin and the expression level of thrombopoietin in the liver were also significantly reduced. Furthermore, increased oxidative stress levels were observed in the iron-overloaded liver and bone marrow. Conclusion:We did not observe any direct effects of excessive iron on hematopoietic cells, but found significant impairment of the hematopoietic microenvironment in the bone marrow of iron-overloaded mice. These results suggest that oxidative stress induced by excess iron could disturb the hematopoiesis-supporting capacity of the bone marrow microenvironment by reducing the expression of many essential molecules. Disclosures:No relevant conflicts of interest to declare.

Age-related changes in peripheral blood counts in humans.

Anaemia has become a common concern in geriatric health. Since its prevalence varies quite significantly among different groups depending on factors such as ethnicity, lifestyle or fitness, the appropriateness of the current WHO definition of anaemia in the elderly may be questioned. We evaluated peripheral blood parameters from 1,724 individuals (908 women aged 18-101 years and 816 men aged 18-96 years), who were treated at the University of Heidelberg Medical Center with no known haematological history. Patients with a known malignant haematological or oncological disease or with chronic infection or inflammation were excluded. Patients with disorders affecting the kidneys, thyroid or stomach, as well as patients with a bleeding history, haemolysis or who had been previously diagnosed with anaemia were excluded from the study. Average haemoglobin levels for men beyond the age of 70 and for women beyond the age of 80 were found to fulfill the WHO criteria for the diagnosis of anaemia. While in our cohort ∼20% of men and women between 60-69 years of age were by definition anaemic, these numbers steadily increased to 63% in females and 76% in males beyond the age of 90. Based on the results of our study and in accordance with the literature on this topic, we suggest age-adjusted criteria for the diagnosis of anaemia in the elderly in conjunction with a geriatric assessment.

Alemtuzumab Treatment of Intermediate-1 (INT-1) Myelodysplasia Patients Is Associated with Sustained Improvement in Blood Counts and Cytogenetic Remissions.

Abstract 116▪▪This icon denotes an abstract that is clinically relevant.Myelodysplastic syndromes (MDS) are a diverse group of disorders characterized by cytopenias and ineffective hematopoiesis. Experimental evidence links bone marrow failure in a portion of MDS patients to a T cell-dominated autoimmune process. Immunosuppressive therapy (IST) can improve cytopenias in selected patients. Features associated with responsiveness to horse ATG in MDS are young age, low IPSS score, and the presence of HLA DR15 (Sloand E et al J Clin Oncol 2008 26(15):2505-11). The addition of cyclosporine to ATG improves response but continued use of this drug is associated with nephrotoxicity. Alemtuzumab, an anti-CD52 antibody, produces more profound lasting immunosuppression compared to ATG that is associated with transient lymphopenia. We conducted a non-randomized, off label, pilot, Phase II study of alemtuzumab (Campath®) in MDS patients who fit the criteria of those likely to respond to IST (Saunthararajah Y et al Blood. 2003 102(8):3025-7). Consecutive patients fulfilling the inclusion criteria received alemtuzumab 10mg IV for 10 days. Primary endpoints were changes in peripheral blood counts (platelets, absolute neutrophil count, reticulocyte count, hemoglobin). Secondary endpoints (in transfusion-dependent patients) included improvement in the transfusion requirements, duration of response, and late effects of treatment, relapse and survival. Median follow-up time was 11 months (range 1-46 months). Fifteen of 16 (93%) int-1 patients and 2 of 5 (40%) int-2 patients responded by 3 months following infusion. All responders were transfusion independent. Five of seven patients with abnormal cytogenetics at start of treatment had complete cytogenetic remission. One patient with monosomy 7 (constituting 65% of his bone marrow mononuclear cells by FISH) had a complete cytogenetic remission that has lasted 3.8 years thus far. Five of 9 (55%) patients evaluable at 9 months had completely normal blood counts. One patient developed immune thrombocytopenic purpura responsive to rituximab. Two int-2 patients developed leukemia and an additional int-2 patient with a history of smoking succumbed to small cell lung cancer. One death occurred in a non-responding int-1 patient due to disease progression. Two patients had declines in their counts (but not to pretreatment levels) and both responded to CsA. We monitored blood for EBV and CMV reactivation by polymerase chain reaction (PCR) weekly and 4 of the 22 patients became transiently positive for EBV but none developed disease and none of our patients had a significant infection while on drug. The response rate of int-1 patients at 3 months was superior to our previous study using ATG (53%; p=0.0071) but comparable to our results with ATG and CsA (93% p=1.0). Relapse was defined as requirement for additional treatment including cyclosporine; relapse-free survival of responders did not differ from those given ATG or ATG and CsA. The results from this pilot study indicate that Alemtuzumab is highly effective in producing durable responses in selected patients with int-1 MDS. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Acquisition of Isochromosome 7 Is a Late Change in the Pathogenesis of Hepatosplenic Lymphoma, Documented On a Case of Adolescent Girl 5 Years After Renal Transplant with Preceding TCR Gamma-Delta Positive LGL Leukemia.

Abstract 5031 BackgroundPost-transplant lymphoproliferative disorders (PTLD) occur in 1-20% of recipients receiving solid organ transplantation. We describe a patient who suffered from hepatosplenic T-cell lymphoma occurring after previous PTLD in a renal transplant recipient. Patient/MethodsAn 11-yr-old girl underwent kidney transplantation for end-stage Fanconi's nephronophthisis in 2002. In October 2006 significant neutropenia (<200/uL) was firstly detected, without any abnormality in bone marrow (BM) aspirate and without hepato- or splenomegaly. Episodes of neutropenia resolved spontaneously or after enhanced immunosuppression and G-CSF. In January 2007 new episodes of neutropenia and newly significant “monocytosis” were detected in peripheral blood (PB) and BM. Percentage of “monocytes” corresponded with immunophenotypically atypical TCR gamma/delta positive T cells (CD7weakposCD5negCD3bright) in PB and BM. Clonal TCR gamma and delta rearrangements were identified which enabled qPCR minimal residual disease (MRD) assessment. No lymphadenopathy was present, slight hepatosplenomegaly was identified by sonography. Conventional and molecular cytogenetic analyses didn't reveal any chromosomal aberration in PB and BM including changes on chromosome 7. No increased levels of EBV and CMV load by PCR were found. Partial increase of granulocytes and slight decrease of atypical TCR gamma/delta T cells were detected after administration of corticosteroid bolus and mercaptopurin. Three months later she presented with fever, rapidly progressive hepatosplenomegaly and pancytopenia, clinically corresponding with hepatosplenic lymphoma. At this time, newly acquired isochromosome 7q was detected by FISH. ResultsInitial therapy with campath and fludarabine was ineffective. She didn't respond to the 2nd line treatment (prednisone, vincristine, daunorubicine,asparaginase) and died 2 weeks later from lymphoma progression. Autopsy identified severe hemophagocytosis in the liver. Retrospectively, we identified identical clonal TCR rearrangements in the PB samples from March 2006 (∼0.03% of lymphoma PB MRD level), when neither changes in PB count nor clinical symptoms were found. ConclusionWe detected a “pre-lymphoma” phase with clonal expansion of atypical TCR gamma/delta T cells more than 1 year before lymphoma manifestation. The presence of isochromosome 7q was a late change during this lymphoma genesis. Grant supportIGA NS/9997-4; IGA NR/9531-3, IGA NS 10480-3, Research Projects MZCR 000064203, MSM0021620813 DisclosuresNo relevant conflicts of interest to declare.

Die Rationale für Knochenmarkuntersuchungen bei Patienten mit entzündlich-rheumatischen Erkrankungen

Inflammatory rheumatic diseases and the applied immunosuppressive treatments can lead to bone marrow depressions and promote hematologic malignancies. Our objective was to explore indications for and results of bone marrow examinations in a large cohort. Between 1990 and 2004 146 bone marrow examinations in 3638 patients were performed due to abnormal laboratory results. Medical history, results of bone marrow examination (morphology, histology) and cytogenetic data were investigated retrospectively. Patients' (67.8% female) mean age at bone marrow examination was 53.5 years (SD 15.5), median disease duration 2.9 years. Indications for bone marrow examination were changes in peripheral blood counts in 81.7%. In 52 patients (35.6%) clinically relevant, partially neoplastic bone marrow changes (5 non-Hodgkin lymphoma, 9 myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) and 3 myeloproliferative neoplasias) were evident. Medication history showed intake of hydroxy-/chloroquine (13.5%), methotrexate (17.3%), cyclosporin (7.7%), sulfasalazine (7.7%), mycophenolatmofetil, gold, leflunomide (each 1.9%), azathioprine (aza, 25.0%) or cyclophosphamide (cyc, 7.7%) prior to bone marrow examination. 7 out of 9 patients, who developed MDS/AML had been treated with either azathioprine alone or additionally with cyclophosphamide (n = 3). One third of our patients showed relevant bone marrow changes that might be associated to therapy. The risk seems to be increased especially in patients with inflammatory rheumatic diseases who had received azathioprine alone or in combination with cyclophosphamide. Health care providers should bear in mind the risk of hematologic malignancies and monitor patients closely in this respect. Bone marrow examination should be performed in case of changes in peripheral blood counts; especially clinically relevant anemia, granulocytes < 2,500/microl, thrombocytes < 100,000/microl and relevant changes over time should lead to bone marrow examinations.

Expanding the Use of Retinoids in Acute Myeloid Leukemia: Spotlight on Bexarotene

In this issue, Tsai et al. ([1][1]) report on a phase-I clinical trial of bexarotene in 27 non-M3 acute myeloid leukemia (AML). AML is characterized by abnormalities in the myeloid line at various stages of commitment and maturation, leading to an accumulation of granulocyte or monocyte precursors.

Hedgehog Signaling in Normal and Malignant Hematopoiesis.

The Hedgehog (Hh) signaling pathway is critical for normal development and dictates the self-renewal, proliferation and differentiation of normal stem cells and progenitors. Aberrant reactivation of Hh signaling has been described in a wide variety of human cancers and its role in normal stem cells suggest that pathway dysregulation contributes to oncogenesis and influences the cell fate decisions in cancer stem cells (CSC). Like their normal counterparts, CSC appear to undergo self-renewal as well as give rise to differentiated progeny, and these properties implicate that CSC are responsible for continual tumor cell production that underlies the initiation, maintenance and progression of clinical disease. Myeloid leukemias have long served as the model system for human CSC, but the cellular processes responsible for regulating these rare biologically distinct cell populations have remained unclear. We hypothesized that Hh pathway activation contributes to the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and studied Hh signaling in these settings. Using both RT-PCR for pathway components and a Gli1 reporter assay, we have found that Hh signaling is active in several human AML derived cell lines (Kasumi-1, KG1, KG1a) and primary AML and MDS samples. Approximately 80% (19/24) of primary AML samples tested express the downstream effectors GLI1 or GLI2 indicative of active Hh signaling. Furthermore, inhibition of Hh signaling with the naturally derived SMOOTHENED antagonist cyclopamine reduces the clonogenic growth of KG1 cells implicating the pathway in self-renewal. In contrast, cyclopamine failed to affect colony growth in the HL-60 cell line that lacks expression of Hh pathway signaling components, confirming that the effect of Hh inhibition is specific. In addition, the ectopic expression of Gli1 in KG1 cells partially rescued the effect of cyclopamine on colony formation further demonstrating the specific nature of this compound. We also studied normal CD34+ bone marrow cells and found that they expressed components of Hh pathway by RT-PCR. However, in contrast to KG1 cells, cyclopamine had little effect on the recovery of either normal hematopoietic progenitors or stem cells in an in vitro long-term culture assay. Therefore, it appears that Hh inhibition may preferentially inhibit myeloid leukemias. We further studied the role of Hh pathway activation on normal hematopoiesis and developed a transgenic mouse model in which SMOOTHENED is conditionally over-expressed in the myeloid lineage via Cre recombinase activity regulated by the Lysozyme promoter. Analysis of these mice demonstrated only subtle changes in peripheral blood counts, but further analysis of cells expressing the transgene revealed a significant reduction in the number of mature myeloid cells. This was confirmed by analyzing blood cells for the granulocyte marker Gr1 and pan-myeloid marker Mac1, both of which were significantly reduced in the SMOOTHENED over-expressing cells. These defects are reminiscent of MDS and further suggest that the Hh signaling pathway plays a role in normal hematopoiesis. Therefore, aberrant Hh pathway activation is a feature of myeloid leukemias and inhibitors such as cyclopamine may have a therapeutic role in the treatment of AML and MDS.

Rapamycin Analogs Reduce mTORC2 Signaling and Inhibit AKT Activation in AML.

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin-insensitive, and was recently shown to regulate the pro-survival kinase AKT by phosphorylation on Ser473 (Sarbassov Science 2005;307 and Mol Cell 2006;22). We investigated the molecular effects of mTOR inhibition by rapamycin analog CCI-779 in AML cells. Unexpectedly, CCI-779 not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased phosphorylation of p70S6K, 4EPB1 and reduction in Glut-1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-Cyclins in AML cell line and 5 of 8 primary AML samples in vitro. Similar observations were made in samples from patients with hematological malignancies who were treated with the rapamycin analogs temsirolimus or everolimus: the levels of Ser473 phosphorylated AKT decreased in 3/5 patient samples at 1 or 24 hour(s) of temsirolimus treatment, and in 6/8 patient samples treated with everolimus. In the 9 samples in which AKT was inhibited, ≥2-fold decrease in Cyclin D1 mRNA was observed in 5, Cyclin D2 in 3, both, Cyclin D1 and D2 in 1 sample, and Glut-1 in 4 patient samples. In 7 of the 9 patients in whom AKT was inhibited, a >50% decrease in peripheral blood absolute blast count (3 AML, 1 ALL) or absolute lymphocyte count (1 CLL) for >1 week duration was documented, and two patients with RAEB-1 had improvements in platelet counts, one fulfilling the criteria for clinical response (hematological improvement). No change in peripheral blood counts or progression of leukemia was seen in 6 patients. Of these, decrease in pAKT was observed in 2, no change in 3 and increase in 1 (Fisher exact two-tailed p= 0.021). Altogether, our study provides first evidence that rapamycin analogs inhibit AKT signaling in primary AML cells both in vitro and in vivo, and support the therapeutic potential of mTOR inhibition strategies in leukemias.

An approach to decreasing dose load in prophylactic X-ray examination

The concept of small radiation dose is uncertain. Usually, doses below 300 mSv are considered as small. In this work, a mathematical approach is used to consider changes in peripheral blood count caused by exposure to an effective dose below 1 mSv. The ACS-ENOFIT device is used to assess the efficiency of treatment of a number of diseases. The results obtained in this work show that an analytical method should be used for determination of the dose value and monitoring of changes in the systems of human body.