Changes In Blood Counts Research Articles

Hepatosplenic T-cell lymphoma associated to thiopurine in Ulcerative Colitis patient from Brazil: A case report

Hepatosplenic T-cell lymphoma (HSTCL) is a rare and usually fatal peripheral T-cell lymphoma that primarily affects men younger than 35 years old. Treatment with thiopurines of patients with inflammatory bowel disease (IBD) has been associated with HSTCL, especially when submitted to a combined therapy with antibodies to tumor necrosis factor (anti-TNFs). Herein, we describe the case of an 25-year-old man with diagnosis of Ulcerative Colitis disease since 2003. He was initially treated with sulfasalazine 6g/dia, interspersed with prednisone during episodes of exacerbation. In July 2007 he started treatment with 50mg of azathioprine after a disease exacerbation. The dose was adjusted by the weight through 150mg/day, maintaining remission. Within 3 years and 3 months under azathioprine therapy he started to show changes in blood count and leukopenia. After an hematology evaluation, there was a diagnose of HSTCL. He died 8 months after diagnosis from chemotherapy-refractory lymphoma. Through a literature review, we identified 16 cases of HSTCL in patients with Inflammatory Bowel Disease treated with azathioprine or 6-mercaptopurine alone. Only 06 of these patients had the diagnosis of Ulcerative Colitis. The median age at diagnosis of HSTCL was 24 years (range, 15-35 years). The median duration of thiopurine exposure was 06 years (range, 3-17 years). 11 of these patients died, with only 04 patients achieving remission. Despite the known benefits in the treatment of IBD, additional research is needed to better understand the role of thiopurines and TNF-alpha inhibitors in promoting HSTCL. The absolute risk and the frequency of HSTCL is very low. With respect to safety, however, special caution in prescribing this therapy to male 35 years-old and younger IBD patients should be recommended, mainly during a period of 24 months or more.

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.

A Novel FVIIa Variant with Increased Potency and Duration of Effect Compared to Wildtype FVIIa. A Study in a Dog Model of Hemophilia A

Abstract 2252Hemophilia patients are treated by replacement therapy, receiving the plasma-derived or recombinant clotting factor in which they are deficient. A significant number of hemophilia patients, however, develop inhibitory antibodies against the factor they receive, becoming refractory to replacement therapy. In these inhibitor patients, hemostasis can be achieved using bypass agents, activated plasma-derived prothombin complex concentrates (aPCC) or recombinant activated Factor VIIa (FVIIa). However, these treatments do not match the efficacy seen in traditional replacement therapy. To overcome this limitation, 813, a modified recombinant human Factor VIIa with enhanced biological properties was developed using a rational protein design approach. 813 was selected from a series of FVIIa variants and is characterized by increased catalytic activity and prolonged duration of effect in vivo. Compared to recombinant wild-type FVIIa, 813 has 7-fold increased catalytic activity, measured by the rate of Factor Xa generation in vitro, both in the presence and absence of tissue factor (TF).813 was studied in Factor VIII (FVIII)-deficient dogs with NovoSeven RT (wt-FVIIa) as comparator. The FVIII-deficient dogs exhibit a severe hemophilia A phenotype, with less than 1% normal coagulant activity. Dogs were dosed intravenously with 813 or wt-FVIIa and blood and plasma were collected at various time points post dosing. Plasma FVIIa levels were measured using a FVIIa-specific clotting assay. Starting from initial peak plasma levels, a time-dependent decrease in FVIIa plasma concentration was observed for wt-FVIIa and variant. Compared to wt-FVIIa, 813 exhibited a smaller volume of distribution (35–48 mL/kg vs. 108–134 ml/kg), a lower rate of clearance (10–18 mL/hr/kg vs. 45 mL/kg/hr) and consequently an increased dose adjusted exposure. Hemophilia A dogs have a prolonged activated partial thromboplastin time (aPTT); treatment with wt-FVIIa or 813 led to a dose-dependent decrease in aPTT. At equivalent doses the peak effect of 813 on decreasing aPTT was approximately two-fold compared to wt-FVIIa. Similar dose-dependent effects were observed when studying whole blood clotting profiles by thromboelastography (TEG). Treatment effects were seen for all TEG parameters monitored (time to clotting onset, rate and strength of clot formation). However, compared to wt-FVIIa, 813 given at equivalent doses caused significantly stronger effects on all TEG parameters; acute peak effects on TEG parameters measured for 813 at 10 ug/kg, were similar to those seen with 50 ug/kg wt-FVIIa. This difference became even more pronounced when monitoring treatment effect over time. All dogs used in this study were monitored throughout the study for possible treatment-related changes; both 813 and wt-FVIIa were well tolerated. No overt adverse events, no significant changes in plasma clinical chemistry or cellular blood counts were observed.In conclusion, 813 is a novel FVIIa protein with increased potency, prolonged duration of effect and a preclinical safety profile comparable to wt-FVIIa. 813 is currently in early clinical development for acute and prophylactic treatment of hemophilia patients with inhibitors. Disclosures:Pittman:Pfizer: Employment. Weston:Pfizer: Employment. Shields:Pfizer: Employment. Parng:Pfizer: Employment. Arkin:Pfizer: Employment. Madison:Catalyst Biosciences: Employment. Nichols:Pfizer: Research Funding. Fruebis:Pfizer: Employment.

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.

Laparoscopic splenectomy is an effective and safe intervention for hypersplenism secondary to liver cirrhosis

Laparoscopic splenectomy has become the standard procedure for the normal to moderately enlarged spleens. We performed this study to investigate the safety, feasibility, and effectiveness of laparoscopic splenectomy for hypersplenism secondary to liver cirrhosis. We performed a retrospective chart review of 24 cases of laparoscopic splenectomy (group 1), 24 cases of open splenectomy (group 2) for hypersplenism secondary to liver cirrhosis, and 68 cases of laparoscopic splenectomy for immune thrombocytopenic purpura (group 3). We performed comparisons between groups 1 and 2 and groups 1 and 3 in terms of demographic, intraoperative, postoperative variables, and changes in blood counts and liver function. Patients in groups 1 and 2 had comparable demographic characteristics, but those in group 1 had less estimated blood loss, fewer complications, and shorter duration of oral intake, and they required less analgesia and shorter post-hospital stays. In both groups, leukocyte and platelet counts increased significantly and transaminase and total bilirubin decreased postoperatively, but not significantly, and there was no significant difference between the two groups. Compared with group 3, patients in group 1 were older, had lower preoperative hemoglobin levels and leukocyte counts, poorer Child-Pugh class, required more operation time, and suffered more estimated blood loss; however, there were no statistically significant differences in terms of conversion rates, transfusion rates, complication rates, and postoperative course. Laparoscopic splenectomy is a safe, feasible, and effective procedure for hypersplenism secondary to liver cirrhosis.

Experimental infection of the deer ked ( Lipoptena cervi) has no negative effects on the physiology of the captive reindeer ( Rangifer tarandus tarandus)

The deer ked ( Lipoptena cervi) is a haematophagous parasitic fly of cervids that spread to Finland in the early 1960's. Presently its northern distribution limit lies at approximately 65°N and it is gradually spreading northwards. In Finland the principal host species has been the moose ( Alces alces), but the deer ked is about to establish contact with another potential host, the semi-domesticated reindeer ( Rangifer tarandus tarandus) causing possible threats to reindeer health and management. The aim of this study was to investigate if the deer ked would have an influence on the welfare of the reindeer. Eighteen adult reindeer were divided into three experimental groups: the control group and two infected groups with 300 deer keds per reindeer introduced in August–September. One of the infected groups was treated with subcutaneous ivermectin in November. To gather comprehensive data on potential health hazards caused by the deer ked a wide array of physiological variables was measured during and at the end of the experiment in December. The keds caused no clear changes in the complete blood count, plasma clinical chemistry, amino acids, endocrinology, energy stores, enzyme activities or tissue fatty acid profiles of the host. The haematological, clinical chemical and endocrinological values displayed changes that could be related to the seasonal physiological adaptations of the species. In conclusion, at the duration and intensity of infection that were employed, the effects of the deer ked on the measured physiological variables of the reindeer were insignificant.

PERFIL HEMATOLÓGICO E ELETROFORÉTICO DE PROTEÍNAS SÉRICAS EM CÃES SOROPOSITIVOS PARA LEISHMANIOSE VISCERAL NO ESTADO DO RIO GRANDE DO NORTE

The present work aimed to determine the changes in complete blood count and electrophoretic separation of serum proteins assays in dogs seropositives to visceral leishmaniasis at Mossoro city, RN, Brazil. It was collected blood samples from 19 dogs with positive serology of visceral leishmaniasis for hemogram and proteinogram. The results revealed that hematological changes were remarkably inconstant at evaluated dogs, most ones presenting unregenerative anemia; another frequent finding was leucopenia. Furthermore, proteinogram from all studied dogs presented some disturbance; the most frequent were hyperproteinemia, hypoalbuminemia, hyperglobulinemia, hypergammaglobulinemia, and reduction on albumin/globulins ratio. The electrophoretic profile revealed polyclonal gammopathy. Thus, it was concluded that hematologic and serum proteins electrophoretic profiles are valuable tools for help on diagnostic of canine visceral leishmaniasis. Keywords: Leishmania, serum proteins, proteinogram, albumin, globulins, hematology.

The changes in complete blood count in thyroid cancer patients treated with radioactive iodine ablation therapy.

The aim of this study was to evaluate the effect of radioactive iodine (RAI) ablation therapy on the complete blood count (CBC) in thyroid cancer patients. One hundred sixty four patients undergoing RAI ablation therapy after total thyroidectomy were included. CBC results were available from the patients' medical records at the time of ablation and at the 1st, 6th, and 12th months after RAI therapy. Hemoglobin (Hb), white blood cell (WBC) and platelet (Plt) values were significantly lower than baseline at 1 month after treatment (p <0.0001). Hb and WBC values were increased at the 6th month and at the 1st year. Plt values increased at the 6th month but had decreased again at the 1st year. The values were usually in normal ranges except in the patients with low pretreatment Hb and WBC values. RAI ablation therapy in thyroid cancer patients is a safe treatment modality without any serious or persistent hematological side effects.

Analysis of Spanish Experience During Imiglucerase Shortage

AbstractAbstract 4725In the last twenty years enzyme replacement therapy (ERT) with imiglucerase has been a clinically effective for Gaucher disease (GD). The recombinant glucocerebrosidase administered intravenously - usually at biweekly intervals by lifelong has improved the quality of life of patients, avoided spleen removal and bone complications. In the last months an acute shortage of imiglucerase manufactured by the Genzyme Corporation (MA, USA) has occurred as a result of viral contamination firstly and other deficiencies in the production facility. In September 2009 a position statement based on the findings of the European Working Group for Gaucher Disease and European Gaucher Alliance, established a set of key recommendations about identification and monitoring of at-risk patients threatened. In Spain the follow-up of patients and the strict complementation of rules of therapy have permitted to obtain a profile of the situation in a group of patients with restricted ERT. Patients and Methods: A total of 50 GD1 patients have been analyzed before and after 6 and 12 months of imiglucerase shortage. Have been excluded for analysis children in order to dose reduction has been minimal as well as patients who have switched to another ERT or miglustat therapy. Results: Gender: 25 males/25 females. Mean age of group: 45.3±15.3 (range:18-84) SSI at diagnosis(Dx): 8.7±3.8 (range:3-19) Chitotriosidase (CT) activity at Dx:13,383±12,783 nM/mL.h; CCL18/PARC at Dx: 767±1,198 ng/mL. 20% of patients were splenectomized and 78% had bone disease at Dx. During shortage 23 patients (46%) discontinued therapy, in this period only one patient suffered a bone crisis and other anaemia as complications. Mean reduction of haemoglobin level: 2.7% (NS), platelet counts: 5.4% (NS). CT activity was increased 135% (p<0.03) and CCL18/PARC 8.2% (p<0.08). In 17 patients (34%) imiglucerase was reduced at 50%, in this period seven patients (41.0%) suffered a bone pain and four patients (23.5%) required support therapy. Mean reduction of haemoglobin level 2.9% (NS), no changes in platelet counts. CT activity increase 48.2% (p<0.03) and not changes in CCL18/PARC concentration was observed. In addition in 3 patients (6%) the reduction was 75% and 7 patients (14%) switched to another ERT (4 patients) or miglustat (3 patients). Conclusions: In our experience, the shortage of imiglucerase in the last months has produced a incidence in bone pain of 20% and one case with anaemia and significant increase of CT activity and no significant changes in blood counts and CCL concentration, 14 % patients has required switch another therapy. Disclosures:Off Label Use: Lenalidomide is not approved for the treatment of smoldering multiple myeloma.

Hematological Testing Is Not Required with Every Dose of Bortezomib In Patients with Adequate Blood Counts at the Start of Each Cycle

Hematological Testing Is Not Required with Every Dose of Bortezomib In Patients with Adequate Blood Counts at the Start of Each Cycle

Early Clinical Trial Results Following Administration of a Low Dose of a Novel Self Complementary Adeno-Associated Viral Vector Encoding Human Factor IX In Two Subjects with Severe Hemophilia B

AbstractAbstract 248We have developed a unique approach for the treatment of hemophilia B (HB) that is currently being tested in the clinic. This open-label Phase I/II clinical trial entails peripheral vein administration of a single dose of our novel self complementary AAV vector encoding a codon-optimised human FIX transgene (scAAV2/8-LP1-hFIXco) into adult subjects with severe HB. Our plan is to evaluate three dose levels, progressing to the intermediate and high doses only in the absence of toxicity in a minimum of two subjects each. Vector is being administered in the absence of immunosuppression. Thus far, two subjects have received peripheral vein infusion at the low dose, each without any side effects. Importantly, there were no adverse reactions during vector infusion and no subsequent evidence of hepatotoxicity. Overall, there were no significant changes in the complete blood count and serum chemistry panel. The longest follow-up is in the first subject, in whom plasma FIX levels increased from a baseline of <1% to between 1.5–2% of normal levels within 2 weeks. This level of transgene expression has been maintained for a period that extends beyond 5 months following vector infusion. Importantly, this subject has not required any treatment or prophylaxis with FIX concentrate over this period and remains free of spontaneous joint bleeds. A robust primary humoral response to AAV8 capsid protein was observed following vector infusion. Capsid-specific cytotoxic T cells were monitored by IFN-g ELISPOT and by staining for IFN-g, TNF-a, and CD107a followed by flow cytometry; this analysis showed no increase in AAV8 specific CD8+ T cells at any of the time points evaluated. Following vector administration there was transient, self-limited shedding of scAAV in some body fluids/excretions but vector was not detected in the semen. The same dose has recently been administered to a second patient without toxicity. The FIX level in this subject, who is on regular prophylaxis, is currently 2% of normal, 13 days after his last dose of prophylaxis which was given the same day as the vector and raised his level to 26%. His levels will be carefully monitored to more conclusively establish evidence of expression of transgenic FIX. A third patient is scheduled for treatment at the intermediate dose in the fall. These early data are highly encouraging and suggest that low doses of scAAV vector, when pseudotyped with serotype 8 capsid can mediate therapeutic levels of FIX for at least several months without provoking an immunological response of the type seen in the previous trial. Disclosures:High:Genzyme, Inc: Consultancy, Patents & Royalties; Third Rock Ventures: Consultancy; Novo-Nordisk: Consultancy; Shire, Inc.: Consultancy.

Alterations In Wnt Signalling In the Megakaryocytic Lineage Leads to Bone Marrow Failure and Myelofibrosis

AbstractAbstract 628 Introduction:Wnt signalling is fundamental in controlling stem cell self-renewal, cell proliferation and development in multicellular organisms. Stabilization of beta catenin or loss of the scaffold protein adenomatis polyposis coli (APC) causes aberrant activation of wnt signalling and often leads to cancer. Mutations to wnt pathway members in haematopoietic stem cells leads to haematopoietic failure and rapid lethality. In this study, we demonstrate that aberrant wnt signalling in the megakaryocyte lineage underlies myelofibrosis. Methods:We created a series of mice with altered wnt pathway signalling in their megakaryocytic lineage using PF4-Cre (platelet factor 4 cre) as follows: Ctnnb1fx(ex3)/wt_ Expresses stabilized active beta catenin henceforth termed PF4bcat+; APCfx/fx_ Loss of APC stabilises beta catenin termed PF4APC-KO; Ctnnb1fx/fx b-catenin knockout termed PF4bcat-KO. Results:By day 40, PF4bcat+ and PF4APC-KO mice are severely underweight, anaemic (wt 6.8+0.2×106/ml v. PF4bcat+ 4.2+0.3×106/ml, PF4APC-KO 5.5+0.5×106/ml), and have a significant reduction in platelet number (wt 1033+37×106/ml v. PF4bcat+ 717+57×106/ml, PF4APC-KO 747+68×106/ml). Furthermore PF4bcat+ and PF4APC-KO mice develop bone marrow fibrosis and consistently die within 50 days of birth. Both populations of mice have splenic extramedullary haemopoiesis with hyperplasia of splenic megakaryocytes, leading to a dramatic increase in spleen to body size ratio. In addition, both PF4bcat+ and PF4APC-KO mice have increased peripheral blood levels of active TGFb, providing a likely molecular basis of the induction in bone marrow fibrosis. PF4APC-KO and PF4bcat+ mutant mice show a dramatic (>10-fold) increase in platelet b-catenin protein levels over wt samples. By comparison, human myelofibrosis patients (n=16) show a 2.7+0.6-fold increase in platelet b-catenin expression over controls. Moreover, overexpression of b-catenin within human patient samples is correlated with a worsening Primary Myelofibrosis prognostic score (Blood, 2009 vol 113 p. 2895), with those patients with a low score (n=7) having a 1.3+/−0.37-fold increase over control, intermediate score (n=4) 4.52+/−1.23-fold, and high score (n=1) 4.56-fold. In contrast PF4bcat-KO mice show no changes in whole blood counts, weight, or evidence of splenic extramedullary haematopoiesis, indicating that b-catenin removal does not adversely affect megakaryocyte development or function. Conclusions:Stabilisation of b-catenin within mouse megakaryocytes leads to a myelodysplastic disorder and myelofibrosis. This finding demonstrates a defined role for aberrant activation of the wnt signalling pathway and marks the wnt pathway and the megakaryocyte lineage as important potential drug targets for the treatment of myelodysplastic disorders. Disclosures:No relevant conflicts of interest to declare.

Evaluation of Spanish Gaucher disease patients after a 6-month imiglucerase shortage

Recently, an acute restriction of imiglucerase has occurred as a result of viral contamination and manufacturing problems. A position statement from the European Working Group for Gaucher Disease and European Gaucher Alliance established a set of key recommendations for identifying and monitoring at-risk patients. In Spain, a profile of the shortage situation was obtained through follow-up of patients with Gaucher disease (GD) and compliance with the therapy recommendations. Here we describe a group of patients, with modified doses of imiglucerase, during the shortage. Fifty adult GD1 patients (25 males/25 females), previously on ERT, were analysed before and after the 6-month shortage. The mean age was 45.3 ± 15.3 years (range: 18–84). The mean Severity Score Index at diagnosis was 8.7 ± 3.8 (range: 3–19); 20% of patients were splenectomized; and 78% had bone disease. During the shortage, 23 patients (46%) discontinued therapy; as complications in this group only one patient suffered a bone crisis and another anaemia (Hb < 10.0 g/dL). The mean reduction of haemoglobin level (−2.7%) and platelet counts (−5.4%) were non-significant. Chitotriosidase (CT) activity was increased 135% ( p < 0.03) and CCL18/PARC 8.2% ( p < 0.08) in this group. Imiglucerase was reduced by 50% in 17 patients (34%) in this group, seven patients (41.0%) suffered bone pain, three of them true bone crisis and four (23.5%) required support therapy. The mean reduction of haemoglobin (−2.8%) and platelet counts (−10.7%), CT activity was increased 48.2% ( p < 0.03) and no changes were observed in CCL18/PARC concentration. In both groups no significant changes in visceral size were observed. In 3 patients (6%), imiglucerase was reduced 75% and 7 patients (14%) needed to switch to another ERT (4 patients) or miglustat (3 patients) due to a restart of symptomatic disease. In Spain the 6 first months shortage of imiglucerase have produced a 20% incidence of bone pain, one case of anaemia, and a significant increase in CT activity. Fourteen percent of patients had to switch to another therapy. No significant changes in blood counts, visceral volumes and CCL18/PARC concentration were observed.

Arthritic presentation of childhood malignancy: beware of normal blood counts

Some children with malignancy (e.g. acute lymphoblastic leukemia) who initially present with musculoskeletal complaints may be misdiagnosed as having a rheumatological disorder. In the literature, importance has been given to subtle changes in blood counts, which may point toward an underlying malignancy. We report 3 children with malignancy, who had an arthritic presentation but had normal blood counts at presentation. Atypical clinical pattern, significant nocturnal pain, pain out of proportion to joint involvement and prominent systemic features in these children prompted us to do a bone marrow examination that revealed a malignancy. Pediatricians must be aware of the arthritic presentation of childhood malignancy. If the clinical features point toward a malignancy, bone marrow examination should always be performed even if the blood counts are normal.

Report of 12 cases of ankylosing spondylitis patients treated with Tripterygium wilfordii

ObjectiveDescription of the clinical response of 12 consecutive cases of disease-active ankylosing spondylitis (AS) treated with the herbal medicine Tripterygium wilfordii Hook f (TwHf; lei gong teng, thunder god vine), which has been reported in controlled studies to be effective in rheumatoid arthritis (RA).MethodsThe clinical status of 12 patients with active AS who were started on 60 mgday-1 of a commercial tablet preparation of TwHf extract. were monitored at weeks 1, 3, and 6.ResultsCompared to baseline, there was significant improvement in mean values of physician assessment, Bath ankylosing spondylitis disease activity index (BASDAI), Bath ankylosing spondylitis functional index (BASFI), and Bath ankylosing spondylitis global score (BAS-G) at weeks 3 and 6, with no changes in liver enzymes or complete blood count (CBC).ConclusionA placebo-controlled double-blind study for Tripterygium is warranted. Until then, this particular report should be considered as case reports and not an endorsement of the use of Tripterygium in clinical practice.

Abstract 2533: Synergistic leukemic killing when combining a small molecule Pim protein kinase inhibitor and standard chemotherapeutic agents

Abstract The serine/threonine Pim protein kinases, originally cloned from a M-MuLV integration site, are up-regulated and associated with poor outcomes in multiple human hematologic malignancies including chronic lymphocytic leukemia, myeloid leukemia, diffuse large B-cell lymphoma, and T-cell lymphoma. We have developed novel benzylidene-thiazolidine-2, 4-diones inhibitors of Pim kinases that kill a wide range of both myeloid and lymphoid cell lines and shown that one of those inhibitors, SMI-4a inhibits subcutaneous tumor growth of T-cell acute lymphoblastic leukemia (T-ALL) in nude mice by an average of 47.9% (p&amp;lt; .05). These SMI-4a treated mice had no change in weight, blood counts, cell morphology, or blood chemistries. Treatment of these leukemic cells demonstrates that SMI-4a induced G1-phase cell cycle arrest secondary to an induction of p27Kip1, apoptosis through the mitochondrial pathway, and inhibition of mTORC1 pathway based on decreases in phosphorylation of p70 S6K and 4E-BP1. Since Pim kinases play an important role in cell survival and resistance to chemotherapy, we investigated the activity of combination treatment with SMI-4a and chemotherapeutic agents including daunorubicin (DNR), cytarabine (Ara-C), and 6-thioguanine (6-TG). Although DNR has been employed to treat both T-ALL and myeloid leukemias and is indispensable for the induction of complete remission, it is known to possess cardiotoxicity leading to cardiac failure, and thus drugs that decrease the dosage of DNR may be useful in therapy. We examined the combination of DNR and SMI-4a in T-ALL cell lines, myeloid leukemic cell lines and primary AML blasts from patients. Although this combination exhibits modest additive effect in T-ALL cells, the combination of 20 nM DNR and 40 μM SMI-4a killed 96% of the FLT3-ITD cell line MV4-11, while as single agents DNR and SMI-4a killed only 15 and 30% respectively. Evaluation of primary AML samples demonstrated that this combination significantly increased cell death compared to each agent alone. In addition to DNR, the combination of Ara-C and SMI-4a proved effective. We found significant killing effects when T-ALL cell lines, myeloid leukemic cell lines, and primary AML blasts were incubated with multiple doses of Ara-C in combination with SMI-4a. These changes were also seen when this inhibitor was combined with 6-TG. Since 6-TG induced hepatotoxicity and neutropenia often limit maintenance therapy, this combination therapy could allow a significant decrease in 6-TG dosing in humans. Thus, SMI-4a, a Pim kinase inhibitor with activity in animal models as a single agent with little overt toxicity, enhances the anti-leukemic effects of standard anti-leukemic drugs DNR, Ara-C, and 6-TG in vitro. If used in humans SMI-4a has the potential to limit the dose of these agents needed for treatment, decreasing their toxicity while maintaining anti-leukemic activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2533.

Anämie und schwere Thrombozytopenie bei Sprue

The frequency of diagnosis of celiac disease has increased since the introduction of serologic testing. The number of patients in whom extraintestinal symptoms reflect the initial manifestation is rising. Common symptoms are changes in blood counts, which can arise from changes in all cell lineages. Among these, iron deficiency anemia is very common, but also thrombocytosis and thrombocytopenia have been reported to manifest themselves within the framework of celiac disease. The authors report on a patient with malnutrition, macrocytic anemia, and pronounced thrombocytopenia. Based on changes of peripheral blood smear and endoscopic result, celiac disease was suspected that could be confirmed by histology of duodenal biopsies. The thrombocytopenia was reversible after initiation of gluten-free diet and folic acid substitution. In addition, clinical symptoms resolved and hematologic reconstitution could be observed. Similar deficiencies have almost exclusively been observed in patients with increased folic acid requirements during pregnancy or intensive care. Celiac disease must be taken into consideration, if changes in blood counts occur associated with gastrointestinal symptoms.

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.

Abstract B249: Small molecule inhibitors of the Pim protein kinases block the growth of T-acute lymphoblastic leukemias

Abstract The Pim protein kinases, first identified as a proviral integration site in c-Myc overexpressor mice, are increased in multiple human hematopoietic neoplasms including myeloid leukemia, diffuse large B-cell lymphoma, and T-cell lymphoma. We have developed novel benzylidene-thiazolidine-2, 4-diones (J. Med. Chem. (2009) 52:74) inhibitors of Pim kinases that kill a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre T-LBL/T-ALL) being the most sensitive. The most potent members of this chemotype have IC50s of 13 nM for Pim-1 and 2.3 M for Pim-2 while some compounds in this chemotype demonstrated selectivity's of more than 2500-fold and 400-fold for Pim-1 or Pim-2 respectively while other congeners had equivalent potency towards both isozymes. Of the 47 additional protein kinases tested, only DYRK1a was sensitive to these agents. Incubation of pre T-LBL cells with one of these Pim inhibitors, SMI-4a, induced G1 phase cell cycle arrest secondary to a dose dependent induction of p27Kip1 and translocation of this protein to the nucleus. Additionally, SMI-4a induced apoptosis in these leukemic cells through the mitochondrial pathway, and inhibited mTORC1 pathway based on decreases in phosphorylation of p70 S6K and 4E-BP1, two substrates of this enzyme. Using immuno-deficient animals, we demonstrate that treatment 5/7 days with 60 mg/kg twice daily by oral gavage of SMI-4a inhibits subcutaneous growth of pre T-LBL tumors by an average of 47.9% (p&amp;lt; .05). These SMI-4a treated mice had no change in weight, blood counts, cell morphology, or blood chemistries. To enhance the killing effect of SMI-4a we have examined a number of potential combination therapies. Because we find in animals and cell culture that single agent SMI-4a treatment stimulates the ERK pathway, and in the spleen and thymus of Pim1/2/3 knock out mice there is increased phosphorylation of ERK1/2, we combined SMI-4a and a MEK1/2 inhibitor, U0126 or PD184352. Our results demonstrate that this combination is highly synergistic in killing pre T-LBL cells in culture. Finally, because SMI-4a inhibits the mTORC1 pathway decreasing the phosphorylation of two mTOR substrates, p70 S6K and 4E-BP1, and because Pim plays an essential role in the FLT3/ITD signaling pathway, we examined the activity of SMI-4a with or without rapamycin in myeloid leukemic MV4–11 carrying both MLL-AF4 and FLT3-ITD. We find that these two agents are highly synergistic in culture. SMI-4a alone inhibited growth 18% and rapamycin 40% but when combined 76% of the cell growth was blocked. Our results demonstrate that unique combinations of a potent Pim inhibitor, SMI-4a, and small molecule blockade of either the mTORC1 or ERK pathways have promise as a novel combination strategy for the treatment of human leukemia. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B249.