ALBERT

Description: In Primary Myelofibrosis, several lines of evidence suggest that pleiotropic cytokine TGF-β1, released by clonal proliferation of pathological megakaryocytes and/or monocytes, plays a prominent role in reticulin fibers deposition. This cytokine is synthesized as a biologically inactive molecule that needs to be activated in order to trigger biological responses. However, the mechanisms involved in local TGF-β1 activation within the hematopoietic environment remain unclear. Since TGF-β1 and thrombospondin-1 (TSP-1) are synthesized and stored within the same organelles in megakaryocytes, one can speculate that the abnormal release of both molecules leads to pathological local TGF-β1 activation that becomes ultimately responsible of fibrosis development in the vicinity of these cells. To investigate the role of TSP-1 in local TGF-β1 activation, we used the TPOhigh murine model of bone marrow (BM) fibrosis. BM cells from wild-type (WT) or Tsp-1-null male littermates were infected with a retrovirus encoding the murine TPO protein and engrafted into lethally irradiated WT or Tsp-1-null female hosts, respectively, leading to the following engraftment combinations, WT/WT (WT TPOhigh mice, n=21) and Tsp-1-null/Tsp-1-null (Tsp-1-null TPOhigh mice, n=17). Lethally-irradiated hosts were engrafted with 4 to 8 × 106 cells in 3 independent experiments. Peripheral blood was analyzed every 4 weeks during 3 months and mice were killed for histological analysis at week 8 and 12 post-engraftment. The magnitude of plasma TPO level increase was comparable regardless of the TPOhigh mice groups. Chimerism levels, analyzed in recipients by FISH on the presence of the donor Y chromosome in whole nucleated BM cells, were more than 90% in either WT or Tsp-1-null TPOhigh mice. We report here that all TPOhigh mice developed a similar myeloproliferative syndrome associated with TGF-β1 overproduction. Surprisingly, we were able to detect the active form of TGF-β1 in BM and spleen extracellular fluids in all mice, including Tsp-1-null TPOhigh mice, suggesting that alternative mechanisms are mainly responsible for local TGF-β1 activation in this murine model of myelofibrosis. We then confirmed that Tsp-1-null platelets are able to activate TGF-β1 in vitro in response to thrombin. As predicted by the detection of the active form of TGF-β1, Tsp-1-null TPOhigh mice developed BM and spleen fibrosis which appears, intriguingly, to be of a greater grade than the one displayed by WT TPOhigh mice. Since TSP-1 is a potent inhibitor of angiogenesis, we investigate whether this increased fibrosis could be correlated with an augmentation of neoangiogenesis. The microvascular density (MVD) in control Tsp-1-null BM were higher than in control WT one (10±4.7 vs 0.6±0.2; p

Description: Interferon-γ (IFN-γ) is recommended as prophylaxis against infections in patients with chronic granulomatous disease (CGD). However, since the optimal dose, the dosing interval, and the mechanisms of action are not well-defined, we studied the effects on CGD neutrophil (PMN) functions ex vivo of interferon-γ (IFN-γ). Evaluations were made on oxidative capacity, measured by superoxide anion production and chemiluminescence after stimulation with f-met-leu-phe (f-MLP) or phorbol-myristate-acetate, the killing of Aspergillus fumigatus hyphae (assessed as conversion of the tetrazolium salt MTT to formazan), and on the expression of FcγRI receptor (CD64). After randomization, 9 CGD patients (4 with gp91phox, 3 with p47phox, 1 with p67phox deficiency and 1 with unspecified CGD) were given IFN-γ, either 50 or 100 μg/m2 subcutaneously on 2 consecutive days after double blinded randomization. Furthermore, one female hyperlyonized X-linked carrier with a CGD phenotype was also studied separately after IFN-γ treatment. Evaluations were made the day before and on days 1, 3, 8, and 18 after IFN-γ administration. The killing of A fumigatus hyphae, being close to zero before IFN-γ, was enhanced on day 3, being 36% higher than pretreatment values in the high-dose CGD group and 17% in the low-dose group. The expression of FcγRI on PMN increased 3.7-fold in the high-dose and 2.3-fold in the low-dose CGD group, being maximal on day 1. Oxidative functions were raised in only selected patients represented by different subtypes of CGD. The hyperlyonized carrier of X-linked CGD responded to IFN-γ with more enhanced oxidative responses and Aspergillus killing of her PMNs than the other patients. This study suggests that a higher dose of IFN-γ than currently recommended confers transient enhancements of certain PMN functions in CGD patients.

Description: Kostmann syndrome, or severe congenital neutropenia (SCN), is an autosomal recessive disorder of neutrophil production. To investigate the potential role of apoptosis in SCN, bone marrow aspirates and biopsies were obtained from 4 patients belonging to the kindred originally described by Kostmann and 1 patient with SCN of unknown inheritance. An elevated degree of apoptosis was observed in the bone marrow of these patients, and a selective decrease in B-cell lymphoma-2 (Bcl-2) expression was seen in myeloid progenitor cells. Furthermore, in vitro apoptosis of bone marrow-derived Kostmann progenitor cells was increased, and mitochondrial release of cytochrome c was detected in CD34+ and CD33+ progenitors from patients, but not in controls. Administration of granulocyte colony-stimulating factor (G-CSF) restored Bcl-2 expression and improved survival of myeloid progenitor cells. In addition, cytochrome c release was partially reversed upon incubation of progenitor cells with G-CSF. In sum, these studies establish a role for mitochondria-dependent apoptosis in the pathogenesis of Kostmann syndrome and yield a tentative explanation for the beneficial effect of growth factor administration in these patients. (Blood. 2004;103:3355-3361)

Description: Transforming growth factor-β1 (TGF-β1) is the most important cytokine involved in the promotion of myelofibrosis. Mechanisms leading to its local activation in the bone marrow environment remain unclear. As a recent study has highlighted the role of thrombospondin-1 (TSP-1) in platelet-derived TGF-β1 activation, we investigated the role of TSP-1 in the TPOhigh murine model of myelofibrosis. Two groups of engrafted mice, WT TPOhigh and Tsp-1–null TPOhigh, were constituted. All mice developed a similar myeloproliferative syndrome and an increase in total TGF-β1 levels in the plasma and in extracellular fluids of marrow and spleen. Surprisingly, we were able to detect the active form of TGF-β1 in Tsp-1–null TPOhigh mice. Accordingly, these mice developed marrow and spleen fibrosis, with intriguingly a higher grade than in WT TPOhigh mice. Our results show that TSP-1 is not the major activator of TGF-β1 in TPO-induced myelofibrosis, suggesting the contribution of another mechanism in the megakaryocyte/platelet compartment.

Description: Introduction An association between thrombocytosis and cancer is well established and several studies have shown that an elevated platelet count at diagnosis implies an inferior prognosis. In ovarian cancer, the pivotal role of platelets in driving the biologic mechanisms of malignant tumors has been demonstrated and paraneoplastic thrombocytosis has been shown to directly fuel tumor growth (Stone et al, N Engl J Med. 2012). In this epidemiological study, we assess the role of prediagnostic platelet levels in primary care patients subsequently diagnosed with gynecological cancer. Methods Using a primary care resource comprising blood differential cell counts from more than 500,000 individuals (Andersen et al, Clin Epidemiol. 2014), we included adults (18-80 years) diagnosed with gynecological cancer (ICD-10 codes C51-C58) as reported in the Danish Cancer Registry (DCR) between July 1, 2003 to January 23, 2010. We analyzed platelet counts in a 3-year period before cancer diagnosis and defined no prediagnostic thrombocytosis as a mean platelet count between 150-400x109/l, mild prediagnostic thrombocytosis as 〉400-550x109/l and severe prediagnosticthrombocytosis as 〉550x109/l. Statistical Analysis We used multivariable logistic regression to compute odds ratios (ORs) with 95% confidence intervals (CIs) for the association between prediagnostic thrombocytosis groups and cancer stage category (localized vs. non-localized) at the time of diagnosis (Table 1). The ORs were adjusted for known and possible confounders such as age (quadratic), year and month of blood sampling, as well as competing comorbid conditions as reported in the Danish National Patient Register. Furthermore, we analyzed time from diagnosis to all-cause mortality (as reported in the Danish Civil Registration System) in Cox regression models. The effects of prediagnostic thrombocytosis were estimated with hazard ratios (HRs) and adjusted for cancer stage category in addition to the above-mentioned confounders. Results A total of 1,083 women were diagnosed in the defined period comprising external female genital organs and vagina (5.1%), cervix uteri (24.8%), corpus uteri (37.2%), ovary, fallopian tube and broad ligament (32.5%) and other and unspecified female genital organs (0.4%). 614 of these patients (57%) had at least one available prediagnostic platelet measurement (mean number of measurements=1.62, SD=1.19, range=1-17) and 109 exhibited prediagnostic thrombocytosis (mild=76%, severe=24%). We observed significant associations between prediagnostic thrombocytosis and the risk of being diagnosed with advanced disease with ORs of 2.19 (1.25-3.84), P=0.006 and 3.80 (1.37-10.57), P=0.0104 for mild and severe prediagnostic thrombocytosis, respectively. The median overall survival among patients with severe prediagnostic thrombocytosis was 0.92 years, as compared with 3.34 years among those with mild prediagnostic thrombocytosis, P

Description: Abstract 5125 We describe features of a previously unpublished 4-generation family with sex-linked thrombocytopenia and large platelets, first investigated in 1993 and followed since then. Whereas there is thrombocytopenia only in 2 males (patient I and III, patelet counts 43 and 29 ×109/l, respectively) a thalassemia trait with elevated HbA2 and HbF is also found in one female. Moderate hitherto non-progressive splenomegaly is present in III. DNA sequencing of exon 4 of the GATA-1 gene was performed in the affected males, and revealed a G to A mutation corresponding to the amino acid change R216Q. This mutation has earlier been shown in 4 other kindred as the cause of XLTT, characterized by thrombocytopenia, splenomegaly and imbalanced globin chain synthesis (Balduini et al Thromb Haemost 2004;91:129-40). Bone marrow findings in our affected males include increased megakaryocytes and morphological abnormalities in megakaryocytes and erythroblasts, confirming the role of GATA-1 for their maturation. A slight reticulin fibrosis (grade 1) is also noted, not hitherto described in other families with XLTT. In an attempt to further characterize this fibrosis and underlying mechanisms we performed CD34 staining which revealed a high microvessel density (MVD) and thus increased angiogenesis. We studied the pericyte coverage of vessels by performing double staining for CD34 and SMA-a, and expression of platelet derived growth factor receptor b (PDGFR-b). Comparisons were made to results in myelofibrosis patients and controls. MVD vessel/HPF Pericyte coverage % PDGFR-β+ pericytes PDGFR-β + cells/HPF BM biopsy from patient I 26.8 6 Yes 50 BM biopsy 1 from patient III 11.4 15.7 No 4.8 BM biopsy 2 from patient III 9.8 4.1 No 0.6 Myelofibrosis patients (n=20) 13±9 92±11 Yes Controls (n=9) 3.7±2 51±20 No Mice carrying the hypomorphic GATA-1low mutation have decreased expression of GATA-1 in megakaryoctes, defective megakaryocyte maturation and develop with age the typical traits of the human myeloproliferative disorder myelofibrosis, including reticulin fibrosis and increased angiogenesis (Vannucchi et al Semin Oncol 2005;32:365-72). In these mice as well as in the human disease myelofibrosis, immature megakaryocytes are retained in the bone marrow and pro-angiogenic and fibroblast stimulating factors are released because of pathologic emperipolesis. We show here that patients with the GATA-1 R216Q mutation also develop reticulin fibrosis and increased angiogenesis. It hasWe have previously been shown that the percentage of pericyte covered vessles is lower in the bone marrow of GATA-1low mice than in control mice, while on the contrary, patients with myelofibrosis have increased pericyte coverage as compared to controls (Zetterberg et al Haematologica 2007;92(5):597-604). We show above thatAs shown above, identical to mice with the GATA-1low mutation, patients with the GATA-1 R216Q mutation have lower pericyte coverage in their bone marrows than controls. Pericytes are cells of mesenchymal origin, important for vessel maturation and recruited to vessels by PDGF. Mice lacking either PDGF or its receptor have no pericyte coverage and die in utero because of hemorrhage from defective vessels. In contrast to malignant primary myelofibrosis, the reticulin fibrosis in our GATA-1 mutation R216Q patients appears non-progressive. We show here that the bone marrow stromal reactions are similar in patients with myelofibrosis and in patients with the GATA-1 R216Q mutation, with exception of pericyte coverage of vessels. Thus, GATA-1 dependent factors regulating pericyte recruitment might also be important for fibrosis progression in myelofibrosis and identifying these factors could forego therapeutic advances. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3053 Poster Board II-1029 In patients with hemophilia, repeated joint bleedings leads to synovitis and bleeding arthropathy is the major cause of morbidity in these patients. The synovitis is characterized by a highly vascular synovial membrane with prominent proliferation of synovial fibroblasts and infiltration by inflammatory cells. This chronic inflammation, as well as a direct toxic effect of blood on chondrocytes ultimately leads to cartilage and bone destruction and a crippling arthropathy. It has recently been shown that. Inflammatory cells isolated from hemophilic joints synthesize pro angiogenic factors (matrix metalloproteas-9, basic fibroblast growth factor and cyclooxygenas-2). Angiogensis in the adult is a complex process involving breakdown of connective tissue, proliferation and migration of endothelial cells but also recruitment of supporting cells, pericytes, important for vessel maturity. The aim of this study was to determine whether repeated joint bleedings in hemophilia patients induce a pro angiogenic reaction [increased micro vascular density (MVD) and expression of vascular endothelial growth factor (VEGF)] in the hemophilic joint and to study if also pericytes are involved in the process. After informed consent, synovial biopsies were collected from patients with severe hemophilia (n=5) when undergoing knee surgery on clinical grounds. As control, synovial biopsies from one patient undergoing diagnostic arthroscopy were used. Biopsies were snap frozen in liquid nitrogen and sectioned by cryotome. After fixation, sections were double stained for CD34 (for detection of endothelial cells) and SMA-a (for detection of pericytes) by immuno fluorescence. Sections were also stained for VEGF by immuno histochemistry. As shown in Table 1 patients with hemophilia had a very high MVD and pericyte coverage as compared to control samples. We also measured levels of VEGF by ELISA in plasma from 23 patients and 4 controls, but in all samples the concentration was below detection level. However, in synovial biopsies from patients with hemophilia, VEGF was clearly expressed by the endothelial cells as well as mononuclear cells present in the section. Table 1 Angiogenesis parameters in patients with hemophilia Synovial biopsies plasma MVD (vessels/HPF) Pericyte coverage (%) VEGF+ (cells/HPF) VEGF (pg/mL Hemophilia patients 17±8 91±0.1 22±19