F A González Fernández

A Villegas

P Ropero

M D Carreño

E Anguita

M Polo

A Pascual

Henández

: M Polo

: A Pascual

: A Henández

Haemoglobinopathies with high oxygen affinity. Experience of Erythropathology Cooperative Spanish Group

Keywords:

Haemoglobinopathies are the world's most frequently found monogenic disorders. In the cases with high oxygen affinity, the decrease in the liberation of the oxygen determines a secondary erythrocytosis. In this work, we present 17 unrelated families of Caucasian race and of Spanish origin, with ten variants of haemoglobin or haemoglobinopathies with high oxygen affinity which were diagnosed in our laboratory. Of the ten haemoglobinopathies, in four (the Hb San Diego, the Hb Johnstown, the Hb Malmö and the Hb Columbia-Missouri), the change of amino acid affects zones of the contact α 1 β 2 ; in two variants (the Hb Strasbourg and the Hb Syracuse), it affects the unions with 2,3-DPG in the central cavity; in the other two (the Hb Badalona and the Hb La Coruña), the cavity of contact with the group haem is affected; in one (Hb Bethesda), it affects the zone of contact α 1 β 1; and in one (Hb Olympia), the position 20 of the chain in the helix B in the surface of the protein is affected. In all cases, the change of amino acid, though of different form, facilitates that the quaternary structure of the haemoglobin becomes stable in its relaxed configuration so the transfer of oxygen and the P 50 value are decreased. All cases were sent to our laboratory because of shown erythrocytosis. In the majority of them, the diagnosis was done during an analysis of routine or for being relatives of the first ones.

Introduction

Haemoglobinopathies are the world's most frequently found monogenic disorders [START_REF] Flint | The population genetics of the haemoglobinopathies[END_REF]. Single mutations, deletions or insertions in the genes that encode globin chains may lead to qualitative alteration of the genes' expression in structural haemoglobinopathies or to a reduced or nonexistent expression, as seen in thalassaemia [START_REF] Huisman | The structure and function of normal and abnormal haemoglobins[END_REF].

So far, over 900 haemoglobin structural variations have been described. The structural alteration is due to a single amino acid replacement in over 95% of these variants. Depending on the replaced amino acid nature and its location, changes will be observed in the haemoglobin molecules' stability, solubility and function that finally lead to clinical haemoglobinopathies. Polyglobulia, generated to compensate for a decrease in the O 2 released into tissues, when the amino acid replacement increases Hb affinity for oxygen (O 2 ), is one of the clinical conditions that can be found [START_REF] Bunn | Hemoglobin: molecular, genetic and clinical aspects[END_REF]. Up to now, 89 haemoglobinopathies that show high oxygen affinity have been described. Only 18 of them affect one of the α genes and 71 in the β chain [4].

Material and methods

In our laboratory, from January 1996 to January 2006, we characterised 17 families, which are not related to each other, that show ten haemoglobin variants or haemoglobinopathies with high oxygen affinity. The haemoglobinopathies were characterised either through electrophoresis in an alkaline pH (8.6 pH) cellulose acetate or an isoelectric focussing in polyacrylamide gel (5.5-8.5 pH) or agar citrate (pH 6.0) electrophoresis or reverse-phase high-performance liquid chromatography (HPLC) for globin chains [START_REF] Riou | Evaluation of cation-exchange high performance liquid chromatography for presumptive identification of hemoglobin variants[END_REF] or ionic-exchange HPLC [START_REF] Shelton | High performance liquid chromatography separation of globin chains on large-pore C 4 column[END_REF]. Haemoglobin stability was determined using the isopropanol test [START_REF] Dacie | Practical Haematology[END_REF], while the function was established through P 50 in the oxygen balance curve plotted by a TCS Haemox-Analyser (TCS Medical Products Co., Huntingdon Valley, PA, USA) [START_REF] Carreño | Poliglobulias secundarias por hemoglobinopatias[END_REF]. The molecular analysis was completed by automatic sequencing of the α and/or β genes' polymerase-chain-reactionamplified products using the ABI Prism TM dRhodamine Terminator Cycle Sequencing Ready reagents kit (PE Applied Biosystems, Foster City, CA, USA) and the sequence was studied in an ABI Prism 310 Genetic Analyser (PE Applied Biosystems) [START_REF] Martín | A novel mutation of the α2-globin causing alpha(+)thalassemia: Hb Plasencia α125(H8)Leu→Arg (α2)[END_REF][START_REF] Ropero | Hb Johnstown [β109(G11)Val→Leu]: second case described and associated for the first time with beta (0)-thalassemia in two Spanish families[END_REF].

Results

All the demographic data, hematrimetric results, P 50 and chromatography findings are summarised in Table 1.

None of the patients in the study had any past or present clinical record of arterial or venous thrombosis at the time of diagnosis. All the referrals to our laboratory were due to erythrocytosis found, most often, during routine tests or through being relatives of the former patients. All patients were ethnically Caucasian and of Spanish origin.

Discussion

In haemoglobinopathies that show high oxygen affinity, the lower release of oxygen leads to tissue hypoxia that prompts erythropoietin output at renal level and induces both an increase in erythropoiesis and an offsetting secondary erythrocytosis [START_REF] Bunn | Hemoglobin: molecular, genetic and clinical aspects[END_REF].

In the haemoglobin structure, the α 1 β 2 bonds, the Cterminal ends of the β and α chains, the bonds to 2,3-DPG in the central cavity and the contact cavity with the haem group are significant zones not only for oxygen uptake but also for the 2,3-DPG, CO 2 and H + allosteric effects over that uptake. Mutations experienced by amino acids enclosed in these zones, which are critical for haemoglobin functions, can therefore determine alterations in affinity to oxygen [11]. Of the ten high-affinity haemoglobinopathies found by our laboratory, in four (Hb San Diego, Hb Johnstown, Hb Malmö and Hb Columbia-Missouri), the amino acid change affects the α 1 β 2 contact zones [START_REF] Nute | Hemoglobinopathic erythrocytosis due to a new electrophoretically silent variant, hemoglobin San Diego (β109(G11)Val→-Met)[END_REF][START_REF] Jones | Hb Johnstown [β109(G11)Val→Leu]: a new electrophoretically silent variant that causes erythrocytosis[END_REF][START_REF] Zak | A hypothesis for the increased oxygen affinity in haemoglobin Malmö[END_REF][START_REF] Perry | Hemoglobin Columbia Missouri or α 2 [88(F9)Ala→-Val]β 2 : a new high-oxygen-affinity hemoglobin that causes erythrocytosis[END_REF], while in two (Hb Strasbourg and Hb Syracuse) the bonds to 2,3-DPG, in the central cavity, are affected [START_REF] Bisse | Hb Strasbourg [β23(B5)Val→Asp]; a high oxygen affinity variant observed in a German family[END_REF][START_REF] Jensen | Hemoglobin Syracuse (α 2 β 2 -143(H21)His leads to Pro), a new high-affinity variant detected by special electrophoretic methods. Observations on the auto-oxidation of normal and variant hemoglobins[END_REF], and in another two (Hb Badalona and Hb La Coruña) the contact cavity to the haem group [START_REF] Juncá | Characterization of a new hemoglobin variant: Hb Badalona β31(B13)Leu→Val[END_REF][START_REF] Ropero | Hb La Coruña [β38(C4)Thr→Ile]: a new hemoglobin variant leading to familial polycythemia[END_REF] is affected and in one (Hb Bethesda) the α 1 β 1 contact zone is affected [START_REF] Bunn | Structural and functional studies on hemoglobin Bethesda (α2β2145His), a variant associated with compensatory erythrocytosis[END_REF]. But, in the Hb Olympia, the amino acid change is located in site 20 of the chain helix B and there is no relation to any of the aforementioned zones, but it is accompanied by erythrocytosis. Replacement at this level of a polar amino acid, such as valine, by a hydrophobic one like methionine will most likely indirectly determine changes in some of those critical zones [START_REF] Stamatoyannopoulos | Hemoglobin Olympia (20 valine leads to methionine): an electrophoretically silent variant associated with high oxygen affinity and erythrocytosis[END_REF]. In this sense, another two haemoglobin variants affecting the site 20 of β chain Hb Trollhättan (Val→Glu) and Hb Uxbridge (Val→Gly) has been reported and in both variants there is an increase in oxygen affinity with mild erythrocytosis in the case of the Hb Trollhättan [START_REF] Wajcman | Hb Uxbridge [β20(B2)Val→Gly]: a new variant with mild increase in oxygen affinity found during a neonatal screening program[END_REF][START_REF] Landin | Hb Trollhattan [β20(B2) Val®Glu] a new haemoglobin variant with increased oxygen affinity causing erythrocytosis[END_REF]. In any event, an amino acid change, even through a different process, helps the haemoglobin quaternary structure to be stabilised in its relaxed configuration, thus reducing oxygen release and therefore lowering P 50 in the oxygen dissociation curve.

Up to now, 89 haemoglobin variants that show high affinity for oxygen have been described: 18 in the α chain and 71 in the β one [4]. But two thirds of them are not accompanied by erythrocytosis, either because the affinity increase is slight or moderate and only found during in vitro studies or when molecular instability is present too, determining a concomitant haemolysis or if the mutating gene expression is low, as occurs in the α chain variations, or is reduced, as in Hb Crete [11]. Some erythrocytosis was found in all the patients reported in this paper or had been referred to our laboratory for that reason. But of the ten haemoglobinopathies, only one was found in an α chain variant. In a family affected by Hb Johnstown, an associated β 0 thalassaemia was found in two members due to IVS-1-nt1 (G→A) mutation. For this reason, all Hb was virtually Hb Johnstown in these patients. Most of the high-affinity haemoglobinopathies appear in heterozygotic individuals, while homozygotic cases are not reported, except for the Hb Tarrant found in an α chain and, for this reason, only 50% was quantified [START_REF] Ibarra | Heterozygosity and homozygosity for the high oxygen affinity hemoglobin Tarrant or alpha 126 (H9) Asp replaced by Asn in two Mexican families[END_REF]. If the case found by our laboratory is added, high-affinity haemoglobinopathies associated to a β-thalassaemia are only found in five instances, all of them in a β chain and, for this reason, the Hb of these patients show high oxygen affinity. In such cases, as was to be expected, larger erythrocytosis levels are described, but significant clinical complications were not reported [11]. The notion that the homozygotic condition of variants with high oxygen affinity is incompatible to life can be questioned based on the aforementioned cases.

Erythrocytosis due to high-affinity haemoglobinopathies are usually fairly well tolerated by young patients, although thrombotic complications have been reported in elderly patients or when other vascular hazard factors are associated. Thrombotic complications have also been described for unstable variants that determine changes in the erythrocytic membrane of splenectomised patients, but none of the 34 patients in the study had either any acute clinical condition nor any thrombotic history [START_REF] Dash | Late emergence of polycythemia in a case of Hb Chandigarh β94(FG1)Asp→Gly[END_REF][START_REF] Kister | Hb Montfermeil [β130(H8)Tyr→Cys]: suggests a key role for the interaction between helix A and H in oxygen affinity of the hemoglobin molecule[END_REF].

Although the high-affinity variants could in animal models determine an increase in foetal losses during pregnancy due to reduced oxygen release in the placenta, an increase in foetal losses has not been reported in families affected by high-affinity haemoglobinopathies.

Evidence of the pathological variant must be produced to diagnose secondary erythrocytosis in high-affinity haemoglobin variants. But over 30 of the aforementioned haemoglobinopathies remain undetected during conventional electrophoretic studies [11] for which reason chromatographic and molecular biology techniques have to be applied. In order to show the variant's high affinity for oxygen, however, a functional haemoglobin study is required. In all the patients studied, P 50 was low but, in six of the ten variants found, the findings of electrophoretic and HPLC ionic-exchange studies were normal and only haemoglobin variants were disclosed by the reversed-phase HPLC.

A haemoglobin variant that shows high affinity for oxygen must be the suspected aetiology in families that report a history of erythrocytosis or young people, when there are no apparent reasons for this. Although it is rarely diagnosed, the 17 families reported that display ten different variants in the same geographic area lead us to think that the percentage of secondary erythrocytosis in haemoglobinopathies of this type could be even higher and therefore their systematic detection should be emphasised in erythrocytosis of unknown origin, more so when a family history is available.

Table 1

 1 Haemoglobin variants with high oxygen affinity diagnosed in the Service of Haematology of Hospital Clínico San Carlos

	Molecular mutation	Hb Olympia [β20(B2) Val→Met; GTG→ATG]	Hb Syracuse [β143(H21)His→Pro; CAC→CCC]	Hb San Diego [β109(G11)Val→Met; GTG→ATG]	Hb San Diego [β109(G11)Val→Met; GTG→ATG]	Hb San Diego [β109(G11)Val→Met; GTG→ATG]	Hb San Diego [β109(G11)Val→Met; GTG→ATG]	Hb Johnstown [β109(G11)Val→Leu; GTG→CTG]	Hb Johnstown [β109(G11)Val→Leu; GTG→CTG]	Hb Johnstown [β109(G11)Val→Leu; GTG→CTG]	Hb Johnstown [β109(G11)Val→Leu; GTG→CTG]	Hb Johnstown + β 0 Tal IVS-1-nt1 (G→A)	Hb Bethesda [β145(HC2)Tyr→His; TAT→CAT]	Hb Badalona [β31(B13)Leu→Val; CTG→GTG]	Hb Strasbourg [β23(B5)Val→Asp; GTT→GAT]	Hb Malmö [β97(FG4)His→Glu; CAC→CAA ]	Hb Columbia-Missouri [α2 88(F9) Ala→Val; GCG→GTG]	Hb La Coruña [β38 (C4)Thr®Ile; ACC→ATC]
	HPLC chains	β X ; β A ; α	β X ; β A ; α	β X ; β A ; α	β X ; β A ; α	β X ; β A ; α	β X ; β A ; α	β A ; β X ; α	β X ; β A ; α	β X ; β A ; α	β X ; β A ; α	β X ; α. No β A	β X ; β A ; α	β X ; β A ; α	β X ; β A ; α	β A ; β X ; α	β A ; α A ; α X	β A ; β X ; α
	HPLC Hb	N	HbX/HbA	N	N	N	N	N	N	N	N	N	HbX/HbA	HbX/HbA	N	HbX/HbA	N	N
	P 50 (mmHg)	13.2	11	21-22	20	14	15	17-19	20	16-19	14-19	13-15	12.5	22	21	17	16-21	16-17
	Hto (%)	58	63.7	44-52	60.2	48.2	55	53-61	45.8	46.6-50.2	53-57.5	60	49.2	48-49	49.5	55	50-55	50-60
	Hb (g/dL)	20	21.3	13-17	18.6	16.7	18.4	17-19	15.3	14.2-17	17.5-19.6	18-19	17.3	17	15.9	18.4	16-18	17-20
	No of patients (age/sex)	1 (50/V)	1 (25/V)	3 (54/V; 26/M; 19/V)	1 (20/M)	1 (52/M)	1 (22/V)	2 (74/V; 47/M)	1 (32/M)	4 (41/M; 11/M; 9/M; 5/M)	4 (52/V; 26/M; 23/V; 21/V)	2 (30/V; 27/M)	2 (23/V; 53/V)	3 (17/M; 35/M; 666/M)	1 (66/V)	1 (25/V)	4 (66/M; 59/V; 35/V; 29/M)	2 (22/V;50/M)
	Fam	1 a	2 a	3 b	4 a	5 a	6 a	7 b	8 a	9 b	10 b	11 a	12 b	13 c	14 a	15 a	16 b	17 b

a One generation b Two generations c Three generations

Acknowledgments The following investigators and clinical departments belonging to the Erythropathology Cooperative Spanish Group have contributed to this study: A. del Arco (H. Doctor Pesset, Valencia); J.M. Vegace (H. Infanta Cristina, Badajoz); K. Atutxa (H. de Galdakao, Bilbao); B. Arrizabalaga (H. de Cruces, Bilbao); M. Ortega (H. Río Ortega, Valladolid); J. Juncá (H. Universitari Germans Trias i Pujol, Badalona); G. Martín-Núñez (H. Virgen del Puerto, Plasencia); A. Remacha (H. San Pau, Barcelona); I. San Roman (H. General Universitario de Guadalajara); P. de Andrés (H. Reína Sofia, Córdoba); S. Garzón (H. General de Jerez); B. Pérez de Mendiguren (H. General Yagüe, Burgos); C. Fernández Lago (H. Universitario Juan Canalejo, A Coruña); J. del Río (H. la Barbanza, A Coruña); M. Barbón (H. Virgen Blanca, León).

Funding from the European Commission for the "eInfrastructure for Thalassaemia Research Network" Coordination Action-Contract no 026539-for which this study was partially funded is gratefully acknowledged.


Georgia Metzgeroth

Dietmar Dinter

Beate Schultheis

Alexandra Dorn-Beineke

Kira Lutz

Oliver Leismann

Rüdiger Hehlmann

Jan Hastka
email: jan.hastka@med3.ma.uni-heidelberg.de

: R Hehlmann

Deferasirox in MDS patients with transfusion-caused iron overload-a phase-II study

Keywords: Myelodysplastic syndromes, Transfusional iron overload, Iron chelator, Deferasirox, LIC assessment by MRI

Blood transfusions represent a main component of supportive care in myelodysplastic syndromes (MDS). To avoid organ damage caused by transfusion-dependent iron overload, an adequate iron chelation therapy is required. Recently, a new oral iron chelator deferasirox (ICL670, Exjade®) has become available. A study was conducted to demonstrate the efficacy and tolerability of deferasirox in transfusion-dependent iron-overloaded patients with MDS. The efficacy of deferasirox was monitored by changes in serum ferritin, bone marrow iron, and liver iron concentration (LIC), as determined by T2*weighted magnetic resonance imaging. Twelve patients with MDS of different subtypes (median age 76 years, range 53-91) were enrolled. Deferasirox administered in a once-daily dose of 20-30 mg/kg for 12 months was effective in reducing median ferritin concentration from 1,515 µg/L (range 665-6,900) to 413 µg/L (range 105-3,052). Within the first 4 weeks of treatment before the continuous decline of ferritin levels, the values markedly rose in eight of 12 patients. The median LIC declined from 315 to 230 µmol/g (p = 0.02) at the end of study, accompanied by a reduction of bone marrow siderosis. The most common adverse events were mild and transient gastrointestinal disturbances, skin rash, nonprogressive transient increases in serum creatinine and urine β2-microglobulin, and a temporary reduction of the creatinine clearance. The renal parameters normalized after end of treatment. No hematologic toxicities were observed. Deferasirox proved to be effective in transfusion-dependent iron overload in MDS by mobilizing iron deposits in liver and at least stabilizing iron stores in bone marrow.

Introduction

The myelodysplastic syndromes (MDS) are clonal disorders of hematopoiesis which are characterized by dysplasia in one or more cell lineages and peripheral cytopenia. For the management of MDS patients, blood transfusions represent the main component of supportive care. Ninety percent of all MDS patients with permanent anemia become dependent on blood transfusions to maintain their quality of life and to survive. With every unit of transfused blood, the patients receive approximately 200 to 250 mg iron [START_REF] Porter | Practical management of iron overload[END_REF]. As there is no physiological mechanism to remove an excess of body iron, regular blood transfusions inevitably lead to an accumulation of iron in various tissues in these individuals. If untreated, iron overload may cause damages in liver, heart, pancreas, brain, joints, and endocrine tissues [START_REF] Britton | Iron toxicity and chelation therapy[END_REF][START_REF] Kushner | Secondary iron overload[END_REF][START_REF] Mclaren | Iron overload disorders: natural history, pathogenesis, diagnosis, and therapy[END_REF], increasing morbidity and the risk of early death [START_REF] Porter | Practical management of iron overload[END_REF]. As phlebotomy is not an option in MDS, the only way to remove toxic iron and to prevent complications of transfusion-dependent iron overload is, to date, rigorous iron chelation therapy [START_REF] Jensen | The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload[END_REF].

The current standard in iron chelation therapy is deferoxamine mesylate (Desferal®). Long-term data collected since the drug was introduced in the early 1960s have demonstrated its safety and high efficacy in reducing stored iron and the complications of iron burden, resulting in decreased mortality in iron-overloaded subjects [START_REF] Aessopos | Cardiac status in welltreated patients with thalassemia major[END_REF][START_REF] Wolfe | Prevention of cardiac disease by subcutaneous deferoxamine in patients with thalassemia major[END_REF]. Unfortunately, deferoxamine has a short plasma half-life and an insufficient oral bioavailability. These features require an intravenous or subcutaneous application of the drug over a period of 8 to 12 h, usually five to seven times a week. Alternatively, deferoxamine can be administered as a subcutaneous bolus injection [START_REF] Borgna-Pignatti | Subcutaneous bolus injection of deferoxamine in adult patients affected by onco-hematologic diseases and iron overload[END_REF]. As a consequence of the cumbersome administration, the compliance of the deferoxamine therapy is often poor, resulting in limited efficacy. In MDS patients, the applicability of deferoxamine is further limited by the fact that the anemia is often associated with severe thrombocytopenia and neutropenia in these individuals.

Due to the risk of bleeding and infection, orally applicable iron chelators are of particular interest in transfused MDS patients. The first oral iron chelator deferiprone (Ferriprox®), introduced already 20 years ago, did not fulfill the expectations. The license of the drug remained limited to second-line therapy of patients with thalassemia major, bearing the risk of neutropenia and agranulocytosis in a frequency of about 0.5-1% [START_REF] Ceci | The safety and effectiveness of deferiprone in a large-scale, 3-year study in Italian patients[END_REF][START_REF] Cohen | Safety profile of the oral iron chelator deferiprone: a multicentre study[END_REF][START_REF] Hoffbrand | Role of deferiprone in chelation therapy for transfusional iron overload[END_REF].

Deferasirox (ICL670, Exjade®) is a once-a-day orally active iron chelator formulated as a dispersible tablet with high iron-binding potency and iron selectivity [START_REF] Cappellini | A phase 3 study of deferasirox (ICL670), a oncedaily oral iron chelator, in patients with beta-thalassemia[END_REF][START_REF] Cappellini | Iron-chelating therapy with the new oral agent ICL670 (Exjade)[END_REF][START_REF] Hershko | ICL670A: a new synthetic oral chelator: evaluation in hypertransfused rats with selective radioiron probes of hepatocellular and reticuloendothelial iron stores and in iron-loaded rat heart cells in culture[END_REF]. Deferasirox binds iron in a 2:1 ratio before being excreted primarily in the bile [START_REF] Nick | Development of tridentate iron chelators: from desferrithiocin to ICL670[END_REF]. A series of studies have demonstrated its efficacy and safety in patients with thalassemia major in a daily dose ranging between 20 and 30 mg/kg [START_REF] Cappellini | A phase 3 study of deferasirox (ICL670), a oncedaily oral iron chelator, in patients with beta-thalassemia[END_REF][START_REF] Galanello | Phase II clinical evaluation of deferasirox, a once-daily oral chelating agent, in pediatric patients with betathalassemia major[END_REF][START_REF] Galanello | Safety, tolerability, and pharmacokinetics of ICL670, a new orally active iron-chelating agent in patients with transfusiondependent iron overload due to beta-thalassemia[END_REF][START_REF] Nisbet-Brown | Effectiveness and safety of ICL670 in iron-loaded patients with thalassaemia: a randomised, doubleblind, placebo-controlled, dose-escalation trial[END_REF][START_REF] Piga | Randomized phase II trial of deferasirox (Exjade, ICL670), a once-daily, orally-administered iron chelator, in comparison to deferoxamine in thalassemia patients with transfusional iron overload[END_REF][START_REF] Porter | Clinical evaluation of deferasirox (Exjade((R)), ICL670)[END_REF][START_REF] Porter | Deferasirox: an effective once-daily orally active iron chelator[END_REF]. So far, experiences regarding treatment with deferasirox in individuals with diseases other than thalassemia are only limited [START_REF] Porter | Clinical evaluation of deferasirox (Exjade((R)), ICL670)[END_REF][START_REF] Porter | Deferasirox: an effective once-daily orally active iron chelator[END_REF][START_REF] Goldberg | Novel treatment options for transfusional iron overload in patients with myelodysplastic syndromes[END_REF].

The aim of the present study was to investigate the efficacy and tolerability of deferasirox in transfusiondependent iron-overloaded patients with MDS.

Patients and methods

The phase-II study was carried out between March 2004 and November 2006 in the III. Medizinische Universitätsklinik, Mannheim, Germany. The aim of the study was to investigate the tolerability and effectiveness of deferasirox (ICL670, Exjade®) in MDS patients with transfusion-induced iron overload. The efficacy of deferasirox was monitored by changes in serum ferritin, bone marrow iron, and liver iron concentration (LIC), the latter parameter determined quantitatively by magnetic resonance imaging (MRI).

MDS patients who received at least 20 blood transfusions and who had a ferritin concentration >500 µg/L were enrolled into the study, irrespective of prior chelation therapy, age, and MDS subtype. Reasons for exclusion were: alanine aminotransferase (ALAT) >250 U/L, increased serum creatinine concentration (>1.3 mg/dL), proteinuria >1,000 mg/day, acute or chronic viral hepatitis, HIV, prolonged corrected QT interval, AV block II-III°, a history of ocular toxicity related to iron chelation therapy, gastrointestinal conditions preventing absorption of an oral medication, alcohol abuse, or chemotherapy. The diagnosis of MDS was performed according to the World Health Organization (WHO) recommendations [START_REF] Jaffe | Pathology and genetics of tumours of haematopoietic and lymphoid tissues[END_REF] by morphological evaluations of a May-Grünwald-Giemsa-stained peripheral blood and bone marrow smears, Prussian blue staining of bone marrow, immunotyping, and cytogenetic analysis.

Treatment with deferasirox was started in all patients at a dose of 20-mg/kg body weight. The drug was given once daily each morning as a suspension in water, half an hour before breakfast. The dose was escalated for 5 mg/kg every 3 months if the ferritin level decreased less than 10% during this period. The maximal allowed dose was 30 mg/ kg. The therapy was discontinued when ferritin decreased below 350 µg/L and reinstituted at 20 mg/kg when ferritin concentration exceeded 500 µg/L again. Treatment was scheduled for 12 months but could be extended at the physicians' discretion when patients had achieved a reduction of iron overload. Clinical response was defined as a reduction of ferritin concentration of at least 10% as compared to baseline values. Blood transfusions were regularly administered during the study period according to the patient's requirement.

Assessments for safety and efficacy were performed at baseline, weekly in the first month after study initiation and after each dose escalation and at monthly intervals afterwards. The check included physical examination and extended laboratory testing including complete blood cell count, serum analysis of electrolytes, zinc, copper, liver enzymes, creatinine, β2-microglobulin, and urine analysis of protein, creatinine, and β2-microglobulin. Iron parameters tested included the serum analysis for ferritin, iron, transferrin, transferrin saturation, and soluble transferrin receptor. The serum ferritin concentration (normal value 35-310 μg/L for men, 25-210 μg/L for women) was measured by an enzyme immunometric assay (Access Ferritin immunoassay, Beckmann-Coulter, Krefeld, Germany) based on enhanced luminescence. Electrocardiograms and ophthalmologic and auditory examinations were performed every 3 months.

Iron stores were monitored by assessment of the iron content in the bone marrow, liver, and heart at baseline and every 3 months during the study. Bone marrow iron stores were assessed semiquantitatively on Prussian-blue-stained marrow smears according to the following scale: grade 0= absent; 1=decreased; 2=normal; 3=increased; 4=markedly increased; 5=massively increased. LIC was determined quantitatively by T2*-weighted MRI as described previously [24][START_REF] Wood | MRI R2 and R2* mapping accurately estimates hepatic iron concentration in transfusion-dependent thalassemia and sickle cell disease patients[END_REF][START_REF] Alexopoulou | R2 relaxometry with MRI for the quantification of tissue iron overload in beta-thalassemic patients[END_REF]. An LIC >35-µmol/g liver tissue was considered as iron overload. The changes in myocardial siderosis were quantified by myocardial T2* MRI [START_REF] Pennell | Randomized controlled trial of deferiprone or deferoxamine in beta-thalassemia major patients with asymptomatic myocardial siderosis[END_REF][START_REF] Wood | Cardiac iron determines cardiac T2*, T2, and T1 in the gerbil model of iron cardiomyopathy[END_REF]. The lower the T2* value in milliseconds (ms), the greater the myocardial iron load (normal value >35 ms; pathologic <25 ms, standard deviation ±20) [START_REF] Tanner | A randomized, placebo-controlled, doubleblind trial of the effect of combined therapy with deferoxamine and deferiprone on myocardial iron in thalassemia major using cardiovascular magnetic resonance[END_REF][START_REF] Westwood | Intercentre reproducibility of magnetic resonance T2* measurements of myocardial iron in thalassaemia[END_REF].

All statistical analyses were performed using the Graph-Pad Prism (Version 4.0) statistical package for Windows. Testing for statistical significance between baseline and end-of-study values was performed using the Wilcoxon matched pairs test, p values<0.05 were considered to be statistically significant. Correlation coefficients were determined by Pearson's correlation test.

The study was approved by the local ethics committee and followed the Helsinki Declaration of 1975, as revised in 1983. Written informed consent was given by all patients.

Results

Twelve MDS patients (RA= 4, RARS = 1, RCMD = 2, RAEB-1=1, 5q -syndrome=4) with transfusion-dependent iron overload were enrolled in this study. The median age of the patients (four males, eight females) was 76 years (range 53-91); nine patients (75%) were older than 70 years. All patients were in a stable-disease phase when entering the study. Median time from diagnosis of MDS to study entry was 15.5 months (range 2-182). From time of diagnosis to study entry, the patients received a median of 36 units (range 20-214) of red blood cells (RBC).

Patient characteristics are shown in Table 1. The individual values of serum ferritin concentration, number of RBC transfusions, dose of deferasirox, LIC, and bone marrow iron in the course of the study are demonstrated in Fig. 1.

Seven patients (nos. 1-7) were treated for 12 months with deferasirox. In the remaining five patients, the study had to be stopped after a median time of 4.7 months (range 1.0-7.5) due to reasons most probably not related to deferasirox therapy. A 54-year-old patient (no. 8) was withdrawn from the study 7.5 months after start of the therapy for allogeneic stem cell transplantation. Another patient (no. 9) developed acute myeloid leukemia 4.9 months after start of the treatment. An 80-year-old woman (no. 10) died from pneumonia 1 month after start with deferasirox. Two patients (nos. 11 and no. 12) died 4.7 and 2.2 months after initiation of the study from fatal bleeding. The bleeding was the result of a nonprogressive thrombocytopenia in one case; the other patient, a 91-yearold male patient (no. 11), died from gastrointestinal bleeding caused by sigma diverticulosis with normal platelet counts. None of the fatal cases was considered as study-drug-related.

In three of the seven patients who completed the study, the deferasirox medication had to be discontinued for adverse events. In one case (no. 1), treatment was interrupted due to pneumonia for 2 weeks. In another patient (no. 5), study medication was stopped for 1 week because of a mild skin rash, which resolved spontaneously. In another case (no. 7), deferasirox was withdrawn after 10 months as this patient developed a severe persistent osteomyelitis of the humerus following an osteosynthesis 1 year before study entry. In the remaining patients, no clinically apparent infection were observed.

Adverse events observed were infrequent and of mild intensity. Nausea was seen in two patients; two patients developed diarrhea occurring early after start of treatment or dose escalation. These symptoms resolved spontaneously without dose interruption in all cases. One patient developed a self-limiting atrial tachycardia after 2 months of treatment at a daily deferasirox dose of 20 mg/kg. Two patients developed a mild hyperbilirubinemia (1.26 and 1.87 mg/dL) which resolved without discontinuation of the study medication. One patient showed a progressive elevation in transaminase levels (ALAT maximal 426 U/L), which lasted for 2 weeks. No abnormalities in serum concentrations of potassium, sodium, or calcium were observed. Serum copper and zinc levels fluctuated during study without decreasing markedly. Hematological parameters were not significantly changed during the study period.

Increased serum creatinine levels (normal 0.7-1.3 mg/dL) were detected in seven patients during the study period, with a maximum of 1.56 mg/dL occurring 1 to 20 weeks after start of treatment at a dose of deferasirox of 20 mg/ kg (n=5) and 25 mg/kg (n=2), respectively. Three of these patients were the only study participants with a pretherapeutic decreased creatinine clearance below 60 mL/min (nos. 5, 11 and 12). The observed creatinine increase was dose independent and transient without requiring any dose adjustment. Eight of 12 patients had a serum creatinine clearance below 100 mL/min at the start of study. During the study period, all patients showed a serum creatinine clearance below 100 mL/min at least at one laboratory check, seven patients a value below 60 mL/min. In all cases, the creatinine clearance returned to at least pretherapeutic values at one of the subsequent revaluation under continued deferasirox treatment.

Eleven patients showed an increase of urine β2-microglobulin at least once in the course of the study. In eight patients, the increase persisted during the whole study. The values observed in the individual patients showed an enormous variability even with unchanged deferasirox dose and normalized in all patients within a few weeks after the study medication was terminated. The median β2-microglobulin concentration in the urine obtained 3 months after study initiation was significantly higher when compared to the baseline value (2,605 vs. 200 mg/L; p=0.02).

The median serum ferritin concentration of all patients at start of study was 1,575 µg/L (range 665-6,900). Within the first 4 weeks, in eight of 12 patients (67%), an increase of the ferritin concentration was observed from a median of 1,382 µg/L (range 665-6,900) to a median of 1,834 µg/L (range 1,093-6,975; p=0.4). In six of these individuals, the increase was detected already at the first control after 1 week of treatment. The highest ferritin levels were measured 4 weeks after the initiation of treatment, being increased by 26.4% (range 8.4-165) when compared to the pretreatment values. In the seven patients receiving deferasirox for 12 months, the median ferritin values decreased by a median of 67.5% (range 20.0-94.7) from 1,575 µg/L (range 665-6,900) to 413 µg/L (range 105-3,052). The observed reduction was not significant due to the rise of ferritin within the first 4 weeks of treatment. However, when comparing the baseline values with the ferritin values obtained after the first 4 weeks of treatment, the difference became significant (1,483 µg/L, range 577-6,975; p=0.017). There were no consistent changes in the levels of other iron tests such as Treatment had to be discontinued after 4.7 months in one patient (no. 6) who showed an excellent response to deferasirox at a dose of 20 mg/kg, with a decrease of ferritin to 190 µg/L. After withdrawal of the chelation therapy due to iron depletion, the serum ferritin rose subsequently to 515 µg/L. This process was paralleled by an increase of iron deposits in bone marrow and liver (Table 1 and Fig. 1).

Bone marrow storage quantified by Prussian blue staining showed a continuing reduction from the median scale of grade 5 (range 4-5) to 3 (range 1-5, p=0.002) at month 12. A decrease of hemosiderin was observed in six of seven patients who received deferasirox for 12 months and in one other case who received chelation therapy for 6 months. LIC as determined by T2*-weighted MRI fell significantly from pretreatment values of 315-µmol/g liver tissue (range 180-350) to 230-µmol/g liver tissue (range 0-350; p=0.015) at month 12. Within the first 6 months, LIC decreased by an average of 13% (range 0-55%). There was no correlation found between serum ferritin levels and the LIC.

In six of eight patients with available cardiac data, the T2* relaxation time increased as a marker of improvement in myocardial iron loading. In two of these patients, values even normalized (T2* relaxation time >35 ms) within 12 months. The lowest cardiac iron concentration was observed after 6 months of the treatment.

Discussion

The presented results show that deferasirox is an effective oral alternative for iron chelation therapy in MDS patients with secondary hemosiderosis. This is obvious from dynamics of serum ferritin and LIC in the individual patients.

Serum ferritin concentration mirrors the total body iron load and is a well-established test to monitor the effect of chelation therapy in transfusional iron overload. In all but one of our MDS patients who finished the study as planned and were treated with deferasirox for 12 months, ferritin concentrations at the end of the study were below baseline values. There was, however, a surprising phenomenon observed during the initiation of treatment: instead of a decrease, there was rather an increase of ferritin observed in about 70% of the patients during the first 4 weeks of deferasirox therapy. The rise was mostly detectable already 1 week after the medication was initiated. The highest ferritin values were measured after 4 weeks, being increased by 26.4% on average when compared to the pretreatment values. A continuous decline of the ferritin right from the start of study without an intermittent rise was observed only in one patient with a low iron load, as demonstrated by a low initial ferritin of 743 mg/L, only moderately increased LIC of 230 µmol/g, and the lack of transfusion requirement during the first 3 months of study.

The initial increase of ferritin observed in our study is of general interest for the management of patients with iron chelation therapy-at least for those receiving deferasirox. Normally, an increase of ferritin in serum leads to dose escalation as it is interpreted as a lack of efficacy. This was also the case in our study design, where the deferasirox dose had to be escalated when the ferritin levels did not decline by at least 10% compared to the ferritin levels obtained at the previously administered dose. Based on our results, which showed a continuous decline of ferritin in responders not before 6-8 weeks after start of treatment, we recommend dose adjustment not until the third month of treatment. This does not only apply for the initial therapy but also for dose escalations later on.

The measurement of LIC by highly T2*-weighted MRI was demonstrated to correlate well with the iron content assessed by invasive liver biopsy [24,[START_REF] Pennell | Randomized controlled trial of deferiprone or deferoxamine in beta-thalassemia major patients with asymptomatic myocardial siderosis[END_REF][START_REF] Rose | Liver iron content assessment by routine and simple magnetic resonance imaging procedure in highly transfused patients[END_REF]. Iron determination by MRI is a reproducible, sensitive, and noninvasive method that can reduce the need for liver biopsies to assess liver iron content especially in thrombocytopenic patients. The changes of LIC during the course of our study confirm the high efficacy of deferasirox to mobilize iron deposits from liver tissue [START_REF] Cappellini | A phase 3 study of deferasirox (ICL670), a oncedaily oral iron chelator, in patients with beta-thalassemia[END_REF][START_REF] Greenberg | Myelodysplastic syndromes: iron overload consequences and current chelating therapies[END_REF][START_REF] Vichinsky | A randomised comparison of deferasirox versus deferoxamine for the treatment of transfusional iron overload in sickle cell disease[END_REF]. Given the fact that the primary goal of chelation therapy is to reduce iron deposits, LIC assessment by MRI seems to be the ideal tool to monitor the efficacy of chelation. In contrast to ferritin, which only indirectly reflects the iron stores and which is influenced by inflammation and liver disorders and obviously also by the chelation therapy itself, the MRI reliably allows a direct control of LIC. Regarding our results which show a LIC decline in responders already after 3-6 months, it is obviously possible to check the success of treatment by this method as early as 3-6 months after start of chelation therapy. From the clinical point of view, before escalating the dose of deferasirox, it may be recommendable to reevaluate body iron by LIC instead of relying on the course of ferritin only.

MRI is not only of interest to determine LIC. A number of studies have demonstrated that by measurement of T2* relaxation time MRI may also be eligible to measure iron overload in the heart, the most critical organ in this respect [START_REF] Wood | Cardiac iron determines cardiac T2*, T2, and T1 in the gerbil model of iron cardiomyopathy[END_REF][START_REF] Mavrogeni | A comparison of magnetic resonance imaging and cardiac biopsy in the evaluation of heart iron overload in patients with beta-thalassemia major[END_REF][START_REF] Pennell | MRI and iron-overload cardiomyopathy in thalassaemia[END_REF]. Deferasirox is assumed to be inferior to deferoxamine and deferiprone for the removal of excess cardiac iron [START_REF] Kontoghiorghes | Deferasirox: uncertain future following renal failure fatalities, agranulocytosis and other toxicities[END_REF][START_REF] Wood | Deferasirox and deferiprone remove cardiac iron in the iron-overloaded gerbil[END_REF][START_REF] Kontoghiorghes | Effects of ICL670 (deferasirox) on cardiac iron concentrations[END_REF], but there are no comparative data that exist in this regard. Removal of cardiac iron was shown experimentally in animals with high doses of deferasirox (100 mg/kg daily) but not in patients at doses of 10-40 mg/kg [START_REF] Wood | Deferasirox and deferiprone remove cardiac iron in the iron-overloaded gerbil[END_REF][START_REF] Kontoghiorghes | Effects of ICL670 (deferasirox) on cardiac iron concentrations[END_REF]. In our study, six out of eight patients with available cardiac data showed a decline of myocardial iron by the end of study. Interestingly, the lowest cardiac iron values were observed at month 6. At this time, seven of the eight patients improved when compared to baseline; in four patients, the iron amount even normalized. The reason for an increase of cardiac iron concentration in most cases during the second half of our study despite dose escalation of deferasirox remains unclear.

The most frequent adverse events with suspected relationship to repeated administration of deferasirox consisted of transient WHO grades I-II gastrointestinal symptoms such as nausea and diarrhea, similarly found in other studies [START_REF] Nisbet-Brown | Effectiveness and safety of ICL670 in iron-loaded patients with thalassaemia: a randomised, doubleblind, placebo-controlled, dose-escalation trial[END_REF][START_REF] Porter | Deferasirox: an effective once-daily orally active iron chelator[END_REF][START_REF] Maggio | Light and shadows in the iron chelation treatment of haematological diseases[END_REF][START_REF] Porter | Relative response of patients with myelodysplastic syndromes and other transfusion-dependent anaemias to deferasirox (ICL670): a 1-yr prospective study[END_REF]. These common side effects may be partly caused by the presence of lactose in the formulation of deferasirox [START_REF] Vichinsky | A randomised comparison of deferasirox versus deferoxamine for the treatment of transfusional iron overload in sickle cell disease[END_REF] and did not require treatment discontinuation in our patients.

Infectious complications observed in our study were most probably not related to the study drug but rather the result of the bone marrow suppression in MDS. Previous studies on thalassemia patients have demonstrated that infections were similarly frequent in iron chelation therapy with deferasirox and deferoxamine [START_REF] Vichinsky | A randomised comparison of deferasirox versus deferoxamine for the treatment of transfusional iron overload in sickle cell disease[END_REF]. The induction of infections through iron chelation therapy would be rather surprising, as a withdrawal of iron is considered as a nonspecific defense mechanism and a common response to systemic infections or generalized inflammatory disorders [START_REF] Weiss | Pathogenesis and treatment of anaemia of chronic disease[END_REF].

Preclinical trials indicated that the kidney would be the organ at risk for deferasirox toxicity. The main toxic effects described were changes of renal tubular cells. The nephrotoxicity was regarded to be caused by decompartmentalization of chelated iron and had been observed in animals that were not heavily iron-overloaded [START_REF] Nick | Development of tridentate iron chelators: from desferrithiocin to ICL670[END_REF]. Based on these observations, a creatinine clearance of <60 mL/ min was determined as an exclusion criterion for deferasirox treatment as recommended by the manufacturer. This is no problem in the cases of young thalassemia patients but of concern in patients with MDS, which is primarily a disease of the elderly, with a median age of 65-75 years [START_REF] Aul | Increasing incidence of myelodysplastic syndromes: real or fictitious?[END_REF]. The creatinine clearance is mostly reduced due to a physiological reduction of kidney weight and a reduced number of glomerula. Several studies have shown that the creatinine clearance allows only a poor estimation of renal function in older individuals. Renal function is often underestimated by this parameter [START_REF] Burkhardt | Creatinine clearance, Cockcroft-Gault formula and cystatin C: estimators of true glomerular filtration rate in the elderly?[END_REF][START_REF] Waller | The accuracy of creatinine clearance with and without urine collection as a measure of glomerular filtration rate[END_REF][START_REF] Walser | Assessing renal function from creatinine measurements in adults with chronic renal failure[END_REF] due to erroneous urine sampling, reduced muscle mass because of reduced physical activity, and low calorie intake in elderly people.

In concordance with studies on patients with thalassemia [13, 16-19, 21, 39], we have shown that the renal function is not permanently affected by deferasirox even in elderly patients with MDS. This was the case in patients both with normal renal function and with initially reduced creatinine clearance. Even in patients with a creatinine clearance of <60 mL/h at baseline, further impairment of the renal parameters was only transitional, with parameters at the end of the study being comparable to pretherapeutic values.

The determination of the β2-microglobulin levels in the urine, considered to be a sign of tubular damage, proved to be especially problematic in this context. Increased values were found in almost all study participants, showing an enormous day-to-day variability with the same deferasirox doses. At the end of the study, the concentrations normalized. Thus, the value of the β2-microglobulin measurement as a sign of tubular damage during deferasirox therapy remains debatable.

Which is the optimal deferasirox dose for treatment of iron overload in patients with MDS? The required dose obviously depends primarily on the number of transfusions required to maintain a stable hemoglobin level. As our results show, a deferasirox dose of 20 mg/kg daily can reduce LIC in many patients who need up to four RBC units per month. There is no reason not to start chelation therapy with this low dose to minimize costs and possible side effects. An increase of the dose by 5 mg/kg is justifiable only after a period of 3 months as described above. The maximum dose of 30 mg/kg as recommended by the manufacturer should not be exceeded, as the use of higher doses in our experience is not necessary.

In contrast to other studies [START_REF] Jensen | The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload[END_REF], we could not observe any significant changes in hemoglobin levels and RBC requirements, no patient became transfusion independent. The lack of improvement of the erythropoietic activity is possibly explained by the relatively low number of patients in our study. Furthermore, the relatively short period of chelation treatment may also contribute to this result, as the hematological improvement was reported to become evident mostly after 12 months [START_REF] Jensen | The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload[END_REF].

The question of which patients should receive chelation therapy is a matter of debate, which is of course influenced by clinical and economical aspects [START_REF] Tefferi | Iron chelation therapy for myelodysplastic syndrome: if and when[END_REF]. Since iron overload has no positive effect but impairs organ functions, chelation therapy has to be considered necessary for all patients whose life expectancy is longer than the time period required for a measurable decrease in tissue iron overload. In light of our data, which show a significant reduction of LIC after 1 year of chelating therapy, there is no reason why patients with a life expectancy of more than 1 year should be deprived of iron chelators beside economical considerations. The consensus conference of Nagasaki [47] recommends chelation therapy for all patients with ferritin levels exceeding 1,000-2,000 μg/L.

In summary, deferasirox is an oral alternative for iron chelation in MDS patients. The drug is well tolerated and effective in decreasing iron overload and in mobilizing iron deposits from liver tissue. The starting dose is 20 mg/kg; dose escalation should be considered not before 3 months of treatment. Suitable parameters of treatment efficacy are serum ferritin and assessment of liver iron by MRI.

Fig. 1

 1 Fig.1 (continued)

Table 1

 1 Clinical and laboratory characteristics of all study participants (n=12). Values are presented as median (range)

	Patient characteristics
	Median age (years)


Hrishi Mittal

Lara Roberts

Gary W Fuller

Sandra O'driscoll

Moira C Dick

Sue E Height

Swee Lay

David C Rees
email: david.rees@kch.nhs.uk

S L Thein

The effects of air quality on haematological and clinical parameters in children with sickle cell anaemia

Keywords: Sickle cell anaemia, Air quality, Nitric oxide, Transcranial Doppler scan, Haemolysis

Sickle cell anaemia (SCA; HbSS) is characterised by its clinical variability, which is only partly explained by known genetic factors. Environmental factors are known to contribute to acute problems but their importance in chronic complications has not been analysed. We have studied 93 children with SCA in a single institution, who underwent transcranial Doppler scanning and steady-state blood tests in 2006. These data were correlated with each individual's exposure to pollution from dust (PM 10 ), nitric oxide (NO) and nitrogen dioxide (NO 2 ). This exposure was derived from patient postcodes and detailed street-level maps of average pollutant levels in 2006. All the pollutants correlated closely with each other. Increased exposure to pollution correlated with a significant reduction in total bilirubin levels, with a trend towards lower levels of lactate dehydrogenase and aspartate transaminase. There was significant correlation between extracranial internal carotid artery blood velocity and PM 10 exposure. These studies suggest that chronic exposure to air pollutants could explain some variability in SCA. The lower levels of bilirubin and other markers of haemolysis with increased exposure to air pollutants could be mediated by increased exposure to NO.

Introduction

Sickle cell anaemia (SCA) is an inherited condition characterised by episodes of acute pain, acute lung problems and progressive organ damage [START_REF] Stuart | Sickle-cell disease[END_REF]. In children, one of the main problems is the increased risk of stroke, typically associated with the narrowing of intracerebral blood vessels [START_REF] Ohene-Frempong | Cooperative study of sickle cell disease: cerebrovascular accidents in sickle cell disease: rates and risk factors[END_REF]. The clinical picture varies widely between individuals, with some children having very few problems and others being severely affected. It is not known why the disease varies so much, although genetic factors are significant and have been extensively studied [START_REF] Steinberg | Predicting clinical severity in sickle cell anaemia[END_REF]. Environmental factors are also likely to be important but have received little attention. We have recently started to analyse these effects on the large population of people with sickle cell disease in South London. We found that windy weather and low humidity were associated with increased hospital admissions [START_REF] Jones | Windy weather and low humidity are associated with an increased number of hospital admissions for acute pain and sickle cell disease in an urban environment with a maritime temperate climate[END_REF]. We have also recently shown that air quality affects acute complications of sickle cell disease, with high levels of ozone (O 3 ) causing more episodes of acute pain. Interestingly, we also found that high levels of carbon monoxide and NO were linked to fewer hospital admissions [START_REF] Yallop | The associations between air quality and the number of hospital admissions for acute pain and sickle-cell disease in an urban environment[END_REF].

Most patients with sickle cell disease in the UK and other developed countries live in urban areas with variable and often poor air quality. It seems likely that air quality has significant effects on the pathophysiology and chronic complications of SCA, as has been demonstrated for many other diseases [START_REF] Cohen | The global burden of disease due to outdoor air pollution[END_REF]. Our previous study looked only at the effect of daily variation of air quality on admission to hospital with acute pain [START_REF] Yallop | The associations between air quality and the number of hospital admissions for acute pain and sickle-cell disease in an urban environment[END_REF]. This study tests the hypothesis that mean annual exposure to PM 10 , NO and NO 2 correlates with some chronic complications in children with sickle cell disease, including frequency of hospital admissions, haematological parameters and cerebrovascular disease. The study involved children less than 16 years of age for two reasons: the cerebrovascular circulation is routinely assessed using transcranial Doppler (TCD) ultrasound in this age group, and children live most of their lives close to home, allowing postcode to act as a reasonable surrogate marker of exposure to pollutants. Understanding the effects of air quality may also allow more evidence-based advice to be given on housing needs and illuminate pathological mechanisms in SCA.

Patients, materials and methods

Patient population

The study was based on children with SCA attending the Paediatric Sickle Cell Clinic at King's College Hospital who also lived in greater London. The data were anonymised prior to analysis and no extra blood samples or tests were performed. The study was discussed with the Research Ethics Committee at King's College Hospital, who decided that formal ethical approval was not necessary. Children were included in the study if they had a confirmed diagnosis of SCA (haemoglobin (Hb), family and DNA studies), underwent routine TCD scan and steady-state blood tests in 2006 and were documented to be living at the same address from 2005 to 2006. Patients receiving regular blood transfusions were excluded, as were those living outside London for whom air quality maps were not available. Patients are seen in clinic routinely every 3-12 months, and steady-state blood tests are performed annually.

Data collection

Relevant data were collected from written and electronic patient records. This included assessment of haemolysis (Hb, reticulocyte count, bilirubin, lactate dehydrogenase (LDH), aspartate transaminase (AST)), steady-state haematology (Hb, white cell, neutrophil and platelet counts), cerebral blood velocities from TCD scans and clinical complications (attendances at Accident and Emergency and days spent in hospital in 2006, use of hydroxyurea). The postcode was recorded routinely at each clinic attendance. The data were recorded anonymously on a secure study database.

Transcranial Doppler scanning

Intracerebral blood velocity was measured according to the protocol used in the STOP study [START_REF] Adams | Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography[END_REF] in the vascular laboratory at King's College Hospital. TCD measurements were first attempted between the ages of 2 and 4 years.

Air quality mapping

The Environmental Research Group at King's College London predicts London's air pollution using a combined modelling-measurement approach. It uses a kernel modelling technique to describe the initial dispersion of pollutants and then calculates the contribution of various sources to total air pollution concentrations by applying the model concentration field and adjusting for the source strength. A number of source types are taken into account: detailed treatment of the road network, gas combustion sources, railway sources and Heathrow airport, plus the effect of the combined road traffic emissions plus all other source types from more distant locations. The model uses hourly sequential meteorological data from the London Heathrow site. Measurements from the London Air Quality Network, which operates a series of air quality monitoring stations across London, are used to validate the model.

Local levels of NO, NO 2 and PM 10 were mapped using the model, although equivalent maps of O 3 levels were not available. These maps were used to assign a mean exposure to pollutants for 2006 to each child, based on the postcode of their home. In the UK, postcodes consist of five to eight numbers and letters, which uniquely designate a small group of houses in the same street.

Analysis

The mean annual exposure to NO, NO 2 and PM 10 was not normally distributed and was analysed by Spearman's rank correlation (ρ), using Statistical Package for Social Sciences (SPSS), version 14.0 (SPSS Inc., Chicago, IL, USA). Mean annual pollution exposure for categorical variables was analysed using the Mann-Whitney U test.

Results

Patients

One hundred seven children with SCA who lived in greater London underwent TCD scanning and blood testing in 2006 at King's College Hospital. Of these, 14 had known cerebrovascular disease and were on transfusion programmes and were not analysed further. The 93 children studied had a mean age of 8.9 years (2.5-16.4 years) and included 38 girls (41%). Ten patients were taking hydroxyurea because of recurrent acute pain or acute chest syndrome and were included in the analyses.

Air quality

Mean annual exposure to the three pollutants analysed varied widely across the different postcodes. The mean annual exposure to pollutants was: NO 2 38.9 μg/m 3 (median 40, range 26-51), NO 24.6 μg/m 3 (median 24.5, range 8-52), PM 10 22.3 μg/m 3 (median 22.2, range 20-26). All three pollutants were very closely correlated with each other (NO 2 vs NO ρ= 0.98, P<0.001; NO 2 vs PM 10 ρ =0.93, P < 0.001; NO vs PM 10 ρ =0.94, P <0.001), suggesting that they are all related to a similar aspect of air quality.

Hospital attendance

The number of days spent in hospital varied from 0 to 26 (median 0, mean 1.96) and the number of A&E attendances ranged from 0 to 4 (median 0, mean 0.37). There was no detectable effect of exposure to pollution for NO, NO 2 or PM 10 on hospital use in 2006 on a number of different analyses. For example, for each pollutant, patients were divided in to two halves: those exposed to lowest levels and those exposed to highest levels. There was no difference in hospital admissions or A&E attendance between these halves for any of the pollutants (Mann-Whitney U test). Taking hydroxyurea is a marker of more severe clinical problems, and there was no difference in the mean annual exposure to pollutants in the ten children taking hydroxyurea compared to those who were not (Mann-Whitney U test).

Routine laboratory measures

There was no significant correlation between exposure to NO, NO 2 or PM 10 and haemoglobin, white cell count, platelet count, and alanine transaminase (Spearman's rank correlation; Table 1).

Measures of haemolysis

Exposure to NO, NO 2 and PM 10 correlated negatively and significantly with total bilirubin levels (Fig. 1). There was a non-significant trend towards increasing pollution exposure being correlated with decreasing levels of LDH, AST and reticulocyte count (Table 1).

Transcranial Doppler measurements

There was significant positive correlation between exposure to all three pollutants and blood velocity in the extracranial internal carotid artery (eICA). There was a non-significant negative relationship between exposure to pollution and intracerebral blood flow in the intracranial carotid artery, middle cerebral, anterior cerebral (ACA) and posterior cerebral arteries. (Table 1).

Conclusions

This study is the first attempt to assess the effects of air quality on steady-state measurements and chronic com- plications of SCA. There are inevitably a number of limitations and confounding factors. Postcode can only act as an approximate estimate of annual exposure to pollutants, and many other factors will be important, including exposure to indoor pollutants such as tobacco smoke [START_REF] Young Rc | Smoking is a factor in causing acute chest syndrome in sickle cell anemia[END_REF]. Equally, this study only looked at pollution exposure over a 12-month period, and some complications may occur over a longer time period and be related to environmental factors several years previously. We were also not able to analyse the effect of O 3 exposure due to unavailability of maps of annual exposure at the time of the study, and we previously found this to have an important effect on increased episodes of acute pain [START_REF] Adams | Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography[END_REF]. However, despite these limitations, this study is the first to try and identify importance of air quality on chronic complications in SCA and showed some potentially important effects. All three pollutants showed significant negative correlation with total bilirubin. The significance of this is unclear, although a similar non-significant trend was seen for other markers of haemolysis, with LDH, reticulocytes and AST all decreasing with increasing exposure to pollution, whilst haemoglobin shows positive correlation with NO. Haemolysis is implicated in the pathogenesis of vascular complications in SCA, with evidence that increased levels of free plasma haemoglobin cause a functional deficiency of NO [START_REF] Reiter | Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease[END_REF], which contributes to a number of complications including cerebrovascular disease, priapism and leg ulcers [START_REF] Kato | Lactate dehydrogenase as a biomarker of hemolysisassociated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease[END_REF][START_REF] O'driscoll | Serum lactate dehydrogenase as a biomarker in children with sickle cell disease[END_REF]. It is interesting that increased exposure to environmental NO is possibly associated with decreasing haemolysis and potentially beneficial effects. This is consistent with clinical evidence of benefit from low-dose NO exposure [START_REF] Weiner | Preliminary assessment of inhaled nitric oxide for acute vaso-occlusive Crisis in pediatric patients with sickle cell disease[END_REF] and our previous study demonstrating decreased episodes of acute pain in SCA on days with high levels of environmental NO [START_REF] Adams | Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography[END_REF].

The other significant finding in our study was a positive correlation between blood flow in the extracranial internal carotid artery and exposure to NO, NO 2 and PM 10 . This relationship was strongest between PM 10 and eICA blood velocity, with a weaker effect for NO and NO 2 , consistent with the primary relationship being with PM 10 . The significance of increased eICA blood velocity in SCA is not established, with no evidence that it is predictive of stroke risk as has been shown for intracranial blood vessels. However, there is evidence that extracranial cerebrovascular disease is a cause of stroke in some children [START_REF] Bhattacharya | Extracranial carotid artery occlusion in children with sickle cell disease[END_REF], and our study suggests that dust exposure may be contributory to this. Consistent with this, previous studies in adults without sickle cell disease have shown an association between increased exposure to PM 10 and increased risk of infarctive stroke [START_REF] Wellenius | Air pollution and hospital admissions for ischemic and hemorrhagic stroke among Medicare beneficiaries[END_REF] and myocardial infarction [START_REF] Peters | Increased particulate air pollution and the triggering of myocardial infarction[END_REF]. It has been proposed that exposure to small particles might contribute to atherosclerotic plaque development and disruption due to demonstrable associations between PM 10 levels and measures of inflammation [START_REF] Riediker | Particulate matter exposure in cars is associated with cardiovascular effects in healthy young men[END_REF], thrombosis [START_REF] Nemmar | Diesel exhaust particles in lung acutely enhance experimental peripheral thrombosis[END_REF] and autonomic excitation [START_REF] Gold | Ambient pollution and heart rate variability[END_REF]. These same mechanisms might reasonably be expected to apply in children with SCA.

This study provides further evidence that air quality and environmental factors contribute to the complications of SCA. Mapping annual exposure to pollutants offers a way of analysing the effects of air quality on complications of SCA, and the findings in this study need to be confirmed in larger, prospective longitudinal studies. These effects may become more important in time due to climate change and deteriorating air quality.

Fig. 1 a

 1 Fig.1a Scatter plot of exposure to nitric oxide (NO in microgramme per cubic metre) against bilirubin (μM). b Scatter plot of dust exposure (PM 10 in microgramme per cubic metre) and blood velocity (cm/s) in the extracranial internal carotid artery (b). The lines were interpolated by linear regression

Table 1

 1 Summary of data showing Spearman rank correlation (r) and two-tailed significance (P) between air pollutants, blood parameters and transcranial Doppler measurements of blood velocity

		Number	Mean	NO 2 ρ	P	NO ρ	P	PM10 ρ	P
	General
	Hb (g/dl)	89	7.7	0.084	0.433	0.086	0.423	0.134	0.212
	WBC (×10 9 per litre)	89	11.7	0.087	0.418	0.100	0.353	0.063	0.558
	Plts (×10 9 per litre)	89	394	-0.065	0.543	-0.052	0.629	-0.091	0.395
	ALT (IU/l)	84	23.4	0.169	0.124	0.159	0.148	0.166	0.131
	Hemolysis
	Bilirubin (μM)	89	53	-0.210	0.048	-0.223	0.036	-0.257	0.015
	LDH (IU/l)	87	582	-0.143	0.186	-0.123	0.257	-0.115	0.288
	AST (IU/l)	88	55	-0.103	0.339	-0.086	0.424	-0.060	0.580
	Retics (×10 9 per litre)	89	301	-0.101	0.346	-0.085	0.430	-0.071	0.508
	TCD
	eICA (cm/s)	93	151	0.219	0.035	0.224	0.031	0.269	0.009
	TAMMV (cm/s)	93	124	-0.127	0.224	-0.140	0.180	-0.118	0.261
	ACA (cm/s)	93	99	-0.113	0.280	-0.117	0.264	-0.143	0.171
	Statistically significant results are shown in bold.


Katja C Weisel
email: katja.weisel@med.uni-tuebingen.de

Sedat K C Yildirim

Eric Schweikle

Lothar Kanz

Robert Möhle

Weisel

Regulation of FLT3 and its ligand in normal hematopoietic progenitor cells

Keywords: FLT3, FL, Hematopoiesis, Expansion

FLT3 and its ligand (FL) are one of the regulators of normal hematopoiesis. Ligand-independent activation of FLT3 occurs in about 30% of acute myeloid leukemia cases and is one goal for selectively targeted therapies. However, the function of FLT3/FL in the regulation of non-malignant immature hematopoietic cells is poorly characterized. In order to elucidate the role of FLT3 in normal hematopoiesis, human adult CD34 + hematopoietic progenitor cells were cultured in cytokinesupplemented liquid culture in the presence or absence of FLT3 inhibition by . Total cell number, lineage-committed, and primitive progenitors and apoptosis were assayed. FLT3 expression and FL secretion in various conditions were analyzed by fluorescent activated cell sorter and enzyme-linked immunosorbent assay. Effects of nonspecific targeting of FLT3 were evaluated with addition of imatinib (Gleevec®) to cell cultures. It is demonstrated that FLT3 inhibition impaired cell and progenitor cell growth and increased the rate in apoptosis. Effects were observed independent of addition of FL. The dose-dependent growth inhibition was partially equalized by inhibiting FL with a neutralizing antibody. FLT3 inhibition resulted in markedly increased production of FL by cultured CD34 + cells as well as upregulation of FLT3 expression. Imatinib mimicked effects of selective FLT3 inhibition. In conclusion, FLT3 and its ligand regulate proliferation of hematopoietic progenitor cells in an autocrine/ paracrine manner Nonspecific inhibition of FLT3 may contribute to hematotoxicity caused by imatinib treatment.

Introduction

Hematopoiesis is an unidirectional multilinear process where hematopoietic stem cells (HSC) differentiate into mature hematopoietic cells by progressive loss of developmental options and restriction to one lineage. Expansion and regeneration of specific lineages are largely regulated by different hematopoietic cytokines [START_REF] Kondo | Biology of hematopoietic stem cells and progenitors: implications for clinical application[END_REF]. Ligand-mediated activation of the FMS-like tyrosine kinase-3 (FLT3) receptor is one of the regulators of HSC self-renewal and differentiation (reviewed in [START_REF] Parcells | FLT3 in normal hematopoiesis and acute myeloid leukemia[END_REF][START_REF] Gilliland | The roles of FLT3 in hematopoiesis and leukemia[END_REF]). FLT3 is member of the type III RTK subfamily that also includes c-kit, c-FMS, and PDGFα/β [START_REF] Mathews | Expression cloning of an activin receptor, a predicted transmembrane serin kinase[END_REF][START_REF] Rosnet | Isolation and chromosomal localization of a novel FMS-like tyrosine kinase[END_REF][START_REF] Rosnet | Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells[END_REF][START_REF] Lyman | c-kit Ligand and Flt3 ligand: stem/progenitor factors with overlapping yet distinct activities[END_REF]. The role of FLT3 differs between the mouse and the human system. In the mouse system, FLT3 expression is correlated with short-term reconstituting HSC (Lin-Sca-1+c-kit+FLT3+), whereas there is no evidence so far that it is also expressed in long-term repopulating (HSC) [START_REF] Adolfsson | Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential. A revised road map for adult blood lineage commitment[END_REF][START_REF] Adolfsson | Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity[END_REF]. In human adult bone marrow (BM), FLT3 expression is restricted to CD34 + cells and a subset of dendritic precursors [START_REF] Small | STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34 + human bone marrow cells and is involved in the proliferation of early progenitor/stem cells[END_REF]. Recently, it was shown that human cord blood CD34 + cells capable of multilineage engrafting express FLT3 [START_REF] Sitnicka | Human CD34 + hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells[END_REF][START_REF] Dao | FLT3 ligand preserves the ability of human CD34 + progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviral transduction[END_REF]. However, cobblestone-area forming cells (CAFCs) [START_REF] Breems | Frequency analysis of human primitive haematopoietic stem cells subsets using a cobblestone area forming cell assay[END_REF] are equally distributed between CD34 + FLT3 + and CD34 + FLT3 -cells [START_REF] Rappold | Functional and phenotypic characterization of cord blood and bone marrow subsets expressing FLT3 (CD135) receptor tyrosine kinase[END_REF]. In general, expression levels of FLT3 decrease with increasing differentiation of multipotent progenitor cells. So far, the detailed role of FLT3 in human HSC regulation still has to be defined.

FLT3 ligand (FL) is a type 1 membrane protein existing in a membrane-bound and soluble isoforms. Soluble forms activate the corresponding FLT3 receptor [START_REF] Gilliland | The roles of FLT3 in hematopoiesis and leukemia[END_REF] Although its messenger RNA is detected in most hematopoietic and nonhematopoietic tissues, protein expression is restricted to T lymphocytes and BM stromal cells composing the hematopoietic microenvironment [START_REF] Hannum | Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs[END_REF][START_REF] Lyman | Cloning of the human homologue of the murine flt3 ligand: a growth factor for early hematopoietic progenitor cells[END_REF][START_REF] Brasel | Expression of the flt-3 receptor and its ligand on hematopoietic cells[END_REF]. Generally, FL acts in combination with other cytokines, resulting in strongly synergistic effects; isolated addition of FL to stem/progenitor cell cultures has no significant effect on proliferation of myeloid and lymphoid progenitors [START_REF] Lyman | The flt3 ligand: a hematopoietic stem cell factor whose activities are distinct from steel factor[END_REF][START_REF] Ray | Flt3 ligand supports the differentiation of early B-cell progenitors in the presence of interleukin-11 and interleukin-7[END_REF][START_REF] Piacibello | Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood[END_REF][START_REF] Petzer | Differential cytokine effects on primitive (CD34 + CD38 -) human hematopoietic cells: novel responses to FLT3-ligand and thrombopoietin[END_REF].

Ligand-independent activation of FLT3 has recently been identified as an important oncogenic event in pathogenesis of acute myeloid leukemia (AML) [START_REF] Birg | The expression of FMS, KIT and FLT3 in hematopoietic malignancies[END_REF][START_REF] Zheng | FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells[END_REF]; activating mutations in the kinase domain correlate with a dismal prognosis and a high risk for relapse [START_REF] Levis | FLT3: it does matter in leukemia[END_REF].

The emerging role of FLT3 in leukemia pathogenesis exposed it as a therapeutic target for specific therapy. Several tyrosine kinase inhibitors have recently been described. CEP-701 (lestaurtinib; Cephalon West Chester, PA, USA) is an indolocarbazole alkaloid which inhibits FLT3 (wild type, ITD, and PM) in an IC 50 of 3 nM [START_REF] Levis | A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo[END_REF]. The most closely related kinases [platelet-derived growth factor (PDGF), FMS, and kit] are inhibited at concentrations of 500-1000 nM or greater. CEP-701 is cytotoxic to primary AML and ALL blast samples and showed clinical activity in a phase I/II trial in patients with relapsed or refractory AML [START_REF] Brown | Pediatric AML primary samples with FLT3/ITD mutations are preferentially killed by FLT3 inhibition[END_REF][START_REF] Brown | FLT3 inhibition selectively kills childhood acute lymphoblastic leukemia cells with high levels of FLT3 expression[END_REF][START_REF] Smith | Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia[END_REF]. In addition, FLT3 is a nonspecific target of various other tyrosine kinase inhibitors. A research group from our institution had previously shown that imatinib (Gleevec™), which is widely used in the therapy of chronic myeloid leukemia, inhibits stem and progenitor cell activity of HSC. Potential signaling pathways responsible for this inhibitory effect were investigated, but at this point, it was excluded that this mechanism is c-kit-or PDGF-dependent [START_REF] Bartolovic | Inhibitory effect of imatinib on normal progenitor cells in vitro[END_REF]. The group discussed FLT3 as a possibly targeted pathway.

In this study, we investigated the effect of FLT3 inhibition on normal CD34 + HSC using CEP-701. This method allowed us to uncover the interaction of FLT3 and its ligand in HSC. In addition, we went back to the results of Bartolovic et al. [START_REF] Bartolovic | Inhibitory effect of imatinib on normal progenitor cells in vitro[END_REF] and compared the effects of FLT3 inhibition with those of imatinib in CD34 + cell culture. From our results obtained, we conclude that FLT3 inhibition most likely contributes to the hematotoxic side effects of imatinib.

Materials and methods

Reagents

CEP-701 (lestaurtinib) was generously provided by Cephalon. For in vitro experiments, stock solutions of lestaurtinib were prepared at 80 and 20 μg/ml by dissolving the compound in dimethyl sulfoxide (DMSO)/X-VIVO 20 medium (BioWhittaker, Walkersville, MD, USA; 1:4) and stored at -20°C.

Cell samples

Peripheral blood mononuclear cells (MNC) were obtained from healthy donors and patients with hematopoietic and non-hematopoietic disorders undergoing stem cell mobilization with G-CSF and were collected after individuals gave their informed consent. MNC were purified using Ficoll-Hypaque density gradient centrifugation (Biochrom, Berlin, Germany). The study was approved by the ethics review board of the medical faculty of the University of Tuebingen (project number 268/2003V, University of Tuebingen).

Purification of stem and progenitor cells CD34 + cells from fresh leukapheresis samples were selected using a Midi-MACS CD34 isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The purity of CD34 + cells ranged between 92% and 99% in all samples as determined by flow cytometry.

Cytokine-supplemented liquid culture Isolated hematopoietic progenitor cells (HPC, 1×10 5 ) were cultured for 1, 2, 4, and 7 days in 5 ml serum-free medium (X-VIVO 20, BioWhittaker, Walkersville, MD, USA) supplemented with a combination of cytokines: SCF 50 ng/ml, IL-6 10 ng/ml and, in selected cultures, FL (50 ng/ml) (all from Tebu, Frankfurt, Germany). Lestaurtinib was added at increasing doses from 0 to 50 nM. Cells were incubated at 37°C in humidified atmosphere with 5% CO 2 in air. In liquid cultures, all cells could be recovered without trypsination.

Flow cytometry

Cells (1-2×10 5 ) were incubated for 20 min at 4°C with the fluorescein isothiocyanate or PE-conjugated monoclonal antibody (mAb) CD34, CD45, and CD38 (Becton-Dickinson, Heidelberg, Germany) or FLT3 (R&D Systems, Minneapolis, MN, USA). An isotype-identical mAb served as a control. Cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson). To calculate the percentage of positive cells, a proportion of ≤1% false positive events was accepted in the negative control sample. Cell acquisition and analysis was performed using the CellQuest software program (Becton Dickinson).

Assay for clonogenic progenitors

One thousand five hundred cells were plated in triplicate in 35-mm tissue culture dishes (Greiner, Frickenhausen, Germany) containing 0.9% methycellulose, 30% fetal bovine serum, 1% bovine serum albumin, 70% Iscove's DMEM (Stem Cell Technologies, Vancouver, Canada), and a combination of five cytokines: human SCF (20 ng/mL, Pepro Tech Hamburg, Gemany), human interleukin-3 (50 ng/ml, Tebu Offenbach, Germany), interleukin-6 (20 ng/ml, Tebu Offenbach), human granulocyte colony-stimulating factor (rhumet-G-SCF, 100 ng/ml, Amgen, Thousand Oaks, CA, USA), and human erythropoietin (6 U/ml, Jannsen-Cilag, Neuss, Germany). In selected cultures, increasing concentrations of CEP-701 (lestaurtinib, 0-20 nM) were added. The plates were cultured for 14 days at 37°C, 100% humidity, and 5% CO 2 . Colonies (>40 cells) were scored using an inverted microscope. By this technique, erythroid (burst-forming and colony-forming units erythroid: BFU-E, CFU-E), myeloid colonies (colony-forming units granulocyte, macrophage, and granulocyte-macrophage: CFU-G, CFU-M, CFU-GM), and mixed colonies were detected.

MTT assay

Cell proliferation and survival were measured by cellular uptake of 3,-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich). Fresh CD34 + cells were resuspended in growth-factor-supplemented serum-free medium at a concentration of 3×10 4 cells/150 μl. Cells were preincubated for 24 h in 96-well flat-bottomed microtiter plates (Becton Dickinson) before CEP-701 (lestaurtinib) was added at increasing concentrations (0-50 nM). All analyses were performed in triplicate. After 48 h, 10 μl MTT solution (10 mg/ml) was added to each well. Plates were incubated for another 2 h before 90 μl MTT lysis solution (15% sodium dodecyl sulfate, 50% dimethylformamide) was added to each well, and the absorbance was measured after 12 h on an enzyme-linked immunosorbent assay (ELISA) plate reader (Dynatech MR7000; Dynatech, Denkendorf, Germany). The IC 50 is defined as the concentration of drug that produces 50% cell growth inhibition and corresponds to the affected fraction (F a value) of 0.5.

Apoptosis (annexin V/propidium iodide) CD34 + cells (1×10 5 cells/well) were cultured in six-well tissue plates under the liquid culture conditions described. Without pre-incubation, lestaurtinib was added at increasing doses from 0 to 20 nM. Cells were incubated either for 24, 48, or 96 h. Propidium iodide (PI) staining solution was used to assess plasma membrane integrity in annexin V apoptosis assays. PI is used in two-color annexin V flow cytometric assays to distinguish cells that are in the early stages of apoptosis (annexin V pos./PI neg) from those that are in the later stages of apoptosis or already dead (annexin V pos./PI pos.). Cells were analyzed after 24, 48, and 96 h.

Cobblestone area formation

For determination of CAFC formation, the contact-inhibited MS-5 murine stromal cell line was used. When stromal layers cultured in T12.5 flasks (Falcon) reached confluency, CD34 + or cultured cells (1×10 5 ) were added. Stromal cultures were incubated at 37°C and 10% CO 2 for 5 weeks in human longterm culture medium consisting of α-MEM (Gibco) supplemented with 12.5% fetal bovine serum (HyClone), 12.5% horse serum (Sigma), 1% L-Glutamin (Gibco), 50 μM hydrocortison (Sigma), 50 mM mercaptoethanol (Gibco), and 10,000 U/ml penicillin/streptomycin (Gibco) with weekly half-medium changes (demidepopulation). At the end of the 5-week period, phase-dark cobblestone areas of a least five cells were counted.

Inhibition of FLT-3 signaling

To investigate whether the growth inhibitory effect of lestaurtinib on normal HPC can be abrogated by addition of FL antibody, 1×10 5 CD34 + were expanded in liquid culture conditions. Expansion was performed with or without the addition of the FL inhibitory antibody according to the manufacturer's instructions (0.1 mg/ml; Peprotech, USA).

Statistical analysis

Data from independent experiments were expressed as mean±SEM (standard error of the mean) of at least three experiments. Differences between groups were compared by the parameter-free Mann-Whitney rank sum test for statistical significance.

Results

Short-term expansion of CD34 + cells in serum-free liquid culture

Mobilized CD34 + cells were expanded for 7 days in serumfree liquid culture starting with 1×10 5 cells/per well supplemented either with SCF and IL-6 or with SCF, IL-6, and FL. CEP-701 was added at day 0 in increasing doses (0-50 nM). Total cell counts after 1 week of culture are shown in Fig. 1a. Cell counts decreased continuously with increasing concentrations of CEP-701 due to induction of apoptosis (see below). The effect observed was independent from addition of FL to the cell culture. Looking on the rates of CD34 + cells after 7 days in culture, CD34 + rates also continuously decreased with increasing concentration of CEP-701 (Table 1).

Effect of FLT3 inhibition on lineage-committed and primitive progenitors

In order to evaluate whether the effect of FLT3 inhibition extends to both, lineage-committed and more primitive HPC, colony and cobblestone area formation was assayed. Again, CD34 + cells were cultured in cytokine-supplemented (IL-6, SCF) liquid culture conditions with increasing concentrations of CEP-701 as described above. For determination of colony formation, cells were transferred to semisolid cytokinesupplemented methylcellulose media without further addition of CEP-701 after 7 days of liquid culture. Colony formation was assayed after 14 days. As shown in Fig. 1b, absolute counts of colony-forming units decrease continuously when cells were exposed to CEP-701 for 7 days in increasing concentrations. The decrease occurs in parallel to Fig. 1 a Absolute cell counts after a 7-day culture period in cytokine-supplemented serumfree liquid culture. Input CD34 + cells were exposed to increasing concentrations of CEP-701 (5-100 nM). Addition of DMSO as the solvent for CEP-701 served as control. Cell counts were obtained using trypan blue dye exclusion. b, c Myeloid and erythroid colony formation of day 7 cells cultured in cytokinesupplemented (SCF, IL-6) liquid cultures under increasing concentrations of CEP-701 (5-100 nM). Absolute and relative CFC numbers per 1,500 input cells are shown. d Colony formation of day 0 input CD34 + cells when CEP-701 was added directly into semisolid methylcellulose cultures with increasing concentrations (5-100 nM). e, f Cobblestone area formation (CAFC) of expanded CD34 + cells, absolute and relative counts. After 96 h in serum-free, cytokine-supplemented liquid culture (IL6, SCF) under addition of CEP-701 (5 and 20 nM) or imatinib, expanded cells were assayed for CAFC formation without any further addition of CEP-701 or imatinib the absolute cell counts measured under different concentrations of CEP-701. However, relative counts of colonyforming units were not significantly different, indicating that non-apoptotic, surviving progenitor cells are not impaired in function (Fig. 1c). However, if CEP-701 is directly added into day 0 methylcellulose cultures, a dose-dependent decrease in colony formation was detected, as it was shown for cell counts in liquid culture system (Fig. 1d).

In addition, we investigated cobblestone area formation of the cultured CD34 + cells to obtain more information about the earlier progenitor cell population [START_REF] Van Hennink | Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice[END_REF]. Similar to the findings in lineage-committed colony-forming units, absolute CAFC counts, assayed after 5 weeks of co-culture of 96 h liquid culture cells with MS5 stroma, decreased with exposure to increasing concentrations of CEP-701 during the liquid culture period (Fig. 1e). Again, relative CAFC counts did not decrease after culturing the cells with 5 nM CEP-701 (Fig. 1f), although this concentration resulted in a significant growth inhibition (Fig. 1a). Using higher concentrations of CEP-701 (20 nM) relative CAFC counts were also affected. With concentrations higher than 20 nM, CAFC assays were not realizable due to increasing amount of apoptotic cells in culture. These results indicate that exposure of HPC to CEP-701 for 7 days reduces the number of CAFC rather than affecting the cloning efficiency of the cells when transmitted to the CAFC assay (Fig. 1e,f). Addition of imatinib (5 μM) leads to a similar effect in absolute but a more pronounced decrease in relative CAFC counts.

Cytotoxicity and apoptosis

A tetrazolium-based MTT assay was used to quantify 50% growth inhibition (IC 50 ) of cytokine-stimulated CD34 + cells after 48 h of exposure to CEP-701. The mean IC 50 (±SD) for CEP-701 on normal CD34 + cells was 56±19 nM.

To determine whether CEP-701 cytotoxicity was due to induction of apoptosis, we performed annexin-V binding flow cytometric assays. With increasing concentrations of CEP-701, an increasing rate of apoptosis in cultures was detected (analysis after 48 h of culture). These data indicate that apoptosis is the predominant mechanism of cytotoxicity of CEP-701 (Fig. 2).

Effects of FLT3 inhibition can be partially equalized by addition of FL Ab

The above findings showed cytotoxic effects of FLT3 inhibition in the early stem and progenitor cell population. To further demonstrate that these effects are specific due to FLT3 inhibition and not due to nonspecific inhibitory events, activation of the FLT3 tyrosine kinase was downregulated by inhibition of its ligand FL with a neutralizing antibody (FL-Ab). When FL-Ab was added into the cytokine-supplemented liquid culture system, inhibitory effects of CEP-701 were partially equalized (Fig. 3). These findings were even present when FL was not added into the culture system, indicating that the FLT3 receptor in this condition is activated by presence of FL in culture obviously produced by the culture cells itself.

FLT3 inhibition results in marked upregulation of FL production in cell culture

Observing the effects of FLT3 inhibition independent of FL substitution, we had to conclude that FL is autonomously produced by input CD34 + cells and their progeny in liquid culture conditions. Measuring FL with ELISA in the supernatant showed a baseline production of 1.3, 2.1, and 0.8 ng/ml FL after 24, 72, and 96 h, respectively. In the case of FLT3 inhibition by CEP-701 (20 nM), FL production by the cultured CD34 + cells markedly increased up to 841, 709, and 511 ng/ml FL after 24, 72, and 96 h. Addition of imatinib, targeting the abl kinase but also having nonspecific inhibitory effects on FLT3, lead to a similar effect (Fig. 4a). After 24 h, virtually all cultured cells were still CD34 + (Fig. 4b), indicating that also CD34 + progenitors and not only their progeny are able to produce FL.

FLT3 inhibition results in upregulation of FLT3 expression

In a next step, we investigated the effect of FLT3 inhibition with CEP-701 of FLT3 expression in liquid-culture CD34 + cells. After 24 h in liquid culture with cytokine supplementation, FLT3 is partially expressed in CD34 + cells assayed by flow cytometry. However, adding CEP-701 into the culture system, FLT3 expression increased (Fig. 4b). Again, a similar effect was observed by adding imatinib to the cell cultures.

Discussion

Hematopoiesis is a complex process in which survival, proliferation, differentiation, and other functions are at least partially regulated through a network of growth factors [START_REF] Kondo | Biology of hematopoietic stem cells and progenitors: implications for clinical application[END_REF][START_REF] Moore | In vitro and in vivo hematopoiesis[END_REF][START_REF] Shivdasani | The transcriptional control of hematopoiesis[END_REF][START_REF] Zhu | Hematopoietic cytokines, transcription factors and lineage commitment[END_REF]. FLT3 and its ligand FL are known regulators in this process, mainly enhancing effects of other cytokines more than having specific effects by itself. Due to the role of FLT3 and its activating mutations in AML, it became also a therapeutic target.

In this study, we investigated the role of FLT3 and its ligand in normal early hematopoiesis. We used a cytokinesupplemented, serum-free liquid culture system that had been shown to be effective for ex vivo expansion of adult CD34 + cells [START_REF] Piacibello | Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood[END_REF]. FLT3 inhibition was performed with CEP-701 (lestaurtinib) which is known to act as a potent inhibitor in nanomolar concentrations.

It was shown that cell counts in the liquid culture system decreased in a significant and dose-dependent fashion in cells treated with CEP-701 even in low nanomolar concentrations (5-10 nM). Dose-dependent inhibition of CD34 + cell growth and expansion was also reflected in investigation of progenitor cell functions (CFC and CAFC formation). Decrease of these colony-or cobblestone-forming units was in parallel with the absolute cell counts in culture. Looking on the relative counts, surviving non-apoptotic progenitor cells showed nearly the same capacity of colony and cobblestone formation. The mechanism CEP-701 acts on CD34 + progenitor cells was mainly apoptotic, as shown using the annexin V staining. Interestingly, the growth inhibitory effects of FLT3 inhibition were independently of FL supplementation in the liquid-culture system. This was the first hint that the cultured cells by themselves produce significant amounts of FL, leading to a continuous tyrosine kinase activation.

To confirm that the observed effects of FLT3 inhibition were specific, we used a neutralizing antibody against FL to block ligand activation of FLT3. With addition of the antibody, inhibitory effects of CEP-701 were partially equalized. This effect was again observed in the absence of supplementation with FL.

Focusing more in detail on the suspicion that FL and FLT3 are acting in an autocrine-or paracrine-independent manner in cell culture, FL production in serum-free liquid culture conditions was analyzed. Using ELISA technique, we did not only show for the first time that FL is produced in these culture conditions by the progenitor cells, we also could show that this production is markedly upregulated by FLT3 inhibition. The same conditions had impact on FLT3 expression itself. Inhibition of FLT3 by CEP-701 results in an upregulation of FLT3 in the CD34 + progenitors. In conclusion, the simultaneous upregulation of FLT3 and FL expression under inhibiting conditions showed a compensatory mechanism in a self-regulated feedback system.

Looking on the inhibitory effect of imatinib on CD34 + hematopoietic progenitor cells, it is comparable to the effect we found with FLT3 inhibition [START_REF] Bartolovic | Inhibitory effect of imatinib on normal progenitor cells in vitro[END_REF]. Here, we demonstrated in a direct comparison that imatinib used in concentrations based on the data of Bartolovic et al. and those usually achieved in patients showed the same effect on FL and FLT3 expression as was observed with CEP-701. Inhibition of progenitor cell function was documented by reduction of CAFC formation. More importantly, imatinib lead to an enhanced production of FL in culture and to an increased expression of FLT3 in CD34 + cells cultured for 24 h, comparable to the data generated with direct FLT3 inhibition. Imatinib is known to have inhibitory effects on the FLT3 tyrosine kinase. It is therefore concluded that the hematotoxicity known as one of the most relevant side effects of imatinib therapy is at least partially mediated by nonspecific FLT3 inhibition.

Fig. 3

 3 Fig. 3 Inhibition of FL by neutralizing antibody. CD34 + cells were cultured for 96 h in serum-free, cytokine supplemented (Il-6, SCF or IL-6, SCF, and FL) liquid culture in the presence of various concentrations of CEP-701 (5-50 nM) with or without a neutralizing antibody against FL (α-FL, n=6). **p<0.01, ns not significant

Fig. 2

 2 Fig. 2 Immunophenotypic analysis of apoptosis. CD34 + cells were cultured for 96 h in cytokine-supplemented liquid culture conditions with increasing concentrations of CEP-701 (5 and 20 nM). Cells were

Fig. 4 a

 4 Fig. 4 a Production of FL by CD34 + cells after 24, 72, and 96 h in serum-free, cytokinesupplemented (IL-6, SCF) liquid culture in presence of various concentrations of CEP-701 (5 and 20 nM) or imatinib (5 μM). Concentrations of FL in the supernatant were determined by ELISA. b Determination of CD34 and FLT3 expression in progenitor cells cultured for 24 h in serum-free, cytokinesupplemented liquid culture conditions under exposure ton CEP-701 (5 and 20 nM) or imatinib

Table 1

 1 Percentage of CD34 + cells after 7 days of liquid culture under supplementation with SCF, IL-6 or SCF, IL-6 and FL

		% CD34 + cells	% CD34 + cells
		(SCF, IL-6)	(SCF, IL-6, FL)
	DMSO	64.2±8.7	57.2±5.5
	CEP-701 5 nM	49.3±8.2	47.7±3.9
	CEP-701 10 nM	41.0±6.6	40.3±2.7
	CEP-701 20 nM	27.8±4.6	18.3±4.1
	CEP-701 50 nM	13.7±3.7	11.2±4.2

Acknowledgements We thank Steven Trusko from Cephalon Inc. for generously providing CEP-701.