Day 2: Thursday, October 22

Keynote Lecture

Thalassemia as a Global Health Problem: Recent Progress Towards Its Control In The Developing Countries

D.J. Weatherall, MD, Weatherall Institute of Molecular Medicine, University of Oxford, UK

It is only in recent years, and particularly in light of data that have been collected for the Global Burden of Disease Program, that a true picture of the enormous global health problem posed by thalassemia in the developing countries, particularly those of Asia, has been appreciated. The major problems that face the high frequency regions include lack of basic information about the complex pathophysiology of some of the commonest forms of thalassemia in Asia, the most appropriate approach to the control and management of severe thalassemia in low-income countries, and the continued lack of appreciation of the importance of these diseases on the part of international health agencies.

While the control and management of thalassemia major presents similar problems for the developing countries as those for richer countries, in many countries the commonest form of severe thalassemia is HbE β thalassemia, a disease with extraordinary phenotypic diversity. Recent work underlines the importance of the environment, variable adaptation to anemia, heterogeneity of response to anemia at different stages of development, and a variety of genetic modifiers as the major reasons for the phenotypic diversity of this disease. A better understanding of these complex interactions is vital for their better management.
Improvement in the control and management of thalassemia in the developing countries will depend on a number of on-going developments, particularly the establishment of North/South and South/South partnerships between countries with genuine expertise in the thalassemia field with those where no such expertise exists. These developments will have to be supported by a greater realization of the global problem of thalassemia on the part of governments, international health agencies, and granting bodies. A vital start in this direction has been made by including genetic disorders of hemoglobin in the Global Burden of Disease Program.

Session I: Iron Regulation and Metabolism

Iron Regulation And Ineffective Erythropoiesis, Jak 2

Luca Melchiori, PhD¹, Sara Gardenghi, PhD¹, Ella Guy¹, MD.², Domenica Cappellini, MD³, MD¹, Robert W. Grady, PhD.¹, Stefano Rivella, PhD¹ , ¹Department of Pediatric Hematology-Oncology, Weill Medical College of Cornell University, New York, NY; ²E. Wolfson Medical Centre, Institute of Hematology, Holon, Israel, ³Centro Anemie Congenite, Fondazione Policlinico, Istituti di Ricovero e Cura a Carattere Scientifico (IRCCS), University of Milan, Milan, Italy

In thalassemia, ineffective erythropoiesis is characterized by apoptosis of the maturing nucleated erythroid cells. Our studies also suggest that limited erythroid cell differentiation plays a role in the development of ineffective erythropoiesis. Some of the major consequences of ineffective erythropoiesis are extra−medullary hematopoiesis (EMH), splenomegaly and systemic iron overload mediated by transfusion therapy and down−regulation of hepcidin. Recall that under conditions of chronic anemia, low levels of hepcidin lead to iron overload. We hypothesized that the protein kinase Jak2, which controls erythroid cell proliferation, plays a major role in exacerbating ineffective erythropoiesis. Therefore, use of Jak2 inhibitors may limit the overproduction of immature erythroid cells in thalassemia, with the potential of reversing extramedullary hematopoiesis and preventing splenectomy. For this reason, we administered a Jak2 inhibitor to mice affected by beta−thalassemia intermedia for 10 days, showing that this treatment was associated with a marked decrease in ineffective erythropoiesis, splenomegaly, no or with little or no reduction of red cell production and no side effects.

There is a clear correlation between the mass of erythroid precursors and suppression of hepcidin. Therefore, administration of a Jak2 inhibitor might also be associated with increased hepcidin synthesis and decreased iron absorption. In order to test this hypothesis we repeated the above-mentioned study in order to evaluate the level of hepcidin expression as well as that of other iron related genes. This analysis clearly indicated that the size of the spleen inversely correlated with hepcidin synthesis. In addition, blood transfusion is a pre-requisite for the management of both thalassemia−major patients and those with thalassemia−intermedia who develop splenomegaly. Therefore, administration of a Jak2 inhibitor, together with blood transfusions, might be a sensible way to further suppress ineffective erythropoiesis and limit splenomegaly/EMH. We conducted an analysis of erythropoiesis and iron metabolism in animals affected by beta−thalassemia major, which require blood transfusion for survival and compared them to transfused animals treated with placebo. Compared to latter controls, the animals treated with the Jak2 inhibitor showed a dramatic decrease in splenomegaly, amelioration of the spleen architecture and complete elimination of EMH in the liver. In addition, due to the reduction of the size of the spleen, the animals treated with the Jak2 inhibitor showed higher levels of hemoglobin at the end of the treatment, again, the spleen size inversely correlating with hepcidin synthesis. These experiments suggest that this class of compounds, if safe, might be an important tool to prevent splenectomy, reverse EMH, limit iron absorption and improve the effectiveness of transfusion therapy in patients with thalassemia.

Hepcidin in Thalassemia

Elizabeta Nemeth, PhD, David Geffen School of Medicine at UCLA, Los Angeles, CA

The iron-regulatory hormone hepcidin controls the plasma iron concentrations by blocking both the absorption of iron from the diet and the release of iron from macrophages recycling old erythrocytes. Pathological hepcidin deficiency, either relative or absolute, results in iron overload, whereas hepcidin excess causes iron restriction and anemia.

Beta thalassemia and other iron-loading anemias are characterized by inappropriately low production of hepcidin due to the strong suppressive effect of ineffective erythropoiesis. Although the precise factors are still incompletely understood, GDF15 and TWSG1 have been suggested as bone marrow-derived proteins causing hepcidin decrease. Hepcidin production is particularly low in untransfused patients, and likely accounts for the excessive iron absorption and development of the severe iron overload. In transfused patients, hepcidin levels are higher than those in untransfused, although still not appropriate for the degree of iron overload. The higher hepcidin production is due to increased iron loading and the partial relief of erythropoietic drive by transfusions. The effect of iron chelation on hepcidin production is not yet known.

The usefulness of hepcidin diagnostics or therapeutics in thalassemia syndromes remains to be demonstrated. Measurement of hepcidin levels may help stratify patients for the risk of greater iron loading or may be help assess the efficacy of therapy. Pharmacologic hepcidin agonists may help restore normal iron homeostasis in thalassemia patients, especially those not requiring transfusions. Mini-hepcidins, small peptide agonists of hepcidin, have been described which display activity in mice. The small size of these peptides may also allow oral bioavailability.

TMPRSS6: A Modifier of Hepcidin Production in Relation to Iron Stores

Matthew Heeney, Dean Campagna, Mark D. Fleming, Children's Hospital Boston, Boston, MA

The peptide hormone hepcidin is a negative regulator of iron efflux from cells. In iron deficiency anemia (IDA), hepcidin levels are ordinarily undetectable, thereby stimulating the release of iron from macrophages and duodenal enterocytes, promoting iron availability for erythropoieisis and increasing iron stores. Rare individuals with lifelong hypoferremia and microcytic anemia unresponsive to enteral and parenteral iron therapy—iron refractory iron deficiency anemia (IRIDA)—have been shown to have mutations in the hepatocyte−specific transmembrane serine protease, TMPRSS6, and that the phenotype can be attributed to excessive hepcidin production.We comprehensively analyzed hematologic and biochemical markers of iron status, inflammation, urinary and plasma hepcidin in individuals with IRIDA and their family members.

Given that the transferrin saturation (TfSat) appears to have a central role in hepcidin regulation, an index relating the hepcidin to transferrin saturation, TfSat/log10hepcidin, was evaluated to attempt to distinguish inappropriate from appropriate hepcidin expression in probands and their family members. Individuals with biallelic TMPRSS6 mutations have elevated absolute urinary and plasma hepcidin concentrations that were profoundly elevated compared to familial wild type controls when normalized for serum transferrin saturation. Furthermore, family members heterozygous for TMPRSS6 mutations had normal iron studies, but had an intermediate TfSat/log10hepcidin, indicative of a codominant phenotype. Several other individuals referred for a clinically milder IRIDA phenotype were found to have heterozygous mutations, suggesting that some heterozygotes may manifest clinically apparent disease. These data provide evidence that even heterozygous mutations in TMPRSS6 can influence systemic iron metabolism.

Session II: Gene Regulation and Therapy

The Identification of Factors Regulating γ-globin Gene Transcription

Frank Grosveld^1,2,3,4, Farzin Pourfarzad¹, Ali Aghajanirefah¹, Ernie de Boer¹, Marieke von Lindern⁵, Jeroen Demmers^3,4, Thamar van Dijk¹, and Sjaak Philipsen^{1 1}Department of Cell Biology, ²Center for Biomedical Genetics, ³The Netherlands Proteomics Centre, ⁴Biomics Department and ⁵Department of Hematology , Erasmus MC, Dr. Molewaterplein 50, 3015GE, Rotterdam, The Netherlands

It is known that the expression of the γ globin genes in sickle cell anemia or γ globin thalassemia patients greatly ameliorates the effects of these diseases. One of the goals of our research is therefore to induce the expression of these genes in patients through the development of novel therapeutics. However before these could be developed on a rational basis, it is necessary to understand how the process of γ globin suppression in postnatal life is coordinated at the molecular level. Association studies on persons with relatively high γ-globin gene expression during adult life have indicated that a number of non-globin loci are involved in the suppression of the γ-globin genes (Lettre et al., 2008; Menzel et al., 2007; Uda et al., 2008; Jiang et al., 2006; Thein and Menzel, 2009).

The most promising of these, Bcl11A, was recently shown to lead to elevated γ-globin gene expression when its activity is suppressed (Sankaran et al., 2008). However the Bcl11A protein binds to a region downstream of the γ-globin genes whereas the promoters of the γ-globin genes were previously identified as the regions responsible for their suppression (Yu M, 2006). Hence we have focused our attention on the γ-globin gene promoters and followed an entirely novel biochemical approach to isolate the proteins that regulate these promoters by purifying the promoter and attached proteins in the suppressed state. We will present the results we have obtained with several putative γ-globin suppressor proteins we have identified via this biochemical approach.

Lentivirus Vector-Based Gene Therapy For Beta-Globinopathies

Punam Malik, MD ^1,2, Ajay Perumbeti, MD², Tomoyasu Higashimoto, PhD¹, Paritha I. Arumugam, PhD¹ and Geetha Puthenveetil, MD³. ¹Div. of Experimental Hematology and Cancer Biology, ²Division of Hematology Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, ³Div. of Hematology Oncology, Childrens Hospital Orange County, CA

Gene therapy for β-globinopathies, particularly β-thalassemia and sickle cell anemia (SCA) hold much promise for the future as a one time cure. High levels of expression of the globin genes have been achieved with HIV-1 based lentivirus (LV) vectors and correction of the disease phenotype has been established in murine and human models. Now the practicalities of taking these vectors into a well designed, safe and beneficial clinical trial and “what it will take to cure these diseases” are being addressed by our group and others, after lessons learnt from a clinical trial in France. Safe methods of collection of adequate hematopoietic stem cells (HSC), toxicity of the chemotherapy conditioning regimen and gene transfer, stability of the transgene construct and potential genotoxicity are some of the hurdles. Gene replacement strategies are also under active investigation by others, using induced pluripotent cell technology.

We have shown correction of human Cooley’s anemia in vitro and in a xenograft model. Using a humanized model of SCA, we have performed studies to determine the gene dosage and HSC dosage required to correct SCA. We have shown that LV delivery of human β-globin gene under β-globin regulatory control elements in HSC results in sufficient post-natal fetal hemoglobin (HbF) expression to SCA in the Berkeley sickle mouse. Upon de-escalating the amount of transduced HSC in transplanted recipients, by using reduced-intensity conditioning and varying gene transfer efficiency and vector copy number, we assessed critical parameters needed for correction. A systematic quantification of functional and hematological RBC indices, organ pathology, and life-span were used to determine the minimal amount of HbF, F-cells, HbF/F-cell, and gene-modified HSC required for correcting the sickle phenotype. We show that amelioration of disease occurred: (a) when HbF exceeded 10%, F-cells constituted two-thirds of the circulating RBC, and HbF/F-cell was one-third of the total hemoglobin in sickle RBC; and (b) when approximately 20% gene-modified HSC repopulated the marrow. Genetic correction was sustained long term. We show a novel model utilizing reduced-intensity conditioning to determine genetically corrected HSC threshold that corrects a hematopoietic disease. The requirement for gene-modified HSC for correction of Cooley’s anemia are likely similar to SCA, but higher and consistent levels of β-globin expression per red cell are likely needed, which can be achieved by insulating the vectors. These studies provide a strong pre-clinical model for “what it would take” to genetically correct SCA, and perhaps Cooley’s anemia, and is a foundation for a human clinical trial.

Cell Therapies In A Humanized Mouse Model Of Cooley’s Anemia

Yongliang Huo, PhD.¹, Sean C. McConnell, BSc.¹, Ting-Ting Zhang, MSc.¹, Rui Yang, MSc.¹, Shan-Run Liu, MD.¹, and Thomas M. Ryan, PhD¹, ¹Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL

Cooley’s Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin gene equivalent. This study reports novel preclinical mouse models of CA that survive solely on human fetal hemoglobin at birth and are blood transfusion dependent for life upon completion of their human fetal to adult hemoglobin switch after birth. These humanized CA mice were generated by targeted gene replacement in embryonic stem cells of the adult mouse αglobin genes with human α globin and the adult mouse β globin genes with a delayed switching human γβ° or γδβ° globin gene cassettes. The nonfunctional human β° globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1. Both wild-type and hereditary persistence of fetal hemoglobin (HPFH) promoter mutations were tested in the human γ globin knock-in allele. Heterozygous knock-in mice exhibit β thalassemia intermedia. Newborn homozygous humanized CA mice express 100% human hemoglobin in their RBCs, suffer from ineffective erythropoiesis, and survive from one day to several weeks after birth, but can survive into adulthood through regular blood transfusion. These CA models are the first to recapitulate the temporal onset of the disease in human patients. Interestingly, humanized CA mice are rescued from lethal anemia by a single intravenous postnatal injection of allogenic bone marrow in the absence of any cytoreductive conditioning of the recipient. Stable hematopoietic chimerism is reached 8 weeks post transplant. Transplanted CA mice have a marked improvement of their anemia, exhibit no growth retardation or graft versus host disease, and are fertile. These novel humanized CA disease models are useful for the study of the regulation of globin gene expression, synthesis, and switching; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.

Identifying cis-acting Elements to Improve Vectors for Gene Therapy of Hemoglobin Disorders

Faith Harrow, Amanda P. Cline, Nancy E. Seidel, David M. Bodine, PhD, Bethesda, MD

Effective gene therapy for β-thalassemia and sickle cell disease requires both expression of the globin gene at ~20% the level of endogenous α-globin gene and delivery of globin genes into `25% of hematopoietic stem cells (HSCs). Although significant progress has been made, neither of these goals have been attained with the globin vectors currently proposed for use in patients. The current vectors all use enhancer elements from the β-globin locus control region (LCR) to increase expression. Unfortunately these vectors are prone to recombination, cryptic splicing and aberrant polyadenylation, resulting in low virus production. Flanking the LCR-globin gene with chicken HS4 insulator element can prevent gene silencing and block the ability of the LCR enhancers to activate neighboring oncogenes. However carrying two copies of chicken HS4 only exacerbates the problems of vector instability and production. To improve safety and efficiency we have developed novel globin vectors that use non-globin promoters and insulators to express a γ-globin gene. The mouse Slc4a1 gene encodes the anion exchanger protein AE1 (Band 3). The Slc4a1 promoter directs γ-globin mRNA and protein expression at levels that are 20% the levels of mouse α-globin per gene copy in transgenic mice, but the Slc4a1 promoter requires barrier elements for uniform expression. To identify cis-acting elements in the Slc4a1 locus, we mapped DNase I sensitivity across a 119 kb region that includes the Slc4a1 locus and 50 kb of upstream and downstream sequence. We identified 13 HSs, 10 upstream of the transcriptional start site (5’ 1-10) and 3 downstream of the transcriptional start site (3’ 1-3), all of which were assayed for enhancer, enhancer blocking and barrier activity. No activity was detected for 5’ HS-3, -4, -6 and -8 and 3’ HS-2. 3’ HS-3 is located at the boundary between the DNase I sensitive chromatin at the 3’ end of the Slc4a1 gene and the DNase I insensitive chromatin of the Ubf-1 gene. 3’ HS-3 had significant barrier element activity (p<0.02). Sites 5’HS - and -2 both showed enhancer activity (4-5 fold increase in expression; p<0.01), while sites 3’HS-1, 5’HS-5 and 5’HS-9 were enhancer blockers (p<0.01). By combining the barrier elements we have identified in the ANK-1 α-spectrin and Slc4a1 loci with the enhancer blockers from the Slc4a1 locus, we have created a series of unique insulators containing a barrier and an enhancer blocker. Because these insulators have different sequences, they are not prone to recombination during virus production or integration. We are testing lentivirus vectors using the Slc4a1 promoter/γ-globin gene flanked by the hybrid insulators we have generated. We predict that the new insulators will prevent silencing of the Slc4a1 promoter, allowing sustained therapeutic levels of γ-globin expression. In addition the insulators should prevent the Slc4a1 enhancers from activating nearby genes, increasing the safety of this approach. The wild type human ANK-1E promoter contains no enhancer elements directs expression of γ-globin mRNA at 4% of the level of mouse α-globin mRNA in transgenic mice. The distal 200 bp of the ANK-1E promoter contains a barrier element, that prevents gene silencing in cultured cells and transgenic mice. The ANK 1E promoter also contains a 9-bp TFIID-binding consensus sequence ([G>T][G/C][G/C]GGNGAG) that is present in 9% of all mammalian promoters and is highly enriched in promoters lacking known consensus elements. A variant of this sequence, (GCGGGTGAG; GC-ANK-1E), expresses 7-fold higher levels of γ-globin mRNA than the wild type ANK-1E promoter (p<0.01) in transgenic mice. Simplified lentiviral vectors containing the GC-ANK-1E/γ-globin gene are produced at high titer, transduce approximately 50% of primitive mouse hematopoietic progenitor cells (CFU-S) and express γ-globin mRNA at levels ranging from 15-21% the level of α-globin mRNA per vector copy

Globin gene transfer in subjects with ß-thalassemia–progess, challenges, and clinical implementation

Michel Sadelain, MD, PhD, Center for Cell Engineering, MSKCC, New York, NY

The transplantation of hematopoietic stem cells (HSCs) harboring functional ß-globin genes is the only means known to cure ß-thalassemia major. While a fraction of all patients may find a matched HSC donor, the vast majority will not and is thus compelled to lifelong transfusion and chelation therapy. The utilization of the patient’s own HSCs, after they have been engineered to express therapeutic levels of ß-globin, is thus the most logical approach to extend the benefit of a curative therapy to potentially all patients. It has been over a decade since we first demonstrated the feasibility of transferring a therapeutic globin gene in HSCs and thereby cured ß-thalassemia in mice (May et al., Nature, 2000). Our findings were subsequently reproduced by several groups, who, as we showed, utilized lentiviral vectors encoding ß-like genes and similar genomic elements, and applied them to various mouse models of thalassemia and/or sickle cell anemia. Rather than rushing to a clinical trial, we spent the ensuing years investigating additional vectors encoding various combinations of proximal and distal transcription control elements, including the until then neglected HS1 element, and thus created a large family of globin vectors which possess distinct efficacy and possibly safety features. Vector safety was investigated in large cohorts of mice that we followed over 12-14 months, in which no single case of leukemia was ever observed. Based on a rational risk/benefit analysis, which we carried out with a large panel of experts in the US, Italy, and Greece, we proposed to the NIH and the FDA a conservative protocol that is based on the transduction of G-CSF-mobilized CD34+ cells and transplantation following non-myeloablative conditioning in subjects of age 15 or more. We obtained a unanimous favorable review of our protocol from the Recombinant DNA Advisory Committee. We next focused on the efficacy of stem cell collection in subjects with ß-thalassemia major and on the efficiency of gene transfer. We opened at MSKCC a clinical trial to mobilize CD34+ cells in adults with ß-thalassemia, which to date strongly supports the safety and efficacy of this process (PI: Dr. Farid Boulad). Studies we performed in late 2007 indicated that clinical grade globin vectors inefficiently transduce human CD34+ cells compared to most other lentiviral vectors, which could jeopardize any study. Over the next 18 months, we managed to substantially increase vector titers, setting the stage for completion of our IND application. Our clinical studies will start at MSKCC (PI: Dr. Farid Boulad) and the NIH (PI: Dr. John Tisdale), which will be followed by extended studies conducted at member centers of an international consortium we have established. In summary, following an extensive optimization of vector design, vector production and stem cell collection, and a rigorous risk/benefit assessment, the first US trial of globin gene transfer and several sister trials are poised to begin shortly.

Conversion To Transfusion Independence With Partial Clonal Dominance After Lentiviral Gene Therapy For Severe Human Beta-Thalassemia

Philippe Leboulch, MD^1,2,3 and the LentiGlobin clinical trial study group, ¹CEA, Institute of Emerging Diseases and Innovative Therapies (iMETI), Fontenay-aux-Roses 92265, France, 2Inserm U962 and University Paris XI, CEA-iMETI, Fontenay-aux-Roses 92265, France, 3Genetics Division, Brigham & Women’s Hospital and Harvard Medical School, Boston, MA

An 18 year old male with severe β^Ε/β°-thalassemia and no HLA-matched sibling donor was transplanted after Busulfex-mediated myeloablation with autologous bone narrow CD34+ cells transduced ex vivo with a lentiviral vector expressing a marked β^A-T87Q-globin gene. Before transplantation, the patient was dependent on monthly transfusions since age 3 (2 to 3 RBC packs each time; 157 ml RBCs/kg the year before transplant) with growth retardation and spontaneous hemoglobin (Hb) levels between 4 and 6 g/dL, splenectomized since age 6, and under iron chelation therapy since age 8. Hydroxyurea therapy was ineffective. Twenty five months after gene therapy, Hb levels are 10 g/dL, of which ~ 3.7 g/dL contains β^A-T87Q-globin, the remainder being HbE and HbF. No transfusion has been provided for the last 13 months, and there is near physiological levels of β^A-T87Q-globin expression on a per gene basis. Half of the therapeutic effect derives from a partially dominant cell clone with vector insertion within the HMGA2 gene, although β^A-T87Q-globin expression output shows little position dependency. While hematopoietic homeostasis is currently maintained, this observation together with recent results of deep sequencing analysis of other gene therapy clinical trials question whether therapeutic potency can be entirely dissociated from vector-mediated genomic effects.

Safety And Efficcacy Of G-Csf Mobilization In Major Beta-Thalassemia

Evangelia Yannaki, MD¹, Thalia Papayannopoulou, MD², Erica Jonlin, PhD², Ioannis Batsis, MD¹, Pamela Becker, MD², Fani Zervou, BSc¹, Angeliki Xagorari, PhD¹, Garyfalia Karponi, BSc¹., Varnavas Constantinou MD¹, Achilles Anagnostopoulos, MD¹., Athanasios Fassas, MD¹, George Stamatoyannopoulos, M.D², ¹George Papanicolaou Hospital, Thessaloniki, Greece, ²University of Washington, Seatlle, WA

In view of human gene therapy (GT) for thalassemia, the optimal source of hematopoietic stem cells (HSCs) for gene-engineering needs to be determined, because high numbers of genetically-modified HSCs are required to effectively compete for niche in the hypercellular thalassemic bone marrow (BM). Mobilized peripheral blood stem cells will probably become the preferable source of HSCs for thalassemia GT due to the higher yields of CD34+cells compared to conventional BM harvest. There is limited information on the mobilization efficacy and safety of adult patients with major β-thalassemia. The rare events of splenic rupture or thrombosis with G-CSF mobilization in normal donors of HSCs, may raise safety concerns for its use in thalassemia where chronic splenomegaly and hypercoagulability exist. Pretreatment with Hydroxyurea (HU) could reduce the risk of splenic rupture or thrombosis by decreasing the splenic hemopoiesis in the non-splenectomized (non-SPL) and the numbers of circulating cells in the splenectomized (SPL) patients before G-CSF initiation.

In an on going mobilization study, conducted at George Papanicolaou Hospital in collaboration with the University of Washington, we aim to assess the safety and efficacy of G-CSF mobilization with or without HU pretreatment in patients with β-thalassemia major. Fifteen patients have been enrolled so far, 8 SPL and 7 non-SPL. Five SPL and 3 non-SPL patients received HU pre-treatment for one month before G-CSF. No severe adverse event was observed. In SPL patients, HU was shown to normalize the high platelet and white blood cell numbers before G-CSF, preventing excessive leukocytosis (max WBCs during mobilization SPL-HU : 45 Χ109/l vs SPL-no HU 75 Χ109/l) and reducing thereby the risk of thrombosis during mobilization. In non-SPL patients, HU was shown to decrease the spleen volume over baseline (306cm3 vs 536cm3) resulting in 9% max increase during mobilization compared to 45% max size increase in non-SPL patients w/o HU pretreatment. HU negatively affected the CD34+yield in both the SPL (mean CD34+yield 0.62X106/kg/2aphereses) and non-SPL patients (mean CD34+yield 1,86X106/kg/2aphereses) when the ‘wash-out’ period before G-CSF was 6-10 days. However, when the interval period from HU stop to G-CSF initiation increased up to18 days in a non-SPL patient (P12), mobilization was successful (CD34+6.5X106/kg/2aphereses). Non-SPL patients w/o HU pretreatment yielded adequate numbers of HSCs (mean CD34+ 5,8 X106/kg/2aphereses). Unexpectedly, CD34+ cell yields were very low in the first 2 non-HU pre-treated SPL patients (mean CD34+0,98X106/kg/2aphereses) due to the development of early excessive leukocytosis (mean max WBCs 79Χ109/l) which necessitated G-CSF dose-hold or reduction. When lower and adjusted to the WBCs G-CSF doses were used and aphereses were initiated 1 day later (day5), mobilization was improved (P15:CD34+cells 4.5X106/kg/2aphereses).
Mobilization of SPL thalassemic patients presents a challenge. Mobilization may be not inherently inefficient in SPL patients but it probably results from the mandatory G-CSF-dose modifications to avoid hyperleukocytosis. Patient-tailored schemes of G-CSF mobilization or alternative ways of mobilization (ie AMD 3100) will be required in the GT setting in order to obtain high numbers of HSCs from SPL patients.

Transcriptional Silencing Of HBF by BCL11A

Stuart H. Orkin, MD, Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School and Howard Hughes Medical Institute, Boston, MA

Despite intensive study the molecular basis for silencing of fetal hemoglobin (HbF) expression has remained elusive until recently. A mechanistic understanding of the fetal-to-adult hemoglobin switch should stimulate targeted approaches to reactivating HbF expression in adults. Recently, we collaborated in the demonstration that genetic variation at a chromosome 2 encoded locus, BLC11A, is associated with the level of HbF in Sardianians and American Blacks in the NIH sickle cell natural history cohort (Uda et al, PNAS, 2008; Lettre et al, PNAS, 2008). The identification of BCL11A by genome-wide association studies provided a candidate regulator of γ-globin expression. This hypothesis was tested directly by down-modulating BCL11A expression in adult human CD34-progenitor derived erythroid precursors by siRNAs and shRNAs (Sankaran et al, Science, 2008). Knockdown of BCL11A expression led to marked reactivation of HbF expression. Moreover, expression of BCL11A correlates inversely with the level of HbF, BCL11A physically interacts with the NuRD chromatin complex as well as GATA1 and FOG1, and BCL11A occupies chromatin within the β-globin complex of adult erythroid precursors.

To test the role of BCL11A in HbF silencing more definitively, we have examined the expression of a β-globin locus YAC transgene in BCL11A knockout mice. Remarkably, loss of BCL11A leads to high-level expression of mouse embryonic β-like globin and human γ-globin RNAs in adult-type (definitive) erythroid cells (Sankaran et al, Nature, 2009). Thus, in addition to maintaining HbF repression in adult cells, BCL11A serves as a major transcriptional regulator of globin gene silencing during development. Further study of the mechanism by which BCL11A silences γ-globin expression should provide a solid foundation on which to explore targeted approaches to HbF reactivation.

Gene Therapy For Sickle Cell Disease And β-Thalassemia Using Lentiviral Vectors To Enhance Fetal Hemoglobin Production

Derek A. Persons¹, Andrew Wilber², Tamara Pestina¹, Huifen Zhao¹, Phillip Hargrove¹, Yoon Sang Kim¹, Matthew Wielgosz¹, Kelli M. Boyd³, Robert Throm¹, John Gray¹, Arthur Nienhuis¹. ¹Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN ²Department of Surgery, Southern Illinois School of Medicine, Springfield, IL, 3Veterinary Core Resource, St. Jude Children’s Research Hospital, Memphis, TN

Increased levels of red cell fetal hemoglobin (α2γ2; HbF) can ameliorate β-thalassemia and sickle cell disease (SCD). During the past decade, our efforts have thus been directed at developing an erythroid-specific, γ-globin lentiviral vector for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing HbF production as a therapeutic modality. Our work can be summarized as follows: 1) In a series of studies, we have shown that a highly engineered γ-globin lentiviral vector can cure murine models of γ-thalassemia intermedia and SCD by providing high levels of HbF expression. 2) We have documented therapeutic levels of HbF expression in differentiated erythroid cells following gene transfer into CD34+ cells from normal and β-thalassemic donors. 3) We have identified ex vivo gene transfer conditions and a vector envelope pseudotype that yield high levels of gene transfer into human primitive cells that engraft in an immunodeficient mouse model. 4) We have performed preclinical safety studies using state of the art genotoxicity assays. 5) We have developed a novel γ-globin lentiviral vector production system for generating GMP vector for a planned clinical trial. Currently, we are conducting gene transfer studies in primates while clinical trial protocol development proceeds. These data will be reviewed and our plans for a clinical trial for β-thalassemia and SCD discussed.

Session III: Iron Overload and Chelation Therapy

Deferiprone

Antonio Piga, MD, Simona Roggero, MD, Ilaria Salussolia, MD, Valeria Orecchia, MS, Filomena Longo, MD Thalassemia Center, University of Turin, Orbassano, Italy

Deferiprone (1,2-dimethyl-3-hydroxypyridin-4-one, or L1, DMHP, Ferriprox®), has been synthesized in 1984 and then extensively evaluated in a wide range of disorders; most of the available data have been obtained in transfusion-dependent thalassemia.
The safety and tolerability profile, as for the other two chelators deferoxamine and deferasirox, is peculiar. Side effects include gastrointestinal problems, raised liver enzymes, weight gain, arthropathy, neutropenia and agranulocytosis. This precludes the use of deferiprone in conditions with bone marrow activity abnormalities and requires close monitoring of blood count.

The efficacy profile is more similar among the three chelators. For deferiprone the choice of dosage is crucial to optimize the effect on liver iron concentration, according to the iron load degree, the transfusional iron input. Differences in hepatic glucuronidation efficiency may account for individual variability. Growing evidence, also from controlled studies, indicates that deferiprone is cardio-protective and effective in removing cardiac iron, alone or in combination with deferoxamine. In some studies, the effect on cardiac function is even more pronounced. These advantages may be explained by its physical characteristics and kinetics.
These data as a whole crop a non marginal role for deferiprone in the management of iron overload. There is a need to weight by randomized controlled trials the relative value of the deferiprone, deferasirox and deferoxamine in the long-term prevention of iron toxicity.

Combined Therapy

Renzo Galanello, MD¹, Annalisa Agus MD¹, Simona Campus MD¹, Fabrice Danjou MD¹, Robert Grady PhD², ¹Pediatric Clinic ² and Thalassemia Unit Asl⁸ - University of Cagliari Italy, ² Weill Cornell Medical Center, New York, NY

Combined therapy consists in the administration of two iron chelators in the same day simultaneously or sequentially. Combined chelation offers several potential advantages, including the concurrent access to multiple iron pools by different chelators, the achievement of iron excretion levels higher than those obtained by either drug alone, the possibility of decreasing the chelator dose while maintaining high iron excretion levels, better tolerability and greater compliance. Several retrospective and some prospective studies have shown that combined therapy with deferiprone and desferrioxamine is effective in reducing cardiac and hepatic iron overload, in improving cardiac function, in the management of iron-induced heart failure and in the reversal of some iron-induced endocrine complications. To date, combined therapy has shown no unanticipated side effects. No studies on other types of combined therapy (i.e. desferrioxamine and deferasirox, deferiprone and deferasirox) have been reported to date.

Deferasirox: An update

John Porter, MD, University College London, London, UK

Over 3000 transfusionally iron overloaded patients have been studied in prospective trials, allowing for evidence-based conclusions about the safety and efficacy of deferasirox across a range of underlying anaemias. Initial pharmacokinetics and metabolic balance studies demonstrated a long plasma half-life, suitable for once daily oral dosing, and a fecal route for iron excretion. Pivotal pre-registration studies established the relationship between dose, iron excretion and tolerability using changes in liver iron concentration (LIC) to measure changes in body iron. The dose allowing the rate of transfusional iron loading to be balanced by iron excretion can now be predicted across a range of underlying diagnoses. Unwanted effects included skin rash, and gastrointestinal disturbances. Serum creatinine increments ≥ 30% were seen in about one third of patients but this was not progressive, rarely exceeding the upper limit of normal. Study extensions of these patients at 4.5 years, including over 160 pediatric patients, show no progression of renal effects and a decrease in the frequency of skin, gastrointestinal and other unwanted effects. The proportion of patients with serum ferritin <1000µg/L has risen from 14% at y1 to 37% at 4.5y without an increase in adverse events. Doses >30mg/kg/day has been given in > 200 patients in trial settings without an increase in adverse events. The large-scale EPIC trial involving > 1600 patients has revealed further insights about the interaction of dose with ferritin trends and safety markers. Cardiac sub-studies of this trial, in >100 patients with established mild to moderate myocardial iron loading by T2*, show a significant reduction in cardiac iron.

Monitoring The Efficiency Of Iron Chelation Therapy

Robert C. Hider, PhD¹, Andre Silva, BSc, Yongmin Ma, PhD, Pharmaceutical Science Division, King’s College London, Franklin-Wilkins Building, 150 Stamford St. London, UK

With the introduction of orally active iron chelators a wider range of patients are now being treated for iron overload. Consequently there is an increased requirement for efficacy monitoring of these therapeutics. The application of MRI to estimate liver and heart iron content and the measurement of serum ferritin levels, transferrin saturation, and hepcidin levels, now offers an effective means of monitoring many of the effects of iron chelation. One parameter which is still not reliable and sufficiently reproducible to be used as a clinical test is the measurement of nontransferrin-bound iron (NTBI). There are a variety of measurements available, some claiming to measure the entire NTBI pool, others the redox active component of the pool. However todate none of these methods have attained general approval at the international level. In this presentation an update will be made on the chemical nature of NTBI and a novel fluorescence-based method for its measurement will be described.

Survival Trends Using Combination Chelation Therapy (CCT)

Paul Telfer, DM, FRCP, Barts and the London School of Medicine and Dentistry, UK

Iron chelation with a combination of deferoxamine (DFO) and deferiprone (DFP) has been used in Europe and the Middle East over the past 10 years for intensification of therapy in patients with thalassemia major who have moderate/heavy total iron load, have significant myocardial iron load, or are unable to adhere to sub cutaneous desferrioxamine at the recommended frequency. Various regimens have been described and most published reports show a more rapid reduction of iron loading compared with monotherapy. Adverse effects are frequent and intensive monitoring is required. Most of these are short term trials, and mortality rates while on CCT have been very low. The chelation protocol adopted in Cyprus provides useful quantitative data on the long-term effects of CCT on survival. A large cohort has been treated with a relatively uniform protocol and analysis has included all eligible patients in the south part of the Island, thereby reducing bias inherent in long-term follow-up of a non-randomised groups. Independent predictors of improved survival were female sex (Hazard Ratio (HR) 0.43, 95% CI 0.25-0.73); Birth >1973 (HR 0.3, 95% CI 0.17-0.51) and CCT (HR 0.14, 95% CI 0.04-0.41 for each year after switching therapy. Patients switched from DFO were 7 times more likely to survive per year of CCT, the majority of the survival benefit was due to reduction in cardiac deaths. This study provides further evidence for the cardioprotective effect of DFP. CCT given as a sequential regime is effective in controlling iron loading and preventing iron-related deaths in patients with iron accumulation after many years of DFO monotherapy, but should be supervised and monitored in a specialised centre.

Current Strategies For Chelation Therapy – How Will We Choose The Best Approaches In Thalassemia?

Ellis J. Neufeld MD, PhD, Division of Hematology/Oncology, Children’s Hospital Boston, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA

The 9th Cooley’s Anemia symposium is a timely occasion to reflect not only progress over the last few years in monitoring and treatment for transfusional iron overload, but also to consider where we are heading. Two key improvements even since the 8th symposium are wide use of non-invasive MRI assessments for iron overload, and regulatory approval and launch of deferasirox as an oral chelator. These advances are presented in many other talks at this symposium. This talk will consider how we can integrate these factors for optimum modern chelation practice. Underlying principles for this discussion include (i) the fact that many patients have suboptimal iron status in 2009; (ii) a consensus that wherever possible, prevention of iron overload and iron-related end-organ toxicity is preferable to “rescue therapy,” and (iii) monotherapy with deferasirox will not be a solution for all patients. The latter principle is based not only upon the evolving post-marketing data from the manufacturer, but also from investigator-initiated trials of deferasirox poor responders (Chirnomas et al, Blood, 2009, in press), and of combination therapy strategies, including deferoxamine with deferiprone for patients with or without heart disease, and deferasirox with deferoxamine. The two oral chelators have not been combined in any active trials to date. Current clinical practice at expert centers is substantially ahead of published clinical trials, in terms of dosing and combinations. Longitudinal observational studies of broader populations, including the NIH-sponsored Thalassemia Longitudinal Cohort study of the Thalassemia Clinical Research Network, may by the fastest and most reliable way to gain knowledge in the field.

Day 4: Saturday, October 24

Nutritional Deficiencies in Patients with Thalassmia

Ellen B. Fung, PhD RD, Elliott Vichinsky, MD, Children’s Hospital & Research Center at Oakland, Oakland, CA

Optimal nutritional status is imperative for achieving the genetic potential for growth and pubertal development in children as well as for robust immune function and bone health in adults. Patients with thalassemia are known to have abnormal growth, altered pubertal development and immune function as well as deficits in bone mineral acquisition. The etiology of these co-morbidites is typically ascribed to the toxic effects of transfusion related iron-overload. Though the relationship of nutritional status to abnormal growth, pubertal development, immune status and bone health has been shown in other chronic diseases, few studies have specifically focused on aspects of nutritional status and its relationship to health in patients with thalassemia.
Over the last decade, our group and others have observed deficiencies of up to 75% of patients studied in vitamins C, D, and selenium and up to 40% of patients appear deficient in copper, zinc, folate and other B vitamins. Typically, depleted circulating levels of nutrients have been observed despite seemingly adequate dietary intake. This disconnect between intake and circulating levels suggest that patients with thalassemia may have increased needs for certain nutrients due to either poor nutrient absorption, elevated losses or increased nutrient turnover.

Dairy intake and weight bearing physical activity are reduced in subjects with thalassemia and are related to poor bone health. Alternatively, higher fat mass has been shown to be protective in both transfused and non-transfused patients. These preliminary findings will be shared, in addition to results from an ongoing placebo controlled trial of zinc supplementation on bone health. Alternative therapies which utilize nutrient and antioxidant supplementation in addition to increased physical activity may improve nutritional status and overall health in this population, as well as decrease long term co-morbidities such as reduced bone mass.

Nutrition and Antioxidant Therapies in Thalassemia

Ashutosh Lal, M.D^1,2, Novie Ko, BS¹, Elliott Vichinsky, MD², Bruce Ames, PhD¹, Jung H Suh, MPH PhD^{1. 1}Children’s Hospital Oakland Research Institute, ²Hematology/Oncology, Children’s Hospital & Research Center at Research Center Oakland, Oakland, CA

The contribution of micronutrient deficiencies to the disease burden experienced by individuals with thalassemia may be considerable. Micronutrient depletion in thalassemia is appropriately considered in the context of iron overload and chelation therapy, and antioxidant defenses have received the most attention. The prevalence of certain deficiencies, particularly vitamin C, vitamin E, vitamin D, zinc and copper, is common, and can promote or worsen pathophysiologic mechanisms such as oxidative stress, immune dysfunction and bone mineral deficits. Chronic increase in free radical production can interfere with metabolism through oxidative modification of proteins and lipids. A metabolomics approach utilizing high-throughput liquid chromatography linked tandem mass spectrometric analysis of metabolites can demonstrate metabolic changes in response to sub-optimal nutrition. Using this approach, differences in the sulfur amino acid metabolism between thalassemia major and control subjects were detected in the plasma and also within the transfused donor red blood cells. The glutathione pool is expanded with accumulation of precursor compounds, but a significantly higher proportion of glutathione and cysteine exist in the oxidized state. Characterization of the compensatory mechanisms would help towards targeted interventions to restore the plasma and red cell redox balance. We propose gauging the utility of antioxidant therapy based on its remediating effects on altered metabolism, instead of specific markers of oxidative damage. Nutritional interventions should occur in the background of stable and effective chelation and transfusion therapies. The eventual goal is to evaluate whether target organ damage in thalassemia can be diminished through maintenance of optimal micronutrient status and the control of systemic oxidative stress.

Ferritin Regulation For Antioxidant Protection, And Iron Nutrition

Elizabeth C. Theil, CHORI (Children's Hospitals and Research Center Oakland), Oakland, USA; University of California, Berkeley, CA

Iron/oxygen reactions, the source of much oxidant damage, are also the substrates for cellular ferritin catalysis used to make the ferritin iron and oxygen mineral, ferrihydrite. Cellular ferritin functions as an oxidant protector by consuming iron and oxygen. Recent data show that ferritin genes are regulated by the same inducers that regulate oxidant protection enzymes such as quinone reductase and thioredoxin reductase.

In normal health, the cellular ferritin mineral i is a biological iron concentrate, inside the protective ferritin protein cage, that is used to synthesize iron–proteins such as hemoglobin and metabolic enzymes. During iron overload cellular ferritin also traps excess iron. Most of the iron in iron overload is in cellular ferritin and degraded ferritin (hemosiderin). Cellualr ferritin synthesis is increased in iron overload by removing a regulator that inhibits ferritin mRNA function. Usually the effect is so small that the iron content of ferritin increases causing protein damage and ferritin conversion to toxic hemosiderin.

Three novel ways to manipulate ferritin that can minimize effects of excess iron are:
1. Increase rates of removal of iron from ferritin by unfolding ferritin protein pores.
2. Increase rates of synthesis by targeting ferritin mRNA.
3. Decrease nutritional iron uptake with ferritin-rich (whole legume) foods, since the absorption mechanism is different than for iron salts or heme.
The stage of development of each of the three approaches will be described and new directions defined. Part Support: NIH, Cooley’s anemia and CHORI Foundations

Session IX: Clinical Syndromes in Thalassemia and Disease Severity

Alpha Thalassemia Syndromes

Elliott Vichinsky, MD, Children’s Hospital & Research Center Oakland, CA

Alpha thalassemia is the most common genetic disorder of hemoglobin synthesis, affecting up to 5% of the world’s population. It represents a group of conditions resulting from reduced or absent synthesis of one to all four of the alpha globin genes. Deletional or non-deletional mutations occur on chromosome 16. The severity of the syndrome ranges from completely asymptomatic to fatal in utero. Hemoglobin H disease, a mutation of three alpha globin genes, is more severe than previously recognized. Anemia, hypersplenism, hemosiderosis, growth failure, and osteoporosis are commonly noted as the patient ages. It can occur in all ethnic groups. In California, one in 9000 births has Hemoglobin H disease. Alpha thalassemia major, a usually fatal in utero disease, is now recognized to have a complex molecular and phenotypic expression with increasing births being reported. Surviving newborns without intrauterine transfusion often have congenital anomalies and neurocognitive injury. Serious maternal complications often accompany pregnancy. Doppler ultrasonography with intrauterine transfusion ameliorates these complications.  The high incidence of this disorder in many populations mandates population screening and prenatal diagnosis of at-risk couples. Universal newborn screening has been adopted in several regions, with necessary DNA confirmatory testing.

Phenotype/Genotype Correlation in Thalassemia Intermedia

C.Borgna-Pignatti, MD, Pediatric Department, University of Ferrara, Ferrara, Italy

Thalassemia intermedia is an heterogeneous clinical syndrome, spanning from an almost asymptomatic condition in which the slight anemia is diagnosed during a routine check-up, to a severe disease, often transfusion-independent only at the price of intense medullary expansion and extramedullary erythropoiesis. The pathophysiology of beta-thalassemia depends on the degree of alpha/non alpha globin chain imbalance. Any factor able to decrease the imbalance in homozygous beta thalassemia will improve the clinical picture. These factors include milder thalassemia alleles, increased production of fetal hemoglobin, concurrent alpha thalassemia. On the other hand, there are individuals with heterozygous beta thalassemia in whom the imbalance, and therefore the clinical picture, is made more severe by the co-inheritance of one or more supernumerary alpha globins. Genetic modifiers can also have a role in the development of complications in patients with thalassemia intermedia. The metabolism of bilirubin is influenced by the common A(TA)(n)TAA promoter mutation at the UGT1A1 locus, that has been demonstrated to be a risk factor for gallstones formation. The inheritance of the C282Y or the H63D polymorphism in the HFE gene can increase the iron absorption and make the hemosiderosis, often present in these patients, more severe. Several mutations at the basis of increased thrombophilia in the non thalassemic population could increase the already present thrombotic predisposition. Other as yet nonconclusive studies have investigated the correlation between osteoporosis and genes involved in bone metabolism, or between cardiac disease and the polymorphisms of apolipoprotein ε-4allele or glutathione S-transferase. There is no doubt, however, that the genetic heterogeneity at the basis of the phenotypic variability thalassemia intermedia will continue to be elucidated

Session X: The Adult Thalassemia Patient

Reproductive Issues in Females with Thalassemia

Sylvia T. Singer, MD¹, Annie Higa¹, Nancy Sweeters PNP^1, Olivia Vega BSc¹, Tiffany Chin BAIB¹, Deborah Trevithick PNP², Marcel Cedars MD²

¹Hematology/Oncology and Clinical Research Center at Children’s Hospital and Research Center Oakland CA; ²Division for Reproductive Endocrinology, UCSF,  Oakland, CA

Achievement of reproductive capacity and creating a family has become a great mission for many thalassemia major (TM) patients. Despite recent progress in assessment of reproductive function–there is limited implementation for females with thalassemia. Though infertility is thought to results primarily from iron toxicity to the pituitary-gonadal axis function, the extent of direct toxicity to the ovaries and amount of the ovarian function preserved in TM females is unknown. Since the natural mechanism of follicle aging is thought to result from oxidative stress, it is likely that in thalassemia females iron-induced oxidative damage can result in earlier follicular aging. Currently, there is no good data on the relation of reproductive potential-both pituitary function and ovarian reserve-to iron overload during puberty and adult life. We studied the predictors of reproductive impairment in 23 adult females with TM (median age 29 years, range 17-42). 20/23 had primary or secondary amenorrhea. FSH, LH and estradiol were low or undetectable in 16/23 women. Ovarian reserve testing (ORT) was determined by ultrasound for an antral follicular count (AFC), obtained in 16 women, and showed a poor AFC for age (4-7 follicles) in 10/16 (66%) women, intermediate AFC (8-12) in 3 and a normal AFC in 3 women. Anti-mullerian-hormone (AMH) levels were low (mean=2.9 ng/ml) and declined with age, correlating with AFC (R2=0.006). These findings demonstrate a low ovarian reserve in a majority of adult females with TM. Elevated LIC and NTBI were more prevalent in women with a low ovarian reserve and hypogonadotropic hypogonadism. Further study on the extent of iron load and toxicity and how it relates to ovarian reserve through puberty and adulthood, are needed.

Thrombosis, Stroke and their Prevention

M. Domenica Cappellini, MD¹, Giovanna Graziadei MD¹ , Khaled M. Musallam, MD^2, Ali T. Taher, MD², ¹Universitá di Milano, Policlinico Foundation IRCCS, Milan, Italy, 2American University of Beirut Medical Center, Beirut, Lebanon

The presence of a high incidence of thromboembolic events (TEE) has led to the identification of a hypercoagulable state in the thalassemia syndromes.¹ However, there are relatively few epidemiological data on the overall frequency of these complications. The largest clinical study to date, by Taher et al.², on 8860 thalassemia patients demonstrated that TEE occurred 4.38 times more frequently in thalassemia intermedia (TI) than thalassemia major (TM) patients, with more venous events occurring in TI and more arterial events (including stroke) occurring in TM. The study described age beyond 20 years, splenectomy, transfusion naïvety, family history and previous thrombotic events as the main risk factors for developing TEE. Moreover, in a study done to assess the rate of brain damage in patients with benign hemoglobinopathies, 37.5% of patients with TI showed asymptomatic brain damage on magnetic resonance imaging (MRI).³ More recently, unpublished data by Taher et al.⁴ on splenectomized TI patients observed a 60% rate of silent brain abnormality by MRI rising to 86.7% when combining MRI to position emission tomography (PET). The main risk factors for the development of silent strokes were increasing age and transfusion naïvety. Autopsy findings in thalassemia patients have confirmed hypercoagulability as a pathologic state. Autopsy series in patients with TM and TI describe the presence of DVT, pulmonary embolism and recurrent arterial occlusion, with thrombi in small and large pulmonary vessels.¹ Current understanding of the underlying pathophysiology describes a procoagulant activity of damaged circulating red blood cells (RBCs), co-inheritance of coagulation defects, depletion of antithrombotic factors, endothelial inflammation and conditions that increase thrombotic burden. These factors have been observed at a higher rate in splenectomized patients.⁵ Several studies have identified a role for transfusion therapy to control the rate of TEE since it decreased numbers of pathological RBC exhibiting indices of membrane damage.¹ The available data on the use of anticoagulants, antiplatelet, or other agents in thalassemia are either lacking or involve small, poorly controlled and/or relatively low-quality studies. However, TI patients who experience TEE and receive aspirin afterwards had a lower recurrence.² Treatment with the fetal hemoglobin-inducing agents, hydroxycarbmide and decitabine, results in decreases in plasma markers of thrombin generation. Hydroxycarbamide may decrease coagulation activation by reducing phospholipid expression on the surface of both RBC and platelets and decreasing RBC adhesion to thrombospondin. In addition to being a nitric oxide donor, hydroxycarbamide may also decrease hemostatic activation by its effect in decreasing the white blood cell count and particularly monocytes that express transcription factor. Another approach would be to correct the reactive oxygen species-induced RBC membrane damage using antioxidants, although this approach has not yet been verified in clinical trials.1 It may also be possible to design a thalassemia-tailored thrombosis risk-assessment model (RAM) to estimate thrombotic risk as a function of intrinsic and extrinsic factors. Moreover, tests for predisposing factors could also be performed, particularly in high-risk patients. If clinically verified, this type of model could serve as a guideline for possible preventative treatment to decrease the incidence of TEE, which can cause significant morbidity and mortality.¹

Osteoporosis in Beta-Thalassemia: Pathophysiology and Management

Evangelos Terpos, MD, PhD¹ & Ersi Voskaridou, MD, PhD^2
1Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece & ²Thalassemia Center, Laikon General Hospital, Athens, Greece

Osteoporosis represents a prominent cause of morbidity in patients of both genders with beta-thalassemia major (TM) or thalassemia intermedia. The pathogenesis of bone loss in thalassemia is multifactorial. The delay in sexual maturation, the presence of diabetes and hypothyroidism, the parathyroid gland dysfunction, the accelerated hemopoiesis with progressive marrow expansion, the direct iron toxicity on osteoblasts, the iron chelators, the deficiency of growth hormone or insulin growth factors have been identified as major causes of osteoporosis in thalassemia.

Osteoporosis is a progressive disease; thus prevention and early diagnosis are very important. Adequate hormonal replacement, effective iron chelation, improvement of hemoglobin levels, calcium and vitamin D administration, physical activity, and no smoking consist the main to-date measures for the management of the disease. However, despite the normalization of hemoglobin levels, adequate hormone replacement and effective iron chelation, patients continue to show an unbalanced bone turnover resulting in seriously diminished bone mineral density (BMD). During the last decade, novel pathogenetic data suggest that the reduced osteoblastic activity, which is believed to be the basic mechanism of bone loss in TM, is accompanied by a comparable or even greater increase in bone resorption. Increased levels of receptor activator of nuclear factor-kappaB ligand (RANKL) and bone resorption markers (N- and C-terminal cross-linking telopeptide of collagen type-I) support the presence of increased osteoclast activity in thalassemia patients. Therefore, the role of bisphosphonates, that are potent inhibitors of osteoclast activation, arises as major in the management of osteoporosis in these patients. Oral alendronate and intravenous pamidronate and zoledronic acid have shown efficacy in increasing BMD in thalassemia patients. However, many aspects have to be clarified before the broad use of bisphosphonates in TM-induced osteoporosis: which one? how long? and at what dose?

Other novel agents that stimulate bone formation such as teriparatide, a recombinant peptide fragment of parathyroid hormone and strontium ranelate, a second anabolic agent that prevents osteoporotic fractures in postmenopausal women are being studied but their effects in TM-induced osteoporosis have not been reported to-date. Antibodies against RANKL, such as denosumab, with proven efficacy in postmenopausal osteoporosis and antibodies against dickkopf-1, which has been found to be increased in TM patients with osteoporosis, or against sclerostin (SOST) may be future agents for the effective management of this difficult complication of thalassemia.