Markus Biburger

Susanne Aschermann

Anja Lux

Falk Nimmerjahn

The role of Fcγ receptors in murine autoimmune thrombocytopenia

Keywords: Autoimmunity, ITP, Immunoglobulin G, Inflammation, Fc receptors M, Biburger : S, Aschermann : A, Lux :

Immune thrombocytopenia (ITP) can become a life-threatening condition that requires immediate medical attention. The loss in platelet numbers during ITP can be induced by a variety of triggers. Anti-platelet antibodies of several isotypes and subclasses are a major cause for ITP and are a hallmark of many complex autoimmune diseases such as systemic lupus erythematosus. Mouse models have been important to understand the effector pathways involved in antibody-mediated platelet depletion. Therapeutic interventions based on these results have been proven successful in treating human ITP, thus validating the use of these model systems. One major problem that remains to be answered is which cell populations are crucial for platelet removal. Targeting these cells directly might be a novel therapeutic strategy and will also be important to understand the underlying biological mechanisms.

Introduction

Per year, roughly 0.05% to 0.1% of the population are estimated to develop immune thrombocytopenia (ITP) in the USA and Europe [START_REF] Psaila | Immune thrombocytopenic purpura[END_REF][START_REF] Cines | Management of adult idiopathic thrombocytopenic purpura[END_REF]. Half of these cases occur in children, where in the majority of cases, ITP is transient and resolves spontaneously after several months. Therefore, there is no urgent need for treatment, and observing platelet counts on a regular basis is usually sufficient. In adults, however, ITP often becomes chronic and requires medical intervention [START_REF] Cines | Management of adult idiopathic thrombocytopenic purpura[END_REF][START_REF] Karpatkin | Autoimmune (idiopathic) thrombocytopenic purpura[END_REF]. Depending on the magnitude of platelet reduction, several mild to life-threatening symptoms, including bleeding at cutaneous, gingival, intracranial, and internal sites, can occur. It is accepted that platelet-specific antibodies of the immunoglobulin (IgG) isotype are a major cause for immune-mediated platelet depletion. Plateletassociated IgG can be detected in approximately 50-70% of the patients with ITP, and more than 50% of newborn children of mothers with ITP will have a transient thrombocytopenia due to the transfer of IgG through the placenta [START_REF] Psaila | Immune thrombocytopenic purpura[END_REF][START_REF] Epstein | Congenial thrombocytopenic purpura; purpura hemorrhagica in pregnancy and in the newborn[END_REF][START_REF] Karpatkin | Cumulative experience in the detection of antiplatelet antibody in 234 patients with idiopathic thrombocytopenic purpura, systemic lupus erythematosus and other clinical disorders[END_REF]. Similarly, infusion of serum from thrombocytopenic patients induced rapid platelet depletion in healthy volunteers [START_REF] Sprague | Platelet transfusions and the pathogenesis of idiopathic thrombocytopenic purpura[END_REF]. Moreover, strategies aiming at depletion of autoantibody-producing B cells such as depletion with monoclonal antibodies (e.g., rituximab) or splenectomy are means to interfere with platelet depletion [START_REF] Karpatkin | Autoimmune (idiopathic) thrombocytopenic purpura[END_REF][START_REF] Garvey | Rituximab in the treatment of autoimmune haematological disorders[END_REF]. It is still unclear what triggers the generation of platelet-specific IgG. As ITP can coincide with certain infections, one possibility might be the generation of crossreactive antibodies during a pathogen-specific immune response recognizing platelet antigens [START_REF] Cines | The ITP syndrome: pathogenic and clinical diversity[END_REF]. The dominant target structures detected by murine and human plateletspecific IgG are glycoproteins (GP) such as GPIb, GPIIb, GPIIIa, or complexes thereof [START_REF] Karpatkin | Autoimmune (idiopathic) thrombocytopenic purpura[END_REF][START_REF] Beardsley | Platelet membrane glycoprotein IIIa contains target antigens that bind anti-platelet antibodies in immune thrombocytopenias[END_REF][START_REF] Fujisawa | Platelet-associated antibody to glycoprotein IIb/IIIa from chronic immune thrombocytopenic purpura patients often binds to divalent cationdependent antigens[END_REF][START_REF] Mcmillan | Autoantibodies and autoantigens in chronic immune thrombocytopenic purpura[END_REF][START_REF] Van Leeuwen | Specificity of autoantibodies in autoimmune thrombocytopenia[END_REF]. Very similar targets are recognized by murine platelet-specific autoantibodies arising in a variety of autoimmune-prone mouse strains such as BXSB mice or BXSB/NZW F1 offspring [START_REF] Mizutani | Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpuraprone (NZW x BXSB)F1 mice[END_REF]. Electron microscopy studies in human ITP patients have demonstrated that platelets can be taken up by monocytes, and injection of radio-labeled platelets showed that the major sites where platelets redistribute to after autoantibody injection are the spleen, the liver, and the lung [START_REF] Karpatkin | Autoimmune (idiopathic) thrombocytopenic purpura[END_REF][START_REF] Coetzee | The effect of monoclonal anti-humanplatelet antibodies on platelet kinetics in a baboon model: IgG subclass dependency[END_REF][START_REF] Heyns | Kinetics, distribution and sites of destruction of 111indium-labelled human platelets[END_REF][START_REF] Schmidt | Kinetics and distribution in vivo of 111In-labelled autologous platelets in idiopathic thrombocytopenic purpura[END_REF][START_REF] Stratton | Platelet destruction in autoimmune thrombocytopenic purpura: kinetics and clearance of indium-111-labeled autologous platelets[END_REF]. These results led to the conclusion that the reticulo-endothelial system is the major site for autoantibodymediated platelet removal. The cells of this system are of hematopoietic and non-hematopoietic origins and express receptors specific for the immunoglobulin G (IgG) Fc fragment. Cross-linking of Fcγ receptors (FcγR) by IgGcoated platelets results in the phagocytosis of these immune complexes and their removal from the circulation [START_REF] Nimmerjahn | Fc gamma receptors as regulators of immune responses[END_REF]. Other mechanisms that are discussed in the literature are complement-mediated lysis of platelets and the involvement of cytotoxic T cells [START_REF] Psaila | Immune thrombocytopenic purpura[END_REF]19]. While all these observations imply some role for these different effector pathways and organs, they cannot distinguish between the importance of the individual pathways for the observed platelet depletion in patients. Thus, further studies in animal models are of major importance to decipher the role of the different effector mechanisms and to develop novel therapeutic concepts. This review will discuss some of the recent insights that have been gained with respect to effector mechanisms leading to platelet depletion in mice and how this knowledge might translate to novel strategies for therapy.

Immunoglobulin G-mediated effector pathways

As has been discussed for humans, mainly two effector pathways were suspected to be important for IgG-mediated platelet removal in mice. Both of these pathways are dependent on the antibody constant domain, which in the case of IgG can bind to cell surface receptors on innate immune effector cells (FcγR) or activate the complement pathway [START_REF] Nimmerjahn | Fc gamma receptors as regulators of immune responses[END_REF][START_REF] Carroll | The complement system in regulation of adaptive immunity[END_REF][START_REF] Hogarth | Fc receptors are major mediators of antibody based inflammation in autoimmunity[END_REF]. FcγRs are widely expressed on cells of the innate (e.g., monocytes, mast cells, neutrophils, NK cells) and adaptive (B cells) immune system. In addition, FcγR expression has been demonstrated on dendritic cells and non-hematopoietic tissues such as endothelial and neuronal cells. Depending on the cell type, cross-linking of these receptors results in a variety of effector functions including antibody-dependent cell-mediated cytotoxicity, phagocytosis, release of pro-inflammatory mediators, or IgG-mediated feedback inhibition of B cells [START_REF] Nimmerjahn | Fc gamma receptors as regulators of immune responses[END_REF][START_REF] Heyman | Regulation of antibody responses via antibodies, complement, and Fc receptors[END_REF]. The family of murine and human FcγRs consists of several members that can be distinguished by two different means. First, by the affinity of the individual receptors for the different IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3 in mice) (Fig. 1). FcγRI (CD64) is the only receptor which can bind to monomeric IgG, whereas all the other receptors can bind IgG only in the form of immune complexes (IC). In general, the higher the affinity of the individual receptor, the narrower its specificity. Thus, FcγRI selectively binds to IgG2a with high affinity, FcγRIV binds IgG2a and IgG2b with intermediate affinity and FcγRIIB and FcγRIII bind IgG1, IgG2a, and IgG2b with low affinity [START_REF] Nimmerjahn | Fcgamma receptors: old friends and new family members[END_REF]. This in vitro binding pattern reflects the importance of the individual FcγRs for the activity of the individual IgG subclasses in different in vivo model systems. Thus, IgG1 is essentially dependent on FcγRIII, whereas IgG2a and IgG2b are additionally dependent on FcγRI and FcγRIV [START_REF] Baudino | Differential contribution of three activating IgG Fc receptors (Fc gamma RI, FcyRIII, and Fc gamma RIV) to IgG2a-and IgG2binduced autoimmune hemolytic anemia in mice[END_REF][START_REF] Giorgini | Fc gamma RIII and Fc gamma RIV Are Indispensable for Acute Glomerular Inflammation Induced by Switch Variant Monoclonal Antibodies[END_REF][START_REF] Hamaguchi | Antibody isotype-specific engagement of Fc gamma receptors regulates B lymphocyte depletion during CD20 immunotherapy[END_REF][START_REF] Hazenbos | Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice[END_REF][START_REF] Meyer | FcgammaRIII (CD16)-deficient mice show IgG isotype-dependent protection to experimental autoimmune hemolytic anemia[END_REF][START_REF] Nimmerjahn | Divergent immunoglobulin G subclass activity through selective Fc receptor binding[END_REF][START_REF] Otten | Experimental antibody therapy of liver metastases reveals functional redundancy between Fc gamma RI and Fc gamma RIV[END_REF][START_REF] Kaneko | Pathology and protection in nephrotoxic nephritis is determined by selective engagement of specific Fc receptors[END_REF]. The second distinguishing feature within the family of FcγRs is whether they transduce activating or inhibitory signals. There is one inhibitory FcγR, FcγRIIB that shows a broad expression pattern on hematopoietic and non-hematopoetic tissues [START_REF] Bolland | Inhibitory pathways triggered by ITIM-containing receptors[END_REF][START_REF] Ravetch | Immune inhibitory receptors[END_REF]. In contrast, FcγRI, FcγRIII, and FcγRIV are activating receptors which have a more restricted cell type expression pattern. None of the activating receptors can signal autonomously but has to associate with signaling adaptor molecules such as the FcR common γ chain (γ chain). On cell types such as monocytes, macrophages, and neutrophils, activating FcγRs are coexpressed with the inhibitory FcγRIIB [START_REF] Baerenwaldt | Immune regulation-Fc gamma RIIB-regulating the balance between protective and autoreactive immune responses[END_REF]. Thus, FcγRs set a threshold for cell activation as ICs will trigger both activating and inhibitory signaling pathways. The affinity of the individual IgG subclasses to the respective activating and the inhibitory FcγRIIB will determine antibody activity. Pro-inflammatory mediators, such as TNF-α, IFN-γ, or C5a, modulate IgG subclass activity by changing the ratio of activating to inhibitory FcγR expression [START_REF] Nimmerjahn | Fcgamma receptors: old friends and new family members[END_REF][START_REF] Schmidt | Fc receptors and their interaction with complement in autoimmunity[END_REF]. In addition to the differential binding of the individual IgG subclasses to FcγRs, there is a differential capacity to activate the complement pathway. Whereas IgG1 is very inefficient in triggering the classical pathway of complement activation by binding to C1q, the other three IgG subclasses can do so efficiently. Therefore, it was suggested that the activity of IgG2a and IgG2b might be dependent on both, cellular FcγRs and the complement pathway.

Effector pathways involved in murine ITP

Several platelet-specific monoclonal antibodies or polyclonal sera specific for mouse platelets can be used to induce ITP [START_REF] Nimmerjahn | Divergent immunoglobulin G subclass activity through selective Fc receptor binding[END_REF][START_REF] Deng | Comparison of the effects of antibody-coated liposomes, IVIG, and anti-RBC immunotherapy in a murine model of passive chronic immune thrombocytopenia[END_REF][START_REF] Nieswandt | Acute systemic reaction and lung alterations induced by an antiplatelet integrin gpIIb/IIIa antibody in mice[END_REF][START_REF] Clynes | Cytotoxic antibodies trigger inflammation through Fc receptors[END_REF][START_REF] Samuelsson | Anti-inflammatory activity of IVIG mediated through the inhibitory Fc receptor[END_REF][START_REF] Teeling | Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia[END_REF][START_REF] Alves-Rosa | Treatment with liposome-encapsulated clodronate as a new strategic approach in the management of immune thrombocytopenic purpura in a mouse model[END_REF]. One common antibody is the CD41-specific rat IgG1 clone MWReg30. As rat IgG1 is very similar to mouse IgG1, it is not efficient in activating mouse complement and can only bind to mouse FcγRIIB and FcγRIII. Consistent with these features, using either a blocking antibody for mouse FcγRIII or the FcγRIII knockout mouse completely prevented MWReg-induced platelet depletion [START_REF] Nieswandt | Acute systemic reaction and lung alterations induced by an antiplatelet integrin gpIIb/IIIa antibody in mice[END_REF][START_REF] Teeling | Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia[END_REF]. Similarly, a mouse IgG1 subclass switch variant derived from the platelet-specific mouse antibody clone 6A6 generated from (NZW×BSXB) F1 animals was completely dependent on FcγRIII [START_REF] Nimmerjahn | Divergent immunoglobulin G subclass activity through selective Fc receptor binding[END_REF]. More importantly, 6A6-IgG2a and IgG2b subclass variants were also fully dependent on cellular FcγRs for their activity despite their capacity to activate the complement pathway. Consistent with this observation, an IgG3 subclass variant, which does not bind to any of the known FcγRs with significant affinity but activates the complement pathway, did not lead to significant platelet depletion [START_REF] Nimmerjahn | Divergent immunoglobulin G subclass activity through selective Fc receptor binding[END_REF].

These results led to the conclusion that the complement system did not seem to be a major player for IgG-mediated platelet depletion in mice. Interestingly, a novel pathway resulting in a complement component C3-independent generation of C5a has been shown to be important for murine autoimmune hemolytic anemia induced by injection of red blood cell-specific IgGs [19,[START_REF] Schmidt | Fc receptors and their interaction with complement in autoimmunity[END_REF]. Although the details of this novel pathway are not entirely clear, it was suggested that C5a could be generated in an FcγRdependent fashion and act as a pro-inflammatory mediator changing the ratio of activating to inhibitory FcγR expression towards a higher level of activating FcγRs. Using either C5a receptor-deficient mice or biological inhibitors of C5a should answer whether this pathway is involved in murine ITP as well and whether this might be a promising novel therapeutic approach for human ITP. Another interesting observation was made with respect to the level of involvement of individual activating FcγRs for IgG2a and IgG2b-mediated platelet clearance. In contrast to the capacity of IgG2a to bind to the high affinity FcγRI, mice with a deletion of this receptor did not have altered rates of IgG2a mediated platelet clearance [START_REF] Nimmerjahn | Divergent immunoglobulin G subclass activity through selective Fc receptor binding[END_REF]. In contrast, blocking antibodies for the medium affinity receptor FcγRIV resulted in an impaired platelet clearance. Similar results were obtained in human ITP patients where blocking antibodies to FcγRIIIA, which is the human ortholog of murine FcγRIV [START_REF] Nimmerjahn | Fcgamma receptors: old friends and new family members[END_REF][START_REF] Hirano | IgEb immune complexes activate macrophages through FcgammaRIV binding[END_REF], but not to human FcγRI were very efficient in blocking platelet depletion [START_REF] Clarkson | Treatment of refractory immune thrombocytopenic purpura with an anti-Fc gamma-receptor antibody[END_REF][START_REF] Ericson | Monoclonal antibody 197 (anti-Fc gamma RI) infusion in a patient with immune thrombocytopenia purpura (ITP) results in down-modulation of Fc gamma RI on circulating monocytes[END_REF][START_REF] Psaila | Fc receptors in immune thrombocytopenias: a target for immunomodulation?[END_REF][START_REF] Wallace | Fc gamma RI blockade and modulation for immunotherapy[END_REF]. Thus, FcγRs are instrumental for IgG-mediated platelet depletion for murine and human ITP and are an attractive target for therapeutic intervention. Indeed, the injection of anti-D antibodies directed against red blood cells is an efficient means to block ITP by generating immune complexes that will compete for binding to activating FcγRs on innate immune effector cells. A problem arising with the use of FcγRIIIA-blocking antibodies in human therapy is the induction of severe neutropenia due to cross-reactivity of the therapeutic antibody with human FcγRIIIB expressed on neutrophils, thereby targeting them for attack via antibody-dependent cellular cytotoxicity. The use of antibody variants with a mutated Fc fragment or F(ab)2 fragments might reduce this unwanted side effect. Recent evidence suggests that a long known therapy for ITP, the intravenous infusion of high doses of immunoglobulin G molecules pooled from thousands of donors (IVIg therapy), might mediate its anti-inflammatory activity by upregulating the inhibitory FcγRIIB in mice and humans, thereby changing the threshold for innate immune effector cell activation [START_REF] Nimmerjahn | Anti-inflammatory actions of intravenous immunoglobulin[END_REF][START_REF] Tackenberg | Impaired inhibitory Fcgamma receptor IIB expression on B cells in chronic inflammatory demyelinating polyneuropathy[END_REF].

Effector cells and organs involved in murine (and human) ITP

As we now have a fairly complete picture of the molecular mechanisms responsible for IgG subclass-dependent platelet depletion, the crucial next question is which cell types express these receptors and where they are located (Fig. 2). Although this sounds trivial at first glance, we are only at the beginning to define the great variety of cell subpopulations in the myeloid lineage for example [START_REF] Gordon | Monocyte and macrophage heterogeneity[END_REF]. In humans, removal of the spleen is one option to stabilize platelet counts. In general, however, only 60-70% of the patients respond to this therapy, and in many cases, this does not lead to a full recovery of platelet numbers but rather stabilizes them on an acceptable level [START_REF] Cines | Management of adult idiopathic thrombocytopenic purpura[END_REF][START_REF] Karpatkin | Autoimmune (idiopathic) thrombocytopenic purpura[END_REF]. Similar results have been obtained with autoimmune-prone mouse strains that develop anti-platelet antibodies spontaneously [START_REF] Mizutani | Effects of splenectomy on immune thrombocytopenic purpura in (NZW x BXSB) F1 mice: analyses of platelet kinetics and anti-platelet antibody production[END_REF]. Of note, removal of the spleen also depletes a considerable number of autoantibody-producing plasma cells and plasma blasts. It is hard to evaluate therefore to what extent removal of splenic phagocytic cells contributes to the overall therapeutic effect. Thus, it is a safe assumption that other organs or cell populations outside the spleen are involved in platelet removal. Interestingly, there seems to be an age-dependent involvement of different organs.

During early age, splenectomy leads to better clinical responses than later in life [START_REF] Karpatkin | Autoimmune (idiopathic) thrombocytopenic purpura[END_REF]. Besides the spleen, the liver was suggested to be involved in removal of antibody-bound platelets. Using radioactively labeled platelets, it has been shown that a proportion of the injected radioactivity ends up in the liver after 24 h [START_REF] Schmidt | Kinetics and distribution in vivo of 111In-labelled autologous platelets in idiopathic thrombocytopenic purpura[END_REF][START_REF] Stratton | Platelet destruction in autoimmune thrombocytopenic purpura: kinetics and clearance of indium-111-labeled autologous platelets[END_REF]. While this is interesting, it does not exclude that cells after having taken up antibody-coated platelets migrate to the liver subsequently.

In fact, injection of anti-platelet antibodies leads to a maximum of platelet depletion within 1 to 4 h in mice and humans [START_REF] Sprague | Platelet transfusions and the pathogenesis of idiopathic thrombocytopenic purpura[END_REF][START_REF] Nimmerjahn | Divergent immunoglobulin G subclass activity through selective Fc receptor binding[END_REF]. As further studies cannot be done in humans, murine model systems are required. The problem here is that a lot of crucial information regarding the mechanism and players involved in IgG-mediated effects have only been identified recently and are still under investigation. For example, the mouse ortholog to human FcγRIIIA has only been identified 4 years ago and is still not fully characterized [START_REF] Hirano | IgEb immune complexes activate macrophages through FcgammaRIV binding[END_REF][START_REF] Nimmerjahn | FcgammaRIV: a novel FcR with distinct IgG subclass specificity[END_REF]. More importantly, monoclonal antibodies specific for all mouse-activating FcγRs have become available only recently. Therefore, there are still many open questions with respect to the exact FcγR expression pattern on individual cell types. In the blood, FcγRIII (important for IgG1-mediated platelet depletion)

and FcγRIV (important for IgG2a/IgG2b-mediated platelet depletion) are largely expressed on monocytes and neutrophils [START_REF] Nimmerjahn | Fc gamma receptors as regulators of immune responses[END_REF][START_REF] Hirano | IgEb immune complexes activate macrophages through FcgammaRIV binding[END_REF]. NK cells, mast cells, eosinophils, and dendritic cells on the other hand largely lack expression of FcγRIV but do express FcγRIII. Besides the blood, most other organs such as the spleen, the liver, the lung, and the kidney contain cell types expressing different FcγRs. One of the dominant sources of FcγR expression in these organs are tissue-specific resident macrophages and endothelial cells. In the liver for example, Kupffer cells seem to express activating FcγRs in the spleen, red pulp macrophages are highly positive for FcγRs and thus could be involved in antibody-mediated platelet depletion [START_REF] Otten | Experimental antibody therapy of liver metastases reveals functional redundancy between Fc gamma RI and Fc gamma RIV[END_REF]. In addition, strong FcγR expression can be detected on endothelial cells, although here, the dominant receptor seems to be the inhibitory FcγRIIB and probably FcγRIII to some degree. Therefore, either tissue-resident macrophages or blood monocytes and neutrophils could be involved in IgG2a/ IgG2b-mediated platelet depletion, whereas IgG1-mediated platelet depletion could be triggered by other cell types as well. One of the cell types that can be excluded are NK cells as they do neither express FcγRIV nor co-express the inhibitory FcγRIIB, which regulates the activity of all IgG subclasses in mice [START_REF] Nimmerjahn | Fc gamma receptors as regulators of immune responses[END_REF]. Ideally mice with cell type-specific deletions of individual activating FcγRs would be required to come to definitive conclusions. Until these animals Fig. 2 The hunters and the prey. Shown are the effector cells and organs, which could be involved in autoantibodymediated platelet depletion in ITP. In the blood, several cell types such as eosinophils, neutrophils, and monocyte subpopulations might be involved in platelet phagocytosis. In organs such as the lungs, the spleen, and the liver, tissueresident macrophages are prime candidates as responsible effector cells. Besides cells of the hematopoietic system, nonhematopoietic cells such as endothelial cells express FcγRs and might phagocytose plateletcontaining immune complexes become available, surrogate systems need to be used. Liposomes containing cytotoxic substances result in the specific depletion of monocytes and macrophages in the blood and in tissues such as the lung, the liver, and the spleen [START_REF] Van Rooijen | Clodronate liposomes: perspectives in research and therapeutics[END_REF]. Indeed, liposome-treated mice showed an impaired platelet clearance mediated by a polyclonal rabbit anti-mouse platelet serum suggesting that monocytes and macrophages are involved in this process. As there are many different subpopulations of monocytes and macrophages, it is unclear which of these are important [START_REF] Gordon | Monocyte and macrophage heterogeneity[END_REF]. In addition, tissue macrophages are sometimes involved in maintenance of tissue organization, and depletion of spleen macrophages often leads to secondary rearrangements in structure, which might be important for antibody-mediated platelet depletion. One step closer to the human system, mice transgenic for human FcγRs have been developed and will be useful to study the impact of human FcγRs in a mouse model in vivo [START_REF] Mckenzie | Humanized mouse models of FcR clearance in immune platelet disorders[END_REF].

Conclusion and outlook

Taken together, recent studies have shed some light on the molecular pathways involved in immunoglobulin G-mediated platelet depletion. Despite the efficient activation of the complement pathway by the most active IgG subclasses, it seems clear that this does not directly contribute to platelet removal but may serve as an enhancing mechanism to modulate activating and inhibitory FcγR expression. In contrast, studies in humans and mice clearly indicate the importance of cellular FcγRs for platelet removal. As there are many different cell types that express the relevant FcγRs, it is unclear which of these contribute to platelet depletion. In addition, many of these cell populations are located in a variety of tissues and organs and thus are difficult to analyze. Although splenic cell populations seem to be involved in antibody-mediated platelet removal, it seems clear that alternative pathways independent of the spleen must exist and need to be characterized in detail to gain a deeper insight into the biology and pathomechanism of ITP.

Fig. 1

 1 Fig. 1 Antibody Fcγ receptor interactions. Shown is the family of FcγRs in mice and their interactions with immunoglobulin G subclasses. The principal distinguishing feature between the family members is the differential affinity for IgG subclasses, the specificity

Acknowledgment This work was supported by grants from the Bavarian Genome Research Network (BayGene) and the German Research Foundation (SFB 643 and FOR832).


A Palumbo
email: appalumbo@yahoo.com

F Davies

M Kropff

J Bladé

M Delforge

F Costa

R Garcia

F Leal Da Costa

Garcia Sanz

S Schey

T Facon

G Morgan

P Moreau

F Leal

R Garcia Sanz

Consensus guidelines for the optimal management of adverse events in newly diagnosed, transplant-ineligible patients receiving melphalan and prednisone in combination with thalidomide (MPT) for the treatment of multiple myeloma

Keywords: Myeloma, MM, Thalidomide, Treatment guidelines

Consensus guidelines for the optimal management of adverse events in newly diagnosed, transplant-ineligible patients receiving melphalan and prednisone in combination with thalidomide (MPT) for the treatment of multiple myeloma

Introduction

Multiple myeloma (MM) is a hematologic cancer of the plasma cells, which results in bone destruction and marrow failure, potentially leading to osteolytic lesions alongside cytopenia-related bacterial infections and anemia [START_REF] Smith | Guidelines on the diagnosis and management of multiple myeloma 2005[END_REF][START_REF] Anderson | NCCN Clinical Practice Guidelines[END_REF]. MM is primarily a disease of the elderly, with 70% of patients 65 years or older at diagnosis [START_REF] Smith | Guidelines on the diagnosis and management of multiple myeloma 2005[END_REF]. This trend has notable implications for the pharmacological management of MM: high-dose chemotherapy and stem cell transplantation are the conventional treatments for MM patients younger than 65 years, with adequate performance status and organ function [START_REF] Smith | Guidelines on the diagnosis and management of multiple myeloma 2005[END_REF], and this approach may not be suitable for older MM patients or those with concomitant disease.

Although the combination of oral melphalan and prednisone (MP) has been the standard front-line therapy for MM for more than four decades in older transplantineligible patients [START_REF] Anderson | NCCN Clinical Practice Guidelines[END_REF], outcomes in this group remain suboptimal. A meta-analysis has suggested that conventional combination chemotherapy offers no survival advantage over MP [START_REF]Combination chemotherapy versus melphalan plus prednisone as treatment for multiple myeloma: an overview of 6,633 patients from 27 randomized trials[END_REF], leading to growing interest in other novel agents such as thalidomide, lenalidomide, and bortezomib.

Following positive results in the relapsed/refractory setting [START_REF] Singhal | Antitumor activity of thalidomide in refractory multiple myeloma[END_REF][START_REF] Barlogie | Extended survival in advanced and refractory multiple myeloma after single-agent thalidomide: identification of prognostic factors in a phase 2 study of 169 patients[END_REF][START_REF] Van Rhee | First thalidomide clinical trial in multiple myeloma: a decade later[END_REF], five phase III studies assessing thalidomide in combination with MP (MPT), in newly diagnosed elderly patients ineligible for transplant, have been undertaken: the Italian Multiple Myeloma Study Group (GIMEMA) study, two studies from the Intergroupe Francophone du Myelome (IFM 99-06 and IFM 01-01), the study conducted by the Nordic Myeloma Study Group and the Dutch Haemato-Oncology Cooperative Group (HOVON) 49 study [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF][START_REF] Hulin | Efficacy of melphalanprednisone-thalidomide (MP-T) to melphalan-prednisone (MP) in patients 75 years of age or older with untreated multiple myeloma (MM). Preliminary results of the randomized, double-blind, placebo controlled IFM 01-01 trial[END_REF][START_REF] Waage | Melphalanprednisone-thalidomide to newly diagnosed patients with multiple myeloma: a placebo controlled randomised phase 3 trial[END_REF][START_REF] Wijermans | Melphalan + prednisone versus melphalan + prednisone + thalidomide in induction therapy for multiple myeloma in elderly patients: final analysis of the Dutch Cooperative Group HOVON 49 study[END_REF][START_REF] Palumbo | Oral melphalan, prednisone and thalidomide in elderly patients with multiple myeloma: updated results of a randomized controlled trial[END_REF]. In these studies, MPT demonstrated significantly improved response rates compared with MP. Moreover, survival was prolonged by approximately 18 months in patients receiving thalidomide compared with MP alone in the IFM studies [START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF][START_REF] Hulin | Efficacy of melphalanprednisone-thalidomide (MP-T) to melphalan-prednisone (MP) in patients 75 years of age or older with untreated multiple myeloma (MM). Preliminary results of the randomized, double-blind, placebo controlled IFM 01-01 trial[END_REF]. However, in the GIMEMA, Nordic and HOVON 49 studies, while improved response rates did not result in an overall survival benefit [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Waage | Melphalanprednisone-thalidomide to newly diagnosed patients with multiple myeloma: a placebo controlled randomised phase 3 trial[END_REF][START_REF] Wijermans | Melphalan + prednisone versus melphalan + prednisone + thalidomide in induction therapy for multiple myeloma in elderly patients: final analysis of the Dutch Cooperative Group HOVON 49 study[END_REF][START_REF] Palumbo | Oral melphalan, prednisone and thalidomide in elderly patients with multiple myeloma: updated results of a randomized controlled trial[END_REF], the addition of thalidomide resulted in a longer median progression-free survival in the GIMEMA study [START_REF] Palumbo | Oral melphalan, prednisone and thalidomide in elderly patients with multiple myeloma: updated results of a randomized controlled trial[END_REF] and a superior eventfree survival in the HOVON 49 study [START_REF] Wijermans | Melphalan + prednisone versus melphalan + prednisone + thalidomide in induction therapy for multiple myeloma in elderly patients: final analysis of the Dutch Cooperative Group HOVON 49 study[END_REF].

On the basis of these data, MPT has been approved by the European Agency for the Evaluation of Medicinal Products for the treatment of newly diagnosed MM patients older than 65 years or ineligible for transplant and is also recommended for this treatment group by the National Comprehensive Cancer Network (NCCN) and Mayo Clinic guidelines [START_REF] Anderson | NCCN Clinical Practice Guidelines[END_REF][START_REF] Dispenzieri | Treatment of newly diagnosed multiple myeloma based on Mayo Stratification of Myeloma and Risk-adapted Therapy (mSMART): consensus statement[END_REF].

In order to optimize the clinical effectiveness of MPT therapy, it is imperative to have a good awareness of the associated adverse events (AEs) and to have strategies in place on how best to manage them. To achieve this, an expert panel of hematologists convened to address the key issues when treating newly diagnosed patients with MPT and to provide recommendations to ensure optimal patient outcomes. This manuscript outlines both evidenceand consensus-based recommendations discussed by the panel, to provide a practical guide for physicians addressing the effective management of newly diagnosed, transplant-ineligible MM patients receiving thalidomide therapy.

Optimizing treatment through side effect management

Thalidomide is likely to be prescribed in combination with MP for up to 18 months [14]. Therefore, optimizing quality of life through a good understanding and management of potential treatment-related AEs is essential.

Thalidomide, in combination with MP, is associated with hematological side effects (neutropenia, anemia, thrombocytopenia), thromboembolic side effects, and nonhematologic side effects, including peripheral neuropathy, Somnolence [START_REF] Ghobrial | Management of thalidomide toxicity[END_REF][START_REF] Dimopoulos | Adverse effects of thalidomide administration in patients with neoplastic diseases[END_REF] • Usually mild and dose-dependent, occurring within 15 days of initiating thalidomide treatment • Limited by taking treatment at bedtime Constipation [START_REF] Dimopoulos | Adverse effects of thalidomide administration in patients with neoplastic diseases[END_REF] • Treated with simple measures such as diet, laxatives, and exercise Impotence • Occurs in around 50% of male patients receiving thalidomide treatment [START_REF] Murphy | Thalidomide induced impotence in male hematology patients: a common but ignored complication?[END_REF] • Discuss with male patients before commencing therapy • Consider prescribing sildenafil or similar agents

Thyroid dysfunction [START_REF] Dimopoulos | Adverse effects of thalidomide administration in patients with neoplastic diseases[END_REF] • Measure thyroid-stimulating hormone at baseline and at regular intervals Edema • Mild peripheral edema may occur in about 15% of patients receiving thalidomide [START_REF] Ghobrial | Management of thalidomide toxicity[END_REF] • Severe edema is less common, occurring in up to 3% of patients [START_REF] Ghobrial | Management of thalidomide toxicity[END_REF] Table 1 Easily manageable adverse events somnolence/fatigue, constipation, arrhythmias [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF], and cutaneous reactions [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF]. In MM patients, some of these AEs (such as neutropenia, thrombocytopenia, fatigue, and thromboembolic events) are also seen with the thalidomide analog, lenalidomide [START_REF] Palumbo | Melphalan, prednisone and lenalidomide treatment for newly diagnosed myeloma: a report from the GIMEMA-Italian multiple myeloma network[END_REF]. Some AEs reported in relation to thalidomide are relatively well known (see Table 1) while others are less common. It is important to be able to recognize these as early as possible so that appropriate management strategies can be implemented and thalidomide treatment can be optimized.

Thromboembolic events

Venous thromboembolism (VTE) is a critical clinical condition manifesting as deep vein thrombosis (DVT) and/or pulmonary embolism (PE) [START_REF] Palumbo | Prevention of thalidomide-and lenalidomide-associated thrombosis in myeloma[END_REF]. The annual incidence is > 1 per 1,000 in the general population [START_REF] Silverstein | Trends in the incidence of deep vein thrombosis and pulmonary embolism: a 25-year population-based study[END_REF] with one third of all cases of VTE presenting as sudden death [START_REF] Heit | Predictors of survival after deep vein thrombosis and pulmonary embolism: a populationbased, cohort study[END_REF]. This is of particular importance in MM patients because, although the risk of VTE is higher among cancer patients versus the general population [START_REF] Sallah | Venous thrombosis in patients with solid tumors: determination of frequency and characteristics[END_REF], it is further increased with hematologic malignancies [START_REF] Blom | Malignancies, prothrombotic mutations, and the risk of venous thrombosis[END_REF].

Thalidomide is a convenient oral therapy that avoids the potential complications of long-term central venous catheter use, such as infection and line-related throm-bosis [START_REF] Boersma | Thrombotic and infectious complications of central venous catheters in patients with hematological malignancies[END_REF]. However, thalidomide treatment is associated with an increased risk of VTE, especially when used in combination with dexamethasone [START_REF] El Accaoui | Thalidomide and thrombosis. A meta-analysis[END_REF] and/or chemotherapy [START_REF] Zangari | Thrombogenic activity of doxorubicin in myeloma patients receiving thalidomide: implications for therapy[END_REF][START_REF] Zangari | Thalidomide and deep vein thrombosis in multiple myeloma: risk factors and effect on survival[END_REF][START_REF] Schutt | Thalidomide in combination with vincristine, epirubicin and dexamethasone (VED) for previously untreated patients with multiple myeloma[END_REF]-the risk is generally greatest in patients treated with thalidomide in the first-line, as opposed to the relapsed/refractory setting (Table 2). There is also evidence to suggest that the risk of developing VTE with thalidomide therapy is influenced by genetic and environmental factors [START_REF] Johnson | Genetic associations with thalidomide mediated venous thrombotic events in myeloma using targeted genotyping[END_REF].

In the IFM 99-06 and GIMEMA studies of MPT in the first-line treatment of newly diagnosed, transplantineligible MM patients, the occurrence of DVT and PE was highest during the first 4 months of MPT therapy [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF]. It is essential to identify patients at risk of thromboembolic complications and use appropriate prophylaxis to reduce their incidence. Risk factors for VTE are summarized in Table 3. It is noteworthy that, since the incidence of MM is increased in the elderly with the majority of patients older than 60 years [START_REF] Smith | Guidelines on the diagnosis and management of multiple myeloma 2005[END_REF], age has not been not included as a risk factor. Thromboprophylaxis is mandatory in patients receiving MPT, who should be assessed for VTE risk factors prior to initiating MPT therapy (Fig. 1). The presence of at least one risk factor in addition to MM confers a high risk of developing thromboembolic complications, and prophylactic low molecular weight heparin (LMWH) is advised in these circumstances. However, as LMWH requires daily intravenous

Hematologic side effects

The occurrence of cytopenias has been noted in patients receiving MP therapy [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF][START_REF] Costa | Melphalan and prednisone: an effective combination for the treatment of multiple myeloma[END_REF]; the addition of thalidomide to this regimen results in grade 3-4 thrombocytopenia in 3-14% of patients, and grade 3-4 neutropenia and anemia in 16-48% and 3-14% of patients, respectively [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF]. Fatigue, bleeding, and infections are associated with anemia, thrombocytopenia, and neutropenia, respectively, and can therefore have a negative impact on patients' quality of life.

The two IFM studies have reported a higher incidence of grade 3-4 neutropenia with MPT compared with MP alone (48% vs 26%, respectively, in the IFM 99-06 study and 23% vs 9%, respectively, in the IFM 01-01 study) [START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF][START_REF] Hulin | Efficacy of melphalanprednisone-thalidomide (MP-T) to melphalan-prednisone (MP) in patients 75 years of age or older with untreated multiple myeloma (MM). Preliminary results of the randomized, double-blind, placebo controlled IFM 01-01 trial[END_REF], although this did not result in an increased infection rate in the IFM 99-06 study [START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF]. On the basis of these data, it is recommended that hematologic counts are undertaken prior to MPT commencement, with treatment postponement and dose reductions in cases of low counts.

Peripheral neuropathy

Peripheral neuropathy (PNP) can be associated with the MM disease process, occurring in approximately 13% of patients prior to initiation of thalidomide therapy [START_REF] Plasmati | Neuropathy in multiple myeloma treated with thalidomide: a prospective study[END_REF]. It is also known to be a side effect of several agents used in the treatment of MM, including thalidomide [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF][START_REF] Hulin | Efficacy of melphalanprednisone-thalidomide (MP-T) to melphalan-prednisone (MP) in patients 75 years of age or older with untreated multiple myeloma (MM). Preliminary results of the randomized, double-blind, placebo controlled IFM 01-01 trial[END_REF] and bortezomib [START_REF] Miguel | A practical update on the use of bortezomib in the management of multiple myeloma[END_REF][START_REF] Richardson | Frequency, characteristics, and reversibility of peripheral neuropathy during treatment of advanced multiple myeloma with bortezomib[END_REF]. The symptoms of PNP noted with thalidomide include numbness, tingling, discomfort, and abnormal coordination or weakness. Careful management is required as these can be irreversible [START_REF] Ghobrial | Management of thalidomide toxicity[END_REF]. Bortezomib-associated PNP is characterized by pain, paresthesia, burning dysesthesia, and numbness, with feet more often affected than hands [START_REF] Miguel | A practical update on the use of bortezomib in the management of multiple myeloma[END_REF]. Prevalence appears to increase with cumulative dose and is irreversible in 29% of patients [START_REF] Richardson | Frequency, characteristics, and reversibility of peripheral neuropathy during treatment of advanced multiple myeloma with bortezomib[END_REF].

With the MPT combination, grade 3-4 PNP has been reported in 6-8% of patients [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF], although in patients≥75 years, the incidence of grade 2-4 PNP has been reported as 18% [START_REF] Hulin | Efficacy of melphalanprednisone-thalidomide (MP-T) to melphalan-prednisone (MP) in patients 75 years of age or older with untreated multiple myeloma (MM). Preliminary results of the randomized, double-blind, placebo controlled IFM 01-01 trial[END_REF]. The incidence and severity are likely to be related to dose and duration of treatment [START_REF] Ghobrial | Management of thalidomide toxicity[END_REF][START_REF] Cavaletti | Thalidomide sensory neurotoxicity: a clinical and neurophysiologic study[END_REF][START_REF] Mileshkin | Development of neuropathy in patients with myeloma treated with thalidomide: patterns of occurrence and the role of electrophysiologic monitoring[END_REF], although this is disputed in some studies [START_REF] Plasmati | Neuropathy in multiple myeloma treated with thalidomide: a prospective study[END_REF]. There is also some debate as to time of onset; in the GIMEMA study of first-line MPT, grade 3-4 PNP was reported after a median of 8 months, while grade 2 PNP led to reductions in thalidomide dose after a median of 5.8 months [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF]. However, there have also been reports of PNP after shorter-term use. In one study of newly diagnosed patients receiving thalidomide therapy alone (200 mg/day), PNP occurred as early as 4 months in approximately 66% of patients and in all patients by 7 months [START_REF] Plasmati | Neuropathy in multiple myeloma treated with thalidomide: a prospective study[END_REF]. It is recommended that patients are assessed prior to and at regular intervals during treatment so that the dose of thalidomide can be reduced if PNP develops (Table 4).

PNP recommendations

• Clinical assessment of all newly diagnosed patients prior to treatment commencement, although nerve conduction studies are not required • Patient education is important to ensure early detection of PNP ○ Patient perceptions are key; different patients are willing to accept varying degrees of PNP at different stages of disease. Physicians should ensure that this is fully discussed • Regular clinical assessment for symptoms of PNP to evaluate changes in patients' symptoms; the majority of patients will develop PNP given sufficient length of treatment with thalidomide [START_REF] Mileshkin | Development of neuropathy in patients with myeloma treated with thalidomide: patterns of occurrence and the role of electrophysiologic monitoring[END_REF] Teratogenicity and the risk management plan for pregnancy Teratogenicity is the most widely known side effect of thalidomide. Thalidomide is absolutely contraindicated in women who are, or could become, pregnant. Therefore, access to the drug in most countries requires participation in a risk management plan to ensure appropriate precautions are taken.

Bradycardia and syncope

Bradycardia is associated with dizziness, weakness, or syncope while more severe forms can lead to convulsions or sudden cardiac death. Thalidomide-induced bradycardia has been observed in patients with comorbidities or concurrent medications that decrease heart rate [START_REF] Kaur | Thalidomide-induced sinus bradycardia[END_REF] and in some situations may require the placement of a pacemaker to alleviate symptoms [START_REF] Fahdi | Bradycardia during therapy for multiple myeloma with thalidomide[END_REF]. Thalidomide is associated with additional non-thrombotic cardiovascular effects [START_REF] Bladé | Thalidomide in refractory and relapsing multiple myeloma[END_REF][START_REF] García-Sanz | The oral combination of thalidomide, cyclophosphamide and dexamethasone (ThaCyDex) is effective in relapsed/refractory multiple myeloma[END_REF] with grade 3-4 cardiac toxicity observed in 7% of patients receiving thalidomide in combination with bortezomib and prednisone [START_REF] Mateos | Bortezomib (Velcade)melphalan-prednisone (VMP) versus velcade-thalidomideprednisone (VTP) in elderly untreated multiple myeloma patients: which is the best partner for velcade: an alkylating or an immunomodulator agent?[END_REF]. A cardiologic examination by the consulting a hematologist is therefore recommended for all patients prior to commencing thalidomide treatment and monitoring should be carried out during therapy so that the dose can be modified if bradycardia is apparent.

Recommendations for bradycardia and syncope • Cardiologic assessment for all patients prior to commencing therapy •Monitor for bradycardia ○ Dose reduction or discontinuation of thalidomide may be required

Cutaneous reactions

Skin rashes are frequent complications with thalidomide treatment [START_REF] Hall | Dermatologic side effects of thalidomide in patients with multiple myeloma[END_REF]; minor-moderate skin reactions have been reported in 46% of patients [START_REF] Hall | Dermatologic side effects of thalidomide in patients with multiple myeloma[END_REF] with case reports of more serious toxic epidermal necrolysis [START_REF] Horowitz | Thalidomide-induced toxic epidermal necrolysis[END_REF][START_REF] Rajkumar | Life-threatening toxic epidermal necrolysis with thalidomide therapy for myeloma[END_REF] and morbilliform rash [START_REF] Nijsten | Thalidomideinduced morbilliform rash: diagnosis and continuation of therapy, premedicated with methylprednisolone[END_REF]. Grade 3-4 dermatologic side effects were reported in 3-6% of patients receiving MPT in the GIMEMA study, with Reduce dose by up to 50%, or interrupt treatment and continue to monitor the patient with clinical and neurological examination. If no improvement or worsening of the neuropathy continues, discontinue treatment. If the neuropathy resolves to Grade 1 or better, the treatment may be restarted at 50% of the last dose, if the benefit/risk is favorable. Grade 3 (interfering with activities of daily living) Discontinue treatment. Grade 4 (neuropathy which is disabling) Discontinue treatment.

one case of toxic epidermal necrolysis [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Palumbo | Oral melphalan, prednisone and thalidomide in elderly patients with multiple myeloma: updated results of a randomized controlled trial[END_REF]. Steroids have been associated with cutaneous reactions [START_REF] Amin | Cutaneous reactions to injectable corticosteroids[END_REF] and should be discounted as causative agents prior to modifying the dose of thalidomide. In situations where cutaneous reactions are related to thalidomide, a dose modification is recommended.

Recommendations for cutaneous reactions • In grade 3 non-hematological AEs, such as cutaneous reactions, treatment should be discontinued until the severity is reduced to grade 1 at which point treatment can be restarted at 50% of the original dose • The exception is cases of toxic reactions such as Stevens-Johnson

Syndrome where treatment with thalidomide should be permanently discontinued [14] Other considerations

Patients with renal impairment

Renal impairment is common in MM patients, occurring in 20-40% of patients at presentation depending on the method used for defining renal function [START_REF] Dimopoulos | Pathogenesis and treatment of renal failure in multiple myeloma[END_REF]. Data from pharmacokinetic studies in MM patients with varying degrees of renal impairment and in patients with end-stage renal disease, suggest that kidney function has no impact on thalidomide pharmacokinetics and therefore dose reductions in patients with renal impairment are not necessary [START_REF] Eriksson | Pharmacokinetics of thalidomide in patients with impaired renal function and while on and off dialysis[END_REF]. However, patients with severe organ impairment should be carefully monitored for adverse reactions. Further investigations are needed in patients with renal impairment. In contrast, lenalidomide is predominantly eliminated via urinary excretion of the unchanged drug with renal clearance directly proportional to renal function [START_REF] Chen | Pharmacokinetics of lenalidomide in subjects with various degrees of renal impairment and in subjects on hemodialysis[END_REF]. Therefore, dose reduction should be considered in patients with moderate/severe renal impairment [START_REF] Chen | Pharmacokinetics of lenalidomide in subjects with various degrees of renal impairment and in subjects on hemodialysis[END_REF]. Administration of thalidomide in patients with renal failure can increase the risk for bradycardia [START_REF] Pineda-Roma | High-dose therapy in patients with plasma cell dyscrasias and renal dysfunction[END_REF].

Dose adjustment for elderly patients (>75 years)

The optimum dose of thalidomide in different patient groups

is not yet well defined [START_REF] Palumbo | A new standard of care for elderly patients with myeloma[END_REF]; this may be an issue in patients aged >75 years where the higher incidence of side effects could be of concern [START_REF] Waage | Melphalanprednisone-thalidomide to newly diagnosed patients with multiple myeloma: a placebo controlled randomised phase 3 trial[END_REF]. A lower thalidomide dose of 100 mg/day in combination with MP in the IFM 01-01 study showed positive results, compared with MP alone, in patients aged >75 years. This suggests a dose of 100 mg/day thalidomide may be appropriate for the treatment of patients >75 years with newly diagnosed MM [START_REF] Hulin | Efficacy of melphalanprednisone-thalidomide (MP-T) to melphalan-prednisone (MP) in patients 75 years of age or older with untreated multiple myeloma (MM). Preliminary results of the randomized, double-blind, placebo controlled IFM 01-01 trial[END_REF].

Recommendations for dose reductions in elderly patients (>75 years)

• 100 mg per day of thalidomide may be the most appropriate dose for patients with MM aged>75 years old ○ Dose reduction to 50 mg/day may be required in the event of adverse effects

Conclusions

MPT is now considered to be an effective standard of care for newly diagnosed, transplant-ineligible patients [START_REF] Anderson | NCCN Clinical Practice Guidelines[END_REF][START_REF] Dispenzieri | Treatment of newly diagnosed multiple myeloma based on Mayo Stratification of Myeloma and Risk-adapted Therapy (mSMART): consensus statement[END_REF]. A number of AEs are associated with MPT, although these are generally manageable. Somnolence and constipation are common side effects associated with MPT [START_REF] Palumbo | Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial[END_REF][START_REF] Facon | Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial[END_REF], while DVT and peripheral neuropathy are potentially the most serious. As clinical trials addressing thromboprophylaxis are limited, the recommendations in this paper are based on consensus opinion. Therefore, prospective randomized trials comparing the use of different thromboprophylactic regimens (aspirin, LMWH, and warfarin) will help determine the optimal prophylactic strategy for patients with MM being treated with MPT.

Other novel agents, such as lenalidomide and bortezomib, have also shown promising results for MM patients. In a recent phase I/II trial, 81% of newly diagnosed MM patients receiving lenalidomide in combination with MP (MPR) achieved at least a partial response, with 1-year event-free survival and overall survival rates of 92% and 100%, respectively [START_REF] Palumbo | Melphalan, prednisone and lenalidomide treatment for newly diagnosed myeloma: a report from the GIMEMA-Italian multiple myeloma network[END_REF]. Bortezomib in combination with MP improved survival compared with MP alone, in a phase III trial of newly diagnosed, transplant-ineligible MM patients [START_REF] Miguel | Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma[END_REF]. It is interesting to note that lenalidomide, in combination with dexamethasone, is effective in patients regardless of prior thalidomide treatment, although overall response rate and complete response rate were higher in thalidomide-naïve patients compared with thalidomideexposed patients with longer median time to progression and longer progression-free survival [START_REF] Wang | Lenalidomide plus dexamethasone is more effective than dexamethasone alone in patients with relapsed or refractory multiple myeloma regardless of prior thalidomide exposure[END_REF]. In patients receiving bortezomib, in combination with pegylated liposomal doxorubicin, the response rates and duration of response are comparable in patients whether or not they have received prior thalidomide treatment [START_REF] Sonneveld | Combined pegylated liposomal doxorubicin and bortezomib is highly effective in patients with recurrent or refractory multiple myeloma who received prior thalidomide/lenalidomide therapy[END_REF]. Therefore, these novel agents could be effective options for the second-line treatment of patients, following MPT therapy.

Cyclophosphamide, thalidomide, and dexamethasone (CTD) is also effective in the treatment of newly diagnosed, transplant-ineligible patients [START_REF] Sidra | Combination chemotherapy with cyclophosphamide, thalidomide and dexamethasone for patients with refractory, newly diagnosed or relapsed myeloma[END_REF]. As with MPT, this contains a combination of an alkylating agent, a steroid and thalidomide. It is therefore likely that the strategies for managing AEs documented here may also be applicable to those associated with CTD.

Studies of cytogenetic abnormalities indicate that MM is heterogeneous with the translocations t(4;14) or t(14;16), deletion of chromosome 13 or p53 deletion defining high-risk prognostic groups [START_REF] Fonseca | Targeted therapeutics for multiple myeloma: the arrival of a risk-stratified approach[END_REF]. Recent data suggest that thalidomide treatment is associated with significantly improved survival in newly diagnosed myeloma patients with metaphase cytogenetic abnormalities compared with control treatment (P=0.02) [START_REF] Barlogie | Thalidomide arm of total therapy 2 improves complete remission duration and survival in myeloma patients with metaphase cytogenetic abnormalities[END_REF]. In the future, it is likely that further studies will identify subgroups of patients who might benefit most from thalidomide-based regimens. These risk-adapted approaches will lead to further improvements in response and survival rates and refine patient management.

After over four decades, thalidomide is the first drug to improve response rates and prolong the duration of remission and survival in patients with MM. The MPT combination offers an effective new approach for transplant-ineligible patients; this efficacy can be maximized through a good awareness of the strategies for optimal patient management. Physician and patient education is key to enabling discussion of practical strategies which would optimize clinical outcomes in transplantineligible patients treated with MPT.

Fig. 1

 1 Fig. 1 Flow chart for thromboprophylaxis in multiple myeloma patients receiving first-line MPT treatment. MPT thalidomide in combination with melphalan and prednisone, LMWH low molecular weight heparin

Table 2

 2 Venous thromboembolism incidence in trials of thalidomide without thromboprophylaxis , aspirin is more suitable for those patients with a low risk of VTE.

	Treatment	Newly diagnosed patients	Relapsed/refractory patients
	regimen
		VTE incidence (%) References	VTE incidence (%) References
	Thalidomide
	Alone	3-4 a	Rajkumar et al. 2003 [58];	2-4	Barlogie et al. 2001 [5]; Neben et al.
			Weber et al. 2003 [59]		2002 [67]; Schey et al. 2003 [68]
	Plus	14-26	Rajkumar et al. 2006 [60]; Cavo et al.	2-8	Anagnostopoulos et al. 2003 [69];
	dexamethasone		2004 [61]; Rajkumar et al. 2002 [62]		Palumbo et al. 2004 [70]
	Plus doxorubicin	10-27	Osman et al. 2001 [63]; Schutt et al.	58 b	Baz et al. 2005 [71]
			2005 [25]; Zervas et al. 2004 [64]
	Plus	3 b -11	Sidra et al. 2006 [53];	4-8	Dimopoulos et al. 2004 [72];
	cyclophosphamide		Wu et al. 2006 [65]		Garcia-Sanz et al. 2004 [38];
					Kropff et al. 2003 [73];
					Suvannasankha et al. 2007 [74]
	Plus melphalan	12-17	Palumbo et al. 2006; [7] Facon et al.	NA
	and prednisone		2007 [8]
	Plus multiagent	3-34	Barlogie et al. 2006 [66];	15	Lee et al. 2003 [75]
	chemotherapies		Zangari et al. 2002 [23]

Note: adapted from Palumbo et al. 2008

[START_REF] Palumbo | Prevention of thalidomide-and lenalidomide-associated thrombosis in myeloma[END_REF]

VTE venous thromboembolism, NA not applicable a Asymptomatic newly diagnosed multiple myeloma patients b Both at diagnosis and relapse injections▪ Aspirin, 100 mg advised although more evidence required

[START_REF] Palumbo | A prospective, randomized, phase III study of enoxaparin versus aspirin versus low-fixed-dose of warfarin in newly diagnosed myeloma patients treated with thalidomide-containing regimens[END_REF]

▪ Duration: as required during the full treatment period plus 30 days after discontinuation • Patients already receiving anticoagulation should remain on their current medication, providing it is appropriate

Table 3

 3 Risk factors for venous thromboembolism in patients diagnosed with multiple myeloma

Table 4

 4 Recommended dose modifications for thalidomide-related neuropathy in first-line treatment of MM[14]

	Severity of neuropathy	Modification of dose and regimen
	Grade 1 (paresthesia, weakness, and/or loss of	Continue to monitor the patient with clinical examination. Consider reducing dose by up to
	reflexes) with no loss of function	50% if symptoms worsen.
	Grade 2 (interfering with function but not with
	activities of daily living)

Acknowledgments Research support was provided by Celgene Ltd. Editorial support was provided by Dr. Marion James from Scope-Medical Ltd, funded by Celgene Ltd.