Experimental models for the autoimmune and inflammatory blistering disease , Bullous pemphigoid Bullous pemphigoid -LRB- BP -RRB- is a subepidermal skin blistering disease characterized immunohistologically by dermal-epidermal junction -LRB- DEJ -RRB- separation , an inflammatory cell infiltrate in the upper dermis , and autoantibodies targeted toward the hemidesmosomal proteins BP230 and BP180 . Development of an IgG passive transfer mouse model of BP that reproduces these key features of human BP has demonstrated that subepidermal blistering is initiated by anti-BP180 antibodies and mediated by complement activation , mast cell degranulation , neutrophil infiltration , and proteinase secretion . This model is not compatible with study of human pathogenic antibodies , as the human and murine antigenic epitopes are not cross-reactive . The development of two novel humanized mouse models for the first time has enabled study of disease mechanisms caused by BP autoantibodies , and presents an ideal in vivo system to test novel therapeutic strategies for disease management . Etiology of bullous pemphigoid In 1953 , Lever -LSB- 28 -RSB- described bullous pemphigoid -LRB- BP -RRB- as a subepidermal blistering disorder primarily seen in the elderly . Lesional/perilesional skin of BP patients exhibits detachment of the basal keratinocytes of the epidermis from the dermis at the level of the lamina lucida -LSB- 55 -RSB- , resulting in tense , fluid-filled vesicles . BP is both an inflammatory disease and an autoimmune disease , characterized by an inflammatory infiltrate at the site of the dermal -- epidermal junction separation and by the deposition of autoantibodies and complement components along the basement membrane zone -LRB- BMZ -RRB- . A number of inflammatory cells are present in the upper dermis and bullous cavity , including eosinophils -LRB- the predominant cell type -RRB- , neutrophils , lymphocytes , and monocytes/macrophages . Both intact and degranulating eosinophils , neutrophils , and mast cells -LRB- MC -RRB- are found in the dermis . Local activation of these cells may occur via the multiple inflammatory mediators present in the lesional skin and/or blister fluids , including -LRB- a -RRB- granular proteins derived from degranulated leukocytes , such as eosinophil cationic protein -LRB- ECP -RRB- , eosinophil major basic protein -LRB- MBP -RRB- , and neutrophil-derived myeloperoxidase -LRB- MPO -RRB- -LSB- 1 , 4 , 8 -RSB- and -LRB- b -RRB- chemoattractants and cytokines , such as C5a fragments , histamine , leukotriene B4 , interleukin-1 , -2 , -4 , 5 , -6 , -8 , -15 , TNF-α , IFN-γ , RANTES , and eotaxin -LSB- 9 , 10 , 21 , 22 , 46 , 47 , 48 , 58 , 62 -RSB- . Additionally , several proteinases are found in BP blister fluid , including plasmin , collagenase , elastase , and 92-kDa gelatinase -LSB- 2 , 14 , 24 , 27 , 44 , 45 , 52 , 57 -RSB- . These proteolytic enzymes may play a crucial role subepidermal blister formation in BP via their ability to degrade extracellular matrix proteins . BP patients generate a polyclonal repertoire of autoantibodies that bind to the BMZ and activate complement , as well as circulating autoantibodies -LSB- 20 -RSB- . These autoantibodies target two major hemidesmosomal antigens of 230 kD -LRB- BP230 or BPAG1 -RRB- and 180 kD -LRB- BP180 , BPAG2 , or type XVII collagen -RRB- -LSB- 25 , 40 , 56 , 57 -RSB- . BP230 , a component of the hemidesmosomal plaque , is an intracellular protein , while BP180 is a type II transmembrane protein -LSB- 19 , 23 , 56 -RSB- . Like BP230 , BP180 's amino-terminal portion localizes to the intracellular hemidesmosomal plaque -LSB- 15 , 18 , 19 -RSB- . Its carboxyl-terminal region extends into the extracellular milieu of the BMZ , making it the preferred target for pathogenic BP autoantibodies . This antigenic extracellular region consists of 15 collagen domains separated from one another by non-collagen sequences . The largest of these non-collagen domains is referred to as NC16A . Epitope mapping studies indicate that BP autoantibodies of IgE and IgG isotypes and IgG1 and IgG4 subclasses recognize multiple epitopes that cluster within BP180 NC16A -LSB- 3 , 11 , 16 , 26 , 63 -RSB- . Serum levels of these autoantibodies are correlated with disease severity -LSB- 11 , 17 , 49 -RSB- . Most BP patients elicit a cell mediated autoimmune response in addition to the humoral response described . Autoreactive CD4 + T lymphocytes recognize epitopes within the extracellular region of BP180 , primarily in the NC16A domain -LSB- 5 , 29 -RSB- . These T cells express memory cell surface markers and exhibit a Th1/Th2 mixed cytokine profile . These studies suggest that BP is a T and B cell-dependent and antibody-mediated skin autoimmune disease . Development of murine IgG passive transfer model of BP The strong correlation between BP disease severity and serum BP180-specific autoantibody levels suggests that BP blister formation is mediated by autoantibodies . Early attempts to demonstrate the pathogenicity of patient autoantibodies via a passive transfer mouse model were unsuccessful because BP autoantibodies that react with an immunodominant and potentially pathogenic epitope in BP180-NC16A fail to cross-react with the murine form of this autoantigen -LRB- mBP180 NC14A -RRB- -LSB- 30 -RSB- . In 1993 , Liu et al. -LSB- 30 -RSB- devised a strategy to overcome this difficulty and generated rabbit polyclonal antibodies raised against a cloned segment of mBP180 NC14A and passively transferred the purified rabbit anti-mBP180 IgG into neonatal BALB/c mice . The injected animals developed a disease that exhibited the following hallmarks of human BP : -LRB- a -RRB- clinical skin lesions ; -LRB- b -RRB- in vivo deposition of rabbit IgG and mouse C3 at the basement membrane by direct IF ; -LRB- c -RRB- dermal-epidermal separation and an extensive inflammatory cell infiltration by H&E staining -LSB- 30 -RSB- . This infiltrate includes neutrophils , lymphocytes , and monocytes/macrophages , with neutrophils being the predominant cells -LSB- 7 , 30 -RSB- . Immunopathogenesis of experimental BP in the murine model Development of an in vivo system to study an experimental BP model has allowed for great progress in defining the etiopathogenesis of disease . Specifically , the roles of pathogenic antibodies , the complement system , inflammatory cells , and proteolytic enzymes have all been elucidated in the context of the murine IgG passive transfer model . Injection of anti-mBP180 IgG initiated subepidermal blister formation , and the levels of circulating anti-mBP180 antibodies completely determine disease onset and severity -LSB- 30 , 34 -RSB- . Epitope mapping studies demonstrated that pathogenic anti-BP180 antibodies recognize a 9 -- 12 amino acid stretch within the murine BP180 NC14A region of the antigen -LSB- 31 -RSB- . This epitope overlaps the region of the human BP180 NC16A that contains the immunodominant epitopes recognized by human BP autoantibodies , supporting the relevance of this murine system as a model for human disease . Using mice deficient in different inflammatory cell types , it has been demonstrated that mast cells , macrophages , and neutrophils , but not T and B lymphocytes , play a direct role in subepidermal blistering in experimental BP -LSB- 7 -RSB- . Anti-mBP180 autoantibodies trigger skin blistering in wild-type mice and mice deficient in T , B , or both T and B cells , but fail to induce lesions in mice deficient in mast cells , macrophages , or neutrophils . Quantification of disease severity in these mice demonstrated that mast cells and neutrophils play a key role in experimental BP . Autoantibodies bind to basement membrane antigens and activate complement in human BP . In the rabbit anti-mBP180 IgG-induced BP , complement activation is absolutely required . Both BALB/c mice depleted of complement -LRB- by pretreatment with cobra venom -RRB- and C5-deficient mice are resistant to experimental BP , and reconstitution of the C5-deficient mice with C5a restores susceptibility -LSB- 32 -RSB- . F -LRB- ab ' -RRB- 2 fragments generated from the pathogenic anti-mBP180 IgG can not induce subepidermal blisters in C5-sufficient mice -LSB- 32 -RSB- . Mice deficient in complement component C4 -LRB- specific for the classical and lectin pathways -RRB- , but not in factor B -LRB- specific for the alternative pathway -RRB- , are resistant to BP -LSB- 41 -RSB- . Wild-type mice depleted of complement component C1q -LRB- specific for the classical pathway -RRB- fail to develop BP skin lesions when injected with pathogenic IgG -LSB- 41 -RSB- . These data suggest that the classical pathway of the complement activation plays a major role in development of disease , namely through the generation of C5a that in turn activates MCs -LSB- 6 -RSB- . Mice injected with pathogenic anti-mBP180 antibodies exhibit extensive MC degranulation in the lesional skin , similar to that observed in human BP -LSB- 6 , 59 -RSB- . MC-deficient mice are resistant to experimental BP , but reconstitution of these mice with MCs restores their susceptibility to disease . MC activation precedes neutrophil infiltration , and either the absence of MCs or the inhibition of MC degranulation prevents neutrophil infiltration and blister formation . However , MC-deficient mice reconstituted locally with neutrophils , or injected locally with neutrophil chemoattractants IL-8 or TNF-α form blisters in response to anti-mBP180 IgG . These results suggest that mast cells release proinflammatory cytokines critical for neutrophil recruitment -LSB- 6 -RSB- . Experimental BP requires neutrophil infiltration , and there is a direct correlation between disease severity and number of infiltrating neutrophils -LSB- 33 -RSB- . Depletion of neutrophils , or disruption of the events upstream of neutrophil infiltration -LRB- complement activation and MC degranulation -RRB- , renders mice resistant to BP blister formation . However , complement system and MC deficiencies only confer protection from the pathogenic effects of BP180 autoantibodies if neutrophil infiltration is blocked as a consequence ; that is , complement activation and MC degranulation are only critical to disease progression in their roles of neutrophil recruitment . This suggests that infiltrating neutrophils are the cells that directly cause tissue injury in the dermal -- epidermal junction , leading to BP skin blisters . Upon activation through molecular interactions between the FcγIIIR on the neutrophil cell surface and the Fc portion of pathogenic anti-mBP180 IgG -LSB- 61 -RSB- , infiltrating neutrophils secrete proteolytic enzymes known to degrade the extracellular matrix , including neutrophil elastase -LRB- NE -RRB- and gelatinase B -LRB- GB -RRB- . A deficit of either of these two enzymes blocks blister formation in mice -LSB- 35 , 36 -RSB- . In vitro , although both GB and NE are capable of degrading the recombinant BP180 protein , only NE produces DEJ separation when incubated with skin sections -LSB- 36 , 52 -RSB- . In vivo , the degradation of BP180 depends on NE activity . Taken together , these findings suggest that GB acts upstream of NE . Specifically , GB proteolytically inactivates the physiological inhibitor of NE -LRB- α1-proteinase inhibitor -RRB- , which allows for unmitigated NE cleavage of extracellular matrix proteins -LRB- including BP180 -RRB- , resulting in DEJ separation -LSB- 37 -RSB- . Development of the humanized murine passive transfer model While the rabbit anti-mBP180 IgG passive transfer model has provided invaluable insight to the key steps in BP disease development , it does not allow for experimentation with BP autoantibodies isolated from human clinical samples . A second animal model , in which rabbit antibodies directed against the extracellular domain of hamster BP180 -LRB- NC16A region -RRB- are transferred into neonatal hamsters , also lacks cross-reactivity with antibodies directed against human BP180 -LSB- 59 -RSB- . To assess the pathogenicity of human anti-BP180 autoantibodies , we generated a novel mouse strain in which the murine BP180NC14A was replaced with the homologous human BP180NC16A epitope cluster region -LSB- 38 -RSB- . The humanized NC16A -LRB- NC16 + / + -RRB- mice injected with anti-BP180NC16A autoantibodies develop BP , and the disease development depends on complement , mast cells and neutrophils -LRB- Fig. 1 -RRB- . Fig. 1Proposed mechanism of subepidermal blister formation in humanized NC16A mouse model of BP . Subepidermal blistering is an inflammatory process involved in following steps : a anti-BP180 autoantibodies are injected into NC16A mice ; b pathogenic antibodies bind to the pathogenic epitope of BP180 antigen in basal keratinocytes -LRB- BK -RRB- ; c the molecular interaction between BP180 antigen and anti-BP180 IgG activates the complement system -LRB- C ′ -RRB- as evidenced by BMZ deposition of mouse C3 ; d C ′ activation products C3a and C5a cause mast cells -LRB- MC -RRB- to degranulate ; e MC activation leads to neutrophil -LRB- PMN -RRB- recruitment ; f infiltrating PMNs bind to the BP180-anti-BP180 immune complex via the molecular interaction between Fcγ receptors on neutrophils and the Fc domain of anti-BP180 IgG . Activated PMNs release proteolytic enzymes ; g proteolytic enzymes degrade BP180 and other extracellular matrix proteins , leading to dermal -- epidermal junction separation ; h pathogenic antibody-injected mice develop clinical blisters Using the NC16A + / + mice and BP patients ' sera , we mapped the pathogenic epitope of BP180 in vivo -LSB- 39 -RSB- . IgG passive transfer experiments identified one pathogenic epitope -LRB- referred to as BP180NC16A2 .5 -RRB- . BP180NC16A2.5-specific autoantibodies induced subepidermal blisters in the NC16A + / + mice , and these blisters were blocked by pretreatment with recombinant BP180NC16A2 .5 . We also found that the NC16A2 .5 pretreated mice had significantly reduced levels of BMZ-bound and circulating pathogenic antibodies and showed reduced complement activation , mast cell degranulation and neutrophil infiltration . These results suggest that targeting the pathogenic epitope specifically could be a new therapeutic strategy to treat BP . A second humanized animal model has been developed by Nishie et al. -LSB- 42 , 43 -RSB- that replaces the mouse BP180 -LRB- COL17 -RRB- with the human analogue . Upon injection of human anti-BP180 autoantibodies , these mice reproduce the DEJ separation at the lamina lucida , the deposition of human IgG along the DEJ , and an inflammatory cell infiltrate consisting of neutrophils and eosinophils seen in human disease . The pathogenic effects of the autoantibody injection are ablated by pretreatment with a 77-amino acid peptide fragment of COL17 NC16A , referred to as R1 . These studies further support the therapeutic potential of employing decoy peptides to block the pathogenic epitope . Relevance of murine and humanized passive transfer models to human BP Both in vitro and in vivo data demonstrate that BP180 is the target for pathogenic autoantibodies in BP -LRB- Table 1 -RRB- . While clinical human BP and experimental murine and humanized murine BP closely mimic each other at the clinical , histological , and immunological levels , the IgG passive transfer models do not reflect the large number of eosinophils typically found in the inflammatory infiltrate of human BP lesional skin . Some patients do exhibit neutrophil-rich pemphigoid , indicating that the neutrophil-mediated blistering observed in these mouse models may be one of several disease mechanisms that contribute to the formation and persistence of subepidermal blisters . The presence of a neutrophil-rich inflammatory infiltrate in the two humanized mouse models , as well as in the aforementioned hamster model , lend credence to a neutrophil-mediated disease mechanism . Furthermore , findings that in vitro DEJ separation induced by human BP autoantibodies specific for BP180NC16A depends on neutrophils -LSB- 51 -RSB- and neutrophil-derived elastase and gelatinase B -LSB- 50 -RSB- , and that BP180 degradation by human BP blister fluid depends on neutrophil elastase activity -LSB- 57 -RSB- support this theory . Taken together , these findings strongly suggest that like the IgG passive transfer model of BP , neutrophils may be responsible for subepidermal blister formation in human BP , at least in those patients who show neutrophil infiltration in their lesional/perilesional skin . The humanized mouse models do not yet offer insight to an eosinophil-mediated mechanism of disease progression , leaving the role of these inflammatory cells , if there is any , yet to be discovered . It is possible that these established mouse models , without additional experimental manipulations , may not be able to fully duplicate human BP pathology related to eosinophil infiltration ; therefore , they may not be appropriate to study the role of eosinophils in BP . Table 1In vitro and in vivo evidence of pathogenicity of anti-BP180 antibodiesSystemAntibodies usedReferenceIn vitro Human skin sectionBP sera -LSB- 13 -RSB- Anti-BP180NC16A autoantibodies -LSB- 51 -RSB- Rabbit anti-BP180NC16A IgGIn vivo Wild-type miceRabbit anti-murine BP180 IgG -LSB- 30 -RSB- HamsterRabbit anti-hamster BP180 IgG -LSB- 60 -RSB- Humanized BP180 miceAnti-BP180NC16A autoantibodies -LSB- 38 , 39 -RSB- Humanized NC16A miceAnti-BP180NC16A autoantibodies -LSB- 42 , 43 -RSB- Zone et al. -LSB- 64 -RSB- reported that IgE hybridoma to LABD97 antigen , a component of the shed ectodomain of BP180 , when injected subcutaneously in SCID mice with engrafted human skin , induced eosinophil infiltration and histological blisters in engrafted human skin . Fairley et al. -LSB- 12 -RSB- also reported that total IgE isolated from BP sera , when injected into human skin grafted onto athymic nude mice , triggered a dermal infiltrate composed of neutrophils , eosinophils , and histological separation of the epidermis from the dermis . These findings implicate eosinophils in BP ; however , a direct link between eosinophil recruitment and blistering in these human skin graft models needs to be established . Conclusions The murine IgG passive transfer model of BP developed by Liu et al. -LSB- 30 -RSB- has provided an invaluable tool for the dissection of the mechanism of disease progression . The development of humanized murine models of BP now provides unprecedented insight to the pathogenic role of the autoantibodies that mediate disease . Initial observations with these humanized models indicate that humanized mouse BP exhibits phenotypic and immunological similarities with murine and human BP . Additionally , these humanized models enabled the first identification of pathogenic epitopes in human disease . Blocking of the pathogenic epitope with decoy peptides , if they do not enhance the autoimmune responses , presents a novel clinical therapeutic strategy that may be very effective in abolishing the pathogenic effects of disease .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

autoimmune disease
basement membrane
humanized animal model


Practical recommendations on the use of lenalidomide in the management of myelodysplastic syndromes Lenalidomide , an oral immunomodulatory agent , has received approval in the USA from the Food and Drug Administration -LRB- FDA -RRB- for the management of myelodysplastic syndromes -LRB- MDS -RRB- classified by the International Prognostic Scoring System -LRB- IPSS -RRB- as low risk or intermediate-1 risk and with a deletion 5q -LRB- del -LRB- 5q -RRB- -RRB- cytogenetic abnormality . Although some patients with del -LRB- 5q -RRB- have a relatively good prognosis , all del -LRB- 5q -RRB- patients will become transfusion-dependent at some point during the course of their disease . The results of two clinical trials in more than 160 patients with MDS have demonstrated clear therapeutic benefits of lenalidomide , with > 60 % of patients achieving independence from transfusion during therapy , irrespective of age , prior therapy , sex , or disease-risk assessment . The recommendations presented in this review will aid the safe administration of lenalidomide for the treatment of patients with low-risk or intermediate-1-risk MDS and a del -LRB- 5q -RRB- cytogenetic abnormality , and they will help physicians avoid unnecessary dose reduction or interruption , thus assuring the best efficacy for patients . Introduction Myelodysplastic syndromes -LRB- MDS -RRB- are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and leading to peripheral cytopenias and a genetic instability with enhanced risk of disease transformation to acute myeloid leukemia -LRB- AML -RRB- . In the general population , MDS affects approximately five in every 100,000 individuals and is regarded as a relatively rare disease -LSB- 1 -RSB- . However , its incidence is highest in people aged > 70 years , and in this age group , MDS is the most frequently occurring malignant hematological disorder : twice as frequent as AML , more common than chronic lymphocytic leukemia , and more common than all other malignant lymphomas combined -LSB- 2 -RSB- . All patients diagnosed with MDS will eventually die from their disease , and in about 30 % , the disease will progress to AML -LSB- 3 -RSB- . Between 40 % and 60 % of patients with primary MDS and about 90 % of patients with secondary MDS have chromosomal abnormalities at the time of diagnosis -LSB- 4 -RSB- . Chromosomal abnormalities may occur as a single abnormality or as part of a complex karyotype ; disease severity increases with the number of abnormalities . The most common chromosomal abnormalities are deletions of chromosome 5 and chromosome 7 and trisomy of chromosome 8 -LSB- 5 -RSB- . In a subgroup of patients who have an isolated deletion 5q -LRB- del -LRB- 5q -RRB- -RRB- , termed 5q-syndrome , the clinical profile is distinct -LSB- 6 -RSB- . Typically , this clinical profile includes macrocytic anemia , a normal to increased platelet count , mild leukopenia , hypolobulated megakaryocytes in the bone marrow , a medullary blast count < 5 % , and an isolated del -LRB- 5q -RRB- abnormality including a common deleted region between 5q31 and 5q33 -LSB- 4 , 7 -RSB- . Consistent with the observation that disease severity is related to the number of chromosomal abnormalities , patients with del -LRB- 5q -RRB- plus additional cytogenetic abnormalities have a worse prognosis than those with an isolated del -LRB- 5q -RRB- abnormality -LSB- 3 , 5 -RSB- . On the basis of the results of an international phase II trial in 148 patients -LSB- 8 -RSB- , the oral immunomodulatory , antiangiogenic , and antineoplastic agent lenalidomide -LRB- Revlimid ® ; Celgene Corporation , NJ , USA -RRB- was approved in December 2005 by the US Food and Drug Administration -LRB- FDA -RRB- for the treatment of patients with transfusion-dependent International Prognostic Scoring System -LRB- IPSS -RRB- classified low-risk to intermediate-1-risk MDS with a del -LRB- 5q -RRB- cytogenetic abnormality , with or without additional cytogenetic abnormalities . The first 46 patients recruited received lenalidomide 10mg/day for 21days every 28-day cycle , for up to 24weeks . After a protocol amendment , the remaining 102 patients received lenalidomide 10mg/day on a continuous dosing schedule -LSB- 8 -RSB- . Overall , 67 % of patients who were initially transfusion-dependent achieved transfusion independence , and another 9 % achieved a decrease -LRB- ≥ 50 % -RRB- in the total number of transfusions required during lenalidomide therapy -LSB- 8 -RSB- . Forty-five percent of the 85 evaluable patients in this study achieved a complete cytogenetic response , irrespective of chromosomal complexity -LSB- 8 -RSB- . In January 2007 , an international group of MDS specialists met to discuss the practical management of lenalidomide in patients with transfusion-dependent , IPSS-classified low-risk or intermediate-1-risk MDS and a del -LRB- 5q -RRB- cytogenetic abnormality . The recommendations presented here are based on the clinical data derived from the current literature -LSB- 8 , 9 -RSB- , from the known clinical profile of lenalidomide in other disorders , and the clinical experience of the panel members in the practical use of this agent . Although lenalidomide has been used in IPSS-classified intermediate-2-risk and high-risk patients and in patients without an associated del -LRB- 5q -RRB- abnormality , the recommendations presented here focus on the current approved indication of the drug in the USA . Selection of patients for lenalidomide therapy The FDA approved lenalidomide for patients with transfusion-dependent anemia due to low - or intermediate-1-risk MDS associated with a del -LRB- 5q -RRB- cytogenetic abnormality , with or without additional cytogenetic abnormalities . This is an important limitation , as patients with a complex karyotype and del -LRB- 5q -RRB- are eligible for treatment only if they have no more than one cytopenia -LRB- i.e. , anemia , neutropenia , or thrombocytopenia -RRB- and their medullary bone marrow blast count is < 5 % . Furthermore , patients with a bone marrow blast count up to 10 % receive a score of 0.5 points in the IPSS classification -LRB- Table 1 -RRB- -LSB- 3 -RSB- . Therefore , in order to remain in the IPSS intermediate-1-risk category , one additional chromosomal abnormality -LRB- not involving chromosome 7 -RRB- or two or more peripheral cytopenias are allowed , but not both . Although teratogenicity has not been reported with lenalidomide , as it is an analogue of thalidomide , women of childbearing potential should have two negative pregnancy tests performed within 14days prior to lenalidomide intake . Table 1International prognostic scoring system for myelodysplastic syndromes : survival and evolution of acute myeloid leukemia Score valuePrognostic variable00 .51.01.52.0 Bone marrow blasts , % < 55 -- 10 -- 11 -- 2021 -- 30KaryotypeaGoodIntermediatePoor Cytopeniasb0/12/3 Scores for risk groups are as follows : Low , 0 ; Int-1 , 0.5 -- 1.0 ; Int-2 , 1.5 -- 2.0 ; and High , ≥ 2.5 aGood : normal , − Y , isolated del -LRB- 5q -RRB- , isolated del -LRB- 20q -RRB- ; poor : ≥ 3 abnormalities or chromosome 7 anomalies ; intermediate : other abnormalitiesbNeutrophils < 1800 × 106/l , hemoglobin < 10 g/dl , platelets < 100 × 109/lTable reproduced with permission from -LSB- 3 -RSB- © The American Society of Hematology Lenalidomide therapy is effective regardless of prior erythropoietin -LSB- 8 , 9 -RSB- or prior thalidomide therapy -LSB- 9 -RSB- , and those previous therapies should not be regarded as contraindications for its use , although concurrent therapy with erythropoietin is not recommended , owing to concerns regarding venous thromboembolism . Clinical data have shown that the frequency of response to lenalidomide is similar across all age groups evaluated -LSB- 8 -RSB- . Importantly , the overall adverse-event burden did not increase in older patients , but serious adverse events were more frequent in patients aged over 65 years compared with younger patients -LRB- 54 % vs. 33 % , respectively -RRB- -LSB- 10 -RSB- . Although the impact of renal impairment on lenalidomide efficacy or toxicity in patients with MDS has not been evaluated , the risk of toxicity is expected to be greater in patients with impaired renal function because lenalidomide is renally excreted -LSB- 11 -RSB- . In patients with known renal impairment , the expert panel support the dosing schedule proposed by Chen et al. , which is based on creatinine clearance -LRB- Table 2 -RRB- -LSB- 11 -RSB- . As advancing age is associated with declining renal function , it is recommended that patients older than 65 years who are prescribed lenalidomide are monitored carefully throughout their treatment -LRB- Table 3 -RRB- . Table 2Recommended dose adjustments for patients with impaired renal functionaRenal impairmentDoseMild10 mg -LRB- full dose -RRB- every 24 h -LRB- 80 > CLcr ≥ 50 ml/min -RRB- Moderate5 mg every 24 h -LRB- 30 ≤ CLcr < 50 ml/min -RRB- Severe5 mg every 48 h -LRB- CLcr < 30 ml/min , not requiring dialysis -RRB- End-stage renal disease5 mg three times a week after each dialysis -LRB- CLcr < 30 ml/min , requiring dialysis -RRB- aRecommendations based on a pharmacokinetic study by Chen et al. -LSB- 11 -RSB- CLcr creatinine clearanceTable adapted from -LSB- 11 -RSB- © 2007 SAGE Publications . Adapted by permission of SAGE Publications , Inc.Table 3Recommendations for laboratory monitoring during treatmentFunctionTestRecommendation -LRB- s -RRB- Kidney functionCreatinineEvery 4 weeks in patients aged 65 years and olderBloodFBCWeekly monitoring of full blood count mandatory for the first 2 months -LRB- it may be continued for 5 months -RRB- . Biweekly or monthly monitoring should be considered thereafter , depending on hematological status.If treatment is interrupted in patients who had a previous episode of neutropenia or thrombocytopenia while on lenalidomide treatment , the same monitoring guidelines apply at re-initiationThyroid functionTSH , T4Monitor every month during the course of treatmentIn case of loss of response during lenalidomide treatmentGonadal functionTestosteroneIn case of loss of response during lenalidomide treatmentDigoxinDigoxinIn patients concomitantly taking Digoxin , the plasma level should be monitored periodicallyPregnancy testUrine testDay − 14 and day 0 at initiation of therapy , monthly thereafter -LRB- in women of childbearing potential -RRB- Bone marrowBM aspiration and cytogenetic testing ; Trephine biopsy optionalAt commencement of therapy.In case of loss of response to rule out progressive disease or cytogenetic evolutionBM Bone marrow , FBC full blood count , T4 thyroxine , TSH thyroid-stimulating hormone Likewise , the safety , efficacy , and pharmacokinetics of lenalidomide therapy have not been fully investigated in patients with evidence of hepatic impairment , or those with frank hepatic dysfunction . Clinical trials conducted to date have excluded patients with inadequate hepatic function , with individual trials using different serum transaminase measures as exclusion criteria ; most trials excluded patients with serum transaminase levels greater than three times the upper limit of normal . Pharmacokinetic studies have revealed that the co-administration of lenalidomide with digoxin may increase the maximal digoxin concentration , although the area under the concentration -- time curve remained unchanged -LSB- 10 -RSB- . Importantly , lenalidomide does not interact with the cytochrome P450 system , and so , can be confidently co-administered with other medications metabolized by this route -LRB- Celgene data on file -RRB- -LSB- 10 -RSB- . This latter finding is particularly relevant for older patients , who may be more likely to be receiving concomitant medications for co-morbidities . The expert panel concurred with the current prescribing information for lenalidomide and recommended that patients taking digoxin should have their digoxin plasma levels monitored periodically -LRB- Table 3 -RRB- . Treatment with lenalidomide in del -LRB- 5q -RRB- MDS The time-to-response during lenalidomide therapy was evaluated in the larger of the two pivotal clinical trials -LSB- 8 -RSB- . For most patients , the median time to initiation of the transfusion-independent period was 4.6 weeks , but responses have been noted to take up to 12 months -LSB- 8 -RSB- . Therefore , the expert panel recommends a treatment duration of at least 4 months in order to obtain an initial response -LRB- Table 4 -RRB- . Patients were followed for a median of 104weeks , but as more than half of them remained transfusion-free at this assessment point , the median duration of transfusion independence could not be calculated . Encouragingly , among the pooled patients with MDS with a del -LRB- 5q -RRB- abnormality from the two studies -LSB- 8 , 9 -RSB- , transfusion independence and major erythroid response were maintained for a median duration of at least 2 years -LSB- 12 -RSB- . Even though there are anecdotal reports of long-lasting erythroid remissions in some patients with hematological complete response who discontinued lenalidomide , the panel recommends continuation of lenalidomide treatment in responders for as long as it is tolerated . In patients where the dose has initially been reduced for mild adverse events , dose escalations may be considered -LRB- Table 4 -RRB- . In responders with high ferritin levels due to long transfusion histories who achieve normal hemoglobin levels , lenalidomide treatment should be continued and therapeutic phlebotomy be considered to reduce the secondary iron overload . In responders who discontinue due to adverse events , the panel recommends checking for response continuation before initiating another treatment -LRB- Table 4 -RRB- . Preliminary data show that prolonged treatment with lenalidomide does not increase the risk of transition to acute myeloid leukemia -LSB- 13 -RSB- . Of note , there are reports of patients acquiring additional cytogenetic abnormalities while on lenalidomide -LSB- 8 -RSB- , but at this time the relevance of this phenomenon is not entirely clear . Until there is data to the contrary , patients with continuous hematological response should remain on the drug . Patients losing their response and developing a complex karyotype should be approached as they would be in the absence of lenalidomide , and alternative therapies , including transplantation , should be considered . Table 4Recommendations for treatment duration Recommendation -LRB- s -RRB- Initial treatmentTreatment should be continued for at least 4 months in order to obtain an initial responsePatients who Have a complete hematological responseContinue lenalidomide therapy for as long as it continues to be well tolerated to avoid relapse -LRB- both erythroid and cytogenetic -RRB- Have a partial responseContinue lenalidomide therapy , and consider an escalation of the lenalidomide dose to a maximum of 10 mg per day , if tolerable Discontinue treatment because of adverse eventsPatients should not begin another therapy immediately ; it is recommended to wait 8 -- 12 weeks to determine whether the response continues Tolerability of lenalidomide in del -LRB- 5q -RRB- MDS Lenalidomide has been shown to be well tolerated in the two clinical trials reported to date -LSB- 8 , 9 -RSB- . The most commonly reported adverse events of grade 3 or 4 severity were neutropenia -LRB- 55 % -RRB- , thrombocytopenia -LRB- 44 % -RRB- , anemia -LRB- 7 % -RRB- , leukopenia -LRB- 6 % -RRB- , rash -LRB- 6 % -RRB- , diarrhea -LRB- 3 % -RRB- , pruritus -LRB- 3 % -RRB- , pneumonia -LRB- 3 % -RRB- , and fatigue -LRB- 3 % -RRB- -LSB- 8 -RSB- . Management of hematological adverse events Hematological adverse events , including neutropenia and thrombocytopenia , were the most common adverse events with lenalidomide therapy and the most frequent reasons for dose adjustment -LSB- 9 -RSB- . As such , thrombocytopenia and neutropenia should be expected to occur in most patients -- in fact , they appear to be associated with a higher likelihood of a response to therapy -LSB- 14 -RSB- . In general , regular monitoring of blood cell counts is recommended -LRB- Table 3 -RRB- . The majority of hematological adverse events -LRB- 62 % -RRB- occurred early in the course of treatment -LRB- within the first 8weeks -RRB- -LSB- 8 -RSB- . Grade 3 or 4 neutropenia was reported in 55 % of patients -LSB- 8 -RSB- . Monitoring and , in some cases , additional treatment with granulocyte colony-stimulating factor -LRB- G-CSF -RRB- is recommended -LRB- Tables 3 and 5 , and Fig. 1 -RRB- . Neutropenic sepsis was the only reported cause of death -LRB- occurring in three patients -RRB- that was regarded as possibly related to the study drug -LSB- 8 -RSB- , therefore , patients should receive clear guidance on how to react in the event of febrile neutropenia -LRB- Table 5 -RRB- . Grade 3 or 4 thrombocytopenia was observed in 44 % of patients -LSB- 8 -RSB- . In some thrombocytopenic patients , interruption of lenalidomide treatment may be necessary -LRB- Table 5 and Fig. 1 -RRB- . Lenalidomide has not been tested in patients with a baseline neutrophil count < 500 × 106/l or baseline platelet count < 50 × 109/l . Fig. 1Recommendations for the management of a neutropenia and b thrombocytopenia . aNo consensus was reached , recommendation is based on the opinion of some of the panel members G-CSF granulocyte colony-stimulating factorTable 5Recommendations for the management of hematological adverse events Recommendation -LRB- s -RRB- NeutropeniaFor treatment recommendations for neutropenia see Fig. 1Febrile neutropeniaProvide patients with clear guidance on how to react in the event of febrile neutropenia -LRB- patient education , specialized hematological care at all times , and broad-spectrum antibiotics within 3 h of fever onset -RRB- ThrombocytopeniaFor treatment recommendations for thrombocytopenia see Fig. 1VTEVTE prophylaxis is not generally recommended in patients with MDS . Combining lenalidomide with erythropoietin is also not recommended.If erythropoietin is used , be aware of a potentially increased risk of VTE . Patients should be informed about the risk of VTE and monitored for symptomsIf VTE does occur , interrupt lenalidomide treatment , treat the VTE , and carefully re-introduce lenalidomide once stable anticoagulation has been establishedPolycythemiaLenalidomide should be continued and phlebotomy considered , depending on ferritin levels . Although polycythemia is usually transient , treatment interruption may be necessary if additional risk factors for VTE are presentMDS myelodysplastic syndromes , VTE venous thromboembolism In the opinion of the panel , patients presenting with a neutrophil count < 1,000 × 106/l prior to the start of lenalidomide therapy can still be treated with lenalidomide if G-CSF is co-administered . When neutropenia occurs during lenalidomide therapy , interruption of the lenalidomide treatment schedule might be necessary -LRB- Table 5 and Fig. 1 -RRB- . When thrombocytopenia -LRB- platelet count < 25 × 109/l -RRB- is diagnosed at presentation , based on the rationale that once a response is achieved thrombocytopenia is likely to resolve , some experts would consider administering lenalidomide with platelet support -LRB- Table 5 and Fig. 1 -RRB- . However , other experts advise against the use of lenalidomide in patients with a platelet count < 50 × 109/l because there is a risk of sustained deterioration of thrombocytopenia , despite an erythroid response . In some patients responding to lenalidomide , polycythemia may occur -LSB- 8 -RSB- ; if appropriate , therapeutic phlebotomy or interruption of lenalidomide therapy may be considered -LRB- Table 5 -RRB- . Grade 3 or 4 venous thromboembolism -LRB- VTE -RRB- was observed in 3 % of patients -LSB- 8 -RSB- . Generally , the risk of VTE is increased if there is a history of superficial vein thrombosis , or previous VTE -LSB- 15 , 16 -RSB- . It is unknown if this applies to patients treated with lenalidomide as well . Nevertheless , these patients should be carefully monitored . In patients with a history of previous VTE , low-molecular-weight heparin should be used to prevent recurrent thrombosis . Although aspirin is effective in the prevention of thrombosis in multiple myeloma , aspirin can not be recommended alongside lenalidomide as lenalidomide frequently leads to grade 3 or 4 thrombocytopenia in patients with MDS with del -LRB- 5q -RRB- . There are insufficient data on low-dose coumadin treatment for the prophylaxis of VTE , and therefore the panel did not recommend its use . Of particular relevance to patients with MDS , VTE risk is increased by concomitant erythropoietin use -LSB- 17 -RSB- . Since the incidence of VTE in patients with lenalidomide-treated MDS is generally low -LSB- 8 -RSB- , prophylaxis in patients without antecedents of VTE is not recommended . If VTE does occur , it should be treated according to standard protocols . Treatment with lenalidomide should be interrupted until stable anticoagulation is achieved and then carefully reintroduced -LRB- Table 5 -RRB- . Once VTE has occurred during lenalidomide therapy , patients must remain on anticoagulation therapy as lenalidomide treatment continues . Management of non-hematological adverse events Unlike thalidomide , lenalidomide does not lead to dose-dependent peripheral neuropathy or somnolence -LSB- 18 -RSB- , but other non-hematological adverse events -LRB- NHAEs -RRB- may occur . The most common NHAEs -LRB- all grades -RRB- associated with lenalidomide therapy are diarrhea -LRB- 49 % -RRB- , pruritus -LRB- 42 % -RRB- , rash -LRB- 36 % -RRB- , and fatigue -LRB- 31 % -RRB- -LSB- 10 -RSB- . The most commonly reported grade ≥ 3 NHAEs were rash -LRB- 6 % -RRB- , fatigue -LRB- 3 % -RRB- , diarrhea -LRB- 3 % -RRB- , and pruritus -LRB- 3 % -RRB- -LSB- 8 -RSB- . In general , should NHAEs arise during lenalidomide therapy , alternative causes should be ruled out and symptomatic treatment initiated -LRB- Table 6 -RRB- . Dry skin and pruritus occur regularly , and itching of the scalp is a characteristic adverse event during the first few weeks of therapy . These NHAEs are usually self-limiting and seldom need treatment . Unselective antihistamines -LRB- e.g. , clemastine -RRB- are usually helpful . In severe cases , a short course of systemic corticosteroids -LRB- 10 mg of prednisone or equivalent -RRB- , or local steroid application may be helpful . Rash often resolves spontaneously with time -LSB- 19 -RSB- , and the panel do not recommend discontinuation of lenalidomide treatment should rash arise ; however , in patients with severe or persistent rash , the temporary interruption of lenalidomide may be necessary -LRB- Table 6 -RRB- . Table 6Recommendations for management of non-hematological adverse events -LRB- NHAEs -RRB- NHAERecommendation -LRB- s -RRB- RashUsually resolves within 2 -- 3 weeks , no interruption of lenalidomide treatment neededIf required treat with unselective antihistamines -LRB- e.g. clemastine -RRB- , topical steroids , or a short course -LRB- 14 days -RRB- of oral 10 mg prednisoneIf rash is severe or persists after treatment , lenalidomide should be interrupted until rash resolves . In the experience of the panel , lenalidomide can be restarted thereafter without recurrence of rashDiarrheaTreat symptomatically after ruling out other underlying causesHypothyroidismIn case of hypothyroidism , thyroid replacement therapy must be initiatedOther NHAEsTreat symptomatically after ruling out other underlying causes such as anemia or autoimmune disorders Diarrhea is a frequent problem and may impact on a patient 's quality of life . Patients with known lactose intolerance should add lactase to their diet , as lenalidomide capsules contain small amounts of lactose . Diverse symptomatic therapies have been reported to be effective in those patients , including , but not limited to , loperamide , pipaverium bromide , uzara root extract , and tinctura opii . Muscle cramps have been reported by a number of patients after a variable time of lenalidomide intake . Magnesium dietary supplements may be tried , but are usually of limited value . Quinine sulphate -LRB- up to 200 mg thrice-daily -RRB- works well , however , physicians should check for other cytochrome-P450-interacting drugs -LRB- e.g. , sotalol , terfenadine , astemizole , voriconazole , erythromycin , rifampicin , and cisapride -RRB- that might prolong QT time in these patients . Patients who experience fatigue will be likely to benefit from counselling -LSB- 20 -RSB- , and in cases of severe fatigue , dose reduction may be needed . Hypothyroidism has been reported in approximately 7 % of patients -LSB- 10 -RSB- . It is almost exclusively of autoimmune cause . Patients should be screened for hypothyroidism every other month during the course of treatment -LRB- Table 3 -RRB- . If hypothyroidism is confirmed , hormone replacement therapy is indicated -LRB- Table 6 -RRB- . Patients losing their response to lenalidomide during treatment may have developed either hypothyroidism or hypogonadism , and should be screened for both conditions . Conclusions The approval of lenalidomide for the treatment of MDS patients with low-risk or intermediate-1-risk disease with a deletion of chromosome 5q represents a significant step forward for this underserved group of patients . The key goal of the strategies presented here is to avoid unnecessary dose delays and reductions , and to maximize probability of response . Patient selection is straightforward , as efficacy does not appear to be influenced appreciably by relevant prognostic factors including age , prior erythropoietin therapy , sex , French -- American -- British type , or IPSS score . Treatment should be continued for a minimum of 4 months to ensure treatment response . Neutropenia and thrombocytopenia are the most common adverse events during lenalidomide therapy but can be managed by the introduction of G-CSF and dose interruption . Other side-effects are generally manageable , and a range of simple -LRB- prophylactic -RRB- interventions are recommended to address the more common and more serious associated adverse events .

O O O O O O B O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

myelodysplastic syndromes
mds
lenalidomide