Thrombocytopenia and hemostatic disorders in chronic graft versus host disease

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1 (2009) 44, & 2009 Macmillan Publishers Limited All rights reserved /09 $ REVIEW Thrombocytopenia and hemostatic disorders in chronic graft versus host disease D Pulanic 1,3, JN Lozier 2 and SZ Pavletic 1 1 Graft-versus-Host and Autoimmunity Unit, Experimental Transplantation and Immunology Branch, National Cancer Institute, Center for Cancer Research, National Institutes of Health, Bethesda, MD, USA; 2 Department of Laboratory Medicine, Hematology Laboratory, Clinical Center, National Institutes of Health, Bethesda, MD, USA and 3 Division of Hematology, University Department of Internal Medicine, Clinical Hospital Center Zagreb, Zagreb, Croatia Chronic graft versus host disease (cgvhd) is a major and frequent late complication in allogeneic stem cell transplantation recipients. Although thrombocytopenia in cgvhd patients is among the most consistent and strongest predictors of poor survival across many cgvhd studies, such correlation is still neither clearly explained nor well understood. Low platelet counts in the setting of cgvhd are associated with an increase in complications and treatment-related mortality, but usually not with higher relapse rate or engraftment failure rate. Bleeding might be occasionally increased along with, paradoxically, thrombosis. Hemostatic disorders in the context of cgvhd are significant complications with multifactorial etiology, including tissue injury with releasing microparticles, cytokine release, macrophage/monocyte clearance, CMV infection, production of transforming growth factor-beta, and low levels of thrombopoietin. Future clinical trials with agents that stimulate megakaryocytopoiesis or influence underlying impaired hemostasis mechanisms should investigate whether such interventions may improve outcomes in patients with cgvhd. (2009) 44, ; doi: /bmt ; published online 17 August 2009 Keywords: thrombocytopenia; chronic graft versus host disease; hemostatic disorders; megakaryocytes Introduction Chronic graft versus host disease (cgvhd) is a major late complication occurring in 25 80% of allogeneic hematopoietic stem cell transplantation (allohsct) recipients. 1 It is a systemic disorder characterized by immune Correspondence: Dr SZ Pavletic, Graft-versus-Host and Autoimmunity Unit, Experimental Transplantation and Immunology Branch, National Cancer Institute, Center for Cancer Research, National Institutes of Health, 9000 Rockville Pike, 10 Center Drive, Room CRC , Bethesda, MD , USA. pavletis@mail.nih.gov Received 8 July 2009; accepted 8 July 2009; published online 17 August 2009 deregulation, immunodeficiency, impaired organ function, development of signs and symptoms of various autoimmune or immunologic disorders, and is associated with decreased survival. 1 4 A low platelet count in cgvhd patients is among the most consistent and strongest negative survival predictors across cgvhd studies in both allogeneic bone marrow transplantation (allobmt) and allogeneic peripheral stem cell transplantation (allopsct). 2,3,5 11 Patients with cgvhd and persistent thrombocytopenia show poorer responses to therapy, experience higher mortality rates from infection or, less often, from hemorrhage. 5,12 Low platelet counts were also reported as a marker for a group of patients with severe cgvhd who have increased incidence of transplantrelated complications and a higher mortality rate. 2,3,5,8,12 15 The thrombocytopenia of cgvhd is not usually associated with disease relapse or graft rejection, but significantly correlates with increased non-relapse mortality, 6 indicating the existence of additional poorly understood pathophysiological mechanisms that could generate the association of thrombocytopenia and negative outcome of cgvhd. Patients with cgvhd may develop several other acquired hemostatic disorders, including also an increased risk for venous thrombosis in spite of higher bleeding risk. This review analyzes and summarizes the characteristics of thrombocytopenia and other hemostatic disorders during cgvhd, focusing on identifying pathophysiological mechanisms associated with decreased platelet count and poor prognosis of cgvhd. Improved understanding of these disorders may lead to better treatments or prevention of cgvhd. Pathophysiology of development of thrombocytopenia Table 1 is a summary of the most important cgvhd studies showing low platelet counts as negative survival indicators. Already known pathophysiological mechanisms for development of thrombocytopenia in a non-transplant setting could also be involved in cgvhd. In general, thrombocytopenia can be caused by factors that decrease the production of platelets in the marrow or increase platelet destruction or sequestration in the peripheral

2 394 Table 1 Chronological summary of studies that defined low platelet counts as negative survival predictor in cgvhd Author (reference)/year First et al. 14 /1985 Sullivan et al. 12 /1988 Anasetti et al. 5 /1989 Akpek et al. 7 /2001 Przepiorka et al. 8 /2001 Akpek et al. 6 /2003 Arora et al. 9 /2003 Pavletic et al. 2 /2005 Arora et al. 10 /2007 Key findings of the study One among the earliest studies, which showed that cgvhd patients with thrombocytopenia, had the worst survival and that low platelet count may reflect more severe cgvhd and could be useful stratification criterion 179 patients with extensive cgvhd were studied, and those with thrombocytopenia less than /ml had increased mortality Important study that concluded that persistent thrombocytopenia after allohsct is most often secondary to increased platelet destruction mediated by multiple mechanisms, and that increased mortality of patients with cgvhd and low platelets may be result of underlying immunodeficiency and immune deregulation In 151 cgvhd patients three risk factors were defined at diagnosis of cgvhd that were significantly associated with increased non-relapse mortality: thrombocytopenia (less than /ml), more than 50% body surface area skin involvement, and progressive type of cgvhd onset Among 116 allohsct patients long-term progression-free survival was 31% for patients without cgvhd, 51% for not thrombocytopenic cgvhd patients, and just 16% for patients with cgvhd and thrombocytopenia (less than /ml) This large multicenter study with a total of 1105 cgvhd patients from four different cohorts showed that thrombocytopenia (less than /ml) was uniformly associated with increased risk of mortality across all test samples 159 cgvhd patients were studied to identify predictors of response and long-term mortality. In multivariate analysis age older than 20 years, progressive onset of cgvhd, gastrointestinal tract involvement and platelets less than /ml were associated with increased mortality Identifies several independent prognostic risk factors for cgvhd incidence and severity comparing allobmt (75 patients) and allopsct (87 patients) patients, suggesting that stem cell source may influence not just the incidence of cgvhd but also its characteristics. Predictive factors for poor survival at 3 years after cgvhd diagnosis in allopsct patients were platelets below /ml and history of agvhd of the liver, and only low platelet count remained predictive for poor survival in allobmt group This is a comparative analysis of clinical presentation and response to treatment in 170 patients with cgvhd (123 after transplant from an unrelated donor and 47 from umbilical cord blood). In both cohorts thrombocytopenia and not achieving complete or partial remission at 2 months were independently associated with increased mortality Abbreviations: agvhd ¼ acute graft versus host disease; allobmt ¼ allogeneic bone marrow transplantation; allohsct ¼ allogeneic hematopoietic stem cell transplantation; allopsct ¼ allogeneic peripheral stem cell transplantation; cgvhd ¼ chronic graft versus host disease. circulation. Table 2 lists possible mechanisms of thrombocytopenia in the cgvdh setting. Transplant-related thrombocytopenia Limited function of the stem cell graft is among the most frequent reasons for decreased platelet production in the post-transplant period. It may be associated with insufficient numbers of transplanted stem cells, relapse of hematological disorder, or persistent marrow fibrosis. 13 However, transplant patients can develop isolated thrombocytopenia in the absence of disease recurrence or graft rejection. 21 A combination of a recovering bone marrow, and simultaneous processes that decrease platelet survival, which might otherwise have been well tolerated, can result in persisting thrombocytopenia or secondary platelet recovery failure after allohsct. 13 First et al. 14 found that among 65 patients who had full engraftment after allohsct, and who survived at least 60 days after transplantation, 24 (37%) developed isolated thrombocytopenia, 9 (14%) with transient, and 15 (23%) with chronic thrombocytopenia (defined as platelet count remaining below /ml through day þ 120). The transient syndrome was not associated with adverse outcome, but patients with chronic thrombocytopenia had increased mortality and an increased risk of having severe acute graft versus host disease (agvhd) and cgvhd. 14 Although bleeding complications in that study contributed directly to death in just two patients with chronic thrombocytopenia, there was a significantly higher mortality among cgvhd patients with chronic thrombocytopenia than in cgvhd patients with only transient or no thrombocytopenia. 14 The authors concluded that observed low platelet count may be a marker for a more severe form of cgvhd. 14 Pavletic et al. 2 found platelets below /ml in cgvhd patients more frequently after allobmt (41%) than after allopsct (27%), but the difference was not statistically significant. Similarly, Flowers et al. 22 also did not find a statistically significant difference in prevalence of thrombocytopenia in cgvhd patients after allobmt or allopsct. Although some authors speculated in earlier works that a graft-versus-stroma effect might contribute to inadequate marrow function resulting in delayed platelet recovery, 23,24 the numbers of megakaryocytes in the marrow were relatively normal in most patients with chronic thrombocytopenia and cgvhd. 5,13,14 It was observed more recently that recipients of unmanipulated allohsct contain only host-type bone marrow stromal cells (BMSC) and mesenchymal stem cells (MSC). 25 As MSCs can be generated as a homogenous population of cells that can be quantified, qualitatively analyzed, and ex vivo manipulated, 25 and have possible clinical importance because of their supportive function for engraftment and immunosuppressive properties, 26,27 transplantation of BMSC are evolving rapidly in different settings There seems to be promise for the beneficial effect in the administration of MSCs in severe steroid refractory agvhd, 27,29 but role of MSCs transplantation in the cgvhd setting is still unclear as it is unknown what the possible impact to thrombocytopenia and hemostatic disorders in cgvhd might be. After primary recovery of peripheral blood counts after allohsct, there can be a late, persistent, isolated decrease

3 Table 2 Proposed mechanisms that may cause thrombocytopenia in cgvhd 395 Mechanism Estimated frequency in cgvhd a Transplant-related thrombocytopenia Inadequate function of the stem cell graft Occasionally Insufficient number of transplanted stem cells Unrelated donor GVHD prophylaxis other than MTX+CSP Earlier acute GVHD Conditioning regimens (combination of busulfan, cyclophosphamide, and TBI) Malignancy relapse Microangiopatic thrombocytopenia Drug-induced thrombocytopenia Immune-mediated thrombocytopenia Hypersplenism Infection Low thrombopoietin Increased TGF-b Other cytokines Occasionally Occasionally Occasionally Often Rare Often Not known Not known Not known Abbreviations: cgvhd ¼ chronic graft versus host disease; CSP ¼ cyclosporine; MTX ¼ methotrexate; TGF ¼ transforming growth factor. a As majority of studies that found thrombocytopenia as a negative survival predictor in cgvhd patients did not analyze separately possible underlying reasons of observed low platelet counts, prevalence of proposed mechanisms that lead to thrombocytopenia in cgvhd is an estimate based on available information from published studies. in platelet counts called secondary failure of platelet recovery (SFPR), also associated with a poor prognosis after transplantation. 21 Bruno et al. 21 reported that among 1401 patients undergoing allohsct after primary recovery of platelet counts, SFPR not because of relapse of underlying disease was observed in 285 (20%) patients, with the median time of onset after transplantation at day 63 (range day ). Among patients who developed SFPR, 141 (51%) were alive at 1 year, among them clinical extensive cgvhd occurred in 71% (101/141) and limited cgvhd in 2.5% (5/141) patients. Hazard ratio of death was significantly higher in patients who developed SFPR (hazard ratio ¼ 2.6 for allohsct and 2.2 for autohsct). No cases of graft rejection were reported in patients with SFPR. 21 The factors significantly associated with SFPR were grafts from unrelated donors, regimens for GVHD prophylaxis other than cyclosporine (CSP) and methotrexate (MTX), grade 2 4 agvhd, impaired renal or liver function, preparative regimen with combination of busulfan, cyclophosphamide, total body irradiation and infections. 21 Microangiopatic thrombocytopenia Batts and Lazarus described HSCT-associated thrombotic microangiopathy as an infrequent but challenging syndrome that may occur in allohsct recipients, and suggested that it is a multifactorial disorder distinct from thrombotic thrombocytopenic purpura (TTP). 30 It is associated with several transplantation-related factors such as conditioning regimens, immunosuppressive agents such as tacrolimus or CSP, GVHD, and opportunistic infections. 30 Hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura (HUS/TTP) has been reported to occur in 13 and 9% of patients after allohsct. 13,31,32 Some studies reported that using sirolimus together with calcineurin inhibitors for the treatment of refractory cgvhd could increase risk for developing thrombotic microangiopathy 33 or renal insufficiency and HUS 34 as toxic side effects. In a retrospective analysis of 477 HSCT patients (364 allogeneic, 83 autologous transplants) Pihusch et al. 35 did not find any microangiopathic hemolytic anemia (MAHA) (defined as the simultaneous occurrence of intravascular hemolysis characterized by appearance of schistocytes in peripheral blood smears and de novo thrombocytopenia) in autologous transplant (autohsct) group, but there were 53 (14.6%) MAHA in allohsct group. MAHA in allohsct group was found among 48 (19.2%) agvhd and just in 6 (3.5%) cgvhd patients. 35 However, in subsequent prospective study from the same group, among 89 allohsct patients MAHA was found in 13 (20.0%) agvhd and in 7 (22.6%) cgvhd patients. 36 Drug-induced thrombocytopenia Drugs such as MTX, ganciclovir, and trimethoprimsulfamethoxazole that are frequently used after allohsct may be associated with delayed platelet engraftment or secondary platelet failure. 14,37 40 Bruno et al. 21 found that regimens for GVHD prophylaxis different from the standard combination with CSP and MTX were associated with SFPR after allohsct. However, other studies did not find differences in platelet transfusion requirements when comparing the combination of CSP and MTX to CSP alone. 38 Mycophenolate mofetil is an established drug for treatment of both acute and chronic GVHD, and it could cause thrombocytopenia as a side effect. 41 Other drugs may cause thrombocytopenia by inducing antibodies against megakaryocytes and/or platelets, such as acetaminophen, amphotericin B, vancomycin, ranitidine, fluconazole, heparin, and others. 42 Many such drugs are used in cgvhd patients, so it is important to consider drug-induced thrombocytopenia as a possible cause of low platelet counts in the cgvhd setting. Immune-mediated thrombocytopenia In addition to drug-induced antiplatelet antibodies and identification of a number of other different autoantibodies

4 396 and alloantibodies in cgvhd patients, 43 development of platelet-specific auto- or allo-antibodies have been reported in cgvhd patients. 5,44 46 Anasetti et al. 5 assessed mechanisms of persistent thrombocytopenia in 20 patients who were between 60 and 649 days (median 90) after allobmt; among them 17 had isolated thrombocytopenia, 10 agvhd, and 6 cgvhd. 5 Platelet survival studies showed that platelets persisted in the circulation for a shorter period of time in patients with GVHD, and in all studied patients a direct relationship between platelet survival and platelet count was observed. 5 Moreover, platelet autoantibodies were found in five of six patients with acute or chronic GVHD, and in none of six patients without GVHD. 5 The investigators concluded that persistent thrombocytopenia after marrow transplantation is most often because of increased platelet destruction mediated by multiple mechanisms and that the immune deregulation accompanying GVHD may produce autoimmune thrombocytopenia. 5 Platelet count increments after transfusions in GVHD patients have also been found to be less than in controls without GVHD, supporting conclusion that GVHD patients may have thrombocytopenia mainly because of decreased platelet survival in the peripheral blood. 13 Hypersplenism Banaji et al. 47 assessed the effect of splenectomy on engraftment and platelet transfusion requirements in 67 allobmt patients with chronic myelogenous leukemia. Comparing with patients splenectomized before transplantation, it was reported that the presence of the spleen was an important factor in delaying recovery of platelet counts after allohsct. However, among unsplenectomized patients the splenic size did not affect the speed of platelet recovery or platelet transfusion requirements. 47 Another study showed a trend for splenectomized patients to have a faster recovery of platelet counts compared with nonsplenectomized controls, with similar incidence of cgvhd among splenectomized and non-splenectomized patients. 48 Anasetti et al. 5 did not find a relationship between platelet count and splenic pooling, and they concluded that hypersplenism did not significantly contribute to thrombocytopenia in their study. Others described two patients who underwent splenectomy because of resistant immune cytopenias after allohsct, resulting in improvement of platelet and other blood counts, but severe late infections compromised the outcome of that approach. 49 Infection It is well known that severe thrombocytopenia may occur in patients with sepsis in the non-transplant setting. 50 As patients with cgvhd have an increased risk for infection in one recently published study 57% of cgvhd patients developed infections with mortality rate of 27% 51 it is possible that some common mechanisms contribute to the thrombocytopenic states found in infections or inflammation and cgvhd. One proposed mechanism is a cytokine storm effect, with increased levels of cytokines that may activate the monocyte/macrophage system, causing increased removal of platelets from circulation. 21,52 54 CMV infection has been shown to be associated with poor marrow graft function after allohsct. 55 CMV has a direct toxic effect on marrow stem cell maturation, 56 and it can disturb effective production of platelets after HSCT. 57 Isolated thrombocytopenia has also been observed during CMV infection. 58 CMV modulates various aspects of immune response, and different mechanisms of marrow suppression have been proposed, such as direct infection of hematopoietic progenitors or stromal cells, abnormal gene expression, and immune reaction against the infected cells More recent studies in longterm bone marrow cultures indicate that BMSC are targets of CMV infection and that CMV replicates in BMSCs. 63 In addition, experiments show that BMSCs are permissive to CMV infection, and that CMV significantly alters the function of BMSCs. 63 It is likely that in the pathobiology of CMV infection, natural killer T cells and CD1d molecules might be protective against CMV-induced suppression of hematopoiesis, according to the results in experimental animals, which may lead to the new strategies in treatment of CMV-induced perturbation of hematopoiesis in immunocompromised patients. 64 In addition to that, other therapeutic approaches to CMV infection and disease have been extensively investigated in recent years Some other viruses may also cause thrombocytopenia by various mechanisms, such as Epstein-Barr virus, human herpes virus-6, parvovirus B CMV or human herpes virus-6 in post-allobmt patients may also cause vascular endothelial injury, which could lead to development of thrombotic microangiopathy. 73 Cytokine-induced thrombocytopenia It is possible that similar mechanisms are responsible for the thrombocytopenia in infection and cgvhd. Serum cytokine levels including granulocyte colony-stimulating factor (G-CSF), macrophage CSF (M-CSF), interleukin- 1a, interleukin-1b, and interleukin-6 may be significantly increased in both conditions, and may lead to the activation of monocytes and macrophages, leading to the increased platelet consumption. 13 In addition, it was observed that both granulocyte macrophage-csf and M-CSF in pharmacologic doses may reduce platelet counts in a dose-dependent manner or delay platelet recovery after chemotherapy. 54,74 76 Additionally, though donor treatment with G-CSF may decrease the ability of donor T cells to induce agvhd, it may also contribute to increase the severity of cgvhd. 77,78 The role of transforming growth factor-beta (TGF-b) in this process was assessed in a murine model of agvhd and cgvhd. It was shown that the production of TGF-b by donor T cells early after HSCT attenuates agvhd and is important for graft versus leukemia effects, and that the production of TGF-b late after HSCT is mostly from mononuclear cells and increases the severity of cgvhd. 1,79 TGF-b suppresses megakaryopoiesis, 80 is a potent immunosuppressive cytokine, and has role in development of tissue fibrosis. 81 Liem et al. 81 found that TGF-b levels correlate significantly with platelet and leukocyte count in BMT recipients and that cgvhd is associated with increased level of serum TGF-b independent of platelet or leukocyte count. Recent work has

5 provided evidence for the activation of TGF-b in a variety of cells and tissues infected by CMV, inducing further downstream signaling that could alter vascular function and lead to development of fibrosis. 82 Such results might suggest another possibly important pathophysiological mechanism that link together cgvhd, CMV infection, TGF-b, and thrombocytopenia. Thrombocytopenia because of low thrombopoietin (TPO) level is generally thought to be relatively rare; however, it has not been thoroughly investigated in the cgvhd setting. Hirayama et al. 83 described two cgvhd patients with low platelet counts and strong correlations of platelet and megakaryocyte numbers with TPO concentrations in peripheral blood. They concluded that low TPO production may be responsible for thrombocytopenia in some cgvhd patients. Thrombophilic and bleeding predisposition in cgvhd Microparticles Pihusch et al. 84 found that patients with cgvhd had a seven-fold increased ratio of circulating platelet microparticles comparing to healthy controls, whereas allohsct patients without cgvhd had a normal ratio of circulating microparticles. Circulating microparticles are membrane vesicles mainly derived from platelets, but also from erythrocytes, leukocytes, and endothelial cells, 85 released after intensive cell activation, which enhance enzymatic reactions of the coagulation cascade by providing a large phospholipid surface, and activating platelets, 90 thereby increasing the risk of thrombosis. Microparticles are, for example, associated with prothrombotic conditions such as heparin-induced thrombocytopenia, 86 TTP, 91 paroxysmal nocturnal hemoglobinuria, 92 and myocardial infarction. 93 Moreover, microparticles produce proinflammatory changes in the endothelium, increase expression of adhesion molecules 90 and have strong mitogenic effects on smooth muscle cells. 94 Leukocytes and endothelial cells might also generate microparticles that have a functional role and may modulate activities of other cells. 95 For example, leukocyte-derived microparticles deliver prothrombotic tissue factor to developing thrombus, and target activating platelets by binding platelet P-selectin. 95 According to a recently published study, leukocyte-derived microparticles also act as a novel platelet agonist targeting GPIba, a platelet-adhesion receptor important for initiation of clot formation by binding von Willebrand factor (vwf); such leukocyte platelet interactions are amplified in situation when neutrophils are also activated. 95,96 It is important to note the link between vwf, endothelial injury, and cgvhd described by Biedermann et al. 97 They found that cgvhd patients have extensive loss of microvessels in affected tissues and increased circulating vwf concentrations, concluding that vwf was released from vascular endothelial cells injured in process of cgvhd. 97 In summary, involvement of microparticles formation in coagulation, inflammation, and vascular problems, as well as in other platelet leukocyte endothelial processes and interactions, 98 could have important roles in the pathophysiology of tissue damage found in cgvhd. 84 Acquired platelet storage pool disease Pihusch et al. 84 observed that platelets were activated in patients after allohsct, but they also found loss of dense platelet granules and a reduced surface expression of the major collagen receptor GP Ia/IIa. Platelet dense bodies contain ADP, ATP, serotonin, and divalent cations, and are essential for primary hemostasis. A defect of dense bodies, termed storage pool deficiency, is usually inborn, but patients following allohsct may have an acquired storage pool deficiency. 84 The integrin receptor GP Ia/IIa is important in the adhesion of platelets to collagen, 99 leading to platelet fixation and activation. A reduced surface expression of this receptor has been described in inborn platelet abnormalities 100 and in myelodysplastic syndromes 101 and leads to an increased bleeding risk. Although the observed HSCT-associated storage pool disease and the GP Ia/IIa deficiency may increase the bleeding tendency in HSCT patients, they have not been associated with cgvhd, platelet count, antiviral, antifungal, or immunosuppressive drugs. Therefore, these investigators suggest that they could be the consequence of a perturbation of platelet synthesis after allohsct. 84 Bleeding and thrombosis in cgvhd In another large study Pihusch et al. 35 retrospectively analyzed hemostatic complications among 447 HSCT patients. The 364 allohsct patients had a higher incidence and severity of hemorrhagic complications than autohsct patients, despite a similar duration of thrombocytopenia in both groups. Subgroup analysis showed that this was due to GVHD: patients with agvhd greater than grade I had a 3-fold higher risk of bleeding, cgvhd patients had a 4-fold higher risk, with an 11-fold risk of severe hemorrhage. Such GVHD-associated bleeding was mostly localized in GVHD-affected organs such as skin and gastrointestinal tract; it correlated with an increased mortality, and could not be explained by demonstrated disturbances in hemostatic mechanisms. The authors concluded that agvhd and cgvhd strongly aggravated bleeding risk by destruction of epithelium and hyperperfusion and proliferation of the blood vessels, which is typical for tissues affected by GVHD. 35 In addition, they observed that hemorrhagic cystitis was strongly associated with both agvhd and cgvhd. In the same study among 447 HSCT patients (364 allohsct), venous thromboembolism (VTE), including catheter thrombosis, extremity thromboses, or pulmonary embolisms, occurred mostly later after transplantation and depended on the type of transplantation. AutoHSCT patients had a 4.8% incidence, allohsct patients with agvhd had a 6.8% incidence, and cgvhd patients had an 8.1% incidence of VTE. 35 cgvhd and treatment with steroids were the most important risk factors for VTE. 35 The authors suggested that cytokines that modulate endothelial hemostatic function could be responsible for VTE in cgvhd. For example, tumor necrosis factor-alpha, which is important in the pathophysiology of GVHD, enhances the production of the prothrombotic plasminogen activator inhibitor I 102 and tissue factor, and decreases the tissue factor pathway inhibitor, thus increasing possibility of the development of 397

6 398 VTE. 35 Other work from the same group of authors prospectively studied the impact of GVHD and of thrombophilic gene mutations on hemostatic complications and on transplant-related toxicity in 89 allohsct patients. 36 They again reported increased risk for venous thrombosis and pulmonary embolism among cgvhd patients, whereas the risk for catheter thrombosis was not affected by GVHD. 36 Risk of bleeding was increased in both agvhd and cgvhd patients, which was explained by differences in the incidence of gastrointestinal bleeding. 36 In that work, thrombophilic gene mutations and polymorphisms did not influence either the incidence of any of the major transplant-related complications, including agvhd and cgvhd, or the bleeding incidence. 36 Among patients with thromboembolic complications those with catheter thromboses and hepatic veno-occlusive disease showed an increased frequency of the 4G allele of the PAI-1 gene, whereas other tested thrombophilic gene mutations and polymorphisms had no impact on the incidence of VTE complications among allohsct patients. Authors concluded that thrombophilic gene mutations have only a moderate influence on hemostatic complications in allohsct patients, and that a possible explanation might be because of major immunologic impact of GVHD on hemostasis in the allohsct setting. 36 In addition to that, in cgvhd patients, the simultaneous presence of a thrombophilic state, because of circulating microparticles or activated platelets, and the HSCT-associated platelet defect, which is attributed to an increased bleeding tendency, may seem paradoxical. However, observed impaired platelet function may mainly disturb a primary or immediate hemostasis, whereas the huge phospholipid surface provided by microparticles mainly activates the plasma coagulation cascade, which is crucial in the pathogenesis of VTE. Therefore, patients with cgvhd may have an increased bleeding tendency and a prothrombotic state at the same time. 35,84 It should be also noted that thalidomide, a drug that is used in the treatment of cgvhd, 103 has prothrombogenic side effects, which may further increase the risk for development of VTE in cgvhd patients. Other acquired hemostatic disorders in cgvhd Other acquired hemostatic disorders in cgvhd patients are rare and are described in several case reports (Table 3). Seidler et al. 16 described a male patient who developed acquired hemophilia A (acquired factor VIII inhibitor) with clinical bleeding diathesis 2 years after allobmt for chronic myelogenous leukemia in association with a flare of cgvhd. 16 The patient had no history of bleeding and had normal coagulation tests in the past. The patient responded well to prednisone therapy with disappearance of FVIII inhibitor, regression of cgvhd, and no further bleeding manifestations. 16 Lazarchick and Green 17 described a patient with acquired von Willebrand s disease and cgvhd. The patient was transplanted because of acute myelogenous leukemia and had worsening of cgvhd 6 months after allohsct with bleeding manifestations. He was found to have no measurable FVIII activity Table 3 Case reports of rare acquired disorders of hemostasis in cgvhd patients Author (reference)/year and a mixing study was compatible with the presence of inhibitor. It was first assumed that he developed acquired hemophilia A. However, further evaluation defined that he had also very low vwf antigen and ristocetin cofactor activity, with antiristocetin cofactor inhibitor and almost complete absence of all vwf multimers, consistent with the diagnosis of acquired von Willebrand s disease. 17 He was treated with combination of inhibitor removal by apheresis followed by infusion of FVIII and vwf concentrate, which resulted in normalization of coagulation parameters and cessation of bleeding. He died of progressive cgvhd with multi-organ system failure. 17 Antiphospholipid syndrome (APS) is characterized by antiphospholipid antibodies, thrombosis, thrombocytopenia, and/or recurrent fetal loss. Antiphospholipid antibodies are also among GVHDassociated autoantibodies, and though some studies have not found them to be pathologic, 104 an occasional case report has found an association between antiphospholipid antibodies and development of APS with thrombotic manifestations in cgvhd. 20 Catastrophic APS, an unusual and potentially lethal variant of the APS has also been described in cgvhd setting. 19 Kasamon et al. 19 described a female patient who developed catastrophic APS after allohsct for acute lymphoblastic leukemia. She developed multi-organ thrombosis and organ failure together with antiphospholipid antibodies and cgvhd and died. Ritchie et al. 18 described passive donor-to-recipient transfer of APS following the onset of cgvhd. In that case, a male patient with CML underwent allohsct. His sister was the donor who had documented systemic lupus erythematous and APS. Six months post transplant he developed cgvhd of liver, and anticardiolipin antibodies, lupus anticoagulant and antinuclear antibody were positive at 8, 12, and 16 months, which was identical to that of the donor. He did not develop any clinical evidence of systemic lupus erythematous, however, at 53 months after transplantation he experienced cerebrovascular thrombosis, which was successfully treated. 18 Future considerations Diagnosis Seidler et al. 16 /1994 Acquired hemophilia A (acquired FVIII inhibitor) in cgvhd Lazarchick and Green 17 /1994 Acquired von Willebrand s disease in cgvhd Sohngen et al. 20 /1994 Antiphospholipid syndrome in cgvhd Kasamon et al. 19 /2005 Catastrophic antiphospholipid syndrome in cgvhd Ritchie et al. 18 /2005 Passive donor-to recipient transfer of antiphospholipid syndrome; antibody appearance was precipitated by the onset of cgvhd Abbreviation: cgvhd ¼ chronic graft versus host disease. Many different mediators are involved in platelet production and activation. These mediators may be of interest in

7 future assessments of coagulation processes and disorders in cgvhd patients. Furthermore, a very recently published paper of Flaumenhaft et al. 105 emphasized the importance of platelet-derived microparticles in many different pathological processes; it should be interesting to investigate their possible role in pathogenesis of cgvhd. In addition, it would be important to study the relationship of thrombocytopenia and disorders of hemostasis in the context of various cgvhd clinical presentations in light of the new NIH cgvhd classification. 106 As a variety of new agents are currently being tested as new options for the management of thrombocytopenia, it would be interesting to determine their effect on thrombocytopenia in cgvhd. Pharmaceutical methods to stimulate the production of platelets from megakaryocytes (analogous to erythropoietin to stimulate red blood cell production or G-CSF to stimulate granulocyte production) have long been sought to treat thrombocytopenia. The first efforts to stimulate platelet production by use of recombinant hormone TPO or a pegylated recombinant partial TPO peptide human megakaryocyte growth and development factor (PEG-rHuMGDG) resulted in faster recovery of normal platelet counts in chemotherapy recipients. 107 However, the use of these drugs was complicated by development of antibodies that cross-reacted to native TPO, and in the case of PEG-rHuMGDG recipients, neutralized TPO (resulting in prolonged thrombocytopenia). 108 For this reason, subsequent drug development in this field has focused on finding molecules that either interact with the TPO receptors on the megakaryocyte surface, or stimulate the downstream intracellular signaling pathway that is activated by TPO. Two such molecules have been identified that act to increase platelet counts by stimulating platelet production in megakaryocytes: eltrombopag, a synthetic molecule with oral bioavailability that interacts non-competitively with the TPO receptor to increase thrombopoiesis, 109 and romiplostim, a novel peptide in which two IgG Fc domains are fused to four copies of a TPO-mimetic peptide. 110 Both romiplostim and eltrombopag seem to be effective in raising platelet counts and have been well tolerated in clinical trials to date It remains to be seen whether use of either of these agents would be of value to patients with thrombocytopenia and cgvhd. Another possible area of interest is testing BMSC infusion in the context of cgvhd and its hemostatic impairments; anecdotal but not well-documented partial responses in cgvhd patients after BMSC transplantation have been already reported. 27 In the context of antiinflammatory and immunosuppressive effect of BMSC it would be interesting to assess its effect in the treatment of cgvhd as well as its effect on low platelet counts and other hemostatic disorders in cgvhd patients. In summary, thrombocytopenia and impaired hemostasis in cgvhd are significant complications. These disorders may be closely implicated in the cgvhd pathogenesis and could represent targets of potential interest for therapeutic intervention. Design and conduct of future clinical trials with agents that stimulate megakaryocytopoiesis or influence underlying hemostatic deregulation should be explored; however, such studies must also consider risks associated with concurrent hemostatic disturbance and complex clinical factors in the context of the cgvhd setting. Conflict of interest The authors declare no conflict of interest. Acknowledgements We thank Drs Georgia Vogelsang and Ronald Gress for their valuable comments and the critical review of the manuscript. This work is supported in part by the intramural program of the National Cancer Institute, Center for Cancer Research. Disclaimer: The opinions expressed here are those of the authors and do not represent the official position of the National Institutes of Health or the US Government. References 1 Baird K, Pavletic SZ. Chronic graft versus host disease. Curr Opin Hematol 2006; 13: Pavletic SZ, Smith LM, Bishop MR, Lynch JC, Tarantolo SR, Vose JM et al. 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