TPO Mimetics Research Articles

The Combination of Intravenous Immunoglobulin (IVIG) and Low Does Recombinant Human Thrombopoietin (rhTPO) for the Management of Corticosteroid/IVIG Monotherapy-Resistant Immune Thrombocytopenia in Pregnancy

Introduction Primary immune thrombocytopenia (ITP) is a common autoimmune disease characterized by a low platelet count resulting from antibody-mediated platelet destruction and insufficient platelet production. ITP, with a prevalence of 1 in 1000-10 000 pregnancies, accounts for approximately 3% of all causes of thrombocytopenia in pregnancy and is reported to be the most common cause of thrombocytopenia in the first and early second trimesters. It has been revealed that severe ITP is associated with devastating outcomes. It remains a challenge to manage pregnant patients with ITP. First-line treatment options in pregnant ITP patients currently include corticosteroids and intravenous immunoglobulin (IVIg). However, previous studies reported that the response rate of pregnant ITP patients to corticosteroids was less than 40% and that to IVIg was 38-56%, which was lower than that of nonpregnant ITP patients. The second-line treatment options are limited for pregnant ITP patients. Recombinant human thrombopoietin (rhTPO), a full-length and glycosylated TPO with a molecular weight of 90 000 Daltons developed by 3SBIO (Shenyang, China), has also been approved by the China State Food and Drug Administration as a second-line option for nonpregnant ITP patients. It cannot cross the placenta theoretically with such a large molecular weight, and thus, it has an apparent theoretical advantage over the two TPO mimetics since it would not impact the fetus. A study investigating the application of rhTPO in pregnancy suggested that rhTPO was a potentially effective and well-tolerated treatment option for ITP patients during pregnancy. It might be explained that the development of ITP was associated with increased platelet destruction and impaired platelet production, and the combination of IVIg and TPO mimetics impacts these two targets since IVIg could reduce platelet destruction, whereas TPO mimetics could increase platelet production. Therefore, we conducted a monocenter, single-arm, open-label study to investigate the efficacy and safety of low-dose rhTPO plus IVIg in pregnant corticosteroid/IVIg monotherapy-resistant ITP patients. Methods This study was registered at www.clinicaltrials.gov as NCT05634824.The following criteria were considered: patients have a diagnosis of primary ITP before or first onset during pregnancy; patients ≥ 18 years; patients complicated with bleeding manifestations and/or have a platelet count < 30 × 10^9/L and failed to respond to initial treatment of corticosteroids or intravenous immunoglobulin (IVIg) monotherapy or relapsed during the tapering or discontinuation of corticosteroids. For those patients, the initial combination therapy consisted of rhTPO at an initial dose of 150 U/kg once daily subcutaneously for 28 days and IVIg 400 mg/kg per day for 5 days. If the platelet count was in the range of 30 to 50 × 10^9/L, the combination therapy was repeated. To reduce the risk for thrombocytosis, patients received maintenance therapy consisting of rhTPO at a dose of 150 U/kg per day if the platelet count rose above 50 × 10^9/L, and treatment was discontinued when platelet counts exceeded 100 × 10^9/L. After delivery, the treatment consisted of rhTPO 150 U/kg per day, and the dose was maintained if the platelet count exceeded 30 × 10^9/L. In addition, IVIg 400 mg/kg per day for 5 days was added if the platelet count could not be maintained above 30 × 10^9/L. If patients did not achieve a count of 30 × 10^9/L within 4 weeks, treatment was also discontinued. Results The study included 19 patients with thrombocytopenia during pregnancy who were admitted to Peking University People's Hospital from December 1, 2022, to May 30, 2023, all of whom had failed hormone and IVIG treatment. The median age of those patients was 34 years old (range 20 to 42 years old), and the median platelet count before treatment was 13×10^9/L (range 1-30×10^9/L). The four-week response rate was 63.2% (12/19), and the median response time was 7.5 days (range 5 to 35 days). There were no treatment-related adverse reactions. Conclusions Low-dose rhTPO plus IVIg in pregnant corticosteroid/IVIg monotherapy-resistant ITP patients is effective and safe. These findings suggest that IVIg plus low-dose rhTPO may represent a potential combination therapy for pregnant patients with ITP.

Applying the Novel EHA/EBMT Grading System for Icaht Following CAR-T Therapy: Comparative Incidence across Disease Entities and Association with Infections and Mortality

Background: Hematological toxicity is a frequent side effect of CAR-T therapy and predisposes for severe infectious complications. Recently, the EHA/EBMT consensus grading for Immune Effector Cell Associated HematoToxicity (ICAHT) was developed ( Rejeski et al. Blood 2023). In contrast to existing grading systems such as the CTCAE, the novel ICAHT grading integrates both the depth and duration of neutropenia. We hypothesized that ICAHT severity is associated with key clinical sequelae such as transfusion requirements, hospitalization, severe infections, and adverse treatment outcomes. Methods: We applied the novel grading of early ICAHT (day 0-30) to a multicenter retrospective real-world cohort of 549 patients treated with BCMA- or CD19-directed CAR T-cells for relapsed/refractory B-cell malignancies (334 large B-cell lymphoma [LBCL], 112 multiple myeloma [MM], 103 mantle cell lymphoma [MCL]) across twelve international sites (Germany, France, Spain, USA). Hematotoxicity endpoints included the total cumulative duration of severe neutropenia (ANC <500/µL, day 0-60) and phenotypes of neutrophil recovery ( Rejeski et al. Blood 2021). Infections were studied until day +90 after CAR-T and were graded on a 5-grade scale as mild, moderate, severe, life-threatening, or fatal. Non-relapse mortality (NRM) was defined as death following CAR-T without evidence of relapse or progression. Kaplan-Meier estimates of progression-free (PFS) and overall survival (OS) were compared using log-rank test. Results: Our study cohort included 424 patients without severe ICAHT (absent or grade 1-2) and 125 patients with severe ICAHT (grade 3-4). The distribution of mild (1°), moderate (2°), severe (3°) and life-threatening (4°) ICAHT was 36%, 29%, 17%, and 5%. Seventy patients (13%) did not develop ICAHT of any grade. When comparing ICAHT grades across disease entities, we noted a numeric trend towards increased severe ICAHT in MCL compared to LBCL or MM patients (Grade ≥3: 28% vs. 23% vs. 15%). Patients that subsequently developed severe ICAHT displayed elevated serum inflammatory markers and pronounced cytopenia at baseline - reflected by increased CAR-HEMATOTOX scores (median 3 vs. 1, p<0.0001). On multivariate binary logistic regression, co-stimulatory domain (CD28z), low platelet count, low ANC, low hemoglobin, increased serum ferritin, and the presence of bone marrow infiltration were independent risk factors for severe ICAHT. We noted a strong positive correlation between ICAHT severity and the cumulative duration of severe neutropenia (r=0.92, p<0.0001). The aplastic phenotype of neutrophil recovery was enriched in patients with grade 3 (73%) and especially grade 4 ICAHT (100%). Severe ICAHT was also associated with coincident multilineage cytopenias, resulting in increased transfusion requirements for both platelets (85% vs. 21%, p<0.0001) and red blood cells (86% vs. 41%, p<0.0001). In terms of management of hematotoxicity, the severe ICAHT group more commonly received G-CSF (72% vs. 44%, p=0.01), TPO mimetics (12% vs. 2%, p<0.0001) and hematopoietic stem cell boosts (6% vs. 0.2%, p<0.0001). Importantly, patients with severe ICAHT displayed a significantly increased rate of severe infections (49% vs. 13%, p<0.0001, Fig. 1a) and severe bacterial infections (36% vs. 8%, p<0.0001). This translated into a markedly increased 1-year NRM rate (14% vs. 4.5%, logrank p<0.0001, Fig. 1b), which was primarily attributed to fatal infections of fungal or bacterial origin. Of note, the ICAHT grading showed superior discrimination for severe infections (C-index 0.73, 95%CI: 0.67-0.78) compared to CTCAE grading (C-index 0.55, n.s.). Prolonged hospitalization was noted in case of severe ICAHT (median 21 vs. 16 days, p<0.0001) and intensive care admission was more common (23% vs. 4%, p<0.001). Finally, we observed inferior PFS (1-year PFS 35% vs. 51%, median 4 vs. 14 months, p<0.0001) and inferior OS (1-year OS 52% vs. 73%, median 12 months vs. not-reached, p<0.0001) in the patients that developed severe ICAHT. Conclusions: These data highlight the clinical relevance of the novel EHA/EBMT ICAHT grading system. Importantly, ICAHT severity was closely associated with transfusion dependence, increased infection incidence, and adverse treatment outcomes with high NRM. Taken together, these findings argue for reporting ICAHT grades in clinical trials evaluating established and novel CAR constructs.

Danazol Treatment for Thrombocytopenia in Myelodysplastic Syndromes: Can an "Old-fashioned" Drug be Effective?

Danazol Treatment for Thrombocytopenia in Myelodysplastic Syndromes: Can an "Old-fashioned" Drug be Effective?

Treatment of Lower Risk Myelodysplastic Syndromes

Purpose of review: Management and Optimization of therapy for lower-risk myelodysplastic syndromes will be reviewed here. Recent findings: Lower-risk MDS typically present with clinical manifestations of anemia, which is the most frequently encountered cytopenia in this setting. While therapy with erythropoietic stimulating agents (ESAs) is used in the vast majority of cases, if correctly selected, some patients do not respond, or become irresponsive to ESAs. Novel agents with very different modes of action show promising clinical results in anemic LR-MDS refractory/relapsed after ESAs. Luspatercept, a TGFbeta family ligand trap, induces nearly 50% of transfusion independence in LR MDS. Another investigational agent showing efficacy and possibly disease modifying activity is the telomerase inhibitor imetelstat. Modulation of dose and schedule of hypomethylating agents, both injectable and oral, is currently being explored, and preliminary results are positive. There is still no standard therapeutic approach for thrombocytopenic and neutropenic LR MDS, although they do represent a smaller proportion of cases. Immunosuppressive treatments, as well as TPO mimetics, could represent a good option in selected MDS cases. Summary: At present, the availability of novel active agents allows the planning of sequential therapy, especially for anemic LR MDS. Better diagnosis and prognostic stratification may allow a more precise and personalized treatment.

Role of Next Generation Sequencing in the Diagnosis of Unexplained Thrombocytopenias- an Experience at a Tertiary Care Hospital

▪BACKGROUNDNext Generation Sequencing (NGS) has been enormously rewarding in the field of diagnostic hematology. In particular, the diagnosis of inherited disorders has progressed in leaps and bounds. These patients tend to remain undiagnosed for a long period of time not only because of unavailability of molecular diagnostics but also due to lack of cognizance and atypical presentations.Thrombocytopenia (TCP) is a common hematological presentation and can lead to chronic hospital visits to life-threatening bleeds. Most of these patients have acquired disorders such as immune TCP, malignancies, liver disease etc. However, some of them are likely to have unidentified inherited causes. We thus intended to study the utility of NGS in the definitive diagnosis of unexplained TCPs with or without other cytopenias to understand the clinicopathologic characteristics of these patients.METHODOLOGYThis was a retrospective descriptive study done at two centres over three years from May 2018 to May 2021. Patients with TCP with one of the following: (a) positive family history (b) clinical/ laboratory clues to an inherited cause (c) chronic TCP with no response to conventional therapies and sent for clinical exome sequencing done by NGS were included in the study. Patients who were negative for germline mutations were excluded.Sequencing of targeted genes was performed on the Illumina platform with a mean coverage of >80-100X. Mutations annotated as pathogenic, likely pathogenic and variant of uncertain significance (VUS) were considered clinically significant. VUS are mutations that are difficult to classify as pathogenic and require clinical validation and family testing.RESULTSOur cohort included 18 patients and were divided into two groups- cases of isolated TCP and cases of TCP with anemia and/or neutropenia.Patients presenting with isolated thrombocytopeniaWe had nine cases of isolated TCP out of which there were three cases of X-linked macrothrombocytopenia with MYH9 mutation, two cases of Wiskott Aldrich syndrome (WAS) and one case each of congenital thrombotic thrombocytopenic purpura (TTP), atypical Hemolytic Uremic Syndrome (HUS), Fanconi anemia (FA) and grey platelet syndrome. The demographic and mutational characteristics are described in Table 1. Clinical and laboratory clues were present in 7 cases, such as chronic kidney disease, micro/ macrothrombocytopenia, neutrophil inclusions etc. Bone marrow examination was carried out in 4 cases- the significant dyspoiesis in FA and myelofibrosis in grey platelet syndrome mislead to a diagnosis of MDS and myelofibrosis respectively. Seven patients had received treatment with steroids, immunosuppressants, splenectomy, danazol and TPO mimetics before the NGS diagnosis.Patients presenting with thrombocytopenia and other cytopeniasThis group consisted of nine cases with two cases each of Dyskeratosis Congenita (DKC) and WAS and one case each of TTP, Ghosal hematodiaphyseal dysplasia, Congenital Amegakaryocytic Thrombocytopenia (CAMT), B-cell immunodeficiency with hypogammaglobulinemia type-25 and double homozygous for FA and DKC. The majority of patients in this group were young and had lower platelet counts (Table 2). The most common associated cytopenia was anemia. Phenotypic clues to diagnosis were present in cases of DKC and WAS. The common differentials considered in this group were inherited bone marrow failure syndromes (IBMFS), congenital immunodeficiency syndromes etc. Bone marrow examination was done more frequently in these patients and showed hypocellular marrow in IBMFS and absent megakaryocytes in CAMT. These patients have also been treated with steroids, IVIg, danazol and TPO mimetics.CONCLUSIONPatients with inherited isolated TCPs have a chronic course and heterogenous causes therefore tend to be diagnosed later in life. However, patients with TCPs and other cytopenias tend to present at a younger age with infrequent family history. IBMFS was the most common disorder identified in this latter group of patients. Positive family history, clinical and laboratory clues and absence of response to conventional therapies should prompt workup of inherited causes by NGS to avoid long term ineffectual treatment. Further, NGS mutations, in particular VUS have to be interpreted with caution with the help of parental study, clinical presentation, in-silico analysis and inputs from molecular and genetic experts. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Refractory Immune Mediated ITP in a Patient with Metastatic Melanoma

Background: Immune check point inhibitors have changed the treatment landscape of many tumors including melanoma. They disrupt the immunoinhibitory signals mediated by programmed cell death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to use the body's immune system to destroy the tumor. Consequently, they also predispose patients to immune related toxicity which may affect any organ, commonly skin, gastrointestinal, hepatic, pulmonary and endocrine. Hematological immune-related adverse events are less common and include neutropenia, autoimmune hemolytic anemia, immune thrombocytopenia (ITP) and aplastic anemia. Herein we present a case of refractory ITP in a patient receiving immunotherapy for metastatic malignant melanoma.Case Presentation: A 54-year-old female with metastatic melanoma harboring the BRAF V600E was started on ipilimumab and nivolumab. After 3 cycles of immunotherapy, she was found to have sudden onset thrombocytopenia requiring hospital admission. Complete blood counts showed platelet counts10k/mcL, hemoglobin 13.0 g/dL and white blood cell 4.1 K/uL. She did not manifest any overt bleeding or bruising on history and exam. There was no evidence of hemolysis on labs and peripheral blood smear; thrombotic microangiopathy was ruled out. She received high dose dexamethasone 40 mg intravenous daily and 1 mg/kg intravenous immune globulin daily for three days for suspected ITP due to immunotherapy. Her platelet counts did not improve and therefore a bone marrow biopsy was performed which revealed a normocellular marrow without evidence of melanoma. Imaging revealed excellent radiological response; it was decided to hold further doses of immunotherapy due to severe thrombocytopenia. She received rituximab 375 mg/m 2, with no response and platelet counts continued to between 2-5 K/mcL and required daily platelet transfusions. Romiplostim was initiated which improved platelet count to 10K/mcL and she was discharged. The following day her platelet counts dropped to 5K/mcL and she was sent back to the emergency room where she received 1 unit of platelet transfusion. Unfortunately, she developed a cold limb and was found to have a complete occlusion of the right distal popliteal artery and left tibio-peroneal trunk. Initiation of heparin infusion was discussed with the patient, however due to the significant bleeding risk from refractory ITP, the patient decided to pursue comfort measures only and was discharged home for hospice care.Discussion: About 0.5% of metastatic melanoma patients experience immune thrombocytopenia, deaths caused by this severe adverse event are even rarer. It is postulated that the activation of CD4+ helper T cells and CD8+ cytotoxic T cells can result in the damage to hematopoietic stem cells. Furthermore, nivolumab induced increased production of platelet-specific IgG autoantibodies also promote platelet destruction. Diagnosis is particularly challenging due lack of specific test or markers, and it remains a diagnosis of exclusion. The risk of bleeding, arterial thromboembolism and venous thrombosis is higher in ITP patients. ITP due to immunotherapy may be profound and refractory. One may consider the use of second line agents, such as TPO mimetics earlier in these patients.Conclusions: Our case highlights refractory ITP as a serious immune related adverse event that failed several lines of treatment strategies. Early diagnosis and combination treatment measures early in the course of the disease is imperative to mitigate associated morbidity and mortality. Combination of steroids immunoglobulins and second line agents may be warranted in some cases, and earlier use of tier 2 agents such as azathioprine should also be considered. DisclosuresNo relevant conflicts of interest to declare.

Effect of Pregnancy on the Clinical Course of ITP Pregnant Women and Their Newborns. Resuts of a Spanish Case Series of 297 Primary ITP Pregnancies

Introduction:Given that there is no large case-series, effect of pregnancy on the course of pre-existing primary immune thrombocytopenia (ITP) patients is unclear1,2. Furthermore, outcome predictors evidence of neonates born to mothers with ITP is very scarce1. Due to obvious ethical reasons, no clinical trial regarding ITP and pregnancy has been reported until recent days3. Nevertheless, the relationship between ITP and pregnancy is considered a “trending topic” nowadays. As it is the, until now, unknown safety and efficacy of new drugs as TPO mimetics in this setting3-6.Aims:To evaluate outcome and global management of pregnancy and delivery on ITP women an on their offspring.Methods:Primary ITP was defined as a platelet count < 100 x 109/L in the absence of other causes or disorders that may be associated with thrombocytopenia.All women diagnosed of primary ITP from 2011 to 2016 in 24 Spanish Hematology Departments who had at least one pregnancy after ITP onset were included in this registry.Results:We included 297 primary ITP pregnancies from 204 women. At pregnancy diagnosis, we observed a majority of chronic ITP cases (71.9 %). At ITP diagnosis, median age of our case-series was 23 years (IQR, 18-31) and median platelet count was 18 x 109/l (IQR, 6-36). Median time from ITP diagnosis to pregnancy was 162 months (IQR, 0-364). Median number of pregnancies prior to ITP diagnosis was 1 (IQR, 0-2) with 1 pregnancy (IQR, 1-2) after ITP diagnosis as a median.51.6% of women received corticosteroids, immunoglobulins (IVIG) (16.6%), rituximab (7.1%) and/or splenectomy (8.7%) as ITP treatments between or before new pregnancies. On the other hand, 26.5% of women needed treatment for ITP during pregnancy, mainly steroids (13.9%) and IVIG (10.2%).The median platelet-count nadir during pregnancy was 73 x 109/l (IQR, 31-174). 135 (45.4%) pregnancies had less than 50 x 109 platelets/l with 77 (25.9%) with less than 30 x 109 platelets/l. 57 women (19.2%) exhibited hemorrhagic symptoms, being 30 (10.1%) of them severe bleedings.Regarding type of delivery, this was vaginal in 187 (62.9%) of pregnancies. Median platelet count at delivery was 111 x 109/l (IQR, 70-187). 47 patients (15.8 %) experienced 60 bleeding episodes. We only observed 52 cases (19.5%) of neonatal thrombocytopenia among 266 living newborns.Conclusions:Our results are comparable to previously reported1,2. No severe bleeding complications during pregnancy and/or delivery were observed in our study. Rate of neonatal thrombocytopenia, and therefore, newborn bleeding is low. DisclosuresNo relevant conflicts of interest to declare.

Practice Patterns and Clinical Outcomes of Platelet Alloimmunization in a Comprehensive Cancer Center

Introduction Platelet transfusion refractoriness (PTR) secondary to human leukocyte antigen (HLA) alloimmunization is a challenge in the treatment of hematology oncology patients and increases the risk of morbidity and mortality from bleeding events. Guidelines for treating PTR have not been clearly described in literature and the use of TPO mimetics or antifibrinolytics in routine clinical practice in the setting of PTR has not been well studied. Aim In this study, we aimed to describe the practice patterns for the management of PTR secondary to HLA alloimmunization, its supportive care interventions and to assess the mortality, thrombosis and bleeding related clinical outcomes at 30 days from the diagnosis of PTR and HLA alloimmunization. Methods A retrospective review was conducted of all PTR in adult hematology oncology patients at MD Anderson Cancer Center between May 2017 and April 2019. PTR was defined as post-transfusion platelet count increment &lt; 10,000 per microliter in at least two consecutive occasions and within 24 hours after platelet transfusion. HLA typing, HLA antibody and platelet transfusion data were obtained from the Blood Bank Electronic records. Patient demographics and clinical data were obtained from the patients electronic medical records. The study was approved by the Institutional Review Board. Results A total of 51 cases of PTR secondary to HLA alloimmunization were analyzed. The majority of patients (98%) had a diagnosis of hematological malignancy of which 88.2% were undergoing active chemotherapy. Clinically relevant bleeding was observed in 33.3% while hemorrhagic shock was diagnosed in 7%. The rate of bleeding related-mortality was estimated at 7.8% (Table 1a). There was great heterogeneity in the management of bleeding risk and events. The use of antifibrinolytics and plasma products (including intravenous immunoglobulin) was more common in cases with major versus non-major bleeding (Table 1b). Grade A or B1U HLA matched products were available in less than half of cases. Conclusions Our study depicts several important observations related to clinical practice and supportive care during HLA platelet alloimmunization and PTR. 1. There is heterogeneity in the management of the bleeding risk and bleeding events during platelet refractoriness. 2. Antifibrinolytics are more commonly used in the patients who suffered severe bleeding. 3. Grade A and BU HLA-matched platelets are not always readily available. 4. The HLA typing and HLA antibody screen/identification testing are not always performed prior to PTR. 5. There is a significant morbidity impact on clinical outcomes due to PTR and its bleeding related complications. Limitations of our retrospective study include the underestimation of actual bleeding outcomes and clinical reasons for the selection of supportive measures (i.e. concern for thrombosis, renal failure). Prospective randomized control trials may be able to determine the safety and efficacy of antifibrinolytics and other supportive measures in the management of PTR. Disclosures No relevant conflicts of interest to declare.

The Thrombopoietin Receptor: Structural Basis of Traffic and Activation by Ligand, Mutations, Agonists, and Mutated Calreticulin.

A well-functioning hematopoietic system requires a certain robustness and flexibility to maintain appropriate quantities of functional mature blood cells, such as red blood cells and platelets. This review focuses on the cytokine receptor that plays a significant role in thrombopoiesis: the receptor for thrombopoietin (TPO-R; also known as MPL). Here, we survey the work to date to understand how this receptor functions at a molecular level throughout its lifecycle, from traffic to the cell surface, dimerization and binding cognate cytokine via its extracellular domain, through to its subsequent activation of associated Janus kinases and initiation of downstream signaling pathways, as well as the regulation of these processes. Atomic level resolution structures of TPO-R have remained elusive. The identification of disease-causing mutations in the receptor has, however, offered some insight into structure and function relationships, as has artificial means of receptor activation, through TPO mimetics, transmembrane-targeting receptor agonists, and engineering in dimerization domains. More recently, a novel activation mechanism was identified whereby mutated forms of calreticulin form complexes with TPO-R via its extracellular N-glycosylated domain. Such complexes traffic pathologically in the cell and persistently activate JAK2, downstream signal transducers and activators of transcription (STATs), and other pathways. This pathologic TPO-R activation is associated with a large fraction of human myeloproliferative neoplasms.

Persistent Thrombopoietin Mimetic Induced Myelofibrosis in a Woman with Chronic Refractory Immune Thrombocytopenia

BackgroundImmune thrombocytopenia (ITP) is a disease caused by autoimmune destruction and impaired production of platelets. It can be primary or secondary in origin. It manifests as isolated thrombocytopenia with platelet counts less than 100 x109/L. Therapy is typically initiated when the platelet counts fall to less than 20 to 30 x 109/L in asymptomatic patients to reduce the risk of bleeding. ITP that persists for greater than 12 months is classified as chronic and ITP that endures despite splenectomy is defined as refractory. Most therapeutic approaches, such as corticosteroids, target the pathologic platelet destruction. Thrombopoietin (Tpo) mimetics, on the other hand, enhance platelet production via stimulation of the Tpo receptor on megakaryocytes. Studies suggest that Tpo mimetics increase reticulin deposition in the bone marrow. Tpo mimetics are postulated to promote reticulin deposition through stimulation of cytokine production by megakaryocytes, including transforming growth factor beta. There are reports of myelofibrosis, with abnormal deposition of collagen and reticulin in bone marrow trephine biopsies of ITP patients treated with these agents. Tpo mimetic induced myelofibrosis however, is described as reversible upon cessation of Tpo based therapy in most reports. Here we present a case of myelofibrosis induced by sequential therapy with two high dose TPO mimetics, which was not reversed six months after discontinuation of the medications.Case ReportAn 87-year-old woman with chronic ITP resistant to multiple lines of therapy (prednisone, dexamethasone, rituximab, dapsone, mycofenylate mofetil, and splenectomy) was treated consecutively with two thrombopoietin (Tpo) mimetics, romiplostim and then eltrombopag. Bone marrow evaluation at diagnosis, prior to treatment with Tpo mimetics, showed megakaryocyte hyperplasia without evidence of fibrosis. There was no definitive evidence of an underlying clonal hematologic disorder.. The patient's course was complicated by gastrointestinal and intracranial hemorrhage. Tpo mimetics were introduced as fourth line therapy. ITP response to romiplostim and then to eltrombopag was initially encouraging but rapidly fleeting.The peak dose of romiplostim used was 10 mcg/kg/week and eltrombopag was used at a dose of 75 mg per day. While on eltrombopag and within six months of romiplostim, therapy, she developed persistent left-shifted leukocytosis and a peripheral blood film showing leukoerythroblastosis and dacrocytic erythrocytes. This prompted a bone marrow evaluation that confirmed myelofibrosis, with a dense and diffuse increase in reticulin content and focal bundles of collagen (MF 2 of 3). There was associated marrow hypercellularity, increased numbers of enlarged megakaryocytes, clustered megakaryocyte topography and extensive bone resorption and remodelling Eltrombopag was promptly discontinued and peripheral blood leukoerythroblastic picture improved. Repeat bone marrow evaluation 6 months after stopping therapy showed partial reversal of the abnormalities seen in the previous marrow, including a reduction in the extent of reticulin fibrosis without any evidence of collagen fibrosis (MF 1-2 of 3). These changes are shown in Figure 1.DiscussionChronic, refractory ITP is a challenging syndrome to treat, and the Tpo mimetics have emerged as an important therapeutic option for patients who have failed traditional therapies. Clinical experience with the relatively novel Tpo mimetic drug class is maturing as we obtain longitudinal data and reports about adverse effects like myelofibrosis. While the myelofibrosis in our patient improved upon cessation of Tpo mimetic therapy, it did not resolve entirely and persisted 6 months after stopping therapy. We hypothesize that the sequential use of high dose Tpo mimetic therapy may have contributed to the development and persistence of myelofibrosis in this patient. This report adds to the body of evidence suggesting that myelofibrosis is a serious and, potentially chronic, adverse effect of treatment with Tpo mimetics.TPO mimetic treatment and B) Grade 1-2 of 3 Myelofibrosis after cessation of TPO mimetic.Semiquantitative grading is based on the 2008 WHO classification.Images from left to right represent hematoxylin andeosin, reticulin, and trichrome stains. [Display omitted] DisclosuresSholzberg:CSL Behring: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding; Amgen: Honoraria.

Thrombopoietin Activates STAT2 Inducing Type I Interferon Effects and Gene Expression: Implications for in Vivo Tpo Treatment and for Myeloproliferative Neoplasms

Thrombopoietin (Tpo) acts via its cell surface receptor (MPL/TpoR) to promote megakaryocyte progenitor survival, proliferation and differentiation via activation of JAK2, STAT5, STAT3, STAT1, ERK1,2-MAPK-kinase and PI-3′-kinase/Akt. In addition, Tpo signaling in the bone marrow niche is required for maintaining hematopoietic stem cells (HSCs). Recently we showed that in cells expressing high JAK2, such as megakaryocytes or engineered cell lines, Tpo induced an anti-proliferative and pro-senescence physiologic effect. Down-modulation of TpoR in myeloproliferative neoplasms (MPNs) progenitors might confer resistance to such anti-proliferative effects in megakaryocytes, thus contributing to their uncontrolled proliferation.Here we report that Tpo activates tyrosine phosphorylation and transcriptional activation of STAT2, and of a complex containing STAT2, STAT1 and IRF-9. This is specific for type I interferon (IFN) and is associated with anti-proliferative and antiviral effects. We detected Tpo induced STAT2 activation in platelets of thrombocytopenia patients treated in vivo with Tpo or Tpo agonists as well as in human CD34+-derived megakaryocytes. In cell lines reconstituted with MPL/TpoR and JAK2, Tpo behaved like an IFN by protecting cells from subsequent viral infection and inducing IFN-stimulated gene expression. We asked whether Tpo-induced STAT2 activation depended on type I IFN receptors, on TYK2 or on STAT1. Using primary cells from mice where these genes have been knocked-out we show that Tpo indeed activates STAT2 via TpoR itself, and does neither require IFNAR1, IFNAR2 nor TYK2, demonstrating that this effect is direct.By using cell lines expressing different levels of JAK2 we show that Tpo exerts a bimodal effect. At low JAK2 levels Tpo induces mainly STAT5, STAT3 and STAT1 activation, while at high JAK2 levels (comparable to those in UT7 cells or primary megakaryocytes) Tpo induces STAT2/STAT1/IRF9 activation, in addition to STAT5/STAT3/STAT1 activation. Interestingly, the STAT2 signaling is dominant and induces an anti-proliferative effect, which can be inhibited by shRNA targeting STAT2, or by mutation of distal receptor tyrosines. Moreover, using STAT2-/- mice we show that STAT2 activation by Tpo is relevant for the steady-state levels of platelets, with knock-out mice having significantly higher platelet numbers. Recovery studies after 5-FU injection and examination of HSCs and early progenitors showed that STAT2 acts as a negative regulator of the stimulatory effects of Tpo on myelopoiesis.We recently identified inactive and active dimeric conformations of TpoR. Here we tested which dimeric receptor conformation activates STAT2 using dimeric fusion proteins where a dimeric coiled coil (Put3) was fused to the transmembrane segment. While cc-TpoR-I was found to recapitulate the classical stimulatory effect of Tpo on STAT5, STAT3 and STAT1 and platelet formation in vivo, here we show that STAT2 activation is induced by another dimeric conformation, represented by cc-TpoR-IV, which is the strongest JAK2 activator. cc-TpoR-IV also induces strong cell-to-cell adhesion possibly reminiscent of the intercellular HSC interactions to the bone marrow niche cells. Increasing JAK2 protein levels leads to stabilization of a Tpo-activated TpoR dimer that assumes a conformation that corresponds to cc-TpoR-IV. Thus, JAK2 levels act on fine-tuning the dimeric conformation of the TpoR dimer, switching it from a classical proliferative cytokine to an IFN-like cytokine, which is anti-proliferative.The new Tpo-induced signaling pathway we describe is of very high relevance for MPN pathogenesis and treatment with IFN-I. Our data indicate that HSCs and megakaryocytes where TpoR-JAK2 V617F signal would be primed for IFN action, and might explain why IFN-I targets for inhibition the mutated HSCs, eventually decreasing the allele burden. Last but not least, autoimmune and viral-induced thrombocytopenia patients are treated with Tpo mimetics, and we could detect STAT2 activation in certain patients after in vivo Tpo treatment. Differences in the ratios of classical versus STAT2 activation might be at the basis of differences in response to Tpo of such patients. DisclosuresConstantinescu:Shire: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Personal Genetics: Scientific Adv Board, Scientific Adv Board Other, Speakers Bureau; Dafra Pharma: Consultancy, Scientific Advisory Board Other.

Clinical and research issues in neonatal anemia and thrombocytopenia

Anemia and thrombocytopenia are the most common hematological problems in neonates. Red blood cell (RBC) and platelet transfusions are the mainstays of therapy, but data to guide neonatal transfusion practices have been sparse. Recombinant hematopoietic growth factors represent another therapeutic alternative, but their use in this population requires a solid understanding of the developmental differences between fetal and adult hematopoiesis. Recently, follow-up studies from children randomized as neonates to either liberal or restrictive RBC transfusion approaches were published. Results of these studies have so far been contradictory and have generated more questions than answers. New developmental stage-specific problems associated with RBC transfusions were also uncovered, such as the transfusion-associated necrotizing enterocolitis. Finally, two thrombopoietin (Tpo) mimetics were approved by the FDA for the treatment of adults with chronic immune thrombocytopenia, thus offering a novel potential therapeutic alternative for thrombocytopenic neonates. In this review, we will discuss the currently available data regarding neonatal RBC and platelet transfusion thresholds, as well as the potential limitations, and concerns associated with the use of erythropoietin and Tpo mimetics in this patient population. Finally, we will point out specific areas wherein additional research is critically needed.

The Effect of Eltrombopag on Platelet Resistance to Apoptosis: The Role of the Bcl-Xl Pathway

Abstract 1158 INTRODUCTION:Immune thrombocytopenia (ITP) is typically characterized by increased platelet destruction and reduced platelet production. Eltrombopag and Romiplostim are thrombopoietin receptor (TPO-R) agonists that are known to increase platelet counts in patients with ITP by stimulating thrombopoiesis. Platelets also express TPO-R on their surface, but it is unknown whether the thrombopoietin mimetics (TPO-M) have a direct effect on the circulating platelets. Although controversial, in a very small number of ITP patients, TPO-M agents may increase platelet counts in 2–5 days, earlier than would be expected from de novo megakaryocytopoiesis. Platelet survival is hypothesized to be mediated by two molecular intermediates in an apoptotic pathway, Bcl-xL and Bak. Bcl-xL/Bak protein expression in megakaryocytes is regulated in part by TPO-mediated activation of Akt pathways through Jak2 and Stat5. We hypothesized that an increase in platelet count in the first week of treatment might be mediated by TPO-R signaling, resulting in decreased platelet apoptosis. This study explored whether Eltrombopag or Romiplostim treatment has anti-apoptotic effects on platelets of patients with ITP. METHODS:Following a treatment wash out period, 75 mg of Eltrombopag once daily or 10 mcg/kg weekly of Romiplostim was initiated for 2 weeks. Blood counts were measured on days 1, 3, 5, 8, 10, 12, and 15. Platelet function and survival was assessed on days 1, 8, and 15 by: immature platelet fraction (IPF), glycocalicin index, Bcl-xL inhibitor (ABT-737) assay, measurement of Bcl-xL by western blot, measurement of several members of the Bcl-xL Akt mediated, apoptotic pathway by flow cytometry (FACS), bleeding score, measurement of thrombin-anti-thrombin complexes (TATs), and quantification of microparticles. RESULTS:Eight of 10 patients responded to treatment with Eltrombopag with a platelet count ≥ 50,000/μL, and 6 of the 8 responders at least doubled their counts during the 2 weeks of treatment. All 3 patients treated with Romiplostim responded with platelet count ≥ 50,000/μL. In both treatment groups there was a significant increase in median platelet count (p<0.001), median large platelet count (p<0.01), and median absolute IPF (A-IPF, p<0.01), while there was no significant change in median % IPF. The dose of ABT-737 required to kill half of the platelets in the sample (IC50) in the Eltrombopag group was lower in patients at day 1 than in non-ITP controls, and there was an increase in resistance to apoptosis between days 1 and 8, but these changes did not reach statistical significance. Between days 8 and 15 the IC50 declined to pre-treatment levels. In the Romiplostim group there was no significant difference in IC50 between the control and the patients over the 2 weeks of study. There was no significant correlation between the platelet counts and the IC50 values. FACS analysis of members of the AKT signal transduction pathway revealed increased activation of each of the markers between days 1 and 8, followed by a decrease between days 8 and 15. The levels of Bcl-xL and phosphor-AKT(308) decreased from day 1 to day 15. The other lab tests are pending. DISCUSSION:Because the A-IPF increased by less than the platelet increase and because the lifespan of the A-IPF is not known, it is unclear if the platelet count increase is solely a result of increased platelet production. Platelet lifespan may be enhanced by Eltrombopag treatment as there was a parallel albeit transient increase in AKT activation markers and platelet apoptosis resistance in the Eltrombopag group. Treatment with Romiplostim did not appear to affect apoptosis resistance although it did result in transient AKT activation. Our data suggest that platelets are more resistant to apoptosis when the levels of anti-apoptotic factors (eg. PTEN, Phospho-GSK3β) involved in the AKT/Bcl-xL pathway are greatest despite a concomitant increase in pro-apoptotic factors (eg. Bak, Bax). Since both the increased AKT activation and apoptotic resistance returned to baseline at day 15, megakaryocytes and platelets already present at the start of treatment may respond differently than those generated de novo in the presence of TPO mimetics. [Display omitted] Disclosures:Bussel:Portola: Consultancy; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cangene: Research Funding; Genzyme: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding.

Eltrombopag for the Treatment of the Inherited Thrombocytopenia Deriving From MYH9 Mutations

AbstractAbstract 2533MYH9-Related Disease (MYH9-RD) is one of the less rare forms of inherited thrombocytopenia. It derives from mutations of the gene MYH9 for the heavy chain of nonmuscle myosin IIA and is characterized by congenital macrothrombocytopenia variably associated with young-adult onset of hearing loss, cataract, and a severe proteinuric nephropathy.Only platelet transfusions are available for increasing platelet counts in this condition, but they expose to the risks of acute reactions, transmission of infectious diseases, and refractoriness to subsequent platelet transfusions. Moreover, this treatment suffers from scarceness of blood donors.Novel thrombopoiesis-stimulating agents have been developed and 2 of them, eltrombopag and romiplostin, have been approved for increasing platelet count in a few forms of acquired thrombocytopenia. Since it has been recently shown that megakaryocytes of patients with MYH9-RD respond in vitro to TPO stimulation, we reasoned that TPO-mimetics could be effective also in this condition and decided to test the effect of eltrombopag.Therefore, we performed a phase II, multicentre, open-label, dose escalation trial. Twelve adult patients with a platelet count lower than 50×10e9/L and a diagnosis of MYH9-RD confirmed by identification of the causative MYH9 mutations received orally eltrombopag 50 mg daily for 21 days (Revolade®, GSK). Patients with platelet counts lower than 100×10e9/L at day 21 increased eltrombopag to 75 mg daily for 21 additional days. Patients with platelet counts between 100 and 150×10e9/L at day 21 continued eltrombopag 50 mg daily for the following 21 days, while patients with more than 150×10e9 platelets/L stopped therapy.The primary endpoints were the achievement of a platelet count over 100×10e9/L or at least three times the baseline value (major response), or at least twice the baseline value but less than major response (minor response). Secondary end points included safety and tolerability, and the reduction of bleeding tendency.After 3 weeks at the eltrombopag dose of 50 mg daily, 3 patients achieved platelet counts of 150×10e9/L or more and stopped therapy. Two had a platelet counts between 100 and 150×10e9/L and continued treatment at the same dosage, while 7 had less than 100×10e9 platelets/L and received eltrombopag 75 mg daily for 3 weeks.A major response was obtained in 8 patients (67%), in 5 of them after 3 weeks of eltrombopag 50 mg daily, and in 3 cases after 3 additional weeks at the dose of 75 mg. Three patients (25%) achieved a minor response, 1 after 3 weeks at 50 mg daily, and 2 after 3 additional weeks at 75 mg. In one patient the treatment resulted in no response.Mean platelet count at the end of treatment was significantly higher than at baseline (105 versus 31×10e9 platelets/L, p=0.0022). In the 11 patients that achieved major or minor responses, mean platelet count was still higher than baseline 15 days after discontinuation of the drug, while it returned to levels near baseline 15 days later.Platelet size did not change either during treatment or after its cessation, and this indicates that the increases in platelet count were paralleled by corresponding increases in total platelet mass.The extent of platelet aggregation was within the normal range after all tested agonists in 5 of the 7 patients that achieved platelet counts higher than 100×10e9/L, while it was slightly reduced after ADP and collagen in 2 patients.Mean serum TPO level was higher than the normal range and this figure did not change neither during treatment with eltrombopag nor after its discontinuation.Ten of 12 patients had grade 1 or 2 bleeding symptoms, as measured by the WHO bleeding scale, at baseline, while 2 were asymptomatic. Upon treatment, bleeding diathesis quickly ameliorated and disappeared in 8 cases, while it remained unchanged in the only patient with no response to eltrombopag and in another one with a minor response. The benefit in terms of bleeding diathesis lasted well beyond treatment discontinuation.Treatment was well tolerated in all cases, with only two patients reporting mild and transient headache and one patient suffering from transient dry mouth at the beginning of treatment.In conclusion, 50–75 mg of eltrombopag per day increased platelet count and reduced bleeding tendency in most patients with MYH9-RD. Further research is required to ascertain whether TPO mimetics are effective also in other forms of inherited thrombocytopenia. Disclosures:Off Label Use: Eltrombopag for MYH9-related disease.

Biology and Chemistry of Thrombopoietic Agents

Endogenous thrombopoietin (eTPO) regulates platelet production by increasing the number, ploidy, and maturation rate of bone marrow megakaryocytes. Early attempts to treat thrombocytopenia by the administration of recombinant TPO were successful but were complicated by the development of antibodies to one of the recombinant proteins. Two new TPO mimetics have recently been approved by the US Food and Drug Administration (FDA) for the treatment of immune thrombocytopenia (ITP). Romiplostim is a peptide TPO mimetic composed of an IgG Fc fragment to which are attached four 14-amino acid TPO peptides that activate the TPO receptor by binding to the extracytoplasmic domain just like eTPO. Romiplostim is administered as a weekly subcutaneous injection. Eltrombopag, a nonpeptide TPO mimetic, is a 442-d drug that binds to a transmembrane site on the TPO receptor and thereby activates it. It is administered daily as an oral tablet. Administration of both romiplostim and eltrombopag to healthy volunteers produced a dose-dependent rise in platelet count beginning on day 5 and peaking at days 12 to 15. Both have been highly effective in increasing the platelet count in patients with ITP and are currently being studied in the treatment of other thrombocytopenic conditions (myelodysplastic syndrome, chemotherapy, liver disease).

Consensus Paper—ICIS Expert Meeting Basel 2009 treatment milestones in immune thrombocytopenia

The rarity of severe complications of this disease in children makes randomized clinical trials in immune thrombocytopenia (ITP) unfeasible. Therefore, the current management recommendations for ITP are largely dependent on clinical expertise and observations. As part of its discussions during the Intercontinental Cooperative ITP Study Group Expert Meeting in Basel, the Management working group recommended that the decision to treat an ITP patient be individualized and based mainly on bleeding symptoms and not on the actual platelet count number and should be supported by bleeding scores using a validated assessment tool. The group stressed the need to develop a uniform validated bleeding score system and to explore new measures to evaluate bleeding risk in thrombocytopenic patients—the role of rituximab as a splenectomy-sparing agent in resistant disease was also discussed. Given the apparently high recurrence rate to rituximab therapy in children and the drug's possible toxicity, the group felt that until more data are available, a conservative approach may be considered, reserving rituximab for patients who failed splenectomy. More studies of the effectiveness and side effects of drugs to treat refractory patients, such as TPO mimetics, cyclosporine, mycophenolate mofetil, and cytotoxic agents are required, as are long-term data on post-splenectomy complications. In the patient with either acute or chronic ITP, using a more personalized approach to treatment based on bleeding symptoms rather than platelet count should result in less toxicity and empower both physicians and families to focus on quality-of-life.

TPO Mimetics: Can More Than One Agent Be Used in a Patient?

Recently, advances in TPO mimetics have expanded therapeutic options for adult patients with chronic ITP. Seven second generation TPO agonists have been reported in the literature. Evidence suggests that non-peptide versus peptide TPO mimetics bind differently to the TPO receptor. Romiplostim, a subcutaneous peptide TPO mimetic has recently been FDA approved. Eltrombopag, Ligand L4665-08, and Aka-Rx 501 are oral non-peptide TPO mimetics undergoing clinical study.The question arises if a patient fails or succeeds one TPO mimetic can the patient be treated with another agent and have a response. We present two cases of patients who were treated with two agents.Patient #1 was initiated on Aka-Rx 501 as she could not tolerate steroids and refused splenectomy. She was able to maintain a platelet count >50,000 for a period of six months without any bleeding. As the study did not have an extension, following a washout period she was enrolled in Ligand L4665-083 with a platelet count of 1,000. Within a week her platelet count rose to 97,000. Her platelet count, on a stable dose of Ligand progressively fell over two weeks and dropped to 2,000 with bleeding. She was then initiated on a higher dose of study medication.The second patient had an eight year history of ITP refractory to IVIG and steroids. She was initially enrolled in AMG 531, and was able to maintain a platelet count greater than 50,000 for over six months. Her platelet count began to progressively decline to zero. Antibody tests were negative and a bone marrow biopsy was normal.After a washout period, the patient was enrolled in AkaRx 501 Cl-003. One week later her platelet count rose to 64,000. She sustained a platelet count of greater than 30,000 and was initiated on the rollover study after four weeks. At week twelve, her platelet count as in the prior study fell to 0 with a concomitant CNS bleed.These two cases demonstrate the use of two different TPO mimetics in a given patient. The first patient was treated with two oral non-peptide TPO mimetics, with a sustained response to one agent and a quick response followed by a rapid loss of response to the second agent. The second patient was treated initially with a peptide TPO mimetic followed by a non-peptide TPO mimetic, both times demonstrating an initial brisk response, long with one and short-lived with the second, which was eventually lost. These data suggest that more than one agent can be used in a given patient.