Extreme Thrombocytosis Under Azacitidine in Patients with Myelodysplastic Syndrome

Abstract Abstract 4961 Background: Platelet increase under azacitidine in patients with myelodysplastic syndrome (MDS) has been acknowledged as an early predictive factor of response to treatment. However, extreme thrombocytosis under azacitidine has not been reported. Methods: We studied consecutive patients with MDS or MDS/myeloproliferative neoplasm (MDS/MPN) who had platelet counts near or over 1, 000 G/L under azacitidine. Results: Four patients, sex ratio 1:1, with median age of 65 years, had extreme thrombocytosis under azacitidine. Baseline characteristics were: WHO classification RAEB-2/CMML-1/CMML-2 in 2/1/1 patients, median platelet count 248 G/L (<400 G/L in all), normal karyotype/+8, −9/−7 in 2/1/1 patients, IPSS low/int-2/high in 1/2/1 patients. None had reticulinic fibrosis or ring sideroblasts>15% at baseline. A median number of 8 cycles of azacitidine was administered. Individual platelet counts reached 2, 960 G/L, 800 G/L, 1, 188 G/L and 2, 740 G/L. Thrombocytosis occured early after treatment onset or resumption (Figure 1). Histologic findings under treatment were: Increased cellularity (N=4), micromegakaryocytes and other signs of megakaryocytic dysplasia (N=4), reticulinic fibrosis grade I and II in 1 and 2 patients, respectively. JAK2 V617F mutation was detected in 1 patient (with maximum platelet count of 2, 900 G/L) and was undetectable in the remaining patients. None had a thrombotic or hemorrhagic event. Two patients had a concomitant increase of WBC count. Response to azacitidine was CR, PR and stable disease in 1/1/2 patients. Three patients received hydroxyurea (HU) in addition to azacitidine and one patient underwent hematopoietic stem cell transplantation (HCT). AML transformation occurred in 1 patient 25 months after azacitidine onset. Median overall survival after azacitidine onset was 25 months. Conclusion: Extreme thrombocytosis of the range of essential thrombocytosis, with megakaryocytic dysplasia and hyperplasia, was noted under azacitidine in 4 patients with MDS-MDS/MPN and normal baseline platelet count. Hypothetically, azacitidine may induce the expression of critical genes of megakaryopoiesis or platelet release in patients with rare mutations. Notably, JAK2 mutation was detected in only one patient. Alternatively, demethylation could unmask an underlying unclassified MDS/MPN similar to RARS-T. Disclosures: No relevant conflicts of interest to declare.

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Calr Mutants Retroviral Mouse Models Lead to a Myeloproliferative Neoplasm Mimicking an Essential Thrombocythemia Progressing to a Myelofibrosis

Blood ◽

10.1182/blood.v124.21.157.157 ◽

2014 ◽

Vol 124 (21) ◽

pp. 157-157 ◽

Cited By ~ 4

Author(s):

Caroline Marty ◽

Nivarthi Harini ◽

Christian Pecquet ◽

Ilyas Chachoua ◽

Vitalina Gryshkova ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

In Vivo Model ◽

Common Mechanism ◽

Loss Of Function ◽

Hematopoietic Stem ◽

Platelet Counts

Abstract Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are malignant homeopathies resulting from the transformation of a multipotent hematopoietic stem cell (HSC). The common mechanism of transformation is the constitutive activation of the cytokine receptor/JAK2 pathway that leads to the myeloproliferation. The acquired point mutation JAK2V617F is the most prevalent (95% of PV and 60% of ET or PMF). In addition, other mutations affecting the same signaling pathway have been described such as JAK2 exon 12 mutations, mutations of MPL affecting W515, and loss-of-function mutations of LNK and also mutations of c-Cbl in 3% of PMF. Recently, whole exome sequencing allowed identifying a new recurrent genetic abnormalities in the exon 9 of the calreticulin gene (CALR) in about 30% of ET and PMF patients. All CALR mutants induce a frameshift of the same alternative reading frame and generate a novel C-terminus tail. To address the role of these new mutants in the pathophysiology of MPN, the goal of this study was to investigate the effect of the CALR mutant (del52 and ins5) expression by a retroviral mouse modeling. For that purpose, we transduced bone marrow cells with retrovirus expressing either CALRdel52, CALRins5, CALRWT or CALRDexon9 and performed a transplantation in lethally irradiated recipient mice (10 mice / group), which were then followed over one year. CALRdel52 expressing mice showed a rapid and strong increased in platelet counts (over 5 x106/mL) without any other changes in blood parameters during 6 months. In contrast, CALRins5 expressing mice presented platelet counts much lower than CALRdel52 but significantly higher than CALRWT or CALRDexon9 expressing mice. After 6 months, CALRdel52 expressing mice showed a decreased in platelets count associated with anemia and development of splenomegaly suggesting the progression to a myelofibrosis. Importantly, the disease was transplantable to secondary recipient for both CALRdel52 and CALRins5 mutants. The bone marrow and spleen were also analyzed over time. We observed a progressive increased in immature progenitors (SLAM cells) as well as a hypersensitivity of the megakaryocytic progenitors (CFU-MK) to thrombopoietin. Altogether, these results demonstrate that CALR mutants are able and sufficient to induce a thrombocytosis progressing to myelofibrosis in retroviral mouse model, thus mimicking the natural history of MPN patients. It will offer a good in vivo model to investigate therapeutic approaches for CALR-positive MPN. Disclosures No relevant conflicts of interest to declare.

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Mpl Expression on AML Blasts Predicts Cytopenia

Blood ◽

10.1182/blood.v126.23.1387.1387 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1387-1387

Author(s):

Philipp J Rauch ◽

Corinne Widmer ◽

Kristin Fritsch ◽

Jana M Ellegast ◽

Jeroen S Goede ◽

...

Keyword(s):

Platelet Count ◽

Negative Correlation ◽

Conflicts Of Interest ◽

Gene List ◽

Receptor Expression ◽

Severe Neutropenia ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Blast Count

Abstract Acute myeloid leukemia (AML) induces profound impairment of healthy hematopoiesis. The production deficit in the bone marrow (BM) leads to development of peripheral anemia, thrombocytopenia and neutropenia, which is a major cause of AML-associated morbidity and mortality. Despite much progress in understanding of AML biology, the mechanisms by which AML blasts interact with elements of normal hematopoiesis to cause cytopenia are unclear. Conventional wisdom has it that blasts infiltrate the marrow and displace normal hematopoiesis. If this concept were to be true, there should be a strong correlation between BM blast count and peripheral cytopenia. Surprisingly, analysis of 223 patients with newly diagnosed AML at a tertiary referral center revealed lack of correlation between initial BM blast count [% of cellularity] and hemoglobin level (ρ=-0.11, P=0.12), platelet count (ρ=-0.00, P=0.53) and absolute neutrophil count (ρ=0.13, P=0.06). This indicates that mechanisms other than displacement of normal hematopoiesis dictate the severity of cytopenia in AML patients. Hematopoiesis is tightly regulated by cytokines. Among them, thrombopoietin (TPO) acts through its receptor c-Mpl as the master regulator of megakaryopoiesis, but also exerts upstream effects on hematopoietic stem and progenitor cells (HSPC). TPO levels are controlled by receptor-mediated scavenging by cells carrying c-Mpl on the surface, with platelets representing the lion's share in a healthy organism. This negative feedback loop results in strong negative correlation between serum TPO concentration and platelet count in the steady state. When we examined this relationship in our AML cohort, TPO levels did not follow the expected negative correlation with platelet counts (ρ=-0.10, P=0.59). Comparison with historic controls with thrombocytopenia induced by chemotherapy for non-hematopoietic malignancy revealed that the lack of correlation was driven by AML cases with severe thrombocytopenia that had lower than expected levels of TPO in the serum. As HSPC are known to express c-Mpl, we hypothesized that HSPC-derived AML blasts may also express the receptor and cause insufficiency of hematopoiesis by means of receptor-mediated TPO scavenging. To test this hypothesis, we compared c-Mpl expression on blasts in AML cases with severe thrombocytopenia and low TPO concentration (potential scavenger cases) to cases with TPO levels adequate for the degree of cytopenia. Both surface flow cytometry and qPCR demonstrated higher c-Mpl expression in potential scavenger cases (3.1-fold, P=0.02). To determine whether this difference in expression translates into increased serum TPO clearance, we incubated AML blasts with high (c-Mpl+) and low (c-Mpl-) receptor expression in serum containing recombinant human TPO at a concentration of 100 pg/mL. After 2h, TPO clearance reached 45 pg per 106 cells in wells with c-Mpl+ blasts, compared to only 4 pg per 106 cells in wells with c-Mpl- blasts (P=0.02). This confirms the hypothesis that AML blasts can lower TPO levels by virtue of their c-Mpl expression. Validation studies in an independent, multi-center Dutch-Belgian-Swiss cohort of 437 AML cases confirmed lack of correlation between initial BM blast count and cytopenia. Ranked gene list correlation analysis of whole genome microarray data proved significant enrichment of the MPL transcript in patients with severe thrombocytopenia when compared to patients with average platelet counts (rank 27/20'589, FDR<10-6). MPL enrichment could also be observed in patients with severe neutropenia (P<0.01), but there was no correlation between MPL transcript level and degree of anemia. Lastly, we asked if MPL expression was related to cytogenetic or molecular AML subtype: indeed, microarray analysis showed higher MPL expression in cases of AML with t(8;21) than in any other subtype (P<10-4). Concurrently, these patients displayed significantly lower platelet count (40 vs 83 x 109/L, P=0.02) when compared to all other AML cases. In summary, our study demonstrates that cytopenia in AML is independent of BM blast count, but strongly correlated with c-Mpl expression on blasts. We show that c-Mpl+ blasts clear TPO, causing insufficient TPO levels and contributing to development of thrombocytopenia and neutropenia. The work may have important ramifications for treatment of AML-induced cytopenia, especially in the relapsed or refractory setting. Disclosures No relevant conflicts of interest to declare.

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Understanding the Mechanisms Underlying Thrombocytopenia in Visceral Leishmaniasis

Blood ◽

10.1182/blood-2019-128133 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2378-2378 ◽

Cited By ~ 1

Author(s):

Gulab Fatima Rani ◽

Olivier Preham ◽

Ian Hitchcock ◽

Paul Kaye

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Visceral Leishmaniasis ◽

Conflicts Of Interest ◽

Experimental Models ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Production ◽

Platelet Counts ◽

Splenic Macrophages

Visceral leishmaniasis (VL) is a neglected tropical parasitic disease caused by Leishmania parasites and only second to malaria in terms of worldwide morbidity and mortality. According to recent WHO report, there are 500,000 cases of VL worldwide leading to ~30,000 deaths per year. VL is endemic in 98 countries but the major disease burden is contributed by Brazil, India and Sudan. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulations and extensive hematological complications. We have shown previously using experimental models of infection that the infiltration of CD4+ T cells results in disruption to the bone marrow environment, resulting in dysfunctional hematopoietic stem and progenitor cells self-renewal (Pinto et al, PLOS Pathogens, 2017) and aberrant medullary erythropoiesis causing pathological anemia (Preham et al, Frontiers in Immunology, 2018). Thrombocytopenia is also dominant hematological feature seen in both human and experimental models that may reflect either reduced platelet production or enhanced clearance. However, the mechanisms of VL-driven thrombocytopenia remain poorly understood. The aim of this study is to explore the possible underlying mechanisms from platelet production to phagocytic cells dependent clearance. Using a murine experimental model of VL, we demonstrate a steady decrease in the platelet count from d14 onwards in infected mice culminating in severe thrombocytopenia on d28 of infection (infected: 225.9 ±35.7 vs naïve: 1005 ±90.6, x 106/µl). Critically, thrombocytopenia is completely reversible after a single dose of liposomal amphotericin B (Ambisome @ 8mg/kg bodyweight, IV) which clears parasites by delivering the drug directly to parasite harbouring tissue macrophages, thereby improving parasite clearance and reducing toxicity. Despite significant thrombocytopenia, the number and gross morphology of bone marrow megakaryocytes (MKs) were not altered, but MK ultrastructure studies using transmission electron microscopy identified significantly reduced demarcation membranes in infected mice compared to naïve. Levels of plasma thrombopoietin (TPO), the key regulator of MK differentiation and platelet production, were decreased in infected vs naïve mice (1254 ± 95.49 vs 3249 ± 125.1 pg/ml) and administration of exogenous TPO resulted in complete recovery of platelet counts. Given that the majority of TPO is produced by the liver, reduction in the levels of circulating TPO during infection is likely due to destruction of liver architecture by parasite loaded hepatic granulomas. Together, these data suggest that despite some changes in MK cytoplasmic maturation, the bone marrow microenvironment remains supportive of MK differentiation capacity during VL. As platelet production is not significantly altered by VL, we next determined effects on platelet clearance. Large number of highly active splenic macrophages are common in VL and are known for their phagocytic properties. Experiments conducted on VL-infected splenectomised mice demonstrated a reduction in thrombocytopenia compared to sham-operated infected mice (685 ±32 vs 297± 16, x 106/µl) and showed a great response to exogenous TPO, implying splenic clearance may be involved in thrombocytopenia. Partial depletion of splenic macrophages in infected mice using clodronate liposomes did not alter platelet count, whereas neutrophil deletion (anti-Gr1 mAb @ 250ug/g IP) in infected mice resulted in a near 2-fold increase in platelet counts. Furthermore, circulating platelets in VL infected mice were IgG coated compared to naive which is likely to further enhance autoimmune platelet clearance. Severe thrombocytopenia and bleeding are important clinical manifestations of VL. Our findings clearly demonstrate that the mechanisms of thrombocytopenia in VL are multifactorial but do not cause permanent long term damage to the BM microenvironment. Critically, these changes could be reversed rapidly by clearing parasitemia, using TPO agonists to increase numbers of circulating platelets and/or by reducing platelet clearance. This highlights the possibility of re-evaluating the current treatment regimen in VL endemic countries by including therapeutic interventions aimed at reversing severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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Clinical Features and Effects of Eradication on Helicobacter Pylori Positive 207 ITP Cases in Japan.

Blood ◽

10.1182/blood.v104.11.2071.2071 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2071-2071

Author(s):

Kingo Fujimura ◽

Masataka Kuwana ◽

Yoshiyuki Kurata ◽

Masahiro Imamura ◽

Hiroshi Harada ◽

...

Keyword(s):

Helicobacter Pylori ◽

Platelet Count ◽

Eradication Therapy ◽

The Other ◽

First Line ◽

Platelet Counts ◽

Count Response ◽

Successful Group ◽

H Pylori ◽

Over 40 Years

Abstract In 1998, Gasbarrini et al reported that in ITP cases with Helicobacter pylori (H.pylori) infection, elevation of platelet counts was observed by eradication of this bacterium. Since then, several reports from Italy and Japan confirmed the elevation of platelet counts after eradication. However, the characteristic background in the H.pylori positive ITP and eradication effects on platelet counts is unclear. On the other hand, reports from Spain, North Europe and USA could not show the evidence that eradication is effective on elevating platelet counts in H.pylori positive ITP. Therefore, we designed a nationwide retrospective study in Japan to evaluate the incidence of H.pylori positive ITP cases and the effects of eradication on platelet counts and to clear above problems. Four hundred and thirty-five ITP cases were enrolled over a period of one and half years (2002. 7~2003.12) from 12 hospitals. H. pylori infection was found in 300 cases(65%), who were significantly (P<0.005) older and showed hyperplastic megakaryocyte in bone marrow (P=0.011) comparison with negative cases. Eradication to H. pylori was performed in 207 H. pylori positive ITP cases and as a whole, the platelet count response was observed in 63% of eradication succeeded group. In the successful group, CR and PR rate were 23% and 42% respectively at 12 months after eradication. The platelet count response was significant in the successfully eradicated group (P<0.005) and the increased platelet count was maintained without ITP treatment for over 12 months. In conclusion, H. pylori infection was involved in most ITP patients over 40 years old in Japan and eradication therapy proved effective for increasing platelet counts even in splenectomy non-responsive cases and the platelet count response appeared one month after eradication. This evidence suggests that eradication therapy is the first line of treatment in H. pylori positive ITP patients.

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Plerixafor (Mozobil ®)Plus G-CSF Is Effective in Mobilizing Hematopoietic Stem Cells in Patients with Concurrent Thrombocytopenia Undergoing Autologous Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.3229.3229 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3229-3229 ◽

Cited By ~ 1

Author(s):

Ivana N Micallef ◽

Eric Jacobsen ◽

Paul Shaughnessy ◽

Sachin Marulkar ◽

Purvi Mody ◽

...

Keyword(s):

Stem Cell ◽

Platelet Count ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Cd34 Cells ◽

Low Platelet Count ◽

Equity Ownership

Abstract Abstract 3229 Poster Board III-166 Introduction Low platelet count prior to mobilization is a significant predictive factor for mobilization failure in patients with non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD) undergoing autologous hematopoietic stem cell (HSC) transplantation (auto-HSCT; Hosing C, et al, Am J Hematol. 2009). The purpose of this study is to assess the efficacy of HSC mobilization with plerixafor plus G-CSF in patients with concomitant thrombocytopenia undergoing auto-HSCT. Methods Patients who had failed successful HSC collection with any mobilization regimen were remobilized with plerixafor plus G-CSF as part of a compassionate use program (CUP). Mobilization failure was defined as the inability to collect 2 ×106 CD34+ cells/kg or inability to achieve a peripheral blood count of ≥10 CD34+ cells/μl without having undergone apheresis. As part of the CUP, G-CSF (10μg/kg) was administered subcutaneously (SC) every morning for 4 days. Plerixafor (0.24 mg/kg SC) was administered in the evening on Day 4, approximately 11 hours prior to the initiation of apheresis the following day. On Day 5, G-CSF was administered and apheresis was initiated. Plerixafor, G-CSF and apheresis were repeated daily until patients collected the minimum of 2 × 106 CD34+ cells/kg for auto-HSCT. Patients in the CUP with available data on pre-mobilization platelet counts were included in this analysis. While patients with a platelet count <85 × 109/L were excluded from the CUP, some patients received waivers and were included in this analysis. Efficacy of remobilization with plerixafor + G-CSF was evaluated in patients with platelet counts ≤ 100 × 109/L or ≤ 150 × 109/L. Results Of the 833 patients in the plerixafor CUP database, pre-mobilization platelet counts were available for 219 patients (NHL=115, MM=66, HD=20 and other=18.). Of these, 92 patients (NHL=49, MM=25, HD=8 and other=10) had pre-mobilization platelet counts ≤ 150 × 109/L; the median platelet count was 115 × 109/L (range, 50-150). The median age was 60 years (range 20-76) and 60.4% of the patients were male. Fifty-nine patients (64.1%) collected ≥2 × 109 CD34+ cells/kg and 13 patients (14.1%) achieved ≥5 × 106 CD34+ cells/kg. The median CD34+ cell yield was 2.56 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 68.5%. The median time to neutrophil and platelet engraftment was 12 days and 22 days, respectively. Similar results were obtained when efficacy of plerixafor + G-CSF was evaluated in 29 patients with platelet counts ≤ 100 × 109/L (NHL=12, MM=10, HD=3 and other=4). The median platelet count in these patients was 83 × 109/L (range, 50-100). The median age was 59 years (range 23-73) and 60.4% of the patients were male. The minimal and optimal cell dose was achieved in 19(65.5%) and 3(10.3%) patients, respectively. The median CD34+ cell yield was 2.92 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 62.1%. The median time to neutrophil and platelet engraftment was 12 days and 23 days, respectively. Conclusions For patients mobilized with G-CSF alone or chemotherapy ±G-CSF, a low platelet count prior to mobilization is a significant predictor of mobilization failure. These data demonstrate that in patients with thrombocytopenia who have failed prior mobilization attempts, remobilization with plerixafor plus G-CSF allows ∼65% of the patients to collect the minimal cell dose to proceed to transplantation. Thus, in patients predicted or proven to be poor mobilizers, addition of plerixafor may increase stem cell yields. Future studies should investigate the efficacy of plerixafor + G-CSF in front line mobilization in patients with low platelet counts prior to mobilization. Disclosures Micallef: Genzyme Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jacobsen:Genzyme Corporation: Research Funding. Shaughnessy:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Marulkar:Genzyme Corporation: Employment, Equity Ownership. Mody:Genzyme Corporation: Employment, Equity Ownership. van Rhee:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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A Case of Clopidogrel Induced TTP Recurring After Four Years

Blood ◽

10.1182/blood.v116.21.4678.4678 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4678-4678

Author(s):

Nanda K. Methuku ◽

Abhinav B. Chandra ◽

Anuradha Belur ◽

Lech Dabrowski

Keyword(s):

Platelet Count ◽

Renal Insufficiency ◽

Peripheral Blood ◽

Blood Smear ◽

Reticulocyte Count ◽

Conflicts Of Interest ◽

Peripheral Blood Smear ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Multiple Organs

Abstract Abstract 4678 Case description - A 61 year old woman was started on clopidogrel after having PTCA with stent placement in February 2006. Four weeks after starting clopidogrel she developed thrombocytopenia with platelet nadir of 17,000. Her LDH was 700 IU/L and she was anemic with hemoglobin of 7.4 gm/dl with elevated reticulocyte count. Peripheral blood smear showed schistocytes and diagnosis of TTP secondary to clopidogrel was made. She did not have renal insufficiency. Clopidogrel was discontinued and patient was started on plasmapheresis with recovery of platelet counts. Early attempts in weaning plasmapheresis resulted in drop in platelet count and Rituximab was given to the patient weekly for four weeks. Subsequently, patient was weaned off plasmapheresis. For four years patient was followed periodically with CBC showing platelet counts greater than 250,000. In May 2010, four years after initial event patient was admitted to hospital for abdominal pain and found to have splenic infarcts. Subsequently, she also developed bilateral cerebral infarcts. Platelet count had decreased to less than 100,000. Her LDH was elevated at 419 IU/L. Reticulocyte count was 2.3%. Peripheral blood smear revealed significant number of schistocytes. There was no renal insufficiency or fever. Trans-esophageal echocardiogram (TEE) was done that did not reveal any vegetations. Patient was diagnosed as having recurrence of TTP and started on plasmapheresis with recovery in platelet counts. Pt was also treated with Rituximab. Discussion- We describe a case of TTP initially occurring within weeks of starting clopidogrel. Patient was treated with plasmapheresis and Rituximab and clopidogrel was discontinued. Patient had recurrence after four years as manifested by infarcts in multiple organs, with mild thrombocytopenia, elevated LDH and significant number of schistocytes on peripheral blood smear. It is very uncommon for clopidogrel associated TTP to recur after such a prolonged period of 4 years. Most cases of clopidogrel associated TTP have mild thrombocytopenia. This patient had severe thrombocytopenia on first presentation of TTP but had mild thrombocytopenia on recurrence. This case illustrates the importance of extended follow up and high index of suspicion for TTP as delays in initiation of plasmapheresis has a poor clinical outcome. Disclosures: No relevant conflicts of interest to declare.

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Eltrombopag Can Stimulate Trilineage Hematopoiesis In Patients with Refractory Severe Aplastic Anemia

Blood ◽

10.1182/blood.v116.21.4427.4427 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4427-4427

Author(s):

Matthew J. Olnes ◽

Yong Tang ◽

Susan Soto ◽

Elaine M Sloand ◽

Philip Scheinberg ◽

...

Keyword(s):

Stem Cell ◽

Platelet Count ◽

Aplastic Anemia ◽

Severe Aplastic Anemia ◽

Successful Outcome ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Transfusion Independence

Abstract Abstract 4427 Severe aplastic anemia (SAA) is characterized by trilineage marrow hypoplasia and a paucity of hematopoietic stem cell progenitors. SAA is treated with immunosuppression or allogeneic stem cell transplantation (SCT), with a successful outcome in a majority. However, 20–40% of patients without a suitable donor for SCT do not respond to immunosuppression and may have persistent severe thrombocytopenia. Thrombopoietin (TPO) is the principal regulator of platelet production, and it exerts its effects through binding the megakaryocyte progenitor TPO receptor mpl, which stimulates production of mature megakaryocytes and platelets. Eltrombopag, a small molecule TPO mimetic that binds to mpl, increases platelet counts in healthy subjects, and in patients with chronic immune thrombocytopenic purpura. Both TPO and eltrombopag stimulate more primitive multilineage progenitors and stem cells in vitro. Patients with SAA and thrombocytopenia have very elevated TPO levels; nevertheless, we asked whether pharmacologic doses of eltrombopag could stimulate hematopoiesis in these patients without other options. We are conducting a pilot phase II study of eltrombopag in SAA patients with severe thrombocytopenia refractory to immunosuppressive therapy. Consecutive eligible adult patients were treated with oral eltrombopag at an initial dose of 50 mg daily, with escalation to a maximum dose 150 mg daily, with the goal of maintaining a platelet count of >20,000/uL above baseline. Treatment response was measured after three months and was defined as platelet count increases to 20,000/uL above baseline, or stable platelet counts with transfusion-independence for a minimum of 8 weeks. Nine patients have been enrolled and six are evaluable for response to date. Two patients did not respond to treatment. Three patients achieved platelet responses by 12 weeks of treatment, and all have sustained their responses (median follow up 10 months). Four patients exhibited improved hemoglobin levels 12 weeks after starting treatment (median hemoglobin increase of 2.1 g/dL) and two patients who were previously dependent on packed red blood cell transfusions have achieved transfusion-independence. Three neutropenic patients exhibited increased neutrophil counts after treatment with eltrombopag (median increase 0.46K cells/uL). These results provide evidence that eltrombopag can improve platelet counts in patients with severe refractory thrombocytopenia, and perhaps more surprisingly, have a clinically relevant impact on erythropoiesis and myelopoiesis. Updated data will be presented at the Society's meeting. Disclosures: Off Label Use: Eltrombopag for thrombocytopenia in refractory severe aplastic anemia patients.

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A Novel Activating JAK2 Mutation, JAK2R564Q, Causes Familial Essential Thrombocytosis (fET) Via Mechanisms Distinct From JAK2V617F

Blood ◽

10.1182/blood.v118.21.123.123 ◽

2011 ◽

Vol 118 (21) ◽

pp. 123-123 ◽

Cited By ~ 4

Author(s):

Leah Etheridge ◽

Lana M Corbo ◽

Kenneth Kaushansky ◽

Edward Chan ◽

Ian S Hitchcock

Keyword(s):

Platelet Count ◽

Growth Factor ◽

Cell Survival ◽

Intracellular Signaling ◽

Double Mutant ◽

Biological Significance ◽

Negative Regulator ◽

Jak2 Mutation ◽

Hematopoietic Stem ◽

Proliferation Assays

Abstract Abstract 123 Activating mutations in JAK2 are responsible for the majority of myeloproliferative diseases (MPDs) by stimulating aberrant signaling and hyperproliferation of one or more cell lineages. Although JAK2V617F is the most common activating mutation, a number of other point mutations also appear to have clinical relevance. Here we describe JAK2R564Q, a novel mutation in the pseudokinase domain that causes fET and determine its biological function in vitro, compared to JAK2V617F. A 6 year old male was referred for evaluation of thrombocytosis (initial platelet count 961k/mcL). The patient was otherwise asymptomatic with no past medical history, no recent illnesses and on no medications. The family history, review of systems and physical examination were unremarkable and workup for secondary thrombocytosis was negative. While the patient did not have the JAK2V617F, MPLW515L/K or S505N mutations, we discovered a novel JAK2 mutation, R564Q. His mother and sister also presented with elevated platelet counts (500–600k/mcL) and were also found to have the JAK2R564Q mutation, whereas the father presented with a normal platelet count and displayed two wild type (WT) JAK2 alleles. The arginine residue at 564 is highly evolutionarily conserved in the autoinhibitory domain of JAK2. To determine the biological significance of JAK2R564Q and compare it to JAK2V617F, we stably expressed WT, R564Q, V617F and R564Q+V617F JAK2 in Ba/F3 cells stably expressing the thrombopoietin receptor c-Mpl (BaF-Mpl). These cells express comparable levels of both JAK2 and c-Mpl. TPO-dependent proliferation assays demonstrated striking differences between the different cell types. JAK2R564Q exhibits significant increases in cell survival in the absence of TPO and at low concentrations compared to WT, while cells expressing JAK2V617F and R564Q+V617F are growth factor independent. Interestingly, the double mutant (R564Q+V617F) exhibits higher maximal cell proliferation than V617F alone, suggesting that R564Q is functioning through alternative mechanisms to that of V617F. Next, we analyzed annexin V expression following growth factor withdrawal to determine the effects of mutated JAK2 on apoptosis. Concurrent with our proliferation assays, JAK2R564Q inhibited apoptosis compared to WT, while JAK2V617F and R564Q+V617F exhibited even less apoptosis. These data suggest that JAK2R564Q is important for cell survival in the absence of cytokines, but it does not elicit the proliferation-promoting effects of JAK2V617F. To elucidate the mechanisms through which JAK2R564Q and JAK2V617F mediate their actions we determined their effects on intracellular signaling. Cells were starved prior to stimulation with TPO. Interestingly, we found that cells expressing JAK2R564Q have considerably higher levels of phospho-JAK2 (Y1007/8) and phospho-STAT5, signals which are normally associated with proliferation, than WT, V617F alone and the double mutant. We also observed differences in the phosphorylation of several other JAK2 tyrosine residues that are important for regulating its activity. Intriguingly, hyperphosphorylation of the negative regulator JAK2Y570, was by far the most robust in JAK2R564Q mutants, which could potentially contribute to the reduced factor-independent proliferation observed, compared to JAK2V617F. Interestingly, we also found differential phosphorylation of JAK2 at Y831, which positively regulates JAK2 signaling via interactions with SH2-Bβ. JAK2Y831 was also hyperphosphorylated in R564Q mutants compared to V617F mutants, especially in the absence of cytokines. Levels of phospho-ERK1/2 and phospho-Akt were comparable in all JAK2 mutants and significantly reduced compared to WT cells, characteristic of cells that fail to undergo starvation induced cell cycle arrest. Taken together, these data demonstrate that the JAK2R564Q mutation causes fET most likely by inhibiting apoptosis in hematopoietic stem cells and megakaryocytic progenitors. Importantly, even though this mutation is localized in the same pseudokinase domain as V617F, its effect on cell survival and signaling in response to TPO is significantly different. This work provides an insight into the functionality of alternative, clinically-relevant JAK2 mutations and how they have separate and additive effects on cell growth and survival. Disclosures: No relevant conflicts of interest to declare.

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Analysis of Platelet Receptor Expression in ITP,

Blood ◽

10.1182/blood.v118.21.3289.3289 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3289-3289

Author(s):

Jianlin Qiao ◽

Huy Tran ◽

Fi-Tjen Mu ◽

Robert K Andrews ◽

Elizabeth E Gardiner

Keyword(s):

Platelet Count ◽

Conflicts Of Interest ◽

Surface Expression ◽

Experimental Models ◽

Receptor Expression ◽

Response To Treatment ◽

Platelet Surface ◽

Healthy Donors ◽

Platelet Receptor ◽

Von Willebrand

Abstract Abstract 3289 In this study we assess platelet receptor expression and shedding in patients with immune thrombocytopenia (ITP) before and during treatment. The aim is to evaluate the potential value of quantitative measurement of platelet receptors for diagnosis and/or monitoring treatment in thrombocytopenia due to immune or other causes. The platelet-specific collagen receptor, glycoprotein (GP)VI, is associated with the Fc receptor γ-chain (FcRγ). GPVI/FcRγ is coassociated on platelet surface with the GPIb-IX-V complex; GPIbα of GPIb-IX-V binds von Willebrand factor and other ligands. Our previous studies showed engagement of platelet FcγRIIa by antiplatelet antibodies induced ectodomain shedding of GPVI, generating soluble ectodomain (sGPVI) in plasma. However, apart from one individual with an anti-GPVI antibody, whether anti-platelet antibodies associated with ITP affect GPVI/GPIb expression/shedding has not been addressed. In this study we used flow cytometry and a sGPVI ELISA to assess 1) whether patients with ITP had dysregulated expression/shedding of GPVI or GPIbα, and 2) whether platelet receptor expression changes prior to recovery of platelet count in individuals undergoing treatment for ITP. In 9 ITP patients (mean age=48.6, range 29–79; 6 female) with platelet count 61±9 × 109/L (range, 33–122 × 109/L), GPVI surface expression (GeoMean±SE, 137±17) was lower than healthy controls (274±26; n=17; platelet count 247±13), and sGPVI in patient plasma was significantly higher (39±4 ng/mL) compared to 17 healthy donors (19±3 ng/mL) (P=0.0006). In longitudinal samples analysed at weekly intervals during 2-month treatment with steroids, decreased GPVI surface expression and increased sGPVI in plasma remained essentially unchanged as the platelet count normalized, consistent with persistent anti-platelet antibody. However, while levels of intact platelet GPIbα were significantly reduced in ITP compared to healthy donors (P=0.0053), they approached healthy levels within 1 week of treatment, preceding improvement in platelet count or other measures. GPIbα expression/cleavage has been previously implicated in platelet clearance in experimental models, and our analysis suggests the proteolytic status of human GPIbα may be a novel early marker for evaluating response to treatment in ITP. Disclosures: No relevant conflicts of interest to declare.

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Thrombopoietin Levels May Predict Responsiveness to Therapy with Thrombopoietin Agonists Among Patients with Immune Thrombocytopenic Purpura,

Blood ◽

10.1182/blood.v118.21.3288.3288 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3288-3288 ◽

Cited By ~ 4

Author(s):

Robert Makar ◽

Olga S. Zhukov ◽

Mervyn A. Sahud ◽

David J. Kuter

Keyword(s):

Platelet Count ◽

Clinical Response ◽

Immune Thrombocytopenic Purpura ◽

Myeloproliferative Disorders ◽

Interquartile Range ◽

Wilcoxon Test ◽

Response To Treatment ◽

Thrombocytopenic Purpura ◽

Platelet Production ◽

Platelet Counts

Abstract Abstract 3288 INTRODUCTION: Thrombopoietin (TPO) is the major regulator of platelet production. In prior clinical studies, thrombopoietin levels have been shown to vary inversely with circulating platelet mass and with the rate of platelet production. Thus, TPO levels may help distinguish between the various disorders of thrombocytopenia. In addition, the introduction of TPO agonists has created an interest in predicting the response of patients to these agents. Determining TPO levels may help predict such treatment responses. METHODS: Sera from 121 patients with a history of abnormal platelet counts were tested using a novel, commercially available ELISA assay that measures TPO levels. The TPO assay detected TPO levels as low as 7 pg/mL and was linear for levels up to 2000 pg/mL. The coefficient of variation ranged from 27% near the lower limit of detection to 9% at a TPO concentration of 669 pg/mL. The reference range for TPO was established in serum samples from 118 apparently healthy individuals (58 males and 60 females) and was 7–99 pg/mL. The Wilcoxon test was used to compare continuous variables and the Fisher's exact test was used to compare categorical variables. RESULTS: The patient population included 40 patients with a consumptive thrombocytopenia (38 with primary or secondary immune thrombocytopenic purpura (ITP), 2 with thrombotic thrombocytopenic purpura), 34 patients with myeloproliferative disorders (23 with essential thrombocytosis, 9 with polycythemia vera, 2 with an ill-defined myeloproliferative disorder), and 47 patients with hypoproliferative thrombocytopenia (29 with chemotherapy-related thrombocytopenia, 19 with primary or secondary bone marrow failure syndromes). Among the 38 patients with ITP, 11 were taking TPO agonists (9 on romiplostim, 2 on eltrombopag), 19 were taking immunomodulatory agents (16 on steroids alone or in combination with other therapies, 2 on azathioprine, 1 on danazol), and 12 were off ITP-specific therapy when the TPO level was measured. 9 out of 38 (24%) patients with ITP had undergone splenectomy and/or been previously treated with rituximab. The median serum TPO level in patients with consumptive thrombocytopenia was 64.5 pg/mL (interquartile range, 48.5–97.5 pg/mL) and the corresponding median platelet count was 68,000/μL (interquartile range, 27,000–144,500) (Figure). While patients with myeloproliferative disorders had similar TPO levels [median 87.0 pg/mL (38.0–125.5)], their platelet counts were significantly higher than those of patients with consumptive thrombocytopenia [median 549,500/mL (431,250–693,000] (P <0.0001). Contrastingly, comparable platelet counts [median 61,000/μL (31,000–118,000)] were observed among patients with hypoproliferative thrombocytopenia, but serum TPO levels were significantly higher than those of patients with consumptive thrombocytopenia [844 pg/mL (409.5–1551.5), P <0.0001]. Among 22 evaluable patients meeting diagnostic criteria for primary or secondary ITP who had taken a TPO agonist for at least 1 month, serum TPO levels appeared to predict responsiveness to the drug. A clinical response to a TPO agonist was defined as achieving a platelet count ≥50,000/μL after starting the drug and maintaining it at or above that count in ≥50% of subsequent complete blood counts from initiation until discontinuation of the drug, loss to follow-up, or 6 months had passed, whichever was longest, without the need for recurrent rescue therapy. Whereas 14 out of 16 (88%) ITP patients with a TPO level <99 pg/mL met our definition for a clinical response to treatment with a TPO agonist, only 1 out of 6 patients (17%) with a TPO level >99 pg/mL responded (P <0.005 for the difference in clinical response to TPO agents.) CONCLUSIONS: TPO levels may have diagnostic utility in discriminating between patients with hypoproliferative and consumptive thrombocytopenia. High TPO levels among patients with ITP may predict a poor clinical response to treatment with TPO agonists. Further studies are required to confirm these data. Disclosures: Zhukov: Quest Diagnostics: Employment. Sahud:Quest Diagnostics: Employment. Kuter:Quest Diagnostics: Consultancy, Research Funding.

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