Jay N. Lozier

Eltrombopag and Improved Hematopoiesis in Refractory Aplastic Anemia

BACKGROUND Severe aplastic anemia, which is characterized by immune-mediated bone marrow hypoplasia and pancytopenia, can be treated effectively with immunosuppressive therapy or allogeneic transplantation. One third of patients have disease that is refractory to immunosuppression, with persistent, severe cytopenia and a profound deficit in hematopoietic stem cells and progenitor cells. Thrombopoietin may increase the number of hematopoietic stem cells and progenitor cells. METHODS We conducted a phase 2 study involving patients with aplastic anemia that was refractory to immunosuppression to determine whether the oral thrombopoietin mimetic eltrombopag (Promacta) can improve blood counts. Twenty-five patients received eltrombopag at a dose of 50 mg, which could be increased, as needed, to a maximum dose of 150 mg daily, for a total of 12 weeks. Primary end points were clinically significant changes in blood counts or transfusion independence. Patients with a response continued to receive eltrombopag. RESULTS Eleven of 25 patients (44%) had a hematologic response in at least one lineage at 12 weeks, with minimal toxic effects. Nine patients no longer needed platelet transfusions (median increase in platelet count, 44,000 per cubic millimeter). Six patients had improved hemoglobin levels (median increase, 4.4 g per deciliter); 3 of them were previously dependent on red-cell transfusions and no longer needed transfusions. Nine patients had increased neutrophil counts (median increase, 1350 per cubic millimeter). Serial bone marrow biopsies showed normalization of trilineage hematopoiesis in patients who had a response, without increased fibrosis. Monitoring of immune function revealed no consistent changes. CONCLUSIONS Treatment with eltrombopag was associated with multilineage clinical responses in some patients with refractory severe aplastic anemia. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT00922883.).

Toxicity of a first-generation adenoviral vector in rhesus macaques

We constructed a first-generation adenovirus vector (AVC3FIX5) that we used to assess the rhesus macaque as a nonhuman primate model for preclinical testing of hemophilia B gene therapy vectors. Although we succeeded in our primary objective of demonstrating expression of human factor IX we encountered numerous toxic side effects that proved to be dose limiting. Following intravenous administration of AVC3FIX5 at doses of 3.4 x 10(11) vector particles/kg to 3.8 x 10(12) vector particles/kg, the animals in our study developed antibodies against human factor IX, and dose-dependent elevations of enzymes specific for liver, muscle, and lung injury. In addition, these animals showed dose-dependent prolongation of clotting times as well as acute, dose-dependent decreases in platelet counts and concomitant elevation of fibrinogen and von Willebrand factor. These abnormalities may be caused by the direct toxic effects of the adenovirus vector itself, or may result indirectly from the accompanying acute inflammatory response marked by elevations in IL-6, a key regulator of the acute inflammatory response. The rhesus macaque may be a useful animal model in which to evaluate mechanisms of adenovirus toxicities that have been encountered during clinical gene therapy trials.

Inflammation, TNFα, and endothelial dysfunction link lenalidomide to venous thrombosis in chronic lymphocytic leukemia

Patients receiving lenalidomide are at an increased risk for deep venous thrombosis (DVT). Here, we prospectively investigated the DVT risk in patients with relapsed chronic lymphocytic leukemia (CLL) treated with lenalidomide (n = 32). Five patients developed six incidents of DVT over 1 year for an annual incidence of 16%. Three of these were considered drug‐related. Median time to DVT was 105 days (range 56–259 days). No pulmonary embolism was detected. Hypercoagulability screen before study entry was negative in all patients who subsequently developed DVTs. Compared to normal volunteers CLL patients had increased baseline levels of D‐dimer, thrombin‐antithrombin, soluble vascular endothelial adhesion molecule 1 (sVCAM‐1), and thrombomodulin (p < 0.001). After 1 week on lenalidomide D‐dimer, thrombomodulin, sVCAM‐1, factor VIII, TNFα, and C‐reactive protein were significantly increased while protein C was decreased (p < 0.001). In patients with lenalidomide‐related DVTs, TNFα, and sVCAM‐1 were more strongly upregulated than in all other patients (p < 0.05) and TNFα and sVCAM‐1 levels were significantly correlated (r = 0.65, p < 0.001). These data link lenalidomide associated DVTs with TNFα upregulation and endothelial cell dysfunction and suggest that aspirin may have a role for DVT prophylaxis in these patients. Am. J. Hematol., 2011.

Interleukin-2 cycling causes transient increases in high-sensitivity C-reactive protein and D-dimer that are not associated with plasma HIV-RNA levels.

Objective:To determine the effects of interleukin (IL)-2 treatment on inflammatory and thrombotic biomarkers in chronically HIV-infected adults receiving antiretroviral therapy. Methods:Cryopreserved plasma was evaluated retrospectively for C-reactive protein (CRP) and D-dimer at baseline, end of an IL-2 cycle, and long-term follow up from two randomized, controlled trials: 57 IL-2-naive adults receiving either three to six cycles of IL-2 as well as antiretroviral therapy (nucleoside analogues) or antiretroviral therapy alone for 12 months, and 40 IL-2-experienced adults on highly active antiretroviral therapy who either interrupted or continued therapy for 6 months after a baseline IL-2 cycle. High-sensitivity CRP (hsCRP) was measured by immunonephelometry (detection limit 0.175 mg/l) and D-dimer by latex agglutination (detection limit 0.20 mg/l). Median within-group differences and pre and post-IL-2 changes between groups were assessed via nonparametric Wilcoxon signed-rank and Mann–Whitney U-tests. Spearmans rank test was used to assess correlations between changes in hsCRP, D-dimer, and HIV-RNA viral load. Results:Significant increases in hsCRP (study 1: 138.6 mg/l; study 2: 58.9 mg/l) and D-dimer (study 1: 3.1 mg/l; study 2: 0.4 mg/l, all P < 0.0001) occurred by the end of the initial IL-2 cycle, returning to baseline by the end of study. No correlations were seen between changes in hsCRP or D-dimer and HIV-RNA, CD4 T-cell count, or proliferation (Ki67 expression). No thrombotic or cardiovascular serious adverse events occurred during these study periods. Conclusion:IL-2 dosing caused transient increases in plasma hsCRP and D-dimer levels, regardless of HIV-RNA viral load, suggesting the possibility of increased risk for thrombotic events.

Elevations in D-dimer and C-reactive protein are associated with the development of osteonecrosis of the hip in HIV-infected adults.

Background:A high incidence of nontraumatic osteonecrosis has been reported in HIV-infected patients. We investigated the levels of D-dimer and C-reactive protein (CRP) in a cohort of HIV-infected adults with and without osteonecrosis of the femoral head. Methods:Forty-three HIV-infected patients with osteonecrosis of the femoral head and a comparison group of 50 HIV-infected patients with negative MRI of the hips and for whom serial plasma samples were available were included. D-dimer and CRP levels were measured prior to and at the time of diagnosis for osteonecrosis patients, at the time of negative MRI of the hips for controls, and at least 6 months later for both groups. Results:Biomarker levels were elevated at the time of diagnosis in the osteonecrosis cohort compared with controls. Median D-dimer value was 0.32 &mgr;g/ml in the osteonecrosis group compared with less than 0.22 &mgr;g/ml in the control group (P = 0.016). For CRP, the corresponding values were 2.52 mg/l and 1.23 mg/l (P = 0.003). Postdiagnosis, D-dimer and CRP levels were also elevated in the osteonecrosis patients compared with controls. Linear regression demonstrated a rise in D-dimer levels from prediagnosis to diagnosis in the osteonecrosis patients whereas CRP levels did not change significantly over time. Conclusion:Compared to controls, patients who developed osteonecrosis had elevated levels of D-dimer and CRP at diagnosis. D-dimer levels increased whereas CRP levels did not change significantly from prediagnosis to diagnosis. These data suggest that patients with higher levels of inflammation are at an increased risk of osteonecrosis.

Antiretroviral Boosting Agent Cobicistat Increases the Pharmacokinetic Exposure and Anticoagulant Effect of Dabigatran in HIV-Negative Healthy Volunteers

Drug interactions between antiretroviral therapy and anticoagulant medications are of particular concern given that ≈50% of the current HIV population is >50 years of age. Moreover, HIV infection is characterized by a hypercoaguable state and premature immunologic aging, in which thromboembolic events may be as much as 10 times more prevalent than in the general population across all age spectra.1 Dabigatran was the first direct oral anticoagulant approved by the US Food & Drug Administration and is the only direct oral anticoagulant with a US Food & Drug Administration -approved specific reversal agent, idarucizumab. Unlike warfarin and many other direct oral anticoagulants, dabigatran is not a substrate, inhibitor, or inducer of cytochrome P450 metabolic enzymes. However, dabigatran is a substrate of Permeability-glycoprotein (P-gp) and renal multidrug and toxin extrusion-1 transporters. Cobicistat is a US Food & Drug Administration -approved antiretroviral-boosting agent that is coformulated with numerous fixed-dose combination antiretroviral products because of its inhibitory effects on cytochrome P450 3A4. Currently, ≈40% of all treatment-naive patients with HIV in the United States are initiated on a cobicistat-boosted antiretroviral regimen. In addition to cytochrome P450 3A4, cobicistat is also an inhibitor of both P-gp and multidrug and toxin extrusion-1 transporters.2 Thus, this study aimed to determine whether the …

Response to Comment on Incidence and Risk Factors of Bleeding-Related Adverse Events in Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib

We thank Drs. Tam and Kamel for their thoughtful comments on our recent publication on the incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia (CLL) treated with the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib.1 We welcome the opportunity to answer their questions and address their concerns. At a median follow-up of 24 months we recorded grade ≤2 bleeding-related adverse events in 55% of 85 patients. No grade ≥3 events occurred. We reported two types of analyses in these patients. First, we prospectively assessed platelet function and coagulation factors at baseline and after 4 weeks of therapy and found that parameters associated with a significantly increased risk of bleeding-related events were present at baseline, including prolonged epinephrine closure time, and lower levels of vWF activity and FVIII. Secondly, we compared platelet aggregation response to collagen and ADP in 30 patients treated with ibrutinib for >6 months to responses in 12 healthy volunteers, 13 patients with treatment-naive CLL, and 3 patients with XLA (who carry loss-of-function mutations in BTK). Cognizant of a prior report that CLL lymphocytes themselves inhibit platelet aggregation,2 we chose to use whole blood for platelet aggregation testing rather than platelet-rich plasma, as others have done, in order to capture the effect of CLL cells on platelet aggregation. While laboratory testing may never fully reflect in vivo situations, whole blood studies arguably more closely reflect the condition of blood circulating in vivo than do studies using platelet rich plasma. Our key findings were that compared to normal controls, response to both agonists was decreased in all patients with CLL, whether on ibrutinib or not. Compared to untreated CLL patients, response to collagen showed a mild further decrement on ibrutinib, while response to ADP improved. Drs. Tam and Kamel rightly point out that the platelet count could influence the analysis of platelet function by aggregation and we appreciate the opportunity to provide the requested additional data and clarifications. Figure 1 shows the distribution of platelet counts in the groups tested: healthy volunteers (median 205 k/μL; IQR 192–229); untreated CLL control subjects (median 161 k/μL; IQR 121–225); and patients on ibrutinib (median 146 k/μL, IQR (118–184). Thrombocytopenia <100 k/μL was present in 4 out of 30 patients on ibrutinib (platelet counts of 72 k/μL, 86 k/μL, 88 k/μL and 97 k/μL, respectively). Drs. Tam and Kamel write that several groups have observed baseline defects in platelet aggregation in CLL that occurs “non-specifically across multiple agonists”, and conclude that this effect was “mainly related to thrombocytopenia” as accurate platelet aggregometry using platelet-rich plasma requires platelet counts greater than 100–150 k/μL.3 We agree that thrombocytopenia would result in a global decrease in aggregation with all agonists; accordingly, the fact that ADP and collagen aggregation were affected in a divergent fashion argues against simple thrombocytopenia (or its reversal by treatment) as the cause for our findings. Additionally, there was no statistically significant difference in platelet counts between healthy controls and untreated CLL patients in our analysis, suggesting that lower platelet counts do not account for the observed baseline aggregation defect in CLL. Furthermore, given that platelet counts did not differ between patients on ibrutinib and CLL controls, we do not believe that treatment related recovery of platelet counts influenced the improvement in ADP response. The significantly lower platelet counts in ibrutinib treated CLL patients (compared to normal subjects) undermines the case for attributing slightly inferior collagen aggregation responses to a drug toxicity, as lower platelet counts confound this inference. Additionally, adjusting for the observed difference in platelet counts would serve only to amplify the relative contribution of ibrutinib to improved ADP-mediated aggregation. Thus, on the whole, we do not believe platelet counts explain the marked differences in collagen and ADP responses which we reported. Figure 1. The distribution of platelet counts in the groups tested. Drs. Tam and Kamel are concerned our results may lead to a perception of “blame the disease [CLL], not the drug”. Clearly, bruising and low-grade bleeding adverse events were more common in patients randomized to ibrutinib compared to the control arm,4 and the drug’s package insert recommends weighing the risks and benefits of interrupting treatment with ibrutinib for invasive procedures. We agree that ibrutinib contributes to platelet dysfunction, and do not argue otherwise. However, prior work by others demonstrated platelet function defects in CLL patients studied,5 and Pulte et al. showed that the addition of CLL lymphocytes to platelet-rich plasma diminishes aggregation responses to ADP.2 In our analysis of whole blood, compared to normal controls platelet aggregation was significantly decreased in untreated CLL patients who had a 43% reduction in response to low dose collagen and 57% reduction in response to high dose ADP. Granule release was similarly impaired, by > 80% in response to collagen and 79% in response to ADP. In summary, our findings and those of others suggest there is some contribution by CLL cells to platelet dysfunction that is not attributable to ibrutinib. Notably, our observation that the risk of bleeding-related adverse events appears to decrease after the first 6 months on ibrutinib is consistent with a contribution of disease factors to overall bleeding risk. Finally, recent studies indicate that the aggregation defect in collagen-evoked signaling in suspension assays may play less of a role in vivo than the initial studies would suggest. For example, by utilizing several methods, including single-cell imaging measuring Ca++ concentrations in individual platelets, Bye et al. found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen compared to in suspension.6 Furthermore, using whole blood they found that initial adhesion to collagen under arterial shear stress was not significantly inhibited by ibrutinib, but stable thrombus formation was strongly inhibited. Combined, all these data suggest that ibrutinib’s effect on platelet function involves several signaling pathways, and a better understanding of the interplay between these factors will contribute to the safe use of ibrutinib, in particular when combinations with anti-platelet agents or anticoagulants have to be considered.

Effect of viral decontamination measures on Wright-stained blood smears

To the editor: Several American health care workers and foreign nationals have been treated at community hospitals and/or specialized treatment units in the United States for Ebola virus infection. The better outcomes seen in patients treated in the United States vs West Africa may depend in large