ALBERT

Description: Background Thrombosis is a major cause of morbidity and mortality in the Philadelphia-chromosome negative myeloproliferative neoplasms (MPN). However, the pathogenesis of thrombosis in patients with MPN is poorly understood. MPNs are associated with elevated levels of inflammatory cytokines and abnormal platelet activation including elevated platelet factor 4 (PF4), B-thromboglobulin (B-TG) and p-selectin levels. However the relationship between these inflammatory markers and the onset of thrombosis is unclear. Current treatment guidelines recommend the use of cytoreductive agents to decrease thrombosis risk based on history of prior thrombosis and age. The effect of cytoreduction on inflammatory markers has only been partially characterized. Furthermore, current clinical guidelines fail to predict thrombotic events in all patients. Thus, more accurate risk stratification is necessary. Aim To determine if levels of p-selectin and PF4 correlate with thrombotic events and if levels of p-selectin and PF4 are affected by cytoreductive therapy. Methods Biobanked samples from 16 patients with a confirmed diagnosis of chronic phase MPN (per WHO 2008 criteria) were included in this study. Seven patients had primary myelofibrosis, five had polycythemia vera, and four had essential thrombocythemia. Median age of patients was 65.5 (range 44-81). Fourteen patients had a JAK2 V617F mutation. Four patients with samples collected before and after thrombotic episodes, four patients with post-thrombotic samples, four without a history of thrombosis, and four patients with samples obtained before and after cytoreduction were analyzed. Patient characteristics are summarized in Table 1. Plasma samples were analyzed for levels of PF4 and p-selectin using commercially-available ELISA assays under standard conditions. Results Measurement of p-selectin levels in MPN patients with a history of a thrombotic event versus disease matched control MPN patients with no history of thrombosis demonstrated an increase in the level of p-selectin in patients with thrombosis. Elevated levels of PF4 were noted in only one patient who had a history of thrombosis, and who was maintained on Aspirin. This suggests that elevated levels of p-selectin may be an indicator of thrombosis risk in patients with MPN. Levels of PF4 and p-selectin were next assessed in patients who had suffered a thrombotic event, both before and after the event had occurred. No changes in p-selectin or PF4 levels pre and post thrombosis were noted. The effect of cytoreduction was measured using samples from patients pre- and post-cytoreduction. No differences in the level of PF4 were noted with cytoreduction. In contrast, reductions in p-selectin were noted in two patients in whom cytoreduction was achieved with Ruxolitinib, but not in two patients in whom cytoreduced with hydroxyurea or pomalidomide. This suggesting that Ruxolitinib may have role in thrombosis risk reduction. Platelet levels were recorded for all 16 patients. There was no difference in platelet levels in each group of patients. In contrast to the changes observed with p-selectin, there was no difference in platelet levels in patients with out a thrombotic event compared to those with a history of thrombosis. This suggests that platelet activation, as measured by p-selectin, may be a stronger indicator of thrombotic risk than platelet levels. Conclusions Improvements in risk stratification of thrombotic risk for patients with MPN are needed. Platelet activation in MPNs is abnormal and may serve as an additional marker of thrombotic risk. Our results suggest that levels of p-selectin, but not PF4, may be associated with increased thrombotic risk. Furthermore, treatment with the JAK1/2 inhibitor Ruxolitinib resulted in reduction in levels of p-selectin, suggesting that the role of Ruxolitinib in preventing thrombotic event should be further investigated. Additional studies with larger numbers are in progress to validate these results. Disclosures: Rampal: Foundation Medicine: Consultancy.

Description: Abstract 1992 The myeloproliferative neoplasms (MPN) can have a variable natural history. Polycythemia vera and essential thrombocythemia, in particular, are conditions that can extend over decades, but some patients have clinical progression to myelofibrosis or acute myeloid leukemia. As first articulated by Warburg, cancers are metabolically distinguished from normal tissues by the use of glycolysis under aerobic conditions. To metabolically characterize the blood cells of patients with myeloproliferative neoplasms, we measured the mitochondrial membrane potential using the cyanine dye, JC-1. In examining cells derived from the blood and/or marrow of 159 patients with primary myelofibrosis, polycythemia vera and essential thrombocythemia, we found that the mitochondrial membrane potential (FL2/FL1=electrochemical potential/mitochondrial mass) was elevated compared to the blood cells of normal individuals. Thirty five percent of patients with polycythemia vera and essential thrombocythemia had normal MMP. In contrast, 97% of patients with primary myelofibrosis, post-polycythemia myelofibrosis, post-essential thrombocythemia myelofibrosis and acute myeloid leukemia following an MPN had evidence of cell populations with higher mitochondrial membrane potential. Cells with distinctly higher mitochondrial membrane potential could be indentified in platelets and polymorphonuclear leukocytes; however the MMP of lymphocytes was normal, indicating that the alteration in metabolic state likely occurred in a multipotential myeloid stem cell. Cell populations were confirmed by co-staining with anti-CD19, -CD45, -GlycophorinA and -β3-integrin antibodies. Sequential analysis of patient samples found that the acquisition of higher mitochondrial membrane potential was stable and persistent over 2 years or more of follow up and that elevated membrane potential predisposed patients to disease progression. The balance of patients (65%) with ET had evidence of increased MMP suggesting the possibility of disease in an early state of evolution to a more aggressive condition. The increased MMP did not correlate with the presence of mutation in JAK2. These results indicate that clinically advanced MPN can be characterized by changes in mitochondrial physiology that might be identified non-invasively by flow cytometric staining with JC-1. In addition, the early nature of these changes may help to identify therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3328 Background: Venous thromboembolism (VTE) is a common cause of morbidity and mortality in cancer patients. Standard of care for treatment is Low Molecular Weight Heparin, but recurrence of VTE remains a concern. We performed a retrospective analysis of our institutional experience, to characterize the patients who had a recurrence of VTE while on therapeutic doses of Dalteparin. Objectives: 1. To determine the VTE recurrence rate for cancer patients on therapeutic Dalteparin. 2. To elucidate potential risk factors for recurrence. 3. To determine the impact of recurrent VTE on overall survival. Methods: Patients beginning treatment for VTE with dalteparin between 1/1/2008 and 12/10/ 2009 were retrospectively identified through the hospital's electronic medical records system and cases of recurrent VTE were characterized. Overall survival was estimated using the Kaplan-Meier method and the influence of VTE recurrence on overall survival was analyzed as a time-dependent covariate using a Cox proportional hazards model. Results: 1,392 patients, treated for VTE with dalteparin were included in this study. 34 recurrent VTE episodes were identified. The overall incidence of recurrent thrombosis by six months was 2.3% (95% CI: 1.7%-3.3%). Older age was significantly associated with recurrence (p=0.04). Lung cancer patients had a significantly elevated risk of recurrence (5.6%, p=0.03). No other cancer types were associated with a significant trend to increased recurrent VTE rates. The incidence of recurrent VTE was higher among females compared to males (3.0% vs. 1.6%), although this trend was not statistically significant (P = 0.08). After adjusting for gender, sex and cancer diagnosis, developing a recurrent VTE was associated with a 3.0-fold hazard ratio of death (

Description: Background: Low-molecular weight heparin (LMWH) has been the standard of care for treatment of venous thromboembolism (VTE) in patients with cancer. LMWH injections are painful and costly. Rivaroxaban, an oral direct factor Xa inhibitor, was FDA approved in 2012 for treatment of pulmonary embolism (PE) and deep vein thrombosis (DVT), but there has been a knowledge gap for its use in patients with cancer-associated thrombosis (CAT). Under a Quality Assurance Initiative (QAI), we established a Clinical Pathway to guide rivaroxaban use for CAT, and began to offer rivaroxaban as an alternative to enoxaparin, in January 2014, for patients who met appropriate clinical criteria. We are tracking all cancer patients with PE or symptomatic proximal DVT, whose full course of anticoagulation is with rivaroxaban (allowing up to 3 days of initial parenteral anticoagulation). We now report the characteristics of the first 200 patients, and an outcome analysis of our first 100 patients, who have been treated for at least 6 months or otherwise reached an endpoint. Materials and Methods: The Clinical Pathway guidelines will be available on request, pending publication. Patients were not treated with rivaroxaban if they had active gastrointestinal or genitourinary lesions, or had undergone gastric resection due to anticipated excess bleeding risk or reduced absorption. This excluded under 5% of patients. The Pathway provided dosing guidelines in the setting of thrombocytopenia, advanced age, transient renal, or hepatic dysfunction. Primary endpoints include new or recurrent PE, symptomatic proximal lower extremity DVT, major bleeding (ISTH definition), clinically-relevant non-major bleeding leading to discontinuation of rivaroxaban, or death. Considering those outcomes as competing risks, the cumulative incidence of each event type was calculated using R 3.2.0 for Windows and package "Survival". Results: The characteristics of our first 200 patients are in Table 1. 70% of the patients had PE. Of the solid tumor patients, 65.6% had metastatic disease. The first 100 patients have completed at least 6 months of rivaroxaban anticoagulation or otherwise reached a primary endpoint. At 6 months, the cumulative incidence of death was 14.4% (95% CI=6.8-21.4%), new or recurrent VTE was 4.3% (95% CI=0.1-8.4%), major bleeding was 1.1% (95% CI=0-3.1%), and clinically relevant non-major bleeding leading to rivaroxaban discontinuation was 7.9% (95% CI=2.1-13.3%). Conclusions: In the analysis of the first 100 patients entered into our QAI program, the rates of major bleeding, and new or recurrent VTE compare favorably to two published studies of LMWH for treatment of cancer associated thrombosis. In the CLOT study (Lee et al, NEJM, 2003) and the Daltecan study (Francis et al, JTH, 2015), the 6-month rates of new or recurrent VTE were approximately 9%, and the rates of major bleeding were 6% and 9.5% respectively. Our final analysis awaits the completion of 6 months follow-up on 200 patients, to be completed in December 2015. But the rates of major bleeding and recurrent VTE at this point suggest safety and efficacy to be at least non-inferior to LMWH, with the advantage of reduced patient burden, and support the ongoing use of our Clinical Pathway. Our low rate of major bleeding likely is influenced by the exclusion of patients with active GI or GU lesions, who would be expected to have a high bleeding risk with an oral direct anticoagulant. However, we estimate this excluded less than 5% of cancer patients with VTE. Further, we anticipated reduced drug clearance in the elderly, and used a reduced dose for patients greater than 75 years of age. This appeared to be associated with no loss in efficacy, and helped maintain a low rate of major bleeding. A randomized trial is the optimal approach to establish non-inferiority or superiority of rivaroxaban to LMWH for cancer associated thrombosis. However, our QAI Clinical Pathway provides guidance and reassurance for rivaroxaban use until a randomized trial is conducted. Table. Baseline Characteristics of Patients Characteristic Number of Individuals Gender Male 82 Female 118 Event Type PE, with or without DVT 140 Proximal, Symptomatic Lower extremity DVT 60 Cancer Type Solid Tumor 186 Hematologic Malignancy 14 Cancer Stage (Of Solid Tumors) No Evidence of Disease (Post-Cancer Surgery) 11 (5.9%) 1 1 (0.5%) 2 4 (2.2%) 3 16 (8.6%) 4 122 (65.6%) Unknown 32 (17.2%) Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Background: Venous thromboembolism (VTE) is a leading cause of mortality and morbidity in cancer patients. The current standard of care is to treat cancer-associated VTE with Low Molecular Weight Heparin (LMWH), which is more effective than vitamin K antagonists, such as warfarin. However, LMWH injections are painful, expensive, and a burden in healthcare resource utilization. Further, many of these patients are referred to an emergency room for education on self-injection techniques, an added health care cost. The Hematology/Anticoagulation Management Service at Memorial Sloan Kettering Cancer Center is developing rivaroxaban as a safe and effective alternative to LMWH for cancer-associated VTE. In this report, we demonstrate a significant reduction in the referral of patients to the MSKCC Urgent Care Center (UCC), our in-house emergency room, to initiate anticoagulation, resulting in a significant reduction in resource utilization. Methods: As a Quality Assessment Initiative, we track all patients with cancer-associated VTE at MSKCC receiving rivaroxaban since January 2014, and have a similar database of cancer-associated VTE from June through December 2013, treated with enoxaparin. For this utilization of resources study we evaluated where anticoagulation was initiated for treatment of a new pulmonary embolism or lower extremity deep vein thrombosis, specifying either a single outpatient visit, two outpatient visits on the same day, a telephone call, or a visit to the UCC. When patients had a second outpatient visit on the same day as diagnosis of the VTE, the second visit was for patient education on injection technique and insurance authorization. The site of anticoagulation initiation was by the judgment and discretion of the physician managing the patient's cancer. Patients who developed a VTE during a hospital stay or were managed at an outside emergency room were not included in this analysis, as our program has no influence in those settings. Statistical analysis was with the Chi-square test. Comparison of safety and efficacy of rivaroxaban and LMWH is the subject of a separate study. Results: As anticipated, changing from a parenteral anticoagulant to rivaroxaban (an oral agent) resulted in significant changes in practice (Table). Significantly fewer rivaroxaban patients required visits to the UCC than with enoxaparin (p=0.009). Fewer patients required a second outpatient, as well. When viewed in the aggregate of UCC or second outpatient visit, 82% of patients treated with enoxaparin required additional medical resources for initiation of anticoagulation, beyond a single outpatient visit, which decreased to 59% with rivaroxaban (p

Description: It is well-established that cancer is associated with activation of the blood coagulation system, with associated thrombosis as a major cause of morbidity and mortality. Increased expression of Tissue Factor (TF) by cancer cells correlates with a more aggressive grade and clinical course. It is widely presumed that activation of coagulation facilitates cancer growth, and in mouse models, anticoagulation can reduce development of lung metastases. Yet primary tumors are not reduced in a fibrinogen knock-out mouse host, and most importantly, anticoagulation has not been shown to reduce tumor growth in cancer patients. We therefore studied the effect of expression of full-length Tissue Factor (FLTF) and alternatively-spliced human Tissue Factor (asHTF) in a mouse model of human pancreatic cancer. Due to the loss of exon 5, asHTF has a truncated extracellular domain with incomplete procoagulant activity. And due to a frame shift, exon 6 does not code for the transmembrane domain and cytoplasmic tail of FLTF, but codes for a novel peptide sequence. asHTF is soluble and of unknown function. We show that 5 of 6 human pancreatic cancer cell lines tested expressed both FLTF as well as asHTF. The MiaPaca-2 line did not express detectable mRNA or protein of either TF isoform. We generated mammalian expression vectors for both FLTF and asHTF, and established Miapaca-2 clones, stably expressing FLTF, asHTF, or control clones with an empty vector. As anticipated, conditioned media from all FLTF clones shortened the whole blood clotting times by approximately 75%. Conditioned media from control cells and asHTF expressing cells had no effect on clotting times. To evaluate the effect of the TF isoforms on primary tumor growth, 5 X 106 cells from three independent clones of stably transfected clones of FLTF, asHTF, or control clones were injected into the flanks of nude mice (4 mice per clone). At 31 days, the mice were sacrificed and tumor mass measured. Tumors grew in 10 of 12 control mice, but were small (mean tumors 90 mg, SEM 21 mg). Interestingly, FLTF was associated with reduced primary tumor growth; only 4 of 12 developed measurable tumors (mean tumors 10 mg, SEM 4 mg, p = 0.002). In contrast, asHTF expression was associated with enhanced tumor growth; 12 of 12 animals developed tumors (mean tumors 390 mg, SEM 102 mg, p=0.018). In animals with asHTF expressing tumors, circulating asHTF protein was observed in the plasma. The asHTF tumors had increased vascular density compared with controls, suggesting a role of asHTF promoting angiogenesis. In contrast to the prevailing paradigm, our data suggest that FLTF, with procoagulant activity, not only fails to promote primary tumor growth, but may actually inhibit tumor growth. In contrast, asHTF, may be the more important TF isoform in the enhancement of tumor growth.

Description: Purpose: Case reports, as well as our anecdotal experience, revealed frequently elevated activated partial thromboplastin time (aPTT) levels in some patients on the direct thrombin inhibitors (DTI) lepirudin and argatroban when dosed according to the FDA-approved guidelines in the Product Inserts. We analyzed our institution’s experience with these agents to determine if dosing guidelines need reconsideration. Methods: A retrospective chart review was conducted under the auspices of the pharmacy quality management team, with IRB approval. Cases were from the pharmacy database of all patients that received lepirudin or argatroban from September 2002 through March 2004. Sixty-six cases were identified and reviewed. Results: Patients were treated with the DTI for Heparin Induced Thrombocytopenia, with or without thrombosis (HIT, HITT), based on a decline of platelet count on heparin by 50% or to