ALBERT

Description: Background Individuals with hemophilia have abnormally low bone density, despite prophylactic factor infusion with the associated reduction in the joint disabling complications of bleeding. Previously, we demonstrated decreased bone mineral density, skeletal strength, and increased bone resorption in factor VIII (FVIII)-deficient mice without differences in physical activity or joint bleeding compared to control mice. Thus, we hypothesize that factor deficiency is an independent risk factor for decreased skeletal health. Methods To test this hypothesis, we evaluated platelet-poor plasma from 80 male subjects with severe FVIII deficiency, 20 male patients with severe factor IX (FIX) deficiency and 52 healthy male controls. Samples were assessed for three markers of bone turnover: receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and carboxy-terminal collagen crosslinks (CTX-1). RANKL binding to the RANK receptor induces the proliferation and differentiation of osteoclasts resulting in increased bone resorption. OPG competitively inhibits RANKL binding to RANK, blocking these osteoclastic effects. CTX-1 is formed through the degradation of bone and thus is a direct marker of bone resorption. Results No differences in RANKL levels are seen between any of the groups. A significant increase in OPG is identified in plasma from FIX-deficient subjects compared to FVIII-deficient subjects (p = 0.0046) and control subjects (p = 0.0095), without differences seen between FVIII-deficient and control subjects (p = 0.920). CTX-1 is higher in samples from FVIII-deficient subjects compared to FIX-deficient subjects (p = 0.020) and control subjects (p = 0.0024), without differences seen between FIX-deficient and control subjects (p = 0.947) samples. In a subset analysis, subjects receiving FVIIII concentrate replacement within 12 hours of plasma collection have decreased CTX-1 (p = 0.022) but not OPG (p= 0.850) compared to those subjects receiving factor concentrate replacement greater than12 hours from plasma collection. We are currently evaluating other plasma markers of bone turnover and cytokine expression in the same cohort of samples. Conclusions Subjects with FVIII deficiency but not FIX deficiency have evidence of increased bone resorption compared to control samples. Increased OPG levels are present in FIX-deficient plasma and may contribute to the differences seen in CTX-1 production between subjects with FIX and FVIII deficiency. Furthermore, FVIII replacement within 12 hours of plasma collection reverses CTX-1 elevation seen in FVIII-deficiency. This study suggests that bone disease is intrinsic to FVIII-deficiency and that aggressive FVIII replacement may ameliorate this pathology. Disclosures: Manco-Johnson: Novo Nordisk: Membership on an entity’s Board of Directors or advisory committees; Biogen Idec: Membership on an entity’s Board of Directors or advisory committees; Baxter BioScience: Membership on an entity’s Board of Directors or advisory committees; CSL Behring: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bayer HealthCare: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Research Funding. Taylor:Novo Nordisk: Research Funding; Baxter BioScience: Research Funding.

Description: Introduction Persons with hemophilia have decreased bone mineral density (BMD) and increased risk of fracture. Our group has demonstrated that mice with factor VIII deficiency have decreased BMD and decreased biomechanical strength. The underlying mechanism for the decreased skeletal health remains undetermined. This study uses small interfering(si) RNA to knockdown prothrombin gene expression to test the hypothesis that thrombin deficiency results in decreased skeletal health. Method Seventeen 3-week old male C57BL/6J wild-type mice were given subcutaneous injections of siRNA against α-prothrombin (1 mg/Kg) and compared to an equal number of mice injected with saline. Mice were injected at 3, 4, 5, 7, 9, 11, 13, 15, 17, and 19 weeks of age with a volume of 200 ul and measurements taken at peak bone mass at 20 weeks of age. In initial dose response studies, this regimen results in 〉85% knockdown of prothrombin gene expression (data not shown). Studies included whole body and femoral BMD, thromboelastography (TEG) of plasma, micro computed tomography (µCT), and three-point breaking. Results TEG studies show significant differences between mice treated with siRNA (n=17) and mice treated with normal saline (n=6) with R, K and α-angle being p 〈 0.02, p 〈 0.014, and p 〈 0.005 respectively. These results confirm that the thrombin is knocked down by the siRNA against α-prothrombin. Comparative analysis of the whole body and femoral BMD between the treatment and saline groups demonstrate no significant differences (p = 0.64 and p = 0.74). There was no significant difference seen in µCT scans of the right femur, including femoral cortical area (p=0.19), cortical thickness (p= 0.63), and cancellus area (p= 0.25). Three-point break data show no significant difference in ultimate force (p = 0.268), strength (p = 0.546), or modulus (p = 0.07). Conclusion This study seeks to understand the underlying mechanism for bone disease in hemophilia. The results indicate that 〉85% prothrombin knockdown in wild-type mice starting at 3 weeks of age does not significantly alter skeletal health. This suggests that either 1) thrombin deficiency is not involved in the mechanism of decreased skeletal health in hemophilia or; 2) the role of thrombin deficiency occurs early in bone development (prior to 3 weeks of age). To evaluate these two potential explanations, thrombin deficiency needs to be evaluated earlier in development. Thrombin deficient mice are embryonically lethal. Therefore our laboratory is currently using a series of transgenic mice to characterize the role of thrombin receptors present osteoblasts and their role in bone metabolism. Disclosures Taylor: Alnylam: Research Funding; Shire: Research Funding; CSL: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees.

Description: Background: Sporadic cases of acquired hemophilia are exceedingly rare, with limited data on the disease incidence, clinical associations, and mortality, particularly in hospitalized patients. The aim of this study is to evaluate the epidemiology and mortality of patients hospitalized with acquired hemophilia from a large national database. Methods: Using the 2012 National Inpatient Sample (NIS), admissions with an International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) codes for Acquired hemophilia (286.52) were extracted. Any patient with a concomitant diagnosis of congenital hemophilia was excluded for quality control. Admission data was extracted and correlated with associated clinical diagnosis, age, gender, length of stay and mortality. Results: Of the 7,296,968 unweighted admissions in the 2012 NIS, 80 admissions met the inclusion criteria (incidence of 0.00001%). Patients admitted with acquired hemophilia tended to be older (mean age 63.6 years, SD 18.7) & male (55%). Admissions were most commonly unplanned (88.7%), lengthy, (mean 8.45 days, SD 7.4 days), and costly (mean charge $280,730.00 USD, SD $617,293 USD). Inpatient mortality was rare (N=3, 3.75%). Packed red cell transfusion, as well as coagulation factor transfusions was common (42.5% and 20% respectively). Admissions involving critical illness were relatively common as well (22.5% with acute renal failure, 10% receiving mechanical ventilation). 30% of patients admitted with acquired hemophilia had an active or previous malignancy diagnosis (the most common being leukemia N=4, breast cancer N=3, and prostate cancer N=3). 50% of admissions were associated with an acute or chronic infectious disease (the most common being viral hepatitis N=10, bacterial infections N=9, and pneumonia N=9). When compared to patients admitted with hemophilia A (N=1645), those with acquired hemophilia were significantly older (mean age 63.6 vs 41.2 years p

Description: Background: We have previously shown that factor VIII deficiency decreases skeletal health in persons with hemophilia and animal mouse models; however, it is uncertain if this is unique to factor VIII deficiency or persists in factor IX (FIX) deficiency as well. Animal models can eliminate genetic and environmental factors of a complex trait like osteoporosis, and offer more invasive testing beyond bone mass density (BMD), studying bone properties using microtomography (μCT), histomorphometry and biomechanics. Decreased skeletal health can be caused by altered signaling between cells that are responsible for bone remodeling, osteoblasts (build bone) and osteoclasts (absorb bone). Understanding mechanisms of bone disease present in factor deficient mice can impact clinically how it is diagnosed and treated in the future. Methods: FIX-deficient and wild-type (WT) littermates were scanned using peripheral dual energy X-ray absorptiometry (DEXA) to obtain measurements for whole body BMD and femoral BMD at 10 and 20 weeks of age. Cortical bone at the midshaft of the femur was analyzed using microtomography (μCT) and biomechanical testing determined by three-point bending. Three-dimensional high resolution μCT analysis of a tibia is analyzed for bone histomorphometry. Important biomarkers of bone remodeling are analyzed on collected mouse serum and include measuring alkaline phosphatase (ALP) activity spectrophotometrically, osteoprotegrin (OPG), receptor activator of nuclear factor kappa-B Ligand (RANKL) and osteocalcin by commercially available ELISA kits. Results: FIX-deficient mice have decreased skeletal health compared to their wild-type (WT) littermate controls at both 10 and 20 weeks of age, without statistical differences in weight or body length. At 10 weeks, FIX-deficient mice (n=11) have lower whole body BMD (p=0.006) and femoral BMD (p=0.015) compared to WT littermates (n=12). Femoral μCT analysis shows parameters related to bone size, including cortical area (p=0.078) and marrow area (p=0.001) are decreased, and overall cross-sectional area (p=0.002). Three-point bending of the bones have decreased in ultimate failure (p=0.042) and stiffness (p=0.039). High resolution μCT of the tibia show decreased total bone volume (p=0.004) and at the midshaft the cortical bone (p=0.004), marrow area (p=0.029), and the total area (p=0.004). At 20 weeks, FIX-deficient mice (n=14) show decreased whole body BMD (p=0.044) and femoral BMD (p=0.053) trends from their WT littermates (n=15). Femoral μCT analysis shows decreased cortical area (p=0.038). Ultimate failure (p=0.053) and stiffness (p=0.002) differences are present in this group. No differences in cortical bone are seen with high resolution μCT, but do have distinct decreased cancellous bone in the tibia's metaphysis. The FIX-deficient bones show decreased bone volume (p=0.05) and structural differences with decreased trabecular number (p=0.03) and trabecular connectivity (p=0.01), and increased trabecular space (p=0.03). Receptor activator of nuclear factor kappa-B ligand (RANKL) located on osteoblasts plays a direct role in bone metabolism with osteoclasts, by binding to RANK causing precursor osteoclasts to mature and begin bone resorption. At 10 weeks of age RANKL was not detectable in our assays. At 20 weeks of age, RANKL was found to be significantly decreased in FIX-deficient mice over WT littermates (p=2.2E-06). OPG is a receptor decoy that inhibits bone resorption by binding to RANKL on osteoblasts. At 10 weeks, the FIX-deficient mice were found to have lower levels of OPG compared to WT controls (p=0.012); however, at 20 weeks, there was a marked increase in OPG levels of the FIX-deficient mice (p=5.7E-05). Bone building biomarkers, ALP activity and osteocalcin showed no measurable differences in serum at 10 or 20 weeks of age (p〉0.5) and (p〉0.2) respectively. Conclusions: FIX deficent mice have bone disease suggesting that alteration of the clotting cascade has a direct impact on skeltal health. The mechanism behind of how factor deficiency alters bone health is unclear, but the RANKL/OPG data is consistent with aberrant osteoblast-osteoclast signaling. Our laboratory is actively investigating the mechanism of decreased skeletal health associated with factor deficiencies. Disclosures Taylor: CSL Behring: Consultancy, Research Funding; Kedrion: Research Funding; Baxalta/Shire: Consultancy, Research Funding; Novo Nordisk: Research Funding.

Description: Introduction: People with Hemophilia (PwH) have decreased bone mineral density (BMD) and increased risk of fracture. Children as well as adults have demonstrably lower BMD than age-matched control populations. Additionally, PwH have increased rates of bone resorption and decreased rates of bone mineralization. This study aims to characterize the impact of factor VIII (FVIII) replacement on biomarkers of bone metabolism in PwH as well as correlate those biomarkers with physical characteristics and Quality of Life. Methods: Twelve participants with severe FVIII-deficiency were screened and enrolled in the single site IRB-approved study. After a 72-hour washout, plasma samples were obtained at 8 time points over the course of five days following FVIII infusions on Day 1 (50 units/Kg) and Day 3 (20 units/Kg). All participants had a physical examination and completed quality of life, activity, pain and factor compliance questionnaires on Day 1. Visual Analog Scale (VAS) via the EQ-5D-5L was utilized to report participants' self-rated health. BMD was measured as Z-scores via Dual-Energy X-ray Absorptiometry (DEXA) Scanning. Joint assessment was performed using the Hemophilia Joint Health Score (HJHS). Plasma cytokine levels were measured using ELISA and magnetic bead multiplex kits. Samples were evaluated for 42 cytokines (Table 1). Results: VAS score increased with Spine L1-L4 Z (SZ)-score (p=0.023, R2=0.42) and Hip Total Z (HTZ)-score (p=0.006, R2=0.54). VAS was lower in PwH with higher HJHS (p=0.006, R2=0.55). HJHS was higher in PwH with lower SZ-scores (p=0.004, R2=0.62). The average ratio of OPG to RANKL increased from 0.9 to 6.2 during the study period (p=0.006, R2=0.75). Plasma vascular endothelial growth factor (VEGF), an angiogenic cytokine that has been implicated in bone development and repair, and IL-8, a chemokine that stimulates osteolysis, inversely correlated with SZ-score. Carboxy-terminal collagen crosslinks (CTX-1), a breakdown product of bone, is elevated in samples from subjects with higher Hip Neck Z (HTZ)-scores. Eotaxin, an eosinophil chemoattractant, and VEGF levels were decreased in samples from subjects with lower HTZ-scores. CTX-1 levels were elevated in subjects with higher HNZ-scores. Parathyroid hormone (PTH), a hormone that stimulates osteolysis, eotaxin, VEGF, and IL-4, a potential osteoblast activity mediator, levels were lower in plasma samples from subjects with higher HNZ-scores. Average plasma eotaxin, VEGF, and IL-12p70, a T-cell stimulating factor, levels were positively correlated with HJHS score. Correlation coefficients, p-values and R2 values for average cytokine values and skeletal health measures are listed in Table 1. Conclusions: PwH with decreased BMD report a lower level of self-rated health. This is evidenced by correlations between VAS, HJHS, and BMD. Plasma CTX-1 is elevated in PwH with higher BMD, suggesting that PwH with improved skeletal health experience higher rates of bone turnover as measured by osteoclast activity. The average OPG/RANKL ratio increased during the study period suggesting that FVIII infusion may have a direct effect on the RANKL-OPG pathway. PTH, eotaxin, and VEGF are inversely correlated with BMD and positively correlated with HJHS. Lower levels of these cytokines appear to be associated with poor skeletal health in PwH. Increased levels of PTH are associated with osteoporosis and VEGF has been implicated in bone development and repair. These data suggest an intricate association between skeletal health, factor administration, and cytokine levels. Continued analysis of these data is ongoing and additional findings will be discussed. Disclosures Lattimore: UniQure BV: Research Funding. Taylor:Bioverativ: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Alnylam: Research Funding; CSL: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: PAC203 is a global multicenter dose-finding study of pacritinib (PAC), an oral JAK2/IRAK1 inhibitor in patients with primary or secondary myelofibrosis refractory or intolerant to ruxolitinib, including patients with severe thrombocytopenia. Patients were randomized 1:1:1 (PAC 100mg QD, 100mg BID or 200mg BID) and stratified by baseline platelet count. The mutational landscape of this patient group is not well characterised, and the impact of mutation status on disease response and hematologic parameters is unknown. We carried out baseline mutational analysis on 105 (of total 164 recruited; 161 treated) patients using an ISO accredited Illumina TruSeq Custom Amplicon Panel, including 32 gene mutation hotspots and exons (~36,000 bp, 287 amplicons). CALR mutation screening was carried out independently. Accepted coverage was achievement of a depth of ≥100 reads per base in ≥95% of targeted bases. Median follow-up time for this cohort was 163 (28-476) days. 57% of patients had a diagnosis of primary myelofibrosis (PMF), whereas 27% and 15% had post-polycythemia vera MF (PPV-MF) and post-essential thrombocythemia MF (PET-MF), respectively. The median baseline platelet count was 64.5 x109/L, with

Description: BACKGROUND: Bone disease is increasingly being clinically recognized in hemophilia. We previously demonstrated bone disease in factor VIII (FVIII)-deficient mice at peak bone mass that was independent of activity, hemarthroses, and weight compared to wild-type littermates. These findings are consistent with the hypothesis that FVIII-deficiency directly impacts skeletal health. However, the impact of FVIII-deficiency on skeletal health at different stages of growth and aging remains unknown. Addressing this issue is critical for understanding the potential underlying mechanisms as well developing therapeutic interventions to ameliorate bone disease in hemophilia. METHODS: The skeletal health of FVIII-deficient and wild-type littermate controls were characterized during growth (10 weeks of age), peak bone mass (20 weeks), and during aging (40 weeks). Characterization included micro computerized tomography (CT) imaging, bone mineral density, biomechanical testing, histomorphometry (quantitative histology), gene expression using reverse transcription polymerase chain reaction (RT-PCR), and bone biomarker evaluation. RESULTS: Femoral bone mineral density in FVIII-deficient mice is decreased compared to wild-type littermate controls with increasing statistical significance with age; p = 0.108 at 10 weeks (12 FVIII-deficient and 15 wild-type mice), p = 0.064 at 20 weeks (19 FVIII-deficient and 22 wild-type mice) and p= 0.017 at 40 weeks (18 FVIII-deficient and 23 wild-type mice) of age. Cortical bone (outer hard bone) thickness differences decrease with age, with FVIII-deficient mice having statistically thinner cortices at 10 weeks (p = 0.0004) compared to 20 (p = 0.008) and 40 weeks (p = 0.079). Trabecular bone (softer inner bone), measured by trabecular number, decreased in FVIII-deficient mice, has a similar trend: 10 weeks (p = 0.035), 20 weeks (p = 0.048), and 40 weeks (p = 0.643). Histomorphometry also shows an age-dependent pattern. At 10 weeks in the FVIII-deficient mice there is higher osteoclast (cells that break down bone) (p = 0.0010) and no change in osteoblast (cells that build bone) numbers; at 20 weeks there is higher osteoclast (p = 0.0011) and lower osteoblast (p = 0.027) numbers; while at 40 weeks there is no difference in osteoclast and lower osteoblast (p = 0.0017) numbers. Dynamic histomorphometry demonstrates lower bone formation rate at 40 weeks (p = 0.002) but no difference at 20 weeks. Biomechanical strength, as measured by ultimate force, is decreased in FVIII-deficient mice at 20 weeks (p = 0.017) and 40 weeks (p= 0.023). 10 week bones were not evaluated for biomechanical strength due to small size. No statistical difference in CTX-1 (biomarker for bone resorption) or osteocalcin (biomarker for bone formation) is observed at any age group. Levels of gene expression using RT-PCR was characterized from the bone marrow cells in the 20 week cohorts evaluating genes related to bone metabolism. Normalized to beta-actin and glyceraldehyde 3-phosphate dehydrogenase, differential gene expression is not seen in osteocalcin, tumor necrosis factor-alpha, receptor activator of nuclear factor-kappa B (RANK), RANK ligand, interferon-beta or tartrate-resistant acid phosphatase. There is lower FBJ osteosarcoma oncogene (c-fos) gene expression (p 〈 0.01), a transcriptional regulator of osteoclast formation, in the bone marrow from FVIII-deficient mice. There is also a trend for lower osteoprotegerin gene expression (p 〈 0.10). CONCLUSIONS: Evidence for bone disease is present in growing, mature and aging bones in FVIII-deficient mice compared to littermate controls but the form of bone disease differs with age. Differences in imaging parameters are most pronounced at 10 weeks while BMD is most pronounced in bones of older mice. Furthermore, FVIII-deficient mice have an age-dependent pattern of osteoblast and osteoclast numbers with a relative increase in osteoclasts during growth and relative decrease in osteoblasts with aging. Even with these changes, the decreases in biomechanical strength, measured against aged-matched wild-type controls, are similar at both 20 and 40 weeks. The observed bone pathology is not associated with global changes in biomarkers of bone formation or bone resorption at any age. These results suggest that FVIII-associated bone disease is a complex age-dependent process that begins during development and continues throughout life. Disclosures Taylor: Baxter BioScience: Research Funding; Novo Nordisk: Research Funding.

Description: Introduction: Hemophilic bone disease is a pathology inherent to factor VIII (FVIII) and factor IX (FIX) deficiency present in both adult and pediatric persons with hemophilia (PWH). This pathology is also present in mouse models. We have previously demonstrated that FVIII replacement restores lost mineralization activity in osteoblast-like cells from FVIII-deficient mice. We hypothesized that bone disease in PWH is secondary to decreased thrombin generation in PWH. Methods: Murine bone marrow stromal cells isolated from FVIII knockout (FVIII-/-) and wild-type (FVIII+/+) C57BL mice and human osteosarcoma (HOS) TE85 cells were cultured in α-MEM with 0-10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 µg/mL Amphotericin B. Ten millimolar ß-glycerophosphate and 50 µg/mL ascorbic acid were added to induce differentiation and mineralization. Mineralized deposits were stained with alizarin red. Alkaline phosphatase (ALP) activity and culture protein content were measured using colorimetric assays. Results: In , the addition of thrombin in serum-free conditions increases cell proliferation ~25% (p=5x10-4, ANCOVA) and decreases mineralization up to 84% (p=1x10-6) (Figure 1). Thrombin's effect on ALP activity in HOS TE85 cells is negative at low cell numbers and positive at high cell numbers (Figure 1). Osteoblast-like cells from FVIII-/- mice mineralize 70% less than those from FVIII+/+ cells (p=3x10-6). Administering FVIII to cells from FVIII-/- mice ameliorates the mineralization deficiency (Figure 1), while mineralization in cells from FVIII+/+ mice does not change (p=0.66). FVIII-/- cells express 30% lower levels of ALP activity than FVIII+/+ (p=3x10-4). FVIII+/+ cells proliferate 26% more rapidly than FVIII-/- cells (p=4x10-7, ANCOVA), but this is not affected by the addition of FVIII in vitro (p=0.45). The addition of FVIII causes a ~33% decrease in ALP activity in osteoblast-like cells from both FVIII-/- (p=2x10-4) and FVIII+/+ (p=5x10-5) mice. Although thrombin impacts osteoblast differentiation and function, it has paradoxical effects on their mineralization, suggesting thrombin deficiency is not the sole driver of reduced bone health in PWH. Deficient mineralization in cells from FVIII-/- mice compared to those from FVIII+/+ mice is corrected with FVIII replacement in vivo or in vitro, suggesting that circulating FVIII has a positive role in these cells' mineralization. More rapid proliferation in cells from FVIII+/+ mice likely contributes to the increase in mineralization, yet, as proliferation is unchanged by FVIII replacement, it appears unlikely that this effect occurs via the same mechanism. Thrombin causes increased proliferation of HOS TE85 cells, yet also causes a significant decline in mineralization activity (Figure 1). Whether this is a paradoxical effect present only in this transformed cell line is yet undetermined. ALP activity increases when HOS TE85 cells are given thrombin in vivo during the log phase of growth (Figure 1), indicating a positive effect on the osteoblast-like function of the cells. However, this notion is undermined by the decrease in mineralization. Identifying the role of FVIII, FIX, thrombin, and active protein C in bone mineralization in PWH is proving to be a challenging undertaking, yet it remains critical, especially as new drugs with the potential to supplant FVIII prophylaxis hit the market. Future experiments will include thrombin and a thrombin inhibitor, a PAR1 agonist and antagonist, a PAR2 antagonist, and an APC neutralizing antibody. Upcoming findings will be discussed. Disclosures Taylor: Baxalta: Research Funding; CTI BioPharma: Employment, Equity Ownership.