ALBERT

Description: Controversy exists regarding management of children newly diagnosed with immune thrombocytopenic purpura (ITP). Drug treatment is usually administered to prevent severe hemorrhage, although the definition and frequency of severe bleeding are poorly characterized. Accordingly, the Intercontinental Childhood ITP Study Group (ICIS) conducted a prospective registry defining severe hemorrhage at diagnosis and during the following 28 days in children with ITP. Of 1106 ITP patients enrolled, 863 were eligible and evaluable for bleeding severity assessment at diagnosis and during the subsequent 4 weeks. Twenty-five children (2.9%) had severe bleeding at diagnosis. Among 505 patients with a platelet count less than or equal to 20 000/mm3 and no or mild bleeding at diagnosis, 3 (0.6%), had new severe hemorrhagic events during the ensuing 28 days. Subsequent development of severe hemorrhage was unrelated to initial management (P = .82). These results show that severe bleeding is uncommon at diagnosis in children with ITP and rare during the next 4 weeks irrespective of treatment given. We conclude that it would be difficult to design an adequately powered therapeutic trial aimed at demonstrating prevention of severe bleeding during the first 4 weeks after diagnosis. This finding suggests that future studies of ITP management should emphasize other outcomes.

Description: Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and while most of the 4,000 new cases of pediatric ITP diagnosed each year self-resolve, 10% of these patients have major bleeding episodes. The platelet count remains the mainstay method for predicting hemorrhage and unfortunately has only proven to be loosely correlated to bleeding severity. Consequently, there currently exists no biomarker that accurately and reliably predicts which patients need immediate medical treatment, all of which have side effects, and which patients only require monitoring. To that end, we leveraged our Platelet Contraction Cytometer (PCC), a versatile system that measures platelet contraction forces at the single-cell level and at high-throughput, to study platelets of patients with ITP prospectively. Buchanan bleeding scores were used to distinguish patients with severe symptoms from asymptomatic patients. With 49 patients, we observed two significant findings: 1) Tracking both single platelet force measurements and platelet count enables stratifying patients into having major, minor, or no risk for bleeding. Accordingly, patients in the major risk category have a combined low platelet force and low platelet count. 2) Longitudinal studies showed that when major risk patients had increased platelet force or higher platelet counts, this was associated with the alleviation of major symptoms. Thus, when utilized together, platelet force and platelet count can more accurately predict bleeding severity in pediatric ITP patients. Platelet Contraction Cytometry: Our PCC utilizes a large array of fibrinogen "microdot" pairs patterned on a flat hydrogel of known stiffness. Thrombin-activated platelets adhere to these microdots, spread to the neighboring microdot, and contract, pulling the microdots closer together. As platelet force is directly proportional to the microdot displacement, only a single microscopy measurement is necessary to determine the force applied by each single platelet (Fig 1). As such, hundreds of platelets are measured in a controlled mechanical and biochemical environment (Myers et al, 2017) Results: With our cohort of 49 ITP patients, we observed that low average single cell platelet contraction forces highly correlate with severe bleeding symptoms (Buchannan scores 2-4). Using a regression tree, we found that an average force of 26nN best separates symptomatic from asymptomatic patients with a diagnostic sensitivity of 100% and specificity of 86.8% (AUC 0.97, 95% CI: 0.9361-1). However, platelets from some asymptomatic patients exerted forces of less than 26nN as well. In a more thorough examination, we found that majority of those asymptomatic patients with low platelet forces had platelet counts 〉 40,000/uL. Although our data shows that platelet count alone is a poor predictor of severe bleeding symptoms (AUC 0.8, 95% CI: 0.6757-0.9189), when a platelet count

Description: Abstract 3681 Background: Pediatric Immune Thrombocytopenia (ITP) has an incidence of 4–6/100,000 with 1/3 of cases becoming chronic. Treatment choice is arbitrary, because few studies are powered to identify predictors of therapy response. Increasingly, rituximab is becoming a treatment of choice in those refractory to other therapies (Neunert CE, et al. Pediatr Blood Cancer 2008; 51(4):513). Previous studies in ITP have not examined predictors of response to rituximab or whether response to prior treatments predicts response. Objective: To evaluate univariate and multivariable predictors of platelet count response to rituximab. Methods: After local IRB approval, 550 patients with chronic ITP enrolled in the longitudinal, North American Chronic ITP Registry (NACIR) between January 2004 and June 2010. Eligibility included: ages 6 months-18 years at ITP diagnosis, clinical diagnosis of ITP, and ITP duration 〉6 months. Primary ITP was defined as isolated thrombocytopenia without associated conditions. Secondary ITP included those patients with immune thrombocytopenia associated with other immune-mediated medical conditions, including Evans Syndrome. Treatment response was defined as a post-treatment platelet count ≥50,000/uL within 16 weeks of rituximab and within 14 days of steroids. Steroids were prescribed as 1–4 mg/kg prednisone or adult equivalent over 4–14 days with or without taper. The NACIR captured treatment responses both retrospectively prior to enrollment and then prospectively, and both periods were included in this analysis. The multivariable logistic regression modeling process utilized SAS 9.1 using binary variables which were either significant in the univariate analysis or clinically important. A backwards elimination procedure was used to select the final model. Results: Seventy-six (13.8%) patients were treated with rituximab. Demographics of the patients treated with rituximab include: 42% male; 81% Caucasian, 17% Black, and 2% Asian. The mean age at diagnosis of ITP was 8.4 ± SD 5.1 years. The median platelet count at diagnosis of acute ITP was 10,000/uL (IQR 5,000-20,000/uL). 19 (25%) patients had secondary ITP or Evans syndrome. Treatment with rituximab had an overall response rate of 63.2% (48/76). Univariate predictors of response to rituximab are shown in Table I. The strongest univariate predictor of response to rituximab was response to steroids. Gender, ethnicity, and race were not predictive of response to rituximab. Furthermore, other variables which did not predict rituximab response include: history of a bleeding score ≥3 (Buchanan and Adix, J Pediatr 2002; 141: 683), symptoms ≥1 month prior to ITP diagnosis, older age (age 〉5 years), platelets ≥20,000/uL at acute ITP diagnosis, and a positive ANA. In multivariable analysis, response to steroids remained a strong predictor of response to rituximab with an OR 6.2 (95% CI 1.8–21.3, p=0.004). Secondary ITP also remained a strong a predictor of a positive response to rituximab with an OR 5.9 (95% CI 1.2–33.3, p=0.03). Conclusion: In the NACIR, response to steroids and secondary ITP were strong predictors of response to rituximab, a finding not previously reported in children or adults. Although this finding requires further validation, this result may provide evidence that rituximab should be most considered in patients previously responsive to steroids. Disclosures: Off Label Use: Rituximab for chronic ITP. Lambert:Cangene: Membership on an entity's Board of Directors or advisory committees. Klaassen:Novartis: Research Funding; Cangene: Research Funding. Neufeld:Novartis, Inc: Research Funding.

Description: Background: The indications for treating children with immune thrombocytopenia (ITP) remain controversial. A valid and reliable bleeding assessment tool could assist in objectively quantifying bleeding and influence treatment decisions. Both the ITP Bleeding Score (IBLS) and the ITP-Bleeding Assessment Tool (BAT) have been developed to assess bleeding severity in ITP. The IBLS scores bleeding severity using an 11-item tool with grading from 0 to 2 and the 18-item BAT grades from 0 to 3 or 4. The BAT includes some types of bleeding not represented on the IBLS, such has intramuscular hematomas. To date, no data describe how these two measures compare when measuring bleeding associated with ITP. Objective: To describe and compare bleeding as assessed by both the IBLS and BAT and to correlate bleeding severity with platelet counts in a cohort of children with ITP. Methods: A longitudinal observational cohort of children ages 〉1 and 〈 18 years with ITP, were enrolled from 2013-2015 in the Pediatric ITP Consortium of North America ICON1 trial. All children were enrolled prior to starting a new second line monotherapy (not IVIG, steroids or anti-D). At enrollment, bleeding was assessed using the IBLS in all children. A subset of children also underwent a BAT assessment. Grades of bleeding were described and compared between tools and agreement in grading was assessed. Severity was correlated with platelet count using Spearman's correlation calculation. Results: 118 children were enrolled from 21 ICON centers. 54% had chronic ITP and the median age was 11.4y (range 1.2-17.8). The mean platelet count was 28 x 109/l (SD 57) and 88% had a baseline platelet count of

Description: Introduction: The phase 3 Kids B-LONG study (NCT01440946) demonstrated the safety, efficacy, and pharmacokinetics of prophylactic recombinant factor IX Fc fusion protein (rFIXFc) for the prevention and treatment of bleeding episodes in children with severe hemophilia B. The ongoing rFIXFc extension study B-YOND (NCT01425723) is evaluating long-term safety and efficacy of rFIXFc in children and adults with hemophilia B. The safety and efficacy data for pediatric subjects from the second B-YOND interim data cut (11 Sep 2015) are reported here. Methods: The Kids B-LONG study enrolled males aged

Description: Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Specifically, using high-throughput platelet contraction cytometry (PCC) measurements of individual platelet forces (Myers et al, 2017), we found that low platelet forces strongly correlate with bleeding symptoms. Unlike existing tests of platelet function that use bulk measurements, our test operates at the single cell level and therefore does not depend on the platelet count, enabling a direct comparison of individual platelet function in health versus disease states. In the broad context of overall function, our findings of impaired force generation agree with previous research demonstrating that impaired platelet function, and not low platelet count, correlates with bleeding in ITP patients (Frelinger et al, 2015). In addition, as the first demonstration of a correlation between single cell force and disease, our novel approach may represent an entirely new class of "physics-based" diagnostics. Platelet Contraction Cytometry (PCC): Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force is calculated from a single fluorescence image of the platelet. Using microfabrication technology, thousands of microdots are created on a single device to enable high-throughput measurements in tightly controlled mechanical, biochemical, and shear microenvironments (Fig 1). Here, gel-filtered platelets from patients are activated (1U/mL thrombin), plated on the device with a moderately stiff surface (75 kPa), and measured as described previously (Myers et al, 2017). Results: In a cohort of ITP patients (n = 27), we observed that patients with bleeding and/or bruising symptoms exhibited significantly lower average platelet contraction forces than asymptomatic patients regardless of platelet count. Using an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. From a mechanistic perspective, our preliminary data also suggests that mean platelet volume does not correlate with platelet force or function, although further studies on a single platelet level are needed to confirm this result. While this data indicates that patients with bleeding symptoms have platelets with low forces, it is unclear whether low forces correlate with bleeding or whether the patient always has had low platelet forces that therefore render them susceptible to bleeding. Our preliminary prospective data suggests that low platelet forces correlate with the bleeding symptoms themselves and return to higher values when bleeding symptoms cease. Importantly, as our approach enables "single-cell" examinations of platelet forces, we identified different platelet subpopulations unique to ITP. Healthy individuals have a distribution of contractile forces with a single peak, while ITP patients tend to have two prominent peaks, one with a lower contractile force and one with a higher one. Surprisingly, patients with bleeding symptoms only have a low force peak, suggesting that the lack of highly contractile platelets may be a biophysical biomarker for bleeding (Fig 2). Ongoing studies are focused on using this finding to further improve specificity as well as elucidating the mechanistic underpinnings of low platelet contraction in ITP. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Anti-GPIbα antibodies exert a pulling force on platelet GPIbα by crosslinking platelets under shear flow. A mechanical feature of an anti-GPIbα antibody, rather than affinity or epitope, determines ability to induce Fc-independent clearance.

Description: Key Points Severe thrombocytopenia is rare and major hemorrhage is uncommon in children with persistent and chronic ITP. In children with persistent or chronic ITP, there is a trend toward reserving drug therapy for those experiencing significant bleeding.

Description: Genome-wide chemical mutagenesis screens in the zebrafish(Danio rerio) have led to the identification of novel genes affecting vertebrate erythropoiesis. In determining if this approach could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in the zebrafish kidney is similar to that in human bone marrow. Zebrafish neutrophils resembled human neutrophils, possessing segmented nuclei and myeloperoxidase-positive cytoplasmic granules. The zebrafish homologue of the human myeloperoxidase (MPO) gene, which is specific to cells of the neutrophil lineage, was cloned and used to synthesize antisense RNA probes for in situ hybridization analyses of zebrafish embryos. Granulocytic cells expressing zebrafishmpo were first evident at 18 hours after fertilization (hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for identifying zebrafish mutants with defects in granulopoiesis. The expression of zebrafish homologues was also examined in 2 other mammalian hematopoietic genes, Pu.1, which appears to initiate a commitment step in normal mammalian myeloid development, andL-Plastin, a gene expressed by human monocytes and macrophages. The results demonstrate a high level of conservation of the spatio-temporal expression patterns of these genes between zebrafish and mammals. The morphologic and molecular genetic evidence presented here supports the zebrafish as an informative model system for the study of normal and aberrant human myelopoiesis.

Description: Background: Children with ITP who are prescribedsecond line treatments vary in terms of their clinical phenotype, prior treatments, and health related quality of life (HRQoL). Objective: To describe the clinical characteristics and HRQoL of North American pediatric patients with ITP initiating second line treatments. Methods: A longitudinal observational cohort of 118 children with ITP starting second line treatments was enrolled from 2013-2015 at 21 ICON centers. Enrollment requirements included age 1-17y and starting a second line treatment (not IVIG, steroids or anti-D) as monotherapy. Baseline demographic and clinical characteristics were recorded, including response to prior treatments, worst bleeding scores, and baseline platelet counts. Fisher's exact test was used to compare treatment with phase of ITP, age cohort, and gender. HRQoL was measured by patient/caregiver report using the Kids ITP Tool (KIT) where 0 is worst and 100 is best, while physicians assessed the perceived effect of ITP on patient HRQoL using a 5-point scale. Spearman correlations were used to test for association between bleeding, HRQoL, and duration of ITP. ANOVA was used to compare the mean KIT scores of the treatment groups. Results: The clinical characteristics of the cohort are shown in the Table. Median age at enrollment was 11.4 y and 15% had newly diagnosed ITP, 31% had persistent ITP, and 54% had chronic ITP. The median number of prior treatments was 3 (range: 1-9). The prior response rate (platelet 〉30 x 109/µL and no bleeding) to prednisone was 59% and IVIG 66%. Fifty-five (47%) patients had received at least one prior second line treatment. At enrollment, physicians reported that ITP had impacted the patients' HRQoL severely in 15%, significantly in 44%, moderately in 38%, and almost not at all in 3%. The mean score of the child KIT report was 71.4 (SD 17.2), the parent proxy KIT was 64.7 (SD 16.4), and the parent impact KIT was 36.1 (SD 19.2). The physician's assessment of the patient's HRQoL significantly correlated with the child report (p