Citations

Objective : Central nervous system (CNS) involvement is a known phenomenon of acute lymphoblastic leukemia. However little is known about the incidence of CNS involvement in acute myeloid leukemia (AML) as routine lumbar punctures are not done if patients are asymptomatic at diagnosis. Our practice has been to obtain lumbar puncture (LP) at or after complete remission (CR) for patients that present with any one of the following high risk features;hyperleukocytosis with WBC >10,000 or blastic crisis, extramedullary disease,monocytic differentiation. Methods: We reviewed our leukemia database at Mayo Clinic to determine the incidence of CNS involvement in patients with high risk features after due IRB approval. We also compared baseline demographic characteristics, blood counts, cytogenetic risk groups, presence of extramedullary disease, relapse rate ,transplant rate and overall survival (OS) among patients with and without CNS involvement. OS estimates were calculated by Kaplan-Meier curves and log-rank testing using JMP v.13. Results: 298 patients with AML were identified with presence of at least 1 risk factor for CNS involvement. The results of LP were only available for 126 (42%) patients. The overall incidence of CNS involvement was 12% (15/126, figure 1). Among the patients with positive disease 3 had CSF examination at diagnosis prior to CR due to the presence of CNS myeloid sarcoma (n=2) and ambiguous lineage myeloid leukemia and were excluded from further evaluation. The incidence of CSF involvement at CR1 was 9.7% (12/123). All patient with CNS positive disease received intrathecal chemotherapy with alternating cytarabine and methotrexate until they cleared their blast and additional 2-4 treatments as per treating physician's discretion .The CNS positive and negative patients were compared and did not show any statistical difference in age, gender, presence of extramedullary disease ,cytogenetic risk group, relapse rate and transplant rate as shown in table1. Molecular data was not available in most of the patients and hence was not included for analysis. The median OS was significantly lower among patients with CNS involvement compared to absence of CNS involvement but was not statistically significant likely due to small sample size (3.9 vs12.8 years (p=0.2), figure 2). The overall CNS relapse rate in patients who did not have a LP done at CR was 5.8%(10/172). Conclusions : CNS evaluation should be done in AML patients with high risk features before declaring CR. CNS disease portends overall a poor prognosis with impaired overall survival and high relapse rate. However the sample size was small and true incidence of CNS disease will need routine diagnostic LP in all high risk patients. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.

Introduction: Patients who develop therapy-related myeloid neoplasms (t-MN) have dismal outcomes. Previous studies reported the incidence and risk factors associated with t-MN development. Lenalidomide, in the setting of oral, but not intravenous, melphalan is associated with a higher risk of t-MN (Palumbo et. al, Lancet Oncology, 2014). We carried out this study to evaluate the clinical and pathologic features of t-MN, therapies employed, and factors that predict long-term survival after diagnosis. Patients and methods: We identified patients who received the first ASCT 1998-2016 at our institution. t-MN was defined per the WHO 2016 classification. Median overall survival (OS) was calculated from the time of t-MN diagnosis to last follow-up or death. Statistical analyses were performed using SAS (JMP v14.1) or GraphPad Prism (v7). Results: Out of 2115 patients that underwent at least one ASCT, 53 (2.5%) developed t-MN. Thirty-five of 53 (66%) patients who developed t-MN had received lenalidomide. Among 2062 patients that did not develop t-MN, 916 (44.4%) patients received lenalidomide. Lenalidomide exposure was associated with development of t-MN (χ2 with Yate's correction 8.9, p=0.003). Ten patients were excluded from further analyses due to lack of follow up. Clinical characteristics are shown in Table 1a (N=43). Median age at t-MN diagnosis was 70 years (range 44-79). Median time from ASCT to t-MN was 5 years (range 1-15). After a median follow-up of 70 months (95% CI, 38-134), the median OS was 12 months (95% CI, 9-17, Figure). Primary causes of death were t-MN (71%), MM (12%), both (6%), and other including infection, GVHD, and unknown (12%). Seven (16%) had t-AML and 36(84%) had t-MDS. Three (42%) of 7 patients with t-AML had pure erythroid phenotype. At the time of last follow-up, 9 (21%) were alive. Seven (17%) underwent two ASCT, 16 (36%) received more than 2 years cumulative dose of lenalidomide. Median number of cycles of alkylator therapy including high-dose melphalan (HDM) used for ASCT was 2 (range 1-6). On univariate analysis, factors predicting OS from t-MN diagnosis were ≥ 2 alkylator vs. < 2 cycles (11 vs. 27 months, p=0.02), ≥10% vs. <10% blast at the time of t-MN diagnosis (5.5 vs. 17 months, p=0.01), and the presence of complex karyotype (CK) vs. not (11 vs. 17 months, p=0.03). There was no difference in survival among t-MDS patients who transformed to t-AML vs. those who did not, those who received 1 vs. 2 ASCT, and those with lenalidomide duration ≥ 2 vs. <2 years. On multivariate analysis, the number of alkylator therapies and CK, but not % blasts predicted OS from t-MN diagnosis (Figure). Eight (19%) patients proceeded to receive an autologous (n=4) or allogeneic (n=4) transplant for t-MN (Table 1b). There was no survival advantage for patients who underwent SCT for t-MN, compared to those who did not (17 vs. 10 months, p=0.3). Similarly, there was no survival advantage for patients who received allo SCT compared to auto SCT (21 vs. 16 months, p=0.7). Survival for t-MDS and t-AML was also not different (15 vs. 6 months, p=0.2). There is only one long-term survivor in the transplant group (s/p allogeneic SCT). Four of seven (57%) patients died of progressive t-MN, 1 died of progressive MM, progressive MM and t-MN, and TRM each. Conclusions: Lenalidomide, in the setting of HDM, is associated with an increased risk of t-MN. Increased exposure to alkylators, a higher tumor burden, and the presence of CK predict poor survival in t-MN. As HDM/ASCT followed by lenalidomide maintenance remains the standard of care for eligible patients, minimizing exposure to other alkylator regimens may be prudent. While considered the standard, only a minority undergo transplant; and post-transplant outcomes remain poor. Studies to identify prognostic genetic and epigenetic risk factors are ongoing. Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Introduction: Germline RUNX1 mutations/deletions result in a Familial Platelet Disorder with propensity to Myeloid Malignancy (FPDMM); an autosomal dominant condition characterized by thrombocytopenia, qualitative platelet defects, and myeloid clonal evolution (MDS and AML). The advent of next generation sequencing (NGS) has allowed detection of clonal cytopenias of unclear significance (CCUS) prior to bone marrow (BM) morphological changes. Somatic RUNX1 mutations (RUNX1MT) can be seen in myeloid malignances, including MDS/MPN overlap syndromes, with an indeterminate prognostic impact. We carried out this study to define the landscape of clonal evolution in FPDMM patients and to compare germline RUNX1MT with somatic mutations. Methods After IRB approval, families with RUNX1MT FPDMM (detected by germline sequencing and deletion/duplication assays) and patients with 2016, WHO-defined MDS/MPN overlap syndromes (except JMML) were included in the study. Index patients with FPDMM underwent BM biopsies and in select cases NGS at diagnosis and then as indicated for follow up, or at disease progression. All MDS/MPN overlap syndrome patients underwent a 29-gene panel NGS assay on BM samples obtained at diagnosis by previously described methods (Patnaik et al., BCJ 2016). Results Eight index patients with FPDMM were included; median age at diagnosis 58 (range, 0-71) years, 3 (38%) males (Figure A). Four (50%) had intragenic deletions involving multiple exons in 3 cases and 2 nucleotides in 1 case, 1 (13%) each had missense, frameshift and splice site mutations, while 1 (13%) patient had a whole gene deletion (Figure C). Notably, in 4 (100%) of 4 patients with large deletions, NGS testing was negative for RUNX1 abnormalities. Four (50%) index patients presented with a myeloid neoplasm; JAK2, SRSF2, ASXL1 mutant CMML, MDS/MPN-U with trisomy 8, AML with del7q and normal karyotype AML (negative NGS). Three (38%) index patients had CCUS at diagnosis with TET2 (67%) being the most common acquired mutation, and others including SRSF2, EZH2, and SF3B1. Cumulatively, myeloid clonal evolution was seen in 7 (88%) patients. Three (38%) index patients (AML-2, CMML-1) underwent allogeneic HCT, and at last follow up were in remission. Median age at myeloid clonal evolution was 61 (range, 4-71) years; median overall survival (OS) from date of myeloid clonal evolution was 12 (range, 7-53) months. For the comparative analysis, 499 patients with MDS/MPN overlap syndromes were included; 382 (77%) with CMML, 48 (10%) with MDS/MPN-RS-T (MDS/MPN with ring sideroblasts and thrombocytosis), 17 (3%) with aCML (atypical CML), and 52 (10%) with MDS-MPN-U (unclassifiable). Median age at diagnosis was 71 (range, 18-99) years, and 329 (66%) were male. RUNX1MT were detected in 57 (11%) patients; 43 (11%) with CMML, 5 (30%) with aCML, and 9 (17%) with MDS-MPN-U. No RUNX1MT were found in MDS-MPN-RS-T. Overall, 62 RUNX1MT were identified in 57 patients, with 52 (39 CMML, 4 aCML, 9 MDS-MPN-U) and 5 (4 CMML, 1 aCML) patients harboring 1 and 2 RUNX1MT, respectively. RUNX1MT spanned the entire coding sequence without specific hotspots (Figure B): 27 (44%) were missense, 2 (3%) involved the splicing sites, 5 (8%) were nonsense and 28 (45%) were frameshift. There was only one RUNX1MT (R273K), seen in both germline and somatic patients (Figures B and C). Compared to wild type patients, RUNX1MT patients with MDS/MPN overlap syndromes had significantly lower platelet counts (median 72 vs 117 x109/L, p<0.0001), without any other phenotypic differences. In the somatic RUNX1MT cohort, co-occurring mutations included ASXL1 (54%), SRSF2 (54%), TET2 (49%), and NRAS (28%), with NRAS (28% vs 13%, p=0.0028), and SRSF2 (54% vs 39%, p=0.0218) mutations clustering with RUNX1MT. Median OS of the entire cohort was 29 (range, 0-170) months. On univariate analysis, while there was a trend for an inferior OS in RUNX1MTvs wild type patients (20 vs 29 months, p=0.0523), the leukemia-free survival was clearly inferior (p=0.0175, Figure D). Conclusion RUNX1 MT FPDMM is a germline predisposition syndrome with a high rate of myeloid clonal evolution (88%), including clonal cytopenias (38%). Germline RUNX1MT have minimal overlap with somatic mutations seen in myeloid malignancies, with NGS testing often missing RUNX1 deletions. Somatic RUNX1MT in myeloid malignancies are significantly associated with thrombocytopenia and an inferior leukemia-free survival. Figure Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders characterized by dysplasia in one or more hematopoietic cell lines, peripheral cytopenia(s), and distinct cytogenetic abnormalities. The presence of certain cytogenetic abnormalities [e.g. 7/del(7q), -5/del(5q), 13/del(13q), i(17p)], in the setting of unexplained persistent cytopenia, has been considered presumptive evidence of MDS, despite definitive morphologic features of MDS. Conversely, chromosomal abnormalities such as isolated 20q- are not considered definitive evidence for MDS, in the absence of dysplasia. Based on our previous findings, patients with isolated 20q- without morphologic features of a myeloid neoplasm have varied clinical outcomes (Jawad MD et al. Am J Hematol.2016). In this study, we investigate the frequency of recurrent somatic gene mutations in these patients, and their relationship to progression to a myeloid neoplasm. Methods We retrospectively reviewed the bone marrow pathology reports of patients evaluated at our institution from 01/2005 to 09/2015 who had isolated 20q- in at least 2 of 20 metaphases. Patients with isolated 20q- and without myeloid neoplasms were included in the study, along with medical record review. A 35-gene next generation sequencing (NGS) panel (OncoHeme) targeting myeloid neoplasms was performed on available archived bone marrow aspirate pellets. Statistical analyses were performed using JMP Pro software version 13. Parametric (t-test) or nonparametric tests such as the Fisher-exact test, chi-square test, or Wilcoxon rank-sum test, were utilized as appropriate for test variables. Statistical significance was based on a 2-sided significance level of .05. Kaplan-Meier analysis was used for analyzing time to progression to myeloid neoplasms and progression-free survival (i.e. time to progression to myeloid neoplasms or death from any cause). Results Fifty-six (42 males, 14 females) patients with isolated 20q- were identified. The average percentage of 20q- was 38.5 % (±29.3). The median age was 68 years (range: 44-90) and the median hemoglobin (Hb), absolute neutrophil count (ANC) and platelet count (Plt) were 11.7 g/dL (range: 8.3-15.1), 2.7 x 10(9)/L (range: 0.6-9.4) and 138 x 10(9)/L (range: 16-392), respectively. The indications for bone marrow biopsies ranged from cytopenia (21), multiple myeloma (20), non-Hodgkin lymphoma (10), amyloidosis (2), CLL (2) and transplant donor evaluation (1). Twenty-three patients harbored pathogenic mutations (15 had one mutation, 1 had two mutations and 7 had three mutations). The identified pathogenic mutations included ASXL1 (8), TET2 (8), SRSF2(3),PHF6 (2), SF3B1 (2), CBL (2), U2AF1 (2), IDH1 (1), IDH2 (1), BCOR (1), JAK2 (1), PTPN11(1), TP53 (1) and RUNX1 (1). Nine patients (16.1%) progressed to myeloid neoplasms (5 MDS, 2 acute myeloid leukemia, 2 chronic myeloid neoplasm-not otherwise specified) at a median follow-up of 36.7 months (range: 1.9-57.4). Of these 9 patients, 5 had one mutation (BCOR, TET2, DNM3TA, CBL, IDH2), 1 had two mutations (SF3B1/CBL) and 3 had three mutations (ASXL1/IDH1/SRSF2,DNM3TA/PTPN11/TET2, PHF6/TET2/TET2). In contrast, only 29.8% of non-progressed cases showed at least 1 mutation. Kaplan-Meier analysis demonstrated the presence of genetic mutations in patients with isolated 20q- carried significantly higher chance of progression to myeloid neoplasms (p<.05, Figure 1A and 1B). However, the number of genetic mutations was not predictive of progression in our relatively small study cohort (p=.66). There was no significant difference between those who did and did not progress with respect to age, gender, Hb, ANC, Plt, 20q- or mutational allele burden. Conclusions Most of the patients with isolated 20q- and without morphologic features of a myeloid neoplasm did not harbor myeloid neoplasm associated gene mutations and further did not develop a myeloid neoplasm during the course of our study. Among patients with pathogenic mutations, nearly one-third showed progression to a myeloid neoplasm. Of the clinical and laboratory variables, the presence of genetic mutation is a significant risk factor for progression to a myeloid neoplasm. This study revealed the underlying molecular heterogeneity of isolated 20q- in the absence of morphologic features of a myeloid neoplasm, and underscored the prognostication value of genetic mutation analysis in these cases. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: Heparin-induced Thrombocytopenia (HIT) is a potentially life and limb threatening pro-thrombotic syndrome triggered by antibodies to platelet factor 4 (PF4)/polyanion complexes. Commonly used screening tests such as the PF4/Heparin ELISA (PF4 ELISA), while easy to perform in the near-patient setting, are highly non-specific. In contrast, more accurate tests such as the serotonin release assay (SRA) are technically complex, require radioactivity and are performed in only a handful of reference laboratories. These diagnostic challenges lead to both unnecessary anticoagulant use as well as avoidable cases of severe bleeding. Recent reports suggest that a relatively simple three-hour flow cytometry-based test that uses PF4-treated normal platelets, the PEA (PF4-dependent p-selectin Expression Assay), is at least as accurate as the SRA for identifying patients who have HIT (Thromb Haemost. 2015 Nov 25;114(6):1322-3; Chest. 2016 Sep;150(3):506-15; Chest. 2017 Oct;152(4):e77-e80). Here we describe results of a large prospective study designed to compare the accuracy of the SRA and PEA for HIT diagnosis in a clinical setting. Methods: The PEA and SRA were performed on serum samples obtained from adult patients (≥18 yrs.) from 2 large tertiary care academic hospitals from May 2016 to Sep 2017. All inpatients for whom PF4 ELISA testing was ordered were included in the study. The PEA was performed with platelets pretreated with PF4 at 3 concentrations: 30mcg/mL (PEA30), 10mcg/mL (PEA10) and 3mcg/mL (PEA3). Sera were batch-tested in both assays (SRA and PEA) using the same target platelets. The central laboratory was blinded to clinical history and to the results of the PF4 ELISA performed by participating hospitals at the time of diagnosis. Both centers performed IgG-specific PF4 ELISAs and utilized manufacturer-recommended cut-offs for positivity. Predefined "more-stringent" and "less stringent" criteria based on clinical findings (4Ts score) and results of PF4 ELISA testing were used to define the likelihood of a patient having HIT (Table 1A). Receiver Operating Curve (ROC) analysis was performed and Area Under the Curve (AUC) of each assay was calculated to assess their diagnostic accuracy. Since high test specificity was desired, positive cut-offs that maximized the sum of sensitivity and specificity in a specificity range of ≥90% and ≥95% were derived. To evaluate non-inferiority of the PEA to the SRA, we assessed whether the lower limit of the one-sided 95% confidence interval of the ΔAUC (PEA-SRA) was higher than a non-inferiority margin of 5% and 10%. Results: A total of 440 patient samples were tested with both assays. Seventeen and 28 patients were classified as having true HIT based on the use of the more- and less-stringent criteria, respectively. When classified by more-stringent criteria, the AUC was numerically greater for each of the three PEA tests than for the SRA (Table 1B). All tests had excellent accuracy (AUC>0.90, "excellent" by the Traditional academic point system). Using less-stringent criteria, the PEA30 had the highest AUC estimate followed by the SRA (Table 1C). Positive cut-offs generated at test specificities of ≥90% and ≥95% using data classified by more stringent criteria are shown in Table 1D. PEA3 was non-inferior to the SRA at a 5% non-inferiority margin, and all PEA tests were non-inferior at the 10% margin using the more stringent classification for true HIT (Fig 1A). In the less-stringently classified data set, the PEA30 was non-inferior to the SRA at a 10% margin (Fig 1B). Conclusions: The PEA, a 3-hour, non-radioactive functional HIT assay demonstrated high diagnostic accuracy and was non-inferior to the SRA using commonly accepted non-inferiority margins for diagnostic testing (5-10%). The technical advantages of the PEA over the SRA may encourage its widespread adoption as a rapid-turnaround, accurate and near-patient diagnostic for patients with suspected HIT. Disclosures Jones: BloodCenter of Wisconsin: Patents & Royalties. Curtis:Ionis Pharmaceuticals: Consultancy. Bougie:BloodCenter of Wisconsin: Patents & Royalties. Aster:BloodCenter of Wisconsin: Patents & Royalties. Garcia:Shingoi: Consultancy; Retham Technologies LLC: Consultancy; Incyte: Research Funding; Daiichi Sankyo: Research Funding; Bristol Meyers Squibb: Consultancy; Portola: Research Funding; Janssen: Consultancy, Research Funding; Pfizer: Consultancy; Boehringer Ingelheim: Consultancy. Padmanabhan:BloodCenter of Wisconsin: Patents & Royalties: Patent/royalties for HIT diagnostic testing; Veralox Therapeutics: Other: Advisory Board; Retham Technologies LLC: Equity Ownership, Other: Retham seeks to develop HITDx(TM), a new assay for HIT diagnosis; TerumoBCT: Consultancy; GE Healthcare: Other: Material support for Clinical Quality Improvement Initiative; Janssen Pharmaceuticals: Consultancy.

Abstract BACKGROUND: Glanzmann's thrombasthenia (GT) is an inherited platelet disorder (IPD) that leads to clinically significant bleeding. Platelets from patients with GT can show qualitative or quantitative defects of platelet membrane glycoprotein (GP) IIb/IIIa complex. Most GT are caused by autosomal recessive genetic defects in ITGA2B and ITGB3 (genes for GPIIb and GPIIIa, respectively) with rare cases showing an autosomal dominant pattern. Accurate diagnosis of GT requires a constellation of both phenotypic and genetic studies. Here we report a unique case of autosomal dominant GT resulted from thorough phenotypic and genetic studies. PATIENTS AND METHODS: A 19 year-old woman was recently evaluated for life-long history of easy bruising and severe menorrhagia that was only partially responsive to medroxyprogesterone. She has severe thrombocytopenia first recognized shortly after birth with a platelet count around 20x109/L. Platelet size was normal. She was presumed to have immune-mediated thrombocytopenia (ITP) since early infancy, but lacking responsiveness to the conventional treatments of ITP including immunomodulation, splenectomy and thrombopoietin receptor agonists. Family history was negative for a bleeding diathesis. Both von Willebrand factor antigen and activity were within normal range. Bone marrow aspirate and biopsy with associated chromosome studies were all normal. Platelet surface glycoprotein assessment by flow cytometry using antibodies to GP IIb, IIIa, Ia, Ib-a, VI, IX, and whole exome sequencing (WES) utilizing a predefined list of 53 clinically significant genes* related to genetically IPDs were performed. Due to thrombocytopenia platelet aggregation studies could not be performed. METHODS/RESULTS: Platelet surface expression of GPIIb (CD41) and GPIIIa (CD61) were markedly decreased suggestive of a variant of GT (Figure). WES performed with Illumina HiSeq 2500 sequencing system by using Agilent SureSelelct CRE kit V1 to capture and target the exonic regions showed presence of a heterozygous mutation in ITGB3 gene (c2213T>G; p.Leu738Arg). By in silico prediction modeling, this mutation is likely to be pathogenic and results in the substitution of hydrophobic leucine with hydrophilic arginine in the transmembrane domain of the β3 subunit of alpha IIb/beta 3 integrin (αIIbβ3), the platelet receptor that binds to fibrinogen. Interestingly, GPVI is also decreased which may be associated with GPIIb and GPIIIa deficiency or due to accelerated shedding since no GPVI mutation was identified. CONCLUSION: We describe a patient with GT associated with a novel heterozygous autosomal dominant mutation in ITGB3 gene with substitution of leucine with arginine (c2213T>G; p.Leu738Arg). This deletion caused a low expression of αIIbβ3 integrin on her platelets surface and severe thrombocytopenia. This case underscores the importance of an integrated phenotyping and genotyping approach to render a definitive diagnosis of an IPD. *Platelet exome gene list: ABCG5, ABCG8, ACBD5, ACTN1, ANKRD26, ANO6, AP3B1, BLOC1S3, BLOC1S6, CYCS, DTNBP1, ETV6, FLI1, FLNA, GATA1, GFI1B, GP1BA, GP1BB, GP6, GP9, HOXA11, HPS1, HPS3, HPS4, HPS5, HPS6, ITGA2B, ITGB3, LYST, MASTL, MPL, MYH9, NBEAL2, ORAI1, PSRX1, P2RY12, PLA2G4A, PRKACG, RASGRP2, RBM8A, RUNX1, STIM1, STX11, STXBP2, TBXA2R, TBXAS1, THPO, TUBB1, UNC13D, VIPAS39, VPS33B, VPS45, WAS Figure Figure. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 3377 Background Bleeding from AVWS may occur in aortic stenosis (AS), hypertrophic cardiomyopathy (HCM), mitral regurgitation (MR), and prosthetic valve dysfunction (PVD) due to loss of von Willebrand factor (VWF) high molecular weight multimers caused by shear stress. Traditional laboratory testing for AVWS, including VWF antigen (VWF:Ag) and ristocetin cofactor activity lack sensitivity in this setting, and VWF multimer analysis, a costly and time-consuming test, must be used for confirmative diagnosis. The goal of this study is to develop a clinical and laboratory approach to predict the likelihood of AVWS, and consequently obviate the need for multimer analysis. Methods 160 patients with or without a bleeding history were prospectively enrolled with mild, moderate, or severe AS (n=65), HCM (n=32), MR (n=25), or with heart valve replacement (n=38). These patients were investigated at the time of clinically indicated echocardiography with VWF latex immunoturbidic activity (VWF:Ltx), VWF:Ag, (abnormal versus normal cutoff of VWF:Ltx/VWF:Ag is <0.80) platelet function analyzer 100 collagen ADP (PFA-CADP) and VWF multimer analysis. Single variable logistic regression analysis was performed to estimate clinical or laboratory variables that were strongly associated with the probability of abnormal multimers, including results of echocardiographic peak velocity and PFA-CADP. Associations with abnormal multimers were summarized with estimated relative risk (RR) and 95% CI. A scoring system was then created to evaluate the risk of abnormal multimers among cardiac patients. Results Overall, 50% of patients had abnormal multimers, indicating likely AVWS. The estimated probability of abnormal multimers showed a significant association between peak velocity, PFA-CADP and VWF:Lx/Ag (Table 1). Logistic regression analysis using forward selection identified only peak velocity (p<0.001) and PFA-CADP (p=0.033) as the factors showing the strongest evidence of association with abnormal multimers. VWF:Lx/Ag was no longer significant after adjusting for these two variables. Estimated relative risk of abnormal multimers for values of peak velocity and PFA-CADP were then derived (Table 2). From these relative risks a scoring algorithm was created in order to determine who should be screened for abnormal multimers. A value of 1 was subtracted from each relative risk and rounded to the nearest integer to obtain a score for peak velocity (<3 m/sec=0, 3.0–3.9 m/sec=3, 4.0–4.9 m/sec=5, >5.0 m/sec=7), and for PFA-CADP (≤120 sec=0, 121–180 sec=2, > 180 sec=3). The score from each of the two variables was added to obtain a total score ranging from 0 to 10. The risk score was categorized into four levels, which are 0, 2–3, 5–7, and 8–10, as shown in Table 3. Conclusion Both hemodynamic severity and PFA-CADP testing results could be incorporated into a scoring system to identify potential AVWS due to loss of HMWM in patients with various cardiovascular conditions. Further studies are needed to validate such a scoring system in a separate set of patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Association of acquired von Willebrand syndrome (AVWS) with various cardiovascular (CV) disorders such as cardiac valve disease and hypertrophic cardiomyopathy (HCM) is well known and documented. The mechanism is thought to be related to shear stress induced loss of high molecular weight multimers (HMWM). The gold standard test to assess for loss of HMWM is von Willebrand protein electrophoresis and then visual assessment of loss of high molecular weight bands. This is both a costly and subjective test. Ratio of von Willebrand factor activity to antigen level is useful in patients with type IIA Von Willebrand Disease caused by loss of HMW multimer, but its sensitivity to detect CV-associated AVWS is unknown. Aim Our aim was to test whether routine VWF laboratory tests could be used to predict which patients with CV conditions are going to have high molecular weight multimer loss. We also aimed to assess whether these tests could be used to predict bleeding risk in patients with CV disorders. Methods We prospectively collected laboratory data of 234 patients with cardiovascular disorders known to be associated with AVWS: aortic stenosis (66), aortic insufficiency(22), aortic and mitral valve prostheses(38), mitral valve regurgitation (51) and hypertrophic cardiomyopathy(57). All patients had Von Willebrand factor antigen (VWF:Ag), Von Willebrand factor activity by latex method (VWF:Ltx), platelet function testing via PFA-100 CADP as well as von Willebrand factor multimers tested. All patients also completed a bleeding questionnaire. We used logistic regression model to calculate the relationship between the VWF:Ltx/VWF:Ag ratio and loss of high molecular weight multimers. Same analysis was performed for PFA-100. We also tested these associations for bleeding risk. Results Mean value for VWF:Ag was 142 IU/dL, VWF:Ltx 121%, PFA-100 151 seconds and 0.86 for the VWF:Ltx/Ag ratio. Over a half of patients (56%) had VWF multimer loss noted on protein electrophoresis testing and a quarter reported bleeding on bleeding questionnaire. The ratio of VWF:Ltx to VWF:Ag had strong correlation with HMW multimer loss (p<0.001) with AUC of 0.77. Correlation with PFA-100 was even stronger with AUC of 0.83. The ratio cut off value of 0.83 had sensitivity of 60% and specificity of 83% in predicting multimer loss. With the cut off of 0.77, specificity reached 95%. With PFA 100 value of 118 seconds, specificity was 76% and sensitivity was 80%. Increasing the cut off to 198 seconds improved the specificity to 95%. The association with bleeding was present for PFA-100 (p=0.01), but did not exist for the Ltx/Ag ratio. Conclusions PFA-100 CADP as well as VWF:Ag and VWF:Ltx can be used to detect acquired Von Willebrand disease in patients with cardiovascular disorders and may decrease the need for costly and time consuming testing of multimers. PFA-100 CADP also correlates with the bleeding risk in these patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Platelet dense granule (DG) deficiencies, including Hermansky-Pudlak syndrome, are relatively common and frequently under-diagnosed hereditary platelet hypofunctional disorders. Among various laboratory tests, platelet whole mount (WM) transmission electron microscopy (TEM) is considered the gold standard method for diagnosing DG deficiencies. Nevertheless, platelet DG TEM testing is still largely a research tool and has not been thoroughly validated and standardized as a clinical test. Furthermore, reference ranges (RR) of DG count/platelet (DGC) have not been adequately established. Aim Our goals were to validate and standardize platelet WM TEM testing and establish reference ranges for platelet DGC. Methods Based on previously established methods (Blood, 33:598-606), we first optimized and standardized various pre-analytical, analytical and post-analytical procedures including sample matrix, sample stability, platelet-rich plasma (PRP) preparation, platelet mounting procedure and DG counting criteria. Then whole blood samples in ACD-B tubes were collected from a total of 128 healthy donors (64 males and 64 females). Platelet DGC, percentage of platelets without any DG (empty platelets), platelet count (PC), mean platelet volume (MPV) and immature platelet fraction (IPF) were measured. In addition, platelet aggregometry analyses were performed on citrated PRP samples from the same donors. Standard statistical analyses were employed in this study. Results DGC and platelet ultra-structure were stable when whole blood samples were stored at room temperature (RT) for up to 4 days. Reproducible DGC and adequate preservation of platelet ultrastructure depended on an optimized mounting method. The DG counting criteria were developed based on previous publications and guidance from Dr. James G. White. Using the same WM TEM images and DG counting criteria, agreements among different technologists (n=5) improved from 60% to 95%. Finally, DGC reference range study using 128 healthy donor samples (age range 18, 72 years) and scoring 100-200 platelets from each donor showed that the DGC distribution of each donor sample was left-skewed with a median of 15% of empty platelets (range: 3, 40). The median DGC was 2.6 with a range of 1.0—5.1. Reference ranges established at the lower 95th percentile for DGC empty platelets is ≤ 31% (95% CI 28, 34) and for the mid-95th percentiles of DGCs between 1.5 and 4.0. DGCs were weakly associated with MPV and IPF (ρ between 0.22 and 0.28); and inversely associated with the percentage of empty platelets (ρ between -0.26 and -0.25). DGC showed no significant association with PC, platelet maximum aggregation or primary slope of aggregation, nor age or gender. Conclusion We validated platelet WM TEM procedures and for the first time established an adult reference range for platelet DGC. The results of association analyses implied that immature platelets may have slightly more DGs. Disclosures: No relevant conflicts of interest to declare.

Abstract Background : Pre-treatment determinants of survival in adult acute myeloid leukemia (AML) include performance status, age, karyotype, the distinction between primary and non-primary AML and FLT3-ITD/NPM1 mutational status. Current literature on AML natural history and risk factors is often based on information derived from stringent protocol studies. In the current series of 1,123 adult patients seen at the Mayo Clinic between 2004 and 2017, we examined long-term survival specified by a number of primarily established risk factors. Methods: Diagnosis of AML and its sub-classification was according to World Health Organization criteria. Study subjects were recruited from the Mayo Clinic AML database. Conventional response criteria were used for CR and CRi assignment; the latter met all criteria for CR with the exception of platelet count <100 x 109/l or absolute neutrophil cunt <1 x 109/l. Standard statistical methods were used for analysis of overall survival, calculated from the initial diagnosis of AML to date of death or last follow-up; analyses were performed both in the absence and in the presence of censoring of survival at time of allogenic stem cell transplant (ASCT). The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: 1,123 adult patients (median age 65 years, range 18-94; 61% males) with non-APL AML were considered; 61% were age >60 years. 351 (31%) patients were seen between 2004 and 2009 and 772 (69%) between 2010 and 2017. AML subcategories were primary (56%), post-myelodysplastic syndromes (post-MDS; 24%), therapy-related (10%), post-myeloproliferative neoplasm (post-MPN; 8%) and post-MDS/MPN (3%). Cytogenetic risk distribution, according to the European LeukemiaNet classification, was favorable in 47 (4%), intermediate in 650 (58%) and adverse risk in 426 (38%) patients. FLT3, NPM1 and CEBPA mutation information was available in 462 (24% mutated), 393 (27% mutated) and 228 (11% mutated) patients, respectively. Treatment included intensive chemotherapy (IC) in 766 (68%) patients, less aggressive chemotherapy, including the use of hypomethylating agents in 144 (13%) and supportive care alone in 213 (19%) patients. ASCT was utilized in 258 (23%) cases, almost all performed after achieving CR/CRi. Complete remission (CR) and CR with incomplete count recovery (CRi) were listed in 333 (30%) and 248 (22%) patients, respectively; the corresponding rates in IC-treated patients were 43% and 32%. Almost all cases of CR/CRi occurred in IC-treated patients; 2 (1.4%) CRs were listed for less aggressive chemotherapy. After a median follow-up of 11 months, 798 (71%) deaths were recorded. Median survival for all 1,123 patients was 14 months with 1-, 3- and 5-year survival rates of 54%, 29% and 23%, respectively; a slight but significant (p=0.01) improvement in survival was apparent in more recent years (Figure 1a). Figures 1b, 1c and 1d depict analysis stratified by AML subcategories, treatment received and response obtained, respectively. Similarly, figures 2a, 2b and 2c depict analysis stratified by karyotype, use of ASCT and age. Multivariable analysis of pre-treatment parameters evaluable in all 1,123 patients identified age >60 years (HR 2.2, 1.9-2.6), adverse karyotype (HR 2.9, 1.9-4.9), intermediate-risk karyotype (HR 1.6, 1.02-2.6), post-MPN AML (HR 1.9, 1.5-2.4) and other secondary AML (HR 1.3 (1.1-1.6), as risk factors for shortened survival; the inclusion in the model of FLT3/NPM1 mutational status in 392 informative cases confirmed the adverse prognostic effect of age >60 years (HR 1.8, 1.4-2.5), adverse karyotype (HR 3.7, 1.4-15.3), post-MPN AML (HR 2.8, 1.6-4.6), other secondary AML (HR 1.4, 1.0-1.9) and FLT3+NPM1- (HR 2.8, 1.6-4.9) and FLT3-NPM1- (HR 1.7, 1.2-2.7) profile. Results were unchanged when survival was censored at time of ASCT. Additional prognostic interaction with treatment strategy and ASCT is further elaborated in an accompanying abstract to be presented. Conclusions: The current study, the largest coming from a single institution, provides practically useful information that should assist with patient consultation on AML prognosis and treatment. The study confirms the primary prognostic importance of age, karyotype and FLT3/NPM1 mutational status, in real-life practice. Novel observations included comparable value of CRi vs CR and the particularly worse prognosis associated with post-MPN AML. Disclosures Al-Kali: Novartis: Research Funding.