ALBERT

Description: Background Thrombosis is a well-described complication of L-asparaginase (asp) treatment in patients with acute lymphoblastic leukemia (ALL), with an incidence of 5-10%. Although asp-associated thrombi can occur anywhere, most are venous and 50% of venous thromboses occur in the central nervous system (CNS); 18% of these are associated with cerebral infarction or stroke. Ninety percent of thrombotic events occur early in the treatment of ALL, during induction and consolidation, consistent with the predominant use of asp during these phases of therapy. Morbidity and mortality have been reported in as many as 50% of patients. Although the mechanism by which asp causes thromboses is thought to be inhibition of protein synthesis resulting in decreased levels of plasma coagulation factors, specific patterns of plasma coagulation factors (including decreases in antithrombotic proteins such as ATIII, protein S, C, and components of the fibrinolytic pathway) have not been good predictors of thrombosis. Risk factors for CNS thrombosis include older age at diagnosis and high risk (HR) disease. Although quantification of proteins in the cerebrospinal fluid (CSF) has been used to identify markers of other diseases, CSF proteomics has not been studied in ALL. We hypothesize that changes in some proteins in the CSF will occur after asp, will be more prominent in patients on HR ALL protocols than in those on standard risk (SR) protocols, and will anticipate CNS thrombosis. In this study, we pilot feasibility and describe serial proteomic analysis of CSF in newly diagnosed patients with ALL or lymphoblastic lymphoma (LL). Methods Sequential patients, ages 0-30 years, with B-cell ALL, T-cell ALL, or LL diagnosed August 2012-August 2013 at the University of North Carolina and treated according to risk stratified Children’s Oncology Group protocols and the US Intergroup C10403 protocol for adolescents and young adults were offered participation. All patients were on a treatment protocol which utilized PEG-asp on Day 4 of therapy. Following consent approved by our Institutional Review Board, 1 mL of CSF was collected at the time of scheduled lumbar punctures (LP) on Days 0, 8, and 29 of induction. Samples were centrifuged at 1500rpm for 10 minutes within an hour of LP, stored in 50mcL aliquots at -800C. and transported on dry ice to the Duke Proteomics Core Facility. Because of cost ($1000/sample), 5 patients (15 samples) were selected for initial study, including the one patient who had a CNS thrombosis on day 22 of consolidation. Thawed batched duplicate samples were immunodepleted using a MARS-14 LC column (Agilent) and Agilent 1000 HPLC to remove high abundance plasma proteins, and quantitative MS analysis was performed in a label-free fashion using one-dimensional liquid-chromatography tandem mass spectrometry on a Synapt G2 HDMS system (Waters Corporation). Data Analysis was performed in Rosetta Elucidator v3.3 (Rosetta Biosoftware). Results Samples were obtained on each of 22 patients (11 male, 11 female). Characteristics of the 5 patient samples set chosen for analysis are shown in Table 1. 636 proteins were identifiable in each sample including several that have potential roles in coagulation. Thirteen proteins met a cut-off of ±1.5-fold change and p

Description: Despite recent advancements, approximately 50% of patients with acute myeloid leukemia (AML) do not respond to induction therapy (primary induction failure, PIF) or relapse after

Description: Background:Polycythemia is the most common adverse effect of testosterone replacement therapy (TRT) and may predispose patients to adverse vascular events. Current Canadian guidelines recommend regular laboratory monitoring and discontinuing TRT or reducing the dose if the hematocrit exceeds 54% (hemoglobin ≥180 g/L). This threshold has been interpreted by some physicians and patients to indicate the need for phlebotomy or blood donation while on TRT. Study Design and Methods: We reviewed all male blood donors in Southwestern Ontario at Canadian Blood Services from December 2013 to March 2016 who self-identified or were found on donor screening to be using TRT in any form. Hemoglobin concentration was measured at the time of donation or clinic visit and with each subsequent appointment in repeat donors. Results:We report a case series of 39 patients on TRT who presented for blood donation over a two-year period. The mean hemoglobin at all donor clinic visits was 173 g/L (range 134-205 g/L, n = 108). Hemoglobin concentrations of ≥180 g/L (calculated hematocrit ≥54%) were measured at 25% of appointments. Of the 27 repeat donors, 12 (44%) had persistently elevated hemoglobin levels (≥180 g/L) at subsequent donations. Conclusions: Hemoglobin concentrations were elevated in blood donors on TRT, with a significant number above levels recommended by current guidelines. These data also suggest that repeat blood donation was insufficient to maintain hematocrit below 54%. Our findings raise concerns about persistent risk of vascular events in these donors, particularly when coupled with the misperception by patients and health care providers that donation has abrogated the risks of TRT-induced polycythemia. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Although recent studies have refined the classification of B-progenitor and T-lineage acute lymphoblastic leukemia into gene-expression based subgroups, a comprehensive integration of significantly mutated genes and pathways for each subgroup is needed to understand disease etiology. Methods: We studied 2789 children, adolescents and young adults (AYA) with newly diagnosed B-ALL (n=2,322 cases) or T-ALL (n=467) treated on Children's Oncology Group (n=1,872) and St. Jude Children's Research Hospital trials (n=917). The cohort comprised childhood NCI standard-risk (41.8%; age range 1-9.99 yrs, WBC ≤ 50,000/ml), childhood NCI high-risk (44.5%; age range ≥10 to 15.99 yrs) and AYA (9.9%; age range 16-30.7 yrs). Genomic analysis was performed on tumor and matched-remission samples using whole transcriptome sequencing (RNA-seq; tumor only; n=1,922), whole exome sequencing (n=1,659), whole genome sequencing (n=757), and single nucleotide polymorphism array (n=1,909). Results: For B-ALL, 2104 cases (90.6%) were classified into 26 subgroups based on RNA-seq gene expression data and aneuploidy or other gross chromosomal abnormalities (iAMP21, Down syndrome, dicentric), deregulation of known transcription factors by rearrangement or mutation (PAX5 P80R, IKZF1 N159Y), or activation of kinase alterations (Ph+, Ph-like). For T-ALL, cases were classified into 9 previously described subtypes based on dysregulation of transcription factor genes and gene expression. In 1,659 cases subject to exome sequencing (1259 B-ALL, 405 T-ALL) we identified 18,954 nonsynonymous single nucleotide variants (SNV) and 2,329 insertion-deletion mutations (indels) in 8,985 genes. Overall, 161 potential driver genes were identified by the mutation-significance detection tool MutSigCV or by presence of pathogenic variants in known cancer genes. Integration of sequence mutations and DNA copy number alteration data in B-ALL identified 7 recurrently mutated pathways: transcriptional regulation (40.6%), cell cycle and tumor suppression (38.0%), B-cell development (34.5%), epigenetic regulation (24.7%), Ras signaling (33.0%), JAK-STAT signaling (12.0%) and protein modification (ubiquitination or SUMOylation, 5.0%). The top 10 genes altered by deletion or mutation in B-ALL were CDKN2A/B (30.1%), ETV6 (27.0%), PAX5 (24.6%), CDKN1B (20.3%), IKZF1 (17.6%), KRAS (16.5%), NRAS (14.6%), BTG1 (7.5%) histone genes on chromosome 6 (6.9%) and FLT3 (6.1%), and for T-ALL, CDKN2A/B (74.7%), NOTCH1 (68.2%), FBXW7 (21.3%), PTEN (20.5%) and PHF6 (18.2%) (Figure 1A). We identified 17 putative novel driver genes involved in ubiquitination (UBE2D3, UBE2A, UHRF1, and USP1), SUMOylation (SAE1, UBE2I), transcriptional regulation (ZMYM2, HMGB1), immune function (B2M), migration (CXCR4), epigenetic regulation (DOT1L) and mitochondrial function (LETM1). We also observed variation in the frequency of genes and pathways altered across B-ALL subtypes (Figure 1B). Interestingly, alteration of SAE1 and UBA2, novel genes that form a heterodimeric complex important for SUMOylation, and UHRF1 were enriched in ETV6-RUNX1 cases. Deletions of LETM1, ZMYM2 and CHD4 were associated with near haploid and low hypodiploid cases. Deletion of histone genes on chromosome 6 and alterations of HDAC7 were enriched in Ph+ and Ph-like ALL. Mutations in the RNA-binding protein ZFP36L2 were observed in PAX5alt, DUX4 and MEF2D subgroups. Genomic subtypes were prognostic. ETV6-RUNX1, hyperdiploid, DUX4 and ZNF384 ALL were associated with good outcome (5-yr EFS 91.1%, 87.2%, 91.9% and 85.7%, respectively), ETV6-RUNX1-like, iAMP21, low hyperdiploid, PAX5 P80R and PAX5alt were associated with intermediate outcome (5-yr EFS 68.6%, 72.2%, 70.8%, 77.0% and 70.9%, respectively), whilst KMT2A, MEF2D, Ph-like CRLF2 and Ph-like other conferred a poor prognosis (55.5%, 67.1%, 51.5% and 62.1%, respectively). TCF3-HLF and near haploid had the worst outcome with 5-yr EFS rates of 27.3% and 47.2%, respectively. Conclusions: These findings provide a comprehensive landscape of genomic alterations in childhood ALL. The associations of mutations with ALL subtypes highlights the need for specific patterns of cooperating mutations in the development of leukemia, which may help identify vulnerabilities for therapy intervention. Disclosures Gastier-Foster: Bristol Myers Squibb (BMS): Other: Commercial Research; Incyte Corporation: Other: Commercial Research. Willman:to come: Patents & Royalties; to come: Membership on an entity's Board of Directors or advisory committees; to come: Research Funding. Raetz:Pfizer: Research Funding. Borowitz:Beckman Coulter: Honoraria. Zweidler-McKay:ImmunoGen: Employment. Angiolillo:Servier Pharmaceuticals: Consultancy. Relling:Servier Pharmaceuticals: Research Funding. Hunger:Jazz: Honoraria; Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Mullighan:Amgen: Honoraria, Other: speaker, sponsored travel; Loxo Oncology: Research Funding; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel.

Description: Thrombin is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. Thrombin activates platelets mainly through protease-activated receptor 1 (PAR1), PAR4, and glycoprotein Ib. Because adenosine diphosphate and thromboxane A2 have been shown to cause platelet aggregation by concomitant signaling through Gq and Gipathways, we investigated whether coactivation of Gq and Gi signaling pathways is the general mechanism by which PAR1 and PAR4 agonists also activate platelet fibrinogen receptor (αIIbβ3).  A PAR1-activating peptide, SFLLRN, and PAR4-activating peptides GYPGKF and AYPGKF, caused inhibition of stimulated adenylyl cyclase in human platelets but not in the presence of either Ro 31-8220, a protein kinase C selective inhibitor that abolishes secretion, or AR-C66096, a P2Y12 receptor–selective antagonist; α-thrombin–induced inhibition of adenylyl cyclase was also blocked by Ro 31-8220 or AR-C66096. In platelets from a P2Y12 receptor–defective patient, α-thrombin, SFLLRN, and GYPGKF also failed to inhibit adenylyl cyclase. In platelets from mice lacking the P2Y12 receptor, neither α-thrombin nor AYPGKF caused inhibition of adenylyl cyclase. Furthermore, AR-C66096 caused a rightward shift of human platelet aggregation induced by the lower concentrations of α-thrombin and AYPGKF but had no effect at higher concentrations. Similar results were obtained with platelets from mice deficient in the P2Y12. We conclude that (1)thrombin- and thrombin receptor-activating peptide–induced inhibition of adenylyl cyclase in platelets depends exclusively on secreted adenosine diphosphate that stimulates Gi signaling pathways and (2) thrombin and thrombin receptor-activating peptides cause platelet aggregation independently of Gi signaling.

Description: The coagulation system is activated in sickle cell disease (SCD) and acute vaso-occlusion may heighten hypercoagulability. Protein C, a natural anticoagulant, has been reported to be low in individuals with SCD. Therefore, the natural anticoagulation pathway may be disrupted in SCD. The objective of this study is to more fully evaluate the protein C pathway in murine and human SCD by examining levels of: coagulation activation; protein C activity; thrombomodulin (TM); and endothelial protein C receptor (EPCR). In order to assess the level of activation of the coagulation system, we measured plasma thrombin/antithrombin (TAT) complex levels in humans and mice. TAT levels were elevated in 22 humans with SCD versus 9 healthy controls at baseline, and levels increased further in 15 individuals with SCD during acute vaso-occlusive events (5.6±1.2 vs. 2.4±0.2 vs. 9.2±1.8ug/L respectively, p=0.02). In order to study acute vaso-occlusive events in mice, we developed a model of acute vaso-occlusion by exposing Berkeley SCD mice to 3 hours of hypoxia (FI02 8–10%) followed by 2, 4, or 21 hours of reoxygenation in room air (HR2, HR4, HR21). In support of our human findings, TAT was elevated in SCD mice compared to HbA mice at baseline, and increased further in SCD mice exposed to HR2 (n=5–14 per group, p

Description: Introduction: Adolescent and young adult (AYA) patients (〉16 years of age) with high-risk B acute lymphoblastic leukemia (HR B-ALL) have inferior outcomes compared to HR B-ALL patients 1-15 years of age, primarily due to relapse and toxicity. In a prior Children's Oncology Group (COG) HR B-ALL study 1961 (1996 - 2002), 12.7% of patients were AYA (ages 16 - 21 years) with 5-year event-free survival (EFS) and overall survival (OS) of 71.5% and 77.5% respectively. Here we report the outcomes of the most recently completed HR B-ALL COG study AALL0232, comparing AYA and younger patients. Methods: COG study AALL0232 was a Phase 3 randomized trial for patients 1-30 years of age with newly diagnosed HR B-ALL utilizing a 2 x 2 factorial design with an augmented intensityBerlin-Frankfurt-Münster (BFM) backbone. Patients were randomized to two weeks of dexamethasone versus four weeks of prednisone during Induction therapy and high dose methotrexate (HD-MTX) versus escalating Capizzi methotrexate plus pegaspargase (C-MTX) during Interim Maintenance I. Between 2004 and 2011, 3,154 patients enrolled, with 3,081 eligible and evaluable for induction. AYA patients comprised 20% (16-21 years, n= 558; 22-30 years, n=47). Results: The study was amended in 2008 due to an excess incidence of osteonecrosis observed in patients older than 10 years of age who were randomized to dexamethasone. Thereafter, they were nonrandomly assigned to prednisone during induction. The dexamethasone delivered during delayed intensification was also rescheduled from continuous (days 1-14) to discontinuous (days 1-8 and 15-22) delivery. 5-year EFS and OS were 65.1% and 76.9% for AYA patients compared to 77.9% and 87.1% for younger patients (p

Description: Background: As many as 30-50% of patients diagnosed with Thrombotic Thrombocytopenic Purpura (TTP) either relapse or become refractory to therapeutic plasma exchange (TPE). There remains a pressing need to evaluate novel treatments for these patients. Rituximab is a chimeric anti-CD20 monoclonal antibody that has demonstrated efficacy potentially by eliminating active B-lymphocytes and production of ADAMTS 13 inhibitor considered central to the pathogenesis of TTP. Intervention: We conducted a prospective multi-center phase II open-label trial in Canadian hospital-based apheresis units to evaluate the efficacy of rituximab in the management of adult patients with relapsed or refractory TTP. Functional and antigenic ADAMTS13 enzyme, inhibitor levels and anti-CD 20 were measured at initiation, 8, 12, 24 and 52-weeks as well as routine hematologic parameters, complete remission, rate of relapse, overall mortality and adverse events. Rituximab was administered intravenously once weekly for a total of 4 doses at 375 mg/m2 in eligible consenting patients. Results: Twenty refractory and 20 relapsing TTP patients were enrolled (N=40). A complete response (CR) was defined by a platelet count 〉150 x 109/L, an LDH

Description: Introduction: Despite a well-established risk of chronic kidney disease (CKD) in 20-30% of patients undergoing AlloHCT, the benefits of treating serious infections, such as cytomegalovirus (CMV), with nephrotoxic drugs often outweigh this risk. Given a lack of consensus on the optimal management of post-transplant Human-Herpes Virus 6 (HHV6) reactivation, our center has taken an aggressive stance toward screening for and treating HHV-6 viremia with foscarnet, a nephrotoxic drug with an unknown impact on long-term renal function. Methods: To clarify the impact of foscarnet exposure on long-term renal function after transplant, we conducted a retrospective cohort study of all adult patients who underwent AlloHCT at Duke from June 2002 - Feb 2016 (n=997). HHV6 viral loads were checked weekly for the first 90 days after umbilical cord blood and haploidentical transplants, and as clinically indicated (e.g. cytopenias, encephalopathy) in others. Foscarnet treatment for HHV6 viremia was given at the physician's discretion. Data were abstracted with a web-based clinical research query tool and manual chart review. Estimated glomerular filtration rate (eGFR) was calculated with the CKD-EPI equation based on repeated measures of serum creatinine (Cr) values at baseline, 90 days (n=839), 6 months (n=720), and 12 months (n=491) after transplant. Acute Kidney Injury (AKI) and Acute Kidney Failure (AKF) were defined as 2 and 3x baseline Cr, or 〉50% and 〉75% decrease in eGFR, respectively. Multivariate logistic regression was used to estimate the association of foscarnet exposure on the probability of 〉30% decline in eGFR at 90 days, 6 months, and 12 months after adjustment for confounders. Results: Of the 997 patients included in the study, 45% (n=448) were treated with foscarnet. Patients treated with foscarnet were slightly older (median age 52yrs vs. 49yrs), less likely to receive myeloablative conditioning, and more likely to be CMV positive, receive an alternative donor graft (umbilical cord blood or haploidentical), and experience acute GvHD. The most frequent indications for treatment were CMV (n=257, 57.4%) and HHV6 (n=140, 31.3%). In the first 90 days post-transplant, when most patients were treated with foscarnet, patients exposed vs. unexposed had similar rates of AKI/AKF: AKI 59.2% vs. 59.2%; p=0.99; AKF 26.1% vs. 27.3%; p=0.67. There was no difference in eGFR at 90 days, but patients treated with foscarnet had significantly lower eGFRs at 6 months and 12 months (Figure 1). There was a significant difference in the decline in eGFR from baseline to 12 months: median -29.1 (mL/min/1.73m2) (interquartile range (IQR) -50.8 to -10.7) vs. -22.2 (-37.4 to -7.4); p=0.002. After adjustment for age, race, acute and chronic GVHD, conditioning regimen, donor type, treatment with alemtuzumab, and HHV6 status, patients treated with foscarnet were more likely to experience a 〉30% decrease in eGFR from baseline to 12 months compared to those who were not (Odds Ratio 1.8 (95% CI 1.11-2.93); p=0.02). In this multivariate model, acute and chronic GVHD were not significant predictors of eGFR decline at 12 months. Unadjusted median survival was 11.9 months (95% CI; 10.1-14.0 months) and 20.8 months (95% CI; 15.8-25.4 months) for patients treated vs. not treated with foscarnet, respectively (p30% in eGFR are strongly associated with a 10-year risk of end-stage renal disease and mortality in 〉60% and 50% of patients in the general population, respectively, (Coresh, et al. JAMA 2014), this information should be considered as one weighs the risks vs. benefits of treating HHV6 viremia following AlloHCT. Disclosures Horwitz: Gamida Cell: Research Funding.