ALBERT

Description: Abstract 3565 Introduction: Birinapant is a small molecule mimetic of Smac that potently and specifically antagonizes multiple inhibitors of apoptosis proteins (IAPs). Birinapant rapidly degrades cIAPs and enables cytokines (TNFα, TRAIL) to activate the extrinsic apoptosis pathway, while it rapidly turns off the canonical NF-κB survival pathway, causing cancer cell death. Preclinical studies using adult cancer models have shown that birinapant causes tumor regressions as a single agent in selected models and that it has potent antitumor activity when combined with chemotherapies and death receptor ligands. Methods: Birinapant was evaluated against the 23 cell lines of the PPTP in vitro panel (including 1 AML and 5 ALL lines) using 96 hour exposure at concentrations from 1.0 nM to 3.0 μM, both as a single agent and in combination with TNFα (10 ng/mL) or TRAIL (10 ng/mL). Birinapant was tested against 2 PPTP solid tumor xenografts (rhabdomyosarcoma, Rh30; Ewing, CHLA-258), an anaplastic large cell lymphoma (ALCL) xenograft (Karpas-299), and 3 B-precursor ALL xenografts (ALL-2, ALL-17, & ALL-19) at a dose of 30 mg/kg administered by the intraperitoneal route using a Q3 day × 5 schedule. Gene expression data for the PPTP cell lines and xenografts was available using both Affymetrix U133 Plus 2.0 and Agilent SurePrint G3 arrays. Results: Birinapant demonstrated limited single agent activity (median relative IC50 (rIC50) 〉 3 μM), with only the AML cell line Kasumi-1 showing Relative In/Out% (Rel I/O%) values 〈 0% with rIC50 of 37 nM. Marked potentiation of birinapant was observed for a subset of cell lines with the addition of TNFα or TRAIL. The 5 ALL cell lines showed a median rIC50 value of 3.6 nM for birinapant in combination with TNFα, with Rel I/O% values between −50% and −100% (indicative of a profound cytotoxic effect). Four of 5 ALL cell lines showed little or no potentiation of birinapant effect with the addition of TRAIL. Among solid tumor cell lines, potentiation of birinapant effect was observed for selected rhabdomyosarcoma, rhabdoid tumor, Ewing sarcoma, and neuroblastoma cell lines with the addition of either TNFα or TRAIL. Birinapant was well tolerated in vivo. Birinapant induced significant differences in event-free survival (EFS) distribution compared to control in 3 of 3 (100%) of the B-precursor ALL xenografts, but in none of the solid tumor or ALCL xenografts. Objective responses were not observed for the solid tumor and ALCL xenografts, whereas for the ALL panel one xenograft (ALL-17) achieved a complete response (CR) and another (ALL-2) achieved a maintained CR, with treated animals remaining in remission at day 42, approximately 30 days after their last treatment with birinapant. Given the mechanism of action of Smac mimetics, TNFα expression was examined. TNFα expression was significantly higher for the PPTP ALL xenografts compared to the PPTP solid tumor xenografts and to 15 normal tissues. TNFα expression in ALL clinical specimens was examined using publicly available datasets, with the observation that its expression is significantly higher for high-risk B-precursor ALL compared to a set of normal tissues, but with a wide range of TNFα expression among ALL cases. Lymphotoxin A and B also show significantly elevated expression in ALL compared to normal tissues. Among the ALL xenografts tested with birinapant, the best responding xenograft (ALL-2) showed the highest TNFα expression. Karpas-299 also showed high TNFα expression, but the two solid tumor xenografts did not. Unlike the ALL cell lines for which exogenous TNFα potentiated birinapant in vitro activity, exogenous TNFα did not potentiate birinapant in vitro activity against Karpas-299. Conclusion: Birinapant showed little single agent in vitro activity against ALL cell lines, though it markedly potentiated the activity of exogenously added TNFα for these cell lines. In vivo, birinapant showed remission-inducing activity against 2 of 3 ALL xenografts, with one of these showing a maintained CR. TNFα is mechanistically associated with the activity of Smac mimetics, and the initial PPTP in vivo data for ALL xenografts are consistent with a relationship between TNFα expression and responsiveness to birinapant. The PPTP results suggest that birinapant may show high level activity against a subset of childhood ALL, and additional in vivo testing is ongoing to better identify predictive markers that can reliably select responsive cases. Disclosures: Chunduru: TetraLogic Pharmaceuticals: Employment, Equity Ownership. Graham:TetraLogic Pharmaceuticals: Employment, Equity Ownership. Geier:TetraLogic Pharmaceuticals: Honoraria. Houghton:TetraLogic Pharmaceuticals: Honoraria.

Description: The macrophage colony-stimulating factor receptor and several other hematopoietic growth factor receptors induce the tyrosine phosphorylation of a 145- to 150-kD protein in murine cells. We have previously cloned a cDNA for the murine 150-kD protein, SHIP, and found that it encodes a unique signaling intermediate that binds the SHC PTB domain through at least one tyrosine phosphorylated (NPXY) site in the carboxyl-terminal region. SHIP also contains several potential SH3 domain-binding sites, an SH2 domain for binding other tyrosine phosphorylated proteins, and an enzymatic activity that removes the phosphate from the 5 position of phosphatidylinositol 3,4,5-phosphate or from inositol 1,3,4,5-phosphate. SHIP has a negative effect on cell growth and therefore loss or modification may have profound effects on hematopoietic cell development. In this study, we have cloned a cDNA for human SHIP and examined mRNA and protein expression of SHIP and related species in bone marrow and blood cells. Flow cytometry indicates that at least 74% of immature CD34+ cells express SHIP cross-reacting protein species, whereas within the more mature population of CD33+ cells, only 10% of cells have similar expression. The majority of T cells react positively with the anti-SHIP antibodies, but significantly fewer B cells are positive. Immunoblotting detects up to seven different cross-reacting SHIP species, with peripheral blood mononuclear cells exhibiting primarily a 100-kD protein and a CD34+ acute myeloblastic leukemia expressing mainly 130-kD and 145-kD forms of SHIP. Overall, these results indicate that there is an enormous diversity in the size of SHIP or SHIP-related mRNA and protein species. Furthermore, the expression of these protein species changes according to both the developmental stage and differentiated lineage of the mature blood cell.

Description: Most patients requiring allogeneic bone marrow transplant (allo-BMT) do not have an HLA-matched sibling donor. A phenotypically matched unrelated donor graft has been made available for approximately 50% of Caucasians and less than 10% of ethnic and racial minorities in need. However, almost all patients have a readily available partially mismatched related donor (PMRD). We summarize our experience with 72 patients who ranged from 1 to 50 years of age (median, 16 years) and who were recipients of a PMRD allo-BMT from haploidentical family members following conditioning therapy using total body irradiation (TBI) and multiagent, high-dose chemotherapy. T-cell depletion and post-BMT immunosuppression were combined for graft-versus-host disease (GVHD) prophylaxis. The probability of engraftment was 0.88 at 32 days. Six of 10 patients who failed to engraft achieved engraftment after secondary transplant. Grade II to IV acute GVHD was seen in 9 of 58 (16%) evaluable patients; extensive chronic GVHD was seen in 4 of 48 (8%) evaluable patients. There was a statistically significant difference in 2-year survival probability between low-risk and high-risk patients (0.55 v 0.27, P = .048). Prognostic factors that affected outcomes in multivariate analysis were (1) a lower TBI dose and 3-antigen rejection mismatch decreased stable engraftment (P = .005 and P = .002, respectively); (2) a higher T-cell dose increased acute GVHD (P = .058); (3) a higher TBI dose increased chronic GVHD (P = .016); and (4) a high-risk disease category increased treatment failure from relapse or death (P = .037). A PMRD transplant can be performed with acceptable rates of graft failure and GVHD. Using sequential immunomodulation, the disease status at the time of transplant is the only prognostic factor significantly associated with long-term successful outcome after PMRD allo-BMT. When allogeneic rather than autologous BMT is indicated, progression in disease status before transplant can be avoided using a PMRD with equal inclusion of all ethnic or racial groups.

Description: Introduction: TRALI is acute lung injury occurring during or within hours of a blood transfusion. The etiology is thought to be infusion of leukocyte antibodies or neutrophil priming and activation caused by biologically active lipids in blood components. We report a TRALI reaction associated with fresh frozen plasma (FFP) and activation of complement in both the unit of FFP and the patient at the time of the reaction. Case History: A 59 year old male with factor XI was admitted to the hospital with hematochezia and given 3 units of FFP. During infusion of the third unit, he developed dyspnea and cyanosis requiring ventilator and O2 support. A chest x-ray showed bilateral diffuse pulmonary infiltrates, CVP was 3 mm Hg, and an echocardiogram was normal. The symptoms resolved in 3 days. Methods: Samples from donors and/or units were screened for the presence of HLA antibodies by ELISA and lymphocytotoxicity and antibodies detected were typed for HLA specificity and antibody class. Reactivity was determined by flow crossmatch. Serologic and molecular HLA typing was completed on donor and patient samples. Priming activity of the implicated FFP, fresh plasma from donor and recipient, and plasma from controls was completed against freshly isolated neutrophils from the three sources. Significant activity was defined as 〉1.5 times the fMLP stimulated superoxide anion (O2−) production. C3aLE, C4aLE, SC5b-9, and Bb were determined by standard techniques. Results: HLA antibodies were only detected in the third unit of FFP. Samples from this unit and the donor exhibited HLA Class I and II reactivity by ELISA but not lymphocytotoxicity. Flow crossmatch cells demonstrated Class II, IgG reactivity of donor serum against recipient DR11, 13. No autologous reactivity was demonstrated. The FFP unit primed the fMLP response in donor, recipient and control neutrophils 2.6, 3.1, and 3.4 fold above baseline. Testing of donor, recipient and control plasma obtained 3 months after the reaction showed no priming against the same battery of cells (priming ratio 0.8–1.3). C4aLE (105%, control range 24–176%); C3aLE (476%, control range 21–180%); and Bb (351%, control range 31–169%) were elevated in recipient samples obtained during the TRALI reaction and SC5b-9 was at the high end of normal (164%, control 0–200%). These returned to normal after the reaction. Strikingly, evidence of complement activation was seen in the FFP unit (C4aLE 214%, C3aLE 402%, C5b-9 213%) but not in subsequent samples from the donor. Conclusion: These studies document a TRALI reaction with symptoms expressed during the administration of FFP. One unit exhibited HLA Class I and II antibodies, the latter of which bound to the recipient’s cells. Priming activity was seen with plasma from the implicated unit, not in subsequent samples from the donor. Laboratory studies document activation of complement in the FFP infused but not donor samples. Plasma from the recipient at the time of the reaction also exhibit activation of complement which became normal after the TRALI resolved. Infusion of the FFP with activated complement capable of priming neutrophils may have induced pulmonary leukostasis and TRALI quite distinct from any subsequent effect of antibodies. Although the cause of FFP complement activation is not defined, these results suggest alternative mechanisms involving complement may be responsible for HLA antibody-associated TRALI.

Description: This article reports metabolic consequences of JAK2-mutant myeloproliferative neoplasms (MPNs) with a therapeutic translational impact: expression of mutant JAK2 induces abnormal metabolic activity of MPN cells, resulting in hypoglycemia, adipose tissue atrophy, and early mortality.

Description: Introduction: Although it is known that children with nephrotic syndrome (NS) are at greater risk for certain complications, the frequency of these complications and predisposing risk factors are poorly defined. In particular, nephrotic syndrome has long been considered a hypercoagulable state. Risk for development of venous thromboembolism (VTE) is known to be increased in the setting of an active infection. The objective of this study was to determine the prevalence of infection and VTE among a cohort of hospitalized children with NS, and the association of these complications on outcomes. Methods: Records of hospitalized children with NS admitted to any of 17 participating pediatric hospitals across North America from 2010-2012 were included. Data including demographics, clinical pattern of NS, renal biopsy results, number of hospitalizations, nephrotoxic medication usage, infection and VTE history were recorded. Descriptive statistics were used to determine prevalence of infection and VTE. Wilcoxon rank sum was used to perform comparisons between groups. Logistic regression analysis was utilized to determine predictors of VTE development. Results: Seven-hundred thirty hospitalizations occurred among 370 children. One-hundred forty-eight children (40%) had at least 1 infection with a total of 211 infectious episodes; 11 (3%) had VTE. Those with infection were more likely to have VTE (p = 0.0457). Infections associated with VTE were C. difficile (1 subject), methicillin sensitive S. Aureus (2), Streptococcus pneumoniae (1), and unknown (3). There were no differences between those with and without infection regarding gender or ethnicity. Those with infection were younger at NS diagnosis (3.0 vs. 4.0 years; p = 0.008), and steroid resistant NS was more highly associated with infection than all other clinical diagnoses (steroid-sensitive NS, steroid-dependent NS, other) (p = 0.003). The most common types of infections encountered included peritonitis (23%), pneumonia (22%), and bacteremia (16%). Bacterial pathogens (Streptococcus pneumoniae 41%, Escherichia coli 16%, Clostridium difficile 10%) were most commonly identified. Children with VTE, infection, or both, also required significantly more days in hospital. The median hospital stay for those without infection was 5 days vs. 10 in those with infection (p〈 0.0001). Similarly, median hospital days for those without VTE were 6 days as compared to 22 in those with VTE (p 〈 0.0001). Of those with infection, 13% had an ICU stay compared with 3.3% of those without. Those with VTE also had a median of 4 days in the intensive care unit as compared to 0 days in those without VTE (p 〈 0.0001). In a logistic regression analysis, only the number of ICU days was predictive of the presence of VTE (OR 1.074, 95% CI 1.013 - 1.138). Conclusions: Children with NS who are hospitalized have high rates of infection. While the rate of VTE was not high in this cohort, presence of VTE was associated with infection. Both infection and VTE were associated with longer hospitalizations and intensive care unit stays. Streptococcus pneumoniae remains the most commonly identified bacterial pathogen in children with nephrotic syndrome, though methicillin sensitive S. Aureus was identified in 2 of 11 patients with VTE. Further studies are needed to identify potentially modifiable risk factors that could minimize these complications in this already high risk population. Disclosures No relevant conflicts of interest to declare.

Description: Most patients requiring allogeneic bone marrow transplant (allo-BMT) do not have an HLA-matched sibling donor. A phenotypically matched unrelated donor graft has been made available for approximately 50% of Caucasians and less than 10% of ethnic and racial minorities in need. However, almost all patients have a readily available partially mismatched related donor (PMRD). We summarize our experience with 72 patients who ranged from 1 to 50 years of age (median, 16 years) and who were recipients of a PMRD allo-BMT from haploidentical family members following conditioning therapy using total body irradiation (TBI) and multiagent, high-dose chemotherapy. T-cell depletion and post-BMT immunosuppression were combined for graft-versus-host disease (GVHD) prophylaxis. The probability of engraftment was 0.88 at 32 days. Six of 10 patients who failed to engraft achieved engraftment after secondary transplant. Grade II to IV acute GVHD was seen in 9 of 58 (16%) evaluable patients; extensive chronic GVHD was seen in 4 of 48 (8%) evaluable patients. There was a statistically significant difference in 2-year survival probability between low-risk and high-risk patients (0.55 v 0.27, P = .048). Prognostic factors that affected outcomes in multivariate analysis were (1) a lower TBI dose and 3-antigen rejection mismatch decreased stable engraftment (P = .005 and P = .002, respectively); (2) a higher T-cell dose increased acute GVHD (P = .058); (3) a higher TBI dose increased chronic GVHD (P = .016); and (4) a high-risk disease category increased treatment failure from relapse or death (P = .037). A PMRD transplant can be performed with acceptable rates of graft failure and GVHD. Using sequential immunomodulation, the disease status at the time of transplant is the only prognostic factor significantly associated with long-term successful outcome after PMRD allo-BMT. When allogeneic rather than autologous BMT is indicated, progression in disease status before transplant can be avoided using a PMRD with equal inclusion of all ethnic or racial groups.

Description: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion death. We hypothesize that TRALI requires 2 events: (1) the clinical condition of the patient and (2) the infusion of antibodies against MHC class I antigens or the plasma from stored blood. A 2-event rat model was developed with saline (NS) or endotoxin (LPS) as the first event and the infusion of plasma from packed red blood cells (PRBCs) or antibodies (OX18 and OX27) against MHC class I antigens as the second event. ALI was determined by Evans blue dye leak from the plasma to the bronchoalveolar lavage fluid (BALF), protein and CINC-1 concentrations in the BALF, and the lung histology. NS-treated rats did not evidence ALI with any second events, and LPS did not cause ALI. LPS-treated animals demonstrated ALI in response to plasma from stored PRBCs, both prestorage leukoreduced and unmodified, and to OX18 and OX27, all in a concentration-dependent fashion. ALI was neutrophil (PMN) dependent, and OX18/OX27 localized to the PMN surface in vivo and primed the oxidase of rat PMNs. We conclude that TRALI is the result of 2 events with the second events consisting of the plasma from stored blood and antibodies that prime PMNs.

Description: We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and show bias for differentiation towards megakaryocytes (Mk). Mouse models of myeloproliferative neoplasms (MPN) expressing JAK2-V617F (VF) or a JAK2 exon 12 mutation (E12) displayed increased frequencies and percentages of the CD41hi versusCD41lo HSCs compared to wildtype controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from MPN patients. CD41hi HSCs produced higher numbers of Mk-colonies HSC in single cell cultures in vitro, but showed reduced long-term reconstitution potential compared to CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, while CD41lo HSCs showed higher gene expression of interferon, JAK/STAT and TNFα/NFkB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in MPN patients. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the numbers of JAK2-V617F positive HSCs in mice and patients with MPN. The shift towards the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.