ALBERT

Description: Key Points Complete genome sequence analysis of 40 DLBCL tumors and 13 cell lines reveals novel somatic point mutations, rearrangements, and fusions. Recurrence of mutations in genes involved in B-cell homing were identified in germinal center B-cell DLBCLs.

Description: Key Points The DNAM-1 adhesion and costimulatory pathway promotes GVHD via effects on regulatory T cells. Effective GVL can still occur in the absence of DNAM-1, making the pathway an attractive therapeutic target.

Description: Attachment of polyubiquitin to substrate proteins generates important biological signaling cues that are inherent to the linkage type of the polyubiquitin chain. For example, K48-linked polyubiquitin chains result in proteasome-mediated degradation of proteins to which they are attached, whereas K63-linked polyubiquitin chains play roles in various intracellular signaling cascades. An important feature of protein ubiquitination is that it is reversible. Substrate-anchored chains may be edited or removed from proteins by highly specialized proteases called deubiquitinating enzymes (DUBs). There are approximately 90 DUBs identified in humans and many have been identified as potential druggable targets because of their involvement in hematological malignancies such as Fanconi Anemia, human follicular lymphomas and diffuse large B-cell lymphomas. DUB activity is regulated by a variety of cues including specificity for a protein substrate(s) to which polyubiquitin chains are conjugated; the presence of protein cofactor(s) that activate or inhibit DUB function; or preference for a specific polyubiquitin linkage type(s). Thus, understanding the mechanisms, regulation, and substrate preferences for deubiquitinases is of great interest, from both academic and clinical viewpoints. To help address these needs, we produced highly purified recombinant deubiquitinase enzymes to facilitate in vitro biochemical studies and drug-discovery efforts. Herein we report the initial characterization of the following, clinically-relevant enzymes: In conclusion, we have investigated the in vitro substrate preferences and kinetic profiles of three deubiquitinases of clinical relevance. This data will be valuable in the design and analysis of assays used to identify small-molecule inhibitors of these highly specialized proteases. Disclosures: Russell: Boston Biochem Inc: Employment. Taylor:Boston Biochem Inc: Employment. Brasher:Boston Biochem Inc: Employment. Melandri:Boston Biochem Inc: Employment.

Description: Introduction Acquired hemophilia A (AHA) is a rare bleeding disorder, resulting from auto-antibodies to human factor VIII (hFVIII). The challenges created by the management of AHA and the co-morbidities present in this typically elderly population, can be managed by a recombinant, highly pure, B-domain deleted, porcine sequence FVIII (OBI-1) that is not generally susceptible to the inhibitory activity of anti-human FVIII antibodies. Treatment with OBI-1 allows for monitoring of FVIII levels which provides a reproducible and objective surrogate predictor of hemostasis. Eradication of hFVIII inhibitors with immunosuppressive therapy is critical for disease management. During immunosuppression, the patient transitions from a bleeding state at initial presentation to a relative hypercoagulable state which can be an issue in patients who are susceptible to thromboembolic events due to their comorbidities. This transition period is of most concern especially when using traditionally utilized bypassing agents that cannot be monitored. OBI-1 enables measurement of FVIII levels, guiding dosing and enhancing treatment safety during this critical period. Methods This global, prospective, multi-center phase 2/3 open label clinical trial investigates the efficacy and safety of OBI-1 in the treatment of serious bleeds in adults with AHA conducted under ICH guidelines and local IRB/Ethics Committee oversight. Primary efficacy endpoint was assessed at 24 hours (eg. effective, partially effective). All subjects (N= 18) presented with a serious bleed and were treated with an initial dose of OBI-1 (200 U/kg), followed by additional doses based on the subject's target factor VIII levels, anti-OBI-1 titer, and clinical factors. Results In all 18 subjects, a positive response (14 effective/4 partially effective) to treatment was observed at 24 hours. This positive response to OBI-1 treatment was seen by 8 hours in 14/18 of the subjects and at 16 hours in 16/18 of the subjects. Median total exposure to OBI-1 per subject was 1782.5 U/kg. The median total first dose was 14,000 U. For subjects who received additional doses of OBI-1, the median dose was reduced from the initial dose, but did not differ considerably over subsequent doses (9180 to 13561 U; median 11000 U). The majority of subjects (17/18) received concomitant immunosuppressive therapies. No related serious adverse reactions occurred. Non-serious adverse events related to treatment were noted in 5/18 (27.8%) subjects. One subject had mild tachycardia, hypotension and constipation. One subject had 2 instances of mild PICC line occlusion. One subject had a mild hypofibrogenemia. All of these adverse effects completely resolved. Three subjects developed anti-porcine inhibitors after infusion of study drug (range 8-108 BU) and two were discontinued from treatment. Anti-porcine inhibitors were detected prior to infusion in 6/18 patients (range 0.8-29 BU). All of these subjects had a favorable clinical response at 24 hours post-OB-1 infusions. Conclusions Data from this prospective study demonstrate OBI-1 as a safe and effective treatment of bleeding episodes in patients with AHA, with the added advantage over other bypass therapies of allowing FVIII monitoring throughout treatment and healing phase. Disclosures: Kruse-Jarres: Baxter Healthcare: Consultancy; Bayer HealthCare: Consultancy; Biogen IDEC: Consultancy; Grifols: Consultancy; Kedrion: Consultancy; Novo Nordisk: Consultancy. St. Louis:CSL Behring: Research Funding; Octapharma: Consultancy, Research Funding; Baxter: Consultancy; Novo Nordisk: Honoraria. Shapiro:Kedrion Biopharma: Consultancy; Chugai Pharma USA: Consultancy; Biogen IDEC: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Bayer HealthCare: Membership on an entity’s Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Baxter Healthcare: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Chowdary:Baxter Healthcare: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Travel grant Other; Novo Nordisk: Honoraria, Research Funding, Travel grant, Travel grant Other; Bayer HealthCare: Honoraria, Travel grant, Travel grant Other; Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Travel grant, Travel grant Other; CSL Behring: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Travel grant Other; Biogen IDEC: Honoraria, Travel, Travel Other. Drebes:Octapharma: Travel grant Other; CSL Behring: Travel grant, Travel grant Other; Leo-pharma: Travel grant, Travel grant Other; Bayer Healthcare: Consultancy, Honoraria. Gomperts:Baxter Healthcare: Consultancy; Asklepios Biopharmaceutoicals Inc: Consultancy; Cangene Inc: Consultancy. Chapman:Baxter Healthcare: Employment. Mo:Baxter Healthcare: Employment. Novack:Baxter Healthcare: Employment. Farin:Baxter Healthcare: Employment.

Description: Background Idelalisib (IDELA) is a first-in-class, selective, oral inhibitor of PI3Kδ that reduces proliferation, enhances apoptosis, and inhibits homing and retention of malignant B cells in lymphoid tissues. Phase 1 trials demonstrated that IDELA is highly active as a single agent or in combination with rituximab (R) in heavily pretreated patients (pts) with CLL. Pts in these trials experienced reductions in disease-associated chemokines, improvement of organomegaly and cytopenias, profound reductions in lymphadenopathy, and durable clinical benefit with an acceptable safety profile (Brown 2013; Barrientos 2013). Patients with early progression and significant co-morbidities have limited treatment options; single-agent rituximab is an option in these pts (NCCN 2013; Zelenetz 2013). Methods This Phase 3 study evaluated the efficacy and safety of IDELA + R vs placebo + R in pts with previously treated CLL. Eligibility criteria included the need for treatment per IWCLL guidelines, measurable lymphadenopathy, and CLL progression 6), renal dysfunction, or cytopenias due to poor marrow reserve. All pts received R at 375 mg/m2 [1st dose] and then 500 mg/m2q2 wks x 4, followed by q4 wks x 3 [8 doses total]) and were randomized to Arm A (n=110; IDELA 150 mg BID continuously) or Arm B (n=110; placebo BID continuously). Primary endpoint was progression-free survival (PFS). Response and progression in both arms were assessed by an independent review committee using standard criteria (Hallek 2008; Cheson 2012). Results were reviewed by an external Data Monitoring Committee (DMC). Results Results are from a pre-specified interim analysis after ∼50% of the total number of 119 planned events of CLL progression or death from any cause. Data cutoff was 30 Aug 2013. Pt characteristics (n=220) included a median age of 71 yrs (78% ≥ 65 yrs); CIRS 〉 6 in 85%; median creatinine clearance of 63.6 mL/min; and presence of anemia (73%), thrombocytopenia (61%), neutropenia (34%). Median time since diagnosis was 8.5 yrs, median number of prior therapies was 3 (range: 1-12), 44% had del(17p)/TP53 mutation, and 84% had unmutated IGHV. PFS in the IDELA + R arm was superior to placebo +R (HR [95% CI] = 0.15 [0.08, 0.28]; p = 3.0 x 1011). Median PFS of pts treated with IDELA + R was not reached and for placebo + R was 5.5 mos. At 24 wks, the PFS rate for IDELA +R was 93% compared to 46% for placebo + R. PFS strongly favored IDELA + R in all subgroups, including those with del(17p)/TP53 or unmutated IGHV. Pts treated with IDELA + R and with ≥1 post-baseline assessment also had a superior overall response rate (ORR) relative to those in the control arm (81% vs. 13%; odds ratio 29.9; p = 3.0 x 1019) and a higher lymph node response (LNR) rate (93% vs. 4%; odds ratio 264.5; p = 1.3 x 10-30). Relative to the control group, pts treated with IDELA +R also had a significant improvement in overall survival (OS): HR (95% CI) = 0.28 (0.09, 0.86), p = 0.018. Adverse events (AEs) occurring in ≥20% of pts (any Gr/Gr ≥3) by arm were: pyrexia (IDELA + R 29%/3%; placebo + R 16%/1%), fatigue (IDELA + R 24%/3%; placebo + R 27%/2%), nausea (IDELA + R 24%/0%; placebo + R 22%/0%), chills (IDELA + R 22%/2%; placebo + R 16%/0%), infusion-related reactions (IDELA + R 16%/0%; placebo + R 28%/4%), and cough (IDELA + R 15%/0%; placebo + R 25%/2%). Other selected AEs (any Gr/Gr ≥3) included diarrhea (IDELA + R 19%/4%; placebo + R 14%/0%) and rash (IDELA + R 10%/2%; placebo + R 6%/0%). Select lab abnormalities (any Gr/Gr ≥3) included ALT elevation (IDELA + R 31%/6%; placebo + R 9%/1%), anemia (IDELA + R 26%/6%; placebo + R 30%/14%), neutropenia (IDELA + R 55%/34%; placebo + R 49%/22%), and thrombocytopenia (IDELA + R 17%/10%; placebo + R 26%/16%). The most common SAEs were pneumonia (6.4%), pyrexia (6.4%), and febrile neutropenia (4.5%) in IDELA + R, and pneumonia (8.4%), febrile neutropenia (5.6%), and dyspnea (3.7%) in placebo + R. AEs led to study drug discontinuation in 9 pts (8.2%) in IDELA + R and 11 pts (10.3%) in placebo + R. Based on a review of efficacy and safety, the DMC recommended stopping the study early. Conclusions IDELA + R demonstrated statistically significant improvement with acceptable safety over placebo + R in PFS, ORR, LNR and OS in heavily pretreated pts with relapsed CLL, including those with adverse genetic features. Disclosures: Furman: Gilead Sciences: Research Funding. Sharman:Gilead Sciences: Consultancy, Research Funding. Coutre:Gilead Sciences: Research Funding. Cheson:Gilead Sciences: Research Funding. Pagel:Gilead Sciences: Research Funding. Hillmen:Gilead Sciences: Research Funding. Barrientos:Gilead Sciences: Research Funding. Zelenetz:Gilead Sciences: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Kipps:Gilead Sciences: Research Funding. Flinn:Gilead Sciences: Research Funding. Ghia:Gilead Sciences: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Eradat:Gilead Sciences: Research Funding. Ervin:Gilead Sciences: Research Funding. Lamanna:Gilead Sciences: Research Funding. Hallek:Gilead Sciences: Research Funding. Coiffier:Gilead Sciences: Research Funding. Pettitt:Gilead Sciences: Research Funding. Ma:Gilead Sciences: Research Funding. Stilgenbauer:Gilead Sciences: Honoraria, Research Funding. Aiello:Gilead Sciences: Employment. Johnson:Gilead Sciences: Employment, Equity Ownership. Miller:Gilead Sciences: Employment, Equity Ownership. Li:Gilead Sciences: Employment. Jahn:Gilead Sciences: Employment. Dansey:Gilead Sciences: Employment, Equity Ownership. O'Brien:Gilead Sciences: Research Funding.

Description: Background Hepatosplenic T-cell lymphoma (HSTL) is a rare form of lymphoma, comprising less than 1% of the cases. However, HSTL extracts a highly disproportionate toll on patients with a median age of diagnosis of 35 years and an expected median survival of less than two years. The vast majority of HSTL patients eventually succumb to their disease. The genetic basis of the disease is largely unknown. Although abnormalities of chromosome 7, including isochromosome 7q occur commonly in the disease, the role of specific genes and genetic mutations to the disease remains essentially unknown. Methods In this study, we sought to define the genetic features of HSTL through the whole genome sequencing and exome sequencing of 32 HSTL tumors and germline DNA (where available) from the same patients. Exome enrichment of DNA was carried out using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes as well as ∼700 human microRNAs. Both whole genome and exome sequencing were performed using the Illumina platform. Results We identified 28 candidate cancer genes that were recurrently mutated in HSTL. Commonly implicated biological processes comprising these genes included signal transduction (e.g. PIK3CD, KRAS) and chromatin modification (e.g. TET1, SETD2 and MLL3), accounting for 16% and 23% of the total genetic events, respectively. Nearly all of these genes have been implicated in HSTL for the first time and provide new insights into the pathogenesis of the disease and potential targets for therapy. Whole genome sequencing confirmed isochromosome 7q as the most common recurrent chromosomal abnormality in HSTL and additional structural genetic alterations in chromosome 7. Conclusion Our study provides the most comprehensive genetic portrait of HSTL to date, and is a significant step in defining the genetic causes of this disease. Disclosures: No relevant conflicts of interest to declare.

Description: Idiopathic Pneumonia Syndrome (IPS) is a fulminant and largely untreatable form of GVHD of the lung following allogeneic stem cell transplantation (SCT). It characteristically develops in the third week after SCT in patients receiving prior total body irradiation (TBI). We have previously shown that IFNγ signaling within lung parenchyma is critical for preventing disease (Burman AC et al, Blood 2007, 110:1064). Using IL-17A-fate reporter grafts (where YFP is expressed if a T cell has ever made IL-17) we now show that Th17 cells preferentially accumulate in the lung (but not other sites) after experimental allo-SCT in recipients in which IFNγ is neutralized (lung Th17 cells: 6.6x104 ± 0.2 vs. 0.8x104 ± 0.01, anti-IFNγ vs. control treated, P = 0.002). To understand whether this accumulation of Th17 cells in the lung and IPS in general was an alloreactive (i.e. GVHD) dependent phenomena, we transplanted luciferase expressing B6 TEa TCR transgenic CD4+ T cells specific for alloantigen (I-Ed) expressed in B6D2F1 recipients in the presence or absence of IFNγ neutralization. Remarkably, allospecific donor TEa T cells again accumulated in the lung in the absence of IFNγ (BLI: 2.2x105 ± 0.6 vs. 0.54x105 ± 0.08 ph/sec/cm2/sr, P = 0.008), but not other sites, excluding responses to other antigens (e.g. pathogen-derived) as a cause for IPS. Critically, we show that donor T cell-derived IFNγ was vital for inhibiting IL-6, TGFβ and IL-21 mRNA expression in lung parenchymal cells after SCT. Protein analysis confirmed that IL-6 was markedly elevated in lung tissue lysate 5 days after allo-SCT in IFNγR–/– recipients compared to WT (128 ± 25 pg/ml vs. 20 ± 5 pg/ml, P = 0.002). In contrast, IL-6 was elevated to equivalent levels in sera of both WT and IFNγR–/– SCT recipients. Studies using IL-6–/– recipients demonstrated that IL-6 was critical for Th17 differentiation within the lung and subsequent IPS. In the absence of IFNγ, IL-6 promoted Th17 cells to preferentially expand in the lungs (14.6x104 ± 0.03 vs. 3.8x104 ± 0.01, WT vs. IL-6–/–, P = 0.04), produce IL-17A (3734 ± 736 pg/ml vs. 521 ± 142 pg/ml, WT vs. IL-6–/–, P = 0.003) and induce IPS (lung pathology scores: 7.3 ± 0.9 vs. 5.0 ± 0.3, WT vs. IL-6–/–; P = 0.02). Consistent with Th17 differentiation as the final pathological pathway, systemic blockade of IL-17A or the transplantation of grafts lacking the IL-17 receptor also profoundly attenuated disease (lung pathology scores: 3.9 ± 0.5 vs. 6.7 ± 0.6, anti-IL-17A vs. control treated, P = 0.002; and 4.5 ± 0.4 vs. 8.1 ± 1.0, IL-17RA–/–- vs. WT, P= 0.003, respectively). Thus IFNγ is the critical cytokine for suppressing an environment conducive to Th17 differentiation within the lung and IPS after allogeneic SCT. To study the clinical relevance of these findings we examined IL-6 dysregulation in a cohort of patients receiving allogeneic peripheral blood stem cell transplantation at our institution. We confirm that plasma levels of IL-6 are also significantly elevated in SCT recipients within the first 2 weeks of transplantation (30.6 ± 8.8 pg/ml vs. 8.3 ± 3.5 pg/ml, day +7 vs. day -7, P = 0.008), particularly in patients receiving Cy/TBI (12 Gy) compared to reduced intensity Flu/Mel (120mg/m2) conditioning (74.4 ± 38.4 pg/ml vs. 23.4 ± 7.1 pg/ml, P = 0.04). Furthermore, standard calcineurin and methotrexate–based immune suppression completely prevented systemic IFNγ generation after SCT, thus inducing an IL-6–high, IFNγ–deplete environment highly conducive to the development of IPS. These data confirm IL-6 and IL-17 as logical therapeutic targets to prevent IPS after clinical SCT utilizing TBI-based conditioning. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points The data demonstrate the complexity of the genetic contribution to inhibitor development in people with hemophilia A. Potentially decisive markers have been identified, indicating the importance of further evaluation of intracellular signaling pathways.

Description: Hepatitis E virus (HEV) is increasingly acknowledged as a cause of hepatitis in healthy individuals as well as immunocompromised patients. Little is known of HEV infection in recipients of allogeneic hematopoietic stem cell transplantation (alloHSCT). Therefore, we set out to study the incidence and sequelae of HEV as a cause of hepatitis in a recent cohort of 328 alloHSCT recipients. HEV RNA was tested in episodes of liver enzyme abnormalities. In addition, HEV RNA and HEV serology were assessed pre- and post-alloHSCT. We found 8 cases (2.4%) of HEV infection, of which 5 had developed chronic HEV infection. Seroprevalence pre-alloHSCT was 13%. Four patients died with HEV viremia, with signs of ongoing hepatitis, having a median time of infection of 4.1 months. The 4 surviving patients cleared HEV after a median period of 6.3 months. One patient was diagnosed with HEV reactivation after a preceding infection prior to alloHSCT. Although the incidence of developing acute HEV post-alloHSCT is relatively low, the probability of developing chronic hepatitis in severely immunocompromised patients is high. Therefore, alloHSCT recipients should be screened pretransplantation by HEV serology and RNA. Furthermore, a differential diagnosis including hepatitis E is mandatory in all alloHSCT patients with severe liver enzyme abnormalities.

Description: Key Points CLEC4M plays a role in the clearance of VWF. CLEC4M polymorphisms contribute to the genetic variability of VWF plasma levels.