Citations

Abstract Gene expression profiling has identified two major subclasses of diffuse large B-cell lymphoma. Cases resembling germinal center B-cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-germinal center cells (ABC-DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here we show that UCH-L1 reflects germinal center lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in germinal center B-cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biologic diversity in this subset of disease. We also find that UCH-L1 synergizes with BCL6 in a mouse model of germinal center B-cell lymphoma, but not with the development of multiple myeloma derived from post-germinal center cells. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in germinal center B-cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL. Disclosures Galardy: Mission Therapeutics: Research Funding.

Abstract Abstract 3691 The mTOR kinase is the core component a master regulatory network that controls nutrient-sensitive mRNA translation and the activity of the Akt growth and survival pathway. The importance of this kinase in cancer has made it the target of several new agents for a variety of cancers. Recently, prospective trials have shown strong responses in patients with refractory B-cell non-Hodgkin's lymphomas treated with mTOR inhibitors. We recently demonstrated that the de-ubiquitiinase UCH-L1 is a potent oncogene that drives the development of B-cell lymphomas in mice and is frequently over-expressed in human B-cell lymphomas of a variety of histologies. UCH-L1 promotes signaling through the Akt pathway, though the molecular basis for this is unknown. Here we describe a novel mechanism by which UCH-L1 regulates the mTOR/Akt signaling pathway and provide data suggesting that UCH-L1 expression predicts a poor response to mTOR inhibition in patients with B-cell lymphoma. Depletion of UCH-L1 in malignant B-cells leads to a dramatic decline in Akt phosphorylation that is not due to changes in the levels of the kinases PDK1, mTOR, or any mTOR complex component. We observe, however, a striking UCH-L1 driven increase in the association of mTOR with the mTORC2 component rictor, and a reciprocal decline in the mTORC1 subunit raptor, suggesting that UCH-L1 promotes mTORC2 assembly. This is verified when we introduce UCH-L1 into HeLa cells and observe an increase in the co-precipitation of rictor with mTOR, with a corresponding decrease in the recovery of raptor. Consistent with these results, we find that mTORC1 activity is greatly reduced in cells with high levels of UCH-L1, and conversely is enhanced in tissues from Uchl1 null mice. This effect is entirely dependent on DUB activity, as expression of a catalytic mutant UCH-L1 construct has no effect on complex assembly. We find that UCH-L1 forms a novel association with the ubiquitin-ligase DDB1 and that the formation of this complex leads to the displacement of DDB1 from raptor, which then leads to the destabilization of mTORC1. As expected, depletion of DDB1 itself produces a phenocopy of UCH-L1 over-expression. We hypothesize that the suppressive effect of UCH-L1 effect on mTORC1 signaling would render malignant cells resistant to mTOR inhibition, as they likely have adapted to low mTORC1 activity. To test this hypothesis, we analyzed data from a recent prospective trial in which patients with refractory mantle cell lymphoma received the mTOR inhibitor temsirolimus along with rituximab. Limiting our analysis to those cases staining positive for phospho-Akt (pAkt), we found a strong trend towards UCH-L1 expression associated with a worse prognosis, despite the small numbers of patients (n = 10 pAkt+/UCH-L1– v. 5 pAkt+/UCH-L1+, median overall survival 913 v. 201 days, respectively; p=0.08). We conclude that UCH-L1 is a physiological regulator of mTOR/Akt signaling and that this function is co-opted in the pathogenesis of B-cell malignancies. Our data suggest that UCH-L1 expression is a negative predictor for responsiveness to mTOR inhibition in B-cell lymphoma, a finding that warrants further evaluation in larger cohorts. Disclosures: Off Label Use: Results from a clinical trial using the drug temsirolimus will be discussed.

Abstract Hemorrhagic complications remain a challenge with surgical procedures in patients with bleeding disorders. Tonsillectomy and adenoidectomy are some of the most common surgical procedures performed in pediatric patients. Adequate hemostasis in patients with bleeding disorders is centered on comprehensive hemostatic support and dexterous surgical technique. To assess our institutional experience with children and young adults with bleeding disorders that underwent tonsillectomy and/or adenoidectomy we performed a retrospective chart review of all such patients (age< 25 years) over duration of 20 years from July 1992 to July 2012. Nineteen patients were identified. The mean age was 10.2 years (Range 2.5 – 23.2 years) with 13 females and 6 males. The cohort included 2 patients with platelet disorders, 5 patients with von Willebrand disease and 12 patients with factor deficiencies (see table 1). Sixteen patients (84%) underwent tonsillectomy and adenoidectomy, while 3 patients (16%) underwent tonsillectomy only. Pre-operative treatment in the form of coagulation factor infusion (with a goal of 100% factor levels prior to surgery) or DDAVP was given to 16 patients (84%). Nine patients (47%) received anti-fibrinolytic agent, aminocaproic acid, starting pre-operatively for an average of 15.5 days (Range 10 – 36 days) post-operatively. Six patients (32%) received aminocaproic acid only post-operatively for an average of 12 days (Range 7-14 days). One patient received Tranexamic acid for 19 days. Intraoperative hemostasis was achieved by electrocautery in 16 patients (84%) and coblation technique in 2 patients (10%). Surgical hemostasis technique for 1 patient was undocumented, however this patient did not have any bleeding complications subsequently. Ten patients (53%) experienced post-operative hemorrhage including 2 patients (10%) with early (<24 hours) bleeding and 8 patients (42%) with delayed (>24 hours) bleeding from surgical site. Bleeding resolved spontaneously in 2 patients while 8 patients (42%) required interventions such as cauterization (4 patients), extended aminocaproic acid dosing (4 patients), DDAVP (1 patient), DDAVP and tranexamic acid (1 patient), recombinant factor VII (1 patient), Humate-P® (1 patient), Factor VIII infusion (1 patient) and Factor IX infusion (1 patient). Three patients (30% of bleeding patients) required transfusions including 1 patient that received platelet transfusions, 1 patient received PRBCs and another patient received FFP. Recurrent bleeding was noted in 3 patients and the rate was significantly higher in older patients amongst those with bleeding complications (p=0.0189).Table 1Age (years, months)GenderDiagnosisSeverity of diseasePost-operative bleeding (Early ≤ 24 hours, Delayed >24 hours)Recurrent bleeding14,5MEssential ThrombocythemiaModerateEarlyYes13,6MFactor VII deficiencyMildDelayedNo6,7FFactor VII deficiencyMildDelayedNo7,6FFactor VII deficiencyMildNo-11,2FFactor XI deficiencyMildEarlyNo8,4MHemophilia ASevereDelayedNo9,4FHemophilia A carrierMildDelayedNo15,2FHemophilia A carrierMildNo-5,0MHemophilia BMildNo-23,2MHemophilia BMildDelayedYes6,1FHemophilia B carrierMildNo-6,8FHemophilia B carrierMildNo-15,2FHemophilia B carrierMildNo-11,4FMay-Hegglin anomalyModerateDelayedNo4,0FType 1 von WillebrandMildDelayedNo13,5FType 2A von WillebrandModerateDelayedYes2,5MType 2A von WillebrandModerateNo-9,9FType 2B von WillebrandModerateNo9,1FType III von WillebrandSevereNo- The rate of bleeding complications in pediatric patients with mild bleeding disorders undergoing adenotonsillectomy has been reported to be similar to that of normal population. In our cohort, delayed bleeding was more common than early bleeding consistent with current literature. We observed a higher rate of bleeding complications (53%) than reported in literature despite aggressive hemostatic support and adequate surgical techniques; however, our sample size was limited. Although there was no association between delayed hemorrhage and age, recurrent bleeding was associated with older age. We conclude that patients with bleeding disorders undergoing adenotonsillectomy are at a higher risk of bleeding and require close monitoring with hemostatic support for a prolonged period of time in post-operative period. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4182 De-ubiquitinating enzymes (DUBs) are important regulators of many cellular processes through their ability to reverse the modifications installed by ubiquitin ligases. Several members of the DUB family are implicated in human cancers, where they may act as oncogenes or tumor suppressors. There is little direct in vivo evidence, however, for their roles in cancer. We recently reported that the DUB UCH-L1 is a potent oncogene that drives the development of spontaneous B-cell lymphoma in transgenic mice, and is required for the survival of malignant B-cells in vitro. The mechanism of these effects involves the strong induction of Akt signaling that is observed in culture and in lymphoid tissues of Uchl1 transgenic mice. Akt is phosphorylated on two distinct sites by the kinases PDK1 and mTOR, with both phosphorylation events required for maximal signaling. The activity of mTOR towards its substrates is dependent on the binding partners to which it is bound. When mTOR binds to raptor, it forms mTORC1, an important regulator of nutrient sensitive protein translation, whereas when complexed with rictor and SIN1, it forms mTORC2, a critical regulator of Akt signaling. It is unclear how the cell regulates the formation of mTORC1/2. Here we show a novel mechanism by which UCH-L1 regulates mTOR-dependent Akt signaling. By co-immunoprecipitating mTOR and its binding partners, we find that UCH-L1 expression promotes the binding of mTOR with rictor at the expense of raptor, without changing the level of either protein. These data suggest a novel ubiquitin-dependent regulatory step in mTOR complex assembly. In addition, UCH-L1 expression leads to a reduction in the levels of the Akt antagonist phosphatase PHLPP1 in vitro and in vivo. This decline is completely dependent on UCH-L1 DUB activity as expression of a catalytic mutant does not affect levels of PHLPP1. We find that the mechanism of this decline is not due to proteasome degradation, nor transcriptional control. We therefore hypothesize that the UCH-L1 associated shift in cellular mTOR activities leads to reduced mTORC1-dependent translation of PHLPP1. To test this, we inhibited mTORC1 activity by depleting raptor using shRNA and found a dramatic decline in PHLPP1 levels, whereas depletion of rictor had no effect on PHLPP1. We conclude that UCH-L1 boosts Akt signaling through a novel two-step mechanism by which it promotes the generation of mTORC2, reduces the levels of mTORC1, and thereby impairs the translation of PHLPP1. These data have important implications for understanding the roles of ubiquitin, mTOR, and Akt, in lymphoma biology and may lead to more intelligent use of next generation mTOR inhibitors in the treatment of lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract The success of proteasome inhibition in multiple myeloma highlights the critical role for the ubiquitin-proteasome system (UPS) in this disease. However, there has been little progress in finding more specific targets within the UPS involved in myeloma pathogenesis. In an unbiased screen of de-ubiquitinase activity, we previously found the ubiquitin hydrolase UCH-L1 to be frequently over-expressed in B-cell malignancies, including myeloma. Using a transgenic mouse model, we also found that aberrant expression of UCH-L1 is sufficient to drive the development of spontaneous B-cell lymphomas and plasmacytomas, demonstrating its oncogenic activity. Whether and how UCH-L1 affects the clinical behavior of myeloma, however, is not known. Here we show that UCH-L1 is a potent negative prognostic factor that is essential for the dissemination and progression of myeloma. We found high levels of UCHL1 to predict early progression in newly diagnosed patients; a finding reversed in studies including bortezomib. We also found high UCHL1 levels to be a critical factor in the superiority of bortezomib over high-dose dexamethasone in relapsed patients. High UCHL1 partially overlaps with, but is distinct from, known genetic risks including 4p16 rearrangement and 1q21 amplification. Using an orthotopic mouse model, we found UCH-L1 depletion delays myeloma dissemination and causes regression of established disease. We conclude that UCH-L1 is a biomarker of aggressive myeloma that may be an important marker of bortezomib response, and may itself be an effective target in disseminated disease. Disclosures Galardy: Mission Therapeutics: Research Funding.

AbstractThe deubiquitinase (DUB) ubiquitin C‐terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small‐molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine‐based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

5031 Background: The phase 2 TRITON2 study (NCT02952534) is evaluating the PARP inhibitor rucaparib in patients with mCRPC who have a deleterious germline or somatic alteration in BRCA ( BRCA1 or BRCA2) or 1 of 13 other DNA damage repair genes. Here we present analyses of tumor genomics and baseline clinical characteristics in mCRPC patients with a deleterious alteration in BRCA. Methods: Plasma (baseline) and tissue (archival or baseline) samples from patients with a deleterious alteration in BRCA were analyzed using Foundation Medicine next-generation sequencing assays. The alterations and zygosity of the alterations that were detected, as well as the somatic/germline status from Color Genomics testing, were summarized. Associations between genomic alterations, DNA yield, allele frequency, and baseline clinical characteristics were investigated. Results: Results are shown in the Table for a cohort of 40 BRCA2 and 5 BRCA1 patients enrolled in TRITON2 (Abida W et al. Presented at ESMO 2018. Abst 793PD). A biallelic alteration was observed in 21 of the 22 BRCA2 patients (95%) for whom alteration zygosity could be determined. Among the 5 BRCA1 patients, 1 alteration was monoallelic and 4 were of unknown zygosity. Co-occurring alterations in cancer-related or DNA damage repair genes were observed in many patients with BRCA alterations. At baseline, cell-free DNA (cfDNA) yield correlated positively with the sum of target lesions (STL; P= 0.04), but not with prostate-specific antigen (PSA) levels ( P= 0.86). No correlation was observed between allele frequency of the BRCA alteration baseline STL ( P= 0.68) or PSA levels ( P= 0.97). Conclusions: Patients with a BRCA mutation enrolled in TRITON2 demonstrate a profile of genomic alterations consistent with that of prior studies of patients with mCRPC. Plasma cfDNA profiling showed a correlation between baseline cfDNA yield and measurable tumor burden, but not baseline PSA. Clinical trial information: NCT02952534. [Table: see text]

Transvenous embolization (TE) has been increasingly applied for arteriovenous malformation (AVM) treatment. Transient cardiac standstill (TCS) has been described in cerebrovascular surgery but is uncommon for endovascular embolization. The authors present a patient with a ruptured thalamic AVM in whom both techniques were applied simultaneously. Surgery was considered, but the patient refused. Transarterial embolization was performed with an incomplete result. The deep-seated draining vein provided sole access to the AVM. A microcatheter was advanced into the draining vein. Under TCS, achieved with rapid ventricular pacing, complete AVM embolization was obtained. One-year magnetic resonance imaging and cerebral angiography demonstrated no residual AVM. The video can be found here: https://youtu.be/CAzb9md_xBU .

Abstract The tolerability of long-term posaconazole use remains poorly defined. We present a patient who developed peripheral neuropathy following long-term exposure to the tablet formulation of posaconazole, which was treated with methylprednisolone and magnesium infusions. The potential role of methylprednisolone and magnesium infusions in managing this potentially irreversible triazole-associated complication requires further study.

Background and Purpose— Mechanical thrombectomy has been shown to improve clinical outcomes in patients with acute ischemic stroke. However, the impact of balloon guide catheter (BGC) use is not well established. Methods— STRATIS (Systematic Evaluation of Patients Treated With Neurothrombectomy Devices for Acute Ischemic Stroke) was a prospective, multicenter study of patients with large vessel occlusion treated with the Solitaire stent retriever as first-line therapy. In this study, an independent core laboratory, blinded to the clinical outcomes, reviewed all procedures and angiographic data to classify procedural technique, target clot location, recanalization after each pass, and determine the number of stent retriever passes. The primary clinical end point was functional independence (modified Rankin Scale, 0–2) at 3 months as determined on-site, and the angiographic end point was first-pass effect (FPE) success rate from a single device attempt (modified Thrombolysis in Cerebral Infarction, ≥2c) as determined by a core laboratory. Achieving modified FPE (modified Thrombolysis in Cerebral Infarction, ≥2b) was also assessed. Comparisons of clinical outcomes were made between groups and adjusted for baseline and procedural characteristics. All participating centers received institutional review board approval from their respective institutions. Results— Adjunctive technique groups included BGC (n=445), distal access catheter (n=238), and conventional guide catheter (n=62). The BGC group had a higher rate of FPE following first pass (212/443 [48%]) versus conventional guide catheter (16/62 [26%]; P =0.001) and distal access catheter (83/235 [35%]; P =0.002). Similarly, the BGC group had a higher rate of modified FPE (294/443 [66%]) versus conventional guide catheter (26/62 [42%]; P <0.001) and distal access catheter (129/234 [55%]; P =0.003). The BGC group achieved the highest rate of functional independence (253/415 [61%]) versus conventional guide catheter (23/55 [42%]; P =0.007) and distal access catheter (113/218 [52%]; P =0.027). Final revascularization and mortality rates did not differ across the groups. Conclusions— BGC use was an independent predictor of FPE, modified FPE, and functional independence, suggesting that its routine use may improve the rates of early revascularization success and good clinical outcomes. Clinical Trial Registration— URL: https://www.clinicaltrials.gov . Unique identifier: NCT02239640.