Citations

Background and Purpose: Clopidogrel is an antiplatelet drug that is metabolized to its active form by the CYP2C19 enzyme. The CHANCE trial (Clopidogrel in High-Risk Patients With Acute Nondisabling Cerebrovascular Events) found a significant interaction between loss-of-function allele status for the CYP2C19 gene and the effect of dual antiplatelet therapy with aspirin and clopidogrel on the rate of early recurrent stroke following acute transient ischemic attack/minor stroke. The POINT (Platelet-Oriented Inhibition in New TIA and Minor Ischemic Stroke Trial), similar in design to CHANCE but performed largely in North America and Europe, demonstrated a reduction in early recurrent stroke with dual antiplatelet therapy compared with aspirin alone. This substudy was done to evaluate a potential interaction between loss-of-function CYP2C19 alleles and outcome by treatment group in POINT. Methods: Of the 269 sites in 10 countries that enrolled patients in POINT, 134 sites participated in this substudy. DNA samples were genotyped for CYP2C19 *2, *3, and *17 alleles and classified as being carriers or noncarriers of loss-of-function alleles. Major ischemia consisted of ischemic stroke, myocardial infarction, or ischemic vascular death. Results: Nine hundred thirty-two patients provided analyzable DNA. The rates of major ischemia were 6.7% for the aspirin group versus 2.3% for the dual antiplatelet therapy group (hazard ratio, 0.33 [95% CI, 0.09–1.21]; P =0.09) among carriers of loss-of-function allele. The rates of major ischemia were 5.6% for the aspirin group versus 3.7% for the dual antiplatelet therapy group (hazard ratio, 0.65 [95% CI, 0.32–1.34]; P =0.25) among noncarriers. There was no significant interaction by genotype for major ischemia ( P =0.36) or stroke ( P =0.33). Conclusions: This substudy of POINT found no significant interaction with CYP2C19 loss-of-function carrier status and outcome by treatment group. Failure to confirm the findings from the CHANCE trial may be because the loss-of-function alleles tested are not clinically important in this context or because the 2 trials had differences in racial/ethnic composition. Additionally, differences between the 2 trials might be due to chance as our statistical power was limited to 50%. Registration: URL: https://www.clinicaltrials.gov . Unique identifier: NCT00991029.

e20004 Background: IB is a Bruton’s tyrosine kinase inhibitor that showed superior progression-free survival compared with BR in CLL patients (pts) 65 years or older in a randomized phase 3 trial (Woyach et al, NEJM 2018). Pts receiving IB had higher rates of atrial fibrillation (AFIB) and hypertension (HTN); BR pts had higher rates of hematologic toxicity. Differences in treatment duration for BR (6 cycles) and IB (until progression) complicated adverse event (AE) comparisons. Here we use an exploratory approach to compare toxicity burden between arms and provide assessment of AFIB, HTN and infections (INF). Methods: AEs were reported for each of cycles 1-6 and then every 3 cycles until progression or nonprotocol CLL therapy. Only grade (gr) 3-4 AEs were reported thereafter until death. A simple global AE score was calculated for each pt by summing grades of all gr 1-4 AEs and dividing by the number of cycles over which AEs were submitted. Results: 537 pts began therapy (176 BR, 361 IB). 68% on BR completed all 6 cycles. At a median follow-up of 38 months, 64% remained on IB. Treatment discontinuation for AE occurred in 10% and 14% of BR and IB pts. Overall, median AE score was 1.8 (interquartile range (IQR) 0.9-3.3) and 3.8 (IQR 2.3-5.9) in BR and IB arms (p < 0.01). For cycles 1-6, median AE score was 6.2 (IQR 3.8-9.0) and 4.8 (IQR 3.0-7.2) in BR and IB arms (p < 0.01). In the IB arm, median AE score post 6 cycles decreased significantly to 3.4 (IQR 1.9-5.6) (p < 0.01). Gr 3 or higher cumulative rates of AFIB, HTN, and INF over time appear in the table. 100 pts (26/176 BR, 74/361 IB) had 137 severe INF (39% respiratory: 16 BR, 37 IB; 25% skin: 3 BR, 31 IB; 12% GU: 3 BR, 13 IB; 25% other: 12 BR, 22 IB). There were 7 gr 5 INF (3 BR, 4 IB), none confirmed fungal. Conclusions: There was no difference in treatment discontinuation rates for AE. Overall toxicity burden was significantly higher for IB, although IB toxicity burden decreased after 6 cycles. Toxicity burden was significantly higher in cycles 1-6 for pts receiving BR. Risk of severe AFIB, HTN, and INF is highest in the first year of IB. A simple AE score provides valuable information, especially when evaluating regimens of varying length. Clinical trial information: NCT01886872 . Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org ; Pharmacyclics; ClinicalTrials.gov Identifier: NCT01886872. [Table: see text]

8049 Background: Our group has demonstrated that combinations of epigenetic modifiers produce potent synergy in pre-clinical models of PTCL and induce the expression of cancer testis antigen, suggesting a role in the addition of the immune-checkpoint inhibitor, pembrolizumab. Methods: This is a phase 1b study of pembrolizumab combined with pralatrexate alone (Arm A), with pralatrexate + decitabine (Arm B), or decitabine alone (Arm C) in patients with relapsed and refractory PTCL and CTCL. A standard 3+3 dose-escalation is applied in the triplet Arm (Arm B) while in the doublet Arms (A and C) de-escalation is applied in case of toxicity. Pharmacokinetic and pharmacodynamic studies are ongoing. Results: We treated a total of 12 patients with 4 patients in each Arm. All patients that received at least one dose of drug were evaluable for toxicity. There was a dose limiting toxicity (DLT) in each arm including prolonged grade 3 thrombocytopenia (Arm A), febrile neutropenia (Arm B), grade 3 hyponatremia, and rash (Arm C). There were no treatment-related deaths. Six patients out of 12 were evaluable for response at the time of this analysis. One patient achieved a complete remission, 2 had partial remission, 1 had stable disease, and 2 experienced progression of disease. Interestingly, all of the responses were seen in the triple combination of pralatrexate, decitabine, and pembrolizumab. Table summarizes the patient characteristics, toxicities, and response rates. Conclusions: These preliminary clinical data suggest that the integration of pembrolizumab on an epigenetic backbone is safe and demonstrates encouraging responses in patient with PTCL and CTCL. Clinical trial information: 03240211 . [Table: see text]

Abstract Introduction: Assays of plasma von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) are essential for the laboratory diagnosis of von Willebrand disease (VWD). However, the current manual platelet aggregation/aggregometry method has relatively poor operating characteristics. Our goal was to develop a simple, accurate and sensitive platelet based assay. Methods: VWF protein from normal or patient plasma was first captured on the polyclonal anti-VWF antibody coated plastic plate surface. VWF:RCo activity was assayed by the binding of mitotracker (a red fluorochrome)-labeled, formalin-fixed normal platelets in the presence of ristocetin (1mg/ml) and under agitation (200 rpm by vortex). The fluorescence from the bound platelets, measured by a fluorescence microtiter plate reader (Fluor4000, BioRad), was related to the calibrator plasma signal (6∼150% or IU/dL), and reported as % or IU/dL. We tested plasma samples from normal donors (n=16) and known VWD patients (type 1, n=6; type 2, n=9) based on clinical history and levels of plasma VWF antigen (VWF:Ag), VWF:RCo activity (manual platelet aggregation/aggregometry method), factor VIII activity and VWF multimer analysis. Results: For both normal donors and VWD patients, VWF:RCo activities by the plate assay vs. manual platelet aggregation/aggregometry method correlated closely (R2=0.84), and VWF:RCo/VWF:Ag ratios did not differ significantly. Platelet binding to VWF-coated plastic wells is both VWF specific and ristocetin dependent. Conclusions: Although this new platelet based VWF:RCo plate assay is still being optimized and validated, the preliminary data suggest that it is simple, accurate and sensitive.

Abstract Studies of the Syk −/− mouse have implicated spleen tyrosine kinase (Syk), a signaling protein with both kinase and scaffolding activities, in platelet signaling following engagement of GPVI and αIIbβ3 by collagen and fibrinogen, respectively. The present study was designed to determine whether specific inhibition of the kinase activity of Syk, without targeting the Syk scaffolding function, affected in vivo arterial thrombosis. In preliminary experiments, blood from wild-type and Syk−/− mice was perfused through collagen-coated capillaries under arterial shear rates to study ex vivo thrombosis. While blood from wild-type mice formed robust thrombi (37±4.7 μm3/μm2), none was observed in Syk−/− mice. Thrombi intermediate in size (16±3.9 μm3/μm2) developed in Syk+/− mice. To achieve specific pharmacological targeting of the kinase activity of Syk, P142-76, a potent (IC50 = 4 nM) and selective Syk kinase inhibitor was utilized. P142-76 was screened against a broad panel of 139 purified kinases at 50 nM. While Syk kinase was inhibited by 92%, all other kinases retained more than 70% of their activity. In washed human platelets, P142-76 inhibited convulxin (CVX)-induced phosphorylation of LAT (linker for activation of T-cells; IC50 = 111 nM) and intracellular calcium increases (IC50 = 31 nM). The GPVI/Syk-specificity of P142-76 activity was confirmed by its inability to inhibit intracellular calcium increases induced by the PAR1 thrombin receptor agonist TRAP. P142-76 also inhibited CVX-induced aggregation of both human washed platelets (IC50 = 87 nM) and platelet-rich plasma (IC50 = 2.5 μM). Considering the controversial data in respect to the participation of GPVI in arterial thrombosis in murine models, the dependence of arterial thrombosis on Syk function was studied in vivo in pigs. Cross-species activity of P142-76 was confirmed in vitro (CVX-induced PRP aggregation IC50= 350 nM; 5 μM P142-76 completely inhibited thrombosis triggered by collagen in the perfusion chamber assay). At a plasma concentration which abolished ex vivo CVX-induced but not ADP-induced pig platelet aggregation, P142-76 significantly inhibited the deposition of [111In]-labeled platelets in a carotid artery crush swine thrombosis model, without compromising primary hemostasis. % aggregation Swine (n=3) Platelet Deposition % inhibition Plasma Conc (ng/ml) Bleed Time (min) Activated Clotting Time (sec) ADP (20 μM) CVX (250 ng/ml) Control Artery 0 0 3±0.9 133±22 100 100 Treated Artery 76±6.5 1343±304 3.5±0.3 130±13 100 0 To clarify further the contribution of the kinase activity of Syk to arterial thrombosis, effects of P142-76 on human blood were evaluated in real time in the collagen-coated perfusion chamber. Low concentrations of P142-76 (0.3 μM) affected thrombus stability, while increasing concentrations (1–5 μM) delayed and then completely inhibited thrombus formation. Furthermore, P142-76 destabilized pre-formed thrombi, indicating a critical role for Syk in conferring strength to platelet-platelet interactions, i.e. αIIbβ3-mediated cohesion. Our data indicate that the kinase activity of Syk acts in arterial thrombosis through at least two distinct mechanisms. First, Syk kinase confers stability to platelet-platelet interactions downstream of αIIbβ3. Second, it initiates thrombus formation on collagen surfaces. This dual activity of the kinase activity of Syk makes it a preferred target for inhibition of arterial thrombosis, as it does not compromise primary hemostasis.

Infection increases propensity for thrombotic events. Gram negative pathogens release lipopolysaccharides (LPS), which initiate intracellular signaling cascades through activation of toll‐like‐receptor 4 (TLR4). Platelets contribute to defense from infection and to thrombosis, but little is known about how LPS affects platelet production, life span and reactivity. Platelet life span was determined by in vivo biotinylation in wild type (C57BL/10SnJ; WT) and TLR4 deficient (C57BL/10ScN; dTLR4) mice before and after a single intravenous injection of LPS (1ng – 500ng/25g body weight). Prior to LPS injection, the number of platelets and percentage of reticulated platelets in dTLR4 mice was less than in WT mice but there were no differences in platelet life span, indicating that loss of TLR4 affects platelet production and not life span. A single injection of LPS dose dependently increased percentages of P‐selectin positive platelets and of reticulated platelets but decreased the number of biotin positive platelets within 24hrs in WT but not in dTLR4 mice. Results suggest that LPS–simulated infection reduces life span of circulating platelet and increases production of more reactive (reticulated) platelets from megakaryocytes through TLR4 mechanisms. Thus, a single exposure to LPS represents a “priming” mechanism which could increase risk for thrombosis with infection. (Funded by NIH‐HL78638 and Mayo Fnd.)

AbstractBackground: Abnormal aggregates of α‐synuclein are pathologic hallmarks of multiple system atrophy (MSA) and Lewy body disease (LBD). LBD sometimes coexists with MSA, but the impact of co‐pathology, particularly diffuse LBD, on presentation of MSA has not been studied. We aimed to determine the frequency and clinicopathologic features of MSA with LBD (MSA+LBD). Methods: Using hematoxylin & eosin and α‐synuclein‐immunostained slides, we assessed the distribution and severity of LBD in 230 autopsy‐confirmed MSA patients collected from 1998 to 2018. Alzheimer‐type pathology was assessed to assign the likelihood of clinical presentations of dementia with Lewy body (DLB) using the consensus criteria for DLB. We reviewed medical records to characterize clinicopathologic features of MSA+LBD. Genetic risk factors for LBD, including APOE ε4 allele and mutations in GBA, SNCA, LRRK2, and VPS35, were analyzed. Results: LBD was observed in 11 MSA patients (5%); seven were brainstem type, three were transitional type, and one was diffuse type. The latter four had an intermediate or high likelihood of DLB. Three of the four had an antemortem diagnosis of Parkinson’s disease with dementia (PDD) or clinically probable DLB. Two patients had neuronal loss in the substantia nigra, but not in striatal or olivocerebellar systems with widespread glial cytoplasmic inclusions, consistent with minimal change MSA. In these cases, LBD was considered the primary pathology, and MSA was considered coincidental. APOE ε4 allele frequency was not different between MSA+LBD and MSA without LBD. Two of nine MSA+LBD patients had a risk variant of GBA (p.T408M and p.E365K). Conclusions: Although rare, MSA with transitional or diffuse LBD can develop clinical features of PDD or DLB. Minimal change MSA can be interpreted as a coincidental, but distinct, α‐synucleinopathy in a subset of patients with diffuse LBD.

TPS7569 Background: Progress in genome technology allows analysis of previously completed trials to identify patients subgroups potentially benefiting from therapy. Enzastaurin is a potent inhibitor of protein kinase C beta (PKC- b) and suppresses the phosphoinositide 3-kinase (PI3K)/AKT pathway. The safety and efficacy of Enzastaurin has been tested in more than 60 clinical trials including 2 major studies in DLBCL: (1) PRELUDE (A phase III maintenance trial of Enzastaurin vs Placebo, N=758) ( Crump, 2016), and (2) S028 (A randomized phase II study of Enzastaurin/R-CHOP vs R-CHOP in frontline intermediate/high-risk DLBCL, N=101) ( Hainsworth, 2016). DNA samples extracted from blood of patients from PRELUDE were retrospectively genotyped using whole genome SNP arrays. From the genome wide screening a novel genetic biomarker, DGM1, was identified showing high correlation with response to Enzastaurin treatment ( Luo, ASH 2018). Importantly, these findings were replicated in the phase II S028 study. In the S028 study the hazard ratio (HR) for OS in high-risk (IPI ≥ 3) DGM1 positive (+) patients who received Enzastaurin/R-CHOP was 0.28 (0.1-0.81) when compared to subjects who received R-CHOP, a benefit favoring Enzastaurin (p=0.018). These data suggest that addition of Enzastaurin to R-CHOP may significantly improve outcome in frontline high-risk DGM1 (+) DLBCL. The ENGINE study was initiated to validate this finding in a prospective study. Methods: Adult patients must have untreated CD20+ DLBCL, IPI ≥ 3. Patients are randomized 1:1 to Enzastaurin/R-CHOP or Placebo/R-CHOP for 6 cycles during combination phase. Each subject’s treatment assignment will be unblinded after response assessment at the end of the combination phase. Subjects randomized to the investigational arm who have a complete or partial response will have the option to continue in the single agent phase to receive Enzastaurin for up to 2 additional years. The study intends to enroll approximately 235 patients with primary endpoint of OS in DGM1 (+) patients. The study is ongoing with 51 sites open in the US and China. As of 22 Jan 2019, 70 patients have been randomized. Clinical trial information: NCT03263026.

8003 Background: Carfilzomib is a novel, irreversible tetrapeptide proteasome inhibitor derived from the natural product epoxomicin. Carfilzomib was well tolerated in preclinical animal studies when administered on a two-week cycle, QDx5; proteasome inhibition one hour after dosing at the MTD was >80%. Two Phase I dose-escalation studies are ongoing, aimed at determining the safety, tolerability, and biological response to carfilzomib. Methods: Carfilzomib was administered according to two different dose-intensive schedules. In PX-171–001, carfilzomib was administered on a two week cycle, QDx5 with nine days rest; in PX-171–002, carfilzomib was administered on a four week cycle, QDx2 weekly for three weeks with 12 days rest. Eligible patients have multiple myeloma (MM), Non-Hodgkin's Lymphoma (NHL), Hodgkin's Disease (HD), or Waldenström's Macroglobulinemia (WM). Results: Thus far, a total of 54 subjects have been enrolled. Although the maximum tolerated dose (MTD) has not yet been identified on either study, responses seen on both protocols have established 11 and 15 mg/m2 as the minimal effective doses (MEDs) on PX-171–001 and 002, respectively. Of 3 patients with MM or WM treated on the 001 protocol, one MM patient has had a Partial Response (PR) and one WM patient had a Minimal Response (MR). Of 8 patients with MM treated on the 002 protocol, 3 patients have had PRs. 6 additional patients have had Stable Disease lasting longer than 6 months and symptomatic improvement has been seen in patients on both protocols. 11 subjects remain on study with stable disease or better. Proteasome inhibition in whole blood at the highest dose levels exceeded 80% one hour after the first dose. Carfilzomib has been well tolerated at doses at and above the MED thus far. There has been no incidence of painful peripheral neuropathy on either study. No dose-limiting toxicities (DLTs) have occurred on PX-171–001; one DLT (Gr 4 anemia and thrombocytopenia) was observed at 27 mg/m2 on PX-171–002. Conclusions: Thus far, intensive dosing with carfilzomib is well-tolerated at proteasome inhibition levels of more than 80%. Five responses have been observed, and several subjects have achieved long lasting SD and/or symptomatic improvement. No significant financial relationships to disclose.

Abstract Background Intronic variant rs564309 in tripartite motif containing 11 (TRIM11 ) is associated with clinical phenotypic differences in progressive supranuclear palsy (PSP), whereby the minor allele (A) is more common in atypical PSP than typical PSP (PSP‐Richardson's syndrome). However, rs564309 has not been investigated relative to neuropathological outcomes. Objective Evaluate the association of rs564309 with the neuropathologically assessed severity of tau pathology, as measured by semi‐quantitative scores for neurofibrillary tangles, tufted astrocytes, neuropil threads, and oligodendroglial coiled bodies. Methods 797 neuropathologically confirmed PSP cases were genotyped for TRIM11 rs564309 and assessed for tau pathology across 20 neuroanatomical regions. Tau pathology measures and age at death were examined for association with TRIM11 rs564309‐A using multivariable linear regression models. Results TRIM11 rs564309‐A was associated with increased neurofibrillary tangles pathology ( P = 0.050), but was not significantly associated with age at death, neuropil threads, coiled bodies, or tufted astrocytes tau pathology scores. Conclusions TRIM11 rs564309 may influence burden of neurofibrillary tangles tau pathology in PSP; further study is warranted. © 2020 International Parkinson and Movement Disorder Society