ALBERT

Description: Introduction: The currently used unfractionated heparin (UFH) and low molecular weight heparins (LMWH) are mostly derived from Porcine mucosal tissue. Since the technology to manufacture heparin has advanced and the quality assurance practices are in place, improved products with high potency and purity are now available. Considering these factors the resourcing of heparins utilizing bovine (cow) and ovine (sheep) tissues is discussed at regulatory and pharmaceutical levels. The purpose of this study is to compare multiple individual batches of UFH obtained from Bovine, Ovine, and Porcine origin and their depolymerized product obtained by benzylation followed by alkaline hydrolysis representing enoxaparins. Materials and Methods: The molecular profile of the heparins and enoxaparins from various sources were determined using the size exclusion chromographic method. The anticoagulant potency was measured using clot based methods such as aPTT and Thrombin Time. The AT mediated anti-Xa and anti-IIa activities were also measured in defined biochemical sysytems. A comercially obtained chromogenic substrate based methods were used to determine the USP potency in terms of anti-Xa and anti-IIa activities (Hyphen Biomedical, Ohio, USA). The relative interaction of the heparins and enoxaparins with heparin induced thrombocytopenia (HIT) antibody induced aggregation of platelets were investigated using serum pool obtained from clinically confirmed HIT cases using aggregometry. Results: The molecular profile of multiple batches of the Bovine, ovine, and porcine heparins and enoxaparin were almost comparable and ranged from 15-18 kDa. The global anticoagulant and amidolytic protease assays for the bovine heparin were consistently lower than porcine and ovine samples. In the purified system the Porcine and Ovine preparations consistently showed lower IC50 values for both the thrombin and Xa inhibition in contrast to the bovine heparin. Similar trends were observed in the anti IIa assays. The USP potency of 28 batches of porcine ranged from 170-210 U/mg, whereas the 21 batches of ovine heparins exhibited comparable potencies in the range of 160-210 U/mL. In contrast the USP potency of 30 batches of bovine mucosal heparin was much lower and ranged from 110-140 U/mg. The anti-Xa - IIa ratio for the porcine and ovine heparins were comparable. However, the anti-Xa - IIa ratio of bovine heparin was somewhat lower. The ovine and porcine enoxaparin exhibited comparable potencies which ranged from 94-110 U/mg whereas bovine enoxaparin was slightly lower, ranging from 80-87 U/mg. However the antiXa and anti-IIa ratios of the enoxaparins derived from various species were comparable. In the HIT screening, there was no difference between the HIT responses in the heparins from different species. Similar results were obtained amongst enoxaparins of different origins. Conclusions: These studies show that heparins from bovine, ovine, and porcine origin exhibit comparable molecular profiles. While the porcine and ovine heparins exhibit similar biological potencies, the bovine heparin was found to be weaker. The enoxaparins derived from these species also exhibit similar molecular profiles, however, in functional assays, they exhibited similar trends where the bovine derived products were weaker. In the HIT screening profile, the heparins from all of these species produced stronger effects in comparison to their enoxaparin counterparts. These results suggest that heparin and enoxaparin derived from ovine mucosal tissue exhibit comparable biosimilar profiles. However, the bovine heparins and enoxaparin derived from bovine mucosal tissues are somewhat weaker. Disclosures Yao: Ronnsi Pharma: Employment.

Description: Background: Most research on prognosis after PE has focused on outcomes such as mortality and PE recurrence, while patient-centered outcomes such as quality of life (QOL), dyspnea and walking capacity have been largely unstudied. To address this knowledge gap, we performed the ELOPE (Evaluation of Longterm Outcomes after PE) study, a prospective, observational, multicenter cohort study of long-term outcomes after acute PE (www.clinicaltrials.gov NCT01174628). Objectives: In this extended analysis of ELOPE data, we assessed clinically meaningful improvement and predictors of change in QOL, dyspnea and walking capacity during the year following PE diagnosis. Methods: Patients aged 18 years or older with a 1st episode of acute PE diagnosed within the previous 10 days screened at 5 Canadian recruiting centers were potentially eligible to participate. Exclusion criteria were subsegmental-only PE, preexisting severe cardiopulmonary comorbidity, previous proximal DVT, contraindication to CT pulmonary angiography, life expectancy

Description: A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP- 2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582–12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb- specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311- 1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.

Description: Key Points Truncating PPM1D mutations confer chemotherapy resistance, leading to the selective expansion of PPM1D-mutant cells in vitro and in vivo. PPM1D inhibitor treatment reverses the chemotherapy-resistance phenotype and selectively kills PPM1D-mutant cells.

Description: Key Points Direct analysis of the HLA-presented peptidome identifies a distinct antigenic signature in MM. T-cell responses for these antigens are detectable exclusively in MM patients and can be induced in vitro in response-naive patients.

Description: Background: AID are seen in 10-30% of MDS and CMML. After initial response to steroids, AID are often poorly controlled and steroid-sparing drugs are difficult to use due to the underlying MDS/CMML. Some case reports suggest a beneficial role of AZA treatment in AID associated to MDS/CMML. Methods: We retrospectively analyzed 22 MDS/CMML patients (pts) with AID who received AZA in French centers between January 2007 and May 2014. Results: Median age of the 22 pts was 70y (range 41-84), including 6F/16M. Diagnosis of MDS/CMML preceded AID (n=8) by a median of 17 months (mo), was concomitant with (n=7) or followed AID (n=7) by a median of 20 mo. 14 pts had lower risk IPSS and 8 higher risk IPSS. AID diagnosis included: Behçet's disease (n=4), polymyalgia rheumatica (n=4), relapsing polychondritis (RP) (n=4), giant cell arteritis (n=2), Sweet's syndrome (n=2), SLE (n=2), Sjögren's (n=1), adult onset Still's disease (n=1), unclassified small vessel vasculitis (n=1) and seronegative polyarthritis (n=1). One pt presented with both RP and Sweet's syndrome. 12 (55%) AID were considered atypical and 10 (45%) fulfilled complete diagnostic criteria. Before AZA, 21 pts (96%) had received steroids and 16 (73%) a median of 2 immunomodulatory drugs (IMD) (range 1-6) in addition to steroids. At AZA onset, AID was still active (n=14), in PR (n=6) and in CR (n=2). 19 pts still reported asthenia (n=17), weight loss (n=8), fever 〉38°5 (n=8), rheumatologic signs (n=15), skin involvement (n=10), oral aphtosis (n=4). 9 pts were steroid dependent or resistant. 20 pts were treated with steroids (91%, median dose of 23 mg/d). 13 pts were non-responders to IMD and 11 received concomitant IMD, without efficacy in 7 pts. AZA was the first-line treatment of MDS/CMML in 13 pts (59%) and was in 9 pts preceded by: intensive chemotherapy (n=3), LEN (n=2), HY (n=2), allo SCT (n=2) and LDaraC (n=2). Median interval between MDS/CMML diagnosis and AZA onset was 9 mo (range 1-93). At AZA onset, IPSS was low (n=4), int-1 (n=7), int-2 (n=8) and high (n=3). Pts received a median of 6 cycles of AZA (range 3-14); 1 pt received 3 cycles, 5 received 5 cycles; 9 pts received more than 6 cycles. Complete remission (CR) of AID was observed in 16 pts (73%), partial remission (PR) in 3 pts (14%) and no effect or worsening of AID in 3 pts (14%). All responses were observed within 3 cycles, but 8 PR of AID after 3 cycles became CR after 6 mo. Discontinuation of steroids was possible in 3 pts (14%) and dose reduction in 14 pts (64%), to a median of 9 mg/d, (range 0-30), (p=0.001). Discontinuation of IMD was possible in 7 pts (32%). Altogether, dose reduction or discontinuation of steroids and/or IMD after AZA was possible in 16 cases (73%). Good response of AID symptoms (including PR and CR) or discontinuation of IST was noted in 19 patients (86%). Median CRP decreased from 40 mg/L to 15 mg/L (p=0.28). Response of MDS/CMML to AZA (IWG2006 criteria) was seen in 12 pts (55%), including CR in 9 pts (41%), PR in 1, SD+HI-E in 1, mCR in 1, SD in 8 and PD in 2. No uncommon side effects to AZA were seen. MDS/CMML and AID evolution was concordant in 13 pts (59%): both improved (n=11) or worsened (n=2). AID improved while MDS worsened (n=8) and vice versa (n=1). With a median FU from response to AZA of 8 mo, only 3 relapses of AID were seen after 3 (n=1) and after 19 mo (n=2), and 5 pts had a response 〉 12 mo. Seven pts (32%) progressed to AML and 10 pts (45%) had died, with 8 deaths occurring in IPSS high or int 2 pts. Median survival from AZA onset was 16 months in IPSS high or int-2 MDS and was not reached in IPSS low and int-1 MDS. Conclusions: AZA frequently seems effective in controlling steroid-dependent AID associated with MDS/CMML, but prospective studies are necessary to confirm those findings. Disclosures Fenaux: JANSSEN: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; NOVARTIS: Honoraria, Research Funding.

Description: Background: Risk-adapted treatment strategies have contributed to excellent outcomes in pediatric acute lymphoblastic leukemia (ALL); however, treatment-associated acute and long-term toxicities persist. Therapy-associated toxicities of note in pediatric ALL are related to a treatment backbone that relies heavily on corticosteroids (prednisone and dexamethasone) and asparaginase (ASP). The most frequently observed toxicities include, but are not limited to, serious infection, pancreatitis, thrombosis and bony morbidities including osteonecrosis (ON) and fracture. Previous studies suggest that children of racial and ethnic minorities are at higher risk for treatment-associated toxicities. We assessed the incidence of treatment-related toxicities in Hispanic and non-Hispanic patients undergoing treatment for pediatric ALL. Patients and Methods: Retrospective cohort study investigating the incidence of treatment-related toxicities including infection, allergy to ASP, pancreatitis, thrombosis and bony morbidities in Hispanic and non-Hispanic children and adolescents with newly diagnosed ALL undergoing therapy on Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocol 05-001. The ethnicity of each patient was designated at the time of study enrollment by research coordinators. Descriptive statistics were calculated, mean +/- SD for continuous variables and frequency and percentages for categorical variables. Toxicity rates were based on number of patients. Comparison between groups was done by Chi-square test or FisherÕs exact test and p -value 〈 0.05 was considered significant. Results: Between 2005 and 2011, 794 children and adolescents (ages 1 - 18 years) were enrolled on Protocol 05-001, 730 of whom were evaluable for this investigation: 150 Hispanic (18%), 580 non-Hispanic (73%). Sixty-four patients did not have ethnicity documented. There was no significant difference in disease-risk group, age or gender between the two groups. Weight was significantly higher in Hispanic patients (31.9 ± 24.4 kg in Hispanic and 26.9 ± 18.7 kg in non-Hispanic, p = 0.021). There was no significant difference in the incidence of ASP-related toxicities (allergy, pancreatitis, thrombosis) between Hispanic and non-Hispanic patients. There was no significant difference in the overall incidence of infection between the two groups (42% in Hispanic and 50% in non-Hispanic, p = 0.081). Non-Hispanic patients had significantly higher rates of opportunistic infection (Pneumocystis pneumonia) than Hispanic patients (0.7% in Hispanic and 4% in non-Hispanic, p = 0.041). A similar difference in the incidence of bacteremia between the two groups approached, but did not reach statistical significance (p = 0.052). The overall incidence of fracture in all patients was 14.5% and was significantly higher in non-Hispanic patients (6% in Hispanic and 16.7% in non-Hispanic, p 〈 0.001). The overall incidence of ON was 8.9% and was significantly higher in non-Hispanic patients (3.3% in Hispanic and 10.3% in non-Hispanic, p = 0.007). (Table 1) Conclusion: The incidence of opportunistic infections and bony morbidities was significantly higher in non-Hispanic patients undergoing treatment for pediatric ALL on the DFCI ALL Consortium Protocol 05-001. The risk for, and impact of therapy-related toxicities varies by a patientÕs treatment tolerance, perhaps as a function of age and gender or as a result of disease biology or genetic polymorphisms affecting drug metabolism. Additionally, non-biologic factors such as medication adherence and nutritional status may also contribute to toxicity incidence in patients undergoing treatment for pediatric ALL. Prospective studies to further investigate our findings are warranted. Table. All Patients, (N = 730) Non-Hispanic, (N = 580) Hispanic, (N = 150) p -value Overall Infection 353/730 (48.4%) 290/580 (50%) 63/150 (42%) 0.081 Bacteremia 289/730 (39.6%) 240/580 (41.4%) 49/150 (9.3%) 0.052 Opportunistic Infection 24/730 (3.3%) 23/580 (4%) 1/150 (0.7%) 0.041 Fracture 106/730 (14.5%) 97/580 (16.7%) 9/150 (6%)

Description: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. The course of disease predisposes patients to the development of secondary malignancies, which might be due to a generalized immunosuppression induced by the disease and/or the therapies used for treatment. Skin cancer, including melanoma, is the most common secondary malignancy reported in these patients. In a recent study we reported on spontaneous memory T cell responses against CLL-associated antigens defined by HLA ligandome analysis in CLL patients. Moreover, retrospective analysis suggests a significant survival benefit for CLL patients showing immune reactions to more than one of the tested antigens (Kowalewski et. al., PNAS 2015). Here we analyzed our CLL patient cohort for the incidence of secondary malignancies, and in particular for skin cancers. A total of 15 CLL-associated HLA-peptides (6 A*02, 3 A*03, 6 B*07) were implemented for stimulation of HLA-matched PBMC obtained from CLL patients. 45 CLL patients (Binet A n=15, Binet B, n=15, Binet C n=15, median age, 33.3% (15/45) with prior CLL specific therapy) were analyzed for antigen-specific memory T-cell responses in IFNγ-ELISPOT assays. 13 patients (28.9%, response cohort) showed specific T cell response against 〉 1 antigen, while 32 patients (71.1%, negative cohort) showed none or only one immune response. The median age was 71 in the response cohort and 73 in the negative cohort. 40.0% (6/15) of CLL patients with Binet stage A and 33.3% (5/15) with stage B were classified as responders whereas only 13.3% (2/15) of patients with stage C showed 〉1 immune responses. Of the included CLL patients with prior therapy only one patient (8%) showed 〉 1 immune responses. In the analyzed CLL patient cohort 7 skin cancers (melanoma: n=2, squamous cell carcinoma: n=2, basal cell carcinoma: n=3), 3 precancerous skin lesions and 9 other secondary malignancies including renal cell carcinoma (n=2), colon (n=3), breast (n=2) and prostate cancer (n=2) were observed. In the negative cohort 34.4% (11/32) of the patients developed a secondary malignancy, while only 15.4% (2/13) in the immune response cohort presented with secondary neoplasms. Strikingly all skin cancers and precancerous skin lesions were observed in the non-immune responder cohort. Furthermore multiple secondary malignancies (e.g. squamous cell carcinoma and colon cancer) were only observed in the cohort that showed no immune response. Taken together our data suggest that a favorable immune signature in CLL patients, i.e. the ability to mount T-cell responses against CLL-associated antigens not only is associated with improved overall survival but also might protect these patients from the development of secondary skin cancers. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Thrombomodulin plays an important role in the regulation of serine proteases and endothelial function. Because of its multiple modulatory action it has a central role in inhibiting thrombogenesis and inflammatory processes in such complex disorders as sepsis associated coagulopathies. A recombinant thrombomodulin (Recomodulin, Asahai Kasei, Tokyo, Japan) is currently used for the management of disseminated intravascular coagulation (DIC) and related disorders in Japan and is currently undergoing a global phase 3 trial in sepsis-induced coagulopathy. Recomodulin (RM) is capable of forming complexes with circulating thrombin generated in sepsis and not only inhibits its coagulant function but also inhibits thrombin generation. The purpose of this study is to determine the effect of RM on thrombin generation in plasma systems and its modulation by both activated and non-activated prothrombin complex concentrates (PCCs). PCCs may have the potential to overcome the anticoagulant effects of RM and may be useful as an antidote. Materials and Methods: RM 12,800 IU (2.02 mg) ampules were commercially obtained. Working solutions of buffered RM were prepared at 100 µg/ml. Tissue factor mediated generation of thrombin and its inhibition in normal human plasma (NHP) (n=50), platelet rich plasma (PRP) (n=50), plasma obtained from patients with DIC (n=25) and various PCCs such as Beriplex, Cofact, Feiba, Konyne, Octaplex, Preconativ, Profilnine, Prothromplex at 1 - 5 IU/ml were investigated. A fluorometric thrombokinetics method (Technoclone, Vienna, Austria) was used to determine thrombin generation and its modulation by RM. In addition such thrombin generation markers as prothrombin fragment (F1.2) and thrombin-antithrombin complex (TAT) were measured. The effect of RM on thrombin mediated fibrinokinetics was also measured using an optical method. Results: RM produced a concentration dependent inhibition of thrombin generation in the plasma based systems. In NHP the IC50 was 0.29±0.06 µg/ml in contrast to PRP where the IC50 was 1.99±0.31 µg/ml. The inhibition of thrombin generation in various PCCs was also concentration and product dependent and only four factor concentrates were found to generate thrombin.. At concentrations of 1 IU/ml marked inhibition of thrombin generation was noted in Preconativ, Prothromplex and Beriplex. RM decreased the generation of F1.2 and TAT in a concentration dependent manner. However, at concentrations 〉2.5 IU/ml the thrombin generated in such PCCs as Prothromplex and Beriplex, markedly higher thrombin was generated overcoming the anticoagulant effect of RM. Proportionately higher levels of thrombin generation markers were formed and were dependant on PCC concentrations. RM also inhibited the thrombin induced formation of fibrin in both PRP and PPP systems in a differential manner. However, at higher concentration of thrombin the inhibitory effects of RM on fibrin formation were overcome. Conclusions: These results suggest that in plasma based systems and in the PCCs at concentrations

Description: Introduction. Upper extremity deep vein thrombosis (UEDVT) is a relatively uncommon event with potentially serious complications. Its clinical outcomes are not well studied. The objective of this study was to assess the incidence of post-thrombotic syndrome (PTS) and functional disability in patients with UEDVT. Patients and methods. This was a pre-specified analysis of a prospective cohort study at 5 Canadian centres. We enrolled adult patients with a symptomatic UEDVT confirmed by compression ultrasound involving the brachial or more proximal veins, with or without a pulmonary embolism (PE). Exclusions included pregnancy, dialysis catheter thrombosis, active or high bleeding risk, platelet count