bluuebunny/crossref_metadata_2025 huggingface dataset:
Clots from FXI-Deficient Patients Display a Reduced Fibrinolytic Resistance
Authors: Peyvandi Flora , Cannavò Antonino , Incampo Francesca , Menegatti Marzia , Semeraro Nicola , Biguzzi Eugenia , Siboni Simona Maria , Colucci Mario
Published: October 2019

Factor XI deficiency (FXI-Def) is associated with mild, unpredictable bleeding tendency. FXI is believed to participate in a thrombin-dependent positive feed-back that leads to the formation of a second burst of thrombin, which, in turn, inhibits fibrinolysis mainly through TAFI activation. It is conceivable, therefore, that FXI-Def leads to the formation of clots that are more susceptible to lysis. Our study was undertaken to test this hypothesis.

Eighteen subjects with FXI-Def (1-58%), and 17 matched controls were studied. All assays were performed on citrate-plasma stored at -80°C.

The fibrinolytic resistance to t-PA (30 ng/ml) of clots generated by 0.5 pM tissue factor was lower in samples from FXI-Def subjects than in controls (lysis time: 61.1 ± 9.7 vs 74.9 ± 14.9 min, P=0.008). Generation of thrombin and TAFIa during fibrinolysis was markedly reduced in FXI-Def samples (<0.0005) and TAFIa10min was significantly correlated with lysis time (rho=0.56, P=0.01). Furthermore, enhancement of TAFI activation by thrombomodulin increased fibrinolytic resistance to normal. However, neutralization of TAFIa by PTCI did not abolish the difference between the two groups, suggesting that both TAFI-dependent and TAFI-independent mechanisms concurred to enhance the lysability of FXI-Def clots. Several findings suggest that FXI-Def is not the main cause of hastened fibrinolysis: 1) lysis time did not correlate with FXI level; 2) a significant difference in fibrinolytic resistance between FXI-Def and controls was observed under conditions that bypassed FXI activation (clots induced by thrombin+CaCl2+FXa-inhibitor or by a 100-fold higher concentration of tissue factor) or after normalization of FXI levels by addition of purified FXI. In conclusion, bleeding in FXI-Def may result from premature lysis of the haemostatic plug, which ensues because of little TAFI activation and other unidentified mechanisms. Moreover, it appears that FXI-Def is not part of the causal pathway leading to hastened fibrinolysis but rather a bystander.

Disclosures

Peyvandi: NovoNordisk: Research Funding, speaker's fee Other; CSL Behring: speaker's fee, speaker's fee Other; LFB: speker's fee, speker's fee Other; Grifols: speaker's fee, speaker's fee Other; Bayer: speaker's fee Other; Baxter: speaker's fee Other; Biotest: Research Funding; Kedrion biopharma: Research Funding. Colucci:Boehringer Ingelheim : Research Funding.
A Systems Biology Model for the Coagulation Network in Non-Bleeding State Describes Baseline Activity of Coagulation
Authors: Lee Dooyoung , Nayak Satyaprakash , Pittman Debra , Arkin Steven , Martin Steven , Heatherington Anne C , Vicini Paolo , Hua Fei
Published: October 2019

Coagulation is a process crucial to stop bleeding from a damaged vessel. The network is comprised of a complex interplay of various pro-coagulant and anti-coagulant factors. Several systems models for the coagulation pathway have been published, as a way to investigate the pathway complexity. Most published models describe the thrombin generation profile (TGA) or prothrombin time (PT) following extrinsic pathway activation by tissue factor, or activated partial prothrombin time (aPTT) through activation of the intrinsic pathway by contact activation. In the clinical setting, thrombin-anti-thrombin complex (TAT) and prothrombin fragment 1+2 (PF1+2) are often used as biomarkers for in vivo coagulation activity in the non-bleeding state. Even in the absence of any treatment, there are detectable levels of PF1+2 and TAT in healthy volunteers indicating low level coagulation activity exists in the normal baseline, non-bleeding state. In this study, we developed a mathematical model for coagulation to describe the baseline activity of PF1+2 and TAT. We then used the model to understand the impact of the coagulation pathway activity during non-bleeding state on thrombin generation activated by tissue factor.

First, a coagulation model from Hockin et al. (J Biol Chem. 2002;277(21):18322-33) was modified to describe internally generated data for thrombin generation and aPTT modulation following addition of various concentrations of recombinant factor VIIa or plasma derived factor Xa to normal or hemophilic human plasma. Next, protein synthesis and degradation were incorporated into the model and platelet-dependent reactions were tuned down to describe the baseline coagulation activity in non-bleeding healthy subjects. Using a simulated annealing algorithm, the new parameters were optimized to fit published data for PF1+2 and TAT in healthy volunteers and changes of PF1+2 following treatment with eptacog alfa (recombinant factor VIIa). In the model, the baseline coagulation activities can be described by a very low level of tissue factor, which is much lower than the detection limit of a regular ELISA method. Using this baseline model, we tested the effect of this baseline coagulation activity on tissue factor-activated thrombin generation, and found that very small baseline levels of activated enzymes significantly shorten the lag time of thrombin generation, but did not affect the peak thrombin. We also performed a global sensitivity analysis to identify key proteins in the coagulation network whose modulation will have the biggest impact on PF1+2 and TAT levels.

Disclosures

Lee: Pfizer Inc.: Employment. Nayak:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment. Arkin:Pfizer Inc.: Employment. Martin:Pfizer Inc.: Employment. Heatherington:Pfizer Inc.: Employment. Vicini:Pfizer Inc.: Employment. Hua:Pfizer Inc: Employment.
Outcomes of Central Venous Catheter Associated Upper Extremity Deep Vein Thrombosis in Cancer Patents
Authors: Delluc Aurelien , Le Gal Gregoire , Scarvelis Dimitrios , Carrier Marc
Published: October 2019

Introduction: Upper extremity deep vein thrombosis (UEDVT) is a frequent complication of central venous catheters in cancer patients. Anticoagulation with low molecular weight heparin (LMWH) for a minimum of 3 months is the currently recommended treatment for catheter-related UEDVT in cancer patients. Following catheter removal and a minimum of three months of anticoagulant therapy, the risk of recurrence of VTE is likely to be low and it might be reasonable to discontinue anticoagulant therapy. However, there are no data available to support these hypotheses. Therefore, we sought to assess the efficacy and safety of LMWH for the treatment of catheter-related UEDVT in cancer patients and determine the risk of recurrence of VTE after discontinuation of anticoagulation.

Material and methods: A retrospective single center cohort study including consecutive cancer outpatients assessed between July 2008 and December 2012 for the management of symptomatic central venous catheter associated proximal UEDVT was conducted.

Results: A total of 99 patients were included. Among them, 89 were treated with one month of full therapeutic weight-adjusted dose of LMWH followed by an intermediate dose. A prophylactic dose of LMWH was given after 3 (n=8), 6 (n=4), or 12 (n=1) months of intermediate dosing of LMWH. The remainder patients continued with intermediate dose of LMWH until discontinuation or last follow up. Median duration of anticoagulation was 124 days (range 40 to 1849). No recurrent VTE and two major bleeding episodes occurred during the first 3 months of treatment. Among the 13 patients who were receiving prophylactic doses of LMWH after completion of 3, 6 or 12 months at full and intermediate doses, 2 (15.4%) had a recurrent VTE event. Others did not recurred while on treatment.

Eighty-three patients discontinued anticoagulation and 80 could be followed-up after anticoagulation discontinuation for a median of 632 days (range 6 to 2495). Central venous line was pulled out in 77 patients (96.2%). Five recurrences were observed during follow up. The cumulative probability of recurrent VTE was higher in patients whose cancer was active at the time of anticoagulation discontinuation as compared with those in remission (22.2% (95% CI: 0 to 40.6) vs. 2.3% (95% CI: 0 to 6.7)) (Figure 1).

Conclusion: LMWH can safely be used to treat catheter associated proximal UEDVT in the setting of cancer. In patients whose central venous line has been pulled and cancer is in remission, anticoagulation therapy can be safely discontinued.

Figure 1: cumulative probability of recurrence according to cancer status at the time of anticoagulation discontinuation

Disclosures

No relevant conflicts of interest to declare.
Transcription Activation of an Antisense Long Noncoding RNA (lnc-Mega1) of RBM15 By RUNX1 Enhances Megakaryocyte Differentiation
Authors: Zhao Xinyang , Tran Ngoc-Tung , Zhou Dewang , Su Hairui , Zhang Li , Townes Tim M.
Published: October 2019

RBM15 encodes an RNA binding protein, which regulates self-renewal and differentiation of hematopoietic stem cells. RBM15 knock-out promotes the proliferation of Mk progenitor cells but blocks the maturation of Mk progenitor cells as measured by polyploidy. RBM15-MKL1 fusion causes pediatric acute megakaryoblastic leukemia without Down’s syndrome. Here, we cloned a long non-coding RNA (we called Lnc-Mega1), which is transcribed in the opposite direction of the human RBM15 gene. Lnc-Mega1 is not transcribed in mice in the RBM15 locus. Blast search also indicates there are no homologues available in mouse. The 5’end of Lnc-Mega1 overlaps with the 5’ end of RBM15 transcript for 180bp. We found that RUNX1 binds to the promoter regions of RBM15 by chromatin immunoprecipitation assay. RUNX1 knock-down reduces the transcription of both RBM15 and Lnc-Mega1. Therefore, RUNX1 controls the transcription of both genes. Nevertheless, the promoter is not always bidirectional. We found in hematopoietic stem cell RBM15 is transcribed more actively than lnc-Mega1. During differentiation, both lnc-Mega-1 and RBM15 are transcribed actively. In addition, ectopic expression of Lnc-Mega1 enhances megakaryocyte differentiation in both leukemic cell lines and human CD34+ cord blood cells. The tertiary structure of the lnc-Mega1 is critical as truncations cannot expedite differentiation. Many genes important for hematopoiesis such as Pu.1, p21, and p53 contain anti-sense transcription, but the mechansims for antisense RNA molecules in regulating their sense counterparts are very different. Although RBM15 does not directly interact with lnc-Mega1, Lnc-Mega1 overexpression enhances the RBM15 protein level via increasing the translation efficiency. Given that RBM15-MKL1 is the causal protein in acute megakaryoblastic leukemia, lnc-Mega1 RNA might contribute to leukemogenesis by increasing the translation efficiency of the fusion protein.

Disclosures

Zhou: Univeristy of Alabama at Birmingham: Employment. Townes:University of Alabama at Birmingham: Employment.
Twist-1 Increases Hematopoietic Stem Cell Self-Renewal and Causes Myeloid Skewing
Authors: Ma Xiaotong , Dong Chengya , Liu Xiaoyan , Wang Nan , Wang Lina , Wang Jianxiang
Published: October 2019

Twist-1 protein belongs to the large family of basic Helix-Loop-Helix transcription factors with diverse physiological functions in mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, there is little information available regarding its expression pattern and functional role in the hematopoietic system. We analyzed Twist-1 expression patterns in different hematopoietic cell populations from normal mouse bone marrow, and found that Twist-1 was most highly expressed in long-term hematopoietic stem cells (LT-HSCs) but showed a low abundance in more differentiated descendants. To investigate Twist-1 gene function, retroviral-mediated overexpression or removal experiments were performed. Competitive repopulation studies demonstrated that enforced expression of Twist-1 in HSC-enriched Lin−c-Kit+Sca-1+ (LKS) cells resulted in an increase in the size of the G0 population, and in their reconstitution ability after the first and a second transplantation. Conversely, removal of Twist-1 in LKS cells impaired their ability to repopulate. In addition, increased Twist-1 expression caused a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activated myeloid lineage-determining factors PU.1 and GATA-1 and down-regulated lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms revealed that Twist-1 overexpression lead to altered expression of Runx1/c-Mpl/Tie2 regulatory pathway, and quiescence-associated N-cadherin and Hes1 in LKS cells, which may contribute to the phenotypes observed in Twist-1-overexpressing mice. These studies shed additional light on the mechanisms involved in the maintenance of normal HSC and myeloid lineage differentiation, and may also provide clues to the mechanisms controlling pathogenesis and preservation of leukemic stem cells.

Disclosures

No relevant conflicts of interest to declare.
Osteoblast Ablation Causes an Imbalance of the HSC Population
Authors: Garcia Mayra
Published: October 2019

The hematopoietic stem cell (HSC) niche is composed of many different types of support cells including endothelial cells, stromal cells, and osteoblast. Studies that ablated mature osteoblast showed that they might be important to support maintenance of HSCs. These studies used Col2.3ΔTK to ablate differentiated osteoblast with daily treatments of ganciclovir after which the samples were analyzed immediately. Two opposing studies have been done using this system one showing a decrease in HSC and another showing an increase thus the role of osteoblast in HSC maintenance is still unclear. We have used a different method of ablating cells using Osteocalcin (Oc) and Osterix (Osx) Cre to drive expression of a diphtheria toxin receptor (iDTR), in mature osteoblast and osteoprogenitors, respectively. We injected eight week old mice with three doses of diphtheria toxin and analyzed the LSK and LT-HSC populations a month after cell ablation. There was no effect when the osteoprogenitors were ablated using Osx-Cre; suggesting ablation at postnatal stages is likely necessary to see an effect using Oxs-Cre. On the other hand, we found that a month after ablation of the Oc expressing cells the osteoblast population had recovered but the LSK and LT-HSC populations were still affected with an increase in both populations, suggesting that being exposed to a defective niche might have long-term effects on the HSC population.

Disclosures

No relevant conflicts of interest to declare.
Long Term Follow up of SWOG S0313: Ibritumomab Tiuxetan Consolidation after 3 Cycles of CHOP Plus Radiotherapy for High Risk Limited Stage Aggressive B-Cell Lymphoma
Authors: Persky Daniel O , Miller Thomas P. , Unger Joseph M , Spier Catherine M. , Puvvada Soham D. , Stea B. Dino , Press Oliver W. , Constine Louis , Barton Kevin , LeBlanc Michael , Smith Sonali , Fisher Richard I. , Friedberg Jonathan W.
Published: October 2019

Introduction: Patients with limited stage aggressive B-cell non-Hodgkin lymphoma (LS-NHL) and at least one stage-modified adverse risk factor have an excessive relapse rate leading to a 5-year overall survival (OS) of 50-77% and 10-year OS of 0-50%. In SWOG S0014 we have shown that the addition of rituximab to 3 cycles of CHOP plus involved field radiation therapy (IFRT) resulted in an improved estimated 4-year progression-free survival (PFS) of 88% and OS of 92%. Relapses were largely systemic (5 of 6 evaluable) and continued to be seen with longer follow-up. Ibritumomab tiuxetan (Zevalin ®) is a radiolabeled anti-CD20 antibody that has excellent single agent activity in diffuse large B-cell lymphoma and could prevent systemic relapse of disease. We now report long term results of SWOG S0313, a phase II study of ibritumomab tiuxetan consolidation after 3 cycles of CHOP plus IFRT in patients with LS-NHL.

Methods: Patients with LS-NHL and at least one stage-modified adverse risk factor (non-bulky stage II, age > 60 years, elevated LDH, or WHO performance status of 2) were treated with CHOP on days 1, 22, and 43, followed 3 weeks later by 40-50 Gy of IFRT. Ibritumomab tiuxetan regimen was initiated 3 – 6 weeks following IFRT.

Results: Forty-six patients were registered and eligible, with median follow-up of 7.3 years. Median age was 61, 37% of patients had elevated LDH, and 20% had systemic symptoms. Grade 4 adverse events occurring more than once included neutropenia (8 patients), leukopenia (5), and lymphopenia (2). Febrile neutropenia was observed in 4 patients. No cases of treatment-related myeloid neoplasms were noted. Eleven patients progressed and 8 patients died. The PFS estimate is 89% at 2 years, 82% at 5 years, and 75% at 7 years. OS estimate is 91% at 2 years, 87% at 5 years, and 82% at 7 years. These outcomes compare favorably to matched cohorts on prior SWOG trials, with 7-year PFS estimate of 68% on S0014 and 65% on S8736 (original pre-Rituximab trial); and 7-year OS estimate of 80% on S0014 and 73% on S8736 cohorts.

Conclusions: Patients with high-risk LS-NHL treated with 3 cycles of CHOP plus IFRT followed by ibritumomab tiuxetan consolidation had outcomes that compare favorably to our historical experience. A US cooperative group study of R-CHOP and response-adapted IFRT followed by consolidative ibritumomab tiuxetan is ongoing.

Disclosures

Off Label Use: ibritumomab tiuxetan in diffuse large B-cell lymphoma.
Antiviral Efficacy Analysis of Telbivudine Concurrent with the First Cycle of Chemotherapy in HBsAg-Positive Lymphoma Patients
Authors: Li Wenyu , Huang Ling , Guo Hanguo , Wei Xiaojuan , Liang Zhanli
Published: October 2019

Background: HBV reactivation is a common complication in HBsAg-positive lymphoma patients with chemotherapy or immunotherapy. There is no standard antiviral treatment for these patients undergoing chemotherapy or immunotherapy. The initial time, the agent and the duration time are confused at present.

Objective: This study aimed to investigate the antiviral efficacy of telbivudine concurrent with initiation of chemotherapy in HBsAg-positive patients with lymphoma.

Study design: Between April 2008 and October 2012, 359 patients with pathological diagnoses of lymphoma were admitted to the hospital. Among those, a cohort of 60 HBsAg-positive cases were enrolled in this retrospective study,and telbivudine was taken at the same day or one day prior to the first cycle of chemotherapy. The rates of HBV reactivation, virological response, undetectable HBV DNA, the relationship between HBV reactivation and its clinical characteristics were investigated.

Results: The rate of HBV reactivation was 11.7% (7/60), while it was 17.5% (7/40) and 75% (3/4) in patients treated with rituximab based chemotherapy and rituximab maintenance therapy, respectively. The fulminant hepatitis rate was 6.6%. The rates of virological response, undetectable HBV DNA and ALT normalization were 88.3%, 61.7% and 83.3%, respectively. HBV reactivation was associated with rituximab (P=0.013), and HBeAg-negativity (P=0.016), but not correlated with age, gender, clinical stages, extranodal disease, bone marrow involvement, B symptoms, HBV viral loads or serum LDH level.

Conclusion: Concurrent telbivudine treatment with initial chemotherapy can effectively reduce HBV reactivation in HBsAg-positive lymphoma patients, and the efficacy is independent of the baseline HBV viral loads.

Disclosures

No relevant conflicts of interest to declare.
Ocular Adnexal Marginal Zone Lymphomas (OAMZL): Lack of Evidence for Chlamydia Psittaci (C. Psittaci) Infection in a Clinicopathologic Study from a Single Institution
Authors: Merchán Brayan Marcel , Jaramillo Anny , Tortola Teresa , Abrisqueta Pau , Castellvi Josep , Medel Ramón , Flores Katia , Hernandez Javier , Gironella Mercedes , Lopez-Hernandez Andres , Bosch Francesc
Published: October 2019

Ocular adnexal lymphomas (OAL) represent up to 55% of all orbital tumors, being extranodal marginal zone lymphoma (OAMZL) the most frequent histological subtype reported in approximately 50% of patients and its etiology and pathogenesis are still not well understood. There are conflicting reports regarding the association between C. Psittaci infection and ocular adnexal lymphoma (OAMZL), in part because this association may vary between different geographical regions. Herein, we analyzed the clinical features and the presence of C. Psittaci infection in a cohort of 28 patients with OAMZL diagnosed from a single Spanish institution. All tumor samples were centrally reviewed by two pathologists, and C. Psittaci infection was determined in tumor specimens by a Semi-nested PCR method. Between January 1984 and April 2009, 28 patients were diagnosed with primary OAMZL. The median age was 70 years (range: 31- 89) and 40% were male. The great majority of patients (79%) were diagnosed in stage I-extranodal (IE), and six patients (21%) had bilateral ocular involvement. IgH rearrangement was confirmed in 25 out of 28 cases with OAMZL. PCR for C. Psittaci infection was negative in all 28 tumor specimens analyzed. Most patients (82%) were treated with immunochemotherapy combinations, being chlorambucil based regimens the most frequently used. Overall, 18 out of 24 patients (75%) achieved a complete response after first-line treatment. Median PFS was of 79 months (IC 95% 36 - 121). Whereas C. Psittaci infection in OAMZLs has been proved in up to 90% of patients in Northern Italy, in this Spanish cohort OAMZLs were not associated with C. Psittaci infection. These findings confirm that the association between C. Psittacci infection and OAML is highly heterogeneous even between the same geographic regions. In these regions where C. Psittaci infection is not prevalent, the search for alternative antigenic stimuli driving the appearance of this lymphoma is warranted.

Disclosures

No relevant conflicts of interest to declare.
Fasudil, a Rho-Associated Protein Kinase (ROCK) Inhibitor, Decreases Disease Burden in a Cbl/Cbl-b Deficiency-Driven Murine Model for Myeloproliferative Disorders
Authors: William Basem M. , Feng Dan , An Wei , Nadeau Scott , Mohapatra Bhopal , Storck Matt , Band Hamid
Published: October 2019

Myeloproliferative disorders (MPDs) are heterogeneous clonal diseases with variable clinical courses. Mutations in the Cbl family genes have been reported in multiple independent studies to be present in about 10% of patients with MPDs and these patients tend of have a poorer prognosis. We have previously demonstrated that Cbl-flox/flox, Cbl-b-null mice rendered Cbl/Cbl-b double knockout (DKO) in the hematopoietic compartment with MMTV-Cre develop a disease phenotypically similar to human MPDs. An in vitro kinase inhibitor screen identified fasudil, a ROCK inhibitor, with relatively selective anti-proliferative activity against Cbl/Cbl-b DKO vs. control murine bone marrow cells. We established a mouse model with a uniform time of MPD onset by transplanting Cbl/Cbl-b DKO bone marrow cells (2x106 per recipient) into 21 busulfan-conditioned NOD/SCID/gamma chain-deficient (NSG) mice. All recipients showed donor cell engraftment. Four weeks post-transplant, we treated 13 mice with 100 mg/kg fasudil daily by gavage. By two weeks of treatment, total white cell and monocyte counts were significantly lower in mice treated with fasudil (p=0.02 and 0.04 respectively). For the entire group, we observed a trend towards improved survival in fasudil-treated mice that didn't reach statistical significance (p=0.07). However, analysis of male recipients only (n=12) revealed a significant survival advantage in fasudil-treated group (p=0.04). The gender difference may stem from a currently unexplained more severe disease phenotype we observed in female recipients. Notably, while all untreated mice succumbed to MPD, prolonged survival was observed in 2 mice beyond 27 weeks, nearly twice the average life-span in the Cbl/Cbl-b DKO MPD model (16 weeks). The 2 long-term survivors had undetectable levels of myosin light chain (MLC), a downstream target of ROCK phosphorylation (figure) suggesting that inhibition of downstream signaling of ROCK is associated with improved disease control and survival. Taken together, our data suggest a therapeutic potential for fasudil in the treatment of MPDs or other mutant Cbl-driven myeloid disorders.

Figure: Fasudil activity in mouse model of Cbl/Cbl-b DKO MPD. Total WBC (A) and monocyte (B) counts over time in untreated and fasudil-treated mice (mean+/-SEM), C: MLC levels by Western Blot in peripheral blood of control vs. fasudil-treated mice; those surviving longer than 27 weeks are indicated. Figure:. Fasudil activity in mouse model of Cbl/Cbl-b DKO MPD. Total WBC (A) and monocyte (B) counts over time in untreated and fasudil-treated mice (mean+/-SEM), C: MLC levels by Western Blot in peripheral blood of control vs. fasudil-treated mice; those surviving longer than 27 weeks are indicated.

Disclosures

No relevant conflicts of interest to declare.
First-Line Prescribing Preferences of U.S. Hematology-Oncology Physicians for Patients with CLL: Impact of Novel Agents
Authors: Green Mark R. , Williams Michael E. , Willey Joanne , Buettner Arden , Neely Douglas , Lankford Maria
Published: October 2019

Background: In November, 2013 obinutuzumab (obin) was approved in combination with chlorambucil (chlor), as 1st line therapy (Rx) for patients (pts) with chronic lymphocytic leukemia (CLL). In February, 2014, ibrutinib was approved in CLL for pts who had received > 1 prior therapy. In April, 2014 ofatumumab (ofa) + chlor was approved as 1st line Rx for pts with CLL for whom fludarabine-based therapy is considered inappropriate. During 2014 we studied PPrefs of 180 U.S-based hematology-oncology physicians (HOPs) for 1stline Rx across a range of clinical scenarios varying by age, co-morbidities, and selected laboratory features.

Methods: PPrefs were assessed through a validated, proprietary, live, case-based market research tool (Challenging Cases®). Assessment dates were 3/08/14 and 4/26/14. Data were acquired using blinded, audience-response technology. A core scenario (CS) and 3 variant scenarios (VS) were utilized to evoke physician PPrefs. CS: 63-year-old male; 1-2 cm diffuse adenopathy, spleen 3 cm below costal margin. Presented with recent fatigue/ low grade fevers; no relevant co-morbidities; CD 38=12%. No FISH abnormalities. PS1. VS1: All equal except 17p deletion and CD 38 = 42%; VS2: CS (age 63; no abnormal FISH) plus co-morbidities (medication-controlled hypertension, type II diabetes, and mild COPD); VS3: VS2 BUT age 73 with same co-morbidities.

Results: PPrefs by scenario shown below: Abstract 4676. Table 1PPref CS VS1 VS2 VS33/8/144/26/143/8/144/26/143/8/144/26/143/8/144/26/14Bendamustine + rituximab[BR]61%63%52%51%72%68%66%64%Bendamustine + Obinutuzumab0%2%2%6%0%1%3%2%Chorambucil + Obinutuzumab1%3%2%3%2%5%8%17%FCR20%16%25%19%5%8%1%2%Ibrutinib + additional agent(s)2%3%11%16%7%8%6%7%Other2%2%4%2%2%2%3%2%Observe14%10%3%3%11%6%12%5%

Conclusions: As of March-April 2014 BR is the 1st line PPref of 51% -72% of US-based HOPs across 4 plausible newly diagnosed CLL scenarios. In VS1 (age 63; 17p deletion) ibrutinib based Rx is emerging despite lack of 1st line indication. In VS 3 (age 73, multiple co-morbidities present, neither high risk FISH nor CD 38 positivity) 1st line PPref for Chlor + Obin is emerging. Prior to ASH 2014, we will assess PPrefs of approximately 180 additional HOPs related to these scenarios (with ofatumumab + chlorambucil added as a treatment option) and have aggregate PPref data (N > 350) across all of 2014 for ASH.

Disclosures

Williams: Pharmacyclics, Janssen: Consultancy, Research Funding.
A Novel PI3K Inhibitor Identified By a High Throughput Virtual Screen Displays Potent Activity Against Multiple Myeloma
Authors: Cao Biyin , Zhu Jingyu , Wang Man , Yu Yang , Qi Huixin , Han Kunkun , Zhang Zubin , Zeng Yuanying , Qiao Chunhua , Zhang Hongjian , Hou Tingjun , Mao Xinliang
Published: October 2019

The phosphatidylinositol-3-kinase (PI3K) has been developed as a promising target for the treatment of many cancers, including hematological malignancies, such as lymphoma, leukemia, and multiple myeloma (MM). Recent clinical trials have demonstrated Idelalisib, a specific PI3Kdelta inhibitor, reached a total 57% of response rate in relapsed indolent lymphoma and 72% of overall complete rate in relapsed chronic lymphocytic leukemia. MM remains fatal as a cancer of plasma cells because of lack of efficient drugs. Therefore, we tried to find novel PI3K inhibitors for MM therapy. To this end, we performed a high throughput virtual screen against 800,000 small molecule compounds using PI3Kγ as the analytic model and identified C98, a function unknown compound, as a potent inhibitor of PI3Ks. The cell-free enzymatic studies showed that C98 inhibited all class I PI3Ks at nano- or low micromolar concentrations but had no effects on AKT or mTOR activity. Molecular docking analysis revealed that C98 interfered with the ATP-binding pockets of PI3Ks by forming H-bonds and arene-H interactions with specific amino residues through which C98 specifically inhibited PI3K/AKT/mTOR signaling pathway, but had no effects on other kinases and proteins including IGF-1R, ERK, p38, c-Src, and STAT3. PTEN is a key negative modulator of the PI3K signaling pathway, but KIF did not show any induction of PTEN, thus further demonstrating that KIF is a selective PI3K inhibitor. Inhibition of PI3K by C98 led to myeloma cell apoptosis as shown activated caspase cascade signaling. Furthermore, oral administration of C98 delayed tumor growth in two independent human myeloma xenograft models in nude mice but did not show overt toxicity. Pharmacokinetic analysis revealed that C98 can be found in MM tissues at an average concentration up to 622.6 ng/g, suggesting that C98 can penetrate into the tumor tissues where it exerts its anti-myeloma activity. It also suggests C98 has a reliable oral activity. Therefore, through a high throughput virtual screen we identified a novel PI3K inhibitor that is orally active against multiple myeloma with great potential for further development.

Disclosures

No relevant conflicts of interest to declare.
Chemotherapy-Induced Senescence Reprograms Lymphoma and Leukemia Cells into Latent Cancer Stem Cells That Are Susceptible to Conceptually Novel Treatments
Authors: Schmitt Clemens A. , Milanovic Maja , Daebritz Henry , Zhao Zhen , Trumpp Andreas
Published: October 2019

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates further expansion of (pre-)malignant cells. Senescence imposes a tumor-suppressive barrier in lymphomagenesis, and acts as an effector program in response to chemotherapy in various hematological malignancies. Interestingly, many key signaling components of the senescence machinery also operate as critical regulators of stem cell functions (collectively termed ‘stemness’), among them the p53 axis and control of lysine 9 trimethylation at histone H3 (H3K9). We investigated here in vitro and in vivo whether chemotherapy-induced senescence may change stem cell-related functionalities in aggressive B-cell lymphomas and acute leukemias of murine and human origin.

Gene expression and functional analyses comparing senescent vs. non-senescent Eµ-myc transgenic B-cell lymphomas unveiled massive upregulation of an adult tissue stem cell signature, activated Wnt signaling, and de novo expression of distinct stem cell markers in senescence. Utilizing Suv39h1- (an H3K9-targeting methyltransferase) and p53-based genetically switchable ‘matched pair’ on/off models of senescence to mimic spontaneous escape (‘previously senescent’, PS), we found PS cells to re-enter the cell-cycle with strongly enhanced and Wnt-dependent clonogenic growth when compared to their equally chemotherapy-exposed but never senescent (NS) counterparts.In vivo, these PS lymphoma cells presented with a much higher tumor initiation potential, which was neutralized upon pharmacological or genetic Wnt inhibition. Strikingly, temporary enforcement of senescence in a p53-regulatable leukemia model reprogrammed non-stem bulk PS leukemia cells into leukemia-initiating stem cells, whose de novo self-renewing potential was also Wnt-dependent. In contrast, equally chemotherapy-exposed NS bulk cells (i.e. p53 always ‘off’) did not acquire stemness potential.

Our data, further supported by consistent findings in various human cancer cell lines and primary patient-derived lymphoma and leukemia samples, characterize senescence as a fundamentally reprogrammed cellular condition, and uncover senescence-associated stemness as an unexpected, cell-autonomous feature that exerts its detrimental potential upon escape from the cell-cycle block. These findings raise concerns about the long-term benefit of senescence-inducing cancer therapies, and provide new mechanistic insights into the plasticity of the “cancer stem cell” condition. In turn, we present synthetic lethal targeting of senescence-associated stemness as a conceptually novel, outcome-improving treatment strategy in lymphoma, leukemia and possibly other cancer entities as well.

Disclosures

No relevant conflicts of interest to declare.
Intravenous Administration of Liposome Encapsulated DFO Efficiently Removes Iron from the Liver and Spleen of Iron Overloaded Mice
Authors: Tran David T , Noble Charles O , Hayes Mark E , Szoka Francis C
Published: October 2019

Introduction: Long-term red blood cell transfusions effectively sustains patients who have β-thalassemia, sickle cell anemia, and myelodysplastic syndromes but they also lead to excess iron accumulation in the body. Iron overload is a major cause of morbidity and mortality in transfusion dependent patients. Chelation therapy reverses iron accumulation but marketed chelators have drawbacks such as: long infusions of deferoxamine (DFO, Novartis), large oral tablets with adverse effects (Exjade, Novartis), or twice daily oral dosing (Ferriprox, ApoPharma). These attributes contribute to poor compliance and poor outcomes in iron overload patients. To overcome long infusions and high doses of current therapies we have devised a stable nanoliposome encapsulated DFO (LDFO) for the treatment of iron overload.

Methods: LDFO composed of saturated soy phosphatidylcholine and cholesterol (3/2 molar ratio) is manufactured using a proprietary remote loading method that provides high encapsulation of DFO in 90 nm diameter liposomes. For pharmacokinetics and bioavailability studies, DFO and lipid concentrations in CF-1 mice plasma and tissues were analyzed by HPLC utilizing an in-house method. For iron removal efficacy studies, CF-1 mice were overloaded with iron dextran and after 10 days washout were treated with 100 mg/kg LDFO or unencapsulated DFO. Animals were sacrificed 5 days post treatment and tissue iron was measured by a ferrozine based spectroscopic assay.

Results: The manufacturing method to prepare LDFO results in a 300 g DFO/mole lipid encapsulation ratio. The formulation has greater than 6 months stability at 4 ºC. LDFO is long circulating and the DFO is bioavailable. At 24 hr post I.V. injection, there is 30% ID DFO in plasma and 10% ID DFO/g in liver whereas unencapsulated DFO is not detectable. Preclinical single dose safety studies in CF-1 mice indicate that LDFO is well tolerated at 300 mg/kg I.V. and 1250 mg/kg I.P. In the iron dextran overload model, LDFO greatly reduces iron levels in the liver and spleen. The absolute efficiency of LDFO is greater than 50% on a mole LDFO injected /mole iron removed from the liver (P<0.0004, n=4) and spleen (P<0.01, n=4). This is corroborated by an elevated iron accumulation in urine and feces from LDFO.

Conclusion: LDFO effectively removes iron from the liver and spleen with an overall molar efficiency > 50%. This high efficacy could lead to a dramatically improved treatment that increases compliance and provides substantially better management of iron overload than current treatments in patients suffering from iron overload conditions.

Disclosures

Tran: ZoneOne Pharma, Inc.: Employment. Noble:ZoneOne Pharma, Inc.: Employment, Equity Ownership. Hayes:ZoneOne Pharma, Inc.: Employment, Equity Ownership. Szoka:ZoneOne Pharma, Inc.: Consultancy, Equity Ownership.
Evaluation of Schistocyte Analysis By a Novel Automated Digital Cell Morphology Application
Authors: Hervent Anne-Sophie , Godefroid Maaike , Cauwelier Barbara , Van Hoof Achiel , Billiet Johan , Emmerechts Jan
Published: October 2019

Introduction: The CellaVision Advanced Red Blood Cell (RBC) Software Application is a new software for advanced morphological analysis of RBC which automatically performs a preliminary characterization and grouping of RBC into 21 morphological categories, including schistocytes. Upon automated classification, the software offers the possibility of reclassification of RBC by the operator. The aim of this study was to evaluate the schistocyte analysis by the CellaVision Advanced RBC Application.

Methods: Schistocyte counts were evaluated comparing the automated count on a CellaVisionTM DM96, both before and after reclassification, with the reference manual microscopic method. 76 samples of both hospitalized patients and healthy donors were analyzed.

Results: Within-run, between-run and between-observer coefficients of variation were lower when counted with the CellaVision compared to the manual microscopic count. The very high sensitivity but rather poor specificity implicates the need for reclassification by the operator, following automated analysis. After reclassification, method comparison studies revealed good agreement with the manual microscopic method, with however slightly higher values of schistocytes for the automated analysis.

Conclusion: The CellaVision Advanced RBC Software Application provides a sensitive, accurate and reproducible measurement of schistocytes in peripheral blood. Furthermore, it is an easy-to-use software and an excellent teaching tool that might contribute to standardization in the investigation of schistocyte-related conditions.

Disclosures

No relevant conflicts of interest to declare.
Could Cobalamin Deficiency Mimic a Clonal Aberration Cytogenetic? a Case Report
Authors: Bellesso Marcelo , Dias Daniela Ferreira , Centrone Renato Torrescasana , Garcia Laura Yolanda Chialanza , Lopes Annelise Correa Wengerkievicz , Santucci Rodrigo , Pinto Ana Paula Almeida Rocha Teixeira , Silva Leniza Pola , Oliveira Veronica Ramos Souza
Published: October 2019

A 32 years old female patient presented to our service complaining asthenia, apathy and loss of her power to work. It was observed, by laboratory test, pancytopenia (Hb: 6.0 g/dL; WBC: 2,100/mm³, platelets 95,000/mm³, Reticulocytes 0.8%) associated with marked elevation of LDH 1,800 U/L (normal range: 240 – 480U/L). Bone marrow aspirate demonstrated morphologic features of megaloblastic anemia. Moreover, low serum concentration of vitamin B12 < 150pg/mL (normal range: 200 – 95-pg/mL), Folic acid 15,9ng/mL (normal range: 3 – 17 ng/mL) confirmed Megaloblastic anemia by cobalamin deficiency. It was not evidenced gastritis. It was initiated the treatment with vitamin B12. However, in the next clinical attendance it was observed an unexpected cytogenetic result: 46,XX,del(9)(q13q22)[3]/76~78,XXX,+1,+3,+4,+9,+11,+12,+17,+20,+21[cp3]/46,XX[34].

It was interpreted that del(9)(q13q22)[3] could be a clonal cytogenetics aberration and the others data due to defects in synthesis of DNA by cobalamin deficiency. It was important, because in diagnosis of Megaloblastic anemia frequently it is not included cytogenetic analysis of bone marrow cells and a new cytogenetic analysis has become necessary due to this data. After two months, a complete hematological recovery was achieved and six months later, bone marrow aspirate and cytogenetic analisys were repeated. Normal morphologic bone marrow cells and normal cytogenetic: 46,XX[20] were evidenced. Therefore, it was really hard to conclude this case. First, cobalamin deficiency could promote this clonal deletion or, as a second hypothesis, a clonal cytogenetic with low proliferative rate was selected due to inefficient hematopoiesis, by vitamin B12 deficiency, and after the recovery of the hematopoiesis, this clonal was suppressed.

Disclosures

No relevant conflicts of interest to declare.
Experience of a Single Latin-American Institution Using a Colloidal Iron Oxide Coated with a Semisynthetic Carbohydrate (ferumoxytol) for Iron Deficiency Anemia (IDA) in Patients with Intolerance or Treatment Failure with Oral Iron
Authors: Ovilla Roberto , Calles-Payan Dannia , Reynolds-Ocampo Lucia A , Ruiz-Reyes Gabriela , Balderas-Delgado Carolina , Montesinos-Ovilla Andres , Cabrera Garcia Alvaro
Published: October 2019

Tolerance to oral iron and low percentage of absorption in the gut has provoked the Iron Deficiency Anemia patients (IDA) fail to treatment or they have symptoms of anemia even more than 12 weeks until recovery. Intravenous iron is an alternative to minimize risk and treatment-related adverse effects and maximize profits in the short and long term. The number of patients who have access to ferumoxytol is reduced in developing countries, therefore experience in the treatment with this type of iron is limited. Patients received ferumoxytol 1020mg (two 510mg 8 days apart) and the response at 4 weeks of treatment by raising hemoglobin and clinical response was evaluated without transfusion support. From a total of 30 patients 76.6% (19) were women of whom 36.8% (7) were postmenopausal. The average age was 45.3 years (range 21-76 years). Before starting treatment, the mean serum iron, ferritin and rate of transferrin saturation was 35.6 mcg (range 8–174 mcg/dL), 13.8 ng/mL (range 1.6–60 ng/mL), 10.8% (range 2-58%) respectively. The mean initial hemoglobin was 10.5 g / dL (range 7.1-13.7g/dL), reaching an average of 13.03 g dL (range 9.4 - 16 _g/dL) at 4 weeks, raising 2.52 g dL (0.3 - 5.4g / _ g/dL) in 28 days. The percentage of patients who achieved an increase of > 2 g/dL of hemoglobin was 70% (21). The initial symptoms attributed to anemia disappeared in 66.6% within the first 2 weeks and the rest in 4-5 weeks. Adverse effects more frequently than 2%: fatigue 16.6% (5), headache 10% (3), nausea and dizziness 10% (3), peripheral edema 6.6% (2) and hypersensitivity 3.3% (1). All previous were easily controlled with medications. Despite the low number of patients treated due to the difficult accessibility of the drug in our population, ferumoxytol was shown to be an attractive option that rapidly improves symptoms with a satisfactory safety profile.

Disclosures

No relevant conflicts of interest to declare.
CD14 Mediates Phagocytic Activity during the Granulocytic Differentiation Process in Acute Promyelocytic Leukemia Cells
Authors: Hsu Hui-Chi , Tsai Wen-Hui , Lin Yu-Chieh
Published: October 2019

All-trans retinoic acid (ATRA) can induce acute promyelocytic leukemia (APL) cells differentiation into mature granulocytes. CD14 and Toll-like receptor 4 (TLR-4) play an important role in the phagocytic activity of macrophage, however, their role during granulopoiesis is still unclear. In this study, we determined the role of CD14/TLR-4 in the development of phagocytic activity in NB4 APL cells after induction into the process of granulocytic differentiation by ATRA. Flow cytometry analysis demonstrate that, during ATRA treatment for 6 days, the phagocytic activity of NB4 cells in engulfing either fluorescein-latex beads or idarubicin-induced apoptotic cells increased in a time-dependent manner, and the level of CD14 expression on NB4 cells was also significantly increased in a time dependent manner, though its level was only minimally expressed in ATRA-untreated NB4 cells. However, TLR-4 was constitutionally expressed in ATRA-untreated cells and its level did not changed significantly during the first 5 days of ATRA treatment. Further study demonstrates that the phagocytic activity of ATRA-NB4 cells was significantly inhibited by pre-treating cells with antibodies specific to either CD14 or TLR-4 before phagocytosis assay. In exploring the role of CD14/TLR4 associated signal transduction mediators, NF-κB and IRF-3, we further demonstrate that the phagocytic activity of ATRA-NB4 cells in engulfing beads was significantly inhibited when cells were pretreated with either a NF-κB inhibitor (BAY 11-7082) or an IRF-3 inhibitor (SP600125). However, this activity in engulfing apoptotic cells was only significantly inhibited by pretreatment with BAY11-7082, but not by pre-treatment with SP600125. Finally, our results indicate that the level of CD14(+) microparticles (MPs) released by ATRA-NB4 cells was significantly enhanced when those cells were induced into the process of apoptosis by pre-treatment with idarubicin. Moreover, by incubation with MPs derived from apoptotic ATRA-NB4 cells, the phagocytic activity of living ATRA-NB4 cells in engulfing apoptotic cells was significantly enhanced, and this phagocytic activity was also significantly inhibited by pre-treating MPs with antibody specific to CD14 before phagocytic assay. We conclude that CD14 contributes to the phagocytic activity of APL cells during the process of granulocytic differentiation.

Disclosures

No relevant conflicts of interest to declare.