bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-Invasive Hemoglobin Estimation in Patients with β-Thalassemia Major
Authors: Al-Khabori Murtadha K. , Al-Riyami Arwa Z. , Al-Huneini Mohammed , Farsi Khalil Al , Al-Hashim Abdulhakeem , Al-Kemyani Nasser , Mohsin Hussain M , Daar Shahina
Published: October 2019

Abstract 5179

Introduction:

Patients with β-thalassemia major receive regular blood transfusions. Non-invasive hemoglobin (Hb) estimation may simplify their care. Masimo Pronto-7 Pulse CO-oximetry device is used to non-invasively estimate the hemoglobin level but has not been previously validated in this group of patients. The primary objective of this study was to validate the pulse CO-oximetry based hemoglobin estimation in children and adults with thalassemia major.

Methods:

We conducted a prospective observational study on 108 children and adults with thalassemia major attending the daycare thalassemia center of a tertiary care hospital over 6 weeks. We estimated a spot Hemoglobin (Sp Hb) level using Masimo Pronto-7 Pulse CO-oximetry device (two measurements per patient) and compared it to a venous sample Hb (Reference Hemoglobin; Ref Hb) measured using Abbott CELL-DYN Sapphire hematology analyzer. We calculated Pearson correlation coefficient and coefficient of determination (R2). The multivariable linear regression model of predicting the estimation differences included age, gender, weight, height, blood pressure and reference hemoglobin.

Results:

We enrolled 108 patients (54 males, 54 females) with a mean age of 21. 6 years (SD 7. 3; 2. 5–38). There were 156 estimation episodes. The mean Ref Hb and SpHb were 9. 4 g/dL (SD 0. 9; 7. 1–12. 3) and 11. 1 g/dL (SD 1. 2; 7. 5–14. 7) respectively. The correlation coefficient between the Sp Hb and Ref Hb was 0. 49 (R2 = 24%) with a mean difference of 1. 7 g/dL (SD 1. 1; −1. 2 to 4. 3). In the multivariable model, Ref Hb level was the only statistically significant predictor of the difference in measurement (p =0. 002). There was a strong correlation between the two CO-oximetry Hb measurements (correlation coefficient 0. 70, R2 = 50%).

Conclusions:

Our results indicate that Masimo Pronto-7 Pulse CO-oximetry device overestimates the hemoglobin level and it cannot be recommended for patients with thalassemia major. Larger prospective studies are needed to confirm these results.

Disclosures:

No relevant conflicts of interest to declare.
Combination Therapy of Deferasirox and Deferoxamine As Paregoric Treatment of Severe Iron Overload in Patients with Thalassemia Major
Authors: Ladis Vassilis , Kyriacopoulou Dimitra , Stokidis Konstantinos , Moira Aggeliki , Kattamis Antonios
Published: October 2019

Abstract 5181

Background:

Despite the recent availability of multiple therapeutic options for chelation therapy, severe iron overload remains a significant cause of morbidity and mortality in a small group of patients with thalassemia major (TM). Every effort to induce negative iron balance for these patients is warranted. In this context, combination of deferasirox (DFX) and deferoxamine (DFO) may be of significant value. In this report, we present the paregoric use of DFX and DFO in severely iron overloaded thalassemic patients.

Patients and Methods:

Five patients (3 Males, 2 females) have been treated with DFX and DFO as a last rescue measure. In these patients previous combined treatment with DFO and DFP had failed. Informed consent and approval for the use of this schema as paregoric therapy was obtained by patients and hospital. The main inclusion criteria for this treatment were: LIC>30 mg/gr d. w. and T2* cardiac <10 msec. Treatment consisted of daily DFX at 30–40 mg/kg/day and DFO at 40–50 mg/kg/day for 3–6 days/week.

Results:

Patients' characteristics, results and toxicity are shown in table 1 and 2. Improvement in iron load status ranged widely during a follow-up from 1 to 3 yrs. LIC reduced in 4/5 patients, cardiac T2* increased in 3/5 patients, while LVEF showed no significant changes. Ferritin levels improved in 2/5 patients. Deterioration of safety parameters necessitated not to discontinue treatment.

Conclusions:

These data in the use of combination therapy of DFX and DFO suggest a potential modality of chelation therapy in severely iron overloaded patients. Response was variable and seemed not to be always related to compliance or the duration of treatment. Increase of serum creatinin and AST in all patients is a considerable problem in patients with persistent significant iron overload and already impaired hepatic and renal function, despite having been treated with different schema of iron chelation. Longer and cautious follow-up is needed to come to reliable conclusions.

Disclosures:

Ladis: Novartis: Consultancy, Honoraria, Research Funding; Apopharma: Consultancy, Honoraria, Research Funding. Kattamis:Apopharma: Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau.
Collagen Receptor-Mediated Mechanochemical Signaling Contributes to Human Pro-Angiogenic Mesenchymal Stem/Progenitor Cell-Induced Neo-Vasculogenesis
Authors: Gimona Mario , Rohban Rokhsareh , Lener Thomas , Peckl-Schmid Doris , Oeller Michaela , Schallmoser Katharina , Strunk Dirk , Rohde Eva
Published: October 2019

Abstract 5196

Background and rationale

Human mesenchymal stem/progenitor cells (MSPCs) are important tools for tissue repair and regenerative approaches. Co-application of autologous pairs of human MSPCs and endothelial colony forming progenitor cells (ECFCs) drives vessel formation in a mouse model. However, a more detailed mechanistic insight into the contribution of MSPCs to vasculogenesis is required prior to a potential application in clinical trials. The formation of perfused blood vessels following the co-injection of MSPC/ECFC pairs requires directed migration of these cell types through the extracellular matrix. For this, cells must interpret and respond to both biochemical cues and physical parameters of the matrix. Here we aimed at identifying changes in early signaling molecules that could potentially mediate these complex behaviors in ECFCs and MSPCs.

Results

By antibody array analysis of biopsies from vasculogenic regions we identified the levels of the discoidin domain receptor 2 (DDR2) to be upregulated about twofold in MSPCs in an MSPC/ECFC mixture compared to MSPC-only implants. Expression of DDR2 was confirmed by flow cytometry analysis. Employing immunofluorescence microscopy we showed components of the mechanotransduction and cytoskeleton machinery (Paxillin, ILK, Src, h1CaP, and cortactin) to be abundantly expressed and correctly localized in MSPCs. MSPCs also responded to manipulation of cytoskeletal integrity with phorbol dibutyrate or Y-27632, demonstrating the presence of a tissue transmigration machinery in MSPCs. Applying various three dimensional cell culture strategies we identified significant alterations in MSPC morphology, as well as in the mRNA levels for DDR1 and DDR2, and for miRNAs 29b, 199a, 331 in response to different matrix conditions.

Conclusions

Our data suggest a mechanosensitive regulation of MSPC function and we thus conclude that direct or indirect (miRNA-mediated) regulation of the collagen receptors DDR1/2 could have a role in modulating MSPC function during stem-cell induced neo-vascularization in vivo.

Disclosures:

No relevant conflicts of interest to declare.
IL-35 Is a Novel Responsive Anti-Inflammatory Cytokine - A New System of Categorizing Anti-Inflammatory Cytokines
Authors: Yang Xiao-Feng , Li Xinyuan , Mai Jietang , Virtue Anthony , Yin Ying , Sha Xiaojin , Wang Hong
Published: October 2019

Abstract 5197

It remains unknown whether newly identified anti-inflammatory/immunosuppressive cytokine interleukin-35 (IL-35) is different from other anti-inflammatory cytokines such as IL-10 and transforming growth factor (TGF)-β in terms of inhibition of inflammation initiation and suppression of full-blown inflammation. Using experimental database mining and statistical analysis methods we developed, we examined the tissue expression profiles and regulatory mechanisms of IL-35 in comparison to other anti-inflammatory cytokines. Our results suggest that in contrast to TGF-β, IL-35 is not constitutively expressed in human tissues but it is inducible in response to inflammatory stimuli. We also provide structural evidence that AU-rich element (ARE) binding proteins and microRNAs target IL-35 subunit transcripts, by which IL-35 may achieve non-constitutive expression status. Furthermore, we propose a new system to categorize anti-inflammatory cytokines into two groups: (1) the house-keeping cytokines, such as TGF-β, inhibit the initiation of inflammation whereas (2) the responsive cytokines including IL-35 suppress inflammation in full-blown stage. Our in-depth analyses of molecular events that regulate the production of IL-35 as well as the new categorization system of anti-inflammatory cytokines are important for the design of new strategies of immune therapies.

Disclosures:

No relevant conflicts of interest to declare.
Dasatinib (Sprycel®) and Low Intensity Chemotherapy for First-Line Treatment in Patients with De Novo Philadelphia Positive ALL Aged 55 and Over: Final Results of the EWALL-Ph-01 Study
Authors: Rousselot Philippe , Coudé Marie Magdeleine , Huguet Françoise , Lafage Marina , Leguay Thibaut , Salanoubat Celia , Witz Francis , Alexis Magda , Janvier Maud , Hunault Mathilde , Agape Philippe , Berthou Christian , Jourdan Eric , Fernandes José , Sutton Laurent , Banos Anne , Reman Oumedaly , Lioure Bruno , Cayuela Jean-Michel , Hayette Sandrine , Gokbuget Nicola , Hoelzer Dieter , Delannoy Andre , Gambacorti-Passerini Carlo , Bassan Renato , Ribera Josep-Maria , Giebel Sebastian , Dombret Herve , Ottmann Oliver G.
Published: October 2019

Abstract 666

Background.

Dasatinib (Sprycel®, Bristol-Myers Squibb) is a potent multi-targeted kinase inhibitor (TKI) of BCR-ABL and SRC family kinases. The EWALL group for adult ALL decided to run a study at the European level evaluating the combination of dasatinib and chemotherapy for Philadelphia positive (Ph+) ALL patients (pts) aged 55 and over.

Aim.

To analyse efficacy of Dasatinib combined to low intensity chemotherapy and to test factors associated with outcome. (EudraCT 2006–005694-21).

Methods.

After prephase, dasatinib was administered at 140 mg QD (100 mg over 70y) during the induction period in combination with weekly vincristine (VCR) 1 mg IV and dexamethasone (DEX) 40 mg for 2 days (20 mg over 70y) for 4 weeks. Consolidation

Disclosures:

Rousselot: BMS, Novartis: Research Funding. Gambacorti-Passerini:BMS, Novartis: Research Funding.
The Clinical Impact of Time to Response in De Novo Accelerated Phase Chronic Myeloid Leukemia (CML-AP)
Authors: Ohanian Maro , Kantarjian Hagop M. , Quintas-Cardama Alfonso , Jabbour Elias J. , Verstovsek Srdan , Borthakur Gautam , Ravandi Farhad , Garcia-Manero Guillermo , Ferrajoli Alessandra , Kadia Tapan M. , O'Brien Susan , Cortes Jorge E.
Published: October 2019

Abstract 72

Background:

Early (3-month) response is important in chronic phase chronic myeloid leukemia (CML-CP). Whether this applies also to CML in accelerated phase (CML-AP) has not been analyzed.

Aim:

To describe the impact of time to response on the outcome of patients (pts) with CML-AP.

Methods:

Frontline tyrosine kinase inhibitor (TKI) therapy was administered on consecutive or parallel clinical trials to 58 CML pts presenting with features of AP at the time of diagnosis, defined as blasts ≥15% (n=8), basophils ≥20%, (n=22), platelets <100×109/L (n=3), cytogenetic clonal evolution (n=22), or more than 1 feature (n=3). 36 pts received imatinib. 22 pts received a 2nd generation TKI (2GTKI) (dasatinib, n = 5 or nilotinib, n= 17).We analyzed the impact of various degrees of molecular and cytogenetic responses at 3, 6, and 12-months on rates of major molecular response (MMR), MR4.5, overall survival (OS), event free survival (EFS), and transformation (to blast phase) free survival (TFS).

Results:

After a median follow-up of 73 months (range 3 to 142) the overall rate of CCyR was 81% (imatinib 75%, 2GTKI 91%), and MMR 69% (imatinib 64%, 2GTKI 77%). At 3 yrs, TFS was 92%, EFS 90% and OS 87%. At 3 months 43 (81%) out of 53 evaluable pts had achieved a major cytogenetic response (MCyR) (71% of pts treated with imatinib, 95% of pts treated with 2GTKI); BCR-ABL/ABL <10% was achieved in 27 (96%) out of 28 evaluable pts (100% with imatinib, 95% with 2GTKI). At 6 months, 44 (86%) out of 51 evaluable pts had achieved a MCyR (83% of pts treated with imatinib, 90% of pts treated with 2GTKI); BCR-ABL/ABL <10% was achieved in 26 (93%) out of 28 evaluable pts (100% with imatinib, 90% with 2GTKI). The probability of achieving various outcomes: MMR, MR4.5, OS, EFS, and TFS by response at 3 and 6 months are shown in Table 1 and 2. The number of pts evaluable for both cytogenetic and molecular responses at 3 and 6 months were 44 and 45 respectively.

Conclusion:

Pts with de novo CML-AP have an excellent outcome with TKIs, particularly 2GTKIs. As for chronic phase, pts with deep responses at 3 months (MCyR or BCR-ABL <10%) have the best probability of a favorable long-term outcome, although a few have not achieved MCyR at 3 months. TKIs should be standard for all pts with AP features at the time of diagnosis.

Disclosures:

Kantarjian: Novartis Pharmaceuticals Corp: Consultancy, Research Funding; BMS: Research Funding; Pfizer: Research Funding. Jabbour:BMS: Honoraria; Novartis: Honoraria; Pfizer: Consultancy. Ravandi:BMS: Honoraria, Research Funding; Novartis: Honoraria. Cortes:Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.
Novel Isoforms of Neutrophil Elastase Produced by Neutropenia-Associated Mutations of the Initiation Codon and an Internal Ribosomal Entry Site (IRES) of ELANE
Authors: Tidwell Timothy , Wechsler Jeremy , Horwitz Marshall S
Published: October 2019

Abstract 9

Severe congenital neutropenia is most commonly caused by mutations in the gene, ELANE, encoding neutrophil elastase, through a mechanism that is not fully understood. Two prevalent hypotheses point to mislocalization of neutrophil elastase or, alternatively, endoplasmic reticulum (ER) stress resulting from protein misfolding. We have recently identified disease-causing mutations in the “Kozak” ribosome-binding sequence and the initiation codon that do not easily fit with either theory. In cell culture models of neutrophil elastase expression, we found that when the ribosome binding site of ELANE is mutated, at least four polypeptides are expressed, all shorter than the wild type. All of the isoforms are reactive to a carboxyl-terminal antibody, indicating that they are likely produced from alternate start sites or represent amino-terminal truncations. ELANE contains three downstream in-frame initiation codons (ATG) that potentially could be utilized as alternate translation start sites, and mutations in the vicinity of these sequences predictably correlate with production of each of the shorter isoforms. Furthermore, a protein-coding region of ELANE mRNA is complementary to the 18s ribosomal RNA subunit and demonstrates internal ribosome entry site (IRES) activity, when tested using a bicistronic luciferase assay. Neutropenia-associated mutations within the identified IRES also affect expression of the shorter isoforms of neutrophil elastase. These findings suggest that some ELANE mutations could cause neutropenia, not through disturbing the primary sequence of full-length neutrophil elastase protein, but instead by altering mRNA-ribosome interactions, thus generating shorter isoforms of normal sequence that are initiated from internal translational start sites. We have therefore biochemically characterized the shorter isoforms. We found that they do not demonstrate proteolytic activity. Because they lack signal sequences and cannot be trafficked through the ER, it is also not surprising that they fail to induce the ER stress response. They do not trigger apoptosis, as measured in several assays. Nevertheless, they do reduce myeloid cell viability in a colony forming test. We propose that these shortened isoforms contribute to the pathogenesis of ELANE-associated neutropenia by impairing myeloid cell development. Because a diversity of ELANE mutations from different patients seem to have identical effects, these shorter neutrophil elastase polypeptides, common to multiple patients regardless of mutation, may offer promise as targets for drug development.

Disclosures:

No relevant conflicts of interest to declare.
Diamond Blackfan Anaemia Patients Have a Higher Rate of Hepatic Iron Accumulation Than Thalassaemia Major Patients Leading to Fibrosis
Authors: Evans Katharine , Goldin Robert , de la Fuente Josu
Published: October 2019

Abstract 997

Diamond Blackfan anaemia is an inherited bone marrow failure syndrome with haematological and systemic manifestations. The classical presentation of the condition is the development of anaemia in infancy, which occurs in approximately 85–90% of the patients. Long-term 40% require transfusions as they fail to maintain erythropoiesis at acceptable doses of steroids and only approximately 10% of the patients go into remission. To investigate the iron load caused by transfusions and its effect we studied the liver biopsies of 13 patients with DBA and compared them with 27 patients suffering from thalassaemia major (TM). The findings were correlated with the number of transfusions, chelation treatment, ferritin level and MR techniques. Image analysis of the degree of fibrosis was performed using NIS-elements software after staining liver biopsy slides with Sirius Red.

TM patients were significantly older [median age: TM 9 years (3–18), DBA 5 years (1–15); p=0.004], which was also reflected in the duration (months) of transfusion [TM 105 (12–198), DBA 52 (12–130); p=0.004]. However, there was no difference in the frequency of transfusions (p=0.51) and in the length of time (months) between starting transfusions and chelation (TM 19, DBA 27; p=0.08). DBA patients received proportionately more chelation per transfusion than TM patients at the time of biopsy (TM 1.24, DBA 1.58; p=0.015). Ferritin levels were higher in TM patients, though not significant [TM 2028 μg/L (1292–4901), DBA 1324 μg/L (535–2300); p=0.16).

Despite having significantly fewer transfusions, the grade of iron deposition was higher in the DBA group (TM 2, DBA 3; p=0.035). This was also reflected in MRI T2* quantitation, which demonstrated a higher hepatic iron load in DBA patients [TM 3 ms (1–12), DBA 2 ms (1–3); p=0.59]. The rate of biochemical iron accumulation (mg/g DW) for every month of transfusion was significantly higher in the DBA group (TM 0.05, DBA 0.11; p=0.005). The rate of fibrosis accumulation was 60% higher in the DBA group, although this was not statistically significant (TM 0.1, DBA 0.16; p=0.07) and could be due to a time lag between iron accumulation and fibrosis formation, particularly as the DBA patients were younger and had had shorter follow up. Biochemical iron, ferritin level and MRI T2* had low, significant correlations with fibrosis in TM patients (0.547, p=0.001; 0.357, p=0.033; −0.430, p=0.011, respectively) and FerriScan in DBA patients (0.75, p=0.05). In conclusion, DBA patients have a higher rate of iron accumulation with a trend to higher hepatic fibrosis.

Disclosures:

No relevant conflicts of interest to declare.
Fanconi Anemia: Emerging Therapeutic Opportunities
Authors: Grompe Markus , Postlethwait John , D'Andrea Alan D. , Olson Susan B. , Zhang Qingshuo , Bagby Grover C.
Published: October 2019

Abstract SCI-1

Fanconi anemia (FA) is a genetically heterogeneous inherited disorder characterized by bone marrow failure, birth defects, and cancer susceptibility. At the cellular level, FA features hypersensitivity to DNA cross-linking agents, cell cycle abnormalities, and abnormal responses to cytokines. Therapy for the hematopoietic defects of FA has consisted of supportive care, stem cell transplantation, and administration of androgens such as oxymetholone. Unfortunately, even successfully transplanted FA patients have a very high risk of subsequent solid tumors, especially squamous cell carcinomas of the oropharyngeal and urogenital areas. The pharmacologic treatment of FA has been unchanged for several decades, and for this reason our group has embarked on a systematic search for small-molecule therapeutics that could benefit FA patients. This effort has been made possible by the cloning of 15 FA genes and biochemical insights into the function of FA proteins. Mouse models, zebra fish models, and FA patient cell lines, including induced pluripotent stem cells, are available for screening and validation. The specific goals of small-molecule interventions are to delay or prevent hematopoietic failure and also to delay or prevent carcinogenesis. To date, we have shown that the superoxide dismutase mimetic drug Tempol can significantly delay tumor formation in FA mice. Similarly, resveratrol clearly improved hematopoiesis in the same model. Medium-throughput screens have revealed numerous new molecules that ameliorate the FA phenotypes of cells, mice, and zebrafish. Using genetic approaches we also have identified the p53 pathway as a therapeutic target in FA. These therapeutic opportunities and progress in FA small-molecule screening will be discussed.

Disclosures:

No relevant conflicts of interest to declare.
Human Dendritic Cell Heterogeneity: Opportunities and Challenges for the Control of Immunity
Authors: Young James W.
Published: October 2019

Abstract SCI-21

Dendritic cells (DCs) comprise a heterogeneous population of potent antigen-presenting cells, which are master orchestrators of the immune response. DCs initiate immunity, and just as importantly, control its quality and outcome. DCs accomplish this by capitalizing on their defined subsets, which exert discrete functions in responding to different types of antigens and factors in the microenvironment. DCs, therefore, provide the immune system with an essential and robust flexibility to discriminate between enormous numbers of harmful and benign agents and to mount functionally distinct and appropriate responses in the contexts of innate and adaptive immunity. Our lab has focused on the contrasting functions of conventional human DC subsets, primarily monocyte-derived DCs (moDCs) versus CD34+ hematopoietic progenitor cell-derived Langerhans-type DCs (LCs). Recent findings that have defined the afferent sensitization of different types of immune responses by these distinct DC subtypes will be discussed. For example, LCs use an endogenous IL-15R-α/IL-15 mechanism to generate potent cytotoxic T lymphocytes and break tolerance against a self-differentiation tumor antigen like WT1. This contrasts with the potency of moDCs in maturing and stimulating NK cells by their production of IL-12p70, whereas LCs can only ensure NK-cell viability. The application of these findings to the holy grail of separating graft-versus-leukemia from the deleterious aspects of graft-versus-host disease in allotransplantation, at the level of afferent sensitization by these distinct DC subtypes, as well as the novel use of small-molecule inhibitors of JAK-STAT signaling in DC-stimulated T cells, will also be addressed.

Disclosures:

Off Label Use: JAK2 inhibitors (potentially several) for inhibition of dendritic cell stimulated alloreactivity - but all data will be in vitro, so perhaps this is not off-label drug use because it is not in vivo in humans.
Therapeutic Targeting of B-Cell Receptor Signaling Pathways
Authors: Byrd John C.
Published: October 2019

Abstract SCI-27

Targeted therapy in hematologic malignancies has achieved significant therapeutic success when relatively selective inhibition is attainable to a target dispensable to the majority of normal cells. The best appreciated example of this is imatinib in chronic myeloid leukemia (CML), in which a single translocation forms a fusion protein involving the tyrosine kinase ABL that molecularly defines the disease. Kinase inhibition of ABL in this setting by imatinib (or other second- and third-generation kinase inhibitors) promotes durable, long-term remission in CML patients. The great majority of B-cell malignancies lack a characteristic translocation, activating mutation, or other aberration that facilitates such targeted therapy as employed in CML. Nonetheless, a common global signaling pathway involving the B-cell receptor (BCR) pathway has emerged as one that might be important to the control of these diseases. Furthermore, mouse models of select kinase targets (phosphoinositide-3 kinase p110 isoform-delta and Bruton's tyrosine kinase [BTK]) have shown that these are dispensable relative to long-term survival. Indeed, knockout or mutation of BTK and p110 delta each have a modest phenotype outside of diminished B-cell development and function. Based upon these preclinical observations, several molecules targeting BCR signaling have come forward to the clinic with exciting results across a wide range of B-cell malignancies. GS-1101 is a selective phosphoinositide-3 kinase p100 delta inhibitor with a very favorable toxicity profile that has shown promising clinical activity in low-grade lymphoma and chronic lymphocytic leukemia (CLL). The toxicity of this orally administered agent is quite modest in the majority of patients and allows sustained continuous dosing. Similarly, ibrutinib is an irreversible inhibitor of BTK and has shown promising clinical potential in an even broader range of B-cell malignancies, including diffuse large B-cell lymphoma, mantle cell lymphoma, low-grade lymphoma, and CLL. Toxicity with ibrutinib has also been modest, allowing long-term continuous dosing. Notably, each of these agents also produces an atypical mobilization of malignant lymphocytes into the blood soon after treatment. This treatment lymphocytosis is BCR-target-related due to diminished CXCR4/SDF-1 interface between tumor cells and stromal cells in the bone marrow, with egress of these cells to the blood. GS-1101 and ibrutinib are now entering phase III studies for regulatory approval and offer great potential to change the treatment paradigm of both CLL and B-cell non-Hodgkin lymphoma (NHL). Questions moving forward with these agents will include molecular predictors of response, feasibility and efficacy of combining with other effective therapies, and mechanisms of resistance. The scientific session presentation will provide an overview of the most promising BCR signaling agents in CLL and NHL clinical trials.

Disclosures:

No relevant conflicts of interest to declare.
Activated Protein C, Sepsis, and the Innate Immune Response to Infection
Authors: Weiler Hartmut , Griffin John H.
Published: October 2019

Abstract SCI-43

The phase 3 PROWESS clinical trial in 2001 resulted in approval of recombinant human activated protein C (APC) using low-dose, 96 hour infusion therapy to reduce mortality for adult severe sepsis linked to bacterial infection (1). In that trial, APC reduced mortality from 30.8% to 24.7%, an absolute mortality reduction of 6.1% (19.4% relative risk reduction), but this therapy carried a risk of serious bleeding (4.0 vs. 1.5%, p=0.06). Trials of APC therapy for less than severe adult or pediatric sepsis failed to show benefit but confirmed increased risk for serious bleeding. Subsequently, 10 years after the PROWESS trial, the similarly designed large trial (PROWESS SHOCK) (2) failed to document therapeutic efficacy in adult severe sepsis, reporting mortalities of 26.4% vs. 24.2% and, curiously, no increased risk of serious bleeding (1.2 vs. 1.0%, p=0.81). Recombinant APC was thus withdrawn from the market in late 2011. A simple comparison of PROWESS to PROWESS-SHOCK is confounded by significant improvements in ICU standard of care for severe sepsis over a decade. These two trials were notable in being limited to therapy using low-dose APC infusion for four days. The success of PROWESS stimulated major research advances in 2002–2012 for understanding APC's in vitro and in vivo mechanisms of action. In preclinical work, APC is highly effective for sepsis, non-infectious inflammatory disease, ischemic stroke and neurodegenerative disorders. This presentation will review current knowledge about mechanisms for APC's antithrombotic and cellular cytoprotection, about new insights that might help explain the absence of efficacy in PROWESS-SHOCK, and about possible avenues towards translating the benefits of APC seen in preclinical studies to patients. This presentation will discuss the rationale for use of second generation APC variants that may reduce the risk of bleeding while retaining beneficial anti-inflammatory and cytoprotective functions. Recent preclinical animal studies indicate that such second-generation APC variants might widen the time-window of opportunity for successful therapies, including for lethal infections. Remarkably, APC reduces mortality caused by high-dose total body radiation in mice by preserving bone marrow cells (3). This presentation will review cellular and molecular mechanisms by which APC affects the response of bone marrow-derived innate immune cells to stress induced by infection or radiation. This presentation will provide evidence for the existence of a previously unrecognized role of coagulation Factor V as a modifier of the response to sepsis therapy with APC. Based on APC's direct cellular effects, one would speculate that the use of second generation APC variants given in high-dose bolus regimens holds great promise for multiple maladies.

Disclosures:

Weiler: BloodCenter of Wisconsin: Patents & Royalties.
Langerhans Cell Histiocytosis: Back to Histiocytosis X
Authors: Allen Carl E.
Published: October 2019

Abstract SCI-8

Langerhans cell histiocytosis (LCH) is a disorder characterized by inflammatory lesions that include pathologic CD207+ dendritic cells. LCH has pleotropic clinical presentations ranging from single lesions cured by curettage to potentially fatal multisystem disease. The first descriptions of LCH, including Hand-Schüller-Christian disease and Letterer-Siwe disease, were based on anatomic location and extent of the lesions. Despite clinical heterogeneity, LCH lesions are generally indistinguishable by histology, which led to the notion that the spectrum of clinical manifestations represents a single disorder, histiocytosis X. The designation “Langerhans cell histiocytosis” was subsequently proposed with discovery of cytoplasmic Birbeck granules in the pathologic infiltrating dendritic cells in histiocytosis X lesions, a feature shared by epidermal Langerhans cells. The etiology of LCH remains elusive, and debate of LCH as an inflammatory versus malignant disorder remains unresolved. However, recent discoveries question the model of LCH arising from transformed or pathologically activated epidermal Langerhans cells. We found cell-specific gene expression signature in CD207+ dendritic cells within LCH lesions to be more consistent with immature myeloid dendritic cell precursors than epidermal Langerhans cells. Furthermore, recent mouse studies demonstrate that CD207+ is more promiscuous than previously appreciated. Langerin (CD207) expression can be induced in many dendritic cell lineages, supporting the plausibility of a spectrum of candidates for an LCH cell of origin, including circulating dendritic cell precursors. Finally, recurrent activating BRAF mutations in LCH lesions suggest a role for a hyperactive RAS pathway in LCH pathogenesis, and possibly in normal dendritic cell development. This presentation will discuss the historical background and recent advances in LCH biology, along with a proposal to reframe “histiocytosis X” as a myeloid neoplasia caused by aberrant maturation and migration of myeloid dendritic cell precursors.

Disclosures:

No relevant conflicts of interest to declare.
Quantitative and Qualtitative Evaluation Of Circulating Cell-Free DNA As a Prognostic Biomarker In Hodgkin Lymphoma
Authors: Hohaus Stefan , Giachelia Manuela , Cupelli Elisa , Cuccaro Annarosa , Massini Giuseppina , Bartolomei Francesca , D'Alò Francesco , Voso Maria Teresa , Leone Giuseppe
Published: October 2019

Tumor tissue is known to release cell-free DNA, that can be detected in peripheral blood. Cell-free DNA (cfDNA) is significantly higher and more fragmented in plasma of cancer patients and levels have been reported to associate with prognosis. The aim of our study was to further explore cfDNA as a biomarker in Hodgkin lymphoma (HL) at diagnosis, during treatment and at follow-up. We studied cfDNA levels in pre-treatment plasma samples of 163 consecutive HL patients diagnosed at our Institution and 64 healthy volunteers. cfDNA was analyzed by quantitative PCR for the beta-globin gene. cfDNA at diagnosis was significantly increased in patients with HL when compared to controls (median, 21.83 vs 12.18 ng/ml; p<0.0001). Using the cfDNA levels of normal volunteers to determine the cut-off point for pathologically elevated cfDNA levels, cfDNA levels were elevated in 62 (38%) patients. Increased cfDNA at diagnosis was associated to parameters indicating an unfavorable prognosis as an IPS score>2 (p=0.0008). In a group of 31 patients and 19 controls, we also evaluated cfDNA fragmentation, amplifying fragments of different length. DNA integrity indices were calculated according to Mouliere et al, PLoS One 2011. At diagnosis, cfDNA was more fragmented in HL patients than in controls resulting in a lower DNA integrity index (median: 0.15 vs 0.22; p=0.016). However, cfDNA in HL patients resulted less fragmented when compared to cfDNA form patients with aggressive tumors like Burkitt lymphoma or solid tumors. Patients received treatment with ABVD (101 patients), BEACOPP (42 young patients with advanced HL) and other types of chemotherapy (17 patients). Seventy patients were also evaluated after two cycles of chemotherapy. cfDNA levels significantly decreased after 2 cycles of chemotherapy in patients with increased pre-treatment levels, while they remained in the normal range in patients with low cfDNA at diagnosis (ratio 0.32 and 1.44, respectively; p<0.0001). Patients with elevated cfDNA levels at diagnosis had an inferior outcome compared to patients with cfDNA levels within the normal range both in univariate and in a multivariate analysis that included IPS and was adjusted for the type of chemotherapy (EFS, p<0.03). In conclusion, our data suggest that cfDNA could serve as a biomarker in HL, that it is frequently elevated at diagnosis of HL, tends to normalize during treatment, and is associated to an inferior outcome. The study of DNA fragmentation suggests that most of the increase of cfDNA in HL is probably due to the tumor microenvironment rather than to the neoplastic cells.

Disclosures:

No relevant conflicts of interest to declare.
CD74 Interferes With The Expression Of Fas Receptor On The Cell Surface
Authors: Berkova Zuzana , Ao Xue , Braun Frank K , Wise Jillian F , Sehgal Lalit , Samaniego Felipe
Published: October 2019

Resistance to Fas-mediated apoptosis manifested by hematopoietic cancers interferes with the efficacy of currently available chemotherapy regimens. We identified CD74, that is known to be overexpressed in hematological malignancies, as one of the factors interfering with Fas-mediated apoptosis.

Through stable suppression of CD74 expression in BJAB and Raji cells using shRNA technology, we have determined that cells overexpressing CD74 are more resistant to FasL-induced apoptosis than control non-target shRNA expressing cells (super FasL 10 ng/mL; 36 ± 3.3% vs.18 ± 0.1% and 35 ± 0.1% vs. 20 ± 1.5%; P< 0.01). Transfection of mice with plasmid encoding full-length CD74 protected mice from a lethal challenge with agonistic anti-Fas antibody Jo2. All 5 of vector-transfected mice died within 6 hours from challenge, while 5 out of 6 CD74-transfected mice survived the challenge (P< 0.05). Suppression of CD74 expression also sensitized BJAB cells to Fas-signaling dependent chemotherapies. Doxorubicin (125nM) killed 27 ± 4% of control and 48 ± 5% of CD74 knock-down cells (P< 0.001). Edelfosine (10 uM) induced apoptosis in 36 ± 7% of control and 64 ± 1% of CD74 knock-down cells (P< 0.001).

A detailed analysis of Fas signaling in cells lacking CD74 and control cells revealed increased cleavage/activation of pro-caspase-8 and corresponding enhancement of caspase-3 activation in the absence of CD74. This result suggested that CD74 affects the immediate early steps in Fas signaling at the plasma membrane. We next analyzed the levels of Fas receptor on the surface of cells by flow cytometry. Cells with suppressed CD74 expression had 30% higher staining of Fas receptor on their surface; the mean fluorescence intensity increased from 4017 ± 704 to 5122 ± 216 (P< 0.003).

We anticipate that specific targeting of the CD74 on the cell surface with milatuzumab will sensitize CD74-expressing cancer cells to Fas-mediated apoptosis and thus will increase effectiveness of chemotherapy regimens for hematological malignancies.

Disclosures:

No relevant conflicts of interest to declare.
HDAC6 Inhibition Increases CD20 Level and Improves The Efficacy Of Anti-CD20 Monoclonal Antibodies
Authors: Bobrowicz Malgorzata , Dwojak Michal , Bojarczuk Kamil , Winiarska Magdalena , Golab Jakub
Published: October 2019

CD20, an integrate membrane protein expressed on the surface of normal and malignant B-cells is widely used as a molecular target for monoclonal antibodies (mAbs) in the therapy of non-Hodgkin’s lymphomas and chronic lymphocytic leukemia (CLL). Accumulating evidence indicates that CD20 can be modulated at several levels, both transcriptional and posttranscriptional and its up-regulation would result in increased efficacy of anti-CD20 mAbs. CD20 antigen has been reported to be regulated epigenetically e.g. by histone deacetylases (HDACs). The results of our preliminary experiments show that use of non-selective HDAC inhibitors as well as blocking the activity of a single HDAC isoform - HDAC6 leads to up-regulation of CD20 protein in B-cell lymphoma cell lines and increase of the efficacy of therapy with anti-CD20 mAbs. Since HDAC6 is engaged mainly in the acetylation of non-histone substrates and the observed up-regulation of CD20 molecule does not seem to rely on transcriptional mechanisms we postulate that HDAC6 is engaged in processes of CD20 trafficking or/and degradation. CD20 being a membrane bound protein is most probably undergoing endocytosis. However, this process and the role of HDAC6 in its regulation has not been explored so far.

The aim of this study was to understand how the inhibition of HDAC6 activity influences CD20 level in normal and malignant B-cells. We wanted to determine the mechanisms underlying this phenomena.

This study required use of B-cell lymphoma cell lines as well as lymphocytes infected with Epstein-Barr virus and normal B- lymphocytes. Several HDAC pan-inhibitors and HDAC6 inhibitors were tested. To assess the membrane level of CD20 antigen, FITC-anti-CD20 staining was performed followed by cytometric analysis. The influence of HDACi on total level of CD20 protein was assessed in Western blotting. The complement-dependent cytotoxicity (CDC) assay was performed using rituximab and ofatumumab as well as human serum as a source of complement. Cell cytotoxicity was assessed by propidium iodide staining followed by cytometric analysis. The influence of HDAC inhibition on the transcription of CD20 was examined by qRT-PCR using SyBR Green and hydrolysis probes. The activity of CD20 promoter after inhibition of HDAC was assessed in Dual Luciferase Assay. The colocalisation of CD20 with other proteins that may influence its trafficking/degradation was assessed using immunocytochemistry with specific antibodies and observed under confocal microscope.

The results of our study strongly suggest that combining HDACi with anti-CD20 antibodies can be an effective therapeutic modality for patients suffering from B-cell malignancies. Extensive experiments aiming at determining what factors are engaged in the regulation of CD20 by HDAC6 are planned.

Disclosures:

No relevant conflicts of interest to declare.
Vegf and bFGF Single-Nucleotide Polymorphisms In Chronic Lymphocytic Leukemia: Impact On Susceptibility
Authors: Ballester Sandra , Pineda Begoña , Tormo Eduardo , Navarro Blanca , Perez Ariadna , Teruel Anabel , Terol Mª José , Solano Carlos , Eroles Pilar
Published: October 2019

Background

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical outcome. Recent studies have identified a number of different molecular prognostic markers (including mutational status of the IgVH gene, ZAP70 and CD38 expression) that allow to discriminate patients in prognostic subgroups. However, different expression patterns of angiogenic factors as VEGF, VEGFR1 and bFGF have been related with B-CLL susceptibility and treatment requirements. We have analyzed the polymorphisms: -710 C/T in VEGFR1, rs1109324, rs1547651, rs3025039 (936C/T) and rs833052 in VEGF and rs1449683 (223 C/T) in bFGF in order to determine the possible association with susceptibility in B-CLL.

Methods

Peripheral blood samples from 230 B-CLL patients and 476 healthy controls were genotyped using probes TaqMan SNP Genotyping Assays. Samples were providing from the Hospital Clinic of Valencia. Four SNPs in the VEGF gene, one SNP in the bFGF gene and one SNP in the VEGFR1 gene were evaluated. Statistical analysis was performed using SNPStats program (Catalan Institute of Oncology) and Fisher's exact test was applied to evaluate the significance.

Results

We have observed an increased frequency of the T allele in the rs1449683 SNP [OR 1.62 (95% CI: 0.98-2.66) p-value =0.063] and in the rs1547651 SNP [OR 0.72 (95% CI: 0.51-1.03), p-value=0.072] in our B-LLC patients when compared to control subjects. Moreover we observed that T allele carriers of rs3025039 (VEGF) have a significant protective effect concerning this disease [OR 0.59 (95% CI: 0.39-0.89) p-value=0.009].

Conclusion

Our data indicate an increased frequency of the T allele in polymorphisms rs1449683 (bFGF) and rs1547651 (VEGF) in the group of patients, which possibly account for the individual susceptibility to develop B-CLL. On the other hand the data provided suggest that the T allele of VEGF rs3025039 is likely important genetic marker of susceptibility to B-CLL.

Further studies regarding the role of pro-angiogenic markers in B-CLL would be beneficial to help elucidate pathogenic pathways in this disease.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy Of Bendamustine and Rituximab In Patients With Relapsed/Refractory Chronic Lymphocytic Leukemia
Authors: Kataeva Elena , Golenkov Anatoly , Triphonova Elena , Dudina Galina , Mitina Tatyana , Lutskaya Tatyana , Vysotskaya Ludmila , Chernykh Yulliya , Zakharov Sergey
Published: October 2019

The objective of the study was to assess the efficacy and safety of the Bendamustine (B) and Rituximab (R) combination therapy in pre-treated patients with relapsed/refractory chronic lymphocytic leukaemia (CLL).

Patients and Methods

13 patients with CLL were examined, out of which 9 were men and 4 women. The average age of the enrolled patients was 62 years (52-77). 5 CLL patients had RAI stage II, 1-III and 7 patients had stage IV. FISH test detected del (17p) in 4 patients (30.8%), del (11q) in 3 patients (13.1%) the immunophenotype test of peripheral blood lymphocytes identified 5 patients (38.5%) with CD38+positive (cut off over 20%).

The patients received prior therapy with COP, CHOP, RFC, FC, RCHOP, FluCam, Alemtuzumab and R courses. The treatment lasted for 23.7 months (12-36). 10.4 (7-20) treatment courses were made on average. Median of Chemotherapy lines in each patient was 4 (2-5). All the patients were resistant to the previous treatment and relapsed. The patients received R through intravenous infusions at a dose of 500 mg/m2 on day 1 of the therapy course and B through intravenous infusions at a dose of 100 mg/m2 on days 2 and 3 of the therapy course, and the course was repeated every 28 days. 4.6 (3-9) therapy courses were administered.

Results

All the patients (100%) responded to the administered therapy by achieving partial remission. Progression-free survival within 6 months is 67%. Median of the examination was 12.3 months (4-33) and 9 patients (69%) are alive. 3 patients (21%) had hematologic toxicity of levels 3 and 4. At the same time 2 patients (14%) had neutropenia, and 1 patient (7%) had thrombocytopenia respectively. 3 patients (23%) had non-haematological toxicity of level III in the form of infections (pneumonia) and 3 patients (27%) had gastroenterological toxicity.

Conclusion

thus, the BR combination has a high anticancer activity in heavily pre-treated patients with relapsed/refractory CLL. It should be noted that the examined patients had pejorative prognostic characters such as del (17p) and del 11(q), and high CD 38+ expression (82.4%). At the same time the toxicity profile was low.

Good anticancer effect and moderate toxicity indicate strict selectivity of the BR program.

Disclosures:

No relevant conflicts of interest to declare.
Higher TRAF6 and Fbxo 3 Expression Is With Progressive Multiple Myeloma and Blockage Of TRAF6 Inhibits Tumor Growth and Bone Resorption
Authors: Li Mingjie , Vardanyan Suzie , Gottlieb Jillian , Wang Cathy , Delijani Kevin , Halleluyan Ran , Ben-Zvi Joseph , Song Mitchell , Sanchez Eric , Bonavida Benjamin , Chen Haiming , Berenson James R.
Published: October 2019

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. F-box proteins such as the E3 ligase component Fbxo3 and another F-box family member Fbxl 2, regulate TRAF protein signaling.

First, we investigated TRAF6 expression in bone marrow mononuclear cells (BMMCs) from multiple myeloma (MM) patients with progressive disease or in remission and healthy subjects.  The results showed higher TRAF6 protein expression among MM patients with progressive disease than among those in remission or healthy subjects. Notably, changes in TRAF6 protein expression in MM BMMCs were found to correlate with response of individual patients to treatment with the proteasome inhibitors bortezomib or carfilzomib. We have previously reported inhibition of MM cell proliferation and increase of apoptosis through regulation of the NF-kB and JNK pathways using a silencing TRAF6 C-domain mRNA construct. In this study, we cloned a 167 amino acid (in residues 333 to 508) portion of the TRAF6 dominant negative (TRAF6dn) and synthesized decoy peptides of the TRAF6 interaction domain with CD40 and the TRAF6-RANK binding domain. Decreases in cell proliferation and increase in cell apoptosis in MM BMMCs treated with TRAF6dn occurred in a concentrationdependent fashion. We also found TRAF6dn markedly inhibited osteoclast cell formation of monocytes induced by RANKL and mCSF in a dose-dependent manner. Next, we examined the effect of TRAF6dn on NF-kB and JNK by determining phosphorylation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. MM BMMCs exposed to the TRAF6dn fragment or TRAF6 decoy peptide showed reduced phosphoNF-kB protein and phosphorylation of JNKK. These studies suggest that the TRAF6dn or combined TRAF6 decoy and CD40 decoy peptide may be excellent agents to block both MM cell growth and osteoclast formation in MM.

We further investigated the protein expression of Fbxo 3, Fbxl 2 and TRAF6 in fresh BMMCs from MM patients with progressive disease or in remission. Results of Western blot analysis showed protein expression of Fbxo 3 and TRAF6 was increased and Fbxl 2 was decreased among patients with progressive disease compared to patients in remission.

Thus, these results may offer a new mechanism through which MM tumor cells are regulated and provide a new therapeutic approach to treat MM and reduce the clinical consequences resulting from enhanced bone loss that commonly occur in these patients.

Disclosures:

No relevant conflicts of interest to declare.