bluuebunny/crossref_metadata_2025_split huggingface dataset:
Therapeutic Targeting of B-Cell Receptor Signaling Pathways
Authors: Byrd John C. 
Published: October 2019

Abstract
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
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
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

Abstract
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

Abstract
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

Abstract
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

Abstract

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

Abstract
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.