bluuebunny/crossref_metadata_2025 huggingface dataset:
A Screen for Drugs Showing Specific Co-Resistance or Hyper-Sensitivity Upon the Acquisition of Ara-C Resistance In AML
Authors: Rathe Susan K , Largaespada David
Published: October 2019

Abstract 1844

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. We hypothesized that the changes required by the cells to become Ara-C resistant might also make them more susceptible to elimination by other drugs. We previously developed two highly resistant murine AML cell lines, B117H and B140H, by introducing increasing concentrations of Ara-C to their parental cell lines, B117P and B140P, respectively. The Ara-C resistant derivatives, B117H and B140H, can tolerate Ara-C concentrations ∼1000X that of their drug sensitive parental cell lines. We previously demonstrated that the down-regulation of deoxycytidine kinase was one mechanism of resistance in the Ara-C resistant B117H and B140H cells, a mechanism that has also been documented in human cases of refractory disease. A high throughput drug screen was conducted to determine if an increase in Ara-C resistance would result in increased sensitivity to other drugs. The screen evaluated 446 FDA approved drugs, which included a variety of drug types, including chemotherapy drugs, analgesics, steroids, antidepressants, immunosuppressives, and antipsychotics. Despite the diverse nature of the parental cell lines and their Ara-C resistant derivatives, two drugs (prednisolone and AM404), showed an increase in sensitivity in both Ara-C resistant cell lines. Follow-up drug assays were performed on both prednisolone and AM404 to establish the 50% inhibitory concentration (IC50) in all four cell lines. Both Ara-C resistant cell lines displayed a greater than 3-fold reduction in the IC50 of prednisolone as compared to their parental lines, and the IC50 of AM404 demonstrated a greater than 2-fold reduction. Prednisolone has been shown to induce differentiation in murine AML cells in vitro, and high-dose methylprednisolone has been found to induce differentiation in various subtypes of childhood AML. The mechanism of differentiation by prednisolone is theorized to be independent of glucocorticoid receptor binding. We believe this model will be useful in elucidating the role of prednisolone in leukemic differentiation, and in suggesting new mechanisms to target refractory AML cells.

Disclosures:

No relevant conflicts of interest to declare.
Adenosine Signaling in Priapism and Novel Therapies
Authors: Ning Chen , Wen Jiaming , Zhang Yujin , Juneja Harinder S. , Qi Lin , Kellems Rodney E. , Xia Yang
Published: October 2019

Abstract 1006

Objective:

Priapism is abnormal prolonged penile erection occurring without sexual interest. The condition is prevalent among men with sickle cell disease (SCD). Priapism is a urological emergency which needs early intervention to avoid the risks of penile fibrosis and eventual erectile dysfunction. Due to poorly understood pathogenesis of priapism no effective approaches to manage the disorder. Recent studies have revealed excess adenosine (Ado) in priapism via Ado A2B receptor (ADORA2B), suggesting novel therapeutic possibilities. Here, we aim to conduct preclinical studies to assess the efficacy and safety of Ado-based therapy in priapism.

Materials and Methods:

ADA-deficient mice (ADA−/−) and SCD Berkeley mice are two independent priapism animal models. We treated both mice with polyethylene glycol-modified ADA (PEG-ADA) to lower penile adenosine level or PSB1115, a selective ADORA2BR antagonist. The erectile function are measured by the changes of intracavernosal pressure (ICP) induced by cavernous nerve stimulation (CNS). Penile fibrosis is evaluated by histological studies and RT-PCR analysis of fibrotic marker gene. In vitro human microvascular endothelial cells (HMECs) were used to determine mechanism underlying ADORA2B-induced priapism.

Results:

Both ADA−/− and SCD mice with elevated Ado levels in penile tissue displayed priapic feature defined by prolonged and heightened erectile in response to CNS. Chronic reduction of accumulation of penile Ado levels by PEG-ADA enzyme therapy or PSB1115 corrected the priapic feature in both priapic animal models and further prevented progression of penile fibrosis. Significantly, both HIF-1α and eNOS mRNA level were elevated but reversed by PSB1115 treatment in penile tissues of ADA−/− mice and SCD mice. Finally, we provide in vitro direct evidence that PSB1115 treatment or siRNA knockdown HIF-1α in HMECs significantly reduced Ado-induced eNOS mRNA, implicating that ADORA2B-mediated HIF-1α induction contributes to elevated eNOS mRNA and underlies Ado-mediated priapism.

Conclusions:

PEG-ADA and PSB1115 are effective and safe to treat priapism and exacerbation of the disease by decreasing penile Ado levels or interfering its signaling. This study provides direct preclinical evidence for the novel and general utility of PEG-ADA enzyme therapy or ADORA2B antagonists for priapism and sets up a solid foundation for future clinical trials to assess the usefulness of Adobased therapeutics to treat priapism.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Inhibition Prevents Against Severe Atherosclerosis Progression in Prothrombotic Mice
Authors: Spronk Henri M.H. , Borissoff Julian I , Soehnlein Oliver , Koenen Rory , van Oerle Rene , Heeneman Sylvia , Daemen Mat J.A.P. , Esmon Charles T , Degen Jay L. , Weiler Hartmut , van Ryn Joanne , Cate Hugo Ten
Published: October 2019

Abstract 103

Background:

Besides its well-known role in initiation and propagation of thrombus formation, thrombin may have multiple pro-atherogenic actions.

Hypothesis:

Pharmacologic thrombin inhibition attenuates the onset and progression of atherosclerosis and favors plaque stability in hypercoagulable mice prone to aggressive atherosclerosis development.

Methods and Results:

Hypercoagulability in TMPro/Pro:ApoE−/− mice resulted in severe atherosclerotic burden formation compared to ApoE−/− mice (389.1*103 μm2 vs. 187.0*103 μm2, p<0.0005). Pro-coagulant mice showed larger necrotic cores, decreased plaque collagen content, vast stenosis (up to 90%) and enhanced outward remodeling. Hypercoagulability markedly increased systemic and vascular inflammation, represented by significantly higher CXCL1, MCP-1 and G-CSF plasma levels, pronounced neutrophilia, enhanced Ly6G+ cells arrest to carotid plaques, increased neutrophil recruitment (IHC/Intravital Microscopy), massive intraplaque hemorrhage and spontaneous plaque ruptures. Genetically diminished prothrombin levels (FII-/WT:ApoE−/− mice) caused a significant decrease in atherosclerosis (72.5*103 μm2 vs. 128.4*103 μm2, p<0.01) with a pronounced stable phenotype with less/smaller necrotic cores, thicker fibrous caps and abundant collagen and vascular smooth muscle cell content. Ly6G+ cell intraplaque recruitment was reduced and white blood cell counts and IL-6 levels (3.8±1.66 vs. 8.8±5.34 pg/mL, p<0.05) were diminished compared to control mice. Remarkably, administration of the oral direct thrombin inhibitor Dabigatran etexilate or the anticoagulant activated protein C (APC) in TMPro/Pro:ApoE−/− mice reduced lesions formation by > 80%, suppressed leukocyte recruitment, enhanced plaque stability, whereas numerous pro-inflammatory cytokines were diminished to levels comparable to non-atherosclerotic mice.

Conclusion:

These data establish a pivotal role for thrombin generation in controlling the level of inflammation related to atherosclerosis development in ApoE−/− mice. The potential clinical impact of long term administration of novel classes of anticoagulants on atherosclerosis merits further study.

Disclosures:

Weiler: BloodCenter of Wisconsin: Patents & Royalties. van Ryn:Boehringer Ingelheim Pharma GmbH & Co KG: Employment.
TMEM16F Ion Channel Regulates Calcium-Dependent PS Exposure, Hemostasis, and Thrombosis
Authors: Kim Andrew Y. , Yang Huanghe , David Tovo , Tien Jason , Coughlin Shaun R , Jan Yuh Nung , Jan Lily
Published: October 2019

Abstract 1111

Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca2+-activated Cl− channels (CaCCs) is linked to Scott Syndrome with deficient Ca2+-dependent lipid scrambling. We generated TMEM16F-knockout mice that exhibit bleeding defects associated with platelet deficiency in Ca2+-dependent phosphatidylserine exposure and procoagulant activity, and lack a Ca2+-activated cation current in the platelet precursor megakaryocytes. TMEM16F channels permeate both monovalent and divalent cations including Ca2+, and exhibit synergistic gating by Ca2+ and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a novel Ca2+-activated channel permeable to Ca2+ and critical for Ca2+-dependent scramblase activity.

Disclosures:

No relevant conflicts of interest to declare.
The Hsa-Let-7a miRNA Enhances Ara-C Induced Apoptosis in Human Acute Myeloid Leukemia Cells
Authors: Chen Ye , Jacamo Rodrigo , Konopleva Marina , Garzon Ramiro , Croce Carlo M. , Andreeff Michael
Published: October 2019

Abstract 1286

CXCR4 surface expression has been reported as an important prognostic marker in AML patients. Our group previously reported that targeting the SDF-1α/CXCR4 axis by CXCR4 inhibition could overcome the resistance of AML cells to chemotherapy both in vitro and in vivo. To further explore the mechanism of targeting CXCR4, we first performed a microRNA microarray platform and revealed that hsa-let-7a as one of the most significantly degraded microRNA in OCI-AML3 cells treated with SDF-1α. Moreover, hsa-let-7a was highly elevated when the OCI-AML3 cells were treated with CXCR4 antagonist. To investigate the role of hsa-let-7a in leukemia cells, we first attempted to transfect human hsa-let-7a mimic into the OCI-AML3 cells by electroporation. When exposed to chemotherapy agent AraC (2.5μM, 48 hours), the hsa-let-7a overexpressed cells exhibited 2 more folds of apoptosis than negative controls (Annexin V positive percentage: 69.8% ± 9.4 % vs 23.2% ± 2.7%, p<0.01). Next, we suppressed CXCR4 in OCI-AML3 cells with lenti-virus delivered CXCR4 shRNA (CXCR4-shRNA-OCI3) to test whether CXCR4 regulates the hsa-let-7a expression. The CXCR4-shRNA-OCI3 cells had ∼70% lower of the cell surface CXCR4 expression as evaluated by Flow cytometry (PE-conjugated 12G5 anti-CXCR4 antibodies) and ∼60% less of migration to SDF-1α compared with the vector control cells. Using RT-PCR, we observed that hsa-let-7a level was elevated in the CXCR4-shRNA-OCI3 cells (∼2.3 folds of level in the NS-shRNA-OCI3 cells). As expected, hsa-let-7a target BCLXL were significantly decreased in CXCR4-shRNA-OCI3 cells in both mRNA and protein levels, which lead to higher sensitivity to AraC treatment (Annexin V positive percentage: 48.1% ± 5.1 % vs NS-shRNA-OCI3 cells 19.1% ± 2.7%, p<0.01). To investigate whether the CXCR4 surface expression is associated with the hsa-let-7a level in patient samples, we collected bone marrow samples from AML patients after informed consent. Mononuclear cells were separated by ficoll and stained with CXCR4 antibody for cell sorting. We observed CXCR4 high expression subpopulation had significantly lower level of hsa-let-7a. c-Myc and BCLXL protein levels were much lower in CXCR4 low expression subpopulation, and it may explain that low CXCR4 expression on AML cells correlated with a better prognosis, resulting in a longer relapse-free and overall survival. Collectively, our results support that CXCR4 antagonists could mobilize AML cells from the protective stromal microenvironments and make them more susceptible to conventional therapy. These findings also showed hsa-let-7a plays an important role in AML cell chemoresistance, and suggested a new mechanism that microRNA is involved in SDF-1α/CXCR4 signaling pathway.

Disclosures:

No relevant conflicts of interest to declare.
Comparison of the Transforming Effect of Different SYK Oncogenes in Vivo Reveals TEL-SYK As Inducer of an MDS/MPS Overlap Syndrome in Mice
Authors: Sprissler Clara , Maurer Helga , Jumaa Hassan , Veelken Hendrik , Dierks Christine
Published: October 2019

Abstract 1310

The spleen tyrosin kinase (SYK) was shown to be overexpressed in various human hematologic malignancies like B-cell lymphomas and acute myeloid leukemias. There are two known translocations involving the SYK kinase: ITK-SYK and TEL-SYK. ITK-SYK was identified in unspecified peripheral T-cell lymphomas, whereas the TEL-SYK fusion had been discovered in a patient with myelodysplastic syndrome (MDS).

Methods:

All SYK containing oncogenes, ITK-SYK, TEL-SYK and SYK wt, were cloned into a pMSCV-IRES-GFP retroviral vector. All oncogenes and a GFP control vector were overexpressed in bone marrow of 5-flurouracil-pretreated mice and oncogene containing bone marrow was transplanted into irradiated recipient mice. Disease development was monitored over 1 year. Additionally the three SYK oncogenes were overexpressed in a myeloid (32D), B-lymphoid (BaF3) and T-cell lineage (Jurkat) to monitor cell context specific differences in cell signaling.

Results:

Here, we demonstrate that the overexpression of 3 SYK containing oncogenes, SYK wt, TEL-SYK and ITK-SYK in a murine bone marrow transplantation model induces the development of 3 different hematologic (pre)diseases. While ITK-SYK exclusively induced T-cell lymphomas in mice, we found that the TEL-SYK fusion induced an MDS/MPS overlapsyndrom. The mice developed an anemia and thrombopenia with signs of dysplasia of megakaryocytes and the erythroid lineage in the bone marrow and spleens. In addition those mice showed a strong expansion of the myeloid lineage, with leucocytosis in the peripheral blood mainly consisting of differentiated neutrophils. The bone marrow shows expansion of differentiated myeloid cells, but also myeloid progenitors and expansion of HSCs. Interestingly, also SYK wt induced a relative expansion of the myeloid compartment without inducing a leucocytosis or a lethal disease indicating a premalignant myeloproliferative state. Only TEL-SYK was able to induce IL-3 independent growth of BaF3- and 32D cells and could activate STAT5, STAT6, ERK, PLCγ2 and SLP76 in those cell lines. SYK and ITK-SYK were able to activate PLCγ2 and SLP76, but no STAT transcription factors. STAT5 activation by TEL-SYK was detectable also in vivo, which might indicate STAT5 as the major driver of TEL-SYK induced transformation.

Our results demonstrate, that the various SYK oncogenes can induce different hematologic diseases in mice and reveales TEL-SYK as the first oncogene inducing an MDS/MPS overlap syndrome in mice.

Disclosures:

No relevant conflicts of interest to declare.
MLL Fusion Driven Activation of CDK6 Potentiates Proliferation in MLL-rearranged Infant ALL
Authors: van der Linden Marieke Hendrika , Willekes Merel , Van Roon Eddy , Schneider Pauline , Pieters Rob , Stam Ronald
Published: October 2019

Abstract 1426

MLL-rearranged acute lymphoblastic leukemia represents a highly aggressive and clinically unfavorable type of childhood leukemia, displaying unique gene expression signatures. Nevertheless, the overwhelming number of differentially expressed genes made it difficult to elucidate the actual “drivers” of the leukemia. However, recent advances demonstrated that MLL fusion proteins recruit the histone methyltransferase DOT1L, leading to H3K79 methylation. Hence, genomic regions displaying aberrant enrichment of H3K79 methylation are prone to mark genes transcriptionally activated by the MLL fusion protein itself. Based on this concept, two independent studies recently identified gene signatures consisting of genes likely to represent direct MLL fusion targets. Yet, functional validation of such genes so far remains unacknowledged. In the present study we confirmed that CDK6 (cyclin-dependent protein kinase 6) represents a direct target of MLL-AF4 in t(4;11)-positive ALL cells. In contrast to its functional homologue CDK4, ChIP-sequencing analysis showed the presence of both MLL and AF4, as well as H3K79 methylation at the CDK6. Moreover, CDK6 mRNA appeared significantly (p<0.001) higher expressed in primary MLL- rearranged infant ALL patient samples when compared with other childhood ALL subtypes without translocations of the MLL gene. Next, using RNA interference, we performed MLL-AF4 and MLL-ENL knockdown experiments in ALL cell lines bearing these corresponding fusion transcripts, resulting in CDK6 down-regulation, whereas CDK4 expression was unaffected. These results emphasize that CDK6 is indeed a genuine transcriptional target of the MLL fusion protein itself. Moreover, direct knockdown of CDK6 itself significantly inhibited proliferation in MLL-rearranged ALL cells, whereas knockdown of CDK4 virtually had no effect on the cell cycle in these cells.

Taken together we conclude that CDK6 up-regulation in MLL-rearranged ALL is directly mediated by the MLL fusion itself and provides these cells with a proliferation advantage.

Disclosures:

No relevant conflicts of interest to declare.
Incidence, Age, and Gender of Children with Thrombotic Thrombocytopenic Purpura (TTP) Associated with Severe, Acquired ADAMTS13 Deficiency.
Authors: Muthurajah Darrshini S. , George James N. , Vesely Sara K. , Terrell Deirdra R.
Published: October 2019

Abstract 2196

Introduction

TTP associated with severe acquired ADAMTS13 deficiency has been rarely reported among children. Specific aims: [1] To identify all published reports of TTP associated with acquired ADAMTS13 deficiency in children <18 years old and document the age and gender distribution. [2] To document the incidence in the Oklahoma TTP-HUS Registry region of TTP associated with acquired ADAMTS13 deficiency (activity <10%) in children <18 years old.

Methods

Systematic literature review using MEDLINE, PubMed, Embase, Cochrane, and CINAHL databases. Comprehensive search terms for TTP, HUS, and children were combined with the term, ADAMTS13. Bibliographies of selected articles were reviewed. Incidence was calculated using person-years of all children < 18 years old in the Registry region (58 counties) over the 16.5 year period (January 1, 1996 to June 30, 2012) using 2000 census data.

Results

The systematic literature search identified 22 publications reporting 53 children with acquired ADAMTS13 deficiency. Their age and gender are presented in the Figure. Two-thirds of children were ages 9–17 years. Among the 18 children ages 0–8 years, 8 (44%) were girls; among the 35 children ages 9–17 years, 24 (69%) were girls. The relative frequency of the older children was greater than younger children (p=0.02). Although there were more girls than boys reported, the difference was not significant (p=0.13). From January 1, 1995 to June 30, 2012, 72 patients with acquired ADAMTS13 deficiency were enrolled in the Oklahoma TTP-HUS Registry; one patient was less than 18 years old. The calculated incidence for the Oklahoma Registry region was 0.10/106 children/year.

Conclusion

The Oklahoma Registry experience documents for the first time the incidence of TTP associated with acquired ADAMTS13 deficiency in children: 0.10/106 children/year, 6% of our previously calculated incidence rate of 1.74/106/year for all patients with TTP and acquired ADAMTS13 deficiency (J Thromb Haemost 2005;3:1432). The age and gender distribution of children in published case reports suggests that TTP associated with acquired ADAMTS13 deficiency in children is not a distinct juvenile disorder, but more likely a continuum of the spectrum of TTP in adults (J Thromb Haemost 2005;3:1432).

Disclosures:

George: Alexion, Inc.: Consultancy; Baxter, Inc.: Consultancy; Amgen, Inc.: Consultancy, PI of clinical trial involving romiplostim, PI of clinical trial involving romiplostim Other, Research Funding. Terrell:Baxter, Inc.: Consultancy; Amgen, Inc.: Consultancy.
Stromal Cells Expressing Hedgehog-Interacting Protein Are One of the Main Regulators of the Hedgehog Signaling System in Myeloid Neoplasms
Authors: Kobune Masayoshi , Horiguchi Hiroto , Kikuchi Shohei , Iyama Satoshi , Takada Kohichi , Murase Kazuyuki , Ono Kaoru , Kamihara Yusuke , Kawano Yutaka , Hayashi Tsuyoshi , Sato Tsutomu , Miyanishi Koji , Sato Yasushi , Takimoto Rishu , Kato Junji
Published: October 2019

Abstract 3840

Aberrant reactivation of Hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasm is unclear. In this study we assessed the expression of Hh-related proteins in normal human CD34+ cells, CD34+ leukemic/dysplastic cells and BM stromal cells. Both Indian hedgehog (Ihh) and its signal transducer, SMO, were expressed in CD34+ acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) derived cells, suggesting that Ihh could be affected in an autocrine manner. Remarkably, expression of the endogenous Hh signaling inhibitor, human hedgehog-interacting protein (HHIP), in AML/MDS-associated stromal cells was significantly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression level in BM stromal cells highly correlated with their tumor-supporting activity of SMO+ leukemic cells. Demethylating agent 5-aza-dC rescued HHIP expression via demethylation of HHIP gene and reduced the leukemia-supporting activity of AML/MDS-associated stromal cells. This effect of 5-aza-dC was negated by HHIP shRNA transfer into stromal cells. These results indicate that suppression of stromal HHIP expression could be involved in the progression of AML/MDS and 5-aza-dC may improve the protective function of BM stromal cells for AML/MDS.

Disclosures:

No relevant conflicts of interest to declare.
ERK1/2 Pathway Is Required for Differentiation of AML Triggered by Anti-CD44 Monoclonal Antibodies
Authors: Al-Madhoun Nour Y. , Gadhoum Samah Zeineb , Merzaban Jasmeen S.
Published: October 2019

Abstract 4334

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by accumulation of immature hematopoietic cells due to a blockage in myeloid differentiation at various stages. Since the current chemotherapy rarely eradicates the leukemic clones entirely, differentiation induction therapy has evolved. Previous experiments have shown that ligation of CD44, a cell surface glycoprotein strongly expressed on all AML cells, with anti-CD44 monoclonal antibodies (mAbs) could reverse the differentiation blockage of leukemic blasts and inhibit their proliferation in most AML subtypes, rendering CD44 a promising target for AML therapy. However, the underlying molecular mechanisms of the induction of differentiation by anti-CD44 mAbs have not been fully elucidated. Here, we investigated the signaling pathways triggered during anti-CD44 mAb (A3D8)-induced granulocytic differentiation of HL60, a human leukemic cell line commonly used as a model of human AML type2/3.

Extracellular signal regulated kinase (ERK1/2) pathway plays a major role in the regulation of differentiation and proliferation for myeloid cells and has been reported to be aberrantly activated in AML. In our studies, we observed an increase of ERK1/2 phosphorylation by mitogen-activated ERK kinase (MEK) following A3D8 treatment. The use of U0126 (a MEK inhibitor) significantly abrogated the anti-CD44 induced granulocytic differentiation of HL60 cells, suggesting that ERK1/2 is critical for the induction of differentiation via CD44 in AML-M2/3. Our work provides the first evidence for the importance of the ERK pathway in granulocytic differentiation via CD44 and adds a new argument to the use of CD44 in differentiation therapy of AML.

Disclosures:

No relevant conflicts of interest to declare.
New Characteristics of Translocation in Anaplastic Large T-Cell ALK-Positive Lymphoma
Authors: Gorenkova Liliya , Kravchenko Sergei , Misurin Andrei , Vinogradova Yulia , Penskaya Ekaterina , Gemdjian Eduard G. , Savchenko Valeri G.
Published: October 2019

Abstract 4419

Background

ALK-Positive Anaplastic Large Cell Lymphoma (ALCL ALK+) represent 5–15% of all non-Hodgkin in children and 5% in adults and characterized by the specific chromosome translocations, which lead to the break of gene ALK. Currently describe 9 translocations. All of them have no prognostic significance.

Purpose

We present a case of ALCL ALK+ with a new chromosomal rearrangement involving locus gene 2p23 and poor response to therapy.

Case presentation. A 24-year old woman presented to our clinic with a month history of right axillar tumor 20×9 sm. Full examination reveals B-symptoms, the involvement of the mammary gland, generalized lymphadenopathy and splenomegaly.

The diagnosis ALCL ALK+ was made on the base of CD30 and ALK expression and ALK gene rearrangement. Cytogenetic analysis showed a normal karyotype including a new translocation t(2;8)(p23;q22). In our centre we provided the pilot study of the efficacy of the protocol NHL BFM-90 in adult ALCL ALK+. Overall five-year survival rate is 90%±6% (n=25).

The patient was treated according to the program, branch K3 (6 cycles AA-BB-C-AA-BB-C, the dose of methotrexate 5 g/m2). The remission was achieved after 4 cycle. CNS prophylaxis was made in the first cycle of the chemotherapy (intrathecal administration of methotrexate 15 mg, cytarabine 30 mg and dexamethasone 4 mg).

A weak after the last cycle of the chemotherapy the patient complained of intensive headache. Neuroleukemia was diagnosed (100 cells in μl large anaplastic cells expressed CD30, ALK, and have ALK rearrangement). Full examination revealed no other tumor lesions. The patient was treated with 10 intrathecal administration of methotrexate, ara-C and dexamethasone. The normalization of the cerebrospinal fluid was after the 7th lumbar puncture. Currently the patient is treated following chemotherapy program for relapsed lymphomas.

Conclusions

we presented a case of primary progressive course of ALCL ALK+ with a new chromosomal rearrangement t(2;8)(p23;q22). To the best of our knowledge and after literature search this appears to be a new cytogenetic abnormality in ALCL ALK+. It was the first case of progression of ALCL ALK+ on the intensive program NHL BFM-90 in adults in our experience. So we propose that this translocation may be associated with unfavorable prognosis. Further trials and study of pathogenesis of t(2;8)(p23;q22) to confirm our observation.

Disclosures:

No relevant conflicts of interest to declare.
The Relationship of Myeloma Cells, Stromal Cells and Monocytes Under Bone Marrow Microenvironment
Authors: Ikeda Hiroshi , Aoki Yuka , Nojima Nasanori , Yasui Hiroshi , Hayashi Toshiaki , Ishida Tadao
Published: October 2019

Abstract 4987

The Bone marrow (BM) microenvironment plays crucial role in pathogenesis of Multiple myeloma(MM). Myeloma cells contacts with bone marrow stromal cells (BMSCs), which secrete factors/cytokines, promoting tumor cell growth and survival. Paracrine secretion of cytokines(i. e., interleukin-6 (IL-6) insulin-like growth factor-1, inflammatory protein-1a) in BM stromal cells promotes multiple myeloma cell proliferation and protects against drug-induced cytotoxicity. These cytokines provide stimulatory signals for multiple myeloma growth and survival.

Bone involvement is a common feature in MM patient, solid and hematologic cancers. MM localizes to the bone in nearly all patients ranges between 40% and 75%. Disease-related skeletal complications result in significant morbidity due to pain, pathologic fractures and spinal cord compression. The bone microenvironment creates a supportive niche for tumor growth. Osteoclasts and bone marrow stromal cells, along with extracellular matrix and cytokines stimulate tumor cell proliferation and confer chemoresistance. Therefore, the reciprocal interactions between tumor cells, osteoclasts, osteoblasts, and bone marrow stromal cells present an important.

In current study, monocyte can directly promote mesenchymal stem cells osteogenic differentiation through cell contact interactions, thus resulting in the production of osteogenic factors by the monocytes. This mechanism is mediated by the activation of STAT3 signaling pathway in the mesechymal stem cells that leads to the upregulation of Osteoblasts-associated genes such as Runx2 and alkaline phosphatase (ALP), and the down-regulation of inhibitors such as DKK1 to drive the differentiation of mesechymal stem cells into osteoblasts.

In this study, we examined the role of monocyte, component of BM cells, as a potential niche component that supports myeloma cells. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BM stromal cells, monocytes, or a combination of BM stromal cells and monocytes. Consistently, we observed increased proliferation of MM cell lines in the presence of either BM stromal cells or monocytes compared to cell line-only control. Furthermore, the co-culture of BM stromal cells plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the BM microenvironment.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Significance of Cell of Origin Subclassification in Diffuse Large B Cell Lymphoma
Authors: Dawod Muath , Gomez-Gelvez Juan , Mattour Ahmad , Inamdar Kedar V. , Janakiraman Nalini
Published: October 2019

Abstract 5081

Background:

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease that has been divided into three different prognostic subgroups: Germinal Center B cell-like (GC), Activated B cell-like (ABC) and type 3 according to gene expression profile using cDNA. Immunohistochemistry (IHC) has been used as surrogate to identify these cell-of-origin subgroups. Data about the prognostic value of IHC has been conflicting.

Patients and methods:

In this retrospective study, we reviewed the charts of 252 patients diagnosed with DLBCL at Henry Ford Hospital from 1999 to 2012. We excluded patients with HIV, transformed lymphomas and unavailable samples. Data was collected on a total of 157 patients. The following data was gathered: age, sex, race, IPI score, disease stage, hemoglobin, white blood and platelet counts, best response achieved and dates of treatment start, relapse, death or last follow up. Tissue microarray slides with the following IHC stains (CD10, MUM1, Bcl6) were prepared and reviewed when needed. Using Hans Algorithm, samples were divided into two major groups (GC-like and non-GC-like). 3-year progression free and overall survivals were compared between all subgroups using a log-rank test. Continuous variables were reported as median and range, and compared using Wilcoxon rank-sum tests. Categorical variables were reported as median and range, and compared using Chi-square tests. Statistical significance was set at p<0. 05.

Results:

Eighty patients (51%) were classified as GC-like, and 77 patients (49%) as non-GC-like. GC-like subgroup had a significantly longer 3-year progression free survival (90% vs 74%, P=0. 024), as compared with the non-GC-like subgroup. There was a trend toward longer overall survival but it didn't reach statistical significance (74% vs 67%, P=0. 161). For all patients, IPI stands as a strong prognostic index with 3-year overall survival of (85% and 46%, P=<. 001) in patients with low IPI (0 to 2) and high IPI (3 to 5) respectively. Interestingly, in patients with low IPI, cell of origin stands as a prognostic factor with 3-year progression free survival of (96% and 81%, P=0. 032) in GC-like and non-GC-like groups respectively. While in patients with high IPI, there was no significant difference in progression free survival in cell-of-origin subgroups.

Conclusion:

Cell of origin subclassification as determined by IHC surrogate markers predict for better progression free survival in GC-like subgroup but not for overall survival. While this prognostic value was maintained in patients with low IPI, there was no prognostic significance in patients with high IPI. IPI is still a valuable prognostic tool in patients with DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Variability in Exposure of Epitope G40-R43 of Domain I in Commercial Anti-β2-Glycoprotein I IgG Elisas Influences the Diagnosis of the Antiphospholipid Syndrome
Authors: Pelkmans Leonie , Kelchtermans Hilde , Ninivaggi Marisa , Lindhout Theo , de Groot Philip G , Pengo Vittorio , De Laat Bas
Published: October 2019

Abstract 632

Introduction.

Diagnosis of the antiphospholipid syndrome (APS) depends on the detection of autoantibodies against phospholipid-bound β2-glycoprotein I (β2GPI), the most prominent antigen in APS. One of the main problems faced is the high variability observed between different commercially available anti-β2GPI assays. Anti-β2GPI antibodies constitute a heterogeneous population, but predominantly antibodies reacting to a cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I of β2GPI are associated with a strong risk to develop thrombosis. b2GPI is present in blood in a native conformation (either circular or S-shaped). After interaction with anionic surfaces it opens up (J-shaped conformation), resulting in exposure of the epitope G40-R43 in domain I. It is therefore important that in diagnostic assays b2GPI is presented in the open conformation enabling antibodies to react with the G40-R43 epitope. We hypothesize that the high variability between different commercial anti-b2GPI ELISA assays arise from variation in exposure of the G40-R43 epitope of b2GPI.

Methods.

Two patient-derived monoclonal antibodies P2-6 and P1-117 were tested for their reactivity towards β2GPI in different conformations. Using both antibodies, we compared exposure of epitope G40-R43 on β2GPI in five different commercial anti-β2GPI IgG assays. 10 patient samples selected for their low to high positivity towards epitope G40-R43, were tested in two anti-β2GPI IgG assays.

Results.

Using neutral versus anionic ELISA plates, we have shown that antibody P1-117 specifically reacts with epitope G40-R43, exposed only in the open conformation, while antibody P2-6 recognizes β2GPI irrespective of its conformation (Fig. 1). In one of the tested anti-β2GPI assays, both antibodies showed equal reactivity towards β2GPI, indicating that all the coated β2GPI exposes epitope G40-R43. In this assay, all ten anti-domain I positive patients tested positive. In other assays P1-117 displayed lower reactivity towards β2GPI than P2-6, demonstrating a reduced exposure of G40-R43. Only 3 out of the 10 patients tested positive in such assay, illustrating that a significant number of patients can be falsely assigned negative in assays characterized by a reduced exposure of epitope G40-R43.

Conclusions.

The exposure of epitope G40-R43 on β2GPI is highly variable in commercial anti-β2GPI assays, and influences the diagnosis of APS. Our results have major implications for the diagnosis of APS, as it provides suitable controls to ensure sufficient exposure of the G40-R43 epitope or suggests the development of alternative assays coating only domain I of β2GPI.

Disclosures:

No relevant conflicts of interest to declare.
New Insights in Development and Function of Human Innate Lymphoid Cells
Authors: Spits Hergen , Bernink Jochem , Peters Charlotte , Mjosberg Jenny
Published: October 2019

Abstract SCI-19

Innate lymphoid cells (ILC) are immune cells that lack a specific antigen receptor, yet possess the capacity to produce an array of effector cytokines that in variety matches that of T-helper-cell subsets. Innate lymphoid cells function in lymphoid organogenesis, tissue remodeling antimicrobial immunity and inflammation, particularly at barrier surfaces. The ability of ILCs to promptly respond to insults inflicted by stress-causing microbes strongly suggests that ILCs are critical in first-line immunological defenses. ILCs are also involved in repair of tissue damage inflicted by pathogenic microbes. The scientific session presentation will include data on developmental requirements, lineage relationship and effector functions of human ILCs. Two families of innate lymphoid cells will be discussed: Rorγt-expressing cells involved in lymphoid tissue formation, mucosal immunity and inflammation, and Type 2 innate lymphoid cells that are important for helminth immunity. In addition, evidence will be presented for the existence of a novel ILC population that is dedicated to producing interferon-γ and which we call type 1 ILC. The potential roles of ILC in the pathology of immunity-mediated inflammatory and infectious diseases, including allergic diseases, will be discussed.

Disclosures:

No relevant conflicts of interest to declare.
The Impact Of Oxidative Stress On The Immune Compartment Of Chronic Lymphocytic Leukemia Patients
Authors: Jitschin Regina , Bricks Juliane , Berger Jana , Eckart Michael J , Mackensen Andreas , Mougiakakos Dimitrios
Published: October 2019

Reactive oxygen species (ROS) such as hydrogen peroxide or superoxide are highly reactive and hyperpermeable. They are continuously produced by mainly the mitochondria during oxidative phosphorylation. Immune cells of especially myeloid origin contribute additionally to the ROS production via the membrane-bound NADPH-oxidase. During inflammation or “inflammatory-like” conditions as often seen in neoplasias ROS production is highly increased. The physiological redox-buffers are overwhelmed leading to the metabolic condition termed oxidative stress. Immune cells, especially NK- and T-cells are very sensitive towards ROS-induced cell death resulting from mitochondrial depolarization. Furthermore, ROS can interfere with nuclear NF-kB translocation thus leading to reduced activation, cytokine production, and cytotoxic activity of immune cells. Functional immune defects at several levels as well as significant shifts in the balance of immune populations have been related to oxidative stress in patients suffering from mainly solid malignant diseases. In this study we sought out to investigate the presence of oxidative stress in patients with chronic lymphocytic leukemia (CLL) and its potential effects on their cellular immune compartment.

Peripheral blood and serum from 65 patients with untreated CLL was collected upon informed consent. Oxidative stress was measured based on the concentration of so-called ROS-surrogate markers in the patients' sera. Therefore oxidized lipids, proteins, and DNA bases were quantified by ELISA. The immune compartment was evaluated using multicolor flow cytometry and the analyses included T-, NK-, myeloid-, and dendritic-cell subsets.

ROS levels were increased in CLL patients. Based on the concentration of ROS-modified molecules we were able to group our patient cohort into a ROShi and ROSlo group. The ROShi group exhibited lower levels of T-cell activation, while there was a shift of the CD56bright/CD56dim NK-cell balance towards the CD56bright subset. We could not detect significant differences in the myeloid- and dendritic-cell compartment or alterations of T- and NK-cell maturation.

Our results implicate that ROS belongs to the tumor immune escape mechanism present in CLL. The use of redox modifying agents in combination with the steadily emerging immune-based approaches could potentially boost their therapeutic efficacy.

Disclosures:

No relevant conflicts of interest to declare.
Kinetics Of Chronic Lymphocytic Leukemia Cells In Tissues and Blood During Therapy With The BTK Inhibitor Ibrutinib
Authors: Wodarz Dominik , Garg Naveen , Komarova Natalia L. , Benjamini Ohad , Keating Michael J. , Wierda William G. , Kantarjian Hagop M. , James Danelle F. , O'Brien Susan M. , Burger Jan A.
Published: October 2019

Bruton's tyrosine kinase (BTK), an essential component of B-cell-receptor (BCR) signaling, is blocked by ibrutinib, a BTK inhibitor that is clinically active in patients with chronic lymphocytic leukemia (CLL). Characteristically, ibrutinib causes CLL cell redistribution from tissue sites into the peripheral blood during the initial weeks of therapy, resulting in a transient surge in lymphocytosis. This activity of ibrutinib is thought to be important for the anti-leukemia activity of ibrutinib in CLL, because it removes CLL cells from the tissue microenvironment and consequently deprives the leukemia cells from growth- and survival-promoting cellular and molecular signals that are present in the tissues. However, clinically it has been noted that the magnitude of lymphocytosis during ibrutinib therapy is lesser than what would be expected from the rapid reduction in overall tissue disease burden, seen during the early weeks of ibrutinib therapy, especially in patients with bulky lymph nodes.

Therefore, we aimed to quantify the relative amount of tissue CLL cells redistributed into the peripheral blood, and to better characterize the dynamics of this redistribution phenomenon. Crucial kinetic parameters were estimated by fitting a two-compartment mathematical model to clinical data that document serial blood lymphocyte counts of CLL patients as well as changes in total tissue burden, calculated from radiological measurements of lymph node and spleen volumes during ibrutinib therapy. This was done in a cohort of ten relapsed CLL patients, treated with single-agent ibrutinib, 420 mg PO daily on the Phase 1b/2 ibrutinib study at MD Anderson Cancer Center. We found that during ibrutinib therapy, on average 1.7 ± 1.1% of the cells die per day in the blood, while in tissue 2.7% ± 0.99% of the cells die per day. These death rates during treatment are approximately 5 times higher than those estimated in previous studies in the absence of treatment. The average fraction of the tissue CLL cells that was redistributed into the blood during ibrutinib therapy accounted for 23.3 ± 17% (range: 1.9% to 52.6%) of the calculated total tissue disease burden, with an average time for achieving a 50% reduction in tissue CLL cells of 20.3 ± 3.6 days. These findings indicate that generally a relatively small fraction of the total tissue CLL cell burden is re-distributed into the blood during ibrutinib therapy, and that a significant amount of drug-induced cell death occurs in tissue compartments.

Disclosures:

Off Label Use: Ibrutinib for CLL therapy. James:Pharmacyclics: Employment, Equity Ownership. O'Brien:Pharmacyclics: Research Funding. Burger:Pharmacyclics: Research Funding.
Role Of PET/CT In The Management Of Burkitt Lymphoma
Authors: Carrillo Estrella , De la Cruz Fatima , Marín Víctor , Solé María , Borrego Isabel , Quiroga Eduardo , Manzanares Marina , Capote Francisco Javier , Ramirez María José , Espigado Ildefonso , Vázquez Ricardo , Jose Antonio Pérez-Simón
Published: October 2019

Introduction

Burkitt lymphoma (BL) is a highly aggressive and chemosensitive B cell non-Hodgkin lymphoma derived from germinal or post-germinal center B cells, which affects commonly extranodal sites. Although studies have described 100% of 18F-FDG avidity in BL, PET/CT is not routinely used in those patients being only recommended in the context of a clinical trial. The aim of the present study is to evaluate retrospectively, the role of PET/CT instead of CT scan alone both to stage newly diagnosed BL patients as well as to evaluate disease response after chemotherapy.

Patients and Methods

53 PET/CT (20 PET/CT at diagnosis, 28 after first line treatment and 5 to monitor residual disease detected in response assessment PET/CT) were performed in 32 patients, between 2006 y 2012. Locations of involved areas were registered comparing staging CT and PET/CT and were classified as discrepancy or not.

Results

Patientxs baseline characteristics are summarized in table 1. At diagnosis, abdominal adenopathies had the highest SUVmax with a mean of 14,83 (3,5-35). Discrepancies were found in 64,7% of patients who had both imaging test available at diagnosis (n=17), almost all of them in extranodal sites. These findings upstaged 12% patients from localized to advanced disease. Sensitivity of PET/CT and CT was 100% and 53%, respectively. Regarding the response assessment, in 5 patients out of 13 (38%) who had both imaging test, the PET/CT after first-line treatment was negative whereas the CT demonstrated residual masses. No relapses were observed in those patients. Among 28 patients with a PET/CT available after first-line treatment, CR was attained in 22 patients; one true-positive and 5 false-positive lesions (FP) (1 nodal and 4 extranodal) were detected after completing treatment. SUVmax of FP single nodal site was 2,6 as compared to a mean of 14,9 for the positive lesions at diagnosis while the mean SUVmax of false-positive extranodal sites was 4,4 as compared to 12,1 at diagnosis. NPV was 100% and PPV 16%. SUVmax value of FP lesion was<60% of diagnosis in all cases. Thus, with a cut-off value SUVmax above 60% of that of diagnosis, the PPV is also 100%.

Conclusion

as compared to CT scan, a more accurate staging in BL can be obtained using PET/CT. Regarding response assessment, NPV reaches 100% and, using a cut-off value for the SUVmax of 60% below the value of diagnosis, also a 100% PPV is observed.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Characteristics and Outcomes Of Waldeyer’s Ring Lymphoma: Nation-Wide Study In Korea
Authors: Shin Ho-Jin , Suh Cheolwon , Il Lee Soon , Kim Won Seog , Lee Won Sik , Kim Hyo Jung , Choi Chul Won , Kim Jin Seok
Published: October 2019

There has been published several papers that analyzed Waldeyer’s ring lymphoma. However, so far, Asian data was rare and WHO classification was not used for classifying subtype of malignant lymphoma.

328 patients who were diagnosed as non-Hodgkin’s lymphoma involved Waldeyer’s ring (WR-NHL) were analyzed retrospectively in Korea.

Male was slightly predominant compared to female. Low risk disease (low to low intermediate by International prognostic index) was 76.8% and localized disease was 64.9%. Most patients had normal level of serum lactate dehydrogenase (85.4%) and were not absent of B symptoms (85.1%). Bone marrow involvement was seen in only 30 patients (9.1%). Diffuse large B cell lymphoma (DLBCL) (73.2%) was most popular, and peripheral T cell lymphoma, NK/T cell lymphoma, and marginal zone B-cell lymphoma were also frequently involved Waldeyer’s ring. Almost subtype of lymphoma including DLBCL involved tonsil most frequently (199/328, 60.7%). Frequently involved extra-Waldeyer’s ring sites were bone marrow, G-I tract, lung/plerura/pericardium, and bone. In DLBCL, 169 patients (70.4%) received chemotherapy, 50 patients (20.8%) received chemotherapy plus radiotherapy, thirteen patients (5.4%) received surgical resection +/- chemotherapy or radiotherapy, and six patients (2.5%) received radiotherapy alone. Surgical resection showed improved PFS, DFS, and OS compared to chemotherapy +/- radiotherapy. In localized disease, all patients who received surgical resection still alive until now and chemotherapy or chemotherapy plus radiotherapy was better PFS and OS than radiotherapy alone (5-year PFS rates, 83.2% and 89.3% vs 66.7%; 5-year OS rates, 77.0% and 75.4% vs 58.9%). PFS, DFS and OS of B-cell lymphoma exhibited improved compared to T-cell lymphoma.

In conclusion, DLBCL was most frequent subtype and tonsil was frequently involved site of WR-NHL. In localized disease, surgical resection should be tried as possible for achieving long-term survival. Especially, in T-cell lymphoma patients, more aggressive treatment was required due to poor outcomes compared with B-cell lymphoma.

Disclosures:

No relevant conflicts of interest to declare.