bluuebunny/crossref_metadata_2025 huggingface dataset:
A Phase I/II Study of Bortezomib in Combination with Doxorubicin and Intermediate-Dose Dexamethasone (iPAD therapy) for Relapsed or Refractory Multiple Myeloma
Authors: Takamatsu Yasushi , Sunami Kazutaka , Muta Tsuyoshi , Tsukada Junichi , Miyamoto Toshihiro , Eto Tetsuya , Higuchi Masakazu , Hata Hiroyuki , Suzumiya Junji , Tamura Kazuo
Published: October 2019

Abstract 5137

[Background]

Bortezomib and doxorubicin have synergistic activity against myeloma cells. However, the combination of bortezomib and cytotoxic agents has not long been permitted in Japan because the safety of the combination therapy has not been determined in Japanese patients. We, therefore, decided to undergo a phase I/II study of bortezomib, doxorubicin and intermediate-dose dexamethasone (iPAD) combination therapy in Japanese patients with relapsed or refractory myeloma.

[Methods]

In a phase I study, the maximally tolerated dose of bortezomib was determined when given in combination with a fixed dose of doxorubicin and dexamethasone. Bortezomib was administered iv on days 1, 4, 8 and 11 at a dose of 1.0 mg/m2 in cohort 1 and 1.3 mg/m2 in cohort 2. Doxorubicin 9 mg/m2 was given iv on days 1–4, and dexamethasone 20 mg po on days 1–2, 4–5, 8–9 and 11–12. After a recommended dose of bortezomib was determined, a phase II study was undertaken to examine overall response rate, survival time and toxicity profiles.

[Results]

The dose-limiting toxicity (DLT), defined as grade 4 hematological toxicity lasting more than 5 days and/or grade 3 or higher non-hematological toxicity, was observed in 2 of 6 patients in cohort 1, indicating this dose was considered tolerable. In cohort 2, 3 of 5 patients developed DLTs including grade 4 thrombocytopenia lasting more than 5 days, grade 3 elevated hepatic transaminase levels and grade 3 ileus in one of each patient, indicating this dose was intolerable. Thus, 1.0 mg/m2 of bortezomib is a recommended dose in iPAD therapy. In a phase II study, 27 patients, aged 40 to 78 (median 63), were enrolled to receive iPAD therapy by using 1.0 mg/m2 dose of bortezomib. Overall response (CR+PR) was achieved in 89% (95% CI, 76–100%), and complete response (CR+nCR) in 30% (95% CI, 11–48%). The median overall survival time was not reached and progression-free survival time was 12.3 months (95% CI, 6.1–18.5 months). One patient died of pneumonia. Grade 3/4 thrombocytopenia, neutropenia and anemia developed in 67%, 41% and 19%, respectively, and febrile neutropenia in 11%. Sensory neuropathy was observed in 78% with grade 3/4 in 22% of the patients. Grade 3/4 hypokalemia developed in 15%, hyponatremia in 11%, and fatigue, nausea and diarrhea in 4%.

[Conclusion]

The iPAD therapy is feasible and effective to treat relapsed or refractory myeloma in Japanese population.

(This clinical study was registered in University hospital Medical Information Network (UMIN) (registration number; UMIN000001210))

Disclosures:

No relevant conflicts of interest to declare.
MSCs From Patients with Myelofibrosis Display a Fibrotic Phenotype
Authors: Schneider Rebekka K , Leisten Isabelle , Ziegler Susanne , Schumacher Anne , Brümmendorf Tim H , Jost Edgar , Ziegler Patrick
Published: October 2019

Abstract 5160

Myeloproliferative neoplasms (MPNs) are characterized by the excessive production of terminally differentiated nonlymphoid cells or platelets in the bone marrow. They represent a heterogenous group of clonal hematologic malignancies and are classified into chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) and other rarer diseases.

MPNs are often associated with extramedullary hematopoiesis and progressive hepatosplenomegaly due to displacement of hematopoietic progenitor cells from the BM to spleen and liver. Progenitor mobilization follows the enhanced deposition of extracellular matrix proteins, which can be found in the bone marrow of MPN patients.

Nonhematopoietic bone marrow stromal cells (BMSCs) and their precursors (mesenchymal stem cells) are believed to be conditioned by abundantly released growth factors from the pathological hematopoietic clone in MPNs and in turn contribute to a modified niche environment which participates in the maintenance of the malignant clone and in disease progression.

We therefore aimed at the comparative analysis of BMSCs from MPN patients (displaying myelofibrosis 0–1) and healthy donors as well as at their matrix remodelling capacity.

BMSCs from PMF patients were obtained by trephine biopsies and isolated by plastic adherence in IMDM, 20% FCS and cortisone. BMSCs from PMF patients fulfilled MSC criteria according to common consensus (Dominici et al., 2007). To compare their potential to support hemato-lymphopoiesis, we analyzed their hemato-lymphotropic cytokine transcription by RT-PCR. MSCs from MPN patients and healthy donors expressed gene transcripts for M-CSF, SDF-1, LIF, FLT3L, Oct-4, SCF, IL-6 and IL-7, suggesting no significant difference in cytokine production. However, when activated through contact with collagen I/III and embedded in three-dimensional scaffolds, MSCs from MPN-patients extensively remodelled the collagenous matrix compared to healthy donors. Under osteogenic and undifferentiated culture conditions, the extracellular matrix (ECM) production by MPN-MSC was strongly enhanced compared to MSCs from healthy controls as shown by RT-PCR and immunohistochemistry for collagen I, IV, fibronectin, laminin and osteopontin. Furthermore, MSCs from MPN patients significantly contracted and densified the collagenous matrix and the ECM deposition by MSCs from MPN patients was highly comparable to the ECM changes observed in corresponding bone punches. We therefore hypothesize that MSCs from MPN patients are primed to produce enhanced ECM proteins, whereas their capacity to support hematopoiesis seems to be unchanged.

Disclosures:

No relevant conflicts of interest to declare.
The Prevalence of HIV-1 DNA in AIDS-Related Lymphoma throughout the Epidemic in the US
Authors: Huysentruyt Leanne C , Kaplan Lawrence D , McGrath Michael S.
Published: October 2019

Abstract 5205

Kaposi sarcoma (KS) and non-Hodgkin lymphoma (NHL) are the two most common AIDS-defining cancers and significantly contribute to HIV-related deaths. While highly active antiretroviral therapy (HAART) has produce a sharp decline in the incidence of KS and AIDS-related NHL (ARL), HIV-infected patients are still at a high risk for developing these malignancies. Recent studies evaluating HIV-1 DNA in 91 AIDS-related KS cases demonstrated a significant decrease in the prevalence of HIV-1 DNA positive KS cases post-HAART (after 1996; 16.7%) as compared to pre-HAART (before 1996; 45%) corresponding to the decrease in incidence of this cancer since the introduction of HAART. Additionally HIV was more prevalent in sites of visceral KS (51.9%) as compared to skin KS (30.7%). We have recently evaluated the HIV sequence evolution in the context of metastatic ARL and identified lymphoma-specific HIV compartmentalized within tumor sites that was genetically distinct from HIV in non-tumor sites suggesting a specific form of HIV arises within individuals who develop ARL. The goal of this research was to determine the prevalence and quantity of HIV DNA in 119 ARL biopsies representing the entire span of the HIV epidemic (1982–2005). Whole genomic amplification was performed on DNA extracted from three 10um paraffin embedded tissue sections and the presence of HIV-1 DNA was determined by quantitative amplification of the HIV gag gene. Of the 119 cases, 45% contained detectable levels of HIV DNA. The HIV antigen was predominantly localized to macrophages. A subset of the ARL cases in this study contained approximately 1 HIV copy per cell which is extremely high considering previously reported data describing HIV-infected lymph nodes contained approximately 1 HIV copy per 1000 cells. Similar to what was observed for KS, the prevalence of HIV-positive ARLs has decreased in the post-HAART era (39%) when compared to pre-HAART (54%). While the prevalence of HIV-1 DNA positive ARLs declined in the post-HAART era, it was not to the same extent as what was observed for KS. Thus, our data is consistent with a decrease in the incidence of both tumor types in the post-HAART era. The higher prevalence of HIV-1 DNA in visceral sites of KS and lymphoma specific-HIV sequences in sites of metastatic lymphoma suggests that HIV, especially HIV infected macrophages, may play a role in the pathogenesis of KS and ARL disease progression.

Disclosures:

No relevant conflicts of interest to declare.
Hemophagocytic Lymphohistiocytosis Is Caused by Engulfment of Hematopoietic Stem Cells by Macrophages Through Stem Cell-Specific Suppression of CD47 Under Influence of Cytokine Deregulation
Authors: Kuriyama Takuro , Takenaka Katsuto , Kohno Kentaro , Yoshimoto Goichi , Kishimoto Junji , Abe Yasunobu , Miyamoto Toshihiro , Akashi Koichi
Published: October 2019

Abstract 522

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by hemophagocytosis in the bone marrow with systemic inflammatory reactions. We have reported that the inflammatory cytokinemia including interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) might play a key role in activating macrophages to induce HLH (Akashi et al. Br J Haematol, 1994). Recent studies have shown that the engulfment of blood cells by macrophages is regulated in part by the interaction of CD47 and its ligand, signal regulatory protein alpha (SIRPA). Blocking the CD47 and SIRPA interaction by using anti-CD47 monoclonal antibodies can induce engulfment of blood cells, suggesting that the SIRPA signaling provide “don't eat me signals” to prevent macrophages from digesting self blood cells. These data led us to hypothesize that HLH is caused by the disruption of self-recognition by macrophages through impairment of the CD47-SIRPA system. To test this hypothesis, we evaluated the expression level of CD47 in bone marrow cells in 24 patients with HLH. Interestingly, the expression of CD47 was significantly downregulated (by ∼2-fold reduction in average at the protein level) in the CD34+CD38− hematopoietic stem cell (HSC) fraction in HLH patients, whereas that of the CD34+CD38+ progenitor and other mature blood cell fractions was unchanged. We then purified the CD34+CD38− HSCs and CD34+CD38+ progenitor cells from HLH patients and normal controls, co-cultured them with human macrophages in the presence of IFN-gamma and lipopolysaccharide, and evaluated the percentage of phagocyting macrophages. The numbers of phagocyting macrophages were significantly higher in cultures of CD34+CD38− HSCs from HLH patients, as compared to those in cultures of normal HSCs and of progenitor populations, suggesting that the expression level of CD47 inversely correlated with the efficiency of hemophagocytosis. Furthermore, normal HSCs but not CD34+CD38+ progenitors or other mature blood cells down-regulated CD47 in vitro in response to IFN-gamma, TNF-alpha, and IL-6, suggesting that these cytokines plays a critical role in down-regulation of CD47 especially in HSCs. In contrast, the expression level of SIRPA did not differ in myeloid cells between HLH patients and normal controls, and mutations of SIRPA were not found in HLH patients. Thus, in HLH, inflammatory cytokines down-regulate CD47 selectively in HSCs, resulting in the engulfment of HSCs by macrophages. Our data strongly suggest that the cytokine deregulation can lead to HLH in human, through disruption of self-recognition guided by the CD47-SIRPA system at the HSC stage.

Disclosures:

No relevant conflicts of interest to declare.
Anaplastic Lymphoma Kinase (ALK)-Positive Diffuse Large B-Cell Lymphoma Presenting with CNS Involvement: A Case Report
Authors: Kute Blakely D , Spalding Aaron , Martin Alvin W. , Sun David
Published: October 2019

Abstract 5225

Introduction

Anaplastic lymphoma kinase (ALK)- positive large B-cell lymphoma is a rare form of large B-cell lymphoma originally described in 19971. Since its initial description, approximately fifty cases have been reported in the literature, none of which describe extranodal involvement of the CNS as we will present below.

Case

The patient is a 30 year old previously healthy male who presented with visual changes described as decreased acuity and headaches. Papilledema was present on exam, but exam was otherwise negative. Imaging showed an enhancing mass posterior to the Torcula Herophili. With the working diagnosis of meningioma, the lesion was embolized and then debulked. Final pathology, however, revealed an ALK-positive large B-cell lymphoma with cytoplasmic granular ALK staining. Immunohistochemistry was positive for CD 138, CD 45, CD 38, CD 4, MUM1, epithelial membrane antigen (EMA) and monoclonal IgA lambda, and negative for CD 20, CD30, CD 3, and CD 8. FISH revealed ALK rearrangement (likely with clathrin, but pending at time of abstract) and c-MYC amplification, but not translocation. Serum immunoglobulins were normal. PET/CT showed skeletal metastasis but no additional visceral disease.

Discussion

ALK-positive large cell lymphomas described previously show immunoblastic/plasmablastic-like phenotypes (CD138+, CD 45+, CD 20-, and cytoplasmic IgA expression), but are CD30-negative and have uniform EMA expression and ALK translocations, as did our patient. The majority of ALK-positive DLBCL exhibit an ALK translocation with the long arm of the clathrin gene to form t(2;17)(p23;q23)2.

ALK-positive large cell tumors typically present nodally, but cases have been reported extranodally in the nasopharynx, tongue, bone, soft tissue, and stomach. There is a 3:1 male predominance and a poor overall prognosis with a median survival of 12.2 months, with isolated cases having longer survival. Most patients were treated with CHOP-based regimens with or without radiation therapy2,3. The need for more intense or biologically directed therapy in these cases, including early intensification with or without autologous stem cell rescue is recognized. Gambacorti-Passerini and Poglinani recently described lymphoma sensitivity with crizotinib, an anaplastic lymphoma tyrosine kinase inhibitor, in relapsed anaplastic large cell lymphoma after standard treatment with promising results. Investigation into the use of crizotinib in ALK-positive large cell lymphomas as salvage therapy is potentially warranted4.

This case presents an interesting treatment dilemma given the ALK mutation, c-myc amplification, and CNS disease. Crizotinib is likely not a salvage option for this patient given unclear CNS availability and the possibility of this tumor being motivated by c-myc as well as ALK.

Disclosures:

No relevant conflicts of interest to declare.
NF-κB Independent Protection Against Apoptosis by Overexpression of A20 in B-Cells
Authors: Alamome Isabelle , Feuillard Jean , Faumont Nathalie
Published: October 2019

Abstract 5233

Introduction

In Activated B-Cell like Diffuse Large B-Cell Lymphomas (ABC DLBCLs) the most commonly affected gene is TNFAIP3, which encodes A20, an ubiquitin modifying enzyme involved in the negative regulation of NF-κB signaling. A20 is itself a target gene of NF-κB and needs to be induced in order to exert its negative feedback effect on NF-κB. Mutations resulting in the inactivation of A20 have been found in a significant proportion of ABC DLBCL cases (Compagno M et al, Nature 2009). The tumor suppressor role of A20 was established by the reintroduction of wild-type A20 in some mutant A20 lymphoma B-cell lines promoting apoptosis, supposedly due to the loss of the ability to inactivate NF-κB. However, a protective effect of A20 against apoptotic stimuli was demonstrated in some cell types. In endothelial cells, A20 protects from TNF- and Fas-mediated apoptosis by inhibiting caspase-8 activation (Daniel S et al, Blood 2004). We thus wanted to understand the relationship between A20 expression, NF-κB activation and protection against apoptosis in B-cells.

Methods

We have cloned cDNAs coding for wild-type A20 (A20wt), for IκBαS32,36A (constitutively active form of IκBα), and for luciferase (as a control) into a doxycycline double-inducible episomal vector pRT-1, and stably transfected these vectors into an EBV positive lymphoblastoid B-cells line. Induction of p53 mediated apoptosis was performed by treatment of cells with Fludarabine*, whereas induction of caspase-8 dependant was performed with agonist antibodies against Fas (CD95).

Results

Both A20 and IκBαS32,36A overexpression suppressed NF-κB activity with downregulation of NF-κB target genes. Loss of NF-κB activation by induction of IκBαS32,36A led to increase of spontaneous apoptosis. Oppositely, loss of NF-κB activation by induction of A20 led to decrease of spontaneous apoptosis. Compared with luciferase transfected cells, no effect of A20 overexpression was seen after treatment of cells with anti-CD95 antibodies. Caspase-9 cleavage was significantly decreased and increase of TP53 protein levels was lower in A20 overexpressing cells after Fludarabine* treatment. By contrast, Fludarabine* treatment of IκBαS32,36A expressing cells led to an increase of both TP53 protein levels and caspase-9 cleavage. Therefore, A20 negatively modulated mitochondrial apoptosis pathway despite NF-κB downregulation.

Conclusions

These results suggest that A20 may exert an anti-apoptotic effect on its own independently of NF-κB activation. We are now looking after the functional properties of A20 natural mutants. Indeed, we can hypothesize that A20 mutants described in ABC DLBCLs have lost their negative feedback effect on NF-κB, but not their protective effect against apoptosis. So, A20 mutants may actively contribute to oncogenesis through their own protective effect on tumor cells.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of a-Catenin(CTNNA1) As a Tumor Suppressor Gene in Acute Myeloid Leukemis
Authors: Zeng Xianwei , Liu Ya , Lv Cui , Hu Zhenbo
Published: October 2019

Abstract 5240

Human a-catenin (CTNNA1) is a 102-kDa cadherin-binding protein that has been recognized as a tumor suppressor gene in solid tumors. Recent studies suggested that a-catenin functional as a tumor suppressor gene in leukemia with 5q deletion as well. In the present study, we speculate that a-catenin may play an important role as a tumor suppressor gene in AML and MDS cases with del(5q), since it is located on chromosome 5, band q31, and within the interval that is consistently deleted in these malignancies. Here, we report the evaluation of a-catenin as a tumor suppressor in myeloid disorders by mutational and functional analysis. Northern blot analysis revealed a very low level of expression of a-catenin mRNA in a panel of leukemia cell lines, whereas it was slightly higher in breast, colon, and prostate cancer lines. Protein truncation analysis in 11 myeloid leukemia cell lines revealed no mutations. By genomic sequencing selected exons in 23 clinical samples of AML/MDS patients with del(5q), we found that 1 of the samples carries a mismatch mutation on exon 5 of the gene. Using pcDNA-catenin expression vector for gene transfection analysis, we found that constitutive expression of a-catenin in the del(5q) leukemia cell line MUTZ-8 inhibited colony formation by 38.7%±6.3%(mea±sd) in the catenin-expressing cells when compared to those with empty vector and caused G1 arrest in the catenin-expressing cells (85.2%±7.3% vs 65.1%±4.6% in control cells) by cell cycle analysis. We conclude that a-catenin causes growth suppression in myeloid leukemia cells, which is consistent with a tumor suppressor gene, and endogenous expression of a-catenin is decreased in most of these cells; However, the finding a-catenin mutation in 1 out of 23 leukemia samples provides limited evidence that it is mutationally inactivated in human leukemias.

Disclosures:

No relevant conflicts of interest to declare.
Characterization of Single Platelet Mass, Volume, and Density in Response to Agonist Stimulation
Authors: Haley Kristina M. , Loren Cassandra P. , Phillips Kevin G. , McCarty Owen J.T.
Published: October 2019

Abstract 5261

An increase in mean platelet volume (MPV) is correlated with platelet activation and subsequent shape change. Pathologic processes marked by increased platelet activity such as myocardial infarction, cerebral vascular accidents, diabetes mellitus, and hypertension are associated with an increased MPV. Elevated MPV in these conditions reflect both a higher level of platelet activation as well as increased platelet turnover secondary to platelet consumption within thrombus formation. Assessment of MPV can be used to risk stratify patients as well as assign them to prognostic categories. However, MPV does not assess platelet heterogeneity or the specific change in single platelet mass, volume, or density. Current methods provide little insight into changes in physical parameters at the single platelet level. In order to overcome this limitation, we developed a quantitative tomographic differential interference contrast (QTDIC) microscopy technique to measure dry mass, volume, and density of platelets at the single-cell level. This technique is based on determining the axially resolved refractive index from a series of through-focus DIC images. Single cell platelet mass was observed to reduce from 1.84 ± 0.14 pg to 1.60 ± 0.13 pg in response to stimulation with thrombin-receptor agonist peptide (TRAP), while single cell platelet volume reduced from 7.28 ± 0.56 fL to 6.03 ± 0.48 fL (mean ± SEM). Single cell platelet density increased from 0.25 ± 0.001 pg/fL to 0.26 ± 0.002 pg/fL (mean ± SEM). Taken together, we have characterized the physical parameters of platelets in response to agonist stimulation. Our data suggest that platelet activation may correlate with decreased mass and volume, perhaps as a consequence of platelet degranulation. Further elucidation of the morphological changes of activated platelets at the single platelet level may allow for better understanding of platelet function and dysfunction in patients affected by platelet granule deficiencies, giant platelet syndromes, and disorders associated with membrane receptorsFigure 1.Characterization of physical parameters of platelets. (a) DIC image of human platelets, (b) refractive index map, (c) dry mass density map determined from refractive index using the Barer calibration, (d) Histogram of platelet dry mass, (e) Histogram of platelet volume, (f) Histogram of platelet density.Figure 1. Characterization of physical parameters of platelets. (a) DIC image of human platelets, (b) refractive index map, (c) dry mass density map determined from refractive index using the Barer calibration, (d) Histogram of platelet dry mass, (e) Histogram of platelet volume, (f) Histogram of platelet density.

Disclosures:

No relevant conflicts of interest to declare.
Successful Therapy of Cold Agglutinin Disease Utilizing Rituximab
Authors: Cholankeril Michelle , Bradley Thomas P. , Devoe Craig , Ghiuzeli Cristina Maria , Kolitz Jonathan E. , Allen Steven L.
Published: October 2019

Abstract 5271

Cold agglutinin disease is an autoimmune hemolytic anemia mediated by cold reactive autoantibodies triggering a complement mediated hemolysis. This condition, when not associated with infection, is characterized by clonal proliferation of CD20+ B cells that produce monoclonal IgM cold agglutinins. Conventional therapies for primary cold agglutinin disease (CAD) are ineffective, but case reports suggest that rituximab, an anti-CD20 monoclonal antibody, may be effective. In this retrospective single institution study, we evaluated the use of rituximab therapy in 6 patients (pts) [1 male, 5 female; median age 70 years (range 62 – 89)]. Three pts had primary CAD, 2 pts had mixed CAD and warm AIHA, and 2 pts had CAD in the setting of CLL. Five pts had received steroid therapy, 2 pts IVIG, 1 pt azathioprine, and 5 pts PRBC transfusion (2–10 units). Five received induction therapy with rituximab 375 mg/m2 IV weekly for four weeks and 1 for seven weeks. Four patients received maintenance rituximab 375 mg/m2 IV every two months (4+ to 12 cycles). All responded to therapy with a median rise in hemoglobin of 1.8 g/dl at 2 months from initiation of induction with further improvement over time (figure 1). Re-induction was performed in 2 pts; both had an initial one year duration of response and both responded following re-treatment. Median duration of response is 3+ yrs (range 1–8+ yrs); the 8+ yr response was in pt 2 who received only induction therapy. Two pts have completed two years of maintenance therapy and remain in remission at 4 months and 2 years post, respectively. Cold agglutinin titers decreased by 4 fold in 2 patients, 1 fold in 1 pt and remained stable in 1. Despite the improvement in hgb in all pts, laboratory evidence of low grade hemolysis persisted in 4 pts. All pts were able to be tapered off steroids and all remain transfusion free. No unexpected adverse events were noted. Rituximab appears to be a well tolerated and effective therapy for cold agglutinin disease. The need for and length of maintenance therapy remains to be determined.

Disclosures:

No relevant conflicts of interest to declare.
Von Willebrand Factor, ADAMTS13 Activity, TNF-Alpha, and Their Relationships in Patients with Chronic Kidney Disease
Authors: Shen Lei , Lu Guoyuan , Dong Ningzheng , Jiang Liqiong , Ma Zhenni , Ruan Changgeng
Published: October 2019

Abstract 5312

Patients with chronic kidney disease (CKD) have increased risks of endothelial dysfunction and thrombosis. Systemic inflammation may contribute to the endothelial dysfunction and accelerated thrombosis observed in CKD patients. von Willebrand factor (VWF), a well-known index of endothelial damage, has been reported to increase both in CKD and inflammatory states. ADAMTS13 is a specific VWF-cleaving protease, and some reports suggested that the ratio of VWF and ADAMTS13 maybe more useful in the diagnosis and treatment evaluation of patients in the prothrombotic state. So we assessed the relationships among endothelial dysfunction, ADAMTS13 activity, and levels of inflammatory cytokines in CKD patients. CKD patients were classified into 3 groups: chronic glomerulonephritis group (CGN, n = 31), idiopathic nephritic syndrome group (NS, n = 32), and lupus nephritis group (LN, n = 41). We measured the plasma levels of TNF-α, VWF antigen (VWF:Ag), and ADAMTS13 activity by using an ELISA-based method in CKD patients (n = 104) and normal controls (n = 32). The ratio of the VWF:Ag level to ADAMTS13 activity was calculated. The VWF:Ag level was significantly higher and the ADAMTS13 activity was significantly lower in all the disease groups than in the controls (P < 0.01). ADAMTS13 activity was lower in the NS group than in the CGN and LN groups (P < 0.05) (Table 1). The TNF-α (pg/mL) level was higher in the CKD group than in the control group (CGN: 3.19 ± 1.01; NS: 3.26 ± 1.18; LN: 3.24 ± 1.24; Control: 2.27 ± 1.23; P < 0.01; P < 0.01). TNF-α was positively correlated with the VWF:Ag level (r = 0.242, P = 0.013) and negatively correlated with the glomerular filtration rate (GFR) (r = −0.193, P = 0.049). ADAMTS13 activity was negatively correlated with the cholesterol level in CKD patients (r = −0.2, P = 0.042). TNF-α level in CKD was positively correlated with the VWF:Ag level and negatively correlated with GFR, which suggests that inflammation might be a major cause of endothelial dysfunction and an index for renal function. The VWF:Ag level increased and ADAMTS13 activity decreased in CKD patients, which indicates that CKD leads to a prothrombotic state.

Table 1. The plasma VWF:Ag level, ADAMTS13 activity, and the VWF/ADAMTS13 ratio in the CGN, NS, LN, and control groups N VWF (%) ADAMTS13 (%) VWF/ADAMTS13 CGN 31 198.25 ± 140.20** 61.93 ± 22.47**# 3.83 ± 3.29** NS 32 149.94 ± 74.50** 48.87 ± 18.63** 3.42 ± 1.59** LN 41 234.75 ± 134.91**## 67.81 ± 22.30**## 3.79 ± 2.52** CONTROL 32 99.55 ± 21.37 94.37 ± 23.66 1.12 ± 0.37 vs. CONTROL ** P < 0.01; vs. NS ## p < 0.01, # P < 0.05

Disclosures:

No relevant conflicts of interest to declare.
Urokinase-Type Plasminogen Activator Receptor (uPAR, CD87) Regulates Integrin Redistribution in Endothelial Cells Via Interaction with Low Density Lipoprotein Receptor- (LDLR-) Like Proteins
Authors: Prager Gerald , Novotny Rene , Unseld Matthias , Poettler Marina , Kalinowska Waclawa , Breuss Johannes , Zielinski Christoph
Published: October 2019

Abstract 5315

angiogenesis by degradation of extracellular matrix proteins as well as induction of intracellular signal transduction. We recently could demonstrate that in VEGF-stimulated endothelial cells pro-uPA becomes activated, which leads to uPAR-complex formation, it's internalization and redistribution of uPAR to newly formed focal adhesions ad the leading edge of migrating endothelial cells. Thereby, uPAR surface expression is tightly transcriptional regulated via the Density Enhanced Phosphatase-1 (DEP-1), but also via the LDLR-family members, which regulate subcellular uPAR distribution.

Here, we describe a mechanisms by which uPAR-internalization regulates integrin redistribution. We have characterized a novel binding motif on uPAR domain 3 for LDLR-protein interaction by using affinity chromatography as well as co-immunoprecipitation experiments. To proof a functional relevance of a direct uPAR/LDLR protein interaction, we reconstituted either uPAR mutants (mutL3/uPAR), lacking the binding site for LDLR-proteins, or wild type uPAR into endothelial cells derived from uPAR−/− mice. Reconstitution of mutL3/uPAR was incapable to redistribute uPAR as well as integrins during VEGF-induced endothelial cell migration when compared to wild type uPAR reconstitutes. The functional importance of uPAR / LDLR interaction was further reflected by the use of an inhibitory peptide (P1) interfering with uPAR/LDLR-protein interaction, which functionally reverted full length uPAR reconstitution, or the chaperon Receptor Associated Protein (RAP), a high affinity ligand for LDLR-proteins, which prevents uPAR/LDLR interactions. Thus, interfering with uPAR/LDLR-protein interaction at different levels led to an impaired endothelial cell spreading behavior on integrin-adhesive matrix proteins as well as a reduced pY576 FAK phosphorylation upon endothelial cell adhesion, leading to an reduced migratory response towards VEGF.

These data suggest a central role of uPAR/LDLR-protein interaction in VEGF-induced endothelial cell migration via induction of integrin redistribution. Thus, uPAR/LDLR interaction might represent a novel therapeutic target in angiogenesis-related diseases.

Disclosures:

No relevant conflicts of interest to declare.
Disruption of MYD88 Pathway Signaling Leads to Loss of Constitutive IRAK1, NF-κβ and JAK/STAT Signaling and Induces Apoptosis of Cells Expressing the MYD88 L265P Mutation in Waldenstrom's Macroglobulinemia
Authors: Yang Guang , Zhou Yangsheng , Liu Xia , Cao Yang , Hunter Zachary , Treon Steven P
Published: October 2019

Abstract 597

Introduction:

Through whole genome sequencing of bone marrow lymphoplasmacytic cells (LPCs) from WM patients, we recently identified a broadly expressed somatic variant (MYD88 L265P) which was present in 90% of WM patients, as well as in BCWM.1 and MWCL-1 WM cells. The presence of this variant has been reported to confer oncogenic activity in cell lines derived from patients with DLBCL ABC subtype lymphomas by induction of IRAK 1/4 kinase activity, and downstream signaling via NF-κβ (Ngo et al, Nature 2011, 470:115-119). We therefore sought to delineate the activity of agents which either disrupt MYD88 homodimerization, a prerequisite to IRAK 1/4 auto-phosphorylation, or by direct inhibition of IRAK 1/4 kinase activity in WM.

Patients and Methods:

Primary WM LPCs were isolated from bone marrow biopsy specimens from WM patients, and CD19+ B-cells were isolated from the peripheral blood of healthy donors. The mutational status of MYD88 was identified by Sanger sequencing, which confirmed the presence of the L265P mutation in BCWM.1 and MWCL-1 cells, and absent in Ramos and MM1S cells. Western blot and phospho-flow studies using phospho-specific antibodies were performed for signaling proteins downstream of MYD88, cell killing evaluated by Annexin V/PI staining and AlamarBlue, and IgM and IL-6 release determined after treatment with and without inhibitors of MYD88 homodimerization (IMG-2005-5, IMGENEX, San Diego CA), and IRAK 1/4 kinase function (407601, EMD, Gibbstown, USA).

Results:

Phosphorylation of the MYD88 downstream signaling proteins IRAK1, IκBα, NFκβ-p65 and STAT3 was greater in MYD88 L265P expressing BCWM.1 and MWCL-1 cells, and primary WM patient LPC, versus MYD88 wild type expressing Ramos, MM1S, and healthy donor CD19+ B-cells. Inhibition of MYD88 homodimerization and IRAK 1/4 kinase activity led to decreased phosphorylation of IRAK1, IκBα, NFκβ-p65 and STAT3 in BCWM.1 and MWCL-1 cells. Importantly, both inhibitors induced robust apoptosis of BCWM.1 and MWCL-1 cells, and primary WM LPCs bearing the MYD88 L265P mutation in comparison to Ramos, MM1S, or healthy donor CD19+ B-cells. Activation of caspase-3 and PARP, decreased BCL-2 expression, and decreased release of IL-6 and IgM accompanied treatment of WM cells following treatment with inhibitors of MYD88 homodimerization and/or IRAK 1/4 kinase activity.

Conclusion:

Disruption of MYD88 pathway signaling leads to loss of constitutive IRAK1, NF-κβ and JAK/STAT activation and induces apoptosis of cells expressing the MYD88 L265P mutation in WM. The results of this study provide novel insights into the pathogenesis of WM, and provide a framework for the clinical investigation of inhibitors of the MYD88 pathway in WM.

Disclosures:

No relevant conflicts of interest to declare.
The Phenotype of Human STK4 Deficiency
Authors: Abdollahpour Hengameh , Appaswamy Giridharan , Beier Rita , Schäffer Alejandro A. , Gertz E. Michael , Kreipe Hans H. , Pfeifer Dietmar , Grimbacher Bodo , Lohrmann Sabine , Sherkat Roya , Klein Christoph
Published: October 2019

Abstract 692

Congenital neutropenia syndromes comprise a heterogeneous group of disorders, whose genetic etiology remains often unknown. We describe a consanguineous pedigree with several affected individuals characterized by predisposition to recurrent and chronic bacterial and viral infections. Affected patients had chronic bronchitis/bronchiectasis, recurrent bacterial and herpes simplex skin infections, and disseminated warts associated with human papillomavirus and molluscum contagiosum virus. One patient developed a lymphoproliferative disorder associated with EBV-infection. Patients had congenital neutropenia with fluctuating absolute neutrophil granulocyte counts (180-4000/μl), yet no evidence of cyclic neutropenia. Immunophenotyping of peripheral blood revealed a paucity of peripheral T- and B-cells. Interestingly all patients showed evidences of autoimmunity.

In addition all affected patients had subtle and hemodynamically not relevant cardiac defects such as ASD-II (P1), patent foramen ovale (P2) and patent foramen ovale associated with mitral, tricuspid and pulmonary insufficiency (P3).

Genome-wide genotyping and linkage analysis of the index family yielded a LOD score of 4.3 on a linkage interval on chromosome 20. Candidate gene sequencing revealed a homozygous nonsense mutation in exon 7 of the gene STK4 (formerly MST1). STK4 is the human ortholog of Drosophila Hippo, the central constituent of a highly conserved pathway controlling cell growth and apoptosis. Isolated STK4-deficient lymphocytes and neutrophils of these patients exhibit enhanced loss of mitochondrial membrane potential and increased susceptibility to apoptosis in response to various proapoptotic stimuli. Lymphopenia and congenital neutropenia may therefore be a consequence of increased loss of peripheral lymphocytes and neutrophils, similar to other well defined monogenetic diseases of the immune system. STK4 deficiency is a novel human primary immunodeficiency syndrome and highlights the role of the HIPPO pathway for the development of the human immune and cardiac systems.

Disclosures:

No relevant conflicts of interest to declare.
Modeling Congenital Amegakaryocytic Thrombocytopenia Using Patient-Specific Induced Pluripotent Stem Cells
Authors: Takayama Naoya , Hirata Shinji , Jono-Ohnishi Ryoko , Nakamura Sou , Hirose Sho-ichi , Endo Hiroshi , Nishi Masanori , Hamazaki Yuhei , Kaneko Shin , Ishii Eiichi , Nakauchi Hiromitsu , Kunishima Shinji , Eto Koji
Published: October 2019

Abstract 703

Patient-specific, induced pluripotent stem cells (iPSCs) enable us to study disease mechanisms and drug screening. To clarify the phenotypic alterations caused by the loss of c-MPL, the thrombopoietin (TPO) receptor, we established iPSCs derived from skin fibroblasts of a patient who received curative bone marrow transplantation for congenital amegakarycytic thrombocytopenia (CAMT) caused by the loss of the TPO receptor gene, MPL. The resultant CAMT-iPSCs exhibited mutations corresponding to the original donor skin. Then using an in vitro culture system yielding hematopoietic progenitor cells (HPCs), we evaluated the role of MPL on the early and late phases of human hematopoiesis.

Although CAMT-iPSCs generated CD34+ HPCs, per se, their colony formation capability was impaired, as compared to control CD34+ HPCs. Intriguingly, both Glycophorin A (GPA)+ erythrocyte development and CD41+ megakaryocyte yields from CAMT-iPSCs were also impaired, suggesting that MPL is indispensable for MEP (megakaryocyte erythrocyte progenitors) development. Prospective analysis along with the hematopoietic hierarchy revealed that, in CAMT-iPSCs but not control iPSCs expressing MPL, mRNA expression and phosphorylation of putative signaling molecules downstream of MPL are severely impaired, as is the transition from CD34+CD43+CD41-GPA- MPP (multipotent progenitors) to CD41+GPA+ MEP. Additional analysis also indicated that c-MPL is required for maintenance of a consistent supply of megakaryocytes and erythrocytes from MEPs.

Conversely, complimentary transduction of MPL into CAMT-iPSCs using a retroviral vector restored the defective erythropoiesis and megakaryopoiesis; however, excessive MPL signaling appears to promote aberrant megakaryopoiesis with CD42b (GPIba)-null platelet generation and impaired erythrocyte production.

Taken together, our findings demonstrate the usefulness of CAMT-iPSCs for validation of functionality in the human hematopoiesis system. For example, it appears that MPL is not indispensable for the emergence of HPCs, but is indispensible for their maintenance, and for subsequent MEP development. Our results also strongly indicate that an appropriate expression level of an administered gene is necessary to achieve curative gene correction / therapy using patient-derived iPSCs.

Disclosures:

No relevant conflicts of interest to declare.
Tissue Factor Pathway Inhibitor Increases Hematopoietic Stem Cell Homing and Engraftment by Glypican-3 Mediated Inhibition of CD26 Activity
Authors: Khurana Satish , Joseph Chacko , Margamuljana Lia , Buckley Shannon , Scouteden Sarah , Verfaillie Catherine M.
Published: October 2019

Abstract 725

Directional migration is an important factor that determines homing of transplanted hematopoietic stem cells (HSC). The SDF-1α-CXCR4 axis is one of the most important determinants of this process. CD26, expressed on various cell types, leads to proteolytic cleavage of SDF-1α, which leads to the inactivation of its chemokine activity. We identified tissue factor pathway inhibitor (TFPI) as an inhibitor of CD26. Culture of murine bone marrow derived c-kit+Lin−Sca-1+ (KLS) cells or human umbilical cord blood derived Lin−CD34+ cells with TFPI significantly inhibited CD26 activity, which resulted in significantly increased migration towards SDF-1α. Moreover, TFPI treatment of murine KLS cells led to significant improvement in homing following intravenous injection and long-term reconstitution upon transplantation in competitive repopulation assays. We found that the effects of TFPI were mediated by the heparan sulphate proteoglycan, Glypican-3 (Gpc3). TFPI directly bound Gpc3; TFPI did not affect CD26 activity, migration or homing of Gpc3−/− KLS cells, but affected Gpc1−/− KLS cells similar to the wild type. Finally, KLS cells from Gpc3−/− but not Gpc1−/− mice homed significantly less following IV injection. Hence, we present a novel molecule that can be used in an HSC specific manner to enhance HSC migration, homing and engraftment.

Disclosures:

No relevant conflicts of interest to declare.
Are Gene Expression Signatures Treatment Specific?
Authors: Shah Parantu K , Minvielle Stephane , Avet-Loiseau Hervé , Li Cheng , Munshi Nikhil C.
Published: October 2019

Abstract 807FN2

Gene expression profiling (GEP) of newly-diagnosed cancer patients is now a routine task in the oncogenomic research using functional genomics platforms like microarray and next generation sequencing. These profiles are then utilized to derive gene expression signatures (GES) that can stratify patients according to survival groups using various statistical methodologies. This is an active area of research with important implications on clinical decision making and patient care. It is important to note that the treatment itself probably plays a major role in influencing outcome in cancer. Therefore, the GES may be specific to a particular treatment and may not be universally applicable in predicting survival of patients treated with different therapeutic regimen.

We evaluated the impact of therapy on GES utilizing two large publicly available gene expression datasets from newly-diagnosed multiple myeloma (MM) patients generated using Affymetrix U133+2 microarrays. The dataset from University of Arkansas Medical Sciences (UAMS; Shaughnessy et al Blood 2007) has gene expression profile (GEP) from 569 patients treated on total therapy (TT)2 and TT3 protocols while the dataset from HOVON-65 trial contains GEO data from 320 patients treated with either the VAD or PAD regimen in equal numbers.

The UAMS dataset was partitioned into training and validation sets. Using a combination of a network inspired univariate ranking procedure and ultra refined methods for variable selection we derived a sparse multivariate survival signature consisting of 40 genes that worked extremely well on the training set (p-value < e-16) as well as the validation set (p-value < e-5). Interestingly we saw the difference of performance between TT2 and TT3 induction arms. The p values were 0.002 for the TT2 induction arm while for the TT3 the p value was 0.02. On applying the signature to the whole HOVON-65 test set our signature worked only moderately well (p-value = 0.003). When the HOVON-65 dataset was split according to the induction treatment arms, the GES worked extremely well (p-value < e-5) in predicting the outcome in patients receiving VAD regimen but had no power to distinguish survival in patients receiving PAD regimen. We have evaluated the results on additional data sets that confirmed our observation from the HOVON study. To our knowledge this is the first clear demonstration of treatment specificity of GES. This data suggest that we may need to derive multiple therapy-specific GES to be applied to the patients to treat the new patient with therapy for which he/she is predicted to have best outcome.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Histone Methylation Arrests Ongoing Graft-Versus-Host Diseases in Mice by Selectively Inducing Apoptosis of Alloreactive Effector T Cells
Authors: He Shan , Wang Jina , Kato Koji , Xie Fang , Varambally Sooryanarayana , Mineishi Shin , Kuick Rork , Mochizuki Kazuhiro , Liu Yongnian , Nieves Evelyn , Mani Ram , Chinnaiyan Arul M. , Marquez Victor E. , Zhang Yi
Published: October 2019

Abstract 820

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment option for patients with hematological malignancies. However, its success is limited by life-threatening graft-versus-host disease (GVHD). Novel approaches are needed to control GVHD. Recent studies have shown the importance of histone methylation in regulating the expression of genes associated with effector T cell differentiation and proliferation. Using several mouse models of allo-HSCT, we report that in vivo administration of the histone methylation inhibitor 3-Deazaneplanocin A (DZNep) arrested ongoing GVHD while preserving graft-versus-leukemia activity (GVL). To assess the therapeutic effect of pharmacologic modulation of histone methylation on GVHD, we administered DZNep to BALB/c mice receiving major histocompatibility-mismatched C57BL/6 mouse T cells 7 days after transplantation, in which GVHD had been fully established. Notably, injection of 12 doses of DZNep controlled the disease in these recipients, with approximately 80% of them surviving long-term without significant clinical signs of GVHD. We found that in vivo administration of DZNep caused selective apoptosis in alloantigen-activated T cells, but did not impair the generation of effector T cells that produced inflammatory cytokines (e.g., TNF-α, IFN-γ and IL-17) and cytotoxic molecules (e.g., granzyme B and Fas ligand). As a result, alloreactive T cells retained potent GVL activity, leading to improved overall survival of the recipients challenged by leukemic cells. These data suggest that DZNep-mediated inhibition of GVHD may be accounted for by reduced number of alloreactive effector T cells. In vitro culture assays showed that DZNep treatment induced apoptosis in T cells activated by anti-CD3/CD28 antibodies but not in naive T cells stimulated by IL-2 or IL-7. This effect was associated with DZNep's ability to selectively reduce trimethylation of histone H3 lysine 27 (H3K27), deplete the histone methyltranferase Ezh2 that specifically catalyzes trimethylation of H3K27, and activate Ezh2-repressed pro-apoptotic gene Bim. Inactivation of Bim partially protected alloreactive T cells from DZNep-mediated apoptosis. Importantly, unlike DNA methylation inhibitors, inhibition of histone methylation by DZNep had no toxicities to hematopoietic cells or impairment on the reconstitution of hematopoiesis and thymopoiesis. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat established GVHD and other T cell-mediated inflammatory disorders in a broad context.

Disclosures:

No relevant conflicts of interest to declare.
Long Term Follow-up of Haploidentical Hematopoietic Stem Cell Transplantation without in Vitro T Cell Depletion for the Treatment of Hematological Malignancies: 9-Year Experience of a Single Center
Authors: Huang Xiao Jun
Published: October 2019

Abstract 839FN2

Many patients who require allogeneic hematopoietic stem cell transplantation (allo-HSCT) lack a human leukocyte antigen (HLA)-matched donor. Recently, we developed a new strategy named GIAC protocol for HLA-mismatched/haploidentical transplantation from family donors that combines granulocyte-colony stimulating factor (G-CSF) primed bone marrow (G-BM) and peripheral blood stem cells (PBSC) without in vitro T-cell depletion (TCD). For the past nine years, promising results for HLA-mismatched allo-HSCT without in vitro TCD have been achieved at our institute using this protocol. From May 2002 to December 2010, 820 patients, including 206 in high-risk group, underwent transplantation from haploidentical family donors. Eight-hundred and eleven patients (99%) achieved sustained, full donor chimerism. The incidence of grade 2–4 acute graft-versus-host disease (GVHD) was 42.9%, and that of grades 3 and 4 was 14.0% which was not associated with the extent of HLA disparity.Figure 1Cumulative incidence of acute GVHD grade 2–4 according to HLA disparity.Figure 1. Cumulative incidence of acute GVHD grade 2–4 according to HLA disparity.Figure 2Probability of LFS after haploidentical HSCT according to disease stage (p =.001).Figure 2. Probability of LFS after haploidentical HSCT according to disease stage (p =.001).

Disclosures:

No relevant conflicts of interest to declare.