bluuebunny/crossref_metadata_2025 huggingface dataset:
An Internet-Based Registry For The Documentation Of Neoplastic Meningitis
Authors: Strik Herwig , Rock Hans
Published: October 2019

Neoplastic involvement of cerebrospinal fluid (CSF) and meninges is a relatively rare and highly heterogeneous disease. Only few randomized studies investigated different treatment strategies and achived no conclusive results. New prospective studies with homogeneous entities are difficult to establish and some initiatives failed recently. Therefore, an open, multicentric registry may be an adequate alternative for a prospective documentation and analysis of a large number of cases of neoplastic meningitis from different primary neoplasms.

To enable an easy multicentric access, an internet-based database was chosen. Secutrial is a professional database which enables pseudonymized registration of clinical courses and meets all requirements for the protection of personal data. Predefined routines enable an immediate analysis of data like survival or toxicity including the most recently documented data. The internet-based access enables a multicentric, international documentation of cases of neoplastic meningitis. Analysis of these data may allow for conclusions on efficacy and toxicity of different treatment strategies in the separate tumor entities.

The configuration of the database provides a separate documentation of the course of primary neoplasm, systemic and central nervous system metastases as well as neoplastic meningitis. Operative, medical treatment and radiotherapy can be assessed as well as results of radiological, histological and cytological examinations including diagnostic and prognostic markers. Also, complications of the disease and toxicity of treatment can be documented. By such, the database is able to discriminate between different causes of clinical deterioration and death. The system is flexible enough to enable the addition of newly established items like biomarkers if needed. Dicomm data of radiological examinations can be uploaded and shared if needed and biobanking organized. The results of tumor boards can be documented as well.

Since multicentric prospective trials are difficult to perform for this rare and heterogeneous disease, an internet based registry appears to be an adequate alternative to collect data on treatment results. We think that the structure and content of the database presented here provides an adequate tool to collect and analyse a large number of cases with neoplastic meningitis.

Disclosures:

Strik: Mundipharma: Consultancy, Honoraria, Speakers Bureau; Medac: Consultancy, Honoraria; Micromet: Consultancy.
N-Glycosylation Of Human ITP Autoantibodies Affects Complement Activation, Platelet Phagocytosis, and Platelet Survival In a Murine Model
Authors: Sachs Ulrich J. , Walek Kathrin , Krautwurst Annika , Rummel Mathias J. , Bein Gregor , Santoso Sentot , Bakchoul Tamam
Published: October 2019

Introduction

Immune thrombocytopenia (ITP) is a bleeding disorder caused by IgG autoantibodies (AAbs) directed against platelets. The IgG effector functions of autoantibodies depend on their Fc-constant region which undergoes posttranslational glycosylation. We investigated the role of Asn279-linked N-glycan of AAbs in vitro and in vivo.

Material and Methods

AAbs were purified from ITP patients (n=15) and controls (n=10) and N-glycans were enzymatically cleaved by endoglycosidase F. The effects of native AAbs and deglycosylated AAbs (deAAbs) were compared in vitro on enhancement of phagocytosis of platelets by monocytes and complement fixation and activation applying flow cytometry, laser scanning microscopy, and a complement consumption assay. The capability of AAbs and deAAbs to eliminate human platelets in vivo was studied in a NOD/SCID mouse model in presence and absence of a complement source.

Results

AAb-induced platelet phagocytosis was inhibited by N-glycan cleavage (median phagocytic activity: 8% vs. 0.8%, p=0.004). Seven out of 15 native AAbs bound C1q and induced complement consumption. N-glycan cleavage significantly reduced C1q binding (MFI 16.4 vs. 4.9, p=0.017) and complement consumption. In vivo survival of human PLTs was assessed after cotransfusion with native or deAAbs in NOD/SCID mice. Injection of AAbs resulted in rapid clearance of human platelets compared to control (platelet clearance after 5h (CL5h) 75% vs. 30%, p<0.001). AAbs that were able to activate complement induced more pronounced platelet clearance in the presence of complement compared to the clearance in the absence of complement (CL5h 82% vs. 62%, p=0.003). AAbs lost their ability to destroy platelets in vivo after deglycosylation (CL5h42%, p<0.001).

Conclusion

Removal of N-glycan from AAbs interferes with Fc-mediated phagocytosis and complement activation and thereby prolongs platelet survival in vivo. Our study provides tools for better characterizing ITP AAbs and sheds light on the heterogeneity of AAbs in ITP. Clinical studies should aim to assess such additional characteristics, since this could lead to the identification of ITP patient subgroups with increased responses to specific or new interventions such as, targetting complement factors.

Disclosures:

No relevant conflicts of interest to declare.
Role Of Monocyte Fcγ Receptors In The Prothrombotic State Of Heparin Induced Thrombocytopenia (HIT)
Authors: Rauova Lubica , Madeeva Daria , Tutwiler Valerie , Bailey Shahara , Hayes Vincent M. , Cines Douglas B. , Poncz Mortimer
Published: October 2019

HIT is an antibody-mediated disorder characterized by thrombocytopenia and thrombosis. Activation of platelet via FcγRIIA by HIT antibodies complexed to surface-bound platelet factor 4 (PF4) contributes to the prothrombotic state. However, using an in vitro microfluidic whole human blood model of HIT, we have found through depletion and repletion studies that activation of monocytes through Syk-kinase is a key step in generating a prothrombotic milieu. Here we define the Fcγ receptor(s) upstream of Syk that mediate the thrombotic signal in monocytes. Monocytes isolated from normal healthy volunteers were incubated with 50 µg/ml of blocking antibodies against FcγRIIA (CD32, clone IV.3) and/or against FcγRI (CD64, clone 10.1) or against FcγRIII (CD16, clone 3G8), washed to remove unbound antibody and added to monocyte depleted-whole blood. These monocyte-repleted samples were then stimulated with PF4 (10 µg/ml) and the HIT like monoclonal antibody KKO (50 µg/ml) for 30 min. After recalcification (5 mmol CaCl2), samples were perfused over a von Willebrand factor-coated BioFlux microfluidic channel at a shear stress of 20 dyne/cm2 at 37°C for 15 min. Platelet adhesion was quantified by fluorescence microscopy after adding Calcein-AM (3 µM), and fibrin was visualized by adding Alexa 561-labeled fibrinogen (1.5 μg/ml) to the whole blood prior to the perfusion. Selective inhibition of FcγRIIA on monocytes led to an ∼40% inhibition of fibrin deposition and comparable reduction in the formation of “coated” platelets relative to samples repleted with non-inhibited monocytes. Flow cytometry studies showed that these observations were not due to leaching of IV.3 antibody off the monocytes with subsequent blocking of platelet FcγRIIA. In contrast, selective blockade of FcγRI or FcγRIII in depletion/repletion studies or in whole blood did not significantly reduce platelet adhesion, formation of coated platelets, or fibrin accumulation. However, blocking FcγRI did decrease tissue factor (TF) activity on monocytes stimulated with PF4 and KKO and this effect was additive to FcγRIIA inhibition. In summary, our data show that monocyte-bound HIT IgG antibodies contribute to the prothrombotic state by activating FcγRIIA directly and indirectly by generating TF though FcγRI. We speculate that this activation of monocytes contributes to platelet activation and monocyte microparticle formation demonstrated previously. Drugs that target FcγRI and FcγRIIA on monocytes may help prevent thrombosis in HIT without causing platelet dysfunction or increasing the risk of bleeding.

Disclosures:

No relevant conflicts of interest to declare.
Characteristics and Outcome Of Children and Young Adults With Early T-Precusor (ETP) ALL Treated On UKALL 2003
Authors: Patrick Katharine , Wade Rachel , Goulden Nicholas , Mitchell Chris , Rowntree Clare , Hough Rachael E , Vora Ajay J
Published: October 2019

Early T-precursor (ETP) ALL is a recently described poor prognostic sub-group of childhood T-ALL identified by a distinctive gene expression profile and immunophenotype (CD1a-, CD5 weak, CD8- and presence of myeloid or stem cell markers). It accounts for around 12% of T-ALL and is associated with a poor outcome in retrospective US and Italian studies with a 5 year event-free survival (EFS) of less than 50% on historical treatment protocols. We sought to determine the outcome for this sub-group on a contemporary MRD stratified treatment protocol, UKALL 2003.

As full immunophenotype data was not collected centrally, individual centres were asked to provide individual patient data retrospectively. Of 387 T-ALL cases in the trial, immunophenotype data to classify ETP status was not available for 148 patients. Of the remaining 239 cases, 35 (15%) were classified as definite/probable (CD8 and 1a neg with stem cell or myeloid antigen expression) and 17 (7%) as possible (CD8 neg and stem cell or myeloid antigen expression, 1a not available) ETP. The definite, probable and possible ETP groups had similar patient characteristics and outcomes and have, therefore, been analysed together. Compared to those without an ETP phenotype, ETP patients were more likely to present with a low (<50x109/l) white cell count (56% vs 26%, p<0.0001) but were less likely to be morphological rapid early responders at day 8/15 (63% vs 83% p =0.002) or MRD low risk at the end of induction (4% vs 27%, p = 0.008). Unlike previous reports, the risk of induction failure was not significantly higher in the ETP group (n= 2 vs 1 among non-ETP cases).

With a median follow-up of 4 years 10 months (range 1 year 4 months - 9 years), the ETP sub-group have a non-significantly inferior EFS (76.0% (95% CI: 64.0-88.0%) vs 84.6% (79.3-89.9%) at 5 years, p = 0.2) and OS (84.1% (73.9-94.3%) vs 90.9% (86.6-95.2%), p = 0.08) compared to the non-ETP sub-group, due primarily to a non-significantly higher relapse rate (17.7% (6.5-28.9%) vs 9.6% (5.1-14.1%), p = 0.1). Although MRD risk group was a determinant of outcome in the non-ETP group (p=0.05), its significance within the ETP group could not be reliably determined since only 2 patients were MRD low risk, of whom 1 suffered a relapse. Interestingly, of the 8 relapses in the ETP group, 7 were isolated marrow relapses and there was one isolated testicular relapse, whereas 12 of the 17 relapses in the non-ETP group involved the CNS (5 isolated and 7 combined).

Although ETP ALL is associated with an adverse outcome compared with non-ETP T-ALL, a 5 year EFS of 76% on a contemporary MRD stratified protocol is not sufficiently poor to warrant experimental treatment or first remission allogeneic transplant for the group universally.

Disclosures:

No relevant conflicts of interest to declare.
In Vivo Analysis of PML-RARA in a Humanized Mouse Model
Authors: Matsushita Hiromichi , Yahata Takashi , Sheng Yin , Nakamura Yoshihiko , Muguruma Yukari , Matsuzawa Hideyuki , Tanaka Masayuki , Hayashi Hideki , Sato Tadayuki , Damdinsuren Anar , Onizuka Makoto , Ito Mamoru , Miyachi Hayato , Pandolfi Pier Paolo , Ando Kiyoshi
Published: October 2019

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia.

Disclosures

No relevant conflicts of interest to declare.
CS-1 Re-Directed Central Memory T Cell Therapy for Multiple Myeloma
Authors: Wang Xiuli , Wong ChingLam W , Urak Ryan , Chang Wen-Chung , Budde Elizabeth E. , Brown Christine , Forman Stephen J.
Published: October 2019

Multiple myeloma (MM), a plasma cell malignancy, accounts for approximately 1 percent of all cancers and slightly more than 10 percent of hematologic malignancies in the United States. Approximately 20,000 new cases will be diagnosed this year and over 11,000 people will die from this disease. Current therapies for MM often induce remission, but nearly all patients eventually relapse and die. T-cell mediated anti-tumor therapies using genetically modify T cells with specific chimeric antigen receptors (CARs) have non-overlapping activity, toxicity and tumor resistance profiles compared to conventional chemotherapeutic agents.

The main challenge in designing a CAR T cell immunotherapeutic approach is identifying the best antigen for tumor targeting. CS-1 is a cell surface glycoprotein of the signaling lymphocyte activation molecule (SLAM) receptor family that is highly and selectively expressed on normal plasma cells and MM cells, with lower expression on NK cells and little or no expression on normal tissues. This unique expression pattern and proven clinical benefit of CS-1 monoclonal antibody for the treatment of relapsed MM makes CS-1 a good target for CAR T cell therapy.

Central memory T cells (TCM) from PBMC were isolated using a two-step process on the AutoMACS device to first deplete CD14+, CD45RA+ and CD25+ cells, then to positively select CD62L+ cells. These TCM undergo anti-CD3/CD28 bead stimulation and transduction with a lentiviral vector encoding CS-1 CAR containing a CD28 co-stimulatory domain and two mutations on IgG4 linker CH-2 portion to ensure enhanced potency and persistence after adoptive transfer. Gene modified CS-1 CAR T cells specifically lysed MM.1S, a MM cell line, in 4-hour 51Cr release assays and all the CAR+ cells expressed 107a upon co-cultured with the MM.1S cells. To investigate the potency of the CS-1CAR T cells, 2x106 MM.1S cells that were engineered to express GFP firefly luciferase were inoculated into NSG mice by tibia injection. 7 days post tumor engraftment, 1x106 CS-1 CAR T cells were intravenously injected into the tumor bearing mice. In contrast to untreated and mock cell treated mice in which tumor progressed rapidly systemically, single intravenous infusion of CS-1 CAR T cells induced dramatic tumor regression and significantly prolonged survival.

In addition to CS-1, CD44v6 and BCMA are antigens that have also been shown to be over-expressed on MM tumor cells. We therefore compared the two CARs with CS-1 CAR for their anti-MM activity. Based on our studies, targeting CS-1 resulted in the best efficacy (Figure 1) and would be an attractive strategy for development of a clinical trial.

Disclosures

No relevant conflicts of interest to declare.
Early and Transient Microchimerism Associated with Complete Remission after Adoptively Transferred Haploidentical NK Cells Against High Risk Myelodysplastic Syndrome and Refractory Acute Myeloid Leukemia
Authors: Björklund Andreas T , Carlsten Mattias , Schaffer Marie , Liu Lisa , Cooley Sarah A. , Miller Jeffrey S. , Watz Emma , Palma Marzia , Hansson Lotta , Wahlin Björn E , Möllgård Lars , Hagglund Hans , Blomberg Pontus , Ljungman Per T. , Hellström-Lindberg Eva , Ljunggren Hans-Gustaf , Malmberg Karl-Johan
Published: October 2019

Introduction: We here report from an ongoing phase I/II study of HLA-haploidentical NK cell therapy to patients with high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) not eligible for standard therapies. The preparative regimen consisted of intermediate doses of Cyklophosphamide (Cy), Fludarabin (Flu) and titrated doses of total lymphoid irradiation (TLI). The trial design excluded systemic IL-2 treatment to avoid expansion of regulatory T cells and to test if in vivo expansion could be obtained without IL-2 support.

Patients:The first 12 patients were treated with Cy/Flu and an escalating dose of TLI (2 Gy and 4 Gy), followed by infusion of short-term IL-2 activated (16 hours) NK cells. Three patients received daily cyclosporine A after the conditioning. Three had relapsed, chemotherapy-refractory, primary AML, seven had secondary relapsed or refractory MDS-AML and two had high risk MDS with fibrosis.

Results: The treatment was well tolerated and no severe non-infectious toxicity could be observed in the patients. The endpoint of expansion (>100 donor NK cells/ul at day 14) was not reached, but six patients had positive microchimerism, NK cells of donor origin detectable by RT-PCR at day 7-14, that thereafter became undetectable within 7-14 days. Four of these six patients achieved complete remission (CR) whereafter they become eligible for and could proceed to allogeneic stem cell transplantation.

None of the patients with negative microchimerism obtained CR. Four patients died from progressive disease and three patients, with minor response and progressive disease, died in infections within three months of therapy.

Discussion: Although the long-term efficacy needs to be evaluated, the results suggest that a combined regimen with mild conditioning followed by NK cell therapy may induce remission in patients with chemo-refractory disease and provide a bridge to allogeneic stem cell transplantation. Notably, clinical responses were observed after only a minimal in vivo NK cell expansion and were independent on KIR-ligand mismatch.

Disclosures

Blomberg: VECURA: Employment. Hellström-Lindberg:Celgene: Research Funding.
Missense SNP rs3731249 Explains the CDKN2A Association with Childhood ALL and Shows Risk Allele Selection in Tumors with Somatic CDKN2A Alterations
Authors: de Smith Adam J. , Walsh Kyle M. , Hansen Helen M. , Barcellos Lisa F. , Chokkalingam Anand P. , Jenkins Robert B. , Wrensch Margaret R. , Wiencke John K. , Metayer Catherine , Wiemels Joseph L.
Published: October 2019

Little is known about the interaction between constitutive and somatic genetic variation in carcinogenesis. Genome-wide association studies (GWAS) of childhood acute lymphoblastic leukemia (ALL) have identified 6 genetic associations, including the single nucleotide polymorphism (SNP) rs3731217, located between the CDKN2A and CDKN2B genes. Somatic deletions of this chromosome 9p21.3 region are common in cancer, and occur in ~30% of childhood ALL.

We replicated the rs3731217 association with childhood ALL in Hispanics (P = 0.021) via a GWAS of 323 childhood ALL cases and 454 controls, using the Illumina OmniExpress SNP array. SNP imputation analysis across chromosome 9p21.3 revealed a stronger association at SNP rs3731249 (P = 6.4x10-4; OR = 2.77), a low-frequency missense variant in exon 2 (p.Ala148Thr) of CDKN2A (MAFcontrols = 2.1%). These associations were replicated in an additional 378 ALL cases and 536 controls using Taqman assays, where the missense variant again had a greater magnitude of effect (Prs3731249 = 6.5x10-3; OR rs3731249 = 2.00 versus Prs3731217 = 0.029; OR rs3731217= 1.38). We assessed the effect of rs3731249 on risk of other cancers with known chr9p21.3 GWAS associations, but observed no association between this variant and either adult glioblastoma or melanoma risk, suggesting an ALL-specific role for rs3731249.

We hypothesized that cases heterozygous for rs3731249 may show allelic imbalance, with preferential loss of the wildtype (WT) allele when hemizygous chr9p21.3 deletions occur in leukemic cells. To test this, we developed a novel method termed “SMART-ddPCR” (Somatic Mutation Allelic Ratio Test using Droplet Digital PCR). Concentration of risk and WT alleles was measured by ddPCR using the rs3731249 Taqman assay in diagnostic bone marrow (i.e. tumor) DNA from 35 known heterozygotes. Allelic imbalance due to somatic CDKN2A alterations was identified in 17 cases, of which 14 had a higher risk:WT allele ratio (P = 0.006, 1-sided binomial significance test), thus demonstrating preferential loss of WT rs3731249 and retention of the missense allele. Subclonal heterogeneity in CDKN2A copy number was also evident, suggesting that CDKN2A alterations are secondary events in leukemogenesis.

Preferential allelic imbalance towards the rs3731249 risk allele in ALL tumor DNA provides strong evidence of an important role for this missense variant in leukemogenesis. We show, for the first time, a direct relationship between constitutive and somatic genetic variants underlying ALL development, suggesting that such constitutive variants can provide an additional substrate for selection during tumor evolution.

Disclosures

No relevant conflicts of interest to declare.
Assessing the Value of Echocardiograms for Patients with Acute Myeloid Leukemia (AML) Receiving Anthracyclines
Authors: Seastone David J. , Nazha Aziz , Pophali Priyanka A , Ornstein Moshe C. , Carraway Hetty E. , Advani Anjali S. , Gerds Aaron T. , Kalaycio Matt E. , Maciejewski Jaroslaw P. , Sekeres Mikkael A.
Published: October 2019

Background

AML is a life-threatening disease that requires prompt administration of anthracycline-based induction chemotherapy, particularly in younger adults. Prior to treatment initiation, patients (pts) undergo routine echocardiography (echo) screening to assess cardiac ejection fraction (EF) as a measure of induction tolerability, despite American Society of Clinical Oncology and National Comprehensive Cancer Center guidelines which state that only pts with prior cardiac disease or exposure to cardiotoxic drugs or radiation therapy require such evaluation This is costly to the health-care system and may be unnecessary. In this study, we assess whether the use of echo prior to anthracycline-based therapy in AML has a major effect on pt outcomes.

Methods

We reviewed clinical data on AML pts treated with anthracycline-based induction chemotherapy regimens at our institution from 2002-2014. EF was obtained by 2D echo prior to induction chemotherapy initiation. Charlson comorbidity index was used to evaluate comorbidities prior to therapy. A cox proportional hazard model was used to analyze the impact of baseline echo on overall survival, with p-values <.05 denoting significance.

Results:

Of the 120 pts included, 60 (50%) were female. At diagnosis, median age was 57 years (range, 23-85), white blood cell count (WBC) 8.9 x 109/L (0.3-227), hemoglobin 9.1g/dL (4.7-13.8), platelets 85 x 103 (8-277), bone marrow blasts 48% (14-95%). Cytogenetic risk categories per CALGB 8461 criteria: 12 pts (10%) were favorable, 63 (53%) indeterminate, and 41 (34%) unfavorable. Charlson morbidity index was > 1 in 23 pts (19%) and 50 (38%) were current or former smokers. All pts received induction chemotherapy with either idarubicin (38%), daunorubicin (38%), or mitoxantrone (23%) combined with cytarabine (7+3). In 116 pts (97%), the baseline echo showed a left ventricular ejection fraction (LVEF) >50% (+/- 5). In the 4 remaining pts (3%), the EF was < 40%. In a multivariable analysis controlling for age, WBC, cytogenetic risk group, Charlson comorbidity index, and smoking exposure, LVEF was not associated with overall survival (HR 1.05 with 95% CI 0.99-1.12 and p=.09). Among the 4 pts with EF<40% (age 57-71 years), all received full dose anthracycline and only the 71-year-old patient experienced congestive heart failure during treatment. Six pts (5%) died during induction therapy (4 weeks) and none of these had an EF <40%.

Conclusion

Among AML pts who received anthracycline-based induction chemotherapy, results from echocardiography did not impact outcome in >99% in this cohort. It is not clear that echocardiography, particularly in younger pts, provides value, and may delay treatment initiation.

Disclosures

No relevant conflicts of interest to declare.
Left Ventricular Ejection Fraction Measurement and Doxorubicin-Based Chemotherapy in Patients with Diffuse Large B-Cell Lymphoma
Authors: Enns Deborah L , Aboulafia David M
Published: October 2019

Purpose:

Doxorubicin-based chemotherapy (DOX) is commonly administered to patients with diffuse large B-cell lymphoma (DLBCL). Prior to chemotherapy, left ventricular ejection fraction (LVEF) is routinely measured to assess left ventricular dysfunction. While LVEF screening is recommended by many national regulatory bodies, evidence supporting the usefulness of LVEF measurement prior to administering DOX is lacking. Our goal was to perform a retrospective analysis of patients with DLBCL to establish (1) how often LVEF was measured prior to administering DOX, and (2) whether the chemotherapy regimen was modified based on LVEF values. As cumulative doses of doxorubicin greater than 400 mg/m2 have been associated with an increased risk of congestive heart failure (CHF), we also determined the incidence of CHF in patients with DLBCL who did receive DOX.

Patients and Methods:

We identified 268 patients diagnosed with DLBCL at Virginia Mason Medical Center between 2001 and 2012 and collected the following data: age at diagnosis; stage of lymphoma; type of chemotherapy given; cumulative doxorubicin dose (mg/m2); LVEF status; and incidence of CHF or cardiac disease. We also compared the number of CHF risk factors between patients who did and did not have LVEF measured. Statistical analyses included a Fischer’s exact or Chi-squared test to compare study groups as well as the number of CHF risk factors. The level of significance was set at a P value of < 0.05.

Results:

LVEF was measured in 238 patients (89%) prior to initiation of chemotherapy. LVEF values were normal in 225 patients (95%) and low (< 50%) in 13 patients (5%). Of the patients with normal LVEF, 193 received DOX (86%), and of these patients, 14 developed CHF post-treatment (7%). For the 13 patients with low LVEF, 8 received DOX (62%) and 1 developed post-treatment CHF (13%). The remaining thirty patients did not have LVEF measured and none received DOX. Of the 268 patients studied, 176 are alive (66%) and 3 were lost to follow-up. The mean follow-up time was 43 months (range 3 d to 12.1 y). The mean number of CHF risk factors did not differ between patients who did and did not have LVEF measured (1.70 vs. 1.65, P = 0.87).

Conclusion:

Our results suggest that the decision to administer DOX was not directly affected by LVEF values. These findings challenge the existing policy of routinely screening patients with DLBCL with echocardiograms or MUGA scans prior to treatment.

Disclosures

No relevant conflicts of interest to declare.
Complement Component 3 (C3) Is a Mediator of Epithelial-Mesenchymal Transition (EMT)
Authors: Cho Min Soon , Hu Qianghua , Rupaimoole Rajesha , Sood Anil , Afshar-Kharghan Vahid
Published: October 2019

We have shown that complement component 3 (C3) is expressed in malignant ovarian epithelial cells and enhances cell proliferation in vitro and tumor growth in vivo. C3 is secreted by cancer cells into the tumor microenvironment and promotes tumor growth through an autocrine loop. To understand the mechanism of upregulation of C3 expression in malignant epithelial cells, we studied the transcriptional regulation of C3, and found that TWIST1, a major regulator of EMT, binds to the C3 promoter and regulates C3 transcription. Knockdown of the TWIST1 gene reduced C3 mRNA, and TWIST1 overexpression increased C3 mRNA.

TWIST1 promotes epithelial-mesenchymal transition (EMT) during normal development and in metastasis of malignant tumors. An important marker of EMT is a reduction in the surface expression of E-cadherin on cells facilitating migration and invasion of these cells. TWIST1 is a transcriptional repressor of E-cadherin; and because TWIST1 increases C3 expression, we investigated whether C3 is also a negative regulator of E-cadherin expression. We overexpressed C3 in ovarian cancer cells by stable transduction of lentivirus carrying C3 cDNA. Overexpression of C3 was associated with 32% reduction in the expression of E-cadherin resulting in enhanced migration ability of cells by 2.3 folds and invasiveness by 1.75 folds, as compared to control cells transduced with control lentivirus.

To investigate whether TWIST1-induced reduction in E-cadherin is C3-mediated or not, we studied the effect of TWIST1 overexpression simultaneous with C3 knockdown in ovarian cancer cells. Overexpression of TWIST1 alone resulted in 70% reduction in E-cadherin mRNA and this was completely reversed after simultaneous C3 knockdown in these cells.

To investigate the correlation between C3 and TWIST1 in vivo, we studied the co-expression of these two proteins in mouse embryos (physiologic EMT) and in malignant tumors (pathologic EMT). Given the role of EMT in embryogenesis we immunostained mouse embryos at different stages of development, using antibodies against TWIST1 or C3. Transverse section of 9.5-day post-coitum (9.5dpc) mouse embryos showed co-expression of TWIST1 and C3 in otocyst (ot) and hindbrain (hb) of neural crest. In the whole-mounted 11.5dpc mouse embryos, C3 and TWIST1 were co-expressed in limb buds.

Given the role of EMT in malignancy, tumors induced in mice after intraperitoneal injection of murine ovarian cancer cells were resected and immunostained for C3 and TWIST1 proteins. TWIST1 and C3 co-localized at tumor edges, where EMT and tumor cells migration occur. Taken together, these data provide evidence that TWIST1 regulates C3 expression, and C3 promotes EMT through E-cadherin.

Disclosures

No relevant conflicts of interest to declare.
Zoledronic Acid Induces the Proliferation of Human Cord Blood Gamma Delta T Cells Ex Vivo
Authors: Tomchuck Suzanne L , He Jin , Perko Ross W. , Evans Scarlett , McKenna Amy , Dash Pradyot , Thomas Paul , Dallas Mari H
Published: October 2019

Cord blood (CB) T cells are known to be naïve cells, but can be activated to respond similar to adult peripheral blood (PB) T cells. Reports indicate that culture with aminobisphosphonate (NBP) stimulates CB gamma delta T cell proliferation ex vivo, specifically the TCRγ9δ2 subset, which has been extensively studied in PB gamma delta T cells. As CB gamma delta T cells are not well described, we compared CB gamma delta T cell proliferation, phenotype and genotype to PB gamma delta T cells when culturing cells with the NBP, Zometa (zoledronic acid), and IL-2. Fourteen days in culture resulted in significant fold increase in the proliferation of gamma delta T cells and in the percent of lymphocytes in both sample types. PB gamma delta T cells proliferated more robustly than CB with a 288.60 versus 21.32 fold increase, respectively. Additionally, in freshly isolated samples, CB gamma delta T cells comprised an average of 1.404% of the lymphocyte population, which was similar to PB gamma delta T cells, with an average of 2.319%. However, by day 14, PB gamma delta T cells increased to 70.15% of lymphocytes whereas CB gamma delta T cells increased to 12.49%. Phenotypically, both CB and PB had similar percent of CD45RA+ and CD45RO+ gamma delta T cell memory subsets in freshly isolated samples. Following culture, PB gamma delta T cells were mostly CD45RO+ memory cells, with significantly fewer CD45RA+ naïve cells, whereas more CB gamma delta T cells were of the intermediate CD45RA+CD45RO+ subset. Further phenotypic analysis of the memory subsets indicated that cultured PB gamma delta T cells were either effector memory cells (CD27-CD45RA-) or central memory cells (CD27+CD45RA-), while CB gamma delta T cells were mostly naïve (CD27+CD45RA+). The cytokines secreted by these cells were also assessed and the culture of PB and CB gamma delta T cells resulted in differing cytokine secretion profiles. After 14 days of culture, PB gamma delta T cells secreted more IFNγ and TNFα, while CB gamma delta T cells secreted more IL-10 and RANTES. We also examined TCRγ9 and TCRδ2 phenotypic expression and found that the TCRγ9δ2 was a common clone in freshly isolated PB gamma delta T cells, which predominated after 14 days in culture. However, while the TCRγ9δ2 variant was expressed in CB gamma delta T cells, it was low before and after culture, suggesting that Zometa may not stimulate gamma delta T cells in CB the same as PB. As limited TCRγδ phenotypic reagents are available, we developed a single cell PCR assay for genotypic analysis of the TCRγδ repertoire. PCR analysis suggests that the TCRγδ repertoire is diverse in both samples, yet TCRγ9δ2 is most prevalent. Further analysis of the variant subsets is warranted and may give insight into how each of these receptor pairings affects function.

Disclosures

No relevant conflicts of interest to declare.
Site-Directed Pegylation of BDD FVIII (BAY 94-9027) Does Not Alter the Ability of the Molecule to Generate Thrombin, Activate Factor X or Become Inactivated By Activated Protein C
Authors: Yegneswaran Subramanian , Jiang Xiaoqiao , Yesantharao Pooja , Kim Ji-Yun , Leong Lilley , Sim Derek S , Patel Chandra , Kauser Katalin Zsofia
Published: October 2019

Introduction: BAY 94-9027 is a rationally designed B Domain Deleted (BDD) FVIII (Mei , B. et al Blood 2010). A single 60 kDa PEG molecule was attached to amino acids 1804 to increase its circulating half-life, reduce the exposure to epitopes reported to cause immunogenicity in the A3 domain (Shima M. Int. J. Haematol. 2006), while preserving full biological function. The PEGylation technology allows for an extended duration of action with the goal of reducing the number of infusions needed while maintaining protection from bleeds. In a recent clinical trial the molecule has shown significant efficacy for prophylactic treatment in hemophilia A patients (NCT01775618). The objective of this study was to further characterize in vitro BAY 94-9027 using Xase kinetics, activated protein C-dependent inactivation of FVIIIa, and thrombin generation assays.

Methods and Results: Biochemical assays to determine apparent binding of the Factor IXa and Factor VIIIa complex and the Factor X activation kinetics revealed identical in vitro properties of BAY 94-9027 to the BDD FVIII comparator, Refacto AFÒ. The molecules also performed similarly in activated protein C (aPC) FVIIIa inactivation studies.

BAY 94-9027 was further characterized by thrombin generation assay (TGA) in human hemophilic plasma. The TGAs were triggered using 1pM TF or 100pM XIa to simulate extrinsic and intrinsic activation. With XIa activation no differences were seen in peak thrombin, ETP or lag time between BDD FVIII and BAY 94-9027. The lag time, peak and ETP values also remained similar using TF activation. The XIa initiated TGA gives a more potent thrombin generation response compared to TF and is more sensitive to FVIII activity as judged by the left shift of the dose response curves. The TGAs were repeated while titrating thrombomodulin (TM) to generate aPC. Both BAY 94-9027 and BDD FVIII showed similar inactivation profile in the TM-TGA.

Conclusion: The data demonstrates that the rationally designed site specific PEGylation of FVIII in BAY 94-9027 does not alter the in vitro coagulation properties, tested by the ability of BAY 94-9027 to generate thrombin, activate FX or inactivation by aPC. BAY 94-9027 represents the next generation of treatment in hemophilia, using PEGylation technology to reduce the number of infusions while maintaining FVIII activity.

Disclosures

Yegneswaran: Bayer Healthcare Pharmaceuticals: Employment. Jiang:Bayer Healthcare Pharmaceuticals: Employment. Yesantharao:Bayer Healthcare Pharmaceuticals: Employment. Kim:Bayer Healthcare Pharmaceuticals: Employment. Leong:Bayer Healthcare: Employment. Sim:Bayer Healthcare: Employment. Patel:Bayer Healthcare Pharmaceuticals: Employment. Kauser:Bayer Healthcare: Employment.
One Year Survey and Implications of an Hemophilic Computerized Alert System in an University Clinic
Authors: Blum Sabine , Guida Linda , Wasserfallen Jean-Blaise , Angelillo-Scherrer Anne
Published: October 2019

Hemophilia patients are confronted to several issues when being treated in urgent and non-urgent situations. Immediate factor substitution can be life or limb preserving. We installed a computerized haemophilia alert system so as to be informed when one of our patients is treated in another department. Patients admitted to the hospital with a bleeding disorder trigger an alert in the hematology department as well as in the department where the patient is actually located.

We defined alerts as being false positive when an alert was triggered by the admission of a patient where interaction with the hematologist wasn’t necessary and as being false negative when a triggered alert for a patient with a bleeding problem or high bleeding risk in need for interaction with the hematologist didn’t reach the hematologist.

We follow 307 patients in our hemophilia consultation. From 01.01.2011 through 31.12.2011, admission of 128 hemophilic patients to our hospital triggered an alert on 232 occasions. Forty-three percent of presenting patients suffered from von Willebrand’s disease type 1, 11.5% from thrombopathies, 9% from severe hemophilia A, 9% from mild hemophilia A, 9% were hemophilia A carrier and 8% had factor XI deficiency.

Out of 232 alerts in 2011, most were identified as false positive. Eighteen alerts were not followed by a contact between the treating physician and the hematology service (8%). Sixteen alerts (7%) were followed by the establishment of a substitution protocol for an intervention that wasn’t planned before, preventing, in consequence, bleeding complications thereafter. Two alerts (1%) were triggered by 2 patients, 1 suffering from von Willebrand’s disease type 1, the other from von Willebrand’s disease type 2B, with massive bleeding, where intervention by the hematologist was potentially life- or limbsaving, even if one of the alerts was only discovered the following morning.

In conclusion, follow up of 232 alerts during 1 year permitted to intervene possibly life- or limbsaving in only 2 cases. In 16 cases a protocol of substitution could be established that wouldn’t have been made without the alert system. All other cases of alerts were followed up without consequences and thus increased work charge unnecessarily. We continue to follow up our patients with the alert system described.

Disclosures

No relevant conflicts of interest to declare.
Gas6 Promotes Pro-Inflammatory (Ly6Chi) Monocyte Recruitment in Venous Thrombosis
Authors: Laurance Sandrine , Bertin Francois-Rene , Ebrahimian Talin , Lehoux Stephanie , Lemarie Catherine A , Blostein Mark D
Published: October 2019

Recent studies have demonstrated that innate immune cells, i.e. neutrophils and monocytes, provide the initiating stimulus for venous thrombus development. Gas6 was found to promote inflammation by inducing interactions between the endothelium and innate immune cells. In addition, we recently showed that Gas6 was involved in venous thrombosis by inducing tissue factor expression in the endothelium. Since Gas6 is expressed in monocytes, we hypothesize that Gas6 may be involved in monocyte recruitment during venous thrombosis. Venous thrombosis was induced in the inferior vena cava of wild type (WT) and Gas6 deficient (-/-) mice using 5% FeCl3. Using ultrasonography, we found that global monocyte depletion by clodronate resulted in the formation of smaller thrombi. Selective depletion of the pro-inflammatory (Ly6Chi) monocyte subset, using an anti-CCR2 antibody, also induced the formation of smaller clots. In addition, MOMA-2 (monocyte-macrophage marker) staining showed a reduced number of monocytes in thrombi from Gas6-/- mice. More importantly, immunofluorescent staining revealed that fewer Ly6Chi monocytes were recruited to the thrombi of Gas6-/- mice compared to WT. However, Ly6Clow (patrolling) monocytes were equivalently recruited between Gas6-/- and WT mice. In vitro, mRNA expression of CCR2 was increased by thrombin in WT but not in Gas6-/- monocytes. The mRNA and protein expression of the CCR2 ligand, CCL2, was also increased by thrombin in WT but not in Gas6-/- endothelial cells. CCL2 secretion, as demonstrate by ELISA, was induced by thrombin treatment in WT but not in Gas6-/- endothelial cells. Conditioned media from WT or Gas6-/- endothelial treated by thrombin was used for monocyte migration experiments. The conditioned media from WT endothelial cells treated with thrombin increased migration of WT monocytes compared to media from untreated or Gas6-/- endothelial cells. Our results demonstrate an important role for Ly6Chi monocytes in thrombus formation and that Gas6 is specifically involved in the recruitment of these monocytes through the expression of CCL2 and CCR2.

Disclosures

No relevant conflicts of interest to declare.
Evaluation of Blood Group Conversion Following ABO-Incompatible Hematopoietic Stem Cell Transplantation (HCT)
Authors: Chan Esther HL , Christofer Thiow , Lim Susan TT , Tan Lip Kun , Poon Michelle
Published: October 2019

Title: Evaluation of blood group conversion following ABO-incompatible Hematopoietic Stem Cell Transplantation (HCT).

Background:

While there have been a number of reports concerning the influence of AB0 incompatibility on hematopoietic transplantation (HCT) outcomes, data evaluating post-HCT blood group conversions following ABO incompatible transplants remains extremely limited.

Methods:

We performed a single centre retrospective analysis of 97 patients undergoing major or minor or bidirectional ABO-mismatched bone marrow (BM), peripheral blood (PB), and cord blood (CB) hematopoietic stem cell transplantation (HCT) between January2005 – April 2014, in whom engraftment had occurred and blood group data was available. The aim of the study was to analyse the red cell ABO phenotype (forward grouping) and levels of anti-A/B antibodies (reverse grouping) in these patients following the ABO-mismatched transplants.

Results:

97 patients transplanted between January 2004 and April 2014 were included in the analysis. There were 40 (41%) matched sibling, 32 (33%) matched unrelated and 25 (26%) CB transplants. Indications for HSCT included both haematological malignancies (n=87) and benign conditions (n=10). Of these donor recipient pairs, there were 44 (45%) minor, 39 (40%) major and 14 (15%) bidirectional ABO mismatches. All patients had achieved full donor type chimerism at the time of analysis.

Analysis of post –HCT blood group conversion was performed in each of the three groups. Amongst the major mismatch patients (n=39), all had conversion of RBC phenotype to donor ABO phenotype and loss of their host derived anti-donor ABO antibodies. Interestingly, in the minor mismatch patients who were tested (n=44), all the patients had conversion of their RBC phenotype, but 43 out of 44 patients failed to produce recipient directed anti-ABO antibodies. Similarly in the bidirectional mismatch group, all 14 patients converted to donor ABO phenotype, but none produced recipient directed anti-ABO antibodies.

Conclusion:

In one of the largest study looking at this issue, our study demonstrated the lack of a development of recipient directed anti A/B antibodies despite complete donor chimerism in the majority (1 out of 58) of patients undergoing minor or bidirectional ABO-mismatched HSCT. We postulate that this phenomenon may be due to the donor B cell tolerance against host ABO antigen due to the presence of host A and B antigens within other body tissues.

Disclosures

No relevant conflicts of interest to declare.
Dissection of BET Protein Function in a Hematopoietic Differentiation Model
Authors: Stonestrom Aaron J , Hsu Sarah C , Jahn Kristen S , Hsuing Chris CS , Huang Peng , Hardison Ross C , Blobel Gerd A.
Published: October 2019

The mammalian Bromodomain and Extra-Terminal motif (BET) proteins associate with acetylated histones and transcription factors including the master erythroid regulator GATA1. Pharmacologic inhibitors that broadly target BET family proteins are being evaluated in clinical trials for hematologic malignancies. Additionally, a rapidly growing number of studies in model organisms and in vitro systems suggests BET modulation is a therapeutic avenue in diverse disorders. However the mechanisms through which BET proteins act as well as the contributions of individual BET family members to biological processes remain mostly uncharacterized.

Previously we showed that BET family members Brd3 and Brd4 can bind to GATA1 in an acetylation-dependent manner and that pharmacologic BET inhibition impairs erythroid maturation (Lamonica et al PNAS 2011). Using a combination of genome-wide occupancy analysis, pharmacologic inhibition, genome editing and knockdown we characterized the mechanism and function of BET proteins in the context of GATA1-driven erythroid differentiation. BET inhibitors prevented GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, GATA1 required BETs both for initial chromatin association and for transcriptional activation following the establishment of GATA1 occupancy. As BET proteins associate with chromatin during mitosis when transcription is globally disrupted, they have been implicated as mitotic “bookmarks”. BET inhibition specifically during mitosis failed to elicit a measurable impact on post-mitotic gene activation, calling into question a role of BET proteins as mitotic bookmarks in this system.

To determine the BET protein(s) most relevant to GATA1-activated transcription, we genomically disrupted BET family members Brd2, Brd3 and Brd4 using the CRISPR/Cas9 tool. Despite being present at nearly all GATA1-occupied sites genome-wide, Brd3 disruption had little impact on GATA1-dependent transcription. In contrast, both Brd2 and Brd4 were individually required for efficient gene activation by GATA1. A Brd3 requirement was only revealed in the setting of Brd2 deficiency, indicating functional compensation among select BET proteins. These results provide a comprehensive definition of the functions of BET proteins in a model of cellular differentiation. They further suggest that pharmacologic BET inhibition should be viewed in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.

Disclosures

No relevant conflicts of interest to declare.