bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying Possible Candidate Factors Influencing the Penetrance of Heterozygous NFKB1 Loss of Function Mutations By Whole Exome Sequencing
Authors: Schipp Cyrill , Borkhardt Arndt , Stepensky Polina , Fischer Ute
Published: October 2019

Introduction

The NFκB signaling pathway is a master regulator of immune and inflammatory responses. Recently we and other groups reported heterozygous NFKB1 loss-of-function mutations in patients with combined variable immunodeficiency (CVID) characterized by recurrent infections, autoimmunity and immunoglobulin deficiency. Pedigree analysis revealed incomplete penetrance of the disease causing mutation in 5 of the 6 analyzed families. While patients showed a severe phenotype including hypogammaglobulinemia, chronic infections and cytopenias, other carriers of the same mutation were unaffected except for slightly perturbed immunoglobulin levels indicating the existence of other factors influencing the penetrance of these mutations.

Methods

To identify genetic factors associated with complete penetrance of dominant NFKB1 mutations, whole exome sequencing was carried out using DNA extracted from blood samples derived from two patients and their families. Sequencing data of two patients and X unaffected carriers of the same NFKB1 mutations (p.R157X and p.I47fsX2) were then screened in silico for single nucleotide variations, small insertions and deletions present in modulators of immune responses in general and the NFκB pathway in particular, employing lists generated based on publicly available data on gene interactions (including e.g. data of the KEGG, and STRING databases).

Results

We detected no deleterious mutations in known modifier genes such as IL10, IL1B, IL6, CCR5, CCL5, RANTES, TGFB1 and others. But strikingly both patients harbored two polymorphisms (g.797C>A, Gly54Asp, Gly57Glu) in the Mannose Binding Lectin 2 (MBL2) gene that were previously reported as disease causing mutations in patients with primary immunodeficiency. These polymorphisms lead to reduced MBL2 expression and are linked with high susceptibility to infections. We hypothesize that low MBL2 expression in an NFKB1 haploinsufficient background may promote disease penetrance or increase the predisposition to infections.

Conclusion

Our combined next-generation sequencing and bioinformatics analyses approach identified MBL2 as an interesting candidate factor whose deficient expression may influence the penetrance of NFKB1 loss-of-function mutations. Further analysis of greater cohorts is needed to reinforce the role of MBL2 in the pathogenesis of NFKB1 haploinsufficiency.

Disclosures

No relevant conflicts of interest to declare.
Loss of NFAT2 Leads to an Acceleration of Clonal Evolution in CLL
Authors: Müller David J , Märklin Melanie , Kanz Lothar , Mueller Martin R , Wirths Stefan
Published: October 2019

Chronic lymphocytic leukemia (CLL) may be defined as a clonal expansion of mature B cells with stereotypic B cell receptors (BCR). Somatic hypermutation of the BCR heavy chains (IGVH) defines subgroups with different prognosis. In up to 10% of patients Richter's transformation to a high grade lymphoma with a mostly dismal prognosis is observed. The TCL1 transgenic mouse is a well accepted model of human CLL. Upon B cell-specific knock out of NFAT2, TCL1 mice develop a disease resembling Richter's syndrome with a significantly more aggressive disease phenotype. While TCL1 B cells exhibit tonic anergic BCR signaling characteristic of human CLL, loss of NFAT2 leads to readily activated BCRs indicating different BCR usage with altered downstream signaling.

Here, we analyzed BCR usage in C57BL/6 wildtype, TCL1, and TCL1-NFAT2-/-mice. Splenocyte cDNA of the respective animals at an age of 7 months was amplified by multiplex PCR covering known heavy chain VDJ alleles. The PCR products were subsequently cloned into standard bacterial plasmids and subjected to conventional DNA sequencing. In addition, PCR products were analyzed by next-generation-sequencing (NGS). For data analysis, the IMGT/HightV-QUEST online tool was applied.

A very diverse and polyclonal BCR usage was found in wild type mice with more than 4000 different clones identified. Although TCL1 mice at 7 months of age exhibit all features of CLL, their BCR usage as analyzed by unambiguously identified VDJ recombination was still polyclonal. Only with respect to VH usage, TCL1 mice were found to use a more limited set of V alleles compared to wild type mice. Loss of NFAT2 by conditional knockout on the contrary, lead to an oligoclonal usage of VDJ recombination, to a further limitation of V alleles, and also to the usage of identical VDJ recombination in different mice with high frequency - indicating BCR selection either by antigen or self-signaling in these mice. Further analyses by sequencing of hypervariable regions showed preferential usage of mutated BCRs in TCL1 mice and of unmutated BCRs in NFAT2 ko mice reflecting benign and aggressive disease in humans.

In summary, the loss of NFAT2 signaling in CLL prefers the selection of unmutated BCRs as well as the preferential usage of certain VDJ recombinations which results in the accelerated development of true oligoclonal disease.

Disclosures

No relevant conflicts of interest to declare.
Anti-FcRH5/CD3 T Cell Dependent Bispecific Antibody (TDB) for the Treatment of Multiple Myeloma
Authors: Li Ji , Stagg Nicola , Johnston Jennifer , Harris Michael , Menzies Sam , DiCara Danielle , Clark Vanessa , Cook Ryan , Slaga Dionysos , Nakamura Rin , Luke McCarty , Sukumaran Siddharth , Luis Elizabeth , Ye Zhengmao , Sumiyoshi Teiko , Danilenko Dimitry , Lee Genee , Totpal Klara , Ellerman Diego , Hötzel Isidro , James John , Junttila Teemu T
Published: October 2019

Bispecific antibodies that retarget cytotoxic T cell activity to kill cancer cells are currently under clinical evaluation. However, the molecular mechanism for how CD3-bispecific antibodies 'trigger' intracellular T cell signaling is not known. We demonstrate that bispecific antibodies invoke an equivalent biophysical mechanism of TCR triggering as that observed for the TCR/pMHC interaction, including target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. However, we demonstrate that rational epitope selection can overcome the spatial inhibition caused by target molecules with a large extracellular domain and result in efficient synapse formation and highly potent T cell triggering. With this insight, we developed a novel T-cell dependent bispecific (TDB) antibody, anti-FcRH5/CD3 TDB, targeting the B cell lineage marker FcRH5 for multiple myeloma. Anti-FcRH5/CD3 TDB demonstrated cytotoxicity against human plasma cells and patient derived myeloma tumor cells at picomolar doses. Very low target expression level is sufficient to induce anti-FcRH5/CD3 TDB mediated killing, indicating broad activity in multiple myeloma where the prevalence of FcRH5 expression is 100%. In primates, anti-FcRH5/CD3 treatment resulted in complete depletion of tissue B cells and bone marrow plasma cells. Anti-FcRH5/CD3 TDB induces immunosuppressive feedback signaling, including PD1 up-regulation, which can be overcome by PD-L1 antibodies. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD1/PDL1 signaling in the treatment of multiple myeloma and other B-cell malignancies.

Disclosures

Li: Genentech: Employment. Stagg:Genentech: Employment. Johnston:Genentech: Employment. DiCara:Genentech: Employment. Clark:Genentech: Employment. Cook:Genentech: Employment. Slaga:Genentech: Employment. Nakamura:Genentech: Employment. Luke:Genentech: Employment. Sukumaran:Genentech: Employment. Luis:Genentech: Employment. Ye:Genentech: Employment. Sumiyoshi:Genentech: Employment. Danilenko:Genentech: Employment. Lee:Genentech: Employment. Totpal:Genentech: Employment. Ellerman:Genentech: Employment. Hötzel:Genentech: Employment. Junttila:Genentech: Employment.
LIGHT (TNFSF14) Increases Survival and Proliferation of Human Bone Marrow-Derived Mesenchymal Stem Cells Via LTβR
Authors: Kim Hawk , Heo Sook-Kyoung , Noh Eui-Kyu , Gwon Gi-Dong , Jo Jae-Cheol , Choi Yunsuk , Park Jinny
Published: October 2019

Background: LIGHT (Homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM)/tumor necrosis factor (TNF)-related 2, HVEM-L, TNFSF14, or CD258) is a member of TNF superfamily. It is expressed as a homotrimer on activated T cells and also on immune dendritic cells, and has three receptors such as HVEM, LT-¥â receptor (LT-¥âR) and decoy receptor 3 (DcR3). So far, three receptors with distinct cellular expression patterns are described to interact with LIGHT. Follicular DCs and stromal cells bind LIGHT through LT-¥âR. We monitored the effects of LIGHT on human bone marrow-derived mesenchymal stem cells (BM-MSCs).

Methods: At first, we checked negative and positive differentiation markers of BM-MSCs. After rhLIGHT treatment, we monitored cell count, viability, proliferation, and cell cycle distribution. Also, PDGF and TGF¥â production by rhLIGHT were examined by ELISA, and biological mechanism were checked by immunoblotting through the rhLIGHT treatment.

Results: FACS analysis result showed that LT-¥âR receptor is expressed in human BM-MSCs, but not HVEM indicating that LIGHT binds only LT-¥âR in human BM-MSCs. (Fig. 1A/B). rhLIGHT and LT-¥âR interaction increased cell numbers in BM-MSCs using an inverted microscope for cell number changes. Cell numbers by rhLIGHT enhanced dose-dependently and time-dependently (Fig. 2 A/B). Cell viability and the expression of p-AKT, Bcl-2 and Bcl-xL by rhLIGHT were significantly increased in BM-MSCs and rhLIGHT-induced IkB-¥á degradation activated NF-kB signal (Fig. 3A/B). rhLIGHT increased cell proliferation by increasing S/G2/M phase in BM-MSCs (Fig. 3C and 4D). And cell cycle regulatory proteins were enhanced by rhLIGHT in BM-MSC including cyclin B1, D1, D3, E, and cyclin dependent kinase (CDK) 1 and 2 while CDK inhibitor, p27 was decreased by rhLIGHT treatment (Fig. 3E). Moreover, rhLIGHT-induced PDGF and TGF¥â production by STAT3 and smad3 activation accelerated BM-MSCs proliferation. And we also confirmed differentiation potential of rhLIGHT on BM-MSCs by the staining for adipogenesis (Oil Red O staining), chondrogenesis (Alcian blue staining) and Osteogenesis (Alizarin red Staining).

Conclusion: LIGHT and LT-¥âR interaction increases survival and proliferation of human BM-MSCs by activation of survival proteins, anti-apoptotic proteins, CDKs and cyclins. Moreover, LIGHT-induced STAT-3 and smad-3 activation causes PDGF and TGF-¥â production, and they enhance LIGHT signals in human BM-MSCs. We proposed the pathway of LIGHT and LT-¥âR interaction in human BM-MSCs. Therefore, LIGHT may play an important role for therapy of stem cells, and contribute to modification of MSCs.

Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3.

Disclosures

No relevant conflicts of interest to declare.
Lower Incidence of Acute Gvhd Is Associated with the Rapid Recovery of CD4+CD25+CD45RA+ Regulatory T Cells in Patients Who Received Haploidentical Allografts from NIMA-Mismatched Donors: A Retrospective (development) and Prospective (validation) Cohort-Based Study
Authors: Wang Yu , Zhao Xiang-Yu , Xu Lan-Ping , Han Wei , Chen Huan , Wang Feng-Rong , Mo Xiao-Dong , Zhang Yuan-Yuan , Zhao Xiaosu , Kong Yuan , Liu Kaiyan , Huang Xiao-Jun , Chang Ying-Jun
Published: October 2019

To investigate the effects of non-inherited maternal antigen (NIMA) on clinical outcomes and immune recovery, especially of regulatory T cells (Tregs), in patients who underwent unmanipulated haploidentical transplantation.A retrospective cohort (n=57) and a prospective cohort (n=88) were included. Reconstitution of immune subsets, including Tregs, was determined using multicolor flow cytometry. In the retrospective cohort, the cumulative incidence of grades II-IV acute GVHD in patients with NIMA-mismatched donors was significantly lower than that of cases with NIPA-mismatched donors (14.8% vs. 43.30%, P=0.018). Patients with higher percentages of CD4+CD25+CD45RA+ T cells (naive Tregs) within CD4+ T cells recovered on day 30 (≥1.55%) experienced a significantly lower incidence of grades II-IV acute GVHD than that of cases with lower percentages of naive Tregs (<1.55%) (13.8% vs. 46.4%, P=0.010). Multivariate analysis showed that NIMA mismatch and the percentages of naive Tregs were associated with the incidence of grades II-IV acute GVHD [P=0.050, and 0.031, respectively]. In the prospective cohort, the association of NIMA mismatch [HR=0.365, 95%CI, 0.169-0.786, P=0.010] or higher percentages of naive Tregs recovered on day 30 (≥1.55%) [HR=0.114, 95%CI, 0.027-0.479, P=0.003] with a lower cumulative incidence of grades II-IV acute GVHD was further demonstrated. No effects of NIMA mismatch on chronic GVHD, transplant-related mortality, relapse, disease-free survival, or overall survival were found. Our results confirmed the role of NIMA mismatch in acute GVHD and provided the first demonstration, based on clinical data, that recovered Tregs may be involved in the effects of NIMA on acute GVHD in a haploidentical transplant setting.

Disclosures

No relevant conflicts of interest to declare.
Characteristics of Early Recovery of Double Negative T-Cell Subpopulations after Haploidentical Hematopoietic Stem Cell Transplantation
Authors: Liu Jiangying , Bian Zhilei , Fu Qiang , Chang Ying-Jun , Huang Xiao-Jun
Published: October 2019

Background: Immune recovery is recognized to be crucial for decreasing morbidity of the subsequent complications in patients who underwent alloHSCT. We recently compared the recoveries of T-cell subpopulations between the haploHSCT recipients with and without EBV-DNAemia and found that lower CD4/CD8 double negative T-cell (DNT) counts were significantly correlated with the increased cumulative incidence of EBV reactivation after transplantation. Multivariate analysis further showed that hampered recovery of DNT cells was an independent risk factor to predict transplant-related mortality. Based on the expression of αβ or γδ T-cell receptors, or NK cell markers, several subpopulations of DNT cells have been identified, e.g. TCR-γ/δ+DNT, TCR-α/β+DNT, NKT cells, and other cells that are incompletely characterized. Early recovery levels of DNT subsets may correlate to the occurrence of HSCT complications. Notably, reconstitution kinetics of these special T-cell subpopulations post-HSCT remains unknown.

Method: A prospective study was conducted to observe the comprehensive characteristics of DNT recovery. Fifteen healthy donors and 60 consecutive patients who were diagnosed as hematological malignancy and underwent haploHSCT at our institute were included. Immnophenotyping analysis was performed to determine the recovery levels of DNT and its subpopulations including α/β+DNT, γ/δ+DNT (Vδ1+ and Vδ2+), and CD56+DNT at 30, 60, and 90 days after transplantation.

Result: Percentages of total DNT, α/β+DNT, and γ/δ+DNT in recipients on day 30 were comparable with those of health donors (defined as normal range). While the percentage of total DNT significantly decreased on day 60 and day 90, percentages of α/β+DNT and γ/δ+DNT were quite stable within the 3 months after haploHSCT. Interestingly, ratio of Vδ1+/ Vδ2+ on day 30 was similar with the normal control, which was significantly reversed on day 60 and day 90. In contrast, the percentage of CD56+DNT on day 30 was 3-fold higher than the normal range, which dropped down to the normal level at 60 and 90 days. In term of the absolute numbers, all detected DNT subpopulations were hampered at 30 days post-transplantation. Only the counts of Vδ1+DNT achieved the normal level at 60 and 90 days whereas numbers of other DNT subpopulations were continuously lower during the evaluation period. The pace of Vδ2+DNT recovery was the slowest compared with others.

Conclusion: Our observations give a picture of the dynamic recovery of DNT subpopulations and contribute to comprehensively understand the characteristics of immune recovery after HaploHSCT. The clinical impact of DNT-subpopulations recovery will be evaluated in the ongoing prospective study. These findings may facilitate the intervention strategies to improve the general outcome of haploHSCT recipients.

Disclosures

No relevant conflicts of interest to declare.
A Novel BRD4 Inhibitor CA2 Suppresses MM Cell Proliferation in an Orthotopic Myeloma Mouse Model
Authors: Imayoshi Natsuki , Yoshioka Makoto , Nakata Susumu , Chauhan Jay , Kado Yoko , Toda Yuki , Fletcher Steven , Strovel Jeffrey , Takata Kazuyuki , Ashihara Eishi
Published: October 2019

We previously demonstrated that a novel BRD4 inhibitor, CA2, suppressed MM cell proliferation in vitro in the 57th ASH Annual Meeting. In the present study, we investigated the inhibitory mechanisms of CA2 on myeloma cell proliferation in vitro and in vivo. CA2 has a unique quinolinone core and inhibited the proliferation of MM cell lines, MM.1s, AMO-1, NCI-H929, OPM-2, and IM-9. As expected, CA2 suppressed the expression of c-MYC mRNA and protein in a dose- and time-dependent manner. In addition, CA2 decreased BRD4 binding to c-MYC promoters and c-MYC enhancers in a ChIP assay using IM-9 cells and an anti-BRD4 antibody (Figure 1). Flow cytometric Annexin-V/propodium iodide staining analyses demonstrated that CA2 induced apoptosis in MM cells. Taken together, CA2 may induce apoptosis in MM cells by inhibiting the binding of BRD4 to promoters and enhancers of c-MYC gene thereby suppressing c-MYC expression.

We next performed a pharmacokinetic study of CA2 in mice. Half-lives (t1/2) of CA2 following oral administration (50 mg/kg) and subcutaneous administration (10 mg/kg) were approximately 61 min and 139.8 min, respectively. The values of area under the curve for oral and subcutaneous administrations were 179,000 hr·ng/mL and 31,000 hr·ng/mL, respectively. Lastly, we assessed the in vivo effects of CA2 using orthotopic MM mouse model, where IM-9Luc cells were inoculated intravenously through the tail veins of SCID mice that had received 2 Gy of irradiation. The mice in the vehicle-control arm died of MM by approximately 30 days after transplantation; IM-9Luc cells engrafted in many organs including bone marrow of spine and femurs, kidneys, ovaries, and stomach. CA2 treatment consisting of 20 cycles of 100 mg/kg/day b.i.d. oral dosing resulted in reduced luminescence intensity of IM-9Luc cells and prolonged the survival of mice (mean 33.7 days) compared to the vehicle-control arm (mean 27.1 days; p=0.029, Figure 2). In conclusion, CA2 is a BRD4 inhibitor with a novel structure and could be a promising molecular targeting-agent against MM.

Disclosures

Yoshioka: ConverGene LLC: Employment. Kado:Japan Community Health Care Organization Kyoto Kuramaguchi Medical Center: Employment. Strovel:ConverGene LLC: Employment.
Genotype in Patients with Thalassemia Major Has No Impact on Risk of Liver Fibrosis
Authors: Al-Khabori Murtadha K. , Daar Shahina , Al-Zedjali Shoaib , Al Omairi Al Ghaliya , Hassan Moez , Al Rahbi Sarah , Ibnouf Abubakr , Al Busaifi Said
Published: October 2019

Introduction and Objectives: Patients with Thalassemia Major (TM) develop various complications related to iron overload and transfusion associated infections. It is unknown as yet if the specific genotype leading to TM has further impact on the development of liver fibrosis. Therefore, we planned to assess the impact of genotypes in patients with TM on the risk of liver fibrosis.

Methods: We included 88 patients with TM with available genotyping data in this study. These patients were followed and transfused in a single tertiary academic center. All those patients had their liver fibrosis assessed by ultrasound elastography (FibroScan device) with a cut off value of 7.8 kPa. The genotyping was done using Amplification Refractory Mutation System (ARMS)-hot start-polymerase chain reaction (PCR) technique. Iron overload was assessed using the average serum ferritin in the 5 years prior to the liver fibrosis assessment. Association was assessed using Chi-squared test and adjustment using multivariable logistic regression.

Results: A total of 88 patients were included with a median age of 27 years (Interquartile range [IQR]: 23-31). Females constituted 54% of patients and 34% of patients were splenectomised. The median alanine transaminase, aspartate transaminase, albumin and total bilirubin were 31 U/L (IQR: 20-58), 30 U/L (IQR: 18-46), 46 g/L (IQR: 43-48) and 23 µmol/L (IQR: 15-34) respectively. The median ferritin and liver iron concentration assessed by MRI T2* were 1472 µg/L (IQR: 741-2760) and 7.0 mg/gdw (IQR: 3.5-15.9) respectively. Thirty seven percent of patients had positive serology for HCV while 1% of patients had positive serology for HBV. The genotypes seen in this study included homozygous IVS-I, 5 (G-C) in 39 patients, homozygous codon 44 (-C) in 19 patients, double heterozygous IVS-I, 5 (G-C)/25 bp deletion in 5 patients and others (≤ 3 patients each) in 25 patients. The proportion of patients with fibrosis was 55%. The proportion of patients with fibrosis in the homozygous IVS-I, 5 (G-C) group was 58% compared to 52% in all other genotypes and the difference was not statistically significant (odds ratio of 1.3 with a p value of 0.6652). The impact of the genotype remained non-significant after adjustment of iron overload in the multivariable logistic regression model. The only statistically significant factor was the iron overload (p values: iron overload 0.006, genotype 0.966).

Conclusion: The risk of liver fibrosis is associated with the iron overload in patients with TM. There is no additional independent impact of the genotype. Further studies with larger sample size to study the impact of various genotypes are required to confirm these results.

Disclosures

No relevant conflicts of interest to declare.
Cardiometabolic and Hemostatic Profiles of Women Carriers of Hemoglobin Variants Using Combined Oral Contraceptives
Authors: Ferreira Junia Raquel Dutra , Guarda Caroline Conceição , Santiago Rayra Pereira , Rocha Luane , Ferreira Jessica SANTOS , Araújo Luís , Mota Barbara , Pitanga Thassila Nogueira , Milena Milena Magalhães , Carvalho Magda Seixas , Adorno Elisangela Vitoria , Goncalves Marilda Souza
Published: October 2019

Introduction: Several studies reported combined contraceptives influence in hemostatic and lipid profile besides the concern about the thromboembolism and cardiovascular risks due to the steroids hormones use. As women with hemoglobin (Hb) variants have a pre-existing inflammatory condition and considering the high frequency of hemoglobin variant worldwide, this study aims to evaluate the association of hematological, lipid, glicemic, inflammatory and hemostatic profiles in women using combined oral contraceptives and carriers of hemoglobin variants.

Methods: We performed a cross-sectional study including 591 women in reproductive-age. We investigated their hemoglobin profile and COCs use. Of them, we included 60 women with HbAA, 21 with HbAC, 25 with HbAS and 7 with HbSC profiles, all of them combined oral contraceptives users. Among those combined oral contraceptives nonusers, 9 were HbAC and 19 HbSC. We evaluated fasting serum glucose, triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), C-reactive protein (CRP), fibrinogen, D-dimer and hematological profile among the studied groups. This study was conducted in accordance and approved by the Research Ethics Committee of the Fundação Oswaldo Cruz - FIOCRUZ, Brazil; and also with the Helsinki Declaration of 1975, and its revisions. Mann-Whitney U tests were used to compare two groups of values within the same variable.

Results: We observed significant differences in some hematological and cardiometabolic parameters in women carriers of different Hb variants and using COCs. We found relevant increases in CRP levels in HbSC and HbAC women that seem to be associated with different types of progestins present in combined oral contraceptives formulations. Also, combined oral contraceptives use seems to be associated with decreased HDL-c levels in HbAC women. Otherwise, D-dimer levels were increased in all women with Hb variants, independently of the contraceptive use.

Conclusions: Although combined oral contraceptive remains an important method to prevent unintended pregnancy, our data suggest that contraception in women carriers of Hb variants, including those in heterozygosis, should be carefully evaluated, especially, considering the pre-existent inflammatory and pro-thrombotic conditions that together with combined oral contraceptive use may result in additional health problems, such as cardiovascular and thromboembolic diseases.

Disclosures

No relevant conflicts of interest to declare.
Studies on the Genetic Mutations of Hereditary Fibrinogen Disorder
Authors: Zhou Rong-Fu , Na Zhou , Jian OuYang
Published: October 2019

Hereditary fibrinogen disorder is a rare kind of bleeding disease, which divided into two types. Type I is a kind of quantity disorder, including afibrinogenemia and hypofibrinogenemia. Type II is a kind of quality disorder, including dysfibrinogenemia. Fibrinogen is a kind of hexameric glycoprotein and consists of two pairs of three chains, which are Aα, Bβ and γchain. FGA、FGB and FGG code for the relevant glycoprotein. The mutations on these genes are responsible for this disorder. In this study, the levels of fibrinogen antigen of 12 cases with low fibrinogen activity were firstly detected. The results showed that one case had low level, and the patient definded as hypofibrinogenemia, and other 11 cases had normal level, thus these patients identified as dysfibrinogenemia. All exons and their flanks of FGA , FGB and FGG were amplified by PCR. THe PCR products were sequenced directly and blasted to normal sequence of corresponding gene to find the mutation. The result showed that among 11 cases with dysfibrinogenemia, five harbored Aα Arg16His heterozygous mutation and one of those also possessed a de novo γAsp185Asn heterozygous mutation ; one Aα Arg16Cys heterozygous mutation; four γArg275Cys heterozygous mutation and one γArg275His heterozygous mutation.The patient with hypofibrinogenemia harbored Aα Cys36Arg heterozygous mutation. Endonuclease restriction digestion was performed to exclude genetic polymorphism for the γAsp185Asn mutation. In molecular modeling, the hydrogen bonds were changed in the mutational variant of γ185Asn. Besides, a new site of glycosylation might appear after the mutation, which might lead to destroy the stability of molecular structure. The alignment of homologous sequence between different species suggested that γAsp185 was a highly conservative site. In a word, the above mutations might be the causes of dysfibrinogenemia or hypofibrinogenemia for these patients.

Disclosures

No relevant conflicts of interest to declare.
Incidence of Venous Thromboembolism in Patients with Resected Biliary Tract Cancers
Authors: Meier Anne , Khorana Alok A. , Sohal Davendra P. S.
Published: October 2019

Background:

Venous thromboembolism (VTE) is an important complication of cancer, with adverse impact on morbidity and mortality. In biliary tract cancer (BTC), there is a paucity of data on incidence of VTE and any prognostic markers that may correlate with VTE.

Objectives:

To determine the incidence of VTE events in patients with resected BTC, as well as identify predictors of VTE.

Methods:

We conducted a retrospective cohort study of consecutive patients with BTC undergoing surgical resection with a curative intent at the Cleveland Clinic between 1/1/2006-6/30/2014. Electronic medical records and tumor registry were reviewed for baseline demographic and laboratory data, the incidence of VTE after initial diagnosis, including deep venous thrombosis (DVT) of the extremities, pulmonary emboli (PE), or intra-abdominal thrombi. Rates of VTE are reported.

Results:

The study population comprised 142 patients with a median age of 62 (range 31-84) years; 53% were women. Location of the tumors included extrahepatic (21%), intrahepatic (45%), hilar (9%), and gallbladder (25%). During follow-up, 33 (23.2%) patients had documented VTE. Twenty-five patients had 1 event each, 6 had 2 events each, 1 had 3 events, and 1 patient had 4 events, for a total of 44 events. Of these 44 events, 19 (43.2%) were lower extremity DVTs, 9 (20.4%) were upper extremity DVTs, 7 (16%) were PE, and 9 (20.4%) were visceral (intra-abdominal) thrombi. Four (12.1%) of the 33 patients experienced VTE prior to surgical resection. Univariable analyses did not reveal any association with clinical variables - including age, sex, race, baseline hemoglobin, white blood cell or platelet counts, albumin, location of the tumor, chemotherapy or radiation, and smoking.

Conclusion:

Nearly one in four patients with biliary tract cancers who have undergone surgical resection develop VTE. Larger prospective studies focusing on risk factors and novel biomarkers are warranted.

Disclosures

Khorana: Sanofi: Consultancy, Honoraria; Leo: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria; Halozyme: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen Scientific Affairs, LLC: Consultancy, Honoraria, Research Funding.
Current Management of Blood Donors with Positive Anti-Hbc in Saudi Arabia
Authors: Al-Momen Abdul Kareem M , Alghamdi Essam , Al-Humaidan Hind , Al-Mishari Mai , Suhaibani Omar , Alnowaiser Abdullah
Published: October 2019

Background: Transfusion Services in Saudi Arabia are mainly hospital-based, where each major hospital recruit donors, test for Hepatitis B service antigen (HBs Ag), Hepatitis C antibody (Anti-HCV), Human Immune Deficiency Virus antibody (Anti-HIV), and Nucleic Acid Testing (NAT) for HBV-DNA, HCV-RNA, and HIV-RNA. In addition, Anti-HBc and Anti-HBs titer are tested for those who are negative for HBs Antigen and NAT, so that blood products from donors with high Anti-HBs titer (>100 u/ml) can be used. The aim of this limited retrospective study is to find out the prevalence of Anti-HBc among blood donors in different blood donation centers in Riyadh city, Saudi Arabia and current policies regarding blood utilization from donors with positive Anti-HBc.

Materials and Methods: We reviewed blood donor records in 4 large hospitals in Riyadh between 2011-2015. Hospital names are not disclosed for confidentiality reasons. Instead, they were named A, B, C, and D

Results: (see Table)

1- The overall prevalence of Anti-HBc positive blood donors is 6.2%, however, there is a significant variation among institutions ranging from 2.8 % to 11.1 % (which needs to be investigated to explain reasons for this variation).

2- Some institutions defer all donors with positive anti-HBc (although they carry out all tests), while others carry out anti-HBs and NAT testing and use blood with high titer anti-HBs (> 100 u/ml).

Conclusion: There is a need for an expert consensus opinion regarding the cost-effectiveness of anti-HBc for either donor deferral or proceeding to NAT and anti-HBs titer testing. Such a consensus is practiced in many countries, based on the prevalence of anti-HBc and the availability of NAT testing

Disclosures

No relevant conflicts of interest to declare.
Lpxn Regulates Proliferation, Adhesion and Invasion of Acute Myeloid Leukemic Cells
Authors: Dai Haiping , Zhu Guohua , Shen Qun , Wu Lili , Wang Qian , Wang Qinrong , Wen Lijun , Yao Hong , Qiu Huiying , Chen Suning , Wu Depei
Published: October 2019

Leupaxin (LPXN) belongs to the focal adhesion-associated adaptor protein family, participating in regulating cell adhesion and metastasis. LPXN is a recurrent fusion partner in translocations identified in AML, namely t(11;12) and t(11;21), highlights its potential leukemogenic role in acute myeloid leukemia (AML). However, the precise mechanisms by which LPXN participate in the pathogenesis and progression of AML remains unknown. In the present study, we found that LPXN had a wide expression among a variety of hematological malignancies, including AML. Stable overexpression of LPXN in K-562 cells highlighted the proliferation advantage induced by this gene observed via colony-forming-assay and nude mice models. Proliferation was believed to be induced by LPXN-driven upregulation of ERK1/2 and pERK1/2 in K562-LPXN cells. Moreover, LPXN-expressing K-562 cells were less inhibited by arsenic trioxide, as compared with control cells, which might due to downregulation of BAX, FAS and upregulation of BCL-2. Adhesion to fibronectin and invasion across matrigel were modulated by overexpression and knockdown of LPXN, respectively. Similarly, activity of MMP-9 and MMP-2, and expression of integrin α4, α5 and β1, correlated with overexpression or silencing of LPXN, respectively. Expression of pP38 also showed a positive correlation with LPXN. In general, our data suggests that LPXN promotes proliferation in vitro and in vivo via the MAPK pathway, as well as enhances adhesion and invasion of AML cells through modulating expression of integrin α4, α5, β1 and MMP-9/MMP-2.

Disclosures

No relevant conflicts of interest to declare.
HSP90 Stabilizes B-Cell Receptor Kinases in a Multi-Client Interactome: PU-H71 Induces CLL Apoptosis in a Cytoprotective Microenvironment
Authors: Wang Yue Lynn , Lu Pin , Lee Jimmy , Zhen Chao Jie , Chiosis Gabriela , Guo Ailin
Published: October 2019

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B-cells in the hematopoietic system and lymphoid tissues. Although inhibitors targeting the B-cell receptor (BCR) pathway have been successful in the treatment of the disease, the underlying mechanisms leading to BCR over-activity in CLL are not fully understood. In this study, we found that HSP90, a highly conserved molecular chaperone, is overexpressed in CLL compared to resting B-cells. HSP90 overexpression is accompanied by the over-expression of several BCR kinases including LYN, SYK, BTK and AKT. Chemical and immune-precipitation demonstrated that these BCR constituents are present in a multi-client chaperone complex with HSP90. Inhibition of HSP90 with PU-H71 destabilized the BCR kinases and caused apoptosis of CLL cells through the mitochondrial apoptotic pathway. Further, PU-H71 induced apoptosis in the presence of stromal co-culture or cytoprotective survival signals. Finally, genetic knock-down of HSP90 client AKT, but not BTK, reduced CLL viability. Overall, our study suggests that the chaperone function of HSP90 contributes to the over-activity of the BCR signaling in CLL and inhibition of HSP90 has the potential to achieve a multi-targeting effect. Thus, HSP90 inhibition may be explored to prevent or overcome drug resistance to single targeting agents.

Disclosures

No relevant conflicts of interest to declare.
A Novel Role of PALB2 in Lymphoid Tumour Development
Authors: Stankovic Tatjana , Byrd Philip J , Stewart Grant S , Taylor Alexander J , Smith Anna , Fooks Peggy , Horsley Robert , Last James I , Janic Dragana , Dokmanovic Lidija , Oliver Antony W , Taylor A.Malcolm R
Published: October 2019

PALB2 is DNA damage response protein that acts in Homologous recombination Repair (HRR) pathway and promotes recruitment of principal HRR components such as BRCA1, BRCA2 and Rad51. Biallelic PALB2 inactivation is associated with the severe form of Fanconi anemia (FA-N), characterised by severe developmental abnormalities and occurrence of embryonal tumours in infancy. Given the rarity of reported patients with biallelic PALB2 inactivation, the full extent of phenotypes associated with PALB2 mutations is still unknown.

Here we describe a family with two inherited PALB2 mutations where the affected individuals presented with few of the clinical features typically exhibited by FA-N patients. The patients, age 19 and 15 were lacking severe developmental abnormalities and displayed impaired growth and mild learning difficulties. Interestingly, however, both individuals developed B cell Non-Hodgkin lymphoma (NHL) at the early age of 12 and 3.5 years respectively, responded well to intensive chemotherapy and stayed in complete remission. Their cellular phenotype included a high level of spontaneous chromosomal damage suggestive of DNA repair defect, but otherwise deviated from the classic FA phenotype revealing an intermediate sensitivity to mitomycin C (MMC) by colony forming assay but an unusually high chromosomal radiosensitivity in G2.

We addressed the possibility that this atypical clinical and cellular phenotype could be related to the functional properties of the mutant PALB2 protein(s) expressed from the affected alleles. Indeed, we demonstrated that the PALB2 protein (T839_K862del) produced from one of the mutant alleles carried by these patients retained both its N- and C-terminal domain required to interact with BRCA1 and BRCA2 respectively. We also showed that this mutant protein preserved some function compared with previously identifiedpatient-associated mutant PALB2 proteins and allowed low level recruitment of Rad51 foci at sites of DNA damage. We propose that the presence of this mutant protein accounts for the milder MMC hyper-sensitivity exhibited by cells from these patients and the lack of FA defining clinical characteristics.

Finally, the shift in tumour spectrum from embryonal tumours towards B cell NHL observed in this family implies PALB2 as a potential suppressor of lymphoid tumourigenesis and raises the possibility that somatic mutations in this gene may contribute to the development of sporadic lymphoid tumours.

Disclosures

Janic: Baxter: Other: Paid Instructor, Research Funding; Pfizer: Other: Paid Instructor, Research Funding; Bayer: Other: Paid Instructor, Research Funding; Octopharma: Research Funding; Novo Nordisk: Other: Paid Instructor, Research Funding.
Role of miRNA in Micro-Environment Mediated Drug Resistance in Acute Promyelocytic Leukemia to Arsenic Trioxide
Authors: Ganesan Saravanan , Lakshmanan Vairavan , Palani Hamenth Kumar , Balasundaram Nithya , Alex Ansu Abu , David Sachin , Korula Anu , George Biju , Balasubramanian Poonkuzhali , Gowda Harsha , Vyas Neha , Palakodeti Dasaradhi , Mathews Vikram
Published: October 2019

Role of stromal microenvironment in drug resistance has been extensively reported for several cancers. We have demonstrated earlier that there is significant micro-environment mediated drug resistance (EM-DR) to arsenic trioxide (ATO) in acute promyelocytic leukemia (APL) and that this was predominantly driven by upregulation of the NF-ⱪB pathway in the malignant cell. In our current study we have probed the molecular mechanism of ATO resistance in further detail. The role of microRNA (miRNA) in mediating this cross talk, if any, has not been reported on. We undertook a study to evaluate the potential role played by miRNA in EM-DR to ATO in APL.

Using NGS based small RNA sequencing we identified two miRNA's that were differentially regulated in NB4 cells upon co-culture with HS-5 stromal cells (FDR corrected p values < 0.05). The two miRNAs were hsa-miR-23a-5p (downregulated) and hsa-miR-125a-3p (upregulated)](Fig 1a). These miRNAs have also been previously reported to be involved in NF-kB regulation, specifically miR125a-3p has been reported to be involved in activation of the NF-kB pathway and miR-23a- 5p can be repressed by the same pathway. These results were consistent with our earlier reported observations that NF-kB pathway is dysregulated and enhances drug resistance to ATO. We also observed miR-23a-5p mimics were able to restore the sensitivity of NB4 cells to ATO even in the presence of stromal cells (Fig 1b).

Consistent with the above small RNA sequencing and our previously reported microarray data, using quantitative proteomics approach we have identified that both NFkB signaling and metallothionein 2A (MT-2A) levels are upregulated in leukemic cells upon stromal co-culture. MT 2A is a known target for hsa-mir-23a-5p. MT's are known to sequester heavy metals such as arsenic and could potentially reduce their cytotoxic effect. The role of metallothionein in ATO resistance in APL and possibly other leukemia's needs further evaluation. This data along with that reported earlier by us illustrates multiple levels of regulation of the NF-kB pathway and resistance to ATO by stromal cell co-culture.

Disclosures

No relevant conflicts of interest to declare.
TSP-A74, a Novel Therapeutic Monoclonal Antibody Against TfR1 for Treatment of Hematologic Malignancies
Authors: Zhang Lilin , Nomura Fumiko , Aikawa Youichi , Sudo Yukio , Morishita Kazuhiro , Kurosawa Yoshikazu
Published: October 2019

Transferrin receptor 1(TfR1) is a type II transmembrane glycoprotein regulating the intracellular uptake of iron and is involved in cell growth, proliferation and survival. TfR1 is highly expressed on malignant cells, including those of hematologic malignancies. Therefore, TfR1 may be an attractive target for therapeutic monoclonal antibodies.

We generated a panel of fully-human, anti-TfR1 monoclonal antibodies and evaluated the anti-tumor effects of these antibodies both in vitro and in vivo. The results led to the selection of TSP-A74, an antibody with potent in vitro and in vivo anti-tumor activity, for further evaluation in several hematologic malignancy models. First, the efficacy of TSP-A74 was evaluated in acute myeloid leukemia (AML) models. Two AML cell lines, Kasumi-1 and HL-60, were subcutaneously inoculated in severe combined immunodeficiency (SCID) mice. After the tumors were grown to a size of 150 mm3, TSP-A74 was administrated intravenously (IV) once weekly for 4 weeks at doses of 0.4, 2 and 10 mg/kg and 1, 3 and 10 mg/kg for the Kasumi and HL60 xenograft models, respectively. TSP-A74 demonstrated complete tumor regression in these two xenograft models at 10 mg/kg and complete tumor growth suppression in the Kasumi model at 2 mg/kg. Even at the low dose of 1 mg/kg, TSP-A74 demonstrated tumor growth inhibition (TGI) of 60% in the HL60 model. Next, the anti-tumor efficacy of TSP-A74 was assessed in an acute lymphoblastic leukemia (ALL) model. The ALL cell line, CCRF-CEM, was engrafted into SCID mice intravenously. After 3 days, TSP-A74 was administrated IV at a dose of 10 mg/kg once weekly for 4 weeks. The control mice (n=10) rapidly developed leukemia and none survived at 42 days after leukemia cell engraftment. However, 7 of 10 (70%) mice treated with TSP-A74 survived to 179 days after engraftment when the study was terminated. Finally, the efficacy of TSP-A74 was evaluated in non-Hodgkin's lymphoma subcutaneous xenograft models. TSP-A74 produced complete regression of established tumors in the SU-DHL-2 (diffuse large B-cell lymphoma) xenograft model at a dose of 3 mg/kg and tumor growth inhibition of 100 % in the HH (cutaneous T cell lymphoma) xenograft model at a dose of 10 mg/kg. These results indicate that the human anti-TfR1 monoclonal antibody, TSP-A74, could be a new therapeutic candidate for hematologic malignancies.

Disclosures

Zhang: Perseus Proteomics Inc.: Employment. Nomura:Perseus Proteomics Inc.: Employment. Aikawa:Perseus Proteomics Inc.: Employment. Sudo:Perseus Proteomics Inc.: Employment. Morishita:Perseus Proteomics Inc.: Research Funding.
Prognostic Stratification of Acute Myeloid Leukemia and Mylodysplastic Syndrome Patients on the Basis of Genetic Variations
Authors: ALI Muhammad , Basit Sulman , Masood Muhammad tariq , Yousafzai Yasar Mehmood , Naseer Asif , Rahman Sadeeq ur , Jelani Musharraf , Khan Imran , Khan Yousaf , Jameel Abid , Ahmed Jawad , Taj Abid Sohail
Published: October 2019

Acute Myeloid Leukemia (AML) is a heterogeneous disorder characterized by the accumulation of acquired genetic alterations in hematopoietic progenitor cells that alter normal mechanisms of self-renewal, and differentiation. Once a rapidly and life fatal disease, the prognosis of AML has improved in the past decades. Recurrent cytogenetic and molecular genetic abnormalities are proven to be of diagnostic and therapeutic value. All cases of AML cannot be explained by the low resolution cytogenetic techniques or biased molecular genetic approaches. Recent advancements in high resolution genomic techniques has allowed detection of additional submicroscopic abnormalities in the AML genome. This study set out to determine the copy number alterations (CNAs) in bone marrow and oral epithelial samples from 08 AML cases by whole genome Single Nucleotide Polymorphism (SNP) genotyping using Illumina OmniExpress-12 v1-1 Bead Chips containing 700,000 SNPs. Additionally, using a targeted approach with TaqMan real-time quantitative polymerase reaction (RT-qPCR), we tested for previously reported SNP (rs11554137) in a group of 47 AML patients.

The group of patients comprised of AML-M1 (n=1), M2 (n=5), M4 (n=1) and M5 (n=1), SNP genotyping data analysis identified 13 acquired CNAs in 05 out 08 AML patients. Deletions were more common than amplifications. These CNAs were distributed across all FAB subtypes except AML-M3.AML-M4 subtypes were found to have more CNAs compared to other subtypes. Notably, in three AML-M2 cases4q24 microdeletion was identified. This region spans TET2 gene which is has been shown to cause microdeletions and copy-neutral loss of heterozygosity (LOH) in a patient with diverse myeloid malignancies. Oral epithelial cells from the same patients were negative for the CNAs. Moreover, the 47 patients tested for known SNPs, 4 patients (8.5%) were found positive for rs11554137. SNP (rs11554137) has been reported to be of prognostic importance for overall survival (OS).

The results from this study shows CNAs in regions implicated in myeloid malignancies. The use of high-resolution genomic array identified significant number of CNAs and SNPs which were not previously documented in AML patients in Pakistani population. Further studies dissecting out their role in AML pathogenesis are required.

Disclosures

No relevant conflicts of interest to declare.
The Isoform II of SRSF1: A Potential Biomarker in the Progression of Pediatric Acute Lymphoblastic Leukemia
Authors: Xu Liting , Zheng Huyong
Published: October 2019

Background: Acute lymphoblastic leukemia (ALL) is the most frequently occurring malignant neoplasm in children. Despite advances in treatment and outcomes for ALL patients, the pathogenesis of the disease remains unclear. Alternative splicing modulates the expression of many oncogene and tumor-suppressor isoforms. SRSF1, a prototypical SR protein has been recognized as an oncoprotein. SRSF1 has been reported to be auto-regulated into multiple isoforms that differ in function in various physiological or pathological conditions.

Methods: Matched, newly diagnosed (ND), complete remission (CR) and relapse (RE) bone marrow samples from 50 patients were collected in order to evaluate the expression patterns of SRSF1 spliced isoforms. The function of isoforms of SRSF1 in leukemogenesis was investigated in leukemia cell lines.

Results: Both in mRNA and protein level, we identified significant up-regulation of the three main spliced isoforms of SRSF1 and the isoform II is the main variant in the ND samples. Importantly, the expression of three isoforms of SRSF1 returned to normal levels after CR. But the protein isoforms rebound in the RE samples, and the isoform II is high expressed a short-term state prior to morphological or immunological change in the RE cases. We also observed the isoform II can promote significantly to the formation of cell clones.

Conclusion: Our results indicate that the alternative splicing of SRSF1 may plays a critical role in leukemogenesis in pediatric ALL, and the isoform II may be a sensitive predictor of relapse and a potential target for the anti-leukemic therapy.

Disclosures

No relevant conflicts of interest to declare.