bluuebunny/crossref_metadata_2025 huggingface dataset:
Fecal Microbiota Transplantation As a Therapeutic Option for Graft-Versus-Host-Disease: A Systematic Review and Future Directions
Authors: Aslam Hafiz muhammad , Veeraballi Sindhusha , Hashmi Shahrukh K.
Published: November 2021

Introduction:

In recent years, there has been an expanding focus on interplay between intestinal microbiotal diversity and outcome of acute graft versus host disease(GVHD) in hematopoietic stem cell transplantation (HCT) recipients. One of the compelling intervention to maintain healthy gut microbiota for better outcome of GVHD in HCT recipients is Fecal microbial transplantation(FMT). Several non randomized small clinical studies and case reports on the efficacy and safety of FMT were reported so far. However, the ultimate role of FMT as a therapeutic option to treat GVHD is yet to be determined due to lack of randomised, large scale, statistically significant studies. Here in, we report a systemic review of literature available so far in an effort to establish a definite role of FMT.

Methodology: A systemic literature search was conducted using various electronic databases. The case reports, case series and clinical studies related to FMT were used as a therapeutic or preventative modality specifically for GVHD are included.

Results: Upon pooling of data, 87 patients from 6 studies and 5 case reports were included in the study in which complete remission(CR) occured in 43.7% and partial remission(PR) occured in 20.7% patients which is equivalent to 64.4% overall response rate in treating GVHD. Furthermore, out of all the species in fecal matter, clostridium was found to be the most valuable species in decreasing the rates of GVHD re-occurrence. Only a limited number of patients had treatment-related mortality

(TRM) from GVHD while few showed mild GI-related (abdominal pain/distention, nausea, regurgitation) and non-GI adverse reactions including infections, anemia, thrombocytopenia, paroxysmal, and atrial fibrillation. We emphasize that the most of the patients did not have any major complications after FMT.

Conclusion:

We conclude that the FMT is a safe and effective strategy for the management of GVHD based on our study. The establishment of gut diversity along with the patient's intrinsic factors like fucosyltransferase 2 (FUT2) secretor status and baseline gut microbial diversity play a major role in the success of FMT. Given the restricted size and absence of randomized data, one cannot portray FMT as a standard of care yet, however, the low or absent toxicity along with improvement in survival justifies this modality to be tested in a randomized fashion. We strongly encourage the transplant community to enroll patients in innovative trials utilizing FMT, as this may be a one of the safest strategies for both prevention and treatment of GVHD

Disclosures

No relevant conflicts of interest to declare.
Anti-HNA-3a Alloantibodies Can Cause Differential Endothelial Damage through a Direct Neutrophil Activation Pathway
Authors: Radenkovic Filip , Chiaretti Sara , Burton Mark , Hassell Penny , Bui Xuan , Flower Robert , Tung John-Paul
Published: November 2021

INTRODUCTION: The human neutrophil antigen (HNA) 3a/b is associated with a single amino acid substitution (R 154Q polymorphism) on the first extracellular loop of choline transporter-like protein 2 (CTL2). Antibodies against HNA-3a have been associated with severe and fatal transfusion-related acute lung injury (TRALI). Epitope mapping suggests the existence of two types of HNA-3a antibodies. Type I antibodies only require first extracellular loop of the CTL2 for expression of the antigen, type II antibodies require at least the first 3 extracellular loops of CTL2 in a native configuration (i.e. expressed on a cell membrane). This study aimed to evaluate the activity of type I and type II anti-HNA-3a antibodies in an in vitro TRALI model.

STUDY DESIGN & METHODS: The granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) were used to confirm anti-HNA-3a activity in two donor sera (Q49 and Q50). Flow cytometry was used to confirm antibody binding to freshly isolated human or mouse neutrophils. A rabbit polyclonal antibody against an epitope in the third extracellular loop of CTL2's was coated onto the wells of an ELISA plate and neutrophil HNA-3a antigen was captured and sera tested. For the TRALI model, human lung microvascular endothelial cells (HLMVECs) were grown to confluence before being treated with lipopolysaccharide (LPS). Freshly isolated neutrophils from HNA-3aa homozygote donors were then added with 10% Q49 or Q50. Microscopic counting of Trypan blue staining was used to measure rate HLMVEC death. Significance was determined using a one-way ANOVA (p<0.05), followed by Dunnett's post-hoc test.

RESULTS AND DISCUSSION: GAT and GIFT confirmed that both Q49 and Q50 contained an anti-HNA-3a antibody, and that neither contained antibodies against any other HNA or HLA molecules. In flow cytometry, both Q49 and Q50 bound to human neutrophils, but only Q49 bound to mouse neutrophils. In the ELISA, signal was detected only for Q49. These data provided evidence that Q49 was a type I antibody and Q50 was a type II antibody. Within the TRALI model, HLVMEC death was observed with both Q49 and Q50 only in the presence of LPS and neutrophils (Table 1). Previously reported HLMVEC permeability in the absence of neutrophils (Bayat et al. Arterioscler Thromb Vasc Biol. 2013) was not observed as in this study the outcome measured was HLMVEC death. In the present study, the effect for Q49 (type I) was significantly larger than for Q50 (type II; Table 1; p-value <0.0001).

CONCLUSIONS: In this model, LPS and neutrophils were required for anti-HNA-3a mediated HLMVEC death thought to reflect events involved in the initiation of TRALI. The observation that type I antibodies elicited a stronger response provides a direction for further research.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
PI3K δ /γ Inhibition Enhances the Expansion and Anti-Tumor Cytotoxicity of CART Cells for CLL Patients
Authors: Wang Shuhua , Funk Christopher R. , Ravindranathan Sruthi , Chen Kevin , Waller Edmund K.
Published: November 2021

Background:

While CD19-targeted chimeric antigen receptor (CAR) based T cell therapy has shown promise in the treatment of chronic lymphocytic leukemia (CLL), overall efficacy is limited due to impaired T-cell fitness. We have previously shown that dual inhibition of PI3Kδ and PI3Kγ enhanced mitochondrial mass and ex vivo expansion of central and stem cell memory T cells from CLL patients(Funk, 2019,Journal for Immunotherapy of Cancer). In this study, we hypothesized that pharmacological inhibition of these pathways during ex vivo culture would increase the expansion and in vivo anti-tumor cytotoxicity.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of CLL patients by ficol hypaque centrifugation. T cells were negatively selected using MACS beads and transduced with CD19 CAR lentivirus (encoding CD28 or 41BB co-stimulatory domains) and stimulated with anti-CD3/CD28 beads in media containing 30 U/mL interleukin-2 with or without the PI3Kδ/γ inhibitor duvelisib (Duv) for 15 days. NOG mice were engrafted with the OSU-CLL cell line for 14 to 18 days with tumor burden measured by flow cytometry of blood samples from the mice, comprising a mean 0.15% of peripheral nucleated cell content. Control-CART or Duv-CART were injected by tail vein injection. Frequencies of CART, OSU-CLL cells and immune checkpoint molecule expression of CART or T cells in blood were measured by serial flow cytometry. Kaplan-Meier survival plots were represented as recipient survival on the indicated days

Results: Treatment with either CD28 or 4-1BB Duv-CART cells led to significantly prolonged survival relative to control-CART (Figure 1, P<0.05). Recipients of Duv-CART cleared circulating OSU-CLL faster than control CART (Figure 2. P<0.05 at day26 for CD28 CART) and exhibited greater peak expansion and persistence of total CART and CD8+ CART. Recipients of Duv-CART cells had significantly greater in vivo persistence and expansion of total CART and CD8+ CART (Figure 3, P<0.001, day14 after CART were infused). Consistent with improved survival, both CD28 Duv-CART and 4-BB Duv-CART show reduced expression of LAG3, TIM3 and PD1 in the CD4 (Figure 4) and CD8+ subsets at earlier time point in vivo. (* p<0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001).

Conclusions: Inhibition of PI3Kd/g during CART cell culture decreased the expression of immune checkpoint molecules and enhanced in vivo expansion leading to greater efficacy in eliminating CLL.

Figure 1 Figure 1.

Disclosures

Waller: Verastem Oncology: Consultancy, Research Funding; Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.
Acute Myeloid Leukemia : Clinical Characteristics and Survival Outcomes in Lebanese Patients
Authors: Nasr Fadi , Diab Saada , Ghauche Ahmad , Yehia Intissar , Nasr Lewis
Published: November 2021

Context The molecular characteristics of this disease remain widely unknown in Lebanon.

Cytogenetics and molecular testing in AML are becoming routine practice for disease classification and prognosis. In Lebanon, a nationwide study showed an incidence of 1.1% among all cancers in 2003, slightly more predominant in males.

Objective The aim of this study was to describe patient's characteristics and outcome in Lebanese patients.

Design 80 patients from a single Lebanese center were included. We reviewed disease characteristics of patients diagnosed with AML between January 2008 and December 2020. Medical files were reviewed and SPSS software was used for statistical analysis.

Results 48.1 % of patients were males, with a median age of 61 years at diagnosis. Cytopenia was present at 30% of patients. Diagnosis was confirmed by bone marrow aspirate in 95% of the patients. Karyotype was missing in 48% of patients. When available, 13%, 54% and 33% were respectively in the low, intermediate, and high risk group. FLT3 was positive in 47.4% of tested cases and NPM1 in 16.7%. No patient was found to have t (16;16) and 5.4% have inversion 16. PML-RARA was positive in 2% of patients. Idarubicin-cytosar was given as induction treatment for 82% of patients. 58% achieved complete remission, 2% were in partial remission and 40% were primary refractory. 51.7% relapsed after remission. PFS was 11 months with a median OS of 17 months. No significant statistical correlation was found between Karyotype on diagnosis and survival [P = 0.3].

Conclusion Our study is the first to describe AML characteristics in Lebanon. Multicenter studies are needed to elaborate a clinical and epidemiological data register for leukemia patients in Lebanon.

Disclosures

No relevant conflicts of interest to declare.
CAR-T Therapy Followed By Unrelated Cord Blood Transplantation for the Treatment of Children and Young Adults Relapsed/Refractory B-Cell ALL: Superior Survival but Relatively High Post-Transplant Relapse
Authors: Sun Guangyu , Tang Baolin , Wan Xiang , Yao Wen , Song Kaidi , Geng Liangquan , Qiang Ping , Wu Yue , Zhu Lijun , Wang Dongyao , Wu Yun , Liu Huilan , Sun Zimin , Zhu Xiaoyu
Published: November 2021

Relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) has poor long-term survival even after chimeric antigen receptor (CAR)-T cell therapy or allogeneic hematopoietic stem cell transplantation (allo-HSCT). Several studies supported that CAR-T therapy followed by allo-HSCT is benefit for improving long-term leukemia free survival (LFS). Since probably a stronger graft versus leukemia (GVL) effect of unrelated cord blood transplantation (UCBT), it is uncertain whether consolidative UCBT is suitable for R/R B-ALL after CAR-T therapy. Here, we conducted a retrospective comparative study for R/R B-ALL patients receiving UCBT in our transplantation center. Totally 43 cases with R/R B-ALL who underwent UCBT were assigned to CAR-T group (patients achieved CR or CRi by CAR-T cell therapy before bridging to UCBT, n = 21) or non-remission (NR) group (n = 22). The median time from CAR-T infusion to UCBT was 62 (range, 42-185) days. All patients achieved neutrophil engraftment by day 42 in CAR-T group. The 180-day cumulative incidence of platelet engraftment was higher in CAR-T group than in NR group (90.5% vs 65.7%, P = 0.16). Incidence of grade II-IV and III-IV acute graft-versus-host disease (GVHD) were 28.6% and 23.8% in CAR-T group, which were both tendency lower than in NR group (45.5% and 31.8%, P = 0.32 and P = 0.63, respectively). No patient suffered from extensive chronic GVHD in CAR-T group, which was lower than in NR group (9.1%, P = 0.23). Lower 2-year cumulative incidence of transplant-related mortality (TRM), and higher probabilities of 2-year overall survival, leukemia free survival (LFS), graft-versus-host disease (GVHD)-free relapse-free survival (GRFS) were found in CAR-T group (4.8% vs 28.2%; 75.0% vs 37.7%; 49.8% vs 23.0%; 42.4% vs 14.8%; P = 0.037, 0.005, 0.028 and 0.017; respectively). However, 2-year cumulative incidence of relapse was comparably high between CAR-T and NR group (26.7% vs 38.3%; P = 0.41). CAR-T therapy followed by UCBT has a superior survival for R/R B-ALL, but remains relatively high post-transplant relapse rate. Prevention of relapse after UCBT is warranted in this patient cohort.

Disclosures

No relevant conflicts of interest to declare.
Kikuchi-Fujimoto Disease: A Case Report
Authors: Hickman Jonathan , An Joseph
Published: November 2021

Kikuchi-Fujimoto: A Case Report

Hickman, JD. MD LT MC USN and An, Joseph, DO. LCDR MC USN

Naval Medical Center Portsmouth

620 Johns Paul John Cir, Portsmouth VA

757-953-2223

The views expressed in this abstract are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the United States Government.

We are military service members and employees of the U.S. Government. This work was prepared as part of my official duties. Title 17 U.S.C. 105 provides that "Copyright protection under this title is not available for any work of the United States Government." Title 17 U.S.C. 101 defines a United States Government work as a work prepared by a military service member or employee of the United States Government as part of that person's official duties.

Kikuchi-Fujimoto disease is a rare benign disorder often presenting with tender cervical lymphadenopathy, fever, and malaise. While first described in Japan, its distribution is worldwide and predominantly seen in young adults. Diagnosis is based on characteristic histopathologic findings of patchy necrosis occupied by karyorrhectic debris and abundant histiocytes on node biopsy. The origin is unclear but associated with a preceding viral illness as well as cutaneous lupus erythematosus. Treatment is generally supportive and focused on managing tender lymph nodes.

We present a case of a 28-year-old female presenting with a 2 month history of night sweats, fever, and weight loss in the setting of painful neck swelling. CT and PET/CT imaging demonstrated numerous hypermetabolic and enlarged nodes in the bilateral cervical and axillary regions. Lab studies were notable for leukopenia, anemia, and elevated inflammatory markers. A COVID-19 screening was negative. Excisional biopsy of a cervical node revealed extensive cortical necrosis and apoptotic debris with scattered histiocytes and plasmacytoid dendritic cells in absence of neutrophils or a monoclonal B cell or T cell population. Treatment was initiated with NSAIDs and close monitoring. The patient exhibited a complete response after two months.

Our case is an important reminder that lymphadenopathy, fever, and night sweats in a young adult are not pathognomonic for lymphoma. Nonetheless, a high suspicion for lymphoma should be maintained and followed with an expedited workup. Kikuchi-Fujimoto can certainly mimic Hodgkin lymphoma or other serious conditions like lupus erythematosus and tuberculosis. The diagnosis is largely one of exclusion following a careful examination of a lymph node histopathology and must be considered in young previously healthy adults to avoid misdiagnosis and unnecessary escalation of treatment.

Disclosures

No relevant conflicts of interest to declare.
The Prevalence of May Thurner Syndrome in Children with Ileo-Femoral Deep Vein Thrombosis- Does It Take 2 to Tango?
Authors: Subburaj Divya , Cox Pamala , Price Victoria E. , Kulkarni Ketan
Published: November 2021

Introduction:

May-Thurner syndrome (MTS) is characterised by compression of the left external iliac vein by overriding right common iliac artery resulting in venous stasis. It carries a higher risk of left iliofemoral deep vein thrombosis (DVT), however the prevalence and management guidelines are unclear in pediatrics. We reviewed the prevalence of MTS in children diagnosed with left iliofemoral DVT at our center, associated prothrombotic risk factors and their clinical outcomes.

Methods:

This is a retrospective audit of pediatric patients (<18 years) with left iliofemoral DVT treated at the IWK Health Center from January 1 2008 to December 31 2020.

Results:

Twelve pediatric patients with left iliofemoral DVT were identified at our center and all patients except one had MRV/CTV during their course of anticoagulation to evaluate for MTS. MTS was diagnosed on imaging in 8 of the eleven evaluable patients with DVT and one patient had an incidental diagnosis of MTS with no DVT. The median age at diagnosis of MTS was 15 years (13-16), male:female ratio of 1:8. The overall prevalence rate was of MTS was 72.72% (8/11) in patients with left iliofemoral DVT. All patients with iliofemoral DVT and MTS had at least one other prothrombotic risk factor- initiation of estrogen containing contraceptive pills in the preceding 3 months of the DVT (n=5), inherited thrombophilia (n=2) and obesity (n=2).

All patients with MTS were referred to vascular surgery. Only one patient required catheter directed thrombolysis and stenting at presentation of DVT, the remaining 7 patients were managed with anticoagulation alone. Complete (n=1) or partial (n=7) resolution of the thrombus was seen in all 8 patients. Median duration of follow up was 3 years. Recurrent DVT was seen in 1 patient and two patients came off anticoagulation at 6 months post therapy. Post thrombotic syndrome was seen in 4 patients, mild in 3 and moderate in 1; as per modified Villalta score.

Conclusion:

We observed a high prevalence of MTS in patients with left iliofemoral DVT which may be due our screening approachwith upfront radiographic evaluation for MTS in all patients with left iliofemoral DVT. A second pro-thrombotic risk factor was identified in all patients, which raises the possibility of a "two hit theory" for the occurrence of DVT in MTS. Compared to published adult studies, majority of our pediatric patients were managed conservatively with anticoagulation therapy alone.

Disclosures

No relevant conflicts of interest to declare.
Analysis of the Tumor Microenvironment By Quantitative Immunohistochemistry Reveals Novel Predictors in Classic Hodgkin Lymphoma Treated with Anti-PD1 Therapy
Authors: Yu Hui , Ni Jiali , Sun Zhenchang , Zhang Mingzhi
Published: November 2021

Classical Hodgkin Lymphoma (cHL) is characterized histologically by the presence of neoplastic Hodgkin and Reed-Sternberg cells surrounded by mixed immune cells. Anti-PD-1 therapy is the principal treatment option for relapsed or refractory cHL. To reveal biomarkers for predicting the cHL patients' responses to immunotherapy, pathological tissues of twenty patients with cHL who have been treated with anti-PD1 therapy were collected. Tumor immune microenvironment was investigated by immunostaining and quantitative measurement of CD68 (for macrophages), CD8 (for cytotoxic T-cells), FoxP3 (for Treg), PD-1, PD-L1 and PD-L2 expression levels. The in situ hybridization for Epstein-Barr virus (EBER-ISH) were also performed. The results showed that the main immune indicators of the tumor microenvironment we had tested were independent of the histological subtypes. Patients who were positive for EBER-ISH had higher mean density of PD-L1 than those were negative for EBER-ISH. Single-factor analysis revealed the high expression of PD-L1 and positive EBER-ISH were associated with significantly increased 2-year PFS in cHL treated with anti-PD1, while CD68, CD8, FoxP3, PD-1 and PD-L2 were not. Therefore, we propose that PD-L1 and EBER-ISH may serve as novel predictors for the treatment outcomes of cHL patients with anti-PD1 therapy.

Disclosures

No relevant conflicts of interest to declare.
P-Selectin Deficiency in Sickle Cell Disease Mice Leads to Senescence Induced Organ Injury
Authors: Vats Ravi , Kaminski Tomasz W. , Gudapati Shweta , Brzoska Tomasz , Sundd Prithu , Pradhan-Sundd Tirthadipa
Published: November 2021

Sickle cell disease (SCD) is caused by a homozygous mutation in the β-globin gene, which leads to erythrocyte sickling, vaso-occlusion, and premature hemolysis . Vaso-occlusion and hemolysis are the two predominant pathophysiologic events in SCD that contribute to chronic organ damage and acute systemic painful vaso-occlusive episode (VOE). Previous studies both in vivo and in vitro have confirmed the role of endothelial and platelet adhesion marker P-selectin in sickle cell vasoocclusive crisis. Moreover, a phase 2 study showed a significant reduction in painful vaso-occlusive episodes among SCD patients receiving the P-selectin-blocking antibody crizanlizumab . However, the long-term effect of crizanlizumab in SCD and associated organ injuries is not known. To understand the roles and regulation of P-selectin dependent vasoocclusion in SCD associated organ injuries, we have introduced the first SCD mice genetically lacking P-selectin in hematopoietic and nonhematopoietic compartments. Using this model (SS-Selp −/−) we have shown that P-selectin deficiency protects SCD mice from lung vaso-occlusion. Here we have used this model to study the liver injury in SCD. Using quantitative liver intravital imaging (qLIM) technique, we show that P-selectin deficiency protects SCD mice from liver vaso-occlusion. However, we found persistent hepatobiliary injury in SS-Selp −/−  mice. Mechanistically, we show that blocking P-selectin causes significant enrichment of circulating inflammatory cells however the organ specific recruitment of inflammatory cells was drastically impaired. Remarkably, impairment of organ specific recruitment of inflammatory cells exacerbated cellular senescence and injury in sickle mouse. We found significant enrichment of senescent markers including P21, P16 and phosphor P53 using both western blot and immunohistochemistry. Colocalization analysis confirmed that hepatocytes, cholangiocytes and macrophages were susceptible to senescence. Moreover, we also found impaired iron trafficking in in SS-Selp −/−  mice. Work is currently underway to understand how p-selectin loss promotes liver senescence and impaired iron trafficking in the liver. In summary, our results reveal a significant defect in iron homeostasis and exacerbated senescence in the liver of SS-Selp −/−  mice suggesting that increased liver senescence might impair iron homeostasis which then contribute to hepatobiliary injury in SCD. Thus, the efficacy of P-selectin inhibition in preventing SCD warrants further studies to determine whether long term inhibition of P-selectin would lead to end stage complications.

Disclosures

Sundd: Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding.
Development and Validation of a Specific and Sensitive Anti-Drug Antibody Assay for Abelacimab That Avoids False Positive Results Due to Factor XI Interference
Authors: Cullen Connie , Cai Xiao-Yan , Dernek Cansu , Freedholm Debra
Published: November 2021

Abelacimab is a fully human IgG1 monoclonal antibody that has dual activity against the inactive zymogen Factor XI (FXI) and the activated Factor XI (FXIa). Clinical trials for this investigational product are ongoing.

As part of the initial assessment of abelacimab safety and efficacy, an anti-drug antibody (ADA) assay was validated and implemented in two single ascending dose studies conducted in the United States and Japan in healthy subjects. In those studies, ADA positive results were detected in up to 31.1% of subjects. There was no apparent impact on PK, PD, or development of immunogenicity-adverse events in study subjects who were ADA positive relative to those who were ADA negative in either study. Further, the rates of ADA positive subjects seemed inconsistent with a single administration of a fully human IgG1 monoclonal antibody therapeutic. Given the homodimeric structure of FXI, it appeared that the ADA assay was returning false positive results due to cross-linking through FXI. To address this issue, a novel ADA assay, following the 3-tiered approach of screening, confirmation, and titer determination was developed and validated. The assay was shown to be sensitive, specific, and did not show false positive interference by FXI. Two subsequent clinical trials (ANT-003 A randomized, subject- and investigator-blinded, placebo-controlled study conducted in healthy subjects and in obese, but otherwise healthy, subjects aged 18 to 60 years of age and ANT-004 A randomized, subject- and investigator-blinded, placebo-controlled, multiple ascending dose study in patients with atrial fibrillation (AF) or atrial flutter aged 18 to 85 years old with a CHA 2DS 2-VASc risk score of 0-1 for men and 1-2 for women and in whom the use of an anticoagulant for stroke prevention was not planned for the duration of the trial) showed no treatment-emergent ADA responses from either trial. The screen positive rate from the two studies was approximately 5% with no screen positive samples confirmed as positive indicating that the new assay format successfully addressed the false positive issue, and that the sensitivity was appropriately set to achieve the FDA-recommended 5% false positive rate for ADA assays.

Disclosures

Cullen: Anthos Therapeutics, Inc: Consultancy. Freedholm: Anthos Therapeutics, Inc: Current Employment, Current holder of stock options in a privately-held company.
Genotype-Phenotype Relationship Among 1200 Unrelated White Patients with Inherited FVII Deficiency: A Three-Center Database Study
Authors: Halimeh Susan , Kenet Gili , Kuta Piotr , Shneyder Maria , Bajorat Tido , Nowak-Göttl Ulrike
Published: November 2021

Background: Congenital factor VII (FVII) deficiency is a rare bleeding disorder caused by mutations in F7 gene with autosomal recessive inheritance. A clinical heterogeneity with poor correlation with FVII:C levels has been described. Aim of this study was to identify genetic defects and to evaluate their relationships with clinical phenotype in a large cohort of 1228 white patients with FVII:C below the age-dependent cut-off %.

Methods: Probands with confirmed inherited factor VII deficiency were i) genotyped (Sanger method & multiplex ligation-dependent probe amplification [MLPA]) for F7 mutations including common polymorphic variants and were ii) classified according to clinical bleeding scores (BC). In addition, common thrombophilic variants and blood groups were determined. Results: Probands included asymptomatic subjects (referred for laboratory work up of recurrent prolonged prothrombin time; n=415; mean age 30 + 19 yrs.; female 52%) and patients who presented with mild, moderate or severe bleeding episodes (n=805; mean age 34 + 18 yrs.; female 70%). Bleeding symptoms included epistaxis, gum bleeding, GI bleeding, hematuria, postoperative and gynecologic hemorrhage. Median FVII activity (entire cohort) measured with clotting-based assays (ACL TOP 750 and/or BCS-XP) was 49% (range 5-78%) and the median ISTH BS recorded within a period of two years prior to work-up was 1(0-17). The latter was significantly higher in women compared with males (2 versus 1; p < 0.001). The corresponding PBAC-Score prior to hormonal treatment was 163 (25-1200). Blood group 0 was present in 40.6% of cases. Known and novel mutations in the F7 gene, including coding regions, exon/intron boundaries and the promoter region, are found in 534 patients (44%) and common polymorphisms were detected in 666 subjects (95%). Logistic regression analysis adjusted for clinical and laboratory data (blood group, FVII activity, presence of F7 gene mutations and /or polymorphisms, thrombophilia status and further factor deficiencies) revealed that older age (increase per year) at referral (odds/95% CI: 1.01/1.007-1.025) and female gender (odds/95% CI: 3.25/2.35-4.50) in part explain differences in clinical bleeding phenotype associated with FVII deficiency.

Conclusion: Overall there is poor correlation between the FVII level and the bleeding phenotype. Older patients and females are more likely to have symptomatic disease as a result of gynecological or muco-cutaneous bleeding.

Disclosures

Kenet: Alnylam: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Opko Biologics: Research Funding; Pfizer: Consultancy, Research Funding; Shire: Research Funding; Novo Nordisk: Consultancy; Roche: Consultancy; Takeda: Consultancy.
A Novel Pathogenic Variant of G6PC3 Gene Presenting As Cyclic Neutropenia in a Pediatric Patient
Authors: Bruni Christian M , de la Rua Wendy , Sadre Sara Y , Nestor Jennifer M , Ahmed Rafat
Published: November 2021

Background: Cyclic neutropenia is a rare hematologic disorder affecting neutrophils. It is classified by recurrent neutropenia (typically every three weeks) with associated symptoms including fever, malaise, and recurrent mucosal and skin infections. Pathogenic variants of ELANE, the gene that encodes neutrophil elastase, are thought to cause these findings, however, other pathogenic variants of different genes have also been noted. The estimated frequency of cyclic neutropenia is 1/10 6 in the general population. It is most commonly diagnosed in children but may rarely be diagnosed in adulthood. There is no known increased prevalence in women as compared to men. Whole Exome Sequencing can be used for diagnosis when genetic concerns are present.

Objective: This case report aims to describe a novel pathogenic variant in a pediatric patient with cyclic neutropenia.

Design/Method: Single subject case report

Results: This is a single case report of a 7-year-old female with past medical history of cyclic neutropenia, anemia, recurrent skin and buccal infections, speech delay, and behavioral problems. The patient initially presented at age 1 with high fevers and skin infections and was found to have decreased neutrophil counts. Work up at that time was consistent with cyclic neutropenia. Bone marrow aspirate and biopsy performed at 1 and 7 years of age showed decreased neutrophil count with normal maturation without any evidence of MDS. Her peripheral blood for bone marrow failure evaluation was non-contributory. Due to persistent neutropenia, normal bone marrow findings, and being on weekly chronic G-CSF administration Whole Exome Sequencing was evaluated. Results of which showed a novel compound heterozygous pattern for two variant copies of the G6PC3 gene, with our patient inheriting a copy from each parent.

Discussion: The G6PC3 gene encodes the expressed glucose-6-phosphate enzyme which catalyzes the final step in glycogenolysis. It is hypothesized that this deficiency causes unregulated levels of glucose, resulting in increased stress of the endoplasmic reticulum leading to apoptosis of neutrophils. Pathogenic variants of G6PC3 cause autosomal recessive G6PC3 deficiency and this can be clinically characterized as severe congenital neutropenia. Classic G6PC3 deficiency includes severe congenital neutropenia as well as cardiovascular abnormalities, urogenital abnormalities, and pulmonary hypertension. This novel pathogenic variant is likely responsible for the cyclic neutropenia observed in our patient. G6PC3 variants are an important differential diagnosis in the work-up of other causes of neutropenia. Whole Exome Sequencing is a cost-effective method for diagnosis and a valuable tool in evaluation and management of complex hematologic disorders.

Disclosures

No relevant conflicts of interest to declare.
Erythropoiesis Stimulating Agents (ESAS) in Supportive Care of Low-Risk Myelodysplastic Syndromes: Recamds Registry FINAL Results
Authors: Cerchione Claudio , Alfinito Fiorella , Vitagliano Orsola , Soriente Ilaria , Danise Paolo , Cerciello Giuseppe , Pane Fabrizio , Martinelli Giovanni
Published: November 2021

Erythropoiesis stimulating agents (ESAs) are the frontline treatment in low-risk anemic MDS patients and an employment of this therapy in the earlier stage of the disease can delay the need for RBC transfusion, hypothetically by slowing the disease course. It's matter of debate whether the clinical response is a result of proliferation and maturation of the dysplastic clone or stimulation of residual normal erythropoiesis by ESAs.

Macrocytosis is one of the cytological hallmarks of dyserithropoiesis in MDS: an analysis of the erythropoietic response to ESAs therapy in a cohort of anemic non trasfusion-dependent MDS patients, enrolled in a retrospective register, RECAMDS, subgroup of Italian register, was performed.

183 patients, treated with standard-dose ESAs, have been retrospectively analyzed. Data analysis was performed, according to IWG 2006 criteria, at the baseline, after 3 and 6 months of continuous treatment, with a subanalysis of the patients according to WHO and R-IPSS risk stratification. ESAs were started at mean Hb concentration of 9.31 g/dl, mean serum EPO concentration: 51 mU/L, after a mean time from diagnosis of 6 months (r.1-118).

ORR was 83.6% (153/183), no difference among WHO and IPSS subgroups was found: 132/183 (72.1%) achieved response after 3 months of treatment, while other 21/183 (11.2%) after 6 months. 19 patients with stable disease

(non-responders, according to IWG criteria), in which treatment was continued, achieved response after 9 months. In the macrocytic-responders group 83.2% exhibits again macrocytosis after 3 months, while 16.8% become normocytic. In the normocytic-responders group 89.8% exhibits again normocytosis, while 10.2% become macrocytic: in these patients, after 3 months, there was a contemporary worsening in neutropenia and thrombocytopenia, with transfusion-dependence, regarded as first signs of progression of disease. Non-responders were 30/183 (16.3%): in the macrocytic non-responders group 89% exhibit again macrocytosis after 3 months, while 11% become normocytic; in the normocytic group 76% exhibits again macrocytosis, while 24% become normocytic.

These preliminary data can suggest that, in the majority of MDS patients responsive to ESAs, the increase of Hb concentration occurs mainly stimulating erythroid production in MDS clones; in the minority of patients probably it happens recruiting residual polyclonal erythropoiesis.

It is interesting to note that stimulating effects of ESAs last even when the expression of dysplasia progresses.

Figure 1 Figure 1.

Disclosures

Martinelli: Roche: Consultancy; Stemline Therapeutics: Consultancy; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Incyte: Consultancy; Daichii Sankyo: Consultancy; Astellas: Consultancy, Speakers Bureau.
Identification of Pre-Gmps and Pre-Meps: Two Novel Types of Committed Progenitor Cells in Bone Marrow
Authors: Mack Ryan , Zhang Lei , Joshi Kanak , Liu Shanhui , Sellin Mark , Wei Wei , Breslin Peter , Zhang Jiwang
Published: November 2021

Elucidating the stepwise differentiation processes that leads from multipotent hematopoietic stem cells to mature effector cells is critical for understanding both normal and neoplastic hematopoiesis. Early studies suggested that common myeloid progenitors (CMPs) are oligo-lineage hematopoietic progenitors that produce all lineages of myeloid cells, including granulocytes, monocytes, erythrocytes and megakaryocytes. CMPs do so by first giving rise to megakaryocyte-erythroid progenitors (MEPs) and granulocyte-monocyte progenitors (GMPs), two types of bi-lineage progenitors. However, this concept was challenged by several recent studies where single cell techniques demonstrated that CMPs, GMPs and MEPs are highly heterogenic. The existence of lineage-restricted subsets within the CMP population leads to questions about whether erythroid and megakaryocytic lineage commitment is actually initiated at the multipotent progenitor or CMP stage. During the past 15 years, several lineage-restricted subsets of progenitors have been separated out from CMP population, including monocyte-dendritic progenitors, megakaryocyte progenitors, and erythroid progenitors based on expression of CD115/Flt3, CD41/CD150, and CD105/CD150, respectively. However, the remaining CMP population is still highly heterogenic. Thus, further separation of functional subsets within the CMP compartment is required.

By screening cell surface markers that can further separate CMPs, we have identified CD27 as a reliable marker to separate all megakaryocyte/erythrocyte-committed progenitors from granulocyte/monocyte-committed progenitors. In addition, we found that CD62L is only expressed on granulocyte/monocyte-committed progenitors. CD27 and CD62L co-staining can separate CMP into CD27 +CD62L +, CD27 +CD62L - and CD27 -CD62L - subsets. Biology and morphology study showed that CD27 +CD62L - cells are closely associated with GMPs, whereas CD27 -CD62L - cells are closely associated with MEPs. In vitro culture and in vivo transplantation functional studies demonstrated that 1) CD27 +CD62L + cells are pre-GMPs that give rise to FcGRII/III + GMPs and only produce granulocytes and monocytes; 2) CD27 -CD62L - cells are pre-MEPs that give rise to MEPs and primarily produce erythrocytes and megakaryocytes with minimal contribution to granulocytes and monocytes; 3) CD27 +CD62L - subset enriches cells with genuine CMP potential capable of producing GMPs, MEPs, and subsequent progeny. Taken together, we have identified two novel populations of committed progenitors that serve as intermediates between CMP-GMP and CMP-MEP commitment pathways. Identification of pre-GMPs and pre-MEPs fills in the gap between CMPs-GMPs and CMPs-MEPs, supporting the hierarchal relationship of myeloid lineage differentiation.

Disclosures

No relevant conflicts of interest to declare.
Dietary Iron Increases Expression of Liver Hepcidin Relative to BMP6 By a Mechanism Involving Transferrin Receptor 2 and Specificity of Transferrin Lobe Iron Occupancy
Authors: Parrow Nermi , Ali Faris , George Nisha , Ginzburg Yelena , Rivella Stefano , Fleming Robert E
Published: November 2021

Introduction: Prior studies have identified two iron signals regulating liver hepcidin expression (PMID 21488083, 21480335). One is mediated by liver sinusoidal BMP6 (and BMP2) and is reflective of liver iron concentration (LIC). The other is independent of BMP expression and reflective of transferrin saturation. The mechanism by which TF regulates hepcidin is unclear but appears to involve hepatocellular transferrin receptor 2 (TFR2). We previously generated mice with TF mutations that block iron binding to either N-lobe (Tf N-bl) or C-lobe (Tf C-bl). These mice demonstrated differences in Epo sensitivity and hepcidin regulation (PMID 31434707). To characterize the differential regulation of Hamp1 in these mice we analyzed the effect of dietary iron in juvenile mice. This model advantages a physiologic low iron state with very low basal Hamp1 and Bmp6 expression.

Methods: Twelve-day old WT, Tf N-bl, Tf C-bland Tfr2 Y245X (functional knockout) mice were gavaged with 4 mg/kg/d FeSO 4 (or saline) in 2 divided doses for 3 days and sacrificed on the fourth day. The primary outcome was liver Hamp1 relative to Bmp6 expression by RT-PCR. Other iron-related parameters included liver Bmp2, serum and tissue non-heme iron, and TIBC. Erythroid parameters included splenic Fam132b (Erfe gene) expression, kidney Epo, blood packed cell volume (PCV), splenic weight index (SWI).

Results: Iron-treated mice demonstrated increased serum iron, TF saturation, LIC, liver Bmp6 in all strains compared to untreated. Liver Hamp1 was increased in all strains with iron-treatment; however, the magnitude was least in the Tf N-bland greatest in the Tf C-bl mice. Splenic Fam132b was unchanged. The iron-treated Tf C-bl had ~16 fold greater Hamp1 compared with iron-treated Tf N-bl mice (P=0.0005), despite similar Bmp6, Bmp2, and splenic Fam132b expression. The iron-treated Tf C-bl mice also had modestly higher splenic index and PCV compared with iron-treated Tf N-bl mice, despite similar kidney Epo. In Tfr2 Y245X mice Hamp1 and Hamp1 indexed to Bmp6 expression was intermediate between Tf N-bland Tf C-bl mice (figure).

Conclusion: Iron treatment increased liver Bmp6 expression as well as Bmp6 sensitivity (Hamp:Bmp6), in association with increases in LIC and in TF saturation. The effect of iron on Bmp6 sensitivity is markedly influenced by which TF lobe iron occupies, as well as the presence of TFR2. These observations support a model in which TF modulates the hepcidin response to BMP6 in either direction, via interaction with TFR2. We speculate that iron occupancy of the N-lobe of transferrin confers an upregulatory signal to hepcidin expression in the hepatocyte, and that its absence confers a downregulatory signal to BMP6 sensitivity in the hepatocyte.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Impairment of Annexin A2-Mediated Cell Surface Fibrinolysis in Nonalcoholic Steatohepatitis Cirrhosis with Portal Vein Thrombosis
Authors: Lim Hana I. , Fortune Brett , Desancho Maria T. , Hajjar Katherine A.
Published: November 2021

The annexin A2 (A2) complex assembles tissue plasminogen activator and plasminogen on cell surfaces, resulting in efficient plasmin generation, and is markedly reduced in some patients with thrombophilia. Nonalcoholic steatosis (NASH) cirrhosis is the leading cause of cirrhosis in developed countries, and patients with NASH cirrhosis are at increased risk of portal vein thrombosis (PVT). However, the exact mechanism for PVT in NASH cirrhosis is poorly understood. Our objective was to determine the potential contribution of A2 to NASH thrombogenesis.

Using semiquantitative immunoblot analysis, we examined A2 expression in peripheral blood mononuclear cell (PBMC) lysates from obese controls and NASH subjects with or without cirrhosis, and in human umbilical vein endothelial cells (HUVECs) treated with platelet-poor plasma (PPP) from patients with varying degrees of NASH cirrhosis. We also analyzed formalin-fixed liver tissue from patients with or without steatosis or NASH cirrhosis for A2 expression by immunofluorescence. Finally, we evaluated A2-dependent PBMC surface plasmin generation using a fluorogenic assay, and systemic fluid-phase fibrinolysis via plasmin-anti-antiplasmin (PAP) complex and D-dimer ELISAs.

Total A2 expression in PBMC lysates did not differ among subjects with a range of severity of NASH cirrhosis versus obese controls. Src kinase-dependent tyrosine phosphorylation of A2, which is critical for its translocation to the cell surface, decreased in HUVECs after co-culture with NASH Child-Turcotte-Pugh C PPP despite no change in total A2. Immunofluorescence of liver tissue from patients with NASH cirrhosis who developed PVT revealed an 85% decrease in microvascular A2 expression compared to NASH cirrhosis patients without PVT. Similarly, PBMC surface plasmin generation decreased as NASH severity worsened, even though d-dimer and PAP complex level increased with worsening disease severity, indicating more activated systemic fibrinolysis in decompensated NASH cirrhosis.

Despite preservation of total A2 and acceleration of systemic fibrinolysis in NASH, A2-mediated cell surface fibrinolysis decreased as NASH cirrhosis worsened. We show for the first time that impaired A2-related fibrinolysis may reflect inhibition of A2 phosphorylation by NASH plasma, and that reduction of A2 expression in the hepatic microvasculature may contribute to PVT in these subjects.

Disclosures

Desancho: Sanofi-Genzyme: Membership on an entity's Board of Directors or advisory committees.
FGFR1 Fusion Genes Combine with RUNX1 Mutations to Drive Leukemogenesis
Authors: Wang Zheng , Wen Lijun , Chen Suning , Wu Depei
Published: November 2021

8p11 myeloproliferative syndrome is a myeloid tumor characterized by FGFR1 fusion gene. Its progression is fast, prognosis is bad and pathogenesis is not clear. We screened 10 cases of FGFR1 rearrangement (1 new fusion gene TFG-FGFR1), and 8 of them performed second generations of target exon capture and resequencing. The results showed that RUNX1 gene mutation occurred in 6 cases, with a mutation rate of 75%. The pretest results showed that FGFR1 fusion gene could significantly activate the downstream signaling pathway of FGFR1, and then promote cell proliferation, while RUNX1 gene mutation could block cell differentiation. It is presumed that FGFR1 gene fusion and RUNX1 mutation could lead to leukemia formation. Based on our previous work, we will explore the synergistic pathogenicity, the differences of variable opposite genes and possible mechanisms of FGFR1 fusion gene and RUNX1 mutation in vitro based on cell biology and animal models in vivo, and explore the therapeutic effect of targeted drugs in such diseases. This topic will help clarify the synergistic pathogenic effect of FGFR1 fusion gene and RUNX1 mutation and its related mechanisms, and provide theoretical and experimental evidence for the diagnosis and treatment of this disease.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.