bluuebunny/crossref_metadata_2025 huggingface dataset:
Remission in Patients with Acute Promyelocytic Leukemia Treated with Arsenic Trioxide (varitrinox) As the First Line in Colombia
Authors: Milone Gustavo , Sarmiento Doncel Samuel , Agudelo Rico Carol , Vizcarra Reyes Fabiola , Diaz Mosquera Gina Alejandra , Duque Ortega Juan Guillermo , Cortes Bernal Javier Mauricio , Meza Cadavid Francisco Javier
Published: November 2020

Acute promyelocytic leukemia (APL) is a subtype of Acute Myeloid Leukemia (AML) in which a chromosomal translocation t (15; 17) (q22; q12) is generated by fusing produces a hybrid PML / RARα gene, generating an altered signal . The combination of transretinoic acid (ATRA) plus arsenic trioxide (ATO) has been shown to be superior to ATRA plus chemotherapy in the treatment of newly diagnosed standard risk patients with acute promyelocytic leukemia (APL) in several countries.

The objective of the present study is to describe the frequency of remission in patients with acute promyelocytic leukemia who were administered as a first line Arsenic Trioxide (varitrinox) during the period from November 2017 to June 2020 in Colombian patients.

Methods: Retrospective observational and descriptive study of 12 patients diagnosed with acute promyelocytic leukemia treated with ATO Arsenic trioxide (Varitrinox) as first line, the source of information was provided by the treating hematologists (medical records) by filling out the technical concept format. Active pharmacovigilance scientist in Colombia, this format keeps the identification information of the patient anonymized and the confidentiality of the data is guaranteed as well as compliance with the rules of good clinical practice.

Results: Twelve patients with age range between 22 and 69 years with a median age of 34.0 were analyzed. It was found in the analysis that 100% had induction hematologic remission with a median of 45 days. 75% of patients received ATO + ATRA and were at low and intermediate risk, the remaining 25% received ATRA + ATO + Chemotherapy and were at high risk, and intermediate risk. 91.7% of molecular remission in consolidation was obtained and it was measured in cycle 3 by means of PCR (undetectable), 8.3% (n = 1) was positive 3% and is finishing consolidation. Regarding the most frequent adverse events, intravascular coagulation (n = 9), neutropenia (n = 6) and thrombocytopenia (n = 6) were observed. 75% of patients are disease-free, 16.7% are on maintenance (they received ATO + ATRA + Induction chemotherapy) and 8.3% are on consolidation. So far, none of the patients under study have died.

Conclusions: Our results support the use of ATO (Varitrinox) in newly diagnosed APL patients (as first line), as a care strategy for low, intermediate and high risk patients. The role of ATRA-ATO is guaranteed in other studies where they manage patients of different risks.

Key words: Arsenic trioxide, leukemia promyelocytic acute, leukemia myeloid acute, remission induction, tretinoin.

Disclosures

No relevant conflicts of interest to declare.
Tet2 Inactivation Enhances the Anti-Tumor Activity of Tumor-Infiltrating Lymphocytes (TILs) to Curtail Melanoma Growth
Authors: Lee Minjung , Li Jianfang , Fang Shaohai , Zhang Joanna , Vo Anh Tran Tram , Han Wei , Zeng Hongxiang , Isgandarova Sevinj , Moczygemba Margarita Martinez , Zhou Yubin , Li Jia , Sun Deqiang , Huang Yun
Published: November 2020

Inactivation of tumor infiltrating lymphocytes (TILs) is one of the mechanisms mitigating anti-tumor immunity during tumor onset and progression. Epigenetic abnormalities are regarded as a major culprit contributing to the dysfunction of TILs within tumor microenvironments. In this study, we used a murine model of melanoma to discover that Tet2 inactivation significantly enhances the anti-tumor activity of TILs, with the efficacy comparable to immune checkpoint inhibition imposed by anti-PD-L1 treatment. Single-cell RNA-seq analysis further revealed that Tet2-deficient TILs exhibit effector-like features. Transcriptomic and ATAC-seq analysis further demonstrated that Tet2 deletion reshapes the chromatin accessibility and favors the binding of transcription factors geared toward CD8+ T cell activation. In summary, our study establishes that Tet2 constitutes one of the epigenetic barriers contributing to dysfunction of TILs, and that Tet2 inactivation could benefit anti-tumor immunity to boost tumor suppression.

Disclosures

No relevant conflicts of interest to declare.
A Retrospective Analysis of Brentuximab Vedotin and Pembrolizumab Combination As Salvage Treatment for Patients with Relapsed or Refractory Hodgkin Lymphoma
Authors: Massaro Fulvio , Meuleman Nathalie , Bron Dominique , Maerevoet Marie
Published: November 2020

Background:The standard treatment for Hodgkin lymphoma (HL) patients presenting a relapsed/refractory (R/R) disease is salvage chemotherapy followed by autologous stem cell transplantation (ASCT). However, with commonly used chemotherapy combinations (such as DHAP, ICE, BEGEV), 25-30% of these patients fail to achieve a complete metabolic response (CMR) and to proceed to ASCT, with subsequently poor outcomes. Single agents brentuximab vedotin (BV) and pembrolizumab have shown efficacy in heavily pretreated HL patients, as reported in previous studies. The combination of BV with nivolumab has been explored in a phase 1/2 study as first salvage treatment for HL patients, showing a CR rate of 61% (Herrera et al 2018). We explored the outcome of BV and pembrolizumab combination as salvage treatment in a series of R/R HL patients.

Methods:We retrospectively collected data of seven HL patients presenting with a high-risk multi-refractory disease (two or more prior treatments) followed at Jules Bordet Institute between May 2019 and July 2020 and treated with a combination of BV and pembrolizumab. Patients were covered by special insurance conditions allowing treatment reimbursement. Treatment proposal was approved by a local multidisciplinary committee. Treatment consisted of 3-week cycles with a combination of BV (1.8 mg/kg IV) and pembrolizumab (200 mg IV fixed dose), an early PET-CT evaluation followed by high-dose chemotherapy and ASCT consolidation for those achieving a Deauville score ≤4. After ASCT, patients received BV as maintenance for a total of 16 administration (including pre-ASCT cycles).

Results:We included 7 patients, 6 male and 1 female, with a mean age of 27.4 (20.6-36.3) years. All patients presented with an advanced-stage disease, with a mean number of 2.56 (2-3) prior treatments. The median follow-up time was 9.7 months, while the average number of received cycles of treatment was 4.2 (range 2-7). Table 1 shows additional patients' characteristics. The overall response rate (ORR) was 87% and particularly a CR was achieved in five patients (72%), with a median time to response of two cycles. One partial response and one disease progression were also observed. Six patients proceeded to ASCT (86%) and subsequent BV maintenance, with one early disease progression. One patient, in complete molecular remission after 2 cycles, locally progressed after 6 cycles and received radiotherapy before ASCT.

Table 2 summarizes the outcome for each patient included in the series.

Conclusions:The BV and pembrolizumab combination is a very effective bridge treatment to ASCT for high-risk R/R HL patients.

Disclosures

No relevant conflicts of interest to declare.
Prophylactic Donor Lymphocyte Infusion Based on Two Different Time for Preventing Relapse in Advanced Acute Leukemia Undergoing Allo-HSCT: Comparison of Two Independent Prospective Cohorts
Authors: Su Qiongqiong , Fan Zhiping , Huang Fen , Xu Na , Nie Danian , Lin Dongjun , Guo Ziwen , Pengcheng Shi , Wang Zhixiang , Jiang Ling , Sun Jing , Jiang Zujun , Liu Qifa , Xuan Li
Published: November 2020

Background: Prophylactic donor lymphocyte infusion (pDLI) can reduce relapse in advanced acute leukemia (AL) undergoing allogeneic hematopoitic stem cell transplantation (allo-HSCT), but the optimal timing of pDLI remains uncertain. Our previous study demonstrated that pDLI on day +60 post-transplant regardless of minimal residual disease (MRD) could reduce relapse, but the incidence and mortality of graft-versus-host disease (GVHD) were high. To reduce GVHD post-pDLI, we modified our pDLI strategy by delaying the time to day +90 unless MRD was positive on day +60.

Methods: This study population came from two prospective multicenter cohorts. In cohort 1, pDLI was given once on day +60 regardless of MRD for all patients without grade II/>II acute GVHD (aGVHD), and then administered based on MRD and GVHD. In cohort 2, for patients who were MRD negative on day +60 and did not experience grade II/>II aGVHD by day +90, pDLI was given once on day +90 and then administered based on MRD and GVHD. For patients with positive MRD on day +60, the application of pDLI was conducted according to cohort 1. The outcomes of pDLI at two different time were compared.

Results: A total of 161 advanced AL undergoing allo-HSCT from January 2012 to December 2017 were enrolled, including 83 from January 2012 to December 2014 in cohort 1 (74 receiving pDLI) and 78 from January 2015 to December 2017 in cohort 2 (69 receiving pDLI). The extensive chronic GVHD (cGVHD) incidence in cohort 2 was lower than that in cohort 1 (10.3% vs 27.9%, P=0.006) and GVHD-free/relapse-free survival (GRFS) in cohort 2 was superior to that in cohort 1 (55.1% vs 41.0%, P=0.042). The 2-year relapse rate, overall and leukemia-free survival were similar between the two cohorts (29.0% vs 28.2%, P=0.986; 63.9% vs 64.1%, P=0.863; 57.8% vs 61.5%, P=0.666). Multivariate analysis revealed that modified pDLI strategy was the only protective factor for GRFS (P=0.015, HR=0.470).

Conclusions:

Delaying pDLI to day +90 based on MRD for advanced AL undergoing allo-HSCT can lower extensive cGVHD incidence and improve GRFS but not increase leukemia relapse.

Disclosures

No relevant conflicts of interest to declare.
DISC-0974, a Novel, First-in-Class, Anti-Hemojuvelin Monoclonal Antibody Is Predicted to be Efficacious at Low Doses in Humans As Determined By Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling in Non-Clinical Studies
Authors: Hixon Mark , Blaustein Cecile , King Christopher , Nguyen Sophia , Savage Will , Beconi Maria G.
Published: November 2020

DISC-0974 is a humanized monoclonal anti-HJV antibody with high binding affinity for human, rat and cynomolgus monkey HJV (100 pM, 240 pM and 110 pM Kd, respectively) [Kovac 2016]. DISC-0974 inhibits the interaction between HJV and the key hepcidin regulatory ligands, bone morphogenetic proteins (BMPs), leading to decreased SMAD phosphorylation (SMAD-P) and reduced hepcidin expression [Kovak 2016].

Elevated hepcidin is a hallmark of and plays a critical role in the pathogenesis of anemia of inflammation (and other forms of anemia) by reducing transferrin saturation and erythroid iron availability, leading to failure of erythropoiesis and decreased hemoglobin synthesis. DISC-0974 is being developed to reduce hepcidin synthesis and treat anemia in these patients. A critical early signal of therapeutic effect is the effect of DISC-0974 on transferrin saturation (TSAT). TSAT has therefore been used as the primary pharmacodynamic marker in a series of non-clinical studies to determine the relationship between drug exposure and pharmacological activity.

In non-human primates (NHP) DISC-0974 elicits a dose-dependent reduction in hepcidin levels leading to increases in serum iron and consequently TSAT. PK/PD responses were examined over 60 days for a single 6 mg/kg intravenous dose of DISC-0974 in three male NHPs and in repeat dose studies occurring at 14 day intervals administered 3 times (vehicle, 0.6 and 3 mg/kg) or two times (60 mg/kg). In these studies, the serum iron response saturated in an exposure-dependent manner over the 0.6-60 mg/kg dose range. The onset of iron elevation lagged compared to the DISC-0974 maximal plasma concentrations by 1- 4 days post dose and persisted beyond what could be predicted by DISC-0974 plasma concentration alone. The observed PD responses were well-described by a delayed response PK/PD model of serum iron which was fit globally to all studies.

We will present single and multiple dose simulations showing that low monthly doses of DISC-0974 are sufficient to elicit a biologically significant and controlled PD modulation (serum iron, TSAT, hepcidin) over a sustained period-of-time (Figure 1). DISC-0974 is under development for the treatment of anemia of inflammation associated with high hepcidin.

Disclosures

Blaustein: Disc Medicine:Current Employment, Current equity holder in private company.King:Disc Medicine:Current Employment, Current equity holder in private company.Nguyen:Disc Medicine:Current Employment, Current equity holder in private company.Savage:Disc Medicine:Current Employment, Current equity holder in private company.Beconi:Disc Medicine:Current Employment, Current equity holder in private company.
Effective Erythropoiesis from Human iPSC-Derived RBC in Immunodeficient Mice
Authors: Deng Jiusheng , Lancelot Moira M. , Jajosky Ryan , Deeb Kristin , Saakadze Natia , Gao Yongixng , Liu Senquan , Stowell Sean R. , Cheng Linzhao , Roback John D
Published: November 2020

Transfusion of red blood cells (RBCs) was the earliest developed form of cell therapy and is still a highly effective life-saving treatment for many patients. Induced pluripotent stem cells (iPSC) can differentiate into RBCs (iPSC-RBCs) and may provide a novel source for blood transfusion and a cellular model for erythroid differentiation. Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. Human iPSC were generated from peripheral blood mononuclear cells of healthy donors by transfection of plasmids containing OCT4, SOX2, MYC, KLF4 and BCL-XL genes. iPSC lines expressed TRA-1-60, SSEA4 and Nanog markers, and showed a normal karyotype. iPSCs were induced to differentiate along the erythroid lineage using a 3-stage culture system requiring 33 days. At the end of the culture period, iPSC-RBCs were CD34-CD235a+CD41+CD43+CD71low; about 10% of cells were enucleated (CD235a+DRAQ5-). iPSC-RBCs were harvested and transfused into immunodeficient NSG mice which had been pretreated with clodronate liposomes and cobra venom factor (CL/CVF). CL/CVF treatment of NSG mice markedly promoted the survival of transfused human iPSC-RBC in vivo, which could be detected with anti-human CD235a antibodies for at least 7 days, although the numbers progressively decreased with time. Interestingly, a large number of transfused iPSC-derived cells homed to bone marrow of NSG mice. In NSG mice that were repetitively treated with CL/CVF every 3 days, nucleated iPSC-derived cells were still detectable in the bone marrow 4 weeks after transfusion. Furthermore, at 3 weeks after transfusion, human iPSC-RBCs reappeared in the peripheral circulation. These circulating iPSC-RBCs were > 90% enucleated and were present at levels more than 4-fold higher than at 1 hour after transfusion. These results suggest that iPSC-RBCs which homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into the peripheral blood. The results offer a new model using human peripheral blood iPSC and CL/CVF-treated NSG mice to investigate the development of human erythroid cells in vivo.

Disclosures

Jajosky: Biconcavity Inc.: Other: CEO and partial owner; BioMarin Pharmaceuticals: Current equity holder in publicly-traded company; Magenta Therapeutics: Current equity holder in publicly-traded company.
Major Infections after Blood Transfusion in Isolated Coronary Artery Bypass Surgery Patients: A Meta-Analysis
Authors: Mosalem Osama , Kherallah Shouq , Mujer Mark , Agarwal Priyal , Treasa Varghese Merryl , Elshafie Ahmed , Garces Christopher C , Abdelsamia Mahmoud , Rayamajhi Supratik
Published: November 2020

BACKGROUND

Blood transfusion in patients undergoing coronary artery bypass surgery (CABG) remains high despite advances in perioperative blood conservation. Several studies have shown poorer clinical outcomes in isolated CABG patients who receive blood products, including an increased rate of renal failure, non-fatal myocardial infarction, and death. Our study sought to determine the impact of blood transfusion on sternal wound infections and other major infections in patients who undergo isolated CABG.

METHODS

We performed a systematic search using PubMed, Google Scholar, EMBASE, SCOPUS, and ClinicalTrials.gov without language restriction until May 15, 2020. A random-effects model was utilized to calculate risk ratio (RR) and mean difference (MD) with 95% confidence interval (CI).

RESULTS

In patients undergoing isolated CABG, sternal wound infections [RR 1.73 (95% CI: 1.35 to 2.20; p<0.0001)] and overall major infections [RR 1.70 (95% CI: 1.39 to 2.08; p<0.0001)] were significantly higher in those who received a periprocedural blood transfusion. Thirty-day mortality was lower in patients who did not receive any transfusion [RR 2.46 (95% CI: 1.09-5.56;p=0.03)]. The test of heterogeneity was low for clinical outcomes aside from 30-day mortality.

CONCLUSION

Periprocedural blood transfusion is associated with an increased risk of sternal wound infections and major infections and higher 30-day mortality in patients who undergo isolated CABG

Disclosures

No relevant conflicts of interest to declare.
Autoimmune Hemolytic Anemia As Initial Presentation of COVID-19 Infection
Authors: Campos-Cabrera Gregorio , Mendez-Garcia Esther , Mora-Torres Maria , Campos-Cabrera Salvador , Campos-Cabrera Virgina , Garcia-Rubio Gabriel , Campos-Villagomez Jose-Luis
Published: November 2020

Introduction: the spectrum of autoimmune complications in patients with SARS-COV2 infection (also known as COVID-19) is broader and includes autoimmune thrombocytopenia and antiphospholipid syndrome among others. (Autoimmunity Reviews 19 (2020) 102597) The reports of autoimmune hemolytic anemia (AIHA) are increasing too, but mainly is an immune dysregulation setting.

Cases: here we report two cases of AIHA in a non immune dysregulation setting. Two females, 35 and 58 years old, presented to emergency service due severe anemic syndrome, severe pallor, dyspnea and mild fever. Work up was started and warm IgG - C3d autoantibodies AIHA was diagnosed. Extension studies included chest X-rays that showed basal infiltrates in both patients; lung CT scan were performed and revealed interstitial pneumonia predominantly in both lung bases. The PCR fo SARS-COV2 was positive for both patients. Treatment with methylprednisolone 1 gr per day for three days and IgG IV 1 gr/kg days 1 and 3. Treatment for COVID-19 was started according to institutional protocols. The evolution in both patients were to partial remission and needs prednisone 20 mg/day and still in it at the time of these report. Images show lung infiltrates and increased 56% reticulocytes count from one of the patients.

Conclusion: the exact etiology of autoimmune diseases still unknown, but there are various factors, including viral infections, that contributes to the trigger of these type of diseases. AIHA has been associated with COVID-19 and it is postulated that molecular mimicry between Ankyrin-1 in the erythrocyte surface and the viral protein spike as the precipitating event. (Br J Haematol 2020;190:e92-e93) There are scanty cases of AIHA in the setting of COVID-19, the majority in the previously immune dysregulated patients (lymphoid neoplasias and autoimmune diseases); only two previously cases of AIHA with no underlaying disorder. We suggest that the work up for every patient with AIHA would include a lung CT Scan and PCR for SARS-COV2. There are not guidelines for treatment, but the lowest dose and time of steroids with IgG IV could be an option as recommended in guidelines for primary immune thrombocytopenia in the setting of COVID-19 infection. (Br J Haematol 2020;189:1038-1043).

Figure

Disclosures

No relevant conflicts of interest to declare.
Multiple Myeloma Cell Defects Erythropoiesis and Results Anemia Via High Level of CCL3 in Bone Marrow Microenvironment
Authors: Liu Lanting , Yu Zhen , Cheng Hui , Sui Weiwei , Deng Shuhui , Wei Xiaojing , Du Chenxing , Huang Wenyang , An Gang , Zhou Wen , Cheng Tao , Qiu Lugui , Hao Mu
Published: November 2020

Background: Anemia is the most common complication of myeloma, and is associated with worse clinical outcomes including diminished quality of life, and increased risk of morbidity and mortality. Bone marrow (BM) is the main location of the growth and differentiation of HSPCs. Although marrow replacement by myeloma cell is widely considered a mechanistic rationale for myeloma-related anemia, however the molecular mechanism has not been fully understood.

Methods: The clinical characteristics and the prognostic impact of myeloma-related anemia were investigated in patients with multiple myeloma. The erythroid differentiation defects of HSPCs were examined by flow cytometry and colony-formation assay. RNA-seq were conducted to clarify the different gene expression and the signaling pathway in HSPCs. Relative protein levels were determined by immunoblotting and relative gene levels were determined by quantitative real-time PCR. 5TGM1 murine myeloma mouse model was utilized.

Results: The data of our large cohort of 1,363 myeloma patients demonstrated that 84.4% of patients had anemia (Hgb<120 g/L), more than 50% of patients had moderate (Hgb=90-120 g/L) or severe anemia (Hgb=60-90 g/L) at the time of diagnosis. Anemia positively correlated with myeloma cell infiltration in the BM and worse outcome of patient. Patient sample and mouse model indicated that the quantity and the erythroid differentiation of HSPCs were affected by myeloma cell infiltration. The number of HPCs was significantly declined in the BM of myeloma, and negatively correlated with the quantity of tumor cells. The master regulator of erythropoiesis GATA1 and KLF1, was obviously down-regulated in myeloma HSPCs cells. However, the gene of chemokine CCL3 was significantly upregulated. Elevated CCL3 in the BM plasma of myeloma further inhibited erythropoiesis of HSPCs through activation of CCL3/CCR1/p38 signaling, and suppressed the expression of GATA1. CCR1 antagonist treatment effectively inhibits CCL3 activity and recovered the expression of GATA1 and rescued the erythropoiesis of HSPCs.

Conclusions: myeloma cell infiltration causes the elevation of CCL3 in microenvironment that suppresses the erythropoiesis of HSPCs and results in anemia by downregulating the level of GATA1 of HSPCs. Thus, our study indicates targeting CCL3 would be a potential strategy against anemia and improvement the survival of myeloma patient.

Disclosures

No relevant conflicts of interest to declare.
Ruxolitinib during Allogeneic Stem Cell Transplantation (allo-SCT) in Primary Myelofibrosis (PMF) Recommendation
Authors: Tan Yaxian , Luo Jie , Zhou Xuan , Liu Xiaoli , Liu Qifa , Xu Na
Published: November 2020

Introduction: Ruxolitinib is the first approved drug for treatment of myelofibrosis ,and also can improve survival outcomes by managing graft-versus-host disease. The aim of this work is to produce Ruxolitinib treatment recommendations during allogeneic stem cell transplantation (allo-SCT) in primary myelofibrosis (PMF).

Methods: A total of 13 patients with intermediate or high-risk myelofibrosis who received allogeneic hematopoietic stem cell transplantation from June 2017 to October 2019 at the Department of Hematology, Southern Hospital were selected.All patients were given a maximum tolerated dose of ruxolitinib for at least 2 months before transplantation, ruxolitinib was discontinued at a median of 7 days (around the start of conditioning) pre-HCT.Decitabine combined with BF conditioning protocol, CSA+MTX+ATG(7.5mg/kg,if Haploid donor)+ruxolitinib (5mg bid ;+6 to +60d) was used to prevent GVHD.Time to hematopoietic reconstruction, rate of improvement in myelofibrosis, change in spleen size, incidence of GVHD, and patient outcomes were observed.

Results: ⑴The median age of the 13 patients was 55 (21-57) years, 10 of whom were male and 3 female. By the MIPSS70 score scale ,11 patients were in the high-risk group and 2 were in the intermediate-risk group.Nine patients carried driver mutations and 3 were triple-negative.All patients received peripheral blood and bone marrow grafts, from a matched sibling donor in 3 patients and a haploidentical-related donor in 10 patient. (2) In 13 patients who received ruxolitinib bridging allogeneic hematopoietic stem cell transplantation,All patients engrafted neutrophils and platelet recovery. The median time to neutrophil engraftment was 12 (10-18) days,the median time to platelet engraftment was 20 (12-35) days; 7 patients developed acute GVHD (6 patients had grade I°and 1 patients had grade 2°) and the median time to acute GVHD was 46 (21-100) days. 6 patients developed chronic GVHD (5 patients were limited and 1 patient was extensive). (3) Three months after transplantation, spleen size improved in all eight patients with splenomegaly; myelofibrosis was stable or improved in all patients and four patients improved to grade 0 . (4) Follow up to July 2020, the non-relapse mortality rate(NRM) for all patients was 0, one patient had a positive MRD conversion and the relapse-free survival rate(RFS) was 92.31%.

Conclusions: Single-center, small-sample data suggest that the use of Decitabine combined with BF conditioning protocol ,and peri-transplant administration of ruxolitinib at 5mg bid dose is safe in myelofibrosis patients, no high relapse incidence founded.

Keywords: myelofibrosis, ruxolitinib, during allogeneic hematopoietic stem cell transplantation;

Disclosures

No relevant conflicts of interest to declare.
Genome-Wide Association Study Identifies Variation in ABO As Risk Factor for Platelet Reactivity in Heparin-Induced Thrombocytopenia
Authors: Giles Jason B , Rollin Jérôme , Shaffer Christian M , Steiner Heidi E , Momozawa Yukihide , Stanaway Ian , Mushiroda Taisei , Pouplard Claire , Heddle Nancy , Kubo Michiaki , Phillips Elizabeth , Warkentin Theodore E. , Gruel Yves , Greinacher Andreas , Roden Dan , Karnes Jason
Published: November 2020

Background: Heparin-induced thrombocytopenia (HIT) is an unpredictable, potentially catastrophic adverse effect of heparin treatment resulting from an immune response to platelet factor 4 (PF4)/heparin complexes. Genome-wide association studies (GWAS) have the potential to identify genetic risk factors and generate insights into biological mechanisms of adverse drug reactions. However, published HIT GWAS are underpowered and do not include confirmation of HIT using functional assays. We aimed to identify genetic associations with HIT using a GWAS approach in two European ancestry populations tested for functional assay and PF4/heparin antibody levels.

Methods: We performed a GWAS with positive functional assay as the outcome in a large discovery cohort of patients, including heparin-induced platelet aggregation (HIPA) positive cases (n=1243), antibody positive but functional assay negative controls (n=1091), and antibody negative controls (n=1718). Logistic regressions were adjusted for age, gender, and first two principal components. Significant associations from the discovery cohort (alpha=5.00x10-8) were then investigated in a replication cohort of serotonin-release assay (SRA)-confirmed HIT cases (n=173), antibody positive controls (n=125), and antibody negative controls (n=488). All patients were genotyped with the HumanOmniExpressExome BeadChip, including 9,065,510 variants after imputation and quality control. Genes near significantly associated loci were resequenced.

Results: In the discovery cohort, a genome-wide significant association was observed between common variation in the ABO gene and HIPA positive status (rs505922 odds ratio 0.738 [0.668-0.816], p=2.813x10-9). This variant was also significantly associated with SRA-confirmed HIT in the replication cohort (odds ratio 0.392 [0.172-0.891], p=0.025). Meta-analysis indicated strong association of ABO variation with positive functional assay (Figure 1). Sequencing and fine-mapping of the ABO locus indicated this effect was driven by ABO blood groups, with the O blood group being a risk factor for HIT (odds ratio 1.42 [1.25-1.61], p=6.11x10-8).

Conclusions: We identify common variation in ABO as a risk factor for platelet reactivity and HIT. Strong association of ABO variation may have important implications for prediction and understanding of HIT pathogenesis.

Figure 1

Disclosures

No relevant conflicts of interest to declare.
Performance of Khorana Score to Predict One-Year Risk of Venous Thromboembolism in over Two Million Patients with Cancer
Authors: Arafah Yasmin , Al-Juhaishi Taha , Abu Zeinah Ghaith , Nayak Lalitha V. , Al-Kindi Sadeer
Published: November 2020

Background: Risk stratification of venous thromboembolism (VTE) in patients with cancer is essential to evaluate the need for extended thromboprophylaxis. The Khorana score is widely used to guide VTE risk stratification in patients with cancer based on clinical parameters: cancer type, platelet count, hemoglobin level, leukocyte count, and body mass index. We sought to validate Khorana score to predict 1-year risk of VTE in a large multi-institutional, diverse, real-world cohort of patients with cancer.

Methods: Using aggregated de-identified electronic medical record data from >300 major hospitals in the in the United States (IBM Watson Explorys), we performed a retrospective cohort study of all patients who had a diagnosis of cancer. We identified cancer type, platelet count, hemoglobin level, leukocyte count, and body mass index (for calculation of Khorana score) within 7 days of cancer diagnosis and followed patients for diagnosis of VTE for 1 year post cancer diagnosis. Logistic regression models were used to evaluate association between patient characteristics and VTE risk. Receiver operating characteristics with area under the curve were used to evaluate model discrimination. All analyses were performed in Statistical Package for Social Sciences.

Results: A total of 2,112,260 patients with cancer were included: 52.9% females, 11.1% African Americans, 44.3% intermediate risk cancers (lymphoma, lung, breast, gynecologic, testicular and bladder), 4.5% high risk cancers (stomach and pancreas), 19.8% had Platelet counts ≥ 350,000/µL, 29% had hemoglobin <10 g/dL, 32.3% had leukocyte count >11,000/µL, and 15.1% had body mass index ≥ 35 kg/m2. Overall, 227,170 (10.8%) had a diagnosis of VTE at 1 year following cancer diagnosis. In multivariable analysis (table 1), VTE risk was associated with cancer type (intermediate vs low risk: OR 1.08 [95% CI: 1.07-1.09]; high vs low risk: OR 1.48 [95% CI: 1.45-1.51]), Platelet ≥ 350,000 (OR 1.27 [95% CI: 1.26-1.29]), hemoglobin <10 g/dL (OR 1.43 [95% CI: 1.41-1.44]), leukocyte count > 11,000 (OR 1.15 [95% CI: 1.14-1.16]), body mass index ≥ 35 kg/m2 (OR 1.29 [95% CI: 1.27-1.30]), table 1. Risk of VTE increased linearly with Khorana score between 7.7% (score = 0) to 18% (score = 6), figure 1. Khorana score had weak to modest discrimination (Area Under Receiver Operating Characteristics 0.565 [95% CI: 0.564-0.566]), figure 2.

Conclusion: In this very large real-world study of diverse patients with cancer, Khorana score was a weak predictor of 1-year risk of VTE, suggesting that VTE prediction in patients with cancer is very complex.

Disclosures

No relevant conflicts of interest to declare.
Myeloma Derived Mitochondrial Damage Associated Molecular Patterns Promote Pro-Tumoral Expansion By Inducing a Pro-Inflammatory Signature in the Bone Marrow Microenvironment
Authors: Jibril Aisha , Mistry Jayna J , Moore Jamie A , Hellmich Charlotte , Willimott Victoria , Bowles Kristian M , Rushworth Stuart A
Published: November 2020

Multiple Myeloma (MM) is a malignancy of antibody producing B cells (plasma cells), leading to the accumulation of tumour cells within the bone marrow (BM) microenvironment. Mitochondrial DNA (mtDNA) is a damage associated molecular pattern (DAMP), the mitochondrial genome contains islands of unmethylated CpG nucleotide motifs that have been shown to activate and promote memory B cell proliferation and antibody secretion.

Recent studies have indicated that mtDNA is elevated in the circulation of trauma and cancer patients and have highlighted the need to determine the functional purpose of elevated mtDNA within the BM microenvironment. Here we investigate the hypothesis that multiple myeloma cells secrete mtDNA and other mtDAMPs to induce a pro-inflammatory bone marrow microenvironment, supportive of the progression and survival of MM. As well as investigating how MM affects the haematopoietic stem cell niche through establishing a syngeneic myeloma mouse model.

We show that immunocompromised NSG mice transplanted with human MM cell lines released higher levels of MM derived mtDNA detected in the plasma by real-time PCR. Furthermore, in vitro experiments showed that pro-inflammatory cytokines IL-1B, IL-6, and IL-8 were elevated in both primary and cell line myeloma samples upon treatment with CpG oligonucleotides. Murine bone marrow derived macrophages (BMDMs) treated with mtDNA or mtDAMPs showed increased expression of pro-inflammatory cytokines. To understand the significance of the mtDAMP proinflammatory induction, C57BL/6 mice were successfully engrafted with murine 5TGM1 myeloma cell line to establish a syngeneic mouse model. Analysis of the haematopoietic stem and progenitor cell populations showed that 5TGM1 induced the expansion of these cell populations when compared to control WT animals. To understand the significance of mtDAMPs in regulating this HSPC expansion we used blocking antibodies to TLR9 (receptor for mtDNA) and FPR1 (receptor for formylated mitochondrial proteins) in 5TGM1 engrafted animals. Results show that myeloma tumour burden was reduced when FPR1 and TLR9 are blocked compared to control animals.

Here we highlight the involvement of mtDAMPs in MM to create a favourable proinflammatory microenvironment which provides the conditions for MM survival and proliferation. This data highlights the importance of mtDAMPs in MM disease progression and suggests targeting these activating receptor pathways may provide a novel therapeutic intervention.

Disclosures

Bowles: Janssen: Research Funding; AbbVie: Research Funding. Rushworth:Janssen: Research Funding; AbbVie: Research Funding.
NGS-Based Screening to Comprehensively Decipher TKIs Resistant Mutations in BCR-ABL1 Positive Leukemias
Authors: Liu Hongxing , Chen Jiaqi , Wang Fang , Fang Jiancheng , Teng Wen , Chen Xue , Zhang Yang , Ma Xiaoli , Cao Panxiang , Nie Daijing , Li Yu , Zhou Xiaosu
Published: November 2020

In BCR-ABL1 positive leukemia, mutations in the BCR-ABL1 kinase domain (KD) are the most common TKIs resistance mechanism. Sanger sequencing (SS) is currently the gold standard for detecting ABL1 KD mutations, but its low detection sensitivity cannot reveal mutations below 20% VAFs (variant allele frequency), and it is challenging to distinguish compound or polyclonal mutations. In recent years, the NGS-based BCR-ABL1 KD mutation screening protocol provides a powerful tool for deciphering complex patterns (compound or polyclonal mutations) and higher detection sensitivity (~2%, hotspot mutation 1%).

Based on an inhouse designed NGS-based BCR-ABL1 KD mutation screening protocol, we retrospectively analyzed 348 specimens that undergone SS (ALL, n=164, CML, n=128, CML-BP, n=33, AML, n=3, other, n=20, other includes MAL, B-LBL). The BCR-ABL1 transcripts of 109 cases were 0, 184 cases were >0.1%, and the remaining 55 cases were between 0-0.1%. NGS screening results showed that the average VAF was 32.81% (range, 1.0%-100%), which was not provided by SS.

Compared with SS, NGS screening provided a higher mutation-positive rate (22.13% vs. 33.05%, P =0.0013), and Gatekeeper T315I remained the most common BCR-ABL1 KD mutation (12.24% vs. 14.06%, P =0.4513). When the BCR-ABL1 transcript was 0, no mutation was detected in SS; while NGS ABL1-KD discovered mutations in 12 cases (0 vs. 11.01%, P =0.0004) with VAFs lower than 10% except for one S438F (VAF 14.9%) mutation,among them are the well-known F359V and E450G, as well as ten rare mutations, including T277A and E494K.

There were also significant differences in mutations between 0 and 0.1% in BCR-ABL1 transcripts (16.36% vs 32.73%, P =0.046). However, when the BCR-ABL1 transcript >0.1%, there was no difference in mutation-positive rate between the two groups (36.96% vs. 46.20%, P=0.072).

NGS screening disclosed more multiple mutations (59 vs 82, P =0.03), with significant advantages in detecting >3 mutations (3 vs 18, P = 0.0009). Compound mutations (CMs) were determined in 8 cases (8/384, 2.08%), with the incidence of 24.24% (8/33) in cases which carrying two or more mutations, of which T315I-including CMs were the most common (6/8, 75%).

CMs or polyclonal mutation analysis is particularly crucial for severe patients who have received multiple consecutive TKIs treatments, and those with multiple background mutations and a higher risk of progression to clinical resistance. Our results showed that the inhouse designed NGS-based screening protocols could decipher TKIs resistant mutations more comprehensively than SS(Fig 1), and worthy of being implicated in clinical practice.

Figure 1

Disclosures

No relevant conflicts of interest to declare.
Zrsr2 Deficient Zebrafish Display Hematopoietic Defects and U12-Type Intron Retention in mRNA Processing Genes
Authors: Weinstein Rachel N. , Broadbridge Elizabeth , Bishop Kevin , Carrington Blake , Yu Kai , Elkahloun Abdel G. , Wu WeiWei , Pei Wuhong , Burgess Shawn M. , Liu Paul P. , Bresciani Erica , Sood Raman
Published: November 2020

ZRSR2 is a small nuclear riboprotein necessary for assembly of the minor spliceosome and subsequent splicing of U12-type introns. It is involved in the recognition of 3' splice sites during the assembly of the spliceosome. Somatic mutations in ZRSR2 gene are present in hematopoietic malignancies, most notably myelodysplastic syndrome (MDS). These mutations are spread throughout the gene, indicating that its function is critical for hematopoiesis. In spite of these clinical associations, the role of ZRSR2 in hematopoiesis has not been well studied. Zebrafish make an excellent animal model for investigating this gene as all of the functional domains in the human protein are conserved with a high level of protein similarity in zebrafish. Additionally, hematopoietic development is well understood in this species. We used CRISPR/Cas9 to generate a zebrafish zrsr2 knockout model with an 11 base pair deletion (zrsr2Δ11) that results in frameshift with premature truncation and loss of all functional domains (p.W167fs*175). The zrsr2Δ11/Δ11 mutants began to appear morphologically different from their siblings by 4 days post fertilization (dpf), showing aberrant development in the mandible and pharyngeal arches. The zrsr2Δ11/Δ11 mutants developed mild to severe edema by 6 dpf and died by 8 dpf. To understand its role in hematopoiesis, we performed o-dianisidine staining and whole mount in situ hybridization (WISH) with lineage-specific markers during embryonic development. Our data showed that zrsr2Δ11/Δ11 embryos exhibit mild anemia by 2 dpf, suggesting that primitive hematopoiesis is defective. WISH showed that zrsr2Δ11/Δ11 mutants have normal early hematopoietic stem cell (HSC) emergence (c-myb) at 36 hours post fertilization. However, these cells are absent through 3 and 5 dpf. At 5 dpf zrsr2Δ11/Δ11 mutants had severely reduced expression of hematopoietic markers for erythroid (hbae1), lymphoid (rag1), and myeloid (mpo) lineages. This verifies that knockout of zrsr2 in zebrafish disrupted normal hematopoiesis and leads to cytopenias - emulating the symptoms of MDS. We then investigated the transcriptional and splicing changes underlying these phenotypes using RNA sequencing on zrsr2Δ11/Δ11 mutant embryos at 3dpf. We found a total of 285 intron retention events in zrsr2Δ11/Δ11 mutants as compared to the control embryos, with about 76% of those events occurring within U12-type introns. Genes with retained introns were associated with mRNA decay and destabilization. We also found that genes related to cell cycle arrest were upregulated in zrsr2Δ11/Δ11 mutants . These results indicate that aberrant mRNA splicing and cell cycle deregulation contribute to arrested hematopoiesis when zrsr2 is knocked out in zebrafish.

Disclosures

No relevant conflicts of interest to declare.
A Report of Fourcases of Severethrombocytopenia with Rh G-CSF Mobilized Allogeneic Peripheral Blood Stem Cell Transplant Donors
Authors: Liu Qingzhen , Yao Jianfeng , Yang Donglin , Pang Aiming , Jiang Erlie , Feng Sizhou , Han Ming-Zhe
Published: November 2020

Background and Objective: Granulocyte colony-stimulating factor (G-CSF) is used to mobilize progenitor cells into the peripheral bloodin healthy donors. Thrombocytopenia is one of common side effects of G-CSFmobilization, but seriousthrombocytopenia is rare. Herein,we report serious thrombocytopeniain 4 allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) healthy donors induced by recombinant human G-CSF (rhG-CSF) mobilization.

Methods: The donor received 10ug/kg/d G-CSF via subcutaneous injection.The blood parameterswere examined every day, Abdominal ultrasound detected the spleen size before, during, and after rhG-CSF mobilization.

Results: From April 2015 to May 2020, 1127 donors received hematopoietic stem cell mobilization, the percentage of serious thrombocytopenia in healthy donors was 0.35%.The count of white blood cell increased to the 4-7 timesbasic level of (35.4x109/L)after rhG-CSF mobilization 4 days, the lowestcount ofplatelet was 22x109/Lafter single postharvest. After mobilization, length of spleen increased 0.6-1.3 cm (median 10.1 cm), thickness ofspleenincreased 0-1.3 cm (median 3.4 cm). There were no obvious bleeding symptomsafter mobilization in four cases of donor.The quality of stem cell collection was relatively poor in 3 of the 4 donors, the number of mononuclear cells (MNC) was 7.47-18.58×108/kg, and the value of CD34+ was 0.82-2.42×106/kg (CD34+ proportion: 0.11-0.16%). The quality of stem cell collection in the other 1 donor was satisfactory, the number of MNC was 9.29-11.75×108/kg, and the proportion of CD34+ was 0.26~0.38%. Platelet count of 4 donors recovered to the basal level 3-10 days after drug withdrawal.

Conclusion:The severe thrombocytopenia caused by rhG-CSF mobilization healthy donor is very rare.while, the mechanismis unknown. Some studies suggested that it may be related to the insufficient expression of megakaryocytes proliferation-related genes, such as PF4 and PTFN4. Other studies indicated that rhG-CSF may aggravate the immune-mediated thrombocytopenia, which leads to hypersplenism and extensive activation of reticuloendothelial cells, thus increasing the consumption of platelets.In addition, the probability of severe thrombocytopenia was increased in allergic donors after rhG-CSF mobilization.In our report, although there was thrombocytopenia, mobilization was not affected.After the mobilization of rhG-CSF, platelets changes should be closely monitored to keep alert of donor bleeding tendency. While ensuring safe collection of hematopoietic stem cells by donors, patients should not be placed in extreme danger of not being able to receive allogeneic hematopoietic stem cells on time.

Key words:Severe thrombocytopenia;Healthy donor; rhG-CSF; Mobilization; Allogeneic peripheral blood stem cell transplantation.

Disclosures

No relevant conflicts of interest to declare.
Hyperglycemia Cooperates with Tet2 Heterozygosity to Induce Leukemia Driven By Pro-Inflammatory Cytokine Induced Lncrna Morrbid
Authors: Cai Zhigang , Kapur Reuben , Lu Xiaoyu , Zhang Chi , Nelanuthala Sai , Kotzin Jonathan , Williams Adam , Henao-Mejia Jorge , Aguilera Fabiola , Hadley Abigail , Fang Fang , Nephew Kenneth
Published: November 2020

Diabetes mellitus (DM) is a risk factor for cancer development. However, the role of DM induced hyperglycemic stress (HG) in the development of blood cancer is poorly understood, largely due to lack of appropriate animal models. Epidemiologic studies show that individuals with DM are more likely to possess higher rate of mutations in genes found in pre-leukemic stem and progenitor cells (pre-LHSC/Ps) including in the epigenetic regulator TET2. TET2-mutant pre-LHSC/Ps require additional hits to evolve into a full-blown leukemia and/or aggressive myeloproliferative neoplasm (MPN). Cell intrinsic mutations have been shown to cooperate with Tet2 to promote leukemic transformation. However, the role of extrinsic factors is poorly understood. Utilizing a novel mouse model bearing haploinsufficiency of Tet2, to mimic the human pre-LHSC/P condition and HG stress, in the form of an Ins2Akita/+mutation, which induces HG and Type-1 DM, we show that the compound mutant mice develop a lethal form of MPN and/or acute myeloid leukemia (AML). RNA-seq revealed that this is in part due to upregulation of pro-inflammatory pathways, thereby generating a feed-forward loop, including the expression of an anti-apoptotic lncRNA Morrbid. Loss of Morrbid in the compound mutants rescues the lethality and mitigates the development of MPN/AML. Our results describe a novel mouse model for age-dependent AML/MPN and suggest that HG stress acts as an environmental driver for myeloid neoplasm, which could be effectively prevented by reducing the expression of inflammation-related lncRNA Morrbid.

Disclosures

No relevant conflicts of interest to declare.
An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion
Authors: Paik Na Yoon , Brown Grace E. , Shao Lijian , Sottoriva Kilian , Hyun James , Khetani Salman R. , Pajcini Kostandin V.
Published: November 2020

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation.

While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC.

Disclosures

No relevant conflicts of interest to declare.