bluuebunny/crossref_metadata_2025_split huggingface dataset:
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&lt;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 &gt;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 &lt;10 g/dL, 32.3% had leukocyte count &gt;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 &lt;10 g/dL (OR 1.43 [95% CI: 1.41-1.44]), leukocyte count &gt; 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 &gt;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 &gt;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 &gt;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.