bluuebunny/crossref_metadata_2025_split huggingface dataset:
Probing Aberrant Splicing in a Novel Model of SF3B1-Mutant Myelodysplastic Syndromes
Authors: Clough Courtnee , Busch Stephanie , Pangallo Joey , Bradley Robert K. , Doulatov Sergei 
Published: November 2019

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is widely appreciated to be a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous mutations in a core U2 spliceosome factor SF3B1 are strongly associated with MDS with ring sideroblasts (MDS-RS). MDS-RS is characterized by iron retention in mitochondria and ineffective erythropoiesis. How aberrant splicing induced by mutant SF3B1 causes ring sideroblasts and erythroid defects remains poorly understood.
There are no genetically accurate models of MDS-RS that recapitulate ring sideroblast formation. Our lab has established an iPSC reprogramming approach to generate an in vitro model of MDS-RS. This method captures genetically distinct MDS subclones as well as normal cells in individual patients enabling internal normalization to the patient's isogenic normal clone. By differentiating iPSCs we generated inducibly immortalized MDS-RS CD34+ progenitor cell lines with robust expansion and erythroid differentiation potential enabling extensive analysis of SF3B1-mutant aberrant splicing throughout erythroid differentiation. We show that SF3B1-mutant lines form abundant ring sideroblasts compared to normal isogenic controls. We next performed RNA-sequencing and splicing analysis at different stages of erythroid differentiation. We show that global splicing patterns are maintained in SF3B1-mutant cells, while revealing distinct subsets of aberrantly spliced transcripts in CD34+ progenitors and erythroblast populations. This suggests that different aberrant splicing events contribute to erythroid progenitor expansion and inefficient erythropoiesis and iron dysregulation in MDS-RS.
Taken together, this novel iPSC model of MDS-RS is a robust tool for studying the role of aberrant splicing in SF3B1-mutant erythroid progenitor expansion, inefficient erythropoiesis and ring sideroblast formation.

Disclosures
No relevant conflicts of interest to declare.
Coexistence of a Huge Venous Thromboembolism and Bleeding Tendency in Cytokine Release Syndrome during CAR-T Therapy
Authors: Liu Haiyan , Yang Ye , Huang Hongming 
Published: November 2019

Chimeric antigen receptor (CAR)-modified T cell therapy is increasingly prescribed, given its growing status as one of the most promising treatments for hematological malignancies. Cytokine release syndrome (CRS) is a common and severe complication of CAR-T therapy that has raised concerns. Herein, we report the case of a 62-year-old male patient who was diagnosed with relapsed and refractory multiple myeloma (RRMM, Isotype IgG-λ; DS stage ⅢB; ISS Ⅲ) and subsequently received consecutive infusions of CD19-CAR-T cells at a total dose of 1×107 (day 1) and BCMA-CAR-T cells at a total dose of 1×107 (days 2 to 3) . After the infusion of CAR-T cells, the patient gradually developed fever, hypotension, hypoxemia, and sinus tachycardia from day 3. At the same time, the patient suffered from sudden severe swelling and painful left lower extremity, with his enlarged left testes measuring 8.7×6.0×5.0 cm. The circumference difference between the man's two thighs was 12.3 cm, and a vascular ultrasound revealed a defect of femoral vein blood flow filling in his left lower extremity, suggesting embolization, which was associated with his platelet count decreasing to 25×109/L. On day 7, the patient's left limb and left edema tests showed aggravation, but the progress with thrombosis was considered after the reexamination of the vascular ultrasound, which showed a more than 10 cm long local thrombus in the left femoral vein, with the level of IL-6 going up continuously. We extracted genomic DNA from the patient's peripheral blood to analyze the polymorphism at the -572C/G site of the promoter of the IL-6 gene with the method of First Generation Sequencing. The result showed that the genotype of -572C/G promoter polymorphism was CC, suggesting that high levels of IL-6 with its -572C/G polymorphism might be associated with the risk of thrombotic disorders. Thus, timely diagnosis and reasonable treatment of coagulopathy are crucial in avoiding CRS-related mortality.

Disclosures
No relevant conflicts of interest to declare.
NUDT15 Genetic Variation Is the Strongest Predictive Marker of Tolerance to 6-Mercaptopurine in Japanese Childhood ALL Patients: A Genome-Wide Association Study
Authors: Tanaka Yoichi , Urayama Kevin Y , Mori Makiko , Hasegawa Daisuke , Noguchi Yasushi , Yanagimachi Masakatsu , Keino Dai , Ota Setsuo , Akahane Koshi , Hangai Mayumi , Kawaguchi Takahisa , Takagi Masatoshi , Koh Katsuyoshi , Matsuda Fumihiko , Manabe Atsushi 
Published: November 2019

Introduction
In Asians, NUDT15 genetic variants have been reported to be associated with low tolerance to 6-mercaptopurine (6-MP). However, variation in 6-MP tolerance is still observed among patients without the NUDT15 variants. Some patients required high doses of 6-MP to obtain treatment effect. We performed a genome-wide association study (GWAS) in Japanese to confirm reported associations as well as to identify additional genetic loci associated with variation of 6-MP tolerance in childhood acute lymphoblastic leukemia (ALL) patients.
Methods
We included ALL patients enrolled into a Tokyo Children's Cancer Study Group (TCCSG) trial who started maintenance therapy by normal protocol dose (30-50 mg/m2/day). Whole genome single nucleotide polymorphism (SNP) microarray genotyping was performed followed by whole genome SNP imputation (Japanese reference genome). Data for over six million SNPs were available for analysis. Association analysis was performed adjusting for age at diagnosis and considering average 6-MP dose during 168 days from maintenance therapy initiation as the outcome.
Results
A total of 224 patients were included in the analysis. The median age at diagnosis was 4.7 (range = 0.9-15.7) years old and boys comprised 56.3%. The average 6-MP dose during the 168 days of maintenance therapy was 41.1 (range = 13.3-78.5) mg/m2/day. Variants representing 10 genomic regions showed potential association (P &lt; 5 x 10-6) with average 6-MP dose, in which one locus was genome-wide significant (13q14.2, rs116855232). Variant rs116855232 is located in the previously identified NUDT15 gene and represents the strongest association with average 6-MP dose within our Japanese study (β = -11.45, P = 8.5 x 10-10). In contrast, the TPMT locus, which is predictive of 6-MP intolerance in populations of European ancestry, was not associated with 6-MP dose in Japanese. Among the suggestive loci requiring further follow-up is a locus residing in AFF3 (2q11.2, P = 2.1 x 10-6), a gene previously implicated in childhood leukemia.
Conclusion
We have validated that NUDT15 rs116855232 is a locus with strong association with 6-MP tolerable dose, and it currently represents the most meaningful clinically predictive marker in Japanese. These findings indicated that NUDT15 genotyping should be considered prior to initiation of 6-MP treatment. Prospects for the identification of additional loci of 6-MP tolerance are high after further validation of suggestive loci observed in this Japanese study.
Figure

Disclosures
No relevant conflicts of interest to declare.
Detection and Analysis of Gene Mutations in Patients with Glanzmann's Thrombasthenia
Authors: Sun Boyang , Zhang Donglei , Li Huiyuan , Dou Xueqing , Yang Renchi 
Published: November 2019

Background: Glanzmann thrombasthenia (GT) is a rare inherited disorder of bleeding, and it is characterized by the impaired or absent platelet aggregation to multiple physiologic agonists such as collagen, adenosine diphosphate (ADP), arachidonic acid(AA), but normal reaction to ristocetin. There is qualitative or quantitative defect in platelet integrin αIIbβ3(GPIIb/IIIa). Pathogenic variants of either αIIb or β3 unit could cause GT. The database of gene mutations is continuously updated on the Internet (http://www.hgmd.org); it totally lists 236 variants of ITGA2B gene and 170 variants of ITGB3 gene.
Aim: To characterize the clinical manifestation and molecular basis of GT patients in China, and update the pathogenic variants database.
Method: Clinical features are evaluated in 104 patients with GT. New generation sequencing was performed with a custom designed panel for the bleeding and platelet disease involving 76 genes, while ITGA2B and ITGB3 were enrolled.
Result: The initial bleeding occurred before 1 age in most patients. Incidence of consanguinity is 12.5%. Symptoms lessened with age in about 30% patients. Female patients suffered more severe bleeding than male patients. Fifty different mutations were detected, among which 15 were novel. Most patients were compound heterozygotes and most mutations detected were missense mutations. Among 15 novel mutations, there were 7 missense mutations, 2 nonsense mutations, 2 splicing mutations, 4 frameshift mutations. Pathogenicity of all novel mutations were evaluated according to the standards and guidelines of ACMG. All variants detected were pathogenic or likely pathogenic. Furthermore, c.1750C>T [p.R584X] and c.2333A>C [p.Q778P] in ITGA2B were detected in 10 and 16 unrelated families, strongly suggesting a founder effect.
Conclusion: Our study reports the largest cohort of GT in China, describing the clinical, laboratory and genetic characteristics of 104 patients. We found 15 novel pathogenic mutations in ITGA2B and ITGB3 causing GT. Theses novel findings expand the GT mutation spectrum.

Disclosures
No relevant conflicts of interest to declare.
SHC014748M, a Novel Selective Inhibitor of PI3Kδ, Demonstrates Promising Pre-Clinical Antitumor Activity in B Cell Lymphomas and CLL
Authors: Fan Lei , Wang Chao , Wang Zhiqiang , Zhang Xian , Cao Lei , Miao Yi , Du Xinyi , Xu Wei , Li Jianyong 
Published: November 2019

Introduction : PI3Kδ, one of the class I PI3Ks, is found expressed primarily in leukocytes and plays an essential role in B-cell development and function. Here, we comprehensively evaluated the in vitro and in vivo antitumor activity and the underlying mechanism of SHC014748M, an oral selective inhibitor of PI3Kδ under Phase I clinical evaluation.
Methods : Biochemical and cell-based assays were used to measure compound potency and selectivity in lymphoma cell lines as well as primary CLL cells, and PI3K/AKT pathway was measured by Western blot assay, Alphalisa and Elisa. Xenograft model was carried out to validate in-vivo antitumor potency of the compound. Besides, chemokines and cytokines derived from blood samples of patients were also detected.
Results: SHC014748M was 125- to 306-fold more selective for PI3Kδ inhibition relative to other class I PI3K enzymes and showed in vitro activity in most of 23 B lymphoma cell lines. We identified that SHC014748M treatment resulted in a 3.1- to 5.5-fold increase in annexin V/7-ADD staining, indicating a significant apoptosis induction. SHC014748M inhibited phosphorylation of AKT, targets downstream of PI3Kδ, in lymphoma cells. Among the 15 primary CLL cells, the 50% inhibitory concentration (IC50) of SHC014748M varies from 850 nM to 37040 nM respectively and expression of phosphorylation AKT decreased to the normal levels in the presence of SHC014748M or positive control, Idelalisib. In-vivo study revealed that SHC014748M significantly reduced lymphoma cell growth in the treatment group compared with control mice. CCL4, CCL17, CCL22 and CXCL13 derived from patients decreased sharply after SHC014748M treatment.
Conclusion: According to the results, SHC014748M appeared to be a novel promising compound in the treatment of B cell lymphomas and CLL.

Disclosures
Wang: Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment. Wang:Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment. Zhang:Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment.
Outcomes in Heparin-Induced Thrombocytopenia Managed with Direct Oral Anticoagulants
Authors: Anusim Nwabundo , Ionescu Filip , Konde Anish S , Jindal Vishal , Gupta Ruby , Huben Marianne Terese , Gaikazian Susanna , Jaiyesimi Ishmael 
Published: November 2019

Background: Heparin-induced thrombocytopenia (HIT) occurs as a result of autoantibodies to the platelet factor 4 (PF4)-heparin complex, which activate the coagulation cascade with subsequent thrombosis. HIT can be fatal if not diagnosed and treated promptly with replacement of all heparin with non-heparin anticoagulants. Classic options are parenteral direct anti-thrombin agents which require intravenous administration and can prolong hospital stay. Direct oral anticoagulants (DOACs) address these inconveniences and are an interesting alternative. However, data regarding their efficacy and safety in HIT is limited.
Methods: We retrospectively identified patients with HIT using ICD code 9: 289.84 and ICD code 10: D75.82 at Beaumont Hospital (Royal Oak) between December 2013 and December 2018. Only patients with HIT confirmed by positive serotonin release assay and managed with DOACs were included. Data regarding diagnostic tests, bleeding and thrombosis during the 30-day follow-up were recorded.
Results: A total of 229 patients were identified using the ICD codes; only 8 patients had confirmed diagnosis of HIT and were treated with DOACs. The average age was 70 years (51-92 years); most were male (5, 62.5%) and Caucasian (6, 75%). The median optical density of PF4-heparin antibody was 1.97 (0.85-3.108). Six patients (75%) had confirmed HIT-associated thrombosis; one had negative Doppler ultrasonography of the lower extremities (upper extremities were not assessed) and one patient was not assessed for thrombosis. Seven patients (87.5%) received apixaban and one patient received rivaroxaban. The lowest platelet count prior to initiating DOAC was 40,000/microL, while three patients started DOACs when their platelet count was above 150,000/microL. Within the follow-up period, none of the patients on apixaban had bleeding episodes or clot progression. The only patient treated with rivaroxaban was re-admitted within one week of discharge for right upper extremity deep vein thrombosis. Unfortunately, this was the one patient who was not evaluated for thrombosis at the time of HIT diagnosis.
Conclusion: In the 30 days following HIT diagnosis, treatment with apixaban resulted in adequate anticoagulation and was not associated with increased bleeding events despite relative thrombocytopenia.

Disclosures
No relevant conflicts of interest to declare.
Variant Pcgf-PRC1 Regulates Susceptibility of PRC2 Mediated H3K27me3 to Safeguard B Cell Fate of Hematopoietic Stem/Progenitor Cells
Authors: Takano Junichiro , Nakajima-Takagi Yaeko , Ito Shinsuke , Koseki Haruhiko , Iwama Atsushi , Ikawa Tomokatsu 
Published: November 2019

Polycomb repressive complex (PRC) resides in two major complexes PRC1 and PRC2. They cooperate with each other to coordinate proper developmental process by silencing target genes; PRC1 posits H2AK119ub1 and PRC2 catalyzes trimethylation of H3K27 (H3K27me3). The PRC1 component BMI1/PCGF4 has long been recognized to be essential in the maintenance of normal and malignant hematopoietic stem cells (HSCs). Recently, diversity of PRC1 has been noticed and PRC1 is now classified into six alternative complexes depending on PCGF proteins. In embolic stem cells, PRC1 which contains PCGF1 (PCGF1-PRC1) has been demonstrated to serve upstream of the BMI1/PCGF4-PRC1. However, the impact of BCOR, which is a component of PCGF1-PRC1 on hematopoiesis is clearly different from BMI1/PCGF4; the mice deficient for BCOR exhibited normal HSC activities and BCOR rather prevented leukemic transformation of HSCs, suggesting the previously unappreciated gene control mechanisms of PCGF1-PRC1. To tackle this issue, we focused on the roles of PCGF1 in hematopoiesis.
Loss of Pcgf1 in hematopoietic stem cells led to severe reduction of B lineage cells with an expansion of myeloid progenitors due to defects in lymphoid-primed multipotent progenitor (LMPP) cells. To explore the molecular mechanisms, we have established Id3-overexpressing hematopoietic progenitor cells (IdHPs) which correspond to LMPP-like cells (Ikawa et al. 2015) from bone marrow of ERT2-Cre Pcgf1 flox mice. The ChIP-seq analysis of normal IdHPs identified 1274 genes whose promoters were associated with PCGF1 peaks and 37% of them exhibited enrichment of H3K27me3 and binding of SUZ12 (PRC2). Deletion of Pcgf1 destabilized H3K27me3 levels, resulting in re-activation of genes associated with PCGF1 and SUZ12 peaks, whereas the chromatin occupancy of SUZ12 was not affected. Intriguingly, proteomic analysis demonstrated that PCGF1 interacts with key factors responsible for the organization of nucleosomes and PCGF1 loss triggered a decline of nucleosome-densities in promoters of genes occupied by PCGF1 and SUZ12 peaks. Since enzymatic activity of PRC2 is dependent on nucleosome-densities, PCGF1 is likely to regulate the susceptibility of H3K27me3 by PRC2 through determination of the nucleosome-densities. Furthermore, genes which were downregulated by PCGF1-nucleosome-H3K27me3 axis entailed many myeloid-related genes and knock down of one of those myeloid genes partially restored the B cell differentiation potential of Pcgf1-KO hematopoietic stem/progenitor cells (HSPCs), supporting the biological significance of the PCGF1-nucleosome-H3K27me3 axis. Collectively, these results indicate PCGF1 determines cellular fate of HSPCs through stabilization of nucleosomal organization.

Disclosures
No relevant conflicts of interest to declare.
Novel Genetic Variations in Acute Myeloid Leukemia in a South Asian Population
Authors: Shahid Saba , Shakeel Dr. Mohammad , Siddiqui Saima , Ahmed Shariq , Sohail Misha , Ahmed Ishtiaq , Shamsi Tahir S. 
Published: November 2019

Abstract:
Introduction:
Acute Myeloid Leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal expansion of blast cells. The prognosis, diagnosis, and treatment of AML have been transformed from histological findings to cytogenetic and genomic testing. We assessed the frequency and clinicopathologic significance of 54 genes in myeloid neoplasm patients by using targeted next-generation sequencing.
Methods:
Blood samples of 26 patients were collected from OPD at National Institute of Blood Diseases (NIBD). The myeloid sequencing panel of 54 genes (complete coding exons of 15 genes and exonic hotspots of 39 genes) was sequenced. The panel total coverage was 141 kb in genomic sequence. TruSight myeloid sequencing (Illumina, CA) libraries were prepared and runs were performed on a MiSeq (Illumina) genome sequencer. The generated data were analyzed by on-instrument software or TruSeq Amplicon® and BaseSpace Apps®.
Results:
The analysis with ANNOVAR showed total 295 variants comprises of 71 nonsynonymous SNVs (including 12 novels), three stop-gained SNVs (including two novel), two novel splice-site SNVs, and eleven frame-shift Indels (including seven novels) in at least one of the patients. Among 71 nonsynonymous, three driver non-synonymous mutations (rs121913535, rs121913529 in KRAS, rs121913250 in NRAS), and a driver non-frame shift insertion in EZH2 were found using the ParsSNP tool. In addition, three high frequency novel variants were also found including a heterozygous indels in FBXW7 in 92% of the cases, a heterozygous SNV in FBXW7 in 54% cases, and a heterozygous SNV in GATA2 in 42% cases.
Conclusion:
Genomic profiling of AML will provide novel insight in the diseases pathogenesis. Our findings demonstrate great heterogeneity of pathogenic/deleterious genetic variation in AML patients of this study. The deleterious alterations vary in different patients of AML. This highlights the usefulness of panel sequencing in cases where prognosis becomes challenging. In our study, the genes EZH2, BCOR, WT1, KRAS, KIT, DNMT3A, and CSF3R were found most frequently mutating. In addition, other genes having single mutation also conferred pathogenicity. These findings will provide a framework for assessing genetic predisposition of AML patients of South Asian region.

Disclosures
No relevant conflicts of interest to declare.
Optimizing the Economic Impact of Sickle Cell Hospitalization By Effective Care Plans
Authors: Ahmed Bilawal , Clements Kelton R , Heidt Jonathan , Trendle Michael , Singh Sindhu 
Published: November 2019

Background: Adults with sickle cell disease (SCD) vaso-occlusive crisis (VOC) frequently seek care in the emergency department (ED) which often results in hospital admissions. The Hematology-Oncology at Ellis Fischel Cancer Center collaborated with organization's ED to develop and implement individualized care plans for adults with SCD presenting to the ED with the goal of adequate outpatient management, reduction in hospital admissions and optimizing the economic impact of sickle cell hospitalization.
Methods: Baseline data was collected from November 2014 through June 2018. Medical Director, Oncology Unit Manager, and Performance Improvement Professional (PIP) collaborated with the ED Medical Director and representatives to develop a disease-specific individualized acute care plan for our adult SCD patient population. 100% case were reviewed by PIP and bi-monthly aggregate case review were done by the Medical Director/Unit Manager. The care plan include pathways for lab collection, pain medication regimens, prescription opioid refill policies, a timeline for outpatient follow-up, and criteria for hospital admission. These care pathways were then transitioned to order sets within the organization's electronic medical record.
Results: Implementation of the SCD care plans resulted in significant improvement of length of stay(LOS) index indicating better management during the hospital stay, resulting in reduction of the inpatient pharmacy cost per encounter in half as mentioned in Table 1. Admissions per patient did not change significantly, but ED visits per patient went down and clinic visits per patient went up pointing to better management outside of acute care as shown in Table 2. Last 4 quarters of this study showed marked decrease in cost per patient as indicated in Table 3.
Conclusions: This study clearly demonstrates that collaboration with the ED and development of care plans can improve the overall patient care for this population resulting in better outpatient management. Implementing this protocol has not only improved the patient experience but also has decreased the financial toxicity for the patient and the institution. We would encourage other institutions to implement such protocol in the ED and in the outpatient setting to meet the patient needs.
Table 3

Disclosures
No relevant conflicts of interest to declare.