bluuebunny/crossref_metadata_2025 huggingface dataset:
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 < 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.
Clinical Impact of 18f-FDG PET/CT As a Valuable Prognostic Tool for the Newly Diagnosed Multiple Myeloma with Extramedullary Disease
Authors: Baek Dong Won , Cho Hee Jeong , Sohn Sang Kyun , Jung Sung-Hoon , chae Moon Hong , Moon Joon Ho
Published: November 2019

Purpose

18F-FDG PET/CT (PET/CT) could be a valuable tool to predict long-term survival outcomes in patients with newly diagnosed multiple myeloma (MM). It has ability to distinguish metabolically active sites such as extramedullary disease (EMD) as well as bone damage with relatively high sensitivity and specificity. In this study, we attempted to evaluate the role of PET-CT as a novel prognostic tool for patients with newly diagnosed MM who have EMD.

Patients and Methods

This study included 211 patients who were newly diagnosed with multiple myeloma from Kyunpook National University Hospital and Chonnam National University Hwasun Hospital. We retrospectively analyzed the medical records of enrolled patients. PET/CT was performed at the diagnosis and EMD was identified in 36 patients (17.1%).

Results

With a median follow-up duration of 21.5 months (range 1.4-67.7), the estimated 2-year PFS and OS rates were 46.1% and 79.6%, respectively. The presence of PET/CT positive EMD and high maximum standardized uptake value (SUVmax) on baseline PET/CT were significantly associated with inferior long-term survivals in terms of PFS (p=0.013, p=0.007) and OS (p=0.002, p=0.004). In addition, patients who underwent autologous stem cell transplantation (auto-SCT) showed superior PFS (p=0.005) and OS (p=0.022) in PET/CT positive EMD group. Meanwhile, Revised-International Staging System (R-ISS) successfully predicted the prognosis in this study. When we modified R-ISS with the presence of EMD, survival outcomes of the R-ISS stage III patients who didn't have EMD were similar to R-ISS II, while patients with PET/CT positive EMD showed even worse prognosis than the R-ISS stage III group. In the multivariate survival analysis, the presence of EMD (hazard ratio (HR), 2.397; 95% confidence internal (CI), 1.281-4.483; p=0.006) and auto-SCT (HR, 0.326; 95% CI, 0.194-0.549; p<0.001) were related to PFS, while LDH (HR, 2.56; 95% CI, 1.221-5.366; p=0.013) level and auto-SCT (HR, 0.398; 95% CI, 0.167-0.953; p=0.039) were independent prognostic factors of OS.

Conclusion

In conclusion, PET/CT positive EMD was a poor prognostic factor in patients with newly diagnosed MM. In addition, PET/CT could be a valuable tool to make better risk-adapted treatment strategies with R-ISS in EMD positive MM patients. Above all, patients with PET/CT positive EMD should be considered auto-SCT to improve long-term survivals.

Figure

Disclosures

No relevant conflicts of interest to declare.
Reduced Number and Function of Endothelial Progenitor Cells in Patients with Beta Thalassemia Major
Authors: Spectre Galia , Stark Pinhas , Steimatzky Arza , Raanani Pia , Lev Dorit , Kornowski Ran , Pazgal Idit
Published: November 2019

There is a high incidence of venous thromboembolic complications in patients with beta thalassemia .Recently, a high incidence of asymptomatic multiple silent cerebral infarcts, presumably of arterial origin, were demonstrated in patients with beta thalassemia major (TM) and intermedia. The etiology remains unclear. Endothelial progenitor cells (EPCs) derive from the bone marrow and contribute to regeneration of endothelial damage. We hypothesized that the change in EPCs contributes to the vascular events in patients with TM and that patients have high markers for arterial thromboembolism.

Study aim: To assess the change in the number and function of EPCs in TM patients.

Methods: Blood samples were drawn from adult patients with TM before blood transfusion and from healthy volunteers. EPCs were isolated from peripheral mononuclear cells by using a Ficoll density-gradient centrifugation. EPCs' levels were detected by flow cytometry, namely by co-expression of VEGFR-2 in CD34 or CD133 positive cells. EPCs were cultured for 7 days after which functional properties were evaluated by: their capacity to form colonies using light microscopy (a colony forming unit (CFU) defined as a cluster of at least 100 flat cells surrounding a cluster of rounded cells) as well as by using a viability assay (using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay). The adhesion molecule P- selectin and the chemokine SDF1 were measured by using Elisa kits according to the manufacturer instructions.

Results: Thirty five patients (median age 35 range 27-47y) and 15 healthy volunteers (median age 34.5 range 30-50y) were included. Lower levels of CD34/VEGFR-2 cells and a trend towards CD133/ VEGFR-2 cells were observed in TM patients (Figure 1A). Viability of EPCs and their ability to perform colonies were markedly reduced in TM patients compared to controls; mean MTT 0.05± 0.05 in TM vs. 0.23±0.08 (p<0.01), median number of CFU = 0, range 0-3 in TM vs. 4, range 1-6 in controls (p<0.001), (a representative picture, Figure 1b). SDF1a and P selectin levels were significantly higher in TM compared to healthy volunteers (p=0.05 and P<0.01 respectively).

Conclusions: Our results suggest that patients with TM have reduced number and function of EPCs. This may contribute to the silent cerebral infarcts and hypercoagulable state in thalassemia. Furthermore, SDF1a and P selectin, both markers for coronary artery disease were higher in TM. Further studies are planned to establish the possible role of other cytokines in this process.

Disclosures

Spectre: Pfizer: Honoraria; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Boeringer ingelheim: Honoraria; Bayer: Membership on an entity's Board of Directors or advisory committees.
Inhibiting the Hippo Signaling Pathway Key Molecule YAP Suppresses Mantle Cell Lymphoma Proliferation By Regulating Multiple Pathogenrelated Signaling Pathways
Authors: Xiong Wenjie , Yi Shuhua , Yan Yuting , Li Zengjun , Liu Wei , Lv Rui , Yu Zhen , Zou Dehui , Qiu Lugui
Published: November 2019

Object: Mantle cell lymphoma (MCL) is a rare hematologic malignancy with heterogeneous course and always resistant to chemotherapy at advance or relapsed stage. Hippo signaling pathway is one of the most popular pathways in solid tumors. The major role of this pathway is regulating cell proliferation, migration, and maintaining the stemness of cells. However, the relationship between this pathway and MCL had been rarely studied. The purpose of this study is to investigate the relationship between MCL and YAP, the key molecule Hippo signaling pathway, to further clarify the mechanism of MCL resistance and to find new therapeutic targets.

Methods: In this study, expression of YAP in B cell non-hodgkin's lymphoma (B-NHL) and mantle cell lymphoma detected by real-time PCR, Western blot analysis, immunofluorescence. Knockdown of YAP by sh-RNA or inhibit the function of YAP using verteporfin (VP). Next, the effects of YAP knockdown and YAP inhibitor on MCL cells were evaluated by fluorescence detection, real-time PCR and Western blot. Cell count, CCK8, Soft Agar Assay and in vitro functional assays were performed to elucidate the function of YAP-mediated cell proliferation, the effect on signaling pathway and the relationship with chemoresistance in MCL.

Results: We detected the expression of YAP in 40 B-NHL patients and 10 healthy donor and found that YAP were overexpressed in relapsed and newly diagnosis patients and it was extremely higher in relapsed patients. Moreover, YAP is extremely high express on MCL. Knockdown YAP by shRNA or YAP inhibitor verteporfin (VP) could not only inhibit the proliferation, induce the apoptosis of MCL cell lines, but also lead to cells stopping in G1 phase. In addition, knockdown YAP could sensitize Z138 cells to the cytotoxics of BTK inhibitors ibrutinib and SYK inhibitor R788.Combined VP and ibrutinib or R788 had obvious synergy. Most importantly, knockdown YAP could obviously reduce the expression of protein involved in G1 phase, inhibit the BCR signaling pathway and PI3K AKT signaling pathway, and promote cell apoptosis and DNA damage signaling pathways. Through regulating multiple signaling pathways, YAP played an important role in cell proliferation and drug resistance in B-NHL.

Conclusion: It was the first time to demonstrate that Hippo signaling pathways may associate with the pathogenesis and drug resistance of MCL. Targeting the key molecular of Hippo signaling pathways may be potential therapeutic targets of MCL patients especially the relapse and refractory patients.

Disclosures

No relevant conflicts of interest to declare.
Maintenance Therapy in Patients with Chronic Lymphocytic Leukemia Who Achieved MRD-Positive Remissions after Induction Therapy
Authors: Kuvshinov Alexey , Voloshin Sergei , Shuvaev Vasily , Belyakova Elena , Mikhaleva Mariia , Garifullin Andrey , Rysev Georgiy , Bogdanov Alexander , Martynkevich Irina , Kleina Elizaveta , Samorodova Alena , Ruzhenkova Iuliia , Vokueva Iuliia , Chechetkin Alexander
Published: November 2019

Introduction. There is strong evidence that the achievement of a negative status for minimal residual disease (MRD) is the single most important factor predictive of final outcome in patients with chronic lymphocytic leukemia (CLL) both in previously untreated and relapsed patients. Maintenance treatment (MT) with the CD20-directed monoclonal antibodies rituximab or ofatumumab yields better progression-free survival (PFS) compared with observation alone in individuals with CLL who have received induction therapy with chemo-immunotherapy. However maintenance therapy of anti-CD20 antibodies, although approved in some other B-cell malignancies, is not yet approved in CLL by regulators.

Aim. We wanted to assess overall and progression-free survival in MRD-positive patients who received maintenance therapy.

Methods. The study included 58 patients (median age 62 years, range 36-82). Male to female ratio - 1.7:1. We have used NCI revised guidelines (Hallek M, et al., 2008) for treatment initiation, assessment of response and MRD. Induction chemotherapy was conducted under the following programs: RB, RFC. The median line of therapy was - 1 (1-5). Evaluation of MRD was performed using 5-color flow cytometry of the bone marrow cells. The maintenance therapy was conducted MRD-positive patients (n=41): Rituximab 500 mg/m2 intravenously every 8 weeks for 2 years. The remaining MRD-negative patients (n=17) were under dynamic observation without therapy. Median observation in study was 51.5 month (15.2-134.8).

Results.The frequency of relapses in the group of patients with MT was 51.2%, in the group of patients without MT - 70.6% (p=0.18). MRD was not detected after 6-12 months of MT in 17.1% (7/41) had previously MRD-positive patients. The medians of PFS and OS were not different in the MRD-positive patients with MT versus in the MRD-negative patients without MT: PFS - 45.9 months and 57 months, respectively (p=0.83); OS - 106 and 128 months, respectively (p=0.47). Significant differences in the incidence of infectious complications between patients with MT and without of MT were not detected (p˃0.05).

Conclusions.Maintenance therapy for MRD-positive patients allows increasing of progression-free and overall survival to the level of patients with MRD-negative status. Maintenance therapy may be a means of control over the minimal residual disease and the method of its eradication. The proposed algorithm need further testing to confirm the initial results.

Figure

Disclosures

Shuvaev: Novartis: Consultancy; Pfize: Honoraria; BMS: Consultancy; Fusion Pharma: Consultancy.
Molecular Characterization and Frequencies of Different Genetic Ameliorating Factors in Transfusion Dependent Thalassemia Patients from District Peshawar
Authors: Mehmood Gulrukh , Taj Abid Sohail , Naz Arshi , Khan Tariq Masood
Published: November 2019

Background

Thalassemia is one of the most common genetic blood disorders worldwide. The carrier rate of β thalassemia in Pakistan is found to be around 5-7%, rendering it a major health concern. Approximately 5000 children are diagnosed with thalassemia major every year in Pakistan due to consanguineous marriages. More than 200 causative molecular defects have been identified in β-globin genes. About 20 mutations account for 90% of the abnormal β-genes. Clinical severity of β-thalassemia is modified by different factors. The important ones include the type of disease causing mutation and the ability to produce α and γ globin chains. A better understanding of these ameliorating factors may have a significant impact on disease management.

Objectives

1. To re-assess diagnoses among local multi-transfused thalassemia syndrome patients.

2. To determine frequency of various genetic determinants of milder phenotypes of thalassemia among study patients.

Methods

Transfusion dependent thalassemia patients, upto 15 years age, were enrolled from Fatimid Foundation, Peshawar Pakistan. A comprehensive questionnaire encompassing demographic and clinical data was filled out for each patient. Genetic analyses for 2 alpha (α) and 13 prevalent beta (β) gene mutations and for polymorphisms at Xmn1-HBG2 and BCL11A were carried out on blood samples of the patients at National Institute of Blood Diseases, Karachi Pakistan. The data collected was analysed at Khyber Medical University (KMU) Peshawar Pakistan.

Results

A total of 54 transfusion dependent thalassemia patients were enrolled into the study. Homozygous or compound heterozygous combinations of β-globin gene mutations were identified in all the study patients. Eleven patients were found to have a co-existing heterozygous α (3.7kb) deletion, two patients had Xmn1-HBG2 polymorphism and 38 had BCL11A polymorphism. Homozygous Fr 8-9 was the most frequent mutation, found in 19 (35.2%) patients. Only 13 patients were found to have isolated β-globin gene mutations. In total, 46 (85.2%) study patients were identified to have an ameliorating genetic factor (a co-existing α-globin gene mutation, an Xmn1-HBG2 polymorphism or a BCL11A polymorphism) besides the main β-globin gene mutation.

Conclusion

It was concluded that co-existing genetic ameliorating factors are frequently found in transfusion dependent β-thalassemia patients of Peshawar District. These factors impart a milder phenotype to an otherwise severe disease. It is hence suggested that β-thalassemia patients from District Peshawar be screened for these factors and due consideration be paid by the physician in devising management plans.

Key Words

Thalassemia, Genetic modifier in thalassemia, Transfusion dependent Anaemia, Xmn1 polymorphism

Disclosures

No relevant conflicts of interest to declare.
Clinical Presentation, Management and Biomarkers of Cytokine Release Syndrome after Anti-CD19 CART-Cell Therapy for r/r ALL
Authors: Li Ping , Zhou Lili , Ye Shiguang , Li Shaoguang , Liang Aibin
Published: November 2019

Chimeric antigen receptor T (CAR-T) cell therapy has emerged as a novel treatment modality for B-cell malignancies. CD19-specific CAR-T cells induce high rates of initial response among patients with relapsed B-cell acute lymphoblastic leukemia (ALL). However, cytokine release syndrome (CRS) is the most common and severe toxicities of CAR T-cell therapy for ALL, and clinical experience is limited. Here, we describe the clinical presentation and management of 30 patients who presented with CRS following CAR-T cell therapy for relapsed/refractory ALL at our hospital. 12 of the 30 patients (40%) developed grade 1-2 CRS, 14 patients (46.7%) presented with grade 3-4 CRS and 2 patients (6.7%) died of grade 5 CRS. Compared with grade 1-2 CRS, grade 3-4 CRS correlated negatively with overall survival and progression-free survival (P =0.02). We found that higher ferritin levels and percentages of CD19 positive cells in blood lymphocytes cells at time of CAR-T cell infusion were associated with more severe CRS. Grade 3-4 neurotoxicity was frequently present in patients with grade ≧3 CRS. We also observed that the organ disfunctions occurred in sequence after fever onset during the period of CRS. Neurotoxicity, cardiovascular disfunction and cytopenia in some patients manifest as biphasic. Compared to use of tocilizumab for CRS ≧ grade 3, early intervention of tocilizumab for hyperpyrexia duration ≧ 6h alleviates the severity of CRS, and no patients died of severe CRS since this management approach was performed. As use of novel CAR-T cell therapy expands, the data from our clinical experience may help others anticipated the clinical course of organ function and manage CRS in CAR-T therapy.

Figure

Disclosures

No relevant conflicts of interest to declare.
In Vitro and In Vivo Characterization of Marstacimab, an Anti-TFPI Antibody
Authors: Parng Chuenlei , Jin Macy , Holsti Matthew , Benard Susan , Rakhe Swapnil , Patel-Hett Sunita R. , Joyce Alison , Webster Rob , Pittman Debra D
Published: November 2019

Hemophilia A and B are X-linked genetic disorders resulting from functional deficiencies of the intrinsic coagulation plasma proteins Factor VIII (FVIII) or Factor IX (FIX), respectively. An approach to achieving hemostatic pharmacology in hemophilia is to augment the extrinsic cascade is to neutralize Tissue Factor Pathway Inhibitor (TFPI). TFPI is a multi-Kunitz (K) domain inhibitor which binds to and inhibits Factor Xa via the K2 domain and Factor VIIa/Tissue Factor activity (K1 domain). Marstacimab is a fully human monoclonal antibody that binds to and neutralizes TPFI activity and is under development for treatment of hemophilia. To support lead identification during late discovery, five anti-tissue factor pathway inhibitor (TFPI) antibodies (EC50<5nM) were evaluated in an in vivo rabbit pharmacokinetics/pharmacodynamics (PK/PD) study and in vitro using rabbit and human hemostatic assays. Marstacimab displayed potent in vitro activities, and potent pharmacology in shortening the dilute prothrombin time (dPT) assay (1.3 nM) and in the thrombin generation assay (TGA) (2.8 nM). Following an intravenous administration in rabbits, marstacimab and the other anti-TFPI antibodies exhibited target-mediated drug disposition (TMDD) with a half-life less than 2 days, when compared to a control IgG. The pharmacokinetics of anti-TFPI antibody can be described using a Michaelis-Menten mechanistic model in which in vivo Km and Vmax were estimated for the observed nonlinear clearance. The estimated pharmacokinetic parameters for marstacimab was 45 and 38 mL/kg for the central and peripheral volume of distribution, 23 nM for Km, 131 nM/kg/hr for Vmax. In addition, the correlation between in vitro Kd, in vivo Km and in vitro/in vivo potency were assessed. Compared to other anti-TFPI antibodies, marstacimab exhibited reduced clearance and a good in vitro-in vivo relationship. Second, the binding epitopes were further characterized using competitive surface plasmon resonance (SPR) and marstacimab has distinct binding epitopes from the other antibodies. Lastly, the potential human pharmacokinetics and efficacy were evaluated using allometry scaling and in vitro potency, the results predict a minimal target occupancy of 76% following a weekly subcutaneous administration of marstacimab. These studies indicate that the differential influence of the TFPI-binding epitopes on the pharmacokinetics, but not on the pharmacodynamics. The selection strategy could be applicable to the other antibody discovery and development.

Disclosures

Parng: Pfizer: Employment. Jin:Pfizer: Employment. Holsti:Pfizer: Employment. Benard:pfizer: Employment. Rakhe:Pfizer Inc.: Employment. Patel-Hett:Pfizer: Employment. Joyce:Pfizer: Employment. Webster:pfizer: Employment. Pittman:Pfizer Inc.: Employment.
Poor Outcomes of HLA-Mismatched Allogeneic Hematopoietic Cell Transplantation and High Ferritin Levels after a Reduced-Intensity Conditioning Regimen in Patients with Advanced Myelofibrosis
Authors: Lee Han Bi , Yoon Jae-Ho , Min Gi June , Park Sung-Soo , Park Silvia , Lee Sung-Eun , Kim Yoo-Jin , Lee Seok , Kim Hee-Je , Min Chang-Ki , Cho Seok-Goo , Lee Jong Wook , Eom Ki-Seong
Published: November 2019

Allogeneic hematopoietic cell transplantation (allo-HCT) preconditioning intensity, donor choice, and graft-versus-host disease (GVHD) prophylaxis for advanced myelofibrosis (MF) have not been fully elucidated. Thirty-five patients with advanced MF were treated with reduced-intensity conditioning (RIC) allo-HCT. We searched for matched sibling (n=16) followed by matched (n=10) or mismatched (n=5) unrelated and familial mismatched donors (n=4). Preconditioning regimen consisted of fludarabine (total 150 mg/m2) and busulfan (total 6.4 mg/kg) with total body irradiation≤ 400cGy. All showed engraftments, but four (11.4%) showed either leukemic relapse (n=3) or delayed graft failure (n=1). Two-year overall survival (OS) and non-relapse mortality (NRM) was 60.0% and 29.9%, respectively. Acute GVHD was observed in 19 patients, and grade III-IV acute GVHD was higher with HLA-mismatch (70% vs. 20%, p=0.008). Significant hepatic GVHD was observed in nine patients (5 acute, 4 chronic), and six of them died. Multivariate analysis revealed inferior OS with HLA-mismatch (HR=6.40, 95%CI 1.6-25.7, p=0.009) and in patients with high ferritin level at post-HCT D+21 (HR=7.22, 95%CI 1.9-27.5, p=0.004), which were related to hepatic GVHD and high NRM. RIC allo-HCT can be a valid choice for advanced MF. However, HLA-mismatch and high post-HCT ferritin levels related to significant hepatic GVHD should be regarded as poor-risk parameters.

Disclosures

Kim: Handok: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Hanmi: Consultancy, Honoraria; AGP: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; SL VaxiGen: Consultancy, Honoraria; Novartis: Consultancy; Janssen: Honoraria; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Otsuka: Honoraria; BL & H: Research Funding; Chugai: Honoraria; Yuhan: Honoraria; Sanofi-Genzyme: Honoraria, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lee:Alexion: Consultancy, Honoraria, Research Funding; Achillion: Research Funding.
Inflammasome Activation Promotes Deep Vein Thrombosis through Pyroptosis
Authors: Zhang Yan , Zhang Guoying , Jian Cui , Wu Congqing , Mackman Nigel , Smyth Susan , Wei Yinan , Li Zhenyu
Published: November 2019

Venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), is one of the most common causes of cardiovascular death worldwide. Monocytic cells (including monocytes/macrophages) and their derived tissue factor have been reported to play important roles in the development of DVT. However, the mechanism by which monocytes contribute to the development DVT is not well elucidated. In this study, we reported a critical role of inflammasome activation and pyroptosis in the development of DVT. Using a flow restriction-induced mouse DVT model in the inferior vena cava, we show that deficiency of caspase-1 protected against flow restriction-induced DVT. Inflammasome activation leads to interleukin (IL)-1b and IL-18 maturation/release and pyroptosis. Recent studies show that caspases-1 cleaves Gasdermin D (GSDMD) and triggers pyroptosis-a form of programmed cell death with similar morphology to necrosis. We tested the hypothesis that GSDMD-dependent pyroptosis drives inflammasome-induced coagulation and DVT using Gsdmd-/- mice. Indeed, flow restriction-induced DVT was inhibited by GSDMD deficiency. After induction of DVT, fibrin was deposited in the vein as detected by Western blot with a monoclonal antibody that specifically recognizes mouse fibrin, which were inhibited in the caspase-1 deficient mice and GSDMD deficient mice. IL-1b was also increased in tissue of vein following induction of DVT, which was inhibited by caspase-1 or GSDMD deficiency. Following inflammasome activation, pyroptotic macrophages release tissue factor (TF), an essential initiator of coagulation cascades. Consistently, flow restriction-induced DVT was flow restriction-induced DVT inhibited in inducible TF deficient mice. Our data reveal a critical role of inflammasome activation and pyroptosis in the development of DVT.

Disclosures

No relevant conflicts of interest to declare.
LSD1 Inhibitor 4SC-202 Inducing Apoptosis in Myelodysplastic Syndrome Cells Via NF-κb-HO-1 Pathway
Authors: Wang Weili , Zhang Zhaoyuan , Kuang Xingyi , Wang Jishi
Published: November 2019

Aims: Human histone lysine-specific demethylase 1(LSD1) inhibit gene transcription and thereby block myeloid differentiation, apoptosis and cell cycle in AML. 4SC-202, a novel inhibitor of LSD1, is potential therapeutic agent for treating Myelodysplastic syndrome, while its mechanism of action remains unclear.

M ainly method : In the study, LSD1 and HO-1 gene expression were detected in MDS/AML patients. After MDS cell line SKM-1 was treated with 4SC-202, cell viability, apoptosis and cell cycle were detected by CCK-8 or FACS. Lastly, western blotting analyzed that the agent caused the change of the related cellular function protein in SKM-1 cells.

K ey findings: We found that LSD1 and HO-1 were overexpression in MDS/AML patients. LSD1 inhibitor 4SC-202 inhibited cell viability. The agent induced apoptosis by up-regulated BAX and cleaved caspase3/9 as well as down-regulated BCL-2, arrested cell cycle in the G2/M phase by down-regulated CDK1 and up-regulated p21, as well as inhibited activation of the NF-κB pathway and decreased HO-1. LSD1 and HO-1 expression were decreased after exposure with the NF-κB inhibitor OBY11-7082. Silencing LSD1 by LSD1 siRNA hardly caused apoptosis in SKM-1 cells, while the combination of Bay11-7082 and silencing LSD1 significantly decreased LSD1 and HO-1 expression and further increased apoptosis. While up-regulated HO-1 significantly limited the 4SC-202-induced down-regulation of BCL-2, up-regulation of cleaved caspase 3/9 and suppressed activation of NF-κB pathway as well as thereby attenuated the agent efficacy.

S ignificant : In conclusion, LSD1 inhibitor 4SC-202 induced apoptosis through the NF-κB-mediated HO-1 pathway. LSD1 might be a potential target for the treatment of MDS.

Disclosures

No relevant conflicts of interest to declare.