bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma Cell-Free DNA Chromosomal Instability As Surrogate Biomarker of Treatment Response and Minimal Residual Diseases for Multiple Myeloma
Authors: Du Juan , Chen Baoan , Qiang Wanting , Jia Yanchun , Lu Jing , Li Lu , Liu Jing , Jiang Hua , Qian Ziliang , Fu Weijun
Published: November 2020

Background: Minimal residual disease (MRD) is becoming standard diagnostic care for multiple myeloma. Here we investigate cell-free DNA chromosomal instability as minimal invasive biomarker for minimal residual disease monitoring.

Methods: 55 patients were recruited, including 47 newly diagnosed and 8 relapsed multiple myelomas. Plasma samples were collected before treatments, end of 2 cycle and 4 cycle of treatments. Treatment response was assessed by using IMWG criteria. Cell-free DNA was analyzed by illumine HiSeq X10, followed by chromosomal instability analyses by a customized bioinformatics workflow, ultrasensitive chromosomal aneuploidy detector (UCAD), and cfDNA CIN responding to treatment was summarized as cfDNA MRD INDEX.

Results: In this cohort study, 53 (96.3%) patients were found with treatment responses after 4 cycle of treatments, including 16 (29.1%) complete, 13 (23.6%) very good partial, 17 (30.9%) partial and 6 (10.9%) marginal responses. The other 2 (3.64%) recurrent MM experiences disease progression.

At baseline, plasma cfDNA chromosomal aberrations were found in 14/17(82.3%) multiple myeloma patients. Less patients were found with detectable chromosomal changes after treatments (58.8% after C2, 41.2% after C4, versus 82.3% before treatments, P<0.01). The treatment responses identified in plasma cfDNA were summarized as cfDNA MRD INDEX. Low cfDNA MRD INDEX predicts better treatment responses (Fisher exact test, Odds ratio=7.3, P=4.69e-05). And it predicts complete response with sensitivity 100% and specificity 83.3%, with cutoff value 0.044.

Furthermore, cfDNA CIN MRD index were found linearly correlated with percentage of malignant plasma cells from bone marrow aspiration as examined by flow cytometry assay (R-square=0.883, P<0.01). Cutoff -0.06 predicts 100% MRD negatives at specificity 100%.

Conclusions: Plasma cfDNA CIN might be used for monitoring multiple myeloma patients treatment responses, especially for predicting minimal residual diseases.

Disclosures

No relevant conflicts of interest to declare.
Preferential Usage of Specific Immunoglobulin Heavy Chain Variable Region Genes with Unmutated Profile and Advanced Stage at Presentation Are Common Features in Patients with Chronic Lymphocytic Leukemia from Senegal
Authors: Sall Abibatou , Amato Teresa , Gozzetti Alessandro , Touré Awa Oumar , Diop Saliou , Leoncini Lorenzo , Piccaluga Pier Paolo , Raphaël Martine
Published: November 2020

Introduction : Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western populations, being rarer in Asian and African people. It has been suggested that patients with CLL from Africa might have a more aggressive disease compared with Causasien patients. In this study, we aimed to identify genetic factors that may account for this difference

Methods: We collected peripheral blood mononuclear cells (PBMCs) from a total of 75 patients with CLL, 25 from Senegal (Africa), and 50 from Siena. Since it is well known that there are differences in germline IGH repertoires between different populations, we also collected PBMCs from five healthy Senegalese individuals as control. We analyzed immunoglobulin heavy chain (IGH) genes mutational status by performing next-generation sequencing in these 2 groups of patients.

Results: We found that Senegalese patients more frequently had adverse prognostic factors and an unmutated profile. Furthermore, we documented that IGHV1 (IGHV1-69), IGHD3, and IGHJ6 were significantly more frequent in Senegalese patients, whereas IGHV3-30 was common and limited to the Italian cohort. Stereotyped receptors commonly detected in the white population were not recorded in our Senegalese series.

Conclusion: The different IGH repertoire we observed in the Senegalese cohort may reflect the diverse genetic and microenvironmental (ie, polymicrobial stimulation) background.

Disclosures

Gozzetti: Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Research Funding.
Ikaros Expression in the Graft Is Associated with Chronic Graft Versus Host Disease
Authors: Pereira Andre Domingues , de Molla Vinicius Campos , Da Fonseca Ana Rita , Novis Yana , Pires Marcela Souza , Popi Ana , Arrais-Rodrigues Celso
Published: November 2020

Introduction: Hematopoietic stem cell transplantation (HSCT) is the only potentially curative treatment for several diseases. Immune reconstitution post HSCT is a complex and extremely variable process. Ikaros transcription factor has an important role in hematopoiesis of several cell lines, especially in the lymphoid compartment. We hypothesized that Ikaros expression, both in the graft and in the recipient after transplant, might influence immune reconstitution and, consequently, the risk of opportunistic infections, relapse, and graft versus host disease (GVHD).

Objectives: To correlate Ikaros expression both in the graft and in the recipient's peripheral blood (PB) after engraftment with the risk of GVHD after allogeneic HSCT outcomes.

Patients and methods: 51 patients were included. Median age was 51 years (19-80), 53% of patients were male, and 57% of them had acute leukemia. Donor were haploidentical in 45% of cases, related identical in 29% and unrelated identical in 26%. Most patients (82%) received reduced-intensity conditioning regimens. Median follow-up was 20 months (10-40 months). Samples were collected from the graft and from the PB of the recipient 3 weeks after neutrophil recovery. Real time polymerase chain reaction (RT-PCR) was performed for analysis of absolute and relative Ikaros expression.

Results: There was no association between Ikaros expression and the risk of acute GVHD, relapse or mortality. However, a significant association was observed in the risk of chronic GVHD. Cumulative incidence (CI) of chronic GVHD and of moderate / severe chronic GVHD (according to National Institute of Health - NIH classification) in two years were 49% and 28%, respectively. Higher Ikaros expression in the graft was associated with a significantly higher risk of moderate/severe chronic GVHD (54% vs. 15%, respectively, P=0.01). Higher Ikaros expression in the recipient's PB after engraftment was also associated with a significantly higher risk of moderate/severe chronic GVHD (65% vs. 11%, respectively, P=0.01).

Conclusions: Ikaros expression in the graft and in the PB of the recipient after transplant seems to be correlated with a higher risk of moderate/severe chronic GVHD and might be evaluated in larger and prospective trials as a potential biomarker.

Disclosures

No relevant conflicts of interest to declare.
TWIST1 Preserves Hematopoietic Stem Cell Function Via Repression of Cacna1b Expression
Authors: Wang Nan , Yin Jing , You Na , Guo Dan , Zhao Yangyang , Ru Yongxin , Liu Xiaoyan , Cheng Hui , Ren Qian , Cheng Tao , Ma Xiao-Tong
Published: November 2020

The mitochondria of hematopoietic stem cell (HSC) play crucial roles in regulating cell fate and in preserving HSC functionality and survival. However, the mechanism underlying its regulation remain poorly understood. Here, we identify transcription factor TWIST1 as a novel regulator of HSC maintenance through modulating mitochondrial function. We demonstrate that Twist1 deletion results in a significantly decreased long-term HSC (LT-HSC) frequency, markedly reduced dormancy and self-renewal capacities and skewed myeloid differentiation in steady-state hematopoiesis. Twist1-deficient LT-HSC are more compromised in tolerance of irradiation and 5 fluorouracil-induced stresses, and exhibit typical phenotypes of senescence and higher levels of DNA damage and apoptosis. Mechanistically, Twist1 deficiency upregulates the expression of voltage-gated calcium channel Cacna1b in HSC, leading to noticeable increases in mitochondrial calcium levels, biogenesis, metabolic activity and reactive oxygen species production. Suppression of voltage-gated calcium channel by a calcium channel blocker largely rescues the phenotypic and functional defects in Twist1-deleted HSCs under both steady-state and stress conditions. Collectively, our data, for the first time, characterize TWIST1 as a critical regulator of HSC function acting through CACNA1B/Ca2+/mitochondria axis, and highlight the importance of Ca2+ in HSC maintenance. These observations provide new insights into the mechanisms for the control of HSC fate.

Disclosures

No relevant conflicts of interest to declare.
Study of the Role of Splicing Factor SRRM2 in Cytarabine Treatment Resistance in Acute Myeloid Leukemia
Authors: Morales María Luz , Garcia-Vicente Roberto , Álvarez Sánchez-Redondo Noemí , Rodríguez García Alba , Ortiz-Ruiz Alejandra , Reyes-Palomares Armando , Hermouet Sylvie , Ayala Rosa , Martinez-Lopez Joaquin , Linares María
Published: November 2020

Introduction. Acute myeloid leukemia (AML) is a very complex and dynamic disease, characterized by clonal expansion of aberrantly differentiated myeloid lineage blasts. Although in recent years the molecular mechanisms involved in the development of the disease have been deeply studied, and as result many new targets have emerged, the treatment has not changed substantially. 7+3 chemotherapy regimens remains as the treatment of choice in most cases. Refractoriness and relapse after reaching complete remission are the main cause of death in the disease. Therefore, to improve current treatments it is crucial not only to know the molecular profile of patients at diagnosis, but also to know the mechanisms of pharmacological resistance that may be developed, in order to propose more appropriate and personalized treatments.

Methods. The analysis of the proteomic profile associated to cytarabine treatment resistance of paired samples from 3 patients with AML was performed by LC-MSMS after IMAC enrichment. The OCI-AML3 cell line was used to generate a model of cytarabine resistance through sustained exposure to increasing doses of the drug, thus generating the OCI-AML3_R line (Figure 1A). Gene expression levels ofSRRM2were studied in the cell model (n=3), as well as in paired samples of AML patients (n=7) by qPCR; in addition to data deposited in public repositories from the TCGA-LAML and GTEx-BM projects. Finally, SR protein phosphorylation levels were studied in paired bone marrow smears by immunohistochemistry (n=7).

Results. Proteomic analysis of paired samples from AML patients has identified an increased in the phosphorylation of SRRM2, among other SR proteins, at the time of cytarabine resistance. Analysis of public data has revealed several mutations and copy number variations in genes related to the spliceosome. However, these studies, as well asin vitroanalysis, have rejected that changes in SRRM2 phosphorylation are due to changes in gene or protein expression (Figure 1B and 1C). Otherwise a significant increase in SR protein phosphorylation has been detected after the development of cytarabine resistance by immunohistochemistry (Figure 1D). This increase in phosphorylation is prominent at the time of diagnosis in patients showing treatment refractoriness.

Conclusions.The results obtained in this study show the involvement of the splicing pathway in AML and in cytarabine resistance. In addition, the immunohistochemistry analysis suggests a role of the phosphorylation levels of SR proteins as a possible predictive biomarker of response to cytarabine treatment.

Disclosures

No relevant conflicts of interest to declare.
Thalidomide Plus Prednisone and Methotrexate for Symptomatic Large Granular Lymphocyte Leukemia: A Prospective, Single-Center, Pilot Study
Authors: Yi Shuhua , Du Jun , Yu Ying , Jiao Yang , Wang Yi , Wang Huijun , Wang Tingyu , Lv Rui , Liu Wei , Xiong Wenjie , Liu Huimin , Huang Wenyang , Zou Dehui , An Gang , Zhao Yaozhong , Wang Jianxiang , Qiu Lugui
Published: November 2020

Background: Large granular lymphocyte leukemia (LGLL) is one type of chronic lymphocytic proliferative disorders, which commonly manifests as infiltration of large granular lymphocytes in both peripheral blood and bone marrow. LGLL now includes two entities with similar clinical course, treatment strategy and outcomes: T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of NK cells. The standard therapy for LGLL is still elusive. Here, we presented the efficacy and safety of combinatorial oral immunoregulatory regimen thalidomide, prednisone, and methotrexate (TPM regimen) in a prospective phase 2 clinical trial. Methods: We designed this phase 2 investigator-initiated clinical trial (NCT04453345) to evaluate the clinical response and safety of the combination of thalidomide, prednisone, and methotrexate in symptomatic treatment naïve LGLL patients. The TPM regimen includes thalidomide 50-100mg per night, prednisone 0.5-1.0mg/kg qod and methotrexate 10mg/m2 per week. This regimen will be administrated for up to 12 months until disease progression or intolerable. Then, thalidomide maintenance will continue for another year or until intolerance. Meanwhile, we set Cyclosporin A (CsA) alone or plus steroids as control. Treatment dosage for CsA was 3-5mg/Kg/day with or without steroids (prednisone) 0.5-1 mg/Kg/day. The primary endpoint of this study was the complete response rate. Results: From Aug 2013, to Jan 2020, twenty-eight patients were enrolled in this study. The median follow-up time was 26 months (range: 7-96). Twenty-five patients (89%) achieved hematologic and symptomatic response. Among them, 21 patients (75%) achieved complete response (CR) and four patients achieved partial response. The median time to best clinical response was 6 months (2-18). The 3-years progression-free-survival (PFS) rate was 90%, and 3-years overall survival (OS) rate was 92%. The median PFS time was not reached in TPM group. The curative effect was better for TPM treatment group, both for overall response (OR) (TPM 89% (25/28) vs CsA 49% (49/99), P=0.000) and CR (TPM 75% (21/28) vs CsA 20% (20/99), P=0.000). Adverse events were uncommon, two patients had grade 1-2 nausea and one had grade 3 nausea. Two patients had grade 1-2 constipation and one patient experienced grade 1-2 peripheral neuritis. Conclusion: The efficacy of this TPM regimen is higher than the history reports with limited adverse events. The multiple-center clinical trial has been initiated to validate this conclusion.

Disclosures

No relevant conflicts of interest to declare.
Investigating the Dual Targeting of BCL-2 and CD38 in Models of Acute Myeloid Leukemia
Authors: Mistry Jayna J , Hellmich Charlotte , Lambert Amelia , Moore Jamie A , Jibril Aisha , Collins Angela , Di Palma Federica , Bowles Kristian M , Rushworth Stuart A
Published: November 2020

Acute myeloid leukemia (AML) is correlated with poor prognosis and a high mortality. Current AML treatment often fails to achieve complete remission and relapse is common, highlighting the need for more targeted treatments. Overexpression of BCL2 is a hallmark of AML progression and is often associated with a poor response to cytotoxic treatment. BCL2 inhibition by the BH3 mimetic Venetoclax has been shown to be effective in promoting AML cell death and recently Venetoclax has received FDA approval for the treatment of AML. However, some patients do not respond or can develop resistance, therefore multiple studies for combinational therapies for Venetoclax have been researched. We have previously demonstrated CD38 inhibition by daratumumab treatment inhibits mitochondrial transfer from mesenchymal stromal cell (MSC) to AML blasts in the bone marrow (BM) microenvironment, which results in a reduction in AML derived oxidative phosphorylation and subsequent reduced leukemia growth and improved animal survival. We therefore investigated the consequences of inhibiting CD38 using daratumumab and BCL2 using Venetoclax on the AML survival.

Primary AML blasts were isolated from patient's BM. CD38 expression and BCL2 expression was assessed by flow cytometry analysis of AML. AML had significantly higher BCL2 expression and a slight increase in CD38 expression compared to CD34+ cells. Venetoclax alone caused a significant decrease in cell viability, however daratumumab or in combination with Venetoclax had no additive effect on AML survival. Since AML is highly reliant on the BM microevironment we cultured AML on MSC with either Venetoclax alone, daratumumab alone, or Venetoclax and daratumumab for 24 hours. Cells were then stained with Annexin V-FITC/PI and analysed using flow cytometry. Cells underwent significantly more apoptosis in the combination Venetoclax and daratumumab treatment when compared to control AML cells.

To determine the effect of Venetoclax and daratumumab treatment in preclinical models we used an NSG xenograft mouse model of AML, we transplanted MV411-luc or patient derived AML and treated the animals with either vehicle control (PBS) daratumumab (5mg/kg) on day 7 and 14 alone, Venetoclax (100mg/kg/day) alone, or both daratumumab and Venetoclax followed by bioluminescence imaging. In vivo, treatment with combination daratumumab and Venetoclax significantly reduced tumor burden and improved survival compared to control and either drug alone in the patient derived AML xenograft mouse model and in MV411.

These data support the further clinical investigation of Venetoclax and Daratumumab combination as a therapeutic approach for the treatment AML.

Disclosures

Bowles: AbbVie: Research Funding; Janssen: Research Funding. Rushworth:Janssen: Research Funding; AbbVie: Research Funding.
Nanoparticle-Directed Targeting of Clustered PSGL-1 Mitigates Neutrophil-Derived Thrombosis in Antiphospholipid Antibody Syndrome
Authors: Sweet David R , Thomas Asha , Lapping Stephanie D , Kalikasingh Kenneth A , Ortel Thomas L. , Jain Mukesh K , Maiseyeu Andrei , Nayak Lalitha V.
Published: November 2020

Multiorgan thromboses and subsequent organ failure are among the more devastating consequences of antiphospholipid antibody syndrome (APS). APS poses a relatively unique risk to patients by virtue of its propensity to cause thrombi of both the arterial and venous vascular systems. While recent studies have explored mechanisms of thrombosis in singular vascular beds, there remains a dearth of targetable effectors of arteriovenous thrombosis in APS. Here, we examine the potential of neutrophils as key players of both arterial and venous thrombosis in APS. From this and previous studies, we have also identified a critical transcriptional regulator in neutrophils whose loss mimics arterial and venous thrombosis susceptibility seen in APS. This factor, Krüppel-like factor 2 (KLF2), is down regulated in APS patients as well as in cells treated with antiphospholipid antibody (APLA) ex vivo. Loss of KLF2 in neutrophils contributes to classic neutrophil activation phenotypes including NETosis, increased tissue factor release, and heightened chemotaxis; remarkably, blocking each of these functions greatly abrogates the thrombotic phenotype. Utilizing the myeloid KLF2 knockout (K2KO) system as a model for aberrant neutrophil activation seen in APS, we have identified clustering of P-selectin glycoprotein ligand 1 (PSGL-1) secondary to actin rearrangement to be operative in neutrophil-mediated thrombosis in arterial and venous systems. While neutralizing antibodies against PSGL-1 have been explored as therapeutic options in APS, there remains a challenge in establishing local specificity to activated immune cells in order to increase efficacy. To circumvent this, we have designed a nanoparticle (NP) based approach that utilizes NPs decorated with anti-PSGL-1 immunoglobulins to better target clustered PSGL-1 on activated neutrophils. PSGL-1 NPs are more effective at inhibiting neutrophil rolling and adhesion in in vitro assays while, incubating activated neutrophils in PSGL-1 NPs prior to infusion attenuates their ability to contribute to both arterial and venous thrombosis. Further, injection of PSGL-1 NPs directly into K2KO mice reduces their extensive thrombotic phenotype. Importantly, this effect occurs at a substantially lower dose of PSGL-1 NPs than that which would be needed of PSGL-1 antibody alone. Finally, PSGL-1 NPs greatly reduce thrombotic burden in mice injected with APLA, diminishing clotting in both arteries and veins. Together, this work identifies key molecular and cellular players in APLA-induced arteriovenous thrombosis. Furthermore, it provides early pre-clinical evidence at specifically targeting clustered PSGL-1 on neutrophils as a viable option in combatting thrombosis in this particularly vulnerable patient population.

Disclosures

No relevant conflicts of interest to declare.
Clonal Hematopoiesis in Patients with Diamond Blackfan Anemia
Authors: Nash Michelle , Vlachos Adrianna , Wlodarski Marcin W. , Lipton Jeffrey Michael
Published: November 2020

Background: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. Patients usually present during infancy or early childhood, but can also be diagnosed as adults. In the vast majority of cases DBA is due to a mutation in a gene encoding a small or large subunit-associated ribosomal protein (RP) leading to RP haploinsufficiency. In a study of 702 patients enrolled in the DBA Registry (DBAR), the observed to expected ratio for acute myeloid leukemia (AML) was 28.8 and for myelodysplastic syndrome (MDS), 352.1 (Vlachos et al, Blood, 2018). The average age of onset for MDS in the DBA cohort was 26 years, compared to 60-70 years in the general population. Evolving clonal hematopoiesis (CH) with age has been observed as a precursor to MDS, with CH rarely observed in individuals younger than 40 years of age. Thus we hypothesized that the young age at the development of MDS in DBA would be presaged by evolving CH.

Objective: The primary objective was to perform whole exome sequencing (WES) specifically screening for previously reported somatic mutations in 56 genes associated with CH (Jaiswal et al, NEJM, 2014).

Design/Method: A total of 69 samples were analyzed from 65 patients, mostly targeting patients older than 18 years (median age 30 years). Multiple samples were run on patients who had available samples in the DBAR Biorepository to determine rate of acquisition of mutations. 468 age- and sex-matched healthy controls were made available from GeneDx who performed the WES for the study. We used a threshold for variant calling of minimum 5% with a minimum of 2 variant reads.

Results: Three of the 65 DBA patients (5%) were found to have somatic mutations in STAG1, U2AF1, SF3B1, and DNMT3A at 8, 20, 41, and 70 years, respectively (Table 1). The patient who was 20 years of age had a sample in the DBAR biorepository from when he was age 8 years which was found to have a different somatic mutation (STAG1) than was found at present (U2AF1). This patient did go on to develop MDS at the age of 21 years. In comparison, of the 468 controls, 4 (0.8 %) had a somatic mutation in SF3B1, LUC7L2, DNMT3A, and LUC7L2 at ages 12, 31, 33 and 40 years, respectively.

Conclusion: Patients with DBA show more somatic mutations as compared to controls (p<0.05). This early acquisition of mutations may be the driving force for their developing MDS at an earlier age than that of the general population. Further studies with more sensitive methods are warranted to accurately determine the prevalence of somatic CH mutations and their potential association with the development of myelodysplastic syndrome in these patients.

Disclosures

No relevant conflicts of interest to declare.
This Is a Title: Single-Center Data Report of Clhag Regimen for R/R-AML Patients
Authors: Jiang Chuanyan , Wang Yu , Zou Mengying , Wang Yijing , Wang Jun , Wu Jiafei , Yang Xi , LI Hui
Published: November 2020

Objectives: To explore the synergistic effect of CLAG combination with HHT in cell experiments and to evaluate the effectiveness and safety of the CLHAG regimen for patients with relapsed/refractory acute myeloid leukemia(R/R-AML).

Methods:In thecell experiments, the Kasumi-1 cell line was treated with a single drug or CLAG combined with HHT. MTT colorimetric method was used to determine the cell growth inhibition rate(GIR). CompuSyn drug cooperation software was used to assess the synergistic effect.In the clinical study,eligible R/ R-AML patients (n=24) were included and CLHAG regimen (cladribine 5mg/m2, d1-5, arabinoside cytosine 2g/m2 q12h, d1-5, homoharringtonine 2mg/m2, d1-d5, GCSF 300μg, d0-d5, WBC>20×109/L disuse) was performed.The efficacy was evaluated by CR, PR, ORR, PFS, OS, and ED. The safety were evaluated by hematological toxicity and non-hematological toxicity.

Results: The GIR value with a combined treatmentregimenwas higher than the sum that with the single drug treatment, showing thata synergistic effect. Twenty-four patients with R/R-AML were included in the clinical study, including 2 cases of sAML (MDS transformation), 8 cases of primary refractory, and 14 cases of relapse.The total ORR was 70.8%; among them,14 cases achieved complete remission, and 3 cases achieved partial remission. Besides, seven patients underwentallo-HSCTsuccessfully. In this study, all patients were well tolerated, and early death was not observed. The IV grade hematological toxicity was the major adverse reaction, and no IV grade non-hematological toxicity occurred.

Conclusions:TheCLHAG regimen is well tolerated and has a high remission rate in R/R-AML patients, and it can be used as a choice of salvage treatment for R/R-AML patients.

Disclosures

No relevant conflicts of interest to declare.
The Role of 5-HT on Proplatelet Formation and Thrombopoietin Production
Authors: Yang Mo , Liang Enyu , Ye Jieyu , Chong Beng H , Li Liang
Published: November 2020

Background: Our previous work confirmed that serotonin (5-HT) promotes the proliferation of hemopoietic stem cells and megakaryocytes (Yang M et al, Stem Cells, 2007; 2014). However, the mechanisms remain indefinite.

Methods: Q-PCR, Flow Cytometry, Western Blot, or Immunofluorescence microscope were used in the receptor and TPO study. MTT/CCK-8, Proplatelet assay, and Flow Cytometry were also used in cell proliferation and apoptosis study. The relationship between 5-HT and TPO was studied in a traumatic stress mice model.

Results: In-vitro study, there was a stimulating effect of 5-HT on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum-free medium with either 5-HT (200nM) or TPO (100 ng/ml) had more proplatelet bearing MKs than the control group (5-HT (12.3 ± 5.0)% vs. Control (6.2 ± 3.5)%, P=0.025; TPO (15.6 ± 2.5)% vs. Control, P=0.04; n=4). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to the TPO group. 5-HT2A, 2B, 2C receptors were detected in the surface of megakaryocytes. The effect of 5-HT on proplatelet formation in MK cells was via 5-HT2 receptors and this effect was reduced by 5-HT2 receptor inhibitor ketanserin. 5-HT acted on cytoskeleton reorganization in MKs via 5-HT2 receptors and ERK1/2 pathway. Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, the polymerization actin level was higher in the 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced. Our data also demonstrated that ERK1/2 was activated in MKs treated with 5-HT for 30 minutes. In a traumatic stress mice model, both of 5-HT and TPO were increased, but the increasing of TPO is posterior to 5-HT. After added LX1606, the synthesis inhibitor of 5-HT, 5-HT was reduced markedly, as well as TPO. The expression of TPO mRNA and the production of TPO protein were increased as compared with the control in this model.

Conclusions: This study suggests that 5-HT promotes thrombopoiesis from two aspects: one is the direct effect on megakaryocytes. 5-HT could promote the proplatelet formation from megakaryocytes. The second is the indirect effect by promoting the production of TPO, which is a paracrine secretion to influence thrombopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Chronic Myeloid Leukemia (CML) in Adolescents & Young Adults (AYA): Single Institution 10 Years Experience with 1st Line Imatinib Mesylate (IM)
Authors: Malhotra Hemant , Yadav Ajay , Gupta Naveen , Soni Satyajeet , Singh Jitender Kumar , Malhotra Bharti
Published: November 2020

Introduction and Aims:

CML is the commonest adult leukemia in India and the disease is diagnosed commonly in the 3rd or 4th decade of life - at least 10 years earlier than in the west, making the disease an important issue in AYA cancers.

The present study reports our 10 year experience (all patients registered after 2005 with minimum of 3 years follow up) with the use of 1st line IM in patients of CML Chronic Phase (CML-CP) in patients aged 15 to 29 years.

Methods:

Patients of CML-CP receiving 1st line IM, aged 15 to 29 were included in the study. Standard demographics; hematological response, molecular responses (RQ-PCR on International Scale) q 6 monthly and toxicity of IM were assessed.

Results

A total of 337 patients of CML-CP were registered, 89 (28.7%) of which were aged between 15 to 29 and were on 1st line IM. These are reported in this study.

Age distribution: 15 to 19 years: 17 (19.1%); 20 to 24: 26 (29.2%); 24 to 29: 46 (51.7%). Male female ratio: 53 (59.5%) to 36 (40.5%).

Sixty one (68.5%) received innovator IM (Glivec) through the GIPAP (Glivec International Patient Assistance Program), 28 (40.5%) generic IM.

At 10 years f/u, 6 (6.7%) patients were lost to f/u, 15 (16.8%) off imatinib (12 sub-optimal molecular response/relapse, and 3 b/o toxicity - 2 hepatitis, 1 renal). Seventy four (74) patients continue on IM, 56 on 400 mg/day and 18 on > 400mg/day. Fifty nine are in deep molecular response, 15 have a bcr/abl between 0.1 to 1.0 but are in complete hematological remission.

Musculo-skeletal toxicity, grade I to II: 11/74 (14.8%), skin toxicity grade I: 27/74 (36.5%) and GI grade I to II: 16/74 (21.6%) were seen.

Discussion and Conclusion

CML is a common cancer in AYA in developing countries. IM, including generic IM, remains the standard 1st line drug for the majority and is effective, well tolerated in most patients. Issues related to treatment-free-remission (TFR), compliance, long-term drug toxicity and fertility need to be studied in this young population.

Disclosures

No relevant conflicts of interest to declare.
Comprehensive RNA Editome Reveals Azin1 As a Functional Regulator in Hematopoietic Stem and Progenitor Cells
Authors: Wang Fengjiao , He Jiahuan , Ma Yanni , Hao Sha , Liu Siqi , Gao Ai , Cheng Hui , Yu Jia , Cheng Tao
Published: November 2020

RNA editing, adenosine (A)-to-inosine (I), plays a vital role in many biological processes. Our previous study has demonstrated that the hematopoietic stem and progenitor cells (HSPCs) deficient in adenosine deaminase acting on RNA 1 (Adar1), an RNA-editing enzyme, cannot reconstitute the irradiated recipients in vivo and form colonies in vitro (Xufeng R et al, PNAS 2009). However, the overall profile of RNA editome in hematopoiesis has not been established and the underlying mechanism how RNA editing governs the function of HSPCs is poorly defined. In this study, we sorted 12 murine adult hematopoietic cell populations and performed RNA sequencing. We depicted the landscape of RNA editome in hematopoietic cells and identified 30,796 editing sites in total. The dynamic landscape of RNA editome comprised of stage/group-specific as well as house-keeping editing patterns. Notably, antizyme inhibitor 1 (Azin1) was uncovered to be highly edited in HSPCs. To understand whether edited Azin1 was required for functioning of HSPCs, we transduced c-Kit+ HSPCs with the lentivirus carrying Azin1 cDNAs with distinct editing frequencies. c-Kit+ cells transduced with fully edited Azin1 showed enhanced reconstitution, compared to that transduced with partially edited or non-edited Azin1. Specifically, inability of RNA editing in Azin1 blocked the differentiation of hematopoietic stem cells (HSCs) in vivo. Moreover, a similar finding was obtained when Azin1 was knocked down. In conclusion, RNA editing of Azin1 (i) results in amino acid change to induce AZIN1 translocation to the nucleus, (ii) enhances AZIN1 binding affinity for DEAD box polypeptide 1 (DDX1) to alter the DDX1 chromatin distribution, and (iii) changes the expression of multiple hematopoietic regulators to ultimately promote HSPC differentiation. This work provides a valuable resource for studying RNA editing and delineates an essential role of Azin1 RNA editing in HSPCs.

Disclosures

No relevant conflicts of interest to declare.
Anlotinib Shows Potent Antileukemia Effects in B-Cell Acute Lymphocytic Leukemia through the Blockage of Angiogenic Related Pathways
Authors: Chen Qiuling , Jiang Yuelong , Chen Qinwei , Liu Long , Xu Bing
Published: November 2020

Acute lymphoblastic leukemia (ALL) derives from the malignant transformation of lymphoid progenitor cells with ~85% being originated from B-cell progenitors (B-ALL). Despite fairly good prognoses for most pediatric B-ALL patients, the outcome is fatal in over 50% of adult patients who have a recurrent or progressive disease and lack of effective therapeutic approaches. Therefore, novel treatment strategies with high efficacy and low toxicity are an unmet need for B-ALL patients, especially those with relapsed or refractory status.

Angiogenesis is a process of new vessel formation that requires the participation of multiple proangiogenic factors (e.g., VEGF, PDGF, and FGF) and their corresponding receptors (e.g., VEGFR, PDGFR, and FGFR). Angiogenesis, a well-established feature of solid tumors, also contributes to leukemia progression and correlates with the involvement of specific sanctuary sites in ALL, highlighting that the perturbation of angiogenesis would be an attractive approach for ALL treatment. Anlotinib is an oral tyrosine kinase (TKI) inhibitor with a broad range of antitumor effects via the suppression of VEGFR, PDGFR and FGFR. Of importance, anlotinib has been approved for the treatment of advanced lung cancer in China. Here, we evaluated the antileukemia activity of anlotinib in preclinical B-ALL models and its underlying molecular mechanisms.

In this study, we observed that anlotinib significantly blunted the capability of cell proliferation and arrested cell cycle at G2 phase in B-ALL cell lines. Subsequently, we found that anlotinib resulted in remarkably enhanced apoptosis in B-ALL in vitro. To assess the in vivo antileukemia potential, we established a B-ALL patient-derived xenograft (PDX) mouse model and then treated the B-ALL PDX model with anlotinib. As a result, oral administration of anlotinib pronouncedly delayed in vivo B-ALL cell growth and reduced leukemia burden with acceptable safety profiles in this model. As for the mechanism of action, the antileukemia effect of anlotinib was associated with the disruption of the role of VEGFR2, PDGFRb, and FGFR3. Moreover, we revealed that this drug blocked the PI3K/AKT/mTOR/ signaling, a pathway that is linked with angiogenesis and its proangiogenic regulators, including VEGFR2, PDGFRb, and FGFR3.

In aggregate, these results indicate that anlotinib is a potent antitumor agent for the treatment of B-ALL via the inhibition of angiogenic relevant pathways, which provide a novel potential treatment intervention for patients with B-ALL who have little effective therapy options.

Disclosures

No relevant conflicts of interest to declare.

OffLabel Disclosure:

Anlotinib originally designed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.
Efficacy and Safety of Lenalidomide, Rituximab Combined with Second-Line Chemotherapy (R2-chemo) in Patients with Relapsed/Refractory Diffuse Large-B Cell Lymphoma
Authors: Xiao Fei , Huang Honghui , Hua Yanni , Fang Yi , Zhang Minyue , Hou Jian
Published: November 2020

Objective: Outcome of patients with relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL) remains poor, highlighting the need for novel treatment regimens. Lenalidomide is an immunomodulatory drug that could reverse rituximab refractoriness in lymphoma patients. This study aimed to explore the efficacy and safety of lenalidomide, rituximab combined with second-line chemotherapy (R2-chemo) in patients with r/r DLBCL.

Methods: In this study, r/r DLBCL patients received R2-chemo as second-line salvage therapy. The treatment plan was as follows: lenalidomide 10mg/day, day 1-14; rituximab 375mg/m2, day 1; chemo-regimen in combination with R2 as per Investigator's discretion. The overall response rate (ORR), complete remission rate (CR rate), duration of remission (DOR), disease-free survival (PFS) and overall survival (OS) were evaluated.

Results: As of Aug 1, 2020, 15 patients with r/r DLBCL were enrolled in this study with median age 65 years (range, 49 to 74 years), and 60% female. 20% (3/15) was transformed DLBCL, 33.3% (5/15) was relapse and 46.7% (7/15) had refractory disease. All received at least 1 prior line of immune-chemotherapy including rituximab, and 1 case was recurrence after autologous hematopoietic stem cell transplantation. 6.7% (1/15) had double-hit lymphoma, 66.7% (10/15) had MYC or BCL-2 high expression by IHC, of whom 26.7% (4/15) had co-expression of the MYC and BCL2. Cell of origin features using the Hans algorithm: 26.7% (4/15) were GCB, and 73.3% (11/15) were non-GCB. The chemo-regimens in combination with R2 were as follows: 5 cases of GEMOX or GCD, 7 cases of DICE or ICE, 2 cases of DHAP, 1 case of CHOPE regimen. As of the cutoff date, 13 cases were evaluable for efficacy. The ORR (CR+PR) was 61.5%, with 53.8% achieving a CR, and the median DOR was 9.7m (range, 2.9 to 23.7m). With a median time to follow-up of 15.3m (95%CI, 12.2 to 18.4m), median PFS and OS had not yet reached. The estimated 2-year PFS and OS rates were 53.8% and 75.2%, respectively. The most common treatment-related adverse event was hematological toxicity, and no treatment-related deaths occurred.

Conclusion: The R2 combined with second-line salvage chemotherapy regimen has a significant efficacy in the treatment of patients with r/r DLBCL, and can be well- tolerated with no significant additive toxicities. The efficacy and safety of this regimen and the choice of chemo-regimen as an optimal combination partner for R2 need to be further verified by prospective clinical studies with a larger sample size.

Figure

Disclosures

No relevant conflicts of interest to declare.
Crispra: A Promising Tool for β-Thalassemia Treatment
Authors: Jia Xiaoqing , Yao Qi , Li Hui , Chen Jieping
Published: November 2020

Induction of hemoglobin γ expression is a reliable strategy to treat β-thalassemia. Gene editing using CRISPR/Cas9 technology has been widely used. However, application in vivo is limited due to the uncertainty on genomic cleavages of Cas9. In contrast, CRISPR/Cas9-based gene activation (CRISPRa) can only locate genomic locus but not interrupt sequence. Here, we use SAM system of CRISPRa to locate and activate HBG1 and HBG2, exploring the great potential of CRISPRa for β-thalassemia treatment.

WWe designed 8 single-guide RNAs (sgRNAs) online and cloned into vector SAM V2, which fused dCas9 and VP64. To test the over-expression efficiency, vector containing sgRNA and MPH (fused HSF1, p65 and MS2) were transfected into 293T cell. After 72h transfection, 293T cells were collected. Q-PCR data showed that two sgRNAs were excellent on activating HBG expression with over 1000-fold increase.

WTo test the activating function in hematological cell and the persistence of hemoglobin γexpression, two screened sgRNA were transfected into NB4 cells using lentivirus system. We harvested NB4 cells at different time-point (3 day, 1 week and 2 weeks), and implemented q-PCR assay. HBG expression were increased 50-hold and 1000-hold, respectively. However, the expression were reducing over time and the intrinsic mechanism is unknown.

WThis study set out to increase HBG without interrupt genome using CRISPRa system. This study has found two sgRNA to activate the expression of HBG in 293T cell and NB4 cell. Further research is required to vertify the efficiency of sgRNA in hematopoietic stem cells and prolong the expression time.

Figure

Disclosures

No relevant conflicts of interest to declare.
Impact of Pulmonary Hypertension on in-hospital outcomes amongst Sickle Cell Disease hospitalizations
Authors: Jhaveri Khushali , Patel Raj , Barnett Christopher , Smith Hedy
Published: November 2020

Introduction: Pulmonary hypertension (PH) is a common and severe complication of Sickle Cell Disease (SCD), and an independent risk factor for mortality. While there is a clear association between SCD and PH, the predictors of PH in SCD and the impact of PH on in-hospital outcomes of SCD hospitalizations remains unknown. In our study, we sought to assess the in-hospital prevalence, predictors, and the impact of PH in SCD hospitalizations.

Methods:

We used the 2016 and 2017 National Inpatient Sample (NIS) to identify all adult hospitalizations with a primary discharge diagnosis of SCD. The sample was then stratified based on the presence or absence of PH. We used the Pearson chi-square test and weighted Student's t-test to analyze categorical and continuous variables, respectively. Multivariate logistic regression analysis was performed to calculate the adjusted odds ratio for various clinical outcomes. SAS was used for the analysis, and the p-value was defined as <0.05.

Results:

We identified n=191,080 weighted hospitalizations for SCD, of which, 5.54% (n=10590) had concomitant PH. Female gender and comorbidities including hypertension, obesity, illicit drug use, hepatic cirrhosis, renal failure, prior venous-thromboembolism, valvular, and congenital heart disease were identified as significant predictors of PH in SCD. PH was associated with increased in-hospital mortality (1.04% vs 0.22%, AOR=2.14, 95% CI 1.15-3.98, p=0.0158). PH in SCD hospitalizations also increased the odds of - acute kidney injury (AKI), need for dialysis, acute respiratory failure (ARF), and need for mechanical ventilation for > 96 hours. The adjusted odds ratio for venous thromboembolism, shock-state, and the need for cardiac catheterization (both right and bilateral) were also higher in patients with PH. Overall hospitalization cost and length of stay increased (7.06±0.16 vs 1.82±0.02 days) in patients with SCD and PH (see table 1).

Conclusion:

In sickle cell disease hospitalizations, PH is independently associated with increased in-hospital morbidity and mortality, with an increased need for in-hospital catheterizations thereby, prolonging the length of stay and overall health care costs. Identifying and treating PH in the SCD population would improve in-hospital outcomes.

Disclosures

No relevant conflicts of interest to declare.
Anti-Inflammatory Activities of Sulfated Non-Anticoagulant Heparin Derivative Beyond Its Anti-Sickling and Anti-Selectin for the Effective Management of Sickle Cell Disease
Authors: Darwish Noureldien , Abdulmalik Osheiza , Mousa Shaker A
Published: November 2020

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by the inheritance of a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases the adhesiveness of RBCs to alter the rheological properties of the blood and trigger inflammatory responses, leading to hemolysis, vasoocclusive crisis, pulmonary complications, plethora, and other pathological sequelae. Glycosaminoglycans, such as low molecular weight heparin (LMWH), have been suggested as treatments to relieve coagulation complications in SCD because of their ability to decrease thrombin generation and sickle cell adhesion. However, they are associated with bleeding complications after repeated dosing.

An alternative, sulfated non-anticoagulant LMWH derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectin-dependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice (1,2). S-NACH has been further engineered to possess an aldehyde moiety, which confers anti-sickling properties primarily due to specific interactions with HbS to increase its affinity for oxygen.

Our in vitro sickling assay under hypoxic conditions using S-NACH at 0.5 - 2 mM demonstrated that S-NACH significantly reduced the sickling of SS cells and in a concentration-dependent manner, with comparable to that of 1 mM GBT440 (Figure 1 A-C). A similar concentration dependent effect on increasing HbS affinity for oxygen using oxygen equilibrium study was documented.

In an vivo animal model using Townes' SCD animals plasma levels of pro-inflammatory cytokines IL-1β, IL-6, IFN-γ, MCP-1, TNF-α, M-CSF, and VEGF were increased in SCD untreated samples in contrast to a significant decrease (*P< 0.001) in S-NACH-treated animals, at both 2 and 6 h. In addition, S-NACH was able to increase the decreased levels of the endogenous anti-inflammatory IL-10 (Figure 1 D).

The above set of novel findings about S-NACH established it to be effective for the management of SCD via the modulation of thromboinflammatory pathways, involved in thromboembolism and end organ damage, beside anti-sickling, anti-selectin and without causing any bleeding risk.

Reference

1. Alshaiban A, Muralidharan-Chari V, Nepo A, Mousa SA. Modulation of Sickle Red Blood Cell Adhesion and its Associated Changes in Biomarkers by Sulfated Non-anticoagulant Heparin Derivative. Clin Appl Thromb Hemost. 2016 ;22(3):230-8.

2. Mousa SA: Compositions and method for anti-sickling of red blood cells in sickle cell disease. US Patent 9,822,190, November 2017.

Disclosures

Mousa: Vascular Vision Pharma Co.: Patents & Royalties.
Role of Roxadustat in Improving Erythropoiesis-Stimulating Agent (ESA)-Resistant Anemia in Patients on Maintenance Hemodialysis
Authors: Funakoshi Satoshi
Published: November 2020

Background: Roxadustat, an oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor, is shown to stimulate erythropoiesis thus improving iron metabolism. Again, while hyperglycemic states are known to be associated with a decreased tissue hypoxia response, to date, roxadustat has not been revaluated for its role in improving anemia in patients with or without diabetes in clinical settings.

Methods: A total of 64 hemodialysis patients being treated with epoetin α (9000 units weekly) participated in the study after giving informed consent. They were switched from intravenous epoetin α to oral roxadustat (100 mg 3 times weekly) therapy and were assessed 8 weeks later for improvements in anemia, as well as for changes in parameters for iron metabolism and C-reactive protein (CRP).

Results: The study included 39 patients without diabetes (mean age, 71.1 ± 12.1 years; mean dialysis vintage, 7.5 ± 7.4 years; mean GA, 16.2 ± 2.9%) and 27 patients with diabetes (mean age, 70.3 ± 10.3 years; mean dialysis vintage, 5.9 ± 5.5 years; mean GA, 24.9 ± 5.5%). As shown in Table, after 8 weeks the Hb concentration was significantly increased from 10.3 ± 0.8 g/dL at baseline to 10.7 ± 1.3 g/dL in patients without diabetes (P = 0.03) but was not increased in patients with diabetes (from 10.4 ± 0.6 at baseline to 10.5 ± 1.1 g/dL). Again, the serum iron, ferritin concentrations and the transferrin saturation ratio were decreased, irrespective of whether or not they had diabetes, with no change shown in serum CRP level.

Conclusion: Switching hemodialysis patients with ESA-resistant anemia from ESA to roxadustat led to improvements in anemia only in those without diabetes, while study results suggested the involvement of mechanisms, other than impaired iron utilization or inflammation, in the impairment of hematopoiesis in those with diabetes.

Table

Disclosures

No relevant conflicts of interest to declare.