bluuebunny/crossref_metadata_2025 huggingface dataset:
Hemoglobin Variants Influence Plasmodium Falciparum Sexual Differentiation
Authors: Flage Bethany , Dent Matthew , Tejero Jesús , Ofori-Acquah Solomon Fiifi
Published: November 2021

It has long been recognized that individuals who express variations of the hemoglobin-A (HbA) protein experience less severe malaria disease. As malaria remains to be one of the most significant infectious diseases in history, this human adaptation has led to the persistence of HbA variants (HbVARs) in the population. The intricate lifecycle of the parasite which causes the most cases of clinical malaria, Plasmodium falciparum, relies on both asexual and sexual reproductive cycles, with host to vector transmission reliant on sexual stage gametocyte formation. Multiple epidemiological studies have shown that HbVARs may influence gametocyte production during P. falciparum infection, with greater gametocyte numbers reported in individuals with hemoglobin variant containing erythrocytes (Hb VAR-Ery) when compared to hemoglobin A containing erythrocytes (Hb A-Ery). Here we provide experimental support for these studies by showing significantly higher sexual differentiation rates among parasites grown in Hb S containing erythrocytes (Hb S-Ery) obtained from sickle cell patients than those differentiated in Hb A-Ery (p=0.038). Because the digestion of hemoglobin is such an integral part of the intraerythrocytic cycle, we then sought to determine whether there was a difference between the hydrolysis efficiencies of HbA and other hemoglobin variants (HbVAR). By using a prominent recombinant P. falciparum hemoglobinase we found the hydrolysis efficiency of HbA to be significantly (p=0.0058) more efficient after 24 hours compared to a HbVAR sample containing mixed amounts of HbA, HbF, and HbS. To further determine whether there is a link between hemoglobin digestion efficiency and sexual differentiation, we therapeutically inhibited the hemoglobin digestion and hemozoin formation process in a culture of P. falciparum using sub-optimal doses of chloroquine diphosphate. We found a significant difference (p<0.001) among gametocyte conversion rates between treated and non-treated cultures, as well as a moderate negative correlation between hemozoin formation and gametocyte conversion rate (Pearson r=0.72, p=0.008). Gene expression analysis also revealed patterns of expression that were consistent with increased gametocytogenesis. We conclude that hemoglobin type plays a significant role in the process of sexual conversion in P. falciparum. Though further studies should be completed in order to confirm these results, these findings may suggest hemoglobin digestion efficiency as a causative factor for sexual differentiation. As individuals with hemoglobinopathies make up approximately 7% of the global population, and malaria infection rates have been shown to differ depending on these genetic dynamics, these findings may support the creation of targeted initiatives to reduce transmission specifically in areas where there is a high percentage of hemoglobinopathy carriage.

Disclosures

No relevant conflicts of interest to declare.
Sickle Cell Disease Bias: An Early Opportunity for Intervention?
Authors: Patel Viral M , Ibrahim Ibrahim F.
Published: November 2021

Background: Sickle cell disease (SCD) is a genetic condition leading to multi-organ dysfunction and chronic pain, with 1 out of 13 African Americans having the trait. Despite its prevalence and debilitating symptoms, SCD remains a disease with limited therapeutic interventions and decreased life expectancy. There is a clinical bias to undertreat the pain of patients with SCD with opioid medications, despite their effectiveness (Zempsky 2009). Early education about SCD may lead to improvements in provider perceptions and disease management.

Methods: An anonymous survey was sent to first-year medical students at an academic institution prior to their hematology course. Data was collected about demographics, prior exposure to SCD, perceptions about opioid use disorder (OUD) in SCD, and baseline knowledge of management. Identical questions were asked for cancer-related pain. The survey was sent again after the completion of the hematology course to assess whether the curriculum was effective at characterizing misperceptions about patients with SCD.

Results: Fifty-five medical students answered the survey prior to the course. Students were 38% male and 62% female. Seventy-five percent of students (N=41) had never encountered a patient with SCD. Forty percent (N=22) answered that OUD in patients with SCD is more likely, 60% as likely (N=33), and 0% less likely than the general population. Seventy-one percent (N=39) believed opiates should play a minor role, 5% (N=3) a major role, and 24% (N=13) said opioids should be avoided when managing SCD-related pain. After course completion, 35 students who had completed the survey prior to the course responded, 29% male and 71% female. Fifty-four percent (N=19) answered that OUD in patients with SCD is more likely, 43% (N=15) as likely, and 3% (N=1) less likely than the general population. Seventy-one percent (N=25) thought opiates should play a minor role, 9% (N=3) a major role, and 20% (N=7) said they should be avoided when managed SCD-related pain.

Conclusion: After course completion, a higher percentage of medical students thought that OUD was more frequent in patients with SCD than the general population, with a similarly high percentage determining that opioid medications should play a minor role or be avoided in SCD. Opioid medications are an important part of treatment for SCD, especially for acute vaso-occlusive pain. Patients with SCD are no more likely to have OUD than the general population and have a lower rate of OUD than other patients with chronic pain conditions (Solomon 2008). This pilot study highlights that there is an opportunity to intervene in misperceptions of patients with SCD in early medical school education.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Harnessing Extracellular Vesicles from Red Blood Cells for Targeted Delivery of Therapeutic Peptides and RNAs for Leukemia Treatment
Authors: Jayasinghe Migara Kavishka , Pirisinu Marco , Chen Huan , Yang Yuqi , Peng Boya , Shi Jiahai , Le Minh TN
Published: November 2021

Extracellular vesicles (EVs) are emerging as a new class of natural drug carriers with intrinsic ability to deliver bioactive cargo at high efficiency and low toxicity. However, clinical applications of EVs are limited by the production scale and the delivery specificity. We have recently established a new platform for purification and surface modification of EVs from red blood cells (RBCs) that are scalable and versatile for targeted delivery of small-molecule drugs and RNA therapeutics (Usman et al, Nat. Com. 2019 and Pham et al, J. Extracell. Vesicles 2021). Here, we describe a new development of the method for conjugation of RBC-EVs to obtain a higher targeting efficiency. We conjugate RBC-EVs, not only with peptides and nanobodies, but also with monoclonal antibodies, by coupling peptide conjugated EVs with streptavidin-bound biotinylated antibodies. Our data demonstrate that the conjugation is stable and does not affect the physicochemical characteristics of EVs. Conjugation of EVs with a cyclic peptide targeting CXCR4 or a monoclonal antibody targeting CD33 promotes specific binding and uptake of the conjugated EVs by leukemia cells expressing the corresponding receptors. We further use CXCR4-targeting RBC-EVs to specifically deliver the pro-apoptotic peptide KLA to CXCR4-expressing leukemia cells. Delivery of KLA using CXCR4-targeting EVs significantly suppresses leukemia burden and increases survival in a leukemia xenografted mouse model. Antibody-conjugated RBC-EVs are used to deliver RNA antisense oligonucleotides to knockdown FLT3 and miR-125b in cell lines and in patient-derived xenograft models of leukemia. Finally, we demonstrate that peptide/antibody conjugated RBC-EVs are biocompatible and nonimmunogenic. Our study provides a new platform for targeted delivery of therapeutic peptides and RNAs that is highly efficient, stable, versatile and biocompatible for potential clinical applications in leukemia treatment.

Migara Kavishka Jayasinghe, Marco Pirisinu, Huan Chen, and Yuqi Yang contributed equally to this work as first authors.

Disclosures

Jayasinghe: Carmine Therapeutics: Patents & Royalties. Shi: Carmine Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Le: Carmine Therapeutics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
Outpatient Based Autologous Hematopoietic Stem Cell Transplantation Is a Feasible Therapeutic Option for Patients with Chronic Inflammatory Demyelinating Polyneuropathy: The Mexican Experience
Authors: Murrieta-Álvarez Iván , Bojalil-Álvarez Lorena , Cantero-Fortiz Yahveth , García-Villaseñor Elizabeth , León-Peña Andrés A. , Priesca-Marin José Manuel , Olivares-Gazca Juan Carlos , González-López Elías , Gomez-De Leon Andres , Ruiz-Delgado Guillermo J. , Gomez-Almaguer David , Ruiz-Argüelles Guillermo J.
Published: November 2021

Introduction

Several autoimmune disorders such as multiple sclerosis, Stiff man syndrome or chronic inflammatory demyelinating polyneuropathy (CIDP) can benefit from autologous hematopoietic stem cell transplantation (HSCT) due to its immunomodulatory effects at short- and long-term. Here we present the results from the conduction of an outpatient HSCT procedures in 16 patients with CIDP.

Methods

Prospective study of HSCT in persons with CIDP recruited since 2015. Patients with and without previous immunomodulatory therapy were enrolled and a 3-month wash-out period was recquired; previous or current use of walking support was not a contraindication. All patients signed a consent before starting the study and the study protocol was approved by Clínica Ruiz IRB. Before the conditioning, all participants underwent a comprehensive evaluation. On day -11 and -10, patients received cyclophosphamide (Cy) at 50 mg/kg, followed by a 7-day course of G-CSF previous to cell collection (day -2) through apheresis using a Mahurkar catheter. Then, on day -2 and -1 a second round of Cy at 50 mg/kg was given (Figure 1). Transfusions and antimicrobial prophylaxis were administered per local protocol. Upon hematological recovery (>500 ANC) all patients received a rituximab infusion at 1000 mg.

Results

Sixteen patients with CIDP underwent screening for transplantation clearance and all of them proceeded to HSCT. Of those, 8 (50%) patients were male. Median age at transplantation was found to be 44 years (SD 10.5). Walking support was employed at the time of HSCT by 7 (44%) patients and most frequent previous therapy was intravenous immunoglobulin in 4 (25%) patients. On average a mean 8.5x10 6/kg (SD 4.2) of CD34 + cells were infused. The mean nadir neutropenia was found to be 0.11x10 9/L (SD 0.13) and the median time of granulocytes recovery was 7.0 days (SD 2.9) while median time of platelet recovery was 5.5 days (SD 3.0). One (6%) patient was admitted to hospital due to febrile neutropenia and 4 (25%) patients required red-blood cell and platelet transfusion support. In 3 of 5 persons the need of walking support reverted.

Conclusions

Our experience suggests that people with CIDP may undergo HSCT with no major complications on an outpatient basis. Hospital admissions, hematological recovery, and infectious complications seem to be relatively low-severity complications that could be manage with standard protocols.

Figure 1 Figure 1.

Disclosures

Gomez-Almaguer: Bristol-Myers-Squibb: Honoraria, Speakers Bureau; Roche: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau.
Momelotinib Is a Highly Potent Inhibitor of FLT3-Mutant AML
Authors: Azhar Mohammad , Kincaid Zachary , Kesarwani Meenu , Wunderlich Mark , Latif Tahir , Starczynowski Daniel , Azam Mohammad
Published: November 2021

Approximately one-third of AML patients harbor kinase activating mutations in FLT3. Several small-molecule first generation FLT3 tyrosine kinase inhibitors (TKIs) have been evaluated in the last two decades, but none could induce a durable response possibly due to poor pharmacokinetics and target selectivity. Second generation FLT3 inhibitors such as, quizartinib, gilteritinib, and crenolanib, were designed for greater selectivity with a narrow kinome-profile and better pharmacokinetic properties, but they failed to induce a durable response. Possibly due to intrinsic resistance conferred by growth factor signaling in the bone marrow resident leukemic cells, which serve as a reservoir to develop resistance resulting to disease relapse. Activation of Jak2 signaling by chemokines and cytokines from the stroma have been reported to confer TKI refractoriness. Recent studies from Martin Carroll's group showed that both GMCSF and IL-3 confer resistance by activating JAK2 signaling, which can be suppressed by combined FLT3 and JAK2 inhibition. However, so far, it is not established whether upfront FLT3/Jak2 inhibition will provide durable response.

For instance, AML patients who achieved complete remission lacking FLT3-ITD clones showed better overall survival than patients with measurable MRD, suggesting that eradicating the FLT3-ITD clones will have a deeper response with better overall survival. Thus, an ideal FLT3 inhibitor should be able to suppress FLT3-ITD resistant mutants, and growth factor activated JAK2 signaling while sparing c-KIT receptor and hERG to avoid myelosuppression and cardiotoxicity, respectively. Here we show that Jak2 inhibitor, momelotinib, is an equipotent type-I FLT3 inhibitor (Fig 1A-D). Biochemical and structural modeling revealed that it binds to an active conformation of FLT3 kinase. Therefore, like gilteritinib, it efficiently suppresses the resistance conferred by activation loop mutations (Fig1 E-H). Moreover, its lack of activity against c-KIT and inhibition of ACVR1 provides additional benefit in alleviating myelosuppression and anemia, which is commonly observed with currently used JAK2 and FLT3 inhibitors (ruxolitinib, fedratinib, and quizartinib). Perhaps more interestingly, momelotinib efficiently suppressed the disease in a preclinical model of AML using NSGS mice which recapitulates cytokine induce refractoriness as usually observed in clinical setting (Fig1 I-L). Our preclinical data provide evidence that momelotinib is an equipotent dual JAK2/FLT3 inhibitor and suppresses resistance conferred by both activation-loop mutations and growth-factor signaling. These data provide evidence that momelotinib treatment will have clinical activity in FLT3-mutated AML. Thus, warrants its clinical evaluation.

Figure 1 Figure 1.

Disclosures

Starczynowski: kurome Inc: Consultancy.
Effect of Thrombospondin-1 on Apoptosis of Human Megakaryocytic Leukemia Cells
Authors: Kong Huimin , Jiang Qianli , Su Weiqing , Luo Yi , Ge Hui , Li Liang , Chong Beng H , Yang Mo
Published: November 2021

Background: Thrombospondin 1 (TSP-1) is an extracellular matrix protein that interacts with a wide array of ligands including cell receptors, growth factors, cytokines, and proteases to regulate various physiological and pathological processes. TSP-1 induces apoptosis of endothelial and cancer cells via its receptor CD36. This study was to investigate the effect of TSP-1 on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism.

Methods: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations, to investigate the effect of TSP-1 on the growth of megakaryocytes. The early apoptosis and the activity of the apoptotic protease caspase-3 were detected by flow cytometry. Bone marrow cells of mice were cultured for CFU-MK and stained with acetylcholine to detect the differentiation of megakaryocytes.

Results: CD36 antigen was detected on the surface of Meg-01 cells by flow cytometry and immunocytochemistry. TSP-1 (5 μg/mL) inhibited the proliferation of Meg-01 cells, but not M-07e cells (CD36 -). After the addition of CD36 antibody FA6-152 (5, 10 and 25 μg/mL), the inhibitory effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5 and 7.5 μg/mL) exerted a pro-apoptotic effect by increasing the expression of Annexin V (P<0.01) and caspase-3 activation (P<0.01). Addition of FA6-152 (25 μg/mL) can significantly reduce the apoptosis induced by TSP-1 in Meg-01 cells. In addition, TSP-1 (5, 10 and 25 μg/mL) repress the formation of CFU-MK in mouse bone marrow cells, while β-TG did not. This repression was relieved efficiently by FA6-152 (25 μg/mL).

Conclusion: TSP-1 could inhibit the proliferation of Megakaryocyte cell line Meg-01 cells, and induce it`s apoptosis. TSP-1 may induce apoptosis of Meg-01 cells via CD36 or caspase-3, which provides a potential new drug choice for clinical treatment of megakaryocytic leukemia.

Disclosures

No relevant conflicts of interest to declare.
IL-1 Via IRAK1/4 Sustains Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse
Authors: Ho Tzu-Chieh , Kawano Hiroki , LaMere Mark W. , Guo Naxin , Chiu Yu-Chiao , Wang Jian , Jordan Craig T. , Dokholyan Nikolay V. , Calvi Laura M. , Singh Rakesh Kumar , Becker Michael W.
Published: November 2021

Current treatment options for relapsed acute myeloid leukemia (AML) are limited and ineffective for the majority of patients. In AML, primitive leukemia stem cells (LSCs) and pre-leukemic populations are able to maintain the disease and drive relapse. Thus, therapies targeting LSC populations may increase the overall survival of AML patients. In this study, we aim to identify the drivers favoring LSC expansion following treatment and relapse and develop potential therapies for AML. The transcriptome analyses of 12 pairs of functionally defined LSC fractions at diagnosis and relapse revealed significant changes of IL-1 signaling in AML patients. We demonstrated that the protein expression levels of interleukin-1 receptor type I (IL1R1) and its complex member interleukin-1 receptor accessory protein (IL1RAP) were both up-regulated in human leukemia stem and progenitor cells (LSPCs) at diagnosis or in relapse compared to normal hematopoietic stem and progenitor cells (HSPCs). Knockdown of IL1R1 and IL1RAP suppressed the clonogenicity and engraftment growth of primary human AML cells but showed low impacts on HSPCs in the normal bone marrow. Additionally, knockout of IL1R1 in leukemia MLL-AF9 mice significantly reduced the LSC frequency and prolonged the overall survival rate. To target IL-1/TLR signaling in LSCs, we performed iterative structure-activity relationship (SAR) guided medicinal chemistry, in silico modeling and leukemia cell line reporter assays to screen and identify a novel interleukin-1 receptor-associated kinase 1/4 (IRAK1/4) inhibitor (termed UR241-2). UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB following IL-1 stimulation. UR241-2 repressed LSPC function as assessed by colony-forming unit assays in primary human AML cells at diagnosis and in relapse while minimally impacting normal HSPC function. Taken together, our findings demonstrate the important role of IL-1/TLR signaling in supporting AML LSC expansion following treatment and relapse and suggest that targeting IL-1/TLR signaling using the novel IRAK1/4 inhibitor, UR241-2, can target LSC function to improve patient outcomes in AML.

Disclosures

No relevant conflicts of interest to declare.
An Autosomal Dominant Form of Ras-Related C3 Botulinum Toxin Substrate 2 (RAC2) Is Associated with Haematopoiesis Failure
Authors: Lagresle-Peyrou Chantal , Olichon Aurélien , Sadek Hanem , Roche Philippe , Tardy Claudine , Sorel Natael , Bessot Boris , Da Silva Cindy , Fischer Alain , Moshous Despina , Collette Yves , Picard Capucine , Casanova Jean-Laurent , André Isabelle , Cavazzana Marina
Published: November 2021

Severe combined immunodeficiencies (SCIDs) constitute a heterogeneous group of life-threatening genetic disorders that typically present in the first year of life. Reticular dysgenesis (RD) is an autosomal recessive form of human Severe Combined Immunodeficiency (SCIDs) characterized by the absence of blood neutrophils and T lymphocytes. This pathology is due to biallelic mutations in the adenylate kinase 2 (AK2) gene, encoding for a mitochondrial protein which regulates the homeostasis of adenine nucleotides.

In three newborns presenting a RD-like clinical phenotype as frequent infections and a profound leukopenia, we identified an heterozygous dominant missense mutation in the gene coding for Rac Family Small GTPase 2 (RAC2) protein (p.G12R). Hematopoietic stem cell transplantation (HSCT) performed soon after birth was successful in two out of the three patients attesting that the inherited defect was intrinsic and not micro-environmental. RAC2 protein belongs to the Rac subfamily of RHO small GTPases. In the inactive GDP-bound state, RAC2 is located in the cytosol and upon stimulation, the active RAC2-GTP-bound form translocates to the plasma membrane. Unlike the other members of the Rac subfamily (RAC1 and RAC3), RAC2 is mostly expressed on hematopoietic cells and during T cell differentiation.

To gain insight into the disease, we transduced human hematopoietic stem and progenitor cells (HSPCs) with a lentiviral construct containing the RAC2 mutated form. The mutation inhibits HSPCs proliferation and differentiation toward the myeloid and lymphoid lineages reproducing the patients' clinical phenotype. In this condition, we also observed high apoptosis level and an alteration of mitochondrial activity and Ros production. In a biochemical model, we demonstrated that the substitution of the glycine (G) amino-acid by a bulky flexible arginine (R) may prevent GTP hydrolysis. Lastly, our findings suggest that RAC2 gene sequencing must be considered in newborn screening programs for SCID detection.

To decipher the mechanisms regulating RAC2 functions, we studied the impact of the p.G12R mutation on a human AML cell line expressing RAC2 protein. Our preliminary data highlight that the cell cycle and mitochondrial activity are disrupted by G12R mutation. By holotomography, we also observed morphological changes and accumulation of lipid droplets into the cells. All these data suggest that RAC2 defective signalling pathway is linked to cell metabolism imbalance and further investigations are ongoing to better understand how RAC2 controls cell energy demand, especially during differentiation.

Disclosures

Cavazzana: Smart Immune: Other: co-founder.
Targeted Therapy in CD38 + Acute Myeloid Leukemia
Authors: Hu Naibo , Gao Chao , Zhang Yuhui , Teng Guangshuai , Wang Yan , Du Chenxiao , Bai Jie
Published: November 2021

The expression rate of CD38 in acute myeloid leukemia is high. It is reported that the expression of CD38 in myeloid leukemia is stable and can be used as a potential therapeutic target for leukemia. Daratumumab is a therapeutic drug targeting CD38. It is widely used in myeloma, but it has not been applied in AML. The prognosis of 4 patients with acute myeloid leukemia treated with CD38 monoclonal antibody combined with demethylating drugs / traditional chemotherapy was reviewed. Patient demographic data and clinical characteristics see table 1. One patient with de novo AML in the high-risk group achieved remission after Dara combined with azacytidine and venetoclax. The other three patients were suffering Relapsed/Refractory AML. Two of them achieved remission after Dara combined with azacytidine and venetoclax. The patient, who was non-remission after Dara combined with IA chemotherapy (Idarubicin, Cytosine Arabinoside), achieved remission after CART targeting CD38. In this paper, four cases were reported, and the rationality of CD38 targeted therapy was further analyzed.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Ruxolitinib Therapy during Hematopoietic Cell Transplantation for Myelofibrosis
Authors: Tan Yaxian , Luo Jie , Luo Dongmei , Liang Hanying , Liu Xiaoli , Liu Qifa , Xu Na
Published: November 2021

Background :The beneficial effects of JAK 1/2 inhibitors (JAKi) ruxolitinib in MF patients have led to control of disease related symptoms,but ruxolitinib has been limited by their inability to deplete the malignant stem cell clones. Allogeneic hematopoietic cell transplantation (HCT) is currently the only treatment with proven curative potential for myelofibrosis (MF). Previous research suggested pre-HCT ruxolitinib may improve post-HCT outcome because of ruxolitinib accelerate engraftment, improve graft function, decrease the risk and severity of graft-versus-host disease (GVHD). However,HCT in PMF with higher non-relapse mortality (NRM),In the present study, we aimed to prospectively assess the effects of Rux therapy during HCT period(NCT04526223).

Objective Preliminarily investigate the safety and efficacy of ruxolitinib in myelofibrosis patients during the peri-transplantation period.

Method: To prospectively analyze the clinical data of 26 patients with myelofibrosis who were used DAC bridge BF as conditioning treatment and treated with ruxolitinib(5mg bid +6d to +60d) during the peri-transplantation period.

Results: 26 patients were eligible if they had primary myelofibrosis (PMF) as defined by the 2008 World Health Organization classification or secondary MF as defined by the IWG for Myeloproliferative Neoplasms Research and Treatment criteria and met the criteria for Intermediate-1 (Int-1), Int-2, or high-risk disease by the DIPSS scoring system at the time of enrollment. Patients consenting to treatment with Rux for at least 8 weeks prior to HCT and could receive Rux (5mg bid +6d to +60d) during the peri-transplantation period. DAC bridge BF as Myeloablative regiment, There have been no episodes of cytokine release syndrome and all patients achieved sustained engraftment. Neutrophil reconstitution was obtained in 26 patients with a median time of 15 (10 to 31) d; platelet reconstitution was obtained in 26 patients with a median time of 26(12 to 68) d. Acute GVHD occurred in 26 patients (46.15%) , of which 1(3.8%) were ш°; chronic GVHD occurred in 9 patients (34.6%), 1 patients with extensive chronic GVHD. There patients relapsed and two patients died because of resistance to alvage treatment . With a median follow-up 336 (70-993) days，no patient died of non-relapse mortality (NRM)., overall survival is 92.3% (95% CI: 0.73, 0.97) and DFS is 88.5% (95% CI: 0.63, 0.96) at1 year post-HCT.

Conclusion: This study demonstrates that Ruxolitinib therapy during HCT is well tolerated and suggests that during-HCT Rux may improve post-HCT outcome.

Keywords: Ruxolitinib;Hematopoietic Cell Transplantation;Myelofibrosis

Disclosures

No relevant conflicts of interest to declare.
Using the Iron Chelator Deferasirox to Overcome Drug Resistance in Mantle Cell Lymphoma
Authors: Samara Aladin , Shapira Saar , Lubin Ido , Raanani Pia , Granot Galit
Published: November 2021

Mantle cell lymphoma (MCL) is a difficult-to-treat B-cell malignancy characterized by the t(11,14) translocation, resulting in cyclin D1 (CD1) overexpression. In addition to CD1 overexpression, pathways such as the B-cell receptor, PI3K/AKT/ GSK3β, NFκB and Wnt have been reported to be deregulated in MCL. Despite advancements in MCL treatment, most patients still relapse. Although the introduction of ibrutinib to relapsed/refractory MCL significantly improved the outcome of MCL patients, ibrutinib resistance has become a clinical obstacle. MCL treatments are thus pursued by studying novel agents with a broad spectrum of targets or by rationally combining existing therapies aiming for synergistic antitumor activities. Deferasirox (DFX) is a clinically approved iron chelator with only few side effects. DFX has been reported to exert anti-tumoral and synergistic effects in several types of cancers by affecting a multitude of targets. We have previously shown that DFX exerts a vigorous anti-tumoral effect via growth inhibition and induction of apoptosis in MCL cells through ROS elevation, triggering of oxidative stress, induction of DNA damage, modulation of PI3K/AKT/GSK3β signaling and most importantly by elimination of CD1 expression. The capacity of DFX to affect a multitude of targets establishes a solid basis for a possible synergistic interaction with other drugs, such that may overcome drug resistance in MCL. In this study we focused on assessing the efficiency of DFX combined with the established therapeutic-agents etoposide, cytarabine and ibrutinib in MCL cell-lines. We found that DFX synergizes with etoposide, cytarabine and ibrutinib, prompting remarkable anti-tumoral effects in MCL cells with combination index (CI) values < 1. Interestingly, the DFX-drug combinations achieved synergism regardless of the innate sensitivity of the cell-lines to the treatment: ibrutinib-resistant cells restored their sensitivity to the drug when it was combined with DFX. In addition, we found that the sensitivity of MCL cells towards the drugs correlated with the drugs ability to induce CD1 degradation. In agreement, DFX co-treatment enhanced CD1 degradation, especially in resistant cells.

We show here that DFX is a putative promising drug-sensitizing agent for the treatment of MCL. DFX-co-treatment not only enhances the efficacy of the tested drugs, but also restores the anti-proliferative activity of the drugs in resistant MCL cells. To the best of our knowledge, this study is the first to provide evidence on the potential of DFX to overcome drug resistance in MCL.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Preliminary Results of Zanubrutinib-Containing Regimens in DLBCL and Cerebrospinal Fluid Distribution of Zanubrutinib
Authors: Zhang Yan , Li Yanan , Zhuang Zhe , Wang Wei , Wei Chong , Zhao Danqing , Li Jian , Zhou Daobin
Published: November 2021

Background: Zanubrutinib is a second-generation Bruton's tyrosine kinase (BTK) inhibitor. The safety and efficacy of zanubrutinib in CNS lymphoma are unknown, as well as its brain distribution through the blood-brain barrier.

Objective: A retrospective study of zanubrutinib-containing regimens was performed in patients with DLBCL to evaluate the efficacy of zanubrutinib. Paired plasma and cerebrospinal fluid (CSF) samples were collected to assess zanubrutinib's permeability through the BBB.

Methods: Consecutive DLBCL patients treated with zanubrutinib-containing regimens from August to December 2020 in PUMCH were recruited. Paired plasma and CSF samples were collected at a fixed time point after zanubrutinib administration. Plasma and brain zanubrutinib quantification was performed by liquid chromatography-tandem mass spectrometry.

Results: Totally 13 patients were enrolled, including primary CNS lymphoma (n=8) and systemic DLBCL (n=5). 53.8% (7/13) were refractory/relapsed, 84.6% (11/13) had CNS involvement. Overall response rates (ORRs) were 84.5% and 81.8% in the whole population and CNS involved cases, respectively. A total of 23 time-matched plasma-CSF sample pairs were collected. The mean peak concentration of zanubrutinib in the CSF was 2941.1 pg/ml (range, 466-9032.0). The corrected mean CSF/plasma ratio by protein binding of 94.0% was 42.7±27.7% (range, 8.6-106.3%), indicating the good penetrating ability through the BBB of free drug. The CSF/plasm ratio was not influenced by sex, DLBCL subtype, co-administrated BBB penetrating drugs or plasma drug concentration.

Conclusion: This preliminary study revealed the efficacy of zanubrutinib-containing regimens in DLBLC, especially CNSL, for the first time. The excellent BBB penetration of zanubrutinib supports its further investigation in CNS lymphomas.

Disclosures

Li: Astellas Pharma, Inc.: Research Funding.

OffLabel Disclosure:

Zanubrutinib is a selective Bruton Tyrosine kinase(BTK) inhibitor and is approved for WM, MCL and CLL/SLL in the Unite States and China. BTK inhibitors such as ibrutinib and tirabrutinib are highly effective in CNS lymphoma and have good penetration ability of brain-blood-barrier. We supposed that zanubrutinib should be effective in DLBCL and CNS lymphomas just as other BTKi,so we prescribed zanubrutinib in this study.
Haplo-Identical Non-Gene Editing CD7 CAR T-Cells Induced Bone Marrow and Extramedullary Remission in a Pediatric Patient with
TP53
Mutated Relapsed and Refractory Early T-Cell Precursor Lymphoblastic Leukemia/Lymphoma after Haploidentical Hematopoietic Stem Cell Transplantation
Authors: Dai Haiping , Lin Yang , Meng Huimin , Cui Qingya , Zhu Wenjuan , Tang Xiaowen , Cui Wei , Wu Depei
Published: November 2021

Patients with relapsed/refractory early T-cell precursor lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) respond poorly to traditional therapy and have dismal prognosis. CD7 expresses in almost all blasts of T-cell lymphoma/leukemia and represents one of the most promising therapeutic targets for T-ALL/LBL by CD7 targeted chimeric antigen receptor modified T cell therapy (CD7-CART). Because of shared CD7 expression in the majority of normal T-cell surfaces, we utilized an non-gene editing strategy by co-transducing CAR-T cells with a CD7 protein expression blocker (PEBL), and successfully overcame the fratricide as well as maintain the proliferation and cytotoxicity of CD7-CART-cells. Here, we presented the efficacy and safety results of CD7-CART therapy in a pediatric patient with TP53 mutated ETP-ALL/LBL. The patient was diagnosed with ETP-ALL/LBL at 2016, achieved and maintained complete remission (CR) for 2 years with traditional chemotherapy. The disease relapsed at a month after discontinuation of chemotherapy. He underwent haploidentical HSCT at the second CR, but suffered relapse again 2 years post haplo-HSCT. TP53 mutation（VAF:96.5%） and extensive extramedullary infiltration was detected at relapse. The patient was resistant to venetoclax combined with decitabine, homoharringtonine, aclarubicin, cytarabine and granulocyte colony stimulating factor (G-CSF), high-dose cytarabine combined with cladribine, G-CSF, chidamide and CD38 CART therapy. Nanobody derived CD7-CART cells were manufactured from lymphocytes of the donor. The CART cells were negative for CD7, CD223 and CD279. 70.5% of blasts in the bone marrow aspirates were observed prior to CAR T-cells infusion. A total of 5×10 6/kg CD7-CART-cells were infused. CR was confirmed at day 30 bone marrow evaluation and maintained at the last followup at day 91. Partial remission was achieved as evaluated by PET-CT scan at day 93. Persistence of CD7-CART-cells can be detected with flowcytometry until day 96 post CAR T-cells infusion. Grade 3 cytokine release syndrome with high fever and hypotension were observed, which was relived by tocilizumab and dexamethosone. No organ dysfuction and immune effector cell-associated neurotoxicity syndrome were observed. In general, we showed for the first time that the nanobody derived CD7-CART with PEBL technology was a potent and safe salvage therapy in a relapsed/refractory ETP-ALL/LBL patient with high tumor burden.

Disclosures

No relevant conflicts of interest to declare.
Development and External Validation of a Prognostic Nomogram for Angioimmunoblastic T-Cell Lymphoma
Authors: Wang Hua , Fu Bibo , Chen Guanjun
Published: November 2021

Introduction: Heterogeneity exists in prognosis of Angioimmunoblastic T-cell lymphoma (AITL) patients. Thus, a personalized prognostic model is crucial for survival prediction for each AITL patient. Nomogram is a powerful mathematical tool to predict survival. In this study, we aimed to develop a prognostic nomogram for AITL based on data from a large clinical database and validate it in an independent external cohort. In addition, we compared the accuracy of the nomogram with previous prognostic models used in AITL including International Prognostic Index (IPI) and Prognostic Index of T-cell lymphoma (PIT) model.

Methods: Totally, 1071 patients were retrieved from the Surveillance, Epidemiology, and End Results (SEER) database as the training cohort; 156 patients diagnosed from 2009-2021 in Sun Yat-Sen University Cancer Center or The First Affiliated Hospital of Guangzhou Medical University were recruited as the external validation cohort. 105 patients with IPI information in the training cohort were used to compare the nomogram and IPI. 156 patients in the external cohort were used to compare the nomogram and IPI or PIT. The Prognostic risk factors in the nomogram were identified by cox proportional hazards model. Concordance index (C-index) and calibration curves were used for internal and external validation. C-index and decision curve analysis (DCA) curves were used to compare the models.

Results: Age, sex, systematic symptoms, Ann Arbor stage and chemotherapy were risk factors finally included to develop the nomogram. C-indexes of the nomogram were 0.676 and 0.652 in the training cohort and the validation cohort. Favorable agreement of nomogram-predicted and actual probability of overall survival (OS) was detected by calibration curves in both training and validation cohorts. In the cohort of 105 patients, C-indexes of the nomogram and IPI were 0.696 vs 0.616 (P<0.05); in the cohort of 156 patients, C-indexes were 0.652 vs 0.597 (P=0.08) of the nomogram and IPI while 0.652 vs 0.616 (P=0.176) of the nomogram and PIT. Decision curve analysis (DCA) showed superiority of the nomogram as compared with the IPI or the PIT model in both 105 and 156 patients' cohorts. A web calculator was published for convenient clinical application. Based on the prognostic scores calculated by the nomogram, three cut points were identified by X-tile program to establish a classification system that could significantly distinguish patients in four risk groups.

Conclusion: We establish a nomogram for survival prediction of AITL, which may assist in treatment strategy making and survival consultation.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Meta-Analysis of D-Dimer, hsCRP and Cytokines in COVID-19 Severe/Critical Patients in China
Authors: Zhang Jie , Zhao Lidong , Wang Gang , Yang Linhua
Published: November 2021

Objective: To analyze the relationship between D-dimer, inflammatory markers, cytokines and disease severity, and the possibility of early identification of COVID-19 critical type patients. Methods: PubMed, EMBASE and CNKI databases were searched by computer, and references of related reviews and systematic reviews were manually searched as supplements. The retrieval deadline is February 9, 2021. According to the inclusion and exclusion criteria, the literatures were screened and the quality was evaluated, and then the data were extracted for meta-analysis. The fixed/random effects model was used to calculate the weighted mean difference (WMD) and 95% CI to evaluate whether the levels of D-dimer, hsCRP, IL-6, IL-8, IL-10 and TNF-α in critical type patients were statistically different from those in severe type patients. If there were statistical differences, logistic regression analysis was used, and establish the receiver operating characteristic curve (ROC) and area under the curve (AUC) of each index for the diagnosis of critical type patients. The best diagnostic value of COVID-19 critical type patients was calculated by Youden index. Results: A total of 3519 literatures entered the screening process. According to the inclusion and exclusion criteria, 40 articles were finally included in this study, and all of them were high-quality studies after evaluation. The results of meta-analysis showed that the levels of D-dimer, hsCRP, IL-6, IL-8 and IL-10 in critical type group were significantly higher than those in severe type group (P<0.05). Based on ROC curve, the AUC of D-dimer was 0.785 (95% CI: 0.671-0.899), AUC of hsCRP was 0.884 (95% CI: 0.632-1.000), AUC of IL-6 was 0.819 (95% CI: 0.700-0.939), which had diagnostic significance for critical type patients (P<0.05). The optimal diagnostic threshold of D-dimer was ≥2.00 mg/L (sensitivity 89.3%, specificity 64.0%); the optimal diagnostic threshold of hsCRP was ≥64.22 mg/L (sensitivity 75.0%, specificity 100%); the optimal diagnostic threshold of IL-6 was ≥33.01 ng/L (sensitivity 68.0%, specificity 92.0%). Conclusion: The levels of D-dimer, hsCRP, IL-6, IL-8 and IL-10 in COVID-19 critical type patients were significantly higher than those in severe type patients. Our results might be helpful in identify and risk reduction of mortality in critical types patients infected with COVID-19.

Disclosures

No relevant conflicts of interest to declare.
Tanshinone Iia Inhibits Megakaryopoiesis in Immune Vasculitis
Authors: Chen Hui , Zhou Min , Shu Huiying , Su Weiqing , Yang Liuming , Li Liang , Chong Beng H , Yang Mo
Published: November 2021

Introduction Tanshinone IIA, an active component of Danshen (Salvia miltiorrhiza), has been used for centuries to treat hypercoagulation-related diseases, which attributed to its anti-platelet and anti-inflammatory effects. However, the role of Tanshinone IIA in megakaryocytes, the precursor of platelet within the bone marrow, remains unclear. Therefore, the present study established a rabbit model with immune vasculitis to examine the effect of Tanshinone IIA on megakaryopoiesis and to identify the underlying mechanism(s).

Methods Immune vasculitis was established in rabbits (3-4 weeks old) by two intravenous injection of 10% bovine serum albumin (2.5 ml/kg) at two-week interval. Those rabbits were randomly treated with Tanshinone IIA (5 mg/kg/d, 7 d, iv) or aspirin (100 mg/kg/d, 7 d, ig). Megakaryocyte count and CFU-MK formation were measured by Wright's and AChE staining, respectively. Human megakaryotic cell lines Meg-01 and CHRF-288-11 were used to examine the effect of Tanshinone IIA on apoptosis by Annexin V-FITC/PI, mitochondrial membrane potential/JC-1 and Caspase-3 activity assays using flow cytometry.

ResultsIn rabbits with immune vasculitis, the platelet count, platelet aggregation and the serum levels of inflammatory cytokines IL-1β, TNF-α and IL-6 were significantly increased when compared to their healthy controls. After 7 days of Tanshinone IIA treatment, all these parameters were significantly reduced, with the inhibitions comparable to those caused by aspirin. In addition, the number of megakaryocytes and the formation of CFU-MK were also statistically increased in rabbits with immune vasculitis, which could be significantly reduced by Tanshinone IIA. In vitro, Tanshinone IIA (1, 3, 10 and 30 μg/ml) also significantly inhibited the formation of CFU-MK of bone marrow cells of BALB/c mice (6-10 weeks) in a dose-dependent manner. In human megakaryocytic cell line Meg-01, Tanshinone ⅡA (10 μg/ml, 72 h) induced apoptosis; both early and late apoptotic rates were significantly increased. In another human megakaryocytic cell line CHRF-288-11, Tanshinone ⅡA (10 μg/ml, 72 h) statistically increased the proportion of depolarized cells, from 9.70% to 14.13%, according to mitochondrial membrane potential using JC-1 assay. The expression of active Caspase-3 in CHRF-288-11 was also significantly increased by Tanshinone ⅡA (10 μg/ml, 72 h) from 5.25% to 15.86%.

Conclusion The present study shows that Tanshinone IIA ameliorates immune vasculitis by inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes, which might explain the anti-platelet and anti-inflammatory effects of Tanshinone IIA.

Disclosures

No relevant conflicts of interest to declare.
A NIR-II Fluorophores for Multiple Myeloma Imaging and Photodynamic Therapy
Authors: Jiang Hongqiang , Ding Qihang , Li Qianqian , Wei Yongchang , Hong Xuechuan , Zhou Fuling
Published: November 2021

Background: Multiple myeloma is a hematological malignant disease in which clonal plasma cells proliferate abnormally. NIR-II fluorophores have great application prospects for cancer treatment.

Methods: Multiple detection techniques were used to evaluate effect and mechanisms of the novel small-molecule NIR-II fluorophores probe on multiple myeloma cells in vitro experiment. In vivo MM.1S Luc+ B-NSG mouse model was built to assess the role of probe in multiple myeloma treatment.

Results: The novel small-molecule NIR-II fluorophores probe shown high affinity with multiple myeloma cells and specific myeloma imaging in vivo. More importantly, the probe could effectively induce tumor death with 808 nm laser irradiation in a dose-dependent manner. Further, Confocal found that the probe was mainly distributed in mitochondria and significantly reduced the mitochondrial membrane potential with laser irradiation. Moreover, Reactive Oxygen Species (ROS) level was notably increased and the up-regulation expression of cleaved poly ADP-ribose polymerase (PARP) was detected after treated with the probe and laser irradiation. Consistent with the previous results, the disease was alleviated obviously in probe laser-treated group.

Conclusions: Our work confirm for the first time that the specific imaging and remarkable photodynamic therapy (PDT) effect of a novel NIR-II fluorophores probe to multiple myeloma. It provides the foundation for application of NIR-II fluorophores from solid tumor to non-solid tumor.

Disclosures

No relevant conflicts of interest to declare.
Development and Validation of a Gamma Globin RT-Ddpcr Exploratory Biomarker for Sickle Cell Phase 1 Clinical Trial Fis 002-2020
Authors: Roth Mark , Yang Jian , Tsavachidou Dimitra , Davis Darren W.
Published: November 2021

FTX-6058, a selective and potent binder of embryonic ectoderm development protein (EED), is being investigated in Sickle Cell Disease (SCD). In-vitro and pre-clinical Townes mouse studies show FTX-6058 upregulates expression of the gamma globulin gene leading to increased levels of fetal hemoglobin (HbF). In human clinical trials, as an early indicator of FTX-6058 induced gamma globulin expression, a reverse transcriptase droplet digital PCR (RT-ddPCR) assay was developed and validated by Precision for Medicine. Versus RT-qPCR, RT-ddPCR gives absolute copy number, does not require a standard curve, and is more precise and reproducible for low expressed targets 1,2. Precision for Medicine developed and validated RT-ddPCR assays for the target globin genes α, β, and γ and for the housekeeping genes TFRC and OAZ1. RNA isolated from 3 healthy and 3 SCD affected individuals was used for development and validation of the assay. The upper and lower limits of quantification were 120,000 and 10.5 copies/20μL reaction, respectively. For samples with >50 copies/20μL reaction, the CV for technical replicates was <20% for all transcripts. Furthermore, Precision for Medicine demonstrated SCD affected individuals express approximately 15-fold more γ globulin versus non affected individuals. Fulcrum utilized the globin RT-ddPCR assay validated by Precision for Medicine in the FTX-6058 phase 1 clinical trial FIS 002-2020.

1 Zhao Y, Xia Q, Yin Y, Wang Z (2016), Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri. PLoS ONE 11(7): e0159004. doi:10.1371/journal.pone.0159004

2 Taylor SC, Laperriere G, Germain H (2017), Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports 7(2409): DOI:10.1038/s41598-017-02217-x

Disclosures

Roth: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Yang: Fulcrum Therapeutics, Inc.: Consultancy. Tsavachidou: Fulcrum Therapeutics, Inc.: Consultancy. Davis: Fulcrum Therapeutics, Inc.: Consultancy.