bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Myeloma Cell-Derived Interleukin-32gamma Increases the Immunosuppressive Function of Macrophages By Promoting Indoleamine 2,3-Dioxygenase (IDO) Expression
Authors: Yan Haimeng , He Donghua , Huang Xi , Fan Zhang En , Huang He , Cai Zhen
Published: October 2019

Background: The interaction of multiple myeloma (MM) cells with macrophages (MΦs) in the bone marrow microenvironment contributes to the pathophysiology of MM. In addition to promoting angiogenesis through vasculogenic mimicry, MM-associated MΦs (mMΦs) protect MM cells from spontaneous and chemotherapy-induced apoptosis. mMΦs therefore represent a potential target for myeloma treatment and it is essential to explore the mechanisms underlying normal MΦ polarization to mMΦs. We previously showed that IL-32 is overexpressed in MM patients and is mainly derived from MM cells. The present study was designed to explore the clinical significance of IL-32 in MM and to further elucidate the molecular mechanisms underlying the IL-32-mediated immune function of MΦs.

Methods: We examined the expression of IL-32 in bone marrow biopsy samples using immunohistochemistry. Quantitative real-time PCR, western blot analysis and immunofluorescence were applied to measure the expression of IL-32, IDO and proteinase 3 (PR3). We obtained the global transcriptional profile of the IL-32γ-treated MΦs by RNA sequencing (RNA-Seq). Immunoprecipitation (IP) and GST pulldown experiments was applied to confirm the binding affinity of PR3 for IL-32. We created IL-32-knockdown MM cells by transfection of IL-32 shRNA and silenced PR3 expression in MΦs using siRNA targeting PR3. CD4+ T cell proliferation and IL-2, IFN-γ and TNF-α production were measured by flow cytometry.

Results: We found that high IL-32 expression in MM patients was associated with advanced clinical stage and high serum β2-microglobulin levels. Several isoforms of IL-32 were detected in MM cells and IL-32γ was the most active subtype. RNA sequencing revealed that IL-32γ significantly induced the production of the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO) in MΦs and this effect was verified at the protein level. Furthermore, IL-32-knockdown MM cells showed less ability than control MM cells to promote IDO expression. As a binding protein for IL-32, PR3 was universally expressed on the surface of MΦs and knockdown of PR3 or inhibition of the STAT3 and nuclear factor κB (NF-κB) pathways hindered the IL-32γ-mediated stimulation of IDO expression. Finally, IDO-positive IL-32γ-educated MΦs inhibited CD4+ T cell proliferation and IL-2, IFN-γ and TNF-α production in response to activation.

Conclusion: Our study showed that MM cell-derived IL-32γ induced IDO production in MΦs through PR3 and the downstream STAT3 and NF-κB pathways, resulting in the suppression of the proliferation and effector function of CD4+ T cells. High IL-32 expression in MM may contribute to an immunosuppressive microenvironment by upregulating IDO production in MΦs and promote MM progression.

Disclosures

No relevant conflicts of interest to declare.
Artesunate Attenuate Chronic Graft-Versus-Host Disease By Regulating Th17/Treg Balance
Authors: Xiaomei Chen , Weng Jianyu , Lai Peilong , Huang Xin , Yulian Wang , Geng Suxia , Guo Liyan , Huang Tian , Zeng Lingji , Du Xin
Published: October 2019

To investigate the effects of Artesunate treatment on chronic graft-versus-host disease (cGVHD). Recipient BALB/cJ mice received 8 × 106 bone marrow cells with 8 × 106 spleen cells from B10.D2 mice. Artesunate solubilized in acetone was injected intraperitoneally every day at the dose of 1 mg/kg at Day 28 after transplatation. The clinical scores, survival times and histopathological damage were analyzed. The frequency of Th17 and Tregs in PB and spleens from the mice was evaluated by flow cytometry. In addition, we cultured the CD4+ T cells from the spleens of mice in vitro, and stimulated these cells with artesunate, the frequency of Th17 and Tregs in these splenocytes was evaluated by flow cytometry. Artesunate administration diminishes clinical and histopathological damage and improved the survival of cGVHD mice. Artesunate contributed to Tregs development (4.45±0.04 vs 8.40±0.23; 6.62±0.24 vs 10.48±0.48; p<0.05) while decreased Th17 cells (4.45±0.04 vs 8.40±0.23; 6.62±0.24 vs 10.48±0.48; p<0.05)expressions both in PB and spleens, and decreased the Th17/Treg ratio (0.34±0.05 vs 0.09±0.03; 0.19±0.03 vs 0.06+0.02; P<0.01. Moreover, artesunate suppressed the Th17 cells expressions (0.82±0.37% vs 3.39±1.22% p<0.01) and contributed to Tregs development (34.63±1.29% vs 14.28±1.69% p<0.001), and also decreased the Th17/Treg ratio 0.24±0.09vs 0.02±0.01; p<0.05) in vitro. Artesunate suppressed the Th17 cells expressions and contributed to Tregs development. These studies provide nowsights into the artemisinin and identify artesunate as a novel drug for cGVHD.

Disclosures

No relevant conflicts of interest to declare.
Stroke in a Pediatric Immune Thrombocytopenia Case
Authors: Leblebisatan Goksel , Cil Metin , Sasmaz Hatice Ilgen , Kilinc Yurdanur , Soyupak Sureyya
Published: October 2019

Objective: Immune thrombocytopenia is a bleeding disorder related to deficiency of the main element in primary hemostasis, platelets. But it rarely results in an opposite clinical complication as thrombosis. The mechanism of this unexpected complication is not fully understood still and the treatment of the patients in this thrombocytopenic and thrombotic condition is critical and hard to manage. Here we present a case of immune thrombocytopenia with cerebral infarct with his clinical management details.

Case: A seven-year old boy diagnosed with Immune thrombocytopenia at the age of 1 in another clinic and treated with IVIG when the platelet levels fell and bleeding symptoms appeared. The patient refered to our clinic with severe refractory thrombocytopenia. After being diagnosed with ITP, IVIG treatment and HDDXM treatment (20 mg/m2/day for 4 days) was given 2 cure in a month then the patient follow up in the clinic. It is learned that the family continued with HDDXM and sometimes IVIG treatment when needed in the local hospital. When the child was 6 years old, because of inability to walk and talk he was referred and it was revealed that he had cerebral infarct in the ICA area with a platelet level of 11000/mm3. He was followed in pediatric ICU. He was treated with thrombocytopenia with replacement and steroids in addition to physical treatment for stroke. Anticoagulation was used as the clinical and laboratory picture allowed.

Result: The patient was discharged with the better clinical Picture and his chronic treatment planned mostly with steroids.

Conclusion: Stroke in the immune thrombocytopenic clinic is a rare but hard to manage condition. Although its mechanism not understand well, the clinician should be identify and manage it carefully.

Disclosures

No relevant conflicts of interest to declare.
Protein Phosphorylation Mechanisms Underlying Chronic Pain in Sickle Cell Disease
Authors: He Ying , Wilkie Diana J. , Molokie Robert E. , DeSimone Joseph , Wang Zaijie J.
Published: October 2019

Pain is one of the most dreadful symptoms in sickle cell disease (SCD) and is often refractory to currently available analgesics. Besides acute painful vaso-occlusive crises, SCD is also accompanied by intractable chronic pain. This persistent, and often unrelieved, pain starts early in childhood and continues throughout life. The neurobiological mechanisms of chronic pain in SCD remain unclear, which markedly limits effective pain management and the quality of life in patients with SCD. Taking advantage of two humanized mouse models of SCD, this study aimed to investigate protein phosphorylation mechanisms for chronic pain in SCD.

We characterized pain in two transgenic mice models of SCD that exclusively express human alleles encoding normal α- and sickle β-globin. Berkeley SCD mice (BERK mice) and Townes' SCD mice (TOW mice) exhibited ongoing spontaneous pain behavior and increased sensitivity to evoked pain stimuli compared with littermate control mice expressing normal human hemoglobins. To investigate the underlying protein phosphorylation mechanisms of chronic pain in SCD, we examined PKC isoform mediated nociceptive signaling. Prominent activation of multiple PKC isoforms were observed in the superficial laminae of the spinal cord dorsal horn in BERK and TOW mice. Functional inhibition and silencing of specific PKC isoforms attenuated spontaneous pain, mechanical allodynia, and heat hyperalgesia in both transgenic SCD mice. Furthermore, employing hematopoietic stem cell transplantation approach, we were able to generate a sickle cell anemia model in PKC-null mice, allowing us to specifically target neuronal PKC in SCD. Neither spontaneous pain nor evoked pain was detected in the mice lacking specific PKC isoform despite the full establishment of SCD phenotypes.

In summary, this study is the first to identify the presence of ongoing spontaneous pain in preclinical sickle cell models, which closely mimic the most prevalent manifestation in patients with SCD. Moreover, we found that spinal PKC is a critical mechanism for the generation and maintenance of ongoing and evoked pain in SCD. These findings offer insights into sickle cell pain mechanisms, which may become a potential target for pharmacological interventions.

Disclosures

No relevant conflicts of interest to declare.
Natural Killer Cell Derived Exosomes As a Novel Therapeutic for Acute Myeloid Leukemia
Authors: Boyiadzis Michael , Hong Chang Sook , Whiteside Theresa L
Published: October 2019

Introduction: Natural killer (NK) cells play a critical role in the innate immune response through their capacity to lyse malignant cells without prior antigen-specific priming. NK cells have been evaluated for safety and efficacy in acute myeloid leukemia (AML), both in the transplant and non-transplant settings. Exosomes, small, 30-150 nm-sized extracellular vesicles originating from the endocytic compartment of parent cells, have recently emerged as a universal intercellular communication system. Exosomes are released by virtually all cells and carry proteins, lipids, and nucleic acids from the parent to recipient cells at short and long distances. The exosome molecular cargo reflects the content of the parent cell and is delivered to recipient cells in a membrane-protected vesicle. We hypothesize that exosomes produced by human activated NK cells carry the machinery necessary for the killing of leukemic cells.

Methods: Venous blood (20-50 mL) was obtained from healthy donors (n=10). NK cells were isolated using Ab-based immunomagnetic selection from the recovered peripheral blood mononuclear cells. NK cells were cultured in the presence of interleukin-2 and interleukin-15, and NK-cell supernatants were used for exosome isolation by size exclusion chromatography. Protein levels, numbers and size (qNano), and exosome morphology (transmission electron microscopy) were determined. Exosome cargos were studied by Western blots and/or flow cytometry for NK cell activating and inhibitory receptors, immune inhibitory molecules and for perforin and granzyme B. Cytotoxicity of the NK cell-derived exosomes for K562 targets, AML cell lines (Kasumi, MLL-1) and primary leukemia blasts was measured using flow cytometry-based assays.

Results: Activated human NK cells produced large quantities of exosomes. PKH-26-labeled NK cell-derived exosomes were avidly taken up by leukemic blasts. NK cell derived exosomes carried activating NK cell receptor NKG2D, natural cytotoxicity receptors, perforin, granzyme B, transforming growth factor beta (TGF-β), killer-cell immunoglobulin-like receptors and PD-1. NK cell derived exosomes mediated anti-leukemia activity against K562 targets, AML cell lines and primary leukemia blasts. Lysis of leukemic blasts by NK cell-derived exosomes was exosome concentration dependent.

Conclusion: We report that NK cell derived exosomes have anti leukemia activity in vitro. These data provide a foundation for the future development of new therapeutic strategies using NK cell-derived exosomes for the elimination of leukemia.

Disclosures

No relevant conflicts of interest to declare.
The Hundred-Person Platelet Releasate Project (100PREPs): A Barcode for Human Health and Disease
Authors: Maguire Patricia , Szklanna Paulina , Parsons Martin , Wynne Kieran , O'Connor Hugh , O'Connell Karen , Egan Karl , McGuigan Christopher , Rickard Scott , Ni Ainle Fionnuala
Published: October 2019

Background:

Upon activation platelets release a host of soluble and vesicular signals, collectively termed the 'platelet releasate' (PR). The contents of this PR play a significant role in haemostasis, wound healing, tissue regeneration, inflammation, and pathologic sequelae. We recently established that the proteome of the platelet releasate was highly reproducible across 32 healthy adult donors, with low variation in secretion levels for individual PR proteins. As there is accumulating evidence that circulating platelets may sense or be 'educated' by their environment, we hypothesised that the PR may store a relevant and bespoke collection of molecules during pregnancy and in platelet-related disease.

Methods:

PR from 18 healthy pregnant women, 22 pregnant women diagnosed with early-onset preeclampsia (EOP) (onset <34 gestational weeks), 15 people with relapsing-remitting multiple sclerosis and 15 people with primary-progressive multiple sclerosis were obtained with informed consent. After trypsin/lys-C double digest, each platelet releasate was individually subjected to label-free quantitative proteomic analysis. A machine learning approach based on differential peptides was used as a training set and then to predict outcome.

Results:

69 PR proteins were found to be differentially released from platelets in healthy pregnancy in comparison to age-matched non-pregnant female controls, including several pregnancy-specific proteins. A further 36 PR proteins were statistically modified in EOP, a disease associated with life-threatening consequences for both mother and baby, and another 26 proteins were altered in multiple sclerosis. Based on a training set of differential PR peptides, we developed several diagnostic algorithms, including a proof-of-concept pregnancy algorithm that could predict outcome with 100% accuracy. Strikingly, our proprietary algorithm based on several platelet releasate peptides could predict the severity of maternal outcome in preeclampsia. Moreover, again based on specific platelet-derived peptides, we could differentiate between clinical subtypes of multiple sclerosis.

Conclusion:

We found that the PR proteome significantly changes in physiologic and pathologic conditions. We conclude that the PR is a barcode for the health status of an individual at a given time and provides an easily accessible discrete set of peptides and proteins that can differentiate disease.

Disclosures

Ni Ainle: Leo Pharma: Research Funding; Actelion: Research Funding; Bayer: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer: Membership on an entity's Board of Directors or advisory committees.
Diffuse Alveolar Hemorrhage Secondary to Direct Oral Anticoagulants: A Systematic Review of Reported Cases
Authors: Abu Rous Fawzi , al-Abcha Abdullah , El-khatib Layan , Shrotriya Shiva , Dhakal Prajwal
Published: October 2019

Introduction:

Direct oral anticoagulants (DOACs), also known as non-vitamin K oral anticoagulants, include rivaroxaban, dabigatran, apixiban, edoxaban, and betrixaban. Guidelines have recently identified DOACs as the first line treatment in venous thromboembolism and nonvalvular atrial fibrillation. As the use of these anticoagulants has gradually increased in last few years, the reports of bleeding-related adverse drug events with these agents has increased. There are very few data related to diffuse alveolar hemorrhage (DAH) with DOACs, a dreaded complication of any anticoagulant.

Objective:

To perform a systematic review of all reported cases of DAH secondary to DOAC use.

Methods:

Using several search terms, we reviewed all cases of DAH associated with DOACs which were published and indexed in English language in PubMed and EMBASE in the last 10 years. The bibliography of each relevant article was searched for additional related reports. Statistical analysis was performed using Statistical Packages for the Social Science (SPSS).

Results:

A total of 34 cases were included in the study. Mean age was 69±15 years; 68% were male, 32% were female. Rivaroxaban, dabigatran, apixaban, and edoxaban were used in 41%, 35%, 21%, 3% of patients respectively. Atrial fibrillation was the commonest indication for DOAC use, 68%. Hemoptysis was the commonest presenting symptom, 74%. DAH was diagnosed with Computed Tomography (CT), Bronchoalveolar Lavage (BAL), and both in 68%, 56%, and 35% respectively. A total of 18 (53%) cases required mechanical ventilation. There was a total of 7 (21%) deaths, while 20 (59%) survived.

Conclusion:

Despite reports of lower risk of major bleeding compared to other anticoagulants, DAH is still a possibility with DOAC use. DOACs' absorption and elimination relies largely on P-glycoprotein (P-gp) efflux transporters, coadministration of P-gp inhibitors such as Amiodarone, a common antiarrhythmic used in atrial fibrillation in conjunction with anticoagulation, may result in disturbance of DOAC's concentrations in the blood, increasing the risk of potential side effects. Autoimmune diseases can lead to DAH independently. Owing to the diseased lung tissue in patients with autoimmune disease, the use of DOAC's may enhance the already present risk of DAH. Renal function plays a role in excretion of DOAC's, AKI may cause accumulation of DOAC's in the body leading to major side effects. Newer cases reported the use of idracuzimab; a newly FDA-approved drug for dabigatran reversal. In conclusion; caution should be taken with the increasing use of DOAC's in management of complex care patients requiring anticoagulation, prompt diagnosis and management is key for survival and prevention of complications, and new reversal agents may play a future role in the management of such complications.

Table. Table.

Disclosures

No relevant conflicts of interest to declare.
Comparative Analysis of Proinflammatory Cytokine Levels in Peripheral Blood and Bone Marrow of Patients with Myeloproliferative Neoplasms
Authors: Chen Pu , Wu Boting , Shao Xia , Liu Chanjuan , Yu Zhenglin , Li Feng , Cheng Yunfeng
Published: October 2019

Background

Myeloproliferative neoplasms (MPNs) are characterized by marker somatic mutations involving JAK2, MPL, and CALR, which lead to constitutive activation of tyrosine kinase signaling cascades, subsequently dysregulated proliferative of myeloid linages, and eventually myelofibrosis or leukemic transformation. Recently, it has been argued that proinflammatory processes are crucial to the pathogenesis and progression of MPNs. A number of proinflammatory cytokines including lipocalin, IL-1, IL-2R, IL-6, IL-8, IL-12, IL-15, and IP-10 have been found elevated in the peripheral blood (PB) of MPN patients. However, there has been limited data on the levels of proinflammatory cytokines in the bone marrow (BM) of MPN patients. The present study determined and compared 40 proinflammatory cytokine levels in the PB and BM plasma of MPN patients with unequivocal molecular background, thus intending to illustrate the proinflammatory features of BM microenvironment as well as to evaluate the credibility of PB cytokine profiles.

Methods

Newly diagnosed MPN patients (n=12, 8 had JAK2 V617F mutation, 4 had CALR mutations) were included in the present study. PB samples were taken within 48 hours of BM samples. Paired PB and BM plasma cytokine profiles were measured as well as 10 health control PB plasma samples in a single procedure by Quantibody Human Inflammatory Array 3 (RayBiotech, Norcross, GA) which permitted detection of 40 inflammation-associated cytokines.

Results

Among 12 MPN patients, 8 had JAK2 V617 mutation (6 males, median age 61.5 years), and 4 had CALR mutations (3 males, median age 53.5 years). Positive linear correlations between PB and BM levels were found in 12 proinflammatory cytokines including BLC (r=0.613, p=0.034), I-309 (r=0.872, p<0.001), IL-1α (r=0.666, p=0.018), IL-1β (r=0.929, p<0.001), IL-12p40 (r=0.642, p=0.024), IL-15 (r=0.608, p=0.036), M-CSF (r=0.906, p<0.001), MIG (r=0.596, p=0.041), MIP-1α (r=0.787, p=0.002), MIP-1δ (r=0.648, p=0.023), sTNFRI (r=0.827, p=0.001), and sTNFRII (r=0.644, p=0.024). Compared to health controls, BM levels of G-CSF, IL-2, IL-4, IL-6R, IL-7, IL-8, IL-10, IL-13, MIP-1β, PDGF-BB, RANTES, and TIMP-1 were markedly elevated (all p<0.01), among which IL-2, IL-4, and IL-7 levels were even higher in patients with CALR mutations than those with classic JAK2 V617F mutation (all p<0.05).

Conclusions

Optimal linear correlation could only be found in limited species of proinflammatory cytokines, especially I-309, IL-1β, M-CSF, and sTNFRI, between PB and BM plasma of MPN patients. Therefore, caution should be recommended during attempts to illustrate the status of inflammation in MPN patients by circulating cytokine markers. A stronger inflammatory component might exist in MPN patients with CALR mutations than those with classic JAK2 V617F mutation.

Disclosures

No relevant conflicts of interest to declare.
Targeted Delivery of CpG-Mir-146a Mimic Oligonucleotides As a Therapeutic Strategy to Reduce NF-Κb-Mediated Pathogenic Inflammation and Myeloid Leukemia Progression
Authors: SU Yu-Lin , Zhang Zhuoran , Mann Mati , Wang Xiuli , Nguyen Le Xuan Truong , Zhang Bin , Li Ling , Swiderski Piotr , Boldin Mark , Forman Stephen J , Marcucci Guido , Kortylewski Marcin
Published: October 2019

NF-kB signaling plays central role in the regulation of immune cell activity. The microRNA-146a-5p (miR-146a) provides negative feedback inhibition of the NF-kB pathway to prevent either excessive immunity, such as cytokine release syndrome. Low expression of miR-146a is also implicated in certain types of leukemia, especially in del(5q)-syndrome myelodysplastic and acute myeloid leukemia (MDS/AML). While miR-146a is a potential therapeutic target, the lack of efficient miRNA delivery methods limits clinical translation. We previously developed a strategy for targeted delivery of oligonucleotide therapeutics, such as siRNA, into human and mouse myeloid cells and B cells, using partly or completely phophorothioated (PS) single-stranded oligodeoxynucleotides containing CpG motif (CpG ODN). Here, we demonstrate that similar strategy, using CpG ODN optimized for targeting human monocytes, can be employed for the delivery of functional miR-146a mimic. The CpG-miR146a mimic conjugate, but not unconjugated miR-146a, was quickly internalized by target human and mouse myeloid cells, such as dendritic cells, macrophages and leukemic cells. In vitro CpG-miR146a mimic reduced protein levels of downstream targets, Irak1 and Traf6, as well as the inhibition of DNA binding and transcriptional activity of NF-kB in myeloid cells. We further verified functional activity of CpG-miR146a mimic against several models of acute inflammation. The intravenous injections of this oligonucleotide reduced myeloproliferation and excessive cytokine response to bacterial endotoxin or to live Listeria challenge in miR-146a-/- mice. Furthermore, CpG-miR146a mimic had similar anti-inflammatory effect also in a human model of cytokine release syndrome induced by CD19 chimeric antigen receptor (CAR) T-cell activity. CpG-miR146a effectively abrogated CAR T-cell induced IL-6 production by human monocytes in vitro. Finally, our studies indicated that CpG-miR146a mimic can target del(5q) MDS and AML cells in vitro and in vivo resulting in growth inhibition of MDSL and HL-60 acute myeloid leukemia. Our results suggest that CpG-miR146a strategy can be provide a new-in-class immunomodulatory therapeutics for treatment of acute inflammatory disorders, myeloproliferative diseases and myeloid leukemia.

Disclosures

Wang: Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Kortylewski:N/A: Patents & Royalties: I am an inventor on the US patent 9,976,147.
RNA Sequencing Based Whole Transcriptome Analysis Detected Precisely All Fusion Transcripts in Leukemias
Authors: Shin Myung Geun , Lee Jun Hyung , Choi Hyun Jung , Kee Seung Jung , Kim Soo Hyun , Shin Jong Hee , Suh Soon Pal
Published: October 2019

Introduction: Fusion transcript is a chimeric RNA encoded by a fusion gene or by two different genes by subsequent trans-splicing. Detection of fusion transcripts is an integral part of routine diagnostics of hematological malignancies. However, most of previous analytical methods couldn't detect all fusion transcripts in leukemia. In this study, we developed accurate fusion transcript detection methood using whole transcriptome sequencing, fusion gene detection software and expression analysis.

Methods: RNA sequencing (RNA-seq) for whole transcriptome was performed in 11 patients with hematological malignancies (4 AML, 2 APL, 2 ALL, and 3 CML) having fusion transcripts detected by multiplex RT-PCR (HemaVision, DNA Diagnostic, Risskov, Denmark). Library were prepared with 1 ug of total RNA for each sample by TruSeq mRNA Sample Prep kit (Illumina, San Diego, USA). The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Santa Clara, USA). Indexed libraries were then sequenced using the HiSeq2500 platform (Illumina). The data obtained from the sequencing was analyzed using STAR-Fusion (v1.2.0). Novel fusion transcripts were confirmed by conventional sequencing.

Results: Using STAR-Fusion, average number of fusion candidates per sample was 949.8 (range, 286-1752). To exclude false positive results and obtain true positive results, we developed the following filtering algorithm. First filtering criterion is to have more than 5 junction reads, the second is to detect more than one number of spanning reads, and the third criterion is to be in-frame fusion, which type of fusion can actually synthesize intact protein. Fusion candidates remaining after applying the above three filtering criteria were 1-3 per sample. All known fusion transcripts (PML--RARA, RUNX--RUNX1T1, CBFB--MYH11, KMT2A--MLLT3, BCR--ABL1, DEK--NUP214, ETV6--RUNX1) by multiplex RT-PCR were also detected in RNA-seq. In addition, 10 novel fusion transcripts (IGKV4-1--IGKC, IGLV1-47--IGLC2, HBA2--HBB, DEFA3--MBNL1, HBB--HBA2, MPO--HBA2, HBS1L--AHI1, HBB--HBA2, IGKV4-1--IGKC, SS18L1--ADRM1) were detected and among them, 6 fusions were confirmed by conventional sequencing.

Conclusions: Whole transcriptome sequencing and optimized filtering algorithms successfully detected all known fusion transcripts and various novel fusions.

Disclosures

No relevant conflicts of interest to declare.
Decitabine Combined with FB3 As Conditional Regimen for Elderly Acute Myeloid Leukemia Patient with Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Gao Shichun , Liu Jia , Peiyan Kong , Gao Li , Zhang Cheng , Liu Yao , Yan Hongju , Zhu Lidan , Zeng Defu , Gao Lei , Zhang Xi
Published: October 2019

Background

Acute myeloid leukemia (AML) is a malignant hematologic disease with high incidence in the elderly peoples. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important method for the treatment of AML. However, elderly patients with AML are unable to receive myeloablative conditioning, and transplant related mortality (TAM) is significantly higher than that of young AML patients. How to reduce TAM in elderly AML patients, improve the efficacy of allo-HSCT, and further expand the applicable population of allo-HSCT is the focus of clinical research.

Patients and methods

According to the clinical trial (ChiCTR-IIR-16008182), elderly patients with AML used Decitabine (Dec) combined with FB3 as conditional regimen (20mg/m2 decitabine on day -11∽-7, 30mg/m2 Fludarabine on day -6∽-2, 130mg/m2 busulfan on day -4∽-2). The primary outcome of the trial was relapse after transplantation, and the secondary outcomes were chronic graft versus host disease (cGVHD) and the safety of treatment.

Results

From Mar 2016 to Jun 2018, 19 elderly patients with AML in our center include in the study. The median age was 62 (60-66) years. According to cytogenetics and molecular mutation, 8 patients were intermediate risk and 11 patients were unfavorable risk. HLA matched HSCT was performed in 7 patients, and HLA haploidentical HSCT in 12 patients. All of the patients achieved complete donor chimerism by day 28 after HSCT. Patients achieved ANC engraftment at 15 (10-19) days and PLT engraftment at 19 (12-26) days. Five patients relapsed after transplantation, and 4 of them died; one patient died of severe acute graft versus host disease (aGVHD) and pulmonary infection. The main adverse reactions during the conditioning treatment were dizziness, nausea, vomiting, diarrhea and oral ulcers. ALL symptoms were relieved after the symptomatic treatment. Three patients had a mild elevated transaminase and improved after liver-protecting treatment. Till now, the leukemia-free survival and overall survival were 73.68%.

Conclusion

Dec combined with FB3 as conditional regimen was well tolerated and can achieve ideal survival. The multicenter, randomized controlled clinical study will conduct to further verify its effectiveness and safety.

Key Words: Decitabine, FB3, Elderly, Acute myeloid leukemia patient, Allogeneic hematopoietic stem cell transplantation

Disclosures

No relevant conflicts of interest to declare.
MDS/Secondary AML Associated with Type I Gaucher Disease
Authors: Okamoto Atsushi , Yamamoto Kenta , Eguchi Go , Kanai Yoshitaka , Yamaguchi Terufumi , Maeda Yasuhiro
Published: October 2019

[Introduction]

Gaucher disease is one of lysosomal diseases, and it is an extremely rare disease characterized by anemia, thrombocytopenia, splenomegaly, multiple pathologic fractures and so on. It has been reported that Gaucher disease often merges with blood malignant diseases. However, there are no reports of myelodysplastic syndrome/secondary AML (MDS/s-AML) concomitant with type I Gaucher disease. Furthermore, we carried out parallel treatment of enzyme replacement therapy for type I Gaucher disease and azacitidine therapy for MDS/s-AML.

[Case]

A 69-year-old woman admitted to our hospital because of multiple abnormal fractures. She was diagnosed of MDS (refractory anemia) in 2011 by a previous doctor. As anemia and thrombocytopenia gradually worsened on May 2017, she was transferred to our department on August 8, 2017. At the time of visit, typical pneumonia determined with chest X-P and CT has been observed and high value of CRP (11.0mg/dl) was existed. Pneumonia improved promptly with antibiotic therapy, however, high value of CRP continued, and bilateral chest and right back pain got worse. We carried out plain CT and MRI (DWIBS). In results, splenomegaly, bilateral rib fracture and osteolytic changes in the right iliac bone were observed. Although bone marrow biopsy was performed twice without significant findings, Gaucher disease has been suspected and glucocerebrosidase activity was measured. As expected, a significant decrease of the enzyme activity was observed. Finally, a diagnosis of type I Gaucher disease was made. We started enzyme replacement therapy for glucocerebrosidase. Therefore, reduction of splenomegaly and independency of transfusion were induced by several replacement therapy. Further, multiple bone fracture lesions have improved. Further, juvenile leukocyte including myeloblasts in peripheral blood (PB) also decreased. Therefore, we suspected that the replacement therapy improved not only several symptoms of Gaucher disease, but several hematological findings of MDS. However, WT1-mRNA in the peripheral blood has gradually increased. Moreover, progression of MDS/s-AML was observed after two months. We selected azacitidine therapy in considering of performance state of the patient. In addition to enzyme supplementation therapy, azacitidine therapy was performed in parallel. After several treatment of azacitidine, blasts in PB were decreased significantly with independency of transfusion.

[Conclusion]

We deeply indicate that the combination of enzyme replacement therapy and azacitidine therapy is useful for cases of MDS/s-AML associated with Gaucher disease. There is a possibility that the reduction of the enzyme is involved not only in Gaucher disease but in the ontogeny of blood diseases such as MDS. The effects of enzyme replacement therapy of Gaucher's disease for MDS/s-AML should be discussed.

Disclosures

No relevant conflicts of interest to declare.
Impact of High Donor-Derived CD4+ CCR7+ T Cell Count on Incidence of Grade II-IV Acute Graft-Versus-Host Disease Following Haploidentical Hematopoietic Cell Transplantation
Authors: Varlet Pauline , Alsuliman Tamim , Trauet Jacques , Demaret Julie , Labalette Myriam , Yakoub-Agha Ibrahim
Published: October 2019

Introduction

Historically the administration of post-transplant high-dose Cyclophosphamide (PTCy) has led to haplo-HCT re-innovation.

Although PTCy has a positive impact towards reducing severe acute graft-versus-host disease (aGVHD), this drug has serious adverse side effects and makes haploidentical-HCT more difficult in some patients-particularly in older patients and/or those presenting other comorbidities.

Considering our previous published experience in the effect of graft lymphocyte composition, this work aims to explore the impact of infused T cell subsets on Overall Survival (OS), Event Free Survival (EFS) and acute GVHD in a retrospective cohort receiving allogeneic haplo-HCT.

Methods

This study retrospectively analyzed 29 adult patients who underwent first allogeneic haplo-HCT for hematologic malignancies at Lille University Hospital (CHRU Lille, France).

Graft samples were analyzed by flow cytometry, and CD4 and CD8 T cell subsets were defined as follows: TN=naïve T cells; , TCM=central memory T cells; TEM=effector memory T cells; TTD =terminally differentiated T cells.

Results

The median follow-up of patients was 11 months (0.4-44.3). The cohort median recipient age was 59 years old. Cumulative incidences of grade 2 to 4 aGVHD were 31%. The rate of grade 3 to 4 severe GVHD was 17%. Eleven patients died with 14% of deaths due to non-relapse mortality.

We found a correlation between high percentage of donor-derived CD4+ CCR7+ T cells (>69.2% for CD4+ T cells-the median value in our study) and aGVHD (p=0.028) without any impact on OS and EFS (Table 1).

In multivariate analysis, only high proportions of donor CD4+ CCR7+ T cells correlated significantly with aGVHD (HR=0.203, 95% CI [0,042-0,980], p=0,047) without any impact on OS and EFS (Figure 1).

Conclusion

Naïve and central memory T cells expressing CCR7 exhibit higher alloreactivity potential than CCR7- T cells. In this study, even with PTCy administration, we observed that a high percentage of donor-derived CD4+ CCR7+ T cells can be considered as a predictive indicator of grade II-IV aGVHD post Haplo-HCT. Thus, selective depletion of CD4+ CCR7+ T cells might be enough to prevent aGVHD in haplo-HCT, enabling the use of low doses of PTCy in order to reduce post haplo-HCT complications.

Disclosures

No relevant conflicts of interest to declare.
The Effect of Ketamine Administration on Pain Control in Painful Crisis of Sickle Cell Anemia Patients during Childhood: A Retrospective Observational Study
Authors: Kilinc Yurdanur , Temizturk Hacer , Yılmaz Hayri levent , Cil Metin , Leblebisatan Goksel , Sasmaz Hatice Ilgen
Published: October 2019

Background: Sickle cell anemia (SCA) is a hemoglobinopathy as a result of substitution of glutamic acid with valine in the 6th position of the β globin chain. The vaso-occlusive crises of patients are often caused by pain that requires in-patient treatment. The attack rate is reported to changed between 0.5-1.0 attacks per year per patient. It could be reach up to 10% in 5% of patients. Identification of the SCA painful crisis and the determination of appropriate treatment is very important for the pain relief. Recently studies support that ketamine treatment is beneficial in patients who do not respond to traditional methods. Ketamine is a non-competitive antagonist at the NMDA receptor. We used retrospective visual analogue scale (VAS) scoring system in acute painful crisis patients and then applied ketamine and tramadol to investigate the effect of treatment and side effects and the maximum change in pain score within the first hour after drug administration retrospectively. The aim of this study is evaluate the level of ketamine effect in painful crises.

Methods: Patients older than 6 years of age with acute painful crisis, defined as VAS of greater or equal to 4 were enrolled.The retrospective data was collected from electronic records and included age, sex, vital signs, changes in pain score within 1 hour, and side effects. Ketamine (0.25 mg / kg / dose) and tramadol (0.1-0.4 mg / kg / h) were administered. Patients with a pain score of <4 or a decrease of ≥3 points in VAS were considered to be responsive.

Results: Ketamine group (n = 16), 6 female (37.5%), 10 male (63.5%) and the mean age was 15.4 (7-21) years. Tramadol group (n = 31) 15 female (48%), 16 male (52%) and the mean age was 15.6 (6-21) years. The age and sex groups were similar (p> 0.05). After drug administration, vital signs were observed in all patients in normal range to their age. In the tramadol and ketamine groups, the number of patients who responded at 20 minutes were 9(29%)-9(56%), 12(38%)-11(68%) (p<0.05) at 60 minutes. Sixteen patients (51.6%) responded in the 6th hour of the tramadol group, no evaluate in ketamin group. No life-threatening side effects was observed in any patient.

Conclusion: In the treatment of painful crises, ketamine administration was found to be more effective than tramadol in the first hour. Due to the longer duration of the initial effect of tramadol treatment, ketamine should be considered as an option in the rapid treatment of painful crises.

Disclosures

No relevant conflicts of interest to declare.
Long Term Outcome Following Splenectomy in Children
Authors: Akca Tugberk , Salcioglu Zafer , Aydogan Gonul , Tahtakesen Güçer Tuba Nur , Bayram Cengiz , Ozdemir Gul Nihal
Published: October 2019

Aim: The aim of the study is to evaluate the efficacy, complications and long term results of splenectomy in children with hematologic diseases.

Patients and Methods: This study includes retrospective analysis of the medical records of 158 children between 0-18 years old who underwent splenectomy due to hematologic diseases from 1989 to 2017. Gender, splenectomy indications, age of diagnosis, splenectomy age, vaccination status, splenectomy outcomes, complications and mortality were evaluated.

Results: Of the cases, 88 (55.7%) were female and 70 were male (44.3%). Indications for splenectomy were hereditary spherocytosis in 59 (37.3%), immune thrombocytopenia (ITP) in 50 (31.6%) and thalassemia major in 40 (25.3%) children. The median age of diagnosis was 3.9 years, the median splenectomy age was 9.2 years, the median time from diagnosis to splenectomy was 5.3 years in all cohort. Mean follow-up time of patients after splenectomy was 7.3±5.2 years. All patients except one were immunized before splenectomy. Responses were assessed based on standart criteria for each disease. Response was seen in 94.7% of patients with hereditary spherocytosis and 80% of ITP patients. Twenty-two percent of splenectomised children with thalassemia major had a decrease in transfusion frequency. Infections following splenectomy were reported in 22.8%. One patient had thrombosis related to splenectomy, none of the cases had major bleeding. There was no death due to sepsis. One patient with ITP died due to Subacute Sclerosing Panencephilitis (SSPE).

Conclusion: Splecetomy is associated with good remission rates following ITP and hereditary spherocytosis. Splenectomy should be performed only in selected cases with transfusion dependent thalassemia. Post-splenectomy sepsis is uncommon. Future studies should focus on health quality of life and cost analysis of splenectomy in children.

Disclosures

No relevant conflicts of interest to declare.
Differential Outcomes of Synchronous and Metachronous Diffuse Large B-Cell Lymphoma Transformed from Follicular Lymphoma
Authors: Chan Natalie Pei Xin , Lim Ryan Mao Heng , Khoo Lay Poh , Cheng Chee Leong , Tan Leonard Kwan Cheong , Poon Eileen Yi Ling , Somasundaram Nagavalli , Farid Mohamad , Tang Tiffany , Tao Miriam , Lim Soon Thye , Chan Jason Yongsheng
Published: October 2019

Aim:

Composite histologies of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) may be present synchronously at diagnosis or metachronously at the time of transformation of a formerly diagnosed FL. The aim of this study was to examine their clinico-pathological characteristics and treatment outcomes.

Method:

Patients who were consecutively diagnosed with composite FL/DLBCL (n=120) and FL (n=346) from 2001-2017 at the National Cancer Centre Singapore were retrospectively analyzed. Chi-squared tests and multivariate logistic regression were performed to evaluate clinico-pathological associations between the two cohorts. Survival analysis was performed using the Kaplan-Meier method and the log-rank test.

Results:

Amongst the FL cases, 21 patients (6.1%) with metachronous transformed FL/DLBCL were identified. The median lag time from diagnosis of FL to DLBCL transformation was 47 months (range, 7.8-168). Clinico-pathological features in synchronous and metachronous FL/DLBCL were similar, with both entities demonstrating a male preponderance (67% male and 33% female). Median age at diagnosis was 67 years (range, 41-81) and 60 years (range, 24-90) for metachronous and synchronous FL/DLBCL, respectively. The cell-of-origin by Han's criteria was similar (metachronous: GCB 52%, ABC 43%, unknown 5%; synchronous: GCB 38%, ABC 57%, unknown 5%; p = 0.21), as were the occurrence of C-MYC/BCL2/BCL6 double-hit rearrangements. However, survival from the time of DLBCL development was significantly worse (median, 3 vs 12 years) for metachronous compared to synchronous FL/DLBCL (HR 2.20, 95%CI 0.88-5.49, p = 0.022). Double-hit, advanced stage, and use of non-RCHOP regimens (OR 7.54, 95%CI 2.84-20.1, p = 0.0001) were associated with lack of complete response to chemotherapy. In metachronous FL/DLBCL, the R-CHOP regimen was less commonly used (77% vs 56%, p = 0.049). Correspondingly, complete response to chemotherapy was less likely in metachronous cases (38% vs 63%, p = 0.037).

Conclusion:

Metachronous and synchronous FL/DLBCL share similar clinico-pathological characteristics.

A preceding diagnosis of FL however, predicts for significantly worse survival outcomes and suboptimal responses to chemotherapy.

Disclosures

No relevant conflicts of interest to declare.
Cell-Free DNA Analysis Demonstrates Comprehensive Genomic Profiling and a Facile Method to Sensitively in Multiple Myeloma
Authors: Du Juan , Lu Jing , Qiang Wanting , Li Lu , Liu Jin , Jiang Hua , Qian Ziliang , Chen Baoan , Fu Weijun
Published: October 2019

Background: Multiple myeloma (MM) is a plasma cell malignancy characterized by complex cytogenetic and molecular abnormalities including translocations involving the immunoglobin heavy chain locus and mutations involving numerous oncogenic signaling pathways. Fluorescence in situ hybridization (FISH) has emerged as the most useful current cytogenetic assessment and provide a new level of insight into the correlation of myeloma prognosis risk model. However, the identification or sorting of malignant cells is required before FISH probes and only involved expansion of the types of probe and number of detectable targets is reached. Cell-free DNA (cfDNA) offers the potential for minimally invasive genome-wide profiling of tumor alterations without tumor biopsy and may be associated with cancer precision medicine and patient prognosis.

Methods: In this retrospective cohort study, we identified 37 patients from 9 relapsed/refractory (RR) and 33 newly diagnosed (ND) patients were analyzed for chromosomal copy number imbalance using the ultrasensitive chromosomal aneuploidy detector (UCAD) platform.

Results: Chromosome copy number aberration (CNA) were frequently (82.6%, N=46) detected in MM plasma cell free DNA. Applying UCAD to cfDNA, FISH in CD138 purified bone marrow aspirates, and some matched bone marrow biopsies, we find concordance in copy number alterations (~81%) between liquid and tumor biopsies. Significant copy number changes, including 1q gains, 13q deletion and 17p deletion could be found in 57.89%, 54.05%, and 16.67% in plasma of MM, which is higher percentage than FISH assay (46.81%, 28.26%, and 8.89%), respectively. Besides, chromosome 6p and 6q were determined the higher frequency aberration from UCAD. Moreover, a higher frequency of copy number aberrations and variations was detected in RR patients than ND (100% vs 78.4%, respectively), obviously CNAs heterogeneity displayed in advanced disease. In the inconsistent some samples, UCAD from the plasma and bone marrow showed the similar results, which indicated the FISH is underdetermined and insensitivity in some patients' routine inspection.

Conclusion: We conclude that cfDNA analysis as an adjunct to BM biopsy represents a noninvasive and broaden the applicability strategy for comprehensive genomic profiling and therapeutic monitoring of MM.

Disclosures

No relevant conflicts of interest to declare.