bluuebunny/crossref_metadata_2025 huggingface dataset:
Toxicity Burden of Bleomycin Treatment in Hodgkin Lymphoma: A Systematic Literature Review
Authors: Fox Kathleen M , Josephson Neil C , Richhariya Akshara
Published: October 2019

Introduction: Bleomycin has been a component of chemotherapeutic regimens for decades, typically among young individuals who may survive for long periods. Yet, toxicity from bleomycin may result in short-term and long-term health problems. The study objectives were to characterize the occurrence and severity of bleomycin toxicities and identify risk factors for bleomycin toxicity among patients with Hodgkin lymphoma.

Methods: A systematic literature review of the burden of bleomycin treatment in cancer patients was conducted using the PRISMA checklist. PubMed, EMBASE, and Cochrane Database were searched for English language articles between 1980 and March 2016 providing data on bleomycin toxicity in studies with >10 patients.

Results: Overall, 18 original research articles of good to moderate quality were included in the systematic review for patients with Hodgkin lymphoma. Pulmonary toxicity ranged from dyspnea, pulmonary fibrosis, and pneumonitis, to acute respiratory distress syndrome and respiratory tract disorders or infections. For Hodgkin lymphoma, the proportion of patients experiencing pulmonary toxicity was >5% in 12 of 17 studies, with 8 studies reporting toxicity in 13% - 28% of patients. Other studies (n=5) reported that 0% - 4.7% of patients experienced pulmonary toxicity. About 4% -5% of Hodgkin lymphoma patients had fatal pulmonary toxicity. Skin toxicity ranged from skin rash to dermatitis and erythema, with 1% - 5% of Hodgkin lymphoma patients experiencing skin toxicity after bleomycin exposure.

In the 4 studies that investigated risk factors for bleomycin-induced pulmonary toxicity among patients with Hodgkin lymphoma, low albumin level (<40 g/dL), treatment with anthracycline containing chemotherapy regimens and use of colony granulocyte stimulating factor concomitant with bleomycin were reported to be significant risk factors. No significant difference was found between patients with and without pulmonary toxicity in terms of exposure to radiation therapy, cumulative bleomycin dose, smoking history, or underlying lung involvement.

Conclusions: Long-term pulmonary toxicity was not consistently evaluated in the studies and may understate the true burden on patients. Many patients treated with bleomycin may experience toxicity and sometimes fatal toxicity. Since Hodgkin lymphoma is a highly curable malignant disease and to further improve patient outcomes, attention needs to be focused on reducing treatment-related toxicities, particularly long-term morbidity and death associated with pulmonary events.

Disclosures

Fox: Seattle Genetics: Research Funding. Josephson:Seattle Genetics: Employment. Richhariya:Seattle Genetics, Inc.: Employment.
Identification of Kinase-Dependent Signaling Pathways Regulated By Rap1b in Murine Platelets
Authors: Paniagua Luis , Wu Xiaogang , Pereckas Michael , Noon Kathleen , White Gilbert
Published: October 2019

Identifying signaling pathways regulated by Rap1b has been challenging using classical biochemical techniques. Using quantitative iTRAQ- and TiO2-based phosphoproteomics, we sought to identify protease-activated receptor 4 (PAR4) thrombin receptor-activated kinase cascades influenced by Rap1b in murine blood platelets. We identified a total of 1,783 phosphopeptides, and after screening for activation dependence, we found 7 phosphopeptides from 4 proteins that were >1.5-fold more abundant in WT compared to Rap1b-null mouse platelets and 5 phosphopeptides from 4 proteins were >1.5-fold more abundant in Rap1b-null. One protein that showed higher phosphorylation in activated WT platelets was Talin, which had 3 sites of increased phosphorylation: T430, S446, and S1201. The functions of these sites have yet to be determined, and the local sequences near these sites do not match any known kinase consensus sequences. Two of these sites, T430 and S446, are in close proximity to the Calpain cleavage site of Talin at Q432-Q433. We found Calpain cleavage of Talin was greater in PAR4-TRAP-activated Rap1b-null platelets compared to WT after 5 minutes of PAR4 stimulation. If phosphorylation inhibits Talin cleavage by Calpain, a possible explanation for the platelet defects seen in Rap1b-null platelets may be that cleavage of Talin reduces Talin clustering, and therefore integrin clustering, leading to reduced aggregation and reduced adhesion under shear. Other proteins identified include RhoGAP 18 (more abundant in WT) and ERK1/2 (more abundant in Rap1b-null). In conclusion, we have 1) used an unbiased proteomic approach to search for downstreams effectors of Rap1b during PAR4-mediated platelet activation, 2) identified 12 phosphopeptides from 8 unique proteins that had >1.5-fold difference in activated platelets from Rap1b-null mice versus WT mice, including Talin, RhoGAP18, and ERK1/2, and 3) identified a difference in Calpain-mediated Talin cleavage between activated plateles from Rap1b-null mice versus WT mice. These results suggest a signaling cascade from Rap1b to Talin, a known regulator of integrin activation. This work is supported by NIH Grant HL45000 and Blood Center Research Foundation.

Disclosures

No relevant conflicts of interest to declare.
The Novel ADCC-Optimized Human CD54 (ICAM-1) Antibody MSH-TP15e Has Potent Anti-Myeloma Activity
Authors: Klausz Katja , Burger Renate , Kellner Christian , Guenther Andreas , Peipp Matthias , Gramatzki Martin
Published: October 2019

Background: Monoclonal antibodies directed against various target antigens have proven efficacy in cancer therapy including hematological malignancies. Recently, daratumumab and elotuzumab became the first antibodies approved for the treatment of multiple myeloma (MM). Due to the substantial number of patients still in need for long-term disease control, the investigation of additional target structures and potent molecule formats for antibody-based therapy of MM remains important.

Methods: Human synthetic single-chain fragment variable (scFv) phage display libraries were subjected to a cellular screening approach. The MSH-TP15 clone was selected based on its binding to various MM cell lines and patient-derived CD138-positive malignant plasma cells. Intercellular adhesion molecule-1 (ICAM-1/CD54), known to be important for the interaction of malignant plasma cells with the bone marrow microenvironment, was identified as target antigen. Based on the scFv sequence, fully human IgG1 antibody variants with selected mutations in the Fc domain were constructed to generate wild-type, antibody-dependent cell-mediated cytotoxicity (ADCC)-optimized and Fc-knockout variants. First, these variants were analyzed for their capacity to kill MM cells in vitro. Apoptosis induction and growth inhibition were tested by flow cytometry and in MTS proliferation assays. ADCC and complement-dependent cytotoxicity were investigated in chromium-release assays using human serum, peripheral blood mononuclear cells or purified NK cells of healthy donors. In vivoefficacy of an ADCC-optimized variant was analyzed in the INA-6 myeloma xenograft model.

Results: The MSH-TP15 antibodies target an epitope on the N-terminal part of the extracellular domain of human ICAM-1/CD54. The antibody variants showed no anti-proliferative effects on patient-derived bone marrow stromal cells, nor directly induced apoptosis or inhibited proliferation of myeloma cells. While complement-dependent cytotoxic activity was generally absent, the Fc-engineered antibody variant MSH-TP15e significantly triggered ADCC against various MM cell lines and freshly isolated patient myeloma cells. The recruitment of human NK cells was crucial for the cytotoxic effects observed in vitro. Importantly, an ADCC-optimized variant completely prevented tumor engraftment in the INA-6 xenograft model.

Conclusions: The novel ADCC-optimized fully human antibody MSH-TP15e directed against ICAM-1/CD54 exerts potent anti-myeloma activity in vitro and in vivo. Therefore, it has promising characteristics and will be further evaluated for MM immunotherapy.

Disclosures

Guenther: Novartis: Consultancy, Honoraria; Celgene: Honoraria; Takeda: Consultancy, Honoraria.
Risk Factors and Outcome of Children with Relapsed/Refractory Acute Leukemia after T-Cell-Rich Haploidentical Hematopoietic Cell Transplantation
Authors: Kikuta Atsushi , Sano Hideki , Mochizuki Kazuhiro , Kobayashi Shogo , Akaihata Mitsuko , Ohara Yoshihiro , Takahashi Yoshinobu , Ohto Hitoshi
Published: October 2019

Background: T-cell rich (TCR) HLA-haploidentical SCT (haplo-SCT) is a form of T-cell therapy that has a high degree of efficacy in hematologic malignancies. Previously we reported the safety profile assessing GVHD prophylaxis that was conducted with anti-human thymocyte immunoglobulin (ATG), tacrolimus, methotrexate (MTX) and prednisolone (PSL) in unmanipulated haplo-SCT (Clin Transplant 2010, Transfus Med 2014). We evaluated efficacy and toxicity of TCR haplo-SCT in children with very high risk refractory/relapsed acute leukemia (VHR-R/R AL).

Methods: VHR-R/R AL were defined as: relapse after SCT, very early or early relapse, induction failure(2 or more) and relapse of risk factor with MLL rearrangement, Ph+, Mo7 and 5q-. From Aug 2000 to April 2014, consecutive 38 patients (pts) with VHR-R/R AL who underwent TCR-haplo-SCT were included. The median age of pts was 8.2(0.3-19.1) years old. The diagnosis included ALL (n=27), AML (n=8), M/NKL (n=3). The disease status at TCR-haplo-SCT were 18 in CR (positive MRD: 8 pts), 20 in non-CR. HLA disparities were 2/8 in 1pt, 3/8 in 9 pts, 4/8 in 28 pts. Donors included fathers (n=21), mothers (n=14), and siblings (n=3). Thirty one pts received myeloablative conditioning (TBI based: 20 pts, Bu based: 11 pts) and 34 pts of them received ATG (rabbit, thymoglobulin 2.5mg/kg) containing regimen. The GVHD prophylaxis was conducted with tacrolimus, MTX and PSL. Thirty four pts received peripheral blood stem cells and 4 pts received BM.

Results: Neutrophil engraftment (defined as >0.5x109/L) was 95% with a median day of 13 (range, 10-15). With a median 1640 days follow-up (range, 320-5510 days) in pts without events, the actuarial 3-year overall survival (OS) and disease-free survival (DFS) were 57% and 39%, respectively. On competing-risk analysis, 1-year cumulative incidences of grade II-IV acute GVHD and chronic GVHD were 71% and 63%, respectively; 3-year cumulative incidences of relapse and non-relapse mortality (NRM) were 40% and 20%. On univariate analysis, 3-year OS in pts with acute GVHD vs. without acute GVHD were 70% vs. 22% (p=.0006), in pts CR vs. non-CR at TCR-SCT were 83% vs. 32% (p=.007), in pts infused CD3 cell doses >=5 x 108/kg vs. <5 x 108/kg were 83% vs. 25% (p=.005), according to age at TCR-SCT <9 vs. >=9 were 79% vs. 34% (.008), respectively. In contrast, the occurrence of acute GVHD had no significant difference in infused CD3 cell doses.

Conclusions: These data suggest that TCR haplo-SCT following low-dose ATG containing conditioning combined with our GVHD prophylaxis is well tolerated, facilitates engraftment, and has significant anti-leukemic activity, particularly in pediatric patients with refractory/ relapsed population.

Disclosures

No relevant conflicts of interest to declare.
Sustained Natural Killer Cell Recovery Post-Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation Predicts Survival in Non-Hodgkin's Lymphoma
Authors: Porrata Luis , Inwards David James , Ansell Stephen M , Micallef Ivana , Johnston Patrick B. , Nevala Wendy , Markovic Svetomir
Published: October 2019

Abstract

Our group recently reported a Phase III clinical trial (Porrata et al. Biology of Blood and Marrow Transplantation 2016; 22: 1017-1023) demonstrating that the infusion of the autograft-absolute lymphocyte count (A-ALC) and autograft natural killer cells (A-NKC) are survival prognostic factors in non-Hodgkin lymphoma (NHL) patients treated with autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). A limitation of our Phase III clinical trial was the short-term follow-up of just two years. Therefore, we evaluated longer follow-up and assessed the role of the infused A-ALC, infused A-NKC, and peripheral blood absolute natural killer cell count (ANKC) recovery on survival post-APBHSCT. Of the 122 patients that participated in the Phase III trial, 111 patients were able to complete APBHSCT and included in the current study. With a median follow-up of 57.2 months (range: 2.1-84.6 months), superior overall survival (OS) and progression-free survival (PFS) was observed in patients infused with an A-ALC ≥ 0.5 x 109 lymphocytes/kg [OS: HR = 0.429, 95%CI, 0.128-0.828, p < 0.01; and PFS: HR = 0.456, 95% CI, 0.177-0.863, p < 0.01] and an A-NKC ≥ 0.09 x 109 cells/kg [OS: HR = 1.58e-3, 95% CI, 1.046e-6-0.179, p < 0.003; and PFS: HR= 1.58e-3, 95% CI, 7.81e-7-0.064, p < 0.003]. Furthermore, patients that maintained an ANKC ≥ 250 cells/µl at 3 months, 6 months, 9 months and 12 months post-APBHSCT also experienced superior OS (Figure 1A) and PFS (Figure 1B)[OS and PFS times in months were evaluated from infusion date of stem cells]. In the multivariate analysis a sustained ANKC ≥ 250 cells/µl was an independent predictor for OS (HR = 0.013, 95% CI, 0.001-0.063, p < 0.0001) and PFS (HR = 0.014, 95% CI, 0.001-0.067, p < 0.0001). This study is an extension of our Phase III clinical trial showing that sustained innate immunity by measuring the ANKC correlated with superior clinical outcomes; thus providing a platform to develop innate immunity immunotherapeutic strategists directing to improve clinical outcomes post-APBHSCT in NHL patients.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Ex Vivo T Cell-Depleted Haploidentical HCT for Children with Acute Leukemia after a Reduced-Intensity Preparative Regimen Containing Low-Dose TBI
Authors: Lim Yeon Jung , Im Ho Joon , Kang Sung Han , Kim Hyery , Koh Kyung-Nam , Choi Eun Seok , Seo Jong Jin , Suh Jin Kyung
Published: October 2019

Background: Recent advances in haploidentical hematopoietic cell transplantation (HHCT) enabled this transplant using haploidentical family donor to be a viable option for pediatric patients lacking matched related or unrelated donor. In our center, HHCT using ex vivo T cell-depleted (TCD) grafts after reduced-intensity conditioning (RIC) was conducted since 2008. The safety and efficacy of this transplantation modality for pediatric with acute leukemia were investigated.

Methods: Thirty-one pediatric patients with acute leukemia received ex vivo T cell-depleted HHCT at Asan Medical Center Children's Hospital between July 2008 and June 2016. Four patients received CD3-depleted grafts and 27 received TCRαβ-depleted stem cells. Among 31 patients, 9 had ALL (3 CR1, 6 CR2-3), 22 had AML (18 CR1-3, 4 NR). Seven patients had relapsed after previous allogeneic HCT. All 31 patients underwent a uniform RIC regimen consisting of low-dose total body irradiation (LD-TBI; 600 cGy), fludarabine (FLU; 180 mg/m2), cyclophosphamide (CY; 100 mg/kg), and rabbit anti-thymocyte globulin (r-ATG; 3 mg/kg).

Results: The median age at HHCT was 14 years (range, 1-19). All 31 patients achieved sustained neutrophil engraftment at a median of 10 days (range, 9-17) post-transplant. The cumulative incidence of acute GVHD grade II-III and III were 30% and 21%, respectively. None developed grade IV. Two of 26 evaluable patients developed extensive chronic GVHD. As of July 2016, 18 of the 31 patients survive free of disease with a median follow-up of 26 months (range, 2-98 months). Ten patients have died. Causes of death were relapse (n=9) and disseminated tuberculosis (n=1). Only one patient died of non-relapse cause, leading to TRM of 5.3% at 1 year. EFS and OS at 2 years for all patients were 51% and 60%, respectively. Sixteen patients with AML who received a first HHCT in any CR showed a favorable outcome (EFS of 85%), whereas, 6 patients with ALL who received a first HHCT in CR showed a poor EFS of 28%. In addition, all patients (6 with AML and 3 with ALL) who received a subsequent HCT in CR or were not in remission developed relapse.

Conclusions: This study demonstrated that our ex vivo T cell-depleted HHCT using RIC is a feasible therapy with low TRM for pediatric patients with acute leukemia. The outcome of patients with AML who received their first transplant in CR was excellent in this treatment modality. However, the outcome of ALL was poor suggesting that more intensified conditioning regimen may be required for those diseases. Furthermore, an innovative treatment strategy is warranted to improve the outcome for patients with relapsed or refractory acute leukemia.

Disclosures

No relevant conflicts of interest to declare.
External Validation of Generic and Cancer-Specific Risk Stratification Criteria for Predicting 30-Day Mortality in Patients Presenting with Pulmonary Embolism and Active Cancer
Authors: Caranfa Jonathan T , Weeda Erin R , Coleman Craig I , Zeichner Simon B. , Kohn Christine G
Published: October 2019

Introduction: The Hestia criteria has been established as a highly sensitive prognostic tool used by clinicians to identify newly diagnosed pulmonary embolism (PE) patients from the general population at low vs. high risk of early (30 day) mortality. Font et al. applied similar risk stratification criteria in a population restricted to PE patients with cancer. We evaluated the performance of the generic Hestia tool and cancer-specific criteria by Font et al. for predicting 30-day post-PE mortality in patients with active cancer.

Methods: We identified consecutive, adult, objectively confirmed PE patients with active cancer presenting to the emergency department at our institution from 11/2010-1/2014. We calculated the proportion of patients categorized as low-risk by each set of criteria and determined the accuracy of the criteria for predicting 30-day all-cause mortality. Mortality was determined through Social Security Death Index searches.

Results: A total of 124 patients with PE and active cancer (mean age 66.2 years, 46.0% with concurrent deep vein thrombosis, 49.2% with metastatic disease and 46.8%, 16.9% and 10.4% receiving chemotherapy, radiation or both, respectively) were included. Mortality at 30-days occurred in 25 (20.2%) patients. The Hestia tool had a sensitivity of 100% but classified <19% of patients as low-risk; while criteria developed by Font et al. had a sensitivity of 88.0% and classified 41.1% of patients as low-risk (Table). The most common reason patients were classified as high-risk by Hestia but not the criteria from Font et al. was the requirement for oxygen (O2) therapy to maintain an O2 saturation >90% for >24 hours (n=19).

Conclusion: In this external validation study, both the generic Hestia tool and cancer-specific Font et al. risk stratification criteria displayed acceptable sensitivity. Compared to Hestia, the Font et al. criteria classified two times as many patients as low-risk. Larger, prospective studies are needed to confirm our results.

Disclosures

Coleman: Janssen Pharmaceuticals: Consultancy, Research Funding; Bayer Pharmaceuticals AG: Consultancy, Research Funding; Boehringer-Ingelheim Pharmaceuticals, inc.: Consultancy, Research Funding.
Desiderosmia (Craving of Olfaction): A Novel Symptom Associated with Iron Deficiency Anemia
Authors: Hansen Bryar , Ravindran Aishwarya , Bottner Wayne A. , Cole Craig E. , Farnen John P. , Go Ronald S.
Published: October 2019

BACKGROUND:In recent years,we saw four patients with iron deficiency anemia (IDA) in our clinical practices who developed a compulsive craving for certain odors including rubber tires, gasoline, Pine-Sol® (a popular household alcohol ethoxylate-based cleaning agent), and "musty odor of the basement." Such behavior resolved after treatment of IDA, and recurred in a patient whose IDA relapsed. This resembled pica, a craving to eat non-nutritive items, which is commonly associated with IDA. Because we had never heard of or read about this phenomenon, we reviewed the English medical literature and World Wide Web to determine if such experience had been previously reported.

METHODS: We searched the PubMed, Google, and Google Scholar to find publications, reports, presentations, or testimonies of individual in online blogs using a combination of search terms including "nasal, olfactory, smell, craving, iron deficiency, and anemia."

RESULTS: We found no scientific publication, report, or presentation similar to our experience in PubMed and Google Scholar. However, using Google search engine, we found several testimonies of individuals in online blog posts (mostly at babycenter.com and medhelp.org), several of which were pregnancy blogs that reported these peculiar symptoms. Individuals were reporting powerful cravings of olfaction, frequently being overtaken by a desire to smell certain odors. The types of substances for which they craved and their associated medical conditions (when reported) are summarized in the Table.

CONCLUSIONS:These preliminary findings and testimonies give suggestive evidence that a condition, entirely separate from pica, exists in certain patients suffering from IDA or during pregnancy. For this, we propose the name, desiderosmia, derived from the Latin word "desiderare" for desire and the Greek word "osme" for smell. In naming this condition, we hope to inspire further investigation, and hopefully shed some light for patients who suffer from this newly described olfactory symptom.

Table Reports of smell cravings and associated medical conditions found in online blogs Table. Reports of smell cravings and associated medical conditions found in online blogs

Disclosures

No relevant conflicts of interest to declare.
Study of Dense Red Blood Cells in Children with Sickle Cell Disease
Authors: Coulon Basil , Di Liberto Gaetana , Wagner Ballon Orianne , Motterlini Roberto , Decrouy Xavier , Moutereau Stephane , Galacteros Frederic , Pirenne France , Bartolucci Pablo , Pondarré Corinne
Published: October 2019

Many Sickle cell disease (SCD) manifestations begin at an early age and with wide inter-individual variability. High Density Red Blood Cells (DRBCs) are a specific feature of SCD, assessed with phthalate density distribution method. RBCs dehydration causes higher polymerisation of hemoglobin S and sickling, and their percentage is associated to chronic organ damage and hemolysis in adults. The spleen is one of the first altered organs, with considered complete asplenia around the age of five.

We assessed the percentage of DRBCs (%DRBCs) in 50 children followed in the Créteil Paediatric Sickle Cell Referral Centre. Eligible patients had hemoglobin SS or Hemoglobin S-β0 thalassemia, without recent blood transfusion. Endpoints were the %DRBCs, its correlation with hemolytic parameters, with splenic function, with clinical manifestation.

In this first prospective study in pediatric population, %DRBCs was associated with age, with particular low rate before the age of four.

%DRBCs was significantly associated with enhanced haemolysis: higher total bilirubin, higher reticulocytes count, and lower hemoglobin. The results concerning LDH and ASAT were more questionable. A higher %DRBCs was associated with a lower hemoglobin F percentage.

The percentage of Pitted cells, reflecting spleen function, was correlated with %DRBCs in a univariate analysis.

There was no association between %DRBCs and vaso-occlusive symptoms, as expected. No statistical correlation has been found between intracranial velocity and %DRBCs. Among the five patients suffering from chronic complications related to hemolysis (kidney disease, priapism), 4 had high %DRBCs.

Thus, DRBCs appearance seems to be related to the impairment of the spleen function. As in SCD adults, anemia and hemolytic markers are associated with high %DRBCs. Longitudinal analysis, with DRBC measurements before any treatment intensification, in a larger population, will be the key to evaluate the prognostic value of DRBCs for organ damage.

Disclosures

No relevant conflicts of interest to declare.
Pedigree Gene Investigation and Parameters of NK Cell Activity, CD107a Degranulation Amd HLH Related Defective Protein Play Significant Role in the Diagnosis of Primary HLH
Authors: Zhang Jia , Wang Zhao
Published: October 2019

Abstract This study aims to evaluate the significance of pedigree genetic screening and rapid immunological parameters in the diagnosis of primary HLH and explore their correlation. Twelve Chinese families of primary HLH with mutations in PRF1, UNC13D, LYST, RAB27A, SH2D1A, BIRC4 were recruited and conducted pedigree investigation, including family genetic screening, NK cell activity, CD107a degranulation and expression of HLH related defective protein. Ten patients were identified with homozygous, compound heterozygous, or hemizygous mutations in PRF1, UNC13D, RAB27A, SH2D1A and BIRC4, including one case of homozygous mutation (c.1349C>T:p.T450M) and four cases of compound heterozygous mutations (frameshift deletion c.65delC:p.P22fs and c.G503A:p.S168N; c.1168C>Tp.R390X and c.1349C>T:p.T450M; frameshift mutation c.1090_1091delCT:p.T364fsX93 and c.1349C>T:p.T450M; non-frameshift deletions c.1083_1094del:p.361_365del and c.T172C :p.S58P) in PRF1, one case of homozygous mutation (c.G2588A:p.G863D) and one compound heterozygous (c.C640T:p.R214X and c.G407A:p.C136Y) in UNC13D, one case of homozygous mutation (c.C244T:p.R82C) in RAB27A, and two male cases of hemizygous mutations in SH2D1A(c.32T>G:p.I11S) and BIRC4(c.G592A:p.V198M). Meanwhile, two patients were identified mutations in two or three different gene, including a male with a hemizygous mutation in SH2D1A(c.7G>T:p.A3S) and a heterozygous mutation(c.127C>A: p.L43M) in PRF1, and a female with three hemizygous mutations respectively in UNC13D(c.G680A:p.R227H), LYST(c.A8368C:p.K2790Q) and BIRC4(c.C962G:p.A321G).The primary HLH patients and their family members presented different levels of decreased NK cell activity. Individuals who were found mutations in PRF1, SH2D1A and BIRC4 showed low expression of perforin, SAP and XIAP. And the patients with homozygous and compound heterozygous mutations in UNC13D and RAB27A showed significant reducing of cytotoxic degranulation function and their family members presented normal or different levels of decreased CD107a. The patient with combination defects involving two genes(UNC13D and LYST) in the degranulation pathway showed decreased of CD107a degranulation which implied a digenic inheritance and diagnosed primary HLH. Pedigree genetic screening and rapid detection of immunological parameters play an important role in the diagnosis of primary HLH and demonstrate good consistency.

Disclosures

No relevant conflicts of interest to declare.
Increased Extracellular Nucleosome Levels and Microparticles in Atrial Fibrillation Patients Compared to the Age-Matched Normal Population
Authors: Hoppensteadt Debra , Jabati Sallu , Phan Trung , Liles Jeffrey , Rowe Timothy , Syed Mushabar , Fareed Jawed
Published: October 2019

Introduction : Atrial fibrillation (AF) is currently the most common cardiac arrhythmia encountered in clinical practice and a major cause of morbidity and mortality among adults. It is estimated that atrial fibrillation affects approximately 2.7 million people in the United States. Extracellular plasma nucleosomes (PNs) are complexes of DNA and histones that are released during cell death. Histones have been shown to function as DAMPs when they are translocated from the nucleus to the extracellular space. It is hypothesized that these extracellular nucleosomes contribute to the observed hypercoagulable state and embolic complications in AF.

Methods : The concentration of PNs in 48 plasma samples of atrial fibrillation patients and 38 aged-matched controls were measured using the Cell Death Detection ELISA PLUS assay (Roche Diagnostics, Mannheim, Germany) and functional microparticle levels were measured using an annexin binding method (Hyphen Biomedical, Paris, France).

Results : When comparing the concentration of nucleosomes in plasma, the atrial fibrillation patients (306.1 ± 328.86 μg/mL) had much higher levels than the aged-matched controls (177.9 ± 146.7 μg/mL), (p = 0.0015). Functional microparticles were also markedly elevated in the AF patients (18.6 ± 4.1 nM) in comparison to the aged-matched controls (4.6 ± 3.1 nM), (p=0.001). A significant correlation was observed between the PN's and microparticles, (p < 0.05).

Conclusion : Both the microparticles and PNs were elevated in atrial fibrillation when compared to aged-matched controls, suggesting increased cell death resulting in the generation of these biomarkers. Extracellular nucleosomes function as DAMPs by binding to receptors and triggering the activation of multiple signal pathways which may contribute to the elevation of microparticles. Due to the involvement of inflammation and thrombosis in the pathology of atrial fibrillation, the simultaneously increased circulating nucleosome and microparticle levels in patients with AF implies their role in the pathogenesis of this syndrome.

Disclosures

No relevant conflicts of interest to declare.
Safety and Efficacy of Thrombopoietin Receptor Agonists in Patients with Previously Treated Chronic Immune Thrombocytopenia: A Systematic Review and Meta-Analysis
Authors: Yamada Yuji , Fujii Takeo , Cromwell Caroline , Shapira Ilan
Published: October 2019

Background: The current American Society of Hematology guideline recommends the use of thrombopoietin receptor agonists, eltrombopag or romiplostim as one of the second-line therapies for chronic immune thrombocytopenia (ITP). The efficacy and safety of those drugs have been tested in several clinical trials. However, the safety profile was not consistent throughout trials and is not yet well understood. We herein conducted a meta-analysis of randomized control trials to compare the safety and efficacy of thrombopoietin receptor agonists; eltrombopag and romiplostim versus placebo in patients with previously treated chronic ITP. Our primary outcome was drug-related adverse events greater than CTCAE grade 3.

Methods: We performed a literature search in MEDLINE, EMBASE, Cochrane library, and the American Society of Hematology website up to September, 2015 by two independent authors according to PRISMA guideline. We included only randomized clinical trials comparing eltrombopag or romiplostim versus placebo. Random-effects model was used to estimate pooled odds ratio (OR) with 95% confidence interval (CI).

Results: A total of eight trials involving 834 participants were included in the analysis. There was no significant difference of grade 3 or higher adverse events between placebo and treatment group (OR=1.01, 95% CI=0.57-1.78). Thromboembolism (OR=0.59, 95% CI=0.20-1.73), elevated ALT (OR=0.68, 95% CI=0.26-1.74), headache (OR=1.26, 95% CI=0.90-1.78), nausea (OR=0.82, 95% CI=0.43-1.55), or fatigue (OR=1.13, 95% CI=0.65-1.91) did not show a significant difference between groups, either. Clinical response, which is defined as platelets ≥50,000/μL at least once on treatment was significantly better in treatment group than in placebo group (OR=0.10, 95% CI=0.07-0.15). Bleeding symptoms (WHO Grades 1-4) were significantly more frequent in the placebo group (OR=1.60, 95% CI=1.14-2.24) during treatment.

Conclusions: Although several studies have suggested clinically significant treatment-related adverse events, such as thromboembolism, this meta-analysis showed that thrombopoietin receptor agonists are safe, well-tolerated, and effective in patients with previously treated chronic ITP.

Disclosures

No relevant conflicts of interest to declare.
Identification of Phenotypic Modulation of Angiotensin-(1-7) in Thrombin-Stimulated Human Aortic Endothelial Cells from iTRAQ Quantitative Proteomics
Authors: Peng Ching-Tien , Wu Kang-Hsi
Published: October 2019

Introduction:

The primary effector of renin-angiotensin system (RAS) system is angiotensin II. RAS activation causes many detrimental effects via AT1 receptor. Angiotensin-(1-7)/ angiotensin-converting enzyme 2/Mas axis is a newly identified counter-regulatory pathway against RAS system. Thrombin plays a critical role in coagulation and inflammation processes in vascular endothelium. Although RAS activation is associated with thrombotic complications, it is unknown whether angiotensin-(1-7) can modulate the pleotropic effects of thrombin. In this study, we investigate the proteomic changes of angiotensin-(1-7) effects on thrombin-stimulated human aortic endothelial cells (HAECs).

Materials and methods:

HAECs were pretreated with 10-7M anigotenion-(1-7) for 1 h and stimulated with 2 units/mL thrombin for additional 5 h. Their proteomes were investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and MetaCoreTMsoftware.

Results:

A total of 653, 717 and 801 proteins were identified in triplicated iTRAQ analyses. MetaCoreTM pathway analysis identified that iTRAQ data showed the consistent pathway alterations (70%) in triplicated experiments. The same altered pathways included "Cytoskeleton remodeling_Cytoskeleton remodeling", "Cell adhesion_Integrin-mediated cell adhesion and migration", "Cell adhesion_Chemokines and adhesion" , "Cytoskeleton remodeling _ Regulation of actin cytoskeleton by Rho GTPases", "LRRK2 in neurons in Parkinson's disease", "Cytoskeleton remodeling_Fibronectin-binding integrins in cell motility", "Cytoskeleton remodeling _TGF, WNT and cytoskeletal remodeling" were among the top 10 statistically significant pathways. Additional experiments validated the phenotypes of angiotensin-(1-7) effects in thrombin-stimulated HAECs.

Conclusions:

Several regulatory pathways are altered by angiotensin-(1-7) in thrombin-stimulated HAECs, with cytoskeleton remodeling, cell adhesion and cell migration (motility) as the dominant altered phenotypes.

Disclosures

No relevant conflicts of interest to declare.
Fluorescent Cell Barcoding As New Flow Cytometric Technique for Multiplexed Phenotyping and Signaling Profiling in Hematologic Patients
Authors: Giudice Valentina , Feng Xingmin , Kajigaya Sachiko , Biancotto Angelique , Young Neal S.
Published: October 2019

Fluorescent cell barcoding (FCB) is a high-throughput multiplexing technique, in which samples from one or more donors can be combined, minimizing staining variability, antibody consumption, and decreasing sample volumes needed. In this study, we optimized FCB technique for routine application in immunophenotyping, phosphoFlow, and intracellular cytokine detection. Human peripheral blood mononuclear cells (PBMCs) from healthy controls and patients were used for optimization, and CBD450 and CBD500, Pacific Orange succinimidyl ester, DyLight 350, and DyLight 800 were tested for barcoding 6 to 36 samples. Working concentrations ranging from 0 to 125 µg/mL were tested, and viability dye staining was also optimized to increase data robustness. We first measured fluorescence intensity (MFIs) of six serial dilutions (0, 0.75, 3.25, 12.5, 62.5 and 125 µg/mL) and the difference in intensity separating them. Second, a 3x3 matrix was prepared using three concentrations of two different dyes, and PMBCs were barcoded with nine different combinations of FCB dye concentrations. When gated with the dyes, nine lymphocyte populations were detected. A 4x(3x3) matrix was also designed using three different FCB dyes (CBD450, DyLight 800, and Pacific Orange) at various concentrations to barcode 36 samples simultaneously. Thus, each lymphocyte population was clearly identified. The separation of each population or purity of deconvolution is based on the distance between MFIs and CVs. When we calculated the purity of deconvolution in our experiments, human PBMCs displayed CVs of 10 - 25%. When MFIs were separated by 2-fold increase, barcoded populations could be identified with a good resolution, higher when MFIs were separated by 3- or more fold increase. The use of viability dyes as LIVE/DEAD Fixable Aqua viability dye together with the FCB showed increase of data robustness due to exclusion of dead cells from gating strategy. In combination with viability dye, we successfully performed a six-color phosphoFlow and a simple four-color stainings using the same FCB dye combinations and cytometer voltages, and also combining one healthy control and two different patient samples. Our methods using FCB dye alone and/or in combination with antibody staining should be useful to efficiently perform multiplex drug screening and lymphocyte characterization. FCB minimizes batches and technical variations, and also increases data robustness, thus improving the immune phenotyping of patients.

Disclosures

Young: GSK/Novartis: Research Funding.
Molecular Characterization of Long Non-Coding RNA CASC15 in Leukemogenesis
Authors: Contreras Jorge R. , Fernando Thilini , Tran Tiffany M , Zampini Matteo , Rodriguez-Malave Norma Iris , Palanichamy Jayanth Kumar , Gajeton Jasmine , Ung Nolan , Casero David , Basso Giuseppe , Pigazzi Martina , Rao Dinesh S.
Published: October 2019

High throughput transcriptome sequencing has uncovered a previously uncharacterized layer of gene regulation by long non-coding RNAs (lncRNAs). LncRNAs are characterized by capped, polyadenylated, and spliced transcripts that lack an open reading frame. Despite the similarities in their genetic organization, they play variety of roles at the cellular level, including regulation of transcription and translation, leading to alterations in gene expression. One of these functions is the regulation of expression of chromosomally adjacent genes. Here, we examined the function of the lncRNA CASC15 that was originally discovered as being dysregulated in in ETV6-RUNX1-translocated B-acute lymphoblastic leukemia. Enforced expression of CASC15 in hematopoietic stem and progenitor cells led to a myeloid bias in development with an overall decrease in engraftment and colony formation. Conversely, using a CRISPR-based approach, CASC15 deletion skewed hematopoietic cell progenitors towards a B cell fate. CASC15 was also demonstrated to regulate cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown in cell lines were enrichment for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. To further characterize this, we queried a functional relationship between YY1 and CASC15. Interestingly, we found that YY1 interacts with CASC15, and that CASC15 enhanced YY1-mediated transcription at the SOX4 promoter. Together these studies represent some of the first functional characterizations of lncRNAs in leukemia and highlight the importance of non-coding regulatory mechanisms in malignant hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
FHL2 Interacts with Iaspp and Impacts the Biological Functions of Leukemia Cells
Authors: Lu Wenting , Yu Tengteng , Liu Shuang , Li Saisai , Li Shouyun , Liu Jia , Xing Haiyan , Tian Zheng , Tang Kejing , Rao Qing , Wang Jianxiang , Wang Min
Published: October 2019

Introduction iASPP played an important role in leukemogenesis in our previous study. In order to clarify its mechanism, a yeast two-hybrid screen was performed to find the binding partner of iASPP. In this study, we reported FHL2 as a novel binding partner of iASPP. The biological functions of the communication between FHL2 and iASPP were detected, and its possible mechanisms were investigated in human leukemia cell lines.

Methods A yeast two-hybrid screen was performed to identify FHL2 as a novel binding partner of iASPP. Immunofluorescence, Co-IP and Western blot analysis were used to confirm the communication between FHL2 and iASPP. After FHL2 or iASPP was knocked down in K562 and Kasumi-1 cells by lentiviral, MTT assay and flow cytometry were performed to detect the proliferation, cell cycle distribution and apoptosis rate of leukemic cells, meanwhile Western blot analysis was used to analyze the level of cell cycle- and apoptotic-related proteins. Dual luciferase assay was conducted to investigate the transcriptional activity of p53 on Bax when iASPP and FHL2 were overexpressed or FHL2 was knocked down.

Results FHL2 was highly expressed in K562 and Kasumi-1 cells. FHL2 and iASPP interacted with each other and co-localized in both nucleus and cytoplasm. Either FHL2 or iASPP silenced could reduce cell proliferation, induce cell cycle arrest at G0/G1 phases, and increase cell apoptosis. Western blot analysis showed that the level of p21 increased and anti-apoptotic protein Bcl-2 was reduced. Interestingly, when FHL2 was knocked down, the protein expression level of iASPP also decreased. Similarly, the expression of FHL2 would reduce when iASPP was silenced. Dual luciferase assay suggested that iASPP could reduce the transcriptional activity of p53 on Bax, furthermore, when FHL2 was knocked down at the same time, the transcriptional activity of p53 was rescued.

Conclusions The interaction between FHL2 and iASPP in AML was observed for the first time. Cell proliferation reducing, cell cycle arresting at G0/G1 phases, and cell apoptosis increasing occurred in either FHL2 knockdown or iASPP knockdown. Moreover, iASPP and FHL2 participated in the regulation of the transcriptional activation function of p53. These results indicated that FHL2 might be a novel potential target for AML treatment.

Disclosures

No relevant conflicts of interest to declare.
Abnormal Expression of the Chemokine (C-C Motif) Ligand 17 Gene in Adult B-Cell Acute Lymphoblastic Leukemia
Authors: Feng Yong-Huai , Wang Wei-Min , Lu Wen-Yi , Wang Shu-Juan , Yao Qiu-Mei , Zhou Jiao , Zhang Jia-Min , Huan Xiao-Jun , Liu Kai-Yan , Ruan Guo-Rui
Published: October 2019

Abstract

Background: Chemokine (C-C Motif) Ligand 17 is a protein coding gene. This chemokine plays important roles in T cell development in thymus as well as in trafficking and activation of mature T cells. Diseases associated with CCL17 include mycosis fungoides, paragonimiasis and some hematologic malignancies such as Hodgkin¡¯s lymphoma, B cell lymphoma, and Nasal natural killer/T cell lymphoma (NNKTL). However, our knowledge of the expression levels of CCL17 in B-cell acute lymphoblastic leukemia (ALL) remains limited.

Aims: The purpose of this study was to investigate the expression levels of human CCL17 messenger RNA in adult B-cell ALL.

Methods: A real-time quantitative reverse transcription-polymerase chain reaction assay based on TaqMan fluorescence methodology was used to quantify the CCL17 mRNA copy number in the bone marrow cells from patients with adult leukemia and in 16 human hematologic malignant cell lines. Normal marrow samples from the allogeneic stem cell transplantation donors were served as control. Informed consent was obtained for every marrow sample.

Results:

Expression levels of the CCL17 gene in leukemic cell lines, leukemia patients and normal donor marrow are shown in Figure 1. These results showed that the relative levels of CCL17 gene expression in marrow from 189 newly diagnosed B-cell ALL(median 0.23%; range 0%¡«69000%) was significantly higher than those of bone marrow from the 43 healthy donors (median 0.05%; range 0%¡«6.87%;P =0.0007). Significant CCL17 mRNA overexpression was found in the de novo B-cell ALL patients compared with 102 treated B-cell ALL patients(median 0.02%; range 0%¡«3339%) who achieved complete remission or 70 de novo AML (median 0.02%; range 0%¡«9.132%;P¡¯s <0.0001). The expression levels of CCL17 was higher in 21 refractory/relapsed B-cell ALL patients (median 0.68%; range 0.003%¡«13490%) than that newly diagnosed B-cell ALL, but no statistical significant difference was observed (P=0.34). Besides, no statistical significant difference was observed in 16 newly diagnosed T-cell ALL(median 0.06%, range 0%¡«9.947%), 70 AML and 43 healthy donors (P¡¯s > 0.05), but it was higher in SupB15 and Nalm-6 cells from B-cell ALL cell lines than in other cells from AML or T-cell ALL cell lines.

Conclusion: These results suggest that abnormal expression of CCL17 in leukemia may be involved in the pathomechanism of B-cell ALL.

Figure 1 A: CCL17 expression levels in B-cell ALL and healthy donors( ** represent P< 0.01). B: CCL17 expression levels in human hematologic malignant cell lines. Figure 1. A: CCL17 expression levels in B-cell ALL and healthy donors( ** represent P< 0.01). / B: CCL17 expression levels in human hematologic malignant cell lines.

Disclosures

No relevant conflicts of interest to declare.
Clinical and Pathological Features and Differential Diagnosis of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): a Series of Cases
Authors: Cesarman-Maus Gabriela , Lome Carmen , Espinosa Karla Adriana , Quezada-Fiallos Carmen Marcela , Rivas Silvia , Quezda Roxana
Published: October 2019

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is a recently recognized highly aggressive malignant proliferation of plasmacytoid dendritic-cell (PDC) precursors which consistently express CD4, CD56 and CD123. Mortality is high despite transplant and the response to treatment is poor, except for preliminary results with conjugated anti-CD123. Clinically, cutaneous involvement is the most common feature with or without the presence of initial bone marrow infiltration, however patients may present with bone marrow-only disease. BPDCN is underdiagnosed, and can be confused with several entities, including acute myeloid leukemia. We describe our experience with 7 cases of BPDCN, their clinical and pathological presentation, and describe possible misdiagnosis and the antibodies that may help in corroborating BPDCN. See table for clinical characteristics at diagnosis. Diagnosis of BPDCN must take into account clinical, morphological and immunohistochemical analysis (IHC), since these tumors variably express markers that may be shared with other neoplasias including CD56 and TCL-1. When IHC is not categorical for BPDCN, the WHO recommends reporting the cases as AML of ambiguous lineage. The typical IHC includes CD4, CD43, CD45RA, CD56, CD123, TCL1, CLA and CD68. The absence of CD56 does not exclude the diagnosis. Markers shared with other hematological tumors include: CD7, CD33, CD2,CD36 and CD38 and TdT. Thus differential diagnosis should be done primarily with A) Skin infiltration by acute myeloid leukemia (myeloperoxidase +, 7- lysozyme +, CD34 +, CD117 +/-) B) Skin Infiltration by T / NK extra nodal lymphoma ( CD8 , cytotoxic cytoplasmic granules [CCG] , granzyme B, perforin, TIA1 and EBER) C) cutaneous peripheral T lymphoma ( +/- CD8, CD2 +/-, +/- CD5, CD7 +/- and variably positive CCG´s). D) Other histiocytic and dendritic cell-neoplasms may also be considered in the differential diagnosis however the histological appearance is usually characteristic. BPDCN is a poorly known entity that should be suspected by both clinician and pathologist in order to make a correct diagnosis.

Table Table.

Disclosures

No relevant conflicts of interest to declare.
The Combination Effect of Homoharringtonine and Ibrutinib on FLT3-ITD Mutant Acute Myeloid Leukemia
Authors: Li Xia , Yin Xiufeng , Wang Huafeng , Huang Jiansong , Yu Mengxia , Ma Zhixin , Li Chenying , Zhou Yile , Yan Xiao , Huang Shujuan , Jin Jie
Published: October 2019

Acute myeloid leukemia (AML) is a highly heterogenous disease and the patients with an internal tandem duplication mutation in FMS-liketyrosine-kinase-3 (FLT3-ITD) have a negative impact on outcome. Finding effective treatment regimens is desperately needed. In this study, we explored the inhibitory effects and mechanism of homoharringtonine (HHT) in combination with ibrutinib on FLT3-ITD mutant AML cells. Consequently, we observed a synergistic inhibitory effect when ibrutinib was combined with HHT to inhibit cell proliferation, induce apoptosis and arrest cell cycle at G0/G1 phase in MV4-11 and MOLM-13 leukemia cells. Our results indicate that the mechanisms of the combination effect are mainly via regulating the STAT5/Pim-2/C-Myc pathway, AKT pathway and Bcl-2 family, activation p21WAF1/CIP1 and inhibiting CCND/CDK complex protein. Interestingly, synergistic cytotoxicity of ibrutinib and HHT was both dependent of FLT3 and BTK. Here we provide a novel effective therapeutic approach for the treatment of AML patients with FLT3-ITD mutation.

Disclosures

No relevant conflicts of interest to declare.