bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of TGF-β Is Decreased in the Bone Marrow of Double Knockout (DKO) SMAD3-/- Fancd2-/- Mice
Authors: Falvello Virginia , Epperly Michael W. , Dixon Tracy , Franicola Darcy , Zhang Xichen , Greenberger Joel S.
Published: October 2019

Homologous deletion recombinant negative DKO mice, missing action of both Fancd2 and SMAD3 proteins were derived to test the hypothesis that abrogation of TGF-β signaling would reverse Fanconi Anemia (FA) stem cell sensitivity to TGF-β. To determine whether DKO mouse tissues demonstrated detectable differences in production of the ligand for TGF-β receptor, tissues from adult six - eight week old DKO mice were compared to those from Fancd2-/- (C57BL/6J background), SMAD3-/- (129/Sv genetic background), or wild type F1 mice (129/Sv X B6) for levels of TGF-β. Tissues examined included bone marrow, intestine, spleen, liver, muscle, and brain. Tissues were homogenized, and analyzed for bone marrow protein by Luminex analysis using a TGF beta 1 multispecies kit for Luminex Platform (Life Technologies).

The results demonstrated decreased but not significant production of TGF-β in the bone marrow of DKO mice (3068 ± 753 pg TGF-β/mg protein) compared to 5211 ± 1662 (p = 0.0761) for wild type F1 mice (129/Sv X B6), and 5192 ± 705 (0.1085) for Fancd2-/- mice. However, TGF-β production in DKO was significantly decreased compared to Smad3-/- (129/Sv) mice (9828 ± 1076, p = 0.0127). The lungs from DKO also had decreased TGF-β production compared to wild type F1 mice (436 ± 125 and 1159 ± 44 pg/mg, respectively, p= 0.0217). Decreased TGF-β production was also seen in the DKO liver compared to F1 wild type liver (13.2 ± 1.7 and 33.5 ± 3.6, respectively, p = 0.0072). Bone marrow stromal cell lines derived from long-term bone marrow cultures of each mouse strain were tested for production of TGF-β. SMAD3-/- bone marrow stromal cells also had an increased production of TGF-β (236 pg/mg) compared to wild type F1, Fancd2-/- and DKO cell lines (117, 136 and 144 pg/ml). Bone marrow CFU-GEMM from each mouse strain was tested for sensitivity to inhibition by increasing concentrations of TGF-β, and both fresh bone marrow from DKO and SMAD3-/- mice demonstrated no TGF-β mediated abrogation of colony formation. In contrast, fresh marrow from wild type and Fancd2-/- mice demonstrated TGF-β concentration dependent inhibition of CFU-GEMM colony formation in vitro. These data indicate that TGF-β production in DKO mice is decreased in bone marrow, lung and liver compared to that in F1 wild type, SMAD3-/- or Fancd2-/- mice, and suggest that the control of TGF-β production by abrogation of TGF-β signaling in the setting of deletion of Fancd2 may be modulated in an organ specific manner.

Supported by research grant NIAID/NIH, U19A168021.

Disclosures

No relevant conflicts of interest to declare.
DNA Cross-Linking Agent Sensitivity of Fanconi Anemia (FA) Cells Is Preserved in Double Knockout (DKO) SMAD3-/- Fancd2-/- Mouse Cell Lines
Authors: Chen Zean , Franicola Darcy , Shields Donna , Epperly Michael W. , Zhang Xichen , Greenberger Joel S.
Published: October 2019

Increased sensitivity to inhibition of hematopoiesis by TGF-β has been hypothesized to be a mechanism of hematopoietic failure in Fanconi Anemia (FA). To determine whether abrogation of TGF-β signaling rescued biologic parameters of hematopoiesis, we derived a novel DKO mouse strain (SMAD3-/- (129/Sv) Fancd2-/- (C57BL/6)) by breeding double heterozygote mice. The DKO mice were small at birth, but achieved normalized growth and development by six - eight weeks. Hematopoiesis in long-term bone marrow cultures derived from DKO mice, ceased generating day 14 CFU-GEMM hematopoietic progenitors by 18 weeks, similar to that observed with Fancd2-/- marrow cultures. In contrast, cultures from SMAD3-/- (129/Sv) demonstrated continuous hematopoiesis for over 30 weeks confirming results with marrow from SMAD3-/- (C57BL/6J) mice (Epperly, et al., Experimental Hematology, 33:353-362, 2005), and significantly longer than that in control 129/Sv X C57BL/6J F1 mouse long-term marrow cultures. Bone marrow stromal cell lines derived from long-term marrow cultures were tested for the diagnostic criterion of FA, increased sensitivity to DNA cross-linking agent, Mitomycin-C. Cells were incubated in Mitomycin-C at concentrations ranging from 0-20 ng/ml for four days and then plated in 4 well tissue culture plates, incubated for 7 days at 37o C in a CO2 incubator, stained with crystal violet, and colonies of greater than 50 cells counted. Similar to Fancd2-/- marrow stromal cells, DKO marrow stromal cells showed increased sensitivity to Mito-C, while marrow stromal cell lines from wild type or SMAD3-/- mouse marrow cultures were relatively resistant (p = 0.0086 comparing Fancd2-/- to control 129/Sv X C57BL/6J F1 cells, p = 0.0156 comparing DKO with control 129/Sv X C57BL/6J F1 cells, and p = 0.4676 comparing Smad3-/- cells to control 129/Sv X C57BL/6J F1 cells). Fresh bone marrow from DKO mice, similar to that from SMAD3-/- mice, demonstrated resistance to inhibition of hematopoietic cell colonies in vitro by increasing concentrations of TGF-β. Therefore, abrogation of TGF-β signaling in DKO cells did not alter the sensitivity of Fancd2-/- marrow stromal cells to Mitomycin-C.

Supported by research grant NIAID/NIH, U19A168021.

Disclosures

No relevant conflicts of interest to declare.
Carbonic Anhydrase IX Promotes the Tumorigenicity of Adult T-Cell Leukemia/Lymphoma-Derived Cells in NOG Mice
Authors: Nasu Kentaro , Yamaguchi Kazunori , Takanashi Yuka , Sugamura Kazuo , Harigae Hideo
Published: October 2019

Carbonic anhydrase IX (CA9) is a membrane-associated carbonic anhydrase that regulates pH through its extra-cellular catalytic domain and thereby fosters tumor cell survival, growth, and invasion. The expression of CA9 is induced by hypoxia via HIF-1α. CA9 overexpression is known to be closely associated with aggressive behaviors and poor prognoses in various types of human solid tumors. In particular, CA9 is a critical mediator of the expansion of breast cancer stem cells in hypoxic niches by sustaining the mesenchymal and 'stemness' phenotypes of these cells. However, as for hematological malignancies, there are few reports suggesting a relation between its tumorigenecity and CA9. Here we demonstrated that CA9 plays a crucial role in promoting the tumorigenicity of the ATL-derived cells.

Recently we established a highly tumorigenic subline, ST1-N6, from an ATL-derived cell line, ST1, by serial transplantation in immunodeficient NOG mice. ST1-N6 cells developed subcutaneous tumors under a shorter duration and smaller cell number than parental ST1 cells. DNA microarray gene expression analysis revealed an upregulated expression of CA9 in ST1-N6 cells as compared with ST1 cells. After serial sorting of ST1 cells by CA9 expression, we obtained ST1-CA9high and ST1-CA9low sublines. Like ST1-N6 cells, ST1-CA9high cells showed stronger tumorigenicity than ST1-CA9low or parental ST1 cells. Knockdown of CA9 expression with shRNAs suppressed the tumorigenicity of ST1-CA9high cells, whereas ST1 cells introduced with CA9 significantly showed enhanced tumorigenicity. Another ATL-derived cell line, TL-Om1 also showed increased tumorigenicity in NOG mice when introduced with CA9. These observations indicate that CA9 contributes to promoting the tumorigenic potential of ATL-derived cells in xenotransplantation.

To investigate CA9 expression on fresh ATL cells, we prepared peripheral blood from six acute ATL patients, and tested for quantitative expression of CA9 in sera of six ATL patients and three healthy donors by ELISA. The amounts of serum CA9 were 31.1~95.4 pg/ml in the healthy donors, while they were 175.6~1, 971.6 pg/ml in the ATL patients. We next prepared ATL tissues from nine ATL patients, and two of them contained a few CA9high cells, which were cells expressing CD25, suggesting that a part of ATL lymphoma cells expressed CA9. These results suggested that the amount of serum CA9 tends to be higher in ATL patients than healthy donors.

Based on these findings, we suppose that CA9 plays an important role in the malignant development of ATL.

Disclosures

No relevant conflicts of interest to declare.
Azatidine Treatment in Very Elderly Patients Diagnosed with Acute Myelogenous Leukemia (AML) or High Risk Myelodysplastic Syndrome (MDS)
Authors: Gutierrez Javier , Morales Elena , Gonzalez Matilde , Herrada Juan
Published: October 2019

Background: The use of azatidine (AZT) is the agent of choice for first line treatment of elderly (age >74 years) patients (pts) with high risk MDS or AML (2015 National Comprehensive Cancer Network Guidelines). The Hematology consultant is frequently involved in the care of very elderly (age >79 years) individuals. Our aim is to report our experience with 3 patient aged 80 or above that received treatment with AZT.

Methods: Retrospective review

Results: We reviewed the medical records of patients treated for high risk MDS or AML at our institution between July 2009 and March 2012. A total of 3 pts aged 80 or above were identified. All three pts were males, and all received AZT 100 mg/m2/day x 7 days subcutaneously in every cycle of 28 days. Patient 1 (aged 81) had 12 % of blasts in the bone marrow (BM), was diagnosed with Refractory Anemia with Excess Blasts-2 (RAEB-2) applying the 2008 World Health Organization Classification (WHO) of MDS. Patient 2 (aged 90) had AML with multilineage dysplasia and 38% blasts in BM. Patient 3 (aged 80) had AML with multilineage dysplasia and 30% blasts in BM. Pts characteristics and results are summarized in Table 1.

Conclusions: In pts diagnosed with high risk MDS or AML, advanced chronological age (> 79 years) alone should not preclude the use of effective and well tolerated therapy. Identification of pts in this geriatric subgroup who may benefit from AZT merits additional research.

Table 1. Diagnosis Age(years) Baseline laboratory Peripheral blood %Blast in Bone Marrow Number of AZT cycles received Response Survival(months) Patient 1 RAEB-2 81 Hb 14 g/dl Platelets 56x10e9/L Leucocytes 1.90x10e9/L (Neutrophils 0.81x10e9/L) 12 11 CR* in BM after 6 cycles 17 Patient 2 AML 90 Hb 8.06 g/dl Platelets 20x10e9/L Leucocytes 1.30x10e9/L(Neutrophils 0.64x10e9/L) 38 15 CR in BM after 7 cycles 22 Patient 3 AML 80 Hb 7.50 g/dl Platelets 30x10e9/L Leucocytes 6.00x10e9/L(Neutrophils 1.44x10e9/L) 30 19 CR in BM after 6 cycles 23 *CR: complete remission (<5% blast in aspirate with spicules)

Disclosures

No relevant conflicts of interest to declare.
Platelets Can Phagocytose Influenza Virus Which May Contribute to the Occurrence of Thrombocytopenia during Influenza Infection
Authors: Jansen A.J. Gerard , Low Hui Zhi , van den Brand Judith , van Riel Debbie , Osterhaus Albert , van der Vries Erhard
Published: October 2019

Thrombocytopenia is a common and sometimes life-threatening symptom in patients with influenza, which indicates that platelets may play a significant role during virus infection. However, the mechanisms underlying influenza associated thrombocytopenia are still unclear to date. In this study, the relationship between platelets and influenza infection was studied in vivo and in vitro.

In the period 2009-2012 we randomly measured viral load and platelet count in laboratory-confirmed influenza patients (A/H1N1 subtype; n=85) presented at the emergency department of Erasmus MC. In a cross-sectional study we found, after excluding patients with HIV infection, chemotherapy treatment and immediate referral to the ICU, that a high viral load was significantly correlated with a low platelet count at presentation (n=36; Spearman correlation coefficient 0.341; P=0.042). To study this relationship further we inoculated ferrets (n=120) with an H3N2 (n=30), pandemic H1N1 (n=30), or H5N1 influenza A virus (n=30) and measured platelet counts in six ferrets per time point at day 0, 1, 2, 3 and 4 after infection. Thrombocytopenia was seen at day 3 in all ferrets compared to the control animals (n=30). The decrease in platelet counts was significant in the H5N1 infected ferrets (p<0.05). For the sialidase and influenza virus inhibitor oseltamivir, we have previously shown that it functions as an inhibitor of platelet degradation. Interestingly, thrombocytopenia in ferrets could also be inhibited by oral administration of oseltamivir. However, these results were not statistically significant.

Finally, we studied the interaction between platelets and influenza viruses by electron microscopy. In a platelet suspension incubated for 1 minute with influenza virus we observed platelets with virus containing vacuoles, suggesting that the platelets had rapidly phagocytosed the viruses. Characteristics of these platelets incubated with influenza virus were measured including membrane expression of the GPIb-IX-V complex, CD62P, and surface glycans sialic acid and GlcNac using flowcytometry and functional capacity employing aggregometry using collagen, thrombin and ADP as agonists. However, no effect was seen in expression of GPIba and GPIX, P-selectin expression and surface expression of sialic acid and GlcNAc after incubation with influenza virus. Also no effect was seen on functional capacity of these platelets.

Our study shows that influenza virus is significantly correlated with a lower platelet count. Phagocytosis of influenza virus by platelets may play an important role in the occurrence of thrombocytopenia during influenza infection and may be a mechanism of virus clearance during infection.

Disclosures

Osterhaus: Viroclinics Biosciences: Other: chief scientific officer and hold certificates of shares.
NCCN-IPI Is Superior to aaIPI and IPI in Predicting Survival of DLBCL in the Rituximab Era
Authors: Wei Yongqiang , Zhang Yuankun , Hao Xiaoxiao , Wei Xiaolei , Huang Weimin , Feng Ru
Published: October 2019

Objective

International prognostic index (IPI) has been widely used for predicting outcome in diffuse large B cell lymphoma. Although the introduction of rituximab to chemotherapy has dramatically improved the outcome of DLBCL, it also changed the prognostic value of IPI. The National Comprehensive Cancer Network IPI (NCCN-IPI) and age-adjusted IPI (aaIPI) were used to evaluate the prognosis for DLBCL in the rituximab era. However which one of them is more powerful in predicting survival remains unknown in Chinese patients.

Patients and Methods

A total of 334 patients with de novo diffuse large B-cell lymphoma diagnosed from 2003 to 2012 were included. All patients were treated with CHOP with or without rituximab. They were divided into CHOP and R-CHOP groups. IPI, NCCN-IPI and aaIPI score were recorded. Survival was performed according to the Kaplan-Meier (K-M) curves using the log-rank test. The predictive abilities of PI, NCCN-IPI and aaIPI were investigated by Harrell's C-statistics.

Result

Compared with the IPI, the NCCN-IPI and the aaIPI had better discrimination in different scores in all patients treated with or without rituximab. Both the NCCN-IPI and aaIPI had power in discriminating low, low-intermediate and high-intermediate risk groups. In comparison, the NCCN-IPI discriminated low-intermediate and high-intermediate risk groups (5-year overall survival [OS]: 74% vs 50%) better than the aaIPI (5-year OS: 80% vs 62%) in all patients and in the R-CHOP group with the NCCN-IPI 5-year OS: 85% vs 67% while the aaIPI: 87% vs 77%. In the CHOP group, the aaIPI discriminated low-intermediate and high-intermediate risk groups better (5-year OS: 70% vs 25%) than the NCCN-IPI (5-year OS: 60% vs 24%). According to the Harrel C statistic, the NCCN-IPI showed higher discrimination of the different scores (C_index: 0.669 ) than the aaIPI (C_index: 0.663 ) in all the patients (C_index: 0.669 vs. 0.663), especially in the R-CHOP group (C_index: 0.681 vs. 0.636). In the CHOP group, the aaIPI had stronger power (C_index: 0.711 ) than the NCCN-IPI (C_index: 0.683 ) in discriminating different scores.

Conclusion

The NCCN-IPI is more powerful than the IPI for predicting survival in the rituximab era. In patients aged 60 or younger and treated with CHOP without rituximab, aaIPI is a preferable tool for evaluating prognosis.

Disclosures

No relevant conflicts of interest to declare.
Pharmacometabolomics for Predicting Variable Busulfan Exposure in Pediatric HSCT Patients
Authors: Kim Bora , Hong Kyung Taek , Lee Ji Won , Yu Kyung-Sang , Jang In-Jin , Park Kyung Duk , Shin Hee Young , Ahn Hyo Seop , Cho Joo-Youn , Kang Hyoung Jin
Published: October 2019

Optimal dosing for busulfan is important for minimization of systemic toxicity from overexposure and graft failure or relapse from underexposure. Herein, we investigated potential markers for predicting individual variation in the pharmacokinetics of busulfan, and suggested possible mechanism for inter-individual variability by using pharmacometabolomics.

Fifty-nine pediatric patients undergoing busulfan-based conditioning chemotherapy for hematopoietic stem cell transplantation were divided into three groups according to the area under the concentration-time curve (AUC) of busulfan; low-, medium-, and high-AUC group. Nontargeted metabolic profiling of baseline urine samples showed that deferoxamine metabolites were abundant, while 2 acylcarnitines and phenylacetylglutamine were significantly lower in high-AUC group, compared with low-AUC group. Higher level of deferoxamine, an iron-chelating agent for the patients with a high serum ferritin level during the conditioning chemotherapy, suggested pharmacokinetic interaction between serum ferritin level and busulfan exposure. Retrospective analysis of the correlation between serum ferritin level and busulfan AUC showed positive correlation in 130 pediatric patients. The optimal busulfan dose to meet the target AUC of 18,750 μg*h/L/day was calculated to be 119.7 ± 30.1 mg/m2 in patients with ferritin < 1,000 ng/mL and 106.1 ± 29.3 mg/m2in patients with ferritin ≥ 1,000 ng/mL (P=0.021).

Mechanismly, previous studies have indicated that ferritin, acylcarnitine and phenylacetylglutamine are closely associated with liver function. Increased serum ferritin is thought to be responsible for an increased production of oxygen free radicals and activation of GSH turnover, which means reduction of GSH levels in both plasma and erythrocytes and this depletion seems to be related to the decrease of busulfan metabolism. Down regulation of acylcarnitines is associated with deregulation of mitochondrial fatty acid β-oxidation by hepatic injury. PAGN, a marker of waste nitrogen scavenger, was down-regulated which indicates ammonia-related metabolism could also be involved in busulfan exposure. Hyperammonemia, a clinical condition of elevated ammonia levels, is mainly lead by liver cell damage.

Taken together, our findings demonstrate that elevated serum ferritin levels are a potential biomarker correlated with busulfan exposure and provide some evidence that busulfan metabolism seems to decrease in the patients with reduced liver function.

Disclosures

No relevant conflicts of interest to declare.
the Role of MSCs in the Development of Acute Myeloid Leukemia
Authors: Al-Matary Yahya Saleh , Botezatu Lacramioara , Fernando Lams Robert , Thivakaran Aniththa , Köster Renata , Schütte Judith , Dührsen Ulrich , Opalka Bertram , Khandanpour Cyrus
Published: October 2019

Acute myeloid leukemia (AML) is a malignant disease of the bone marrow. The prognosis of AML is still poor with only 25% of patients living longer than 5 years. Thus, new therapeutic approaches are warranted to improve patients' survival. The growth of AML cells not only depends on cell intrinsic factors, but is also supported by stroma cells, among them mesenchymal stem cells (MSCs). We reported here that MSCs derived from human AML patients or murine models of human AML better supported the in vitro growth of leukemic cells than MSCs from non-leukemic patients or mice and this is dependent on cell-to-cell contact. Similarly, immortalized MSCs lines derived from AML patients supported the growth of human AML cell lines better than a MSC line originating from a healthy donor. In addition, the frequency of MSCsin the BM of leukemic mice was increased. On a molecular level, the polarization of MSCs towards an AML-supporting state depended on the expression of the transcription factor Growth factor independence 1 (Gfi1). Loss of Gfi1 abrogated the tumor-supporting state of AML-associated MSCs in vitro and in vivo. In summary, MSCs from AML patients support the growth of AML cells in vitro in a Gfi1-dependent manner which could open the path to new therapeutic approaches.

Disclosures

Dührsen: Amgen: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Khandanpour:Hospital of Essen university: Research Funding; Max-Eder: Research Funding.
Modeling Clonal Hematopoietic Disorders in Zebrafish Using Color Barcoding
Authors: Avagyan Serine , Henninger Jonathan E , Moore Jessica , Mannherz William , Zon Leonard I.
Published: October 2019

Myeloid neoplasms, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), are clonal hematopoietic disorders that arise from abnormal hematopoietic stem and progenitor cells (HSPCs)with acquired somatic mutations. To follow the hematopoietic output of HSPC clones endogenously, we have developed a novel marking strategy in which each HSPC clone is labeled in a unique fluorescent hue. The transgenic zebrafish line Zebrabow uses a Cre/lox system where stochastic recombination of multiple transgenic insertions harboring three fluorescent proteins (dTomato, YFP and CFP) provide stable and heritable color barcodes. Tissue-specificity is ensured by tamoxifen-induced Cre activation under the draculin promoter which is highly expressed in zebrafish HSPCs throughout development. The result of Zebrabow labeling is a collection of colored blood cells, which are grouped by their hue, and largely cluster in various groups, each representing a clonal population of cells that arise from a specific HSPC. To induce clonal hematopoietic states in zebrafish assessed by color dominance in hematopoietic cells, we introduced combinations of known genetic mutations founds in MDS and AML into Zebrabow embryos. Embryos at the 1-cell stage were injected with combinations of mutations and HSPC color labeling was performed at 36 to 48 hours post fertilization, during peak emergence of definitive blood stem cells from the dorsal aorta. The injected mutations were grouped by known co-occurence in patients with MDS and AML. The transgenic overexpression of Jak2V617F, FLT3ITD, IDH2R140Q, HOXA9 or HOXB4 under the zebrafish blood-specific promoter draculin, was combined with somatic mutagenesis of endogenous tet2, asxl1, dnmt3a, ezh2, ptena/b and tp53 via the CRISPR/Cas9 system. Mosaic combinations of these mutations resulted in the expansion of single color clones, contributing to ≥30% of granulocytes, with concomittant myeloid expansion in a third of these zebrafish. The majority of zebrafish exhibiting clonal expansion harbored frame-shift mutations in asxl1 with >25% variant allelic frequency identified by next-generation sequencing. Similarly, combinations including Jak2V617F, tet2, or asxl1 mutations lead to single color expansion in kidney marrow cells in 30% of injected zebrafish, with the occasional presence of a blast-like population of cells in the marrow and spleen. We have successfully modeled early stages of clonal hematopoietic expansion with myeloproliferation in the presence of Jak2V617F and asxl1 deletion using the unique color barcoding system in zebrafish.

Disclosures

Zon: Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.
Deferasirox Associated to Liver Failure and Death in a Sickle Cell Anemia Patient Homozygous for the -1774delG Polymorphism in the ABCC2 Gene Encoding Multidrug Resistance Protein 2 (MRP2)
Authors: Braga Caroline Cordeiro Barroso , Benites Bruno Deltreggia , de Albuquerque Dulcineia M , Alvarez Marisa Claudia , Costa Fernando F , Gilli Simone C O , Saad Sara T.O.
Published: October 2019

Introduction: Regular blood transfusions may result in hemochromatosis, leading to damage of various organs. Deferasirox exhibits good efficacy as iron chelation therapy, however changes in liver parameters may occur, as well as, despite being rare, more serious complications such as liver failure. Multidrug resistance protein 2 (MRP2) is a member of the ATP-binding cassette (ABC) transporter family of membrane proteins encoded by the ABCC2 gene and is expressed in the canalicular part of the hepatocyte. MRP2 acts in the biliary transport of several endogenous compounds and many drugs, including Deferasirox. Thus, genetic polymorphisms related to the ABCC2 gene, which encodes the MRP2 protein, may influence individual susceptibility to hepatotoxicity related to Deferasirox.

Methods: Two functional genetic variants, rs369192412 (-1774delG) and rs717620 (-24C> T), of the ABCC2 gene, were studied using the DNA sequencing technique in 31 genomic DNA samples from patients with iron overload who were currently using or had previously used Deferasirox. The sequencing results were compared to clinical data related to the presence or absence of hepatotoxicity during the use of this drug.

Results: Of all patients studied, 9 (29%) of them developed hepatotoxicity based on elevation of AST and/or ALT >5 X ULN or elevation of bilirubins >3 X ULN, which occurred in an average of 239 (14-983) days after initiation of treatment. From the 9 (29%) patients with hepatotoxicity, only 2 (22.2%) of them had the polymorphism -24C> T in only one allele, and the remaining 7 (77.8%) patients did not (OR = 0.77, 95% CI = 0.06 to 5.91, p = 1). Despite no association was detected between the -17774delG polymorphism and hepatotoxicity (OR = lower, 95% CI = 0.06 to bottom, p = 0.29), this polymorphism was present in homozygosis in 1 patient with sickle cell anemia, who, coincidently, had fulminant hepatitis leading to death, with no other causes for liver toxicity and despite all introduced therapies.

Conclusions: Our study showed no correlation between ABCC2 polymorphisms (-24C> T, -1774delG) and hepatotoxicity to Deferasirox, however, the rs369192412 (-1774delG) polymorphism was present in homozygosis in the only patient who died due to fulminant hepatitis secondary to the drug. This data may indicate a possible influence of the presence of this polymorphism in the severity of liver damage related to Deferasirox.

Disclosures

No relevant conflicts of interest to declare.
Cell-Free DNA Is Elevated after Acute Arterial Injury in Infants
Authors: Kim Jina , Naqvi Ibtehaj , Nag Uttara P. , Leraas Harold J. , Otto James C. , Borst Alexandra J. , Hill Kevin D. , Fleming Gregory A. , Sullenger Bruce A. , Tracy Elisabeth T.
Published: October 2019

Purpose: Circulating cell-free DNA in blood may originate from cell necrosis, apoptosis, or NETosis. NETosis is a recently described inflammatory process in which neutrophils release a meshwork of DNA fibers called neutrophil extracellular traps (NETs) to attack microorganisms. Higher levels of cell-free DNA have been observed in patients with sepsis and malignancy, and it may be linked to arterial thrombosis as well. We examined changes in cell-free DNA in infants who experienced acute, iatrogenic thrombosis of the femoral artery after arterial catheterization to better understand the role of cell-free DNA in clot formation.

Methods: Plasma samples were collected from infants < 1 year of age with congenital heart disease during cardiac catheterization. Post-procedurally, femoral artery thrombus formation at the site of sheath insertion was detected by ultrasound. Cell-free DNA was quantified at two time points (before and after arterial catheterization) in three infants who developed femoral artery thrombus after catheterization and three age-matched controls. Cell-free DNA was also quantified from plasma of normal adults as a comparison.

Results: At baseline,cell-free DNA level for the infant cohort was similar to that in normal adults (13.9 ng/mL vs. 10.3 ng/mL, p = 0.20, Figure). After arterial catheterization, cell-free DNA was increased from baseline in all infants (51.0 ng/mL vs. 13.9 ng/ml, p = 0.004). However, infants who developed femoral artery thrombus after catheterization did not demonstrate higher cell-free DNA after arterial injury, compared to the control group (47.1 ng/mL vs. 54.9 ng/ml, p = 0.83).

Conclusions: Cell-free DNA is significantly and consistently elevated in infants after arterial catheterization. As part of NETosis, cell-free DNA may serve as an inflammatory mediator in infants who experience arterial injury. We plan to confirm the findings of this pilot study in a larger human infant cohort as well as in a piglet model of vascular injury.

Figure Cell-free DNA levels in infants, before and after arterial catheterization, in comparison to normal adults. Figure. Cell-free DNA levels in infants, before and after arterial catheterization, in comparison to normal adults.

Disclosures

No relevant conflicts of interest to declare.
Intravital Microscopy Reveals Fundamental Differences in the Interaction of Stem Cells and T Acute Lymphoblastic Leukaemia with the Bone Marrow Microenvironment
Authors: Lo Celso Cristina , Hawkins Edwin D , Duarte Delfim , Akinduro Olufolake , Khorshed Reema A , Passaro Diana , Nowicka Malgorzata , Straszkowski Lenny , Scott Mark K , Rothery Steve , Ruivo Nicola , Foster Katie , Waibel Michaela , Johnstone Ricky W , Harrison Simon J. , Westerman David A , Quach Hang , Gribben John , Robinson Mark D , Purton Louise E , Bonnet Dominique
Published: October 2019

Haematopoiesis is the complex developmental process maintaining the turnover of all blood cell lineages, i.e. erythrocytes, immune (white) cells and platelets. It takes place primarily in the bone marrow, where it critically depends on the correct functioning of rare, quiescent haematopoietic stem cells (HSCs) and more numerous HSC-derived, highly proliferative and differentiating haematopoietic progenitor cells (HPCs). This is regulated by complex interactions between stem cells and the bone marrow microenvironment, and it was suggested to change in settings involving stress. Indeed, using intravital microscopy, we previously showed that HSCs injected in irradiated recipients steadily engage with endosteal niches. Moreover, if phenotypically identical HSCs are harvested from mice harbouring a natural infection, such as that caused by T.spiralis, they show a unique 'stop and go' migratory behaviour, engage larger niches, yet remain in the proximity of osteoblastic cells.

Based on these and other studies, it has been hypothesised that also malignant haematopoietic cells rely on interactions with specific bone marrow microenvironments to grow, and especially to develop chemoresistance. To investigate this further, we performed longitudinal imaging of mice injected with murine and human T acute lymphoblastic leukaemia (T-ALL) cells over the course of hours or days, including during response to multiple chemotherapy treatments. Strikingly, we observed a very different behaviour from that of HSCs, with T-ALL cells showing a highly migratory behaviour throughout all stages of disease, from initial bone marrow infiltration to response to chemotherapy. Interestingly, in untreated mice and in patients trephine biopsies we observed rapid loss of osteoblastic cells by apoptosis, an event that likely contributes to the dramatic loss of healthy haematopoiesis observed in mice and patients. Together, these finds indicate that novel therapeutic strategies should target the ability of T-ALL cells to entertain short-lived and promiscuous interactions with any neighbouring cells, and aim to strengthen the bone marrow microenvironment to improve support for residual healthy haematopoiesis.

Disclosures

Harrison: AbbVie: Research Funding; Janssen Cilag: Research Funding, Speakers Bureau. Quach:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.
Decitabine Combined with Haag Regimen Is an Effective Salvage Treatment for Advanced Acute Myeloid Leukemia
Authors: Tang XiaoWen , Cui Wei , Jin Zhengming , Zhu Xiaming , Sun Aining , Qiu Huiying , Miao Miao , Cao Jing , Guo Dandan , Wu Depei
Published: October 2019

Objective To evaluate the clinical efficacy and safety of decitabine (DAC) in combination with HAAG regimen [homoharringtonine (HHT), cytarabine (Ara-C), doxorubicin (Acla) and recombinant human granulocyte colony stimulating factor (G-CSF)] for advanced patients with acute myeloid leukemia (AML). Methods Thirty-six patients with advanced AML receiving DAC combined with HAAG chemotherapy in our center from December 2012 to August 2015 were enrolled in this study. Eighteen of them were refractory or relapsed AML, and another 18 patients were those who didn't achieve complete remission (CR) after a course of induction chemotherapy. The therapeutic responses, side effects and long-time survival were retrospectively analyzed. Results After a course of treatment, the rate of CR and partial response (PR) was 58.3% (21/36) and 22.2% (8/36) respectively, while the overall response rate (ORR) was 80.6% (29/36) in the cohort. For the patients with refractory or relapse AML, CR was 61.0% (11/18), PR was 22.2% (4/18), and ORR was 83.3% (15/18). While for the other not getting CR after a course of induction chemotherapy, CR was 55.6% (10/18), PR was 22.2% (4/18), and ORR was 77.8% (14/18). Grade 4 hematological toxicities were observed in all patients, and 72.2% cases experienced infection. And all non hematological side effects were mild and well-tolerated. With a median follow-up of 7.5 (0.5~33.3) months, the 1-year overall survival (OS) rate was 43.3%, 24.2% for the refractory or relapsed AML patients, and 61.6% for those not achieving CR after a course of induction chemotherapy. The difference was significantly (P=0.01). Conclusion DAC combined with HAAG regimen is safe and effective salvage treatment for advanced stage AML patients.

Disclosures

No relevant conflicts of interest to declare.
Identification of Genetic Alterations Associated with Mutant IL7Ralpha in T-ALL
Authors: Rodrigues Gisele O. L. , Li Wenqing , Cramer Sarah , Campos Livia W. , Zenatti Priscila P. , Laranjeira Angelo A.B.A , Hixon Julie A. , Senkevitch Emilee , Andrews Caroline , Noronha Elda P. , Pombo-de-Oliveira Maria , Brandalise Silvia , Lobo Francisco P. , Yunes Jose Andres , Durum Scott
Published: October 2019

The IL7/IL7R mediated signaling is essential for normal development and homeostasis of T cell precursors. Early studies have shown that around 10% of patients with Acute lymphoblastic leukemia T cell (T-ALL) have mutations in the alpha chain of the receptor for IL7 (IL-7Ralpha) driving constitutive signaling via JAK1 and independent of IL-7, gamma-chain or JAK3. Some genetic changes are important factors to initiate leukemia, but in many cases these changes are insufficient to achieve the complete leukemic phenotype, suggesting that collaborative oncogenic mutations may be present. To identify candidate mutations that work in collaboration with the oncogenic IL7R, we performed exome sequencing and SNP-CNV-Array assay on a group of eight primary T-ALL samples carrying the IL7R mutation (T-ALL-IL7Rmut). The microarray was performed using Cytoscan HD - Affymetrix and CNVs were detected by ChAs software, version 2.0.1.2. For exome sequencing we used Illumina Hiseq2000 platform and Agilent SureSelect V4 51M Capture kit (mean sequencing depths of 80X / 50X for leukemia and remission samples, respectively). Somatic Single Nucleotide Variants (SNVs) and Small Insertion/Deletion (InDels) were detected using VarScan, and mutations were functionally annotated using ANNOVAR. All somatic mutations detected were manually curated. We found 17 genes recurrently mutated (in ≥ 2 cases) and chose five of them for further analyses due to their previous involvement in ALL (PHF6, RB1, CTCF, SGK223 and DNM2). Ongoing experiments are being conducted to determine whether these recurrent mutations can collaborate functionally with mutIL7R by co-transfection into immature murine thymocytes, transplanting into mice and determining incidence of leukemia.

Disclosures

No relevant conflicts of interest to declare.
Concomitant Myeloproliferative Disorders (MPD) and Amyloidosis
Authors: Pallante Angeline J , Leung Nelson , Kyle Robert A. , Tefferi Ayalew , Russell Stephen J. , Habermann Thomas M. , Lacy Martha Q. , Hayman Suzanne R. , Al-Kali Aref , Reeder Craig , Dispenzieri Angela , Gertz Morie A. , Buadi Francis K , Hook C. Christopher , Dingli David , Pardanani Animesh
Published: October 2019

Background: The combination of MPD and amyloidosis is rare, and this patient population's clinical outcome is not well studied.

Methods: Pts with a MPD and amyloidosis were identified via an clinical note search engine that searches through the electronic medical records of patients seen at Mayo Clinic Rochester, Jacksonville, and Scottsdale between 1990 and 2016. Terms used included amyloid or amyloidosis, chronic myelogenous leukemia or CML, essential thrombocytopenia or ET, and polycythemia vera or PV. Demographic and clinical data were abstracted from the medical record. Pts with both disorders were analyzed and their mortality rates along with median time to death were calculated. Prevalences at the Mayo Clinic were calculated for the years 2014 and 2015.

Results: Twenty-three pts diagnosed with both a MPD and amyloidosis were identified. Thirteen (56.5%) were male, 10 (43%) were female. Eleven (47.8%) were initially diagnosed at the Mayo Clinic. Types of amyloidosis were as follows: Eleven (47%) had immunoglobulin light-chain (AL), four had localized (17%), two (8.7%) had wild-type transthyretin (ATTR), one (4.3%) had mutant ATTR, and five (21.8%) were unknown. Types of MPD were as follows: Seven (30%) had polycythemia vera (PV), seven (30%) had chronic myelogenous leukemia (CML), five (22%) had myelofibrosis, and four (17%) had essential thrombocytosis (ET). Fifteen (65%) were initially diagnosed with a MPD. Median time to last follow-up from second diagnosis was 1.7 years, and median time to death following second diagnosis was 1.4 years. The mortality rate was 87% in the total population. The median time to death for AL and PV was 2.7 years, AL and CML 1.0 years, and AL and ET 1.17 years. Myelofibrosis did not occur with AL. The most common combination was AL and PV, which accounted for five (22%) of the cases.

Treatment regimens for the patients with AL were varied. Multiple drugs were combined with dexamethasone including melphalan, velcade, pomalidomide, lenalidomide, doxorubicin, and revlimid. Cyclophosphamide, bortezomib, and dexamethasone (CyBorD) were used in two cases. The mean time to diagnosis of amyloidosis from symptom onset was 10.3 months.

Prevalences of AL and a MPD in 2014 and 2015 at the Mayo Clinic were 2.8% and 1.5%, respectively.

Conclusions: The mortality rate for the combined diagnoses is high. The most common combination of diagnoses was that of AL and PV, which was associated with a mean time to death of 2.7 years.

Disclosures

Al-Kali: Celgene: Research Funding; Onconova Therapeutics, Inc.: Research Funding.
Th17 Cells Exhibit Anti-Tumor Effects in MDS Possibly through Augmenting Functions of CD8+ T Cells
Authors: Shao Zonghong , Li Jing , Yue Lanzhu , Wang Huaquan , Liu Chunyan , Liu Hui , Jinglian Tao , Qi Weiwei , Wang Yihao , Zhang Wei , Fu Rong
Published: October 2019

Myelodysplastic syndromes (MDS) are a common malignant hematological disease.Th17 cells are a newly found subset of distinct CD4+ Th effector cells family and are found to play an important role in cancers. Intracellular staining and flow cytometric analysis were used to detect the percentage of Th17 cells .Here, we showed that both the percentage and the function of Th17 cells were elevated in low-risk MDS while decreased in high-risk MDS. To determine the concentration of IL-17, IL-6 and IL-23, the serum were analyzed by ELISA. Levels of upstream molecules of Th17 cells, IL - 6 and IL - 23, were higher in low-risk MDS but lower in high-risk MDS patients. The abnormal percentage of Th17 cells was closely related to clinical parameters including karyotype, morphologic blast percentage of bone marrow, peripheral absolute neutrophil count and hemoglobin concentration. Furthermore, the expression of IL-17, RORγt , STAT-3 mRNA were determinated of by qRT-PCR .Expression rates of perforin and granzyme B in BM CD3+CD8+ cells (cytotoxic T lymphocyte, CTL) positively correlated with levels of IL-17 but negatively correlated with BM blast percentage, and could be significantly increased after stimulation with human recombinant IL-17 (rhIL-17). Our results suggested Th17 cells might play an antitumor effect in the pathogenesis of MDS through IL-17/CTL pathway.

Disclosures

No relevant conflicts of interest to declare.
Autocrine and Paracrine Interactions Between Multiple Myeloma Cells and Bone Marrow Stromal Cells By Growth Arrest-Specific Gene 6 Crosstalk with Interleukin-6
Authors: Furukawa Miki , Hiroshi Ohkawara , Ogawa Kazuei , Ikeda Kazuhiko , Koki Ueda , Shichishima-Nakamura Akiko , Ito Emi , Imai Jun-ichi , Yanagisawa Yuka , Honma Reiko , Watanabe Shinya , Waguri Satoshi , Ikezoe Takayuki , Takeishi Yasuchika
Published: October 2019

The pathogenesis of multiple myeloma (MM) has not yet been fully elucidated. Our microarray analysis and immunohistochemistry revealed significant upregulation of growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein with a structural homology with protein S, in bone marrow (BM) cells of MM patients. ELISA showed that the serum levels of soluble Gas6 and interleukin (IL)-6were significantly increased in the MM patients when compared with healthy controls. Gas6 and TAM receptor tyrosine kinases Mer were overexpressed in human CD138-positive MM cell line RPMI-8226. Exogenous Gas6 and its signaling through Mer suppressed apoptosis induced by serum deprivation and enhanced cell proliferation of the MM cells. The conditioned medium from human BM stromal cell line HS-5 induced anti-apoptosis and cell proliferation of the MM cells with ERK, Akt and NF-kappaB phosphorylation, which were reversed by the neutralizing antibody to Gas6, IL-6 or Mer siRNA. Furthermore, the Gas6 neutralizing antibody reduced the upregulation of IL-6 and intercellular cell adhesion molecule-1 induced by a HS-5 cell-conditioned medium in MM cells. The present study provides new evidence that autocrine and paracrine stimulation of Gas6 in concert with IL-6 contributes to the pathology of MM, suggesting that Gas6/Mer signaling may be a promising novel target for treating MM.

Disclosures

No relevant conflicts of interest to declare.
Contribution of High-Density Neutrophils to Multiple Myeloma Microenvironment Dysregulation
Authors: Romano Alessandra , Giallongo Cesarina , Parrinello Nunziatina L , Tibullo Daniele , Puglisi Fabrizio , Camiolo Giusy , La Cava Piera , Parisi Marina , Consoli maria Letizia , Conticello Concetta , Di Raimondo Francesco , Luca Vanella
Published: October 2019

Background and objective: Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Previous reports in murine models and our previous work in MM patients, showed that myeloid cells, both granulocyte-myeloid derived suppressor cells (G-MDSC) and neutrophils sedimenting on top of red cells after density gradients, defined as high-density neutrophils (HDN) are increased and exert an immunosuppressive activity. Moreover, we showed that myeloid precursors could be activate and acquire G-MDSC phenotype if cultures in presence of MM mesenchymal cells.

Thus, we characterized functionally HDN to gauge their contribution in immunesuppression, angiogenesis and bone resorption in MM microenvironment.

Methods:

In HDN freshly-isolated from a series of 60 newly-diagnosed MM, 30 smoldering MM and 30 MGUS we investigated by RT-PCR, flow-cytometry, western-blot and immunofluorescence genes and proteins related to immunesuppression (Arg-1, IDO), angiogenesis (BV8, VEGF, MMP9) and bone resorption (IL1-b, MMP9, CHI3L).

Results:

First, MM-HDN exhibited an increased expression of immunesuppressive molecules Arg-1 and IDO compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity, immunostaining and WB, positively correlated with advanced disease.

Second, MM-HDN showed increased expression of BV8 and MMP9 (p=0.01, p=0.05) but not VEGF, as confirmed by immunofluorescence. In line with this observation, by using migration assay (CIM plate) MM-HDN induced neo-vasculogenesis in vitro by forming more and aberrant tubes than healthy or MGUS-HDN, thus to disclosure a novel proangiogenic mechanism driven by BV8.

Third, MM-HDN showed increased expression of IL1-b, MMP9, CHI3L, associated to increased bone resorption activity, evaluated by dentin-disk assay. The same findings could be obtained treating bone matrix with MM-HDN-conditioned media, suggesting the crucial role of molecules released by MM-HDN but no from MGUS-HDN.

Conclusion: taken together, our observations disclosure the contribution of aberrantly activated HDN in MM clinical features, contributing to immunosuppression, angiogenesis and bone resorption.

Disclosures

No relevant conflicts of interest to declare.
Overcoming Innate Resistance to a Beta-Catenin Inhibitor-BC2059- By Manipulating Autophagy in Multiple Myeloma
Authors: Savvidou Ioanna , Khong Tiffany T. , Horrigan Stephen K. , Spencer Andrew
Published: October 2019

Introduction: Chemoresistance is a major challenge in the development of new therapies in multiple myeloma [MM]. Inhibition of autophagy has been shown to restore chemosensitivity in several tumors. We have previously validated a beta-catenin inhibitor (BC2059) which targets the Wnt/beta-catenin signaling pathway.

Aim: In the present study we aim to overcome innate resistance to BC2059 by manipulating the autophagic pathway.

Methods: Autophagic flux was estimated by measurement of LC3II/LC3I in the absence and presence of hydroxychloroquine [HQ] by Western blot [WB]. Induction of autophagy was measured by the increase of LC3II/LC3I by WB, and concomitant drop of p62 expression by Flow Cytometry [FC]. Combination Indices [CI] were calculated using Calcusyn software.

Results: BC2059 induces apoptosis in a dose-dependent manner by induction of both the intrinsic and extrinsic apoptotic pathways, (increase of active -caspase-8, -caspase-9 and -caspase-3 measured by FC and cleaved PARP by WB) in all human myeloma cell lines [HMCL] tested. All HMCL tested have significant autophagic flux at baseline. Chemical inhibition of autophagy has an anti-proliferative effect, decreasing the relative cell numbers from 40% (NCI-H929) to 23% (KMS12BM) at 24hr. In parallel BC2059 is able to induce autophagy in a dose dependent manner. Induction of autophagy is BC2059 specific as treatment with melphalan or bortezomib at relative equal anti-proliferative doses did not increase LC3II/LC3I. Further autophagic inhibition by HQ was synergistic for all HMCL with CI of 0.7-0.3 (CI<1.1 indicates synergy). Interestingly, inhibition of autophagy halved the LD50 of BC2059 in a resistant HMCL (LP1). Other autophagy inhibitors (3-MA, bafilomycin A1 and NH4Cl) were also synergistic with BC2059 in LP1.

Conclusion: BC2059 exerts cytotoxicity mainly by induction of apoptosis but also induces cyto-protective autophagy. Autophagy inhibition was able to overcome innate resistance to the drug, ameliorating its cytotoxic effect. This study warrants further investigation.

Disclosures

Horrigan: betacatpharmaceuticals: Employment.