The Molecular Basis of Long First Remissions in Normal Karyotype AML Patients

Currently, it is not clear why some patients with acute myeloid leukemia (AML) can be "cured" with chemotherapy alone; are they living with small amounts of disease that is held in check by immunologic (or other) mechanisms, or is their disease really eradicated? The percentage of cytogenetically normal AML patients who have long (>5 years) first remissions (LFRs) after chemotherapy alone is low (about 9.1% in patients <60 years and 1.6% in >60 years1). For this reason, most intermediate risk patients are offered allogeneic transplantation to decrease their risk for relapse. To better understand mechanisms of chemotherapy sensitivity in AML, we performed an analysis of the mutation landscape and persistence, using samples from 8 normal karyotype LFR patients (without CEBPA mutations) who received standard "7+3" induction and high dose cytarabine consolidation as their only therapy. The mean age at diagnosis was 43.5 years, and the mean follow up in first remission is 7.6 years; none of these patients has relapsed to date. For each case, we performed enhanced exome sequencing at diagnosis (235x coverage of the entire exome, and ~1008x coverage of recurrently mutated AML genes). Each case had at least one documented AML driver mutation, with a median of 29 somatic mutations in the exome space. We created probes for 225 mutations (mean 28 per case), and performed error-corrected sequencing (Haloplex) for all available remission samples. The mean depth of Haloplex coverage was 1607x, and each sample had at least one AML-specific mutation assayed, with a sensitivity of 1 cell in 1,750 (0.06%). 7/8 patients demonstrated complete clearance of all mutations in all remission samples tested, which was confirmed with digital droplet PCR for 5 cases, with a sensitivity of detection of 1 cell in 100,000. In one case, we detected a persistent ancestral clone harboring DNMT3AR882H, which can be associated with long first remissions for some patients2. Strikingly, the founding clone in all 8 cases had one or more somatic mutations in genes known to drive cell proliferation (e.g. MYC, FLT3, NRAS, PTPN11, Figure 1 top panel). These are usually subclonal mutations that occur late during leukemic progression, suggesting that the presence of a "proliferative hit" in the founding clone might be important for chemotherapy clearance of all the AML cells in a given patient. To support this hypothesis, we analyzed the mutational clearance of 82 AML cases with paired diagnosis and day 30 post-chemotherapy bone marrow samples. We observed that, whether present in the founding clone or in subclones, mutations in MYC, CEBPA, FLT3, NRAS, and PTPN11 cleared after induction chemotherapy in all samples, while other mutations were often persistent at day 30 (e.g. DNMT3A, IDH1, IDH2, NPM1, TET2; Figure 1 bottom panel). Compared to other published sequencing studies of AML, MYC and NRAS mutations were significantly enriched in this small cohort (MYC p= 0.002, and NRAS p= 0.034), with MYC enrichment being particularly striking (37.5% versus 1.8%). All MYC mutations were canonical single base substitutions occurring in the highly conserved MYC Box 2 domain at the N-terminus of MYC (p.P74Q or p.T73N). Overexpression of MYCP74Q in murine hematopoietic progenitors prolonged MYC half life (89 min vs. 44 min for wild type), and enhanced cytarabine sensitivity at all concentrations tested (range 10-1000 nM, p=0.0003), both in vitro and in a MYC-driven leukemia model in vivo. MYC expression measured with flow cytometry in the blasts of the LFR samples was significantly higher (p=0.045) compared to unfavorable risk (complex karyotype) or other intermediate risk categories, but similar to good risk AML (biallelic CEBPA mutations, core binding factor fusion-associated AML, and AML with isolated NPMc), suggesting that activation of the MYC pathway may represent a shared feature of chemosensitive patients. Taken together, these data suggest that some intermediate patients who are effectively "cured" with chemotherapy alone may not have persistent subclinical disease, nor retained ancestral clones that could potentially contribute to relapse. Importantly, these patients often have mutations driving cell proliferation in the founding clone, indicating that the presence of specific mutations in all malignant cells may be critical for complete AML cell clearance with chemotherapy. 1. Blood Adv. 2018 Jul 10; 2(13): 1645-1650 2. N Engl J Med 2018; 378:1189-1199 Disclosures No relevant conflicts of interest to declare.

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Evidence for Complete Mutation Clearance in Normal Karyotype AML Patients with Very Long (> 5 years) Remissions after Chemotherapy Alone

Blood ◽

10.1182/blood-2018-99-117288 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1503-1503

Author(s):

Francesca Ferraro ◽

Christopher A Miller ◽

Nichole Helton ◽

Robert S Fulton ◽

Catrina Fronick ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Normal Karyotype ◽

Small Subset ◽

High Dose ◽

Primary Therapy ◽

Intermediate Risk ◽

Mutational Landscape ◽

First Remission ◽

The Mean

Abstract The fraction of patients with cytogenetically normal acute myeloid leukemia (AML) who have long (>5 years) first remissions after chemotherapy alone is estimated to be about 9.1% in young (<60 years) and 1.6% in older (>60 years) patients1; very few intermediate risk patients are therefore "cured" with chemotherapy alone. At this time, it is not yet clear whether these long remission patients are "living with disease", or whether all AML cells have been eliminated. Many people in complete morphologic remission have evidence for persistence of their ancestral and/or founding clones within the first 2-3 years after treatment, but genomic studies of very long first remissions have not yet been described. To determine whether patients with very long first remissions with chemotherapy alone clear their mutations, or are living with persistent disease, we performed an analysis of the mutational landscape and mutation persistence of 8 cases of normal karyotype AML with remissions of >5 years, and who received standard "7+3" induction and high dose cytarabine consolidation (3 to 4 cycles) as their primary therapy. All patients were diagnosed and treated at Washington University in Saint Louis; patient characteristics are summarized in Table 1. The mean age of patients at initial diagnosis was 43.5 years (range 19-63), and the mean time of follow up in first remission is currently 92 months (7.6 years) (range 62-146 months). To the best of our knowledge, none of the patients has relapsed to date. In every case, the day +14 bone marrow demonstrated ablation, and the day +28 bone marrow showed complete morphologic remission. For each case, we defined the mutational landscape at presentation with enhanced exome sequencing (235x coverage of the entire exome, and ~1008x coverage of recurrently mutated AML genes, as defined in the TCGA AML study). Each case had one or more recognized AML driver mutations, with a mean of 44 (range 31-64) somatic exome mutations (Table 1). The mutational spectrum at diagnosis was not statistically different from larger cohorts of intermediate risk AML patients with standard outcomes: 2 cases had mutations in DNMT3A, 6 had NPMc, 4 had NRAS, 3 had FLT3, 2 had WT1, 2 had STAG2, 2 had SLC43A3, and 2 had PTPN11 mutations. We were able to create probes for 225 mutations (mean of 28 per case, range 17-41) for analysis on the Haloplex platform; we used this platform to perform deep, error-corrected sequencing in one or more remission samples obtained from the bone marrow or peripheral blood of each patient after more than 1 year of remission. The mean depth of Haloplex coverage for all variants for all 8 samples was 1607x (range: 102-12,538x). Importantly, each sample had at least one AML-specific mutation assayed with a coverage depth greater than 3500x, yielding a sensitivity to detect 1 cell in 1,750 (0.06%). In the remission samples, 7/8 patients demonstrated complete clearance of all mutations in all samples tested, strongly suggesting that all AML cells had been eliminated by standard induction and consolidation therapies. In one case (868442), we detected a persistent ancestral clone in morphologic remission samples, harboring DNMT3AR882H (VAF 10.19% in long remission) and IRS2D106Y mutations (VAF 12.82% in long remission). These data suggest that a small subset of normal karyotype AML patients treated with chemotherapy alone may able to completely eradicate all AML cells. Although the mechanism is not yet clear (increased susceptibility to chemotherapy, immunologic surveillance of neoantigens, etc.), these data suggest that some patients in long remissions after chemotherapy do not have persistent subclinical disease, nor do they retain ancestral clones that could potentially contribute to relapse. In ongoing studies, our aim is to define biomarkers that can be used to prospectively identify this subset of patients, so that they can avoid the risk of allotransplantation in first remission. Reference:Vasu, Sumithira et al., Blood Advances 2.13 (2018): 1645-1650. Web. 31 July. 2018. Disclosures No relevant conflicts of interest to declare.

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EBV-Encoded Mirnas Facilitate Inflammation and Tumor Formation Through Both Intra- and Inter-Cellular Regulation In Vivo Mouse Model

Blood ◽

10.1182/blood.v122.21.3735.3735 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3735-3735

Author(s):

Natsuko Yamakawa ◽

Jun Ogata ◽

Takashi Yahata ◽

Jun Lu ◽

Kazuaki Yokoyama ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cells ◽

Small Rnas ◽

Conflicts Of Interest ◽

Functional Characterization ◽

Tumor Formation ◽

Sequencing Analysis ◽

Immunodeficient Mice

Abstract Introduction EB virus (EBV) is associated with heterogeneous lymphomas. Hodgkin's lymphoma (HL) cells are embedded in non-neoplastic bystanders: B, T cells, and macrophages. Without these bystander cells, the lymphoma cells are incapable of being engrafted in immunodeficient mice. In this context, the bystanders are tumor-supportive “inflammatory niche”. Recently, EBV-infected cells produce exosomes that contain EBV specifically encoded miRNAs (EBV-miRNAs). The miRNAs are transferred to cells, and involved in tumor metastasis. However, the detailed mechanism is unknown. Accordingly, we hypothesized that exosomal EBV-miRNAs might redirect tumor surrounding immune cells from tumor reactive into tumor-supportive “inflammatory niche”. Methods We evaluated the expression of EBV-miRNAs in EBV+HL clinical specimens by in situ hybridization, their functional characterization in vitro, and their effects on persistent infection and tumor development in vivo humanized NOG mice model. Moreover, in order to clarify its sorting mechanism, trans factor and cis factor which determined secreted and non-secreted miRNAs was analyzed by use of mass-spectrograhy and next-generation sequencing. Results and Discussion The EBV-miRNAs effects were potent on monocyte/macrophage Mo/Mf in inducing CD69, IL-10, and TNF, suggesting that EBV-miRNAs might polarize Mo/Mf into tumor associated Mf (TAM). EBV-miRNAs suppress tumor cell proliferation in vitro, implying that it works as tumor-suppressor in the tumor cells, while they are required to develop LPD in vivo, which seems contradict to the result in vitro. These results suggest that EBV-miRNAs intra-cellularly regulate the tumor cells to adjust to the surrounding circumstances, for example, to escape from immune surveillance, and inter-cellularly regulate Mo/Mf to support the tumor survival or development. Most importantly, exosomal EBV-miRNAs derived from the tumor cells were transferred to Mf in human EBV+ HL samples. Interestingly, one EBV coded miRNA was not secreted at all, though it abundantly expresses in the cells. The miRNA has been reported to strongly promote cell proliferation in EBV infected tumor cells. It made us hypothesized that the sorting system of secretary and non-secretary miRNAs is critical in the formation of “inflammatory niche”. In order to clarify the mechanism of the sorting, the chimeric miRNA was constructed then, we determined the sequence, which regulates secretion and non-secretion, and purified the protein complex, which specifically bound to the sequence. Mass spectrography and successive knockdown assay, the trans factor which inhibits secretion was identified. Moreover, the next sequencing analysis for the small RNAs revealed that abundant EBV-coded small RNAs occupied RNA-induced silencing complex (RISC), and that non-secreted EBV-miRNA was specifically modified. It is now under investigation whether the modification is involved in the sort mechanism between secretary and non-secretary miRNAs. Taken together, EBV-miRNAs have critical roles in intra- and inter-cellular manner. Especially, the functions as an inter-cellular communicator might be important in the tumor formation and the mechanism needs further investigation. Disclosures: No relevant conflicts of interest to declare.

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CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization

Blood ◽

10.1182/blood.v126.23.2974.2974 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2974-2974

Author(s):

David R Fooksman ◽

Amitabha Mazumder ◽

Mark McCarron

Keyword(s):

Plasma Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Early Stage ◽

Serum Starvation ◽

High Dose ◽

Rapid Reduction ◽

Myeloma Cells

Abstract Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations. Disclosures No relevant conflicts of interest to declare.

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Androgens MAY BOOST Hematologic Response to ANTI-Thymocyte Globulin in Acquired Aplastic Anemia

Blood ◽

10.1182/blood.v128.22.3900.3900 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3900-3900 ◽

Cited By ~ 8

Author(s):

Sabrina Giammarco ◽

Maria Teresa van Lint ◽

Teresa Lamparelli ◽

Francesca Gualandi ◽

Carmen Di Grazia ◽

...

Keyword(s):

Aplastic Anemia ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Liver Function Tests ◽

High Dose ◽

Unrelated Donor ◽

Gradual Improvement ◽

Androgen Treatment

Abstract Background: Androgens have shown encouraging results in the treatment of marrow failure, both in the animal model (Nouv Rev Fr Hematol. 1984, 26;391) and in patients with aplastic anemia (Blood. 1990;76: 2222). A first trial comparing ATG with or without androgens , failed to show improved response and survival in the androgens group (Blood. 1985; 66:184). However, in a second randomized trial , females receiving ATG+androgens had significantly superior response rates (BJH 1993,83:145). High dose ATG (twice the conventional dose) and androgens, was given to 87 patients, with 77% response rate and 78% survival at 5 years (Ann Hematol 2006;85:711). More recently sex hormones have been shown to increase telomerase activity, resulting in elongated telomeres in vitro [ Blood. 2009 114:2236), and in vivo [ N Engl J Med. 2016 19;374]. Aim of the study: The aim of this study was to evaluate the outcome of androgen treatment in patients with Aplastic Anemia (AA) who had received a previous course of ATG and were persistently cytopenic. Patients and methods: 78 AA patients were selected as having received oral testosterone 40 mg/day 5-7 days/week , because of persistent cytopenia, in 1-3 cell lines. Typically testosterone was added to the ongoing treatment with cyclosporine. Median age was 37 years (9-72) ; there were 28 males and 50 females.. The median Hb level at start of testosterone, was 8.8 gr/dl( 3-14), the median neutrophil counts 1.7 x10^9/L (0.0-8) and the median platelet count 27x10^9/L (1-128). The median interval between ATG treatment and testosterone was 5 months (0-148); the median duration of androgen treatment was 517 days (range 122- 8984) . Patients were classified as non responders ( no hematologic improvement and/or transfusion dependent), partial responders (transfusion independent and improved peripheral blood counts), or complete responders (normalized peripheral blood counts). Result: Androgen treatment , at this relatively low dose, was well tolerated, with no abnormalities in liver function tests, and no apparent virilization. One patient discontinued testosterone because of intolerance (1,28%). Of the 78 patients treated, 33% (n=23) were classified as non responders, 29% (n=20) as partial responders and 38% (n=26) as completer responders. The median hemoglobin level at 90, 180 and 365 days after androgen therapy were respectively: 9.1; 10.6 and 11.4 g/dl; the median neutrophil counts were respectively 2.0; 2.0 and 3.0 x10^9/L; the median platelets were respectively 38, 54, 75 x10^9/L. There was therefore a gradual improvement of peripheral blood counts with time.With a median follow up of 871 days (range 27-8392), the actuarial 5 year survival, free from transplantation or a second course of immunosuppressive therapy, is 85% (Fig.1); it is 96% for responders and 45% for non responders (Fig2); fifteen out 23 non responders were allografted. Conclusion: The addition of androgen to cyclosporine , improved peripheral blood counts in 67% of our patients, who had failed, or partially failed first line therapy with ATG, with low or absent detectable toxicity. Our results confirm a role for androgens in patients with marrow failure, and this may be relevant for patients eligible for an unrelated transplant, or for older patients discussing an HLA identical sibling transplant. A 3 months course of androgens is still less hazardous as compared to an unrelated donor graft, and may offer the patient an opportunity to achieve transfusion independence without a graft. Disclosures No relevant conflicts of interest to declare.

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Synthesis of Novel Derivatives of Quinazoline Schiff base Compound Promotes Epithelial Wound Healing

Current Pharmaceutical Design ◽

10.2174/1381612824666180130124308 ◽

2018 ◽

Vol 24 (13) ◽

pp. 1395-1404

Author(s):

Elham Bagheri ◽

Kamelia Saremi ◽

Fatemeh Hajiaghaalipour ◽

Fadhil Lafta Faraj ◽

Hapipah Mohd Ali ◽

...

Keyword(s):

Cell Proliferation ◽

Wound Healing ◽

Biological Activities ◽

Inflammatory Cells ◽

Negative Control ◽

High Dose ◽

Sprague Dawley ◽

Wound Tissue

Quinazoline is an aromatic bicyclic compound exhibiting several pharmaceutical and biological activities. This study was conducted to investigate the potential wound healing properties of Synthetic Quinazoline Compound (SQC) on experimental rats. The toxicity of SQC was determined by MTT cell proliferation assay. The healing effect of SQC was assessed by in vitro wound healing scratch assay on the skin fibroblast cells (BJ-5ta) and in vivo wound healing experiment of low and high dose of SQC on adult Sprague-Dawley rats compared with negative (gum acacia) and positive control (Intrasite-gel). Hematoxylin and Eosin (H&E), Masson’s Trichrome (MT) staining and immunohistochemistry analysis were performed to evaluate the histopathological alterations and proteins expression of Bax and Hsp70 on the wound tissue after 10 days. In addition, levels of antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase), and malondialdehyde (MDA) were measured in wound tissue homogenates. The SQC significantly enhanced BJ-5ta cell proliferation and accelerated the percentage of wound closure, with less scarring, increased fibroblast and collagen fibers and less inflammatory cells compared with the negative control. The compound also increases endogenous enzymes and decline lipid peroxidation in wound homogenate.

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RFWD2 Induces Cellular Proliferation and Proteasome Inhibitor Resistance By Mediating p27 Ubiqutinaiton in Multiple Myeloma

Blood ◽

10.1182/blood-2019-127888 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3068-3068

Author(s):

Ye Yang ◽

Mengjie Guo ◽

Chunyan Gu

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Drug Resistance ◽

Proteasome Inhibitor ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Scid Mice ◽

Rpmi 8226

Purpose: In recent years, with the emergence of targeted proteasome inhibitors (PIs), the treatment of multiple myeloma (MM) has made great progress and significantly improves the survival rate of patients. However, MM remains an incurable disease, mainly due to the recurrence of drug resistance. The constitutive photomorphogenic 1 (RFWD2, also known as COP1), is closely related to the occurrence and development of tumors, but its role in MM is largely unknown. This study was aimed to explore the mechanism of RFWD2 on cell proliferation and resistance to proteasome inhibitor in MM. Experimental Design: Using gene expression profiling (GEP) samples, we verified the relation of RFWD2 to MM patients' survival and drug-resistance. The effect of RFWD2 on cell proliferation was confirmed by MTT and cell cycle analysis in RFWD2-overexpressed and RFWD2-knockdown MM cells. MTT and apoptosis experiments were performed to evaluate whether RFWD2 influenced the sensitivity of MM cells to several chemotherapy drugs. MM xenografts were established in immunodeficient NOD/SCID mice by injecting wild-type or RFWD2 over-expression MM cells with drug intervention. The mechanism of drug resistance was elucidated by analyzing the association of RFWD2 with E3 ligase of p27. Bortezomib-resistant RPMI 8226 cells were used to construct RFWD2 knockdown cells, which were injected into NOD/SCID mice to assess the effect of RFWD2 on bortezomib resistance in vivo. Results: RFWD2 expression was closely related to poor outcome, relapse and bortezomib resistance in MM patients' GEP cohorts. Elevated RFWD2 induced cell proliferation, while decreased RFWD2 inhibited cell proliferation and induced apoptosis in MM cells. RFWD2-overexpression MM cells resulted in PIs resistance, however, no chemotherapy resistance to adriamycin and dexamethasone was observed in vitro. In addition, overexpressing RFWD2 in MM cells led to bortezomib resistance rather than adriamycin resistance in myeloma xenograft mouse model. RFWD2 regulated the ubiquitination degradation of P27 by interacting with RCHY1 ubiquitin ligase. The knockdown of RFWD2 in bortezomib-resistant RPMI 8226 cells overcame bortezomib resistance in vivo. Conclusions: Our data demonstrate that elevated RFWD2 induces MM cell proliferation and resistance to PIs, but not to adriamycin and dexamethasone both in vitro and in vivo through mediating the ubiquitination of p27. Collectively, RFWD2 is a novel promising therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

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Quantification of the Active Decitabine-Triphosphate (DAC-TP) Metabolite: A Novel Pharmacoanalytical Endpoint for Optimization of Hypomethylating Therapy in Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v120.21.3578.3578 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3578-3578

Author(s):

Hongyan Wang ◽

Ping Chen ◽

Jiang Wang ◽

Ramasamy Santhanam ◽

Josephine Aimiuwu ◽

...

Keyword(s):

Mononuclear Cells ◽

Conflicts Of Interest ◽

Dna Methyltransferases ◽

Analytical Methodology ◽

In Vivo Experiments ◽

Accuracy And Precision ◽

Coefficients Of Variation ◽

The Mean

Abstract Abstract 3578 Decitabine (DAC) is successfully used for treatment of patients (pts) with myelodysplastic syndromes and AML. Following cellular uptake, DAC is thought to be activated to DAC-TP and incorporated into DNA. The DAC-TP/DNA complex binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately anti-leukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated pts has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a sensitive and specific LC-MS/MS method for quantification of DAC-TP. The assay exhibited excellent accuracy and precision. The accuracy values were 83.7–109.4%, as determined by calculating the percentage of measured DAC-TP relative to the respective nominal concentrations (50, 500 and 5,000 nM) of the quality control samples. The within-day coefficients of variation (CVs) were 19.9 % (n=6) at 50 nM and 4.7–7.0 % between 500–5,000 nM; the between-day CVs (n=3) were 15.2 % at 50 nM and 7.5–10.2 % between 500–5,000 nM. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant MV4–11, and in THP-1 and FDC-P1/Kitmut cells (in vitro); and in bone marrow (BM) and spleen of normal and FDC-P1/Kitmut-driven AML mice (in vivo). DAC-TP reached peak levels (0.8, 1.4 and 0.5 pmol/106 cells) in 1–4 hours and declined to 20 % of its peak concentration after 24 hours incubation with 2.5 μM DAC in MV4–11, THP-1 and FDC-P1/Kitmut cells, respectively. Inhibition of hENT1 that mediates DAC transport into the cells and dCK that phosphorylates DAC into DAC-TP by NBTI and 2-thio-2′-deoxycytidine, respectively, significantly inhibited DAC-TP accumulation in AML cells. DAC-TP decay was instead blocked by tetrahydrouridine (THU)-induced inhibition of CDA, the catabolizing enzyme for cytidine and deoxycytidine and analogs. Consistent with these results, low dCK and hENTs but not CDA expression were detected in DAC-resistant MV4–11 cells, which showed 60 % decrease in DAC-TP levels as compared to their parental counterparts. DAC/DAC-TP-mediated downregulation of DNMT proteins (preferentially DNMT1 and DNMT3a) was also demonstrated in the AML cells even at DAC-TP concentrations as low as 0.1–1.3 pmol/106 cells in vitro after 4 hours DAC incubation. In the in vivo experiments, DAC-TP levels in leukemic mice were comparable to that in normal C57BL/6 mice, 0.3 pmol/106 cells in BM and 199.2 pmol/g tissue in spleen at 4-hours and 0.2 pmol/106 cells in BM and 165.3 pmol/g tissue in spleen at 24-hours following an i.v. bolus of 6.5 mg/kg DAC. In BM of leukemic mice, not only DNMT1 and DNMT3a but also DNMT3b protein expression reduced 80 % (DNMT3a) or diminished (DNMT1 and DNMT3b). The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells (PBMC) from AML pts treated with a 10-day regimen of DAC given 20 mg/m2/day i.v. over 1 hour. In BM samples, the mean DAC-TP levels were 0.8 ± 0.6 (Day 1) and 0.9 ± 0.5 pmol/106 cells (Day∼5) in complete responsive (CR) pts (n=4); and 0.4 ± 0.3 (Day 1) and 0.12 ± 0.02 pmol/106 cells (Day∼5) in non-responsive (NR) pts (n=3). In PBMC samples, the mean DAC-TP levels were 0.5 ± 0.2 (Day 1) and 1.2 ± 0.4 pmol/106 cells (Day∼5) in CR pts (n=3); and 0.02 ± 0.02 (Day 1) and 0.21 ± 0.04 pmol/106 cells (Day∼5) in NR pts (n=3). These data suggested that higher levels are seemingly associated with clinical response, but a larger number of pts need to be tested. In conclusion, monitoring the intracellular concentration of DAC-TP is feasible, and DAC-TP levels correlate with DNMT downregulation and may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens. Disclosures: No relevant conflicts of interest to declare.

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FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina

Blood ◽

10.1182/blood.v124.21.5318.5318 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5318-5318

Author(s):

Cristina N. Alonso ◽

Patricia L. Rubio ◽

Adriana Medina ◽

Silvia Eandi Eberle ◽

Andrea Bernasconi ◽

...

Keyword(s):

Conflicts Of Interest ◽

Prognostic Significance ◽

Normal Karyotype ◽

Prognostic Impact ◽

Sequencing Analysis ◽

Rt Pcr ◽

Genetic Abnormalities ◽

Childhood Aml ◽

The Mean ◽

Cebpa Mutations

Abstract Background: Mutations of FLT3, NPM1 and CEBPA are found in 25 to 35% of adult-AML. These mutations correlate with outcome, especially in AML with normal karyotype. There are few reports concerning the incidence and prognostic significance of these mutations in childhood-AML and there is no data from Argentina. Objectives: To describe the prevalence of FLT3, NPM1 and CEBPA mutations and to analyze the prognostic impact in the outcome in our setting. Methods: Samples from 195 children treated with AML protocols were retrospectively analyzed. The mean age at diagnosis was 6.8 [0.0-17.9] years, including 65 patients younger than 2 years of age. FAB subtypes were M2: 18%, M3: 15%, M4: 12%, M5: 34%, M6: 3%, M7: 10%, while 16 cases (8%) disclosed an ambiguous lineage immunophenotype. Genetic abnormalities of AML cases were characterized by cytogenetic analysis (97%) and/or RT-PCR for AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF4, MLL-AF9, MLL-ENL and MLL-AF10 fusion transcripts (95%). The distribution of the genetic abnormalities was: AML1-ETO: 11%, PML-RARA: 15%, CBFB-MYH11: 6%, MLL/11q23: 23%, other abnormalities: 25% and normal karyotype: 16%. Detection of NPM1 and CEBPA mutations was performed by Gene-scanning; FLT3-ITD and FLT3-TKD were studied by RT-PCR and RFLP respectively. Positive cases were further characterized by sequencing analysis. Results: The prevalences of the studied mutations were: FLT3-ITD: 10.3%, FLT3-TKD: 8.2%, NPM1mut: 4.6% and CEBPAmut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1mut: 25.0% and CEBPAmut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1mut: 12 years and CEBPAmut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPAmut. FLT3-ITD and FLT3-TKD showed significant association with the presence of PML-RARA (p<0.00001 and p=0.055 respectively). Eight out of nine patients with NPM1mut and 4/4 patients with CEBPAmut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITDmut: M3 (n:10/20; p<0.00001), FLT3-TKDmut: M5 (n:8/16; p=n.s.), NPM1mut: M2 (n:4/9; p=0.062) and CEBPAmut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITDmut, NPM1mut and CEBPAmut were significantly higher (p<0.00001, p=0.006 and p=0.033, respectively). FLT3-TKD was the only mutation detected in 5/45 (11%) of patients younger than 1 year of age. The five-years leukemia-free survival probabilities (pLFS) and standard error (SE) were: Total AML: 49 (4)%, FLT3-ITDmut:68 (12)%, FLT3-TKDmut:46 (17)%, NPM1mut: 75 (15)%, CEBPAmut: 100 (0)% and NPM1mut/CEBPAmut/FLT3-ITDneg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITDneg and NPM1mut or CEBPAmut was 88 (12)% (p=0.066). Conclusions: This is the first report of the frequencies of FLT3, NPM1 and CEBPA mutations in childhood AML in our country. The incidences of NPM1mut and CEBPAmut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1mut/FLT3-ITDneg and CEBPAmut/FLT3-ITDneg genotypes, especially in cases with normal karyotype. The present results support the notion that this group should be considered as a new AML subset with better outcome. This group of AML patients with better outcome could be included in the standard risk group, thus avoiding intensive treatments and related toxicity. Disclosures No relevant conflicts of interest to declare.

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Analysis Of The Molecular Mechanisms Leading To Significantly Higher Serum Hepcidin and More Severe Anemia In Multicentric Castleman’s Disease (MCD) Patients Than In Rheumatoid Arthritis (RA) Patients

Blood ◽

10.1182/blood.v122.21.2181.2181 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2181-2181

Author(s):

Soken-Nakazawa J. Song ◽

Hiroshi Kawabata ◽

Kazuyuki Yoshizaki

Keyword(s):

Iron Homeostasis ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Severe Anemia ◽

Negative Regulators ◽

Hepcidin Expression ◽

Serum Hepcidin ◽

The Mean

Abstract Background Hepcidin is a key regulator of body iron homeostasis and its increase in synthesis is implicated in anemia of inflammation (AI), which is commonly observed in patients with chronic inflammatory disorders such as MCD and RA. Inflammatory cytokines, mainly interleukin-6 (IL-6), play a central role in hepcidin induction during inflammation. However, tumor necrosis factor-a (TNF-a) does not induce but rather inhibits hepcidin expression in vitro and in vivo. The bone morphogenetic proteins (BMP) and erythropoietin (EPO) are the known positive and negative regulators of hepcidin expression. Our preliminary data has showed that MCD patients have more severe anemia and higher serum hepcidin-25 concentration than did RA patients. To clarify the mechanisms resulting in this difference, the activated patterns of hepcidin-regulating cytokines and their associations with serum hepcidin-25 levels and severity of anemia were analyzed in MCD and RA patients. Methods 42 patients with AI (14 with MCD and 28 with RA) treated with tocilizumab (an anti-IL-6 receptor antibody) were enrolled in this study. Major iron-related parameters including serum hepcidin-25, and serum levels of cytokines including IL-6, TNF-a, BMP and EPO were measured and correlations with hepcidin-25 as well as Hb were evaluated. Effects of cytokines on IL-6-induced hepcidin expression were analyzed in hepatoma cells by quantitative real-time PCR. Results The mean levels of hepcidin-25 at baseline was significantly higher (44.6 ng/ml), and Hb was significantly lower (9.2 g/dL) in MCD, than those in RA (28.6 ng/ml for hepcidin-25, 11.2 g/dL for Hb). There were significant and positive correlations of serum hepcidin-25 levels with serum ferritin and CRP in both groups (r=0.67 and 0.68 for ferritin, and r=0.41 and 0.36 for CRP, p< 0.001 respectively for both). In contrast, serum hepcidin-25 levels did not show any significant correlation with the levels of serum IL-6 or BMP or TNF-a (p > 0.05, respectively for both groups). The mean values of IL-6, TNF-a, BMP2, BMP4 and hepcidin-25 at baseline were elevated in two groups as compared to healthy control. Of note, we found that MCD patients showed significantly lower serum TNF-a (mean 195 pg/ml) and higher serum BMP4 (mean 193 pg/ml) concentrations than did RA patients (TNF-a= 241 pg/ml, BMP4= 92 pg/ml), although the two patient groups showed comparably elevated values for IL-6, BMP2 and EPO (p> 0.05, respectively). Significant improvements in anemia and systemic symptoms, and reductions in serum hepcidin-25 levels were observed within 2 weeks in both groups after tocilizumab treatment. In in vitro experiments, IL-6-induced hepcidin mRNA expression in hepatocytes was completely inhibited with tocilizumab and partially with TNF-a, but enhanced by BMP4 as well as MCD patient's serum. These results suggest that the negative effect of TNF-a on the IL-6-induced hepcidin was more pronounced in the RA than in MCD, in contrast, the positive effect of BMP was stronger in MCD than in RA. In addition, the finding that IL-6-induced hepcidin in hepatocytes was enhanced only by adding MCD patients’ serum but not RA patients’ serum, indicating the activated pattern of serum hepcidin-regulating factors in the MCD different from it in RA. Conclusions Our results suggest that the difference between MCD and RA in serum hepcidin-25 levels is partially due to the different activated patterns of positive and negative regulators of hepcidin expression. By the evidence that treatment with tocilizumab can reduce serum hepcidin and improve AI in patients with MCD and RA, we believe that IL-6 plays an essential role in the induction of hepcidin which leads to AI in MCD and RA, although multiple factors affect hepcidin levels. Disclosures: No relevant conflicts of interest to declare.

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Antithymocyte Globulin at Clinically Relevant Concentration Kills Leukemic Blasts

Blood ◽

10.1182/blood.v126.23.5481.5481 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5481-5481

Author(s):

Rosy Dabas ◽

Rachelle Lee ◽

Maria Theresa Servito ◽

Poonam Dharmani Khan ◽

Monica Modi ◽

...

Keyword(s):

Antithymocyte Globulin ◽

Conflicts Of Interest ◽

Annexin V ◽

Leukemic Cells ◽

High Dose ◽

Acute Leukemias ◽

Patient Specimen ◽

Blast Cells

Abstract Background/Rationale: Success of allogeneic hematopoietic cell transplantation (HCT) is limited due to graft-versus-host disease (GvHD) and leukemia relapse. Contrary to small molecule immunosuppressive drugs like cyclosporine or methotrexate, rabbit antithymocyte globulin (ATG) used for GvHD prophylaxis with myeloablative HCT conditioning does not increase relapse. The mechanism of the anti-GvHD effect of ATG has been studied extensively, however, the reason for ATG not increasing relapse is not known. We hypothesized that ATG has an anti-leukemic activity. Here we investigated the anti-leukemic activity of ATG in vitro at concentrations achieved in vivo with our standard ATG dose of 4.5 mg/kg (median 10 mg per liter of serum; range, 1-25). We focused on complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC). Methods: Blood or bone marrow from patients newly diagnosed with acute myeloid leukemia (AML, n=25) or acute lymphoid leukemia (ALL, n=4), obtained before induction chemotherapy, was used as the source of leukemic cells. We measured CDC and CIC of ATG, specifically against leukemic blasts at clinically achieved concentrations (1-25 mg/L) and an achievable concentration (50 mg/L). The leukemic blasts were identified as CD45dim/neg and side scatter low (SSclow) cells in case of AML-M0, M1 or M2 (according to British-American-French classification) or ALL. In case of AML-M4 or M5, we gated on CD45dim/neg and SSclow/intermediate cells. These cells could contain both leukemic and normal blasts and some post-blast (maturing/mature) cells. We minimized the fraction of normal blasts and post-blast cells included by introducing a "dump channel". The dump channel consisted of antibodies against antigens expressed by cells of lineages other than the leukemia lineage (except for antigens aberrantly expressed by the leukemic cells) and by post-blast cells. For most AML patients, the dump channel consisted of CD10, CD19, CD41a, CD235a, CD14 and CD16. For most ALL patients, the dump channel consisted of CD117, CD33, CD41a, CD235a, CD14 and CD16. Thus, leukemic blasts were defined as mononuclear cells that were CD45dim/neg and SSclow (or SSclow/intermediate for M4/M5) and did not express dump channel antigens. CDC was induced by incubation of the patient specimen with human serum (source of complement). CIC was induced by incubation of the patient specimen with heat-inactivated serum (no complement). For CDC dead cells were identified as 7-amino-actinomycin D positive (7AAD+). For CIC dead/dying cells were identified as Annexin V positive (Annexin V+). Results: ATG induced death of leukemic blasts both via CDC and CIC. Median 0.3%, 3.0%, 12.6% and 41.7% blasts were killed with 1, 10, 25 and 50 mg/L ATG, respectively, via CDC. Median 2.1%, 8.0%, 11.4% and 12.6% blasts were killed after 4 hour incubation with 1, 10, 25 and 50 mg/L ATG, respectively, via CIC. For both CDC and CIC, the percents were significantly higher than background (p<0.05). There was a high variability in the sensitivity of the leukemic blasts to ATG. The adjusted (background subtracted) percent of blasts killed ranged from 2.6% to 93.8% via CDC (Figure 1) and from 1.5% to 69.9% via CIC (Figure 2). When we arbitrarily defined a patient with resistant leukemic blasts as <10% (adjusted) leukemic blasts dead after exposure to 50 mg/L ATG, there were 3/28 patients with resistant leukemic blasts in case of CDC (Figure 1) and 9/23 patients in case of CIC (Figure 2). Conclusion: ATG at clinically relevant concentrations kills primary leukemic blasts in vitro. Some acute leukemias are highly sensitive and others relatively resistant to ATG. Whether this applies to in vivo needs to be determined. If yes, and given that the killing of leukemic cells is ATG concentration-dependent, conditioning regimens with high dose ATG could be developed for patients with leukemia sensitive to ATG to maximize the concurrent anti-GvHD and anti-relapse effects. Figure 1. Sensitivity of leukemic blasts to ATG induced CDC is highly variable. Percents 7AAD+ (dead) leukemic blasts shown are adjusted percents. In case of a negative value of the adjusted percent, zero value is displayed here. Figure 1. Sensitivity of leukemic blasts to ATG induced CDC is highly variable. Percents 7AAD+ (dead) leukemic blasts shown are adjusted percents. In case of a negative value of the adjusted percent, zero value is displayed here. Figure 2. Sensitivity of leukemic blasts to ATG induced CIC is highly variable. Percents Annexin V+ (dying or dead) leukemic blasts shown are adjusted percents. In case of a negative value of the adjusted percent, zero value is displayed here. Figure 2. Sensitivity of leukemic blasts to ATG induced CIC is highly variable. Percents Annexin V+ (dying or dead) leukemic blasts shown are adjusted percents. In case of a negative value of the adjusted percent, zero value is displayed here. Disclosures No relevant conflicts of interest to declare.

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