Bone Marrow Mononuclear Cells Samples Research Articles

Chimeric Antigen Receptor T-Cell Therapy Shapes T Cell Repertoire in Chinese Patients with B-Cell Acute Lymphocyte Leukemia

Introduction: Chimeric antigen receptor (CAR) T-cell therapy has displayed potent anti-leukemia activity in refractory/relapsed acute lymphocytic leukemia (ALL). However, the influence of CAR-T therapy on host systemic and local immunity has not been well examined. We therefore applied high-throughput T cell receptor β chain sequencing to track dynamic change of T-cell repertoire in vivo induced by CAR-T therapy in B-cell acute lymphocytic leukemia patients.Patients and Methods: Six patients with 45 samples were under observation. The samples obtained to be tested were the peripheral blood mononuclear cell (PBMC) samples and bone marrow mononuclear cell (BMMC) samples before and after CAR-T administration, as well as the CAR-transduced autologous T cell samples on the day when they were to be infused to patients. The information of samples and patients was summarized in Table 1. The TCR full length mRNAs of these samples were deeply sequenced using the ImmunHubTM TCR profiling system (ImmunQuad Biotech). Briefly, a 5'RACE unbiased amplification protocol was used. An algorithm was applied to raw sequencing data for PCR and sequencing errors correction and V, D, J, C gene segments mapping with IMGT®. The inverse Simpson index and the clonality index was calculated to estimate TCRβ clonotype diversity and the state of clonal proliferation of T cells. The donut chart and clone tracking heat map were generated by R.Result: Compared to preinfusion TCR diversity, we observed inverse Simpson's index of 4 of 6 patients' PBMC samples (Fig. 1A) and of 3 of 4 patients' BMMC samples (Fig. 1B) had been increasing to day 7 after CAR-T treatment. As time went by, a decline of TCR diversity from day 7 to day 28 was detected in both PBMC and BMMC samples (Fig. 1). Of note, a relative rising tide of TCR diversity was observed after the decline in PBMC samples (Fig. 1A). The decreased TCR diversity leaded us to test the change of TCR clonality. As shown by Fig. 2A and 2B, in comparison with the pre-treatment and the CAR-T samples, we detected highly clonal proliferation of T cells in both blood and bone marrow. Next, we applied the clonality index to quantitatively define T cell clonal expansion (Fig. 2C and 2D). The clonality index was raised from 0.16±0.07 and 0.14±0.05 before treatment to 0.27±0.09 and 0.3±0.15 at last time point post infusion in PBMCs and BMMCs, respectively (mean ± SD, student's t test, P=0.03 and P=0.17, respectively).CAR-T-induced T-cell clonal expansion triggered us to trace back the original clonal source. For tracking clonal evolution, we selected the 100 most prevalent T cell clonotypes found at the last time point in both PBMCs and BMMCs and tracked their frequencies at earlier time points. As was displayed by the heat map, the top 100 T cell clones dominating in PBMCs and BMMCs at the last time point were barely found in CAR-T cell pool and were the low-frequency clonotypes in preinfusion samples (Fig. 3A and 3B). In order to quantitatively describe this phenomenon, we compared the total frequency of top 10 T cell clones in PBMC and BMMC samples at the last time point to their corresponding total frequency in preinfusion samples and the infused CAR-T pool (Fig. 3C, 3D, 3E and 3F). The total frequency of the top 10 T cell clones was 23%±11% and 27%±14% at last time point, 4%±6% and 8%±7% before treatment and 0.05%±0.09% and 0.01%±0.01% in the infused CAR-T samples in PBMCs and BMMCs, respectively (mean ± SD, Fig. 3C, 3D, 3E and 3F; student's t test, P=0.01, P=0.01, P=0.08 and P=0.03, respectively). Only one patient's (patient 6's) top 10 T cell clones in PBMCs at last time point were not found in both CAR-T pool and preinfusion sample, which may be caused by the limitation of sequencing detection (Fig. 3C).Conclusion: For the first time, we demonstrated CAR-T therapy could stimulate the clonal proliferation of endogenous non-CAR T cells in patients. Along with other groups' animal results (Barber A et al. J Immunol. 2009) indicating that CAR-T therapy could facilitate the infiltrating of tumor antigen-specific T cells, these expanded T cell clones of patients in our trial are most likely tumor antigen specific, which could provide synergistic anti-tumor effect following CAR-T adoptive transfer. The trial was registered in Chinese Clinical Trial Registry and the registration number is ChiCTR-OCC-15007008. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Metformintreatment Overcomes ATRA-Resistance in Acute Promyelocytic Leukemia and Increases FOXO3A Expression

Introduction:FOXO3A is a transcription factor shown to be involved in all-trans retinoic acid (ATRA)-induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL). Its biological activity may be significantly enhanced upon metformin, raising the possibility that ATRA and Metformin may act synergistically; which could be useful to overcome ATRA resistance. Despite progress in APL treatment, approximately 10-15% of patients will relapse after treatment with ATRA and anthracyclines and frequently present with ATRA resistance. Relapsed patients respond well to arsenic trioxide (ATO) treatment, but the cost of ATO is a significant barrier in many countries. Aims: Here, we investigated the effects of in vitro treatment with ATRA plus metformin in APL cell lines and primary blasts, and quantified FOXO3A expression and correlated its levels with treatment outcome in a cohort of patients enrolled in the International Consortium on Acute Leukemia (ICAPL2006) study. Finally, we evaluated the effect of induced overexpression of FOXO3A gene in regards to cell viability and proliferation. Methods: Primary leukemic blasts from hCG-PMLRARα transgenic mice (TM; n=4) and APL patients (age, 25-47y; n=4) were treated with metformin alone (5mM/ 10mM) or in combination with ATRA (1µM) and evaluated for cell viability. In addition, 106 patients (age, 18-82y) with newly diagnosed APL enrolled in the ICAPL2006 study were included. As controls, eight samples of bone marrow mononuclear cells (BMMC) from healthy volunteers (age, 18-60y) were analyzed. To validate our data, FOXO3A transcript levels from an independent cohort was used (31 patients from Amazonia!, Probe n. 204131_s_at, and five normal BMMC samples included in the same databank). NB4/NB4R2 (ATRA-resistant) cell lines were transduced with FOXO3A or empty vector (control). After synchronization using double thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For dose-response curves, cells were treated with graded concentrations of ATRA, ATO and metformin. For the apoptosis analysis, cells were treated with ATRA (1µM), metformin (5mM) and combination for 24, 48 and 72 hours. The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b surface levels. Results: In primary cells (from TM/APL patients) a 2-fold reduction in viable cells was observed after metformin and metformin plus ATRA treatment (P Disclosures Tallman:BioSight: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; Orsenix: Other: Advisory board. Ganser:Novartis: Membership on an entity9s Board of Directors or advisory committees. Lowenberg:Editorial Board "European Oncology & Haematology": Membership on an entity9s Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity9s Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity9s Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity9s Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity9s Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity9s Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity9s Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity9s Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity9s Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees; Astex: Consultancy; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity9s Board of Directors or advisory committees; Celgene: Membership on an entity9s Board of Directors or advisory committees.

Gene expression and risk of leukemic transformation in myelodysplasia

AbstractMyelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic disorders with a highly variable prognosis. To identify a gene expression–based classification of myelodysplasia with biological and clinical relevance, we performed a comprehensive transcriptomic analysis of myeloid neoplasms with dysplasia using transcriptome sequencing. Unsupervised clustering of gene expression data of bone marrow CD34+ cells from 100 patients identified 2 subgroups. The first subtype was characterized by increased expression of genes related to erythroid/megakaryocytic (EMK) lineages, whereas the second subtype showed upregulation of genes related to immature progenitor (IMP) cells. Compared with the first so-called EMK subtype, the IMP subtype showed upregulation of many signaling pathways and downregulation of several pathways related to metabolism and DNA repair. The IMP subgroup was associated with a significantly shorter survival in both univariate (hazard ratio [HR], 5.0; 95% confidence interval [CI], 1.8-14; P < .001) and multivariate analysis (HR, 4.9; 95% CI, 1.3-19; P = .02). Leukemic transformation was limited to the IMP subgroup. The prognostic significance of our classification was validated in an independent cohort of 183 patients. We also constructed a model to predict the subgroups using gene expression profiles of unfractionated bone marrow mononuclear cells (BMMNCs). The model successfully predicted clinical outcomes in a test set of 114 patients with BMMNC samples. The addition of our classification to the clinical model improved prediction of patient outcomes. These results indicated biological and clinical relevance of our gene expression–based classification, which will improve risk prediction and treatment stratification of MDS.

Abstract 3634: Simultaneous isolation of genomic DNA and total RNA using the Qiagen AllPrep® method

Abstract Introduction: The purification of genomic DNA (gDNA) and total RNA (RNA) from various specimen types using a single sample is currently in high demand in genomic settings due to the limitation of available biological material. There is an increased need for simultaneous isolation of nucleic acids in cancer genetics for use in downstream applications such as genotyping, microarrays or next generation sequencing. The Qiagen AllPrep® technique provides a comprehensive solution for extraction of gDNA and RNA from a single specimen. In this study, we tested the AllPrep® kits for concurrent isolation of gDNA and RNA from five different sample types. Materials and Methods: Genomic DNA and RNA were extracted simultaneously according to Qiagen AllPrep® manufacturer's instructions from five specimen types: bone marrow mononuclear cells (BMMC), cryopreserved cultured cell lines, whole blood, fresh frozen and FFPE samples. Quantity and quality of both nucleic acids were assessed by the NanoDrop 2000 and Qubit fluorometer 2.0. Purified gDNA from BMMC, fresh frozen, whole blood and FFPE samples was further evaluated by library preparation, targeted hybridization capture and sequencing on an Illumina NextSeq500. FastQ files were submitted to a Clinical Genome Analytics (CGA) pipeline for quality metrics analysis. Results: The AllPrep® technique resulted in effective isolation of high quality gDNA and RNA (Table1). All set wet lab and sequencing QC metrics were met for each sample type. Conclusion: We have successfully verified the AllPrep® method can be used for simultaneous extraction of both gDNA and RNA using the same input material without compromising the yield or purity of both final products. This simple procedure based on spin column technology not only gave the advantage of generating high quality results using limited specimen resources, but also decreased the hands on time by 3.5 hours and cost when compared to extraction done with two separate kits. Table 1.Represents average QC results and SD for each specimen type. **n = 8 for sequencing metricsgenomic DNAgenomic DNAtotal RNAtotal RNASample TypeOD260/280Total Yield (ng)OD260/280Total Yield (ng)Mean Target Coverage% DuplicationBMMC (n = 3)1.853033.31.9413855206.34%Whole Blood (n = 2)1.818936.51.881335566.96.47%Fresh Frozen (n = 2)1.69312752.0432375345.44.02%FFPE Specimen (n = 16)1.665133.41.9410524.1**748.2**6.10%Cultured Cell Lines (n = 5)1.8442981.9810489N/AN/ATotal Average (n = 28)1.736898.141.9510443624.75.92%Standard Deviation (SD).249180.90.110616.2228.30.013% Citation Format: Aleksandra Ras, Samantha Helm, Kevin Kelly, Vanessa Spotlow, Guruprasad Ananda, Gregory Tsongalis. Simultaneous isolation of genomic DNA and total RNA using the Qiagen AllPrep® method. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3634.

Clinical Meanings of Quantitative PCR of Patient-Specific Immunoglobulin VDJ Regions Using Various Materials of Myeloma Patients

Introduction; The PCR with patient-specific, allele-specific oligonucleotide primers for individual IgH VDJ regions (ASO-PCR) is considered the most sensitive method to detect minimal residual disease (MRD) levels in patients with multiple myeloma (MM). In this study, we quantified the ASO-PCR products by using peripheral blood mononuclear cells (PBMNCs) as well as bone marrow mononuclear cells (BMMNCs). We also quantified the ASO-PCR products in mRNAs from CD20+38- B-cells in BM to examine whether there are clonogenic cells in relatively earlier B-cell fraction as well as cell-free DNAs from the sera to examine whether there are DNA fragments from MM cells in PB.Materials and methods; We have analyzed 30 MM cases. After an informed consent, patient-oriented PCR primers were designed from sequence information of immunoglobulin heavy chain (IgH) variable regions of myeloma cells of each patient.Results; The median age in this cohort was 64.5 years (36-83); the ratio of men to women was 16:14; and the numbers of IgG-, IgA-, and Bence Jones protein (BJP)- type patients were 18, 7, and 5, respectively. The patient-specific ASO primers could be designed in 25 cases, but not in 3 BJP-type cases or 2 IgG-type cases. We could quantify the ASO-PCR products in 20 of 30 BMMNCs samples at diagnosis. The ASO-PCR levels (IgH/β−actin levels)in BMMNCs correlated with those in PBMNCs, but not to the percent of plasma cells in BM or the values for M-protein. However, the ASO-PCR levels were decreased after the treatment and reflected tumor burden well individually. The ASO-PCR levels in PBMNCs showed a statistically significant correlation with those in BMMNCs at diagnosis (Spearman’s ρ= 0.98, P<0.001), 6 months (Spearman’s ρ= 0.83, P = 0.020) and 12 months (Spearman’s ρ= 1.90, P = 0.001). Therefore, ASO-PCR using PBMNCs as well as BMMNCs is suitable for MRD evaluation. We could detect the patient-specific IgH DNA sequences in cell-free DNA extracted from the sera and quantify the ASO-PCR products. The sequences of the ASO-PCR product were identical to the originally designed sequence, suggesting that detection of the ASO-PCR products in cell-free DNA could reflect the persistence of myeloma cells somewhere in body. The ASO-PCR products for CD20+CD38- B-cells in BM were relatively low but were clearly detected in 17 cases at diagnosis. The ASO-PCR levels in CD20+CD38- B-cells in BM showed good correlation with both values of ASO-PCR in BMMNCs (Spearman’s ρ= 1.35, P <0.001) and in PBMNCs (Spearman’s ρ= 1.09, P <0.001). Thus, the evaluation of relatively earlier B-cell stages of myeloma cells seems to be of interst, including for the possible existence of MM clones in CD20+CD38- B-cell population in BM.Discussion; Our findings that we could quantify the ASO-PCR products in PBMNCs, BM CD20+CD38- B-cells, cell-free DNA as well as BMMNCs strongly suggest wide sources of clinical materials to analyze. There were statistically significant correlations in values of the ASO-PCR products between BMMNCs and PBMNCs, therefore suggesting the possibility that clonogenic plasma cells or myeloma precursor cells might circulate in peripheral blood. We expect the possibility that PBMNCs will be a good source to monitor MRD. We could also detect and quantify the ASO-PCR products in cell-free DNA from the sera. To the best of our knowledge, this is the first report that IgH DNA fragments from MM cells circulate in the sera. Furthermore, we could detect and quantify the ASO-PCR products in CD20+CD38- B-cells in BM at diagnosis. These results indicated that clonogenic MM cells could exist not only in the CD20-CD38high plasma cell fraction but also in the CD20+CD38- B-cell fraction, which might include myeloma stem or initiating cells. Thus, we could consider the treatment strategies to include anti-CD20 antibodies against the clonogenic CD20+CD38- B-cell population. In conclusion, our ASO-PCR using various clinical materials is supposed to be useful for detecting MRD in the patients with MM as well as for clarifying the pathogenesis of MM. DisclosuresSata:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Shibayama:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Habuchi:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Fukushima:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Fujita:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Ezoe:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Tadokoro:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Maeda:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Mizuki:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Oritani:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Kanakura:Janssen: Research Funding; Takeda: Research Funding; Celgen: Research Funding.

Pyrosequencing as a Fast and Reliable Method in Detecting the MYD88 p.L265P Mutation in Decalcified Formalin-Fixed and Paraffin-Embedded Tissues

Niklas Gebauer, M.D., Veronica Bernard, Ph.D., Claudia Röhner, M.D., Manuela Krokowski, M.D., Hartmut Merz, M.D., Alfred C. Feller, M.D., and Christoph Thorns, M.D.. Ann Lab Med 2014;34:170-3. https://doi.org/10.3343/alm.2014.34.2.170

Pretreatment long interspersed element (LINE)-1 methylation levels, not early hypomethylation under treatment, predict hematological response to azacitidine in elderly patients with acute myeloid leukemia

BackgroundEpigenetic modulations, including changes in DNA cytosine methylation, are implicated in the pathogenesis and progression of acute myeloid leukemia (AML). Azacitidine is a hypomethylating agent that is incorporated into RNA as well as DNA. Thus, there is a rationale to its use in patients with AML. We determined whether baseline and/or early changes in the methylation of long interspersed element (LINE)-1 or CDH13 correlate with bone marrow blast clearance, hematological response, or survival in patients with AML treated with azacitidine.MethodsAn open label, phase I/II trial was performed in 40 AML patients (median bone marrow blast count was 42%) unfit for intensive chemotherapy treated with azacitidine 75 mg/m2/day subcutaneously for 5 days every 4 weeks. Bone marrow mononuclear cell samples were taken on day 0 (pretreatment) and day 15 during the first treatment cycle; LINE-1 and CDH13 methylation levels were quantified by methylation-specific, semiquantitative, real-time polymerase chain reaction.ResultsTreatment with azacitidine significantly reduced LINE-1 but not CDH13 methylation levels over the first cycle (P < 0.0001). Absolute LINE-1 methylation levels tended to be lower on day 0 (P = 0.06) and day 15 of cycle 1 (P = 0.03) in patients who went on to achieve subsequent complete remission, partial remission or hematological improvement versus patients with stable disease. However, the decrease in LINE-1 methylation over the first treatment cycle did not correlate with subsequent response (P = 0.31). Baseline methylation levels of LINE-1 or CDH13 did not correlate with disease-related prognostic factors, including cytogenetic risk, relapsed/refractory AML, or presence of NPM1 or FLT3 mutations. No correlation was observed between LINE-1 or CDH13 methylation levels and overall survival.ConclusionAnalysis of baseline LINE-1 methylation levels may help identify elderly AML patients who are most likely to respond to azacitidine therapy.

A Regulatory Promoter Polymorphism and Hypermethylation of Intron 1 Are Associated with Reduced Expression of KLF5 and Inferior Survival in AML

AbstractAbstract 3508 Background:Krüppel-like factor 5 (KLF5) is a transcription factor with regulatory roles in cell growth, survival, and differentiation. Our previous studies have indicated that KLF5 is required for myeloid blood lineage differentiation and is a target of genomic methylation in Acute Myeloid Leukaemia (AML) which is associated with reduced expression. Further, we have shown that treatment of leukaemia cell lines, displaying KLF5 hypermethylation, with demethylating agents is associated with induction of KLF5 expression, supporting an inhibitory role of methylation on expression (Diakiw et al, Leuk Res 36:110). Other recent studies have shown that KLF5 expression is reduced by the presence of a heterozygous genotype at a promoter SNP (rs3812852) and plays a role in pathogenic processes in heart and brain. We investigated both methylation and SNP genotype as mechanisms of KLF5 gene regulation in AML and their relationship to patient outcome. Methods:We analysed 232 diagnostic bone marrow mononuclear cell samples from a retrospective cohort of patients with AML. The genotype of the rs3812852 SNP was determined by Sequenom MassARRAY genotyping. DNA methylation of the KLF5 gene locus (intron 1) was assessed using Sequenom MassARRAY epityper. KLF5 gene expression was determined for 85 of these samples using quantitative real-time PCR. Of the 232 patients, 161 received treatment with induction chemotherapy and were included in survival analysis. Results:We found that 28 (12%) patients were heterozygous (G/A) for the SNP (rs3812852) in the KLF5 promoter, with one patient having the rare homozygous genotype (G/G) and 204 (88%) patients the common homozygous genotype (A/A). KLF5 gene expression correlated with the SNP genotype, with the heterozygous patients displaying significantly reduced KLF5 expression compared to normal controls (CD34 cells from normal donors, p=0.044). Consistent with our previous study on a smaller test cohort we observed that hypermethylation of this region of KLF5 is common and is present in 50% of samples in our larger cohort. AML patients with high methylation of KLF5 had significantly reduced KLF5 expression compared to normal controls and AML patients with low KLF5 methylation (p=0.032 and p=0.048 respectively). The correlation of gene expression with the SNP genotype and KLF5 methylation groups was also true when considered together, as patients with the heterozygous genotype (A/G) and high methylation had lower KLF5 expression than patients with the common homozygous genotype (A/A) and low KLF5 methylation (Figure 1A).Cox multi-variate analysis (variables of age, WCC and mutational status of FLT3-ITD, IDH1/2, NPM1) of survival of patients treated with induction chemotherapy showed that the presence of a G/A genotype at the KLF5 promoter SNP was not an independent predictor of overall survival (p= 0.502). In contrast hypermethylation of KLF5 intron 1 was a highly significant independent predictor of poorer overall survival (median survival 10.4 months versus 20.7 months, HR 1.5, p=0.001, Figure 1B). Importantly, when the heterozygous SNP genotype (A/G) and high methylation status were combined, this group of patients had even poorer OS (median survival 5.2 months, HR 3.6, Figure 1C) and this was significant compared to the group with homozygous SNP genotype (A/A) and low methylation (p=0.002). Conclusions:We have shown that both methylation of KLF5 intron 1 and a heterozygous genotype at a promoter polymorphism are associated with reduced KLF5 expression in AML. KLF5 hypermethylation is an independent predictor of poor prognosis and combined with the heterozygous SNP genotype defines a group with particularly poor survival indicating a connection with KLF5 gene expression and response to treatment. Importantly, as the majority of the effect is associated with hypermethylation of KLF5, these patients (50%) may benefit from treatment with demethylating agents. [Display omitted] Disclosures:Wei:Celgene: Research Funding.

Methylation of a Single CpG in the GADD45A Proximal Promoter Is Associated with Poor Survival in Acute Myeloid Leukemia,

Abstract 3540▪▪This icon denotes a clinically relevant abstract Background:GADD45A is a tumor suppressor gene that plays cell-type dependent roles in cellular stress, coordinating DNA repair and de-methylation, cell cycle arrest, and pro-apoptotic or pro-survival responses. GADD45A expression is normally rapidly induced in response to radiation and cytotoxic drugs1 and ectopic expression of GADD45A in the M1 leukemic cell line sensitises cells to stress-induced apoptosis in response to a range of genotoxic agents.2 Silencing of GADD45A by promoter methylation is a hallmark of many tumors,3–5 however to date there have been no investigations to determine whether this is associated with response to therapy. In AML, we have shown GADD45A expression is broadly down-regulated6 and here we investigate the mechanism of GADD45A repression in AML and its clinical significance. METHODS:We analysed 131 diagnostic bone marrow mononuclear cell samples from a retrospective cohort of patients with de novo AML. 93 of these patients were treated with induction chemotherapy. Patients 60 years or under were treated with chemotherapy regimens containing idarubicin and high dose cytarabine, and patients older than 60 received idarubicin and standard dose cytarabine. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom MassARRAY) to analyze methylation of 4 GADD45A promoter CpG dinucleotides previously shown to be associated with silencing of GADD45A in breast and prostate cancer.4,5 We determined association of CpG hyper-methylation with outcome in our treated patient cohort. For AML cells with GADD45A hyper-methylation, and for those with normal levels of GADD45A promoter methylation, we also determined the response to cytotoxic agents in vitro in the presence and absence of hypo-methylating agents. RESULTS:We observed hyper-methylation of the 4 CpG residues in the proximal promoter of GADD45A in 49 of 131 (37%) de novo AML patients and in 6 AML cell lines. Multivariable analysis showed that methylation of a single CpG (CpG1) was an independent predictor of poor survival in AML overall (median survival 281 days versus 794 days, HR 2.25, p=0.009), in normal karyotype AML (NK-AML) (median survival of 281 versus 793 days, HR 5.77, p=0.03, >250 days), and in the elderly patient group (>60 years, median survival of 218 versus 392 days, HR 2.64, p=0.03)(see Figure 1). Additionally, treatment of AML cell lines and patient blasts with decitabine resulted in selective induction of GADD45A mRNA in samples with GADD45A hyper-methylation, and this was associated with increased sensitivity to daunorubicin. [Display omitted] CONCLUSIONS:DNA methylation of the GADD45A proximal promoter is an independent predictor of poor outcome particularly in AML patients with normal karyotype and in the elderly group. Our biological data shows that induction of GADD45A mRNA expression with decitabine in hyper-methylated samples is associated with increased response to cytotoxic agents. Thus GADD45A promoter CpG methylation represents a new biomarker that may provide prognostic information in the heterogeneous NK-AML group, and in elderly patients. Given that recent trials are combining Azacitidine with chemotherapy and other agents for initial induction treatment of AML9, this may represent a marker to help define patients that will benefit from this approach. Elderly patients with GADD45A hyper-methylation may benefit from treatment with hypo-methylating agents which are associated with less toxicity (see Refs 7,8). Disclosures:Wei:Celgene: Honoraria, Research Funding.

Kidney injury and cell therapy: Preclinical study

The aim of this study is to show histological and immunofluorescence analysis of renal parenchyma of agoutis affected by gentamicin-induced renal disease after the infusion of bone marrow mononuclear cells (BMMC) stained with Hoechst®. Nine agouti's males were divided into three groups: Test group (TG): renal disease by gentamicin induced (n = 3), cell therapy group (CTG): renal disease by gentamicin induced and BMMC infusion (n = 3), and control group (CG): nonrenal disease and BMMC infusion (n = 3). TG and CTG were submitted to the protocol of renal disease induction using weekly application of gentamicin sulfate for 4 months. CG and CTG received a 1 × 108 BMMC stained with Hoechst and were euthanized for kidney examination 21 days after BMMC injection and samples were collected for histology and immunofluorescence analysis. Histological analysis demonstrated typical interstitial lesions in kidney similarly to human disease, as tubular necrosis, glomerular destruction, atrophy tubular, fibrotic areas, and collagen deposition. We conclude that histological analysis suggest a positive application of agouti's as a model for a gentamicin inducing of kidney disease, beyond the immunofluorescence analysis suggest a significant migration of BMMC to sites of renal injury in CTG.

Diagnostic microRNAs in myelodysplastic syndrome

The myelodysplastic syndromes (MDS) are aging-associated disorders characterized by ineffective maturation of hematopoietic elements, which are often diagnostically challenging. This study identifies microRNAs (miRNA) and miRNA targets that might represent diagnostic markers for MDS. This study utilized a total of 42 MDS samples and 45 controls. A discovery set of 20 frozen bone marrow mononuclear cell samples (10 MDS, 10 controls) was profiled on a custom Agilent miRNA microarray. Classifier miRNAs were validated in a separate set of 49 paraffin-embedded particle preparations by real-time polymerase chain reaction (24 MDS, 25 controls). Target prediction analysis was compared to a de novo transcriptional profile of MDS derived from the Microarray Innovations in Leukemia study. c-Myb and Sufu were further investigated by immunohistochemical stains on a set of 26paraffin-embedded samples. We identified 13 miRNAs of interest from the discovery set, 8 of which proved statistically significant on real-time polymerase chain reaction verification. These eight miRNAs were then examined in an independent real-time polymerase chain reaction validation set. Notably, hsa-miR-378, hsa-miR-632, and hsa-miR-636 demonstrated particularly high discrimination between MDS and normal controls. Target prediction identified potential targets of miRNA regulation that correspond to many of the genes that characterize MDS. Immunohistochemical staining performed on a third validation set confirmed that c-Myb and Sufu are differentially expressed in MDS. Our data utilize both discovery and validation sets and two complementary platforms to identify miRNAs associated with MDS. We have analyzed predicted targets and identified c-Myb and Sufu as potential diagnostic markers of MDS.

Abstract 1791: Stat3 signaling in acute myeloid leukemia: Ligand-dependent and -independent activation, impact on prognosis, and induction of apoptosis by a novel Stat3 inhibitor

Abstract Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite very toxic chemotherapy. Newly developed therapies for AML are targeted at signal transduction pathways known to support blast survival. One such potential target is Stat3, a signaling intermediate that is activated by tyrosine phosphorylation in response to a number of cytokines active in myeloid cells, such as G-CSF. Aberrant activation of Stat3 has been demonstrated in many malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were to 1) characterize Stat3 signaling patterns in AML cells lines and primary pediatric AML samples, and 2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We measured tyrosine-phosphorylated Stat3 (pStat3) in 7 AML cell lines by FACS and Western blot. We found that in 6 of the 7 cell lines, at least 20% of cells had pStat3 (by FACS) in the absence of stimulation, and thus were considered constitutively activated. Likewise, 6 of 7 cell lines demonstrated at least a 2-fold increase in the mean fluorescence intensity (MFI) of pStat3 after G-CSF stimulation, and thus were considered responsive to G-CSF. We next analyzed primary pediatric AML samples (from the Children's Oncology Group AML Reference Lab) and normal bone marrow mononuclear cells (BMMCs) for constitutive and G-CSF-induced pStat3. We found 6 of 18 AML samples and 1of 5 BMMCs showed constitutive pStat3 (pStat3+), and 16/18 AML and 3/5 BMMC samples were responsive to G-CSF. Event-free survival analysis revealed that constitutive pStat3 was significantly associated with inferior outcome. Specifically, 4/6 pStat3+ patients died of relapsed or refractory disease, while 2/12 pStat3- patients died (p = 0.019). These results support the hypothesis that aberrant Stat3 activity promotes an aggressive phenotype in pediatric AML. In this regard, we evaluated the effect of a novel small molecule Stat3 inhibitor that has been developed by us, C188-9, to block G-CSF-induced Stat3 phosphorylation and to induce apoptosis in AML cells. The IC50s of C188-9 to inhibit Stat3 activation in AML cell lines were in the range of 4-7 μM, and in primary AML samples the IC50s were in the range of 8-18 μM. For apoptosis studies, AML cell lines and primary samples were treated for 24 hours with the compound, then apoptotic cells were quantified by FACS analysis for annexin V-labeled cells. The EC50s for apoptosis induction were quite variable, ranging from 6 μM to over 50 μM. These results indicate that there is a subset of AML samples that are dependent on Stat3 signaling for survival and are killed by inhibition of Stat3. Our data suggest that aberrant Stat3 activity may be both a valuable new prognostic indicator and an important target for novel therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1791.

Increased inducible nitric oxide synthase expression and nitric oxide concentration in patients with aplastic anemia.

Nitric oxide (NO) is a biological mediator that is synthesized from L-arginine by the nitric oxide synthase (NOS) family. We investigated the expression of iNOS in bone marrow (BM) mononuclear cells (MNCs) using a reverse transcriptase polymerase chain reaction (RT-PCR) assay and the concentration of NO from BM serum by measuring the metabolite NO(2)(-) in 13 patients with aplastic anemia (AA) compared with 10 normal controls who were donors for allogeneic bone marrow transplantation (BMT). All samples of BM MNCs in patients with AA expressed iNOS mRNA, but iNOS was not expressed in patients who were treated successfully with allogeneic BMT. Normal control samples and samples from leukemia patients who had bone marrow aplasia after chemotherapy did not show significant iNOS expression. When we measured the density of bands for both iNOS and beta(2)-microglobin expressed as the iNOS/beta(2)-microglobin density ratio, there was a significant difference in the ratio between AA and normal controls (0.88+/-0.15 vs 0.26+/-0.05, P<0.001). The BM serum NO(2)(-) concentration in the patients with AA was significantly higher than that of normal controls (88.1+/-32.8 microM vs 48.8+/-8.6 microM, P=0.002). In addition, there was a significant correlation between the NO(2)(-) concentration and the calculated iNOS/beta(2)-microglobin density ratio (r=0.567, P=0.01). These findings suggest that upregulation of iNOS expression for local NO production may contribute in part to the pathogenesis of AA.

In vivo interleukin 6 gene expression in the tumoral environment in multiple myeloma

Whether interleukin 6 (IL 6) is an autocrine or paracrine myeloma cell growth factor in vivo remains unresolved. To identify which cells are producing IL 6 in vivo, we have studied the IL 6 gene expression in bone marrow mononuclear cells (BMMC) of 19 patients with multiple myeloma (MM) and in peripheral blood mononuclear cells (PBMC) of 9 patients with plasma cell leukemia (PCL). We found that the IL 6 gene was transcribed by BMMC of most patients with MM (79%). Further, IL 6 mRNA was not produced by purified myeloma cells from patients with either MM (5 patients) or PCL, but by the bone marrow environment, mainly by monocytes and myeloid cells (CD13+CD15+ cells). For 2 patients with PCL, for whom PBMC and BMMC samples were available, IL 6 mRNA could be detected in BMMC but not in PBMC. Finally, no IL 6 mRNA was detected in five freshly established IL 6-dependent myeloma cell lines. The present data give a clear-cut demonstration of the paracrine origin of IL 6 in vivo in human MM.