Analysis Of Acute Myeloid Leukemia Research Articles

Investigation of screening method for DNMT3A mutations by high‐resolution melting analysis in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease associated with various genetic abnormalities. Somatic mutations in nucleophosmin 1 (NPM1), fms-related tyrosine kinase 3 (FLT3), and DNA methyltransferase 3 alpha (DNMT3A) are the most frequent mutations associated with AML. However, because DNMT3A mutations are broadly distributed, they are challenging to analyze in routine laboratory tests. Hence, we developed a rapid screening method for DNMT3A mutations by high-resolution melting (HRM) analysis for clinical use at the point of AML diagnosis. The detection limit for DNMT3A mutations from exons 8-23 by an HRM analysis was investigated using plasmid mixtures. In 69 patients with AML, somatic mutations in NPM1, FLT3-internal tandem duplication (ITD), FLT3-tyrosine kinase domain (TKD), DNMT3A, and isocitrate dehydrogenase 1/2 were screened using HRM analysis, and direct sequencing was performed for positive samples. High-resolution melting analysis enabled complete mutation detection in samples with 20% mutant alleles in all regions. In a clinical laboratory test, DNMT3A mutations were detected in 12 cases (17.3%), and we identified five novel mutations. Simultaneous NPM1, FLT3-ITD, and DNMT3A mutations constituted the most common pattern (30%) in de novo cytogenetically normal AML. High-resolution melting analysis has sufficient performance for the detection of DNMT3A mutations in AML. This approach can facilitate rapid AML genotyping at diagnosis in clinical settings.

The Integrated Stress Response Activity Marks Stem Cells in Normal Hematopoiesis and Leukemia

Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Perturbations of cellular homeostasis such as nutrient deprivation, irradiation, and endoplasmic reticulum stress can result in HSC loss. However, HSCs must survive low-level stressors in order to sustain lifelong replenishment of the hematopoietic system. It is poorly understood how human HSCs balance apoptosis with survival in the context of basal stress, and how adaptive signalling is regulated in leukemia stem cells (LSCs). The Integrated Stress Response (ISR) is an adaptive pathway that can protect cells against stressors such as ROS, nutrient deprivation and misfolded proteins.To assess the expression levels of key ISR pathway components, we analyzed the proteome of purified human HSCs and progenitor cells from cord blood (CB). Quantitative label-free mass spectrometry revealed lower expression of eIF2α, eIF2β and eIF2γ subunits in HSCs compared to downstream progenitors. Furthermore, activated-transcription factor 4 (ATF4) mRNA is highly expressed in HSCs compared to progenitors. Similar to our findings in normal CB cells, analysis of acute myeloid leukemia (AML) patient samples revealed lower protein levels of eIF2α, eIF2β and eIF2γ in phenotypically primitive (CD34+CD38-) compared to differentiated (CD34+CD38+) AML cell populations. These results suggest that primitive cells in normal hematopoiesis and AML are primed for ISR activation.To assess ISR activity in human HSPCs, we used an ATF4 lentiviral reporter (ATF4rep) that measures ISR-induced ATF4 translation. We subjected ATF4rep-transduced CD34+ CB cells to hypoxia and amino acid deprivation, and found that valine depletion strongly induced ATF4rep activity. ATF4rep upregulation was abolished in the presence of an eIF2αS52A mutant that cannot be phosphorylated. Furthermore, knockdown of eIF2α, eIF2β or eIF2γ subunits in CD34+ CB cells increased ATF4rep activity. Thus, low levels of eIF2α, eIF2β or eIF2γ result in efficient ATF4 translation, and nutrient deprivation upregulates ATF4 through eIF2α phosphorylation.We assessed the effect of ATF4 upregulation on CB cell proliferation and survival. Following knockdown of ATF4 mRNA in CD34+ CB cells, the cells were incubated in valine deficient media to induce translational upregulation of ATF4. Valine depletion of shCTRL-transduced cells for 2 days did not affect proliferation or apoptosis, as measured by EdU incorporation or Annexin-V. In contrast, valine depletion of shATF4-transduced cells resulted in decreased proliferation (2-fold, P = 0.0004) and increased apoptosis (4-fold, P < 0.0001,). Thus, ATF4 promotes survival of primitive CD34+ CB cells undergoing valine depletion.We performed in vivo xenograft studies to examine the ISR activity in the best available setting to approximate homeostatic conditions for human HSPCs. Transplantation of ATF4rep-transduced CB cells showed that human HSPCs in the mouse bone marrow maintained a 2.4-fold higher ATF4rep activity compared to downstream progenitors (P = 0.0002). ATF4rep activity further declined in mature monocytes, granulocytes and B-cells (13-fold, P < 0.0001). To determine if high ISR activity is associated with improved HSC function, we transplanted lin- CB cells expressing high ATF4rep activity (GFP-high) and low ATF4rep activity (GFP-low) into mice. The level of engraftment as well as the number of engrafted mice was increased from GFP-high cells compared to GFP-low cells (P = 0.001). The hierarchical structure of normal hematopoiesis is partially maintained in AML. We evaluated ATF4rep expression in the malignant hierarchy and found that 4/5 patient samples had higher ATF4rep expression in CD34+ cells compared to CD34- cells. Furthermore, serial transplantation of ATF4rep-transduced cells showed higher engraftment from GFP-high compared to GFP-low cells ( P < 0.0001). Thus, primary human AML cells that possess high ISR activity are enriched for LSC function.Our data establish that the adaptive ISR pathway plays a key role in maintaining homeostasis of normal and malignant stem cells. We show that Amino acid deprivation activates the ISR in human HSPCs resulting in ATF4-dependent pro-survival signals. In an unperturbed state, HSCs are in a state of primed ISR activity, mechanistically maintained by eIF2 scarcity and high ATF4 levels. DisclosuresNo relevant conflicts of interest to declare.

Analysis of acute myeloid leukemia (AML) incidence and geographic distribution in Canada from 1992 to 2010

Analysis of acute myeloid leukemia (AML) incidence and geographic distribution in Canada from 1992 to 2010

Minimal residual disease assessed with deep sequencing of NPM1 mutations predicts relapse after allogeneic stem cell transplant in AML

Mutations in NPM1 can be used for minimal residual disease (MRD) analysis in acute myeloid leukemia (AML). We here applied a newly introduced method, deep sequencing, allowing for simultaneous analysis of all recurrent NPM1 insertions and thus constituting an attractive alternative to multiple PCRs for the clinical laboratory. We retrospectively used deep sequencing for measurement of MRD pre- and post-allogeneic hematopoietic stem cell transplantation (alloHCT). For 29 patients in morphological remission at the time of alloHCT, the effect of deep sequencing MRD on outcome was assessed. MRD positivity was defined as variant allele frequency ≥0.02%. Post-transplant MRD status was significantly and independently associated with clinical outcome; 3-year relapse-free survival 20% vs 85% (p < .001), HR 45 (95% CI 2–1260), and overall survival 20% vs 89% (p < .001), HR 49 (95% CI 2–1253). Thus, the new methodology deep sequencing is an applicable and predictive tool for MRD assessment in AML.

Cytogenetic analysis of acute myeloid leukemia with t(8;21): Its clinical correlation with loss of X Chromosome and Del (9q)

BACKGROUND: Translocation (8;21), t(8;21), is one of the most common cytogenetic abnormalities in adult de novo acute myeloid leukemia (AML) patients. It is usually associated with secondary chromosomal abnormalities; however, it's unclear whether these abnormalities affect the clinical characteristics of t(8;21) patients. OBJECTIVES: To investigate the effect of additional aberrations; loss of X chromosome and deletion of the long arm of chromosome 9 (del 9q)on the clinicopathological and immunophenotypic characteristics and prognostic behavioral of t(8;21) de novo AML. METHODS: Fifty six adults with de novo AML-M2 were enrolled. Detection of loss of X-chromosome and del 9q were performed using fluorescent in situ hybridization (FISH). RESULTS: More than half of the patients (53.6%) harbored a secondary chromosomal abnormality in addition to t(8;21). Del 9q was found in 17.9% of the patients. A significant association was found between this chromosomal aberration and age, hepatomegally, high total leucocytic count and low platelets count (P< 0.05). Most patients had poor clinical outcome, high tendency for resistance to therapy and significantly shorter survival. On the other hand, loss of X chromosome was found in 25% of the studied patients and was not related to clinicopathological features or prognostic markers except for high platelets count and low expression of aberrant CD19 (P< 0.05). CONCLUSIONS: 9q deletion associated t(8;21) could be considered as an adverse prognostic predictor being associated with poor disease outcome and shorter survival of the patients. Thereafter the use of these cytogenetic aberrations would be recommended to guide therapeutic regimens.

Diagnostic and Prognostic Utility of Fluorescence In situ Hybridization (FISH) Analysis in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematologic neoplasia consisting of incompletely differentiated hematopoietic cells of the myeloid lineage that proliferate in the bone marrow, blood, and/or other tissues. Clinical implementation of fluorescence in situ hybridization (FISH) in cytogenetic laboratories allows for high-resolution analysis of recurrent structural chromosomal rearrangements specific to AML, especially in AML with normal karyotypes, which comprises approximately 33-50% of AML-positive specimens. Here, we review the use of several FISH probe strategies in the diagnosis of AML. We also review the standards and guidelines currently in place for use by clinical cytogenetic laboratories in the evaluation of AML. Updated standards and guidelines from the WHO, ACMG, and NCCN have further defined clinically significant, recurring cytogenetic anomalies in AML that are detectable by FISH. FISH continues to be a powerful technique in the diagnosis of AML, with higher resolution than conventional cytogenetic analysis, rapid turnaround time, and a considerable diagnostic and prognostic utility.

LncRNA co-expression network model for the prognostic analysis of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy with great variability of prognostic behaviors. Previous studies have reported that long non-coding RNAs (lncRNAs) play an important role in AML and may thus be used as potential prognostic biomarkers. However, thus use of lncRNAs as prognostic biomarkers in AML and their detailed mechanisms of action in this disease have not yet been well characterized. For this purpose, in the present study, the expression levels of lncRNAs and mRNAs were calculated using the RNA-seq V2 data for AML, following which a lncRNA-lncRNA co-expression network (LLCN) was constructed. This revealed a total of 8 AML prognosis-related lncRNA modules were identified, which displayed a significant correlation with patient survival (p≤0.05). Subsequently, a prognosis-related lncRNA module pathway network was constructed to interpret the functional mechanism of the prognostic modules in AML. The results indicated that these prognostic modules were involved in the AML pathway, chemokine signaling pathway and WNT signaling pathway, all of which play important roles in AML. Furthermore, the investigation of lncRNAs in these prognostic modules suggested that an lncRNA (ZNF571-AS1) may be involved in AML via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway by regulating KIT and STAT5. The results of the present study not only provide potential lncRNA modules as prognostic biomarkers, but also provide further insight into the molecular mechanisms of action of lncRNAs.

High-Dimensional Analysis of Acute Myeloid Leukemia Reveals Phenotypic Changes in Persistent Cells during Induction Therapy.

The plasticity of AML drives poor clinical outcomes and confounds its longitudinal detection. However, the immediate impact of treatment on the leukemic and non-leukemic cells of the bone marrow and blood remains relatively understudied. Here, we conducted a pilot study of high dimensional longitudinal monitoring of immunophenotype in AML. To characterize changes in cell phenotype before, during, and immediately after induction treatment, we developed a 27-antibody panel for mass cytometry focused on surface diagnostic markers and applied it to 46 samples of blood or bone marrow tissue collected over time from 5 AML patients. Central goals were to determine whether changes in AML phenotype would be captured effectively by cytomic tools and to implement methods for describing the evolving phenotypes of AML cell subsets. Mass cytometry data were analyzed using established computational techniques. Within this pilot study, longitudinal immune monitoring with mass cytometry revealed fundamental changes in leukemia phenotypes that occurred over time during and after induction in the refractory disease setting. Persisting AML blasts became more phenotypically distinct from stem and progenitor cells due to expression of novel marker patterns that differed from pre-treatment AML cells and from all cell types observed in healthy bone marrow. This pilot study of single cell immune monitoring in AML represents a powerful tool for precision characterization and targeting of resistant disease.

Heat induced aggregation of gold nanorods for rapid visual detection of lysozyme

Gold nanorods have been nominated as propitious candidates for nanobiodiagnostic applications. Herein, a technique has been introduced for rapid visual detection of lysozyme, as its high level of excretion in biological fluids is a characteristic sign of leukemia and kidney disorders. Gold nanorods were biofunctionalized with lysozyme aptamer and characterized with UV–Visible and FTIR spectroscopy, zeta potential analyzer and transmission electron microscopy. Exposure of the nanoprobe to nano molar levels of lysozyme (20nmoll−1) lead to dictated aggregation of the nanostructures at ambient temperature; which was significantly improved by heat induced morphological perturbations and rapid detection by the naked eye (down to pico molar level). Qualitative analysis of Acute myeloid leukemia, Acute lymphocytic leukemia and Lymphoma blood serums showed sensitivity and specificity of the fabricated aptasensor under both temperature conditions. This report encourages utilization of heat-induced aggregation of gold nanorods as a promising nanodiagnostic technique for the emerging nanotechnologies.

SKIP Is Underexpressed in AML Leading to Sphingosine Kinase Hypofunction

Background: Sphingosine kinase interacting protein (SKIP) has been shown to be mostly silenced by hypermethylation in AML [1]. SKIP interacts with and regulates the function of sphingosine kinase (SK) enzyme. SK activity results in phosphorylation of sphingosine (SPH) to form sphingosine 1 phosphate (S1P), which promotes cell survival and resistance to apoptosis. On the other hand, S1P precursors ceramide (CER) and SPH mediate antiproliferative and apoptotic responses. SKIP has been reported to negatively regulate SK1 activity in fibroblasts. Therefore, we investigated the consequences of SKIP silencing in primary AML cells. In addition, we studied the effects of SKIP re-expression in leukemic cell lines.Methods: CTS and K562 cells were transfected with SKIP gene using standard techniques. SKIP is normally silenced in both cell lines. Intracellular and extracellular S1P, SPH and CER were measured by UPLC-MS/MS. In addition, intracellular SK activity was determined based on C17 S1P production from C17 SPH substrate. Chemosensitivity to doxorubicin, Imatinib and Ara-C in transfected cells was also studied.Results: In Primary AML cells, intracellular S1P and CER concentrations were reduced compared to G-CSF-mobilized peripheral blood mononuclear cells. In addition, SK activity was found to be downregulated in AML primary cells. When we transfected leukemic cell lines with SKIP gene, S1P and CER showed at least 2 fold increase in intracellular and extracellular basal levels compared to vector alone control (Figure 1). Further studies confirmed a significant increase in intracellular SK activity in SKIP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase in S1P and CER was associated with increasing apoptotic signals as evidenced by Annexin V staining and cleaved PARP expression. Moreover, chemosensitivity to Imatinib and AraC was significantly increased in SKIP transfected cell lines. These experiments confirm the regulation of SK1 function by SKIP.Conclusion: These data indicate that SKIP is downregulated in AML leading to reduced SK activity, which ultimately inhibits the apoptosis response. [Display omitted] 1. Saied, M.H., et al., Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats. PLoS One, 2012. 7(3): p. e33213.EAG and PS contributed equallyJG and DT contributed equally DisclosuresNo relevant conflicts of interest to declare.

Clinical analysis of acute myeloid leukemia with t(8;21) (q22;q22) and loss of Y chromosome

This study was aimed to investigate the clinical characteristics of acute myeloid leukemia (AML) with t (8;21) (q22;q22) and loss of Y chromosomes. Clinical data of 267 cases of AML were collected from January 2010 to June 2013. Among 267 AML, there were 13 cases with t (8;21) (q22;q22) and loss of Y chromosomes. The clinical data including clinical indicators, treatment protocols, curative effect and prognosis were analyzed retrospectively. The results showed that after normalized chemotherapy, there were 4 patients with complete remission at the first cycle of treatment, 4 patients with complete remission at the second cycle, 4 patients with complete remission at the third cycle, but one patient without complete remission after 4 cycles. There were 6 patients who did not relapse during consolidation and intensive therapy. Among these 6 patients, 4 cases accepted chemotherapy combined with transplantation, other 2 cases accepted chemotherapy. In the remainder 6 patients, 4 cases relapsed once, one cases relapsed twice, 1 cases relapsed for three times. Moreover, 2 cases who accepted the chemotherapy and auto-hematopoietic stem cell trans-plantation, were diagnosed as relapse, after accepted allo-hematopoietic stem cell transplantation, currently are in disease-free status. In follow-up period, the relapse-free survival (RFS) time was 4.67 ± 3.45 months in chemotherapy group, the RFS time is 34.17 ± 21.37 months in chemotherapy and transplantation group. The chemotherapy combined with transplantation extended the RFS time (P < 0.05). It is concluded that the NCCN guide indicates that AML with t (8;21) ( q22;q22) showed a good prognosis. but the clinical course of treatment confirmed that the prognosis of AML patients with t (8;21) (q22;q22) and loss Y chromosomes is poor, including uneasy remission and easy relapse, for improving the prognosis of these patients, the hematopoietic stem cell transplantation should be recommended.

HLA Class I Ligandome Analysis In Acute Myeloid Leukemia – Novel T-Cell Epitopes For Peptide-Based Immunotherapy

Data regarding the graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion strongly suggest that T lymphocytes play a major role in the rejection of leukemic cells. Immunotherapy directed against leukemia- associated antigens might elicit specific immune responses that may serve to eliminate minimal residual disease after chemotherapy, or enhance the GVL effect after SCT. To achieve this goal there is need to identify appropriate leukemia associated HLA ligands, which are able to induce specific T cell responses. We here aimed to characterize the HLA class I ligandome in AML patients to provide novel tumor associated antigens (TAA) for peptide-based immunotherapy employing our recently implemented approach of direct isolation and identification of naturally presented HLA ligands by affinity chromatography and mass spectrometry (LC-MS/MS) in AML (Stickel et.al., abstract in Blood 2012). Absolute HLA surface expression on AML cells and autologous monocytes and granulocytes was quantified by flow cytometry. HLA class I ligands were isolated from AML cells as well as bone marrow and peripheral blood mononuclear cell (BMNCs/PBMCs) of healthy donors. LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived TAA (LiTAA) and to select peptide vaccine candidates. The most abundantly detected peptide candidates were checked for immunogenicity by ELISpot and confirmed by intracellular interferon-g staining of CD8+ T-cells. Meanwhile 15 AML patients (8 FLT3-ITD mutant) and 35 healthy donors were analyzed. We observed overexpression of HLA class I and II on AML cells as compared to autologous monocytes and granulocytes, with the level of significance reached for HLA class II (p=0,04). A total of more than 12,000 AML derived HLA ligands representing >6,000 different source proteins were identified; of which 2,220 were exclusively represented in AML, but not in healthy PBMC/BMNC. Data mining for broadly represented LiTAA pinpointed 98 TAA as most promising targets. HLA ligands derived from these TAA were presented exclusively on more than 33% of all analyzed AML samples, amongst them already described TAA (e.g. JUP, FAF1) as well as several new leukemia-associated proteins (e.g. MTCH2, METTL7A). Subset analysis of the FLT3-ITD positive AML cohort revealed 21 LiTAA presented exclusively on more than 50% of FLT3-ITD positive AML cases. Additional screening for HLA ligands derived from described leukemia associated antigens revealed overrepresentation for e.g. FLT3, NUSAP, RHAMM and RGS5. Specific CD8+ T cell responses were detected against two A*03 epitope pools (pool 1: APLP2, DKGZ, FAF1, MTCH2; pool 2: KLF2, METTL7A, VCIP1, WIPI1) in AML patients. Notably, the chosen A*03 epitope pools did not elicit specific responses of CTL from healthy donors. Taken together, our HLA class I ligandome analysis in AML for the first time identified naturally presented HLA ligands from patients including a vast array of new leukemia associated antigens representing promising targets for a multipeptide-based immunotherapy approach in AML. Disclosures:No relevant conflicts of interest to declare.

Mapping Cellular Hierarchy by Single-Cell Analysis of the Cell Surface Repertoire

Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method, we analyzed over 1,500 single cells throughout the mouse hematopoietic system and illustrate its utilityfor revealing important biological insights. The comprehensive single cell data set permits mapping of the mouse hematopoietic stem cell differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems.

Clinicopathologic analysis of acute myeloid leukemia arising from chronic myelomonocytic leukemia

Acute myeloid leukemia arising from chronic myelomonocytic leukemia is currently classified as acute myeloid leukemia with myelodysplasia-related changes, a high-risk subtype. However, the specific features of these cases have not been well described. We studied 38 patients with chronic myelomonocytic leukemia who progressed to acute myeloid leukemia. We compared the clinicopathologic and genetic features of these cases with 180 patients with de novo acute myeloid leukemia and 34 patients with acute myeloid leukemia following myelodysplastic syndromes. We also examined features associated with progression from chronic myelomonocytic leukemia to acute myeloid leukemia by comparing the progressed chronic myelomonocytic leukemia cases with a cohort of chronic myelomonocytic leukemia cases that did not transform to acute myeloid leukemia. Higher white blood cell count, marrow cellularity, karyotype risk score, and Revised International Prognostic Scoring System score were associated with more rapid progression from chronic myelomonocytic leukemia to acute myeloid leukemia. Patients with acute myeloid leukemia ex chronic myelomonocytic leukemia were older (P<0.01) and less likely to receive aggressive treatment (P=0.02) than de novo acute myeloid leukemia patients. Most cases showed monocytic differentiation and fell into the intermediate acute myeloid leukemia karyotype risk group; 55% had normal karyotype and 17% had NPM1 mutation. Median overall survival was 6 months, which was inferior to de novo acute myeloid leukemia (17 months, P=0.002) but similar to post myelodysplastic syndrome acute myeloid leukemia. On multivariate analysis of all acute myeloid leukemia patients, only age and karyotype were independent prognostic variables for overall survival. Our findings indicate that acute myeloid leukemia following chronic myelomonocytic leukemia displays aggressive behavior and support placement of these cases within the category of acute myeloid leukemia with myelodysplasia-related changes. The poor prognosis of these patients may be related to an older population and lack of favorable-prognosis karyotypes that characterize many de novo acute myeloid leukemia cases.

Normal karyotype mosaicism in adult AML patients with adverse-risk and undefined karyotype: preliminary report of treatment outcomes after hematopoietic stem cell transplantation

Karyotype analysis in acute myeloid leukemia (AML) is one of the powerful prognostic factors for complete remission (CR), relapse, and overall survival (OS). Cytogenetic mosaicism is considered to be one of the important characteristics in expression of phenotypic manifestations. However, it has not come into focus due to emerging molecular biological approaches and the results of a number of mutation studies. Clinical correlates and prognostic relevance of mosaicism were evaluated in 163 AML patients [adverse-risk karyotypes (n=72) and undefined karyotypes (n=91)]. All patients were treated by induction and consolidation chemotherapies and finally went on hematopoietic stem cell transplantations (HSCT). Patients were divided into two subgroups, either with or without normal karyotype (NK) mosaicism. Seventy patients exhibited NK mosaicism and 93 did not. There were no significant differences in age, gender, chemotherapy cycles to achieve CR, HSCT donor type, source or intensity properties between the two subgroups. We found that NK mosaicism remaining in adverse-risk and undefined karyotype at diagnosis significantly correlates with better OS (p=0.001) and lower CIR (p=0.021) rate after HSCT. Our data show that the poor prognostic properties of unfavorable risk karyotype can be overcome to a great extent by allogeneic HSCT and chronic GVHD, especially in the subgroup with NK mosaicism. Cytogenetic mosaicism at initial diagnosis can be an influential factor for survival outcomes, even after HSCT.

Clinical analysis of acute myeloid leukemia with CBFB-MYH11-positive

The study was aimed to investigate the clinical characteristics of acute myeloid leukemia (AML) with CBFB-MYH11 gene. The clinical data of 12 cases were analyzed retrospectively, including age, clinical characteristics, immunophenotype, treatment protocols and efficacy as well as the prognosis. The results indicated that 12 patients with CBFB-MYH11 were detected in 293 AML patients. The median age of the 12 patients was 32.5 (21 - 57) years old. According to French-American-British (FAB) classification, 66.7% (8/12) patients was diagnosed as M4Eo and 33.3% patients was diagnosed as M4. At new diagnosis, the median WBC count was 19.8×10(9)/L (2.46 - 164.30×10(9)/L). The WBC count > 100×10(9) was found in 16.7% patients (2/12). The complete remission (CR) rate after 1 and 2 cycles of induction chemotherapy were 83.3% and 16.7% respectively, so the total CR rate was 100%. Estimated 5-year relapse-free survival (RFS) and overall survival (OS) were 80% and 83%, respectively. It is concluded that patients with CBFB-MYH11 are usually M4Eo and M4. Patients with this fusion gene are often associated with high frequency of CD33, CD34, CD117, HLA-DR, CD15, CD64 and CD14 expression. Patients with CBFB-MYH11 have a tendency of higher CR rate, longer RFS and OS.

HLA Ligandome Analysis of Acute Myeloid Leukemia (AML) Revealed Novel Tumor-Associated Antigens for Immunotherapy.

AbstractAbstract 3012 Introduction:Eradication of minimal residual disease (MRD) by antigen-specific immunotherapy after conventional chemotherapy and also after allogeneic stem cell transplantation may improve overall survival in acute myeloid leukemia (AML). To achieve this goal there is a need for novel leukemia-associated HLA-presented peptides, which are able to induce a tumor-specific T cell response. Accordingly we here aimed to identify novel leukemia associated antigens employing, for the first time, the approach of direct elution and analysis of naturally processed and presented HLA ligands from the surface of primary AML cells. In particular we focused on peptides derived from AML specific mutated proteins, which are produced only by leukemia cells, only.Methods: HLA class I ligands were isolated from MACS-purified PBMCs of AML patients using immunoprecipitation. Liquid chromatography tandem mass spectrometry (LC-MS/MS) based peptide sequencing was performed to identify HLA presented peptides. The obtained data were mined for leukemia associated peptides by comparison of HLA ligandomes of AML cells with that of PMBCs and granulocytes from healthy donors, investigation of gene expression databases and literature research. All acquired peptide sequences were blasted against the COSMIC database (http://www.sanger.ac.uk/genetics/CGP/cosmic/), to identify sequences from AML specific mutated proteins. Identified mutations were confirmed by PCR. In addition, HLA quantification experiments on the cell surface of AML cells and autologous healthy monocytes and granulocytes were performed using a flow cytometric indirect immunofluorescence assay. Results:Quantitative preliminary results showed similar amounts of HLA class I molecules on AML cells as compared to autologous healthy monocytes and granulocytes lymphocytes (p=0.56, unpaired t-test). For HLA class II we could show a clear upregulation on AML cells compared to autologous healthy leukocytes, not reaching level of significance due to the small cohort examined till now (p= 0.12, unpaired t-test).We were able to identify a total of more than 10.000 peptides from 6 AML patients, including 2 NPM1 mutated AMLs, 1 FLT3-ITD mutated AML, 1 JAK2 mutated AML from osteomyelofibrosis and two AMLs from myelodysplastic syndrome without any cytogenetical aberrations. Using a new approach of spectral counting the obtained sequences could be compared semi-quantitatively to the same amount of peptides obtained from HLA matched healthy volunteers. Thus several new naturally processed and presented leukemia-associated peptides (LEUMAPs) were identified. This comprised, amongst others, ligands of p53, c-myc oncogene, PRAME, RHAMM and NPM1.The sequences of identified LEUMAPs were all verified by LC-MS/MS-based peptide sequencing of their synthetic counterparts. Testing for immunogenicity by ELISPOTs-assay and an in vitro T cell priming approach is presently ongoing. Conclusions:Our analysis revealed several new leukemia associated antigens, which were, for the first time, directly obtained from the HLA ligandomes of AML patients. Quantitative HLA analysis showed no loss or downregulation of HLA molecules on the surface of AML cells, thereby excluding a potential major obstacle for successful immunotherapy. Our results may pave the way for future peptide based immunotherapy approaches to eradicate MRD after obtaining a remission by presently available conventional therapies in AML patients. Disclosures:No relevant conflicts of interest to declare.

Comparative proteome analysis of acute myeloid leukemia with and without maturation

Acute myeloid leukemia (AML) is a severe, rapidly progressing disease triggered by blocking granulocyte or monocyte differentiation and maturation. Because of its heterogeneity, AML is divided into a number of subtypes. Unfortunately, so far very few correlations have been found between AML classification and its clinical course or patient response to treatment. In addition, as yet only a few subtype-specific AML biomarkers have been discovered. To solve these problems here, we focused on two AML subtypes M1 and M2 that are especially difficult to differentiate. Using 2D electrophoresis and mass spectrometry, we analyzed the protein profiles of peripheral blood (PB) and/or bone marrow (BM) samples collected from 38 AML-M1/M2 patients and 17 healthy volunteers. Comparative analysis of AML-M1/M2 and control PB/BM cells revealed 25 proteins that accumulated differentially. Hierarchical clustering of proteomic results clearly divided the AML samples into 2 groups (M1 and M2). Annexin III, L-plastin and 6-phosphogluconate dehydrogenase were found only in the M2 group. We also observed that the levels of annexin I and actin gamma 1 were correlated with resistance to treatment and the time of relapse. It appears that these five proteins can serve as potential AML biomarkers.

Multiplex high-throughput gene mutation analysis in acute myeloid leukemia

Classification of acute myeloid leukemia increasingly depends on genetic analysis. However, the number of known mutations in acute myeloid leukemia is expanding rapidly. Therefore, we tested a high-throughput screening method for acute myeloid leukemia mutation analysis using a multiplex mass spectrometry-based approach. To our knowledge, this is the first reported application of this approach to genotype leukemias in a clinical setting. One hundred seven acute myeloid leukemia cases were screened for mutations using a panel that covers 344 point mutations across 31 genes known to be associated with leukemia. The analysis was performed by multiplex polymerase chain reaction for mutations in genes of interest followed by primer extension reactions. Products were analyzed on a Sequenom MassARRAY system (San Diego, CA). The multiplex panel yielded mutations in 58% of acute myeloid leukemia cases with normal cytogenetics and 21% of cases with abnormal cytogenetics. Cytogenetics and routine polymerase chain reaction-based screening of NPM1, CEBPA, FLT3-ITD, and KIT was also performed on a subset of cases. When combined with the results of these standard polymerase chain reaction-based tests, the mutation frequency reached 78% in cases with normal cytogenetics. Of these, 42% harbored multiple mutations primarily involving NPM1 with NRAS, KRAS, CEBPA, PTPN11, IDH1, or FLT3. In contrast, cases with abnormal cytogenetics rarely harbored more than 1 mutation (1.5%), suggesting different underlying biology. This study demonstrates the feasibility and utility of broad-based mutation profiling of acute myeloid leukemia in a clinical setting. This approach will be helpful in defining prognostic subgroups of acute myeloid leukemia and contribute to the selection of patients for enrollment into trials with novel inhibitors.

Functional pathway analysis in acute myeloid leukemia using single cell network profiling assay: Effect of specimen source (bone marrow or peripheral blood) on assay readouts

Single cell network profiling (SCNP) is used to simultaneously measure the effects of modulators on signaling networks at the single cell level. SCNP-based biomarker assays predictive of response to induction therapy and relapse risk in acute myeloid leukemia (AML) patients are being developed. Such assays have typically used bone marrow (BM) as the sample source of blasts. Because circulating peripheral blasts are detectable in ∼65% of AML patients and peripheral blood (PB) sampling is less invasive than BM sampling, this study was performed to assess the effect of sample source on AML blasts signaling as measured in SCNP assay. SCNP using multiparametric flow cytometry was used to evaluate the activation state of intracellular signaling molecules in leukemic blasts under basal conditions and after treatment with modulators in 46 pairs of BM mononuclear cells/PB mononuclear cells. The relationship between readouts of modulated intracellular proteins ("nodes") was measured using linear regression, Bland-Altman method, and Lin's concordance correlation coefficient. The majority (156/161) of signaling nodes show strong correlations between paired PB and BM samples independently from the statistical method used. Notable exceptions were two PB samples with almost undetectable levels of circulating blasts compared with paired BM samples. Our results demonstrate that specimen source (BM or PB) does not significantly affect proteomic signaling in patients with AML and circulating blasts. The ability to use PB as a sample source will facilitate the monitoring of cellular signaling effects following administration of targeted therapies and at time points when BM aspirates are not clinically justifiable.