Profiling Of Acute Myeloid Leukemia Research Articles

The Transmembrane Protein CD37 Is Not a Promising Therapeutic Target in Acute Myeloid Leukaemia

Acute myeloid leukaemia is a disease of the haematopoietic stem and progenitor cells distinguished by an impairment of proliferation, propagation and differentiation of the haematopoietic lineage[1]. AML is a heterogenous disease, where patient stratification and personalised therapeutic strategies are of importance[2]. The transmembrane protein CD37 has been associated with regulation of cell to cell interaction and proliferation in both B and T cells. Mice knockout studies have suggested that CD37 is not crucial for B cell development, however, it is highly expressed in malignant and suboptimal conditions and thus represent a good target for B-cell malignancies[3]. Pereira et al. reported that their anti-CD37 antibody-drug conjugate was a potential therapeutic in AML[4]. Their study revealed anti-leukemic effect in primary AML material and cell lines bothin vivoandin vitro. An investigation of CD37 expression in AML patients is needed to understand the relevance of CD37 immunotherapy. To investigate the relevance of targeted therapy of CD37 the chimeric mAb NNV003 anti-CD37 antibody was evaluated in AML samples. Single-cell time-of-flight mass cytometry (CyTOF), an established tool to analyse the immune signalling in bone marrow and peripheral blood, was used to investigate CD37 expression in 59 AML patient samples. A panel was designed to categorise hierarchical landscape of AML and the leukemic stem cell population (LSCs). CyTOF identified CD37 expression exclusively in the B-cell population of our 59 patients, whereas the AML blast population was negative for CD37 expression. Flow cytometry, however; identified 5/59 patients with CD37 expression in the leukemic blast population. Although previous studies show that an anti-CD37 drug conjugate showed anti-leukemic effectin vivoandin vitro, our results do not support that it is a promising therapeutic biomarker. We have uncovered that malignant expression of CD37 is not a feature of the AML patients investigated. We do not consider CD37 to be a promising and good target for precision therapy of AML. Kayser, S. and M.J. Levis, Advances in targeted therapy for acute myeloid leukaemia. Br J Haematol, 2018. 180(4): p. 484-500.Patel, J.P., et al., Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med, 2012. 366(12): p. 1079-89.Payandeh, Z., et al., Anti-CD37 targeted immunotherapy of B-Cell malignancies. Biotechnol Lett, 2018. 40(11-12): p. 1459-1466.Pereira, D.S., et al., AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody-Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML. Mol Cancer Ther, 2015. 14(7): p. 1650-60. Disclosures Heyerdahl: Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Dahle:Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Gjertsen:Novartis:Consultancy;KinN Therapeutics AS:Current equity holder in private company;Pfizer Inc:Consultancy;BerGenBio AS:Consultancy, Research Funding;Alden Cancer Therapy AS:Current equity holder in private company.Mccormack:KinN Therapeutics AS:Current equity holder in private company.

Abstract B63: Immune gene expression profiling of acute myeloid leukemia identifies predictors of survival and actionable targets for treatment

Abstract Background: Acute myeloid leukemia (AML) is characterized by clonal expansion of poorly differentiated myeloid precursors, resulting in impaired hematopoiesis and often bone marrow (BM) failure. The general therapeutic approach in patients with AML has not changed substantially in more than 30 years. Investigation of new strategies, including immunotherapy, remains a priority. Tumor phenotypes are dictated not only by major oncogene drivers, but also by the tumor immunologic microenvironment (TIME) which is inherently immunosuppressive, is equipped to hamper effector T-cell function and includes immune and inflammatory cells, soluble mediators such as interferon (IFN)-gamma and extracellular matrix components. Herein, we profiled the TIME of AML to identify gene signatures that are reflective of general immune status and predictive of antileukemia immune potential. Methods: We used the hybridization-based nCounter® system (NanoString Technologies®, Seattle, WA) and the RNA Pan-Cancer Immune Profiling PanelTM to analyze BM aspirates from 290 adults and 40 children with newly diagnosed, nonpromyelocytic AML. Data were normalized to a set of reference genes and log2-transformed for bioinformatics analysis. The clinical relevance of the association between immune gene signatures and patient outcome was further validated in silico using publicly available cancer transcriptomic datasets (The Cancer Genome Atlas; TCGA). Results: We identified distinct sets of co-expressed genes corresponding to innate immunity, adaptive immunity and IFN-gamma signaling. BM samples with IFN-gamma-dominant gene expression profiles showed up-regulation of actionable immune checkpoints, such as B7-H3, PD-L1 and CTLA-4. A set of 4 differentially expressed immune genes between children and adults with AML (FDR<0.005) was associated with adverse cytogenetic features, leukemia relapse and shorter patient survival, and also stratified survival in the TCGA cohort of adult patients with AML. Finally, all four genes were amplified, deleted or mutated in 7% of 51,175 TCGA solid tumors, with the highest frequency of mutations being detected in primary CNS lymphoma (40%) and diffuse large B-cell lymphoma (25%). Conclusions: Our study identified distinct immune gene programs in the TIME of patients with newly diagnosed AML. From a translational standpoint, immune-enriched and IFN-gamma-dominant AMLs may benefit from immune checkpoint blockade and from new immunotherapy approaches, including dual-affinity T-cell redirecting antibodies targeting CD123. Grant support: John and Lucille van Geest Foundation, UK; Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). Citation Format: Sergio Rutella, Jayakumar Vadakekolathu, Tressa Hood, Stephen Reeder, Sarah E. Warren, Patrick Danaher, Jan Davidson-Moncada, Alessandra Cesano, Joseph M. Beechem, Sarah K. Tasian, Mark D. Minden. Immune gene expression profiling of acute myeloid leukemia identifies predictors of survival and actionable targets for treatment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B63.

Advances in Genomic Profiling and Risk Stratification in Acute Myeloid Leukemia.

To review the current state of molecular and genetic profiling of acute myeloid leukemia (AML) and its implications. Peer-reviewed journal articles. Significant advances in the understanding of the pathology of acute myeloid leukemia have led to refined risk stratification of patients and application of novel targeted therapies based on genetic profiles. Minimal residual disease testing allows for highly sensitive disease surveillance that can be used to predict relapse and assess treatment response. Accurate prognostication and therapeutic decision-making for patients with acute myeloid leukemia is dependent on molecular profiling. Being knowledgeable of the implications of minimal residual disease testing is critical for patient-centered care.

Protein Post-Translational Modification Crosstalk in Acute Myeloid Leukemia Calls for Action.

Post-translational modification (PTM) crosstalk is a young research field. However, there is now evidence of the extraordinary characterization of the different proteoforms and their interactions in a biological environment that PTM crosstalk studies can describe. Besides gene expression and phosphorylation profiling of acute myeloid leukemia (AML) samples, the functional combination of several PTMs that might contribute to a better understanding of the complexity of the AML proteome remains to be discovered. By reviewing current workflows for the simultaneous enrichment of several PTMs and bioinformatics tools to analyze mass spectrometry (MS)-based data, our major objective is to introduce the PTM crosstalk field to the AML research community. After an introduction to PTMs and PTM crosstalk, this review introduces several protocols for the simultaneous enrichment of PTMs. Two of them allow a simultaneous enrichment of at least three PTMs when using 0.5-2 mg of cell lysate. We have reviewed many of the bioinformatics tools used for PTM crosstalk discovery as its complex data analysis, mainly generated from MS, becomes challenging for most AML researchers. We have presented several non-AML PTM crosstalk studies throughout the review in order to show how important the characterization of PTM crosstalk becomes for the selection of disease biomarkers and therapeutic targets. Herein, we have reviewed the advances and pitfalls of the emerging PTM crosstalk field and its potential contribution to unravel the heterogeneity of AML. The complexity of sample preparation and bioinformatics workflows demands a good interaction between experts of several areas.

Clinical Profile of Acute Myeloid Leukemia in North India and Utility of Nontransplant Measures in its Management

AbstractIntroduction: Acute myeloid leukemia (AML) is a clonal accumulation of myeloid precursors in body tissues, which ultimately leads to bone marrow failure. This is an 8-year prospective, observational study in which 254 patients were enrolled. Aim of the Study: To document the clinical profile of AML and differential outcome in M3 versus non-M3 phenotype and to see impact of different variables on its survival. Methods: Patients enrolled in the study were examined, evaluated, and given standard 3:7 induction protocol, and acute promyelocytic leukemia (APML) patients were given the ICAPL 2006 protocol. Results: In our study, males outnumbered females and most of our patients were in 20–60 years of age group. The better prognosis was in patients who were in the second decade of life. Total leukocyte count and platelet count had a significant impact on the survival of the a patient. Bone marrow morphology of M3 type has extremely good prognosis and was the most common FAB type seen in our study. Flow cytometric markers such as CD15, CD33, CD117, and myeloperoxidase had positivity among 90% of patients. Overall survival is around 40% in whole-study group, 87% in APML group, and 16.5% in non-M3 group. There are still unmet needs in managing the non-M3 patients in resource-constraint countries where allogenic transplant and newer drugs have the least access. For improving the outcome in M3 AML, further newer molecules such as Flt3 and PIK3 inhibitors are being used in trials. Conclusion: There are still unmet needs in managing the non-M3 patients in resource-constraint countries where allogenic transplant and newer drugs have the least access. For improving the outcome in M3 AML, further newer molecules such as Flt3 and PIK3 inhibitors are being used in trials.

Targeted Metabolomic Profiling in Acute Myeloid Leukemia with IDH2R140 and IDH2R172 Mutations

IntroductionAcute myeloid leukemia (AML) represents an aggressive bone marrow malignancy with diverse genetic abnormalities. The tumor cells, to support their own growth and proliferation, may alter metabolism 1) to accelerated glycolysis to provide energy/biosynthetic precursors and 2) to produce intermediates by active TCA cycle for synthesis of essential biomolecules [1]. Mutations of isocitrate dehydrogenase (IDH1/2) as frequent mutations (affecting approximately 20% of AML patients) cause the reduction of α-ketoglutarate to D-2-hydroxyglutarate instead of oxidative decarboxylation of isocitrate to α-ketoglutarate. IDH2 mutations occur almost exclusively in hematopoietic tumors. Whereas IDH2R140 mutation is frequently accompanied by normal cytogenetics and NPM1 mutations, IDH2R172 is frequently the only mutation detected in AML. In addition, IDH2R172 confers a poor prognosis in patients with AML [2, 3].The aim of this study was to characterize and uncover the differences in metabolism of AML patients with or without IDH2 mutations in diagnosis (the initial stage), in remission (after chemotherapy treatment), and before transplantation (after conditioning).MethodsSerum metabolomics profiles were generated with samples obtained at diagnosis, in remission, and before transplantation from patients (n=20) treated in the Institute of Hematology and Blood Transfusion, Prague, Czech Republic. Patients were assigned in IDH2WT (n=7) and IDH2R140/ IDH2R172 (n=7/6) groups. Targeted metabolomic profiling of 19 metabolites related mainly to TCA cycle and glycolysis was performed by LC-MS/MS.ResultsUsing non-parametric randomized block analysis of variance (Friedman test) we found significant differences in levels of 13 metabolites among treatment periods for all AML patients. Moreover, when comparing each treatment periods for all patients, we found significantly decreasing levels of 12 metabolites between the initial stage and the period before transplantation. If we considered the division of patients into groups according to IDH2 mutations, we found significantly different levels of 4 metabolites among treatment periods for samples of patients with IDH2R140,namely 2 metabolites of TCA cycle: isocitrate, succinate; and 3-hydroxybutyrate and urate. We obtained significantly different levels in the same number of other metabolites for IDH2WT. In samples of patients with IDH2R172 we found significantly different levels of 8 metabolites among treatment periods, namely 5 metabolites of TCA cycle: citrate, 2-hydroxyglutarate, succinate, fumarate, malate; 2 metabolites of glycolysis: 3-phosphoglycerate, phosphoenolpyruvate; and pyroglutamate of glutathione metabolism. Furthermore, using Nemenyi post-hoc test we ascertained significantly decreasing levels of named metabolites in IDH2R140 and IDH2R172 samples before transplantation with respect to the initial stages of AML (diagnosis).ConclusionOverall, this study identified significant changes in several metabolites of TCA cycle and glycolysis between initial and final treatment periods and also between remission and period before transplantation in AML patients. With respect to IDH2 mutations we found more significant changes in metabolites specifically in TCA cycle for IDH2R172 patients relative to IDH2R140. The results of our preliminary targeted metabolomic profiling of AML patients with IDH2 mutations are corresponding with the already described differences in morphological and genetic patterns in the patients and, moreover, support the classification of IDH2R172 as separate AML subtype. Metabolomic profiling thus seems to be a new valuable tool providing additional important information.AcknowledgmentThis work was supported by the European Regional Development Fund and the state budget of the Czech Republic (project AIIHHP: CZ.02.1.01/0.0/0.0/16_025/0007428, OP RDE, Ministry of Education, Youth and Sports), by the project of the Ministry of Health, Czech Republic, 00023736, by Grant from the Academy of Sciences, Czech Republic, P205/12/G118, and by ERDF OPPK CZ.2.16/3.1.00/24001.[1] DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. Cell Metab. 2008;7(1):11-20.[2] Rakheja D, Konoplev S, Medeiros LJ, Chen W. Hum Pathol. 2012;43(10):1541-51.[3] Meggendorfer M, Cappelli LV, Walter W, Haferlach C, Kern W, Falini B, Haferlach T. Leukemia. 2018;32(5):1249-1253. DisclosuresNo relevant conflicts of interest to declare.

MiR-425 expression profiling in acute myeloid leukemia might guide the treatment choice between allogeneic transplantation and chemotherapy

BackgroundAcute myeloid leukemia (AML) is a highly heterogeneous disease. MicroRNAs function as important biomarkers in the clinical prognosis of AML.MethodsThis study identified miR-425 as a prognostic factor in AML by screening the TCGA dataset. A total of 162 patients with AML were enrolled for the study and divided into chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT) groups.ResultsIn the chemotherapy group, patients with high miR-425 expression had significantly longer overall survival (OS) and event-free survival (EFS) compared with patients with low miR-425 expression. In multivariate analyses, high miR-425 expression remained independently predictive of a better OS (HR = 0.502, P = 0.005) and EFS (HR = 0.432, P = 0.001) compared with patients with low miR-425 expression. Then, all patients were divided into two groups based on the median expression levels of miR-425. Notably, the patients undergoing allo-HSCT had significantly better OS (HR = 0.302, P < 0.0001) and EFS (HR = 0.379, P < 0.0001) compared with patients treated with chemotherapy in the low-miR-425-expression group. Mechanistically, high miR-425 expression levels were associated with a profile significantly involved in regulating cellular metabolism. Among these genes, MAP3K5, SMAD2, and SMAD5 were predicted targets of miR-425.ConclusionsThe expression of miR-425 may be useful in identifying patients in need of strategies to select the optimal therapy between chemotherapy and allo-HSCT treatment regimens. Patients with low miR-425 expression may consider early allo-HSCT.

Region-Specific Methylation Profiling in Acute Myeloid Leukemia

Alteration of DNA methylation level in cancer diseases leads to deregulation of gene expression—silencing of tumor suppressor genes and enhancing of protooncogenes. There are several tools devoted to the problem of identification of CpG sites' demethylation but majority of them focuses on single site level and does not allow for quantification of region methylation changes. The aim was to create an adaptive algorithm supporting detection of differentially methylated CpG sites and genomic regions specific for acute myeloid leukemia. Knowledge on AML methylation fingerprint helps in better understanding the epigenetics of leukemogenesis. Proposed algorithm is data driven and does not use predefined quantification thresholds. Gaussian mixture modeling supports classification of CpG sites to several levels of demethylation. p value integration allows for translation from single site demethylation to the demethylation of gene promoter and body regions. Methylation profiles of healthy controls and AML patients were examined (GEO:GSE63409). The differences in whole genome methylation profiles were observed. The methylation profile differs significantly among genomic regions. The lowest methylation level was observed for promoter regions, while sites from intergenic regions were by average higher methylated. The observed number of AML related down methylated sites has not substantially exceeded the expected number by chance. Intergenic regions were characterized by the highest percentage of AML up methylated sites. Methylation enhancement/diminution is the most frequent for intergenic region while methylation compensation (positive or negative) is specific for promoter regions. Functional analysis performed for AML down methylated or extreme high up methylated genes showed strong connection to the leukemic processes.

Abstract PR09: Single-cell transcriptional profiling of acute myeloid leukemia identifies self-renewing stem cells

Abstract Acute myeloid leukemia (AML) is a lethal cancer with a survival of less than 50%. Standard cytotoxic therapies frequently induce complete remission, but patients frequently relapse and die of their disease. Leukemia stem cells (LSCs) are the leukemia cells with self-renewal potential and ability to recapitulate the disease. Most anticancer therapies are designed to inhibit proliferation. Yet, in hematopoietic stem cells, the mechanisms of proliferation are distinct from self-renewal (Li et al. Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to target LSCs and eradicate AML. Our goal is define the self-renewing LSCs in order to develop therapeutics that target them and eliminate AML relapse. We previously showed that activated NRAS (NRASG12V) facilitates self-renewal in the LSC-enriched subpopulation of a transgenic mouse model of AML (Mll-AF9/NRASG12V) (Sachs et al. Blood 2014; Kim et al. Blood 2009). We hypothesize that self-renewal capacity and the NRAS-activated pathways required for self-renewal are limited to a subpopulation of LSCs. We used single-cell RNA sequencing to identify the self-renewing cells among the LSC-enriched subgroup in this model (Mac1LowKit+Sca1+, “MKS”). We identified three discrete transcriptional profiles among the LSC-enriched subpopulation and found that that two of these profiles (Profile 1 and Profile 2) are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted the MKS LSCs based on CD36 and CD69 expression. Sorted LSC subsets were transplanted into recipient mice to compare their ability to transfer leukemia as a measure of their self-renewal capacity. We found that MKS-CD36-CD69+ cells (consistent with Profile 1) rapidly transferred leukemia with high penetrance in 20 of 22 mice. In contrast, MKS-CD36+CD69- cells (Profile 2) failed transfer leukemia in most mice; only 2 of 25 of these mice developed AML (p &amp;lt; 0.004). In our previous work, we demonstrated that the NRASG12V-activated self-renewal gene expression profile that we identified in our murine model was expressed in human AML, suggesting that the gene expression behavior of LSCs from this model may recapitulate the gene expression behavior of human LSCs (Sachs et al. Blood 2014). In order to determine if the single-cell transcriptional profiles of our murine AML can be found in primary human AML precursors, we performed single-cell RNA sequencing on CD34+ human AML cells obtained from a diagnostic bone marrow specimen. Analogous to our murine model, we found that these human AML cells express 2 distinct single-cell transcriptional profiles and they differentially express RAS-activated gene expression profiles and profiles of hematopoietic differentiation. Next, we used our murine single-cell self-renewal transcriptional profile to define a 96-gene panel consisting of 88 genes from this profile and 8 housekeeping genes. We sorted primary, diagnostic human AML cells for leukemia stem and progenitor cells (CD34+CD38-) and performed single-cell qPCR on these cells using our 96-gene panel. We found that a subset of these cells preferentially expresses Profile 1, the self-renewal gene expression profile that we identified in our murine model, and another subset preferentially expresses Profile 2 (the profile associated with no leukemia-reconstituting capacity). In these experiments, we use a murine model of AML to define the LSC self-renewal gene expression profile at the single-cell level and functionally validate this profile in vivo. Analogous to the murine model, a subset of human AML stem and progenitor cells expresses this LSC self-renewal gene expression profile at the single-cell level. These data suggest that single-cell gene expression profiling can delineate leukemia cells with true self-renewal capacity. This abstract is also being presented as Poster 43. Citation Format: Klara E. Noble-Orcutt, Karen Sachs, Connor Navis, Alexandria Hillesheim, Ian Nykaza, Rebecca S. LaRue, Conner Hansen, Ngoc Ha, Michael A. Linden, David A. Largaespada, Zohar Sachs. Single-cell transcriptional profiling of acute myeloid leukemia identifies self-renewing stem cells [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR09.

Genome-wide characterization of lncRNAs in acute myeloid leukemia.

Long noncoding RNAs (lncRNAs) are a large family of noncoding RNAs that play a critical role in various normal bioprocesses as well as tumorigenesis. However, the expression patterns and biological functions of lncRNAs in acute leukemia have not been well studied. Here, we performed transcriptome-wide lncRNA expression profiling of acute myeloid leukemia (AML) patient samples, along with non-leukemia control hematopoietic samples. We found that lncRNAs were differentially expressed in AML samples relative to control samples. Notably, we identified that lncRNAs upregulated in AML (relative to the control samples) are associated with a lower degree of DNA methylation and a higher ratio of being bound by transcription factors such as SP1, STAT4, ATF-2 and ELK-1 compared with those downregulated in AML. Moreover, an enrichment of H3K4me3 and a depletion of H3K27me3 were observed in upregulated lncRNAs in AML. Expression patterns of three types of lncRNAs (antisense, enhancer and intergenic lncRNAs) have previously been characterized. Of the identified lncRNAs, we found that high expression level lncRNA LOC285758 is associated with the poor prognosis in AML patients. Furthermore, we found that LOC285758 regulates proliferation of AML cell lines by enhancing the expression of HDAC2, a key factor in carcinogenesis. Collectively, our study depicts a landscape of important lncRNAs in AML and provides novel potential therapeutic targets and prognostic markers for AML treatment.

Single-cell genomic profiling of acute myeloid leukemia for clinical use: A pilot study.

Although bulk high-throughput genomic profiling studies have led to a significant increase in the understanding of cancer biology, there is increasing awareness that bulk profiling approaches do not completely elucidate tumor heterogeneity. Single-cell genomic profiling enables the distinction of tumor heterogeneity, and may improve clinical diagnosis through the identification and characterization of putative subclonal populations. In the present study, the challenges associated with a single-cell genomics profiling workflow for clinical diagnostics were investigated. Single-cell RNA-sequencing (RNA-seq) was performed on 20 cells from an acute myeloid leukemia bone marrow sample. Putative blasts were identified based on their gene expression profiles and principal component analysis was performed to identify outlier cells. Variant calling was performed on the single-cell RNA-seq data. The present pilot study demonstrates a proof of concept for clinical single-cell genomic profiling. The recognized limitations include significant stochastic RNA loss and the relatively low throughput of the current proposed platform. Although the results of the present study are promising, further technological advances and protocol optimization are necessary for single-cell genomic profiling to be clinically viable.

Clinical and Hematological Profile of Acute Myeloid Leukemia (AML) Patients of Sindh

In Acute Myeloid Leukemia (AML) patients, a diversity of clinical and hematological parameters has been examined for possible value in calculating treatment response and survival. The study was conducted at National Institute of Bone Diseases, Karachi (NIBD), Sind, Pakistan, a tertiary care and teaching hospital, affiliated with Liaquat University of Medical and Health Sciences, Jamshoro. This study included 107 cases of de novo AML, both male and female patients of ages between 10-60 years were selected randomly. The clinical features included fever, weakness, loss of weight, vomiting, bleeding gums, liver and spleen enlargement, lymph node enlargement. The Laboratory investigations included CBC (Complete Blood Count) specifically heamoglobin percentage, total leucocyte count, platelet count and blast count: peripheral blood smears, bone marrow aspiration biopsy, trephine biopsy, cytochemistry (Myeloperoxidase Staining, Immunohistochemistry (CD markers)) and DNA extraction for genetic sequencing. Percentage, validity and cumulative index were calculated of all parameters. It was found that about 70% of AML patients were adults and FAB subtype M2 of acute myeloid leukemia was more prevalent than other subtypes, followed by AML, M1.

Single Cell-Level Signaling Profiling of Acute Myeloid Leukemia Following Treatment with Axl Kinase Inhibitor BGB324

Axl is a member of the Tyro3, Axl, Mer (TAM) receptor tyrosine kinase family that regulate a wide range of cellular functions, including cell survival, proliferation, migration/invasion and adhesion. Axl has been shown to play a key role in the survival and metastasis of many tumors, and has also been found to be upregulated and constitutively active in human AML. Indeed, Axl has been reported as an independent prognostic marker and a potential novel therapeutic target in AML.BGB324 is a first-in-class highly selective small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in clinical safety studies in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in phase I b clinical trials for AML and non-small cell lung cancer.In this study, we use phosphoflow cytometry to measure changes in signal transduction nodes in single AML cells treated with BGB324. We are applying this approach to monitor signaling profiles in primary AML cells harvested from patients undergoing BGB324 treatment.Results:The human AML cell line MOLM13 was treated in vitro with BGB324 (0.5 and 1µM for 1 hour) and analyzed for signal transduction changes by phosphoflow cytometry. We found a significant reduction in phosphorylation of Axl (pY779), Akt(pS473), Erk1/2(pT202/Y204) and PLCɣ1(pY783).Next we established a systemic MOLM13 preclinical AML model in NOD/SCID mice. The mice were treated with 25 or 50 mg/kg BGB324 until moribund (up to 16 days). We found a dose-dependent and significant increase in overall survival in BGB324-treated mice. We further investigated intracellular signaling in BGB324-treated cells in vivo. Mice carrying systemic AML disease (MOLM13) were treated with BGB324 at 50mg/kg for 4 days, and we monitored CD33/45-positive MOLM13 cells harvested from spleen and bone marrow by flow cytometry. BGB324-treated mice showed a significant reduction in pErk and pPLCɣ1 relative to mice in the control group.PBMCs from peripheral blood of AML patients treated with BGB324 400 mg x1 at day 1 and 2, and thereafter 100 mg daily were collected for single cell signal profiling of signal transduction changes by conventional flow cytometry (phospho-flow) and mass cytometry (CyTOF). Preliminary phopho-flow analyses show decrease of pAkt(T308) and pPLCgamma1(Y783) in one patient. Further analyses are ongoing and will be presented. [Display omitted] [Display omitted] [Display omitted] DisclosuresHellesøy:BerGenBio AS: Other: Previous employee. Stock option holder. Wnuk-Lipinska:BerGenBio AS: Employment. Boniecka:BerGenBio AS: Employment. Nævdal:BerGenBio AS: Employment. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding. Cortes:Teva: Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Lorens:BerGenBio AS: Employment, Equity Ownership. Micklem:BerGenBio AS: Employment, Equity Ownership. Gausdal:BerGenBio AS: Employment. Gjertsen:Haukeland University Hospital: Research Funding.

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs.

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.

Mass Cytometric Functional Profiling of Acute Myeloid Leukemia Defines Cell-Cycle and Immunophenotypic Properties That Correlate with Known Responses to Therapy.

Acute myeloid leukemia (AML) is characterized by a high relapse rate that has been attributed to the quiescence of leukemia stem cells (LSC), which renders them resistant to chemotherapy. However, this hypothesis is largely supported by indirect evidence and fails to explain the large differences in relapse rates across AML subtypes. To address this, bone marrow aspirates from 41 AML patients and five healthy donors were analyzed by high-dimensional mass cytometry. All patients displayed immunophenotypic and intracellular signaling abnormalities within CD34(+)CD38(lo) populations, and several karyotype- and genotype-specific surface marker patterns were identified. The immunophenotypic stem and early progenitor cell populations from patients with clinically favorable core-binding factor AML demonstrated a 5-fold higher fraction of cells in S-phase compared with other AML samples. Conversely, LSCs in less clinically favorable FLT3-ITD AML exhibited dramatic reductions in S-phase fraction. Mass cytometry also allowed direct observation of the in vivo effects of cytotoxic chemotherapy. The mechanisms underlying differences in relapse rates across AML subtypes are poorly understood. This study suggests that known chemotherapy sensitivities of common AML subsets are mediated by cell-cycle differences among LSCs and provides a basis for using in vivo functional characterization of AML cells to inform therapy selection.

Integrating Genomics Into Prognostic Models for AML

The use of genomic profiling in acute myeloid leukemia (AML) has led to an improved understanding of disease pathogenesis. Genomic profiling has given rise to fundamental observations about the biology of AML and has served to better define clinical outcomes for patients based on somatic mutational status. As additional mutations are identified with a known or postulated role in AML pathogenesis, the challenge ahead will be learning how to integrate these findings into clinical practice in such a way that they have a meaningful impact on patient care and, ultimately, on patient outcomes. Potential goals include using genomic information for refined risk stratification and clinical decision making, and to identify genetic lesions that guide the use of molecularly targeted therapies. The development of next-generation sequencing technologies has made genomic profiling a viable option for use in clinical practice because it can provide robust, high-coverage sequencing data for multiple genes in 1 assay, within a clinically reasonable time frame. The present article discusses recent candidate gene sequencing studies, the development of prognostic models based on these studies, and the current and potential future uses of next-generation sequencing technologies in making treatment decisions for patients with AML.

Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia

Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia

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.

Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML.MethodsWe performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients.ResultsWe identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P=0.001 for FLT3-ITD, P=0.009 for MLL-PTD, P=0.05 for ASXL1, and P=0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P=0.05 for CEBPA and P=0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P=0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P=0.67).ConclusionsWe found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.)

Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia

Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia