Heterogeneity within AML with CEBPA mutations; only CEBPA double mutations, but not single CEBPA mutations are associated with favourable prognosis

CCAAT/enhancer binding protein alpha (CEBPA) mutations in AML are associated with favourable prognosis and are divided into N- and C-terminal mutations. The majority of AML patients have both types of mutations. We assessed the prognostic significance of single (n=7) and double (n=12) CEBPA mutations among 224 AML patients. Double CEBPA mutations conferred a decisively favourable overall (P=0.006) and disease-free survival (P=0.013). However, clinical outcome of patients with single CEBPA mutations was not different from CEBPA wild-type patients. In a multivariable analysis, only double – but not single – CEBPA mutations were identified as independent prognostic factors. These findings indicate heterogeneity within AML patients with CEBPA mutations

Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia

Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML

A one-mutation mathematical model can explain the age incidence of acute myeloid leukemia with mutated nucleophosmin (NPM1).

Acute myeloid leukemia with mutated NPM1 gene and aberrant cytoplasmic expression of nucleophosmin (NPMc(+) acute myeloid leukemia) shows distinctive biological and clinical features. Experimental evidence of the oncogenic potential of the nucleophosmin mutant is, however, still lacking, and it is unclear whether other genetic lesion(s), e.g. FLT3 internal tandem duplication, cooperate with NPM1 mutations in acute myeloid leukemia development. An analysis of age-specific incidence, together with mathematical modeling of acute myeloid leukemia epidemiology, can help to uncover the number of genetic events needed to cause leukemia. We collected data on age at diagnosis of acute myeloid leukemia patients from five European Centers in Germany, The Netherlands and Italy, and determined the age-specific incidence of AML with mutated NPM1 (a total of 1,444 cases) for each country. Linear regression of the curves representing age-specific rates of diagnosis per year showed similar slopes of about 4 on a double logarithmic scale. We then adapted a previously designed mathematical model of hematopoietic tumorigenesis to analyze the age incidence of acute myeloid leukemia with mutated NPM1 and found that a one-mutation model can explain the incidence curve of this leukemia entity. This model fits with the hypothesis that NPMc(+) acute myeloid leukemia arises from an NPM1 mutation with haploinsufficiency of the wild-type NPM1 allele

Analogue peptides for the immunotherapy of human acute myeloid leukemia

Accepted manuscript. The final publication is available at: http://link.springer.com/article/10.1007%2Fs00262-015-1762-9The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies

Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the FLT3 internal tandem duplication

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin−, HLA-DR+, CD11c+, CD86+) and plasmacytoid DC (pDC: Lin−, HLA-DR+, CD123+, CD86+) in diagnostic samples of 47 FLT3-ITD− and 40 FLT3-ITD+ AML patients. The majority of ITD+ AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD− AML samples. Interestingly, mDCs and pDCs sorted out from ITD+ AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD+ AML (n = 11) and ITD− AML (n = 12) samples analyzed for mixed lineage DCs (Lin−, HLA-DR+, CD11c+, CD123+). ITD+ AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression

Distinct Patterns of DNA Damage Response and Apoptosis Correlate with Jak/Stat and PI3Kinase Response Profiles in Human Acute Myelogenous Leukemia

BACKGROUND:Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML) disease subtypes based on survival, DNA damage response and apoptosis pathways. METHODOLOGY AND PRINCIPAL FINDINGS:Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct "DNA damage response (DDR)/apoptosis" profiles: 1) AML blasts with a defective DDR and failure to undergo apoptosis; 2) AML blasts with proficient DDR and failure to undergo apoptosis; 3) AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the "DDR/apoptosis" proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents. CONCLUSIONS AND SIGNIFICANCE:Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in combination with chemotherapy

Prognostic or predictive role of gene mutations in chronic lymphocytic leukemia: results from the pivotal phase III study COMPLEMENT1

Next generation sequencing studies in Chronic lymphocytic leukemia (CLL) have revealed novel genetic variants that have been associated with disease characteristics and outcome. The aim of this study was to evaluate the prognostic value of recurrent molecular abnormalities in patients with CLL. Therefore, we assessed their incidences and associations with other clinical and genetic markers in the prospective multicenter COMPLEMENT1 trial (treatment naive patients not eligible for intensive treatment randomized to chlorambucil (CHL) vs. ofatumumab-CHL (O-CHL)). Baseline samples were available from 383 patients (85.6%) representative of the total trial cohort. Mutations were analyzed by amplicon-based targeted next generation sequencing (tNGS). In 52.2% of patients we found at least one mutation and the incidence was highest in NOTCH1 (17.0%), followed by SF3B1 (14.1%), ATM (11.7%), TP53 (10.2%), POT1 (7.0%), RPS15 (4.4%), FBXW7 (3.4%), MYD88 (2.6%) and BIRC3 (2.3%). While most mutations lacked prognostic significance, TP53 (HR2.02,p<0.01), SF3B1 (HR1.66,p=0.01) and NOTCH1 (HR1.39,p=0.03) were associated with inferior PFS in univariate analysis. Multivariate analysis confirmed the independent prognostic role of TP53 for PFS (HR1.71,p=0.04) and OS (HR2.78,p=0.02) and of SF3B1 for PFS only (HR1.52,p=0.02). Notably, NOTCH1 mutation status separates patients with a strong and a weak benefit from ofatumumab addition to CHL (NOTCH1wt:HR0.50,p<0.01, NOTCH1mut:HR0.81,p=0.45). In summary, TP53 and SF3B1 were confirmed as independent prognostic and NOTCH1 as a predictive factor for reduced ofatumumab efficacy in a randomized chemo (immune)therapy CLL trial. These results validate NGS-based mutation analysis in a multicenter trial and provide a basis for expanding molecular testing in the prognostic workup of patients with CLL. ClinicalTrials.gov registration number: NCT0074818

Functional Characterization of FLT3 Receptor Signaling Deregulation in Acute Myeloid Leukemia by Single Cell Network Profiling (SCNP)

Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative.Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management.These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations

Current findings for recurring mutations in acute myeloid leukemia

The development of acute myeloid leukemia (AML) is a multistep process that requires at least two genetic abnormalities for the development of the disease. The identification of genetic mutations in AML has greatly advanced our understanding of leukemogenesis. Recently, the use of novel technologies, such as massively parallel DNA sequencing or high-resolution single-nucleotide polymorphism arrays, has allowed the identification of several novel recurrent gene mutations in AML. The aim of this review is to summarize the current findings for the identification of these gene mutations (Dnmt, TET2, IDH1/2, NPM1, ASXL1, etc.), most of which are frequently found in cytogenetically normal AML. The cooperative interactions of these molecular aberrations and their interactions with class I/II mutations are presented. The prognostic and predictive significances of these aberrations are also reviewed

Expression of myeloperoxidase and gene mutations in AML patients with normal karyotype: double CEBPA mutations are associated with high percentage of MPO positivity in leukemic blasts.

The percentage of myeloperoxidase (MPO)-positive blast cells is a simple and highly significant prognostic factor in AML patients. It has been reported that the high MPO group (MPO-H), in which >50% of blasts are MPO activity positive, is associated with favorable karyotypes, while the low MPO group (≤50% of blasts are MPO activity positive, MPO-L) is associated with adverse karyotypes. The MPO-H group shows better survival even when restricted to patients belonging to the intermediate chromosomal risk group or those with a normal karyotype. It has recently been shown that genotypes defined by the mutational status of NPM1, FLT3, and CEBPA are associated with treatment outcome in patients with cytogenetically normal AML. In this study, we aimed to evaluate the relationship between MPO positivity and gene mutations found in normal karyotypes. Sixty AML patients with normal karyotypes were included in this study. Blast cell MPO positivity was assessed in bone marrow smears stained for MPO. Associated genetic lesions (the NPM1, FLT3-ITD, and CEBPA mutations) were studied using nucleotide sequencing. Thirty-two patients were in the MPO-L group, and 28 patients in the MPO-H group. FLT3-ITD was found in 11 patients (18.3%), NPM1 mutations were found in 19 patients (31.7%), and CEBPA mutations were found in 11 patients (18.3%). In patients with CEBPA mutations, the carrying two simultaneous mutations (CEBPA (double-mut)) was associated with high MPO expression, while the mutant NPM1 without FLT3-ITD genotype was not associated with MPO activity. Both higher MPO expression and the CEBPA (double-mut) genotype appeared to be associated with improved overall survival after intensive chemotherapy. Further studies are required to determine the importance of blast MPO activity as a prognostic factor, especially in CEBPA wild-type patients with a normal karyotype