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Recurrent somatic internal tandem duplications (ITD) in the FMS‐like tyrosine kinase 3 (FLT3) gene characterise approximately one third of patients with acute myeloid leukaemia (AML), and FLT3‐ITD mutation status guides risk‐adapted treatment strategies. The aim of this work was to characterise FLT3‐ITD variant distribution in relation to molecular and clinical features, and overall survival in adult AML patients. We performed two parallel retrospective cohort studies investigating FLT3‐ITD length and expression by cDNA fragment analysis, followed by Sanger sequencing in a subset of samples. In the two cohorts, a total of 139 and 172 mutant alleles were identified in 111 and 123 patients, respectively, with 22% and 28% of patients presenting with more than one mutated allele. Further, 15% and 32% of samples had a FLT3‐ITD total variant allele frequency (VAF), Internal tandem duplications (ITD) in the FMS‐like tyrosine kinase 3 (FLT3) gene is present in 20–30% of acute myeloid leukaemia (AML) patients. Here, we perform molecular profiling of FLT3‐ITD mutations in 263 AML patients. We provide a comprehensive overview of the heterogeneity and impact of FLT3‐ITD mutations by assessing various molecular features and the relationship with clinical features and overall survival.

With evolving knowledge and with the development and implementation of precision oncology related practices the meaning of cancer is rapidly changing. With the shift towards a molecular understanding and classification of cancer the relationship between cancer as a disease and cancer as an illness and sickness gradually dissolve. This chapter explore these developments by examining the scientific and clinical challenges that are emerging in the attempt to understand and manage FLT3 mutated acute myeloid leukaemia.

Ideas about precision medicine found its way into cancer research around the turn of the twentieth century and resulted in the imaginary of precision oncology. This chapter presents the emergence of the imaginary as well as its historical background. It furthermore argues that the imaginary is not well suited to take full biological complexity into account. This gives rise to conceptual limitations as well as practical risks as ambitions of precision are translated into practice.

We describe here a simple and efficient antibody titration approach for cell‐surface markers and intracellular cell signaling targets for mass cytometry. The iterative approach builds upon a well‐characterized backbone panel of antibodies and analysis using bioinformatic tools such as SPADE. Healthy peripheral blood and bone marrow cells are stained with a pre‐optimized “backbone” antibody panel in addition to the progressively diluted (titrated) antibodies. Clustering based on the backbone panel enables the titration of each antibody against a rich hematopoietic background and assures that nonspecific binding and signal spillover can be quantified accurately. Using a slightly expanded backbone panel, antibodies quantifying changes in transcription factors and phosphorylated antigens are titrated on ex vivo stimulated cells to optimize sensitivity and evaluate baseline expression. Based on this information, complex panels of antibodies can be thoroughly optimized for use on healthy whole blood and bone marrow and are easily adaptable to the investigation of samples from for example clinical studies. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

SUMMARYAcute myeloid leukaemia (AML) is a highly heterogeneous disease. Here, we decipher the disease composition of a single AML patient through longitudinal sampling scrutinized by high-resolution genetic and phenotypic approaches, including sequencing, immunophenotyping, ex vivo drug sensitivity testing and establishment of patient-derived xenograft models. Throughout the disease course we identified patterns of both divergent and convergent molecular evolution within the leukemic compartment. We identified at least six discrete leukaemia initiating cell populations, of which five were characterised by known recurrent mutations in AML. These populations partly correlated with immunophenotypically defined cell subsets, drug sensitivity profiles and population-specific potential for engraftment in immunodeficient mice. Our results indicate that the genetic and phenotypic development are closely intertwined, and that diversity in the leukaemic gene-environment likely influences disease trajectories.SIGNIFICANCENovel therapeutic approaches in AML are characterised by targeting molecular mechanisms thought to drive leukemogenesis, but emergent evidence suggests that intra-leukemic composition may be more diverse than previously appreciated. Through in-depth genetic and phenotypic characterization of the disease course of a single AML patient, we demonstrate a high degree of inter-individual complexity that exceeds the prevailing disease conception. The temporal molecular landscape of this patient suggests that leukemogenic transitions may not be categorically monoclonal. Patterns of converging molecular evolution further imply that higher levels of biological organisation than the molecular machinery of single cells may influence leukemogenic trajectories. Disease dynamics, relational properties and causal contribution from several levels of biological organization comes into conflict with the linear monocausal explanatory model on which precision oncology is largely built. This may have implications for current precision oncology oriented prectices, including molecular categorization, molecular therapeutic targeting and predictive models.

Little attention has been directed at untangling sex-related molecular and phenotypic differences in AML. While increased incidence and poor risk is generally associated with a male phenotype, FLT3-ITD, NPM1 and DNMT3A mutations are overrepresented in female AML. Here, we have investigated the relationship between sex and FLT3-ITD mutation status by comparing clinical data, mutational profiles, gene expression and ex vivo drug sensitivity responses in four cohorts: the Beat AML cohort, the LAML-TCGA cohort and two independent HOVON/SAKK clinical trial-associated cohorts, comprising a total of 1755 AML patients. We found that sex-associated molecular differences were prevalent in FLT3-ITD mutated AML. Co-occurrence of FLT3-ITD, NPM1 and DNMT3A mutations was overrepresented in females, while males with FLT3-ITDs were characterised by additional mutations in genes involved in RNA splicing and epigenetic modification. Female and male FLT3-ITD mutated AML had diverging expression of multiple leukemia-associated genes, as well as discrepant ex vivo drug-responses, suggestive of discrete functional properties. Surprisingly, we found significant prognostication of FLT3-ITD only in female patients. Thus, we suggest optimisation of FLT3-ITD mutation status as a clinical tool in a sex-adjusted manner. We further hypothesize that prognostication, prediction and development of therapeutic strategies in AML can be improved by including sex-specific considerations.

Acute myeloid leukemia (AML) is characterized by extensive clinical and biological heterogeneity. Despite vast advances in understanding the molecular pathology in AML during the last two decades few new AML therapeutics have been approved by the European Medicines Agency. Since 2005 only the epigenetic modulators decitabine and azacytidine, as well as histamine (plus interleukin- 2) have been approved against AML. None of these have outstanding efficiency, and decitabine and azacytdine have only been incorporated in frontline therapy of AML with limited enthusiasm. The majority of AML patients are frail and elderly, and lack of mild but effective agents for this patient cohort constitutes a major unmet need as overall survival remains poor. Along with the recent advancements in the molecular characterization of AML, numerous targeted therapies have been tested in clinical trials. In this review, we discuss the biological rationale for a selection of these novel therapeutic approaches, including epigenetic modifiers, agents targeting signalling pathways and inhibitors of nuclear-cytoplasmic shuttling. Further we discuss some of the possible shortcomings in current trial design that could explain the apparent incoherence between our improved biological knowledge and the lack of progress in therapy development of AML.

Internal tandem duplications (ITDs) of the gene encoding the Fms-Like Tyrosine kinase-3 (FLT3) receptor are present in approximately 25% of patients with acute myeloid leukemia (AML). The mutation is associated with poor prognosis, and the aberrant protein product has been hypothesized as an attractive therapeutic target. Various tyrosine kinase inhibitors (TKIs) have been developed targeting FLT3, but in spite of initial optimism the first generation TKIs tested in clinical studies generally induce only partial and transient hematological responses. The limited treatment efficacy generally observed may be explained by numerous factors; extensively pretreated and high risk cohorts, suboptimal pharmacodynamic and pharmacokinetic properties of the compounds, acquired TKI resistance, or the possible fact that inhibition of mutated FLT3 alone is not sufficient to avoid disease progression. The second-generation agent quizartinb is showing promising outcomes and seems better tolerated and with less toxic effects than traditional chemotherapeutic agents. Therefore, new generations of TKIs might be feasible for use in combination therapy or in a salvage setting in selected patients. Here, we sum up experiences so far, and we discuss the future outlook of targeting dysregulated FLT3 signaling in the treatment of AML.