Abstract
FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.
Highlights
FMS-like tyrosine kinase 3 receptor (FLT3) receptor mutations are among the most common somatic mutations in acute myeloid leukemia (AML), with FLT3 internal tandem duplications (ITDs) occurring in 20–35% of adult AML [1,2,3,4,5,6,7] and,5–15% of pediatric AML [8,9,10]
None of the variables examined in either study, including age, gender, race, white blood cell count or blast percent at presentation, or NPM1 mutational status were associated with FLT3 ITD mutational status (Table 1)
The homogeneity of FLT3 pathway signaling abnormalities observed in FLT3 ITD AML samples was verified and shown to be correlated with FLT3 ITD mutational load [27]
Summary
FMS-like tyrosine kinase 3 receptor (FLT3) receptor mutations are among the most common somatic mutations in acute myeloid leukemia (AML), with FLT3 internal tandem duplications (ITDs) occurring in 20–35% of adult AML [1,2,3,4,5,6,7] and ,5–15% of pediatric AML [8,9,10]. While the presence of FLT3 ITD is not predictive of outcome to induction chemotherapy, it has consistently been shown to be associated with significantly shorter disease-free (DFS) and relapse-free survival [11]. Molecular characterization of the FLT3 receptor and NPM1 mutational status in cytogenetically normal AML has been incorporated into clinical guidelines based on correlations of these markers with overall survival [17]. Patients with NPM1 mutation but FLT3 WT are thought to be of good prognostic risk, while FLT3 ITD but NPM1 WT patients are at high risk for disease relapse and should be considered for stem cell transplant (SCT) in first complete remission (CR). AML with NPM1 WT/FLT3 WT or carrying both NPM1 mutation and FLT3 ITD are considered to have intermediate-risk disease [17]. Clinical heterogeneity remains in these cytogenetically and molecularly defined AML subgroups [18]
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