Identification of Driver and Passenger Mutations of FLT3 by High-Throughput DNA Sequence Analysis and Functional Assessment of Candidate Alleles

SummaryMutations in the juxtamembrane and kinase domains of FLT3 are common in AML, but it is not known whether alterations outside these regions contribute to leukemogenesis. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in AML patients without known FLT3 mutations and experimentally tested the consequences of each candidate leukemogenic allele. This approach identified gain-of-function mutations that activated downstream signaling and conferred sensitivity to FLT3 inhibition and alleles that were not associated with kinase activation, including mutations in the catalytic domain. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation and underscore the importance of functional studies to distinguish “driver” mutations underlying tumorigenesis from biologically neutral “passenger” alterations

ASPP2 mRNA expression in acute leukemia.

<p>qRT-PCR based mRNA expression levels are displayed after normalizing to a healthy blood donor (set as 1) on a logarithmic scale. Cohort analysis reveals significant lower ASPP2 levels for an acute leukemia population compared to a healthy peripheral blood and bone marrow donor cohort (A). Comparison of prognostic risk groups confirms lower ASPP2 expression levels for the good-risk as well as higher-risk cohort when compared to a healthy donor population – whereas attenuated ASPP2 expression levels are more pronounced and statistically significantly different for the higher-risk cohort (B). Analysis of therapy responders (i.e. achievement of complete remission after one cycle of induction chemotherapy) demonstrates significantly lower ASPP2 levels for the therapy-failure population when compared to the responder cohort (including good-/higher-risk pts.) (C). ROC curve analysis defining the ideal threshold to distinguish a definite non-responding sub-population is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080193#pone-0080193-g001" target="_blank">figure 1D</a> (i.e. patients with attenuated ASPP2 expression levels ≤0.8 are likely not to respond to induction chemotherapy (with no single falsely positive tested patient at this threshold). P-values are provided as indicated by an asterix. Patient characteristics, including definitions of the prognostic risk groups, are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080193#pone-0080193-t001" target="_blank">Table 1</a> and 2.</p

Patient Characteristics (mRNA Assay): Good-Risk Cohort.

<p>The prognostic good-risk population is segregated from the total cohort according to ELN-guidelines²²; this includes Core-binding Factor Leukemia (CBFL), Acute Promyelocytic Leukemia (APL) and Nucleophosmin1-mutated AML.</p><p>The CBFL group includes KIT-mutated cases, which have an adverse prognosis in some studies³⁸. Induction therapy was based on anthracycline plus cytarabine chemotherapy. Complete Remissions include cases with complete remission with incomplete hematopoietic recovery (CRi).</p