Controversy exists whether internal tandem duplication of FMS-like tyrosine 
kinase 3 (FLT3-internal tandem duplication [ITD]) allelic ratio (AR) and/or 
length of the ITD should be taken into account for risk stratification of 
pediatric acute myeloid leukemia (AML) and whether it should be measured on RNA 
or DNA. Moreover, the ITD status may be of relevance for selecting patients 
eligible for FLT3 inhibitors. Here, we included 172 pediatric AML patients, of 
whom 36 (21%) harbored FLT3-ITD as determined on both RNA and DNA. Although 
there was a good correlation between both parameters ARspearman = 0.62 (95% 
confidence interval, 0.22-0.87) and ITDlengthspearman = 0.98 (95% confidence 
interval, 0.90-1.00), only AR ≥ 0.5 and length ≥48 base pairs (bps) based on RNA 
measurements were significantly associated with overall survival (AR: Plogrank = 
.008; ITDlength: Plogrank = .011). In large ITDs (>156 bp on DNA) a remarkable 
90-bp difference exists between DNA and RNA, including intron 14, which is 
spliced out in RNA. Ex vivo exposure (n = 30) to FLT3 inhibitors, in particular 
to the FLT3-specific inhibitor gilteritinib, showed that colony-forming capacity 
was significantly more reduced in FLT3-ITD-AR ≥ 0.5 compared with ITD-AR-low and 
ITD- patient samples (P < .001). RNA-based FLT3-ITD measurements are recommended 
for risk stratification, and the relevance of AR regarding eligibility for 
FLT3-targeted therapy warrants further study.