Impact of FLT3-ITD Mutational Burden in 469 Acute Myeloid Leukemia (AML) Patients
Authors: Hirsch Pierre , Labopin Myriam , Marzac Christophe , Mamez Anne-Claire , Tang Ruoping , Isnard Françoise , Lapusan Simona , Vekhoff Anne , Gorin Norbert C , Marie Jean-Pierre , Mohty Mohamad , Legrand Ollivier 
Published: October 2019

Abstract
Background:
FLT3-ITD mutational status is a well known prognostic factor in AML patients. However, its prognostic value in case of low mutational burden is currently under debate, especially in patients with intermediate karyotype and NPM1 mutations. Indeed some studies suggested that patients with an allelic ratio (i.e FLT3-ITD/FLT3 WT) lower than 50 % might have the same prognosis as unmutated patients.
Patients and methods:
Four hundred and sixty nine (469) patients diagnosed with AML in a single center from 1995 to 2014 and who received intensive chemotherapy based on anthracycline and cytarabine were included. Patients with t(15;17)(q24;q21) or t(9;22)(q34;q11) translocations were excluded. 134 patients underwent allogeneic stem cell transplantation. FLT3-ITD allelic burden was determined using fresh or frozen cells drawn at time of diagnosis in all patients. The mutational status for FLT3-ITD was determined using high-resolution sizing of fluorescent dye-labeled PCR amplification of exons 14-15. Beyond its contribution to mutation detection, high-resolution sizing also allows to estimate the allelic burden by measuring peak height ratios (mutant/wild type) on a fluorescence scale. Low allelic burden was defined by an allelic ratio lower than 50%. NPM1 and CEBPA mutational status were determined in most patients (n=409 and n=349 respectively). Median follow-up time for patients who were still alive was 37 months.
Results:
Median age was 56 years (range 16-81). Karyotype was unfavorable in 110 (24%) patients, favorable in 45 (10%), intermediate in 298 (63%) patients and normal in 229 (48 %). Overall, FLT3-ITD was detected in 83 (17%) patients. Twenty three patients (5%) had a low allelic ratio and 60 (13%) had a high allelic ratio. NPM1 mutation was found in 100 (24%) patients and bi-allelic CEBPA mutations in 21 (6%).
In the overall population, patients with FLT3-ITD had poorer outcome compared to other patients (OS at 3 years: 33+/-5% vs. 52 +/-3%, p =0.02). There was no difference between patients with low allelic burden or patients with high allelic burden for leukemia-free survival (7+/-7% vs. 38+/-8%, p=0.12), overall survival (25+/-5% vs. 37+/-6%, p=0.67) or CR reaching (70% in the two groups). In multivariate analysis, factors independently associated with OS were cytogenetic risk group, age, leukocyte count at diagnosis, NPM1 status, CEBPA status and FLT3-ITD status. There were no difference between patients with high vs. low allelic burden (HR=1.20 [0.59-2.42]).
Results were the same when focusing on patients with intermediate karyotype. FLT3-ITD was associated with lower survival (p=0.003), but the mutational burden was not (OS at 3 years 30+/-12% vs. 36+/-7%, for low or high burden respectively, p=0.86). In multivariate analysis, factors independently associated with OS were age, leukocyte count, NPM1 status, CEBPA status and FLT3-ITD status. Again, there were no difference between patients with high versus low allelic burden (HR=0.98[0.45-2.08].
Conclusions:
In this series, low FLT3-ITD allelic burden had a similar prognostic significance compared to a higher allelic burden. Therefore, patients with low FLT3-ITD allelic burden should likely be considered as having the same prognosis as patients with high burden, and FLT3-ITD detection, but not FLT3-ITD allelic burden, should be the major factor to be considered for therapeutic decisions.

Disclosures
No relevant conflicts of interest to declare.