Study of the Clinical Significance of the Length of FLT3-ITD in Acute Myeloid Leukemia Patients
Authors: Castaño Tamara , Ayala Rosa , Llamas Sillero Maria Pilar , Martinez Lopez Joaquin , Salgado Rocio N , Atance Mireia , Lopez Lorenzo Jose Luiz , Tomas Jose F. , Blas Carlos , Alonso Dominguez Juan Manuel 
Published: October 2019

Abstract
Introduction
Acute myeloid leukemia (AML) is a clonal disease characterized by multiple genetic anomalies. The internal tandem duplications (FLT3-ITD mutations) in the juxtamembrane domain of the receptor occur in approximately 15-35% of de novo AML . This mutation is one of the most frequent genetic alterations, and confers an increased risk of treatment failure and a reduced disease-free survival (DFS). There are scarce data regarding the possible implication of the length of the FLT3-ITD fragment in the clinical outcome . We sought to shed light on the possible prognostic relevance of the length of the FLT3-ITD fragment in AML patients.
Methods
Twenty five patients (n=25) diagnosed with de novo AML in Hospital Universitario 12 de Octubre between 2005-2017 were included in the study. The median follow-up was 10 months from diagnosis (range 0.5-138 months). The median age was 65 years (range 1-85 years) with 72% of female patients. Bone marrow or blood samples were analyzed by fragment length analysis in an ABI3100 sequencer in order to detect the FLT3-ITD mutation.
A ROC curve was plotted to predict death for any reason. Overall survival (OS) was estimated by Kaplan-Meier curves and groups were compared by a stratified log-rank test based on allelic burden of FLT3 (≥ 0.5 vs <0.5). Additionally, Fisher test was employed in order to compare the proportion of patients with negative minimal residual disease.
Results
Twenty four percent of patients (n = 6) were acute leukemias secondary to previous hematologic neoplasms (n = 2) or prior therapies received (n = 4). In forty four percent of patients (n = 11), hyperleukocytosis was observed at diagnosis (median leukocytes 126.000/mm3, range 1400-209.000/mm3). Fifty six percent of patients (n = 14) showed a normal karyotype and only two of the individuals a complex karyotype. Thirty two percent of patients (n = 8) have achieved negative minimal residual disease at any time-point.med Seventy two percent of the patients died (n = 18). Sixty percent of the total (n=15) presented additional genetic alterations NPM1: 53% (n = 8), PML-RARA: 7% (n = 1), RUNX1/RUNXT1: 13% (n = 2), WT-1: 66% (n = 9).
The allelic burden of the ITD mutation expressed as a mutated/unmutated ratio oscillates between 0.05-1.96 with a median of 0.5. The length range of the ITD fragments analyzed ranged between 18-71 pairs of bases (pb) with a median of 38 pb.
The ROC curve showed an area under the curve of 0.58 (IC 95%: 0,37-0,78). A cutoff point of 33.5 pb was chosen with a sensitivity for prediction of death for any cause of 41.2%, specificity 87.5%, positive predictive value 87.5% and negative predictive value 41.2%.
Fifty two percent of patients (n = 13) had an allelic burden ≥ 0.5 of which 85% (n = 11) presented a length of the fragment affection ≥ 33.5 pb (total n = 17) and 48% of patients (n = 12) an allelic burden < 0.5 of whom 50% (n = 6) presented a length of the fragment affection < 33.5 pb (total n = 8). In terms of survival analysis, the P-value of the stratified test log-range was 0.3 (Figure 1). Fisher's exact test for MRD prediction was not significant either (P = 0.182).
Conclusion
To the best of our knowledge, the largest report on this topic included seventy three patients and showed that the length of the FLT3-ITD was clinically relevant. We have conducted this study and found only a trend towards significance probably due to the reduced number of patients analyzed. A multicenter collaboration is currently being carried out in order to gather a larger number of patients and try to shed light on this controversial topic.
Figure. Figure.

Disclosures
Martinez Lopez: Celgene: Research Funding, Speakers Bureau; Jansen: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; BMS: Research Funding, Speakers Bureau.