Prevalence Of FLT3 Mutations In Acute Myeloid Leukemia: A Multicenter Latin America Study
Authors: Cuervo-Sierra Jorge , Gómez-Almaguer David , Jaime-Pérez Jose Carlos , Martínez-Hernández Ramón Alejandro , García Sepúlveda Ricardo David , Sánchez-Cárdenas Mónica , Ortiz-López Rocio , Ignacio-Ibarra Gregorio , Muciño-Hernández Gabriel , Arana-Trejo Rosa Maria , Ruiz-Arguelles Guillermo J. , Ruiz-Delgado Guillermo J. , Lutz-Prestno Julia , Jiménez-Mejía Angélica , Vásquez-Palacio Gonzalo , Camargo-Guerrero Mauricio 
Published: October 2019

Abstract
In acute myeloid leukemia (AML), FLT3 mutations are associated with a poor prognosis, particularly the internal tandem duplication (ITD/FLT3). In Latin America there are few epidemiological data about these mutations.This study assessed the prevalence of FLT3 mutations in patients with AML at four reference hematology centers from Latin America. We included 138 samples of patients attending the Hematology Service of three Mexican University Hospitals (Monterrey,México D.F. and Puebla) and one Colombian center (Medellín) with a diagnosis of AML from different morphologic subgroups according to the French-American-British classification from 2006 to 2011. AML was diagnosed by morphology according to the FAB classification, by immunohistochemical staining and/or by immunophenotype according to each particular case. For sample processing DNA was extracted from peripheral blood or bone marrow with the automatic Maxwell®16 System (Promega Corporation, Madison, WI) using the principle of cellular lyses and binding nucleic acids to magnetized silice particles or the QIAAmp DNA Blood Kit (QIAGEN, Mexico City). The quality and DNA concentration was evaluated with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). Then internal tandem duplication and kinase domain mutations detection was performed with the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA), through amplification of exons 14,15 with specific primers of FLT3 gen region, using the Seeplex® FLT3 Genotyping kit (Seegene, Rockville, MD, USA) or according to Kottaridis et al (1). Later, an electrophoretic analysis of the amplified products was made in a 2% agarose gel stained with ethidium bromide or in polyacrylamide gel electrophoresis (PAGE) and were observed by transilumination. In the Puebla samples the products were analyzed by capillary electrophoresis (ABI3130, Applied Biosystems, Foster City, CA). For detection of D835X mutations the exon 20 amplicom was subjected to digestion with EcoRV and analyzed by 4.5% PAGE. The patients were cytogenetically classified into three risk groups: favorable, intermediate, and adverse.

Results
We analyzed 138 samples of AML patients and found FLT3 mutations in 28 patients, for a prevalence of 20.3%. The median age was 47 years (5-96). Only four patients had the KD FLT3 mutation (3% of total population). The FLT3 mutation positive group was older than the negative (47 vs. 39 years), had higher WBC/mm3 (66.0 vs. 56.4 x 109/dl), higher hemoglobin values (9.3 vs. 8.6 g/dl), and lower platelet counts (72.6 vs. 92.5 x109/dl), although there were not statistically significant differences. Thirteen patients had AML M2, nine the monocytic variety, four had M3 and two M1. On cytogenetics 25% , 62.5% and 12.5% had favorable, intermediate and unfavorable risk karyotypes respectively. The rate response to standard Induction Chemotherapy was 78.3 % for the FLT3 positive group vs. 74.1 % for the non-mutated group. Nineteen of 28 patients (67.8%) with FLT3 mutations died, compared to 47 of 100 (42.72%) in those without the mutation. The median survival was 4.9 months for the FLT3 mutated group vs 20.4 months for the FLT3 negative group, P= 0.009. The cytogenetic intermediate risk group (62.5%) was further analyzed and no statistically significant difference in overall survival between FLT3 non-mutated and FLT3 mutated patients was found (P= 0.22). Younger patients (<55 years) had a higher mortality in the FLT3 positive group (P = 0.023).The presence or absence of the FLT3 mutation in patients who had the morphologic subtype M3 did not impact mortality (P = 0.28), but in non M3 subtypes, it did (P= 0.017). As conclusion, in this Latin American population the FLT3 mutation conferred a bad prognosis.

References
1. Kottaridis PD, Gale RE,Frew ME et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds importatnt prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood.2001; 98: 1752-1759
2. Emerenciano M, Meneses J, Vasquez ML et al, Brazilian Collaborative Study Groupof Infant Acute L. Clinical relevance of FLT3 gene abnormalities in Brazilian patients with infant leukemia.Leuk Lymphoma. 2008; 49 (12):2291-2297.

Disclosures:
No relevant conflicts of interest to declare.