Heterogeneous Patterns of FLT3 Asp⁸³⁵ Mutations in Relapsed de Novo Acute Myeloid Leukemia: A Comparative Analysis of 120 Paired Diagnostic and Relapse Bone Marrow Samples

Purpose: We analyzed Asp⁸³⁵ mutations of FLT3 on paired marrow samples at diagnosis and relapse from 120 adult patients with de novo acute myeloid leukemia (AML) to determine the role of FLT3 Asp⁸³⁵ mutation in the relapse of AML. Experimental Design: Asp⁸³⁵ mutation was analyzed by DNA PCR amplification of exon 20 of FLT3 gene followed by EcoRV digestion. All of the mutations were confirmed by sequence analysis. Mutant to wild-type allelic ratio was determined by Genescan analysis. The Expand Long Template PCR System was used to determine the allelic location of internal tandem duplication of FLT3 (FLT3/ITD) and Asp⁸³⁵ mutations. Results: Thirteen patients had Asp⁸³⁵ mutations at diagnosis, of them 8 lost the mutations at relapse, and the remaining 5 patients carrying Asp ⁸³⁵ mutations at diagnosis relapsed with the identical mutation types. Another 6 patients acquired Asp⁸³⁵ mutations at relapse. Five samples harbored both FLT3/ITD and Asp⁸³⁵ mutations that were found on different alleles by cloning analysis in the 3 patients studied. There were no differences in WBC count, French-American-British subtype, percentage of marrow blasts, or circulating blasts between patients with and without Asp ⁸³⁵ mutations, whereas the difference in the prevalence of Asp ⁸³⁵ mutations among cytogenetic/molecular subgroups was statistically significant (P = 0.025). Conclusions: The present study showed that patients with AML had heterogeneous patterns of FLT3 Asp⁸³⁵ mutations, either acquisition or loss of the mutations at relapse. Asp ⁸³⁵ mutant clone may develop as a secondary event in a subset of patients with AML.