Mutational Shift in FLT3 and NPM1-Positive Acute Myeloid Leukemia (AML) Relative to Therapy and Disease Progression
Authors: Velu Priya D , Hsiung Chris C.S. , Salafian Kiarash , Bagg Adam , Morrissette Jennifer J.D. , Luger Selina 
Published: October 2019

Abstract
Introduction:
Clonal myeloid malignancies such as AML are often characterized and treated based on specific mutation profiles identified at diagnosis. Routine use of clinical cancer next-generation sequencing (NGS) in diagnosis and disease monitoring has resulted in sequential mutation profiles of individual patients. On NGS profiling at interval follow-ups, mutations identified at diagnosis may change, or shift, through disease course. Here we track FLT3 and NPM1 mutations in the context of therapy, disease progression, clinical course, and pathology.
Methods:
Patients with AML or MDS whose blood or marrow specimens were sequenced using the Penn hematological-NGS panel (68 genes with full exon coverage) on at least 2 occasions and found at least once to have FLT3 or NPM1 mutations were included. An R script was written to track mutations and allele frequencies over time. These data were integrated with corresponding pathology and clinical data to evaluate mutation profiles in relation to disease status, therapy and progression. This study was approved by the institutional review board.
Results:
Review of clinical NGS data identified 37 patients with pathogenic mutations in FLT3 (n=15), NPM1 (n=5), or FLT3 and NPM1 (n=17) in samples characterized as de novo AML (AML), recurrent AML (rAML), myelodysplastic syndrome (MDS), recurrent MDS (rMDS), AML transformed from MDS (tAML), or AML in remission (R). Patients were divided into 3 mutational groups: FLT3 only, FLT3 and NPM1, and NPM1 only. The average number of pathogenic mutations (including mutations in genes other than FLT3 or NPM1) at initial and second testing was 1.9/3.1; 3.5/3.4 and 3.2/1.8, respectively.
Tracking of FLT3 and NPM1 allele frequencies (>10% change) revealed changes in populations of multiple subclones at different time points. In the FLT3 and FLT3 and NPM1 groups, the original FLT3 and/or NPM1 mutation often became undetectable at subsequent testing. This frequently coincided with the emergence of new clones with different mutations. Nineteen patients showed loss of an original FLT3 mutation at subsequent testing, with 11 of these patients showing emergence of a new FLT3 clone with a different mutation. All 11 patients with loss of an original NPM1 clone had a new NPM1 clone with a different mutation appear. The NPM1 only group did not show loss of the original NPM1 clone unless it was at remission. All five patients in the NPM1 only group (3 AML, 2 rAML at initial CPD testing) achieved clinical and histologic remission with concordant loss of NPM1 mutations. In four patients, the detectable mutations remaining at remission were exclusively from the original clone (DNMT3A, DNMT3A/TET2, DNMT3A/IDH2/TET2, IDH2). The fifth patient had a newly detected SRSF2 mutation in addition to the original IDH2 mutation. All five patients are alive, and do not have overt morphologic evidence of recurrent AML on bone marrow biopsies that were performed within the last year.
In four AML patients with FLT3 mutations, of which three were on FLT3 inhibitor therapy, the loss of a FLT3 mutation was followed by the emergence of a new NRAS mutation. Three patients achieved remission, but two died soon after of complications from graft versus host disease. The fourth patient achieved remission and had a detectable mutation only in IDH2, but on subsequent NGS testing showed reemergence of the previous NRAS mutation and relapsed three months later.
Conclusions:
Monitoring of FLT3 and NPM1 mutations in AML patients by NGS rather than single gene assays is crucial due to the frequent findings of other pathogenic co-mutations, mutational shift in response to disease progression and to therapy, and emergence of new disease associated clones at recurrence.

Disclosures
No relevant conflicts of interest to declare.