FLT3 gene internal tandem duplication (ITD) mutations in patients with acute myeloid leukemia (AML)

Reviewer: John P. Plastaras, MD, PhD
Abramson Cancer Center of the University of Pennsylvania
Last Modified: March 23, 2007

Presenter: Arana-Trejo, RM
Presenter's Affiliation: Genética, Hospital General de México and Laboratorio de OncoHematologia, SC, Mexico
Type of Session: Scientific

Background

FMS-like tyrosine kinase 3 (FLT3) is a Class III receptor tyrosine kinase, that is mainly expressed by early myeloid and lymphoid progenitor cells. FLT3 consists of 2 peptides, a 156-160 kDa transmembrane domain and a 130-143 kDa cytoplasmic domain.
FLT3 mutations are the most frequent genetic lesion seen in acute myeloid leukemia (AML). Two distinct types of activating mutations have been identified in FLT3, and both have been associated with poor clinical outcome:
internal tandem duplication (ITD) in the juxtamembrane domain which allows for ligand-independent receptor dimerization and constitutive activation
point mutation within the activation loop of the tyrosine kinase domain (e.g. D835 or I836) leading to constitutive activation
FLT3 is potentially an excellent target for therapy with tyrosine kinase inhibitors.
The goal of this study was to examine the ITD FLT3 gene mutations in patients with AML and determine the relationship between the mutations and prognosis.

Materials and Methods

Design: Single institution, prospective laboratory-based study.
Patients:
- 20 patients with de novo AML
DNA was obtained and stably stored until analysis in DNAzol solution
Mutations were studied using a single-strand conformational polymorphism (SSCP) assay
ITD mutations were identified using PCR amplification of the juxtamembrane domain from exons 11 and 12.
The PCR products were denatured and loaded into 7.5% polyacrylamide gel and after electrophoresis, the gels were stained with a silver stain.
ITD DNA fragments were detected as two bands of higher molecular weight than the wild-type FLT3 fragment (at 240 and 330 bp).
These higher molecular weight fragments were then isolated and sequenced.
Clinicopathologic correlates:
- Leukemias were karyotyped
- Complete remission and relapses were characterized
- The treatment was the same in all cases

Results

ITD mutations were detected in three of the 20 cases (15%).
Sequence analysis of ITD mutation samples showed tandem duplications, confirming and characterizing the ITD in these samples.
Outcomes in patients with ITD mutations:
- 1. Normal karyotype, complete remission >35 months
- 2. Normal karyotype, complete remission >12 months
- 3. t(6,11) translocation, relapsed after 8 months

Author's Conclusions

The incidence of FLT3/ITD positive mutations was low (15%) compared with other reports.
In AML with normal karyotype, FLT3/ITD mutations can be an important factor predicting for relapse.
The importance of FLT3/ITD mutations is being studied in a large cohort of Mexican patients.

Clinical/Scientific Implications

FLT3 is a potentially interesting “druggable” target in AML. It may be inhibited by a variety of tyrosine kinase inhibitors in development (e.g. PKC 412, AG1295, CEP-701) as well as the FDA-approved multikinase inhibitor, sorafenib.
Importantly, in patients with ITD mutations, the kinase domain is not mutated and should therefore be sensitive to inhibitors that target the kinase domain.
This study showed that the incidence of FLT3 ITD mutations are relatively infrequent in the Mexican AML population (15% compared with 20-30% in other studies), however, the number of patients is small. A more accurate estimation of the frequency of this mutation will be possible in the expanded study that the authors propose.
The good prognosis of patients with normal karyotypes and FLT3 ITD mutation is intriguing, however more patients and longer follow-up will be required to validate this claim.