Study on the Pathogenesis and Clinical Significance of FLT3 and C-KIT Mutations in Core-Binding Factor AML

Objective

1. To analyze the clinical and laboratorial characteristics of 176 patients with de novo CBF-AML who received cytogenetics and molecular genetics analysis in the First Affiliated Hospital of Soochow University.

2. To clone two kinds of rare FLT3-TKD mutants in CBF-AML and explore their roles in leukemogenesis via in vitro experiments.

3. To investigate the frequency of C-KIT mutation and prognosis in t(8;21) AML with trisomy 4.

Methods

1. Cytogenetic studies were performed on 176 patients' BM samples at their diagnosis. The karyotype obtained was described in accordance with the recommendations of the ISCN 2005.

2. PCR was performed to acquire two rare FLT3-TKD mutations which was afterwards inserted to a transfer plasmid-LV5, respectively. The mutants were transfected into BaF3 cells by DNA-calcium phosphate, respectively.

3. This retrospective study was conducted from February 2005 to January 2013. 145 AML patients with t(8;21) were included in the present study.

Results

1. The clinical and laboratorial characteristics of 176 newly diagnosed patients with CBF-AML

We investigated the clinical and laboratorial characteristics of 176 CBF-AML patients including 139 patients with RUNX1-RUNX1T1 and 37 with CBFβ-MYH11 fusion transcripts. The patients consisted of 101 males and 75 females with a median age of 35 (range, 11-77).

174 cases had cytogenetics analyzed successfully and 84 cases had additional chromosomal aberration. The most frequent additional chromosomal aberration was loss of X/Y identified in 55 (31.3%) patients, followed by del(9q) (n = 10, 5.7%), trisomy 22 (n = 10, 5.7%), trisomy 4 (n = 7, 4.0%) and trisomy 8 (n = 2, 1.1%).

Overall, 73 patients (41.5%) had no detected mutations, a single gene was mutated in 97 (55.1%) patients, and more than one gene in six (3.4%) patients. The frequency of C-KIT mutation was 34.7% (n = 61) and most of the mutation occurred in exon 17 (90.2%, 55/61). FLT3 mutation was presented in 12.5% of the patients (22/176), among which FLT3-ITD was 40.9% (9/22) and FLT3-TKD was 59.1% (13/22), respectively. Mutation of FLT3-TKD was predominantly occurred in exon 20 (84.6%, 11/13), resulting in variable amino acid changes (D835Y, D835E or D835H). More t(8;21) AML patients suffered C-KIT mutation than patients with inv(16) (39.6% vs. 16.2%) (P<0.01). We also identified two rare FLT3-TKD mutants, namely FLT3 N676K and FLT3 H671Q, respectively.

2. Molecular cloning and function analysis of the two rare FLT3-TKD in leukemogenesis

The mutated FLT3 was cloned and ligated to the LV5 vector plasmid. BaF3 cell lines stably expressing various FLT3 constructs were established. Through cell viability detection, we observed that the mutants could accelerate the proliferation of BaF3 cell and make the cells grow cytokine-independently to some extent.

3. Frequency of C-KIT mutation and prognosis in t(8;21) AML with trisomy 4

In 145 t(8;21) AML patients with trisomy 4, 91.7% (11/12) of patients were identified with C-KIT mutation, which was significantly higher than those without trisomy 4 (26.3% for the latter, P < 0.01). More importantly, the follow-up data showed that the patients with trisomy 4 correlated with a lower 3-year overall survival (OS) (15% vs. 56%, P < 0.01) and disease-free survival (DFS) (0% vs. 51%, P < 0.01) when compared with patients without trisomy 4. Furthermore, the subgroup of t(8;21)AML patients with both trisomy 4 and C-KIT mutation had a worse OS and DFS than the patients without trisomy 4 and C-KIT mutation (P < 0.05).

Conclusion

1. Loss of X/Y chromosome is the most frequent additional chromosomal aberration and C-KIT mutation is the most frequent gene mutation in CBF-AML, especially in t(8;21) AML. We also identified two rare FLT3-TKD mutants, namely FLT3 N676K and FLT3 H671Q.

2. We successfully cloned the mutants and constructed the stable transfected cell lines. We observed that the mutants could accelerate the proliferation of BaF3 cell and make the cells grow cytokine-independently to some extent.

3. We firstly identified high frequency of C-KIT mutation in t(8;21) AML patients with trisomy 4. And patients with trisomy 4 demonstrate an adverse implication in t(8;21) AML patients, which indicates it may define a distinctive subtype of t(8;21) AML.