Frequency of FLT3 Internal Tandem Duplications in Adult Syrian Patients with Acute Myeloid Leukemia and Normal Karyotype

Objective: Activating mutations of the fms-like tyrosine kinase 3 gene (FLT3) by internal tandem duplications (ITDs) in the juxtamembrane domain (JMD) have been reported in ~30% of adult acute myeloid leukemia (AML) patients with cytogenetically normal karyotype (CN). However, FLT3/ITD mutations are frequently accompanied with leukocytosis, high percentage of blasts in bone marrow (BM), and increased the risk of treatment failure in AML patients. FLT3-ITD mutated AML patients mainly with normal karyotype have higher relapse probability and shorter duration of complete remission (CR) after chemotherapy, so FLT3-ITD mutation is considered as an independent poor prognostic factor in AML. Methods: FLT3-ITD and FLT3-KTD were studied by polymerase chain reaction (PCR) and restriction fragment length polymorphism- PCR (RFLP-PCR) in 44 adults AML patients with cytogenetically normal karyotype (AML-CN) at diagnosis to characterize FLT3 status. The results were correlated with the prognostic factors. Results: In this study, FLT3-ITD mutations were identified in 7 (15.9%) of the 44 AML-CN patients. Among the 7 patients with FLT3/ITD mutations, 6 patients revealed a typical ITDs mutation (fragment size was 329 bp) and one patient showed untypical ITD mutation (fragment size was ~400 bp). Whereas 37 patients (61.7%) were FLT3-ITD. None of all AML-CN patients examined showed FLT3-KTD mutations. Conclusions: Our results support that FLT3-ITD are independent adverse prognostic factors for elderly AML-CN patients and are associated with low overall survival (OS), low rate of CR, high relapse rate (RR), and high percentage of BM blast at diagnosis. We concluded, FLT3 mutation analysis should be performed as a routine test in AML-CN patients.


Introduction
In patients with acute myeloid leukemia (AML) genetic diagnostics was performed in the past mainly by cytogenetics and molecular cytogenetics. In recent years also tumor markers were added, which can be uncovered only by molecular genetic methods (Kirtonia et al., 2020).
The fms-like tyrosine kinase 3 (FLT3) gene encodes a class III receptor tyrosine kinase for the FLT3 ligand, which is normally expressed in CD34+ hematopoietic stem/progenitor cells and plays a fundamental role in both normal and leukemic hematopoiesis (Stirewalt et al., 2003). Internal tandem duplications (ITDs) of the FLT3 gene (FLT3-ITDs) represent one of the most common molecular abnormalities in patients with AML, detectable in around 25%-30% of all patients (Schnittger et al., 2002;Patnaik 2018). ITDs consist of in-frame insertions of duplicated sequences localized in the juxtamembrane domain (JMD) of the FLT3 molecule. Their presence results in a constitutive, ligand independent activation of the tyrosine kinase activity of the FLT3 receptor; this is responsible for abnormal proliferation and differentiation of leukemia stem cells (Stirewalt et al., 2003). Constitutive activation of kinase domain is due to disruption of autoinhibitory interaction between JMD and the activation loop in AML, which normally stabilizes inactive kinase, and at the same time protects ATP binding pocket (Griffith et al., 2004;Chan 2011). Also, FLT3-ITDs protect leukemia cells from damaging chemotherapeutic agents (Lagunas-Rangel and Chávez-Valencia, 2017).
Presence of FLT3-ITDs has been associated with an increase an initial peripheral white blood cell (WBC) count, a high percentage of bone marrow blast cells, reduced disease-free survival (DFS) and overall survival (OS), and increased relapse rate (RR) with an overall adverse prognosis. However, rate of complete remission (CR) was not significantly affected (Kottaridis et al., 2001;Yanada et al. 2005;Canaani et al., 2018). Thus, a prognostic significance of FLT3/ITDs has been suggested (Stirewalt et al., 2006) and according to National Comprehensive Cancer Network and European LeukemiaNet (ELN) 2017, cases with FLT3-ITD mutation plus cytogenetically normal karyotype have a poor prognosis.
Whereas, the less frequent FLT3-TKD mutations are of unclear prognostic relevance have been observed in ~7% of AML patients (Bacher et al., 2008;Yamamoto et al., 2001;Kim 2010). However, the incidence of ITD and TKD mutations in FLT3 vary slightly according to age, clinical risk groups, and cytogenetic profile (Levis, 2013). Moreover, adult AML patients usually have a higher prevalence of FLT3-ITDs than pediatric patients. This observation may partially explain why adult AML has a poorer clinical outcome than pediatric AML (Gregory et al., 2009). Many clinical studies have shown that patients with an ITD at diagnosis have frequent disease relapses and a short duration of survival when compared to patients without an ITD (Schnittger et al., 2002;Kim et al., 2004).
In this study, we investigated both the prevalence and prognostic significance of FLT3-ITDs in adult Syrian AML patients with cytogenetically normal (AML-CN) and could show, it has an impact on the therapy outcomes of AML.

Subjects
The present study comprised 44 newly patients diagnosed with de novo AML between October 2018 and February 2020 were included in this study. Patients without previous treatment were included in the study; patients with normal karyotype were selected for molecular analysis and patients with history of exposure to chemotherapy/radiotherapy, and secondary AML patients, were excluded. AML diagnosis was made according to French-American-British (FAB) classification. Their initial bone marrow (BM) or peripheral blood (PB) samples were collected for use in the study. Patients consisted of 23 men and 21 women; the median age was 35.3±12.4 years (range, 18-77 years) (Table 1). This study was approved by the Ethics Committee in Syrian Ministry of High Education and written informed consent was obtained from all the participants.

Treatment protocol
The majority of patients received (3+7) standard induction chemotherapy, which consisted of daunorubicin at 45 mg/m 2 for 3 days and cytarabine at 100-200 mg/m2 for 7 days, followed by high doses of a cytarabine-based consolidation phase (cytarabine at mg/m2 3 every 12 h for 3 days, repeated for 2 to 3 cycles). Patients with acute promyelocytic leukemia (M3) received all-trans retinoic acid plus anthracycline. Patients received conventional induction chemotherapy and were followed for 14 months. BM aspiration was performed between 21 and 28 days after initiation of chemotherapy. The patients were followed up once every 3 months with clinical examination and complete blood counts. A BM aspiration was performed if there was any suggestion of relapse on clinical examination or peripheral smear.

Sample collection
Genomic DNA was isolated from PB or BM samples from de novo AML patients using the QIAamp DNA Blood Mini kit (Qiagen, Germany) according to the manufactures instructions and was stored at -20°C. The total DNA of each sample was measured by using a spectrophotometer followed by quantity ultraviolet light absorbance
on 3% agarose gel stained with DNA SafeStain Dye and visualized under UV light. Samples with additional longer PCR products were identified as FLT3-ITD+. All mutant samples were verified by direct sequencing using the ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The cycle-sequencing reaction was performed in a 10-μl volume containing 1 μl of the terminator ready reaction, 5 pmol of either the forward or reverse primer and 10 ng of purified PCR product (ExoSAP-IT kit; Amersham BioSciences, Piscataway, NJ, USA). The thermal cycle protocol was 95˚C for 4 min followed by 30 cycles at 96˚C for 10 sec, 50˚C for 5 sec and 60˚C for 4 min (ABI GeneAmp PCR System 9700, Applied Biosystems). Centri-Sep columns (Princeton Separations, Adelphia, NJ, USA) were used for the effective and reliable removal of excess dye terminators (DyeEx 2.0, Qiagen, Germany) from completed DNA sequencing reactions. Data were compared and aligned with different sequences using the NCBI BLAST Assembled Genomes tool (http://blast.ncbi.nlm.nih.gov/ Blast.cgi).

Analysis of the FLT3-TKD mutation
For detection of the FLT3-TKD mutation, the specific forward primer 5'-CCGCCAGGAACGTGCTTG-3' and reverse primer 5'-GCAGCCTCACATTGCCCC-3' were used (Rezaei et al., 2017). The PCR reaction was performed in a total volume of 15 μl with similar reagents as used for the FLT3-ITD mutation, except for the primers. PCR conditions were also the same, except for the annealing temperature, which was 65 °C for 30 s. The amplification reaction was conducted in a PCR T100 thermocycler (Applied Biosystems). The 119-bp PCR products were then digested with 2 U of EcoRV at 37°C for 17 h, run on 3% agarose gel stained with DNA SafeStain Dye, and visualized under UV light. The presence of an undigested PCR product was an indication of a mutant sample.

Statistical analysis
The comparison of qualitative data such as age, WBC count, platelet count, hemoglobin level and blast count percentage between FLT3-ITD+ and FLT3-ITD-patients were statistically evaluated using Fisher exact and chisquare tests. OS and DFS were estimated for patients who received at least one induction course of therapy using the Kaplan-Meier method. p<0.05 was considered to be of statistical significance. All analyses were performed using SPSS Statistics 19 software (SPSS, Chicago, IL, USA).

Discussion
We evaluated the prevalence and prognostic FLT3-ITD+ in 44 Syrian adult patients with AML-CN newly diagnosed.
The clinical impact of the characteristics of patients with and/or without FLT3-ITD demonstrated no statistically significant difference between FLT3-ITD+ and FLT3-ITD− patients regarding to a gender, our findings which are an agreement with those have been reported (Bao et al., 2006;Zwaan et al., 2003). Clinically, AML patients with FLT3-ITD+ tend to have higher WBC counts and an increased percentage of leukemic blasts (Kottaridis et al., 2001;Yanada et al., 2005;Canaani et al., 2018). In our study, a positive association has been found between FLT3-ITD+ mutation versus BM blasts count (Kottaridis et al., 2001;Yanada et al., 2005;Canaani et al., 2018).
Although the clinical significance of this FLT3 mutation especially in AML-CN not clear yet. However, several studies showed that FLT3-ITD+ mutation is a strong adverse prognostic factor in AML patients (Stirewalt et al., 2008;Kiyoi and Naoe 2006;Zheng and Small 2005) with reduced DFS and OS and increased RR (Kottaridis et al., 2001;Yanada et al., 2005;Canaani et al., 2018). In our study, median OS was 4.0 months for FLT3-ITD+ patients and 8.2 months for FLT3-ITD-patients (p= 0.03), and DFS was also worse for FLT3-ITD+ than FLT3-ITD-patients (p= 0.02) because of a higher RR in FLT3-ITD+ patients. Also, high leucocytes count, high blast cells count in peripheral blood and resistance to therapy confers a poor prognosis. This has led to the development of a number of small molecule tyrosine kinase inhibitors (TKI) with activity against FLT3 (Small 2006;Leung et al., 2013). Moreover, patients with low or absent levels of FLT3-ITD, consistent with homozygosity for the FLT3-ITD allele, appear to have a particularly dismal outcome (Thiede et al., 2002).
A failure to achieve post-induction remission was observed in 57.1% (4/7) of evaluable pediatric patients with FLT3-ITD+, as opposed to 13.5% (5/37) of FLT3-ITD-patients (P= ˂0.01), our findings in accordance with Kumiko et al. (Kumiko et al., 2005). Most of patients with FLT3-ITD+ were found to be resistant to initial chemotherapy and failed to achieve CR (Xu et al., 2000;Arrigoni et al., 2003). Older patients (age less than 60 years) with FLT3-ITD+ mutation have a significantly association with increased RR (Kottaridis et al., 2003). Schnittger et al., (2018) could show that decreased FLT3-ITDs positively correlates with older age. However, other studies that did not reveal any age-dependency of FLT3-ITD, being performed in cohorts including other cytogenetic groups, smaller patient numbers of AML-CN, or restriction to patients up to 60 years of age (Thiede et al., 2002;Fröhling et al., 2002;Gale et al., 2005). In our study, patients who had FLT3-ITD+ were older than FLT3-ITD-(P=0.002).
In Conclusion, we report here for the first time the frequency and prognosis of the presence of FLT3-ITD+ mutations in adult Syrian patients with AML-CN. The frequency of FLT3 mutations in our study was lower (15.9%) than in previous studies; however, some reports agree with our observation, and that these mutations are an important adverse prognostic factor. Overall, this report supports the view that FLT3-ITD+ is a strong prognostic factor in AML patients and is associated with low CR, high RR, resistance to therapy, low OS, low DFS and confers a poor prognosis. Thus, FLT3 mutation analysis should be performed as a routine test in AML-CN patients.

Author Contribution Statement
IA provided all cases, a clinical data and a chemotherapy plan; AW and WA did primary cytogenetic and main part of the FISH-tests; FA and BA performed the molecular cytogenetic analyses; IA scientific supervisor of the IA student and put of the work plan. IA and AW drafted the paper and all authors worked on the final version of the paper. All authors read and approved the final manuscript.