FMS-Like Tyrosine Kinase 3 Internal Tandem Duplication and the Patterns of Its Gene Sequence in 207 Chinese Patients With De Novo Acute Myeloid Leukemia

Genomic DNA was extracted from cells obtained from ethylenediaminetetra-acetic acid (EDTA)–buffered peripheral blood samples of 207 consecutive patients newly diagnosed with AML (1 with subtype M0, 28 with M1, 78 with M2, 31 with M3, 20 with M4, 43 with M5, and 6 with M6). Bone marrow samples were used when available. The median age of patients was 38 years, with a range from 14 to 77 years (112 males and 94 females, with a male to female ratio of 1.19∶1). The AML diagnosis was established with standard morphologic, immunologic, and molecular evaluations. Cytochemical staining for myeloperoxidase, nonspecific esterase, and periodic acid–Schiff were performed when appropriate. This study was approved by the ethics board of the West China Hospital. Each patient gave written informed consent to participate in the study. The control group consisted of 110 healthy volunteers.

Polymerase chain reaction (PCR) was performed on genomic DNA by using published primer molecules.8 In brief, 1 µl of DNA was amplified in a volume of 50 µl containing 50 mM potassium chloride, 10 mM Tris-HCl, pH 8.3, 1.5 mM magnesium chloride, 0.001% (weight per volume) gelatin, 200 µM deoxyribonucleotide triphosphates, 0.5 µM of each oligonucleotide (FLT3-14F: 5′-GC AATTTAGGTATGAAAGCCAGC-3′ and FLT3-15R: 5′-CTTTCAGCATTTTGA CGGCAA CC-3′) (Invitrogen, Carlsbad, California), and 1 U Taq DNA polymerase (Takara, Kyoto, Japan). The PCR consisted of an initial incubation step at 94°C for 150 seconds, followed by 35 cycles at 94°C for 30 seconds, 57°C for 1 minute, 72°C for 2 minutes, and a final elongation step at 94°C for 30 seconds and 60°C for 10 minutes. Cellular DNA of healthy subjects and empty template DNA were taken as double negative controls. Polymerase chain reaction products were analyzed on standard 3% agarose gels (Biosystems, Foster City, California). The samples with FLT3- ITD were analyzed by using GeneScan.

For GeneScan analysis, the PCR primer of FLT3-14F8 was labeled with 6-FAM (Invitrogen). The PCR setup was identical to that of standard PCR. The DNA concentration was determined and adjusted to 5 ng. The ratio was determined by dividing the peak area of mutation by that of wild-type. Polymerase chain reaction conditions were the same as above. We analyzed DNA from peripheral blood of 21 healthy subjects with this technique and did not detect any additional FLT3 signals. One µl of PCR product was mixed with deionized formamide and size standards (TAMRA) per the manufacturer's protocol, heated to 95°C for 5 minutes, and placed on ice for at least 1 minute before electrokinetic injection to the 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, California).

Polymerase chain reaction products were purified by using QIAquick columns (Qiagen, Hilden, Germany) and cycle sequenced by using Big Dye, version 2 (Applied Biosystems), according to the manufacturer's protocol. Sequences were compared to the wild-type (wt) sequence (accession No. E970630). DNA sequence was analyzed with the basic local alignment search tool, BLAST.

All of the patients were classified into 3 groups: FLT3-wt, ITD/wt < 0.43, or ITD/wt > 0.43. Thereafter, clinical characteristics at presentation were compared across the 3 groups. Categoric variables, such as immunophenotype and leukemic involvement of liver and spleen, were compared by using the Fisher exact test. Continuous variables, such as white blood cell count and hemoglobin, were compared by using the Kruskal-Wallis test. Overall survival (OS) was measured from the protocol on-study date until date of death, regardless of cause of death, censoring for patients who were alive. Kaplan-Meier curves were used for determining 2-year OS in the 3 groups. The log-rank test was carried out to determine whether there was a significant difference between the survival curves.

Finally, to adjust for potential confounding covariates, a Cox proportional hazards model was built with a forward stepwise (likelihood ratio) variable selection procedure to determine the significant prognostic factors.

As predicted, the wild-type FLT3 PCR product was seen as a 329-bp band. The ITD bands were seen as larger DNA fragments and varied in size from 355 bp to 386 bp (Figure 1). All samples with evidence of mutation by PCR assays were repeated twice to confirm the findings. A total of 207 patients with de novo AML were evaluated for FLT3-ITD. An ITD within the FLT3 gene was identified in 42 of 207 patients (20.3%). In our results, as shown in Table 1, FLT3-ITD occurred equally in M1 to M6 AML subtypes. M2 subtype seemed to have a relatively higher proportion of FLT3-ITD, but the result was not statistically significant.

A quantitative assay based on GeneScan analysis was carried out. The ITD mutation peak height divided by the wild-type peak height provided the ratio. Genomic DNA from 42 patients with FLT3-ITD was analyzed (Figure 2, a and b). The ratio of mutant to wild-type FLT3 ranged from 0.03 to 3.78, with a median of 0.43. Therefore, 0.43 was chosen as the boundary to divide all patients into 3 groups (FLT3-wt, ITD/wt < 0.43, and ITD/wt > 0.43). There were no significant differences with respect to hemoglobin level, platelet count, percentage of peripheral blasts and bone marrow blasts, and other clinical characteristics such as lymphadenopathy, splenomegaly, or hepatomegaly across the 3 groups (Table 2). The median white blood cell count increased from 14.29 × 10³/µL in the FLT3-wt group to 72.33 × 10³/µL in the group with ITD/wt ratio < 0.43 and was the highest in the group with ITD/wt ratio > 0.43 (213.23 × 10³/µL, P  =  .01). There was evidently a much higher expression rate for CD7, CD19, and CD56 in the ITD/wt ratio > 0.43 group. In our 2 years of follow-up, the trend of a decrease in cancer remission rate in the 3 groups did not reach statistical significance (P  =  .70; Table 2). Patients with ITD/wt ratio greater than 0.43 had a significantly lower OS compared to patients with a ratio less than 0.43 and to the FLT3-wt group (Figure 3). By pairwise comparisons of outcome for the 3 groups, OS was significantly shorter for the ITD/wt > 0.43 group than for the FLT3-wt group (χ²  =  60.709, P < .001) and the ITD/wt < 0.43 group (χ²  =  9.151, P < .001). The OS of the ITD/wt < 0.43 group was borderline shorter than the FLT3-wt group (χ²  =  5.042, P  =  .02).

Forty-two patients with ITDs were identified and the mutations confirmed by DNA sequence analysis. The FLT3-ITD mutations ranged in size from 26 to 57 nucleotides (median, 43 bp) and led to in-frame alterations. Twenty-six mutations were seen in the stretch between codons 591 and 612, which were present in 63% of the ITDs. Sixteen mutations clustered in the stretch from codon 626 to codon 644. By DNA sequence analysis, 2 types of mutations were found in patients with FLT3-ITD (Figure 4, a and b): in the first, the duplicated fragment was immediately adjacent to the normal sequence, without any additional base pairs, and was named type I; in the second, the duplicated fragment followed the normal sequence, but had an extra interval of 12 to 30 bp, and was named type II. The sequence of the interval was unrelated to the FLT3 gene sequence but exclusively contained a GAAAAG hexanucleotide. The median value of the FLT3-ITD to wild-type ratio for type I is 1.58, whereas it is 0.64 for type II. Patients with type I mutation showed worse survival rates than patients with type II mutation (Figure 5).

Cox regression analysis was performed to search for an independent prognostic factor. Several known risk factors, such as white blood cell count, immunophenotype (CD7, CD19, and CD56), and FLT3 status (ITD/wt ratios, gene patterns) were included. Cox regression analysis showed ITD to wild-type ratio was the strongest independent prognostic factor. Gene pattern and CD7 status were also independent prognostic factors (Table 3).

FLT3-ITD is a somatic mutation in the juxtamembrane region of the FLT3 gene and is believed to cause constitutive activation of the cell proliferation and differentiation-related proteins.9 The JM domain, which has been shown to be crucial for kinase autoinhibition, is disrupted by gene mutation caused by ITDs of varying sizes and insertion sites in 28% to 34% of cytogenetically normal AMLs.5 A number of studies10–13 published to date have indicated that the presence of an FLT3-ITD is a poor prognostic indicator in adult AML, whether favorable molecular changes in NPM1 or PML-RARα are present or not.13 Our study has analyzed the frequency of FLT3 gene mutations in 207 Chinese patients with AML. The overall incidence of FLT3-ITD was 20.3%, which is consistent with results reported by other groups.14 A previous report5 has shown that FLT3-ITD is more common in patients with FAB (French, American, British classification) M5 subtype of AML. Our data indicate that FLT3-ITD could be found equally in M1 to M6 subtypes, including some favorable subtypes such as M3.

Thiede et al5 have reported that quantitative determination of the ratio between mutant and wild-type FLT3 alleles may be of major prognostic importance. They identified a threshold (0.78) that represented the median ratio and distinguished prognostic subgroups. In our patients, the ITD to wild-type ratio is lower, and the median ratio was 0.43. The median survival of patients with FLT3-wt is evidently longer than that of patients with FLT3-ITD. Furthermore, patients in the ITD/wt > 0.43 group have the shortest survival and poorest outcome. We observed that FLT3-ITD has a major impact on the prognostic relevance, which is consistent with other groups' reports.15 The levels of FLT3-ITD mutant allele are quite different in distinct populations of patients with AML; we suggest that a threshold of 0.43 is useful as a risk stratification factor for Chinese patients with AML. Patients with FLT3-ITD usually have some distinct clinical features. In the present study, this group was more frequently associated with abnormal CD7 and CD56 expression, higher white blood cell counts, an obviously decreased cancer remission rate, and a significantly shortened overall survival. Cruse et al16 reported that FLT3/ITD mutation was associated with aberrant CD7 and CD56 expression and with low complete remission rate and biologic aggressiveness in a significant proportion of acute leukemias. In a recent report, Rausei-Mills et al17 suggested that FLT3-ITD mutations are closely associated with CD7 expression in myeloblasts and that FLT3/ITD-mediated leukemic transformation occurs at an earlier stage in myeloid progenitor cells. Signal transduction by constitutively active FLT3-ITD is complex. It may increase reactive oxygen species production, DNA damage, and frequency of misrepair, contributing to malignant transformation and chemoresistance.18 Molecular target therapy with FLT3 tyrosine kinas inhibitors appears to have promising therapeutic effects for those patients with ITD mutations.19 

Our DNA sequence data show that the length and the location of insertion of tandem duplicated fragments are similar to those observed in a previously published report.20 In our study, about half of the patients with ITD mutations had a simple duplication, accompanied with a higher ITD to wild-type ratio. Nearly half of the patients with ITD mutation had an inserted interval containing the hexanucleotide GAAAAG, with a significantly lower ITD to wild-type ratio. It is possible that the inserted GAAAAG interval may disturb the codon sequence and affect transcription. Meshhinchi et al21 reported that internal tandem duplications occurred in the JM zipper (JM-Z, D593-W703) motif, by crystal structure modeling, and demonstrated that the JM-Z region seems to play an important role in directing the orientation of the autophosphorylation “switch” residues Y589 and Y591 and in maintaining the autoinhibited conformation. Stirewalt et al22 observed that an increase in ITD size was associated with decreasing OS, and it is suggested that ITD size may have prognostic significance. Our results show that ITD size, as well as the gene mutation pattern, may both affect prognosis. The latter may involve a frameshift and affect transcriptional activity, especially for some critical codons.23 

In summary, our study indicates that FLT3-ITD is a common genetic alteration in Chinese adult patients with AML. Multivariate analysis shows that the ITD to wild-type ratio, the gene patterns, and CD7 expression status appear to be independent prognostic indices for patients with AML. Detection of FLT3 mutation is fast, easy, and inexpensive, and mutation analysis should be performed as a routine test. Furthermore, we suggest that GeneScan analysis of mutant to wild-type ratio is helpful for detailed risk stratification. DNA sequence analysis is more precise for confirming and evaluating the mutation pattern and is advisable for some patients. However, further studies are needed to explore the pathogenesis mechanism of the gene mutation.

We acknowledge the cooperation of all patients, nurses, and physicians who contributed to the collection of samples for this study. We thank Jing Yuan, PhD; Liang Liang Ma, PhD; Qin Zheng, PhD (Department of Hematology and Laboratory Medicine, West China Hospital, Sichuan University) for their technical work. Min Zheng, PhD, (West China School of Public Health, Sichuan University) kindly gave advice on statistics.