Tandem duplication of the FLT3 gene is infrequent in infant acute leukemia

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TO THE EDITOR

Fms-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase (RTK) which is associated with the proliferation and differentiation of normal hematopoietic stem cells.1 In recent years, an internal tandem duplication within JM/TK-1 domains as a somatic mutation of the FLT3 gene has been reported in five (16.7%) of 30 adult patients with acute myeloid leukemia (AML),2 in 15 (20.3%) of 74 adult patients with acute promyelocytic leukemia (APL),3 in seven (7.6%) of 92 adult patients with myelodysplastic syndrome (MDS) and AML with trilineage myelodysplasia,4 but in none of 20 patients with chronic myelogenous leukemia (CML)5 or 50 patients with acute lymphoblastic leukemia (ALL). The patients with this duplication have been associated with hyperleukocytosis3 and a poor prognosis.6 In childhood hematological malignancies, we found the tandem duplication of the FLT3 gene in 12 (13.8%) of 87 AML patients, all of whom died within 47 months. We also found two (4%) of 50 ALL patients who had mixed lineage phenotype, but none among patients with MDS or juvenile chronic myelogenous leukemia (JCML).7 There were, however, no reports of FLT3 duplication in infant leukemia. Here, we examined tandem duplication of the FLT3 gene in infant acute leukemia, which is known to often have myeloid phenotype and a poor prognosis.8

One hundred and eight infant acute leukemia patients under 1 year of age including 23 with AML, 82 with ALL, and three with acute mixed lineage leukemia according to the French–American–British (FAB) classification were examined in this study. Cytogenetic analysis of these infant leukemia patients demonstrated various abnormal karyotypes, including 32 patients with t(4;11) (q21; q23), 14 patients with t(11;19)(q23;p13), six patients with t(9;11)(p22;q23), five patients with other 11q23 translocations, and 20 patients with other chromosomal abnormalities. Eleven patients were judged as having normal chromosomes and 20 patients as having unknown karyotype. Among them, MLL gene rearrangement was found in 66 (64.1%) of 103 patients examined, including 14 AML, 50 ALL, and two mixed lineage leukemia patients.

High molecular weight DNA was extracted from fresh samples from the above 108 patients using the proteinase-K phenol-chloroform method.7 One hundred nanograms (ng) of genomic DNA was amplified in a total volume of 50 μl of reaction mixture.7 Genomic PCR was performed with primers of the 11F and 11R combination2 which was located at exon 11 as described in a previous study.7

The sequences of the primers used are as follows: 11F (sense), 5′-CAATTTAGGTATGAAAGCC-3′; 11R (antisense), 5′-CAAACTCTAA ATTTTCTCT-3′. Ten μl of the amplified PCR products were electrophoresed on a 3% agarose gel, stained with ethidium bromide, and photographed with a polaroid camera under UV light. Abnormal PCR product was cloned into a TA cloning vector, and then subjected to sequencing7 with primers T7 and M13-Reverse, using the Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Urayasu, Japan) on an Applied Biosystems DNA sequencer (model ABI 310).

The tandem duplication of the FLT3 gene was found in only one (4.3%) of 23 AML patients by genomic PCR (Figure 1). Sequence analysis of this one sample revealed a tandem duplication of 19 bp (nt 1777˜1795) with a 2 bp insertion within exon 11 (Figure 1). This AML patient with the duplication was a 9-month-old boy, and had FAB-M4Eo morphology (17% bone marrow eosinophil) with karyotype of 46, XY, inv(16)(p13q23). He was treated by AML-oriented therapy but relapsed 6 months after diagnosis. Then, he received a CD34-positive transplant from his mother but died 1 month later. The duplication was not found in 82 infant ALL patients or three acute mixed lineage leukemia patients. Notably, 66 infant acute leukemia patients with MLL gene rearrangement, including 50 ALLs and 14 AMLs, had no duplication.

Figure 1
figure1

 (a) Detection of FLT3 tandem duplication in infant leukemia samples by genomic PCR method. M, size marker φX174 HaeIII. N, normal peripheral lymphocyte; lanes 1 and 2 are AML, lanes 4 to 6 are ALL. Lane 3 shows the longer product in addition to 133 bp wild-type FLT3. The size of the longer product is 154 bp. (b) Partial sequence of the duplicated region. Arrows indicate the duplicated sequences.

Recently, tandem duplication within JM/TK-1 domains was reported in 17˜20% of adult AML patients and 12.3% of childhood AML patients.7 However, in our present study, the tandem duplication was found in only one (4.3%) of 23 infant AML patients. Thus, the frequency of duplication in infant AML was lower than that in adult and childhood AML patients reported to date.267 The reason why there was a close relationship between age and the duplication is unknown. One possibility is that about half of the infant AML had MLL rearrangements, which might not be associated with the FLT3 duplication. No tandem duplications of the FLT3 gene were detected in 82 infant ALL patients, including 50 patients with MLL gene rearrangements in this study. Notably, 66 infant leukemia patients with MLL gene rearrangement had no duplication. The one AML patient with the duplication died within 7 months of diagnosis. These results suggest that tandem duplication is rarely involved in infant AML, but not in infant ALL, and that infant AML patients with the duplication may have a poor prognosis, although inv(16) is usually considered to be a biologically good prognostic factor. These findings of infant acute leukemia are compatible with those of childhood and adult acute leukemia.237

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Acknowledgements

This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan, a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

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Correspondence to Y Hayashi.

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Xu, F., Taki, T., Eguchi, M. et al. Tandem duplication of the FLT3 gene is infrequent in infant acute leukemia. Leukemia 14, 945–947 (2000) doi:10.1038/sj.leu.2401760

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