Myeloid Leukemia

196 Kiyoi and Naoe
cell-lines have both wild-type and ITD alleles, but the latter lacks the wild-type
allele (see Note 4). KOCL-33 and KOCL-48 have D835 mutations (14).
3. It has been reported that ITD mutations consist of 3–400 bp of additional
sequence. Generally, agarose gel electrophoresis is sufficient for separating the
ITD band from the wild-type band. However, electrophoresis using polyacrylamide
gels will give rise to better results than agarose gels in some samples, especially
those with short duplications (15).
4. The importance of the genotype in which ITD mutations occurred on one allele
and the other wild-type allele was deleted, was demonstrated by the Cancer and
Leukemia Group B (CALGB) (16). In that report, it was shown that the hemizygous
FLT3 ITD/- genotype was an adverse prognostic factor in AML. A subsequent
study has also demonstrated that the ratio of ITD to wild-type alleles is an independent
prognostic factor in AML (6). The allelic ratio can be semi-quantitatively
assessed by using densitometry, while more sensitive and quantitative
methods using fluorescent-labeled PCR have also been developed (6). For this
method, fluorescent-labeled primers are required. Amplified products, which are
generated from the fluorescent-labeled primers, are subjected to gene scanning
procedures, and the intensities of ITD and wild-type products are determined by
laser excitation.
References
1. Gilliland, D. G. and Griffin, J. D. (2002) The roles of FLT3 in hematopoiesis and
leukemia. Blood 100, 1532–1542.
2. Kiyoi, H. and Naoe, T. (2002) FLT3 in human hematologic malignancies. Leuk.
Lymphoma 43, 1541–1547.
3. Stirewalt, D. L. and Radich, J. P. (2003) The role of FLT3 in haematopoietic
malignancies. Nat. Rev. Cancer 3, 650–665.
4. Nakao, M., Yokota, S., Iwai, T., et al. (1996) Internal tandem duplication of the
flt3 gene found in acute myeloid leukemia. Leukemia 10, 1911–1918.
5. Yamamoto, Y., Kiyoi, H., Nakano, Y., et al. (2001) Activating mutation of D835
within the activation loop of FLT3 in human hematologic malignancies. Blood
97, 2434–2439.
6. Thiede, C., Steudel, C., Mohr, B., et al. (2002) Analysis of FLT3-activating mutations
in 979 patients with acute myelogenous leukemia: association with FAB
subtypes and identification of subgroups with poor prognosis. Blood 99, 4326–
4335.
7. Spiekermann, K., Bagrintseva, K., Schoch, C., Haferlach, T., Hiddemann, W.,
and Schnittger, S. (2002) A new and recurrent activating length mutation in exon
20 of the FLT3 gene in acute myeloid leukemia. Blood 100, 3423–3425.
8. Piccaluga, P. P., Bianchini, M., and Martinelli, G. (2003) Novel FLT3 point mutation
in acute myeloid leukaemia. Lancet Oncol. 4, 604.
9. Stirewalt, D. L., Meshinchi, S., Kussick, S. J., et al. (2004) Novel FLT3 point
mutations within exon 14 found in patients with acute myeloid leukaemia. Br. J.
Haematol. 124, 481–484.