Frascella et al: FLT3-ITD: technical approach Frascella E, Rondelli R, Pigazzi M, Zampieron C, Fagioli F, Favre C, Lippi AA, Locatelli F, Luciani M, Menna G, Micalizzi C, Rizzari C, Testi AM, Pession A, Basso G (2004) Clinical features of childhood acute myeloid leukaemia with specific geneRearrangements. Leukemia 18, 1427-1450. Gaymes TJ, Mufti GJ, Rassool FV (2002) Myeloid leukemias have increased activity of the nonhomologous end joining pathway and concomitant DNA misrepair that is dependent on the Ku70/86 heterodimer. Cancer Res. 62: 2791-2797. Kiyoi H, Naoe T, Yokota S, Nakao M, Minami S, Kuriyama K, Takeshita A, Saito K, Hasegawa S, Shimodaira S, Tamura J, Shimazaki C, Matsue K, Kobayashi H, Arima N, Suzuki R, Morishita H, Saito H, Ueda R, Ohno R (1997) Internal tandem duplication of the FLT3 associated with leukocytosis in acute promyelocytic leukemia. Leukemia Study Group of the Ministry of Health and welfare (Kohseisho). Leukemia 11, 1447-1452. Kiyoi H, Towatari M, Yokota S, Hamaguchi M, Ohno R, Saito H, Naoe T (1998) Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product. Leukemia 12, 1333-1337. Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA, Walker H, Wheatley K, et al (2001) The presence of an internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 98, 1752-1759. Libura M, Asnafi V, Tu A, Delabesse E, Tigaud I, Cymbalista F, Bennaceur-Griscelli A, Villarese P, Solbu G, Hagemeijer A, Beldjord K, Hermine O, Macintyre E (2003) FLT3 and MLL intragenic abnormalities in AML reflect a common category of genotoxic stress. Blood 102, 2198-2204. Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, Sonoda Y, Fujimoto T, Misawa S (1996) Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 10, 1911-1918. Rosnet O, Buhring HJ, Marchetto S, Rappold I, Lavagna C, Sainty D, Arnoulet C, Chabannon C, Kanz L, Hannum C, Birnbaum D (1996) Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. Leukemia 10, 238-248. Sambrook J, Fritsh EF, and Maniatis T (1989) Molecular Cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, New York, USA. Schnittger S, Schoch C, Dugas M, Kern W, Staib P, Wuchter C, Loffler H, Sauerland CM, Serve H, Buchner T, Haferlach T, Hiddemann W (2002) Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood 100, 59-66. Stirewalt D and Radich JP (2003) The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer 3, 650-665. Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, Platzbecker U, Wermke M, Bornhauser M, Ritter M, Neubauer A, Ehninger G, Illmer T (2002) Analysis of FLT3activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtype and identification of subgroups poor prognosis. Blood 99, 4326-4335. van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, Gottardi E, Rambaldi A, Dotti G, Griesinger F, Parreira A, Gameiro P, Diaz MG, Malec M, Langerak AW, San Miguel JF, Biondi A (1999) Standardized RT-PCR
Microcon-YM column, represents the most effective and fast method (Table 1 number 12). The Genescan analysis allowed for the identification of normal and mutated transcripts even if present in very low amounts. In addition it allowed the study of mutant level. In our series 20% of AML carried an FLT3-ITD and according with previous report all the internal tandem duplication found were in-frame. The high frequency of FLT3-ITD could be due to the retrospective nature of the study (Frascella et al. 2004) and the high number of acute promyelocytic leukaemia (52/261). In contrast with data regarding adult population (Withman et al, 2001), in our paediatric series the absence of the WT transcript seems to be very rare. In 3 out of 4 cases a low quantity of WT FLT3 transcript was found but we suppose that this small amount could originate from residual bone marrow normal cells. Finally we individuate a subset of patients carrying more than one FLT3-ITD. Among these cases we identify 2 cases carrying two internal tandem duplications with the same length but different nucleotide sequence. These cases were discovered by polyacrilamide gel because the ITDs appeared as a unique band on agarose gel and as a unique peak with the Genescan analysis. It is to note that, in this group, only in one case the lack of WT FLT3 might suggest lost of heterozygosity or biallelic mutation. In these patients the structure of the couple of ITDs found could be classify in 3 group based on the region involved in the duplication: group 1- different ITDs (M167, M218); group 2 - partially overlapped ITDs (M375, M380); group 3 - completely overlapped ITDs, in which all the nucleotide involved in the shorter one are included also in the longer (M397, M447) (Figure 2). Until now no definitive hypothesis regarding FLT3-ITD origin exists. Some authors suggested that binding sites for Topoisomerase II, identified in the region interested by duplication, could cause breaks to double strand of the DNA (Libura et al, 2003). These breaks are normally repaired by either non-homologous or homologous repair systems. In some AML a decreased efficiency of the nothomologous repair system has been reported (Gaymes et al, 2002; Zhong et al, 1999), and it could contribute to the creation of the FLT3-ITD in consequence of the loop formation, (Kiyoi et al, 1998). In our series we could hypothesize different mutation in group 1 patients cases while an evolution of the first mutation could be suggested in group 2 and 3 cases.
Acknowledgments We thank Dr C. Case for manuscript preparation. This research was supported by Fondazione CittĂ della Speranza and AIL.
References Abu-Duhier FM, Goodeve AC, Wilson GA, Care RS, Peake IR, Reilly JT (2001) Genomic structure of human FLT3: implication for mutational analysis. Br J Haematol 113, 1076-1077.
170