FLT3 mutations in childhood acute lymphoblastic leukemia

Abstract

IntroductionThe FMS-related tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase expressed in early hematopoietic progenitors that plays an important role in hematopoietic development. 1,2 Multiple studies have shown that activating mutations of FLT3 are common in blasts from patients diagnosed with acute myelogenous leukemia (AML) but are rarely found in adult patients with acute lymphoblastic leukemia (ALL). [2][3][4] We have recently shown that FLT3 is consistently highly expressed in MLL rearranged acute lymphoblastic leukemias (MLLs). 5 This prompted analysis for FLT3 mutations in MLL, where we found approximately 15% contain activating mutations in the receptor activation loop. 6 A large gene expression analysis of childhood ALL samples has shown that high-level FLT3 expression is also found in ALL samples containing more than 50 chromosomes (hyperdiploid ALL). 7 Here we show that activating FLT3 mutations are found in diagnostic specimens from 6 of 25 patients with hyperdiploid ALL, but only 1 of 29 patients with TEL/AML1-rearranged ALL. Also, 3 of 16 patients with leukemia who would ultimately have a relapse harbored FLT3 mutations. Most of the mutations are previously described activation loop mutations, but 4 are new mutations consisting of either small deletions or insertions in the juxtamembrane region of the receptor. These data suggest that patients with hyperdiploid or relapsed ALL should be considered possible candidates for therapy with recently described small-molecule FLT3 inhibitors. [8][9][10][11] Study design Patient samplesLeukemia samples were obtained from either bone marrow or peripheral blood at diagnosis from patients with ALL. The peripheral blood samples all had more than 10% blasts at diagnosis. Patients were treated according to Dana Farber Cancer Institute protocols 91-001, 95-001, and 00-001. Informed consent was obtained from all patients after approval of the Institutional Review Board. Standard cytogenetic analysis was performed on all samples. TEL-AML1 translocations were determined by florescence in situ hybridization (FISH) or polymerase chain reaction (PCR) as previously described. 12 Mutation detectiongDNA was extracted from leukemia samples with Trizol (Invitrogen, Carlsbad, CA). Mutations in the juxtamembrane domain were identified by amplifying a region spanning exons 14 and 15 with primers 14F (TGTA-AAACGACGGCCAGTCAATTTAGGTATGAAAGCC) and 15R (GAG-GAAACAGCTATGACCCTTTCAGCATTTTGACGGCAACC). The upstream primer in this reaction was fluorescently labeled (6-FAM) to allow sizing of all products when electrophoresed on a sequencing apparatus (Model 377, Applied Biosystems, Foster City, CA). The area under the curve of a variant product was divided by the total area under all curves to approximate the percentage of variant alleles in a sample. When variant products were determined to constitute more than 30% of the signal, the sample was directly sequenced following the initial PCR. Samples with no detectable length mutations were also directly sequenced. For a sample with ...

Mutations of FLT3, NRAS, KRAS, and PTPN11 are frequent and possibly mutually exclusive in high hyperdiploid childhood acute lymphoblastic leukemia

“…Although mutations in the FLT3, NRAS, KRAS, and PTPN11 genes have been suggested to be particularly common in high hyperdiploid childhood ALLs (Armstrong et al, 2004;Taketani et al, 2004;Tartaglia et al, 2005;Wiemels et al, 2005), the previous studies have been small, with Armstrong et al (2004), Taketani et al (2004), and Wiemels et al (2005) investigating only 25, 19, and 56 high hyperdiploid cases, respectively. Tartaglia et al (2004) included 102 pediatric BCP ALLs with a DNA index >1.0, but did not specifically delineate the high hyperdiploid subgroup.…”

“…In fact, several gene mutations have been suggested to be quite specifically associated with this genetic subgroup. For example, the FLT3 gene at 13q12 has been reported to harbor activating point mutations in 20-25% of high hyperdiploid cases when compared with 9-14% in pediatric ALLs in general (Armstrong et al, 2004;Taketani et al, 2004). Furthermore, mutations in the NRAS (at 1p13) or KRAS (at 12p12) genes, which are found in 15% of childhood ALLs, were recently reported in 30% of high hyperdiploid cases, and PTPN11 (at 12q24) mutations have been detected in 14% of BCP ALLs with a DNA index of >1, which is three times higher than the frequency in cases with DNA indices 1 Shu et al, 2004;Tartaglia et al, 2004;Wiemels et al, 2005).…”

Prognostic significance of FLT3 mutational status and expression levels in MLL-AF4+ and MLL-germline acute lymphoblastic leukemia

“…[1][2][3][4] Gene expression profiling showed that the FMS-related tyrosine kinase-3 (FLT3) is highly expressed in MLL-rearranged acute lymphoblastic leukemia (ALL), 5 leading to the characterization of FLT3 mutations as potential secondary cooperating events. 6 Accordingly, Ono et al 7 and Yamaguchi et al 8 have reported that constitutively activating FLT3 mutations cooperate with MLL-ENL and MLL-AF4, respectively, to induce leukemia in murine models.…”

“…Several groups reported the presence of FLT3/tyrosine kinase domain (TKD) mutations in 3-20% of MLL-rearranged ALLs, 6,[12][13][14] whereas Stam et al 15,16 together with Bardini et al, 17,18 using high-resolution genomic analysis, showed that FLT3 mutations in MLL-rearranged ALLs do not occur. Despite this emerging controversy, FLT3 is consistently highly expressed in MLLrearranged ALL, and therefore it is not fully resolved whether the constitutive activation of FLT3 in MLL-rearranged ALL is due to the presence of activating FLT3 mutations, or simply due to a transcriptional increased expression of FLT3 in an attempt to provide blast cells with survival and proliferative advantage.…”

Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML

“…6 An increased frequency of FLT3 mutations has also been associated with mutations involving the mixed-lineage leukemia (MLL) gene. 7 ITD and AL mutations result in constitutive FLT3 kinase activity. When FLT3 receptors harboring such mutations are introduced into mammalian cells, downstream signaling pathways are activated that lead to factor-independent growth in vitro and to leukemogenesis in vivo.…”