Abstract
Acute promyelocytic leukemia (APL) is characterized by the specific PML-RARα fusion gene resulting from translocation t(15;17) (q22;q12). Internal tandem duplication (ITD) of the FLT3 gene has been observed in approximately 35% of APLs, and large-scale studies have identified the presence of ITD as an adverse prognostic factor for acute myeloblastic leukemia (AML) patients. Aberrant expressions of surface antigens, such as CD2, CD34, and CD56, have been found in APL, but the implications of this are not well understood. We investigated the incidence of the FLT3/ITD mutation and FLT3/D835 (I836) point mutation in 25 APL patients. Incidence ratios of FLT3/ITD, D835 (I836), and both FLT3/ITD and D835 (I836) were 36%, 36% and 8%, respectively. FLT3/ITD+ cases showed a predominance of the bcr3 isoform (P=0.008) and M3v morphology (P<0.001). We found that all FLT3/ITD+ cases expressed CD2 (9 of 9) more frequently than that of FLT3/ITD− (1 of 16) (P<0.001), while only one of the CD2+ cases (1 of 10, 10%) did not harbor FLT3/ITD, and all CD2+CD34+ cases (5 of 5, 100%) harbored FLT3/ITD. In addition, quantitative polymerase chain reaction analysis showed that FLT3 mRNA was more abundantly expressed in FLT3/ITD+ than that in FLT3/ITD− (P=0.025), while there was no difference between D835(I836) + and D835(I836)− with regards to aberrant surface-antigen expression, expression levels of FLT3 mRNA, M3v morphology, and the bcr3 isoform of PML-RARα mRNA. This study demonstrates that the presence of FLT3/ITD, but not D835 (I836), is closely related to aberrant CD2 expression and high expression levels of FLT3 mRNA. Our findings also suggest that FLT3/ITD as a secondary genetic event may block differentiation at the immature stage of APL.
Highlights
Of the fms-like tyrosine kinase 3 (FLT3) gene has been observed in approxm imately 35% of Acute promyelocytic leukemia (APL), and large-scale studies have identified the presence of Internal tandem duplication (ITD) as an m adverse prognostic factor for acute myeloblaso tic leukemia (AML) patients
Aberrant expresc sions of surface antigens, such as CD2, CD34, - and CD56, have been found in APL, but the n implications of this are not well understood. o We investigated the incidence of the FLT3/ITD N mutation and FLT3/D835 (I836) point mutation acid (ATRA), features three types of heterogeneity: clinical [white blood cell (WBC) counts, age, and treatment outcome], morphological, and molecular.[1,2]
Results duplicated FLT3/ITD fragments varied from aberrant surface-antigens was confirmed in 6 21bp to 57bp, and the reading frame was of the 9 cases with D835/I836+ (66.7%), and 2
Summary
RT-PCR (AMV) (Roche Diagnostics, Tokyo, Immunophenotypic analysis was performed Japan) according to the manufacturers’ Detection of the FLT3 mutations with FACSCalibur CA, USA), using commercially available script, cDNA was amplified with two-step nest- FLT3/ITD. The panel of phycoerythrin (PE)- or ed PCR in 2 mM MgCl2, 200 mM dNTP, domain were amplified, using the specific fluorescein isothiocyanate (FITC)-conjugated GeneAmp 10X PCR Gold Buffer containing 15 primers R5 and 12R as described by Kiyoi et monoclonal antibodies against: i) myeloid lin- mM Tris-HCL (pH 8.0) and 50 mM KCl, and al.[24] FLT3/ITD was detected as an additional eage-associated antigens, CD11b, CD13, CD14, 1.25 U AmpliTaq Gold To detect D835 and I836 mutations, exon 20, which corresponds to TKD2, was amplified with the primers 17F and 17R as described by Yamamoto et al.[25] D835 and I836 amino acids ial us Table 1. Clinical characteristics of 25 acute myeloid leukemia patients with PML/RARa mRNA
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