FLT3-ITD: technical approach and characterization of cases with double duplications.

FLT3-Internal Tandem Duplication (ITD) of the juxtamembrane domain is one of the most common genetic alterations in acute myeloid leukemia (AML) and in some FAB subgroups seems to represent an unfavorable prognostic factor. Thus, its correct identification is critical. We analyzed 261 AML cases to individuate FLT3-ITD by RT-PCR and we compare different techniques (agarose and polyacrilamide gel electrophoresis, sequence and Genescan of PCR products) to define FLT3-ITD presence, length and number. All 53 positive cases were identified by electrophoresis on agarose gel. The sequence of the FLT3-ITD amplicons eluted from polyacrilamide gel was successfully performed while failing from agarose gel. We compared different methods of purifying PCR products from polyacrilamide gel to identify the fastest and most effective one. Genescan analysis was used to confirm the presence and the length of the ITD and to study the rate between ITD/WT transcripts. In our experience electrophoresis on 2% agarose gel is adequate for identifying FLT3-ITD, while purification from polyacrilamide gel is suggested for sequencing. In our series we found 20% of positive cases, 7.5% of these lacked FLT3 wild-type transcript and 13.2% showed two different FLT3-ITDs. In addition we identify 2 cases carrying 2 FLT3-ITD with the same length but different nucleotide sequence