Mechanism and Characterization of FLT3 Internal Tandem Duplications in AML by Massively Parallel Sequencing
Mohammad O Hussaini, Eric Duncavage. Washington University, St. Louis, MO
Background: FLT3 internal tandem duplications (ITD) are one of the most common molecular mutations in acute myeloid leukemia (AML) and are associated with poor prognosis. While most studies have focused on the functional and prognostic significance of the FLT3 ITD, little is known about the mechanism by which FLT3 ITDs occur in DNA.
Design: A set of 21 previously characterized FLT3 ITD+ AMLs was identified. DNA was capture enriched for all FLT3 exons plus 200 bp of flanking intronic segments and sequenced in multiplex on a next generation sequencing instrument with 2 X 101 bp paired-end reads. Sequence variation including insertions and ITDs were called with PINDEL. Sequences adjacent to ITDs/insertions were computationally evaluated for a variety of structural motifs with Emboss and Imperfect Microsatellite Extractor (IMEx).
Results: True ITDs containing tandem insertions were only found in 6 cases (28%, 84-186 bp, mean= 108) with the remaining cases showing variably-sized, non-tandem insertions (18-72 bp, mean=45) containing adjacent FLT3 exon 14 sequences. Non-templated sequence additions were rare (3/20 cases). Multiple ITDs/insertions were seen in 6 cases. The 20-30 bp sequence 5' to insertion/ITD was analyzed. No L1 or Alu insertion motifs, CpG islands, or stretches of non-templated sequence were observed. However, multiple palindromic complementary pairs (each partner ranging from 3-4 bp) were present in all insertion cases and 50% of ITD cases with an average of 2.4 pairs per 20 bp segment. In addition, TCATAT, CAATTC, and TGC repeats were found in 76% of cases.
Conclusions: True ITDs were seen in only 28% of FLT3+ AMLs; the remaining cases consisted of insertions of adjacent non-tandem DNA. Multiple palindromic sequences were frequently found 5' to insertion sites suggesting that secondary DNA structural changes may be involved in ITD formation. In addition, microsatellite repeats were frequently observed suggesting the possibility of slipped strand mispairing as a basis for generating ITDs. DNA damage repair mechanisms, however, do not appear to be involved in FLT3 ITD formation.
Monday, March 4, 2013 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 213, Monday Morning