Simultaneous Detection of FLT3/ITD and NPM Mutations in AML with Normal Cytogenetics Using Formalin-Fixed, Paraffin-Embedded Bone Marrow Biopsies
Q Huang, C Chen, W Chen, LM Weiss. City of Hope National Medical Center, Duarte, CA
Background: Acute myeloid leukemia (AML) with normal cytogenetics represents approximately 40-50% of de novo AML. Stratified prognostic determinants are required to predict which patients in this category have increased risk of relapse, resistance to therapy or long term disease outcomes. Recently, we developed a one step multiplex PCR assay for simultaneous detection of FLT3/ITD and NPM mutations in AML patients in fresh specimens. Now we report that we modify and validated the methodology and apply it to formalin-fixed, paraffin embedded bone marrow biopsies, enabling us for retrospective studies by using archived materials.
Design: Multiplex PCR was performed on genomic DNA using formalin-fixed, paraffin embedded bone marrow biopsies with specific primer sets designed to detect the presence or absence of FLT3/ITD and NPM gene 4 bp insertion mutations in patients with AML with normal cytogenetics. A specific set of b-globin primers was used simultaneously with FLT3 and NPM primers to ensure DNA integrity and PCR amplification. The results were compared and correlated with that of tests by using fresh materials.
Results: Bone marrow trephine biopsy and clot sections from 39 AML patients with normal cytogenetics were included in this study. The expected amplified products for wild type FLT3, wild type NPM and b-globin were at 152 bp, 168 bp and 282 bp, respectively. FLT3/ITD mutations produced larger peaks ranging from 167 to 225 bp, while NPM mutation yield a single peak at 172 bp, 4 bp larger than the wild type. In the study, wild type FLT3, NPM and internal control were successfully amplified in 25/39 cases (64%). Bone marrow clot sections yielded excellent results while trephine biopsy with decalcification process had a marked impact on PCR amplification. Of 25 successfully amplified cases, 14 cases of FLT3/ITD and 12 cases of NPM mutations were identified. Ten of 25 cases were positive for both FLT3/ITD and NPM mutations. A100% correlation in regarding to the mutational status and the size of mutations of FLT3 and NPM genes was obtained by comparing the results from fresh materials and tissue in the cases studied.
Conclusions: The multiplex PCR method provides a one step, rapid assay for detection of FLT3/ITD and NPM mutation using archived tissue. The test demonstrates excellent correlation with the results from fresh materials and provides a valuable tool for retrospective studies. However, bone marrow biopsies with decalcification have major impact on the reliability of the assay.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 184, Wednesday Afternoon