Analysis of IDH1 and IDH2 Mutations in Myeloid Neoplasms Using Archived Bone Marrow Frozen Cell Pellet and Pyrosequencing Technology
Nichole Steidler, James Gale, Mohammad Vasef. University of New Mexico, Albuquerque, NM; TriCore Reference Laboratories, Albuquerque, NM
Background: Isocitrate dehydrogenase (IDH) mutations occur in 20-30% of myeloid neoplasms and are associated with a poor prognosis. In myeloid neoplasms, IDH1 and IDH2 mutations are often detected by Sanger sequencing or high resolution melting analysis of DNA amplified products from fresh bone marrow aspirates or formalin-fixed, paraffin-embedded tissues. We report the successful use of several years old frozen cell pellets from bone marrow monolayer cultures of myeloid neoplasms for analysis of IDH1 and IDH2 mutation status by pyrosequencing technology.
Design: Frozen cell pellets of bone marrow cultures stored for 4-5 years in cytogenetic freezing media were selected for this study. A total of seventy-two cases of suspected myeloid neoplasms were analyzed for IDH1 and IDH2 mutations. Briefly, cell pellets were thawed and then, samples treated with protein kinase buffer during white blood cell lysis, prior to DNA precipitation. After DNA isolation and polymerase chain reaction, PCR products were analyzed for mutations involving codon 132 of IDH1 and codon 140 1nd 172 of IDH2 by pyrosequencing.
Results: Forty-nine of 72 cases (68%) were from diagnostic pre-treatment myeloid neoplasms including AML, MDS, and MDS/MPN. The remaining 23 cases (32%) were composed of AML in remission, acute lymphoblastic leukemia, and non-specific, non-neoplastic disorders. The IDH1 and IDH2 mutational analysis were successful in 69/72 cases (96%). IDH1 or IDH2 mutations were identified in 11 out of 46 myeloid neoplasms (23%). These consisted of four IDH1 mutations (8%) at codon 132 with substitutions of cysteine (2 cases) or histidine (2 cases) and seven IDH2 mutations (14%) at codon 140 with substitutions of glutamine (6 cases) and codon 172 with the substitution of lysine (1 case). Eight out of 11 (72%) IDH1 or IDH2 mutated cases had a normal karyotype.
Conclusions: Our results indicates that the archived long term frozen bone marrow cultures are valuable resources for successful analysis of genetic abnormalities including IDH1 and IDH2 mutations using pyrosequencing technology. The 23% rate of IDH1 and IDH2 mutations in myeloid neoplasms detected in our study is comparable with previous published data. Additionally, none of the non-neoplastic samples in our study showed IDH mutations. This observation suggests that detection of IDH mutations in a borderline case may prove helpful in separating a neoplastic myeloid proliferation from a non-neoplastic process in subset of cases.
Monday, March 19, 2012 1:00 PM
Poster Session II # 290, Monday Afternoon