A Gene Panel NGS-Based Strategy for Genomic Characterization of Acute Myeloid Leukemias (AMLs)

AMLs are clonal disorders characterized by high genomic heterogeneity and several chromosomal and molecular alterations affecting patients' outcome. In about 40% of AML patients who do not show any citogenetic alteration, sequencing analysis identified different gene mutations which play a pivotal role in leukemogenesis and have a negative prognostic impact: FLT3, ASXL1, TET2, IDH1, IDH2, RUNX1, CBL, CEBPα, DNMT3A and TP53. Conventional Sanger sequencing may detect clones representing more than 20% of the total tumor population, whereas Next Generation Sequencing (NGS) can identify mutations in less than 1% of leukemic cell burden. The detection of these variants is relevant because they can play an important role in driving drug resistance and disease relapse and for biologic risk assessment.

To that purpose, we designed a 23-genes panel including: FLT3, DNMT3A, RUNX1, ASXL1, IDH1, IDH2, BCOR, NRAS, KRAS, TET2, TP53, U2AF1, ZRSR2, SF3B1, SRSF2, CBL, CEBPα, EZH2, NPM1, TERT, TERC, ETV6, GATA2. By using a 454 GS Junior by Roche Diagnostics with an amplicon-based sequencing approach and performing three sequencing runs per sample in order to reach a sensitivity of at least 1%, we settled an investigative strategy in order to assess the genomic profile of AMLs at diagnosis and possibly at the time of relapse or resistance. By this approach, we were able to confirm all the variants (i.e. FLT3, NPM1) previously documented with conventional tests; to reveal the presence of variants under the Sanger threshold of 20% in the genes resulted as wild type with routinely analysis; to evaluate the AML clonal heterogeneity, by assessing the coexistence of several mutated clones which have been described to be related to drug resistance, poor prognosis or to prior myelodysplastic syndrome.

This gene panel NGS-based strategy may be proposed as a highly accurate and sensitive approach for genomic characterization of acute myeloid leukemias and as an useful tool for planning a target and personalized AML therapy.