Evaluation of Minimal Residual Disease in Acute Myeloid and Juvenile Myelomonocytic Leukemia Using Allele-Specific Real-Time PCR
Purpose. To develop an approach for monitoring minimal residual disease in acute myeloid leukemia and juvenile myelomonocytic leukemia using next generation sequencing and allele-specific real-time PCR. Materials and methods. Bone marrow samples from patients with acute myeloid leukemia and juvenile myelomonocytic leukemia were obtained at diagnosis and therapy stages. The search for clonal gene mutations in tumor cells was carried out using high- throughput targeted sequencing and Sanger sequencing. Evaluation of gene mutations frequency at therapy stages was carried out by semi-quantitative allele-specific real-time PCR. Results. At least one clonal gene mutation of tumor cells was found in each case. The limit of quantification of allele-specific PCR was 0.05% in all cases and the sensitivity was 0.005% in 7 out of 8 cases. The change in clonal gene mutations frequency in all cases corresponded to the clinical response, as well as to the dynamics of other markers of minimal residual disease, such as AML1/ETO expression and the number of cells with a leukemia-associated immunophenotype. Conclusion. The presented approach is suitable for determining minimal residual disease at the genetic level in the absence of standard targets of leukemic cells such as chimeric oncogenes, and demonstrates high sensitivity and specificity.