Projects & Grants
| Increasing the sensitivity of determining residual measurable disease in patients with acute myeloid leukemia by NGS (next-generation sequencing) analysis of mutation frequency in the CD34+ cell subpopulation. | |
|---|---|
| Project Id | SGS04/LF/2025 |
| Main solver | MUDr. Barbora Dluhošová |
| Period | 1/2025 - 12/2025 |
| Provider | Specifický VŠ výzkum |
| State | solved |
| Anotation | Determination and monitoring of measurable residual disease (MRD) in acute myeloid leukemia (AML) is of fundamental importance for prognosis, early detection of relapses, and choice of treatment procedure, including allogeneic hematopoietic transplantation. Due to the heterogeneity of AML, it is recommended to combine flow cytometry and molecular genetics techniques when monitoring MRD. Approximately one-third of AML pts have detectable and traceable mutations only with NGS. Although NGS can detect mutations with a sensitivity of 10E-4 to 10E-6 if the sequencing depth is sufficient, the practical limits of mutation detection by NGS are determined by the input amount of material, the method of library preparation, and the type of sequencing technology. For most clinical NGS platforms, the lower limit of detection (LOD) ranges between 2-5 %. However, the method's sensitivity at the level of at least 0.01% is necessary for MRD monitoring. There are two approaches to increasing the sensitivity of NGS: amplicon scanning and target enrichment. Both approaches subsequently require sufficient depth of sequencing, which proportionally increases the cost of analysis. The desired increase in NGS sensitivity to 10E-4 can be achieved in AML differently. AML blasts almost always express the CD34 antigen, so if DNA obtained from a subpopulation of CD34+ cells (in remission pts around 1% of bone marrow cells) were used for NGS, there would be a 100-fold increase in the expected sensitivity. The proposed work aims to verify and standardize the procedure detection of MRD by NGS in patients with AML in a subpopulation of CD34+ cells selected from bone marrow aspirate by MACS (MAgnetic Cell Separation). If we use NGS sensitivity at LoD of 0.1% in combination with input material obtained from separated CD34+ cells, NGS sensitivity will increase to 10E-4. This procedure will make it possible to achieve the required sensitivity with a minimal increase in costs. |
