Medizinische Universität Graz - Research portal
Selected Publication:
Mueller, M.
Detection of AML-specific TP53 mutations in bone marrow–derived mesenchymal stromal cells cultured under hypoxia conditions.
Humanmedizin; [ Diplomarbeit ] Graz Medical University; 2020. pp. 22
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- Authors Med Uni Graz:
- Advisor:
- Hrzenjak Andelko
- Sill Heinz
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- Abstract:
- Acute myeloid leukaemia (AML) is a heterogeneous, aggressive malignancy affecting mostly elderly patients. The disease is characterised by malignant transformation of hematopoietic stem and progenitor cells (HSPCs) leading to a block in their differentiation capacity. Malignant transformation of single HSPCs is caused by a multitude of genetic as well as epigenetic alterations forming the basis of AML subentities like acute promyelocytic leukemia, AML with NPM1 or TP53 mutations. Over the last years, the role of the bone marrow microenvironment in AML has increasingly been elucidated with respect to both, pathogenesis and therapeutic resistance. In the following thesis, we focused on genetic analysis of mesenchymal stromal cells (MSCs), an essential part of the bone marrow microenvironment, in patients with TP53 mutated AML - a subentity showing an exceedingly poor outcome with 3-year survival rates of <10%. Diagnostic bone marrow specimens from a total of 14 patients with TP53 mutated AML were analyzed – 13 with somatic mutations and 1 with a germline mutation serving as control specimen. These specimens were characterized cytogenetically and by targeted deep sequencing of a panel of 39 myeloid-associated genes. Ex vivo cultures of mononuclear bone marrow cells were performed under low oxygen conditions with the addition of human platelet lysates. Adherent cells representing MSCs were further subjected to FACS sorting to obtain pure cell populations. Using error-corrected, high-resolution next-generation sequencing, patient-specific TP53 and cooperating mutations were analyzed. The bone marrow specimens showed a complex karyotype in 12/14 (86%) cases, whereas cooperating mutations were rare (median, 1; range, 0-3). MSCs were cultivated up to 4 passages and their adipogenic, chondrogenic, and osteogenic differentiation capacity was demonstrated. In purified MSCs, no cooperating gene mutations were detectable. However, the leukemia-specific TP53 mutation was detected in 2/13 specimens at low variant allele frequencies (VAFs) (0.25 and 0.1%, respectively) and confirmed using biological replication. As expected, MSCs with the germ-line TP53 mutation showed a VAF of 47.1%. The data presented here further confirm that TP53 mutations are early events in acute myeloid leukemogenesis, possibly having their origin in a common mesodermal ancestral cell. Clinically, they may also have consequences as MSCs generated from AML bone marrows are increasingly used as a source of constitutional material.