Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Daga, S.
Leukemic stem cells and a novel method of minimal residual disease detection in acute myeloid leukemia
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2019. pp. 133
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Höfler Gerald
- Sill Heinz
- Wölfler Albert
- Altmetrics:
- Abstract:
- Acute myeloid leukemia (AML) is an aggressive malignant disorder of hematopoietic stem and precursor cells (HSPCs) characterized by an adverse clinical outcome due to frequent relapse after initial response to therapy. AML is driven by a minor fraction of leukemic stem cells (LSCs), which predominantly reside within the CD34+38- subpopulation and whose persistence is considered being the primary cause of disease relapse. In the first part of this study we aimed to characterize surface markers to identify leukemia cells that reflect LSC activity at diagnosis. Among 16 markers analyzed by multicolour flow-cytometry (MFC), GPR56 and CLL-1 were found to be the most prominently differently expressed markers within the CD34/38 subcompartments. While GPR56 was highest expressed within the LSC-enriched CD34+38- population as compared to CD34+38+ and CD34- leukemic bulk cells, CLL-1 expression was lowest in CD34+38- leukemic cells and highest on CD34- blasts. Furthermore, high GPR56 surface expression in CD34+38- leukemic cells correlated with a recently published LSC gene expression signature and was associated with decreased overall survival in patients receiving intensive chemotherapy. In contrast, CLL-1 expression correlated inversely with the LSC gene signature and was not informative on outcome. Our data therefore strongly support GPR56 as a promising clinically relevant marker for identifying leukemic cells with LSC activity at diagnosis in CD34-positive AML. In the second part of study, we aimed to develop a highly sensitive and broadly applicable method for detection of minimal residual disease in AML by combining MFC-based leukemic cell enrichment followed by mutational analysis using either targeted deep sequencing or digital PCR. A combination of antibodies against CLL-1, TIM-3, CD123 and CD117 was identified to perform best for leukemic cell enrichment by enabling staining of >90% of leukemic cells in 134 of 146 diagnostic AML samples (91.7%). In dilution experiments using NPM1-mutated samples and normal BM, leukemic cell enrichment by these markers followed by mutational analysis showed a sensitivity of 10-5 for residual disease detection. For validation, BM samples of 41 patients in complete remission (CR) after induction chemotherapy were prospectively collected and analyzed for MRD using this newly developed two-step detection method. In 39 samples DNA quality of sorted cells was sufficient for sequencing. Twenty-one samples tested MRD positive, whereas 18 were negative. With a median follow-up of 559 days 71% of MRD positive (15/21) and 28% (5/18) of MRD negative patients relapsed (p=0.0065). Accordingly, median relapse free survival was significantly shorter in MRD positive patients (283 vs. not reached, p=0.0031). Furthermore, in multivariate analysis MRD positivity as detected by this method was the most informative parameter for cumulative incidence of relapse (hazard ratio 7.07). In conclusion, MFC-based leukemic cell enrichment using antibodies against CLL-1, TIM-3, CD123 and CD117 followed by mutational analysis is feasible for MRD detection with high sensitivity and informative on relapse risk in AML patients. Further multi-center clinical trials to standardize and to compare this promising novel method to other approaches of MRD detection are warranted.