Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Gaksch, L.
Next generation deep sequencing for minimal residual disease detection in acute myeloid leukemia
Humanmedizin; [ Diplomarbeit ] Graz Medical University; 2017. pp. 54
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Kashofer Karl
- Wölfler Albert
- Altmetrics:
- Abstract:
- Introduction: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and despite achieving complete remission after intensive therapy. Most patients with cytogenetically normal AML relapse due to the persistence of submicroscopic residual disease. In this diploma thesis we asked whether broadly available targeted sequencing of DNA isolated from bone marrow slides displaying complete remission allows the detection of persisting AML-associated mutations and whether this persistent molecular disease is associated with increased risk for relapse and less-favorable clinical outcomes. Methods: We included 34 de novo AML patients of whom diagnostic material and slides after at least one cycle of consolidation were available. Isolated DNA was screened for mutations in 19 genes recurrently mutated in AML using an Ion Torrent target sequencing platform. In addition, the variant allelic frequency of distinct mutations was validated by digital PCR and parallel sequencing using a barcoding approach. Results: Three patients each revealed either no mutation in the diagnostic material or the somatic origin of the identified mutations could not be proven. In addition, DNA isolated from four slides was not sufficient for sequencing. Therefore, in total 27 patients could be analyzed for mutation clearance. In these patients we identified 68 somatic mutations (median of 3 mutations per patient, range 1-5) and 22 of these were still detected in 16 patients after consolidation therapy (median one mutation, range 0-3). The determined variant allelic frequencies of NPM1 or DNMT3A correlated well compared with either digital PCR or a barcoding parallel sequencing approach. The most frequent non-cleared mutations were found in DNMT3A (n=10 pts). Since these are known to persist in preleukemic stem cells without any impact on relapse risk, we performed survival and relapse risk analysis excluding DNMT3A mutations. Importantly, persistence of non-DNMT3A mutations was significantly associated with a higher risk of AML relapse (7 out of 8 pts versus 6/19 pts; p=0,013) and with a shorter relapse-free survival (333 days vs. not reached; log-rank p=0,0236). In addition, there was a trend for worse overall survival (log-rank p=0,050). Conclusions: Persistence of non-DNMT3A mutations after consolidation therapy as detected by broadly available targeted deep sequencing is associated with an increased risk of relapse in cytogenetically normal AML.