The diagnostic potentials of extracellular vesicles in pediatric acute myeloid leukemia

Kontopoulou, Evangelia GND

Pediatric Acute Myeloid Leukemia (AML) has a high relapse rate of >30%. Therefore, new methods are needed to precisely measure the mutational changes between the different stages of the disease, which could provide useful information regarding the disease’s progress. Extracellular vesicles (EVs) are released by both healthy and malignant cells. Due to their abilities to carry and transfer information of the parental cells, as well as to mediate changes in the microenvironment, their mutational profile can serve as a valuable diagnostic tool.
In this thesis, we aimed to establish a new detection method for AML-specific mutations in plasma-derived EV-RNA and EV-double stranded DNA (dsDNA) from primary pediatric AML samples. For this purpose, ultracentrifugation was performed to obtain EVs from the plasma of 29 pediatric AML patients at different time points of the disease, starting at the stage of diagnosis and during therapy. Afterwards, mutational analysis for the AML-specific mutations was performed using next generation sequencing (NGS) and GeneScan-based fragment-length analysis. NPM1 and FLT3-ITD mutations were detectable in the EV-RNAs of before-treatment samples, where RT-PCR and GeneScan-based fragment-length analysis were performed, respectively. The outcome was similar to the results of the mutational analysis we obtained from the genomic DNA (gDNA) of the same samples, supporting a potential use of EV-RNAs in pediatric AML diagnostics. However, the same results were not observed in the analysis of the after-treatment samples, implying an issue of reduced sensitivity. The mutational analysis of the EV-dsDNA from the initial samples mirrored the same AML-specific mutations found in the gDNA. Nonetheless, the mutations in the majority of the after-treatment samples were undetectable, suggesting again the limitation of low sensitivity. Finally, the highlight of the study was that the mutational background appeared to play an important role in the levels of the EV-RNA and EV-dsDNA as well as in the number of EVs. In conclusion, our findings support the potential of using EV-RNA and EV-dsDNA as diagnostic tools in complementation with the already existing clinical methods, leading to a more comprehensive analysis and monitoring of pediatric AML.

Die akute myeloische Leukämie (AML) im Kindesalter hat mit >30 % eine hohe Rezidivrate. Deshalb sind neue Methoden notwendig, die die mutationsbedingten Änderungen zwischen den verschiedenen Stadien der Erkrankungen präzise messen und so nützliche Aussagen über den Progress der Erkrankung bereitstellen können. Extrazelluläre Vesikel (EVs) werden sowohl von gesunden als auch von malignen Zellen freigesetzt. Aufgrund ihrer Fähigkeit, Informationen ihrer Ursprungszellen zu tragen und zu übertragen sowie Veränderungen in der Mikroumgebung zu vermitteln, könnte ihr Mutationsprofil als wertvolles diagnostisches Hilfsmittel dienen.

In dieser Arbeit sollte  eine neue Nachweismethode für AML-spezifische Mutationen in aus Plasma gewonnener EV-RNA und doppelsträngiger EV-DNA (dsDNA) aus initialen pädiatrischen AML Proben zu etablieren. Zu diesem Zweck, führten wir zur Gewinnung der EVs eine Ultrazentrifugation von Plasmaproben durch, die von 29 pädiatrischen AML Patienten zu unterschiedlichen Erkrankungszeitpunkten, beginnend im Stadium der Diagnose und während der Therapie, stammten. Im Anschluss wurde die Analyse auf AML-spezifische Mutationen mittels Next Generation Sequencing (NGS) und der GeneScan-basierten Fragmentlängen Analyse durchgeführt. NPM1 und FLT3-ITD Mutationen ließen sich in der EV-RNA der initialen Proben detektieren, wofür wir die Verfahren der RT-PCR und der GeneScan-basierten Fragmentlängen Analyse nutzten. Diese Ergebnisse glichen denen der Mutationsanalysen, welche wir zuvor mit der genomischen DNA (gDNA) derselben Proben durchgeführt hatten, was den potenziellen Nutzen der EV-RNA in der pädiatrischen AML-Diagnostik unterstützt. Allerdings konnten wir diese Ergebnisse in der Analyse der Proben nach Therapiebeginn nicht beobachten, was auf eine mangelnde Sensitivität hindeutet. Die Mutationsanalyse der EV-dsDNA der initialen Proben wies ebenso die gleichen AML-spezifischen Mutationen auf, welche zuvor in der gDNA gefunden wurden. Nichtsdestotrotz waren die Mutationen in dem Großteil der Proben nach Therapiebeginn nicht detektierbar, was erneut darauf hinweist, dass diese Methode aufgrund der geringen Sensitivität begrenzt ist. Allerdings spielt der Mutationshintergrund offenbar eine wichtige Rolle bei den Niveaus der EV-RNA und EV-dsDNA sowie bei der Anzahl der EVs. Zusammenfassend lässt sich sagen, dass unsere Ergebnisse das Potenzial der Verwendung von EV-RNA und EV-dsDNA als diagnostische Hilfsmittel in Ergänzung zu den bereits bestehenden klinischen Methoden unterstützen, was zu einer umfassenderen Analyse und Überwachung der pädiatrischen AML führt.

 

ABSTRACT

Pediatric Acute Myeloid Leukemia (AML) has a high relapse rate of >30%. Therefore, new methods are needed to precisely measure the mutational changes between the different stages of the disease, which could provide useful information regarding the disease’s progress. Extracellular vesicles (EVs) are released by both healthy and malignant cells. Due to their abilities to carry and transfer information of the parental cells, as well as to mediate changes in the microenvironment, their mutational profile can serve as a valuable diagnostic tool.  

In this thesis, we aimed to establish a new detection method for AML-specific mutations in plasma-derived EV-RNA and EV-double stranded DNA (dsDNA) from primary pediatric AML samples. For this purpose, ultracentrifugation was performed to obtain EVs from the plasma of 29 pediatric AML patients at different time points of the disease, starting at the stage of diagnosis and during therapy. Afterwards, mutational analysis for the AML-specific mutations was performed using next generation sequencing (NGS) and GeneScan-based fragment-length analysis. NPM1 and FLT3-ITD mutations were detectable in the EV-RNAs of before-treatment samples, where RT-PCR and GeneScan-based fragment-length analysis were performed, respectively. The outcome was similar to the results of the mutational analysis we obtained from the genomic DNA (gDNA) of the same samples, supporting a potential use of EV-RNAs in pediatric AML diagnostics. However, the same results were not observed in the analysis of the after-treatment samples, implying an issue of reduced sensitivity. The mutational analysis of the EV-dsDNA from the initial samples mirrored the same AML-specific mutations found in the gDNA. Nonetheless, the mutations in the majority of the after-treatment samples were undetectable, suggesting again the limitation of low sensitivity. Finally, the highlight of the study was that the mutational background appeared to play an important role in the levels of the EV-RNA and EV-dsDNA as well as in the number of EVs. In conclusion, our findings support the potential of using EV-RNA and EV-dsDNA as diagnostic tools in complementation with the already existing clinical methods, leading to a more comprehensive analysis and monitoring of pediatric AML.

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Kontopoulou, E., 2020. The diagnostic potentials of extracellular vesicles in pediatric acute myeloid leukemia. https://doi.org/10.17185/duepublico/73311
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