Background: Acute promyelocytic leukemia (APL) is considered a hematologic emergency due to high risk of bleeding and fatal hemorrhages being a major cause of death. Despite lower death rates reported from clinical trials, patient registry data suggest an early death rate of 20%, especially for elderly and frail patients. Therefore, reliable diagnosis is required as treatment with differentiation-inducing agents leads to cure in the majority of patients. However, diagnosis commonly relies on cytomorphology and genetic confirmation of the pathognomonic t(15;17). Yet, the latter is more time consuming and in some regions unavailable. - Methods: In recent years, deep learning (DL) has been evaluated for medical image recognition showing outstanding capabilities in analyzing large amounts of image data and provides reliable classification results. We developed a multi-stage DL platform that automatically reads images of bone marrow smears, accurately segments cells, and subsequently predicts APL using image data only. We retrospectively identified 51 APL patients from previous multicenter trials and compared them to 1048 non-APL acute myeloid leukemia (AML) patients and 236 healthy bone marrow donor samples, respectively. - Results: Our DL platform segments bone marrow cells with a mean average precision and a mean average recall of both 0.97. Further, it achieves high accuracy in detecting APL by distinguishing between APL and non-APL AML as well as APL and healthy donors with an area under the receiver operating characteristic of 0.8575 and 0.9585, respectively, using visual image data only. - Conclusions: Our study underlines not only the feasibility of DL to detect distinct morphologies that accompany a cytogenetic aberration like t(15;17) in APL, but also shows the capability of DL to abstract information from a small medical data set, i. e. 51 APL patients, and infer correct predictions. This demonstrates the suitability of DL to assist in the diagnosis of rare cancer entities. As our DL platform predicts APL from bone marrow smear images alone, this may be used to diagnose APL in regions were molecular or cytogenetic subtyping is not routinely available and raise attention to suspected cases of APL for expert evaluation.

La 5-azacytidine (AZA) est un médicament approuvé pour le traitement des leucémies myéloïdes aiguës des patients qui ne sont pas éligibles à une greffe de cellules souches hématopoïétiques. Bien que l’AZA est augmenté significativement le pronostic des patients, le mécanisme d’action précis de l’AZA demeure nébuleux. En plus de son activité d’hypométhylation, il a été montré que l’AZA a aussi des effets immunologiques. Des études précédentes suggèrent que ces réponses immunitaires sont causées par des modifications du répertoire de peptides présentés par le CMH-I (MAPs), dont l’expression de MAPs dérivés de rétroéléments endogènes (EREs) et des cancer-testis antigens (CTAs). Ces gènes sont généralement réprimés par la méthylation de l’ADN. Dans cette thèse, nous avons testé cette hypothèse à l’aide de séquençage à haut débit et de spectrométrie de masse appliqués à quatre lignées cellulaires d’AML différentes. Notre approche protéogénomique d’avant-garde a révélé que l’AZA induit la présentation de MAPs dérivés de CTAs, mais pas d’EREs, malgré le fait que ces deux groupes de séquences soient surexprimés au niveau transcriptomique. Ces résultats indiquent que les réponses des lymphocytes T observées chez les patients suite au traitement à l’AZA dépendent probablement des MAPs dérivés des CTAs, et non pas des EREs. Les EREs stimulés par l’AZA ont tout de même un impact sur la réponse immunitaire en formant des ARN double-brins menant à une activation de l’immunité innée. L’incorporation de l’AZA et l’inhibition subséquente de la DNMT2 mène cependant à des agrégats protéiques et à l’autophagie, qui dégrade les transcrits EREs et limite leur surexpression. Nous avons démontré que les effets immunologiques de l’AZA peuvent être amplifiés par un traitement combiné de l’AZA et d’inhibiteurs de l’autophagie. De plus, le travail contenu dans cette thèse a montré que bien qu’elles soient un modèle expérimental pratique, les lignées cellulaires ont des limitations et doivent être utilisés avec prudence. Des différences majeures ont été observées entre des lignées cellulaires supposément identiques provenant de fournisseurs établis. Nos analyses ont permis de démontrer quelle lignée cellulaire était la plus similaire à la lignée parentale. Ainsi, ce travail fourni des recommandations pour améliorer les lignes directrices d’utilisation des lignées cellulaires en recherche. / 5-azacytidine (AZA) is approved for the treatment of acute myeloid leukemia (AML) patients ineligible for hematopoietic cell transplantation. Although AZA treatment has substantially improved patient outcomes, there remains a lack of clear understanding of the mechanisms driving these responses. In addition to its hypomethylating activity, AZA has been shown to have immunological effects. Previous reports suggest that these immune responses occur due to alterations in the repertoire of MHC-I-associated peptides (MAPs), including the expression of MAPs deriving from endogenous retroelements (EREs) and cancer-testis antigens (CTAs). These genes are typically silenced by methylation. With this thesis, we aimed to test this hypothesis using high-coverage RNA sequencing and mass spectrometry in four different AML cell lines. Our state-of-the-art proteogenomic approach uncovered that AZA treatment induced MAPs deriving from CTAs, but not EREs, despite both being upregulated at the RNA level. This indicates that T-cell responses post-AZA treatment are more likely to be dependent on CTA- than ERE-derived MAP presentation. AZA-induced EREs produced at the RNA level still contributed to immune responses by forming double-stranded RNA leading to a state of viral mimicry. However, AZA incorporation into RNA and subsequent DNMT2-inhibition led to protein aggregation and autophagy responses. These responses were responsible for degrading EREs, which limited their upregulation. We further demonstrate that the immune effects of AZA can be enhanced by the combination of AZA with autophagy inhibitors. Additionally, the work in this thesis has shown that although a practical model, cell lines have their caveats and must be used with caution. This work has highlighted the grave discrepancies between supposedly identical cell lines supplied by established repositories. Moreover, our analyses determine which of the two is closer to the parental cell line. Finally, this work provides recommendations for improving the current guidelines for cell line-based research.

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NKmediated specific lysis of tumor cells was maintained at stable levels for three days postirradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

Die myeloische Entwicklung wird durch die Familie der Transkriptionsfaktoren CCAAT/Enhancer-Binding-Protein (C/EBP) reguliert. Eine aberrante Expression oder Funktion von C/EBPs stört die normale myeloische Differenzierung und wird bei vielen Arten hämatopoetischer Malignome beobachtet. Mutationen von CEBPA führen zu einem veränderten Expressionsanteil der verkürzten Isoform C/EBPa p30 und werden bei etwa 15% der AML-Patienten (akute myeloische Leukämie) nachgewiesen. Obwohl die verkürzte Isoform C/EBPα p30 als Onkogen identifiziert wurde da sie die Proliferation myeloischer Vorläufer fördert, behält sie dennoch eine Differenzierungsfunktion. Unser Interesse gilt der Frage, wie diese beiden Funktionen von C/EBPα p30 reguliert werden. Die C/EBP-Familie gehört der Gruppe intrinsisch ungeordneter Proteine an, die zudem viele posttranslationale Modifikationen (PTMs) aufweisen. PTMs auf C/EBPα verändern seine biologische Funktionsweise stark. Frühere Forschungsarbeiten haben drei Argininreste am N-Terminus von C/EBPα p30 identifiziert, die aufgrund des Methylierungsstatus differentiell mit anderen Proteinen interagieren. In dieser Arbeit untersuchen wir den Einfluss der C/EBPα p30 Arginin-Methylierung auf seine pro-leukämische Aktivität sowie dessen Fähigkeit zur Neuausrichtung der hämatopoietischen Differenzierungslinie. Mit Hilfe von Aminosäuresubstitutionen fanden wir heraus, dass C/EBPα p30 Mutanten der Methylierungsmimesis oder Ladungsabschaffung die myeloische Differenzierung verstärkt, während Ladungserhalt-Mutanten die Erneuerung und Proliferation hämatopoetischer Stamm-/Vorläuferzellen unterstützt. Transkriptionelles Profiling von Zellen, die mutierte C/EBPα -p30-Varianten exprimieren, deutet auf potenzielle Ziele der methyliertem bzw. unmethyliertem C/EBPα p30 hin. Die Ergebnisse legen nahe, dass der Arginin-Methylierungsstatus das Leukämie- und Differenzierungs-Potenzial von C/EBPα p30 verändert und somit ein neues Ziel der Leukämietherapie darstellen könnten. / Myeloid development is regulated by the family of transcription factors CCAAT/enhancer-binding-protein (C/EBP). Aberrant expression or functioning of C/EBPs disturbs normal myeloid differentiation and is found in many types of hematopoietic malignancies. Mutations of CEBPA lead to imbalanced expression of the truncated isoform C/EBPα p30 and are found in approximately 15% of AML (acute myeloid leukemia) patients. Yet, how C/EBPα participates in leukemic progression remains to be discovered. More specifically, the truncated isoform C/EBPα p30, although being identified as an oncogenic isoform that promotes proliferation of myeloid progenitors, still retains differentiation function. The question of how both functions of C/EBPα p30 are regulated, is of our interest. C/EBP family also represents a group of intrinsically disordered proteins, which contain many post-translational modifications (PTMs). PTMs on C/EBPα greatly alter its functioning. Previous works have identified three arginine residues at the N-terminus of C/EBPα p30 that interact differently with others protein dependent on their methylation status. We hypothesize, that methylation of these arginine residues plays important roles in the biology of C/EBPα p30. In this study, we used a lymphoid-to-myeloid transdifferentiation (LMT) system to investigate the influence of arginine-methylation on C/EBPα-induced lineage switch and its pro-leukemic activity. Using amino acid substitution, we found that C/EBPα p30 mutants that resemble arginine-methylated p30 enhanced myeloid differentiation, while the charge-retention mutant, resembling arginine-unmethylated p30, supported renewability and proliferation of hematopoietic progenitors. Transcriptional profiling of cells expressing C/EBPα p30 variants suggested potential targets of either methylated or unmethylated p30. The results implied that arginine methylations alter C/EBPα p30’s leukemic potential and might comprise novel targets of leukemia therapy.

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La 1-β-D-arabinofuranosylcytosine (ara-C) demeure l’agent anticancéreux principalement utilisé dans le traitement de la leucémie myéloblastique aiguë (LMA), malgré sa dégradation et son élimination rapide après une administration parentérale. Son encapsulation dans des vecteurs pharmaceutiques, majoritairement des liposomes, a permis de surmonter ces inconvénients. L’objectif général de ce projet de doctorat était de développer deux systèmes à libération prolongée, à base de phospholipides, de cholestérol et de poly(éthylène glycol) (PEG) afin d’encapsuler l’ara-C et ultimement, d’améliorer son efficacité dans le traitement de la LMA. Des Sphérulites® (vésicules multilamellaires d’un type particulier) ont d’abord été étudiées pour leur forte capacité d’encapsulation, due à leur mode de préparation. Par la suite, une formulation liposomale capable, d’une part de cibler spécifiquement les cellules leucémiques et, d’autre part, de promouvoir la libération intracellulaire de l’ara-C grâce à sa sensibilité au pH, a été mise au point. Les deux formulations se devaient d’avoir un faible diamètre, une stabilité en présence de fluides biologiques et des temps de circulation prolongés chez l’animal. Une préparation de Sphérulites®, composée de Phospholipon 90G, de Solutol HS15 et de cholestérol, a permis d’obtenir des vésicules de 300 nm de diamètre. Un dérivé lipidique de PEG a pu être fixé à leur surface, sans modifier la disposition concentrique des lamelles, ni changer leur stabilité. Les Sphérulites® PEGylées ont été chargées d’ara-C et injectées chez le rat par la voie intraveineuse. Elles ont démontré des temps de circulation significativement prolongés comparativement aux Sphérulites® sans PEG. Cependant, l’ara-C s’est retrouvée éliminée de la circulation sanguine très rapidement, révélant une libération précoce du principe actif à partir de ces vésicules. Les liposomes sensibles au pH (~150 nm) ont été obtenus suite à l’insertion d’un copolymère à base de dioctadécyle, de N-isopropylacrylamide (NIPAM) et d’acide méthacrylique. L’anticorps anti-CD33, soit complet soit son fragment Fab’, a été fixé à la surface des liposomes afin de cibler les cellules leucémiques. Les essais in vitro ont démontré la spécificité de la formulation pour différentes cellules leucémiques (CD33+), sa stabilité en présence de protéines plasmatiques et la libération intracellulaire d’un marqueur fluorescent et de l’ara-C. Enfin, des études menées chez la souris saine et immunodéprimée inoculée de cellules HL60 ont montré que la formulation exposant le fragment Fab’ possédait un profil pharmacocinétique et une biodistribution semblables à ceux des liposomes contrôles non-ciblés. L’encapsulation de l’ara-C a permis d’améliorer grandement ses temps de circulation après une administration intraveineuse. Cependant, bien que les immunoliposomes ont permis de prolonger la survie des souris leucémiques comparativement à l’ara-C libre, l’addition du polymère sensible au pH n’a pas permis d’apporter de réel avantage à la formulation lorsque administrée in vivo. Les résultats obtenus dans ce travail de thèse ont, dans un premier temps, mis en évidence que les Sphérulites® pourraient s’avérer utiles dans la vectorisation d’agents anticancéreux si leur capacité à retenir le principe actif in vivo était améliorée. Dans un second temps, les données présentées avec les immunoliposomes suggèrent qu’ils pourraient apporter un bénéfice notable dans le traitement de la LMA. / Despite its rapid degradation and fast elimination in vivo, 1-β-D-arabinofuranosylcytosine (ara-C) is the main anticancer agent used in the treatment of acute myeloid leukemia (AML). The encapsulation of this drug into nanocarriers such as liposomes has been shown to improve its stability, pharmacokinetic profile and, consequently, the treatment efficacy. The purpose of this doctoral work was to develop two nanocarriers employing phospholipids, cholesterol and poly(ethylene glycol) (PEG) to encapsulate ara-C, with the ultimate goal of developing more efficient treatments for AML. The first formulation relied on Spherulites®, which are multilamellar vesicles possessing high entrapment yields due to their fabrication method. In a second part, pH-sensitive immunoliposomes were optimized to target specifically the leukemia cells and promote the release of the loaded ara-C at the desired intracellular site. Both formulations required a small diameter, stability in the presence of biological fluids and long circulation time properties when injected intravenously. An optimized formulation of Spherulites® was developed. It was composed of Phospholipon 90G, Solutol HS15 and cholesterol. The vesicles (300 nm) were able to accommodate PEG-lipid derivatives at their surface without altering their concentric lamellar shape and their in vitro stability. The PEGylated Spherulites® were loaded with ara-C and injected intravenously into rats. The surface-modified vesicles exhibited longer circulation times compared to uncoated Spherulites®. However, most of the loaded-drug was cleared from the systemic circulation very rapidly, reflecting rapid leakage of ara-C from the vesicles. The pH-sensitive immunoliposomes (~150 nm) were obtained by including a terminally-alkylated copolymer made of dioctadecyl, N-isopropylacrylamide (NIPAM) and methacrylic acid in the liposome bilayer. The whole monoclonal antibody anti-CD33 or its Fab’ fragment were grafted on liposomes to target leukemic cells. In vitro assays revealed that this formulation was really specific for the various CD33+ leukemic cell lines, stable in presence of blood proteins, and able to promote the intracellular release of an encapsulated fluorescent probe as well as ara-C. In vivo studies in naïve Balb/c and immunodeprimed (SCID) mice inoculated with HL60 cells confirmed that the anti-CD33 Fab’ targeted formulation possessed pharmacokinetic and biodistribution profiles similar to those of the non-targeted liposomes. The encapsulation of ara-C in this formulation improved substantially its circulation time after intravenous injection. However, although ara-C-loaded immunoliposomes were able to prolong the survival of leukemic mice compared to the free drug, the addition of pH-sensitive polymer did not add any benefit to the formulation. Although these formulations require some optimization, the first part of this work pointed out that Spherulites® could be used to deliver anticancer agents provided that leakage is reduced in vivo. On the other hand, the data obtained with the targeted immunoliposomes suggest that these carriers could be beneficial in the treatment of AML.

Hematooncological diseases are often accompanied by dietary restriction, especially in cytotoxic therapy. The main purpose of the work was to assess the effect of high-dose chemotherapy on the change of nutritional status in two groups of hematooncological patients. A total of 16 patients were enrolled. Changes of the body composition were evaluated using bioelectrical impedance analysis supplemented by monitoring of biochemical nutritional indicators. Observations showed that in both groups the majority lost weight. In the first group of eight patients with acute myeloid leukemia observed during three consecutive hospitalizations, the median of change of body weight was -3.7 kg (-4.3%). Loss of lean body mass with a median value of -4.8 kg (-7.2%) was detected at all patients. Body fat was reduced at half of the patients. In some cases, with length of observation, there was an increase in fat mass along with visceral fat. In the second group, which included eight patients (after autologous hematopoietic stem cell transplantation) at whom one hospitalization was evaluated, body weight was reduced at six patients. The medianof change of body weight was -2.1 kg (-2.3%). At five patients, the treatment representeda loss of active metabolic mass. The change of the weight of the lean body mass was shown...

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