Improving prognostication for patients with myelodysplastic syndromes

Background and aims: MDS constitute a heterogenous group of myeloid malignancies mainly characterized by dysfunctional hematopoiesis. Although cytopenia, dysplastic features and evidence of clonality are essential criteria for the diagnosis of all MDS, the several subtypes of the disease have a highly variable prognosis. The increasing quality and accessibility of DNA sequencing techniques have enabled huge advances for molecular characterization of the disease, and the prognostic impact of specific molecular markers in MDS is now well established. Several prognostic scoring systems have been developed during the last two decades but none of these tools accounted for the effect of molecular markers on outcome. MDS with RS is easily recognizable by the intra-cellular presence of iron-loaded mitochondria and this subtype reflects the heterogeneity of MDS. Hence, while RS are classically associated with SF3B1 mutations and an indolent disease course, RS are sometimes found in aggressive subtypes of MDS or AML. Patients and diseases change over time, and evolution patterns themselves can tell us something about disease biology and outcome. Clinicians account for these variations in practice, but current prognostic models do not. This may partly explain remaining discrepancies between observed and predicted prognosis. Hence, in this thesis we aimed to i) develop a novel prognostic score including molecular markers to refine prognosis prediction at diagnosis, ii) study the prognostic impact of combined gene mutation and gene expression in MDS with RS and iii) assess whether changes in erythrocyte (E) transfusion patterns during the early disease course can refine outcome prediction. Methods: Study I – an international cohort of 2957 patients with MDS, MDS/myeloproliferative neoplasms (MPN) were retrospectively collected. DNA sequencing with a panel of 152 genes known to be involved in myeloid malignancies was performed on all samples. Clinical data, cytogenetic and molecular features were retrieved and their association with outcomes was studied. A Cox multivariable model was used to estimate relative weights of selected explanatory variables. The score was validated on an independent cohort of 754 Japanese patients with MDS. Study II – A total of 129 patients with MDS and RS (MDSRS+) was assembled. All samples underwent DNA sequencing according to study I and thereafter RNA sequencing of CD34 sorted bone marrow mononuclear cells. Supervised/unsupervised clustering analysis and digital sorting were performed. A Cox multivariable model was used to assess association between clinical and genomic/transcriptomic patterns and outcome. Study III – a cohort of 677 Swedish patients was gathered from study I. We collected complete information on administered E-transfusions through the nationwide SCANDAT3-S database. Cox regression analyses were used to assess associations between clinical, molecular and transfusion data, and outcome. A Markov multistate model was used to assess association between changes in transfusion patterns and outcome. Results: Study I – TP53multi, MLL-PTD and FLT3 mutations were shown to be predictive of dismal outcome. In contrast, SF3B1 mutations were associated with favorable prognosis, however this effect was significantly influenced by the co-mutation patterns. A total of 22 variables (clinical, cytogenetic, and molecular markers) were used to build the IPSS-M score, each of them carrying a specific mathematic weight according to their individual impacts on endpoints. The calculation of the IPSS-M resulted in a unique score for individual patients and assigned each case to one of the 6 IPSS-M risk categories. When compared to the IPSS-R, the IPSS-M score clearly improved outcome prediction and led to the restratification of 46% of patients. The IPSS-M is validated both in MDS/MPN with WBC count below 13x109/L and in therapy related MDS (t-MDS). Study II – Most (~90%) MDSRS+ cases were found to have a mutation in SF3B1, SRSF2 or TP53multi. Overall, TP53multi and splice factors mutations were mutually exclusive, and SF3B1 and SRSF2 mutations cooccurred in only 3% of the patients. The three genetic subgroups were shown to have very different outcomes. Supervised transcriptome analysis confirmed the distinction between SF3B1-, SRSF2- and TP53multi-mutated MDS with RS. Unsupervised clustering analysis found three transcriptomic groups, each with distinct erythroid/megakaryocytic progenitor fraction, which predicted OS independently of IPSS-M. Study III – Whereas TP53multi, poor cytogenetic and higher bone marrow blasts predicted shorter time to first E-transfusion event, higher hemoglobin level and SF3B1alpha only were associated with longer time to first E-transfusion event. Next, E-transfusion state at 8 months after diagnosis was shown to be a strong predictor of OS independently of IPSS-M. Our model based on E-transfusion state at 8 months and IPSS-M (model 2) improved significantly prognostic prediction compared to IPSS-M only (model 1). Finally, a multistate model revealed that individual transfusion trajectories during the early disease course impacted both future transfusion requirement and OS. Conclusion: This thesis provides evidence that integration of genomic data to clinical characteristics improves greatly prognostication in MDS and we suggest that the novel IPSS-M prognostic score is implemented in clinical practice to provide further guidance in therapeutic decision-making. Our work also indicates that the heterogeneity of outcome in MDS cannot be explain by genetic profiling only and that studies of gene expression and integration of dynamic parameters among other techniques will contribute to a better understanding of the clinical course. In general, this thesis advocates for the need of a holistic approach of the disease to deepen our understanding of underlying mechanisms and ultimately to improve the care of patients with MDS. Enormous efforts are currently put in the field of precision medicine in cancer. Future integrative multiomics studies will hopefully improve individualized care to increase survival and quality of life of patients with MDS

A Single-Cell Taxonomy Predicts Inflammatory Niche Remodeling to Drive Tissue Failure and Outcome in Human AML

Cancer initiation is orchestrated by an interplay between tumor-initiating cells and their stromal/immune environment. Here, by adapted single-cell RNA sequencing, we decipher the predicted signaling between tissue-resident hematopoietic stem/progenitor cells (HSPC) and their neoplastic counterparts with their native niches in the human bone marrow. LEPR + stromal cells are identified as central regulators of hematopoiesis through predicted interactions with all cells in the marrow. Inflammatory niche remodeling and the resulting deprivation of critical HSPC regulatory factors are predicted to repress high-output hematopoietic stem cell subsets in NPM1-mutated acute myeloid leukemia (AML), with relative resistance of clonal cells. Stromal gene signatures reflective of niche remodeling are associated with reduced relapse rates and favorable outcomes after chemotherapy across all genetic risk categories. Elucidation of the intercellular signaling defining human AML, thus, predicts that inflammatory remodeling of stem cell niches drives tissue repression and clonal selection but may pose a vulnerability for relapse-initiating cells in the context of chemotherapeutic treatment.</p

Study of genetic factors in treatment-related complications in patients with childhood acute lymphoblastic leukemia and post transplantation of hematopoietic stem cells

La leucémie lymphoblastique aiguë (LLA) est le cancer le plus fréquent chez les enfants. Malgré le fait que plus de 80% des enfants atteints de LLA sont aujourd'hui guéris de leur maladie, ce succès a toutefois un prix élevé, car l’exposition aux médicaments cytotoxique et/ou à l’irradiation pendant une période vulnérable du développement de l’enfant peut entraîner des conséquences à long terme. En effet, environ 60% des enfants ayant survécu à une LLA devront vivre avec des problèmes de santé liés au traitement, également appelés effets indésirables tardifs (late-adverse effects, LAEs). Parmi ces derniers, on notera des problèmes métaboliques, l’ostéoporose, une altération des fonctions cognitives ou cardiaques, ainsi que la dépression et l’anxiété. Si certains survivants ne présentent aucune de ces complications, d'autres peuvent en avoir plusieurs. Différents facteurs peuvent contribuer à cette variabilité, notamment le traitement reçu, les caractéristiques de la maladie, les habitudes de vie et, surtout, la constitution génétique du patient. Ce projet s'est concentré sur les biomarqueurs génétiques permettant d'identifier les individus les plus susceptibles de souffrir de LAEs. Récemment, une étude exhaustive (évaluations cliniques, psychosociales et biochimiques) s’est déroulée au CHU Sainte-Justine pour caractériser chacune de ces morbidités chez 250 survivants de la LLA de l'enfant (cohorte PETALE). De plus, on a obtenu le profil génétique de chaque participant. Nous avons utilisé cet ensemble de données et des outils statistiques et bio-informatiques pour réaliser des études d'association comparant la fréquence des variants génétiques chez les survivants ayant développé ou non des LAEs; en particulier, les complications cardiovasculaires et neurocognitives, ainsi que les troubles de l'humeur tels que l'anxiété et la dépression. D'autres facteurs de risque tels que les caractéristiques de traitement et/ou de la leucémie ont été pris en compte lors de l'analyse pour dériver les meilleurs prédicteurs génétiques. Ainsi, en utilisant l'approche des gènes candidats, nous avons identifié les variants communs des gènes MTR, PPARA, ABCC3, CALML5, CACNB2 et PCDHB10 qui étaient associés à des déficits de performance des tests neurocognitifs, tandis que les variants des gènes SLCO1B1 et EPHA5 étaient associés à l'anxiété et à la dépression. Deux variants, rs1805087 dans le gène MTR et rs58225473 dans le gène CACNB2 sont particulièrement intéressants, car ces associations ont été validées dans la cohorte de réplication SJLIFE (St. Jude Children's Research Hospital, Memphis, USA). Les analyses d'association ont été complémentées par une étude d'association à l'échelle de l'exome, qui a identifié plusieurs gènes supplémentaires comme des modulateurs potentiels du risque de développer des complications neurocognitives liées au traitement (gènes AK8 et ZNF382), ainsi que l'anxiété et la dépression (gènes PTPRZ1, MUC16, TNRC6C-AS1, APOL2, C6orf165, EXO5, CYP2W1 et PCMTD1). Le variant rs61732180 du gène ZNF382 a ensuite été validé dans la cohorte de réplication SJLIFE. Également, nous avons effectué des analyses d’association concernant les complications cardiaques liées au traitement qui ont identifié plusieurs nouveaux marqueurs associés à ces complications dans les gènes TTN, NOS1, ABCG2, CBR1, ABCC5, AKR1C3, NOD2 et ZNF267. De plus, nous avons résumé les connaissances actuelles sur les marqueurs pharmacogénomiques qui ont été associés aux effets de cardiotoxicités, induites par les anthracyclines, qui affectent les patients atteints de cancer pédiatrique. Nous avons également inclus un aperçu de l'applicabilité des résultats rapportés, notamment ceux qui ont été validés dans la cohorte PETALE. Par ailleurs, nous nous sommes intéressés aux complications qui surviennent après une greffe de cellules souches hématopoïétiques. Nous avons appliqué des approches bio-informatiques et statistiques similaires pour obtenir un profil plus complet de la composante génétique derrière ces complications potentiellement mortelles. Ainsi, une étude d'association à l'échelle de l'exome a été réalisée dans une cohorte de patients pédiatriques subissant une greffe de cellules souches hématopoïétiques après un régime de conditionnement contenant du busulfan. Nous avons identifié de nouvelles variations génétiques conférant un risque plus élevé de syndrome d'obstruction sinusoïdale (notamment dans les gènes UGT2B10, BHLHE22, et KIAA1715) et de maladie aiguë du greffon contre l'hôte (dans les gènes ERC1, PLEK, NOP9 et SPRED1), qui pourraient être utiles pour des stratégies personnalisées de prévention et de traitement. Ces travaux contribuent à la compréhension de l'influence des facteurs génétiques sur le risque de développer des complications liées au traitement, tant au cours du traitement qu'à long terme. De plus, les marqueurs génétiques signalés ainsi que d'autres facteurs de risque connus peuvent conduire à des modèles de prédiction identifiant les patients à risque accru de ces complications.Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Even though more than 80% of children with ALL are now cured of their disease, this success comes at a high price as exposure to cytotoxic drugs and/or radiation during a vulnerable period of child development may have long-term consequences. In fact, approximately 60% of children who survive ALL will have to live with treatment-related health problems, also called late-adverse effects (LAEs). These include metabolic problems, osteoporosis, impaired cardiac or cognitive functions, as well as depression and anxiety. While some survivors do not have any of these complications, others may have more than one. Different factors can contribute to this variability, in particular, the treatment received, the characteristics of the disease, the lifestyle, and, above all, the genetic makeup of the patient. This project focused on genetic biomarkers capable of identifying the individuals most likely to suffer from LAEs. Recently, an exhaustive study (clinical, psychosocial, and biochemical evaluations) took place at Sainte-Justine University Health Center (Montreal, Canada), with the goal to characterize each of these morbidities in 250 survivors of childhood ALL (PETALE cohort). In addition, the genetic profile of each participant was obtained, and we used statistical and bioinformatics tools to perform association studies on this dataset in order to compare the frequency of genetic variants in survivors with or without LAEs. We evaluated cardiovascular and neurocognitive complications, as well as mood disorders such as anxiety and depression. Other risk factors, such as treatment and/or leukemia characteristics were also considered during the analysis to derive the best genetic predictors. Thus, using the candidate gene approach, we identified common variants in the MTR, PPARA, ABCC3, CALML5, CACNB2, and PCDHB10 genes that were associated with deficits in neurocognitive tests performance, whereas variants in the SLCO1B1 and EPHA5 genes were associated with anxiety and depression. Two variants, rs1805087 in the MTR gene and rs58225473 in the CACNB2 gene, are of particular interest since these associations were validated in an independent SJLIFE replication cohort (St. Jude Children's Research Hospital, Memphis, USA). The association analyses were complemented by an exome-wide association study, which identified several additional genes as potential modulators of the risk of developing treatment-related neurocognitive complications (genes AK8 and ZNF382), as well as anxiety and depression (genes PTPRZ1, MUC16, TNRC6C-AS1, APOL2, C6orf165, EXO5, CYP2W1, and PCMTD1). Variant rs61732180 in the ZNF382 gene was further validated in the replication SJLIFE cohort. To a great extent, we performed association analyses regarding treatment-related cardiac complications which identified several novel markers associated with these toxicities in the TTN, NOS1, ABCG2, CBR1, ABCC5, AKR1C3, NOD2, and ZNF267 genes in survivors of childhood ALL. In addition, we summarized the current knowledge on pharmacogenomic markers related to anthracycline-induced cardiotoxicity affecting pediatric cancer patients. We also included a brief overview of the applicability of reported findings to the PETALE cohort, validating several of them. Besides, we were interested in the complications that arise after a hematopoietic stem cell transplantation. We applied similar bioinformatics and statistical approaches to gain a more complete insight into the genetic component behind these life-threatening complications. Thus, an exome-wide association study was performed in a cohort of pediatric patients undergoing hematopoietic stem cell transplantation following a conditioning regimen containing busulfan. Our results identified new genetic variations conferring a higher risk of sinusoidal obstruction syndrome (notably in the UGT2B10, BHLHE22, and KIAA1715 genes) and acute graft-versus-host disease (ERC1, PLEK, NOP9, and SPRED1 genes), which could be useful for personalized prevention and treatment strategies. This work contributes to the understanding of the influence of genetic factors on the risk of developing treatment-related complications, both during treatment and in the long term. Furthermore, the reported genetic markers along with other known risk factors can lead to prediction models identifying patients at increased risk for these complications

Characterization of lymphoid compartments and hematopoiesis in a TSLP transgenic mouse model

Immunodeficiencies are severe diseases, which are inherited or acquired after cytotoxic treatment or radiotherapy. Hematopoietic stem cell transplantation (HSCT) has become a common treatment for patients suffering from immunodeficiency, but especially the re-establishment of a functional T cell pool is often delayed and patients suffer from infections and relapse of malignancies. The generation of an adequate T cell pool can be achieved by de novo generation and peripheral expansion of donor T cells. However, T cell development after pretransplantational treatment is often impaired due to damage of the thymic environment. To improve thymic engraftment and preserve the thymic microenvironment, current treatments involve application of cytokines, which have often severe side effects. Therefore it is important to find alternative ways to improve T cell reconstitution after HSCT. LTi cells are members of the family of innate lymphoid cells group 3 (ILC3s), which promote lymphoid tissue generation and are involved in tissue remodelling in primary and secondary lymphoid organs. In my study, I wanted to investigate the impact of ILC3s and secondary lymphoid organs on T cell reconstitution after HSCT. Therefore immunodeficient Rag2-/-γc-/- mice, which have reduced numbers of ILC3s and severe defects in LN development and K14 TSLP+/- Rag2-/- γc-/- mice with increased numbers of ILC3s were compared before and after transplantation with hematopoietic stem cells or mature T cells. Characterization of the recipient mice showed that TSLP overexpression in Rag2-/-γc-/- mice increases the number of double negative thymocytes DN2 and DN3s, and improves the thymic architecture with the development of mTECs and Aire expression. Reconstitution of the hematopoietic system with fetal liver (FL) HSCs from TSLPR-/- mice showed that TSLP overexpression results in an accelerated T cell reconstitution in the thymus and peripheral organs. The reconstitution of B cells did not differ between Rag2-/-γc-/- and K14 TSLP+/- Rag2-/-γc-/- mice, suggesting that the reconstitution itself was equally efficient. T cells depend on IL-7, as this cytokine promotes T cell proliferation and survival. I could show that the IL-7 expression was significantly higher in the spleen of K14 TSLP+/- Rag2-/-γc-/- compared to Rag2-/-γc-/- mice most likely as a result of higher numbers of dendritic cells (DCs) expressing IL-7. Finally, I could demonstrate that TSLP overexpression increases T cell proliferation in secondary lymphoid organs. Taken together, these data suggest, that TSLP overexpression accelerates T cell reconstitution by improving de novo T cell development in the thymus and T cell expansion in secondary lymphoid organs

Role of clinical markers of inflammation in assessing chronic graft versus host disease activity and severity

Chronic graft versus host disease (cGVHD) remains the major cause of non-relapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Currently there are no accepted measures of cGVHD activity to aid in clinical management and disease staging. We performed this study in a cohort of cGVHD patients highly enriched for those with established, severe and previously heavily treated disease. All patients were evaluated, and at a single time-point in their disease trajectory, the sera samples were well-annotated using a multidimensional battery of cGVHD descriptors. We analyzed clinical markers of inflammation in the sera of patients with established cGVHD and correlated those with definitions of disease activity. 189 adult patients with cGVHD (33% moderate and 66% severe according to NIH global scoring) were consecutively enrolled into a cross-sectional prospective cGVHD natural history study. At the time of evaluation, 80% were receiving systemic immunosuppression and failed a median of 4 prior systemic therapies for their cGVHD. This study identified a number of laboratory indicators of inflammation differing between patients with primarily established, moderate, or severe cGVHD and non-cGVHD transplanted controls, suggesting ongoing tissue inflammation in the patient cohort. We also identified several laboratory markers associated with the clinician's assessment of disease activity or severity. Lower albumin (p<0.0001), higher CRP (C-reactive protein; p=0.043), higher platelets (p=0.030) and higher number of PST (p<0.0001) were associated with active disease defined as clinician's intention to intensify or alter systemic therapy due to the lack of response. Higher platelet count (p=0.021) and higher number of PST (p<0.0001) were associated with more severe disease as defined by NIH global score. In the Cox proportional hazards model, better Karnofsky performance status (>= 80; p=0.0008; Hazard ratio=0.33; 95 CI: 0.17-0.63), higher FEV1 (>57; p=0.0028; Hazard ratio=0.35; 95% CI: 0.18-0.70) and higher absolute lymphocyte count (>0.65; p=0.017; Hazard ratio=0.43 (95% CI: 0.22-0.86) were associated with better survival. We developed a prognostic model and prediction equations for active and severe disease. Using this model (Table 10.), the equation for predicting disease activity was established. Based on this model, 71% of patients with active disease and 79 % of those with non-active disease would be correctly classified. Also, the equation for predicting disease severity was made and based on the developed equation, 76% of patients with severe disease and 74% of those with moderate disease would be correctly classified. This study identified common laboratory indicators of inflammation that can serve as markers of cGVHD activity and severity

Hodgkin's Lymphoma

Hodgkin's Lymphoma is the book consisting of 11 chapters: Recent insights into the biology of Hodgkin's lymphoma, including historical aspects, epidemiology, pathophysiology, genetic defects, and prognostic indicators are explained in the intro chapters. After a translational chapter from tumor microenvironment to immunotherapeutic approach, treatment of early stage, advanced, and refractory Hodgkin's lymphoma are explained in the following chapters. MALT lymphoma and adverse effects of chemotherapy and radiotherapy in the affected patients are discussed in the subsequent chapters, while the final chapter is focused on survivorship in Hodgkin's lymphoma. The book is intended to present recent advances in the pathophysiology of Hodgkin's lymphoma as well as practical approach to diagnosis and management in clinical practice, which is hoped to be welcomed by the physicians, who wish to learn more about Hodgkin's lymphoma