Carmelo Gurnari

Simona Pagliuca

Pedro Henrique Prata #

Jacques-Emmanuel Galimard

Luiz Fernando B Catto

Lise Larcher

Ph.D Marie Sebert

Vincent Allain

Bhumika J Patel

Arda Durmaz

Andre L Pinto

MSc Mariana C B Inacio

Lucie Hernandez

Nathalie Dhedin

Sophie Caillat-Zucman

Ph.D Flore Sicre De Fontbrune

Maria Teresa Voso

Valeria Visconte

Ph.D Régis Peffault De Latour

Jean Soulier

Rodrigo T Calado

Ph.D Gérard Socié

Jaroslaw P Maciejewski

Clinical and Molecular Determinants of Clonal Evolution in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Keywords: aplastic anaemia, paroxysmal nocturnal haemoglobinuria, Fanconi Anaemia, acute leukaemia, myelodysplastic syndromes, clonal evolution, clonal haematopoiesis

on developing a model of genetic reversion in FA mice. We aimed to test the fitness of gene-corrected or preleukemic subclones in the Fanconi context. This work will contribute to understand the intrinsic and extrinsic mechanisms underlying clonal dominance and progression towards MDS/AML in BMF, helping to adapt follow-up and treatment strategies, reducing morbidity.

Titre : Dominance clonale dans les insuffisances médullaires

Résumé :

Les insuffisances médullaires sont des maladies rares qui se manifestent principalement par pancytopénie. L'incapacité du tissu hématopoïétique à maintenir l'hématopoïèse peut être acquise, principalement l'aplasie médullaire idiopathique (AM), ou des défauts génétiques, tels que le déficit de réparation de l'ADN de l'anémie de Fanconi (AF). L'hématopoïèse clonale est très fréquente chez les patients atteints d'une insuffisance médullaire et l'évolution vers un syndrome myélodysplasique ou leucémie aiguë myéloïde (SMD/LAM) est un événement préoccupant. Certaines données publiées indiquent que l'hématopoïèse clonale affecterait le risque de progression. Notre travail s'est concentré sur l'émergence de clones avec des mutations somatiques chez AM et AF et leur effet clinique.

Dans la première partie, nous nous sommes concentrés sur l'aplasie acquise. Près d'un tiers des patients AM présente une hémoglobinurie paroxystique nocturne (HPN) concomitante. La HPN est une maladie rare et monogénique qui se manifeste par une hémolyse intravasculaire. Elle est causée par des mutations somatiques du gène PIGA, entraînant une susceptibilité cellulaire au système du complément. Outre le gène PIGA, les anomalies du HLA de classe I sont fréquentes chez les patients AM. Ces deux groupes de cellules échapperaient à la destruction immunitaire, contribuant ainsi à soutenir l'hématopoïèse. De plus, certains allèles HLA de classe I sont surreprésentés dans l'AM. Cela pourrait indiquer que l'HLA joue un rôle dans la physiopathologie de l'AM. Jusqu'à présent, une prédisposition héréditaire à l'HPN est inconnue. Nous avons constaté que des variantes constitutionnelles du facteur H du complément étaient surreprésentées chez les patients atteints d'HPN. Certaines caractéristiques constitutionnelles semblent donc influencer la dominance clonale chez les patients atteints d'une aplasie acquise.

Nous avons étudié comment les caractéristiques cliniques, cytogénétiques et moléculaires des patients AM/HPN affectent la transformation en SMD/LAM. Nous avons confirmé que l'absence de réponse aux traitements non-greffe, l'âge et les mutations myéloïdes conféraient un risque plus élevé de progression. Par ailleurs, nous avons observé que les mutations PIGA/HLA, prédominantes au départ, étaient remplacées par des mutations myéloïdes classiques lors de la transformation.

Pour la deuxième partie, nous avons voulu explorer l'impact du mosaïcisme somatique dans l'AF sur la transformation en néoplasies myéloïdes. Près de 30% des patients atteints d'AF développeront un cancer du sang au cours de leur vie. Globalement, les complications hématologiques représentent près de 70 % des décès dus à l'AF. Diversement, certains patients présentent un phénotype hématologique moins grave : des cellules hématopoïétiques spontanément corrigées coexistent avec des cellules Fanconi, soutenant hématopoïèse. Notre équipe a précédemment étudié une cohorte de 350 patients FA, décrivant les caractéristiques cliniques et moléculaires de 17 révertants, et définissant les mécanismes génétiques expliquant la réversion. Jusqu'à présent, nous n'avons pas identifié de patient révertant évolué vers SMD/LAM. La correction fonctionnelle d'un certain nombre de cellules souches hématopoïétiques pourrait conférer un avantage écologique, mitigeant le risque de dominance clonale de clones pré-malins.

Inspirés par le mosaïcisme somatique, des essais cliniques ont montré des résultats prometteurs en utilisant des cellules souches hématopoïétiques corrigées génétiquement -thérapie génique -pour traiter l'insuffisance médullaire chez les patients AF. Cependant, l'effet sur le risque de transformation en SMD/LAM est inconnu pour l'instant. Nous avons travaillé sur le développement d'un modèle de réversion génétique chez des souris AF. Nous avons voulu tester des sous-clones corrigés génétiquement ou pré-leucémiques dans le contexte de Fanconi et le risque d'évolution.

Ce travail contribuera à la compréhension des mécanismes qui participent de la progression vers les SMD/LAM dans les insuffisances médullaires, pouvant aider à adapter les stratégies de suivi et de traitement, réduisant ainsi la morbidité.

Mots-clefs :

aplasie medullaire, heḿoglobinurie paroxystique nocturne, aneḿie de Fanconi, leuceḿie aigue, syndrome myeĺodysplasique, heḿatopoïese clonale Título: Dominância clonal nas falências medulares

Resumo:

As falências medulares são doenças raras que se manifestam principalmente por pancitopenia. A incapacidade do tecido hematopoiético de manter a hematopoiese pode ser adquirida, como na aplasia imune, ou constitucional, como a deficiência de reparo do DNA da anemia de Fanconi (AF). A hematopoiese clonal é muito comum em pacientes com falência da medula óssea e progressão para síndrome mielodisplásica ou leucemia mielóide aguda (SMD/LMA) é um evento preocupante. Existem evidências de que a hematopoiese clonal pode afetar o risco de progressão. Nosso trabalho concentrou-se no aparecimento de clones com mutações somáticas em aplasia imune e AF e seu efeito clínico.

Na primeira parte, concentramo-nos na aplasia imune. Cerca de um terço dos pacientes com aplaisa tem hemoglobinúria paroxística noturna concomitante (HPN). A HPN é uma doença rara que se manifesta principalmente por hemólise intravascular. É causada por mutações somáticas no gene PIGA, resultando em suscetibilidade celular ao sistema do complemento. Além do gene PIGA, anormalidades do HLA de classe I são comuns em pacientes com aplasia imune. Pensa-se que ambos os grupos de células (HLA e PIGA) escapariam do ataque imunológico, ajudando a manter a hematopoiese. Além disso, certos alelos HLA de classe I estão sobre-representados na aplasia imune, podendo indicar que o HLA desempenha um papel na fisiopatologia da aplasia. Por enquanto, uma predisposição constitucional para a HPN é desconhecida. Curiosamente, descobrimos que as variantes constitucionais do fator H do complemento estavam sobre-representadas em pacientes com HPN. Assim, certas características constitucionais parecem influenciar o domínio clonal em pacientes com aplasia adquirida.

Para aprofundar, investigamos como as características clínicas, citogenéticas e moleculares dos pacientes aplasia/HPN afetam a transformação para SMD/LMA. Confirmamos que a resposta sub-ótima aos tratamentos não transplante, a idade ao diagnóstico e mutações mielóides conferiram um maior risco de progressão. Além disso, observamos que as mutações PIGA/HLA, que eram predominantes no diagnóstico, foram substituídas por mutações mielóides clássicas durante a transformação.

Na segunda parte, exploraramos o impacto do mosaicismo somático na AF na transformação para neoplasia mielóide. Quase 30% dos pacientes com AF desenvolverão um câncer de sangue durante sua vida. Em geral, as complicações hematológicas são responsáveis por quase 70% das mortes de AF. Diversamente, alguns pacientes apresentam um fenótipo hematológico menos grave: células hematopoiéticas corrigidas espontaneamente (reverantes) coexistem com células de Fanconi, suportando a hematopoiese. Nossa equipe estudou previamente uma coorte de 350 pacientes da FA, descrevendo as características clínicas e moleculares de 17 revertantes e definindo os mecanismos genéticos que explicam a reversão. Até o momento, não identificamos nenhum paciente revertante que tenha progredido para SMD/LMA. É possível que a correção funcional de algumas células-tronco hematopoiéticas confira uma vantagem ecológica, reduzindo a probabilidade de que células pré-malignas atinjam dominância clonal.

Inspirados pelo mosaicismo somático, ensaios clínicos mostraram resultados promissores usando células-tronco hematopoiéticas corrigidas geneticamenteterapia gênica -para tratar a falência da medula óssea em pacientes com AF. No entanto, o efeito sobre o risco de transformação para MDS/AML é desconhecido no momento. Desenvolvemos um modelo de reversão genética em camundongos AF para testar subclones geneticamente corrigidos ou pré-leucêmicos no contexto de Fanconi e o risco de progressão.

Estes trabalhos contribuirão para a compreensão dos mecanismos envolvidos na progressão para SMD/LMA na falência da medula óssea, podendo ajudar a adaptar as estratégias de acompanhamento e tratamento, reduzindo assim a morbidade.

Palavras-chave:

aplasia medular, hemoglobinúria paroxística noturna, anemia de Fanconi, leucemia aguda, síndrome mielodisplásica, hematopoiese clonal Title: Clonal Dominance in Bone Marrow Failure Syndromes

Abstract:

Bone marrow failure syndromes (BMF) are a diverse group of rare diseases that manifest chiefly with pancytopenia. The incapacity of the haematopoietic tissue to sustain haematopoiesis can be acquired, chiefly immune-mediated aplastic anaemia (AA), or inherited intrinsic cellular defects, such as the DNA repair deficiency in Fanconi anaemia (FA). BMF severely impairs patients' quality of life and complications can be fatal. Besides, clonal haematopoiesis is very frequent among BMF patients, and progression into MDS/AML is a concerning event with a dismal prognosis. Despite a long-known association between BMF and myeloid malignancies, the precise mechanisms involved in clonal dominance are unclear. Some published data indicate that clonal haematopoiesis affects the MDS/AML progression risk. This work focused on the emergence of clones bearing somatic mutations in AA and FA and their clinical effect.

In the first part of this work, we focused on acquired BMF. Nearly a third of AA patients have concomitant paroxysmal nocturnal haemoglobinuria (PNH). PNH is a rare, monogenic disease that manifests as intravascular haemolysis. It is caused by somatic mutations in the PIGA gene, resulting in impaired cellular defence against the complement system. Besides PIGA, HLA class I abnormalities are frequent among AA patients. Such cells would evade the immune-mediated destruction and expand, helping to sustain haematopoiesis. Moreover, some HLA class I alleles are overrepresented in aplastic anaemia irrespective of ethnic background. The overrepresentation of specific class I alleles among AA patients strongly supports its influence on AA pathophysiology. Thus far, an inherited predisposition to PNH is unknown. We found that rare germline complement factor H variants were overrepresented among PNH patients. Germline characteristics seem to influence clonal dominance among acquired BMF patients.

We investigated how baseline clinical, cytogenetic, and molecular characteristics in AA/PNH patients affected AML/MDS transformation. We confirmed that lack of response to non-transplant treatments, older age, and myeloid driver mutations at marrow failure onset conferred a higher risk of progression. Besides, we observed that PIGA/HLA-mutations, predominant at baseline, were replaced by classic myeloid drivers at the transformation.

For the second part of this work, we were interested in exploring the impact of somatic mosaicism in FA on AML/MDS transformation. Nearly 30% of FA patients will develop a myeloid malignancy during their lifetime. Overall, hematologic complications account for almost 70% of FA deaths. Diversely, a few patients present a milder hematologic phenotype: a mosaicism of spontaneously corrected cells coexist with FAdeficient cells. Our team has previously studied a cohort of FA patients, described revertants' clinical and molecular characteristics, and defined the genetic mechanisms that explained reversion. Thus far, revertant patients have not progressed to MDS/AML. The functional correction of a few hematopoietic stem cells might confer an ecologic advantage, mitigating the risk of pre-leukemic clonal dominance.

Allogeneic hematopoietic stem cell transplantation is the only curative treatment for haematologic FA manifestations. This modality carries high treatment-related mortality but abrogates the risk of AML/MDS. Inspired by revertant somatic mosaicism, clinical trials showed promising results using autologous ex vivo gene-corrected hematopoietic stem cells -gene therapy -to rescue marrow failure in FA patients. However, the effect on AML/MDS transformation risk is unknown thus far. We worked I am grateful to my parents, Luiz Carlos and Beatriz, for their love, kindness, and altruism. They lead by example, and I am honoured to be their son.

My sisters and brothers Cynthia, Karen, Diana, Lídia, Raphael, Daniel and Joana: I am blessed to be among them. Each of them faces their own challenges. I also admire their examples of resilience and faith. Thank you for helping me pursue my aims! Léo, thank you for giving your advice when I came up against difficult decisions. I would like to thank, My mentors Dr Rodrigo Calado, Dr Régis Peffault de Latour, and Dr Jean Soulier who believe in my potential and invest their time in me. I express my gratitude for your trust and patience in guiding me towards becoming a physician-scientist. I am especially grateful for your support regarding the personal challenges I have come through during these last four years.

Dr Gérard Socié for this advice, opportunities, and constructive feedback.

Dr Marlène Pasquet and Dr Sara Terezinha Olalla Saad, thank you for being the reporters for this work. Dr Fabíola Traina, Dr Orianne Wagner-Ballon, Dr Lydie Da Costa, and Dr Antonio Risitano, thank you for being examiners. I am most grateful to the jury members for taking the time to read this manuscript, give feedback, and attend my defence.

My vocabulary is insufficient to express my gratitude to my lab colleagues. I have very much appreciated working with you. Stéph and Loïc, you were promoters in all aspects of my lab experience, helping me go beyond Kaplan-Meiers and navigate wet lab and pre-clinical studies. Lulu, thank you for your patience in teaching me the basics of lab life. Rathana, Marie Passet, Marie de Tersant, Lise and Sam: thank you for helping find me the solution to many questions. I am grateful to the Centre de référence des aplasies médullaires for all its support. Isabelle, thank you for helping me with regulations, MTAs, and IRBs. Flore, thank you for helping identify the cases and samples.

Jacques-Emmanuel, I am indebted to you for answering my endless statistical questions. Thank you for your availability and patience, so I understand what we are doing (or why we should not do it!).

I show my gratitude to the patients, for their well-being is the unique objective of our research. In particular, I would like to thank the Association HPN France -Aplasie Médullaire for their support.

I have written this thesis, but my path towards becoming a physician-scientist is ongoing. I am grateful to the American Society of Hematology for trusting me and investing in my training. The ASH Global Research Award allowed me to have 100% protected time to research during the thesis. The Clinical Research Training Institute has been a fantastic experience. I am honoured to serve as faculty in the Latin America programme.

Table of contents

Résumé en Français

Le sang est un tissu liquide complexe vital pour la vie des vertébrés. Le compartiment cellulaire du sang est constitué de globules rouges, de globules blancs et de plaquettes. En résumé, les globules rouges, ou érythrocytes, sont responsables du transport de l'oxygène et du dioxyde de carbone des poumons vers les tissus ; les globules blancs, ou leucocytes, sont le pilier du système immunitaire ; les plaquettes ne sont pas des cellules à part entière, mais des fragments d'une autre cellule spécialisée (les mégacaryocytes) et sont des acteurs majeurs de la coagulation sanguine et de la réparation vasculaire.

Les cellules sanguines dérivent d'un type cellulaire unique dans la moelle osseuse : la cellule souche hématopoïétique (CSH). Le processus hiérarchique de différenciation des cellules souches jusqu'aux cellules matures circulantes est connu sous le nom d'hématopoïèse. Lorsqu'une mutation génétique a lieu dans une CSH et que cette mutation confère un avantage écologique à cette cellule de façon à ce que le clone muté contribue de manière disproportionnée et persistante à la formation des cellules sanguines, la condition est appelée hématopoïèse clonale. Cela est un facteur de risque d'évolution en néoplasies hématologiques, notamment des syndromes myélodysplasiques et leucémies aiguës myéloïdes (SMD/LAM).

L'hématopoïèse clonale est très fréquente chez les patients atteints d'une insuffisance médullaire et l'évolution vers un SMD/LAM est un événement préoccupant. Les insuffisances médullaires sont des maladies rares qui se manifestent principalement par une insuffisance globale de production de cellules du sang appelée pancytopénie. L'incapacité du tissu hématopoïétique à maintenir l'hématopoïèse peut être acquise, principalement l'aplasie médullaire idiopathique, ou causée par des défauts génétiques, tels que le déficit de réparation de l'ADN de l'anémie de Fanconi.

Les objectifs de mon travail étaient de présenter l'effet de l'hématopoïèse clonale dans les insuffisances médullaires type aplasie acquise et anémie de Fanconi et leur impact sur l'évolution des SMD/LAM. Les patients atteints d'une insuffisance médullaire et d'une hématopoïèse clonale présentent des histoires naturelles très différentes. Je me suis concentré sur deux questions : l'étude des déterminants de la transformation dans les aplasies acquises et l'impact de la correction génétique spontanée dans le tissu hématopoïétique dans l'anémie de Fanconi (réversion) sur le risque de progression dans un modèle murin.

Dans une première partie, je me suis concentré sur l'aplasie acquise. Près d'un tiers des patients aplasiques présente une hémoglobinurie paroxystique nocturne (HPN) concomitante. La HPN est une maladie rare et monogénique qui se manifeste par une hémolyse intravasculaire. Elle est causée par des mutations somatiques du gène PIGA, entraînant une susceptibilité cellulaire au système du complément. Outre le gène PIGA, les anomalies du HLA de classe I sont fréquentes chez les patients aplasiques. Ces deux groupes de cellules échapperaient à la destruction immunitaire, contribuant ainsi à soutenir l'hématopoïèse. De plus, certains allèles HLA de classe I sont surreprésentés dans l'aplasie. Cela pourrait indiquer que l'HLA joue un rôle dans la physiopathologie de l'aplasie.

Mon objectif initial était de rechercher si des anomalies d'expression du HLA de classe I dans les cellules hématopoïétiques chez les patients atteints d'une aplasie médullaire acquise était associées à un risque augmenté de transformation vers SMD/LAM. Nous avons donc mené une étude pilote et avons séquencé les gènes HLA des classes I et II de dix patients (neuf LAM et une SMD avec excès de blastes) qui avaient transformé à partir d'une aplasie acquise. Nous n'avons pas mis en évidence d'anomalie génomique des gènes HLA (mutation de perte de fonction ou de perte d'hétérozygotie). Ces résultats n'ont pas conforté notre hypothèse initiale. De ce fait, nous avons établi une collaboration multicentrique (Hôpital Saint-Louis en France, Université de Sao Paulo au Brésil et Clinique de Cleveland aux États-Unis).

Pour infirmer notre hypothèse, nous avons interprété ensemble les séquences HLA de 10 patients français et de 10 patients américains. Chaque fichier de séquençage a été vérifié par l'autre centre compte tenu de la difficulté d'une telle analyse (quantité de polymorphismes connus et inconnus et de nombreux pseudogènes). Après cette analyse, nous avons confirmé l'absence de telles lésions HLA dans les échantillons de SMD/LAM secondaires à une aplasie médullaire. Nous avons conclu que les anomalies HLA ne jouent pas de rôle majeur dans la progression.

À la suite de ces résultats, nous avons étudié comment les caractéristiques cliniques, cytogénétiques et moléculaires des patients aplasie acquise/HPN affectent la transformation en SMD/LAM. Nous avons confirmé que l'absence de réponse aux traitements, l'âge et les mutations myéloïdes conféraient un risque plus élevé de progression. Par ailleurs, nous avons observé que les mutations PIGA/HLA, prédominantes au départ, étaient remplacées par des mutations myéloïdes classiques lors de la transformation. Ce travail, dans lequel je suis co-premier auteur, est publié dans le Journal of Clinical Oncology en Septembre 2022. Ce papier est inclus dans son intégralité dans ce manuscrit.

Avec un suivi médian supérieur à 8 ans, aucun patient greffé (n=117) n'a évolué vers un SMD/LAM, ce qui suggère que l'hématopoïèse du donneur l'emporte sur les clones pré-leucémiques du receveur qui pourraient exister au moment de la transplantation. Parmi les patients atteints d'une forme grave d'aplasie médullaire, nous avons confirmé qu'une réponse sous-optimale à l'immunosuppression et l'âge au moment de l'apparition de l'aplasie étaient des facteurs pronostiques indépendants pour la survenue d'une néoplasie myéloïde secondaire.

Les mutations les plus fréquemment trouvées au moment du diagnostic de l'aplasie étaient en accord avec la littérature et se chevauchaient avec l'hématopoïèse clonale liée à l'âge : BCOR, BCORL1, TET2 et ASXL1. La présence de mutations myéloïdes au diagnostic de l'aplasie prédisait un risque plus élevé de progression par rapport à l'absence de mutations ; les mutations isolées PIGA/HLA prédisaient le risque le plus faible.

Nous avons pu comparer le profil moléculaire des patients dont le séquençage a été effectué au diagnostic de l'aplasie et au diagnostic de l'évolution (analyse transversale). Les fréquences alléliques de PIGA ou de HLA ont eu tendance à augmenter chez ceux qui n'ont pas progressé et à diminuer chez ceux qui ont transformé en SMD/LAM, remplacés par une augmentation des mutations de gènes myéloïdes.

Comme on pouvait s'y attendre, le pronostic des patients transformés était mauvais et comparable aux résultats publiés. Le nombre de blastes au moment du diagnostic de SMD/LAM et l'âge au diagnostic de l'aplasie prédisaient indépendamment une mauvaise survie.

Les SMD secondaires à l'aplasie différaient cytogénétiquement des SMD primaires, présentant fréquemment des caryotypes à haut risque. Les anomalies du chromosome 7 et les caryotypes complexes conféraient un mauvais pronostic. Contrairement au R-IPSS, un caryotype normal au moment du diagnostic de SMD/LAM ne permettait pas de prédire un bon pronostic. L'intervalle entre l'aplasie et le SMD/LAM était plus court et le caryotype normal était associé à des mutations dans des gènes associés aux LAM tels que NPM1, FLT3, IDH1/2 et U2AF1.

Pour la deuxième partie, nous avons voulu explorer l'impact du mosaïcisme somatique dans l'anémie de Fanconi sur la transformation en néoplasies myéloïdes. Globalement, les complications hématologiques représentent près de 70 % des décès. Près de 30% des patients atteints d'anémie de Fanconi développeront une SMD/LAM au cours de leur vie. L'évolution clonale vers SMD/LAM semble être linéaire dans l'anémie de Fanconi. L'anomalie la plus précoce et la plus fréquente est la trisomie 1q (état pré-malin). La trisomie 3q, des anomalies du chromosome 7 et des mutations du gène RUNX1 sont plus tardives et prédisent une évolution vers SMD/LAM. Néanmoins, certains patients présentent un phénotype hématologique moins grave : des cellules hématopoïétiques spontanément corrigées coexistent avec des cellules Fanconi soutenant hématopoïèse -on appelle ce phénotype « révertants ». Parmi 350 patients Fanconi de la cohorte française, notre équipe a identifié 17 cas de réversion : aucun de ces patients n'a évolué vers de SMD/LAM. Inspirés par la réversion, des essais cliniques ont montré des résultats prometteurs en utilisant des cellules souches hématopoïétiques corrigées génétiquement -thérapie génique -pour traiter l'insuffisance médullaire chez les patients Fanconi. Cependant, l'effet sur le risque de transformation en SMD/LAM est inconnu pour l'instant. J'ai commencé un projet sur le développement d'un modèle de réversion génétique chez des souris Fancg -/-. Nous testons des sous-clones corrigés génétiquement ou pré-leucémiques dans le contexte de Fanconi et le risque d'évolution en SMD/LAM. L'objectif est de comparer l'aptitude des cellules souches et progénitrices corrigées ou pré-leucémiques, en modélisant le phénotype révertant et l'évolution clonale. La correction fonctionnelle d'un certain nombre de cellules souches hématopoïétiques pourrait conférer un avantage écologique, mitigeant le risque de dominance clonale de clones pré-malins. Dans l'hématopoïèse, les cellules révertantes écraseraient les cellules pré-malignes (1q+, 3q+).

Pour tester cette hypothèse, j'ai procédé à des expériences de greffe en compétition entre les cellules Fancg -/-génétiquement corrigées ex vivo et cellules Fancg -/-pré-leucémiques (Fancg -/-transgéniques sur-exprimant Mdm4, comme un marqueur de substitution pour le 1q+ et Fancg -/-sur-exprimant Evi1, comme un marqueur de substitution 3q+).

La validation de notre hypothèse peut avoir des implications thérapeutiques. Chez les patients atteints de Fanconi qui sont greffés, le tissu hématopoïétique est remplacé par un tissu sain. En plus de réduire la charge de cellules oligoclonales, la surveillance du nouveau système immunitaire allogène détecte et élimine les cellules résiduelles. Cependant, cet avantage a un coût : les patients atteints de Fanconi sont très sensibles aux régimes de conditionnement pour la greffe. Outre la toxicité à court terme du conditionnement, les patients transplantés sont exposés à la maladie du greffon contre l'hôte. Malgré cela, les résultats cliniques de la greffe pour les patients atteints de Fanconi se sont considérablement améliorés au cours des dernières décennies et la survie est excellente pour les jeunes patients transplantés avant la transformation.

L'hématopoïèse clonale fait partie du vieillissement physiologique et est plus fréquente chez les patients atteints d'une insuffisance médullaire que dans la population générale. Cependant, les patients atteints d'une insuffisance médullaire avec hématopoïèse clonale semblent présenter des histoires naturelles très différentes. La présence de mutations des gènes myéloïdes spécifiques ou l'augmentation de la taille du clone semblent conférer un risque plus élevé de transformation en SMD/LAM. Des mécanismes de réversion sont observées dans l'ensemble des insuffisances médullaires d'origine génétique (outre Fanconi, GATA2, maladie des télomères et Shwachman-Diamond), et la transformation en SMD/AML semble exceptionnelle.

Une surveillance étroite comprenant au moins une numération-formule sanguine, un myélogramme et une cytogénétique conventionnelle doivent être faits à intervalles réguliers pour tous les patients atteints d'une insuffisance médullaire. Nous ne disposons pas encore de suffisamment de preuves pour recommander le séquençage de routine pour tous les cas -une anomalie moléculaire isolée ne changera guère la thérapeutique. Le NGS et les études fonctionnelles doivent être envisagés au cas par cas.

Prologue

I was interested in the mechanism involving the emergence of clones bearing somatic mutations in bone marrow failure syndromes. We started a partnership between my mentors, Rodrigo Calado, Régis Peffault de Latour, and Jean Soulier, through a joint doctoral programme between the Universities of São Paulo (Brazil) and Paris-Cité (France). This work encouraged international and multi-institutional collaborations in bone marrow failure clinical and translational research.

I performed the experiments, data collection and interpretation at the "Institut de Recherche Saint Louis" (IRSL) within the team of Jean Soulier. Upon starting this doctoral programme, I had a background and training focused on clinical practice and research. I am most grateful to the lab team and mentors for their will and patience in training me in Science, preclinical models, and wet lab techniques. I am also very grateful to the American Society of Hematology for their financial (ASH Global Research Award) and training support (Clinical Research Training Institute).

We conducted independent, novel, clinical, and translational research to understand the mechanisms contributing to clonal evolution in marrow failure syndromes, from the "protective" somatic mosaicism to premalignant and malignant progression. The results help perceive what underlies clonal selection of different subsets, and may help better understand malignant evolution. An adequate risk assessment of MDS/AML would arguably impact therapy choice, from novel options such as gene therapy in Fanconi Anaemia to adapting the timing of consolidated strategies as allogeneic hematopoietic stem cell transplantation for Aplastic Anaemia. I have worked full-time in research for the last three years, except for the unprecedented sanitary crisis. At that moment, as a physician, I felt it necessary to resume hospital clinical activities to join the collective effort to assist the overflow of patients for a few months.

My lab journey was a wonderful and rich experience, and my research skills improved significantly from the beginning. During these years, I have learnt how to perform bibliographical research, especially in the PubMed database. I have learnt statistics applied to clinical and preclinical studies. I gained experience in medical and scientific writing and received good feedback from my peers and mentors. I wrote research proposals and applied to grants, some more successful than others. I have learnt about data management, visualisation, and analysis using R. I have submitted all my research projects to institutional review boards and got their approval. I have learnt how to ask scientific questions, test hypotheses, and conduct experiments in the laboratory and animal facility, as I will present in this manuscript. I could lead my projects and collaborate with colleagues' research. I also had the opportunity to present my research progress to my colleagues periodically in lab meetings in France and Brazil (videoconference) and congresses (EBMT and ASH).

My initial research project was entitled: "Clonal evolution in acquired aplastic anaemia: immune escape and somatic genomic alterations". Our primary objective was to investigate if the reduction or loss of HLA (HLA -clone) in hematopoietic cells in acquired aplastic anaemia patients was associated with AML/MDS transformation.

As a pilot study, we sequenced HLA classes I and II of ten patients (nine acute myeloid leukaemias and one myelodysplastic syndrome with excess of blasts type 2) transformed from severe aplastic anaemia. We failed to demonstrate any loss of function mutation or loss of heterozygosity at the moment of the myeloid neoplasm diagnosis. This pilot study did not support our hypothesis that the loss of HLA expression in hematopoietic cells in acquired aplastic anaemia patients is linked to AML/MDS-EB transformation. However, we did not have enough power to affirm negative results and established a collaboration with the Cleveland Clinic to increase the sample size.

To overturn our postulated hypothesis, we have interpreted together the HLA sequences of 10 patients from France and ten from the United States, each FASTQ file cross-checked by the other centre considering the difficulty of such an analysis (massive amount of known and unknown polymorphisms and many pseudogenes). We confirmed the absence of such lesions. We concluded that HLA -loss-of-function mutations or 6p copy-neutral loss of heterozygosity is not a major player in the progression to MDS/AML. I present the theoretical basis and the continuation of this successful tripartite collaboration between Saint-Louis Hospital, University of Sao Paulo and Cleveland Clinics in Chapter 2, section 2.1 of this manuscript. I have included a discussion and proposition for a continuation study we are about to start.

Given the preliminary negative results of the work mentioned above, my mentors and I developed an alternative plan to secure an experimental PhD work. Therefore, I worked on developing a murine model of somatic mosaicism (genetic reversion) in Fanconi Anaemia mice (Fancg -/-already available at the animal facility). The aim was to test the fitness of gene-corrected or preleukemic stem and progenitor cells, mimicking the revertant phenotype and the early stages of bone marrow progression. I present the theoretical basis, methods, pitfalls and preliminary results in Chapter 2, section 2.2. I have included a discussion and the next steps towards completing this project.

Chapter 1 presents a general introduction to clonal haematopoiesis, preleukemic states, and the use of animal models in the study of blood diseases. Chapter 1 also introduces the subject of bone marrow failures, focusing on acquired aplastic anaemia and Fanconi anaemia. I have included the ancillary and collaborative projects as annexes.

I tried to avoid heavy writing while presenting my progress, reasoning, and steps undertaken. From the many lessons I have learnt from this journey, first and foremost, I have learnt to adapt to new environments and to overcome adversities, both on the professional and personal sides. Ultimately, I have become somatic mosaicism: germline Brazilian and somatically French. I hope you will enjoy reading it.

Chapter 1: Introduction

The blood and polyclonal haematopoiesis

The blood is a complex, liquid tissue vital for animal life. The blood's cellular compartment consists of red and white cells and platelets. In short, red blood cells, or erythrocytes, are responsible for oxygen and carbon dioxide transportation from lungs to tissues; white blood cells, or leukocytes, are the mainstay of the immune system; platelets are not cells by themselves, but they are fragments of another specialised cell (megakaryocytes) and are major players in blood coagulation and vascular repair.

Despite very diverse functions and structures, blood cells derive from a unique cell type in the bone marrow: the haematopoietic stem cell (HSC). 1 Stem cells can divide and give rise to identical copies of themselves -a process called self-renewalbut at the same time, to differentiated cells that will further differentiate and divide and so on. 2 One single HSC is the ancestor of thousands of mature cells. 1,3 The hierarchic process of production and differentiation, from stem to circulating mature cells, is known as haematopoiesis.

Vertebrate haematopoiesis starts with a few active and many quiescent multipotent HSC. The classical representation of haematopoiesis (Figure 1) consists of a hierarchical process with many oligopotent intermediates. Downstream intermediates no longer have self-renewal capacities and go through many lineagecommitting steps until becoming mature cells. Understanding the process though which multipotent cells generate blood is crucial for the perception of quantitative (e.g. bone marrow failure -BMF) or qualitative diseases (e.g., myelodysplastic syndromes -MDS) of blood cells.

Insufficient or abnormal production of blood might severely impair patients' quality of life because of infections, bleeding, and frequent trips to healthcare facilities for blood transfusions. Depending on the degree of abnormality, marrow neoplastic or failure complications can be fatal. LT-HSC: long-term haematopoietic stem cell; ST-HSC: short-term haematopoietic stem cell; MPP: multipotent progenitors; CMP: common myeloid progenitor; MEP: megakaryocyte-erythrocyte progenitor; GMP: granulocyte-macrophage progenitor; CLP: common lymphoid progenitor. In red, intrinsic transcription factors and in blue, extrinsic cytokines that regulate differentiation. The immunophenotypic markers used for population enrichment are inside the pink boxes purple.

Men and mice

Studying normal and pathological haematopoiesis in humans is difficult because the HSC remains an elusive cell. Studies based on different animal models contributed significantly to today's knowledge. A game-changer happened in the aftermath of WWII: James Till and Ernest McCulloch were interested in hematopoietic stem cells as a countermeasure against the toxicity of nuclear weapons. They demonstrated that donor bone marrow cells could rescue haematopoiesis in mice treated with supralethal irradiation. 1,3 From that moment on, haematopoietic cell transplantation experiments in mice became the backbone of studies in haematopoiesis and much of what we know today became from mouse studies.

Mice are so close but far away. Phylogenetically, we are about 50 million years apart 5 (Figure 2). It might seem much, but if we compiled the life history on planet Earth in 365 days (the Big Bang happened on the 1 st of January) 6 , the development gap between mice and man would fit in only a few hours. Being so close, it is no surprise that numerous evolutionary mechanisms, including facets of haematopoiesis and DNA repair, are similar.

The human and mouse genomes contain nearly twenty thousand genes, among which nearly 70% are shared between both species. These one-to-one orthologs have, on average, 80% homology. 7 On the other hand, 20% of human genes have no matching ortholog on mice and vice-versa. The remaining 10% resulted from duplications or further sequence change, rendering the genetic copy number and functions different between species. 8 The similarities between man and mice enable biomedical research.

The description of such a didactic system of blood cells' differentiation (Figure 1) is based significantly on mouse studies. HSCs are divided according their capacities of sustaining haematopoiesis: long and short-term (16 weeks). Multipotent progenitors (MPP), no longer capable of self-renewal, are still ancestors to all mature blood cells. In this model, the first critical commitment step occurs at the stage of common myeloid (CMP) or lymphoid progenitors (CLP): at this stage, progenitor cells are already lineage-committed. Intrinsic (i.e., transcription factors) and extrinsic (cytokines) regulators strictly control the differentiation steps. 4 The isolation of each population depends on its surface markers identified using flow cytometry.

Subsequent mice studies have identified more intermediate populations with different self-renewal and differentiation capacities. For example, MPPs can be further divided based on surface markers into MPP1 to 4 with distinct self-renewal, gene expression, and differentiation potentials (Figure 3). 9,10 Also, human MPPs, CMPs, and MEPs are heterogeneous populations. The red bar represents the LSK population whereas Lin -all but the light gray (mature effector cells) bars.

However, haematopoiesis schematic representations were based on predefined surface markers. Along with technological evolution (e.g, multiomics and scRNA-seq), more heterogeneity is uncovered and the model represented in Figure 1 seems insufficient. Most likely, each cell undergoes its own differentiation process, without hierarchical boundaries. 12 In homeostasis, committed haematopoietic cells would emerge directly from HSCs (Figure 4). 11,12 In special, in both human and mouse, megakaryocytes precursors seem to emerge directly from HSCs, and MEP would generate chiefly RBCs, as represented in Figure 1. 4,11 Although differentiation seem to be a continuous process, to further study haematopoiesis and the characteristics of each cell compartment, researchers need to enrich samples with target populations. In studying diseases, we are more interested in hematopoietic stem and progenitor cells (Figures 1 and3). For practical reasons, many research teams use a pool of cells lacking surface markers of commitment to a specific mature lineage (lineage-negative I cells or Lin -, around 2% of marrow's mononucleated cells, Figures 1 and3), or, for more enriched HSPCs, the LSK population (around 0.05% -HSCs and MPP1-4, 9 Figure 3). The true HSCs are very rare and difficult to isolate (less than 0.01%). We can highly enrich samples by selecting a group of cells within the LSK population (SLAM markers, signalling lymphocyte and activation molecule: CD150 + CD244 -CD48 -CD41 -. I have worked with Lin -and LSK cells for the mice studies I present in this manuscript.

By definition, models are imperfect approximations, often reductionist. Differences exist, and we must not simply extrapolate conclusions from mice models to human diseases. Some species-specific evolutionary needs -different body sizes (70kg vs 20g) and life span (80 vs two years) -affect the daily needs for blood cells (proportionally, the ratio of active and quiescent HSCs are ten times higher in mice than humans). 13 Hematopoietic stem cells face, then, different selective pressures concerning replication, DNA repair, and tumorigenesis. For instance, while the CD34 marker is helpful for distinguishing stem and progenitor cells in humans, its expression is dim or absent in mice. On the other hand, sca-1 and SLAM, very useful for mice studies (Figure 3), is absent or less useful for human studies. Another significant difference is telomere length, which is much higher in mice. 13 Therefore, mice studies are a guide, not a mirror for human haematopoiesis.

Clonal haematopoiesis

During mitosis, the DNA must be duplicated. Cells have complex machinery to copy the nearly three billion base pairs of their genome. Although this replication process is exact, tens of thousands of errors might occur during each mitotic cycle. Therefore, the accumulation of replication errors is an inevitable fate for any ageing organism. Furthermore, some environmental factors such as sunlight, pollutants, radiation, and chemicals increase manifold the occurrence of DNA lesions. Fortunately, evolution granted cells mechanisms to correct such mistakes. In superior vertebrates, the apparatus is diverse and highly efficient. If such systems are dysfunctional -as in some diseases -the accumulation of errors can trigger cells' programmed death (apoptosis) or be a risk factor for cancers due to the accumulation of errors.

However, even with the DNA-repair apparatus in perfect function, some mistakes might slip the proofreading process and be transmitted to daughter cells. In other words, the DNA sequence of daughter cells will be slightly different from mother cells. In most cases, these differences will not affect the cell because they happen to be in places of the DNA that are not needed for the normal functioning of that cell or because the variants do not confer a selective advantage. The American College of Medical Genetics defines mutation as a permanent change in the nucleotide sequence" . 14 In current medical practice, the word "mutation" is reserved for changes entailing a deleterious effect on the organism. When the effect is not known, the change is considered a "variant of unknown significance" (VUS). Beneficial changes at an organism level are unusual and less evidently noticed. When a living organism acquires a permanent genetic change, the variants are called somatic (from Greek, sōma 'body'). These variants are therefore restrained to the tissue where it happened in the body (e.g., the haematopoietic tissue). When the variant is transmitted from one generation to another or occurs in germ cells that become a new individual, they are called constitutional or germline. These variants are therefore present in all the cells of the individual.

I Lineage-negative cells are negative for the following markers: CD5 -CD45R -CD11b -CD5 -Gr-1 -7-4 - Ter-199 - Gene abnormalities are detected using molecular biology techniques. In the last twenty years, a significant breakthrough, the next-generation sequencing (NGS), allowed physicians and researchers to study in-depth many genes (a panel) at the same time. NGS enables the detection of somatic and germline genetic abnormalities that would hardly be seen otherwise. Haematology pioneered the use of NGS in research and clinical care chiefly because of the ease of access (blood and marrow samples as opposed to tissue biopsies).

The frequency of detected somatic variants can be expressed as percentages: each cell has two alleles for each gene (but for genes located in X and Y chromosomes in men). The variant allele frequency (VAF) measures the proportion of individual variants in the DNA sample. For example, when a patient inherited one mutated allele from his or her progenitor and another healthy one, the expected VAF for the mutant allele is close to 50% (not precisely 50% because of measurement variation). On the other hand, the replication error can occur in one hematopoietic stem cell and give some replicative or survival advantage. This mutated HSC will self-renewal and differentiate, producing thousands of clones. When the variant is acquired and expands during patients' life, the VAF can assume any value from 0 to nearly 100%. When a mutated HSC clone contributes disproportionally and persistently to the blood cells' formation (frequently defined as a mutation detected in blood with a VAF ≥ 2%the lower limit detectable using whole-exome sequencing, WES), the condition is called clonal haematopoiesis (CH).

As replication errors are part of physiological ageing, clonal haematopoiesis can be detected in about 12.5% of individuals in all age groups (>50% in the elderly) 15,16 and is not a synonym for disease. Besides errors, base substitutions occur because of thermodynamics: for instance, methylated cytosines can undergo hydrolysis (Figure 5), becoming thymidine (C>T transition), i.e., age-associated mutations. 17 Hydrolysis in biology are exergonic processes, therefore, spontaneous. In other words, somatic mutations are an unavoidable fate of all living beings. Although CH has been linked to a higher risk of coronary artery disease 18 and a tenfold increased risk of blood cancers compared to the general population, the absolutes risks are still low. 19 Furthermore, the risks and diseases associated with different somatic variants are unequal, and some CH are secondary to neutral drift II instead of an adaptative advantage. 15 For some patients, the presence of CH might be a reflex, a biomarker of stressed haematopoiesis (i.e., the more replication cycles, the more errors) that is a risk factor to malignant transformation and not the cause in itself. Therefore, clinicians must interpret the meaning of CH adequately. Clonal haematopoiesis and non-haematologic disorders 20 are out of the scope of this work.

Overall, mutations in epigenetic regulators are frequent in clonal haematopoiesis, 16,19 and clones steadily expand with age. Faster clone growth rate indicates a higher risk of malignant progression. 16 Some other features of CH might confer a higher risk for myeloid neoplasm. Notably, the diagnosis of CH in the presence II Neutral drift: variants with neutral effect occur and expand not because of gain in adaptation skills, but because of random genetic drift.

of cytopaenias carries a high risk of progression and deserves close monitoring. Furthermore, specific genes seem to play a more significant role than others. Splicing factors III , TP53, IDH1/2, PHF6, RUNX1, the total number of mutations, and VAF ≥ 10% conferred a higher hazard ratio to progression than DNMT3A, ASXL1 or TET2. 21,22 Cancer evolution is a multistep process IV in which the accumulation of DNA lesions in tissues' indolent self-renewing stem cells induces clonal expansion. Once sufficient driver mutations V happen in subclones, an overt neoplasm develops. Eventually, additional somatic lesions in neoplastic cells lead to an aggressive disease.

Neoplastic cells evolve from premalignant to malignant. These concepts help to face CH as a multistep process, from a non-malignant predisposing condition to an overt myeloid neoplasm through a pre-myelodysplastic stage (Figure 6). Single-cell analysis showed that founder HSC mutations are silent and followed by serial acquisition of clones and mutations, preceding overt myeloid malignancy. 23 Although single-cell studies are not yet included in routine clinical practice, NGS is accessible and used in the workup of cytopaenias. Clinicians needed new nosologic entities to categorise CH patients without sufficient criteria for myeloid neoplasm who are, nonetheless, premalignant: ARCH: age-related clonal hematopoiesis 19 . It is defined as the expansion of an HSC clone harbouring "specific, disruptive, and recurrent genetic variants in individuals without a clear diagnosis of haematological malignancies" 24 . Its definition overlaps with clonal haematopoiesis of indeterminate potential (CHIP). The absolute risk of malignant transformation is low.

CHIP: identification of a clonal mutation associated with hematologic neoplasms VI with a VAF ≥ 2% in an individual with normal or near-normal full blood counts not meeting WHO 2016 criteria for hematologic neoplasm, PNH, monoclonal gammopathy of undetermined significance, or monoclonal B lymphocytosis. 25 The risk of progression is nearly 1%/year. 21 CCUS: clonal cytopaenias of undetermined significance. It is generally assumed as CHIP with at least one cytopaenia. 25 However, some authors prefer to use a higher VAF cut-off (20%) to separate from other cytopaenias that co-occur with incidental and not causal small CHIP. 26 ICUS: idiopathic cytopenia of undetermined significance. It describes a condition without evidence of clonality not fulfilling the WHO-2016 criteria for MDS. 27 III SF3B1, SRSF2, U2AF1, ZRSR2, SF1, PRPF8 IV Knudson hypothesis or two-hit theory: Alfred Knudson was the first to hypothesis that cancer develops in a multi-step genetic process. He proposed that sequential genetic "hits" / mutations were needed to the development of cancer 174 . V Driver mutations: "changes in the DNA sequence of genes that cause cells to become cancer cells and grow and spread in the body" 175 as opposed to passenger mutations, which do not confer any adaptative advantage. Discriminating between driver and passenger mutations can be very difficult and relies on the sequencing method, analytical pipeline, filtering, annotation and in silico prediction of pathogenicity 26 .

VI ASXL1, BCOR, BCORL1, BRAF, CALR, CDKN2A, CREBBP, CBL, CUX1, DNMT3A, EZH2, ETV6, FLT3, GATA2, GNAS, GNB1, IDH1, IDH2, IRF1, JAK2, JARID2, KDM6A, KIT, KRAS, KTM2A/MLL, MPL, MYD68, NOTCH1, NPM1, NRAS, PHF6, PPM1D, PTEN, PTPN11, PRPF8, RUNX1, SETBP1, SETDB1, SETD2, SF1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2 21,24,25 . This list is constantly evolving and is not exhaustive.

CHOP: clonal haematopoiesis with substantial oncogenic potential. It is CHIP with high-risk and persistent disease-determining abnormalities (e.g., JAK2, KIT, KRAS, HRAS, FLT3). 28 CHOP is still an evolving definition.

MDS: myelodysplastic syndromes. CH with cytopaenias and dysplastic changes in at least 10% of cells of one lineage in the bone marrow. Different syndromes confer different risks to progression into acute myeloid leukaemia (AML). MDS diagnostic criteria are defined by the 2016 revision of the World Health Organization classification of myeloid neoplasms and acute leukemia. 29 Several tools assess the risk of MDS transformation into AML. The most widely used (Revised International Prognostic Scoring System -R-IPSS) combines clinical and cytogenetic data. 30 In 2022, a newer revision of the scoring system included molecular data (IPSS-M). 31 Thus far, most of the molecular data available derive from bulk analyses and data from single-cell studies for CH/CHIP/ARCH is still scarce -the need for longitudinal samples over a long follow-up being a crucial limitation. scDNAsequencing confirmed that DNMT3A and TET2 are the most frequent driver mutations and that the number of unique clones in overt myeloid malignancies is higher than in CH. 32 On the other hand, the number of mutations within the dominant clone is not different, indicating that the mutation burden is not the main driver of leukemogenesis. 32,33 The overrepresentation of DNMT3A, TET2, and ASXL1 among CH suggests that epigenetic regulators mediate clonal dominance. Interestingly, the coexistence of such mutations is rare within the same clone in CH and frequent in overt myeloid malignancies. 32 For instance, clones with both DNTMT3A and TET2 mutations in CH showed higher VAFs. 34 These data support that the combination of some mutations have a synergistic effect, driving clonal dominance from premalignant to malignant states.

Bone marrow failure syndromes

The hallmark of bone marrow failure syndromes is pancytopenia because of the incapacity of the haematopoietic tissue to sustain haematopoiesis. Historically, this group of diseases was called aplastic anaemia. The first documented cases date from the late XIX and early XX centuries, without any aetiopathological distinction.

Thus far, three main aetiopathologies have been described: direct cellular toxicity: drugs, radiation, or chemicals.

• Idiopathic: autoimmune.

• Inherited: intracellular biological pathway dysfunctions (DNA repair, ribosome or telomere biology).

I will focus on idiopathic and inherited bone marrow failure syndromes in this work.

Idiopathic aplastic anaemia

This condition is also known as immune / acquired aplastic anaemia, or plainly, aplastic anaemia. Aplastic anaemia (AA) is a rare disease (ORPHA 88), with an incidence of 2 cases per one million inhabitants and a prevalence of 0.0004% in Western countries. 35,36 For unknown reasons, it is two to three times more frequent in Asia. 37 AA affects both men and women equally and has a bimodal age distribution, with the first peak among young adults and a second peak among patients over 50. The classic clinical findings are secondary to pancytopenia: fatigue, bleeding, and infection.

Pathophysiology

The first evidence of immune pathophysiology for aplastic anaemia came from a clinical observation: Mathé and Schwarzenberg noted an autologous reconstitution following allogeneic cell transplantation with anti-thymoglobulin (ATG) and concluded that immunosuppression was responsible for the response 38 .

The first functional demonstration of the immune pathophysiology came from the Louis Wasserman team in 1977: peripheral blood lymphocytes from five out of seven aplastic anaemia patients induced approximately 80% reduction in erythroid colony formation in semi-solid co-culture assay 39 . The demonstration of cytotoxic Tcell mediated destruction of autologous bone marrow mononuclear cells further strengthened the immune hypothesis [40][START_REF] Nakao | Isolation of a T-cell clone showing HLA-DRB1*0405-restricted cytotoxicity for hematopoietic cells in a patient with aplastic anemia[END_REF][START_REF] Risitano | In-vivo dominant immune responses in aplastic anaemia: Molecular tracking of putatively pathogenetic T-cell clones by TCR β-CDR3 sequencing[END_REF] . These autoreactive T cells, enriched in the patient's bone marrow environment, [START_REF] Melenhorst | T cells selectively infiltrate bone marrow areas with residual haemopoiesis of patients with acquired aplastic anaemia[END_REF] synthesise TNF-a and IFN-g, cytokines that inhibit haematopoiesis and upregulate Fas expression in haematopoietic cells [START_REF] Maciejewski | Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia[END_REF] , favouring apoptosis. Thus far, the triggers of the immune process leading to bone marrow failure are unknown.

Treatment

Aplastic anaemia can be fatal if left untreated. First-line treatment consists of haematopoietic cell transplantation for eligible patients younger than 40 years of age for whom a fully matched HLA sibling is apt to donate 37,[START_REF] Bacigalupo | How I treat acquired aplastic anemia[END_REF][START_REF] Peffault De Latour | Transplantation for bone marrow failure: current issues[END_REF] . However, such a donor is unavailable for nearly one-third of patients. Furthermore, many patients are older or not eligible for upfront transplantation. Triple therapy with horse ATG and cyclosporine [START_REF] Bacigalupo | How I treat acquired aplastic anemia[END_REF][START_REF] Scheinberg | Horse versus rabbit antithymocyte globulin in aplastic anemia [5[END_REF] , and, more recently, eltrombopag [START_REF] Peffault De Latour | Eltrombopag Added to Immunosuppression in Severe Aplastic Anemia[END_REF] yielded the best results: nearly two-thirds of overall response.

Relapsed or refractory patients may respond to a second cycle of immunosuppression. However, the results are disappointing. Eltrombopag alone can restore some degree of haematopoiesis and render 40% of patients transfusionindependent. [START_REF] Townsley | Eltrombopag restores trilineage hematopoiesis in refractory severe aplastic anemia that can be sustained on discontinuation of drug[END_REF][START_REF] Olnes | Eltrombopag and Improved Hematopoiesis in Refractory Aplastic Anemia[END_REF] A matched sibling or unrelated transplantation is the preferred choice if eligible. [START_REF] Bacigalupo | How I treat acquired aplastic anemia[END_REF] If a matched donor is unavailable, an alternative transplant is an option. We and others reported good results using haploidentical transplantation using posttransplant cyclophosphamide for relapsed/refractory patients (Annexe III). [START_REF] Prata | Haploidentical transplantation and posttransplant cyclophosphamide for treating aplastic anemia patients: a report from the EBMT Severe Aplastic Anemia Working Party[END_REF][START_REF] Esteves | Haploidentical BMT and post-transplant Cy for severe aplastic anemia: a multicenter retrospective study[END_REF][START_REF] Arcuri | Impact of CD34 Cell Dose and Conditioning Regimen on Outcomes after Haploidentical Donor Hematopoietic Stem Cell Transplantation with Post-Transplantation Cyclophosphamide for Relapsed/Refractory Severe Aplastic Anemia[END_REF][START_REF] Clay | Nonmyeloablative Peripheral Blood Haploidentical Stem Cell Transplantation for Refractory Severe Aplastic Anemia[END_REF][START_REF] Dezern | Haploidentical BMT for severe aplastic anemia with intensive GVHD prophylaxis including posttransplant cyclophosphamide[END_REF] Prospective phase 2 trials are currently ongoing.

Patients who fail salvage treatment have a poor prognosis, and infection is the leading cause of death. [START_REF] Valdez | Decreased infection-related mortality and improved survival in severe aplastic anemia in the past two decades[END_REF]

Aplastic anaemia animal models

As an acquired disease of elusive aetiology, the use of animal models is less reliable than for FA. To mimic pancytopenia, some models were based on exposure to chemical agents, but they were insufficient to study the classic mechanism of AA, the immune-mediated destruction of HSCs. Therefore, models based on mismatched hematopoietic cell transplantation resulted in cytopenias and were used to understand marrow destruction better. [START_REF] Scheinberg | Aplastic Anemia_ What Have We Learned From Animal Models and From the Clinic[END_REF] However, one confounding factor is graft-versus-host disease. Considering the mouse models available thus far for studying immune AA, we based our study on clonal evolution on direct observation of patient data.

Inherited bone marrow failure syndromes

The inherited bone marrow failure syndromes (IBMF) comprise different genetic diseases, affecting distinct biological pathways that converge to cellular stress and apoptosis. Marrow failure is only one aspect of otherwise systemic syndromes. The most frequent are Fanconi Anaemia (FA), Shwachman-Diamond syndrome (SDS) Diamond-Blackfan Anaemia (DBA), and telomere biology diseases (TBD, e.g., Dyskeratosis Congenita). The true incidence of these diseases is unknown: variable penetrance, limited availability of genetic testing, misleading beliefs that the clinical picture must start at childhood, and somatic mosaicism contribute to underestimate the real epidemiology of IBMF (Table 1).

The correct diagnosis of IBMF prior to treatment initiation is of utmost importance, for IST is ineffective; haematologists must adapt HSCT conditioning regimen and donor screening accordingly. The initial work-up includes, at least, chromosomal breakage tests (FA), pancreatic function tests (SDS), and telomere length (TBD). Targeted genetic sequencing to look for germline mutations are becoming widely available and some centres defend haematologists must screen all patients with BMF. Unrecognised IBMF would affect a significant number of transplanted patients and be a risk factor for treatment related mortality. 58

Table 1: Estimated incidences of different BMF

Syndrome

Incidence (x10 6 newborns) FA [START_REF] Dokal | Inherited bone marrow failure syndromes[END_REF] 11.4 SDS [START_REF] Minelli | Incidence of Shwachman-Diamond Syndrome in Italy[END_REF] 6 DBA [START_REF] Dokal | Inherited bone marrow failure syndromes[END_REF] 5 TBD [START_REF] Dokal | Clinical utility gene card for: Dyskeratosis congenita -update 2015[END_REF] 1 AA 35,36 2 (general population)

Some IBMF increase the risk of solid malignancies and all of them are associated with clonal haematopoiesis and progression to MDS/AML, the archetype being Fanconi Anaemia.

Fanconi Anaemia

Guido Fanconi described pancytopenia and physical anomalies within a family in 1927. [START_REF] Fanconi | Familiare infantile perniziosaartige anamie (pernizioses blutbild und konstitution)[END_REF] Besides marrow failure and physical abnormalities, the presenting sing of FA can also be solid tumours. The clinical picture might be heterogeneous within the family, suggesting other factors might influence disease course (epigenetic, environmental).

FA pathway is crucial for interstrand crosslinks DNA-repair. To date, 22 different proteins and many other associated proteins play a role in this pathway (Tables 2 and3). Figure 7 represents the FA-core complex, an assembly of eight FA-proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FAAP100), [START_REF] Shakeel | Structure of the Fanconi anaemia monoubiquitin ligase complex[END_REF] that is recruited by FANCM to DNA crosslinks and, in conjunction with FANCT, monoubiquitinates two proteins (FANCD2 and FANCI). This ubiquitinated heterodimer recruits the FA-downstream proteins, that correct the crosslink by nucleotide excision repair and homologous recombination (HR). Biallelic inactivations in any of the 22 FA genes disrupt the pathway and cause chromosome instability and accelerated cellular aging. It is the most frequent cause of bone marrow failure syndrome in the world. Other hallmarks of the disease are cancer predisposition and malformations. The diagnosis relies on the presence of chromosome breakages in cytogenetic studies performed under genotoxic stress and the sequencing of the FANC genes in order to identify the causal mutations. FANCA is, by far, the most affected gene, accounting for more than 70% of cases of FA.

Nearly all FA patients will present hematologic abnormalities impacting prognosis and quality of life. [START_REF] Butturini | Hematologic abnormalities in Fanconi anemia: an International Fanconi Anemia Registry study[END_REF] Progressive haematopoietic failure starts before birth, and the median age of bone marrow failure diagnosis is eight years. [START_REF] Peffault De Latour | How I treat MDS and AML in Fanconi anemia[END_REF][START_REF] Risitano | Twenty years of the Italian Fanconi Anemia Registry: Where we stand and what remains to be learned[END_REF][START_REF] Garaycoechea | Why does the bone marrow fail in Fanconi anemia?[END_REF] FA hematopoietic cells undergo a p53/p21 response to stress and DNA damage, leading to progressive marrow failure since an very early age. [START_REF] Garaycoechea | Why does the bone marrow fail in Fanconi anemia?[END_REF][START_REF] Ceccaldi | Bone marrow failure in Fanconi anemia is triggered by an exacerbated p53/p21 DNA damage response that impairs hematopoietic stem and progenitor cells[END_REF] Reactive oxygen species (ROS) and especially aldehydes are an important source of endogenous DNA damage. [START_REF] Garaycoechea | Why does the bone marrow fail in Fanconi anemia?[END_REF] Inflammatory cytokines, such as TNFa and IFNg, contribute also to marrow failure [START_REF] Garaycoechea | Why does the bone marrow fail in Fanconi anemia?[END_REF] . Nearly half of non-transplanted FA patients will develop a myeloid neoplasm during their lifetime. The prognosis of FA MDS/AML patients is unfavourable. [START_REF] Ayas | Allogeneic hematopoietic cell transplantation for fanconi anemia in patients with pretransplantation cytogenetic abnormalities, myelodysplastic syndrome, or acute leukemia[END_REF][START_REF] Giardino | Outcome of patients with Fanconi anemia developing myelodysplasia and acute leukemia who received allogeneic hematopoietic stem cell transplantation: A retrospective analysis on behalf of EBMT group[END_REF] Overall, hematologic complications account for nearly 70% of FA deaths. [START_REF] Risitano | Twenty years of the Italian Fanconi Anemia Registry: Where we stand and what remains to be learned[END_REF] The cumulative incidence of solid tumours by the age of 30 is about 30% and account for significant morbi-mortality. [START_REF] Risitano | Twenty years of the Italian Fanconi Anemia Registry: Where we stand and what remains to be learned[END_REF] Solid malignancies in FA are out of the scope of this work.

Standard treatment

The sole curative treatment for FA-associated haematologic complications is HSCT. [START_REF] Peffault De Latour | How we diagnose and treat MDS and AML in Fanconi anemia[END_REF] Historically, transplantation in the FA setting resulted in significant early (organ failure) and late (secondary malignancies) because of the conditioning regimen and graft-versus-host disease. However, reduced intensity regimens, adapted donor choice, and better immunosuppression has improved survival. [START_REF] Bonfim | Long-term Survival, Organ Function, and Malignancy after Hematopoietic Stem Cell Transplantation for Fanconi Anemia[END_REF] Transplantation yields better survival if performed before myeloid malignancy and 14 years of age. [START_REF] Peffault De Latour | How we diagnose and treat MDS and AML in Fanconi anemia[END_REF] Androgens are an alternative to patients unsuitable for transplantation or with milder marrow failure [START_REF] Dufour | How I manage patients with Fanconi anaemia[END_REF] . However, it cause liver adenoma and might boost premalignant clones, and progression towards MDS/AML is a risk. [START_REF] Paustian | Androgen therapy in Fanconi anemia: A retrospective analysis of 30 years in Germany[END_REF] Transplantation replaces recipient's defective haematopoiesis by donors' healthy cells, reducing the risk of progression. [START_REF] De Latour | Allogeneic hematopoietic stem cell transplantation in Fanconi anemia: The European Group for Blood and Marrow Transplantation experience[END_REF]

Fanconi anaemia models

After discovering the Fanconi anaemia pathway and the genetic aetiology of the disease, many researchers looked for orthologs among other species. Ancestor FA pathways are present since lower eukaryotes, such as funghi 76 and plants [START_REF] Knoll | The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis[END_REF] , followed by progressively more advanced systems in nematodes 78 , arthropods [START_REF] Marek | Drosophila homologs of FANCD2 and FANCL function in DNA repair[END_REF] , and chordates [START_REF] Stanley | The Simple Chordate Ciona intestinalis Has a Reduced Complement of Genes Associated with Fanconi Anemia[END_REF] . Among vertebrates, the FA pathway is strikingly conserved 81 (Table 2). The FA-core complex evolved later in the phylogenetic tree. Of the most frequently used animal models, the rodent's FA-pathway is closest to man's. All 22 human FANC genes are also present in mice with over 70% homology (Table 3). Therefore, scientists developed transgenic knock-out mice for nearly all FANC genes to study the FA pathway and disease [START_REF] Dong | Update of the human and mouse fanconi anemia genes[END_REF] . Mice studies have been central to the study of FA, allowing not only to grasp the intricacies of protein interactions [START_REF] Van De Vrugt | Evidence for complete epistasis of null mutations in murine Fanconi anemia genes Fanca and Fancg[END_REF] , but also the development of newer and promising therapeutics, such as gene therapy [START_REF] Río | Engraftment and in vivo proliferation advantage of gene-corrected mobilized CD34+ cells from Fanconi anemia patients[END_REF] . Broadly, although the cells reproduce the FA phenotype (e.g., chromosomal breakage under genotoxic stress and hypersensitivity to mitomycin), most models do not present the classical hallmarks of FA disease (spontaneous bone marrow failure and cancer development). All FA mice models are hypofertile, and many have a high embryonic or perinatal mortality, and it is associated with the background. The C57BL/6 background, the most widely used inbred strain in haematopoiesis studies, is associated with a more severe lethality phenotype [START_REF] Van De Vrugt | Embryonic lethality after combined inactivation of Fancd2 and Mlh1 in mice[END_REF] . Curiously, FA-mouse models do not present spontaneous, severe, and progressive bone marrow failure in most FA patients, except Brca2 D27/D27 86 and Fancp -/-. [START_REF] Crossan | Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia[END_REF] Thus far, the precise mechanisms by which FA mouse models sustain normal haematopoiesis and fail to present spontaneous tumorigenesis even upon ageing is unknown. Arguably, the clean environment of research animal facilities might play a protective role. Notwithstanding, our team induced BMF in a C57BL/6J Fancg -/-by treating mice with chronic exposure to polyinosinic-polycytidylic acid (pI:pC), a synthetic analogue of double-stranded RNA that stimulates inflammation (Sebert et al., in revision). Both human and murine FANCG have 14 exons, present nearly 80% homology, and encode the FANCG protein (622 and 623 amino acids, respectively). Both human and mouse FANCG are ~65kDa proteins with four tetratricopeptides (TPRs, proteinprotein interaction sites). [START_REF]UniProt Blast: Fancg_mouse[END_REF] The murine model I used in this project (Fancg -/-) was previously described and backcrossed into C57BL/6J Ly5.2 over 20 times at the IRSL. In short, the exons 1-4 were deleted, creating a Fancg knock-out model (Figure 8). [START_REF] Koomen | Reduced fertility and hypersensitivity to mitomycin C characterize Francg/Xrcc9 null mice[END_REF] These mice have reduced fertility, and heterozygous couples give rise to less than 10% KO instead of the expected 25% -KO mice are infertile. This submendelian ratio can be explained by gonadal abnormalities and perhaps high embryonic mortality between E12.5 and E14.5. Besides, we observed that C57BL/6J Fancg KO are smaller and have craniofacial and ophthalmic abnormalities at birth (Figure 9). Haematologywise, these mice, like most FA mouse models, do not present spontaneous cytopaenias, or any kind of neoplasm, up to the age of 24 months. However, they do become cytopenic under chronic inflammation (Sebert M et al., in revision) as the Fanca -/-model. [START_REF] Walter | Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells[END_REF] Although this Fancg -/-presents normal blood and marrow counts, it has 30% less LT-HSC than its wild-type counterparts. In vitro, Fancg -/-CFU-assays demonstrated a proliferative defect (smaller and fewer colonies). In vivo, competitive transplantation experiments (Fancg -/-vs WT) injected into lethally irradiated mice showed that WT outcompeted KO cells at 24 weeks. [START_REF] Barroca | Impaired functionality and homing of fancg-deficient hematopoietic stem cells[END_REF] Together, these data indicate a haematopoietic defect of Fancg -/-mice. Fancg exons 1-4 were deleted by homologous recombination in a 129/Ola embryonic stem cell. The ES129/Ola Fancg -/-was implanted in pseudopregnant females and inbred in the FVB background. [START_REF] Koomen | Reduced fertility and hypersensitivity to mitomycin C characterize Francg/Xrcc9 null mice[END_REF] Subseqyently, FVB Fancg -/-were outcrossed with C57BL/6j and more than 20 backcrosses for obtaining coisogenic strains. Clonal evolution into MDS/AML is the most concerning late event in severe acquired aplastic anaemia, and it occurs in approximately 15% of long-term responders to immunosuppressive therapy [START_REF] Scheinberg | How I treat acquired aplastic anemia[END_REF][START_REF] Socie | Malignant tumors occurring after treatment of aplastic anemia. European Bone Marrow Transplantation-Severe Aplastic Anaemia Working Party[END_REF] . In 1967, William Dameshek asked: "What do Aplastic Anemia, Paroxysmal Nocturnal Hemoglobinuria, and Hypoplastic Leukemia Have in Common?". Despite a long-known association, the precise mechanisms involved in malignant clonal evolution are not fully elucidated. While chromosomal abnormalities such as monosomy 7 and trisomy 8 are considered drivers of transformation, [START_REF] Maciejewski | Distinct clinical outcomes for cytogenetic abnormalities evolving from aplastic anemia[END_REF][START_REF] Yoshizato | Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia[END_REF] the potential role of somatic genetic variants is unclear to date.

Species D2/I heterodimer

Homo sapiens A B C D E F G L M T yes D1 J N O P Q R S U V W Mus musculus A B C D E F G L M T yes D2 J N O P Q R S U V W Danio rerio A B C D E F G L M T yes D2 J N O P Q R U V W Drosophila melanogaster L M T yes O P Q R U V Caenorhabditis elegans T yes J O P Q R S Saccharomyces cerevisiae M T no J O P Q R FA core complex Downstream proteins
Half to a third of acquired aplastic anaemia patients have a detectable mutated clone at SAA diagnosis [START_REF] Peffault De Latour | Eltrombopag Added to Immunosuppression in Severe Aplastic Anemia[END_REF][START_REF] Yoshizato | Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia[END_REF][START_REF] Ogawa | Clonal hematopoiesis in acquired aplastic anemia[END_REF] . As expected, the prevalence of mutations increased with age. At baseline, the most frequent mutations are among PIGA, BCOR, BCORL1 or HLA genes (Figure 10). Among genes associated with myeloid neoplasms, ASXL1, RUNX1, and DNMT3A are frequently found with VAF ≤ 10%. 21 Data suggest that PIGA, BCOR or BCORL1 would be associated with a better response to immunosuppressive treatment. [START_REF] Yoshizato | Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia[END_REF] On the other hand, the univariate analysis indicated that baseline myeloid disease-associated gene mutations would be associated with worst survival in a fashion similar to the aged bone marrow found in ARCH/CHIP. [START_REF] Ogawa | Clonal hematopoiesis in acquired aplastic anemia[END_REF][START_REF] Kulasekararaj | Somatic mutations identify a subgroup of aplastic anemia patients who progress to myelodysplastic syndrome[END_REF] Less than a half of AA patients treated with the state-of-the-art immunosuppression (horse ATG + cyclosporine + eltrombopag) present a complete remission. [START_REF] Peffault De Latour | Eltrombopag Added to Immunosuppression in Severe Aplastic Anemia[END_REF] Therefore, for most AA patients, some degree of cytopenia will persist for many years. The absence of complete response is a risk factor for progression. [START_REF] Socie | Malignant tumors occurring after treatment of aplastic anemia. European Bone Marrow Transplantation-Severe Aplastic Anaemia Working Party[END_REF] Stress haematopoiesis and chronic inflammation of the marrow's microenvironment might profit myeloid-disease-associated mutants, as demonstrated in animal models. [START_REF] Muto | TRAF6 functions as a tumor suppressor in myeloid malignancies by directly targeting MYC oncogenic activity[END_REF][100][101][102][103] Because of the rarity of SAA, populational studies are not feasible. Thus far, no study confirmed nor rejected the findings for progression in CHIP (near-normal haematopoiesis, analogy to complete response) nor CCUS (persistent cytopenias, analogy to absence of complete response). Among CCUS patients, the 5-year risk of progression for patients with mutations among U2AF1, ZRSR2, SRSF2, JAK2, or RUNX1 can be as high as 80%. 104 For other CHIP/ARCH-associated mutations (TET2, DNMT3A, or ASXL1), the risk is inferior to 50%. 104 Therefore, CCUS and nontransplanted AA patients must regularly be screened for MDS/AML. Cytogenetics-wise, monosomy 7 is much more frequent among transformed AA patients than primary MDS and confers a high risk of evolution to AML. [START_REF] Maciejewski | Distinct clinical outcomes for cytogenetic abnormalities evolving from aplastic anemia[END_REF][START_REF] Ogawa | Clonal hematopoiesis in acquired aplastic anemia[END_REF] Other cytogenetic abnormalities confer different risks of AML transformation between primary MDS and progressed AA: del(13q), a WHO 2016 MDS-defining cytogenetic abnormality confers intermediate risk for MDS patients, 29 whereas it seems favourable among AA patients and linked to PNH and immune escape. 31,105 On the other hand, del(7q) seems to confer a poor prognosis among progressed AA patients (Cleveland Clinic, personal communication, manuscript to be submitted), whereas it is classified as intermediate for primary MDS. 30

Clonal evolution and immune escape: the Luzzatto-Rotoli hypothesis

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, monogenic disease that manifests classically as intravascular hemolysis, bone marrow failure, and thrombosis. It is caused by somatic mutations in the PIGA gene, resulting in impaired glycosylphosphatidylinositol (GPI) biosynthesis. 106 GPI anchors many proteins on cells' surfaces, including the decay-accelerating factor (CD55) and the membrane inhibitor of reactive lysis (CD59) -modulators of the alternative and terminal complement pathway. Impaired GPI synthesis (GPI -) leads to complement-mediated hemolysis, the hallmark of PNH. 107 Nearly 30% of AA patients have a detectable PNH clone, and PIGA mutation can also be found among healthy individuals. In other words, acquiring a PIGA mutation alone is insufficient for PNH disease. The mechanisms underlying GPI -clone expansion are unclear since GPI deficiency does not seem to confer any survival or proliferative advantage in animal models 108 .

The most widespread hypothesis to justify GPI -clonal dominance is the bottleneck effect VII : GPI -cells would resist the immune-mediated haematopoietic cell destruction seen in AA. The remaining PIGA-mutated clones would therefore expand, helping to sustain haematopoiesis. [START_REF] Ogawa | Clonal hematopoiesis in acquired aplastic anemia[END_REF]109 This immune escape theory is known as the Luzzatto-Rotoli hypothesis (Figure 11). 110 Although the immune escape explains the clonal dominance of GPI -in the AA micro-environment, it is less satisfactory to explain classic PNH patients (haemolysis without AA), since there is no evident bottleneck effect. Classic putative intrinsic drivers of clonal evolution as telomere shortening 111 and somatic mutations are more frequent in GPI -clones 112 and might help contribute to GPI -clonal dominance. Thus far, other factors such as environmental or inherited predisposition to PNH are unknown.

VII Bottleneck effect: the way in which a reduction and subsequent increase in a population's size affects the distribution of genetic variation among its individuals 176 . We described for the first time that rare complement factor H (CFH) variants were overrepresented among PNH patients and that such variants were associated with suboptimal response to anti-C5 treatment. Factor H is a negative regulator of the alternative pathway. 113 Specific CFH variants may downsize factor H activity, upregulating the alternative pathway. The functional consequences of the reported CFH variants and their possible role in GPI -clones clonal dominance are incompletely known to date. Our data supported that CFH plays a pathophysiological role in PNH, and its protection is necessary for the positive selection of GPI -cells. I submitted this work to Blood (brief communication), which is currently under revision. I include a nonpeer-reviewed version of the manuscript in Annexe I. Noteworthy, none of the patients with a rare CFH variant evolved to a myeloid malignancy (median follow-up of 5.8 years).

PNH is a risk factor for myeloid malignancies, with more than 100-fold risk compared to an age-matched population 114 . A PNH clone can be found in up to 20% of patients at primary MDS diagnosis 115 AA/PNH had a much higher risk than classic PNH patients (HR 48.8 vs 2.2, p<0.001). The overall 10-year cumulative incidence of MDS/AML was 3.8% (95% CI; 0.1-7.5). 114 Based on the data mentioned above (association of PNH with myeloid malignancies and putative drivers of clonal evolution in GPI -cells), one should expect that the premalignant clone arises directly from PNH cells. Surprisingly, the evidence goes otherwise: cytogenetic abnormalities found among PNH patients occur in GPI + cells, 116 GPI -and MDS/AML clones seem to originate from distinct founders, 117 and the presence of PIGA-mutated clone grants a better progression-free and overall survival. [START_REF] Yoshizato | Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia[END_REF] So far, the data supports that the leukemic cells derive from a PIGA-wt (GPI+) clone (Figure 12). In other words, premalignant and PNH clones co-exist, and the former eventually outcompete the latter.

The Luzzatto-Rotoli hypothesis: beyond GPI-deficient cells

Besides PNH, the immune escape hypothesis also explains another frequent clonal evolution in AA. Nearly one-third of AA patients present clones with loss of HLA class I expression (HLA -) either by chromosome 6p loss of heterozygosity or loss-offunction mutations at diagnosis (Figure 13). 119,120 The HLA -cells would similarly escape T cell-mediated cytotoxicity as GPI -cells, explaining clonal haematopoiesis. Moreover, some HLA class I alleles are overrepresented in aplastic anaemia irrespective of the genetic background (HLA*B 40:02, for instance). [120][121][122] The overrepresentation of specific class I alleles among AA patients consists of strong evidence of its influence on AA pathophysiology. 123 The link between HLA expression and cancer is known for solid malignancies such as melanoma, and lymphoid neoplasms, such as Hodgkin lymphoma and chronic lymphocytic leukemia. 124-126 Also, researchers have described HLA class I loss in de novo AML/MDS, and this was allegedly related to progression from MDS to AML. 127 Vago and colleagues identified AML relapses after a haploidentical hematopoietic stem cell transplantation who have lost the non-identical HLA haplotype. 128 These leukemic cells would have a selective advantage, escaping immunologic pressure from alloreactive donor T cells. In like manner, Christopher and colleagues identified patients with chronic myeloid leukaemia who relapsed after transplantation that have a down-regulated HLA class II expression at relapse. 129 It was initially proposed that such HLA alleles or abnormalities could be associated with a more severe clinical course and evolution to MDS in the context of AA. 121,130 Although GPI -and HLA -clones can expand through the natural history of AA patients, driver mutations are hardly seen among HLA-cells. 122 Nakao's team studied 633 patients with AA and found that 127 had HLA -clones. Interestingly, none of these patients progressed to a myeloid malignancy after a follow-up of 4.2 years. In contrast, 17 patients without HLA -clones transformed to MDS/AML. HLA -was an independent negative predictor for progression (HR: 2.82×10 -5 , p-value < 0.001). 131

A B

In summary, HLA -or GPI -somatic mosaicism VIII can expand in the context of AA, help rescue blood counts, and are probably not drivers of transformation (Figure 13). Clonal haematopoiesis is frequent among IBMF and patients have an elevated risk of progression to MDS/AML. However, as for acquired marrow failures, CH confer different risks. Each syndrome has its own particularities, depending on the germline biological deficit. Cell competition can sometimes be protective: some patients might spontaneously acquire a genetic correction of the abnormal cellular pathway (a reversion) 133 or another somatic event can attenuate the cellular impact of the genetic deficit (a compensation). 133 Clones acquiring mutations that restore cellular function might be less susceptible to DNA error accumulation, i.e., lower risk of progression. Therefore, CH is not synonym to cancer.

CH in DKC frequently occurs without driver mutations, perhaps reflecting neutral drift or unknown adaptative mechanisms. 133 Indeed, the most frequent events detected in TBD are reversion or compensation (e.g., TERT promoter mutations leading to enhanced telomerase activity). 134 In SDS, patients frequently acquire i(7q) or del(20q). The first encompasses SBDS, increasing transcription, and the latter induces EIF6 haploinsufficiency. 135 In both cases, the near normal ratio SBDS:EIF6 ameliorates HSPCs fitness. i(7q) and del(20q) seem not to be pre-malignant in SDS; somatic TP53 drive transformation in SDS. 136 The risk of MDS/AML is extremely high in non-transplanted FA patients. 137 Leukemogenesis seems to be somewhat linear. [START_REF] Peffault De Latour | How we diagnose and treat MDS and AML in Fanconi anemia[END_REF] The earliest, and most frequent, VIII Somatic mosaicism: the occurrence of two genetically distinct populations of cells derived from a somatic mutation within an individual affecting a single tissue (e.g., the blood) 177 . cytogenetic abnormality is trisomy 1q (pre-neoplastic state). Trisomy 3q, chromosome 7 abnormalities, and RUNX1 mutations occur later in the disease course and harbinger myeloid malignancies (Sebert M. et al, in revision, 2022). 138 On the other hand, a minority of patients present a milder hematologic phenotype: a mosaicism of spontaneously corrected cells coexist with FA-deficient cells. 139 Our team has previously studied a cohort of 350 FA patients, described revertants' clinical and molecular characteristics, and defined the genetic mechanisms that explained reversion. 140,141 Thus far, revertant patients not only have subnormal full blood counts, but also have not progressed to MDS/AML (Figure 15).

The diagnosis of functional reversion in FA is straightforward: the chromosomal breakage test correction proves that the pathway is restored. 139 Furthermore, demonstrating FANCD2 monoubiquitination in a FA-core patient proves the functionality of the pathway. 140,141 FA-core patients are unable to monoubiquitinate FANCD2 and, the presence in blood and the absence in cultured fibroblasts of monoubiquinated FANCD2 demonstrates the somatic mosaicism (Figure 14). For some FA patients, the underlying biological mechanism of phenotype attenuation is still unknown. In FA cell lines, SLFN11 depletion reduced cell cycle arrest and lower sensitivity to mytomicin. 143 Likewise, haematopoietic cells from patients with attenuated phenotype present abrogation of the G2 arrest and have lower expression of CHK1 and p53, protecting cells from apoptosis. Diversely from revertants, the attenuated patients evolve to MDS/AML. 142 Our team is currently identifying 8 patients with an attenuated phenotype for pairwise whole genome sequencing (blood and cultured fibroblasts), trying to identify a genomic mechanism of compensation / attenuation.

In mice models of FA, TBD, or GATA2, constitutive cells were capable of sustaining haematopoiesis during homeostasis. However, in competitive transplantation with wt cells, constitutive cells lost clonal competition and were eliminated. [START_REF] Navarro | Hematopoietic Dysfunction in a Mouse Model for Fanconi Anemia Group D1[END_REF][START_REF] Barroca | Impaired functionality and homing of fancg-deficient hematopoietic stem cells[END_REF]133,144 The functional correction of a few hematopoietic stem cells might confer an ecologic advantage, mitigating the risk of pre-leukemic clonal dominance.

Gene therapy mimics somatic mosaicism in Fanconi anaemia

Inspired by Nature's somatic mosaicism, gene therapy is an advanced therapy medicinal product (ATMP) with very promising results for monogenic diseases. The use of an autologous cellular source overcomes the burden of graft-versus-host disease, rejection, and the need of long-lasting immunosuppression. The FDA and EMA have already approved ATMPs for thalassemia, adenosine deaminase severe combined immunodeficiency, and metachronic leukodystrophy.

The first platform used for gene-therapy in haematologic disorders used γ retroviral vectors (γRV derived from Moloney murine leukaemia virus -MLV) for gene delivery. This strategy was rather genotoxic (insertional mutagenesis IX ) and was deem causal for 19 acute leukaemias / MDS. 145 γRV integrates non randomly, preferentially IX Insertional mutagenesis happens when viral cDNA integrates in host genomic DNA, deregulating neighbour genes. When it deregulates oncogenes or tumour suppressor, insertional mutagenesis can lead to haematologic malignancies. 178 LV vetors are less genotoxic and, 147 thus far, have not been causal to secondary leukaemia. 145 Notwhitstanding, at least two patients developed AML following LV-gene therapy with busulfan conditioning regimen for sickle cell disease, and neoplastic cells contained the LV insert in one of these cases (close to the vesicle-associated membrane protein 4, VAMP4 -no known association with myeloid malignancies). Strikingly, both cases presented similar features (-7, PTPN11, and RUNX1 mutations), but the abnormalities were not present at the time of gene therapy. In the case that leukaemic cells contained the viral insert, the genomic and transcriptional data were not in favour of gene-therapy causality. 148 The main hypothesis so far is that the leukaemia developed independently from the gene therapy and might be secondary to busulfan conditioning regimen.

Thus far, three clinical trials studied the safety and efficacy of gene therapy in FA: NCT01331018 (Fred Hutchinson, n=3), NCT03157804 (Madrid, n=9), and NCT03814408 (Rocket Pharmaceuticals, n=2). All included patients received ex vivo gene-corrected autologous CD34 + using self-inactivating PGK X lentivirus and no conditioning regimen -no reported deaths thus far. The Spanish team reported progressive engraftment of gene-edited HSPCs on 4/4 patients and a halt on the marrow failure process; they did not observe any genotoxic events (Figure 16). 149 The best timing for gene-therapy in FA is not yet defined. The FA patients treated with gene-therapy had already some degree of marrow failure at the time of treatment. However, FA haematopoietic dysfunction starts early in the ontogenesis, i.e., the multistep leukaemogenic process of clonal haematopoiesis is likely already ongoing at the BMF onset even if not detected with conventional cytogenetics. We do not know if the functional correction of a few hematopoietic stem cells confers an ecologic advantage, in other words, if the gene-corrected cells would outcompete the pre-existing undetected preleukemic clones, mitigating the risk of pre-malignant clonal dominance.

Gene-therapy arguably offers many advantages over HSCT for FA patients, notably reduced toxicity, no risk of GvHD, and perhaps standard risk of solid tumours. However, transplantation undeniably reduces the risk of evolution to MDS/AML. It is unclear whether gene-therapy would be the best therapeutic option to all FA patients considering the current good results of HSCT. Therefore, it is crucial to demonstrate that the gene-therapy's low treatment related mortality is followed by an also low risk of MDS/AML (Figure 17).

X Phosphoglycerate kinase is major player in the glycolytic pathway. This promoter, as housekeeping, allow for constitutive gene expression.

Thesis aims

I was interested in mechanisms explaining the emergence of clones bearing somatic mutations in bone marrow failure syndromes. From completing my clinical fellowship to starting the doctoral programme, I had many changes in my life, objectives, and thesis subject. All the proposals I present below resulted from discussions with my mentors.

My first project was entitled: "ER-stress: a potential therapeutic target in paroxysmal nocturnal haemoglobinuria". GPI-deficient cells, normally synthesise these proteins, though the endoplasmic reticulum does not adequately fold and use them; the exact mechanism of these proteins' disposal is unknown, but they may be either ubiquitinated and metabolized by the proteasome, processed by lysosomes or exported to the extracellular matrix. We hypothesized that the GPI-anchored proteins that are not anchored are processed by the ubiquitin-proteasome pathway, and we would evaluate whether the proteasome blockade induced apoptosis in human GPIdeficient cells. 150 Understanding what happens to the unused GPI-AP in GPI-deficient cells could allow us to understand better how these cells strive instead of being driven to apoptosis. Moreover, showing that GPI-deficient cells are more susceptible to apoptosis induced by proteasome blockade than non-GPI deficient cells could be of therapeutic interest, since new targets could be addressed in this pathway towards eradicating the PNH clone. We did not carry on this proposal because it was outside the scope of host laboratories.

My second proposal was to study the "Emergence of TERT promoter somatic mutations to rescue haematopoiesis in bone marrow failure syndromes". Acquired TERT promoter mutations have been initially described in solid tumours and mantle cell lymphoma. They are the most common non-coding acquired mutation observed in cancer. These mutations increase telomerase function, elongate telomeres in cancer cells, and modulate tumour proliferation and aggressiveness. Acquired TERT promoter mutations have been detected in leukocytes of approximately 5% of patients with idiopathic pulmonary fibrosis carrying a constitutional telomerase gene mutation. The study aimed to screen bone marrow failure patients carrying constitutional pathogenic telomere-associated gene mutations and their family members for somatic mutations in the TERT promoter. We planned to correlate the presence of somatic TERT promoter mutations with bone marrow cellularity, peripheral blood counts, telomere length, and evolution to MDS/AML. This proposal was completed by a collaborating team. 151 I continued to be interested in the emergence of clones bearing somatic

Hypothesis

Donor's hematopoiesis mutations to rescue haematopoiesis in bone marrow failure syndromes. In the light of papers Nakao's team had recently published (2017 / 2018), I have written the proposal entitled Clonal evolution in acquired aplastic anaemia: immune escape and somatic genomic alterations.

Hematopoietic cells that lost HLA class I expression in AA patients, by chromosome 6p loss of heterozygosity (6pLOH) or loss-of-function mutations in HLA genes, especially in HLA-B*40:02 are frequent among acquired aplastic anaemia patients. 119,120 These cells (HLA -) would escape T cell cytotoxicity, helping sustain haematopoiesis. In consonance with their findings, Babushok et al. showed that HLA-A*68:01, HLA-A*33:03, HLA-B*14:02, and HLA-B*40:02 alleles are overrepresented among AA patients and associate with a more severe clinical course and evolution to myelodysplastic syndromes (MDS). 8 Together, these results pointed to immune selection as central in the pathogenesis and clonal progression of acquired AA. Whereas chromosomal abnormalities such as monosomy 7, telomere attrition, and mutations are drivers of evolution from AA to AML/MDS, the potential role of immune escape was unknown. [10][11][12] The link between HLA and cancer is known for solid cancers and lymphoid neoplasms [13][14][15] . Researchers have described the HLA class I lost in de novo AML/MDS, allegedly related to progression from MDS to AML. 16 Vago et al. identified AML relapses after an HSCT that lost the non-identical HLA haplotype or deregulated T-cell recognition pathways. 17,18 These leukaemic cells would escape immunologic pressure from donor T cells. Likewise, Christopher and colleagues identified patients with chronic myeloid leukaemia who relapsed after HSCT that have a down-regulated HLA class II expression at relapse. 19 Altogether, we hypothesized that HLA played a role in malignant clonal evolution in AA. Our primary objective was to investigate whether the reduction or loss of HLA expression in hematopoietic cells in acquired aplastic anaemia patients is associated with AML/MDS transformation. As secondary aims, I would:

• identify the HLA alleles that were overrepresented among AA patients in our participating centres; • identify risk HLA alleles that were linked to malignant evolution in AA patients;

• determine which AML-associated genes were somatically mutated in the AML cells occurring after AA, in addition, or alternate to the known frequent events such as monosomy 7; • determine the gene expression profile of AML cells after AA, looking for immune-escape mechanisms (frozen samples).

Therefore, we conducted a pilot study in collaboration with the Histocompatibility laboratory of Saint-Louis Hospital, sequencing HLA classes I and II (NGSgo®-MX11-3, GenDx, Utrecht, The Netherlands -methods are in Supplementals of the JCO paper) of ten patients (nine acute myeloid leukaemias and one myelodysplastic syndrome with an excess of blasts type 2) who transformed from aplastic anaemia. Using the existing HLA pipeline, no mutation or loss of heterozygosity was found at the transformation into myeloid neoplasm. Lacking power to any conclusion, we collaborated with the Cleveland Clinic to increase the sample size and work out the analysis pipeline. Altogether, we have interpreted the HLA sequences of 10 patients from France and 10 from the United States. Each centre cross-checked the results: we confirmed that no mutation nor LOH was found among blastic patients. The hypothesis that the loss of HLA expression would not play a significant role in progression was further corroborated by Nakao's team's research published when we found our results. 131 Following these results, we have adapted our proposal, and I present the study in Chapter 2, section 2.1.

Given the negative results of the pilot study, my mentors and I developed an alternative plan to secure experimental work. I would develop a mice model of somatic mosaicism (genetic reversion) in Fanconi Anaemia. I aimed to test the fitness of various revertant or preleukemic subclones in Fanconi, mimicking in vivo the early stages of bone marrow progression. I present this project and the experiments I carried out in Chapter 2, section 2.2.

My proposals changed and evolved for various reasons, but always within the scope of clonal haematopoiesis in marrow failure syndromes and their impact on the evolution of MDS/AML. BMF patients with CH present very different natural histories, and I have focused on two questions: looking into the determinants of transformation in SAA/PNH and the impact of gene correction in FA on the risk of progression.

Chapter 2: Methods and Results

Clinical and molecular determinants of clonal evolution in aplastic anemia and paroxysmal nocturnal hemoglobinuria

Aim of the work

We and others studied the genomic landscape of patients with malignant evolution, focusing on genomic lesions known to have a role in myeloid neoplasms and bone marrow failures. A sub-analysis concerned the mutational study of the HLA region for all samples already sequenced for class I and II HLA genes. The potential role of immune evasion secondary to HLA loss and the evolution of myeloid malignancies was unknown.

The scientific question

In aplastic anaemia (AA)/paroxysmal nocturnal haemoglobinuria (PNH) patients, how do baseline clinical, cytogenetics, and molecular characteristics influence the risk of secondary myeloid neoplasm?

Results

We have established a collaboration between Cleveland Clinics in the USA, Saint-Louis Hospital, Paris, and University of Sao Paulo, Ribeirao Preto, Brazil. All three Institutional Review Boards approved this work.

I have included a non-edited version of the manuscript, in which I am co-first, with its supplementals in this chapter. This manuscript was accepted for publication in the Journal of Clinical Oncology on the 6 of July 2022 and published online on the 2 nd of September 2022.

INTRODUCTION

Aplastic anemia (AA) is a prototypic bone marrow (BM) failure syndrome manifesting with pancytopenia of autoimmune origin. 1 While hematopoietic stem cell transplant (HSCT) represents a curative option for younger cases, older patients and those lacking a suitable donor are treated with immunosuppressive therapy (IST). 2 Particularly in such patients, the refinement of IST during the last decades has dramatically improved survival outcomes. 3 Nevertheless, with increased numbers of survivors, a higher risk for clonal evolution to both myeloid neoplasia (MN) and paroxysmal nocturnal hemoglobinuria (PNH) emerged, corroborating the hypothesis described by Dr. Dameshek's riddle 50 years ago as to the intertwining relationship of such entities: "what do aplastic anemia, paroxysmal nocturnal hemoglobinuria and "hypoplastic" leukemia have in common?". 4,5 Indeed, retrospective studies with long follow-up revealed that up to 20% of patients with AA treated with IST may develop either myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). 6,7 These events are distinguished by dismal outcomes and absence of obvious clinical predictors. Furthermore, as it has happened for G-CSF in the past 8,9 , the use of thrombopoietin-receptor agonists has recently raised concerns as to the increased incidence of clonal evolution, further stressing the importance of studies aiming at the identification of predictors of such deadly complication. 10 SNP array karyotyping, and more recently deep massive parallel sequencing (NGS) revealed somatic mutations with a wide range of frequencies (5% up to 72%) in patients with AA and PNH according to the testing timing (diagnosis vs. follow-up), the number of interrogated genes, and technique (targeted vs. whole exome/genome sequencing). 6,[11][12][13][14][15][16][17] In addition to PIGA mutations, responsible for the emergence of PNH clone(s), the most frequently mutated genes are DNMT3A, ASXL1, and BCOR/L1, whose effects on AA and PNH clonal dynamics are far from being completely understood. 5,11 In the paucity of hematopoietic stem cells (HSC) surviving after AA immune attack, mutations generating a survival advantage such as PIGA or myeloid driver hits (e.g., RUNX1, SETBP1) may arise, or acquire clonal dominance if already present. 18 These observations suggest that the pathogenesis of secondary MN (sMN) may be linked to clonal hematopoiesis (CH), characterized by somatic mutations typical of MN with a peculiar MDS-related signature. 6,11,12,17 However, low burden mutations may be transient events and not contributory to later evolution while others may persist, thereby heralding progression. This is reminiscent of CHIP (CH of indeterminate potential) found in healthy individuals, or CCUS (clonal cytopenia of undetermined significance). [19][20][21] Unlike leukemic driver mutations, PIGA hits produce an escape phenotype ("blessing in disguise") in the context of AA immune-mediated attack. 22 The theory of "clonality as escape" 23 posits that PIGA-mutant HSCs do not significantly sustain BM hematopoiesis unless the T cell-mediated immune attack selectively targets the PIGA-wild-type HSCs. 24 Likewise, somatic hits in the human leukocyte antigen (HLA) genomic region have also been identified in AA and tentatively assigned to an immune escape of the stressed BM, paralleling findings observed in cancer. 16,17,[25][26][27] The rarity of AA and the timing of clonal evolution have made prospective controlled studies aiming to characterize factors associated with evolution difficult. Perhaps the long-term follow-up of the recently completed RACE trial 3 may be able to do so; but it will take years to get the full picture. Thus, retrospective analyses including large numbers of patients followed for extended periods of time are the sole able to decipher this complex field. Herein, we performed a comprehensive study of late clonal complications in 1008 consecutive patients with AA and PNH from a multicenter international cohort in order to define predictors of malignant progression. Furthermore, we examined clinical and molecular features of progression, being able to track the dynamics of clonal evolution in a subgroup of patients in a cross-sectional and longitudinal fashion.

PATIENTS AND METHODS

Study design and patients

We devised a retrospective, multicenter study to investigate the influence of baseline characteristics of patients with AA and PNH on the risk of late clonal complications (e.g., sMN and secondary PNH) and to characterize the clinical and genomic features of evolution. All consecutive patients diagnosed with acquired AA and/or PNH between 1972 and 2020 at the three participating centers were enrolled in this study after obtaining written informed consent (Table 1, Figure 1). The review of medical records was approved by the internal Institutional Review Boards of each center in agreement with the Declaration of Helsinki. Full details on study design, patients and definitions are available in the Supplementary Appendix.

Genomic Studies

Samples were collected and subjected to multiple NGS platforms as previously described [28][29][30] and according to the manufacturer's protocols. Full details are provided in the Supplementary Appendix (Table S1, Figure S1).

Statistical analysis

Cumulative incidences of clonal complications were calculated in a competing risk setting, where death and HSCT were considered competing events. Non-adjusted effect was evaluated using Log-Rank and Gray's tests, respectively for overall survival (OS) and cumulative incidence outcomes. Multivariate impact of baseline variables was evaluated using multivariate Cox-cause specific model. The risk proportionality of the Cox regression models was tested and the assumption was not violated (i.e., no global test was significant) while a potential center effect was taken into account using a random effect term (method also known as frailty).

All statistical tests were two-sided, and a p-value <0.05 was considered statistically significant. All analyses and data visualization were generated using the statistical computing environment R (4.0.0 R Core Team, R Foundation for Statistical Computing, Vienna, Austria), Excel Microsoft Office 365 and GraphPad Prism (8.4.0). Further details on statistics are provided in the Supplementary Appendix.

RESULTS

Determinants of late clonal complications and survival

A total of 1008 patients were assessed for inclusion. Because of absence of follow-up data (n=3) and overt diagnosis of MN at PNH presentation (n=6), 9 patients were excluded from further analysis (Table 1, Figure 1). In agreement with our previous report 7 , none of the patients undergoing HSCT upfront (n=117, Table S2) experienced late clonal complications (either sMN or secondary PNH) at a median follow-up time of 8.2 years (IQR, 5.7 -10.6), registering a 10-year OS of 86.7% (77.8 -92.1).

We thus studied non-transplanted patients with AA, AA/PNH overlap, and classical hemolytic PNH (n=882, Table S3). Their 10-year OS reached 76.4% (median follow-up time 8.6 years, Figure S2) and the 10-year cumulative incidence of sMN was 11.6% (Figure S3). A trend towards a higher risk of malignant progression was observed in AA (12.8%) and AA/PNH (13.1%) as compared to classical hemolytic PNH (3.4%, both p=0.06, Figure 2A). Consequently, classic PNH cases had the best OS with 95.2% alive at 10 years from diagnosis vs. 72.1% (p<0.001) and 88.8% (p=0.99) in patients with AA and AA/PNH, respectively (p<0.001 globally by Log-Rank, Figure S4A).

Besides the presence of PNH hemolytic clones, outcomes of AA were also affected by disease severity. While showing similar rates of sMN progression (13.6 vs. 12.4%, p=0.86; Figure 2B), non-severe cases had better OS (81.3 vs. 71.0%, p<0.001) but a slightly higher risk of secondary PNH (15 vs. 9%, p=0.12) when compared to patients with severe disease (Figure S4B,S5).

In severe AA (n=504, Table S4), the risk for malignant evolution was higher in older (>35 years) cases (20 vs. 2). When studying patients with available molecular information (n=302), overall the presence of myeloid mutations at disease onset did not seem to affect evolution (Table S5). The presence of a small PNH clone characterized patients with better survival outcomes (83.9 vs. 70.7% at 10 years; p=0.02) and was strongly associated with evolution to secondary hemolytic PNH (19.8 vs. 4.7%; p=<0.001; Table S5). A lower incidence of secondary PNH evolution was instead observed in poor IST responders (5 vs.

15.8%, p<0.001) and in male patients (6.0 vs. 12.1% in females; HR=0.49 [95%CI 0.27-0.89]; p=0.02 in a multivariate setting).

A similar analysis on non-severe AA (n=192, Table S6) identified non-treated patients (accouting for 34% of cases) as the group with the highest risk of sMN evolution (21.9 vs. 8.9 and 8.3% at 10 years in non-treated, poor and good IST responders, respectively; p=0.04; Figure S6). A slightly increased risk was also observed in AA patients with hemolytic PNH (16.4 vs. 11.5%; p=0.03). It is noteworthy that in non-severe cases the risk of sMN progression was not affected by age, which instead still influenced survival outcomes as well as did the presence of PNH clones (Table S7).

Clinical and molecular features of secondary myeloid neoplasms

After a median time of 4.5 years (IQR 1.8 -7.7), 94 patients (M:F ratio 1.61; median age 61 years, IQR 34 -69) evolved to sMN. The majority of progressed cases were patients with severe AA or those not registering complete responses to either first or salvage therapies. Of note is that nearly all patients undergoing second-line treatment (excluding cases rescued with HSCT) showed poor responses, with only one patient achieveing a complete response (Table S8-9). MDS (75%) was the most frequent diagnosis at evolution, followed by AML (18%) and MDS/myeloproliferative neoplasm (7%; Figure S7).

With a median follow-up of 4.7 years, the 5-year OS after sMN diagnosis reached 40% (Figure S8) and was independently influenced by blast >5% (HR= 3.64 [95%CI 1.82 -7.30]; p<.001; Figure 3A-B;Table S10-11).

In constrat to the usual characteristics of the MDS population, the majority of MDS cases had higher R-IPSS scores due to the overrepresentation of poor cytogenetic risk groups. Overall, myeloid mutations (N=82/94) were found in at least 80% of sMN with 61% of mutants harboring > 2 lesions (Figure 3C). Apart from cases uneligible/unfit for active treatment (44.4%), patients received chemotherapy (12.2%), hypomethylating agents (18.9%) or HSCT (36.2%), either upfront (21.1%) or as a consolidation strategy (15.1%).

Analysis of molecular characteristics resulted in the distinction of four different sMN groups (Figure S9-10). In particular, cases harboring del7/7q and complex karyotype (CK) had a similar mutational burden and survival outcomes to those with normal karyotype (NK), differing instead from patients carrying other cytogenetic alterations. ASXL1, SETBP1, RUNX1 and RAS pathway gene mutations constituted the unique signature of del7/7q carriers, classical leukemogenic drivers such as DNMT3A, FLT3, and NPM1 were typical of NK, whereas cases classified as "others" and CK had diverse molecular configurations (Figure 3D-E).

To further establish the discrete peculiarities of myeloid disorders arising from AA, we then applied a propensity score matching to reduce any possible confounding effects between our sMN cases (n=94) and an internal control cohort (n=94 matched patients with a 1:1 ratio from 3599 de novo cases) by using age, gender, bone marrow blast percentage and disease type as relevant baseline covariates (confounders). As expected, we found that higher-risk R-IPSS scores (54% vs.

37%) and chromosome 7 aberrations (53% vs. 11%) as well as ASXL1 (24% vs. 12%) and RUNX1 (21% vs. 8%) mutations were more frequent in sMN as compared with de novo cases, emphasizing that MN arising post-AA constitutes a distinct nosologic entity (Figure S11).

Immuno-molecular dynamics of clonal evolution

DISCUSSION

Over the course of 10 years from diagnosis, one-fifth of patients with AA treated with IST will experience late clonal complications (e.g., sMN, secondary PNH) . Early studies on X-chromosomal inactivation and karyotyping already indicated that CH may be present in a substantial number of AA cases, possibly as a prodromal sign of the increased risk for clonal evolution. 17,31 Among these clonal cytogenetic aberrations, partial or total loss of chromosome 7 represents the most frequent and worrisome alteration. 31 Subsequently, a systematic application of NGS further underscored how widely spread CH occurs in AA. 3,11 To that end, diverging theories on AA oligoclonality have been proposed, ranging from contraction of normal HSC pool and bystander CHIP-like phenomena to immunologic tumor surveillance reactions triggered by abnormal clones. 5 Despite progress, these issues remained unresolved due to the rarity of AA, and the long latency of sMN evolution, whose today study is amenable to only retrospective analyses of large cohorts of patients, waiting for the long-term follow-up of the randomized RACE trial in which prospective sampling has been performed. 3 In the present study, we have now precisely estimated the incidence, timing and clinical associations of late clonal complications including response to IST. Furthermore, the availability of a large cohort of patients followed for a median of 8.6 years enabled us to characterize the clinical and genetic features of sMN evolution, by compiling the largest cohort of cases so far described.

Our results confirm that older age at diagnosis is an independent risk factor for malignant progression. 7,32 An interesting implication on this association stems from the correlation between age and CHIP in healthy individuals and the lower incidence of CH in pediatric AA cases, paralleling findings on age-related CH. 17,40 For instance, AA cases presenting at an older age may already carry somatic age-related mutations, which could be positively selected after multiple rounds of IST because of the inherent increased fitness advantage of oligoclonal over normal HSCs. On the other hand, the differential diagnosis with hypoplastic MDS (without excess blasts) occurring in older age is not trivial, even in expert centers. Whatever the scenario is, this shares mechanistic analogies to therapy-related MN, where pre-existing clones are preferentially selected, and is also reminiscent of anecdotal PNH clones expansion/contraction under specific environmental conditions. 30,33 In line with previous studies, we found that myeloid hits were present in older patients and heralded sMN progression. 11 Indeed, the acquisition of mutations in ASXL1 and RUNX1 genes may provide increased HSC self-renewal capabilities offering a fertile soil for clonal sweeping in the context of an "immunologically stressed" hematopoiesis. 11,34 Besides age, poor response to standard IST also was found independently associated with an increased risk for myeloid evolution, typically accompanied by a history of failing multiple lines of treatment. Our results show that AA progression to sMN has unique molecular characteristics with the distinction of different subgroups characterized by discrete cytogenetic and molecular features. The high frequency of chromosome 7 abnormalities, ASXL1, RUNX1 and SETBP1 mutations, and their dynamics with other lesions putatively deriving from AA immune-pressure (PIGA, HLA mutations) indicate a peculiar signature of clonal evolution, different from that of other MN subtypes. 5 It is noteworthy that sMN cases acquiring monosomy 7 or del(7q) have been reported to have a shorter time to progression and a lower tendency towards association with del(5q) or TP53 disruption as opposed to primary or treatment-related MN. 6 Indeed, we found that the majority of our cases who progressed to sMN within 5 years from the initial diagnosis carried chromosome 7 alterations. Remarkably, the aforementioned deletion 7 gene signature (ASXL1, RUNX1, SETBP1) has been recently found associated with sMN arising in children with SAMD9/SAMD9L syndromes 36 and in other congenital BM failure syndromes, MN developing post-autologous HSC transplantation or chemotherapy, and recently in the context of expanding hematopoiesis following gene therapy. 37,38 Besides somatic myeloid mutations, other players have been identified as possible determinants of AA and PNH clonal dynamics. We have previously shown that germline HLA configurations imprint different risk of clonal progression (to both secondary PNH and sMN) and survival outcomes. 39 In this scenario, immune-privileged lesions or dysregulation of immune pathways may favor the evasion from T cell attack and provide a sustainable balance in the context of CH, cooperating with myeloid genes in intricate ways reported as a "war of clones" "struggling for dominance". 18,27,40 In conclusion, we propose that AA malignant evolution may be the consequence of a relentless autoimmune attack producing a maladaptive response to immunosuppression (Figure S17). Our results call for a close monitoring of patients with AA with classical BM smear examination and cytogenetics. Furthermore, information deriving from targeted deep NGS at regular intervals throughout the entire follow-up time must be integrated in the survivorship plan of such patients to allow early interventions. That said, although morphological progression to MDS and occurrence of monosomy 7 are obvious triggers to discuss transplantation, the occurrence (or expansion) of isolated molecular alterations as an indication for transplant is, nowadays, highly debated (except for TP53 mutations) and must be judiciously interpreted within the context of other variables such as age, donor's availability and response to IST. 10.0 -15.9) for AA, 13.1% (6.0 -22.9) for AA/PNH overlap syndrome, and 3.4% (0.9 -9.0) for PNH, respectively (p=0.13 globally; p=0.47 in AA vs. AA/PNH; p=0.06 in AA vs. PNH and AA/PNH vs. PNH by Gray's test).

TABLES

According to disease severity, the 10-year cumulative incidence of malignant progression was 12.4% (9.4 -15.8) for severe and 13.6% (8.6 -19.6) for non-severe cases (p=0.86 by Gray's test, panel B). Panels C and D show cumulative incidence curves for myeloid progression according to treatment response and age at diagnosis. At 10 years the cumulative incidence of progression was 15.7% (11.5 -20.6) in patients with partial or no response to treatment vs. 8.5% (4.4 -14.2) in cases achieving complete response (p=0.02). At 10-year the cumulative incidence of progression was 6.6% (3.7 -10.8) in younger cases (aged <35) vs. 20.0% (14.3 -26.2) in older patients (p<0.001). Numbers at risk are color-coded and indicated below each curve. shows the survival curves of main cytogenetic risk groups (numbers at risk for each subgroup are color-coded and provided below the curves). In panel E, a circle plot illustrates the relationship between mutations and cytogenetics. Three cases failed the cytogenetic testing and thereby were removed from the panels including cytogenetics features. -Patients initially diagnosed with acquired AA but who were later re-classified as inherited bone marrow failure syndrome -Clinical suspicion of inherited bone marrow failure syndrome

Objectives

Primary endpoint and main objective:

Cumulative incidence of secondary myeloid neoplasm and its characteristics (competing events: death and HSCT)

Definitions:

AA diagnosis was established according to standard guidelines 1 and severity defined according to Camitta criteria. 2,3 Myeloid neoplasm diagnosis was defined according to the 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemias and, when not already indicated as such, reclassified accordingly after abstraction of data from medical records. 4 Baseline PNH: positive Ham Dacie's test (historical cases) or flow cytometry PNH clone ≥1%.

Classical hemolytic PNH: PNH granulocyte clone size >20% and lactate dehydrogenase >x2.5 upper limit of normal. 5 AA/PNH overlap syndrome: intravascular hemolysis in the presence of a PNH granulocyte clone size >20% and/or positive Ham Dacie's test and at least two cytopenias. 6 Secondary PNH: new onset of intravascular hemolysis in the presence of a PNH granulocyte clone size >20% and/or positive Ham Dacie's test during the follow-up of a patient with AA and no previous clinical evidence of intravascular hemolysis.

Landmark time was defined as six months after AA diagnosis for severe or untreated patients, or six months after firstline treatment for non-severe patients. For the landmark analysis on the cumulative incidence of secondary myeloid neoplasia, all patients with follow-up inferior to 6 months from diagnosis (dead or alive), event (myeloid progression), or competing events (HSCT or death) were excluded.

Complete response was defined as recovery of hemoglobin levels >10 g/dL, absolute neutrophil count >1,000/ mm 3 , and platelet count >100,000 / mm 3 , as previously established. 7,8 Partial response was defined as the achievement of transfusion independence with no longer meeting the criteria for severe disease (see above). 8 and >98% of targeted regions showed over 100X coverage. Variants were classified according to established guidelines 15 , and internal bio-analytic pipelines to identify somatic mutations using sequences derived from controls and mutational databases such as dbSNP138, 1000 Genomes or ESP 6500 database, and Exome Aggregation Consortium (ExAC). [16][17][18][19][20][21] Furthermore, in silico predictions scores were used to assess potential pathogenicity and clinical relevance according to VarSome classifiers (https://varsome.com/) . Variants were retained when a strong and/or potential clinical relevance was ascertained after sorting for potential artifacts and according to general population frequencies, previous literature reports, pathogenicity prediction and availability of serial samples.

Approximately 4% of the variants detected for which controversial clinical evidence exist (Extended Data) were included based on clinical and genomic criteria, given the rarity of sequencing data in AA/PNH, the inherent differences as compared to classical myeloid neoplasm and the fact that their presence/absence did not affect the results of our analyses.

Deep Targeted PIGA Sequencing. Genomic DNA was amplified with primers designed to cover all exons of the PIGA gene and sequenced on an Illumina MiSeq as previously shown for patients enrolled at The Cleveland Clinic. 5,22 HLA sequencing and analysis. The TruSight HLA v2 (Illumina, San Diego CA) was utilized to sequence 11 HLA loci (Class I HLA-A, B, and C; Class II HLA-DRB1/3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1) according to the manufacturer's instructions. The DNA libraries were then loaded directly onto a MiSeq system for sequencing. (Figure S1) For 10 samples at evolution from the French cohort, the NGSgo®-AmpX (GenDx) was used.

As previously described 23,24 , after obtaining a full 8-digit typing by means of NovoHLA (Novocraft Technologies, Malaysia), paired-end reads were directly aligned on a per-patient HLA reference with selected alignment options favoring intermediate clipping, high mismatch and gap penalty, multi alignment, and high seed length. After sorting, marking duplicates and indexing procedures, variant calling was performed using VarScan in tumor-only mode. Highquality calls (present in both read mates) with a coverage>20 reads (for the reference base) and 4 reads (for the alternative allele) were prioritized. Non-polymorphic calls were retained after filtering by means of a customized javabased filter, exploiting the multi-alignment files provided by the HLA-IPD/IMGT database. 25 Both coding and noncoding variants were topographically and functionally annotated. Only pathogenic or likely pathogenic variants were retained for further analysis.

Unbalanced allelic HLA loss was imputed from sequencing data exploiting read cover ratio as previously described. 26 The following formula (where Ci and Cz represent the read coverage for each homologous allele) shows the method used to impute allelic loss:

!"#2 %& (Σ%&, %*)/2
An adjustment for structurally similar alleles was included using the sequence variation defined as "Variant coverage", which is provided by the NovoHLA pipeline. Based on a previous internal validation study on 234 healthy controls, Log2 ratios <-1.5 were retained as confident allelic loss.

Statistical analysis

Statistical analysis was performed by a third-party statistician (JE) from the European Blood and Marrow Transplant (EBMT) Group, who was the only person able to access and handle de-identified clinical data of the merged cohort, within the respect of the European General Data Protection Regulation (GDPR, 2017/679). As a first step, the three cohorts were tested as to potential center-related differences in terms of rate and timing of secondary myeloid neoplasm (sMN) progression (main outcome), overall survival (OS) and secondary PNH evolution. While no centerrelated effects were observed for the main outcome (sMN evolution) and secondary PNH evolution, a significant difference was found for OS. In order to take into account the center effect, we introduced a random effect or frailty for each center into the multivariate models (see below).

Quantitative variables were expressed as median, interquartile range, minimum and maximum. Qualitative variables were expressed as numbers and percentages. For all relevant comparisons, comparative analyses between two groups have been performed by two-sided Wilcoxon test for quantitative variables. Fisher test or Chi-square were used for testing independence between groups, for qualitative variables. OS was defined as the time from diagnosis to last follow-up or death for any cause, and was calculated using Kaplan-Meier estimation. 27 Cumulative incidences of sMN and secondary PNH were defined as the time from diagnosis to the acquisition of cytogenetic alterations/evidence of bone marrow dysplasia as per WHO 2016 diagnostic criteria 4 and new-onset of intravascular hemolysis (see definitions), respectively, and were calculated in a competing risk setting, with death and non-first line HSCT as competing events. Median follow-up was calculated using the reverse Kaplan-Meier estimation and the 95% confidence interval.

Non-adjusted effect of baseline variables was evaluated using Log-Rank and Gray's tests, respectively for OS and cumulative incidence outcome. 28 Multivariate impact of baseline variables was evaluated using a multivariate Coxcause specific model. The risk proportionality of the Cox regression models was tested and the assumption was not violated (i.e., no global test was significant). 29 The potential center effect was taken into account in the multivariate Cox models using a random effect term (method also known as frailty). 30 The impact of variables was evaluated on AA and separately between non-severe and severe cases. To evaluate the treatment response, landmark analyses were performed at 6 months after the diagnosis and treatment initiation for severe and non-severe AA, respectively. A landmark time of 6 months after diagnosis was used for non-treated cases.

Moreover, to account for a possible time period-related effect, we adjusted our analysis based on the year of diagnosis. Considering all the included patients with available information, a Cox-proportional hazard function adjusting for age was fit to estimate baseline sMN progression hazard rates and generate progression-free survival (PFS) curves. Cases were classified based on features (PIGA/PNH clone presence and BCOR/L1, no mutation, and myeloid mutations only -see list of our myeloid gene panel in Table 1) previously hypothesized to be associated with sMN evolution.

Determinants of Clonal Evolution in Aplastic Anemia

Gurnari C. et al. 2022

32

In a second part, the OS and impact of variables on OS of the patients with sMN were estimated from the time of sMN onset to death or last follow-up. The impact of consecutive HSCT post progression on OS was estimated as time-varying covariate in the Cox multivariate model. Also in this case, a random effect or frailty for each center was introduced.

Finally, a 1:1 matched-pair analysis based on nearest neighbor propensity score matching (PSM) was used to compare post-AA MN cases of this study and matches from a cohort of de novo myeloid neoplasm (n=3599) using age, gender, bone marrow blast percentage and disease type as relevant baseline covariates.

All statistical tests were two-sided, and a p-value < 0.05 was considered statistically significant. All analyses and data visualization were generated using the statistical computing environment R (4.0.0 R Core Team, R Foundation for Statistical Computing, Vienna, Austria), Excel Microsoft Office 365 and GraphPad Prism (8.4.0). MDS: myelodysplastic syndrome; AML: acute myeloid leukemia; WHO: World Health Organization; MDS-MLD: myelodysplastic syndrome with multilineage dysplasia; MDS-RS-MLD: myelodysplastic syndrome with ringed sideroblasts and multilineage dysplasia; MDS-SLD: myelodysplastic syndrome with single lineage dysplasia; MDS-RS-SLD: myelodysplastic syndrome with ringed sideroblasts and single lineage dysplasia; MDS-EB-1/2:myelodysplastic syndrome with excess blasts-1/2; AML-MRC: acute myeloid leukemia with myelodysplasia-related changes; MDS-U: myelodysplastic syndrome-unclassifiable; AML-NOS: AML not otherwise specified; CMML: chronic myelomonocytic leukemia; MDS/MPN-U: myelodysplastic/myeloproliferative neoplasms-unclassifiable; MF: primary myelofibrosis. pie chart illustrating the percentage of mutations shared at both time points. Lines between the two bar charts associate shared mutations. In panel C, two exemplificative patients are shown. On the left, the fish plot showcases a 28 year-old male with severe AA treated with conventional immunosuppression (anti-thymocyte globulin and cyclosporine A) achieving a partial response and subsequently evolving after 3 years to a myelodysplastic syndrome (MDS) with excess blasts type 1. The ASXL1 mutation present at AA onset (p.G642fs, variant allelic frequency, VAF 3%) constituted the bulk of disease at MDS evolution (VAF 19%) with monosomy 7 (45,XX,-7 [20]) and an additional PTPN11 (p.A72T, VAF 13%) mutation acquired at evolution. On the right, a case of a 67 year-old man with severe AA is illustrated. The patient had an initial complete response to standard immunosuppressive treatment, subsequently relapsed and eventually progressed to a MDS with multilineage dysplasia after 8 years from initial diagnosis. At AA onset, the patient harbored CUX1 (p.I517fs, VAF 5%) and NF1 mutations (p.W777C, VAF 26%) while at the time of progression he lost the CUX1 lesion and the NF1 mutation increased in VAF (35%). Moreover, the patient acquired monosomy 7 (46,XX,-7,+mar [20]) and two additional alterations in SETBP1 (p.D868N, VAF 35%) and ETV6 (c.163+1G>A splice site variant, VAF 36%) genes.

Supplementary Figures

PNH clone (N=83

0 (0) 0 (0) others 2 (1.3) 2 (1.4) 0 (0) 0 (0) ASXL1 CEBPA ETV6 IDH2 MPL PRPF8 SF3B1 TP53 BCOR CUX1 EZH2 JAK2 NF1 PTPN11 SMC3 U2AF1 BCORL1 DDX41 FLT3 KDM6A NPM1 RAD21 SRSF2 U2AF2 CALR DNMT3A GATA2 KIT NRAS RUNX1 STAG2 WT1 CBL EP300 IDH1 KRAS PHF6 SETBP1 TET2 ZRSR2
*indicated is the available sample size due to missing data. AA: aplastic anemia; PNH: paroxysmal nocturnal hemoglobinuria; HSCT: hematopoietic stem cell transplant; F: female; M: male; y:years. For categorical variables, numbers in parentheses indicate percentages.

Table S3. Characteristics of patients not undergone upfront HSCT (N=882).

Variables

N. of myeloid mutations (N=302) * 0

*indicated is the available sample size due to missing data. AA: aplastic anemia; PNH: paroxysmal nocturnal hemoglobinuria; F: female; M: male; ATG: anti-thymocyte globulin; CSA: cyclosporine A. For categorical variables, numbers in parentheses indicate percentages.

First line treatment (N=500)* ATG/CSA based 453 (

Discussion and limitations

With a median follow-up of more than 8 years, no transplanted patient (n=117) progressed to MDS/AML, suggesting that donor hematopoiesis outcompete recipient premalignant clones that might exist at the moment of transplantation [median age 23.3 (3.1 -52.1)] years. Of note, conditioning regimens for AA are rather immunosuppressive than myeloablative. [START_REF] Bacigalupo | How I treat acquired aplastic anemia[END_REF]154,155 In other words, conditioning itself does not kill LT and ST-HSCs.

Among severe AA (SAA), we have confirmed that suboptimal response to immunosuppression and age at BMF onset were independent prognostic factors for secondary myeloid malignancy and worst survival. 156 Likewise, the prevalence of CHIP increases with age. Older patients might already carry premalignant clones that are positively selected under stress.

Among non-severe cases (nSAA), we observed that untreated patients had the higher cumulative incidence of secondary myeloid malignancy. However, we could not adjust for confounding variables. As for CCUS and SAA with poor response to immunosuppression, stress haematopoiesis might help select premalignant clones.

We observed that classic PNH had a lower cumulative incidence of MDS/AML than AA/PNH and AA alone, although we were underpowered to show a significant difference. Arguably, a less stressed haematopoiesis might play a protective role in clonal selection. Classic PNH patients had fewer myeloid mutations at diagnosis. The presence of PNH clone in aplastic patient did not affect the risk of secondary myeloid malignancy.

The most frequently found mutations at AA diagnosis were in consonance with the literature and overlapped with CHIP: BCOR, BCORL1, TET2, and ASXL1. 19,[START_REF] Yoshizato | Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia[END_REF][START_REF] Ogawa | Clonal hematopoiesis in acquired aplastic anemia[END_REF] The presence of any mutation at AA onset did not affect the risk of MDS/AML evolution. However, myeloid driver mutations predicted higher risk of progression when compared to absence of mutations; isolated PIGA/HLA mutations predicted the lowest risk. These findings also confirm previously published data. [START_REF] Yoshizato | Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia[END_REF][START_REF] Kulasekararaj | Somatic mutations identify a subgroup of aplastic anemia patients who progress to myelodysplastic syndrome[END_REF]156 Patients bearing myeloid mutations need close monitoring.

We could compare the molecular profile of patients sequenced at AA baseline and evolution in a cross-sectional analysis: PIGA or HLA mut VAFs tended to increase among non-progressors and decrease in progressors, followed by an increase myeloid genes. The inverted clonal dynamics supports that PIGA or HLA mut are not drivers of transformation. Among progressors, we could backtrack the driver mutation at AA for 4/25.

Expectedly, the prognosis of progressors was poor and comparable to published results. 157 Blast count at MDS/AML diagnosis and older age predicted independently worst survival.

MDS secondary to AA differed cytogenetically from primary MDS, presenting worse karyotypes. Most post-AA MDS patients had at least one detectable myeloid mutation and nearly two thirds, ≥2.

Chromosome 7 abnormalities and complex karyotypes conferred poor prognosis. Diversely from R-IPSS, normal karyotype at MDS/AML diagnosis did not predict good prognosis. The interval between AA and MDS/AML was shorter and normal karyotype was associated with classic leukemic drivers such as NPM1, FLT3, IDH1/2, and U2AF1.

According to the 2016 WHO revision for myeloid malignancies, isolated del(13q) is sufficient for MDS diagnosis. Historically, in the context of AA, del(13q) is considered to confer a good prognosis. 105,158 From 44 published del(13q) post-AA, five progressed to high-risk disease (monosomy 7 or excess of blasts) [158][159][160] We did not have enough cases of isolated del(13q) to draw conclusions. Such patients might need close monitoring.

Despite having published the largest cohort on AA/PNH so far, we were underpowered to assess the contribution of individual mutations to the risk of progression. Besides, we observed a higher risk of myeloid malignancy among treatment-naïve nSAA in univariate analysis, but again we were underpowered to adjust for confounding factors.

The availability of NGS (the first patient included in the model was diagnosed in 1972), the indications for sequencing, and the evolution of panels used throughout the years made that we did not have complete molecular data in over half of the cohort, precluding some analyses. We must be cautious interpreting molecular observations. On the other hand, we had excellent completeness of clinical data.

Lessons learnt from this study

I gained experience in bibliographical research, literature review, project and grant writing, grant management, IRB approval (Brazil and France), statistical analysis plan co-writing with a statistician, establishment of international collaborations, and teamwork. Collaborative research is an excellent learning tool for combining different points of view towards a common interest.

Working with co-PIs and a statistician, I have learnt how to create tidy databases and improve preexisting ones. We had the chance to discuss the strengths and weaknesses of our statistical plan, data analyses and visualisation, and draw datadriven conclusions.

In parallel, I have learnt how to identify the cases fulfilling the inclusion criteria, collect clinical data, retrieve biological samples and extract DNA (blood and marrow frozen samples, FFPE marrow biopsies, blood and marrow smears). I did not perform the NGS myself but worked closely with the molecular biology, histocompatibility, and bioinformatics teams.

Perspectives

Molecular profiling using next-generation sequencing is currently performed using heterogeneous panels at non-standardised time points during AA natural history. However, clinicians still have no tool to stratify such patients before first-line treatment by the risk of secondary myeloid neoplasm (evolution. The effect of specific myeloid gene mutations at diagnosis (treatment-naïve patients) and its associations with other factors (e.g., age at onset, conventional cytogenetics, PNH clone, and disease severity) is currently unknown.

We aim to combine clinical, cytogenetic, and molecular characteristics at baseline (treatment-naïve) to develop the first tool for sMN risk-stratification in immune AA.

We propose the development of a novel tool for stratifying AA patients. AA is a rare disease, and the studies published so far did not have enough patients and power to develop a risk score. Studies have described that somatic mutations are frequent events in the natural history of aplastic anaemia and can be already seen in patients' first evaluation [START_REF] Yoshizato | Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia[END_REF][START_REF] Kulasekararaj | Somatic mutations identify a subgroup of aplastic anemia patients who progress to myelodysplastic syndrome[END_REF][161][162][163][164][165][166] . The study is relevant because the predictive value of such mutations in AA is unknown. A score will help stratify the risk of malignant transformation and allow clinicians to adapt the follow-up strategy for high-risk patients (closer monitoring to assess the best moment for treatment modification).

Because of the rarity of the disease, the most effective strategy to fill this gap is establishing a sizeable multicentric consortium. Based on available data (please see the table below) and considering database updates, we estimate this study is feasible.

* Risk of overlapping patients with Gurnari and Groarke **Risk of overlapping patients with Gurnari and Yoshizato We propose a multicentric cohort study. We plan to study a discovery cohort with sufficient clinical and biological data to develop the so-called International Consortium on Aplastic Anemia Risk Score (ICAARIS) model; we will validate the model internally using the bootstrap method and externally on an independent validation cohort.

Aim of the work

Today, FA patients with BMF undergo HSCT if eligible. The 15-year OS reaches 80%, [START_REF] Bonfim | Long-term Survival, Organ Function, and Malignancy after Hematopoietic Stem Cell Transplantation for Fanconi Anemia[END_REF] and transplantation reduces the risk of progression towards MDS/AML. [START_REF] De Latour | Allogeneic hematopoietic stem cell transplantation in Fanconi anemia: The European Group for Blood and Marrow Transplantation experience[END_REF] Revertant patients seem not to progress to MDS/AML and gene therapy mimics this functional reversion. Gene therapy has the advantages of not needing any conditioning regimen and not causing GvHD. However, it is unknown if gene therapy modifies the risk of progression. Considering that HSCT provides excellent results for the haematologic complications of FA and neither reduces the risk of solid cancers, we asked ourselves if somatic functional reversion reduces the risk of progression to MDS/AML.

The scientific question

We would need long follow-up with many patients measure the impact of gene therapy on leukemogenesis. Therefore, we worked on developing a model of genetic reversion in Fancg -/-mice. We aimed to test the fitness of gene-corrected or preleukemic subclones in the Fanconi context. Our main objective is to demonstrate that functionally corrected Fancg -/-HSPCs have an adaptative advantage over noncorrected premalignant Fancg -/-HSPCs in vivo.

Methods

Mice

C57BL/6J Fancg (Figures 8 and9) were kept in specific opportunist pathogen free (SOPF) environment and mating scheme was het x het. All animals used for experiences were older than 7 weeks. All experiments were carried according to European regulations (APAFIS#28841-2021010510188985v2 -Modélisation du mosaïcisme somatique et selection clonale dans un modèle murin de l'Anémie de Fanconi).

Cell harvesting, lineage-negative and LSK purification

C57BL/6J Fancg with the appropriate genotype were euthanized by cervical dislocation, and had their lower limbs dissected for femur, tibia, and, later during my thesis, hip retrieval. Under laminar flow, individual mononuclear cell suspensions were prepared after bones were crushed, filtered, and purified from red blood cells (Red Blood Cell Lysing Buffer Hybri-Max TM , Sigma Life Sciences, Saint-Louis, MO, #R7757-100mL). Lin -were obtained using a lineage cell depletion kit (Miltenyi, Bergisch Gladbach, Germany, #130-090-858) and magnetically sorted (Miltenyi, #130-090-976, #130-042-401) according to the manufacturer's instructions. LSK (Lin -, Sca1 + , c-kit + ) were purified from the magnetically sorted Lin -by flow cytometry (Aria III, Becton and Dickinson, Franklin Lakes, NJ).

Murine embryonic fibroblasts

Ten cages containing each one adult C57BL/6J Fancg -/+ male and two adult C57BL/6J Fancg -/-females were set up for mating at 5PM. The next morning, all females were checked for a copulation plug and weighted. On the day E12.5, females gaining at least two grams were euthanized by cervical dislocation and dissected for uterus retrieval. Each embryo was individually genotyped, dissected under a microscope, and had its head and red organs removed. Under laminar flow, embryos of the same genotype were grouped by three. Cells suspensions were obtained by repeated pipetting back and forth using 3mL/embryo ice-cold 0.05% Trypsin-EDTA. Then, fragments were further dissociated using scalpels in a 10cm cell-treated Petri dish. Twenty-seven (9mL per 1mL of trypsin) millilitres of DMEM supplemented with decomplemented 10% foetal bovine serum, and 1% penicillin/streptomycin were added and kept at 37°C and hypoxia for 48h. Then, cells were retrieved using 0.25% Trypsin-EDTA (Gibco, #25200-056), washed with PBS and re-seeded or frozen with FBS + 10% DMSO in liquid nitrogen for further use.

2.0 x 10 6 cells were seeded in cell-treated T75 flasks in DMEM (Gibco, #41966-029) supplemented with decomplemented 10% foetal bovine serum, and 1% penicillin/streptomycin and kept at 37°C and hypoxia until confluency.

Lentiviral production and titration

I did not carry out all the steps described in this paragraph myself (I was not habilitated to access the biosafety level 3 facility). Lentiviral virions were produced using jetPRIME reagents (Polyplus-transfection, France) by co-transfection of viral production plasmids (VSV-G, PAX, and transfer plasmid) in T150 cm 2 tissue culture flasks containing 2x10 6 HEK-293T cells by flask in Advanced DMEM + 1% penicillin/streptomycin + 1% L-glutamine + 10 foetal bovine serum, according to the manufacturer's protocol. Viral supernatants were filtered through a 0.45um HV Durapore membrane (Merck Millipore) and concentrated by ultra-centrifugation at 24,000 rpm for two hours at 4 °C, and stored at -80 °C.

Transfer plasmids and lentiviral transduction

All the plasmids I used in this project were already available in the lab and I profited from an already existing transduction protocol. pLenti-hEF1a-mFancg-tRFP, pLenti-hEF1a-mFancg-eGFP, pLVX-hEF1a-mEvi1-ZsGreen, pLVX-hEF1a-mock-ZsGreen, pRRL-hPGK-shFHL1-mCherry (Figure 18) were transduced overnight for 16 hours with the lentiviral supernatants and polybrene 4µg/L with different multiplicities of infection (range 0->1000). For in vivo experiments, treated cells were harvested and washed at day 1. Transduction efficacy was measured by flow cytometry (FACS Aria III cell sorter, Beckton Dickinson) or Fortessa Analyzer, Beckton Dickinson). For in vitro experiments, cells were re-seeded for another 24h. Gene overexpression was checked using specific immunoblot, RT-qPCR, and flow cytometry We chose Evi-1 because it is located in the long arm of chromosome 3. EVI-1 is frequently amplified among de novo and Fanconi AML (because of +3q). Using Evi1, the team from Hildelberg had 100%-penetrant AML in Fanca -/-B6 model (data presented in the 1 st Translational Research Conference: from the cell to the cure of the disease, 2020).

Cell culture

Haematopoietic cells About 1 x 10 6 Lin -cells were seeded in round-bottom 96-well plates in StemSpan TM (Stem Cell Technologies, Vancouver, BC, #09600) supplemented with 1% pennicilin/streptomycin for lentiviral transduction, 100µL/well. MEFs About 2.0 x 10 6 cells were seeded in cell-treated T75 flasks in DMEM (Gibco, #41966-029) supplemented with decomplemented 10% foetal bovine serum, and 1% penicillin/streptomycin and kept at 37°C and hypoxia until confluency. Then, cells were retrieved using 0.25% Trypsin-EDTA (Gibco, #25200-056), washed with PBS and reseeded for experiments, or frozen as cell pellets for Western Blot or RT-qPCR. NIH 3T3 cell lines and HEK 293T cell lines About 0.9 x 10 6 mycofree cells were seeded in cell-treated T150 flasks in DMEM (Gibco, #41966-029) supplemented with decomplemented 10% foetal bovine serum, and 1% penicillin/streptomycin and kept at 37°C and normoxia until confluency. Then, cells were retrieved using 0.25% Trypsin-EDTA (Gibco, #25200-056), washed with PBS and re-seeded or frozen as cell pellets for Western Blot or RT-qPCR.

RNA extraction and RT-qPCR

DNA was extracted and purified from frozen cell pellets using the RNeasy Plus Micro kit (Qiagen, Hilden, Germany, #74034) following the manufacturer's instructions. RNA was quantified using the NanoDrop One (Thermo Fischer, Waltham, Massachusetts, MA). cDNA was synthesized from purified RNA using the SuperScript Vilo Master Mix (Thermo Fischer, Carlsbad, CA, #11755-050) following the manufacturer's instructions. The protocol was 10min of annealing at 25°C, 60min of retrotranscription Quantitative PCR was performed using the PowerSYBR Green PCR Master Mix (Applied Biosciences, Waltham, Massachusetts, MA, #4367659) following the manufacturer's instructions in the StepOne Plus PCR System (Applied Biosciences, Waltham, Massachusetts, MA). Except for Fancg, the protocol used was 10min holding stage 95°C, 50 cycles 15s 95°C and 50s 60°C, 15s 95°C, 1min 60°C and 15s 95°C melting curve. For Fancg, the second step of the cycling stage was at 61.5°C. Gene expression was normalized with the expression of Gapdh. Western Blots were performed using a standard protocol. The antibodies used were against murine Fancg (sc-393382, Santa Cruz Biotechnology; PA5-27117, Thermo Fisher; ab-151516, Abcam), human EVI1 C50E12, Cell Signaling Technology), actin (Santa Cruz) and vinculin (Santa Cruz).

Competitive transplantation experiments

C57BL/6J Ly5.1 (CD45.1) wild-type mice were lethally irradiated (9Gy singledose) one day before transplantation. A total of 5x10 5 lineage-negative cells suspended in 200µL of phosphate buffered saline (PBS) were injected in the tail vein. The number of effectively transduced cells varied from 2x10 4 to 7x10 4 for each condition and were kept at a 1:1 ratio between groups. For some experiments, 5x10 5 to 1x10 6 whole bone marrow Fancg -/-cells were co-injected to support haematopoiesis during the first weeks after lethal irradiation. For one experiment, transduced cells were sorted (Aria III, BD) prior to injection. Engraftment was followed monthly by marrow or blood sampling (CD45.1-PE, CD45.2-APC from BD Biosciences, eGFP, and RFP). Mice were euthanized if signs of discomfort or after 6 months of follow-up. Flow cytometry analyses were performed on Aria III or Fortessa Analyzer.

Colony-forming unit assays

3000 lineage-negative or 100-300 LSK cells were plated and cultured in MethoCult 3434 medium (Stem Cell Technologies) and colonies were counted using standard criteria on the 7th day. Cells were treated or not with different doses of mytomicin (MMC)(Roche).

Results

Mouse model validation

First of all, we have confirmed our model was suitable for experiments. Lineage negative-or LSK cells harvested from C57BL/6J are sensitive to low doses of mytomicin, whereas paired wild-type cells were resistant (Figure 19). Fancg mRNA was absent from different tissues from Fancg -/-mice. Western blot experiments were not specific enough to compare Fancg protein between genotypes (data not shown). I tested three different antibodies, including one targeting amino-acids 1-160 (deleted 1-163). Fancg -/-mice have high embryonic lethality and the birth rate of the KO genotype was inferior about 10%. Therefore, the number of KO animals was a limitation for my experiments. To mitigate sample availability, I tested if collecting the hip bone would increase Lin -retrieval (Figure 20). Collecting femurs, tibias, and hips did not increase the dissection time and increased significantly the number of viable Lin -. Comparison of the number of viable lineage-negative cells retrieved from femurs, tibias, and anterior limbs (orange) against femurs, tibias and, hips (green).

Transduction and plasmid expression

Next, I tested the transduction protocol in Lin -cells. In this early stage of my thesis, I had not controlled the multiplicity of infection (I used 10µL of the lentiviral supernatant per 10 5 cells) and, therefore, had significant variability from one experiment to another. The mean transduction efficiency in Lin -for Lenti-RFP plasmids was 55% of single cells; for pLVX-ZsGreen, 21% at day one.

Despite variability, transduction increased many hundred-fold the expression of Fancg in KO Lin -cells normalized to untreated wild-type (Figure 21).

The increase in Fancg expression was also detectable at the protein level (Figure 21). For Fancg Western blot, I used KO MEF because of the number of nonspecific bands present when using Lin -. The mRNA expression for Evi-1 was increased over 50-fold in MEF compared to control. This increase was seen in the protein level (Figure 22).

Competitive experiments

Following the validation of cell harvest and transduction, I have tried to mimic gene therapy. I have transduced KO Lin -CD45.2 either with Lenti-EF1a-Fancg-RFP ou Lenti-EF1a-control-eGFP and re-injected in competition (1:1 ratio) into 5 lethally irradiated CD45.1 wt. In the first experiment, all recipient mice developed skin ulcers from D+40 and were euthanized. The median CD45.2 engraftment was 98.2% (range: 96.9 -99.2). There were no detectable RFP or eGFP cells. I repeated the experiment, however, had to sacrifice mice because of primary graft failure.

Because of the negative results, I have re-assessed the Lin -culture and transduction steps. After exchanging with colleagues that performed similar experiments, we have noticed a 40% differentiation of Lin -at 24h in culture. A possible cause was the change in the culture medium lot. My colleagues tested different culture conditions and, from this moment on, I have not added cytokines to the culture medium (lower differentiation rate).

Then, I focused on developing a model of clonal dominance between KO Lin - transduced with Lenti-EF1a-Fancg-RFP (gene-corrected) or pLVX-EF1a-Evi1-ZsGreen (premalignant). Following Lin -harvest, purification, and transduction, a 1:1 cell mix was injected in the tail vein of lethally irradiated wt CD45.1 mice. Transplanted mice were monthly followed up to 6 months.

In the first attempt to carry out the experience represented in Figure 23 (n=10), four mice died or were euthanized within 15 days from irradiation because of graft failure. In the second (n=6) and third (n=6) attempts, I added KO whole bone marrow supporting cells. This strategy allowed me to overcome graft failure; however, one mouse was euthanized in each experiment because of skin ulcers. Ten other mice were received only Evi1--transduced cells.

Fancg x Evi1

Control x Evi1

Lin -harvesting and enrichment

Donor KO

In total, I have transplanted 42 mice and 21 reached 6 months follow-up (9 primary graft failure, 7 euthanized because of skin ulcers, 5 of unknown causes. Transduced cells were not detectable in blood or marrow in any analysed mice. On the other hand, donor engraftment was excellent (Figure 24). Therefore, the transplantation steps were functional (9Gy irradiation and KO support whole bone marrow). I have discussed with the animal facility team about my results and, specially, the skin ulcer clinical picture. Serial sampling assessments for infectious organisms in the animal facility were negative and the experimental plan is not in favour of graft-versus-host disease (inbred strain and co-isogenic backgrounds). Thus far, the aetiology is skin ulcers is not clear.

Notwithstanding, the absence of transduced cells in all analysed mice intrigued me. So, I investigated why it did not work.

Colony-forming unit assay

The first question I asked myself was how the transduced cells behaved in culture. So, I transduced Lin -with pLVX-EF1a-Evi1-ZsGreen or pLVX-EF1a-control-ZsGreen and put in methylcellulose culture, bulk and sorted. Each week I measured the proportion of ZsGreen-positive cells in flow cytometry. Astonishingly, the proportion dropped below 5% in all conditions by day 14 (Figure 25). My hypothesis was of a viral failure or a negative selection of transduced cells.

Transduced MEF

To assess if the loss of RFP or GFP/ZsGreen signal was tissue specific, I transduced wt MEF with different constructions and kept them in culture for 4 passages (Figure 26). Contrary to what I observed for Lin -cells, the signal remained stable. These results strongly supported that viral integration worked. Therefore, negative selection was the main possibility.

Multiplicity of infection, efficacy, and viability

The first uncontrolled variable I had in mind was the viral supernatant. We had an important batch-to-batch lentiviral titration variability, reaching 4 log between some batches (Figure 27). In other words, the number of virions per cell was unknown. So, to choose the best ratio, I transduced KO and WT Lin -with different multiplicities of infection and measured the efficiency by flow cytometry. At MOI<50, the increase in efficiency was evident with the increase in viral concentration (Figure 28). However, it seems of no benefit to go beyond 50. Next, I measured apoptosis in transduced cells using annexin V. Surprisingly, nearly 80% of cells transduced with pLenti-RFP constructions were apoptotic from MOI 1 and higher MOIs had little or no effect, whereas more than 70% of nontransduced cells from a same sample were viable (internal control, Figure 29). Therefore, the high MOI could not explain the negative selection, at least not alone. Within a same sample, the viability of transduced cells (red dots) was much lower than nontransduced cells (blue dots).

Intrigued by such observations, I tested apoptosis in KO and WT cells. Viral integration causes double strand breaks and might be too toxic to Fanconi cells. Furthermore, I tested different constructions to assess if results were associated with a viral factor. Surprisingly, nearly all transduced cells with pLVX or pLenti were apoptotic, regardless of the genotype (Figure 30). Based on these observations, the construct seems to play a major role on cellular toxicity.

Discussion and lessons learnt from this study

My main objective was to investigate if the functional correction of HSPCs was protective against the evolution into AML (Figure 17). We have observed that, among revertant patients, none presented overt MDS/AML. Our hypothesis was that functionally revertant HSPCs would outcompete FA-deficient cells in the haematopoietic niche. Revertant cells would, therefore, halt the process of premalignant clonal dominance seen in FA patients (1q+, 3q+, MDS/AML).

The validation of our hypothesis can have therapeutic consequences: in transplanted FA patients, the FA haematopoietic tissue is replaced by a healthy one. Besides reducing the burden of oligoclonality by debulking possible pre-existing premalignant clones, the new allogenic immune system's surveillance detects and eliminates residual cells. However, this benefit comes with a cost: FA patients are very sensitive to genotoxic stress and transplant conditioning regimens highly depend on alkylating agents (irradiation is rarely used for Fanconi patients). Besides conditioning short-term toxicity, transplanted patients are at risk of graft-versus-host disease. GvHD might further increase the risk of solid cancers among FA patients. Notwithstanding, HSCT clinical outcomes for FA patients improved greatly in the last decades and survival is excellent for young patients transplanted before transformation.

Gene therapy offers an alternative: ex vivo corrected autologous cells overcame the risk of GvHD and engrafted without any prior conditioning regimen, reflecting an ecologic advantage over Fanconi cells in vivo.

To test our hypothesis, I worked on a preestablished C57BL/6J Fancg -/-Ly5.2 available at IRSL animal facility. The team was knowledgeable on the model and on the style of experiences I would perform. I am grateful for the competence transfer. I could also profit from an already validated transduction protocol and viral constructs that were already available at the lab.

After validating the pre-transplantation steps, I have started in vivo experiences one after another. When I noticed the absence of ZsGreen or RFP cells in the followup, I had concluded that it was too early to detect transduced cells. It was after reaching 6 months FU for the first experiment that I concluded the others would not work as well.

I have encountered some experimental difficulties along the process. First of all, Fancg -/-are born at sub-mendelian rates and we had to increase the number of het x het couples (KO mice are infertile). Besides, I changed the strategy of cellular harvesting to increase yield.

Another problem was early mortality following irradiation. Undoubtedly, one factor that progressed through time was my skill of injecting, and I am confident that it has affected my first results. However, I still observed mortality within 15 days following irradiation after having mastered the technique. Co-injecting untreated whole bone marrow cells surmounted early mortality. Some mice presented skin ulcers and the aetiology remains unclear. Initially, I thought it would be GvHD, but after discussing with many other researchers, this hypothesis is unlikely. Infection does not seem to be the case either. C57BL/6J can spontaneously develop ulcerative dermatitis. 167 It is my diagnosis of exclusion.

Contrary to competitive transplantation against WT cells, 91 Fancg -/-Lin -result in excellent engraftment when injected alone (Figure 24). Yet, I could not understand the absence of transduced cells following donor engraftment with 6 months follow-up. I controlled all the other variables I have identified (Lin -differentiation, integration, gene expression, transduction efficacy, multiplicity of infection). Surprisingly, I found out that nearly all the cells transduced with the constructions I used in this project died within 48h (Figure 30), and, likely, the cells that were still alive at that moment died soon after. Cells had good viability with a third construct that a colleague uses in her project. The mortality following Lenti-hEF1-tRFP and pLVX-EF1-ZsGreen explained my results, both in vivo and in vitro.

What both plasmids have in common that is different from the third plasmid is the promoter: hEF1, human elongation factor 1 is a strong promoter used for geneoverexpression. Indeed, I noticed that Fancg or Evi1 were many hundred-fold overexpressed in transduced cells. Such promoters have the advantage of robust expression and are less frequently silenced in hematopoietic cells than other promoters. However, strong promoters can also increase the expression of cis-neighbouring genes to the viral insertion site. Perhaps unwanted genetic overexpression was toxic for the Lin -.

Perspectives

I will test different constructs with different promoters with Fancg / Evi1. I will validate the transduction and gene expression as I did for the first couple of constructions. I will also test if the addition of retronectin allows for higher transduction efficacy at lower MOIs and, therefore, lower viral toxicity. After choosing the most adequate construction and MOI, I will perform CFU assays to test the persistence of transduced cells after passages. I will test the functional reversion of the cellular FA phenotype by demonstrating that gene-corrected Fancg -/-became resistant to MMC (CFU).

Another difference from the original protocol is that I will use LSK instead do Lin - because they are more enriched in true stem cells and have lower differentiation (data not shown).

Once these steps work (a viral construction with a less powerful promoter and LSK cells), I will perform another in vivo experiment.

Chapter 3: Concluding remarks

CH is part of physiological ageing. Surveillance is important to adapt / modify the therapeutic strategy before progression to AML/MDS regardless the BMF status. However, CH is more frequent among BMF patients than in the general population. In the presence of unfavourable intrinsic (i.e., DNA-repair deficiency and consequent p53/p21 response) or extrinsic (i.e, immune aplastic anaemia or radio-chemotherapy) haematopoiesis can be sustained by more fit clones. Such clones can have built-in proliferative advantages (arguably pre-malignant, as in CHIP), or simply be more adapted to the environmental (protective, as in reversion) and mediate homeostasis. Somatic genetic rescue is not restricted to the haematopoietic tissue: in inflammatory bowel disease, clones resistant to IL-17 help maintain intestines' function not taking part in tumorigenesis; on the contrary, tumour formation was reduced in a mice model. 168,169 BMF patients with CH seem to present two very different natural histories: lower risk of MDS/AML as reversion in IBMF; higher risk to MDS/AML as specific myeloid driver mutations in BMF (Figure 31). Although GPI -or HLA -seem to confer a protection at the cellular level, it seems not to confer a competitive advantage over the remaining GPI + or HLA + on the long run. Indeed, MDS/AML can stem from such cells in patients with PNH or HLA loss of function mutations or 6p-LOH. On the other hand, reversion or compensation are seen across IBMD (besides FA, GATA2, 170 TBD, 134,151,171 and SDS 136 ), and transformation to MDS/AML seem exceptional. Close surveillance including at least complete blood counts, marrow morphology and conventional cytogenetics at regular intervals is mandatory for all BMF patients. We still have insufficient evidence to recommend indiscriminate NGS -an isolated molecular abnormality will hardly change therapeutics. NGS and functional studies (as FANCD2 Western Blot) be considered on an individual basis.

Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease that manifests classically as intravascular hemolysis, bone marrow failure, and thrombosis. It is caused by somatic mutations in the PIGA gene, resulting in impaired glycosylphosphatidylinositol (GPI) biosynthesis. 1 GPI anchors many proteins on cells' surfaces, including the decay-accelerating factor (CD55) and the membrane inhibitor of reactive lysis (CD59) -which are modulators of the alternative and terminal complement pathway. Impaired GPI synthesis leads to complement-mediated hemolysis, the hallmark of PNH. 2 The most widely available treatments for PNH act as terminal-complement inhibitors, binding to C5, halting hemolysis, and reducing the occurrence of thrombosis. [3][4][5] The clinical response varies from complete to none. 6,7 C5 polymorphisms, red blood cell C3d opsonization, and genetic variants in complement regulatory genes might partly explain this variability. [8][9][10] Complement factor H (CFH) is a crucial alternative pathway regulator acting on the host-cell surface or in plasma, protecting them from complement damage. Impaired regulatory functions of factor H, caused by genetic abnormality or autoantibodies, have been linked to kidney diseases such as atypical hemolytic uremic syndrome and C3 glomerulopathy. 11 Both membrane-bound regulators and soluble complement inhibitors, particularly factor H and factor I, provide efficient protection from unwanted complement-mediated damage, especially under conditions with strong complement activation.

Therefore, we aimed to screen PNH patients for constitutional variants in complement regulatory genes, compare variant frequencies from PNH patients with population data and a geographically-matched healthy control group, and correlate the presence of such variants with response to anti-C5 treatment. We searched if these variants were associated with transfusion dependence six months after eculizumab initiation.

4

deficiency in the patient's plasma as previously described for complement regulatory genes). 13 Other variants were classified as variants of uncertain significance (VUS). 13,14 We confirmed variants' constitutional nature by sequencing DNA extracted from buccal swab samples or from cultured skin fibroblasts.

Statistical analyses

Qualitative variables are presented as numbers and percentages and compared with Fisher's exact test. Quantitative variables are presented as medians with ranges and interquartile ranges and compared with the Wilcoxon test. The distribution of rare variants in this cohort was compared to a control group of 80 healthy individuals and public data (1000 Genomes).

Transfusion dependence was the primary endpoint, defined as more than two red blood cell transfusion episodes during six months as a binary outcome. The secondary outcome was the event-free survival (EFS), defined as the first event of red blood cell transfusion after 28 days from eculizumab initiation (loading dose), thrombosis, hematopoietic stem cell transplantation (HSCT), evolution to a WHO 2016-defined myeloid neoplasia or death. Patients free of events were censored at the last followup. The EFS was estimated using the Kaplan-Meier estimator. The impact of baseline variables on the primary and secondary outcome was tested using Fisher or Wilcoxon tests and log-rank tests, respectively. We used the reverse Kaplan-Meier method to estimate the median follow. All tests were bilateral with a 5% level. We present EFS with 95% confidence intervals. We used the R software (R Core Team 2021. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/) for statistical analyses and figures.

All clinical and genetic data are presented in the main text or supplementary material. Requests for additional information (deidentified data) should be sent to the corresponding author.

Results and discussion

We found that rare germline CFH variants were overrepresented among PNH patients and associated with suboptimal response to anti-C5 treatment (based on transfusion requirements).

Eighty-four patients were eligible for the genetic variant screening, and 83 were evaluable for the primary and secondary objectives. Supplemental Figure 1 shows the patients' inclusion flow chart. Patients' characteristics are described in Supplemental Table 1. PNH diagnoses ranged from 1978 to 2019. Two patients started eculizumab after relapsed for PNH following HSCT for aplastic anemia, and with autologous reconstitution. Patients bearing a CFH variant were older at PNH diagnosis. Other baseline characteristics and PNH clinical picture at diagnosis were not different between variant CFH and wild-type (WT) CFH. During follow-up, seven patients presented a myeloid neoplasm: six myelodysplastic syndromes, one acute myeloid leukemia, and one chronic myelomonocytic leukemia. Three patients died: two progressions of myeloid malignancy and one of hemorrhage. None of these patients had a rare variant in the studied genes.

Twenty-eight patients had a thrombosis before eculizumab initiation: nine Budd-Chiari syndromes, six calf/femoral deep vein thrombosis, three strokes, two pulmonary 5 embolisms, two cerebral sinus thrombosis, one mesenteric artery, one splenic, one cochlear thrombosis, and three unspecified sites.

We identified 16 patients with a rare variant in complement regulatory genes: CFH (n=9), C3 (n=4), and CFI (n=3) (Supplemental Table 2). Among these rare variants, four were deemed pathogenic. We confirmed the germline nature of variants in 10 patients using DNA from buccal swab samples (n=2) or cultured skin fibroblasts (n=8). CFH variants were significantly overrepresented among PNH patients in our study when compared to healthy controls (p=0.02) or public data (p<0.001) (Table 1).

CFH variants or previous transfusions affected transfusion independence at six months after eculizumab initiation in univariate analysis (33.3% vs. 75.7% for CFH-variant and CFH-WT, respectively, p=0.015, Supplemental Table 3); no other baseline characteristics impacted transfusion dependence.

The median follow-up was 5.8 years [95%CI: 5.2 -7.3]. The 6-year EFS was 39.5 [95%CI: 27.7-51.0] and 0% for the CFH-standard and CFH-variant groups, respectively (Figure 1 and Supplemental Table 4). All but one CFH-variant patient received transfusions after eculizumab loading dose (Supplemental Table 4). One patient remained transfusion independent (never transfused, eculizumab started after a thrombotic event). Two patients had thrombosis under eculizumab therapy. No CFHvariant patient fulfilled the criteria for severe aplastic anemia from the initiation of eculizumab. Overall, patients younger at PNH diagnosis, without a CFH variant, and transfusion independent before eculizumab treatment had a better 6-year EFS (Supplemental Table 4).

Our data support that FH protection is necessary for the survival of PNH cells even during the C5 cleavage inhibition. Factor H is a negative regulator of the alternative pathway, binding to C3b. 15 Among CFH-variant patients, we found that the LDH (n=6) was mildly higher, whereas hemoglobin levels (n=7) were lower (Supplemental Figure 2) from day 28 to 6 months. These observations are compatible with extravascular hemolysis caused by C3d opsonization (Supplemental Figure 3). Specific CFH variants may downsize factor H activity, upregulating the alternative pathway and regulating the C3 and C5 convertases. In patients under anti-C5 treatment, the upstream C3b accumulation might aggravate hemolysis and contribute to poor response to eculizumab. Not all identified CFH variants are pathogenic using stringent criteria of functional deficiency. The role of FH on the PNH patient RBC surface is unclear. However, few studies suggested that FH plays an important RBCprotective role and that variation of FH levels may impact the erythrocytes' lifespan. 16 A limitation of this study was the heterogeneity of biological data during follow-up (reticulocyte count and CH50 activity), precluding analyses. We acknowledge the possibility of referral bias (national reference center). We were underpowered to perform subgroup or multivariate analyses because of the rarity of the disease.

Besides anti-C5, the FDA recently approved an anti-C3. 17 Moreover, a factor B inhibitor presented promising results in a phase 2 study. 18 Patients bearing these CFH variants might respond differently to proximal complement inhibitors.

The functional consequences of CFH variants and their possible role in GPI-deficient clones' positive selection are incompletely known to date. Our findings may help better understand the heterogeneity of PNH clinical course and response to anti-C5 treatment. NGS is becoming progressively accessible in clinical care, and patients' molecular stratification in choosing the treatment is standard for many diseases. PNH 6 drugs are expensive. Tailored treatments could reduce costs and morbidity, ultimately improving patients' quality of life. Our results need validation and justify a collaborative study to confirm the impact of CFH variants on response to PNH treatment. • EGF331-L: BRCA2 -/- (positive control) • EGF088-L: FANCA -/- • EGF192-L: FANCA -/- • EGF248-L: FANCA -/-BRCA2 -/+ • EGF409-L: FANCM -/- Survival assay EBV cell lines were plated at a density of 1x10 4 cells per well in opaque flat bottom 96well plates and exposed to increasing doses of talazoparib (0, 1n 5, 10nM). Cell viability was assessed after 3 days of culture using the Cell Titer Glo Luminescent Cell Viability Assay (G7573, Promega).

Results and Discussion

The treatment for FA-related myeloid neoplasms, especially AML is an unmet clinical need: in the era of precision medicine, patients with a significant DNA-repair deficiency still depend highly on traditional genotoxic chemotherapy. The sole curative treatment option is allogeneic hematopoietic stem cell transplantation. This modality carries a high treatment-related mortality in this genetically sensitive population. Therefore, we investigated the underlying mechanisms driving progression towards MDS/AML in Fanconi anaemia. We extracted the FA cases from the National Reference Center for Inherited Bone Marrow Failure Syndromes cohort (n=335, probably all FA patients followed in France over 18 years) and found that nearly onethird developed a hematopoietic clonal evolution (somatic cytogenetic abnormality or myeloid neoplasm). We pair-wise (AML cells and cultured fibroblasts for germline benchmarking) studied samples of 15 patients (10 FA core and 5 BRCA2/FANCD1). Interestingly, we found that AML cells from the BRCA2/FANCD1 and the FA-core genotype patients displayed an HR-deficient, BRCA-like signature (COSMIC 3) compared to a TARGET AML control. Signature 3 is associated with failure of DNA double-strand break repair by homologous recombination. This signature is strongly associated with cancers with somatic or germline BRCA1/2 mutations. BRCAness is of clinical interest as it might guide treatment choice in HR-deficient cancers. The poly(ADP-ribose) polymerase (PARP) is an early and versatile player in DNA repair, acting in single and doublestrand breaks. It contributes to HR and alternative end-joining and inhibits nonhomologous end-joining repair. The inhibition of PARP in HR-deficient cells induces synthetic lethality. Currently, at five four PARP inhibitors have FDA/EMA approval for treating HR-deficient / BRCA-like / COSMIC 3 signature solid cancers -Talazoparib, Olaparib, Rucaparib, and Niraparib.

Studies testing PARP inhibition in Fanconi anemia are scarce and contradictory. Following our observations, we wondered if FA-deficient cells were more sensitive to PARPi than FA-competent cells in vitro. I tested a potent PARP inhibitor, talazoparib, in human immortalized EBV-lymphoblasts bearing biallelic inactivations in BRCA2 (n=1), FANCA (n=2), FANCA + BRCA2 het (n=1), and FANCM, and normal. I found that FANCA-deficient cell lines do not seem to present increased sensitivity to PARP inhibition, contrary to BRCA2 (Figure 32). On the other hand, the FANCM cell line was very sensitive to PARP inhibition. Adding daunorubicin, cytarabine, or mitomycin is not synergistic in Fanconi lines (data not shown). We will validate our findings in other complementation groups and cell lines. Our preliminary data does not support PARP inhibition as a target in FA-core associated MDS/AML. Diversely, haematopoietic malignancies are not the rule for FANCM -for such patients, solid cancers is the major concern. 172 Germline heterozygous patients that develop a solid cancer (e.g., breast, ovary), BRCAness is restricted to the neoplastic tissue and PARP inhibition is beneficial. On the other hand, FA patients are present a DNA-repair deficiency in all his / her cells. Thus far, we do not know if toxicity would be prohibitive. The preclinical studies were based on patient-derived xenografts and cell-derived xenografts, i.e., the mice DNArepair was intact.

We are currently establishing a colony of BRCA2 mut mice. 173 These mice's HSPCs present a proliferation defect and spontaneous chromosomal instability but not spontaneous cytopaenias. [START_REF] Navarro | Hematopoietic Dysfunction in a Mouse Model for Fanconi Anemia Group D1[END_REF] Then, we will backcross into the background C57BL/6J CD45.2. Our aim is to develop a BRCA2-associated leukaemia using viral transduction (EV1 as presented in Chapter 4), test leukaemia's cells sensitive to PARP inhibition, and toxicity in a constitutively deficient subject. We will investigate a therapeutic window for PARP inhibition in FA-AML.

  EN FRANÇAIS .......................................................................................................................... PROLOGUE ............................................................................................................................................... CHAPTER 1: INTRODUCTION ................................................................................................................ 1.1 THE BLOOD AND POLYCLONAL HAEMATOPOIESIS .................................................................................................... 1.2 MEN AND MICE .............................................................................................................................................. 1.3 CLONAL HAEMATOPOIESIS ................................................................................................................................. 1.4 BONE MARROW FAILURE SYNDROMES ................................................................................................................. 1.4.1 Idiopathic aplastic anaemia ............................................................................................................... 1.4.1.1 Pathophysiology ............................................................................................................................................ 1.4.1.2 Treatment ...................................................................................................................................................... 1.4.1.3 Aplastic anaemia animal models ...................................................................................................................

  ................................................................................................................................................. CHAPTER 2: METHODS AND RESULTS .............................................................................................. 2.1 CLINICAL AND MOLECULAR DETERMINANTS OF CLONAL EVOLUTION IN APLASTIC ANEMIA AND PAROXYSMAL NOCTURNAL HEMOGLOBINURIA ................................................................................................................................................. 2.1.

  myeloid leukaemia ARCH: age-related clonal haematopoiesis ASH: the American Society of Hematology BMF: bone marrow failure CCUS: clonal cytopenia of undetermined significance CFU: colony-forming unit CH: clonal haematopoiesis CHIP: clonal haematopoiesis of indeterminate potential CHOP: clonal haematopoiesis of oncogenic potential DBA: Diamond-Blackfan Anaemia EBMT: European Society for Blood and Marrow Transplantation EMA: European Medicine Agency FDA: Food and Drug Administration GPI: glycosylphosphatidylinositol GvHD: graft-versus-host disease HLA: human leukocyte antigen HSC: haematopoietic stem cell HSCT: haematopoietic stem cell transplantation HSPC: haematopoietic stem and progenitor cells IBMF: inherited bone marrow failure ICUS: idiopathic cytopenia of undetermined significance KO: knock-out Lin -: lineage-negative haematopoietic cells LSK: lineage-negative, Sca-positive, Kit-positive haematopoietic cells MDS: myelodysplastic syndrome NGS: next-generation sequencing pI:pC: polyinosinic : polycytidylic acid PNH: paroxysmal nocturnal haemoglobinuria SDS: Schawcham-Diamond syndrome TBD: telomere biology diseases VAF: variant allele frequency VUS: variant of unknown / uncertain significance WT: wild-type

Figure 1 :

 1 Figure 1: A schematic representation of the hierarchical process of haematopoiesis 4

Figure 2 :

 2 Figure 2: Schematic representation of the last common ancestor between different animal models and humans (adapted from 5 ).

11

 11

Figure 3 :

 3 Figure 3: Revised murine haematopoietic tree 10

Figure 4 :

 4 Figure 4: Lineage-committed cells would emerge directly from multipotent cells.11

11

 11 Figure 4: Lineage-committed cells would emerge directly from multipotent cells.11

Figure 5 :

 5 Figure 5: 5-methylcytosine hydrolysis into thymidine 18

Figure 6 :

 6 Figure 6: Schematic representation of the stepwise evolution from CHIP to AML though the accumulation of multiple hits in the stem cell compartment 26

Figure 7 :

 7 Figure 7: Schematic representation of the FA core complex and I/D2 monoubiquitination process 63

Figure 8 :

 8 Figure 8: Schematic representation of the steps for obtaining the Fancg KO mouse model

92

 92

Figure 9 :

 9 Figure 9: Two sisters Note the smaller size and absence of the right eye in Fancg -/-mouse (left) compared to her wt sister (right) -both are four weeks old.

Figure 10 :

 10 Figure 10: Distribution of the most frequently found genomic abnormalities across ARCH, AA, and MDS 80

Figure 11 :

 11 Figure 11: The Luzzatto-Rotoli hypothesis Panel A: normal individuals might bear a small PNH clone without clinical significance. Panels B, C, and D represent different scenarios of PNH clones in the context of BMF. Panel E represents acute leukaemia emerging following BMF. 110

Figure 12 :

 12 Figure 12: Clonal dynamics in patients' natural history A) Schematic representation of MDS and PNH clones' co-existence. MDS and PNH originate from different founders 118 B) the malignant clone outcompeted PNH cells 117

Figure 13 : 1 . 6 Clonal haematopoiesis in Fanconi Anaemia 1 . 6 . 1

 1316161 Figure 13: The immune escape hypothesis HLA -or GPI -HSCs would escape T-cell mediated cytotoxicity and achieve clonal dominance because of a bottleneck effect (Luzzatto-Rotoli hypothesis). The immune escape clones would co-exist with premalignant clones (intrinsic proliferative advantage) that could or not progress to myeloid malignancy.132

Figure 14 :

 14 Figure 14: FANCD2 western blot of FA patients Non-FA patients monoubiquitinate FANCD2 constitutively and the 162kDa band is present in both the haematopoietic tissue and fibroblasts. FA-core patients fail to monoubiquitinate FANCD2 and the 162kDa band is absent. On the other hand, revertant (REV) patients are capable of monoubiquitinating FANCD2 within the haematopoietic tissue but fail elsewhere. The compensation mechanisms underlying some attenuated phenotypes (ATT) are unclear 142

Figure 15 :

 15 Figure 15: Clinical representation of revertant patients' natural history -French cohort

Figure 16 :

 16 Figure 16: Engraftment dynamics following gene therapy 149

Figure 17 :

 17 Figure 17: Schematic representation of clonal dominance in untreated, transplated, and revertant / gene-therapy FA patients.

Figure 1 .Figure 2 .

 12 Figure 1. Study design. CONSORT flowchart of patients' inclusion (A) with details of sequenced patients for cross-sectional and longitudinal analysis (B). MN=myeloid neoplasia

Figure 3 .

 3 Figure 3. Features of malignant progression. Panel A shows the 5-year overall survival according to bone marrow blast percentage at sMN onset (58.2% [41.1 -71.9] in patients with <5% vs. 20.2% [8.6 -35.3] in those with >5%). Forrest plot in panel B illustrates multivariate analysis for overall survival using a multivariate Cox-cause specific model. In panel C, a waterfall plot depicts the genomic landscape of progressors (N=82 patients with available molecular information). Disease subtype, cytogenetic risk groups according to R-IPSS, R-IPSS risk group (higher >3.5; lower <3.5), and age categories (aged <60/>60 years) are color-coded on top while on the right side frequencies of mutations for each gene are shown. Panel D

Figure 4 .-oo

 4 Figure 4. Clonal dynamics in aplastic anemia. In panel A, the pie chart shows the frequency of mutants for PIGA and myeloid mutations with a breakdown of cases according to characteristics at onset and treatment response (n=302). Bar charts in panel B illustrate the difference of molecular spectra between baseline and myeloid progression with frequencies estimated on the total of mutants (n=302 at baseline; n=82 at evolution). Panel C shows the dynamics of changes in molecular lesions (HLA, PIGA, myeloid) in patients sequenced at baseline (D=diagnosis), follow-up (F) and evolution (E, see Figure S13 for details). Frequencies are shown as solid lines on the left, clonal burdens (median VAFs at each time point) are shown as dashed lines on the right with each color representing a different set of mutations. In panel D, a stratified Cox-proportional hazard function adjusting for age was fit to estimate baseline progression hazard rates and generate progression-free survival (PFS) curves of patients with AA and AA/PNH (N=264). Patients were classified based on features (myeloid driver mutations only=Group 1; PIGA/PNH clone presence and BCOR/L1=Group 2, no mutation=Group 3)previously hypothesized to be associated with myeloid evolution (see also Supplementary Appendix; numbers at risk are indicated below the curves). Particularly, N=7 patients (1 evolved to sMN) with concomitant PIGA/PNH clone and myeloid mutations were considered as belonging to Group 2. The bar chart on the lower right illustrates the difference in age at onset in an analysis of patients with pure AA phenotype (n=58).

3. 2

 2 Secondary objectives: -Overall survival: time from diagnosis to last follow-up or death -Secondary PNH incidence: time from AA diagnosis to occurrence of secondary PNH (competing events: death and HSCT) -Risk-factors for secondary myeloid neoplasm or PNH -Characterization of the genomic landscape of patients with evolution, focusing on genomic lesions known to have a role in myeloid neoplasms and bone marrow failure syndromes -Comparison of mutational patterns observed in secondary myeloid neoplasms with the distribution of genetic lesions seen in primary patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), focusing on chromosome 7 abnormalities

  Figure S1. Bio-informatic pipeline for HLA mutation detection. Details are provided above.

Figure S5A -

 S5A Figure S5A-B. Cumulative incidence of secondary PNH evolution in patients non-transplanted upfront

Figure S6 .

 S6 Figure S6. Cumulative incidence of malignant evolution in non-severe AA cases according to treatment and treatment response

Figure S7 .

 S7 Figure S7. WHO 2016 subtypes of secondary MN cases. The circle diagram showcases the diagnoses

Figure S8 .

 S8 Figure S8. Overall Survival of evolved patients from sMN onset

Figure S10 .

 S10 Bar charts illustrating the frequency (A) and type (B) of mutations according to the identified cytogenetic groups in sMN cases.

Figure S9 .

 S9 Figure S9. Cytogenetics at evolution and time elapsed from initial bone marrow failure diagnosis

Figure S10

 S10 Figure S10 Bar charts illustrating the frequency (A) and type (B) of mutations according to the identified cytogenetic groups in sMN cases.

Figure S11 .

 S11 Figure S11. Matched-pair comparison of secondary with primary MN cases.

Figure S12 .

 S12 Figure S12. Waterfall plot showcasing the mutational landscape at baseline of patients with available information.

Figure S13 .

 S13 Figure S13. Details on the cross-sectional analysis of immune-molecular dynamics.

Figure S14 .

 S14 Figure S14. Details on treatment response according to mutational status.

Figure S14 .

 S14 Figure S14. Details on treatment response according to mutational status

Figure S16 .

 S16 Figure S16. Longitudinal molecular studies of 25 serial cases.

Figure 18 :

 18 Figure 18: Examples of plasmid backbones used in this work.

  pAdenoX-CMV Linear pAdenoX-DsRed-Express Linear pAdenoX-PRLS-DsRed-Express Linear pAdenoX-PRLS-ZsGreen1 Linear pAdenoX-PRLS Linear pAdenoX-Tet3G Linear pAdenoX-ZsGreen1 Linear pAd/BLOCK-iT-DEST pAd/CMV/V5-DEST pLVX-rHom-Sec1 pLVX-Tet-Off Advanced pLVX-Tet-On Advanced pLVX-Tet3G pLVX-TetOne pLVX-TetOne-Puro pLVX-Tight-Puro pLVX-TRE3G pLVX-TRE3G-Hom1 pLVX-TRE3G-IRES at 42°C, and 5min of enzymatic inactivation at 85°C (Veriti Thermal Cycler, Applied Biosciences, Waltham, Massachusetts, MA).

Figure 19 :

 19 Figure 19: Validating the mouse model Upper left: CFU assay of KO vs WT lineage-negative cells treated with MMC. Upper right: CFU-assay of KO vs WT LSK cells treated with MMC. Absence of colonies in KO at 50nM. Bottom left and right: Absence of Fancg mRNA in different tissues of Fancg KO mice.

Figure 20 :

 20 Figure 20: Harvesting Lin -

Figure 21 :

 21 Figure 21: Fancg overexpression. Left panel: RT-qPCR comparing Fancg expression at 48 in RFP-sorted KO cells by plasmid. Right panel: Western blot of KO MEF transduced with Fancg plasmid or control.

Figure 22 :

 22 Figure 22: Evi1 protein expression after transduction

  Figure represents schematically the next three experiments.

Figure 23 :

 23 Figure 23: Schematic representation of competitive transplantation experiments.

Figure 24 :

 24 Figure 24: Engraftment Nearly all of recipients' mononuclear marrow haematopoietic cells were of donor origin (CD45.2) at 6 months.

Figure 25 :

 25 Figure 25: CFU-assay of transduced KO Lin-cells

Figure 26 :

 26 Figure 26: Proportion of transduced MEF through time

Figure 27 :

 27 Figure 27: qPCR of viral titration, pooled constructions.

Figure 28 :

 28 Figure 28: Efficiency x multiplicity of infection Left panel: Transduction efficiency with different constructions at increasing MOIs. Right panel: Transduction efficiency by multiplicity of infection using the Lenti-EF1a-RFP construction.

Figure 29 :

 29 Figure 29: Viability of lineage-negative cells by transduction status.

Figure 30 :

 30 Figure 30: Viability x MOI x plasmid x genotype A) Viability by multiplicity of infection, transduction status, construction, and genotype. B) Representative flow cytometry histogram of transduced and non-transduced cells at MOI of 5.

Figure 31 :

 31 Figure 31: Different natural histories for BMF patients

Figure 32 :

 32 Figure 32: EBV cell line sensitivity to PARP inhibition

  1.4.2 Inherited bone marrow failure syndromes ......................................................................................... 1.4.2.1 Fanconi Anaemia ........................................................................................................................................... 1.4.2.2 Standard treatment ....................................................................................................................................... 1.4.2.3 Fanconi anaemia models ............................................................................................................................... 1.5 CLONAL HAEMATOPOIESIS IN APLASTIC ANAEMIA ................................................................................................... .6 CLONAL HAEMATOPOIESIS IN FANCONI ANAEMIA ..................................................................................................

1.5.1 The Dameshek's Riddle ....................................................................................................................... 1.5.2 Clonal evolution and immune escape: the Luzzatto-Rotoli hypothesis .............................................. 1.5.3 The Luzzatto-Rotoli hypothesis: beyond GPI-deficient cells ................................................................ 11.6.1 Different pathways: reversion, premalignancy, and MDS/AML ......................................................... 1.6.2 Gene therapy mimics somatic mosaicism in Fanconi anaemia .......................................................... 1.7 THESIS AIMS ..

Table of figures

 of FIGURE 1: A SCHEMATIC REPRESENTATION OF THE HIERARCHICAL PROCESS OF HAEMATOPOIESIS 4 FIGURE 2: SCHEMATIC REPRESENTATION OF THE LAST COMMON ANCESTOR BETWEEN DIFFERENT ANIMAL MODELS AND HUMANS (ADAPTED FROM 5 ).

	FIGURE 3: REVISED MURINE HAEMATOPOIETIC TREE 10
	FIGURE 4: LINEAGE-COMMITTED CELLS WOULD EMERGE DIRECTLY FROM MULTIPOTENT
	CELLS. 11
	FIGURE 5: 5-METHYLCYTOSINE HYDROLYSIS INTO THYMIDINE 18
	FIGURE 6: SCHEMATIC REPRESENTATION OF THE STEPWISE EVOLUTION FROM CHIP TO
	AML THOUGH THE ACCUMULATION OF MULTIPLE HITS IN THE STEM CELL
	COMPARTMENT 26
	FIGURE 7: SCHEMATIC REPRESENTATION OF THE FA CORE COMPLEX AND I/D2
	MONOUBIQUITINATION PROCESS 63
	FIGURE 8: SCHEMATIC REPRESENTATION OF THE STEPS FOR OBTAINING THE FANCG KO
	MOUSE MODEL
	FIGURE 9: TWO SISTERS
	FIGURE 10: DISTRIBUTION OF THE MOST FREQUENTLY FOUND GENOMIC ABNORMALITIES
	ACROSS ARCH, AA, AND MDS 80
	FIGURE 11: THE LUZZATTO-ROTOLI HYPOTHESIS
	FIGURE 12: CLONAL DYNAMICS IN PATIENTS' NATURAL HISTORY
	FIGURE 13: THE IMMUNE ESCAPE HYPOTHESIS
	FIGURE 14: FANCD2 WESTERN BLOT OF FA PATIENTS
	FIGURE 15: CLINICAL REPRESENTATION OF REVERTANT PATIENTS' NATURAL HISTORY -
	FRENCH COHORT
	FIGURE 16: ENGRAFTMENT DYNAMICS FOLLOWING GENE THERAPY 149
	FIGURE 22: EVI1 PROTEIN EXPRESSION AFTER TRANSDUCTION
	FIGURE 23: SCHEMATIC REPRESENTATION OF COMPETITIVE TRANSPLANTATION
	EXPERIMENTS.
	FIGURE 24: ENGRAFTMENT
	FIGURE 25: CFU-ASSAY OF TRANSDUCED KO LIN-CELLS
	FIGURE 26: PROPORTION OF TRANSDUCED MEF THROUGH TIME
	FIGURE 27: QPCR OF VIRAL TITRATION, POOLED CONSTRUCTIONS.
	FIGURE 28: EFFICIENCY X MULTIPLICITY OF INFECTION
	FIGURE 29: VIABILITY OF LINEAGE-NEGATIVE CELLS BY TRANSDUCTION STATUS.
	FIGURE 30: VIABILITY X MOI X PLASMID X GENOTYPE
	FIGURE 32: DIFFERENT NATURAL HISTORIES FOR BMF PATIENTS
	FIGURE 33: EBV CELL LINE SENSITIVITY TO PARP INHIBITION

FIGURE 17: SCHEMATIC REPRESENTATION OF CLONAL DOMINANCE IN UNTREATED, TRANSPLATED, AND REVERTANT / GENE-THERAPY FA PATIENTS. FIGURE 18: EXAMPLES OF PLASMID BACKBONES USED IN THIS WORK. FIGURE 19: VALIDATING THE MOUSE MODEL FIGURE 20: HARVESTING LIN - FIGURE 21: FANCG OVEREXPRESSION.

Table 2 :

 2 Orthologs of the 22 FA genes in different speciesAdapted from[START_REF] Dong | Update of the human and mouse fanconi anemia genes[END_REF] and updated based on https://www.alliancegenome.org.

Table 3 :

 3 High homology between men and mice FANC orthologs

*per cent identity in https://blast.ncbi.nlm.nih.gov/

Does somatic functional reversion reduce the risk of myeloid clonal evolution in MDS/AML in Fanconi patients ? Main objective: to

  demonstrate that functionally corrected Fanconi cells have an adaptative advantage over non-corrected Fanconi premalignant cells in vivo

	Allo-SCT
	Reversion / gene therapy	?	?	? ?	?	?	?	? ?	? ?	? ?	? ?	? ?	?	?	? ?	? ?	?	?	?	?	?	? ?	?	? ?	?	?	? ?	?

  7,35 In either case, progression of CHIP, age-related CH acceleration, or Darwinian selection phenomena in the context of AA immune responses may be invoked to explain the relationship of age and IST responses with regards to myeloid evolution. One could even speculate that the same mutation may hold a diverse prognostic significance when occurring in two patients with different ages at disease onset (e.g., 20 vs. 60 years). Besides biological implications, in such a scenario therapeutic choice would be strongly influenced by age, with patients undergoing HSCT possibly protected from the development of sMN because of the

complete subversion of BM hematopoiesis (e.g., from oligoclonal in AA to normal, polyclonal after HSCT). This is the reason why sMN is a problem typical of the non-transplant setting, where clonal trajectories are sculpted by the re-expansion of the individual patient's hematopoiesis after IST (so-called bottleneck effect), which not only increases the number of HSCs replications with a non-negligible BM stress/telomere attrition but generates processes of clonal dominance if mutations are already present (selection of the fittest clone).

Table 1 . Main clinical characteristics of the study cohort.

 1

	Variables	Modalities	N=999	AA (N=817)	AA/PNH (N=75)	PNH (N=107)
		Female	502 (50.3)	399 (48.8)	43 (57.3)	60 (56.1)
	Gender	Male	497 (49.7)	418 (51.2)	32 (42.7)	47 (43.9)
	Age at diagnosis	median [IQR]	34.0 [20.1-54.3]	34 [19.1-56.7]	30.5 [20.5-43.9]	36.1 [27.3-46.9]
	PNH clone at	no	433 (52)	433 (66.5)	0 (0)	0 (0)
	diagnosis (N=832)*	yes	399 (48)	218 (33.5)	74 (100)	107 (100)
	PNH clone (percentage)	median [IQR]	11 [2.4-61.4]	3.3 [1.2-8]	48 [30.8-82.7]	83.5 [61.4-94.5]
	Camitta criteria	non-severe severe		176 (21.5) 641 (78.5)	48 (64) 27 (36)
		Normal	725 (93)	598 (91.6)	64 (100)	63 (100)
	Karyotype at diagnosis (N=780)*	Failure	43 (5.5)	43 (6.6)	0 (0)	0 (0)
		Others**	12 (1.5)	12 (1.8)	0 (0)	0 (0)
		RPH	116 (11.6)	98 (12)	14 (18.7)	4 (3.7)
	Cohort	CCF	396 (39.6)	336 (41.1)	17 (22.7)	43 (40.2)
		SLS	487 (48.7)	383 (46.9)	44 (58.7)	60 (56.1)

*indicated is the available sample size due to missing data;** including sex chromosome abnormalities and age-related findings not configuring an alternative diagnosis (see also Supplementary Appendix). CCF: Cleveland Clinic Foundation; SLS: Hôpital Saint-Louis; RPH: Riberão Preto Hospital; AA: aplastic anemia; PNH: paroxysmal nocturnal hemoglobinuria.

Table 2 . Multivariate analysis for severe AA cases (N=504)

 2

	Variables		Overall Survival	sMN incidence		sPNH incidence*
			HR (95% CI)	p-value	HR (95% CI)	p-value	HR (95% CI)	p-value
		F	1		1		1
	Gender
		M	1.00 (0.69-1.45)	0.99	1.39 (0.82-2.33)	0.22	0.49 (0.27-0.89)	0.02
	Response to	CR	1		1		1
	treatment	No CR	4.45 (2.54-7.77) <0.001	2.60 (1.39-4.84)	0.003	0.76 (0.41-1.41)	0.39
	Age (10y effect)	1.29 (1.16-1.43) <0.001	1.37 (1.20-1.56)	<0.001	0.88 (0.75-1.02)	0.1
	Year at transplant (10y effect) 1.35 (0.96-1.88)	0.08	1.14 (0.76-1.72)	0.52	1.47 (0.95-2.28)	0.09
	Center effect as frailty		0.015		0.97		0.02
	*only SAA. sPNH: secondary paroxysmal nocturnal hemoglobinuria; F: female; M: male; CR: complete response; sMN: secondary
	myeloid neoplasia; HR: hazard ratio; 95% CI: 95% confidence intervals.

Table S4 . Main baseline, treatment, and response characteristics of patients with severe AA (N=504)

 S4

	Variables	Modalities	All (N=504)	SAA (N=481)	SAA/PNH (N=23)
	PNH clone	no	265 (63.9)	265 (67.4)	0 (0)
	(N=415)*	yes	150 (36.1)	128 (32.6)	22 (100)
	PNH percentage	median [IQR]	4 [1.3-11]	3 [1.2-6.5]	37.4 [27-90]
	(N=415)*	(range)	(0.1-99)	(0.1-84)	(0.4-99)
	Age	median [IQR] (range)	34.9 [19.1-58.1] (3-85.4)	35.2 [19.1-58.5] (3-85.4)	29.4 [20.4-40.9] (9.6-78.6)
	Gender	F M	240 (47.6) 264 (52.4)	225 (46.8) 256 (53.2)	15 (65.2) 8 (34.8)
	Karyotype (N=431)*	normal failure other	394 (91.4) 25 (5.8) 12 (2.8)	371 (90.9) 25 (6.1) 12 (3.0)	23 (100) 0 (0) 0 (0)
	N. of myeloid mutations (N=155)*	0 1 2 3	124 (80) 25 (16.1) 5 (3.2) 1 (0.6)	118 (79.2) 25 (16.8) 5 (3.4) 1 (0.7)	6 (100) 0 (0) 0 (0) 0 (0)
	PIGA mutant	No	97 (79.5)	94 (80.3)	3 (60)
	(N=122)*	Yes

Table S6 . Main baseline, treatment and response characteristics of patients with non-severe AA (N=192)

 S6

	Variables	Modalities	nSAA (N=192)	nSAA (N=151)	nSAA/PNH (N=41)
	PNH clone	no	78 (46.2)	78 (60.9)	0 (0)
	(N=169)*	yes	91 (53.8)	50 (39.1)	41 (100)
	PNH percentage	median [IQR]	14 [4.2-44.2]	5.4 [2-12]	63 [37-84]
	(N=169)*	(range)	(0.1-99)	(0.1-27)	(14-99)
	Age	median [IQR] (range)	37.2 [22-58.8] (5.1-89.4)	38.7 [22.1-60.2] (5.1-89.4)	33.1 [21-44.6] (5.8-86.7)
	Gender	F M	113 (58.9) 79 (41.1)	90 (59.6) 61 (40.4)	23 (56.1) 18 (43.9)
	Karyotype (N=169)*	normal failure other	163 (96.4) 3 (1.8) 3 (1.8)	127 (96.2) 3 (2.3) 2 (1.6)	36 (97.3) 0 (0) 1 (2.7)
	N. of myeloid	0	68 (81.9)	59 (79.7)	9 (100)
	mutations	1	14 (16.9)	14 (18.9)	0 (0)
	(N=83)*	2	1 (1.2)	1 (1.4)	0 (0)
	PIGA mutant	no	42 (65.6)	40 (71.4)	2 (25)
	(N=64)*	yes	22 (34.4)	16 (28.6)	6 (75)
		None	65 (33.9)	45 (29.8)	20 (48.8)
	First line	ATG/CSA based	28 (
	treatment
					)
	Response to	CR	176 (35.5)	169 (35.7)	7 (31.8)
	treatment (N=496)*	no CR	320 (64.5)	305 (64.3)	15 (68.2)
	Pts. receiving >1	no	250 (50.2)	236 (49.7)	14 (60.9)
	line of treatment (N=498)*	yes	248 (49.8)	239 (50.3)	9 (39.1)
		1	250 (50.3)	236 (49.8)	14 (60.9)
	N. of lines received (N=497)*	2 3 4	155 (31.2) 61 (12.3) 20 (4)	151 (31.9) 58 (12.2) 20 (4.2)	4 (17.4) 3 (13) 0 (0)
		>5	11 (2.2)	9 (1.9)	2 (8.7)
	Eltrombopag	no	382 (76.2)	363 (75.9)	19 (82.6)
	(N=501)* /&	yes	119 (23.8)	115 (24.1)	4 (17.4)
	Eculizumab	no	444 (88.6)	438 (91.6)	6 (26.1)
	(N=501) * /&	yes	57 (11.4)	40 (8.4)	17 (73.9)
	Subsequent	no	380 (75.4)	362 (75.3)	18 (78.3)
	transplant	yes	124 (24.6)	119 (24.7)	5 (21.7)

Table S8 . Main characteristics at secondary myeloid progression (N=94)

 S8

	Variables				Modalities	N=94
	Gender				F	36 (38.3)
					M	58 (61.7)
	Age at progression		median [IQR] (range)	61.0 [34.4-69.3] (11-87.5)
	Time from diagnosis to	median [IQR]	4.5 [1.8-7.7]
	progression (years)		(range)	(0.7-33.1y)
	BM blasts				median [IQR] (range)	3.5 [1-8.8] (0-66)
					AML	17 (18.1)
	MN diagnosis			MDS MDS/MPN	71 (75.5) 5 (5.3)
					MPN	1 (1.1)
					AML-MRC	12 (12.8)
					AML-NOS	3 (3.2)
					AML with mutated NPM1	1 (1.1)
					CMML	3 (3.3)
					MDS-del(5q)	1 (1.1)
					MDS-EB-1	14 (14.9)
					MDS-EB-2	7 (7.4)
					MDS-MLD	28 (29.8)
					MDS-RS-MLD	2 (2.1)	)
	Median time to	median [IQR]	0.37 [0-1] MDS-RS-SLD	0.4 [0-1]	0.5 [0.1-0.9] 1 (1.1)
	Treatment (years) Response to treatment (N=188)* WHO 2016 Pts. receiving >1 line of treatment (N=189)*	(range) CR No CR No TTT no yes	(0-30.6) MDS-SLD 35 (18.6) 88 (46.8) MDS-U MDS/MPN-U 65 (34.6) MF 142 (75.1) MPN-U 47 (24.9) complex del(7/7q)	(0-17) 31 (20.8) 73 (49) 45 (30.2) 110 (73.3) 40 (26.7)	4 (4.3) (0-30.6) 4 (10.3) 14 (14.9) 2 (2.1) 15 (38.5) 1 (1.1) 20 (51.3) 1 (1.1) 32 (82.1) 9 (9.8) 7 (17.9) 42 (44.6)
	Cytogenetics		0	65 (34.4) normal	45 (30)	20 (51.3) 18 (19.1)
	N. of lines received* (N=189) R-IPSS (MDS cases)	1 2 3 4	77 (40.7) other 16 (8.5) failure 17 (9) 5 (2.6) higher lower	65 (43.3) 14 (9.3) 14 (9.3) 5 (3.3)	12 (30.8) 22 (23.4) 2 (5.1) 3 (3.1) 3 (7.7) 0 (0) 37 (57.8) 27 (42.2)
				>5	9 (4.7) 0	7 (4.7)	2 (5.1) 16 (19.5)
	Eltrombopag (N=191)* /& N. of myeloid mutations no yes Eculizumab (N=190)* /& no (N=82)* yes no Treatment (N=90)*	150 (78.5) 41 (21.5) 1 137 (72.1) 2 53 (27.9) >2 171 (89.1) no CHT	112 (74.7) 38 (25.3) 133 (88.7) 17 (11.3) 135 (89.4)	38 (92.7) 26 (31.7) 3 (7.3) 15 (18.3) 4 (10) 25 (30.5) 36 (90) 40 (44.4) 36 (87.8) 11 (12.2)
					HMA	17 (18.9)

Table S9 . Baseline characteristics of patients subsequently progressed to sMN (N=94)

 S9

	Variables	Modalities	N=94
		AA	77 (81.9)
	Diagnosis of BMF	AA/PNH	11 (11.7)
		PNH	6 (6.4)
	PNH clones (N=74)*	no yes	37 (50) 37 (50)
	PNH percentage at	median [IQR]	7 [3.2-47]
	diagnosis (N=74)*	(range)	(0.3-99.5)
	Age at diagnosis	median [IQR] (range)	56.5 [28.4-65.5] (7.6-86.7)
	Gender	F M	36 (38.3) 58 (61.7)
	Severity	non-severe severe	25 (28.4) 63 (71.6)
	N. of myeloid mutations (N=25)*	0 1 2	18 (72) 6 (24) 1 (4)
	PIGA mutations (N=18)*	No Yes	16 (88.9) 2 (11.1)
		None	18 (19.1)
		ATG/CSA based	55 (58.5)
	First-line treatment	CSA based	13 (13.8)
		Eltrombopag	2 (2.1)
		Other	6 (6.4)
	Response to first-line treatment (N=93)*	CR No CR No TTT	16 (17.2) 59 (63.4) 18 (19.4)
		0	18 (19.4)
	Number of lines received	1	39 (41.9)
	(N=93)*	2	18 (19.4)
		>3	18 (15.5)
	Second-line treatment	no	38 (50.7)
	received (N=93)*	yes	37 (49.3)
		ATG+CSA	13 (41.9)
		ATG+CSA+Epag	1 (3.2)
	Second-line treatment	CSA	6 (19.4)
	(N=31)*	CSA+Epag	1 (3.2)
		Other	9 (29)
		Other+Epag	1 (3.2)
	Response to second line	CR	1 (3.8)
	(N=26)*	NR	14 (53.8)

Table S11 . Univariate analysis for overall survival according to mutational status (most frequent mutations) in progressed cases (N=82 patients with molecular information)

 S11

		Overall survival from MN progression
	Marker	N=82	Univariate HR (95% CI)	p-value (Wald)
	ASXL1	20	1.49 (0.74-3.00)	0.26
	RUNX1	17	1.35 (0.66-2.73)	0.4
	SETBP1	11	1.51 (0.67-3.42)	0.32
	DNMT3A	10	Nonproportional risk	0.02 (Log-Rank)
	U2AF1	10	0.78 (0.30-1.99)	0.6
	TET2	7	1.83 (0.71-4.70)	0.21
	EZH2	5	0.27 (0.04-1.98)	0.2
	CBL	4	0.29 (0.04-2.13)	0.23
	ETV6	4	1.12 (0.27-4.67)	0.87
	FLT3	4	0.77 (0.11-5.66)	0.8
	TP53	4	12.03 (3.67-39.44)	<0.001

reporting molecular data on aplastic anemia non transplanted upfront

	2.2
	Gurnari Studies Yoshizato* Groarke Negoro** Park Babushok Huang		Kulasekararaj	Heuser	Lane Total
	Journal	JCO	Leukemia	Blood	BJH Cancer Genetics Haematologica		NEJM		Blood Haematologica	Leukemia
	Year	2022	2022	2017	2017	2015	2015		2015		2014	2014	2013
	Technique	NGS	NGS	WES / NGS	NGS	WES	NGS		WES / NGS		NGS	NGS	Sanger
	Centers	Paris, Cleveland, Brazil	NIH Cleveland, Basel	Seoul	U. Penn	Peking	Cleveland	NIH	Japan	King's	Hannover Dana Farber
	n (AA + AA/PNH)	779	642	199	210	22	138	24	256	159	150	38	33 2650
	Age at BMF onset	37.2	31.9	52	42	14.5	30.7	46.9	29	60	41.6	30	34.8
	Male (%)	383 (49.2)	362 (56.4)	71 (53.4)	105 (50)	9 (41)	81 (58.7)	15 (62.5)	157 (61.3)	72 (45.3)	71 (54.6)	18 (47) 22/39 (46)
	PNH (positive/total, %)	264/646 (40.9) 140/366 (38.2)	-15/105 (14.3)	10 (45.5)	20 (14.5)	16 (66.7)	146 (57)	95 (59.7) 61/150 (40.6)	17/35 (48.6) 4/31 (12.9)
	Severe (%)	566 (70.8)	-	48/133 (36.1)	57 (27.1)	21 (95.4)	81 (58.7)	21 (87.5)	256 (100)	67 (42.1)	74 (52.8)	27 (71)	20 (74.3)
	Cytogenetics (%)	650 (83.4)	-	-	210 (100)	22 (100)	138 (100)	-	-	-	143 (95)	25 (65.8)	33 (100)
	Baseline molecular data (%)	256 (32.8)	-	59	-	13 (59.1)	-	5	0	35	63 (18.2)	-	-431
	sMN events	88	93	35	18	0	9/100 (9)	5/24 (20.8)	36/256 6/141 (42.6)	17	1 (2.6)	-308
	Median FU (years)	8.6	4.7	-	-	1.2	-	5.9	4.9	6.3	3.8	7	-

Developing a mouse model of somatic mosaicism in Fanconi Anaemia

  Protein extracts were prepared from frozen cell pellets with a RIPA lysis and extraction buffer (Thermo Fisher). Samples were dosed using the QuantiPro TM BCA Assay Kit (Sigma Life Sciences, ref: 1002803688) according to manufacturer's instructions. Absorbance was measured by CLARIOstar Plate Reader (BMG Lab Tech, France).

	The primers used for PCR reactions were:
	Evi-1 forward:	ATTTCATCGGGAACAGCAAC
	Evi-1 reverse:	ACGAGTCACCCCTAGCTCCT
	Fancg forward:	TGCTAGCAAGTCGATGCCTG
	Fancg reverse:	GGGAGAACCTTGTCTCTGAGC
	Gapdh forward:	CATCACTGCCACCCAGAAGACTG
	Gapdh reverse:	ATGCCAGTGAGCTTCCCGTTCAG
	2.2.3.8 Protein extraction and Western Blot

Table 1 . Prevalence of rare variants (MAF<0.1%) among PNH patients and controls

 1 : n: number of individuals carrying rare variant. None of the tested patients carry rare variants in MCP and CFB genes. a https://gnomad.broadinstitute.org

Figure 1: Event-free survival of PNH patients bearing a CFH variant (red) versus without a CFH variant (black

  ). Events were first red blood cell transfusion after the eculizumab loading dose, thrombosis, HSCT, evolution to myeloid neoplasia, or death. The 6-year EFS for the no CFH group was 39.5 months [95%CI: 27.7-51.0].

		PNH	French controls	1000 Genomes controls	p (PNH vs French	p (PNH vs 1000
		(n=84)	(n=80)	(n=503)	a	controls)	genomes)
	Individuals with rare variants	16 (19)	5 (6.3)	35 (7.0)	0.02	0.001
	Individuals with at least one pathogenic variant	4 (4.8)	0 (0)	2 (0.4)	0,12	0,005
	CFH rare variant, n (%)	9 (10.7)	1 (1.3)	8 (1.6)	0.02	<0.001
	Pathogenic variant, n (% of CFH rare variant)	3 (3.6)	0 (0)		0 (0)	0.25	0.003
	CFI rare variant, n (%)	3 (3.6)	0 (0)	7 (1.4)	0,25	0.16
	Pathogenic variant, n (% of CFI rare variant)	1 (1.9)	0 (0)	2 (0.4)	1	0.37
	C3 rare variant, n (%)	4 (4.8)	4 (5)	13 (2.6)	1	0.29
	Pathogenic variant, n (% of C3 rare variant)	0 (0)	0 (0)		0 (0)	Not tested	Not tested
							10

Figure S2. Overall Survival in patients non-transplanted upfront

Determinants of Clonal Evolution in Aplastic AnemiaGurnari C. et al. 2022

Acknowledgements

First and foremost, I dedicate this work to my beloved wife, Clarinda. She is my role model of courage, resilience, tenderness, and perseverance. I am the happiest man alive to be her husband and the father of Lucas and Alice. You three are the force that makes me move forward and strive to be better.

ACKNOWLEDGMENTS

We wish to dedicate this work to Dr. Neal S. Young, a pioneer in the field of aplastic anemia, erudite, our mentor and a friend from whom we learned so much. (J.P.M., R.T.C. and R.PdeL.) This project was supported by the HENRY & MARILYN TAUB FOUNDATION, grants R01HL118281, R01HL123904, R01HL132071, R35HL135795 (all to JPM), AA&MDSIF (to VV, SP and JPM), VeloSano Pilot Award, Vera, and Joseph Dresner Foundation-MDS (to VV), Foundation For Rare Diseases (FFRD) and Association HPN France-Aplasie Médullaire, Italian Societies of Hematology and Experimental Hematology (to SP), The Italian Foundation for Cancer Research (AIRC) 5 × 1000 project entitled "Metastatic disease: the key unmet need in oncology" to MYNERVA, award number 21267 (MYeloid NEoplasms Research Venture to MTV), and Fondation Laurette-Fugain (to JS). PHP was supported by the American Society of Hematology Global Research Award and the Association HPN France-Aplasie Médullaire. CG was supported by the American-Italian Cancer Foundation Post-Doctoral Research Fellowship.

Acknowledgments: This study was partly supported by research funding from the Association HPN France -Aplasie Médullaire and Fondation Maladies Rares to PHP.

PRIOR PRESENTATION

Presented in part as an oral abstract at the 2021 American Society of Hematology Annual Meeting, Atlanta, GA, 2021.

DATA SHARING STATEMENT

The authors have included all clinical and genetic data in the main text of the article and Supplementary Appendix.

Requests for additional information should be sent to the corresponding author.

CONFLICT OF INTEREST

The authors declare no competing financial interests. ABSTRACT PURPOSE: Secondary myeloid neoplasms (sMN) remain the most serious long-term complications in patients with aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH). However, sMN lack specific predictors, dedicated surveillance measures and early therapeutic interventions.

PATIENTS AND METHODS:

We studied a multicenter, retrospective cohort of 1008 patients (median follow-up 8.6 years) with AA and PNH to assess clinical and molecular determinants of clonal evolution.

RESULTS:

Whereas none of the patients transplanted upfront (n=117) developed clonal complications (either sMN or secondary PNH), the 10-year cumulative incidence of sMN in non-transplanted cases was 11.6%. In severe AA, older age at presentation and lack of response to immunosuppressive therapy were independently associated with increased risk of sMN whereas untreated patients had the highest risk among non-severe cases.

Elapsed time from AA to sMN was 4.5 years. sMN developed in 94 patients. Five-year overall survival reached 40% and was independently associated with bone marrow blasts at sMN onset. Myelodysplastic syndrome with high-risk phenotypes, del7/7q and ASXL1, SETBP1, RUNX1 and RAS pathway gene mutations were the most frequent characteristics.

Cross-sectional studies of clonal dynamics from baseline to evolution revealed that PIGA/HLA lesions decreased over time, being replaced by clones with myeloid hits. PIGA and BCOR/L1 mutations carriers had a lower risk of sMN progression whereas myeloid driver lesions marked the group with a higher risk.

CONCLUSIONS:

The risk of sMN in AA is associated with disease severity, lack of response to treatment and patients' age. sMN display high-risk morphological, karyotypic and molecular features. The landscape of acquired somatic mutations is complex, and incompletely understood, and should be considered with caution in medical management.

Determinants of Clonal Evolution in Aplastic Anemia

Gurnari C. et al. 2022

CONTEXT SUMMARY Key Objective

Secondary myeloid neoplasms (sMN) are concerning late events in non-transplanted acquired aplastic anemia (AA) or paroxysmal nocturnal hemoglobinuria (PNH) patients. This retrospective, multinational study (n=1008) reports how clinical, cytogenetic, and molecular characteristics influence the risk of sMN, suggesting rational diagnostic approaches.

Knowledge Generated

sMN presented high-risk morphological, cytogenetic, and molecular features. Cross-sectional analyses revealed an inversion in clonal dynamics: PIGA/HLA-mutations, predominant at baseline, were overwhelmed by myeloid drivers at transformation. Lack of response to non-transplant treatments, older age, and myeloid driver mutations at AA/PNH onset conferred higher risk of progression.

Relevance

Patients with AA or PNH need close, prolonged monitoring with marrow smear and cytogenetics. Morphological progression to sMN or occurrence of monosomy 7 should prompt clinicians to consider transplantation in eligible patients.

The landscape of somatic mutations is complex and incompletely understood. Clinicians should carefully interpret the molecular profile with morphological and cytogenetic data for therapeutic decisions.

Determinants of Clonal Evolution in Aplastic Anemia Gurnari C. et al. 2022 10

In a cross-sectional study of clonal dynamics from baseline (n=302, Figure S12) to evolution, overall 18% of patients harbored myeloid mutations at primary disease onset with a higher frequency of mutant cases found among patients with AA as opposed to PNH (Figure 4A). After exclusion of PIGA, mutated in up to one-third of cases, the most recurrent alterations at baseline were found in BCOR/L1, ASXL1, and TET2 genes (Figure 4B).

In cases sequenced at baseline, follow-up and evolution, the frequency of PIGA mutations dramatically decreased at sMN progression, consistent with a reciprocal expansion of PNH clones and evolution to secondary hemolytic PNH in nonprogressors. The same trend was observed for somatic HLA aberrations that decreaed during follow-up and at progression, whereas an opposite effect was noticed for myeloid lesions. These quantitative changes were paralleled by dynamics of mutational burden (Figure 4C,S13).

We then classified our patients based on features (myeloid driver mutations only =Group The results of previous cross-sectional analysis have been confirmed longitudinally in 25 serial cases (AA onset and sMN evolution; Figure S15). As expected, when compared to baseline we found higher mutational (median number of mutations 0 [IQR 0-1] vs. 2 [1-2], p<0.001) and clonal burdens (median VAF 20% [IQR 5-27] vs. 30% [19-44]; p=0.01) at myeloid progression (Figure S16A). Interestingly, the dissection of the genomic architecture of such cases showed that 10% of mutations, which constituted the bulk of disease at evolution, were ancestral events already present at the time of BM failure onset or early in its course. Conversely, we noticed that CUX1 mutations disappeared at progression with concomitant acquisition of deletions affecting chromosome 7 (Figure S16B-C). Nevertheless, no difference in time to progression was observed in patients with and without mutations shared at both time points.

Determinants of Clonal

Supplementary Appendix

Clinical and Molecular Determinants of Clonal Evolution in

Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria diagnosis at evolution and other clinical complications were collected. PNH clone was defined according to the percentage of GPI-deficient granulocytes detected by flow cytometry as previously described (see below). 10,11 Specimens were obtained in accordance with ethical committee approvals of participating institutions. In total, our study cohort included 1008 patients with a diagnosis of AA, classical hemolytic PNH and AA/PNH overlap syndrome as per previously published criteria and definitions described above. 6

Flow Cytometry

Cells were acquired on a XL-MCL (Beckman Coulter) or FC500 cytometer. A 5-color cocktail was used to detect and quantify PNH granulocytes clone size as per established guidelines. 5,10,12 Conventional Cytogenetics G-banded metaphase cytogenetic analysis using standard techniques was performed on bone marrow (BM) aspirates with a median number of metaphases analyzed of 20. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature. 13

Genomic studies

Myeloid panel. When possible, patients were routinely tested for somatic mutations at diagnosis, when clinically indicated and/or to rule out the possibility of myeloid evolution using the diagnostic panel currently available at the participating institutions. When available, samples from our biorepository were also used to retrospectively study longitudinal clonal dynamics. Briefly, DNA was extracted from peripheral blood or BM specimens. Sequencing libraries were generated according to a paired-end library protocol (Illumina or Agilent Technologies) and sequenced on an Illumina instrument (San Diego, CA) with paired end, and 150x2 cycle reads. A panel of 40 genes (see Table S1) was interrogated. Reads were aligned to the reference human genome (Genomic Build GRCh37/hg19). Variants were annotated using Annovar

Annexes

Annexe I: Rare Germline Complement Factor H Variants in Patients with Paroxysmal Nocturnal Hemoglobinuria

Pedro Henrique Prata, Jacques-Emmanuel Galimard, Flore Sicre de Fontbrune, Paula Vieira Martins, Stephane Roncelin, Pierre-Édouard Debureaux, Anne-Claire Lepretre, Lise Larcher, Jean Soulier, Gérard Socié, , Véronique Fremeux-Bacchi, and Régis Peffault de Latour This manuscript is under revision as Brief report in Blood.

2

Key point

• Rare germline CFH variants are overrepresented among PNH patients

Abstract

The response to eculizumab among paroxysmal nocturnal hemoglobinuria patients is variable. In this cohort study, we used next-generation sequencing to screen 84 PNH patients treated with eculizumab for complement regulatory gene variants, compared the allele frequencies with populational data and a geographically-matched control group, and looked for an association between the presence of variants and response to treatment. Transfusion independence was evaluated at six months. Sixteen patients presented rare variants, 9 in the CFH gene. Germline CFH variants were more frequent among PNH patients than in healthy controls (p=0.02) or public data (p<0.001) and were more likely to be transfusion dependent at six months after eculizumab initiation (p=0.015). With a median follow-up of 5.8 years, all but one CFH-variant patient received transfusions, and two developed a thrombosis. No CFH-variant patient fulfilled the criteria for severe aplastic anemia from the initiation of eculizumab. We show for the first time that CFH variants are overrepresented among PNH patients and that they were associated with suboptimal response to eculizumab. Patients' molecular stratification to tailored treatments, e.g., proximal complement inhibition, could reduce costs and morbidity.

Study design

Data collection

We studied patients diagnosed with PNH by flow cytometry or Ham Dacie's test, who received treatment with eculizumab, were enrolled in the , and had available blood samples for molecular profiling with next-generation sequencing (NGS). Patients were excluded if follow-up was inferior to 6 months, insufficient clinical data for transfusion independence, inclusion in a clinical trial within six months from the start of eculizumab therapy, or upfront clinical trial. All participants signed informed consent. This research was carried on by the Helsinki Declaration. The research, registry, and biobank were approved by the Comité de protection des personnes Ouest VI.

Gene sequencing

Genomic DNA was extracted from blood, buccal swabs, and fibroblasts samples. All coding and flanking intronic sequences of CFH, CFI, MCP and C3 genes were analyzed by NGS as reported.11,12 We defined rare when a variant minor allele frequency was below 0.1% in the general population. We considered pathogenic those in which the genetic change affected the protein function (by in vitro functional studies supporting a damaging effect on the gene product), found in a disease-related functional domain, or affected protein expression (nonsense, frameshift, canonical ±1 or two splice sites variants, well-demonstrated lack of in vitro synthesis, or quantitative Authorship contributions: PHP helped elaborate the project, participated in patient recruitment, collected, analyzed, interpreted clinical and molecular data, and wrote the manuscript; JEG performed statistical analysis on clinical data; FSF participated in patient recruitment and collected samples; PED participated in patient recruitment and collected clinical data. PVM and SR performed molecular analyses; ACL collected clinical data; LL and JS prepared and biobanked samples and gave helpful intellectual insights. GS participated in patient recruitment, gave helpful intellectual insights, and edited the manuscript. VFB performed molecular analyses, interpreted molecular data, helped elaborate and sponsor the project, and edited the manuscript. RPL helped elaborate and sponsored the project, participated in patient recruitment, analyzed, interpreted clinical and molecular data, and edited the manuscript. All authors read and approved the final version of the manuscript and are accountable for all aspects of the work.

Conflicts of Interest Disclosures:

The authors declare no competing financial interests.

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Supplemental appendix

Supplemental Figure 1

Abstract

In the absence of an HLA-matched donor, the best treatment for acquired aplastic anemia patients refractory to immunosuppression is unclear. We collected and analyzed data from all acquired aplastic anemia patients who underwent a haploidentical transplantation with posttransplant cyclophosphamide in Europe from 2011 to 2017 (n = 33). The cumulative incidence of neutrophil engraftment was 67% (CI95%: 51-83%) at D +28 and was unaffected by age group, stem cell source, ATG use, or Baltimore conditioning regimen. The cumulative incidence of grades II-III acute GvHD was 23% at D +100, and limited chronic GvHD was 10% (0-20) at 2 years, without cases of grade IV acute or extensive chronic GvHD. Two-year overall survival was 78% (64-93), and 2-year graft-versus-host disease-free survival was 63% (46-81).

In univariate analysis, the 2-year OS was higher among patients who received the Baltimore conditioning regimen (93% (81-100) versus 64% (41-87), p = 0.03), whereas age group, stem cell source, and ATG use had no effect. Our results using unmanipulated haploidentical transplantation and posttransplant cyclophosphamide for treating refractory AA patients are encouraging, but warrant confirmation in a prospective study with a larger number of patients and longer follow-up.

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Annexe

Abstract

Steroid-refractory graft versus host disease (GVHD) represents a fearsome complication after allogeneic hematopoietic stem cell transplantation (HSCT). We conducted a retrospective study on outcomes and risk factors associated with acute and chronic steroid-refractory GVHD in a large cohort of 1207 patients receiving HSCT in Saint Louis Hospital between 2007 and 2017. Among patients who developed an acute and/or a chronic GVHD, the cumulative incidences of acute and chronic steroidrefractory disease were 31% and 48%, respectively, at day +100 and 1-year post-HSCT. Through a multivariable analysis we selected several risk factors associated with the development of a steroid-refractory disease. For acute GVHD steroid refractoriness, we identified (1) a very high disease risk index, (2) an unrelated donor, (3) the absence of in vivo T-depletion as GVHD prophylaxis, and (4) a reduced intensity conditioning regimen. For chronic GVHD, (1) the use of peripheral blood stem cells, (2) unrelated donors, and (3) absence of in vivo T-depletion were more likely associated with a steroid-refractory disease. After the construction of a multistate dynamic model, we found that the probability of being alive without relapse after the resolution of all GVHD episodes was about 36% in the long term.

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Categorizing hematological response to eculizumab in paroxysmal nocturnal hemoglobinuria: a multicenter real-life study