Uncovering the chromosomal abnormalities associated with human malignancy can provide significant insights into the molecular basis of tumorigenesis, as well as identifying potential targets for therapy. The present study set out to examine the genetic characteristics of t(2;7)(p11-12;q21-22) translocations arising in conjunction with chronic B-cell neoplasia. Using long-range PCR, a t(2;7) was initially mapped in an individual presenting with the preclinical entity CD5- monoclonal B-cell lymphocytosis. This revealed a breakpoint at 2p11.2 localized to the recombination signal of the immunoglobulin kappa (IGK) variable gene IGKV3-15, and a breakpoint at 7q21.2 located 520 bp upstream of cyclin dependent kinase 6 (CDK6). The same approach was subsequently employed to elucidate near-identical t(2;7) breakpoints in 4 additional cases presenting with chronic lymphocytic leukemia or indolent non-Hodgkin lymphomas. The remarkable consistency of these translocations implicates the dysregulation of CDK6 via translocation to IGK as a recurrent pathomechanism during the emergence of B-cell lymphoproliferative disorders.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) both belong to the group of mature B-cell malignancies. However, MCL is typically clinically aggressive while the clinical course of CLL varies. CLL can be divided into prognostic subgroups based on IGHV mutational status and into multiple subsets based on closely homologous (stereotyped) B-cell receptors. In paper I we investigated 31 MCL cases using high-density 250K single-nucleotide polymorphism arrays and gene expression arrays. Although most copy-number aberrations (CNAs) were previously reported in MCL, a novel deletion was identified at 20q (16%) containing the candidate tumor suppressor gene ZFP64. A high proliferation gene expression signature was associated with poor prognosis, large CNAs, 7p gains and 9q losses. Losses at 1p/8p/13q/17p were associated with increased genomic complexity. In paper II we sequenced exons 4 to 8 of the TP53 gene in 119 MCL cases. 17p copy-number status was known from previous studies or determined by real-time quantitative polymerase chain reaction. TP53 mutations were detected in 14% of cases and were strongly associated with poor survival while 17p deletions were more common (32%) but did not predict survival. In papers III and IV we applied high-resolution genomic 27K methylation arrays to 20 MCL and 39 CLL samples. In paper III MCL displayed a homogenous methylation profile without correlation with the proliferation signature whereas MCL was clearly separated from CLL. Gene ontology analysis revealed enrichment of developmental genes, in particular homeobox transcription factor genes, among targets methylated in MCL. In paper IV we compared three different stereotyped CLL subsets: #1 (IGHV unmutated), #2 (IGHV3-21) and #4 (IGHV mutated). Many genes were differentially methylated between each two subsets and immune response genes (e.g. CD80 and CD86) were enriched among genes methylated in subset #1 but not in subsets #2/#4. In summary, CNAs were frequent and not random in MCL. Specific CNAs correlated with a high proliferation gene expression signature or genomic complexity. TP53 mutations predicted short survival whereas 17p deletions did not. A high proliferation signature was not associated with differential DNA methylation in MCL, which demonstrated a homogeneous methylation pattern. In contrast, genomic methylation patterns differed between MCL and CLL and between stereotyped CLL subsets.

Les neuropathies à anticorps anti-MAG sont secondaires à une gammapathie monoclonale IgM dirigée contre la MAG des gaines de myéline des nerfs périphériques. Le traitement est celui de l’hémopathie sous‐jacente. Même si les thérapeutiques sont de plus en plus efficaces, les hémopathies restent le plus souvent incurables. Le rituximab est couramment utilisé dans le traitement des neuropathies à anticorps anti‐MAG, mais son efficacité n’a pas pu être clairement démontrée dans deux études contrôlées. Le FTY720 ou fingolimod est un sphingolipide, analogue de la sphingosine, qui inhibe les récepteurs de la sphingosine-1-phosphate (S1P). Il est utilisé comme immunosuppresseur dans la Sclérose en Plaques. Des études ont également rapporté un effet cytotoxique du FTY720 dans des hémopathies sans toutefois clairement expliquer son mécanisme d’action. L’objectif de ce travail est d’élucider les mécanismes moléculaires de l’effet cytotoxique du FTY720 dans un modèle d’hémopathie B, la leucémie lymphoïde chronique (LLC). Des cellules leucémiques primaires de LLC et une lignée cellulaire MEC1 ont été utilisées comme modèle expérimental in vitro. Le FTY720, comme la sphingosine, entraîne une cytotoxicité dose‐dépendante dans la LLC. Cet effet, médié par la forme non phosphorylée de FTY720, est indépendant des récepteurs au S1P. Le FTY720 induit l’expression de marqueurs d’apoptose: exposition de la phosphaJdylsérine, clivage de PARP et de caspase 3. Cependant sa toxicité apparaît indépendante des caspases. La lipidation accrue de LC3 et la formation d’autophagolysosomes indiquent que le FTY720 augmente également le flux autophagique. Cependant, des inhibiteurs de l’autophagie ne permettent pas de bloquer la mort cellulaire induite par le FTY720, suggérant que l’autophagie a ici un rôle protecteur vis à vis de la toxicité du FTY720. Plusieurs éléments permettent de conclure que le FTY720 est responsable d’une nécrose cellulaire : aspect morphologique de nécrose en microscopie électronique, perméabilisation membranaire précoce avec relocalisation cytoplasmique de HMGB1, libération extracellulaire de LDH, perméabilisation de la membrane lysosomale associée à une activation des cathepsines. Au niveau moléculaire, l’action du FTY720 n’est pas bloquée par la nécrostatine 1, indiquant que la nécrose induite par le FTY720 est indépendante de RIPK1 (receptor interacJng protein 1), une kinase clef des voies extrinsèques de nécrose cellulaire programmée. Par contre, nos travaux ont établi l’implication de DRP1 (dynamin related protein), une enzyme régulatrice de la fission mitochondriale, dans le processus de nécrose induite par le FTY720. En plus d’une relocalisation précoce de DRP1 à la mitochondrie accompagnée d’une augmentation de sa phosphorylation sur des sites régulateurs de son activité, nos expériences montrent que la suppression de son expression par interférence à ARN dans les cellules leucémiques réduit fortement la mort cellulaire induite par le FTY720. Le FTY720 est donc responsable dans la LLC d’une nécrose cellulaire programmée dépendante de DRP1. Nos résultats illustrent l’implication des sphingolipides dans la régulation de la survie cellulaire et dans les voies de nécrose programmée. Le FTY720 a un mode d’action original différent de l’apoptose induite par les chimiothérapies classiques. Le FTY720 pourrait donc être une alternative thérapeutique dans les néoplasies B résistantes aux chimiothérapies usuelles et dans certaines manifestations auto‐immunes des hémopathies comme les neuropathies à anticorps anti‐MAG. / Fingolimod (FTY720) is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of haematological malignancies. Previous studies have indicated a role for FTY720 in inducing autophagy and caspase-independent cell death in cancer cells through incompletely characterized molecular mechanisms. Our study thus aims at a beeer understanding of the way of action of FTY720. In chronic lymphocytic leukaemia (CLL) cells, FTY720 induced cell death with typical features of apoptosis, including phosphatidylserine exposure and caspase-3 activation, and features of autophagy, including LC3 conversion, autophagolysosome formation and lysosomal cathepsins activation. However, neither caspase nor autophagy blockade prevented the cytotoxic effect of FTY720, suggesting another mechanism of cell death. Using electron and fluorescence microscopy, flow cytometry and biochemical analyses, we found that FTY720 treatment increased a fraction of annexin V-/7-AAD+ cells both in primary and transformed leukemic cells and induced morphological changes representative of necrosis, including oncosis, mitochondrial and plasma membrane alteration. FTY720 treatment resulted in increased plasma membrane permeability as shown by the extracellular translocation of the nuclear high mobility group box 1 (HMGB1) protein and by the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH). Interestingly, cell death induced by FTY720 was not prevented by pharmacological inhibition of RIPK1 and PP2A. In contrast, FTY720--‐induced necrosis was accompanied by an early relocation to the mitochondria of Dynamin Related Protein 1, DRP1. Importantly, FTY720 stimulation led to ma tior changes in the phosphorylation of serine residues associated with the mitochondrial fission activity of DRP1. Finally, siRNA--‐mediated knockdown of DRP1 significantly reduced necrotic cell death induced by FTY720. In this study, we thus demonstrate that in leukemic cells the cytotoxic effect of the immunosuppressive drug Fingolimod involves a DRP1--‐dependent regulated necrosis. These observations are important in line of the future development of Fingolimod as a new therapeutic agent in haematological malignancies.

Cytogenetic analysis of cells of chronic lymphocytic leukemia (CLL) is difficult because of their low proliferative activity. To obtain sufficient number of mitoses for performing chromosomal analysis a suitable stimulation of cell division is needed. Using DSP30/IL2 stimulated cultivation 391 CLL samples were investigated in 5 years' period. The cultivation was showed to have high success rate (96%; 375/391) with also high rate of detection of pathological clones by both karyotype and metaphase FISH analyses (in 84% of samples; 329/391). Almost in half of samples (44%; 171/391) other aberrations than recurrent FISH (i.e. 13q14 deletion, trisomy 12, TP53, ATM genes deletions) were found. Also high frequency of translocations (37%; 144/391), complex karyotypes (28%; 111/391) and clonal evolution, which was detected in one third of all samples (34% of samples with presence of more than two clones; 133/391) and like a new event in disease duration even more frequently (in 39% of samples repeatedly investigated after stimulated cultivation; 21/54), was revealed. The presence of translocations, complex karyotypes and clonal evolution was associated with progressive form of disease (P 0,000003, resp. P 0,0002 and P 0,05/P 0,04). In cases of the recurrent deletions the detailed analysis of metaphase...

L’évaluation de l’effet cytotoxique de différents jus de fruits naturellement riches en polyphénols vis-à-vis de quatre lignées de cancer colorectal a montré que le jus de la canneberge est particulièrement actif. En effet, les polyphénols de la canneberge induisent l’apoptose associée à une surexpression des deux facteurs de transcription pro-apoptotiques p73 et FOXO3a. Cette mort programmée est aussi associée à une diminution de l’expression de SIRT1 le déacétylateur de protéines non histone telles que p73, KU70, ou FOXO. D’autres événements précoces comme la production de ROS et les dommages à l’ADN connus pour réguler l’expression de SIRT1 ont été confirmés. Une deuxième étude avait pour objectif de valider le potentiel anticancéreux in vivo chez la souris Balb/C injectée de cellules d’adénocarcinome colique murin. Pour cela nous avons choisi le jus d’aronie noire qui a montré in vitro un profil de cytotoxicité intéressant. L’analyse des tumeurs a montré que l’administration de jus d’aronie entraine une réduction de la prolifération des cellules tumorales. Enfin, l’augmentation significative de la mobilité de LC3 suggère l’activation d’une mort cellulaire autophagique. Afin d’évaluer l’utilisation clinique des polyphénols, nous avons évalué les effets cytotoxiques des polyphénols de myrtille sur des lymphocytes, de patients atteints de LLC. Nos résultats montrent que l’extrait polyphénolique induit une apoptose dépendante du stress oxydant et impliquant aussi des protéines pro-apoptotiques dans des cellules de patients atteints de LLC mais pas dans les cellules de sujets sains. / The evaluation of the cytotoxic effect of different fruit juices, naturally rich in polyphenols, on four different colorectal cancer cell lines proved that cranberry juice was the most active. Indeed, cranberry polyphenols induce apoptosis associated with the overexpression of two important proapoptotic transcription factors, p73 and FOXO3a on one hand. Furthermore, it has been also correlated with a decrease in the expression of SIRT1, the deacetylase of several non-histone proteins such as p73, KU70 and FOXO. Other early events such as ROS production and DNA damage, which are known to regulate the expression of SIRT1 were confirmed. The second study aims at validating the potential anticancer effects in an in vivo model of colorectal cancer in BALB/c mice injected subcutaneously of murine colon adenocarcinoma cells. Accordingly, we chose the black chokeberry juice, which showed an interesting cytotoxic profile in vitro. The analysis of tumors demonstrated that the administration of chokeberry juice leads to a reduction in tumor cell proliferation. Finally, the significant increase in the mobility of LC3 suggests the activation of autophagic cell death. To validate the clinical use of polyphenols, we evaluated the cytotoxic effects of blueberry polyphenols on lymphocytes of CLL patients. Our results show that the polyphenolic extract induces an oxidative stress-dependent apoptosis that involve various pro-apoptotic proteins in cells of patients with CLL but not in healthy subjects.

La leucémie lymphoïde chronique (LLC) est particulièrement associée aux cytopénies auto-immunes (AIC). L’expression de ZAP-70 dans la LLC est un facteur pronostique,due à une forte signalisation par le BCR. Nous décrivons une nouvelle population B normale (LyBn) qui exprime ZAP-70. Ces LyBn ZAP-70+ ne semble pas appartenir à une sous-population LyB particulière, et elles n’ont pas un phénotype activé. L’expression de ZAP-70 dans les LyB naïves suggère que ce phénomène est précoce. Il y a une bonne corrélation dans le niveau de ZAP-70 entre les LyBn et les LyB de LLC. Les LyBn ZAP-70+ ont été retrouvé dans tous les cas de AIC associée à la LLC.L’analyse des anticorps monoclonaux prévenant de LyB ZAP-70+ et des souris ZAP-70KI, réalisés pendant ce travail, définiront les conséquences de la surexpression de ZAP-70 dans les LyB dans la pathogénèse de l’auto-immunité et de la lymphoprolifération. / Chronic lymphocytic leukemia (CLL) is particularly associated with autoimmune cytopenia (AIC). The expression of ZAP-70 in CLL cells is a prognostic factor, through increased BCR signaling. We described a novel normal B cell population (LyBn) that expresses ZAP-70. ZAP-70+ LyBn do not seem to belong to a certain subset of B cells, nor seem to have an activated phenotype. The presence of ZAP-70 in the naïve B cell subset suggests that this is an early process, which probably occurs before malignant transformation. There is a good correlation in the level of ZAP-70 expression, between normal B cells and CLL B cells. We found a significant percentage of ZAP-70+ LyBn in all AIC-associated CLLs. Analysis of monoclonal antibodies and of conditional ZAP-70KI mouse model synthesized in this work will clarify the consequences of aberrant ZAP-70 expression in B cells on autoimmunity and lymphoproliferation.

La leucémie lymphoïde chronique (LLC) est la plus fréquente des leucémies de l'adulte, caractérisée par l'accumulation de lymphocytes B CD5+ anormaux dans le sang, la moelle osseuse, et les organes lymphoïdes secondaires. Les événements oncogéniques à l'origine du développement de la LLC sont peu connus. L'identification de nouveaux gènes dérégulés est importante afin d'améliorer notre compréhension du développement et de l'évolution de la LLC, et de proposer de nouvelles stratégies ciblées. Tout d'abord, nous avons montré que la LLC se développe à partir de progéniteurs hématopoïétiques multipotentes pré-leucémiques portant des mutations somatiques. J'ai également analysé la délétion 14q dans une large série de patients, et nous avons conclu que la del(14q) est associé à la trisomie 12 et à des facteurs pronostiques péjoratifs : IGHV non mutée, mutations NOTCH1, et une survie sans traitement plus courte. Enfin, la partie principale de ma thèse portrait sur la caractérisation du gain du bras court du chromosome 2 (2p). J'ai identifié une région minimale de gain chez les patients LLC/2p+ et démontré que CRM1/XPO1 (Exportin-1) se trouve dans cette région, et muté de façon récurrente dans la LLC. J'ai démontré que le gain 2p provoque la résistance aux traitements RFC, Ibrutinib, R-Idélalisib, et au Selinexor. Nos résultats révèlent également que le Selinexor est inefficace pour induire l'apoptose dans les cellules LLC-B muté pour XPO1. Au total, mon travail préconise l'évaluation du gain 2p et des mutations de XPO1 avant de décider d'un traitement de la LLC. / Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by an accumulation of abnormal CD5+ B-lymphocytes in the peripheral blood, bone marrow, and secondary lymphoid organs. The origin and pathogenic mechanisms of CLL are not fully understood. Identifying the deregulation of new genes is important to improve our understanding about CLL development and evolution, and to propose new targeted strategies. First, we have shown that CLL develops from pre-leukemic multipotent hematopoietic progenitors carrying somatic mutations. I have also analyzed the deletion 14q in a large series of patients, and we have concluded that del(14q) was associated with trisomy 12 and with pejorative prognostic factors: unmutated IGHV, NOTCH1 mutations, and a short treatment-free survival. Finally, the main part of my thesis was about the characterization of the short arm of chromosome 2 (2p). I identified a minimal region gained in 2p+/CLL patients and demonstrated that CRM1/XPO1 (Exportin-1) is located in this region, and recurrently mutated in CLL. I demonstrated that 2p gain provokes FCR, Ibrutinib, R-Idelalisib, and Selinexor drug resistance. Our results also reveal that Selinexor is inefficient in inducing apoptosis in CLL B-cells with mutations in XPO1. Altogether, my work advocates for the assessment of the 2p+ and XPO1 mutations before deciding a CLL therapy.

La leucémie lymphoïde chronique (LLC) est le lymphome avec phase circulante le plus fréquent chez l’adulte dans les pays occidentaux. Elle est caractérisée par une grand hétérogénéité dans son évolution naturelle avec des formes indolentes ne nécessitant jamais de traitement spécifique et des formes agressives requérant une chimiothérapie rapidement après le diagnostic. Dans ce travail de thèse, nous avons posé la question du rôle des anomalies moléculaires et en particulier des gènes des immunoglobulines dans l’histoire naturelle de la maladie, tant au plan mécanistique que pour le pronostic. Nous avons étudié trois remaniements atypiques impliquant un gène des immunoglobulines et un partenaire inconnu dans la LLC/lymphome lymphocytique. Les points de cassure ont pu être identifiés et nous ont permis de mettre en évidence l’implication dans 2 cas d’ARN longs non codants en 17q25 et 8q24. De plus, deux cas ont des points de cassure dans une région chromosomique restreinte (espacés de 200 kb en 17q25). Il pourrait s’agir d’un locus important dans la lymphomagénèse, de même que pour la région 8q24 contenant MYC et de nombreux gènes non codants jusqu’ici peu explorés. Par ailleurs, dans l’ère du séquençage haut débit, de nombreux marqueurs pronostiques moléculaires sont décrits dans la LLC. Nous avons démontré que l’électrophorèse des protéines sériques normale (immunoglobulines polyclonales sans hypogammaglobulinémie) au diagnostic, un marqueur simple et peu couteux, reste dans l’ère du NGS, un marqueur indépendant de bon pronostic dans la LLC. Sa combinaison avec un statut IGHV muté identifie un groupe de patients qui n’auront probablement jamais besoin de traitement spécifique. Ce travail nous a conduit à mettre au point un outil performant de détection des anomalies de nombre de copies par séquençage haut débit. Celui-ci permet la mise en évidence de disomies uniparentales dont la signification pronostique n’est actuellement pas établie dans la LLC. Ces anomalies pourraient être le reflet d’une instabilité chromosomique et il serait intéressant d’étudier leur impact pronostique dans la LLC. / Chronic lymphocytic leukemia (CLL) is the most frequent lymphoma with leukemic phase in the elderly in Western countries. It is characterized by a great heterogeneity in its natural history with indolent forms never requiring any specific treatment and aggressive forms needing chemotherapy rapidly after diagnosis. In this work of thesis, we asked the question of the role of molecular abnormalities, and in particular of the immunoglobulin genes in the natural history of the disease, at mechanistic level and for prognosis. We studied three atypical rearrangements implicating an immunoglobulin gene and an unknown partner in CLL/lymphocytic lymphoma. The breakpoints have been identified and the implication of long non coding RNA was highlighted in two cases in 17q25 and 8q24. Moreover, two cases harboured breakpoints in a restricted chromosomic region (200 kb spaced in 17q25). It could be an important locus in lymphomagenesis, as is 8q24 region containing MYC and numerous other non coding genes poorly characterized by now. Furthermore, in high throughput sequencing (HTS) era, many molecular prognosis markers have been described in CLL. We demonstrated that normal serum protein electrophoresis (polyclonal immunoglobulin without hypogammaglobulinemia) at diagnosis, a simple and unexpensive marker, stays in HTS era an independent good prognosis marker in CLL. Its combination with unmutated IGHV genes status identifies a group of patients who will probably never require any specific treatment. This work led us to develop an efficient tool to detect copy number variations by THS. This tool allows to highlight uniparental disomy whose prognosis signification is not established in CLL. These abnormalities could reflect chromosomal instability and it could be interesting to study their prognosis impact in CLL.

Cytogenetic analysis of cells of chronic lymphocytic leukemia (CLL) is difficult because of their low proliferative activity. To obtain sufficient number of mitoses for performing chromosomal analysis a suitable stimulation of cell division is needed. Using DSP30/IL2 stimulated cultivation 391 CLL samples were investigated in 5 years' period. The cultivation was showed to have high success rate (96%; 375/391) with also high rate of detection of pathological clones by both karyotype and metaphase FISH analyses (in 84% of samples; 329/391). Almost in half of samples (44%; 171/391) other aberrations than recurrent FISH (i.e. 13q14 deletion, trisomy 12, TP53, ATM genes deletions) were found. Also high frequency of translocations (37%; 144/391), complex karyotypes (28%; 111/391) and clonal evolution, which was detected in one third of all samples (34% of samples with presence of more than two clones; 133/391) and like a new event in disease duration even more frequently (in 39% of samples repeatedly investigated after stimulated cultivation; 21/54), was revealed. The presence of translocations, complex karyotypes and clonal evolution was associated with progressive form of disease (P 0,000003, resp. P 0,0002 and P 0,05/P 0,04). In cases of the recurrent deletions the detailed analysis of metaphase...

The overall aim of this thesis is to improve knowledge about the prevention of infectious and bleeding complications in patients with hematological malignancies, primarily in those with chronic lymphocytic leukemia (CLL) and myelodysplatic syndrome (MDS). Hypogammaglobulinemia, impaired production of immunoglobulins (Ig), is an established risk factor for infection, but the impact of IgG pure subclass deficiency (IgG subclass deficiency with adequate production of IgG, IgA, and IgM) has been debated. In a retrospective single institution study, we concluded that pure IgG subclass deficiency in CLL patients is rare and is not associated with an increased risk of infection. Hence, routine analysis of IgG subclasses in patients with CLL is not warranted. There is no consensus on recommending vaccination against Streptococcus pneumoniae to CLL patients mainly because comparative studies are lacking. In our randomized trial, the efficacy of a conjugated pneumococcal vaccine on immune response was superior or equal to a polysaccharide vaccine for all pneumococcal serotypes common for the two vaccines. A conjugate pneumococcal vaccine should therefore be included in vaccination programs for patients with CLL. Bronchoalveolar lavage (BAL) is a well-established invasive method to identify the cause of pulmonary infiltrates in immunocompromised patients. In a retrospective trial, we have studied the diagnostic yield of BAL in patients with hematological malignancies. We concluded that BAL is highly useful in either verifying or excluding some of the important respiratory tract infections affecting these patients, particularly invasive pulmonary aspergillosis (IPA) and Pneumocystis jirovecii pneumonia (PJP). However, standardized procedures for BAL sampling should be continually revised to avoid unnecessary microbiological tests. Thrombocytopenia, an adverse prognostic factor in patients with MDS, can be aggravated by azacitidine, first-line treatment for high-risk MDS. Eltrombopag, a thrombopoietin-receptor agonist (TPO-R), alleviates thrombocytopenia in patients with immune thrombocytopenic purpura (ITP). In a phase I clinical trial, we concluded that the combination of eltrombopag and azacitidine in high-risk MDS patients with thrombocytopenia is feasible and well tolerated in doses up to 200 mg eltrombopag daily.