Human acute nonlymphoblastic or myelogenous leukemia (ANLL or AML) is a malignant disease of the bone marrow involving hemopoietic (blood-forming) cells of the myeloid lineage. ANLL is a complex neoplastic disease, whose fundamental nature is only partially understood despite intensive research. The disease is complicated by its apparent heterogeneity in terms of the degree of differentiation of hemopoietic stem cell involvement in different patients and the cellular expression of immunologically defined surface markers. The presence of a common antigen in myelogenous leukemia (CAMAL) has been previously identified. This thesis examines the expression of the CAMAL marker in or on bone marrow (BM) and peripheral blood (PB) cells using a monoclonal antibody-based indirect immunoperoxidase slide test. Increased numbers of CAMAL-positive cells were found in or on BM and PB of myeloid leukemia patients (with acute or chronic forms of the disease) compared with those found in normals or most lymphoid malignancies. Results presented herein have demonstrated that fluctuations in CAMAL BM values (% positive cells) correlated with survival time prior to relapse. In a blind study, ANLL patients Whose CAMAL BM values decreased post-chemotherapy had significantly (p < 0.025) longer first remission times (x = 19.2 months) than patients with increasing or static CAMAL BM values (x = 6.8 months). CAMAL BM values were often observed to increase during remission, prior to relapse, suggesting the presence of residual subclinical disease. Addition of excess purified leukemia-derived CAMAL to an in vitro myeloid progenitor cell assay caused profound inhibition of normal CFU-c growth but had no inhibitory effect on CFU-c growth from myeloid leukemia patients in active disease states. Depletion of CAHAL from normal plasma and conditioned media (sources of numerous hemopoietic growth regulatory factors) caused significant inhibition of normal, but not myeloid leukemic, CFU-c growth. These results indicated that myeloid leukemic cells possessed apparent differences in responsiveness to CAMAL-mediated hemopoietic regulation compared to normal cells. Lack of responsiveness to inhibition by leukemia-derived CAMAL may facilitate dominance of the malignant clone over normal cells. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 121-132).

This report describes a 75-year-old man with chronic myelomonocytic leukaemia (CMML) and marked marrow fibrosis associated with t(3;6)(q12;24). Although structural abnormalities of 3q occur in haematological neoplasia, this particular chromosomal translocation has not been previously described in CMML. Karyotypic abnormalities involving 3q and marrow fibrosis may affect prognosis in CMML.

Systemic mastocytosis refers to a heterogeneous group of clinical disorders characterized by excessive mast cell accumulation in one or multiple organs. Mastocytosis is now considered as a separate disease category in the 2016 WHO classification of myeloid neoplasm and acute leukemia. It is no longer considered as a subgroup of meyloproliferate neoplasms. The clinical presentation of mastocytosis is heterogeneous ranging from skin-limited disease (cutaneous mastocytosis) to a more aggressive form with extra cutaneous presentation (systemic mastocytosis) with or without skin involvement. We are presenting a case of systemic mastocytosis that aroused in a patient who carried diagnosis of CMML for almost 2 years. The worsening B symptoms along with worsening splenomegaly were the driving factor for further investigations including Bone Marrow biopsy which revealed the diagnosis. A 74 year old Caucasian male with past oncology history of Chronic myelomoncytic leukemia diagnosed after persistant monocytosis on complete blood count . Patient presented with gradual onset of low grade fever , weight loss and night sweating , CT abdomen showed hepatosplenomegaly. core biopsy of the liver showed portal and lobular infiltrate consistent with involvement by mastocytes and extra medullary hematopoiesis. The infiltrate was positive for CD117, CD33, CD68, myeloperoxidase and CD163. Patient had bone marrow biopsy which showed increased CD117 positive cells consistent with involvement by systemic mastocytosis. The core biopsy showed multifocal nodules of spindle cells with fibrosis which was morphologically consistent with abnormal mast cells. Immunohistochemistry for CD117 was strongly positive in the spindle cell nodules and scattered polygonal cell nodules. KIT D816V mutation was detected. Patient met criteria for diagnosis of systemic mastocytosis with presence of previous diagnosis of CMML and classified as Systemic mastocytosis with an associated hematologic neoplasm (SM-AHN). Due to patient multiple comorbiditeis , he was not a candidate for Allo HCT. In an attempt to control his disease , patient was started on dose reduced Dacogen, but his functional status continued to delined and eventually dacogen was discontinued and patient was placed on best supportive car Conclusion Systemic mastocytosis is a rare entity with heterogeneous clinical presentation, highly variable disease course and consequently survival rates.Though recent advances in understanding genetic and molecular basis of disease, bone marrow transplantation remains the only treatment with possible curative potential in patients with advanced form of mastocytosis though carrying substantial mortality risk .Further understanding of Kit mutation might be able to offer a highly effective medication with durable response in a fashion similar to the success story of gleevac with CML treatment .

Thesis (M.D. with Distinction in Research) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 25-28.

Une infiltration médullaire par des cellules plasmocytoïdes CD123+ est présente chez certains patients atteints de leucémie myélomonocytaire chronique (LMMC), mais les mécanismes aboutissant à la génération de ces cellules, et leur impact sur l'évolution de la maladie n'ont jamais été explorés. En cytométrie en flux, nous avons détecté un excès de cellules mononucléées négatives pour les marqueurs de lignée lymphocytaires, monocytaires et granulocytaires, et exprimant CD123, HLA-DR, BDCA-2, BDCA-4 et CD4 dans la moelle de 39/161 patients(24%) . L'analyse de ces cellules en microscopie conventionnelle et électronique, en cytométrie en flux et leur analyse transcriptomique identifient ces cellules comme d'authentiques cellules dendritiques plasmocytoïdes (pDCs). Ces pDCs répondent à la stimulation par des agonistes de Toll-like receptor 9 (TLR9) et de TLR7 en produisant respectivement de faibles quantités d'interféron alpha et de grandes quantités d'interleukine 8. Le séquençage d'exome complet de monocytes et de pDCs triés détecte une ou plusieurs mutations qui activent constitutivement la voie Ras chez tous les patients riches en pDCs, avec un certain niveau d'hétérogénéité sous-clonale. Les cellules CD34+ de patients LMMC riches en pDCs génèrent de grandes quantités de pDCs en culture ex vivo, y compris en l'absence de FMS-like tyrosine kinase 3-ligand (Flt3-L). Dans des expériences de coculture, les pDCs extraites de moelles de LMMC riches en pDC diminuent la prolifération des cellules CD34+ de manière dose-dépendante. L'augmentation des pDCs est associée à une expansion des lymphocytes T régulateurs (Tregs). L'analyse rétrospective d'une cohorte de 212 patients atteints de LMMC a montré un effet mitigé de l'infiltration médullaire par des cellules CD123+ TCL1+ sur la survie, avec une tendance à une meilleure survie globale chez les patients riches en pDCs, mais également un risque accru de transformation en leucémie aigüe. / Bone marrow infiltration with plasmacytoid CD123high cells was identified in a fraction of patients with a chronic myelomonocytic leukemia (CMML), but the mechanisms promoting the generation of these cells and their impact on disease evolution remain poorly known. Using a multiparametric flow cytometry assay, we detect an excess of lineage-negative mononucleated cells expressing CD45, CD123, HLA-DR, BDCA-2, BDCA-4 and CD4 in the bone marrow of 39/161 (24%) CMML patients. Conventional and electron microscopy, flow cytometry and gene expression analyses identify these cells as authentic plasmacytoid dendritic cells (pDCs). These pDCs respond to Toll-like receptor-9 (TLR9) and TLR7 agonists by producing low levels of interferon alpha and high levels of interleukin-8 (IL-8), respectively. Whole exome sequencing of sorted monocytes and pDCs detects one or several mutations that constitutively activate the Ras pathway in every pDC-rich patient, with some subclonal heterogeneity. CD34+ cells from pDC-rich CMML produce high level of pDCs in ex vivo culture, even in the absence of FMS-like tyrosine kinase 3 ligand (FLT-3L). In co-culture experiments, pDCs collected from the bone marrow of pDC-rich CMML decrease the proliferation of CD34+ cells in a dose-dependent manner. pDC increase is associated with an expansion of CD4+ regulatory T cells (Tregs). Retrospective analysis of a cohort of 216 CMML patients detected a mitigated effect of bone marrow infiltration with CD123high, TLC1+ cells on disease outcome, including a trend for a better overall survival of patients with a pDC excess but also an increased risk of leukemic transformation.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser. På omsl. felaktigt " ... daunorobicin ..."

La LMMC est une pathologie clonale de la cellule souche hématopoïétique dont les caractéristiques la classent parmi les syndromes myélodysplasiques/myéloprolifératifs (SMD/SMP). L’invalidation conditionnelle de Tif1γ au niveau hématopoïétique chez la Souris (Tif1γΔ/Δ) est responsable du développement d’un SMD/SMP mimant la LMMC humaine lorsque la souris atteint l’âge de 6 mois, faisant de Tif1γ un gène suppresseur de tumeur. Par ailleurs, malgré une monocytose, la population macrophagique péritonéale de ces souris est diminuée.Les objectifs de mon travail étaient de caractériser chez les souris malades la population myéloïde et d’étudier la différenciation macrophagique. Nous avons identifié chez les souris homozygotes Tif1γΔ/Δ une population morphologiquement immature, associant des caractéristiques granulocytaires et monocytaires. Les propriétés phénotypiques et moléculaires de cette population évoquent celles des cellules myéloïdes suppressives granulocytaires de type PMN-MDSC, faisant de Tif1γ un régulateur négatif de son développement. En sus, la différenciation in vitro des cellules myéloïdes médullaires en macrophages sous l’effet du CSF-1 est altérée. La baisse d’expression du CSF-1R n’explique pas à elle seule ces altérations puisque celle des cellules dendritiques est également perturbée sans modification de l’expression du GM-CSFR. Nous émettons l’hypothèse que l’augmentation d’expression de S100A8 et S100A9 chez les souris malades induit le développement des progéniteurs myéloïdes en cellules proches des PMN-MDSC au détriment des différenciations dendritique et macrophagique. En conclusion, Tif1γ est un régulateur majeur de la myélopoïèse. / Chronic myelomonocytic leukemia (CMML) is a hematologic stem cell disease whose characteristics correspond to myelodysplastic/myeloproliferative syndroms (MDS/MPS). Hematopoietic conditional deletion of Tif1γ in mice leads to the development of a MDS/MPS, mimiking human CMML, when age is comprised between 6 to 10 months, defining Tif1γ as a tumour suppressor gene. Moreover, peritoneal macrophage population in these mice is decreased despite a monocytosis.The aims of my work were first to characterize in sick mice the myeloid population, and second to study macrophage differentiation. The myeloid population in Tif1γΔ/Δ mice is morphologically immature, with granulocytic and monocytic features. We demonstrated that phenotypic and molecular characteristics of this population are close to those observed in PMN-MDSC (polymorphonuclear myeloid-derived suppressor cells), suggesting that Tif1γ is a negative regulator gene of this myeloid subset. Furthermore, we showed that in vitro macrophage differentiation of myeloid progenitors upon CSF-1 treatment is altered. Decreased expression of CSF1-R (CSF-1 receptor) does not totally explain this alteration since dendritic cell differentiation is also abnormal, without alteration in GM-CSFR expression. Therefore, we hypothesize that S100A8 and S100A9 hyperexpression in Tif1γΔ/Δ mice is able to promote PMN-MDSC-like differentiation at the expense of macrophage and dendritic differentiations. In conclusion, Tif1γ is a major myelopoiesis regulator gene.

La leucémie myélomonocytaire chronique (LMMC) est une pathologie clonale de la cellule souche hématopoïétique qui touche principalement les personnes âgées. Le seul traitement curatif de cette maladie est la greffe allogénique de cellules souches hématopoïétiques, souvent difficile à mettre en oeuvre. Les patients qui ne peuvent être greffés et dont la maladie présente des critères de gravité se voient proposer un agent déméthylant de l'ADN. Chez 30 à 40% d'entre eux, ce traitement induit une réponse objective dont le bénéfice en termes de survie n'est pas démontré. Le séquençage de gènes candidats a identifié une trentaine de gènes mutés de façon récurrente. Il s'agit de gènes codant des régulateurs épigénétiques, des facteurs d'épissage, des facteurs de transcription, et des protéines de la signalisation intracellulaire. Cette approche ne donnait qu'une vision partielle des événements génétiques associés à la maladie.Le premier objectif de cette thèse a été de recenser l'ensemble des mutations touchant les régions codantes et non codantes de l'ADN dans les cellules leucémiques des patients.Le séquençage de l'exome de cellules malades et de cellules contrôles a été réalisé chez 49 patients. Nos analyses ont montré qu'en moyenne, un patient porte 14 mutations somatiques dans les régions codantes. Nous avons confirmé que les mutations récurrentes les plus fréquentes affectaient les gènes TET2, SRSF2 et ASXL1. Nous avons aussi identifié 8 nouveaux gènes mutés de façon récurrente à une faible fréquence. En moyenne, 3 des 14 mutations affectent des gènes touchés de façon récurrente.Le séquençage du génome de cellules malades et de cellules contrôles a été réalisé chez 17 patients. L'analyse réalisée a détecté 475 mutations par patient dans les régions non répétées du génome. Dans l'exome, comme dans le reste du génome, les altérations principales sont des transitions. Deux signatures mutationnelles ont été identifiées et sont observées dans de nombreux cancers, traduisant probablement des altérations de la méthylation des cytosines au cours du vieillissement. Une troisième signature, jamais observée jusqu'alors et de signification indéterminée, a été détectée chez 2 patients.Nous avons alors répété l'analyse de l'exome dans les monocytes triés de 17 patients prélevés de façon séquentielle sur plus de 2 années : 6 n'ont pas été traités et 11 ont été traités par un agent déméthylant, parmi lesquels 6 sont restés stables et 5 ont montré une réponse clinique et biologique objective. L'analyse montre que 1) l'accumulation de mutations est un événement rare ; 2) l'hétérogénéité génétique du clone malade est limitée ; 3) la charge allélique des mutations reste inchangée, même chez les répondeurs ; 4) de nouvelles mutations peuvent apparaître alors que le patient est répondeur.Nous avons alors sélectionné 9 patients, 3 non traités, 3 stables sous traitement sans réponse objective, et 3 répondeurs. Nous avons collecté leurs monocytes avant tout traitement et quelques mois plus tard, alors que 6 d'entre eux étaient traités par un agent déméthylant. Nous avons analysé l'expression des gènes et la méthylation globale de l'ADN à ces deux temps. Chez les patients non traités, nous avons observé une remarquable stabilité de l'expression des gènes et de la méthylation de l'ADN. Chez les patients répondeurs, le traitement induit un changement significatif du niveau d'expression d'environ 500 gènes et la déméthylation d'environ 35,000 régions de l'ADN. Chez les patients stables sous traitement, le traitement induit un changement d'expression d'une soixantaine de gènes et du niveau de méthylation d'une centaine de régions seulement. Ces résultats suggèrent que les agents déméthylants n'affectent l'expression des gènes et la méthylation de l'ADN que chez les répondeurs, fournissant un argument important pour un effet essentiellement épigénétique et très peu cytotoxique de ces médicaments. / Chronic myelomonocytic leukemia is a clonal disorder of the hematopoietic stem cell, affecting mainly the elderly. The only curative therapeutic is allogeneic stem cell transplantation, which is rarely feasible. When transplantation is not an option, patients with a severe disease can be treated with a demethylating agent. Thirty to 40% of these patients show hematological improvement, but it remains unknown if these drugs increase overall survival. Analysis of candidate genes by Sanger sequencing, then by New Generation Sequencing, identified about thirty genes that are frequently mutated. These genes encode epigenetic regulators, splicing factors, transcription factors and cell signalling regulators. However, this approach catched only part of the genetic events that characterize this disease.The first objective of this study was to determine the mutational landscape of CMML cells by analyzing the coding and non coding regions of leukemic cell genome.We first performed whole exome sequencing analysis of leukemic and control cells in 49 patients. These analyses showed that in average, a patient carries 14 somatic mutations in its coding regions. We confirmed that the most frequent mutations were in TET2, SRSF2 and ASXL1 genes. We identified also recurrent mutations in 8 new genes, these recurrent mutations occurring at a low frequency. In average, 3 out of the 14 mutations identified in each patient affected recurrently mutated genes.Secondly, we performed whole genome sequencing of leukemic and control cells in 17 patients. These analyses showed that in average, a patient carries 475 somatic mutations in the non repeated regions of the genome. In both the coding and non coding sequences, alterations were observed to be mainly transitions. As a signature of CMML, two mutational processes were identified in all 17 patients and are found in various other cancer types, most likely resulting from the cytosine methylation observed with ageing. A third process, never seen before and without known significance, was also detected in two patients.We collected several samples from 17 patients on a more than two year period: 6 of these patients remained untreated whereas 11 were treated with demethylating agent, among which 6 showed a stable disease and 5 fulfilled criteria of hematological improvement. These sequential analyses showed that 1) the occurence of new mutations is a relatively rare event ; 2) the genetic heterogeneity of the malignant clone is limited ; 3) the mutation allele burden remains unchanged under treatment, whatever the response ; 4) new mutations can appear, even in responding patients.We selected 9 patients, 3 untreated, 3 stable on therapy and 3 responders. We collected monocytes before treatment and a few months later and we analyzed gene expression and DNA methylation in sorted monocytes at these two time points. We did not detect any significant change in gene expression and DNA methylation pattern in untreated patients. In those who responded to treatment, we noticed significant changes in both gene expression, with about 500 deregulated genes, and the DNA methylation pattern, with about 35,000 demethylated regions. In stable patients, the treatment had a limited effect with changes in the expression of about 60 genes, and in the DNA methylation pattern of about 100 regions. These results show that demethylating agents affect gene expression and DNA methylation of responding patients only, suggesting they have mostly an epigenetic effect rather than a cytotoxic one.

No / Recent studies have revealed extensive cortical demyelination in patients with progressive multiple sclerosis (MS). Demyelination in gray matter lesions is associated with activation of microglia. Macrophages and microglia are known to express myeloperoxidase (MPO) and generate reactive oxygen species during myelin phagocytosis in the white matter. In the present study we examined the extent of microglial activation in the cerebral cortex and the relationship of microglial activation and MPO activity to cortical demyelination. Twenty-one cases of neuropathologically confirmed multiple sclerosis, with 34 cortical lesions, were used to assess microglial activation. HLA-DR immunolabeling of activated microglia was significantly higher in demyelinated MS cortex than control cortex and, within the MS cohort, was significantly greater within cortical lesions than in matched non-demyelinated areas of cortex. In homogenates of MS cortex, cortical demyelination was associated with significantly elevated MPO activity. Immunohistochemistry revealed MPO in CD68-positive microglia within cortical plaques, particularly toward the edge of the plaques, but not in microglia in adjacent non-demyelinated cortex. Cortical demyelination in MS is associated with increased activity of MPO, which is expressed by a CD68-positive subset of activated microglia, suggesting that microglial production of reactive oxygen species is likely to be involved in cortical demyelination.