Dissertations / Theses on the topic 'Lymphomas - Genetic aspects'

NK-cell malignancies (NKLL) consist of two separate entities: Extranodal NK-cell lymphoma, nasal type (ENKL) and aggressive NK-cell leukemia (ANKL). ENKL is the second most common group of extranodal lymphomas in Hong Kong. Deletions in the 6q21 region in ENKL have been consistently reported in the literature and differential expression data indicated that the transcription factor PRDM1 (PR domain containing 1, with ZNF domain) located at 6q21-q22.1 is a candidate TSG in NK-cell neoplasms. PRDM1 exists as 2 isoforms generated from the same gene by alternative transcription promoter. PRDM1- differs from PRDM1-βin that it lacks the amino-terminal 101 amino acids with a disrupted PR domain. As the PRDM1- is functionally impaired, with a loss of repressive function on multiple target genes while maintaining normal DNA-binding activity, we hypothesize that the  -isoform, which is overexpressed in NKLLs, may act as a negative regulator of the tumor suppressive α-isoform in NKLLs. In this study, we investigated the possible role of PRDM1- as a negative regulator of tumor suppressor PRDM1-α in NK-cell lymphoma by using a gene silencing technique. Short hairpin-RNA (sh-RNA) construct with sequence targeting to PRDM1- purchasing from biotechnology company was used to knockdown of the gene expression. Series of functional assays were then performed to evaluate the effect of the PRDM1-  knockdown in two NK cell line, YT and NKYS, which xpress endogenous PRDM1-. Comparison was made between the 1) shRNA targeting to nt65-nt94 of PRDM1- sequence, sh-PRDM1 -pGFP-V-RS (shV2), and 2) scrambled-pGFPV-RS (scrambled shRNA), negative control with a non-effective shRNA cassette in pGFP-VRS plasmid. Western blot analysis was performed to examine the efficiency of shRNA in knockdown the expression of PRDM1-  in 293T cells (normal human embryonic kidney cells). The protein expression level for ectopic PRMD1- was reduced in cells expressing shV2 when compared with the negative control. NKYS cell line expressed with shV2 showed a significant reduction in the number of colonies. Percentage of dead cells was found higher in these cells. The proliferation rate of shV2 expressing cells started to retarded significantly on the third day of measurement in the MTS proliferation assay. The cell also underwent G1 cell cycle arrest and had lower proliferation rate, as indicated by cell cycle analysis. For YT cell line expressed with shV2, significant reduction in both colony number and size in methylcellulose colony formation assay was observed. Base on the results obtained from the two NK-cell lines, we suggest that the shV2 inhibit the tumor cell growth. The knockdown of the PRDM1-  lead to an increase level of PRDM1- α. PRDM1- α is a tumor suppressor gene with suppressive function by preventing damaged cells from proliferation or inhibiting the clonogenecity of the tumor cells. An imbalance expression of PRDM1- and PRDM1-αplay an important role in tumor growth and formation, and PRDM1 could possibly be the new tumor suppressor gene in NK-cells lymphoma.
published_or_final_version
Pathology
Master
Master of Medical Sciences

It is known that many types of leukemias and lymphomas are of viral origin. A new strain of immunologically deficient mice, the BALB/c x C57B1/6 beige nude mice, has been observed to develop spontaneous lymphomas of unknown origin at a high frequency. It is possible the tumors originate from a retroviral infection, which we attempted to show by detection of viral reverse transcriptase (RT) activity. We measured the (RT) activity in the supernatants of cocultures from the spleen and lymph node tissues of the beige nude animals by two methods, tritiated thymidine triphosphate incorporation in a standard RT assay, and the commercially available RT-DetectTM (DuPont) method. Of all supernatants tested, none showed a significant amount of RT activity compared with a cell line that was known to be actively producing the retrovirus MuLV. Upon electron microscopic analysis of the tumor-like cells grown in coculture, no viral particles were observed. Flow cytometric analysis of the tumor-like cells showed two general phenotypes; one predominately of a helper T cell type, and the other of a less differentiated immature thymocyte type.
Department of Biology

L'infection par le virus d'Epstein-Barr (EBV) touche plus de 90% de la population mondiale et est dans la majorité des cas asymptomatique dans l'enfance. Certains individus, souvent à l'adolescence, développent une primo-infection symptomatique appelée mononucléose infectieuse. L'EBV peut également entraîner chez des individus immunodéprimés des désordres lymphoprolifératifs, des lymphomes ou syndromes d'activation lymphohistiocytaire. Depuis une trentaine d'années plusieurs déficits immunitaires primitifs entraînant une susceptibilité particulière à l'infection par l'EBV ont été identifiés ; parmi ceux-ci figurent les déficits en SAP, XIAP, ITK, MAGT1, CTPS1, CD27 ou CD70. Leur caractérisation a permis de mettre en évidence de nouveaux mécanismes immunitaires impliqués dans la réponse anti-EBV. L'objectif de ce travail a donc été d'identifier de nouveaux défauts génétiques entraînant une susceptibilité particulière à l'infection par l'EBV. Au sein de deux familles consanguines, trois patients ont développé des lymphomes B liés à l'EBV ainsi que des épisodes de lymphoproliférations également liées à ce virus. Deux mutations homozygotes dans RASGRP1 entraînant un stop prématuré, A638GfsStoXp16 et S314X ont été respectivement identifiées par séquençage d'exome (WES) chez ces deux familles. Sur le plan immunologique ces patients sont caractérisés par une lymphopénie CD4+, un défaut de cellules T naïves, une accumulation de cellules T effectrices mémoires et une absence de cellules MAIT et iNKT. RASGRP1 est une protéine de la famille des facteurs d'échange nucléotidiques fortement exprimée dans les lymphocytes T et NK. Elle active la petite protéine G Ras qui elle-même va activer la cascade des kinases Raf-MEK-ERK (ou cascade des MAP kinases). L'analyse des cellules du patient ou de cellules de contrôles sains dans lesquelles l'expression de RASGRP1 a été inhibée par RNA interférents a permis de mettre en évidence le rôle fondamental de RASGRP1 dans la prolifération lymphocytaire T et l'expression de gènes impliqués dans cette prolifération tels que CTPS1, PCNA ou RECQL4. A l'inverse, RASGRP1 semble être un régulateur négatif du facteur de transcription EOMES impliqué dans la différenciation des lymphocytes T. EOMES est retrouvé surexprimé dans les lymphocytes T en l'absence de RASGRP1, pouvant expliquer le phénotype effecteur mémoire et sénescent des lymphocytes des patients déficients en RASGRP1. Au sein d'une autre famille consanguine, chez deux patients ayant développé une primo-infection à l'EBV symptomatique, dont l'un a nécessité un traitement par anti-CD20 et corticoïdes, a été identifiée une mutation homozygote non-sens dans IL27RA entraînant un codon stop précoce (G96X) et une absence d'expression protéique dans les cellules T des patients. IL-27RA code pour la sous-unité alpha du récepteur à l'IL-27 impliqué dans la prolifération des lymphocytes T et le développement Th1 des lymphocytes CD4+ via la cascade des JAKs/STATs. Dans les lymphocytes T des patients, l'activation de la voie JAK/STAT par l'IL-27 est complètement abolie et l'IL-27 n'augmente pas leur prolifération en réponse à une stimulation anti-CD3 (au contraire des cellules contrôles issues de donneurs sains). De plus, un défaut fonctionnel de la voie Th1 est retrouvé chez un des deux patients. Ces résultats démontrent que la voie dépendante de l'IL-27RA est déficiente chez ces deux patients et que ce défaut génétique rend vraisemblablement compte de leur immunodéficience. La description de ces deux nouveaux déficits immunitaires caractérisés par une susceptibilité à l'EBV a permis de confirmer le rôle fondamental dans l'étape de prolifération et d'expansion des lymphocytes T au cours de la réponse immune anti-EBV, mais également de mettre en évidence de nouveaux mécanismes et facteurs impliqués dans cette étape
Epstein-Barr virus (EBV) is a gamma-herpes virus that infects 90% of humans without any symptoms in most cases. Some individuals, mostly adolescents, can develop infectious mononucleosis. In immunocompromised individuals, EBV can lead to lymphoproliferative disorders, lymphomas or virus-associated hemophagocytic syndrome. In the past 30 years, several primary immunodeficiencies associated with a high risk to develop EBV-associated disorders have been identified, including SAP, XIAP, ITK, MAGT1, CTPS1, CD27 or CD70 deficiencies. Their characterization has highlighted specific pathways required for efficient immunity to EBV. The objective of this work was to identify new genetic defects associated to a peculiar susceptibility to EBV infection. In two consanguineous families 3 patients developed EBV-associated B cell lymphomas and other EBV-associated lymphoproliferative disorders. By while exome sequencing (WES) we identified two homozygous mutations in RASGRP1 leading to a premature stop codon (A638GfsX16 and S314X). Immunologically these patients presented with CD4+ lymphopenia, low number of naïve T cells and absence of MAIT and iNKT cells. RASGRP1 codes for a diacylglycerol-regulated exchange factor preferentially expressed in T and NK cells, which acts as an activator of the small G protein RAS and the downstream RAF-MEK-ERK kinases cascade (or MAP kinases pathway). Analysis of patients' T cells or control T cells in which RASGRP1 expression was downregulated by short-hairpin RNA technique has highlighted the crucial role of RASGRP1 in T cell proliferation and in the expression of genes known to be involved in cell proliferation or replication such as CTPS1, PCNA or RECQL4. Furthermore, RASGRP1 seems to be a negative regulator of the transcription factor EOMES involved in T cell differentiation. EOMES was found overexpressed in T cells in the absence of RASGRP1. This might explain the skewed effector-memory and exhausted phenotype observed in RASGRP1-deficient patients. In another large consanguineous family two patients developed symptomatic EBV primary infection requiring for one or them anti-CD20 and corticosteroids treatment. Homozygous nonsense mutation leading to a premature stop codon in IL-27RA (G96X) was identified by exome sequencing. No protein expression could be detected in patients' cells. IL-27RA codes for the subunit of IL-27 receptor involved T cell proliferation and Th1 CD4+ development through JAKs/STATs pathway. Stimulation of patients' T cells with IL-27 led to absent JAK/STAT activation pathway and did not enhance their proliferation after anti-CD3 stimulation (contrary to healthy control T cells). Furthermore, Th1 functional defect was found in one patient. These results demonstrate that IL-27RA pathway is deficient is these two patients and that this genetic defect causes their immunodeficiency. Characterization of these two new primary immunodeficiencies associated with a high susceptibility to EBV infection has confirmed the crucial role of T cell proliferation and activation in EBV immune response but has also highlighted new pathways involved in T cell expansion

Previously a t(2;14)(p13;q32) translocation was characterized in four unusually aggressive cases of B cell chronic lymphocytic leukemia (B-CLL). A gene located near the 2p13 breakpoint, B cell lymphoma/leukemia 11A (BCL11A), was shown to overexpress 3 isoforms (BCL11A-XL, L and S). Bcl11a knockout mice are severely impaired in B cell development at the early (pro-B) stage. I have further characterized BCL11A, focusing on the most abundant and evolutionarily conserved isoform, BCL11A-XL (XL). I demonstrated that XL resides in the nuclear matrix, is modified by ubiquitination, and is destabilized by B cell antigen receptor ligation. I identified domains within XL required for its localization within nuclear paraspeckles and for its transcriptional repression. While BCL11A-XL represses model promoters in non-B cells, its biologically relevant targets in B lymphocytes were unknown. I have identified and confirmed a number of XL targets which are both up- and down-regulated by XL over-expression in B cell lines. A number of these genes have been implicated in B cell function, including the V(D)J recombination activating (RAG) genes. Both RAG1 and RAG2 transcripts were up-regulated by XL. XL binds to the RAG1 promoter and RAG enhancer (Erag) in vivo as well as in vitro. Unexpectedly, XL repressed RAG1 transcription in non-B cells, indicating that additional B cell-specific factors are required for activation. Overexpression of XL in a V(D)J recombination-competent pre-B cell line markedly induced RAG expression and VDJ recombination. IRF4 and IRF8, transcription factors previously shown to be required for early B cell development, were also induced by BCL11A-XL. I propose that the early B cell progenitor block in Bcl11a knockout mice is, at least in part, a direct result of BCL11A-XL regulation of V(D)J recombination. Further experiments are required to establish how other XL targets promote B cell lineage development and how malignant transformation such as in B-CLL may corrupt BCL11A function.

Chung, Po Yin.
Thesis submitted in: December 2006.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 128-155).
Abstracts in English and Chinese.
Thesis Abstract --- p.i
論文摘要 --- p.iv
Acknowledgements --- p.vi
Abbreviations --- p.vii
Thesis Content --- p.xi
List of Figures --- p.xv
List of Tables --- p.xvii
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1. --- Normal Hematopoiesis --- p.1
Chapter 1.2. --- Hematological Malignancy and the Aberrant Development of Blood Cells --- p.2
Chapter 1.3. --- Leukemia and Its Classification --- p.3
Chapter 1.4. --- Childhood Acute Lymphoblastic Leukemia (ALL) --- p.5
Chapter 1.4.1. --- Epidem iology --- p.5
Chapter 1.4.2. --- Causes and Risk Factors --- p.6
Chapter 1.4.3. --- Molecular Pathophysiology --- p.7
Chapter 1.4.4. --- Clinical Presentation --- p.9
Chapter 1.4.5. --- Classification --- p.10
Chapter 1.4.5.1. --- Immunophenotyping --- p.10
Chapter 1.4.5.2. --- French-American-British (FAB) Classification --- p.12
Chapter 1.4.6. --- Diagnosis and Prognosis --- p.14
Chapter 1.4.6.1. --- Morphological and Cytochemical Analysis --- p.15
Chapter 1.4.6.2. --- Cytogenetic and Molecular Genetic Characterizations --- p.16
Chapter 1.4.7. --- Treatment --- p.19
Chapter 1.5. --- Overview of Epigenetics --- p.21
Chapter 1.6. --- Concepts ofDNA Methylation --- p.23
Chapter 1.6.1. --- CpG Islands --- p.23
Chapter 1.6.2 --- Mechanisms of DNA Methylation --- p.24
Chapter 1.6.3 --- Physiological Roles of DNA Methylation --- p.28
Chapter 1.6.4 --- Initiation of Aberrant DNA Methylation --- p.30
Chapter 1.7. --- DNA Methylation in Tumorigenesis --- p.31
Chapter 1.7.1. --- Regional Hypermethylation --- p.33
Chapter 1.7.2 --- Global and Regional Hypomethylation --- p.34
Chapter 1.7.3 --- Microatellite Instability and Oncogeneic Mutation --- p.35
Chapter Chapter 2 --- Literature Review --- p.37
Chapter 2.1. --- Aberrant DNA Methylation in Childhood ALL --- p.37
Chapter 2.1.1. --- Cell Cycle --- p.39
Chapter 2.1.2. --- Apoptosis --- p.41
Chapter 2.1.3. --- Tissue Invasion and Metastasis --- p.42
Chapter 2.1.4. --- Transcription Factors and Metabolic Enzymes --- p.44
Chapter 2.1.5. --- Putative Tumor Suppressor Genes --- p.44
Chapter 2.1.6. --- Chromosome Instability --- p.46
Chapter 2.2. --- Methodologies in DNA Methylation Analysis --- p.50
Chapter 2.2.1. --- Principle of Methylation-sensitive Arbitrarily Primed PCR (MS-AP PCR) --- p.50
Chapter 2.2.2. --- Combined Bisulfite Restriction Analysis (COBRA) --- p.53
Chapter 2.2.3. --- Cloned Bisulfite Sequencing --- p.55
Chapter 2.2.4. --- Experimental Use of Demethylating Agents --- p.55
Chapter Chapter 3 --- Background of Research --- p.58
Chapter 3.1. --- Current Methylation Studies in Childhood ALL --- p.58
Chapter 3.2. --- Objectives of Research --- p.60
Chapter 3.3. --- Study Approach and Experimental Design --- p.61
Chapter Chapter 4 --- Materials and Methods --- p.63
Chapter 4.1. --- Clinical Samples and ALL Cell Lines --- p.63
Chapter 4.1.1. --- Clinical Samples from Pediatric Patients with ALL and Normal Healthy Donors --- p.63
Chapter 4.1.2. --- ALL Cell Lines --- p.63
Chapter 4.2. --- Genomic DNA Isolation from Clinical Samples and Cell Lines --- p.64
Chapter 4.2.1. --- Ficoll Gradient Centrifugation --- p.64
Chapter 4.2.2. --- DNA Extraction --- p.64
Chapter 4.3. --- MS-AP PCR --- p.65
Chapter 4.3.1. --- Methylation-sensitive Restriction Enzyme Digestion of Genomic DNA --- p.65
Chapter 4.3.2. --- Arbitrarily Primed Polymerase Chain Reaction --- p.66
Chapter 4.3.3. --- Isolation of Differentially Methylated DNA Fragments --- p.69
Chapter 4.4. --- Cloning of Differentially Methylated DNA Fragments --- p.70
Chapter 4.4.1. --- TA Cloning --- p.70
Chapter 4.4.2. --- Screening of Positive Clones --- p.71
Chapter 4.4.3. --- Preparation of Plasmid DNA by Alkaline Lysis Method --- p.72
Chapter 4.5. --- DNA Sequence Analysis of Differentially Methylated DNA Fragments --- p.72
Chapter 4.5.1. --- Dye-terminator Cycle Sequencing --- p.72
Chapter 4.5.2. --- CpG islands Analysis of Differentially Methylated Sequences --- p.73
Chapter 4.6. --- DNA Methylation Analysis --- p.74
Chapter 4.6.1. --- Sodium Bisulfite Modification --- p.74
Chapter 4.6.2. --- Combined Bisulfite Restriction Analysis --- p.75
Chapter 4.6.3. --- Cloned Bisulfite Genomic Sequencing --- p.76
Chapter 4.7 --- Gene Expression Study --- p.76
Chapter 4.7.1. --- RNA Extraction from Clinical Samples and ALL Cell Lines --- p.76
Chapter 4.1.2. --- Reverse Transcription PCR --- p.77
Chapter 4.7.3. --- Semi-quantitative RT-PCR --- p.78
Chapter 4.7.4. --- 5-aza-2 '-deoxycytidine Demethylation Treatment --- p.79
Chapter Chapter 5 --- Results --- p.80
Chapter 5.1. --- Generation of DNA Methylation Pattern by MS-AP PCR --- p.80
Chapter 5.1.1. --- Differential Methylation Patterns of Childhood ALL --- p.84
Chapter 5.1.2. --- Methylation Patterns of B and T lineages Childhood ALL --- p.86
Chapter 5.2. --- UCSC BLAT Analysis of Differential Methylated DNA Sequences
Chapter 5.3. --- Identification of Candidate Gene --- p.89
Chapter 5.4. --- Fibrillin 2 --- p.90
Chapter 5.4.1. --- FBN2 CpG Islands: UCSC BLAT Search Analysis --- p.90
Chapter 5.4.2. --- Verification ofFBN2 by ALL Cell Lines --- p.91
Chapter 5.4.2.1. --- Semi-quantitative RT-PCR --- p.91
Chapter 5.4.2.2. --- COBRA --- p.92
Chapter 5.4.2.3. --- Cloned Bisulfite Sequencing --- p.94
Chapter 5.4.2.4. --- Demethylation Treatment Resorted FBN2 mRNA Expression in ALL Cell Lines --- p.98
Chapter 5.4.3. --- Studies ofFBN2 in Childhood ALL --- p.99
Chapter 5.4.3.1. --- Methylation Analysis --- p.99
Chapter 5.4.3.2. --- Semi-quantitative RT-PCR --- p.105
Chapter Chapter 6 --- Discussion --- p.107
Chapter 6.1. --- Genome-wide Screening Approach: MS-AP PCR --- p.107
Chapter 6.2. --- Sample Selection in this Study --- p.109
Chapter 6.2.1. --- MS-AP PCR --- p.109
Chapter 6.2.2. --- Methylation Studies --- p.109
Chapter 6.2.3. --- Studies in ALL Cell Lines --- p.110
Chapter 6.3. --- Methylation Patterns in Childhood ALL --- p.111
Chapter 6.4. --- Candidate Genes Selection Strategies in MS-AP PCR --- p.112
Chapter 6.5. --- Fibrillin 2: mRNA Expression and Methylation Studies --- p.113
Chapter 6.5.1 --- ALL Cell Lines --- p.113
Chapter 6.5.2 --- Childhood ALL --- p.113
Chapter 6.5.2.1 --- mRNA Expression and Methylation Studies --- p.113
Chapter 6.5.2.2 --- Statistical Analysis --- p.115
Chapter 6.5.3. --- Possible Roles of FBN2 in Leukemogenesis --- p.116
Chapter 6.6. --- Clinical Application of FBN2 Aberrant Methylation --- p.119
Chapter 6.6.1. --- Tumor Markers --- p.119
Chapter 6.6.2. --- Use of Demethylating Drugs in Chemotherapy --- p.121
Chapter 6.7. --- Limitations of Methylation Studies --- p.122
Chapter 6.7.1. --- MS-AP PCR --- p.122
Chapter 6.7.2. --- Techniques Used in Methylation Study --- p.122
Chapter 6.7.3. --- Problems in Methylation Study --- p.123
Chapter 6.8. --- Future Studies --- p.125
Chapter Chapter 7 --- Conclusion --- p.127
References --- p.128
Appendix --- p.155