Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Oncology, Department of Oncology. Title from pdf file main screen (viewed on July 30, 2009). Includes bibliographical references.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

by Kwok Fung, Lo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 89-97). / Acknowledgement --- p.i / Table of Content --- p.ii / Abstract --- p.vi / List of Abbreviation --- p.viii / List of Tables and figures --- p.ix / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The immunoglobulin --- p.1 / Chapter 1.1.1 --- Immunoglobulin structure --- p.1 / Chapter 1.1.2 --- Immunogloblulin genes --- p.1 / Chapter 1.1.3 --- Immunogloblulin gene recombination --- p.3 / Chapter 1.1.4 --- Antibody diversity --- p.4 / Chapter 1.1.4.1 --- Imprecise joining --- p.5 / Chapter 1.1.4.2 --- N region addition --- p.5 / Chapter 1.1.4.3 --- Somatic mutation --- p.6 / Chapter 1.2 --- Hypermutation --- p.6 / Chapter 1.2.1 --- Features of hypermutation --- p.6 / Chapter 1.2.2 --- Germinal centre & Affinity maturation --- p.8 / Chapter 1.2.3 --- Mutational Hotspots --- p.9 / Chapter 1.2.4 --- Intrinsic characteristics of hypermutation --- p.10 / Chapter 1.2.5 --- Models for the mechanism of hypermutation --- p.11 / Chapter 1.2.5.1 --- DNA replication --- p.11 / Chapter 1.2.5.2 --- DNA repair --- p.12 / Chapter 1.2.5.3 --- Gene conversion --- p.13 / Chapter 1.2.5.4 --- Transcription --- p.15 / Chapter 1.2.5.5 --- Homologous recombination of reverse transcribed mRNA --- p.16 / Chapter 1.2.5.6 --- Transcription-coupled repair --- p.17 / Chapter 1.3 --- Scope of investigation --- p.17 / Chapter Chapter 2 --- Material and Method --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.2 --- Methods (first generation of LMPCR) --- p.21 / Chapter 2.2.1 --- Animal and cell lines --- p.21 / Chapter 2.2.2 --- Oxazolone antigen immunization --- p.21 / Chapter 2.2.2.1 --- Preparation of Bordetella pertussis --- p.21 / Chapter 2.2.2.2 --- Coupling of phenyloxazolone with CSA or BSA --- p.22 / Chapter 2.2.2.3 --- Preparation of aluminium hydroxide adjuvant --- p.23 / Chapter 2.2.2.4 --- Mice immunization --- p.23 / Chapter 2.2.3 --- Detection of anti-phOx antibody by enzyme-linked immunosorbent assay (ELISA) --- p.24 / Chapter 2.2.3.1 --- Reagents --- p.24 / Chapter 2.2.3.2 --- Assay procedure --- p.24 / Chapter 2.2.4 --- Extraction of genomic DNA (mice/cell line) --- p.25 / Chapter 2.2.4.1 --- Reagents --- p.25 / Chapter 2.2.4.2 --- Isolation of DNA from cell line (NQ2.12.4 & NQ5.4.3) --- p.25 / Chapter 2.2.4.3. --- DNA extraction from mice --- p.26 / Chapter 2.2.5 --- Ligation-mediated polymerase chain reaction (LMPCR) --- p.26 / Chapter 2.2.5.1 --- Procedure --- p.26 / Chapter 2.2.5.1.1 --- First primer extension --- p.29 / Chapter 2.2.5.1.2 --- Ligation --- p.29 / Chapter 2.2.5.1.3 --- PCR amplification --- p.30 / Chapter 2.2.5.1.4 --- Labelling of LMPCR product --- p.30 / Chapter 2.2.6 --- Marker preparation --- p.31 / Chapter 2.2.7 --- Polyacrylamide gel electrophoresis --- p.32 / Chapter 2.2.7.1 --- Reagents --- p.32 / Chapter 2.2.7.2 --- Procedure --- p.32 / Chapter 2.2.8 --- Southern blot hybridization --- p.33 / Chapter 2.2.8.1 --- Reagents --- p.33 / Chapter 2.2.8.2 --- DNA blotting --- p.34 / Chapter 2.2.8.3 --- Preparation of 32P labelling DNA probe --- p.34 / Chapter 2.2.8.4 --- Prehybridization and Hybridization --- p.35 / Chapter 2.2.9 --- Simplified protocol for the first generation of LMPCR --- p.36 / Chapter 2.3 --- Method (second generation of LMPCR) --- p.37 / Chapter 2.3.1 --- Excess linker removal --- p.37 / Chapter 2.3.1.1 --- Exonuclease III Treatment --- p.37 / Chapter 2.3.1.2 --- Mung bean nuclease Treatment --- p.37 / Chapter 2.3.1.3 --- Chroma Spin Treatment --- p.37 / Chapter 2.3.2 --- HindIII digestion after LMPCR --- p.38 / Chapter 2.3.3 --- DNA sequencing --- p.38 / Chapter 2.3.3.1 --- Cloning of amplified sequences to M13mpl9 plasmid --- p.38 / Chapter 2.3.3.2 --- Plaque hybridization --- p.39 / Chapter 2.3.3.3 --- Preparation of single-stranded templates --- p.39 / Chapter 2.3.3.4 --- Sanger dideoxy sequencing of single-stranded DNA --- p.40 / Chapter 2.3.4 --- Simplified protocol for second generation of LMPCR --- p.41 / Chapter Chapter 3 --- First generation of LMPCR --- p.42 / Chapter 3.1 --- General design --- p.42 / Chapter 3.1.1 --- LMPCR protocol and its modification --- p.42 / Chapter 3.1.2 --- Oligonucleotide design --- p.44 / Chapter 3.1.3 --- Experimental design --- p.48 / Chapter 3.2 --- Result --- p.50 / Chapter 3.2.1 --- Anti-phOx Ig level in normal and immunized mice --- p.50 / Chapter 3.2.2 --- LMPCR analysis of the sense strand of VkOxl --- p.50 / Chapter 3.2.2.1 --- Overall patterns of the LMPCR signals --- p.50 / Chapter 3.2.2.2 --- Southern hybridization --- p.50 / Chapter 3.2.2.3 --- Distribution of signals --- p.57 / Chapter 3.2.2.4 --- LMPCR analysis of the VkOxl-Jk5 anti-phOx transgene --- p.61 / Chapter 3.2.2.5 --- Effect of the number of cells carrying the VkOxl-Jk5 gene on LMPCR --- p.61 / Chapter 3.2.3 --- LMPCR analysis of the antisense strand of VkOxl --- p.64 / Chapter 3.3 --- Discussion --- p.64 / Chapter Chapter 4 --- Second generation of LMPCR --- p.72 / Chapter 4.1 --- Introduction(experi mental modification) --- p.72 / Chapter 4.1.1 --- Tagging the specific LMPCR products by addition of a Hin dIII site in the linker --- p.72 / Chapter 4.1.2 --- "Removal of excess linker, OXUH" --- p.72 / Chapter 4.1.2.1 --- Exonuclease III treatment --- p.73 / Chapter 4.1.2.2 --- Chroma spin treatment --- p.73 / Chapter 4.1.2.3 --- Mung Bean Nuclease treatment --- p.75 / Chapter 4.1.3 --- Other modifications in LMPCR --- p.75 / Chapter 4.2 --- Results --- p.75 / Chapter 4.2.1 --- Effect of including Exonuclease III treatment --- p.75 / Chapter 4.2.2 --- Effect of including Mung Bean Nuclease treatment --- p.76 / Chapter 4.2.3 --- Effect of including Chroma spin treatment --- p.76 / Chapter 4.2.4 --- Strand break positions detected at the sense strand --- p.76 / Chapter 4.2.5 --- DNA sequence analysis of the antisense strand LMPCR products --- p.82 / Chapter 4.3 --- Discussion --- p.84 / References --- p.89 / Appendix I --- p.98

<p>Recent studies have shown that the somatic mutation status of the immunoglobulin (Ig) V_H genes can divide chronic lymphocytic leukemia (CLL) into two prognostic subsets, since cases with mutated V_H genes display superior survival compared to unmutated cases. Biased V_H gene usage has also been reported in CLL which may reflect antigen selection.</p><p>We performed V_H gene analysis in 265 CLL cases and confirmed the prognostic impact of the V_H mutation status. Preferential V_H gene usage was also demonstrated in both the mutated and unmutated subset. Interestingly, CLL cases rearranging one particular V_H gene, V_H3-21, displayed poor outcome despite that two-thirds showed mutated V_H genes. Many of the V_H3-21 cases expressed λ light chains, rearranged a V_λ2-14 gene, and had homologous complementarity determining region 3s (CDR3s), implying recognition of a common antigen epitope. We believe that the V_H3-21 subset comprises an additional CLL entity.</p><p>To further explore the B-cell receptors in CLL, we analyzed the V_H gene rearrangements and, specifically, the heavy chain CDR3 sequences in 346 CLL cases. We identified six new subgroups with similar HCDR3 features and restricted V_L gene usage as in the V_H3-21-using group. Our data indicate a limited number of antigen recognition sites in these subgroups and give further evidence for antigen selection in the development of CLL.</p><p>Different cutoffs have been suggested to distinguish mutated CLL in addition to the 2% cutoff. Using three levels of somatic mutations, i.e. <2%, 2-5% and >5%, we divided 323 CLLs into subsets with divergent survival. This division revealed a low-mutated subgroup (2-5%) with inferior outcome that would have been masked using the traditional 2% cutoff. </p><p>A 1513A/C polymorphism in the P2X₇ receptor gene was reported to be more frequent in CLL, but no difference in genotype frequencies was revealed in our 170 CLL cases and 200 controls. However, CLL cases with the 1513AC genotype showed superior survival than 1513AA cases and this was in particular confined to CLL with mutated V_H genes.</p><p> In summary, we could define new prognostic subgroups in CLL using Ig gene rearrangement analysis. This also allowed us to gain insights in the biology and potential role of antigen involvement in the pathogenesis of CLL.</p>

Recent studies have shown that the somatic mutation status of the immunoglobulin (Ig) VH genes can divide chronic lymphocytic leukemia (CLL) into two prognostic subsets, since cases with mutated VH genes display superior survival compared to unmutated cases. Biased VH gene usage has also been reported in CLL which may reflect antigen selection. We performed VH gene analysis in 265 CLL cases and confirmed the prognostic impact of the VH mutation status. Preferential VH gene usage was also demonstrated in both the mutated and unmutated subset. Interestingly, CLL cases rearranging one particular VH gene, VH3-21, displayed poor outcome despite that two-thirds showed mutated VH genes. Many of the VH3-21 cases expressed λ light chains, rearranged a Vλ2-14 gene, and had homologous complementarity determining region 3s (CDR3s), implying recognition of a common antigen epitope. We believe that the VH3-21 subset comprises an additional CLL entity. To further explore the B-cell receptors in CLL, we analyzed the VH gene rearrangements and, specifically, the heavy chain CDR3 sequences in 346 CLL cases. We identified six new subgroups with similar HCDR3 features and restricted VL gene usage as in the VH3-21-using group. Our data indicate a limited number of antigen recognition sites in these subgroups and give further evidence for antigen selection in the development of CLL. Different cutoffs have been suggested to distinguish mutated CLL in addition to the 2% cutoff. Using three levels of somatic mutations, i.e. &lt;2%, 2-5% and &gt;5%, we divided 323 CLLs into subsets with divergent survival. This division revealed a low-mutated subgroup (2-5%) with inferior outcome that would have been masked using the traditional 2% cutoff. A 1513A/C polymorphism in the P2X7 receptor gene was reported to be more frequent in CLL, but no difference in genotype frequencies was revealed in our 170 CLL cases and 200 controls. However, CLL cases with the 1513AC genotype showed superior survival than 1513AA cases and this was in particular confined to CLL with mutated VH genes. In summary, we could define new prognostic subgroups in CLL using Ig gene rearrangement analysis. This also allowed us to gain insights in the biology and potential role of antigen involvement in the pathogenesis of CLL.

Biased immunoglobulin heavy variable (IGHV) gene usage and distinctive B-cell receptor (BCR) features have been reported in chronic lymphocytic leukaemia (CLL), which may reflect clonal selection by antigens during disease development. Furthermore, the IGHV gene mutation status distinguishes two clinical entities of CLL, where patients with unmutated IGHV genes have an inferior prognosis compared to those with mutated IGHV genes. Recently, one subgroup of CLL patients expressing the IGHV3-21 gene was found to display highly similar immunoglobulin (IG) gene features, even within the heavy chain complementarity-determining region 3 (HCDR3). Patients in this subgroup typically had a poor prognosis. In paper I, we aimed to identify further subgroups with restricted BCR features among 346 CLL cases. Six subsets were defined which carried virtually identical BCRs in terms of rearranged heavy and light chain (LC) IG genes and CDR3 length and composition. In paper II, we investigated 90 IGHV3-21 cases from diverse geographical locations. We confirmed the highly restricted HCDR3 characteristics in 56% of patients and a biased usage of the IGLV3-21 gene in 72% of cases. Survival analysis also confirmed the poor outcome of this group, irrespective of IGHV gene mutation status and geographical origin. Papers III and IV involved a large-scale analysis of IGH and IG kappa and lambda (IGK/L) gene rearrangements, to define subsets with ‘stereotyped’ BCRs and also to systematically examine the somatic hypermutation (SHM) features of the IG genes in CLL. We studied a cohort of 1967 IGH and 891 IGK/L gene sequences from 1939 patients from 6 European institutions. Over 5300 IGH and ~4700 IGK/L sequences from non-CLL B cells were used as a control data set. In total, 110 CLL stereotyped subsets were defined according to HCDR3 homology. Striking IGK/L gene biases were also evident within subsets, along with distinctive K/LCDR3 features, such as length and amino acid composition. At cohort level, the patterns of mutation appeared to be consistent with that of a canonical SHM mechanism. However, at a subgroup level, certain stereotyped subsets, e.g. IGHV3-21/IGLV3-21 and IGHV4-34/IGKV2-30 CLL, deviated from this pattern. Furthermore, recurrent ‘stereotyped’ mutations occurred in cases belonging to subsets with restricted HCDR3s, in both IGHV and IGK/LV genes, which were subset- and CLL-biased when compared to non-CLL B cells. In conclusion, our findings implicate antigen selection as a significant factor in the pathogenesis of CLL, particularly in cases carrying stereotyped BCRs. The presence of stereotyped mutations throughout the VH and VL domain also indicates involvement of IG regions other than the CDR3 in antigen recognition. Finally, biased IGK/L gene usage and specific K/LCDR3 features are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains.

Thesis (Ph. D.)--University of Chicago, Committee on Immunology. / Includes bibliographical references. Also available on the Internet.

<p>B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (V_H) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin. </p><p>Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by V_H3-21/V_λ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the V_H3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin.</p><p>In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases. </p>