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Array-based Characterization of Chronic Lymphocytic Leukemia: - with Focus on Subsets Carrying Stereotyped B-cell Receptors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Molecular Hematology)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In chronic lymphocytic leukemia (CLL), the presence of multiple subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has implicated antigen(s) in leukemogenesis. These stereotyped subsets display similar immunoglobulin (IG) gene usage, almost identical complementarity determining region 3’s and may share clinical features. For instance, subsets #1 (IGHV1/5/7/IGKV1-39) and #2 (IGHV3-21/IGLV3-21) have inferior outcome compared to non-subset patients, whereas subset #4 (IGHV4-34/IGKV2-30) display a favourable prognosis. The aim of this thesis was to investigate genomic aberrations, gene expression patterns and methylation profiles in stereotyped subsets and compare epigenetic profiles in CLL and mantle cell lymphoma (MCL).

In paper I, we investigated genomic aberrations in subsets #2, #4 and #16 and in non-stereotyped samples (n=101) using high-density 250K SNP arrays. Subset #2 and non-subset #2 IGHV3-21 cases displayed a higher frequency of aberrations than subset #4 cases. The high incidence of del(11q) in both subset #2/non-subset #2 may reflect the adverse survival reported for IGHV3-21 patients. In contrast, the lower frequency of genetic events and lack of poor-prognostic aberrations in subset #4 may partially explain their indolent disease. In paper II, we analysed the global RNA expression in subset #4, #16 and non-subset IGHV4-34 CLL patients (n=25). Subsets #4 and 16 showed distinct gene expression profiles, where genes involved in cell regulatory pathways were significantly lower expressed in subset #4, in line with their low-proliferative disease. In paper III, a genome-wide methylation array was applied to investigate methylation profiles in subsets #1, #2 and #4 (n=39). We identified differential methylation patterns for all subsets and found affected genes to be involved in e.g. apoptosis and therapy resistance. When performing functional annotation, a clear enrichment of genes involved in adaptive immunity was observed. These genes were preferentially methylated in subset #1 when compared to either subset #2 or #4, possibly due to different antigen responses. In paper IV, the genome-wide methylation profiles for 30 CLL and 20 MCL patients were investigated. Distinct methylation profiles were observed, where MCL displayed a more homogeneous profile. Homeobox transcription factor genes showed a higher degree of methylation in MCL, while apoptosis-related genes and proliferation-associated genes were methylated in CLL.

In summary, this thesis demonstrates that stereotyped CLL subsets display differences in gene expression profiles, genetic aberrations and methylation patterns, underscoring the functional relevance of subgrouping according to BCR stereotypy. The distinct methylation profiles of CLL and MCL suggests that different epigenetic mechanisms are involved in the pathogenesis of these B-cell malignancies.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis Uppsala , 2010. , p. 73
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 617
Keywords [en]
chronic lymphocytic leukemia, array-based characterization, stereotyped B-cell receptors, subsets, antigens, SNP array, gene expression array, methylation array
National Category
Medical Genetics
Research subject
Clinical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-132895ISBN: 978-91-554-7936-7 (print)OAI: oai:DiVA.org:uu-132895DiVA, id: diva2:359582
Public defence
2010-12-10, Rudbecksalen, Rudbeckslaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2010-11-18 Created: 2010-10-28 Last updated: 2018-01-12Bibliographically approved
List of papers
1. High-density screening reveals a different spectrum of genomic aberrations in chronic lymphocytic leukemia patients with 'stereotyped' IGHV3-21 and IGHV4-34 B-cell receptors
Open this publication in new window or tab >>High-density screening reveals a different spectrum of genomic aberrations in chronic lymphocytic leukemia patients with 'stereotyped' IGHV3-21 and IGHV4-34 B-cell receptors
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2010 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 95, no 9, p. 1519-1525Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The existence of multiple subsets of chronic lymphocytic leukemia expressing 'stereotyped' B-cell receptors implies the involvement of antigen(s) in leukemogenesis. Studies also indicate that 'stereotypy' may influence the clinical course of patients with chronic lymphocytic leukemia, for example, in subsets with stereotyped IGHV3-21 and IGHV4-34 B-cell receptors; however, little is known regarding the genomic profile of patients in these subsets.

DESIGN AND METHODS: We applied 250K single nucleotide polymorphism-arrays to study copy-number aberrations and copy-number neutral loss-of-heterozygosity in patients with stereotyped IGHV3-21 (subset #2, n=29), stereotyped IGHV4-34 (subset #4, n=17; subset #16, n=8) and non-subset #2 IGHV3-21 (n=13) and non-subset #4/16 IGHV4-34 (n=34) patients.

RESULTS: Over 90% of patients in subset #2 and non-subset #2 carried copy-number aberrations, whereas 75-76% of patients in subset #4 and subset #16 showed copy-number aberrations. Subset #2 and non-subset #2 patients also displayed a higher average number of aberrations compared to patients in subset #4. Deletion of 13q was the only known recurrent aberration detected in subset #4 (35%); this aberration was even more frequent in subset #2 (79%). del(11q) was more frequent in subset #2 and non-subset #2 (31% and 23%) patients than in subset #4 and non-subset #4/16 patients. Recurrent copy-number neutral loss-of-heterozygosity was mainly detected on chromosome 13q, independently of B-cell receptor stereotypy.

CONCLUSIONS: Genomic aberrations were more common in subset #2 and non-subset #2 than in subset #4. The particularly high frequency of del(11q) in subset #2 may be linked to the adverse outcome reported for patients in this subset. Conversely, the lower prevalence of copy-number aberrations and the absence of poor-prognostic aberrations in subset #4 may reflect an inherently low-proliferative disease, which would prevent accumulation of genomic alterations.

Keywords
chronic lymphocytic leukemia, stereotyped B-cell receptors, antigens, leukemogenesis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-132887 (URN)10.3324/haematol.2009.021014 (DOI)000281933200014 ()
Available from: 2010-10-28 Created: 2010-10-28 Last updated: 2017-12-12Bibliographically approved
2. Distinct gene expression profiles in subsets of chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B cell receptors
Open this publication in new window or tab >>Distinct gene expression profiles in subsets of chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B cell receptors
Show others...
2010 (English)In: Haematologica (online), ISSN 0390-6078, E-ISSN 1592-8721, Vol. 95, no 12, p. 2072-2079Article in journal (Refereed) Published
Abstract [en]

Background Numerous subsets of patients with chronic lymphocytic leukemia display similar immunoglobulin gene usage with almost identical complementarity determining region 3 sequences. Among IGHV4-34 cases, two such subsets with "stereotyped" B-cell receptors were recently identified, i.e. subset #4 (IGHV4-34/IGKV2-30) and subset #16 (IGHV4-34/IGKV3-20). Subset #4 patients appear to share biological and clinical features, e.g. young age at diagnosis and indolent disease, whereas little is known about subset #16 at a clinical level. DESIGN AND METHODS: We investigated the global gene expression pattern in sorted chronic lymphocytic leukemia cells from 25 subset/non-subset IGHV4-34 patients using Affymetrix gene expression arrays. RESULTS: Although generally few differences were found when comparing subset to non-subset 4/16 IGHV4-34 cases, distinct gene expression profiles were revealed for subset #4 versus subset #16. The differentially expressed genes, predominantly with lower expression in subset #4 patients, are involved in important cell regulatory pathways including cell-cycle control, proliferation and immune response, which may partly explain the low-proliferative disease observed in subset #4 patients. Conclusions Our novel data demonstrate distinct gene expression profiles among patients with stereotyped IGHV4-34 B-cell receptors, providing further evidence for biological differences in the pathogenesis of these subsets and underscoring the functional relevance of subset assignment based on B-cell receptor sequence features.

Keywords
Chronic lymphocytic leukemia, Gene expression, IGHV4-34, Stereotyped BCR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-132891 (URN)10.3324/haematol.2010.028639 (DOI)000285571400013 ()20801898 (PubMedID)
Available from: 2010-10-28 Created: 2010-10-28 Last updated: 2017-12-12
3. Chronic lymphocytic leukemia subsets with stereotyped B cell receptors are distinguished by unique methylation patterns
Open this publication in new window or tab >>Chronic lymphocytic leukemia subsets with stereotyped B cell receptors are distinguished by unique methylation patterns
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Epigenetic modifications, such as DNA methylation, are crucial for both normal and tumor cell development. In chronic lymphocytic leukemia (CLL), multiple subsets have been reported that carry closely homologous, “stereotyped” B cell receptors (BCRs) with varying clinical outcome. To compare the methylation profiles of stereotyped CLL subsets, we here applied high-resolution methylation arrays (27,578 CpG sites) to 3 major subsets, i.e. poor-prognostic subsets #1 (IGHV1/5/7/IGKV1-39/1D-39, n=15) and #2 (IGHV3-21/IGLV3-21, n=9) and favorable-prognostic subset #4 (IGHV4-34/IGKV2-30, n=15). Overall, our results demonstrated significantly different and unique methylation profiles between these stereotyped subsets. Of particular interest was the observation that genes involved in apoptosis (e.g. RIPK3, AATK and TCF3) were methylated in subset #1 compared to subset #4, whilst genes involved in therapy resistance (e.g. ABCB11 and ITGAM) were unmethylated in subset #2 when compared to subset #4. Functional annotation of the differentially methylated genes revealed marked differences in genes involved in the adaptive immune response (e.g. CD80 and CD86), which generally remained unmethylated in subsets #2 and #4 in contrast to subset #1, indicating potential differences in antigenic response between subsets. Taken together, this data convincingly illustrates the distinct methylation profiles amongst stereotyped CLL cases and highlights a key role for epigenetics in disease pathogenesis.

Keywords
Chronic lymphocytic leukemia
Research subject
Clinical Genetics
Identifiers
urn:nbn:se:uu:diva-132884 (URN)
Available from: 2010-10-28 Created: 2010-10-28 Last updated: 2011-01-13
4. Genome-wide array-based methylation profiling reveals preferential methylation of homeobox transcription factor genes in mantle cell lymphoma and pro-apoptotic genes in chronic lymphocytic leukemia
Open this publication in new window or tab >>Genome-wide array-based methylation profiling reveals preferential methylation of homeobox transcription factor genes in mantle cell lymphoma and pro-apoptotic genes in chronic lymphocytic leukemia
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are B-cell malignancies that differ with regards to biological and clinical characteristics. Aberrant DNA methylation has been described for each disorder, but comparison between the two entities is lacking. We employed high-resolution methylation microarrays (27,578 CpG sites) to compare methylation profiles in 20 MCL (10 each with high/low proliferation signature) and 30 CLL (15 IGHV mutated, stereotyped subset #4 and 15 IGHV unmutated, stereotyped subset #1) samples. Distinct differences in methylation patterns were revealed between MCL and CLL, where MCL displayed a more homogenous profile. When comparing MCL and CLL/subsets, 51 genes were consistently identified as differentially methylated in all comparisons. Among 19 genes methylated in MCL, six were homeobox transcription factors (e.g. HLXB9, HOXA13), whereas unmethylated loci in MCL included genes enhancing proliferation and tumor progression (e.g. MERTK and CAMP). In contrast, apoptosis-related genes were prominent among genes methylated in CLL (e.g. DYRK2 and CYFIP2). Correlation between promoter methylation and gene expression was confirmed for selected genes using RQ-PCR. Notably, homeobox genes were not expressed in either entity, implying different gene silencing mechanisms in MCL and CLL. In summary, the differences in global methylation profiles between MCL and CLL have unfolded distinct epigenetic mechanisms operating in each disease.  

Keywords
mantle cell lymphoma, chronic lymphocytic leukemia, Genome-wide array-based methylation profiling
Identifiers
urn:nbn:se:uu:diva-132886 (URN)
Available from: 2010-10-28 Created: 2010-10-28 Last updated: 2011-01-13

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