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Molecular Genetic and DNA Methylation Profiling of Chronic Lymphocytic Leukaemia: A Focus on Divergent Prognostic Subgroups and Subsets
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Molecular Haematology)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Advancements in prognostication have improved the subdivision of chronic lymphocytic leukaemia (CLL) into diverse prognostic subgroups. In CLL, IGHV unmutated and IGHV3-21 genes are associated with a poor-prognosis, conversely, IGHV mutated genes with a favourable outcome. The finding of multiple CLL subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has suggested a role for antigen(s) in leukemogenesis. Patients belonging to certain stereotyped subsets share clinical and biological characteristics, yet limited knowledge exists regarding the genetic and epigenetic events that may influence their clinical behaviour. This thesis aimed to, further investigate Swedish IGHV3-21-utilising patients, screen for genetic and DNA-methylation events in CLL subgroups/subsets and study DNA methylation over time and within different CLL compartments.

In paper I, IGHV gene sequencing of 337 CLL patients from a Swedish population-based cohort revealed a lower (6.5%) IGHV3-21 frequency relative to previous Swedish hospital-based studies (10.1-12.7%). Interestingly, this frequency remained higher compared to other Western CLL (2.6-4.1%) hospital-based cohorts. Furthermore, we confirmed the poor-outcome for IGHV3-21 patients to be independent of mutational and stereotypy status.

In paper II, genomic events in stereotyped IGHV3-21-subset #2, IGHV4-34-subset #4 and subset #16 and their non-stereotyped counterparts were investigated via SNP arrays (n=101). Subset #2 and non-subset #2 carried a higher frequency of events compared to subset #4. A high frequency of del(11q) was evident in IGHV3-21 patients particularly subset #2 cases, which may partially explain their poor-prognosis. In contrast, the lower prevalence of aberrations and absence of poor-prognostic alterations may reflect the inherent low-proliferative disease seen in subset #4 cases.

In papers III and IV, differential methylation profiles in IGHV mutated and IGHV unmutated patients were identified using DNA-methylation microarrays. CLL prognostic genes (CLLU1, LPL), tumor-suppressor genes (TSGs) (ABI3, WISP3) and genes belonging to TGF-ß and NF-kB/TNFR1 pathways were differentially methylated between the subgroups. Additionally, the re-expression of methylated TSGs by use of methyl and deacetyl inhibitors was demonstrated. Interestingly, analysis of patient-paired diagnostic/follow-up samples and patient-matched lymph node (LN) and peripheral blood (PB) cases revealed global DNA methylation to be relatively stable over time and remarkably similar within the different compartments.

Altogether, this thesis provides insight into the aberrant genomic and DNA methylation events in divergent CLL subgroups. Moreover this thesis helps distinguish the extent to which DNA methylation changes with respect to time and microenvironment in CLL.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 102 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 745
Keyword [en]
DNA methylation, Chronic lymphocytic leukemia, SNP, array, IGHV3-21, IGHV4-34
National Category
Hematology Medical Genetics
Research subject
Biology with specialization in Molecular Biology; Genetics; Medical Genetics; Molecular Biology; Molecular Genetics; Oncology
Identifiers
URN: urn:nbn:se:uu:diva-168945ISBN: 978-91-554-8289-3 (print)OAI: oai:DiVA.org:uu-168945DiVA: diva2:506809
Public defence
2012-04-13, Rudbecksalen, Rudbeck Laboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-03-22 Created: 2012-02-20 Last updated: 2012-03-29
List of papers
1. IGHV3-21 Gene Frequency in a Swedish Cohort of Patients With Newly Diagnosed Chronic Lymphocytic Leukemia
Open this publication in new window or tab >>IGHV3-21 Gene Frequency in a Swedish Cohort of Patients With Newly Diagnosed Chronic Lymphocytic Leukemia
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2012 (English)In: Clinical Lymphoma, Myeloma & Leukemia, ISSN 2152-2650, E-ISSN 2152-2669, Vol. 12, no 3, 201-206 p.Article in journal (Refereed) Published
Abstract [en]

The IGHV3-21 gene has been shown to be overrepresented in Scandinavian patients with chronic lymphocytic leukemia (CLL). By investigating a population-based cohort of 337 Swedish patients with CLL, a lower (6.5%) IGHV3-21 frequency was determined relative to our previous hospital-based studies (10.1%-12.7%), yet this frequency remained higher compared to other Western CLL cohorts (2.6%-4.1%). Furthermore, we confirmed the poor outcome for patients with IGHV3-21 to be independent of mutational and stereotypy status. Background: Scandinavian patients with CLL have shown an overrepresentation of the poor-prognostic IGHV3-21 gene. Furthermore, approximately 50% of patients with IGHV3-21 carry stereotyped B-cell receptors, which implicate antigen selection in leukemogenesis. These patients have also been reported to have shorter time to progression than patients with nonstereotyped IGHV3-21. Materials and Methods: To investigate the IGHV3-21 frequency and the clinical impact of IGHV3-21 stereotypy, 337 newly diagnosed Swedish CLL patients from a population-based cohort were analyzed. Results: Interestingly, the IGHV3-21 frequency was indeed lower (6.5%) in this indolent patient cohort than in our previous hospital-based cohort studies (10.1%-12.7%). Hence, a selection bias of more-aggressive cases rendered a higher proportion of IGHV3-21 cases in our original studies. Nevertheless, the Swedish IGHV3-21 frequency still remained higher when compared with other larger European or American studies (2.6%-4.1%). Finally, we confirmed the poor outcome for IGHV3-21 patients to be independent of mutational status and found stereotypy to have no impact on survival or time to treatment. Conclusion: The Swedish geographic bias in IGHV3-21 gene frequency was validated albeit at a lower frequency than previously reported. Moreover, no prognostic value could be attributed to IGHV3-21 stereotype status.

Keyword
IGHV3-21, stereotypy, Chronic lymphocytic leukemia, Immunoglobulin heavy-chain variable 3 21 gene frequency, Prognosis, Stereotyped B-cell receptors
National Category
Hematology Medical Genetics
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-168942 (URN)10.1016/j.clml.2012.01.009 (DOI)000304495800008 ()
Available from: 2012-02-27 Created: 2012-02-20 Last updated: 2017-12-07Bibliographically approved
2. 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, 1519-1525 p.Article 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.

Keyword
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
3. Differential genome-wide array-based methylation profiles in prognostic subsets of chronic lymphocytic leukemia
Open this publication in new window or tab >>Differential genome-wide array-based methylation profiles in prognostic subsets of chronic lymphocytic leukemia
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2010 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 115, no 2, 296-305 p.Article in journal (Refereed) Published
Abstract [en]

Global hypomethylation and regional hypermethylation are well-known epigenetic features of cancer; however, in chronic lymphocytic leukemia (CLL), studies on genome-wide epigenetic modifications are limited. Here, we analyzed the global methylation profiles in CLL, by applying high-resolution methylation microarrays (27,578 CpG sites) to 23 CLL samples, belonging to the immunoglobulin heavy-chain variable (IGHV) mutated (favorable) and IGHV unmutated/IGHV3-21 (poor-prognostic) subsets. Overall, results demonstrated significant differences in methylation patterns between these subgroups. Specifically, in IGHV unmutated CLL, we identified methylation of 7 known or candidate tumor suppressor genes (eg, VHL, ABI3, and IGSF4) as well as 8 unmethylated genes involved in cell proliferation and tumor progression (eg, ADORA3 and PRF1 enhancing the nuclear factor-kappaB and mitogen-activated protein kinase pathways, respectively). In contrast, these latter genes were silenced by methylation in IGHV mutated patients. The array data were validated for selected genes using methylation-specific polymerase chain reaction, quantitative reverse transcriptase-polymerase chain reaction, and bisulfite sequencing. Finally, the significance of DNA methylation in regulating gene promoters was shown by reinducing 4 methylated tumor suppressor genes (eg, VHL and ABI3) in IGHV unmutated samples using the methyl-inhibitor 5-aza-2'-deoxycytidine. Taken together, our data for the first time reveal differences in global methylation profiles between prognostic subsets of CLL, which may unfold epigenetic silencing mechanisms involved in CLL pathogenesis.

National Category
Hematology Medical Genetics
Identifiers
urn:nbn:se:uu:diva-121108 (URN)10.1182/blood-2009-07-232868 (DOI)000273622600021 ()19897574 (PubMedID)
Available from: 2010-03-18 Created: 2010-03-18 Last updated: 2017-12-12Bibliographically approved
4. 450K DNA methylation analysis of chronic lymphocytic leukaemia reveals DNA methylation to be relatively stable over time and remarkably similar in cells derived from resting and proliferation compartments
Open this publication in new window or tab >>450K DNA methylation analysis of chronic lymphocytic leukaemia reveals DNA methylation to be relatively stable over time and remarkably similar in cells derived from resting and proliferation compartments
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(English)Manuscript (preprint) (Other academic)
Keyword
DNA methylation, Chronic lymphocytic leukemia
National Category
Medical Genetics
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-169308 (URN)
Available from: 2012-02-27 Created: 2012-02-27 Last updated: 2012-03-22

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