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Prognostic markers and DNA methylation profiling in lymphoid malignancies
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University. (Molecular Hematology)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In recent years, great progress has been achieved towards identifying novel biomarkers in lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), at the genomic, transcriptomic and epigenomic level for accurate risk-stratification and prediction of treatment response. In paper I, we validated the prognostic relevance of a recently proposed RNA-based marker in CLL, UGT2B17, and analyzed its expression levels in 253 early-stage patients. Besides confirming its prognostic impact in multivariate analysis, we could identify 30% of IGHV-mutated CLL (M-CLL) cases with high expression and poor outcome, which otherwise lacked any other poor-prognostic marker. In paper II, we investigated the prognostic impact of a previously reported 5 CpG signature that divides CLL patients into three clinico-biological subgroups, namely naive B-cell-like CLL (n-CLL), memory B-cell-like CLL (m-CLL) and intermediate CLL (i-CLL), in 135 CLL patients using pyrosequencing. We validated the signature as an independent marker in multivariate analysis and further reported that subset #2 cases were predominantly classified as i-CLL, although displaying a similar outcome as n-CLL. In paper III, we investigated the methylation status and expression level of miR26A1 in both CLL (n=70) and MCL (n=65) cohorts. High miR26A1 methylation was associated with IGHV-unmutated (U-CLL) and shorter overall survival (OS) in CLL, while it was uniformly hypermethylated in MCL. Furthermore, overexpression of miR26A1 resulted in significant downregulation of EZH2 that in turn led to increased apoptosis. In paper IV, we performed DNA methylation profiling in 176 CLL cases assigned to one of 8 major stereotyped subsets (#1-8) in relation to non-subset CLL (n=325) and different normal B-cell subpopulations. Principal component analysis of subset vs. non-subset CLL revealed that U-CLL and M-CLL subsets generally clustered with n-CLL and m-CLL, respectively, indicating common cellular origins. In contrast, subset #2 emerged as the first defined member of the i-CLL subgroup, which in turn alludes to a distinct cellular origin for subset #2 and i-CLL patients. Altogether, this thesis confirms the prognostic significance of RNA and epigenetic-based markers in CLL, provides insight into the mechanism of miRNA deregulation in lymphoid malignancies and further unravels the DNA methylation landscape in stereotyped subsets of CLL.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 77
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1363
Keywords [en]
Lymphoid malignancies, CLL, MCL, prognostic markers, micro-RNA, methylation, stereotyped subsets
National Category
Hematology
Identifiers
URN: urn:nbn:se:uu:diva-328616ISBN: 978-91-513-0053-5 (print)OAI: oai:DiVA.org:uu-328616DiVA, id: diva2:1136447
Public defence
2017-10-19, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2017-09-26 Created: 2017-08-28 Last updated: 2017-10-17
List of papers
1. UGT2B17 expression: a novel prognostic marker within IGHV-mutated chronic lymphocytic leukemia?
Open this publication in new window or tab >>UGT2B17 expression: a novel prognostic marker within IGHV-mutated chronic lymphocytic leukemia?
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2016 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 101, no 2, p. E63-E65Article in journal, Letter (Refereed) Published
Keywords
CLL; UGT2B17; prognostic markers
National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-271300 (URN)10.3324/haematol.2015.136440 (DOI)000379156300007 ()26589911 (PubMedID)
Available from: 2016-01-07 Created: 2016-01-07 Last updated: 2019-04-01
2. Prognostic impact of epigenetic classification in chronic lymphocytic leukemia: The case of subset #2
Open this publication in new window or tab >>Prognostic impact of epigenetic classification in chronic lymphocytic leukemia: The case of subset #2
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2016 (English)In: Epigenetics, ISSN 1559-2294, E-ISSN 1559-2308, Vol. 11, no 6, p. 449-455Article in journal (Refereed) Published
Abstract [en]

Based on the methylation status of 5 single CpG sites, a novel epigenetic classification of chronic lymphocytic leukemia (CLL) was recently proposed, classifying CLL patients into 3 clinico-biological subgroups with different outcome, termed memory like CLL (m-CLL), naive like CLL (n-CLL), and a third intermediate CLL subgroup (i-CLL). While m-CLL and n-CLL patients at large corresponded to patients carrying mutated and unmutated IGHV genes, respectively, limited information exists regarding the less defined i-CLL group. Using pyrosequencing, we investigated the prognostic impact of the proposed 5 CpG signature in a well-characterized CLL cohort (135 cases), including IGHV-mutated and unmutated patients as well as clinically aggressive stereotyped subset #2 patients. Overall, we confirmed the signature's association with established prognostic markers. Moreover, in the presence of the IGHV mutational status, the epigenetic signature remained independently associated with both time-to-first-treatment and overall survival in multivariate analyses. As a prime finding, we observed that subset #2 patients were predominantly classified as i-CLL, probably reflecting their borderline IGHV mutational status (97-99% germline identity), though having a similarly poor prognosis as n-CLL patients. In summary, we validated the epigenetic classifier as an independent factor in CLL prognostication and provide further evidence that subset #2 is a member of the i-CLL group, hence supporting the existence of a third, intermediate epigenetic subgroup.

Keywords
CLL, epigenetic classification, methylation, prognosis
National Category
Cancer and Oncology Medical Genetics
Identifiers
urn:nbn:se:uu:diva-303408 (URN)10.1080/15592294.2016.1178432 (DOI)000380902700006 ()27128508 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2016-09-19 Created: 2016-09-19 Last updated: 2018-01-10Bibliographically approved
3. Epigenetic silencing of miR-26A1 in chronic lymphocytic leukemia and mantle cell lymphoma: Impact on EZH2 expression
Open this publication in new window or tab >>Epigenetic silencing of miR-26A1 in chronic lymphocytic leukemia and mantle cell lymphoma: Impact on EZH2 expression
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2016 (English)In: Epigenetics, ISSN 1559-2294, E-ISSN 1559-2308, Vol. 11, no 5, p. 335-343Article in journal (Refereed) Published
Abstract [en]

Downregulation of miR26A1 has been reported in various B-cell malignancies; however, the mechanism behind its deregulation remains largely unknown. We investigated miR26A1 methylation and expression levels in a well-characterized series of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). From 450K methylation arrays, we first observed miR26A1 (cg26054057) as uniformly hypermethylated in MCL (n = 24) (all >75%), while CLL (n = 18) showed differential methylation between prognostic subgroups. Extended analysis using pyrosequencing confirmed our findings and real-time quantitative PCR verified low miR26A1 expression in both CLL (n = 70) and MCL (n = 38) compared to normal B-cells. Notably, the level of miR26A1 methylation predicted outcome in CLL, with higher levels seen in poor-prognostic, IGHV-unmutated CLL. Since EZH2 was recently reported as a target for miR26A1, we analyzed the expression levels of both miR26A1 and EZH2 in primary CLL samples and observed an inverse correlation. By overexpression of miR26A1 in CLL and MCL cell lines, reduced EZH2 protein levels were observed using both Western blot and flow cytometry. In contrast, methyl-inhibitor treatment led to upregulated miR26A1 expression with a parallel decrease of EZH2 expression. Finally, increased levels of apoptosis were observed in miR26A1-overexpressing cell lines, further underscoring the functional relevance of miR26A1. In summary, we propose that epigenetic silencing of miR26A1 is required for the maintenance of increased levels of EZH2, which in turn translate into a worse outcome, as shown in CLL, highlighting miR26A1 as a tumor suppressor miRNA.

Keywords
Chronic lymphocytic leukemia, DNA methylation, mantle cell lymphoma, microRNA, tumor suppressor
National Category
Cancer and Oncology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-298924 (URN)10.1080/15592294.2016.1164375 (DOI)000377274700002 ()27052808 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2016-07-12 Created: 2016-07-12 Last updated: 2017-11-28Bibliographically approved
4. Genome-wide DNA methylation profiling in chronic lymphocytic leukemia patients carrying stereotyped B-cell receptors
Open this publication in new window or tab >>Genome-wide DNA methylation profiling in chronic lymphocytic leukemia patients carrying stereotyped B-cell receptors
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(English)Manuscript (preprint) (Other academic)
National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-328613 (URN)
Available from: 2017-08-28 Created: 2017-08-28 Last updated: 2017-09-06

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