Change search
ReferencesLink to record
Permanent link

Direct link
Genomic and transcriptomic sequencing in chronic lymphocytic leukemia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Richard Rosenquist Brandell)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Identification of recurrent mutations through next-generation sequencing (NGS) has given us a deeper understanding of the molecular mechanisms involved in chronic lymphocytic leukemia (CLL) development and progression and provided novel means for risk assessment in this clinically heterogeneous disease. In paper I, we screened a population-based cohort of CLL patients (n=364) for TP53, NOTCH1, SF3B1, BIRC3 and MYD88 mutations using Sanger sequencing, and confirmed the negative prognostic impact of TP53, SF3B1 or NOTCH1 aberrations, though at lower frequencies compared to previous studies. In paper II, we assessed the feasibility of targeted NGS using a gene panel including 9 CLL-related genes in a large patient cohort (n=188). We could validate 93% (144/155) of mutations with Sanger sequencing; the remaining were at the detection limit of the latter technique, and technical replication showed a high concordance (77/82 mutations, 94%). In paper III, we performed a longitudinal study of CLL patients (n=41) relapsing after fludarabine, cyclophosphamide and rituximab (FCR) therapy using whole-exome sequencing. In addition to known poor-prognostic mutations (NOTCH1, TP53, ATM, SF3B1, BIRC3, and NFKBIE), we detected mutations in a ribosomal gene, RPS15, in almost 20% of cases (8/41). In extended patient series, RPS15-mutant cases had a poor survival similar to patients with NOTCH1, SF3B1, or 11q aberrations. In vitro studies revealed that RPS15mut cases displayed reduced p53 stabilization compared to cases wildtype for RPS15. In paper IV, we performed RNA-sequencing in CLL patients (n=50) assigned to 3 clinically and biologically distinct subsets carrying stereotyped B-cell receptors (i.e. subsets #1, #2 and #4) and revealed unique gene expression profiles for each subset. Analysis of SF3B1-mutated versus wildtype subset #2 patients revealed a large number of splice variants (n=187) in genes involved in chromatin remodeling and ribosome biogenesis. Taken together, this thesis confirms the prognostic impact of recurrent mutations and provides data supporting implementation of targeted NGS in clinical routine practice. Moreover, we provide evidence for the involvement of novel players, such as RPS15, in disease progression and present transcriptome data highlighting the potential of global approaches for the identification of molecular mechanisms contributing to CLL development within prognostically relevant subgroups.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 63 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1259
Keyword [en]
chronic, lymphocytic, leukemia, CLL, genomics, transcriptomics, DNA, RNA, mutations, NGS, whole-exome, sequencing, prognostic, markers, TP53, SF3B1, RPS15, relapse, stereotyped, subsets.
National Category
Cancer and Oncology
Identifiers
URN: urn:nbn:se:uu:diva-303703ISBN: 978-91-554-9702-6OAI: oai:DiVA.org:uu-303703DiVA: diva2:972818
Public defence
2016-11-11, Rudbecksalen, Dag Hammarskjölds väg 20, 75237 Uppsala University, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-10-20 Created: 2016-09-22 Last updated: 2016-11-22
List of papers
1. On the way towards a 'CLL prognostic index': focus on TP53, BIRC3, SF3B1, NOTCH1 and MYD88 in a population-based cohort.
Open this publication in new window or tab >>On the way towards a 'CLL prognostic index': focus on TP53, BIRC3, SF3B1, NOTCH1 and MYD88 in a population-based cohort.
Show others...
2014 (English)In: Leukemia, ISSN 0887-6924, E-ISSN 1476-5551, Vol. 28, no 3, 710-713 p.Article in journal, Letter (Refereed) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-214248 (URN)10.1038/leu.2013.333 (DOI)000332845700029 ()24217197 (PubMedID)
Note

Mansouri and Rosenquist contributed equally as senior authors to this work.

Available from: 2014-01-08 Created: 2014-01-08 Last updated: 2016-09-22Bibliographically approved
2. Targeted next-generation sequencing in chronic lymphocytic leukemia: a high-throughput yet tailored approach will facilitate implementation in a clinical setting
Open this publication in new window or tab >>Targeted next-generation sequencing in chronic lymphocytic leukemia: a high-throughput yet tailored approach will facilitate implementation in a clinical setting
Show others...
2015 (English)In: Haematologica (online), ISSN 0390-6078, E-ISSN 1592-8721, Vol. 100, no 3, 370-376 p.Article in journal (Refereed) Published
Abstract [en]

Next- generation sequencing has revealed novel recurrent mutations in chronic lymphocytic leukemia, particularly in patients with aggressive disease. Here, we explored targeted re- sequencing as a novel strategy to assess the mutation status of genes with prognostic potential. To this end, we utilized HaloPlex targeted enrichment technology and designed a panel including nine genes: ATM, BIRC3, MYD88, NOTCH1, SF3B1 and TP53, which have been linked to the prognosis of chronic lymphocytic leukemia, and KLHL6, POT1 and XPO1, which are less characterized but were found to be recurrently mutated in various sequencing studies. A total of 188 chronic lymphocytic leukemia patients with poor prognostic features ( unmutated IGHV, n= 137; IGHV3- 21 subset # 2, n= 51) were sequenced on the HiSeq 2000 and data were analyzed using well- established bioinformatics tools. Using a conservative cutoff of 10% for the mutant allele, we found that 114/ 180 ( 63%) patients carried at least one mutation, with mutations in ATM, BIRC3, NOTCH1, SF3B1 and TP53 accounting for 149/ 177 ( 84%) of all mutations. We selected 155 mutations for Sanger validation ( variant allele frequency, 10- 99%) and 93% ( 144/ 155) of mutations were confirmed; notably, all 11 discordant variants had a variant allele frequency between 11- 27%, hence at the detection limit of conventional Sanger sequencing. Technical precision was assessed by repeating the entire HaloPlex procedure for 63 patients; concordance was found for 77/ 82 ( 94%) mutations. In summary, this study demonstrates that targeted next- generation sequencing is an accurate and reproducible technique potentially suitable for routine screening, eventually as a stand- alone test without the need for confirmation by Sanger sequencing.

National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-251831 (URN)10.3324/haematol.2014.109777 (DOI)000351279900027 ()25480502 (PubMedID)
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2015-04-27 Created: 2015-04-24 Last updated: 2016-09-22Bibliographically approved
3. Whole-exome sequencing in relapsing chronic lymphocytic leukemia: clinical impact of recurrent RPS15 mutations
Open this publication in new window or tab >>Whole-exome sequencing in relapsing chronic lymphocytic leukemia: clinical impact of recurrent RPS15 mutations
Show others...
2016 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 127, no 8, 1007-1016 p.Article in journal (Refereed) Published
Abstract [en]

Fludarabine, cyclophosphamide and rituximab (FCR) is first-line treatment for medically fit chronic lymphocytic leukemia (CLL) patients, however despite good response rates many patients eventually relapse. Whilst recent high-throughput studies have identified novel recurrent genetic lesions in adverse-prognostic CLL, the mechanisms leading to relapse after FCR therapy are not completely understood. To gain insight into this issue, we performed whole-exome sequencing of sequential samples from 41 CLL patients who were uniformly treated with FCR but relapsed after a median of 2 years. In addition to mutations with known adverse-prognostic impact (TP53, NOTCH1, ATM, SF3B1, NFKBIE, BIRC3) a large proportion of cases (19.5%) harbored mutations in RPS15, a gene encoding a component of the 40S ribosomal subunit. Extended screening, totaling 1119 patients, supported a role for RPS15 mutations in aggressive CLL, with one-third of RPS15-mutant cases also carrying TP53 aberrations. In most cases selection of dominant, relapse-specific subclones was observed over time. However, RPS15 mutations were clonal prior to treatment and remained stable at relapse. Notably, all RPS15 mutations represented somatic missense variants and resided within a 7 amino-acid evolutionarily conserved region. We confirmed the recently postulated direct interaction between RPS15 and MDM2/MDMX and transient expression of mutant RPS15 revealed defective regulation of endogenous p53 compared to wildtype RPS15. In summary, we provide novel insights into the heterogeneous genetic landscape of CLL relapsing after FCR treatment and highlight a novel mechanism underlying clinical aggressiveness involving a mutated ribosomal protein, potentially representing an early genetic lesion in CLL pathobiology.

National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-257013 (URN)10.1182/blood-2015-10-674572 (DOI)000373397800011 ()26675346 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research CouncilEU, European Research CouncilSwedish National Infrastructure for Computing (SNIC)
Note

De två första författarna delar förstaförfattarskapet.

De två sista författarna delar sistaförfattarskapet.

Available from: 2015-06-29 Created: 2015-06-29 Last updated: 2016-09-22Bibliographically approved
4. Differential expression of coding/non-coding transcripts and splice variants in stereotyped subsets of chronic lymphocytic leukemia
Open this publication in new window or tab >>Differential expression of coding/non-coding transcripts and splice variants in stereotyped subsets of chronic lymphocytic leukemia
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-303702 (URN)
Available from: 2016-09-22 Created: 2016-09-22 Last updated: 2016-10-06

Open Access in DiVA

fulltext(881 kB)96 downloads
File information
File name FULLTEXT01.pdfFile size 881 kBChecksum SHA-512
4bc290b290f0dd6b3389b8668660e503e9a8b267e1cfcc768d94359a915d22a2d86b01d3ea876d2c7b315b685c3bf192a1980e9a211185b87b45cdeebf5aa0de
Type fulltextMimetype application/pdf
Buy this publication >>

Search in DiVA

By author/editor
Cortese, Diego
By organisation
Department of Immunology, Genetics and Pathology
Cancer and Oncology

Search outside of DiVA

GoogleGoogle Scholar
Total: 96 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 1282 hits
ReferencesLink to record
Permanent link

Direct link