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Whole-exome sequencing in relapsing chronic lymphocytic leukemia: clinical impact of recurrent RPS15 mutations
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
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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.

Place, publisher, year, edition, pages
2016. Vol. 127, no 8, 1007-1016 p.
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-257013DOI: 10.1182/blood-2015-10-674572ISI: 000373397800011PubMedID: 26675346OAI: oai:DiVA.org:uu-257013DiVA: diva2:828070
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: 2017-12-04Bibliographically approved
In thesis
1. Exploring next-generation sequencing in chronic lymphocytic leukemia
Open this publication in new window or tab >>Exploring next-generation sequencing in chronic lymphocytic leukemia
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Next-generation sequencing (NGS) techniques have led to major breakthroughs in the characterization of the chronic lymphocytic leukemia (CLL) genome with discovery of recurrent mutations of potential prognostic and/or predictive relevance. However, before NGS can be introduced into clinical practice, the precision of the techniques needs to be studied in better detail. Furthermore, much remains unknown about the genetic mechanisms leading to aggressive disease and resistance to treatment. Hence, in Paper I, the technical performance of a targeted deep sequencing panel including 9 genes was evaluated in 188 CLL patients. We were able to validate 143/155 (92%) selected mutations through Sanger sequencing and 77/82 mutations were concordant in a second targeted sequencing run, indicating that the technique can be introduced in clinical practice. In Paper II we screened 18 NF-κB pathway genes in 315 CLL patients through targeted deep sequencing which revealed a recurrent 4 base-pair deletion in the NFKBIE gene. Screening of NFKBIE in 377 additional cases identified the mutation in ~6% of all CLL patients. We demonstrate that the lesion lead to aberrant NF-κB signaling through impaired interaction with p65 and is associated with unfavorable clinical outcome. In Paper III we sought to delineate the genetic lesions that leads to relapse after fludarabine, cyclophosphamide, and rituximab treatment. Through whole-exome sequencing of pre-treatment and relapse samples from 41 cases we found evidence of frequent selection of subclones harboring driver mutations and subsequent clonal evolution following treatment. We also detected mutations in the ribosomal protein RPS15 in 8 cases (19.5%) and characterization of the mutations through functional assays point to impaired p53 regulation in cells with mutated RPS15. Paper IV aimed at characterizing 70 patients assigned to three major subsets (#1, #2, and #4) through whole-genome sequencing. Besides recurrent exonic driver mutations, we report non-coding regions significantly enriched for mutations in subset #1 and #2 that may facilitate future molecular studies. Collectively, this thesis supports the potential of targeted sequencing for mutational screening of CLL in clinical practice, provides novel insight into the pathobiology of aggressive CLL, and demonstrates the clinical outcome and cellular effects of NFKBIE and RPS15 mutations. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1251
Keyword
CLL, next-generation sequencing, clonal evolution, stereotypy, RPS15, NFKBIE
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-302026 (URN)978-91-554-9674-6 (ISBN)
Public defence
2016-10-14, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-09-22 Created: 2016-08-29 Last updated: 2016-10-11
2. Genomic and transcriptomic sequencing in chronic lymphocytic leukemia
Open this publication in new window or tab >>Genomic and transcriptomic sequencing in chronic lymphocytic leukemia
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
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:nbn:se:uu:diva-303703 (URN)978-91-554-9702-6 (ISBN)
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

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Ljungström, ViktorCortese, DiegoYoung, EmmaPandzic, TatjanaMansouri, LarryBaliakas, PanagiotisHöglund, MartinSjöblom, TobiasRosenquist, Richard

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Ljungström, ViktorCortese, DiegoYoung, EmmaPandzic, TatjanaMansouri, LarryBaliakas, PanagiotisHöglund, MartinSjöblom, TobiasRosenquist, Richard
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Experimental and Clinical OncologyScience for Life Laboratory, SciLifeLabDepartment of Immunology, Genetics and PathologyHaematology
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