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Exploring next-generation sequencing in chronic lymphocytic leukemia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
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 [en]
CLL, next-generation sequencing, clonal evolution, stereotypy, RPS15, NFKBIE
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-302026ISBN: 978-91-554-9674-6OAI: oai:DiVA.org:uu-302026DiVA: diva2:956044
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: 2016-09-22
List of papers
1. 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
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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
2. Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia.
Open this publication in new window or tab >>Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia.
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2015 (English)In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 212, no 6Article in journal (Refereed) Published
Abstract [en]

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-279237 (URN)10.1084/jem.20142009 (DOI)25987724 (PubMedID)
Available from: 2016-02-29 Created: 2016-02-29 Last updated: 2016-08-29
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
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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.

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. Whole-genome sequencing in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors
Open this publication in new window or tab >>Whole-genome sequencing in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-302024 (URN)
External cooperation:
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2016-08-28 Created: 2016-08-28 Last updated: 2016-08-29

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