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DISSECTING RESISTANCE MECHANISMS IN CHRONIC LYMPHOCYTIC LEUKEMIA USING WHOLE-EXOME SEQUENCING: IMPACT OF RECURRENT RPS15 MUTATIONS ON P53 DYSREGULATION
Author(s): ,
Viktor Ljungström
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Diego Cortese
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Emma Young
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Tatjana Pandzic
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Larry Mansouri
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Karla Plevova
Affiliations:
Department of Molecular Medicine,CEITEC, Masaryk University,Brno,Czech Republic;Department of Internal Medicine – Hematology and Oncology,University Hospital Brno and Faculty of Medicine, Masaryk University,Brno,Czech Republic
,
Lesley-Ann Sutton
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Niki Stavroyianni
Affiliations:
Hematology Department and HCT Unit,G. Papanicolaou Hospital,Thessaloniki,Greece
,
Andreas Agathangelidis
Affiliations:
Division of Molecular Oncology and Department of Onco-Hematology,San Raffaele Scientific Institute,Milan,Italy;Università Vita-Salute San Raffaele,Milan,Italy
,
Davide Rossi
Affiliations:
Division of Hematology, Department of Translational Medicine,Amedeo Avogadro University of Eastern Piedmont,Novara,Italy
,
Martin Höglund
Affiliations:
Department of Medical Sciences, Section of Hematology,Uppsala University,Uppsala,Sweden
,
David Oscier
Affiliations:
Department of Haematology,Royal Bournemouth Hospital,Bournemouth,United Kingdom
,
Gianluca Gaidano
Affiliations:
Division of Hematology, Department of Translational Medicine,Amedeo Avogadro University of Eastern Piedmont,Novara,Italy
,
Frederic Davi
Affiliations:
Laboratory of Hematology and Universite Pierre et Marie Curie,Hopital Pitie-Salpetriere,Paris,France
,
Christiane Pott
Affiliations:
Second Medical Department,University Hospital Schleswig-Holstein,Kiel,Germany
,
Livio Trentin
Affiliations:
Department of Medicine, Hematology and Clinical Immunology Branch,Padua University School of Medicine,Padova,Italy
,
Sarka Pospisilova
Affiliations:
Department of Molecular Medicine,CEITEC, Masaryk University,Brno,Czech Republic;Department of Internal Medicine – Hematology and Oncology,University Hospital Brno and Faculty of Medicine, Masaryk University,Brno,Czech Republic
,
Paolo Ghia
Affiliations:
Division of Molecular Oncology and Department of Onco-Hematology,San Raffaele Scientific Institute,Milan,Italy;Università Vita-Salute San Raffaele,Milan,Italy
,
Kostas Stamatopoulos
Affiliations:
Hematology Department and HCT Unit,G. Papanicolaou Hospital,Thessaloniki,Greece;Institute of Applied Biosciences,CERTH,Thessaloniki,Greece;Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Tobias Sjöblom
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
Richard Rosenquist
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
EHA Library. Ljungström V.
Jun 12, 2015; 103153
Disclosure(s): Uppsala University
Dept of Immunology, Genetics and Pathology
Viktor Ljungström
Viktor Ljungström
Contributions
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Abstract
Abstract: S121

Type: Oral Presentation + travel grant

Presentation during EHA20: From 12.06.2015 11:30 to 12.06.2015 11:45

Location: Room A8

Background
Fludarabine, cyclophosphamide and rituximab (FCR) is the gold-standard first-line regimen in medically fit patients with chronic lymphocytic leukemia (CLL); however, despite good response rates most patients will eventually relapse. Besides TP53 aberrations, the mechanisms leading to relapse after FCR treatment are currently poorly understood.

Aims
To characterize the genetic mechanisms underlying relapse following treatment with FCR using whole-exome sequencing (WES).

Methods
Forty-one CLL patients receiving FCR with either a partial response (PR, with ≥4 cycles of treatment completed) or a complete response (CR, ≥1 cycle of treatment completed) were selected. Pre-treatment and relapse samples (mean time to relapse 3.2 years, range 0.7 – 10.9), together with matched germline DNA for 28 patients, were analyzed by WES. Well-established bioinformatics tools and pipelines were used to process raw sequencing reads, enabling the identification of somatic mutations and also facilitating the analysis of copy-number aberrations (CNA) and absolute cancer cell fractions (CCF).

Results
Amongst the 28 patients with matched germline DNA, 1191 somatic variants (>10% allele frequency) were found in the pre-treatment samples and 1334 in the relapse samples, with an average of 15.2 (range, 3-24) and 17.6 (range, 2-32) non-silent mutations per case, respectively. Mutations were predominantly missense substitutions (81%) and less frequently frameshift or in-frame insertions/deletions (14%) or nonsense mutations (5%). As expected, at relapse, a high proportion of cases harbored mutations in genes previously linked to adverse prognosis in CLL: TP53 (n=8; 19.5%), NOTCH1 (n=8; 19.5%), ATM (n=7; 17%), SF3B1 (n=6; 14.6%), NFKBIE (n=4; 9.8%), EGR2 (n=4; 9.8%) and BIRC3 (n=3; 7.3%). Intriguingly, a large proportion of cases also harbored mutations in RPS15 (n=8; 19.5%), a gene encoding a component of the 40S ribosomal subunit. High allele frequencies were observed for RPS15 mutations at both time points (range, 29% - 56%), and all mutations were missense variants residing within a 7 amino-acid evolutionarily conserved region. Besides its role in protein translation, RPS15 has been shown to stabilize p53 by interfering with the MDM2-p53-MDMX network and inhibiting MDM2-mediated p53 degradation.  Characterization of two recurrent RPS15 mutations in the HCT116 colorectal cancer cell line transiently expressing either wild-type (wt) or mutant RPS15 revealed impaired ability of RPS15P131S and RPS15G132A in regulating endogenous p53. As both mutations map within the region that interacts with MDM2, this finding strongly suggests that binding of RPS15P131S and RPS15G132A to MDM2 is less efficient compared to wt protein thus leading to more pronounced p53 degradation. Finally, by calculating the absolute CCF for all mutations at both time points allowed monitoring of clonal heterogeneity over time. All 24 cases with available exome-derived CNA data showed mutations expanding ≥0.3 in CCF between the time points (mean 7.4 mutations, range 1-21). Among recurrently mutated genes, i) RPS15 remained stable over time, ii) TP53, EGR2, NOTCH1 and BIRC3 mutations expanded or remained stable, and iii) for SF3B1 and ATM mutations both increasing and decreasing CCFs were observed.

Summary
We provide novel insights into the heterogeneous genetic landscape of CLL relapsing after FCR treatment with our most prominent finding being recurrent RPS15 mutations (19.5%) and with in vitro studies of RPS15 mutations pointing to a novel mechanism for p53 dysregulation in CLL.

Keyword(s): Chronic lymphocytic leukemia, Mutation analysis



Session topic: CLL - Biology: Interacting determinants of CLL ontogeny and evolution
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