| 1. |
- Baliakas, Panagiotis, et al.
(författare)
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Recurrent mutations refine prognosis in chronic lymphocytic leukemia
- 2015
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Ingår i: Leukemia. - : Springer Science and Business Media LLC. - 0887-6924 .- 1476-5551. ; 29, s. 329-336
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Tidskriftsartikel (refereegranskat)abstract
- Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods.
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| 2. |
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| 3. |
- Baliakas, Panagiotis, 1977-, et al.
(författare)
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Tailored approaches grounded on immunogenetic features for refined prognostication in chronic lymphocytic leukemia
- 2019
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 104:2, s. 360-369
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Tidskriftsartikel (refereegranskat)abstract
- Chronic lymphocytic leukemia (CLL) patients with differential somatic hypermutation status of the immunoglobulin heavy variable genes, namely mutated or unmutated, display fundamental clinico-biological differences. Considering this, we assessed prognosis separately within mutated (M-CLL) and unmutated (U-CLL) CLL in 3015 patients, hypothesizing that the relative significance of relevant indicators may differ between these two categories. Within Binet A M-CLL patients, besides TP53 abnormalities, trisomy 12 and stereotyped subset #2 membership were equivalently associated with the shortest time-to-first-treatment and a treatment probability at five and ten years after diagnosis of 40% and 55%, respectively; the remaining cases exhibited 5-year and 10-year treatment probability of 12% and 25%, respectively. Within Binet A U-CLL patients, besides TP53 abnormalities, del(11q) and/or SF3B1 mutations were associated with the shortest time-to-first-treatment (5- and 10-year treatment probability: 78% and 98%, respectively); in the remaining cases, males had a significantly worse prognosis than females. In conclusion, the relative weight of indicators that can accurately risk stratify early-stage CLL patients differs depending on the somatic hypermutation status of the immunoglobulin heavy variable genes of each patient. This finding highlights the fact that compartmentalized approaches based on immunogenetic features are necessary to refine and tailor prognostication in CLL.
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| 4. |
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| 5. |
- Cortese, Diego
(författare)
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Genomic and transcriptomic sequencing in chronic lymphocytic leukemia
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
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| 7. |
- Ljungström, Viktor, et al.
(författare)
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Whole-exome sequencing in relapsing chronic lymphocytic leukemia : clinical impact of recurrent RPS15 mutations
- 2016
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 127:8, s. 1007-1016
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Mansouri, Larry, et al.
(författare)
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Functional loss of I kappa B epsilon leads to NF-kappa B deregulation in aggressive chronic lymphocytic leukemia
- 2015
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 212:6, s. 833-843
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Tidskriftsartikel (refereegranskat)abstract
- NF-kappa B 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-kappa B pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes I kappa B epsilon, a negative regulator of NF-kappa B 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 I kappa B epsilon 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 I kappa B epsilon 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-kappa B deregulation during lymphomagenesis.
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| 9. |
- Mansouri, Larry, et al.
(författare)
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Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia.
- 2015
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 212:6, s. 833-843
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Sutton, Lesley-Ann, et al.
(författare)
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Targeted next-generation sequencing in chronic lymphocytic leukemia : a high-throughput yet tailored approach will facilitate implementation in a clinical setting
- 2015
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 0390-6078 .- 1592-8721. ; 100:3, s. 370-376
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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