| 1. |
- Agathangelidis, Andreas, et al.
(författare)
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Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia : From Patient Material To Sequence Interpretation
- 2018
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Ingår i: Journal of Visualized Experiments. - : JOURNAL OF VISUALIZED EXPERIMENTS. - 1940-087X. ; :141
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Tidskriftsartikel (refereegranskat)abstract
- During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a 'molecular timeline' since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.
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| 2. |
- Agathangelidis, Andreas, et al.
(författare)
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Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia : a molecular classification with implications for targeted therapies
- 2012
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 119:19, s. 4467-4475
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Tidskriftsartikel (refereegranskat)abstract
- Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1: 2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.
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| 3. |
- Baliakas, Panagiotis, et al.
(författare)
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Chromosomal translocations and karyotype complexity in chronic lymphocytic leukemia : A systematic reappraisal of classic cytogenetic data
- 2014
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Ingår i: American Journal of Hematology. - : Wiley. - 0361-8609 .- 1096-8652. ; 89:3, s. 249-255
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Tidskriftsartikel (refereegranskat)abstract
- The significance of chromosomal translocations (CTRAs) and karyotype complexity (KC) in chronic lymphocytic leukemia (CLL) remains uncertain. To gain insight into these issues, we evaluated a series of 1001 CLL cases with reliable classic cytogenetic data obtained within 6 months from diagnosis before any treatment. Overall, 320 cases were found to carry 1 CTRAs. The most frequent chromosome breakpoints were 13q, followed by 14q, 18q, 17q, and 17p; notably, CTRAs involving chromosome 13q showed a wide spectrum of translocation partners. KC (3 aberrations) was detected in 157 cases and significantly (P<0.005) associated with unmutated IGHV genes and aberrations of chromosome 17p. Furthermore, it was identified as an independent prognostic factor for shorter time-to-first-treatment. CTRAs were assigned to two categories (i) CTRAs present in the context of KC, often with involvement of chromosome 17p aberrations, occurring mostly in CLL with unmutated IGHV genes; in such cases, we found that KC rather than the presence of CTRAs per se negatively impacts on survival; (ii) CTRAs in cases without KC, having limited if any impact on survival. On this evidence, we propose that all CTRAs in CLL are not equivalent but rather develop by different processes and are associated with distinct clonal behavior. Am. J. Hematol. 89:249-255, 2014. (c) 2013 Wiley Periodicals, Inc.
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| 4. |
- Baliakas, Panagiotis, et al.
(författare)
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Clinical effect of stereotyped B-cell receptor immunoglobulins in chronic lymphocytic leukaemia: a retrospective multicentre study
- 2014
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Ingår i: The Lancet Haematology. - 2352-3026. ; 1:2, s. 74-84
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Tidskriftsartikel (refereegranskat)abstract
- Background About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptor immunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggests that B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never been explored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn. Methods For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogenetic data were available. These patients were followed up in 15 academic institutions throughout Europe (in Czech Republic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collected between June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptor immunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressing unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome of our analysis was time to first treatment, defined as the time between diagnosis and date of first treatment. Findings 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 major subsets. Stereotyped subsets showed significant differences in terms of age, sex, disease burden at diagnosis, CD38 expression, and cytogenetic aberrations of prognostic significance. Patients within a specific subset generally followed the same clinical course, whereas patients in different stereotyped subsets-despite having the same immunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status-showed substantially different times to first treatment. By integrating B-cell receptor immunoglobulin stereotypy (for subsets 1, 2, and 4) into the well established Dohner cytogenetic prognostic model, we showed these, which collectively account for around 7% of all cases of CLL and represent both U-CLL and M-CLL, constituted separate clinical entities, ranging from very indolent (subset 4) to aggressive disease (subsets 1 and 2). Interpretation The molecular classification of chronic lymphocytic leukaemia based on B-cell receptor immunoglobulin stereotypy improves the Dohner hierarchical model and refines prognostication beyond immunoglobulin mutational status, with potential implications for clinical decision making, especially within prospective clinical trials.
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| 5. |
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| 6. |
- Baliakas, Panagiotis, et al.
(författare)
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Not all IGHV3-21 chronic lymphocytic leukemias are equal: prognostic considerations.
- 2015
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 125:5, s. 856-859
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Tidskriftsartikel (refereegranskat)abstract
- An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2. Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non-subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non-subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.
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| 7. |
- 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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| 8. |
- Bikos, Vasilis, et al.
(författare)
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An Immunogenetic Signature of Ongoing Antigen Interactions in Splenic Marginal Zone Lymphoma Expressing IGHV1-2*04 Receptors
- 2016
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Ingår i: Clinical Cancer Research. - 1078-0432 .- 1557-3265. ; 22:8, s. 2032-2040
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Prompted by the extensive biases in the immunoglobulin (IG) gene repertoire of splenic marginal-zone lymphoma (SMZL), supporting antigen selection in SMZL ontogeny, we sought to investigate whether antigen involvement is also relevant post-transformation. Experimental Design: We conducted a large-scale subcloning study of the IG rearrangements of 40 SMZL cases aimed at assessing intraclonal diversification (ID) due to ongoing somatic hypermutation (SHM). Results: ID was identified in 17 of 21 (81%) rearrangements using the immunoglobulin heavy variable (IGHV) 1-2*04 gene versus 8 of 19 (40%) rearrangements utilizing other IGHV genes (P = 0.001). ID was also evident in most analyzed IG light chain gene rearrangements, albeit was more limited compared with IG heavy chains. Identical sequence changes were shared by subclones from different patients utilizing the IGHV1-2*04 gene, confirming restricted ongoing SHM profiles. Non-IGHV1-2*04 cases displayed both a lower number of ongoing SHMs and a lack of shared mutations (per group of cases utilizing the same IGHV gene). Conclusions: These findings support ongoing antigen involvement in a sizable portion of SMZL and further argue that IGHV1-2*04 SMZL may represent a distinct molecular subtype of the disease.
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| 9. |
- Chatzouli, Maria, et al.
(författare)
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Heterogeneous Functional Effects of Concomitant B Cell Receptor and TLR Stimulation in Chronic Lymphocytic Leukemia with Mutated versus Unmutated Ig Genes
- 2014
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 192:10, s. 4518-4524
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Tidskriftsartikel (refereegranskat)abstract
- We recently reported that chronic lymphocytic leukemia (CLL) subgroups with distinct clonotypic BCRs present discrete patterns of TLR expression, function, and/ or tolerance. In this study, to explore whether specific types of BCR/TLR collaboration exist in CLL, we studied the effect of single versus concomitant BCR and/or TLR stimulation on CLL cells from mutated (M-CLL) and unmutated CLL (U-CLL) cases. We stimulated negatively isolated CLL cells by using anti-IgM, imiquimod, and CpG oligodeoxynucleotide for BCR, TLR7, and TLR9, respectively, alone or in combination for different time points. After in vitro culture in the absence of stimulation, differences in p-ERK were identified at any time point, with higher p-ERK levels in U-CLL versus M-CLL. Pronounced p-ERK induction was seen by single stimulation in U-CLL, whereas BCR/TLR synergism was required in M-CLL, in which the effect was overall limited in scale. An opposite pattern was observed regarding induction of apoptosis, as studied by Western blotting for the cleaved fragment of poly(ADP-ribose) polymerase, and the active isoform of caspase-8, with M-CLL responding even to single stimulation, contrasting with U-CLL that showed minimal response. Our findings suggest that concomitant engagement of BCR and TLR leads to differential responses in CLL depending on the mutational status of the BCR. Differential intensity and duration of responses in M-CLL versus U-CLL indicates that the differences in signal transduction between the two subgroups may be primarily quantitative rather than qualitative.
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| 10. |
- Hadzidimitriou, Anastasia, et al.
(författare)
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Evidence for the significant role of immunoglobulin light chains in antigen recognition and selection in chronic lymphocytic leukemia
- 2009
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 113:2, s. 403-411
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Tidskriftsartikel (refereegranskat)abstract
- We analyzed somatic hypermutation (SHM) patterns and secondary rearrangements involving the immunoglobulin (IG) light chain (LC) gene loci in 725 patients with chronic lymphocytic leukemia (CLL). Important differences regarding mutational load and targeting were identified in groups of sequences defined by IGKV/IGLV gene usage and/or K/LCDR3 features. Recurrent amino acid (AA) changes in the IGKV/IGLV sequences were observed in subsets of CLL cases with stereotyped B-cell receptors (BCRs), especially those expressing IGHV3-21/IGLV3-21 and IGHV4-34/IGKV2-30 BCRs. Comparison with CLL LC sequences carrying heterogeneous K/LCDR3s or non-CLL LC sequences revealed that distinct amino acid changes appear to be "CLL-biased." Finally, a significant proportion of CLL cases with monotypic LC expression were found to carry multiple potentially functional LC rearrangements, alluding to active, (auto) antigen-driven receptor editing. In conclusion, SHM targeting in CLL LCs is just as precise and, likely, functionally driven as in heavy chains. Secondary LC gene rearrangements and subset-biased mutations in CLL LC genes are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains. Therefore, CLL is characterized not only by stereotyped HCDR3 and heavy chains but, rather, by stereotyped BCRs involving both chains, which generate distinctive antigen-binding grooves.
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| 11. |
- 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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| 12. |
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| 13. |
- 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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| 14. |
- 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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| 15. |
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| 16. |
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| 17. |
- Murray, Fiona, et al.
(författare)
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Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia : implications for the role of antigen selection in leukemogenesis
- 2008
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 111:3, s. 1524-1533
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Tidskriftsartikel (refereegranskat)abstract
- Somatic hypermutation (SHM) features in a series of 1967 immunoglobulin heavy chain gene (IGH) rearrangements obtained from patients with chronic lymphocytic leukemia (CLL) were examined and compared with IGH sequences from non-CLL B cells available in public databases. SHM analysis was performed for all 1290 CLL sequences in this cohort with less than 100% identity to germ line. At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged from the analysis of certain subgroups of CLL sequences defined by: (1) IGHV gene usage, (2) presence of stereotyped heavy chain complementarity-determining region 3 (HCDR3) sequences, and (3) mutational load. Recurrent, "stereotyped" amino acid changes occurred across the entire IGHV region in CLL subsets carrying stereotyped HCDR3 sequences, especially those expressing the IGHV3-21 and IGHV4-34 genes. These mutations are un-derrepresented among non-CLL sequences and thus can be considered as CLL-biased. Furthermore, it was shown that even a low level of mutations may be functionally relevant, given that stereotyped amino acid changes can be found in subsets of minimally mutated cases. The precise targeting and distinctive features of somatic hypermutation (SHM) in selected subgroups of CLL patients provide further evidence for selection by specific antigenic element(s).
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| 18. |
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| 19. |
- Ntoufa, Stavroula, et al.
(författare)
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B Cell Anergy Modulated by TLR1/2 and the miR-17 similar to 92 Cluster Underlies the Indolent Clinical Course of Chronic Lymphocytic Leukemia Stereotyped Subset #4
- 2016
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 196:10, s. 4410-4417
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Tidskriftsartikel (refereegranskat)abstract
- Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BCR Ig) display a particularly indolent disease course. Immunogenetic studies of the clonotypic BCR Ig of CLL subset #4 suggested a resemblance with B cells rendered anergic through chronic autoantigenic stimulation. In this article, we provide experimental evidence that subset #4 CLL cells show low IgG levels, constitutive ERK1/2 activation, and fail to either release intracellular Ca2+ or activate MAPK signaling after BCR cross-linking, thus displaying a signature of B cell anergy at both biochemical and functional levels. Interestingly, TLR1/2 triggering restored BCR functionality, likely breaching the anergic state, and this was accompanied by induction of the miR17 similar to 92 cluster, whose members target critical BCR-associated molecules, including MAPKs. In conclusion, we demonstrate BCR anergy in CLL subset #4 and implicate TLR signaling and the miR-17 similar to 92 cluster in the regulation of the anergic state. This detailed signaling profiling of subset #4 has implications for advanced understanding of the complex regulation of intracellular signaling pathways in CLL, currently a major therapeutic target of the disease.
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| 20. |
- Papakonstantinou, Nikos, et al.
(författare)
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Integrated epigenomic and transcriptomic analysis reveals TP63 as a novel player in clinically aggressive chronic lymphocytic leukemia
- 2019
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 144:11, s. 2695-2706
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Tidskriftsartikel (refereegranskat)abstract
- Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness. What's new? In chronic lymphocytic leukemia (CLL), cases with unmutated immunoglobulin receptors (U-CLL) are generally associated with inferior outcome, albeit still displaying considerable heterogeneity. Might such differences in CLL progression be explained by epigenetics? In this study, the authors found that an unusually aggressive subset of CLLs called subset #8 has a distinctive DNA-methylation profile. They also found that p63 is a novel pro-survival factor for CLL cells. These molecular studies may lead to new prognostic biomarkers, and possibly new therapeutic targets, for CLL.
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| 21. |
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| 22. |
- Polychronidou, Eleftheria, et al.
(författare)
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Automated shape-based clustering of 3D immunoglobulin protein structures in chronic lymphocytic leukemia
- 2018
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Ingår i: BMC Bioinformatics. - : Springer Science and Business Media LLC. - 1471-2105. ; 19
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Tidskriftsartikel (refereegranskat)abstract
- Background: Although the etiology of chronic lymphocytic leukemia (CLL), the most common type of adult leukemia, is still unclear, strong evidence implicates antigen involvement in disease ontogeny and evolution. Primary and 3D structure analysis has been utilised in order to discover indications of antigenic pressure. The latter has been mostly based on the 3D models of the clonotypic B cell receptor immunoglobulin (BcR IG) amino acid sequences. Therefore, their accuracy is directly dependent on the quality of the model construction algorithms and the specific methods used to compare the ensuing models. Thus far, reliable and robust methods that can group the IG 3D models based on their structural characteristics are missing. Results: Here we propose a novel method for clustering a set of proteins based on their 3D structure focusing on 3D structures of BcR IG from a large series of patients with CLL. The method combines techniques from the areas of bioinformatics, 3D object recognition and machine learning. The clustering procedure is based on the extraction of 3D descriptors, encoding various properties of the local and global geometrical structure of the proteins. The descriptors are extracted from aligned pairs of proteins. A combination of individual 3D descriptors is also used as an additional method. The comparison of the automatically generated clusters to manual annotation by experts shows an increased accuracy when using the 3D descriptors compared to plain bioinformatics-based comparison. The accuracy is increased even more when using the combination of 3D descriptors. Conclusions: The experimental results verify that the use of 3D descriptors commonly used for 3D object recognition can be effectively applied to distinguishing structural differences of proteins. The proposed approach can be applied to provide hints for the existence of structural groups in a large set of unannotated BcR IG protein files in both CLL and, by logical extension, other contexts where it is relevant to characterize BcR IG structural similarity. The method does not present any limitations in application and can be extended to other types of proteins.
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| 23. |
- Sutton, Lesley-Ann, et al.
(författare)
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Antigen selection in B-cell lymphomas : tracing the evidence
- 2013
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Ingår i: Seminars in Cancer Biology. - : Elsevier BV. - 1044-579X .- 1096-3650. ; 23:6, s. 399-409
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Forskningsöversikt (refereegranskat)abstract
- While signaling through the B cell receptor (BcR) facilitates B cell development and maintenance, it also carries intertwined risks for the development of lymphomas since malignant B cells can exploit these pathways in order to trigger and fuel clonal expansion. This corruption of the normal B cell response to antigens, leading to sustained BcR signaling, has given great impulse to investigate in detail the role of antigen in lymphomas. Suffice it to conclude from such studies, largely immunogenetics based, that the evidence implicating antigens (exogenous or self) in lymphoma development is substantial and that lymphomagenesis is functionally driven and dynamic, rather than a simple stochastic process. As the paradigm of antigen-driven lymphoma evolves, further investigation will be paramount to the identification of the inciting agent(s) that may be responsible for immunoproliferative neoplasms and also for the development of therapeutic agents targeting effectors of the BcR signaling pathway.
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| 24. |
- Sutton, Lesley-Ann, et al.
(författare)
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Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors
- 2016
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 0390-6078 .- 1592-8721. ; 101:8, s. 959-967
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Tidskriftsartikel (refereegranskat)abstract
- We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets# 2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s).
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| 25. |
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| 26. |
- 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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| 27. |
- Vardi, Anna, et al.
(författare)
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IgG-Switched CLL Has a Distinct Immunogenetic Signature from the Common MD Variant : Ontogenetic Implications
- 2014
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Ingår i: Clinical Cancer Research. - 1078-0432 .- 1557-3265. ; 20:2, s. 323-330
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE:IgG-switched chronic lymphocytic leukemia (G-CLL) is a rare variant of CLL, whose origin and ontogenetic relationship to the common IgM/IgD (MD-CLL) variant remains undefined. Here we sought for clues regarding the ontogeny of G-CLL versus MD-CLL by profiling the relevant IG gene repertoires.EXPERIMENTAL DESIGN:Using purpose-built bioinformatics methods, we performed detailed immunogenetic profiling of a multinational CLL cohort comprising 1256 cases, of which 1087 and 169 expressed IG mu/delta and gamma heavy chains, respectively.RESULTS:G-CLL has a highly skewed IG gene repertoire that is distinct from MD-CLL, especially in terms of: (i) overuse of the IGHV4-34 and IGHV4-39 genes; and, (ii) differential somatic hypermutation (SHM) load. Repertoire differences held also when comparing subgroups with similar SHM status and were mainly attributed to the exclusive representation in G-CLL of two major subsets with quasi-identical (stereotyped) B-cell receptors. These subsets, namely #4 (IGHV4-34/IGKV2-30) and #8 (IGHV4-39/IGKV1(D)-39), were found to display sharply contrasting SHM and clinical behavior.CONCLUSIONS:G-CLL exhibits an overall distinct immunogenetic signature from MD-CLL, prompting speculations about distinct ontogenetic derivation and/or immune triggering. The reasons underlying the differential regulation of SHM among G-CLL cases remain to be elucidated.
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| 28. |
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| 29. |
- Xochelli, Aliki, et al.
(författare)
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Chronic Lymphocytic Leukemia with Mutated IGHV4-34 Receptors : Shared and Distinct Immunogenetic Features and Clinical Outcomes
- 2017
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Ingår i: Clinical Cancer Research. - 1078-0432 .- 1557-3265. ; 23:17, s. 5292-5301
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication. Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes. Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management.
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| 30. |
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| 31. |
- Xochelli, Aliki, et al.
(författare)
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Disease-biased and shared characteristics of the immunoglobulin gene repertoires in marginal zone B cell lymphoproliferations
- 2019
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Ingår i: Journal of Pathology. - : WILEY. - 0022-3417 .- 1096-9896. ; 247:4, s. 416-421
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Tidskriftsartikel (refereegranskat)abstract
- The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments.
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| 32. |
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| 33. |
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| 34. |
- Xochelli, Aliki, et al.
(författare)
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Immunoglobulin heavy variable (IGHV) genes and alleles : new entities, new names and implications for research and prognostication in chronic lymphocytic leukaemia
- 2015
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Ingår i: Immunogenetics. - : Springer Science and Business Media LLC. - 0093-7711 .- 1432-1211. ; 67:1, s. 61-66
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Tidskriftsartikel (refereegranskat)abstract
- Ieext generation sequencing studies in Homo sapiens have identified novel immunoglobulin heavy variable (IGHV) genes and alleles necessitating changes in the international ImMunoGeneTics information system (IMGT) GENE-DB and reference directories of IMGT/V-QUEST. In chronic lymphocytic leukaemia (CLL), the somatic hypermutation (SHM) status of the clonotypic rearranged IGHV gene is strongly associated with patient outcome. Correct determination of this parameter strictly depends on the comparison of the nucleotide sequence of the clonotypic rearranged IGHV gene with that of the closest germline counterpart. Consequently, changes in the reference directories could, in principle, affect the correct interpretation of the IGHV mutational status in CLL. To this end, we analyzed 8066 productive IG heavy chain (IGH) rearrangement sequences from our consortium both before and after the latest update of the IMGT/V-QUEST reference directory. Differences were identified in 405 cases (5 % of the cohort). In 291/405 sequences (71.9 %), changes concerned only the IGHV gene or allele name, whereas a change in the percent germline identity (%GI) was noted in 114/405 (28.1 %) sequences; in 50/114 (43.8 %) sequences, changes in the %GI led to a change in the mutational set. In conclusion, recent changes in the IMGT reference directories affected the interpretation of SHM in a sizeable number of IGH rearrangement sequences from CLL patients. This indicates that both physicians and researchers should consider a re-evaluation of IG sequence data, especially for those IGH rearrangement sequences that, up to date, have a GI close to 98 %, where caution is warranted.
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| 35. |
- Xochelli, Aliki, et al.
(författare)
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Molecular Evidence for Antigen Drive in the Natural History of Mantle Cell Lymphoma
- 2015
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Ingår i: American Journal of Pathology. - : Elsevier BV. - 0002-9440 .- 1525-2191. ; 185:6, s. 1740-1748
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Tidskriftsartikel (refereegranskat)abstract
- To further our understanding about antigen involvement in mantle cell Lymphoma (MCL), we analyzed the expression levels of activation-induced cytidine deaminase (AID), a key player in B-cell responses to antigen triggering, in 133 MCL cases; assessed the functionality of AID by evaluating in vivo class switch recombination in 52 MCL cases; and sought for indications of ongoing antigen interactions by exploring intraclonal diversification within 14 MCL cases. The AID full-length transcript and the most frequent splice variants (AID-Delta E4a, AID-Delta E) were detected in 128 (96.2%), 96 (72.2%), and 130 cases (97.7%), respectively. Higher AID full-Length transcript levels were significantly associated (P < 0.001) with Lack of somatic hypermutation within the clonotypic immunoglobulin heavy variable (IGHV) genes. Median AID transcript levels were higher in lymph node material compared to cases in which peripheral blood was analyzed, implying that clonal behavior is influenced by the microenvironment. Switched tumor-derived IGHV-IGHD-IGHJ transcripts were identified in 5 of 52 cases (9.6%), all of which displayed somatic hypermutation and AID-mRNA expression. Finally, although most cases exhibited low levels of intraclonal diversification, analysis of the mutational activity revealed a precise targeting of somatic hypermutation indicative of an active, ongoing interaction with antigen(s). Collectively, these findings strongly allude to antigen involvement in the natural history of MCL, further challenging the notion of antigen naivety.
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| 36. |
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| 37. |
- Young, Emma, 1990-, et al.
(författare)
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EGR2 mutations define a new clinically aggressive subgroup of chronic lymphocytic leukemia
- 2017
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Ingår i: Leukemia. - : Springer Science and Business Media LLC. - 0887-6924 .- 1476-5551. ; 31:7, s. 1547-1554
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Tidskriftsartikel (refereegranskat)abstract
- Recurrent mutations within EGR2 were recently reported in advanced-stage chronic lymphocytic leukemia (CLL) patients and associated with a worse outcome. To study their prognostic impact, 2403 CLL patients were examined for mutations in the EGR2 hotspot region including a screening (n = 1283) and two validation cohorts (UK CLL4 trial patients, n = 366; CLL Research Consortium (CRC) patients, n = 490). Targeted deep-sequencing of 27 known/postulated CLL driver genes was also performed in 38 EGR2-mutated patients to assess concurrent mutations. EGR2 mutations were detected in 91/2403 (3.8%) investigated cases, and associated with younger age at diagnosis, advanced clinical stage, high CD38 expression and unmutated IGHV genes. EGR2- mutated patients frequently carried ATM lesions (42%), TP53 aberrations (18%) and NOTCH1/FBXW7 mutations (16%). EGR2 mutations independently predicted shorter time-to-first-treatment (TTFT) and overall survival (OS) in the screening cohort; they were confirmed associated with reduced TTFT and OS in the CRC cohort and independently predicted short OS from randomization in the UK CLL4 cohort. A particularly dismal outcome was observed among EGR2-mutated patients who also carried TP53 aberrations. In summary, EGR2 mutations were independently associated with an unfavorable prognosis, comparable to CLL patients carrying TP53 aberrations, suggesting that EGR2-mutated patients represent a new patient subgroup with very poor outcome.
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| 38. |
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