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- Ljungström, Viktor, 1986-
(författare)
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Exploring next-generation sequencing in chronic lymphocytic leukemia
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Young, Emma, 1990-
(författare)
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Recurrent Genetic Mutations in Lymphoid Malignancies
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In recent years, the genetic landscape of B-cell derived lymphoid malignancies, including chronic lymphocytic leukemia (CLL), has been rapidly unraveled, identifying recurrent genetic mutations with potential clinical impact. Interestingly, ~30% of all CLL patients can be assigned to more homogeneous subsets based on the expression of a similar or “stereotyped” B-cell receptor (BcR). Considering that biased distribution of genetic mutations was recently indicated in specific stereotyped subsets, in paper I, we screened 565 subset cases, preferentially assigned to clinically aggressive subsets, and confirm the SF3B1 mutational bias in subset #2 (45%), but also report on similarly marked enrichment in subset #3 (46%). In contrast, NOTCH1 mutations were predominantly detected in subsets #1, #8, #59 and #99 (22-34%). This data further highlights a subset-biased acquisition of genetic mutations in the pathogenesis of at least certain subsets. Aberrant NF-κB signaling due to a deletion within the NFKBIE gene previously reported in CLL warranted extended investigation in other lymphoid malignancies. Therefore, in paper II, we screened 1460 patients with various lymphoid malignancies for NFKBIE deletions and reported enrichment in classical Hodgkin lymphoma (27%) and primary mediastinal B-cell lymphoma (PMBL) (23%). NFKBIE-deleted PMBL cases had higher rates of chemorefractoriness and inferior overall survival (OS). NFKBIE-deletion status remained an independent prognostic marker in multivariate analysis. EGR2 mutations were recently reported in advanced stage CLL patients; thus, in paper III we screened 2403 CLL patients for mutations in EGR2. An overall mutational frequency of 3.8% was reported and EGR2 mutations were associated with younger age, advanced stage and del(11q). EGR2 mutational status remained an independent marker of poor outcome in multivariate analysis, both in the screening and validation cohorts. Whole-genome sequencing (WGS) of 70 CLL cases, assigned to poor-prognostic subsets #1 and #2 and indolent subset #4, were investigated in Paper IV and revealed a similar skewing of SF3B1 mutations in subset #2 and NOTCH1 mutations in subset #1 to that reported in Paper I. Additionally, an increased frequency of the recently proposed CLL driver gene RPS15 was observed in subset #1. Finally, novel non-coding mutational biases were detected in both subset #1 and #2 that warrant further investigation.
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| 3. |
- Baliakas, Panagiotis, 1977-
(författare)
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Reappraising prognosis in chronic lymphocytic leukemia
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chronic lymphocytic leukemia (CLL) exhibits remarkable clinical heterogeneity likely reflecting the underlying biological heterogeneity. The genetic landscape of CLL has been recently enriched with mutations within a number of genes proposed as novel prognostic markers. Mounting evidence also supports the pivotal role of the clonotypic B-cell receptor immunoglobulin (BcR IG) in the natural history of CLL. Interestingly, almost 30% of all CLL patients can be assigned to different patient subsets, each defined by expression of a distinct stereotyped BcR IG. Whether stereotyped subsets exhibit distinct clinical behavior is still an issue of debate. The aim of this thesis was to evaluate the prognostic relevance of recurrent gene mutations and to assess the clinicobiological associations and clinical impact of BcR IG stereotypy in CLL. In a cohort of 3490 patients, NOTCH1, SF3B1 and TP53 mutations were enriched within clinically aggressive cases carrying unmutated IGHV genes (U-CLL), frequently co-occurring with trisomy 12, del(11q) and del(17p), respectively. Of note, SF3B1 mutations increased in parallel with increasing timespan between diagnosis and mutational screening. NOTCH1 mutations, SF3B1 mutations and TP53 abnormalities (TP53abs, deletions and/or mutations) correlated with shorter time-to-first-treatment among early stage cases, while in multivariate analysis, only SF3B1 mutations and TP53abs retained independent significance. In a series of 8593 CLL patients, stereotyped subsets showed marked differences in demographics, clinical presentation, cytogenetic aberrations and gene mutational spectrum. Patients within a specific subset generally followed similar clinical courses, whereas patients in different stereotyped subsets—even when displaying similar IG somatic hypermutation status— experienced significantly different clinical outcome. In particular, subset #2 (IGHV3-21/IGLV3-21), the largest overall, was found to exhibit (i) a remarkably high incidence of SF3B1 mutations (44%), alluding to subset-biased acquisition of genomic aberrations, in the context of particular antigenic stimulation; and, (ii) a dismal clinical outcome, distinct from the remaining IGHV3-21 CLL. Our findings strongly support the adverse clinical impact of SF3B1 mutations in CLL in addition to TP53abs. BcR IG stereotypy also emerges as prognostically relevant, further highlighting that an immunogenetic sub-classification of CLL based on BcR IG configuration could refine patient risk stratification.
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| 4. |
- Bergh, Ann-Charlotte, 1980-
(författare)
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Importance of microenvironment and antigen in the regulation of growth and survival of CLL cells
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chronic lymphocytic leukemia (CLL) cells rapidly die when put in culture implying that microenvironmental signals delivered by accessory cells confer CLL cells with a growth advantage. Recent findings show that CLL cells are antigen experienced and antigen binding play a critical role in the pathogenesis of the disease. The overall aim of this thesis was to study the influence of the microenvironment and antigen binding in CLL.In paper I, we studied the influence of the small redox-regulatory molecule thioredoxin (Trx) on CLL cell survival and proliferation. We found Trx to be highly expressed in CLL lymph nodes (LNs), secreted from stromal cells surrounding proliferating CLL cells in proliferation centers, indicating growth promoting properties. Secreted Trx was also shown to protect CLL cells from apoptosis.In paper II, oxidized LDL was added to subset #1 CLL cells. However, in contrast to our hypothesis, we could not observe activation and proliferation of CLL cells. Instead subset #1 CLL cells were unresponsive/anergic through the B cell receptor (BcR). This anergic state could however be overcome by “wash out” of bound antigen or addition of toll-like receptor 9 stimulation in some patients.Gene expression profiles differ between groups of CLL patients and in peripheral blood (PB) and LN compartment, due to different microenvironments. However, it is not known whether these differences also apply for DNA methylation. In paper III, we identified various genes that were alternatively methylated between IGHV mutated (M) and unmutated (UM) groups. For example prognostic genes, CLLU1 and LPL, genes involved in B cell signaling, IBTK, as well as numerous TGF-β and NF-κB/TNF pathway genes.The intensity and duration of BcR signals are fine-tuned by enhancing or inhibitory coreceptors. SHP-1 inhibits BcR-signals by dephosphorylation. In paper IV, we compared the expression and activity of SHP-1 in CLL cells from LN with matched PB samples. However, in contrast to our hypothesis, SHP-1 activity/phosphorylation status in PB and LN, did not differ significantly.This thesis, add another piece to the puzzle, on how the microenvironment and antigens influence CLL pathogenesis. Since great variations among individuals are seen, further studies in different groups of patients are necessary to elucidate the importance of antigen for the development of CLL.
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| 5. |
- Bhoi, Sujata
(författare)
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Prognostic markers and DNA methylation profiling in lymphoid malignancies
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In recent years, great progress has been achieved towards identifying novel biomarkers in lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), at the genomic, transcriptomic and epigenomic level for accurate risk-stratification and prediction of treatment response. In paper I, we validated the prognostic relevance of a recently proposed RNA-based marker in CLL, UGT2B17, and analyzed its expression levels in 253 early-stage patients. Besides confirming its prognostic impact in multivariate analysis, we could identify 30% of IGHV-mutated CLL (M-CLL) cases with high expression and poor outcome, which otherwise lacked any other poor-prognostic marker. In paper II, we investigated the prognostic impact of a previously reported 5 CpG signature that divides CLL patients into three clinico-biological subgroups, namely naive B-cell-like CLL (n-CLL), memory B-cell-like CLL (m-CLL) and intermediate CLL (i-CLL), in 135 CLL patients using pyrosequencing. We validated the signature as an independent marker in multivariate analysis and further reported that subset #2 cases were predominantly classified as i-CLL, although displaying a similar outcome as n-CLL. In paper III, we investigated the methylation status and expression level of miR26A1 in both CLL (n=70) and MCL (n=65) cohorts. High miR26A1 methylation was associated with IGHV-unmutated (U-CLL) and shorter overall survival (OS) in CLL, while it was uniformly hypermethylated in MCL. Furthermore, overexpression of miR26A1 resulted in significant downregulation of EZH2 that in turn led to increased apoptosis. In paper IV, we performed DNA methylation profiling in 176 CLL cases assigned to one of 8 major stereotyped subsets (#1-8) in relation to non-subset CLL (n=325) and different normal B-cell subpopulations. Principal component analysis of subset vs. non-subset CLL revealed that U-CLL and M-CLL subsets generally clustered with n-CLL and m-CLL, respectively, indicating common cellular origins. In contrast, subset #2 emerged as the first defined member of the i-CLL subgroup, which in turn alludes to a distinct cellular origin for subset #2 and i-CLL patients. Altogether, this thesis confirms the prognostic significance of RNA and epigenetic-based markers in CLL, provides insight into the mechanism of miRNA deregulation in lymphoid malignancies and further unravels the DNA methylation landscape in stereotyped subsets of CLL.
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| 6. |
- Liljeholm, Maria, 1973-
(författare)
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Congenital Dyserythropoietic Anemia type III (CDA III) : diagnostics, genetics and morbidity
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Congenital Dyserythropoietic Anemias (CDA) are rare hereditary hemolytic disorders with large bi- to multi-nucleated erythroblasts in the bone marrow. Hemolysis is negative in a direct antiglobulin test (DAT). Based on morphology and clinical picture, three major forms of CDAs, type I, II, and III have been defined. CDA III, dominantly inherited, constitutes the rarest type with a majority of cases belonging to a family in Västerbotten, Sweden. The genetic background of CDA I and CDA II has been linked to mutations in CDAN1 and SEC23B respectively. The mutation of CDA III has been linked to 15q22 in earlier studies.In this project we have defined the causative genetic lesion in two families with CDA III. The novel mutation KIF23 c.2747C>G (p.P916R) was shown to segregate with CDA III in the Swedish and American CDA III families and was absent in 356 healthy controls. KIF23 encodes mitotic kinesin-like protein 1 (MKLP1), which plays a central role in the last step of cytokinesis. RNAi-based knock-down and rescue experiments demonstrated that the p.P916R mutation causes cytokinesis failure in HeLa cells, resulting in increasing number of bi-nuclear cells, consistent with appearance of large multinucleated erythroblasts in CDA III patients. We conclude that CDA III is caused by a mutation in KIF23, encoding MKLP1, a conserved mitotic kinesin crucial for cytokinesis.Flow cytometry with eosin-5´-maleimide (EMA), anti-CD55 and anti-CD59 is commonly used when investigating non-autoimmune hemolytic anemias. Reduced fluorescence of EMA, typically detected in hereditary spherocytosis, is also seen in CDA II, while reduction of CD55 and CD59 characterizes paroxysmal nocturnal hemoglobinuria (PNH). We studied the flow cytometric profile of EMA, CD55, and CD59 on erythrocytes in CDA III. We found no abnormality of the erythrocyte membrane in CDA III and concluded that standard flow cytometry cannot be used to discriminate between CDA III and normal controls.In CDA I and CDA II a majority of patients, including those who are not transfusion dependent, suffer from iron overload, which, according to earlier studies, is not the case in CDA III. We found that individuals of the Västerbotten CDA III family carry mutations in the hemochromatosis (HFE) gene. Three CDA III patients with heterozygous or compound HFE mutations need treatment with phlebotomy due to iron overload. One of them carries heterozygous H63D mutation, which is not reported to lead to iron overload by itself in otherwise healthy individuals. We propose that molecular genetic testing of the HFE gene is indicated in all patients with CDA, including CDA III.
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