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1
Baliakas, Panagiotis. "Reappraising prognosis in chronic lymphocytic leukemia." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-280943.
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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.
2
Shen, Yandong. "The Chronic Lymphocytic Leukemia (CLL) Microenvironment and Novel Targeted Therapies." Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/20805.
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Activation of the B-cell receptor (BCR), and subsequent signalling via the Bruton's tyrosine kinase (BTK), phosphoinositide-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK), plays a significant role in the pathogenesis of CLL. This thesis aimed to better understand the role of the CLL microenvironment and to investigate novel treatment strategies for targeting CLL cells in the lymph nodes or bone marrow. We demonstrated using the DotScan cluster of differentiation (CD) antibody microarray, that immunophenotypic changes induced on CLL cells by co-culture with fibroblasts expressing the CD40 ligand can be blocked by ibrutinib or idelalisib. These data provide insight on the mechanisms underlying the lymphocytosis observed in patients treated with these agents. We demonstrated that as a single agent the MEK1/2 inhibitor, binimetinib was effective against CLL cells under certain in vitro conditions and that the drug was effective and synergistic with the AKT inhibitor, MK2206, but not idelalisib. These data suggest that this combination of drugs may represent a novel therapeutic option for CLL effective against CLL cells in the tumour microenvironment. Next, we demonstrated efficacy of the dual PI3K/PIM inhibitor, IBL-202 and showed high synergy with the Bcl-2 inhibitor, venetoclax against CLL cells under conditions that mimic the tumour microenvironment and against a TP53 knock-out cell line we derived from the OSU-CLL cell line using the CRISPR-Cas9 system. This combination was synergistic in terms of apoptosis and inhibition of both the proliferative and migratory capacities of CLL cells. These data suggest that IBL-202 in combination with venetoclax may be an effective treatment option for high risk CLL disease. Collectively, the data presented highlight several pathways and novel drugs that may contribute to the development of therapeutic strategies for CLL patients.
3
Beckwith, Kyle Addison. "Novel Immunotherapeutic Strategies for Chronic Lymphocytic Leukemia." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461203257.
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4
Imam, Hasan. "Effects of protein kinase inhibitors on chronic lymphocytic leukemia (CLL) cells." Thesis, Linköpings universitet, Institutionen för klinisk och experimentell medicin, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-73883.
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B cell Chronic lymphocytic leukemia (B-CLL) is a neoplastic disorder characterized by accumulation of B lymphocytes due to uncontrolled growth and resistance to apoptosis. Src family kinases (SFKs) are non receptor tyrosine kinases present in the cytosol, which couple with downstream B cell receptor signaling and thus mediate growth, survival, proliferation and antiapoptosis. In CLL cells SFKs are remarkably overexpressed, especially Lyn kinase. This gives the rational to use SFKs inhibitor to treat CLL. Addition of the specific pharmacological inhibitors of SFKs, bosutinib and saracatinib, inhibited the global tyrosine phosphorylation as well as the basal auto-phosphorylation of SFKs. Mechanistically, inhibition of SFKs is coupled to apoptosis induction via decreased protein levels of the anti-apoptotic proteins Bcl-2, Mcl-1 and survivin, which were demonstrated by Western blotting. To assess apoptosis induction, annexin V binding to freshly isolated CLL cells with or without treatment with kinase inhibitors was measured flow cytometrically. Using the inhibitors at a concentration of 10 μM the average percentages of annexin V-positive, apoptotic cells in 11 CLL samples increased from 24 % in untreated controls to 55 %, 45 % and 37 % after treatment with bosutinib, saracatinib and dasatinib, respectively. The response to each of the inhibitors showed a high but comparable degree of variation among the investigated CLL samples. On the average bosutinib induced apoptosis with significantly higher efficiency than dasatinib, which calls for further investigation of its pre-clinical potential for treatment of CLL.
5
Ljungström, Viktor. "Exploring next-generation sequencing in chronic lymphocytic leukemia." Doctoral thesis, Uppsala universitet, Experimentell och klinisk onkologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-302026.
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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.
6
Browning, Rebekah L. "Combination Therapies with Interleukin-21 in Chronic Lymphocytic Leukemia." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429719855.
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7
Mohamed, Ahmed. "Deciphering the ontogeny of unmutated and mutated subsets of Chronic Lymphocytic Leukemia." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17286.
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Chronic Lymphocytic Leukemia (CLL) is a type of cancer that affects the B cells of the immune system causing problems in the process of producing antibodies. It can be sorted into mutated and unmutated CLL based on the percentage of somatic mutations in the Immunoglobulin Heavy chain Variable region (IgHV). The B cells of healthy individuals can be sorted into three groups; CD27dull memory B cells (MBCs), CD27bright MBCs and naïve B cells. The hypothesis for the project was that the unmutated CLL subset originates from CD27dull MBCs and the mutated CLL subset originates from CD27bright MBCs. RNA-sequencing data from healthy individuals were acquired from a collaboration partner in Rome and CLL-patients were collected from public datasets available online. Several bioinformatic tools were used to analyze the data. First, the quality of the data files was checked, then adapter sequence from the sequencing process and low-quality bases were removed (trimming). Good quality of the files was confirmed after the trimming. Secondly, these files were mapped against the human reference genome (GRCh38/hg38) for alignment, then the resulted data was used to check for genes that showed differential expression between the different groups. Results were analyzed and visualized using Venn diagrams, Principal Component Analysis (PCA) and heatmap plots and random forest. A list of 85 genes was generated based on the different comparisons and was used in one PCA plot that showed clear separation between the different groups. The SWAP70 gene was analyzed for single nucleotide polymorphisms (SNPs). The study concluded five genes that could be used as biomarkers for CLL and the diagnosis of its subtypes where some of them were discussed in previous studies. Also, the mutated CLL subset showed a similar behavior to the healthy individuals and this could validate the original hypothesis and justifies the better disease prognosis for this subtype.
8
Lanemo, Myhrinder Anna. "Restricted antigen recognition in B cell chronic lymphocytic leukemia." Licentiate thesis, Linköping University, Linköping University, Faculty of Health Sciences, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-16355.
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Chronic lymphocytic leukemia (CLL) cells are considered to be derived from antigen-exposed B cells. To further explore the antigen-driven selection behind the leukemogenesis of CLL, we performed immunoglobulin (Ig) specificity screening of 7 CLL cell lines and 23 primary CLL clones from patient peripheral blood. We also included a recombinant monovalent monoclonal antibody (mAb) belonging to a subset of CLL cases with identical or semiidentical heavy chain complementarity determining region 3 (HCDR3) of the IGHV3-21 gene rearrangement. We found CLL mAb specificities against vimentin, filamin B, cofilin-1, proline-rich acidic protein 1, cardiolipin, oxidized low density lipoprotein and Streptococcus pneumoniae polysaccarides. These molecules are functionally associated with microbial infection and/or apoptotic cell removal. An antigen-driven selection would therefore imply that CLL B cell precursors are involved in the elimination and scavenging of pathogens and apoptotic cells, which could trigger the development of the disease.
The limited in vitro survival of CLL cells makes Epstein-Barr virus (EBV) immortalization of CLL cells a useful experimental model for studies on antibody-specificity screening. Considering the intricate procedure of EBV transformation of CLL cells and the many false cell lines used worldwide, we also wanted to characterize and evaluate the authentic origin of several previously established CLL cell lines and their normal lymphoblastoid counterparts. Three of the CLL cell lines tested were truly authentic (I83-E95, CLL-HG3 and CII), two had features of a biclonal Ig expression (232B4 and WaC3CD5+), one was only tentatively verified (PGA-1), whereas one cell line could not be verified (EHEB) due to lack of original patient cells for comparison. Two of the presumed normal lymphoblastoid cell lines tested were shown to be a neoplastic CLL clone. This study emphasizes the importance of proper cell line authentication and we will continue to verify additional cell lines not yet proven authentic.
In conclusion, we provide evidence for natural Ab production by CLL cells and suggest that these cells might be derived from B cell precursors involved in the innate immunity and, thus, providing a first-line-defence against pathogens and in elimination of apoptotic cells.
9
Niu, Suli. "Quantitative Determination of Surface Markers on B-cell Chronic Lymphocytic Leukemia (CLL) Cells." Thèse, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/30982.
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To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.
10
Beiggi, Sara. "Epidemiological study of chronic lymphocytic leukemia (CLL) in the province of Manitoba, Canada." British Journal of Cancer (Nature Group), 2013. http://hdl.handle.net/1993/23508.
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A previous population-based study of survival in Chronic Lymphocytic Leukemia (CLL) patients in the province of Manitoba demonstrated a lower five-year relative survival among CLL patients compared with the age- and gender-adjusted general population. This decreased relative survival was most pronounced among elderly male CLL patients.
In this study, we have demonstrated that the reduced five-year relative survival observed in CLL patients compared to the general population of Manitoba may partially be attributed to increased risk of second cancers and non-referral to specialized CLL clinics.
The increased risk of second cancers in CLL patients compared to Follicular Lymphoma (FL), a similar indolent B cell malignancy, was only observed after CLL diagnosis indicating that a CLL-specific factor may be responsible for the increased risk of second cancers in these patients. The risk of second cancers is independent of treatment and surveillance bias but is further increased with chemotherapy.
A superior outcome in CLL patients who have been referred to the CancerCare Manitoba (CCMB) specialized CLL clinic was observed that was independent of age, gender, treatment and history of previous cancers. This superior outcome was most pronounced in the elderly CLL patients.
We propose that CLL patients should be referred to CLL-specific hematologists and, where not possible, that guidelines created by such experts be followed. Appropriate screening for second cancers should be performed during routine follow up of CLL patients.
11
Morrison, Eleshia JP. "Psychological Distress and Symptom Burden: Vulnerabilities in Chronic Lymphocytic Leukemia Patients." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366305005.
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12
Trimarco, Valentina. "Role of Nocodazole on the survival of chronic lymphocytic leukemia B cells." Doctoral thesis, Università degli studi di Padova, 2012. http://hdl.handle.net/11577/3422967.
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B-cell Chronic Lymphocytic Leukemia (B-CLL) is the most common leukemia in adults and is characterized by the accumulation of clonal CD19+/CD5+/CD23+ B lymphocytes, due to uncontrolled growth and resistance to apoptosis. Leukemic cells from B-CLL show reduced crosslink with specific molecules and high susceptibility to microtubule disrupting drugs, which suggest cytoskeletal alterations.
Microtubules play a crucial role in the vital functions of neoplastic cells, including mitosis, motility and cell-cell contact, and for this reason they became an important target in cancer therapies. In particular, tubulin, a cytoskeletal member, is the target of specific drugs, named microtubule inhibitors. Among these inhibitors, nocodazole induces tubulin depolimerization, mitotic process blocking and shows an apoptotic effect in B leukemic cells.
The aim of this study was to define the effects of nocodazole on B-CLL cells.
First of all, we verified nocodazole capability to favour the depolymerization of tubulin cytoskeleton in different cell types. In addition, we tested nocodazole-induced apoptosis in normal and leukemic B cells, in cell lines (Jurkat, Raji, and K562), in mesenchymal stromal cells (MSCs), and in T lymphocytes of B-CLL patients. Our data pointed out the high specificity of nocodazole for B-CLL cell apoptosis (leukemic cells: 57±25% vs normal B cells: 98±6%, p<0.0001; data are expressed as mean±standard deviation (SD) of percentage of viable cells after treatment with nocodazole) and the absence of toxicity to others cell types.
Growing evidence suggests that the marrow microenvironment, where MSCs are present, protects B-CLL cells from conventional anti-neoplastic drugs. The cultures of neoplastic B cells with MSCs and nocodazole demonstrated that nocodazole is able to overcome MSC protective effect, even after survival signal supplemental, such as CD40L or plasma from the same patients.
The action mechanism of nocodazole in B-CLL cells is still under investigation. However, we observed that nocodazole is able to turn off the increased basal tyrosine phosphorylation of leukemic cells mediated by Src-kinase Lyn through the down-modulation of Lyn active site. Since the specific inhibition of Lyn induces B-CLL cells apoptosis, this linking will be further investigated.
The results obtained in this study suggest a future role of nocodazole as a possible agent for treatment of B-CLL, for its extreme selectivity, the absence of toxicity and its ability to counteract the protective effect provided by marrow microenvironment.La Leucemia Linfatica Cronica di tipo B (LLC-B) è la forma più comune di leucemia nell’adulto ed è caratterizzata dall’accumulo clonale di piccoli linfociti B CD19+/CD5+/CD23+, dovuto sia ad una crescita incontrollata che ad una resistenza all’apoptosi. Le cellule leucemiche di LLC-B presentano inoltre alcune anomalie, come ridotta capacità di legare specifiche molecole e suscettibilità a farmaci che distruggono i microtubuli, che indicano la presenza di alterazioni a livello citoscheletrico. Il ruolo cruciale che i microtubuli rivestono nelle funzioni vitali delle cellule neoplastiche, quali mitosi, motilità e contatti cellula-cellula, li ha resi un importante target nelle terapie anti-tumorali. In particolar modo la tubulina, componente dei microtubuli, è il bersaglio di una categoria specifica di farmaci anti-tumorali, gli inibitori dei microtubuli; di questa famiglia fa parte anche il nocodazolo, un agente sintetico che induce la depolimerizzazione della tubulina, arresta il processo mitotico ed ha una peculiare specificità nell’indurre l’apoptosi nelle cellule B di LLC-B. Sulla base di queste considerazioni, abbiamo voluto approfondire gli effetti ed il meccanismo d’azione del nocodazolo sulle cellule di LLC-B. Dopo aver verificato che il nocodazolo sia effettivamente responsabile della depolimerizzazione dei filamenti di tubulina citoscheletrica in numerosi tipi cellulari, abbiamo valutato l’effetto apoptotico indotto dal nocodazolo in cellule B normali e di LLC-B, in linee cellulari (Jurkat, Raji e K562), in cellule stromali mesenchimali (MSC) e nei linfociti T residui di pazienti affetti da LLC-B. I risultati ottenuti evidenziano l’estrema selettività del nocodazolo nell’indurre l’apoptosi nelle sole cellule B di LLC-B (linfociti B di LLC-B: 57±25% vs B normali: 98±6%, p<0,0001; dati espressi come media±deviazione standard (DS) della percentuale di cellule vive dopo trattamento con nocodazolo) e l’assenza di tossicità nei confronti delle altre popolazioni cellulari prese in esame. Studi recenti suggeriscono che il microambiente midollare, in cui si trovano anche le MSC, sia in grado di proteggere le cellule leucemiche dall’azione dei farmaci chemioterapici convenzionali. La co-coltura di MSC e cellule B di LLC-B in presenza di nocodazolo ha dimostrato che tale inibitore è in grado di annullare l'effetto protettivo esercitato dalle MSC, nonostante la presenza di segnali di sopravvivenza quali CD40L o plasma ricavato dagli stessi pazienti. I meccanismi d’azione del nocodazolo rimangono ancora da chiarire, tuttavia abbiamo osservato come nelle cellule leucemiche di LLC-B il nocodazolo sia in grado di ridurre l’aumentata fosforilazione tirosinica basale mediata dalla Src-chinasi Lyn, mediante down-regolazione del sito attivatorio di Lyn. Dal momento che abbiamo dimostrato che l’inibizione specifica di Lyn induce apoptosi nelle cellule di LLC-B, questi primi risultati diventano rilevanti e dovranno essere ulteriormente indagati. In conclusione, i risultati ottenuti in questo studio hanno evidenziato l’estrema selettività del nocodazolo nell’indurre apoptosi nei linfociti B leucemici, l’assenza di tossicità in vitro e la capacità di contrastare l’effetto protettivo fornito dal microambiente midollare, suggerendo un futuro ruolo di questa sostanza quale possibile agente terapeutico per la cura della LLC-B.
13
Cortese, Diego. "Genomic and transcriptomic sequencing in chronic lymphocytic leukemia." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-303703.
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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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Santanam, Urmila. "Role of microRNA-29 in the Pathogenesis of B-Cell Chronic Lymphocytic Leukemia." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1281447982.
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Favini, Chiara. "Biological and clinical implications of BIRC3 mutations in chronic lymphocytic leukemia." Doctoral thesis, Università del Piemonte Orientale, 2020. http://hdl.handle.net/11579/114875.
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The current shift of therapy of chronic lymphocytic leukemia (CLL) towards novel targeted agents mandates the identification of molecular predictors to inform on who can still benefit from chemoimmunotherapy and who can be instead early considered for novel targeted agents. Fludarabine, cyclophosphamide, and rituximab (FCR) is the most effective chemoimmunotherapy regimen for the management of CLL and represents the current standard of care for young and fit patients devoid of TP53 disruption. A retrospective multicenter cohort of 287 untreated patients receiving first-line FCR was analyzed by targeted next generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients failing FCR (median progression free survival: 2.2 years, p < 0.001) similar to cases harboring TP53 mutations (median progression free survival: 2.6 years, p < 0.0001). BIRC3 mutations maintained an independent association with an increased risk of progression with a hazard ratio of 2.8 (95% confidence interval 1.4-5.6, p = 0.004) in multivariate analysis adjusted for TP53 mutation, 17p deletion and IGHV mutation status. The functional implications of BIRC3 mutations are largely unexplored and little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first line FCR. By immunoblotting analysis, we showed that the non-canonical NF-kB pathway is active in BIRC3 mutated cell lines and in primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3 mutated primary CLL cells are less sensitive to fludarabine. If validated, BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel frontline therapeutic approaches.
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Kiaii, Shahryar. "T cells in patients with B-cell chronic lymphocytic leukemia (B-CLL) and multiple myeloma (MM) : an immunological study /." Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-050-3/.
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Giacopelli, Brian John. "Global DNA methylation analysis of chronic lymphocytic leukemia and acute myeloid leukemia reveals distinct clinically relevant biological subtypes." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1591114255694166.
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Gärtner, Kathrin Christina [Verfasser], and Josef [Akademischer Betreuer] Mautner. "Engineered extracellular vesicles for immunotherapy of chronic lymphocytic leukemia (CLL) / Kathrin Christina Gärtner ; Betreuer: Josef Mautner." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1163534471/34.
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Gorgone, Ausilia Giuseppa. "Correlazione tra dati biologici e prognosi in pazieti affetti da B-CLL." Doctoral thesis, Università di Catania, 2012. http://hdl.handle.net/10761/1138.
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Gli studi di questi anni hanno dimostrato che i fattori biologici giocano un ruolo fondamentale per la stratificazione del rischio come elementi predittivi del treatment-free survival e dell' overall survival nei pazienti affetti da CLL in stadio iniziale.La maggioranza di questi markers prognostici ,anche se ormai in uso quotidiano nella pratica clinica, non è ancora incluso nelle linee guida internazionali che si basano ancora su criteri esclusivamente clinici e individuano la valutazione biologica da usare in combinazione ai parametri clinici.Il loro impiego può certamente contribuire al miglioramento del clinical management dei pazienti affetti da CLL .
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Chen, Shih-Shih. "Transcriptional Silencing Of Foxd3 Is An Early Event Mediating Epigenetic Silencing In Tcl1 Positive Chronic Lymphocytic Leukemia." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1214923446.
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Yoon, Ju-Yoon. "Elderly patients with chronic lymphocytic leukaemia (CLL): predicting their survival and managing their disease with valproic acid and fludarabine." Informa Healthcare, 2012. http://hdl.handle.net/1993/23317.
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Chronic Lymphocytic Leukaemia (CLL) is a disease of B-lymphocytes that
account for significant morbidity and mortality in mostly elderly patients (aged ≥ 70
years). The relative survival of patients with CLL has been shown to decrease with
patient age, and this age-related reduction in survival was found to correlate with the
levels of two inflammatory cytokine levels in the patients’ plasma. The levels of two
inflammatory cytokines, interleukin-6 and -8 (IL-6, IL-8) were found to correlate
positively with patient age, and increased levels were associated with lower overall
survival. Addition of IL-6 or IL-8 to a co-culture system of CLL cells with bone marrow
stromal cells increased the CLL-stromal cell adhesion, and co-culturing increased IL-8
secretion. In a search of a treatment regimen that may be effective and readily tolerated
by elderly patients, we examined the combination of fludarabine with valproic acid
(VPA), an epileptic that was found to inhibit histone deacetylases (HDACs). The
combination was synergistic against human leukaemic cells, including primary CLL cells.
In a phase II clinical trial where six elderly patients with relapsed, previously treated CLL
were enrolled (half of whom were clinically refractory to fludarabine), the VPAfludarabine
combination induced reduction in the peripheral and lymph node tumour
loads. Mechanistically, the fludarabine treatment induced disruption of the lysosomes,
while VPA induced increase in the level and activity of cathepsin B, a lysosomal protease.
The VPA-induced increase in cathepsin B levels was observed in in cell lines (in vitro),
primary CLL cells (ex vivo) and in patients treated with VPA (in vivo). Chemical
inhibition of cathepsin B was sufficient to dampen the VPA-fludarabine cytotoxicity, and
the addition activated cathepsin B to leukaemic cell lysates was sufficient to induce
caspase cleavage and reduction in anti-apoptotic protein levels. The VPA-fludarabine
combination also lowered phospho-Akt levels and ATM activation, which also
contributed to the VPA-fludarabine synergy, and VPA treatment lowered ATM levels
and phospho-Akt levels in vivo.
In summary, there lies a biological explanation for the poor survival observed
with elderly patients, and the VPA-fludarabine may be a useful regimen for these patients.
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Herman, Sarah Elizabeth May. "MANIPULATION OF KINASE SIGNALING IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE EFFECT ON DISEASE STATE." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1291046222.
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Rezvany, Mohammad Reza. "Natural specific T cell immunity in patients with B-cell chronic lymphocytic leukaemia (B-CLL) : (a clinical and immunological study) /." Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4841-0/.
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FARINELLO, DIEGO. "A retinoic acid-dependent stroma-leukemia crosstalk promotes tissue remodelling and disease progression in a mouse model of chronic lymphocytic leukaemia." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/153230.
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Il compartimento stromale di origine non ematopoietica gioca un ruolo cruciale nello sviluppo della leucemia linfatica cronica, e lo fa attraverso l'attivazione delle cellule tumorali, l'aumento della loro sopravvivenza e l'espansione del clone leucemico. Ancora ad oggi, l'identità delle cellule stromali coinvolte in questo processo e le vie di segnalazione che dirigono questa comunicazione bi-direzionale tra stroma e leucemia rimangono largamente non chiarite. Utilizzando il modello murino di LLC Eμ-Tcl1, abbiamo dimostrato come la progressione della malattia sia associata all'espansione di uno stroma follicolare che manca dei marker convenzionali di superficie propri delle FDC e che esprime CXCL13, una tra le più importanti chemochine per il richiamo dei linfociti B. Successivamente, grazie ad analisi estensive di espressione genica svolte sullo stroma a contatto con la leucemia in vitro, abbiamo scoperto la deregolazione di geni legati ad acido retinoico e deputati alla sintesi ed alla regolazione della trascrizione. Sia il blocco farmacologico, sia la deplezione dei precursori di RA tramite una dieta carente determina un prolungamento della sopravvivenza di topi leucemici. Oltretutto, RA promuove la secrezione di chemochine di richiamo della B-LLC e l'accumulo di queste cellule maligne all'interno di nicchie promuoventi la sopravvivenza di esse; queste nicchie comprendono gli agglomerati linfoidi associati al grasso del mesentere, che rappresentano un contesto di espansione per la leucemia. Questi dati stabiliscono un nuovo ruolo della via di segnalazione dell'acido retinoico nella patogenesi della B-LLC e suggerisce nuove strategie terapeutiche che consentano di bersagliare il microambiente stromale per contenere e controllare la leucemia.In B-cell chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment (SM) plays a critical role in promoting tumor cell activation, survival and expansion. However, the identity of the stromal cells involved and signaling pathways underlying the stroma-leukemia cross-talk remain largely unknown. Using the Eμ-Tcl1 CLL mouse model, we showed that leukemia progression is associated with expansion of follicular stromal cells lacking conventional FDC markers, and expressing the homing chemokine CXCL13. Gene expression profile analysis of stromal cells cultured with CLL cells revealed increased expression of genes regulating retinoic acid (RA) synthesis and signaling. Reducing retinoic acid precursors from the diet or blocking pharmacologically RA-signaling prolong survival of Eμ-Tcl1 mice. Accordingly, RA signaling promotes chemokine secretion and accumulation of CLL cells into survival niches, including fat-associated lymphoid clusters of the mesentery that represent gut-associated niches for leukemia expansion. These findings establish a novel role for retinoic acid signaling in CLL pathogenesis, and provide new therapeutic strategies aiming to target the stromal microenvironment to control leukemia progression.
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Gupta, Sneha Veeraraghavan. "Targeting Protein Metabolism in B-cell Malignancies." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1343169973.
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Dai, Yuntao. "CHARACTERIZATION OF TCL1-MURINE B-1A CELL TRANSCRIPTOME DYNAMICS REVEALS NOVEL INSIGHTS INTO CHRONIC LYMPHOCYTIC LEUKEMIA ONSET." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1434632288.
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Rossmann, Eva D. "Targets for immune mediated killing of tumor cells and T cell functions in B-CLL /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-622-7/.
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Blanco, Ares Gonzalo 1989. "Molecular characterization of the microenvironment in CLL-like monoclonal B cell lymphocytosis and early-stage chronic lymphocytic leukemia." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/664506.
Full textAbstract:
The analysis of the microenvironment in CLL-like monoclonal B cell lymphocytosis (MBL) and early-stage chronic lymphocytic leukemia (CLL) is relevant for understanding the natural history of CLL. To this end, a total of 58 MBL, 54 early-stage CLL and 31 healthy subjects were extensively characterized by various immunological and molecular methods. Purified CD4+ and CD8+ mononuclear cells from peripheral blood were subjected to gene expression studies and T cell receptor (TR) repertoire analysis, whereas cytokine immunoassays were performed in serum samples. Gene expression studies in CD4+ cells revealed increased cytotoxic and inflammatory pathways, which were higher in MBL than in early-stage CLL. Gene dysregulation was not remarkable in CD8+ cells. Increased serum levels of cytokines such as IL8, IFNγ and TNFα were also observed in MBL, while early-stage CLL generally displayed lower cytokine levels, especially amongst cases bearing somatically hypermutated IGHV genes. TR analysis demonstrated oligoclonality in both entities with persisting T cell clones over time and increasing clonality within CD4+ T cells concurrently with the expansion of neoplastic B cells. Besides, identical T cell clonotypes were identified in different MBL/CLL cases. All these findings implicate inflammatory processes and antigenic elements in the immune background of CLL, whose effects are significantly altered during progression from MBL to CLL.El estudio del microambiente en la linfocitosis B monoclonal (LBM) de tipo LLC y en estadios iniciales de la leucemia linfática crónica (LLC) tiene gran relevancia para entender la historia natural de la enfermedad. Con este objetivo se caracterizaron 58 casos de LBM, 54 de LLC en fases iniciales y 31 sujetos sanos. Se analizó la expresión génica y el repertorio del receptor de células T (TR) en fracciones de células mononucleares CD4+ y CD8+ purificadas a partir de sangre periférica. Además, se realizaron inmunoensayos para medir niveles de citoquinas en suero. Los estudios de expresión génica revelaron patrones citotóxicos e inflamatorios aumentados en células CD4+, superiores en LBM con respecto a LLC en fases iniciales. En las células CD8+ no se observó ninguna disfunción remarcable en la expresión génica. Se detectaron niveles aumentados de citoquinas como IL8, IFNγ y TNFα en sueros de sujetos con LBM, mientras que en LLC en fases iniciales los niveles de citoquinas fueron generalmente inferiores, principalmente debido a los casos con hipermutaciones del gen IGHV. El análisis del TR mostró la existencia de oligoclonalidad en ambas entidades y de clones T persistentes en el tiempo, así como niveles de clonalidad en la fracción T CD4+ que aumentan conjuntamente con la expansión de las células B malignas. Asimismo, se identificaron clonotipos comunes en diferentes casos con LBM/LLC. Todos estos hallazgos implican un papel clave de los procesos inflamatorios y de los elementos antigénicos desde las etapas más tempranas de la enfermedad, cuyos efectos varían notablemente durante la progresión desde LBM a LLC.
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MELANDRI, AURORA. "CLINICAL AND BIOLOGICAL MEANING OF GENOMIC COMPLEXITY IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AND COMPLEX KARYOTYPE (CK)." Doctoral thesis, Università degli studi di Ferrara, 2021. http://hdl.handle.net/11392/2488207.
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Chronic Lymphocytic Leukemia (CLL) is the most common form of leukemia in western countries. CLL’s clinical course can range from an indolent condition, with a slow progression, to an aggressive form, which can lead to an early death. The discovery of new and more accurate prognostic markers is crucial for identifying patients in immediate need of treatment. Complex Karyotype (CK) (≥ 3 chromosome aberrations in the same clone) has emerged as an independent negative prognostic marker. CK includes a variety of cytogenetic aberrations, from numerical to structural abnormalities. In this study, it was performed a cytogenetic analysis on 90 treatment-naïve CLL patients with CK. It was evaluated the prognostic impact of each cytogenetic abnormality for the Overall Survival (OS) and Time To First Treatment (TTFT). Among all the abnormalities, unbalance translocations had displayed an independent prognostic impact, with a worse OS and TTFT. It was performed a gene expression profile analysis on 23 of the 90 patients, representative of the entire cohort, 11 with unbalance translocation and 12 without. It emerged that the presence of unbalanced translocation identified a subset of patients with distinct molecular features.
Among the differentially expressed genes, the attention was focused on SLAMF1, a gene involved in lymphocyte activation, apoptosis, and cell cycle control. It was decided to analyze the expression of SLAMF1 in all CLL onset (349) stored in the Ferrara laboratory from 2009 to 2018. Through a droplet digital PCR, it was studied the expression of SLAMF1. The housekeeping gene, β-actin, was used as a standard control. The patients were divided into three different groups based on SLAMF1 expression. Patients with an expression above 6,24 belonged in the high-expression group. A value below 2,8 was considered low, while the rest of the patients were classified as an intermediate group. This partition inversely overlaps with the division of patients based on their prognostic risks. Patients with lower expression of SLAMF1 had a shorter TTFT. and O.S. In CLL miRNAs play an important role in the pathogenesis of the disease. The deletion of the miR-15a and miR-16-1, involved in the Bcl2 pathway, causes the development of CLL. It could be possible that miRNAs are responsible for the downregulation of SLAMF1 in CLL patients. To clarify the role played by these miRNAs in the regulation of SLAMF1 expression it was performed a luciferase assay. The miRNAs selected as possibly responsible for SLAMF1 regulation were the ones up-regulated in CLL: miR-17/92, miR132 and miR-148a. The results of the luciferase assay show no difference in SLAMF1 expression based on the actions of miRNAs. This indicates that miRNAs are not responsible for the dysregulation of SLAMF1. The mechanisms remain unknown. Due to the clinical and molecular heterogeneity of CLL, a better understanding of the molecular pathway that regulates the development of CLL is important for highlighting new possible treatment targets. Although CK is a negative prognostic marker, in this study, it was proven that specific cytogenetic anomalies could worsen the CK prognostic significance. Also, the expression of SLAMF1, easy to measure on the surface of B lymphocytes, could represent a new prognostic marker. These findings will be helpful to separate the patients into risk categories and to select the best treatment.La leucemia linfatica cronica (LLC) è la leucemia più diffusa nei Paesi occidentali. Il decorso clinico della LLC può spaziare da una condizione indolente, a progressione lenta, ad una forma più aggressiva che può portare a una morte precoce. È importante, per identificare i pazienti che necessitano di immediato trattamento clinico, la continua ricerca di nuovi e più accurati marcatori prognostici. Tra questi, il cariotipo complesso (CK) (definito dalla presenza di più di 3 anomalie nello stesso clone) è emerso come un indipendente marcatore prognostico negativo. Il CK include una varietà di anomalie citogenetiche, da quelle numeriche a quelle strutturali. In questo studio è stata eseguita l’analisi citogenetica su 90 pazienti con la LLC e il cariotipo complesso che non sono mai stati trattati farmacologicamente. È stato valutato l’impatto prognostico di ogni singola anomalia citogenetica in relazione alla sopravvivenza globale (O.S) e il tempo al primo trattamento (TTFT). Tra tutte le anomalie, le traslocazioni sbilanciate hanno mostrato di avere un indipendente impatto prognostico, con un peggioramento dell’OS e un più breve TTFT. È stata eseguita anche l’analisi dei profili di espressione genica su 23 dei 90 pazienti, 11 con traslocazioni sbilanciate e 12 senza. La presenza di traslocazioni sbilanciate ha identificato una categoria di pazienti con diverse caratteristiche geniche. Tra i geni differentemente espressi, l’attenzione è stata focalizzata su SLAMF1, un gene coinvolto nei processi di attivazione dei linfociti B, apoptosi e controllo del ciclo cellulare. L’espressione di SLAMF1 è stata analizzata in tutti gli esordi di LLC (349), conservati a Ferrara tra il 2009 e il 2018, attraverso l’uso della droplet digital PCR, usando la β-actina come gene di controllo. I pazienti sono stati divisi in tre diversi gruppi in base all’espressione di SLAMF1. Un livello di espressione superiore a 6,24 è stato considerato come alto, mentre i pazienti con livello inferiore a 2,8 sono stati inseriti nel gruppo con espressione considerata bassa. I restanti pazienti sono stati posizionati nel gruppo a espressione intermedia. Questa divisione è risultata inversamente sovrapponibile a una divisione dei pazienti basata sul loro rischio prognostico. Quelli con una bassa espressione di SLAMF1 sono risultati avere un TTFT più breve e un peggiore OS. Nella patogenesi della LLC i miRNA giocano un ruolo importante. La delezione dei miR-15a e miR-16-1, coinvolti nel pathway di Bcl2, può causare l’insorgenza della LLC. È ipotizzabile che i miRNAs possano essere la causa della down-regolazione di SLAMF1 nei pazienti con LLC e traslocazioni sbilanciate. Per capire meglio il ruolo che questi miRNAs svolgono nel regolare l’espressione di SLAMF1, è stato eseguito un saggio di luciferasi analizzando i miRNA normalmente over-espressi in casi di LLC, sospettati di essere i responsabili della down-regolazione di SLAMF1, ovvero i miR-17/92, miR132 e miR-148a. I risultati della luciferasi non hanno mostrato differenze nell’espressione di SLAMF1 a seguito dell’azione di questi miRNA. Questo indica che i miRNA non sono responsabili per la down-regolazione di SLAMF1 e che questo meccanismo rimane tutt’ora sconosciuto. Capire i meccanismi molecolari che regolano lo sviluppo della malattia permetterebbe di identificare nuovi target terapeutici. Il CK è un marcatore prognostico negativo, ma la presenza di specifiche anomalie citogenetiche, le traslocazioni sbilanciate, possono modificare il suo valore prognostico. L’espressione di SLAMF1 sulla superficie dei linfociti B può rappresentare un nuovo marcatore prognostico veloce e facile da misurare. Queste scoperte possono avere una grande rilevanza clinica per stratificare i pazienti in categorie di rischio, permettendo di selezionare il trattamento clinico più adatto.
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Fabris, S. "GENOMIC AND EPIGENETIC APPROACHES IN THE CLINICAL AND PROGNOSTIC STRATIFICATION OF CHRONIC LYMPHOCYTIC LEUKEMIA." Doctoral thesis, Università degli Studi di Milano, 2011. http://hdl.handle.net/2434/150561.
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Chronic lymphocytic leukemia (CLL) is characterized by highly clinical heterogeneity; the identification of factors that could predict the clinical course of early-stage CLL represents a crucial objective in this malignancy. The aim of the study is to identify novel biological markers that may be clinically relevant to envisage specific risk subgroups of CLL patients using both genomic (integrated FISH and microarray technology) and an epigenetic approach in highly purified B-cell populations obtained from early-stage CLLs (Binet stage A). The availability of information concerning the follow-up of the analyzed patients allowed the investigation of a potential prognostic significance associated with the biological markers identified. Genome-wide DNA profiling and FISH were used to perform a deletion-mapping analysis of 17p in a subset of 18 CLLs with TP53 deletion and in all the investigated cases, the breakpoints were scattered along the 17p10–p11.2 region. Additionally, gene expression profile (GEP) analysis revealed specific transcriptional patterns and altered molecular pathways associated with 17p aberrations. SNP-array technology and gene expression profiling data were also applied to investigate the 13q14 deletion occurring in a panel of 100 CLLs representative of the major genetics, molecular and biological features of the disease. Based on SNP-array, our study shows the presence of two different molecular groups of patients with del(13)(q14) based on the deletion size and the presence of biallelic deletions. Notably, global gene expression profiling identified a significant transcriptional deregulation specifically associated with the two groups. The genomic complexity detected by SNP-array approach indicates that a relatively high degree of genomic alterations is associated with early-stage CLLs. As regards the genomic changes, the most important and novel finding is the occurrence of 2p gain in a recurrent fraction of early-stage CLLs, which appears to represent an independent prognostic factor for treatment occurrence.
An epigenetic approach was used to investigate global DNA hypomethylation affecting repeated sequences, such as long interspersed nuclear elements-1 (LINE-1), Alu and satellite α DNA (SAT-α DNA), reported to be associated with chromosomal instability. Our analysis was performed in a panel of 77 CLLs and 7 healthy donors using robust quantitative Pyrosequencing methodology to detect the methylation status of the three repetitive elements. For the first time, we reported a significant association between Alu, LINE-1 and SAT-α hypomethylation and the occurrence of the 17p13 deletion; our data also indicate that SAT-α hypomethylation may represent an independent negative prognostic marker significantly correlated with the need of starting treatment. Overall, our data further support: i) the use of microarray technology to characterize well-known lesions for a better prognostic stratification of the disease as well as to investigate genomic changes in CLL, allowing the definition of novel aberrations with pathogenetic and prognostic implications; ii) the use of an epigenetic approach to identify the potential clinical relevance of specific repetitive elements hypomethylation, which may be used as a novel prognostic indicator of unfavorable disease progression.
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Konstandin, Nikola [Verfasser], and Heinrich [Akademischer Betreuer] Leonhardt. "Whole exome sequencing of chronic lymphocytic leukemia (CLL) : extending the number of recurrently mutated genes in CLL and detailed analysis of the putative CLL driver mutations in XPO1 / Nikola Konstandin ; Betreuer: Heinrich Leonhardt." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2015. http://d-nb.info/1137227168/34.
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Abrams, Zachary. "A Translational Bioinformatics Approach to Parsing and Mapping ISCN Karyotypes: A Computational Cytogenetic Analysis of Chronic Lymphocytic Leukemia (CLL)." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461078174.
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Chiodin, Giorgia. "Chronic Lymphocytic Leukemia: analysis of microenvironmental influence on neoplastic clone survival and IgM signaling during Ibrutinib therapy." Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3422771.
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Chronic Lymphocytic Leukemia (CLL) is characterized by the monoclonal expansion of mature CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow and lymphoid tissues. Surface IgM (sIgM) signaling is key to CLL behavior and is a therapeutic target of the BTK-inhibitor Ibrutinib. SIgM levels and signaling capacity are variable in CLL and correlate with the behavior of the disease. In CLL, the microenvironment also plays an important role in disease support and progression. In this thesis two projects are presented: the analysis of the microenvironmental influence on neoplastic clone survival in different in vitro culture conditions, and the study of the effects that Ibrutinib in vivo therapy exerts on sIgM in CLL patients.
Mesenchymal Stromal Cells (MSCs), which represent the major component of the stromal microenvironment, were isolated from the marrow aspirate of CLL patients and co-cultured with leukemic cells. After 7 days, we observed a relevant extended survival of leukemic cells in respect to the B cells cultured alone, and the behavior of the neoplastic clones could be differently dependent on the signals coming from the stromal cells. MSCs were able to counteract the cytotoxic effect of Fludarabine/Cyclophosphamide in vivo administration, confirming the important role played by the microenvironment during therapy. However, the kinase inhibitors Ibrutinib and Bafetinib could induce apoptosis of leukemic cells co-cultured with MSCs, and inhibited CLL B cell CD49d-mediated adhesion and pseudoemperipolesis, suggesting that the new kinase inhibitors are effective in targeting the pro-survival cross-talk between leukemic lymphocytes and stromal cells.
In patients, Ibrutinib treatment induces a rapid redistribution of CLL cells into the blood. In this study, the expression and function of sIgM was analyzed in 12 CLL patients after 1 week of Ibrutinib therapy. At this time point, the expression of sIgM increased significantly (P=0.001), accompanied by full N-glycan maturation of sIgM heavy-chain, indicating recovery from antigen engagement. In addition, the sIgM levels correlated with increased sIgM-mediated SYK phosphorylation. The data suggest that Ibrutinib could prevent antigen encounter, thus favoring sIgM expression and maturation.La leucemia linfatica cronica (LLC) e’ caratterizzata dall’accumulo di linfociti B maturi con fenotipo CD19+/CD5+/CD23+ nel sangue periferico, nel midollo osseo e nei tessuti linfatici. I segnali mediati dalle immunoglobuline M di superficie (sIgM) sono fondamentali per il comportamento dei linfociti di LLC, e sono divenuti target di inibitori chinasici come Ibrutinib. I livelli di sIgM e la capacita’ di mediare segnali intracellulari sono variabili nei cloni tumorali e si associano al comportamento della malattia. Inoltre, anche il microambiente tumorale ricopre un ruolo importante nel supporto e nella progressione della LLC. In questa tesi sono presentati due progetti: l’analisi dell’influenza del microambiente sulla sopravvivenza del clone neoplastico in diverse condizioni di coltura in vitro, e lo studio degli effetti della terapia con Ibrutinib su signalling e funzionalita’ delle sIgM in pazienti di LLC. Nella prima parte dello studio, le cellule mesenchimali stromali (MSCs) sono state isolate da aspirati midollari da pazienti affetti da LLC e sono state poste in co-coltura con cellule B neoplastiche. Dopo 7 giorni di incubazione, abbiamo osservato un rilevante incremento della sopravvivenza delle cellule leucemiche poste in co-coltura con MSCs rispetto alle cellule poste in coltura singola; abbiamo osservato che cloni diversi mostrano comportamento diverso in termini di sopravvivenza, in base alle caratteristiche intrinseche dei cloni stessi. Le MSCs, inoltre, sono in grado di contrastare l’effetto citotossico della terapia Fludarabina/Ciclofosfamide quando somministrata in vivo in pazienti con LLC, a conferma dell’importante ruolo svolto dal microambiente. Tuttavia, i risultati ottenuti hanno mostrato che gli inibitori chinasici Ibrutinib e Bafetinib sono invece in grado di indurre apoptosi nelle cellule tumorali anche in presenza di MSCs e di inibirne l’adesione mediata da CD49d e la pseudemperipolesi, suggerendo che gli inibitori del signalling del BCR sono efficaci nel bloccare il cross-talk tra linfociti neoplastici e cellule stromali. Nei pazienti affetti da LLC il trattamento con Ibrutinib induce una rapida ridistribuzione delle cellule tumorali nel sangue. In questo studio, l’espressione e la funzione delle sIgM e’ stata analizzata in 12 pazienti con LLC dopo 1 settimana di terapia con Ibrutinib. A questo time point, l’espressione di IgM sulla superficie delle cellule neoplastiche e’ risultata significativamente aumentata (P=0.001); allo stesso tempo abbiamo anche osservato un aumento della forma matura della catena pesante delle sIgM, indicativa di un mancato incontro con l’antigene. Inoltre, i risultati ottenuti hanno mostrato una correlazione tra l’incremento dei livelli di sIgM e l’aumentata fosforilazione di SYK mediata da IgM. I dati suggeriscono che Ibrutinib potrebbe prevenire l’incontro con l’antigene, favorendo quindi espressione e maturazione delle sIgM nelle cellule di LLC.
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Orlandi, Veronica. "Effects of p66shc expression on bioenergetics and cell viability of b-cell chronic lymphocytic leukemia." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3423953.
Full textAbstract:
During tumorigenesis many oncogenic pathways are reprogrammed and modulate mitochondrial metabolism, decreasing oxidative phosphorylation (OXPHOS) activity. They inhibit ATP synthesis and ROS production from OXPHOS complexes favoring metabolic switching toward a more glycolytic metabolism and tumor growth. This metabolic rewiring is well established in solid tumor but very little is known about this event in non-solid tumors such as leukemias. Indeed, it has been demonstrated that in chronic lymphocytic leukemia (B-CLL), cells show a high OXPHOS activity and elevated ROS levels. Notably, expression of the p66Shc protein, whose mitochondrial fraction modulates cell bioenergetics and increases ROS generation, is lost during B-CLL tumorigenesis. I investigate the effects of the expression of p66Shc on bioenergetics and viability of B-CLL cells. After p66Shc expression in MEC-1 cells, a B-CLL cell model, a fraction of it is in the intermembrane space of mitochondria. p66Shc expression decreases both basal and maximal respiration, decreases mitochondrial membrane potential, total ATP levels and renders cells less dependent from OXPHOS to ATP supply without changes mitochondrial mass. I propose that decreased mitochondrial respiration is due to decreased activity of respiratory complex I and II. In more detail, there is a decrease in complex I assembly with a consequent inhibition of its activity, and a decrease in complex II activity caused by a multi-protein complex in which TRAP1 and active ERK are involved. p66Shc expression in MEC-1 cells increases mitochondrial ERK activation. The mitochondrial fraction of ERK contribute to tumor cell survival by inhibiting opening of the permeability transition pore (PTP), a mitochondrial channel whose opening irreversibly commits cells to death. I check the effect of p66Shc expression on cell viability. Although its pro-apoptotic function well described in many cell models, in MEC-1 cells p66shc protects cells from death induced by mitochondrial-derived oxidative stress derived from starvation, EM20-25 and CisPlatin treatments. In depletion conditions, p66Shc expression correlates with more active mitochondrial ERK and the inhibition of ERK decreases cell viability. Inhibition of mitochondrial chaperones activity with Cyclosporine A, an inhibitor of CyP-D, or 17AAG, an inhibitor of TRAP1, also affects cell viability under depletion condition, thus supporting the hypothesis that mitochondrial ERK is involved in survival mechanism induced by p66Shc expression.
Taken together these data indicate that regulation of RC complex activity can be linked to regulation of cell survival. We hypothesize that the mitochondrial branch of the Ras-ERK signaling pathway regulates mitochondrial bioenergetics by affecting SDH activity and resistance to apoptosis of B-CLL cells. Through the activation of mitochondrial ERK and inhibition of SDH activity, p66Shc can decrease OXPHOS activity and ROS production, a condition that is necessary in the early stages of tumorigenesis. It can also support tumour cell growth making mPTP less sensitive to opening by activating mitochondrial ERK.Durante lo sviluppo tumorale, molte vie di segnale delle cellule vengono riprogrammate cambiando il metabolismo mitocondriale e diminuendo l’attività della fosforilazione ossidativa. In questa maniera viene diminuita la produzione di ATP e di ROS da parte della fosforilazione ossidativa favorendo un metabolismo glicolitico e la crescita tumorale. I meccanismi alla base dello sviluppo di questo fenotipo sono ben caratterizzati nelle cellule di tumori solidi, ma poco si sa a riguardo delle leucemie. Nella leucemia linfocitica cronica, le cellule presentano elevata attività OXPHOS e alti livelli di ROS. È noto che la proteina p66Shc, di cui una frazione si trova nello spazio intermembrana del mitocondrio, modula la bioenergetica cellulare e induce la produzione di ROS. Durante il processo di tumorigenesi della B-CLL l’espressione di p66Shc viene persa. Quindi, sono andata ad analizzare l’effetto della espressione di p66Shc sulla bioenergetica e la vitalità cellulare della leucemia linfocitica cronica. Dopo l’espressione di p66Shc nel modello cellulare di leucemia linfocitica cronica di tipo B, MEC-1, una frazione della proteina è stata trovata nello spazio intermembrana dei mitocondri. L’espressione di p66Shc nelle cellule diminuisce la respirazione mitocondriale, massima e basale, ma anche il potenziale di membrana e i livelli totali di ATP. Ho proposto che queste differenze sono dovute alla diminuzione dell’attività dei complessi respiratori: complesso I e complesso II indotta dalla espressione di p66Shc nelle cellule MEC-1. In particolare, il complesso I è meno assemblato con la conseguente diminuzione della sua attività mentre il complesso II diminuisce la sua attività a causa di un complesso multi proteico in cui sono coinvolti la proteina chinasi ERK e lo sciaperone mitocondriale TRAP1. L’espressione di p66Shc nelle MEC-1 aumenta l’attivazione della frazione mitocondriale di ERK. Questa frazione, contribuisce alla sopravvivenza delle cellule tumorali inibendo l’apertura del poro di transizione di permeabilità (mPTP), un canale mitocondriale la cui apertura porta alla morte cellulare. Ho quindi analizzato l’effetto dell’espressione di p66Shc sulla vitalità cellulare delle MEC-1. Sebbene, p66Shc ha una attività pro-apoptotica, ho osservato che la sua espressione nelle MEC-1 protegge le cellule dalla morte cellulare indotta dallo stress ossidativo prodotto dal mitocondrio da trattamenti come EM20-25, CisPlatino e assenza di glucosio. In assenza di glucosio, l’espressione di p66Shc correla con una maggiore attivazione di ERK nel mitocondrio. L’inibizione di ERK diminuisce la vitalità cellulare. In assenza di glucosio, anche l’inibizione degli sciaperoni mitocondriali CyP-D e TRAP1 influenzano la vitalità cellulare. Quindi, la frazione mitocondriale di ERK è coinvolta nella regolazione delle vie di segnale che proteggono le cellule dalla morte cellulare dopo l’espressione di p66SHhc. Questi dati, indicano che la regolazione dell’attività dei complessi respiratori è legata alla regolazione della sopravvivenza cellulare. Abbiamo ipotizzato che nella leucemia linfocitica cronica la parte mitocondriale della via di segnale RAS-ERK può regolare la bioenergetica influenzando l’attività enzimatica del complesso II e la resistenza alla morte cellulare. Inoltre, abbiamo ipotizzato anche una nuova funzione per p66Shc in cui, attraverso l’attivazione di ERK e l’inibizione dell’attività del complesso II può diminuire l’attività della fosforilazione ossidativa e i livelli di ROS, una condizione necessaria nelle prime fasi della tumori genesi. p66Shc può anche sostenere la crescita tumorale rendendo mPTP meno sensibile all’apertura attraverso l’attivazione della frazione mitochondrial di ERK.
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CARDILLO, MARTINA. "Expression of IL12 and IL23 receptors and cytokines in Chronic Lymphocytic Leukemia and normal B cells." Doctoral thesis, Università degli studi di Genova, 2021. http://hdl.handle.net/11567/1044948.
Full textAbstract:
The mechanisms of clonal expansion of CLL are only partially understood. Several interactions of neoplastic cells with accessory cells and cytokines potentially sustaining neoplastic B cell clone survival and proliferation have been described. Recently, a paracrine/autocrine loop has been reported, involving the upregulation of the IL23R complex and IL23 secretion by CLL cells. This loop drives CLL cell clonal expansion in vitro and in xenografted NSG mice. Furthermore, in situ observations on tissue sections demonstrate that infiltrating IL23 secreting CLL cells interact with macrophages and CD40L expressing T cells. Although inducible in vitro by co-culturing CLL cells with T cells or CD40L expressing cells, the IL23 loop is not observed following stimulation of CLL cells via surface Ig or contact with nurse like cells or bone marrow stromal cells.
In this study, we investigated whether the IL23 loop could be induced following Toll-like receptor 9 (TLR9) engagement which influences leukemic cell survival, activation proliferation albeit in a heterogeneous manner. In addition, we explored the possible existence of an autocrine/paracrine loop mediated by IL12 which shares similarities and surface receptors with IL23 although with a likely opposite outcome in term of the possibility to sustain leukemic cell growth .
IL23R and IL12R complexes (IL23R/IL12Rβ1, IL12β2/IL12Rβ1) expression were evaluated by flow-cytometry following stimulation with CpG oligodeoxynucleotide (ODN) that binds the TLR9 on CLL, showing that CLL cells are able to express the IL23R complex on membrane and, at lower extent, the IL12R complex.
These receptors were assessed also in normal B cells by flow cytometry after 72h of stimulation with CpG and CpG+IL15. In this setting, normal B cells were less capable of IL23R complex expression compared to CLL cells. A further striking difference observed was related to the limited expression of IL12Rß2 receptor chain in stimulated CLL cells compared to normal B cells.
Supernatants of CLL cells and normal B cells were both tested for the production of these cytokines after stimulation. The results showed a low level of IL23p19 secretion for both CLL cells and normal B cells, which is significant after CD40L stimulation (used as positive control), and a higher production of IL12p70 which is more pronounced in normal B cells compared to CLL. In another series of tests, CLL cells were stimulated with CpG for 72h, and subsequently exposed to IL12 or IL23. Exposure to IL12 and IL23 induced the expression of pSTAT1 and pSTAT3. Collectively our data corroborate the notion that IL23R complex act as a pro-survival factor for CLL cells. In contrast, the restricted IL12R complex expression in CLL cells compared to normal B cells indicated that the suppression of the expression of this receptor may favor the survival of the leukemic clones. The possibility of a reciprocal competition of the shared receptor chains is discussed.
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Hellqvist, Eva. "Antigen interaction with B cells in two proliferative disorders : CLL and MGUS." Doctoral thesis, Linköping : Department of Clinical and Experimental Medicine, Linköping University, 2010. http://www2.bibl.liu.se/liupubl/disp/disp2010/med1158s.pdf.
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Kodipad, Ahad Ahmed. "XPO1 mutations are a novel predictor of shorter time to first treatment in early stage CLL patients." Doctoral thesis, Università del Piemonte Orientale, 2021. http://hdl.handle.net/11579/128430.
Full textAbstract:
In approximately 70% of newly diagnosed cases, CLL presents at an early clinical stage and is managed with a watch & wait strategy. Until now, few clinical and molecular inform on the risk of treatment requirement. On these grounds, we aimed at identifying new molecular markers that may predict early treatment requirement and may help clinicians to better plan the watch & wait strategy in asymptomatic early stage CLL patients. 295 Binet A CLL patients who referred to our institution were subjected to next-generation-sequencing (NGS) in a panel of recurrently mutated genes in CLL. Two validation multicenter cohorts of 402 treatment naïve Binet A CLL patients (Binet A validation cohort) and 395 untreated Rai 0 CLL patients (Rai 0 validation cohort) were also included and analyzed for XP01 mutations. The primary endpoint was time to first treatment (TTFT). In the training cohort, NGS mutational analysis showed that XP01 was mutated in 7 (2.4%) patients. In univariate analysis, trisomy 12 (p=0.001), unmutated IGHV genes (p<0.0001) and mutations of XP01 (p<0.0001), NOTCHI (p<0.001) and SF3B1 (p=0.022) were associated with a shorter TTFT. By multivariate analysis, XPOI mutations (p=0.002) and unmutated IGHV genes (p<0.0001) maintained an independent association with a shorter TTFT. XPO1 mutational analysis was subsequently investigated in 2 independent multicenter cohorts of early stage CLL
patients. In the Binet A validation cohort (N=402 patients), XPO1 was mutated in 15 (3.7%) patients and was associated with a shorter TTFT (p=0.004). Similarly, also in the Rai 0 validation cohort, (N=395 patients), XP01 was mutated in 8 (2.0%) patients and was associated with a shorter TTFT (p<0.001). By combining the training and the validation cohorts (N=1092 patients), a total of 30 somatically acquired XP01 mutations were identified (2.7% of patients). More precisely, 27
(90.0%) mutations affected XP01 codon E571 and 3 (10.0%) codon D624. From a clinical perspective, patients carrying either XPO1 E571 or D624 mutations showed superimposable outcome in terms of TTFT (p=0.345). Based on these results, XP01 mutational analysis might be incorporated in other prognostic scores and help clinicians to refine the management of the watch and wait strategy for early stage CLL.
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Adhinaveni, Ramesh. "Molecular analysis of different clinical presentations of chronic lymphocytic leukemia reveals novel molecular predictors and molecular heterogenety of different anatomical compartments." Doctoral thesis, Università del Piemonte Orientale, 2022. http://hdl.handle.net/11579/144065.
Full textAbstract:
Chronic lymphocytic leukemia (CLL) and Small lymphocytic lymphoma (SLL) are different manifestation of the same lymphoproliferative disease. Our study aims at evaluating molecular markers that predispose to chemorefractoriness in CLL and to identify molecular landscape of the different anatomical compartments of SLL. Fludarabine, cyclophosphamide, and rituximab (FCR) is the most effective chemoimmunotherapy regimen for young and fit CLL patients devoid of TP53 disruption. A cohort of 287 patients receiving first-line FCR was analyzed by next generation sequencing (NGS). By univariate analysis, BIRC3 mutations identify a poor prognostic subgroup of patients failing FCR (p<0.001) as cases harboring TP53 mutations (p<0.001). BIRC3 mutations maintained an independent association with an increased risk of progression (p=0.004) in multivariate analysis. By immunoblotting analysis, we showed that the non-canonical NF-KB pathway is active in BIRC3 mutated cell lines and in primary CLL samples. In vitro results indicate that BIRC3 mutated primary CLL cells are less sensitive to fludarabine. BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel drugs. Regarding SLL, we investigated 12 SLL patients, provided with: cell free DNA (CfDNA) from plasma, genomic DNA (gDNA) from LNF biopsies, gDNA from CD19+ cells and from CD3+ cells, by using NGS. By comparing mutations identified, SLL genotyping on the cfDNA does not recapitulate SLL genetics and lacks 65.2% identified in the PB CD19+ cells and in the LNF. By considering the 44 mutations identified in the LNF and in the PB CD19+ cells, 20.4% of mutations were unique to the LNF biopsy, 36.4% were unique to the PB CD19+ cells and only 43.2% were shared. The analysis of the LNF only or of the PB CD19+ cell only may miss mutations with potential clinical relevance, suggesting that both these two compartments should be tested to have a comprehensive view of the SLL genetics relevance.
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Pagano, Mario Angelo Primo. "Dismantling the aberrant signaling network in chronic lymphocytic leukemia: PP2A and SHP-1 as promising targets for drug discovery." Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3426201.
Full textAbstract:
Reversible protein phosphorylation is a fundamental post-translational modification by which virtually all cellular events are regulated, enabling cells to properly respond to intra- and extracellular cues. Protein kinases and protein phosphatases are the principal factors involved in this in this dynamic process and are placed at the different levels of cellular signalling, and, albeit traditionally considered as functionally opposed to one another, not rarely act in an interplay to finely orchestrate and appropriately drive signal transduction. The imbalance of their expression and function affects the cell life and fate, which frequently underlies the onset and progression of a plethora of diseases. B cell chronic lymphocytic leukemia (CLL), the most common leukemia in the Western world, is no exception to this paradigm, many studies having highlighted a crucial role of kinases in the sustained signals from the signalosome downstream of the B cell receptor (BCR), and having led to the development of the promising second-line drugs, but also the blockade of a number of phosphatases underlying pro-survival and anti-apoptotic signals. In this regard, Protein Phosphatase 2A (PP2A) and the Src homology 2 domain-containing phosphatase 1 (SHP-1) exhibit a marked functional inhibition in this disease, which can be properly circumvented by a pharmacological approach, thereby inducing apoptosis of cancer cells. Nintedanib and MP-0766, a drug acting as an angiokinase inhibitor and a new fingolimod analogue devoid of immunosuppressive action, activating respectively SHP-1 and PP2A have enabled for the discovery of a signalling axis that when activated provokes massive cell death, and might provide a new paradigm for the treatment of CLL, which now endorses kinase inhibitors.La fosforilazione proteica è una fondamentale modificazione post-traduzionale che regola virtualmente tutti i processi cellulari, permettendo alla cellula di rispondere a stimoli intra- ed extracellulari. Le protein chinasi e le protein fosfatasi sono i fattori principali coinvolti in questo processo dinamico e si localizzano a diversi livelli del signaling cellulare, e, sebbene tradizionalmente considerate opposte le une alle altre sotto il profilo funzionale, non raramente compartecipano per finemente modulare e opportunamente dirigere la trasduzione del segnale. Uno squilibrio di espressione e/o funzione di questi fattori si riflette sulla vita e il destino della cellula, cosa che frequentemente è alla base dell’insorgenza nocnhé l’evoluzione di un gran numenro di patologie. La leucemia linfatica cronica a cellule B (B Chronic Lymphocytic Leukemia, CLL), la più comune leucemia in occidente, non fa eccezione a tale paradigma e, sebbene la ricerca per lo più si è concentrata sull’anomala attività di diverse protein chinasi con lo sviluppo di promettenti farmaci di seconda linea, sempre più di frequente viene confermata l’ipotesi che la sopravvivenza e la resistenza all’apoptosis delle cellule tumorali dipende anche dalla ridotta espressione o funzionalità delle protein fosfatasi. A questo riguardo, la protein fosfatasi 2A (Protein Phosphatase 2A, PP2A) e la fosfatasi 1 contenente domini Src homology 2 (Src Homology 2 domain-containing phosphatase 1 (SHP-1) in questa patologia si dimostrano funzionalmente inibiti, ma che, quando opportunamente attivate farmacologicamente, inducono morte delle cellule tumorali. Nintedanib, un farmaco che agisce come inibitore 'angiochinasico”, e MP-0766, un nuovo analogo del fingolimod privo di azione immunosoppressiva, si sono dimostrati in grado di attivare SHP-1 e PP2A rispettivamente, permettendo inoltre di individuare un asse di signaling cellulare che provoca la morte di celle cellule leucemiche, potenzialmente rappresentando un nuovo paradigma per il trattamento della CLL, che ad oggi privilegia gli inibitori chinasici.
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CASTELLI, MONICA. "INCREASED SURVIVAL OF CLL B CELLS IN THE PRESENCE OF MARROW MESENCHYMAL STROMAL CELLS: A NOVEL MODEL TO DEFINE NEW TARGETS FOR THERAPY." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424459.
Full textAbstract:
Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western World, accounting for about 30% of adult leukemia, and it is characterized by the clonal expansion and accumulation of mature CD19+/CD5+/CD23+ B lymphocytes in the peripheral blood, bone marrow and secondary lymphoid organs. Despite their apparent longevity in patients, in vitro CLL leukemic B cells rapidly undergo spontaneous apoptosis. The selective survival advantage is due both to intrinsic defects on apoptosis mechanism and to signals delivered by accessory cells at the active site of the disease. Previous studies demonstrated that mesenchymal stromal cells (MSCs), derived from bone marrow, and CD68+ nurse-like cells, derived from peripheral blood, are involved in CLL clone longevity and migration, suggesting a crucial role of MSCs on favouring disease progression.
Therefore, in this thesis we evaluated the effect of MSCs, the main stromal population in the bone marrow of CLL patients, on the survival of leukemic B cells and their role in drug resistance.
MSCs were isolated from the bone marrow of 46 CLL patients; their immunophenotypic characterization was based on the expression of CD105, CD73 and CD90 and the negativity of CD14, CD34, CD45 and CD31. Co-culturing MSCs and CLL B cells, we confirmed that MSCs are able to support malignant B cell survival, providing an in vitro culture system that closely approximates CLL microenvironment in vivo. We observed that different leukemic clones demonstrated a large variety in the pro-survival effect. Evaluating the cleavage pattern of PARP, we revealed two subsets of CLL clones with different sensitivity to MSCs pro-survival signals. Our results indicate that both cell-cell contact and soluble molecules are actors in the relationship between malignant B cells and the MSCs, promoting CLL B cell survival and migration.
Later, we evaluated the role of the MSCs on CLL B cells during the most common cytotoxic therapy used in clinical practice. Our data demonstrate that MSCs are able to protect leukemic B cells from apoptosis during Fludarabine and Cyclophosphamide treatment, both in vitro and in vivo. We tested MSCs protective role also during CLL B cells treatment with Ibrutinib, a novel inhibitor of Btk involved in the BCR signaling pathway, and we found that the treatment counteracts the MSC pro-survival effect. To better understand the effect of Ibrutinib on the cross-talk between CLL B cells and MSCs, we evaluated its role on leukemic B cell migration, also analyzing the expression levels of CCR7 and CXCR4, two chemokine receptors that are central in the homing of the neoplastic clone. We demonstrated that malignant B cell migration is not significantly affected by the Btk inhibitor; since cell-cell contact with MSC is crucial for CLL B cell survival, we analyzed the adhesion of leukemic B cells to MSCs after treatment with Ibrutinib. We found a significant reduction in leukemic B cells and MSCs interactions mediated by the CD49d integrin.
In this thesis, we demonstrate that MSCs enhance the survival of leukemic B cells through the release of soluble factors and cell-cell direct contact and that each CLL clone reveals a peculiar response to the anti-apoptotic signals delivered by MSCs. These observations could be relevant to identify patients more responsive to druggable targets on marrow microenvironment and also to find putative new strategies for CLL therapy. A better understanding on the complexity of the cross-talk between CLL cells and their microenvironment during CLL therapy could also help to define mechanisms of drug resistance and treatment failure, as well to plan randomized clinical trials comparing new compounds and their combinations with standard chemo-immunotherapy.La Leucemia Linfatica Cronica (LLC) è considerate la più comune leucemia del mondo occidentale, rappresentando circa il 30% delle leucemie dell’adulto, ed è caratterizzata dalla proliferazione clonale e dall’accumulo nel sangue periferico, nel midollo osseo e negli organi linfatici secondari di linfociti B maturi CD19+/CD5+/CD23+. Nonostante i linfociti B leucemici mostrino un’aumentata sopravvivenza nei pazienti affetti da LLC, in vitro vanno rapidamente incontro ad apoptosi. Il vantaggio sulla sopravvivenza è legato sia a difetti intrinseci del meccanismo di apoptosi sia a segnali forniti da cellule accessorie, presenti nel sito attivo della malattia. Precedenti studi hanno dimostrato che le cellule mesenchimali stromali (MSC) e le cellule accessorie (“nurse-like”) derivate rispettivamente dal midollo osseo e dal sangue periferico, sono coinvolte nell’aumentata longevità e mobilità del clone leucemico, suggerendo un ruolo cruciale delle MSC nel favorire la progressione della malattia. In questa tesi abbiamo valutato l’effetto delle MSC, la principale popolazione stromale nel midollo osseo dei pazienti affetti da LLC, sulla sopravvivenza dei linfociti B neoplastici e il loro ruolo sulla resistenza ai farmaci. Le MSC sono state isolate da campioni di sangue midollare provenienti da 46 pazienti affetti da LLC; la loro caratterizzazione immunofenotipica è stata effettuata sulla base dell’espressione di CD105, CD73 e CD90 e sulla negatività di CD14, CD34, CD45 e CD31. Allestendo co-colture di MSC e linfociti B leucemici, abbiamo confermato la capacità delle MSC di incrementare la sopravvivenza delle cellule B neoplastiche, fornendo un sistema di coltura in vitro che mima profondamente il microambiente della LLC in vivo. Abbiamo osservato una grande varietà sulla vitalità dimostrata dai diversi cloni leucemici e, mediante la valutazione del frammento clivato della proteina PARP, abbiamo identificato due differenti gruppi di cloni di LLC, con una diversa sensibilità ai segnali di stimolo provenienti dalle MSC. I nostri risultati indicano che sia il diretto contatto cellula-cellula che la presenza di molecole solubili sono coinvolte nell’interazione tra le cellule B leucemiche e le MSC, promuovendo la sopravvivenza e la migrazione della cellula B leucemica. Successivamente, abbiamo valutato l’effetto delle MSC sui linfociti B neoplastici durante un trattamento chemioterapico di uso comune nella pratica clinica. I nostri dati hanno dimostrato che le MSC sono in grado di proteggere le cellule B leucemiche dall’apoptosi durante il trattamento con Fludarabina e Ciclofosfamide, sia in vitro che in vivo. Abbiamo esaminato il ruolo protettivo delle MSC anche durante il trattamento dei linfociti neoplastici con Ibrutinib, un nuovo inibitore della Btk, una chinasi coinvolta nella cascata del segnale del BCR, e abbiamo dimostrato che il trattamento delle cellule B con Ibrutinib è in grado di contrastare l’effetto anti-apoptotico delle MSC. Per meglio definire l’azione di Ibrutinib nell’interazione tra le cellule B di LLC e le MSC, abbiamo valutato il suo ruolo sulla mobilità dei linfociti B leucemici, analizzando inoltre i livelli di espressione di CCR7 e CXCR4, due recettori chemiochinici fondamentali nella migrazione del clone neoplastico. Abbiamo dimostrato che la migrazione delle cellule B neoplastiche non è significativamente influenzata dall’inibitore del Btk; inoltre, considerando che il diretto contatto cellula-cellula con le MSC è di notevole importanza per la sopravvivenza dei linfociti B leucemici, abbiamo analizzato l’adesione delle cellule B alle MSC dopo il trattamento con Ibrutinib, evidenziando che la loro adesione era significativamente ridotta. In questa tesi abbiamo dimostrato che le MSC incrementano la sopravvivenza delle cellule B neoplastiche attraverso il rilascio di fattori solubili e mediante il diretto contatto cellula-cellula, e che ogni clone leucemico rivela una peculiare risposta ai segnali anti-apoptotici rilasciati dalle MSC. Queste osservazioni potrebbero essere determinanti al fine di identificare i pazienti più sensibili a trattamenti mirati a colpire il microambiente midollare ed a trovare potenziali nuove strategie terapeutiche per la LLC. Una migliore comprensione della complessità delle interazioni tra i linfociti leucemici e il loro microambiente nel corso del trattamento potrà inoltre aiutare a chiarire i meccanismi di chemioresistenza e refrattarietà, così come a pianificare studi clinici randomizzati che confrontino nuovi farmaci e la loro combinazione con i trattamenti chemio-immunoterapici già in uso.
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Veronese, Lauren. "Leucémie lymphoïde chronique : étude des marqueurs du pronostic et de l'instabilité génomique." Thesis, Clermont-Ferrand 1, 2013. http://www.theses.fr/2013CLF1MM09/document.
Full textAbstract:
La leucémie lymphoïde chronique (LLC), hémopathie lymphoïde fréquente, se caractérise par une évolution clinique extrêmement variable. Bien que les marqueurs de pronostic soient nombreux dans la LLC, aucun n'est univoque. Dans ce contexte, identifier de nouveaux facteurs prédictifs et comprendre la pathophysiologie de marqueurs pronostiques déjà établis constituent deux objectifs importants pour améliorer la prise en chargethérapeutique de cette hémopathie. Nous avons tout d'abord choisi d'étudier la valeur pronostique et les mécanismes de régulation de l'expression du gène anti-apoptotique MCL1. Nous avons montré que l'expression de MCL1 est un marqueur prédictif de la survie globale parmi l'ensemble despatients et parmi les stades précoces ; ce marqueur est également prédictif de la survie sans traitement des patients en stade A. Ainsi, l'expression de MCL1 permet d'identifier précocement les formes de LLC à haut risque et faible risque d'évolution défavorable. Nous avons également démontré que l'expression de MCL1 est fortement corrélée à l'expression de VEGF, confirmant le rôle de cette voie de signalisation dans la survie des lymphocytes tumoraux et suggérant que VEGF pourrait réguler positivement l'expression de MCL1 selon un mode autocrine. Nous avons ensuite exploré la fonction télomérique en rapport avec les anomalies chromosomiques à valeur pronostique, reflets de l'instabilité génomique. Notre travail a contribué à démontrer la relation entre l'instabilité génomique et le statut télomérique, évalué par la longueur des télomères et l'expression de hTERT et des gènes du complexe shelterin. Nous avons ainsi mis en évidence trois groupes de patients présentant des profilscytogénétiques et télomériques distincts : le premier groupe combine une cytogénétique favorable, des télomères longs, une expression faible ou absente de hTERT et une expression forte des gènes du complexe shelterin ; le troisième groupe se caractérise par de multiples aberrations chromosomiques (notamment délétions 17p et 11q), une augmentation de l'expression de hTERT et une diminution de la longueur des télomères et des niveaux d'expression de TRF1, TRF2 et POT1 ; le deuxième groupe est intermédiaire. Ces résultats confirment l'existence d'un lien entre statut télomérique et instabilité génomique au cours de la LLC et soulignent le rôle de la perte de TP53 ou ATM dans cette dysfonction télomérique. L'altération du statut télomérique est par ailleurs associée à des caractéristiques de pronostic défavorable, comme l'absence de mutation des IgVH, l’expression de CD38 et le doublement rapide de la lymphocytose. Enfin, nous avons évalué l’intérêt de la technique de MLPA pour la mise en évidence des anomalies cytogénétiques récurrentes à valeur pronostique de la LLC. Nous avons montré qu'il existe une bonne concordance entre la technique de référence et la MLPA, qui constitue une approche rapide et peu coûteuse pour la recherche d'anomalies génomiques présentes dans une majorité de cellules malignes. Nous avons cependant mis en évidence des cas intéressants de faux-positifs et de faux-négatifs avec la MLPA, indiquant que cette méthode ne peut pas remplacer les techniques classiques, mais constitue une approche complémentaire permettant une évaluation simultanée de divers déséquilibresChronic lymphocytic leukemia (CLL) is a frequent lymphoid hemopathy characterized by an extremely variable clinical course. Although there are numerous prognostic markers in CLL, none is univocal. In this context, identifying new predictive factors and understanding the pathophysiology of previously established prognostic markers represent two important aims to improve therapeutic management of this hemopathy. We first chose to study the prognostic value and mechanisms of regulation of antiapoptotic MCL1 gene expression. We showed that MCL1 expression is a predictive marker of overall survival within the whole patient cohort and among early stages; this marker is also a predictor of treatment free survival of stage A patients. Thus, MCL1 expression allows early identification of CLL forms with high risk and low risk of unfavourable evolution. We alsodemonstrated that MCL1 expression is strongly correlated to VEGF expression, confirming the role of this signalling pathway in tumour lymphocytes survival and suggesting that VEGF may be a positive autocrine regulator of MCL1 expression. We then explored telomeric function regarding prognosis-related chromosomal anomalies, reflecting genomic instability. Our work contributed to demonstrate the relationship between genomic instability and telomeric status, evaluated by telomere length and expression of hTERT and shelterin complex genes. We described three groups of patients with distinct cytogenetic and telomeric profile: first group combines good-prognosis cytogenetics, long telomeres, low or negative hTERT expression and high expression of the shelterin complex genes; third group displays multiple chromosome aberrations (particularly 17p and 11q deletions), increased hTERT expression and decreased telomere length and TRF1, TRF2 and POT1 expression levels; second group is intermediate. These results confirm the relationship between telomeric status and genomic instability in CLL and underline the role of TP53 or ATM loss in this telomeric dysfunction. The alteration of telomeric status is also associated with poor-prognosis features, such as unmutated IgVH, CD38 expression and rapid lymphocytosis doubling time. Finally, we evaluated the contribution of MLPA approach for detection of recurrent prognosis-related cytogenetic anomalies. We found a good concordance between the goldstandard technique and MLPA, which represent a time and cost-effective approach for the detection of genomic aberrations affecting most malignant cells. We however described interesting MLPA false-positive and false-negative cases, indicating that this method may not replace classic techniques, but may constitute a complementary approach allowingsimultaneous evaluation of various imbalances
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PUGLISI, FRANCESCA. "Studio dei fattori responsabili dell’alterazione del metabolismo osseo nella Leucemia Linfatica Cronica: specifico ruolo di HGF nel microambiente stromale." Doctoral thesis, Università degli studi di Genova, 2020. http://hdl.handle.net/11567/1005358.
Full textAbstract:
Chronic Lymphatic Leukemia (CLL) is the most common leukemia in the Western world and is characterized by the proliferation and accumulation of mature CD5 + B lymphocytes in peripheral blood, lymph nodes and bone marrow. CLL leukemic cells in vitro don’t survive, undergoing apoptosis, preventing the availability of reliable experimental models. On the contrary, in vivo it is precisely long-term survival in the lymph node and bone marrow districts that cause disease relapses and make the disease difficult to eradicate. One of the consequences of this accumulation and disease progression is an alteration of the trabecular structure of the bone, highlighted by tomographic analyzes in patients at intermediate and advanced stages of the pathology. These premises lead to the hypothesis that the medullary microenvironment, and in particular its figurative component of the Bone Marrow Stromal Cells (BMSC), through cell-cell contacts and secreted cell signals, is able to allow and promote the survival of leukemic clones; on the other hand, leukemia cells seem to influence the surrounding environment, thus altering bone metabolism and its normal homeostasis. Thus, a cross-exchange of signals appears to occur between the leukemic cells and the cells of the medullary niche, with substantial changes in both directions. The aim of this project was to investigate the nature of this cross-talk, in particular on the one hand by investigating the role of the Hepatocyte Growth Factor (HGF) and its regulation in the survival of the CLL cell, on the other by verifying through which mechanisms and signals the leukemic clone is able to alter bone metabolism. Overall, the data collected so far lead to indicate how the bone erosion and the weakening of the trabecular structure, present in the intermediate and advanced stages of CLL, can be attributed to an alteration of the balance, present in physiological conditions, between the bone formation and reabsorption. This seems to be due to a decrease in the number of stromal cells that differentiates into osteoblasts, with a consequent decrease in the deposition of ECM, and to a simultaneous increase in the number of monocytes that differentiates into osteoclasts, with a consequent increase in the reabsorption of the bone matrix itself. Both of these phenomena seem to be in close correlation with the proximity of CLL B cells present in the bone marrow, which exert their influence on stromal cells and monocytes through the release of some cytokines, among which they seem to play a main role IL-11, IL -6 and especially TNFα. In investigating the role played by HGF in the survival of the leukemic clone, however, the studies carried out so far have identified the following possible TFs of interest on the promoter of the HGF gene: PPARγ, Ikaros, ERα and AP-2α. By then using lysate samples from cell lines having different HGF expression, it was possible to verify, through the use of Chromatin Immunoprecipitation, that TF Ikaros doesn’t seem to be involved in gene regulation.
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Fernandes, Margareth. ""Expressão de Zap-70 e CD38 em leucemia linfocítica crônica (LLC) e sua correlação com prognóstico"." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/5/5136/tde-30052006-160411/.
Full textAbstract:
Atualmente, a Leucemia Linfocítica Crônica (LLC) pode ser dividida em dois grupos: um com mutações somáticas no gene da região variável da cadeia pesada da imunoglobulina (MIgVH) e outro sem mutações (NMIgVH). Alguns estudos mostraram que a expressão de CD38 na superfície das células B de LLC pode estar correlacionada com o estado mutacional do gene VHIg, entretanto, esses controversos. Estudos recentes mostraram que a expressão da proteína tirosina quinase Zap-70 está melhor associada com o estado mutacional do gene IgVH. O objetivo deste estudo foi avaliar a expressão de Zap-70 e CD38, por citometria de fluxo, nas células CD19+ de pacientes com LLC e correlacioná-los com o estádio clínico (EC), sobrevida livre de tratamento (SLT) e sobrevida global (SG). A expressão de Zap-70 e CD38 foi avaliada, em 144 de pacientes com LLC classificados nos estádios clínicos A, B e C de acordo com os critérios de Binet: 59 (41%) do EC-A, 38 (26%) do EC-B e 47 (33%) do EC-C. Foi observada menor positividade para Zap-70 e CD38 nos pacientes do EC-A do que nos EC-B e C. Quando avaliada a SLT nos pacientes do EC-A, os casos Zap-70+ assim como os CD38+ apresentaram menor SLT. A média de SG dos pacientes Zap-70+ e CD38+ foi menor quando comparado com os Zap-70- e CD38- entretanto quando correlacionada com o EC não foi observada diferença estatisticamente significante entre a expressão desses marcadores e o EC-A, B ou C. Pela analise combinada de CD38 e Zap-70, dividimos os pacientes em dois grupos (Zap-70-/CD38- e Zap-70+ ou CD38+). Observamos que a expressão positiva desses dois marcadores estava associada ao EC, uma vez que a grande maioria dos pacientes dos estádios B (74%) e C (66%) expressam Zap-70 ou CD38. Entretanto, os pacientes do EC-A, Zap-70+ ou CD38+, apresentaram SG menor quando comparado com os Zap-70-/CD38-. Essa diferença não foi observada nos pacientes do EC-B e do EC-C. Também foi observada menor SLT nos pacientes no EC-A, Zap-70+ ou CD38+. Esses resultados sugerem que análise combinada de Zap-70 e CD38 podem ser empregadas na avaliação dos pacientes do EC-A para se acompanhar a evolução clinica desse grupo de pacientes. Porém, estudos adicionais devem ser realizados para se validar a utilização clínica desses marcadores.Actually, chronic lymphocytic leukemia (CLL) can be divided in two subsets: one with somatically mutated immunoglobulin heavy-chain variable-region genes (MIgVH) and other with unmutated sequences. (UMIgVH). Some studies have shown that CD38 expression in CLL cells are correlated with IgVH mutational status. However, the value of CD38 as surrogate IgVH mutational status is controversial. Recent studies, have found that Zap-70 protein tyrosine kinase expression is strongly associated with the mutational status IgVH. The aim of this study was to evaluate the Zap-70 and CD38 expression, for flow cytometry, in CD19+ LLC cells and correlate with the Binets staging system, treatment-free survival (TFS) and a overall survival (OS). Zap-70 and CD38 was evaluated, in 144 CLL patients that was classified in A, B and C Binets staging system: 59 (41%) in stage A, 38 (26%) in B and 47 (33%) in C. We observed low Zap-70 and CD38 expression in stage A patients than in stage B and C cases. When we analyzed the TFS in stage A patients Zap-70+ and CD38+ patients showed shorter TFS than Zap-70- and CD38-. Then we observed that the OS of Zap-70+ and CD38+ patients was, also, shorter than Zap-70- and CD38- cases. However, statistical differences was not found when Zap-70 and CD38 expression was correlated with stage A, B or C Binets staging system. To understand the associated Zap-70 and CD38 expression, we divided the CLL patients in two subgroups (Zap-70-/CD38 - and Zap-70+ or CD38+). We observed that CD38+ or Zap-70+ was associated Binets staging system, once most of stage B (74%) and C (66%) patients are Zap-70+ or CD38+. However, stage A patients, Zap-70+ or CD38+, showed shorter OS than Zap-70-/CD38-. These differences were not observed in stage B and C patients. Shorter TFS was also observed in the Zap-70+ or CD38+ stage A patients. These results suggest that combined analysis of Zap-70 and CD38 can be used to evaluate stage A patients to observe the clinical evolution of the disease. Nevertheless, other studies must be carried to confirm the clinical use of these markers.
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Silvestrov, Pavel. "Computational Investigation of DNA Repair Enzymes: Determination and Characterization of Cancer Biomarkers and Structural Features." Thesis, University of North Texas, 2018. https://digital.library.unt.edu/ark:/67531/metadc1157566/.
Full textAbstract:
Genomic integrity is important for living cells' correct functioning and propagation. Deoxyribonucleic acid as a molecule is a subject to chemical reactions with agents that can come from environment as well as from internal metabolism processes. These reactions can induce damage to DNA and thus compromise the genetic information, and result in disease and death of an organism. To mitigate the damage to DNA, cells have evolved to have multiple DNA repair pathways. Presented here is a computational study of DNA repair genes. The structure of the Homo sapiens direct DNA repair gene ALKBH1 is predicted utilizing homology modeling methods and using AlkB and DBL proteins as templates. Analysis of the obtained structure and molecular dynamics simulations give insights into potentially functionally important residues of the protein. In particular, zinc finger domains are predicted, and lysines that could perform catalytic activities are investigated. Subsequent mutagenesis experiments revealed the effect of the residues predicted to form zinc fingers on activity of ALKBH1. Structure and dynamics of AlkD, a Bascillus cereus base excision DNA repair protein is also studied. This protein has been shown to bind DNA with large alkyl adducts and perform excision catalysis without base flipping which is characteristic to other enzymes in the same family. MD simulations of AlkD revealed that B helix, which interacts with DNA, has higher fluctuations when AlkD is not bound to DNA, and thus could have a role in binding and recognition of DNA. For the purpose of finding biomarkers and to further our understanding of a mode of action of DNA repair genes, statistical methods were applied to identify mutations that are linked to cancer phenotypes. Analysis was based on case-control studies of patients with cancers of prostate, breast, pancreas, lung as well as chronic lymphocytic leukemia from NCBI dbGAP database. Those mutations that result in missense mutations were further investigated. In particular, extensive MD simulations and experimental investigations were performed on the mutation in the ALKBH7 gene that was found to be linked to prostate cancer.
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CASTAGNA, Monica. "Targeting CD38 antigen as a therapeutic strategy for hematological malignancies." Doctoral thesis, 2013. http://hdl.handle.net/11562/537549.
Full textAbstract:
Il successo di terapie convenzionali come la chemioterapia e la radioterapia per il trattamento delle neoplasie è stato limitato a causa di diversi fattori come la chemioresistenza ai farmaci e la tossicità periferica causata dalla mancanza di specificità di questi approcci. Per questo motivo l’interesse per le terapie selettive che prevedono l’uso di immunotossine, specialmente per il trattamento di tumori ematologici, è in aumento. Le immunotossine sono proteine chimeriche costituite da un ligando selettivo per la cellula bersaglio (dominio di origine anticorpale, citochina o fattore di crescita) che media il legame e l’internalizzazione della porzione tossica legata chimicamente o fusa geneticamente, generalmente rappresentata da una tossina di origine vegetale o batterica che agisce interferendo con la sintesi proteica.
In questo lavoro viene descritta la costruzione di nuove proteine di fusione ad uso terapeutico progettate per indurre apoptosi selettivamente in neoplasie umane dei linfociti B e la valutazione dell’effetto potenziante ottenuto attraverso l’associazione delle immunotossine con farmaci coinvolti in meccanismi metabolici intracellulari. Il dominio di legame delle nostre immunotossine è rappresentato da frammenti anticorpali a singola catena (scFv) diretti verso l’antigene CD38, una molecole di superficie espressa ad alti livelli dai linfociti B di un sottogruppo particolarmente aggressivo di Leucemia Linfatica Cronica (CLL) che evolve in una patologia dall’esito prognostico sfavorevole, nota come Sindome di Richter, e dalle plasmacellule tumorali immature nel Mieloma Multiplo (MM). L’scFv è fuso ad una porzione tossica che agisce inibendo il meccanismo della sintesi proteica negli organismi eucarioti e nel caso delle nostre immunotossine è rappresentato da una forma tronca della Esotossina A prodotta dal batterio Pseudomonas aeruginosa (PE40) o in alternativa dalla tossina di origine vegetale saporina.
Abbiamo inizialmente progettato una immunotossina con PE40 ed una con saporina contenenti un scFv derivato da un anticorpo monoclonale (mAb) sviluppato e caratterizzato nel nostro laboratorio. Tutti i costrutti ricombinanti sono stati prodotti nel sistema di espressione di origine batterica Escherichia coli e purificati da corpi di inclusione tramite IMAC. Tuttavia, l’scFv 1E8 non ha consentito di preservare l’efficienza di legame dell’anticorpo parentale. Inoltre, le immunotossine ricombinanti ottenute dalla fusione dell’scFv 1E8 con PE40 o saporina hanno mostrato una bassa affinità di legame nei confronti delle cellule bersaglio esprimenti la molecola CD38 e, di conseguenza, è stata rilavata solo una trascurabile attività citotossica.
Con la progettazione della forma divalente dell’scFv 1E8, il nostro scopo è stato quello di aumentare l’affinità di legame dei costrutti. Nonostante i risultati sconfortanti del saggio di legame in citometria a flusso, la molecola DIV1E8-SAP ha dimostrato di inibire la sintesi proteica di cellule CD38-positive con una IC50 nell’ordine del sub-nanomolare.
Successivamente abbiamo progettato due immunotossine ricombinanti dirette verso l’antigene CD38, il cui dominio di legame era costituito da un scFv derivato da un mAb con una specificità epitopica diversa da quella del precedentemente descritto 1E8. Le immunotossine AT13/5-PE e AT13/5-SAP hanno dimostrato buone proprietà di legame con una elevata affinità e specificità per l’antigene CD38 espresso sulla superficie di cellule derivate da Linfoma di Burkitt e cellule di mieloma.
Abbiamo dimostrato l’abilità si queste immunotossine di inibire la sintesi proteica nelle linee cellulari studiate e ne abbiamo chiaramente dimostrato un effetto dose-risposta. Il blocco della sintesi proteica causato dalle immunotossine derivate da AT13/5 ha determinato infine l’innesco del processo di apoptosi e la morte cellulare. Attraverso saggi di apoptosi abbiamo dimostrato la capacità di AT13/5-PE e AT13/5-SAP di indurre apoptosi in cellule Daudi e RPMI8226.
Abbiamo perciò provato che l’associazione delle nostre immunotossine con molecole terapeutiche che agiscono su diversi bersagli dalla cascata di traduzione del segnale coinvolta nella crescita cellulare, nella sopravvivenza e nella proliferazione, potrebbe essere sinergica in alcune linee cellulari. In particolare abbiamo osservato che farmaci coinvolti nell’inibizione di Bcl-2, Bcl-xL e Bcl-w (noti come BH3-mimetics) possono aumentare la potenza delle nostre immunotossine.
Abbiamo infine dimostrato una prima prova di concetto riguardo l’efficacia delle immunotossine derivate da AT13/5 su linfociti B derivati da pazienti affetti da CLL, tuttavia questo studio necessita di essere implementato con una casistica più ampia.The success of conventional chemotherapy and radiotherapy for the treatment of cancer has been limited due to several factors like chemoresistance to drugs and peripheral toxicity caused by the lack of specificity of these approaches. For this reason the interest in targeted therapies using immunotoxins (ITs) especially for the treatment of hematological malignancies is increasing. Immunotoxins are chimeric proteins with a cell-selective ligand (antibody-derived domain, cytokine or growth factor) which drives the binding and internalization of a chemically linked or genetically fused toxic portion, generally represented by a plant or bacterial toxin which acts by interfering with protein synthesis. Here we report on the construction of novel therapeutic fusion proteins designed to induce target antigen-restricted apoptosis in human B-cell neoplasias and the evaluation of the potentiating effect obtained by the association of the ITs with drugs involved in intracellular metabolic pathways. The binding portion of our ITs is represented by a single-chain antibody fragment (scFv) directed against CD38 antigen, a surface molecule highly expressed by B lymphocytes of a particularly aggressive sub-group of Chronic Lymphocytic Leukemia (CLL) leading to the prognostically unfavorable Richter’s Syndrome and by the neoplastic immature plasma cells in Multiple Myeloma (MM). The scFv is fused to a toxic portion which acts by inhibiting the mechanism of protein synthesis in eukaryotes and in our ITs is represented by a truncated version of the bacterial toxin Pseudomonas aeruginosa Exotoxin A (PE40) or alternatively by the plant toxin saporin. We firstly designed a PE40- and a saporin-based IT comprising a scFv derived from a monoclonal antibody (mAb) developed and characterized in our laboratory. All the recombinant constructs were produced in the bacterial expression system E. coli and purified from inclusion bodies by IMAC. However, the scFv format (1E8) did not allow to preserve the binding efficiency of the parental monoclonal. Moreover, the recombinant ITs created by the fusion of 1E8 scFv with PE40 or saporin showed a low binding affinity to the CD38 target cells and, as a consequence, only negligible citotoxic activity was detected. With the creation of the divalent form of the 1E8 scFv, our purpose was to increase the binding affinity of the constructs. Despite the discouraging results of the flow-cytometric binding assay, DIV1E8-SAP demonstrated to inhibit protein synthesis of CD38-positive cells with an IC50 in the sub-nanomolar range. Then we designed two anti-CD38 recombinant ITs whose binding portion was a scFv derived from a mAb with an epitope specificity different from that of the previously described 1E8. AT13/5-PE and AT13/5-SAP showed good binding properties with a high affinity and specificity for CD38 antigen expressed on the surface of Burkitt’s lymphoma cells and myeloma cells. We proved the ability of these ITs to inhibit protein synthesis in the cell lines studied and we clearly demonstrated a dose-response effect of the ITs. The arrest of protein synthesis caused by the AT13/5-derived ITs finally leads to the triggering of the apoptotic cascade and to cell death. By using apoptosis assays we demonstrated the capability of AT13/5-PE and AT13/5-SAP to induce apoptosis of Daudi and RPMI8226 cells. Then we proved that the association of our ITs with therapeutic molecules acting on different targets of the signal transduction cascade involved in cell growth, survival and proliferation, could be synergistic in some cell lines. In particular we observed that drugs involved in the Bcl-2, Bcl-xL and Bcl-w inhibition (BH3-mimetics) can increase the potency of our ITs. Finally we demonstrated a first proof of concept about the efficacy of AT13/5-derived ITs on B-lymphocytes derived from CLL patient, but this study needs to be implemented with a wider number of cases.
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Wong, Karrie. "CD200: A Novel Therapeutic Target for Chronic Lymphocytic Leukemia." Thesis, 2012. http://hdl.handle.net/1807/34969.
Full textAbstract:
The ability of cancer cells to escape anti-tumor immune responses is acknowledged as one of the hallmarks of cancer. Overexpression of immunoregulatory molecules is one mechanism responsible for the immunsuppressive network that is characteristic of the tumor microenvironment.
In this thesis, we investigated the role of CD200, a potent immunoregulatory molecule, in Chronic Lymphocytic Leukemia. We showed that functional blockade of CD200 on lymphoma cells or primary CLL cells, both of which express CD200 at high levels, augmented cytotoxic killing of these cells by effector CD8+ T cells in vitro. We also identified and characterized a previously unrecognized soluble form of CD200, sCD200, present in elevated levels in CLL plasma when compared to plasma from controls.
The data reported show that patients with high sCD200 levels have more aggressive disease, inferring that sCD200 may be a novel prognostic marker for CLL. The in vivo function of sCD200 was investigated for its ability to support engraftment of CLL splenocytes in NOD.SCID mice. Infusion of sCD200hi CLL plasma, but not sCD200lo normal plasma, enhanced engraftment of CLL-splenocytes in vivo, an effect which was abrogated by depletion of sCD200 from CLL plasma. The prolonged engraftment of CLL cells seen in this model (>6 months) suggests these mice represent a useful pre-clinical model for drug screening. The effect of CD200 blockade was tested in this model, and was found to be as effective in eliminating engrafted CLL cells as rituximab. Investigation of the mechanisms leading to the release of sCD200 from CLL cells showed that sCD200 was produced following ectodomain shedding by ADAM proteases and MMPs.
Results from studies reported in this thesis support the hypothesis that CD200 plays a major role in CLL biology, and suggests it may represent a novel therapeutic target for CLL.
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Graham, Bonnie Anne. "Mechanism of death receptor 5 (DR5) gene expression : applications to chronic lymphocytic leukemia (CLL)." 2006. http://hdl.handle.net/1993/20377.
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Mao, Zhengrong. "Clonality analysis in B-Cell Chronic Lymphocytic Leukemia (B-CLL) associated with Richter's syndrome." Doctoral thesis, 2006. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-20596.
Full textAbstract:
Die chronische lymphatische Leukämie vom B-Zell-Typ (B-CLL) macht ca. 90% der chronischen lymphatischen Leukämien aus. Der klinische Verlauf der B-CLL ist heterogen, und bei 3-5% der Patienten kommt es im Verlauf der Erkrankung zu einer Transformation in ein aggressives Lymphom, meist in ein diffuses großzelliges B-Zell-Lymphom (DLBCL) oder in ein Hodgkin-Lymphom (HL). Eine solche Transformation wird als Richter-Syndrom bezeichnet und ist mit einer ungünstigen klinischen Prognose assoziiert. In B-Zell-Lymphomen (B-NHL) ermöglicht der Mutationsstatus der variablen Anteile der Immunglobulinschwerkettengene (IgVH) eine Aussage über das Entwicklungsstadium der B-Zelle, in dem sich die Transformation zu einem B-Zell-Lymphom ereignet hat. In der B-CLL stellt der Mutationsstatus auch einen bedeutenden prognostischen Faktor dar, wobei die Erkrankung bei Patienten mit unmutierten IgVH Genen meist einen ungünstigen klinischen Verlauf zeigt. Die wenigen bisher durchgeführten molekularen Analysen an Fällen von Richter-Syndromen deuten darauf hin, dass ein Transformationsereignis sowohl bei B-CLL-Patienten mit mutierten als auch mit unmutierten IgVH Genen vorkommen kann, und dass die Tumorzellen des DLBCL oder HL sowohl klonal identisch mit dem B-CLL-Klon sein können als auch unabhängig als sekundäres Lymphom entstehen können. Um die klonale Beziehung zwischen DLBCL- bzw. Hodgkin/Reed-Sternberg (HRS)-Zellen und den Zellen der vorbestehenden B-CLL zu analysieren, den Mutationsstatus des IgVH Gens sowie mögliche prognostische Faktoren in B-CLL-Fällen mit Richter-Transformation zu identifizieren, wurde eine größere Fallserie mit Hilfe eines PCR-basierten GeneScan Ansatzes mit anschließender Sequenzierung der IgVH-Gene untersucht. In Fällen mit HRS bzw. HRS-ähnlichen Zellen wurden die CD30-positiven Tumorzellen mittels Laser-Capture Mikrodissektion (LCM) isoliert. Weiterhin erfolgte eine morphologische und immunhistochemische Analyse der Fälle. Insgesamt wurden 48 Patientenproben untersucht, darunter 40 Fälle mit Richter-Syndrom sowie weitere 8 B-CLL-Fälle mit CD30-positiven HRS-ähnlichen Zellen. Unter den 40 Proben mit Richter-Syndrom zeigten 34 B-CLL-Fälle eine Transformation in ein DLBCL, in 6 Fällen erfolgte eine Transformation in ein HL. In 23 Fällen mit B-CLL und DLBCL wurde eine Sequenzierung der IgVH Gene durchgeführt. In 18 Fällen waren B-CLL und DLBCL klonal identisch, in 5 Fällen war das DLBCL als klonal unabhängige Neoplasie entstanden. Unter den klonal verwandten Paaren zeigten 11 von 15 Paaren unmutierte IgVH-Gene sowohl in der B-CLL als auch im DLBCL, wohingegen 5 von 6 Fälle mit Transformation einer B-CLL in ein HL mutierte IgVH Gene trugen. HRS-Zellen in 2 Fällen und HRS-ähnliche Zellen in einem Fall waren klonal verschieden vom B-CLL-Zellklon. Die VH-Gene VH3-23, VH3-74, VH1-2 und VH3-9 waren überproportional häufig in B-CLL Fällen mit einer späteren Transformation in ein DLBCL vertreten, wohingegen VH4-34 und VH3-48 in mehr als der Hälfte der Fälle mit Transformation zu einem HL nachgewiesen werden konnten. Der immunhistochemische Nachweis von ZAP70 zeigte eine signifikante Assoziation mit unmutierten IgVH-Genen in B-CLL mit nachfolgender Richter-Transformation. Bei 24 Patienten konnte der klinische Verlauf eruiert werden. Die mediane Überlebenszeit von Patienten mit B-CLL mit stattgehabter Transformation in ein DLBCL oder ein HL betrug 7 beziehungsweise 21 Monate. Es fanden sich weder signifikante Unterschiede der Überlebenszeit zwischen klonal verwandten und nicht verwandten Fällen, noch zwischen IgVH-mutierten und -unmutierten Fällen. Zusammenfassend kann festgestellt werden, dass bei der Richter-Transformation einer B-CLL in ein DLBCL dieses einerseits aus dem präexistenten B-CLL-Klon entstehen kann, andererseits aber auch als unabhängige, klonal nicht verwandte Neoplasie auftreten kann. In der Mehrzahl der Fälle (78% in dieser Serie) sind B-CLL und DLBCL jedoch klonal identisch und nur gelegentlich entsteht ein DLBCL ohne klonale Beziehung zur B-CLL als unabhängige, sekundäre Neoplasie. Eine klonale Transformation in ein DLBCL tritt vorwiegend bei B-CLL-Patienten mit unmutierten IgVH-Genen auf, wohingegen die Mehrzahl der B-CLL-Patienten mit einer Transformation in ein HL oder CD30-positive HRS-ähnliche Zellen mutierte IgVH-Gene aufweist. Dieser Befund lässt auf unterschiedliche Transformationswege der beiden Subtypen des Richter-Syndroms schließen. Weiterhin existieren vermutlich wesentliche Unterschiede in der Pathogenese zwischen DLBCL-Fällen, die sich aus einer vorbestehenden B-CLL entwickelt haben, und de novo DLBCL-Fällen, da de novo DLBCL zumeist durch mutierte IgVH-Gene charakterisiert sindB-cell chronic lymphocytic leukemia (B-CLL) comprises 90% of chronic lymphoid leukemias in Western countries and patients with B-CLL have a heterogeneous clinical course. Approximately 3-5% of B-CLL patients encounter transformation to an aggressive lymphoma, mainly diffuse large B-cell lymphoma (DLBCL) or Hodgkin’s lymphoma (HL) which has been defined as Richter’s syndrome and is associated with a poor clinical outcome. The mutational status of the immunoglobulin heavy chain variable region (IgVH) gene not only implies the developmental stage at which the neoplastic transformation occurs in a given B-cell lymphoma, but also constitutes an important prognostic factor in B-CLL, since B-CLL patients with unmutated IgVH genes usually have a poor clinical outcome. Sparse molecular analyses performed in Richter’s syndrome so far suggest that it can occur in B-CLL patients carrying mutated or unmutated IgVH genes, and tumor cells in DLBCL or HL can be clonally identical to the B-CLL clone or arise as an independent, secondary lymphoma. To determine the clonal relationship between DLBCL or Hodgkin/Reed-Sternberg (HRS) cells and pre-existing B-CLL cells in a larger series, to identify the IgVH gene usage and the mutational status and to explore possible prognostic factors in B-CLL undergoing Richter’s transformation, we utilized a PCR-based GeneScan approach with subsequent sequencing of the IgVH genes. In cases with HRS/HRS-like cells laser capture microdissection (LCM) was employed to isolate these cells. In addition, a thorough morphological and immunohistochemical analysis was performed. In total, specimens from 48 patients were investigated including 40 cases of Richter’s syndrome and additional 8 cases of B-CLL cases with the presence of CD30-positive HRS-like cells. Among 40 cases of Richter’s syndrome, 34 B-CLL cases showed transformation to DLBCL and 6 cases transformed from B-CLL to HL. Sequencing was performed in 23 paired B-CLL and DLBCL cases. In 18 cases, B-CLL and DLBCL were clonally identical, whereas DLBCL developed as a clonally independent neoplasm in 5 patients. Among the clonally related pairs, 11 out of 15 cases carried unmutated IgVH genes in both the B-CLL and DLBCL component, whereas 5 of 6 B-CLL cases that showed transformation to HL carried mutated IgVH genes. HRS cells in two samples and HRS-like cells in one sample were clonally distinct from the B-CLL clone and infected by EBV, whereas one sample of HRS-like cells was related to the clone from the surrounding B-CLL cells and did not express latent membrane protein-1 (LMP1). The VH genes VH3-23, VH3-74, VH1-2 and VH3-9 were overused in B-CLL cases that transformed to DLBCL, whereas VH4-34 and VH3-48 were used in over half of the B-CLL cases with transformation to HL. Immunohistochemical staining of ZAP70 was significantly associated with unmutated IgVH genes in B-CLL cases undergoing Richter’s transformation. Clinical follow-up data could be obtained from 24 patients. The median survival times of B-CLL patients with transformation to DLBCL or HL were 7 and 21 months, respectively. No significantly different survival times were found between clonally related or unrelated cases, or between IgVH-mutated or -unmutated cases. We conclude that in Richter’s transformation, DLBCL can evolve by clonal transformation of the pre-existing B-CLL clone or occur as an independent, clonally unrelated neoplasm. In the majority of cases (78% in our series), B-CLL and DLBCL are clonally identical. In a subset of patients, however, DLBCL develops as an independent secondary neoplasm that is not clonally related to the B-CLL. Clonal transformation into DLBCL predominantly occurs in B-CLL patients with unmutated IgVH genes, whereas most B-CLL patients that show transformation to HL or CD30-positive HRS-like cells carry mutated IgVH genes. The tendency that IgVH-unmutated B-CLL transforms to DLBCL and IgVH-mutated B-CLL transforms to HL implies different transformation pathways in the two subtypes of Richter’s syndrome. In addition, important pathogenetic differences are likely to exist between DLBCL cases derived from a pre-existing B-CLL as compared to de novo DLBCL cases, since de novo DLBCL is usually characterized by mutated IgVH genes. The biased usage of IgVH genes in the two subtypes of Richter’s syndrome suggests a possible role for antigen involvement in tumorigenesis also in B-CLL cases that undergo Richter’s transformation. Finally, EBV-association in the HL variant of Richter’s syndrome occurs more frequently in clonally unrelated secondary malignancies
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Ruppel, Melanie. "Elucidation of the epigenetic code of the chromosomal region 13q14.3 in Chronic Lymphocytic Leukemia (CLL)." Phd thesis, 2009. https://tuprints.ulb.tu-darmstadt.de/1354/2/Ruppel_Melanie_Dissertation.pdf.
Full textAbstract:
B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world with a median age of 72 years of CLL patients at diagnosis. The most common genomic aberration in CLL is the deletion of a critical region in chromosomal band 13q14.3, which is deleted in more than 50 % of patients. The high frequency of deletions of 13q14.3 in CLL and other tumors points to a tumor suppressor mechanism localized in the critical region. The candidate genes localized in the critical region in 13q14.3 have been shown to be monoallelically expressed. Towards elucidation of the tumor suppressor mechanism in 13q14.3, the epigenetic modifications of genes and CpG-islands in the region were analyzed in this thesis. Monoallelic expression of almost all genes in the critical region in 13q14.3 in non-malignant cells was detected to be marked by epigenetic modifications at promoter and exonic regions of these genes. A chromatin pattern specific for monoallelically expressed and imprinted genes, which is promoter-restricted enrichment of dimethylated lysine 4 of histone H3 (H3K4me2), was detected at the genes DLeu2/BCMSUN and C13ORF1. Detection of this specific chromatin pattern led to identification of a novel monoallelically expressed gene in the region: C13ORF1. Here, C13ORF1 was in addition to RFP2 and DLeu2/BCMSUN, shown to be monoallelically expressed in B- and T-cells from healthy donors. The chromatin immunoprecipitation (ChIP) methodology was established in order to quantify two chromatin modifications at the critical region. ChIP was technically established and optimized to analyze enrichment of the histone mark H3K4me2 that is correlated with an active transcription state, and of the histone variant macroH2A, which was shown to be allelically enriched at monoallelically silenced loci, at 13q14.3. In addition, also DNA-methylation of the CpG-islands was analyzed. Thus, an epigenetic code of the candidate genes that correlates with their transcription status was determined in non-malignant hematopoietic and CLL cells. In non-malignant cells, the epigenetic code identifies one active and one inactive copy of the critical region in 13q14.3. Thereby, the genes DLeu2/BCMSUN and C13ORF1 are monoallelically expressed from the epigenetically active allele and their expression status is marked by a distinct chromatin pattern. In contrast, the distinct chromatin pattern could not be detected at the gene RFP2 that was earlier shown to be monoallelically expressed. The quantification of H3K4me2 and macroH2A occurrence at RFP2 points to allelic differences of enrichment at the gene locus, although this could not be further defined by the chosen methods. However, allelic enrichment of chromatin marks could be detected at two distinct loci in the 5’-regions of the large noncoding RNA genes, BCMS and DLeu2/BCMSUN, in samples derived from healthy probands. Thereby, in this study, two candidate locus control regions (LCRs) were identified that are located in the CpG-islands D and E in the promoter region of the two large ncRNAs and showed differential DNA-methylation and histone modifications. The differential epigenetic code and the localization of the candidate LCRs suggest their involvement in the regulation of expression of the two large ncRNA genes that span the critical region in 13q14.3. In summary these findings suggest a model of the epigenetically regulated tumor suppressor mechanism in 13q14.3 that leads to monoallelic expression of the candidate genes in the region. This model consists of the features that i) 13q14.3 is present in one active and one silent chromosome copy in non-malignant healthy cells, ii) the large ncRNAs BCMS and BCMSUN are likely involved in the regulation (in cis) of expression of the other genes in 13q14.3, and iii) differential epigenetic modifications of candidate LCRs control transcription of the large ncRNA genes. Accordingly, the epigenetic status of the LCRs is critical for transcription of the ncRNAs that in turn regulate the expression of the candidate genes in the critical region. Surprisingly, the most evident epigenetic aberration at 13q14.3 was the generally higher enrichment of the active chromatin mark at the region in CLL cells. However, also the inactive chromatin mark was increased at 13q14.3 in CLL cells. Significant differences of enrichment of H3K4me2 and macroH2A between non-malignant and CLL cells, were detected at CpG-islands B, C and E and at the exonic region of DLeu2/BCMSUN, which clearly showed that the aberrant modifications in CLL are most evident at the promoter regions of the genes C13ORF1, RFP2 and DLeu2/BCMSUN, respectively. The chromatin pattern that marks monoallelic expression at C13ORF1 and DLeu2/BCMSUN remained unchanged in the analyzed CLL cells. However, in CLL cells derived from patients with different genomic aberrations, the deletion of genomic material from 13q14.3 and loss of active chromatin marks could be correlated. This suggests that in del(13q) CLL patients, indeed the active copy of 13q14.3 is lost, which would explain downregulation of expression of the genes in the critical region observed in these patients. In line with the proposed model of the tumor suppressor mechanism in 13q14.3, the most evident aberrant epigenetic modifications of 13q14.3 in CLL cells were detected at the two candidate LCRs, where distinct epimutations were detected. Thus, an epimutation in 13q14.3 was shown that could be responsible for the inactivation of the epigenetic regulatory mechanism of 13q14.3 in CLL cells. Further, these findings suggest that the regulatory impact of the ncRNAs, the LCR or another yet unidentified part of the critical region in 13q14.3 is affected by this epimutation, which leads to deregulation and inactivation of the tumor suppressive function of 13q14.3. In conclusion, a specific epigenetic code at the genes in the critical region in 13q14.3 was determined that is correlated with their transcriptional state. Evidence was obtained for a model of epigenetic regulation that implies differential chromatin packaging of the two copies of 13q14.3 and results in monoallelic expression of the candidate genes in healthy B- and T-cells. Furthermore, two candidate LCRs were identified in the critical region in 13q14.3 in non-malignant cells that showed distinct epimutations in CLL cells. The localization and the detected epimutations of the LCRs in CLL point to their role in regulation of the ncRNA genes that most likely regulate expression of the other genes in the critical region. On the basis of these findings, further characterization of epigenetic features at 13q14.3 will help to fully elucidate the complex epigenetic regulatory network that controls the tumor suppressor mechanism in 13q14.3. The findings presented here will help to understand the pathogenicity of (epigenetic) inactivation of the tumor suppressor mechanism in CLL and thereby provide the basis for development of more efficient and specific therapies for CLL in the future.
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Paul, James Traquair. "Correlation of the D-type cyclins with clinical features and survival in chronic lymphocytic leukemia (CLL)." 2002. http://hdl.handle.net/1993/19637.
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