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| 2. |
- Papakonstantinou, Nikos, et al.
(författare)
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Integrated epigenomic and transcriptomic analysis reveals TP63 as a novel player in clinically aggressive chronic lymphocytic leukemia
- 2019
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 144:11, s. 2695-2706
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Tidskriftsartikel (refereegranskat)abstract
- Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness. What's new? In chronic lymphocytic leukemia (CLL), cases with unmutated immunoglobulin receptors (U-CLL) are generally associated with inferior outcome, albeit still displaying considerable heterogeneity. Might such differences in CLL progression be explained by epigenetics? In this study, the authors found that an unusually aggressive subset of CLLs called subset #8 has a distinctive DNA-methylation profile. They also found that p63 is a novel pro-survival factor for CLL cells. These molecular studies may lead to new prognostic biomarkers, and possibly new therapeutic targets, for CLL.
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| 3. |
- Agathangelidis, Andreas, et al.
(författare)
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Highly similar genomic landscapes in monoclonal B-cell lymphocytosis and ultra-stable chronic lymphocytic leukemia with low frequency of driver mutations
- 2018
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 0390-6078 .- 1592-8721. ; 103:5, s. 865-873
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Tidskriftsartikel (refereegranskat)abstract
- Despite the recent discovery of recurrent driver mutations in chronic lymphocytic leukemia, the genetic factors involved in disease onset remain largely unknown. To address this issue, we per-formed whole-genome sequencing in 11 individuals with monoclonal B-cell lymphocytosis, both of the low-count and high-count subtypes, and 5 patients with ultra-stable chronic lymphocytic leukemia (>10 years without progression from initial diagnosis). All three entities were indistinguishable at the genomic level exhibiting low genomic complexity and similar types of somatic mutations. Exonic mutations were not frequently identified in putative chronic lymphocytic leukemia driver genes in all settings, including low-count monoclonal B-cell lymphocytosis. To corroborate these findings, we also performed deep sequencing in 11 known frequently mutated genes in an extended cohort of 28 monoclonal B-cell lym phocytosis/chronic lymphocytic leukemia cases. Interestingly, shared mutations were detected between clonal B cells and paired polymorphonuclear cells, strengthening the notion that at least a fraction of somatic mutations may occur before disease onset, likely at the hematopoietic stem cell level. Finally, we identified previously unreported non-coding variants targeting pathways relevant to B-cell and chronic lymphocytic leukemia development, likely associated with the acquisition of the characteristic neoplastic phenotype typical of both monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia.
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| 4. |
- Dasenaki, Marilena, et al.
(författare)
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Simultaneous Determination of Free Cortisol, Cortisone and their Tetrahydrometabolites in Urine by Single Solvent Extraction and Liquid Chromatography-Tandem Mass Spectrometry
- 2019
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Ingår i: Analytical Letters. - : Taylor & Francis. - 0003-2719 .- 1532-236X. ; 52:17, s. 2764-2781
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Tidskriftsartikel (refereegranskat)abstract
- A fast, efficient and low-cost high performance liquid chromatography-tandem mass spectrometry methodology was developed and validated for the simultaneous determination of free urinary cortisone, cortisol and their tetrahydro-metabolites. The developed method comprises a simple liquid-liquid extraction with CH2Cl2, followed by reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) in positive mode. The baseline chromatographic separation of the analytes, including the stereoisomers tetrahydrocortisol (THF) and allo-THF, was achieved on a Hypersil Gold C-18 column with a mobile phase consisting of 0.05%v/v formic acid in water-acetonitrile, using a gradient elution program. The influence of the mobile phase composition and the ESI parameters on the sensitivity of the method was extensively studied. Sample preparation was also optimized, testing two techniques: solid phase extraction (SPE) and liquid-liquid extraction (LLE). Recoveries ranged from 74.7% (a-THF) to 93.5% (cortisol) and the method limits of detection (MLD) ranged from 0.34 ng mL(-1) (cortisol) to 1.37 ng mL(-1) (THF). Intra- and inter-day coefficient of variation of the assay varied from1.5% (allo-THF) to 13% (tetrahydrocortisone) and from 3.6% (allo-THF) to 14.9% (tetrahydrocortisone), respectively. The method was applied for the analysis of urine samples from 53 healthy individuals with a mean age of 13.96 years in order to estimate the concentration of the five corticosteroids and the ratio of the metabolites. Associations between urinary cortisol/cortisone and serum cortisol/cortisone values were also characterized.
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| 5. |
- Ghia, Paolo, et al.
(författare)
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Crystallographic evidence of autologous recognition by a clonotypic B cell receptor in chronic lymphocytic leukemia
- 2015
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Ingår i: Leukemia and Lymphoma. - IRCCS Sci Inst San Raffaele, Div Expt Oncol, Milan, Italy. [Degano, Massimo] IRCCS Sci Inst San Raffaele, Dept Immunol Transplantat & Infect Dis, Biocrystallog Unit, Milan, Italy. [Duehren-von Minden, Marcus; Schneider, Dunja; Alkhatib, Alabbas] Univ Freiburg, Ctr Biol Signaling Studies, Fac Biol, D-79106 Freiburg, Germany. [Belhart, Rudolf; Jumaa, Hassan] Univ Ulm Klinikum, Ulm, Germany. [Ntoufa, Stavroula; Stamatopoulos, Kostas] Ctr Res & Technol, Inst Appl Biosci, Thessaloniki, Greece. [Chiorazzi, Nicholas] Feinstein Inst Med Res, Manhasset, NY USA. [Stamatopoulos, Kostas] Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden. [Ghia, Paolo] Univ Milan, Osped San Raffaele, Dept Oncohematol, I-20127 Milan, Italy. [Ghia, Paolo] Univ Vita Salute San Raffaele, Milan, Italy.. - 1042-8194 .- 1029-2403. ; 56:S1, s. 110-111
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 6. |
- Gounari, Maria, et al.
(författare)
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Excessive antigen reactivity may underlie the clinical aggressiveness of chronic lymphocytic leukemia stereotyped subset #8
- 2015
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 125:23, s. 3580-3587
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Tidskriftsartikel (refereegranskat)abstract
- Subset #8 is a distinctive subset of patients with chronic lymphocytic leukemia (CLL) defined by the expression of stereotyped IGHV4-39/IGKV1(D)-39 B-cell receptors. Subset #8 patients experience aggressive disease and exhibit the highest risk for Richter transformation among all CLL. In order to obtain biological insight into this behavior, we profiled the antigen reactivity and signaling capacity of subset #8 vs other clinically aggressive stereotyped subsets, namely subsets #1 and #2. Twenty-seven monoclonal antibodies (mAbs) from subsets #1, #2, and #8 CLL clones were prepared as recombinant human immunoglobulin G1 and used as primary antibodies in enzyme-linked immuno-sorbent assays against representatives of the major classes of established antigenic targets for CLL. Subset #8 CLL mAbs exhibited broad polyreactivity as they bound to all antigens tested, in clear contrast with the mAbs from the other subsets. Antigen challenge of primary CLL cells indicated that the promiscuous antigen-binding activity of subset #8 mAbs could lead to significant cell activation, again in contrast to the less responsive CLL cells from subsets #1 and #2. These features constitute a distinctive profile for CLL subset #8, supporting the existence of distinct mechanisms of aggressiveness in different immunogenetic subsets of CLL.
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| 7. |
- Minici, Claudia, et al.
(författare)
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Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukaemia
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Cell-autonomous B-cell receptor (BcR)-mediated signalling is a hallmark feature of the neoplastic B lymphocytes in chronic lymphocytic leukaemia (CLL). Here we elucidate the structural basis of autonomous activation of CLL B cells, showing that BcR immunoglobulins initiate intracellular signalling through homotypic interactions between epitopes that are specific for each subgroup of patients with homogeneous clinicobiological profiles. The molecular details of the BcR-BcR interactions apparently dictate the clinical course of disease, with stronger affinities and longer half-lives in indolent cases, and weaker, short-lived contacts mediating the aggressive ones. The diversity of homotypic BcR contacts leading to cell-autonomous signalling reconciles the existence of a shared pathogenic mechanism with the biological and clinical heterogeneity of CLL and offers opportunities for innovative treatment strategies.
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| 8. |
- Ntoufa, Stavroula, et al.
(författare)
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B Cell Anergy Modulated by TLR1/2 and the miR-17 similar to 92 Cluster Underlies the Indolent Clinical Course of Chronic Lymphocytic Leukemia Stereotyped Subset #4
- 2016
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 196:10, s. 4410-4417
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Tidskriftsartikel (refereegranskat)abstract
- Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BCR Ig) display a particularly indolent disease course. Immunogenetic studies of the clonotypic BCR Ig of CLL subset #4 suggested a resemblance with B cells rendered anergic through chronic autoantigenic stimulation. In this article, we provide experimental evidence that subset #4 CLL cells show low IgG levels, constitutive ERK1/2 activation, and fail to either release intracellular Ca2+ or activate MAPK signaling after BCR cross-linking, thus displaying a signature of B cell anergy at both biochemical and functional levels. Interestingly, TLR1/2 triggering restored BCR functionality, likely breaching the anergic state, and this was accompanied by induction of the miR17 similar to 92 cluster, whose members target critical BCR-associated molecules, including MAPKs. In conclusion, we demonstrate BCR anergy in CLL subset #4 and implicate TLR signaling and the miR-17 similar to 92 cluster in the regulation of the anergic state. This detailed signaling profiling of subset #4 has implications for advanced understanding of the complex regulation of intracellular signaling pathways in CLL, currently a major therapeutic target of the disease.
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