122 research outputs found
Non-coding NOTCH1 mutations in chronic lymphocytic leukemia; their clinical impact in the UK CLL4 trial
No full textThe SF3B1 inhibitor spliceostatin A (SSA) elicits apoptosis in chronic lymphocytic leukemia cells through downregulation of Mcl-1
Get PDFThe pro-survival Bcl-2 family member Mcl-1 is expressed in chronic lymphocytic leukemia (CLL), with high expression correlated with progressive disease. The spliceosome inhibitor spliceostatin A (SSA), is known to regulate Mcl-1 and so here we assessed the ability of SSA to elicit apoptosis in CLL. SSA induced apoptosis of CLL cells at low nanomolar concentrations in a dose- and time-dependent manner, but independently of SF3B1 mutational status, IGHV status and CD38 or ZAP70 expression. However, normal B and T cells were less sensitive than CLL cells (P=0.006 and P<0.001, respectively). SSA altered the splicing of anti-apoptotic MCL-1L to MCL-1s in CLL cells coincident with induction of apoptosis. Overexpression studies in Ramos cells suggested Mcl-1 was important for SSA-induced killing since its expression inversely correlated with apoptosis (P=0.001). IL4 and CD40L, present in patient lymph nodes, are known to protect tumor cells from apoptosis and significantly inhibited SSA, ABT-263 and ABT-199 induced killing following administration to CLL cells (P=0.008). However, by combining SSA with the Bcl-2/Bcl-xL antagonists ABT-263 or ABT-199, we were able to overcome this pro-survival effect. We conclude that SSA combined with Bcl-2/Bcl-xL antagonists may have therapeutic utility for CL
Frequent mutation of the polycomb-associated gene ASXL1 in the myelodysplastic syndromes and in acute myeloid leukemia.
No full textThe identification of those genes that are frequently mutated in malignancies is essential for a full understanding of the molecular pathogenesis of these disorders, and often for the provision of markers for the study of disease progression. Recently, mutation of the ASXL1 (additional sex combs 1) gene has been reported in 4 out of 35 patients (11%) with myelodysplastic syndromes (MDS) and in 17 out of 39 patients (43%) with chronic myelomonocytic leukemia (CMML), a disease classified as MDS/myeloproliferative disorder
NUP98 is fused to HOXA9 in a variant complex t(7;11;13;17) in a patient with AML-M2
No full textThe t(7;11)(p15;p15.4) has been reported to fuse the NUP98 gene (11p15), a component of the nuclear pore complex, with the class-1 homeobox gene HOXA9 at 7p15. This translocation has been associated with myeloid leukemias, predominantly acute myeloid leukemia (AML) M2 subtype with trilineage myelodysplastic features, and with a poor prognosis. The derived fusion protein retains the FG repeat motif of NUP98 N-terminus and the homeodomain shared by the HOX genes, acting as an oncogenic transcription factor critical for leukemogenesis. We report here a new complex t(7;11)-variant, i.e., t(7;11;13;17)(p15;p15;p?;p1?2) in a patient with AML-M2 and poor prognosis. The NUP98-HOXA9 fusion transcript was detected by RT-PCR, suggesting its role in the malignant transformation as it has been postulated for other t(7;11)-associated leukemias. No other fusion transcripts involving the NUP98 or HOXA9 genes were present, although other mechanisms involving several genes on chromosomes 13 and 17 may also be involved. To our knowledge, this is the first t(7;11) variant involving NUP98 described in hematological malignancies
Extraction From Striped Weakfish (cynoscion Striatus) Wastes With Pressurized Co 2: Global Yield, Composition, Kinetics And Cost Estimation
No full textExtraction with pressurized carbon dioxide was applied to recover polyunsaturated fatty acids (PUFA) from striped weakfish (Cynoscion striatus) wastes. Extraction yield was evaluated at temperatures from 30 to 60 °C and pressures from 20 to 30 MPa. Temperature and pressure of 30 °C and 20 MPa were selected to evaluate process kinetics. A scale-up was performed by keeping the ratio constant between solid mass and solvent flow rate. Mathematical models were applied to the extraction curves, and the broken and intact cell model provided good fits, being applied to multiple curves simultaneously. Manufacturing cost of the extraction was estimated, and an optimal extraction time was observed by the end of the convective period. Finally, the extracts were analyzed by gas chromatography, and over 20% of the oil was composed by PUFA, whose concentration increased with extraction time. 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Longitudinal copy number, whole exome and targeted deep sequencing of 'good risk' IGHV-mutated CLL patients with progressive disease
No full textDisease progression in IGHV-M CLL with 'good-risk' cytogenetics is frequently associated with co-evolution of 'poor risk' driver mutations and DNA methylation changes.Drug resistance in IGHV-M CLL may be consequent upon the emergence of an IGHV-U cloneThe biological features of IGHV-M CLL responsible for disease progression are still poorly understood. We undertook a longitudinal study close to diagnosis, pre-treatment and post relapse in thirteen patients presenting with cMBL or Stage A disease and good risk biomarkers (IGHV-M genes, no del(17p) or del(11q) and low CD38 expression) who nevertheless developed progressive disease, of whom ten have required therapy. Using cytogenetics, FISH, genome-wide DNA methylation and copy number analysis together with whole exome, targeted deep- and Sanger sequencing, at diagnosis we identified mutations in established CLL driver genes in nine (69%), non-coding mutations (PAX5 enhancer region) in three, and genomic complexity in two patients. Branching evolutionary trajectories predominated (n=9/13), revealing intra-tumoural epi- and genetic heterogeneity and sub-clonal competition prior to therapy. Of the patients subsequently requiring treatment, two had sub-clonal TP53 mutations that would not be detected by standard methodologies, three qualified for the very-low risk category defined by integrated mutational and cytogenetic analysis and yet had established or putative driver mutations and one patient developed progressive, therapy-refractory disease associated with the emergence of an IGHV-U clone. These data suggest that extended genomic and immunogenetic screening may have clinical utility in patients with apparent good risk disease.Leukemia accepted article preview online, 05 February 2016. doi:10.1038/leu.2016.10
Recurrent mutations refine prognosis in chronic lymphocytic leukemia
No full textThrough the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods
Surface IgM expression and function associate with clinical behavior, genetic abnormalities and DNA methylation in CLL
No full textChronic lymphocytic leukemia (CLL) with unmutated (U-CLL) or mutated (M-CLL) IGHV displays different states of anergy, indicated by reduced surface Immunoglobulin M (sIgM) levels and signaling, consequent to chronic (super)antigen exposure. The subsets also differ in the incidence of high-risk genetic aberrations and in DNA methylation profile, preserved from the maturational status of the original cell. We focused on sIgM expression and function, measured as intracellular Ca(2+) mobilization following stimulation, and probed correlations with clinical outcome. The relationship with genetic features and maturation status defined by DNA methylation of an 18-gene panel signature was then investigated. SIgM levels/signaling were higher and less variable in U-CLL than in M-CLL and correlated with disease progression between and within U-CLL and M-CLL. In U-CLL, increased levels/signaling associated with +12, del(17p) or NOTCH1 mutations. In M-CLL, there were fewer genetic lesions, while the methylation maturation status, generally higher than in U-CLL, varied and was increased in cases with lower sIgM levels/signaling. These features revealed heterogeneity in M-CLL and U-CLL with clear clinical correlations. Multivariate analyses with phenotype, genetic lesions, or DNA methylation maturation status identified high sIgM levels as a new potential independent factor for disease progression. Multiple influences on sIgM include the cell of origin, the clonal history of antigen encounter in vivo and genetic damage. This simple marker compiles these different factors into an indicator worthy of further investigations for prediction of clinical behavior, particularly within the heterogeneous M-CLL subset
Proteomics profiling of CLL versus healthy B-cells identifies putative therapeutic targets and a subtype-independent signature of spliceosome dysregulation
Get PDFChronic lymphocytic leukaemia (CLL) is a heterogeneous B-cell cancer exhibiting a wide spectrum of disease courses and treatment responses. Molecular characterisation of RNA and DNA from CLL cases has led to the identification of important driver mutations and disease sub-types, but the precise mechanisms of disease progression remain elusive. To further our understanding of CLL biology we performed isobaric labelling and mass spectrometry proteomics on 14 CLL samples, comparing them with B-cells from healthy donors (HDB). Of 8694 identified proteins, ~6000 were relatively quantitated between all samples (q<0.01). A clear CLL signature, independent of subtype, of 544 significantly overexpressed proteins relative to HDB was identified, highlighting establishedhallmarks of CLL (eg. CD5, BCL2, ROR1 and CD23 overexpression). Previously unrecognised surface markers demonstrated overexpression (eg. CKAP4, PIGR, TMCC3 and CD75) and three of these (LAX1, CLEC17A and ATP2B4) were implicated in B-cell receptor signalling, which plays an important role in CLL pathogenesis. Several other proteins (eg. Wee1, HMOX1/2, HDAC7 and INPP5F) were identified with significant overexpression that also represent potential targets. Western blotting confirmed overexpression of a selection of these proteins in an independent cohort. mRNA processing machinery were broadly upregulated across the CLL samples. Spliceosome components demonstrated consistent overexpression (p=1.3x10-21) suggesting dysregulation in CLL, independent of SF3B1 mutations. This study highlights the potential of proteomics in the identification of putative CLL therapeutic targets and reveals a subtype-independent protein expression signature in CLL
Scale-up Of Extraction Processes
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