Dynamic variation of CD5 surface expression levels within individual chronic lymphocytic leukemia clones.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonally derived mature CD5high B cells; however, the cellular origin of CLL is still unknown. Patients with CLL also harbor variable numbers of CD5low B cells, but the clonal relationship of these cells to the bulk disease is unknown and can have important implications for monitoring, treating, and understanding the biology of CLL. Here, we use B-cell receptors (BCRs) as molecular barcodes to first show by single-cell BCR sequencing that the great majority of CD5low B cells in the blood of CLL patients are clonally related to CD5high CLL B cells. We investigate whether CD5 state switching was likely to occur continuously as a common event or as a rare event in CLL by tracking somatic BCR mutations in bulk CLL B cells and using them to reconstruct the phylogenetic relationships and evolutionary history of the CLL in four patients. Using statistical methods, we show that there is no parsimonious route from a single or low number of CD5low switch events to the CD5high population, but rather, large-scale and/or dynamic switching between these CD5 states is the most likely explanation. The overlapping BCR repertoires between CD5high and CD5low cells from CLL patient peripheral blood reveal that CLL exists in a continuum of CD5 expression. The major proportion of CD5low B cells in patients are leukemic, thus identifying CD5low B cells as an important component of CLL, with implications for CLL pathogenesis, clinical monitoring, and the development of anti-CD5-directed therapies

System size and centrality dependence of the balance function in A + A collisions at sqrt s NN = 17.2 GeV

Electric charge correlations were studied for p+p, C+C, Si+Si and centrality selected Pb+Pb collisions at sqrt s_NN = 17.2$ GeV with the NA49 large acceptance detector at the CERN-SPS. In particular, long range pseudo-rapidity correlations of oppositely charged particles were measured using the Balance Function method. The width of the Balance Function decreases with increasing system size and centrality of the reactions. This decrease could be related to an increasing delay of hadronization in central Pb+Pb collisions

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies.

The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers

Leukemia-associated somatic mutations drive distinct patterns of age-related clonal hemopoiesis.

Clonal hemopoiesis driven by leukemia-associated gene mutations can occur without evidence of a blood disorder. To investigate this phenomenon, we interrogated 15 mutation hot spots in blood DNA from 4,219 individuals using ultra-deep sequencing. Using only the hot spots studied, we identified clonal hemopoiesis in 0.8% of individuals under 60, rising to 19.5% of those ≥90 years, thus predicting that clonal hemopoiesis is much more prevalent than previously realized. DNMT3A-R882 mutations were most common and, although their prevalence increased with age, were found in individuals as young as 25 years. By contrast, mutations affecting spliceosome genes SF3B1 and SRSF2, closely associated with the myelodysplastic syndromes, were identified only in those aged >70 years, with several individuals harboring more than one such mutation. This indicates that spliceosome gene mutations drive clonal expansion under selection pressures particular to the aging hemopoietic system and explains the high incidence of clonal disorders associated with these mutations in advanced old age

Genetic modification of primary human B cells to model high-grade lymphoma

Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models

Genetic modification of primary human B cells to model high-grade lymphoma

Abstract: Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models

Two-particle correlations in azimuthal angle and pseudorapidity in inelastic p + p interactions at the CERN Super Proton Synchrotron

Results on two-particle ΔηΔϕ correlations in inelastic p + p interactions at 20, 31, 40, 80, and 158 GeV/c are presented. The measurements were performed using the large acceptance NA61/SHINE hadron spectrometer at the CERN Super Proton Synchrotron. The data show structures which can be attributed mainly to effects of resonance decays, momentum conservation, and quantum statistics. The results are compared with the Epos and UrQMD models.ISSN:1434-6044ISSN:1434-605

Synthesis and evaluation of l-phenylalanine ester-based chiral ionic liquids for GC stationary phase ability

Several l-phenylalanine alkyl ester-based cations formed ionic liquids after coupling with bis-(perfluoroethylsulfonyl) imide ([(C2F5SO2)2N]) and bis-(trifluoromethanesulfonyl) amide ([(CF3SO2)2N]). The relationship between the structure of l-phenylalanine alkyl ester cations and perfluorinated anions and properties of subsequently synthesized chiral ionic liquids was investigated. The ionic liquids were obtained in good yields and were liquid at room temperature. The ionic liquids having a methyl and ethyl substituent on the cation unit exhibited the highest thermal decomposition temperatures that ranged from 280 °C to 296 °C among the synthesized ionic liquids. These ionic liquids displayed the lowest glass transition temperatures of between − 45 °C to − 51 °C. The introduction of a different alkyl substituents on the cation caused the melting point and viscosity of the ionic liquids to vary slightly. Ionic liquids based on the [(C2F5SO2)2N] anion presented higher melting points than those based on the [(CF3SO2)2N] anion, for each corresponding cation. The neat chiral ionic liquids showed different fluorescence spectral behavior when excited at the same wavelength. Steady-state fluorescence spectroscopic measurements revealed the chiral discrimination of l-Phenylalanine ethyl ester bis-(perfluoroethylsulfonyl) imide for pure enantiomers of 2,2,2-trifluoroanthryl ethanol

Genetic modification of primary human B cells to model high-grade lymphoma

Abstract: Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models