Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis

Genome-wide association analysis implicates dysregulation of immunity genes in chronic lymphocytic leukaemia

Several chronic lymphocytic leukaemia (CLL) susceptibility loci have been reported; however, much of the heritable risk remains unidentified. Here we perform a meta-analysis of six genome-wide association studies, imputed using a merged reference panel of 1,000 Genomes and UK10K data, totalling 6,200 cases and 17,598 controls after replication. We identify nine risk loci at 1p36.11 (rs34676223, P=5.04 × 10−13), 1q42.13 (rs41271473, P=1.06 × 10−10), 4q24 (rs71597109, P=1.37 × 10−10), 4q35.1 (rs57214277, P=3.69 × 10−8), 6p21.31 (rs3800461, P=1.97 × 10−8), 11q23.2 (rs61904987, P=2.64 × 10−11), 18q21.1 (rs1036935, P=3.27 × 10−8), 19p13.3 (rs7254272, P=4.67 × 10−8) and 22q13.33 (rs140522, P=2.70 × 10−9). These new and established risk loci map to areas of active chromatin and show an over-representation of transcription factor binding for the key determinants of B-cell development and immune response

Genome-wide association analysis implicates dysregulation of immunity genes in chronic lymphocytic leukaemia

Several chronic lymphocytic leukaemia (CLL) susceptibility loci have been reported; however, much of the heritable risk remains unidentified. Here we perform a meta-analysis of six genome-wide association studies, imputed using a merged reference panel of 1,000 Genomes and UK10K data, totalling 6,200 cases and 17,598 controls after replication. We identify nine risk loci at 1p36.11 (rs34676223, P=5.04 × 10−13), 1q42.13 (rs41271473, P=1.06 × 10−10), 4q24 (rs71597109, P=1.37 × 10−10), 4q35.1 (rs57214277, P=3.69 × 10−8), 6p21.31 (rs3800461, P=1.97 × 10−8), 11q23.2 (rs61904987, P=2.64 × 10−11), 18q21.1 (rs1036935, P=3.27 × 10−8), 19p13.3 (rs7254272, P=4.67 × 10−8) and 22q13.33 (rs140522, P=2.70 × 10−9). These new and established risk loci map to areas of active chromatin and show an over-representation of transcription factor binding for the key determinants of B-cell development and immune response

Bioactivity-Guided Synthesis: In Silico and In Vitro Studies of <i>β</i>-Glucosidase Inhibitors to Cope with Hepatic Cytotoxicity

The major cause of hyperglycemia can generally be attributed to β-glucosidase as per its involvement in non-alcoholic fatty liver disease. This clinical condition leads to liver carcinoma (HepG2 cancer). The phthalimides and phthalamic acid classes possess inhibitory potential against glucosidase, forming the basis for designing new phthalimide and phthalamic acid analogs to test their ability as potent inhibitors of β-glucosidase. The study also covers in silico (molecular docking and MD simulations) and in vitro (β-glucosidase and HepG2 cancer cell line assays) analyses. The phthalimide and phthalamic acid derivatives were synthesized, followed by spectroscopic characterization. The mechanistic complexities associated with β-glucosidase inhibition were identified via the docking of the synthesized compounds inside the active site of the protein, and the results were analyzed in terms of the best binding energy and appropriate docking pose. The top-ranked compounds were subjected to extensive MD simulation studies to understand the mode of interaction of the synthesized compounds and binding energies, as well as the contribution of individual residues towards binding affinities. Lower RMSD/RMSF values were observed for 2c and 3c, respectively, in the active site, confirming more stabilized, ligand-bound complexes when compared to the free state. An anisotropic network model was used to unravel the role of loop fluctuation in the context of ligand binding and the dynamics that are distinct to the bound and free states, supported by a 3D surface plot. An in vitro study revealed that 1c (IC50 = 1.26 µM) is far better than standard acarbose (2.15 µM), confirming the potential of this compound against the target protein. Given the appreciable potential of the candidate compounds against β-glucosidase, the synthesized compounds were further tested for their cytotoxic activity against hepatic carcinoma on HepG2 cancer cell lines. The cytotoxicity profile of the synthesized compounds was performed against HepG2 cancer cell lines. The resultant IC50 value (0.048 µM) for 3c is better than the standard (thalidomide: IC50 0.053 µM). The results promise the hypothesis that the synthesized compounds might become potential drug candidates, given the fact that the β-glucosidase inhibition of 1c is 40% better than the standard, whereas compound 3c holds more anti-tumor activity (greater than 9%) against the HepG2 cell line than the known drug

Cell surface phenotype profiles distinguish stable and progressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is clinically heterogeneous. While some patients have indolent disease for many years, 20-30% will progress and ultimately die of their disease. CLL may be classified by the Rai or Binet staging system, mutational status of the immunoglobulin variable heavy-chain gene (IGVH), ZAP-70 overexpression, cytogenetic abnormalities (13q-, + 12, 11q-, 17p-) and expression of several cell surface antigens (CD38, CD49d) that correlate with risk of disease progression. However, none of these markers identify all cases of CLL at risk. In a recent review, we summarized those CD antigens known to correlate with the prognosis of CLL. The present study has identified surface profiles of CD antigens that distinguish clinically progressive CLL from slow-progressive and stable CLL. Using an extended DotScan? CLL antibody microarray (Version 3; 182 CD antibodies), and with refined analysis of purified CD19 + B-cells, the following 27 CD antigens were differentially abundant for progressive CLL: CD11a, CD11b, CD11c, CD18, CD19, CD20 (two epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD180, CD196 and CD270. The extensive surface profiles obtained provide disease signatures with an accuracy of 79.2%, a sensitivity of 83.9% and a specificity of 72.5% that could provide the basis for a rapid test to triage patients with CLL according to probability of clinical progression and potential earlier requirement for treatment.8 page(s

Myogenesis and Analysis of Antimicrobial Potential of Silver Nanoparticles (AgNPs) against Pathogenic Bacteria

The widespread and indiscriminate use of broad-spectrum antibiotics leads to microbial resistance, which causes major problems in the treatment of infectious diseases. However, advances in nanotechnology have opened up new domains for the synthesis and use of nanoparticles against multidrug-resistant pathogens. The traditional approaches for nanoparticle synthesis are not only expensive, laborious, and hazardous but also have various limitations. Therefore, new biological approaches are being designed to synthesize economical and environmentally friendly nanoparticles with enhanced antimicrobial activity. The current study focuses on the isolation, identification, and screening of metallotolerant fungal strains for the production of silver nanoparticles, using antimicrobial activity analysis and the characterization of biologically synthesized silver nanoparticles by X-ray diffraction (XRD) spectroscopy, energy-dispersive X-ray spectroscopy (EDX), and scanning electron microscopy (SEM). In total, 11 fungal isolates were isolated and screened for the synthesis of AgNPs, while the Penicillium notatum (K1) strain was found to be the most potent, demonstrating biosynthetic ability. The biologically synthesized silver nanoparticles showed excellent antibacterial activity against the bacteria Escherichia coli (ATCC10536), Bacillus subtilis, Staphylococcus aureus (ATCC9144), Pseudomonas aeruginosa (ATCC10145), Enterococcus faecalis, and Listeria innocua (ATCC13932). Furthermore, three major diffraction peaks in the XRD characterization, located at the 2θ values of 28.4, 34.8, 38.2, 44, 64, and 77°, confirmed the presence of AgNPs, while elemental composition analysis via EDX and spherical surface topology with a scanning electron microscope indicated that its pure crystalline nature was entirely composed of silver. Thus, the current study indicates the enhanced antibacterial capability of mycologically synthesized AgNPs, which could be used to counter multidrug-resistant pathogens

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis. Introduction Chromosomal translocations to the immunoglobulin heavy chain (IGH) or light chain (IGL or IGK) loci, and less frequently to non-IG loci represent well known mechanisms of oncogene activation in B-cell neoplasms. 1,2 Many of the genes deregulated by chromosomal translocations in these neoplasms are involved in important cellular processes like cell-cycle control (CCND1, CCND3, BCL6, and CDK6), apoptosis (BCL2), proliferation (MYC), or signal transduction (BCL3 and MALT1). Methods Patient samples The features of B-cell malignancies showing aberrations in the TERT region (5p1) are listed in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). The study was performed as part of the &quot;Molecular Mechanisms in Malignant Lymphoma&quot; Network Project for which approval from the Institutional Review Board of the Medical Faculity of the Christian-Albrechts-University Kiel has been obtained. FISH Fluorescence in situ hybridization (FISH) was performed on fixed cells from bone marrow, peripheral blood, or lymph node cell suspensions as described previously. 7 Generation of FISH probes as well as FISH procedures are described in supplemental Methods. A list of FISH probes used is shown in supplemental Array CGH to custom-designed arrays The microarrays were designed using the eArray software from Agilent (https://earray.chem.agilent.com/earray/) and the 4 ϫ 44k format. The experimental procedures were performed according to the manufacturer&apos;s instructions. Arrays were scanned with the GenePix4000B Scanner (Axon Instruments) and log ratios obtained with the comparative genomic hybridization (CGH) Analytics Version 3.5.14 software (Agilent). All microarray data are available to be viewed at ArrayExpress under accession number E-MEXP-2675 (http://www.ebi.ac.uk/miamexpress/). Quantitative reverse transcription PCR RNA was extracted from tumor cell-containing samples and control tissue, using the RNeasy Mini Kit (QIAGEN) and transcribed into cDNA with the QuantiTect Rev. Transcription Kit (QIAGEN). TERT transcripts were amplified and detected as described elsewhere. 8 TRAP assay The PCR-based telomeric repeat amplification protocol (TRAP) assay was performed as described previously. 9,10 Protein from tumor cell containing samples and control tissue was isolated according to Kim et al. 11 Additional information about materials and methods is available in supplemental Methods. Results and discussion By FISH screening of B-cell neoplasms for translocations affecting the IGH locus we observed a cytogenetically cryptic translocation t(5;14)(p15;q32) involving the IGH locus in 3 cases of chronic lymphocytic leukemia (CLL) and one case of precursor B-cell acute lymphoblastic leukemia (ALL; The IGH locus harbors strong transcriptional enhancers, which can activate oncogenes by translocation on both der(14) and der(5) chromosomes 12 (supplemental To investigate the frequency of rearrangements in the TERT-CLPTM1L region we screened additional cases by FISH (supplemental None of the additional 6 cases with translocations detected by FISH showed juxtaposition to any of the 3 IG loci. Cytogenetically, in 4 of the 6 variant translocations (ie, non-IG), 7p11, 9q31ϳ33, 10q25 and 19p13 were identified as partners of 5p15. In a splenic marginal zone lymphoma (SMZL, case 5) with t(5;7)(p15.33;p11), FISH and tiling array CGH again confirmed a breakpoint centromeric to TERT with loss of CLPTM1L similar to that in the t(5;14)-positive CLL mentioned above ( The inactivation of CLPTM1L by deletion and disruption in 3/10 cases with break at the TERT-CLPTM1L region and also the fact that in precursor B-cell ALL with IGH translocation oncogene activation has always been assigned to the der14 chromosome 13 (supplemental To investigate the effect of the identified chromosomal aberrations on TERT expression, we performed qRT-PCR in cases with t(5;14)/TERT-IGH juxtaposition (n ϭ 3; 1318 NAGEL et al BLOOD, 26 AUGUST 2010 ⅐ VOLUME 116, NUMBER 8 We next assessed whether the up-regulation of TERT mRNA was also associated with increased telomerase activity. Using the TRAP assay, cases of CLL (cases 1 and 6) and MCL (cases 8 and 9) with chromosomal breakpoints at the TERT-CLPTM1L locus showed markedly higher telomerase activity compared with appropriate disease and tissue controls ( Taken together, we show that the TERT-CLPTM1L region in 5p15 is recurrently targeted by chromosomal translocations in B-cell neoplasms. Based on the finding that CLPTM1L is recurrently lost or disrupted by the mentioned aberrations, it does not seem to contribute to the process of lymphomagenesis. Interestingly, germ line sequence variants in the TERT-CLPTM1L region were recently shown to be associated with predisposition for several cancer types. 14-17 All 6 additional translocations and both amplifications identified herein were of somatic origin as cells without the aberration were always present. The pattern of IGH translocations as well as the increased TERT expression and telomerase activation in cases with 5p15 translocations strongly suggest TERT to be the candidate gene involved in lymphomagenesis. Telomerase activity, which is clearly detectable in up to 90% of human tumors, including hematologic neoplasias, but not in most normal somatic cells, is in part explained by changes in chromatin structure or amplification of the TERT locus

Defining the prognosis of early stage chronic lymphocytic leukaemia patients

Approximately 70% of chronic lymphocytic leukaemia (CLL) patients present with early stage disease, therefore defining which patients will progress and require treatment is a major clinical challenge. Here, we present the largest study of prognostic markers ever carried out in Binet stage A patients (n = 1154) with a median follow-up of 8 years. We assessed the prognostic impact of lymphocyte doubling time (LDT), immunoglobulin gene (IGHV) mutation status, CD38 expression, ZAP-70 expression and fluorescence in situ hybridization (FISH) cytogenetics with regards to time to first treatment (TTFT) and overall survival (OS). Univariate analysis revealed LDT as the most prognostic parameter for TTFT, with IGHV mutation status most prognostic for OS. CD38 expression, ZAP-70 expression and FISH were also prognostic variables; combinations of these markers increased prognostic power in concordant cases. Multivariate analysis revealed that only LDT, IGHV mutation status, CD38 and age at diagnosis were independent prognostic variables for TTFT and OS. Therefore, IGHV mutation status and CD38 expression have independent prognostic value in early stage CLL and should be performed as part of the routine diagnostic workup. ZAP-70 expression and FISH were not independent prognostic markers in early stage disease and can be omitted at diagnosis but FISH analysis should be undertaken at disease progression to direct treatment strategy

HHV-8-unrelated primary effusion-like lymphoma associated with clonal loss of inherited chromosomally-integrated human herpesvirus-6A from the telomere of chromosome 19q

Primary effusion lymphomas (PEL) are associated with human herpesvirus-8 (HHV-8) and usually occur in immunocompromised individuals. However, there are numerous reports of HHV-8-unrelated PEL-like lymphomas with unknown aetiology. Here we characterize an HHV-8-unrelated PEL-like lymphoma in an elderly woman who was negative for human immunodeficiency viruses 1 and 2, and hepatitis B and C. The woman was, however, a carrier of an inherited-chromosomally-integrated human herpesvirus-6A (iciHHV-6A) genome in one 19q telomere. The iciHHV-6A genome was complete in blood DNA, encoding a full set of protein-coding genes. Interestingly, the entire iciHHV-6A genome was absent from the HHV-8-unrelated-PEL-like lymphoma cells despite retention of both copies of chromosome 19. The somatic loss of the 19q-iciHHV-6A genome occurred very early during lymphoma development and we propose it occurred via telomere-loop formation and excision to release a circular viral genome that was subsequently lost. Whether release of the HHV-6A genome from the telomere contributed to lymphomagenesis, or was coincidental, remains unclear but this event may have deregulated the expression of HHV-6A or 19q genes or else disrupted telomere function. To establish the frequency and importance of iciHHV-6 loss from telomeres, the HHV-6 copy number should be assessed in tumours that arise in iciHHV-6 carriers