MicroRNA expression in multiple myeloma is associated with genetic subtype, isotype and survival

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited - Copyright @ 2011 Chi et al.Background: MicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. However, the role of microRNAs in the pathogenesis of multiple myeloma (MM) is only poorly understood. We therefore used microarray analysis to elucidate the complete miRNome (miRBase version 13.0) of purified tumor (CD138+) cells from 33 patients with MM, 5 patients with monoclonal gammopathy of undetermined significance (MGUS) and 9 controls. Results: Unsupervised cluster analysis revealed that MM and MGUS samples have a distinct microRNA expression profile from control CD138+ cells. The majority of microRNAs aberrantly expressed in MM (109/129) were up-regulated. A comparison of these microRNAs with those aberrantly expressed in other B-cell and T-cell malignancies revealed a surprising degree of similarity (~40%) suggesting the existence of a common lymphoma microRNA signature. We identified 39 microRNAs associated with the pre-malignant condition MGUS. Twenty-three (59%) of these were also aberrantly expressed in MM suggesting common microRNA expression events in MM progression. MM is characterized by multiple chromosomal abnormalities of varying prognostic significance. We identified specific microRNA signatures associated with the most common IgH translocations (t(4;14) and t(11;14)) and del(13q). Expression levels of these microRNAs were distinct between the genetic subtypes (by cluster analysis) and correctly predicted these abnormalities in > 85% of cases using the support vector machine algorithm. Additionally, we identified microRNAs associated with light chain only myeloma, as well as IgG and IgA-type MM. Finally, we identified 32 microRNAs associated with event-free survival (EFS) in MM, ten of which were significant by univariate (logrank) survival analysis. Conclusions: In summary, this work has identified aberrantly expressed microRNAs associated with the diagnosis, pathogenesis and prognosis of MM, data which will prove an invaluable resource for understanding the role of microRNAs in this devastating disease.This work was funded by grants from Leukaemia and Lymphoma Research (JC, EB, X-HC, DT, JB and JSW) and the Julian Starmer-Smith Memorial Fund (CHL). The authors acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Oxford Radcliffe NHS Trust

Definition of the zebrafish genome using flow cytometry and cytogenetic mapping

<p>Abstract</p> <p>Background</p> <p>The zebrafish (<it>Danio rerio</it>) is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data.</p> <p>Results</p> <p>Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG) chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC) clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescence <it>in situ </it>hybridization (FISH) and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed.</p> <p>Conclusion</p> <p>The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models.</p

Characterization of gene mutations and copy number changes in acute myeloid leukemia using a rapid target enrichment protocol

Prognostic stratification is critical for making therapeutic decisions and maximizing survival of patients with acute myeloid leukemia. Advances in the genomics of acute myeloid leukemia have identified several recurrent gene mutations whose prognostic impact is being deciphered. We used HaloPlex target enrichment and Illumina-based next generation sequencing to study 24 recurrently mutated genes in 42 samples of acute myeloid leukemia with a normal karyotype. Read depth varied between and within genes for the same sample, but was predictable and highly consistent across samples. Consequently, we were able to detect copy number changes, such as an interstitial deletion of BCOR, three MLL partial tandem duplications, and a novel KRAS amplification. With regards to coding mutations, we identified likely oncogenic variants in 41 of 42 samples. NPM1 mutations were the most frequent, followed by FLT3, DNMT3A and TET2. NPM1 and FLT3 indels were reported with good efficiency. We also showed that DNMT3A mutations can persist post-chemotherapy and in 2 cases studied at diagnosis and relapse, we were able to delineate the dynamics of tumor evolution and give insights into order of acquisition of variants. HaloPlex is a quick and reliable target enrichment method that can aid diagnosis and prognostic stratification of acute myeloid leukemia patients.This project was funded by the Wellcome Trust. NB is a fellow of the European Hematology Association and was supported by the Academy of Medical Sciences. EP is a European Hematology Association Advanced Research Fellow. GV is a Wellcome Trust Senior Fellow in Clinical Science. IV is funded by Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal

JAK2 V617F hematopoietic clones are present several years prior to MPN diagnosis and follow different expansion kinetics.

TO THE EDITOR: The JAK2 V617F mutation is the most common somatic mutation in the classical myeloproliferative neoplasms (MPNs), present in >95% of cases of polycythemia vera (PV) and ∼50% of essential thrombocythemia (ET) and myelofibrosis (MF).1⇓⇓-4 It is usually the sole identifiable driver mutation in MPNs5 and was recently also identified as a driver of age-related clonal hemopoiesis in healthy individuals.6⇓⇓-9 In order to investigate the preclinical clonal evolution of MPNs, we identified 12 individuals with a JAK2 V617F mutant MPN, who 4.6 to 15.2 years previously (median 10.2 years) had also donated blood to register with the Cyprus Bone Marrow Donor Registry at the Karaiskakio FoundationThis work was supported by the Wellcome Trust Sanger Institute (WT098051). T. McKerrell is funded by a Wellcome Trust Clinician Scientist Fellowship (100678/Z/12/Z). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA), and work in his laboratory is also funded by Cancer Research UK, Bloodwise, the Kay Kendall Leukaemia Fund, and Celgene. I.V. is supported by the Spanish Ministerio de Economía y Competitividad, Programa Ramón y Cajal

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

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Network Pharmacology and Experimental Validation Reveal the Effects of Chidamide Combined With Aspirin on Acute Myeloid Leukemia-Myelodysplastic Syndrome Cells Through PI3K/AKT Pathway

International audienceChidamide (CDM), a novel histone deacetylase inhibitor, is currently used for patients with peripheral T-cell lymphoma. Aspirin (ASA), an anti-inflammatory drug, has been shown to exert anticancer activity. Herein, we investigated the effect of CDM combined with ASA on myelodysplastic syndromes-derived acute myeloid leukemia (AML-MDS) cells and explored the underlying mechanism. The putative targets of CDM and ASA were predicted by network pharmacology approach. GO functional and KEGG pathway enrichment analyses were performed by DAVID. Furthermore, experimental validation was conducted by Cell Counting Kit-8 assay, Flow cytometry and Western blotting. Network pharmacology analysis revealed 36 AML-MDS-related overlapping genes that were targets of CDM and ASA, while 10 hub genes were identified by the plug-in cytoHubba in Cytoscape. Pathway enrichment analysis indicated CDM and ASA significantly affected PI3K/AKT signaling pathway. Functional experiments demonstrated that the combination of CDM and ASA had a remarkable synergistic anti-proliferative effect by blocking the cell cycle in G0/G1 phase and inducing apoptosis. Mechanistically, the combination treatment significantly down-regulated the phosphorylation levels of PI3K and AKT. In addition, insulin-like growth factor 1 (IGF-1), an activator of PI3K/AKT pathway, reversed the effects of the combination treatment. Our findings suggested that CDM combined with ASA exerted a synergetic inhibitory effect on cell growth by inactivating PI3K/AKT pathway, which might pave the way for effective treatments of AML-MDS