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Fractions Enriched in Recently Born/Divided and Older/Quiescent Cells

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Fractions Enriched in Recently Born/Divided and Older/Quiescent Cells Intraclonal Complexity in Chronic Lymphocytic Leukemia: Fractions Enriched in Recently Born/Divided and Older/Quiescent Cells Carlo Calissano,1 Rajendra N Damle,1,2 Sonia Marsilio,1 Xiao-Jie Yan,1 Sophia Yancopoulos,1 Gregory Hayes,3 Claire Emson,3 Elizabeth J Murphy,3,4 Marc K Hellerstein,3 Cristina Sison,5 Matthew S Kaufman,6 Jonathan E Kolitz,1,2 Steven L Allen,1,2 Kanti R Rai,1,6 Ivana Ivanovic,7 Igor M Dozmorov,7 Sergio Roa,8 Matthew D Scharff,8 Wentian Li,1 and Nicholas Chiorazzi1,2,9 1The Feinstein Institute for Medical Research, North Shore–LIJ Health System, Manhasset, New York, United States of America; 2Department of Medicine, North Shore University Hospital, North Shore–LIJ Health System, Manhasset, and Hofstra North Shore–LIJ School of Medicine, Hempstead, New York, United States of America; 3KineMed, Inc., Emeryville, California, United States of America; 4Department of Medicine, University of California, San Francisco, California, United States of America; 5Biostatistics Unit, The Feinstein Institute for Medical Research, North Shore–LIJ Health System, Manhasset, New York, United States of America; 6Department of Medicine, Long Island Jewish Medical Center, North Shore–LIJ Health System, New Hyde Park, and Hofstra North Shore–LIJ School of Medicine, Hempstead, New York, United States of America; 7Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America; 8Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America; and 9Department of Molecular Medicine, Hofstra North Shore–LIJ School of Medicine, Hempstead, New York, United States of America The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lym- phocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium (2H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4dimCD5bright (proliferative) fraction contained more 2H-labeled DNA and hence divided cells than the CXCR4brightCD5dim (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle–associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4dimCD5bright and CXCR4brightCD5dim fractions indicated higher levels of pro-prolifer- ation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunopheno- type was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, en- riched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two pop- ulations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more ro- bust understanding of the development and clonal evolution of this currently incurable disease. © 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.org Online address: http://www.molmed.org doi: 10.2119/molmed.2011.00360 INTRODUCTION tients live for long periods with the dis- with genomic changes (3), suggesting Chronic lymphocytic leukemia (CLL) ease, many undergo progressive decline, that clonal evolution is a key factor in the is a relatively frequent, incurable B-cell leading to demise. Progression to a more disease. malignancy (1,2). Even though some pa- aggressive disease is often associated We previously found that CLL clones are composed of subpopulations of cells that proliferate at different rates (4), as Address correspondence and reprint requests to Nicholas Chiorazzi, The Feinstein Institute measured by in vivo deuterium (2H)- for Medical Research, 350 Community Drive, Manhasset, NY 11030. Phone: 516-562-1090; incorporation into newly synthesized Fax: 516-562-1011; E-mail: [email protected]. DNA of dividing cells (5,6). The most Submitted September 22, 2011; Accepted for publication September 22, 2011; Epub proliferative fraction of a cancer clone is (www.molmed.org) ahead of print September 23, 2011. of major interest for several reasons. 1374 | CALISSANO ET AL. | MOL MED 17(11-12)1374-1382, NOVEMBER-DECEMBER 2011 RESEARCH ARTICLE First, the “proliferative compartment” 2H Measurements by Gas USA). Purified a-RNA was quantified, may contain cells that developed new Chromatography/Mass Spectrometry and the fragment size was ascertained on structural DNA abnormalities leading to and Calculation of the Fraction of a Bioanalyzer (Agilent Technologies) more lethal disease. Furthermore, the Labeled Cells using an Agilent RNA 6000 Nano Kit. most recently born fraction may be prog- Peripheral blood mononuclear cells Labeled a-RNA was hybridized to a eny of putative leukemic stem cells. Fi- were separated from heparinized venous Human WG-6 v3.0 Expression BeadChip nally, such cells would be potential tar- blood and leukocyte-enriched fractions containing 48,804 probes and stained gets for therapies to abort clonal by density gradient centrifugation using using streptavidin-Cy3 as per the manu- evolution. Ficoll-Paque (Pharmacia LKB Biotechnol- facturer’s instructions. The signal gener- We therefore studied the kinetic com- ogy, Piscataway, NJ, USA) and cryo - ated was detected by high-performance plexity of individual CLL clones to deci- preserved until use. Calculation of the laser optics of the BeadArray Reader (Il- pher fundamental insights about the fraction of newly divided cells was per- lumina, San Diego, CA, USA). Data were pathophysiology of the disease. In partic- formed after determination of 2H enrich- normalized using quantile normalization ular, we focused on further characteriz- ment in plasma or saliva and of 2H en- by BeadStudio software (Illumina). To ing the proliferative and resting compart- richment in deoxyadenosine of genomic determine differentially expressed genes, ments using differences in the densities DNA as described (4). the CXCR4dimCD5bright versus CXCR4bright of a surface membrane molecule upregu- CD5dim expression value ratio was com- lated after normal B-cell activation (clus- Isolation of Cell Fractions on the Basis puted for each patient and log-trans- ter designation 5 [CD5]) and another in- of Expression of CXCR4 and CD5 formed, and a Student t test was per- volved in maintaining B-cell contact with Peripheral blood mononuclear cells formed using R (www.Rproject.org). stromal elements of solid lymphoid tis- were incubated with murine anti–human Significant gene differences had 1.3 or sues (chemokine [C-X-C motif] receptor 4 monoclonal antibodies (mAbs): CD5- greater fold change and P < 0.01; for fold [CXCR4]). Using samples from patients FITC (fluorescein isothiocyanate), change, we used the nth root of the prod- for which CLL cells had been labeled in CXCR4-PE (phycoethrin), CD3–peridinin- uct of all individual ratios, or the geo - vivo with 2H, we divided clones into sub- chlorophyll-protein (CD3-PerCP) and metric mean of the ratio, for n samples. fractions enriched in the most prolifera- CD19-allophycocyanin (CD19-APC) (all Genes were assigned to specific func- tive and most quiescent compartments. from BD Biosciences, San Jose, CA, tional categories using Ingenuity Path- These fractions were then further charac- USA). After gating on CD19+CD3–CD5+ way Analysis (IPA, www.ingenuity.com), terized by comparing expression of events, cells were sorted with a BD Panther Classification System (Panther, genes encoding molecules usually upreg- FACSAria™ (Becton Dickinson Immuno- www.pantherdb.org), DAVID Bioinfor- ulated in dividing or resting populations. cytometry Systems, San Jose, CA, USA) matics Resources 6.7, National Institute of Finally, to provide a robust membrane on the basis of intensity of CXCR4 and Allergy and Infectious Diseases (NIAID)/ map of these compartments that might CD5 (Figures 1A, B), and cell pellets National Institutes of Health (NIH) be used for further characterization and were stored at –80°C until performing (DAVID, http://david.abcc.ncifcrf.gov/ therapeutic targeting in patients, an ex- gas chromato graphy/mass spectrometry home.jsp) and GeneCards Batch Queries tended surface phenotype was defined or RNA extraction. (GeneALaCart, www.genecards.org/ with a larger patient cohort. BatchQueries/index.php). Significantly Gene Expression Profiling and Gene different gene expression values were MATERIALS AND METHODS Expression Data Analyses clustered hierarchically on the basis of av- The quality of RNA extracted from erage linkage on a correlation-based dis- Patients sorted cells using the RNeasy minikit tance (defined at http://arxiv.org/abs/ The Institutional Review Board of the (Qiagen, Valencia, CA, USA) was as- cs/ 0402061). Heatmaps of gene expres- North Shore–LIJ Health System approved sessed using an Agilent RNA 6000 Pico sion values for identified relevant func- these studies. After obtaining informed Kit (Agilent Technologies, Colorado
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