769-778 24/3/06 12:54 Page 769 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 17: 769-778, 2006 769 Genomic abnormalities in chronic lymphocytic leukemia influence gene expression by a gene dosage effect JOHN D. DICKINSON1, AVADHUT JOSHI1, JAVEED IQBAL2, WARREN SANGER3, PHILIP J. BIERMAN4 and SHANTARAM S. JOSHI1 1Department of Genetics, Cell Biology, and Anatomy, 2Department of Pathology and Microbiology, 3Center for Human Genetics, 4Oncology/Hematology Section, Department of Internal Medicine, Center for Research in Leukemia and Lymphoma, University of Nebraska Medical Center, Omaha, NE, USA Received November 16, 2005; Accepted December 21, 2005 Abstract. This work describes the identification and impact lymphocytes and is identified by co-expression of CD5, 19, of somatic genomic abnormalities in human chronic lympho- and 23 cell surface markers (3,4). CLL has been characterized cytic leukemia (CLL). Using molecular cytogenetics (FISH) by broad clinical and biological heterogeneity and not all and G-banding cytogenetic analysis, chromosome abnormalities patients require immediate therapy (3,5,6). Much of the were detected in 37 of 46 (80.4%) CLL patients. 13q14 research in the area of CLL has focused on identifying prog- deletion was the most common finding followed by trisomy nostic factors that can predict which patients will ultimately 12 and 11q22.3 deletion. 17p13 deletion was also detected as undergo tumor progression and require therapy. In addition, were several less frequent chromosome abnormalities. The elucidating the mechanisms of tumor progression in order to presence of these abnormalities significantly influenced the develop new molecular therapies and adjuvant therapies to period of treatment-free survival as well as other clinical induce long-term remissions is an important goal of CLL characteristics. In particular, CLL samples with trisomy 12 research. and 11q22.3 deletion were associated with shorter treatment- Genomic abnormalities have been detected in CLL for free survival. In order to identify the under-lying molecular decades but no single abnormality has been identified which differences among CLL subgroups with different chromosome is common to all CLL cases as in other similar hematological abnormalities, gene expression profiling was performed on a malignancies (7-10). The more precise definition of CLL on custom DNA microarray consisting of 10,000 human gene- the basis of its cell surface phenotype and the increased specific oligonucleotides. A gene dosage effect was observed resolution of detection due to the use of fluorescent in situ where the expression of genes at the genetic loci of the sites hybridization (FISH) led to reassessment of the role of genomic of the somatic genomic abnormality was altered in a fashion abnormalities in CLL. Deletions of 13q14, 11q22.3, and 17p13, according to the type of genomic change. This phenomenon and trisomy 12 are all recognized to be common genomic was particularly evident in CLL samples with trisomy 12 and aberrations in CLL (8,11). These findings have led to increased 17p13 deletion. Thus, this study demonstrates that genomic interest in the role of these genomic abnormalities in the abnormalities influence gene expression in CLL by a dosage pathogenesis and progression of this malignancy. We have effect. previously demonstrated, in this subset of CLL samples, that the presence of distinct genomic abnormalities is correlated Introduction with the immunoglobulin heavy chain variable region (IgVH) mutation status of the malignant B cell (12). CLL samples Chronic lymphocytic leukemia (CLL) is the most common with trisomy 12 or 11q22.3 deletion were found almost entirely leukemia in North America and Europe and yet it remains in the unmutated IgVH subset. Thus, the presence of certain largely incurable (1,2). CLL is derived from malignant B chromosome abnormalities may be indicative of particular tumor development pathways with differing clinical outcomes. The purpose of this study was to characterize the gene _________________________________________ expression patterns among CLL samples in different genomic abnormality subgroups using a custom DNA microarray Correspondence to: Professor Shantaram S. Joshi, Department spotted with 10,000 human oligonucleotides. Here, we found of Genetics, Cell Biology, and Anatomy, University of Nebraska that the presence of different genomic abnormalities in CLL Medical Center, 986395 Nebraska Medical Center, Omaha, NE was associated with treatment-free survival and that they had 68198-6395, USA a specific impact on gene expression by a gene dosage effect. E-mail: [email protected] Materials and methods Key words: genomic abnormalities, chronic lymphocytic leukemia Isolation of mononuclear tumor cells from peripheral blood. Peripheral blood samples from 46 CLL patients were collected 769-778 24/3/06 12:54 Page 770 770 DICKINSON et al: GENOMIC ABNORMALITIES IMPACT CLL GENE EXPRESSION by venipuncture under Institutional Review Board (IRB) (Invitrogen Carlsbad, CA). Each cDNA was then incubated approval with informed consent from the Nebraska Medical with the appropriate nucleotide linked fluorescent marker in Center, Alegent Health System, and Methodist Hospital in the dark for 30 min. An indirect labeling method was used to Omaha, NE. Mononuclear cells were isolated from whole couple the Cy5 dye to the CLL cDNA and the Cy3 dye to the blood by density centrifugation with Accu-Prep™ (Accurate human reference cDNA after reverse transcription. The labeled Chemical & Scientific Corp., Westbury, NY) lymphocyte cDNA was then washed and purified by standard protocols. separation medium. In order to assess the quantity and purity The two cDNAs were mixed together and denatured at 95˚C of the CLL tumor cells from peripheral blood, the cell surface for 3 min followed by a quick chill on ice. The probe mixture phenotype was assessed by detection of fluorescent labeled was added to the microarray glass slide under a cover slip in antibodies using flow cytometry as previously described (12). the hybridization chamber. After 16 h of hybridization (over- night) at 42˚C, the slide was then washed three times in Detection of genomic abnormalities. Conventional cytogenetic solutions with successively lower amounts of SSC and SDS. analysis with G-banding was performed on CLL samples GenePix Pro 5.1 microarray image analysis software (Axon using either unstimulated cultured bone marrow cells or Instruments, Union City, CA) was then used to quantify the phytohemmagluttin (PHA) stimulated peripheral blood cells. image intensity and provide gene identifiers to each spot on Cells were cultured for 24-48 h at 37˚C in CHANG BMC the array image. Low intensity spots were filtered out if the media (Irvine Scientific, Irvine, CA) as described previously signal intensity in both channels was <100 and the signal to (13). Briefly, the cultured cells were harvested after 20-min noise ratio (SNR) was <2.0. Spots with non-specific binding incubation at 37˚C in a hypotonic solution of 0.4% KCL, plus or contamination were labeled as outliers, flagged, and trypsin and 0.08 mg/ml colcemid, which arrests the cells in removed. The median of ratio expression value (Rm) was metaphase. Up to 20 metaphases were used for analysis then log2 transformed. after the cells had been fixed and prepared for G-banding. Normalization of the raw intensities from different arrays Fluorescent in situ hybridization (FISH) was performed on was performed by the median normalization method using all samples. For each CLL cell sample, four hybridization BRB Array tools, version 3.2. The dataset was filtered so that areas were prepared on a Thermatron drying chamber at 25˚C genes missing >50% of the median of ratio values were and 47% humidity. FISH probes were mixed in a proportion removed from subsequent analyses. Clustering of the gene of 7 μl lysis buffer with 2 μl of probe and 1 μl of sterile expression data for CLL samples was performed using water. The cellular DNA and FISH probes were denatured at hierarchical cluster with Pearson's correlation and average 75˚C for 1 min and then allowed to hybridize for 18 h at 37˚C linkage. One-way analysis of variance (ANOVA) was used in a HyBrite hybridization oven (Vysis Inc., Downers Grove, to compare variance in gene expression across the five CLL IL). The fluorescein-labeled anti-digoxigenin detection agent subgroups with different chromosome abnormalities relative was then added to the hybridization chamber and allowed to variation in gene expression within groups (14,15). In to incubate for 10 min at 37˚C. The samples were then order to identify differentially expressed genes in pairwise examined with an Olympus BX-60 fluorescent microscope. comparisons of genomic abnormality groups, non-parametric Two hundred cells were observed for genomic abnormalities SAM (significance analysis of microarrays) was used in a and the average percentage of a given abnormality was taken manner previously described (16). In order to reduce the from two observations. The FISH panel included CEP 12- number of genes selected and increase the robustness of the trisomy 12 DNA probe, which spans the centromere from comparison, SAM analysis was used on genes previously 12q11 to 12p11, LSI IGH dual color breakpoint probe for selected by t-test in a manner as described by Shen et al (17). 14q32, D13S25 13q14.3 DNA probe, LSI 17p-p53 DNA probe at 17p13.1, and LSI 11q22.3-ATM DNA probe which spans Gene expression analysis by PCR. Total RNA was isolated 4 genes at 11q22.3 (Cul5, ACAT, NPAT, and ATM) (Vysis from CLL cells by TRIzol™ reagent (Invitrogen). RNA pellets Inc.). were dissolved in HPLC grade H2O (Sigma, St. Louis, MO) and stored at -80˚C. The quality and quantity of RNA were 10,000 human oligonucleotide arrays. This research describes determined by the measured 260/280 nm spectroscopic ratio the analysis of gene expression data from CLL samples on as well as running the total RNA on a 1% TAE (Tris-actetate- a 10,000 (10K) gene microarray.