Defining Ploidy-Specific Thresholds in Array Comparative Genomic Hybridization to Improve the Sensitivity of Detection of Single Copy Alterations in Cell Lines
2006; Elsevier BV; Volume: 8; Issue: 4 Linguagem: Inglês
10.2353/jmoldx.2006.060033
ISSN1943-7811
AutoresGrace Ng, Jingxiang Huang, Ian Roberts, Nicholas Coleman,
Tópico(s)Gene expression and cancer classification
ResumoArray comparative genomic hybridization (CGH) is being widely used to screen for recurrent genomic copy number alterations in neoplasms, with imbalances typically detected through the application of gain and loss thresholds. Review of array CGH publications for the year 2005 showed that a wide range of thresholds are used. However, the effect of sample ploidy on the sensitivity of these thresholds for single copy alterations (SCAs) has not been evaluated. Here, we describe a method to evaluate the detection accuracy of thresholds for detecting SCAs in cell line array CGH data. By applying a hidden Markov model-based method, we segmented array CGH data from well-karyotyped cell lines and generated ploidy-specific sensitivity-specificity plots, from which we identified optimum thresholds relevant to sample ploidy. We demonstrate that commonly used nonploidy-specific thresholds are suboptimal in their ability to call SCAs, particularly when applied to hypertriploid or tetraploid cell lines. We conclude that the use of ploidy-specific thresholds improves the sensitivity of thres-hold-based array CGH for detecting SCAs in cell lines. Because polyploidy is a common feature of cancer cells, the application of ploidy-specific thresholds to cell lines (and potentially to clinical samples) may improve the detection sensitivity of SCAs of biological significance. Array comparative genomic hybridization (CGH) is being widely used to screen for recurrent genomic copy number alterations in neoplasms, with imbalances typically detected through the application of gain and loss thresholds. Review of array CGH publications for the year 2005 showed that a wide range of thresholds are used. However, the effect of sample ploidy on the sensitivity of these thresholds for single copy alterations (SCAs) has not been evaluated. Here, we describe a method to evaluate the detection accuracy of thresholds for detecting SCAs in cell line array CGH data. By applying a hidden Markov model-based method, we segmented array CGH data from well-karyotyped cell lines and generated ploidy-specific sensitivity-specificity plots, from which we identified optimum thresholds relevant to sample ploidy. We demonstrate that commonly used nonploidy-specific thresholds are suboptimal in their ability to call SCAs, particularly when applied to hypertriploid or tetraploid cell lines. We conclude that the use of ploidy-specific thresholds improves the sensitivity of thres-hold-based array CGH for detecting SCAs in cell lines. Because polyploidy is a common feature of cancer cells, the application of ploidy-specific thresholds to cell lines (and potentially to clinical samples) may improve the detection sensitivity of SCAs of biological significance. The acquisition of genomic DNA copy number alterations and corresponding changes in expression of genes involved in cellular growth and survival pathways are key events in the development and progression of human cancers. Array comparative genomic hybridization (CGH) represents an efficient approach to screening entire genomes for regions with DNA copy number alterations by providing global information on characteristics of the genome structure. There is considerable interest in applying the technique to identify copy number alterations in neoplasms, using cell lines and clinical samples. With the emergence of increasing array CGH data sets, there is a critical need for an approach that identifies copy number alterations with high sensitivity. In a typical array CGH experiment, genomic DNA is isolated from test and reference samples, differentially labeled, and hybridized to DNA microarrays containing elements mapped to the genome sequence.1Pinkel D Segraves R Sudar D Clark S Poole I Kowbel D Collins C Kuo WL Chen C Zhai Y Dairkee SH Ljung BM Gray JW Albertson DG High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.Nat Genet. 1998; 20: 207-211Crossref PubMed Scopus (1774) Google Scholar The addition of Cot-1 DNA suppresses the hybridization of highly repetitive sequences. Relative differences in signal intensity ratios between test and reference DNA reflect copy number alterations in the test DNA. Before analysis, the data are usually normalized by setting the median of the intensity ratios from the entire genome to 1 on a linear scale.2Pollack JR Sorlie T Perou CM Rees CA Jeffrey SS Lonning PE Tibshirani R Botstein D Borresen-Dale AL Brown PO Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors.Proc Natl Acad Sci USA. 2002; 99: 12963-12968Crossref PubMed Scopus (951) Google Scholar After normalization, the most commonly used method to identify regions of gain or loss is to set thresholds, either arbitrarily or at multiples of the SD (log2 ratio value) of the mean from normal-normal hybridizations.3Veltman JA Fridlyand J Pejavar S Olshen AB Korkola JE DeVries S Carroll P Kuo WL Pinkel D Albertson D Cordon-Cardo C Jain AN Waldman FM Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors.Cancer Res. 2003; 63: 2872-2880PubMed Google Scholar Table 1 summarizes our review of array CGH publications within the year 2005 and the thresholds used therein to define gains and losses. Although threshold-based analysis is in widespread use, justification for the choice of thresholds used is frequently neglected in array CGH publications. Verification of ratio profiles may be limited to the use of fluorescence in situ hybridization (FISH) on a few loci to show the absence of false-positives, whereas the potential presence of false-negative results is not addressed. Importantly, the accuracy (particularly the sensitivity) of commonly used thresholds at calling single copy gains and losses has not been evaluated adequately. Because the linear relationship between intensity ratio and copy number is dependent on the ploidy of the sample,4Mohapatra G Moore DH Kim DH Grewal L Hyun WC Waldman FM Pinkel D Feuerstein BG Analyses of brain tumor cell lines confirm a simple model of relationships among fluorescence in situ hybridization, DNA index, and comparative genomic hybridization.Genes Chromosom Cancer. 1997; 20: 311-319Crossref PubMed Scopus (32) Google Scholar we would expect the thresholds to be ploidy-dependent. As such, true single copy gains and losses may be missed by arbitrarily selected thresholds. This limitation in detection sensitivity is of critical importance in applying threshold-based array CGH to screening cells from neoplasms because polyploidy is a common feature of malignancies and premalignancies.Table 1Thresholds Used to Call Gains and Losses in Array CGH Publications in the Year 2005Reference numberThresholds used (log2 ratio values unless otherwise stated)Reason for selection13Knijnenburg J van der Burg M Nilsson P Ploos van Amstel HK Tanke H Szuhai K Rapid detection of genomic imbalances using micro-arrays consisting of pooled BACs covering all human chromosome arms.Nucleic Acids Res. 2005; 33: e159Crossref PubMed Scopus (4) Google Scholar±0.25None identified14Magnani I Ramona RF Roversi G Beghini A Pfundt R Schoenmakers EF Larizza L Identification of oligodendroglioma specific chromosomal copy number changes in the glioblastoma MI-4 cell line by array-CGH and FISH analyses.Cancer Genet Cytogenet. 2005; 161: 140-145Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 15Veltman I Veltman J Janssen I Hulsbergen-van de Kaa C Oosterhuis W Schneider D Stoop H Gillis A Zahn S Looijenga L Gobel U van Kessel AG Identification of recurrent chromosomal aberrations in germ cell tumors of neonates and infants using genomewide array-based comparative genomic hybridization.Genes Chromosom Cancer. 2005; 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44: 37-51Crossref PubMed Scopus (56) Google Scholar±0.520Tagawa H Karnan S Suzuki R Matsuo K Zhang X Ota A Morishima Y Nakamura S Seto M Genome-wide array-based CGH for mantle cell lymphoma: identification of homozygous deletions of the proapoptotic gene BIM.Oncogene. 2005; 24: 1348-1358Crossref PubMed Scopus (263) Google Scholar, 21Tagawa H Suguro M Tsuzuki S Matsuo K Karnan S Ohshima K Okamoto M Morishima Y Nakamura S Seto M Comparison of genome profiles for identification of distinct subgroups of diffuse large B-cell lymphoma.Blood. 2005; 106: 1770-1777Crossref PubMed Scopus (187) Google Scholar, 22Nakashima Y Tagawa H Suzuki R Karnan S Karube K Ohshima K Muta K Nawata H Morishima Y Nakamura S Seto M Genome-wide array-based comparative hybridization of natural killer cell lymphoma/leukemia: different genomic alteration patterns of aggressive NK-cell leukemia and extranodal NK/T-cell lymphoma, nasal type.Genes Chromosom Cancer. 2005; 44: 247-255Crossref PubMed Scopus (158) Google Scholar±0.2±2 SDs from normal hybridizations23Reis-Filho JS Simpson PT Jones C Steele D Mackay A Iravani M Fenwick K Valgeirsson H Lambros M Ashworth A Palacios J Schmitt F Lakhani SR Pleomorphic lobular carcinoma of the breast: role of comprehensive molecular pathology in characterization of an entity.J Pathol. 2005; 207: 1-13Crossref PubMed Scopus (170) Google Scholar±0.08±3 SDs from normal hybridizations24Coe BP Henderson LJ Garnis C Tsao MS Gazdar AF Minna J Lam S Macaulay C Lam WL High-resolution chromosome arm 5p array CGH analysis of small cell lung carcinoma cell lines.Genes Chromosom Cancer. 2005; 42: 308-313Crossref PubMed Scopus (39) Google Scholar±0.1325Bejjani BA Saleki R Ballif BC Rorem EA Sundin K Theisen A Kashork CD Shaffer LG Use of targeted array-based CGH for the clinical diagnosis of chromosomal imbalance: is less more?.Am J Med Genet A. 2005; 134: 259-267Crossref PubMed Scopus (181) Google Scholar±0.326van Duin M van Marion R Watson JE Paris PL Lapuk A Brown N Oseroff VV Albertson DG Pinkel D de Jong P Nacheva EP Dinjens W van Dekken H Collins C Construction and application of a full-coverage, high-resolution, human chromosome 8q genomic microarray for comparative genomic hybridization.Cytometry A. 2005; 63: 10-19Crossref PubMed Scopus (17) Google Scholar±0.4227Hughes S Damato BE Giddings I Hiscott PS Humphreys J Houlston RS Microarray comparative genomic hybridisation analysis of intraocular uveal melanomas identifies distinctive imbalances associated with loss of chromosome 3.Br J Cancer. 2005; 93: 1191-1196Crossref PubMed Scopus (56) Google Scholar, 28Takada H Imoto I Tsuda H Nakanishi Y Ichikura T Mochizuki H Mitsufuji S Hosoda F Hirohashi S Ohki M Inazawa J ADAM23, a possible tumor suppressor gene, is frequently silenced in gastric cancers by homozygous deletion or aberrant promoter hypermethylation.Oncogene. 2005; 24: 8051-8060Crossref PubMed Scopus (58) Google Scholar±2 SDs of each sample profile±2 SDs of each sample profile29Snijders AM Schmidt BL Fridlyand J Dekker N Pinkel D Jordan RC Albertson DG Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma.Oncogene. 2005; 24: 4232-4242Crossref PubMed Scopus (236) Google Scholar±2.5 SDs of each sample profile±2.5 SDs of each sample profile30Van Esch H Hollanders K Badisco L Melotte C Van Hummelen P Vermeesch JR Devriendt K Fryns JP Marynen P Froyen G Deletion of VCX-A due to NAHR plays a major role in the occurrence of mental retardation in patients with X-linked ichthyosis.Hum Mol Genet. 2005; 14: 1795-1803Crossref PubMed Scopus (95) Google Scholar±3 SDs of all clones±3 SDs of all clones31Davison EJ Tarpey PS Fiegler H Tomlinson IP Carter NP Deletion at chromosome band 20p12.1 in colorectal cancer revealed by high resolution array comparative genomic hybridization.Genes Chromosom Cancer. 2005; 44: 384-391Crossref PubMed Scopus (25) Google Scholar±3 SDs of the normal regions of each sample±3 SDs of the normal regions of each sample32Rubio-Moscardo F Climent J Siebert R Piris MA Martin-Subero JI Nielander I Garcia-Conde J Dyer MJ Terol MJ Pinkel D Martinez-Climent JA Mantle-cell lymphoma genotypes identified with CGH to BAC microarrays define a leukemic subgroup of disease and predict patient outcome.Blood. 2005; 105: 4445-4454Crossref PubMed Scopus (164) Google ScholarGain 0.39; loss −0.5None identified33Koolen DA Reardon W Rosser EM Lacombe D Hurst JA Law CJ Bongers EM van Ravenswaaij-Arts CM Leisink MA van Kessel AG Veltman JA de Vries BB Molecular characterisation of patients with subtelomeric 22q abnormalities using chromosome specific array-based comparative genomic hybridisation.Eur J Hum Genet. 2005; 13: 1019-1024Crossref PubMed Scopus (40) Google Scholar± 0.4Gaussian modeling34de Stahl TD Hartmann C de Bustos C Piotrowski A Benetkiewicz M Mantripragada KK Tykwinski T von Deimling A Dumanski JP Chromosome 22 tiling-path array-CGH analysis identifies germ-line- and tumor-specific aberrations in patients with glioblastoma multiforme.Genes Chromosom Cancer. 2005; 44: 161-169Crossref PubMed Scopus (27) Google Scholar, 35Ammerlaan AC de Bustos C Ararou A Buckley PG Mantripragada KK Verstegen MJ Hulsebos TJ Dumanski JP Localization of a putative low-penetrance ependymoma susceptibility locus to 22q11 using a chromosome 22 tiling-path genomic microarray.Genes Chromosom Cancer. 2005; 43: 329-338Crossref PubMed Scopus (22) Google ScholarLoss by comparison with X chromosome controls; gains using arbitrary valuesX chromosome controls for losses36Callagy G Pharoah P Chin SF Sangan T Daigo Y Jackson L Caldas C Identification and validation of prognostic markers in breast cancer with the complementary use of array-CGH and tissue microarrays.J Pathol. 2005; 205: 388-396Crossref PubMed Scopus (126) Google ScholarGain 1.2; loss 0.69 (absolute ratio values)FISH validation of true positives37Tsubosa Y Sugihara H Mukaisho K Kamitani S Peng DF Ling ZQ Tani T Hattori T Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization.Cancer Genet Cytogenet. 2005; 158: 156-166Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 38Schleiermacher G Bourdeaut F Combaret V Picrron G Raynal V Aurias A Ribeiro A Janoueix-Lerosey I Delattre O Stepwise occurrence of a complex unbalanced translocation in neuroblastoma leading to insertion of a telomere sequence and late chromosome 17q gain.Oncogene. 2005; 24: 3377-3384Crossref PubMed Scopus (21) Google Scholar, 39Le Caignec C De Mas P Vincent MC Boceno M Bourrouillou G Rival JM David A Subtelomeric 6p deletion: clinical, FISH, and array CGH characterization of two cases.Am J Med Genet A. 2005; 132: 175-180Crossref Scopus (32) Google ScholarGain 1.2; loss 0.8 (absolute ratio values)None identified40Wakui K Gregato G Ballif BC Glotzbach CD Bailey KA Kuo PL Sue WC Sheffield LJ Irons M Gomez EG Hecht JT Potocki L Shaffer LG Construction of a natural panel of 11p11.2 deletions and further delineation of the critical region involved in Potocki-Shaffer syndrome.Eur J Hum Genet. 2005; 13: 528-540Crossref PubMed Scopus (31) Google ScholarGain 1.5; loss 0.5 (absolute ratio values)41Kawaguchi K Honda M Yamashita T Shirota Y Kaneko S Differential gene alteration among hepatoma cell lines demonstrated by cDNA microarray-based comparative genomic hybridization.Biochem Biophys Res Commun. 2005; 329: 370-380Crossref PubMed Scopus (15) Google ScholarAmplified 1.8; deleted 0.55 (absolute ratio values)The literature search was done via the National Library of Medicine (NCBI) search engine (), using the search term ′array CGH.′ This search resulted in 90 studies (up to the end of December, 2005). The studies were surveyed for their relevance by reading the abstracts, when available. Twenty-nine relevant studies were found, and these were assessed in more detail. Open table in a new tab The literature search was done via the National Library of Medicine (NCBI) search engine (), using the search term ′array CGH.′ This search resulted in 90 studies (up to the end of December, 2005). The studies were surveyed for their relevance by reading the abstracts, when available. Twenty-nine relevant studies were found, and these were assessed in more detail. In this report, we describe a method to evaluate the detection accuracy of thresholds for single copy alterations (SCAs) in array CGH data from carcinoma cell lines. We identify optimum thresholds relevant to sample ploidy and evaluate the accuracy of these in comparison to standard thresholds (arbitrarily set at ±3 SDs of the mean log2 ratio value from normal-normal hybridizations) at calling SCAs known to exist in well-karyotyped cell lines. We verify that these thresholds are more accurate than standard thresholds at calling SCAs in cell lines. Moreover, our preliminary observations in tumor tissue indicate that these ploidy-specific thresholds may be applicable to clinical samples of known ploidies. We used six cervical keratinocyte cell lines derived from squamous cell carcinomas (SCCs) of the uterine cervix (Table 2). All were obtained from the American Type Culture Collection (Manassas, VA) and cultured as described by the American Type Culture Collection. We also used snap-frozen and formalin-fixed, paraffin-embedded tissue from a cervical SCC that had been shown previously by interphase FISH using centromeric probes to be hypertriploid or tetraploid. The tissue was obtained from the Department of Histopathology, Addenbrooke's Hospital, Cambridge, UK, with local research ethics committee approval. In the frozen sample of the tumor, at least 80% of the cells were malignant.Table 2Details of Cervical Squamous Cell Carcinoma Cell Lines UsedCell lineATCC numberModal chromosome numberC4ICRL-159445C4IICRL-159546ME180HTB-3362SiHaHTB-3569SW756CRL-1030280CaSkiCRL-155080The table shows the cell line name and modal chromosome number determined from cytogenetic analysis (N. Foster, manuscript in preparation). ATCC, American Type Culture Collection. Open table in a new tab The table shows the cell line name and modal chromosome number determined from cytogenetic analysis (N. Foster, manuscript in preparation). ATCC, American Type Culture Collection. Genomic DNA (gDNA) was isolated by conventional phenol/chloroform extraction. gDNA from peripheral blood lymphocytes of a healthy male was used as the reference for normal gene copy numbers. DNA concentrations and quality were determined using the Nanodrop UV spectrophotometer (Nanodrop Technologies, Wilmington, DE). The arrays used were kindly provided by Professor Barbara Weber, University of Pennsylvania (Philadelphia, PA), and contained 4134 bacterial artificial chromosome (BAC) clones that covered the human genome at 1 MB resolution. DNA labeling and hybridization were performed as described previously.5Vissers LE de Vries BB Osoegawa K Janssen IM Feuth T Choy CO Straatman H van der Vliet W Huys EH van Rijk A Smeets D van Ravenswaaij-Arts CM Knoers NV van der Burgt I de Jong PJ Brunner HG van Kessel AG Schoenmakers EF Veltman JA Array-based comparative genomic hybridization for the genomewide detection of submicroscopic chromosomal abnormalities.Am J Hum Genet. 2003; 73: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar One μg each of test DNA from cervical cell lines or tissue sample and reference DNA from normal male peripheral blood lymphocytes were labeled with Cy3-dCTP or Cy5-dCTP (with dye-swapping) using random-prime labeling (BioPrime Plus array CGH labeling module; Invitrogen Ltd., Paisley, UK). Hybridization was performed at 37°C in a shaking water bath for 72 hours. Hybridized arrays were washed in 2× standard saline citrate, 50% formamide, pH 7.0, at 45°C for 15 minutes and 2× standard saline citrate, 0.1% sodium dodecyl sulfate at 45°C for 30 minutes before a final wash in 0.2× standard saline citrate at room temperature for 15 minutes. The arrays were dried in a slide centrifuge before being scanned using an Axon 4000B scanner (Axon Instruments, Burlingame, CA). The acquired Cy3 and Cy5 images were preprocessed with GenePix Pro 4.1 imaging software (Axon Instruments, Foster City, CA). Differences in overall signal intensity between the Cy3 and Cy5 channels were adjusted by normalizing all signal intensities to a 1:1 ratio. For each spot, the median pixel intensities minus the median local backgrounds for both dyes were used to obtain the log2 value of test to reference copy number ratio. Fluorescence ratios of the clones were calculated as the average of paired dye-swapped arrays. Cells lines of various ploidies were selected for the investigation. In accordance with commonly used procedures,6Harris CP Lu XY Narayan G Singh B Murty VV Rao PH Comprehensive molecular cytogenetic characterization of cervical cancer cell lines.Genes Chromosom Cancer. 2003; 36: 233-241Crossref PubMed Scopus (90) Google Scholar we defined the ploidy of each cell line as the copy number of the majority of the genome; that is, present in at least 70% of 50 metaphases examined (N. Foster, I. Roberts, M. R. Pett, N. Coleman, manuscript in preparation). On this basis, we determined the cell lines to be diploid (C4I, C4II), triploid (ME180, SiHa), and hypertriploid (SW756, CaSki), in keeping with published American Type Culture Collection findings. The cytogenetic data from the diploid and triploid cell lines and SW756 were examined to identify regions showing SCAs in more than 70% of metaphases. Whole chromosomes or chromosome arms with 1-copy loss, 1-copy gain, and normal copies relative to the base ploidy of each cell line were selected for further investigation. Only chromosomes with unambiguous karyotype data were used. For more accurate definition of the boundaries of SCAs in the selected chromosome arms, segmentation was performed on the array CGH ratios from the selected chromosomes using the aCGH package for the R statistical language from Bioconductor (/). This package contains a hidden Markov model7Fridlyand J Snijders AM Pinkel D Albertson DG Jain AN Hidden Markov models approach to the analysis of array CGH data.J Multivar Anal. 2004; 90: 132-153Crossref Scopus (271) Google Scholar-based method that assigns clones to states with constant copy number. Clones lying within the segments defined as showing SCAs were selected for further analysis. Because of the limitations of segmentation, some clones within the defined segments remained stateless. To ensure accuracy of the selection, any clone showing focal (ie, isolated) aberrations after segmentation, together with those lying within three positions of the end of each defined SCA segment, were therefore excluded from the analysis. The ability of threshold-based array CGH to detect SCAs at different ploidies was evaluated by comparing receiver-operating characteristic (ROC) curves. For each ploidy, ROC curves were generated for single copy gains and losses by entering the appropriate array fluorescent intensity ratios into the statistical software SPSS 11.5 for Windows (SPSS Inc., Chicago, IL). Sensitivity and specificity at a range of gain and loss thresholds were calculated and plotted to identify optimum thresholds. We evaluated the accuracy of standard thresholds and compared this to the performance of optimum thresholds, when applied to cell lines of different ploidy. Standard thresholds were arbitrarily set at ±3 SDs (log2 ratio value) of the mean of normal-normal hybridizations. These normal hybridizations were of gDNA from normal cervical squamous epithelium, or normal female placenta, versus gDNA from normal male peripheral blood lymphocytes. To evaluate the accuracy of the optimum thresholds for detecting true single copy gains and losses in cell lines, additional array CGH data from the hypertriploid cell lines SW756 and CaSki were analyzed. For the cell line SW756, the chromosomes selected for validation were different from those used in identifying optimum thresholds, whereas CaSki had not been used to identify optimum thresholds. SCAs were called from raw unsegmented data, without exclusion of any clone. A random sampling of clones that were identified as gained or lost by the optimum thresholds, but not the standard thresholds, were selected for verification by BAC-FISH. Metaphase spreads of the cell lines were prepared using standard procedures and FISH was performed as described by Hoglund and colleagues.8Hoglund M Johansson B Pedersen-Bjergaard J Marynen P Mitelman F Molecular characterization of 12p abnormalities in hematologic malignancies: deletion of KIP1, rearrangement of TEL, and amplification of CCND2.Blood. 1996; 87: 324-330PubMed Google Scholar BAC clones were obtained from BACPAC Resources () and DNA was extracted as described previously.9Shing DC McMullan DJ Roberts P Smith K Chin SF Nicholson J Tillman RM Ramani P Cullinane C Coleman N FUS/ERG gene fusions in Ewing's tumors.Cancer Res. 2003; 63: 4568-4576PubMed Google Scholar BAC DNA was labeled with biotin 16-dUTP (Roche, East Sussex, UK) or digoxigenin 11-dUTP (Roche) using nick translation (Vysis, Downers Grove, IL) according to the manufacturer's instructions. After hybridization, probes labeled with biotin were detected with avidin-Cy5 (Amersham Biosciences, Uppsala, Sweden) (1:400) and biotin anti-avidin (1:300), whereas probes labeled with digoxigenin were detected with anti-digoxigenin rhodamine (Roche) (1:200). FISH images were captured with a fluorescence microscope equipped with a charge-coupled device camera, controlled by a Macintosh computer running the SmartCapture (Vysis) software. We performed a preliminary experiment to examine whether ploidy-specific thresholds would improve the sensitivity for detecting SCAs in clinical samples as well as cell lines. Array CGH data for gDNA from a frozen primary cervical SCC was analyzed with optimized thresholds selected using the approach described above and the results were compared with those obtained using the standard thresholds. The sample was known to be hypertriploid or tetraploid, as determined by FISH using centromeric probes on chromosomes 9 and 10, in which more than 70% of the cells examined had three or four centromeric signals (data not shown). Randomly selected clones identified as gained using both thresholds or using the optimum threshold only were used in interphase FISH on formalin-fixed paraffin-embedded sections from the same SCC. FISH on interphase nuclei was performed as described previously.9Shing DC McMullan DJ Roberts P Smith K Chin SF Nicholson J Tillman RM Ramani P Cullinane C Coleman N FUS/ERG gene fusions in Ewing's tumors.Cancer Res. 2003; 63: 4568-4576PubMed Google Scholar Test and control probes were labeled with Spectrum Orange (Vysis) and biotin 16-dUTP, respectively, using nick translation (Vysis). Avidin-Cy5 (1:400) (Amersham) and biotin anti-avidin (1:300) were used to detect biotin-labeled probes. FISH images were captured as before. The karyograms of the diploid (C4I and C4II), triploid (ME180 and SiHa), and hypertriploid (SW756) cell lines were examined, and chromosome segments with SCAs in more than 70% of metaphases were selected. The array CGH ratio profiles of the selected chromosomes were partitioned into copy number states using a hidden Markov model-based method implemented in the R statistical language. The segmentation allowed more precise definitions of the ends of the selected chromosome segments and verified the changes identified by cytogenetic analysis (Figure 1). BAC clones from two or three whole chromosomes or segments at each state (1-copy loss, 1-copy gain, normal) were selected from each cell line. Clones at the same state, from different cell lines of the same ploidy (ie, C4I and C4II; ME180 and SiHa), were pooled in downstream analysis. Pooling was done after Student's t-tests showed no significant difference in the intensity ratio values of clones at the same state from cell lines of the same ploidy (Table 3).Table 3Results of Student's t-tests Performed on Fluorescence Intensity Ratios of BAC Clones at the Same State (1-Copy Loss, 1-Copy Gain, Normal) from Cell Lines of the Same PloidyPloidyCopy number stateCell lineMean ratio value (n)P valueDiploid1-Copy lossC4I0.8164 (85)0.15C4II0.7086 (90)Normal copiesC4I1.0156 (410)0.21C4II1.0067 (478)1-Copy gainC4I1.3370 (23)0.99C4II1.3366 (116)Triploid1-Copy lossME1800.7847 (207)0.16SiHa0.7777 (281)Normal copiesME1801.0279 (189)0.067SiHa1.0081 (349)1-Copy gainME1801.2762 (75)0.070SiHa1.2403 (77)No significant difference between ratio values was detected (P > 0.05). Ratio values of clones at the same state within each ploidy were therefore pooled in downstream analyses. Open table in a new tab No significant difference between ratio values was detected (P > 0.05). Ratio values of clones at the same state within ea
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