High-Resolution Analysis of Paraffin-Embedded and Formalin-Fixed Prostate Tumors Using Comparative Genomic Hybridization to Genomic Microarrays
2003; Elsevier BV; Volume: 162; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63873-4
ISSN1525-2191
AutoresPamela L. Paris, Donna G. Albertson, Janneke C. Alers, Armann Andaya, Peter R. Carroll, Jane Fridlyand, Ajay N. Jain, Sherwin Kamkar, David Kowbel, Pieter-Jaap Krijtenburg, Daniel Pinkel, Fritz H. Schröder, Kees J. Vissers, Vivienne J.E. Watson, Mark F. Wildhagen, Colin C. Collins, Herman van Dekken,
Tópico(s)Gene expression and cancer classification
ResumoWe have used prostate cancer, the most commonly diagnosed noncutaneous neoplasm among men, to investigate the feasibility of performing genomic array analyses of archival tissue. Prostate-specific antigen and a biopsy Gleason grade have not proven to be accurate in predicting clinical outcome, yet they remain the only accepted biomarkers for prostate cancer. It is likely that distinct spectra of genomic alterations underlie these phenotypic differences, and that once identified, may be used to differentiate between indolent and aggressive tumors. Array comparative genomic hybridization allows quantitative detection and mapping of copy number aberrations in tumors and subsequent associations to be made with clinical outcome. Archived tissues are needed to have patients with sufficient clinical follow-up. In this report, 20 formalin-fixed and paraffin-embedded prostate cancer samples originating from 1986 to 1996 were studied. We present a straightforward protocol and demonstrate the utility of archived tissue for array comparative genomic hybridization with a 2400 element BAC array that provides high-resolution detection of both deletions and amplifications. We have used prostate cancer, the most commonly diagnosed noncutaneous neoplasm among men, to investigate the feasibility of performing genomic array analyses of archival tissue. Prostate-specific antigen and a biopsy Gleason grade have not proven to be accurate in predicting clinical outcome, yet they remain the only accepted biomarkers for prostate cancer. It is likely that distinct spectra of genomic alterations underlie these phenotypic differences, and that once identified, may be used to differentiate between indolent and aggressive tumors. Array comparative genomic hybridization allows quantitative detection and mapping of copy number aberrations in tumors and subsequent associations to be made with clinical outcome. Archived tissues are needed to have patients with sufficient clinical follow-up. In this report, 20 formalin-fixed and paraffin-embedded prostate cancer samples originating from 1986 to 1996 were studied. We present a straightforward protocol and demonstrate the utility of archived tissue for array comparative genomic hybridization with a 2400 element BAC array that provides high-resolution detection of both deletions and amplifications. Array comparative genomic hybridization (aCGH) affords high-resolution quantitative information regarding DNA copy number changes within tumor genomes. Recurrent deletions and amplifications reveal loci encoding tumor suppressor genes and oncogenes, respectively, and their identification is now facilitated by completion of the human genome sequence. Moreover, it is increasingly evident that differing clinical behavior of histologically similar tumors is because of distinct and recurrent patterns of genomic alterations and that these may define distinct subsets of disease and thus be prognostic.1Bepler G Gautam A McIntyre LM Beck AF Chervinsky DS Kim YC Pitterle DM Hyland A Prognostic significance of molecular genetic aberrations on chromosome segment 11p15.5 in non-small-cell lung cancer.J Clin Oncol. 2002; 20: 1353-1360Crossref PubMed Scopus (81) Google Scholar, 2Elo JP Harkonen P Kyllonen AP Lukkarinen O Vihko P Three independently deleted regions at chromosome arm 16q in human prostate cancer: allelic loss at 16q24.1-q24.2 is associated with aggressive behavior of the disease, recurrent growth, poor differentiation of the tumor and poor prognosis for the patient.Br J Cancer. 1999; 79: 156-160Crossref PubMed Scopus (44) Google Scholar, 3Kressner U Inganas M Byding S Blikstad I Pahlman L Glimelius B Lindmark G Prognostic value of p53 genetic changes in colorectal cancer.J Clin Oncol. 1999; 17: 593-599PubMed Google Scholar, 4Sisley K Rennie IG Parsons MA Jacques R Hammond DW Bell SM Potter AM Rees RC Abnormalities of chromosomes 3 and 8 in posterior uveal melanoma correlate with prognosis.Genes Chromosom Cancer. 1997; 19: 22-28Crossref PubMed Scopus (291) Google Scholar aCGH is ideally suited to delineating these genomic differences and for the development of biomarkers. However, the coupling of aCGH and clinical data for biomarker discovery and disease stratification requires patients with significant clinical follow-up, meaning that in most cases archived tumor material must be used to identify associations between copy number changes and clinical outcome. In aCGH, microarrayed BAC DNA targets are co-hybridized with differentially fluorophore-labeled DNAs from normal reference and tumor test genomes.5Pinkel D Segraves R Sdar D Clark S Poole I Kowbel D Collins C Kuo W-L Chen C Zhai Y Dairkee SH Ljung B-M 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 (1790) Google Scholar, 6Wessendorf S Fritz B Wrobel G Nessling M Lampel S Goettel D Kuepper M Joos S Hopman T Kokocinski F Dohner H Bentz M Schwaenen C Lichter P Automated screening for genomic imbalances using matrix-based comparative genomic hybridization.Lab Invest. 2002; 82: 47-60Crossref PubMed Scopus (77) Google Scholar Gene copy number along the genome is proportional to the ratio of the fluorescent intensities. In this study, we used a whole genome-scanning array containing DNA from 2460 BAC clones, which provides an average resolution of ∼1.4 Mb.7Snijders AM Nowak N Segraves R Blackwood S Brown N Conroy J Hamilton G Hindle AK Huey B Kimura K Law S Myambo K Palmer J Ylstra B Yue JP Gray JW Jain AN Pinkel D Albertson DG Assembly of microarrays for genome-wide measurement of DNA copy number.Nat Genet. 2001; 29: 263-264Crossref PubMed Scopus (774) Google Scholar An advantage of this array is that all of the clones have been mapped on the UCSC genome assembly (http://genome.ucsc.edu/index.html) and thus can be computationally linked to the underlying and annotated genome sequence. Paraffin-embedded tissue has recently been reported to be suitable for aCGH; however the array in their study only consisted of 58 BACS containing known oncogenes.8Daigo Y Chin SF Gorringe KL Bobrow LG Ponder BA Pharoah PD Caldas C Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers: a new approach for the molecular analysis of paraffin-embedded cancer tissue.Am J Pathol. 2001; 158: 1623-1631Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar In addition, their technique required degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification of the tumor material. In this report, we show that archived prostate tissue can be used with a whole genome-scanning array without a probe amplification step, eliminating the possibility of introducing artifactual copy number gains and losses that can result from biased amplification. In contrast to the expensive and laborious method of laser microdissection, we demonstrate that the use of a bore coupled to a microscope for the dissection step works well. Our extraction procedure also includes a xylene treatment, RNase treatment, and protein precipitation in addition to the lysis step. Our method for aCGH on paraffin-embedded tissue allows the identification of DNA copy number changes at high resolution including detection of single copy losses and gains. These genome-wide copy number abnormalities (CNAs) in turn can provide valuable insight into critical genomic clones and genes that may serve as biomarkers. Detection of CNAs was confirmed through comparison to chromosome CGH (some previously published)9Alers JC Rochat J Krijtenburg PJ Hop WCJ Kranse R Rosenberg C Tanke HJ Schröder FH van Dekken H Identification of genetic markers for prostate cancer progression.Lab Invest. 2000; 80: 931-942Crossref PubMed Scopus (157) Google Scholar, 10Alers JC Krijtenburg PJ Vis AN Hoedemaeker RF Wildhagen MF Hop WCJ van der Kwast TH Schröder FH Tanke HJ van Dekken H Molecular cytogenetic analysis of prostatic adenocarcinomas from screening studies: early cancers may contain aggressive genetic features.Am J Pathol. 2001; 158: 399-406Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar using the same tumor DNA and through use of quantitative microsatellite analysis (QuMA), a quantitative PCR technique related to TaqMan. Archival prostate cancer specimens were obtained between 1986 and 1996. Four metastases (two brain, one regional lymph node, and one bone metastasis; samples 1 to 4) and 16 primary tumors from patients who underwent radical prostatectomy alone (samples 5 to 20) were included in this preliminary study. The tumors were pathologically staged according to the pTNM classification11Hermanek P Hutter RVP Sobin LH TNM Atlas IUAC. ed 4. Springer, New York1997Crossref Google Scholar and graded according to the Gleason grading system.12Gleason DF Histologic grading of prostate cancer: a perspective.Hum Pathol. 1992; 23: 273-279Abstract Full Text PDF PubMed Scopus (849) Google Scholar Isolation of DNA from the formalin-fixed, paraffin-embedded tumor material was performed as described by Alers and colleagues.13Alers JC Krijtenburg PJ Rosenberg C Hop WCJ Verkerk AM Schröder FH van der Kwast TH Bosman FT van Dekken H Interphase cytogenetics of prostatic tumor progression: specific chromosomal abnormalities are involved in metastasis to the bone.Lab Invest. 1997; 77: 437-448PubMed Google Scholar Briefly, the tissue blocks were counterstained in 4′,6-diamidino-2-phenylindole (DAPI) and placed under a fluorescence microscope, enabling a precise selection of the tumor area. Microdissection of the tumor areas was performed using a hollow bore coupled to the microscope. In the case of very small tumor areas, manual microdissection of the selected areas was performed by scraping successive hematoxylin-stained 10-μm tissue sections using a needle under a stereomicroscope. Lower boundaries were checked for the presence of tumor on 4-μm hematoxylin and eosin-stained tissue sections. Isolation of DNA from the formalin-fixed, paraffin-embedded material was performed using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) in accordance with the manufacturer's instructions, except that the DNA was hydrated with 25 μl of 10 mmol/L Tris, pH 8. Genomic target DNA was isolated from bacterial cultures and arrayed as described previously,5Pinkel D Segraves R Sdar D Clark S Poole I Kowbel D Collins C Kuo W-L Chen C Zhai Y Dairkee SH Ljung B-M 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 (1790) Google Scholar except chromium-coated slides were used (Nanofilm, Westlake Village, CA). Each array consists of 2460 BACS spotted in triplicate with ∼1.4 Mb average resolution.7Snijders AM Nowak N Segraves R Blackwood S Brown N Conroy J Hamilton G Hindle AK Huey B Kimura K Law S Myambo K Palmer J Ylstra B Yue JP Gray JW Jain AN Pinkel D Albertson DG Assembly of microarrays for genome-wide measurement of DNA copy number.Nat Genet. 2001; 29: 263-264Crossref PubMed Scopus (774) Google Scholar One μg each of test and reference male (Promega, Madison, WI) genomic DNA was labeled by random priming using a Bioprime Labeling Kit (Invitrogen, Carlsbad, CA). The manufacturer's protocol was followed with the following concentration changes; 120 μmol/L of dATP, dGTP, and dCTP; 30 μmol/L of dTTP; and 40 μmol/L of CY3-dUTP or CY5-dUTP (Amersham Pharmacia Biotech, Piscataway, NJ). Random DNA octamers served as the primers (Invitrogen). Unincorporated nucleotides were removed using a Qiagen QIAquick PCR Purification Kit (Qiagen, Valencia, CA) according to manufacturer's instructions. DNA was eluted in 100 μl of EB buffer (provided with kit). Labeled test and reference DNAs were co-precipitated in the presence of Cot-1 DNA (Roche, Indianapolis, IN) with ethanol. The precipitated DNA was redissolved in a hybridization solution containing 50% formamide, 10% dextran sulfate, 2× standard saline citrate, 4% sodium dodecyl sulfate, and 10 μg/μl of yeast tRNA. The probes were denatured at 72°C for 10 minutes and then preannealed with a 1-hour incubation at 37°C. Each array was surrounded by a wall of rubber cement on the chromium-coated slide and then was cross-linked (Stratagene UV Stratalinker, Stratagene, La Jolla, CA, 2600 × 100 μJ). The hybridization mixture (60 μl) was added to each array. A rubber gasket and a glass microscope slide fastened to the slide provided an enclosed chamber for the hybridization. A 48-hour hybridization at 37°C was performed on a unidirectional tilting platform (3 rpm) within an incubator. Slides were washed for 15 minutes in 50% formamide, 2× standard saline citrate, pH 7.0 at 45°C, 2× standard saline citrate, 0.1% sodium dodecyl sulfate for 20 minutes at 45°C, and once in 0.1 mol/L sodium phosphate buffer, 0.1% Nonidet P-40, pH 8, for 10 minutes. The array was counterstained with a 1 μg/μl of DAPI solution. A charge-coupled device camera equipped with filters for CY3, CY5, and DAPI was used to capture the aCGH images. The imaging set up and custom software are described elsewhere.5Pinkel D Segraves R Sdar D Clark S Poole I Kowbel D Collins C Kuo W-L Chen C Zhai Y Dairkee SH Ljung B-M 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 (1790) Google Scholar Imaging processing was performed with SPOT version 1.2 and SPROC version 1.1.1 software packages.14Jain AN Tokuyasu TA Snijders AM Segraves R Albertson DG Pinkel D Fully automatic quantification of microarray image data.Genome Res. 2002; 12: 325-332Crossref PubMed Scopus (297) Google Scholar Log2 ratios of either greater than or less than 0.5 were defined as chromosomal gains or losses, respectively. Array validation was performed by hybridization with normal human female against normal reference human male DNA. The female DNA was extracted from paraffin-embedded tissue as described in the subsection above entitled "Specimens." The male reference was the same reference used in all experiments. Tumor DNA with a fragment size of <1 kb was chemically labeled with biotin-universal linkage system (ULS) or fluorescein-ULS (Kreatech Diagnostics, Amsterdam, The Netherlands). Tumor DNA with larger DNA fragment sizes was labeled by nick translation with biotin (Nick Translation System; Life Technologies, Inc., Gaithersburg, MD) or with green fluorescent nucleotides (Molecular Probes, Leiden, The Netherlands). Likewise, male reference DNA (Promega) was labeled by nick translation with digoxigenin or red fluorescent nucleotides (both from Boehringer Mannheim, Indianapolis, IN). The reaction time and the amount of DNase were adjusted to obtain a matching probe size for reference and tumor DNAs. The labeled DNAs were hybridized onto normal male metaphase chromosomes (Vysis Inc., Downers Grove, IL), as described previously.9Alers JC Rochat J Krijtenburg PJ Hop WCJ Kranse R Rosenberg C Tanke HJ Schröder FH van Dekken H Identification of genetic markers for prostate cancer progression.Lab Invest. 2000; 80: 931-942Crossref PubMed Scopus (157) Google Scholar, 10Alers JC Krijtenburg PJ Vis AN Hoedemaeker RF Wildhagen MF Hop WCJ van der Kwast TH Schröder FH Tanke HJ van Dekken H Molecular cytogenetic analysis of prostatic adenocarcinomas from screening studies: early cancers may contain aggressive genetic features.Am J Pathol. 2001; 158: 399-406Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 15Alers JC Krijtenburg PJ Hop WCJ Bolle WABM Schröder FH van der Kwast TH Bosman FT van Dekken H Longitudinal evaluation of cytogenetic aberrations in prostatic cancer: tumours that recur in time display an intermediate genetic status between non-persistent and metastatic tumours.J Pathol. 1998; 185: 273-283Crossref PubMed Scopus (8) Google Scholar, 16Alers JC Rochat J Krijtenburg PJ van Dekken H Raap AK Rosenberg C Universal linkage system (ULS): an improved method for labeling archival DNA for comparative genomic hybridization.Genes Chromosom Cancer. 1999; 25: 301-305Crossref PubMed Scopus (29) Google Scholar After washing of the slides, fluorescent detection of the biotin- and digoxigenin-labeled DNA probes was accomplished with avidin-fluorescein isothiocyanate and anti-digoxigenin rhodamine, respectively. CGH analysis was performed with Quips XL software version 3.1.1 (Vysis). Loss of DNA sequences was defined as chromosomal regions in which the mean green:red ratio was below 0.85, whereas gain was defined as chromosomal regions in which the ratio was more than 1.15. These threshold values were based on a series of normal controls (data not shown). A QuMA assay was developed to cover the region spanning from the WWOX gene at 16q23.1 to the telomere at 16q24.3. The copy number of the test locus was assessed using quantitative, real-time PCR, and related to a pooled reference. Experimental design and procedure was performed as detailed in Ginzinger and colleagues.17Ginzinger DG Godfrey TE Nigro J Moore DH Suzuki S Pallavicini MG Gray JW Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis.Cancer Res. 2000; 60: 5405-5409PubMed Google Scholar Control primers were a pool of oligos designed to amplify loci in regions where CNA is not observed in prostate cancer (DG Ginzinger, unpublished results). The aim of this study was to determine whether DNA extracted from paraffin-embedded prostate tumors (up to 16-years-old) could be used for mapping CNAs, both gains and losses, by aCGH. Hybridization of female DNA extracted from paraffin with the male reference DNA used in all of the experiments, detected as expected a gain of chromosome X (Figure 1). Chromosome CGH profiles were available for the 20 tumors analyzed and these were directly compared to the aCGH copy number profiles. A graphical representation of one such comparison for chromosome 8 is presented in Figure 2. Table 1 contains a list of CNAs found by both techniques.Figure 2Comparing CGH with aCGH for chromosome 8 in sample 2. A comparison of chromosomal CGH (top) with aCGH (bottom) for chromosome 8 in prostatic cancer sample 2. In the CGH figure, loss is displayed as a bar to the left of the chromosome 8 ideogram, gain is seen as a bar to the right. In the aCGH graph, a log2 scale is used, values below 0 signify loss, above 0 gain of DNA sequences. Note good concordance between the two CGH techniques.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1CGH Results Compared to aCGH Findings*Bolded text denotes regions found by both CGH and aCGH.†(1) signifies that an isolated BAC, i.e. locus, showed gain or loss.Sample numberCGH resultsaCGH resultsA. Deletions11q31-qter, 5q11.2-q13, 6q12-q22, 8p11.2-pter1p36 (1), 1p32.1 (1), 1q31-q41, 1q43-44, 2p12-p13 (1), 5q11.2-q12, 5q22-23 (1), 5q31-q31.3, 6q12-15,7p21.1b (1), 8p12-p23, 10q22.3-q23, 15q11.2 (1), 16q22-q24, 18p11.31-p11.32, 20q11.2-12 (1), 21q22.2-q22.3, Xp11.3 (1)24q24-q26, 5q11.2-q23, 6q14-q22, 8p11.2-pter, 9p13-p21, 10q21-qter, 12p11.2-p13, 12q23-qter Xq21-q224q25-26, 5q11-q34, 6q14-q22.3, 7q11.2 (1), 7q36.2-q36.3, 8p11.2-p23.3, 9p21.1-23, 10q21.1-q26.3, 11q13.5 (1), 12p11.2-13.3, 12q23-24.3, 13q12.1 (1), 16q22-24, 17p12 (1), 19p13.3 (1)31q31-qter, 2q21-q22, 4q12-qter, 5q13-q23, 6q15-q22, 10q11.2-q22, 10q23-q24, 11p13-pter, 11q22-qter, 13q12-qter, 15q14-qter, 16q21-qter, 17p12-pter, Y1q21-22 (1), 1q32, 1q41, 4p16, 4q21-23, 5q13 (1), 5q21 (1), 5q23-31, 6q21-22.1, 7p21, 8q21.1 (1), 9q33, 10q22.3 (1), 10q26.3 (1), 11p12-p15.5, 11q22-q25, 13q14.1 (1), 13q21.1 (1), 13q31.1 (1), 13q34 (1), 15q11.2, 15q14-15, 15q21-q26.2, 16q13-24, 17p12-13, 18p11.31p11.32 (1), 18q22 (1), Xp21.1 (1), Yqter42q14.2-q24, 5q12-q22, 6q12-q22, 13q13-q221p21-31, 2q22 (1), 2q24, 2q32 (1), 4p15 (1), 4q21-q23, 4q26-q31, 4q35, 5q12-q15, 5q21-q23, 5q32-q34, 6p12 (1), 6p22 (1), 6q11-q22, 7p21 (1), 8q21 (1), 8q24 (1), 9p21-p23, 10p11-p14, 10q21-q26, 11p12-p15, 11q14 (1), 11q22, 12q21-q22 (1), 13q13-q14, 13q21-q31, 14q22 (1), 18p11 (1), 19p11-q11 (1), 20p12-p13, 21q11 (1), 21q21, Xq21513q2114q-tel61p21-p22, 3q24-q25, 5q14-q21, 13q14-q313q24-q25, 5q12, 9p24 (1), 13q14-q3176q14-q162q32.1, 4q28-q31 (1), 6q15, 7p13 (1), 7q11.1-q11.2 (1), 7q33, 9p21 (1), 11p15.4 (1), 21q22.2-q22.3 (1), 22q12 (1)88p11.2-pter, 16q23-qter1q22-q23 (1), 3p14.3 (1), 3q13, 4q21, 5p15 (1), 7q11.22 (1), 8p12-p22, 9p24, 10p15 (1), 10q24 (1), 11q14 (1), 12q23 (1), 15q23 (1), 18q22 (1), 20q13.3 (1), 21q21 (1)98p12-p22, 16q22-qter3p21.2 (1), 8p11-p23, 14q-tel (1), 16q23.1 (1), 16q24 (1), 21q11.1-q11.2, Xq27-28 (1)104p12-p14, 5q11.2-q21, 10q23-q24, 13q21, 14q22-q24, 15q11.2-q21, 16q12.1-q22, 18, Xq21-q262p12-2p11.2 (1), 2q24.3, 4p12-p14, 4q32 (1), 5p15 (1), 5q14 (1), 6p21 (1), 7p12-p13 (1), 7q21, 7q33-35 (1), 9q31118p11.2-pter, 12p11.2-pter8p23.2, 8p22, 12p12128p11.2-pter, 18q11.2-qter8p23.1-p23.2, 18q12, 18q221313q21-q223p14, 6q26 (1), 16q23-q24146q16-q226q15-q16.1, 6q22.2-q22.3, 6q26 (1)152q21-q22, 5q12-q13, 6q12-q21, 13q14-q212q21.2-q22, 4q27-q31.1, 6q15, 8p22-p23.2, 12p13.1168p12-pter, 17p11.2-p122q37.3, 6q21-q22.1, 8p21.2-p23.2, 8p11.2-p12, 16q23-q24, 17p12, 17q11.2, 18p11.21176q14-q22, 8p11.2-p215q21-q31, 6q14, 8p23.3, 8p12-p21.3, 10q23, 15q23-q24, 17p12182q21-q22, 13q14-q222q21.1-q23, 18q22, Xp11.3-p11.4195q15-q22, 6q13-q22, 8p11.2-pter, 11q14-q23, 12q15-q21, 13q14-q214q26-q28, 4q33, 5q21-q23, 6q12-q22, 8p12, 8p23, 10q24-q25, 11q21-q24, 12q21, 13q-q14, 13q21206q12-q24, 13q13-q324p15.1, 4q33, 6q12-q13, 6q22, 7q11.22, 7q31-q32, 9q21.3, 11q23, 13q14-q22, 13q31, 14q23, 16q21B. Gains13q13.3-qter, 12q21-qter, 20q12-qter3p23-24 (1), 3q13.3 (1), 3q24-25, 3q26.1-26.2, 3q28, 3q29 (1), 8q12.1, 8q21.1-21.2, 8q22.2 (1), 12q21.1-q22, 12q2327p15-pter, 7q31-q34, 8q11.2-qter7p21.1-p22.1, 7q33-q35, 8q11-q24.2, 18p11.31-p11.32, 20p13 (1), 22q12-qtel31q21-q22, 5p14-pter, 6p21.1, 7q31-qter, 11q12-q14, 17q11.2-qter1q12 (1), 1q21, 3p14.3 (1), 3q24-q25 (1), 3q26 (1), 3q28 (1), 4q28-q31 (1), 5p14.3-p15.3, 6p22 (1), 7q32 (1), 7q34-q35, 9p12-p13 (1), 9q23.3 (1), 9q32-q33 (1), 11p11.2-p13, 11q13-q14, 11q21 (1), 11q22 (1), 17p12 (1), 17q21-q2543p13-pter, 3q13.3-q21, 3q26.3-qter, 7pter-qter1p31 (1), 1p36, 1q12 (1), 1q25 (1), 2p25 (1), 2q35-q36, 3p21-p26, 3q13, 3q21 (1), 13q23-q25, 3q26 (1), 3q29 (1), 4q13-q21, 4q31-q32, 5ptel (1), 7p11-p15, 7p21-p22, 7q11 (1), 7q21-qtel, 9p13-p21 (1), 9q21 (1), 9q34 (1), 10p11.2 (1), 10q21-q23, 11p11-p15, 11q13.4 (1), 12p13 (1), 12q13 (1), 12q24 (1), 14q23 (1), 14q31-q32, 15q11.2 (1), 16q24 (1), 17p12-p13, 17q12-q21, 17q24-q25, 18q21, 20q11-q13, Xq11-q12 (1)53p14.3 (1), 7q36.3 (1), 9q34 (1), 11q12-q13 (1), 15q26.1 (1)66p21.1-p21.3, 14q24-qter, 20q11.2-qter1q34 (1), 3p13-p14 (1), 5q31 (1), 6p21.1-p21.3, 7q36 (1), 10p11.2-p12 (1), 11p13-p14 (1), 12q13 (1), 14q24.1 (1), 18p11.21, 20q13.3 (1), Xp22.3 (1)717q23-q241p36 (1), 3p25 (1), 6p21.3 (1), 9q34 (1), 17p12 (1)81p12 (1), 3p21.2 (1), 4q12 (1), 7q34 (1), 11p15.2-11p15.3 (1), 11p14 (1), 13q12 (1), 13q21.1, 21q22.1 (1)91p36 (1), 1q42-q43 (1), 2q35-q36 (1), 3p25 (1), 4q31.1 (1), 5q33.2 (1), 7p12 (1), 7p14, 7q21.12 (1), 7q36.3 (1), 8q22.2 (1), 9q34.2 (1), 10q26.2 (1), 11p14.1 (1), 11q23.2 (1), 12q13.2 (1), 12q21.3-q22 (1), 14q11.2 (1), 14q31-q32.1 (1), 18p11.21-p11.3, 18q11-12 (1), 18q23 (1), 19p13.3 (1), 22q13.1 (1), Xq26 (1)101q31-qter, 3p14-p21, 3q13.3-q21, 3q26.2-qter, 8q23-qter, 9q22-qter, 17q23-qter, 20q11.2-qter, Xp11.2-p22.2, Xq12-q131q41 (1), 3p22 (1), 3p26 (1), 3q21 (1), 4q21 (1), 4q31.3-4q32 (1), 5p13 (1), 5q21 (1), 7p12.2, 7p21.1 (1), 7q21.1 (1), 7q31, 7q36 (1), 8q21 (1), 11q12-q13 (1), 13q-tel (1), 15q26 (1), 17p12 1(1), 17p13 (1), 18q21(1), 20q13, Xp11-p22, Xq11-q13, Xq23(1)112p22.1, 17q11.2126p23 (1), 11p15.3-p15.4, 13q341319p13.3-pter2q11.2, 2q35, 8p21.3, 11p15.3-p15.4, 11p13, 11q12-q13, 11q23.2, 14q11.2, 17qtel142p21, 10q21.3-q22.1, 11p15.3-p15.4, 15q15, 17p13.315204p13, 7q36, 11p13, 11q12-q13, 14q11.2, 17qtel, 20p11, 20q13.116173q26.2, 5q33.2, 10q26.2, 13q32, 13q34, 15q15189q32-qter193q12-qter, 7p11.2-pter, 7q11.2-q31, 8q24.1-qter3q21-q29, 7p12-p14, 7p22, 7q11, 7q21-q31, 9q34, 10q26.3, 11p15.2 (1), 15q11.2, 18p11.21203q27-qter3q26.3-q27, 4p16.3, 9q31* Bolded text denotes regions found by both CGH and aCGH.† (1) signifies that an isolated BAC, i.e. locus, showed gain or loss. Open table in a new tab The first description of aCGH demonstrated its utility by measuring copy number changes along chromosome 20 in breast cancer cell lines and primary tumors.5Pinkel D Segraves R Sdar D Clark S Poole I Kowbel D Collins C Kuo W-L Chen C Zhai Y Dairkee SH Ljung B-M 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 (1790) Google Scholar Using an array spanning 7 Mb of chromosome 22 with 90% coverage, aCGH methodology has been shown to be useful for rapid and comprehensive detection of heterozygous and/or homozygous deletions as small as 40 kb at the NF2 locus.18Bruder CE Hirvela C Tapia-Paez I Fransson I Segraves R Hamilton G Zhang XX Evans DG Wallace AJ Baser ME Zucman-Rossi J Hergersberg M Boltshauser E Papi L Rouleau GA Poptodorov G Jordanova A Rask-Andersen H Kluwe L Mautner V Sainio M Hung G Mathiesen T Moller C Pulst SM Harder H Heiberg A Honda M Niimura M Sahlen S Blennow E Albertson DG Pinkel D Dumanski JP High resolution deletion analysis of constitutional DNA from neurofibromatosis type 2 (NF2) patients using microarray-CGH.Hum Mol Genet. 2001; 10: 271-282Crossref PubMed Scopus (136) Google Scholar Another report of the utility of the aCGH technology for the detection of gene amplifications in glioblastomas and nasopharyngeal carcinomas has recently been published.19Hui AB Lo KW Yin XL Poon WS Ng HK Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization.Lab Invest. 2001; 81: 717-723Crossref PubMed Scopus (178) Google Scholar, 20Hui AB Lo KW Teo PM To KF Huang DP Genome wide detection of oncogene amplifications in nasopharyngeal carcinoma by array based comparative genomic hybridization.Int J Oncol. 2002; 20: 467-473PubMed Google Scholar The targets spotted on the array they describe were BAC clones containing 58 oncogenes commonly amplified in various human cancers compared to the array used in this study, which covers the whole genome. In the first report of aCGH being used to analyze DNA from paraffin-embedded tissue, the DNA required amplification by DOP-PCR.8Daigo Y Chin SF Gorringe KL Bobrow LG Ponder BA Pharoah PD Caldas C Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers: a new approach for the molecular analysis of paraffin-embedded cancer tissue.Am J Pathol. 2001; 158: 1623-1631Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar The arrays were similar to those used by Hui and colleagues,19Hui AB Lo KW Yin XL Poon WS Ng HK Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization.Lab Invest. 2001; 81: 717-723Crossref PubMed Scopus (178) Google Scholar, 20Hui AB Lo KW Teo PM To KF Huang DP Genome wide detection of oncogene amplifications in nasopharyngeal carcinoma by array based comparative genomic hybridization.Int J Oncol. 2002; 20: 467-473PubMed Google Scholar consisting of oncogenes, and therefore not well suited for detection of deletions. We have established that in this tumor cohort direct random-prime labeling of DNA extracted from paraffin-embedded tissue yields quantitative data (ie, good signal to noise) and gives results comparable to established techniques such as CGH. This is important because it eliminates the expense and labor of PCR as well as eliminating the introduction of copy number artifacts resulting from amplification bias. A female-male control hybridization (Figure 1) only resulted in 0.3% false copy number calls (six false gains and zero false losses). The robustness of our labeling and extraction techniques is demonstrated here by our success with the analysis of prostate tissue that is inherently heterogeneous in nature (ie, comprised of normal, mixed grade tumor, and stroma). Additionally, we showed that these techniques work well on arrays that contain greater than 2000 BACs spanning the entire genome and we show that our technique allows detection of single copy number gains and losses. Verification of CNAs with techniques such as CGH and QuMA is important because prostate tumors are inherently heterogeneous and we are using DNA extracted from paraffin-embedded tissue. The aCGH technology was able to reproduce and extend the findings from CGH. With our analytical criteria, we detected ∼80% of the CNAs found by CGH. On closer examination of the aCGH profiles, we were 90% concordant with the CGH results. For all of the discordant loci, the aCGH change fell below our log2 ratio cutoff value and also occurred in a region where few BACs were represented either because of poor printing on that array or a poor hybridization. A stringent log2 ratio cutoff value of 0.5 was intentionally, but arbitrarily selected (∼0.5 = gain of one copy and −1.0 = loss of one copy) to determine amplification or deletion of DNA. A significant number of new CNAs were identified because of the very high resolution of aCGH (Table 1). Some of these changes involved only single BAC clones implying an inter
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