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Detection of Gene Amplification by Genomic Hybridization to cDNA Microarrays

Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892 [M. A. H., M. L. B., Y. C., J. K., J. M. T., P. S. M.], and DuPont NEN Life Science Products, Inc., Boston, Massachusetts 02118 [K. E. A.]

	ABSTRACT

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Gene amplification is one of the major mechanisms of oncogene activation in tumorigenesis. To facilitate the identification of genes mapping to amplified regions, we have used a technique based on the hybridization of total genomic DNA to cDNA microarrays. To aid detection of the weak signals generated in this complex hybridization, we have used a tyramide-based technique that allows amplification of a fluorescent signal up to 1000-fold. Dilution experiment suggests that amplifications of 5-fold and higher can be detected by this approach. The technique was validated using cancer cell lines with several known gene amplifications, such as those affecting MYC, MYCN, ERBB2, and CDK4. In addition to the detection of the known amplifications, we identified a novel amplified gene, ZNF133, in the neuroblastoma cell line NGP. Hybridization of NGP cDNA on an identical array also revealed over expression of ZNF133. Parallel analysis of genomic DNA for copy number and cDNA for expression now provides rapid approach to the identification of amplified genes and chromosomal regions in tumor cells.

	Introduction

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Chromosomal anomalies resulting in gain or loss of genetic material are frequent in tumor cells. These changes may result in alterations in the level of expression of numerous genes. Indeed, gene amplification is one of the most common mechanisms for oncogene activation in solid tumors. It is therefore of considerable interest to develop strategies for identifying amplified genes and determining their expression levels in cancer. Until recently, gene amplification has been detected by DNA-based techniques (PCR or Southern blot) or by molecular cytogenetic techniques (FISH² with gene-specific probes). These techniques are inherently restricted to the analysis of previously known amplified genes. In contrast, genome-wide scanning of amplified chromosomal regions with CGH (1) has become an important technique for the detection of amplified regions in tumor DNA. However, CGH has limited sensitivity and resolution (2) . In addition, the identification of the specific target gene within an amplicon defined by CGH remains daunting because of the limited mapping resolution provided by the metaphase chromosomes.

DNA microarray technology offers the possibility to replace the target metaphase chromosomes with arrays of DNA clones on a microscope slide. Arrayed fragments of cloned genomic DNAs have been used for this purpose (3 , 4) . These CGH microarray techniques allow amplification detection on the resolution level equal to the length of the arrayed DNA clones (100 kb). Genomic microarrays have been applied to amplicon mapping (4) , but the technique can also be used for rapid surveys of known copy number alterations in tumor samples. In principle, a further increase in resolution can be obtained by using arrayed cDNAs rather than genomic DNA. This approach is particularly attractive because of the availability of thousands of accurately mapped cDNAs. Furthermore, expression analysis can be carried out in parallel on the same microarray slides, enabling a correlation of copy number and gene expression. However, signal intensities in genomic hybridizations are proportional to the length of the target DNA (4) . Reproducibly achieving a measurable hybridization signal from total genomic DNA hybridized to targets covering only 0.5–2 kb is difficult and requires a signal detection system with high sensitivity and low background. An approach for CGH on cDNA microarrays was reported recently by Pollack et al. (5) using directly labeled fluorochrome probes. We report here an alternative technique that also uses cDNA arrays prepared for expression studies. Our results indicate that this technique is reproducibly capable of detecting gene amplifications of 5-fold or higher. Finally, the suitability of the technique for genome-wide screening of amplified and overexpressed genes was tested by hybridizing both genomic neuroblastoma DNA and mRNA on a microarray containing 1400 genes. In addition to known amplifications on 2p and 12q, a previously unrecognized amplicon on 20p containing a zinc finger gene (ZNF133) was identified.

	Materials and Methods

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Preparation of cDNA Microarrays
cDNA microarrays were prepared as described previously (6) . To validate the technique, microarrays containing 14 different cDNAs representing genes known to be amplified in cancer cell lines were printed (CDK4, MDM2, OS4, OS9, MYCN, MYC, MYCL, EGFR, AKT2, ERBB2, AIB1, IGFR1, CLND, and SAS.) For screening of unknown amplified genes, an array containing 1400 cDNAs was used (6) .

Genomic Hybridization, Signal Detection, and Amplification
Total genomic DNA was labeled with biotin by nick translation for 2.5 h at 15°C. The fragment size of the labeled probe was between 400-2000 bp. Unincorporated nucleotides were removed using Micro Bio-Spin 6 Chromatography Columns (Bio-Rad, Hercules, CA). Hybridization mixture (10 µl) was composed of 1 µg of biotinylated probe, 40 µg of Cot-1 DNA (Life Technologies, Inc., Rockville, MD) and 8 µg of poly dA in 3X SSC (0.15 M NaCl, 0.015 M sodium citrate)/0.01% SDS. After denaturation, the hybridization mixture was added on the slide and hybridized in a hybridization chamber at 65°C over night. Slides were washed in 0.5X SSC/0.01% SDS, 0.06x SSC/0.01% SDS, and 0.06X SSC at room temperature for 5 min each. Hybridization signals were developed using tyramide reagents (Renaissance TSA-indirect ISH; DuPont NEN Life Science Products). Slides were blocked using 10% goat serum in TN blocking buffer [0.1 M Tris (pH 7.6), 0.15 M NaCl]. Hybridization was detected by first incubating the slide with streptavidin conjugated with horseradish peroxidase (1:100 in TN-10% goat serum), followed by signal amplification with biotinyl tyramide (1:50 in reaction buffer with 1% blocking reagent). Biotinyl tyramide was detected by streptavidin conjugated with Cy3 (1:500 in TN-10% goat serum). Between and after incubations, slides were washed with TNT buffer [0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, and 0.05% Tween 20] 3 x 1 min. All of the incubations and washings were done at room temperature.

cDNA Hybridization for Gene Expression Analysis
Hybridization of NGP cDNA on a microarray containing 1400 genes has been described in detail previously (6) .

Image Analysis and Outlier Detection
Amplification Intensity.
A gray scale fluorescent image for each microarray slide was obtained from a confocal scanning microscope. DNA target segmentation and signal detection methods were then used to determine the actual target regions, average signal intensities, and local background intensities (7) . The background subtracted average signal intensity was reported as the hybridization intensity.

Fold Increase.
An iterative amplification intensity outlier detection algorithm was then applied as follows:

(a) Assuming there were N cDNA targets presented in microarray slides, we first sorted all intensities I_k in ascending order, I₁ > I₂ >... > I_N. We then partitioned intensities into two groups (I₁, ... , I_N–m) and (I_N–m+1, ... , I_N). Initially, we chose m = N/2.

(b) The discordance test (for a single outlier in a normal sample with m and s unknown) was performed for the first amplification intensity from the second group:

where

and

and _b is the SD of local background at the same location of I_N-m+1. The test statistic T can be converted to Student’s t test with N - 2 degrees of freedom [Barnett and Lewis (8) ]. As an example, the critical value for n = 40 and = 1% discordance, t must be >3.24.

3) If the I_N-m+1 was not an outlier, let m m + 1, and then repeat step 2 until the first outlier intensity was obtained.

If at least one outlier was obtained, the amplification intensities were partitioned into two groups: (a) negative targets that exhibit no signification amplification intensities; and (b) positive targets of which intensities were statistically different from those from the negative group. The fold increase r_k was then calculated for all genes in the positive target group (I_N-m+1, , I_N) by r_k = I_k/µ₁ for k = N - m + 1, , N.

P Value.
To further assess the significance of each reported positive amplification intensity, P from the aforementioned discordance t test statistic (8) can be calculated.

In Situ Hybridization
A BAC clone specific for ZNF133 (169o05) was screened from a human BAC library (Research Genetics, Inc. Huntsville, AL) using primers specific for marker W1–18789.³ The probe was labeled with Spectrum Orange by random priming (BioPrime DNA Labeling System; Life Technologies, Inc.). FISH-based copy number determination for the ZNF133 region included a fluorescein-labeled satellite probe for chromosome 20 (Oncor).

	Results

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We initially tested tyramide signal detection on microarrays containing 14 genes amplified in various cancers by hybridizing genomic DNA from a neuroblastoma with a 100-fold amplification of the MYCN oncogene (9) . MYCN was specifically identified as an outlier in this hybridization. To determine the sensitivity of the procedure, tumor DNA was diluted with normal DNA prior to labeling. Seven dilutions ranging from 100 to 2% of NGP DNA were hybridized to cDNA microarrays (Fig. 1) . Four normal control hybridizations were also included in the experiment. The MYCN signal intensities were normalized to the average signal intensity of all of the nonamplified spots on the array. The results revealed a decrease in the MYCN cDNA signal intensity with dilution of the NGP DNA. In the hybridization containing 2% NGP DNA, the MYCN signal intensity was increased 2.5-fold relative to the nonamplified spots, indicating that amplifications of 5-fold can be detected. In this experiment, the signal intensities were not directly proportional to the gene copy number. The lower signal intensity of the undiluted NGP hybridization compared with the first dilution (75% of NGP) is likely explainable by the increased self annealing of the probe, which limits the hybridization of the probe to the target cDNA.

View larger version (62K): [in this window] [in a new window]   Fig. 1. Dilution of NGP neuroblastoma cell line DNA with normal DNA. The X-axis shows the different dilutions of tumor DNA, from 100% of NGP (representing 100-fold amplification of MYCN) down to 2% of NGP DNA in the labeling reaction. The MYCN ratio on the Y-axis was calculated by dividing the MYCN signal intensity with the average intensity of all of the nonpositive spots on the array. Four hybridizations of the same normal DNA used for NGP dilution were included in the experiment. The average ratio of the MYCN spot to the other spots on the array in four normal control hybridizations was 0.66 (SD, 0.70).

View larger version (51K): [in this window] [in a new window]   Fig. 2. Two examples of the genomic hybridization on cDNA arrays. A, high-level amplification of MYC in colon cancer cell line Colo320. Signal intensity of MYC is 16-fold higher than the nonamplified signal intensity in this hybridization. B, hybridization of breast cancer cell line SKBR3. In this hybridization, the signal intensity ratios were 9-fold for MYC and 22-fold for ERBB2. The level of ERBB2 amplification has been determined to be 6-fold and MYC amplification 32-fold by interphase FISH.4 The grid indicates the pattern of cDNAs printed on the microarray.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Amplification and overexpression of zinc finger gene ZNF133 in NGP neuroblastoma cell line. A, section of a 1400-element cDNA microarray containing ZNF133 (boxed) after hybridization of NGP total genomic DNA and tyramide detection. The intense ZNF133 signal (8-fold) suggests amplification of ZNF133. B, corresponding section of an identical microarray after cDNA hybridization with NGP cDNA (pseudocolor red) and normal fibroblast DNA (pseudocolor green). The red:green ratio of 3.37 for ZNF133 (boxed) shows that the amplified copies of ZNF133 are highly overexpressed. C, interphase FISH on NGP confirming amplification of ZNF133 (red) relative to a chromosome 20 satellite (green).

	Discussion

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Compared with the previously published CGH microarray techniques where genomic DNA is hybridized on arrayed genomic clones (3 , 4) , hybridization on cDNA arrays offers significant advantages. The most important is the possibility to directly identify amplified genes rather than amplified genomic regions. Another advantage is the ability to do expression studies on the same arrays using the standard cDNA microarray approach. Finally, thousands of mapped cDNAs are readily available, which facilitates amplicon mapping and identification of new cancer genes.

The complexity of the probe and the small sizes of the arrayed target cDNAs (0.5–2 kb) place high demands on the sensitivity of the system. Using tyramide-based signal amplification (14) , it is possible to enhance fluorescent signals up to 1000-fold. Deposition of biotin tyramides has been applied previously for the amplification of in situ hybridization signals (15) . We show here that the peroxidase-mediated deposition of biotin tyramide can also be applied on high sensitivity detection of gene amplification on cDNA microarrays to detect gene amplifications of 5-fold or greater. Although we have tested the addition of a second tyramide reagent to provide two-color CGH, there was excessive cross-talk between tyramide reagents under the conditions necessary for genomic hybridization. Nonetheless, the tyramide method described here consistently generates significant signal intensities necessary for a screening technique for gene amplification.

In the hybridization of NGP DNA to a 1400-element cDNA microarray, we identified the known 2p and 12q amplicons as well as a novel 20p amplicon containing a zinc finger gene ZNF133. This gene belongs to the family of Kruppel-related zinc finger genes that have been connected with transcriptional repression (13) . Amplification and overexpression of ZNF133 have not been reported previously, but the amplification of this chromosomal region has been detected by CGH in several different types of malignancies. In a CGH study of 58 primary gastric cancers, 20p gain was detected in 38% of cases (16) . In chondrosarcomas, gain of 20p was observed in 31% of the analyzed tumors (17) . 20p amplification has also been reported in ductal carcinoma in situ of the breast, in bladder tumors, as well as in osteosarcoma, ovarian cancer, adenocarcinoma of gastroesophageal junction, squamous cell carcinoma, small cell lung cancer, and non-small cell lung cancer (18) . Although it is impossible to delineate the size and genetic composition of the 20p amplicon from the small microarray used in this study, larger arrays will provide an amplicon map at higher density. This information should prove extremely useful for focusing efforts to identify amplification target genes. With sufficient cDNA density, it should prove possible to map core regions of amplification in multiple tumor specimens. This information can then be further correlated with expression patterns determined across the amplified region. When integrated with the rapidly emerging human genome sequence, this approach should greatly accelerate the discovery of genes amplified during tumor progression.

	ACKNOWLEDGMENTS

 
We thank Kimberly Gayton, William Giasi, Gerald Gooden, and John Lueders for excellent technical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, Cancer Genetics Branch, National Human Genome Research Institute, NIH, 49 Convent Drive, MSC 4470, Bethesda, MD 20892.

² The abbreviations used are: FISH, fluorescence in situ hybridization; CGH, comparative genomic hybridization.

³ Internet address: http://www.genome.wi.mit.edu.

⁴ A. Kallioniemi, personal communication.

Received 9/22/99. Accepted 1/ 3/00.

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