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The recent announcement that cancer has become the number one killer of Americans under the age of 85 likely came as no surprise to scientists studying the disease—research increasingly shows that cancer is caused by many different genetic factors and that the genome itself is far more complex than anyone imagined.
Identifying the genes that contribute to cancer requires scientists to sift through the 3.1 billion molecules that make up our genome and find the handful of genes that are mutated, missing or of which we have too many copies. The task is daunting and progress has been slow, but two search methods—called chromosome copy number analysis and loss of heterozygosity analysis—are generating promising new results.
While both methods have been around for a while, by using a new tool to conduct the experiments—the GeneChip® Mapping 100K Set—scientists are able to search the genome faster and in greater detail than ever before. Researchers at institutes such as the Dana Farber Cancer Research Center, University of California Los Angeles and the Max-Delbruck Center in Germany are using this Affymetrix technology in cancer studies and have already published over a dozen new discoveries.
"The results from the publications that have used LOH and copy number methods to find genes associated with cancer are very encouraging," said Greg Yap, Vice President of DNA Analysis Products at Affymetrix. "Because the 100K can scan the genome for more than 100,000 genetic variations, it's a perfect tool for conducting these types of complex studies. It's like a digital camera - higher resolution provides clearer pictures."
To understand the progress researchers have made using microarrays to study cancer genetics, it's first necessary to explain the types of mutations they're looking for:
Chromosomal Copy Number Changes
Normally, you get one chromosome from mom, and one from dad. This means you have two copies of the chromosome. Changes in copy number occur when a chromosome—or even a part of a chromosome, like a gene—is deleted or amplified. For example, Down's syndrome occurs when a person has an extra copy of chromosome 21-a condition called trisomy 21.
Loss of Heterozygosity
One reason for having two copies of each chromosome is so that they can back each other up. If one is mutated (making it not function properly) often the other one can compensate. Even though you have two of each chromosome, they are not identical sequences because your mom and your dad are not identical people. For example, the gene for eye color—your dad may have given you a brown-eye version and your mom a blue-eye version. Having two variants of the same gene is called heterozygosity. Loss of heterozygosity is a type of mutation that happens when one chromosome is lost, leaving you with only one working chromosome. If this single remaining copy becomes mutated, you no longer have a backup that is working properly.
The Cancer Tool Box
For many cancers, finding a cure starts with finding the genetic changes—copy number alterations or loss of heterozygosity—that cause a cell to grow wildly out of control. Most often, these changes activate cancer-promoting genes (called oncogenes) or inactivate the genes that stifle cancer (called tumor-suppressing genes). To study copy number of these genes, researchers have been limited to measuring comparative genomic hybridization (CGH) over relatively large regions, making it difficult to find small deletion or amplifications. And to study LOH, researchers used separate experiments that were limited to genotyping relatively few microsatellite markers.
The GeneChip Mapping 100K Set enables researchers to assess copy number and genotype across the entire genome in just one experiment. By measuring over 100,000 single nucleotide polymorphisms (SNPs), the array can detect more detailed copy number changes than conventional CGH, and offers many times more markers than microsatellite analysis for LOH studies. This high-density combined approach offers researchers the quickest way yet to search the genome for the many different and often, difficult to detect mutations that can cause cancer.
Early Microarray Studies in Cancer Genetics
Using microarrays to search for tumor gene copy number changes and loss of heterozygosity actually started five years ago. Dr. Matthew Meyerson and Dr. Bill Sellers of the Dana Farber Cancer Research Institute led one of the first groups to publish a technical report in Nature Biotechnology1 validating a first generation Affymetrix genotyping array for tumor gene copy number studies. These researchers found that the microarray provided a way to probe the entire genome at much higher resolution than ever before. Because tumor-associated genetic changes could be virtually anywhere in a genome over 3 billion molecules long, the more places you're able to scan for cancer genes, the more likely you are to find them.
Since then, researchers have used second and third generation Affymetrix SNP microarrays in studies to help locate tumor genes responsible for cancers of the breast2-6, bladder7,8, prostate9,10, bone11, mouth12 and lung1,3,13. The majority of these published studies have used a second generation microarray capable of scanning 10,000 SNPs across the genome.
Finding Disease Genes: Lung & Breast Cancer
In May of 2004, Meyerson's research group reported using the Mapping 10K Set to simultaneously detect cancer-specific DNA copy number changes and LOH, two major causes of neoplastic growth. In a study of 7 lung cancer cells3, the team used the 10K to detect twice the LOH regions as microsatellite analysis, identifying the already-known deletions, as well as 14 previously unknown LOH regions on 9 different chromosomes. In a second study of 18 lung and breast cancer cell lines, Meyerson's research team found copy number amplifications (encompassing known proto-oncogenes) and deletions (encompassing TSGs), and distinguished LOH caused by a DNA deletion coupled with a gene mutation13.
"Over the last year, we have been using the Mapping 10K Array to identify regions of chromosomal amplification, deletion, and loss of heterozygosity in the same experiment, resulting in multiple publications," said Meyerson. "GeneChip array-based genotyping has significantly strengthened our ability to identify cancer causing genes, and molecular rearrangements that correlate with clinical outcomes."
Meyerson says that rapid detection of cancer genome mutations will allow earlier cancer diagnosis, assessment of cancer predisposing markers, characterization of tumors for tailored chemotherapies, and enable new insights into the molecular basis of cancer.
Meyerson is now collaborating with Dr. Bill Sellers at the Dana Farber, characterizing cancer-specific genetic anomalies with the new Mapping 100K Set. He says a publication on this work is forthcoming.
Finding Disease Genes: Mouth Cancer
In the September 2004 issue of Human Genetics, Dr. David Wong of the University of California, Los Angeles reported using the Mapping 10K to identify four regions of the genome associated with mouth cancer. The study highlights the need to track both chromosomal copy number changes along with loss of heterozygosity. For example, on chromosome 3, the team used copy number analysis to find DNA amplification and deletion mutation shared by the premalignant and malignant cells. But finding the mutations that converted those premalignant cells into malignant cancers required looking for a different type of mutation—LOH. When the team analyzed the 10K data for LOH, they found two regions of chromosome 3 that had mutated in the malignant cells.
"Our results demonstrate the utility of the Mapping 10K Array for genome-wide concordant loss of heterozygosity and copy number abnormality studies of oral squamous cell carcinoma," said Wong. "Identifying important new candidate genes that associate with malignancy progression will help us better understand the genetic underpinnings of oral cancer."
Finding Disease Genes: Beyond Cancer
In addition to cancer, gene deletions and amplifications are also a major cause of developmental defects, like Down's syndrome. In the December 2004 issue of the Journal of Medical Genetics, researchers from the Friedrich-Alexander University, Max-Delbruck Center and Affymetrix reported using the Mapping 10K Array to study chromosomal amplifications and deletions that cause a variety of mental retardation syndromes14.
"The GeneChip Mapping 10K Array proved highly sensitive and specific, allowing for detection of aberrations as small as 700 kilobases," said Dr. Peter Nuernberg, Director of the Gene Mapping Center at the Max-Delbruck Center in Berlin and senior author on the publication. "We anticipate that this method will allow the identification of further genomic regions that, when deleted, contribute to human pathology."
Forward Spin
By combining Affymetrix genotyping arrays with gene expression arrays, researchers can do even more. When researchers find copy number changes with genotyping arrays, they can immediately check the impact of those changes with expression arrays because genes that are amplified or deleted will produce more or less RNA. Likewise, if researchers see changes in RNA levels with expression arrays, they can check for corresponding genetic changes with genotyping arrays. Next-generation arrays capable of genotyping 500,000 SNPs will provide even more information about our genome and offer the promise to demote cancer from its new-found, but ignominious "number one" ranking.
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References:
1. Lindblad-Toh, K. et al. Loss-of-heterozygosity analysis of small-cell lung carcinomas using single-nucleotide polymorphism arrays. Nat Biotechnol 18, 1001-5 (2000).
2. Huang, J. et al. Whole genome DNA copy number changes identified by high density oligonucleotide arrays. Hum Genomics 1, 287-99 (2004).
3. Zhao, X. et al. An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays. Cancer Res 64, 3060-71 (2004).
4. Schubert, E. L. et al. Single nucleotide polymorphism array analysis of flow-sorted epithelial cells from frozen versus fixed tissues for whole genome analysis of allelic loss in breast cancer. Am J Pathol 160, 73-9 (2002).
5. Wang, Z. C. et al. Loss of heterozygosity and its correlation with expression profiles in subclasses of invasive breast cancers. Cancer Res 64, 64-71 (2004).
6. Paez, J. G. et al. Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. Nucleic Acids Res 32, e71 (2004).
7. Primdahl, H. et al. Allelic imbalances in human bladder cancer: genome-wide detection with high-density single-nucleotide polymorphism arrays. J Natl Cancer Inst 94, 216-23 (2002).
8. Hoque, M. O., Lee, C. C., Cairns, P., Schoenberg, M. & Sidransky, D. Genome-wide genetic characterization of bladder cancer: a comparison of high-density single-nucleotide polymorphism arrays and PCR-based microsatellite analysis. Cancer Res 63, 2216-22 (2003).
9. Lieberfarb, M. E. et al. Genome-wide loss of heterozygosity analysis from laser capture microdissected prostate cancer using single nucleotide polymorphic allele (SNP) arrays and a novel bioinformatics platform dChipSNP. Cancer Res 63, 4781-5 (2003).
10. Dumur, C. I. et al. Genome-wide detection of LOH in prostate cancer using human SNP microarray technology. Genomics 81, 260-9 (2003).
11. Wong, K. K. et al. Allelic imbalance analysis by high-density single-nucleotide polymorphic allele (SNP) array with whole genome amplified DNA. Nucleic Acids Res 32, e69 (2004).
12. Zhou, X., Mok, S. C., Chen, Z., Li, Y. & Wong, D. T. Concurrent analysis of loss of heterozygosity (LOH) and copy number abnormality (CNA) for oral premalignancy progression using the Affymetrix 10K SNP mapping array. Hum Genet 115, 327-30 (2004).
13. Janne, P. A. et al. High-resolution single-nucleotide polymorphism array and clustering analysis of loss of heterozygosity in human lung cancer cell lines. Oncogene 23, 2716-26 (2004).
14. Rauch, A. et al. Molecular karyotyping using an SNP array for genomewide genotyping. J Med Genet 41, 916-22 (2004). |
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