November 1, 2010 (Vol. 30, No. 19)
K. John John Morrow Jr. Ph.D. President Newport Biotech
Improved and Reconfigured Platforms Help Researchers Maximize Genomics Studies
Microarray technology has come of age as a powerful approach to revealing the genome and its dynamic expression through the application of gene-hybridization or gene-expression microarrays. While the former are aimed at detecting the presence of specific genes in a sample, the latter are designed to measure the level of activity of genes in particular tissues.
Because gene-expression microarrays display the transcriptional activity of the tissue from which they are derived, they have the potential to reveal new and unanticipated gene functions, including unforeseen disease subtypes, new diagnostic markers, and mechanisms determining disease susceptibility.
As the technology has developed over the years, earlier problems of lack of consistency and repeatability have been addressed, and the data generated by these investigations is much more robust and comprehensive than in the early discovery period of this technology. A number of companies are moving forward on different fronts to make these improved and reconfigured platforms available to their customer base.
Much of the activity in microarray design is based on the custom requirements of investigators. The starting point is the basic material used in building microarrays, mainly oligonucleotides and peptides. Sigma Life Sciences, a division of Sigma-Aldrich, has substantial involvement in this technology with facilities throughout the world dedicated to their manufacture, according to Stacey Hoge, product manager for custom DNA and PEPscreen, and Carlos I. Martinez, Ph.D., head of global technology development.
“Given that we have many manufacturing sites, it is important that our technology be normalized and harmonized,” Hoge explains. “Everything is made to order, but at the same time, we have to rapidly meet the needs of our customers.”
Synthetic peptides can be important components of disease-monitoring technologies. Sigma produces overlapping peptides that represent strong HIV epitopes. They can be incorporated into 96-well microarrays and used to screen patient sera for HIV progression, as evidenced by the production of anti-HIV antibodies.
“Frequently, customers are looking to improve the quality of printing when they design microarrays, so we may use an amine linkage to achieve better printing quality,” explains Dr. Martinez.
Monitoring and repairing errors that may be inadvertently introduced into the sequences of the reagents is an important component of the Sigma services, especially since the work is time-sensitive. Peptides and oligonucleotides are quality controlled through the use of MALDI-TOF and electrospray ionization.
“Analytical instrumentation is similar to the computer industry, in that it is constantly changing. The machines cost hundreds of thousands of dollars, so we can’t be constantly replacing them; however we are adding minor upgrades and improved software all the time.”
In fact, the lifespan of a typical instrument is about 10 years. “The benefits of this evolving technology are greater simplicity and lower operating costs, but this requires orchestration because all our centers must have identical approaches.”
Affymetrix offers platforms for DNA copy number, SNPs, and RNA-expression analysis, according to Dara Wright, vp for clinical marketing.
“Recently, we commercialized the OncoScan FFPE Express cancer copy-number array service.” This platform is designed to profile FFPE samples on the company’s GeneChip microarray platform. These genetic signatures aid in clinical trial stratification and may ultimately lead to companion diagnostic and other clinical test development, Wright adds.
The technology requires a modest quantity of input DNA and yields copy number, allelic ratio, and somatic mutation data from the samples. The intention is also to develop an off-the-shelf product that customers can run in their own lab.
The challenges inherent in building microarray-based tests that can achieve FDA approval are well-known. “There is a growing body of literature and clinical studies detailing how multiparametric gene signatures can significantly improve diagnostic yield as compared to traditional techniques such as karyotyping.
“There is a consensus building in the industry concerning the optimal approach to validation and development of oversight guidelines for lab-based interpretations of complex findings. I believe it is likely that many new genetic tests for cancer diagnosis and prognosis will reach commercialization in the next few years.”
To expedite the improvement of array-based clinical assays, the company has introduced the Powered by Affymetrix Program, which enables partners to build and commercialize tests for disease diagnosis and stratification.
A collaboration with The Medical Prognosis Institute (MPI) to develop microarray-based tests to determine which cancer patients have the greatest likelihood of benefiting from a particular drug exemplifies this approach. Using the FDA-approved Affymetrix GeneChip® System 3000 Dx v.2 and custom microarrays, MPI’s Drug Response Predictor pairs its algorithms with a proven platform to help guide clinical validation and patient treatment.
In another partnership, Affymetrix is working with Signature Diagnostics, a German clinical diagnostic company with two microarray-based tests for colorectal cancer. Signature Diagnostics plans to launch these tests in Europe and will subsequently seek U.S. regulatory approval.
As a component of its partnership with the scientific community, Affymetrix participates in relevant scientific societies, consortia, and government-sponsored symposia. Through these efforts, the company helps promote the application and standardization of microarray technologies.
One recent example is its work with the MicroArray Quality Control (MAQC-II) initiative. “This is the second phase of a study that involved more than 400 representatives working to establish best practices for development and validation of predictive models based on microarray gene expression and genotyping data for personalized medicine.”
Agilent Technologies offers a family of microarray products that can answer any nucleotide question you may wish to pose in comparative genomics or gene expression, according to Glenda Anderson, director of genomic diagnostics.
The company has an online customization tool, known as eArray, through which customers can design microarrays from a database of probes or choose their own specific oligonuclotide sequences, thus enabling researchers to investigate diagnostic questions of import.
A feature of eArray, known as the Probe Score, predicts the probes’ expected performance in a given hybridization setting. The capability has reportedly been put to use by a number of research consortia around the world, investigating genomic questions in developmental disorders, autism, IVF, and oncology.
An example of how the eArray technology is applied comes from the ISCA consortium, a worldwide group of 130 laboratories investigating developmental disorders. Founded by David Ledbetter, Ph.D., the ISCA team developed a microarray design and a set of probes targeted to regions known to be involved in pediatric developmental delay.
Anderson believes it is important to distinguish between DNA-based tests used to detect polymorphisms and copy-number changes as opposed to RNA-based measures of gene expression, since the former are inherently more reproducible. “The reliability of microarray comparative genomic hybridization data points a clear path to FDA clearance, whereas the issue is much more nuanced in the case of gene-expression data.”
Agilent has an active program of clinical discovery, Anderson says. “Many of our customers are clinical researchers with whom we are forging coalitions to drive discovery. We see our role as that of a catalyst, playing a supporting function to help bring together groups of clinical researchers so that they may share their data.
“We just launched a CGH + SNP microarray product for looking at copy-number changes and simultaneously identifying regions where loss of heterozygosity has occurred. Detecting copy-neutral LOH is important to clinical researchers seeking to understand the DNA changes that influence developmental disorders and many cancers.”
While advances in array technology hold great promise for the understanding gene function and the development of therapies derived from these findings, careful controls and monitoring are essential for success. “We view consumer-based microarray tests with caution because the interpretation of this complex data really belongs in the hands of board-certified professionals.”
Pursuing the Genome
The NimbleGen array platform was designed to help researchers better understand the genome and the affect of genetics on disease, according to Xinmin Zhang, Ph.D., senior product manager at Roche NimbleGen.
The NimbleGen Sequence Capture Exome technology is focused on the most biologically relevant part of the genome, the exome, capturing only the coding exons that are the most functionally relevant and the best understood 1% of the genome.
The company recently introduced the SeqCap EZ Human Exome v2.0, which allows researchers to capture all RefSeq coding exons and miRNA genes in a single tube. It employs empirical rebalancing, a probe-optimization method, in order to enhance capture uniformity. This process is based on real capture and sequencing data derived from optimization of probe density and coverage for all regions.
Because the platform reduces probe density on regions with high-capture efficiency while increasing it in regions with low-capture efficiency, it provides uniformity leading to detection of over 90% of variants in the exome with a single lane (~3 Gb) of sequencing.
Other recent introductions for the cytogenetics research market include NimbleGen CGX cytogenetic arrays, according to Emily A. Rorem, director for product management. The CGX design is optimized for constitutional chromosome analysis to identify copy-number variation in the genome, which may be associated with recognized and unknown syndromes.
The assay interrogates the entire genome and has a high resolution of coverage over targeted chromosomal regions, including over 200 recognized genetic syndromes, 675 functionally significant genes, and the subtelomeres and pericentromeric regions, she adds. The CGX array portfolio specifically addresses cytogenetic applications and provides scalability with three different array formats to process 3, 6, or 12 samples per slide, she adds.
“In the future, we will expand the capabilities of our long oligonucleotide array platform to include SNP detection, providing accuracy and flexibility,” Dr. Zhang reports. “This technology will aid researchers in validation and follow up on SNPs detected through whole-genome or whole-exome sequencing.”
The company also intends to achieve GMP compliance followed by subsequent submission for clearance of select array products for the cytogenetics market to the FDA, Rorem explains. The SNP program described by Dr. Zhang will also play an important role in the areas of oncology research.
Dr. Zhang emphasizes that bringing new products through the approval process requires new content, which is not always readily available and requires significant research and advanced tools.
“With the recent progress in genomic technologies, most notably in next-generation sequencing, many more genetic and epigenetic markers will be discovered and validated for diagnostic use. We expect that arrays will be an ideal platform to develop tests for this novel content and new markers.
“For many genomic studies, researchers are discovery-driven and not necessarily hypothesis-driven. Therefore, the focus and end results of the research do not always establish the link between diseases and genes. This doesn’t necessarily reflect on the technology as long as arrays continue to deliver accurate and comprehensive data. The key bottleneck to this issue is in the experimental design and data analysis.”
“The Omni 2.5 and Omni 5 are the most complex chips in the market today, containing 2.5 and 5 million markers, respectively,” states Jay Flatley, CEO of Illumina. These arrays, derived from the Thousand Genomes Program, will reportedly permit screening for rare variants, providing quality data through robotic automation. “The 2.5 array is a whole new generation that will get at human variation at a much deeper level than was historically possible.”
Illumina is also in the process of submitting its products for FDA approval, including chips for cytogenetic analysis. Flatley believes that the market is moving toward arrays of much higher complexity, and this is reflected in the FDA’s evaluation process. The rapid pace of development has spurred the agency to work with industry to develop appropriate regulations governing these advanced technologies.
The new cytogenetic arrays are revolutionizing clinical cytogenetics, and in three to five years, the vast majority of these studies will be done with arrays, and only a small fraction of specialized tests will be carried out with traditional karyotyping platforms. This is reflected in a recently released set of guidelines from the American College of Medical Genetics, changing the standards of care for application of SNP-based cytogenetics. The new technology is less labor-intensive, faster, and more accurate. While this will mandate retraining for professional cytogeneticists, it clearly will provide greater accuracy, speed of analysis, and cost-savings.
Pharmacogenetic markers are also an expanding area of microarray analysis. These markers are already providing better predictive accuracy in disease association including diabetes and prostate cancer for which there are many putative markers under scrutiny.
“While some of this data does not currently provide patients and their physicians with clear options for treatment, the bulk of knowledge is rapidly expanding, and in the near future microarray descriptions of patients profile will be much more informative,” says Flatley.
These advances are requiring updating and retraining for physicians, as well as cytogeneticists. “The new technologies are complex and until arrays are more widely adopted, it will be necessary to work with physicians to educate and inform them regarding the tests that they are already prescribing.”
Array technology operates on the basis of collaborative effort, which may involve clinicians, research scientists, and software designers. The companies profiled in this article have programs designed to advance drug discovery through large-scale interactive programs that bring together diverse groups of scientists searching for a common goal. Given the scope of these studies, it is clear that successful drug discovery will be predicated on this coordinated response.
K. John Morrow Jr., Ph.D. ([email protected]), is president of Newport Biotech and a contributing editor for GEN.