October 15, 2006 (Vol. 26, No. 18)
Overcoming Inherent Experimental Variability and Impurities Will Aid in Acceptance
Microarrays have been a mainstay of the scientific research community for nearly a decade but given the more stringent demands for accuracy and reliability, have not been as closely embraced in the clinical arena. Arrays are, however, finding clinical applications in increasing numbers, and the growth of companies developing specific solutions for various stages of the array pipeline suggests that they are making progress toward meeting the requirements of the clinical and regulatory communities.
The recent CHI “Microarrays in Medicine” conference brought together key speakers in the clinical microarray technology arena, providing expertise in areas ranging from overcoming technical challenges in designing array platforms to describing successful clinical applications of microarrays.
Several of the platform presentations focused on array comparative genomic hybridization (aCGH). Comparative genomic hybridization is a general term that applies to the comparison of test and reference genomic DNA samples prepared from a tissue biopsy or blood in order to detect a presumptive aberration in the test sample.
CGH consists of three principal components: the test, reference DNA samples, and an immobilized substrate. “In earlier forms of CGH, the immobilized substrate was represented by chromosomes,” said Mansoor Mohammed, Ph.D., president and COO, CombiMatrix Molecular Diagnostics (www.cmdiagnostics.com). More recently, the place of chromosomes has been taken by arrayed sequences of DNA specifically selected to represent selected regions of the human genome. “Array CGH is the modern incarnation of traditional CGH,” said Dr. Mohammed.
In a typical CGH experiment, the test and reference DNA samples are differentially labeled with fluorescent dyes of differing colors. They are mixed and applied to the array. With the identity of the sequence at each point on the array and the relative amounts of fluorescence at those points both being known, inferences can then be made regarding the relative levels of specific sequences in the test sample.
An excess of the test sample at a specific sequence might indicate an amplification of that region in the test DNA. Conversely, a deficit of fluorescence from the test sample at another sequence might indicate a deletion or recombination event that prevented the binding of the test sample DNA to that probe sequence.
Resolution: BAC vs. Oligo Arrays
The first set of variables encountered in array CGH platforms concerns the selection, design, and production of the array platform. Broadly speaking, these are all a function of the specific information required from the test. One of the first considerations in aCGH is whether or not the mutation is in a known mutation or not and, if so, what size of aberration it represents.
“Choosing the DNA source and microarray type depends on the chromosomal changes and resolution that are to be detected by the assay,” said Todd Martinsky, executive vp, TeleChem International(www.arrayit.com). Telechem provides a variety of technical solutions to developing custom array-based clinical tests. “Our NanoPrint microarrayer, SpotLight scanner, surface chemistries, and other sample preparation products play a key role in any microarray experimental lifecycle.”
Early low-resolution approaches using fixed chromosomes have been superseded by BAC (Bacterial Artificial Chromosome)-based arrays, which perform a low-resolution interrogation of the test sample and are typically deployed to detect large-scale gross genomic rearrangements, deletions, insertions, or amplifications. A further refinement of the technique, oligonucleotide-based arrays, can detect smaller mutations in the test sample and offer the highest resolution in aCGH.
“With oligonucleotides, it is possible to tile across a gene at specific lengths at a defined resolution,” said Martinsky. This type of custom design affords a greater degree of resolution than can be obtained by spotting larger, less well-defined sequences of DNA.
“Oligonucleotide arrays are typically used when you don’t see gross deletions with BAC arrays or FISH,” said Henry Sadowski, Ph.D., senior scientist and director of product applications, Enzo Life Sciences (www.enzo.com).
Besides the resolution factor, another distinct advantage of aCGH is the speed at which custom chips can be designed, manufactured, and delivered. “CombiMatrix offers a range of custom array design and manufacturing services,” said Dr. Mohammed. “In some cases the turnaround on these arrays can be overnight.”
The type of resolution needed in the test dictates many of the technical parameters required for the success of the application. “If the assay is designed to discover new chromosomal disease associations, higher density microarrays are required,” said Martinsky. “These require more expensive microarray detection systems because of the required resolution.”
Conversely, microarray platforms designed to detect specific known mutations are typically of lower density and require less sophisticated detection systems. “With fewer than 2,000 BACs spotted into a microarray, it is possible to detect more than 70 recognized microdeletion or microduplication syndromes,” Martinsky pointed out.
The enormous logistical advantages that aCGH provides over traditional cytogenetic diagnostics can be appreciated by considering the situation that occurs when infants present at birth or in early childhood development with defined mental or physical defects, which physicians use as the basis for the tentative diagnosis of a specific syndrome.
In order to make a specific diagnosis they typically order a series of genetic tests. Traditionally, these would have taken the form of serial tests using some form of in situ hybridization in order to identify or rule out a known large-scale genetic change.
“In practical terms this can mean that using FISH, a clinician may have to spend thousands and thousands of dollars and many weeks to iteratively look at a small number of disorders and at the end may or may not have identified an abnormality,” said Peter J. Welch, Ph.D., director of gene-expression profiling R&D at Invitrogen (www.invitrogen.com). The advent of array technology, however, greatly increases the options open to clinicians seeking a diagnosis.
“Arrays offer two dramatic improvements over FISH-based CGH analysis,” said Dr. Welch. “Much finer resolution interrogation of the genome is possible with the use of specific probes on the arrays targeted to smaller regions of the genome.” The second area of improvement, Dr. Welch pointed out, is the ability of an array CGH platform to interrogate multiple regions of the genome at a time. “Array CGH converts a serial methodology to a parallel methodology,” Dr. Mohammed observed.
Technical Variables in CGH
Perhaps the most critical set of variables in aCGH experiments concern the preparation, purification, and quantitation of the test genomic DNA. “Reliable genomic DNA purification and quantification approaches are key to obtaining accurate results,” said Dr. Welch. “This is particularly the case for cancer aCGH applications.”
Typically, tumor biopsies are highly vascularized and fibrotic, making extraction and purification of DNA difficult. The bar is set even higher for retrospective studies, in which formalin fixing and paraffin embedding severely limit the amount of DNA that can be extracted. “The integrity and purity of isolated genomic DNA is key to efficient and balanced dye labeling and detection,” Dr. Welch stressed.
“Accurate quantification of isolated genomic DNA is also important,” he said. “Methods employed in aCGH require precise input of equal quantities of genomic DNA into the aCGH dye-labeling reaction in order to reliably resolve twofold gene copy number differences.” Invitrogen provides several solutions in the area of genomic DNA purification and quantification, from traditional silica-based PureLink™ purification products to the Quant-iT™ line of products designed for determination of yield and concentration from small amounts of double-stranded genomic DNA.
In cases where low DNA yield is a problem, amplification of the DNA prior to labeling is required to have a sufficient signal for the results to be interpreted. Dr. Sadowski described a novel genomic DNA amplification protocol for use in formalin-fixed, paraffin-embedded samples. “One of the advantages of this protocol is that it can be used as an index of the quality of a DNA sample, thereby giving the person running the test an idea of the usefulness of the information that the test will yield.
“It is more rapid than a PCR-based amplification. This method of whole-genome amplification can generate greater than 10 micrograms of DNA from 10 nanograms of high-quality genomic DNA in less than four hours.”
Purity is also a consideration. “Because PCR is exponential in nature, any contaminants present in the starting material will be present in significant quantities in the final reaction, which can impact the quality of the DNA to be used in the array.”
Purity of the test sample is another crucial factor in determining success in diagnostic CGH. When a biopsy is obtained during a surgical procedure, normal tissue is often removed along with the diseased tissue. When the biopsy is processed into DNA, a mosaic of normal and abnormal DNA is obtained. “Given the diluting effect of normal cells, we at CombiMatrix have found that at least 50% of the genomic DNA sample needs to represent the abnormality you are trying to detect,” said Dr. Mohammed.
Several microarray-based tests have progressed further along the regulatory pipeline. Drug-drug interaction is an issue of concern for physicians. Roche’s (www.roche.com) AmpliChip CYP450 is designed to discern genetic variation in the coding sequences of a series of genes encoding enzymes that play a major role in metabolizing up to 25% of all prescription drugs. The chip provides important information for physicians seeking to prescribe appropriate drug dosages on an individual basis.
Citing leukemia as a case in point, Walter Koch, Ph.D., head of research at Roche Molecular Diagnostics, emphasized the ability of microarray platforms to perform simultaneous tests to rapidly and accurately stratify the genetic changes underlying a disease state and to provide a molecular signature that can be translated into a customized therapeutic approach.
“Currently, analyses to determine leukemia sub-types are performed using up to six separate and often labor-intensive tests,” he said. “Microarray-based tests, such as the AmpliChip Leukemia Test now in development at Roche, may shorten this by offering a single assay designed to deliver the same results in a shorter time.”
What are the barriers to the increased acceptance of microarrays in the clinic? “Arrays are far from routine in the clinical setting due to their inherent experimental variability,” said Dr. Welch. “They are most often used as a first screening step, with the actual diagnostic carried out using an FDA-approved ISH test.”
Rick Hockett, M.D., director of genomic medicine at Eli Lilly (www.lilly.com), agreed. “The assay is not amendable to widespread use, as it is too cumbersome, and the entry-level cost for instrumentation is too high for most laboratories.”
Clearly, more information is needed in defining and improving the sources of variability in the platform before its acceptance on a wider basis in the clinical setting. Research being carried out by Dr. Hockett’s group at Eli Lilly is tackling this specific question.
The Affymetrix (www.affymetrix.com) microarray platform has been widely used in the research setting for hypothesis generation, but has yet to find widespread application in the clinical setting. A study by Dr. Hockett’s group was designed to mimic potential clinical applications using multiple operators, machines, and reagents, in order to determine the principal sources of variability in the platform. “The single largest source of variability is in the chip,” said Dr. Hockett. “The second largest source of variability is the lot-to-lot variation of chip production.”
The study, published last year in the Journal of Molecular Diagnostics, suggested that the validation protocol could be used to improve quality control in potential clinical applications of microarray platforms.