Roche is preparing to introduce an Affymetrix-based ASR to the market, the first commercial product to emerge from a multi-year licensing collaboration announced in January.
The company will roll out the AmpliChip CYP450 microarray for lab use in the US in May. Roche will later seek approval for IVD, and CE mark approval in the US and Europe. The AmpliChip will be sold as an analyte-specific reagent in Roche’s initial step towards bringing microarrays into the world of personalized medicine, a visionary’s land where genetic tests help create patient-specific treatments.
It has been almost three months since Affymetrix announced an 18-year technology licensing deal with Roche that included a $70 million one-time payment amortized over the next five years, as well as payments for chips, royalties, and milestone incentives (BioArray News, Feb. 7, 2003).
This initial product of the deal is based on cytochrome P450 liver enzymes that functions in drug metabolism. Isoenzymes encoded by several of these genes are critical in the research related to the metabolism of key therapeutics, including beta blockers for cardiovascular disease, selected anticonvulsant medications, and a large class of antidepressants. Patients lacking the ability to metabolize drugs using this pathway may suffer adverse reactions due to build-up of medications in their bloodstream.
While it is based on the Affymetrix platform, the AmpliChip designed by Roche is not your father’s GeneChip.
“It is a significant extension beyond what Affymetrix has as a research product,” said Walter Koch, director of pharmacogenetics for Roche Molecular Diagnostics of Pleasanton, Calif., the US arm of Roche Diagnostics. Affymetrix manufactures the chip in its Sacramento, Calif., plant and Roche puts together the final version in its Branchburg, NJ, facilities.
The AmpliChip array, with its “powered by Affymetrix,” label, queries the CYP2D6 and CYP2C19 genes. Arrayed at 20 microns, the chip probes for more than 30 polymorphisms in the CYP2D6 area, and two in CYP2C19, and it also detects inherited deletions or duplications. Roche is also using a long-PCR multiplex amplification procedure with a novel DNA polymerase that allows UNG enzymatic sterilization for carry-over prevention, and a different labeling protocol than that used by Affymetrix.
The AmpliChip builds on the research platform that Affymetrix launched as its GeneChip CYP450 assay in November 1997. As described on the Affymetrix website, the GeneChip CYP450 microarray is a DNA probe-based assay for the simultaneous detection of 18 known genetic variants of two human cytochrome P450 genes. It defines 10 alleles of the 2D6 gene and two alleles of the 2C19 gene, which encode some of the cytochrome p450 enzymes. This assay discriminates homozygous and heterozygous genotypes, while providing sense and antisense strand analysis in a single hybridization experiment. Affymetrix began designing a CYP450 array in 1993 and at one point developed one for Hewlett-Packard to sell. That deal did not reach fruition and Affy then released its own in 1997 to compete with genotyping methods such as conventional dideoxy sequencing and restriction fragment length polymorphism analysis.
Changing the Protocols
To create a method for distinguishing between deletions and duplications of CYP2D6, both of which generate a unique 3.5-kilobase PCR product and cannot be co-amplified in the same reaction, Roche uses two reactions that separate the primers. The duplication-specific product, when present, is amplified together with 2.8 and 3.1 kilobase amplicons encompassing the entire CYP2D6 coding and promoter regions. The gene deletion-specific amplicon, when present, is amplified together with a 1.4 kilobase product that encompasses exons 4 and 5 of CYP2C19. The two reactions are amplified under identical cycling conditions, and the products are pooled and fragmented and labled, then hybridized to the array for detection by a conventional Affymetrix scanner.
However, Roche has also cre-ated a different labeling protocol than that used by Affymetrix, using phycoerythrin, which provides a 10-fold improvement in fluorescence intensity, Koch said.
Selling targets: Clinical Diagnostic Labs
The initial US market for the AmpliChip and the specific primers will be CLIA-certified high complexity labs, which will use the AmpliChip ASR set as part of their homebrew test. The laboratories are responsible for validating the performance of their test.
Roche does intend to seek FDA approval, according to Koch, “but we did not want to delay making the test available to reference labs,” he said.
While Roche is not giving a specific time frame for seeking FDA approval for the AmpliChip, there are steps that need to be taken with an eye to agency approval and that process is ongoing.
Probing the Populations
“We spent several years on this assay and essentially tested it with, if not hundreds, upwards of a thousand unique samples, and uncovered polymorphisms that were under primers, or under particular probes that could lead to infrequent but aberrant results,” said Koch. “We studied globally diverse populations to ensure that we had the most robust assay possible.”
The company integrated many of the polymorphisms that had been discovered since Affymetrix created its array, including those for populations from Asia and Africa.
It also introduced a long-PCR procedure to detect for gene deletion, as well as gene duplication, Koch said.
“These deletions and duplications can only be detected through long-PCR,” he said.
With assay results in hand, clinicians could infer a genetically inherited phenotype and adjust treatment accordingly.
The 30-member-strong family comprising the cytochrome P450 liver enzymes is involved in the metabolism of around a quarter of all drugs. So far, scientists have identified 49 genetic variants that cause deviations in the structure or expression of these enzymes. For CYP2D6, there are at least 70 polymorphisms now known. This gene is involved in the metabolism of over 50 drugs — from cough remedies to antihypertensive agents.
Some of the first patients that may benefit from phenotypic profiling using an assay such as this may be those who suffer from depression. The enzymes are important in the metabolism of several antidepressants.
But Koch doesn’t expect these assays to be adopted quickly.
“There is a time lag where new ways of doing medicine need to be validated, its effectiveness proven, and clinicians and patients educated on how this can help them,” he said.
Microarray technology, he noted, is much more complex. But, “we see many scenarios in the future where a clinician likely would want to perform a diagnostic test prior to using a drug,” he said.
This chip is the first of a list of assays that Roche will develop on this platform, said Tom Metcalfe, Roche's senior vice president for genomics and strategic business development. The AmpliChip roadmap, given a timeframe of the next decade, includes: a number of genotyping assays, particularly for risk assessment in association with complex diseases; infectious agent genotyping assays; pharmacogenetic assays similar to CYP450 and assays for stratifying complex diseases; and tests looking at the expression profiles of cancer tumors (to assist with therapy prediction), Metcalfe said.
“We have several pieces of key technology, and competitive access to these technologies,” Metcalfe said.
“We have a significant first-mover advantage. There are other arrays, sequencing and other ways of getting the same information. We will have to compete with those. We are certainly taking some risks, but they are all calculated, driven by a belief in the way that this market will develop.
“We are working like beavers to get products out the door.”
Roche has not made a pricing decision, he said. What the company hopes to provide with AmpliChip, he added, is information to make a better clinical decision, “helping people use therapies without fear of adverse reaction,” Metcalfe said.