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454, Intelligent Bio-Systems to Test Their Sequencers’ Dx Chops on Rx-Resistant HIV

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Roche subsidiary 454 Life Sciences and Intelligent Bio-Systems are testing the diagnostic capabilities of their respective sequencing technologies by applying them to HIV drug resistance, the groups said independently last week.
 
In addition, at least two other research groups have used 454’s platform to test for drug-resistant HIV variants, In Sequence has learned.
 
IBS, which is located in Waltham, Mass., is at a much earlier stage with its project than 454, which has been working with HIV researchers at Yale University since 2005.
 
Both companies aim to improve the sensitivity of current commercial assays, which rely on Sanger sequencing and cannot detect drug-resistant HIV variants that make up less than 20 percent of a patient’s viral population.
 
Yale and 454
 
To get in the game, Yale and 454 must prove that their test is better than existing ones, has clinical relevance, and will be competitively priced with current resistance tests, which cost between $300 and $500, according to experts.
 
Last week, Michael Kozal, an associate professor of medicine at Yale University School of Medicine and director of the HIV program at the VA Connecticut Healthcare System, presented results from a retrospective study that his team conducted with 454. The researchers showed that 454’s FLX instrument can detect low-level drug-resistance variants in antiretroviral-naïve HIV-positive patients. Moreover, these mutations can predict failure of antiretroviral therapy (see In Sequence 3/20/2007).
 
454 and Kozal, who presented the group’s findings last week at the XVI International HIV Drug Resistance Workshop in Barbados, sequenced two viral genes in 258 blood samples taken from long-term infected patients prior to drug treatment. The samples came from the FIRST study, a multi-center five-year trial comparing three different approaches to antiretroviral therapy. The researchers will soon submit their results to a journal for publication, according to Kozal.
 
Screening the sequences for almost 150 known drug-resistance mutations, Kozal and his colleagues detected some that were only present in 1 percent of a patient’s viral population. Overall, they found mutations in more than twice as many patients than standard resistance testing methods.
 
Many of the patients that harbored resistance mutations quickly failed drug therapy once they started a regimen. “If the clinicians knew that, they probably would have put [these patients] on alternate regimens to suppress those resistant variants,” Kozal told In Sequence last week.
 
While real-time PCR methods that test for single mutations can reach a similar level of sensitivity, none can test so many mutations in parallel, he said. The 250-base-pair reads from 454’s FLX instrument also enable the researchers to determine whether several mutations are linked in the same virus. “Sometimes that’s important ..., so you would know how to treat that one strain,” he said.
 
Kozal, who started collaborating with 454 in 2005, believes that if the new test was used in more clinical trials and found to be useful, the HIV field would accept it and implement it. “I don't think the assay is ready for prime time as is,” he said in an e-mail message this week.
 
A Roche spokesman told In Sequence by e-mail that the company has no immediate plans to develop the assay into a clinical test, noting that the Genome Sequencer is marketed for research use only at the moment. Under that label, clinicians can use the platform for “an aspect” of clinical trials, he said. He also noted that clinicians have adopted “research only” technologies for patient care in the past, but it was up to them to obtain “the necessary approvals.”
 
Other researchers are cautious as well. “A lot more work needs to be done before this will be used clinically,” said Robert Shafer, an assistant professor of medicine at Stanford University and medical director of the HIV antiretroviral drug testing program at Stanford University Hospital.
 
For example, researchers need to conduct more clinical studies to prove that knowing about minor drug-resistance variants in advance actually helps treat patients better. “You might think that’s obvious, but those studies still need to be done before anyone is willing to pay the extra cost,” Shafer said.
 
Secondly, the price of the assay needs to be kept low. “There are a lot of adjustments that could be made to the technology, and we are working on some of those that could lower the cost,” he said. “I think the cost will come down pretty quickly, it will be the clinical relevance studies that will take longer.”
 
According to the Roche spokesman, one HIV assay costs about $500 at the moment, based on an analysis of 16 samples in parallel in an $8,000 instrument run.
 
Shafer and his colleagues at Stanford have been using 454’s GS 20 in-house for over a year to analyze HIV drug-resistance variants, and will publish their results in an upcoming issue of Genome Research.
 
Though he was not able to discuss the results of his study in detail, he noted that “personally, I think 454 has much greater potential than [real-time] PCR assays,” which test for single point mutations. The reason is that 454 sequencing assays many different mutations at once and that it does not depend on hybridization, which can differ markedly depending on the sequence context, and can thus lead to false positives or negatives, he said.
 

“There are a lot of adjustments that could be made to [454’s assay], and we are working on some of those that could lower the cost.”

Another researcher who has tested 454 sequencing for HIV genotyping is Randall Lanier, a senior investigator in the department of clinical virology at GlaxoSmithKline in Research Triangle Park, NC. He compared “a very laborious traditional ‘clonal analysis’ to 454 and found the results were similar, but that 454 was relatively cheaper, faster, and more informative,” he told In Sequence by e-mail last week. That clonal method, though, would not be suitable for clinical use, he added.
 
Lanier, who had 454 perform sequencing for him, noted that the company “needs to reduce cost and increase throughput to become a real clinical tool.”
 
IBS
 
A low-cost assay that can detect mutations present in 1 percent of a viral population is what Intelligent Bio-Systems wants to develop as well. “We expect that our test is going to cost just a few hundred dollars,” IBS CEO Steven Gordon told In Sequence.
 
Last week, IBS said it has won a two-year, $600,000 phase I SBIR grant from the National Institute of Allergy and Infectious Disease to apply its next-generation sequencing platform to HIV resistance testing.
 
During their SBIR research, the company, which licensed a sequencing-by-synthesis chemistry from Jingyue Ju’s lab at Columbia University last year (see GenomeWeb Daily News 12/12/2006, In Sequence’s sister publication), plans to “prove out the use of the technology and the feasibility of having a low-cost test,” Gordon said. Later on, the company, which collaborates with Daniel Kuritzkes, a professor of medicine at Harvard Medical School and director of AIDS research at Brigham & Women's Hospital, plans to validate a prototype of its platform using clinical samples.
 
IBS has not yet determined a timeline for developing an HIV-resistance test, but the project “demonstrates how our technology can be used in the diagnostics world,” Gordon said.
 

The company still plans to place early-access versions of its sequencing instrument before the end of the year. Last month, the company closed a several-million-dollar Series A funding round from angel investors that will enable it to complete development of its technology, according to Gordon.

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