By Monica Heger
Sequencing could be used to detect whether Pfizer's HIV drug Selzentry will be effective for individual patients, according to researchers from the Institute for Immunology and Genetics in Kaiserslautern, Germany.
Testing the ability of Roche's 454 GS FLX to detect viral tropism in HIV, the team sequenced the viruses of 14 patients, selecting specifically for the V3 loop of the virus, the region that allows it to infect human immune cells.
Viral tropism is particularly important for drugs such as maraviroc, marketed by Pfizer as Selzentry, which only works in patients harboring so-called R5 viruses, which enter their immune cells via the coreceptor CCR5. Selzentry binds to CCR5 and is therefore not effective against viruses that enter cells through the CXCR4 coreceptor, or so-called X4-viruses.
Reporting in Genome Biology this month, the team found that sequencing can predict which patients will respond to maraviroc as well as the current "gold standard" — a phenotypic assay developed by Monogram Biosciences called Trofile.
Martin Däumer, lead author of the study, said he and his team wanted to see whether sequencing could be a good alternative to the Trofile test.
He noted that the main advantage of sequencing is that "you also get a quantitative output" regarding the proportion of X4 virus, which could potentially be used to "establish a clinically relevant cutoff" for determining how much X4 virus a patient can have before he or she does not respond to maraviroc.
To perform the sequencing, the researchers first extracted RNA using the Viral RNA mini kit from Qiagen. They then amplified the V3 loop region using PCR primers. All 14 patients were tested with the Trofile assay, population-based Sanger sequencing, and deep sequencing on the 454 GS FLX.
The team generated about 7,600 reads per isolate, detecting an average of 280 variants in the V3 loop per sample. Comparing the X4 isolates, which will not respond to maraviroc, to the R5 viruses, which will respond, the team did not find any differences in terms of the number of reads generated, the viral load, or the number of variants in the V3 loop. Compared to the Trofile assay, their results were concordant in all but on isolate.
Däumer said that the sequencing assay would have been more sensitive than the Trofile assay since it could detect minor variants below 1 percent frequency. However, the researchers applied an artificial cutoff of 5 percent presence of X4 virus for determining whether a patient would respond to maraviroc — a value that matched the proposed sensitivity limit of the Trofile assay used in the analysis.
The authors noted that the Trofile assay they used in the study has been replaced by an enhanced version that is able to detect minor variants with frequencies as low as 0.3 percent.
Harlan Robins, a computational biologist at the Fred Hutchinson Cancer Research Center and co-founder of immunorepertoire sequencing company Adaptive TCR, said that while the study was scientifically interesting, it was hard to see the practical applications of a sequencing-based test for viral tropism in HIV patients when a proven test already exists.
"It has potential," he said, "but you're competing with something that is commonly used and works."
Additionally, for the test to be commercially viable it would likely need to be validated in a large clinical trial and compared with the Trofile assay to determine whether it is better at predicting which patients respond to maraviroc and which do not.
It would be difficult to find funding to support such a trial given that the drug has already been approved by the US Food and Drug Administration and the Trofile assay is already being used as a companion diagnostic, with good results. Particularly for drug companies, there would be no motivation to fund a study that could result in fewer patients being prescribed the drug.
"If the best you can do is say, 'Most of the time we reproduce this gold standard,' it will be hard to convince someone to switch over," Robins added.
Däumer acknowledged that there would be significant hurdles to launching a sequencing-based tropism assay that would compete against the Trofile assay and said that his team is currently not looking to commercialize such a test.
He said for such a test to be commercialized, costs would have to come down and there would need to be significant automation to the 454 sequencing protocol. Currently, "the workflow is still very complex and only applicable for labs that are highly experienced in the 454 technology," Däumer said. "The technology itself is very expensive and labor intensive."
He said he is now looking to see whether a similar tropism test can be developed for other viruses for which a test is not already available, such as the hepatitis C virus, which demonstrates a "similar variability" and may be affected by tropism similarly to HIV.
The studies done by his team are driven by "focusing on a research interest, but always with one eye on a routine diagnostics application."
For instance, Däumer said that he is also collaborating with Roche to use sequencing to determine drug resistance in HIV patients.
"I'm absolutely convinced," he said, that next-generation sequencing has already "changed the view on diagnostics" and "will be a very big brick in the wall of personalized medicine."
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