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Quest Publishes Validation Study of HIV Tropism Test Combining 454 and Sanger Sequencing


Quest Diagnostics has published a validation study of its laboratory-developed HIV tropism test that uses a combination of Sanger sequencing and next-generation sequencing on Roche's 454 GS Junior platform.

Quest began offering the test this summer for patients with HIV to determine whether they are eligible to receive the drug maroviroc, marketed by Pfizer as Selzentry.

In the study, published in PLoS One last month, the Quest team retrospectively tested 327 blinded samples from two clinical trials of Selzentry and compared their test to the current standard, a phenotypic assay developed by Laboratory Corporation of America subsidiary Monogram Biosciences called Trofile.

A tropism test is required before receiving Selzentry because the drug is effective only in patients harboring so-called R5 viruses, which use the CCR5 chemokine coreceptor to infect cells. Selzentry binds to CCR5, so is not effective against X4 viruses, which enter the immune cells through the CXCR4 coreceptor.

Ron Kagan, director of bioinformatics at Quest, said the company decided to develop a test combining both Sanger and next-gen sequencing because each technology offers distinct advantages.

Sanger sequencing is done as a first pass, said Kagan. It targets the third variable, or V3, loop of the HIV-1 gene.

About 30 percent to 40 percent of patient samples harbor the X4 virus, he said, and those can be readily detected with Sanger sequencing. "At that point, we can report them out very rapidly," he said.

Samples that are predicted by Sanger sequencing to be R5 then go on to be confirmed via 454 sequencing because Sanger sequencing is not sensitive enough to detect minor non-R5 variants, Kagan said.

For the next-gen sequencing portion, Quest pools 16 samples, sequencing the V3 loop of the HIV-1 gene.

The combination helps reduce both turnaround time and cost, compared to the Trofile test, Kagan said.

All told, the Sanger step has a turnaround time of about seven days, while the combination screen takes around 10 days total, faster than Monogram's phenotypic Trofile test, which has a turnaround time of about two weeks, Kagan said.

Additionally, the Sanger sequencing portion of the test costs $800 while the combo costs $1,300. If patients are confirmed as X4 by the initial Sanger sequencing test, they are only charged the $800, says Kagan. Meantime, the Trofile assay runs around $2,800, he said.

In the PLoS One study, the Quest team demonstrated that the Sanger/next-gen combination assay, dubbed Reflex, performed similarly to Monogram's Trofile test for predicting which patients would respond to Selzentry.

The team tested samples at two different time points — eight weeks after receiving maroviroc and 24 weeks after receiving maroviroc.

At eight weeks, the Reflex assay and the Trofile assay had similar positive predictive values of 65 percent and 66 percent, respectively, as well as similar negative predictive values of 58 percent and 59 percent, respectively.

Similarly, at 24 weeks, both assays had increased negative predictive values — 71 percent and 73 percent, respectively, and lowered positive predictive values — 40 percent and 42 percent, respectively.

While the Trofile assay has been in use for around five years as a companion diagnostic for determining which patients should receive Selzentry, Kagan said that the "issue of rapid turnaround time and cost, coupled with clinical validation, provides strong evidence for clinicians" to use Quest's tropism test.

Other researchers have also sought to develop genotypic tropism tests, including a group from the Institute for Immunology and Genetics in Kaiserslautern, Germany, which used the 454 GS FLX alone to detect viral tropism (CSN 5/24/2011).

While that team, too, found that the next-gen test performed as well as the Trofile test, Martin Däumer, who led the study, said that there were significant hurdles toward commercializing an assay based solely on next-gen sequencing, including cost and the complexity of the workflow.

Quest's Reflex test thwarts some of these issues by first doing a Sanger sequencing screen, and Kagan said that currently, Quest is not considering dropping the Sanger sequencing portion of the assay.

"We don't have plans to remove Sanger sequencing at this point because it provides an advantage," he said, including "a more rapid turnaround time and [being] more cost-effective."

Moving forward, however, Quest is planning to develop other next-gen sequencing-based diagnostics.

Andrew Grupe, the senior director of pharmacogenomics in Quest's Celera business previously told Clinical Sequencing News that the company is evaluating the Illumina MiSeq, Roche's 454 GS Junior, and Life Technologies' Ion Torrent PGM, and that it plans to develop tests in the areas of oncology and infectious disease (CSN 7/11/2012).

Kagan declined to elaborate further on these plans.