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Why Proviral DNA Drug Resistance Testing is Useful in Guiding Treatment Decisions for HIV-1 Patients

Why Proviral DNA Drug Resistance Testing is Useful in Guiding Treatment Decisions for HIV-1 Patients

The management of human immunodeficiency virus (HIV) has come a long way since the infection was first identified in the early 1980s. While there is no cure for HIV, current treatments are able to keep the infection in check by suppressing viral replication to reduce viral loads.

Sustained viral suppression is achieved through antiretroviral (ARV) therapy, which often consists of a cocktail of two or more ARV drugs that work to inhibit viral replication. There are various classes of ARVs, which include nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) that block HIV reverse transcriptase, non-nucleoside reverse transcriptase inhibitors (NNRTIs) that allosterically block HIV reverse transcriptase, protease inhibitors (PIs) that block the function of HIV protease, integrase inhibitors that inactivate HIV protease and entry or fusion inhibitors which prevent binding of HIV to cells.

ARVs have been quite successful in managing HIV as a chronic condition. While ARV-based treatments work well to keep the virus suppressed and patients healthy — some for many years — resistance to one or more drugs in a treatment regimen can develop in some patients. HIV drug resistance has ben a problem since ARV drugs were first introduced in the late 1980s. While newer generation of drugs are less likely to induce resistance, the problem still persists.

The prompt and effective identification of viral mutations is therefore critical in switching patients off of failing treatment regimens due to drug resistance. In light of this, HIV drug resistance testing has become a routine part of HIV diagnosis and treatment monitoring alongside tests for CD4 T cell counts and viral loads.

Apart from the issue of drug resistance, clinicians and patients may also consider changing treatment regimens for other reasons, including replacing older drugs with new and improved ones or reducing the total number of drugs in an ARV treatment regimen to reduce the risk of long-term toxicity and cost.

In a recent webinar, experts from Labcorp/Monogram Biosciences discussed how proviral HIV-1 drug resistance testing can effectively guide treatment adjustments or regimen switches that may be required due to adverse events, regimen intolerance or drug-drug interactions.

Compared to standard plasma HIV-1 RNA drug resistance profiling, which requires a viral load of at least 500 copies per mL, proviral HIV-1 testing can be performed on samples with much lower virion counts of <50 copies per mL (below the detectable limit in RNA testing). This is of particular benefit for virally suppressed patients who have low viral loads; however, the technology can also be used in patients with viremia.

The panel of experts from Labcorp/Monogram Biosciences included Charles Walworth, MD, Associate VP, Medical Affairs and Education; Dusica Curanovic, PhD, Medical Science Liaison; Sharon Martens, MN, Medical Science Liaison; and Milka Rodriguez, PhD, Medical Science Liaison.

The panel spoke about the utility of proviral HIV-1 drug resistance testing in guiding treatment regimen changes for patients with long-term viral suppression and also those who may be treatment-experienced. They addressed some common questions about proviral HIV-1 DNA drug resistance testing, discussed the power of the GenoSure Archive proviral DNA testing platform, presented clinical studies and case studies and also discussed some challenges and key considerations when switching treatment regimens for HIV patients based on proviral DNA drug resistance mutational testing.

HIV Drug Resistance Testing

HIV drug resistance occurs when virions develop mutations that make them less susceptible to given drugs. ARV combination or cocktail treatment regimens typically involve drugs from different classes; this is ideal because if the virus develops a mutation against a certain drug, it is likely it will be resistant to all drugs in that class. Therefore, drugs from different classes are combined in a treatment regimen.

Dr. Curanovic explains that in situations where drug concentrations are subtherapeutic, perhaps due to poor treatment adherence, the virus will continue to replicate and the drugs that remain in the patient’s system will select for viral variants that replicate just in their presence. This is how drug resistance develops, leading a patient to fail their treatment regimen.

As the viral variant replicates, some of it enters long-lived CD4-positive T cells and becomes part of the latent reservoir; the resistance mutations it encodes are stored within this reservoir. If the treatment is discontinued, those drug resistance mutations can eventually be lost and wild type virus can rebound from the latent reservoir and outcompete drug resistant variants.

Therefore, the DHHS HIV Treatment Guideline states that drug resistance testing on plasma virus should be performed within four weeks of treatment discontinuation because if more time elapses, the variants may no longer be detectable in the plasma by conventional plasma HIV-1 RNA drug resistance testing. However, the variant viral genomes will still persist in the latent reservoir as provirus and can be detected with proviral DNA testing.

Moreover, resistance mutations are archived within the latent compartment and can re-emerge upon appropriate selective pressure. Drug resistance is therefore cumulative over a patient’s course of treatment.

Due to improvements in HIV-1 ARV drugs, many patients experience complete and sustained suppression of virus replication. Because of this, resistance mutations may be missed in virally suppressed patients with low viral loads. Conventional mutational testing involves detection of resistance mutations in plasma HIV-1 RNA, which requires viral loads of at least 500 virion copies per mL.

Therefore, for patients with virologic suppression or low-level viremia (those with less than 1,000 copies per mL), standard plasma RNA drug resistance testing may not be effective because there is simply not enough RNA in samples for amplification and sequencing. In contrast to HIV RNA, HIV proviral DNA remains present in a patient’s cells even during virological suppression. Thus, a plausible solution to this is to look for drug resistance mutations in the integrated viral DNA, known as proviral DNA, of latently infected CD4 cells.

Importantly, ARV treatment does not affect the proviral DNA genome that persists in latently infected cells.

Proviral DNA Genotyping

Proviral DNA drug resistance testing involves interrogating proviral DNA sequences in infected cells. In cells, HIV-1 RNA is reverse-transcribed to DNA by HIV reverse transcriptase, after which HIV integrase mediates the integration of the viral DNA into the host cell genome. The resulting integrated HIV DNA is known as proviral DNA and may remain latent or be transcribed to produce new viral particles.

The proviral DNA in these cells represents an archive of previous viral replication, including viral mutations. Therefore, any previous resistance to a drug should be present as drug-resistant virus in proviral DNA. In this way, proviral DNA can provide a historical record of a patient’s drug resistance mutations, offering information on viral mutations that emerged through the course of a patient’s infection.

Moreover, drug-resistant virus in proviral DNA could lead to viral rebound if latently infected cells containing drug-resistant proviral DNA become activated.

As such, sequencing proviral DNA for mutational detection is becoming an increasingly attractive approach in drug resistance testing. In the HIV space, proviral DNA resistance testing is the newest genotypic resistance testing to be incorporated into the day-to-day management of patients living with HIV, explains Dr. Walworth. And although the assay was originally intended for use in patients with undetectable viral loads or low-level viremia when standard plasma resistance testing cannot be carried out, the DHHS guidelines have expanded the clinical application of proviral DNA testing, with recent clinical data supporting the expansion.

Clinicians can use proviral HIV-1 DNA drug resistance testing technology to identify ARV drugs that are active against the virus. This technology is particularly useful in patients with low level viremia due to complete and sustained suppression of virus replication owing to their current treatment regimens, but who may need regimen modification due to sudden adverse events, drug-drug interactions or trying new and improved drugs with less toxicity.

However, the experts from Labcorp/Monogram Biosciences say regimen modification should be made cautiously because archived drug resistant viruses within latently infected cells could emerge given the opportunity under appropriate selective drug pressure. Therefore, it is critical to review the full ARV treatment history of patients prior to selecting a new treatment regimen.

Information gained from HIV-1 DNA resistance testing, such as the GenoSure Archive assay, may be used in combination with historical plasma RNA resistance test results to guide regimen switching in the setting of viral suppression. This testing may also provide additional information regarding archived drug resistance when prior resistance test results are unavailable or incomplete.

GenoSure Archive

GenoSure Archive is a proviral DNA testing platform that can be used to genotype HIV-1 variants present in latent CD4 positive T cell reservoirs, detecting drug resistance mutations archived in proviral HIV. The test is performed by sequencing HIV DNA extracted from infected cells and identifying mutations associated with drug resistance.

Some key questions that the expert presenters addressed during the webinar included:

  • What is the concordance between GenoSure Archive results and historical resistance reports?
  • Why might GenoSure Archive miss a mutation?
  • How much of a role do sampling bias in viral decay play?
  • Can GenoSure Archive be useful in patients with Viremia?
  • How do I interpret results of GenoSure Archive that indicates my patient is on a failing regimen but is fully suppressed?
  • What actions should be taken if a result indicates a failing regimen?

Case studies and clinical data were presented that demonstrate successful, continued viral suppression when GenoSure Archive is used to facilitate a regimen change.

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Related: Proviral HIV-1 DNA Drug Resistance Testing: Your Questions Answered

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GenoSure Archive: Concordance and Clinical Data

The panelists explain how one of the most common questions about GenoSure Archive is whether the proviral assay can detect all of the drug resistance mutations in a patient’s history. In other words, what is the concordance between proviral resistance results and the historical archive by traditional virus genotyping?

Dr. Curanovic presented results from a study by scientists at Monogram in collaboration with Dr. Stephen Deeks at the University of California San Francisco (UCSF), which was presented at ICAAC in 2015. The study was conducted on a subset of the SCOPE cohort, which is the study of the consequences of the protease inhibitor Era led by Dr. Deeks. Patients in the study have been closely followed for a long time and have been through monotherapy and ineffective dual therapy, and during treatment with non-suppressive regimens, they have developed extensive drug resistance but are now virologically suppressed.

The study aimed to see how many historical resistances the assay would be able to detect. The results showed that the GenoSure Archive assay yielded an overall assay sensitivity of 89 percent in its ability to detect historical drug resistance. With respect to specific resistance mutations, the assay sensitivity was 85 percent. For example, historical drug resistance variants at position M184 were found in 92 percent of cases.

Another study conducted by a group at Weill Cornell Medical College and presented at IDWeek in 2016 showed that across a cohort of 140 virologic supressed patients for whom GenoSure Archive was ordered over the course of one year, 163 drug resistance mutations were found on the patients’ historical tests. The Archive assay found a total of 197 drug resistance mutations, with an overlap between Archive and the historical tests being 153. This translates to nearly 94 percent of all historical drug resistance mutations in the cohort being captured on the GenoSure Archive assay, with an additional 44 mutations only being found on Archive and not in the historical drug resistance records.

Proviral DNA HIV Drug Resistance Testing: Factors to Consider and Viremia

The assay has been shown to not only have utility for virologically suppressed patients, but also patients with viremia when comparing mutations found in standard plasma testing versus proviral testing.

In a study of 89 patients with viremia for whom both plasma virus and proviral DNA drug resistance tests were done on samples collected on the same day, it was found that 92 percent of plasma virus mutations were also found in the proviral DNA.

The presenters also discussed instances where the GenoSure Archive test may miss a mutation, or when a drug resistance mutation identified previously in plasma RNA is not detected by GenoSure Archive. This could be linked to factors such as viral load, viral decay and sampling bias.

For example, Dr. Rodriguez outlined a study by De La Cruz et al., and researchers at Stanford University, which found that the likelihood of detecting a resistance mutation in proviral DNA is better at higher viral load.

Decay of proviral DNA due to natural cell turnover in a latent reservoir also results in loss of drug resistance mutations. Moreover, there is a significant negative correlation between re-detection of drug resistance mutations and time between testing, where as the time interval between testing increases, the percentage of drug resistance mutations re-detected by the assay decreases.

Use in Viremic Patients to Inform Regimen Switches

While GenoSure Archive was developed based on clinician request for use in patients with suppressed virus, or low-level viremia, one of the considerations was whether the assay could be additionally helpful in elucidating resistance patterns in individuals who present for care with detectable viremia and limited treatment history, explains Dr. Martens.

New guidelines for use of ARV agents in adults and adolescents with HIV recommend incorporation of proviral archive testing when conventional HIV RNA drug resistance testing is unsuccessful or unavailable for patients initiating therapy. However, all resistance testing should be interpreted with caution including standard plasma resistance testing, which is generally only reflective of the selective drug pressure of the current ARV regimen and may not capture mutations that have fallen below the levels for reporting or have reverted to wild type, explains Dr. Martens.

For patients who engage in intermittent care or who have breaks in adherence pose unique challenges to clinicians, she explains. In these instances, there may be patients with viremia who were previously suppressed; these cases often necessitate plasma resistance testing to evaluate for regimen failure.

Dr. Martens presented several studies that support the use of GenoSure Archive in clinical practice to help guide potential changes in treatment regimens. In most cases, regimen simplification prompted the use of GenoSure Archive, explains Dr. Martens.

Although the cohorts are small in some of the studies, says Dr. Martens, overall, successful maintenance of viral load suppression persisted at follow-up time points in 85 to 98 percent of participants whose regimens were change based on GenoSure Archive results.

Not every patient will require GenoSure Archive testing prior to a regimen change, but it is certainly a useful tool in cases where solely relying on historical plasma HIV RNA drug resistance testing isn’t enough as it fails to capture the complete archive of a patient’s drug resistance mutations.

For further information on proviral HIV-1 DNA drug resistance testing, including answers to important questions about the new technology, watch this on-demand webinar: Proviral HIV-1 DNA Drug Resistance Testing: Your Questions Answered.