Issues to Consider in HIV Resistance Testing Jason Tokumoto, MD National HIV/AIDS Clinicians’ Consultation Center

Issues in resistance testing How ”beneficial” is resistance testing? What resistance test to use? Resistance testing in the multi-drug resistant patient. “When to switch” vs “How to switch” Genotype/phenotype discordance. Phenotypic hypersusceptibility. Replication capacity.

Current guidelines for resistance testing DHHS IAS-USA Acute infection Yes Yes Chronic infection Consider Yes (< 2 years) Treatment failure Yes Yes

“Benefit” How beneficial is resistance testing(genotype, phenotype, virtual phenotype)?

Clinical trials of resistance testing Genotype vs no testing: Viradapt, ARGENTA, GART, Havana. Phenotype vs no testing: VIRA3001, CCTG 575. Genotype vs phenotype vs no testing: CERT, NARVAL. Genotype + phenotype vs genotype: ERA. Virtual phenotype vs measured phenotype: GenPheRex, Realvirfen.

“Benefit” Virologic benefit derived from the use of genotypic resistance tests “relatively small.” At 6 months, genotypic testing increased proportion of patients who achieved undetectable viral load by 11% compared with no resistance test. Mean viral load reduction was 0.25 log 10. No clear evidence of benefit associated with phenotype vs no test. Pandou E T. Limited benefit of antiretroviral resistance testing in treatment-experienced patients: a meta-analysis. AIDS 2004

“Benefit” Some of the phenotype studies used fold change cut- offs that have been replaced by more accurate cut- offs(clinical cut-offs). Some patients had very advanced disease with few antiretroviral options and achieving undetectable viral load was unlikely no matter what method was used to select subsequent regimens. In patients failing initial or early regimens, clinicians might have found it straightfoward to select a new regimen without the use of resistance testing making it harder to detect a difference between arms. Many of these studies were short term and thus long- term benefits of resistance testing not known.

“Benefit” TORO 1 and 2 studies which assessed efficacy of T-20 showed that resistance testing was beneficial. These studies demonstrated that the number of drugs in the “optimized background regimen” to which the virus was sensitive as determined by genotype or phenotype was highly predictive of response to therapy.

“Benefit” Resistance test results can help to avoid using drugs that may be of no benefit and thereby avoid unnecessary toxicity.

“Benefit” Conclusion: There is short term virological benefit from resistance test vs no test; long term benefits(clinical benefits) unclear.

Which resistance test to order? Advantages/disadvantages. But which resistance test should one use?

Which test? Based on available clinical data, cannot be easily answered.

Which test? Many experts feel that genotype is adequate in initial or early failure. Assessment of NNRTI resistance can readily be assessed by genotype. Many experts prefer phenotype(plus genotype) in multi-drug resistant patients.

Issues in genotype interpretation in the multi-drug resistant patient In highly experienced patients, the multitude of accumulated mutations may be complex. Mutations may interact with each other making interpretation difficult. “Archived” mutations.

Issues in phenotype interpretation in the multidrug resistant patient Phenotype Cut-off: fold change above which a virus is not susceptible to an antiretroviral agent. Technical cut-off: based on a single reference virus. Biological cut-off: based on clinical samples from treatment-naïve patients. Clinical cut-off: based on virological response to antiretroviral drugs in clinical trials.

Resistance testing in the multi-drug resistant patient Help identify agents that are likely to retain the most activity(even if that activity is only partial compared with activity against the wild-type). In this setting, a phenotype may be more useful. Help to assess whether a new agent can be used with adequate support from the background regimen or whether its use should be deferred until it can be combined with other new agents. In other words, resistance testing in this setting might be used to determine “WHEN TO SWITCH” rather than “HOW TO SWITCH.”

When to switch? DHHS Guidelines(2004): “There is no consensus on the optimal time to change therapy for virological failure. The most aggressive approach would be to change for any repeated, detectable viremia…” But, “…a decision to change regimens might reduce future treatment options for that patient. This consideration can influence the clinician to be more conservative when deciding to change therapy.”

The big question If the decision is made not to switch based on resistance test results(and of course CLINICALLY), “WHAT TO DO IN THE MEANTIME?”

Genotype-phenotype discordance In the multi-drug resistant patient, you may have both genotype and phenotype results. In analyzing the data, you realize there is discordance between the genotype and phenotype.

Phenotype/genotype discordance Not uncommon. In one study(ViroLogic), which included over 30,000 matched genotypes and phenotypes: 67% of viruses discordant for 1 or > drugs 45% of viruses discordant for 2 or > drugs 30% of viruses discordant for 3 or > drugs

Reasons for genotype-phenotype discordance Presence of mixtures. Incomplete genotype interpretation data(not accounting for novel or unknown mutations or unrecognized effects of mutations). “Re-sensitization” caused by a specific mutations.

Case 44 year old male with AIDS with an initial CD4 nadir of 50 and viral load of 100,000. Has been on multiple antiretroviral regimens in the past. The specific regimens are not known but he has been on AZT, 3TC, DDI, abacavir, sustiva. He may have been on crixivan, saquinavir, nelfinavir in the past. He is currently on tenofovir, ddi-ec, kaletra with an HIV viral load of 50,000. You obtain both a genotype and phenotype.

Genotype/Phenotype discordance Nucleoside analogues Genotype: K65K/R M184M/V Resistant to abacavir, ddi, 3tc, tenofovir. PhenoSense: Sensitive to abacavir(1.18), ddi(1.29), 3tc(2.98), d4t, azt, tenofovir(0.78) IS THE VIRUS RESISTANT or SENSITIVE to abacavir, ddi, 3tc, tenofovir?

Genotype/phenotype discordance Mixtures: Genotype will report as resistant. Phenotype may or may not call it resistant; it depends on the proportion of the mixture. When there are mixtures,the genotype is likely to be the more reliable. Therefore, virus probably resistant to abacavir, ddi, 3tc, tenofovir.

Genotype/phenotype discordance Protease inhibitors Genotype: L10F, K20I, L24I, M46I, L63P, V77V/I Resistant to atazanavir, crixivan Sensitive to amprenavir, lopinavir, nelfinavir, ritonavir, saquinavir Phenotype(fold change): Resistant to amprenavir(13), crixivan(22), lopinavir(22),nelfinavir(7.01), ritonavir(8.23) Sensitive to atazanavir(2.10), saquinavir(1.58) IS THE VIRUS SENSITIVE OR RESISTANT TO AMPRENAVIR, ATAZANAVIR, LOPINAVIR, NELFINAVIR, RITONAVIR?

Genotype/Phenotype discordance Incomplete genotypic data Genotype not accounting for novel or unknown mutations or for unrecognized effects of mutations. In cases where the phenotype demonstrates resistance but the genotype indicates susceptibility, the phenotype is more likely to be the accurate of the two. Virus resistant to amprenavir,lopinavir, nelfinavir, ritonavir; virus sensitive to atazanavir.

Genotype-phenotype discordance “Re-sensitization” caused by a specific mutation. Genotype: M184V, M41L, K70R, L210W Tenofovir-resistant Phenotype: Sensitive to tenofovir

Phenotypic hypersuceptibility Clinical significant hypersuceptibility M184V affecting azt, d4t, tenofovir K65R hypersensitizing azt TAMS causing hypersusceptibility to NNRTIs.

Replication capacity(RC) Viral fitness: ability of entire virus to replicate in a defined environment. Replication capacity: an in-vitro measure of a single replication cycle of the plasma HIV-derived pol gene.

Replication capacity May have prognostic value: Overall better CD4 cell response when RC <65%. Among multidrug resistant patients whose HIV viral load not suppressed, >65% RC correlated with worse virologic response at 3 months. (CROI 2005) However, the role of RC in the management of antiretroviral therapy remains to be defined.