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  International HIV and Hepatitis Drug Resistance Workshop
June 8-12, 2010,
Dubrovnik Croatia
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Genotypic Analysis of V3 in Patients Experiencing Virologic Failure on Maraviroc-containing Regimens and Correlation with Pre-treatment "Deep" Sequencing Results - poster
  ResisWksp: Genotypic Analysis of V3 in Patients Experiencing Virologic Failure on Maraviroc-containing Regimens and Correlation with Pre-treatment Deep Sequencing Results - (06/11/10)
Reported by Jules Levin
Intl HIV/Hepatitis Drug Resistance Wkp, June 8-12 2010, Croatia
Luke C Swenson1, Celia KS Chui1, Chanson J Brumme1, Dennison Chan1, Conan K Woods1, Theresa Mo1, Winnie Dong1, Douglass Chapman2, Marilyn Lewis2, Jim Demarest3, Ian James2, Simon Portsmouth2, Jayvant Heera2, Hernan Valdez2, P. Richard Harrigan11 BC Centre for Excellence in HIV/AIDS, Vancouver, Canada; 2 Pfizer, Inc. New York, NY; 3 ViiV Healthcare, Research Triangle Park, NC
Author Conclusions
Genotypic and phenotypic tropism assays can detect tropism changes upon failure of maraviroc-containing regimens
Early MVC VF with R5 HIV is not associated with obvious mutations in V3
Even simple algorithms (11/25rule) seem capable of identifying nonR5 VF sequences following MVC treatment, since many sequences are "highly" non-R5
Most non-R5 virologic failures show outgrowth of pre-existing minority variants that could be detected earlier with "deep" sequencing
· Virologic failure (VF) on CCR5 antagonists can occur by selection of non-R5 and/or drug-resistant R5 variants
· Limited information is available regarding genotypic changes in the HIV V3 loop following maraviroc (MVC) exposure
· 184 individuals were examined from the MOTIVATE trials of maraviroc in treatment-experienced patients
· All patients were R5 at screening by the original Trofile assay, and went on to experience VF on a maraviroc-containing regimen . Earliest follow-up sample was generally notprotocol-defined failure sample*
* (1) An increase to at least 3 times the baseline pVL at week 2 or thereafter. (2) pVL <0.5 log10 decrease from baseline on 2 consecutive visits starting at week 8. (3) pVL <1.0 log10 decrease from baseline on 2 consecutive visits starting at week 8, in a patient who previously achieved a ≥2 log10 decrease from baseline. (4) Increase in pVL to ≥5000 c/mLon2 consecutive visits in subjects previously confirmed to have pVL <400 c/mL on 2 consecutive visits.
· "Deep" V3-loop sequencing with a Roche/454 GS-FLX was performed on the screening sample of these patients, as was standard, population-based sequencing
· Population-based V3 sequencing was subsequently also performed at follow-up
· HIV tropism was inferred using the geno2pheno algorithm (cutpoints: 3.5 for "deep" sequencing; 5.75 for population-based sequencing)
· The V3 sequences obtained at VF were evaluated for phylogenetic relatedness to pre-treatment "deep" 454 sequencing data using ClustalX neighbor-joining trees and Figtree
Genotyping at follow-up
· Standard V3 sequencing was 91% concordant with Trofile in determining tropism at VF: 168/184 cases

-- 87% sensitivity, 97% specificity
· Both methods indicated non-R5 in 91 VF samples, & R5 in 77
-- 16 samples were discordant, with Trofile indicating non-R5 in 14, and genotyping indicating non-R5 in 2
-- 15/16 patients with discordant samples had evidence of non-R5 HIV by Trofile at other weeks, plus 12 patients that had R5 by both methods at the particular time-point tested
· Non-R5 HIV rapidly emerged (median 4 weeks) faster than virologic rebounds with R5 HIV (median 8 weeks)
· Follow-up non-R5 variants usually had very low (i.e., very non-R5) g2p false positive rates (fpr) (median 1.1), much lower than the median value at screening (23.9)
Figure 1: Low g2p fpr are selected despite their low prevalence at screening by standard or deep sequencing

g2p false positive rates

g2p false positive rates
· The distribution of g2p fpr at screening and VF across all concordant non-R5 VF patients plus the mean proportion of 454 sequences present at various g2p scores. Low g2p fprs are common at failure despite having low prevalence at screening by standard and deep sequencing.
· 65 patients had R5 virus by both methods at all weeks tested and likely experienced VF due to either resistance to MVC or to a component of their background regimen (Figs 5 & 6)
· Few mutations and Insufficient phenotypic MVC-resistance data to assess MVC-resistant R5 HIV in this population
Deep sequencing
· 454 identified 28% of all patients (51/184) as already having ≥2% non-R5 at screening
· 68% of cases (63/93), non-R5 sequence(s) closely related to follow-up sequence were detected pre-treatment by 454 (Figs 2-4)
-- Suggests outgrowth of pre-existing non-R5 variants
· Patients who experienced non-R5 VF had a mean of 15% non-R5 virus present at screening by 454 vs. 1% for patients with R5 VF
Clinical Response
· Patients who experienced non-R5 VF by genotyping had poorer clinical outcomes on MVC-containing regimens than those with R5 at follow-up
· Smaller median plasma viral load (pVL) decline from baseline to week 8: 0.4 log vs 2.0 log.
-- This trend held for the decline at follow-up sampling. Median (Q1, Q3): non-R5: -0.5 (-1.0,-0.04); R5 : -0.9 (-1.8, -0.1)
· Patients with non-R5 at follow-up had lower median CD4 cell gains between baseline and week 24: 34 cells vs 90 cells