Genotypic prediction of viral co-receptor tropism: Correlation with enhanced Trofile Angela L Strang, Jane Cameron, Clare Booth, Ana Garcia, Anna Maria Geretti Royal Free Hospital & UCL Medical School, London
Background � Phenotypic testing to predict HIV-1 co-receptor tropism can reliably guide use of CCR5 antagonists in clinical practice • Labour intensive, time consuming, expensive, limited availability • Viral load, plasma volume � Genotypic prediction methods based on V3 sequences offer an alternative testing tool • Growing but still limited data on correlation with phenotypic testing
Objectives � To evaluate genotypic tropism prediction for clinical use in a routine setting � To assess its concordance with results obtained by the enhanced Trofile assay � To analyse the clinical characteristics of the patient population according to the tropism prediction � To analyse the quasispecies of samples showing discordant phenotypic / genotypic tropism predictions (ongoing)
Methods � Plasma samples from 106 patients in routine care � Enhanced Trofile at Monogram (Pfizer programme) � Genotypic testing in house (population env sequences) � Interpretation of population sequence by Geno2Pheno • Clonal model (trained using clonal data from naïve patients) • Clinical model (input: CD4 n and %, CD8 n, viral load) • Varying false positive rate (FPR) to adjust sensitivity/specificity � Clonal analysis and 454 analysis of discordant and unusual samples (ongoing)
Study population Total 106 Subtype B n (%) 69 65% Non-B n (%)* 37 35% HAART Naïve n (%) 56 53% Experienced n (%) 50 47% Current CD4 n Median (range) 453 2-1067 Current CD4 % Median (range) 21 1-50 Nadir CD4 n Median (range) 361 2-950 Nadir CD4% Median (range) 21 2-47 Viral load log 10 Median (range) 4.6 2.8-6.0 *Non-B subtypes: A (6), C (16), D (2), CRF01 (2), CRF02 (8), CRF10 (1), cpx (2)
Enhanced Trofile R5 D/M NR Subtype B n 54 10 5 Non-B n 26 3* 8 HAART Naïve n 45 6 5 Experienced n 35 7 8 Current CD4 n Median (range) 455 (2-1067) 331 (22-894) 498 (289-688) Current CD4% Median (range) 23 (1-41) 15 (2-28) 23 (12-50) Nadir CD4 n Median (range) 366 (2-950) 288 (22-576) 390 (76-648) Nadir CD4% Median (range) 23 (4-47) 15 (2-28) 24 (5-33) Viral load log 10 Median (range 4.5 (3.2-6.0) 4.8 (4.4-5.9) 4.1 (2.8-5.5) Total n (%) 80 (76%) 13 (12%) 13 (12%) *A, C, CRF02
Genotypic tropism � Pre-validated across major subtypes/CRFs (n=50) In this study: � 99/106 samples successfully amplified � 7/106 (6.6%) assay failures • 4 B, 1 CRF02, 1 CRF01, 1 cpx • Median viral load 4.4 (range 3.5-4.9)
Concordance analysis: G2P vs enhanced Trofile � 87 samples with matched results FPR % Interpretation model Clonal Clinical 1 77/87 87% 77/87 88% 5 76/87 87% 77/87 88% 10 70/87 80% 79/87 91% 15 63/87 72% 78/87 90% 20 60/87 69% 77/87 88%
Concordance analysis: G2P vs enhanced Trofile � 11 samples with matched results and D/M call by enhanced Trofile FPR % Interpretation model Clonal Clinical 1 0/11 (0%) 2/11 (18%) 5 6/11 (55%) 3/11 (27%) 10 6/11 (55%) 6/11 (55%) 15 7/11 (64%) 7/11 (64%) 20 8/11 (73%) 8/11 (73%) Concordance for X4:
Sensitivity and specificity analysis for the detection of X4 virus* Clonal model Clinical model FPR Sensitivity Specificity Sensitivity Specificity 1 52% 100% 55% 99% 5 69% 93% 58% 97% 10 69% 86% 69% 96% 15 73% 79% 73% 94% 20 79% 76% 79% 92% *Analysis uses enhanced Trofile as comparator
Discordant samples: Enhanced Trofile vs Geno2Pheno with clonal interpretation at FPR 5% Pt Sub CD4 CD4 CD4 n CD4 VL ART Trofile G2P G2P clinical type n % nadir % Clonal FPR FPR nadir FPR 5% 10% 15% 1 B 288 13 288 13 4.5 Exp DM R5 R5 X4 2 B 331 18 331 18 4.8 Naïve DM R5 X4 X4 3 B 894 26 459 22 4.9 Naïve DM R5 R5 R5 4 B 472 20 277 28 4.9 Naïve DM R5 R5 R5 5 B 91 6 91 6 4.8 Exp DM R5 R5 R5 6 A 494 27 410 24 4.5 Naïve R5 X4 R5 R5 7 B 504 17 433 16 4.8 Naïve R5 X4 R5 R5 8 B 426 25 257 18 3.9 Exp R5 X4 X4 X4 9 D 343 25 301 23 4.0 Exp R5 X4 R5 X4 10 B 781 28 677 27 3.8 Exp R5 X4 R5 R5 11 B 474 29 474 29 4.2 Exp R5 X4 R5 R5
Discordant samples: Enhanced Trofile vs Geno2Pheno with clinical interpretation at FPR 10% Pt Sub CD4 CD4 CD4 n CD4 VL ART Trofile G2P G2P clinical type n % nadir % Clonal FPR FPR FPR nadir FPR 5% 10% 15% 20% 1 B 288 13 288 13 4.5 Exp DM R5 R5 X4 X4 12 B 642 28 576 27 4.7 Naïve DM X4 R5 R5 X4 3 B 894 26 459 22 4.9 Naïve DM R5 R5 R5 R5 4 B 472 20 277 28 4.9 Naïve DM R5 R5 R5 R5 5 B 91 6 91 6 4.8 Exp DM R5 R5 R5 R5 13 CRF02 338 13 118 8 3.4 Exp R5 R5 X4 X4 X4 14 C 2 1 13 4 5.9 Exp R5 R5 X4 X4 X4 8 B 426 25 257 18 3.9 Exp R5 X4 X4 X4 X4
Analysis of 100 clones from 2 patients Subtype Trofile FRP G2P G2P % X4 clones* Clonal Clinical model model B R5 1 R5 R5 94/100 R5 5 X4 10 X4 R5 15 X4 R5 20 X4 X4 D R5 1 R5 R5 79/100 5 X4 R5 10 X4 R5 15 X4 X4 20 X4 X4 *G2P clonal model FPR 5%
Conclusions � In this pilot evaluation we found excellent correlation between enhanced Trofile and Geno2Pheno • Best overall with clinical interpretation, FPR 10-15% • Best for X4 with highest FPR � Sensitivity/Specificity 79%/92% with clinical interpretation at FPR 20% � Use can be tailored to the patient’s treatment history in terms of optimal FPR settings � Geno2Pheno provides a valid tropism prediction tool
Acknowledgement � Rolf Kaiser, University of Cologne � Linos Vandekerckhove, Chris Verhofstede, University of Gent � Lieven Stuyver, Virco, Belgium � Patients and staff at ICDC clinic, Royal Free Thank you
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