HIV DNA Genotyping Helen King, MD May 5th, 2017 UCSD HIV & Global Health Rounds

2. Helen King, MD
AIDS Clinical Rounds
May 5, 2017
HIV DNA Genotyping

3. Overview
• HIV Resistance Testing Techniques
• Purpose of DNA Genotype
• Literature comparing DNA and RNA Genotype Results
• UCSD Study: Clinical Outcomes after DNA Genotype Testing

4. HIV Resistance Testing
• HIV RNA genotype
• Phenotype
• Phenosense GT
• Ultradeep sequencing
• Tropism assay
• HIV DNA genotype (GenoSure ArchiveSM)

5. http://www.thebody.com/content/art46024.html

6. When to order resistance testing?
• DHHS Guidelines (July 14, 2016)
- To guide selection of initial regimen for ARV-naïve patients
- When changing regimens in patients with virologic failure (HIV RNA
>1000 copies/mL)
- Consider in patients with HIV RNA >500 and < 1000 copies/mL (may
be unsuccessful)
- When managing suboptimal viral load reduction

7. HIV viral compartments
• HIV genotype testing is based on 2
different viral compartments:
1) HIV-1 DNA
PBMC-derived
“Archived” virus
2) HIV-1 RNA
“Plasma” virus
Circulating virus

8. HIV RNA Compartment
• Actively replicating virus circulating in plasma
• The most current form of the virus
• Often wild type virus, if there is no selective drug pressure
- Not representative of viral variants that may be present in the viral archive
• 10% of persons get infected with transmitted resistance

9. HIV RNA Genotype
• Most common method to detect ARV resistance
mutations
• Only useful when detectable virus (HIV RNA >
500 copies/mL)
• Primarily detects mutations associated with
recent drug exposure
• Mutations present on previously archived
viruses are often not identified in the
circulating viruses despite their presence in the
latent cellular reservoir

11. Caveats to RNA resistance testing
• Note that when patients go off meds, within the first 4-6 weeks
wild-type virus often reemerges as the predominate viral
population.
• Drug-resistance variants that are less than 10-20% of the
circulating population may not be detected by standard RNA
population sequencing.
- These variants may be archived in the latent cellular reservoir and re-
emerge with re-introduction of medications.

12. 11
HIV Life Cycle
Cytoplasm
Nucleus
Furtado MR, et al. N Engl J Med. 1999;340:1614-1622.

13. 1212
Long-Term Persistence of Latently Infected
CD4+ T Cells in Patients on ART
• HIV-1 infected adults on ART with
ongoing HIV RNA suppression and
no treatment failure (n=62)
– Mean time on ART: 45.4 months
• Half-life decay of reservoir: 44
months (27.4-114.5 months)
• Time on ART to eradication of a
reservoir size of 106 cells
– Whole cohort: 73.4 years
– With viral blips: 95.8 years
– Without blips: 51.2 years
Frequency of Latently Infected CD4+ T Cells
Harboring Replication-Competent HIV-1
LatentlyInfectedCells(IUPM)
0 1 2 3 4 5 6 7 8
Time on ART
(years)
10,000
100
1.0
0.01
0.0001
Siliciano JD, et al. Nat Med. 2003;9:727-728.

14. HIV DNA Compartment
• “Archived” virus
• Cell-associated virus
• Virus transmitted and archived in the host cells during viral replication
• May be DNA integrated into the host cell genome (pro-viral) OR
cytoplasmic, unintegrated viral DNA

15. HIV DNA Resistance Testing
• PBMCs are a circulating reservoir of
latently HIV-infected cells
- source of virus for DNA genotypic
resistance testing.
https://smhs.gwu.edu/timetoendhiv/cure

16. GenoSure ArchiveSM

17. http://www.monogrambio.com/hiv-tests/suppression-management/genosure-archive

18. When and why should we order DNA resistance testing?
• Switching regimens
• Low level viremia
• No access to prior RNA tests

19. Why switch if a patient is virologically suppressed?

20. Why switch if a patient is virologically suppressed?
- Newer drugs can simplify the regimen
- Improve side effect and long term adverse effect profiles
- Fewer potential drug-drug interactions
- Single dose tablets convenient and preferred by most patients

21. Is DNA resistance testing necessary?

22. SwitchMRK
• Evaluated switching virologically suppressed patients on a PI-based
(lopinavir-ritonavir) ART regimen to an INSTI-based (raltegravir) regimen.
• Stopped early due to a higher rate of virologic failure in the patients who
switched to raltegravir compared to the group that remained on lopinavir-
ritonavir
• Primary reason for the higher virologic failure rate was the presence of
unrecognized pre-existing resistance mutations to NRTIs that were part of
the new treatment regimen.
• Study design did not account for results from prior RNA resistance testing
Eron, et al. “Switch to a Raltegravir-Based Regimen versus Continuation of a Lopinavir-Ritonavir-Based Regimen in
Stable HIV-Infected Patients with Suppressed Viraemia (SWITCHMRK 1 and 2): Two Multicentre, Double-Blind,
Randomised Controlled Trials.” The Lancet 375, no. 9712 (February 5, 2010): 396–407.

23. DNA Resistance Testing Efficacy

24. The EASIER trial
• ANRS (Agence Nationale de Recherche sur le SIDA) study
• 48 week noninferiority, randomized, multicenter trial
• Assessed the efficacy and safety of a switch from enfuvirtide to
raltegravir in highly treatment-experienced patients on a
suppressive enfuvirtide-containing regimen
• 169 enrolled
• Findings:
- switch to raltegravir was safe, well tolerated and virologically non-inferior
to continuing enfuvirtide.

25. • Substudy with the objective of comparing resistance
mutation profiles between prior RNA resistance
tests to current DNA resistance tests.

26. Compilation of prior RNA tests
• Results of all available prior RNA resistance tests were
compiled for each patient “Cumulative” RNA genotype
• Viral DNA was extracted from frozen stored whole blood
specimens (200 μl)
- RT-PCR and then nested PCR used to amplify the RT and PR genes
- Population sequencing was performed

27. NRTI Mutations
Dark gray = RNA resistance
tests
Light gray = DNA resistance
tests

28. NNRTI mutations
Dark gray = RNA resistance
tests
Light gray = DNA resistance
tests

29. PI mutations
Dark gray = RNA resistance
tests
Light gray = DNA resistance
tests

30. SCOPE Cohort
• Toma et al, ICAAC 2015 Abstract
• Monogram
• 48 HIV patients with known prior HIV resistance now on
effective ART
• Compared DNA genotype (ArchiveSM) results with prior RNA
genotypes

31. 89% Sensitivity to detect historical drug
resistance

32. 85% Sensitivity to Detect Major Resistance-
Associated Variants

33. Low False Omission Rate

34. SCOPE Cohort Conclusions
• GenoSure Archive drug resistance testing had:
- Sensitivity 89% to detect drug resistance (calls) and 85% to detected major
drug-resistance mutations found in historical RNA resistance tests
• Caveat: Stored PBMC samples rather than whole blood samples
were used for the assay
- Much higher concentration of PBMCs (i.e. cellular DNA) in the samples than
for the commercially available assay

35. SPIRIT Study
• Switch study designed to reduce regimen complexity
• Patients on a PI/r + FTC/TDF were switched to a single tablet
regimen of rilpivirine + FTC/TDF
• Monogram provided DNA-based resistance information which was
matched with historical resistance information
Porter et al. Clinical Outcomes of Virologically-Suppressed Patients with Pre-existing HIV-1 Drug
Resistance Mutations Switching to Rilpivirine/Emtricitabine/Tenofovir Disoproxil Fumarate in the
SPIRIT Study. HIV Clin Trials. 2016 Feb;17(1):29-37.

36. SPIRIT Study Design
Study excluded patients with documented resistance to study drugs by
their historical genotype, including but not limited to the RT
resistance mutations K65R, K101E/P, E138G/K/R/Q, Y181C/I/V,
M184V/I, or H221Y

37. Snapshot Analysis

38. SPIRIT Archived Mutations Substudy
• All patients had historical genotype records at baseline
• Monogram provided viral DNA-based mutational information for 56
patients from baseline PBMC samples

39. Historical vs DNA Genotypes
• Good concordance for RT and PR drug
resistance mutations in historical
genotypes vs. baseline DNA-based
genotypes
• DNA genotype identified additional
NRTI and NNRTI resistance mutations

40. Historical vs DNA Genotypes
• Yellow rows indicate mutations
associated with RPV treatment
• *= M184I and Y181Y/C were present
in the same sample – this patient
was switched to RPV+FTC/TDF STR
and failed.

41. SPIRIT Conclusions
• Monogram HIV-1 DNA genotyping assay using PBMC samples had
good agreement with historical plasma RNA genotypes
- In some cases, DNA genotype identified additional mutations
- One patient with archived Y181C and M184I at baseline

42. Zaccarelli Study
• Zaccarelli M, et al. J Clin Vir 2016
• Italy 2006-2014
• Treatment-experienced patients with suppressed viral loads or
low-level viremia
• Evaluated prevalence of resistance mutations in DNA genotype
from stored PBMC samples compared to historical RNA genotypes

43. Zacarrelli DNA Resistance Mutations

45. Comparison of DNA to RNA Genotype:
PI Mutations

46. Comparison of DNA to RNA Genotype:
NRTI Mutations

47. Comparison of DNA to RNA Genotype:
NNRTI Mutations

48. Zacarrelli Study Conclusions
• Detection of primary resistance mutations in DNA resistance
testing was less frequent than in historical RNA genotypes
• PRMs that were mostly found in PBMCs (archived mutations) were
related to drugs previously administered and/or drugs with a low
genetic barrier, e.g. NNRTI mutation K103N
• 20% did have primary resistance mutations detected in DNA
genotype and not historic RNA genotype – suggests that there may
be some added benefit to having this information

49. Archive HIV DNA resistance testing - issues
• The test may not detect all mutations that have occurred over time
• Sampling bias - most whole blood samples have DNA copy number <10
• Not all latent HIV-1 compartments may be adequately represented in peripheral blood
• Role of the regimen? CD4 count? Role of length of viral suppression?
• Consequently, positive test results are likely more helpful than negative test
results
• Are certain types of mutations more reliably found than others?
• If the test is not fully sensitive, could ART changes lead to treatment failure?
• If unexpected resistance to the current regimen is discovered by this test, does
the current regimen need to be changed or intensified?

50. HIV treatment guidelines and DNA resistance
testing
Per the DHHS guidelines:
“A commercially available test amplifies viral DNA in
whole blood samples to detect the presence of archived
resistance mutations in patients with suppressed HIV RNA.
Its value in clinical practice is still being evaluated.”

51. DNA Genotype: No data on clinical outcomes
• There is no data published to date on clinical outcomes after using
DNA genotype testing to assist in ARV switch decisions
• We only have data on concordance of mutations detected with
DNA genotype resistance and mutations noted on cumulative prior
RNA resistance tests

52. UCSD Owen Clinic Study
• Research question:
• Does the use of DNA genotype testing improve clinical outcomes when
considering ARV switch in patients with HIV RNA suppression?
• Outcome measure:
• HIV RNA suppression 4-12 months after the genotype results are available to
provider OR after ARV switch is made
• HIV RNA suppression defined as HIV RNA < 50 copies/mL
• Comparison group:
• Historical controls from the SWITCHMRK studies where ARV switch was made
without access to DNA resistance data.

53. SWITCHMRK 1 & 2:
Study Design
• Identical, multicenter,
double-blind, randomized,
active-controlled studies
• Enrolled pts with HIV RNA <50
c/mL on LPV/r BID regimen in
combination
with at least 2 NRTIs
− No limit on number of prior ART
regimens
− Prior virologic failure not an
exclusion
− No lipid lowering therapy
for at least 12 weeks
• Randomized (1:1) to continue
LPV/r or switch
to RAL
Eron J, et al. 16th CROI, Montreal, Canada, 2009. Abst. 70aLB.
SWITCHMRK 1 SWITCHMRK 2
RAL
(N=174)
LPV/r
(N=174)
RAL
(N=176)
LPV/r
(N=178)
HIV RNA
≤ 50 c/mL
94.3% 92.5% 96.0% 95.5%
Mean CD4
(cells/mm3)
478 508 471 482
LPV/r ≤ 1 yr 16.7% 17.8% 17.6% 18.5%
Median yrs
prior ART
(min, max)
3.3
(0.3, 22.3)
3.6
(0.5, 20.2)
3.7
(0.5,19.2)
4.6
(0.6,16.3)
Median #
prior ART
(min, max)
5.0
(4.0, 16.0)
5.0
(2.0, 5.0)
5.5
(3.0,13.0)
6.0
(4.0,14.0)

54. • Statistically significant improvements in total cholesterol, non-HDL cholesterol and triglycerides
were observed following switch to RAL
• Further Analysis underway to assess factors associated with failure after switch to RAL
− Previous Resistance: 84% with confirmed HIV RNA >50 c/mL) in the RAL group were not on 1st ART regimen;
66% with history of VF on prior regimen(s)
− NRTI Backbone: Virologic failure rate higher on ABC/3TC than TDF/FTC
SWITCHMRK:
Week 24 Outcomes
*HIV RNA <50 copies/mL: did not meet non-inferiority criteria.
Eron J, et al. 16th CROI. Montreal, 2009. Abstract 70aLB.
Patients(%)
HIV RNA <50 Copies/mL (NC=F)*
81%
SWITCHMRK 1 SWITCHMRK 2
87% 88%
94%
CD4CellChange(cells/mm3)
CD4 Cell Change
5
17
SWITCHMRK 1 SWITCHMRK 2
5
7
 Raltegravir
 LPV/r

55. UCSD Study Objectives
• Primary Objective:
• To compare the rate of HIV RNA suppression (<50 copies/mL) in virally
suppressed patients for whom an DNA genotype was completed to that of
historical controls who were randomized to switch without DNA resistance
data.
• Secondary Objective:
• Similar comparison using a higher cutoff for HIV RNA suppression of <200
copies/mL
• To account for increased sensitivity of current viral load assays compared to
those used for SWITCHMRK controls

56. Study Population
• All Owen Clinic patients with DNA genotype resistance test
ordered between November 3, 2014 and December 4, 2015
• Primary analyses were restricted to patients:
1) with an available DNA genotype result
2) HIV RNA <50 copies/mL at the time of DNA genotype testing
3) Availability of HIV RNA (viral load) results on tests done 4-12 months after
management decisions based on DNA genotype results were made

57. * Lost-to-follow-up = no follow-up HIV RNA within 1 year of DNA genotype or ARV switch

58. DNA Genotype Cohort
(N=188)
SWITCHMRK RAL
Group (N=350)
SWITCHMRK LPV-
RTV Group (N=352)
N
Age, median (IQR) 188 53 (47-58) 43 (37-48) 42 (36-49)
Sex, male (%) 188 162 (86%) 283 (81%) 267 (76%)
Duration of ART (years), median (IQR) 169 15 (10-19) 3.4 (2.0-7.3) 4.1 (2.1-7.4)
Number of drugs, median (IQR) 153 8 (5-11) 5 (4-7) 5 (4-7)
CD4, median (IQR) 187 488 (315-751) 436 (298-637) 454 (315-642)
Baseline HIV RNA <50 copies/mL (%) 188 188 (100%) 333 (95%) 331 (94%)
Baseline ART Regimen (n) 188
INSTI-NRTI 11 (6%)
NNRTI-NRTI 10 (5%)
PI-NRTI 93 (49%) 350 (100%) 352 (100%)
2-Class NRTI-sparing 4 (2%)
3+ Classes 70 (37%)
BASELINE CHARACTERISTICS

59. ARV Changes After DNA Genotype

60. ARV Class Switches After DNA Genotype

61. Sustained HIV RNA suppression after DNA resistance testing
compared to rates from the SWITCHMRK studies
HIV RNA Suppression = HIV RNA < 50 copies/mL

62. Sustained HIV RNA suppression after DNA resistance
testing compared to rates from the SWITCHMRK studies
HIV RNA Suppression = HIV RNA < 200 copies/mL

63. Comparison of DNA Genotype to Historic RNA
Genotype Results
• 63% had at least 1 primary resistance mutation detected in DNA
genotype
• 47% NRTIs, 31% PIs, 30% NNRTIs
• The most commonly detected mutations were in reverse transcriptase:
• 38% M184V and 24% K103N/S
• 51 patients had historic RNA genotype results available:
• 80% had primary resistance mutations detected in either DNA or RNA genotypes
• 31 with mutations in both RNA and DNA genotypes
• 7 with mutations detected in DNA but not in RNA
• 3 with mutations detected in RNA but not in DNA
• 10 had no mutations identified in either DNA or RNA resistance testing

64. UCSD Study Limitations
• Retrospective
• Lack of randomization with historic controls
• Intention-to-treat for historic controls vs As-treated analysis

65. Conclusions from UCSD DNA Genotype Study
• Compared to a study population that switched treatment without guidance from
DNA resistance testing, the current cohort experienced improved virologic
suppression rates 4-12 months after DNA resistance test results were available to
the clinician (94.7% versus 87.6%; p-value 0.005)
• Improved virologic outcomes were seen for:
• all patients having HIV DNA testing
• whether or not an ART switch was made
• the subset who switched from PI-NRTI to INSTI-NRTI regimens
• Our findings suggests that this test favorably affects clinical outcomes in patients
who are being considered for ART switches

66. Using the DNA Genotype in Clinical Practice
• DNA genotype testing can be a helpful tool when considering ARV
switch in patients who have achieved viral suppression on their
current ARV regimen.
• The DNA genotype should be used IN CONJUNCTION with a careful
review of ARV history and historical RNA genotype data, when
available.

67. THANKS to the study team and contributors!
UCSD:
Charles Hicks (PI)
Bradford Colwell
Craig Ballard
Sonia Jain
Shelly Sun
Monogram:
Charles Walworth
Jeanette Whitcomb
Special thanks to:
The SWITCHMRK study sponsor for
providing unpublished data used for
our comparison with the SWITCMRK
historical control population
BMS Virology Research Fellows
Award
UCSD CFAR

68. Thoughts? Questions?