1. Prevalence and clinical implications of NRTI- and NNRTI-associated minority variant drug-resistance mutations in ARV-naïve patients with and without transmitted drug resistance
IDWeek
October 29th, 2016
Dana Clutter
Stanford University School of Medicine
Division of Infectious Diseases and Geographic Medicine
Good afternoon. My name is Dana Clutter and I am an ID fellow at Stanford. Today I will be describing our project entitled “Prevalence and clinical implications of NRTI- and NNRTI-associated minority variant drug-resistance mutations in ARV-naïve patients with and without transmitted drug resistance”

2. Financial disclosures
D Clutter: Bristol-Myers Squibb Virology Fellows Research Program
Co-investigators:
R Swanstrom: Co-inventor of Primer ID
R Shafer: Research funding from Gilead Sciences, Bristol-Myers Squibb, Merck, and Vela Diagnostics
These are the financial disclosures for myself and my co-investigators.

3. Background
I will start with some background information on minority variants.

4. Minority variants
Limit of detection for drug resistance mutations (DRMs) by Sanger sequencing is 15-20%
Additional low-abundance HIV-1 variants below Sanger threshold are “minority variants (MVs)”
MVs require ultrasensitive methods for detection (allele-specific PCR, NGS)
Sanger sequencing is routinely used to detect HIV drug resistance mutations. The limit of detection of the Sanger method is reported to be near 15-20%.
Any low-abundance variant occurring below this threshold and escaping detection by Sanger sequencing is termed a Minority Variant.
The detection of minority variants requires ultrasensitive methods such as allele-specific PCR and NGS methods.

5. Clinical implications of pre-therapy MVs
Prevalence of transmitted drug resistance (TDR) is ~10-20%
An additional 10-20% of ART-naïve individuals harbor MVs bearing DRMs (MV-DRMs)
MV-DRMs associated with increased virological failure (VF)
Transmitted drug resistance occurs when a person becomes infected with a drug resistant virus, which is typically detected by Sanger sequencing. In the United States, the prevalence of TDR is reported to be about 10-20% in most areas.
When ultrasensitive methods are used to detect MVs bearing DRMs, which we refer to as MV-DRMs, drug-resistant viruses are found in an additional 10-20% of ART-naïve individuals. Use of these ultrasensitive methods increases the detection of DRMs from 5 to 300% relative to Sanger sequencing alone depending on the DRM and assay used.
The clinical importance of these MV-DRMs has been investigated by multiple studies. An individual level metaanalysis pooling data from 10 studies and including nearly 1000 patients showed that MV-DRMs in ART-naïve individuals are associated with a greater than 2-fold increase in the risk of VF when initiating an NNRTI-based regimen.
Li et al. JAMA 2011; Panichsillapakit et al. JAIDS 2016; Rhee et al. PLOS Med

6. Are patients with isolated NNRTI TDR at high risk of having MV-DRMs?
NRTI DRMs occur commonly in combination with NNRTI DRMs during VF
Following transmission of multiple DRMs, low-fitness DRMs decay
M184V >> NNRTI DRMs > 215 revertants
Several small uncontrolled studies have shown that patients with TDR frequently have MV-DRMs
Identifying groups at high-risk for MV-DRMs may be important because they are not routinely detected, yet may negatively affect clinical outcomes.
The rationale making us question whether NNRTI TDR is associated with MV-DRMs is that NRTI DRMs commonly occur in combination with NNRTI DRMs in individuals failing a NNRTI-based regimen. Following transmission of a virus bearing multiple DRMs, the less fit DRMs (such as the NRTI DRM M184V) may decay to MV levels – leaving only the higher fitness DRMs (such as the NNRTI DRM K103N) to persist at sanger-detectible levels that will be detected on a baseline genotype to assess for TDR.
Empirically, several small uncontrolled studies have shown that patients with TDR frequently have patients with TDR frequently have MV-DRMs.
MV-DRMs common in individuals with TDR
Toni 2009: All patients with TDR had MV-DRMs
Varghese 2009: 7 of 13 patients with NNRTI TDR had MV-DRMs
Jain et al. JID 2011; Castro et al. JID 2013; Toni et al. AAC 2009; Varghese et al. JAIDS 2009

7. Aim
Determine if NRTI and NNRTI MV-DRMs occur more commonly in individuals with isolated NNRTI TDR compared to those without
Particularly

8. Methods

9. Study population ART-naïve Kaiser Permanente Northern California
Baseline genotype (Sanger): 2004 – 2012
Cryopreserved plasma sample available
HIV RNA >10,000 copies/mL
Groups:
TDR: consecutively sampled, isolated NNRTI DRM(s)
Wild type (WT): matched by HIV RNA level, CD4 count, sampling year, and ART regimen where possible
Individuals included in our study were ART-naïve, entrolled in routine HIV care at KPNC, had a baseline genotype performed using Sanger sequencing methods between , had a cryopreserved sample available with HIV RNA >10K for repeat sequencing using an NGS method as I will describe later.
Our TDR group was assembled by consecutively sampling patients meeting the above criteria who also had isolated NNRTI DRMs defined as the presence of one or more NNRTI DRMs from 2009 list of surveillance DRMs.
Our control group was assembled using controls with no DRMs detected by sanger sequencing who were matched by HIV RNA level, CD4 count, sampling year, and ART regimen where possible

10. Primer ID-Illumina MiSeq sequencing
Accurate quantification of MV-DRMs
Distinguish MV-DRMs from technical artifact
Our co-investigators at UNC performed Primer ID-Illumina Miseq sequencing, a type of NGS sequencing, on all patient samples to look for MV-DRMs.
The primer ID method uses a unique barcoded primer (the Primer ID) for reverse transcription. Because each primer ID corresponds to just one initial viral template in the starting material, resampling can be identified which allows for accurate quantification of MV-DRMs. Additionally, a consensus sequence can be generated for each starting template that can correct for PCR and sequencing errors – this allows MV-DRMs to be distinguished confidently from such technical artifacts.
Zhou et al. J Virol 2015; Jabara et al. PNAS 2011; Keys et al. AIDS Res Hum Retrovirus 2015

11. Statistical analysis Definitions: Analysis:
MV-DRM: NNRTI or NRTI surveillance DRM detected only by Primer ID Illumina sequencing
VF: HIV RNA > 50 copies/mL at week 48
Analysis:
Compare proportions with MV-DRMs between groups
Compare outcomes (VF, regimen change) between groups
Describe outcomes when MV-DRMs conferred resistance to ART regimen (HIVDB v 7.0)
intermediate or high-level resistance to a regimen component according to HIVDB 7.0 – recently updated.
Bennett et al. PLOS ONE 2009; HIVDB

12. Results

13. Baseline characteristics
WT n = 49
TDR n = 33
Age, median (IQR)
42 (35-49)
42 (34-46)
Male, n (%)
46 (94)
28 (85)
Race/ethnicity, n (%)
White
Black
Hispanic
Other
25 (51)
10 (20)
8 (16)
6 (12)
20 (61)
5 (15)
6 (18)
2 (6)
Baseline log viral load, median (IQR)
4.8 ( )
4.7 ( )
Baseline CD4 count, median (IQR)
339 ( )
339 ( )
Initial anchor drug, n (%)
RAL
ATVr
21 (43)
16 (33)
12 (24)
13 (39)
Initial NRTIs, n (%)
FTC/TDF
3TC/ABC
41 (84)
4 (8)
31 (94)
1 (3)
Sample year, median (IQR)
2010 ( )
2009 ( )
The two groups were similar with a median age 42, predominantly white males with log VL of 4.8, CD ART regimens frequently included boosted ATV or RAL plus FTC/TDF.

14. MV-DRM prevalence and between-group comparisons
Overall 60% had MV-DRMs
Abundance: 0.2 ( )%
No between-group differences
MV-DRMs conferred resistance to ART regimen in 10 individuals:
M184V/I: 7
G190E: 2
K65R: 1
P = 0.25
P = 0.65
P = 0.06

15. Clinical outcomes
Outcome
WT n = 49
n (%)
TDR n = 33
n(%) P
Regimen change
13 (27)
3 (9)
0.09 VF
5 (10)
1.00
VF with emergent resistance
1 (2)
1 of 10 with MV-DRMs conferring resistance to ART regimen developed VF:
MV-DRM: M184V
Regimen contained 3TC and ABC
No repeat genotype available at VF
M184V: 4 RAL + FTC/TDF, 1 DRV/r + FTC/TDF, 1 ATV/r + FTC/TDF, 1 ATV/r + 3TC/ABC/TDF (VF was on this regimen)
G190E: 2 EFV + FTC/TDF
K65R: 1 RAL + FTC/TDF

16. Conclusions MV-DRMs common in this Northern California cohort
MV-DRMs were not associated with isolated NNRTI TDR
Despite high prevalence of MV-DRMs, responses to ART were favorable
This high prevalence may be either due to transmitted variants that had faded to low levels or
background HIV-1 RT enzymatic errors.
May be because there is no association, could also be related to populations studied. If onward transmission, or naïve to naïve transmission of TDR is common, one would expect MV-DRM prevalence to be more similar to WT patients than if TDR was transmitted by a patient failing therapy. Preliminary phylogentic analysis in this cohort does suggest that onward trasmission occurs commonly in this cohort.
Also our numbers were low to study this outcome, particularly given the low rate of failure. The favorable responses may be related to the relatively low abundance of the MV-DRMs detected, and also that many of the MV-DRMs did not confer resistance to their regimen.

17. Acknowledgments Participants UNC Kaiser Permanente Northern California
Shuntai Zhou
Ronald Swanstrom
Kaiser Permanente Northern California
Daniel Klein
Jeffrey Fessel
Leo Hurley
Michael Silverberg
Stanford
Vici Vargese
Soo-yon Rhee
Robert Shafer
Benjamin Pinsky
This work was conducted with support from a KL2 Mentored Career Development Award of the Stanford Clinical and Translational Science Award to Spectrum (NIH KL2 TR ) and (UL1 TR )

18. Questions?

19. Thank you!