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Pre-Existing L74V is a Risk for Virologic Non-Response and Development of K65R in Patients Taking Tenofovir
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Reported by Jules Levin
Mike Miller (Gilead Sciences) presented this study in poster 158 at the XIII International HIV Drug Resistance Workshop (June 8-12, 2004, Tenerife, Canary Islands, Spain). Here is his report.
Notes from Jules Levin: With the advent of two Fixed Dose Combinations which are both expected to be FDA approved around the same time, by September 2004, patients & medical care providers will make a choice for firstline therapy. The two choices will be the FDC containing abacavir+3TC one pill once daily and the FDC containing tenofovir+FTC one pill once daily.
There is a chance that you could acquire the L74V mutation from prior use of abacavir. There is a risk of acquiring the K65R mutation due to Tenofovir use. The Gilead poster examined 14 patients with detectable viral load who developed the K65R in a tenofovir study after intensifying their regimen with tenofovir (see study data below). Standard genotype testing found 4 of the 14 had the L74V before TDF intensification and sensitive genotype testing found that 2 of the 4 also harbored the K65R at low levels before starting TDF. One of the 4 patient's blood sample was not available for followup testing with the sensitive test. Gilead's Miller suggests that development of L74V may encourage development of K65R at low levels; that prior use of ABC may dispose patients to developing the L74V, which might encourage low levels of K65R undetectable with standard genotype, which in turn may result in TDF failure. In speaking with GSK at this conference they feel that the development of L74V is an infrequent occurrence; they said this is what their data in-house shows them; and they feel that if the L74V develops this virus would be more sensitive to HIV therapy than a virus with K65R; that is, subsequent response to therapy is more likely to be successful than if the K65R develops.
In general, the emergence of the K65R mutation appears to be a low occurrence. The mutation does not develop easily. As you see below in the intensification study K65R does not develop often. But if it does develop it could lead to pan-resistance to NRTIs. In study 903 presented at several HIV conferences, patients received EFV/TDF/3TC or EFV/3TC/d4T. The regimen was very effective as 80%+ had <50 copies. 9.7% were virologic failures in the TDF group and 24% of these viral failures developed the K65R. Therefore, 2.7% of patients in the study developed the K65R. These patients were switched to other regimens and 5/7 achieved <50 copies/ml. You can read a review of this study at: http://www.natap.org/2003/Jan/010903_3.htm
The hypersensitivity reaction associated with abacavir is a consideration. It occurs to about 4% of patients but about 8-10% of the time other types of medical situations appear that look like HSR, such as a fever or flu-like symptom. This creates another barrier to use of abacavir. It is easier and more convenient to use tenofovir because you don't have to deal with HSR. After you get past the potential for HSR occurring within the first weeks following initiation of abacavir therapy, the use of abacavir appears pretty clean. Both drugs, abacavir and tenofovir, are potent. Tenofovir is approved for once daily one pill use. Abacavir studies using it once daily have been presented at conferences showing it to be effective & it's once daily use is expected to be approved within the next few months. At the Pharmacology Workshop this past Spring Trevor Hawkins presented a study showing good tenofovir levels for 48 hours and good levels of abacavir for 24 hours. Of course, you need to also consider PK variability between individuals. Also to consider are the potential for development of yet unknown long-term adverse events associated with these or any drugs. At this point, it is difficult to look into the future & predict long-term adverse events that might emerge from these drugs. We have, however, familiarity with the short-term adverse event and side effects profiles of tenofovir & abacavir.
Regarding the study discussed in this report, the Mike Miller, the study author, concluded: prior abacavir or ddI therapy may result in selection of L74V, K65R, or a mixture of both mutations in RT: minor species of K65R may not be detected by standard population sequencing; SGS (sensitivie genotyping) detected K65R in 2.6% and 6.3% of HIV genomes from two patients with L74V and undetectable K65R by population sequencing at baseline (GSK told me informally at Tenerife that they see L74V about 1% in patients using EFV/ABC/3TC, presumably using standard genotyping); K65R and L74V were always observed on separate genomes; baseline phenotype showed full susceptibility to TDF based on the dominant L74V genotype. Subsequent TDF therapy showed expansion of the K65R quasispecies, loss of L74V, and a poor virologic response to TDF. The selection and sequencing of NRTIs should consider the development of resistance mutation mixtures, potentially including undetectable quasispecies, that may limit future treatment options.
INTRODUCTION
DDI or abacavir (ABC) can select for either the K65R or L74V mutations in reverse transcriptase (RT); tenofovir (TDF) only selects for the K65R.
Note from Jules Levin: I spoke with GSK in Tenerife and they said that in their database of about 1000 patients taking EFV/ABC/3TC about 1% of patients developed the L74V. Presumably, this is using the standard commercially available genotype test, not a sensitive genotype test. It appears to me that the L74V emerges first and the K65R can follow later.
L74V shows full susceptibility to TDF in vitro; however, patients with L74V have shown significantly reduced response to TDF therapy (Miller et al, JID, 2004); Masquelier et al, Antiviral Therapy, 2004).
Resistance mutations that are undetectable by bulk population sequencing have been shown to limit NNRTI treatment response (Mellors et al, 12th Resistance Workshop, 2003).
The current study assesses whether treatment-experienced patients with detectable L74V at baseline who go on to develop K65R also had low-level K65R at baseline.
Studies 902 and 907 were treatment intensification studies of TDF added to stable but failing background antiretroviral therapy: a virology substudy genotypically analyzed 50% of patients at baseline and during therapy by population-based sequencing (Virco).
Of 14 patients who developed K65R during the studies, 4 patients had L74V at baseline. Three of these patients had sufficient baseline sample (blood) for further analysis by single genome sequencing (SGS).
DEVELOPMENT of K65R by BASELINE RESISTANCE GENOTYPE
TAMS are AZT-like mutations. A number of studies show, including this study, that the presence of TAMS can prevent or delay the K65R mutation from developing. The 3TC mutation, M184V, may also have a positive affect in increasing TDF antiviral activity.
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Baseline Genotype
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Number
Developing
K65R
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TAMS/no L74V
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0/277*
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TAMS + L74V
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0/34*
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No TAMS/no L74V
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10/116 (8.6%)
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p=0.014
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No TAMS + L74V
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4/10 (40%)
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*Presence of baseline TAMS negatively predicts K65R development (P<0.001)
TREATMENT REGIMENS and GENOTYPES of PATIENTS WHO DEVELOP K65R
*patients with L74V at baseline
| Pt | Regimen | Baseline RT Genotype | Genotype with K65R
(study week) | | 1 | AZT/ABC/3TC/TDF | A62V M184V | A62V K65R/K M184V (W48 mix) | | 2 | AZT/3TC/TDF | M184V/M | K65R (w24) | | 3 | AZT/3TC/NVP/TDF | -- | K65R V106A (w16 mix) | | *4 | d4T/3TC/EFV | L74V K103N M184V | K65R L74I/L K103N M184V (w24 mix) | | 5 | d4T/3TC/NFV/TDF | M184V | K65R M184V (w48) | | 6 | d4T/ddI/RTV/TDF | -- | A62V K65R (W48) | | 7 | d4T/ddI/NFV/TDF | V75S | K65R V75S (W30) | | *8 | ddI/EFV/SQV/TDF | L74V K103N Y181C | K65R K103N Y181C (W4 mix) | | 9 | ABC/NFV/NVP/TDF | K103N | K65R Y181C (W24) | | *10 | ABC/3TC/APV/TDF | L74V V118I M184V | K65R V118I Y115F M184V (W4 mix) | | 11 | ABC/ddI/3TC/EFV/TDF | -- | K65R D67G G190V (W48) | | 12 | ABC/ddI/3TC/EFV/TDF | M184V | K65R K103N Y115F M184V K219N (W80 mix) | | *13 | ABC/ddI/3TC/TDF | L74V Y115F M184V | K65R Y115F M184v (W72) | | 14 | ABC/ddI/NFV/TDF | A62V V75I F116Y | M41L A62V K65R V75I F116Y Q151M | | | Q151M M184V G190A | M184V G190A (W12) |
Note from Jules Levin: as stated 4 patients had the L74V and developed the K65R. But the other 10 patients also developed the K65R and did not have the L74V detected at baseline. SGS, sensitive genotyping, was not reported for the other 10 patients so we don't know if K65R was present at low levels for them at baseline.
Single Genome Sequencing was performed on the 4 patients with L74V at baseline.
SUMMARY of SINGLE GENOME SEQUENCING ANALYSIS
You will note that in 2 of the patients with L74V at baseline K65R was also detected at baseline using SGS, a sensitive genotype. They did not test the other 10 patients using SGS to look for K65R.
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SINGLE GENOME SEQUENCING CLONAL FREQUENCY
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Pt
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Time Pt
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Population
65/74 Genotype
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K65R
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L74V/I
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Wild-type
at 65/74
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4
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basel
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L74V
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0/91
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91/91
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0/91
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wk 24
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K65R/k+L74V/I
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12/20
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18/20*
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0/20
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8
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basel
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L74V
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2/77
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58/77**
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17/77
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wk4
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K65R/K+L74V/L
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13/28
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15/28**
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0/28
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wk 8
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K65R/K+L74V/L
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13/17
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4/17**
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0/17
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10
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basel
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L74V
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4/63
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57/63**
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2/63
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wk 4
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K65R+L74V/L
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20/36
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15/36**
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1/36
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wk 8
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K65R/K+L74V/L
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16/24
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8/24**
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0/24
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13
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basel
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L74L
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no sample available for analysis
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*all clones with L74I expressed K65R (n=10), all clones with L74V were K65R wild-type (n=8).
**all clones contained L74V and were K65R wild-type.
Mutational Linkage Analysis
K65R andL74V were never present on the same genome: K65R found associated with L74I in one patient
Baseline SGS genotypes showed a single major RT quasispecies with L74V that represented 27-41% of clones:
--multiple minor genotypic groups (4-7 per patient) with unique patterns of substitutions (3-10% frequency per group)
--remaining quaispecies were singly represented in the sample (about 1-2% each) with a unique set of substitutions
--each patient had a core cluster of mutations and polymorphisms that were unique to the patient and present in all quasispecies
In both patients 10 (prior ABC+3TC+APV, added TDF) and 8 (prior ddI+EFV+SQV/r, added TDF) K65R was present at baseline at low levels using SGS (2.6% for patient 8 and 6.3% for patient 10) but not detected using standard genotyping. L74V was prevalent at baseline for both patients. As time passed the prevalence on K65R increased and L74V decreased.
PHENOTYPIC ANALYSIS OF BASELINE L74V AND DEVELOPMENT OF K65R
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Mean Fold Change from Wild-Type
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Pt
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Time Pt
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Population 65/74
genotype
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TFV
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AZT
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d4T
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ddI
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ABC
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3TC
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4
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basel
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L74V+M184V
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0.4
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0.4
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0.6
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2.2
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6.8
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>30
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8
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basel
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L74V
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1.1
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1.8
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2.6
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4.2
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3.8
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5.4
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10
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basel
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L74V+M184V
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0.9
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0.3
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1.2
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1.8
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6.1
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>50
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mean (n=3)
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basel
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L74V±M184V
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0.8
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0.8
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1.5
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2.7
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5.6
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>30
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mean (n=3)
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wk 24
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K65R±M184V
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1.8
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1.1
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1.7
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3.6
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6.3
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>30
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fold change from baseline
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2.3
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1.4
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1.1
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1.3
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1.1
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--
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