Utilization of Genotype and Phenotype Resistance Tests in Patient Management Richard Haubrich, MD Professor of Medicine University of California San Diego

Utilization of Genotype and Phenotype Resistance Tests in

Patient Management

Richard Haubrich, MDProfessor of Medicine

University of California San Diego

To what is Condi referring?

To what is Condi referring?

Case 41 y/o GWM HIV history (all distant)

– zoster– thrush– bacterial pneumonia– cutaneous KS

Medications– azithromycin – fluconazole– oxandralone– sulfamethoxazole./ trimeth

Case

CD4 = 9 (4%)

HIV RNA = 78,900

Current ARV (02- present)

– ABC

– 3TC

– TDF

– SQV

– RTV

Past ARV

– ZDV/ ddC 93-4

– d4T/ 3TC/ IDV 96-01

– ABC/ ddI/ EFV/ APV/r 01-02

NRTI

When considering TDF Susceptibility

1. The current phenotype is important in deciding to use TDF

2. Past treatment history and resistance tests suggests reduced TDF activity

3. The M184V mutation re-sensitizes the virus to TDF

4. All of the above

ZDV Resistance and Resensitization

100I

L74V/I

Model of NRTI Resistance

Parikh et al. JID 2006; 194: 651

215Y/F + other TAMs


Excision


65R 215Y/F + other TAMs


Decreased Incorporation

Excision





Excision

Phenotypic AntagonismCounter selection




Multi-NRTI Resistance


Excision

Phenotypic AntagonismCounter selection

Q151M Complex


NNRTI

V90I A98G L100I K101E K101P K101Q K103H K103N K103S

K103T V106A V106I V106M V108I E138G E138K E138Q V179D

V179E V179F V179G V179I Y181C Y181I Y181V Y188C Y188H

Y188L V189I G190A G190C G190E G190Q G190S H221Y P225H

F227C F227L M230I M230L P236L K238N K238T Y318F

DUET: Identification of the ETR Resistance Mutations

• Of 44 NNRTI mutations studied, 26 NNRTI mutations were present in 5 patients

• 13 TMC125 RAMs were identified at baseline to have a significant impact on virological response

ETR RAM

RAM Present

0

20

40

60

80

Response according to number of ETR RAMs

0 1No NNRTI mutations (reference)

2 3 4 5

Number of ETR RAMs

52 161 121 64 32 19 9Patients (n)

Data are pooled from DUET-1 and -2; Vingerhoets J, et al. Antiviral Therapy 2007:12:S34;RAMs = resistance-associated mutations

VL

< 5

0

Overall placebo group

414

0

20

40

60

80



2 3 4 5

Number of TMC125 RAMs

52 161 121 64 32 19 9Patients (n)


VL

<50


414

0

20

40

60

80



2 3 4 5

Number of TMC125 RAMs

52 161 121 64 32 19 9Patients (n)


VL

<50


414

CASE = 100I; 103N

Impact of Specific Mutations

*No detectable baseline NNRTI RAMs from the list of 44

Nomut

(ref)*

G190A+ 0

ETR RAMs

G190A+ 1

G190A+ 2

G190A+ 3

G190A+ 4

52 18 46 25 17 9

G190A1.00.90.80.70.60.50.40.30.20.1

0

Vingerhoets J, et al. 16th IHDRW 2007, #32.

Res

pons

e re

lativ

e to

the

subg

roup

with

no

NN

RT

I RA

Ms

Nomut

(ref)*.

1.00.90.80.70.60.50.40.30.20.1

0Y181C

+ 0

ETR RAMs

Y181C+ 1

Y181C+ 2

Y181C+ 3

Y181C+ 4

52 23 36 26 17 8Patients (n)

Y181C

Impact of Specific Mutations

*No detectable baseline NNRTI RAMs from the list of 44Vingerhoets J, et al. 16th IHDRW 2007, #32.

Res

pons

e re

lativ

e to

the

subg

roup

with

no

NN

RT

I RA

Ms

Nomut

(ref)*.

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0Y181C

+ 0

ETR RAMs

Y181C+ 1

Y181C+ 2

Y181C+ 3

Y181C+ 4

52 23 36 26 17 8Patients (n)

Y181C

ETR: Determination of clinical cutoffs

Winters B, et al. 15th CROI, Boston 2008, #873

Cutoffs based on response rates in DUET

Clinical cutoffs (CCOs) for ETR of 1.6 and 27.6 FC defined for the vircoTYPE report (20% and 80% loss of wt ETR response)









PI

TPV RESIST: Change in VL at Week 24 According to TPV Score Mutations

Med

ian

log 1

0 H

IV R

NA

cha

nge

from

bas

elin

e

Shapiro. 12th CROI; 2005: 104

21 mutations at 16 amino acid loci identified:10V, 13V, 20M/R/V, 33F, 35G, 36I, 43T, 46L, 47V, 54A/M/V, 58E, 69K, 74P, 82L/T, 83D, and 84V

-2.1

-1.4

-0.6 -0.5 -0.4

-0.7-0.5

-0.2-0.4

-0.2 -0.2 -0.3

-2.5

-2

-1.5

-1

-0.5

0

1 2 3 4 5 >=6

Number of TPV score mutations

TPV/r CPI/r

PI Mutations: 10I, 13V, 33F, 46I, 47V, 54M, 63P, 77I, 84V, 90M

TPV RESIST: Change in VL at Week 24 According to TPV Score Mutations

Med

ian

log 1

0 H

IV R

NA

cha

nge

from

bas

elin

e

Shapiro. 12th CROI; 2005: 104

21 mutations at 16 amino acid loci identified:10V, 13V, 20M/R/V, 33F, 35G, 36I, 43T, 46L, 47V, 54A/M/V, 58E, 69K, 74P, 82L/T, 83D, and 84V

Our case = 5 TPV Mutation

-2.1

-1.4

-0.6 -0.5 -0.4

-0.7-0.5

-0.2-0.4

-0.2 -0.2 -0.3

-2.5

-2

-1.5

-1

-0.5

0

1 2 3 4 5 >=6

Number of TPV score mutations

TPV/r CPI/r

PI Mutations: 10I, 13V, 33F, 46I, 47V, 54M, 63P, 77I, 84V, 90M

Stanford Algorithm

Protease Inhibitors

ATV High-level resistance

DRV Intermediate resistance

FPV High-level resistance

IDV High-level resistance

LPV High-level resistance

NFV High-level resistance

SQV High-level resistance

TPV High-level resistance

Stanford Algorithm

ATV DRV FPV IDV LPV NFV SQV TPV

M46I 15 6 12 12 12 25 5 8

I47V 5 12 20 5 10 5 5 10

I54M 10 12 20 10 12 15 10 5

I84V 25 12 35 25 12 35 35 25

L90M 20 6 15 20 10 50 35 6

L10I 2 2 2 2 2 2 2 2

L33F 5 5 5 0 5 0 0 8

Total: 82 55 109 74 63 132 92 64

http://hivdb.stanford.edu/cgi-bin/GetResiData.cgi?pos=54&drug=IDV&class=PI&drug1=any

http://hivdb.stanford.edu/cgi-bin/GetResiData.cgi?pos=54&drug=LPV&class=PI&drug1=any

http://hivdb.stanford.edu/cgi-bin/GetResiData.cgi?pos=54&drug=NFV&class=PI&drug1=any

http://hivdb.stanford.edu/cgi-bin/GetResiData.cgi?pos=54&drug=SQV&class=PI&drug1=any

http://hivdb.stanford.edu/cgi-bin/GetResiData.cgi?pos=54&drug=TPV&class=PI&drug1=any

http://hivdb.stanford.edu/cgi-bin/GetResiData.cgi?pos=84&drug=ATV&class=PI&drug1=any

http://hivdb.stanford.edu/cgi-bin/GetResiData.cgi?pos=84&drug=DRV&class=PI&drug1=any

http://hivdb.stanford.edu/cgi-bin/GetResiData.cgi?pos=84&drug=FPV&class=PI&drug1=any






























Number of mutations associated with a diminished response to DRV/r and virological response HIV RNA <50

6450

42

2210

42

0

20

40

60

80

100

0(67)

1(94)

2(113)

3(58)

≥4(41)

All (373)

Number of DRV mutations (Number of patients)

Pat

ien

ts w

ith

VL

HIV

RN

A <

50 c

op

ies/

mL

at

Wee

k 24

(%

)

MP de Bethune. 15th DRW 2006, #73

Clinical Cutoffs Applying Breakpoints to a Continuum of SusceptibilityP

rob

ab

ilit

y o

f re

sp

on

se

Fold change at baseline


rob

ab

ilit

y o

f re

sp

on

se


“Traditional” lower clinical cutoff…fold change above which the probability of clinical response begins to decrease


rob

ab

ilit

y o

f re

sp

on

se



Upper clinical cutoff…fold change beyond which the probability of clinical response is low.


rob

ab

ilit

y o

f re

sp

on

se



Upper clinical cutoff…fold change beyond which the probability of clinical response is low. “ Intermediate

Probability of Response”

Pat

ien

ts W

ith

HIV

-1 R

NA

< 5

0 c/

mL

at

Wee

k 24

(%

)

DUET-1 and -2: Response (< 50 c/mL) According to DRV Fold Change

DRV FC 10-40 DRV FC > 40

71

DRV FC < 10

56 57

30

44

2

100

75

50

25

246/345

200/358 73/129 39/132 31/71 1/67

0

Cahn P, et al. ICAAC 2007. Abstract H-717.

Placebo + OBRETR + OBR

Pat

ien

ts W

ith

HIV

-1 R

NA

< 5

0 c/

mL

at

Wee

k 24

(%

)

DUET-1 and -2: Response (< 50 c/mL) According to DRV Fold Change

DRV FC 10-40 DRV FC > 40

71

DRV FC < 10

56 57

30

44

2

100

75

50

25

246/345

200/358 73/129 39/132 31/71 1/67

0

Cahn P, et al. ICAAC 2007. Abstract H-717.

Placebo + OBRETR + OBR

Our case = DRV FC = 42

Continuous Phenotypic Susceptibility Score (cPSS)

• Activity of drug defined by relation to cut-off• Maximum score = 1 for FC < Lower cut off• Minimum = 0 for FC > Upper cut off• For drugs where FC > lower but < upper cut off,

get partial score (between 0 and 1):

Score = Upper CO – patient FC Upper CO – Lower CO

Continuous Phenotypic Susceptibility Score (cPSS)

• Activity of drug defined by relation to cut-off• Maximum score = 1 for FC < Lower cut off• Minimum = 0 for FC > Upper cut off• For drugs where FC > lower but < upper cut off,

get partial score (between 0 and 1):

Score = Upper CO – patient FC Upper CO – Lower CO

LPV 55 – 34 = 0.46 55-9

PI Scoring for the Case

Drug FC Lower CO

Upper CO

cPSS

DRV 42 10 40 0

DRV 42 10 90 0.6

LPV 34 9 55 0.46

TPV 5.75 2 8 0.375

PI Scoring for the Case

Drug FC Lower CO

Upper CO

cPSS

DRV 42 10 40 0

DRV 42 10 90 0.6

LPV 34 9 55 0.46

TPV 5.75 2 8 0.375

CCR5 ANTAGONISTS

R5 Tropism

CD4 + CCR5 CD4 + CXCR4

R5 Tropism


X4 Tropism


X4 Tropism


Dual Co-receptor Tropism


Dual Co-receptor Tropism


R5/X4 (Mixed) Co-receptor Tropism


R5/X4 (Mixed) Co-receptor Tropism


HIV-1 Expression Vector: pHIVlucU3

P A+

HIV envelope

a/b c/d

Indicator Gene

U5gag

pol

Luciferase

P R

envP

RA+

gp120 gp41

U3

Envelope Expression Vector: pHIVenv

Tropism Assay

Transfection

HIV envexpression

vector

++++

HIV genomicluc vector


P A+

HIV envelope

a/b c/d

Indicator Gene

U5gag

pol

Luciferase

P R

envP

RA+

gp120 gp41

U3


Tropism Assay

Transfection

HIV envexpression

vector

++++



P A+

HIV envelope

a/b c/d

Indicator Gene

U5gag

pol

Luciferase

P R

envP

RA+

gp120 gp41

U3


CD4 +CXCR4 +

X4 Virus

Tropism Assay

Transfection

HIV envexpression

vector

++++


CD4 +CCR5 +


P A+

HIV envelope

a/b c/d

Indicator Gene

U5gag

pol

Luciferase

P R

envP

RA+

gp120 gp41

U3


CD4 +CXCR4 +

X4 Virus

R5 Virus

Tropism Assay

Tropism Assay

Percentage of HIV Co-receptor Usage

Study/Source Population N R5 D/M X4

Homer cohort1 Naive 979 82% 18% <1%

C & W cohort2 Naive 402 81% 19% <1%

Pfizer 10264 Naive142

885% 15% <1

MOTIVATE 1 & 24 Experienced256

056% 41% 3%

TORO 1/25 Experienced 612 50% 46% 4%

ACTG 52116 Experienced 391 49% 47% 4%

1Brumme ZL, et al. J Infect Dis. 2005;192:466-474.2Moyle GJ, et al. J Infect Dis. 2005;191:866-872.

4Coakley E, et al. 2nd Viral Entry Workshop, Abstract 85Melby et al 13th CROI 2006 Abstract 233.&Wilkin T, et al. CROI 2006. Abstract 655.

Percentage of HIV Co-receptor Usage

1Brumme ZL, et al. J Infect Dis. 2005;192:466-474.2Moyle GJ, et al. J Infect Dis. 2005;191:866-872.

4Coakley E, et al. 2nd Viral Entry Workshop, Abstract 85Melby et al 13th CROI 2006 Abstract 233.&Wilkin T, et al. CROI 2006. Abstract 655.

Population CCR5 Only

Naïve ~80%

Experienced ~50%

MERIT: Week 48 virologic response by tropism

MVC (300 mg BID) vs EFV (600 mg QD) + ZDV/3TC in naïve patients (n=721)

All pts screened in as R5 tropic

25 (3.5%) changed from R5 at screening to D/M at baseline

Heera J, et al. 15th CROI, Boston, 2007, #40LB

% Patients with HIV RNA <50 c/mL

n= 339 / 331 11 / 14

%

68.0

7.1

54.6

69.3

0

20

40

60

80

R5 D/M

MVC +ZDV/3TC EFV +ZDV/3TC

MERIT: Week 48 virologic response by tropism

MVC (300 mg BID) vs EFV (600 mg QD) + ZDV/3TC in naïve patients (n=721)

All pts screened in as R5 tropic

25 (3.5%) changed from R5 at screening to D/M at baseline

Heera J, et al. 15th CROI, Boston, 2007, #40LB

% Patients with HIV RNA <50 c/mL

n= 339 / 331 11 / 14

%

68.0

7.1

54.6

69.3

0

20

40

60

80

R5 D/M

MVC +ZDV/3TC EFV +ZDV/3TC

Enhanced Assay Detects CXCR4 Use Enhanced Assay Detects CXCR4 Use Prior to Standard AssayPrior to Standard Assay

Reeves, et al. ICAAC 2007. Abstract H-1026

Possible Mechanisms of MVC ResistancePossible Mechanisms of MVC Resistance

• Emergence of CXCR4 virusEmergence of CXCR4 virus

– Pre-existing low level populationsPre-existing low level populations

– Viral mutation from CCR5 to CXCR4Viral mutation from CCR5 to CXCR4

• Non-competitive resistance through mutationNon-competitive resistance through mutation

• Failure of MVR most likely due emergence to CXCR4 or D/M Failure of MVR most likely due emergence to CXCR4 or D/M

– 60% of MVC60% of MVC

– 6% control6% control

• Origin likely preexisting low frequency CXCR4 virus rather than Origin likely preexisting low frequency CXCR4 virus rather than tropism switch tropism switch

– detailed clonal analysis from 20 (16 MVC, 4 placebo arm) and detailed clonal analysis from 20 (16 MVC, 4 placebo arm) and analysis of amino acid sequence differences and phylogenetic analysis of amino acid sequence differences and phylogenetic data, suggests emergence and not tropism switchdata, suggests emergence and not tropism switch

– Low level CXCR4 not detected by the tropism assay prior to Low level CXCR4 not detected by the tropism assay prior to treatmenttreatment

-100 0 100 200 300

0

100

200

300

400

500

CXCR4-using env Clones Were Detected at Low Frequency in the Baseline Sample

Time Since First Administration (Day)

• CXCR4-using clones detected at baseline (7%)

• No CCR5-tropic clones on treatment

R5 R5 DM DM DM DM DM DM R5 R5

1

2

3

4

5

6

HIV

-1 R

NA

(lo

g10

co

pie

s/m

L)

CD

4 C

ou

nt

(ce

lls/m

cL)

Patient T6

Lewis M et al. XVI IDRW, 2007, Abstract 56

-100 0 100 200 300

0

100

200

300

400

500






1

2

3

4

5

6

HIV

-1 R

NA

(lo

g10

co

pie

s/m

L)

CD

4 C

ou

nt

(ce

lls/m

cL)

Patient T6R5 tropic

Dual tropicX4 tropic

Non-functional clone


-100 0 100 200 300

0

100

200

300

400

500






1

2

3

4

5

6

HIV

-1 R

NA

(lo

g10

co

pie

s/m

L)

CD

4 C

ou

nt

(ce

lls/m

cL)

Patient T6R5 tropic




-100 0 100 200 300

0

100

200

300

400

500






1

2

3

4

5

6

HIV

-1 R

NA

(lo

g10

co

pie

s/m

L)

CD

4 C

ou

nt

(ce

lls/m

cL)

Patient T6R5 tropic




Non-competitive Inhibition & Resistance

CC

R5

CC

R5

resistancenon-comp.inhibition

ENV

virus membraneC

CR

5

cell membrane


CC

R5

CC

R5

resistance

ENV

non-comp.inhibition

ENV

virus membraneC

CR

5

cell membrane


CC

R5

CC

R5

resistance

ENV

ENV

non-comp.inhibition

ENV

virus membraneC

CR

5

cell membrane

CCR5 Resistance Mutations

• 8 subjects from ACTG 5211 phase IIb study with confirmed virologic failure on VCV analyzed– No tropism shifts observed– Mutations in V3 loop of gp120 identified in all

patients

• 1 patient underwent detailed analysis of viral inhibition at various time points– Progressive susceptibility to VCV until reaching

plateau– When VCV therapy was removed at Week 28,

virus became susceptible to VCV again and plateau effect was reversed

Tsibris AMN, et al. HIV Resistance Workshop 2007. Poster 13.

Clonal 07J SusceptibilitiesClonal 07J Susceptibilities

0 1 2 3

-50

0

50

100Week 0Week 16Week 19Week 28

VCV (log nM)

Per

cen

t In

hib

itio

n (

%)

Following d/c of VCV, phenotypic susceptibility returned by wk 48

Tsibris, Resistance Workshop, 6/07, abstract #13

INTEGRASE INHIBITORS

Partial Analysis of Raltegravir Resistance in BENCHMRK-1 and BENCHMRK-2

• Virologic failure on Raltegravir vs. placebo: 76 (16%) vs. 121 (51%)

• Raltegravir failure was generally associated with one of two genetic pathways: N155H or Q148K/R/H

• Additional mutations were observed with both pathways

N155H + (E92Q,V151I, T97A, G163R, L74M)

Q148K/R/H + (G140S/A, E138K)

• Other pathways? Y143R/C + (L74A/I, E92Q, T97A, I203M, S230R)

• Longitudinal analyses and associations are in progress

Partial analysis based on genotyping 41 Raltegravir failures32 with integrase changes, 9 with no consistent changes from baseline

1. Cooper D, et al. 14th CROI, Los Angeles 2007, #105aLB; 2. Steigbigel R, et al. ibid, #105bLB

Mutation Pathways Leading to Resistance to RAL Identified

RAL failures in a phase II study analyzed Mutations:

– Near active site

– Similar to mutations selected with other integrase inhibitors in cell culture

2 genetic pathways appear to confer resistance to RAL

Hazuda DJ, et al. HIV Resistance Workshop 2007. Abstract 8.







Path 1

Path 2







Path 1

Path 2

Catalytic residues

How many new/ active drugs do you need?

• Unanswered question• Multiple new agents from different classes

entering into expanded access• Guidelines suggest addition of at least 2

active agents• cPSS may help to ‘add up’ how many drugs

to include (i.e. include agents until cPSS > 2)

• Perhaps can leave the NRTI out?• A testable hypothesis (ACTG 5241)

MOTIVATE 1 and 2: VL < 50 c/mL at Week 24 by Active Drugs in OBR

No. of active drugs in OBR 0 1 2 ≥ 3

n =

010

2030

4050607080

90100

35 51 56 44 130 134 59 88 104 64 132 121

3

18

29

9

43 43

19

52 53 5561 58

Pat

ien

ts (

%)

Combined Analysis: MOTIVATE 1 & 2

Placebo + OBR MVC QD + OBR MVC BID + OBR

Nelson M, et al. CROI 2007. Abstract 104aLB. Lalezari J, et al. CROI 2007. Abstract 104bLB.

Regimens with cPSS of >2.0

Resistance test

cPSS >2.0 cPSS ≤2.0

Site selects regimenObservational armRegimen + NRTIs

Virologic failure

RegimenwithoutNRTIs

Regimen+

NRTIs

Virologic failure or permanent discontinuation of NRTI strategy

Randomize

ACTG 5241: OPTIONS

K Tashima, Chair


Resistance test



Virologic failure

RegimenwithoutNRTIs

Regimen+

NRTIs


Randomize

K Tashima, Chair


Resistance test



Panel of agentsT20RAL

DRV/rTPV/rETRMVC

Virologic failure

RegimenwithoutNRTIs

Regimen+

NRTIs


Randomize

K Tashima, Chair

TIME TO GO….

Documents

Utilization of Genotype and Phenotype Resistance Tests in Patient Management Richard Haubrich, MD Professor of Medicine University of California San Diego