advanced resistance the clinician 4 raffanti.pdfraltegravir elvitegravir (gsk364735) genotype assay...

Advanced Resistance Issues for the ClinicianStephen Raffanti MD MPH

CMO, Comprehensive Care CenterAssociate DirectorHIV Clinical Services

Vanderbilt University School of Medicine

Goals of this talkGoals of this talk

• Work with the essential components of retroviralWork with the essential components of retroviral resistance in a pragmatic, clinical approach;

• Discuss (yes that means audience participation!)Discuss (yes that means audience participation!) real life cases;

• Come away with a feasible approach to• Come away with a feasible approach to maximizing available knowledge and resources to get the best outcomes for your patients.get the best outcomes for your patients.

• Have a little fun.

First some important terms (and ones you can ignore)

• Resistance: how we describe the changes theResistance: how we describe the changes the virus makes to replicate in the presence of external factors:

• Clinically significant mutations in appropriate genes;• Clinically insignificant mutations in appropriate genes (polymorphs);(polymorphs);

• Net phenotypic change from above mutations;• Changes in receptor tropism;• Immunologic al escape mechanisms;*• Efflux mechanisms;*

» *not extremely helpful for clinicians at this point» *not extremely helpful for clinicians at this point

Retrovirus Life CycleC t CD4Co-receptor, CD4 binding inhibitorsMaravirocVicrivirocTNX 355

Protease inhibitorsSQV IDVTNX 355RTV NFVFPV LPVATV TPVDRV

Fusion InhibitorsEnfuvirtide

Reverse transcriptase inhibitorsZDV NVPddI DLVddC EFVddC EFVd4T 3TCFTC ABCTDF EtravirineRilpivirine

Integrase inhibitorsRaltegravirp gElvitegravir(GSK364735)

Genotype AssayGenotype Assay

Viral PR/RT gene isolation

PATIENTPLASMA(>200 µL)

total RNA cDNAPR/RT GENES(amplicon)

/ g

extraction RT PCR

C T A G C A C GTC T A G C A C GTAutomated DNA sequencingSequence interpretation

AAA GAC AGT

Codon

AAA AAC AGC

Mutation Silent mutation

LysLys AspAsp SerSer LysLys AsnAsn SerSer

Phenotype AssayResistance Test Vector DNA

Phenotype Assay

Patient‐derived Segment

PR RT IN

Luciferase

++++

Indicator Gene

A‐MLV envDNA

Resistance Test Vector DNA

Transfect vector I f tiDNA into cell Infection

PR Inhibition

RT Inhibition

New dx, ART naive

Current Rx: ZDV/3TC, NVPHIV RNA 45,000, CD4+ 350

hPhenotype

ResistanceResistance

Remember no matter how you measure it, you areRemember no matter how you measure it, you are probably talking about the dominant strain in a swarm of strains.

It is only one of the ones that matter now.


• Fitness: a measure of how well a virusFitness: a measure of how well a virus replicates in a certain environment or with certain changes (mutations):certain changes (mutations):

Ask me about

my MG…


• Fitness can be measured:Fitness can be measured:– in the lab:

– Replicative capacityReplicative capacity

– in the patient– Current viral load

• It can explain some phenomena:– Meds that shouldn’t be active having an impact:eds a s ou d be ac e a g a pac

• 3TC, maybe NRTI’s, maybe PI’s,

– Duration of resistance mutations of meds.

Partial Treatment Interruptions (N= 65)(Change in Viral Load)(Change in Viral Load)

1.01.0

Change 0.5ChangePlasmaHIV RNA

0.0(week 2)

NRTI PI NNRTI Fusion-0.5

Discontinued Treatment Class Deeks 2005

FitnessFitness

• For the most part measuring it is notFor the most part measuring it is not important.

“forget about it”


• Minority variants and dominant strains:Minority variants and dominant strains:– HIV replicates at very high rates: 1010‐1011 virions a day;– HIV polymerases have a very high error rate: approx. 1 mutation per

genome per cycle;genome per cycle;– In addition to mutations, recombinations occur between diploid

genomes in each virion;– Every possible mutation and many double mutations are generated in y p y g

each individual every day.– New assays are available that can detect minor variants down to <1%

of replicating strains (point mutation assays, ultradeep)pyrosequencing);

– The presence and magnitude of some minor drug resistant variants has been associated with treatment failure in naïve and treated patientspatients.


• Current role of supersensitive assays for minorCurrent role of supersensitive assays for minor variants:

• Not yet ready for prime timeNot yet ready for prime time.

Six easy rules to Select the Best fRegimen for your patient

• Know the history; ( no help here‐just good old fashionedKnow the history; ( no help here‐just good old fashioned medicine, old records)

• Know the math; (all sorts of tools, charts and websites); ( , )

• Know the meds; (keep updated, especially drug‐drug interactions)

• Know the patient; (you are probably very good at this)

• Create your virus; (make the dreaded MDR HIV)Create your virus; (make the dreaded MDR HIV)

• Test your hypothesis. (treat the patient, monitor response and if necessary regroup)and if necessary, regroup)

James, energetic motivational speaker and d idrodeo rider

• It is 1991 James has been diagnosed with HIVIt is 1991. James has been diagnosed with HIV for 5 years he has been treated off and on with two meds His current CD4 count iswith two meds. His current CD4 count is 362/22%– Who is Secretary of State?– Who is Secretary of State?

– What is the number one pop hit?

What meds was he probably exposed to?– What meds was he probably exposed to?

– Any ideas on the likelihood of resistance mutations and what they might be?mutations and what they might be?

1991

• (Everything I Do) I Do it for you. Bryan Adams

Mutations Selected by nRTIs

Abacavir

Did iDidanosine

Emtricitabine

Lamivudine

Stavudine

Tenofovir

Zidovudine

James, successful real estate agent and isongwriter

• It is 1996. In 1994 James developed hypertension and is being p yp gtreated for depression. He was screened for a new clinical trial comparing two drugs (AZT/3TC) with 3 drugs (AZT/3TC/Indinavir)(AZT/3TC/Indinavir).

• He is enrolled in study, stays on meds for 34 weeks until he develops kidney stones and drops out of care.

• Now his CD4 count is 262/18%, HIV‐1 RNA is 64,320.

• What is the most important information we can get without a ?genotype?

• Do we need him to be unblinded?

James and the Clinical TrialJames and the Clinical Trial

• Patient was unblinded: randomized toPatient was unblinded: randomized to AZT/3TC and Indinavir

D CD4 C CD4 % HIV 1 RNADate CD4 Count CD4 % HIV‐1 RNA

2/1994 207 14 94,330

6/1994 284 17 1,243

10/1994 323 19 2,240

1/1995 362 19 1120

6/1995 303 18 83,200

James and the Clinical TrialJames and the Clinical Trial

• Any idea about possible resistance mutations?

Mutations Selected by nRTIs

Abacavir

Did iDidanosine

Emtricitabine

Lamivudine

Stavudine

Tenofovir

Zidovudine

James, Round ThreeJames, Round Three

• James undergoes genotypic testing after beingJames undergoes genotypic testing after being off meds for about 7 months:– RT: 44D 41L 215V– RT: 44D, 41L, 215V,

– PRO: 36I, 77I,

Wh t th t ti i d b t?• What other mutations are you worried about?

James, Round ThreeJames, Round Three

• Most likely significant background mutation:Most likely significant background mutation:– 1) RT K65R

– 2) RT 103N

– 3) RT 184V

– 4) PRO 90M

The Special Case of M184VThe Special Case of M184V

• M184V emerges rapidly in nonsuppressive regimens containing 3TC or FTC

• Associated with high‐level phenotypic resistance to 3TC/FTC in vitro but also reduction in replication

b lcapacity, HIV RNA < baseline

• Increases susceptibility to ZDV, d4T, TDF

• Decreases susceptibility (slightly) to ABC, ddI

• Clinical impression: all NRTIs retain some activity

M184V I S tibilit tM184V Increases Susceptibility to d4T, ZDV, and TDF

3TC Alone vs Treatment Interruption in i ili dPatients Failing 3TC‐Based Rx

1 L)

Weeks

Mean CD4+ Decrease (ITT)Mean VL Increase (ITT)

2.0 3TC (n = 29)4 12 24 36 48

ange

in H

IV-1

0co

pies

/mL

n C

D4+

s/

mm

3 )

-100-50

0P = .0015

1.0

1.5

( )TI (n = 29)

-300

Mea

n C

haR

NA

(log 1

an C

hang

e in

l Cou

nt (c

ell

04 12 24 36 48 P = NS-250

-200-1500.5

3TC (n = 29)TI (n = 29)300

Mea

Cel

lWeeks

In contrast to treatment interruption arm, 3TC alone resulted in– Smaller recovery in replication capacity

( )

Castagna A, et al. AIDS. 2006;20:795-803.

S a e eco e y ep cat o capac ty– No further selection of resistance mutations

James, Round FourJames, Round Four

• James is started on stavudine, didanosine andJames is started on stavudine, didanosine and nelfinavir.

• He remains on this regimen until his leg pain g g pbecomes so severe that he stops all meds.

• He sees you in 2003. He has been off meds for 4 yyears. He is doing poorly, recently discharged from the hospital after an admission for esophagitis and pneumonia.

• He is homeless, living in a shelter.

James, Round FourJames, Round Four• James sees you in clinic and is eager to get back on ART His meds include fluconazoleback on ART. His meds include fluconazole, dapsone and amitriptyline.

ld d ?• Would you order resistance testing?Date CD4 count CD4 % HIV‐1 RNA

O1/1996 345 17 67,290

06/1997 402 21 <400

11/1997 422 22 <400

02/2002 88 6 94 24002/2002 88 6 94,240

James, Round FourJames, Round Four

• No resistance testing is availableNo resistance testing is available.

• What regimen would you choose?St di t f i k l t– Stavudine, tenofovir, kaletra;

– Tenofovir, didanosine, efavirenz;

– Tenofovir, lamivudine, kaletra;

– Tenofovir, lamivudine, efavirenz.

James, Round FiveJames, Round Five

• James stayed on his current regimen off and on for another y gfour years.

• He is successfully employed as a motivational speaker and has t l d t i ltraveled extensively.

• In 2008 he suffered an acute MI. He stopped his “HAART” when he started his “heart” meds.

• His current meds include lisinopril, pravastatin, glypizide and plavix.

• It is 2009, he wants to start a new regimen, had some diarrhea with the last.

• He has been off HAART for 5 months• He has been off HAART for 5 months.


• Is it worth doing resistance testing?Is it worth doing resistance testing?

Date CD4 Count CD4 % HIV‐1 RNA

3/2007 172 12 1,320

/4/2007 442 23 <50

2/2008 513 25 <50

1/2009 412 21 86,234


• James genotype shows the following:James genotype shows the following:– RT: 103N, 188L, 41L, 44D, 69S, 74V, 215V;

PRO: 32I 36I 47V 50V 77I 82A;– PRO: 32I,36I, 47V, 50V, 77I, 82A;

• What do you do now?What do you do now?– Hint: you can call a friend

Darunavir (Prezista) mutation scoreDarunavir (Prezista) mutation score

• 3 or more baseline mutations from a list of 113 or more baseline mutations from a list of 11 specific mutations

Virologic Response by Number of PREZISTA Resistance‐associated Mutations at Baseline

80

100

associated Mutations at Baselineop

ies/

mL

60

80

50%RN

A <5

0 co

k 24

20

40

22%

%) w

ith H

IV-1

at

wee

0

20

0-2 mutations 3 mutations 4 mutations

10%

Patie

nts

(%

Based on supportive analyses of POWER 1, 2, and 3 examining patients achieving viral load <50 copies/mL at week 24.

Etravirine Resistant ScoresEtravirine Resistant Scores

1st Gen

2nd Gen

1st Gen1st Gen

Response by Number of DUET 1 + 2

80Etravirine RAMs

80 Y181C G190A

60

50 c

/mL 60

40

med

VL

<5

40

0

20

pts

conf

irm

0

20

0

% 0

Nomut*(ref)*

+ 0Etravirine RAMs

+ 1 + 2 + 3 + 4

52 23 36 26 17 8P ti t ( )

Nomut*(ref)*

+ 0Etravirine RAMs

+ 1 + 2 + 3 + 4

52 18 46 25 17 9

Y181C G190A

52 23 36 26 17 8Patients (n) 52 18 46 25 17 9

Vingerhoets J, et al. Antiviral Therapy 2007;12:S34. Abstract 32.

*no detectable baseline NNRTI RAMs from the list of 44


• What do you recommend for the nextWhat do you recommend for the next regimen?– get a Trofile assay and represent;– get a Trofile assay and represent;

– lopinavir/rtv, raltegravir, etravirine;

atazanavir/rtv raltegravir etravirine;– atazanavir/rtv, raltegravir, etravirine;

– darunavir/rtv, raltegravir, etravirine;


i b b d hi i• James is about to be started on his regimen:– Darunavir/rtv, etravirine and raltegravir

• Anything else you want to do?y g y

Putting It All togetherPutting It All together

• How can all these factors be taken intoHow can all these factors be taken into account when designing a regimen?– Patient factors: concurrent diagnoses tolerances– Patient factors: concurrent diagnoses, tolerances, adherence, insurance status;

– Viral factors: phenotype of current dominantViral factors: phenotype of current dominant strain, archived strains, viral fitness;

– Medication factors: dosing, drug interactions,Medication factors: dosing, drug interactions, short and long term toxicities.

Comprehensive Care Center ART Conference

• All patients either starting, stopping orAll patients either starting, stopping or changing antiretroviral therapy;

• Presentation includes past regimens with labs,Presentation includes past regimens with labs, tolerances, genotypes, phenotypes;

• All co‐morbidities included;All co morbidities included;• Patient preferences included;• Wide participation of clinical staff• Wide participation of clinical staff.• Available at www.compclinic.org

ART Conference QuestionsART Conference Questions

• Should patient be treated?Should patient be treated?

• What are the goals of treatment?

h h i f i i i b f• What other information is important before designing an effective regimen?

• What is the greatest obstacle to success?

• What should be done and in what order?

Final ThoughtsFinal Thoughts

• Prior and current phenotypes and genotypesPrior and current phenotypes and genotypes are critically important components of designing a successful regimendesigning a successful regimen.

• A complete review of past meds, labs, co‐morbidities and tolerances is essentialmorbidities and tolerances is essential.

• Once a predictably successful regimen is d id d i i h i hdecided upon, giving the patient the appropriate time and support to succeed is

ll iequally important.

AIDS 1985‐ One Patient’s Experience

• 322 IV insertions 8 intubations322 IV insertions 8 intubations

• 14 hospital admissions 4 lumbar punctures

• 11 months of hospital stay 3 bone marrows11 months of hospital stay 3 bone marrows

• 60 phlebotomies 5 cycles of chemo

• 32 chest x rays 2 lymph node bx• 32 chest x‐rays 2 lymph node bx

• 5 CT scans of head

• 3 abdominal ct scans• 3 abdominal ct scans

• 6 bronchoscopies

Useful HIV Websiteswww.compclinic.org www.aidsetc.orghttp://hivdb.stanford.edu/www.hivatis.org (DHHS, USPHS/IDSA Guidelineswww.cdc.gov/nchstp/hiv aids.htmwww.cdc.gov/nchstp/hiv_aids.htmwww.hiv-web.lanl.govwww.niaid.nih.govwww AIDS medscape comwww.AIDS.medscape.comwww.hopkins-aids.eduwww.iapac.orgwww.igm.govwww.acis.orgwww.centerwatch.comce te atc cowww.ucsf.edu/medicalwww.virology.net

GerryGerry

• 46 year old male with chronic renal lithiaisis46 year old male with chronic renal lithiaisis, hypertension, depressive disorder and recurrent HSVrecurrent HSV.

• His current medications include acyclovir, ramipril hydrochlorothiazide tmp smxramipril, hydrochlorothiazide, tmp‐smx, hydrocodone, emtricitabine, darunavir, ritonavir enfuvirtideritonavir, enfuvirtide.

• He would like to discontinue his T 20 i j iinjections.

GerryGerry

• Patient discontinues the T‐20 and continuesPatient discontinues the T 20 and continues on the darunavir /rtv and FTC. He begins raltegravir and etravirineraltegravir and etravirine.

• His HAART pill burden is five pills in the morning and six pills in the eveningmorning and six pills in the evening.

• His CD4 count is 732/24% and HIV‐1 RNA is 48 i / l<48 copies/ml.

ErikaErika

• 43 year old female with depressive disorder43 year old female with depressive disorder, treated breast cancer, hypertension, and obesity is presented for virologic failureobesity is presented for virologic failure.

• Her current medications include: valsartan, dapsone mirtazapine gabapentindapsone, mirtazapine, gabapentin, amitriptyline, didanosine, nevirapine and kaletrakaletra.

Etravirine Resistance ScoreEtravirine Resistance Score

• V90I = 1 A98G = 1

• L100I = 2.5 K101E = 1

• K101H = 1 K101P = 2.5

• V106I = 1.5 E138A = 1.5

• V179D = 1 V179F = 1.5

• V179T = 1 Y181C = 2.5

• Y181I = 3 Y181V = 3

• G190A = 1 G190S = 1 5• G190A = 1 G190S = 1.5

• M230L = 2.5 erikas’s score = 3.5 (50%)

ErikaErika

• Patient discontinues her current regimen.Patient discontinues her current regimen.

• Her sulfa allergy was considered but it was associated with a questionable, mild skin rash.q ,

• She begins darunavir/rtv, raltegravir and maraviroc.

• She is seen by the CPS team and has weekly calls toShe is seen by the CPS team and has weekly calls to review meds and adherence.

• Her current labs are CD4 count of 407/22% and HIV‐/1 RNA is <40 copies/ml.

advanced resistance the clinician 4 raffanti.pdfraltegravir elvitegravir (gsk364735) genotype assay...

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