Research

JAMA Cardiology | Original Investigation

ADCY9 Genetic Variants and Cardiovascular Outcomes

With Evacetrapib in Patients With High-Risk Vascular Disease
A Nested Case-Control Study
Steven E. Nissen, MD; Sreekumar G. Pillai, PhD; Stephen J. Nicholls, MBBS, PhD; Kathy Wolski, MPH;
Jeffrey S. Riesmeyer, MD; Govinda J. Weerakkody, PhD; Wendra M. Foster, BS; Ellen McErlean, MSN; Lin Li, PhD;
Pallav Bhatnagar, PhD; Giacomo Ruotolo, MD, PhD; A. Michael Lincoff, MD

Editor's Note
IMPORTANCE A pharmacogenetic analysis of dalcetrapib, a cholesteryl ester transfer protein Supplemental content
inhibitor, reported an association between a single-nucleotide polymorphism (SNP) in the
ADCY9 gene (rs1967309) and reduction in major adverse cardiovascular events despite a
neutral result for the overall trial.

OBJECTIVE To determine whether the association between the SNP in the ADCY9 gene and a
reduction in major adverse cardiovascular events could be replicated for another cholesteryl
ester transfer protein inhibitor, evacetrapib, in patients with high-risk vascular disease.

DESIGN, SETTING, AND PARTICIPANTS A nested case-control study examining the rs1967309
SNP in 1427 cases and 1532 matched controls selected from the 12 092–patient Assessment
of Clinical Effects of Cholesteryl Ester Transfer Protein Inhibition with Evacetrapib in Patients
at a High Risk for Vascular Outcomes (ACCELERATE) trial, a randomized, double-blind,
placebo-controlled phase 3 trial conducted in patients with high-risk vascular disease
randomized from October 2012 through December 2013. The genotyping was conducted
from January 2017 to March 2017, and the data analyses were conducted from July 2017 to
November 2017.

EXPOSURES Evacetrapib, 130 mg, or matching placebo.

MAIN OUTCOMES AND MEASURES The primary analyses used a conditional logistic regression
model to assess the odds ratio (OR) for major adverse cardiovascular events for evacetrapib
compared with placebo for each genotype. The basic model included adjustment for age, sex,
and the top 5 principal components. An additional model included cardiovascular risk factors to
adjust for potential bias in selecting control patients. The primary major adverse cardiovascular
event end point was the composite of death from cardiovascular causes, myocardial infarction,
stroke, coronary revascularization, or hospitalization for unstable angina.

RESULTS For patients with the AA genotype reported to demonstrate a beneficial effect from
dalcetrapib, the OR for evacetrapib compared with placebo was 0.88 (95% CI, 0.69-1.12). For Author Affiliations: The Cleveland
patients with the AG genotype, the OR was 1.04 (95% CI, 0.90-1.21). For patients with the Clinic Coordinating Center for Clinical
GG genotype reported to show evidence for a harmful effect from dalcetrapib, the OR for Research, Department of
Cardiovascular Medicine, Cleveland
evacetrapib was 1.18 (95% CI, 0.98-1.41). The interaction P value among the 3 genotypes was
Clinic, Cleveland, Ohio (Nissen,
P = .17 and the trend P value was P = .06. When adjusted for cardiovascular risk factors, the Wolski, McErlean, Lincoff); Eli Lilly,
OR for evacetrapib was 0.93 (95% CI, 0.73-1.19) for the AA genotype, 1.05 (95% CI, 0.91-1.22) Indianapolis, Indiana (Pillai,
for the AG genotype, and 1.02 (95% CI 0.85-1.24) for the GG genotype; interaction P = .71 and Riesmeyer, Weerakkody, Foster,
Bhatnagar, Ruotolo); South Australian
trend P = .59.
Heart and Medical Research Institute,
University of Adelaide, Adelaide,
CONCLUSIONS AND RELEVANCE Pharmacogenetic analysis did not show a significant Australia (Nicholls); School of Medical
association between the ADCY9 SNP (rs1967309) and cardiovascular benefit or harm for the Sciences, University of New South
Wales, Sydney, Australia (Nicholls);
cholesteryl ester transfer protein inhibitor evacetrapib. BioStat Solutions Inc, Frederick,
Maryland (Li).
Corresponding Author: Steven E.
Nissen, MD, Cleveland Clinic,
JAMA Cardiol. doi:10.1001/jamacardio.2018.0569 9500 Euclid Ave, Cleveland, OH
Published online March 11, 2018. 44195 (nissens@ccf.org).

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 Research Original Investigation
L
arge cardiovascular outcome trials have studied the ef-
fects of 4 drugs that inhibit cholesteryl ester transfer pro-
tein (CETP): torcetrapib, dalcetrapib, evacetrapib, and
anacetrapib.1-4 These CETP inhibitors markedly raised circu-
lating levels of high-density lipoprotein cholesterol (HDL-C),
with increases ranging from approximately 30% to 130%. With
the exception of dalcetrapib, these drugs also reduced low-
density lipoprotein cholesterol (LDL-C), with decreases rang-
ing from 21% to 37%. Dalcetrapib and evacetrapib had no sig-
nificant effect on major adverse cardiovascular events (MACE),
torcetrapib increased MACE, and anacetrapib produced a small
reduction in MACE, which was consistent with the expected
event reduction based on meta-analyses of trials of other agents
that reduce levels of atherogenic lipoproteins.5
A post hoc pharmacogenetic analysis of the dalcetrapib trial
suggested an association between a single-nucleotide poly-
morphism (SNP) in the ADCY9 gene on chromosome 16
(rs1967309) and cardiovascular outcome (P = 2.41 × 10−8).6 Pa-
tients with the AA genotype who received dalcetrapib showed
a 39% reduction in the primary composite cardiovascular end-
point compared with placebo, whereas patients with the GG
genotype showed a 27% increase in the primary end point. Re-
sults for the AG genotype were neutral. Supporting evidence
for a pharmacogenetic relationship included an analysis of the
relationship between genotype status and carotid intimal me-
dial thickness in a separate clinical trial but for a different SNP
(rs2238448) in the ADCY9 gene.6 A subsequent analysis sug-
gested a beneficial relationship between the genotype and
change in 2 biomarkers, high-sensitivity C-reactive protein
(hsCRP), and cholesterol efflux capacity.7 On the basis of these
findings, a new cardiovascular outcome trial (NCT02525939),
currently underway, was designed to study the cardiovascular
effects of dalcetrapib compared with placebo only in patients
with the AA genotype.
In this analysis, we sought to replicate the pharmacoge-
netic findings from the dalcetrapib analysis in patients en-
rolled in the Assessment of Clinical Effects of Cholesteryl Ester
Transfer Protein Inhibition with Evacetrapib in Patients at a
High Risk for Vascular Outcomes (ACCELERATE) trial, which
assessed the effect of evacetrapib on cardiovascular outcome
in patients with high-risk vascular disease.3
Methods
The ACCELERATE trial
The ACCELERATE trial was a multicenter, randomized, double-
blind, placebo-controlled, phase 3 trial conducted at 543 sites
in 36 countries (NCT01687998). The trial design has been
previously described.3,8 Approval was obtained from local
institutional review boards and all patients enrolled in the
ACCELERATE Trial provided written informed consent.
Institutional review boards also approved the pharmacogenetics
analyses. The genetic analyses were conducted in those patient
samples for patients who provided written informed consent
for participation in the genetics study. Enrolled patients had
preexisting vascular disease with high-risk features, defined as
the presence of at least 1 of the following conditions: an acute
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ADCY9 Genetic Variants and Cardiovascular Outcome With Evacetrapib
Key Points
Question Does the rs1967309 single-nucleotide polymorphism,
reported to show a strong interaction for dalcetrapib, influence
cardiovascular outcomes for patients treated with the cholesteryl
ester transfer inhibitor evacetrapib?
Findings In this nested case-control study, this single-nucleotide
polymorphism was examined in 1427 cases and 1532 matched
controls selected from the 12 092–patient evacetrapib
cardiovascular outcome trial. The conditional logistic regression
odds ratio for major adverse cardiovascular events for
evacetrapib-treated patients with the AA genotype was not
significant.
Meaning Although directionally similar to the dalcetrapib
analysis, there was no significant interaction between genotype
and cardiovascular outcome with evacetrapib.
coronary syndrome within the previous 30 to 365 days,
cerebrovascular atherosclerotic disease, peripheral arterial
disease, or diabetes with coronary artery disease. Patients were
randomly assigned in a 1:1 ratio to receive oral evacetrapib
at a dose of 130 mg or matching placebo. The primary
efficacy end point was MACE, defined as the first occur-
rence of any component of the composite of death from
cardiovascular causes, myocardial infarction, stroke, coronary
revascularization, or hospitalization for unstable angina
(5-component MACE). A key secondary efficacy end point was
the composite of death from cardiovascular causes, myocardial
infarction, or stroke (3-component MACE). The trial was
terminated prematurely for futility at the recommendation of
the data monitoring committee after 82% of the planned 1670
primary events had occurred.
Pharmacogenetic Study Design
DNA samples were extracted from the frozen whole blood from
the patients who consented for genetic analyses. All samples
were genotyped using the Axiom Biobank genotyping array,
version 2 (Affymetrix). Patient samples, along with dupli-
cates and HapMap controls, were genotyped, and data on
717 345 SNPs were generated. Standard metrics for genome-
wide association study (GWAS) quality control were used, and
634 382 SNPs passed quality-control screening. Genotype call
rate for the key ADCY9 SNP of interest (rs1967309) was 99.2%,
with a minor allele frequency of 41%. The rs1967309 SNP did
not deviate from Hardy-Weinberg equilibrium. The pharma-
cogenetic sample information is illustrated in eFigure 1 in the
Supplement.
The pharmacogenetic analysis used a nested case-
control design that genotyped all patients with cardiovascu-
lar events and matched control patients. eFigure 2 in the
Supplement shows the flow of patients in the analysis. From
779 patients in the evacetrapib treatment group with a posi-
tively adjudicated cardiovascular event for the primary end
point, genotype data for genetic analysis were available in 719
patients. The frequency of the AA, AG, and GG genotypes were
compared with 763 control patients without a primary cardio-
vascular end point matched for age, sex, and race/ethnicity.
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 ADCY9 Genetic Variants and Cardiovascular Outcome With Evacetrapib
For the placebo treatment group, genotype data were avail-
able for 708 of 776 patients with a cardiovascular event and
compared with 769 matched control patients. The primary end
point for the analysis was the odds ratio (OR) for 5-compo-
nent MACE comparing the evacetrapib treatment group with
the placebo treatment group within each genotype. A sensi-
tivity analysis assessed the OR for 3-component MACE, the key
secondary end point in the ACCELERATE trial. Additional
analyses assessed the OR for the effect per minor allele for eva-
cetrapib and placebo for both 5-component and 3-compo-
nent MACE (additive genetic model). The relationship be-
tween genotype and biomarkers associated with cardiovascular
disease, including high-sensitivity C-reactive protein (hsCRP),
was also determined.
Statistical Analyses
Conditional logistic regression methods were used to account
for the matching of patients with events (cases) to patients with-
out events (controls). To test the additive genetic effect for the
minor allele, using 1° of freedom, the common alleles (GG) were
coded as 0 (reference), heterozygotes (AG) were coded as 1, and
minor allele homozygotes (AA) were coded as 2. To correct for
any potential population substructure, principal component
analysis was performed on linkage disequilibrium-pruned GWAS
data, and top 5 principal components captured most of the ge-
netic variability in our data set. Principal components plots show-
ing the race/ethnicity distribution using principal component
1 and principal component 2 are shown in eFigure 3 in the
Supplement, and a scree plot showing individual and cumula-
tive percent genomic variance explained by top 10 principal com-
ponents is shown in eFigure 4 in the Supplement. The primary
analyses used a conditional logistic regression model that in-
cluded treatment, genotype, and genotype-by-treatment terms
and adjusted for the age, sex, and the top 5 principal compo-
nents. An additional model included cardiovascular risk factors
to adjust for potential bias in selecting control patients. The mod-
els were used to assess the effect of evacetrapib compared with
placebo within each genotype category and to assess genetic ef-
fect within each treatment group. To understand the heteroge-
neity of treatment effects across the genotypes, a genotype-
by-treatment interaction test was conducted using a likelihood
ratio test with 2° of freedom. An alternative analysis examined
the trend across genotypes after coding the genotypes as the
number of minor alleles in the conditional logistic model, report-
ing the corresponding genotype-by-treatment interaction P val-
ues. A sensitivity analysis using only white patients was also con-
ducted. For the primary outcome, the study was 87% powered
to detect the treatment by SNP interaction hazard ratio (HR) re-
ported for dalcetrapib. The P value level of significance was .05,
and all P values were 2-sided.
ADCY9 Biomarker Analyses
Linear regression analyses were used to assess the genetic as-
sociation of the ADCY9 SNP with the change from baseline of
several biomarkers of interest (HDL-C, LDL-C, hsCRP, and sys-
tolic and diastolic blood pressure). The model was adjusted for
baseline level, and evaluations were separately performed in
both the evacetrapib and placebo treatment groups.
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Original Investigation Research
Results
Baseline Characteristics
Table 1 reports the baseline characteristics for both
evacetrapib- and placebo-treated patients who experienced
and who did not experience a primary end point within the
ACCELERATE pharmacogenetic analyses. eTable 1 in the
Supplement shows the baseline characteristics for patients
who underwent genotyping and the full study population.
These comparisons show similar characteristics with the
exception of geographic region. For comparative purposes,
baseline characteristics for the dalcetrapib outcomes (dal-
OUTCOMES) genetic analysis patients are shown in eTable 2
in the Supplement. By trial design, all dalcetrapib-treated
patients experienced a recent acute coronary syndrome,
whereas slightly more than half of the ACCELERATE patients
had experienced a prior acute coronary syndrome. In the
evacetrapib study, a higher percentage of patients had diabe-
tes compared with the dalcetrapib-treated patients. In the
evacetrapib analysis nearly, all of the matched control
patients resided in North America compared with approxi-
mately half of the dalcetrapib-treated patients.
Genotype Frequencies
The genotype frequencies in the evacetrapib (ACCELERATE)
and dalcetrapib (dal-OUTCOMES) trial for all patients who un-
derwent genotyping are reported in eTable 3 in the Supple-
ment. Frequencies are shown for placebo-treated and CETP in-
hibitor–treated patients who did and did not experience a
cardiovascular event.
Logistic Regression Analysis
Table 2 shows the associations for the 3 genotypes based on
conditional logistic regression analysis for evacetrapib (both
5-component MACE and the 3-component MACE sensitivity
analysis). The ORs for 5-component MACE and 3-component
MACE showed no significant difference between evacetrapib
and placebo for any of the 3 genotypes. Patients with the AA
genotype for the rs1967309 SNP that was reported to show evi-
dence for a beneficial effect from dalcetrapib showed an OR
of 0.88 (95% CI, 0.69-1.12) for 5-component MACE and an OR
of 0.92 (95% CI, 0.67-1.27) for 3-component MACE. Patients
with the AG genotype showed an OR of 1.04 (95% CI, 0.90-
1.21) for evacetrapib treatment compared with placebo for
5-component MACE and 1.05 (95% CI, 0.86-1.28) for 3-com-
ponent MACE. Patients with the GG genotype reported to show
evidence for a harmful effect from dalcetrapib showed an OR
of 1.18 (95% CI, 0.98-1.41) for evacetrapib treatment com-
pared with placebo for 5-component MACE and 0.92 (95% CI,
0.72-1.16) for 3-component MACE. For 5-component MACE, the
interaction P value for the primary analysis among the AA, AG,
and GG genotypes was P = .17. An alternative analysis exam-
ining the trend across the 3 genotypes showed no significant
interaction for 5-component MACE (P = .06). For 5-compo-
nent MACE, analyses adjusted for cardiovascular risk factors
also did not show significant differences for evacetrapib-
treated patients compared with placebo for either the AA (OR,
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 Research Original Investigation ADCY9 Genetic Variants and Cardiovascular Outcome With Evacetrapib

0.93; 95% CI, 0.73-1.19) or GG genotype (OR, 1.02; 95% CI, 0.85-
1.24); interaction P = .71; trend P = .12 (Table 2) For 3-compo-
nent MACE, analyses adjusted for cardiovascular risk factors
also did not show significant differences for the AA genotype
(OR, 1.06; 95% CI, 0.77-1.46) or GG genotype (OR, 0.81; 95%
CI, 0.63-1.03; interaction P = .17; trend P = .12).
For comparative purposes, the HRs based on the Cox propor-
tional hazards model reported for dalcetrapib (5-component
MACE) are illustrated in eTable 4 in the Supplement. The Figure
shows forest plots for the key analyses for the primary end point
for the 3 genotypes for both dalcetrapib-treated and evacetrapib-
treated patients, and eFigure 5 in the Supplement shows forest

Table 1. Baseline Characteristics of Patients Selected for Pharmacogenetic Analysis in ACCELERATE Trial

No. (%)
Patients With Events Patients Without Events
Evacetrapib-Treated Evacetrapib Placebo Evacetrapib-Treated Evacetrapib Placebo
Characteristic (n = 719) (n = 708) (n = 763) (n = 769)
Age, mean (SD), y 65.6 (9.6) 65.7 (9.1) 65.3 (8.5) 66.0 (8.6)
Female sex 158 (22.0) 175 (24.7) 166 (21.8) 182 (23.7)
White 612 (85.1) 628 (88.7) 650 (85.2) 675 (87.8)
Geographic region
North America 396 (55.1) 395 (55.8) 689 (90.3) 695 (90.4)
Europe 199 (27.7) 200 (28.2) 34 (4.5) 32 (4.2) Abbreviations: ACCELERATE,
Assessment of Clinical Effects of
Asia 36 (5.0) 29 (4.1) 15 (2.0) 24 (3.1) Cholesteryl Ester Transfer Protein
Other 88 (12.2) 84 (11.9) 25 (3.3) 18 (2.3) Inhibition with Evacetrapib in
BMI, mean (SD) 30.9 (5.8) 31.1 (5.9) 32.0 (6.9) 31.5 (5.8) Patients at a High Risk for Vascular
Outcomes; ACS, acute coronary
Hypertension 673 (93.6) 659 (93.1) 697 (91.3) 716 (93.1) syndrome; ApoA, apolipoprotein A;
Diabetes 523 (72.7) 544 (76.8) 583 (76.4) 583 (75.8) ApoB, apolipoprotein B; BMI, body
Current smoker 126 (17.5) 107 (15.1) 104 (13.6) 100 (13.0) mass index (calculated as weight in
kilograms divided by height in meters
ACS 448 (62.3) 435 (61.4) 414 (54.3) 408 (53.1) squared); CABG, coronary artery
PAD 184 (25.6) 155 (21.9) 121 (15.9) 125 (16.3) bypass grafting; HDL-C, high-density
Previous MI 470 (65.4) 440 (62.1) 420 (55.0) 426 (55.4) lipoprotein cholesterol; hsCRP,
high-sensitivity C-reactive protein;
Previous PCI 509 (70.8) 517 (73.0) 524 (68.7) 487 (63.3) LDL-C, low-density lipoprotein
Previous CABG 227 (31.6) 231 (32.6) 281 (36.8) 286 (37.2) cholesterol; MI, myocardial infarction;
Any statin 676 (94.0) 670 (94.6) 720 (94.4) 740 (96.2) PAD, peripheral arterial disease;
PCI, percutaneous coronary
HDL-C, mean (SD), 44.8 (11.4) 44.8 (11.6) 43.6 (11.3) 43.5 (11.3) intervention.
mg/dL
SI conversion factor: To convert
LDL-C, mean (SD), 84.3 (30.0) 83.7 (29.2) 80.0 (26.5) 80.8 (26.4)
mg/dL cholesterol levels to millimoles per
liter, multiply by 0.0259;
ApoB/ApoA ratio 1.82 (0.57) 1.84 (0.58) 1.87 (0.59) 1.85 (0.56)
high-sensitivity C-reactive protein to
hsCRP, mean (SD), 4.5 (8) 4.1 (8) 4.2 (13) 3.2 (7) nanomoles per liter, multiply by
mg/L 9.524.

Table 2. Treatment Effects of Evacetrapib Compared With Placebo Within Each Genotype of rs1967309

Conditional Logistic Regressiona Conditional Logistic Regression Adjusted for Cardiovascular Risk Factorsb
Evacetrapib (5-Component MACE) Evacetrapib (3-Component MACE) Evacetrapib (5-Component MACE) Evacetrapib (3-Component MACE)
vs Placebo vs Placebo vs Placebo vs Placebo
P Value P Value P Value P Value
OR OR OR OR
Genotype (95% CI) Interactionc Trendd (95% CI) Interactionc Trendd (95% CI) Interactionc Trendd (95% CI) Interactionc Trendd
AA 0.88 0.92 0.93 1.06
(0.69- (0.67- (0.73- (0.77-
1.12) 1.27) 1.19) 1.46)
AG 1.04 1.05 1.05 1.08
(0.90- .17 .06 (0.86- .65 .83 (0.91- .71 .59 (0.88- .17 .12
1.21) 1.28) 1.22) 1.32)
GG 1.18 0.92 1.02 0.81
(0.98- (0.72- (0.85- (0.63-
1.41) 1.16) 1.24) 1.03)
Abbreviations: MACE, major adverse cardiovascular events; OR, odd ratio; smoking status in an interaction model with treatment and SNP main effects
SNP, single-nucleotide polymorphism. and treatment-by-SNP interaction effect.
a c
Adjusted for age, sex, and top 5 principal components in an interaction model P value for heterogeneity of treatment effects across the genotypes, using the
with treatment and SNP main effects and treatment-by-SNP interaction effect. likelihood ratio test with 2° of freedom.
b d
Adjusted for age, sex, top 5 principal components, and risk factors, including Trend test performed after coding the genotypes as the number of minor
baseline apolipoprotein B, history of cerebral vascular disease, history of alleles in the conditional logistic model and the corresponding
peripheral arterial disease, history of prior percutaneous coronary genotype-by-treatment interaction tested.
intervention, history of prior myocardial infarction, region, race/ethnicity, and

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 ADCY9 Genetic Variants and Cardiovascular Outcome With Evacetrapib Original Investigation Research

Figure. Treatment Effects of Cholesteryl Ester Transfer Protein (CETP) Inhibition Compared With Placebo
for 5-Component Major Adverse Cardiac Events (rs1967309 Single-Nucleotide Polymorphism)

Study and OR Interaction P Value CETP-Inhibitor Placebo

Genotype (95% CI) P Valuea For Trendb Better Better
ACCELERATE .17 .06 ACCELERATE indicates Assessment
AA 0.88 (0.69-1.12) of Clinical Effects of Cholesteryl Ester
AG 1.04 (0.90-1.21) Transfer Protein Inhibition with
GG 1.18 (0.98-1.41) Evacetrapib in Patients at a High Risk
for Vascular Outcomes;
0.1 1.0 10 dal-OUTCOMES, dalcetrapib
OR (95% CI) outcomes; HR, hazard ratio;
OR, odds ratio.
a
Study and HR Reduction, Interaction P Value CETP-Inhibitor Placebo P value for heterogeneity of
Genotype % (CI) P Valuea For Trendb Better Better treatment effects across the
dal-OUTCOMES NA .001 genotypes, using the likelihood ratio
AA 0.61 (0.41-0.92) test with 2° of freedom.
b
AG 0.94 (0.77-1.16) Trend test performed after coding
GG 1.27 (1.02-1.58) the genotypes as the number of
minor alleles in the conditional
0.1 1.0 10 logistic model and the
HR (95% CI) corresponding genotype-by-
treatment interaction tested.

Table 3. Results From the Conditional Logistic Regression Analyses Within Treatment Group for the Additive
Genetic Effect of the Minor Allele of rs1967309

OR (95% CI)
Patients Patients
Treatment Genotype With Events Without Events Unadjusted Adjusteda
5-Component MACE
Evacetrapib AA 125 130 Abbreviations: MACE, major adverse
AG 349 379 0.99 (0.89-1.10) 1.02 (0.91-1.14) cardiovascular events; OR, odd ratio.
a
GG 245 254 Adjusted for sex, top 5 principal
components, and risk factors,
Placebo AA 143 129
including baseline apolipoprotein
AG 353 378 1.15 (1.03-1.27) 1.06 (0.95-1.19) B, history of cerebral vascular
GG 212 262 disease, history of peripheral arterial
3-Component MACE disease, history of prior
Evacetrapib AA 74 181 percutaneous coronary
intervention, history of prior
AG 191 537 1.03 (0.90-1.20) 1.02 (0.88-1.18) myocardial infarction, region,
GG 131 368 race/ethnicity, and smoking status in
Placebo AA 81 191 an interaction model with treatment
AG 191 540 1.01 (0.88-1.17) 0.87 (0.75-1.00) and SNP main effects and
treatment-by-SNP interaction
GG 143 331
effect.

plots for evacetrapib-treated patients adjusted for cardiovascu- Biomarker Analyses

lar risk factors. eTable 5 in the Supplement shows results for con-
ditional logistic regression analysis in white patients, and eTable
6 in the Supplement shows genotype frequencies for various
racial/ethnic subgroups. eTable 7 in the Supplement shows the
summary of United Kingdom phenome-wide association study
and data from public consortia for rs1967309, in which the P value
was less than .05.
Table 3 shows the results for conditional logistic regres-
sion analysis for the effect of each A allele in both evacetrapib
and placebo treatment groups. No significant associations were
demonstrated within the evacetrapib treatment group for
either 5-component or 3-component MACE. However, the ef-
fect of each A allele in the placebo treatment group showed a
nominally significant adverse effect on 5-component MACE
(OR, 1.15; 95% CI 1.03-1.27), which was not significant when
adjusted for cardiovascular risk factors (OR, 1.06; 95% CI, 0.95-
1.19), and also not significant for 3-component MACE (unad-
justed OR, 1.01; 95% CI, 0.88-1.17) (Table 3).
Table 4 shows the least square mean and 95% confidence
intervals for changes for the 3 genotypes in biomarkers (LDL-C,
HDL- C, and hsCRP) and blood pressure during the
ACCELERATE trial in both the evacetrapib and placebo treat-
ment groups. There were no significant differences in the
changes in these variables for the 3 rs1967309 genotypes.
Discussion
Genome-wide association study analyses are increasingly per-
formed within large clinical outcome trials studying pharmaco-
logical therapies. A key rationale for conducting such studies is
the possibility that a specific subgroup determined via GWAS
might identify patients who show a more favorable response to
the studied therapy or identify subgroups who experience ad-
verse events. A GWAS analysis of the dal-OUTCOMES trial, a car-
diovascular outcome trial studying the CETP inhibitor dalcetra-

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 Research Original Investigation ADCY9 Genetic Variants and Cardiovascular Outcome With Evacetrapib

Table 4. Effect of rs1967309 Genotype on Biomarkers and Blood Pressure in the ACCELERATE Trial

LSM (95% CI)

Treatment
Biomarker Group Total No. GG/AG/AA, No. AA Genotype AG Genotype GG Genotype P Value
Change, Evacetrapib 1411 474/698/239 −26.4 (−29.3 to −23.4) −25.4 (−27.16 to −23.63) −25.24 (−27.32 to −23.15) .81
LDL-C,
mg/dL Placebo 1413 451/706/256 5.19 (2.37 to 8.01) 3.05 (1.33 to 4.78) 4.1 (1.95 to 6.24) .40
Change, Evacetrapib 1411 474/698/239 123 (117 to 130) 126 (122 to 130) 123 (118 to 127) .50a
HDL-C, %
Placebo 1413 451/706/256 0.4 (−2.3 to 3.2) 0.2 (−1.5 to 1.9) 0.01 (−2.1 to 2.1) .97a
Change, Evacetrapib 1097 373/528/196 16 (3 to 32) 14 (5 to 23) 10 (1 to 20) .74b
hsCRP, %
Placebo 1078 341/537/200 6 (−6 to 19) 8 (−0.2 to 16) 6 (−3 to 16) .95b
Change, DBP, Evacetrapib 1445 488/712/245 0.68 (−0.36 to 1.72) 1.17 (0.55 to 1.8) 0.55 (−0.18 to 1.29) .38
mm Hg
Placebo 1443 464/714/265 −1.15 (−2.14 to −0.16) −0.12 (−0.73 to 0.5) 0.34 (−0.41 to 1.1) .06
Change, SBP, Evacetrapib 1445 488/712/245 0.83 (−0.96 to 2.62) 2.04 (0.96 to 3.11) 1.49 (0.22 to 2.75) .48
mm Hg
Placebo 1443 464/714/265 −1.84 (−3.55 to −0.14) −0.31 (−1.37 to 0.74) −0.38 (−1.68 to 0.92) .28
a
Abbreviations: ACCELERATE, Assessment of Clinical Effects of Cholesteryl Ester A sensitivity analysis using Kruskal-Wallis rank sum test yielded a P value of
Transfer Protein Inhibition with Evacetrapib in Patients at a High Risk for .40 for testing the difference in change of HDL-C in the evacetrapib group and
Vascular Outcomes; DBP, diastolic blood pressure; HDL, high-density a P value of .68 in the placebo group.
lipoprotein cholesterol; hsCRP, high-sensitivity C-reactive protein; b
A sensitivity analysis using Kruskal-Wallis rank sum test yielded a P value of
LDL-C, low-density lipoprotein cholesterol; LSM, least square mean; .94 for testing the difference in change of hsCRP in the evacetrapib group and
SBP, systolic blood pressure. a P value of .49 in the placebo group.
SI conversion factor: To convert cholesterol levels to millimoles per liter,
multiply by 0.0259; high-sensitivity C-reactive protein to nanomoles per liter,
multiply by 9.524.

pib, generated considerable scientific interest when it reported
a 39% relative risk reduction for a variant within the ADCY9 gene
(rs1967309) despite a neutral overall result for the trial. An un-
usual finding was the observation that the AA genotype was as-
sociated with benefit, whereas the GG genotype was associated
with harm. On the basis of these findings, a new cardiovascular
outcome trial (NCT02525939) was initiated to study only
patients with the AA genotype. We conducted a cardiovascular
outcome trial (ACCELERATE) with the CETP inhibitor
evacetrapib, which also showed no cardiovascular benefits for
the primary end point, 5-component composite MACE,
compared with placebo (HR, 1.01; 95% CI, 0.91-1.11; P = .91).3 This
trial provided the opportunity to determine whether the
pharmacogenetic finding reported for dalcetrapib could be
replicated for a different CETP inhibitor.
Our analysis showed no significant association between
rs1967309 variant and cardiovascular outcomes using several
analytical methods. For evacetrapib-treated patients compared
with control patients, the primary analysis (5-component MACE)
showed an OR of 0.88 (95% CI, 0.69-1.12) for patients with the
AA genotype, 1.04 (95% CI, 0.90-1.21) for patients with the AG
genotype, and 1.18 (95% CI, 0.98-1.41) for patients with the GG
genotype (interaction P = .17). An alternative analysis examin-
ing the trend across genotypes for the relationship between
rs1967309 variant and the association of evacetrapib with car-
diovascular outcomes, showed a nonsignificant trend for
5-component MACE (P = .06) (Table 2 and Figure). Neither ap-
proach to interaction testing showed a significant relationship
when the primary 5-component analysis was adjusted for car-
diovascular risk factors, AA genotype (OR, 0.93; 95% CI, 0.73-
1.19), and GG genotype (OR, 1.02; 95% CI, 0.85-1.24) (interaction
P = .71; trend P = .59). A sensitivity analysis assessing
3-component MACE showed an OR of 0.92 (95% CI, 0.67-1.27)
for the AA genotype, 1.05 (95% CI, 0.86-1.28) for the AG geno-
type, and 0.92 (95% CI, 0.72-1.16) for the GG genotype (interac-
tion P = .65 and trend P = .83) (Table 2; eFigure 5 in the Supple-
ment). The observed ORs in the ACCELERATE trial, although di-
rectionally similar, contrast with the significantly lower HR
reported for dalcetrapib-treated patients with the AA genotype
(0.61; 95% CI, 0.41-0.92) and significantly higher HR for patients
with the GG genotype (1.27; 95% CI, 1.02-1.58) (Figure).
Additional evacetrapib analyses also did not show a sig-
nificant association between the ADCY9 SNP and cardiovas-
cular outcomes. A per allele analysis showed an OR of 0.99
(95% CI, 0.89-1.10) in the evacetrapib treatment group for each
A allele for 5-component MACE and an OR of 1.03 (95% CI, 0.90-
1.20) for the sensitivity analysis based on 3-component MACE.
Analysis of biomarkers, including hsCRP and blood pressure,
showed no association between evacetrapib rs1967309 geno-
type and change from baseline (Table 4).
There are several potential explanations for the differences
between evacetrapib pharmacogenetic analyses and findings re-
ported for dalcetrapib. Although both drugs are CETP inhibitors,
there may be differences in mechanism of action with respect to
either the on-target or off-target effects. Dalcetrapib is a weaker
CETP inhibitor, showing approximately a 35% increase in HDL-C
compared with 130% for evacetrapib, and dalcetrapib does not
lower LDL-C. The patient populations studied in the 2 trials were
somewhat different, including only recent acute coronary syn-
drome in dal-OUTCOMES and patients during a more chronic
phase of the coronary disease in ACCELERATE. The 2 analyses
used different statistical approaches. The dal-OUTCOMES analy-
sis included 5749 patients selected from a 15 871–patient clini-
cal trial and calculated an HR for each genotype based on a Cox
proportional hazard model. The ACCELERATE analysis used a
nested case-control design studying 1427 patients with events
and 1532 matched control patients using conditional logistic re-
gression analysis to calculate an OR for each genotype. The ob-

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 ADCY9 Genetic Variants and Cardiovascular Outcome With Evacetrapib Original Investigation Research

served differences in the results from dal-OUTCOMES and
ACCELERATE trials may be confounded by association of the SNP
with some measured or unmeasured cardiovascular risk factors
(mediation). To explore this possibility, we conducted a search
of publicly available UK Biobank and consortia data sets. The as-
sociation summary is shown in eTable 7 in the Supplement. There
were no associations with genome-wide significance for any clini-
cal phenotypes and rs1967309 in any of the data sets.
Statistical considerations represent an important issue in
pharmacogenetic studies. The large number of analyzed SNPs
in GWAS analyses (5.5 million in the dalcetrapib analysis) creates
the potential for false discovery. Although the results from the
dalcetrapib analyses within treatment reached genome-wide sig-
nificance, the results comparing the effects within the genotypes
and between treatments (genotype by treatment interaction)
were only nominally significant. A best practice involves iden-
tification of potential genetic effects via a discovery cohort, with
subsequent confirmation using a replication cohort. For dalce-
trapib, the replication cohort was a small imaging study, and the
end point was carotid intima medial thickness, not morbidity-
mortality. The SNP identified in the replication cohort was dif-
ferent (rs2238448) from the discovery cohort, although it was
in linkage disequilibrium with rs1967309 (r2 = 0.8). Because the
number of patients with primary end point events in the dalce-
trapib discovery cohort was low, the genetic substudy used a
broader secondary end point that included unanticipated coro-
nary revascularizations. Even using this broader end point, the
significant genetic results from the dal-OUTCOMES study were
driven by relatively sparse number of events in the dalcetrapib-
treated AA genotype subgroup (38 events).
Strengths and Limitations
This analysis has strengths and limitations. Strengths
include greater number of events in genotyped patients in
the evacetrapib analysis (1427 events) compared with the
dalcetrapib analysis (788 events), providing potentially
more reliable estimates of the genotype frequencies. Limita-
tions of our study include the retrospective nature of the
analyses and the differences in the studied CETP inhibitors,
the patient population, study design (nested case-control
analysis), and relatively short follow-up for the evacetrapib
study. Neither the dalcetrapib nor the evacetrapib analysis
could be confirmed via a large replication study because
only a single outcome trial exists for each drug.
Conclusions
In summary, our analysis does not demonstrate a sig-
nificant association between the ADCY9 SNP (rs1967309)
and clinical benefits from the CETP inhibitor evacetrapib.
A nonsignific ant trend was observed for the associ-
ation between the ADCY9 SNP (rs1967309) and clinical
benefits from the CETP inhibitor evacetrapib but was far
less in magnitude than observed in the pharmacogenetic
study with dalcetrapib. This trend did not persist when
examining only harder cardiovascular outcomes or when
adjusting for cardiovascular risk factors. The completion of
the dalcetrapib pharmacogenetics outcome trial should
clarify whether this is a false signal or a paradigm-shifting
discovery.

ARTICLE INFORMATION
Accepted for Publication: February 20, 2018.
Published Online: March 11, 2018.
doi:10.1001/jamacardio.2018.0569
Author Contributions: Drs Nissen and Pillai
contributed equally to this article as co–first
authors.
Study concept and design: Nissen, Pillai, Nicholls,
Riesmeyer, Weerakkody, Ruotolo, Lincoff.
Acquisition, analysis, or interpretation of data: All
authors.
Drafting of the manuscript: Nissen, Pillai, Foster,
McErlean, Bhatnagar, Ruotolo.
Critical revision of the manuscript for important
intellectual content: Nissen, Pillai, Nicholls, Wolski,
Riesmeyer, Weerakkody, Li, Bhatnagar, Ruotolo,
Lincoff.
Statistical analysis: Wolski, Weerakkody, Li, Bhatnagar.
Obtained funding: Nissen, Pillai, Nicholls, Riesmeyer.
Administrative, technical, or material support:
Nissen, Pillai, Nicholls, Riesmeyer, Foster, McErlean,
Bhatnagar, Lincoff.
Study supervision: Nissen, Pillai, Nicholls,
Riesmeyer, Bhatnagar.
Conflict of Interest Disclosures: All authors have
completed and submitted the ICMJE Form for
Disclosure of Potential Conflicts of Interest.
Dr Lincoff reports grants from Eli Lilly during the
conduct of the study; grants from Astra Zeneca,
CSL, AbbVie, and Esperion outside the sumbmitted
work; and personal fees from Novo Nordisk outside
the submitted work. Dr Nicholls reports grants from
Eli Lilly during the conduct of the study; grants from
AstraZeneca, Amgen, Anthera, Eli Lilly, Novartis,
Cerenis, The Medicines Company, Resverlogix,
InfraReDx, Roche, Sanofi-Regeneron, and
LipoScience outisde the submitted work; and
personal fees from AstraZeneca, Eli Lilly, Anthera,
Omthera, Merck, Takeda, Resverlogix,
Sanofi-Regeneron, CSL Behring, Esperion, and
Boehringer Ingelheim outside the submitted work.
Dr Nissen reports grants and nonfinancial support
from Eli Lilly during the conduct of the study. Drs
Bhatnagar, Foster, Pillai, Riesmeyer, Ruotolo, and
Weerakkody are employees of Eli Lilly. No other
disclosures were reported.
Funding/Support: Eli Lilly and Company,
Indianapolis, Indiana.
Role of the Funder/Sponsor: The funding source
was involved in the design and conduct of the
study; collection, management, analysis, and
interpretation of the data; and preparation and
review of the manuscript; however, final approval
of the manuscript and the decision to submit the
manuscript for publication was the responsibility of
the academic authors.
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