Cardiovascular Disease

1. MULTI INSTITUTIONAL COHORT TO
FACILITATE CARDIOVASCULAR DISEASE
BIOMARKER VALIDATION USING EXISTING
BIOREPOSITORY SAMPLES – A PILOT
STUDY
Deanna Cross, PhD
Porat Erlich, PhD, MPH
“Any honest test of a theory is an attempt to refute it”
--Carl Popper

2. INTRODUCTION
 Acute myocardial infarction (AMI) is a leading cause of death world wide and
the number one cause of death in the developed world
 Even when non-fatal, AMI aftermath is devastating to the patient and costly to
society
 Etiology:
• Proximal cause: coronary artery thrombosis at sites of arterial stenosis
• Primary cause: atherosclerosis (lipid oxidation; inflammation; reverse cholesterol transport)
 Primary prevention relies on risk stratification and mitigation
 After 25 years of population-scale prevention, cardiac events still persist
many of which occur in individuals without detected risk

3. LIMITATIONS OF
CURRENT APPROACHES
Risk Calculation
 Framingham baseline data were collected 40 years ago (1971-1974)
 13 years before statins (first statin marketed in 1987)
 The mean BMI was 25.8 as compared to 30.6 in this cohort
 Cannot be re-parameterized
 ATP III operational guidelines for prescribing statin are more flexible but…
 Driven by LDL
The goal of this pilot study is to incorporate emerging biomarkers into the risk
factor framework for myocardial infarction

4. METHODS
 Design: retrospective cohort
 Setting: integrated healthcare systems with broad-consent biorepositories linked to EHR:
 Marshfield Clinic (Wisconsin)
 Geisinger Health System (Pennsylvania)
 Observation period 1/1/2004 to 05/30/2010 (dynamic entry; exit upon AMI, death or 05/30/2010)
 One year of lead time for exclusion of prevalent CVD
 Site differences:
 Biorepository initial contact: Marshfield=mailing; Geisinger=in clinic
 Baseline definition: MC=blood draw date; GHS=first encounter
 Person time at risk starts: MC=blood draw; GHS=1 year after 1st encounter
 Primary outcomes: incident AMI and statin indication (ATP III)
 Predictors (at baseline): age, sex, smoking history, blood pressure, overt hypertension, overt diabetes
mellitus, BMI, total-C, LDL-C, HDL-C
 Predictive modeling and evaluation of competing model performance
 Logistic regression with ten-fold cross validation
 Receiver Operating Characteristics analysis (ROC-AUC)
 Molecular methods
 Targeted genotyping
 Kinetic spectroscopy (paraoxonase)

5. COHORT CHARACTERISTICS
Marshfield Geisinger
Clinical variables Gender MI No MI All MI No MI All
N at closure (%females) N/A 382(40.3) 10161(59.1) 10543(58.4) 424(37.5) 7362(57.7) 7786(56.6)
Mean age Male 63.0 55.7 56.1 63.5 58.8 59.1
Female 64.7 55.9 56.2 66.0 58.1 58.3
Length of record [person-years] Male 6.0 5.8 5.8 6.0 5.9 5.9
Female 5.9 5.8 5.8 5.9 6.0 6.0
Mean BP diastolic Male 76.3 76.3 76.3 75.6 78.0 77.8
Female 74.1 74.7 74.7 74.6 76.7 76.6
Mean BP systolic Male 133.5 128.4 128.7 133.7 133.0 133.1
Female 132.6 126.5 126.7 134.3 131.0 131.2
Mean HDL Male 47.2 48.8 48.7 44.7 47.5 47.3
Female 55.8 61 60.9 52.0 57.3 57.1
Mean LDL Male 119.1 122.8 122.6 105.4 111.3 110.8
Female 120.7 119.2 119.3 108.6 118.2 117.8
Mean cholesterol Male 194 195.7 195.6 183.1 192.9 192.1
Female 204.1 201.2 201.3 201.3 207.6 207.4
% ever a smoker Male 71.5 54.5 55.4 80.8 63.9 65.3
Female 50.0 39.8 40.1 52.8 40.9 41.4
% diabetic Male 33.3 17.7 18.5 43.8 35.7 36.4
Female 39.0 14.6 15.2 52.8 31.5 32.3
% hypertensive Male 83.8 48.2 50.0 89.4 73.0 74.3
Female 89.6 47.7 48.7 91.8 70.1 70.9
 Number of subjects: 18,329 (58% females)
 Number of incident AMI cases: 806 (incidence rate 743 per 100,000 person-years)
 Mean age at baseline: 57.2 years old
 Mean record length: 5.9 years
 Smoking history (current or former): 49.8%
 Overt hypertension: 59%
 Overt diabetes mellitus 24%
 Statin indication (ATP III) 46.7%
 Mean time from baseline to event 2.4 years

6. RISK FACTORS FOR AMI IN THE JOINT
COHORT
Variable Mean(SE) or Odds ratio of MI in the
Frequency(column%) Joined Cohort
MI No MI All
No. of subjects 806 17522 18329
Age [years] 64.1 (0.37) 56.9(0.09) 57.2(0.09) 1.04 (1.03,1.05; 7.2E-23)
Male 493(61.2) 7273(41.5) 7767(42.4) 1.6 (1.3,1.8; 1.8E-07)
Female 313(38.8) 10249(58.5) 10562(57.6) Reference
HDL [mg/dL] 49.0(0.5) 54.8(0.1) 54.5(0.1) 0.98 (0.97,0.99; 3.2E-11)
Overt hypertension 712(88.3) 10117(57.7) 10829(59.1) 3.3 (2.6,4.3; 4.7E-22)
No overt HT 94(11.7) 7406(42.3) 7500(40.9) Reference
Ever a smoker 538(67.3) 8387(49.0) 8925(49.8) 1.8 (1.5,2.1; 6.4E-12)
Never a smoker 261(32.7) 8739(51.0) 9000(50.2) Reference
Overt diabetes mellitus 336(41.7) 4063(23.2) 4399(24.0) 1.4 (1.1,1.6; 2.6E-04)
No overt DM 470(58.3) 13460(76.8) 13930(76.0) Reference
BMI 30.5(0.3) 30.6(0.1)) 30.5(0.1) NS
LDL 112.8(1.3) 118.3(0.26) 118.0(0.25) NS
Total cholesterol 193.8(1.5) 200.0(0.3) 199.7(0.3) NS
Dia. blood pressure [mmHg] 75.3(0.4) 76.2(0.08) 76.1(0.08) NS
Sys. blood pressure [mmHg] 133.5(0.7) 129.3(0.13) 129.3(0.13) NS
Marshfield Clinic 382(47.4) 10161(58.0) 10543(57.5) NS
Geisinger Health System 424(52.6) 7362(42.0) 7786(42.5)
Length of record [person-years] 5.9(0.03) 5.9(0.01) 5.9(0.01) NS
N.S.: not significant (p>0.01)

7. PERFORMANCE CHARACTERISTICS OF A
SIMULATED BIOMARKER
HDL
Model Dichotomization sensitivity% specificity% PPV%
percentile S T1-T10 S T1-T10 S T1-T10
Risk factor >=90th 28.6 29.0 91.0 91.0 13.2 13.4
=0.42
model >=75th 59.4 59.6 76.7 76.6 11.0 11.0
>=50th 86.7 86.5 51.7 51.8 8.0 8.0
Risk factor >=90th 62.5 61.8 92.5 92.5 28.9 28.5
model + >=75th 81.6 81.5 77.7 77.7 15.1 15.1
biomarker >=50th 92.7 92.7 52.0 52.0 8.6 8.6
S: fitted and tested on the full cohort.
T1-T10: trained and fitted using 10-fold validation
Decision rule
ATPIII Practice Risk factor model Risk factor model
Guidelines + biomarker
% overall indicated at baseline 46.7 46.7 46.7
% of incident cases indicated at baseline 54.9 80.7 92.6
% of non-cases indicated at baseline 46.3 45.1 44.5
Net number of cases correctly reclassified 0 (reference) 200 cases 279 cases
•Biomarker simulated with delta=0.5 STDDEV
•cutoff set to the 53.3 percentile

8. ROC-AUC
DISEASE
+ -
+
TEST
-
RISK SCORE

9. DEPENDENCE ON BIOMARKER
INFORMATIVENESS
38%
13%

10. DEPENDENCE ON CASE-TO-CONTROL RATIO
Sensitivity, specificity and ROC-AUC
dependence on the case-to-control ratio
16% 50%
specificity gain 45%
14%
ROC-AUC gain
Percent gain over RF model
40%
12%
sensitivity gain 35%
10%
30%
8% 25%
20%
6%
15%
4%
10%
2%
5%
0% 0%
Case-to-control ratio

11. NESTED SAMPLE CHARACTERISTICS
Variable Odds ratio in the nested Odds ratio in the full
CC sample Cohort
Number of subjects 1540 18,329
Age [years] NS 1.04 (1.03,1.05; 7.2E-23)
Sex [male versus female] NS 1.6 (1.3,1.8; 1.8E-07)
HDL [mg/dL] 0.98 (0.97,0.983; 2.1E-06) 0.98 (0.97,0.99; 3.2E-11)
Overt hypertension [yes versus no] 3.9 (3.36,4.63;1.0E-18) 3.3 (2.6,4.3; 4.7E-22)
Smoker [ever versus never] 1.9 (1.6,2.1; 4.4E-07) 1.8 (1.5,2.1; 6.4E-12)
Overt DM [yes versus no] 1.3 (1.1,1.4; 0.07[N.S.]) 1.4 (1.1,1.6; 2.6E-04)
BMI NS NS
LDL NS NS
Total cholesterol NS NS
Dia. blood pressure [mmHg] NS NS
Sys. blood pressure [mmHg] NS NS
Site [MC versus GHS] NS NA
Length of record [person-years] NS NS
N.S.: not significant (p>0.01)

12. PON1,2,3 Anti-oxidation
Reverse cholesterol efflux
APOE Cholesterol transport
INITIAL SNP SCREEN 9p21 Uncertain function
CTLA4 Inflammatory response
IL10 Inflammatory response
CFTR Mendelian disorder with
cardiac manifestations
2.00% 6
[simulated biomarker versus risk factors]
1.00% 5
0.00% 4
-Log10(p-value)
APOE
ROC-AUC
9p21
-1.00% 3
P-value=(0.05/27) Delta ROC-AUC
-2.00% Significance level 2
-3.00% 1
-4.00% 0
rs662
rs7493
rs231775
rs213950
rs1800872
rs429358
rs854565
rs978903
rs854560
rs2375005
rs987539
rs2383206
rs2237582
rs2269829
rs2299255
rs1157745
rs1859121
rs2074352
rs3757708
rs2072200
rs3735586
rs10487133
rs6961624
rs2299263
rs1034809
rs2286233
rs2299267
Single Nucleotide Polymorphisms Screened

13. APOE (RS429358)
 Introduction
 Main apoprotein of the chylomicron
 Well established association with AMI and other vascular
outcomes
 Results in this cohort
 Association with AMI confirmed
 51% increase in the odds of AMI with each copy of allele ‘A’
(frequency(A)=2.1%)

14. 9P21 (RS2383206)
 Introduction
 Association with AMI
 Functionality and etiological role - unclear
 Results in this cohort
 Association with AMI confirmed
 38% increase in the odds of AMI with each copy of allele
‘G’ (frequency(G)=27.4%)

15. PARAOXONASE
 Introduction
 Lipoprotein-associated ester hydrolase
 Attracted considerable attention as a candidate biomarker
for atherosclerosis-driven vascular outcomes
 Results in this cohort
 Serum activity associated with lipid traits
 total-C, HDL-C & TG
 No evidence for association with AMI

16. CONCLUSIONS
 The Marshfield-Geisinger biorepository cohort can be
used for evaluating the clinical usability of emerging
biomarkers for AMI
 Single nucleotide polymorphisms in APOE and the
9p21 locus have discriminatory potential that merits
further investigation
 Serum paraoxonase is a potential therapeutic target
for hyperlipidemia

17. LIMITATIONS
 Continuous enrollment assumed
 Missing 1.5 plates of genotyping results
 Source populations are >98% Caucasian

18. ACKNOWLEDGEMENTS
 Marshfield:  Geisinger:
 Catherine McCarty  Walter (Buzz) Stewart
 Lynn Ivacic  Steven Steinhubl
 Rob Strenn  Glenn Gerhard
 Joe Finamore  Xin Chu
 CVRN (KP Northern  Ryan Kissinger
California):  Jessica Webster
 Allan Go
 Sue Hee Sung
This study was conducted within the Cardiovascular Research Network, a consortium of research
organizations affiliated with the HMO Research Network and sponsored by the
National Heart Lung and Blood Institute (NHLBI) (U19 HL91179-01).