“Any honest test of a theory is an attempt to refute it”--Carl Popper

MULTI INSTITUTIONAL COHORT TO FACILITATE CARDIOVASCULAR DISEASE BIOMARKER VALIDATION USING EXISTING BIOREPOSITORY SAMPLES – A PILOT STUDY

Deanna Cross, PhDPorat Erlich, PhD, MPH

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

LIMITATIONS OFCURRENT 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

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)

COHORT CHARACTERISTICSMarshfield Geisinger

Clinical variables Gender MI No MI All MI No MI AllN 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 58.3Length 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 6.0Mean 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 76.6Mean BP systolic Male 133.5 128.4 128.7 133.7 133.0 133.1

Female 132.6 126.5 126.7 134.3 131.0 131.2Mean HDL Male 47.2 48.8 48.7 44.7 47.5 47.3

Female 55.8 61 60.9 52.0 57.3 57.1Mean LDL Male 119.1 122.8 122.6 105.4 111.3 110.8

Female 120.7 119.2 119.3 108.6 118.2 117.8Mean 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

RISK FACTORS FOR AMI IN THE JOINT COHORT

Variable Mean(SE) orFrequency(column%)

Odds ratio of MI in the Joined Cohort

MI No MI AllNo. 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)

MaleFemale

493(61.2)313(38.8)

7273(41.5)10249(58.5)

7767(42.4)10562(57.6)

1.6 (1.3,1.8; 1.8E-07)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 hypertensionNo overt HT

712(88.3)94(11.7)

10117(57.7)7406(42.3)

10829(59.1)7500(40.9)

3.3 (2.6,4.3; 4.7E-22)Reference

Ever a smokerNever a smoker

538(67.3)261(32.7)

8387(49.0)8739(51.0)

8925(49.8)9000(50.2)

1.8 (1.5,2.1; 6.4E-12)Reference

Overt diabetes mellitusNo overt DM

336(41.7)470(58.3)

4063(23.2)13460(76.8)

4399(24.0)13930(76.0)

1.4 (1.1,1.6; 2.6E-04)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 ClinicGeisinger Health System

382(47.4)424(52.6)

10161(58.0)7362(42.0)

10543(57.5)7786(42.5)

NS

Length of record [person-years] 5.9(0.03) 5.9(0.01) 5.9(0.01) NSN.S.: not significant (p>0.01)

PERFORMANCE CHARACTERISTICS OF A SIMULATED BIOMARKER

Model Dichotomization percentile

sensitivity% specificity% PPV%S T1-T10 S T1-T10 S T1-T10

Risk factor model

>=90th 28.6 29.0 91.0 91.0 13.2 13.4>=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 model + biomarker

>=90th 62.5 61.8 92.5 92.5 28.9 28.5>=75th 81.6 81.5 77.7 77.7 15.1 15.1>=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 ruleATPIII Practice Guidelines

Risk factor model Risk factor model + 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 44.5Net 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

HDL

=0.42

ROC-AUC

+

+

-

-

DISEASE

TES

T

RISK SCORE

DEPENDENCE ON BIOMARKER INFORMATIVENESS

38%

13%

DEPENDENCE ON CASE-TO-CONTROL RATIO

1::1

1::2

1::3

1::4

1::5

1::6

1::7

1::8

1::9

1::10

1::11

1::12

1::13

1::14

1::15

1::16

1::17

1::18

1::19

1::20

1::21

1::22

0%

2%

4%

6%

8%

10%

12%

14%

16%

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

Sensitivity, specificity and ROC-AUCdependence on the case-to-control ratio

specificity gain

ROC-AUC gain

sensitivity gain

Case-to-control ratio

Perc

ent

gain

over

RF m

odel

NESTED SAMPLE CHARACTERISTICS

Variable Odds ratio in the nested CC sample

Odds ratio in the full Cohort

Number of subjects 1540 18,329Age [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 NSLDL NS NSTotal cholesterol NS NSDia. blood pressure [mmHg] NS NSSys. blood pressure [mmHg] NS NSSite [MC versus GHS] NS NALength of record [person-years] NS NSN.S.: not significant (p>0.01)

INITIAL SNP SCREEN

rs23

1775

rs23

8320

6

rs42

9358

rs22

6982

9

rs22

9925

5

rs85

4565

rs20

7435

2

rs97

8903

rs20

7220

0

rs10

4871

33

rs98

7539

rs22

9926

3

rs22

8623

3

rs74

93-4.00%

-3.00%

-2.00%

-1.00%

0.00%

1.00%

2.00%

0

1

2

3

4

5

6

Delta ROC-AUCSignificance level

Single Nucleotide Polymorphisms Screened

RO

C-A

UC

[sim

ula

ted b

iom

ark

er

vers

us r

isk f

acto

rs]

-Log10(p

-valu

e)

PON1,2,3 Anti-oxidationReverse cholesterol efflux

APOE Cholesterol transport

9p21 Uncertain function

CTLA4 Inflammatory response

IL10 Inflammatory response

CFTR Mendelian disorder withcardiac manifestations

P-value=(0.05/27)

APOE9p21

APOE (RS429358)

Introduction Main apoprotein of the chylomicron Well established association with AMI and other

vascular outcomes Results in this cohort

Ö Association with AMI confirmedÖ 51% increase in the odds of AMI with each copy of

allele ‘A’ (frequency(A)=2.1%)

9P21 (RS2383206)

Introduction Association with AMI Functionality and etiological role - unclear

Results in this cohortÖ Association with AMI confirmedÖ 38% increase in the odds of AMI with each copy of

allele ‘G’ (frequency(G)=27.4%)

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

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

LIMITATIONS

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

ACKNOWLEDGEMENTS

Marshfield: Catherine McCarty Lynn Ivacic Rob Strenn Joe Finamore

CVRN (KP Northern California): Allan Go

Sue Hee Sung Geisinger:

Walter (Buzz) Stewart Steven Steinhubl Glenn Gerhard Xin Chu Ryan Kissinger Jessica Webster

This study was conducted within the Cardiovascular Research Network, a consortium of research organizations affiliated with the HMO Research Network and sponsored by theNational Heart Lung and Blood Institute (NHLBI) (U19 HL91179-01).